EP3191505A2 - Antigènes supérieurs du papillomavirus présentant des propriétés immunologiques supérieures et vaccin les contenant - Google Patents

Antigènes supérieurs du papillomavirus présentant des propriétés immunologiques supérieures et vaccin les contenant

Info

Publication number
EP3191505A2
EP3191505A2 EP15808033.3A EP15808033A EP3191505A2 EP 3191505 A2 EP3191505 A2 EP 3191505A2 EP 15808033 A EP15808033 A EP 15808033A EP 3191505 A2 EP3191505 A2 EP 3191505A2
Authority
EP
European Patent Office
Prior art keywords
hpv
protein
human papilloma
vaccine
gene
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP15808033.3A
Other languages
German (de)
English (en)
Inventor
Gaurav Gupta
Viviana Giannino
Reinhard Glueck
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Zydus Lifesciences Ltd
Original Assignee
Cadila Healthcare Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Cadila Healthcare Ltd filed Critical Cadila Healthcare Ltd
Publication of EP3191505A2 publication Critical patent/EP3191505A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5258—Virus-like particles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20023—Virus like particles [VLP]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20051—Methods of production or purification of viral material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20071—Demonstrated in vivo effect
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host

Definitions

  • 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.
  • HPV Human papillomavirus
  • the HPV genome encodes for non-structural-proteins (El, E2, E4, E5, E6 and E7) and structural proteins (LI and L2).
  • 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.
  • HPV 16 and HPV 18 types contribute to nearly 80% of the entire uterine cervical cancer incidence in India.
  • 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 Guttraum Institute, the HPV vaccine "is widely considered one of the greatest health care advances for women in recent years.”
  • bivalent HPV vaccine is composed of only two antigens: HPV types 16 and 18.
  • HPV types 16 and 18 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.
  • 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.
  • VLPs virus-like particles
  • 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 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.
  • 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.
  • LI major capsid protein
  • VLPs 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.
  • 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.
  • codon-optimization of HPV L2 gene, early antigens of HPV (E6 and E7) can be generated to develop the vaccine against HPV antigens.
  • 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 FiveTM) 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.
  • 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
  • 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.
  • inventors have used two adjuvants namely aluminum hydroxide and 3D MPL in formulation. These adjuvants help in inducing immunogenicity of the antigens.
  • 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.
  • 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.
  • 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.
  • Yeast Sacehromyces cervisiae
  • Baculovirus expression system Baculovirus expression system
  • 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).
  • 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.
  • 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.
  • HPV serotypes preferably HPV 16 LI, HPV 18 LI, HPV 6 LI and HPV 1 1 LI
  • 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.
  • 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.
  • different proteins of HPV include major capsid protein LI, minor capsid protein L2 and early antigens E6 and E7.
  • 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.
  • 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.
  • the present invention provides host cell transformed with the said genes encoding different proteins of HPV.
  • the present invention provides host cell with high copy number of genes encoding different proteins of HPV of various serotypes.
  • the present invention provides a vector containing genes encoding different proteins of HPV of various serotypes.
  • 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.
  • the current invention provides a vaccine against human papilloma virus with significantly higher immunogenicity.
  • the current invention provides a vaccine against HPV antigens with improved immunization schedule wherein vaccine dosage regimen is reduced to single time.
  • the current invention provides a vaccine against HPV antigens with substantially reduced amount of antigens.
  • the current invention provides the vaccine against HPV antigens using yeast as an expression system.
  • the current invention provides the vaccine against HPV antigens using P. pastoris as an expression system.
  • 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.
  • the present invention provides suitable host cell preferably P. pastoris with high copy number of genes encoding various HPV antigens of several serotypes.
  • the current invention provides vaccine against HPV antigens with improved immunization schedule wherein vaccine dosage regiment and amount of antigen dose are reduced.
  • the current invention provides an immunogenic composition containing proteins of HPV antigens with suitable adjuvant(s).
  • 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)
  • 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.
  • different proteins of HPV include major capsid protein LI, minor capsid protein L2 and early antigens E6 and E7.
  • 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 .
  • 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.
  • the present invention provides host cell transformed with the said genes encoding different proteins of HPV.
  • host cell can be termed as an expression system in which genes transformed with a suitable vector will express.
  • 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.
  • host cell according to the present invention is yeast cell preferably P. pastoris.
  • P. pastoris strains X-33, GS 1 15, KM71, SMD1168 or others can be used as an expression system.
