DESC:
A combination of vaccine formulation against human papillomavirus and hepatitis E virus infection

Field of the Invention
A present invention relates to vaccine formulation that comprises human papillomavirus (HPV) virus like particles in combination with hepatitis E virus (HEV) antigen.

Background of the invention
HPV, or human papillomavirus, is a human DNA virus of the papillomavirus family of viruses. Similar to other papillomaviruses, HPVs infects keratinocytes of the skin and mucous membranes. Typically, HPV infections are subclinical and cause little to no physical symptoms. Infections can become clinical and lead to the development of benign papilloma, also called warts or squamous cell papilloma. Clinical infections can also become cancerous developing into cancer of, for example, the throat and reproductive tissues such as the cervix. [1] Only a few HPV vaccines, including Cervarix®, Cecolin®, Walrinvax®, Cervavac®, Gardasil® have received licenses so far. Each prevents infection caused by various respective HPV serotypes. These HPV strains are believed to be responsible for over seventy percent of cervical cancers. [1] The HPV vaccine targets the HPV types that most commonly cause cervical cancer and some cancers of the vulva, vagina, anus, and oropharynx. It also protects against the HPV types that cause most genital warts. The HPV vaccine is highly effective in preventing the targeted HPV types infection in young females with child bearing age and protect them from the dreaded disease.
Hepatitis E virus (HEV) is a leading cause of acute viral hepatitis in developing countries. Acute hepatitis E can be severe and result in fulminant hepatitis (acute liver failure) with higher risk of death. Pregnant women with hepatitis E, particularly those in the second or third trimester, are at increased risk of acute liver failure, fetal loss and mortality. Currently, one clinical trial (NCT05415345) already going in China to evaluate the immunogenicity and safety of human papillomavirus vaccine (Cecolin) designed to protect against HPV types 16 and 18 co-immunized with recombinant hepatitis E virus ORF2 vaccine (Hecolin). [2] The said clinical trial discloses co-administration of two separate vaccines i.e. HPV types 16 and 18 (Cecolin) and hepatitis E virus ORF2 vaccine (Hecolin).
Present invention provides vaccine formulation comprising combination of human papillomavirus (HPV) L1 VLPs and hepatitis E virus (HEV) ORF2 antigen. Combining these two will have great benefit to the people especially young and child bearing age females as this combination of HPV and HEV will protect them with these two diseases in one vaccine with single shot. As with all vaccines, the vaccine formulation of the present invention needs to be stabilized during transportation and storage and therefore will contain stabilizers and other suitable excipients.

Summary of the invention
Present invention provides a vaccine formulation comprising human papillomavirus (HPV) L1 virus like particles (VLPs) in combination with hepatitis E virus ORF2 antigen (HEV) and suitable excipients. The present invention provides a vaccine formulation comprises at least one HPV L1 serotypes selected from HPV6L1, HPV11L1, HPV16L1, HPV18L1, HPV31L1, HPV33L1, HPV45L1, HPV52L1, HPV58L1 or any other HPV L1 antigen in combination with hepatitis E virus ORF2 antigen with suitable excipients. In other aspect, the present invention provides a method of preventing an infection and / or disease comprising administering a therapeutically effective amount of the vaccine formulation of the present invention.

Brief description of figures of the present invention
Figure 1: depicts vector map of pPICZ HPV6L1
Figure 2: depicts vector map of pPICZ HPV11L1
Figure 3: depicts vector map of pPICZ HPV16L1
Figure 4: depicts vector map of pPICZ HPV18L1
Figure 5: depicts vector map of pET30a (+) HEV ORF2
Figure 6: depicts SDS PAGE Analysis of purified HPV 6L1, HPV 11L1, HPV 16L1, HPV 18L1 and HEV antigen.
Figure 7: depicts transmission electron microscopy (TEM) analysis of HPV6L1, HPV11L1, HPV16L1 and HPV18L1 VLPs
Figure 8: depicts immunogenicity study of HPV 6L1, HPV 11L1, HPV 16L1, HPV 18L1 by ELISA in mice injected with HPV formulation comprising alum.
Figure 9: depicts immunogenicity study of HEV antigen by ELISA in mice injected with HEV formulation comprising alum.
Figure 10: depicts immunogenicity study of HPV 6L1, HPV 11L1, HPV 16L1, HPV 18L1 by ELISA in mice injected with HEV and HPV combination formulation comprising alum and Sepivac SWE™.
Figure 11: depicts immunogenicity study of HEV antigen by ELISA in mice injected with HEV and HPV combination formulation comprising alum and Sepivac SWE™.

Brief description of sequences of the present invention
SEQ ID No 1: depicts amino acid sequence of human papilloma virus 6L1 (HPV 6L1)
SEQ ID No 2: depicts amino acid sequence of human papilloma virus 11L1 (HPV 11L1)
SEQ ID No 3: depicts amino acid sequence of human papilloma virus 16L1 (HPV 16L1)
SEQ ID No 4: depicts amino acid sequence of human papilloma virus 18L1 (HPV 18L1)
SEQ ID No 5: depicts amino acid sequence of hepatitis E virus (HEV) ORF2 antigen.

