Method of separating rotavirus variants and live attenuated rotavirus vaccine

(74) Agent

GlaxoSmithKline Biologicals SA, Rue de Institute 89.1330 RIXENSART, BE

Georges Thiry, Rixensart, BE

Brigitte Desiree Alberta Colau, c / o GlaxoSmithKline Biologicals SA,

Rue de llnstitut 89.1330 RIXENSART, BE

Frangoise Denamur, Rixensart, BE

Isabelle Knott, Rixensart, BE

Annick Poliszczak, Rixensart, BE

Vincent Vande Velde, c / o GlaxoSmithKline Biologicals SA, Rue de llnstitut 89.1330 RIXENSART, BE Zacco Norway AS, PO Box 2003 Vika, 0125 OSLO (54) Name (56) Published publications (57) Summary

Impaired human rotavirus population, method of preparing it, as well as vaccine composition comprising the attenuated rotavirus population, method of producing rotavirus vaccine and use of attenuated human rotavirus for vaccine preparation

MIDTHUN K et al. Single gene substitution rotavirus reassortants containing the major neutralization protein (VP7) of human rotavirus serotype 4. J Clin Microbiol. 1986 Nov; 24 (5): 822-6, US 4571385 A, US 4341763 A, Garbag-Chenon A et al. Reactogenicity and immunogenicity of rotavirus WC3 vaccine in 5-12 month old infants ”. Res Virol. 1989 May-Jun; 140 (3): 207-17., WO 9208786 A1, US 4624850 A, ATCC, Animal Virology collection, VR-2104, VR-2018, VR-2417

The invention provides an attenuated Rotavirus population comprising a single variant or essentially a single variant defined by a nucleotide sequence encoding at least one of the major virus proteins designated VP4 and VP7. More particularly, the invention provides a Rotavirus population designated P43. The invention further provides a novel formulation of a Rotavirus vaccine which is in the form of a fast soluble tablet for immediate dissolution by placement on the tongue.

BACKGROUND OF THE INVENTION The present invention relates to attenuated human rotavirus population, method of preparation thereof, as well as vaccine composition comprising attenuated human rotavirus population, method of preparing rotavirus vaccine and use of attenuated human rotavirus for preparation of vaccine.

Acute, infectious diarrhea is a major cause of illness and many deaths in many parts of the world. In developing countries, the incidence of diarrheal disease is overwhelming. For Asia, Africa and Latin America, it has been estimated that between 3-4 billion cases of diarrhea occur each year, and of these cases, approximately 5-10 million cause deaths (Walsh, JA et al .: N. Engl. J. Med., 301: 967-974 (1979)).

Rotavirus has been recognized as one of the leading causes of severe diarrhea in infants and young children (Estes, MK, Rotaviruses and Their Replication in Fields Virology, 3rd edition, edited by Fields et al., Raven Publishers, Philadelphia, 1996). It is estimated that rotavirus disease is the cause of more than one million deaths annually. Rotavirus-induced disease most commonly affects trees at the age of 6 to 24 months, and the disease generally occurs most frequently during the coolest months in temperate climates and year-round in tropical areas. Rotavirus is typically transmitted from person to person from faeces to mouth with an incubation time of approximately one to approximately three days. Unlike infections in the age group from 6 months to 24 months, newborns are generally asymptomatic or have only mild disease. Contrary to the severe disease normally observed in young chi ldren, most adults are protected from previous rotavirus infection, so most adult infections are mild or asymptomatic (Offit, PA et al., Comp. Ther., 8 (8 ): 21-26, 1982).

Rotaviruses are generally spherical, and the name is derived from their distinctive outer and inner shells or bald capsid structure. The double-bellied capsid structure of a rotavirus typically surrounds an inner shell or nucleus containing the genome. The genome of a rotavirus consists of 11 segments of double-stranded RNA that encode at least 11 distinct viral proteins. Two of these viral proteins, designated VP4 and VP7, are arranged on the outside of the double-shell capsid structure. The inner capsid of the rotavirus comprises a protein, the rotavirus protein designated VP6. The relative importance of these three particular rotavirus proteins in triggering the immune response following a rotavirus infection has not yet been established. Nevertheless, the VP6 protein determines the group and subgrou p antigen, while the VP4 and VP7 protein are determinants of serotype specificity.

The VP7 protein is a 38,000 molecular weight glycoprotein (34,000 molecular weight when not glycosylated) that is the translational product of genomic segment 7, 8, or 9, depending on the strain. This protein stimulates the formation of the most important neutralizing antibody after rotavirus infection. The VP4 protein is a non-glycosylated protein of molecular weight approximately 88,000 which is the translation product of genome segment 4. This protein also stimulates neutralizing antibody formation after rotavirus infection.

Since the VP4 and VP7 proteins are the viral proteins to which neutralizing antibodies are targeted, they are believed to be major candidates for the development of rotavirus vaccines that can provide protection against rotavirus disease.

Natural rotavirus infection during early childhood is known to trigger protective immunity. Thus, a live attenuated rotavirus vaccine is highly desirable. This should preferably be an oral vaccine as this is the natural route of infection for the virus.

Early vaccine development for the prevention of rotavirus infections began in the 1970s following the discovery of the virus. Initially, attenuated strains from animals and humans were studied, and these yielded varying or disappointing results. Recent trials have focused on human-animal reassortants, which has been more successful.

Midthun et al., J. Clin. Microbiol. 1986 relates to isolation of rotavirus reassortants derived from co-infection of the cell cultures with a bovine rotavirus of a serotype 6 and with a human rotavirus of serotype 3.

US 4,571,385 relates to a selection method for separating rotavirus reassortants derived from co-infection of cell cultures with an animal rotavirus and with a human rotavirus.

US 4,341,763 relates to the use of live attenuated or inactivated bov ine rotaviruses as a human vaccine.

Garbag-Chenon et al., Res. Virol. 1989 relates to the safety and immunogenicity studies of a rotavirus vaccine candidate comprising a live attenuated bovine strain after its administration to neonates.

US 4,624,850 relates to a culture method for human rotavirus strains and the preparation of corresponding live attenuated human rotavirus strains used for vaccine production.

Animal Biology Collection, VR-2104, VR-2018 and VR-2417 filed with ATCC show the product description of several isolated human rotaviruses.

A rotavirus strain designated 89-12 has been described by Ward; see U.S. Pat.

474 773 and Bernstein, DL et al., Vaccine, 16 (4), 381,387,1998. Strain 89-12 was isolated from a stool sample collected from a 14-month-old child with natural rotavirus disease in 1988. According to U.S. Patent No. 5,474,773, human rotavirus HRV 89-12 was then adapted for culture by 2 passages in pri mary African kidney cells. , green monkey (AGMK), and 4 passages in MA-104 cells, as described by Ward in J. Clin. Microbiol., 19,748-753,1984. The virus was then plaque-purified 3 times in MA-104 cells (to passage 9) and cultured after a further 2 passages in these cells. Another passage was made (passage 12) for deposition with ATCC under the accession designation ATCC VR 2272. The deposited strain is designated 89-12C2.

The 1998 article in Vaccine by Bernstein et al. is described below the Vaccine (1998) article. The article describes the safety and immunogenicity of a live human rotavirus vaccine candidate, administered orally. This vaccine was obtained from strain 89-12, attenuated by passage without plaque purification 26 times in primary AGMK cells and then an additional 7 times in an established AGMK cell line (a total of 33 passages).

Subsequently, the above described material which was given 26 serial passes will be designated P26. and the material that has been given 33 series passes will be designated P33. In general, rotavirus derived by passage of 89-12 n times will be designated Pn.

In the following examples, the P33 material was given an additional 5 passages in Vero cells. This preparation is designated P38.

The P26 and P33 isolates described in the Vaccine (1998) article were neither deposited in a culture collection nor analyzed to determine their genetic characteristics.

It has now been found that the P26 population described in the literature comprises a mixture of variants. This has been determined by genetic characterization as described herein below (see the examples). Therefore, P26 is not a reliable, sustained population for additional passengers, especially for the preparation of vaccine portions. Similarly, P33 comprises a mixture of variants and is not reliably stable for the production of vaccine portions.

It has been found that the P26 material is a mixture of at least three VP4 gene variants. P33 5 and P38 are similarly a mixture of two variants. In the case of neutralizing epitopes, these variants appear to be antigenically different from the 8912C2 strain deposited with the ATCC by an evaluation of the serum neutralizing antibody titer from children vaccinated with P33 against these variants. This is shown in Figure 3.

Furthermore, it has been found that when P33 material is administered to infants, two identified variants are replicated and excreted. Of 100 vaccinated infants, only two showed gastroenteritis due to rotavirus infection, while 20% of a placebo group was infected. These findings suggest that the identified variants are associated with protection against rotavirus disease. The present invention provides new rotavirus variants and an improved live attenuated rotavirus vaccine.

Accordingly, the present invention provides, in a first aspect, attenuated human rotavirus population, characterized in that it comprises a single variant or a substantially single variant, wherein said variant is designated P43 and is deposited under accession number ECACC 99081301.

