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WO2007018049A1 - Antigène capable d’induire un anticorps neutralisant pour le papillomavirus humain à haut risque - Google Patents

Antigène capable d’induire un anticorps neutralisant pour le papillomavirus humain à haut risque Download PDF

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WO2007018049A1
WO2007018049A1 PCT/JP2006/314919 JP2006314919W WO2007018049A1 WO 2007018049 A1 WO2007018049 A1 WO 2007018049A1 JP 2006314919 W JP2006314919 W JP 2006314919W WO 2007018049 A1 WO2007018049 A1 WO 2007018049A1
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epitope
amino acid
capsid
amino acids
protein
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Tadahito Kanda
Kazunari Kondo
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Japan Health Sciences Foundation
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    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • 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

Definitions

  • Antigens that induce neutralizing antibodies of high-risk group human papillomaviruses
  • the present invention relates to an antigen that induces a neutralizing antibody of a high-risk group human papillomavirus.
  • the present invention relates to a chimeric protein obtained by inserting a human papillomavirus L2 epitope into a human papillomavirus (HPV) type 16 L1 protein, and a capsid which is an assembly of the chimeric protein.
  • HPV human papillomavirus
  • the capsid of papillomavirus is an icosahedral particle composed of 360 L1 proteins and 12 L2 proteins.
  • viruses with humans, lions, deer, magpies, etc. as the host have been found and infect the mucous membranes to form proliferative lesions.
  • inactivated virus particles particles formed only by L1 protein (L1-capsid), particles formed by L1 protein and L2 protein (L1 / L2-capsid), in E. coli It has been shown that immunization using one of the L2 proteins produced as a vaccine antigen can protect against infection.
  • Human papillomavirus has more than 80 genotypes.
  • types that infect the mucous membrane at least 12 types (high risk types: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 68) It is known to cause cervical cancer.
  • high risk types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 68
  • cervical cancer Despite efforts to detect cervical cancer early through mass screening in Japan, 15,000 cases and 2500 deaths are reported annually.
  • some penile cancer, vulvar cancer, tongue cancer, pharyngeal cancer, laryngeal cancer, and esophageal cancer are also thought to be caused by HPV infection! /. WHO estimates that 11% (450,000) of malignancies in women worldwide are affected by HPV infection and that there are 300 million HPV carriers.
  • HPV particles have a structure of icosahedral capsid composed of 72 L1 protein pentamers and 12 molecules of L2 protein that wraps around the DNA genome! (Fig. 1). Capsid-like structures (L1-capsids) can be obtained when recombinant DNA technology is applied to express only L1 protein in large quantities. If L2 protein is also expressed at the same time, capsid (L1 / L L2-capsid) is formed. L1-capsid has been shown to be highly immunogenic and induces both antibody production and cellular immunity without adjuvant when inoculated into animals. This immunogenicity is specific to the HPV type, and type 16 L1-capsid immunization results in a type 16 specific response.
  • HPV L 1 / L2-capsid Since there is no cultured cell line that allows HPV growth, HPV L 1 / L2-capsid has been devised with an infectious pseudovirus that incorporates an expression plasmid for a marker gene into HPV. Yes. HPV neutralizing antibodies have been measured by inhibiting infection with this infectious pseudovirus.
  • Non-Patent Document 1 Koutsky et al .: A controlled trial of a human papillomavirus type 16 vaccine.N Eng J Med, 347 (21), 1645-1651, 2002 (Non-Patent Document 2) ))
  • This first generation vaccine antigen has a very high type specificity, for example, type 16 L1-capsidactin can only prevent type 16 infection. Therefore, it is desired to develop a vaccine antigen that induces a common high-risk type neutralizing antibody.
  • the inventors of the present invention have previously studied a mouse (BALB / c) monoclonal antibody (MAb5, 13) that recognizes an epitope (L2-epitope) in the amino acid 108-120 region of HPV16 type L2 protein, and Antisera obtained by immunizing BALB / c with a synthetic peptide having the same sequence as the amino acid 108-120 region of HPV16 type L2 protein shows that it inhibits infections of 6, 11 and 6 types as well as type 16. (Kawana. K, et al .: ommon neutralization epitope in minor capsid protein L2 of human papillomavirus types 16 and 6. J. Virology., 73, 6188-6190, 1999 (Non-Patent Document 3)).
  • the present inventors connect the amino acid 108-126 region of the HPV16 type L2 protein to the fluorescent protein and add it to the cell culture medium at 4 ° C. It was found that when heated to C (Kawana. Y, et al .: Human papillomavir us type 16 minor capsid protein 12 N— terminal region containing a common neutralization epitope binds to the cell surface and enters the cytoplasm J. Virology., 75, 23 31-2336, 2001 (see Non-Patent Document 5)).
