Influenza virus vaccines and uses thereof.
Abstract
The present invention provides influenza hemagglutinin stem domain polypeptides comprising (a) an influenza hemagglutinin HA1 domain that comprises an HA1 N-terminal stem segment, covalently linked by a linking sequence of 0-50 amino acid residues to an HA1 C-terminal stem segment, and (b) an influenza hemagglutinin HA2 domain, wherein on or more amino acids in the HA2 domain have been mutated. Also provided are nucleic acids encoding the polypeptides, compositions comprising the polypeptides and/or nucleic acid molecules, as well as methods of their use, in particular in the detection, prevention and/or treatment of influenza.

Term
6.2 yearsleft in the term
Expires 27 November 2032.
- Priority
- Filed
- Granted
- Today
- Expires
12 claims: 4 independent, 8 dependent
- 1REIVINDICACIONES 1. Un polipéptido que comprende (a) un dominio HAl de la hemaglutinina de la influenza, que comprende un segmento de tallo N-terminal de HAl que 10 comprende los aminoácidos 1 a 52 de HAl, ligados covalentemente por una secuencia de ligación de 0 a 10 residuos de aminoácidos a un segmento del tallo C-terminal de HAl que comprende los aminoácidos 321 hasta el extremo de HAl, caracterizado porque los aminoácidos 53 a 320 se 15 eliminan, y (b) un dominio HA2 de la hemaglutinina de la influenza, en donde el residuo de aminoácido C-terminal del segmento de tallo C-terminal HAl es cualquier aminoácido diferente a arginina (R) o Usina (K) ; 20 en donde uno o más aminoácidos en las posiciones 406, 409, 413 y 416 han mutado en un aminoácido seleccionado del grupo que consiste en S, Τ, N, Q, R, Η, K, D, E y G, y en donde los polipéptidos comprenden un puente disulfuro entre los aminoácidos de la posición 324 y 436; Μ Η· 280 Va. TiV’I INSTITUTO MLXICAHO '/'¿l·' C i ·,! Dt LA PROPIEDAD ' INDUSTRIAL ~'· en donde el dominio HAl y el vdominio HAZ—se- der-i-v-an—del virus la influenza tipo A subtipo Hl; y en donde la numeración se basa en la numeración de los aminoácidos de la cepa de influenza H1N1 A/Brisbane/59/2007 (SEQ ID NO:1).
- 2El polipéptido de conformidad con la reivindicación 1, caracterizado porque caracterizado porque el polipéptido está glicosilado.
- 3El polipéptido de conformidad con la reivindicación 1, caracterizado porque el polipéptido comprende una o más mutaciones adicionales en el dominio HAl y/o el dominio HA2.
- 4El polipéptido de conformidad con la reivindicación 1, caracterizado porque el polipéptido no comprende la secuencia de señal.
- 5El polipéptido de conformidad con la reivindicación 1, caracterizado porque el polipéptido no comprende la secuencia intracelular y de transmembrana de HA. I
- 6El polipéptido de conformidad con la reivindicación 5, caracterizado porque él polipéptido no comprende la parte C-terminal del dominio Hl HA2 que abarca desde el residuo de aminoácido de lá posición 520, 521, 522, 523, 524, 525, 526, 527, 528, 529 o 530 hasta el aminoácido C-terminal de H1HA2. ΊUn método para producir un polipéptido caracterizado porque comprende:$ PI A i 281 proveer una secuencia de aminoácidos de influenzaHAO;eliminar el sitio de escisión entre HAl y HA2, por la mutación del aminoácido C-terminal de HAl en un aminoácido diferente de arginina (R) o Usina (K) ;remover la secuencia de aminoácidos que comprende los residuos de aminoácidos desde la posición 53 hasta y con la inclusión del residuo de aminoácido en la posición 320 del dominio de cabeza globular de la secuencia HAO y juntar las partes remanentes de la secuencia directamente o mediante la introducción de una secuencia de ligación de 1 a 10 aminoácidos de largo;introducir una o más mutaciones en la secuencia de aminoácidos que une al residuo C-terminal de la hélice A con el residuo N-terminal de la hélice CD;e introducir al menos un puente disulfuro en el polipéptido del dominio del tallo de HA entre los aminoácidos 324 y 436 (numeración de conformidad con la SEQ ID NO:1).
- 78. Una composición inmunogénica, caracterizada porque comprende el polipéptido de conformidad con la reivindicación 1.
- 89. Una composición inmunogénica, caracterizada porque comprende el polipéptido de conformidad con la reivindicación 1 en una cantidad suficiente para inducir una respuesta inmune al sujeto. 282 INSTITI r DE I.A PROPIEDAD ikduto.u
- 910. El polipéptido de conformidad con la reivindicación 1, caracterizado porque el residuo de aminoácido C-terminal del segmento de tallo C-terminal HAl es glutamina (Q).
- 1011. El polipéptido de conformidad con la reivindicación 1 caracterizado porque el polipéptido comprende dominios de tallo de hemaglutinina de o basados en HA del virus de influenza A/Brisbane/59/2007 (SEQ ID NO:1).
- 1112. El polipéptido de conformidad con la reivindicación 1 caracterizado porque el polipéptido comprende SEQ ID NO:84 en las posiciones 419 a 433.
- 1213. El polipéptido de conformidad con la reivindicación 1, caracterizado porque el polipéptido se une selectivamente a los anticuerpos CR6261 y/o CR9114 y no se une al_ anticuerpo CR8057. 283
Independent claims12
2,527 paragraphs in 498 sections, as filed
(54) Title: VACCINES AGAINST THE INFLUENZA VIRUS AND ITS USES. (54) Title: INFLUENZA VIRUS VACCINES AND USES THEREOF.
(57) Summary
The present invention relates to Polypeptides comprising influenza hemagglutinin stem domain polypeptides, wherein said polypeptides comprise (a) an influenza hemagglutinin HA1 domain, comprising an N-terminal stem segment of HA1, covalently linked by a 0-50 amino acid linker sequence to a segment of the C-terminal stem of HA1, and (b) an HA2 domain of influenza hemagglutinin in which one or more amino acids have been mutated. Nucleic acids encoding such polypeptides, compositions comprising polypeptides and / or nucleic acid molecules are also provided; as well as methods for using them, particularly in the detection, prevention and / or treatment of influenza.
(57) Abstract
The present invention provides influenza hemagglutinin stem domain polypeptides comprising (a) an influenza hemagglutinin HA1 domain that comprises an HA1 N-terminal stem segment, covalently linked by a linking sequence of 0-50 amino acid residues to an HA1 C-terminal stem segment, and (b) an influenza hemagglutinin HA2 domain, where on or more amino acids in the HA2 domain have been mutated. Also provided are nucleic acids encoding the polypeptides, compositions comprising the polypeptides and / or nucleic acid molecules, as well as methods of their use, in particular in the detection, prevention and / or treatment of influenza.
IMPI
<img file="MX357009B_D0001.tif" />
PATENT TITLE No. 357009
Headlines): JANSSEN VACCINES & PREVENTION BV
Address: Archirnedesweg 4, NL-2333, CN Leiden, NETHERLANDS
Denomination: VACCINES AGAINST THE INFLUENZA VIRUS AND ITS USES.
Classification:
Inventor (s):
CIP: C07K14 / 11; A61K39 / 12; „A61K39 / 145; C07K16 / 10; C12N7 / 00
CPC: C07K14 / .11; A6í1K ^ 12 | <A61K¿39 / 145; C07K16 / 1018; C12N7 / 00;
A61K2039 / 5258; Á6tkÉ039 «^ 22; £ 07X2317 / 33; * *
JAN WILEM MEUBERG; ANTONIETTA IMPAGB1AZZO; RONALD VOGELS; ROBERT HEINZ EDWARD FRIESEN, PfitÜPP & ^ LARD; STÉFAN LOVERIX; KATARINA RADOSÉVIC
Number:
MX / a / 20147006396
Country:
EP
US
<img file="MX357009B_D0002.tif" />
international:
what 2012
Number:
11191003.0 5«,086
Validity: Twenty years
Winding Date of 27 March 2032 3
Exned | c ^ on Date: ¿ejifiio 208 ^
The patent of reference «». Ot <r | a with foundation éíM®íFÍehsjj | úos U, 2<sup>6</sup> frícc ^ tV S<sup>Q</sup> féccij ^ ii, fSSjdéWtey of the fet-qsp ^ ací Industrial.
Pursuant to the article ie Law dMa + Wptedatf Industriadla preísnjp MWf ^ íeít®1 (mM «0eícia de veinfc adfccfmprorrogaWes, counted from the date of presentation of the international affcity y-esíariS eefjwa pa ^ jM ^ to rate pa | a «Loe-rights people.
Who subscribes to the present title is the b asis of the erijo diabuesto Ttoíadb artlculi »6'tóeawnes III í7 * .Hs Λ of the Industrial Property Law (Official Gazette of the Federation (D.OdF)« 7/0 & 199T, tefamiAa el fe ( fe / l994, fc / MMsW 26M2 / W7, 17 / 0S / 1999, 01/26/2004, 06/16/2005, 01/25/2006, 06/05/2009, 06/01/2010, IWOWMrtO / te / oe / Se'lQ 27 / lfr / tel2 vt> 9¡0 # 2jM), articles 1 °, * ° V'inciso a), 4 ° and 12 ° fractions I and of the Regulations of the Mexican Institute of Ι <Εΐ ^ βΐ ^ ΜύΜΜ (ί4 ^ (Ο.θΤΓ3 ^ 9τ999, reforfria ^^ lPI Q1 / Q £ / 2ÓÓíd 07/15/2004, 07/28/2004 and 09/07/2007): articles 1, 3, 4, 5, section V Subsection a), 16 (MHÓWÓs HrvjjIRÍel tstjti
12/27/1999, amended on 10/10/2002, 07/29/2004, Ο ^ ββΛΛΜ '
Deputy Generals, Coordinator. DivisiorafesrTItAj Directors
<img file="MX357009B_D0003.tif" />
'1
MfckÓHan:
Departmental and other subordinates of the Instituto Mexicano de la Tro ^ Jetyp ^ tetrial. (DOF 12/15/1999, 08/04/2004 and 09/13/2007).
'Carenes III 26 | 2 1 °. ly oiro / 2o8: utowlexicano of Industrial Property (DOF. Agreement that delegates powers to the final Directors, Divisional Deputy Directors. Coordinators amended on 02/04/2000, 07/29/2004,
This letter is signed with an advanced electronic signature (FIEL), based on articles 7 BIS 2 of the Industrial Property Law; 3 of its Regulations, and 1 section III, 2 section V, 26 BIS and 26 TER of the Agreement establishing the guidelines for the use of the Electronic Payment and Services Portal (PASE) of the Mexican Institute of Industrial Property, in the procedures indicated.
THE DIVISIONAL DIRECTOR OF PATENTS
NAHANNY CANAL REYES
<img file="MX357009B_D0004.tif" />
Original string:
NAHANNY MARISOL CANAL REYES | 00001000000403252793 | Tax Administration Service695 || MX / 2018/52228 | MX / a / 2014/006396 | Patent title PCT | 1223 | GAGV | Pág (s) 2 | kESfVU4aoBwwNVRXJn7h8P
Digital stamp:
hJJ97Pqt4u30dv3nFEYEjUa + mSUYt1YFkC4LfJccCKPD2mc7PbXLE2TGZMgPCmLblc + flkRTOjNfmojz9¡9Od5ZLa5 yd5Omplzsup99H¡MRT95NNdtGYUCQQNLYItwbWINXD3RQT / jCDY3IQVEvlnpOTNoenmFhll8GK0¡FvfKyv3UAR2c1A nunBI74IGhotUbqqLgx7tPJeOE8oRRXv6W3G1 ueBNcMkjF + + + 8T9ZpNk43TCPVnlu8Vg2wKPVIJvfgK3i3XkjPx HIW bcgNM + NV9ICzgPlsDarRgY8eaTBzx8TydENJjfKKrvNJcKfcgypD7BTogwjwCNRFfm7tJ2ZA == * Additional information on the back
Arenal No. 550, Floor 1, Pueblo Sarita Maria Tepepan, Xochimilco, 16020, Mexico City.
(55) 53340700 www.gob.mx/impl
<img file="MX357009B_D0005.tif" />
MX / 2018/52228
2 and
<img file="MX357009B_D0006.tif" />
<img file="MX357009B_D0007.tif" />
'7, -ι
VACCINES AGAINST INFLUENZA VIRUSES AND SUBJECTS
FIELD OF THE INVENTION
The invention relates to the field of medicine.
Influenza hemagglutinin stem domain polypeptides are provided herein; methods of providing hemagglutinin stem domain polypeptides; compositions comprising the same; vaccines comprising the same; and methods of using them, in particular for the detection, prevention and / or treatment of influenza.
BACKGROUND OF THE INVENTION
Influenza viruses are important human pathogens, which cause a respiratory disease (commonly known as influenza or the flu) whose severity varies in a range between a subclinical infection and a primary viral pneumonia that can result in death. The clinical effects of infection vary with the virulence of the strain and exposure to influenza, with the history, age, and immune status of the host. It is estimated that approximately one billion people worldwide will contract an infection with the influenza virus each year, including 3-5 million cases
<img file="MX357009B_D0008.tif" />
they will develop severe illness and around — ete — 3'ΘΦ ·: Θ-ΘΌ—
500,000 will correspond to influenza-related deaths. Most of these infections can be attributed to influenza A viruses that comprise the hemagglutinin Hl or H3 subtypes, and a lower contribution by influenza B viruses, therefore all three are represented in the seasonal vaccine. Current immunization practice is based on the early identification of influenza viruses in circulation for the scheduled production of an effective seasonal influenza vaccine. In addition to the inherent difficulties in predicting strains that will dominate during the next season, antiviral resistance and immune leakage also play a role in the failure of current vaccines to prevent morbidity and mortality. In addition to this, the possibility of a pandemic caused by a highly virulent viral strain originating in animal reservoirs and redistributed in such a way as to increase dispersion between humans, represents a significant and real threat to global health.
Influenza A viruses are widely distributed in the wild and can infect a variety of birds and mammals. Influenza viruses are enveloped RNA viruses that belong to the Ortomixoviridae family. Their genomes consist of eight single-stranded RNA segments
IMPI
INSTITUTO MEXICANO DI LA MOHEDA!)
INDUSTRIAL encoding 11 different proteins, one myeloprotein (NP), three polymerase proteins (PA, PB1 and PB2), two matrix proteins (MI and M2), three non-structural proteins (NS1, NS2 and PB1-F2) and two external glycoproteins: hemagiutinin (HA) and neuraminidase (NA). Viruses are classified based on differences in the antigenic structure of HA and NA proteins, where different combinations represent unique viral subtypes, which in turn are classified into specific strains of the influenza virus. Although all known subtypes are found in birds, the influenza HlNl and H3N2 subtypes
A human are those found in circulation. Phylogenetic analyzes have shown that hemagglutinins are subdivided into two large groups: among others, subtypes Hl, H2, H5 and H9 in phylogenetic group 1 and for example subtypes H3, H4 and H7 in phylogenetic group 2.
Strains of influenza virus type B are strictly human. The antigenic variation in HA between strains of influenza B virus is less than that observed among strains of type A. There are two genetically and antigenically distinct lineages of influenza B virus in circulation in humans, represented by B lineages. / Yamagata / 16/88 (also called B / Yamagata) and B / Victoria / 2/87 (B / Victoria) (Ferguson et al.,
2003). Yes
<img file="MX357009B_D0009.tif" />
Although the spectrum of disease caused by influenza B viruses is generally milder than the spectrum caused by influenza A viruses, severe diseases requiring hospitalization with influenza B infection are still frequently observed.
Antibodies that neutralize influenza virus are known to be directed primarily against hemagglutinin (HA). Hemagglutinin or HA is a trimeric glycoprotein that is anchored to the viral coating and has a dual function: it is responsible for the binding of sialic acid to the cell surface receptor and, after 'uptake, it is involved in the fusion of viral membranes and endosomal which leads to the release of viral RNA into the cytosol of the cell. HA comprises a large head domain and a smaller stem domain. Binding to the viral membrane is mediated by a C-terminal anchor sequence connected to the stem domain. The protein is cleaved after translation in a loop resulting in two polypeptides, HAl and HA2 (the entire sequence is called HAO). The distal head region in the membrane derives primarily from HA1 and the proximal stem region in the membrane derives primarily from HA2 (Figure 1).
The reason why influenza vaccines
<img file="MX357009B_D0010.tif" />
Seasonal should be updated every year is 1 to large »variability of the virus. In the hemagglutinin molecule this variation manifests itself particularly in the head domain, where antigenic drift and shift have resulted in a large number of different variants. Data that this is also the area that is immunodominant, most of the neutralizing antibodies are directed against this domain and act by interference with receptor binding. The combination of immunodominance and large variation in head domain also explains why an infection with a particular strain does not lead to immunity for other strains: the antibodies generated by the first infection only recognize a limited number of strains closely related to the virus of the primary infection.
Recently, influenza hemagglutinin stem domain polypeptides, where all or substantially all of the globular head domain of influenza hemagglutinin is missing, have been described and used to elicit an immune response to one or more conserved epitopes of the stem domain of the polypeptide. The epitopes of the stem domain polypeptide are believed to be less immunogenic than the highly immunogenic regions of a globular head domain, therefore the absence of ϊ> ·
D h Λ // i 7 'i' ·
... <? T&T 'MEXICAN ·
FROM PROPERTY 0 ^ ~ £ & -, ί INDU. <Tilil * * ^ * £ 1 A globular head domain in the polypeptide-_del__ domipio could allow the development of an immune response against one or more epitopes of the polypeptide of the stem domain (Steel et al., 2010). Steel et al. they have then created a new molecule by deletion of amino acid residues 53 to 276 of HA1 from strains A / Puerto
Rico / 8/1934 (H1N1) and A / Hong Kong / 1968 (H3N2) of the HA primary sequence, and replacing them with a short flexible GGGG linker sequence. Vaccination of mice with the H3 HK68 construct did not generate a cross-reactivity antiserum with group 1 HAs. Furthermore, as shown later in the Examples, the stem domain polypeptides were highly unstable and did not adopt the correct conformation as evidenced by the lack of binding of the antibodies that had demonstrated their binding to conserved epitopes in the stem region .
Furthermore, Bommakanti et al., (2010) described a HA2-based polypeptide comprising amino acid residues 1-172 of HA2, a 7-amino acid linker (GSAGSAG), amino acid residues 7-46 of HA1, a connector of 6 GSAGSA amino acids, followed by residues
290-321 of HA1, with mutations V297T, I300E, Y302T and
C305T on HA1. The design was based on the H3 HA sequence (A / Hong Kong / 1968). The polypeptide not only had
<img file="MX357009B_D0011.tif" />
cross-protection against another strain of influenza virus · within subtype H3 (A / Phil / 2/82) but did not present against subtype Hl (A / PR / 8/34).
The need therefore remains for a safe and effective universal vaccine capable of stimulating the production of a broadly neutralizing and robust antibody response and offering protection against a broad range of current and future strains of influenza virus (both seasonal and pandemic). , in particular to provide protection against one or more influenza A virus subtypes within phylogenetic group 1 and / or phylogenetic group 2, for effective prevention and therapy of influenza.
SUMMARY OF THE INVENTION
Stem domain polypeptides of influenza hemagglutinin are provided herein; methods for obtaining the stem domain polypeptides; compositions comprising the same; vaccines comprising the same; and methods for its use.
In a first aspect, the present invention provides novel immunogenic polypeptides that comprise a stem domain but not the globular head of influenza hemagglutinin, called influenza hemagglutinin stem domain polypeptides (HA). Polypeptides
<img file="MX357009B_D0012.tif" />
they have the ability to induce an immune response.
when administered to a subject, particularly a human subject. The polypeptides of the invention display conserved epitopes of the HA membrane proximal stem domain molecule for the immune system in the absence of dominant epitopes that are present in the head domain distal to the membrane. To this end, part of the primary sequence of the HAO protein that makes up the head domain is removed and the amino acid sequence is reattached, either directly or, in some modalities, by introducing a flexible short linker sequence (' connector ') to restore the continuity of the amino acid chain. The resulting sequence is modified by introducing specific mutations that stabilize the native three-dimensional structure of the remaining part of the HAO molecule. Immunogenic polypeptides do not comprise HA1 and / or full-length HA2 from an influenza virus.
The influenza hemagglutinin stem domain polypeptides are based on HA strains of influenza viruses that are generally used in the production of vaccines against human influenza. In particular, the polypeptides are based on the HA of influenza A viruses of subtypes Hl, H5 and / or H3.
<img file="MX357009B_D0013.tif" />
Μ Ρ1
INSTITUTO «MEXICANO DE LA 1 ROFlt '-' AÜ IND'Jf.TRÍAL
In certain embodiments, the invention "provides polypeptides from the influenza hemagglutinin stem domain comprising (a) an HAl influenza hemagglutinin domain comprising a covalently linked segment of the N-terminal stem of HAl by means of a linker sequence of 0-50 amino acid residues to a segment of the C-terminal stem of HAl, and (b) an HA2 domain of influenza hemagglutinin, wherein the hemagglutinin stem domain polypeptides are resistant to cleavage by proteases at the junction between HAl and HA2, and wherein one or more of the amino acids in the amino acid sequence binding to the A helix and the CD helix HA2 was mutated when compared to a wild type influenza HA2 domain. Preferably, the HAl and HA2 domains are derived from an influenza A virus selected from the group consisting of the Hl, H5 and H3 subtypes.
The polypeptides of the invention comprise one or more mutations in the HA2 amino acid sequence that joins the C-terminal residue of the A helix with the N-terminal residue of the CD helix, as indicated in Figure 1. In certain embodiments, one or more hydrophobic amino acids has been substituted in said amino acid sequence
HA2 by hydrophilic amino acids, such as polar and / or charged amino acids, or the flexible amino acid glycine (G).
MEXICAN INSTITUTE ΜΙ
OELAP «.<sub>Wle! M</sub>¿
IΜ ΡI a - ». ™
In certain embodiments, the segment of T £ alT <5 ~ Tr = - ·· terminal of HAl comprises amino acids 1-x of HAl, and the segment of the C-terminal stem of HAl comprises amino acids and-th terminals (i.e. a Cterminal amino acid of HAl) of HAl. Accordingly, in certain embodiments, deletion in the HAl segment comprises the amino acid sequence between the amino acid at the x + 1 position to and including the amino acid at the y-1 position. In certain embodiments, the polypeptides do not comprise the signal sequence. Accordingly, in certain embodiments, the N-terminus HAl segment comprises the px amino acid of HAl, where p is the first amino acid of the mature HA molecule (eg p = 18 in the case of SEQ ID NO: 1). The specialist will be able to prepare the polypeptides described herein without the signal peptides (for example, amino acids 1-17 of SEQ ID NO: 1). In certain embodiments, the polypeptides of the invention contain the intracellular sequences of HA and the transmembrane domain. In other embodiments, the polypeptides of the invention do not comprise the intracellular sequences of HA and the transmembrane domain. In certain embodiments, the intracellular sequence and the transmembrane sequence have been removed, for example the amino acid sequence from position (or its equivalent) 523, 524, 525, 526, 527, 526 i ívjl r λ
INSTITUTE McXlvAN'7 l! T! .A arohedau iNr / USTW.L
528, 529, or 530 from the HA2 domain to the Gt-ernrfrrsL end of the HA2 domain.
The polypeptides of the invention do not comprise full-length HA1.
In certain embodiments, the polypeptides are glycosylated.
In certain embodiments, the immunogenic polypeptides are substantially less than HAO, preferably without all or substantially all of the globular head of HA. Preferably, the immunogenic polypeptides are not more than 360, preferably not more than 350, 340, 330, 320, 310, 305, 300, 295, 290, 285, 280,
275 or 270 amino acids in length. In certain embodiments, the immunogenic polypeptides are between about 250 and about 350, preferably between about 260 and about 340, preferably between about 270 and about
330, preferably between about 270 and about 330 amino acids in length.
In certain embodiments, the polypeptides further comprise one or more additional mutations in the HA1 domain and / or
HA2, compared to the amino acid sequence of HA on which the HA 1 and HA2 domains are based.
The invention also provides methods for supplying influenza hemagglutinin stem polypeptides, ----
<img file="MX357009B_D0014.tif" />
which comprise the general steps of:
<td>(to)</td><td>provide</td><td>an amino acid sequence</td><td>HAO's</td><td>the</td>
<td colspan="2">influenza;</td><td></td><td></td><td></td>
<td>(b)</td><td>remove</td><td>the cleavage site between HAl and</td><td>HA2;</td><td></td>
<td>(c)</td><td>remove</td><td>the amino acid sequence of the</td><td>domain</td><td>of</td>
<td>head</td><td>globular</td><td colspan="2">of the HAO sequence, in particular</td><td>the</td>
amino acid sequence between positions x + 1 and y-1;
(d) introducing one or more mutations in the amino acid sequence that joins the C-terminal residue of helix A with the N-terminal residue of helix CD; and (e) introducing one or more disulfide bridges into the HA stem domain polypeptide.
The polypeptides that can be obtained by such methods also form part of the present invention.
In certain embodiments the polypeptides comprise conserved epitopes from the stem domain of group 1 cross neutralizing antibodies, CR6261 (disclosed in
W02008 / 028946) and / or the CR9114 antibody (as described below and in copending application EP 11173953.8), an antibody capable of binding and neutralizing group 1 and group 2 of influenza A viruses, as well as viruses of influenza B. Accordingly, another aspect of the invention is to provide stem domain polypeptides of the
<img file="MX357009B_D0015.tif" />
HA, where said polypeptides bind to anti-s-uer-pG-GR & Sé-l · and / or to CR9114 antibody. In one embodiment, the polypeptides do not bind CR8057 (described in WO 2010/130636), a monoclonal antibody that only binds to H3 influenza viruses. In certain embodiments, the polypeptides bind to the CR8020, CR8043, and / or CR9114 antibodies. The influenza hemagglutinin stem domain polypeptides provided herein are suitable for use in immunogenic compositions (eg, vaccines) capable of eliciting immune responses against a plurality of influenza A and / or B virus strains . In one embodiment, influenza hemagglutinin stem domain polypeptides can elicit immune responses against strains of influenza A viruses of phylogenetic group 1 and / or 2, in particular against strains of influenza viruses of both phylogenetic groups 1 and 2. In one embodiment, the polypeptides can elicit an immune response against homologous strains of influenza virus. In one embodiment, the polypeptides can elicit an immune response against heterologous strains of influenza viruses of the same subtypes and / or different subtypes. In a further embodiment, the polypeptides can elicit an immune response against influenza virus strains of both phylogenetic groups 1 and 2 and against strains of
<img file="MX357009B_D0016.tif" />
influenza Β.
The polypeptides according to the invention can be used, for example, in a therapy and / or prophylaxis and / or autonomous diagnosis of a disease or condition caused by an influenza virus, in particular an influenza A virus phylogenetic group 1 or 2 and / or an influenza B virus, or in combination with other prophylactic and / or therapeutic treatments, such as vaccines (existing or future), antiviral agents and / or monoclonal antibodies.
In a further aspect, the present invention provides the nucleic acid molecules encoding the influenza HA stem domain polypeptides.
In yet another aspect, the invention provides vectors comprising nucleic acids encoding immunogenic polypeptides.
In a further aspect, the invention provides methods of inducing an immune response in a subject, where the methods comprise administering to the subject a polypeptide and / or a nucleic acid molecule according to the invention.
In another aspect, the invention provides immunogenic compositions comprising a polypeptide and / or a nucleic acid molecule according to the invention. The
<img file="MX357009B_D0017.tif" />
Immunogenic compositions provided in the container can be found in any form that allows the compositions to be administered to a subject, for example mice, ferrets or humans. In a specific embodiment, the immunogenic compositions are suitable for administration to humans.
Polypeptides, nucleic acid molecules, and compositions can be used in methods to prevent and / or treat disease caused by an influenza virus and / or for diagnostic purposes. The compositions may further comprise a pharmaceutically acceptable carrier or excipient. In certain embodiments, the compositions described herein comprise or are administered in combination with an adjuvant.
In another aspect, the invention provides polypeptides, nucleic acids and / or immunogenic compositions for use as a vaccine. The invention relates in particular to immunogenic polypeptides, nucleic acids and / or immunogenic compositions for use as a vaccine in the prevention and / or treatment of a disease or condition caused by a subtype of phylogenetic groups 1 and / or 2 of influenza A virus and / or an influenza B virus.
The various modalities and uses of the polypeptides according to the invention will become clear from the following detailed description of the invention.
<img file="MX357009B_D0018.tif" />
BRIEF DESCRIPTION OF THE FIGURES____
Figure 1: Shows a model of the HA monomer in the prefusion state present in the native trimer. HA 1 is shown light gray, HA2 is shown dark gray. Helix A (an important part of the epitope of
CR6261) and the CD helix (part of the trimer interface), as well as the loop that joins these elements of the secondary structure.
Figures 2A-2B: Binding of monoclonal antibodies to full-length HA and stem domain HA polypeptides according to the invention analyzed by
FACS. Fig. 2A: Percentage of positive cells after staining. Fig. 2B: Average fluorescence intensity. Hl of
<td colspan="2">full length</td><td colspan="2">(SEQ ID N °: 1), miniHA-cll</td><td colspan="3">(SEQ ID N °:</td><td> 3) ,</td>
<td>miniHA-cll + 2</td><td>(I KNOW THAT</td><td>ID N °:</td><td>4), miniHA-cll + 3</td><td>(I KNOW THAT</td><td>ID</td><td>N °:</td><td> 5) ,</td>
<td>miniHA-cll + 4</td><td>(I KNOW THAT</td><td>ID N °:</td><td>6) miniHA-cll + 2 + 3</td><td>(I KNOW THAT</td><td>ID</td><td>N °:</td><td> 7) ,</td>
miniHA-cll + 2 + 3 + 4 (SEQ ID N °: 8).
Figures 3A-3B: Binding of monoclonal antibodies to full-length HA and stem domain HA polypeptides analyzed by FACS. Fig. 3A: Percentage of positive cells after staining. Fig. 3B: Average fluorescence intensity. Hl full length (SEQ ID No.: 1), miniHA (SEQ ID No.: 2), miniHA-cll (SEQ ID No.: 3)
Figures 4A-4B: Binding of serum antibodies to HEK293F
<img file="MX357009B_D0019.tif" />
<img file="MX357009B_D0020.tif" />
expressed full-length HA and the He ™ Ta ™ invention polypeptides. Fig. 4A: Average fluorescence intensity.
Fig. 4B: Percentage of positive cells after staining. Hl-FL (SEQ ID No.: 1), CL1 (SEQ ID No.: 3), CLl + 2 (SEQ
ID N °: 4) and CL1 + 4 (SEQ ID N °: 6). cM2 is a negative control.
Figure 5: Binding of monoclonal antibodies to full-length HA and stem domain HA polypeptides analyzed by FACS. Top: Percentage of positive cells after staining. Bottom:
Fluorescence average intensity. H1 full length (SEQ ID No.: 1), miniHA-cll (SEQ ID No.: 3), Hl-minil-clll (SEQ
ID No.: 9), Hl-mini2-clll (SEQ ID No.: 10), Hl-mini3-clll (SEQ
ID N °: 11) ,, Hl-mini4-clll (SEQ ID N °: 12), Hl-minil-clll + 5 (SEQ ID N °: 13), Hl-mini2-clll + 5 (SEQ ID N ° : 14), H1 mini315 clll + 5 (SEQ ID N °: 15) and Hl-mini4-clll + 5 (SEQ ID N °: 16).
Figures 6A-6B: Binding of serum antibodies to the full-length HA / Brisbane ectodomain
59/2007 after immunization im with DNA encoding HA from A / Brisbane / 59/2007 (SEQ ID No.: 1), MiniHA-clusterl (SEQ ID 20 No.: 3), Mini2-clusterll (SEQ ID No. : 10), Minil-clusterll + 5 (SEQ ID N °: 13), Mini2-clusterll + 5 (SEQ ID N °: 14) and cM2 (consensus sequence M2) or immunization with DNA gene coding for HA from A / Brisbane / 59/2007 (SEQ ID No.: 1), Mini2-clusterll + 5 (SEQ ID No.: 14) and cM2 (sequence
<img file="MX357009B_D0021.tif" />
M2 consensus). Fig.6A: 28 days after — ia — pximexa __________ immunization. Fig. 6B: after 49 days of immunization.
Figure 7: Binding of monoclonal antibodies to full-length HA and stem domain HA polypeptides analyzed by FACS. Top: Percentage of positive cells after staining. Bottom: Average fluorescence intensity. Full-length H1 (SEQ ID No.: 1), miniHA (SEQ ID No.: 2), Hl-minÍ2-clll + 5 (SEQ
ID N °: 14), Hl-mini2-cll +5 (SEQ ID N °: 48), Hl-mini2-cll + 5 + 6 (SEQ ID N °: 46), Hl-mini2-clll + 5 + 6 (SEQ ID No.: 47), Hl-mini2cll + 5 + 6-trim (SEQ ID No.: 44), Hl-mini2-cll + 5 + 6-GCN4 (SEQ ID
N °: 45).
Figures 8A-8B: Binding of monoclonal antibodies to full-length HA and stem domain HA polypeptides analyzed by FACS. Fig. 8A: Percentage of positive cells after staining. Fig. 8B: Average fluorescence intensity.
Figures 9A-9B: Binding of monoclonal antibodies to full-length HA and stem domain HA polypeptides analyzed by FACS. Fig. 9A: Percentage of positive cells after staining. Fig. 9B: Average fluorescence intensity.
Figures 10A-10B: Expression of constructs based on Hong Kong / 1/1968 on the cell surface.
<img file="MX357009B_D0022.tif" />
Figures 11A-11F: Analysis by SDS-PAGE- (Fig.HAFig.llD) and Western blot (Fig.llE-Fig.11F) of the purification of various polypeptides of the invention. For Western blot an antibody directed against the his-mark is used for detection.
Figures 12A-12C: Binding of monoclonal antibody CR9114 (Fig.l2A), CR8020 (Fig.l2B) and anti-HA collclonal serum (Flg.l2C) to various polypeptides of the invention detected by Elisa.
Figures 13A-13B: Analysis by SDS-PAGE (Fig.l3A) and Western blot (Fig.l3B) of the glycosylation of the polypeptides of the invention. With deglycosylation, bands in the expected molecular weight are observed. For Western blotting, polyclonal serum directed against
HA Hl for detection
Figure 14: SEC-MALS analysis of the polypeptides of the invention. The plots indicate SEQ ID N °.
Figures 15A-15B: Fig. L5A: Transfer analysis
Western supernatant from cells expressing SEQ ID
N °: 145. For Western blot an antibody directed against the his-mark was used for detection. Fig. L5B:
Binding of monoclonal antibody CR9114 (squares), CR6261 (circles), CR8020 (triangles pointing upwards) and
Fl6v3 (triangles pointing down) to SEQ ID N °: 145
<img file="MX357009B_D0023.tif" />
<img file="MX357009B_D0024.tif" />
detected by Elisa.
Figure 16: Elution profile for purification of SEQ ID No.: 145 from the culture supernatant on a His trap column. The polypeptide of the invention elutes at
100 (peak A) and 200 mM (peak B) of imidazole, respectively.
Figures 17A-17D: Fig.17A-Fig.17B: Elution profile for purification of SEQ ID No.: 145 by size exclusion chromatography (Superdex 200). Both peaks A and B contain polypeptides of the invention. Fig. L7C: Analysis by
Native PAGE of fractions from size exclusion chromatography. Most purified proteins run at a molecular weight consistent with a monomeric form of the protein. Fig. L7D: SDS PAGE analysis of size exclusion chromatography fractions.
Figure 18: Time course of the response of
IgG into the full-length homologous protein ectodomain as a result of the immunization schedule with
DNA that is described in this application.
Figures 19A-19B: IgG responses at week 7 after initial immunization of individual mice against the full-length hemagglutinin ectodomain of the homologous strain H1N1 A / Brisbane / 59/2007 (Fig. L9A) and the heterologous strain H1N1 A / California / 07/2009 (Fig.l9B). Open symbols correspond to values below the limit of
<img file="MX357009B_D0025.tif" />
assay detection.
Figures 20A-20D: IgG responses at week 7 after initial immunization of individual mice against the full-length hemagglutinin ectodomain of the homologous strain H1N1 A / Brisbane / 59/2007 (Fig. 20A) and the heterologous strain HlNl A / California / 07/2009 (Fig.20B) the heterosubtypal strain H5N1 A / Vietnam / 1203/2004 (Fig.20C) and the heterosubtypal strain H3N2 A / Hong Kong / 1/1968 (Fig.20D). Open symbols correspond to values below the detection limit of the assay.
Figures 21A-21B: FACS assay of HA H3 based stem domain polypeptides. The average fluorescence intensity (Fig.21A) and the% of positive cells (Fig.21B) are shown.
Figures 22A-22C: IgG responses at week 7 after initial immunization of individual mice against the full-length hemagglutinin ectodomain of the homologous strain HlNl A / Brisbane / 59/2007 (Fig. 22A) and the heterologous strain HlNl A / California / 07/2009 (Fig. 22B) and the heterosubtypal strain H5N1 A / Vietnam / 1203/2004 (Fig. 22C). Open symbols correspond to values below the detection limit of the assay.
Figure 23: FACS analysis of the binding of the CR6261 mAbs,
CR9114, CR8020 and CR9020, as well as polyclonal anti-Hl serum
<img file="MX357009B_D0026.tif" />
to the full-length HA and the corresponding - of the invention. Top: Average fluorescence intensity. Bottom: percentage of positive cells. Solid bars represent full-length proteins, striped bars represent the polypeptides of the invention. Full-length HA and the corresponding polypeptide of the invention derived from said sequence have the same background color.
Figures 24A-24C: Kaplan-Meier survival curves (Fig. 24A), changes in weight (Fig. 24B) and median clinical scores (Fig. 24C) for the influenza attack experiment described in example 21.
Figure 25: Alignment of selected H1N1 sequences according to Example 22.
Figure 26: FACS assay with HA H1 based stem domain polypeptides selected according to Example 22. Average fluorescence intensity is shown.
Figures 27A-27C: H1 binding kinetics of full-length HA (SEQ ID N °: 149) in its trimeric and monomeric form and s-Hl-mini2-clusterl + 5 + 6-GCN4 (SEQ ID N °: 145) to the immobilized monoclonal antibodies CR6261 (Fig. 27A),
CR9114 (Fig.27B) and CR8020 (Fig.27C) determined by i
biolayer interferometry.
<img file="MX357009B_D0027.tif" />
<img file="MX357009B_D0028.tif" />
Figures 28A-28B: S-Hl-mini2-clusterl + 5 + 6-GCN4 (SEQ ID No. 145) binding state titration to immobilized antibodies CR6261 (Fig. 28A) and CR9114 (Fig. 28B) followed by biolayer interferometry.
Figure 29: IgG response against HA ectodomain from A / Hong Kong / 1/1968 at day 49 determined by ELISA. The details of the experiment are described in Example 24. The open symbols correspond to values below the detection limit of the assay.
Figures 30A-30C: Kaplan-Meier survival curves (Fig.30A), changes in weight (Fig.30B) and median clinical scores (Fig.30C) for the influenza attack experiment described in example 24.
Figure 31: FACS assay with HA Hl-based stem domain polypeptides selected according to Example 25. Average fluorescence intensity is shown.
Figures 32A-32C: IgG response after 49 days against the HA ectodomain determined by the HA of
A / Wisconsin / 67/2005 (Fig. 32A), A / Hong Kong / 1/1968 (Fig. 32B) and
A / Perth / 16/2009 (Fig. 32C). The details of the experiment are described in Example 26. The open symbols correspond to values below the detection limit of the assay.
<img file="MX357009B_D0029.tif" />
Figure 33: IgG response after 4 9 -di ^ s · oonfe-ra-el · ------- HA ectodomain of A / Hong Kong / 1/1968 determined by
ELISAs. The details of the experiment are described in Example 27. The open symbols correspond to values below the detection limit of the assay.
Figure 34: FACS assay with HA Hl-based stem domain polypeptides selected according to Example 28. Average fluorescence intensity is shown.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
Definitions of the terms as used in the present invention are given below.
An amino acid according to the invention can be any of the twenty natural amino acids (or 'standard' amino acids) or variants thereof such as, for example, D-proline (the D-enantiomer of proline), or any variant that does not it is naturally seen in proteins such as, for example, norleucine. Standard amino acids can be divided into several groups based on their properties. Important factors are load, hydrophilicity or hydrophobicity, size, and functional groups.
These properties are important for the structure of the
<img file="MX357009B_D0030.tif" />
protein and for protein-protelirer interactions: —A-lgune-s— amino acids have special properties such as cistern, which can form covalent disulfide bonds (or disulfide bridges) with other cysteine residues; proline cycled with the backbone of the polypeptide;
and glycine which is more flexible than other amino acids. In the
Table 5 shows the abbreviations and properties of the standard amino acids.
The term "amino acid sequence identity" refers to the degree of identity or similarity between a pair of aligned amino acid sequences, usually expressed as a percentage. Percent identity is the percentage of amino acid residues in a candidate sequence that are identical (i.e., amino acid residues at a given position in the alignment are the same residue) or similar (i.e., amino acid substitution in a given position in the alignment is a conservative substitution, as will be described later), to the corresponding amino acid residue in the peptide after aligning the sequences and introducing mismatches or gaps, if necessary, to achieve the maximum percentage of sequence homology. Sequence homology, including percentages of sequence identity and sequence similarity, are determined using
<img file="MX357009B_D0031.tif" />
alignment of well-known sequences in ~ the ~ "aTCS7 ·· such as by visual inspection and mathematical calculation or, more preferably, the comparison is made by comparing the sequence information using a computer program.
As an example, a preferred computer program is the Wisconsin package program, version 10.0, Genetics
Computer Group (GCG; Madison, Wis.), 'GAP' (Devereux et al., (1984))
A conservative substitution refers to the replacement of an amino acid of one class by another amino acid of the same class. In particular embodiments, a conservative substitution does not alter the structure or function, or both, of a polypeptide. Amino acid classes for conservative substitution include hydrophobic amino acids (eg, Met, Ala, Val, Leu), neutral hydrophilic amino acids (eg, Cys, Ser, Thr), neutral acidic amino acids (eg, Asp, Glu), basic amino acids (eg Asn, Gln, His, Lys, Arg), conformation-interrupting amino acids (eg Gly, Pro) and aromatic amino acids (eg Trp, Tyr, Phe).
As used herein, the terms disease and disorder are used interchangeably to refer to a condition in a subject. In some modalities, the condition is a viral infection, particularly an infection
<img file="MX357009B_D0032.tif" />
<img file="MX357009B_D0033.tif" />
INSTITUTO MtXICANO Dt LA VOFItOAD ¡NDUSTaIAL
<img file="MX357009B_D0034.tif" />
by the influenza virus. In specific embodiments, the term "disease" refers to the disease state resulting from the presence of the virus in a cell or subject, or due to invasion into a cell or subject by the virus. In certain embodiments, the condition is a disease in a subject, the severity of which decreases with the induction of an immune response in the subject by administration of an immunogenic composition.
As used herein, the term "effective amount," in the context of administering a therapy to a subject, refers to the amount of a therapy that has prophylactic and / or therapeutic effect (s). . In certain embodiments, an effective amount, in the context of administering a therapy to a subject, refers to the amount of a therapy that is sufficient to achieve a reduction or relief of the severity of a virus infection of the influenza, a disease or symptom associated therewith, such as, but not limited to, a reduction in the duration of an influenza virus infection, a disease or a symptom associated therewith; preventing the progression of an influenza virus infection, illness, or a symptom associated with it; preventing the development or onset or recurrence of a virus infection
<img file="MX357009B_D0035.tif" />
influenza, of a disease or a symptom of the_ same; preventing or reducing the spread of an influenza virus from one subject to another; reducing a subject's hospitalization and / or the length of hospitalization; an increased survival of a subject with an influenza virus infection or an illness associated with it; elimination of an influenza virus infection or an illness associated with it; inhibition or reduction of influenza virus replication; reducing the titer of the influenza virus; and / or an increase and / or an improvement in the prophylactic or therapeutic effect (s) of another therapy. In certain embodiments, the effective amount does not result in complete protection against influenza virus disease, but results in a lower titer or reduced amount of influenza virus compared to an untreated subject. The benefits of a reduction in the titer, amount, or total burden of influenza virus include, but are not limited to, less severe symptoms of infection, fewer symptoms of infection, and a reduction in the length of the virus. disease associated with. the infection.
The term host, as used herein, refers to an organism or a cell in which
<img file="MX357009B_D0036.tif" />
<img file="MX357009B_D0037.tif" />
MEXICAN INSTITUTE _ _ L »E LA PKO?; £ L? Alj
9 INDUSTRIAL has introduced a vector such as a cloning vector -g-tar expression vector. The organism or the cell can be prokaryotic or eukaryotic. Preferably, the host comprises isolated host cells, for example cultured host cells. The term host cells merely means that the cells were modified for the (over) expression of the polypeptides of the invention. It will be understood that the term host is intended not only to refer to the particular subject organism or cell but also to the progeny of said organism or cell. Given that certain modifications can occur in successive generations either due to mutations or environmental influences, it is possible that said progeny is not, in fact, identical to the organism or the progenitor cell, but it is still included within the scope of the term host as used herein.
The term included or including, as used herein, must be followed by the words non-exhaustively.
As used herein, the term infection means the invasion, multiplication, and / or presence of a virus in a cell or in a subject. In one embodiment, an infection is an active infection, that is, one in which the virus replicates in a cell or in a subject. Bliss
IMPI
MEXICAN INSTITUTE OF THE E'ROHIEDAD INLUSTklAt.
<img file="MX357009B_D0038.tif" />
Infection is characterized by the spread of the dC'WtTcre virus to cells, tissues, and / or organs from the cells, tissues, and / or organs initially infected by the virus. An infection can also be a latent infection, that is, one in which the virus does not replicate. In certain modalities, an infection refers to the disease state resulting from the presence of the virus in a cell or subject, or due to
<td>invasion into a cell</td><td>or on a subject</td><td>by the virus.</td>
<td>Viruses</td><td>influenza se</td><td>classifies into types of</td>
<td>influenza virus</td><td>: the genders</td><td>A, B and C. The term</td>
<td>virus subtype</td><td>the flu</td><td>, as used in the</td>
<td>present, refers to</td><td>variants of</td><td>influenza A virus that</td>
they are characterized by combinations of the viral surface proteins hemagglutinin (H) and neuramidase (N). In accordance with the present invention, influenza virus subtypes can be designated by their H number such as, for example, influenza virus comprising HA of the subtype
H3, influenza virus of subtype H3 or influenza H3, or by a combination of an H number and an N number such as, for example, the H3N2 influenza virus subtype or
H3N2. The term subtype specifically includes all the individual strains of each subtype, which are usually the result of mutations and have different pathogenic profiles, including isolated natural forms.
<img file="MX357009B_D0039.tif" />
as well as human-made mutants or regroupeds and the like. Such strains can also be designated as various isolated forms of a viral subtype. Accordingly, as used herein, the terms strains and isolated forms can be used interchangeably.
The current nomenclature for strains or isolated forms of the human influenza virus includes the type (genus) of the virus, i.e. A, B or C, the geographical location of the first isolation, the number of the strain and the year of isolation, usually with the antigenic description of HA and NA indicated in parentheses, for example A / Moscow / 10/00 (H3N2).
Non-human strains also include the host of origin in the nomenclature. Influenza A virus subtypes can be further classified with reference to their phylogenetic group. Phylogenetic analyzes have shown that hemagglutinins are subdivided into two large groups: among others, the Hl, H2, H5 and H9 subtypes in the phylogenetic group (group 1 influenza virus) and, among others, the H3, H4 subtypes , H7 and H10 from phylogenetic group 2 (group 2 influenza virus).
As used herein, the term influenza virus disease refers to the disease state resulting from the presence of an influenza virus, for example an influenza A or B virus in a cell or subject
IMPI
INSTITUI 'MEXICANO: · £ LA PROFIEvAO V ** íb £> Í¿L-> T>
NÍ7USTWAL or invasion of a cell or subject by an influenza virus. In specific embodiments, the term refers to a respiratory illness caused by an influenza virus.
As used herein, the term nucleic acid includes DNA molecules (eg, cDNA or genomic DNA) and RNA molecules (eg, mRNA), and analogs of DNA or
RNA generated using analog nucleotides. The nucleic acid can be single or double chain. Nucleic acid molecules can be chemically or biochemically modified or can contain unnatural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the natural nucleotides for an analogue, internucleotide modifications such as uncharged bonds (for example, methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), bonds charged (eg, phosphorothioates, phosphorodithioates, etc.), pendant groups (eg, polypeptides), intercalators (eg, acridine, psoralen, etc.), chelators, alkylating and modified linkages (eg, alpha-anomeric nucleic acids, etc.). Reference to a nucleic acid sequence encompasses its complement unless otherwise specified.
IMPI
<img file="MX357009B_D0040.tif" />
way. Accordingly, reference to a nucleic acid molecule having a particular sequence should encompass its complementary strand, with its complementary sequence. The complementary strand is also useful, for example, for antisense therapy, hybridization probes, and primers for PCR primers.
As used herein, in certain embodiments the amino acid numbering in HA is based on the amino acid numbering in HAO of a wild type influenza virus, for example the amino acid numbering of strain A / Brisbane / 59/2007 HlNl influenza (SEQ ID N °: 1). As used in the present invention, the phrase "amino acid at position x in HA" means that the amino acid corresponding to amino acid at position x in HAO of the particular wild-type influenza virus, for example A / Brisbane / 59/2007 (SEQ ID
N °: 1; where the amino acids of the HA2 domain are indicated in italics). The specialist will understand that the amino acid equivalents in other strains and / or subtypes of the influenza virus can be determined by aligning multiple sequences (see, for example, the
Table 8). It should be noted that, in the numbering system used in this application, 1 refers to the N-terminal amino acid of an immature HAO protein (SEQ ID
<img file="MX357009B_D0041.tif" />
Ν °: 1). The mature sequence begins, for example, at position 18 of SEQ ID NO: 1. In certain embodiments, the numbering of the equivalent amino acids is based on the numbering of amino acids in H3 HAO, in particular the numbering of the amino acids of influenza A / Wisconsin / 67/2005 H3N2 strain (SEQ ID No.: 89). Equivalent amino acids in other H3 HA sequences can be determined by alignment. The skilled person will understand that the leader sequence (or signal sequence) that directs the transport of a protein during production (for example, corresponding to amino acids 1-17 of SEQ ID NO: 89), is generally not present in the polypeptide final that is used for example in a vaccine. In certain embodiments, the polypeptides according to the invention then comprise an amino acid sequence without the leader sequence, ie the amino acid sequence is based on the HAO amino acid sequence without the signal sequence.
