JP4825958B2 - Antigens that induce neutralizing antibodies to high-risk group human papillomaviruses - Google Patents

Description

  The present invention relates to an antigen that induces a neutralizing antibody of a high-risk group human papillomavirus. In particular, the present invention relates to a chimeric protein in which the human papillomavirus L2 epitope is inserted into the human papillomavirus (HPV) type 16 L1 protein, and a capsid that is an assembly of the chimeric protein.

  The papillomavirus capsid is an icosahedral particle composed of 360 L1 proteins and 12 L2 proteins. So far, viruses with humans, cows, deer, rabbits, etc. as hosts have been found, and infects the huff and mucous membranes to form proliferative lesions. For bovine and rabbit papillomaviruses, inactivated virus particles, particles formed only by L1 protein (L1-capsid), particles formed by L1 protein and L2 protein (L1 / L2-capsid), made from E. coli It has been shown that immunization with one of the L2 proteins as a vaccine antigen can protect against infection.

  There are over 80 genotypes of human papillomavirus. Of the types that infect the mucous membrane (mucosa-directed HPV), at least 12 types (high-risk types: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 68) are uterus It is known to cause cervical cancer. Despite efforts to detect cervical cancer early by mass screening in Japan, 15000 cases of onset and 2500 deaths are reported annually. In addition, it is thought that HPV infection may cause some of penile cancer, vulvar cancer, tongue cancer, pharyngeal cancer, laryngeal cancer, and esophageal cancer. 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 in which a capsid of icosahedral composed of 72 L1 protein pentamers (capsomeres) and 12 molecules of L2 protein wraps the DNA genome (Fig. 1). Capsid-like structures (L1-capsids) can be formed by applying recombinant DNA technology to express only L1 protein in large quantities. If the L2 protein is also expressed at the same time, a capsid (L1 / L2-capsid) with the same composition as the virus particle is formed. L1-capsid has been shown to be highly immunogenic and induce 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 reaction.

  Since there is no cultured cell line that allows HPV growth, an infectious pseudovirus in which an expression plasmid of a marker gene is incorporated into the L1 / L2-capsid of HPV has been devised to monitor HPV infection. HPV infection neutralizing antibodies have been measured by inhibiting infection with this infectious pseudovirus.

  Since animal papillomavirus can prevent infection with a vaccine, it is expected that HPV infection can also be prevented with a vaccine. Vaccines that use HPV6, 11, 16, and 18 L1-capsids as antigens have been developed in the United States and are undergoing clinical trials. (Luisa Vilola et al .: Prophylactic quadrivalent human papillomavirus types 6, 11, 16, and 18) L1 virus-like particle vaccine in young women: a randomised double-blind placebo-controlled multicentre phase II efficacy trial. Lanset Oncology6, 271- 278, 2005 (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 is very type specific, for example, type 16 L1-capsid vaccine only prevents type 16 infection. Therefore, development of a vaccine antigen that induces a common high-risk type neutralizing antibody is desired.

  In the previous studies, the present inventors have investigated a mouse (BALB / c) monoclonal antibody (MAb5, 13) and HPV16 L2 that recognize an epitope (L2-epitope) in the amino acid 108-120 region of the HPV16 L2 protein. Antisera obtained by immunizing BALB / c with a synthetic peptide having the same sequence as the amino acid 108-120 region of the protein has been shown to inhibit not only type 16 but also type 6, 11 and type infections (Kawana K, et al .: Common neutralization epitope in minor capsid protein L2 of human papillomavirus types 16 and 6. J. Virology., 73, 6188-6190, 1999 (Non-patent Document 3)).

  Since the amino acid sequence of this L2 region is very similar in the mucosa-directed HPV group, it has been pointed out that antibodies that recognize the L2-epitope may prevent infection of the mucosa-directed HPV group widely. Furthermore, it was shown that this synthetic peptide is safe even when nasally inoculated into healthy human volunteers, and antibodies are induced in some human sera, neutralizing at least types 16 and 52 (Kawana). K, et al .: Safety and immunogenicity of a peptide containing the cross-neutralization epitope of HPV16 L2 administered nasally in healthy volunteers. Vaccine., 21: 4256-4260, 2003 (non-patent document 4)).

