EP3191505A2 - Superior human papilloma virus antigens with superior immunological properties and vaccine containing it - Google Patents

Abstract

The present invention provides genes encoding various HPV antigens of several serotypes. The gene of the present invention produce virus like particles with an improved immunological quality and quartnary structure. The present invention also provides suitable host cell preferably P.pastoris with high copy number of genes encoding various HPV antigens of several serotypes. The present invention further provides vaccine against HPV antigens with improved immunization schedule wherein vaccine dosage regiment and amount of antigen dose are reduced. The present invention also provides an improved human papilloma virus vaccine composition using yeast.

Description

SUPERIOR HUMAN PAPILLOMA VIRUS ANTIGENS WITH SUPERIOR IMMUNOLOGICAL PROPERTIES AND VACCINE CONTAINING IT

FIELD OF THE INVENTION

The present invention is directed to genes encoding human papilloma virus antigens and use of it in the development of vaccine, in particular for the prevention of human papilloma virus infection in human beings. The present invention also provides an improved human papilloma virus vaccine composition using yeast.

BACKGROUND OF THE INVENTION

Worldwide, cervical cancer is the second most common cancer in women, accounting for 274,000 deaths each year. The incident rate of cervical cancer in India is approximately 132,000 per year; which is nearly 52% of the recorded incidence of the disease in the Asia-Pacific region. Nearly 73,000 women die of cervical cancer each year in India; thereby it holds the dubious distinction of accounting for nearly a quarter of all cervical cancer deaths recorded globally. It is estimated that by the year 2025, the incidence rates in the developing world would account for nearly 80% of the global rates. Human papillomavirus (HPV) is an epitheliotropic double stranded DNA virus of approximately 8 kb in size. The HPV genome encodes for non-structural-proteins (El, E2, E4, E5, E6 and E7) and structural proteins (LI and L2). There are over 120 HPV types reported. Persistent infection by high-risk HPV types is the single most important factor for the induction of the cervical cancer. Amongst high-risk HPV types, HPV16 and 18 are the genotypes most frequently associated with cervical cancer across the world. Overall HPV type prevalence in cervical cancer in India was found to be in the following order, HPV 16, 18, 31, 33, 35, 39, 45, 52, 56, 58, 59 and HPV68. Together, HPV 16 and HPV 18 types contribute to nearly 80% of the entire uterine cervical cancer incidence in India.

Currently, there are two cervical cancer vaccines used globally in the market: quadrivalent HPV (qHPV) vaccine, produced by Merck & Co. Inc. (Gardasil®, Merck, Rahway, NJ, USA); and bivalent HPV vaccine, produced by GlaxoSmithKline pic. (CervarixTM, GSK, Middlesex, UK). Gardasil was approved by the U.S. Food and Drug Administration (FDA) in June 2006, which protects against four strains of HPV - HPV 6, 11, 16 and 18. Although there are over 100 strains of HPV, certain types are particularly likely to be linked to cancer; strains 16 and 18 are associated with 70 percent of all cervical cancer cases in the U.S. Strains 6 and 1 1 are associated with 90 percent of genital warts. In clinical trials, the vaccine showed high efficacy in preventing persistent infection, precancerous lesions and external genital lesions from the four strains ^"among females who had not been previously infected. A subset of women from the trial have been followed for five years and have shown vaccine efficacy of 95.8 percent against persistent infection or disease and 100 percent efficacy against precancerous and external lesions. Further follow-up studies are needed to determine the duration of protection, but research has demonstrated that younger adolescent females aged 10-14, show a stronger immune response to the vaccine than females aged 15-24, suggesting that earlier vaccination may result in longer antibody persistence. Other recent research has shown that the vaccine also provides protection against vaginal and vulvar cancer. According to the Alan Guttmacher Institute, the HPV vaccine "is widely considered one of the greatest health care advances for women in recent years."

Unlike qHPV vaccine, bivalent HPV vaccine is composed of only two antigens: HPV types 16 and 18. For the synthesis of its LI proteins, genes are cloned into the baculovirus expression vector and the manufactured vector is infected into the insect cell, Trichoplusiani cell (High Five), followed by incubation in the serum-free media for two days; then the cells are harvested.

The currently available and evaluated HPV vaccines target preventing infection by HPV types 16 and 18. HPV vaccines are prepared from virus-like particles (VLPs) produced by recombinant technology and are given as three 0.5 ml intramuscular injections over a six-month period. Recent results indicate that HPV vaccines are highly immunogenic inducing high levels of serum antibodies in almost all vaccinated women, and have conferred a high degree of protection against HPV- 16/ 18 infection and thus the associated precancerous cervical lesions in fully vaccinated women.^' A quadrivalent (HPV 6/11/16/18) vaccine, evaluated in 27,000 women in 33 countries, has proved to be effective in preventing more than 99 per cent of persistent infections.

The vaccine, according to the present invention, is produced using recombinant technology as used in the development of earlier HPV vaccines. Major steps involved to get the desired protein product by using recombinant technology are cloning of desired gene into an expression vector, expression of gene into host cell (it can be referred to as an expression system.), purification and characterization of protein product obtained from the inserted gene. Methods and components generally used at each above mentioned steps are well available in the prior art. All these mentioned steps have some limitations and dependences on components which are generally used therein such as an expression vector, host cell, nucleotide sequences, etc. A person skilled in the art has to analyze compatibility of every component with each other in the recombinant technology. The present invention provides human papilloma virus vaccine composition with novel codon optimized gene encoding HPV protein of late HPV proteins of four different HPV types HPV 16, HPV 18, HPV 6 and HPV 11 using yeast as an expression system. It is well known that combination of strain HPV 16 and HPV 18 may protect only 70 % of all circulating HPV strains. It is therefore advisable to add additional antigens to a vaccine. Since there are epidemiological differences from one region to another in the world, it is advisable to include more serotypes providing broader protection in the relevant region of the world (For India, HPV 33 and HPV 45). It has been described that L2 antigens together with LI antigens will broader the coverage of protection. In vivo seroconversion response of the Human Papilloma vaccine developed according to the present invention is found much superior to existing reference product.

In the present field of the invention, it is available in the art that P.pastoris (KM71 strain) transformed with the same plasmid containing the wild-type HPV 16 LI gene did not express the LI protein indicating the need for codon optimization for HPV 16 LI expression in P.pastoris.

[Bazan et al, Arch Virol. 2009; 154(10); pl-16] This article describes codon optimization of HPV 16 LI gene and cloned it into a non-integrative vector and used further to transform competent Pichia pastoris cells. Bazan et al discloses that the VLPs appeared more regular and defined (45-50 nm). It is known that for large-scale production episomal vectors (non-integrative vector) would not be advantageous since the transformed yeast can lose these vectors after successive mitotic divisions because they are not integrated into the genome and thus can be lost in the absence of selection pressure. Codon-optimization genes are required to get the expression in selected expression system. This technique is well known in the art for any antigen with suitable expression system. For example, N. Hanumantha Rao et al, Vaccine 29 (201 1) 7326- 7334 describes an expression of codon optimized major capsid protein (LI) of human papillomavirus type 16 and 18 in Pichia pastoris. Also, Hanumantha Rao et al disclose that "The purified VLPs of HPV 16 and HPV 18 showed variable particle size having a mean of approximately 53 nm. ^v Here, HPV genes are cloned and expressed in P.pastoris strain GS115.

These articles do not provide information regarding improvisations in immunogenic properties of their VLPs. Increased immunogenicity of VLPs can be achieved by increasing the size of VLPs which allows higher number of repetitive antigen presentation. Ultimately, it will provide better immune response against HPV antigens.

Therefore, the current invention provides codon-optimization of HPV LI gene for different serotypes which provides self-assembled VLPs with improved immunological quality and finally substantially higher immunogenic response against HPV antigens. In the same manner, codon-optimization of HPV L2 gene, early antigens of HPV (E6 and E7) can be generated to develop the vaccine against HPV antigens.

Although selecting optimized genes for the expression of said gene in a specific host is known technique in the art, there are still a lot of possible variations in the optimization. Methods for codon-optimization are not universal. Thus, every gene has unique codon optimization method. We have mentioned here below few patent applications which are part of the prior art of the present invention.