A polypeptide refers to a polymer of amino acids linked by amide bonds, as is known to those of skill in the art. As used herein, the term can refer to a single chain of polypeptides linked by covalent amide bonds. The term can also refer to multiple polypeptide chains
IMPI
MEXICAN INSTITUTE "> OF INDUSTRIAL PROPERTY
<img file="MX357009B_D0042.tif" />
associated by non-covalent interactions such as ionic contacts, hydrogen bonds, Van der forces
Waals and hydrophobic contacts. Those skilled in the art will understand that the term includes polypeptides that were modified, for example by post-translational processing, such as cleavage of a signal peptide, formation of disulfide bonds, glycosylation (eg, N-linked glycosylation), cleavage by proteases and lipid modification (eg, S-palmitoylation).
A stem domain polypeptide refers to a polypeptide comprising one or more polypeptide chains that make up a stem domain of a natural (or wild-type) hemagglutinin (HA). Typically, a stem domain polypeptide comprises a single polypeptide chain (i.e., corresponding to the stem domain of a hemagglutinin HAO polypeptide) or two polypeptide chains (i.e., corresponding to the stem domain of a hemagglutinin HAl polypeptide). associated with a hemagglutinin HA2 polypeptide). In accordance with the invention, a stem domain polypeptide comprises one or more mutations compared to the wild type HA molecule, in particular one or more amino acid residues of the wild type HA may have been replaced by other amino acids, not natural, in the corresponding position of
<img file="MX357009B_D0043.tif" />
.MEXICAN INSTITUTE OF THE MUPIEL'AD
INDUSTRIAL
<img file="MX357009B_D0044.tif" />
a HA of a particular natural type. The po3J.pept.I.doñ .riel.
Stem domain according to the invention may further comprise one or more connecting sequences, as will be described later.
The term vector refers to a nucleic acid molecule into which a second nucleic acid molecule can be inserted for introduction into a host where it will replicate and, in some cases, be expressed. In other words, a vector can carry a nucleic acid molecule that was attached to it. As used herein, the term vector contemplates cloning vectors as well as expression vectors. Vectors include, but are not limited to, plasmids, cosmids, bacterial artificial chromosomes (BACs), and yeast artificial chromosomes (YACs) and vectors derived from bacteriophages or viruses from plants or animals (including humans). The vectors comprise an origin of replication that is recognized by the proposed host and, in the case of expression vectors, a promoter and other regulatory regions recognized by the host. Certain vectors are able to replicate autonomously in the host into which they have been introduced (eg, vectors with a bacterial origin of replication can replicate in bacteria). Other vectors can be integrated into
IMPIOS
MLXIC'AN INSTITUTE
PROPERTY Cm- •. • MttSÍ 'the genome of a host when introduced into eX - KiÍ «íft © T - pejt ™ ie ——— which replicate together with the genome of the host.
As used herein, the term wild type in the context of a virus refers to influenza viruses that are prevalent, natural in the circulation, and that produce the typical outbreaks of the disease.
Description
Influenza viruses have a significant impact on public health globally, causing millions of cases of severe illness each year, thousands of deaths, and considerable economic losses. Current trivalent influenza vaccines generate a potent neutralizing antibody response to closely related strains and isolated forms of vaccines, but rarely extend to the most divergent strains within one subtype or other subtypes. Furthermore, selecting the appropriate vaccine strains provides many challenges and often results in suboptimal protection. Furthermore, prediction of the next pandemic virus subtype, including when and where it appears, is currently impossible.
Hemagglutinin (HA) is the most important glycoprotein in the envelope of influenza A viruses, which is the
<img file="MX357009B_D0045.tif" />
The main target of the nentralized-hes antibodies— hehemagglutinin fulfills two main functions during the entry process. First, hemagglutinin is involved in binding the virus to the surface of the target cells through interactions with sialic acid receptors.
Second, after virus endocytosis, hemagglutinin subsequently triggers fusion of the viral and endosomal membranes to release its genome into the cytoplasm of the target cell. HA comprises a large ectodomain of ~ 500 amino acids that is cleaved by host-derived enzymes to generate 2 polypeptides that remain attached via a disulfide bond. Most of the N-terminal fragment (HAl, 320-330 amino acids) forms a distal globular domain for the membrane that contains the receptor binding site and most of the determinants recognized by neutralizing antibodies
<td>of the virus.</td><td>Serving</td><td>C-terminal</td><td>plus</td><td>little (</td><td>HA2, -180</td>
<td>amino acids)</td><td>form a</td><td>structure</td><td>type</td><td>stem that</td><td>serves as</td>
<td>anchor for</td><td>The Dominion</td><td>globular</td><td>to</td><td>membrane</td><td>cell or</td>
viral. The degree of sequence homology between the subtypes is less among the HAl polypeptides (34% - 59% of homology between the subtypes) than among the HA2 polypeptides (51% - 80% homology). The most conserved region is the sequence around the cleavage site, particularly the 23
<img file="MX357009B_D0046.tif" />
N-terminal amino acids of HA2, which is conserved among all subtypes of influenza A virus (Lorieau et al.,
2010). A part of this region is exposed as a surface loop in the HA precursor molecule (HAO), but becomes inaccessible when HAO cleaves HA1 and HA2.
Most neutralizing antibodies bind to the loops that surround the receptor binding site and interfere with receptor binding and binding. Since these loops are highly variable, most of the antibodies directed to these regions are strain-specific, which would explain why current vaccines generate such limited strain-specific immunity. However, fully human monoclonal antibodies to influenza virus hemagglutinin have recently been generated with broad cross-neutralizing potency. Functional and structural analyzes have revealed that these antibodies interfere with the membrane fusion process and are directed against highly conserved epitopes in the stem domain of the protein.
Influenza HA (Throsby et al., 2008; Ekiert et al.,
2009, WO 2008/028946, WO 2010/130636).
In accordance with the present invention, the new HA stem domain polypeptides were designed to contain these epitopes in order to create a vaccine
<img file="MX357009B_D0047.tif" />
Universal epitope-based inducer protection against a wide range of influenza strains. Essentially, the highly variable and immunodominant part, ie the head domain, of the full-length HA molecule is first removed to create a stem domain polypeptide, also called mini-HA. In this way, the immune response will be redirected towards the stem domain where the epitopes of the widely neutralizing antibodies are located. The previously mentioned broadly neutralizing antibodies were used to investigate the correct folding of the newly created molecules, and to confirm the presence of the neutralizing epitopes.
The stem domain polypeptides of this invention can present conserved epitopes of the HA membrane proximal stem domain molecule to the immune system in the absence of dominant epitopes present in the head domain distal to the membrane. To that end, part of the primary sequence of the HAO protein that makes up the head domain is removed and reattached either directly or, in some modalities, by introduction of a flexible short linker sequence ('linker') to restore the continuity of the amino acid chain. The resulting polypeptide sequence is modified by introduction of specific mutations that stabilize the / 1 INSTITUTO MEXICANO
VVajJSUS 'PROPERTY
INDUSTRIAL - <Your.ii_ 'native three-dimensional structure of the remnant part of_the HAO molecule.
The present invention then provides polypeptides comprising (a) an influenza hemagglutinin HAl domain comprising an N-terminal stem segment of
HAl, covalently linked via a linker sequence of 0-50 amino acid residues to a segment of the C-terminal stem of HAl, and (b) an HA2 domain of influenza hemagglutinin, where one or more have been mutated amino acids in the HA2 domain. In the polypeptides of the invention, the HA2 domain then comprises one or more mutations compared to the HA2 domain of a wild type influenza hemagglutinin on which the HA stem domain polypeptide is based.
The influenza hemagglutinin stem domain polypeptides are based on influenza A virus HA subtypes that are generally used in human influenza virus vaccines. In preferred embodiments, the stem domain polypeptides are based on the HA of an influenza virus comprising the subtype
Hl, H5 and / or H3 of HA.
The present invention in particular provides polypeptides from the stem domain of influenza hemagglutinin comprising (a) a HAl domain of the hemagglutinin of influenza
IMPI
<img file="MX357009B_D0048.tif" />
influenza comprising a stem segment — N Leruiineri — dS ”
HAl, covalently linked via a 0-50 amino acid residue linker sequence to a C-terminal stem segment of HAl, and (b) an HA2 domain of influenza hemagglutinin, wherein the stem domain polypeptide of hemagglutinin is resistant to cleavage by proteases at the junction between HAl and HA2, and wherein one or more of the amino acids in the HA2 helix A and CD helix CD amino acid sequence was mutated when compared to a wild type influenza HA2 domain. Preferably, the HAl and HA2 domains are derived from an influenza A virus subtype selected from the group consisting of Hl, H5, and H3.
The polypeptides of the invention then comprise one or more mutations in the HA2 amino acid sequence linking
<td>to the residue</td><td>C-terminal</td><td>the</td><td>propeller A with</td><td>the</td><td>residue</td><td>N-</td>
<td>terminal</td><td>the CD propeller,</td><td>how</td><td>indicated in</td><td>the</td><td>Figure 1.</td><td>In</td>
<td>certain</td><td>modalities,</td><td>I know</td><td colspan="2">has replaced</td><td>one or</td><td>plus</td>
hydrophobic amino acids in said amino acid sequence
HA2 by hydrophilic amino acids, such as polar and / or charged amino acids, or the flexible amino acid glycine (G).
The polypeptides of the invention do not comprise full-length HAl.
In certain modalities the polypeptides
MEXICAN INSTITUTE * ¿*** - ¿1 ^ - ^ Μ
11 * 1 Γ 1 immunogenic are
4- \ in.imuiumtAivAr <v ySf * «* - · Τ3. -<sub>T</sub>
J OF EROULTAD
INDUSTRIAL fe * substantially less than HAO, preferably without all or substantially all of the globular head of HA. Preferably, the immunogenic polypeptides are not more than 360, preferably not more than 350, 340, 330, 320, 310, 305, 300, 295, 290, 285, 280,
275 or 270 amino acids in length. In certain embodiments, the immunogenic polypeptides are between about 250 and about 350, preferably between about 260 and about 340, preferably between about 270 and about
330, preferably between about 270 and about 330 amino acids in length.
In certain embodiments, the polypeptides further comprise one or more additional mutations in the HAl domain and / or
HA2, compared to the amino acid sequence of HA from which the HAl and HA2 domains are derived. Accordingly, the stability of the stem polypeptides is further increased.
According to the invention, the N-terminal segment of
HAl refers to a segment of polypeptide that corresponds to the amino-terminal portion of the HAl domain of an influenza hemagglutinin (HA) molecule. In certain embodiments, the N-terminus segment of the HAl polypeptide comprises amino acids between position 1 and position x of the HAl domain, where the amino acid at position x is
<img file="MX357009B_D0049.tif" />
ΙΜΡΙ ~ an amino acid residue in HAl. The term Cterminal segment of HAl refers to a polypeptide segment that corresponds to the carboxyl-terminal portion of an HAl domain of influenza hemagglutinin. In certain embodiments, the C-terminal polypeptide segment of the HAl comprises amino acids between position and up to and including the C-terminal amino acid of the HAl domain, wherein the amino acid at position y is an amino acid residue of
HAl. According to the invention y is greater than x, therefore a segment of the HAl domain has been removed between the N-terminal segment of HAl and the C-terminal segment of HAl, i.e. between the amino acid at position x and the amino acid at the y position of HAl and, in some embodiments, it was replaced by a linker sequence.
In certain embodiments, the N-terminal stem segment of HAl comprises amino acids 1-x of HAl, and the C-terminal stem segment of HAl comprises the y-terminal amino acids of HAl. Accordingly, in certain embodiments, deletion in the HAl segment comprises the amino acid sequence between the amino acid at the x + 1 position to and including the amino acid at the y-1 position.
In certain embodiments, the polypeptides do not comprise the signal sequence.
Therefore in
<img file="MX357009B_D0050.tif" />
In certain IMPI modalities, the N-terminal segment HA1 comprises the px amino acid of HA1, where p is the first amino acid of the mature HA molecule (for example p = 18 in the case of SEQ ID No.: 1). The specialist will be able to prepare the polypeptides described herein without the signal peptides (for example, amino acids 1-17 of SEQ ID NO: 1). In certain embodiments, the polypeptides of the invention contain the intracellular sequences of HA and the transmembrane domain. In other embodiments, the polypeptides of the invention do not comprise the intracellular sequences of HA and the transmembrane domain. In certain embodiments, the intracellular sequence and the transmembrane sequence have been removed, for example the amino acid sequence from position (or its equivalent) 523, 524, 525, 526, 527, 526,
528, 529, or 530 from the HA2 domain to the C-terminus of the HA2 domain.
According to the invention, hemagglutinin stem domain polypeptides are resistant to cleavage by proteases at the junction between HA1 and HA2. It is now known to those in the art that the Arg (R) -Gly (G) sequence spanning HA1 and HA2 is a recognition site for trypsin and trypsin-like proteases and is typically cleaved for hemagglutinin activation. Since the HA stem domain polypeptides described in
IM F
<img file="MX357009B_D0051.tif" />
should not be activated, the pnl i pept-i dns dpi stem domain of influenza hemagglutinin of the invention are resistant to cleavage by proteases. According to the invention, either the protease cleavage site or the protease site encompassing HA1 is removed and HA2 is mutated into a sequence that is resistant to protease cleavage.
In certain embodiments, the C-terminal amino acid residue of the C-terminal stem segment of HA1 is any amino acid other than arginine (R) or Usin (K). In certain embodiments, the C-terminal amino acid of HA1 is glutamine (Q), serine (S), threonine (T), asparagine (N), aspartic acid (D), or glutamic acid (E). In certain embodiments, the C-terminal amino acid residue of the C-terminal stem segment of HA1 is glutamine (Q).
In certain embodiments, the polypeptides are glyosylated.
Stem domain polypeptides of influenza hemagglutinin can be based on the HA of any hemagglutinin of a natural influenza A virus of a subtype used in human influenza vaccines. Influenza A virus subtypes generally used in influenza vaccines are the Hl, H3, or H5 subtypes of influenza A virus. The term "based on" means that
<img file="MX357009B_D0052.tif" />
<img file="MX357009B_D0053.tif" />
INSTITUTO MEXICANO Pfc LA FrFí> '| EOaL> INDUSTRIAL the N-terminal segments, and / or C-terminal segments of the HAl domain and / or HA2 domains have at least 70%,
75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with corresponding segments
N-terminals and / or C-terminals of the HAl and / or HA2 domains of any natural influenza hemagglutinin of a subtype
Hl, H3 and / or H5 known to those of skill in the art or later discovered. In certain embodiments, the influenza hemagglutinin stalk domain polypeptides are based on an influenza A virus group 1 influenza hemagglutinin. In certain embodiments, the influenza hemagglutinin stalk domain polypeptides are based on a hemagglutinin from influenza A group 2 influenza A virus In some embodiments, the influenza hemagglutinin stem domain polypeptide is a hybrid or chimeric polypeptide that comprises or consists of segments and / or domains of a plurality of influenza strains or subtypes. For example, a polypeptide from the influenza hemagglutinin stalk domain may comprise N-terminal HAl and C-terminal HAl stem segments and / or domains.
HA2 from different HA subtypes of influenza virus A.
In certain embodiments, the polypeptides are based on Hl HA. In a particular embodiment, the polypeptides
<img file="MX357009B_D0054.tif" />
comprise hemagglutinin stem domains of ..... o-based ·· in<sup>1</sup> HA from an influenza A virus comprising the Hl subtype of HA, such as from influenza virus
A / Brisbane / 59/2007 (H1N1) (SEQ ID No.: 1), as will be described later. The skilled person will understand that other influenza A viruses comprising the Hl subtype of HA according to the invention can also be used. In certain embodiments, the polypeptides comprise the HA-based hemagglutinin stem domains of an Hl domain of influenza A virus selected from the
Table 7.
In certain embodiments, the polypeptides comprise a segment of the HA1 N-terminal polypeptide that comprises the amino acids between position 1 and position x of the HA1 domain of Hl, where x is any amino acid between the amino acid at position 46 and the amino acid at position 60, such as amino acid at position 4 7, 48,
49, 50, 51, 52, 53, 54, 55, 56, 57, 58 or 59, preferably where x is 52, 53, 55 or 59. Preferably, the polypeptides comprise an N-terminal segment of HA1 without the sequence signal, i.e. an N-terminal segment of HA1 comprising the amino acids between position 18 (for example, for Hl HA, such as SEQ ID No.: 1), or an equivalent position in other Hl strains of the virus of the influenza, and
<img file="MX357009B_D0055.tif" />
x position of the HAl domain. In certain embodiments, the N-terminus segment of HAl then comprises the amino acids between position p (where p = 18 for HI HA in SEQ ID
N °: lo an equivalent position in other HI HA), and the x position of the HAl domain.
In certain modalities, the polypeptide segment
C-terminal HAl comprises the amino acids between position and up to and including the C-terminal amino acid of Domain HI
HAl, where y is any amino acid between the amino acid at position 290 and the amino acid at position 325 of HI
HAl, preferably where y is 291, 303, 318 or 321. According to the invention, the HA2 domain comprises one or more mutations in the HA2 amino acid sequence that binds to the C-terminal residue of helix A with the residue N-terminal of the CD helix (Figure 1). In certain embodiments, one or more hydrophobic amino acids in said HA2 amino acid sequence has been replaced by hydrophilic amino acids, such as polar and / or charged amino acids. In certain modalities (for example for HI HA, such as SEQ ID
N °: l), the amino acid sequence of HA2 that joins the residue
C-terminal of helix A and N-terminal residue of helix CD comprises the amino acid sequence between residues 402-418 of influenza HA2. In certain embodiments, the HA2 amino acid sequence that binds to
<img file="MX357009B_D0056.tif" />
<img file="MX357009B_D0057.tif" />
C-terminal residue of helix A and N-terminal residue of__ helix CD comprises the amino acid sequence
MNTQFTAVGKEFN (H / K) LE (K / R) (SEQ ID N °: 17).
In certain modes, x is 59 and y is 291.
In certain embodiments, x is 52 and y is 321.
In certain modalities x is 53 and y is 303.
In certain modalities x is 55 and y is 318.
In one embodiment, the amino acid sequence linking the C-terminal residue of helix A with the N-terminal residue of helix CD corresponds to the amino acid sequence between the amino acid at position 402 and the amino acid at position 418 of HA2 of SEQ ID NO: 1, wherein the polypeptides comprise one or more mutations in the amino acid sequence ranging from amino acids 402 to 418 of SEQ ID NO: 1. The amino acid sequence between residues 402-418 of influenza HA serotype Hl comprises the amino acid sequence
MNTQFTAVGKEFN (H / K) LE (K / R) (SEQ ID N °: 17). In certain embodiments, the amino acid sequence between residues
402-418 of influenza HA serotype Hl comprises the amino acid sequence MNTQX! TAX<sub>2</sub>GKEX<sub>3</sub>N (H / K) X<sub>4</sub>E (K / R).
In certain embodiments, the polypeptides then comprise one or more of the mutations in the Hl HA2 domain as shown in Table 6. In certain embodiments,
<img file="MX357009B_D0058.tif" />
one or more of the amino acids at position 406, 409, 413 and__
416, that is, one or more of the amino acids Χχ, X<sub>2</sub>, X3 and X4 were mutated (numbering refers to SEQ ID No.: 1).
In certain embodiments, the amino acid at the position
406, i.e. X<sub>x</sub>, was exchanged for an amino acid selected from the group consisting of S, Τ, N, Q, R, Η, K, D, E and G, preferably S. In certain embodiments, the amino acid at position 409, i.e. X<sub>2</sub>, was changed to an amino acid selected from the group consisting of S, Τ, N, Q, R, Η, K,
D, E and G, preferably T, Q or G. In certain.
Modalities, the amino acid at position 413, i.e. X3, was changed to an amino acid selected from the group consisting of S, Τ, N, Q, R, Η, K, D, E, G, preferably S.
In certain embodiments, the amino acid at the position
416, i.e. X<sub>4</sub>, was exchanged for an amino acid selected from the group consisting of S, Τ, N, Q, R, Η, K, D, E, G, preferably S. Combinations of these mutations are also possible.
In certain embodiments, the HAl Nterminal stem segment comprises amino acid residues 1-59 of
HAl, and the C-terminal stem segment of HAl comprises amino acid residues 291-343 of, wherein the amino acid at position 343, i.e. R343, has been mutated and is an amino acid other than R, preferably it is glutamine ( Q).
ί. Μ ΡI ^ ί · η Mexican Institute
AXIS UA KOW »® • χ ^ '-' ϋ ·· '· ^ ·'<sup>7</sup>
INDUiTRiAU '-<sup>1</sup>-—
In certain embodiments, the N-terminal segment of HAl consists of amino acid residues 1-59 of HAl and the C-terminal segment of HAl consists of amino acid residues 291-343 of HAl. It should be noted that the amino acid numbering is based on the amino acid numbering of HAO Hl, in particular the amino acid numbering of influenza strain HlNl A / Brísbane / 59/2007 (SEQ ID N °: 1 ). In addition, it should be noted that since the HA sequences of different influenza subtypes / strains may contain insertions or deletions in the head region compared to each other, the numbering is not always the same. The specialist will be able to determine the equivalent amino acid positions in the HA sequences of different strains and / or subtypes of influenza virus by sequence alignment.
In certain modalities, the polypeptide segment
N-terminus of HAl does not comprise the signal sequence. In preferred embodiments, the N-terminus segment of HAl comprises the amino acids between position 18 and the position of the HAl domain. In certain modalities, the segment
N-terminus of HAl consists of amino acids 18-59 of the HAl domain.
In some embodiments, the polypeptides of the invention comprise one or more additional mutations, i.e.
<img file="MX357009B_D0059.tif" />
IN'SI amino acid substitutions, in the HAi-domain and / or · -en-ei ”HA2 domain. In certain embodiments, the HAl domain then further comprises one or more of the following L58T, V314T, and I316T mutations. Again it should be noted that the amino acid numbering is based on the HAO Hl amino acid numbering, in particular the amino acid numbering of the HlNl influenza strain
A / Brisbane / 59/2007 (SEQ ID N °: 1). The specialist will be able to determine the equivalent amino acids in the HA of other H1 influenza viruses and therefore will be able to determine the equivalent mutations.
In a specific embodiment, the HAl domain comprises the L58T, V314T and I316T mutations, and the HA2 domain comprises one or more of the following F406S, V409T and L416S mutations.
In certain embodiments, the HAl domain further comprises the K321C mutation and / or the HA2 domain further comprises one or more of the following Q405C, F413C, E421C and
Y502S.
In a specific embodiment, the HAl domain comprises the L58T, V314T, I316T and K321C mutations and the HA2 domain comprises the Q405C, F406S, V409T and L416S mutations.
In a specific embodiment, the HAl domain comprises the L58T, V314T and I316T mutations, and the HA2 domain
MPH
INSTITUTO McXICAN ': F DE LA í' (lGPIr ') AL) \ INDUSTRIAL includes mutations F406S, V409T, F413C, L41- & S — and— & 424ÍY—
In a specific embodiment, the HAl domain comprises the L58T, V314T, and I316T mutations, and the HA2 domain comprises the F406S, V409T, L416S, and Y502S mutations.
In a specific embodiment, the HAl domain comprises the L58T, V314T, I316T and K321C mutations and the HA2 domain comprises the Q405C, F406S, V409T, F413C, L416S and
E421C.
In a specific embodiment, the HAl domain comprises the L58T, V314T, I316T and K321C mutations and the HA2 domain comprises the Q405C, F406S, V409T, F413C, L416S mutations,
E421C and Y502S.
In other embodiments, the HA2 domain further comprises one or more of the M420I and V421I mutations, or equivalent mutations.
In a specific embodiment, the HAl domain comprises the L58T, V314T and I316T mutations, and the HA2 domain comprises one or more of the following F406S mutations,
V409T, L416S, M420I and V421I.
In certain embodiments, the HAl Nterminal stem segment comprises amino acid residues 1-52 of
HAl, preferably amino acid residues 18-52 of
HAl, and the C-terminal stem segment of HAl comprises amino acid residues 321-343 of HAl, where the
<img file="MX357009B_D0060.tif" />
amino acid at position 343, ie R343, fua- 'used and is an amino acid other than R, preferably it is glutamine (Q), where the HA2 domain comprises the F406S mutations,
V409T, L416S, M420I and V421I. In certain embodiments, the N-terminal stem segment of HA1 consists of amino acid residues 1-52 of HAl, preferably amino acid residues 18-52 of HAl, and the C-terminal stem segment of HAl consists of residues of amino acids 321-343 of
HAl.
In certain embodiments, the HAl Nterminal stem segment comprises amino acid residues 1-53 of
HAl, preferably amino acid residues 18-53 of
HAl, and the segment of the C-terminal stem of HAl comprises amino acid residues 303-343 of HAl, where the amino acid at position 343, i.e. R343, was routed and is an amino acid other than R, preferably it is glutamine ( Q). In certain embodiments, the N-terminal stem segment of HAl consists of amino acid residues 1-53 of HAl, preferably amino acid residues 18-53 of HAl
HAl, and the C-terminal stem segment of HAl consists of amino acid residues 303-343 of HAl. In a specific embodiment, the HAl domain comprises V314T mutations and
I316T, and the HA2 domain comprises one or more of the following F406S, V409T, L416S, M420I and V421I mutations. In a
<img file="MX357009B_D0061.tif" />
Preferred embodiment, the polypeptide comprises the-, sequence, • -damino amino acids of SEQ ID NO: 11.
In certain embodiments, the HAl Nterminal stem segment comprises amino acid residues 1-55 of
HAl, preferably amino acid residues 18-55 of
HAl, and the segment of the C-terminal stem of HAl comprises amino acid residues 318-343 of HAl, where the amino acid at position 343, i.e. R343, was mutated and is an amino acid other than R, preferably it is glutamine ( Q). In certain embodiments, the N-terminal stem segment of HAl consists of amino acid residues 1-55 of HAl, preferably amino acid residues 18-55 of HAl
HAl, and the C-terminal stem segment of HAl consists of amino acid residues 318-343 of HAl. In one embodiment, the HA2 domain comprises the F406S, V409T, L416S mutations,
M420I and V421I.
In certain embodiments, the polypeptides further comprise the R324C mutation in the HAl domain and T436C in the HA2 domain.
In a specific embodiment, the HAl domain comprises the L58T, V314T, I316T and R324C mutations and the HA2 domain comprises one or more of the following F406S mutations,
V409T, L416S, M420I, V421I and T436C.
In one embodiment, the HAl domain comprises the mutation
<img file="MX357009B_D0062.tif" />
INDUSTkíAL
R324C, and the HA2 domain comprises the F406S mutations,
V409T, L416S, M420I, V421I and T436C.
In another embodiment, the HAl domain comprises the V314T, I316T and R324C mutations, and the HA2 domain comprises one or more of the following F406S, V409T, L416S mutations,
M420I, V421I and T436C.
In one embodiment, the HAl domain comprises the mutation
R324C, and the HA2 domain comprises the F406S mutations,
V409T, L416S, M420I, V421I and T436C.
In certain embodiments, the polypeptides of the invention contain the intracellular sequences of HA and the transmembrane domain. In other embodiments, the intracellular and transmembrane sequences have been removed, for example the amino acid sequence between position (or its equivalent)
523, 524, 525, 526, 527, 526, 528, 529, or 530 of the HA2 domain and the C-terminus of the HA2 domain (numbering according to SEQ ID NO: 1). In certain embodiments, the polypeptides are further stabilized by introduction of a sequence known to form trimeric structures, ie AYVRKDGEWVLL (SEQ ID No.: 143) ('foldon' sequence), optionally linked by means of a linker. The connector may optionally contain a cleavage site for further processing according to protocols well known to those of skill in the art. To facilitate
IMPIIO / Yes
ΙΚΚΤίΤΊ ΙΤίΊ ΜΓΪΙΓΑΝΙΊ VS<sup>-</sup>^ * ® *. *? ^ »** ./-Ί
INSTITUTO MEXICANO Γ> Ε INDUSTRIAL PROPERTY purification of the soluble form can be added by adding a »sequence of a mark, for example a his mark (HHHHHHH) connected through a short connector, for example EGR. In some embodiments the linker and his tag sequence are added in the absence of a foldon sequence.
In certain embodiments, the amino acid sequence between position (or its equivalent) has been removed
530 of the HA2 domain and the C-terminus of the HA2 domain (numbering according to SEQ ID N °: 1). In certain embodiments, the intracellular and transmembrane sequences have been replaced by the amino acid sequence AGRHHHHHHH (SEQ
ID N °: 81) or SGRSLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHHH (SEQ ID N °: 82).
In certain embodiments, the polypeptides selectively bind to the CR6261 and / or CR9114 antibodies. In one embodiment, the polypeptide does not bind to CR8057 antibody. In one embodiment, CR6261 comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 20 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 21;
CR9114 comprises a variable region of the heavy chain comprising the amino acid sequence of SEQ ID NO: 18 and a variable region of the light chain comprising the amino acid sequence of SEQ ID NO: 19. In a
<img file="MX357009B_D0063.tif" />
ín ^ tiyuto v.lxicano Lt LA PRO? .EDAD \ ivnr: c '.', »ii ·>. · .-. i + modality, CR8057 comprises a variable region of. is, heavy chain comprising the amino acid sequence of SEQ ID NO: 22 and a variable region of the light chain comprising the amino acid sequence of SEQ ID NO: 23.
As previously described, the polypeptides comprise an HAl domain of influenza hemagglutinin comprising a segment of the N-terminal stem of HAl covalently linked via a linker sequence of 0-50 amino acid residues to the segment of the C-terminal stem of
HAl. The linker sequence does not appear in wild type or wild type HA. In certain embodiments, the linker is a peptide comprising one amino acid residue, two or fewer amino acid residues, three or fewer amino acid residues, four or fewer amino acid residues, five or fewer amino acid residues, ten or fewer amino acid residues , 15 or less amino acid residues or 20 or less amino acid residues or 30 or less amino acid residues or 40 or less amino acid residues or 50 or less amino acid residues. In a specific embodiment, the linker sequence is a sequence selected from the group consisting of G, GS, GGG, GSG, GSA, GSGS, GSAG, GGGG, GSAGS,
GSGSG, GSAGSA, GSAGSAG and GSGSGSG.
The present invention also provides methods for providing the polypeptides of the invention, in particular for
<img file="MX357009B_D0064.tif" />
I2V1 Ρ ϊ
MEXICAN INSTITUTE
FROM THE INDUSTRIAL PROPERTY gQ provide the stem domain polypeptide ΊΓΓ HA according to the invention, as well as the polypeptides that can be obtained or are obtained by these methods. In certain modalities, the methods comprise the steps of:
(a) providing an influenza HAO amino acid sequence, in particular an amino acid sequence of influenza HAO serotype Hl;
(b) eliminate the cleavage site between HAl and HA2, preferably by mutation of the C-terminal amino acid of HAl into an amino acid other than arginine (R) or Usin (K);
(c) deleting the amino acid sequence from the globular head domain of the HAO sequence. This is done by removing a segment of the HAl domain between the amino acid at position x and an amino acid at position y, and reattaching the N-terminal segment (spanning from amino acid at position 1 to and including amino acid at position x of HAl) and the C-terminal segment of HAl (spanning from the amino acid to the C-terminal amino acid of HAl) thus obtained, optionally via a 0-50 amino acid linker sequence. In certain embodiments, x is an amino acid at any position between positions y 60, preferably the amino acid at position 52, 53, or 59 of HAl and where y is an amino acid at any
<img file="MX357009B_D0065.tif" />
INSTITUTO MEXICANO Dt LA HiOHEPAO INDUSTRIAL position between positions 290 and 325, prpfqpj bl pmpnt-p nn.
amino acid at position 291, 303, 318 or 321 of HAl.
Again, the numbering used refers to SEQ ID N °: 1.
The specialist will understand that the leader sequence (or signal sequence) that directs the transport of a protein during production (for example corresponding to amino acids 1-17 of SEQ ID N °: 1), in general will not be present in the polypeptide final, to be used, for example, in a vaccine. In certain embodiments, the polypeptides according to the invention then comprise a segment
N-terminus of HAl without leader sequence.
(d) increase the stability of the prefusion conformation and destabilize the post-fusion conformation of the modified HA, preferably by introducing one or more mutations in the amino acid sequence that binds to the C-terminal residue of helix A with the N-terminal residue of the CD helix, preferably in the amino acid sequence encompassing amino acids 402-418 of SEQ ID
No. 1, in particular comprising the amino acid sequence MNTQFTAVGKEFN (H / K) LE (K / R) (SEQ ID No.: 17). Mutations preferably comprise the substitution of hydrophobic amino acid residues by hydrophilic amino acid residues.
(e) introducing one or more disulfide bridges into the HA stem domain polypeptide.
<img file="MX357009B_D0066.tif" />
According to the invention, elimination of the cleavage site between HAl and HA2 can be achieved by mutation of R (in a few cases of K) by Q at the Pl position (see, for example, Sun et al, 2010, for an explanation of the cleavage site nomenclature (position 343 in SEQ ID NO: 1) The Q mutation is preferred but S, T, N, D or E are other alternatives.
Elimination of the head domain can be accomplished, for example, by deletion of amino acids 53 to 320 SEQ ID NO: 1, or at equivalent positions in the HA of other influenza viruses. Those skilled in the art will be able to easily determine the equivalent positions by aligning the sequences using a suitable algorithm, such as for example Clustal or Muscle. The remaining parts of the sequence can be directly attached or, alternatively, a flexible connector can be inserted. The connecting sequences can be from 1 to 50 amino acids in length. Flexible connectors of limited length (less than or equal to 10 amino acids) are preferred, for example GGG,
GGGG, GSA, GSAG, GSAGSA, GSAGSAG or the like. The length of the deletion may also vary, for example starting the deletion at position (x) (or an equivalent thereof), for example at position 54, 55, 56, 57 or 58, or for
<img file="MX357009B_D0067.tif" />
IMPI <or INSTITUTO MEXICANO
OO DE LA l'ROPIEPAD
INDUSTRIAL increase the length of the suppression, by cutting in position. 47, 48, 49, 50, 51 or 52. Similarly, the last amino acid to be deleted can be found at position (y) (or an equivalent thereof), such as 315,
316, 317, 318, or 319, or to increase the length of the suppression at the position (or an equivalent thereof) 321,
322, 323, 324 or 325. It is important to consider that changes in the length of the deletion can be compensated in part by matching the length of the connector sequence, that is, a larger deletion can be matched with a larger connector and vice versa. These polypeptides are also included in the invention.
According to the invention, it increases the solubility of the loop between helix A and helix CD. This loop is made up of (equivalents of) residues 402 to 418 in Hl
A / Brisbane / 59/2007 (SEQ ID N °: 1). Accordingly, the stability of the prefusion conformation is increased and the post-fusion conformation of the modified HA is destabilized. This loop is highly conserved in Hl sequences, as can be seen later in
Table 6. This can be accomplished, for example, by replacing amino acids I, L, F, or V in said loop with hydrophilic counterparts. Equivalent positions can be readily determined by those skilled in the art by alignment
<img file="MX357009B_D0068.tif" />
of the sequences using a suitable algorithm, such as for example Clustal or Muscle. Glycine mutations destabilize the post-fusion conformation since the great flexibility of this amino acid leads to a decrease in the stability of the post-fusion helix that will form with this part of the HA sequence. The consensus sequence that describes the loop between residues 402-418 of influenza HA serotype Hl is (SEQ ID No.: 17) MNTQFTAVGKEFN (H / K) LE (K / R). In the polypeptides of the invention, the amino acid at positions 406, 409, 413 and / or
416 (or its equivalents, determined by sequence alignment) is a polar (S, Τ, N, Q), charged (R, Η, K, D, E) or flexible (G) amino acid. Combinations of mutations at these sites are also possible, for example
F406S, V409T, L416S. In some cases, a mutation is preferred to restore the consensus amino acid, for example where V or M is at position 404 (for Τ), V at 408 (for A) or 410 (for G), or I at 414 (for N); the incidence of sequences with these particular amino acids is very low. Table 6 shows a summary of the previously described mutations that characterize the polypeptides of the invention.
In accordance with the invention, one or more disulfide bridges are introduced into the stem domain polypeptides,
<img file="MX357009B_D0069.tif" />
IMPI preferably between the amino acids of (or de-equív-aientede) position 324 and 436 in Hl A / Brisbane / 59/2007. Those skilled in the art will be able to easily determine the equivalent positions by sequence alignment using a suitable algorithm such as Clustal, Muscle etc.
The manipulated disulfide bridges are created by mutation of at least one (if the other is already a cysteine), but usually two spatially close residues in cysteine, which will spontaneously or by oxidation form a covalent bond between the sulfur atoms of these residues.
Native HA exists as a trimer on the cell surface. Most of the interactions between the individual monomers that hold the trimer together are in the head domain. After removal of the head, the tertiary structure is destabilized, and therefore a strengthening of the interactions between the monomers in the truncated molecule will increase stability. In the stem domain trimerization is mediated by the formation of a trimeric coiled-loop motif. By reinforcing this motif a more stable trimer can be created. In accordance with the invention, a consensus sequence can be introduced for the formation of a trimeric coiled loop, for example
IEAIEKKIEAIEKKIE (SEQ ID N °: 83), in a polypeptide of the
Ή 1
<img file="MX357009B_D0070.tif" />
IM invention in (the equivalent of) the position — Ήτ8 — a — 4337 ErT ”~ 'certain modalities, the sequence MKQIEDKIEEIESKQ (SEQ
ID No.: 84), derived from GCN4 and also known for its ability to trimerize, is entered at (the equivalent of) position 419-433. In certain modalities, the trimer interface is stabilized by modifying M420, L423,
V427, G430 for isoleucine.
In certain embodiments, the polypeptides of the invention contain the intracellular Hl HA sequences and the transmembrane domain. In other embodiments, the intracellular and transmembrane sequences have been removed, for example the amino acid sequence between position (or its equivalent) 523, 524, 525, 526, 527, 526, 528, 529 or 530 of the HA2 domain and the end C-terminus of the HA2 domain (numbering according to SEQ ID No.: 1) to produce a soluble polypeptide after expression in cells. In certain embodiments, the polypeptides are further stabilized by introducing a sequence known to form trimeric structures, i.e., AYVRKDGEWVLL (SEQ ID NO:
80), optionally connected by means of a connector. The connector may optionally contain a cleavage site for further processing according to protocols well known to those of skill in the art. To facilitate the purification of the soluble form you can ιτΛΛΤώ »juartcare TTc-tffnx67
<img file="MX357009B_D0071.tif" />
<img file="MX357009B_D0072.tif" />
ιν.πτππυ 'Mexican OE LA FROPILOAP INDUSTRIAL
<img file="MX357009B_D0073.tif" />
add a sequence of a mark, for example a his mark (HHHHHHH) attached through a short connector, for example
EGR. In some embodiments the linker and his tag sequence are added in the absence of a foldon sequence.
In certain embodiments, the intracellular and transmembrane sequences have been replaced by the amino acid sequence AGRHHHHHHH (SEQ ID No.: 97) or
SGRSLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHHH (SEQ ID NO:
82) .
Applicants have previously identified widely neutralizing antibodies isolated from primary human B cells from vaccinated individuals, some of which were specific for group 1 influenza viruses (eg, CR6261, described in WO 2008/028946) and others were specific. for group 2 influenza viruses (eg CR8020, described in WO 2010/130636). A detailed analysis of the epitopes of these monoclonal antibodies has revealed the reason for the lack of cross-reactivity of these specific antibodies. In both cases, the presence of glycans in group 1 or group 2 HA molecules at different positions explains at least in part the fact that the antibodies are group specific. By identifying type CR9114 antibodies that cross-react with many HA molecules of groups 1 and
<img file="MX357009B_D0074.tif" />
2, as will be described later, it has become possible for the human immune system to generate widely neutralizing antibodies against influenza viruses.
However, given the need for an annual vaccination schedule, these antibodies do not appear to be generated, or only to a very limited extent, after infection or (seasonal) vaccination with the Hl and / or H3 subtypes of influenza virus. . In certain embodiments, the present invention then provides polypeptides that present the stem region of HA in a correct conformational manner so that epitopes that generate broadly neutralizing antibodies are presented to the immune system in the absence of immune dominant variable regions. Since it is known that the glycan pattern differs between Hl and H3 from HA, and that this difference can lead to a more restricted antibody response to the group, in different modalities the polypeptides of the invention are based on group 2 molecules of HA (for example, HA of
H3). As shown in Example 3 below, the in vitro neutralizing capacity of CR9114 is higher for the Hl subtypes compared to the H3 subtypes. Therefore, it has been hypothesized that the CR9114 epitope is more accessible on Hl compared to H3 HA molecules which could be due to a glycan on N38 in HAl which is
ΙΜ
INSTITUTE Μ PE LA
INDUSTRIAL
.. Λ- JI. GOES
M EX ICA N. ·· y:> feK «CíS ÍROFIEUAD V *» »2W? Z
MDUSTRIAI 'Ug.
common to many subtypes of group 2 HA. Without confirming this particular theory, it can be speculated that if a polypeptide of the invention is Hl-based, the resulting antibodies are more likely to be hindered by glycan in
N38 of group 2 HA molecules and therefore would be somewhat less active in group 2 influenza viruses.
Therefore, in order to generate broadly neutralizing antibodies that act on influenza viruses of both groups 1 and 2 with good activity, in certain modalities the stem domain polypeptides of the invention are based on the H3 HA subtypes.
Humans are frequently infected with seasonal influenza viruses that comprise HA of the Hl subtype or
H3. Apparently, despite exposure to these influenza viruses, widely neutralizing antibodies are not very often generated in the wild. One of the reasons for this, in addition to the presence of the variable head region in HA, could be that exposure to a new subtype closely related to a previously existing one makes the response less extensive. Therefore it would be preferable to expose the individual to a sequence of a less related subtype. Thus , in yet another embodiment, the stem domain polypeptides of the invention are based on HA of a subtype of group 2 that
IMPI
MEXICAN INSTITUTE OF PROPERTY
INDUSTRIAL
<img file="MX357009B_D0075.tif" />
it contains an asparagine (N) at position 38 in HA-1— (N38 ··) -; - and it is not a subtype H3.
In certain embodiments, the polypeptides are based on a subtype of the influenza A virus. In certain embodiments, the polypeptides are not based on HA H7.
As previously described, the polypeptides of the invention are not only designed on the basis of parental HA sequences of subtypes of group 1 of influenza virus vaccines (such as, for example, Hl and H5), but also they can be based on HA sequences of influenza group 2 subtypes, in particular influenza virus group 2 subtypes that are used for influenza vaccines, such as H3. In accordance with the invention, polypeptides that conserve the CR8020 and CR8043 epitope were constructed because these antibodies can neutralize a wide range of group 2 strains (W02010 / 130636). In these polypeptides, the beta sheet at the base of the stem region and its vicinity should be as conserved as possible since this is the region where CR8020 and CR8043 bind to H3 HA.
In certain embodiments, HA domains are of an H3 subtype, preferably A / Wisconsin / 67/2005 (SEQ ID No.: 89) or A / Hong Kong / 1/1968 (SEQ ID No.: 121). The specialist will understand that other viruses can also be used
<img file="MX357009B_D0076.tif" />
IMPI influenza A comprising the H3 subtype of HA, ... do you,. According to the invention.
In certain embodiments, the polypeptides comprise or consist of a HAl N-terminal polypeptide segment comprising the amino acids between position 1 and position x of the HAl H3 domain, preferably amino acids between position p and position x of the domain
HAl, where x is any amino acid between positions y 69, such as 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67 or 68, of H3 HAl, preferably where x is 61 , 62, 63 or
68. In certain modalities, the polypeptide segment
C-terminal HAl comprises the amino acids between position and up to and including the C-terminal amino acid of the HAl domain
H3, where y is any amino acid between and including positions 292 and 325, of H3 HAl, preferably where y is 293, 306, 318 or 323.
In certain embodiments, HA domains are of an H3 subtype, preferably A / Wisconsin / 67/2005 (SEQ ID No.: 89) or A / Hong Kong / 1/1968 (SEQ ID No.: 121).
In accordance with the invention, the head domain was removed by deleting a large part of the HAl sequence and rejoining the N- and C-terminal sequences by means of a short linker. The length of the deletion may vary, but it is preferred that the last residue of the
MEXICAN INSTITUTE OF THE PR'jFíEÜAD
INDUSTRIAL
<img file="MX357009B_D0077.tif" />
* _ _ MEXICAN INSTITUTE
OF THE PROPERTY
INDUSTRIAL N-terminal sequence of HAl and the first residue of the C-terminal sequence are spatially close to avoid the introduction of tension through the connecting sequence. Deletions can be inserted into the H3 sequence at (the equivalent positions of) S62-P322,
S63-P305 and T64-T317. Those skilled in the art will be able to easily determine the equivalent positions by aligning the sequences using a suitable algorithm, such as · for example Clustal or Muscle. The remaining parts of the sequence can be directly attached or, alternatively, a flexible connector can be inserted. The connecting sequences can be from 1 to 50 amino acids in length. Flexible connectors of limited length (less than or equal to 10 amino acids) are preferred, for example GGG,
GGGG, GSA, GSAG, GSAGSA, GSAGSAG or the like. The length of the suppression may also vary, for example by decreasing the amount of residue in the suppression by starting at (the equivalent of) position 63, 64, 65, 66, 67, or by increasing the length of the suppression, by trim at position 57, 58, 59, 60 or 61. Similarly, the last amino acid to be deleted may be at (the equivalent of) position 317, 318, 319, 320 or 321, or by increasing the length of the deletion at (the equivalent of) position
323, 324, 325, 326 or 327. It is important to consider that the
<img file="MX357009B_D0078.tif" />
<img file="MX357009B_D0079.tif" />
Changes in the length of the deletion can be compensated in part by matching the length of the connector sequence, i.e. a larger deletion can be matched with a larger connector and vice versa. These polypeptides are also included in the invention.
In certain modalities x is 61 and y is 323.
In certain modes, x is 62 and y is 306.
In certain modalities x is 63 and y is 318.
In certain modes, x is (an equivalent of) position 62, 63, 64, 65, 66 or position 56, 57, 58, 59 or
60.
In certain modalities, y is (an equivalent of) position 306, 318, 319, 320, 321 or 322, or (an equivalent of) position 324, 325, 326, 327 or 328.
In one embodiment, the amino acid sequence linking the C-terminal residue of helix A with the N-terminal residue of helix CD corresponds to the amino acid sequence between the amino acid at position 400 and the amino acid at position 420 of HA2 of SEQ ID No.: 89, or amino acid residues at equivalent positions in other H3 virus strains, where the polypeptides comprise one or more mutations in the amino acid sequence that binds the residue
C-terminal of helix A with the N-terminal residue of helix CD, i.e. the amino acid sequence encompassing
<img file="MX357009B_D0080.tif" />
<img file="MX357009B_D0081.tif" />
INSTITIITO MKICAM) OF LA PPOPIEDAD
INDUSTRIAL amino acids 400-420 of SEQ ID NO: 89, or · residues — equivalent deamino acids in other H3 strains of influenza virus.
In certain embodiments, the amino acid sequence that links the C-terminal residue of helix A to the residue
N-terminal of the helix CD of influenza HA serotype H3 comprises the amino acid sequence of SEQ ID
N °: 104.
According to the invention, the polypeptides comprise one or more mutations in the amino acid sequence that links the C-terminal residue of the A helix to the N-terminal residue of the CD helix. In certain embodiments, the polypeptides comprise one or more of the mutations in the
Table 8, or equivalent mutations in other strains of influenza virus subtype H3.
In accordance with the invention, the cleavage site between HAl and HA2 has been removed. In certain embodiments, removal of the cleavage site at position 345 (numbering refers to SEQ ID No.: 89) was mutated (R345Q) to prevent the formation of HAl and HA2 from HAO.
Optionally, residues 347 can also be deleted.
351 (IFGAI, part of the fusion peptide) to minimize exposure of hydrophobic residues to the aqueous solvent. The positive charge on cleavage is 100% conserved in H3 and
<img file="MX357009B_D0082.tif" />
therefore it is possible to apply this mutation in all ids sequences.
The deletion of the head domain leaves loop B between residues 400-420 that were now exposed to the aqueous solvent. In H3 HAs this loop is highly conserved (see Table 9). The consensus sequence is 401
I (E / G) KTNEKFHQIEKEFSEVEGR 421 (SEQ ID No.: 104; numbering refers to SEQ ID No.: 89). To increase the solubility of this loop for the polypeptides of the invention in the prefusion conformation and to destabilize the post-fusion conformation, it is necessary to modify some hydrophobic residues in polar amino acids (S, Τ, N, Q), charged amino acids (R , Η, K, D, E), or flexibility must be increased by mutation in G. Specifically, mutations at positions 401, 408, 411, 415, 418, (numbering refers to SEQ ID No.: 89) will contribute to the stability of a polypeptide of the invention.
To stabilize the prefusion conformation of the polypeptides of the invention, a covalent bond is introduced between two distant parts in the primary sequences but close to the folded prefusion conformation. To that end, a disulfide bridge can be manipulated in the polypeptides of the invention, preferably between (the equivalent of) position 326 and 438 in H3 A / Wisconsin / 67/2005 (SEQ ID No.: 89).
<img file="MX357009B_D0083.tif" />
Art specialists will be able to easily determine<sup>1</sup> IcTS equivalent positions by aligning the sequences using a suitable algorithm, such as Clustal or
Muscle: The manipulated disulfide bridges are created by mutation of at least one (if the other is already a cysteine), but usually two spatially close residues in cysteine, which will spontaneously or by oxidation form a covalent bond between the sulfur atoms of these. waste. An alternative cysteine bridge can be created between (the equivalent of) position 334 and 393 in H3 A / Wisconsin / 67/2005 (SEQ ID No.: 89) by mutation of these residues in cysteine. In some cases, the cysteine at (the equivalent of) position 321 is modified by a glycine to prevent the formation of unwanted disulfide bridges.
In certain embodiments, the polypeptides comprise one or more of the following mutations F408S, I411T, F415S,
V418G, I401R, K326C, S438C, T334C, I393C, C321G.
Native HA exists as a trimer on the cell surface. Most of the interactions between the individual monomers that hold the trimer together are in the head domain. After removal of the head, the tertiary structure is destabilized, and therefore a strengthening of the interactions between the monomers in the truncated molecule will increase stability. In the domain of
<img file="MX357009B_D0084.tif" />
<img file="MX357009B_D0085.tif" />
Stem trimerization is mediated by the formation of a trimeric coiled loop motif. By reinforcing this motif a more stable trimer can be created. A consensus sequence for the formation of a trimeric coiled loop, IEAIEKKIEAIEKKIEAIEKK, is introduced at (the equivalent of) position 421 to 441. To avoid interference with the formation of the disulfide bridge between positions 326 and
438, an alternate shorter sequence was also used
IEAIEKKIEAIEKKI at (the equivalent of) positions 421 to
435. An alternative is to enter the sequence
RMKQIEDKIEEIESKQKKIEN, derived from GCN4 and known for its ability to trimerize, at position 421-441 or the shorter sequence RMKQIEDKIEEIESK at position 421 to 435.
