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WO2024218165A1 - Vaccin à vecteurs viraux contre le vph - Google Patents

Vaccin à vecteurs viraux contre le vph Download PDF

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Publication number
WO2024218165A1
WO2024218165A1 PCT/EP2024/060445 EP2024060445W WO2024218165A1 WO 2024218165 A1 WO2024218165 A1 WO 2024218165A1 EP 2024060445 W EP2024060445 W EP 2024060445W WO 2024218165 A1 WO2024218165 A1 WO 2024218165A1
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hpv
viral vector
prime
boost
vaccine
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PCT/EP2024/060445
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English (en)
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Thomas Evans
Sarah SEBASTIAN
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Barinthus Biotherapeutics (Uk) Limited
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Publication of WO2024218165A1 publication Critical patent/WO2024218165A1/fr

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    • 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
    • A61P31/20—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
    • A61K2039/5256—Virus expressing foreign proteins
    • 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
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • 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
    • A61K2039/70—Multivalent vaccine
    • 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
    • C12N2710/10011—Adenoviridae
    • C12N2710/10041—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2710/24011—Poxviridae
    • C12N2710/24041—Use of virus, viral particle or viral elements as a vector
    • C12N2710/24043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • This invention relates to viral-vectored vaccines for use in a vaccination against HPV infection.
  • Human papillomavirus infection is an infection by human papillomavirus (HPV). Most HPV infections cause no symptoms and resolve spontaneously. However, in some cases they persist and this can result in the development of warts or precancerous lesions. The precancerous lesions increase the risk of cancer of the cervix, vulva, vagina, penis, anus, mouth, or throat. There are approximately 0.5 million cases of HPV-attributable cervical cancer that occur annually worldwide, and over half of these are fatal. About 85% of cases occur in low/middle income countries where there is limited or no treatment available. Women who have not received a prophylactic vaccine require 3-yearly screening to identify and treat cervical intra-epithelial neoplasia (CIN).
  • CIN cervical intra-epithelial neoplasia
  • HPV vaccines that have been developed for therapy of existing HPV infection include Inovio - VGX-3100 (DNA encoding E6, E7) similarly Genexine (GX-188E); Janssen - Ad26/Ad35 +/-MVA encoding E2, E6, E7 fusion protein; Synthetic long peptides (E6, E7) and similar eg.
  • an aim of the present invention is to provide an improved vaccine for HPV infection.
  • the present invention relates to a multi-HPV immunogen viral vector, a vaccine, compositions, methods and dosage regimes for use in medicine, optionally wherein the use may be the treatment of human papillomavirus (HPV) infection including prophylactic use to prevent HPV infection and/or cancer, including inducing an improved immune response and improvement in the performance of therapeutic vaccines.
  • the multi-HPV immunogen viral vector vaccine has an immunogen expression cassette encoding a polypeptide antigen.
  • the use of a specially designed antigen having segments from the early proteins of HPV (immunogenic HPV protein fragments) can provide protection against multiple important genotypes, such as HPV 16, HPV 18 and HPV 33. References herein to methods of treatment of the human or animal body are to be understood as references to medicaments for use in a method of treatment.
  • the invention provides a viral vector encoding an antigen comprising two or more HPV protein fragments of each of the HPV proteins E1 , E2, E6 or E7 wherein each HPV protein fragment is linked with a linker sequence.
  • the viral vector can comprise an immunogen expression cassette wherein the immunogen expression cassette encodes the antigen.
  • the expression of a protein encoded by the expression cassette can be arranged to be driven by a promoter.
  • Each HPV protein fragment can be linked with a linker sequence that is cleavable.
  • the antigen can further comprise two or more HPV protein fragments of each of the HPV proteins E4 and E5.
  • the antigen can comprise from 20 to 50 HPV protein fragments.
  • the antigen can comprise 46 HPV protein fragments.
  • the antigen can comprise 15 to 30 HPV fragments.
  • the antigen can comprise 15 to 25 HPV protein fragments.
  • the antigen can comprise 19 to 23 HPV protein fragments.
  • the antigen can comprise 19 or 23 HPV protein fragments.
  • the HPV protein fragments can be from 12 to 125 amino acids.
  • the HPV protein fragments can be from 16-74 amino acids.
  • the HPV protein fragment length can be from 23-122 amino acids.
  • the antigen can comprise HPV fragments from one or more of HPV genotypes 16, 18, 31 , 33, 45, 52 and 58.
  • the antigen can comprise one or more HPV protein fragments from each of HPV genotypes 16, 18, 31 , 45, 52 and 58.
  • the antigen can comprise one or more HPV protein fragments from each of HPV genotypes 16, 18, and 33.
  • an antigen such as HRV3.2 comprising two or more HPV protein fragments of each of the HPV proteins E1 , E2, E6 or E7 wherein each HPV protein fragment is linked with a linker sequence induces T cells specific to each HPV protein.
  • the invention provides a prime HPV viral vector for use in a prime-boost method which requires administration of a prime vaccine and a boost vaccine, comprising an HPV viral vector described herein, optionally wherein the prime viral vector is a simian adenovirus, preferably wherein the prime viral vector is ChAdOxl .
  • the prime viral vector may also referred to as a prime HPV viral vector.
  • the invention provides a boost viral vector vaccine for use in a prime-boost method which requires administration of a prime vaccine and a boost vaccine, comprising an HPV viral vector as described herein.
  • the boost viral vector may also referred to as a boost HPV viral vector.
  • the boost viral vector can be a poxvirus.
  • the boost viral vector can be a Modified Vaccina Virus Ankara (MVA).
  • MVA Modified Vaccina Virus Ankara
  • Studies in an animal model (mice) have shown that for an antigen such as HRV3.2 comprising two or more HPV protein fragments of each of the HPV proteins E1 , E2, E6 or E7 wherein each HPV protein fragment is linked with a linker sequence administered using a prime-boost method, the T-cell response to each HPV protein was increased after administration of the boost.
  • a composition (also referred to as a vaccine composition) comprising a viral vector as described herein and a pharmaceutically acceptable carrier.
  • the invention further provides a method for making an HPV viral vector for use in a prime-boost method which requires administration of a prime vaccine and a boost vaccine, comprising providing a prime HPV viral vector and a boost viral vector, wherein the HPV protein fragments encoded by the prime HPV viral vector and the boost HPV viral vector are the same.
  • the linker sequences in the prime HPV immunogen viral vector vaccine and in the boost HPV viral vector can be different.
