Vaccine combinations

Abstract

Vaccine combinations which comprise at least two or more of the following antigens: DTap-HEV-HepB-HPV suitable for administration in humans. A number of variations in the combination of these antigens have been disclosed that is suitable for concomitant administration. The methods of preparing the vaccine combinations are disclosed. Nucleic acids encoding the antigens, as well as methods for their production and use are provided.

Claims

1. A combination vaccine composition comprising: (i) a diphtheria toxoid, D; (ii) a tetanus toxoid, T; (iii) pertussis antigens aP; (iv) recombinant human papillomavirus L1 antigens of HPV16 and HPV18; and (v) a chimeric fusion protein comprising an HPV L2 epitope in fusion with Hepatitis E ORF2 (HEV ORF2) protein wherein the chimeric fusion protein is expressed as virus like particles.

2. The combination vaccine composition according to claim 1, wherein the HPV L2 epitope is fused at the N-terminus of HEV ORF2 protein.

3. The combination vaccine composition according to claim 2, wherein the amino acid sequence of the chimeric fusion protein is set forth in SEQ ID NO: 6 or SEQ ID NO: 10.

4. The combination vaccine composition according to claim 2, wherein the nucleotide sequence encoding the chimeric fusion protein is set forth in SEQ ID NO: 5 or SEQ ID NO: 9.

5. The combination vaccine composition according to claim 1, wherein the HPV L2 epitope is fused at the C-terminus end of HEV ORF2 protein.

6. The combination vaccine composition according to claim 5, wherein the amino acid sequence of the chimeric fusion protein is set forth in SEQ ID NO: 8 or SEQ ID NO: 12.

7. The combination vaccine composition according to claim 5, wherein the nucleotide sequence encoding the chimeric fusion protein is set forth in SEQ ID NO: 7 or SEQ ID NO: 11.

8. The combination vaccine composition according to claim 1, wherein the chimeric fusion protein comprises SEQ ID NO: 6, wherein SEQ ID NO: 6 is encoded by SEQ ID NO: 5 that is codon optimized for enhanced expression as a virus like particle in Pichia pastoris.

9. The combination vaccine composition according to claim 1, wherein the chimeric fusion protein comprises SEQ ID NO: 8, wherein SEQ ID NO: 8 is encoded by SEQ ID NO: 7 that is codon optimized for enhanced expression as a virus like particle in Pichia pastoris.

10. The combination vaccine composition according to claim 1, wherein the chimeric fusion protein comprises SEQ ID NO: 10, wherein SEQ ID NO: 10 is encoded by SEQ ID NO: 9 that is codon optimized for enhanced expression as a virus like particle in Pichia pastoris.

11. The combination vaccine composition according to claim 1, wherein the chimeric fusion protein comprises SEQ ID NO: 12, wherein SEQ ID NO: 12 is encoded by SEQ ID NO: 11 that is codon optimized for enhanced expression as a virus like particle in Pichia pastoris.

12. The combination vaccine composition according to claim 1, wherein the recombinant antigens are cloned, expressed and purified from yeast cells Pichia pastoris.

13. The combination vaccine composition according to claim 1, further comprising an adjuvant.

14. The combination vaccine composition according to claim 13, wherein the adjuvant is alum, selected from aluminum hydroxide or aluminum phosphate, and gamma inulin and algammulin.

15. A kit comprising the combination vaccine composition according to claim 1.

16. A kit comprising the combination vaccine composition according to claim 1, wherein DTaP is formulated in aluminum hydroxide.

17. A kit comprising the combination vaccine composition according to claim 1, wherein, the kit comprises: (a) a first component comprising HEV, HPV and chimeric HEV or HBsAg antigens; and (b) a second component comprising DTaP; wherein contents of the first component and the second component are mixed prior to administration.

18. The combination vaccine according to claim 1, further comprising a recombinant HEV ORF 2 antigen.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

(2) FIG. 1: PCR amplification of (1A) approximately 1.494 Kb of HEV ORF2 gene (1B) 110 bp (that includes the gene sequence, restriction enzyme sequence and the 5 overhang) corresponding to the amino acid SATQLYKTCKQAGTCPPDIIPKVEG of HPV16 L2 and 60 bp (that includes the gene sequence, restriction sequence and the 5 overhang) corresponding to the amino acid sequence LVEETSFIDAGAP of the HPV L2 epitopes by PCR using gene specific primers described in Example 1. The size of the amplified gene fragment is shown against the 1 Kb ladder fore HEV ORF2 and against 100 bp ladder for HPVL2 fragment.