The polypeptides of the invention can contain the intracellular sequences of HA and the transmembrane domain so that the resulting polypeptides are presented on the cell surface when expressed in cells. In other modalities, the cytoplasmic sequence and the transmembrane sequence are removed from (the equivalent of) the position
522 to the C-terminus, so that a secreted (soluble) polypeptide is produced after expression in cells. Optionally, some additional residues may be included in the soluble protein by deletion of the sequence of (the equivalent of) 523, 524, 525, 526, 527, 528
MEXICAN INSTITUTE V<sup>2</sup> Ut M I'ROPIÍOAO
INDUSTRIAL VV or 529. The soluble polypeptide can stabilize se — srdefftá-s — fíor .... ^.
introduction of a sequence known to form trimeric structures, ie AYVRKDGEWVLL (SEQ ID No.: 143) ('foldon' sequence), optionally linked by means of a linker. The connector may optionally contain a cleavage site for further processing according to protocols well known to those of skill in the art. To facilitate purification of the soluble form, a one-label sequence, for example a his (HHHHHHH) label attached through a short linker, for example, may be added.
EGR. In some embodiments the linker and his tag sequence are added in the absence of a foldon sequence.
In accordance with the present invention, the amino acid sequence between position 530 (numbering according to SEQ ID No.: l) · and the C-terminal amino acid of the HA2 domain can be removed and replaced by the following sequences:
EGRHHHHHHH (SEQ ID N °: 81) or
SGRSLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHHH (SEQ ID NO:
82) .
In certain embodiments, the HAl Nterminal stem segment does not comprise the signal sequence. The specialist will understand that the leader sequence (or signal sequence) that directs the transport of a protein during production (for example, corresponding to the
IMPI
MEXICAN INSTITUTE t <- DE LA P! <;;? IrsAU V7<sup>K</sup>'-', r *<sup>,</sup>j £ o ^
INDUSTRIAL amino acids 1-17 of SEQ ID N °: 89), generally-ne-estaTá '*' present in the final polypeptide which is used for example in a vaccine. In certain embodiments, the polypeptides according to the invention then comprise an amino acid sequence without the leader sequence.
According to the invention, the polypeptides are not based on influenza B HA molecules. Influenza B virus strains are strictly human. The antigenic variation in HA between strains of influenza virus type B is less than that observed among strains of type A. There are two genetically and antigenically distinct lineages of influenza B virus in circulation in humans, represented by the lineages B / Yamagata / 16/88 (also called B / Yamagata) and B / Victoria / 2/87 (B / Victoria) ( Ferguson et al., 2003). Although the spectrum of illness caused by influenza B viruses is generally milder than the spectrum caused by influenza A viruses, severe illnesses requiring hospitalization with influenza B infection are still frequently observed.
In accordance with the present invention, polypeptides are provided that mimic the specific epitopes of CR6261 and
CR9114, and which can be used as immunogenic polypeptides, for example to generate cross neutralizing antibodies
<img file="MX357009B_D0086.tif" />
when administered in vivo, either alone or in —-- · * - ----------- with other prophylactic and / or therapeutic treatments. Cross neutralizing antibodies are antibodies that have the ability to neutralize at least two, preferably at least three, four, or five different subtypes of influenza A viruses belonging to phylogenetic group 1, and / or at least two, preferably at least three, four or five different influenza A virus subtypes belonging to phylogenetic group 2, and / or at least two different influenza B virus subtypes, in particular at least all virus strains that are neutralized by CR6261 and CR9114.
The polypeptides of the invention do not comprise full-length HAl. In certain embodiments, immunogenic polypeptides are substantially less than
HAO, preferably without all or substantially all of the globular head of HA. Preferably, the immunogenic polypeptides are not more than 360, preferably not more than 350, 340, 330, 320, 310, 305, 300, 295, 290, 285, 280,
275 or 270 amino acids in length. In one embodiment, the immunogenic polypeptide is between about 250 and about 350, preferably between about
260 and about 340, preferably between about 270 and about 330, preferably
<img file="MX357009B_D0087.tif" />
between about 270 and about 330 amino acids in length.
In certain embodiments, the polypeptides selectively bind to the CR6261 and / or CR9114 antibodies. In one embodiment, the polypeptide does not bind to CR8057 antibody. In one embodiment, CR6261 comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 20 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 21;
CR9114 comprises a variable region of the heavy chain comprising the amino acid sequence of SEQ ID NO: 18 and a variable region of the light chain comprising the amino acid sequence of SEQ ID NO: 19. In one embodiment, CR8057 comprises a variable region of the heavy chain comprising the amino acid sequence of SEQ ID NO: 22 and a variable region of the light chain comprising the amino acid sequence of SEQ ID NO: 23.
As previously described, the polypeptides comprise an influenza hemagglutinin HA1 domain comprising an N-terminal stem segment of HA1 covalently linked by means of a linker sequence of 0-50 amino acid residues to the C-terminal stem segment. of
HA1. The linker sequence does not appear in wild type or wild type HA. In certain embodiments, the connector is a
<img file="MX357009B_D0088.tif" />
INDUSTRIAL peptide comprising one amino acid residue, amino acid residues, three or fewer amino acid residues, four or fewer amino acid residues, five or fewer amino acid residues, ten or less amino acid residues, 15 or less amino acid residues or 20 or less amino acid residues or 30 or less amino acid residues or 40 or less amino acid residues or 50 or less amino acid residues. In a specific embodiment, the linker sequence is a sequence selected from the group consisting of G, GS, GGG, GSG, GSA, GSGS, GSAG, GGGG, GSAGS,
GSGSG, GSAGSA, GSAGSAG and GSGSGSG.
The present invention also provides methods for providing the polypeptides of the invention, in particular for providing the amino acid sequence of the HA stem domain polypeptide according to the invention, as well as the polypeptides that can be obtained or are obtained by these methods. In certain modalities, the 'methods comprise the steps of:
providing an influenza HAO amino acid sequence, for example a sequence of influenza HAO serotypes Hl, H5 or H3;
eliminate the cleavage site between HAl and HA2, preferably by mutation of the C-terminal amino acid of HAl into an amino acid other than arginine (R) or lysine (K);
Λ Ρ I ίτϋ> 5, instituto mkxican ·. » ΡΆά? ~ ΤΤ ··>) '\ OF PROPERTY;
INDUSTRIAL ''
- deleting the amino acid sequence from the globular head domain of the HAO sequence. This is done by removing a segment of the HAl domain between the amino acid at position x and an amino acid at position y, and reattaching the N-terminal segment (spanning from amino acid at position 1 to and including amino acid at position x of HAl) and the C-terminal segment of HAl (spanning from the amino acid to the C-terminal amino acid of HAl) thus obtained, optionally via a 0-50 amino acid linker sequence.
increase the stability of the prefusion conformation and destabilize the post-fusion conformation of the modified HA, preferably by introducing one or more mutations in the amino acid sequence that joins the C-terminal residue of helix A with the N- residue. helix terminal CD, preferably in the amino acid sequence encompassing amino acids 402-418 of Hl HA, in particular comprising the amino acid sequence MNTQFTAVGKEFN (H / K) LE (K / R) (SEQ ID No.: 17 ) or
I (E / G) KTNEKFHQIEKEFSEVEGR 421 (SEQ ID No.: 104) for H3 HA.
Mutations preferably comprise the substitution of hydrophobic amino acid residues by hydrophilic amino acid residues.
introduce one or more disulfide bridges into the
<img file="MX357009B_D0089.tif" />
HA stem domain polypeptide. . __________
According to the invention, elimination of the cleavage site between HAl and HA2 can be achieved by mutation of R (in a few cases of K) by Q at the Pl position (see, for example, Sun et al, 2010, for an explanation of the cleavage site nomenclature (position 343 in SEQ ID
N °: 1). The Q mutation is preferred but S, Τ, N, D or E are other alternatives.
Elimination of the head domain can be achieved, for example, by deletion of amino acids 53 to 320 of SEQ ID NO: 1. Those skilled in the art will be able to easily determine the equivalent positions by sequence alignment using a suitable algorithm , such as for example Clustal or Muscle. The remaining parts of the sequence can be directly attached or, alternatively, a flexible connector can be inserted. The connecting sequences can be from 1 to 50 amino acids in length. Flexible connectors of limited length (less than or equal to 10 amino acids) are preferred, for example GGG, GGGG, GSA,
GSAG, GSAGSA, GSAGSAG or the like. The length of the suppression can also be varied, for example starting the suppression at position (x) (or an equivalent thereof), for example at position 54, 55, 56, 57 or 58, or to increase the length of the suppression, by means of a cut in the
<img file="MX357009B_D0090.tif" />
<img file="MX357009B_D0091.tif" />
<img file="MX357009B_D0092.tif" />
position.47, 48, 49, 50, 51 or 52. Similarly, the last amino acid to be deleted can be found at position (y) (or an equivalent thereof), such as 315,
316, 317, 318, or 319, or to increase the length of the suppression at the position (or an equivalent thereof) 321,
322, 323, 324 or 325. It is important to consider that changes in the length of the deletion can be compensated in part by matching the length of the connector sequence, that is, a larger deletion can be matched with a larger connector and vice versa. These polypeptides are also included in the invention.
According to the invention, it increases the solubility of the loop between helix A and helix CD. This loop is made up of (equivalents of) residues 402 to 418 in HI
A / Brisbane / 59/2007 (SEQ ID N °: 1). Accordingly, the stability of the prefusion conformation is increased and the post-fusion conformation of the modified HA is destabilized. This loop is highly conserved in Hl sequences, as can be seen later in
Table 6. This can be accomplished, for example, by replacing amino acids I, L, F, or V in said loop with hydrophilic counterparts. Those skilled in the art will be able to easily determine the equivalent positions by aligning the sequences using a suitable algorithm, such as by
<img file="MX357009B_D0093.tif" />
Clustal or Muscle example. Mutations ._ ^ pQX ^ _gJjxihaj ..
they destabilize the post-fusion conformation since the great flexibility of this amino acid leads to a decrease in the stability of the post-fusion helix that will form with this part of the HA sequence. The consensus sequence that describes the loop between residues 402-418 of influenza HA serotype Hl is (SEQ ID No.: 17) MNTQFTAVGKEFN (H / K) LE (K / R). In certain polypeptides of the invention, the amino acid at positions 406, 409, 413 and / or
416 (or its equivalents, determined by sequence alignment) is a polar (S, Τ, N, Q), charged (R, Η, K, D, E) or flexible (G) amino acid. Also possible
<td>combinations</td><td>of</td><td>mutations</td><td>in these</td><td>sites,</td><td>by</td><td>example</td>
<td>F406S, V409T,</td><td colspan="3">L416S as shown in</td><td>the SEQ ID</td><td>N °:</td><td>10 and in</td>
<td>SEQ ID N °:</td><td> 14 .</td><td>In some</td><td>cases, it</td><td>prefer</td><td>a</td><td>mutation</td>
to reset the consensus amino acid, for example where V or M is at position 404 (for Τ), V at 408 (for A) or 410 (for G) or I at 414 (for N); the incidence of sequences with these particular amino acids is very low. Table 6 shows a summary of the previously described mutations that characterize the polypeptides of the invention.
In accordance with the invention, one or more disulfide bridges are introduced into the stem domain polypeptides, preferably between the amino acids of (or the equivalent
<img file="MX357009B_D0094.tif" />
de) position 324 and 436 in Hl A / Brisbane / 59 / 200-7; SEQ ΙΟΝ<sup>0</sup>: 13-16. Those skilled in the art will be able to easily determine the equivalent positions by sequence alignment using a suitable algorithm such as Clustal,
Muscle etc. The manipulated disulfide bridges are created by mutation of at least one (if the other is already a cysteine), but usually two spatially close residues in cysteine, which will spontaneously or by oxidation form a covalent bond between the sulfur atoms of these residues.
The polypeptides that can be obtained by said method also form part of the present invention.
Native HA exists as a trimer on the cell surface. Most of the interactions between the individual monomers that hold the trimer together are in the head domain. After removal of the head, the tertiary structure is destabilized, and therefore a strengthening of the interactions between the monomers in the truncated molecule will increase stability. In the stem domain trimerization is mediated by the formation of a trimeric coiled-loop motif. By reinforcing this motif a more stable trimer can be created. In accordance with the invention, a consensus sequence can be introduced for the formation of a trimeric coiled loop, for example
IMPIOS
MEXICAN INSTITUTE V'fe'OE LA PftOPIEDAD V »* INDUSTRIAL
IEAIEKKIEAIEKKIE (SEQ ID No.: 83), in a polypeptide of the invention at (the equivalent of) position 418 to 433. In certain embodiments, the sequence MKQIEDKIEEIESKQ (SEQ
ID No. 84), derived from GCN4 and also known for its ability to trimerize, is entered at (the equivalent of) position 419-43. In certain modalities, the trimer interface is stabilized by modifying M420, L423,
V427, G430 for isoleucine.
In certain embodiments, the polypeptides comprise an amino acid sequence selected from the group consisting of SEQ ID No.: 3-16, SEQ ID No.: 44-53, SEQ ID No.: 111-114, SEQ ID No.: 119-120, SEQ ID No.: 125, 126, 130, SEQ ID No.: 144-175 and SEQ ID No.: 177-187.
In certain embodiments, the polypeptides are selected from the group consisting of SEQ ID NO: 45, the
SEQ ID N °: 113 and SEQ ID Ν ': 130.
The skilled person will understand that the leader sequence (or signal sequence) that directs the transport of a protein during production (for example, corresponding to amino acids 1-17 of SEQ ID NO: 1), is not present in the final polypeptide which is used for example in a vaccine. In certain embodiments, the polypeptides according to the invention then comprise an amino acid sequence without the leader sequence.
<img file="MX357009B_D0095.tif" />
IMPI
Influenza taphth-1-a —------- hemagglutinin domain polypeptides can be prepared according to any technique considered suitable to the specialist, including the techniques described below.
Accordingly, the immunogenic polypeptides of the invention can be synthesized as DNA sequences by standard methods known in the art and cloned and then expressed, in vitro or in vivo, using suitable restriction enzymes and methods known in the art. The present invention then also relates to nucleic acid molecules encoding the previously described polypeptides. The invention further relates to vectors comprising nucleic acids encoding the polypeptides of the invention. In certain embodiments, the nucleic acid molecule according to the invention forms part of a vector, for example a plasmid. Such vectors can be easily manipulated by methods well known to the person skilled in the art, and can be designed, for example, so that they can replicate in prokaryotic and / or eukaryotic cells. Furthermore, many vectors can be used directly or in the form of a desired isolated fragment thereof, in the transformation of eukaryotic cells and will be wholly or in part integrated into the
<img file="MX357009B_D0096.tif" />
genome of said cells, resulting in stable host cells that comprise the desired nucleic acid in their genome. The vector used can be any vector suitable for cloning DNA and which can be used in the transcription of a nucleic acid of interest. When using host cells it is preferred that the vector be an integrating vector. Alternatively, the vector may be an episomal replication vector.
The person skilled in the art will be able to choose the appropriate expression vectors, and insert the nucleic acid sequences of the invention in a functional way. To obtain expression of the nucleic acid sequences encoding the polypeptides, those skilled in the art know that sequences capable of directing expression can be functionally linked to nucleic acid sequences encoding the polypeptide, resulting in recombinant acid molecules. nucleic acids that encode a protein or a polypeptide in a format that can be expressed. In general, the promoter sequence is located 5 'with respect to the sequences that should be expressed. There are many expression vectors available in the art, for example Invitrogen's pcDNA and pEF vector series; pMSCV and pTK-Hyg from BD Sciences; Stratagene pCMV-Script, etc., which can be used to obtain suitable promoters and / or
<img file="MX357009B_D0097.tif" />
transcription terminator sequences, sequences ,,, p.cJ.iA ··. and the like. When the sequence encoding the polypeptide of interest is appropriately inserted with reference to the sequences that govern transcription and translation of the encoded polypeptide, the resulting expression cassette is useful in producing the polypeptide of interest, termed expression. Expression directing sequences can include promoters, enhancers, and the like, and combinations thereof. They should have the ability to function in the host cell, thereby directing the expression of nucleic acid sequences functionally linked thereto. The skilled artisan will understand that various promoters can be used to achieve expression of a gene in host cells. Promoters can be constitutive or regulated, and can be obtained from a variety of sources, including viral, prokaryotic, or eukaryotic sources, or can be artificially engineered. The expression of the nucleic acids of interest can be from the natural promoter or derived from it or from a completely heterologous promoter (Kaufman, 2000). Some well known and widely used promoters for expression in eukaryotic cells include promoters derived from viruses, such as adenoviruses, for example the E1A promoter, promoters derived from cytomegalovirus (CMV),
<img file="MX357009B_D0098.tif" />
such as the immediate early CMV (IE) promoter (referred to as the CMV promoter herein) (obtainable, for example, from pcDNA, Invitrogen), promoters derived from simian virus 40 (SV40) (Das et al, 1985) and the like. Suitable promoters can also be derived from eukaryotic cells, such as the metallocythein (MT) promoters, the elongation factor promoter la (EF-la) (Gilí et al., 2001), the ubiquitin C promoter or
UB6 (Gilí et al., 2001), the actin promoter, an immunoglobulin promoter, heat shock promoters and the like. Evaluations of promoter function and promoter strength are a matter of routine for the skilled artisan, and in general may encompass, for example, cloning a test gene, such as the lacZ gene, the luciferase gene , the GFP gene, etc. behind the promoter sequence, and then evaluate the expression of said test gene. It is of course possible to alter the promoters by deletion, addition, mutation of the sequences thereof and then evaluate their functionality to obtain new, attenuated or improved promoter sequences. In accordance with the present invention, strong promoters that provide high levels of transcription in eukaryotic cells of choice are preferred.
Constructs can be transfected into cells
<img file="MX357009B_D0099.tif" />
eukaryotes (eg, plant, fungal, yeast, or animal cells) or in suitable prokaryotic expression systems, such as in E. coli, using methods well known to those skilled in the art. In some cases, you can add a sequence of a suitable 'mark' (such as, but not limited to, a his, myc, strep or flag mark) or a complete protein (such as, for example, but non-exhaustively, a maltose or glutathione S transferase binding protein) to the sequences of the invention to allow purification and / or identification of polypeptides from cells or the supernatant. Optionally, a sequence containing a specific proteolytic site can be included so that the tag can then be removed by proteolytic digestion.
Purified polypeptides can be analyzed by art-known spectroscopic methods (eg, circular dichroism spectroscopy, Fourier transform infrared spectroscopy, and NMR spectroscopy or X-ray crystallography) to investigate the presence of desired structures, such as propellers and beta sheets. . ELISA, Octet, and FACS and the like can be used to investigate the binding of the polypeptides of the invention to the broadly neutralizing antibodies previously described (CR6261,
CR9114, CR8057). Therefore, you can select
<img file="MX357009B_D0100.tif" />
polypeptides according to the invention of Lamn + correct statement.
The invention further relates to immunogenic compositions comprising a therapeutically effective amount of at least one of the polypeptides and / or nucleic acids of the invention. In certain embodiments, the compositions comprise polypeptides comprising hemagglutinin stem domains of (or based on) the HA of a
<td>subtype</td><td>of</td><td>influenza,</td><td>by</td><td colspan="2">HA-based example of</td><td>a</td>
<td>virus</td><td>the</td><td>influenza</td><td>than</td><td>comprises the</td><td>HA for example</td><td>of the</td>
<td>subtype</td><td>H1</td><td>or H7.</td><td>In</td><td>certain</td><td>modalities,</td><td>the</td>
Compositions comprise polypeptides comprising HA-based hemagglutinin stem domains from two or more different influenza subtypes, for example compositions comprising both polypeptides comprising HA-based haemagglutinin domains of the H1 subtype and polypeptides comprising domains of hemagglutinin stem based on HA of subtype H7.
The immunogenic compositions preferably further comprise a pharmaceutically acceptable carrier. In the present context, the term "pharmaceutically acceptable" means that the vehicle, at the dosages and concentrations used, will not cause undesired or harmful effects on the subjects to whom it will be administered.
<img file="MX357009B_D0101.tif" />
<img file="MX357009B_D0102.tif" />
Such acceptable pharmaceutically acceptable vehicles and excipients are well known in the art (see Remington's
Pharmaceutical Sciences, 18<sup>to</sup> edition, AR Gennaro, Ed.,
Mack Publishing Company [1990]; Pharmaceutical Formulation
Development of Peptides and Proteins, S. Frokjaer and L.
Hovgaard, Eds., Taylor & Francis [2000]; and Handbook of
Pharmaceutical Excipients, 3<sup>to</sup> edition, A. Kibbe, Ed.,
Pharmaceutical Press [2000]). The term vehicle refers to a diluent, adjuvant, excipient, or vehicle with which the composition will be administered. Saline solutions and aqueous solutions of dextrose and glycerol can be used, for example, as liquid carriers, particularly in injectable solutions. The exact formulation should suit the mode of administration. Polypeptides and / or nucleic acid molecules are preferably formulated and administered as a sterile solution. Sterile solutions are prepared by sterile filtration or by other methods known per se in the art.
The solutions can then be lyophilized or filled in pharmaceutical dosing containers. The pH of the solution is generally in the range of pH
3.0 and 9.5, for example pH 5.0 to 7.5.
The invention also relates to methods of inducing an immune response in a subject, where the
<img file="MX357009B_D0103.tif" />
The method comprises administering to a subject, a d <-> polypeptide, a nucleic acid molecule and / or an immunogenic composition previously described. A subject according to the invention is preferably a mammal that can be infected with an agent causing an infectious disease, in particular an influenza virus, or that can otherwise benefit from the induction of an immune response, said subject for example a rodent, for example a mouse, a ferret or a domestic or farm animal, or a non-human primate or a human. Preferably the subject is a human being. The invention then provides methods of inducing an immune response to an influenza virus hemagglutinin (HA), in particular a group 1 and / or group 2 influenza A virus, such as an influenza virus comprising the HA of subtype Hl, H2, H3, H4, H5, H7 and / or
H10, and / or from an influenza B virus, in a subject, using the polypeptides, nucleic acids and / or immunogenic compositions described herein. In some modalities, the induced immune response is effective in preventing and / or treating an influenza virus infection caused by subtypes of group 1 and / or group 2 influenza A viruses and / or influenza B viruses . In some embodiments, the immune response induced by the polypeptides, nucleic acids, and / or compositions
<img file="MX357009B_D0104.tif" />
Immunogenic agents described herein are effective for, preventing, and / or treating an infection with influenza A and / or B virus caused by two, three, four, five, or six subtypes of influenza A and / or B viruses.
Since it is well known that proteins and / or small nucleic acid molecules do not always effectively induce a potent immune response, it may be necessary to increase the immunogenicity of polypeptides and / or nucleic acid molecules by addition of an adjuvant. In certain embodiments, the immunogenic compositions described herein comprise or are administered in combination with an adjuvant. The adjuvant for combined administration with a composition described herein can be administered before, concomitantly, or after administration of said composition. Examples of suitable adjuvants include aluminum salts such as aluminum hydroxide and / or aluminum phosphate;
oil emulsion compositions (or oil-in-water compositions), including squalene-water emulsions, such as MF59 (see, eg, WO 90/14837); saponin formulations, such as for example QS21 and Complexes
Immunostimulants (ISCOMS) (see, eg, US
5,057,540; WO 90/03184, WO 96/11711, WO 2004/004762, WO
2005/002620); bacterial or microbial derivatives, being
<img file="MX357009B_D0105.tif" />
IMPI
MEXICAN INSTITUTE λ q DE LA ^ RORIEíjAO
0 INDUSTRIAL examples of the same monophosphoryl lipid A (M-PL ·), MPL 3, -0 .- =.
deacylated (3dMPL), oligonucleotides containing the motif
CpG, ADP ribosilating bacterial toxins or mutants thereof, such as heat-labile enterotoxin LT from E.
coli, cholera toxin CT, Pertussis toxin PT or tetanus toxoid TT, Matriz M (Isconova). In addition, immunopotentiating technologies, such as by fusion of the polypeptides of the invention with proteins known in the art, can be used to enhance the immune response (eg, tetanus toxoid, CRM197, rCTB, bacterial flagellins, or others) or by inclusion of the polypeptides in virosomes or combinations thereof. Other non-exhaustive examples that can be used are disclosed, for example, in
Coffman et al., (2010).
In one embodiment, the influenza hemagglutinin stem domain polypeptides of the invention are incorporated into viral-like particle vectors (VLPs). The
VLPs generally comprise one or more viral polypeptides, typically derived from one or more structural proteins of a virus. VLPs preferably do not have the ability to replicate. In certain embodiments, VLPs may lack the entire genome of a virus or may comprise a portion of the genome of a virus. In some modalities, VLPs do not have the ability to infect a
<img file="MX357009B_D0106.tif" />
cell. In some embodiments, VLPs express “~ 5Utüf ^ '' their 'surface one or more viral (eg, virus surface glycoprotein) or non-viral targeting groups (eg, an antibody or protein) known to a art specialist.
In a specific embodiment, the polypeptide of the invention is incorporated into a virosome. A virosome containing a polypeptide according to the invention can be produced using techniques known to those skilled in the art. For example, the virosome can be produced by disrupting a purified virus, removing the genome, and reassembling the particles with viral proteins (eg, a influenza hemagglutinin stem domain polypeptide) and lipids to form virus particles. lipid containing viral proteins.
The invention also relates to the previously described polypeptides, nucleic acids and / or immunogenic compositions for inducing an immune response in a subject against influenza HA, in particular for use as a vaccine. The influenza hemagglutinin stem domain polypeptides, the nucleic acids encoding said polypeptides, or the vectors comprising said nucleic acids or polypeptides described herein can then be used to generate antibodies
100
IMPLO®.
INSTITUTO MFXICaK ni LA ΜΟΤίεΟΛ ·) CVací »,
INDUSTRIAL '' A'UTL neutralizers against influenza viruses, for example, __ against the stem region of the influenza virus hemagglutinin. The invention relates in particular to the previously described polypeptides, nucleic acids and / or immunogenic compositions for use as a vaccine in the prevention and / or treatment of a disease or condition caused by an influenza A virus of the phylogenetic group 1 and / or from phylogenetic group 2 and / or by an influenza B virus. In one embodiment, the vaccine can be used in the prevention and / or treatment of diseases caused by two, three, four, five, six or more different subtypes of phylogenetic groups 1 and / or 2 and / or virus influenza B. The polypeptides of the invention can be used after synthesis in vitro or in a suitable cellular expression system, including bacterial and eukaryotic cells or, alternatively, they can be expressed in vivo in a subject in need, by expression of an acid nucleic encoding the immunogenic polypeptide. Such nucleic acid vaccines can take any form, including naked DNA, plasmids, or viral vectors, including adenoviral vectors.
Administration of the polypeptides, nucleic acid molecules and / or immunogenic compositions according to the invention can be carried out using the
<img file="MX357009B_D0107.tif" />
101 standard administration routes. Non-limiting examples-examples include parenteral administration, such as intravenous, intradermal, transdermal, intramuscular, subcutaneous, etc., or administration by mucosa, for example intranasal, oral and the like. The specialist will be able to determine the various possibilities to administer the polypeptides, nucleic acid molecules and / or immunogenic compositions according to the invention, in order to induce an immune response. In certain embodiments, the polypeptide, nucleic acid molecule, and / or immunogenic composition (or vaccine) is administered more than once, i.e., in the so-called homologous priming-priming regimen. In certain embodiments where the polypeptide, nucleic acid molecule, and / or immunogenic composition is administered more than once, administration of the second dose may be performed after a time interval of, for example, one week or more later from the administration of the first dose, two weeks or more after the administration of the first dose, three weeks or more after the administration of the first dose, one month or more after administration of the first dose, six weeks or more after administration of the first dose, two months or more after administration of the first dose, 3 months
102
IMPIí ^ Mexican industrial institute or more after the administration of the primara_dose, 4 months or more after the administration of the first dose, etc., up to several years after the administration of the first dose of the polypeptide, of the nucleic acid molecule and / or of the immunogenic composition. ' It is also possible to administer the vaccine more than twice, for example three times, four times, etc. so that the first administration of sensitization is followed by more than one booster administration. In other embodiments, the polypeptide, nucleic acid molecule, and / or immunogenic composition according to the invention is only administered once.
Polypeptides, nucleic acid molecules, and / or immunogenic compositions can also be administered either as a priming or as a boost, in a heterologous priming-boost regimen.
The invention further provides methods of preventing and / or treating influenza virus disease in a subject using the polypeptides, nucleic acids, and / or compositions described herein. In a specific embodiment, the method of preventing and / or treating influenza virus disease in a subject comprises administering to a subject in need an effective amount of a polypeptide, a nucleic acid and / or a
<img file="MX357009B_D0108.tif" />
103
IMPI
INSTl IUTO MEXICANO OE INDUSTRIAL PROPERTY immunogenic composition, as previously described ^ - A therapeutically effective amount refers to an amount of the polypeptide, nucleic acid and / or composition defined herein that is effective in preventing, alleviating and / or treat a disease or condition resulting from an infection with an influenza A virus of groups 1 or 2, and / or with an influenza B virus. Prevention includes inhibiting or reducing the spread of the influenza virus or inhibiting or reducing the onset, development, or progress of one or more of the symptoms associated with an infection with an influenza virus. Relief, as used herein, may refer to the reduction of visible or perceptible symptoms of the disease, a viraemia, or any other manifestation of measurable influenza infection.
Those in need of treatment include those already suffering from a condition resulting from infection with a group 1 or 2 influenza A virus, or an influenza B virus, as well as those in whom infection with the virus should be prevented. influenza virus. The polypeptides, nucleic acids and / or compositions of the invention can then be administered to a naive subject, i.e., a subject who is not suffering from a disease caused by an infection with the influenza virus or has not yet been, and who is
<img file="MX357009B_D0109.tif" />
104
IMPIí ~ is not currently infected with an influenza virus infection, or subjects who are already and / or who have already been infected with an influenza virus.
In one embodiment, prevention and / or treatment may be directed at groups of patients who are susceptible to infection with the influenza virus. Such patient groups include, but are not limited to, for example, the elderly (for example h 50 years of age, D 60 years of life and preferably D 65 years of life), young people (for example, ñ 5 years of age, year of life), hospitalized patients and patients who were treated with an antiviral compound, but who have demonstrated an inadequate antiviral response.
In another embodiment, polypeptides, nucleic acids, and / or immunogenic compositions can be administered to a subject in combination with one or more additional active agents, such as existing or future influenza vaccines, monoclonal antibodies, and / or antiviral agents, and / or antibacterial and / or immunomodulatory agents. Said one or more additional active agents may be beneficial in the treatment and / or prevention of an influenza virus disease or may alleviate a symptom or condition associated with an influenza virus disease.
105
<img file="MX357009B_D0110.tif" />
influenza. In some embodiments, additional active agents are analgesics, antipyretic medications, or therapies that relieve or assist breathing.
The dosage regimens of the polypeptides and / or nucleic acid molecules of the invention can be adjusted to provide the desired optimal response (eg, a therapeutic response). A suitable dosage can vary in a range of, for example,
0.1-100 mg / kg of body weight, preferably 1-50 mg / kg of body weight, preferably 0.5-15 mg / kg of body weight. The precise dosage of the polypeptides and / or nucleic acid molecules to be used will depend, for example, on the route of administration, and on the severity of the infection or disease caused by it, and should be determined according to the professional judgment. in charge and the circumstances of each subject. For example, effective doses vary depending on the target site, the patient's physiological status (including age, body weight, health), and whether the treatment is prophylactic or therapeutic. Usually the patient is human, but non-human mammals including transgenic mammals can also be treated. Treatment dosages are optimally titled to
106
<img file="MX357009B_D0111.tif" />
optimize safety and effectiveness.
The polypeptides of the invention can also be used to verify the binding of monoclonal antibodies identified as potential therapeutic candidates. Furthermore, the polypeptides of the invention can be used as diagnostic tools, for example to assess the immune status of an individual, to establish if there are antibodies in the serum of said individual that are capable of binding to the polypeptide of the invention. Accordingly, the invention also relates to an in vitro diagnostic method for detecting the presence of an influenza infection in a patient, wherein said method comprises the steps of a) bringing a biological sample obtained from said patient into contact with a polypeptide. according to the invention; and
b) detecting the presence of antigenic antibody complexes.
The polypeptides of the invention can also be used to identify new binding molecules or to enhance existing binding molecules, such as monoclonal antibodies and antiviral agents.
The invention is further illustrated in the following examples and in the figures. These examples are not intended to limit the scope of the invention in any way.
107
INSTITUTE ΜΕΧ: Ο.Ν <DLLAPS 'í'IEDAD INDUSTRIAL
EuiaMPLOS
Example 1: Identification of a new group 1 and group 2 cross neutralization antibody: CR9114
Peripheral blood from normal healthy donors was collected by venipuncture in sample tubes with EDTA anticoagulant. ScFv phage display libraries were obtained essentially as described in WO 2008/028946, which is incorporated by reference herein. Screening was carried out against influenza A recombinant hemagglutinin (HA) subtype Hl (A / New Caledonia / 20/99), H3 (A / Wisconsin / 67/2005), H4 (A / Duck / Hong Kong / 24 / 1976), H5 (A / Pollitoen / Vietnam / 28/2003), H7 (A / Netherlands / 219/2003) and H9 (A / HongKong / 1073/99). Two consecutive rounds of selection were made prior to isolation of the single chain single phage antibodies. After the second round of selection, the individual colonies of
E.coli to prepare monoclonal phage antibodies. Selected supernatants were validated containing single chain phage antibodies that were obtained in the screens that were previously described in ELISA for specificity, that is, binding to different HA antigens. For this purpose, Maxisorp ™ ELISA plates were coated with HA Hl (A / New Caledonia / 20/99), H3 (A / Wisconsin / 67/2005), H5
108
<img file="MX357009B_D0112.tif" />
(A / Vietnam / 1203/04) H7 (A / Paises Rajos / ?! 9/2003). and _ — B, (B / Ohio / 01/2005) recombinants expressed in baculovirus (Protein Sciences, CT, USA). Of the single chain phage antibodies that were obtained, the single chain phage antibody SC09-114 showed specific binding to Hl, H3, H5,
Recombinant H7 influenza A and HA influenza B. The binding and specificity of SC09-114 were validated by FACS analysis. For this purpose, recombinant influenza A HAs of full length subtypes Hl (A / New Caledonia / 20/1999), H3 (A / Wisonsin / 67/2005) and H7 (A / Netherlands / 219/2003) were expressed. on the surface of cells
PER.C6. SC09-114 showed specific binding to influenza HA HA subtypes Hl, H3 and H7. The light and heavy chain variable regions of the scFv were cloned as previously described (WO 2008/028946). The resulting expression constructs were transiently expressed by encoding the IgGl heavy and light human chains in combination in 293T cells and supernatants containing the human IgGl CR9114 antibody were obtained and produced using standard purification procedures.
<td>The sequences</td><td>of</td><td>amino acids from</td><td>the CDRs</td><td>of</td><td>the</td><td>chains</td>
<td colspan="2">heavy and light</td><td>from CR9114 se</td><td>they hit the</td><td colspan="2">Table</td><td>1. The</td>
<td>sequences of</td><td colspan="2">nucleotides and</td><td>amino acids</td><td>and</td><td>the</td><td>of the</td>
heavy and light chain variable regions are given more
109
IMPI
MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY
<img file="MX357009B_D0113.tif" />
ahead.
Example 2: CR9114 cross-binding reactivity
CR9114 was validated in ELISA for binding specificity, that is, binding to different HA antigens. For this purpose, Maxisorp ™ ELISA plates were coated with HA of Hl (A / New Caledonia / 20/1999), H3 (A / Wisconsin / 67/2005), H5 (A / Vietnam / 1203/04, H7 ( A / Netherlands / 219/2003) and recombinant baculovirus H9 (A / HongKong / 1073/99) (Protein Sciences, CT, USA). As a control, an unrelated IgG CR4098 was used. CR114 was shown to have heterosubtypical cross-binding activity for all recombinant HAs tested. See Table 2.
In addition, the antibody for heterosubtypal binding was tested by FACS analysis. For this purpose, full length recombinant HAs of influenza A subtypes Hl (A / New Caledonia / 20/1999), H3 (A / Wisonsin / 67/2005) and H7 (A / Countries) were expressed.
<td>Bass / 219/2003)</td><td>on</td><td>the</td><td>surface of</td><td>cells</td><td>PER.C6.</td><td>I know</td>
<td>incubated</td><td>cells</td><td>with</td><td>CR9114 during</td><td>1 hour</td><td>Following</td><td>by</td>
<td>three steps of</td><td>washed</td><td>with</td><td>PBS + 0.1% of</td><td>BSA. I know</td><td>detected</td><td>the</td>
antibody bound using anti-human antibody conjugated to
PE. As a control, PER.C6 cells without transfection were used.
CR9114 showed influenza A cross-binding activity to H1, H3 and HA subtypes of H7 but not to cell-type
IMPIí ~ INSTITUTO MEXICANO I
11U OF IA l'ROi IEOaO
INDUSTRIAL natural PER.C6. See Table 2. ____________
Example 3: CR9114 Cross Neutralization Activity
In order to determine if CR9114 was capable of blocking multiple strains of influenza A, additional in vitro virus neutralization (NAV) assays were carried out.
VNAs were carried out on MDCK cells (ATCC CCL-34). MDCK cells were cultured in cell culture medium
MDCK (MEM medium supplemented with antibiotics, 20 mM Hepes and 0.15% (w / v) sodium bicarbonate (complete MEM medium, supplemented with 10% (v / v) fetal bovine serum). Strains Hl (A / WSN / 33, A / New Caledonia / 20/1999, A / Solomon Islands / IVR-145 (high-growth gene rearrangement of A / Islands
Solomon / 3/2006), A / Brisbane / 59/2007, A / NYMC / X-181 (high-growth gene rearrangement of A / California / 07/2009), H2 (A / Env / MPU3156 / 05), H3 (A / Hong Kong / 1/68, A / Johannesburg / 33/94,
A / Panama / 2000/1999, A / Hiroshima / 52/2005, A / Wisconsin / 67/2005 and
A / Brisbane / 10/2007), H4 (A / WF / HK / MPA892 / 06), H5 (PR8-H5N1-HK97 (6: 2 genetic rearrangement of A / Hong Kong / 156/97 and A / PR / 8 / 34) and A / Eurasian Mallard / MPF461 / 07), H6 (A / Mallard
Eurasian / MPD411 / 07), H7 (NIBRG-60 (6: 2 genetic rearrangement of A / Azulón / Netherlands / 12/2000) and PR8-H7N7-NY (7: 1 genetic rearrangement of A / New York / 107 / 2003 (H7N7) and A / PR / 8/34)), H8 (A / Eurasian Mallard / MPH571 / 08) H9 (A / Hong Kong / 1073/99 and
A / Chick / HK / SSP176 / 09), H10 (A / Chick / Germany / N / 49) and H14
<img file="MX357009B_D0114.tif" />
111 MEXICAN INSTITUTE
OF THE PROPERTY
INDUSTRIAL X (PR8-H14N5 (6: 2 genetic rearrangement of
A / Azulón / Astracán / 263/1982 (H14N5) and A / PR / 8/34)) that were used in the test were all diluted to a titer of 5.7 xlO<sup>3 </sup>TCID50 / ml (50% infective dose of tissue culture per ml), with the titer calculated according to the Spearman method and
Karber. IgG preparations (200 pg / ml) were diluted
<td>serially in the middle (1: 2</td><td> - 1:512)</td><td>in the middle</td><td>MEM</td><td>full</td><td>in</td>
<td>quadruple wells</td><td colspan="2">25 μΐ mixed</td><td>of</td><td colspan="2">dilution</td>
<td>respective IgG with 25</td><td>μΐ of</td><td>suspension</td><td>of</td><td>virus (</td><td> 100</td>
<td>TCID50 / 25 μΐ) and incubated</td><td>during</td><td>an hour to</td><td> 37°</td><td>C. Then</td><td>I know</td>
<td>transferred suspension</td><td colspan="2">quadrupled</td><td colspan="2">to plates</td><td> 96</td>
wells with confluent cultures of MDCK in 50 μΐ of complete MEM medium. Before use, MDCK cells were seeded at 3xl0<sup>4 </sup>Cells per well in MDCK cell culture medium were grown until the cells reached confluence, washed with between 300 and 350 µΐ of PBS, pH 7.4, and finally 50 µ completo of complete MEM medium was added to each well. Inoculated cells were cultured 3-4 days at 37 ° C and observed daily for development of cytopathogenic effect (ECP).
The ECP was compared with the positive control.
CR9114 was shown to have heterosubtypical cross-neutralizing activity against representative strains of all tested influenza virus subtypes A Hl, H2, H3, H4, H5,
H6, H7, H8, H9 and H10. See Table 3.
112
IMPI
INOTITUTC MEXICANO DE LA HKCHIEUAD INDUSTRIAL.
<img file="MX357009B_D0115.tif" />
Example 4: Design of a stem domain polypeptide comprising conserved stem domain epitopes from
H6 HA-based CR6261 and CR9114
Complete human monoclonal antibodies against influenza virus hemagglutinin with broad cross-neutralizing potency were previously identified. CR6261 (as described in WO 2008/028946) was shown to have broad cross-neutralizing activity against influenza A viruses of phylogenetic group 1. In addition, CR9114, previously described, has been shown to be able to bind and neutralize influenza A virus from both phylogenetic group 1 and 2, as well as influenza B virus. Functional and structural analysis has revealed that these antibodies interfere with the membrane fusion process and are directed against highly conserved epitopes in the stem domain of the protein
Influenza HA (Throsby et al. (2008); Ekiert et al. (2009)
W02008 / 028946, and copending application no. EP11173953.8).
In the research leading to the present invention, new molecules comprising the HA stem domains containing these epitopes were designed with the aim of creating universal epitope-based immunogenic polypeptides that can be used, for example as a vaccine that induces protection against a wide range of strains of
<img file="MX357009B_D0116.tif" />
113 influenza. Essentially, the highly variable and immunodominant part, that is the head domain, is first removed from the full-length HA molecule to create a HA stem domain polypeptide, also referred to as mini-HA. In this way, the immune response will be redirected to the stem domain where it is l the epitopes for the broadly neutralized antibodies are located. Antibodies CR6261 and CR9114 were used to test the correct folding of the newly created molecules, and to confirm the presence of neutralizing epitopes.
The polypeptides of this invention therefore present conserved epitopes from the stem proximal to the membrane domain of the HA molecule to the immune system in the absence of dominant epitopes that are present in the head domain distal to the membrane. To this end, part of the primary sequence of the HAO protein that forms the head domain is removed and reconnected, either directly or by introducing a flexible short linker sequence ('linker') to restore the continuity of the chain. The resulting sequence is further modified by introducing specific mutations that stabilize the native three-dimensional structure of the remaining part of the HAO molecule.
The role of the HA molecule in the virus is binding
<img file="MX357009B_D0117.tif" />
to cell surface receptor sialic acid and, depulping uptake into endosomes, mediates the fusion of viral and endosomal membranes leading to the release of viral RNA into the cell. An essential step in the fusion process is a major conformational change in the HA molecule that rearranges the secondary structure elements of the molecule so that the fusion peptide is exposed. As a consequence, there are two conformations (pre- and post-fusion) of the HA molecule that are very different in terms of its tertiary structure. Because protein
Viral HA is exposed primarily to the immune system in the pre-fusion stage, it is important to ensure that the polypeptide of the invention adopts this conformation. This requirement can be achieved by stabilizing the pre-melt conformation and at the same time destabilizing the post-melt conformation. This stabilization / destabilization is a necessity because the pre-fusion conformation is metastable and the adoption of the post-fusion conformation results in a stable conformation, that is, a minimum of low energy (Chen et al., 1995).
In this example, HA from H1N1 A / Brisbane / 59/2007 (SEQ ID NO: 1) is taken as the primary (or wild-type) sequence to create the polypeptides of the invention.
<img file="MX357009B_D0118.tif" />
IMPI „„ „MEXICAN INSTITUTE
115 Say LA I'lOi'ÍEUAD
INDUSTRIAL
In a first step, the polypeptides of the 1st invention are constructed by removing the 'HAl sequences between positions 59 and 291 (numbering refers to the position in the HAO sequence, as shown in SEQ ID
NO: 1. In certain embodiments, the HAl part comprises amino acids 18 to 343 and the HA2 part includes amino acid residues 344 to 565; because SEQ ID NO: 1 comprises the signal peptide, and that the HAl part starts at the position
18). This results in the removal of residues 60 to 290 of HAO. These residues were replaced by a GGGG linker sequence. Then, the accessible surface area of each residue was calculated in both the pre and post fusion conformation with the help of Brugel (Delhaise et al.,
1984). The degree of exposure and concealment of each residue was determined as described in Samantha et al. (2002), where emphasis was placed on residues that are exposed in the pre-fusion conformation and that were hidden in the post-fusion conformation. Further analysis of these residues indicated that some of these amino acid residues can be mutated in such a way that the mutation does not have an effect on the pre-fusion conformation but destabilizes the post-fusion conformation. These residues generally have a hydrophobic side chain and are involved in the formation of the coiled helix in the conformation of
<img file="MX357009B_D0119.tif" />
116 post fusion. Mutation of these amino acid residues to * a hydrophilic amino acid will damage the properties of the wound helix - the contacts between the helices in a wound helix. They are generally hydrophobic - and therefore destabilize the post fusion conformation.
Following this reasoning, some mutations were introduced in the HA2 part of the sequence: Phe 406 a Ser (F406S), Val 409 a Thr (V409T), Leu 416 a Ser (L416S) and Tyr
502 to be (Y502S). These are mutations that remove a hydrophobic residue from the HA surface. It should be noted that the L416 mutation to either S or T also introduces an N-glycosylation consensus site (the consensus sequence is NX (S / T). Glycosylation at this position will further increase the solubility of this region. In addition, Leu 58 to Thr (L58T), Val 314 to Thr (V314T) and lie 316 to Thr (I316T) were mutated; these mutations are all in the HAl domain, that is, the part of the sequence that corresponds to HAl after cleavage of the native HAO chain. The last two mutations maintain the beta branch of the side chain but remove a hydrophobic residue from the surface. As will be shown below, some of these mutations were introduced into all variants, others were tested on separate polypeptides to investigate whether mutations influence each other in a non-way.
<img file="MX357009B_D0120.tif" />
117
IMPW
MEXICAN INSTITUTE
OF THE DESIRABLE KVINDUSTRIAL PISDAD.
To increase the stability of the polypeptides, two disulfide bridges were investigated to block HA in the pre-fusion conformation. Disulfide bridges are formed between residues that are spatially at an appropriate distance from each other (in the full-length HA molecule) and that already have their C beta atom in the correct position to form a disulfide bridge. The first proposed disulfide bridge is between position 321 (domain HA1) and position
405 (HA2 domain). Within the HA2 domain, a disulfide bridge was created between positions 413 and 421.
Because cleavage of HA at position R343 is an essential step for conformational change to take place, in the polypeptides of the invention, the cleavage site was removed by introducing an Arg (R) mutation to a Gln (Q). Another solution according to the invention is to change the Arg to a Gln and remove residues 345 to 350, a small part of the eHA2 fusion peptide. Elimination of this (hydrophobic) sequence will further stabilize the polypeptide.
In certain embodiments, the polypeptides of the invention contain the intracellular sequences of HA and the transmembrane domain. In other modalities, the cytoplasmic and transmembrane sequences were removed from the
<img file="MX357009B_D0121.tif" />
<img file="MX357009B_D0122.tif" />
118 position (or the equivalent thereof) 523, 524, 525, 526,
<td> 526, 527,</td><td>528, 529, or</td><td>HA2 530</td><td>or the C-terminal</td><td>of</td><td>HA2</td>
<td>(numeration</td><td>agree</td><td>to SEQ ID</td><td>NO: 1) so</td><td>than</td><td>I know</td>
<td>produced a</td><td>polypeptide</td><td>(soluble)</td><td>secreted after</td><td>of</td><td>the</td>
expression in cells, which can be used, for example, in a vaccine. The soluble polypeptide was further stabilized by introducing a sequence known to form trimeric structures, namely AYVRKDGEWVLL (SEQ ID NO:
143), optionally connected via a connector. The connector may optionally contain a cleavage site for further processing according to protocols well known to those of skill in the art. To facilitate purification of the soluble form, a tag sequence, eg, a his tag (HHHHHHH) connected via a short link, eg EGR, may be added. In some embodiments the connector's his tag sequence is added without the folding sequence present. In accordance with the present invention, the amino acid sequence between position 530 (numbering according to SEQ ID NO: 1) and the C-terminal amino acid of the domain was removed
HA2 and was replaced by the following sequences:
EGRHHHHHHH (SEQ ID NO: 81), comprising a short plug and his tag, or
SGRSLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHHH (SEQ ID NO:
IMPI
MEXICAN INSTITUTE OF THE INDUSTRIAL fMOPIEDAP
82), comprising a thrombia cleavage site, trimerization domain, and his tag.
The previously described mutations were grouped into clusters according to their function and location in the three-dimensional structure of HA stem polypeptides. All polypeptides contain the HA Hl sequence between 1 and 59 and between 291 and 565 and the mutation
R343Q, with the following additional mutations: L58T,
V314T, I316T, F406S, V409T, L416S (SEQ ID NO: 3; call pool 1). In addition variants were made that have additional mutations:
<img file="MX357009B_D0123.tif" />
<td>Group 2: K321C,</td><td>Q405C</td><td>(SEQ ID NO:</td><td> 4)</td>
<td>Group 3: F413C,</td><td>E421C</td><td>(SEQ ID NO:</td><td> 5)</td>
<td colspan="3">Group 4: HA2 Y502S (SEQ ID NO: 6)</td><td></td>
<td>Furthermore, they were made</td><td colspan="2">two variants that</td><td>contained the</td>
<td>grouping sequence</td><td>iy</td><td>also the</td><td>mutations of</td>
<td>grouping 2 and 3 (SEQ ID</td><td>NO: 7)</td><td colspan="2">or grouping 2, 3 and 4</td>
(SEQ ID NO: 8).
Genes encoding the above protein sequences were synthesized and cloned into the pcDNA2004 expression vector using methods generally known to those of skill in the art. For comparison purposes, the full length sequence (SEQ ID NO: 1) was included in. the experiment as well as the
<img file="MX357009B_D0124.tif" />
L
MP
120 sequence described by Steel et al
ID NO: 24), which is based on
Rico / 8/1934.
(2010) (Hl-PR8-dHl; SEQ sequence H1N1 A / Port
Suspension cells HEK293F (Invitrogen) were transfected (10<sup>6</sup> cells / ml, 30 ml) with expression vectors (1 pg / ml) using 40 µΐ of 293transfectin as a transfection agent and allowed to further propagate for 2 days. Cells were harvested, aliquoted (0.3 ml, approximately 3 * 10<sup>5</sup> cells) and aliquots were treated with either Hl HA polyclonal serum to mark expression or with a HA specific monoclonal antibody (5 micrograms / ml) and a secondary antibody was used for staining. Cells were then analyzed by fluorescence associated cell selection (FACS) for expression of the membrane-bound stem domain HA polypeptides of the invention using polyclonal serum made against Hl HA to mark expression. A panel of monoclonal antibodies of known specificity binding to the full-length protein was used (eg
CR6261 and CR9114) to mark the presence of the conserved epitopes and, by inference, the correct folding of the full-length HA and mini-HA polypeptides of the invention. The results are expressed as percentage of positive cells and average fluorescence intensity and are
121
IMPI
MEXICAN INSTITUTE OF THE PK) «INDUSTRIAL AGE shown in Figures 2A-2B.