1066 MUMNP/2010 discloses codon-optimized genes encoding major capsid protein LI of human papilloma virus. A macromolecule with immunogenicity, which is produced by expression of the codon-optimized genes encoding major capsid protein LI of human papilloma virus in yeast cells, the uses and the composition thereof are provided. VLPs obtained by the method described in this application have size in the range of about 50 to 80 nm

IN 203333 discloses a vaccine composition comprising virus like particles containing LI proteins or functional LI protein derivatives from human papilloma virus 16, human papilloma virus 18, human papilloma virus 31 and human papilloma virus 45 genotypes, wherein the antibody immune response generated by the vaccine is at a level similar to that for each human papilloma virus, virus like particle formulated alone. This patent discloses that HPV 16/18 proteins were expressed in Trichoplusia ni (High Five™) cells (at a density of- 350000 cells/ml) infected with recombinant Baculdvirus (MOI of 0.3) encoding the HPV 36 or 18 LI gene of interest. Here, preferred adjuvant is aluminum hydroxide in combination with 3D-MPL is disclosed. Use of an insect cell includes risk of impurities such as like host cell DNA and host cell proteins.

IN 245189 discloses an immunogenic composition comprising VLPs or capsomers from HPV 16 and 18 and at least one other HPV cancer type, the other cancer type being selected from the list consisting of HPV types 31, 45 and 52, wherein the dose of the VLP or capsomer of the at least one other cancer type is reduced relative to that of HPV 16 or 18. Here, inventors have used two adjuvants namely aluminum hydroxide and 3D MPL in formulation. These adjuvants help in inducing immunogenicity of the antigens.

The above mentioned prior patent applications depicts that lot of work has been done in the development of vaccine against HPV antigens using yeast as an expression system. P. pastoris is the second most used system for heterologous expression after the bacteria Escherichia coli. Still selection of P. pastoris as a host to express desired antigen is not enough to get the higher expression of desired antigen and subsequently one good candidate for the development of vaccine against HPV. It is well understood from the reference mentioned above [Bazan et al]. Therefore, there is need of codon-optimization of desired antigen according to host cell to get the expression of inserted gene of interest.

Thus, there are several challenges and uncertainties that need to be resolved before HPV vaccination can be widely implemented in low-resource countries. The challenges include: current high costs of the available vaccines, feasibility, acceptability, logistics of vaccine delivery (in view of the need for three doses spread over 6 months, improved strategies and vaccine platforms to reach out to pre- or early-adolescent girls), long-term immunogenicity and efficacy in preventing cervical neoplasia, cross-protection against HPV infections not targeted by the vaccine antigens and the need for more logistically feasible dose regimes in inducing and maintaining immunogenicity and long- term protection against cervical neoplasia. These issues are critical for adequate support for a global acceptance and momentum for the introduction of HPV vaccines in public health services.

Inventors of the current invention have tried to solve majority of the problems related to expression yield, immunogenicity, dose regimes and cost of HPV vaccine. In the present invention, it is shown that the vaccine preparation that delivers higher immunogenicity with lower doses of HPV LI antigens by only one time immunization in mice. The present invention provides novel genes encoding LI protein of various serotypes of HPV which will ultimately provide surprisingly higher immune response against HPV antigens. Thus, these candidates can be used in the development of vaccine against HPV that will solve major issues related to HPV vaccine. The current invention also provides an immunogenic composition comprising virus like particles containing LI proteins from HPV 16, HPV 18, HPV 6, HPV 11 and at least one another genotype selected from HPV 31, HPV 52, HPV 58 and HPV 45 genotypes with only one adjuvant. Furthermore, the current invention does not use an insect cell as an expression system. So, it reduces risk of insect derived host cell impurities like host cell DNA and host cell proteins which can be of safety concern (allergy). The immunogenic composition for other serotypes of HPV such as HPV 35, HPV 39, HPV 56, HPV 59 and HPV 68 can be prepared according to the method described in the present invention.

Moreover, the current invention provides VLPs with the higher size and with improved immunological quality which will provide substantially higher immune response against HPV antigens.

HPV vaccine commercially available are based on VLP's formation by HPV proteins either expressed in Yeast (Sacehromyces cervisiae ) or in Baculovirus expression system and their immunogenicity is directly correlated with their quaternary structure formation. [ Henryk Mach et al, Journal of pharmaceutical sciences, Vol. 95, No. 10, p 2195-2206] This article discloses that the expression of HPV type 6, 11 and 16 LI VLP proteins in Sacehromyces cervisiae yielded irregularly shaped, broadly distributed VLPs smaller in size (30-50 nm) than expected (60 nm).

In our invention, inventors have optimized dis/reassembly conditions to form improved VLPs by forming better quaternary structures leading to higher immunogenicity in different serotypes of HPV preferably HPV 16 LI, HPV 18 LI, HPV 6 LI and HPV 11 LI.

To achieve this, genes are codon optimized for different HPV serotypes (HPV serotypes preferably HPV 16 LI, HPV 18 LI, HPV 6 LI and HPV 1 1 LI) to express in yeast. The certain regions of genes were modified (as described herein below in the detailed description of the present application) to achieve post translational conformation dependent quaternary structure changes in VLPs.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavor to which this specification relates.

OBJECTS OF THE INVENTION

In first aspect, the present invention provides an isolated gene encoding different proteins of HPV of various serotypes, wherein said gene is codon optimized according to host cell.

In one of the aspects, different proteins of HPV include major capsid protein LI, minor capsid protein L2 and early antigens E6 and E7.

In another aspect, the present invention provides an isolated gene encoding LI protein of various serotypes preferably HPV 16, HPV 18, HPV 6, HPV 11 and the like.

In second aspect, the current invention provides virus like particles with improved immunological quality obtained as a result of codon optimized genes encoding different HPV antigens which are integrated into host cell.

In third aspect, the present invention provides host cell transformed with the said genes encoding different proteins of HPV.

In another aspect, the present invention provides host cell with high copy number of genes encoding different proteins of HPV of various serotypes.

: In further aspect, the present invention provides a vector containing genes encoding different proteins of HPV of various serotypes.

In fourth aspect, the present invention provides virus like particles with an improved quaternary structure obtained as a result of codon optimized genes encoding different HPV antigens which are integrated into host cell.

In fifth aspect, the current invention provides a vaccine against human papilloma virus with significantly higher immunogenicity.

In sixth aspect, the current invention provides a vaccine against HPV antigens with improved immunization schedule wherein vaccine dosage regimen is reduced to single time.

In another aspect, the current invention provides a vaccine against HPV antigens with substantially reduced amount of antigens.

In further more aspect, the current invention provides the vaccine against HPV antigens using yeast as an expression system.

In a preferred aspect, the current invention provides the vaccine against HPV antigens using P. pastoris as an expression system.

SUMMARY OF THE INVENTION

The present invention provides codon-optimized genes encoding various HPV antigens of several serotypes. Such genes further produce virus like particles with an improved immunological quality and quaternary structure. Such VLPs produce substantially higher immune response against HPV antigens.

Moreover, the present invention provides suitable host cell preferably P. pastoris with high copy number of genes encoding various HPV antigens of several serotypes.

Further, the current invention provides vaccine against HPV antigens with improved immunization schedule wherein vaccine dosage regiment and amount of antigen dose are reduced.

Furthermore, the current invention provides an immunogenic composition containing proteins of HPV antigens with suitable adjuvant(s).