  • HPV capsid is an icosahedral structure consisting of 72 L1 protein pentamers (capsomer)
  • L1-capsid has a structure with 12 L2 proteins inserted. Part of the L2 molecule appears on the capsid surface and is thought to play an important function in the early stages of infection.
  • the present inventors have clarified that a particle is formed even when a short peptide is inserted into three L1 protein sites, and the inserted peptide exists on the particle surface.
  • a chimera particle can present 1080 epitopes. The antiserum obtained by immunizing mice with the chimera particles neutralized multiple high-risk HPVs, so file a patent application as a vaccine antigen candidate!
  • the present inventors have so far developed a vaccine antigen using a high-risk type common neutralizing epitope in the amino acid 108-120 region of the HPV16 L2 protein.
  • the amino acid sequence of this epitope is about 60% and 75% homologous between high-risk L2 proteins, and the induced antibody binds to multiple high-risk HPVs, but compared to HPV16. The binding ability is inferior. Therefore, the appearance of antigens with higher type commonality is desired.
  • an object of the present invention is to provide an antigen that can cope with at least all types of high-risk types and has higher type commonality.
  • the present invention for solving the above problems is as follows.
  • a chimeric protein in which at least one L2 epitope of human papillomavirus L2 epitopes (hereinafter referred to as 64-81 L2 epitopes) is inserted into the loop site of human papillomavirus (HPV) type 16 L1 protein. Capsid is an aggregate,
  • L2 Epitopu group SGTGGRTGYIPLGTRPPT, SGTGGRTGYIPLGGRSNT, SG TGGRTGWPLSTRPST ⁇ SGSGGRTGYVPIGTDPPT, SGTGGRSGYVPLGTTPPT, T GTGGRTGYIPLGGRPNT, SGSGGRTGYVPLGGRSNT, SGSGGRTGYIPLGGGGRP, AGSGGRAGYVPLSTRPPT, TGSGGRAGYVPLGSRPST, SGTGGRTGYVPLGSTPP S or Kiyapushido consisting of an amino acid group having an amino acid sequence represented by SGTGGRTGYIPLGGKPNT,.
  • the L2 epitope group is an HPV antigen having an amino acid strength in which one or several amino acids are deleted, substituted, or added in the amino acid sequence, and having an L2 epitope based on the capsid.
  • the capsid according to [1] which also has a group strength of amino acids that imparts the same antigenicity as the sex.
  • the loop site for inserting the 64-81 L2 epitope is at least one force between amino acids 56 and 57, amino acids 140 and 141, amino acids 266 and 267, and amino acids 430 and 433. Capsid according to [1] or [2].
  • the loop site for inserting the 109-117 L2 epitope is at least one of amino acids 56 and 57, amino acids 140 and 141, amino acids 266 and 267, and amino acids 430 and 433.
  • L2 Epitopu group SGTGGRTGYIPLGTRPPT, SGTGGRTGYIPLGGRSNT, SG TGGRTGWPLSTRPST ⁇ SGSGGRTGYVPIGTDPPT, SGTGGRSGYVPLGTTPPT, T GTGGRTGYIPLGGRPNT, SGSGGRTGYVPLGGRSNT, SGSGGRTGYIPLGGGGRP, AGSGGRAGYVPLSTRPPT, TGSGGRAGYVPLGSRPST, SGTGGRTGYVPLGSTPP S, or chimeric protein comprising the amino acid group having an amino acid sequence represented by SGTGGRTGYIPLGGKPNT.
  • the L2 epitope group is an HPV having an L2 epitope in a capsid that also has an amino acid strength in which one or several amino acids are deleted, substituted, or added in the amino acid sequence and that also constitutes the chimeric protein strength.
  • the chimeric protein according to [7] which also has an amino acid group power that imparts antigenicity similar to that of.
  • the loop site that inserts the 64-81L2 epitope is at least one of the strongest positions between amino acids 56 and 57, between amino acids 140 and 141, between amino acids 266 and 267, and between amino acids 430 and 433.
  • the chimeric protein according to [7] or [8].
  • At least one L2 epitope (hereinafter referred to as 109-117 L2 epitopes) of the L2 epitope group consisting of amino acid groups having the amino acid sequence represented by VEETSFIDA, VEESGIVDV, IEETTFIES, IEESSFIDA, IEDSSWTS ⁇ or IE ESAIINA Inserted [7 The chimeric protein according to any one of [9] to [9].