By a population comprising a single variant or substantially a single variant is meant a rotavirus population containing no more than 10%, preferably less than 5%, and most preferably less than 1% of one or more other variants.

Virus populations can be purified to homogeneity or substantially homogeneity by passage in suitable cell types or by performing a series of one or more cloning steps.

An advantage of the invention is that a population comprising a single variant is more suitable for formulating a consistent vaccine production. Specific variants, defined by nucleotide sequences encoding the major virus protein, may also be associated with increased efficacy in the prevention of rotavirus infection.

In a preferred embodiment, the rotavirus variant designated P43 is a rotavirus variant, characterized by being designated P43 and deposited under accession number ECACC 99081301, its rotavirus progeny and immunologically active derivatives thereof and materials obtained therefrom.

In a further embodiment, the human rotavirus variant is characterized in that it comprises at least one antigen or at least one segment of the rotavirus variant P43 according to claim

2.

The present invention also provides a method for producing rotavirus as defined in any one of claims 1-3, characterized in that it comprises:

Passage of a rotavirus preparation into a suitable cell line;

io · if desired, selection of homogeneous culture using either:

o boundary dilution; or o single-plate insulation; and • controlling the presence of substantially a single variant by sequencing a suitable region of the VP4 and / or VP7 gene sequence.

Advantageously, the sequencing can be performed by a quantitative or semi-quantitative hybridization technique, for example, slot-blot hybridization or plaque hybridization.

Preferably, the selected variant is a variant that is replicated and secreted when the starting rotavirus preparation is administered to a human, preferably a child.

The resulting cloned virus population resulting from the method of the invention can be amplified by additional passages in a suitable cell line.

Suitable cell types for passage of the rotavirus population in the method above include kidney cells from African Green Monkey (AGMK cells), which may be established cell lines or primary AGMK cells. Suitable AGMK cell lines include, for example, Vero (ATCC CCL-81), DBS-FRhl-2 (ATCC CL-160), BSC-1 (ECACC 85011422) and CV-1 (ATCC CCL-70). Also suitable are the cell lines MA-104 (rhesus monkey) and MRC-5 (human ATCC CCL-171). Vero cells are particularly preferred for amplification purposes. Passage in Vero cells provides high viral yield.

Techniques for checking whether there is a single variant in a virus population obtained by the method and for determining the properties of this single variant include standard sequencing or hybridization procedures known in the art and described herein below.

In a preferred embodiment, the method of the invention is carried out using a suitable rotavirus, preferably a rotavirus having the properties of the 89-12 strain or a derivative thereof obtained by passage.

A particularly preferred single variant population is P43, which was obtained from P33 (an isolated human rotavirus with 33-fold passage in culture in suitable cell types) at a series of boundary dilution cloning steps followed by passage of the cloned material into Vero cells for amplification.

io A P43 population was deposited with the European Collection of Animal Cell Cultures (ECACC), Vaccine Research and Production Laboratory, Public Health Laboratory Service, Center for Applied Microbiology and Research, Porton Down, Salisbury, Wiltshire, SP4 OJG, United Kingdom on 13. August 1999 under the deposit number 99081301, under the terms of the Budapest Convention.

While this disclosed public availability is the easiest way to obtain the human rotavirus P43, it is not entirely impossible or unlikely that similar and functionally substantially identical rotavirus can be prepared by these or other methods in light of the teachings of the present invention. Such functionally, substantially identical rotavirus is considered biologically equivalent to the human rotavirus P43 of the invention and is therefore within the general scope of the present invention. Therefore, it will be understood that the invention encompasses rotavirus populations having the characteristics of the P43 variant as described herein.

It will also be understood that the invention encompasses materials derived from the deposited P43 ECACC 99081301 by further processing thereof, for example, by continuing it by additional passages, cloning or other methods utilizing the live virus, or by modifying P43 in some way. , including by genetic modification or reassortant techniques. Such steps and techniques are well known in the art.

Materials derived from the deposited P43 encompassed by the invention include protein material and genetic material. Of particular interest are reassorted rotaviruses comprising at least one antigen or at least one segment from P43, for example, reassortants comprising a virulent rotavirus strain wherein one or part of one of the

II genome segments have been replaced with the genome segment or part thereof from P43. In particular, a rotavirus reassortant, wherein the segment or sub-segment encoding NSP4 is a P43 segment or sub-segment, may have useful properties. Reassorted rotaviruses and techniques for their preparation are well known (Foster, RH and Wagstaff, AJ Tetravalent Rotavirus Vaccine, a review, ADIS drug evaluation, BioDrugs, Gev, 9 (2), 155-178,1998).

Materials of particular interest are progeny from P43 and immunologically active derivatives of P43. Immunologically active derivatives mean materials obtained from or with the P43 virus, more specifically, antigens from the virus, which can trigger an immune response that produces a response to rotavirus when injected into a host animal.

When adapting the rotavirus to a suitable cell line, such as Vero cells, it may be necessary to treat the virus to get rid of any possible contaminants, such as any randomly introduced agents that may be present and which would otherwise cause contamination. In the case of ether-sensitive contaminating viruses, treatment can be done by ether treatment as described herein below. The present invention also relates to the introduction of such ether treatment as an optional step in the general method of obtaining an attenuated live rotavirus or a vaccine formed therefrom.

Also within the scope of the invention are mixtures of P43 with other rotavirus variants, for example other cloned variants, or with other viruses, preferably other attenuated viruses. Such compositions are useful in the vaccines of the invention described herein below.

The present invention also provides a vaccine composition, characterized in that it comprises a live attenuated virus according to any one of claims 1 to 3 admixed with a suitable pharmaceutical carrier or adjuvant.

The rotavirus vaccine of the invention is preferably a vaccine adapted for oral administration.

The rotavirus vaccine of the invention is preferably a monovalent rotavirus vaccine comprising a single rotavirus strain.

The present invention is particularly advantageous in providing a live rotavirus vaccine in which the live attenuated rotavirus is a human rotavirus that does not provide intussusception.

Suitable pharmaceutical carriers for use in the vaccine of the invention include carriers known in the art as suitable for oral administration, particularly to trees. Such carriers include, but are not limited to, carbohydrates, polyalcohols, amino acids, aluminum hydroxide, magnesium hydroxide, hydroxyapatite, talc, titanium oxide, iron hydroxide, magnesium stearate, carboxymethylcellulose, hydroxypropylmethylanicellulose cellulose cyclodextrin.

The invention also provides a method for preparing a rotavirus vaccine according to any one of claims 8-22, characterized in that it comprises mixing a attenuated human rotavirus with a suitable pharmaceutical carrier or adjuvant.

Ice It may also be advantageous to design the virus of the invention in lipid-based carriers, for example, virosomes or liposomes, in oil in water emulsions or with carrier particles. Alternatively or additionally, immunostimulants, for example agents known in the art of oral vaccines, may be included in the composition. Such immunostimulants include bacterial toxins, preferably cholera toxin (CT) in the form of the holotoxin (whole molecule) or only the B chain (CTB), and the heat-labile enterotoxin from E. coli (LT). Mutated LT (mLT) which has less tendency to convert to its active form than native LT is described in WO 96/06627, WO 93/13202 and US Patent No. 5,182,109.

Additional immunostimulants which may be advantageously included are saponin derivatives such as QS21 and monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipid A (3D-MPL). Purified saponins as oral adjuvants are described in WO 98/56451. Saponins and monophosphoryl lipid A can be used alone or in combination (see, for example, WO 94/00153) and can be designed in adjuvant systems with other agents. 3D-MPL is a well-known adjuvant manufactured by Ribi Immunochem, Montana, and the preparation is described in GB 2122204.

A general discussion of carriers and adjuvants for oral immunization can be found in Vaccine Design, The Subunit and Adjuvant Approach, edited by Powell and

Newman, Plenum Press, New York, 1995.

The invention also provides the use of an attenuated human rotavirus for the preparation of a vaccine according to any one of claims 10-22 for the prevention of rotavirus infection in a human.

In a preferred embodiment, the vaccine composition of the invention is formed with an antacid preparation to minimize inactivation of the vaccine by gastric acid. Suitable acid neutralizing ingredients include inorganic antacids, for example aluminum hydroxide A1 (OH)₃ and magnesium hydroxide Mg (OH) 2. Commercially available antacids suitable for use in the invention include Mylanta (trademark), which contains aluminum hydroxide and magnesium hydroxide. These are insoluble in water and are given in suspension.

Aluminum hydroxide is a particularly preferred component of a vaccine composition according to the invention, as this not only provides an acid neutralizing effect but also an adjuvant effect.

Also suitable for use as antacids in the vaccine of the invention are organic antacids, for example, organic carboxylic acid salts. A preferred antacid in the vaccine composition of the invention contains an organic carboxylic acid salt, preferably a citric acid salt such as sodium citrate or potassium citrate.

A particularly preferred antacid which can be used in the vaccine composition of the invention is the insoluble inorganic salt calcium carbonate (CaCO₃). Calcium carbonate can be associated with the rotavirus and rotavirus activity is maintained while the virus is associated with calcium carbonate.

In order to prevent the precipitation of calcium carbonate during the filling step, viscosity agents are preferably present in the composition.