(3) FIG. 2: 2A represents Expression of HEV ORF2 of SEQ ID NO. 4 in Pichia pastoris at 24 hrs and 120 hrs after induction (I) with methanol showing the expression of 54 kDa protein; UIuninduced cells; 2B represents Expression of HEV-HPVL2 of SEQ ID NO. 6 in Pichia pastoris at 48 hrs and 120 hrs after induction (I) with methanol showing the expression of the 56 kD protein; UIuninduced cells; Mis the medium range molecular size marker (Genei, Bangalore).

(4) FIG. 3: 3A represents Purified recombinant 54 kD HEV ORF2 protein of SEQ ID NO.4 indicated by thick arrow; and 3B represents the purified recombinant chimeric 56 kD HEV-HPV16 L2 protein is indicated by thick arrow. The size of the molecular size markers is shown.

(5) FIG. 4: Represents Transmission electron microscopy (TEM) of the chimeric HEV-HPV16L2 protein stained with uranyl acetate shows the assembly of the protein spontaneously into virus like particles. The size of the bar is 200 nm as shown in the bottom of the picture.

DETAILED DESCRIPTION OF THE INVENTION

(6) Detailed embodiments of the present invention are disclosed herein below. However, it is to be understood that the disclosed embodiments are merely exemplary of the invention, which can be embodied in various forms. The scope of the invention is not limited to the disclosed embodiments and terms and phrases used herein are not intended to be limiting but rather to provide an understandable description of the invention. The invention is defined by claims appended hereto.

(7) The invention relates to vaccine composition that includes a combination of antigens of human papillomavirus (HPV) and hepatitis E virus (HEV) for the prophylaxis and diagnosis of HPV and Hepatitis E virus infections in mammals, particularly humans. Addition of Hepatitis B antigen to the above combination is included in the scope of the invention. Furthermore, the combination of tetanus toxoid and/or Tdap (tetanus toxoid, reduced diphtheria toxoid, and acellular pertussis) vaccine to the aforementioned vaccine combination is also within the scope of the invention. Such vaccine compositions are administered by mixing the antigens extemporaneously in a single container before administering to a host as an injection, or can be concomitantly administered in humans in two or more separate formulations that can be injected parenterally at different sites for eliciting immune response. A combination vaccine of the aforementioned antigens is supplied in a single container such as a vaccine vial or in a pre-filled syringe ready for injection. The combination of the aforementioned vaccines with other viral and bacterial antigens is also within the scope of the invention.

(8) The invention also provides a process for preparing a combination vaccine that comprises (i) a hepatitis E (HEV) virus like particle, (ii) a chimeric protein of hepatitis B virus surface antigen HbsAg with HPV L2 protein (HbsAg-HPVL2), and/or chimeric HEV-HPV L2 chimeric protein (iii) HPV16 L1 and HPV18 L1 antigens (iv) tetanus toxoid (T), (iii) acellular pertussis antigens (aP), (vi) diphtheria toxoid (D) and the process includes the steps of (a) combining the HEV component to the chimeric HEV-HPVL2 component to get a bivalent component (b) combining the HEV antigen to the chimeric HbsAg-HPVL2 to get a bivalent component (a) combining the HEV component to HPV16 L1 and HPV18 L1 components to get a trivalent HEV-HPV16L1-HPV18L1 component (a) combining a trivalent HEV-HPV16L1-HPV18L1 component with a monovalent chimeric HEV-L2 component, to get a tetravalent HEV-HPV16L1-HPV18L1-HEV-L2 component; (b) combining a trivalent HEV-HPV16L1-HPV18L1 component with chimeric HbsAg-HPVL2 to get a tetravalent HEV-HPV16L1-HPV18L1-HbsAg-L2 component and (c) mixing the D-T-aP-component with either of the bivalent/trivalent/tetravalent components for a combination vaccine/or concomitantly administering the D-T-aP vaccine along with the viral vaccines parenterally to a mammalian host.