  • the present invention provides a vector containing genes encoding different proteins of HPV of various serotypes.
  • pPICZ, pPIC6, pGAPZ, pA0815 or other similar vectors can be used as a vector in the present invention. These vectors are commercially available.
  • 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.
  • 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.
  • VLPs according to the present invention are of 50- 500 nm, preferably, 50-400 nm, more preferably 100-400 nm.
  • 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.
  • the present invention provides method of preparing human papilloma vaccine comprising the following steps:
  • 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.
  • purification process according to the current invention is column chromatography, sterile filtration, diafiltration or suitable combination thereof.
  • 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, ⁇ - mercaptoethanol, tween 80 or others like), salts (NaCl or KC1), amino acids and acidic or basic buffers.
  • suitable reducing agents DTT, ⁇ - mercaptoethanol, tween 80 or others like
  • salts NaCl or KC1
  • amino acids amino acids and acidic or basic buffers.
  • 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.
  • the current invention provides a vaccine against HPV antigens with improved immunization schedule wherein vaccine dosage regimen is reduced.
  • 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.
  • the present invention provides an immunogenic composition containing proteins of HPV antigens with suitable adjuvant.
  • MPL Mono Phosphoryl Lipid analogues such as GLA
  • glucose lipid adjuvant glucopyranosyl lipid adjuvant
  • monatide cytokine inducers
  • squalene based adjuvants squalene based adjuvants
  • lipophilic adjuvants squalene based adjuvants and others like
  • the present invention provides a pharmaceutical composition comprising an immunogenic composition with pharmaceutically acceptable carrier or excipient.
  • SEQ ID NO. 1 Nucleotide sequence of HPV 16 LI antigen (Gene bank No. gblGO423063. il " )
  • 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.
  • 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 GCTGCTAACGCTGGTGTTGATA
  • SEP ID NO. 3 Nucleotide sequence of HPV 18 LI antigen
  • SEP ID NO. 4 Nucleotide sequence of HPV 6 LI antigen
  • 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:
  • ⁇ 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.
  • ⁇ * ⁇ 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.
  • 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.
  • yeast extract peptone dextrose medium YPD
  • 80 ⁇ of the cells from the above step was mixed with 5-10 g of linearized pPICZa DNA (in 5-10 ⁇ 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 ⁇ , 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 ⁇ 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).
  • 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.
  • the pooled fractions were diafiltered through appropriate PES membrane filter against phosphate buffer to remove excess amount of sodium chloride from protein 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.
  • 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.
  • the bulk drug substance was stored at -80 ° C in non-pyrogenic containers.
  • Aluminium phosphate with 0.8-20 mg/ 0.5 ml of phosphate buffer saline was added to the 160 ⁇ g 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.
  • CFA Complete Freund's adjuvant
  • IFA Incomplete Freund's adjuvant
  • montanide MF 59
  • Adjuvant 65 bacterially derived adjuvants or combination thereof.
  • 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
  • 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.
  • 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.
  • 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
  • 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.
  • rDNA Human Papillomavirus
  • ELISA Enzyme Linked Immuno-Sorbent Assay
  • HPV 16 LI or HPV 18L1 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 50 ⁇ 1 of each sample was added in the pre-determined wells and kept for incubation at 37°C for 1 hour.
  • PBST Phosphate Buffer Saline- Tween 20
  • 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.
  • rDNA Human Papillomavirus
  • HPV vaccine formulations containing 20 g each for HPV 6L1 & HPV 18 LI and 40 ⁇ g.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 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.
  • 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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Virology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Molecular Biology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Microbiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Oncology (AREA)
  • Communicable Diseases (AREA)
  • Epidemiology (AREA)
  • Mycology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Biotechnology (AREA)
  • Engineering & Computer Science (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne des gènes codant pour divers antigènes du papillomavirus de plusieurs sérotypes. Les gènes de la présente invention permettent de produire des particules de type virus présentant une amélioration en termes de qualité immunologique et de structure quaternaire. La présente invention concerne également une cellule hôte adaptée, de préférence P.pastoris, avec un nombre élevé de copies de gènes codant pour divers antigènes de papillomavirus de plusieurs sérotypes. La présente invention porte en outre sur un vaccin contre des antigènes du papillomavirus avec programme d'immunisation amélioré, le régime posologique du vaccin et la quantité de dose d'antigène étant réduits. La présente invention concerne également une composition de vaccin améliorée du virus du papillomavirus au moyen de levure.
EP15808033.3A 2014-09-11 2015-09-11 Antigènes supérieurs du papillomavirus présentant des propriétés immunologiques supérieures et vaccin les contenant Withdrawn EP3191505A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IN2905MU2014 2014-09-11
PCT/IN2015/000355 WO2016038625A2 (fr) 2014-09-11 2015-09-11 Antigènes supérieurs du papillomavirus présentant des propriétés immunologiques supérieures et vaccin les contenant

