Rapid and prolonged immunologic-therapeutic.
Abstract
The present invention shows that intranasal administration of E1 / E3 defective adenovirus particles can confer rapid and comprehensive protection against viral and bacterial pathogens in a variety of disease settings. Protective responses last for many weeks on a single dose regimen in animal models. When a pathogen-derived antigen gene is inserted into the E1 / E3-defective adenovirus genome, the antigen-induced protective immunity against the specific pathogen is induced before the adenovirus-mediated protective response declines by elimination, and thus provides fast, long-lasting protection without protections against pathogens. In addition to E1 / E3-defective adenovirus, other biomanipulated non-replicating vectors encoding pathogen-derived antigens can also be developed in a new generation of rapid and long-term immunologic therapeutics (RAPIT).

Term
5.5 yearsleft in the term
Expires 21 March 2032.
- Priority
- Filed
- Granted
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- Expires
17 claims: 9 independent, 8 dependent
- 1REIVINDICACIONES Habiéndose descrito la invención como antecede, se —, «.y T »11^· g reclama como propiedad lo contenido en las siguientes reivindicaciones:1. Un adenovirus suprimido en El y E3 para usarse en un método para inducir una respuesta protectora contra patógenos respiratorios en un sujeto mamífero en necesidad del mismo, que está adaptado para ser administradle intranasalmente, 1 ó 2 días antes de exponer al patógeno respiratorio, una cantidad eficaz de adenovirus suprimido en El y E3, con o sin codificación de un antígeno derivado de patógeno.
- 2El adenovirus suprimido en El y E3 para usarse de conformidad con la reivindicación 1, en donde el adenovirus suprimido en El y E3 es un adenovirus vacío que no codifica un antígeno derivado de patógeno.
- 3El adenovirus suprimido en El y E3 para usarse de conformidad con la reivindicación 1, en donde el patógeno respiratorio es un virus, o una bacteria.
- 4El adenovirus suprimido en El y E3 para usarse de conformidad con la reivindicación 3, en donde el virus es un virus de influenza, un virus sincicial respiratorio (RSV), un virus del resfriado común o un virus del sarampión.
- 5El adenovirus suprimido en El y E3 para usarse de conformidad con la reivindicación 4, en donde el virus del 142 IMPI INSTITUTO ΜίΧΙΟΛΝΟ D * la nent:AD INÜUSTIUA1 resfriado común es un rinovirus o un coronaviTusT
- 6El adenovirus suprimido en El y E3 para usarse de conformidad con la reivindicación 3, en donde la bacteria se selecciona del grupo que consiste de Bacillus, Mycobacterium, Staphylococcus, Streptococcus, Pseudomonas, Klebsiella, Haemophilus y Mycoplasma.
- 7El adenovirus suprimido en El y E3 para usarse de conformidad con la reivindicación 6, en donde la bacteria es Bacillus anthracis.
- 8El adenovirus suprimido en El y E3 para usarse de conformidad con la reivindicación 1, en donde el patógeno respiratorio es un hongo.
- 9El adenovirus suprimido en El y E3 para usarse de conformidad con la reivindicación 8, en donde el hongo es Aspergí 11us.
- 10El adenovirus suprimido en El y E3 para usarse de conformidad con cualquiera de las reivindicaciones 1-8, en donde la cantidad eficaz es por lo menos 10 7 unidades infecciosas (ifu) de adenovirus suprimido en El y E3 con o sin codificación de un antígeno derivado de patógeno, preferiblemente por lo menos 10 8 unidades infecciosas (ifu) de adenovirus vacío defectuoso o suprimido en El y E3, incluso más preferiblemente por lo menos 10 9 unidades infecciosas (ifu) de adenovirus vacío defectuoso o suprimido en El y E3. 143 •NSTlTln·,. * -Λ • N ° u riUAi
- 11El adenovirus suprimido en El y E3 para usar56~^— de conformidad con cualquiera de las reivindicaciones 1-10, en donde el sujeto en necesidad del mismo es un adulto.
- 12El adenovirus suprimido en El y E3 para usarse de conformidad con cualquiera de las reivindicaciones 1-10, en donde el sujeto en necesidad del mismo es un niño.
- 13El adenovirus suprimido en El y E3 para usarse de conformidad con cualquiera de las reivindicaciones 1-12, en donde el sujeto en necesidad del mismo es un paciente inmunocomprometido.
- 14El adenovirus suprimido en El y E3 para usarse de conformidad con cualquiera de las reivindicaciones 1-13, que comprende por lo menos dos etapas de administración, en donde la administración es con un intervalo de 40 días entre ellas, de 41 días entre ellas, de 42 días entre ellas, de 43 días entre ellas, de 44 días entre ellas, de 45 días entre ellas, de 46 días entre ellas, de 47 días entre ellas, de 48 días entre ellas, de 49 días entre ellas, o de 50 días entre ellas.
- 15El adenovirus suprimido en El y E3 para usarse de conformidad con cualquiera de las reivindicaciones 1-14, en donde el adenovirus es un adenovirus humano, un adenovirus bovino, un adenovirus canino, un adenovirus primate no humano, un adenovirus de pollo o un adenovirus porcino o de cerdo. 144 IMPI •¿«UTUTO MEX'CAN industrial
- 16El adenovirus suprimido en El y E3 para jasarse--—— de conformidad con cualquiera de las reivindicaciones 1-15, en donde la respuesta protectora se provoca dentro de 24 horas.
- 17El adenovirus suprimido en El y E3 para usarse de conformidad con cualquiera de las reivindicaciones 1-16, en donde la respuesta protectora dura de 1 día a 47 días. - 145 IMPI INSTITUTO MEXiON 1 C£ LA FKOPILPaD INUU.'-TklAL
Independent claims17
861 paragraphs in 52 sections, as filed
(54) Title: RAPID AND PROLONGED THERAPEUTIC IMMUNOLOGICAL SUBSTANCE.
(54) Title: RAPID AND PROLONGED IMMUNOLOGIC-THERAPEUTIC.
(57) Summary
The present invention relates to the intranasal administration of E1 / E3 defective adenovirus particles that can confer rapid and comprehensive protection against viral and bacterial pathogens in a variety of disease settings. Protective responses last for many weeks on a single dose regimen in animal models. When a pathogen-derived antigen gene is inserted into the E1 / E3-defective adenovirus genome, the antigen-induced protective immunity against the specific pathogen is induced before the adenovirus-mediated protective response declines by elimination, and thus provides fast, long-lasting protection without protections against pathogens. In addition to E1 / E3-defective adenovirus, other biomanipulated non-replicating vectors encoding pathogen-derived antigens can also be developed in a new generation of Rapid and Extended Therapeutic Immunologic Substances (RAPIT).
(57) Abstract
The present invention shows that intranasal administration of E1 / E3 -detective adenovirus particles may confer rapid and broad protection against viral and bacterial pathogens in a variety of disease settings. Protective responses lasted for many weeks in a single-dose regimen in animal models. When a pathogen-derived antigen gene was inserted into the EI / E3defective adenovirus genome, the antigen-induced protective immunity against the specific pathogen was elicited before the adenovirus-mediated protective response declined away, thus conferring rapid, prolonged, and seamless protection against pathogens . In addition to E1 / E3 -detective adenovirus, other bioengineered non-replicating vectors encoding pathogen-derived antigens may also be developed into a new generation of rapid and prolonged immunologic-therapeutic (RAPIT).
Μ Ρ1 '!: Η: ^ ΒΙΜ »
PATENT TITLE No. 348172
Owner (s): ALTIMMUNE INC.
Address: 19 Firstfield Road, Gaithersburg, Maryland, 20878, USA
Denomination: RAPID AND PROLONGED THERAPEUTIC IMMUNOLOGICAL SUBSTANCE.
Classification: CIP: A61K39 / 145; A61K39 / 00; A61K39 / 07; A61K39 / 12; A61K39 / 23; A61K39 / 155;
C12N15 / 86
CPC: A61K39 / 145; A61K39 / 00; A61K39 / 07; A61K39 / 12; A61K39 / 155; C12N15 / 86;
A61K2039 / 58; A61K2039 / 543: * * ...
Inventor (s): DE-CHU C. TANG,
REQUEST
Number: International Presentation Date:
MX / a / 2013/010794 March 21, 2012
PRIORITY
Country: ......... Date: Number:
US March 21, 2011 61 / 454,819
US December 7, 2011. 61 / 568,054
Validity: Twenty years
Expiration Date: March 21, 2032
Issue Date: June 2, 2017
The reference patent is granted <sup>with</sup> based on articles 1 “, 2nd section V, 6th section III, and 59 of the Industrial Property Law.
In accordance with article 23 of the Industrial Property Law, this patent is valid for twenty years, which cannot be extended, counted from the date of presentation of the application and will be subject to payment of the fee to keep the rights. '
Whoever signs this title does so based on the provisions of articles 6 ° sections III and 7 ° bis 2 of the Industrial Property Law (Official Gazette of the Federation (BO.F.) 27/96/1991, amended on 0206/1994, 10/25/1996, 12/26/1997, 17) 05/1999, 01/26/2004, 06/16/2005, 01/25/2006, 05/06/2009, 06/01 / 2010, 06/18/2010, 06/28/2040,27/01/2012 and 09/94/2012); Articles 1 », 3rd section V subsection a), 4th and 12th sections I and III of the Regulations of the Mexican Institute of Industrial Property (DOF 12/14/1999, amended on 07/01/2002, 07/15/2004 , 07/28/2004 and 09/07/2007); Articles 1, 3, 4, 5 fraction V beginning a), 16 fractions.! and lf! and 3® of the Organic Statute of the Mexican Institute of Industrial Property (DOF 12/27/1999, amended on 10/10/2002, 07/29/2004, 08/04/2004 and 09/13/2007); 1st, 3rd and 5th initiation a) of the Agreement which delegates powers to the Deputy General Directors, Coordinator, DMSional Directors, Office Holders · »Begtonates, Divisional Deputy Directors, Departmental Coordinators and other subordinates of the Mexican Institute of Industrial Property . (DOF 12/15/1999, amended on 02/04/2000, 07/29/2004, 08/04/2004 and 09/13/2007). This document 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 Payment and Electronic Services Portal (PASE) of the Mexican Institute of Industrial Property, in the procedures indicated.
<td></td><td></td><td>THE DIVISIONAL DIRECTOR OF PATENTS</td>
<td></td><td></td><td>NAHANNY CANAL REYES</td>
<td> 0^</td><td></td><td>Original string:</td>
<td></td><td></td><td>NAHANNY MARISOL CANAL REYES | 00001000000403252793 | Administration Service Tax | 1695 || MX / 2017/43607 | MX / a / 2013/010794 | PCT patent title | 1488 | IAR | Page (s) 2 | 15OZcScpHYFp / XR1RuH7LhJJXZ8 =</td>
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• Additional information on the back
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RAPID AND PROLONGED THERAPEUTIC IMMUNOLOGICAL SUBSTANCE
FIELD OF THE INVENTION
The following applications and all the documents mentioned in the present or during its prosecution (documents mentioned in the application) and all the documents mentioned or referred to in the documents mentioned in the application, and all the mentioned documents that are referenced herein (documents mentioned herein) and all documents mentioned or referenced in documents mentioned herein, together with any instructions, descriptions, product specifications, and manufacturer's product sheets for any of the products mentioned herein or in any document incorporated by reference herein are incorporated herein by reference, and may be used in practice. of the invention. More specifically, all referenced documents are incorporated by reference to the same extent so that each inhibitor document is specifically and individually indicated as incorporated by reference.
The present invention relates generally to the fields of immunology and therapeutic technology. The present invention also relates to
REF: 243660 <sup>2</sup>'IMPÍ ^
MEXICAN INSTITUTE
Dt THE PROPERTY ______. ,. . . ,. . INDUSTRIAL> <=! methods for inducing rapid-prolonged innate immune responses and uses thereof. ———
BACKGROUND OF THE INVENTION
The disease-fighting ability of immunological substances (eg vaccines) and therapeutic substances (eg drugs) has had a health bonanza that has been evidenced in a global reduction in mortality and morbidity. The objective is to further amplify the capacity of medical intervention that requires the development of a new generation of rapid response immunological substances that can be mass-produced at low costs and administered in a general way by personnel without medical training; as well as a new generation of therapeutic substances that can confer prolonged protection preferably without being impaired by drug resistance. New immunological and therapeutic substances must also be endowed with a greater margin of safety compared to conventional vaccines and medicines.
The use of conventional drugs against microbial pathogens often builds drug resistance over time because microbes are constantly evolving under natural pressure. This invention illustrates that an antiviral or antibacterial state can be rapidly induced in animals after
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‘<sup>3</sup>'MAID
INGGPITO McX.Can Of LA PR <fie; <sub>TO</sub>| intranasal administration of a particle of ^ 'acle] defective in E1 / E3 (ΔΕ1Ε3) by changing the habitat in 1'átJ airways that prevents the growth of pathogens. Since adenovirus particles do not directly attack the pathogen, there is little likelihood that this novel therapeutic substance will induce drug resistance. In addition, the adenovirus-induced anti-pathogen state may persist for many weeks in animals, a period long enough to overlap with the induction of protective immunity induced by a pathogen-derived antigen expressed from the adenovirus, if a pathogen-derived antigen is inserted into the adenovirus genome as a vaccine. It is conceivable that the non-replicating adenovirus particle could be co-administered with other mucosal vaccines as a therapeutic adjuvant.
The vaccine that uses non-replicating adenovirus as the vector holds promise in enhancing vaccine coverage because the vector can be rapidly manufactured in serum-free suspension cells in response to a surge in demand. Furthermore, pre-existing immunity to adenovirus does not appreciably interfere with the potency of a nasal vaccine using adenovirus as a vector. In addition to human vaccination, animals can also be massively immunized by this class of targeted vaccines.
MAID
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> Al Ai.
There are a lot of lawsuits for my young vaccines. Although vaccination has proven to be the cost-effective method of disease prevention, a devastating push for booster vaccine coverage remains a driving goal in the movement toward improved public health around the world. Current vaccines that have been licensed for commercialization include whole killed microorganisms, live attenuated microorganisms, microbial extracts, purified or recombinant proteins, DNA vaccines, and virus-like particles. Although many diseases have been defeated by the wide distribution of these vaccines, the goal of generating immunity to a community (herds) in a wide variety of disease settings still remains elusive due to the large number of problems in current vaccination programs.
Specifically, adverse side effects associated with vaccines range from a local and systemic inflammatory response, fever, platelet activation, cardiac autonomic dysfunction, anaphylactic reaction (induced by needle injection of certain vaccines) [Solomon ME, Halperin R, Yee J Evaluation of the two-needle strategy for reducing reactions to DPT vaccination. Am. J. Dis. Child. 141, 796-798 (1987), Lanza GA, Barone L, Scalone G et al. Inflammationrelated effects of adjuvant influenza A vaccination on platelet activation and cardiac autonomic function. J.
Intern. Med. 269, 118-125 (2011), Jae SY, HeJ_f ernan JKS ^ ParF SH et al. Does an acute inflammatory response temporariíy --attenuate parasympathetic reactivation? Clin. Auton. Res. 20, 229-233 (2010) and Sever JL, Brenner Al, Gale AD et al.
Safety of anthrax vaccine: an expanded review and evaluation of adverse events reported to the Vaccine Adverse Event Reporting System (VAERS). Pharmacoepidemiol. Drug Saf. 13, 825-840 (2004)] to the rare occurrence of paralytic poliomyelitis (mediated by ingestion of the oral polio vaccine) [Minor P. Vaccine-derived poliovirus (VDPV): impact on poliomyelitis eradication. Vaccine 27, 2649-2652 (2009)], myopericarditis (induced by inoculation of the Dryvax smallpox vaccine) [Poland GA, Grabenstein JD, Neff JM. The US smallpox vaccination program: a review of a large modem was smallpox vaccination implementation program. Vaccine 23, 2078-2081 (2005)] and Bell's palsy (induced by a bacterial toxin nasal adjuvant) [Lewis DJ, Huo Z, Bamett S et al. Transient facial nerve paralysis (Bell's palsy) following intranasal delivery of a genetically detoxified mutant of Escherichia coli heat labile toxin. PLoS ONE 4, e6999 (2009) and Couch RB. Nasal vaccination, Escherichia coli enterotoxin, and Bell's palsy. N. Engl. J. Med. 350, 860-861 (2004)].
In 2010, a surge in narcolepsy has been reported among vaccinated people in some countries after water injection of an H1N1 pandemic influenza vaccine containing the adjuvant squarfeso-r Squalene injection can only induce rheumatoid in animals [Carlson BC , Jansson AM, Larsson A,
Bucht A, Lorentzen JC. The endogenous adjuvant squalene can induce a chronic T-cell-mediated arthritis in rats. Am. J. Pathol. 156, 2057-2065 (2000)]. Evidence has emerged showing that chronic low-grade inflation is associated with cardiovascular disease [Finch CE, Crimmins EM.
Inflammatory exposure and historical changes in human life-spans. Science 305, 1736-1739 (2004)], with obesity [Gregor MF, Hotamisligil GS. Inflammatory mechanisms in obesity. Annu. Rev. Immunol. 29, 415-445 (2011)], with diabetes [Gregor MF, Hotamisligil GS. Inflammatory mechanisms in obesity. Annu. Rev. Immunol. 29, 415-445 (2011)], with cancer [O'Callaghan DS, O'Donnell D, O'Connell F, O'Byrne KJ. The role of inflammation in the pathogenesis of non-small cell lung cancer. J. Thorac. Oncol. 5, 2024-2036 (2010)] and with neurological disorders [Witte ME, Geurts JJ, de Vries HE, van der Valk P, van Horssen J. Mitochondrial dysfunction: a potential link between neuroinflammation and neurodegeneration? Mitochondrion 10, 411-418 (2010)], vaccine-induced inflammation now needs focused attention.
If an acute inflammatory reaction induced by injection of a vaccine complex of an immunostimulatory adjuvant [Solomon ME, Halperin R, Yee J. Evaluation
INSTITUTE Me ^ CAI ·. ·. 'J}
DE LA rtll'riEPAD of the two-needle strategy for reducin ^^ 'Pé'áct ibri £! to DPT vaccination. Am. J. Dis. Child. 1Í17 · 798 --- 7-98 "ΤΤ987), Lanza GA, Barone L, Scalone G et al. Inflammation-related effects of adjuvant influenza A vaccination on platelet activation and cardiac autonomic function. J. Intern. Med. 269, 118-125 (2011) and Jae SY, Heffernan KS, Park SH et al. Does an acute inflammatory response temporarily attenuate parasympathetic reactivation? Clin. Auton. Beef. 20, 229-233 (2010)] can evolve into chronic low-grade inflammation and activate any of these discomforts in a subset of vaccinated over time is of primary public health importance; however, this potential risk has not been rigorously investigated. Since the concept of vaccine safety is evolving from protection against pathogen-induced diseases to without the possibility of inducing adverse consequences, any of the known foreign agents, the residual toxicity and virulence found in a vaccine should not be allowed and any possibility should be avoided. to induce unknown side effects (eg inflammation of vital organs).
Mucosal and systemic immune responses are induced and regulated with a considerable degree of independence, and most vaccines have been administered invasively by intramuscular injection.<sup>8</sup>’
INIT! TU »C 'MÜICANO □ € la mohidao which induces good systemic immunity but with weak mucosal immunity which is crucial in crumb *'« —— »against mucosal pathogens (eg influenza virus , Mycobacterium tuberculosis and human immunodeficiency virus) [Gallichan WS, Rosenthal KL. Long-lived cytotoxic T lymphocyte memory in mucosal tissues after mucosal but not systemic immunization. J. Exp. Med. 184, 1879-1890 (1996) and Saurer L, MeCullough KC, Summerfield A. In vitro induction of mucosa-type dendritic cells by all-trans retinoic acid. J.
Immunol. 179, 3504-3514 (2007)]. Efficient induction of mucosal immunity usually utilizes nasal or oral vaccination due to the unique ability of resident mucosal dendritic cells (DCs) to induce switch to IgA and to impart mucosal-specific ecotaxic receptors (e.g. , CCR9 and integrin α4β7) on lymphocytes [Saurer L, MeCullough KC, Summerfield A. In vitro induction of mucosa-type dendritic cells by all-trans retinoic acid. J. Immunol. 179, 3504-3514 (2007) and Molenaar R, Greuter M, van der Marel AP et al. Lymph node stromal cells support dendritic cell-induced gut-homing of T cells.
J. Immunol. 183, 6395-6402 (2009)].
In addition to the weak mucosal immunity induced by an injectable vaccine, the syringe needle as a vaccine delivery device also poses serious problems through unintentional reuse or
IMP ^ 5
INSTITUTE ΜIXΙΟΚ <sup>1 </sup>inadvertent non-sterile, water stem damage<sup>F</sup>j ^^^ fee & «£ É ^ of inappropriate waste as well as injection service ______ limited by authorized medical personnel during a crisis [Tang DC, Van Kampen KR. Toward the development of vectored vaccines in compliance with evolutionary medicine. Expert Rev. Vaccines 7 (4), 399-402 (2008)]. Public fear of pointed needles (aichmophobia) plays another role in avoiding vaccine coverage. Some people in this way prefer the probability of acquiring a disease versus the probability of receiving pain, harm or death from systemic vaccination. Since the goal of vaccination programs is to reduce the overall probability of infection by generating immunity in the community (herd), vision will be impaired by the exclusion of vaccination due to public fear of risks. Currently, enabling technologies to reverse negative perceptions by developing a new generation of rapid-response vaccines that are safe, effective, painless, and inexpensive are emerging on the horizon.
Citations or identifications of any document in this application is not an admission that such document is available as prior art for the present invention.