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 depicts expression vector map pPICZa containing HPV 16L1 gene Figure 2 depicts in-process analysis of HPV 16 LI at various stages of production Figure 3 depicts identity analysis of HPV 16 LI by western blot method

Figure 4 depicts size-distribution analysis of HPV 16 LI VLPs by dynamic light scattering method (Average diameter of VLPs: 238.9 nm)

Figure 5 depicts final VLP formation by electron microscopy through negative staining method

Figure 6 depicts antibody response after four weeks by single immunization for HPV 16 LI antigen

Figure 7 depicts antibody response after four weeks by single immunization for HPV 18 LI antigen

Figure 8 depicts antibody response after four weeks by single immunization for HPV 11 LI antigen

Figure 9 depicts antibody response after four weeks by single immunization for HPV 6 LI antigen

Figure 10 depicts antibody titer against HPV 16L1 antigen measured by ELISA obtained after Primate immunization with the HPV vaccine of the present invention and reference vaccine compared

Figure 1 1 depicts antibody titer against HPV 18L1 antigen measured by ELISA obtained after Primate immunization with the HPV vaccine of the present invention and reference vaccine compared

Figure 12 depicts size-distribution analysis, of HPV 18 LI VLPs by dynamic light scattering method (Average diameter of VLPs: 388.4 nm)

Figure 13 depicts size-distribution analysis of HPV 6 LI VLPs by dynamic light scattering method (Average diameter of VLPs: 365 nm)

Figure 14 depicts size-distribution analysis of HPV 11 LI VLPs by dynamic light scattering method (Average diameter of VLPs: 307.9 nm)

DETAILED DESCRIPTION OF THE INVENTION

In one of the embodiments, the present invention provides an isolated gene encoding different proteins of HPV of various serotypes, wherein said gene is codon optimized according to host cell.

In another embodiment, different proteins of HPV include major capsid protein LI, minor capsid protein L2 and early antigens E6 and E7.

In a preferred embodiment, the present invention provides an isolated gene encoding LI protein of various serotypes such as HPV 16 LI, HPV 18 LI, HPV 6 LI and HPV 11 LI . In further embodiment, the current invention provides virus like particles with improved immunological quality obtained as a result of codon optimized genes encoding different HPV antigens which are integrated into host cell. Such VLPs deliver enhanced immune response for protection against HPV infections by different serotypes. VLPs with an improved immunological quality help to reduce the use of adjuvant(s) in the preparation of vaccine.

In one of the embodiments, the present invention provides host cell transformed with the said genes encoding different proteins of HPV. According to the present - invention, host cell can be termed as an expression system in which genes transformed with a suitable vector will express.

In another embodiment, the present invention provides host cell with high copy number of genes encoding different proteins of HPV of various serotypes. It leads to higher expression of HPV antigens of different serotypes.

In a preferred embodiment, host cell according to the present invention is yeast cell preferably P. pastoris.

In a more preferred embodiment, P. pastoris strains X-33, GS 1 15, KM71, SMD1168 or others can be used as an expression system.

In one of the embodiments, the present invention provides a vector containing genes encoding different proteins of HPV of various serotypes.

In a preferred embodiment pPICZ, pPIC6, pGAPZ, pA0815 or other similar vectors can be used as a vector in the present invention. These vectors are commercially available. In a preferred embodiment, the current invention provides vector transformed with the genes encoding different proteins of HPV can be selected from vectors having accession number MTCC 5969, MTCC 5970, MTCC 5971 and MTCC 5972. These vectors have been deposited by the applicant of the current invention under Budapest treaty. The said deposited vectors have genes developed according to the present invention. MTCC 5969, MTCC 5970, MTCC 5971 and MTCC 5972 refer to pPICzHPV 6L1, pPICzHPV 11L1 pPICzHPV 16L1 and pPICzHPV 18L1 respectively.

In further embodiment, the present invention provides virus like particles with an improved quaternary structure obtained as a result of codon optimized genes encoding different HPV antigens which are integrated into host cell. Such VLPs are of high diameter around 80-100 nm along with the VLPs of an average diameter between 50-80 nm. In a preferred embodiment, VLPs according to the present invention are of 50- 500 nm, preferably, 50-400 nm, more preferably 100-400 nm. In a preferred embodiment, the present invention provides vaccines containing immunogenic composition of the present invention for various antigens of HPV. These vaccines can be administered in conventional routes and dosages.

In one of the embodiments, the present invention provides method of preparing human papilloma vaccine comprising the following steps:

a. Synthesis of gene encoding major capsid protein of human papilloma virus b. Construction of expression vector for gene encoding major capsid protein of human papilloma virus

c. Selection of clone with high copy number of transformed gene of said protein of human papilloma Virus

d. Expression analysis of transformed gene of said protein of human papilloma virus

e. Purification of protein encoded by gene of said protein of human papilloma virus

f. Preparation of bulk solution of VLPs of said protein of human papilloma virus

g. Characterization of said protein in host cell

h. Preparation of vaccine containing said protein of human papilloma virus Here, major capsid protein according to the present invention is LI protein of various serotypes such as HPV 16 LI, HPV 18 LI, HPV 6 LI and HPV 1 1 LI . Host cell according to the present invention is yeast cell, preferably Pichia, more preferably Pichia pastoris.

In further embodiment, purification process according to the current invention is column chromatography, sterile filtration, diafiltration or suitable combination thereof.

In one of the embodiments, the present invention provides optimized dis/reassembly conditions to form improved VLPs by forming better quaternary structures leading to higher immunogenicity in different serotypes of HPV. VLPs received from polishing chromatography step are subjected to suitable pre-formulation buffer to get improved VLPs with better quaternary structure. The preferred pre- formulation buffer according to the present invention is phosphate buffer.

Pre-formulation buffer can be defined as buffer used to provide stability to VLPs for maintaining their structures for further formulation development with different adjuvants and combination thereof.

Such pre-formulation buffer may further comprise of suitable reducing agents (DTT, β- mercaptoethanol, tween 80 or others like), salts (NaCl or KC1), amino acids and acidic or basic buffers. -

In furthermore embodiment, the current invention provides a vaccine against human papilloma virus with significantly higher immunogenicity. The vaccine according to the present invention elicits protective immune response against different HPV serotypes.

In one of the embodiments, the current invention provides a vaccine against HPV antigens with improved immunization schedule wherein vaccine dosage regimen is reduced.

In another embodiment, the current invention provides a vaccine against HPV antigens with substantially reduced amount of antigens. It helps to reduce cost of preparation of the vaccine which makes it feasible to use at commercial level in developing countries.

In one of the embodiments, the present invention provides an immunogenic composition containing proteins of HPV antigens with suitable adjuvant.

Salts of aluminium, Mono Phosphoryl Lipid (MPL) analogues such as GLA

(glucopyranosyl lipid adjuvant), monatide, cytokine inducers, squalene based adjuvants, lipophilic adjuvants and others like may be used.

In a furthermore embodiment, the present invention provides a pharmaceutical composition comprising an immunogenic composition with pharmaceutically acceptable carrier or excipient.

EXAMPLES

The following non-limiting examples describe codon optimization of genes encoding LI protein of HPV of various serotypes expressively of HPV 16, HPV 18, HPV 6 and HPV 11. It will be appreciated that other immunogenic compositions with different antigens can be prepared and such immunogenic compositions are within the scope of a person skilled in the art and are to be included within the scope of the present invention. Example 1: Synthesis of codon-optimized gene for HPV 16 LI

Nucleotide sequences for HPV 16 LI antigens available at Genebank were collected. Alignment of amino acid sequence obtained from each nucleotide sequence collected from Genebank was done. After that, consensus sequence which is the most representative of amino acid sequence of HPV 16 LI protein was chosen. Nucleotide sequence was_. determined from chosen consensus amino acid sequence to use it further for codon-optimization according to host cell. This sequence is defined as SEQ ID NO. 1. SEQ ID NO. 1 : Nucleotide sequence of HPV 16 LI antigen (Gene bank No. gblGO423063. il^")