  • the order of the HPV protein fragments in the prime viral vector can be different to the order of the HPV protein fragments in the boost viral vector.
  • the invention further provides a combination of viral vectors for use in a method of treatment, wherein the combination comprises a prime HPV viral vector and a boost HPV viral vector, the method comprising administering the vaccine boost composition at least 7 days after administration of the vaccine prime composition.
  • the HPV protein fragments encoded by the prime viral vector and the boost viral vector are the same.
  • the linker sequences can be different in the prime HPV vector vaccine and in the boost viral vector.
  • the order of the HPV protein fragments in the prime viral vector can be different to the order of the HPV protein fragments in the boost viral vector.
  • the linker sequences can be different in the prime HPV viral vector and in the boost HPV viral vector and the order of the HPV protein fragments in the prime viral vector can be different to the order of the HPV protein fragments in the boost viral vector.
  • the invention further provides a method of treating a subject in need thereof, wherein the method comprises administering a viral vector as described herein, e.g. as a vaccine composition.
  • the HPV viral vector can be a prime HPV viral vector.
  • the HPV viral vector can be a boost HPV viral vector.
  • the viral vector can be in the form of a composition comprising a HPV viral vector such as a prime viral vector or a boost viral vector and pharmaceutically acceptable carrier.
  • the invention further provides a method of treating a subject in need thereof, wherein the method comprises administering a prime HPV viral vector, and administering a boost HPV viral vector at least 7 days after the administration of the prime HPV viral vector.
  • the HPV protein fragments encoded by the prime viral vector and the boost viral vector are the same.
  • the linker sequences can be different in the prime HPV viral vector and in the boost HPV viral vector and/or the order of the HPV protein fragments in the prime viral vector can be different to the order of the HPV protein fragments in the boost viral vector.
  • the invention further provides a viral vector as described herein for use in a method of treatment.
  • the treatment can be for HPV.
  • the treatment can be for cancer.
  • the treatment can be prophylactic treatment for HPV.
  • the treatment can be prophylactic treatment for cancer.
  • the subject to be treated can suffer from a HPV infection.
  • the HPV infection can be persistent cervical high-risk HPV (hrHPV).
  • the HPV infection can be low-grade (Cl N 1 ) cervical lesions.
  • the subject to be treated can suffer from cancer.
  • the cancer can be a HPV- associated cancer.
  • the cancer can be cervical cancer.
  • kits comprising a HPV viral vector.
  • the kit can comprise a HPV viral vector in the form of a vaccine composition comprising a HPV viral vector vaccine such as a prime viral vector vaccine or a boost viral vector and pharmaceutically acceptable carrier.
  • kits comprising a combination of compositions wherein the combination comprises a prime viral vector, e.g. as vaccine composition and a boost viral vector, e.g. as a vaccine composition.
  • the HPV protein fragments encoded by the prime viral vector and the boost viral vector are the same.
  • the linker sequences can be different in the prime viral vector and in the boost viral vector and/or the order of the HPV protein fragments in the prime viral vector can be different to the order of the HPV protein fragments in the boost viral vector.
  • the polypeptide is a fusion polypeptide.
  • fusion polypeptide used herein is understood to mean a polypeptide comprising a combination of sequences derived from different gene products (for example different HPV proteins) or combinations of sequences from the same gene product (for example a single HPV protein), wherein the sequences are from distinct/separate regions of the wildtype gene product.
  • the fusion polypeptide may comprise combinations of sequences which are normally separated by other sequence segments in wild-type, and the separating sequence(s) have been removed.
  • FIG. 2 A) Baseline IFN-y SFC in clinical trial participants. B) Frequency of IFN-y SFC in clinical trial participants immunised with VTP-200. Participants were dosed with a placebo or a ChAdOxI - HPV and MVA-HPV primer-boost regime. ChAdOx1-HPV was administered at day 0 and boosted with MVA-HPV at day 28. Immunogenicity results are shown for day 35 (peak response), when peripheral blood samples were taken from the participants to measure the magnitude of response by ELISpot.
  • FIG. 3 A) Baseline CD8+ T cell responses in patients receiving placebo. B) Frequency of CD8+ T cell responses in patients immunised with VTP-200. Participants were dosed with a placebo or a ChAdOx1-HPV and MVA-HPV primer-boost regime. ChAdOx1-HPV was administered at day 0 and boosted with MVA-HPV at day 28.
  • FIG. 4 A) Baseline CD4+ T cell responses in patients receiving placebo. B) Frequency of CD4+ T cell responses in patients immunised with VTP-200. Participants were dosed with a placebo or a ChAdOx1-HPV and MVA-HPV primer-boost regime. ChAdOx1-HPV was administered at day 0 and boosted with MVA-HPV at day 28.
  • FIG. 5 the antigen sequence of antigen HPV2.
  • the HPV protein fragments are shown in rectangles below the sequence, and the HPV protein (E1 , E2, E4, E5, E6, or E7) and the HPV genotype from which the fragment is derived is included in the name of the fragment (16, 18,31 , 45, 52, 58).
  • the additional polypeptide sequences between the HPV protein fragments are linker sequences.
  • Design of the HPV peptide pools used to assess the number of IFN-y spot-forming cells (SFC) by ELISpot is indicated. Pool 1 polypeptides are derived from amino acids 1-630. Pool 2 polypeptides are derived from amino acids 631 - 1260. Pool 3 polypeptides are derived from amino acids 1261-1819.
  • FIG. 6 A) IFN-y SFC frequencies in cells restimulated with HPV peptide pool 1.
  • CD1 mice Four groups of CD1 mice were immunised with ChAdOxI and/or MVA vectors encoding the HPV2 antigen.
  • Group 1 comprised mice immunised with ChAdOxI -HPV2 only, Group 2 with MVA-HPV2, Group 3 with a ChAdOx-HPV2 and MVA-HPV2 prime-boost regime, and Group 4 with a phosphate buffered saline.
  • the frequency of antigen-specific T cells were measured by ELISpot are represented as the number of IFN-y SFC per million cells.
  • FIG. 7 A) IFN-y SFC frequencies in cells restimulated with HPV peptide pool 3.
  • CD1 mice Four groups of CD1 mice were immunised with ChAdOxI and/or MVA vectors encoding the HPV2 antigen.
  • Group 1 comprised mice immunised with ChAdOxI -HPV2 only, Group 2 with MVA-HPV2, Group 3 with a ChAdOx-HPV2 and MVA-HPV2 prime-boost regime, and Group 4 with a phosphate buffered saline.