Embodiments of the invention
In one embodiment, the present invention provides a vaccine composition comprising human papilloma (HPV) virus like particles (VLPs) in combination with hepatitis E virus antigen (HEV) and suitable excipients. The human papillomavirus (HPV) antigen according to the present invention is HPV L1 serotype. In one embodiment, the present invention provides a vaccine formulation (composition) comprising human papillomavirus (HPV) L1 virus like particles (VLPs) in combination with hepatitis E virus ORF2 antigen (HEV) and suitable excipients. In one embodiment, the vaccine formulation of the present invention comprises at least one HPV L1 serotype selected from HPV6L1, HPV11L1, HPV16L1, HPV18L1, HPV31L1, HPV33L1, HPV45L1, HPV52L1 HPV58L1 or any other HPV L1 serotype or any combination thereof combined with hepatitis E virus (HEV) ORF2 antigen. At least one HPV L1 serotype includes, but not limited to, one HPV L1, two HPV L1, three HPV L1, four HPV L1, five HPV L1, six HPV L1, seven HPV L1, eight HPV L1, nine HPV L1 serotype selected from HPV6L1, HPV11L1, HPV16L1, HPV18L1, HPV31L1, HPV33L1, HPV45L1, HPV52L1 HPV58L1. At least one HPV L1 serotype may also include more than nine HPV L1 serotype. In one embodiment, the vaccine formulation of the present invention comprises at least two HPV L1 serotype selected from HPV6L1, HPV11L1, HPV16L1, HPV18L1, HPV31L1, HPV33L1, HPV45L1, HPV52L1, HPV58L1 or any other HPV L1 serotype or any combination thereof combined with hepatitis E virus (HEV) ORF2 antigen. In one embodiment, the vaccine formulation of the present invention comprises at least three HPV L1 serotype selected from HPV6L1, HPV11L1, HPV16L1, HPV18L1, HPV31L1, HPV33L1, HPV45L1, HPV52L1, HPV58L1 or any other HPV L1 serotype or any combination thereof combined with hepatitis E virus (HEV) ORF2 antigen. In one embodiment, the vaccine formulation of the present invention comprises at least four HPV L1 serotype selected from HPV6L1, HPV11L1, HPV16L1, HPV18L1, HPV31L1, HPV33L1, HPV45L1, HPV52L1, HPV58L1 or any other HPV L1 serotype or any combination thereof combined with hepatitis E virus (HEV) ORF2 antigen. In one embodiment, the vaccine formulation of the present invention comprises at least five HPV L1 serotype selected from HPV6L1, HPV11L1, HPV16L1, HPV18L1, HPV31L1, HPV33L1, HPV45L1, HPV52L1, HPV58L1 or any other HPV L1 serotype or any combination thereof combined with hepatitis E virus (HEV) ORF2 antigen. In one embodiment, the vaccine formulation of the present invention comprises at least six HPV L1 serotype selected from HPV6L1, HPV11L1, HPV16L1, HPV18L1, HPV31L1, HPV33L1, HPV45L1, HPV52L1, HPV58L1 or any other HPV L1 serotype or any combination thereof combined with hepatitis E virus (HEV) ORF2 antigen. In one embodiment, the vaccine formulation of the present invention comprises at least seven HPV L1 serotype selected from HPV6L1, HPV11L1, HPV16L1, HPV18L1, HPV31L1, HPV33L1, HPV45L1, HPV52L1, HPV58L1 or any other HPV L1 serotype or any combination thereof combined with hepatitis E virus (HEV) ORF2 antigen. In one embodiment, the vaccine formulation of the present invention comprises at least eight HPV L1 serotype selected from HPV6L1, HPV11L1, HPV16L1, HPV18L1, HPV31L1, HPV33L1, HPV45L1, HPV52L1, HPV58L1 or any other HPV L1 serotype or any combination thereof combined with hepatitis E virus (HEV) ORF2 antigen. In one embodiment, the vaccine formulation of the present invention comprises at least nine HPV L1 serotype selected from HPV6L1, HPV11L1, HPV16L1, HPV18L1, HPV31L1, HPV33L1, HPV45L1, HPV52L1, HPV58L1 or any other HPV L1 serotype or any combination thereof combined with hepatitis E virus (HEV) ORF2 antigen. In one embodiment, the vaccine formulation of the present invention comprises more than nine HPV L1 serotype selected from HPV6L1, HPV11L1, HPV16L1, HPV18L1, HPV31L1, HPV33L1, HPV45L1, HPV52L1, HPV58L1 or any other HPV L1 serotype or any combination thereof combined with hepatitis E virus (HEV) ORF2 antigen. In one embodiment, the vaccine formulation of the present invention comprises HPV L1 serotype selected from HPV6L1, HPV11L1, HPV16L1, HPV18L1, HPV31L1, HPV33L1, HPV45L1, HPV52L1, HPV58L1 or any other HPV L1 serotype or any combination thereof combined with hepatitis E virus (HEV) ORF2 antigen having amino acids 368 to amino acids 606 of HEV ORF 2 of genotype 1 strain of hepatitis E virus (HEV). The structure of the HEV ORF 2 of genotype 1 strain of hepatitis E virus (HEV) has been disclosed in the prior art. [3] The HEV ORF2 of the present invention having amino acid sequence as set forth in SEQ ID No: 5. In a preferred embodiment, the present invention provides the vaccine composition comprises human papilloma virus (HPV) antigen and hepatitis E virus (HEV) antigen wherein HPV antigen comprises HPV6L1, HPV11L1, HPV16L1, HPV18L1 and HEV antigen comprises hepatitis E virus open reading frame 2 (HEV ORF2).
In a more preferred embodiment, the present invention provides the vaccine composition comprises human papilloma virus (HPV) antigen and hepatitis E virus (HEV) antigen wherein HPV antigen comprises HPV6L1 having amino acid sequence as set forth in SEQ ID No: 1, HPV11L1 having amino acid sequence as set forth in SEQ ID No: 2, HPV16L1 having amino acid sequence as set forth in SEQ ID No: 3, HPV18L1 having amino acid sequence as set forth in SEQ ID No: 4 and HEV antigen comprises HEV ORF2 having amino acid sequence as set forth in SEQ ID No: 5.
In one embodiment, the vaccine formulation (composition) according to the present invention comprises HPV L1 serotype(s) as disclosed in the present invention in combination with hepatitis E virus (HEV) ORF2 antigen and suitable excipients wherein suitable excipients are selected from adjuvants, preservatives, stabilizing agents and pharmaceutically acceptable carriers or any combination thereof. In one embodiment, the vaccine formulation according to the present invention comprises HPV L1 serotype(s) as disclosed in the present invention in combination with hepatitis E virus (HEV) ORF2 antigen and adjuvants selected from alum, alum in combination with MF-59, TLR3 agonist selected from Polyinosinic:polycytidylic acid [Poly(I:C)], TLR 4 agonist selected from Monophosphoryl Lipid A like 3-O-desacyl-4’-monophosphoryl lipid A (MPL) or GLA and like, TLR5 agonist selected from Flagellin, TLR7 agonist selected from Gardiquimod and Imiquimod, TLR7/8 agonist selected from R848 (Resiquimod), Nucleotide-binding Oligomerization Domain 2 (NOD2) agonist selected from N-glycolyl-MDP, CpG-containing nucleic acid (where the cytosine is unmethylated), QS21 (saponin adjuvant), stable oil in water emulsions like Sepivac SWE™, interleukins, beta-sitosterol and the like that induces both humoral and durable effector T-Cell responses. In one embodiment, the vaccine formulation according to the present invention comprises HPV L1 serotype(s) as disclosed in the present invention in combination with hepatitis E virus (HEV) ORF2 antigen and preservative(s) selected from but not limited to thiomersal, 2- Phenoxyethanol, sodium borate, antibiotics, gelatin, monosodium glutamate. In one embodiment, the vaccine formulation according to the present invention comprises HPV L1 serotype(s) as disclosed in the present invention in combination with hepatitis E virus (HEV) ORF2 antigen and stabilizing agent(s) selected from one or more gelatins selected from animal / human derived gelatins such as human collagen, and / or fish gelatin or any combination thereof, one or more amino acids selected from glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenyl alanine, tryptophan, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine and histidine or any combination thereof, or one or more carbohydrates selected from dextrose, sucrose, fructose, dextran, glucose, glycerol, erythritol, threitol, avabitol, xylitol, ribitol, mannitol, galactitol, fucitol, iditol, inositol, volemitol, isomalt, lacitol, sorbitol, maltotriitol, maltotetraitol, polyglyitol or dextrin, or isosorbide or any combination thereof, polysorbate such as polysorbate 20 or polysorbate 80 or any combination thereof, sodium glutamate, sodium chloride, buffers selected from acetate buffer, phosphate buffer, succinate buffer, tris buffer, histidine buffer, preferably phosphate buffer. The phosphate buffer comprises di sodium hydrogen phosphate and potassium dihydrogen phosphate according to the present invention.
In one embodiment, the vaccine formulation according to the present invention comprises HPV L1 serotype(s) as disclosed in the present invention in combination with hepatitis E virus (HEV) ORF2 antigen and pharmaceutically acceptable carriers selected from the group consisting of alcohol, propylene glycol, fatty alcohols, triglycerides, fatty acid esters, mineral oils, liquid petrolatum, isopropyl palmitate, polyethylene ethanol, sodium lauryl sulfate, an anti-oxidant, a humectant, a viscosity stabilizer or modifier, a colorant or a flavouring agent.
In one embodiment, the vaccine formulation according to the present invention comprises HPV L1 serotype(s) as disclosed in the present invention in combination with hepatitis E virus (HEV) ORF2 antigen and suitable excipients wherein suitable excipients are selected from adjuvants, preservatives, stabilizing agents and pharmaceutically acceptable carriers or any combination thereof wherein adjuvants selected from alum, alum in combination with MF-59, TLR3 agonist selected from Polyinosinic:polycytidylic acid [Poly(I:C)], TLR 4 agonist selected from Monophosphoryl Lipid A like 3-O-desacyl-4’-monophosphoryl lipid A (MPL) or Glucopyranosyl Lipid Adjuvant (GLA) and like, TLR5 agonist selected from Flagellin, TLR7 agonist selected from Gardiquimod and Imiquimod, TLR7/8 agonist selected from Resiquimod (R848), Nucleotide-binding Oligomerization Domain 2 (NOD2) agonist selected from N-glycolyl muramyl dipeptide (N-glycolyl MDP), CpG-containing nucleic acid (where the cytosine is unmethylated), QS21 (saponin adjuvant), stable oil in water emulsions like Sepivac SWE™, interleukins, beta-sitosterol and the like; preservative(s) selected from thiomersal, 2- Phenoxyethanol, sodium borate, antibiotics, gelatin, monosodium glutamate stabilizing agent(s) selected from one or more gelatins selected from animal-derived gelatins such as human collagen, and / or fish gelatin or any combination thereof, one or more amino acids selected from glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenyl alanine, tryptophan, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine and histidine or any combination thereof, or one or more carbohydrates selected from dextrose, sucrose, fructose, dextran, glucose, glycerol, erythritol, threitol, avabitol, xylitol, ribitol, mannitol, galactitol, fucitol, iditol, inositol, volemitol, isomalt, lacitol, sorbitol, maltotriitol, maltotetraitol, polyglyitol or dextrin, or isosorbide or any combination thereof, polysorbate such as polysorbate 20 or polysorbate 80, sodium glutamate, sodium chloride or any combination thereof, buffers selected from acetate buffer, phosphate buffer, succinate buffer, tris buffer, histidine buffer, preferably phosphate buffer; and pharmaceutically acceptable carriers selected from the group consisting of alcohol, propylene glycol, fatty alcohols, triglycerides, fatty acid esters, mineral oils, liquid petrolatum, isopropyl palmitate, polyethylene ethanol, sodium lauryl sulfate, an anti-oxidant, a humectant, a viscosity stabilizer or modifier, a colorant or a flavouring agent.
In one embodiment, the vaccine formulation embodied herein the present invention has a pH between 6.0 to 8.0. In one embodiment, the vaccine formulation embodied herein the present invention is liquid or lyophilized powder.
In one of the embodiments, the present invention provides a vaccine formulation embodied herein wherein an amount of human papillomavirus (HPV) HPV L1 serotype in the vaccine formulation of the present invention is between about 10 µg to about 100 µg. In one embodiment, the present invention provides a vaccine formulation embodied herein wherein total amount of human papillomavirus (HPV) HPV L1 virus like particles (VLPs) is between about 20 µg and about 1000 µg, preferably between about 50 µg and about 1000 µg. Total amount of HPV L1 VLPs refers amount of individual HPV L1 serotype in totality. HPV L1 serotypes are selected from HPV6L1, HPV11L1, HPV16L1, HPV18L1, HPV31L1, HPV33L1, HPV45L1, HPV52L1, HPV58L1 or any other HPV L1 serotype or any combination thereof.
In one of the embodiments, the present invention provides a vaccine formulation embodied herein wherein an amount of hepatitis E virus (HEV) ORF2 antigen in vaccine formulation of the present invention is between about 10 µg to about 60 µg.
In a preferred embodiment, the present invention provides a vaccine composition comprises HEV antigen 30 µg per dose, HPV 6L1 antigen 20 µg per dose, HPV 11L1 antigen 40 µg per dose, HPV 16L1 antigen 40 µg per dose, HPV 18L1 antigen 20 µg per dose, Di Sodium Hydrogen Phosphate 4.887 mM per dose, Potassium Dihydrogen Phosphate 4.41 mM per dose, Sodium Chloride 9.56 mg per dose, Dextrose 50 mg per dose, Aluminium in form of Aluminium Hydroxide adjuvant 0.8 mg per dose, Polysorbate 80 50 µg per dose, Sepivac SWE™ 0.25 mL and Water for injection q. s. to 0.5 mL.
In second embodiment, the present invention provides a method of treating or preventing an infection and / or disease comprising administering a therapeutically effective amount of the vaccine of the present invention to a mammal in need thereof. The mammal according to the present invention is human having age group between 2 years to 65 years, preferably between the age 9 years and age 45 years. The mammal includes an infant, a toddler, an adolescent, an adult or a senior. In one embodiment, the method of treatment or preventing an infection and / or disease comprising determining the therapeutically effective amount of the vaccine to be administered, and administering that amount of the vaccine to the mammal. In some embodiment, the vaccine composition is used in the treatment of any infection or disease occur by hepatitis E virus and human papilloma virus. In one embodiment, the method of treatment or preventing an infection and / or disease comprising reconstitution of lyophilized vaccine to an aqueous or non-aqueous liquid prior to administration to the patient. In one of the embodiments, the route of administration of the vaccine of the present invention is intra-muscular, intradermal, subcutaneous, intra-peritoneal, or intra-venous, preferably intra-muscular. In another embodiment, the present invention provides a method for the manufacture of a vaccine formulation embodied herein the present invention. In another embodiment, the present invention provides dose frequency of the vaccine formulation embodied here in the present invention. The frequency of administration can vary depending on any of a variety of factors, e.g., severity of the symptoms, degree of immunoprotection desired, whether the composition is used for prophylactic or therapeutic purposes, etc. The dose frequency of the vaccine could be either three dose, two dose or one dose. In one embodiment, the vaccine of the present invention is administered in two dose wherein second dose is administered either 1 month or 6 month from initial (first) dose. In one embodiment, the vaccine of the present invention is administered in three dose wherein second dose is administered in 1 month from initial dose and third dose is administered in 6 month from initial dose. In one embodiment, the vaccine of the present invention is administered in single dose only. Initial dose is the first dose of the vaccine of the present invention administered into subject. Subject includes human having age between year 2 to year 65 preferably between the age 9 years and age 45 years. In some embodiment, the present invention provides the vaccine composition wherein two dose of vaccine composition is administered in a human to treat any infection or disease occur by hepatitis E virus and human papilloma virus wherein second dose is administered 1 month after first dose. In some embodiment, the present invention provides the vaccine composition wherein two dose of vaccine composition is administered in a human to treat any infection or disease occur by hepatitis E virus and human papilloma virus wherein second dose is administered 6 month after first dose.
Abbreviations used herein the present application
The following abbreviations are employed in the examples and elsewhere herein:
µg : Microgram
DO: Dissolved oxygen
HEV ORF2: Hepatitis E virus open reading frame 2
HEV: Hepatitis E virus
HPV: Human papillomavirus
IB: Inclusion bodies
IPTG: Isopropyl-ß-D-thiogalactopyranoside
kDa: Kilodalton
Kpsi: Kilo pound per square inch
LB: Luria-Bertani broth
LB: Lysate buffer
N-glycolyl MDP: N-glycolyl muramyl dipeptide
NOD2: Nucleotide-binding Oligomerization Domain 2
OD: Optical density
PBS: Phosphate buffer saline
PBST: Phosphate Buffer saline with Tween 20
PCR: Polymerase chain reaction
PMSF: Phenyl methyl sulfonyl fluoride
Poly(I:C): Polyinosinic:polycytidylic acid
R848: Resiquimod
SDS-PAGE: sodium dodecyl sulfate–polyacrylamide gel electrophoresis
SFB: Sterile filtered bulk
TEM: Transmission electron microscopy
TFF: Tangential flow filtration
TLR: Toll-like receptor
VLPs: Virus like particles
YEPD: Yeast extract Peptone Dextrose
YEPDS: Yeast extract Peptone Dextrose Sorbitol
YPD: Yeast Extract–Peptone–Dextrose