1 ATGTCTTTGT GGTTGCCATC TGAAGCTACT GTTTACTTGC CAGCAGTTCC AGTTTCTAAG

61 GTTGTTTCTA CTGATGAATA CGTTGCTAGA ACTAACATTT ACTACCATGC TGGTACTTCT

121 AGATTGTTGG CTGTTGGTCA CCCATACTTT CCAATTAAGA

AGCCAAACAA CAACAAGATT

181 TTGGTTCCAA AGGTTTCTGG TTTGCAATAC AGAGTTTTTA

GAATCCATTT GCCAGATCCA

241 AACAAGTTTG GTTTTCCAGA TACTTCTTTT TACAACCCAG

ATACTCAAAG ATTGGTTTGG

301 GCTTGTGTTG GTGTTGAAGT TGGTAGAGGT CAACCATTGG

GTGTTGGTAT TTCTGGTCAC

361 CC ATTGTTG A AC AAGTTGG A TG ATACTGAA AACGCTTCTG

CTTACGCTGC TAACGCTGGT

421 GTTGATAACA GAGAATGTAT TTCTATGGAT TACAAGCAAA

CTCAATTGTG TTTGATTGGT

481 TGTAAGCCAC CAATTGGTGA ACATTGGGGT AAGGGTTCTC

CATGTACTAA CGTTGCTGTT

541 AACCCAGGTG ATTGTCCACC ATTGGAATTG ATTAACACTG

TTATTCAAGA TGGTGATATG

601 GTTGATACTG GTTTTGGTGC TATGGATTTT ACTACTTTGC

AAGCTAACAA GTCTGAAGTT

661 CCATTGGATA TTTGTACTTC TATTTGTAAG TACCCAGATT

ACATTAAGAT GGTTTCTGAA

721 CCATACGGTG ATTCTTTGTT TTTTTACTTG AG AAGAGAAC

AAATGTTTGT TAGACACTTG

781 TTTAACAGAG CTGGTGCTGT TGGTGAAAAC GTTCCAGATG

ATTTGTACAT TAAGGGTTCT

841 GGTTCTACTG CTAACTTGGC TTCTTCTAAC TACTTTCCAA CTCCATCTGG TTCTATGGTT

901 ACTTCTGATG CTC AAATTTT TAACAAGCCA TACTGGTTGC

AAAGAGCCCA AGGTCATAAC

961 AACGGTATTT GTTGGGGTAA CCAATTGTTT GTTACTGTTG TTGATACTAC TAGATCTACT

1021 AACATGTCTT TGTGTGCTGC TATTTCTACT TCTGAAACTA

CTTACAAGAA CACTAACTTT

1081 AAGGAATACT TGAGACACGG TGAAGAATAC GATTTGCAAT

TTATTTTTCA ATTGTGTAAG

1141 ATTACTTTGA CTGCTGATGT TATGACTTAC ATTCATTCTA

TGAACTCTAC TATTTTGGAA

1201 GATTGGAACT TTGGTTTGCA ACGACCACCA GGTGGTACTT

TGGAAGATAC TTACAGATTT

1261 GTTACTTCTC AAGCTATTGC TTGTC AAAAG CACACTCC AC

CAGCTCCAAA GGAAGATCCA

1321 TTGAAGAAGT ACACTTTTTG GGAAGTTAAC TTGAAGGAAA

AGTTTTCTGC TGATTTGGAT

1381 CAATTTCCAT TGGGTAGAAA GTTTTTGTTG C AAGCTGGTT

TGAAGGCTAA GCCAAAGTTT

1441 ACTTTGGGTA AGAGAAAGGC TACTCCAACT ACTTCTTCTA

CTTCT ACTAC TGCT A AG AG A

1501 AAGAAGAGAA AGTTGTAATA G Here, in the present invention, P.pastoris is used as a host cell. Other yeast organism preferably various species of Pichia genus can be used as host cell for the codon-optimization process. Nucleotide sequence of HPV 16 LI protein defined as SEQ ID NO. 1 was further modified as described below.

Modifications were done at position between 61-300, 361-840 and/or at 961-1520 of nucleotide sequence of HPV 16 LI given in SEQ ID NO. l. These modifications were done in such a manner that amino acid sequence obtained after codon-optimization of nucleotide sequence will be 100% homologous with the primary structure of HPV 16 LI protein. This codon-optimized sequence provides subsequently better quaternary structure of VLP in terms of size and immunogenicity. After repetitive modification in the said nucleotide sequence, at said position, inventors had found one novel altered codon optimized sequence for HPV 16 LI antigen which is defined here as SEQ ID NO.2.

SEQ ID NO. 2 : Nucleotide sequence of HPV 16 LI antigen 1 ATGTCTTTGTGGTTGCC ATCTGAAGCTACTGTTTACTTGCCACCA 46 GTTCCAGTTTCTAAAGTTGTTTCCACTGACGAATACGTTGCTAGA 91 ACTAACATCTACTACCACGCTGGTACTTCTAGATTGTTGGCTGTT 136 GGTC ATCCAT ACTTCCC A ATTAAGAAGCC A AAC A AC A AC AAG ATT 181 TTGGTTCCAAAGGTTTCCGGATTGCAATACAGAGTTTTCAGAATC 226 CATTTGCCAGATCCAAACAAGTTTGGTTTCCCAGATACTTCTTTC 271 TACAACCCAGACACTCAAAGACTTGTTTGGGCTTGTGTTGGTGTT 316 GAAGTTGGTAGAGGTCAACCATTGGGTGTTGGTATTTCTGGTCAC 361 CCATTGTTGAAC AAGTTGGACGATACTGAAAACGCTTCTGCTTAC 406 GCTGCTAACGCTGGTGTTGATAACAGAGAATGTATTTCTATGGAC 451 TACAAGCAAACTC AATTGTGTTTGATTGGTTGTAAGCC ACCAATT 496 GGTGAACATTGGGGAAAGGGTTCTCCATGTACTAATGTTGCTGTT 541 AACCCTGGTGATTGTCCACCATTGGAATTGATTAACACTGTTATT 586 CAAGACGGTGATATGGTTGATACTGGTTTCGGTGCTATGGATTTC 631 ACTACTTTGCAAGCTAACAAGTCTGAAGTTCCATTGGACATTTGT 676 ACTTCCATCTGTAAGTACCCAGACTACATTAAGATGGTTTCTGAA 721 CC ATACGGTGATTCTTTGTTCTTCTACTTGAG AAG AG AAC AAATG 766 TTTGTTAGACACTTGTTCAACAGAGCTGGTGCTGTTGGTGAAAAC 81 1 GTTCCAGATGACTTGTACATTAAGGGTTCTGGTTCTACTGCTAAC 856 TTGGCTTCTTCT AACTACTTTCC AACTCC ATCTGGTTCT ATGGTT 901 ACTTCTGACGCTCAAATTTTCAACAAGCCATACTGGTTGCAAAGA 946 GCACAAGGTCATAACAACGGTATTTGTTGGGGTAACCAATTGTTC 991 GTTACTGTTGTTGACACTACTAGATCCACTAACATGTCCTTGTGT 1036GCTGCTATTTCTACTTCTGAAACTACTTACAAGAAC ACTAACTTC 1081 AAAGAGTACTTGAGAC ACGG AGAAGAATACGACTTGC AATTC ATT 1126TTCC AATTGTGT A AG ATTACTTTG ACTGCTGACGTTATG ACTTAC 1171 ATTCACTCTATG AACTCTACTATTTTGGAAGATTGGAACTTCGGA 1216TTGCAACCACCACCAGGTGGTACTTTGGAAGATACTTAC AGATTC 1261 GTTACTTCTC AAGCTATTGCTTGTCAAAAGCATACTCC ACCTGCT 1306CCAAAAGAAGATCCATTGAAGAAGTAC ACTTTCTGGGAAGTTAAC 135 lTTGAAAGAAAAGTTCTCTGCTGATTTGGATCAATTCCCATTGGGT 1396AGAAAGTTTTTGTTGC AAGCTGGATTGAAGGCTAAACCAAAGTTC 1441 ACTTTGGGAAAGAGAAAGGCTACTCCAACTACTTCTTCTACTTCT 1486 ACTACTGCTAAGAGAAAGAAGAGAAAATTGTAA 1518 In the same manner, codon-optimized sequence for HPV 18L1, HPV 6L1 and HPV 11 LI were generated. The codon-optimized sequence of HPV 18L1, HPV 6L1 and HPV 11L1 are given as SEQ ID NO. 3, 4 and 5, respectively.