  • Spleens were harvested at 42 days post-immunisation, splenocytes were recovered and restimulated with an HPV peptide pool.
  • the frequency of antigen-specific T cells were measured by ELISpot and represented as the number of IFN-y SFC per million cells.
  • FIG. 8 Prime-boost immunisation induces high frequencies of HPV-specific T cells.
  • IFN-y ELISpot was carried out on splenocytes from CD1 mice immunised with ChAdOx1-HPV2 (Group 1), MVA- HPV2 (Group 2) or ChAdOx1-HPV prime and MVA-HPV boost (Group 3).
  • the splenocytes were restimulated with a mixture of HPV2 peptide pools 1 to 3.
  • the frequency of antigen-specific T cells was represented as spot-forming units (sfu) per million cells.
  • FIG. 9 IFNy ELISpot data.
  • Figure 10 shows a first amino acid sequence encoded by the immunogen expression cassette in HPV3.2 e.g. for use in the ChAdOxI viral vector.
  • the N-terminal sequence shown in italics is the tPA leader sequence.
  • the HPV protein fragments are underlined and the linker sequences are shown in bold.
  • HPV protein fragments are, in order, (fragment_HPV genotype_HPV protein): 1_16_E6; 2_16_E6; 3_16_E6; 4_33_E2; 5_16_E2; 6_18_E6; 7_16_E7; 8_33_E6; 9_33_E6; 10_16_E2; 11_18_E2; 12_33_E6; 13_16_E2; 14_16_E1 ; 15_16_E7; 16_18_E6; 17_18_E7; 18_33_E7; 19_18_E6.
  • Figure 11 shows a second amino acid sequence encoded by the immunogen expression cassette in HPV3.2 e.g. for use in the MVA viral vector.
  • the N-terminal sequence shown in italics is the tPA leader sequence.
  • the HPV protein fragments are underlined and the linker sequences are shown in bold.
  • HPV protein fragments are, in order, (fragment_HPV genotype_HPV protein): 1_16_E6; 2_18_E6; 3_16_E1 ; 4_18_E6; 5_18_E6; 6_33_E6; 7_16_E6; 8_16_E2; 9_33_E6; 10_18_E7; 11_16_E6; 12_16_E2; 13_33_E7; 14_16_E7; 15_33_E6; 16_33_E2; 17_18_E2; 18_16_E7; 19_16_E2.
  • Figure 12 shows a first amino acid sequence encoded by the immunogen expression cassette in HPV3.1 e.g. for use in the ChAdOxI viral vector.
  • the N-terminal sequence shown in italics is the tPA leader sequence.
  • the HPV protein fragments are underlined and the linker sequences are shown in bold.
  • HPV protein fragments are, in order, (fragment_HPV genotype_HPV protein): 1_18_E2; 2_16_E6; 3_16_E4; 4_33_E2; 5_16_E2; 6_33_E6; 7_18_E4; 8_16_E6; 9_16_E6; 10_18_E6; 11_16_E7; 12_16_E7; 13_16_E4; 14_16_E1 ; 15_16_E2; 16_18_E7; 17_33_E7; 18_18_E4; 19_18_E6; 20_33_E6; 21_33_E6; 22_18_E6; 23_16_E2.
  • Figure 13 shows a second amino acid sequence encoded by the immunogen expression cassette in HPV3.1 e.g. for use in the MVA viral vector.
  • the N-terminal sequence shown in italics is the tPA leader sequence.
  • the HPV protein fragments are underlined and the linker sequences are shown in bold.
  • HPV protein fragments are, in order, (fragment_HPV genotype_HPV protein): 1_18_E2; 2_16_E7; 3_33_E6; 4_33_E2; 5_16_E1 ; 6_18_E6; 7_18_E6; 8_16_E6; 9_18_E4; 10_16_E7; 11_16_E2; 12_16_E6; 13_33_E6; 14_18_E4; 15_16_E2; 16_33_E6; 17_18_E7; 18_16_E6; 19_16_E2; 20_33_E7; 21_18_E6; 22_16_E4; 23_16_E4.
  • Figure 14 A shows a schematic of a prime -boost vaccination method used for administration of immunogen cassettes HPV1 and HPV3.2 to mice.
  • Figure 14 B shows stacked bar plots of HPV peptide interferon gamma responses.
  • the Y-axis shows SFU per 1 x 10 6 splenocytes.
  • Response to E1 is shown in white.
  • the response to E2 is shown in black.
  • the response to E4 is shown with vertical hatching.
  • the response to E5 is shown with diagonal markings from bottom left to top right.
  • the response to E6 is shown in grey.
  • the response to E7 is shown with diagonal markings from top left to bottom right.
  • the first column shows the response of mice treated with ChAdOx1-HPV1/MVA-HPV1
  • the second column shows the response of mice treated with PBS
  • the third column shows the response of mice treated with ChAdOx1-HPV1/MVA-HPV1.
  • the fourth column shows the response of mice treated with PBS.
  • Figure 15 shows scatter plots of HPV peptide interferon gamma responses for the junctional peptides (ChAdOxI -/MVA-HPV1) and the linkers (ChAdOxI -/MVA-HPV3.2) in the study described in Example 4.
  • the Y-axis shows SFU per 1 x 10 6 splenocytes.
  • ChAdOx1/MVA-HPV1 junction responses are shown with a solid circle.
  • ChAdOx-HPV3.2 spacer responses are shown with a solid square.
  • MVA-HPV3.2 spacer responses are shown with a solid triangle.
  • Figure 16 provides a bar chart showing the T-cell responses to each HPV protein following administration of ChAdox1-HPV3.2 prime vaccination alone or in combination with MVA- HPV3.2 boost vaccination in 6-8 week old female C57L/6J mice. Mean SFU per 1 x 10 6 cells are shown on the Y-axis. From the left, the response to HPV3 E1 , HPV3 E2, HPV3 E6 and HPV3 E7 are shown as a bar representing the average.
  • Figure 17 provides a scatter plot showing T-cell responses to junction regions in the HPV3.2 antigen after administration of ChAdox1-HPV3.2 prime vaccination alone or in combination with MVA-HPV3.2 boost vaccination in 6-8 week old female C57L/6J mice.
  • Mean SFU values per 1 x 10 6 cells are shown for: ChAdOx1-HPV3.2 spacer-specific T-cells in mice receiving ChAdOx- HPV3.2 only (solid circle), ChAdOx1-HPV3.2 spacer-specific T-cells in mice receiving ChAdOxl- HPV3.2 followed by MVA-HPV3.2 (C; solid triangle), and MVA-HPV3.2 spacer-specific T-cells in mice receiving ChAdOx1-HPV3.2 followed by MVA-HPV3.2 (M; solid triangle).