Possible viscosity agents which may be used include pseudoplastic excipients. A pseudoplastic solution is defined as a solution having higher viscosity upon standing, compared to the viscosity with stirring. Excipients of this type are natural polymers such as gum arabic, adhesive rubber, agar-agar, alginates, pectins or semi-synthetic polymers such as carboxymethylcellulose (Tyloses C), methylcellulose (Metocels A, Viscotrans MC, Tylose MH and MB), hydroxyp , and hydroxypropyl methyl cellulose (Metocels E and K, Viscontrans MPHC). Generally, these pseudoplastic excipients are used with thixotropic agents. Alternative viscosity agents that can be used are low flow pseudoplastic excipients. At sufficient concentration, these polymers give rise to a structural liquid arrangement which results in a very viscous solution with low flowability upon standing. A certain amount of energy must be supplied to the system to achieve flow and mass transfer. External energy (stirring) is necessary for the temporary destruction of the structural fluid arrangement to achieve a liquid solution. Examples of such polymers are Carbopols and xanthan gum.

Thixotropic excipients form a gel structure upon standing, while stirring to form a liquid solution. Examples of thixotropic excipients are Veegum magnesium-aluminum silicate and Avicel RC (approximately 89% microcrystalline cellulose and 11% carboxymethyl cellulose-Na).

The vaccine composition of the present invention preferably comprises a viscosity agent selected from xanthan gum and starch.

Thus, the vaccine composition of the present invention is preferably formed with a combination of calcium carbonate and xanthan gum.

Other ingredients of a composition used in the invention preferably include sugars, for example, sucrose and / or lactose.

The vaccine composition of the invention may contain additional ingredients, such as flavoring agents (preferably for an oral vaccine) and bacteriostatic agents.

Various embodiments of the vaccine composition of the invention are envisaged.

In a preferred embodiment, the vaccine is administered as a liquid preparation. The liquid preparation is preferably reconstituted prior to administration from at least the following two components:

i) virus component; ii) fluid component.

In this embodiment, the virus component and the liquid component are normally contained in separate containers, which may advantageously be separate portions of a single container, or separate containers which can be interconnected in such a way that the final vaccine preparation is reconstituted without being exposed to air.

Prior to reconstitution, the virus may be in dry or liquid form. The virus component is preferably freeze-dried, Freeze-dried virus is more stable than virus in an aqueous solution. The freeze-dried can advantageously be reconstituted using a liquid antacid preparation to produce a liquid vaccine preparation. Alternatively, the freeze-dried virus may be reconstituted with water or an aqueous solution, in which case the freeze-dried virus preparation preferably contains an antacid component.

The vaccine composition preferably comprises a viral constituent formed with calcium carbonate and xanthan gum in one compartment or container, and this is reconstituted with water or an aqueous solution present in the other compartment or container.

In another preferred embodiment, the vaccine composition is a solid composition, preferably a lyophilized mass suitable for immediate dissolution when placed in the mouth. Freeze-dried preparations may advantageously be in the form of tablets in a pharmaceutical plastic package.

In another embodiment, the invention provides a vaccine composition according to any one of claims 10 to 18, characterized in that the live attenuated virus is formed with the antacid preparation and lyophilized in a plastic package.

In another embodiment, the invention provides a vaccine composition according to claim 20, characterized in that the live attenuated virus and the antacid composition are in separate containers for formulation as a liquid vaccine preparation prior to administration.

In another embodiment, the invention provides a vaccine composition according to claim 20, characterized in that the live attenuated virus and the antacid preparation are in the same container for formulation as a lyophilized vaccine preparation for reconstitution with an aqueous solution prior to administration.

In another embodiment, the invention provides a rotavirus vaccine in the form of a rapidly dissolving oral administration tablet.

In another embodiment, the invention provides a composition comprising a live attenuated rotavirus strain, preferably a human rotavirus strain, wherein the composition is a freeze-dried solid which can be dissolved immediately when placed in the mouth.

The fast-dissolving tablet of the invention preferably dissolves in the patient's mouth sufficiently fast to prevent swallowing of the undissolved tablet. This approach is particularly advantageous for pediatric rotavirus vaccines.

Preferably, the virus is a live attenuated rotavirus formed with an inorganic antacid, such as calcium carbonate, and a viscosity agent, such as xanthan gum.

A further embodiment of the present invention is to provide a freeze-dried composition wherein the virus component is any rotavirus strain formed with calcium carbonate and xanthan gum.

Vaccines according to the invention are used as medicine and can be designed and administered by known techniques using a plurality of live vims suitable for obtaining effective protection against rotavirus infection without significant undesirable side effects for typical vaccine recipients. A suitable amount of live virus will normally be between 10⁴ and 10⁷ fifu pr. dose. A typical dose of the vaccine may comprise 10⁵-10⁶ ffu pr. dose and can be given in multiple doses over a certain period of time, for example in two doses given at two-month intervals. However, advantages can be obtained by using more than two doses, for example a three or four dose dosage form, especially in developing countries. The interval between doses can be more or less than two months long. An optimal amount of live virus for a single dose or for a multiple dose schedule and optimal dosing times can be established by standard studies that include antibody titre observation and other patient responses.

The vaccine of the invention may also comprise other suitable live viruses for protection against other diseases, such as polioviruses. Alternatively, other suitable live virus vaccines for oral administration may be administered in a separate dose, but at the same time as the rotavirus vaccine composition of the invention.

Text for Figure 3

Serum from twelve 4 to 6 month old infants vaccinated with P33 material as described in Vaccine (1998) article was analyzed for neutralization of P33, P38, P43 and 89-12C2.

The neutralizing titre range for the analyzed sera is similar for P33, P38 and P43. The statistical analysis shows no significant difference in the total neutralizing titer against these three viruses. This suggests that conformational and non-conformational neutralizing epitopes in P33, P38 and P43 are equally well recognized by the anti-P33 sera of P33 vaccinated infants. This observation indirectly suggests that the neutralizing epitopes recognized in this in vitro test do not differ between P33, P38 and P43.

However, the neutralizing titre range of P89-12C2 was significantly different from the range of P33, P38 and P43. This observation suggests that the conformational and nonconformational neutralizing epitopes in P33, P38 and P43 are not recognized as well by anti-P33 serum from P33 vaccinated infants. This observation indirectly suggests that the neutralizing epitopes recognized in this in vitro analysis have been altered in 89-12C2, compared with P33, P38 and P43.

The following examples illustrate the invention.

EXAMPLES

Example 1: Demonstration that strain 89-12 at passage 26 (P26) is a mixture of variants

Sequencing of the VP4 and VP7 genes from different passages

Sequencing of the VP4 and VP7 gene from passage P26 (primary AGMK cells), passage P33 (established (as opposed to primary) AGMK cell line), passage P41, and passage P43 were performed. Extracted total RNA was reverse transcribed and amplified by PCR in one tube / one step.

Primers Rota 5bis and Rota 29bis amplified the entire VP4 gene, and primers Rota 1 and Rota 2bis amplified the entire VP7 gene. The PCR material has been sequenced using different primers (see Table 1).

The sequence from passage P26 differed from the sequence from passage P33 in three bases (in positions 501, 788 and 802bp from the start codon) in VP4 and in three bases in VP7 (108,605 and 897 bp from the start codon).

Passage P26 sequence analyzes of VP4 and VP7 indicate, in mutated positions, the presence of the passage P33 sequence as background. Thus, it can be observed that passage P26 is a mixture of at least two variants.

Passage P33 sequence analyzes appear homogeneous for VP4 and heterogeneous for VP7 (see Table 2).

Passage P38 (derived from passage P33) was given five times passage in Vero cells and showed the same set of VP4 and VP7 sequences as passage P33 (AGMK cell line). Thus, there was no significant change in the populations between P33 and P38.