SUMMARY OF THE INVENTION
The present invention is based on the inventor's unforeseen finding that a particle emptied of
IΜ ΡI $ 1¾¾ adenovirus ΔΕ1Ε3 without transgene or a vector d £<sup>ST</sup>S¿féaw ^
INP16TJUAI encoding a pathogen-derived antigen can induce a rapid-long-extended protective response against pathogens in a variety of disease settings when administered intranasally.
Without wishing to be bound by any limitation, the applicant hypothesizes that adenovirus may be involved in activating specific arms of innate immunity that prevent the growth of respiratory mucosa pathogens.
The present invention relates to a method of inducing a response in a patient in need thereof which may comprise administering to the patient an adenovirus that is defective or that is suppressed in its El and / or E3 regions in an amount effective to induce the answer. In an advantageous embodiment the patient can be a mammal.
In one embodiment, the adenovirus does not contain or express a transgene.
In another embodiment, the adenovirus can contain and express a nucleic acid molecule that encodes a gene product. In particular, the adenovirus may comprise an exogenous or heterologous nucleic acid molecule that encodes a pathogen-derived gene product that induces protective immunity. The exogenous or heterologous nucleic acid molecule may encode the epitope of interest. In particular, exogenous or heterologous nucleic molocul-a-de-Te ido can encode for one or more influenza viruses, respiratory syncytial virus (RSV); Bacillus anthracis; or other epitopes derived from pathogen of interest and / or one or more influenza antigens.
In an advantageous embodiment, the adenovirus can be a human adenovirus. In another embodiment, the immune response can be induced within 24 hours. In another embodiment, the administration resulting in a protective response is from about one day to about 47 days.
Consequently, an objective of the invention is not to encompass within the invention any product, process or product elaboration or method of use of the product previously known so that the applicants reserve the right and hereby describe an exclusion of any product , previously known process or method. It is further noted that the invention is not intended to encompass within the scope of the invention any product, process or manufacture of the product or method using the product which does not satisfy the written description and licensing requirements of the USPTO (35 USC
§ 112, first paragraph) and of the EPO (Article 83 of the EPC), by right and hereby that the
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describe the exclusion of any product, process or product manufacturing or product use method previously described.
It is noted that in this description and particularly in the claims and / or paragraphs, terms such as comprising, comprehended, comprising and the like may have the meanings attributed thereto in US patent law; for example, they can mean includes, included, including, and the like; and terms such as "consists essentially of and" consists in essence of have the meaning ascribed to them in US patent law; for example, they allow the items to not be explicitly mentioned but exclude foods that are found in the prior art or that affect a basic or novel feature of the invention.
These and other modalities are described or are obvious from what is encompassed in the following Detailed Description.
BRIEF DESCRIPTION OF THE FIGURES
The following detailed description, provided by way of example, but not intended to limit the invention only to the specific embodiments described, should be better understood in conjunction with the accompanying figures.
Figure 1 shows the protection of mice against exposure to influenza viruses by intranasal administration of adenovirus particles.
Figure 2 shows rapid protection of mice against anthrax by intranasal administration of adenovirus particles.
Figure 3A and Figure 3B show prophylactic treatment against fatal challenge by A / PR / 8/34 (PR8) in mice. Prophylactic treatment was performed by in-administration of Ad5 particles shortly before exposure to PR8. AdE / in / -2 and AdE * / in / -2, in administration of AdE on day -2; AdE / in / + l, AdE in administration 1 day after exposure to PR8; AdE / im / -2, im injection of AdE on day -2; AdNC / in / -2 and AdNC * / in / -2, administration in of AdNC.Hl.l on day -2; AdNC / im / -2, im injection of AdNC.Hl.l on day -2; untreated control, untreated Balb / c mice prior to PR8 challenge; all groups were inoculated with AdE or AdNC.Hl. at a dose of 1.7 X 10<sub>6</sub> i<sub>fu </sub>except the AdE * / in / -2 and AdNC * / in / -2 groups that received a dose of 1.7 X 10<sup>6</sup> ufi; all groups were exposed by instillation in of 4 X DL<sub>50</sub> of PR8 on day 0; body weights were recorded daily for 18 days after exposure where 30% loss of body weight taken as the disease endpoint; the numbers in parentheses represent the number of animals in each group. -
Figure 4A and Figure 4B show protection of mice by Ad5-mediated prophylactic treatment and vaccination against a higher challenge dose of PR8. AdNC / in / -47, in administration of AdNC.Hl.l on day -47; AdE / in / -47, AdE in administration on day -47;
AdE / in / -47-2, AdE in administration on day -47 followed by a booster application on day -2;
AdE / in / -l, AdE in administration on day -1;
wtAd / in / -l, in administration of natural Ad5 particles The<sup>+</sup>/ E3<sup>+</sup> on day -1; all groups were inoculated with Ad5 particles at a dose of 1.2 X 10<sup>8</sup> ifu followed by exposure of 10 X DL<sub>50</sub> of PR8 on day 0; body weights were recorded daily for 14 days after challenge; the other symbols and procedures are the same as those described in the legend of Figures 3A-3B.
Figure 5 shows the health status of animals exposed to PR8 as shown by loss of body weight. Post-challenge body weights are represented by mean% body weight taking the individual mouse body weight on day 1 as 100%. The symbols and display procedures are the same as those described in the legends of Figures 3A-3B and Figures 4A-4B. Although the AdE / in / -47-2 and AdNC / in / -47 animals lost less weight than the mice in the other groups, the
IMPI ^ á. (Ντπτυτ · mmicam · -., „, * LA ΜβΝΧΒΛϋ Cs ^ SHLJ difference did not reach statistical significance tp ^ P<sup>| A</sup>ANO * 2ffc® one way with multiple comparison of Turkey poatüiluí · —a — Las. tests; the untreated control group was excluded in the statistical analysis due to early completion of the data points).
Figure 6A through Figure 6H shows the lung histopathology induced by PR8 infection. (A and E) Lung excised from untreated control mice (Figure 3) 19 days after PR8 challenge. (B and F) Lungs excised from normal Balb / c mice as a control. (C and G) lung excised from AdE / in / -2 mice (figure 3) 19 days after PR8 challenge, · each section is representative of three mice. (D and H) lung excised from a AdNC / in / -2 mouse (Figure 3) 19 days after PR8 challenge; each section is representative of three mice. The lung sections were examined on a Zeiss Axioskop2 plus microscope using a 2X (AD) or 10X (EH) objective in conjunction with an Axiocam digital camera.
Figure 7 shows PR8 titers in lungs after exposure. AdE particles (1.2 X 10<sup>8</sup> ifu per 50 ml) in mice on day -2 followed by challenge control and mice challenged towards AdE with 4.6 X 10<sup>s</sup> pfu of PR8 on day 0. On day 5, PR8 titers in lungs excised from control mice days after PR8 challenge; AoE-day 5, titles
IMPI ^
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'I HEARD THE MORf »AC of PR8 in lungs excised from exposed mice<sup>5</sup>™ ^ Adt 5 days after exposure to PR8; day, LILUl'05 give 'PR8 in lungs excised from control mice 7 days after PR8 challenge; AdE-day 7, PR8 titers in lungs excised from mice exposed to AdE 7 days after PR8 challenge; triangle and circle, log<sub>2</sub> (pfu of PR8) / g of lung in individual mice; bar, geometric mean of PR8 titers in lungs. No PR8 titers were detected in lungs excised from control mice that were not challenged with PR8. The difference between day 7 and AdE day 7 reached statistical significance (by one-way ANOVA with multiple Turkey comparison after tests).
Figure 8 shows protection against lethal challenge by pandemic CA04 in mice. They were instilled in particles of AdE or AdNC.Hl.l (2.5 X 10<sup>8</sup> ifu for 50 μΐ) in mice at variable time points followed by exposure to CA04. AdE / in / -22, AdE in administration on day -22; AdNC / in / -22, in administration of AdNC.Hl.l on day -22; placebo control, in administration of 50 μΐ of saline solution on day -22; the animals were exposed by instillation in of 3 x DL<sub>50</sub> pandemic CA04 on day 0; the other symbols and procedures are the same as those described in the legend of figure 3.
Figures 9A-9B show the architecture of the adenovirus. (Fig. 9A) Structure of a particle which is an icosahedral non-enveloped virus DNA. The highly coiled DNA gnome is packaged within the hexagonal protein capsid. (Fig. 9B) distribution of the AD5 vector ΔΕ1Ε3. The AD5 ΔΕ1Ε3 vector has been used extensively in a large number of gene therapy as well as vaccine trials. Its high immunogenicity is considered an impediment to re-administration; however, the problem has been lessened by recent evidence showing that nasal vaccines with an AD5 ΔΕ1Ε3 vector can overcome pre-existing AD5 immunity. Ad: Adenovirus; LITR: left inverted terminal repeat; Promoter: Common promoter A for activating transgene expression is the cytomegalovirus early promoter; polyA +: a common polyadenylation site is the SV40 polyadenylation signal; RITR: right inverted terminal repeat sequence.
Figure 10 shows a progeny diagram of biomanipulated non-replicating adenovirus vectors. Both human and animal Ad have been biomanipulated into a wide variety of non-replicating Ad vectors for the delivery of exogenous genes in or subject to animals and humans. Ad: adenovirus.
Figure 11 shows the seamless protection conferred by intranasal administration of a drug-vaccine duo having Ad5 as vector. It has recently been shown that instillation in (an empty Ad5 Δ sin1Ε3 particle without transgene)
<img file="MX348172B_D0006.tif" />
(an AD4 vector
ΔΕ1Ε3 encoding for
<img file="MX348172B_D0007.tif" />
Caledonia / 20/99 HA1) can confer almost immediate protection to mice against exposure to live influenza virus [Zhang et al. PLoS ONE 6, e22605 (2011)]. AdE-induced prophylactic treatment lasts in mice for at least 22 days, with partial decline in potency observed 47 days after AdE administration. The protection induced by AdNC.Hl.l is solid after 47 days. Solid line: solid protection time frame; dashed line: partial protection time frame. Since AdE-induced complete protection is observed for 22 days while partial protection is observed 47 days after administration, the assumption is made that the effects of the DVD drug initiate decline after 22 days, as shown by the dashed line after the solid line. A vaccine vectoring Ad5 has been reported to induce protective immunity as early as two weeks after immunization [Boyer et al. Hum. Gene Ther. 16, 157-168 (2005)] as shown by a solid line starting on day 14 for DVD vaccine effects when the Ad5 particle is inoculated on day 0. The results show that seamless protection against influenza can be obtained in mice intranasal DVD that has as vector
<img file="MX348172B_D0008.tif" />
Protective immunity can be induced by the vaccine before the effects of the drug wane. Ad: adenovirus:
DVD: drug-vaccine duo.
Figure 12 shows mice protected with nasal vaccine having as vector Ad5 against avian influenza virus A / VN / 1203/04 (H5N1). Mice were immunized i .n. on day 0 and exposed to A / VN / 1203/04 (H5N1) at a dose of 10 X 10 x MDL<sub>50</sub> (10<sup>4</sup>’<sup>4</sup> I KNOW<sub>50</sub>) to SRI on day 63. HA, Ad encodes HA1 + HA2; HA1, Ad codes for HA1; E7, 10<sup>7</sup> pv; E10, 10<sup>10</sup> pv; H1, GMT of serum HI titres on day 49.
Figure 13 shows ferrets protected with nasal vaccine having as vector Ad5 against avian influenza virus A / VN / 1203/04 (H5N1). Ferrets were immunized i .n. on day 0; and exposed with A / VN / 1203/04 at a dose of 10 FDL<sub>50</sub> (10<sup>2</sup> I KNOW<sub>50</sub>) in SRI on day 56. HA, Ad encodes HA1 + HA2; HA1, Ad encodes HA1; E10, 10<sup>10</sup> pv; HI, GMT of serum titers HI at day 51.
Figure 14 shows the prophylactic treatment against anthrax by intranasal instillation of adenovirus particles shortly before exposure to spores. AdVAV / -2, AdVAV particles in instilled 2 days prior to exposure at a dose of 1.3 X 10<sup>8</sup> ufi; AdE / -2, particles. ΙΜ, ΡΙ ^ of AdE instilled in 2 days before ®Kpo®j, atÓjQ traine:
DE LA POPISIMO vVZ ~ HF industrial 1.3 X 10 dose<sup>8</sup> ufi; AdE * l-2, AdE particles instilled in 2 days before exposure at a dose of 1.3 X ΙΟ<sup>5</sup> ifu (100-fold dilution in PBS); AdE / -l, AdE particles instilled in 1 day before exposure at a dose of 1.3 X 10<sup>8</sup> ufi; Control, untreated control mice; the numbers in parentheses represent the number of animals in each group.
Figure 15 shows post-challenge anthrax treatment by instillation of AdVAV particles. AdVAV / D-2, AdVAV particles instilled in 2 days prior to exposure at a dose of 1.3 X 10<sup>8</sup> ufi; AdVAV / DO, AdVAV particles instilled in 1 hour after exposure at a dose of 1.3 X 10<sup>8</sup> ufi; AdVAV / Cipro / DO, AdVAV particles instilled in 1 hour after exposure at a dose of 1.3 X 10<sup>8</sup> ifu together with ip injection of ciprofloxacin; Cipro / DO, ip injection of ciprofloxacin; Control, untreated control mice without pre-challenge treatments ; the numbers in parentheses represent the number of animals in each group.
Figure 16 shows the effect of administering AdE intranasally in RSV-Tracy nasal wash as well as virus titers in lung lavage on day +4. Group 1: 6 CR prophylactically (day -2) treated intranasally with vehicle (buffer A195), Group 2: 6 CR prophylactically
- 21 (day -30) treated intranasally with
Group 3: 6 CR prophylactically
IM p} (ΝΓπτι / τη, ^ r ~ V <sup>no</sup> ia soon> Γ ·<sup>Λ</sup>*; · Ι.
2.4 X 10<sup>8</sup>¾¾^
<img file="MX348172B_D0009.tif" />
intranasally with 2.4 X 10<sup>8</sup> ifu of AdE, Group 4: 6 CR prophylactically (days -30 and -2) treated intranasally with 2.4 X 10<sup>8</sup> AdE ifu during each treatment cycle (prime / boost) and group 5: 6 CR prophylactically (-5 h) treated intranasally with 2.4 X 10<sup>8</sup> ifu from AdE.
DETAILED DESCRIPTION OF THE INVENTION
The invention is based, in part, on the inventor's discovery that as quickly as one day after administration of an empty Ad vector (deleted in E1 / E3 without insert) mice are protected from exposure to influenza . The mechanism for this protection is currently unknown but it is a very broad based protection. Mice are protected from exposure to seasonal influenza, exposure to swine influenza, exposure to avian influenza, exposure to RSV, and even exposure to anthrax. This protection lasts from about 1 day to about 47 days. Wild-type Ad controls did not provide any protection and vaccines delivered intramuscularly did not provide any protection. Protection occurred even when a gene was inserted into the El region, although there seems to be some interference when the gene is an influenza HA gene but interestingly, there is increased protection when the gene is
<img file="MX348172B_D0010.tif" />
protector against anthrax. In addition to<sup>ra1</sup>??] ing, protected cotton rats against RSV exposures following intranasal administration of AdE particles either 2 days or 30 days prior to exposure.
Embodiments of the invention using adenovirus recombinants can include El-defective, E3-defective, and / or E4-defective adenovirus vectors. The El mutation generates the vector safety margin because El-defective adenovirus mutants are incapable of replication in non-permissive cells. The E3 mutation increases the immunogenicity of the antigen by breaking the mechanism by which the adenovirus down-regulates class I CPH molecules. The E4 mutation reduces the immunogenicity of the adenovirus vector by suppressing the expression of the late gene. Specific sequence motifs such as the RGD motif can be inserted into the HI loop of the adenovirus vector to increase its infectivity. A recombinant adenovirus is constructed by cloning specific transgenes or transgene fragments into any of the adenovirus vectors such as those described above.
Generation of transgene-free Ad5 ΔΕ1Ε3 void particles can be carried out as described in Tang DC, Zhang J, Toro H, Shi Z, Van Kampen KR (2 009) Adenovirus
IMPJ ^ as a carrier for the development of inf
INDUSTRIAL avian influenza vaccines. Expert Rev Vaccines 8: 469-481.
The term "viral vector", as used herein, includes but is not limited to retroviruses, adenoviruses, adeno-associated viruses, alphaviruses, and herpes simplex viruses.
The adenovirus can be any adenovirus such as, but not limited to, bovine adenovirus, canine adenovirus, non-human primate adenovirus, chicken adenovirus, or a porcine or pig adenovirus.
The term human adenovirus as used herein is intended to encompass all adenoviruses of the Adenoviridae family which include members of the Mastadenovirus genera. To date, more than fifty-one human adenovirus serotypes have been identified (see, for example, Fields et al., Virology 2, Ch. 67 (3d ed., Lippincott-Raven Publishers)). The adenovirus can be serogroup A, B, C, D, E, or F. Human adenovirus can be serotype 1 (Adl), serotype 2 (Ad2), serotype 3 (Ad3), serotype 4 (Ad4), serotype 6 (Ad6), serotype 7 (Ad7), serotype 8 (Ad8), serotype 9 ( Ad9), serotype 10 (AdlO), serotype 11 (Adll), serotype 12 (Adl2), serotype 13 (Adl3), serotype 14 (Adl4), serotype 15 (Adl5), serotype 16 (Adl6), serotype 17 (Adl7), serotype 18 (Adl8), serotype19 (Adl9), serotype 19a (Adl9a), serotype 19p (Adl9p), serotype 20 (Ad20), serotype 21 (Ad21), serotype 22 (Ad22), serotype
<td> 23</td><td>(Ad23),</td><td>serotype</td><td> 24</td><td>(Ad24),</td><td>serotype</td><td>MAID INDUSTRY</td><td> 1</td>
<td> 26</td><td>(Ad26),</td><td>serotype</td><td> 27</td><td>(Ad27),</td><td>serotype</td><td>28 (Ad28),</td><td>serotype</td>
<td> 29</td><td>(Ad29),</td><td>serotype</td><td> 30</td><td>(Ad30),</td><td>serotype</td><td>31 (Ad31),</td><td>serotype</td>
<td> 32</td><td>(Ad32),</td><td>serotype</td><td> 33</td><td>(Ad33),</td><td>serotype</td><td>34 (Ad34),</td><td>serotype</td>
<td> 35</td><td>(Ad35),</td><td>serotype</td><td> 36</td><td>(Ad36),</td><td>serotype</td><td>37 (Ad37),</td><td>serotype</td>
<td> 38</td><td>(Ad38),</td><td>serotype</td><td> 39</td><td>(Bd3 9),</td><td>serotype</td><td>40 (Ad40),</td><td>serotype</td>
<td> 41</td><td>(Ad41),</td><td>serotype</td><td> 42</td><td>(Ad42),</td><td>serotype</td><td>43 (Ad43),</td><td>serotype</td>
<td> 44</td><td>(Ad44),</td><td>serotype</td><td> 45</td><td>(Ad45),</td><td>serotype</td><td>46 (Ad46),</td><td>serotype</td>
<td> 47</td><td>(Ad47),</td><td>serotype</td><td> 48</td><td>(Ad48),</td><td>serotype</td><td>49 (Ad49),</td><td>serotype</td>
(Ad50), serotype 51 (Ad51), or, preferably serotype (Ad5), but not limited to these examples.
Also contemplated by the present invention are receptor binding ligands, recombinant vectors, drug-vaccine compositions, and recombinant adenovirus that may comprise subviral particles of more than one adenovirus serotype. For example, it is known that adenovirus vectors may exhibit altered tropism for specific tissues or cell types (Havenga, MJE et al., 2002), and therefore it may be advantageous to mix and match different adenoviral capsids, i.e. fiber protein or penton from various adenoviral serotypes. Modification of adenoviral capsids, including fiber and penton, can result in an adenoviral vector with a tropism that is different from unmodified adenovirus. Adenovirus vectors that are modified and
<img file="MX348172B_D0011.tif" />
- 25 INOUSTMIAL optimized in their ability to infect target cells can allow a significant reduction in ' <sup>1</sup> Dosí ^ "" '· therapeutic or prophylactic resulting in reduced local and disseminated toxicity.
Viral vector gene delivery systems are commonly used in gene transfer in gene therapy applications. Different viral vector systems have their own unique advantages and disadvantages. Viral vectors that can be used to express a pathogen-derived ligand of the present invention include but are not limited to adenoviral vectors, adeno-associated viral vectors, alphavirus vector, herpes simplex viral vector, and retroviral vectors, described in greater detail below. next.
Adenovirus vectors have many characteristics which are ideal for gene delivery, especially delivery in the respiratory tract. Examples of these features include:
(a) ability of adenovirus vectors to transduce mitotic and post-mitotic cells in situ;
(b) existing technology to prepare concentrates containing high virus titers [greater than 10<sup>12</sup> ifu (infectious units) per mi] to transduce cells in situ with a high multiplicity of infection (MOI),
MAID
INSTO << i '. .UiCani C- * í ·· '^ .. / · \. * T
M LA MOPIEOAD C> *, .- VúSW .- 'y,,, ifír.uSTxiAL (c) inhalation of adenovirus in accordance with evolutionary medicine (Tang and Van Kampen, 2000); '(d) the potency of an adenovirus vector administered intranasally does not interfere with pre-existing immunity to adenovirus (Hoelscher et al., 2006; Shi et al., 2001; Van Kampen et al., 2005); Although not wishing to be bound by theory this can be attributed to the high efficiency of gene delivery, high level of transgene expressions and a high degree of antigen presentation along the mucosal barrier in the airways;
(e) ability of the adenovirus to induce high levels of transgene expression (at least as an initial shock); and (f) ease with which replication-defective adenovirus vectors can be biomanipulated.