Results show that all constructs are expressed on the cell surface because the reaction with polyclonal serum (poly anti-Hl) results in 75% or more of all cells tested as positive compared to approximately 4% for non-cells. transected. This is confirmed by the mean fluorescence intensity (MFI) values, which is similar for all constructs after treatment with polyclonal serum.
Control experiments in the absence of IgG, using only the
Labeled anti-human IgG or an irrelevant mAb are all negative. Both full-length HA proteins A / Brisbane / 59/2007 and A / Puerto Rico / 8/1934 are recognized by the monoclonal antibodies CR6261, CR6254, CR6328 (all known to bind and neutralize HA from Hl; Throsby et al. .
(2008), WO 2008/028946), CR9114 (previously described),
CR8001 (binds to HA of Hl, but does not neutralize Hl; described in W02010 / 130636), but not CR8057 (binds only some strains of H3, also described in W02010 / 130636) and CR6307 20 (Throsby et al. (2008), WO 2008/028946).
Considering the discontinuous and conformational character of the CR6261 epitope (Ekiert et al. 2009), it is concluded that both full-length proteins are present in their native three-dimensional conformation. For the polypeptides of
<img file="MX357009B_D0125.tif" />
122
INi .USTKIAL the newly designed invention which is tested ep this.
experiment, the same pattern of recognition by the panel of monoclonal antibodies was observed: binding by
CR6261, CR6254, CR6328, CR9114 and CR8001 but not by CR6307 and
CR8057. This is most evident in the data for the percentage of positive cells, but is also observed in the data for the average intensity of fluorescence data. Better results are obtained with miniHA-grouping both with respect to the% of positive cells as well as the average intensity of fluorescence.
The addition of other modifications, such as the previously mentioned disulfide bridges (grouping 2 and
3) and the Y502S mutation in cluster 4 (or combinations thereof) resulted in lower percentages of positive cells and lower mean intensities. The construct described in Steel et al. (2010) (SEQ ID NO: 24) containing the deletion of the head domain, but lacking other modifications is not recognized above the noise level by any of the antibodies that were used in this experiment. Therefore, it is concluded that after DNA transfection this protein is not exhibited by the native three-dimensional conformation that it has in HA.
The results described previously point to the importance of group 1 mutations that
<img file="MX357009B_D0126.tif" />
123 they increase the hydrophilicity of the loop formed by residues 4.02 to 418 that connect the A helix and the long-skeleton helix (CD) of the HA molecule and the area around it. To better establish the beneficial effect of clustering mutations on the stability and folding of the polypeptides of the invention, miniHA (SEQ ID NO: 2; polypeptide according to Steel, but based on A / Brisbane) and miniHA_agrupamientol (polypeptide) were compared according to the invention; SEQ ID NO: 3) in a separate experiment (Figures 3A-3B).
While approximately 60% of the cells transected with the miniHA-cluster are positive after binding to CR6261, CR6254, CR6328 and CR9114, transfection with miniHA (polypeptide according to Steel, but based on A / Brisbane; SEQ ID NO : 2) leads to levels very close to noise values (between 1 and
3%). The inventors conclude that clustering mutations contribute favorably to the proper folding and stability of polypeptides according to the present invention, compared to the unmodified miniHA protein (SEQ ID NO: 2) lacking these mutations.
Steel et al. created a new molecule by removing HAl amino acid residues 53-276
124
IΜ Ρ ΐ in <titi ιτη mexican · j PE LA l'R JPIU'AU INDUSTRIAL of the Hl A / Puerto Rico / 8/1934 strain and H3 — HK63 the primary sequence, and its replacement by a short flexible connector. As shown in this Example this results in a highly unstable molecule that does not conform to the correct conformation, as evidenced by the lack of binding to antibodies that were previously shown to bind to conserved epitopes in the stem region.
Incorrect folding is caused by solvent exposure to a large area that is normally shielded by the globular head on the full-length HA molecule. Because this area is hydrophobic in nature the molecule is not more stable and therefore some adaptations are necessary.
The exchange of hydrophobic residues for hydrophilic residues as has been done in the polypeptides of the invention compensates for this effect and stabilizes the stem domain HA polypeptides. Further stabilization of the three-dimensional native folding of the stem domain is achieved by introducing disulfide bridges at appropriate locations to connect close to residues that are spatially close in the native tertiary structure but are separated in the primary structure.
IMPI
125
MEXICAN INSTITUTE.,
DE LA CROPIEDAÜ I'V industrial
Example 5: Immunogenicity of the stem domain HA polypeptides of Example 4
In order to assess the immunogenicity of the stem domain polypeptides, mice were immunized with the expression vectors encoding the full-length Hl of A / Brisbane / 59/2007 (SEQ ID NO: 1), the miniHA-grouping (SEQ ID NO: 3), the miniHA- grouping + 2 (SEQ ID NO: 4) and the miniHA- grouping + 4 (SEQ ID NO: 6). For comparison purposes, the miniHA design by Steel et al. Was also included in the experiment. (2010) (mini-PR8; SEQ ID NO: 24) and the corresponding full-length protein HA from A / Puerto Rico / 8/1934.
An expression vector encoding cM2 was also included as a negative control.
Groups of 4 mice (BALB \ c) were immunized with 50 pg of construct + 50 pg of adjuvant (pUMCVl-GM-CSF) im on day 1, 21 and 42. On day 49, terminal bleeding was performed and serum was collected. Serum was analyzed by FACS assay. Suspension cells HEK293F (Invitrogen) were transected (10<sup>6</sup> cells / ml, 30 ml) with the expression vectors (1 microgram / ml) using 40 microliters of
293transfectin as a transfection agent and allowed to spread for another 2 days. Cells were harvested,
<img file="MX357009B_D0127.tif" />
<img file="MX357009B_D0128.tif" />
126 aliquoted (0.3 mi, approximately 3 * 10<sup>5 </sup>cells) and aliquots were treated with the specific sera for the construction, stained with secondary antibodies and analyzed by fluorescence associated cell selection. The results are shown in the
Figures 4A-4B.
As expected, cM2-specific serum (negative control) recognizes cM2, but neither full-length HA or stem domain polypeptides as evidenced by% positive cells and MFI. In contrast, full-length HA-specific serum stains cells expressing the corresponding full-length HA (SEQ ID NO: 1), but also miniHAgrouping (SEQ ID NO: 3), miniHA-clustering + 2 (SEQ ID
NO: 4) and miniHA-clustering + 4 (SEQ ID NO: 6), although at a lower level (approximately 40% of positive cells versus approximately 80% for full length, MFI of approximately 1000 versus approximately 7000 for full length ). The converse is also true: the specific sera for miniHA-grouping (SEQ ID NO: 3), miniHA-grouping + 2 (SEQ ID NO: 4) and miniHA-grouping + 4 (SEQ ID NO: 6) recognize cells expressing the corresponding construct as well as the full-length HA (SEQ ID NO: 1). The results are summarized
<img file="MX357009B_D0129.tif" />
GAVE
127 in Table 4, below. ___________________
Contrary to the previous result for miniHA groupingol (SEQ ID NO: 3), miniHA-groupingol + 2 (SEQ ID
NO: 4) and miniHA-clustering + 4 (SEQ ID NO: 6), serum obtained from mice immunized with full-length PR8 did not bind HlPR8-dHl transfected cells very well (SEQ ID NO: 24). The percentage of positive cells was around 20%, compared to between 40 and 50% for the miniHA-grouping (SEQ ID NO: 3) and miniHA10 grouping 1 + 2 (SEQ ID NO: 4). The results are also reflected in the observed mean fluorescence intensity that is just above the background level.
In conclusion, the data shows that the polypeptides of the invention are capable of inducing a full-length HA-directed immune response. In particular the modifications in the region between the residues
402 and 418 (numbering according to SEQ ID NO: 1) are important in creating a stable molecule.
Example 6: Second Generation Preparation of Stem Domain Polypeptides
The mean fluorescence intensities for the stem domain polypeptides described in
Example 4 in all cases are less than observed for
<img file="MX357009B_D0130.tif" />
128 the corresponding proteins of length compleqt ^. · in fact the best design, the miniHA-cluster 1 (SEQ ID NO: 3), has an intensity that is in the order of 10% of the average intensity of the full-length construct after of binding to monoclonal antibodies. This indicates that the expression and / or folding of the stem domain polypeptides on the cell surface is. smaller than those observed for full-length proteins and that designs can be further improved. The results obtained from the first generation show that the improvement of the first generation constructions is possible and therefore a second design round was started.
The polypeptides described in Example 4 were based on the same deletion of the HAO chain, that is residues L60 to K290 (minil; numbering refers to the position in the full length HAO of HlNl
A / Brisbane / 59/2007; SEQ ID NO: 1). This strategy creates an unstructured long loop that is now no longer tied to the head domain. It was reasoned that this loop is not contributing to the overall stability of the protein and that it can be shortened considerably without affecting the folding of other parts of the polypeptide. Three additional deletions were designed and replaced with a linker sequence
IMPI
129
<img file="MX357009B_D0131.tif" />
li-JS'JS'íR1AL
GGGG as before, and combined with the 1 ^ grouping mutations previously described. The deletions are S53 to P320 (mini2), H54 to 1302 (mini3), G56 to G317 (mini4). Additional modifications identical to previous grouping 1 were introduced (L58T, V314T, I316T, F406S, V409T,
L416S).
Some of the residues belonging to this cluster are part of the deleted sequences and therefore cannot be further modified (see below). Furthermore, two additional mutations were created in the long C helix which forms a trimeric coiled helix in the pre-fusion state. It is well known in the art that trimeric coiled helices are stabilized by lie at positions 420 a and d of the repetitive sequence of septets that is the hallmark of this structural motif (Suzuki et al., (2005); Woolfson et al. (2005 )). This knowledge was applied by introducing lie at positions 420 (M420I) and 427 (V427I). The combination of these two mutations and the group 1 mutations were designated as group 1; for clarification, the modifications are listed below:
Minil: deletion from L60 to K290 grouping loll: M420I, V427I, L58T, V314T, I316T, F406S,
V409T, L416S
IMPI
MEXICAN INSTITUTE OF Industrial FROi'IEDAD
<img file="MX357009B_D0132.tif" />
Mini2: grouping deletionoll:
Mini3: grouping deletionoll:
from S53
M420I, from H54
M420I
130 to P320
V427I, F406S, V409T to 1302
V427I, V314T,, L416S
I316T, F406S,
V409T, L416S
Mini4: deletion from G56 to G317 groupingoll: M420I, V427I, F406S, V409T, L416S
To further stabilize the pre-fusion state of the stem domain polypeptides an additional disulfide bridge was introduced between position 324 and 436 (cluster 5: R324C, T436C) and combined with the different deletion mutants. The following combinations were synthesized and tested for binding in the FACS assay as previously described:
<td> 15</td><td>Minil-groupingoll</td><td>(I KNOW THAT</td><td>ID</td><td>NOT:</td><td> 9)</td>
<td></td><td>Mini2-grouping</td><td>(I KNOW THAT</td><td>ID</td><td>NOT:</td><td> 10)</td>
<td></td><td>Mini3-grouping</td><td>(I KNOW THAT</td><td>ID</td><td>NOT:</td><td> 11)</td>
<td></td><td>Mini4-groupingoll</td><td>(I KNOW THAT</td><td>ID</td><td>NOT:</td><td> 12)</td>
<td></td><td>Mini1-grouping11 + 5</td><td>(I KNOW THAT</td><td>ID</td><td>NOT:</td><td> 13)</td>
<td> 20</td><td>Mini2-grouping11 + 5</td><td>(I KNOW THAT</td><td>ID</td><td>NOT:</td><td> 14)</td>
<td></td><td>Mini3-grouping11 + 5</td><td>(I KNOW THAT</td><td>ID</td><td>NOT:</td><td> 15)</td>
<td></td><td>Mini4-grouping11 + 5</td><td>(I KNOW THAT</td><td>ID</td><td>NOT:</td><td> 16)</td>
For comparison purposes miniHA-clustering was also included in the experiment (SEQ ID NO: 3). The ♦ I · »ΊΒ
<img file="MX357009B_D0133.tif" />
MEXICAN INSTITUTE OF PROPERTY
INDUSTRIAL
131 Results are shown in Figure 5.. , --------------- In all cases the stem domain polypeptides were present on the cell surface after transfection of the expression vectors into cells
HEK293F, as evidenced by the percentage of positive cells (90% or more) after treatment with polyclonal anti-Hl serum.
All of the stem domain HA polypeptides in this experiment were recognized by CR6261, CR6254, CR6328 and
CR9114, but not by CR8057; the latter was expected because this mAb is specific for H3 HA. However, there are clear differences in the percentages of positive cells and the MFI for the different antibodies. The best characterized antibody is CR6261, of which the epitope is known in detail. The epitope is discontinuous and conformational, and binding to this antibody can therefore be seen as demanding evidence of correct folding of stem domain HA polypeptides. CR9114 is widely neutralizing, covering strains of both groups 1 and 2 (Table
3) . Fewer details are known of the epitopes of CR6328 and CR6254, but based on the highest values found for% positive cells and MFI, as well as a small dispersion in the data, the binding of these antibodies appears to be a less sensitive test the
<img file="MX357009B_D0134.tif" />
132 correct folding compared to CR6261.
By comparing the percentage of positive cells (taking into account the data for all the antibodies) the Minil to 4 constructs can be classified (% higher than lower).
Mini2> Minil> Mini4> Mini3 for combinations with the grouping and
Mini2> Minil = Mini4> Mini3 for combinations with groupingoll + 5
This classification is also reflected by the MFI data, and leads to the conclusion that the deletion of the Mini2 construct, S53 to P320, leads to the highest level of proteins exhibited on the cell surface in the correct conformation in this group.
Comparing MiniHA-grouping (SEQ ID NO: 3) with mini-grouping (SEQ ID NO: 9), the additional M420I, V427I mutations did not appear to lead to further stabilization of the construct; If there is something, it is that they lead to lower percentages of positive cells and MFI values, but the differences are small.
The introduction of the R324C, T436C (cluster 5) disulfide bridge leads to an increase in correctly folded protein on the cell surface for mini2grouping (SEQ ID NO: 10) and mini4-grouping (SEQ ID
133
<img file="MX357009B_D0135.tif" />
NO: 12), but little or no improvement - for-irrinlT groupingoll (SEQ ID NO: 9) and mini3-groupingoll (SEQ ID
NO: 11). The best overall results are obtained with mini2-groupingoll + 5 (SEQ ID NO: 14). In particular this is evident from the MFI values which for this construction are approximately 50% of the value for the full length construction.
In certain embodiments, the polypeptides of the invention contain the intracellular sequences of HA and the transmembrane domain. In other embodiments, the cytoplasmic sequence and the transmembrane sequence between position (or the equivalent thereof) 523, 524, 525, 526, 526, 527 are removed,
528, 529, or 530 of HA2 and the C-terminus of HA2 (numbering according to SEQ ID NO: 1), and are optionally replaced by the introduction of a sequence known to form trimeric structures, i.e. AYVRKDGEWVLL (SEQ ID NO: 143), optionally connected through a connector. The connector can optionally contain a cleavage site for further processing according to protocols well known to those of skill in the art. To facilitate purification of the soluble form, a tag sequence, for example a his tag, can be added.
HHHHHHH connected via a short connector, for example
EGR. According to the present invention, the sequence of
134
MEXICAN INSTITUTE ¡Jt LA PROPfrÜAO
INDUSTRIAL amino acids between position 530 (numbering according to
SEQ ID NO: 1) and the C-terminal amino acid of the HA2 domain and was replaced by SEQ ID NO: 81 or SEQ ID NO: 82.
Example 7: Immunogenicity of Stem Domain HA Second Generation Polypeptides
In order to assess the immunogenicity of the stem domain polypeptides of Example 6, mice were immunized with the expression vectors encoding the full-length Hl of
A / Brisbane / 59/2007 (SEQ ID NO: 1), miniHA-grouping (SEQ
ID NO: 3), Mini2-groupingoll (SEQ ID NO: 10), Mini-groupingoll + 5 (SEQ ID NO: 13), Mini2-groupingoll + 5 (SEQ
ID NO: 14). An expression vector encoding cM2 was also included as a negative control.
Groups of 4 mice (BALB \ c) were immunized with 50 pg of construct + 50 pg of adjuvant (pUMCVl-GM-CSF) im on day 1, 21 and 42. On day 49, terminal bleeding was performed and serum was collected. Full-length HA0 (SEQ ID NO: 1), cM2 negative control and
Mini2-grouping + 5 (SEQ ID NO: 14) to separate groups of mice by gene tripping, using approximately 10 pg of construct + approximately 2 pg of adjuvant (pUMCVl-GM-CSF) and the same immunization schedule. I know
<img file="MX357009B_D0136.tif" />
<img file="MX357009B_D0137.tif" />
135 they analyzed the sera by Elisa using the____jaeixtdominiQ.
Recombinant HA full-length strain
A / Brisbane / 59/2007 (obtained from Protein Sciences Corporation,
Meriden, CT, USA) as an antigen. Briefly, 96-well plates were coated with 50 ng HA overnight at 4 ° C, followed by incubation with blocking buffer (100 µΐ PBS, pH 7.4 + 2% skim milk) for 1 hour at room temperature. . Plates were washed with PBS + 0.05% Tween-20, and 100 µΐ of a serial dilution to the medium in blocking buffer, beginning with addition of a 20-fold dilution of serum. Bound antibody is detected using HRP-conjugated goat anti-mouse IgG, using standard protocols established in the art. Titers are compared to a standard curve using mAb 3AH1 InA134 (Hytest, Turku,
Finland) to give units of Elisa / mi (EU / ml).
ELISA results after 28 and 49 days are shown in Figures 6A and 6B, respectively. Serum obtained from mice immunized with DNA encoding Mini2-grouping + 5 (SEQ ID NO: 14) exhibits clear binding to the full-length HA ectodomain after 28 and days after immunization using gene shots and also after 49 days when IM were immunized. For
Mini2-groupingoll (SEQ ID NO: 10) and Minil-groupingoll + 5
<img file="MX357009B_D0138.tif" />
136 (SEQ ID NO: 13) a response was detected for 1 of 4 mice, __ „while passing miniHA-grouping (SEQ ID NO: 3) no binding was detected.
In conclusion, the data shows that the polypeptides of the invention are capable of inducing a full-length HA-directed immune response. In particular the modifications in the region between the residues
402 and 418 (numbering according to SEQ ID NO: 1), deletion of
S53 to P320 in combination with the R324C disulfide bridge,
T436C are important to create a stable molecule.
Example 8: Preparation of third generation stem domain polypeptides
To further improve the design of the stem domain polypeptides a third design round was implemented. An additional mutation was introduced to increase the hydrophilicity of the hidden surfaces in the full-length HA, but not the stem domain polypeptides, at position 413, F413G (numbering according to SEQ ID NO: 1), and was named grouping 6. This grouping was combined with the deletion of mini-2 (S53 a
P320), the disulfide bridge of group 5 (R324C, T436C) and mutations either of group 1 (that is F406S,
V409T, L416S; SEQ ID NO: 46) or group 11
137
<img file="MX357009B_D0139.tif" />
INSTITUTO MEXICAN E »E LA FROriEIOAU industrial (M420I, V427I, F406S, V409T, L416S; SEQ ID 'W: ^ Ττ · —iracombination of the deletion mini-2 (S53 to P320) with grouping 1 (F406S, V409T, L416S ) and cluster 5 (R324C, T436C) was also included in this experiment for reference (SEQ ID NO: 48).
Native HA exists as a trimer on the cell surface. Most of the interactions between the individual monomers that conserve the trimer all together are located in the head domain. After removal of the head the tertiary structure is destabilized in this way
<td>and therefore</td><td>the</td><td>reinforcement of</td><td colspan="3">the interactions between what</td>
<td>monomers in</td><td>the</td><td>molecule</td><td>truncated</td><td>will increase</td><td>the</td>
<td>stability. In</td><td>the</td><td>domain of</td><td>stem the</td><td>trimerization</td><td>this</td>
mediated by the formation of a trimeric coiled helix motif. By reinforcing this motif a more stable trimer can be created. According to the invention, a consensus sequence for the formation of a trimeric coiled helix, IEAIEKKIEAIEKKIE (SEQ ID NO: 83), is introduced into a polypeptide of the invention at (the equivalent of) position 418 to 433 (SEQ ID NO : 44) in Hl A / Brisbane / 59/2007 (numbering according to SEQ ID NO: 1). An alternative is to introduce the sequence MKQIEDKIEEIESKQ (SEQ ID NO: 84), which is derived from GCN4 and known to trimerize, at position 419 to 433 (SEQ ID NO: 45).
<img file="MX357009B_D0140.tif" />
i ο of the PR <VÍ £ DAD
-LOO INDUSTRIAL '* ^ £ 7 * 57
In the case of the Ha tAllo domain polypeptides described by SEQ ID NO: 44 to SEQ ID NO: 48 all the proteins were present on the cell surface after transfection of the expression vectors in HEK293F cells, as evidenced by the percentage of positive cells (90% or more) after treatment with polyclonal anti-Hl serum. The results are shown in Figure.
All stem domain HA polypeptides in this experiment, with the exception of miniHA (SEQ ID NO: 2), were recognized by CR6261, CR6328, and CR9114, but not by
CR8020; the latter was expected because this mAb is specific for H3 HA. The percentage of positive cells is around 80% for stem domain polypeptides using CR6261, CR6328 and CR9114 for staining, with the exception of miniHA, which is only recognized by anti-Hl polyclonal serum. Again, this is indicative of a lack of correct folding of this particular construction. There are, however, clear differences in the MFI for the different antibodies. The best characterized antibody is CR6261, of which the epitope is known in detail. The epitope is discontinuous and conformational, and binding to this antibody can therefore be seen as a demanding test of correct folding of HA domain polypeptides.
<img file="MX357009B_D0141.tif" />
139 stem. CR9114 is widely neutralizing, covering strains of both groups 1 and 2 (Table 3). Fewer details are known of the CR6328 epitope but competition with CR6261 is observed in full length HA binding experiments.
The MFIs for Hl-mini2-clll + 5 (SEQ ID NO: 14), Hl-mini2cll + 5 (SEQ ID NO: 48), Hl-mini2-cll + 5 + 6 (SEQ ID NO: 46) and Hlmini2- clll + 5 + 6 (SEQ ID NO: 47) are very similar, regardless of the monoclonal antibody used in the experiment. Inclusion of the consensus trimerization domain (SEQ ID NO: 44) reduces MFI by a factor of 3 to 4 compared to the equivalent sequence without the trimerization domain being
Hl-mini2grouping l + 5 + 6; SEQ ID 46), but the result is still clearly better than in the absence of modifications to the stem polypeptide after deletion of the head domain (cf miniHA results). The addition of the GCN4 trimerization sequence (SEQ ID NO: 45) increases the MFI to levels comparable to the full-length protein.
Example 9: Design of other stem domain polypeptides comprising conserved stem domain epitopes of CR6261 and CR9114
The polypeptides of the invention following the procedure previously described can be
140
<img file="MX357009B_D0142.tif" />
further modify to increase the dad—
Said modifications can be introduced to increase the formation of trimeric forms of the polypeptides of the invention over monomeric and / or dimeric species.
As previously described, native HA exists as a trimer on the cell surface. Most of the interactions between the individual monomers that conserve the trimer all together are located in the head domain.
After removal of the head the tertiary structure is destabilized in this way and therefore the enhancement of interactions between the monomers in the truncated molecule will increase stability. In the stem domain trimerization is mediated by the formation of a trimeric coiled helix motif. By reinforcing this motif a more stable trimer can be created.
According to the invention, a consensus sequence was introduced-for the formation of a trimeric coiled helix,
IEAIEKKIEAIEKKIE (SEQ ID NO; 83), in a polypeptide of the invention at (the equivalent of) position 418 to 433 (SEQ
ID NO: 44) in Hl A / Brisbane / 59/2007 (numbering according to
SEQ ID NO: 1). As an alternative you can enter
IEAIEKKIEAIEKKI (SEQ ID NO: 85) at 419 to 433 (SEQ ID NO: 49) or IEAIEKKIEAIEKK (SEQ ID NO: 86) at 420 to 433 (SEQ ID NO:
fifty) . An alternative is to enter the sequence
<img file="MX357009B_D0143.tif" />
141
MKQIEDKIEEIESKQ (SEQ ID NO: 84) which is derived from GCN4 and which
IMPI
INSTITUTO MEXICANO DE LA ERÚFIEUAP industrial knows that it trimerizes, in position 419 to 433 (SEQ ID NO: 45).
As an alternative MKQIEDKIEEIESK (SEQ ID NO: 87) can be entered in position 420 to 433 (SEQ ID NO: 51) or
RMKQIEDKIEEIESKQK (SEQ ID NO: 88) at position 417 to 433 (SEQ ID NO: 52). Similarly, the trimer interface is strengthened by modifying M420, L423, V427,
G430 to Isoleucine. (SEQ ID NO: 53).
All peptides were shown to bind CR9114 and
CR6261.
In certain embodiments, the polypeptides of the invention do not contain the signal sequence and / or intracellular sequences and the transmembrane domain of HA, as previously described.
Example 10: Design of a stem domain polypeptide comprising conserved stem domain epitopes of
CR6261 and CR9114 based on HA from H7
The procedure previously described to design the polypeptides of the invention was also applied to H7. This example describes the design of a polypeptide of the invention based on the H7 serotype. HA from influenza virus H7 A / Azulón / Netherlands / 12/2000 was used (SEQ ID
NO: 31) as a parental sequence, but people with
142
Wicked
MEXICAN INSTITUTE
MEXICAN INSTITUTE
PROPERTY ζ> INDUSTRIAL experience in art will understand that the use of sequence.
other than H7 would have been equally possible because the sequences are well conserved, particularly in the stem region.
The first modification in the sequence is the removal of the cleavage site at position 339 (the numbering refers to SEQ ID. NO: 31 by mutating R to Q (R339Q) to prevent HAl formation and
HA2 from HAO. Optionally additionally 341 to 345 (LFGAI, part of the fusion peptide) can be removed from the residue to minimize exposure of hydrophobic residues to the aqueous solvent. The positive charge is 100% preserved in the cleavage at H7 and therefore this mutation can be applied in all sequences.
The second modification is the removal of the head domain by removing a large part of the HAl sequence and reconnecting the C and Nterminal sequences through a short linker. The deletion can vary in length, but it is preferred that the last residue of the N-terminal sequence of HAl and the first residue of the C-terminal sequence are spatially very close to avoid introducing tension through the connecting sequence.
Deletions can be introduced into the H7 sequences at (the equivalent positions of) R53-P315 (mini2; SEQ ID NO:
143
<img file="MX357009B_D0144.tif" />
MEXICAN INSTITUTE OF PROPERTY
INDUSTRIAL £ <** • 73,
33) in H7 A / Azulón / Netherlands / 12/2000 (SEQ ID NO: 31). Equivalent positions can be readily determined by those skilled in the art by aligning the sequences using an appropriate algorithm such as for example Clustal or Muscle. The other parts of the sequence can be attached directly or alternatively a flexible connector can be inserted. The connecting sequences can be between 1 and 50 amino acids in length. Flexible connectors of limited length (less than or equal to 10 amino acids) are preferred, for example GGG,
GGGG, GSA, GSAG, GSAGSA, GSAGSAG or the like.
SEQ ID NO: 40 describes said polypeptide of the invention containing the T54-C314 deletion (mini5; SEQ ID NO: 40). The deletions previously described ensure that unstructured regions that are formed by residues
280 310 are also eliminated; this is beneficial for the overall stability of the polypeptides of the invention. A similar effect was observed for the polypeptides of the invention derived from an Hl sequence (see above).
The deletion of the head domain leaves the loop between residues 394 to 414 now exposed to the aqueous solvent. In H7 HAs this loop is highly conserved (see
Table 7). The consensus sequence is: LI (E / D / G) KTNQQFELIDNEF (N / T / S) E (I / V) E (Q / K) (SEQ ID NO: 32).
<img file="MX357009B_D0145.tif" />
144
To increase the solubility of this loop in the pre-fusion conformation and to destabilize the post-fusion conformation, some hydrophobic residues were modified to polar amino acids (S, T, N, Q), charged (R, H, K, D, E), or flexibility was increased by mutation to G. Specific mutations at positions 402, 404, 405, 409,
412 (the numbering refers to SEQ ID NO: 31) will contribute to the stability of a polypeptide of the invention.
For positions F402 and F409 mutation to S is preferred but other polar (T, N, Q), charged (R, H, K, D, E) and highly flexible (G) amino acids will have the same effect. For position 404 (96% L), mutation to N or S is preferred; the last of the amino acids also exists naturally, although in low frequency, and the mutation in this position is in those cases unnecessary. Other highly flexible (R, H, K, D, E) and highly flexible (G) polar (T, Q) amino acids will have the same effect. For position 405 (99% I) mutation to T or D is preferred. D also exists naturally and mutation in this position is therefore unnecessary. Other polar (S, N, Q), charged (R, Η, K,) and highly flexible (G) amino acids will have the same effect.
For position 412 (I or V) mutation to N is preferred but other polar residues (S, T, Q), charged (R, Η, K, D, E) or
MEXICAN INSTITUTE ·· »333
OF PROPERTY -. <.
INDUSTRIAL -1 ---- 145
Flexible IMPI (G) are also possible. Said ρ or 1 ip é pt Idoa-. · ™, - · »- - contain at least one of the mutations previously described. Combinations of more than one mutation have also been applied, as shown for example in SEQ ID NOs: 34 to
39 and 41 to 43.
To stabilize the pre-fusion conformation of the polypeptides of the invention, a covalent bond was introduced between two parts distant in the primary sequence but close to each other in the folded pre-fusion conformation. For this purpose, a disulfide bridge was assembled in the polypeptides of the invention at, preferably between (the equivalent of) position 319 to 432 in A / Azulón / Netherlands / 12/2000 of H7 (SEQ ID NO: 36-39, 42, 43). Equivalent positions can be readily determined by those skilled in the art by aligning the sequences using an appropriate algorithm such as Clustal,
Muscle etc. Armed disulfide bridges were created by mutating at least one (if the other is already a cysteine), but usually two residues that are spatially close, to cysteine, which spontaneously or by active oxidation form a covalent bond between the sulfur atoms of this waste.
As previously described, native HA exists as a trimer on the cell surface. Most of the
<img file="MX357009B_D0146.tif" />
146 Interactions between the individual monomers that ^ xxuxaexJíaju-, w.-_ ^. »- the trimer all together are located in the head domain.
After removal of the head the tertiary structure is destabilized in this way and therefore the enhancement of interactions between the monomers in the truncated molecule will increase stability. In the stem domain trimerization is mediated by the formation of a trimeric coiled helix motif. By reinforcing this motif a more stable trimer can be created. It is well known in the art that trimeric coiled helices are stabilized by lie at positions a and d of the repetitive septet sequence which is the hallmark of this structural motif. Here this knowledge was applied to introduce lie at (the equivalent of) positions 419,
423, 426 and 430 (SEQ ID NO: 38, 43). Alternatively, a consensus sequence for the formation of a trimeric coiled helix, EAIEKKIEAI, is introduced at (the equivalent of) position 417 to 426 (SEQ ID NO: 39).
These sequences (SEQ ID NO: 33-43) were subjected to the Fluorescence-Associated Cell Selection assay described previously. However, no binding of the monoclonal antibodies CR8020, CR8043, CR9114 or
CR8957. The inventors conclude that these sequences do not present the epitopes of these antibodies and as
<img file="MX357009B_D0147.tif" />
<img file="MX357009B_D0148.tif" />
147 Consequently, proteins as they are present in the cell membrane are not folded in their native three-dimensional structure.
Example 11; Design of stem domain polypeptides comprising conserved stem domain epitopes from
CR8020, CR8043 and CR9114 based on H3 HA
In a first step, a sequence representing a polypeptide of the invention was constructed analogously to that described by Steel et al. (Steel et al. 2010) using HA from H3 A / Wisconsin / 67/2005 as the parent sequence ( SEQ ID NO: 89). The HA head is removed by deleting a portion of HAl from amino acid D69 to amino acid K292.
These residues can be replaced by 3 or 4 Gly. The 4 Gly linker was tested by Steel and coworkers and gave good expression results and was adopted here to create the mini-H3 (SEQ ID NO: 90). To prevent cleavage of the polypeptide chain, a normal post-translational processing step was mutated for the full-length HA protein, the cleavage site at position 345 (arginine) to a glutamine (R345Q).
Then, the accessible surface area of each residue was calculated in both the constructed mini-HA and the
<img file="MX357009B_D0149.tif" />
148 post-merger conformation with the help of Brugel. The degree of exposure and concealment of each residue was determined as described in Samantha et al. (Samantha et al., 2002). It focused on the residues that are exposed in the pre-fusion conformation and that are hidden in the post-fusion conformation. Further analysis of these residues indicates that some of them can be modified in such a way that the mutation does not have an effect on pre-fusion but destabilizes the post-fusion conformation. In general these residues have a hydrophobic side chain and are involved in the formation of the coiled helix in the post-fusion conformation. Mutation of these residues to include a hydrophilic side chain will damage the properties of the wound helix - the contacts between the helices in a wound helix are generally hydrophobic - and therefore destabilize the post-fusion conformation. The residues that go from exposed in the pre-fusion to hidden in the post-fusion conformation and that are expected to have a destabilizing effect on the latter conformation after the mutation are L397, 1401 and L425 (numbering according to
<td>SEQ ID</td><td>NOT:</td><td>89). Here</td><td>I know</td><td>include</td><td>L397K and I401T.</td><td></td>
<td>The</td><td colspan="2">loop (loop</td><td>B,</td><td>waste</td><td>401 to 420) which</td><td>connects to the</td>
<td>propeller</td><td>TO</td><td>(residue</td><td> 383</td><td>to 400)</td><td>with the propeller</td><td>CD central</td>
<img file="MX357009B_D0150.tif" />
149 (residues 421 to 470) changes conformation ctrarrüD<sup>-</sup>Adopt<sup>1</sup>'crowded post merger; it becomes helical and is part of an extended trimeric coiled helix. To stabilize the conformation of the pre-fusion loop of this connector and / or to destabilize its post-fusion conformation, it was reasoned that it would be sufficient to mutate all the residues that are involved in the formation of the core of the wound helix. For the N405 position, several mutations are designed, particularly in residues that carry a negative charge (Asp and Glu, N405D, N405E) because this extra charge will reinforce the ionic network observed in the pre-fusion conformation. Also included in this study is a mutation in the Neutral Wing (Ν405Ά). The inventors also mutated Phe 408 to Thr, His 409 to Ser, and Val 418 to
Ser (numbering according to SEQ ID NO: 89; F408T, H409S,
V418S) to further increase the solubility of the new exposed area after removal of the head domain.
Five disulfide bridges were designed to block HA in the pre-fusion conformation. These bridges are formed between residues that are spatially at an appropriate distance from each other and that have their Οβ already in the correct position to form a disulfide bridge. They are entered between positions 320 and 406 (A320C, E406C;
<img file="MX357009B_D0151.tif" />
150 numbering according to SEQ ID NO: 89), 326 and— <L3 £ - (4432607
S438C) and between 415 and 423 (F415C, Q423C). The first two intersect between the HAl and HA2 parts of the chain, while the latter covalently connects the top of loop B. The K326C, S438C disulfide bridge is accompanied by the Asp 435 to Ala (D435A) mutation. Disulfide bridges
F347C / N461C and S385C / L463C were taken from the publication of
Bommakanti et al. (2010) and were also used in this study.
To eliminate the new hydrophobic residues exposed to the solvent, several additional mutations are designed. The lie at position 67 (numbering according to SEQ ID NO: 89) is mutated to a Thr (I67T). This mutation maintains the beta branch of the side chain but removes hydrophobic residue from the surface. The same can be said for the lie mutation.
298 to Thr (I298T). Another mutation is introduced into the position
316, isoleucine in the native sequence. Intuitively, one would propose to mutate this residue to a Thr to maintain the beta branch but remove hydrophobicity from the surface. However, this mutation would result in the introduction of an extra N-glycosylation site (position 314 is an Asn) and therefore a Gln (I316Q) mutation is introduced.
Gly 495 was also mutated to Glu (G495E). This mutation is designed to introduce an ion bridge because there is a positive charge in the surroundings. Nature has already provided
151
<img file="MX357009B_D0152.tif" />
some of the H3 strains of a Glu in this position: ----- An important HA residue is position 345 (Arg) because this is the position where the cleavage of the protease occurs to competently render the protein fusion . Mutation of this Arg to a Gln (R345Q) prevents cleavage from occurring thereby blocking the protein in the pre-fusion state.
The previously described mutations were regrouped as follows:
Group 1: I67T, I98T, I316Q, F408T, H409S,
V418S
Group 2: A320C, E406C
Group 3 X326C, D435A, S438C
Group 4 L397K, I401T
Group 5 N405D or N405E or N405A
Group 6 F415C, Q423C
Group 7 G495E
Group 8 F347C, S385C, N461C, L463C
To achieve the polypeptides of the invention, the pools were combined with the D69 deletion to K292 and the R345Q mutation according to the scheme described below.
H3 Mini-HA grouping 1 (SEQ ID NO: 91)
H3 Mini-HA grouping 1 + 2 (SEQ ID NO: 92)
<img file="MX357009B_D0153.tif" />
152
<img file="MX357009B_D0154.tif" />
<img file="MX357009B_D0155.tif" />
MEXICAN INSTITUTE or the ι'χορίΕΟΑυ
INDUSTRIAL
<td></td><td>H3</td><td>Mini-HA</td><td>grouping</td><td> 1+3</td><td>(SEQ ID NO: 93)</td><td></td>
<td></td><td>H3</td><td>Mini-HA</td><td>grouping</td><td> 1 + 4</td><td>(SEQ ID NO: 94)</td><td></td>
<td></td><td>H3</td><td>Mini-HA</td><td>grouping</td><td> 1+5</td><td>N405A (SEQ ID NO:</td><td> 95)</td>
<td></td><td>H3</td><td>Mini-HA</td><td>grouping</td><td> 1 + 5</td><td>N405D (SEQ ID NO:</td><td> 96)</td>
<td> 5</td><td>H3</td><td>Mini-HA</td><td>grouping</td><td> 1 + 5</td><td>N405E (SEQ ID NO:</td><td> 97)</td>
<td></td><td>H3</td><td>Mini-HA</td><td>grouping</td><td> 1 + 6</td><td>(SEQ ID NO: 98)</td><td></td>
<td></td><td>H3</td><td>Mini-HA</td><td>grouping</td><td> 1 + 7</td><td>(SEQ ID NO: 99)</td><td></td>
H3 Mini-HA grouping 1 + 2 + 3 + 4 + 5 + 6 + 7-N405E (SEQ ID NO:
100)
H3 Mini-HA grouping 1 + 2 + 3 + 4 + 5 + 6 + 7-Ν4Ό5Α (SEQ ID NO:
101) H3 Mini-HA grouping 1 + 2 + 3 + 4 + 5 + 6 + 7-N405D (SEQ ID NO:
102)
H3 Mini-HA grouping 1 + 8 (SEQ ID NO: 103).
Genes encoding the above protein sequences were synthesized and cloned into the expression vector pcDNA2004 using methods generally known to those of skill in the art. For comparison purposes the full length HA sequence of H3 A / Wisconsin / 67/2005 was included in the experiment as well as the full length HA sequence of Hl
A / Brisbane / 59/2007 containing the R343Q cleavage site mutation.
Suspension cells HEK293F (Invitrogen) were transfected (10<sup>6</sup> cells / ml, 30 ml) with the vectors of
153 ϊ Μ. Ρ ί
INSTITUTO MEXICANO DE LA AOPIFDAP iHCrilST'.UAL expression (1 pg / ml) using 40 μΐ of 293transfecb ± iT '~ txJnrcr · transfection agent and allowed to propagate for another 2 days. Cells were harvested, aliquoted (0.3 ml, approximately 3 * 10<sup>5</sup> cells) and aliquots were treated either with polyclonal serum made against H3 HA (Protein Sciences Corp, Meriden, CT, USA) to mark the expression or with a specific monoclonal antibody for
HA (5 micrograms / ml) and a secondary antibody was used for staining. Cells were then analyzed by fluorescence associated cell selection (FACS) for expression of the membrane-bound stem domain HA polypeptides of the invention using polyclonal serum made against H3 HA or Hl HA to mark expression. A panel of monoclonal antibodies of known specificity binding to the full-length protein (CR8020, CR8043, and CR9114) was used to mark the presence of conserved epitopes and, by inference, correct folding of the full-length HA and miniHA polypeptides of the invention. Monoclonal antibodies CR6261 (known to bind to H3 HA) were also used and
CR8057 (joins the HA head domain of
A / Wisconsin / 67/2005) in the experiment. Results are expressed as a percentage of positive cells and are shown in Figures 8A-8B.
<img file="MX357009B_D0156.tif" />
154
IMPÍ
The results show that all sp ------- constructs express on the cell surface because the reaction with polyclonal H3 serum results in between 80 and 90% of all cells analyzed as positive for H3-based sequences. and more than 50% for the full length Hl sequence compared to less than 4% for untransfected cells. Using the polyclonal anti-Hl antibody, between 60 and 70% of all cells are positive, except for the full-length Hl sequence that approaches
100% Control experiments in the absence of IgG, using only labeled anti-Human or anti-rabbit IgG are all negative. Both full-length HA proteins
A / Wisconsin / 67/2005 and A / Brisbane / 59/2007 are recognized by the monoclonal antibody CR9114, which is known to be capable of neutralizing both strains. Full-length HA
A / Wisconsin / 67/2005 further binds to CR8020, CR8043, and CR8057 (binds only to some strains of H3, described in
W02010 / 130636), but not to CR6261 (Throsby et al. (2008), WO 2008/028946). For the full-length HA of
A / Brisbane / 59/2007 is the opposite: it joins CR6261 but not CR8020, CR8043 and CR8057.
Polypeptides as described in SEQ ID NO: 91 a
SEQ ID NO: 103 are unable to bind CR8020, CR8043 and
CR9114 in none of the cases as evidenced by the lack
<img file="MX357009B_D0157.tif" />
ΪΜΡΙ „_ _ MEXICAN INSTITUTE
155 OF THE PROPERTY
INDUSTRIAL signal above noise in Figures 8A-8B, It was therefore concluded that these sequences do not represent the epitopes of these antibodies and as a consequence that proteins as they are present on the cell surface do not fold in their structure three-dimensional native.
Example 12: Design of other stem domain polypeptides comprising conserved stem domain epitopes of CR8020, CR8043 and CR9114 based on HA from H3
This example describes the design of other polypeptides of the invention based on serotype H3. The H3 influenza viruses A / Wisconsin / 67/2005 (SEQ ID NO: 89) and A / Hong Kong / 1/1968 (SEQ ID NO: 121) were used as the parental sequence.
The first modification in the sequence is the removal of the cleavage site at position 345 (the numbering refers to SEQ ID NO: 89 by mutating R to Q (R345Q) to prevent HAl formation and
HA2 from HAO. Optionally, it can be additionally removed from residue 347 to 351 (IFGAI, part of the fusion peptide) to minimize exposure of hydrophobic residues to the aqueous solvent. The positive charge is 100% conserved in the H3 cleavage and therefore this mutation can be applied in all sequences.
<img file="MX357009B_D0158.tif" />
156
The second modification in removing the head domain by removing a large part of the HAl sequence and reconnecting the C and Nterminai sequences through a short linker. The deletion can vary in length, but it is preferred that the last residue of the N-terminal sequence of HAl and the first residue of the C-terminal sequence are spatially very close to avoid introducing tension through the connecting sequence.
Deletions can be introduced into the H3 sequence at (the equivalent positions of) S62 to P322 (mini2; SEQ ID NO:
105), S63 to P305 (mini3; SEQ ID NO: 119) and T64 to T317 (mini4;
SEQ ID NO: 120. Equivalent positions can be easily determined by those skilled in the art by aligning the sequences using an appropriate algorithm such as for example
Clustal or Muscle. The other parts of the sequence can be attached directly or alternatively a flexible connector can be inserted. The connecting sequences can be from 1 to 50, amino acids in length. Flexible connectors of limited length (less than or equal to 10 amino acids) are preferred, for example GGG, GGGG, GSA, GSAG, GSAGSA,
GSAGSAG or the like. You can also vary the length of the deletion, for example by decreasing the number of residues in the deletion starting at (the equivalent of) position 63,
<img file="MX357009B_D0159.tif" />
157
64, 65, 66, 67, or increase the length of -Ta '' deléúiórTT by cutting at position 57, 58, 59, 60 or 61. Similarly, the last amino acid in (the equivalent of) can be removed position 317, 318, 319, 320 or 321, or increase the length of the deletion at (the equivalent of) position 323, 324, 325, 326, or 327. It is important to realize that changes in deletion length can be partially offset by matching the length of the connector sequence, that is, a longer deletion can be matched to a longer connector and vice versa.
These polypeptides are also included in the invention.
The deletion of the head domain leaves loop B between residues 400 and 420 now exposed to the aqueous solvent. In H3 HAs this loop is highly conserved (see
Table 9). The consensus sequence is: 401
I (E / G) KTNEKFHQIEKEFSEVEGR 421 (SEQ ID NO: 104; numbering refers to SEQ ID NO: 89). To increase the solubility of this loop for the polypeptides of the invention in the pre-fusion conformation and to destabilize the post-fusion conformation, some hydrophobic residues have to be modified to polar amino acids (S, T, N, Q), charged (R , H, K, D, E), or flexibility must be increased by mutation to G. Specifically, mutations at positions 401, 408, 411, 415, 418, (the
<img file="MX357009B_D0160.tif" />
158
IMPÍ
MEXICAN INSTITUTE OF THE INDUSTRIAL PkOHEÜAÍ numbering refers to SEQ ID NO: 89) will contribute to the stability of a polypeptide of the invention.
For positions F408 and F415 the S mutation is preferred but other polar (T, N, Q), charged (R, H, K, D, E) and highly flexible (G) amino acids will have the same effect. For the 411 (I) position, the mutation to
T; Other highly flexible (R, H, K, D, E) and highly flexible (G) polar (S, N, Q) amino acids will have the same effect and are therefore also included in the invention. For the position
418 (V) mutation to G is preferred. Other charged polar (S, Τ, N, Q) amino acids (R, Η, K, D, E) will have the same effect and are therefore also included in the invention. For position 401 (I) the mutation to
R but other polar residues are also possible (S, Τ, N,
Q), loaded (Η, K, D, E) or flexible (G). Therefore, the polypeptides of the invention contain at least one of the mutations previously described. Combinations of more than one combination are also possible, as shown for example in SEQ ID NOs 123 to 127 and 129 to 131.
To stabilize the pre-fusion conformation of the polypeptides of the invention, a covalent bond is introduced between two parts distant in the primary sequence but close to each other in the folded pre-fusion conformation. For this purpose, a disulfide bridge is engineered in the
<img file="MX357009B_D0161.tif" />
<img file="MX357009B_D0162.tif" />
159 polypeptides of the invention preferably —- intereequivalent to) position 326 and 438 at H3
A / Wisconsin / 67/2005 (SEQ ID NO: 89). Equivalent positions can be readily determined by those skilled in the art by aligning the sequences using an appropriate algorithm such as
Clustal, Muscle etc. Armed disulfide bridges were created by mutating at least one (if the other is already a cysteine), but usually two residues that are spatially close, to cysteine, which either spontaneously or by active oxidation will form a covalent bond between the atoms of sulfur from these residues. An alternative cysteine bridge can be created between (the equivalent of) position 334 and 393 in H3 A / Wisconsin / 67/2005 (SEQ ID NO: 89) by mutating these residues to cysteine. In some cases, cysteine at (the equivalent of) position 321 is modified to a glycine to prevent the formation of unwanted disulfide bridges.
Native HA exists as a trimer on the cell surface. Most of the interactions between the individual monomers that conserve the trimer all together are located in the head domain. After the removal of the head the tertiary structure is destabilized in this way and therefore the reinforcement of the interactions between the
<img file="MX357009B_D0163.tif" />
160
ΙΜΡΪ
MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY
<td>monomers</td><td>in</td><td>the</td><td>molecule</td><td>truncated</td><td>will increase</td><td> —</td>
<td>stability.</td><td>In</td><td>the</td><td>domain of</td><td>stem the</td><td>trimerization</td><td>this</td>
<td>mediated by</td><td>the</td><td colspan="2">formation of a</td><td>reason</td><td colspan="2">coiled propeller</td>
trimeric. By reinforcing this motif a more stable trimer can be created. A consensus sequence is introduced for the formation of a trimeric coiled helix,
IEAIEKKIEAIEKKIEAIEKK, at (the equivalent of) position 421 to 441. To avoid interference with disulfide bridge formation between positions 326 and 438, an alternate shorter sequence IEAIEKKIEAIEKKI was also used at (the equivalent of) positions 421 to 435. An alternative is to enter the RMKQIEDKIEEIESKQKKIEN sequence, which leads to GCN4 and is known to trimerize, at position 421 to 441 or the shorter RMKQIEDKIEEIESK sequence at position 421 to 435.
The polypeptides of the invention can contain the intracellular sequences of HA and the transmembrane domain such that the resulting polypeptides are present on the cell surface when expressed in cells. In other embodiments, the cytoplasmic sequence and the transmembrane sequence between (the equivalent of) position 522 and the C-terminus are removed so that a secreted (soluble) polypeptide is produced after expression in cells. Optionally some additional residues may be included in the soluble protein by removing
<img file="MX357009B_D0164.tif" />
161 the sequence from (the equivalent of) 523,
ΙΜΡΓ
MEXICAN INSTITUTE
OF INDUSTRIAL PROPERTY
<img file="MX357009B_D0165.tif" />
.526^
527, 528 or 529. The soluble polypeptide can be further stabilized by introducing a sequence known to form trimeric structures, ie AYVRKDGEWVLL (SEQ ID NO: 143) ('foldon' sequence), optionally connected via a connector. The connector can optionally contain a cleavage site for further processing according to protocols well known to those of skill in the art. To facilitate purification of the soluble form, a tag sequence, eg, a his tag (HHHHHHH) connected via a short connector, eg EGR, may be added. In some embodiments, the connector and tag sequence are added without the foldon sequence being present.
An important HA residue is position 345 (Arg) because this is the position where protease cleavage occurs to give the competent fusion protein. Mutation of this Arg to a Gln (R345Q) prevents cleavage from occurring thereby blocking the protein in the pre-fusion state.