Detailed description of the invention

The HPV vaccine targets the HPV types that most commonly cause cervical cancer and some cancers of the vulva, vagina, anus, and oropharynx. It also protects against the HPV types that cause most genital warts. The HPV vaccine is highly effective in preventing the targeted HPV types infection in young females with child bearing age and protect them from the dreaded disease. Hepatitis E virus (HEV) is a leading cause of acute viral hepatitis in developing countries. Acute hepatitis E can be severe and result in fulminant hepatitis (acute liver failure) with higher risk of death. Pregnant women with hepatitis E, particularly those in the second or third trimester, are at increased risk of acute liver failure, fetal loss and mortality. The proposed invention relates to a combination vaccine for intervention against the infection caused by different serotypes of Human Papillomavirus and Hepatitis E virus. The combination may include one, two, three, four, five, six, seven, eight, nine or more serotypes of HPV L1 VLPs of HPV6L1, HPV11L1, HPV16L1, HPV18L1, HPV31L1, HPV33L1, HPV45L1, HPV52L1, HPV58L1 or any other HPV L1 serotypes and HEV ORF2 protein adsorbed to aluminium and formulated with suitable excipients.
The objective of developing HPV and HEV combination vaccine is to prevent infections caused by hepatitis E virus (HEV) and human papillomavirus serotypes such as HPV 6L1, HPV11L1, HPV16L1, and HPV18L1. The main objective of the present invention is to put a greater emphasis on women's health, particularly preventing the death of pregnant women from HEV infection and cancers related to the human papillomavirus (HPV) in the vaginal, vulvar, and cervical regions. The existing dosing regimen requires atleast two dose of parenteral administration (intravenous or intramuscular) to treat HPV infections and atleast three dose of parenteral administration (intravenous or intramuscular) to treat HEV infection to provide protection against both diseases, hence in total atleast five doses are required to treat HPV infection and HEV infection. Therefore, multiple punctures (injections) of needle are being done in patient’s body for administration of HEV and HPV vaccines separately. These doses regimen is painful and inconvenient for the patients. The combination vaccine that comprises HPV and HEV of the present invention provides protection against both HPV and Hepatitis E virus (HEV) infection by parenteral administration of administering atleast only two doses. The second dose can be administered either after 1 month from initial dose or the second dose can be administered after 6 month from initial dose based on the immunogenicity and efficacy outcomes of HEV and HPV tetravalent vaccines of the present invention. The dose regimen of the present invention requires only few punctures of needle, preferably only two punctures of needle, in patient’s body for administration of the vaccine of the present invention. Thus, vaccine of the present invention is convenient and less painful for patients as compared to existing treatment or therapy of HPV and HEV infections / diseases. The various HPV L1 serotypes are known to the skilled person. The HPV L1 serotypes are disclosed in the prior arts. [4] The hepatitis E virus (HEV) ORF 2 antigen is also known to the skilled person and disclosed in the prior art. [3] The amino acid sequences of HPV L1 serotypes and HEV ORF2 antigen of the present invention are herein mention in table 1.
Table 1
Sequence No Amino acid sequence
1 MWRPSDSTVYVPPPNPVSKVVATDAYVTRTNIFYHASSSRLLAVGHPYFSIKRANKTVVPKVSGYQYRVFKVVLPDPNKFALPDSSLFDPTTQRLVWACTGLEVGRGQPLGVGVSGHPFLNKYDDVENSGSGGNPGQDNRVNVGMDYKQTQLCMVGCAPPLGEHWGKGKQCTNTPVQAGDCPPLELITSVIQDGDMVDTGFGAMNFADLQTNKSDVPIDICGTTCKYPDYLQMAADPYGDRLFFFLRKEQMFARHFFNRAGEVGEPVPDTLIIKGSGNRTSVGSSIYVNTPSGSLVSSEAQLFNKPYWLQKAQGHNNGICWGNQLFVTVVDTTRSTNMTLCASVTTSSTYTNSDYKEYMRHVEEYDLQFIFQLCSITLSAEVMAYIHTMNPSVLEDWNFGLSPPPNGTLEDTYRYVQSQAITCQKPTPEKEKPDPYKNLSFWEVNLKEKFSSELDQYPLGRKFLLQSGYRGRSSIRTGVKRPAVSKASAAPKRKRAKTKR
2 MWRPSDSTVYVPPPNPVSKVVATDAYVKRTNIFYHASSSRLLAVGHPYYSIKKVNKTVVPKVSGYQYRVFKVVLPDPNKFALPDSSLFDPTTQRLVWACTGLEVGRGQPLGVGVSGHPLLNKYDDVENSGGYGGNPGQDNRVNVGMDYKQTQLCMVGCAPPLGEHWGKGTQCSNTSVQNGDCPPLELITSVIQDGDMVDTGFGAMNFADLQTNKSDVPLDICGTVCKYPDYLQMAADPYGDRLFFYLRKEQMFARHFFNRAGTVGEPVPDDLLVKGGNNRSSVASSIYVHTPSGSLVSSEAQLFNKPYWLQKAQGHNNGICWGNHLFVTVVDTTRSTNMTLCASVSKSATYTNSDYKEYMRHVEEFDLQFIFQLCSITLSAEVMAYIHTMNPSVLEDWNFGLSPPPNGTLEDTYRYVQSQAITCQKPTPEKEKQDPYKDMSFWEVNLKEKFSSELDQFPLGRKFLLQSGYRGRTSARTGIKRPAVSKPSTAPKRKRTKTKK
3 MSLWLPSEATVYLPPVPVSKVVSTDEYVARTNIYYHAGTSRLLAVGHPYFPIKKPNNNKILVPKVSGLQYRVFRIHLPDPNKFGFPDTSFYNPDTQRLVWACVGVEVGRGQPLGVGISGHPLLNKLDDTENASAYAANAGVDNRECISMDYKQTQLCLIGCKPPIGEHWGKGSPCTNVAVNPGDCPPLELINTVIQDGDMVDTGFGAMDFTTLQANKSEVPLDICTSICKYPDYIKMVSEPYGDSLFFYLRREQMFVRHLFNRAGAVGENVPDDLYIKGSGSTANLASSNYFPTPSGSMVTSDAQIFNKPYWLQRAQGHNNGICWGNQLFVTVVDTTRSTNMSLCAAISTSETTYKNTNFKEYLRHGEEYDLQFIFQLCKITLTADVMTYIHSMNSTILEDWNFGLQPPPGGTLEDTYRFVTSQAIACQKHTPPAPKEDPLKKYTFWEVNLKEKFSADLDQFPLGRKFLLQAGLKAKPKFTLGKRKATPTTSSTSTTAKRKKRKL
4 MALWRPSDNTVYLPPPSVARVVNTDDYVTRTSIFYHAGSSRLLTVGNPYFRVPAGGGNKQDIPKVSAYQYRVFRVQLPDPNKFGLPDNSIYNPETQRLVWACAGVEIGRGQPLGVGLSGHPFYNKLDDTESSHAATSNVSEDVRDNVSVDYKQTQLCILGCAPAIGEHWAKGTACKSRPLSQGDCPPLELKNTVLEDGDMVDTGYGAMDFSTLQDTKCEVPLDICQSICKYPDYLQMSADPYGDSMFFCLRREQLFARHFWNRAGTMGDTVPQSLYIKGTGMRASPGSCVYSPSPSGSIVTSDSQLFNKPYWLHKAQGHNNGICWHNQLFVTVVDTTRSTNLTICASTQSPVPGQYDATKFKQYSRHVEEYDLQFIFQLCTITLTADVMSYIHSMNSSILEDWNFGVPPPPTTSLVDTYRFVQSVAITCQKDAAPAENKDPYDKLKFWNVDLKEKFSLDLDQYPLGRKFLVQAGLRRKPTIGPRKRSAPSATTSSKPAKRVRVRARK
5 IALTLFNLADTLLGGLPTELISSAGGQLFYSRPVVSANGEPTVKLYTSVENAQQDKGIAIPHDIDLGESRVVIQDYDNQHEQDRPTPSPAPSRPFSVLRANDVLWLSLTAAEYDQSTYGSSTGPVYVSDSVTLVNVATGAQAVARSLDWTKVTLDGRPLSTIQQYSKTFFVLPLRGKLSFWEAGTTKAGYPYNYNTTASDQLLVENAAGHRVAISTYTTSLGAGPVSISAVAVLAPHSA