Publications (1)

Publication Number Publication Date
EP3191505A2 true EP3191505A2 (fr) 2017-07-19

Family

ID=54843870

Family Applications (1)

Application Number Title Priority Date Filing Date
EP15808033.3A Withdrawn EP3191505A2 (fr) 2014-09-11 2015-09-11 Antigènes supérieurs du papillomavirus présentant des propriétés immunologiques supérieures et vaccin les contenant

Country Status (10)

Country Link
EP (1) EP3191505A2 (fr)
JP (1) JP2017528137A (fr)
CN (1) CN107002085A (fr)
AR (1) AR101840A1 (fr)
AU (1) AU2015313756A1 (fr)
BR (1) BR112017004181A2 (fr)
CA (1) CA2958222A1 (fr)
EA (1) EA201790365A1 (fr)
MA (1) MA40624A (fr)
WO (1) WO2016038625A2 (fr)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104845985B (zh) * 2014-02-18 2020-02-07 上海泽润生物科技有限公司 重组人乳头瘤病毒蛋白表达
CA3002323A1 (fr) 2015-10-19 2017-04-27 Cadila Healthcare Limited Nouvel adjuvant et composition vaccinale le contenant
CN109750049B (zh) * 2017-11-07 2023-08-18 上海泽润生物科技有限公司 重组人乳头瘤病毒52亚型蛋白表达
WO2019096796A1 (fr) 2017-11-14 2019-05-23 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Papillomavirus non humains pour administration de gènes in vitro et in vivo
BR102020006846A2 (pt) * 2020-04-03 2021-12-07 Imunoscan Engenharia Molecular Ltda Peptídeos sintéticos miméticos a proteína l1 do hpv, método de diagnóstico de hpv, sistema de diagnóstico de hpv, composição farmacêutica e uso dos mesmos no tratamento ou profilaxia do hpv