  • the loop site for inserting the 109-117 L2 epitope is at least one of amino acids 56 and 57, amino acids 140 and 141, amino acids 266 and 267, and amino acids 430 and 433.
  • Keyhole limpet hemocyanin and human papillomavirus L2 epitope (hereinafter referred to as 64-81 L2 epitope) complex, which is SGTGGR TGYIPLGTRPPT, SGTGGRTGYIPLGGRSNT, SGTGGRTGYVPLSTRPST, SGSGGRPTGTG , TGTGGRTGYIPLGGRPNT ⁇ SGSGG RTGYVPLGGRSNT, SGSGGRTGYIPLGGGGRP, AGSGGRAGYVPLSTRPPT, TGS GGRAGYVPLGSRPST, SGTGGRTGYVPLGSTPPS, or a complex having an amino acid sequence represented by SGTGGRTGYIPLGGKP NT.
  • the present invention in addition to having a strong immunity-inducing activity in the particle structure, it is possible to strongly present HPV L2-epitopes and to provide antigens with higher type commonality. As a result, it is possible to provide an L1-capsid vaccine that can handle at least all high-risk types.
  • amino acid 64-81 region and amino acid 131-144 region of HPV16 type L2 protein contain an epitope of infection neutralizing antibody, and the amino acid sequence is extremely well conserved in the high-risk HPV group.
  • the best mode for carrying out the invention is capable of simultaneously inhibiting infection of high-risk HPV groups by crossing neutralizing activity
  • the L1-capsid of the present invention is a loop of human papillomavirus (HPV) type 16 L1 protein. It is a capsid that is an assembly of chimeric proteins in which at least one L2 epitope (64-81 L2 epitopes) of the human papillomavirus L2 epitope group is inserted at the site.
  • HPV human papillomavirus
  • HPV particles form 72 icosahedron capsids by collecting 72 capsomers assembled from 5 L1 proteins.
  • L1 protein When L1 protein is highly expressed in cells, L1 proteins gather in the nucleus and form capsids autonomously. Capsid formed only by L1 protein is called L1-capsid or virus-like particle (VLP).
  • VLP virus-like particle
  • L1 protein and L2 protein are expressed at the same time, 12 L2 proteins are incorporated into L1-capsid to form L1 / L2-capsid (or L1 / L2-VLP).
  • L1-capsid and L1 / L2-capsid cannot be distinguished by electron microscopy.
  • the L1-capsid of the present invention is a capsid based on an HPV16 type L1-capsid. Since HPV has many genotypes, it is generally written with the type on the display such as HPV16 or HPV58. HPV16 type L1-capsid is described as 16L1-capsid, HPV52 type L1 / L2-capsid is described as 52L1 / L2-capsid.
  • the L1-capsid of the present invention is an aggregate of chimeric proteins in which HPV L2 epitopes are inserted into HPV16 type L1 protein.
  • the chimeric protein means “a chimeric protein in which at least one L2 epitope of the L2 epitope group of human papillomavirus is inserted into the loop site of human papillomavirus (HP V) type 16 L1 protein”, and the present invention relates to This chimeric protein is also included.
  • the L2 epitope inserted into the L1 protein to produce the chimeric protein of the present invention has 18 amino acid strengths in the amino acid 64-81 region of the HPV16 type L2 protein.
  • amino acid residues of protein amino acids are also numbered in order, for example, the 50th amino acid is described as amino acid 50.
  • HPV52 and 58 type L1 / L2-capsids were used as antigens to examine the cross-reactivity of antibodies.
  • HPV infection is 72 hours after infecting 293TT cells by creating a pseudovirus in which the expression plasmid of the secretory alkaline phosphatase gene is incorporated into L1 / L2-capsid.
  • the subsequent culture was monitored by alkaline phosphatase activity.
  • the neutralization of the infection by the antibody was examined by reducing the alkaline phosphatase activity after mixing the infectious pseudovirus and the antibody and then infecting the cells (Fig. 5).
  • Anti-64-81, 107-122, and 131-144 antibodies inhibited infection of cultured cells with HPV type 16 pseudovirus. Among them, the anti-6481 antibody showed the most excellent neutralizing activity.
  • the amino acid sequence of the 64-81 region is very well conserved among multiple carcinogenic HPVL2s (homology rate of 75% or higher), and there is a possibility that a common type neutralizing epitope exists here (Fig. 6).
  • the anti-641-81 antibody showed neutralizing activity against HPV type 18 as expected (FIG. 7).