  • Figure 18 shows T-cell responses that are directed to HPV proteins (HPV) and directed to spacers (junctions or spacers) following administration of ChAdOx1-HPV1 and MVA-HPV1 (Figure 18A) or ChAdOxI -HPV3.2 and MVA-HPV3.2 ( Figure 18B) to mice.
  • HPV HPV proteins
  • spacers junctions or spacers
  • Prior art vaccines include a multi-HPV viral vaccine HPV001 consisting of 59 protein fragments joined end-to-end (no linkers), fragments are 9-55 amino acids in length and are derived from consensus sequences of five early HPV genes (E1 , E2, E4, E5, E6, E7) from six different high-risk genotypes (HPV16, 18, 31 , 52, 53, 58). The same HPV1 antigen was inserted into both ChAdOxI and MVA viral vectors.
  • ChAdOxI prime I MVA boost regimen was assessed in the HPV001 clinical trial: Antigen-specific T cell responses were generally low and primarily against E1 , E2, E4, and E6. A significant T cell response directed towards junctional epitopes was seen after ChAdOxI prime, which was further boosted by MVA. This constituted -50% of the total T cell response against the HPV1 antigen.
  • the current invention provides an improved HPV immunogen viral vector vaccine where the antigenspecific T cell responses are improved and the T cell response directed towards junctional epitopes is reduced.
  • the invention provides an HPV immunogen viral vector vaccine comprising a viral vector comprising an immunogen expression cassette, wherein the immunogen expression cassette encodes HPV protein fragments, and wherein the HPV protein fragments comprise immunogenic fragments of E1 , E2, E6 and E7 and where each HPV protein fragment is linked with a linker sequence.
  • the HPV immunogen viral vector vaccine is a multi-HPV immunogen viral vector vaccine.
  • the immunogen expression cassette expresses an antigen (polypeptide) that consists of 15 or more protein fragments that are all joined by linkers.
  • the fragments are immunogenic fragments from so- called early HPV genes which are expressed early in the viral life cycle, such as HPV proteins E1 , E2, E4, E5, E6 and E7.
  • the antigen comprises two or more fragment sequences from each of HPV protein E1 , E2, E6 and E7.
  • the fragment sequences are from three or more different high risk genotypes selected from the group consisting of HPV genotype 16, 18, 31 , 33, 45, 52 and 58.
  • the fragment sequences can be selected from the three HPV genotypes 16, 18 and 33.
  • the fragment sequences can be selected from the five HPV genotypes 16, 18, 31 , 52 and 58.
  • the immunogen expression cassette is a polynucleotide sequence in the viral vector that comprises a polynucleotide sequence that encodes an HPV antigen.
  • the antigen is a polypeptide comprising HPV protein fragments. Preferably each HPV protein fragment is joined to another HPV protein fragment by a polypeptide linker.
  • the HPV antigen can comprise from 15 to 50 HPV protein fragments.
  • the HPV antigen can comprise 46 HPV protein fragments.
  • the HPV antigen can comprise 15 to 30 HPV fragments.
  • the HPV antigen can consist of 23 HPV protein fragments.
  • the HPV antigen can consist of 19 HPV protein fragments.
  • the order of the HPV fragments in the antigen encoded by a first viral vector can be different from the order of the HPV fragments in the antigen encoded by a HPV viral vector (such as a boost HPV viral vector).
  • the order of the HPV fragments refer to the position where each HPV fragment is located in the polypeptide sequence, e.g. from the N-terminus to the C-terminus of the polypeptide, such as where the fragment closest to the N-terminus is fragment 1 , and so on.
  • Suitable antigens include the sequences of Table 1 , which are illustrated in Figures 5, and 10-13.
  • Each HPV protein fragment (also referred to herein as a fragment) is a contiguous amino acid sequence, e.g. 12 to 125 amino acids long, chosen independently from HPV protein E1 , E2, E4, E5, E6 or E7.
  • the HPV protein fragments can be chosen from E1 , E2, E6 and E7.
  • the fragment length can be 12 to 125 amino acids.
  • the fragment length can be 12 to 100 amino acids.
  • the fragment length can be 16 to 74 amino acids.
  • the fragment length can be 20 to 125 amino acids.
  • the fragment length can be 23 to 122 amino acids.
  • a suitable HPV protein fragment can be chosen from a segment in the amino acid sequence of relevant HPV protein where the sequence is substantially conserved across two or more genotypes, e.g. HPV16 and HPV18, or across three or more genotypes.
  • Suitable HPV fragments include one or more peptide sequences chosen from Table 2, or variants thereof (SEQ ID NO:s 6 to 24 and 71 to 75).
  • Table 2 P refers to the protein (antigen) from which the peptide sequence is derived.
  • the antigen can consist of or comprise the antigen peptide sequences in Figure 10 or variants thereof, where the antigen peptide sequences are joined by linker sequences.
  • the antigen can consist of or comprise the antigen peptide sequences in Figure 11 or variants thereof, where the antigen peptide sequences are joined by linker sequences.
  • the antigen can consist of or comprise the antigen peptide sequences in Figure 12 or variants thereof, where the antigen peptide sequences are joined by linker sequences.
  • the antigen can consist of or comprise the antigen peptide sequences in Figure 13 or variants thereof.
  • the antigen peptide sequences may be joined with the linker sequences, where the antigen peptide sequences are joined by linker sequences.
  • a linker sequence can comprise 2 to 15 amino acids.
  • a linker sequence can have at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, The linker can have less than 15, 14, 13, 13, 11 , 10, 9, or 8 amino acids.
  • Preferably a linker sequence comprises from 2 to 10 amino acids.
  • the polypeptide linker can be cleavable.
  • the polypeptide linker may comprise random amino acid sequences, or amino-acids that have been selected to be non-immunogenic based on epitope prediction computer programs or experiments in animal models. For example, a linker may not be considered if it is predicted or known to be an epitope (i.e. in order to avoid an immune response to epitopes, e.g.
  • the linker may be flexible.
  • the linker may comprise or consist of K, G, P or S amino acid residues, or combinations thereof.
  • the linker may comprise or consist of G and/or P amino acid residues.
  • the linker residues may be between 1 and 10 amino acids in length.
  • the linker residues may be between 2 and 8 residues in length.
  • the linker residues may be between 1 and 6 residues in length.