Family Cites Families (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2237224C (fr) * 1995-11-15 2006-06-27 Steven Ludmerer Particules synthetiques de type viral hpv11
EP0973546B1 (fr) * 1997-04-08 2004-03-17 Merck & Co., Inc. Formulations stabilisees du virus du papillome humain
DK1015561T3 (da) * 1997-09-05 2006-11-13 Medimmune Inc In vitro-fremgangsmåde til adskillelse/gensamling af papillomavirus-lignende partikler (VLP'er)
MY140664A (en) * 2003-09-29 2010-01-15 Merck Sharp & Dohme Optimized expression of hpv 45 l1 in yeast
MY139500A (en) * 2003-11-12 2009-10-30 Merck Sharp & Dohme Optimized expression of hpv 58 l1 in yeast
CN101115766B (zh) * 2004-06-18 2013-05-08 印度免疫有限公司 用于抗人乳头瘤病毒型16的salmonella疫苗菌株的密码子经优化的hpv16l1
RU2494106C2 (ru) * 2007-11-23 2013-09-27 Шанхай Зеран Биотекнолоджи Ко., Лтд. Гены, кодирующие главный капсидный белок l1 вируса папилломы человека, и их применение
CN101487009B (zh) * 2008-01-15 2012-11-21 上海泽润生物科技有限公司 用毕赤酵母表达系统制备抗人乳头瘤病毒16型感染的疫苗的方法
PT2318042E (pt) * 2008-07-31 2014-11-05 Glaxosmithkline Biolog Sa Vacina contra hpv
KR101181907B1 (ko) * 2009-06-19 2012-09-11 아이진 주식회사 자궁경부암 백신
CN102154325B (zh) * 2011-01-01 2013-08-21 上海生物制品研究所有限责任公司 针对人乳头瘤状病毒的疫苗及其制法和用途
US9833504B2 (en) * 2011-05-13 2017-12-05 Folia Biotech Inc. Virus-like particles and process for preparing same
CA2834618C (fr) * 2011-06-24 2020-11-03 Merck Sharp & Dohme Corp. Formulation de vaccin contre le papillomavirus comprenant un adjuvant a l'aluminium, et procede de production correspondant
KR101559622B1 (ko) * 2012-07-30 2015-10-13 중앙대학교 산학협력단 인유두종바이러스 바이러스 유사입자의 고효율 정제방법
CN103667319B (zh) * 2012-09-10 2017-09-26 同济大学 抗人乳头瘤病毒的三价疫苗及其制法和用途

Also Published As

Publication number Publication date
CA2958222A1 (fr) 2016-03-17
BR112017004181A2 (pt) 2017-12-05
EA201790365A1 (ru) 2017-07-31
AR101840A1 (es) 2017-01-18
JP2017528137A (ja) 2017-09-28
MA40624A (fr) 2016-03-17
AU2015313756A1 (en) 2017-03-09
WO2016038625A2 (fr) 2016-03-17
WO2016038625A3 (fr) 2016-04-28
CN107002085A (zh) 2017-08-01

Similar Documents

Publication Publication Date Title
US9428555B2 (en) Truncated L1 protein of Human Papillomavirus type 16
JP3863559B2 (ja) 乳頭腫ウィルスワクチン
US9249193B2 (en) Truncated L1 protein of human papillomavirus type 33
EP3191505A2 (fr) Antigènes supérieurs du papillomavirus présentant des propriétés immunologiques supérieures et vaccin les contenant
EP0817852B1 (fr) Adn de synthese hybride hpv6/11 l1 codant pour des proteines de papillomavirus humain de type 11 l1
KR20050002868A (ko) 인간 파필로마 바이러스의 바이러스 유사 입자
EP2589604B1 (fr) Protéine l1 tronquée du papillomavirus humain de type 52
US7897155B2 (en) Method for producing yeast expressed HPV types 6 and 16 capsid proteins
US9738691B2 (en) Truncated L1 protein of human papillomavirus type 58
US9364529B2 (en) Truncated L1 protein of human papillomavirus type 18
US9034340B2 (en) Genes encoding major capsid protein L1 of human papilloma virus
US20240000915A1 (en) C-terminally modified human papillomavirus type 11 l1 protein and use thereof
JP7290258B2 (ja) ヒトパピローマウイルス５１型のｌ１タンパク質の変異型
JP6916115B2 (ja) ＨＰＶＬ２ペプチド／ＨＢｓキメラタンパク質を有効成分とするＨＰＶ感染症用ワクチン
CA2216827C (fr) Adn de synthese hybride hpv6/11 l1 codant pour des proteines de papillomavirus humain de type 11 l1

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20170330

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

AX Request for extension of the european patent

Extension state: BA ME

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION HAS BEEN WITHDRAWN

18W Application withdrawn

Effective date: 20170919