Additional general features of adenovirus are those in which the biology of the adenovirus is characterized in detail: adenovirus is not associated with severe human pathology; adenovirus is extremely efficient at introducing its DNA into the host cell; adenovirus can infect a wide variety of cells and has a wide host range; adenovirus can be produced in large numbers relatively easily; and the adenovirus can become defective in its replication and / or not
IMPI _. . 'ΝΑ-πτυτο .Μ LXICA, Ni, replicating by deletions in the early region 1 viral genome. - _ _
Reference is made to US Patent No. 5,990,091 issued November 23, 1999, Einat et al. or Quark Biotech, Inc., WO 99/60164, published November 25, 1999 from PCT / US99 / 11066, filed May 14, 1999, Fischer or Rhone Merieux, Inc., WO98 / 00166, published on January 8, 1998 from PCT / US97 / 11486, filed June 30, 1997 (claiming priority for US applications Serial numbers 08 / 675,556 and 08 / 675,566), van Ginkel et al., J. Immunol 159 (2): 685-93 (1997) (Adenoviral gene delivery elicits distinct pulmonary-associated T helper cell responses to the vector and to its transgene), and Osterhaus et al., Immunobiology 184 (2-3): 180- 92 (1992) (Vaccination against acute respiratory virus infections and measles in man), for information regarding expressed gene products, antibodies and uses thereof, vectors for in vivo and in vitro expression of exogenous nucleic acid molecules, promoters for activating expression or for operatively binding to nucleic acid molecules to be expressed, method and documents for producing these vectors, compositions comprising these vectors or nucleic acid molecules or antibodies, dosages and modes and / or routes of administration (including compositions for
<img file="MX348172B_D0012.tif" />
Nasal administration), for example, the dulYl pugdé P'ser INSTITUTE -JÓ'C ^ O L'l LA 1'koHi DAD. • T 'i π -. · T. . industrial used in the practice of this invention; and therefore US Patent No. 5,990,091 issued November 23, 1999, Einat et al. or Quark Biotech, Inc., WO 99/60164, published November 25, 1999 from PCT / US99 / 11066, filed May 14, 1999, Fischer or Rhone Merieux, Inc., WO98 / 00166, published on January 8, 1998 from PCT / US97 / 11486, filed June 30, 1997 (claiming priority for US application serial numbers 08 / 675,556 and 08 / 675,566), van Ginkel et al., J. Immunol 159 (2): 685-93 (1997) (Adenoviral gene delivery elicits distinct pulmonary-associated T helper cell responsos to the vector and to its transgene), and Osterhaus et al., Immunobiology 184 (2-3): 180- 92 (1992) (Vaccination against acute respiratory virus infections and measles in man) and all documents mentioned or referred to herein and all documents mentioned or referred to in documents mentioned in each of the US patent Number 5,990,091 issued November 23, 1999, Einat et al. or Quark Biotech, Inc., WO 99/60164, published November 25, 1999 from PCT / US99 / 11066, filed May 14, 1999, Fischer or Rhone Merieux, Inc., WO98 / 00166, published on January 8, 1998 from PCT / US97 / 11486, filed on June 30, 1997 (claiming priority of applications for
INSTm ITO Μ í XICA NC Γς Dt LA INDUSTRIAL CURRENCY
USA Serial Numbers 08 / 675,556 and 08 / 675,566), van Ginkel et al., J. Immunol 159 (2): 685-93 (1997) (Adenoviral gene delivery elicits distinct pulmonary-associated T helper cell responsos to the vector and to its transgene), and Osterhaus et al., Immunobiology 184 (2-3): 180-92 (1992) (Vaccination against acute respiratory virus infections and measles in man) are incorporated herein by reference. The information in the US patent Number 5,990,091 issued November 23, 1999, WO 99/60164, WO98 / 00166, van
Ginkel et al., J. Immunol 159 (2): 685-93 (1997), and Osterhaus et al., Immunobiology 184 (2-3): 180-92 (1992) can be taken as a basis for the practice of this invention (e.g., expressed products, antibodies and uses thereof, vectors for in vivo and in vitro expression of exogenous nucleic acid molecules, exogenous acid molecules nucleic agents that encode epitopes of interest or antigens or therapeutics and the like, promoters, compositions comprising vectors or nucleic acid molecules or expressed products or antibodies, dosages, for example).
It is noted that the immunological products and / or expressed antibodies and / or products obtained in accordance with this invention can be expressed in vitro and can be used in a manner in which the immunological and / or expressed products and / or antibodies are use regularly,
IMP * <sup>or</sup>· the
<img file="MX348172B_D0013.tif" />
and the cells that express these immunophilic and / or expressed products and / or antibodies
<img file="MX348172B_D0014.tif" />
in vitro and / or ex vivo applications, for example uses and applications such as diagnosis, testing, ex vivo treatment (for example, where cells expressing the gene product and / or its immune response are expanded in vitro and reintroduced into the host or animal), etc., see US Patent No. 5,990,091, WO 99/60164 and
WO 98/00166 and documents mentioned therein.
Furthermore, expressed antibodies or gene products that are isolated by the methods herein or that are isolated from cells expanded in vitro following the methods of administration herein can be administered in compositions, similar to the administration of subunit epitopes or antigens or therapeutic substances or antibodies to induce immunity, stimulate a therapeutic response and / or stimulate passive immunity. The amount to be administered will vary for the patient (host) and the condition being treated and will vary from one to a few or a few hundred or thousands of microorganisms, for example 1 pg to 1 mg, from about 100 ng / kg body weight 100 mg / kg of body weight per day and preferably will be 10 pg / kg to 10 mg / kg per day.
A vector can be administered to a patient or host in an amount to achieve the amounts established for gene product compositions (eg, epitope, antigen, therapeutic, and / or antibody). Of course, the invention considers dosages below and above what is exemplified herein and for any composition to be administered to an animal or human, including the components thereof and for any particular method of administration, it is prefers to determine for the same: toxicity, for example when determining the lethal dose (DL) and DL<sub>50</sub> in a suitable animal model, for example a rodent such as a mouse; and the dosage of one or more of the compositions, the concentration of components in the present and the timing of administration of one or more of the compositions, which induces an adequate response, for example by serum titrations and analysis thereof, by example by ELISA and / or serum neutralization analysis. These determinations do not require undue experimentation based on the knowledge of experts in the field, this description and the documents mentioned herein. Furthermore, the invention also encompasses the sequential administration of inventive compositions or sequential performance of the methods herein, for example periodic administration of compositions of the invention such as in the course of therapy or treatment for a condition and / or booster administration of immunological compositions<sup>r </sup>INDUSTRIAL priming-reinforcement; furthermore, the time and manner for sequential administrations can be determined without undue experimentation.
The dosage of the adenovirus of the present invention can be from about 10<sup>6</sup> ifu up to about 10<sup>10</sup> ufi. The dosage can be from approximately 10<sup>6</sup> ufi, about 10<sup>7</sup> ufi, about 10<sup>8</sup> ufi, about 10<sup>9</sup> ifu or about 10<sup>10</sup> ufi. In an advantageous embodiment, the dosage is approximately 10<sup>6</sup> ufi, about 10<sup>7</sup> ifu or about 10<sup>8</sup> ufi.
In a particularly advantageous embodiment, multiple dosages of the adenovirus of the present invention. In particularly advantageous embodiments, approximately two doses are administered. In a mode
<td>advantageous the</td><td>dose</td><td>they are administered</td><td>with</td><td>a seperation</td><td>from</td>
<td>approximately</td><td> 20</td><td>days, with</td><td>a</td><td>separation</td><td>from</td>
<td>approximately</td><td> 25</td><td>days, with</td><td>a</td><td>separation</td><td>from</td>
<td>approximately</td><td> 30</td><td>days, with</td><td>a</td><td>separation</td><td>from</td>
<td>approximately</td><td> 35</td><td>days, with</td><td>a</td><td>separation</td><td>from</td>
<td>approximately</td><td> 40</td><td>days, with</td><td>a</td><td>separation</td><td>from</td>
<td>approximately</td><td> 45</td><td>days, with</td><td>a</td><td>separation</td><td>from</td>
<td>approximately</td><td> 50</td><td>days, with</td><td>a</td><td>separation</td><td>from</td>
<td>approximately</td><td> 55</td><td>days, with</td><td>a</td><td>separation</td><td>from</td>
<img file="MX348172B_D0015.tif" />
<img file="MX348172B_D0016.tif" />
approximately 60 approximately 65 administered with a day or with a Α ?! INSTITU '_ _,
Ofc THE INDUSTRIAL FaCPltPAL days. Advantageously, the doses are approximately 40 days apart, approximately 41 days apart, with a
<td colspan="3">separation of approximately</td><td>42 days,</td><td>with</td><td>a seperation</td><td>from</td>
<td>approximately</td><td> 43</td><td>days,</td><td>with</td><td>a</td><td>separation</td><td>from</td>
<td>approximately</td><td> 44</td><td>days,</td><td>with</td><td>a</td><td>separation</td><td>from</td>
<td>approximately</td><td> 45</td><td>days,</td><td>with</td><td>a</td><td>separation</td><td>from</td>
<td>approximately</td><td> 46</td><td>days,</td><td>with</td><td>a</td><td>separation</td><td>from</td>
<td>approximately</td><td> 47</td><td>days,</td><td>with</td><td>a</td><td>separation</td><td>from</td>
<td>approximately</td><td> 48</td><td>days,</td><td>with</td><td>a</td><td>separation</td><td>from</td>
<td>approximately</td><td> 49</td><td>days</td><td>or with</td><td>a</td><td>separation</td><td>from</td>
<td>approximately</td><td>50 days</td><td> .</td><td></td><td></td><td></td><td></td>
Furthermore, the invention encompasses compositions and methods for making and using vectors that include methods for producing gene products and / or immunological products and / or antibodies in vivo and / or in vitro and / or ex vivo (for example, the latter is, for For example, after isolation of the same cells from a host having a non-invasive administration according to the invention, for example after optional expansion of these cells) and uses for such genes and / or immunological products and / or antibodies, including in diagnostics, assays, therapies, treatments and the like. Vector compositions are formulated by mixing the vector with a suitable carrier or diluent. In addition, the
- 34 - 'Λ / ί O <sup>1</sup> ’· '<sup>:</sup> -· <sup>ν</sup>'.; gene product and / or immunological product and / or 'the antibody compositions are likewise formulated - mixing the gene and / or immunological product and / or antibody with a suitable carrier or diluent; see, for example, US patent Number 5,990,091, WO 99/60164, WO 98/00166, documents mentioned herein, and other documents mentioned herein and other teachings herein (eg, with respect to carriers, diluents, and the like).
In an advantageous embodiment, the vector expresses a gene encoding an influenza antigen, an RSV antigen, an HIV antigen, an SIV antigen, an HPV antigen, a HCV antigen, a HBV antigen, a CMV or a Staphylococcus antigen. The flu can be swine flu, seasonal flu, avian flu, an H1N1 antigen, or H5N1 flu.
In another advantageous embodiment, the vector expresses a gene which codes for influenza hemagglutinin, influenza nuclear protein, influenza M2, tetanus toxin fragment C, anthrax protective antigen, anthrax fatal factor, rabies glycoprotein, antigen of surface HBV, HIV gp 120, HW gp 160, human carcinoembryonic antigen, malaria CSP, malaria SSP, malaria MSP, malaria pfg, Mycobacterium tuberculosis HSP or a mutant thereof.
In one embodiment of the invention, the answer ______________,. . ..
inmumtana in the animal is induced by v ^ pQ ^ e ^,
I * L) <S ^ iAL ^ * - 21 —- that express genes that code for antigens of interest in the cells of the animal. The antigens of interest can be selected from any of the antigens described herein.
In another embodiment of the method, the cells of the animals are epidermal cells. In another embodiment of the method, the immune response is against a pathogen or a neoplasm. In another embodiment of the method, the genetic vector is used as a prophylactic vaccine or as a therapeutic vaccine. In another embodiment of the invention, the genetic vector comprises genetic vectors capable of expressing an antigen of interest in the cells of an animal. In a further embodiment of the method, the animal is a vertebrate.
With regard to exogenous DNA for expression in a vector (e.g. encoding an epitope of interest and / or an antigen and / or a therapeutic substance) and documents providing such exogenous DNA, as well as with regard to the expression of factors of transcription and / or translation to increase the expression of nucleic acid molecules and regarding terms such as epitope of interest, therapeutic, immune response, immune response, protective immune response, immunological composition, immunogenic composition and
IMPI, INSTITUTO MEXICANO composition of vaccines, for example, has been ^^^^ r US patent number 5,990,091 issued on November 23, 1999, and documents WO 98/00166 and WO 99/60164, and the documents mentioned in the present and the registration documents in the prosecution of those patents and those of the PCT applications; all of which are incorporated herein by reference. In this way, the US patent No. 5,990,091 and documents WO 98/00166 and WO 99/10164 and documents mentioned in those documents and documents or records in the prosecution of that patent and of those PCT applications and other documents mentioned or otherwise incorporated in the same for reference may be consulted in the practice of this invention; furthermore, all of the exogenous nucleic acid molecules, promoters, and vectors mentioned herein can be used in the practice of this invention. Mention is also made in this regard of US Patent Numbers 6,004,777; 5,997,878;
5,989,561; 5,976,552; 5,972,597; 5,858,368; 5,863,542; 5,833,975; 5,863,542; 5,843,456; 5,766,598; 5,766,597; 5,762,939; 5,756,102; 5,756,101; 5,494,807.
In another embodiment of the invention, the animal is advantageously a vertebrate such as a mammal, bird, reptile, amphibian or fish; more advantageously a human or a
<img file="MX348172B_D0017.tif" />
pet or domesticated animal or an animal to produce
- 37 foods or that produces races or sports such feed or
<img file="MX348172B_D0018.tif" />
and <sup>1HG</sup>'η La üt UA * · * CTUIAl like a cow, a peri ^ im cat, a goat, a sheep, a pig, a horse1 poultry such as turkeys, ducks or chickens. In another especially advantageous embodiment of the invention, the vertebrate is a human.
In another embodiment of the invention, the genetic vector is a viral vector, a bacterial vector, a protozoan vector, a retrotransposon, a transposon, a virus coat, or a DNA vector. In another embodiment of the invention, the viral vector, the bacterial vector, the protozoan vector, and the DNA vector are recombinant vectors. In another embodiment of the invention, the immune response is against influenza A. In another embodiment of the invention, the immune response against influenza A is induced by the genetic vector that expresses a gene that codes for an influenza hemagglutinin, an influenza nuclear protein, an influenza M2 or a fragment thereof in cells of the animals. In another embodiment of the invention, the genetic vector is selected from the group consisting of viral vector and plasmid DNA.
In another embodiment of the invention, the genetic vector is an adenovirus. In another embodiment of the invention, the adenovirus vector is defective in its El region. In another embodiment of the invention, the adenovirus vector is defective in its E3 region. In otr * AncfcláliñatCi ^ Se JSa INSlnuTr. HAHiCAN. 't' · * .., · 'J, Of LA ι · ικνι · 0Α,' ς 'íy •'. -1, -, <- 'N ^ USTUIAL' ♦ 'By the invention, the adenovirus vector is defective in its El and / or E3 regions. In another embodiment of the invention 'he'DNA' is in plasmid form, In another embodiment of the invention, the contacting step further comprises placing the genetic vector containing the gene of interest on a delivery device and applying the device that has the genetic vector that contains the gene of interest in it to the skin of the animal. In another embodiment of the invention, the genetic vector codes for an immunomodulatory gene, as a costimulatory gene or as a cytokine gene. In another embodiment of the invention, the vector has all viral genes deleted. In another embodiment of the invention, the genetic vector induces an antitumor effect in the animal. In another embodiment of the invention, the genetic vector expresses an oncogene, a tumor suppressor gene, or a tumor-associated gene.
Representative examples of antigens which can be used to elicit an immune response using the methods of the present invention include influenza hemagglutinin, influenza nuclear protein, influenza M2, tetanus toxin fragment C, anthrax protective antigen, lethal factor of anthrax, rabies glycoprotein, HBV surface antigen, HIV gp 120, HIV gp 160, human carcinoembryonic antigen, malaria CSP, malaria SSP, MSP of malaria, pfg of
MAID
Ηίτττυτυ macano
OF THE FLOPieUA » <sub>r</sub> malaria and Mycobacterium tuberculosis HSP, the protective immune response against neoplasms or infectious pathogens will be more preferable.
In another embodiment of the present invention, the vector further contains a gene that is selected from the group consisting of costimulatory genes and cytokine genes. In this method the gene is selected from the group consisting of a gene for GM-CSF, a gene for B7-1, a gene for B7-2, a gene for interleukin-2, a gene for interleukin-12 and genes for interferon. .
The recombinant vectors and methods of the present invention can be used in the treatment or prevention of various respiratory pathogens. These pathogens include, but are not limited to influenza viruses, severe acute respiratory syndrome coronavirus (SARS-CoV), human rhinovirus (HRV), and respiratory syncytial virus (RSV). for its acronym in English).
Furthermore, the present invention encompasses the use of more than one therapeutic ligand, immunogen, or antigen in the vectors and methods described herein, delivered either in separate recombinant vectors or together in a recombinant vector so as to provide a multivalent vaccine or a immunogenic composition that stimulates or modulates the immunogenic response to one or more strains of
<img file="MX348172B_D0019.tif" />
influenza and / or hybrids. Furthermore, the presentI j ^ Lepc Jó ^^ teár ^
ΙΝ * ΤΙ ΠΠ <> MhXiCANÓ IH LA the use of a therapeutic ligand, immunogen 'b' '' ^ htígerro — of more than one pathogen in vectors and ntítudus — qrre — here— described, supplied either in separate recombinant vectors or together in a recombinant vector.
Embodiments of the invention using DNA / adenovirus complexes can have plasmid DNA that forms a complex with adenovirus vectors using a suitable agent therefor, such as either PEI (polyethyleneimine) or polylysine. The adenovirus vector within the complex can be either alive or killed by UV irradiation. The UV-inactivated adenovirus vector as a receptor binding ligand and an endosomolysis agent to facilitate DNA-mediated transfection (Cotten et al., 1992) can increase the margin of safety of the vaccine carrier. The DNA / adenovirus complex is used to transfect epidermal cells of a vertebrate in a non-invasive manner for use as an immunizing agent.
The genetic vectors provided by the invention can also code for immunomodulatory molecules which can act as an adjuvant to elicit a humoral and / or cellular immune response. These molecules include cytokines, costimulatory molecules, or any other molecule that can change the course of an immune response. One can conceive of ways in which
IMPT ^
JMWUK ..Ah * · '<' '_ ^ · ϊ: Ok, which technology can be modified to further increase the immunogenicity of antigens. Jn feémiiiius — StU χ ^ terminology used herein, an immunologically effective amount is an amount or concentration of the genetic vector encoding the gene of interest that, when administered to an animal, produces an immune response to the gene product of interest .
Various epitopes, antigens, or therapeutic substances can be delivered topically by expressing them at different concentrations. Generally, useful amounts for adenovirus vectors are at least about 100 pfu and for plasmid DNA at least about 1 ng of DNA. Other amounts can be determined from this description and knowledge in the field, including documents mentioned and incorporated herein by reference, without undue experimentation.
The methods of the invention can be appropriately applied to prevent diseases as a prophylactic vaccination to treat diseases as a therapeutic vaccination.
The vaccines of the present invention can be administered to an animal either alone or as part of an immunological composition.
Beyond the human vaccines described, the method of the invention can be used to immunize
<img file="MX348172B_D0020.tif" />
groups of animals. The term animal means<sup>l</sup>'^^ iiAitipFCr4ie animal include human. The examples of animals —inel-uygTr ~ humans, cows, dogs, cats, goats, sheep, horses, pigs, turkeys, ducks and chickens, etc. Since the immune systems of all vertebrates operate in a similar way, the applications described can be implemented in all vertebrate systems.
The present invention also encompasses combinations of vectors, in particular adenovirus vectors. For example, an empty adenovector (deleted in E1 / E3 without an insert) can be administered sequentially or simultaneously to a patient in need of the same together with another vector, such as an adenovector which can be deleted in E1 / E3 with an insert such as an exogenous gene as described herein. Without wishing to be bound by theory, the empty adenovector (deleted in E1 / E3 without the insert) can initially induce a rapid immune response where a vector expressing an exogenous gene, such as an antigen or epitope, can induce an additional protective response. .
Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations may be made herein without thereby departing from the spirit and scope of the invention as defined in the appended claims.
<img file="MX348172B_D0021.tif" />
I Λ APJ _ 43 - 1 1 ^ .1 1 λ
INSTITUTE '': <VICAN OF THE r'kOrifc! >.> INDUSTRIAL
The present invention will be further illustrated in the following examples which are provided for illustration purposes only and are not intended to limit the invention in any way.