  • HPV16 pseudovirus was prepared by introducing amino acid substitution mutations into this region, and the infectivity was examined (Fig. 8). Replacing 69th arginine and 72nd Thai mouth sine with alanine significantly reduced the infectivity. Therefore, this region of the L2 protein plays an important role when HPV infects cells, and it is thought that HPV infection is inhibited by the binding of antibodies.
  • the amino acid 64-81 region of the HPV16 type L2 protein can be applied as a vaccine antigen for preventing infection of a high-risk type HPV group.
  • the L2 epitope (64-81 L2 epitope) group in the amino acid 64-81 region of HPV16 type L2 protein is SGTGGRTGYIPLGTRPPT (SEQ ID NO: 2), SGTGGR TGYIPLGGRSNT (SEQ ID NO: 3), SGTGGRTGYVPLSTRPST (SEQ ID NO: 4), SGSGGRTG YVPIGTDPPT (SEQ ID NO: 5), SGTGGRSGYVPLGTTPPT (SEQ ID NO: 6), TGTGGRTGYI PLGGRPNT (SEQ ID NO: 7), SGGRTGYVPLGGRSNT (SEQ ID NO: 8), SG ), AGSGGRAGYVPLSTRPPT (SEQ ID NO: 10), TGSGGRAGYV PLGSRPST (SEQ ID NO: 11), SGTGGRTGYVPLGSTPPS (SEQ ID NO: 12), or SGTGG RTGYIPLGGKP
  • SGTGGRTGYIPLGGRSNT (SEQ ID NO: 3) is an HPV group 18 type L2 epitope
  • SGTGGRTGYVPLSTRPST (SEQ ID NO: 4) is an HPV group 31 type L2 epitope
  • SGSG GRTGYVPIGTDPPT (SEQ ID NO: 5) is an HPV group.
  • Type 33 L2 epitope SGTGGRSG YVPLGTTPPT (SEQ ID NO: 6) is HPV group type 35 L2 epitope, TGTGGRTGYIPL GGRPNT (SEQ ID NO: 7) is HPV group type 39 L2 epitope, SGSGGRTGYVPLGGRS NT (SEQ ID NO: 8) Is an HPV group 45-type L2 epitope, SGSGGRTGYIPLGGGGRP (SEQ ID NO: 9) is an HPV group 51-type L2 epitope, AGSGGRAGYVPLSTRPPT (SEQ ID NO: 10) is an HPV group 52-type L2 epitope, and TGSGGRAGYVPLGSRPST (sequence) No.
  • HPV group 56 type L2 epitope is the HPV group 56 type L2 epitope
  • SGTGGRTGYVPLGSTPPS is the HPV group type 58 L2 epitope
  • SGTGGRTGYIPLGGKPNT is the HPV group type 68 L2 epitope.
  • the 64-81 L2 epitope group has one or several amino acid strength deletions, substitutions, or additions in the amino acid sequence, that is, the amino acid sequence described in any one of SEQ ID NOs: 2 to 13.
  • the capsids of the present invention can also have an amino acid group strength that imparts the same antigenicity as that of HPV having the L2 epitope as a base.
  • the 64-81 L2 epitope group has an amino acid strength that is deleted, substituted, or added to one or several amino acid strengths in the amino acid sequence represented by SGTGGRTGYIPLGTRPPT (SEQ ID NO: 2), and
  • the capsid of the present invention may include a variant of L2 epitopes having an amino acid sequence that confers HPV16 type antigenicity.
  • Such variants include HPV16 / T66A, HPV16 / R69A, HPV16 / Y72A, HPV16 / P74A, HPV16 / R78A, as shown in FIG. And HPV16 / R69A / Y72A. These mutations can be similarly applied to the following HPV type 18 and the like.
  • the 64-81 L2 epitope group has one or several amino acid strength deletions, substitutions or additions in the amino acid sequence represented by SGTGGRTGYIPLGGRSNT (SEQ ID NO: 3).
  • the capsid of the invention may include a variant of L2 epitopes having an amino acid sequence conferring HPV18 type antigenicity.
  • the 64-81 L2 epitope group has an amino acid force in which, in the amino acid sequence represented by SGTGGRTGYVPLSTRPST (SEQ ID NO: 4), several amino acids are deleted, substituted, or added,
  • the capsid of the present invention may include a variant of L2 epitopes having an amino acid sequence that confers HPV31 type antigenicity.
  • the 64-81 L2 epitope group consists of amino acids in which several amino acids are deleted, substituted, or added in the amino acid sequence represented by SGSGGRTGYVPIGTDPPT (SEQ ID NO: 5).
  • the capsid of the present invention may include a variant of L2 epitope having an amino acid sequence that confers HPV 33 type antigenicity.