TABLE 1: Oligonucleotides used for RT-PCR and sequencing

<td></td><td>name</td><td>Sequence</td><td>position</td>

<td>VP7</td><td>Rota 1</td><td>GGC TTT AAA AGA GAG AAT TTC CGT CTG G</td><td>-49 to -22</td>

<td></td><td>Rota Ibis</td><td>GGT TAG CTC CTT TTA ATG TAT GGT A</td><td>-16 to 10</td>

<td></td><td>Rota 2bis</td><td>GGT CAC ATC GAA CAA TTC TAA TCT AAG</td><td> 1014-988</td>

<td></td><td>Rota 7</td><td>CAA GTA CTC AAA TCA ATG ATG G</td><td> 266-287</td>

<td></td><td>Rota 12</td><td>TGT TGA TTT TTC TGT CGA TCC AC</td><td> 372-394</td>

<td></td><td>name</td><td>Sequence</td><td>position</td>

<td>VP7</td><td>Rota 46</td><td>GGT TGC TGA GAA TGA GAA ATT AGC TAT AGT GG</td><td> 651-682</td>

<td></td><td>Rota 18</td><td>CCA CTA TAG CTA ATT TCT CAT TCT CAG CAA CC</td><td> 682-651</td>

<td>VP4</td><td>Rota 5</td><td>TGG CTT CGC CAT TTT ATA GAC A</td><td> 2-23</td>

<td></td><td>Rota 6</td><td>ATT TCG GAC CAT TTA TAA CC</td><td> 878-859</td>

<td></td><td>Rota 5bis</td><td>TGG CIT CAC TCA TTT ATA GAC A</td><td> 2-23</td>

<td></td><td>Rota 6bis</td><td>ATT TCA GAC CAT TTA TAA CCT AG</td><td> 878-856</td>

<td></td><td>Rota 25</td><td>GGA GTA GTA TAT GAA AGT ACA AAT AAT AG</td><td> 268-296</td>

<td></td><td>Rota 26</td><td>CTA TTA TTT GTA CTT TCA TAT ACT ACT CC</td><td> 296-268</td>

<td></td><td>Rota 27bis</td><td>TCG ATA CAG TAT AAG AGA GCA CAA G</td><td> 721-745</td>

<td></td><td>Rota 28</td><td>TTC ATT AAC TTG TGC TCT CTT ATA CTG</td><td> 753-727</td>

<td></td><td>Rota 31</td><td>GTA TAT GTA GAC TAT TGG GAT G</td><td> 1048-1070</td>

<td></td><td>Rota 32</td><td>CAT CCC AAT EIGHT CTA CAT ATA C</td><td> 1070-1048</td>

<td></td><td>Rota 45</td><td>TGT AAC TCC GGC AAA ATG CAA CG</td><td> 1205-1227</td>

<td></td><td>Rota 53</td><td>CGT TGC ATT TTG CCG GAG TTA CA.</td><td> 1227-1205</td>

<td></td><td>Rota 54</td><td>GTA AGA CAA GAT TTA GAG CGC CA.</td><td> 1465-1487</td>

<td></td><td>Rota 55</td><td>TGG CGC TCT AAA TCT TGT CTT AC</td><td> 1487-1465</td>

<td></td><td>Rota 40</td><td>CTT GAT GCT GAT GAA GCA GCA TCT G</td><td> 1703-1727</td>

<td></td><td>Rota 39</td><td>CAG ATG CTG CTT CAT CAG CAT CAA G</td><td> 1727-1703</td>

<td></td><td>Rota 33</td><td>CGA TCA TAT CGA ATA TTA AAG GAT G</td><td> 2008-2032</td>

<td></td><td>Rota 34</td><td>CAT CCT TTA ATA TTC GAT ATG ATC G</td><td> 2032-2008</td>

<td></td><td>Rota 29bis</td><td>AGC GTT CAC ACA ATT TAC ATT GTA G</td><td> 2335-2311</td>

TABLE 2: Oligonucleotides Used in Hybridization

<td></td><td>name</td><td>Sequence</td><td>position</td>

<td>VP7</td><td>Rota 41</td><td>ATT AT TTA TAC TAT AGT AGA TTA TAT TAA TC</td><td> 882-913</td>

<td></td><td>Rota 42</td><td>AGE ATT TTA TAC TAT GGT AGA TTA TAT TAA TC</td><td> 882-913</td>

<td>VP4</td><td>Rota 15</td><td>ATC CCC ATT ATA CTG CAT TCC TTT C</td><td> 807-783</td>

<td></td><td>Rota 16</td><td>ATC CCT ATT ATA CTG CAT TTC TTT C</td><td> 807-783</td>

<td></td><td>Rota 35</td><td>ATC CCC ATT ATA CTG CAT TTC TTT C</td><td> 807-783</td>

<td></td><td>Rota 36</td><td>ATC CCT ATT ATA CTG CAT TCC TTT C</td><td> 807-783</td>

The bases shake with highlighted font in Table 2 are sites for specific sequence variation in VP4 and VP7.

TABLE 3: Sequence variation in the VP4 and VP7 gene

3.1

<td colspan="4">VP4</td><td colspan="3">VP7</td>

<td></td><td>501 bp 167 amino acid</td><td>788 bp 263 amino acid</td><td>802 bp 268 amino acid</td><td>108 bp 36 amino acid</td><td>605 bp 202 amino acid</td><td>897 bp 299 amino acid</td>

<td>P26 (AGMK)</td><td>A</td><td>GO</td><td>GO</td><td>A</td><td>C / T</td><td>A</td>

<td>P33 (AGMK)</td><td>T</td><td>A</td><td>A</td><td>GO</td><td>T / C</td><td>A / G</td>

<td>P38 (VERO)</td><td>T</td><td>A</td><td>A</td><td>A / G</td><td>T</td><td>GO</td>

<td>P43 (VERO)</td><td>T</td><td>A</td><td>A</td><td>S</td><td>T</td><td>A</td>

io NB In another clone from the three clones that were developed to production level, the nucleotide is at 897 bp in VP7 G rather than A contained in the P43 selected clone. This leads to methionine instead of isoleusin in the amino acid sequence. Variants corresponding to both the selected P43 clone and the clone in which there is a G in VP7 at position 897 bp from the start codon were excreted in feces in children vaccinated with the P3315 material.

In Table 3.1, where there are two alternative bases in a given position, the first of these represents the base that exists in the main population, while the second is the base that occurs in a smaller part of the population. Main populations and smaller populations are assessed based on signal strength in sequencing.

3.1

<td colspan="4">VP4</td><td colspan="3">VP7</td>

<td></td><td>501 bp 167 amino acid</td><td>788 bp 263 amino acid</td><td>802 bp 268 amino acid</td><td>108 bp 36 amino acid</td><td>605 bp 202 amino acid</td><td>897 bp 299 amino acid</td>

<td>P26 (AGMK)</td><td>A</td><td>GO</td><td>GO</td><td>A</td><td>C / T</td><td>A</td>

<td>P33 (AGMK)</td><td>T</td><td>A</td><td>A</td><td>GO</td><td>T / C</td><td>A / G</td>

<td>P38 (VERO)</td><td>T</td><td>A</td><td>A</td><td>A / G</td><td>T</td><td>GO</td>

<td>P43 (VERO)</td><td>T</td><td>A</td><td>A</td><td>S</td><td>T</td><td>A</td>

3.2

<td colspan="4">VP4</td><td colspan="3">VP7</td>

<td></td><td>501 bp 167 amino acid</td><td>788 bp 263 amino- • acid</td><td>802 bp 268 amino acid</td><td>108 bp 36 amino acid</td><td>605 bp 202 amino acid</td><td>897 bp 299 amino acid</td>

<td>P26 (AGMK)</td><td>Leu</td><td>Gly / Glu</td><td>Gly / Arg</td><td>Arg</td><td>Thr / Met</td><td>Ile</td>

<td>P33 (AGMK)</td><td>Phe</td><td>Glu</td><td>Arg</td><td>Arg / Arg</td><td>Met / Thr</td><td>Ile / Met</td>

<td>P38 (VERO)</td><td>Phe</td><td>Glu</td><td>Arg</td><td>Arg / Arg</td><td>Met</td><td>Met / He </td>

<td>P43 (VERO)</td><td>Phe</td><td>Glu</td><td>Arg</td><td>Arg</td><td>Met</td><td>Ile</td>

Table 3.2 shows the amino acid changes resulting from the nucleotide differences between the variants.

TABLE 4

<td rowspan="2"></td><td colspan="4">VP4 (heading 788-802)</td><td colspan="2">VP7 (heading 897)</td>

<td>GG</td><td>AA</td><td>AG</td><td>GO</td><td>A</td><td>G</td>

<td>probe</td><td>Rota 15</td><td>Rota 16</td><td>Rota 35</td><td>Rota 36</td><td>Rota 41</td><td>Rota 42</td>

<td>passage</td><td colspan="6"></td>

<td>P26</td><td> -</td><td> +</td><td> +</td><td> +</td><td>nd</td><td>nd</td>

<td>P33</td><td> -</td><td> +</td><td> -</td><td> -</td><td> ++</td><td> +</td>

<td>P38</td><td> -</td><td> +</td><td> -</td><td> -</td><td> +</td><td> ++</td>

<td>P43</td><td> -</td><td> +</td><td> -</td><td> -</td><td> +</td><td> -</td>

Slot-blot hybridization

The population changes between passage P26 and passage P33 in AGMK cells have been further confirmed by slot blot hybridization. The VP4 and VP7 gene fragments formed by RI7PCR were hybridized with oligonucleotide probes specific to each variant (see Tables 3.1 and 3.2). Unlike P26, which hybridized with Rota 16, Rota 35 and Rota 36 and not with Rota 15, the VP4 PCR fragment from the P33 material at positions 788 and 802 hybridized only with Rota 16, and neither with Rota 15, Rota 35 or Rota 36. These results determined the presence of at least three variants in P26 (see Table 4).

For the VP7 PCR fragment from the P33 material, position 897 hybridized with Rota 41 and Rota 42. These results determined the presence of at least two variants in the P33 material.

Example 2: Isolation and characterization of the P43 clone

For isolation of P33 constituents as a homogeneous virus population, three end point dilutions of P33 / AGMK in Vero cells were performed and the resulting virus was used to infect Vero cells.