EXAMPLES
EXAMPLE 1: ADENOVIRUS PARTICLE AS A BROAD SPECTRUM DRUG AGAINST RESPIRATORY PATHOGENS
Although vaccination is an effective approach to preventing infectious disease when given weeks or months in advance, it is too slow to protect animals or people who are at immediate risk. An agent capable of reducing the severity of an infection when ingested shortly before or after an infection is of primary public health importance. Tamiflu (oseltamivir phosphate) and Relenza (zanamivir) are often effective in preventing influenza virus infections; however, these neuraminidase inhibitors can generate drug-resistant strains of influenza viruses over time (Poland et al., 2009). Similar to viruses, drug resistant bacteria have also commonly been generated by drug overuse (Davies and Davies, 2010). Thus, it is urgent to develop additional drugs because medical personnel will have the option of using another drug in the line of suppressing pathogens when a
IMPI medication in use is deteriorated by resiÍBÍSotika me d aments. -
Applicant has shown that intranasal instillation of E1 / E3-defective adenovirus (Ad) particles 1-2 days prior to intranasal challenge with a lethal dose of influenza virus can confer rapid protection against influenza in mice (Figure 1) . The applicant has also shown that intranasal administration of Ad 1-2 days before intranasal challenge with a lethal dose of Bacillus anthrancis Sterne spores is also capable of protecting mice against anthrax (Figure 2). It is conceivable that intranasal administration of Ad rapidly induces a range of reactions that prevent the growth of other microbes in the respiratory tract.
Since Ad has been biomanipulated in a non-replicating vaccine carrier with an excellent safety profile in animals and humans (Tang et al., 2009), it is conceivable that an Ad vector encoding a pathogen-derived antigen could be developed in a drug-vaccine duo (DVD) which is capable of conferring rapid and comprehensive protection against a variety of pathogens before adaptive immunity is induced; followed by induction of specific protective immunity against pathogens such as a vaccine; in a single package, the
IMPIOS figure 1 and figure 2 show that unicardCTX ^; rJ ^^ s major Ad (AdE) free of transgene can confer rapid protection against viruses and bacteria as a drug but also their counterparts that encode antigens derived from pathogens are capable of suppress pathogens like a drug.
Methods of Figure 1. Purified AdNC.Hl.l vectors encoding influenza virus HA1 or A / NC / 20/99 (H1N1) and its transgene-free counterpart (AdE) were administered dropwise in a volume of 0.05 ml. into the nostril of each young Balb / c mouse (2 months old) using a mechanical pipette as described (Shi et al., 2001) prior to challenge. One or two days after Ad administration, mice are challenged intranasally with a lethal dose (0.2 HA units) of influenza A / PR / 8/34 (H1N1) virus and monitored daily for survival.
Results of Figure 1. Seventy percent of the mice (7/10) were protected against a lethal dose of live influenza A / PR / 7/34 virus by intranasal instillation of the AdNC.Hl vector at a dose of 1.7 X 10<sup>8 </sup>infectious units (ufi) 2 days before exposure (AdEin-2); 100% of the mice (10/10) were protected against influenza by intranasal instillation of the AdE vector at a dose of 1.7 X 10<sup>6</sup> ifu 2 days before exposure (AdEin-2);
- 46 20% of
<img file="MX348172B_D0022.tif" />
were protected against influenza by more intranasal AdE vector at a dose of 1.7 X 10<sup>6</sup> ifu 2 days before exposure (AdE * in-2); 90% of the mice (9/10) were protected against influenza by intranasal instillation of the AdE vector at a dose of 1.7 X 10<sup>8</sup> ifu 1 day before exposure (AdEin-1); all control mice (previously unexposed controls) died within 10 days of challenge. Data were plotted as% survival versus days after exposure. The numbers in parentheses represent the number of animals in each group.
Significance of Figure 1. Protection of animals against influenza by intranasal administration of an Ad vector 1 or 2 days before exposure to live influenza virus shows that the Ad particle is capable of rapidly inducing an antiviral state in the respiratory tract. . Since the Ad vectors code for influenza viruses. HA has been developed in influenza vaccines (Hoelscher et al., 2006; Tang et al., 2009; Van Kampen et al., 2005) and the vector AdNC.Hl.l encoding HA A / NC / 20/99 still confer rapid protection as a drug against influenza before adaptive immunity is induced, this is a test suggesting that this regimen represents a drug-vaccine duo (DVD) that can confer broad and rapid protection as an antiviral followed by induction of protective immunity as an antiviral vaccine, in a single package.
Methods of figure 2. The purified AdPA83 vectors encoding the protective antigen of Bacillus anthracis and its transgene-free counterpart (AdE) were administered dropwise in a volume of 0.05 ml into the nostril of each A / J (2 months old) using a mechanical pipette as described (Shi et al., 2001) before challenge. One to two days after Ad administration, mice were challenged intranasally with a lethal dose [1 X 10<sup>5</sup> colony-forming units (ufe)] of B. anthracis Sterne spores and was monitored daily for survival.
Results of figure 2. Sixty-seven percent of the mice (6/9) were protected against a lethal dose of anthrax spores by intranasal instillation of the vector AdPA83 at a dose of 1.3 X 10<sup>8</sup> ifu 2 days before exposure (AdPAin-2); 30% of the mice (3/10) were protected against anthrax by intranasal instillation of the AdE vector at a dose of 1.3 X 10<sup>8</sup> ifu 2 days before exposure (AdEin-2); no mice (0/9) were protected against anthrax by intranasal instillation of the AdE vector at a dose of 1.3 X 10<sup>6</sup> ifu 2 days before exposure (AdE * in-2); 22% of mice (2/9)
-48- IMPI ^ <Ν «ΤΤΓ> T <.> .Ítx ICA NC r> i the aromdac inpustriai were protected against anthrax by intranasal instillation of the AdE vector at a dose of 1.3 X 10<sup>8</sup> ifu 1 day before exposure (AdEin-1); all control mice (previously unexposed controls) died within 4 days post challenge. Data were graphed as% survival versus days after exposure. The numbers in parentheses represent the number of animals in each group.
Significance of Figure 2. Protection of animals against anthrax by intranasal administration of an Ad vector 1 or 2 days before exposure to anthrax spores shows that the Ad particle is capable of rapidly inducing an antibacterial state in the respiratory tract. Since PA-encoding Ad vectors have been developed in anthrax vaccines (McConnell et al., 2007) and PA-encoding vector AdPA83 still confer rapid protection as a drug against anthrax before adaptive immunity is induced, there is conclusive evidence that this regimen represents a drug-vaccine duo (DVD) that can confer rapid protection as an antibacterial drug followed by induction of protective immunity as a single-packaged antibacterial vaccine.
REFERENCES
Davies, J., and Davies, D. (2010). Origins and evolution of antibiotic resistance.
Microbiol Mol Biol Rev
- 49 74, 417-433.
IMPT tNRTITVW MEXICAN
OF THE PRC'MEBaL
INDUSTRY'
<img file="MX348172B_D0023.tif" />
Hoelscher, MA, Garg, S., Bangari, ώ. S., Belser,
JA, Lu, X., Stephenson, I., Bright, RA, Katz, JM, Mittal, SK, and Sambhara, S. (2006). Development of adenoviral-vector-based pandemic influenza vaccine against antigenically distinct human H5N1 strains in mice. Lancet 367,
475-481.
McConnell, MJ, Hanna, PC, and Imperiale, M.
J. (2007). Adenovirus-based prime-boost immunization for rapid vaccination against anthrax. Mol Ther 15, 203-210.
Poland, GA, Jacobson, RM, and Ovsyannikova, IG (2009). Influenza virus resistance to antiviral agents: a plea for rational use. Clin Infect Dis 48, 1254-1256.
Shi, Z., Zeng, M., Yang, G., Siegel, F., Cain, L.
J., Van Kampen, KR, Elmets, CA, and Tang, DC (2001). Protection against tetanus by needle-free inoculation of adenovirus-vectored nasal and epicutaneous vaccines. J Virol 75, 11474-11482.
Tang, DC, Zhang, J., Toro, H., Shi, Z., and Van Kampen, KA (2009). Adenovirus as a carrier for the development of influenza virus-free avian influenza vaccines. Expert Rev Vaccines 8, 469-481.
Van Kampen, KR, Shi, Z., Gao, P., Zhang, J., Foster, KW, Chen, DT, Marks, D., Elmets, CA, and Tang, DC (2005). Safety and immunogenicity of adenovirus50
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MAID <sup>, NST</sup>™7' .
OF LA RM,> F |<sub>£ A)</sub>. 'noustuial vectored nasal and epicutaneous influenza vaccines in humans.
Vaccine 23, 1029-1036.
EXAMPLE 2: DUO MEDICINE-VACCINE THAT HAS AS A VECTOR
ADENOVIRUS, AS A RAPID RESPONSE TOOL FOR
CONFER SEAMLESS PROTECTION AGAINST INFLUENZA
Some other diseases carry as great a burden of suffering as influenza. The applicant reports here that intranasal (in) administration of defective adenovirus particles in E1 / E3 (ΔΕ1 / Ε3) serotype 5 (Ad5) rapidly induces an anti-influenza state as a means of prophylactic treatment which lasts for several weeks. in mice. By encoding an influenza virus (IFV), the HA1 domain of hemagglutinin (HA), an Ad5-HA1 vector confers rapid protection as a prophylactic drug followed by induction of sustained protective immunity as a vaccine to induce protection Influenza seamless as a drug-vaccine duo (DVD) in a single package. Since Ad5 particles induce a complex set of responses in the host, which can suppress influenza by activating a specific arm of innate immunity to prevent the growth of IFV in the airways, it is conceivable that this branch influenza DVD multiples may escape the fate of drug resistance that harms current influenza drugs.
MSMCAN INSTITUTE.
Influenza is a disease that has germinated virtually without pn «i h 1-i ^ τ-radiate the causative virus which activates a seasonal influenza as well as a pandemic. As a zoonotic disease with the potential to make both animals and humans sick [1], an IFV designer can generate rapidly using reverse genetics [2] and can be spread by terrorists to harm agriculture, public health and the economy within of a target region. Although this highly contagious and life-threatening disease has been partially controlled by vaccination, licensed influenza vaccine is difficult to mass-produce [1] and is unable to provide timely as well as broad protection against heterosubtypical IFV strains [3]. Another line of defense against influenza is the use of influenza medications [eg, oseltamivir (Tamiflu); zanamivir (Relenza)]; however, this option is limited by the emergence of drug-resistant IFV due to selection under mutational pressure [4, 5].
To develop a rapid-response anti-influenza agent, we have fortuitously shown that a nasal influenza vaccine that has Ad5 as a vector can confer rapid protection against influenza in a drug-like manner. A replication competent adenovirus (RCA) free Ad5 vector '<sup>52</sup>' &
It encodes for pathogen antigens from this marilffiz ^ pu ^ rai potentially conferring protection without eos lili s ~ ^ - against mucosal pathogens as a DVD in a wide variety of clinical settings. RCA-free Ad5 vectors can be rapidly mass-produced in serum-free PER.C6 suspension cells; they can be administered in bulk painlessly by nasal spray [1], followed by induction of innate as well as adaptive immune responses to pre-existing Ad5 immunity. In the case of an influenza DVD, the probability of generating drug resistant IFV is minimal since Ad5 particles conceivably induce an anti-influenza state without directly attacking IFV. In contrast to a live attenuated IFV vaccine (LAIV), a DVD vectoring Ad5 is non-replicating and does not reassign with wild-type IFV. Nasal pressure from an influenza DVD vectoring Ad5 is expected to confer broad protection against heterosubtypical IFV strains for several weeks as a prophylactic drug; followed by induction of strain-specific protective immunity as a vaccine for months or even years before drug-induced protection wanes. This novel regimen can add a rapid response tool to the public health arsenal against influenza and other diseases if the protective effects of DVD are replicated in human subjects. wustmm
The particle Ad5 ΔΕ1Ε3 is an agent-aiitl lili lllenza.
The transgene-free Ad5 ΔΕ1Ε3 (AdE) empty particle and its counterpart DNAC.H1.1 encoding the HA1 domain of IFV A / New Caledonia / 20/99 H1N1 (NC20) are generated in PER.C6 cells as described. [one] . As shown in Figure 3, the 1.7 X 10 in.<sup>8</sup> Infectious Units (ufi) of AdE 2 days (day -2) before challenge protects 100% (10/10) of mice against a lethal dose of live IFV A / Puerto Rico / 8/34 H1N1 (PR8); only 20% (2/10) of the animals are protected when the AdE dose is reduced 100 times to 1.7 X 10<sup>6</sup> ifu and no protection when given 1.7 X 10<sup>8</sup> ifu of AdE in mice by instillation in 1 day after exposure to PR8 or by injection in on day -2. The insertion of the NC20 HA1 domain in the AdE genome slightly interferes with the ability of Ad5 ΔΕ1Ε3 to induce an anti-influenza state since only 70% (7/10) of the animals were protected when 1.7 X 10<sup>8 lfu</sup> of AdNC.Hl.l was administered in to mice on day -2. Similar to AoE, nor instillation in 1.7 X 10<sup>6</sup> ifu nor injection in of 1.7 X 10<sup>8</sup> AdNC.Hl.l ifu confers some protection against PR8 when administered on day -2 (Figure 3). The protection provided by the in administration of AdE (P, 0.0001) or AdNC.Hl.l (P = 0.0077) at a dose of 1.7 X 10<sup>8</sup> ifu in the day
<img file="MX348172B_D0025.tif" />
IMPI ΐΝ / πτυτυ mlxican.
Df THE HvrifDAt induítaiai
-2 reached statistical significance when compared with that of the untreated control group (by logarithmic classification tests).
Intranasal administration of AdE on day -47 (47 days before exposure to PR8) protects 70% of animals (7/10) showing that the anti-influenza state induced by AdE can persist for several weeks (figure 4 ). Intranasal instillation of AdNC.Hl.l on day -47 protects 100% (10/10) of the mice (Figure 4) probably due to adaptive immunity induced by HA1 of NC20, which produced a cross-reaction with PR8 although they were not detectable serum hemagglutination inhibition (HI) antibodies to PR8 (Table 1). Unlike immunization with AdNC.Hl.l on day -47 which induced high HI antibody titers for NC20 and undetectable titers for PR8, exposure with PR8 induced high HI antibody titers for PR8 and low titers for NC20 in the survivors and the administration of either AdE or AdNC.Hl.l on day -2 did not induce HI titers for NC20 or PR8 (Table 1). The protection provided by the administration in of AdNC.Hl.l on day -47 (P, 0.0001), AdE on day -47 (P = 0.0032), the double-dose AdE regimen (day -47 followed by a booster application on day - 2 (P = 0.0001), AdE on day -1 (P, 0.0001) or -2 (P = 0.0005) at a dose of 1.2 X 10<sup>8</sup> ifu all reached
<img file="MX348172B_D0026.tif" />
ΛίΤΓΠΠΟ-IxiCANe De LA ^ kü * t £ QAp 'NDU ^ KIAI statistical significance when compared to the untreated control group.
TABLE 1. HI SERIC ANTIBODY TITLES INDUCED BY
ADNC.HI IMMUNIZATION AND EXPOSURE TO PR8
<td></td><td></td><td></td><td></td><td></td><td></td><td></td>
<td>immunization</td><td>n</td><td>Whey collection day</td><td>Log<sub>2</sub> [anti- NC20 HI GMT] (+ SD)</td><td>Seroconversion to NC20 (%)</td><td>Log<sub>2</sub> [anti-PR8 HI GMT] (+ SD)</td><td>Seroconversion to PR8 (%)</td>
<td><sup>to</sup>AdNC / a 2 + pr8</td><td> 7</td><td> 19</td><td> 7.9(+0.5)</td><td> 100</td><td> 8.9 (+0.5)</td><td> 100</td>
<td><sup>to</sup>AdE / in-2 + PR8</td><td> 10</td><td> 19</td><td> 5.3( + 0.7)</td><td> 100</td><td> 7.5 (+0.6)</td><td> 100</td>
<td>"AdNC / in / 47</td><td> 10</td><td> -1</td><td> 10.2(+1.7)</td><td> 100</td><td> 2.3 (+0)</td><td> 0</td>
<td>° AdNC / in / -2</td><td> 10</td><td> -1</td><td> 2.3( + 0)</td><td> 0</td><td> 2.3( + 0)</td><td> 0</td>
<td>"AdE / in / -2</td><td> 10</td><td> -1</td><td> 2.3( + 0)</td><td> 0</td><td> 2.3( + 0)</td><td> 0</td>
<td>"Control no treaty</td><td> 10</td><td> -1</td><td> 2.3 ( + 0)</td><td> 0</td><td> 2.3 (±0)</td><td> 0</td>
HI antibodies were measured against respective IFV with titers expressed as GMT on a Log scale<sub>2</sub>; a Log title<sub>2</sub> out of 23 were arbitrarily assigned to samples with undetectable titers, each serum sample was run in triplicate wells;
<sup>to</sup>Animals described in Figure 1 with sera collected 19 days after PR8 challenge;
^ The animals described in figure 2 with sera collected 1 day before exposure to PR8. Seroconversion was defined as "> 4-fold increase in HI titer above baseline before immunization; n, number of animals; GMT, geometric mean title, SD, standard deviation.
Although various regimens protect mice against influenza-mediated mortality, the double-dose AdE regimen tends to confer stronger protection than its single-dose counterpart (day -47 or day -2), as shown
<img file="MX348172B_D0027.tif" />
shows for less weight loss
ÍMPl ^ • í LA MOhldab ____ -i <sup>, N</sup>»VSTitAL? body after exposure to
PR8 although the difference was not statistically significant (Figure 5). To induce an anti-influenza state, it is essential to suppress El and / or E3 since natural Ad5 El<sup>+</sup>/ E3<sup>+</sup> it is unable to suppress influenza after in-administration to mice under identical conditions (Figures 4A-4B).
Ad5-induced protection of the lung against influenza. As shown by lung histopathology after PR8 challenge, in-administration of AdE or AdNC.Hl.l on day -2 protects mice against influenza by preventing the development of severe lung damage. Intranasal instillation of PR8 without the protection of Ad5 induces massive lung inflammation 19 days after exposure, one-way ANOVA test and Turkey's multiple comparison post-test) 7 days after PR8 exposure.
Protection against a pandemic IFV strain. To demonstrate that Ad5 ΔΕ1Ε3 particles can protect mice against not only PR8 but also a more clinically relevant strain of IFV, it was administered in 2.5 X 10<sup>8 </sup>ifu of AdE or AdNC.Hl.l in mice followed by exposure of the animals with a lethal dose of the swine influenza isolate H1N1 2009 pandemic A / California / 04/2009 (CA04), as shown in Figure 8, 100% (10/10) of the animals were protected by in-instillation of AdlrOE AdNC.Hl.l on day -2 and AdNC.Hl.l on day -22, 90 were protected by in-administration of AdE on day - 22. The protection obtained against CA04 in all these groups exposed to Ad5 reached statistical significance when compared with that of the placebo control group (P, 0.0001).
The non-replicating Ad5 ΔΕ1Ε3 vector has been biomanipulated in a nasal influenza vaccine carrier with high potency and excellent safety profile [1]. In addition to elucidating protective immunity as a vaccine, we here demonstrate that this class of vaccine also confers prophylactic treatment against influenza before adaptive immunity is induced. Administration of Ad5 ΔΕ1Ε3 particles in mice has been documented to rapidly induce the production of a wide distribution of inflammatory cytokines and chemokines [6] including type I interferon (IFN-a and IFN-b) [7]; impairs lung dendritic cells [8]; activates natural killer cells [9]; induces antiviral nitric oxide production [10]; activates multi-faceted interactions between Ad5 and blood proteins, platelets, macrophages, endothelial cells, and respective parenchyma cells [6]. The inhibition of inflammation associated with Ad5 by proteins E1A, E1B and E3 [11] suggests that the incompetence of Ad5 El<sup>+</sup>/ E3<sup>+</sup> to induce an anti-influenza state (Figures 4A-4B) can be attributed to the suppression of inflammation, other mechanisms since they induce many responses although not
<img file="MX348172B_D0028.tif" />
<img file="MX348172B_D0029.tif" />
immune as well as non-immune, and some reactions remain undefined in animals [12]. It is conceivable that multiple reactions induced by Ad5 ΔΕ1Ε3 particles can be integrated to establish an anti-influenza state in the airways and in this way a multidimensional defense barrier is generated that can hardly be overcome by an IFV. This hypothesis is supported by the finding that the IFN-α / β receptor provides protection against influenza in a deliverable manner which demonstrates that animals have evolved bypassing mechanisms to respond to influenza [13]. Furthermore, the Balb / c mice exposed in this study have a defective allele of the IFN-α / β-induced influenza resistance factor, Mxl [14], which implies that the production of type I IFN induced by Ad5 ΔΕ1Ε3 [7] may not play a major role during the establishment of an anti-influenza status in this strain of mice.
The finding that in-administration of AdE 1 day after PR8 exposure is unable to suppress influenza (Figure 3A-3B) suggests that IFV can induce a pro-influenza state that is not disrupted by the Ad5 ΔΕ1Ε3 particle when the The first enters the airways _ »* 4 4 '^ * ¢ £ 5 before the last, similar to the<sup>N,</sup>'<Jsreeta ^ ®.
Ad5-induced anti-influenza which cannot be ex ^ ex ^ ed by an IFV when AdE particles are administered before PR8 or CA04 (Figures 3A-3B to Figure 9). To further develop the Ad5 Δ 1Ε3-based prophylactic drug into a post-exposure influenza drug, it is crucial to characterize the antagonistic reactions induced by the two types of viruses in the respiratory tract.
Previous exposure to Ad5 has been associated with loss of Ad5 potency when this vector is injected im [15]. However, emerging evidence shows that a nasal vaccine using Ad5 as a vector can overcome pre-existing immunity to Ad5 in mice [15], macaques [16] and humans [17] probably due to the delivery of a highly efficient gene within cells in the surface layer along the mucosal barrier along with a potent antigen presentation associated with this immunocompetent interface tissue. The synergy between the primary and booster applications induced by a double dose regimen of AdE (Figures 4A-4B and Figure 5) show that the rapid anti-influenza responses induced by AdE are additive in the presence of pre-existing immunity to Ad5. These findings support the promise that this nasal flu DVD is not only capable of inducing rapid protection
<img file="MX348172B_D0030.tif" />
I ñy Ip -, ry sustained against influenza on a single regimen but can also be administered repeatedly (for example when different HAs are required for its vaccine component) without losing potency.