SEP ID NO. 3 : Nucleotide sequence of HPV 18 LI antigen

1 ATGGCTCTTTGGAGACCATCCGACAACACTGTTTACTTGCCACCA

46 CCATCCGTTGCTAGAGTTGTTAACACTGACGACTACGTTACTAGA

91 ACTTCCATCTTCTACCACGCTGGTTCTTCCAGATTGTTGACTGTT

136 GGTAACCCATACTTCAGAGTTCCAGCTGGAGGTGGTAACAAGCAA 181 GACATCCCAAAGGTTTCCGCTTACCAGTACAGAGTTTTCAGAGTT

226 CAGTTGCCAGACCCAAACAAGTTTGGATTGCCAGACAATTCCATC

271 TACAACCCAGAGACTCAGAGACTTGTTTGGGCTTGTGCTGGTGTT

316 GAAATGGGT AG AGG AC AGCC ATTGGGTGTTGGTTTGTCTGGTC AC

361 CCATTCTACAACAAGTTGGACGACACTGAATCTTCTCACGCTGCT

406 ACTTCTAACGTTTCCGAGGATGTTAGAGACAACGTTTCCGTTGAC

451 TACAAGCAGACTCAGTTGTGTATCTTGGGTTGTGCTCCAGCTATT

496 GGTGAACATTGGGCTAAGGGTACTGCTTGTAAGTCCAGACCATTG

541 TCTC AGGGAGATTGTCC ACCATTGGAGTTGAAGAAC ACTGTTTTG

586 GAGGACGGTGATATGGTTGATACTGGTTACGGTGCTATGGACTTC

631 TCTACTTTGCAGGACACTAAGTGTGAAGTTCCATTGGACATCTGT

676 CAGTCCATCTGTAAGTACCCAGACTACTTGCAAATGTCCGCTGAT

721 CCATACGGTGACTCTATGTTCTTCTGTTTGAGAAGAGAGCAGTTG

766 TTCGCTAGACACTTCTGGAACAGAGCTGGTACTATGGGTGACACT

811 GTTCCACAATCCTTGTACATCAAGGGTACTGGAATGAGAGCTTCT

856 CCTGGTTCTTGTGTTTACTCTCCATCTCCATCCGGTTCCATTGTT

901 ACTTCCGACTCCCAGTTGTTCAACAAGCCATACTGGTTGCATAAG

946 GCTCAAGGTCACAACAACGGTATTTGTTGGCACAACCAGTTGTTC

991 GTTACTGTTGTTGACACTACTAGATCCACTAACTTG ACTATCTGT

1036 GCTTCC ACTC AATCTCC AGTTCC AGGAC A AT ACG ACGCTACT AAG 1081 TTCAAGC AGTACTCCAGACACGTTGAAGAGTACGACTTGC AGTTC 1126 ATCTTCCAGTTGTGTACTATCACTTTGACTGCTGATGTTATGTCC

1171 TACATCCACTCTATGAACTCCTCCATTTTGGAGGATTGGAACTTC

1216 GGTGTTCCACCACCACCAACTACTTCATTGGTTGACACTTACAGA .

1261 TTCGTTC AGTCCGTTGCTATC ACTTGTC AAA AGG ACGCTGCTCC A 1306 GCTGAAAACAAGGACCC ATACGAC AAGTTGAAGTTCTGGAACGTT 1351 GACTTGAAAGAGAAGTTCTCCTTGGACTTGGACCAATACCCATTG 1396 GGTAGAAAGTTTTTGGTTCAGGCTGGATTGAGAAGAAAGCCAACT 1441 ATCGGTCCAAGAAAGAGATCAGCTCCATCCGCTACTACTTCATCC 1486 AAGCC AGCTAAGAGAGTTAGAGTTAGAGCTAGAAAGTAG 1524

SEP ID NO. 4 : Nucleotide sequence of HPV 6 LI antigen

1 ATGTGGAGACCATCCGACTCCACTGTTTATGTTCCACCACCAAAC 46 CCAGTTTCCAAGGTTGTTGCTACTGACGCTTACGTTACTAGAACT 91 AATATCTTCTACCACGCTTCCTCATCCAGATTGTTGGCTGTTGGT 136 C ACCCATACTTCTCC ATC AAGAGAGCTAAC AAGACTGTTGTTCC A 181 AAGGTTTCCGGTTACCAGTACAGAGTTTTTAAGGTTGTTTTGCCA 226 GACCCAAACAAGTTCGCTTTGCCAGACTCTTCCTTGTTCGACCCA 271 ACTACTCAGAGATTGGTTTGGGCTTGTACTGGTTTGGAGGTTGGT 316 AGAGGTCAGCCATTGGGTGTTGGTGTTTCTGGTCACCCATTCTTG 361 AACAAGTACGACGACGTTGAGAACTCTGGTTCTGGTGGTAACCCA 406 GGTCAGGACAACAGAGTTAACGTTGGTATGGACTACAAGCAGACT 451 CAGTTGTGTATGGTTGGTTGTGCTCCACCATTGGGTGAACATTGG 496 GGTAAGGGTAAGCAGTGTACAAACACTCCAGTTCAGGCTGGTG AC 541 TGTCCACCTTTGGAGTTGATCACTTCCGTTATTC AGGACGGTGAC 586 ATGGTTGACACTGGTTTTGGTGCTATGAACTTCGCTGACTTGCAG 631 ACTAACAAGTCCGACGTTCCAATCGACATCTGTGGTACTACTTGT 676 AAGTACCCAGACTACTTGCAGATGGCTGCTGATCCATACGGTGAC 721 AGATTGTTCTTCTTCTTGAGAAAAGAACAGATGTTCGCTAGACAC 766 TTCTTCAACAGAGCTGGTGAAGTTGGTGAGCCAGTTCCAGACACT 81 1 TTGATCATTAAGGGTTCCGGTAACAGAACTTCCGTTGGTTCCTCC 856 ATCTACGTTAACACTCCATCCGGTTCCTTGGTTTCTTCTGAGGCT 901 CAGTTGTTCAACAAGCCATACTGGTTGCAGAAGGCTCAGGGTCAC 946 AACAACGGTATCTGTTGGGGTAACCAGTTGTTCGTTACTGTTGTT 991 GACACTACTAGATCCACTAATATGACTTTGTGTGCTTCCGTTACT 1036 ACTTCCTCCACTTACACTAACTCCGACTACAAAGAGTACATGAGA 1081 CACGTTGAAGAGTACGACTTGCAGTTCATCTTCCAGTTGTGTTCC 1126 ATCACTTTGTCCGCTGAGGTTATGGCTTACATCCACACTATGAAC 1171 CCATCCGTTTTGGAGGACTGGAACTTCGGTTTGTCTCCACCACCT 1216 AACGGTACTTTGGAGG AC ACTT AC AGATACGTTC AGTCCC AGGCT 1261 ATCACTTGTC AGAAGCCAACTCC AGAGAAAGAGAAGCC AGACCCT 1306 TACAAGAACTTGTCCTTCTGGGAGGTTAACTTGAAAGAGAAGTTC 1351 TCCTC AG AGTTGGACC AGTACCC ATTGGGT AGAAAGTTCTTGTTG 1396 C AGTCCGGTTAC AGAGGTAGATCCTCCATCAGAACTGGTGTTAAG 1441 AGACCAGCTGTTTCCAAGGCTTCTGCTGCTCCAAAGAGAAAGAGA 1486 GCTAAGACTAAGAGATAG 1503 SEP ID NO. 5 : Nucleotide sequence of HPV 11 LI antigen