  • the 64-81 L2 epitope group has one or several amino acid strength deletions, substitutions or additions in the amino acid sequence represented by SGTGGRSGYVPLGTTPPT (SEQ ID NO: 6).
  • the capsid of the invention may include a variant of L2 epitopes having an amino acid sequence conferring HPV35 type antigenicity.
  • the 64-81 L2 epitope group has one or several amino acid strength deletions, substitutions or additions in the amino acid sequence represented by TGTGGRTGYIPLGGRPNT (SEQ ID NO: 7).
  • the capsid of the invention may include a variant of L2 epitopes having an amino acid sequence conferring HPV39 type antigenicity.
  • the 64-81 L2 epitope group has one or several amino acid strength deletions, substitutions, or additions in the amino acid sequence represented by SGSGGRTGYVPLGGRSNT (SEQ ID NO: 8).
  • the capsid of the invention may include a variant of L2 epitopes having an amino acid sequence that confers HPV45 type antigenicity.
  • the 64-81 L2 epitope group has one or several amino acid strength deletions, substitutions, or additions in the amino acid sequence represented by SGSGGRTGYIPLGGGGRP (SEQ ID NO: 9).
  • the L2 epitope mutant having an amino acid strength and having an amino acid sequence imparting HPV51 type antigenicity to the capsid of the present invention can be included.
  • the 64-81 L2 epitope group has one or several amino acid strength deletions, substitutions, or additions in the amino acid sequence represented by AGSGGRAGYVPLSTRPPT (SEQ ID NO: 10).
  • the capsid of the invention may include a variant of L2 epitopes having an amino acid sequence conferring HPV52 type antigenicity.
  • the 64-81 L2 epitope group has one or several amino acid strength deletions, substitutions, or additions in the amino acid sequence represented by TGSGGRAGYVPLGSRPST (SEQ ID NO: 11).
  • the capsid of the invention can include a variant of L2 epitopes having an amino acid sequence conferring HPV56 type antigenicity.
  • the 64-81 L2 epitope group has one or several amino acid strength deletions, substitutions or additions in the amino acid sequence represented by SGTGGRTGYVPLGSTPPS (SEQ ID NO: 12).
  • the capsid of the invention may include a variant of L2 epitopes having an amino acid sequence conferring HPV58 type antigenicity.
  • the 64-81 L2 epitope group has one or several amino acid strength deletions, substitutions, or additions in the amino acid sequence represented by SGTGGRTGYIPLGGKPNT (SEQ ID NO: 13).
  • the capsid of the invention may include a variant of L2 epitopes having an amino acid sequence conferring HPV68 type antigenicity.
  • the L1 protein into which the 64-81 L2 epitope is inserted is a loop site of the L1 protein.
  • the loop site of the L1 protein is, for example, the amino acid 52-62, 85-97, 133-152, 171-183, 266-294, 315-325, 346-360, and 426-446 regions of the L1 protein.
  • the loop site of the L1 protein into which the 64-81 L2 epitope is inserted is at least between amino acids 56 and 57, between amino acids 140 and 141, between amino acids 266 and 267, and between amino acids 430 and 433. It is preferable that it is a single place.
  • the amino acid sequence of HPV16 type L1 protein is shown in SEQ ID NO: 1.
  • the present inventors compared the amino acid sequences of HPV16, 18, and 6 L1 proteins side-by-side, and considered that the common part between the types is likely to be related to the structure. Inappropriate. A region with a large difference in amino acid sequence depending on the type was predicted to be not essential for capsid formation itself, and was selected as an insertion candidate region for L2 epitopes.
  • L1-small particles small icosahedral particles smaller than capsids formed by the assembly of proteins excluding 10 amino acids of the amino terminal force of HPV16 L1 protein, and neutralization
  • L2 epitope it is possible to insert L2 epitope outside L1 protein.
  • the regions that can be recognized as one antigen are amino acids 52-62 (BC loop in HPV16 type L1-small particles), 85-97 (CD loop), 133-152 (DE loop), 171-183 (EF loop), It was estimated to be 266-294 (FG loop), 315-325 (GH loop), 346-360 (HI loop), and 426-446 (carboxy terminal) region.
  • the cited documents are listed at the end of the examples.
  • Site A In the region of amino acids 52-62, a report (cited reference 3) in which a hepatitis B virus epitope was inserted between amino acids 56 and 57 was helpful.
  • Amino acid 50 is ferulalanin
  • 51 is proline
  • 52 is isoleucine and hydrophobic amino acid
  • 53 and 54 are lysine
  • 56, 57 and 58 are asparagine
  • 59 is lysine and almost all hydrophilic amino acids
  • 60 becomes hydrophobic with isotocin it was assumed that the L2 epitope inserted between 56 and 57 appeared on the surface, and was selected as the insertion site.