  The inventors linked the amino acid 108-126 region of the HPV16 type L2 protein to the fluorescent protein and added it to the cell culture at 4 ° C. This fusion protein bound to the cell surface, and when heated to 37 ° C, (Kawana. Y, et al .: Human papillomavirus type 16 minor capsid protein l2 N-terminal region containing a common neutralization epitope binds to the cell surface and enters the cytoplasm. J. Virology., 75 , 2331-2336, 2001 (non-patent document 5)).

  Based on these findings, the present inventors have found that a type-common neutralizing epitope exists in the amino acid 108-120 region of the HPV16 type L2 protein (FIG. 1), and this epitope is expressed in three locations of the L1 protein. We have already filed a patent application, indicating that the chimera capsid produced by incorporating the above into the above is a candidate for a second generation vaccine antigen.

The HPV capsid has a structure in which 12 L2 proteins are inserted into an icosahedral structure (L1-capsid) in which 72 L1 protein pentamers (capsomeres) are assembled. 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 positions of the L1 protein, and the inserted peptide exists on the particle surface. We identified the presence of a common neutralizing epitope in the amino acid 108-120 region of the HPV16 L2 protein that neutralizes the infection of the high-risk HPV group, and inserted this region into the L1 protein to create a chimeric particle. The chimeric particle can display 1080 epitopes. Since antisera obtained by immunizing mice with chimeric particles neutralized multiple high-risk HPVs, patent applications have been filed as vaccine antigen candidates.
Luisa Vilola et al .: Prophylactic quadrivalent human papillomavirus types 6, 11, 16, and 18) L1 virus-like particle vaccine in young women: a randomised double-blind placebo-controlled multicentre phase II efficacy trial. Lanset Oncology6, 271-278 , 2005 Koutsky et al .: A controlled trial of a human papillomavirus type 16 vaccine.N Eng J Med, 347 (21), 1645-1651, 2002 Kawana.K, et al .: Common neutralization epitope in minor capsid protein L2 of human papillomavirus types 16 and 6. J. Virology., 73, 6188-6190, 1999 Kawana.K, et al .: Safety and immunogenicity of a peptide containing the cross-neutralization epitope of HPV16 L2 administered nasally in healthy volunteers.Vaccine., 21: 4256-4260, 2003 Kawana.Y, et al .: Human papillomavirus type 16 minor capsid protein l2 N-terminal region containing a common neutralization epitope binds to the cell surface and enters the cytoplasm.J. Virology., 75, 2331-2336, 2001

  Immunization of animals with HPV L1-capsid induces a type-specific immune response. Interim results from clinical trials using type 16 L1-capsid vaccines have shown effectiveness in preventing type 16 infection, while having little effect on other types of prevention. Therefore, in order to prevent the development of cervical cancer with a vaccine, it is necessary to develop a vaccine antigen that can cope with at least all high-risk types.

  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 type L2 protein. However, the amino acid sequence of this epitope is about 60 to 75% homologous among high-risk L2 proteins, and the induced antibody binds to multiple high-risk HPVs, but has a binding capacity compared to HPV16. Is inferior. Therefore, the appearance of an antigen with higher type commonality is desired.