The previously described mutations were regrouped as follows:
Group 9 F408S, I411T, F415S
Grouping 10
V418G ϊ
Λ »X
162
INSTITUTO MfiXíCz'.NO Df LA PROr'E; 'A · ·
INDUSTRIAL
Grouping 11
Grouping 12
Grouping 13
Grouping 14
GCN4 RMKQIEDKIEEIESKQKKIEN in position 421 to 441
I401R
K326C, S438C
T334C, I393C
C321G or RMKQIEDKIEEIESK in position 421 to 435 tri IEAIEKKIEAIEKKIEAIEKK in position 421 to 441 or IEAIEKKIEAIEKKI in positions 421 to 435
Using the H3N2 full-length HA sequence
A / Wisconsin / 67/2005 as a starting point, the clusters previously described with the S62 deletion to P322 (mini2; SEQ ID NO: 105) were combined to achieve the polypeptides of the invention
<td></td><td>SEQ ID NO:</td><td> 105:</td><td>H3-mini2</td>
<td> 15</td><td>SEQ ID NO:</td><td> 106:</td><td>H3-mini2-cl9 + 10</td>
<td></td><td>SEQ ID NO:</td><td> 107:</td><td>H3-mini2-cl9 + ll</td>
<td></td><td>SEQ ID NO:</td><td> 108 :</td><td>H3-mini2-cl9 + 10 + 11</td>
<td></td><td>SEQ ID NO:</td><td> 109:</td><td>H3-mini2-cl9 + 10 + ll-tri (tri sequence in</td>
<td></td><td>position 421</td><td colspan="2">to 441)</td>
<td> 20</td><td>SEQ ID NO:</td><td> 110:</td><td>H3-mini2-cl9 + 10 + ll-GCN4 (sequence GCN4</td>
<td></td><td>in position</td><td>421 to</td><td> 441)</td>
<td></td><td>SEQ ID NO:</td><td> 111:</td><td>H3-mini2-cl9 + 10 + 11 + 12</td>
<td></td><td>SEQ ID NO:</td><td> 112 :</td><td>H3-mini2-cl9 + 10 + 12</td>
SEQ ID NO: 113: H3-mini2-cl9 + 10 + ll + 12-GCN4 (sequence
<img file="MX357009B_D0166.tif" />
163
GCN4 cuts at position 421 to 435).
SEQ ID NO: 114: H3-mini2-cl9 + 10 + ll + 12-tri (tri short sequence at position 421 to 435)
SEQ ID NO: 115: H3-mini2-cl9 + 13
SEQ ID NO: 116: H3-mini2-cl9 + 10 + ll + 13
SEQ ID NO: 117: H3-mini2-cl9 + 10 + ll + 13-GCN4 (sequence
GCN4 at position 421 to 441)
SEQ ID NO: 118: H3-mini2-cl9 + 10 + ll + 13-tri (tri sequence at position 421 to 441)
In addition, deletions S63-P305 (mini3) and
T64-T317 (mini4) with clusters 9, 10, 11, and 14 to create the polypeptides of the invention
SEQ ID NO: 119: H3-mini3-cl9 + 10 + ll + 12 + 14
SEQ ID NO: 120: H3-mini4-cl9 + 10 + ll + 12 + 14
Using the H3N2 full-length HA sequence
A / Hong Kong / 1/1968 as a starting point, the groupings previously described were combined with the deletion
S62 to P322 to achieve the polypeptides of the invention
SEQ ID NO: 121: A / Hong Kong / 1/1968 H3 full length
<td>I KNOW THAT</td><td>ID</td><td>NOT:</td><td> 122:</td><td>HK68</td><td>H3m2-cl9</td>
<td>I KNOW THAT</td><td>ID</td><td>NOT:</td><td> 123:</td><td>HK68</td><td>H3m2-cl9 + 10</td>
<td>I KNOW THAT</td><td>ID</td><td>NOT:</td><td> 124 :</td><td>HK68</td><td>H3m2-cl9 + 10 + 11</td>
SEQ ID NO: 125: HK68 H3m2-cl9 + 10 + 12
164
ΙΜΡΙί ^ 5
MEXICAN INSTITUTE% *>! && /, EU PROPERTY CSszTít>
INDUSTRIAL
SEQ ID NO: 126: HK68 H3m2-cl9 + 10 + ll + 12 __
SEQ ID NO: 127: HK68 H3m2-cl9 + 10 + ll + 13
SEQ ID NO: 128: HK68 H3m2-cl9 + 10 + ll + 12-tri (tri short sequence at position 421 to 435)
SEQ ID NO: 129: HK68 H3m2-cl9 + 10 + ll + 13-tri (tri sequence at position 421 to 441)
SEQ ID NO: 130: HK68 H3m2-cl9 + 10 + ll + 12-GCN4 (sequence
GCN4 cuts at position 421 to 435)
SEQ ID NO: 131: HK68 H3m2-cl9 + 10 + ll + 13-GCN4 (sequence
GCN4 at position 421 to 441).
Genes encoding the above protein sequences were synthesized and cloned into the expression vector pcDNA2004 using methods generally known to those of skill in the art. For comparison purposes we included the full-length HA sequence from H3 A / Wisconsin / 67/2005 and / or H3 A / Hong
Kong / 1/1968 in the experiment.
Suspension cells HEK293F (Invitrogen) were transfected (10<sup>6</sup> cells / ml, 30 ml) with expression vectors (1 pg / ml) using 40 µΐ of 293transfectin as a transfection agent and allowed to propagate for another 2 days. Cells were harvested, aliquoted (0.3 ml, approximately 3 * 10<sup>5</sup> cells) and aliquots were treated with either polyclonal serum made against H3 HA
<img file="MX357009B_D0167.tif" />
<img file="MX357009B_D0168.tif" />
165 (Protein Sciences Corp, Meriden, CT, USA) to mark the expression or with a specific monoclonal antibody for
HA (5 micrograms / ml) and a secondary antibody was used for staining. Cells were then analyzed by fluorescence associated cell selection (FACS) for expression of the membrane-bound stem domain HA polypeptides of the invention using polyclonal serum made against H3 HA or Hl HA to mark expression. A panel of monoclonal antibodies of known specificity binding to the full-length protein (CR8020, CR8043, and CR9114) was used to mark the presence of conserved epitopes and, by inference, correct folding of the full-length HA and miniHA polypeptides of the invention. Monoclonal antibodies CR6261 (known to bind to H3 HA) were also used and
CR8057 (which binds to the HA head domain of
A / Wisconsin / 67/2005) in the experiment. Results are expressed as a percentage of positive cells and are shown in Figures 9A-9B for H3 HA based on sequences of
A / Wisconsin / 67/2005 and in Figures 10A-10B for H3 HA based on sequences from A / Hong Kong / 1/1968.
The results show that all constructs based on A / Wisconsin / 67/2005 (FIG. 9) are expressed on the cell surface since the reaction with serum H3
\ .ύ'Χ / 3 '„<7¡ \
MEXICAN INSTITUTE „L ι
OF PROPERTY Ί
INDUSTRIAL ^ '166
ΜΡ polyclonal results in approximately 80% o · ΐΒ & 5 · -Óβ ·· ΐΑ @ · cells tested positive compared to less than 5% for untransfected cells. Control experiments in the absence of IgG, using only the labeled anti-human or anti-rabbit IgG are all negative. Full-length HA from A / Wisconsin / 67/2005 is recognized by monoclonal antibodies CR8020, CR8043, CR8057 (it binds only some strains of H3, described in W02010 / 130636) and CR9114 is known to have the ability to bind to this protein, but not by mAb CR6261. In contrast, most of the stem domain polypeptides are not recognized by CR8020, CR8043, or CR9114 with some notable exceptions.
Polypeptides that comprise the cluster mutation were recognized by CR8020 and / or CR8043. The H3-mini2C19 + 10 + 11 + 12 (SEQ ID NO: lll), H3-mini2-cl9 + 10 + 12 (SEQ ID NO:
112), H3-mini2-cl9 + 10 + ll + 12-GCN4 (SEQ ID NO: 113) and H3-mini2cl9 + 10 + ll + 12-tri (SEQ ID NO: 114) exhibit recognition for
CR8020 (% percentage of positive cells that vary in a range between approximately 10 and 60) and CR8043 (between 40 and
70%) (indicated by arrows). Of the 4 positive constructs, H3-mini2-cl9 + 10 + ll + 12-GCN4 (SEQ ID NO: 113) exhibits the highest response in this assay. The same results are obtained from the average fluorescence intensity shown in panel B (FIG. 9). H3-mini2-
<img file="MX357009B_D0169.tif" />
167
C19 + 10 + 11 + 12 (SEQ ID NO: 111), H3-mini2-cl9 + 10 + 12d-SEQ ™ W ~ ^ Oh112), H3-mini2-cl9 + 10 + ll + 12-GCN4 (SEQ ID NO : 113) and H3-mini2cl9 + 10 + ll + 12-tri (SEQ ID NO: 114) exhibit medium fluorescence intensity well above the background signal after exposure to CR8020 and CR8043 and staining, with the highest of the responses for H3-mini2-cl9 + 10 + ll + 12-GCN4 (SEQ ID NO:
113). None of the HA-based polypeptides from
A / Wisconsin / 67/2005 has the ability to recognize CR9114.
Figures 10A-10B show that all constructs based on A / Hong Kong / 1/1968 (FIGS. 10A-10B) are expressed on the cell surface since reaction with H3 polyclonal serum results in most of constructs approximately 40 to 60% of all cells tested positive compared to less than 5% for untransfected cells. Control experiments in the absence of IgG, using only labeled anti-Human or anti-rabbit IgG are all negative. The percentage of positive cells for the full length protein of A / Hong Kong / 1/1968 after treatment with polyclonal serum is low (approximately 10%), but the strong signals obtained from the binding of CR8020, CR8043 and
CR9114 indicate that the protein is present on the cell surface. CR8057 does not recognize A / Hong Kong / 1/1968 based sequences, only full length protein
168
<img file="MX357009B_D0170.tif" />
Γ> £ LA ',' R · SKIN'Ai '· vJ<sup>:</sup>®'<sup>w</sup>5T «* tRS<sup><</sup>S3 ^ 'IN DU STR1A L of A / Wisconsin / 67/2005. Four constructs (containing the cluster 12 mutation) are recognized by CR8020 and
CR8043, i.e.HK68 H3m2-cl9 + 10 + 12 (SEQ ID NO: 125), HK68
H3m2-cl9 + 10 + ll + 12 (SEQ ID NO: 126), HK68 H3m2-cl9 + 10 + ll + 12tri (SEQ ID NO: 128 containing the shortened trisequence at position 421-435) and HK68 H3m2-cl9 + 10 + ll + 12-GCN4 (SEQ ID
NO: 130, which contains the GCN4 sequence cut at position
421-435), as indicated by the% of positive cells (15% or greater) and MFI clearly above the background signal. The strongest signals (MFI) are obtained from HK68 H3m2C19 + 10 + 11 + 12-GCN4 (SEQ ID NO ·: 130); this construct of the stem domain polypeptide also shows binding to
CR9114 that can be detected.
In conclusion the inventors have shown that by following the method described above, stem domain polypeptides of the invention can be obtained for group 2 serotypes, in particular influenza A viruses of subtype H3.
Example 13: Design, expression and partial purification of soluble stem domain polypeptides comprising conserved epitopes from the stem domain
In certain embodiments, the polypeptides of the invention contain the intracellular sequences of HA and the domain
169
<img file="MX357009B_D0171.tif" />
transmembrane that cause the resulting polypeptides to broaden on the cell surface when expressed in cells. In other modalities, the cytoplasmic sequence and the transmembrane position sequence (or the equivalent
<td>de) 523,</td><td> 524</td><td> , 525</td><td> , 526, 527, 528, 529</td><td>or 530</td><td>to the</td><td>extreme</td><td>C</td>
<td>terminal</td><td>of</td><td>HA2 (</td><td>numbering according</td><td>to SEQ</td><td>ID</td><td>NO: 1)</td><td>I know</td>
<td>removed</td><td>of</td><td>mode</td><td>that the expression in</td><td colspan="2">cells</td><td>generate</td><td>the</td>
secreted (soluble) polypeptide that can be used for example in a vaccine. The soluble polypeptide can be further stabilized by introducing a sequence known to form trimeric structures (also known as foldon), i.e. AYVRKDGEWVLL (SEQ ID NO: 143) optionally connected via a linker (eg.
GSGYIPEAPRDGQAYVRKDGEWVLLSTFL). The linker may optionally contain a cleavage site for post-purification processing according to protocols well known to those of skill in the art. To facilitate purification of the soluble form, a labeling sequence can be added, for example a histidine label (six or seven consecutive histidines) connected by means of a short linker, for example EGR. In some embodiments, the linker or histidine tag is added without the foldon sequence being present.
In accordance with the present invention, the
170
<img file="MX357009B_D0172.tif" />
amino acid sequence from position 530 _de._ la, ... hA, ^ full length from H1N1 A / Brisbane / 59/2007 (numbering according to SEQ ID NO: 1) to the amino acid of the C-terminus of the HA2 domain and replaced by the following EGRHHHHHHH sequences (SEQ ID NO: 81) comprising a short linker and a histidine tag. This exchange was applied to SEQ ID NO: 44: Hl-mini2-grouping + 5 + 6-trim (resulting in SEQ ID NO 144: s-Hl-mini2 grouping + 5 + 6-trim), SÉQ ID NO: 45: Hl-mini2grouping l + 5 + 6-GCN4 (resulting in SEQ ID NO: 145:
s-Hl-mini2-grouping + 5 + 6-GCN4), SEQ ID NO: 46: mini2 grouping + 5 + 6 (A / Brisbane / 59/2007) (resulting in SEQ ID NO: 146: s-Hl-mini2 -grouping + 5 + 6), SEQ ID NO: 47:
mini2-groupingoll + 5 + 6 (A / Brisbane / 59/2007) (resulting in SEQ ID NO: 147: s-Hl-mini2-groupingoll + 5 + 6), SEQ
ID NO: 48: mini2-grouping + 5 (A / Brisbane / 59/2007) (resulting in SEQ ID NO: 148: s-Hl-mini2-grouping + 5).
Similarly, for comparison reasons the exchange was applied to SEQ ID NO 1: full length Hl (A / Brisbane / 59/2007) and the HA cleavage site was further deteriorated by modification of Arginine 343 to a
Glutamine (R343Q mutation) to obtain SEQ ID NO: 149: full-length R343Q s-Hl). Additionally, two polypeptides of the invention were created with a different connector between
<img file="MX357009B_D0173.tif" />
171 the N-terminal and C-terminal parts of ΗΑ1Ί s-Hl-mini2-
<td>grouping l + 5 + 6-nl</td><td>(I KNOW THAT</td><td>ID</td><td>NOT:</td><td> 150)</td><td>and s-Hl-mini2-</td>
<td>grouping l + 5 + 6-n! 2</td><td>(SEQ ID</td><td>NOT:</td><td> 151).</td><td> •</td><td></td>
<td colspan="2">The genes that encode</td><td>for</td><td>the</td><td colspan="2">protein sequences</td>
The above were synthesized and cloned into the expression vector pcDNA2004neo using methods generally known to those of skill in the art. HEK293F cells in suspension (Invitrogen) were transected with the expression vectors using 293transfectin as a transfection agent following protocols well known in the art and further propagated for 7 days. Cells were separated from the culture medium by centrifugation and discarded, while the supernatant containing the soluble polypeptides of the invention was collected for further processing. The supernatant was purified by immobilized metal affinity chromatography on a Ni-NTA column to bind the His-tagged polypeptides of the invention to the resin, and the effluent was collected. The column was washed with between 3 and 10 column volumes of 20mM sodium phosphate pH 7.4, 500mM NaCl, 10mM imidazole (wash), between 5 and 15 column volumes of 20mM sodium phosphate pH 7.4, NaCl 500 mM, 100mM imidazole (thorough washing) and eluted with 20mM sodium phosphate pH 7.4, 500mM NaCl, 500mM imidazole. In individual cases the compositions of
172
<img file="MX357009B_D0174.tif" />
Buffers or nsaHns volumes were adapted.
To increase purity or yield, a linear gradient was used instead of a step gradient. Fractions were collected from start to finish and analyzed by SDS-PAGE and Western blot, using a HA antiH1 polyclonal serum for detection (see Figures 11A-11F). The results indicate a clear enrichment of the polypeptides of the invention in the eluates compared to the materials of
<td>departure.</td><td></td><td></td><td></td><td></td><td></td>
<td>With the</td><td>objective</td><td>of</td><td>confirm the</td><td>folding</td><td>and</td>
<td>functionality</td><td>appropriate</td><td>of</td><td>the polypeptides</td><td>purified</td><td>of</td>
<td>the invention,</td><td>it was evaluated</td><td>the</td><td colspan="3">monoclonal antibody binding</td>
CR9114. To this end, a standard 96-well plate was coated with a monoclonal antibody capable of binding a His tag (6 or 7 consecutive histidines) to the C-terminus of a protein by application of 100 microliters of an antibody solution 1 pg / ml to each well and incubation overnight at 4 ° C. After removing the excess solution and washing, the plate was blocked with 150 microliters of a 2% skim milk solution for 1 hour at room temperature. After removing the blocking agent and washing, 100 microliters of a 1 pg / ml solution of the polypeptides of the invention were added, as well as the ectodomain of the corresponding protein of
<img file="MX357009B_D0175.tif" />
173
IMPI full length (SEQ ID NO: 149) and incubated for T hours at room temperature. After removing the excess of the polypeptides of the invention, the mAb CR9114, mAb was added
CR8020 (negative control) or polyclonal serum directed against
Hl HA in rabbits (positive control) at concentrations ranging from 2 to 20 pg / ml and incubated for 2 hours at room temperature. Binding was detected with HRP-conjugated anti-human antibody using protocols well known in the art.
The results (Figures 12A-12C) show that the CR9114 monoclonal antibody binds to the purified soluble polypeptides of the invention as well as the full-length ectodomain (Figure 12A), whereas the CR8020 monoclonal antibody does not (Figure 12B). Anti-Hl polyclonal serum also binds the polypeptides of the invention and the full-length ectodomain in a very similar way (Figure 12C). Therefore, it is concluded that the widely neutralizing epitope of CR9114 is preserved in the polypeptides of the invention, and taking into account the discontinuous and conformational nature of this epitope, that the stem domain is appropriately folded and adopts an equal or very three-dimensional conformation. similar to the conformation in native full-length HA.
The preparations of the polypeptides of the invention
174 ri
INSTITUTO MEXICANO 'p.' 'ÜE LA PRi l'IEOAD Cja INDUSTRIAL> 1 were heterogeneous in size determined from SDS-PAGE and Western blot results. The inventors hypothesized that the variation is due to variation in the glycosylation patterns of proteins between individual protein molecules. To confirm this, small aliquots of the protein preparations were treated with 3 units of N-glycosidase F (an enzyme that removes carbohydrate groups bound to
N of the Asparagine residues) for 18 hours at 37 ° C and analyzed by SDS-PAGE and Western blot. The results (Figures 13A and 13B) show that the N-glycosidase treatment targets the fuzzy bands of the polypeptides of the invention to a single band at the expected molecular weight calculated from the amino acid sequence. This is clear evidence that the observed heterogeneity in size does indeed arise from variation in glycosylation patterns.
The preparations of the polypeptides of the invention were further characterized by HP-SEC. For this purpose, approximately 40 pg of the polypeptides of the invention were applied in a volume between 43 and 63 pl (polypeptide concentration between 0.64 and 0.93 mg / ml) to a Tosoh TSKgel G2000 SWxl column connected to a multi-angle light scattering detector. . The results are shown in Figure
175
YEAR
MEXICAN INSTITUTE .. ... _,
OF PROPERTY (Jas .- ^ - 'S INDUSTRIAL
14. The main peak (retention time of approximately ..... 8 minutes) arises from the polypeptides of the invention, and is well separated from the larger species, indicating that further purification can be achieved. Based on the data from the multi-angle light scattering detector, the main peaks correspond to the molecular species with a molecular weight between 50 and 80 kiloDalton (see Table 9) depending on the polypeptide of the invention under study. Based on the heterogeneity of size and variety in glycosylation of the polypeptides described above, as well as the dependence of the results on the hydrodynamic form of the molecules, these numbers should only be taken as an indication.
Example 14: Expression and partial purification of a soluble stem domain polypeptide comprising the conserved stem domain epitope of monoclonal antibodies CR9114, CR6261 and FI6v3
In order to obtain a highly pure preparation of a polypeptide of the invention, HEK293F cells were transected with the expression vector pcDNA2004 that contains the gene coding for s-Hl-mini2grouplingol + 5 + 6-GCN4 (SEQ ID NO: 145 ). The experienced person will understand that a leader sequence (or sequence
176 . .<sub>v</sub>-, P j
INSTITl JTG MEX'CANO Vz /, * - ^ <sup>Λ</sup> - * <V / · D ¿LA PXCItfOW V ¿'sirv, ·' .c'T · ?; · I Ύ '' -. * .. “i
IN0üST¿X »l signal) that directs the transport of a protein '^ düránte''Ta' production (which corresponds to amino acids 1-17 of SEQ ID
NO: 145) will not be present in the final secreted polypeptide. For this purpose, 1.0 * 10 were sown<sup>6</sup> vc / mL by centrifugation of HEK293F cells (Invitrogen) at 300 g for 5 minutes and resuspension in 300 mL of medium
Freestyle ”preheated by SF1000. This culture was incubated
<td>for 1</td><td>hour to</td><td> 37</td><td>'C,</td><td> 10%</td><td>CO<sub>2</sub> to 110</td><td>rpm</td>
<td>multitron.</td><td>Then</td><td>of</td><td> 1</td><td>hour</td><td>he took</td><td>with</td>
<td>10 plasmid</td><td>at 9.9</td><td>mL</td><td>of</td><td>means, medium</td><td>Optimem a</td><td>a</td>
1.0 pg / mL in the 300 mL culture volume. In parallel, 440 pL of 293fectin® in 9.9 mL of Optimem medium were pipetted and incubated for 5 minutes at room temperature. After 5 minutes the Plasmid DNA / Optimem mixture was added to
293fectin® / Optimem and incubated at room temperature for 20 minutes. After incubation the plasmid DNA / 293fectin® mixture was added dropwise to the cell suspension. The transfected culture was incubated at 37 ° C, 10% CO<sub>2</sub> and
110 rpm in a multitron incubator. On day 7 the cells were separated from the culture medium by centrifugation (30 minutes at 3000 g), while the supernatant containing the soluble polypeptides of the invention was filtered through the mixture with a 0.2 µm bottle top filter for processing.
IMPI (/ ¾
177
MEXICAN INSTITUTE í $ p * *,? ·
OF THE ri.DílECAI 'V »_ INDUSTRIAL additional.
To verify the presence of the polypeptide of the invention, a small aliquot of the supernatant was analyzed by Western blot, using a monoclonal antibody directed against the His tag for detection (Figure
15A). Various bands were observed at an apparent molecular weight between 37 and 50 kDa, which is close to or greater than the calculated molecular weight based on the amino acid composition of the protein. Heterogeneity is caused by variation in glycosylation patterns, since previous experiments have shown that treatment of this protein with N-glucosidase F to remove N-linked glucans from the protein results in the concentration of the band in the expected molecular weight.
The presence of the broadly neutralizing epitopes in the polypeptide of the invention was confirmed by ELISA, using the broadly neutralizing antibodies CR6261,
CR9114 and FI6v3 as probes. For comparison reasons the monoclonal antibody CR8020 was also included as a negative control in the experiment; this antibody has the ability to bind to HA virus molecules of group 2 (for example HA H3 and H7), but not of group 1 (for example HA Hl and
H5). To this end, it was coated with a monoclonal antibody capable of binding His marks (6 or 7 histidines
178
<img file="MX357009B_D0176.tif" />
) at the C-terminus of a standard 96-well plate plate protein by applying 100 microliters of a 1 pg / ml antibody solution to each well and incubating overnight at 4 ° C. After removing the excess solution and washing, the plate was blocked with 150 microliters of a 2% skim milk solution for 1 hour at room temperature. After removing the blocking agent and washing, 100 microliters of the supernatant was added and incubated for 2 hours at room temperature. After removal of excess polypeptides of the invention, mAb CR9114 was added, in 1: 2 dilution series starting at a concentration of 5pg / ml, and incubated for 2 hours at room temperature. Binding was detected by HRP-conjugated anti-human antibodies using protocols well known in the art. Clear binding of CR9114, Fl6v3 and to a lesser extent CR6261 to the polypeptide of the invention is observed, while for CR8020 no response is observed indicating that the binding observed is specific for the monoclonal antibodies tested (Figure 15B).
For purification purposes 250 ml of culture supernatant was applied to a 5 ml His-trap column, washed with 75 ml of wash buffer (20mM TRIS, 500mM NaCl, pH 7.8), and eluted with gradients in steps <ί na .ir: ι V.-í f μ '<sup>; i</sup>vv?. 'gSS
179 of imidazole (10, 50, 100, 200, 300 and 500 mM .- © e-seltiei-err buffer for washing). The chromatogram (fig. 16) exhibits multiple peaks, with the polypeptides of the invention eluting with 100mM imidazole (peak A) and 200mM imidazole (peak
B). Both peaks were collected, concentrated, and applied to a size exclusion column for further purification (Superdex 200). The elution profiles are shown in Figures 17A and 17B. Fractions were collected and analyzed by SDS-PAGE (Figures 17C and
D). Fraction 3 derived from both peaks A and B contained the highly pure polypeptide of the invention. The final yield was approximately 10 pg / ml of culture supernatant. The purified batches are endotoxin-free (dosage at 5mg / kg; <1 EU / mg); LAL chromogenic) and biological load is less than 1 CFU / 50pg.
Example 15. Design of a stem domain polypeptide comprising the conserved epitope of the stem domain of
Influenza B HA-based CR9114
The procedure described above to design polypeptides of the invention was also applied to Influenza B.
In this example, the polypeptides of the invention are described based on HA sequences taken from virus strains of both known lineages, i.e.B / Florida / 4/2006
<img file="MX357009B_D0177.tif" />
(lineage B / Yamagata) and B / Malaysia / 2 50 6 / 2-9O4— ~ - («li-na-j eB / Victoria). Those of skill in the art will understand that the use of other Influenza B HA sequences is also possible since the sequences are well conserved, particularly in the stem region. Therefore polypeptides derived from other Influenza B HA sequences according to the description below are also encompassed by the invention.
The first modification in the HA sequence of
B / Florida / 4/2006 was the removal of the cleavage site at position 361 (numbering refers to SEQ ID NO:
132) by mutation of R (or in a limited number of K cases) to
Q (R361Q) to prevent the formation of HAl and HA2 from HAO.
Optionally the residue between 363 and 367 (GFGAI, part of the fusion peptide) can be further removed to minimize exposure of hydrophobic residues to the aqueous solvent. The positive charge on cleavage is 100% conserved in Influenza B HA and this mutation can therefore be applied in all sequences.
The second modification is the deletion of the head domain by deletion of a large part of the sequence of
HAl and the reconnection of the terminal N and C sequences by means of a short connector. The removal may vary in length, but it is preferred that the last residue of the
IMPIAS
181 Mexican institute - / - Ν, /.
FROM PROPERTY Qr? ^ Or-'v2> '
INDUSTRY!
N-terminal sequence of HAl and the first residue. .C terminal sequence are spatially close to each other to avoid introducing stress through the junction sequence. In B sequence deletions can be introduced at (the equivalent positions of) P51-I336 (m2; SEQ ID NO: 133) in
B / Florida / 4/2006 (SEQ ID NO: 132). Equivalent positions can be easily determined by those skilled in the art by aligning the sequences using a suitable algorithm such as for example Clustal or
Muscle. The remaining parts of the sequence can be attached directly or alternatively a flexible connector can be inserted. Connector sequences can be between 1 and 50 amino acids in length. Flexible connectors of limited length (less than or equal to 10 amino acids) are preferred, for example GGG, GGGG, GSA, GSAG, GSAGSA,
GSAGSAG or the like.
SEQ ID NO: 133 describes said polypeptide of the invention that contains the P51-I332 deletion (m2; SEQ ID
NO: 133). The deletions described above ensure that unstructured regions formed by residues between P51 and N58 and between E306 and 1337 are also removed; this is beneficial for the overall stability of the polypeptides of the invention. A similar effect was observed for the polypeptides of the invention derived from an Hl sequence.
<img file="MX357009B_D0178.tif" />
182
IMPI (see above). ____
Elimination of the head domain leaves a loop between residues 416 and 436 now exposed to the aqueous solvent. In
HA of B this loop is highly conserved (see table
10). The consensus sequence is: LSELEVKNLQRLSGAMDELHN.
To increase the solubility of this loop in the prefusion conformation and to destabilize the post-fusion conformation, some hydrophobic residues were modified by polar amino acids (S, T, N, Q), charged (R, H, K, D, E), or flexibility had to be increased by mutation to G.
Specifically mutations at positions 421, 424,
427, 434 (the numbering refers to SEQ ID NO: 132) will contribute to the stability of a polypeptide of the invention.
For positions V421 and L427, the T mutation is preferred but other polar (S, N, Q), charged (R, H, K, D, E) and highly flexible (G) amino acids will have the same effect. For position 424, the S mutation is preferred.
Other polar amino acids (N, T, Q), charged (R, H, K, D, E) and highly flexible (G) will have the same effect. For the L434 position, the mutation to G is preferred. Other charged polar amino acids (S, Τ, N, Q) (R, Η, K, D, E) will have the same effect. Polypeptides containing at least one of the mutations described above were made. The
<img file="MX357009B_D0179.tif" />
• i f.
183 '..X .X combinations of more than one mutation are also - pesífeiee<sub>T</sub>as shown for example in SEQ ID NOs: 134-136.
To stabilize the prefusion conformation of polypeptides of the invention, a covalent bond was introduced between two distant parts in the primary but close sequences in the folded prefusion conformation. For this purpose, a disulfide bridge was designed in the polypeptides, preferably between (the equivalent of) position K340 and
S454 in HA of B / Florida / 4/2006 (SEQ ID NO: 134-136). Equivalent positions can be easily determined by those of skill in the art by sequence alignment using a suitable algorithm such as Clustal,
Muscle etc. Designed disulfide bridges are created by mutation of at least one (if the other is already a cysteine), but usually two residues that are spatially close to cysteine, which will form a covalent bond spontaneously or by active oxidation between the sulfur atoms of these waste.
In the stem domain trimerization is mediated by the formation of a trimeric coiled helix motif.
By strengthening this motif a more stable trimer can be created. Sequences supporting this formation of a GCN4-derived coiled helix are introduced at position (the equivalent of) 436 to 452 RRMKQIEDKIEEILSKI (SEQ
<img file="MX357009B_D0180.tif" />
184
IMPI
ID NO: 135), or alternatively RMKQIEDKIEEHifiírí — at — ta ------------ position 436 to 451 (SEQ ID NO: 136).
The same procedure was followed for HA from
B / Malaysia / 2506/2004 (SEQ ID NO: 137) to provide polypeptides. Compared to HA from B / Florida / 4/2006 this HA has an additional asparagine residue inserted at position 178 as can be easily seen in an alignment of the two sequences. Consequently the cleavage site is at position 362, and the corresponding mutation to prevent cleavage is R362Q. The removal to remove the head region in this case is for example P51 to 1337 (m2; SEQ
ID NO: 138). Again the remaining parts of the sequence can be joined directly or alternatively a flexible connector can be introduced. Connector sequences can be between 1 and 50 amino acids in length. Flexible connectors of limited length (less than or equal to 10 amino acids) are preferred, for example GGG, GGGG, GSA,
GSAG, GSAGSA, GSAGSAG or the like.
SEQ ID NO: 138 describes said P51-I332 deletion-containing polypeptide (m2; SEQ ID NO: 138). The deletions described above ensure that unstructured regions formed by residues between P51 and N58 and between E307 and 1338 are also removed; this is beneficial for the overall stability of the polypeptides of
185 the invention. A similar effect was observed for pplipeptides of the invention derived from an Hl sequence (see above).
Elimination of the head domain leaves the loop between residues L420 and H436 now exposed to the aqueous solvent.
In HAs of B this loop is highly conserved (see Table x). The consensus sequence is: LSELEVKNLQRLSGAMDELHN.
To increase the solubility of this loop in the prefusion conformation and to destabilize the post-fusion conformation, some hydrophobic residues were modified by polar amino acids (S, T, N, Q), charged (R, H, K, D, E), or flexibility has to be increased by mutation to G. Specific mutations were tested at positions 422, 425,
428, 435 (numbering refers to SEQ ID NO: 137).
For positions V422 and L428, the T mutation is preferred but other polar (S, N, Q), charged (R, H, K, D, E) and highly flexible (G) amino acids will have the same effect. For position 425, the S mutation is preferred.
Other highly flexible (R, H, K, D, E) and highly flexible (G) polar (N, T, Q) amino acids will have the same effect. For position L435, mutation to G is preferred. Other charged polar amino acids (S, T, N, Q) (R, Η, K, D, E) will have the same effect. Polypeptides containing at least one of the mutations described above were made. They are also
Τ Tk / ίΓ ^ 5 X
X XV X 1 X
IWÍ1 IT'JT '·! MEXICAN · C i
M The LLnArL PROPERTY,. · ';
INDUSTRIAL 'AjXSX' '
186 possible combinations of more than one mutacj οη ^ 4; ηιηο, ^. 5 & shown for example in SEQ ID NOs: 139-141.
To stabilize the prefusion conformation of the polypeptides of the invention, a covalent bond is introduced between two distant parts in the primary but close sequences in the prefusion folded conformation. For this purpose, a disulfide bridge is designed in the polypeptides of the invention, preferably between (the equivalent of) the K341 and S455 position in HA of
B / Malaysia / 2506/2004 (SEQ ID NO: 139-141). Equivalent positions can be easily determined by those of skill in the art by sequence alignment using a suitable algorithm such as Clustal, Muscle, etc.
<td>The</td><td>bridges</td><td>disulfide</td><td>I know</td><td>create</td><td>by mutation of</td><td>at least one</td>
<td>(yes</td><td>the other</td><td>it's already a</td><td colspan="2">cysteine),</td><td>but usually</td><td>two residues</td>
<td>than</td><td>is it so</td><td colspan="2">spatially</td><td>near</td><td>, to cysteine,</td><td>what will form</td>
spontaneously or by oxidation activates a covalent bond between the sulfur atoms of these residues.
As previously described, native HA exists as a trimer on the cell surface. Most of the interactions between the individual monomers that hold the trimer together are located in the head domain. After removal of the head, the tertiary structure is consequently destabilized and
<img file="MX357009B_D0181.tif" />
187
<img file="MX357009B_D0182.tif" />
MEXICAN INSTITUTE OF PROPERTY
INDUSTRIAL therefore reinforcing interactions—<sup>pn7r</sup>P ins monomers in the truncated molecule will increase stability.
The trimerization of the stem domain is mediated by the formation of a trimeric coiled helix motif. By reinforcing this pattern, a more stable trimer can be created. Sequences supporting the formation of a GCN4-derived trimeric coiled helix are introduced at (the equivalent of) position 437 to 453 RRMKQIEDKIEEILSKI (SEQ ID
NO: 135), or alternatively RMKQIEDKIEEILSKI in position
437 at 452 (SEQ ID NO: 136).
Influenza hemagglutinin-based polypeptides
B, SEQ ID NO: 133-136 and 138-141 were assayed for the presence of the CR9114 epitope by Fluorescence-associated cell selection as described above. However, non-binding of CR9114 mAb was observed for these constructs.
Example 16: Immunogenicity of HA Generation 3 Stem Domain Polypeptides
In order to assess the immunogenicity of stem domain polypeptides, mice were immunized with the expression vectors encoding for the full length H1 of A / Brisbane / 59/2007 (SEQ ID NO: 1),
Mini3-groupingoll (SEQ ID NO: 11), Mini2-groupingoll + 5
188
<img file="MX357009B_D0183.tif" />
MEXICAN INSTITUTE OE LA I KOPIF.DAI)
INDUSTRIAL
<img file="MX357009B_D0184.tif" />
(SEQ ID NO: 14), mini2-grouping + 5 (SBQ — ID MO; —- 4B).
mini2-grouping + 5 + 6 (SEQ ID NO: 46), mini2-groupinglent + 5 + 6-GCN4 (SEQ ID NO: 45) and mini2-grouping + 5 + 6-nl (SEQ ID NO: 152). An expression vector encoding cM2 was also included as a negative control.
Groups of 4 mice (BALB \ c) were immunized with 50 pg of construct + 50 pg of adjuvant (pUMCVl-GM-CSF) im on day 1, 21 and 42. On day 49 a final bleed was performed and collected the serum. Sera were analyzed by Elisa using the recombinant ectodomains of full-length HA from strains A / Brisbane / 59/2007 and
A / California / 07/2009 (obtained from Protein Sciences
Corporation, Meriden, CT, USA) as the antigen.
Briefly, 96-well plates were coated with 50 ng of
HA overnight at 4 ° C, followed by incubation with buffer for blocking (100 pl PBS, pH 7.4 +
2% skim milk) for 1 hour at room temperature. The plates were washed with PBS + 0.05% Tween-20, and 100 pl of a 2-fold dilution series in buffer buffer was added, starting from a 20-fold dilution of the serum. Bound antibody is detected using HRP-conjugated anti-mouse IgG, using standard protocols well known in the art. The titles
<img file="MX357009B_D0185.tif" />
189
MEXICAN INSTITUTE OF PROPERTY
INDUSTRIAL
<img file="MX357009B_D0186.tif" />
they are compared to a standard curve using the mAb InAl3A.
(Hytest, Turku, Finland) to derive in units of
ELISA / ml (EU / ml).
The time course of the IgG response against the ectodomain of the full-length homologous protein induced by the immunization scheme described above is shown in Figure 18. A high response can already be observed for mice immunized with DNA (SEQ ID NO: 1) that codes for the full length protein after 4 weeks. The response increases with an enhancer injection, as shown by the increased titer at 7 weeks. Immunization with DNA encoding the polypeptides of the invention mini2-grouping + 5 (SEQ ID
NO: 14), mini2-grouping + 5 (SEQ ID NO: 48), mini215 grouping + 5 + 6 (SEQ ID NO: 46), mini2-grouping + 5 + 6GCN4 (SEQ ID NO: 45) and mini2-grouping + 5 + 6-nl (SEQ ID NO:
152) leads to intermediate titers that are further increased with an enhancing immunization as evidenced by the titers at week 7. Immunization with DNA encoding
Mini3-grouping (SEQ ID NO: 11) and cM2 negative control result in a response that cannot be detected in this assay.
Figures 19A-19B show IgG responses at week 7 after initial immunization for mice.
190
<img file="MX357009B_D0187.tif" />
IMPI
INSTITUTO MtXICANO DE LA ÉROPIEDAí)
INDUSTRIAL individual against the hemagglutinin ectodomain.
full length of the homologous strain H1N1 A / Brisbane / 59/2007 (panel A) and the heterologous strain H1N1 A / California / 07/2009. DNA-induced antibodies encoding polypeptides of the invention mini2-grouping + 5 (SEQ ID NO: 14), mini2-grouping + 5 (SEQ ID NO: 48), mini2-grouping + 5 + 6 (SEQ ID NO: 46) , mini2-grouping + 5 + 6GCN4 (SEQ ID NO: 45) and mini2-grouping + 5 + 6-nl (SEQ ID NO:
152) bind equally well to the hemagglutinin ectodomain derived from the homologous and heterologous strain. In contrast, immunization with DNA encoding the full-length protein (SEQ ID NO: 1) results in high titers against the homologous hemagglutinin (more than an order of magnitude greater than the observed titers for DNA immunization that encodes for the polypeptides of the invention), but gives lower titers against the heterologous hemagglutinin ectodomain. Immunization with DNA encoding Mini3-grouping (SEQ ID NO: 11) and negative control cM2 results in a response that cannot be detected against any of the hemagglutinin ectodomains in this assay.
In conclusion, the antibodies directed against the polypeptides of the invention mini2-grouping + 5 (SEQ ID
NO: 14), mini2-grouping l + 5 (SEQ ID NO: 48), mini2191
Hee .-ϊ<sup>1</sup>- * “ΙΝ'ΤΠ'ΠΤΟ Mexican p'¿ LA PHOFíECAl;
INDJSTüJAL
<img file="MX357009B_D0188.tif" />
grouping + 5 + 6 (SEQ ID NO: 46), mini2-groupingQ £ x> l - k54-ÉM * —- ·
GCN4 (SEQ ID NO: 45) and mini2-grouping l + 5 + 6-nl (SEQ ID NO:
152) have the ability to recognize full-length hemagglutinin. Its epitopes are necessarily located in the hemagglutinin stem domain and are conserved among the full-length hemagglutinins of
H1N1 A / Brisbane / 59/2007 and HlNl A / California / 07/2009.
Example 17: Immunogenicity of the stem gene domain polypeptide mini2-grouping + 5 + 6-GCN4 <· of third generation
In order to further assess the immunogenicity of the stem domain polypeptides of the invention, mice were immunized once with the expression vector encoding mini2-grouping + 5 + 6-GCN4 (SEQ
ID NO: 45) (sensitization) and was potentiated twice with the purified protein s-Hl-mini2-grouping + 5 + 6-GCN4 (SEQ ID
NO: 145) at three week intervals. For comparison reasons, groups separated three times at immunization intervals of three weeks were immunized with the expression vectors encoding mini2-cluster + 5 + 6-GCN4 (SEQ ID NO: 45) as well as full length Hl of
A / Brisbane / 59/2007 (SEQ ID NO: 1). An expression vector encoding cM2 was also included as a control.
192 __ í PI
MEXICAN INSTITUTE · · negative faith.
Groups of 4 mice (BALB \ c) were immunized intramuscularly (im) with 1000 pg of construct encoding mini2-cluster + 5 + 6-GCN4 (SEQ ID NO: 45) +
100 pg of adjuvant (pUMCVl-GM-CSF) on day 1 and with s-Hlmini2-grouping + 5 + 6-GCN4 (SEQ ID NO: 145; 100 pg of purified protein) with added as adjuvant of 10 pg of Matrix- M on day 21 and 42. One group received a 2<sup>gives</sup> and 3<sup>ra </sup>sc immunization, while another received 2<sup>gives</sup> and 3<sup>ra </sup>im immunizations A third group was again sensitized with 100 pg of construct encoding mini2-cluster + 5 + 6-GCN4 (SEQ ID NO: 45) + 100 pg of adjuvant (pUMCVl-GM-CSF) on day 1 as above and received enhancer immunizations on day 21 and 41 of sHl-mini2-grouping + 5 + 6-GCN4 (SEQ ID NO: 145; 100 pg of purified protein) with aggregate as Montanide adjuvant
ISA-720 (1: 1 volume by volume). For comparison, groups of 4 mice (BALB \ c) im were immunized on day 1, 21 and 42 with 100 pg of construct encoding mini2grouping + 5 + 6-GCN4 (SEQ ID NO: 45), full length Hl de A / Brisbane / 59/2007 '(SEQ ID NO: 1) or cM2, with an adjuvant of 100 pg of adjuvant (pUMCVl-GMCSF) added.
On day 49, a final bleed was performed and
<img file="MX357009B_D0189.tif" />
<img file="MX357009B_D0190.tif" />
MEXICAN INSTITUTE <sub>Λ</sub> _ Of the property
193 INDUSTXIAL collected the serum. Sera were analyzed by full-length recombinant ELI3AusartdoHA from H1N1 strains
A / Brisbane / 59/2007, H1N1 A / California / 07/2009, H5N1
A / Vietnam / 1203/2004 and H3N2 A / Hong Kong // 1968 (obtained from
Protein Sciences Corporation, Meriden, CT, USA) as an antigen. Briefly, 96-well plates were coated with 50 ng HA overnight at 4 ° C, followed by incubation with buffer for blocking (100 µΐ of
PBS, pH 7.4 + 2% skim milk) for 1 hour at room temperature. The plates were washed with PBS + 0.05% Tween20, and 100 µΐ of a series of fold dilutions in buffer buffer was added, starting from a 20 fold dilution of the serum. Bound antibody was detected using goat anti-mouse IgG conjugated to
HRP, using standard protocols well established in the art. Titers are compared to a standard curve consisting of a serial dilution of a mouse monoclonal antibody that binds to the HA antigen and expressed as ELISA units per ml (EU / ml). Figures 20A-20D show IgG responses at week 7 after initial immunization for individual mice against the full length hemagglutinin ectodomain of the homologous strain H1N1 A / Brisbane / 59/2007 (panel A), heterologous H1N1
A / California / 07/2009 (panel Β), the heterosubtypal strain H5N1
<img file="MX357009B_D0191.tif" />
194
<img file="MX357009B_D0192.tif" />
INSTIT! ΠΌ MEXICANO Γ) Ε LA FROP.'EOAD
INDUSTRIAL
A / Vietnam / 1203/2004 (panel C) and the heterosubtypical strain H3N2 A / Hong Kong / 1/1968 (panel D). Antibodies induced by immunization with DNA encoding the polypeptide of the invention mini2-grouping + 5 + 6-GCN4 (SEQ ID NO: 45) have the ability to recognize HA from H1N1 A / Brisbane / 59/2007,
H1N1 A / California / 07/2009 and to a lesser extent H5N1
A / Vietnam / 1203/2004. Antibodies produced in response to immunization with DNA encoding full-length Hl from A / Brisbane / 59/2007 (SEQ ID NO: 1) recognize the homologous protein very well, but much less the
Heterologous HA from H1N1 A / California / 07/2009, and heterosubtypical HA from H5N1 A / Vietnam / 1203/2004, as evidenced by the lower titles in Figures 17B and 17C. The group of mice immunized with DNA encoding mini2grouping + 5 + 6-GCN4 (SEQ ID NO: 45; priming) followed by enhancer immunizations with the sHl-mini2-grouping protein + 5 + 6-GCN4 (SEQ ID NO: 145 ) exhibits high titers against ectodomains of HA derived from H1N1 homologous A / Brisbane / 59/2007, heterologous H1N1
A / California / 07/2009 and heterosubtypical H5N1
A / Vietnam / 1203/2004.
Figure 20D shows IgG responses at one week against the H3N2 A / Hong Kong / 1/1968 HA ectodomain. Unlike mini2-clustering + 5 + 6-GCN4 (SEQ ID NO: 45) and
ΙΜΡΪ
195
MEXICAN INSTITUTE U¿ LA FR JPíEOáD
INDUSTRIAL ¡> «-rc / i _ecll
<img file="MX357009B_D0193.tif" />
s-Hl-mini2-cluster + 5 + 6-GCN4 (SEQ ID NO: 145) which are derived from HA from H1N1 A / Brisbane / 59/2007, a strain belonging to Influenza group 1, H3N2 A / Hong Kong / 1/1968 belongs to group 2 of Influenza and is therefore phylogenetically distant from the parental sequence used for the design of the polypeptides of the invention used in this experiment. Immunization three times with DNA encoding mini2-grouping + 5 + 6-GCN4 (SEQ ID NO: 45) or
Full-length HA from H1N1 A / Brisbane / 59/2007 (SEQ ID
NO: 1) does not give IgG levels as a result that can be detected by ELISA against this antigen. In contrast, immunization with DNA encoding mini2grouping + 5 + 6-GCN4 (SEQ ID NO: 45) followed by two enhancer immunizations with the purified protein s-Hlmini2-grouping + 5 + 6-GCN4 (SEQ ID NO: 145) results in high titles against HA from H3N2 Ά / Hong Kong / 1/1968.
This result is obtained regardless of the immunization route used (ie intramuscular vs. subcutaneous) or the adjuvant added to protein enhancing immunizations (Matrix-M or Montanide ISA-720).
In conclusion, immunization with polypeptides of the invention can produce in response IgGs that have the ability to recognize HA from a wide range of influenza strains, including homologous, heterologous, and
196 τ and $ ρ a
one. Á + .Λ AA
INSTIT * rn> MEXICAN Ul THE INDUSTRIAL PROPERTY
<img file="MX357009B_D0194.tif" />
heterosubtypes of influenza group 1 as well as a strain of group 2 influenza. Conversely, immunization with full-length HA results in high titers against HA of the homologous strains, low titers against heterologous and heterosubtypical strains, and levels of
IgG below the detection limit for the influenza group strain.
Example 18: Design of other stem domain polypeptides comprising conserved epitopes from the stem domain of CR6261 and CR9114
The polypeptides of the invention designed according to the procedure described above can be further modified to increase stability. Said modifications can be introduced to improve the formation of trimeric forms of the polypeptides of the invention with respect to monomeric and / or dimeric species. As described, native HA exists as a trimer on the cell surface. Many of the interactions between the individual monomers that hold the trimer together are located in the head domain. After removal of the head the tertiary structure is then destabilized and therefore reinforcing the interactions between the monomers in the truncated molecule will increase the
IMPI
<img file="MX357009B_D0195.tif" />
197 stability of the trimeric form. Trimerization is mediated by the formation of a trimer. By strengthening the coiled helix motif in the stem domain a more stable trimer form can be obtained.
According to the invention, a consensus sequence is introduced for the formation of a trimeric coiled helix,
IEAIEKKIEAIEKKIE (SEQ ID NO: 83), in a polypeptide of the invention at (the equivalent of) position 418 to 433 (SEQ
ID NO: 44) in Hl A / Brisbane / 59/2007 (numbering according to
SEQ ID NO: 1). Alternatively, you can enter
IEAIEKKIEAIEKKI (SEQ ID NO: 85) at 419-433 (SEQ ID NO: 49) or
IEAIEKKIEAIEKK (SEQ ID NO: 86) at 420-433 (SEQ ID NO: 50).
An alternative is to introduce the sequence MKQIEDKIEEIESKQ (SEQ ID NO: 84), derived from GCN4 and known to form trimer, at position 419-433 (SEQ ID NO: 45). As an alternative, you can enter MKQIEDKIEEIESK (SEQ ID NO:
87) at position 420-433 (SEQ ID NO: 51) or
RMKQIEDKIEEIESKQK (SEQ ID NO: 88) at position 417-433 (SEQ
ID NO: 52). Similarly, the trimer interface could be strengthened by modification of M420, L423, V427, G430 to
Isoleucine (SEQ ID NO: 53).
In certain embodiments, the polypeptides of the invention contain intracellular sequences of HA Hl and the transmembrane domain. In other modalities, the sequence ίί Κ ·.; 4 Ο
198
I: * 5?: R: jTO MEXICANO · r¿ the ['Rowuw VVtoíJaLg INDUSTRIAL cytoplasmic and the transmembrane sequence of the position (or the equivalent thereof) 523, 524, 525, 526, 526, 527,
528, 529, or 530 from HA2 to the C-terminus of HA2 (numbering according to SEQ ID NO: 1) are removed so that a secreted (soluble) polypeptide is produced. The soluble polypeptide can be further stabilized as previously described.
Connector Variants Description
Genes encoding the above protein sequences were synthesized (SEQ ID NO: 44 to 46; SEQ
ID NO: 49 to 53 and SEQ ID NO: 152-157) and were cloned into the expression vector pcDNA2004 using methods generally known to those of skill in the art. For comparison reasons, an expression vector encoding the full length sequence (SEQ ID NO: 1) as well as cM2 was included in the experiment.