1 ATGTGGAGACCATCCGACTCCACTGTTTATGTTCCACCACCAAAC 46 CCAGTTTCCAAGGTTGTTGCTACTGACGCTTACGTTAAGAGAACT 91 AATATCTTCTACC ACGCTTCCTC ATCCAGATTGTTGGCTGTTGGT 136 C ACCCTTACTACTCC ATC A AGA AGGTTAAC AAGACTGTTGTTCC A 181 AAGGTTTCCGGTTACCAGTACAGAGTTTTTAAGGTTGTTTTGCCA 226 GACCCAAACAAGTTCGCTTTGCCAGACTCTTCCTTGTTCGACCCA 271 ACTACTCAGAGATTGGTTTGGGCTTGTACTGGTTTGGAGGTTGGT 316 AGAGGTCAGCCATTGGGTGTTGGTGTTTCTGGTCACCCTTTGTTG 361 AACAAGTACGACGACGTTGAGAACTCCGGTGGTTATGGTGGTAAC 406 CCAGGTCAAGACAACAGAGTTAACGTTGGTATGGACTACAAGCAG 451 ACTCAGTTGTGTATGGTTGGTTGTGCTCCACCATTGGGTGAACAT 496 TGGGGTAAGGGTACTCAGTGTTCCAACACTTCCGTTCAGAACGGT 541 GACTGTCCACCTTTGGAGTTGATCACTTCCGTTATTCAGGACGGT 586 GACATGGTTGACACTGGTTTTGGTGCTATGAACTTCGCTGACTTG 631 CAGACTAACAAGTCCGACGTTCCATTGGACATCTGTGGTACTGTT 676 TGTAAATACCCAGACTACTTGCAGATGGCTGCTGATCCATACGGT 721 GACAGATTGTTCTTCTACTTGAGAAAAGAACAGATGTTCGCTAGA 766 CACTTCTTCAACAGAGCTGGTACTGTTGGTGAGCCAGTTCCAGAT 811 GACTTGTTGGTTAAGGGTGGTAACAACAGATCCTCCGTTGCTTCC 856 TCCATCTACGTTCATACTCCATCCGGTTCCTTGGTTTCTTCTGAG 901 GCTCAGTTGTTCAACAAGCCATACTGGTTGCAGAAGGCTCAGGGT 946 CACAACAACGGTATTTGTTGGGGTAACCACTTGTTCGTTACTGTT 991 GTTGACACTACTAGATCCACTAATATGACTTTGTGTGCTTCCGTT 1036 TCC AAGTCCGCTACTT AC ACT AACTCCG ACTAC AAAG AGT AC ATG 1081 AGACACGTTGAAGAGTTCGACTTGCAGTTCATCTTCCAGTTGTGT

1126 TCCATCACTTTGTCCGCTGAGGTTATGGCTTACATCCACACTATG

1171 AACCCATCCGTTTTGGAGGACTGGAACTTCGGTTTGTCTCCACCA

1216 CCTAACGGTACTTTGGAGGAC ACTTACAGATACGTTCAGTCCCAG 1261 GCTATC ACTTGTCAGAAGCC AACTCCAGAGAAAGAGAAGC AGGAC 1306 CCATACAAGGACATGTCTTTCTGGGAGGTTAACTTGAAAGAGAAG 1351 TTCTCCTCAGAGTTGGACCAGTTCCCATTGGGTAGAAAGTTCTTG

1396 TTGCAGTCCGGTTACAGAGGTAGAACTTCCGCTAGAACTGGTATC 1441 AAAAGACCAGCTGTTTCCAAGCCATCCACTGCTCCAAAGAGAAAA 1486 AGAACTAAGACTAAGAAGTAG 1506

Example 2: Construction of expression vector for HPV 16 LI

The expression vector pPICZa was used for expression of HPV16L1 gene. pPIC6, pGAPZ, pA0815 or other like vector can be used for the expression of HPV 16L1. pPICZa is a 3.6 kb vector used to express and secrete recombinant proteins in Pichia pastoris. It has the following features:

^ A 942 bp fragment containing the AOX1 promoter that allows methanol- inducible, high level expression of the gene of interest in Pichia. Targets plasmid integration to the AOX1 locus.

^ AOX1 transcription termination (TT) region allows native transcription termination and polyadenylation signal from AOX1 gene (-260 bp) that permits efficient 3 ' mRNA processing, including polyadenylation, for increased mRNA stability.

^ Zeocin resistance gene allows selection of transformants in E. coli and Pichia.

EM7 promoter that drives constitutive expression of the Zeocin resistance gene in E. coli.

■*· TEF1 promoter from Saccharomyces cerevisiae that drives expression of the Zeocin resistance gene in Pichia.

A pUC origin allows replication and maintenance of the plasmid in E. coli.

The HPV16L1 expression vector was constructed by inserting the BstBI/ otI fragment encoding HPV16L1 into the multiple cloning site of the vector pPICZa. The resulting expression vector is designated pPICZ-HPV16Ll . Its physical map is shown in Figure 1. Methods used for construction are basically as in Sambrook and Russell (2001). In the same manner, expression vectors containing HPV 18 LI, HPV 6 LI, and HPV 11 LI were constructed.

Example 3: Selection of clone with high copy number of transformed gene of HPV 16 LI

a) Preparation of competent cells

Pichia pastoris strain was grown in yeast extract peptone dextrose medium (YPD) at 30°C overnight. 500 ml of fresh medium in a 2 liter flask was inoculated with 0.1-0.5 ml of the overnight culture. It was grown overnight again till OD600 = 1.3—1.5. Cells were centrifuged and resuspended the pellet with ice-cold, sterile water once or twice. Cells were centrifuged and resuspended the pellet in 20 ml of ice-coldlM sorbitol. Centrifuge the cells and resuspend the pellet in 1 ml of ice-cold 1 M sorbitol for a final volume of approximately 1.5 ml. Keep the cells on ice and use that day.

b) Transformation

80 μΐ of the cells from the above step was mixed with 5-10 g of linearized pPICZa DNA (in 5-10 μΐ sterile water) and transfer them to an ice-cold 0.2 cm electroporation cuvette. The cuvette with the cells was incubated on ice for 5 minutes. Aliquots of competent yeast cells were thawed on ice and 600 ng of circular plasmid DNA was added to each aliquot. The suspensions were transferred to pre-cooled electroporation cuvettes. Electroporation was carried out in a BIORAD GenePulser II at 1.7 kV, 25 μΡ, 200 Ohm. Immediately 1 ml of ice-cold 1 M sorbitol was added to the cuvette. The cuvette contents were transferred to a sterile 15 ml tube. The tube was incubated at 30°C without shaking for 2 hours. 200 μΐ of tube was spreaded on each separate, labeled YPD plates containing the concentration of Zeocin. The plates were incubated for 3 days at 30°C until colonies form. Pick 10— 20 colonies and purify (streak for single colonies) on fresh YPD plates containing the appropriate concentration of Zeocin.

The primary selection of the clones was done on the basis of the copy number of the gene integrated into the pichia genome. This correlates with the expression of the clones. The screening was done using a semi quantitative PCR which is specific for the corresponding specific gene (HPV16L1, HPV18L1, HPV6L1 or HPVl 1L1).

Different parameters of the quantitative colony PCR assay were optimized using different sets of primers. These primers were further tested for their cross reactivity with the Pichia genome and its sensitivity to pick the lowest copy number of the specific gene.

After the quantitative colony PCR, the HPV16L1 clones were analysed for the gene copy number using RT PCR and the clones with the highest gene copy number were selected. Example 4: Expression analysis of transformed gene of HPV 16 LI

The clones which were selected from the RT PCR results were inoculated into BGY medium and their expression was analyzed after methanol induction of the cultures. The cultures were lysed and the resulting supernatant of transformant pools was analysed by Western blot. Final clones were selected on the basis of the intensity of the expressed bands on western blot. These clones were again analyzed by SDS PAGE and western blotting for the selection of the best producing clones for HPV proteins. SDS PAGE gel after commasie brilliant blue staining is shown as Figure 2. Identification of HPV serotype 16 LI by western blot is shown as Figure 3.