Although prophylactic therapy against influenza can be performed by in-administration of complex bacterial uses [18] or bacterial toxins [19], the anti-influenza state induced by bacterial components is very transient with its protective effects diminishing within a period of a few days after treatment [18, 19]. The finding that the protective effects induced by AdE can last for at least 3 weeks (Figure 6) and up to 47 days (Figures 4A-4B) in a single dose regimen suggests that the underlying mechanisms between induced anti-influenza states by bacterial components or by Ad5 they may differ. Furthermore, only the latter allow sufficient time for the DVD vaccine component to induce adaptive immunity before the effects of the drug wear off. Furthermore, replicating natural Ad5 is a benign respiratory virus and its non-replicating counterpart used in this study is even safer; Notably, the safety profile of a nasal influenza vaccine using Ad5 as a vector has been shown [17] in human subjects. Like a common respiratory virus, the immune system in the human mucosa is
IMPI ^,,, IMTJTUTU MÜUON · • X ^ ieÉr '* familiar with Ad5 particles and must specific protective mechanisms for Ad5. In contrast, the administration of a bacterial toxin associated to the digestive tract into the respiratory tract as an influenza drug [19] would surprise the immune system, and this unnatural regimen has been associated with the induction of Bell's palsy in human subjects [20 ].
IFV is insidious in mutating into drug-resistant strains when attacked by an influenza drug [eg, the M2 ion channel blocker (amantadine; rimantadine) or the neuraminidase inhibitor (oseltamivir; zanamivir)] [5]. Unlike contemporary influenza drugs, the Ad5 vector DVD conceivably changes the habitat in the airways without directly attacking IFV; therefore, DVD does not confer mutational pressure to induce drug resistance. In contrast to an oseltamivir-induced suppression of mucosal immunity with the risk of increasing vulnerability to subsequent mucosal pathogen infections [21], the Ad5 vector DVD increases innate mucosal immunity against at least a subset of mucosal pathogens. The efficacy of DVD is further enhanced by its vaccine component that induces sustained adaptive immunity before its effects as a drug completely wear off (figure 2 a licensed (for example,
- 62 figure 8).
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contains live IFV [1], co-administration of LAIV with an influenza drug may be counterproductive because the drug will disable the vaccine by destroying live IFV. The DVD that has Ad5 as a vector is not only compatible with the authorized influenza medicine but also confers prophylactic treatment as a medicine in itself, in addition to its capacity as a vaccine.
Emerging evidence shows that a number of nasal vaccines induce a weaker systemic adaptive immune response than their parenteral counterparts [22-26], although nasal vaccines confer more robust protection against a mucosal respiratory pathogen by inducing an immune response. more powerful adaptive mucosa [22, 25]. The applicant provides evidence that not only adaptive immunity but also innate immunity can be induced with a focus on the airways against mucosal pathogens when the Ad5 ΔΕ1Ε3 particle is administered in but not im as shown by the survival percentage provided through the in and im paths respectively (figure 3). It remains to be seen whether nasal DVD that has Ad5 as a vector can confer protection against influenza induced by other routes (eg, oral infection).
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The finding that management in of AdNC.Hl.l on day -47 induces more robust protection against exposure to PR8 compared to its counterpart inoculated on day -2 or administered by AdE on day -47 (Figure 3 and Figure 4) suggesting that Animals in the AdNC / in / -47 group may be protected by an adaptive immune response mediated by ΗΑ1 NC20 that cross-reacts with PR8 47 days after immunization in the absence of detectable serum HI antibody to PR8 (Table 1). The data corroborate other reports that serum HI antibody titer is an inadequate surrogate marker for predicting nasal influenza vaccine-induced protective immunity [24, 26].
Findings that Ad5 vector DVD can confer prophylactic treatment in conjunction with vaccination in a single package provides rationale for the development of a novel anti-influenza agent that can be mass-produced in cultured cells, painlessly administered by spray nasal, with the ability to bypass pre-existing immunity to Ad5 and mobilize innate as well as adaptive immune repertoires toward a rapid and sustained beneficial response against influenza, without the potential to generate drug-resistant strains of IFV.
Adenovirus. To generate the AdE particle, homologous recombination was performed between the pAdHigh shuttle and
IMPT ^ s * backbone plasmids pAdEasy-1<sup>1</sup>/ Escherichia coli BJ5183 cells subsequent to generation of a RCA-free AdE particle in PER.C6 cells (provided by Crucell Holland BV; Leiden, The Netherlands) as described [1]. AdE therefore is Ad5 ΔΕ1Ε3 with an expression cassette in its El [1] region without coding for any transgene. To generate the AdNC.Hl.l vector, the HA NC20 gene is synthesized in GENEART (Regensburg, Germany) with codons optimized to match the pool of tRNA found in human cells along with the insertion of a eukaryotic ribosomal binding site immediately towards the 5 'end from the ATG start codon [27]. The HA1 fragment of NC20 contains 347 amino acids and was amplified from a synthetic HA template by polymerase chain reaction (PCR) using primers 5'-CACAGGTACCGCCACCATGAAGGCCAAGCTG-3<sup>1</sup> and 5'GAGTCTAGATTATCAGCCGAACAGGCCTCTGCTCTGG-3 '. The KpnlXbal fragment containing the amplified HA1 fragment with a stop codon added in frame was inserted into the Kpnl-Xbal site of pAdHigh in the correct orientation under transcriptional control of the human cytomegalovirus (CMV) early promoter. An RCA-free Ad5 vector encoding HA1 NC20 (AdNC.Hl.l) is subsequently generated in PER.C6 cells as described above. Both AdE and AdNC.Hl.l are validated by DNA sequencing; produced in
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mass in PER.C6 cells; purified by ultracentiTU ^ acr & S on a cesium chloride gradient as "described" [27]; dialyzed in A195 buffer [28] with titers (ufi per ml) determined in 293 cells [17] by the Spearman-Karber method [29] after staining of Ad5-infected monolayers with anti-Ad5 exon antibody conjugated to horseradish peroxidase (HRP) and 3,3'-diaminobenzidine (DAB) substrate (Clontech Laboratories, Inc .; Mountain View, CA). The natural Ad5 The<sup>+</sup>/ E3<sup>+ </sup>(VR-1516) is obtained from the American Type Culture Collection (ATCC, Manassas, VA).
Influenza virus. PR8 (VR-95) is obtained from ATCC and grown in Madin Darby dog kidney cells (MDCK) in the presence of TPCKtrypsin as described [17] with titers determined by plaque analysis [ 30]. Mouse adapted CA04 is generated by Natalia A. Ilyushina and provided by Elena Govorkova at St. Jude Children's Research Hospital (Memphis TN). The CA04 virus adapts to replication in the lungs of Balb / c mice by 9 sequential passages through mouse lungs. The virus is plaque purified in MDCK cells and a virus concentrate is prepared by growing in 10-day-old embryonated chicken eggs and then in MDCK cells as described [31] with titers expressed as infectious cell culture dose ( CCID<sub>50</sub>) how has it <sup>66</sup> IMPI ΐΜ'ΠΤυΐ · '. «EuCA.N the FnoPieoAt <INtmTIlAl described [32]. NC20 is provided by the Center for Disease Control (CDC; Atlanta, GA). ~ '
Exposure studies. Intranasal administration and im injection of 50 µΐ of Ad5 particles into young female Balb / c mice (approximately 2 months old) was performed as described [27]. Mice were challenged by instillation in of 50 μΐ of PR8 containing either 1.4 x 10<sup>6</sup> plaque-forming units (pfu) [equivalent to approximately 4 x LD50 (lethal dose 50%)] or 3.5 x 10<sup>6</sup> pfu (equivalent to approximately 10 x LD50) at University of Alabama at Birmingham (UAB) as well as 90 μΐ of CA04 containing 26105 CCID<sub>50</sub> (equivalent to approximately 3 x DL<sub>50</sub>) at Utah State University (USU). All experiments using mice were performed in accordance with the approval of the Institutional Animal Care and Use Committees at UAB and USU (UAB approval ID, # 7705; UAB animal welfare assurance number, A3255-01; USU approval ID, # 552; USU Animal Welfare Assurance Number, A3801-01). The animal facilities at both UAB and USU have been accredited by AAALAC.
PR8 titers in lungs after exposure. AdE particles were administered in to young female Balb / c mice at a dose of 1.2 x 10® ifu in a volume of 50 µΐ on day -2. Five to seven days after instillation in ED 4.6 X 10<sup>6</sup> ufp in PR8 on day 8,
<img file="MX348172B_D0033.tif" />
: ITUTO A., '<> (»·. * AdE-exposed mouse lungs and controls se cótt ^' ^^ on. ^ S. ** · <sup>;</sup>immediately on dry ice after excision and stored at 280 pC (sic) until analysis. After rewarming, a fraction from each lung was weighed and homogenized in cold phosphate buffered saline (PBS) as a 10% (w / v) suspension. Tissue debris was removed by centrifugation and the supernatant was transferred to another sterile tube for virus titration. IFV plaque analysis was performed as described [30].
Hemagglutination-inhibition analysis. Sera were tested for activity against PR8 or NC20 by a standard assay for HI after pretreatment of the sera with receptor-destroying enzyme, as described [17]. Each serum sample is tested starting with a 1:10 dilution. All sera are blind or code-tagged tested from paired samples before and after immunization. Animals are considered seronegative and assigned an H1 antibody titer of 5 (2.3 on a Log2 scale) if their serum specimen has an HI titer of <10.
Lung histopathology analysis. Mouse lungs were fixed by perfusing 10% buffered formalin through the trachea. Paraffin-embedded tissues are cut into 5 µm thick slices followed by staining of the sections with hematoxylin and eosin.
Statistic analysis.
All analyzes
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5.04 (GraphPad Software, San Diego, CA). Logarithmic classification tests were performed to compare Kaplan-Meier survival curves; and post-test Turkey multiple comparison one-way ANOVA were performed to compare body weight loss as well as PR8 titers in lungs. Statistical significance was established at P, 0.05.
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7. Yamaguchi T, Kawabata K, Kouyama E, Ishii KJ, Katayama K, et al. (2010) Induction of type 1 interferon by adenovirus-encoded small RNAS. Proc Nati Acad Sci USA 107: 17286-17291.
8. Thiele AT, Sumpter TL, Walker JA, Xu Q, Chang CH, et al. (2006) Pulmonary immunity to viral infection: adenovirus infection of lung dendritic cells renders T cells nonresponsive to interleukin-2. J Virol 80: 1826-1836.
9. Zhu J, Huang X, Yang Y (2008) A critical role for type I IFN-dependent NK cell activation in innate immune elimination of adenoviral vectors in vivo. Mol Ther 16: 1300-1307.
10. Higashimoto Y, Yamagata Y, Itoh H (2006) Complex effect of adenovirus early region proteins on innate immune system. Inflamm Allergy Drug Targets 5: 229-237.
eleven. Schaack J, Bennett ML, Colbert JD, Torres AV, Clayton GH, et al. (2004) E1A and E1B proteins inhibit inflammation induced by adenovirus. Proc Nati Acad Sci USA 101: 3124-3129.
12. Rhee EG, Blattman JN, Kasturi SP, Kelley RP,
Kaufman DR, et al. (2011) Multiple innate immune pathways
- 70 contribute to the immunogenicity of vaccine vectors. J Virol 85: 315-323.
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INSTIT.ITO '<sup>,F</sup> W m · ί · <ΠΛΓ · '^' OUjTRlA,
<img file="MX348172B_D0035.tif" />
recombinant adenovirus
13. Goodman AG, Zeng H, Proll SC, Peng X, Cilloniz C, et al. (2010) The alpha / beta inferred receptor provides protection against influenza virus replication but is dispensable for inflammatory response signaling. J Virol 84: 2027-2037.
14. Tumpey TM, Szretter KJ, Van Hoeven N, Katz JM, Kochs G, et al. (2007) The Mxl gene protects mice against the pandemic 1918 and highly lethal human H5N1 influenza viruses, J Virol 81: 10818-10821.
fifteen. Croyle MA, Patel A, Tran KN, Gray M, Zhang Y, et al. (2008) Nasal delivery of an adenovirus-based vaccine bypasses pre-existing immunity to the vaccine carrier and improves the immune response in mice. PLoS ONE 3: e3548.
16. Song K, Bolton DL, Wilson RL, Camp JV, Bao S, et al. (2010) Genetic immunization in the lung induces potent local and systemic immune responses. Proc Nati Acad Sci USA 107: 22213-22218.
17. Van Kampen KR, Shi Z, Gao P, Zhang J, Foster KW, et al. (2005) Safety and immunogenicity of adenovirusvectored nasal and epicutaneous influenza vaccines in humans. Vaccine 23: 1029-1036.
18. Tuvim MJ, Evans SE, Clement CG, Dickey BF, Gilbert BE (2009) Augmented lung inflammation protects against
- 71 influenza A pneumonia. PLoS ONE 4: e4176.
<img file="MX348172B_D0036.tif" />
19. Norton EB, Clements JD, Vece TG ,. ... Carmenas-
Freytag L (2010) Prophylactic administration of bacterially derived immunomodulators improves the outcome of influenza virus infection in a murine model. J. Virol. 84: 2983-2995.
twenty. Couch RB (2004) Nasal vaccination, Escherichia coli enterotoxin, and Bell's palsy. N. Engl. J. Med 350: 860-861.
twenty-one. Takahashi E, Kataoka K, Fujii K, Chida J, Mizuno D, et al. (2010) Attenuation of inducible respiratory immune responses by oseltamivir treatment in mice infected with influenza. A virus. Microbes Infect 12: 778-783.
22. Wang J, Thorson L, Stokes RW, Santosuosso M, Huygen K, et al, (2004) Single mucosal, but not parenteral, immunization with recombinant adenoviral-based vaccine provides potent protection from pulmonary tuberculosis. J Immunol 173: 6357-6365.
2. 3. Lemiale F, Kong WP, Akyurek LM, Ling X, Huang Y, et al. (2003) Enhanced mucosal immunoglobulin A response of intranasal adenoviral vector human immunodeficiency virus vaccine and localization in the central nervous system. J Virol 77: 10078-10087.
24. Hoelscher NA, Garg S, Bangari DS, Belser JA, Lu X, et al. (2006) Development of adenoviral-vector-based pandemic influenza vaccine against antigenically distinct human H5N1 strains in mice. Lancet 367: 475-481.
.<sub>72</sub>. IMPI ^ pcrrm πτ. mlx ¡c year Pf THE UNPUSTUAL PROPERTY ^ »7 **
25. Price GE, Soboleski MR, Lo CY, Misplon JA, Quirion MR, et al. (2010) Single dose mucosal immunization with a candidate universal influenza vaccine provides rapid protection from virulent H5N1, H3N2 and H1N1 viruses. PLoS ONE 5: el3162.
26. Clemente ML, Betts RF, Tiemey EL, Murphy BR (1986) Serum and nasal wash antibodies associated with resistance to experimental challenge with influenza A wildtype virus. J Clin Microbiol 24: 157-160.
27. Shi Z, Zeng M, Yang G, Siegel F, Cain IJ, et al. (2001) Protection against tetanus by needle-free inoculation of adenovirus-vectored nasal and epicutaneous vaccines. J Virol 75: 11474-11482.
28. Evans RK, Nawrocki DK, Isopi LA, Williams DM, Casimiro DR, et al. (2004) Development of stable liquid formulations for adenovirus-based vaccines. J Pharm Sci 93: 2458-2475.
29. Lynn DE (2001) Effects of temperature on the susceptibility of insect cells to infection by baculoviruses. Methods Cell Sci 23: 221-225.
30. Gaush CR, Smith TF (1968) Replication and plaque assay of influenza virus in an established line of canine kidney cells. Appl Microbiol 16: 588-594.
31. Ilyushina NA, Khalenkov AM, Seiler JP, Forrest HL, Bovin NV, et al. (2010) Adaptation of pandemic H1N1
IΜ ρ J '“•' SAW Ίο .aericanv '- <11. _- ,. . . . ,<sup>Gl u</sup> λ.
influenza viruses m mice. J Virol 84: 8607-8616.
32. Barnard DL, Wong MH, Bailey KJ — T * r? Ry — PW<sub>V</sub>· ... gjLHwa Ί 1 RW, et al. (2007) Effect of oral gavage treatment with ZnAL42 and other metallo-ion formulations on influenza A H5N1 and H1N1 virus infections in mice. Antivir Chem Chemother 18: 125-132.
EXAMPLE 3: DUO MEDICATION-VACCINE THAT HAS ADENOVIRUS AS A VECTOR AS A POTENTIAL ACTIVATOR TO CONFER MASS PROTECTION AGAINST INFECTIOUS DISEASES
The disease-fighting ability of vaccines has been a public health bonanza credited with reducing worldwide mortality and morbidity. The objective is to further amplify its potency by strengthening vaccine coverage that requires the development of a new generation of rapid response vaccines that can be mass-produced at low costs and administered massively by non-medical personnel. New vaccines also need to have a greater margin of safety than conventional vaccines. The vaccine that has as a vector non-replicating adenovirus holds promise in enhancing vaccine coverage because the vector can be rapidly manufactured in cells in serum-free suspension in response to a sudden increase in demand and can be administered non-invasively by nasal spray in human subjects in compliance with the
IΜ ΡI · evolutionary medicine. In contrast to palliate injection, non-invasive mucosal vaccination minimizes a · - systemic inflammation. Furthermore, pre-existing immunity to adenovirus does not appreciably interfere with the potency of a nasal vaccine vectoring adenovirus. Nasal administration of adenovirus vectors encoding pathogen antigens is not only worry-free and painless but also confers rapid and sustained protection against mucosal pathogens as a drug-vaccine duo since the adenovirus particles alone without the Expression of a transgene can induce an anti-influenza state in the respiratory tract. In addition to human vaccination, animals can also be massively immunized by this class of targeted vaccines.
A lot of demands for better vaccines.
Although vaccination has proven to be the most cost-effective method of disease prevention, a sweeping crackdown for booster vaccine coverage remains a driving goal in the movement toward improved public health around the world. Current vaccines that have been licensed for commercialization include whole killed microorganisms, live attenuated microorganisms, microbial extracts, purified or recombinant proteins, DNA vaccines, and particles similar to
<img file="MX348172B_D0037.tif" />
virus. Although many diseases have been of ^ F ^ t'á ^ s;! ^ Wide distribution of these vaccines, the goal of generating immunity in a community (herd) in a wide variety of disease settings remains elusive due to the large number of problems in current vaccination programs. Specifically, adverse side effects associated with vaccines range from a local and systemic inflammatory response, fever, platelet activation, cardiac autonomic dysfunction, anaphylactic reaction (induced by needle injection of certain vaccines) [1-4] to the rare presentation of crippling poliomyelitis (mediated by ingestion of oral polio vaccine) [5], myopericarditis (induced by inoculation of the Dryvax smallpox vaccine) [6] and Bell's palsy (induced by a nasal adjuvant for bacterial toxin) [7, 8]. In 2010, a sudden increase in narcolepsy was reported among vaccinated persons in some countries after water injection of an H1N1 pandemic influenza vaccine containing adjuvant squalene [201]. Squalene injection alone can induce rheumatoid arthritis in animals [9]. As evidence is being generated, chronic low-grade inflammation is associated with cardiovascular disease [10], obesity [11], diabetes [11], cancer [12] and neurological disorders [13], vaccine-induced inflammation now you need focused attention. The fact that an injection-induced acute inflammatory reactionjd | ^ ú © vaccine-immunostimulating adjuvant [1-3] can & e ^ e ^^ jú ^ íohMl ^^ chronic low-grade inflammation and activate any of these discomforts in a subset of people vaccinated over time is of primary importance in public health; however, this potential hazard has not been thoroughly investigated. Since the concept of vaccine safety has evolved from protection against pathogen-induced diseases to zero possibility of inducing adverse consequences, no known foreign agent, toxicity or residual virulence found in a vaccine is allowed and any possibility of inducing side effects should be avoided. unknown (for example, inflammation in vital organs).
Both mucosal and systemic immune responses are induced and regulated with a considerable degree of independence, and most vaccines have been administered invasively by intramuscular injection, inducing good systemic immunity but often weak mucosal immunity that is crucial. in defense against mucosal pathogens (eg, influenza viruses, Mycobacterium tuberculosis and HIV) [14,15]. Efficient induction of mucosal immunity usually utilizes nasal or oral vaccination due to the unique ability of mucosal resident dendritic cells (DCs) to 'NSTir ,, · ,,. «Induce IgA change and print mucosal-specific receptors ee © teáticq ^ í« C ^ P (for example, CCR9 and
Jal) on lymphocytes
[15,
16]. In addition to weak mucosal immunity induced by an injectable vaccine, the needle of a syringe as a vaccine delivery device also poses serious problems from unintentional or inadvertent non-sterile reuse, needle stick damage, waste disposal of inappropriate mode as well as limited injection service by authorized medical personnel during a crisis [17]. The public fear of pointed needles (aichmophobia) plays another role in avoiding vaccine coverage. Some people in this way may prefer the probability of acquiring a disease compared to the probability of inflicting pain, harm or death by systemic vaccination. Since the goal of vaccination programs is to reduce the overall likelihood of infection by generating immunity in the community (herd), the mission is impaired by not being vaccinated due to public concern about the risks. Today, technologies being developed to reverse negative perceptions by developing a new generation of rapid-response vaccines that are safe, effective, painless, and inexpensive are on the horizon.