(9) The HPV capsid antigens of the combination vaccine are highly immunogenic when expressed as recombinant proteins in a eukaryotic expression system and are purified therefrom. The HPV antigens are derived from the major capsid protein L1 that is expressed as virus like particles (VLPs). HPV antigens include two or more antigens selected from a list of oncogenic HPV types including HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68 etc and could include the HPV L2 capsid protein from any of the above mentioned HPV types Chimeric protein of HPV L2 capsid and HbsAg is a good vaccine candidate for use in combination vaccine with HEV and other HPV antigens. Additions of heterologous viral and bacterial epitopes that are fused to the aforementioned vaccine antigens also augment the immunogenicity of the HPV vaccine.

(10) The hepatitis E vaccine antigens are those encompassing the full length or the truncated sequence of Open Reading Frame 2 (ORF2) of protein which is a structural protein of the Hepatitis E virus. Truncations of the HEV full length ORF2 protein were made to result in better expression with increased stability and without compromising the immunogenicity of the candidate antigen to assemble into VLPs. Truncated sequences of the HEV ORF2 such as 112-608 amino acid region and alternatively 368-606 amino acids were so designed to accommodate heterologous viral sequences without compromising the structural conformity required for eliciting strong immune response. From the full length sequences of HEV ORF2 protein such as in SEQ ID NO.1 and the full length protein of HPV16 L2 such as in SEQ ID NO. 2, different truncated gene fragments that encompass the major neutralizing epitopes of both the antigens were generated by PCR and several chimeric constructs were made by ligating the fragments together. The HEV gene sequences can be derived from any HEV genotype such as genotypes 1-4. The different sequences of the chimeric antigens are within the scope of the invention. Such constructs code for the chimeric proteins of different molecular sizes. No antigenic interference was observed with the combination of aforementioned antigens. It was found that the vaccines of the invention can be prepared extemporaneously at the time of use by mixing together two components: (a) a first component comprising HEV, HPV and chimeric HEV or HBsAg antigens; and (b) a second component comprising Tdap. The two components are filled separately, and thus in general the invention provides a kit comprising: (a) a first component comprising HEV, HPV and chimeric HEV or HBsAg antigens; and (b) a second component comprising Tdap. Alternatively Tdap can be formulated in aluminum hydroxide and used as a single component vaccine along with the viral antigens. If packaged separately, the mixing step will typically take place at the time of use. Thus the invention provides a process for preparing a vaccine composition of the invention, comprising the steps of (a) providing a first component comprising HEV, HPV and chimeric HEV or HBsAg antigens and (b) providing a second component comprising Tdap antigens (c) mixing the first and second components to give the vaccine.

(11) With the aforementioned combination vaccine it was possible, to obtain antibody titers against HPV and HEV virus approaching or equivalent to, or in excess of 100% of the titer obtained when the antigen is administered in isolation to animals. The combination vaccine elicited robust antibody levels against both HPV and HEV virus infections and no antigenic interference was observed. All the methods described in the invention are applicable to any genotype or genotypic variants/serotypes/strains of HPV and Hepatitis E virus. Within the scope of the invention are the compositions and methods of preparing and using the HPV and HEV antigens of defined sequence expressed as recombinant proteins or recombinant virus like particles in any prokaryotic or eukaryotic expression system. The gene sequences are the native viral sequences or synthetic genes that are codon optimized for expression in the host cells. The eukaryotic expression system of choice includes mammalian cells; baculovirus mediated expression in insect cells, and yeast cells of any species, most preferably Pichia pastoris or Saccharomyces cerevisiae. Pichia pastoris was found to express the aforementioned antigens to a high level. Optimization of the sequence for eukaryotic expression includes the presence of Kozak's consensus sequence such as (gcc)gccRccATGG at the 5 end of the gene, where the ATG is the initiation codon, or the use of yeast consensus sequence (5-[A/T]TTTAT[A/G]TTT[A/T]-3). For expression in Pichia pastoris, the synthetic gene sequences were codon optimized to further increase the expression levels. The aforementioned proteins were expressed either as intracellular protein and purified therefrom. Alternatively they can be expressed as secretary proteins and purified. Any suitable expression vector is selected from the list that includes but is not limited to pPIC3.5, pPIC3.5K, pA018, pPIC9, pPIC9K, PHIL D2 etc. for expression in yeast cells. Any plasmid vector suitable for expression in Saccharomyces cerevisiae or any other species of yeast are used. The yeast cells include the following but are not limited to Pichia pastoris, Saccharomyces spp, Hanensula polymorpha, Schizosaccharomyces spp, kluveromyces spp. Either of the Pichia pastoris strains such as GS115, KM71 and protease deficient strains such as SMD1168 or SMD1163 is used for recombinant expression. The aforementioned genes are cloned either in single copy or in more than one copy. The antibodies against the HPV and HEV virus antigens are used for developing methods for estimation of antigen content by ELISA and also for use as a reference standard while estimating vaccine antibody titer. They find application in diagnosis also.