Example 5: Purification of HPV 16 LI protein

Purification process of HPV 16 LI protein is given below:

Each unit operation step involved in the purification and recovery process of each HPV protein has been demonstrated with one representative batch material.

a) Lysis of the washed cell pellet

Batch was harvested at the end of production phase. From each fermentor around

12L of culture broth was collected. Cells were separated primarily by centrifugation in order to separate the culture cells containing protein from the spent media. The washed pellet was dissolved in lysis buffer. Cells were lysed using a high pressure homogenizer. b) Clarification

After cell lysis, the supernatant containing the product was separated from the cell debris by centrifugation. The supernatant was further clarified using a 0.45 μιη filter. c) Capture step chromatography

The clarified lysate was loaded onto a suitable resin. Elution of the protein was carried out by increasing the sodium chloride concentration in the buffer.

d) Polishing step chromatography

Pooled fractions containing the protein of interest are loaded onto a suitable resin. Elution of the protein was carried out by decreasing the sodium chloride concentration in the buffer.

e) Buffer exchange by diafiltration

The pooled fractions were diafiltered through appropriate PES membrane filter against phosphate buffer to remove excess amount of sodium chloride from protein solution.

f) Sterile filtration

After the diafiltration step, the purified protein solution was subjected to for the preparation of its bulk solution. Example 6: preparation of bulk solution of VLPs of HPV 16 LI

The purified protein obtained after above mentioned purification process was subjected to dis/reassembly conditions to obtain final stable VLPs with improved quaternary structure. Dis/reassembly of the expressed VLPs was done in the pre- formulation buffer. This pre-formulation buffer was diluted to around 5-10 times for the purpose of reassembly of the expressed VLPs. Salt such NaCI or KCl was added with the concentration from 500mM - 2M to the pre-formulation buffer used at the disassembly of the expressed VLPs.

Disassembly buffer contains reducing agents and amino acids. Reassembly buffer includes higher concentration of salts along with the components of disassembly buffer.

The size of VLPs obtained was analyzed by dynamic light scattering method. The homogenous VLPs of HPV 16 LI obtained after this step had mean diameter of 90 nm. It is depicted in the Figure 4. It is depicted in the Figure 4. The homogenous VLPs of HPV 18 LI, HPV 6 LI and HPV 11 LI obtained after this step are depicted in Figure 12, Figure 13 and Figure 14 respectively. -·.■.^■_ -

The purified protein solution was filtered through a 0.22 micron filter under aseptic conditions and stored as bulk drug substance.

Example 7: Characterization of HPV-16 LI capsid protein in Pichia pastoris

Partial N-terminal sequence of the protein has been confirmed by Edman degradation and primary structure (tryptic maps) has been observed. Batch purified antigens does not show any free thiol (-SH) groups in the structure, as assessed by DTNB assay. The batch purified antigens were found to be free from any aggregates as determined by Nanoparticle Tracking Analysis. The batch purified antigens were found to be free from host cell protein by ELISA and DNA contaminant by RT-PCR based method. The preparation was found to be free from endotoxins as assessed by LAL test. To confirm structural stability of the VLPs, electron microscopy was done where inventors had found particles having mean diameter ~ 90 nm of these VLPs. Negative staining of HPV 16 LI VLPs are shown in Figure 5.

All the techniques mentioned in this example are well available in the art.

The bulk drug substance was stored at -80 ° C in non-pyrogenic containers.

Example 8: Preparation of vaccine containing HPV 16 LI

The purified antigens were adsorbed onto aluminium based adjuvant and filled aseptically into vials. Aluminium phosphate with 0.8-20 mg/ 0.5 ml of phosphate buffer saline was added to the 160 μg of HPV 16 LI protein for final formulation. Aluminium based adjuvant can be replaced by mineral salt adjuvants such as salt of calcium, iron and zirconium, Complete Freund's adjuvant (CFA), Adjuvants emulsions such as Incomplete Freund's adjuvant (IFA), montanide, MF 59 and Adjuvant 65, bacterially derived adjuvants or combination thereof. In the same manner, a skilled person can prepare vaccine containing LI protein of other serotypes such as 18, 6, 1 1 and combination thereof.

Example 9: Mice immunization and determination of immunogenicity of the expression product

Method:

The study was conducted based on British Pharmacopoeia 2012 on one representative batch of test sample with adjuvant, without adjuvant and reference product. They were analyzed for in vivo efficacy using Balb/c mice (6-8 weeks old female mice) using experimental design mentioned in Table 1.

Table 1 : Experimental design for mice immunogenicity studies for FTPV vaccines

0.5 ml of diluted Reference product / Test vaccine(s) was injected into each mouse subcutaneously (10 mice for each dilution group). 0.5 ml of vaccine diluent was administered in the same manner in 10 mice. These are the PBS controls and maintain the animals for 28 days. On 29^th day, blood was collected from each mouse by ocular vein puncture and left at room temperature for 1 hour to clot. The tubes were centrifuged at 4,000 rpm at 4°C for 10 minutes and collected the sera into separate labeled microfuge tubes.

All sera samples were tested at a time using the following ELISA kits from ADI (Alpha Diagnostic International), USA.

Mouse anti HPV 16L1 ELISA kit (#550-316-PMG)

Mouse anti HPV 18L1 ELISA kit (#550-318-PMG)

Mouse anti HPV6L1 ELISA kit (#550-306-PMG)

Mouse anti HPV1 1L1ELISA kit (#550-31 1-PMG)

The VLPs based vaccine according to the present invention was compared with the VLPs preparation without adjuvant and with the reference standard. The result obtained by ELISA assay is shown in figures 6, 7, 8 & 9 & Table 2. response against various serotypes

Example 10: Identification and determination of the end point of antibodies produced against to Human Papillomavirus vaccine in rats

Method:

HPV 16 LI or HPV 18L1 proteins were immobilized first on the wells of a micro titer plate. The plates were blocked further with Skim milk powder to avoid non-specific interaction. The sera samples from rats having antibodies against HPV I6L1 and HPV 18L1 were diluted in sample diluent buffer to achieve specific binding with the respective antigens coated on the respective plates with either proteins (HPV 16 LI/ HPV 18 LI). Anti-Rat HRP labeled was added as secondary antibody. The immunological reactions result in formation of complex in between anti-rat Antibody and specific antibody (HPV 16 LI/ HPV 18L1). In every step of washing unbound reactants were removed. Substrate OPD was then reacted. The amount of substrate read out of color reaction on microtiter plates reader is directly proportional to the concentration of respective antibodies present in the serum samples.

Experimental design

Groups of 10 male and 10 female rats per group were treated with Human Papillomavirus (rDNA) vaccine formulation injected intramuscularly once in two weeks at three dose levels of 0.25 ml/animal (Low), 0.5 ml/animal (Mid) and 1.0 ml/animal (High) to Wistar rats for a period of 3 months. There was independent control group as vehicle control group to receive placebo alone in this study. In addition, recovery groups were maintained at high dose level along with concurrent

vehicle control for a period of one month post treatment to observe the reversibility, persistence or delayed occurrence of adverse effects. Blood samples were collected for immunogenicity at pre-treatment, post-treatment and post-recovery period and then antiHPVs was determined by Enzyme Linked Immuno-Sorbent Assay (ELISA), for vehicle control (1.0 ml placebo/rat), low dose (0.25 ml vaccine/rat), mid dose (0.5 ml vaccine/rat) and high dose (1.0 ml vaccine/rat) with pre-treatment groups.

Table 3: Treatment details

Determination of titer

Different plates for HPV 16 LI or HPV 18L1 were coated by making antigen concentration 50ng/well and kept for over-night incubation at 2-8°C. Then, plates were kept in 5% blocking solution (skimmed milk in PBST) and were kept in incubation at 37°C for 2 hours. All the samples from each group were pooled separately and diluted 50 times in PBST (Phosphate Buffer Saline- Tween 20) Buffer. Plates were washed and 50μ1 of each sample was added in the pre-determined wells and kept for incubation at 37°C for 1 hour. Plates were washed with PBST and then 50 μΐ of Anti-Rat IgG HRP was added to each well and kept for incubation for 60 minutes at 37°C. Then plates were washed again with PBST and 50 μΐ of substrate (OPD dissolved in citrate phosphate buffer and H202) was added to each well and kept the plates for 15minutes in dark at room temperature. Then, 25 μΐ Stop solution (5.4ml H₂S0-₄ in lOOjnl WFI) was added to each well and plates were read at 490nm on SpectraMAX 190 micro plate reader.