Further, the process of preparing HPV L1 serotypes virus like particles (VLPs) are known in the art. [4] The present invention claims vaccine formulations comprising combination of atleast one HPV L1 serotypes and Hepatitis E virus (HEV) ORF 2 antigen along with suitable excipients. The suitable excipients includes, but not limited to, adjuvants, preservatives, stabilizing agents and pharmaceutically acceptable carriers or any combination thereof. The examples of the adjuvants are, but not limited to, alum, alum in combination with MF-59, TLR3 agonist selected from Poly(I:C), TLR 4 agonist selected from Monophosphoryl Lipid A like 3-O-desacyl-4’-monophosphoryl lipid A (MPL) or GLA and like, TLR5 agonist selected from Flagellin, TLR7 agonist selected from Gardiquimod and Imiquimod, TLR7/8 agonist selected from R848, NOD2 agonist selected from N-glycolyl-MDP, CpG-containing nucleic acid (where the cytosine is unmethylated), QS21 (saponin adjuvant), stable oil in water emulsions like Sepivac SWE™, interleukins, beta-sitosterol and the like. The amount of adjuvants according to the present invention is in the range of 10 µg to about 2000 µg. The amount range between 10 µg and about 2000 µg includes, but not limited to, between 10 µg to about 50 µg, between 25 µg to about 70 µg, between 32 µg to about 77 µg, between 45 µg to about 90 µg, between 60 µg to about 127 µg, between 79 µg to about 140 µg, between 100 µg to about 150 µg, between 140 µg to about 200 µg, between 170 µg to about 217 µg, between 250 µg to about 500 µg, between 250 µg to about 1000 µg, between 250 µg to about 400 µg, between 270 µg to about 450 µg, between 300 µg to about 400 µg, between 330 µg to about 470 µg, between 370 µg to about 500 µg, between 410 µg to about 500 µg, between 450 µg to about 520 µg, between 480 µg to about 570 µg, between 575 µg to about 675 µg, between 565 µg to about 665 µg, between 590 µg to about 700 µg, between 700 µg to about 800 µg, between 740 µg to about 835 µg, between 820 µg to about 920 µg, between 900 µg to about 1000 µg, between 990 µg to about 1100 µg, between 1100 µg to about 1200 µg, between 1120 µg to about 1250 µg, between 1200 µg to about 1300 µg, between 1310 µg to about 1420 µg, between 1420 µg to about 1530 µg, between 1530 µg to about 1630 µg, between 1620 µg to about 1720 µg, between 1715 µg to about 1835 µg, between 1827 µg to about 1943 µg, between 1850 µg to about 1963 µg, between 1870 µg to about 1973 µg, between 1890 µg to about 2000 µg. The amount range according to the present invention disclosure include each integer and non-integer number between a particular ranges. Further, the recitations of numerical ranges as disclosed here in the present description by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).
The examples of the preservatives are, but not limited to, thiomersal, 2- Phenoxyethanol, sodium borate, antibiotics, gelatin, monosodium glutamate. The preservatives could be added in the formulation in the amount range from 2.5 mg to about 5 mg per single human dose. The amount range from 2.5 mg to about 5 mg per single human dose includes, but not limited to, 2.5 mg per single human dose, 2.6 mg per single human dose 2.7 mg per single human dose, 2.8 mg per single human dose, 2.9 mg per single human dose, 3.0 mg per single human dose, 3.1 mg per single human dose, 3.2 mg per single human dose, 3.3 mg per single human dose, 3.4 mg per single human dose, 3.5 mg per single human dose, 3.6 mg per single human dose, 3.7 mg per single human dose, 3.8 mg per single human dose, 3.9 mg per single human dose, 4.0 mg per single human dose, 4.1 mg per single human dose, 4.2 mg per single human dose, 4.3 mg per single human dose, 4.4 mg per single human dose, 4.5 mg per single human dose, 4.6 mg per single human dose, 4.7 mg per single human dose, 4.8 mg per single human dose, 4.9 mg per single human dose, 5.0 mg per single human dose.
The examples of stabilizing agents are, but not limited to, one or more gelatins selected from animal / human-derived gelatins such as human collagen, and / or fish gelatin or any combination thereof, one or more amino acids selected from glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenyl alanine, tryptophan, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine and histidine or any combination thereof, or one or more carbohydrates selected from dextrose, sucrose, fructose, dextran, glucose, glycerol, erythritol, threitol, avabitol, xylitol, ribitol, mannitol, galactitol, fucitol, iditol, inositol, volemitol, isomalt, lacitol, sorbitol, maltotriitol, maltotetraitol, polyglyitol or dextrin, or isosorbide or any combination thereof, polysorbate such as polysorbate 20 or polysorbate 80 or any combination thereof, sodium glutamate, sodium chloride. The amount of stabilizing agents according to the present invention is in the concentration range of 0.001 % w / v to 10 % w / v. The concentration range of 0.001 % w / v to 10 % w / v includes, but not limited to, between 0.001 % w / v to 0.005 % w / v, between 0.003 % w / v to 0.007 % w / v, between 0.007 % w / v to 0.03 % w / v, between 0.01 % w / v to 0.04 % w / v, between 0.04 % w / v to 0.08 % w / v, between 0.05 % w / v to 1.0 % w / v, between 0.07 % w / v to 1.5 % w / v, between 1.0 % w / v to 2.2 % w / v, between 1.2 % w / v to 2.7 % w / v, between 2.0 % w / v to 3.0 % w / v, between 2.6 % w / v to 3.7 % w / v, between 3.0 % w / v to 4.0 % w / v, between 3.5 % w / v to 4.5 % w / v, between 4.4 % w / v to 5.7 % w / v, between 5.0 % w / v to 6.3 % w / v, between 5.5 % w / v to 7.0 % w / v, between 7.0 % w / v to 8.0 % w / v, between 7.5 % w / v to 8.5 % w / v, between 7.9 % w / v to 9.1 % w / v, between 8.2 % w / v to 9.7 % w / v, between 9.0 % w / v to 10.0 % w / v. The amount of stabilizing agent could also between ranges of 0.05 mg to 5 mg in single human dose. The amount range from 0.05 mg to about 5 mg per single human dose includes, but not limited to, 0.05 mg per single human dose, 0.07 mg per single human dose, 0.09 mg per single human dose, 0.1 mg per single human dose, 0.2 mg per single human dose, 0.3 mg per single human dose, 0.4 mg per single human dose, 0.5 mg per single human dose, 0.6 mg per single human dose, 0.7 mg per single human dose, 0.8 mg per single human dose, 0.9 mg per single human dose, 1.0 mg per single human dose, 1.1 mg per single human dose, 1.2 mg per single human dose, 1.3 mg per single human dose, 1.4 mg per single human dose, 1.5 mg per single human dose, 1.6 mg per single human dose, 1.7 mg per single human dose, 1.8 mg per single human dose, 1.9 mg per single human dose, 2.0 mg per single human dose, 2.1 mg per single human dose, 2.2 mg per single human dose, 2.3 mg per single human dose, 2.4 mg per single human dose, 2.5 mg per single human dose, 2.6 mg per single human dose 2.7 mg per single human dose, 2.8 mg per single human dose, 2.9 mg per single human dose, 3.0 mg per single human dose, 3.1 mg per single human dose, 3.2 mg per single human dose, 3.3 mg per single human dose, 3.4 mg per single human dose, 3.5 mg per single human dose, 3.6 mg per single human dose, 3.7 mg per single human dose, 3.8 mg per single human dose, 3.9 mg per single human dose, 4.0 mg per single human dose, 4.1 mg per single human dose, 4.2 mg per single human dose, 4.3 mg per single human dose, 4.4 mg per single human dose, 4.5 mg per single human dose, 4.6 mg per single human dose, 4.7 mg per single human dose, 4.8 mg per single human dose, 4.9 mg per single human dose, 5.0 mg per single human dose. The amount range according to the present invention disclosure include each integer and non-integer number between a particular ranges. Further, the recitations of numerical ranges as disclosed here in the present description by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).
The examples of the pharmaceutically acceptable carriers are, but not limited to, alcohol, propylene glycol, fatty alcohols, triglycerides, fatty acid esters, mineral oils, liquid petrolatum, isopropyl palmitate, polyethylene ethanol, sodium lauryl sulfate, an anti-oxidant, a humectant, a viscosity stabilizer or modifier. The amount of pharmaceutically acceptable carriers according to the present invention is in the concentration range of 0.001 % w / v to 10 % w / v. The concentration range of 0.001 % w / v to 10 % w / v includes, but not limited to, between 0.001 % w / v to 0.005 % w / v, between 0.003 % w / v to 0.007 % w / v, between 0.007 % w / v to 0.03 % w / v, between 0.01 % w / v to 0.04 % w / v, between 0.04 % w / v to 0.08 % w / v, between 0.05 % w / v to 1.0 % w / v, between 0.07 % w / v to 1.5 % w / v, between 1.0 % w / v to 2.2 % w / v, between 1.2 % w / v to 2.7 % w / v, between 2.0 % w / v to 3.0 % w / v, between 2.6 % w / v to 3.7 % w / v, between 3.0 % w / v to 4.0 % w / v, between 3.5 % w / v to 4.5 % w / v, between 4.4 % w / v to 5.7 % w / v, between 5.0 % w / v to 6.3 % w / v, between 5.5 % w / v to 7.0 % w / v, between 7.0 % w / v to 8.0 % w / v, between 7.5 % w / v to 8.5 % w / v, between 7.9 % w / v to 9.1 % w / v, between 8.2 % w / v to 9.7 % w / v, between 9.0 % w / v to 10.0 % w / v. The amount of pharmaceutically acceptable carriers could also between ranges of 0.05 mg to 5 mg in single human dose. The amount range from 0.05 mg to about 5 mg per single human dose includes, but not limited to, 0.05 mg per single human dose, 0.07 mg per single human dose, 0.09 mg per single human dose, 0.1 mg per single human dose, 0.2 mg per single human dose, 0.3 mg per single human dose, 0.4 mg per single human dose, 0.5 mg per single human dose, 0.6 mg per single human dose, 0.7 mg per single human dose, 0.8 mg per single human dose, 0.9 mg per single human dose, 1.0 mg per single human dose, 1.1 mg per single human dose, 1.2 mg per single human dose, 1.3 mg per single human dose, 1.4 mg per single human dose, 1.5 mg per single human dose, 1.6 mg per single human dose, 1.7 mg per single human dose, 1.8 mg per single human dose, 1.9 mg per single human dose, 2.0 mg per single human dose, 2.1 mg per single human dose, 2.2 mg per single human dose, 2.3 mg per single human dose, 2.4 mg per single human dose, 2.5 mg per single human dose, 2.6 mg per single human dose 2.7 mg per single human dose, 2.8 mg per single human dose, 2.9 mg per single human dose, 3.0 mg per single human dose, 3.1 mg per single human dose, 3.2 mg per single human dose, 3.3 mg per single human dose, 3.4 mg per single human dose, 3.5 mg per single human dose, 3.6 mg per single human dose, 3.7 mg per single human dose, 3.8 mg per single human dose, 3.9 mg per single human dose, 4.0 mg per single human dose, 4.1 mg per single human dose, 4.2 mg per single human dose, 4.3 mg per single human dose, 4.4 mg per single human dose, 4.5 mg per single human dose, 4.6 mg per single human dose, 4.7 mg per single human dose, 4.8 mg per single human dose, 4.9 mg per single human dose, 5.0 mg per single human dose. The amount range according to the present invention disclosure include each integer and non-integer number between a particular ranges. Further, the recitations of numerical ranges as disclosed here in the present description by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).
The various embodiments of the vaccine formulations and other embodiments related to vaccine formulations of the present invention are described here in the present specification. The term “vaccine” and “vaccine formulation(s) can be used interchangeably here in the present invention. In one embodiment, the vaccine formulation embodied herein the present invention has a pH between 6.0 to 8.0. The pH range of about 6.0 to about 8.0 according to present disclosure include each integer and non-integer number between a particular ranges. For Example, the pH range between 6.0 to 8.0 encompasses pH 6.0, pH 6.2, pH 6.5, pH 6.8, pH 7.0, pH 7.3, pH 7.6, pH 7.9 and pH 8.0. In one of the embodiments, the present invention provides a vaccine formulation embodied herein wherein an amount of human papillomavirus (HPV L1) serotypes in the vaccine formulation of the present invention is between about 10 µg to about 100 µg. The amount range between about 10 µg to about 100 µg includes, but not limited to, between about 10 µg to about 18 µg, between about 15 µg to about 25 µg, between about 20 µg to about 33 µg, between about 30 µg to about 45 µg, between about 40 µg to about 57 µg, between about 52 µg to about 72 µg, between about 60 µg to about 80 µg, between about 65 µg to about 75 µg, between about 80 µg to about 95 µg, between about 83 µg to about 99 µg, between about 90 µg to about 100 µg. The amount range according to the present invention disclosure include each integer and non-integer number between a particular ranges. In one embodiment, the present invention provides a vaccine formulation embodied herein wherein total amount of human papillomavirus (HPV) HPV L1 virus like particles (VLPs) is between about 20 µg and about 1000 µg per dose. The volume of dose of the vaccine of the present invention is in the range between 0.5 ml and 1 ml. In one embodiment, the volume of dose of the vaccine of the present invention is 0.5 mL. Total amount of HPV L1 VLPs refers amount of individual HPV L1 serotype in totality. HPV L1 serotypes are selected from HPV6L1, HPV11L1, HPV16L1, HPV18L1, HPV31L1, HPV33L1, HPV45L1, HPV52L1, HPV58L1 or any other HPV L1 serotype or any combination thereof. The amount range between about 20 µg to about 1000 µg includes, but not limited to, between about 20 µg to about 40 µg, between about 40 µg to about 65 µg, between about 65 µg to about 130 µg, between about 80 µg to about 100 µg, between about 95 µg to about 135 µg, between about 137 µg to about 190 µg, between about 190 µg to about 240 µg, between about 225 µg to about 275 µg, between about 280 µg to about 355 µg, between about 355 µg to about 450 µg, between about 450 µg to about 550 µg, between about 555 µg to about 665 µg, between about 671 µg to about 777 µg, between about 740 µg to about 825 µg, between about 800 µg to about 917 µg, between about 920 µg to about 973 µg, between about 900 µg to about 1000 µg. The amount range according to the present invention disclosure include each integer and non-integer number between a particular ranges. Further, the recitations of numerical ranges as disclosed here in the present description by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).
In one embodiment, the present invention provides a vaccine formulation embodied herein wherein an amount of hepatitis E virus (HEV) ORF2 antigen in vaccine formulation of the present invention is between about 10 µg to about 60 µg per dose. The volume of dose of the vaccine of the present invention is in the range between 0.5 ml and 1 ml. In one embodiment, the volume of dose of the vaccine of the present invention is 0.5 mL. The amount range between about 10 µg to about 60 µg includes, but not limited to, between about 10 µg to about 22 µg, between about 13 µg to about 25 µg, between about 18 µg to about 30 µg, between about 20 µg to about 33 µg, between about 23 µg to about 38 µg, between about 28 µg to about 50 µg, between about 35 µg to about 55 µg, between about 37 µg to about 57 µg, between about 40 µg to about 60 µg, between about 50 µg to about 60 µg. The amount range according to the present invention disclosure include each integer and non-integer number between a particular ranges.
As used herein, "carrier" includes medium, vehicle, coating, diluent, antibacterial, and/or antifungal agent, isotonic agent, absorption delaying agent, buffer, carrier solution, suspension, colloid, and the like. The use of such media and/or agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions. As used herein, "pharmaceutically acceptable" refers to a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the immunogen without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. The pharmaceutical composition may be formulated in a variety of forms adapted to a preferred route of administration. Thus, a composition can be administered via known routes including, for example intradermal, subcutaneous, intramuscular, intravenous, intraperitoneal, etc. In one embodiment, the pharmaceutical composition comprising combination of HPV and HEV antigens is administered by intramuscular route. A formulation may be conveniently presented in unit dosage form or multi-dose sealed containers, such as ampules and vials, pre-filled syringe (PFS) and may be prepared by methods well known in the art. Methods of preparing a composition with a pharmaceutically acceptable carrier include the step of bringing with a carrier that constitutes one or more accessory ingredients. As used herein, the term "treat" or variations thereof refer to reducing, limiting progression, ameliorating, or resolving, to any extent, the symptoms or signs of disease or infection. A "treatment" may be therapeutic or prophylactic. "Therapeutic" and variations thereof refer to a treatment that ameliorates one or more existing symptoms or clinical signs associated with a condition. "Prophylactic" and variations thereof refer to a treatment that limits, to any extent, the development and/or appearance of a symptom or clinical sign of a condition. Generally, a "therapeutic" treatment is initiated after disease or infection manifests in a subject, while "prophylactic" treatment is initiated before disease or infections manifests in a subject. As used herein, the term "symptom" refers to any subjective evidence of disease or of a patient's condition, while the term "sign" or "clinical sign" refers to an objective physical finding relating to a particular condition capable of being found by one other than the patient. As used herein, the term "ameliorate" refers to any reduction in the extent, severity, frequency, and/or likelihood of a symptom or clinical sign characteristic of disease or infections.
In the preceding description, the term "and / or" means one or all of the listed elements or a combination of any two or more of the listed elements; the terms "comprises," "comprising," and variations thereof are to be construed as open ended— i.e., additional elements or steps are optional and may or may not be present; unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably and mean one or more than one; and the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.). In the preceding description, particular embodiments may be described in isolation for clarity. Unless otherwise expressly specified that the features of a particular embodiment are incompatible with the features of another embodiment, certain embodiments can include a combination of compatible features described herein in connection with one or more embodiments. For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously. The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.
Preferred embodiments of the present invention:
In one embodiment, present invention provides a vaccine composition comprising human papilloma (HPV) virus like particles (VLPs) in combination with hepatitis E virus antigen (HEV) and suitable excipients.
In one embodiment, present invention provides the vaccine composition as embodied herein the present invention wherein human papilloma virus (HPV) antigen is HPV L1.
In one embodiment, present invention provides the vaccine composition as embodied herein the present invention wherein human papilloma virus (HPV) antigen is selected from atleast one serotypes of HPV L1 selected from HPV6L1, HPV11L1, HPV16L1, HPV18L1, HPV31L1, HPV33L1, HPV45L1, HPV52L1 or HPV58L1 or any suitable combinations thereof.
In one embodiment, present invention provides the vaccine composition as embodied herein the present invention wherein human papilloma virus (HPV) antigen is selected from atleast two serotypes of HPV L1 selected from HPV6L1, HPV11L1, HPV16L1, HPV18L1, HPV31L1, HPV33L1, HPV45L1, HPV52L1 or HPV58L1 or any combination thereof.
In one embodiment, present invention provides the vaccine composition as embodied herein the present invention wherein human papilloma virus (HPV) antigen is selected from atleast three serotypes of HPV L1 selected from HPV6L1, HPV11L1, HPV16L1, HPV18L1, HPV31L1, HPV33L1, HPV45L1, HPV52L1 or HPV58L1 or any combination thereof.
In one embodiment, present invention provides the vaccine composition as embodied herein the present invention wherein human papilloma virus (HPV) antigen is selected from atleast four serotypes of HPV L1 selected from HPV6L1, HPV11L1, HPV16L1, HPV18L1, HPV31L1, HPV33L1, HPV45L1, HPV52L1 or HPV58L1 or any combination thereof.
In one embodiment, present invention provides the vaccine composition as embodied herein the present invention wherein human papilloma virus (HPV) antigen is selected from atleast five serotypes of HPV L1 wherein HPV L1 is selected from HPV6L1, HPV11L1, HPV16L1, HPV18L1, HPV31L1, HPV33L1, HPV45L1, HPV52L1 or HPV58L1 or any combination thereof.
In one embodiment, present invention provides the vaccine composition as embodied herein the present invention wherein human papilloma virus (HPV) antigen is selected from atleast six serotypes of HPV L1 wherein HPV L1 is selected from HPV6L1, HPV11L1, HPV16L1, HPV18L1, HPV31L1, HPV33L1, HPV45L1, HPV52L1 or HPV58L1 or any combination thereof.
In one embodiment, present invention provides the vaccine composition as embodied herein the present invention wherein human papilloma virus (HPV) antigen is selected from atleast seven serotypes of HPV L1 wherein HPV L1 is selected from HPV6L1, HPV11L1, HPV16L1, HPV18L1, HPV31L1, HPV33L1, HPV45L1, HPV52L1 or HPV58L1 or any combination thereof.
In one embodiment, present invention provides the vaccine composition as embodied herein the present invention wherein human papilloma virus (HPV) antigen is selected from atleast eight serotypes of HPV L1 wherein HPV L1 is selected from HPV6L1, HPV11L1, HPV16L1, HPV18L1, HPV31L1, HPV33L1, HPV45L1, HPV52L1 or HPV58L1 or any combination thereof.
In one embodiment, present invention provides the vaccine composition as embodied herein the present invention wherein human papilloma virus (HPV) antigen is selected from atleast nine serotypes of HPV L1 wherein HPV L1 is selected from HPV6L1, HPV11L1, HPV16L1, HPV18L1, HPV31L1, HPV33L1, HPV45L1, HPV52L1 or HPV58L1 or any other HPV L1 serotype or any combination thereof.
In one embodiment, present invention provides the vaccine composition as embodied herein the present invention wherein human papilloma virus (HPV) antigen comprises HPV6L1, HPV11L1, HPV16L1 and HPV18L1.
In one embodiment, present invention provides the vaccine composition as embodied herein the present invention wherein human papilloma virus (HPV) antigen comprises HPV6L1 having amino acid sequence as set forth in SEQ ID No: 1, HPV11L1 having amino acid sequence as set forth in SEQ ID No: 2, HPV16L1 having amino acid sequence as set forth in SEQ ID No: 3 and HPV18L1 having amino acid sequence as set forth in SEQ ID No: 4.
In one embodiment, present invention provides the vaccine composition as embodied herein wherein hepatitis E virus antigen (HEV) is hepatitis E virus open reading frame 2 (HEV ORF2).
In one embodiment, present invention provides the vaccine composition as embodied herein wherein hepatitis E virus antigen (HEV) is HEV ORF2 having amino acid sequence as set forth in SEQ ID No: 5.
In one embodiment, present invention provides the vaccine composition as embodied herein wherein vaccine composition comprises human papilloma virus (HPV) antigen and hepatitis E virus (HEV) antigen wherein HPV antigen comprises HPV6L1 having amino acid sequence as set forth in SEQ ID No: 1, HPV11L1 having amino acid sequence as set forth in SEQ ID No: 2, HPV16L1 having amino acid sequence as set forth in SEQ ID No: 3, HPV18L1 having amino acid sequence as set forth in SEQ ID No: 4 and the HEV antigen is HEV ORF2 having amino acid sequence as set forth in SEQ ID No: 5.
In one embodiment, present invention provides the vaccine composition as embodied herein wherein suitable excipients are selected from adjuvants, preservatives, stabilizing agents and pharmaceutically acceptable carriers or any combination thereof.
In one embodiment, present invention provides the vaccine composition as embodied herein wherein suitable excipients comprises adjuvants wherein the adjuvants are selected from aluminum hydroxide gel, alum, alum in combination with MF-59, TLR3 agonist Polyinosinic:polycytidylic acid, TLR 4 agonist selected from Monophosphoryl Lipid A like 3-O-desacyl-4’-monophosphoryl lipid A or Glucopyranosyl Lipid Adjuvant, TLR5 agonist Flagellin, TLR7 agonist selected from Gardiquimod and Imiquimod, TLR7/8 agonist Resiquimod, Nucleotide-binding Oligomerization Domain 2 agonist N-glycolyl muramyl dipeptide, TLR9 agonist CpG-containing nucleic acid, saponin adjuvant QS21, stable oil in water emulsions like Sepivac SWE™, interleukins and beta-sitosterol.
In one embodiment, present invention provides the vaccine composition as embodied herein wherein suitable excipients comprises preservatives wherein the preservatives are selected from thiomersal, 2- Phenoxyethanol, sodium borate or any combination thereof.
In one embodiment, present invention provides the vaccine composition as embodied herein wherein suitable excipients comprises stabilizing agents wherein the stabilizing agents are selected from one or more gelatins selected from animal-derived gelatins such as human collagen, and / or fish gelatin or any combination thereof, one or more amino acids selected from glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenyl alanine, tryptophan, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine and histidine or any combination thereof, or one or more carbohydrates selected from dextrose, sucrose, fructose, dextran, glucose, glycerol, erythritol, threitol, avabitol, xylitol, ribitol, mannitol, galactitol, fucitol, iditol, inositol, volemitol, isomalt, lacitol, sorbitol, maltotriitol, maltotetraitol, polyglyitol or dextrin, or isosorbide or any combination thereof, polysorbate such as polysorbate 20 or polysorbate 80, sodium glutamate, sodium chloride, buffers selected from acetate buffer, phosphate buffer, succinate buffer, tris buffer, histidine buffer, preferably phosphate buffer.
In one embodiment, present invention provides the vaccine composition as embodied herein wherein suitable excipients comprises pharmaceutically acceptable carriers wherein the pharmaceutically acceptable carriers are selected from the group consisting of alcohol, propylene glycol, fatty alcohols, triglycerides, fatty acid esters, mineral oils, liquid petrolatum, isopropyl palmitate, polyethylene ethanol, sodium lauryl sulfate, an anti-oxidant, a humectant, a viscosity stabilizer or modifier, a colorant or a flavouring agent.
In one embodiment, present invention provides the vaccine composition as embodied herein wherein vaccine composition has pH between 6.0 to 8.0.
In one embodiment, present invention provides the vaccine composition as embodied herein wherein the vaccine composition comprises human papilloma virus (HPV) in an amount between about 10 µg to about 100 µg.
In one embodiment, present invention provides the vaccine composition as embodied herein wherein the vaccine composition comprises hepatitis E virus (HEV) in an amount between about 10 µg to about 60 µg.
In one embodiment, present invention provides the vaccine composition as embodied herein wherein the vaccine composition comprises total amount of human papilloma virus (HPV) and hepatitis E virus (HEV) is between about 50 µg and about 1000 µg.
In one embodiment, present invention provides the vaccine composition as embodied herein comprises HEV antigen at 30 µg per dose, HPV 6L1 antigen at 20 µg per dose, HPV 11L1 antigen at 40 µg per dose, HPV 16L1 antigen at 40 µg per dose, HPV 18L1 antigen at 20 µg per dose, Di Sodium Hydrogen Phosphate at 4.887 mM per dose, Potassium Dihydrogen Phosphate at 4.41 mM per dose, Sodium Chloride at 9.56 mg per dose, Dextrose at 50 mg per dose, Aluminium in form of Aluminium Hydroxide adjuvant at 0.8 mg per dose, Polysorbate 80 at 50 µg per dose, Sepivac SWE™ 0.25 mL and Water for injection q. s. to make upto 0.5 mL.
In one embodiment, present invention provides the vaccine composition as embodied herein wherein vaccine composition is a liquid or a lyophilized powder.
In one embodiment, present invention provides the vaccine composition as embodied herein wherein vaccine composition is a lyophilized powder which is reconstituted to an aqueous or non-aqueous liquid prior to administration to the patient.
In one embodiment, present invention provides the vaccine composition as embodied herein wherein the vaccine composition is used in the treatment of any infection or disease caused by hepatitis E virus and human papilloma virus.
In one embodiment, present invention provides the vaccine composition as embodied herein wherein atleast two dose of vaccine composition is administered in a human to treat any infection or disease occur by hepatitis E virus and human papilloma virus.
In one embodiment, present invention provides the vaccine composition as embodied herein wherein the second dose is administered atleast 1 month after the first dose.
In one embodiment, present invention provides the vaccine composition as embodied herein wherein the second dose is administered 6 months after the first dose.
In one embodiment, present invention provides the vaccine composition as embodied wherein the vaccine composition is administered via intra-muscular, intra-peritoneal, or intra-venous routes, preferably intra-muscular route.