HEK293F cells in suspension (Invitrogen) were transected (10<sup>6</sup> cells / ml, 30 ml) with expression vectors (1 pg / ml) using 40 µΐ of 293-transfectin with transfection agent and further propagated for 2 days. The cells were collected; aliquoted (0.3 ml, approximately 3 * 10<sup>5</sup> cells) and aliquots were treated with polyclonal serum directed against HA
199
IMPI.
INSTIT 'ITO MÍXICANO
Say LA PROKIÍDAP Vce-jí-í'.í-<sup>1</sup>
INDUSTRIAL - Hl for expression of the probe or an HA-specific monoclonal antibody (5 micrograms / ml) and a secondary antibody used for staining. Cells were then analyzed by fluorescence-associated cell selection (FACS) for expression of membrane-bound HA stem domain polypeptides of the invention using Hl HA-targeted polyclonal serum for probe expression. A panel of monoclonal antibodies of known specificity binding to the full-length protein (CR6261, CR9114, CR9020 and CR8020) was used to investigate the presence of conserved epitopes and, by inference, correct folding of full-length HA and the HA mini-polypeptides of the invention. Results are expressed as percentage of positive cells and average fluorescence intensity and are shown in FIGS. 21A-21B. <
> The results show that all the variants tested are expressed on the cell surface as evidenced by the positive response of the anti-Hl polyclonal serum.
The H3 HA-specific antibody CR8020 does not recognize any of the constructs included in the experiment, while CR9020, which binds to the Hl HA head domain, only clearly recognizes the full-length protein. All the polypeptides of the invention as well as the full length protein are recognized by CR6261 and
<img file="MX357009B_D0196.tif" />
200
CR9114, indicating that the corresponding epitopes are.
present in the polypeptides of the invention in the same conformation as in the wild type protein. Among the polypeptides of the invention with an additional trimerization motif included in the CD helix (see FIG. 1), SEQ ID
NO: 45, 51 and 52 containing the sequences derived from
GCN4 SEQ ID NO: 84, 87, and 88 result in responses equal to or greater (MFI) than SEQ ID NO: 44, 49, and 50 respectively, containing the consensus trimerization sequences of SEQ ID NO: 83, 85, and 86 .
Variation in the composition of the connector connecting amino acids 52 and 321 (numbering refers to SEQ
ID NO: 1) in the polypeptides of the invention does not lead to major changes in recognition of monoclonal antibodies CR6261 and CR9114. The largest change is seen when GGGG in SEQ ID NO: 4 6 is replaced by HNGK, resulting in SEQ ID NO: 152, which leads to a slightly lower response to CR6261, but does not affect the response to CR9114. Elimination of the connector and introduction of amino acids
53-56 of SEQ ID NO: 1 (SHNG), i.e. creating a polypeptide of the invention without a linker in SEQ ID NO: 46 (resulting in SEQ ID NO: 153), SEQ ID NO: 45 (resulting SEQ ID NO: 154) or SEQ ID NO: 50 (resulting in SEQ ID NO: 155) has no impact on the response in
<img file="MX357009B_D0197.tif" />
201
IMPI
MEXICANC INSTITUTE ·
OF THE FR iriEDAT)
INDUSTRIAL FACS test, indicating that the connector sequence is not critical.
SEQ ID NO: 156 is derived from SEQ ID NO: 46 by introduction of the I337N, I340N and F352Y mutations, while SEQ ID NO: 157 contains an additional mutation at position 353, i.e. I353N. These mutations do not lead to an improved response to CR6261 and CR9114 in the FACS assay shown in FIGS. 21A-21B.
In certain embodiments, the polypeptides of the invention contain the intracellular sequences of HA and the transmembrane domain. In other embodiments, the cytoplasmic sequence and the transmembrane sequence are removed from positions (or the equivalent thereof) 523, 524, 525, 526, 526,
527, 528, 529, or 530 from HA2 to the C-terminus of HA2 (numbering according to SEQ ID NO: 1), and are optionally replaced by introduction of a sequence known to form trimeric structures, i.e. AYVRKDGEWVLL (SEQ
ID NO: 143), optionally connected via a connector. The connector may optionally contain a cleavage site for further processing according to protocols well known to those of skill in the art. To facilitate the purification of the soluble form a labeling sequence can be added, for example a His HHHHHHH label connected by means of a linker
<img file="MX357009B_D0198.tif" />
202 short, for example EGR. According to the present invention ,.
the amino acid sequence from position 530 (numbering according to SEQ ID NO: 1) to the amino acid of the C-terminus of the HA2 domain was removed and replaced by SEQ ID
NO: 81 or SEQ ID NO: 82.
Example 19: Immunogenicity of Third Generation HA Stem Domain Polypeptides
In order to assess the immunogenicity of the stem domain polypeptides, mice were immunized with the expression vectors encoding for full-length Hl from A / Brisbane / 59/2007 (SEQ ID NO: 1), Mini3grouping (SEQ ID NO : 11), Mini2-groupingoll + 5 (SEQ
ID NO: 14), mini2-grouping l + 5 + 6 (SEQ ID NO: 46), mini2-
<td>grouping + 5 + 6-GCN4</td><td>(I KNOW THAT</td><td colspan="2">ID NO: 45),</td><td>mini2-</td>
<td>grouping1 + 5 + 6-nl</td><td>(I KNOW THAT</td><td>ID</td><td>NO: 152),</td><td>mini2-</td>
<td>grouping l + 5 + 6-nl2</td><td>(I KNOW THAT</td><td>ID</td><td>NO: 153),</td><td>mini2-</td>
<td colspan="3">grouping l + 5 + 6-nl2s-GCN4 (SEQ</td><td>ID NO: 154),</td><td>mini2-</td>
<td>grouping l + 5 + 6-GCN4t2</td><td>(I KNOW THAT</td><td></td><td>ID NO: 51),</td><td>mini2-</td>
<td>grouping l + 5 + 6-GCN4t3</td><td>(I KNOW THAT</td><td></td><td>ID NO: 52),</td><td>mini2-</td>
<td>grouping l + 5 + 6 + 12 (</td><td colspan="2">; seq ID</td><td>NO: 156) and</td><td>mini2-</td>
<td>grouping l + 5 + 6 + 12 + 13</td><td>(SEQ ID</td><td>NOT:</td><td>157). I also know</td><td>included</td>
<td>an expression vector</td><td colspan="3">which encodes for cM2 as</td><td>control</td>
negative.
IMPÍ (* 5>
Mexican Institute of Property ¿UO INDUSTRIAL
Groups of 4 mice (BALB ^) - »^ oa ^ lXLQ«., Pg of construct + 100 pg of adjuvant (pUMCVl-GM-CSF) were immunized im on day 1, 21 and 42. On day 49 it was performed a final bleed and serum was collected. Sera were analyzed by ELISA using recombinant full-length HA from H1N1 strains.
A / Brisbane / 59/2007, H1N1 A / California / 07/2009 and H5N1
A / Vietnam / 1203/2004 (obtained from Protein Sciences
Corporation, Meriden, CT, USA) as an antigen. Briefly, 96-well plates were coated with 50 ng HA overnight at 4 ° C, followed by incubation with blocking buffer (100 µl PBS, pH 7.4 + 2% skim milk) for 1 hour at room temperature. . The plates were washed with PBS + 0.05% Tween-20, and added
100 pl from a series of 2-fold dilutions in blocking buffer, starting from a 50-fold dilution of the serum. Bound antibody was detected using HRP-conjugated goat anti-mouse IgG, using standard protocols well established in the art.
Titers were compared to a standard curve consisting of a serial dilution of a mouse monoclonal antibody that binds to the HA antigen and were expressed as units of
ELISA for me (EU / ml).
Figures 22A-22C show IgG responses at week 7 after initial immunization for mice.
IΜ Ρ1
<img file="MX357009B_D0199.tif" />
204 ”Individual tLA against the full length hemagiutinin of the homologous strain HlNl A / Brisbane / 59/2007 (panel A), the heterologous strain HlNl A / California / 07/2009 (panel B) and the heterosubtypical strain H5N1 A / Vietnam / 1203/2004 (panel C). Antibodies induced by immunization with DNA encoding the polypeptides of the invention Mini2grouping + 5 (SEQ ID NO: 14), mini2-grouping + 5 + 6 (SEQ ID NO: 46), mini2-grouping + 5 + 6- GCN4 (SEQ ID NO:
<td colspan="2">45), mini2-grouping l + 5 + 6-nl</td><td>(I KNOW THAT</td><td>ID NO</td><td> : 152),</td><td>mini2-</td>
<td>grouping l + 5 + 6-nl2</td><td colspan="2">(SEQ ID</td><td>NOT:</td><td> 153) ,</td><td>mini2-</td>
<td colspan="2">grouping l + 5 + 6-nl2s-GCN4 (SEQ</td><td>ID</td><td>NOT:</td><td> 154) ,</td><td>mini2-</td>
<td>grouping l + 5 + 6-GCN4t2</td><td>(I KNOW THAT</td><td>ID</td><td>NOT:</td><td> 51) ,</td><td>miní2-</td>
<td>grouping l + 5 + 6-GCN4t3</td><td>(I KNOW THAT</td><td>ID</td><td>NOT:</td><td> 52) ,</td><td>mini2-</td>
NOT:
156) mini2grouping l + 5 + 6 + 12 (SEQ ID clustering l + 5 + 6 + 12 + 13 (SEQ ID NO: 157) bind equally well to the ectodomain of hemagiutinin derived from the HlNl strain
A / Brisbane / 59/2007 homologated and HlNl A / California / Q7 / 2009 heterologous (Figure 22A and 22B). The highest titers are observed for mini2-grouping + 5 + 6-GCN4 (SEQ ID NO: 45), mini2-grouping + 5 + 6-GCN4t2 (SEQ ID NO: 51) and mini2-
<td>grouping l + 5 + 6-nl2S-GCN4</td><td>(I KNOW THAT</td><td>ID</td><td>NOT:</td><td> 154) .</td><td>By</td><td>the</td>
<td>contrary, immunization</td><td>with</td><td>DNA</td><td>than</td><td>encode</td><td>for</td><td>the</td>
<td colspan="2">full length protein</td><td>(I KNOW THAT</td><td>ID</td><td>NO: 1)</td><td colspan="2">give as</td>
high titers against homologous hemagiutinin resulted (more
IMPI
<img file="MX357009B_D0200.tif" />
205
MEXICAN INSTITUTE
Dt THE INDUSTRIAL PROPERTY of an order of magnitude greater than the titers oby e 1 v addu s for ·· immunization with DNA encoding the polypeptides of the invention), but low titers against the ectodomain of heterologous hemagglutinin (more than one order of magnitude). Immunization with DNA encoding Mini3-grouping (SEQ ID NO: 11) and cM2 negative control gives an undetectable response against any of the hemagglutinin ectodomains in this assay.
Titers against the ectodomain of heterosubtypical hemagglutinin from H5N1 A / Vietnam / 1203/2004 (Figure 22C) indicate a clear response for mini2-grouping + 5 + 6GCN4t2 (SEQ ID NO: 51). Observable titers are also obtained for 2 out of 4 mice after immunization with
DNA encoding the mini2-cluster + 5 + 6-GCN4 (SEQ ID
NO: 45) and for 1 of 4 mice for mini2-grouping + 5 + 6-nl (SEQ ID NO: 152), mini2-grouping + 5 + 6-nl2 (SEQ ID NO:
153), mini2-grouping l + 5 + 6-nl2s-GCN4 (SEQ ID NO: 154).
Surprisingly, the inventors also find that titers can be detected after immunization with DNA encoding Mini3-grouping (SEQ ID NO 11) and Mini2-grouping + 5 (SEQ ID NO: 14). The first construct did not induce any detectable homologous and heterologous Hl HA antibody titre, while the latter induced only moderate responses (Figures 22A and 22B). The
206
LA ΚHUI · ItUAi>
INDUSTRIAL comparison of the sequences of all constructs in_ this experiment and HA of H5 points to a putative linear epitope located at the distal end of the long CD helix membrane (see Figure 1). Mutation of methionine to isoleucine at position 175 in SEQ ID NO: 11 and at position 156 in SEQ ID NO: 14 results in the linear sequence ERRIENLNKK (position 172 to 181 in SEQ ID NO: 11;
position 153 to 162 in SEQ ID NO: 14). This sequence is also present in HA of H5N1 A / Vietnam / 1203/2004, but not in HA of HlNl A / Brisbane / 59/2007 and HlNl A / California / 07/2009, where the corresponding ERRMENLNKK and
EKRIENLNKK, respectively.
In conclusion, the antibodies directed against the
<td colspan="2">polypeptides of the invention</td><td>mini2</td><td colspan="2">-groupingoll + 5</td><td>(SEQ ID</td>
<td colspan="3">NO: 14), mini2-grouping l + 5 + 6</td><td>(I KNOW THAT</td><td>ID NO: 46)</td><td>, mini2-</td>
<td>grouping + 5 + 6-GCN4</td><td>(I KNOW THAT</td><td colspan="3">ID NO: 45),</td><td>mini2-</td>
<td>grouping l + 5 + 6-nl</td><td>(I KNOW THAT</td><td>ID</td><td>NOT</td><td> : 152),</td><td>mini2-</td>
<td>grouping l + 5 + 6-nl2</td><td>(I KNOW THAT</td><td>ID</td><td colspan="2">NO: 153),</td><td>mini2-</td>
<td colspan="2">grouping l + 5 + 6-nl2s-GCN4</td><td>(I KNOW THAT</td><td>ID</td><td>NO: 154),</td><td>mini2-</td>
<td>grouping l + 5 + 6-GCN4t3</td><td colspan="2">(I KNOW THAT</td><td>ID</td><td>NO: 52),</td><td>mini2-</td>
<td>grouping l + 5 + 6-GCN4t2</td><td colspan="2">(I KNOW THAT</td><td>ID</td><td>NO: 51),</td><td>mini2-</td>
<td>grouping l + 5 + 6 + 12</td><td>(I KNOW THAT</td><td>ID</td><td>NOT:</td><td>156) and</td><td>mini2-</td>
<td>grouping l + 5 + 6 + 12 + 13</td><td colspan="2">(SEQ ID NO:</td><td> 157)</td><td>they have the</td><td>capacity</td>
to recognize full length hemagglutinin. The
<img file="MX357009B_D0201.tif" />
207 epitopes of these antibodies must be located in the stem domain of hemagglutinin and are conserved among the full-length hemagglutinins of HlNl
A / Brisbane / 59/2007 and HlNl A / California / 07/2009. Antibodies elicited a response by immunization with
DNA encoding the mini2-cluster + 5 + 6-GCN4t2 (SEQ ID
NO: 51), and to a lesser extent mini2-groupingoll + 5 (SEQ ID NO:
14), mini2-grouping + 5 + 6-GCN4 (SEQ ID NO: 45), mini2-grouping + 5 + 6-nl (SEQ ID NO: 152), mini2-grouping + 5 + 6-nl2 (SEQ ID NO: 153), mini2groupling + 5 + 6-nl2s-GCN4 (SEQ ID NO: 154) and mini2grouping + 5 + 6 + 12 (SEQ ID NO: 155) also have the ability to recognize the HA ectodomain of H5N1
A / Vietnam / 1203/2004. The Mini3grouping polypeptide of the invention (SEQ ID NO: 11) has the ability to induce antibodies that recognize HA from H5N1 A / Vietnam / 1203/2004.
Example 20. General method for the design of stem domain polypeptides comprising conserved epitopes from the stem domain of CR6261 and CR9114.
Based on the results described above, a general method is defined for creating a polypeptide of the invention from an HAO sequence of influenza virus, in particular from a HAO sequence of influenza serotype
<img file="MX357009B_D0202.tif" />
208
ΙΜΡΪ
Hl. The method comprises the steps:
one. Elimination of the cleavage site between HAl and HA2. This can be done by mutation of R (in a small number of K cases) by Q at position Pl (see for example Sun et al., 2010 for an explanation of cleavage site nomenclature (position 343 in SEQ ID NO : 1) A mutation to Q is preferred but S, Τ, N, D or E are alternatives.
2. Elimination of the head domain by elimination of amino acids 53 to 320 of SEQ ID NO: 1, or at equivalent positions in HA of other influenza viruses. Equivalent positions can be easily determined by those skilled in the art by aligning the sequences using suitable algorithms such as for example Clustal or Muscle. The remaining parts of the sequence can be joined directly or alternatively by introducing a flexible connector. The linker sequences can be between 1 and 50 amino acids in length. Preferred flexible linkers are of limited length (less than or equal to 10 amino acids), for example GGG, GGGG, GSA, GSAG, GSAGSA, GSAGSAG or the like. The length of the deletion can also be varied, for example by starting the deletion (the equivalent of) at position 54, 55, 56, 57 or 58, or for
<img file="MX357009B_D0203.tif" />
209 increase the length of the deletion, by eerlte — at — iT position 47, 48, 49, 50, 51, or 52. Similarly, the last
<td>amino acid</td><td>to</td><td>To be removed</td><td>can be</td><td>in</td><td>the</td><td>(the</td>
<td>equivalent</td><td>of:</td><td>i position 315,</td><td>316, 317, 318 or</td><td> 319,</td><td>or</td><td>for</td>
<td colspan="2">increase the</td><td colspan="2">removal length</td><td>in</td><td>the</td><td>(the</td>
<td>equivalent</td><td>of</td><td>) position 321,</td><td> 322, 323, 324,</td><td>or</td><td> 325</td><td>Is</td>
It is important to realize that changes in the length of the deletion can be partly compensated by equalizing the length of the connector sequence, that is, a greater elimination can be equaled with a longer connector and vice versa. These polypeptides are also encompassed in the invention.
3. Increase the solubility of the loop (between the helix-A and the helix CD) formed by (the equivalent of) the residues
402 at 418 in Hl A / Brisbane / 59/2007 (SEQ ID NO: 1) to increase the stability of the prefusion conformation and destabilize the postfusion conformation of the modified HA. This loop is highly conserved in Hl sequences, as can be seen in Table 6 below. This for example can be achieved by replacing residues I, L, F or V in said loop by their hydrophilic counterparts. Equivalent positions can be easily determined by those of skill in the art by sequence alignment using a «ΛΤ4
<img file="MX357009B_D0204.tif" />
MEXICAN INSTITUTE OF PROPERTY
INDUSTRIAL
<img file="MX357009B_D0205.tif" />
210
<img file="MX357009B_D0206.tif" />
Glycine mutations destabilize the post-fusion conformation since the high flexibility of this amino acid leads to a decrease in the stability of the post-fusion helix to be formed by this part of the HA sequence. The consensus sequence describing the loop between residues 402-418 of influenza HA serotype Hl is (SEQ ID NO: 17) MNTQFTAVGKEFN (H / K) LE (K / R).
In the polypeptides of the invention the amino acid at positions 406, 409, 413 and / or 416 (or its equivalents, determined from a sequence alignment) is a polar amino acid (S, T, N, Q), charged ( R, H, K, D, E) or flexible (G). It should be noted that the L416 to S or T mutation also introduces an N-glycosylation consensus site (the consensus sequence is NX (S / T)). Glycosylation of asparagine in this position will further increase the solubility of this region. Combinations of mutations at these sites are also possible, for example F406S, V409T,
L416S as in SEQ ID NO: 10 and SEQ ID NO: 14. In some cases a mutation is preferred to restore the consensus amino acid, for example where V or M is at position 404 (a Τ), V at 408 (a A) or 410 (a G) heard at 414 (a N); the incidence of sequences with these particular amino acids is very low. Table 6 gives a summary
211
IMPI
MEXICAN INSTITUTE
- / 3 l> h THE MUiPIXDAÜ Ο "*. 5% '43 © Γ industrial of the mutations described above than the polypeptides of the invention.
Four. Introduce a disulfide bridge in the polypeptides of the invention, preferably between amino acids of the (the
Hl
436 in equivalent of) position 324 and
A / Brisbane / 59/2007; SEQ ID NO: 13-16. Equivalent positions can be readily determined by those skilled in the art by aligning the sequences using a suitable algorithm such as
Clustal, Muscle, etc. Disulfide bridges are created by mutation of at least one (if the other is already a cysteine), but usually two residues that are spatially close to a cysteine, which will spontaneously or by oxidation form a covalent bond between the sulfur atoms of these waste.
Using the general method according to the invention, described above, the polypeptides of the invention were created based on. the HAO sequences of H1N1
A / California / 04/2009 (SEQ ID NO:
A / California / 07/2009 (SEQ ID NO:
Rico / 8/1934 (SEQ ID NO: 78), and H1N1 A / Texas / 36/1991 (SEQ ID
NO: 64). Additionally, the method was applied to HA of another subtype that is part of group 1, that is, H5, using HA of
H5N1 A / Vietnam / 1203/2004 (SEQ ID NO: 158).
159), H1N1
56), H1N1 A / Port
INSTITIITO MEXICANO «T = SÍÍS Λ
OF THE I KOFIEilAD
212
INDUSTRIAL
Hl mini-HA A / California / 07/2009 (SEQ ID N0 -: - 1604 — is ... created.
as of Hl FL HA A / California / 07/2009 (SEQ ID NO: 56) by
one. Cleavage site deletion: R344Q mutation (numbering refers to SEQ ID NO: 56).
2. Elimination of K53 to P321 residues and introduction of a GGGG connector between D52 and K322 (the numbering refers to SEQ ID NO: 56).
3. Introduction of a serine residue in the position
407, 417 (F407S, L417S; numbering refers to SEQ ID NO: 2), Threonine at position
410 (V410T; numbering refers to SEQ ID
NO: 56) and a glycine residue at position 414 (F414G; numbering refers to SEQ ID NO:
2) in the loop between helix A and helix CD (residues 403-419 in SEQ ID: NO: 56).
Four. Introduce a disulfide bridge by mutation of residues Lysine 325 and Threonine 437 to cysteine (K325C, T437C; numbering refers to SEQ
ID NO: 56).
5. Introduce an additional stabilizing element by replacing 419KRIENLNKKVDDGFLD434 (the numbering refers to SEQ ID NO: 56) by the sequence
RMKQIEDKIEEIESKQ.
The mini-HA sequence based on the HA in length
213
<img file="MX357009B_D0207.tif" />
Complete A / California / 04/2009 (SEQ ID NO: 159) can be created in the same way and is identical to the Hl miniHA sequence A / California / 07/2009 (SEQ ID NO: 160).
Similarly, Hl mini-HA A / Puerto Rico / 8/1934 (SEQ ID: NO:
161) is created from Hl FL HA A / Puerto Rico / 8/1934 (SEQ ID NO: 78) by
one. Cleavage site deletion: R343Q mutation (numbering refers to SEQ ID NO: 78).
2. Elimination of residues S53 to P320 and introduction of a GGGG connector between D52 and K321 (the numbering refers to SEQ ID NO: 78).
3. Introduce a Serine residue at position 406,
416 (F406S, L416S; numbering refers to
SEQ ID NO: 78), Threonine at position 409 (V409T;
numbering refers to SEQ ID NO: 78) and a Glycine residue at position 413 (F413G; numbering refers to SEQ ID NO: 78) in the loop between helix A and helix CD (residues
402-418 in SEQ ID NO: 78)
Four. Introduce a disulfide bridge by mutation of residues Arginine 324 and Threonine 436 to a cysteine (R324C, T436C; numbering refers to SEQ
ID NO: 78).
5. An additional stabilizing element was introduced
<img file="MX357009B_D0208.tif" />
<img file="MX357009B_D0209.tif" />
214 by replacing 418KRMENLNNKVDDGFLD433 (the numbering refers to SEQ ID NO: 78) by the sequence RMKQIEDKIEEIESKQ.
Another difference between mini-HA Hl A / Puerto Rico / 8/1934 (SEQ ID: NO: 161) and HA Hl FL A / Puerto Rico / 8/1934 (SEQ ID NO:
78) is at position 397, which is a Serine in the full length protein (SEQ ID: NO: 78) but a Threonine in the polypeptide of the invention of SEQ ID: NO: 161 (mutation
S397T). This is a naturally occurring variation on the sequence of A / Puerto Rico / 8/1934, and therefore sequences containing this mutation are also included in the invention.
Hl mini-HA A / Texas / 36/1991 (SEQ ID NO: 162) is created from HA Hl FL A / Texas / 36/1991 (SEQ ID NO: 64) by
one. Cleavage site deletion: R344Q mutation (numbering refers to SEQ ID NO: 64).
2. Elimination of residues S53 to P321 and introduction of a GGGG connector between D52 and K322 (the numbering refers to SEQ ID NO: 64).
3. Introduction of a Serine residue in the position
407, 417 (F407S, L417S; numbering refers to SEQ ID NO: 64), Threonine at position 410 (V410T; numbering refers to SEQ IE NO: 64) and a Glycine residue at ϊ
Τ · \ F
1V1
ΡΙ
Saw
215
MEXICAN INSTITUTE OF THE ROPIEPAI. '
INDUSTRIAL position 414 (F414G; numbering refers.
to SEQ ID NO: 64) in the loop between helix A and helix CD (residues 403-419 in SEQ ID: NO: 64).
Four. Introduce a disulfide bridge by mutation of residues Arginine 325 and Threonine 437 to a cysteine (R325C, T437C; numbering refers to SEQ
ID NO: 64).
5. An additional stabilizing element was introduced by replacing 419RRMENLNKKVDDGFLD434 (the numbering refers to SEQ ID NO: 64) by the sequence RMKQIEDKIEEIESKQ.
H5 mini-HA A / Vietnam / 1203/2004 (SEQ ID NO: 163) is created from HA H5 FL A / Vietnam / 1203/2004 (SEQ ID NO: 158) by
one. Elimination of the cleavage site. Since HA of
H5 FL A / Vietnam / 1203/2004 (SEQ ID NO: 158) contains a polybasic cleavage site (341RRRKKR346). A single site mutation is not sufficient to prevent protein cleavage. Instead 341RRRKK345 is removed and a mutation is introduced
R346Q.
2. Removal of K52 to P319 residues and introduction of a GGGG connector between K51 and K320 (numbering refers to SEQ ID NO: 158).
3. Introduce a Serine residue at position 409,
<img file="MX357009B_D0210.tif" />
<img file="MX357009B_D0211.tif" />
216
419 (F409S, L419S; the numbering refers to _____
SEQ ID NO: 158), Threonine at position 412
<td></td><td>(V412T;</td><td>the</td><td>numeration</td><td>refers to SEQ ID</td><td>NOT:</td>
<td></td><td>158) and</td><td>a</td><td>residue of</td><td>Wisteria in position</td><td> 416</td>
<td> 5</td><td>(F416G;</td><td>the</td><td>numeration</td><td>refers to SEQ ID</td><td>NOT:</td>
<td></td><td>158) in</td><td>the</td><td colspan="2">loop between propeller A and propeller</td><td>CD</td>
(residues 405-421 in SEQ ID: NO: 158)
Four. Introduce a disulfide bridge by mutation of Lysine 323 and Threonine 439 residues to a cistern (K323C, T439C; numbering refers to SEQ
ID NO: 158).
5. A stabilizing element was introduced by replacing 421rrienlnkkmedgfldv437 (the numbering refers to SEQ ID NO: 158) by the sequence
RMKQIEDKIEEIESKQI.
Genes encoding the protein sequences of SEQ ID: NO: 56, 160, 78, 161, 162, 158 and 163 were synthesized and cloned into pcDNA2004 expression vector using methods generally known to those of skill in the art. For comparison reasons the H3 A / Hong Kong / 1/1968 full-length HA sequence (SEQ ID NO: 121), as well as the H1 A / Brisbane / 59/2007 full-length HA sequence ( SEQ ID NO: 1) with additional mutation of the R343Q cleavage site were included
217
Jfc MEXICAN INSTITUTE
OE LA »ROPIEDA! '<' ^ • τγμΓ *
INDUSTRIAL? B »in the experiment.
HEK293F cells in suspension (Invitrogen) were transfected (10<sup>6</sup> cells / ml, 30 ml) with expression vectors (1 pg / ml) using 40 µΐ of 293transfectin as the transfection agent and allowed to further propagate for 2 days. Cells were collected, aliquoted (0.3 ml, approximately 3 * 10<sup>5</sup> cells) and aliquots were treated with Hl HA-directed polyclonal serum (Sino Biological Inc. Beijing, China) to study expression or with a HA-specific monoclonal antibody (5 micrograms / ml) and a secondary antibody was used for staining. The cells were then analyzed by fluorescence associated cell selection (FACS) for expression of the membrane-bound HA stem domain polypeptides of the invention on the cell surface. A panel of monoclonal antibodies of known specificity binding to the stem domain in the full-length protein (CR6261, CR9114) was used to study the presence of conserved epitopes and, by inference, correct for the folding of full-length HA and the mini-HA polypeptides of the invention. Monoclonal antibodies CR8020 (known to not bind to Hl and H5 HA) and CR9020 (bind to HA head domain of Hl A / Brisbane / 59/2007) were also included in the experiment. The results are
<img file="MX357009B_D0212.tif" />
218
IMPI expressed as percentage of positive cells and ia-theasity-mean defluorescence (MFI) and are shown in FIG. 2. 3.
Treatment of the transfected cells with polyclonal anti-Hl serum results in between 20 and 80% full-length HA positive cells (solid bars) and between 40 and 50% mini-HA positive cells. FL H3 A / Hong Kong / 1/1968 and cM2 negative controls only show very low numbers of positive cells. This is reflected by the average fluorescence intensity (bottom panel) that clearly shows a signal that can be detected for all full-length Hl HA proteins. The signal for ia HA of full length of H5 remains low; however, this can be explained by a smaller number of transfected cells in combination with reduced recognition by polyclonal Hl serum. The negative controls FL A / Hong Kong / 1/1968 and cM2 show intensities the level of the background signal.
Both CR6261 and CR9114, known to be group 1 stem binders, recognize all full-length group 1 HA and mini-HA proteins as indicated by high numbers of positive cells (approximately 50 to approximately 95%) and high MFI . This is strong evidence that the neutralizing epitopes of these antibodies are in the mini-HA proteins, indicating a structure
IMPI
MEXICAN INSTITUTE »
Q of property O »<sub>W</sub>X3Í * · ^ industrial Yes »three-dimensional that closely resembles the native structure of the HA stem domain in full-length HA. As expected, the negative control CR8020 (specific for group 2 HA) does not bind to full-length Hl and H5 HA or mini-HA of Hl and H5, indicating that the observed binding of neutralizing antibodies CR6261 / CR9114 to mini-HA proteins does not arise from specific protein-protein interactions. The junction between HA of H3 full length
A / Hong Kong / 1/1968 and CR9114 or CR8020 clearly shows the percentage of positive cells and MFI, according to previous observations and that provides the functionality of these monoclonal antibodies. Similarly, the antibody
CR9020 negative control (HA head binder for
A / Brisbane / 59/2007) does not recognize full-length mini-HA or HA proteins, with the exception of HA from A / Brisbane / 59/2007, further highlighting the specificity of the binding observed between CR6261 and CR9114.
In conclusion, there have been four novel HA-derived polypeptides of the invention that have been shown to contain epitopes recognized by neutralizing antibodies
CR6261 and CR9114 in the absence of the HA head domain.
Example 21: Protection with the polypeptides of the invention against a lethal attack by influenza in mice
MEXICAN INSTITUTE, M
I 'E LA PR- IPIEDAD
INDUSTRIAL> Hn ** <“«!
220
In order to determine if the polypeptides <sup>1</sup> Of the invention they have the ability to induce an immune response that protects mice from death after exposure to the influenza virus that would otherwise be fatal, an influenza attack experiment was conducted. Mice were immunized with expression vectors encoding SEQ ID NO: 78, 161, 45 and 6, as well as full-length HA from A / Brisbane / 59/2007 (SEQ ID NO: 1) containing an additional R343Q mutation to remove the cleavage site. An expression vector encoding cM2 was included as a negative control. Immunization was performed using 50 pg of expression construct + 50 pg of adjuvant (pUMCVl-GM-CSF) according to the study protocol below:
<td>Protocol</td><td>study</td>
<td>Day 1</td><td>Bleeding</td>
<td>Day 0</td><td>Vaccine administration (im).</td>
<td>Day 21</td><td>Vaccine administration (im).</td>
<td>Day 28</td><td>Bleeding</td>
<td>Day 42</td><td>Vaccine administration (im).</td>
<td>Day 47</td><td>Bleeding</td>
<td>Day 48</td><td>Measurement of weight, temperature, classification</td>
and lethality.
221
IMPI
INSTITUTE MiXiCANf DE LA PROi'IEDAI
INDUSTRIAL
Day 49 Attack with lethal dose of infection — Jira! pop influenza (nasal route).
Day 49 The remaining inoculum retrotitling the virus.
used for weight, temperature,
Day 49-70 Daily measurement of clinical classification and case fatality.
Animals with clinical classification 3 are monitored twice a day.
Animals with clinical classification> 4 or temperature
32 ° C, whichever occurs first, are immediately removed from the study.
Day 70
Slaughter of all mice.
Group 1-6: Attack by PR8 (A / Puerto Rico8 / 34, H1N1)
<td> 15</td><td>Group Group</td><td> 1: 2:</td><td>I KNOW THAT I KNOW THAT</td><td>ID ID</td><td>NOT: NOT:</td><td> 78 161</td>
<td></td><td>Group</td><td> 3:</td><td>I KNOW THAT</td><td>ID</td><td>NOT:</td><td>1 R343Q</td>
<td></td><td>Group</td><td> 4:</td><td>I KNOW THAT</td><td>ID</td><td>NOT:</td><td> 45</td>
<td></td><td>Group</td><td> 5:</td><td>I KNOW THAT</td><td>ID</td><td>NOT:</td><td> 6</td>
<td> 20</td><td>Group</td><td> 6:</td><td colspan="2">vector</td><td colspan="2">empty</td>
mice per group. 60 mice in total. BALB / c.
Materials and methods:
Virus strain and source:
IMPI
222
MEXICAN INSTITUTE '· A PROPERTY'
INDUSTRIAL
The PR8 strain of the Influenza virus (A / Puerto Rico8 / 34, H1N1) was obtained from Virapur (San Diego). Stock solution lxl0e8 pfu / ml
Lot # E2004B.
Storage conditions. -75 ° C ± 10 ° C. Freezer: -86 ° C
UCT freezer. Thermo Form. Fisher Scientific.
Animals:
Mouse, BALB / c (Specific pathogen free; SPF), female.
Between 6 and 8 weeks of age per day of the study 0 approximately between 17 and 19 grams. They were obtained from Charles River Laboratories and identified with ear identification. All animals were acclimatized and kept for 11 days before the start of the experiment.
DNA administration
Inoculum reconstitution method
Appropriate DNA formulations were prepared, as listed above, aliquoted and stored at -20 ° C. To construct an aliquot, it was thawed at room temperature immediately prior to injection, loaded into a syringe, and injected. The remainder of each aliquot was discarded after finishing all injections from each immunization round.
<img file="MX357009B_D0213.tif" />
f ri
223 by injection (Graeub E Dr. AG
Dose level and method of administration
Mice were anesthetized intraperitoneally with 9.75mg of Xylasol (www.graeub.com); Cat: 763.02) and 48.75mg of Ketasol (Graeub E
Dr. AG (www.graeub.com); Cat: 668.51) per kg of body weight. 50 µΐ of DNA solution was injected using a 0.5 ml syringe with a G29 needle intramuscularly (im) into the quadriceps muscle of each hind paw, providing a total volume of 100 µΐ injected per mouse. The remainder of each aliquot was discarded after all injections from each immunization round were completed.
Virus administration:
Inoculum reconstitution method
Virus material was stored at -75 ° C ± 10 ° C and thawed before administration. Once thawed, the material was diluted in cold PBS (4 ° C) corresponding to 5 LD50 / 50 µΐ for attacks by A / PR / 8/34. The diluted virus was kept on ice until administration to the mice.
Dose level and method of administration
Animals were anesthetized by intraperitoneal injection with 9.75mg of Xylasol and 48.75mg of Ketasol
<img file="MX357009B_D0214.tif" />
224
<img file="MX357009B_D0215.tif" />
NSTIT1 'Τ <') MEXICAN DF LA PROmDAD
INDUSTRIAL per kg of body weight and each animal received 50 μΐ of virus solution intranasally. The unused material was returned to the laboratory for back titration.
Blood collection and serum preparation
On the days specified in the Study Protocol, above, blood samples were taken (intermediate bleeding: between 100 and 150 μΐ by means of retroorbital cannulation, terminal bleeding by means of cardiac puncture: approximately between 300 and 500 μΐ). Serum was isolated from this blood by centrifugation for 5 minutes at 14000g and stored at -20 ° C until shipped on dry ice.
Clinical classification
Clinical signs after virus attack were classified using a classification system (1 point for a healthy mouse; 2 points for a mouse showing signs of discomfort, including mild hairy erection, slightly modified gait, and increased ambulation; 3 dots for a mouse showing signs of strong hairy erection, contracted abdomen, altered gait, periods of inactivity, increased respiratory rate, and sometimes rales (clicking / sizzling noise); 4 points for a mouse with
<img file="MX357009B_D0216.tif" />
225
Τ
I,?;
~ Γκ «Τ - * Γ *
Mr
ΤίΤΙ-τα MEXICAN U? LA Ι'ΙλΉΕΙχΑΠ industrial enhanced characteristics of the group previq,. ^ - pass -— qtre show little activity and become dying; 5 points for a dead mouse). Animals were inspected twice a day while receiving a rating of 3. The rating was performed by a single investigator and the symptomatic mice partially represented by two ratings were rated +/- 0.5.
Determination of weight
All animals were weighed daily, beginning on day 48 (authorization number 2216). Animals were also weighed before the end of the study in the event of death, ie upon removal from the study. Body weight was recorded in grams (g)
Retyping of viruses
The dose of virus administered was determined by titration of 8 samples per replicate of the remaining inoculum after the inoculation of the animals was completed. For viral retrotitulation, the TCID50 measurement was used following the protocol detailed in Current Protocols in
Immunology, Animal Models of Infectious Disease 19.11.7.
<img file="MX357009B_D0217.tif" />
ΙΜΪ
226
INSTIT'JTl MEXICANI
DE IA ΡηΟΠΚΟΑΡ .. - INDUSTRIAL
Results:
The study was carried out without technical difficulties and according to the defined study protocol. Re-titration of the inoculums of the PR8 strain of the Influenza virus (A / Puerto
Rico8 / 34, HlNl) resulted in the following TCID50: PR8 (A / Puerto Rico8 / 34, HlNl): 3.2xl0<sup>4</sup> TCID50 / ml
Figure 24A shows the survival curves of
Kaplan-Meier for this experiment. Immunization with DNA encoding polypeptides of the invention SEQ ID NO: 45, and 161 results in a survival of 50, 40, and 40% of mice infected with a lethal dose of influenza, respectively, indicating that immunization with the polypeptides of the invention can indeed induce a protective immune response. In contrast, all animals immunized with the empty control vector succumbed to infection 8 days after the viral attack. The
<td>immunization</td><td>with</td><td>DNA</td><td>than</td><td colspan="2">code for</td><td>the HA of</td><td>length</td>
<td colspan="2">full homolog</td><td>to</td><td>strain</td><td>Of attack</td><td>(I KNOW THAT</td><td>ID NO: 78</td><td>) protects</td>
<td>completely</td><td colspan="2">everybody</td><td>the</td><td>animals</td><td>(is</td><td>say</td><td>100% of</td>
<td>survival)</td><td colspan="2">against</td><td>the</td><td colspan="2">lethal attack,</td><td>While</td><td>that</td>
<td>immunization</td><td>with</td><td>DNA</td><td>than</td><td>encode</td><td colspan="2">for HA's</td><td>length</td>
complete derived from heterologous strain A / Brisbane / 59/2007 (SEQ ID NO: 1) containing an additional cleavage site mutation (R343Q; numbering refers to SEQ ID
<img file="MX357009B_D0218.tif" />
227
ΙΜΡΪ
NO: 1) leads to 90% survival of infected _ animals.
The results obtained from the survival curves are also reflected in the mean change in body weight and median of clinical classifications for each group shown in Figures 24B and 24C. Animals immunized with the polypeptides of the invention SEQ ID NO: 45, 6 and 161 exhibit weight loss of up to 25-30%, but animals that survive after day 9 post-infection show an increase in weight. For animals immunized with SEQ ID NO: 45 a drop in clinical classification of 4 to 3 is also observed. Animals immunized with DNA coding for full-length HA homologous to the attack strain (SEQ ID NO:
78) show no weight loss, while animals immunized with DNA encoding full-length HA derived from the heterologous strain A / Brisbane / 59/2007 (SEQ ID NO:
1) containing an additional excision site mutation (R343Q; numbering refers to SEQ ID NO: 1) experience weight loss and clinical symptoms but survivors fully recover. Weight loss and the largest clinical symptoms are observed for the control group immunized with an empty vector, according to the lack of survival of these animals.
In conclusion, the polypeptides of. the invention SEQ ID
<img file="MX357009B_D0219.tif" />
228
<img file="MX357009B_D0220.tif" />
NO: 6, 45 and 78 have the ability to induce a response
<td>protective</td><td colspan="2">against an attack</td><td>lethal</td><td colspan="2">by H1N1 A / Port</td>
<td>Rico / 8/1934</td><td>in mice.</td><td>I know</td><td>should</td><td>mention that</td><td>the</td>
<td>polypeptides</td><td>of the invention</td><td>I KNOW THAT</td><td>ID NO:</td><td>6 and 45 derive from</td><td>a</td>
<td>5 molecule</td><td>HA heterologous of</td><td>the</td><td>strain of</td><td>attack while</td><td>than</td>
SEQ ID NO: 78 is derived from the homologous strain of influenza. Therefore, the polypeptides of the invention can induce protection against homologous and heterologous influenza infection.
Example 22. Selection of a panel of representative H1N1 HA sequences and design of polypeptides of the invention based on these sequences.
In order to show the wide applicability of the design method described in Example 20, the method was applied to a panel of selected HAO sequences that cover a large percentage of the natural variation found for H1N1 viruses. Selection of a panel of representative HA sequences from the set of known sequences of
<td>2 0 HA of H1N1 of</td><td>human in</td><td>this</td><td>example</td><td>has</td><td>by object</td>
<td>select a</td><td>number</td><td colspan="2">minimum of</td><td>strains</td><td>with a</td>
<td>representativeness</td><td>maximum.</td><td>For</td><td>achieve</td><td>this,</td><td>all</td>
<td>differences between</td><td colspan="2">the sequences</td><td>of HA of</td><td>virus</td><td>influenza</td>
Human H1N1 present in the Sequence Database
<img file="MX357009B_D0221.tif" />
<img file="MX357009B_D0222.tif" />
229 Influenza viruses have been quantified, the structure has been investigated in these differences and homogeneous subgroups have been identified. From each of these groups, the most representative sequence has been selected to contribute to the panel.
The main step in the procedure is the quantification of the difference between each pair or sequences in the sequence database considered. Matrix
Reverted PAM250 (rPAM250) (Xu, 2004) is used to quantify the difference at each amino acid position. Then the Euclidean addition is used to quantify the total difference for that pair. All differences between pairs are used to form a symmetric matrix of n * n of differences, where n is equal to the number of virus strains considered.
Principal Coordinate Analysis (PCA) is used to structure the difference matrix (Higgins, 1992). PCA is based on downsizing. The input matrix is considered a distribution in the dimensional space n (where n is equal to the number of strains considered). The variability is then analyzed and structured in such a way that a minimum dimensionality is required to cover most (or all) of the variability. The result is a dimensional coordinate system of m (where m is the number of dimensions to cover most or all of the ϊ Μ ΡI Mexican institute
PROE / EUAD fl » <sup>J</sup> ‘
INDUSTRIAL
<img file="MX357009B_D0223.tif" />
230 variability) with the greatest variation in the first axis and which then decreases. All the considered sequences are positioned in the coordinate system. In the case where only 2 or 3 dimensions are needed, the result can be completely plotted on a 2D or 3D graph, respectively, in which the difference between the strains can be visualized. In the case where more dimensions are needed, you can also build a 3D graph for the first 3 axes, but that graph does not cover all the variability, since part of the variation is in the fourth dimension and larger.
The sequences in the m dimensional space are then grouped, using hierarchical grouping and k-means. Group average indexing is used to obtain groups of similar internal variability, and to avoid a large proportion of single strain clusters. Clustering is done at all levels, starting at 1 (all strains in a cluster) to n (each strain forming its own). From each grouping the most central strain is selected as the most representative. The set of most central strains then forms the panel of representative strains for that level of grouping. For each grouping level, the coverage (or percentage of variation explained) is estimated by computing the sum of the square of the distances of each strain from its strain
ΙΜ
<img file="MX357009B_D0224.tif" />
231
MEXICAN INSTITUTE OF CENTRAL INDUST PROPERTY as a comparison to the sum of the squares of the distances of each strain from the center of the coordinate system.
A minimum level of coverage to be achieved is then adjusted to be the smallest required size of the representative panel.
In addition to the Xu rPAM250 matrix, small values were assigned for the difference when one of the two sequences had a mismatch at a certain position (due to insertions or deletions). A weighting factor was also included in the procedure, to take into account the large differences in the numbers of isolates over the years. This variation was considered as a partially true variation in occurrence, partially given by different levels of vigilance / knowledge. Therefore, the weighting factor was adjusted to one divided by the square root of the number of observations in a particular year. This weighting factor was taken into account when the dimensional space m was constructed, during the grouping analysis and selection of the central points, and in the estimation of the level of variation covered.
In this example, the constructed sequences are used, consisting of HA portions encoding the polypeptide of the invention. Two different sets of constructed sequences were created. In the first set the sequences
232
MEXICAN INSTITUTE <sub>x</sub> , -<sub>F</sub> i '-' THE PROn'OAD i NO<sup>1</sup>. ηττρ-ΐ a «.
natural except the signal sequence (for example-amino acidsf ·
1-18), amino acids 53 to 320 (the HA head domain), the transmembrane sequence (amino acid 530 to the C-terminal amino acid) (numbering refers to SEQ ID NO: 1) or the equivalents of these positions in other sequences They were considered. In the second set of amino acids at position (or equivalent position) 406, 409, 416, 324, 436,
413 Neither were they considered, since they are modified according to the general method described in Example 20.
Additionally, positions (equivalents of) 419-433 were also not considered in the second set reflecting the addition of a GCN4-based stabilization sequence in the polypeptides of the invention as described in Example 9.
Using the method described above, HA sequences were selected from set 1 of constructed sequences, covering 75% of the sequence variation and 8 HA sequences from set 2 of sequences constructed covering the
74% of the sequence variation. The strains are listed in Table 10. Three of the selected sequences appear in both sets, therefore 13 unique HA sequences remain. These sequences were used to design the polypeptides of the invention according to the method described in Example 20. Additionally, the GCN4 sequence is introduced.
IMPI
INSTITUTO MSXICANO ^ l'i
OF THE RRl'HSDAD '
INDUSTRIAL
233 stabilizer MKQIEDKIEEIESKQ (SEQ ID NO: —84 ~) -in eX equivalent of position 419-433 (numbering refers to SEQ ID NO: 1), as described in Example 9.
The designed polypeptides of the invention based on the HA sequences of H1N1 A / Memphis / 20/1978 and H1N1
A / USSR / 92/1977 are identical, as are the designed polypeptides of the invention based on the HA sequences of
A / Wisconsin / 629-D01415 / 2009 and H1N1 A / Sydney / DD3-55 / 2010. Thus a total of 10 unique polypeptides of the invention were designed, and an alignment of these sequences is shown in Figure 25.
Expression vectors containing DNA encoding the polypeptides of the invention SEQ ID NO: 154 to SEQ ID NO: 173, as well as the polypeptide of the invention SEQ ID NO: 45 based on the HA sequences of
A / Brisbane / 59/2007 and the corresponding full-length HA SEQ ID NO: 1 with additional R343Q mutation of the cleavage site in the expression vector pcDNA2004 were used for transfection of HEK293F cells and the cells were analyzed by FACS as above . In addition to human monoclonal antibodies CR6261, CR9114, and CR8020, the mouse monoclonal antibody C179 which is known to neutralize the Hl and H2 strains of
Influenza A (Okuna et al., 1993). The results are shown
ΙΜΡΪ
0 ^ 4 MEXICAN INSTITUTE
OF THE PROPERTY
INDUSTRIAL in Figure 26.
<img file="MX357009B_D0225.tif" />
<td>Everybody</td><td>the</td><td>polypeptides</td><td>of</td><td>the</td><td>invention as well as</td><td>the</td>
<td>sequence</td><td>of</td><td colspan="2">full length</td><td>of</td><td>A / Brisbane / 59/2007</td><td>I know</td>
<td colspan="2">express about</td><td>the surface</td><td colspan="2">mobile</td><td>and they are recognized</td><td>by</td>
the broadly neutralizing antibodies CR6261, CR9114 and
C179, but not by CR8020. The latter is known to bind only to Influenza A group 2 HA. Binding of antibodies CR6261, CR9114 and C179 indicates that broadly neutralizing epitopes are well preserved in the polypeptides of the invention. Considering the sequence variation covered in these sequences is clear evidence of the general applicability of the inventors' design method to generate widely neutralizing epitopes-containing polypeptides of the invention.
Example 23. Characterization of the polypeptide of the invention s-Hl-mini2-grouping + 5 + 6-GCN4 (SEQ ID NO: 145)
The polypeptide of the invention s-Hl-mini2 clustered + 5 + 6-GCN4 purified (SEQ ID NO: 145) was obtained as described in Example 13. To confirm the presence of the conformational epitopes of CR6261 and CR9114, binding was studied of these antibodies with the protein purified by bilayer interferometry (Octet Red<sup>384</sup> , Forte Bio). For this purpose CR6261, CR9114 and CR8020 were immobilized
<img file="MX357009B_D0226.tif" />
235 Biotinylated on streptavidin-coated sensors, the sensors were exposed first to a solution of the purified polypeptide (250 nM) of the invention to measure the rate of association and then to a wash solution to measure the rate of dissociation. For comparison reasons the experiment was repeated with the full length protein (SEQ ID NO 14 9) in its trimeric and monomeric forms. The results are shown in Figures 27A-27C.