Noninvasive vaccination as a means of reinforcing vaccine coverage. Non-invasive vaccination without needles
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Of LA l '»ONEOAD CV — Industrial TOajíC still stands as a promise of changing the public's attitude from being forced to receive a needle stick to proactively seeking vaccination without fear. The vaccines can be administered non-invasively by oral ingestion [5], nasal spray [18, 19] as well as topical application of a skin patch [19-23] in a painless manner. Non-invasive vaccination by administering vaccines at the interface between the internal body and the external environment not only confers a high degree of comfort towards the vaccine but can also generate a qualitatively superior immune response compared to conventional systemic vaccination. Mucocutaneous surfaces are covered by a highly immunocompetent epithelium that serves as a physical barrier and that ensures that antigens that penetrate the surface layer are efficiently retained and presented to the immune system. Logically, animals and humans must develop more competent immune cells along the surface barrier to eliminate infections since it could be counterproductive to keep these professional immune soldiers in deep tissues where they will rarely encounter invading pathogens. Professional antigen-presenting cells (APCs) include multiple DC subsets [24, 25], gdT cells [26], and others can be found in high II - A -; [í TT \. .- · ·. . i 'ii densities along the mucóéútáfo & á surface.
Subset of mouse bone marrow cells expressing retinoic acid synthesizing enzyme are capable of delivering retinoic acid to DC precursors to induce mucosal DC functions, including generation of Foxp3 + regulatory T cells, IgA-secreting B cells, and ecotaxic receptors mucosal specific [27]. The vaccination route has been shown to critically affect not only the magnitude but also the phenotype and trafficking of antigen-specific CD8 + T lymphocytes in mice. Intramuscular injection of a vaccine using adenovirus (Ad) as a vector induces robust local transgene expression and induces polyfunctional CD8 + T lymphocytes with high frequency that carry out extensive traffic to both systemic and mucosal compartments. In contrast, intranasal instillation of a vaccine having Ad as a vector has led to similarly robust local transgene expression but generating low-frequency monofunctional CD8 + T cells with limited anatomical trafficking patterns [28]. Noninvasive vaccination in this manner takes advantage of an existing biological pathway that catapults the ability of the immune system to respond at superficial but immunocompetent tissue site along the mucocutaneous surface to induce localized protective immunity against mucosal pathogens in the portal without inducing a
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Although it is required to aseptically manufacture vaccines under current good manufacturing practices, contamination by unknown microorganisms or contamination below detection by modern instruments and high-throughput testing can still occur. Co-administration of these contaminants with a non-invasive vaccine on the mucocutaneous surface may pose little danger to the vaccine since the mucocutaneous immune system is well versed in countering microbial invasions at all times since the interface is in constant contact with microbes. In contrast, the injection of a contaminated vaccine into deep tissues can theoretically trigger an exponential growth of microorganisms within the body in the absence of a timely immune response or, conversely, an immune storm induced by an overreactive immune system. In general, the induction of protective immunity along the mucocutaneous surface is a daily routine, animals and humans have evolved with adequate mechanisms to win in daily battles (daily microbial invasion) without losing the war (general health). Noninvasive vaccination utilizes the daily operation of the immune system along the interface without suppressing the immune system by the physical delivery of deep immunostimulatory adjuvant-vaccine complexes where immunocompetence is low.
The zigzag pathway to develop an adenovirus in a vaccine carrier. Adenovirus belongs to a family of non-enveloped icosahedral DNA viruses with a linear DNA genome of 30-68 kb (size varies from group to group), packed in inverted terminal repeating units. An Ad particle contains a highly coiled DNA genome packaged within a hexagonal protein capsid (Figure 9A). The Ad genome contains both early genes encoding regulatory proteins as well as late genes encoding structural proteins [29]. Multiple serotypes of Ad are commonly found in animals and humans, and these can be significant differences in pathogenicity and disease course between the different serotypes; some are very benign in immunocompetent human hosts (eg human Ad serotype 5 [30] while others can cause diseases that usually range from moderate to self-limiting. A number of notable reasons warrant development of Ad in a vaccine carrier. Specifically, oral vaccines based on human Ad4 and Ad7 (a type of non-invasive vaccine) have been shown to be safe as well as effective during mass immunization of recruits in the military [31].
Potentially, the replication of Ad4 or Ad7 can be
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further biomanipulated in vamna carriers to induce immunity against other pathogen-derived antigens. However, it is difficult to quantitatively release a replicating biomanipulated vector representing a genetically modified organism in a controlled manner. The introduction of a genetically modified organism into the ecosystem is also undesirable in the public perception. A vector not replicating in this way may be safer and more acceptable than its replicating counterpart. Although the non-replicating Ad5 vector was developed nearly three decades ago by cutting its El region (Figure 9B) [32], a critical issue for the El-defective Ad5 vector (ΔΕ1) produced in human 293 cells is intrinsic contamination by Ad replication competent (RCA) that arises through homologous recombination between overlapping sequences framed by the El locus shown by transected 293 cells and the vector backbone [33]. RCA represents a biohazard because it can replicate in an infected host with the capacity for horizontal transmission by bystanders through virus shedding [30]. To eliminate this RCA problem, RCA-free Ad vectors have been generated in human PER.C6 cells using PER.C6-compatible shuttle vectors that do not contain overlapping sequences with the PER.C6 genome [34, 35]. Unlike replicating Ad4 and Ad7, Ad5 does not
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L1 LA? K PIEDAD V »· ™ industrial replicant does not immunize animals efficiently when wa 11i'iw <- administered orally due to their inability to undergo virus amplification and their susceptibility to low pH, gastric and pancreatic proteases and extracellular mucins [36].
Despite the problems observed after oral administration, non-replicating E1 / E3 (ΔΕ1Ε3) defective Ad5 has developed (Figure 9B) and has been used as a parenteral gene therapy vector in a large number of therapeutic trials due to to its high capacity to host transgenes, high production titer, high efficiency in gene delivery and high level of transgene expression (at least in an initial download) [35]. However, Ad5 is not an ideal vector for classical gene therapy because the expression of a transgene is transient [35]. Therefore, the use of an Ad5 vector does not satisfy a primary goal of gene therapy, which usually requires sustained transgene expression. Furthermore, the common presence of pre-existing Ad5 immunity in human populations [37-39] and the rapid development of an anti-Ad5 immune response after the first inoculation with the vector [40, 41] has prevented its clinical use by limiting efficiencies. gene transfer. Strategies to overcome this drawback include serotype switching, capsid modification, and development of non-Ad vectors.
IMPIOS ΐΝΓΠτυτη MetíCANO ,, Γ4Ι.Α Ρ ^ 'Ρ ^ ΓΑΩ humans, on the assumption that another vector Ad<sup>ST, i,</sup>pueae —--- substitute when initial is disabled p ^ t> - preexisting immunity — Ad. Although human Ad3, Ad4, Ad35, Ad41 or chimeric Ad5 containing the Adll or Ad35 fibers have been biomanipulated in non-replicating Ad vectors (Figure 10), Ad5 is still more potent and safer than other serotypes in preclinical animal models [42]. Numerous non-human Ads such as bovine Ad [43], porcine Ad [43], and non-human primate Ad [44] have been developed to expand their repertoire of Ad vectors (Figure 10). Although the porcine Ad vector vaccine may be at least as potent as its Ad5 counterpart in mice [45], human Ad5 is still the premier gene transfer vector due to the risk of inducing unpredictable human disease by non-human Ad [46]. The human Ad5 genome is remarkably stable in the field even after coinfection with other Ad serotypes [47]. Furthermore, the Ad5 vector has been further developed to display foreign antigens on the surface after fusion of pathogenic epitopes to pIX [48] or exon capsid proteins [49] in addition to encoding pathogenic antigens in its DNA genome (Figure 10). The least immunogenic Ad5 vectors have been developed by deletion of E2b [50, 51] or almost complete Ad5 sequences except for the inverted terminal repeat sequences and the packaging signal (Ad without gut) (figure 10)
[52] .
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<img file="MX348172B_D0040.tif" />
sophisticated strategies have yet to provide profound clinical improvement.
In contrast to intramuscular or intravenous injection of Ad5, it has been shown that intranasal administration (the natural route of infection of Ad5) can allow a vaccine vectoring with Ad5 ΔΕ1Ε3 to bypass pre-existing Ad5 immunity without appreciable loss of potency in mice [41, 53], non-human primates [54], and humans [19]. These observations are conceivably attributable to the high efficiency of gene delivery, robust transgene expression, and potent antigen presentation along the mucosal barrier in the airways. Anti-Ad5 immunity in this way is not an insurmountable limiting factor and refinement of biomanipulated Ad vectors may no longer be a sine qua non for further development of Ad vector vaccines.
The reputation of the Ad5 vector has been shattered multiple times during its development. In addition to pre-existing immunity to Ad5, death in a patient with partial ornithine transcarbamylase (OTC) deficiency after infusion of a high dose of an Ad5-OTC vector into his hepatic artery during a human gene therapy trial [55] I mark Ad5 as a dangerous vector
<img file="MX348172B_D0041.tif" />
- 86 IMPI in public perception. Evidence shows that the injection of Ad particles into the circulatory system (an unnatural pathway by Ad) is an unsafe approach because Ad particles rapidly induce post-injection systemic inflammation [56, 57] and a variety of Ad serotypes elicit coagulation activation, possibly through interaction with platelets [42]. During a large-scale human trial (study stage) of an HIV vaccine that has Ad5 as a vector, administration by intramuscular injection did not decrease the HIV viral load and vaccination was associated with an increased risk of HIV infection. in humans in Ad5-seropositive subjects [58, 59]. The counterintuitive results can again be attributed to a misuse of the vector since the potency of a vaccine that has Ad5 as a vector surpasses that of other vaccine platforms based on other viruses or based on substances that are not viruses in inducing cellular immunity [60]; consequently, the Ad5-induced expansion of CD4 + T cells may exacerbate this peculiar disease since CD4 + T cells are the specific targets for HIV infection. [61]. In addition, human subjects were immunized by intramuscular injection of Ad5 particles during the study stage [58, 59] which are not very potent in inducing mucosal immunity against a mucosal pathogen such as HIV [14, 62, 63].
’<sup>87</sup>IMPI ^ <sup>Ν</sup> οι LA · * ΡΙ £ ΜΛπ ,, __. ^ .___ · -_ J _<sub>c</sub>. -U η jj π η INDUSTRIAL
Verification of reliability of vaccines that have current adenovirus as vector. To develop the next generation of vaccines that are safe and effective, it is crucial that the vaccine induces protective immunity quickly with a high benefit-to-risk ratio. The manufacture, distribution, and administration of the vaccine should be easy, fast, and inexpensive. Furthermore, the inherent stability of the formulated vaccine and the final filled product needs to allow long term storage without a cold temperature chain.
As shown in Table 2, protective immunity against a wide variety of pathogens has been induced in mice, guinea pigs, chickens, hamsters, cotton rats, raccoons, skunks, pigs, and non-human primates after immunization with vector vaccines. to Ad. In general, Ad vector vaccines can confer faster and more robust protection against live pathogens compared to other types of vaccines in animal models.
Although multiple human clinical trials of vaccines using Ad vector have been conducted, some immunized human subjects have been challenged with a live virulent pathogen (Table 3). Notably, a subset of human volunteers immunized by intramuscular injection of DNA and malaria vaccines have
<img file="MX348172B_D0042.tif" />
MAID
ΚΤΓΠΙΤ * MEXICAN • Ε LA MOHEDAL 'INDUSTRIAL an Ad5 vector (DNA-primed / Ad5-boosted) protected against exposure to live sporocyte of post-mosquito feeding malaria in human subjects seronegative to Ad5. DNA-only vaccination without Ad5 boost has been shown not to protect humans against malaria; whether vaccination with Ad5 alone can confer protection remains to be explored [64]. Although immunized humans have not been challenged with live pathogens during most human trials (Table 3), they have provided a comprehensive safety database for the use of Ad vectors in humans.
Potency and safety of nasal vaccines that have adenovirus as a vector. As described above, nasal vaccination induces potent mucosal immunity in a needle-free manner. Airway DCs form a continuous subepithelial network within the naso-respiratory tract, forming a bridge between innate and acquired immunity. The density of DCs within the airways is higher in those areas exposed to greater amounts of inhaled antigen [65]. The nasopharyngeal-associated lymphoid tissue, which constitutes Waldeyer's ring in humans, is a unique inducing site for B-lymphocyte responses and plasma cell generation. In this way, nasal vaccination is an activator for the induction of humoral immunity that includes the formation of IgA antibody.
<img file="MX348172B_D0043.tif" />
- 89 secretory IMPI within the respiratory tract · * induced humoral immune responses, in nasal, vaginal and salivary secretions after intranasal administration of vaccines that have Ad as a vector within non-human primates [67]. A nasal vaccine using Ad5 as a vector induces specific IgA responses to higher antigens in mucosal secretions and serum in mice than its injectable counterpart [68]. In addition to humoral immunity, cellular immune responses are observed in mucosal and systemic immune compartments shortly after mice are immunized with a herpes vaccine having Ad vector regardless of the route of inoculation; however, anamnestic cytotoxic T cell responses were exclusively divided to mucosal or systemic lymphoid tissues after mucosal or systemic immunization, respectively, several months post-immunization [14].
Although the DNA-evaded / Ad5-boosted malaria vaccine induced protection against exposure to live malaria sporozoites in Ad5-seronegative human subjects, it failed to protect five Ad5-seropositive human volunteers [64] which can be attributed to pre-existing immunity against Ad5 [37, 38, 40, 41]. As described above, one approach to overcome this problem is to inoculate vaccines that have Ad as a vector.
<img file="MX348172B_D0044.tif" />
by nasal administration, which apunjaj ^ l ^
INSTITUTE MüXICaN. ·:
OS W «uriTOAM disadvantage for injectable vaccines and a '^ WTffajá for noninvasive mucosal vaccines without nelucid efficacy, 1 subsequent re-administration of Ad5 [19, 41, 53, 54]. A nasal vaccine vectoring Ad5 can induce mucosal immunity focused on the airways, as shown by the findings that intranasal immunization, but not systemic immunization, induces long-lived cytotoxic T lymphocytes in mucosal tissues [ 14]. In addition, nasal vaccines using Ad5 as a vector can protect animals against mucosal pathogens when systemic immunization fails, although the latter induces a more robust systemic immune response [63, 69, 70]. The hypothesis that the focused mucosal immune response induced by nasal vaccination can greatly reduce the systemic burden (e.g., systemic inflammation) to unaffected internal tissues and organs has been generated by the finding that CD103 + mucosal DC may dampen inflammatory responses by harboring the conversion of previously unexposed T cells to Foxp3 regulatory T cells<sup>+</sup>.
Common adverse effects induced by systemically delivered Ad particles are liver damage and systemic toxicity due to the sequestration of Ad particles to the liver in large quantities after <sup>91</sup> IMPI ^
ΙΝΓΠΤυΤΟ MEXICAN * ot la MoriiMAi IN »UST * iAL --injection [72]. In contrast to parenteral injection, the biodistribution of Ad is limited to the lung after intranasal administration [73] with no inflammation observed in any of the internal organs [68].
Due to the proximity of the nasal cavity to the brain, it is crucial to determine whether Ad5 particles can induce inflammation and toxicity in the brain after nasal spray. Unlike influenza, which is associated with neurological disorders in humans [74], a natural Ad5 infection has not been reported to induce encephalitis in humans. Intranasal administration of Ad5 ΔΕ1Ε3 vectors in mice does not mediate the expression of transgenes bypassing the olfactory bulb, nor does it induce inflammation in the brain [68]. In this way, it is conceivable that significant amounts of Ad5 cannot enter the brain after nasal delivery. Although a small number of Ad5 particles can infiltrate the brain as they occur, it is likely that non-replicating Ad5 does less damage than its replicating natural counterpart due to its inability to amplify adverse effects through replication and delayed gene expression. The safety profile of the live attenuated influenza virus (LAIV; known as FluMist<sup>MR</sup> in the United States) [75] corroborates the hypothesis that influenza virus-induced encephalitis [74] can be attributed to viral replication in the brain since LAIV can only replicate where the temperature is lower but not within? ToiX, '<sub>l</sub> c ^ jeBKjjí where there is too much heat for T.AW> ... adapted to cold. The induction of herpes simplex encephalitis in TLR-3 deficient patients [76] suggests that there may be a common event for a small number of viruses to enter the brain through the olfactory pathways and that there is an effective defense mechanism in immunocompotent people to suppress the virus before it replicates uncontrollably within the brain. Since natural infection by replication of natural Ad5 is not associated with encephalitis, nasal spraying of the non-replicating Ad5 vector therefore represents an impetus in the search for a safe carrier for vaccine delivery.
Although there may be an error in immunizing humans by intramuscular injection of an HIV vaccine with Ad5 vector [58, 59], the ability of Ad5 to mobilize the CD4 + T cell repertoire may in part be the activator to induce potent protective immunity against other pathogens [41, 53, 63, 77-79]. To date, intranasal administration of an influenza vaccine containing Ad5 as a vector has induced seroconversion in human subjects without causing serious side effects in the presence of pre-existing immunity to Ad5 [19]. The induction of sterile immunity against malaria [64] and the
<img file="MX348172B_D0045.tif" />
<sup>93</sup> MAID
INSTITUTE Μ * ICAN · υι the rtomuAn seroconversion against influenza [19] in humans (táEYa in conjunction with robust protective immunity mdüdldd. Pur models in multiple animals (Table 2) collectively demonstrates the value of vaccines using Ad as a vector to prevent disease .
LAIV has been licensed to immunize a subset of human populations (2-49 years of age in the United States) [75]. Like LAIV, it is conceivable that a nasal vaccine vectoring Ad may not be licensed to immunize the very young and elderly, at least for the initial period before its safety profile is well established through trials. large-scale field testing. Also, nasal vaccination may not be recommended for people with respiratory diseases (eg asthma). It remains to be determined whether pregnant persons are susceptible to nasal vaccination using non-replicating Ad particles.
Leaflet for the commercialization of vaccines that have Ad as a vector and other vaccines based on recombinant Ad. The vaccine having a non-replicating Ad5 ΔΕ1Ε3 vector without RCA contamination [35] can be classified as a variant of DNA vaccines because it consists of a linear DNA genome embedded in a protein capsid (figure 9A) without the capacity replication in non-permissive cells. Unlike naked DNA vaccines that need to be inoculated by trained personnel using a penetration device such as the gene gun [80, 81], the syringe needle [82], or the electroporator [83], Ad -can autonomously penetrate cells along the mucosal barrier subsequent to nasal delivery [35]. Only a decade ago, DNA vaccines were an unproven novelty with limited acceptance in the scientific community, although DNA vaccines have come a long way with regard to potential safety concerns relative to contemporary vaccines and recombinant DNA technology can generate new vaccines quickly and creatively at low costs [80, 82]. To date, four naked DNA vaccines have been licensed for commercial animal use [84]. An RCA-contaminated Ad5 vector encoding p53 produced in 293 cells has been licensed to treat a large number of cancer patients in China since 2004 [85]. Given that the clinical picture is beginning to unfold as a result of the harms of increased use and careful patient monitoring, it is conceivable that promising data could accompany an aged recombinant DNA-based vaccine with a vaccine that has Ad vector as one of the tools. essential in the public health arsenal against infectious diseases.
Maintenance of the viability of the Ad vector during storage. In addition to safety and efficacy,
IMPI ^ the next generation of vaccines must go down a chain of cold facilities to ensure a wide dissemination of vaccines to the least accessible populations in the world. Until now, novel formulations have allowed Ad vectors to be stored in a liquid buffer at 4 ° C for at least one year [86]; at 45 ° C in carbohydrate glass for at least 6 months [87]; or at 4 ° C for at least one year as a lyophilized dry powder [88]. Registered technologies for storing Ad particles at room temperature in either liquid or lyophilized form have also been developed at Stabilitech [202]. In summary, RCA-free Ad5 vectors can be rapidly manufactured in serum-free PER.C6 suspension cells, easily purified by column chromatography, and formulated as final fillings that can be stored and shipped without a cold temperature chain (Figure 5).
Drug-vaccine duo that has adenovirus as a vector to confer rapid, sustained and seamless protection against pathogens. The applicant has recently demonstrated that intranasal administration but not intramuscular injection of Ad5 ΔΕ1Ε3 particles, with or without a pathogenic antigen encoded in the Ad5 genome can confer prophylactic treatment against influenza before adaptive immunity is induced [89]. An Ad5 vector that encodes pathogen antigens in this way can. . . .... ΙΜΡΓ «^ induce rapid and sustained protection without cO> atrura> x, X2Qn £ i ^ RVn> If l'l * Ά r«.
pathogen as a drug-vaccine duo (DVD). An influenza DVD that has Ad5 as a vector confers numerous advantages when compared to licensed influenza vaccines (Table 3) and medications (Table 5). It has been documented that the administration of Ad5 ΔΕ1 Se3 particles in mice rapidly induces the production of a wide distribution of inflammatory cytokines and chemokines [56] including type I interferon (IFN-α and IFN-β) [90], impairs the DC of lung [91], activates natural killer cells [92] induces antiviral nitric oxide production [57] and activates multi-facet interactions between Ad5 and blood proteins, platelets, macrophages, endothelial cells and respective parenchyma cells [56]. it is conceivable that multiple particle-induced reactions
Ad5 ΔΕ1Ε3 can combine to establish an anti-influenza status in the respiratory tract, thus generating a multidimensional defense barrier that cannot be easily overcome by an influenza virus.