Examples
Example 1: Clone development for HPV antigens
Genes for major surface antigen L1 of HPV subtypes 6, 11, 16 and 18 were synthesized separately by GeneArt (Germany), constructed by de novo synthesis based on the amino acid sequence. The nucleotide sequences were codon optimized for expression in Pichia pastoris. Pichia pastoris strain was inoculated in YEPD (Yeast extract Peptone Dextrose) media and grown overnight on shaker incubator at 30 ºC. This grown strain culture was re-inoculated at 2 % inoculum density to YEPD media and this was grown at above mentioned condition for 20 hrs. Electro-competent cells were prepared and used for transformation and production of desired protein. Gene for major surface antigen L1 of HPV for subtypes 6, 11, 16 and 18 were sub-cloned separately in to pPICZa vector. The respective HPV L1 expression vector was constructed by inserting the BstBI / NotI digested fragment encoding HPV L1 into the multiple cloning site of the vector pPICZa, resulting expression vector was designated as pPICZ-HPV respective (6, 11, 16 & 18) L1. Figure 1, 2, 3 and 4 represents vector map of HPV6L1, HPV 11L1, HPV 16L1 and HPV 18L1 respectively. All four HPV expression vectors pPICZ-HPV16L1, pPICZ-HPV18L1, pPICZ-HPV6L1 and pPICZHPV11L1 were propagated in E. coli and plasmid was purified using kit method. This individual plasmid DNA was transformed into the Pichia pastoris host strain KM71. For transformation of pPICZ- HPVL1 in Pichia pastoris, the plasmid was linearized using Enzyme SacI. Transformation was done in Pichia pastoris KM71 by electroporation. Clones were selected using Zeocin antibiotic. Colonies were selected and master plate was made using YEPDS agar (Yeast extract Peptone Dextrose Sorbitol). Selection of high expression clones was done on the basis of copy number of gene integrated into Pichia pastoris KM71. Quantitative Colony PCR assay was used for screening.