Positive wells were selected based on two criteria: Growth, shown by the largest number of foci detected in the wells and the most isolated positive wells in the plates, as is done in a classical manner. After three boundary dilution passages in 96-well microtiter plates, ten positive wells were amplified one after another in Vero cells and evaluated for yield.

Based on the yield, three clones were developed to production level. Immune recognition by polyclonal antibodies was shown to be similar between the three clones and between the clones and P33. The homogeneity of the clones was estimated by slot-blot hybridization. The final selection of a single clone was based on yield and sequence.

The selected clone was amplified by successive passages in Vero cells to form a main suspension, a working suspension and final production portions.

The selected clone was genetically characterized at different passage levels by sequencing VP4 and VP7 (identity) and by specific slot-blot hybridization with VP4 and VP7 (homogeneity) from the PCR amplified materials. The sequence of the VP4 and VP7 gene in the P43 material is given in Figure 1 and 2, respectively, and is identical to P41.

The homogeneity of the selected clone was estimated by selective hybridization using oligonucleotide probes that recognize nucleotide changes in the VP4 and / or VP7 region of each of the variants identified during sequencing of P26 / primary AGMK (see Table 4).

The VP4 fragment hybridized with Rota 16 and not with Rota 15, Rota 35 or Rota 36.

The VP7 fragment hybridized with Rota 41 and not with Rota 42.

These results confirmed that P43 is a homogeneous population.

Example 3: Removal of any additional virus

Ether was added to P33 (grown in AGMK) to a final concentration of 20% for one hour. The ether was then bubbled with N₂ for 35 minutes. No effect on the titre in the P33 suspension was observed.

Example 4: Design of a live attenuated vaccine

The production portions described above are designed for oral administration to infants by the following procedure.

1, Freeze-dried virus

Standard techniques are used to prepare virus doses. Frozen, purified virus pulp is thawed and diluted with a suitable medium, in this case Dulbecco's modified r π

Eagle's Medium, up to a desired standard viral concentration, in this case 10 'ffu / ml. The diluted virus is then further diluted with freeze-drying stabilizer (4% sucrose, 8% dextran, 6% sorbitol, 4% amino acids) up to the desired virus titer, in this case 10^s’⁶ FFII / dose. Extract of 0.5 ml of the stabilized virus preparation is transferred septically to 3 ml ampoules. The vials are then partially closed with a rubber stopper, the sample is freeze-dried under vacuum, the vial is completely closed and an aluminum cap is clamped in place around the vial to hold the cork in place.

Prior to use, the virus is reconstituted using one of the following acid neutralizing constituents:

(a) Citrate reconstitution agent

Sodium citrate is dissolved in water, sterilized by filtration and aseptically transferred to reconstitutive containers in 1.5 ml quantities at a concentration of 544 mg of NajSitrate. 2H₂O pr. 1.5 ml dose. For example, the reconstitution agent vials may be 3 ml ampoules, 4 ml ampoules or 2 ml syringes, or collapsible soft plastic capsules for oral administration. As an alternative to using sterile ingredients under sterile conditions, the final container can be autoclaved.

(b) Al (OHh reconstitution)

An aseptic aluminum hydroxide suspension (Mylanta trademark) is aseptically diluted with sterile water and aseptically transferred to reconstitute container (e.g. 2 ml syringes or collapsible soft plastic capsules) in 2 ml quantities each containing 48 mg Al (OH) 3. An alternative to using sterile ingredients under sterile conditions is to γ-irradiate the aluminum hydroxide suspension (preferably at a diluted stage).

Standard ingredients are included to prevent suspension from settling. Such standard ingredients include, for example, magnesium stearate, carboxymethyl cellulose, hydroxypropyl methyl cellulose, microcrystalline cellulose, and silicone polymers. Bacteriostatic agents, for example, butylparaben, propylparaben or other standard bacteriostatic agents used in foods, and flavorings may also be included.

2. Freeze-dried virus with AKOHh in a liquid preparation

Standard techniques are used to prepare virus doses. Frozen, purified virus pulp is thawed and diluted with a suitable medium, in this case Dulbecco's modified Eagle's Medium, to a desired standard virus concentration, in this case 10⁶’² ffu / ml. Aluminum hydroxide suspension is added to a final dose of 48 mg / dose and the virus preparation is diluted with freeze-drying stabilizer (4% sucrose, 8% dextran, 6% sorbitol, 4% amino acids) up to the desired virus titre, in this case 10^{3 * 5}’⁶ ffu / dose. Aseptically, withdrawal of 0.5 ml of the stabilized virus preparation is transferred to 3 ml ampoules. Freeze drying and sealing of the vials is performed as described in Part 1.

3, Freeze-dried virus with AKOHE for delivery in plastic containers

Standard techniques are used to prepare virus doses. Frozen, purified virus pulp is thawed and diluted with a suitable medium, in this case Dulbecco's modified Eagle's Medium, to a desired standard virus concentration, in this case £ f ffu / ml. Aluminum hydroxide suspension is added to a final dose of 48 mg / dose and the virus preparation is diluted with freeze-drying stabilizer which may be sucrose, dextran or 4% amino acids, or gelatin, vegetable peptone or xanthan, up to the desired virus titre of 10⁵’⁶ ffu / dose. An aseptic filling method is used to transfer doses of 0.5 ml, or preferably less, to the voids in the plastic container. The preparation is freeze-dried and the cavities in the plastic container are sealed by heat treatment.

If desired, standard ingredients are included to prevent the aluminum hydroxide suspension from settling. Such standard constituents include, for example, magnesium stearate, carboxymethyl cellulose, hydroxypropyl methyl cellulose, microcrystalline cellulose and silicone polymers. Flavors can also be included.

Example 5: Titration of viruses in various preparations

<td colspan="4">5.1: Comparison between lactose and sucrose based preparations:</td>

<td>Portion no.</td><td>Composition of preparations</td><td>Virus titer before freeze drying</td><td>Virus titer after freeze-drying and 1 week at 37 ° C</td>

<td>98G06 / 01</td><td>Lactose: 2%; Dextran: 4%; Sorbitol: 3% Amino acid: 2%</td><td> 10 ^W2</td><td> 10^{4, b /}</td>

<td>98G06 / 03</td><td>Sucrose: 2%; Dextran: 4%; Sorbitol: 3% Amino acid: 2%</td><td> 10^5,28</td><td> 10^4,92</td>

P43 rotavirus either with sucrose or with lactose as shown in the table above.

The virus titrated prior to freeze-drying is virus-titrated in the finished liquid (containing sucrose, dextran, sorbitol and amino acids) and without the freeze-drying step.

Good results are results in which a &lt;0.5 log reduction in the freeze-drying step and &lt;0.5 log reduction over a week at 37 ° C (accelerated stability analysis) is achieved.

The precision of titrating the virus is approximately +/- 0.2 log.

The results suggest that sucrose may be used in place of lactose.

5.2; Effect of arginine and replacement of sorbitol with maltitol;

<td>Portion no.</td><td colspan="2">Composition of preparations</td><td>Virus titer at time = zero after freeze drying</td><td>Virus titer after freeze-drying and 1 week at 37 ° C</td>

<td>98L16 / 01</td><td>Lactose Dextran Sorbitol Amino Acid</td><td> 2% 4% 3% 2%</td><td> 10⁴¹⁸</td><td>io⁷^</td>

<td>98L16 / 02</td><td>sucrose dextran Sorbitol Amino Acid arginine</td><td> 2% 4% 3% 2% 3%</td><td> 10^4,8</td><td> 10^{4, y}</td>

<td>98L16 / 04</td><td>lactose dextran Maltitol Amino Acid</td><td> 2% 4% 3% 3%</td><td>io ^⁷</td><td>Ϊθ3</td>

The results show that the addition of arginine (which is known to improve the stability of virus during freeze-drying and also provides a basal medium that compensates for the acidity of the stomach) retains the virus titer.

Sorbitol tends to reduce the glass transfer temperature of the freeze-dried pulp to a large extent. This can be overcome by using maltitol instead of sorbitol as shown above, and the virus titer is still maintained.

o

5.3: Design of various preparations ¹⁵

This experiment shows that a variety of designs are possible.