Immobilized CR6261 recognizes the monomeric and trimeric forms of the full-length HA ectodomain of H1N1
A / Brisbane / 59/2007 as evidenced by the clear responses after exposure to these proteins in solution (Figure
27A). The observed response for the trimeric protein is greater than that observed for the monomer (approximately 1.3 nm vs. 0.9) with the same sequence, an effect that is caused (at least in part) by the smaller size of the monomer compared to the trimer. Binding of CR6261 to s-Hl-mini2 clustering + 5 + 6-GCN4 (SEQ ID NO: 145) results in a maximum response of approximately 0.25 nm. Upon exposure to the wash solution, the dissociation of the complex for the three analytes is observed with the fastest release observed for the polypeptide of the invention s-Hlmini2-grouping + 5 + 6-GCN4 (SEQ ID NO: 145), and the smallest for the trimeric form of the ectodomain of HA of length
236
IMri
MEXICAN INSTITUTE L'E LA PRCll'IEDAI l INDUSTRIAL complete of H1N1 A / Brisbane / 59/2007 (SEQ ID NO L43X .________
The immobilized CR9114, similar to CR6261, also recognizes the trimeric and monomeric forms of the full-length HA ectodomain of H1N1 A / Brisbane / 59/2007, as well as the polypeptide of the invention s-Hl-mini2agrupamientol + 5 + 6- GCN4 (SEQ ID NO: 145). The response is stronger for all three analytes compared to CR6261 (1.5,
1.4 and 0.8 nm for monomeric, monomeric (SEQ ID NO: 149) full-length HA and stem domain polypeptide of s-Hl-mini2-clustering + 5 + 6-GCN4 (SEQ ID NO: 145), respectively) and With exposure of the complex to the wash buffer, antigen release is minimal or cannot be detected in any of the three cases. For CR8020, no responses were observed for any of the analytes, according to the specificity of the stem domain of influenza group 2 of this antibody.
To further characterize the binding of CR6261 and
CR9114 to the purified stem domain polypeptide was titrated. To this end, sensors containing immobilized CR6261 were exposed to solutions of s-Hl-mini2 clustering + 5 + 6-GCN4 (SEQ ID NO: 145) at concentrations of
500, 250, 125, 63, 31, 16, and 8 nM, respectively, and the final response was recorded after 14000 s. The responses were plotted as a function of the concentration of the
<img file="MX357009B_D0227.tif" />
237
ΙΜΡΙ
Stem domain polypeptide, MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY, and a 1: 1 union model was performed in the steady state, generating a dissociation constant K<sub>d</sub> approximately 190 nM for the CR6261 / stem domain polypeptide complex, FIG 28A.
Similarly, the modified sensors with the immobilized CR9114 were exposed to s-Hl-mini2-cluster + 5 + 6GCN4 (SEQ ID NO: 145) at concentrations of 80, 40, 20, 10, 5,
2.5 and 1.3 nM, respectively, and the final response was recorded after 10800 s. The adjustment of the final responses as a function of the concentration of the stem domain polypeptide generates a K value.<sub>d</sub> 5.4 nM for the CR9114 / stem domain polypeptide complex (FIG. 28B).
In conclusion, the polypeptide of the invention s-Hlmini2-grouping + 5 + 6-GCN4 (SEQ ID NO: 145) has the ability to bind monoclonal antibodies CR6261 and
CR9114 broadly neutralizing, confirming the presence of the corresponding neutralizing epitopes in this stem domain polypeptide.
Example 24: Protection with the polypeptides of the invention against a lethal attack by influenza in mice
In order to determine if the polypeptides of the invention have the ability to induce an immune response that protects mice from death by a
ΙΜΡΪ
<img file="MX357009B_D0228.tif" />
238 Exposure to influenza virus that could otherwise be fatal, an influenza attack experiment was conducted. Mice were immunized im with expression vectors encoding for full-length H3 A / Hong Kong / 1/1968 5 (SEQ ID NO: 121), HK68 H3m2-cl9 + 10 + ll (SEQ ID NO: 124) and HK68
H3m2-cl9 + 10 + ll + 12-GCN4 SEQ ID NO: 130. Immunization was performed using 50 pg of expression construct + 50 pg of adjuvant (pUMCVl-GM-CSF) according to the study protocol below
Study protocol
Day -1 Bleeding.
Day 0 Vaccine administration (im)
Day 21 Vaccine administration (im).
Day 28 Bleeding.
Day 42 Vaccine administration (im).
Day 47 Bleeding.
Day 48 Measurement of weight, temperature, clinical classification and case fatality.
Day 49 Attack with lethal dose of viral influenza infection (nasal route).
Day 49 The remaining inoculum is used for virus re-titration.
Day 49-70
Daily measurement of weight, temperature,
239 'Ί PI
MEXICAN INSTITUTE Λ
OE LA PROriECAO
INDUSTRIAL ^ »- a clinical classification and case fatality.
Animals with clinical classification 3 monitored twice a day.
Animals with clinical classification á4 or temperature 32 ° C, whichever occurs first, are immediately withdrawn from the study.
Day 70 Slaughter of all mice.
I know
Group 7-10: Attack by HK68 (A / Hong Kong / 1/68, H3N2)
<td>10 Group</td><td> 7:</td><td>I KNOW THAT</td><td>ID</td><td>NOT:</td><td> 121</td>
<td>Group</td><td> 8:</td><td>I KNOW THAT</td><td>ID</td><td>NOT:</td><td> 130</td>
<td>Group</td><td> 9:</td><td>I KNOW THAT</td><td>ID</td><td>NOT:</td><td> 124</td>
Group 10: empty vector mice per group. Total 40 mice. BALB / c.
Materials and methods:
Strain and source of virus:
Influenza virus strain HK68 (A / Hong Kong / 1/68) was provided by Prof J. Katz (Center for Disease Control and
Prevention, Atlanta, GA, USA) followed by spread by
Virapur (San Diego). The virus has undergone multiple passages in mouse lungs to enhance virulence in mice.
A suitable reference for this virus is: Frace et al., Vacuna 1999; 17: 2237. 3xl0e stock solution<sup>8</sup> pfu / ml. Lot
<img file="MX357009B_D0229.tif" />
240
<img file="MX357009B_D0230.tif" />
<img file="MX357009B_D0231.tif" />
# F1109A.
Storage conditions. -75 ° C ± 10 ° C
UCT freezer. Thermo Form. Fisher Scientific.
Freezer: -86 ° C
Animals:
Mouse, BALB / c (Specific pathogen free; SPF), female. Between 6 and 8 weeks of age on Day 0 approximately between 17 and 19 grams. Obtained from Charles River
Laboratories and were identified with identification on ears. All animals were acclimatized and kept for 11 days before the start of the experiment.
DNA administration
Inoculum reconstitution method
Appropriate DNA formulations were prepared, as listed above, aliquoted and stored at -20 ° C. To construct an aliquot, it was thawed at room temperature immediately prior to injection, loaded into a syringe, and injected. The remainder of each aliquot was discarded after finishing all injections from each immunization round.
Dose level and method of administration
Mice were anesthetized by injection
241 intraperitoneal with 9.75mg of Xylasol (Graeub E Dr. AG (www.graeub.com); Cat: 763.02) and 48.75mg of Ketasol (Graeub E
Dr. AG (www.graeub.com); Cat: 668.51) per kg of body weight. 50 µΐ of DNA solution was injected using a 0.5 ml syringe with a G29 needle intramuscularly (im) into the quadriceps muscle of each hind paw, providing a total volume of 100 µΐ injected per mouse. The remainder of each aliquot was discarded after all injections from each immunization round were completed.
Virus administration:
Inoculum reconstitution method
Virus material was stored at -75 ° C ± 10 ° C and thawed before administration. Once thawed, the material was diluted in cold PBS (4 ° C) corresponding to 10
LD50 / 50 μΐ for attacks by A / HK / 1/68. The diluted virus was kept on ice until administration to the mice.
Dose level and method of administration
Animals were anesthetized by intraperitoneal injection with 9.75mg of Xylasol and 48.75mg of Ketasol per kg of body weight and each animal received 50 µΐ of virus solution intranasally. The unused material was returned to the laboratory for back titration.
242
IMPIAS
MEXICAN INSTITUTE OF PROPERTY
INDUSTRIAL
Blood collection and serum preparation
On the days specified in the Study Protocol, above, blood samples were taken (intermediate bleeding: between 100 and 150 μΐ through retroorbital cannulation, terminal bleeding through cardiac puncture: approximately between 300 and 500 μ y). Serum was isolated from this blood by centrifugation for 5 minutes at 14000g and stored at -20 ° C until shipped on dry ice.
Clinical classification
Clinical signs after virus attack were classified using a classification system (1 point for a healthy mouse; 2 points for a mouse showing signs of discomfort, including mild hairy erection, slightly modified gait, and increased ambulation; 3 dots for a mouse showing signs of strong hairy erection, contracted abdomen, altered gait, periods of inactivity, increased respiratory rate, and sometimes rales (clicking / sizzling noise); 4 points for a mouse with enhanced characteristics from the previous group, but show little activity and become dying; 5 points
<img file="MX357009B_D0232.tif" />
243 for a dead mouse). Animals were inspected twice a day while receiving a rating of 3. The rating was performed by a single investigator and the symptomatic mice partially represented by two ratings were rated +/- 0.5.
Determination of weight
All animals were weighed daily, beginning on day 48 (authorization number 2216). Animals were also weighed before the end of the study in the event of death, ie upon removal from the study. Body weight was recorded in grams (g)
Retyping of viruses
The dose of virus administered was determined by titration of 8 samples per replicate of the remaining inoculum after the inoculation of the animals was completed. For viral retrotitulation, the TCID50 measurement was used following the protocol detailed in Current Protocols in
Immunology, Animal Models of Infectious Disease 19.11.7.
Results:
The study was carried out without technical difficulties and according to the defined study protocol. Re-titration
244
IMPÍ ^^
MEXICAN INSTITUTE
OF THE PROPERTY '' '
INDUSTRIAL inoculum of Influent virus strain HK68, (A / .Hong
Kong / 1/68, H3N2) resulted in the following TCID50: HK68 (A / Hong Kong / 1/68): lxlO<sup>3</sup> TCID50 / ml.
Figure 29 shows the IgG response against the HA / Hong Kong / 1/1968 HA ectodomain at day 49 determined by ELISA. Immunization with DNA encoding the polypeptides of the invention SEQ ID NO: 124 and SEQ ID NO: 130 induces a response that is clearly detectable against that of the H3 HK68 HA ectodomain, while no response is detected for the vector negative control vacuum. As expected, the largest responses are seen for immunization with full-length H3 A / Hong Kong / 1/1968 (SEQ ID NO: 121).
Figure 30A shows the survival curves of
Kaplan-Meier for this experiment. Immunization with DNA encoding the polypeptides of the invention SEQ ID NO:
124 and 130 results in the survival of 40 and 20% of mice infected with a lethal dose of influenza, respectively, indicating that immunization with the 20 polypeptides of the invention can indeed induce a protective immune response. In contrast, all animals immunized with the empty control vector succumbed to infection 10 days after the viral attack. Immunization with DNA encoding for HA from
245
IMPI
INSTITUTO MEXICANO full length (SEQ ID NO: 121) fully protects all animals (i.e. 100% survival) against lethal attack.
The results obtained from the survival curves are also reflected in the mean change in body weight and median of clinical classifications for each group shown in Figures 30B and 30C. Animals immunized with the polypeptides of the invention SEQ ID NO: 124 and 130 exhibit weight loss of up to 20%, but animals that survive after day 9 post-infection show an increase in weight.
Animals immunized with DNA coding for full-length HA (SEQ ID NO: 121) show no loss of body weight. Weight loss and major clinical symptoms were observed for the control group immunized with an empty vector, coinciding with the lack of survival of these animals.
In conclusion, the polypeptides of the invention SEQ ID
NO: 124 and 130 are immunogenic and have the ability to induce a protective response against a lethal attack by
H3N2 A / Hong Kong / 1/1968 in mice.
Example 25. Design and characterization of H3 HA-based stem domain polypeptides
<img file="MX357009B_D0233.tif" />
246
<img file="MX357009B_D0234.tif" />
MEXICAN INSTITUTE OF HICHirCAD industrial
To further enhance the stem domain polypeptides described in Example 12, an additional set of constructs was designed. Additional sets of cysteine mutations were designed to allow stabilizing disulfide bridges to form at position 53 and 334 (T53C, G334C; cluster 16) and position 39 and 51 (G39C-E51C;
grouping 17) (numbering refers to SEQ ID NO:
121). Furthermore, two sequences to be inserted between position 420-421, that is to say on the N-terminal side of the helix
Long CD (see Figure 1). The insertion sequences have been designed so as to facilitate the formation of intermonomeric disulfide bridges between individual monomers in the trimeric molecule. Two different sequences have been designed, ie NATGGCCGG (Cluster 18) and GSGKCCGG (Cluster 19). The sequence in cluster 18 also comprises a sequence that introduces a glycosylation site (ie NAT) into the stem domain polypeptide. In some cases a glycosylation site is also introduced at position 417-419 by mutation to NAT.
Using the A / Hong full-length HA sequence
Kong / 1/1968 as a starting point, the modifications described above were combined with the elimination of
S62-P322 to obtain the following stem domain polypeptides:
<img file="MX357009B_D0235.tif" />
247
<img file="MX357009B_D0236.tif" />
MEXICAN INSTITUTE OF PROPERTY
INDUSTRIAL
SEQ ID NO: 174: H3 HK mini2a-connector + cl9 + 10 + 11 + 12 + GCN4T-CG7-1_
SEQ ID NO: 175: HK68 H3mini2a-connector + cl9_ + 10 + 12 + 18 + GCN4T
SEQ ID NO: 176: HK68 H3mini2a-connector + cl9_ + 10 + 12 + 16 + CG7-GCN4T
SEQ ID NO: 177: HK68 H3mini2a-connector + cl9_ + 10 + 12 + 19 + GCN4T
SEQ ID NO: 178: HK68 H3mini2a-connector + cl9_ + 10 + 12 + 17 + CG7-GCN4T
SEQ ID NO: 179: H3 HK68 mini2a-connector2 + cl9_ + 10 + 12 + GCN4T
Genes encoding the protein sequences described above were synthesized and cloned into the pcDNA2004 expression vector using methods generally known in the art. Cell surface expression and monoclonal antibody binding were analyzed by fluorescence associated cell selection as described above. For comparison, H3N2 full-length HA was also included in the experiment.
A / Hong Kong / 1/1968 (SEQ ID NO: 121), which additionally contains an R345Q mutation at the cleavage site, and
SEQ ID NO: 130 (HK68 H3m2-cl9 + 10 + ll + 12-GCN4), as well as the negative control cM2.
Figure 31 shows the results of this experiment.
and
All constructs are expressed on the cell surface * as evidenced by the responses (MFI, panel A;
percentage of positive cells, panel B) observed for polyclonal anti-H3 serum. The binding of CR8043 and CR8020 is observed for SEQ ID NO: 174, 175, 176, 177, 178, 179, 121 and
<img file="MX357009B_D0237.tif" />
248
<img file="MX357009B_D0238.tif" />
130, indicating that mutations in<img file="MX357009B_D0239.tif" /> They contribute to stabilize the conformational epitopes of these antibodies. For the CR9114 mAb binding above the background signal can only be observed for SEQ ID NO: 174: H3 HK mini2a-connector + cl9 + 10 + 11 + 12 + GCN4T-CG7-1 and to a lesser extent SEQ ID NO : 177: HK68 H3mini2acnector + cl9_ + 10 + 12 + 17 + CG7-GCN4T. Both sequences contain an additional glycosylation site in loop B, indicating the stabilizing effect of this modification on the conformational neutralizing epitope of CR9114.
In conclusion the inventors have shown that by following the method described above, the stem domain polypeptides of the invention can be obtained for serotypes of group 2, in particular H3 subtypes. Further stabilization of these stem domain polypeptides can be accomplished by introducing a glycosylation site into loop B. These sequences are also encompassed by the invention.
Example 26: Immunogenicity of H3 HA-based HA stem domain polypeptides
In order to assess the immunogenicity of stem domain polypeptides, mice were immunized with expression vectors encoding for H3 length
249
MIXICAN INSTITUTE D7 LA rROHlUAD
Complete OIISTRIAL IN A / Wisconsin / 67/2005 (SEQ ID NO: 8 9), ._.,. SEQ _.... IJQ, .. JiQp_
105: H3-mini2, SEQ ID NO: 108: H3-mini2-cl9 + 10 + ll, SEQ ID NO:
112: H3-mini2-cl9 + 10 + 12, SEQ ID NO: lll: H3-mini2C19 + 10 + 11 + 12, SEQ ID NO: 114: H3-mini2-cl9 + 10 + ll + 12-tri, SEQ
ID NO: 113: H3-mini2-cl9 + 10 + ll + 12-GCN4, SEQ ID NO: 119: H3mini3-cl9 + 10 + ll + 12 + 14, SEQ ID NO: 120: H3-mini4cl9 + 10 + ll + 12 + 14. An expression vector encoding cM2 was also included as a negative control.
Groups of 4 mice (BALB \ c) were immunized with 50 pg of construct + 50 pg of adjuvant (pUMCVl-GM-CSF) im on day 1, 21 and 42. On day 49 a final bleed was performed and collected serum. A negative control cM2 plasmid was administered with a gene gun, using approximately 10 pg of construct + approximately 2 pg of adjuvant (pUMCVl15 GM-CSF) and the same immunization schedule. Sera were analyzed by ELISA using recombinant full-length HA from A / Wisconsin / 67/2005 and A / Hong Kong / 1/1968 (obtained from Protein Sciences Corporation, Meriden, CT,
USA) as the antigen. Briefly, 96-well plates were coated with 50 ng HA overnight at 4 ° C, followed by incubation with blocking buffer (100 µl PBS, pH 7.4 + 2% skim milk) for 1 hour at room temperature. . The plates were washed with PBS + 0.05% Tween-20, and 100 pl of a series were added
250
<img file="MX357009B_D0240.tif" />
2-fold dilutions in blocking buffer, starting from a 50-fold dilution of the serum. Bound antibody was detected using HRP-conjugated goat anti-mouse IgG, using standard protocols well established in the art. Titers were compared to a standard curve consisting of a serial dilution of a mouse monoclonal antibody that binds to the HA antigen and were expressed as ELISA units per ml (EU / ml).
ELISA results using HA from
A / Wisconsin / 67/2005, A / Hong Kong / 1/1968 and A / Perth / 16/2009 after 49 days are shown in Figures 32A, 32B and 32C, respectively. Sera obtained from mice immunized with DNA encoding the stem domain polypeptides included in this experiment have the ability to recognize full-length homologous HA from
A / Wisconsin / 67/2005 and to a similar extent the full-length heterologous HA from A / Hong Kong / 1/1968. In contrast, sera obtained from mice immunized with the full-length HA HA from A / Wisconsin / 67/2005 (SEQ ID NO: 89) show a greater response to homologous HA than to heterologous HA from A / Hong Kong / 1/1968 and A / Perth / 16/2009.
In conclusion, the data shows that the polypeptides of the invention derived from HA from H3 have the ability to induce an immune response directed towards HA from
<img file="MX357009B_D0241.tif" />
251
ΜΡΪ
ILSTITliT ·· »MEXICAN DE LA FROí'IEDaP industrial full length.
Example 27: Immunogenicity of A / Hong Kong / 1/1968 H3 HA-based HA stem domain polypeptides
In order to assess the immunogenicity of stem domain polypeptides, mice were immunized with expression vectors encoding for full length H3 from A / Hong Kong / 1/1968 (SEQ ID NO: 121), SEQ ID NO :
124: HK68 H3m2-cl9 + 10 + ll, SEQ ID NO: 125: HK68 H3m2C19 + 10 + 12, SEQ ID NO: 126: HK68 H3m2-cl9 + 10 + ll + 12, SEQ ID NO:
128: HK68 H3m2-cl9 + 10 + ll + 12-tri, SEQ ID NO: 130: HK68 H3m2cl9 + 10 + ll + 12-GCN4. An expression vector encoding cM2 was also included as a negative control.
Groups of 4 mice (BALB \ c) were immunized with 100 pg of construct + 100 pg of adjuvant (pUMCVl-GM-CSF) im on day 1, 21 and 42. On day 49 final bleeding was performed serum was collected . Negative control plasmid of cM2 was administered with a gene gun, using approximately 10 pg of construct + approximately 2 pg of adjuvant (pUMCVl-GMCSF) and the same immunization schedule. Sera were analyzed by ELISA using recombinant full-length Ha from A / Hong Kong / 1/1968 (obtained from Protein
Sciences Corporation, Meriden, CT, USA) as the antigen.
Briefly, 96-well plates were coated with 50 ng of
<img file="MX357009B_D0242.tif" />
252 ί Μ Ρ ί
MEXICAN INSTITUTE (> industrial THE INDUSTRIAL PROPERTY
HA overnight at 4 ° C, followed by incubation D_. With blocking buffer (100 μΐ PBS, pH 7.4 +
2% skim milk) for 1 hour at room temperature. The plates were washed with PBS + 0.05% Tween-20, and 100 µΐ of a series of 2-fold dilutions in buffer buffer was added, starting from a 50-fold dilution of the serum. Bound antibody was detected using HRP-conjugated goat anti-mouse IgG, using standard protocols well established in the art.
Titers were compared to a standard curve consisting of a serial dilution of a mouse monoclonal antibody that binds to the HA antigen and were expressed as ELISA units per ml (EU / ml). ELISA results using HA from A / Hong Kong / 1/1968 after 49 days are shown in the
Figure 33. Sera obtained from mice immunized with DNA encoding the stem domain polypeptides included in this experiment have the ability to recognize the full-length homologous HA from A / Hong Kong / 1/1968. As expected, immunization with DNA encoding full-length HA leads to high antibody titers against the homologous HA protein, while immunization with the negative control expression vector encoding cM2 does not induce antibodies that recognize HA full length A / Hong
IMPI
253
MEXICAN INSTITUTE OF PROPERTY
INDUSTRIAL
<img file="MX357009B_D0243.tif" />
Kong / 1/1968.
In conclusion, the data shows that the polypeptides of the invention derived from H3 HA have the ability to induce a targeted immune response against full-length HA from H3.
Example 28: Design of another stem domain polypeptide based on an HA of Hl with the capacity to produce antibodies that neutralize group 1 and group 2 influenza viruses
Examples 4 and 6 describe Hl sequence-based polypeptides that stably expose the epitope of the broadly neutralizing antibody CR6261. Since CR6261 exclusively neutralizes influenza viruses of phylogenetic group 1, polypeptides designed for this epitope may not produce a strong reaction against influenza viruses of phylogenetic group 2. Another way to design polypeptides according to the invention that induce such antibodies with broad cross-neutralizing action is to use Hl HA sequence variants that are more closely similar to H3 HA sequences in terms of structural and biochemical characteristics of amino acids. important in the epitope. Based on comparison between group specific antibody and molecule structures (CR6261, FIO and HB36) and FI6 antibody
254
<img file="MX357009B_D0244.tif" />
MEXICAN INSTITUTE OF PROPERTY
INDUSTRIAL
<img file="MX357009B_D0245.tif" />
pan-influenza crystallized (Corti et al., 20111 -, - L&& inventors found that the group T4 9N (HA2) mutation
1-group 2 can be accommodated only by FI6 without the introduction of spherical impediments. Asparagine at position 49 of HA2 exists in two group 1 viruses in the NCBI influenza database: A / pig / Hubei / Sl / 2009 (ACY06623) and
A / pig / Haseluenne / IDT2617 / 2003 (ABV60697). Therefore, in one embodiment the Hl sequences that form the basis of the invention as described in Examples 4, 6 and 9 is one of these N4-containing HA sequences 9. Alternatively, the sequences according to the invention as described in Examples 4, 6 and 9 have an additional mutation at position 4 9 in HA2 to change amino acid T to N.
Table 7 shows a sequence alignment of Hl HA exemplary sequences that can be used as starting sequences for the polypeptides of the invention.
SEQ ID NO: 180 is derived from SEQ ID NO: 45 by the T392N mutation (numbering refers to SEQ ID NO:
one) . T392 in SEQ ID NO: 1 corresponds to Threonine at position 49 in HA2 as described above. The gene that codes for this polypeptide. of the invention was synthesized and cloned into a pcDNA2004 expression vector using methods well known to those of skill in the art. The presence of neutralizing epitopes of CR9114 and
255
IMPIOUS"
MEXICAN INSTITUTE. Z, r-.r) THE PROHEUAL '
--------.... ν<sub>4></sub>.<sub>Λ</sub> λ, Α ”·· INDUSTRIAL '· *» ·
CR6261 was confirmed by fluorescence associated cell selection as described above. The results are shown in Figure 34. The MFI for SEQ ID NO: 180 is comparable to the observed MFI for SEQ ID NO: 45 and SEQ ID
NO: 1 for binding of CR6261 and CR9114, while CR9020 (known to bind to the head region of the full-length HA molecule) only recognizes SEQ ID NO: 1, and CR8020 (HA-specific for Influenza A from group 2) does not recognize
SEQ ID NO: 180, SEQ ID NO: 1 or SEQ ID NO: 45. The cM2 negative control is not recognized by any of the monoclonal antibodies used in this experiment.
In conclusion, SEQ ID NO: 180, which contains the T392N mutation, comprises the neutralizing epitopes of
CR6261 and CR9114.
Example 29. Design of additional polypeptides of the invention lacking the transmembrane sequence.
Influenza HA in its native form exists as a trimer in the cell or virus membrane. In certain modalities the intracellular and transmembrane sequence is removed so that a secreted (soluble) polypeptide is produced upon expression in cells. Methods for expressing and purifying HA secreted ectodomains have been described (see for example Dopheide et al. 2009;
<img file="MX357009B_D0246.tif" />
256
<img file="MX357009B_D0247.tif" />
HOTO MEXICANO lc LA / ROh'EDaZ
Ekiert et al. 2009, 2011; Stevens et al. 2004, 200fu ... WiXgorp et al. 1981). A person skilled in the art will understand that these methods can also be applied directly to the stem domain polypeptides of the invention with the aim of achieving expression of the secreted (soluble) polypeptide. Therefore these polypeptides are also encompassed in the invention.
For example, in the case of a polypeptide of the invention derived from an influenza virus group 1 HA sequence, a soluble polypeptide of the invention can be created from removal of the polypeptide sequence from the residue (the equivalent of ) 514 to the C terminal (numbering according to SEQ ID NO: 1). Alternatively, additional residues can be included in the polypeptide of the invention, for example by deletion of the sequence from residue 515, 516, 517, 518, 519, 520, 521 or 522. Optionally, a sequence of his dialing (HHHHHH or
HHHHHHH), for purification purposes, optionally connected via a connector. Optionally, the linker may contain a proteolytic cleavage site to remove the his tag after purification. The soluble polypeptide can be further stabilized by introducing a sequence known to form trimeric structures, such as the foldon sequence. Polypeptides obtained as
<img file="MX357009B_D0248.tif" />
257
JHSTITUTO MEXICANO {··> £ LA; Τχί'Γ! ΕΓ7ΑΡ INDUSTRIAL described above are also covered — in, invention.
SEQ ID NO: 181 to 185 show polypeptide sequences of the soluble invention derived from the HA sequence of H1N1 A / Brisbane / 59/2007. Similarly, the
SEQ ID NO: 186 to 187 show soluble polypeptide sequences of the invention derived from the HA sequence of H3N2
A / Hong Kong / 1/1968. A person skilled in the art will understand that equivalent sequences can be designed for polypeptides of the invention derived from other sequences of
HA from other influenza A vaccine strains of eg the Hl, H3, H5 subtypes. It will also be clear to a person skilled in the art that the 6 C-terminal histidines are linked for purification purposes. Since there are other purification methods that do not use this label, the sequence of 6 histidines is optional, and sequences that lack this label for purification are also encompassed in the invention.
Table 1. CDR regions of antibodies. SEQ IDs are NOT given in parentheses.
<td>Ab</td><td>HC CDRl</td><td>HC CDR2</td><td>HC CDR3</td><td>LC CDR1</td><td>LC CDR2</td><td>LC CDR3</td>
<td> 9114</td><td>GGTSNN</td><td>ISPIFG</td><td>ARHGNY</td><td>DSNIGR</td><td>SND</td><td>AAWDDS</td>
<td></td><td>ALREADY</td><td>ST</td><td>YYYSGM</td><td>RS</td><td> (29)</td><td>LKGAV</td>
<img file="MX357009B_D0249.tif" />
<img file="MX357009B_D0250.tif" />
258
MSXICAN INSTITUTE OF PROPERTY
INDU-TKiAL
<td></td><td> (25)</td><td> (26)</td><td>DV (27)</td><td> (28)</td><td></td><td> (30) ........</td>
Table 2. CR9114 Cross-Binding Reactivity, Measured By
ELISA and FACS. Hl = soluble recombinant HA of Hl A / Novel
Caledonia / 20/1999; H3 = HA from soluble recombinant H3
A / Wisconsin / 67/2005; H5 = HA of recombinant soluble H5 of
A / Vietnam / 1203/04; H7 = soluble recombinant H7 HA from
A / Netherlands / 219/2003; H9 = HA of recombinant soluble H9 of
A / Hong Kong / 1073/99; B = soluble recombinant influenza B HA from B / Ohio / 01/05; Rabies = rabies glycoprotein;
PER.C6 = PER.C6 cells not transfused (control); mHl = HA of
Hl of A / New Caledonia / 20/1999 expressed in PER.C6; mH3 = HA from H3 from A / Wisconsin / 67/2005 expressed in PER.C6; mH7 = HA of
H7 of A / Netherlands / 219/2003 expressed in PER.C6; ND = no
<td></td><td colspan="7">IgG Elisa</td><td colspan="4">IgG Facs</td>
<td></td><td> 1</td><td> 3</td><td> 5</td><td> 7</td><td> 9</td><td>B</td><td>Rage</td><td>PerC6</td><td>Hl</td><td>H3</td><td>H7</td>
<td>CR9114</td><td> +</td><td> +</td><td> +</td><td> +</td><td> +</td><td> +</td><td> -</td><td> -</td><td> +</td><td> +</td><td> +</td>
<td>CR4098</td><td></td><td> -</td><td> -</td><td> —</td><td> -</td><td> —</td><td> +</td><td> —</td><td> —</td><td> —</td><td> —</td>
done. + = junction (> 10x background signal); +/- = low junction (2lOx background signal) - = no junction can be detected.
Table 3. CR9114 Cross Neutralizing Activity; The titers (indicated in pg / ml) are geometric mean values of IC50 determined according to the Spearman-Karber method of at least experiments- in duplicate; > 100 = no
A '<d-5f> · / 77
259
Τ7 Τ χ iVi D 1
INSTITUTO MEXICANO Dt LA PRGP'IiuAi) INDUSTRIAL neutralizing to the highest concentration tested (1.00 pg / ntl) ..
<td></td><td>Subtype</td><td>Strain</td><td>CR9114</td>
<td rowspan="12">Group i</td><td rowspan="5">Hl</td><td>A / WSN / 33</td><td> 1.1</td>
<td>A / New Caledonia / 20/99</td><td> 3.7</td>
<td>A / Solomon Islands / 3/2006</td><td> 1.8</td>
<td>A / Brisbane / 59/2007</td><td> 2.6</td>
<td>A / California / 7/2009</td><td> 0.3</td>
<td>H2</td><td>A / Env / MPU3156 / 05</td><td> 8.8</td>
<td rowspan="2">H5</td><td>A / Hong Kong / 156/97</td><td> 0.4</td>
<td>A / EW / MPF461 / 07</td><td> 10.5</td>
<td>H6</td><td>A / EW / MPD411 / 07</td><td> 10.5</td>
<td>H8</td><td>A / EW / MPH571 / 08</td><td> 8.8</td>
<td rowspan="2">H9</td><td>A / Hong Kong / 1073/99</td><td> 4.4</td>
<td>A / Ck / HK / SSPl76 / 09</td><td> 6.3</td>
<td rowspan="11">Group II</td><td rowspan="6">H3</td><td>A / Hong Kong / 1/68</td><td> 19</td>
<td>A / Johannesburg / 33/94</td><td> 21.9</td>
<td>A / Panama / 2007/1999</td><td> 39.9</td>
<td>A / Hiroshima / 52/2005</td><td> 12.5</td>
<td>A / Wisconsin / 67/2005</td><td> 32.4</td>
<td>A / Brisbane / 10/2007</td><td> 5.6</td>
<td>H4</td><td>A / WF / MPA 892/06</td><td> 0.8</td>
<td rowspan="2">H7</td><td>A / Azulón / Countries Low / 12/2000</td><td> 4.8</td>
<td>A / New York / 107/2003</td><td> > 100</td>
<td>H10</td><td>A / Chick / Germany / N / 49</td><td> 15.7</td>
<td>H14</td><td>A / Azulón / Astracán / 2 63/1982</td><td> > 100</td>
Table 4. Binding of serum obtained from mice immunized with
Full-length HA or mini-HA constructs analyzed
260
<img file="MX357009B_D0251.tif" />
by FACS. Data are average values (n ^ Ή-— pays * -the percentage of positive cells after staining followed by mean fluorescence intensity (in parentheses).
Hl-FL (SEQ ID NO: 1), CL1 (SEQ ID NO: 3), CL1 + 2 (SEQ ID NO:
4) and CL1 + 4 (SEQ ID NO: 6). cM2 is a negative control.
<td>Immunization (serum)</td><td>cM2</td><td>H1- FL</td><td>CL1</td><td>CL1 + 2</td><td>CL1 + 4</td>
<td>Transfected DNA</td><td></td><td></td><td></td><td></td><td></td>
<td>cM2</td><td> 47.5(1817)</td><td> 4.8 (404)</td><td> 0.9 (272)</td><td> 0.7 (263)</td><td> 0.7 (269)</td>
<td>H-IFL</td><td> 2.1 (389)</td><td> 84.1 (7130)</td><td> 40.0(1324)</td><td> 38.7(1195)</td><td> 45.5(1618)</td>
<td>CL1</td><td> 2.1 (368)</td><td> 39.1 (1124)</td><td> 43.9(1763) ¡</td><td>ND</td><td>ND</td>
<td>CL1 + 2</td><td> 1.7(348)</td><td> 47.9(1616)</td><td>ND</td><td> 51,4 (2472)</td><td>ND</td>
<td>CL1 + 4</td><td> 1.7 (342)</td><td> 19.6 (787)</td><td>ND</td><td>ND</td><td> 30.0(1047)</td>
% PE pos (geometric MFI, n = 4)
Table 5. Standard amino acids, abbreviations and properties.
<td>Amino acid</td><td>3- Letters</td><td>one- Lyrics</td><td>Polarity of side chain</td><td>Load of lateral (pH</td><td>chain 7.4)</td>
<td>to the girl</td><td>To</td><td>TO</td><td>non polar</td><td>Neutral</td><td></td>
<td>arginine</td><td>Arg</td><td>R</td><td>polar</td><td>Positive</td><td></td>
<td>asparagine</td><td>Asn</td><td>N</td><td>polar</td><td>Neutral</td><td></td>
<td>acid aspartic</td><td>Asp</td><td>D</td><td>polar</td><td>Negative</td><td></td>
<td>cysteine</td><td>Cys</td><td>C</td><td>non polar</td><td>Neutral</td><td></td>
<td>acid glutamic</td><td>Glu</td><td>AND</td><td>polar</td><td>Negative</td><td></td>
<td>glutamine</td><td>Gln</td><td>Q</td><td>polar</td><td>Neutral</td><td></td>
<td>glycine</td><td>Gly</td><td>G</td><td>non polar</td><td>Neutral</td><td></td>
<img file="MX357009B_D0252.tif" />
261
<img file="MX357009B_D0253.tif" />
INSTITUTO MEXICANO DI · THE PROPERTY
INDUSTRIAL
<td rowspan="2">histidine</td><td rowspan="2">His</td><td rowspan="2">H</td><td rowspan="2">polar</td><td>Positive (10%)</td>
<td>Neutral (90%)</td>
<td>isoleucine</td><td>lie</td><td>I</td><td>non polar</td><td>Neutral</td>
<td>leucine</td><td>Leu</td><td>L</td><td>non polar</td><td>Neutral</td>
<td>lysine</td><td>Lys</td><td>K</td><td>polar</td><td>Positive</td>
<td>methionine</td><td>Met</td><td>M</td><td>non polar</td><td>Neutral</td>
<td>phenylalanine</td><td>Phe</td><td>F</td><td>non polar</td><td>Neutral</td>
<td>proline</td><td>Pro</td><td>P</td><td>non polar</td><td>Neutral</td>
<td>serine</td><td>To be</td><td>S</td><td>polar</td><td>Neutral</td>
<td>threonine</td><td>Thr</td><td>T</td><td>polar</td><td>Neutral</td>
<td>tryptophan</td><td>Trp</td><td>W</td><td>non polar</td><td>Neutral</td>
<td>tyrosine</td><td>Tyr</td><td>AND</td><td>polar</td><td>Neutral</td>
<td>valine</td><td>Val</td><td>V</td><td>non polar</td><td>Neutral</td>
Table 6. Consensus sequence for Hl 402-418 (SEQ ID NO: 17), other natural variants, and mutations that stabilize the polypeptides of the invention. One or more mutations in the parental sequence are present in the polypeptides of the invention.
<td>Posi tion</td><td>Not me acid</td><td>Conserve vacation (%)</td><td>Others aa of origin natural</td><td>Mutac ion prefe laugh</td><td>Others mutation ones polare s</td><td>Others mutation ones load ace</td><td>Others mutation ones flexib them</td>
<td> 402</td><td>M</td><td> 99.88</td><td>YOU</td><td></td><td></td><td></td><td></td>
<td> 403</td><td>N</td><td> 99.88</td><td>T, D</td><td></td><td></td><td></td><td></td>
<td> 404</td><td>T</td><td> 96.24</td><td>S, A, M, N , I, V</td><td></td><td></td><td></td><td></td>
<td> 405</td><td>Q</td><td> 99.92</td><td>H, K</td><td></td><td></td><td></td><td></td>
<td> 406</td><td>F</td><td> 99.92</td><td>L</td><td>S</td><td>Τ, N,</td><td>R, H, K,</td><td>G</td>
<img file="MX357009B_D0254.tif" />
according to particular embodiments of the invention
<td> 1.</td><td>To / Islands</td><td>Solomon / 6/2003 (HlNl)</td><td>(I KNOW THAT</td><td>ID</td><td>NOT:</td><td> 54)</td>
<td> 2.</td><td colspan="2">A / Brisbane / 59/2007 (HlNl)</td><td>(I KNOW THAT</td><td>ID</td><td>NOT:</td><td> 1)</td>
<td><sup>20</sup> 3.</td><td>To new</td><td>Caledonia / 20/1999 (HlNl)</td><td>(I KNOW THAT</td><td>ID</td><td>NOT:</td><td> 55)</td>
Four. A / California / 07/2009 (HlNl) (SEQ ID NO:
56)
5. A / pig / Hubei / Sl / 2009 (HlNl) (SEQ ID NO: 57)
6. A / pig / Haseluenne / IDT2617 / 2003 (HlNl) (SEQ ID NO:
263
ΪΜΡΙ
....... .
MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY
58'
<td></td><td> 7 .</td><td>A / New York / 8/2006 (HlNl)</td><td></td><td>(I KNOW THAT</td><td>ID</td><td>NOT:</td><td> 59)</td>
<td></td><td> 8.</td><td>A / Solomon Islands / 3/2006 (HlNl)</td><td></td><td>(I KNOW THAT</td><td>ID</td><td>NOT:</td><td> 60)</td>
<td></td><td> 9.</td><td>A / New York / 146/2000 (HlNl)</td><td></td><td></td><td colspan="3">(SEQ ID NO</td>
<td> 5</td><td> 61)</td><td></td><td></td><td></td><td></td><td></td><td></td>
<td></td><td> 10.</td><td>A / New York / 653/1996 (HlNl)</td><td></td><td></td><td colspan="3">(SEQ ID NO</td>
<td></td><td> 62)</td><td></td><td></td><td></td><td></td><td></td><td></td>
<td></td><td> 11.</td><td>A / Beijing / 262/1995 (HlNl)</td><td></td><td>(I KNOW THAT</td><td>ID</td><td>NOT:</td><td> 63)</td>
<td></td><td> 12.</td><td>A / Texas / 36/1991 (HlNl)</td><td></td><td>(I KNOW THAT</td><td>ID</td><td>NOT:</td><td> 64)</td>
<td> 10</td><td> 13.</td><td>A / Singapore / 6/1986 (HlNl)</td><td></td><td>(I KNOW THAT</td><td>ID</td><td>NOT:</td><td> 65)</td>
<td></td><td> 14.</td><td>A / Chile / 1/1983 (HlNl)</td><td></td><td>(I KNOW THAT</td><td>ID</td><td>NOT:</td><td> 66)</td>
<td></td><td> 15.</td><td>A / Baylor / 11515/1982 (HlNl)</td><td></td><td>(I KNOW THAT</td><td>ID</td><td>NOT:</td><td> 67)</td>
<td></td><td> 16.</td><td>A / Brazil / 11/1978 (HlNl)</td><td></td><td>(I KNOW THAT</td><td>ID</td><td>NOT:</td><td> 68)</td>
<td></td><td> 17.</td><td>A / USSR / 90/1977 (HlNl)</td><td>(I KNOW THAT</td><td colspan="2">ID NO:</td><td> 69)</td><td></td>
<td> 15</td><td> 18.</td><td>A / New Jersey / 8/1976 (HlNl)</td><td></td><td></td><td colspan="3">SEQ ID NO</td>
<td></td><td> 70)</td><td></td><td></td><td></td><td></td><td></td><td></td>
<td></td><td> 19.</td><td>A / Denver / 1957 (HlNl)</td><td></td><td>(I KNOW THAT</td><td>ID</td><td>NOT:</td><td> 71)</td>
<td></td><td> 20.</td><td>A / Albany / 4835/1948 (HlNl)</td><td></td><td>(I KNOW THAT</td><td>ID</td><td>NOT:</td><td> 72)</td>
<td></td><td> 21.</td><td>A / FortMonmouth / 1/1947 (HlNl)</td><td></td><td>(I KNOW THAT</td><td>ID</td><td>NOT:</td><td> 73)</td>
<td> 20</td><td> 22.</td><td>A / Cameron / 1946 (HlNl)</td><td>(I KNOW THAT</td><td colspan="2">ID NO:</td><td> 74)</td><td></td>
<td></td><td> 23.</td><td>A / Weiss / 1943 (HlNl)</td><td></td><td>(I KNOW THAT</td><td>ID</td><td>NOT:</td><td> 75)</td>
<td></td><td> 24.</td><td>A / Iowa / 1943 (HlNl)</td><td></td><td>(I KNOW THAT</td><td>ID</td><td>NOT:</td><td> 76)</td>
<td></td><td> 25.</td><td>A / Bellamy / 1942 (HlNl)</td><td></td><td>(I KNOW THAT</td><td>ID</td><td>NOT:</td><td> 77)</td>
<td></td><td> 26.</td><td>A / PuertoRico / 8/1934 (HlNl)</td><td></td><td>(I KNOW THAT</td><td>ID</td><td>NOT:</td><td> 78)</td>
264
<img file="MX357009B_D0255.tif" />
27. A / WSN / 1933 (HlNl)
28. A / South Carolina / 1/1918 (HlNl) (SEQ ID NO: 79) (SEQ ID NO:
80)
one. MKVKLLVLLC TETATYADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDSHNGKLCL 60
2. MKVKLLVLLC TETATYADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL ENSHNGKLCL 60
3. MKAKLLVLLC TETATYADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDSHNGKLCL 60
Four. MKAILWLLY TEATANADTL CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDKHNGKLCK 60
5. MEAKLFVLFC AETALKADTF CVGYHANYST HTVDTILEKN VTVTHSVNLL ENSHNGKLCS 60
6. MEAKLFVLFC AFTALKADTI CVGYHANNST DTVDTILEKN VTVTHSINLL ENNHNGKLCS 60
7. MKVKLLVLLC TETATYADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDSHNGKLCL 60
8. MKVKLLVLLC TETATYADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDSHNGKLCL 60
9. MKAKLLVLLC AETATYADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDSHNGKLCR 60
10. MKAKLLVLLC AETATYADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDSHNGKLCR 60
eleven. MKAKLLVLLC TFTATYADII CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDSHNGKLCL 60
12. MKAKLLVLLC AETATYADII CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDSHNGKLCR 60
13. MKAKLLVLLC AFTATDADII CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDSHNGKLCR 60
14. MKAKLLVLLC ALSATDADII CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDNHNGKLCK 60
fifteen. MKAKLLVLLC ALSATDADII CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDSHNGKLCR 60
0 16. MKAKLLVLLC ALSATDADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDSHNGKLCR 60
17. MKAKLLVLLC ALSATDADTI CIGYHANNST DIVETVLEKN VTVTHSVNLL EDSHNGKLCR 60
18. MKAKLLVLLC AETATDAETI CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDSHNGKLCR 60
19. MKAKLLILLC ALSATDADII CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDSHNGKLCR 60
twenty. MKAKLLVLLC ALSATDADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDSHNGKLCR 60
265
twenty-one. MKAKLLILLC ALTATDADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL
22. MKAKLLILLC ALSATDADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL
2. 3. MKARLLVLLC ALAATDADTI CIGYHANNST DTVDTILEKN VTVTHSVNLL
24. MKARLLVLLC ALAATDADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL
25. MKARLLVLLC AIAATDADTI CIGYHANNST DTVDTILEKN VTVTHSVNLL
26. MKANLLVLLC ALAAADADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL
27. MKAKLLVLLY AFVATDADTI CIGYHANNST DTVDTIFEKN VAVTHSVNLL
28. MEARLLVLLC AEAATNADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL ★ · ★ · · ♦ · · · ★★★ · ★ ★★★★★ · · ★★★ ★ · ★★★★★★★★ · * · ·