Although prophylactic therapy against influenza can be carried out by intranasal administration of complexes of bacterial lists [93] or bacterial toxins [94], the anti-influenza status induced by bacterial components can be very transient, with its protective effects diminishing within some days later
<img file="MX348172B_D0046.tif" />
<img file="MX348172B_D0047.tif" />
to treatment [93, 94]. The finding that the protective effects induced by Ad5 can last for at least 3 weeks with only a partial decline observed on day 47 on a single dose regimen [89] suggests that the underlying mechanisms between component-induced anti-influenza states Bacterial or Ad5-induced ΔΕ1 indu3 may differ. Notably, only Ad5-mediated treatment can allow sufficient time for the DVD vaccine component to induce adaptive immunity before its effects as a drug wane [Figure 11). Furthermore, the administration of an associated bacterial toxin to the digestive tract within the respiratory tract as an influenza drug [94] violates a fundamental principle in evolutionary medicine by surprising the immune system, and this unnatural regimen has been associated with the induction of Bell's palsy in human subjects [7,8].
Influenza virus is insidious in mutating into drug-resistant strains when inhibited by an influenza drug (eg, M2 ion channel blocker [amantadine, rimantadine] or neuraminidase inhibitor [oseltamivir, zanamivir]) [95 ]. Unlike contemporary influenza drugs, the Ad5 vector DVD conceivably changes the habitat in the respiratory tract without directly altering the influenza virus; therefore, the Ad5 particle does not
-<sup>98</sup>- IMPI ^
JNSTIUíTr 'MEXICANA Of LA fKOflíüAr 44 .NDusTniAi confers mutational pressure on influenza virus to induce drug resistance. In contrast to the oseltamivir-induced suppression of mucosal immunity with the risk of increasing vulnerability to subsequent infections by pathogens in the mucosa in animals to which the drug has been administered [96], the DVD that has Ad5 as vector increases protective innate immunity in the mucosa, at least in the setting of influenza [89]. Since licensed nasal LAIV (eg, FluMist) contains live influenza virus [35], co-administration of LAIV with an influenza drug is counterproductive because the drug can disable the vaccine by killing the live influenza virus. The DVD that has Ad5 as a vector is not only compatible with the authorized influenza drug due to its lack of drug targets (for example the ion channel or neuraminidase) (Table 4) but also confer prophylactic treatment as a drug by itself in addition to its vaccine capacity (table 5) [89].
Influenza is unlikely to be the only disease that can be suppressed by Ad5 particles, it is also unlikely that Ad5 can counteract all diseases as a panacea. The findings simply show that a single intranasal administration of DVD having Ad5 vector can confer prophylactic therapy against at least a subset of respiratory pathogens.
99- IMPI ^ iNSTrrvre méxican oí iam> fi »í ad INDUSTRIAL - of the mucosa for many weeks in a preclinical animal model and the use of DVD may be incapable of inducing drug resistance. Subsequent elucidation of the protective immunity sustained by the DVD vaccine component enhances efficacy. The development of a DVD platform conceivably accommodates the development of novel clinical strategies in a wide variety of disease settings.
Mass immunization of animals with adenovirus vector vaccines. As shown in the table
2, vaccines have been developed using Ad as a vector to massively immunize farm animals as well as wildlife. Notably, chickens can be immunized against avian influenza (possibly also other poultry diseases) by intramuscular injection [78] by administration into the egg [97-101] or aerosol spray [102, 103] of Ad5 vectors humans that code for the hemagglutinin of the avian influenza virus. The versatility of Ad5 vector vaccines in mass immunization of poultry in this manner is superior to other poultry vaccines. Pigs have also been successfully immunized with Ad5 vector vaccines [104, 105]. A rabies vaccine has been developed using oral canine Ad vector as tallow for mass immunization of wildlife [106]. In general, vaccines that have Ad<sup>100</sup> 'MAID
INSmUTi MEXICAN. Of the 'CFIEDAI, they are emerging as a promising tool' ^ in mass immunization programs.<sup>11</sup>
Conclusion. Tests show that the Ad vector vaccines and the new DVD provide a potentially revolutionary approach, enabling precise design, easy fabrication, and highly effective DVDs for fast and sustained, seamless protection in humans and animals towards a wide variety of disease settings without the side effect profile, shelf instability, or manufacturing challenges that other approaches have presented.
Expert commentary. To further strengthen vaccine coverage around the world, there is an urgent need to develop a new generation of vaccines that can be manufactured rapidly at low costs and are administered massively by non-medical personnel without the requirement of a cold chain. Vaccines with Ad5 as a vector meet these criteria. The development of a DVD platform can potentially change the medical landscape by consolidating vaccines and drugs into a single package that does not deteriorate due to drug resistance.
Five-year outlook: Two phase I human clinical trials of nasal-administered influenza vaccines using Ad5 vector have been completed, with promising results. The Challenge of Human Subjects with Live Post-Spray Influenza Viruses
<img file="MX348172B_D0048.tif" />
-<sup>101</sup> ΙΜΤ »«> Ή> Μ »xiCán * Μ I * <V-2ÍJL1 £, • '» i> ir <rnnAL W nasal of a DVD that has Ad5 as vector is expected to be made in the next 5 years. Poultry vaccines using Ad5 vector are expected to enter the commercial market in the next 5 years.
Key issues. There is an urgent need to develop a new generation of vaccines that can be rapidly manufactured and mass administered by non-medical personnel during a crisis. Replication competent adenovirus (RCA) -free adenovirus (Ad) 5 vaccines can be rapidly produced at low cost from their PER.C6 suspension cells in serum-free medium in response to an increase in demand. RCA-free Ad5 vector vaccines can be massively administered to humans by nasal spray, as well as to poultry by automated in ovo administration and aerosol spray. Wildlife animals can be massively immunized by tallow containing oral vaccines that have canine Ad as vector. Vaccines that vector Ad can induce highly specific immune interventions based on well-defined antigens that are the focus of specific immune reactivity. There should be no safety concern for nasal administration of an RCA-free Ad5 vector in humans.
- 102
IMPI fNSTTnn Q MtXlCAN · L * PROmOAO
<img file="MX348172B_D0049.tif" />
since the vector is non-replicating and the prncedim-i ent-a --- complies with evolutionary medicine. There should also be no safety concerns for mass immunization of poultry by an Ad5 vector since chicken cells do not support replication of human Ad5, it is conceivable that chickens shed Ad5 rapidly after the immune repertoire it has been mobilized towards beneficial post-vaccination immune protection. An Ad5-boosted / DNA-primed malaria vaccine has successfully protected human subjects against exposure to live malaria sporozoites after mosquito feeding. A nasal vaccine vectoring Ad5 has surprisingly been shown to confer rapid protection against influenza in a drug-like manner. Developing a drug-vaccine duo that consolidates both drug and vaccine in a single package that does not deteriorate due to drug resistance can fundamentally change the way influenza drugs and vaccines are prepared. In general, more and more adverse effects induced by systemic vaccination have been identified. Non-invasive mucosal immunization is safer and more effective in conferring protection against mucosal pathogens compared to its systemic counterpart.
103
<img file="MX348172B_D0050.tif" />
TABLE 2. EXAMPLES OF INDIVIDUAL PROTECTIVE IMMUNITY
VACCINES THAT HAVE CONlTOt-ftajOGENOS AS A VECTOR ADENOVIRUS
LIVE IN ANIMAL MODELS
<td>Vaccine</td><td>Pathogenic antigen expressed from Ad</td><td>Via</td><td>Animal model</td><td>Exposition</td><td>Reference</td>
<td>primed with Ad26 / reinforced with Ad35</td><td>ebola GP virus</td><td>im</td><td>primate no human</td><td>Ebola virus</td><td> [107]</td>
<td>Bd5</td><td>ebola GP virus</td><td>im</td><td>primate no human</td><td>Ebola virus</td><td> [108]</td>
<td>Bd5</td><td>Angola virus Marburg GP</td><td>im</td><td>primate no human</td><td>Angola virus Marburg</td><td> [109]</td>
<td>Primed in BCG / reinforced with Ad5</td><td>Bg85A</td><td>in, in.</td><td>guinea pig</td><td>Mycobacterium tuberculosis</td><td> [110]</td>
<td>Bd5</td><td>PA</td><td>im</td><td>mouse</td><td>spore of Bacillus anthracis Stars</td><td> [111]</td>
<td>Bd5</td><td>H5 HA avian influenza virus</td><td>in, in</td><td>mouse</td><td>avian influenza virus</td><td> [77, 78]</td>
<td>primed with vaccine influenza / reinforced with Bd5</td><td>influenza HA virus</td><td>im</td><td>mouse</td><td>heterosubtypical influenza virus</td><td> [112]</td>
<td>Bd5</td><td>avian influenza virus H5 HA</td><td>im. in ovo ocular</td><td>chicken</td><td>avian influenza virus</td><td> [78, 97, 98, 102]</td>
<td>Bd5</td><td>influenza virus HA / nucleoprotein</td><td>im</td><td>pork</td><td>swine influenza virus</td><td> [104]</td>
<td>Bd5</td><td>Hanta / GP virus nucleocapsid</td><td>im</td><td>Hamster</td><td>Hanta virus</td><td> [113]</td>
<td>Bd5</td><td>botulinum neurotoxin fragment C</td><td>in</td><td>mouse</td><td>botulinum neurotoxin</td><td> [114]</td>
<td>Bd5</td><td>measles virus / HA fusion protein</td><td>in, in</td><td>cotton rat</td><td>virus measles</td><td> [70]</td>
<td>Canine ad</td><td>rabies virus GP</td><td>oral</td><td>raccoon, skunk</td><td>rabies virus</td><td> [106]</td>
<td>Bd5</td><td>antigen V</td><td>im</td><td>mouse</td><td>Yersinia pestis</td><td> [115]</td>
Ad: adenovirus; BCG bacillus of Callmette-Guerin; GP: glycoprotein; HA: hemagglutinin; im: intramuscular, in: intranasal, PA: protective antigen.
104
SIZE 3:
ÍMPH
ΙΗ5ΤΪΉΠΎ) MÍXICAN '
EXAMPLES OF CLINICAL TRIALS IN HUMANS
AND VACCINES THAT HAVE ADENOVIRUS AS A VECTOR
<td>Vaccine</td><td>Pathogenic antigen expressed from Ad</td><td>Via</td><td>Exposition</td><td>Reference</td>
<td>Bd4 and Bd7</td><td>none</td><td>oral</td><td>natural Ad infection</td><td> [31]</td>
<td>primed with DNA / reinforced with Bd5</td><td>CSP / AMA1</td><td>M</td><td>sporozoite of malaria</td><td> [64]</td>
<td>Bd5</td><td>HIV-1 gag / pol / nef</td><td>im</td><td>natural HIV-1 infection</td><td> [58, 59]</td>
<td>primed with DNA / reinforced with Bd5</td><td>HIV-1 gag / pol / env</td><td>im</td><td>none</td><td> [203]</td>
<td>primed with Bd5 / reinforced with Bd5</td><td>HIV-1 gag</td><td>im</td><td>none</td><td> [203]</td>
<td>primed with Bd5 / reinforced with NYVAC; NYVAC primed / Ad5 boosted</td><td>HIV-1 gag / pol / env / nef</td><td>im</td><td>none</td><td> [203]</td>
<td>Bd5</td><td>influenza virus H1HA</td><td>in and skin patch</td><td>none</td><td> [203]</td>
<td>primed with Bd4 / reinforced with Bd4</td><td>H5HA avian influenza virus</td><td>oral</td><td>none</td><td> [203]</td>
<td>encapsulated and Ad5 as adjuvant</td><td>H5HA avian influenza virus</td><td>oral</td><td>none</td><td> [203]</td>
<td>Bd5</td><td>Mycobacterium tuberculosis 85A</td><td>im</td><td>none</td><td> [203]</td>
<td>primed with BCG / reinforced with Ad35</td><td>M. tuberculosis 85A / 85B / 10.4</td><td>im</td><td>none</td><td> [203]</td>
Ad: adenovirus; AMA1 apical membrane antigen 1; BCG: Callmette-Guerin bacillus; CSP: circumsporosoite protein; HA: hemagglutinin; im: intramuscular; in: intranasal.
- 105 TABLE 4.
MAID
OMSTnyrc, muicand
O £ LA F »UFI £ DAD industxme
<img file="MX348172B_D0051.tif" />
RATIONALE FOR DEVELOPING A DUO OF INFLUENZA VACCINE DRUGS THAT HAVE ADENOVIRUS AS A VECTOR
SEROTYPE 5 TAKING INFLUENZA VACCINES INTO CONSIDERATION
AUTHORIZED
<td>Vaccine</td><td>requirement to spread a virus influenza</td><td>requirement to use embryonated chicken eggs such as substratum</td><td>production speed</td><td>post-administration replication</td><td>mode of administration</td><td>concomitant use with influenza medications authorized</td>
<td>TIV</td><td>yes</td><td>yes</td><td>slow</td><td>no<sup>+</sup></td><td>needle injection</td><td>Yes<sup>+</sup></td>
<td>LAIV *</td><td>yes</td><td>yes</td><td>slow</td><td>yes</td><td>nasal spray</td><td>no</td>
<td>DVD ίθ9]</td><td>no<sup>+</sup></td><td>no<sup>+</sup></td><td>Quick*</td><td>no<sup>+</sup></td><td>nasal spray</td><td>yes<sup>+</sup></td>
When developing an influenza DVD that is vectored by adenovirus serotype 5 (Ad5), the multiple problems associated with preparing a live influenza virus from qualified chicken eggs [35] can be eliminated.
<sup>+</sup> desirable results * known as FluMist<sup>MR</sup> in the U.S; a LAIV produced in Madin Darby dog kidney cells (MDCK) was licensed in Europe although its license in the United States has not yet been approved by the US FDA due to the association of MDCK cells<sup>+</sup> with tumorigenicity and oncogenicity [116].
DVD: drug-vaccine duo: LAIV: live attenuated influenza virus vaccine; TIV: trivalent inactivated virus vaccine.
- 106 Continuation of table 4
<img file="MX348172B_D0052.tif" />
MAID
MUICAN INSTITUTE
DS LA Μ · ΝΕΟΑΓ
INDUSTRIAL
<td>reassignment with wild-type influenza viruses</td><td>systemic inflammation; platelet reactivity; cardiac autonomic dysfunction; narcolepsy</td><td>almost immediate protection against influenza</td><td>broad protection against heterosubtypical strains</td><td>adverse effects after co-administration with undetectable contaminating pathogens</td>
<td>no<sup>+</sup></td><td>if [2, 201]</td><td>no</td><td>no</td><td>it can be serious</td>
<td>yes</td><td>no<sup>+</sup></td><td>no</td><td>no</td><td>light to none<sup>+</sup></td>
<td>no<sup>+</sup></td><td>no<sup>+</sup></td><td>yes<sup>+</sup></td><td>yes<sup>+</sup></td><td>light to none<sup>+</sup></td>
When influenza DVD vector is developed with adenovirus serotype 5 (Ad5), the multiple problems associated with the spread of a live influenza virus in qualified chicken eggs [35] can be eliminated.
<sup>+</sup> desirable results * known as FluMist<sup>MR</sup> in the U.S; a LAIV produced in Madin Darby dog kidney cells (MDCK) licensed in Europe although its license in the United States has not been approved by the US FDA due to the association of MDCK cells<sup>+</sup> with tumorigenicity and oncogenicity [116].
DVD: drug-vaccine duo: LAIV: live attenuated influenza virus vaccine; TIV: trivalent inactivated virus vaccine.
Table 5. Rationale for developing an influenza drug-vaccine duo using Ad-5 as a vector taking into consideration authorized influenza drugs
107
<img file="MX348172B_D0053.tif" />
<td></td><td></td><td></td><td></td>
<td>associated problems</td><td>blocker ion channel M2</td><td>epheneuraminidase inhibitor</td><td>drug vaccine [89]</td>
<td>requirement for administer dose multiple</td><td>yes</td><td>yes</td><td>no<sup>+</sup></td>
<td>potential to induce drug resistance</td><td>yes [95]</td><td>yes [95]</td><td>no<sup>+</sup></td>
<td>prophylactic treatment</td><td>yes<sup>+</sup></td><td>yes<sup>+</sup></td><td>yes<sup>+</sup></td>
<td>post-exposure treatment</td><td>partial·</td><td>partial·</td><td>no</td>
<td>regularization of mucosal immunity</td><td>unknown</td><td>Oseltamivir suppresses mucosal immunity in animals [96]</td><td>mucosal protective immunity is increased [89] *</td>
<td>sustained protection for a few months</td><td>no</td><td>no</td><td>yes<sup>+</sup></td>
<img file="MX348172B_D0054.tif" />
* Desirable results
The main reason for developing an influenza drug-vaccine duo that has Ad5 (DVD) as vector based on authorized influenza drugs is its potential not to be deteriorated by drug resistance, in addition to the convenience of consolidating the drug and the vaccine in a single package [89]. Please note that DVD development is still in its stages.
0 early; It remains to be seen whether prophylactic treatment can be produced in human subjects and whether post-exposure treatment can be developed. The M2 ion channel blocker includes amantadine and rimantadine; the neuraminidase inhibitor includes oseltamivir (Tamiflu) and zanamivir (Relenza); DVD represents
108
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INSTITUTO MEXICANO »f LA FIONt · * · the duo of medicine-influenza vaccine that '<sup>NEITHER</sup>'tT ^ he vector Ad5 [89].
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Toro H, Tang DC, Suarez DL, Sylte MJ, Pfeiffer J, Van Kampen KR. Protective avian influenza in ovo vaccination with nonreplicating human adenovirus vector. Vaccine 25, 2886-2891 (2007).
Zhang J. Tarbet EB, Feng T, Shi Z, Van Kampen KR, Tang DC. Adenovirus-vectored drug-vaccine duo as a rapidresponse tool for conferring seamless protection against influenza. PLoS ONE 6, e22605 (2011).
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EXAMPLE 4: RAPID AND LONG-TERM THERAPEUTIC IMUNOLOGICAL SUBSTANCES OF INFLUENZA THAT HAVE ADENOVIRUS AS VECTOR
The objective of RAPIT of influenza that has Ad5 as vector is to develop a rapid and prolonged immunological-therapeutic substance (RAPIT, for its acronym in English) for influenza that can be produced in large quantities at low costs and that can be administered massively by personnel not medical; with the ability to confer rapid / sustained protection against influenza but without the potential to induce drug resistance and
126 reassignment
INSTITUTE with a natural influenza virus
<img file="MX348172B_D0067.tif" />
request to spread a virus of. £ 1 llliiiii? and nn h ^ v requirement to need injection by authorized medical personnel.
Influenza vaccine using Ad5 vector can be rapidly generated without growing influenza viruses. In an influenza virus, growth varies from strain to strain, some strains are fatal and are susceptible to reassignment and mutation events, and there is low-titer protection in eggs. In an Ad vector encoding influenza HA, there are more consistent growth rates, the vector is benign, there are no reassignment events, there is high-titer protection in cells
PER.C6 and a new RCA free Ad can be generated by the AdHigh system within a period of one month.
Influenza vaccines vectoring with Ad5 in cultured suspension cells can be mass-produced. For an influenza vaccine that has Ad vector, cloning influenza HA into Ad does not require the growth of influenza virus, a 500 liter wave bioreactor can produce 1016 Ad particles at one time from PER.C6 suspension cells in serum-free medium, Ad particles can be purified by chromatography in column and the production of influenza vaccines that have Ad as vector can be installed in
127
ΙΜΡΙ @? ^ Rapid response to an increase in derfSBíJíg'H ^ pas ^^ itó ^ iNoumiM conventional influenza vaccine, some influenza virus strains do not grow well in eggs, the average yield is approximately one dose per egg, the Contamination is more difficult to identify in eggs than in cell cultures, egg-associated allergies may exist, and processing is cumbersome.
Ad5-mediated gene therapy and nasal vaccination can be compared as follows. In gene therapy, a therapeutic protein is expressed from Ad and a biological effect is directly induced by a dose co right of therapeutic protein expressed from Ad in cells subjected to transduction. In nasal vaccination, the antigen protein is expressed from Ad, the antigen is presented and an immune response is induced through a cascade of reactions activated by antigen expressed from Ad in transduced cells. Reports that support the hypothesis that pre-existing immunity to Ad does not interfere with the potency of nasal vaccines using Ad as a vector include Shi Z et al. J. Virol. 75: 11474, 2001 (mice), Hoelscher MA et al. Lancet 367: 475, 2006 (mice), Croyle MA et al. PLoS ONE 3: e3548, 2008 (mice), Song K et al. PNAS 107: 22213, 2010 (macaques) and Van Kampen KR et al. Vaccine 23: 1029, 2005 (humans).
The study design of a phase I clinical trial in
128 humans from an avian influenza vaccine
<img file="MX348172B_D0068.tif" />
has as vector Ad5 is as follows. A vector
AdhVN1203 / 04, H5 encoding fallen by HA1 + HA2 of avian influenza virus A / VN / 1203/04 (H5N1). The study is a randomized, double-blind, placebo-controlled, single-site study. There are three groups in an increasing dose of ΙΟ<sup>8</sup>, 10<sup>9</sup> and 10<sup>10</sup> pv. Doses are administered by nasal spray in two doses, on days 0 and 28. There are a total of 48 healthy volunteers, aged 19 to 49. There are sixteen human subjects per dose group, which includes four placebo controls per group. The cell culture is an RCA-free cell culture based on the manufacture of PER.C6 suspension cells in serum-free medium. Adverse events in the respiratory system in 30% or more of subjects include rhinorrhea, nasal irritation, nasal congestion, cough, and / or sore throat.
EXAMPLE 5 ADENOVIRUS PARTICLE AS A SUBSTANCE
RAPID AND PROLONGED IMMUNOLOGICAL-THERAPEUTIC (RAPIT) OF
WIDE SPECTRUM AGAINST PATHOGENS RES PIRATORIES
Figure 14 shows prophylactic anthrax treatment by intranasal instillation of adenovirus particles shortly before spore exposure.