  • the linker may comprise residues A, N or D, or combinations thereof, such as ADN, AD, or DN.
  • the linker may comprise resides M, W, or A, or combinations thereof, such as MWA or WA.
  • a linker may comprise or consist of any of the sequences in Figures 5, 10, 11 ,12 and 13 i.e. the sequences that link the HPV protein fragments identified by rectangles below the antigen sequence in Figure 5 or shown in bold in figures 10-13.
  • the linker sequence can be selected from the following group of sequences (SEQ ID NO:s 77-126):
  • AD AD; ADAKCD; ADAKVD; ADAKVL; ADAQVD; ADAQVL; ADAYVD; ADDKVD; ADEQVD; ADFKTD; ADFQVD; ADGQVD; ADIQVD; ADLCL; ADLQD; ADLVL; ADMKVD; ADNCL; ADNCVW; ADNKCL; ADNKTW; ADNKVW; ADNNTL; ADNVCW; ADNVL; ADNVQL; ADNVTW; ADNVVL; ADNVVW; ADN W; ADNYTL; ADNYVL; ADPKVD; ADVQD; ADYVL; AINVVW; MW; MWAKQW; MWAW; MWCW; MWGKQD; MWGWCW; MWKQW; MWMKFW; MWQD; MWQW; MWRD; MWTW; MWWQW; and NDNVVCW.
  • the linker sequences in an immunogen cassette can be a linker sequence where the first two amino acids are AD (e.g. SEQ ID NO:s 105-109, 111-122, 124 -126).
  • the linker sequences in an immunogen cassette can be a linker sequence where the first two amino acids are MW (E.g. SEQ ID NO:s 77-83, 99-104).
  • the linker sequences in an immunogen cassette can be selected from the group of linker sequences where the first two amino acids are AD (e.g. SEQ ID NO:s 105-109, 111- 122, 124 -126).
  • the linker sequences in an immunogen cassette can be selected from the group of linker sequences where the first two amino acids are MW (E.g.
  • Each linker sequence may occur three or less times in an immunogen cassette sequence. Preferably each linker sequence appears six or less, five or less, four or less, three or less, two or less times in an immunogen cassette sequence.
  • the linker sequences in each of the more than one immunogen cassettes are preferably unique to that immunogen cassette. Without wishing to be bound by theory, by having a different set of linker sequences in different vaccines, e.g. in a prime and boost regimen, any immune reaction to the linker sequences is therefore not reinforced.
  • linkers can avoid generation of peptides with homology to human proteome (which could potentially generate immune response to self-antigen) and they avoid immune-dominant artificial epitopes. Additionally, linkers can provide a flexible hinge between segments of protein, so that they can fold into their native conformation.
  • Linkers may be varied between different immunogen expression cassettes. Using different linkers avoids boosting of any T-cell response created to any potential artificial epitopes (i.e. by changing linkers or changing junctions). For example, when vaccines are used in prime boost vaccination strategies, changing the linkers or the order of proteins within the immunogen layout, helps to overcome boosting of artificial epitope response.
  • linker sequences may be different in the antigens encoded by different immunogen expression cassettes. Different linker sequences include linker sequences with polypeptide sequences in which do not have 100% amino acid identity. Different linker sequences include linker sequences with polypeptide sequences which have less than 80% amino acid identity. Different linker sequences include linker sequences with polypeptide sequences which are longer or shorter.
  • a prime-boost regimen is method of vaccination involving the sequential administration of two vaccines, e.g. viral vectored vaccines, spaced by an interval of days or weeks.
  • the ‘prime’ vaccine is the first administered vaccine.
  • the prime vaccine can comprise an antigen (immunogen cassette) as described herein.
  • the boost vaccine is administered after the prime vaccine.
  • the boost vaccine can comprise an antigen (immunogen cassette) as described herein.
  • the prime can comprise an immunogen cassette with sequence SEQ ID NO: 1 or SEQ ID NO:3 or variants thereof.
  • the boost can comprise an immunogen cassette with sequence SEQ ID NO: 2 or SEQ ID NO:4 or variants thereof.
  • the prime can comprise an immunogen cassette with sequence SEQ ID NO: 1 or a variant thereof and the boost can comprise an immunogen cassette with sequence SEQ ID NO: 2 or a variant thereof or vice versa.
  • the prime can comprise an immunogen cassette with sequence SEQ ID NO: 3 or a variant thereof and the boost can comprise an immunogen cassette with sequence SEQ ID NO: 4 or a variant thereof or vice versa.
  • the boost vaccine can be administered at any time after the prime vaccine has initiated the primary immune response.
  • the boost vaccine is administered at least 7 days after the prime vaccine.
  • Preferably the boost vaccine is administered 14 days after the prime vaccine.
  • a variant of an amino acid or nucleic acid sequence may comprise a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% identity with that sequence.
  • a variant of an amino acid or nucleic acid sequence may comprise a sequence having at least 1 , 2, 3, 4 or 5 amino acid or nucleic acid substitutions with that sequence.
  • a variant of an amino acid or nucleic acid sequence may include additional amino acid or nucleic acid residues (such as 1 to 5, 1 to 20 additional amino acid or nucleic acid residues respectively for an amino acid or nucleic acid sequence) at the beginning or end of the sequence, e.g. where the additional residues are taken from the reference antigen sequence (e.g. E1 , E2, E6, or E7). Unless otherwise stated, for each of the amino acid or nucleic acid sequences described herein, a variant may be also be used..
  • a vaccine prime composition is the first administered vaccine composition, e.g. the first vaccine composition administered in a prime-boost regimen.
  • the vaccine prime composition is preferably a viral vectored vaccine encoding one or more target antigens.
  • the viral vector may be a nonreplicating adenovirus.
  • the non-replicating adenovirus may be of simian origin, such as chimpanzee adenovirus.
  • the adenovirus may be the ChAdOxI vector described in WO2012/172277, which is incorporated herein by reference.
  • the ChAdOxI virus has been engineered to be replication deficient.
  • a boost viral vector or a vaccine boost composition is a vaccine composition that is administered second after a prime viral vector or a vaccine prime composition, e.g. in a prime-boost regimen.
  • the boost viral vector or vaccine boost composition is administered at least 7 days after the prime viral vector or vaccine prime composition.
  • the vaccine boost composition preferably comprises a viral vectored vaccine encoding one or more target antigens.
  • the one or more target antigens comprise the one or more target antigens encoded by the vaccine prime composition.
  • the viral vectored vaccine does not replicate in the subject.