(12) Purification of the virus was achieved by physical or chemical means and preferably by a combination of both. Physical methods utilize the physical properties of the virus like particles such as density, size, mass, sedimentation coefficient etc. and include any of the following techniques but are not limited to: ultracentrifugation, density gradient centrifugation, ultrafiltration etc. Purification through chemical means employs methods such as adsorption/desorption through chemical or physiochemical reactions such as ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, gel filtration chromatography, hydroxyapatite matrix, salting with inorganic salts one such example being ammonium sulphate, and by the use of proprietary Himax technology, organic salts, organic solvents, aluminum phosphate, aluminum hydroxide and organic compounds such as polyethylene glycol. Purification of the recombinant antigens of the virus was achieved by either one or a combination of two or more of the above mentioned methods.

(13) The antigenic compositions of the above mentioned HPV-HEV vaccine and HPV-HEV and tetanus toxoid and Tdap vaccines were formulated in pharmaceutically acceptable carrier that is suitable for immunization in human. Furthermore, for adjuvanted vaccine formulations, suitable adjuvants were selected from the following list, which includes but is not limited to: aluminum hydroxide, aluminum phosphate; calcium phosphate; inulin of any polymorphic form, preferably gamma inulin; adjuvants containing inulin in combination with other organic and inorganic compounds such as aluminum hydroxide, aluminum phosphate, aluminum sulphate phosphate and calcium phosphate; liposomes, chitosan and complex carbohydrates such as dextran, dextrins, starch, mannans and glucomannans, galactomannans, beta-glucans, heparin, cellulose, pectins and pectinates, lectins and any other carbohydrates either synthetic or derived from any source, any biodegradable and biocompatible polymers, such as poly lactide and poly(lactide co-glycolides; PLG) or PLGA; any emulsions including but not limited to oil in water emulsions, other squalene based adjuvants, oil in water emulsion containing cholecalciferol, cholecalciferol by itself as an adjuvant, any water in oil emulsion; liposomes prepared with cholecalciferol as one of the ingredients along with other lipid soluble compounds; liposomes of other compositions; RIBI adjuvant systems, saponins including but not limited to QS-21, QuilA, tomatine, ISCOMs, ISCOMATRIX etc, lipopeptides, glycopeptides, lipopolysaccharides, muramyl dipeptides and any peptide based adjuvants, oligonucleotides, any TLR ligands as adjuvants, any cytokine, vitamins and non-toxic bacterial toxins etc. In addition to the above, any other organic and inorganic substances that have good immunopotentiating activity can be used as adjuvants either singly or in combinations to enhance the immunogenicity of the HPV-HEV combination vaccines. The vaccine combinations with alum elicited strong immunological response when administered parenterally, in animals. The methods and vaccine compositions containing vaccine adjuvants are within the scope of the invention.

(14) For potency testing of the vaccine, the vaccine formulations were tested in mice and guinea pigs. The resultant serum was assayed in vitro by estimation of antibody titer by ELISA. Toxin neutralization test was used for evaluation of potency of tetanus toxoid and diphtheria toxoid by procedures well described in literature. Seroconversion was observed in the animals immunized with the vaccine formulations described in the present invention. No antigenic interference either due to combination of different antigens or by adjuvant was observed. The buffer used in the formulations is phosphate buffer Phosphate-citrate buffer or any other pharmaceutically acceptable buffer of appropriate pH can be used. 2-phenoxyethanol was used vaccine preservative; thiomerosol or any other vaccine preservative can be used. Alternatively the vaccine combination can be formulated without any vaccine preservative. The vaccines optionally contain stabilizer(s) etc. The excipients were selected from a list that includes but is not limited to non-reducing sugars, sugar alcohols such sorbitol and mannitol, glycerol, amino acids, human serum albumin, and a choice of adjuvant from the aforementioned list of adjuvants. A stable formulation of the immunogen either in a liquid or in a lyophilized form and after reconstitution in a pharmaceutically acceptable buffer or water is suitable for administration parenterally in human host. More specifically, for antigens of the present invention, a diagnostic assay with high sensitivity and specificity for detecting infection are provided. The methods in the scope of the invention are applicable to any genotype/genotypic variants/serotype/strain of HPV and hepatitis E viruses.