  Therefore, 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.
[1] A collection of chimeric proteins in which at least one L2 epitope of the human papillomavirus L2 epitope group (hereinafter referred to as 64-81 L2 epitope) is inserted into the loop site of human papillomavirus (HPV) type 16 L1 protein. A capsid,
The L2 epitope group, SGTGGRTGYIPLGTRPPT, SGTGGRTGYIPLGGRSNT, SGTGGRTGYVPLSTRPST, SGSGGRTGYVPIGTDPPT, SGTGGRSGYVPLGTTPPT, TGTGGRTGYIPLGGRPNT, SGSGGRTGYVPLGGRSNT, SGSGGRTGYIPLGGGGRP, AGSGGRAGYVPLSTRPPT, TGSGGRAGYVPLGSRPST, SGTGGRTGYVPLGSTPPS or capsid consisting of a group of amino acids having the amino acid sequence represented by SGTGGRTGYIPLGGKPNT,.
[2] The L2 epitope group consists of amino acids in which one or several amino acids are deleted, substituted, or added in the amino acid sequence, and the antigenicity of HPV having the original L2 epitope in the capsid The capsid according to [1], comprising an amino acid group that confers antigenicity similar to the above.
[3] The loop site for inserting the 64-81 L2 epitope is at least one position 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. Capsid according to [1] or [2].
[4] At least one L2 epitope (hereinafter referred to as 109-117 L2 epitope) of the L2 epitope group consisting of an amino acid group having an amino acid sequence represented by VEETSFIDA, VEESGIVDV, IEETTFIES, IEESSFIDA, IEDSSVVTS, or IEESAIINA The capsid according to any one of [1] to [3], which is inserted.
[5] The loop site for inserting the 109-117 L2 epitope is at least one position 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. And the capsid according to [4], wherein the 64-81 L2 epitope is not inserted.
[6] The capsid according to any one of [1] to [5], which is used for producing an L1-capsid vaccine.
[7] A chimeric protein in which at least one L2 epitope (hereinafter referred to as 64-81 L2 epitope) of the L2 epitope group of human papillomavirus is inserted into the loop site of human papillomavirus (HPV) type 16 L1 protein,
The L2 epitope group, SGTGGRTGYIPLGTRPPT, SGTGGRTGYIPLGGRSNT, SGTGGRTGYVPLSTRPST, SGSGGRTGYVPIGTDPPT, SGTGGRSGYVPLGTTPPT, TGTGGRTGYIPLGGRPNT, SGSGGRTGYVPLGGRSNT, SGSGGRTGYIPLGGGGRP, AGSGGRAGYVPLSTRPPT, TGSGGRAGYVPLGSRPST, SGTGGRTGYVPLGSTPPS, or chimeric protein comprising the amino acid group having an amino acid sequence represented by SGTGGRTGYIPLGGKPNT.
[8] The L2 epitope group is an HPV having one or more amino acids deleted, substituted or added in the amino acid sequence, and having an L2 epitope on a capsid composed of the chimeric protein. The chimeric protein according to [7], comprising an amino acid group that confers antigenicity similar to that of
[9] The loop site for inserting the 64-81L2 epitope is at least one position between amino acids 56 and 57, amino acids 140 and 141, amino acids 266 and 267, and amino acids 430 and 433 [ The chimeric protein according to [7] or [8].
[10] At least one L2 epitope (hereinafter referred to as 109-117 L2 epitope) of the L2 epitope group consisting of an amino acid group having an amino acid sequence represented by VEETSFIDA, VEESGIVDV, IEETTFIES, IEESSFIDA, IEDSSVVTS, or IEESAIINA The chimeric protein according to any one of [7] to [9], which is inserted.
[11] The loop site for inserting the 109-117 L2 epitope is at least one position 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 [10], wherein the 64-81 L2 epitope is not inserted.
[12] The method for producing a capsid according to any one of [1] to [5], comprising forming a capsid by assembling the chimeric protein according to any one of [7] to [11].
[13] A complex of keyhole limpet hemocyanin and human papillomavirus L2 epitope (hereinafter referred to as 64-81 L2 epitope), and the 64-81 L2 epitope is SGTGGRTGYIPLGTRPPT, SGTGGRTGYIPLGGRSNT, SGTGGRTGYVPLSTRPST, SGSGGRTGYVPIGTTGTG, TGTGGRSGGTGTGTGTGTGSG , SGSGGRTGYIPLGGGGRP, AGSGGRAGYVPLSTRPPT, TGSGGRAGYVPLGSRPST, SGTGGRTGYVPLGSTPPS, or a complex having an amino acid sequence represented by SGTGGRTGYIPLGGKPNT.
[14] The complex according to [13], wherein keyhole limpet hemocyanin and 64-81 L2 epitope of human papillomavirus are bound via cysteine.

  According to the present invention, in addition to the strong immunity-inducing activity of the particle structure, HPV L2-epitope can be strongly presented as an antigen, and an antigen with higher type commonality can be provided. As a result, it is possible to provide an L1-capsid vaccine that can handle at least all high-risk types.