Example 2: Fermentation process for production of HPV antigens
The clones of HPV antigens as prepared in example 1 were transferred in fermenter for scale up the production. Production of individual HPV antigen in fermenter was performed in fed-batch mode at 15 L scale. In the process, 1 mL of culture from one master cell bank was inoculated into 100 mL of YPD media and this culture was incubated at 30 °C at 200 RPM for 24 hours. This pre-seed culture was used to inoculate the seed in 400 mL of sterile defined medium. These freshly grown yeast cells generated with seed culture were inoculated in to 15L production fermenter. Cells were grown at 30± 2 °C; pH: 4.8 ± 0.2, in the presence of chemically defined media, in fed-batch mode. Feeding was started in a controlled manner once optical density (OD) reached at 15 to 20, agitation between 300 and 900 rpm, DO (Dissolved oxygen) set point was 40 %, air was maintained at 1 vvm in fermenter for 24 ± 2 hrs. The unit 'vvm' is a volume of air sparged per unit of medium per minute. After achieving OD > 350, induction was started with methanol for 72 hrs, maintaining same production parameters. The induction batch was harvested after 72 hrs with final volume of about 13 L. Harvested cell suspension was centrifuged at 4500 - 4700 rpm for 45 to 60 minutes at temperature 2 to 8 °C. Obtained cell pellet was stored in appropriate containers at minus 70 °C (-70 °C) for further downstream purification process to purify respective HPV VLPs.

Example 3: Purification of human papilloma virus like particles (HPV VLPs)
The cell pellet as prepared in example 2 was washed in three different washing steps- 1st wash: cell pellet was suspended in 2L of 1x PBS buffer containing detergent. 2nd wash: Collected cell mass from 1st wash was re-suspended in 2L of 1x PBS buffer containing higher concentration of NaCl. 3rd wash: the cell mass of 2nd wash was re-suspended in 2L of only 1x PBS buffer. After washing, the cell pellets were resuspended in 4L of buffer containing PMSF, pepstatin A, glycerol and Polysorbate 80, pH 7.5 ± 0.3. The intact whole cells were disrupted with two passes through a high-pressure homogenizer at 23-25 kpsi in cold conditions. After disruption, the cell lysate was centrifuged at 8500 to 10000 rpm at temperature 2 to 8 °C for 20 to 30 minutes to separate supernatant from cell debris. Clarified cell lysate was collected and the cell debris was discarded. Assembly buffer (20mM Tris-Cl, 150mM NaCl, 0.5mM Calcium chloride and Glycerol 5% pH 6.5 – 6.7, conductivity 14 – 18 mS/cm) along with adequate endonuclease enzyme was added to the clarified lysate at pH of 7.0 ± 0.3, under stirring conditions in a ratio of 1:1.5 (v/v) for maturation of VLP particles. This lysate suspension was incubated overnight for about 12 to 18 hours at 25 to 35 °C to assist self-assembly of VLP. The lysate supernatant after incubation was centrifuged at 8500 to 10000 rpm at 2 to 8 °C for 20 to 30 minutes. Series of column chromatographies such as cation exchange column chromatography followed by mixed mode column chromatography were performed to purify centrifuged clarified lysate (VLPs). Finally, diafiltration was performed with tangential flow filtration (TFF) system using 500 kDa cut off membrane to obtained purified VLPs followed by 0.2 µm filtration.
Sterile Filtered Bulk(SFB) was prepared by addition of Al(OH)3 gel at a ratio of 1:1 (w/w) with total protein in SFB (HPV VLPs) to adsorb the antigens (HPV VLPs) on Al(OH)3 gel. Then required quantity of Dextrose solution was added to get final concentration of dextrose to 10 % w/v. After that Polysorbate 80 was added to a final concentration of 0.01%(w/v). The adsorbed SFB (Drug substance) was stored at 2 to 8 °C.

Example 4: Clone development for hepatitis E virus (HEV) antigen
The immunodominant region of pORF2 of wild type hepatitis E virus was used for constructing the gene of interest. The nucleotide sequence was codon optimized and synthesized by Gene Art (Germany). The gene was cloned between NdeI and BamHI restriction sites of pET30a (+) vector. The figure 5 represents pET30a (+) vector. For transformation of recombinant plasmid into E. coli B21 (DE3) cell, the competent B21 (DE3) cells were thawed on ice and transformation with the plasmids was performed by heat shock method. 100 µL of Luria-Bertani broth (LB) media was added and incubated in shaking condition for 37 ºC at 180 rpm for 2 hours. E. coli B21 (DE3) cells were plated on the LB agar plates with kanamycin (50 µg/mL) and incubated overnight at 37 ºC. Positive clones were selected using colony PCR. The master cell bank of selected clones of HEV antigens was prepared.

Example 5: Fermentation for hepatitis E virus (HEV) antigen production
The scale up production of the HEV clones as prepared in example 4 was performed in fed batch mode. 1 mL of culture from one master cell bank vial was inoculated in 100 mL primary seed containing defined media and was incubated at 37°C and 200 rpm for 12 to 14 hours to achieve an OD of = 2.0. 40 mL from this primary seed was used to inoculate secondary seed containing 800 mL of defined media and this was incubated at 37 °C and 200 rpm for 7 to 10 hours to achieve an OD of = 3.0. The secondary seed was inoculated in 20 L fermenter containing defined medium. The culture was grown at 37°C at controlled pH of 6.8 with dissolved oxygen concentration maintained at 40% by a cascade of agitation and oxygen enrichment. Once OD reaches about 20 to 22, feed was started to maintain the exponential growth. Once OD reaches =80, culture was induced with 1mM IPTG. Batch was harvested 4 hours post induction.