<td>Portion no.</td><td>Composition of preparations</td><td>Virus titer before freeze drying</td><td>Virus titer after freeze-drying and 1 week at 37 ° C</td>

<td>99C11 / 01</td><td>Sucrose: 2% Dextran: 4% Sorbitol: 3% Amino acid: 2%</td><td> 10 ^W4</td><td>_{3) UV}</td>

<td>99C11 / 02</td><td>Sucrose: 2% Dextran: 4% Maltitol: 3% Amino acid: 2%</td><td>_Wi.uy</td><td> 10^{4, yz}</td>

<td>99C11 / 04</td><td>Dextran: 4% Maltitol: 3% Amino acid: 3%</td><td> 10 4.89</td><td> 10 ⁵’^uh</td>

<td>Portion no.</td><td>Composition of preparations</td><td>Virus titer at time = zero after freeze drying</td><td>Virus titer after freeze-drying and 1 week at 37 ° C</td>

<td>99C17 / 01</td><td>Sucrose: 2% Dextran: 4% Sorbitol: 3% Amino acid: 2%</td><td> 10^3,4U</td><td> 10^3,41</td>

<td>99C17 / 02</td><td>Sucrose: 2% Dextran: 4% Sorbitol: 1.5% Amino acid: 2%</td><td> 10 ³’^JU</td><td>ίο ™</td>

<td>99C17 / 03</td><td>Sucrose: 2% Dextran: 4% Amino acid: 2%</td><td>1O³’^{J |}</td><td> 10^3,24</td>

<td>99C17 / 04</td><td>Sucrose: 2% Dextran; 4% Maltitol: 3% Amino acid: 2%</td><td> 10^4,42</td><td> 10⁴¹⁴³</td>

<td>99C17 / 05</td><td>Sucrose: 2% Dextran: 4% Maltitol: 1.5% Amino acid: 2%</td><td></td><td>iF⁴⁰</td>

<td>99C17 / 06</td><td>Sucrose: 2% Dextran: 4% Sorbitol: 3%</td><td>1F⁴⁴</td><td> 10^{4&gt; y}'</td>

<td>99C17 / 07</td><td>Sucrose: 2% Dextran: 4% Sorbitol: 1.5%</td><td> 10³’¹¹</td><td>LF *⁹</td>

5.4: Association between Rotavirus and Al (OH)₃-antacider:

<td>rotavirus</td><td>A1 (OH)₃</td><td>H₂O</td><td>Contact time at room temperature</td><td>Spin</td><td>Supernatant virus titer ffu / ml</td><td>Centrifuged virus titer ffu / ml</td>

<td> 10^{3, ΰ}ffu / ml</td><td>48mgi 0.240 ml</td><td>0.76 ml</td><td>30 min</td><td>8000 rpm, 10 min</td><td>l₀J, bb</td><td></td>

<td> 10^{3&gt; b}ffu / ml</td><td>48 mg i 0.240 ml</td><td>0.76 ml</td><td>30 min</td><td>8000 rpm, 10 min</td><td>1F⁴¹</td><td></td>

<td> 10³¹⁶ffu / ml</td><td>12 mg i 0.120 ml</td><td>1,380 ml</td><td>30 min</td><td>8000 rpm, 10 min</td><td></td><td></td>

<td>Freeze-dried rotavirus</td><td>12 mg i 0.120 ml</td><td>1,380 ml</td><td>30 min</td><td>8000 rpm, 10 min</td><td>below the detection limit</td><td>iF⁹</td>

A1 (OH)₃ used as antacid. This shows that Rotavirus is associated with the insoluble inorganic salt (AL (OH) 3) since it is centrifuged with A1 (OH)₃ (reduced virus activity in the supernatant).

5.5: Resolution of Al (OH)₃antacid with sodium citrate before virus titration

<td>virus Trial</td><td>solvent</td><td>Conditions</td><td>virus titer</td>

<td>99B10 / 06 liquid preparation before freeze drying:</td><td>1.5 ml of Na₃citrate</td><td>24 hours at room temperature</td><td> 10^5,11</td>

<td>99B10 / 06 freeze-dried: 10⁵’⁴³</td><td>1.5 ml of Na₃citrate</td><td>24 hours at room temperature</td><td> 10⁴¹⁵³</td>

When Rotavirus is associated with A1 (OH)₃ it is possible to freeze-dry everything (including Al (OH) 3). After freeze-drying, it is possible to recover the Rotavirus by solving A1 (OH)₃in sodium citrate. This step does not damage the Rotavirus and maintain the activity after this resolution step.

5.6: Infectivity to Rotavirus after release from the A1 (OH) 3-Rotavirus Complex:

The mechanism of virus release (upon dissolution of the carrier) may well occur in vivo. In fact, aluminum hydroxide becomes completely soluble below pH 6 and thus the Rotavirus will be released into the stomach.

A1 (OH)₃ + 3H⁺ - - - -► Al⁺⁺⁺ (water soluble) + 3H₂O

In the stomach, Al is not absorbed⁺⁺⁺ ions (JJ Powell, R. Jugdaohsingh, and RPH Thompson, The regulation of mineral adsorption in the gastrointestinal track, Proceedings of the Nutrition Society (1999), 58, 147-153).

Intestinal forms of insoluble aluminum (A1 (OH))₃ or AIPO4) due to elevated pH and removed naturally.

It is not known whether the newly formed precipitate of A1 (OH) 3 (or AIPO4) will be able to re-associate with free Rotavirus. This raises the question of the infectiousness of A1 (OH)₃The rotavirus complex itself.

Release of Rotavirus from Al (OH)₃The rotavirus complex by other mechanisms is also possible. For example, lysine interferes with virus adsorption to A1 (OH)₃. Other anions such as borate, sulfate, carbonate and phosphate are known to be adsorbed specifically to aluminum hydroxide, so it should be theoretically possible to displace (in competition for the adsorption site) Rotavirus from Al (OH)₃'Rotavirus complex.

DRVC003A46 +

mg A1 (OH)₃in 0.120 ml 65 mg lysine 1.380 ml H₂O +

my room temp.

+ centrifugation

<img file="NO328112B1_D0001.tif" />

Centrifuged material Supernatant +

solution in citrate below the detection limit 3.8

Rotavirus can thus be released from Rotavirus - Al (OH)₃complex, and the released Rotavirus remains active.

The release can be performed either by dissolving A1 (OH)₃ by HCI in the stomach or with io Na₃Citrate in vitro, or by displacing the Rotavirus with a basic amino acid (lysine).

5.7: Infectiousness of Al (OH)₃-Rotavirus complex

A single dose of freeze-dried Rotavirus was reconstituted with water and split in two. The ice first part, which is the reference part, was given a further volume of water. The second part was added 24 mg of A1 (OH)₃ suspended in 0.240 ml of water (preclinical virus titration).

DRVC003A4 +

1.5 ml of H₂0

<img file="NO328112B1_D0002.tif" />

<td>0.750 ml</td><td>0.750 ml</td>

<td> +</td><td> +</td>

<td>0.240 ml</td><td>24 mg</td>

<td>H₂O</td><td>A1 (OH)₃</td>

<td></td><td>in 0.240 ml</td>

<td>1 hour</td><td>1 hour</td>

<td> 5,55</td><td> 6,22</td>

When A1 (OH) 3 is present, Rotavirus is active and the virus titer value is higher than in the reference sample.

This experiment was repeated without dividing the freeze-dried dose and by the addition of 12 mg of A1 (OH)₃ or 24 mg A1 (OH)₃.

Here, the reference sample was the sample reconstituted with a citrate bicarbonate buffer. The virus titer is again higher in the presence of Al (OH) 3.

<td>DRVC003A46</td><td>DRVC003A46</td><td>DRVC003A46</td>

<td> +</td><td> +</td><td> +</td>

<td>1.5 ml WL-</td><td>12 mg</td><td>24 mg</td>

<td>buffer</td><td>A1 (OH)₃</td><td>A1 (OH)₃</td>

<td></td><td>in 0.120 ml</td><td>in 0.240 ml</td>

<td></td><td> +</td><td> +</td>

<td></td><td>1,380 ml of H₂O</td><td>1,260 mlH₂0</td>

<td> 5,34</td><td> 6,24</td><td> 6,05</td>

<td> 5,32</td><td> 5,95</td><td> 6,26</td>

ice As in the example above, Rotavirus is associated with the Al (OH) 3 particles since the virus can be removed by centrifugation. DRVC003A46 is a physio-dried Rotavirus (Sucrose: 2%; Dextran: 4%; Sorbitol: 3%; Amino Acids: 2%).

DRVC003A46 +

12mgAl (OH)₃ in 0.120 ml +

1,380 ml of H₂O + centrifugation 8000 rpm. 10 min \

<img file="NO328112B1_D0003.tif" />

DRVC003A46 +

MgAl (OH)₃in 0.240 ml +

1,260 ml of H₂O +

spin 8000 rpm. 10 min

<img file="NO328112B1_D0004.tif" />

<td>pelleted</td><td colspan="2">supernatant down-centrifuged</td>

<td>material</td><td></td><td>material</td>

<td> +</td><td></td><td> +</td>

<td>1.5 ml</td><td></td><td>1.5 ml</td>

<td>SDSAA</td><td></td><td>SDSAA</td>

<td> 5,78</td><td> &lt;1,44</td><td> 5,92</td>

<td> 5,96</td><td> &lt;1,44</td><td> 6,11</td>

supernatant &lt;1.44 &lt;1.44

SDSAA = 2% sucrose, 4% dextran, 3% sorbitol, 2% amino acid.