EXAMPLES

(15) The invention is further described in following examples.

Example-1: Cloning and Recombinant Expression of Hepatitis E (HEV) Viral and Chimeric HEV-HPVL2 Antigens

(16) A synthetic gene of the Hepatitis E ORF2 gene of amino acid sequence of SEQ ID NO.1 was amplified by PCR with 5primer and 3 primer containing EcoR1 and Not1 restriction sites respectively to obtain a 1494 bp PCR fragment of SEQ ID NO.3 that encodes the protein of SEQ ID NO. 4. (see FIG. 1). The primers used for PCR amplification of the HEV gene are 5-CATTTGAATTCACCATGGCCGTTGCACCTGCTCATGATAC-3 and 5-TATCGCGGCCGCTCATTAAGCCAATGCAGAATGAGGAGC-3.

(17) The conditions for PCR amplification were denaturation at 94 C. for 45 seconds, annealing at 60 C. for 40 seconds and extension at 72 C. for 2.5 minutes for 30 cycles followed by final extension at 72 C. for 10 minutes. The reaction mix consisted of 1 Taq DNA polymerase buffer, 0.25 mM dNTPS, 20 picomoles of each primer and 1 U of Taq DNA polymerase (Genei) and 1 U of Pfu DNA polymerase (New England Biolabs). The PCR fragment was digested with EcoR1 and Not1, gel purified and used for ligation to EcoR1 and Not1 digested yeast vector pPIC3.5K (Invitrogen, Carlsbad, USA). The 1494 bp PCR fragment was used for the chimeric fusion with gene sequence for HPV16 L2 epitopes consisting of 14-37 amino acids of the sequence SATQLYKTCKQAGTCPPDIIPKVEG and/or 108-120 amino acids of the sequence LVEETSFIDAGAP of the HPV16 L2 protein of SEQ ID NO 2 Chimeric fusion of the gene sequence for HPV16 L2 epitope 14-37 amino acids generated SEQ ID NO. 5 encoding SEQ ID NO.6 when ligated to the 5end of HEV gene, and SEQ ID NO.7 encoding SEQ ID NO.8 when it was ligated to the 3 end of the of the HEV ORF2 PCR fragment. The ligation was achieved with a BamH1 site located at either end of the primers for HEV or HPV L2 sequence. The following primers were used for amplification of 14-37 amino acids of the HPV16 L2 sequence for fusion at the N-terminus of the HEV ORF2 fragment: 5-ATGTCGAATTCACCATGGCTGCAACTCAGTTGTATAAAAC-3 and 5-ATCGAGGATCCTTGAACCTTTGGGATAATGTC-3. For amplification and fusion at the C-terminus of the HEV ORF2 fragment, the following primers were used for HPV16 L2 PCR of the above region: 5-ATGTCGGATCCTCTGCAACTCAGTTGTATAAAAC-3 and 5-ATCGAGCGGCCGCTCATTAACCTTCAACCTTTGGGATAATGTC-3. PCR primers that were designed to amplify the 5 TTGGTTGAAGAAACCTCCTTTATTGACGCTGGTGCTCCA-3 region of HPV16 L2 gene corresponding to the protein sequence LVEETSFIDAGAP also contained a BamH1 site for ligation of the two gene fragments. Chimeric fusion of the gene fragment encoding LVEETSFIDAGAP to the C-terminus of HEV ORF2 resulted in SEQ ID NO. 9 encoding SEQ ID NO.10. The SEQ ID NO.11 is nucleotide sequence of HEV ORF2 with HPV L2 14-37 amino acid region at the N-terminus and HPV16 L2 108-120 amino acids at the C-terminus. The corresponding protein sequence is SEQ ID NO.12. Similarly, for generating chimeric constructs of HEV ORF2 with the aforementioned HPV L2 sequences, PCR primers for amplification of the 1494 bp fragment also included a BamH1 site either at the end of the 5 region or at the 3end of the gene fragment to facilitate ligation to the HPV L2 epitopes. Alternatively construction of chimeric constructs of Hepatitis E with HPV L2 protein, a 714 bp fragment (from 368-606 amino acids) of ORF2 was amplified by PCR and ligated to HPV16 L2 epitopes (region 14-37 amino acids and/or 108-120 amino acids) either at the N-terminus or at the C-terminus of the HEV PCR fragment. For ligation at the N-terminal end, the HEV fragment was amplified with the primer 5-ACGTCGGATCCATCGCCCTTACATTGTTTAATTTG-3 and 5-TATCGCGGCCGCTCATTAAGCCAATGCAGAATGAGGAGC-3 and for ligation at the C-terminal end of the HEV fragment, the HEV gene was amplified with 5-AGCTCGAATTCACCATGGCCCTTACATTGTTTAATTTG-3 and 5-ACTCAGGATCCTTATCAAGCCAATGCAGAATGAGGAGC-3. The conditions for PCR amplification were denaturation at 94 C. for 45 seconds, annealing at 56 C.-62 C. for 40 seconds for various primer combinations and extension at 72 C. for 2 minutes for 30 cycles followed by final extension at 72 C. for 10 minutes with reagents of the same concentration as mentioned above for 1494 bp HEV ORF2 amplification. For the generation of the chimeric genes of HPV L2 and HEV, the PCR amplified genes were ligated after BamH1 digestion, further digested with EcoR1 and Not1 and ligated into the EcoR1 and Not1 digested pPIC3.5K yeast vector and transformed in E. coli DH5. The recombinant plasmid DNA was isolated from transformed E. coli selected on 50 g/ml ampicillin. The recombinant plasmid containing the HEV-HPVL2 chimeric gene was linearized with BglII and transformed in Pichia pastoris GS115 cells as per the standard yeast transformation protocols (Invitrogen, Carlsbad, USA).