  • the viral vector may be a non-replicating pox virus, such as Modified Vaccinia virus Ankara (MVA) as described in W001/21201 , which is incorporated herein by reference.
  • MVA Modified Vaccinia virus Ankara
  • the viral vectored vaccine may be an RNA vectored vaccine.
  • the RNA vectored vaccine may be a self-amplifying RNA.
  • the vaccine boost composition may be the same as the vaccine prime composition (a homologous prime-boost method).
  • the vaccine boost composition may be different from the vaccine prime composition (a heterologous prime-boost method).
  • a viral vector may comprise a virus.
  • the viral vector may be an attenuated viral vector.
  • the viral vector may comprise an adenovirus, such as a human or simian adenovirus.
  • the viral vector comprises an adenovirus, such as a group E simian adenovirus, when used in a prime vaccine of a prime boost regime.
  • the viral vector may comprise a group E simian adenovirus.
  • the viral vector may comprise ChAdOxI (a group E simian adenovirus, like the AdCh63 vector used safely in malaria trials) or ChAdOx2.
  • the skilled person will be familiar with ChAdOxI based viral vectors, for example from patent publication W02012172277, which is herein incorporated by reference.
  • the viral vector may comprise AdCh63.
  • the viral vector may comprise AdC3 or AdH6.
  • the viral vector is a human serotype.
  • the viral vector comprises Modified Vaccinia Ankara (MVA).
  • the viral vector may comprise Adeno-associated virus (AAV) or Lentivirus.
  • AAV Adeno-associated virus
  • the viral vector may comprise any of Vaccinia virus, fowlpox virus or canarypox virus (e.g. members of Poxviridae and the genus Avipoxvirus), or New York attenuated vaccinia virus (Tartaglia et al. Virology. 30 1992 May;188(1):217-32, which is herein incorporated by reference).
  • the viral vector may comprise any of Herpes simplex virus, Cytomegalovirus (e.g.
  • human cytomegalovirus Measles virus (MeV), Sendai virus (SeV), Flavivirus (e.g. Yellow Fever Virus — 17D), or alphavirus vectors, such as Sindbis virus (SINV), Venezuelan equine encephalitis virus, or Semliki forest virus.
  • Measles virus Measles virus
  • Sendai virus SeMV
  • Flavivirus e.g. Yellow Fever Virus — 17D
  • alphavirus vectors such as Sindbis virus (SINV), Venezuelan equine encephalitis virus, or Semliki forest virus.
  • the prime viral vector or vaccine prime composition and the boost viral vector or vaccine boost composition are administered at least 7 days apart.
  • the vaccine boost composition may be administered up to 56 days after the vaccine primer.
  • the vaccine boost composition may be administered about 28 days after the vaccine prime composition. Tn.
  • the method and dosage regimes may include more than one administration of the vaccine boost composition, such as administration of a further (second) dose of a vaccine boost composition on up to day 84, preferably up to day 56, and optionally a further (third) dose of a vaccine boost composition on up to day 84..
  • the invention provides methods of treatment, compositions for use in a method of treatment and dosage regimes.
  • Treatment can mean a cure of the disease, e.g. HPV or cancer, an alleviation of symptoms or a reduction or slowing of severity in the disease or symptoms of the disease.
  • the antigens, compositions, viral vectors (including HPV prime viral vectors and HPV boost viral vectors), and vaccine compositions described herein can be used medicine, e.g. in a method of treatment.
  • compositions of the invention which include a vaccine composition comprising a viral vector as described herein, such as a vaccine prime composition or a vaccine boost composition, may comprise one or more additional active ingredients, an adjuvant, a pharmaceutically acceptable carrier, diluent and/or excipient.
  • Suitable carriers and/or diluents are well known in the art and include pharmaceutical grade starch, mannitol, lactose, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, saccharose (or other sugar), magnesium carbonate, gelatin, oil, alcohol, detergents, emulsifiers or water (preferably sterile).
  • the composition may be a mixed preparation of a composition or may be a combined preparation for simultaneous, separate or sequential use (including administration).
  • Suitable adjuvants are well known in the art and include incomplete Freund's adjuvant, complete Freund's adjuvant, Freund's adjuvant with MDP (muramyldipeptide), alum (aluminium hydroxide), alum plus Bordatella pertussis and immune stimulatory complexes (ISCOMs, typically a matrix of Quil A containing viral proteins).
  • composition according to the invention for use in the aforementioned indications may be administered by any convenient method, for example by oral (including by inhalation), parenteral (including by injection and by infusion), mucosal (e.g. buccal, sublingual, nasal), rectal ortransdermal administration and the compositions adapted accordingly.
  • a liquid formulation will generally consist of a suspension or solution of the compound or physiologically acceptable salt in a suitable aqueous or non-aqueous liquid carrier(s) for example water, ethanol, glycerine, polyethylene glycol or oil.
  • a suitable aqueous or non-aqueous liquid carrier(s) for example water, ethanol, glycerine, polyethylene glycol or oil.
  • the formulation may also contain a suspending agent, preservative, flavouring or colouring agent.
  • Typical parenteral compositions consist of a solution or suspension of the compound or physiologically acceptable salt in a sterile aqueous or non-aqueous carrier or parenterally acceptable oil, for example polyethylene glycol, polyvinyl pyrrolidone, lecithin, arachis oil or sesame oil.
  • a sterile aqueous or non-aqueous carrier or parenterally acceptable oil for example polyethylene glycol, polyvinyl pyrrolidone, lecithin, arachis oil or sesame oil.
  • the solution can be lyophilised and then reconstituted with a suitable solvent just prior to administration.
  • the pharmaceutical composition is preferably sterile. It is preferably pyrogen-free. It is preferably buffered e.g. at between pH 6 and pH 8, generally around pH 7. Preferably, the composition is substantially isotonic with humans.
  • the pharmaceutical compositions of the present invention deliver an immunogenically or pharmaceutically effective amount of a viral vector.
  • a pharmaceutically effective dose of a ChAdOxI -vectored vaccine composition comprises 1 x 10 7 to 1 x 10 12 viral particles, preferably 2 x 10 8 to 1 x 10 11 particles. More preferably, a pharmaceutically effective dose of a ChAdOxI -vectored vaccine composition comprises 2.5 x 1O 10 viral particles.
  • a pharmaceutically effective dose of an MVA-vectored vaccine composition comprises 1 x 10 5 to 1 x 10 11 plaque forming units (pfu), preferably 1 x 10 7 to 1 x 10 9 pfu. More preferably, a pharmaceutically effective dose of an MVA-vectored vaccine composition comprises 1 x 10 8 pfu.