Table 4: Pre-treatment - Male

Table 5: Pre-treatment Recovery- Male

Table 6: Post-Treatment Male

Table 7: Post- recovery Male

Table 8: Pre Treatment-Female III 0.5 ml lOlF-110 F Not reactive Not reactive

Mid

vaccine/animal

IV 1.0 ml; 111F-120F Not reactive Not reactive

High

vaccine/animal

Table 9: Pretreatrrient Recovery Female

Table 10: Post treatment -Female

Table 11 : Post recovery -Female

SUMMARY

5 The results of pre-treatment samples show that the absorbance values which are almost equal to the control rats and non-reactive in both male and female rats. The^same groups of male and female rats were treated with Human Papillomavirus (rDNA) vaccine for three months at low dose (0.25 ml/animal), mid dose (0.5 mV animal) and hig t dose (1.0 ml/animal). At the end of three month treatment the results show the higher 0 absorbance values highly reactive to the vaccine. The reactivity measured with ELISA titers as 3880.417 for HPV 16 LI & 522.338 for HPV 18 LI in low dose, 6377.327 for HPV 16 LI & 632.235 for HPV 18 LI in mid dose and 25832.417 for HPV 16 LI & 1198.762 for HPV 18 LI for high dose in male groups, and the reactivity measured with ELISA titers as 15567.343 for HPV 16 LI & 770.180 for HPV 18 11 in low dose, 5 24445.245 for HPV 16 LI & 4058.034 for HPV 18 LI in mid dose and 26283.248 for HPV 16 LI & 4100.000 for HPV 1 8 LI in high dose in female groups. At the end of one month recovery period, serum samples show reactivity as ELISA titers of 25416.265 for HPV 16 LI & 1042.376 for HPV 1 8 LI in males and 30410.085 for HPV 16 LI & 951.940 for HPV 18 LI in female rats.

0 CONCLUSION

The end point study results reveals that the Human Papilloma Virus (rDNA) vaccine, manufactured by Cadila Healthcare Ltd. is immunogenic by producing antibodies for low, mid and high doses are measurable up to highest titer in ELISA as 30410.085 in HPV 16 LI and 4100.000 in HPV 18 LI.

5 Example 11: Primate (Rhesus monkeys) immunization and determination of titer

HPV vaccine formulations containing 20 g each for HPV 6L1 & HPV 18 LI and 40 μg.each for HPV 16 LI & HPV 1 1 LI was prepared in Alum Phosphate gel. The formulated vaccine stored at 2-8 degree C till it use. The vaccine, was immunized as 1ml per animal in rhesus monkeys equivalent to one human dose by single dose intramuscular administration on day 0 followed by booster injection on day 21 , day 180, and additionally also at day 342 in reference Vaccine group & negative control groups. Before the initiation of experiment all the nonhuman primates were subjected to thorough physical, veterinary examination and analysis of clinical chemistry parameters.

The Primates were immunized as per following immunization_, schedule with following vaccines at various time intervals

Table 12: Immunisation schedule for primate studies

The blood samples were collected for serum at day 21 ,33,46,63,126, 186, 193, 342, 372 and 433 for determination of antibody titers by ELISA. The serum samples were collected as per experimental design from monkeys at predefined intervals. The sera samples were stored further at -20 degree C for antibody measurement by ELISA.

The ELISA was performed after overnight coating with Purified proteins of HPV 16 Ll/HPV LI over ELISA plate as 50-100 ng per well. The unbound protein was washed with PBS tween buffer and blocking buffer containing 1 % BSA was added to plate for 1 hour to block non-specific sites. The serum samples were diluted in PBS appropriately and added to wells. The unbound sample was washed with PBS tween buffer and anti- Monkey IgG labeled with HRPO was added for 45 minutes. The plate was washed with wash buffer again and peroxidase substrate was added. It was incubated in dark for 25 minutes. Finally 25ul of Stop Solution was added into each test well. Read the plate at 405nm within 15 minutes. The standard curve was plotted using different dilutions of pool of positive reference vaccine sera, O.D. values verses their assigned ELISA titers based on considering end point titer after ELISA cut off O.D (Mean of O.D from blank & negative samples+3S.D). The antibody titers measured by ELISA were shown below in table 13 and 14 and figure 10 and 11. Table 13: Anti- HPV 16 LI ELISA titer (in Log 10 Geometric Mean)

Table 14: Anti- HPV 18 LI ELISA titer (in Log 10 Geometric Mean)

5

RESULT

The test vaccine prepared according to the present invention provides initially comparable response with the reference vaccine. Afterwards, immune response obtained by the test vaccine persist for longer period of time whereas, reference vaccine requires booster dose 0 to achieve similar immune response. Similarly, to confirm the neutralization antibody response in HPV 16 LI and HPV 18 LI, we also found comparable immune response by Pseudovirus neutralization assay. Our vaccine was found comparable in terms of total antibody response measured by ELISA for HPV 16 LI and HPV 18 LI with reference vaccine with 3 doses in total than 4 doses of reference vaccine over a period of 433 days 5 post vaccination. Thus, it provides strong evidence of our vaccine superiority than Reference vaccine for efficacy against HPV 16 and 18 LI in Monkeys.

Claims

We claim

1. An isolated gene encoding major capsid protein of human papilloma virus wherein the said gene is selected from SEQ ID NO. 2, SEQ ID NO.3, SEQ ID N0.4 and SEQ ID NO.5.

2. A vector comprising gene encoding major capsid protein of human papilloma virus wherein the said gene is selected from SEQ ID NO. 2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and combination thereof.

3. The vector as claimed in claim 2 is selected from pPICZ, pPIC6, pGAPZ and pA0815z.

4. The vector as claimed in claim 2 having the MTCC accession numbers selected from MTCC 5969, MTCC 5970, MTCC 5971 or MTCC 5972.

5. A host cell transformed with the gene as claimed in claim 1.

6. A host cell comprising vector as claimed in claim 2.

7. The host cell as claimed in claim 5 and 6 is yeast cell preferably P. pastoris.

8. The host cell as claimed in claim 6 is selected from P. pastoris strains X-33, GS115, KM71 and SMD1168.

9. Virus like particles obtained by using the genes as claimed in claim 1.

10. The virus like particles as claimed in claim 9 having a diameter in the range of 50- 500 nm, preferably, 50-400 nm, more preferably 100-400 nm.

11. A human papilloma virus vaccine comprising at least one gene encoding capsid protein from those claimed in claim 1 which can elicit immune response against HPV antigen.

12. The human papilloma vaccine as claimed in claim 1 1, further comprising gene encoding L2, E6 or E7 antigen of single or different serotypes.

13. The serotype as clalimed in claim 12 is selected from HPV 16, HPV 6, HPV 18, HPV 1 1, HPV 31, HPV 33, HPV 45, HPV 52, HPV 58 and combination thereof.

14. An immunogenic composition containing at least one protein of HPV antigens as claimed in claim 1 with suitable adjuvant(s).

15. The adjuvant as claimed in claim 14 is selected from salts of aluminium, Mono hosphoryl Lipid analogues such as glucopyranosyl lipid adjuvant, monatide, cytokine inducers, squalene based adjuvants, lipophilic adjuvants and combination thereof.

16. A method of preparing human papilloma vaccine as claimed in claim 11 comprising following steps:

a. Synthesis of gene encoding major capsid protein of human papilloma virus Construction of expression vector for gene encoding major capsid protein of human papilloma virus

Selection of clone with high copy number of transformed gene of said protein of human papilloma virus

Expression analysis of transformed gene of said protein of human papilloma virus

Purification of protein encoded by gene of said protein of human papilloma virus

Preparation of bulk solution of VLPs of said protein of human papilloma virus

Characterization of said protein in host cell

h. Preparation of vaccine containing said protein of human papilloma virus.

17. The method of preparing human papilloma vaccine as claimed in claim 16, wherein the host cell is yeast cell preferably P. pastoris.

18. The major capsid protein as claimed in any preceding claim is HPV LI of single or different serotypes.

19. The HPV LI protein as claimed in claim 18 is selected from HPV 16 LI, HPV 18 LI, HPV 6 LI, HPV 1 1 LI, HPV 31 LI , HPV 33 LI, HPV 45 LI , HPV 52 LI, HPV 58 L 1 and combination thereof, preferably HPV 16 L 1 , HPV 18 L 1 , HPV 6

LI, HPV 11 LI and combination thereof.