Example-2: Purification of the HEV and HEV-HPV L2 Chimeric Proteins

(18) The Pichia pastoris GS115 clones expressing the recombinant proteins of HEV and the HEV-L2 chimeric proteins were grown for 96 hours at 280 C and induced with 1% methanol every 24 hours and harvested at the end of 120 hours (FIG. 2). The cells were washed once with 50 mM Tris-HCl buffer pH 8.0 containing 2 mM EDTA, 10 mM NaCl, and lysed in the same buffer containing 0.05% Triton X-100. The cell lysate was centrifuged at 4000 rpm for 20 minutes and the cell supernatant containing the proteins were purified on a Q-sepharose column equilibrated with the same buffer. The proteins were eluted with a salt gradient containing 50 mM to 750 mM NaCl in the same buffer and the fractions containing the protein were pooled and checked on SDS-PAGE gel for the presence of 54 kDa band for the HEV protein and 56 kDa band for the chimeric HEV-HPV16 L2 protein. The fractions containing HEV proteins were concentrated using TFF system and the concentrate was loaded on Captocore700 gel filtration column equilibrated with 50 mM phosphate buffer, pH 7.2 containing 154 mM NaCl. The viruses like particles were collected in the flow through. The purity of the protein was checked on SDS-PAGE gel and found to be more than 95% pure (FIG. 3). The purified proteins were further characterized.

Example-3: Transmission Electron Microscopy

(19) The purified viruses like particles (VLPs) were visualized by Transmission Electron microscopy (TEM) after negative staining with 2% uranyl acetate and is depicted in FIG. 4.