  The amino acid 64-81 region and amino acid 131-144 region of HPV16 type L2 protein contain the epitope of the neutralizing antibody for infection, and the amino acid sequence is very well preserved in the high-risk HPV group. Sex can simultaneously inhibit infection in high-risk HPV groups.

(L1-capsid)
The L1-capsid of the present invention is an assembly of chimeric proteins in which at least one L2 epitope (64-81 L2 epitope) of the L2 epitope group of human papillomavirus is inserted into the loop site of human papillomavirus (HPV) type 16 L1 protein. Capsid is the body.

  As described above, HPV particles form 72 icosahedron capsids with 72 capsomeres assembled from 5 L1 proteins. When L1 protein is highly expressed in cells, L1 proteins gather in the nucleus and form capsids autonomously. The capsid formed only by the L1 protein is called L1-capsid or virus-like particle (VLP). When 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). However, L1-capsid and L1 / L2-capsid cannot be distinguished by electron microscopy.

  The L1-capsid of the present invention is a capsid based on the HPV16 type L1-capsid. Since HPV has many genotypes, HPV16 and HPV58 are generally indicated along with the type. Here, HPV16 type L1-capsid is described as 16L1-capsid, and 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 the HPV L2 epitope is inserted into the HPV16 type L1 protein. Hereinafter, 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 (HPV) type 16 L1 protein", and the present invention includes: This chimeric protein is also included.

  The L2 epitope inserted into the L1 protein to produce the chimeric protein of the present invention consists of 18 amino acids in the amino acid 64-81 region of the HPV16 type L2 protein.

  In the present invention, amino acid residues are numbered in order from the amino terminus of the protein. For example, the 50th amino acid is described as amino acid 50.

  Focusing on the amino acid 14-27, 64-81, and 131-144 regions as regions with many hydrophilic amino acids in the HPV16 L2 protein, rabbits were immunized with synthetic peptides having these amino acid sequences (Figure 2). . All peptides induced specific antibodies (Figure 3).

  An ELISA using HPV16 capsid as an antigen was used to examine whether the antigen was presented on the particle surface. For the analysis, anti-107-122 antibody, which has been focused on so far, was added. None of the antibodies bound to L1-capsid (ELISA absorbance of about 0.1 is the background of this measurement system). For L1 / L2-capsid, not only anti-107-122 antibody but also anti-64-81 and 131-144 antibodies were bound, indicating that a region containing these epitopes was present on the particle surface. . The anti-14-27 antibody did not bind to the L1 / L2-capsid, indicating that this region exists within the capsid (FIG. 4).

  Furthermore, antibody cross-reactivity was examined using HPV52 and 58 L1 / L2-capsids as antigens (FIG. 4). The anti-64-81 antibody showed clear crossing with both antibodies. As for the anti-107-122 antibody, one antibody had a low response to type 58, and the cross property of the anti-131-144 antibody was largely different among rabbits.

Since there is no culture cell line that allows HPV to grow, HPV infection is a culture solution 72 hours after infecting 293TT cells by making a pseudovirus incorporating a secreted alkaline phosphatase gene expression plasmid in L1 / L2-capsid. Were monitored for alkaline phosphatase activity. Infection neutralization by antibody was examined by reducing alkaline phosphatase activity after infecting cells after mixing this infectious pseudovirus and antibody (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-64-81 antibody showed the most excellent neutralizing activity.
The amino acid sequence of the 64-81 region is extremely well conserved among multiple carcinogenic HPVL2s (homology rate of 75% or more), and there may be a type-common neutralizing epitope (Fig. 6).

  As expected from the homology of the amino acid sequence, the anti-64-81 antibody also showed infection neutralizing activity against HPV type 18 (FIG. 7).

  We considered that the 64-81 region plays an important role in the establishment of infection, and made HPV16 pseudovirus with amino acid substitution mutations in this region, and examined the infectivity (Fig. 8). Replacing the 69th arginine and 72nd tylosin 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 binding of antibodies.

  From the above results, the amino acid 64-81 region of the HPV16 type L2 protein can be applied as a vaccine antigen for preventing infection of the high-risk HPV group.