Methods. AdE particles (E1 / E3 defective empty vector Ad5, no transgene) and AdVAV (E1 / E3 defective Ad5 vector encoding protective antigen
129
IMPI í «HMSTm./T (.! MEXICAN from Bacillus anthracis) intranasaUfi ^ jJ ^^ ia drops are administered into the nostrils of young female A / J mice in a volume of 0.05 ml on a regimen. single dose just before exposure in with 1 X 10<sup>5</sup> die (~ 25 x DL<sub>50</sub>) of spores of Bacillus anthracis Sterne. The exposed animals are monitored for survival on a daily basis for 14 days.
Results. AdVAV particles administered 2 days before challenge protect 67% of mice against anthrax; AdE particles administered 2 days prior to ex position protect 30% of mice against anthrax; AdE particles administered 1 day before challenge protect 22% of mice against anthrax; Untreated control mice and mice given dilute AdE particles all died of anthrax within 5 days. AdVAV / -2, AdVAV particles instilled in 2 days prior to exposure at a dose of 1.3 x 10<sup>8</sup> ufi; AdE / -2, AdE particles instilled in 2 days prior to exposure at a dose of 1.3 x 10<sup>8</sup> ufi; AdE * / - 2, AdE particles instilled in 2 days prior to exposure at a dose of 1.3 x 10<sup>6</sup> ifu (100-fold dilution in PBS); AdE / -l, AdE particles instilled in 1 day before exposure at a dose of 1.3 x 10<sup>8</sup> ufi; control, untreated control mice; the numbers in parentheses represent the number of animals in each group.
<img file="MX348172B_D0069.tif" />
AdE or AdVAV particles can confer prophylactic treatment against anthrax in a drug-like manner, probably by activating a specific arm of innate immunity that prevents the growth of Bacillus anthracis in infected animals. The data suggest that the PA gene expressed from AdVAV may confer synergy with AdE-mediated protection against anthrax. It is conceivable that the nasal spray of AdVAV particles may confer more rapid protection against anthrax than other anthrax vaccines during an attack.
Figure 15 shows post-anthrax exposure treatment by instillation of AdVAV particles.
Methods. AdVAV particles are administered in drops into the nostrils of young (2 months old) female A / J mice in a volume of 0.05 ml in a single dose regimen, either before or after exposure in with 4 x 10<sup>5</sup> die (~ 100 x DL<sub>50</sub>) Bacillus anthracis spore
Sterne. Ciprofloxacin is given by ip injection at a dose of 30 mg / kg (1 injection per day for 2 days; injected 1 and 24 hours after exposure). Exposed animals are monitored for survival on a daily basis for 14 days.
Results. AdVAV particles administered 2 days prior to challenge protect 40% of mice
- 131 -
<img file="MX348172B_D0070.tif" />
against anthrax (confirmation of the results of the figure
one) ; AdVAV particles administered 1 hour after exposure delayed death but did not improve survival rate; ciprofloxacin injected 1 hour after exposure also delays death without success in improving survival rate; AdVAV particles administered together with injection of ciprofloxacin 1 hour after challenge protect 56% of mice against anthrax; all untreated control mice died within 5 days. AdVAV / D-2, AdVAV particles instilled in 2 days prior to exposure to a 1.3 x 10 dose<sup>8</sup> ufi; AdVAV / DO, AdVAV particles instilled in 1 hour after exposure at a dose of 1.3 x 10<sup>8</sup> ufi; AdVAV / Cipro / DO, AdVAV particles instilled in 1 hour after exposure at a dose of 1.3 x 10<sup>8</sup> ifu together with ip injection of ciprofloxacin; Cipro / DO, ip injection of ciprofloxacin; control, untreated control mice with no pre-challenge treatments; the numbers in parentheses represent the number of animals in each group.
Significance. Data suggest that AdVAV particles can confer post-anthrax treatment in conjunction with antibiotic treatments. In this experiment synergy between AdVAV and antibiotics was revealed. It is conceivable that the nasal spray of
<img file="MX348172B_D0071.tif" />
132
MAID
Mexican OMsrnvru DE LA PLOFIIDAD
INDUSTRIAL AdVAV particles may be able to reduce the requirement of antibiotic use in a field after exposure.
EXAMPLE 6 ADENOVIRUS PARTICLES AS A SUBSTANCE
RAPID AND PROLONGED IMMUNOLOGICAL-THERAPEUTIC (RAPIT) OF
WIDE SPECTRUM AGAINST RESPIRATORY PATHOGENS
It has recently been shown that intranasal (in) administration of adenovirus type 5 (Ad5) ΔΕ1Ε3 particles, with or without pathogenic antigen encoded in the Ad5 genome, can confer prophylactic therapy against influenza before adaptive immunity is induced. An Ad5 vector that encodes pathogen antigens in this way can induce seamless rapid and sustained protection against a pathogen as a drug-vaccine duo (DVD). Administration of Ad5 ΔΕ1Ε3 particles in mice has been documented to rapidly induce the production of a wide range of inflammatory cytokines and chemokines including type I interferon (IFN-α and IFN-β); impairs lung dendritic cells, activates natural killer cells; induces antiviral nitric oxide production; activates the multi-faceted interactions between Ad5 and the respective blood, platelet, macrophage, endothelial cell, and parenchymal cell proteins. It is conceivable that multiple reactions induced by
133
<img file="MX348172B_D0072.tif" />
MAID
INSTITUTE M EXICANv •<sup>£</sup> THE MerilOAD particles Ad5 ΔΕ1Ε3 can be combined to establish ^ é® ^<sup>1</sup> anti-inf luenza state in the airways and in this way a multidimensional defense barrier is generated that can hardly be overcome by an influenza virus. Influenza is unlikely to be the only disease that can be suppressed by Ad5 particles; Ad5 particles are also unlikely to be able to counteract all diseases as a panacea. The findings simply show that an administration in Unique AdE particles can confer prophylactic therapy against at least a subset of respiratory pathogens. mucosal oss for many weeks in mice and the use of DVD may be inappropriate for inducing drug resistance because AdE particles change habitat in the airways without directly conferring mutational pressure toward other viruses. Subsequent elucidation of sustained protective immunity by the DVD vaccine component enhances efficacy. The development of a DVD platform will conceivably accommodate the development of numerous clinical strategies in a wide variety of disease settings.
The aim of this example is to evaluate prophylactic intranasal treatment with Vaxin's AdE (an empty Ad5 vector without an RSV transgene) on cotton (CR) rats infected with respiratory syncytial virus (RSV). The criteria of this study are demonstrated by
134
IMPJ virus titers reduced in fluids of 1 avac ^ a ^^^ uMSW (3 ml) and nasal wash (2 ml) of infected cotton rats I 111 'I <sup>1 1</sup>' ' <sup>11</sup> - - (apr approximately 60-125 g of weight) compared to untreated cotton rats, virus quantification will be performed by plaque reduction analysis.
Prophylactic effectiveness in the RSV-cotton rat model: (cotton rats (60-125 g body weight):
Group 1: 6 CR treated prophylactically (day -2) intranasally with vehicle (buffer A195)
Group 2: 6 CR treated prophylactically (day -30) intranasally with 2.4 x 10<sup>8</sup> ifu from AdE.
Group 3: 6 CR treated prophylactically (day -2) intranasally with 2.4 x 10<sup>8</sup> ifu from AdE.
Group 4: 6 CR treated prophylactically (days -30 and -2) intranasally with 2.4 x 10<sup>8</sup> ifu of AdE during each treatment cycle (prime / boost).
Group 5: 6 RCs treated prophylactically (-5 h) intranasally with 2.4 x 10<sup>8</sup> ifu from AdE.
Challenge virus: RSV-Tracy (P3 wp 1/20/12 growing in Hep-2 cells), 2.25 x 10<sup>5</sup> PFU intranasally (100 μΐ) in cotton rats (60-125 g) lightly anesthetized with isof-lurane. Concentrate: 2.25 x 10<sup>6</sup> PFU / ml.
AdE vector: vehicle (Al95 buffer) and AdE concentrations of 2.4 x 10<sup>9</sup> are stored at -80 ° C.
Just before use the materials are warmed up to
<img file="MX348172B_D0073.tif" />
<sup>135</sup> IMPI • NSTmm> mercan, I heard LA Pí.jritDA '· <sup>rNri</sup>t / <7 ^^ room temperature. At least 0.8 ml is needed for each treatment for each group (6 CR / group x 0.1 mr ^ of ~ -. Inoculum). Material that is not used is kept at -80 ° C.
Collection of organs and samples. After euthanasia with CO<sub>2</sub>, each cotton rat is weighed and sex and age are recorded. The left lung and one of the large right lobes are removed, rinsed in sterile water to remove external blood contamination, and weighed. The left lobe is subjected to transplural lavage using 3 ml of Iscove's medium with 15% glycerin mixed with 2% FBSMEM (1: 1, v: v) in a 3 ml syringe with a 26 g 3/8 needle when injecting at multiple sites to fully inflate the lobe. The lavage fluid is recovered by lightly pressing the inflated lobe by flattening it, and the right lobe is used for transplural lavage following the same technique. The wash fluid is collected and stored on ice until it is titrated. For nasal washes of the upper respiratory tract, the jaws are disarticulated, the head is then excised and 1 ml of Iscove's medium with 15% glycerin mixed with 2% FBS-MEM (1: 1, v: v) is propelled through of each nostril (total of 2 mi). Effluent is collected from the posterior opening of the shoulder blade and stored until titrated. Samples are not frozen prior to titration which occurs at the end of sample collection.
136
RSV lung lavage titers
IMPI the nomPAp! NnV $ TWIM lung) and title or nasal wash (total PFU).
Plating analyzes were performed using 24-well tissue culture plates containing nearly confluent monolayers (20 to 40 x 10<sup>4</sup> cells / well) of HEp-2 cells prepared in 10% FCS 24 h before starting the analysis.
At the beginning of each analysis, dilutions are made (usually serialized in logi units).<sub>0</sub>) of the test samples. A 0.2 ml sample of each is then added to the wells in duplicate and allowed to absorb for 90 min with occasional light agitation. After the inoculum is removed, the monolayers are then overlaid with 0.75% methylcellulose in 2% FBS-MEM containing antibiotics, vitamins, and other nutrients. Tissue culture and virus positive controls are included in each test. Plates are placed in an incubator at 36 ° C, C0<sub>2 </sub>5%. Day 6 ± 1 day later, the plates are stained with 0.1% crystal violet / formalin solution at 10% (1.5 ml / well) and allowed to stir for 24-48 h at room temperature. The wells are rinsed with water. Plates, when present, are easily visible (light circles on a very dark blue background). All plates in wells containing between 20 and 80 plates will be numbered, averaged and virus titers calculated as total log PFU.<sub>10</sub> for nasal wash fluid or logi<sub>0</sub> PFU / g of tissue
<img file="MX348172B_D0074.tif" />
137 for lungs or other organs. The limit
MAID
INSTITUTE MLX1CAN Pt THE lower INDUSTRIAL CURRENCY of detection by this method is approximately 1.5 logi<sub>0 </sub>PFU / g of tissue.
Antibody response to AdE: Blood is collected from the orbital plexus of groups 2 and 4 (3 CR / group) on day -30 and groups 2 and 4 (6 CR / group) on day -2. Blood will be collected from groups 1-5 on day +4. Sera are stored at -20 ° C.
Reserve samples . Aliquots of nasal lavage and lung lavage fluids (groups 1-5) are saved, stored at -80 ° C. Serum samples from day +4 are saved, stored at -80 ° C.
Table 6: Proposed study plan:
<td>group<sup>1</sup></td><td>treatment</td><td>via</td><td>volume (mi)</td><td>AdE particles (ufi / CR)</td><td>treatment procedure</td><td>harvest</td><td>endpoint</td>
<td> 1</td><td>buffer day -2</td><td> ...</td><td> 0</td><td> 0</td><td>Day 2</td><td rowspan="5">day +4</td><td rowspan="5">virus titer in lung lavage fluid and nasal lavage by UPF</td>
<td> 2</td><td>AdE, day - 30</td><td>i.n.</td><td> 0.100</td><td>2.4x10“</td><td>Day 2</td>
<td> 3</td><td>AdE, day -2</td><td>i.n.</td><td> 0.100</td><td>2.4 x10<sup>a</sup></td><td>day 30</td>
<td> 4</td><td>AdE, days 30, -2</td><td>i.n.</td><td> 0.100</td><td>2.4 x10<sup>a</sup></td><td>days -30 and -2</td>
<td> 5</td><td>AdE, -5 hours</td><td>i.n.</td><td> 0.100</td><td>2.4 x10<sup>to</sup></td><td>hour -5</td>
abbreviations: in, intranasal; UFP, plaque forming units,<sup>X</sup>N = 6 animals / group; 30 animals in total.<sup>2</sup>All animals have been exposed in (100 μΐ) with RSV-Tracy (approximately 2.25 x 10<sup>5</sup> UFP) on day 0.
- 138 Table 7: Daily procedure:
<img file="MX348172B_D0075.tif" />
JMPI • N ^ TUTO MEXICANO
OR! THE «ΟΝΕΓ-αΠ INDUSTRIAL
<td>day 30</td><td>Day 2</td><td>day 0</td><td>day +1 to +3</td><td>day +4</td><td>- day Tb “+ 16</td>
<td>Treat groups 2 and 4 with AdE i. n. ; bleed groups 2 and 4 (3 CR / gp)</td><td>Treat group 1 with vehicle i. n. ; groups 3 and 4 with AdE in; bleed groups 2 and 4 (6 CR / gp)</td><td>at -5 hours, treat group 5 with AdE in At 0 hours, infect 15 in groups with RSVTracy</td><td>monitor animals</td><td>collect wash or nasal and lavage of 2 lobes of the lungs for virus titers; collect blood from groups 1-5</td><td>monitor degrees for UFP</td>
abbreviations: in, intranasal; UFP, plaque-forming units; gp, group.
Timeline day 0:
am => treat group 5 AdE in
pm => infect all groups
Lung and body weight and dosage on day +4:
Table 8. Lung and body weights on day +4
<td rowspan="2">group</td><td rowspan="2">treatment</td><td rowspan="2">AdE dose (ufi / cr)</td><td colspan="2">lung lobe weight (g)<sup>1</sup></td><td colspan="2">body weight (g)<sup>2</sup></td>
<td>half</td><td>SD</td><td>half</td><td>SD</td>
<td> 1</td><td>buffer day -2</td><td>2.4 x 10<sup>8</sup></td><td> 0.31</td><td> 0.02</td><td> 163.0</td><td> 16.1</td>
<td> 2</td><td>AdE, day -30</td><td>2.4 x10<sup>to</sup></td><td> 0.35</td><td> 0.05</td><td> 155.9</td><td> 12.1</td>
<td> 3</td><td>AoE, day -2</td><td>2.4 x 10<sup>to</sup></td><td> 0.36</td><td> 0.05</td><td> 139.8</td><td> 19.9</td>
<td> 4</td><td>AoE, days 30, -2</td><td>2.4 x 10<sup>8</sup></td><td> 0.37</td><td> 0.03</td><td> 141.7</td><td> 19.1</td>
<td> 5</td><td>AdE, - 5 hours</td><td>2.4 x 10<sup>to</sup></td><td> 0.38</td><td> 0.04</td><td> 146.1</td><td> 8.7</td>
There is a statistically difference
- 139 significant between groups 1 v 3, 4
<img file="MX348172B_D0076.tif" />
0.004, respectively.
There is a statistically significant difference between groups 1 and 5; P = 0.047.
Plaque reduction titers of nasal lavage and lung lavage after RSV-Tracy
Table 9. RSV-Tracy titers in nasal wash fluids on day +4
<td rowspan="2">group</td><td rowspan="2">treatment</td><td colspan="8">RSV title (log<sub>10</sub> total PFU) in cotton rat</td><td rowspan="2">change (log<sub>10</sub>)</td><td rowspan="2">T / 2 test v. Gp Γ</td>
<td>TO</td><td>B</td><td>C</td><td>D</td><td>AND</td><td>F</td><td>half</td><td>SD</td>
<td> 1</td><td>buffer day -2</td><td> 4.99</td><td> 5.40</td><td> 4.98</td><td> 4.98</td><td> 5.09</td><td> 5.02</td><td> 5.08</td><td> 0.16</td><td> —</td><td> ___</td>
<td> 2</td><td>AdE, day -30</td><td> 5.04</td><td> 5.09</td><td> 4.86</td><td> 5.07</td><td> 5.00</td><td> 4.69</td><td> 4.96</td><td> 0.16</td><td> -0.12</td><td> 0.230</td>
<td> 3</td><td>AoE, day -2</td><td> 4.86</td><td> 5.51</td><td> 5.39</td><td>dead</td><td> 4.94</td><td> 5.55</td><td> 5.25</td><td> 0.32</td><td> 0.17</td><td> 0.280</td>
<td> 4</td><td>AoE, days 30, -2</td><td> 5.45</td><td> 5.23</td><td> 4.99</td><td> 5.13</td><td> 5.10</td><td> 5.03</td><td> 5.15</td><td> 0.17</td><td> 0.08</td><td> 0.425</td>
<td> 5</td><td>AdE, -5 hours</td><td> 5.56</td><td> 5.35</td><td> 5.51</td><td> 5.45</td><td> 5.51</td><td> 5.13</td><td> 5.42</td><td> 0.16</td><td> 0.34</td><td> 0.0042</td>
* Detect minimum tion = 0.7 log<sub>10</sub> of total UFP. For statistical analysis (Student's T test, two-tailed) the minimum detection (0 plaques) was considered as 0.35 logio of the total PFU. Additional significant P values: group 5 v 2, 4, P <0.02.
140
<img file="MX348172B_D0077.tif" />
IMPI iNsrmmj mexigan OF THE PROPERTY Table 10. RSV-Tracy titers in washing fluids ¿ré<sup>us</sup>last day +4
<td rowspan="2">group</td><td rowspan="2">treatment</td><td colspan="8">RSV title (log,<sub>0</sub> of PFU / g of lung) in cotton rat</td><td rowspan="2">change (logio)</td><td rowspan="2">T / 2 test v. GP 1 *</td>
<td>TO</td><td>B</td><td>C</td><td>D</td><td>AND</td><td>F</td><td>half</td><td>SD</td>
<td> 1</td><td>buffer day -2</td><td> 5.06</td><td> 5.18</td><td> **</td><td> 5.06</td><td> 5.01</td><td> 5.03</td><td> 5.07</td><td> 0.07</td><td> —</td><td> ...</td>
<td> 2</td><td>AdE, day -30</td><td> 4.74</td><td> 4.59</td><td> 4.44</td><td> 4.52</td><td> 4.65</td><td> 4.52</td><td> 4.58</td><td> 0.11</td><td> -0.49</td><td> 0.000010</td>
<td> 3</td><td>AoE, day -2</td><td> 5.03</td><td> 4.26</td><td> 4.60</td><td>dead</td><td> 4.60</td><td> 4.49</td><td> 4.60</td><td> 0.28</td><td> -0.47</td><td> 0.0063</td>
<td> 4</td><td>AoE, days 30, -2</td><td> 3.95</td><td> 5.01</td><td> 4.14</td><td> 4.54</td><td> 4.65</td><td> 4.68</td><td> 4.49</td><td> 0.39</td><td> -0.57</td><td> 0.0098</td>
<td> 5</td><td>AdE, -5 hours</td><td> 5.02</td><td> 5.34</td><td> 4.53</td><td> 3.88</td><td> 5.45</td><td> 4.65</td><td> 4.81</td><td> 0.59</td><td> -0.26</td><td> 0.357</td>
* Minimum detection = 1.3 logio / g of lung. ** No plates though there is virus in nasal fluid. Therefore the assumption is made that the lungs were not infected or that there was a technical error. These data were not included in the analysis. For statistical analysis (Student's t-test, two-tailed) the minimum detection (0 plates) was counted as 1.1 logi<sub>0</sub>/ g of lung. There were no additional significant P values.
Having thus described in detailed preferred embodiments of the present invention, it is understood that the invention defined by the preceding paragraphs is not limited to the particular details set forth in the preceding description since many variations thereof are possible without departing from the spirit or scope of the present invention.
It is noted that in relation to this date, the best method known to the applicant for putting the aforementioned invention into practice is the one that is clear from the present description of the invention.
141
<img file="MX348172B_D0078.tif" />
IΜ ΡI
Contents52
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Numbers
- Publication
- 348172
- Publication, DOCDB
- 348172
- Publication, EPODOC
- MX348172
- Application
- 2013010794
- Application, DOCDB
- 2013010794
- Application, EPODOC
- MX20130010794
Titles2
- Spanish
- SUSTANCIA INMUNOLOGICA TERAPEUTICA RAPIDA Y PROLONGADA.
- English
- RAPID AND PROLONGED THERAPEUTIC IMMUNOLOGICAL SUBSTANCE.
Classification
- CPC, 25
- A61K39/00
- A61K39/07
- C12N15/86
- A61K2039/5254
- A61K2039/5256
- A61K2039/543
- C12N2710/10034
- A61K39/145
- A61K39/155
- A61K39/12
- A61K2039/58
- C12N2710/10043
- C12N2760/16134
- A61P11/00
- A61P31/00
- A61P31/04
- A61P31/06
- A61P31/08
- A61P31/10
- A61P31/12
- A61P31/14
- A61P31/16
- A61P31/20
- A61P37/04
- Y02A50/30
- IPC, 7
- A61K39 145
- A61K39 00
- A61K39 07
- A61K39 12
- A61K39 23
- A61K39 155
- C12N15 86