  • Site B In the region of amino acids 133-152, a report in which amino acids 140 and 141 were inserted into hepatitis B virus epitope (Cited document 3) was helpful. Since this region contains many hydrophilic amino acid residues, it was assumed that the inserted L2 epitope appeared on the surface. We decided to insert it between amino acids 140 and 141, avoiding the immediate vicinity of the 146th cysteine residue.
  • Site C In the amino acid 266-294 region, 267 palin and 269 glutamine coincided with the palin at the amino terminal of the L2 epitope, and the third glutamine, so it was inserted between 266 and 267. Even if it matches the amino acid sequence of the L1 protein, it is easy to form a capsid. is there. At the same time, a report on the insertion of a hepatitis B virus epitope (Cited document 3) was also referenced.
  • Site D In the amino acid 426-446 region, 428 cysteine residues were known to be necessary for capsid formation (Reference 4), so 428 was avoided.
  • the amino acids 431 and 432 are HP V16, 18 and 6 type L1 proteins, and amino acids are mixed, and insertion of L2 epitopes is considered to have little effect on capsule formation, so it was inserted between amino acids 430 and 433. .
  • the chimeric L1 protein (16L1-430VA) inserted with L2 epitopes formed capsids.
  • the chimeric L1 protein (16L1-140 / 266 VA) in which L2 epitopes were simultaneously inserted into site 2 and site 3 formed capsids.
  • 64-81 L2 epitopes inserted into the L1 protein can be one in one L1 protein, but two or more identical 64-81 L2 epitopes are at different loop sites in one L1 protein. It can also be inserted, or it can be inserted into different loop sites of at least two different types of 64-81 L2 epitobu L1 proteins.
  • the capsid of the present invention may further contain an L2 epitope other than the 64-81 L2 epitope.
  • L2 epitope other than the 64-81 L2 epitope.
  • VEETSFIDA SEQ ID NO: 14
  • VEESGIVDV SEQ ID NO: 15
  • IEETTFIES SEQ ID NO: 16
  • IEESSFIDA SEQ ID NO: 17
  • IEDSSWTS SEQ ID NO: 18
  • IEESAIINA SEQ ID NO: 19
  • a capsid in which at least one L2 epitope (109-117 L2 epitope) in the L2 epitope group having an amino acid sequence is inserted in addition to the 64 -81 L2 epitope is also a capsid of the present invention.
  • V EETSFIDA (SEQ ID NO: 14) is a HPV group type 16 L2 epitope, which is particularly preferred.
  • VEESGIVDV (SEQ ID NO: 15) is the HPV group type 31 L2 epitope
  • IEETTFIES (SEQ ID NO: 16) is the HPV group type 52 L2 epitope
  • IEESSFIDA (SEQ ID NO: 17) is the HPV group type 58
  • IEDSSWTS SEQ ID NO: 18
  • IEESAIINA (SEQ ID NO: 19) is a 6-type and 11-type L2 epitope.
  • the 109-117 L2 epitope is similar to the 64-81 L2 epitope in the loop site of the L1 protein.
  • the loop site to be inserted is at least one of amino acids 56 and 57, amino acids 140 and 141, amino acids 266 and 267, and amino acids 430 and 433. It can be a place where the force is applied and the 64-81 L2 epitopes are not inserted.
  • 109-117 L2 epitopes inserted into the L1 protein can be one in one L1 protein, but two or more identical 109-117 L2 epitopes can be found at different loop sites in one L1 protein. It can also be inserted, or it can be inserted into different loop sites of at least two different types of 109-117 L2 Epitopeca S1 L1 protein. In either case, the L1 protein contains one or more 64-81 L2 epitopes.
  • FIG. 9 shows an example of a combination of the 64-81 L2 epitope inserted into the L1 protein and the 109-117 L2 epitope and the insertion position. However, these are merely examples and are not intended to limit the combination of these.
  • the L2 peptide (amino acids 64-81 or 109-117) was synthesized by the Fmoc solid-phase method using a 96-column automated peptide synthesizer by a conventional method.
  • This peptide is KLH (keyhole limpet hemocyanin) -conjugated for the purpose of antibody production, and a cysteine residue is added to the N-terminus to prevent the peptide region from being masked by KLH. .