Example 6: Purification of recombinant hepatitis E virus (HEV) antigen
The harvested cell culture of HEV antigen (cell pellets of HEV antigen) as prepared in example 5, was resuspended in Lysis buffer (20 mM Tris-HCl, 10 mM EDTA, 0.5% Tween 20, pH 8.50) and lysed with a cell disruptor using two passes at pressure of 16 –18 Kpsi. To collect inclusion bodies (IB), the lysed pellet was centrifuged at 8000 to 8500 rpm for 35 to 45 minutes at 2 to 8°C. The IB was washed with water and then solubilized in solubilization buffer containing 50 mM Tris-HCl and 6.5M Urea (pH 8.5) added in a ratio of 1:5 followed by incubation at 4 to 10 °C for 45 minutes and performed centrifugation. After centrifugation, supernatant was loaded on anion exchange (AEX) column chromatography. The elutes obtained after AEX, was diluted with 1 X PBS for urea removal and incubated for 10 to 20 hours at 2 to 8 °C. Finally, diafiltration was performed with 1 X PBS with tangential flow filtration (TFF) System using 10 kDa cut off membrane to obtained purified recombinant HEV antigen. Purified recombinant HEV antigen was filtered using 0.22 µ filter and stored at -70 °C (minus 70 degree celcius).

Example 7: Characterization studies
SDS PAGE Analysis of purified antigens
Denaturing polyacrylamide gel electrophoresis (PAGE) was used to analyze the purity of the HPV and HEV antigen molecules (antigens). The molecular weight of HPV L1 monomer is ~55,000 Da and HEV antigen is ~ 26000 Da. All antigens were analysed in a 12 % SDS- PAGE under reduced condition. For preparation of antigen sample for SDS-PAGE, required amount of antigen was mixed with reducing dye .The sample were heated at 95 °C for 5 minutes. The samples were loaded on to SDS-PAGE and the gel was run constant volt of 80 V. After that the bands were visualized by silver staining using standard protocol. The molecular weight of a protein was estimated on the basis of protein marker loaded along with the samples and its purity was estimated by analysing the gel by densitometry. The figure 6 represents SDS PAGE gel of HPV and HEV antigens.
TEM Analysis of HPV6L1, HPV11L1, HPV16L1 and HPV18L1 VLPs:
3µl of the sample at a concentration of 0.05 µg/µl was applied on to a carbon-coated grid and negatively stained with 2% uranyl acetate for transmission electron microscopy (TEM). Grids were analyzed using a transmission electron microscope CM200 FEG (FEI) operating at 200 kV. Pictures were taken at a magni?cation of 100,000X using a slow scan charge-coupled-device (2,000 X 2,000) camera. Figure 7 represents TEM analysis of HPV antigens.

Example 8: Preparation of vaccine formulations
Steps of Formulation: Hepatitis E antigen to be formulated one day in advance before making combination with HPV antigens. Next day first HPV antigen to be taken followed by the Hepatitis E antigen addition for making the formulations or vice versa. After that Aluminum concentration to be adjusted as required followed by addition of other excipients. The pH of the formulation can be set between 6.0 to 8.0. Various vaccine formulations can be prepared from following Table 2:
Table 2
Category Ingredients Amount (Unit)
Drug substance 1 One or more of HPV6L1, HPV11L1, HPV16L1, HPV18L1, HPV31L1, HPV33L1, HPV45L1, HPV52L1 , HPV58L1 or any other HPV L1 serotype 10 µg to about 100 µg in one single human dose. (Total amount of all HPV L1 antigen together is in the range of 20 µg to about 1000 µg)
Drug substance 2 HEV antigen 10 µg to about 100 µg in one single human dose
Excipient Aluminium Between about 250 µg to about 1000 µg in one single human dose.
Excipient MF-59, TLR3 agonist selected from Poly(I:C), TLR 4 agonist selected from Monophosphoryl Lipid A like 3-O-desacyl-4’-monophosphoryl lipid A (MPL) or GLA and like, TLR5 agonist selected from Flagellin, TLR7 agonist selected from Gardiquimod and Imiquimod, TLR7/8 agonist selected from R848, NOD2 agonist selected from N-glycolyl-MDP, CpG-containing nucleic acid (where the cytosine is unmethylated), QS21 (saponin adjuvant), stable oil in water emulsions like Sepivac SWE™ SWE™ that induces both humoral and durable effector T-Cell responses, interleukins, beta-sitosterol and the like. 10 µg to about 2000 µg.
Excipient 2-Phenoxyethanol, thiomersal, sodium borate, antibiotics, gelatin, monosodium glutamate. 2.5 mg to about 5 mg per single human dose.
Excipient one or more gelatins selected from animal/human-derived gelatins such as human collagen, and / or fish gelatin or any combination thereof, one or more amino acids selected from glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenyl alanine, tryptophan, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine and histidine or any combination thereof, or one or more carbohydrates selected from dextrose, sucrose, fructose, dextran, glucose, glycerol, erythritol, threitol, avabitol, xylitol, ribitol, mannitol, galactitol, fucitol, iditol, inositol, volemitol, isomalt, lacitol, sorbitol, maltotriitol, maltotetraitol, polyglyitol or dextrin, or isosorbide or any combination thereof, polysorbate such as polysorbate 20 or polysorbate 80 or any combination thereof, sodium glutamate, sodium chloride. 0.001 % w / v to 10 % w / v
and / or
0.05 mg to 5 mg in single human dose.
Excipient One or more alcohol, propylene glycol, fatty alcohols, triglycerides, fatty acid esters, mineral oils, liquid petrolatum, isopropyl palmitate, polyethylene ethanol, sodium lauryl sulfate, an anti-oxidant, a humectant, a viscosity stabilizer or modifier. 0.001 % w / v to 10 % w / v
and / or
0.05 mg to 5 mg in single human dose.

Example 9: Preparation of combination of HPV6L1, HPV11L1, HPV16L1 and HPV 18L1 alum adsorbed formulation

Components Concentration per dose
HPV 6L1 antigen 20 µg
HPV 11L1 antigen 40 µg
HPV 16L1 antigen 40 µg
HPV 18L1 antigen 20 µg
Di Sodium Hydrogen Phosphate 4.887 mM
Potassium Dihydrogen Phosphate 4.41 mM
Sodium Chloride 19 mg
Dextrose 50 mg
Aluminium in form of Aluminium Hydroxide adjuvant 0.5 mg
Polysorbate 80 50 µg
Water for injection q. s. to 0.5 mL

The pre-sterilized glass bottle was taken and a sterile magnet bar was put under Biosafety cabinet. Adsorbed bulk of HPV 6L1, HPV 11L1, HPV16L1 and HPV18L1, mentioned in example were taken and added to the bottle in the required quantity followed by addition of dextrose and Polysorbate 80 to final concentration of 10% and 50 µg per dose respectively. After that, additional amount of Aluminium Hydroxide adjuvant and Sodium chloride solution were added to the final desired concentration of 0.5 mg and 19 mg per dose. The phosphate buffered was added to make up the final volume. The formulated bulk was kept on magnetic stirrer with continuous stirring at 100-200 RPM in cold room (2-8°C) for not less than 12 hours. The formulated bulk was then filled in vaccine vials and stored at temperature between +2° C and +8°C.

Example 10: Preparation of HEV monovalent alum adsorbed formulation

Components Concentration
HEV antigen 30 µg
Disodium Hydrogen Phosphate 4.887 mM
Potassium Dihydrogen Phosphate 4.41 mM
Sodium Chloride 4.25 mg
Potassium Chloride 0.2 mg
Aluminium adjuvant 0.8 mg
Water for injection q. s. to 0.5 mL

The required quantity of recombinant Hepatitis E Virus (HEV) antigen was thawed at 2-8°C before the formulation activity. The pre-sterilized glass bottle was taken and a sterile magnet bar was put under Biosafety cabinet. At first, required amount of Aluminium hydroxide gel was added to the bottle followed by required quantity of Hepatitis E Virus (HEV) antigen polypeptide. After that, required quantity of phosphate buffered saline was added to the bottle as per formulation calculation sheet. The formulated bulk was kept on magnetic stirrer with continuous stirring at 100-200 RPM in cold room (2-8°C) for not less than 12 hours. The formulated bulk was then filled in vaccine vials and stored at temperature between +2° C and +8°C.

Example 11: Preparation of vaccine formulation in alum and Sepivac SWE™
Components Concentration per dose
HEV antigen 30 µg
HPV 6L1 antigen 20 µg
HPV 11L1 antigen 40 µg
HPV 16L1 antigen 40 µg
HPV 18L1 antigen 20 µg
Di Sodium Hydrogen Phosphate 4.887 mM
Potassium Dihydrogen Phosphate 4.41 mM
Sodium Chloride 9.56 mg
Dextrose 50 mg
Aluminium in form of Aluminium Hydroxide adjuvant 0.8 mg
Polysorbate 80 50 µg
Sepivac SWE™ 0.25 mL
Water for injection q. s. to 0.5 mL

A pre-sterilized glass bottle was taken and a sterile magnet bar was put under Biosafety cabinet. At first, required amount of Aluminium hydroxide gel was added to the bottle followed by required quantity of Hepatitis E Virus (HEV) antigen polypeptide. The formulation container was kept at 2 to 8°C for overnight stirring. After that required amount of dextrose solution and polysorbate 80 solution was added to the adsorbed HEV component followed by addition of required quantity of HPV 6L1, HPV 11L1, HPV 16L1 and HPV 18L1 antigen in order with continuous stirring. Finally, required amount of Sepivac SWE™ emulsion (contains squalene 4.1% w/w, Sorbitan trioleate 0.5% w/v, Polysorbate 80 0.5% w/v, Sodium Citrate buffer 10 mM, pH 6.5) adjuvant was added to the HEV-HPV formulation. The formulated bulk was kept at stirring for at least 15 minutes after addition of Sepivac SWE™ emulsion. As per formulation calculation, 1X phosphate buffer containing 0.3M NaCl was added to make up the volume to final batch size while keeping the formulation container at stirring. After completion of formulation process, the vials were aseptically filled with HPV-HEV (adsorbed) combination vaccine while keeping the formulation container on stirring during the entire filling procedure. The vaccine vials were stored at 2 to 8 °C.

Example 12: In-vivo Immunogenicity study of HPV and HEV antigens of combination vaccine
To perform the mouse potency test for the combination vaccines and control vaccines as prepared in above examples 9, 10 and 11, the vaccine sample was diluted at 1:4 (used for calculating HEV potency) and 1:10 (used for calculating HPV potency) with diluent and injected in 6 to 8 weeks old female BALB/c mice. Each mouse was given a 0.5-mL injection subcutaneously. A preimmunization serum sample was taken prior to inoculation, and a final bleed was taken on Day 28 to collect serum samples. The sample were diluted using placebo. The resulting sera were tested in using in-direct ELISA assay. Type-specific HPV VLPs and HEV antigens were diluted in phosphate buffered saline (PBS) and 100 µL of the diluted HPV or HEV antigens were added to each well of a 96-well microtiter (Nunc Maxisorp Immunoplates) for coating. The plate was incubated overnight at 4 °C and washed thrice using PBST buffer. The plate was then blocked with 200 µL per well of 5 % skimmed milk prepared in PBS buffer. Once the test plates were prepared, serum samples were diluted 1:100 in PBS containing 1% skimmed milk. Both pre and post-immunization serum samples were analyzed to calculate fold increase. The samples were allowed to incubate for approximately one hour at 37 °C. The plates were then washed three times in PBST. Next, 100 µL of goat-anti-mouse IgG-horse radish peroxidase conjugate was added to each well at a dilution of 1:2000 in PBST containing 1% skimmed milk and allowed to incubate for one hour at 37°C. The plates were washed again three times and 100 µL of tetramethylbenzidine (TMB) was added. TMB was allowed to react with the horseradish peroxidase for 15 minutes at room temperature before the reaction was stopped by the addition of 100 µL of 0.2 N sulfuric acid. The Plates were read at an optical density at 450 nm. The fold change for the each of the samples were calculated using pre-immune sera as cut-off. The significant antibody titers were observed against all HPV antigens i.e. HPV 6L1, HPV 11L1, HPV 16L1 and HPV 18L1 and HEV antigen as mentioned in figures 10 to 11. Further, the titre was comparable to the control formulation of HPV and HEV antigens (prepared as explained in example 9 and 10), results of which are shown in figures 8 and 9. This shows that there is no interference of immune responses upon combining both types of antigens to prepare the HPV and HEV combination vaccine.