  • the vaccine prime composition or vaccine boost composition is in unit dose form such as a capsule or ampoule.
  • the viral vectors and/or vaccine compositions of the present invention are capable of eliciting, inducing or boosting an antigen-specific immune response.
  • the immune response is a strong T cell immune response, for example a strong CD8+ T cell response and optionally a CD4+ T cell response.
  • the T cell immune response is a protective T cell immune response.
  • the T cell immune response is long lasting and persists for at least 1 , 2, 5, 10, 15, 20, 25 or more years.
  • the viral vectors, vaccine compositions, compositions and dosage regimens of the invention may be used to treat HPV infection.
  • the viral vector vaccines, compositions and dosage regimens of the invention may be used to treat persistent cervical high-risk HPV (hrHPV).
  • the viral vector vaccines, compositions and dosage regimens of the invention may be used to treat low-grade (CIN1) cervical lesions.
  • the viral vector vaccines, compositions and dosage regimens of the invention may be used to prevent or treat cancer, optionally wherein the cancer is a HPV-associated cancer, preferably wherein the cancer is cervical cancer.
  • the compositions, dosage regimes and methods can be used to treat or prevent infection by human papillomavirus and lesions, such as cervical lesions, caused by human papillomavirus.
  • compositions and dosage regimens preferably may be used to treat conditions which require induction of a CD8+ T cell response.
  • induction of a CD8+ T cell response to HPV can be used to treat HPV including persistent cervical high-risk HPV (hrHPV).
  • the T cell response optionally the CD8+ T cell response, is induced by administering a vaccine prime composition, e.g. including a replication incompetent adenoviral vector, followed by administering a vaccine boost composition, e.g. including an attenuated poxvirus vector.
  • a vaccine prime composition e.g. including a replication incompetent adenoviral vector
  • a vaccine boost composition e.g. including an attenuated poxvirus vector.
  • Conditions which may be treated include cancer, conditions (E.g. including cancer) caused by human papillomavirus.
  • the subject being treated using the method of treatment may be a patient at risk of or a patient suffering from a viral infection caused by human papilloma virus (HPV).
  • HPV human papilloma virus
  • the subject being treated using the method of treatment may suffer from chronic infection such as a chronic HPV infection.
  • the subject being treated using the method of treatment may have undergone therapy for the condition being treated, such as antiviral therapy, prior to administering the vaccine prime composition.
  • the subject may have undergone therapy for at least a month, at least 3 months, at least 6 months, at least 9 months, or least 12 months prior to administering the vaccine prime composition, most preferably at least 12 months.
  • compositions herein may be used to treat or to induce and/or boost an immune response against human papillomavirus.
  • the prime-boost regimen can produce a cross reactive response.
  • a prime boost regimen comprising ChAdOx1-HPV and MVA-HPV, comprising polypeptide antigen sequences from HPV16 and HPV18, can be used to treat or to induce and/or boost an immune response for one or more further HPV genotypes, such as 31 , 33, 45, 52, 53, 58.
  • the term boost as used herein relates to increasing the immune response.
  • the immune response can be measured for example by measuring levels of antigen-specific antibodies or T-cells in the blood of a subject using ELISA or ELISpot assays respectively.
  • an immune response can be measured by measuring the levels of a target antigen in the blood of the subject following administration of the compositions or combinations of the invention according to the methods of the invention.
  • kits are provided for use in treatment of HPV, comprising a vaccine or a vaccine composition as described herein.
  • a kit is provided for use in treatment of HPV comprising a prime composition and a vaccine boost composition which are administered separately.
  • the kit may suitably include instructions for use.
  • Kits are provided comprising two or more of the viral vectors or vaccine compositions described herein, such as a prime vaccine and a boost vaccine, as a combined preparation for separate, simultaneous or sequential use in a method of treatment of a viral infection or cancer.
  • a kit may comprise a prime HPV viral vector and a boost HPV viral vector, e.g. in the form of a vaccine composition comprising a pharmaceutically acceptable excipient..
  • the kits may be used with the methods of treatment described herein.
  • kits may be used in methods of treatment of HPV infection described herein, including prophylatic use.
  • the kits are used in methods comprising administering the boost viral vector or boost vaccine composition at least 7 days after administration of the prime viral vector or prime vaccine composition.
  • the viral vector may comprise nucleic acid comprising the sequence of SEQ ID NO: 127 and 128 (ChAdOxI) or a variant thereof.
  • a variant of SEQ ID NO: 127 and 128 may comprise a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% identity with SEQ ID NO: 127 and 128.
  • the variant of SEQ ID NO: 127 and 128 may encode a viral vector that substantially retains the function of the viral vector of SEQ ID NO: 127 and 128 (ChAdOxI).
  • the viral vector may comprise nucleic acid comprising the sequence of SEQ ID NO: 129 and 130 (ChAdOx2) or a variant thereof.
  • a variant of SEQ ID NO: 3 and 4 may comprise a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% identity with SEQ ID NO: 129 and 130.
  • the variant of SEQ ID NO: 129 and 130 may encode a viral vector that substantially retains the function of the viral vector of SEQ ID NO: 129 and 130 (ChAdOx2).
  • VTP-200 is a heterologous ChAdOx1-HPV prime and MVA-HPV boost regimen of two viral vectors that contain 59 conserved regions from all early proteins of 5 common high-risk HPV genotypes, arranged contiguously (end to end without linkers) in a polypeptide antigen.
  • Groups of 10 CD1 mice were primed intramuscularly with ChAdOxI -HPV (1 x 10 8 infectious units) and then boosted two weeks later with MVA-HPV (1 x 10 6 plaque-forming units). Spleens were harvested at 42 days postimmunisation, splenocytes were recovered and restimulated with an HPV peptide pool.
  • the frequency of antigen-specific T cells were measured by ELISpot and represented as the number of IFN-y SFC per million cells. Frequency of IFN-y SFC specific to the HPV proteins E1 , E2, E4, E5, E6 and E7 was assessed ( Figure 1). HPV proteins E1 , E2, E6 and E7 show the best response.
  • Example 2 The APOLLO trial (also known as HPV001) is evaluating the safety, immunogenicity and efficacy of VTP-200 in participants with persistent cervical high-risk HPV (hrHPV) infection and coexisting low- grade (Cl N 1 ) cervical lesions, or HPV-related change only (LSIL/ASCUS).