  • the present invention is a complex of keyhole limpet hemocyanin and human papillomavirus 64-81 L2 epitope, which is SGTGGRTGYIPLGTRPPT (SEQ ID NO: 2), SGTGGRTGYIPLGGRSNT (SEQ ID NO: 3), SGTGGRTGYVPLSTRPST ( SEQ ID NO: 4), SGSGGRTGYVPIGTDPPT (SEQ ID NO: 5), SGTGGRSGYVPLGTTPPT (SEQ ID NO: 6), TGTGGRTGYIPLGGRPNT (SEQ ID NO: 7), SGGRTGYVPLGGRSNT (SEQ ID NO: 8), SGSGGRTGYIPLGGGGRP (SEQ ID NO: 9), SGSGGRTGYIPLGGGGRP (SEQ ID NO: 9) AGSGGRAGYVGRSTRPPT No.
  • SGTGGRTGYVPLGSTPPS SEQ ID NO: 12
  • a complex having an amino acid sequence represented by SGTGGRTGYIPLGGKPNT SEQ ID NO :.
  • Keyhole limpet mosyanin and human papillomavirus L2 epitope can be bound via cysteine.
  • SGTGGRTGYIPLGTRPPT SEQ ID NO: 2 is shown in FIG. [0062] (Preparation of LI protein inserted with L2 epitopes (chimeric protein))
  • a chimeric L1 gene was prepared and expressed using a baculovirus vector to prepare a chimeric L1 protein and a chimeric capsid.
  • the chimeric L1 gene was prepared by PCR. Details are as follows.
  • the present invention includes a method for producing a capsid, which comprises forming a capsid by assembling the chimeric protein of the present invention.
  • a chimeric L1 gene is produced and expressed using a baculovirus vector to produce a chimeric L1 protein.
  • a chimeric capsid (capsid of the present invention) is autonomously formed.
  • Expression using a baculovirus vector can be performed by a conventional method, but it is preferable to set conditions from the viewpoint of promoting autonomous formation of a chimeric capsid.
  • the antigen used was a chemically synthesized peptide combined with KL H (keyhole limpet hemocyanin).
  • KL H keyhole limpet hemocyanin
  • Synthetic peptides were used as antigens. Antigen was added to the ELISA plate at 0.5 g / 100 1 / well, and allowed to stand at 4 ° C to bind to the antigen well.
  • the antigen solution was removed, washed twice with a washing solution (0.2% Teen20 in PBS), filled with well with the washing solution, and allowed to stand at room temperature for 2 hours.
  • the 16L1, 16L1 / L2, 52L1, 52L1 / L2, 58L1, and 58L1 / L2 genes were expressed in a recombinant baculovirus system.
  • a recombinant virus was prepared using the Bac-to-Bac baculovirus expression system (GIBCO-BRL Inc., New York, NY) and expressed in s! 9 cells (night stealing-derived cells).
  • the 16L1 (as well as 52L1 and 58L1) gene was cloned into the pFastbacl vector to obtain pFsatbacl / 16Ll.
  • 16L1 / L2 (52L1 / L2, 58L1 / L2) was cloned into pFastbac dual vector to obtain pFastbac dual / 16Ll / L2.
  • each cFunged pFas tbac Veguta ⁇ DH10BAC Otsuki! 3 ⁇ 4 (jVlax Efficiency competent cell containing baculovirus DNA and helper plasmid, GIBCO BRL) was introduced to prepare Bacmid.
  • Bacmid DNA was introduced into sf-9 using Effectene Transfection Reagent (QIAGEN GmbH, Hilden, Germany) to obtain a recombinant baculovirus expressing capsid protein.
  • the recombinant baculovirus was infected with sf_9, and the cells were collected 72 hours later. Suspend infected cells in 0.5% NP40 solution, let stand at room temperature for 10 minutes, then centrifuge (9000 rpm, 15 minutes, 4 ° C) for nuclear fractionation (Precipitation) and cytoplasmic fraction. The nuclear fraction was suspended in a 1.28 g / ml salt / cesium-PBS solution, sonicated (Sonifier250, Branson), and then ultracentrifuged with SW50.1 roter (Beckman Coulter Inc., Fulleron, CA). 34000 rpm, 20 hours, 20 ° C). The protein collected at a specific gravity of about 1.28 g / ml in a cesium chloride gradient was collected and dialyzed with 0.5 M NaC ⁇ PBS to obtain a capsid protein solution.
  • HPV16L1 protein expression plasmid HPV16L2 protein expression plasmid
  • secretory alkaline phosphatase expression plasmid were transfected into 293TT cells using Lipofectamine2000. (Use 18L1 plasmid and 18L2 plasmid for preparation of type 18 infectious pseudovirus.) Cells were collected 72 hours after transfection.