Example-4: Vaccine Formulations for Potency Testing

(20) For potency testing, the following vaccine combinations each consisting of the purified vaccine antigen of the below mentioned concentrations and adjuvant(s) were prepared for immunization in mice: a) 30 g of HEV ORF2 54 kDa protein, 40 g HPV16 L1 protein, 20 g HPV18 L1 protein, and 30 g HbsAg-HPV L2/HEV-HPV L2 chimeric antigen all adsorbed on 1.5 mg of aluminum hydroxide and Tdap (tetanus toxoid, reduced diphtheria toxin and acellular pertussis) comprising formaldehyde detoxified 2.5 Lf units of diphtheria toxin (DT), 5 Lf units of tetanus toxoid (TT) and 8 g of detoxified Pertussis toxin (PT), 8 g filamentous hemagglutinin (FHA) and 2.5 g of pertactin (PRN) all adsorbed individually on aluminum hydroxide. Alternatively aluminum phosphate at 1.5 mg can be used for the adsorption of all the above antigens except tetanus toxoid that is adsorbed in aluminum hydroxide. The tetanus toxoid (TT) and detoxified Pertussis toxin (PT) are GMP products obtained from the production facility of Bharat Biotech Intl Ltd. The acellular pertussis antigens were purified from the culture supernatant of Bordetella pertussis though steps involving concentration of the culture supernatant, salt fractionation, diafiltration, column chromatography and buffer exchanged into 1PBS, pH 7.2. The HPV16 and HPV18 L1 antigens are recombinant proteins expressed and purified from Pichia pastoris cells. The method of production of recombinant HPV proteins and HbsAg-HPV16 L2 is previously described in WO2010/001409. Addition of other adjuvants further enhanced the potency of the vaccine preparation. Addition of polymorphic forms of inulin, particularly the gamma inulin enhanced the potency of the vaccines. Gamma inulin comprises particles with a molecular weight of above 8000 daltons and is virtually insoluble in water at 37 C. The high molecular weight inulin OraftiHPX was obtained from Beneo Orafti, Belgium. Gamma inulin fractions were prepared by the methods outlined in the prior art (U.S. Pat. No. 4,954,622; PCT/AU86/00311) and the preparation of algammulin was as per the methods described by Cooper and Steele, 1991. The gamma inulin and algammulin preparations at a concentration of 2 mg/ml were used in the vaccine formulations. The vaccine antigens were mixed with adjuvants for two hours at ambient temperature and used immediately for injections. Similar formulation was also tested with phosphate-citrate buffer of pH 6.8-7.2 with adverse no effect on stability. Adsorption of the vaccine antigens to alum was tested by centrifuging the formulation and supernatant was checked by SDS-PAGE for protein bands of expected size for different antigens. All the aforementioned antigens adsorbed efficiently to alum. The vaccine formulations were tested in mice for potency.

Example-5: Potency Testing of the Vaccine Formulations

(21) Six Balb/c mice were used per group for testing different viral antigen combinations described in Example 4. The highest vaccine antigen concentration is described in Example 4. Four two-fold dilutions of the vaccine formulations were injected intramuscularly in 6-8 week old mice of 18-20 g body weight. The dilutions were made in 1PBS (50 mM phosphate buffer, pH 7.2 containing 150 mM NaCl) and containing the appropriate amount of adjuvant in order to maintain a constant adjuvant concentration. 2-phenoxyethanol was used as a vaccine preservative. For control animals, equal volume of buffer containing the same quantity of adjuvant was injected without the viral antigens. A booster injection was given on day 14 after the administration of the first vaccine dose. Blood was collected from retro-orbital sinus 7 days after administration of the booster dose, serum separated, heat inactivated at 560 C for 30 minutes and stored frozen at 800 C until used for serological assays. For testing the potency of diphtheria toxin, guinea pigs were used as described below in Example 6.

Example-6: Determination of Antibody Titer by ELISA and by Toxin Neutralization Assay