According to the virus titration performed on the supernatant, the amount of A1 (OH)₃ necessary for adsorption of Rotavirus out to be low (based on a freeze-dried dose 5.7 log) scaling up of virus titration:

<td>A1 (OH)₃</td><td>adsorption</td><td>titer in supernatant</td>

<td colspan="3"></td>

<td>12 mg</td><td>1 hour RT</td><td> 2,7</td>

<td>24 mg</td><td>1 hour RT</td><td> 3,4</td>

<td>48 mg</td><td>1 hour RT</td><td> 3,4</td>

<td>72 mg</td><td>1 hour RT</td><td> 2,0</td>

<td>96 mg</td><td>1 hour RT</td><td>below the detection limit</td>

<td colspan="3"></td>

<td>12 mg</td><td>over the night</td><td> 2,7</td>

<td>24 mg</td><td>over the night</td><td>below the detection limit</td>

<td>48 mg</td><td>over the night</td><td> 2,5</td>

<td colspan="3"></td>

<td>12 mg</td><td>immediately</td><td>below the detection limit</td>

<td>24 mg</td><td>immediately</td><td> 2,0</td>

<td>48 mg</td><td>immediately</td><td>below the detection limit</td>

Time required for adsorption of Rotavirus to A1 (OH)₃ appears to be short: A dose of freeze-dried Rotavirus was reconstituted in the presence of 24 mg A1 (OH)₃ and centrifuged after 0.15 and 60 minutes, and after 24 hours. Down-centrifuged material was resuspended in SDSAA before virus titration:

<td>Time</td><td>Centrifuged material</td><td>supernatant</td>

<td>0 min</td><td> 5,26</td><td> 3,17</td>

<td>15 min</td><td> 5,34</td><td> &lt;1,44</td>

<td>60 min</td><td> 5,96</td><td> &lt;1,44</td>

<td>24 hours</td><td> 6,13</td><td> &lt;1,44</td>

5.8: Use of CaCO₃ as antacid

To avoid aluminum in the vaccine, the antacid A1 (OH) was₃ replaced with another insoluble, ice inorganic salt: CaCO₃ (Calcium carbonate).

The observations with CaCO₃ corresponds to those described for A1 (OH)₃:

- Association between the Rotavirus and the inorganic salt;

- Maintaining Rotavirus activity in complex with the inorganic salt;

- Possible release of the Rotavirus from the complex by dissolving the inorganic salt with an acid;

- Possible simultaneous freeze-drying of the antacid and Rotavirus.

Complex between CaCOa and Rotavirus

In a first experiment, freeze-dried Rotavirus (virus titer 5.7) was reconstituted with an io suspension of CaCO₃ in water (50 mg in 1.5 ml) and then centrifuged and the virus titrated in the supernatant was compared with the titrated in the down-centrifuged material.

DRVC003A46 +

mg of CaCO₃ in

1.5 mlH₂0 +

centrifugation

8000 rpm. 10 min / \

Centrifuged Supernatant Material +

1.5 ml

SDSAA

5,83' 4,46

DRVC003A46 +

mg of CaCO₃ in

1.5 mlH₂0

4sentrifugering

8000 rpm. 10 min / \

Centrifuged Supernatant Material +

1.5 ml

Na Citrate

5,88 4,33

This indicates that more than 90% of the Rotavirus is associated with CaCO₃.

When the virus was present in the complex, it was also possible to perform the titration and recover the original virus amounts.

The virus titer is also somewhat higher than the titer obtained without CaCO₃.

DRVC003A46

1.5 mlH₂O +

centrifugation

8000 rpm. 10 min

<img file="NO328112B1_D0005.tif" />

DRVC003A46 +

1.5 ml WL Buffer

Centrifuged Supernatant Material

4,99 5,03

5,35

Amount of complex between CaCO₃ and Rotavirus

Freeze-dried Rotavirus was reconstituted with a CaCO₃suspension in water (1.5 ml): 10 mg mg

100 mg and then centrifuged, and the virus titrated in the supernatant was compared with the down-centrifuged material.

<td>CaCO₃</td><td colspan="2">Extempo + centrifugation</td><td colspan="2">1 hour + spin</td>

<td></td><td>centrifuged material</td><td>supernatant</td><td>centrifuged material</td><td>supernatant</td>

<td>100 mg</td><td> 4,57</td><td> 3,01</td><td> 4,79</td><td> 3,09</td>

<td>50 mg</td><td> 4,17</td><td> 4,15</td><td> 4,22</td><td> 3,86</td>

<td>10 mg</td><td> 3,17</td><td> 4,77</td><td> 3,87</td><td> 4,87</td>

Thus, it is obvious that the more CaCO 2, the more virus is associated and the less virus is present in the supernatant. However, the complete dose is not recovered (a total of at least 5.3, or even 5.8 as observed previously - see above).

Protection of Rotavirus with CaCOi during Baby Rosett-Rice antacid titration

Using 1 dose of freeze-dried Rotavirus (DRVC003A46) and 50 mg of CaCO₃, two types of baby Rossett-Rice titration were performed:

In a classic Rossett-Rice titration, the antacid is mixed with Rotavirus and HCl poured into this mixture.

In the inverse baby Rossett-Rice, the situation is the opposite: Antacid is added

HCl portion (as in vivo).

<td colspan="4">Classic baby Rossett-Rice titration</td>

<td>Freeze-dried Rotavirus stored by:</td><td>buffer</td><td>Theoretical virus titer</td><td>Measured virus titer</td>

<td>4 ° C</td><td>60 mg CaCl3</td><td> 5,3</td><td> 4,6</td>

<td>-80 ° C</td><td>60 mg CaCCb</td><td> 5,3</td><td> 4,6</td>

<td>4 ° C</td><td>24mgAl (OH)₃</td><td> 5,4</td><td> &lt;2,9</td>

<td>-80 ° C</td><td>24mgAl (OH) 3</td><td> 5,4</td><td> &lt;2,9</td>

<td colspan="4">Inverse baby Rossett-Rice titration</td>

<td>Freeze-dried Rotavirus stored by:</td><td>buffer</td><td>Theoretical virus titer</td><td>Measured virus titer</td>

<td>4 ° C</td><td>60mgCaCO₃</td><td> 5,3</td><td> 4,6</td>

<td>-80 ° C</td><td>60 mg CaCO₃</td><td> 5,3</td><td> 4,6</td>

<td>4 ° C</td><td>24mgAl (OH)₃</td><td> 5,4</td><td> &lt;2,9</td>

<td>-80 ° C</td><td>24mgAl (OH)₃</td><td> 5,4</td><td> &lt;2,9</td>

Thus, in this in vitro experiment, calcium carbonate can protect approximately 20% of the rotavirus from the presence of HCl while aluminum hydroxide does not protect.

5.9: Freeze-drying of Rotavirus in the presence of CaCO3 antacids:

<td>Serving no.</td><td>Preparation</td><td>Virus titer at time = zero after freeze drying</td><td>Virus titer after freeze-drying and 1 week at 37 ° C</td>

<td>99K08 / 01</td><td>sucrose: 2% dextran: 4% sorbitol: 3% amino acids: 2% CaCO₃: 50 mg</td><td> 10^5,28</td><td> 10^{3, lu}</td>

<td>99K08 / 02</td><td>sucrose: 2% dextran: 4% sorbitol: 3% amino acids: 2% CaCO₃: 60 mg</td><td>Two³³³</td><td> 10</td>

<td>00C24 / 01</td><td>sucrose: 2% dextran: 4% sorbitol: 3% amino acids: 2% CaCO₃: 60mg xanthan: 0.3%</td><td>Ίο³⁷⁵⁷</td><td> 10⁴’^6y</td>

<td>00C24 / 03</td><td>sucrose: 2% dextran: 4% sorbitol: 3% amino acids: 2% CaCO₃: 60 mg xanthan: 0.3%</td><td> 10 5,07</td><td> 10 ^w</td>

<td>00E09 / 25</td><td>sucrose: 2%, dextran: 4% sorbitol: 3% amino acids: 2% CaCO₃: 60mg xanthan: 0.25%</td><td> 10^w</td><td> 10^{4&gt; yi}</td>

<td>00E09 / 30</td><td>sucrose: 2% dextran: 4% sorbitol: 3% amino acids: 2% CaCO₃: 60 mg xanthan: 0.30%</td><td> 10^{5, ul}</td><td> 10</td>

<td>00F26 / 06</td><td>sucrose: 2% dextran: 4% sorbitol: 3% amino acids: 2% CaCO₃: 60 mg starch: 2%</td><td>ίο⁴·⁵⁰</td><td> 10^4JU</td>

This is all in one-freeze-drying of Rotavirus and Antacid (CaCO₃) together in the same vial. To prevent the precipitation of CaCO₃ during the filling step, viscosity agents are necessary. Examples of such viscosity agents include xanthan gum and starch. Rotavim activity is maintained even in the presence of xanthan gum and starch.

5.10: Freeze-dried tablets for rapid disintegration when placed in the mouth:

The subsequent preparations demonstrate the lyoc concept. That is, rapid dissolution of the freeze-dried mass in the mouth.