<img file="MX357009B_D0256.tif" />
EDSHNGKLCR 60
EDSHNGKLCR 60
EDSHNGKLCR 60
EDSHNGKLCR 60
EDSHNGKLCR 60
EDRHNGKLCK 60
EDSHNGKLCK 60 ★★★★★★
one. LKGIAPLQLG NCSVAGWILG NPECELLISR ESWSYIVEKP NPENGTCYPG HEADYEELRE 120
2. LKGIAPLQLG NCSVAGWILG NPECELLISK ESWSYIVEKP NPENGTCYPG HEADYEELRE 120
3. LKGIAPLQLG NCSVAGWILG NPECELLISK ESWSYIVETP NPENGTCYPG YEADYEELRE 120
Four. LRGVAPLHLG KCNIAGWILG NPECESLSTA SSWSYIVETP SSDNGTCYPG DFIDYEELRE 120
5. LNGKIPLQLG NCNVAGWILG NPKCDLLLTA NSSSYIIETS KSKNGACYPG EEADYEELKE 120
6. LNGKAPLQLG NCNVAGWILG NPECDLLLTV DSWSYIIETS NSKNGACYPG DFADYEELRE 120
7. LKGIAPLQLG NCSVAGWILG NPECELLISK ESWSYIVETP NPENGTCYPG YEADYEELRE 120
8. LKGIAPLQLG NCSVAGWILG NPECELLISR ESWSYIVEKP NPENGTCYPG HEADYEELRE 120
9. LKGTAPLQLG NCSIAGWILG NPECESLFSK ESWSYIAETP NPKNGTCYPG YEADYEELRE 120
10. LKGTAPLQLG NCSVAGWILG NPECESLFSK ESWSYIAETP NPENGTCYPG YEADYEELRE 120
0 11. LKGIAPLQLG NCSVAGWILG NPECESLISK ESWSYIVETP NPENGTCYPG YEADYEELRE 120
12. LKGIAPLQLG NCSVAGWILG NPKCESLFSK ESWSYIAETP NPENGTCYPG YEADYEELRE 120
13. LKGIAPLQLG NCSIAGWILG NPECESLFSK KSWSYIAETP NSENGTCYPG YEADYEELRE 120
14. LKGIAPLQLG KCSIAGWILG NPECESLFSK KSWSYIAETP NSENGTCYPG YEADYEELRE 120
fifteen. LKGIAPLQLG KCSIAGWILG NPECESLFSK KSWSYIAETP NSENGTCYPG YEADYEELRE 120
<img file="MX357009B_D0257.tif" />
266
16. LKGIAPLQLG KCSIAGWILG NPECESLFSK KSWSYIAETP
17. LKGIAPLQLG KCNIAGWILG NPECESLFSK KSWSYIAETP
18. LKGIAPLQLG NCSIAGWILG NPECESLFSK KSWSYIAETP
19. LKGKAPLQLG NCNIAGWVLG NPECESLLSN RSWSYIAETP
twenty. LKGIAPLQLG KCNIAGWILG NPECESLFSK KSWSYIAETP
twenty-one. LKGIAPLQLG KCNIAGWILG NPECESLLSK RSWSYIAETP
22. LKGIAPLQLG KCNIAGWILG NPECESLLSK RSWSYIAETP
2. 3. LKGIAPLQLG KCNIAGWILG NPECESLLSE RSWSYIVEIP
24. LKGIAPLQLG KCNIAGWILG NPECESLLSE RSWSYIVETP
25. LKGIAPLQLG KCNIAGWILG NPECESLLSE RSWSYIVETP
26. LKGIAPLQLG KCNIAGWLLG NPECDPLLPV RSWSYIVETP
27. LKGIAPLQLG KCNITGWLLG NPECDSLLPA RSWSYIVETP
28. LKGIAPLQLG KCNIAGWLLG NPECDLLLTA SSWSYTVETS k · -k • k-k'k · -kk · 9c ★ ★★★★★ 9c
NSENGTCTPG ~ WWECRE
NSENGTCYPG YFADYEELRE 120
NSENGTCYPG YEADYEELRE 120
NSENGTCYPG DEADYEELRE 120
NSENGTCYPG YEADYEELRE 120
NSENGACYPG DEADYEELRE 120
NSENGACYPG DEADYEELRE 120
NSENGTCYPG DETDYEELRE 120
NSENGTCYPG DETDYEELRE 120
NSENGTCYPG DFIDYEELRE 120
NSENGICYPG DFIDYEELRE 120
NSENGACYPG DFIDYEELRE 120
NSENGTCYPG DFIDYEELRE 120 ** **** * *******
one. QLSSVSSFER EEIFPKESSW PNHTTT-GVS ASCSHNGESS FYKNLLWLTG KNGLYPNLSK 179
2. QLSSVSSFER EEIFPKESSW PNHTVT-GVS ASCSHNGESS FYRNLLWLTG KNGLYPNLSK 179
3. QLSSVSSFER EEIFPKESSW PNHTVT-GVS ASCSHNGKSS FYRNLLWLTG KNGLYPNLSK 179
Four. QLSSVSSFER FEIFPKTSSW PNHDSNKGVT AACPHAGAKS FYKNLIWLVK KGNSYPKLSK 180
5. QLSTVSSFER FEIFPKAISW PDHDATRGTT VACSHSGVNS FYRNLLSTVK KGNSYPKLSK 180
6. QLSTVSSFER FEIFPKATSW PNHDTTRGIT ISCSHSGANS FYRNLLWTVK KGNSYPKLSK 180
7. QLSSVSSFER EEIFPKESSW PNHTVT-GVS ASCSHNGKSS FYRNLLWLTG KNGLYPNLSK 179
8. QLSSVSSFER EEIFPKESSW PNHTTT-GVS ASCSHNGESS FYKNLLWLTG KNGLYPNLSK 179
9. QLSSVSSFER FEIFPKDSSW PNHTVTKGVT ASCSHNGKSS FYKNLLWLTE KNGLYPNLSK 180
<img file="MX357009B_D0258.tif" />
267
INSTITUTO MEXICANO or LA l'KOPIECAU INDUSTRIAL
10. QLSSVSSFER FEIFPKESSW PNHTVTKGVT
eleven. QLSSVSSFER FEIFPKESSW PNHTVT-GVT
12. QLSSVSSFER FEIFPKESSW PNHTVTKGVT
13. QLSSVSSFER FEIFPKESSW PNHTVTKGVT
14. QLSSVSSFER FEIFPKESSW PKHNVTKGVT
fifteen. QLSSVSSFER FEIFPKESSW PKHSVTRGVT
16. QLSSVSSFER FEIFPKERSW PKHNITRGVT
17. QLSSVSSFER FEIFPKERSW PKHNVTRGVT
18. QLSSVSSFER FEIFPKESSW PNHTVTKGVT
19. QLSSVSSFER FEIFPKERSW PNHTTR-GVT
twenty. QLSSVSSFER FEIFPKERSW PKHNITRGVT
twenty-one. QLSSVSSFER FEIFPKERSW PKHNITRGVT
22. QLSSVSSFER FEIFPKGRSW PEHNIDIGVT
2. 3. QLSSVSSFER FEIFPKESSW PKHNTARGVT
24. QLSSVSSFER FEIFSKESSW PKHTTG-GVT
25. QLSSVTSFER FEIFPKETSW PKHNTTKGVT
26. QLSSVSSFER FEIFPKESSW PNHNTN-GVT
27. QLSSVSSLER FEIFPKESSW PNHTFN-GVT
28. QLSSVSSFER FEIFPKTSSW PNHETTKGVT kkkk · k · -k · ★ kk k «k
ASCSHNGKSS FYKNLLWLTE KNGLYPNLSK 180
ASCSHNGKSS FYRNLLWLTE KNGLYPNLSN 179
TSCSHNGKSS FYRNLLWLTK KNGLYPNVSK 180
ASCSHKGRSS FYRNLLWLTK KNGSYPNLSK 180
AACSHKGKSS FYRNLLWLTE KNGSYPNLSK 180
ASCSHKGKSS FYRNLLWLTE KNGSYPNLSK 180
ASCSHKGKSS FYRNLLWLTE KNGSYPNLSK 180
ASCSHKGKSS FYRNLLWLTE KNGSYPNLSK 180
ASCSHKGRSS FYRNLLWLTK KNGSYPNLSK 180
AACPHARKSS FYKNLVWLTE ANGSYPNLSR 179
AACSHKGKSS FYRNLLWLTE KNGSYPNLNK 180
AACSHAGKSS FYKNLLWLTE TDGSYPKLSK 180
AACSHAGKSS FYKNLLWLTE KDGSYFNLNK 180
AACSHAGKSS FYRNLLWLTE KDGSYPNLKN 180
AACSHAGKSS FYRNLLWLTE KDGSYPNLNN 179
AACSHAGKCS FYRNLLWLTE KDGSYPNLNN 180
AACSHEGKSS FYRNLLWLTE KEGSYPKLKN 179
VSCSHRGKSS FYRNLLWLTK KGDSYPKLTN 179
AACSYAGASS FYRNLLWLTK KGSSYPKLSK 180 »k kk · ★ ★ · XrXr kk · ·
SYANNKEKEV
SYANNKEKEV
SYVNNKEKEV
SYINDKGKEV
LVLWGVHHPP
LVLWGVHHPP
LVLWGVHHPP
LVLWGIHHPS
NIGDQRALYH
NIGNQKALYH
NIGNQRALYH
TSADQQSLYQ
KENAYVSWS
TENAYVSWS
TENAYVSWS
NADAYVEVGS
SHYSRKFTPE
SHYSRKFTPE
SHYSRRFTPE
SRYSKKFKPE
IAKRPKVRDQ 239
IAKRPKVRDQ 239
IAKRPKVRDQ 239
IAIRPKVRXX 240
268
MEXICAN INSTITUTE ¿C - <sub>ώ </sub>DE LA FR 'PIEDAD
5. SYTNNKGKEV LVIWGVHHPP TDSVQQTLYQ NKHTYVSVGS SKYYKRFTPE IVARIWTOQ 240 .....
6. SYTNNKGKEV LVIWGVHHPP TDSDQQTLYQ NNHTYVSVGS SKYYQRFTPE IVTRPKVRGQ 240
7. SYANNKEKEV LVLWGVHHPP NIGDQRALYH TENAYVSWS SHYSRRFTPE IAKRPKVRDQ 239
8. SYANNKEKEV LVLWGVHHPP NIGDQRALYH KENAYVSWS SHYSRKFTPE IAKRPKVRDQ 239
9. SYVNKKGKEV LVLWGVHHPS NMGDQRAIYH KENAYVSVLS SHYSRRFTPE IAKRPKVRDQ 240
10. SYVNNKEKEV LVLWGVHHPS NIGDQRAIYH TENAYVSWS SHYSRRFTPE ITKRPKVRDQ 240
eleven. SYVNNKEKEV LVLWGVHHPS NIRDQRAIYH TENAYVSWS SHYSRRFTPE IAKRPKVRGQ 239
12. SYVNNKEKEV LVLWGVHHPS NIGDQRAIYH TENAYVSWS SHYSRRFTPE IAKRPKVRDQ 240
13. SYVNNKEKEV LVLWGVHHPS NIGDQRAIYH TENAYVSWS SHYNRRFTPE IAKRPKVRDQ 240
14. SYVNNKEKEV LVLWGVHHPS NIEDQKTIYR KENAYVSWS SHYNRRFTPE IAKRPKVRNQ 240
fifteen. SYVNDKEKEV LVLWGVHHPS NIEDQKTIYR KENAYVSWS SHYNRRFTPE IAKRPKVRDQ 240
16. SYVNNKEKEV LVLWGVHHPS NIEDQKTIYR KENAYVSWS SNYNRRFTPE IAKRPKVRGQ 240
17. SYVNNKEKEV LVLWGVHHPS NIEDQKTIYR KENAYVSWS SNYNRRFTPE IAERPKVRGQ 240
18. SYVNNKEKEV LVLWGVHHPS NIGDQRAIYH TENAYVSWS SHYNRRFTPE IAKRPKVRDQ 240
19. SYVNNQEKEV LVLWGVHHPS NIEEQRALYR KDNAYVSWS SNYNRRFTPE IAKRPKVRDQ 239
twenty. SYVNNKEKEV LVLWGVHHPS NIEDQKTLYR KENAYVSWS SNYNRRFTPE IAERPKVRGQ 240
twenty-one. SYVNNKEKEV LVLWGVHHPS NIEDQKTLYR KENAYVSWS SNYNRRFTPE IAERPKVRGQ 240
22. SYVNKKEKEV LILWGVHHPP NIENQKTLYR KENAYVSWS SNYNRRFTPE IAERPKVRGQ 240
2. 3. SYVNKKGKEV LVLWGVHHPS SIKEQQTLYQ KENAYVSWS SNYNRRFTPE IAERPKVRDQ 240
24. SYVNKKGKEV LVLWGVHHPS NIKDQQTLYQ KENAYVSWS SNYNRRFTPE IAERPKVRGQ 239
25. SYVNKKGKEV LVLWGVHHPS NIKDQQTLYQ KENAYVSWS SNYNRRFTPE IAERPKVRGQ 240
26. SYVNKKGKEV LVLWGIHHPP NSKEQQNLYQ NENAYVSWT SNYNRRFTPE IAERPKVRDQ 239
27. SYVNNKGKEV LVLWGVHHPS SSDEQQSLYS NGNAYVSVAS SNYNRRFTPE IAARPKVKDQ 239
28. SYVNNKGKEV LVLWGVHHPP TGTDQQSLYQ NADAWSVGS SKYNRRFTPE IAARPKVRDQ 240
269
<img file="MX357009B_D0259.tif" />
INSTITUTO MEXICANO Ct LA PROPIEDAD industrial
<img file="MX357009B_D0260.tif" />
★ ★ ★ · kkk ★ · kkk · · ★ · · k »kkk k · * k · · ~ k kk k · kkkk · ·· · · ·» ··· ·· · ·····, .-,<sub>B</sub>
one. EGRINYYWTL LEPGDTIIFE ANGNLIAPRY AEALSRGFGS GIINSNAFMD ECDAKCQTPQ 299
2. EGRINYYWTL LEPGDTIIFE ANGNLIAPRY AEALSRGFGS GIINSNAFMD KCQAKCQTPQ 299
3. EGRINYYWTL LEPGDTIIFE ANGNLIAFWY AEALSRGFGS GIITSNAFMD ECDAKCQTPQ 299
Four. EGRMNYYWTL VEPGDKITFE ATGNLWPRY AEAMERNAGS GIIISDTPVH ECNTTCQTPK 300
5. AGRMNYYWTL FDQGDTITFE ATGNLIAPWH AEALKKGSSS GIMLSDAQVH NCTTKCQTPH 300
6. AGRMNYYWTL LDQGDTITFE ATGNLIAPWH AEALNKGPSS GIMISDAHVH NCTTKCQTPH 300
7. EGRINYYWTL LEPGDTIIFE ANGNLIAPRF AEALSRGFGS GIITSNAFMD ECDAKCQTPQ 299
8. EGRINYYWTL LEPGDTIIFE ANGNLIAPRY AEALSRGFGS GIINSNAFMD ECDAKCQTPQ 299
9. EGRINYYWTL LEPGDTIIFE ANGNLIAFWY AEALSRGFGS GIIISNASMG ECDAKCQTPQ 300
10. EGRINYYWTL LEPGDTIIFE ANGNLIAFWY
eleven. EGRINYYWTL LEPGDTIIFE ANGNLIAFWY
12. EGRINYYWTL LEPGDTIIFE ANGNLIAFWY
13. EGRINYYWTL LEPGDTIIEE ANGNLLAEWY
14. EGRINYYWTL LEPGDTIIEE ANGNLIAFWY
fifteen. EGRINYYWTL LEPGDTIIFE ANGNLIAFWY
16. EGRINYYWTL LEPGDTIIFE ANGNLIAFWY
17. AGRINYYWTL LEPGDTIIFE ANGNLIAFWH
18. EGRINYYWTL LEPGDTIIEE ANGNLIAFWY
19. SGRMNYYWTL LEPGDTIIFE ATGNLIAPWY
twenty. AGRINYYWTL LEPGDTIIFE ANGNLIAFWH
twenty-one. AGRINYYWTL LEPGDTIIEE ANGNLIAFWY
22. AGRINYYWTL LEPGDTIIFE ANGNLIAFWY
2. 3. AGRMNYYWTL LEPGDTIIEE ANGNLIAFWY
AEALSRGFGS GIIISNASMG ECDAKCQTPQ 300
AEALSRGFGS GIITSNAPMN ECDAKCQTPQ 299
AEALSRGFGS GIITSNASMD ECDAKCQTPQ 300
AEALSRGFGS GIITSNASMD ECDAKCQTPQ 300
AEALSRGFGS GIITSNASMD ECDAKCQTPQ 300
AEALSRGFGS GIITSNVSMD ECDAKCQTPQ 300
AEALSRGFGS GIITSNASMD ECETKCQTPQ 300
AFALNRGFGS GIITSNASMD ECDTKCQTPQ 300
AEALSRGFGS GIITSNASMD ECDAKCQTPQ 300
AFALSRGPGS GIITSNAPLD ECDTKCQTPQ 299
AEALSRGFGS GIITSNASMD ECDTKCQTPQ 300
AEALSRDEGS GIITSNASMD ECDTKCQTPQ 300
AEALNRGIGS GIITSNASMD ECDTKCQTPQ 300
AEALSRGFGS GIITSNASMH ECDTKCQTPQ 300
To the."
270 lf Λ / J ρ! ί
-i. J · - ν Λ. .I ♦ N sTJTUTl * MüXJC / .NO 9? ¡.A iT.VüóA · · •• íVl'TXiA '+24' .I # ·
24. AGRINYYWTL LKPGDTIMFE
25. AGRMNYYWTL LEPGDTIIFE
26. AGRMNYYWTL LKPGDTIIFE
27. HGRMNYYWTL LEPGDTIIFE
28. AGRMNYYWTL LEPGDTITFE ★ ★ · kkkkkk · · kkk ★ ★ ★ ★
ANGNLIAPWY AFALSRGFGS
ANGNLIAPWY AFALSRGFGS
ANGNLIAEMY AFALRRGEGS
ATGNLIAFWY AEALSRGFES
ATGNLIAPWY AFALNRGSGS kkk · k kkk · ★ Ά · kkk
GIITSNASMH ECDTKCQTPQ 299
GIITSNASMH ECNTKCQTPQ 300
GIITSNASMH ECNTKCQTPL 299
GIITSNASMH ECNTKCQTPQ 299
GIITSDAFVH DCNTKCQTPH 300
Ά · · · k · ♦ kkkk
PKYVRSAKLR MVTGLRNIPS
PKYVRSAKLR MVTGLRNIPS
PKYVRSAKLR MVTGLRNIPS
PKYVKSTKLR LATGLRNIPS
PKYVKSTQLR MATGLRNIPS
PKYVKSTQLR MATGLRNIPS
PKYVRSAKLR MVTGLRNIPS
PKYVRSAKLR MVTGLRNIPS
PKYVRSTKLR MVTGLRNVPS
PKYVRSTKLR MVTGLRNIPS
PKYVRSTKLR MVTGLRNIPS
PKYVRSTKLR MVTGLRNIPS
PKYVRSTKLR MVTGLRNIPS
PKYVRSTKLR MVTGLRNIPS
PKYVRSTKLR MVTGLRNIPS
PKYVRSTKLR MVTGLRNIPS
PKYVRSTKLR MVTGLRNIPS
PKYVRSTKLR MVTGLRNIPS
one. GAINSSLPFQ NVHPVTIGEC
2. GAINSSLPFQ NVHPVTIGEC
3. GAINSSLPFQ NVHPVTIGEC
Four. GAINTSLPEQ NIHPITIGKC
5. GALKNNLPLQ NVHLFTIGEC
6. GALKSNLPFQ NVHPSTIGEC
7. GAINSSLPEQ NVHPVTIGEC
8. GAINSSLPEQ NVHPVTIGEC
9. GAINSSLPFQ NVHPVTIGEC
10. GAINSSLPFQ NVHPVTIGEC
eleven. GAINSSLPFQ NVHPVTIGEC
12. GAINSSLPFQ NVHPVTIGEC
13. GAINSSLPEQ NVHPVTIGEC
14. GAINSSLPFQ NVHPVTIGEC
fifteen. GAINSSLPEQ NVHPVTIGEC
16. GAINSSLPFQ NVHPVTIGEC
17. GAINSSLPFQ NIHPVTIGEC
18. GAINSSLPFQ NVHPVTIGEC
IQSRGLFGAI AGFIEGGWTG 359
IQSRGLFGAI AGFIEGGWTG 359
IQSRGLFGAI AGFIEGGWTG 359
IQSRGLEGAI AGFIEGGWTG 360
IQSRGLFGAI AGFIEGGRTG 360
IQSRGLEGAI AGFIEGGWTG 360
IQSRGLEGAI AGFIEGGWTG 359
IQSRGLFGAI AGFIEGGWTG 359
IQSRGLFGAI AGFIEGGWTG 360
IQSRGLFGAI AGFIEGGWTG 360
IQSRGLEGAI AGFIEGGWTG 359
IQSRGLFGAI AGFIEGGWTG 360
IQSRGLFGAI AGFIEGGWTG 360
IQSRGLFGAI AGFIEGGWTG 360
IQSRGLFGAI AGFIEGGWTG 360
IQSRGLEGAI AGFIEGGWTG 360
IQSRGLFGAI AGFIEGGWTG 360
IQSRGLFGAI AGFIEGGWTG 360
<img file="MX357009B_D0261.tif" />
PI
MEXICAN INSTITUTE
OF THE PROPERTY .......
271 INDUSTRIAL ''
19. GAINSSLPFQ NIHPVTIGEC PKYVRSTKLR MVTGLRNIPS VQSRGLFGAI '• aSFIEGCTffTG ·' 359'—
twenty. GAINSSLPFQ NIHPVTIGEC PKYVRSTKLR MVTGLRNIPS IQSRGLEGAI AGFIEGGWTG 360
twenty-one. GAINSSLPFQ NIHPVTIGEC PKYVKSTKLR MVTGLRNIPS IQSRGLFGAI AGFIEGGWTG 360
22. GAINSSLPFQ NIHPFTIGEC PKYVRSTKLR MVTGLRNIPS IQSRGLFGAI AGFIEGGWDG 360
2. 3. GAINSSLPFQ NIHPVTIGEC PKYVRSTKLR MVTGLRNIPS IQSRGLFGAI AGFIEGGWTG 360
24. GAINSSLPFQ NIHPVTIGEC PKYVRSTKLR MVTGLRNIPS IQSRGLFGAI AGFIEGGWTG 359
25. GAINSSLPFQ NIHPVTIGEC PKYVRSTKLR MVTGLRNIPS IQSRGLFGAI AGFIEGGWTG 360
26. GAINSSLPYQ NIHPVTIGEC PKYVRSAKLR MVTGLRNIPS IQSRGLFGAI AGFIEGGWTG 359
27. GSINSNLPPQ NIHPVTIGEC PKYVRSTKLR MVTGLRNIPS IQYRGLPGAI AGFIEGGWTG 359
28. GAINSSLPFQ NIHPVTIGEC PKYVRSTKLR MATGLRNIPS IQSRGLFGAI AGFIEGGWTG 360 ★ ·· * · * ·· · * · ★ ♦ ★ * · ♦ * · * · * ·· * ★ ★ ★ · 'k ★★★★★★★ ★★★★★ ★★ · * · ★
<img file="MX357009B_D0262.tif" />
one. MVDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI EKMNTQFTAV GKEFNKLERR 419
2. MVDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI EKMNTQFTAV GKEFNKLERR 419
3. MVDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI EKMNTQFTAV GKEFNKLERR 419
Four. MVDGWYGYHH QNEQGSGYAA DLKSTQNAID EITNKVNSVI EKMNTQFTAV GKEFNHLEKR 420
5. MIDGWYGYHH QNEQGSGYAA DQKSTQIAID GINNKANSVI GKMNIQLTSV GKEFNSLEKR 420
6. MIDGWYGYHH QNEQGSGYAA DQKSTQIAID GINNKVNSII EKMNTQFTSV GKEENDLEKR 420
7. MVDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI EKMNTQFTAV GKEFNKLERR 419
8. MVDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI EKMNTQFTAV GKEENKLERR 419
9. MIDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSII EKMNTQFTAV GKEENKLEKR 420
10. MIDGWYGYHH QNEQGSGYAA DQKSTQNAID GITNKVNSVI EKMNTQFTAV GKEFNKLERR 420
eleven. MMDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI EKMNTQFTAV GKEFNKLERR 419
12. MIDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI EKMNTQFTAV GKEFNKLERR 420
13. MIDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI EKMNTQFTAV GKEFNKLERR 420
IMPIDO
272
14. MIDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSII
fifteen. MIDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI
16. MIDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI
17. MIDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI
18. MIDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI
19. MMDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI
twenty. MIDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI
twenty-one. MIDGWYGYHH QNEQGSGYAA DQKSTQNAIN WITNKVNSVI
22. MIDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI
2. 3. MIDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI
24. MIDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI
25. MIDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI
26. MIDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI
27. MIDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI
28. MIDGWYGYHH QNEQGSGYAA DQKSTQNAID GITNKVNSVI
IKSTITIJT! MEX1C..NO '' ~ M
C £ LA R? O?! 5KAO
To go! RUSTRI AL _——
EKMNTQETAV GKEENKLEKR 420
EKMNTQETAV GKEENKLEKR 420
EKMNTQETAV GKEENKLEKR 420
EKMNTQETAV GKEENKLERR 420
EKMNTQETAV GKEENKLEKR 419
EKMNTQETAV GKEENKLEKR 420
EKMNTQETAV GKEENKLEKR 420
EKMNTQETAV GKEENKLEKR 420
EKMNTQFTAV GKEENNLEKR 420
EKMNTQETAV GKEENNLEKR 419
EKMNTQETAV GKEENNLEKR 420
EKMNIQETAV GKEENKLEKR 419
EKMNTQETAV GKEENNLEKR 419
EKMNTQETAV GKEENNLERR 420
*.******** ********** * *******. *******.* **** ***** *****.**.*
one. MENLNKKVDD GFIDIWTYNA ELLVLLENER TLDFHDSNVK NLYEKVKSQL KNNAKEIGNG 479
2. MENLNKKVDD GFIDIWTYNA ELLVLLENER TLDFHDSNVK NLYEKVKSQL KNNAKEIGNG 479
3. MENLNKKVDD GFLDIWTYNA ELLVLLENER TLDFHDSNVK NLYEKVKSQL KNNAKEIGNG 479
Four. IENLNKKVDD GFLDIWTYNA ELLVLLENER TLDYHDSNVK NLYEKVRSQL KNNAKEIGNG 480
5. KENLNKTVDD RELDVWTENA ELLVLLENQR TLEFHDLNIK SLYEKVKSHL RNNDKEIGNG 480
6. IENLNKKVDD GELDVWTYNA ELLILLENER TLDFHDENVK NLYEKVKSQL RNNAKEIGNG 480
7. MENLNKKVDD GFLDIWTYNA ELLVLLENER TLDFHDSNVK NLYEKVKSQL KNNAKEIGNG 479
8. MENLNKKVDD GFIDIWTYNA ELLVLLENER TLDFHDSNVK NLYEKVKSQL KNNAKEIGNG 479
273 : \.? Γι?;. * 7Ό MEXICANO TS 4.A l-XOPÍf.VD ί '· ί *? Ϋ3ΤϋΙ Al
9.
10.
11.
12,
13,
14,
15,
1.
2.
3.
MENLNKKVDD GFLDIWTYNA ELLVLLENER TLDFHDLNVK NLYEKVKNQL KNNñKEIGNG ~ 4SQ ~
MENLNKKVDD GELDIWTYNA ELLVLLENER
MENLNKKVDD GELDIWTYNA ELLVLLENER
MENLNKKVDD GELDIWTYNA ELLVLLENGR
MENLNKKVDD GELDIWTYNA ELLVLLENER
MENLNKKVDD GELDIWTYNA ELLVLLENER
MENLNKKVDD GELDIWTYNA ELLVLLENER
MENLNKKVDD GELDIWTYNA ELLVLLENER
MENLNKKVDD GELDIWTYNA ELLVLLENER
MENLNKKVDD GELDIWTYNA ELLVLLENER
MENLNKKVDD GEMDIWTYNA ELLVLLENER
MENLNKKVDD GELDIWTYNA ELLVLLENER
MENLNKKVDD GFLDIWTYNA ELLVLLENER
MENLNKKVDD GFLDIWTYNA ELLVLLENER
MENLNKKVDD GELDIWTYNA ELLILLENER
MENLNKKVDD GELDIWTYNA ELLVLLENER
MENLNKKVDD GELDIWTYNA ELLVLLENER
MENLNNKVDD GELDIWTYNA ELLVLLENER
MENLNKKVDD GELDIWTYNA ELLVLLENER
IENLNKKVDD GELDIWTYNA ELLVLLENER • á · Ά - A - Á- · ** · -k -kk · 'k-k'k'k'k'k'k' k'k'k · 'k-k'k 'k -k
CFEEYHKCND ECMESVKNGT YDYPKYSEES
CFEEYHKCND ECMESVKNGT YDYPKYSEES
CEEFYHKCNN ECMESVKNGT YDYPKYSEES
TLDEHDSNVK NLYEKVKTQL KNNAKEIGNG 480
TLDEHDSNVK NLYEKVKSQL KNNAKEIGNG 479
TLDEHDSNVK NLYEKVKSQL KNNAKEIGNG 480
TLDEHDSNVK NLYEKVKSQL KNNAKEIGNG 480
TLDEHDSNVK NLYEKVKSQL KNNAKEIGNG 480
TLDEHDSNVK NLYEKVKSQL KNNAKEIGNG 480
TLDEHDSNVK NLYEKVKSQL KNNAKEIGNG 480
TLDEHDSNVK NLYEKVKSQL KNNAKEIGNG 480
TLDEHDSNVK NLYEKVKSQL KNNAKEIGNG 480
TLDEHDSNVK NLYEKVKNQL RNNAKELGNG 479
TLDEHDSNVK NLYEKVKSQL KNNAKEIGNG 480
TLDEHDSNVK NLYEKVKNQL RNNAKEIGNG 480
TLDEHDSNVK NLYEKVKNQL RNNAKEIGNG 480
TLDEHDSNVK NLYEKVKSQL RNNAKEIGNG 480
TLDEHDSNVK NLYEKVKNQL RNNAKEIGNG 479
TLDEHDSNVK NLYEKVKSQL RNNAKEIGNG 480
TLDEHDSNVK NLYEKVKSQL KNNAKEIGNG 479
TLDEHDLNVK NLYEKVKSQL KNNAKEIGNG 479
TLDEHDSNVR NLYEKVKSQL KNNAKEIGNG 480
-kk · Ά · ★ Ά ”· ★ ★
KLNREKIDGV KLESMGVYQI
KLNREKIDGV KLESMGVYQI
KLNREKIDGV KLESMGVYQI *****. ***
LAIYSTVASS
LAIYSTVASS
LAIYSTVASS
539
539
539
<img file="MX357009B_D0263.tif" />
MEXICAN INSTITUTE <sup>1 </sup>nk IA RR .j? l £ DAi>
Four. CFEFYHKCDN
5. CFEFYHKREN
6. CFEFYHKCDN
7. CFEFYHKCND
8. CFEFYHKCND
9. CFEFYHKCNN
TCMESVKNGT
ECLECVKNGT
ECMESVKNGT
ECMESVKNGT
ECMESVKNGT
ECMESVKNGT
YDYPKYSEEA
YNYPKYSEES
YNYPKYSEES
YDYPKYSEES
YDYPKYSEES
YDYPKYSKES
274
KLNREEIDGV
KENREEIVGV
KLNREKIDGV
KLNRERIDGV
KLNREKIDGV
KLNREKIDGV
KLESTRIYQI IAI'Y0TVADg-54fr
KLESM3IHQI LAIYSTVASS 540
KLESMGVHQI LAIYSTVASS 540
KLESMGVYQI LAIYSTVASS 539
KLESMGVYQI LAIYSTVASS 539
KLESMGVYQI LAIYSTVASS 540
<td>10. CFEFYHKCNN</td><td>ECMESVKNGT</td><td>YDYPKYSEES</td><td>KLNREKIDGV</td><td>KLESMGVYQI</td><td>LAIYSTVASS</td><td> 540</td>
<td>11. CFEFYHKCNN</td><td>ECMESVKNGT</td><td>YDYPKYSEES</td><td>KLNREKIDGV</td><td>KLESMGVYQI</td><td>LAIYSTVASS</td><td> 539</td>
<td>12. CFEFYHKCNN</td><td>ECMESVKNGT</td><td>YDYPKYSEES</td><td>KLNRGKIDGV</td><td>KLESMGVYQI</td><td>LAIYSTVASS</td><td> 540</td>
<td>13. CFEFYHKCNN</td><td>ECMESVKNGT</td><td>YDYPKYSEES</td><td>KLNREKIDGV</td><td>KLESMGVYQI</td><td>LAIYSTVASS</td><td> 540</td>
<td>14. CFEFYHKCNN</td><td>ECMESVKNGT</td><td>YDYPKYSEES</td><td>KLNREKIDGV</td><td>KLESMGVYQI</td><td>LAIYSTVASS</td><td> 540</td>
<td>15. CFEFYHKCNN</td><td>ECMESVKNGT</td><td>YDYPKYSEES</td><td>KLNREKIDGV</td><td>KLESMGVYQI</td><td>LAIYSTVASS</td><td> 540</td>
<td>16. CFEFYHKCNN</td><td>ECMESVKNGT</td><td>YDYPKYSEES</td><td>KLNREKIDGV</td><td>KLESMGVYQI</td><td>LAIYSTVASS</td><td> 540</td>
<td>17. CFEFYHKCNN</td><td>ECMESVKNGT</td><td>YDYPKYSEES</td><td>KLNREKIDGV</td><td>KLESMGVYQI</td><td>LAIYSTVASS</td><td> 540</td>
<td>18. CFEFYHKCNN</td><td>ECMESVKNGT</td><td>YDYPKYSEES</td><td>KLNREKIDGV</td><td>KLESMGVYQI</td><td>LAIYSTVASS</td><td> 540</td>
<td>19. CFEFYHKCDN</td><td>ECMESVKNGT</td><td>YDYPKYSEES</td><td>KLNREKIDGV</td><td>KLESMGVYRI</td><td>LAIYSTVASS</td><td> 539</td>
<td>20. CFEFYHKCNN</td><td>ECMESVKNGT</td><td>YDYPKYSEES</td><td>KLNREKIDGV</td><td>KLESMGVYQI</td><td>LAIYSTVASS</td><td> 540</td>
<td>21. CFEFYHKCNN</td><td>ECMESVKNGT</td><td>YDYPKYSEES</td><td>KLNREKIDGV</td><td>KLESMGVYQI</td><td>LAIYSTVASS</td><td> 540</td>
<td>22. CFEFYHKCNN</td><td>ECMESVKNGT</td><td>YDYPKFSEES</td><td>KLNREKIDGV</td><td>KLESMGVYQI</td><td>LAIYSTVASS</td><td> 540</td>
<td>23. CFEFYHKCNN</td><td>ECMESVKNGT</td><td>YDYPKYSEES</td><td>KLNREKIDGV</td><td>KLESMGVYQI</td><td>LAIYSTVASS</td><td> 540</td>
<td>24. CFEFYHKCNN</td><td>ECMESVKNGT</td><td>YDYPKYSEES</td><td>KLNREKIDGV</td><td>KLESMGVYQI</td><td>IAIYSTAASS</td><td> 539</td>
<td>25. CFEFYHKCNN</td><td>ECMESVKNGT</td><td>YDYPKYSEES</td><td>KLNREKIDGV</td><td>KLESMGVYQI</td><td>LAIYSTVASS</td><td> 540</td>
<td>26. CFEFYHKCDN</td><td>ECMESVRNGT</td><td>YDYPKYSEES</td><td>KLNREKVDGV</td><td>KLESMGIYQI</td><td>LAIYSTVASS</td><td> 539</td>
27. CFEFYHKCDN ECMESVRNGT YDYPKYSEES KLNREKIDGV KLESMGVYQI LAIYSTVASS 539
<img file="MX357009B_D0264.tif" />
28. CFEFYHKCDD ACMESVRNGT YDYPKYSEES KLNREEIDGV KLESMGVYQI LMY-SWASS-Me—
Ar-ArArAc-ArArATAr ·· ArArArArAr »ArArAr ★★★★★ · ★ · ★ · Ar Ar Ar Ar · Ar Ar Ar AA ** · Ar · Ar
AAAAAA.Ar Ar Ar
<td> 1.</td><td>LVLLVSLGAI</td><td>SFWMCSNGSL i</td><td>2CRICI</td><td> 565</td>
<td> 2.</td><td>LVLLVSLGAI</td><td>SFWMCSNGSL i</td><td>QCRICI</td><td> 565</td>
<td> 3.</td><td>LVLLVSLGAI</td><td>SFWMCSNGSL '</td><td>QCRICI</td><td> 565</td>
<td> 4.</td><td>LVLVVSLGAI</td><td>SFWMCSNGSL <</td><td>2CRICI</td><td> 566</td>
<td> 5.</td><td>LVLLVSLGAI</td><td>SFWMCSNGSL '</td><td>QCRVCI</td><td> 566</td>
<td> 6.</td><td>LVLLVSLGAI</td><td>SFWMCSNGSL '</td><td>QCRICI</td><td> 566</td>
<td> 7.</td><td>LVLLVSLGAI</td><td>SFWMCSNGSL '</td><td>QCRICI</td><td> 565</td>
<td> 8 .</td><td>LVLLVSLGAI</td><td>SFWMCSNGSL '</td><td>QCRICI</td><td> 565</td>
<td> 9.</td><td>LVLLVSLGAI</td><td>SFWMCSNGSL '</td><td>QCRICI</td><td> 566</td>
<td> 10</td><td>. LVLLVSLGAI</td><td>SFWMCSNGSL</td><td>QCRICI</td><td> 566</td>
<td> 11</td><td>. LVLLVSLGAI</td><td>SFWMCSNGSL</td><td>QCRICI</td><td> 565</td>
<td> 12</td><td>. LVLLVSLGAI</td><td>SFWMCSNGSL</td><td>QCRICI</td><td> 566</td>
<td> 13</td><td>. LVLLVSLGAI</td><td>SFWMCSNGSL</td><td>QCRICI</td><td> 566</td>
<td> 14</td><td>. LVLLVSLGAI</td><td>SFWMCSNGSL</td><td>QCRICI</td><td> 566</td>
<td> 15</td><td>. LVLLVSLGAI</td><td>SFWMCSNGSL</td><td>QCRICI</td><td> 566</td>
<td> 16</td><td>. LVLLVSLGAI</td><td>SFWMCSNGSL</td><td>QCRICI</td><td> 566</td>
<td> 17</td><td>. LVLLVSLGAI</td><td>SFWMCSNGSL</td><td>QCRICI</td><td> 566</td>
<td> 18</td><td>. LVLLVSLGAI</td><td>SFWMCSNGSL</td><td>QCRICI</td><td> 566</td>
<td> 19</td><td>. LVLLVSLGAI</td><td>SFWMCSNGSL</td><td>QCRICI</td><td> 565</td>
<td> 20</td><td>. LVLLVSLGAI</td><td>SFWMCSNGSL</td><td>QCRICI</td><td> 566</td>
<td> 21</td><td>. LVLLVSLGAI</td><td>SFWMCSNGSL</td><td>QCRICI</td><td> 566</td>
276
<td> 22.</td><td>LVLLVSLGAI.</td><td>SFWMCSNGSL</td><td>QCRICI</td><td> 566</td>
<td> 23.</td><td>LVLLVSLGAI</td><td>SFWMCSNGSL</td><td>QCRICI</td><td> 566</td>
<td> 24.</td><td>LVLLVSLGAI</td><td>SFWMCSNGSL</td><td>QCRICI</td><td> 565</td>
<td> 25.</td><td>LVLLVSLGAI</td><td>SFWMCSNGSL</td><td>QCRICI</td><td> 566</td>
<td> 26.</td><td>LVLLVSLGAI</td><td>SFWMCSNGSL</td><td>QCRICI</td><td> 565</td>
<td> 27.</td><td>LVLLVSLGAI</td><td>SFWMCSNGSL</td><td>QCRICI</td><td> 565</td>
<td> 28.</td><td>LVLLVSLGAI</td><td>SFWMCSNGSL</td><td>QCRICI</td><td> 566</td>
★★★★★★
<img file="MX357009B_D0265.tif" />
Table 9. Consensus sequence for H3 401-421 (SEQ ID NO: 104), other natural variants, and mutations that stabilize the polypeptides of the invention. One or more mutations in the parental sequence are present in the polypeptides of the invention.
<td>Posi tion</td><td>Not me acid</td><td>Preserves tion (%)</td><td>Other Natu ral</td><td>Muta tion Prefe laugh</td><td>Polar</td><td>Loaded</td><td>Flexi ble</td>
<td> 401</td><td>I</td><td> 99.26</td><td>V</td><td>R</td><td></td><td>K</td><td></td>
<td> 402</td><td>E / G</td><td> 56.2/40.3</td><td>K</td><td></td><td></td><td></td><td></td>
<td> 403</td><td>K</td><td> 94.85</td><td>R</td><td></td><td></td><td></td><td></td>
<td> 404</td><td>T</td><td> 99.88</td><td>TO</td><td></td><td></td><td></td><td></td>
<td> 405</td><td>N</td><td> 99.88</td><td>S</td><td></td><td></td><td></td><td></td>
<td> 406</td><td>AND</td><td> 10.0</td><td></td><td></td><td></td><td></td><td></td>
<td> 407</td><td>K</td><td> 100</td><td></td><td></td><td></td><td></td><td></td>
<td> 408</td><td>F</td><td> 100</td><td></td><td>S</td><td>T, N, Q</td><td>R, H, K, D ,AND</td><td>G</td>
Η * ΤΓ
3/33
277
IMP
Mexican INSTITUTE 0Ϊ LA PEOI'IÍUAI industrial
<img file="MX357009B_D0266.tif" />
amino acid composition of the processed polypeptide of the invention, ie after the leader peptide has been cleaved off. The additional mass arising from glycosylation has not been taken into account.
<td>SEQ ID NO</td><td>Molecular weight calculated (Da)</td><td>Molecular weight experimental (kDa)</td>
<td> 144</td><td> 29137</td><td> 75</td>
<td> 145</td><td> 29257</td><td> 50</td>
<td> 146</td><td> 29203</td><td> 56</td>
<td> 147</td><td> 29119</td><td> 60</td>
278
4 <ΤΙΤυΤ () MEXICAN OF THE PKüMfc'DAP iUD'jnUlAL
<td> 148</td><td> 29293</td><td> 66</td><td></td>
<td> 150</td><td> 29411</td><td colspan="2"> 64</td>
<td> 151</td><td> 29283</td><td colspan="2"> 79</td>
Table 11.
<td colspan="2">Selected strains</td>
<td>Set 1</td><td>Set 2</td>
<td>H1N1 A / Maryland / 12/1991</td><td>HlNl A / Maryland / 12/1991</td>
<td>H1N1 A / Henry / 1936 /</td><td>H1N1 A / Henry / 1936 /</td>
<td>H1N1 A / AA / Marton / 1943</td><td>HlNl A / AA / Marton / 1943</td>
<td>H1N1 A / Memphis / 20/1978</td><td>HlNl A / USSR / 92/1977</td>
<td>H1N1 A / New York / 607/1995</td><td>HlNl A / New York / 629/1995</td>
<td>H1N1 A / New Jersey / 11/2007</td><td>HlNl A / Virginia / UR06- 0549/2007</td>
<td>H1N1 A / Wisconsin / 629-D01415 / 2009</td><td>HlNl A / Texas / UR06-0526 / 2007</td>
<td></td><td>HlNl A / Sydney / DD3-55 / 2010</td>
<img file="MX357009B_D0267.tif" />
<img file="MX357009B_D0268.tif" />
279
NOVELTY OF THE INVENTION
Having described the present invention, it is considered as a novelty, and therefore the content of the following is claimed as property:
Contents498
374 sheets
Sheet 1 Sheet 2 Sheet 3 Sheet 4 Sheet 5 Sheet 6 Sheet 7 Sheet 8 Sheet 9 Sheet 10 Sheet 11 Sheet 12 Sheet 13 Sheet 14 Sheet 15 Sheet 16 Sheet 17 Sheet 18 Sheet 19 Sheet 20 Sheet 21 Sheet 22 Sheet 23 Sheet 24 Sheet 25 Sheet 26 Sheet 27 Sheet 28 Sheet 29 Sheet 30 Sheet 31 Sheet 32 Sheet 33 Sheet 34 Sheet 35 Sheet 36 Sheet 37 Sheet 38 Sheet 39 Sheet 40 Sheet 41 Sheet 42 Sheet 43 Sheet 44 Sheet 45 Sheet 46 Sheet 47 Sheet 48 Sheet 49 Sheet 50 Sheet 51 Sheet 52 Sheet 53 Sheet 54 Sheet 55 Sheet 56 Sheet 57 Sheet 58 Sheet 59 Sheet 60 Sheet 61 Sheet 62 Sheet 63 Sheet 64 Sheet 65 Sheet 66 Sheet 67 Sheet 68 Sheet 69 Sheet 70 Sheet 71 Sheet 72 Sheet 73 Sheet 74 Sheet 75 Sheet 76 Sheet 77 Sheet 78 Sheet 79 Sheet 80 Sheet 81 Sheet 82 Sheet 83 Sheet 84 Sheet 85 Sheet 86 Sheet 87 Sheet 88 Sheet 89 Sheet 90 Sheet 91 Sheet 92 Sheet 93 Sheet 94 Sheet 95 Sheet 96 Sheet 97 Sheet 98 Sheet 99 Sheet 100 Sheet 101 Sheet 102 Sheet 103 Sheet 104 Sheet 105 Sheet 106 Sheet 107 Sheet 108 Sheet 109 Sheet 110 Sheet 111 Sheet 112 Sheet 113 Sheet 114 Sheet 115 Sheet 116 Sheet 117 Sheet 118 Sheet 119 Sheet 120 Sheet 121 Sheet 122 Sheet 123 Sheet 124 Sheet 125 Sheet 126 Sheet 127 Sheet 128 Sheet 129 Sheet 130 Sheet 131 Sheet 132 Sheet 133 Sheet 134 Sheet 135 Sheet 136 Sheet 137 Sheet 138 Sheet 139 Sheet 140 Sheet 141 Sheet 142 Sheet 143 Sheet 144 Sheet 145 Sheet 146 Sheet 147 Sheet 148 Sheet 149 Sheet 150 Sheet 151 Sheet 152 Sheet 153 Sheet 154 Sheet 155 Sheet 156 Sheet 157 Sheet 158 Sheet 159 Sheet 160 Sheet 161 Sheet 162 Sheet 163 Sheet 164 Sheet 165 Sheet 166 Sheet 167 Sheet 168 Sheet 169 Sheet 170 Sheet 171 Sheet 172 Sheet 173 Sheet 174 Sheet 175 Sheet 176 Sheet 177 Sheet 178 Sheet 179 Sheet 180 Sheet 181 Sheet 182 Sheet 183 Sheet 184 Sheet 185 Sheet 186 Sheet 187 Sheet 188 Sheet 189 Sheet 190 Sheet 191 Sheet 192 Sheet 193 Sheet 194 Sheet 195 Sheet 196 Sheet 197 Sheet 198 Sheet 199 Sheet 200 Sheet 201 Sheet 202 Sheet 203 Sheet 204 Sheet 205 Sheet 206 Sheet 207 Sheet 208 Sheet 209 Sheet 210 Sheet 211 Sheet 212 Sheet 213 Sheet 214 Sheet 215 Sheet 216 Sheet 217 Sheet 218 Sheet 219 Sheet 220 Sheet 221 Sheet 222 Sheet 223 Sheet 224 Sheet 225 Sheet 226 Sheet 227 Sheet 228 Sheet 229 Sheet 230 Sheet 231 Sheet 232 Sheet 233 Sheet 234 Sheet 235 Sheet 236 Sheet 237 Sheet 238 Sheet 239 Sheet 240 Sheet 241 Sheet 242 Sheet 243 Sheet 244 Sheet 245 Sheet 246 Sheet 247 Sheet 248 Sheet 249 Sheet 250 Sheet 251 Sheet 252 Sheet 253 Sheet 254 Sheet 255 Sheet 256 Sheet 257 Sheet 258 Sheet 259 Sheet 260 Sheet 261 Sheet 262 Sheet 263 Sheet 264 Sheet 265 Sheet 266 Sheet 267 Sheet 268 Sheet 269 Sheet 270 Sheet 271 Sheet 272 Sheet 273 Sheet 274 Sheet 275 Sheet 276 Sheet 277 Sheet 278 Sheet 279 Sheet 280 Sheet 281 Sheet 282 Sheet 283 Sheet 284 Sheet 285 Sheet 286 Sheet 287 Sheet 288 Sheet 289 Sheet 290 Sheet 291 Sheet 292 Sheet 293 Sheet 294 Sheet 295 Sheet 296 Sheet 297 Sheet 298 Sheet 299 Sheet 300 Sheet 301 Sheet 302 Sheet 303 Sheet 304 Sheet 305 Sheet 306 Sheet 307 Sheet 308 Sheet 309 Sheet 310 Sheet 311 Sheet 312 Sheet 313 Sheet 314 Sheet 315 Sheet 316 Sheet 317 Sheet 318 Sheet 319 Sheet 320 Sheet 321 Sheet 322 Sheet 323 Sheet 324 Sheet 325 Sheet 326 Sheet 327 Sheet 328 Sheet 329 Sheet 330 Sheet 331 Sheet 332 Sheet 333 Sheet 334 Sheet 335 Sheet 336 Sheet 337 Sheet 338 Sheet 339 Sheet 340 Sheet 341 Sheet 342 Sheet 343 Sheet 344 Sheet 345 Sheet 346 Sheet 347 Sheet 348 Sheet 349 Sheet 350 Sheet 351 Sheet 352 Sheet 353 Sheet 354 Sheet 355 Sheet 356 Sheet 357 Sheet 358 Sheet 359 Sheet 360 Sheet 361 Sheet 362 Sheet 363 Sheet 364 Sheet 365 Sheet 366 Sheet 367 Sheet 368 Sheet 369 Sheet 370 Sheet 371 Sheet 372 Sheet 373 Sheet 374
70 members in 25 offices
Priority claims34
| Document | Office | Kind | Date |
|---|---|---|---|
| 11191003 | European Patent Office (EPO) | A | |
| 11191003 | European Patent Office (EPO) | A | |
| 111910030 | European Patent Office (EPO) | – | |
| 11191009 | European Patent Office (EPO) | A | |
| 11191009 | European Patent Office (EPO) | A | |
| 111910097 | European Patent Office (EPO) | – | |
| 201161564086 | United States of America | P | |
| 201161564086 | United States of America | P | |
| 201161564198 | United States of America | P | |
| 201161564198 | United States of America | P | |
| 61564086 | United States of America | – | |
| 61564198 | United States of America | – | |
| 12166268 | European Patent Office (EPO) | A | |
| 12166268 | European Patent Office (EPO) | A | |
| 121662688 | European Patent Office (EPO) | – | |
| 201261720281 | United States of America | P | |
| 201261720281 | United States of America | P | |
| 61720281 | United States of America | – | |
| 2012073706 | European Patent Office (EPO) | W | |
| 2012073706 | European Patent Office (EPO) | W | |
| 111910030 | – | – | – |
| 111910097 | – | – | – |
| 121662688 | – | – | – |
| 61564086 | – | – | – |
| 61564198 | – | – | – |
| 61720281 | – | – | – |
| EP20110191003 | – | – | – |
| EP20110191009 | – | – | – |
| EP20120166268 | – | – | – |
| PCTEP2012073706 | – | – | – |
| US201161564086P | – | – | – |
| US201161564198P | – | – | – |
| US201261720281P | – | – | – |
| WO2012EP73706 | – | – | – |
Members70
| Document | Office | Kind | |
|---|---|---|---|
| CA2857087A1 | Canada | A1 | |
| WO2013079473A1 | World Intellectual Property Organization (WIPO) | A1 | |
| TW201329101A | Taiwan Province of China | A | |
| CA2857550A1 | Canada | A1 | |
| WO2013105113A1 | World Intellectual Property Organization (WIPO) | A1 | |
| TW201331234A | Taiwan Province of China | A | |
| WO2013105113A4 | World Intellectual Property Organization (WIPO) | A4 | |
| SG11201402792UA | Singapore | A | |
| AU2012343981A1 | Australia | A1 | |
| MX2014006406A | Mexico | A | |
| MX2014006396A | Mexico | A | |
| AR089005A1 | Argentina | A1 | |
| AU2012365382A1 | Australia | A1 | |
| PH12014501142A1 | Philippines | A1 | |
| PH12014501142B1 | Philippines | B1 | |
| PH12014501118A1 | Philippines | A1 | |
| PH12014501118B1 | Philippines | B1 | |
| KR20140097265A | Republic of Korea | A | |
| KR20140099515A | Republic of Korea | A | |
| CN104066446A | China | A | |
| SG11201402633UA | Singapore | A | |
| CU20140061A7 | Cuba | A7 | |
| EP2785372A1 | European Patent Office (EPO) | A1 | |
| EP2785780A1 | European Patent Office (EPO) | A1 | |
| US2014357845A1 | United States of America | A1 | |
| US2014364660A1 | United States of America | A1 | |
| JP2015500355A | Japan | A | |
| AU2012365382B2 | Australia | B2 | |
| JP2015502353A | Japan | A | |
| CN104350095A | China | A | |
| EA201491051A1 | Eurasian Patent Organization (EAPO) | A1 | |
| HK1199211A1 | Hong Kong, China | A1 | |
| EP2785780B1 | European Patent Office (EPO) | B1 | |
| IN4742CHN2014A | India | A | |
| ES2548553T3 | Spain | T3 | |
| ZA201404795B | South Africa | B | |
| PL2785780T3 | Poland | T3 | |
| RU2014126229A | Russian Federation | A | |
| MX339245B | Mexico | B | |
| KR101625041B1 | Republic of Korea | B1 | |
| JP5952914B2 | Japan | B2 | |
| CN104350095B | China | B | |
| US9452211B2 | United States of America | B2 | |
| NZ625973A | New Zealand | A | |
| TWI560203B | Taiwan Province of China | B | |
| US2016355553A1 | United States of America | A1 | |
| US2016362455A1 | United States of America | A1 | |
| CA2857550C | Canada | C | |
| BR112014012681A2 | Brazil | A2 | |
| BR112014012681A8 | Brazil | A8 | |
| RU2625300C2 | Russian Federation | C2 | |
| US9725649B2 | United States of America | B2 | |
| AU2012343981B2 | Australia | B2 | |
| IL232780A | Israel | A | |
| CN104066446B | China | B | |
| JP6294828B2 | Japan | B2 | |
| TWI618715B | Taiwan Province of China | B | |
| JP2018052953A | Japan | A | |
| US9969778B2 | United States of America | B2 | |
| MX357009BThis record | Mexico | B | |
| MY167439A | Malaysia | A | |
| KR101983989B1 | Republic of Korea | B1 | |
| EP2785372B1 | European Patent Office (EPO) | B1 | |
| MY170927A | Malaysia | A | |
| EA033386B1 | Eurasian Patent Organization (EAPO) | B1 | |
| EP3566714A1 | European Patent Office (EPO) | A1 | |
| SG10201908920QA | Singapore | A | |
| BR112014013283B1 | Brazil | B1 | |
| CA2857087C | Canada | C | |
| ZA201404797B | South Africa | B |
2 legal events, as the office reported them to INPADOC
Over the term
Point at a mark for the eventEvents
| Event | Code | |
|---|---|---|
| Grant or registrationFG | FG | |
| Change of company name or juridical statusHC | HC |
Numbers
- Publication
- 357009
- Publication, DOCDB
- 357009
- Publication, EPODOC
- MX357009
- Application
- 2014006396
- Application, DOCDB
- 2014006396
- Application, EPODOC
- MX20140006396
Titles2
- Spanish
- VACUNAS CONTRA EL VIRUS DE LA INFLUENZA Y SUS USOS.
- English
- INFLUENZA VIRUS VACCINES AND USES THEREOF.
Classification
- CPC, 22
- C07K14/005
- A61K39/12
- C07K2319/21
- C07K2319/35
- C07K2319/50
- C07K2319/73
- C12N2760/16122
- C12N2760/16134
- A61K2039/55522
- A61P31/14
- A61P31/16
- A61K39/00
- A61K2039/5258
- C07K16/108
- A61K39/145
- C12N7/00
- C12N2760/16151
- C12N2760/16171
- C07K2317/33
- C07K2317/76
- A61K2039/575
- C07K2319/40
- IPC, 5
- C07K14 11
- A61K39 12
- A61K39 145
- C07K16 10
- C12N7 00