  • the collected supernatant is layered on 27%, 33%, 39% optiprep (AXIS-SHIELD PoC AS) solution (diluted with PBS) and ultracentrifuged at 43700 rpm for 3 hours at 16 ° C. did.
  • Example 3 the same procedure as in Example 3 was performed, except that instead of the HPV16L2 protein expression plasmid, an L2 mutant HPV16L2 protein expression plasmid introduced with mutations at 64-81 shown in FIG. 6 was used. The results showing the relationship between the L2 mutant type and infectivity are shown in FIG.
  • Non-Patent Documents 1 to 5 are as follows. lj Matsumoto. K, et al .: Antibodies to human papillomavirus ID, 18, 58, and D b major capsid proteins among Japanese females. Jpn. J. Cancer Res., 88, 369—375, 1997.
  • Particles from chimeric proteins in which amino acids 108-120 of HPV16 L2 protein are inserted into HPV16L1 protein have strong immunogenicity and neutralizing antibody-inducing ability common to high-risk HPV groups (patent pending) ).
  • a vaccine antigen capable of inducing a stronger type-common neutralizing antibody can be produced.
  • FIG. 1 is a schematic diagram of a chimeric capsid for which a patent application was previously filed.
  • FIG. 2 Region of the peptide antibody produced.
  • the peptide was combined with KLH to obtain an immunizing antigen.
  • FIG. 3 shows the titer (ELISA) of anti-peptide antibodies based on the reactivity of antisera and peptides obtained by immunizing 2 rabbits with each peptide (the results of 1 animal were shown).
  • ELISA antigens are peptides only. Serum contained specific antibodies.
  • FIG. 4 shows the results of binding (ELISA) of anti-peptide antibodies and HPV capsids based on the reactivity of each peptide antibody to L1- or L1 / L2-capsid.
  • ELISA antigens are HPV type 16 L1 / L2-capsid (16L1 / L2), L1-capsid (16L1), etc.
  • FIG. 5 Neutralizing activity of each peptide antibody against HPV type 16 (HPV 16 type infectious pseudovirus (0.0 2 1)). Anti-14-27 antibody that does not bind to the surface region of L2 protein and anti-52 type L1 antibody with high type specificity did not show neutralizing activity. Anti-64-81, 107-122 and 131-144 antibodies showed neutralizing activity. [Fig. 6] Comparison of amino acid sequences of L2 (64-81) region. Representative anti-risk HPVL2 protein was shown.
  • FIG. 7 Neutralization of HPV18 infectious pseudovirus by anti-6481 antibody. The two doses of pseudovirus used were efficiently neutralized.
  • FIG. 8 Infectivity of HPVl type 6 pseudovirus having a mutant with amino acid substitution in L2 (64-81) region.
  • FIG. 9 shows an example of the insertion position and the combination of 64-81 L2 epitope inserted into L1 protein and 109-117 L2 epitope.

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Abstract

Le problème à résoudre dans le cadre de l'invention est de fournir un antigène pouvant être utilisé contre au moins un papillomavirus humain (HPV) à haut risque et qui soit un antigène vaccinal très courant. La solution proposée consiste à fournir une capside comprenant un ensemble d’une protéine chimère ayant au moins un épitope L2 sélectionné parmi des épitopes L2 du papillomavirus humain insérés dans le site en boucle de la protéine L1 de type 16 du papillomavirus humain, une protéine chimère ayant au moins un épitope L2 sélectionné parmi les épitopes L2 du papillomavirus humain insérés dans le site en boucle de la protéine L1 de type 16 du papillomavirus humain ; et un procédé pour fabriquer une capside comprenant un ensemble de la protéine chimère, le procédé comprenant l'assemblage de la protéine chimère de sorte à former la capside.
PCT/JP2006/314919 2005-08-10 2006-07-27 Antigène capable d’induire un anticorps neutralisant pour le papillomavirus humain à haut risque Ceased WO2007018049A1 (fr)

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RU2471807C2 (ru) * 2007-06-26 2013-01-10 Джапан Хелт Сайенсес Фаундейшн Вакцинный антиген, способный индуцировать перекрестно реагирующее и нейтрализующее антитело против папилломавируса человека с высоким риском
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EA022213B1 (ru) * 2009-06-25 2015-11-30 Глаксосмитклайн Байолоджикалс С.А. Новые конструкции белка вируса папилломы человека (hpv) и их применение в предупреждении заболевания, вызываемого hpv
CN103582651A (zh) * 2010-12-27 2014-02-12 公益财团法人日本健康科学振兴财团 识别人类乳突病毒(hpv)l2蛋白质的单克隆抗体及使用其的测定hpv中和抗体的效价的方法
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