  • HPV001 also known as HPV001
  • HrHPV high-risk HPV
  • Cl N 1 low- grade
  • LSIL/ASCUS HPV-related change only
  • the primary objective is to determine the safety and tolerability of VTP-200.
  • the trial will also determine the effect on the hrHPV infection and lesion(s), as well as select the appropriate dose for further development.
  • the main phase is a blinded, randomised, placebo-controlled trial investigating 3 dose levels of ChAdOx1-HPV (Day 0) and 2 dose levels of MVA-HPV (Day 28), with a 12-month follow-up period.
  • PBMC peripheral blood mononuclear cell
  • Insert-specific cytokine production was assessed by intracellular cytokine staining (ICS) using thawed, rested PBMC samples.
  • PBMCs were stimulated for 6 hours using a pool of 107 overlapping peptides representing the antigen sequences of HPV (“insert”), or with a pool of 54 “junctional” peptides (16-mers with 8 amino acids on both the amino and carboxy terminus end of the antigen fusion site), as well as with positive (SEB and extended CEF peptide pools) and negative (DMSO) controls.
  • Stimulations were performed in the presence of Golgi transport blockers, monensin and brefeldin A, along with a fluorescently labelled antibody specific for the degranulation marker CD107a.
  • CD3+CD4+ or CD3+CD8+ T-cell population and their respective memory subsets i.e. excluding naive CD45RA+CCR7+ cells.
  • a qualified IFNy ELISpot assay was used to resolve the specificity of T-cell responses to the 107 constituent peptides. Peptides were divided into six separate HPV pools representing the E1 , E2, E4, E5, E6, and E7 early protein sequences and these were used to stimulate PBMC overnight resulting in IFNy production as measured by ELISpot.
  • FIG. 3 shows Day 35 CD8 responses to any cytokine (Gamma interferon, IL2, TNF alpha) separated into specific responses to an HPV component or a junctional sequence. The data reveals that robust CD8+ T cell responses make up most of the ELISpot responses shown in Figure 2. Baseline responses were essentially negative.
  • Figure 4 shows Day 35 CD4 responses to any cytokine (Gamma interferon, IL2, TNF alpha) are shown separated into specific responses to an HPV component or a junctional sequence. Baseline responses were essentially negative.
  • the junctional responses are of similar magnitude to the HPV responses (8/11 cases), which is consistent with the data from the lead-in phase.
  • FIG. 5 shows the antigen sequence of HPV2.
  • the HPV protein fragments are shown in rectangles below the sequence, and the HPV protein (E1 , E2, E4, E5, E6, or E7) and the HPV genotype from which the fragment is derived is included in the name of the fragment (16, 18,31 , 45, 52, 58).
  • the additional polypeptide sequences between the HPV protein fragments are linker sequences.
  • Design of the HPV peptide pools used to assess the number of IFN-y spot-forming cells (SFC) by ELISpot is indicated.
  • Pool 1 polypeptides are derived from amino acids 1-630.
  • Pool 2 polypeptides are derived from amino acids 631 - 1260.
  • Pool 3 polypeptides are derived from amino acids 1261-1819.
  • Group 1 comprised mice immunised with ChAdOx1-HPV2 only, Group 2 with MVA-HPV2, Group 3 with a ChAdOx-HPV2 and MVA-HPV2 prime-boost regime, and Group 4 with a phosphate buffered saline. Spleens were harvested at 42 days post-immunisation, splenocytes were recovered and restimulated with an HPV peptide pool.
  • the frequency of antigen-specific T cells were measured by ELISpot and represented as the number of IFN-y SFC per million cells
  • Figure 6 shows the number of IFN-y SFC per million cells for peptide pools 1 and 2.
  • Figure 7 shows the number of IFN-y SFC per million cells for peptide pool 3 and peptide pool 4.
  • Figure 8 shows the frequency of antigen-specific T cells represented as spot-forming units (sfu) per million cells for groups 1 to 3.
  • mice were immunised with an HPV primer boost regime (ChAdOx1-HPV1/MVA-HPV1 , ChAdOx3.2- HPV1/MVA-HPV3.2, or PBS) according to the schedule in Figure 14 A, Briefly, ChAdOx1-HPV1 or ChAdOx1-HPV3.2 prime vaccinations were administered on day 0, MVA-HPV1 or MVA-HPV3.2 boost vaccinations were administered on day 14, and PBS was administered to control animals on days 0 and 14. Spleens were harvested on day 28, and HPV peptide interferon gamma responses were measured using the methods described in Example 3.
  • Splenocytes from mice treated with ChAdOx1-HPV1 I MVA-HPV1 have a significant response to junctional peptides compared to cells from mice treated with PBS. No significant responses were seen to spacer peptide pools for HPV3.2-treated mice.
  • mice age 6 to 8- weeks old were used. Groups of 8 mice were used for each study. 2 x 10 5 splenocytes were stimulated for 18 hours ex vivo with HPV3 E1 , HPV3 E2, HPV3 E6 or HPV3 E7 peptide pools. Release of interferon-gamma from activated antigen-specific splenocytes was detected by ELISPOT as described above.
  • HPV3.2 is immunogenic in C57BL/6 mice. MVA (‘boost’ vaccination) was shown to boost the response after ChAdOxI (‘prime’ vaccination).
  • Example 6 The HPV3.2 immunogen cassette is designed such that the peptides appear in the antigen in a different order. In addition, no spacer from the ‘prime’ vaccination immunogen cassette is used in the ‘boost’ vaccination immunogen cassette.
  • Mice receiving ChAdOx-HPV3.2 only were tested for immune responses against the ChAdOx-HPV3 spacer peptide pool. Very low responses ( ⁇ 100 SFU per 1 x 10 6 cells) were seen in 3 out of 7 animals.
  • Mice receiving ChAdOx-HPV3.2 followed by MVA- HPV3.2 were tested for immune responses against the ChAdOx-HPV3 spacer and the MVA-HPV3 spacer (Figure 17). Very low responses to the ChAdOx-HPV3 spacer ( ⁇ 100 SFU per 1 x 10 6 cells) were seen in 3 out of 8 animals. No responses were seen towards the MVA-HPV3 spacer.

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Abstract

La présente invention concerne un immunogène multi-VPH contenant des vecteurs viraux, des vaccins, des compositions, des procédés et des régimes posologiques destinés à être utilisés en médecine, l'utilisation pouvant être pour le traitement d'une infection par le papillomavirus humain (VPH), notamment une utilisation prophylactique pour prévenir une infection par le VPH et/ou un cancer.
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