Incorporation by reference
The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes.

Equivalents
The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

References
1. “VLP stabilized vaccine compositions” International patent publication no. WO2017041083; published on March 09, 2017.
2. “Immunogenicity and Safety of Co-immunization With Cecolin and Hecolin”; ClinicalTrials.gov Identifier: NCT05415345; Phase 4; Last update posted on June 13, 2022.
3. Shao W. Li et al. “A bacterially expressed particulate hepatitis E vaccine: antigenicity, immunogenicity and protectivity on primates” Vaccine 23 (2005) 2893–2901.
4. “Optimized expression of HPV 52 L1 in yeast” International patent publication no. WO2005097821; published on October 20, 2005.

,CLAIMS:We claim:

1. A vaccine composition comprising human papilloma (HPV) virus like particles (VLPs) in combination with hepatitis E virus antigen (HEV) and suitable excipients.

2. The vaccine composition as claimed in claim 1 wherein human papilloma virus (HPV) antigen is HPV L1.

3. The vaccine composition as claimed in claim 1 wherein human papilloma virus (HPV) antigen is selected from atleast one serotypes of HPV L1 selected from HPV6L1, HPV11L1, HPV16L1, HPV18L1, HPV31L1, HPV33L1, HPV45L1, HPV52L1 or HPV58L1 or any suitable combinations thereof.

4. The vaccine composition as claimed in claim 1 wherein human papilloma virus (HPV) antigen is selected from atleast two serotypes of HPV L1 selected from HPV6L1, HPV11L1, HPV16L1, HPV18L1, HPV31L1, HPV33L1, HPV45L1, HPV52L1 or HPV58L1 or any combination thereof.

5. The vaccine composition as claimed in claim 1 wherein human papilloma virus (HPV) antigen is selected from atleast three serotypes of HPV L1 selected from HPV6L1, HPV11L1, HPV16L1, HPV18L1, HPV31L1, HPV33L1, HPV45L1, HPV52L1 or HPV58L1 or any combination thereof.

6. The vaccine composition as claimed in claim 1 wherein human papilloma virus (HPV) antigen is selected from atleast four serotypes of HPV L1 selected from HPV6L1, HPV11L1, HPV16L1, HPV18L1, HPV31L1, HPV33L1, HPV45L1, HPV52L1 or HPV58L1 or any combination thereof.

7. The vaccine composition as claimed in claim 1 wherein human papilloma virus (HPV) antigen is selected from atleast five serotypes of HPV L1 wherein HPV L1 is selected from HPV6L1, HPV11L1, HPV16L1, HPV18L1, HPV31L1, HPV33L1, HPV45L1, HPV52L1 or HPV58L1 or any combination thereof.

8. The vaccine composition as claimed in claim 1 wherein human papilloma virus (HPV) antigen is selected from atleast six serotypes of HPV L1 wherein HPV L1 is selected from HPV6L1, HPV11L1, HPV16L1, HPV18L1, HPV31L1, HPV33L1, HPV45L1, HPV52L1 or HPV58L1 or any combination thereof.

9. The vaccine composition as claimed in claim 1 wherein human papilloma virus (HPV) antigen is selected from atleast seven serotypes of HPV L1 wherein HPV L1 is selected from HPV6L1, HPV11L1, HPV16L1, HPV18L1, HPV31L1, HPV33L1, HPV45L1, HPV52L1 or HPV58L1 or any combination thereof.

10. The vaccine composition as claimed in claim 1 wherein human papilloma virus (HPV) antigen is selected from atleast eight serotypes of HPV L1 wherein HPV L1 is selected from HPV6L1, HPV11L1, HPV16L1, HPV18L1, HPV31L1, HPV33L1, HPV45L1, HPV52L1 or HPV58L1 or any combination thereof.

11. The vaccine composition as claimed in claim 1 wherein human papilloma virus (HPV) antigen is selected from atleast nine serotypes of HPV L1 wherein HPV L1 is selected from HPV6L1, HPV11L1, HPV16L1, HPV18L1, HPV31L1, HPV33L1, HPV45L1, HPV52L1 or HPV58L1 or any other HPV L1 serotype or any combination thereof.

12. The vaccine composition as claimed in claim 1 wherein human papilloma virus (HPV) antigen comprises HPV6L1, HPV11L1, HPV16L1 and HPV18L1.

13. The vaccine composition as claimed in claim 1 wherein human papilloma virus (HPV) antigen comprises HPV6L1 having amino acid sequence as set forth in SEQ ID No: 1, HPV11L1 having amino acid sequence as set forth in SEQ ID No: 2, HPV16L1 having amino acid sequence as set forth in SEQ ID No: 3 and HPV18L1 having amino acid sequence as set forth in SEQ ID No: 4.

14. The vaccine composition as claimed in claim 1 wherein hepatitis E virus antigen (HEV) is hepatitis E virus open reading frame 2 (HEV ORF2).

15. The vaccine composition as claimed in claim 1 wherein hepatitis E virus antigen (HEV) is HEV ORF2 having amino acid sequence as set forth in SEQ ID No: 5
16. The vaccine composition as claimed in claim 1 wherein vaccine composition comprises human papilloma virus (HPV) antigen and hepatitis E virus (HEV) antigen wherein HPV antigen comprises HPV6L1 having amino acid sequence as set forth in SEQ ID No: 1, HPV11L1 having amino acid sequence as set forth in SEQ ID No: 2, HPV16L1 having amino acid sequence as set forth in SEQ ID No: 3, HPV18L1 having amino acid sequence as set forth in SEQ ID No: 4 and the HEV antigen is HEV ORF2 having amino acid sequence as set forth in SEQ ID No: 5.

17. The vaccine composition as claimed in claim 1 wherein suitable excipients are selected from adjuvants, preservatives, stabilizing agents and pharmaceutically acceptable carriers or any combination thereof.

18. The vaccine composition as claimed in claim 1 wherein suitable excipients comprises adjuvants wherein the adjuvants are selected from aluminum hydroxide gel, alum, alum in combination with MF-59, TLR3 agonist Polyinosinic:polycytidylic acid, TLR 4 agonist selected from Monophosphoryl Lipid A like 3-O-desacyl-4’-monophosphoryl lipid A or Glucopyranosyl Lipid Adjuvant, TLR5 agonist Flagellin, TLR7 agonist selected from Gardiquimod and Imiquimod, TLR7/8 agonist Resiquimod, Nucleotide-binding Oligomerization Domain 2 agonist N-glycolyl muramyl dipeptide, TLR9 agonist CpG-containing nucleic acid, saponin adjuvant QS21, stable oil in water emulsions like Sepivac SWE™, interleukins and beta-sitosterol.

19. The vaccine composition as claimed in claim 1 wherein suitable excipients comprises preservatives wherein the preservatives are selected from thiomersal, 2- Phenoxyethanol, sodium borate or any combination thereof.

20. The vaccine composition as claimed in claim 1 wherein suitable excipients comprises stabilizing agents wherein the stabilizing agents are selected from one or more gelatins selected from animal-derived gelatins such as human collagen, and / or fish gelatin or any combination thereof, one or more amino acids selected from glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenyl alanine, tryptophan, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine and histidine or any combination thereof, or one or more carbohydrates selected from dextrose, sucrose, fructose, dextran, glucose, glycerol, erythritol, threitol, avabitol, xylitol, ribitol, mannitol, galactitol, fucitol, iditol, inositol, volemitol, isomalt, lacitol, sorbitol, maltotriitol, maltotetraitol, polyglyitol or dextrin, or isosorbide or any combination thereof, polysorbate such as polysorbate 20 or polysorbate 80, sodium glutamate, sodium chloride, buffers selected from acetate buffer, phosphate buffer, succinate buffer, tris buffer, histidine buffer, preferably phosphate buffer.

21. The vaccine composition as claimed in claim 1 wherein suitable excipients comprises pharmaceutically acceptable carriers wherein the pharmaceutically acceptable carriers are selected from the group consisting of alcohol, propylene glycol, fatty alcohols, triglycerides, fatty acid esters, mineral oils, liquid petrolatum, isopropyl palmitate, polyethylene ethanol, sodium lauryl sulfate, an anti-oxidant, a humectant, a viscosity stabilizer or modifier, a colorant or a flavouring agent.

22. The vaccine composition as claimed in claim 1 wherein vaccine composition has pH between 6.0 to 8.0.

23. The vaccine composition as claimed in claim 1 wherein the vaccine composition comprises human papilloma virus (HPV) in an amount between about 10 µg to about 100 µg.

24. The vaccine composition as claimed in claim 1 wherein the vaccine composition comprises hepatitis E virus (HEV) in an amount between about 10 µg to about 60 µg.

25. The vaccine composition as claimed in claim 1 wherein the vaccine composition comprises total amount of human papilloma virus (HPV) and hepatitis E virus (HEV) is between about 50 µg and about 1000 µg.

26. The vaccine composition as claimed in claim 1 comprises HEV antigen at 30 µg per dose, HPV 6L1 antigen at 20 µg per dose, HPV 11L1 antigen at 40 µg per dose, HPV 16L1 antigen at 40 µg per dose, HPV 18L1 antigen at 20 µg per dose, Di Sodium Hydrogen Phosphate at 4.887 mM per dose, Potassium Dihydrogen Phosphate at 4.41 mM per dose, Sodium Chloride at 9.56 mg per dose, Dextrose at 50 mg per dose, Aluminium in form of Aluminium Hydroxide adjuvant at 0.8 mg per dose, Polysorbate 80 at 50 µg per dose, Sepivac SWE™ 0.25 mL and water for injection q. s. to make upto 0.5 mL.

27. The vaccine composition as claimed in claim 1 wherein vaccine composition is a liquid or a lyophilized powder.

28. The vaccine composition as claimed in claim 1 wherein vaccine composition is a lyophilized powder which is reconstituted to an aqueous or non-aqueous liquid prior to administration to the patient.

29. The vaccine composition as claimed in claim 1 wherein atleast two dose of vaccine composition is administered in a human to treat any infection or disease occur by hepatitis E virus and human papilloma virus.

30. The vaccine composition as claimed in claim 29 wherein the second dose is administered atleast 1 month after the first dose.

31. The vaccine composition as claimed in claim 29 wherein the second dose is administered 6 month after the first dose.

Dated this 13th day of June 2024.

(HARIHARAN SUBRAMANIAM)
IN/PA-93
Of SUBRAMANIAM & ASSOCIATES
ATTORNEYS FOR THE APPLICANTS