<td>Portion no.</td><td>Composition of preparations</td><td>Virus titer before freeze drying</td><td>Virus titer after freeze-drying and 1 week at 37 ° C</td>

<td>99B10 / 06</td><td>Sucrose: 4% Sodium Glutamate: 3.7% A1 (OH)₃ 48 mg</td><td> 10⁵’^{I 11}</td><td> 10+53</td>

<td>99C11 / 12</td><td>Maltitol: 3% A1 (OH)₃: 48 mg Hydroxypropylmethyl cellulose: 1%</td><td>ΙΟ⁴’¹⁶</td><td> 10 3·⁷⁹</td>

<td>Portion no.</td><td>Composition of preparations</td><td>Virus titer at time = zero after freeze drying</td><td>Virus titer after freeze-drying and 1 week at 37 ° C</td>

<td>00C24 / 05</td><td>Sucrose: 2% Dextran: 4% Sorbitol: 3% Amino Acids: 2% CaCO₃: 60 mg Xanthan: 0.3%</td><td> 10^5,02</td><td> 10^4,54</td>

<td>00C24 / 06</td><td>Sucrose: 2% Dextran: 4% Sorbitol: 3% Amino Acids: 2% CaCO₃: 60 mg Xanthan: 0.3%</td><td> 10^4,86</td><td> 10⁴’⁵⁶</td>

<td>00F26 / 11</td><td>Sucrose: 1% Dextran: 2% Sorbitol: 1.5% Amino Acids: 1% CaCO₃: 60 mg Starch: 2%</td><td> 10^4,70</td><td>10 mo</td>

In the lyoc concept, both xanthan and starch can be used (while maintaining the fast dissolving properties of the freeze-dried mass).

Example 6: Use of Calcium Carbonate as Antacid for Rotavirus Vaccine Preparation

If a suspension of CaCO₃ in water used as an antacid for Rotavirus, it is a problem that calcium carbonate particles settle rapidly upon addition of water, since the density of the virus is close to 2.6 and the average particle size is 30 µm. The sedimentation rate can be reduced by:

1. to increase the density of the surrounding medium

2. to increase the viscosity of the surrounding medium

3. to reduce the particle size

4. keeping the particles apart

6.1: Increased density in the surrounding medium:

If CaCO₃- the water suspension (when placed in the syringe) is placed on the freeze-dried mass (containing 2% sucrose, 4% dextran, 3% sorbitol and 2% amino acids), the density of the surrounding medium is increased, but the sedimentation rate of CaCO₃ is not very different from the speed of CaCO₃water suspension.

6.2: Increased viscosity in the surrounding medium:

Pseudoplastic excipients

A pseudoplastic solution is defined as a solution having higher viscosity upon standing, compared to the viscosity with stirring.

Common excipients of this type are:

natural polymers, for example:

gum arabic adragant gum agar-agar alginates pectins semisynthetic polymers, for example:

carboxymethyl cellulose (Tyloses C) methyl cellulose (Methocels A, Viscotrans MC, Tylose MH and MB) hydroxypropyl cellulose (Klucels) hydroxypropyl methyl cellulose (Methocels E and K, Viscotrans MPHC)

Generally, these pseudoplastic excipients are used with thixotropic agents.

Low flowability pseudoplastic excipients

At sufficiently high concentrations, these polymers give rise to a structural liquid arrangement which results in a solution with high viscosity and low flowability upon standing. A certain amount of energy must be supplied to the system to achieve flow and mass transfer.

External energy (stirring) is necessary for the temporary destruction of the structural fluid arrangement, in order to obtain a liquid liquid solution.

Examples of such polymers are Carbopols and xanthan gum.

Thixotropic excipients

With these excipients, a gene structure is obtained upon standing, while a liquid solution is obtained with stirring.

Examples of thixotropic excipients are Veegum (magnesium aluminum silicate) and Avicel RC (approximately 89% microcrystalline cellulose and 11% carboxymethyl cellulose Na).

6.3 Reduction of particle size

A reduction in the CaCCh particle size led to a reduction of the acid neutralizing ability of the compound.

6.4 Keeping the particles away from each other

This is the case for Veegum and Avicel, for which insoluble particles smaller (approximately 1 µm) than the CaCCh particles are placed between the CaCCh particles to prevent aggregation.

Example 7: Product Design

The following diagrams show examples of possible product designs.

7.1 CaCO 2 in the syringe

Since clinical portions of Rotavirus already exist in lyophilized ampoules, the antacid may be placed in the reconstitution fluid contained in the syringe.

1.3 ml syringe

CaCl 3 (60 mg / ml)

IN

cannula

<img file="NO328112B1_D0006.tif" />

sublimated

rotavirus

In this product design, sedimentation of CaCCh must be under control, not only during the filling step, but also during the complete shelf life of the product (at least 2 years).

7.2 CaCO 2 in the freeze-dried vial

Spray with

1.3 ml of water

cannula

<img file="NO328112B1_D0007.tif" />

Freeze-dried vial Rotavirus + CaCCh (60 mg) Xantan

7.3. Freeze drying in a plastic package

In this case, Rotavirus, CaCO3 and Xanthan gum are freeze-dried together, directly in the plastic package.

Example 8: Freeze Drying of Different Rotavirus Strains

<td>Portion no.</td><td>rotavirus Strain</td><td>Composition of preparations</td><td>Virus titer at t = zero after freeze-drying</td><td>Virus titer after freeze-drying and 1 week at 37 ° C</td>

<td>00F26 / 01</td><td>Gl SB purifn ° 61 PRO / 0232</td><td>Sucrose: 2% Dextran: 4% Sorbitol: 3% Amino Acids: 2%</td><td>io</td><td> 10^4&gt;/</td>

<td>00F26 / 02</td><td>G2 (DS-1)</td><td>Sucrose: 2% Dextran: 4% Sorbitol: 3% Amino Acids: 2%</td><td> 10^4,4</td><td> 10^4,4</td>

<td>00F26 / 03</td><td>G3 (P)</td><td>Sucrose: 2% Dextran: 4% Sorbitol: 3% Amino Acids: 2%</td><td> 10^4,6</td><td> 10^4,3</td>

<td>00F26 / 04</td><td>G4 (VA-70)</td><td>Sucrose: 2% Dextran: 4% Sorbitol: 3% Amino Acids: 2%</td><td> 10⁴¹⁸</td><td>10 M</td>

<td>00F26 / 05</td><td>G9 (W161)</td><td>Sucrose: 2% Dextran: 4% Sorbitol: 3% Amino Acids: 2%</td><td> 10 4.6</td><td> 10⁴¹³</td>

Strains DS-1, P and VA70 are described as reference strains of human Rotavirus for serotypes G2, G3 and G4, respectively, on page 1361 of Fields Raven press 1990.2. issue.^{15 * *}

In this experiment, various Rotavirus strains have been lyophilized.

For all, virus titer was preserved during freeze-drying, and accelerated stability (one week at ° C) has been shown.

Example 9: Phase I safety study in adults with oral administration of the Rotavirus vaccine.

A phase I study was performed to estimate the safety and reactivity of a single oral dose of 1O^6,0 ffu of the P43 vaccine in healthy adults 18 to 45 years of age.

Three clinical trials were double blind and randomized. It was placebo controlled and independent. The study was conducted in a single center in Belgium.

Volunteers

A total of 33 individuals, 11 in the placebo group, and 22 in the vaccine group were included, and all completed the study. All volunteers were Europeans. The average age at the time of vaccination was 35.3 years, within the limits of 18 to 44 years. The trial began in January and lasted for just over a month.

Materials

Vaccine

Clinical portions of the P43 vaccine were prepared, purified, formulated and lyophilized according to Good Manufacturing Practices. The portions were approved by Quality Control and Quality Assurance. Each vaccine ampoule contained the following ingredients:

Active ingredient:

Strain P43 Min. 10⁵’⁸ ffu

Excipients, stabilizers:

sucrose

dextran

sorbitol

Amino acids mg mg

13.5 mg mg

placebo

Placebo ampoules were prepared and distributed. Each placebo pill contained the following ingredients:

Excipients, stabilizers:

<td>sucrose dextran sorbitol Amino acids</td><td>9 mg 18 mg 13.5 mg 9 mg</td>

Thinner

Injection water was used as a diluent for vaccine and placebo reconstitution.

supply

Approximately 10 to 15 minutes before administration of vaccine or placebo, participants in both groups were given 10 ml of Mylanta orally. Mylanta is a registered antacid. The antacid increases the pH of the stomach and prevents inactivation of the Rotavirus during passage through the stomach.

To prepare the vaccine, two vials of freeze-dried P43 containing 10 were added⁵’⁸ ffu pr. vial reconstituted with 1.5 ml of diluent for injection water. This gave a calculated virus titer of 10⁶’¹ ffu pr. dose. The reconstituted vaccine was administered immediately as a single oral dose.

For the preparation of the placebo preparation, two vials of freeze-dried placebo were reconstituted with 1.5 ml of injection water and administered orally as a single dose.

Safety and reactivity

The following safety and reactivity criteria applied:

General symptoms that were requested were fever, diarrhea, vomiting, nausea, abdominal pain and lack of appetite. These were noted within 8 days of administration. Unexpected symptoms were noted within 30 days of administration.

Serious adverse events were noted throughout the trial period.

Diarrhea samples should be collected for less than 8 days after administration.

The results were:

No expected symptoms, no unexpected symptoms and no serious adverse events were reported during the specified observation periods.

No cases of diarrhea were reported.

conclusions

SB Biological's P43 vaccine was safe relative to the placebo by double-blind oral administration as a single dose of 10⁶’¹ ffu to healthy, grow volunteers between 18 and 44 years.