(22) For estimation of the antibody titer to HEV, and the HEV chimeric antigen with HPV16 L2, the HEV antibodies were estimated by indirect ELISA. About 1 g of the 54 kD vaccine antigen was coated on 96-well plate in carbonate buffer, pH 9.6 and incubated overnight at 2-8 C. Similarly for estimation of antibody titer to HPV16 and HPV18 L1 antigens and the HPV16 L2 epitopes, 1 g of the purified HPV16 L1 and HPV18 L1 and HPV16 L2 antigens respectively were coated in 96-well plates in carbonate buffer, pH 9.6 incubated overnight at 2-8 C. The rest of the procedure for the above HEV and HPV antigens are same as described below. After discarding the plate contents, the wells were blocked with 3% skimmed milk powder in phosphate buffered saline, pH 7.4. The plates were incubated at 37 C. for one hour and were washed with the washing buffer containing 0.05% Tween-20 in PBS (PBST) five times. Serial two fold dilutions of the test sera were added and the plates were incubated at 37 C. for one and half hours. The plates were washed eight times with the wash buffer. The mouse anti-IgG HRPO conjugate was added at 1:2500 dilution and incubated at 37 C. for one hour. The wells were washed five times with wash buffer and three times with PBS and the color developed by adding OPD (O-phenylenediamine, Sigma Aldrich, USA) and H.sub.2O.sub.2. The readings were taken 10 min after the addition of 1N sulfuric acid. Antibody titer as a measure of seroconversion in animals was estimated as the reciprocal of the serum dilution that is above that of control animal+3 standard deviation of the control value. Antibody titers to HbsAg were estimated using the Axsym Ausab kit (Abbot Laboratories) as per the kit protocol provided by the manufacturer. A titer of 10 mIU/mL was considered as the cut-off for seroconversion. The antibody concentrations were expressed as mIU/mL. Diptheria antitoxins were estimated by the toxin neutralization test (TNT) in guinea pigs. Six animals were used per test group, control group and for the reference standard group. The reference standards were obtained from NIBSC. The dilutions of the vaccine and the reference standard were injected subcutaneously into the guinea pig and the control group received equal volume of the vehicle containing aluminum hydroxide. After four weeks the vaccinated animals were challenged with 100 LD50 of the diphtheria toxin. The control group was challenged with 1 LD50 of the toxin. The animals were observed for mortality in the different dilution groups and scored against the reference standard group. The cut off for potency was 30 IU/dose. All the animals seroconverted for the diphtheria toxin. The tetanus toxoid potency was assayed in Balb/c mice. Six animals were included in each test, reference standard and the control group. Four two-fold serial dilutions of the TT were injected in a volume of 0.5 ml subcutaneously in each mouse. After four weeks the vaccinated animals (both the test vaccine and the reference standard groups) were challenged with 100 LD50 of tetanus toxin in a volume of 0.5 ml. Control group were challenged with 1/50 to 1/200 dilution of the tetanus toxin. The animals were observed for mortality. The potency of the results was expressed in IU/ml (Table 1). The cut off for potency is 60 IU/dose. All the animals that were concomitantly administered the vaccines seroconverted and no antigenic interference of the vaccines antigens or alum was observed. The potency of the acellular pertussis components were assayed by ELISA against the antibody titers elicited by the detoxified pertussis toxin using reference standard from NIBSC. Antibody titer against pertactin proteins were also determined

(23) TABLE-US-00001 TABLE 1 Potency testing of concomitantly administered vaccine antigens antibody titer (average Method for Vaccine antigens Animals for testing % seroconversion of 6 animals) antibody assay HEV + HbsAg-HPVL2 Mice, Balb/c 100% Anti-HbsAg (as ELISA, (Axsym (or HEV-HPVL2)@ + Mice, Balb/c HbsAg-L2) = 32 mIU/ml Ausab, Abbot HPV16 L1 + HPV18 Guinea pigs Laboratories) L1 + Tdap Anti HEV = 128,000* in-house indirect Tdap components: (measures HEV ELISA Tetanus antibody to both HEV Diptheria and HEV-L2 antigens) Acellular pertussis Anti- in-house indirect antigens: HPV16L1 = 64,000* ELISA detoxified pertussis Anti- in-house indirect toxin HPV18L1 = 128,000* ELISA Pertactin Anti-HPV16 L2 = in-house indirect FHA 12,800* (measures L2 ELISA antibody to both HEV- L2 and/or HbsAg-L2 antigens. Tetanus antitoxin = 75 IU/ml Toxin neutralization test Diptheria antitoxin = Toxin 45 IU/ml neutralization test Detox pertussis in-house ELISA antitoxin = 12800* Anti-pertactin = 6400* in-house ELISA Anti-FHA not determined *Antibody titer was estimated as the reciprocal of the serum dilution that is above that of control animal + 3 standard deviation of the control value. @administration of either of the HbsAg-L2 or HEV-L2 elicited immune response against L2 epitope without interference from other antigens.

REFERENCES

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