  In particular, in the present invention, the L2 epitope (64-81 L2 epitope) group in the amino acid 64-81 region of HPV16 type L2 protein includes 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), SGSGGRTGYVPLGGRSNT (SEQ ID NO: 8), SGSGGRTGYIPLGGGGRP (SEQ ID NO: 9), AGSGGRAGYVPLSTRPPT (SG No. , SGTGGRTGYVPLGSTPPS (SEQ ID NO: 12), or SGTGGRTGYIPLGGKPNT (SEQ ID NO: 13). SGTGGRTGYIPLGTRPPT (SEQ ID NO: 2) is a type 16 L2 epitope of the HPV group and is particularly preferred.

  SGTGGRTGYIPLGGRSNT (SEQ ID NO: 3) is the HPV group type 18 L2 epitope, SGTGGRTGYVPLSTRPST (SEQ ID NO: 4) is the HPV group type 31 L2 epitope, and SGSGGRTGYVPIGTDPPT (SEQ ID NO: 5) is the HPV group type 33 L2 epitope. SGTGGRSGYVPLGTTPPT (SEQ ID NO: 6) is the HPV group type 35 L2 epitope, TGTGGRTGYIPLGGRPNT (SEQ ID NO: 7) is the HPV group type 39 L2 epitope, SGSGGRTGYVPLGGRSNT (SEQ ID NO: 8) is the HPV group type 45 L2 SGSGGRTGYIPLGGGGRP (SEQ ID NO: 9) is an HPV group type 51 L2 epitope, AGSGGRAGYVPLSTRPPT (SEQ ID NO: 10) is an HPV group type 52 L2 epitope, and TGSGGRAGYVPLGSRPST (SEQ ID NO: 11) is an HPV group type 56 It is an L2 epitope, SGTGGRTGYVPLGSTPPS (SEQ ID NO: 12) is an HPV group type 58 L2 epitope, and SGTGGRTGYIPLGGKPNT (SEQ ID NO: 13) is an HPV group type 68 L2 epitope.

  The 64-81 L2 epitope group is derived from an amino acid in which one or several amino acids are deleted, substituted, or added in the amino acid sequence, that is, the amino acid sequence described in any of SEQ ID NOs: 2 to 13. And an amino acid group conferring antigenicity similar to that of HPV having the original L2 epitope on the capsid of the present invention.

  That is, the 64-81 L2 epitope group consists of amino acids in which one or several amino acids are deleted, substituted, or added in the amino acid sequence represented by SGTGGRTGYIPLGTRPPT (SEQ ID NO: 2), and the capsid of the present invention. May include a variant of the L2 epitope having an amino acid sequence that confers HPV16 type antigenicity. Examples of such mutants include HPV16 / T66A, HPV16 / R69A, HPV16 / Y72A, HPV16 / P74A, HPV16 / R78A, and HPV16 / R69A / Y72A shown in FIG. These mutations can be similarly applied to the following HPV type 18 and the like.

  Similarly, the 64-81 L2 epitope group consists of amino acids in which one or several amino acids are deleted, substituted, or added in the amino acid sequence represented by SGTGGRTGYIPLGGRSNT (SEQ ID NO: 3), and Variants of the L2 epitope having an amino acid sequence conferring HPV18 type antigenicity to the capsid can be included.

  Similarly, the 64-81 L2 epitope group consists of amino acids in which one or several amino acids are deleted, substituted, or added in the amino acid sequence represented by SGTGGRTGYVPLSTRPST (SEQ ID NO: 4), and Variants of L2 epitopes having an amino acid sequence conferring HPV31 type antigenicity to the capsid can be included.

  Similarly, the 64-81 L2 epitope group consists of amino acids in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SGSGGRTGYVPIGTDPPT (SEQ ID NO: 5), and Variants of L2 epitopes having amino acid sequences that confer HPV 33 type antigenicity to the capsid can be included.

  Similarly, the 64-81 L2 epitope group consists of amino acids in which one or several amino acids are deleted, substituted, or added in the amino acid sequence represented by SGTGGRSGYVPLGTTPPT (SEQ ID NO: 6). Variants of the L2 epitope having an amino acid sequence conferring HPV35 type antigenicity to the capsid can be included.

  Similarly, the 64-81 L2 epitope group consists of amino acids in which one or several amino acids are deleted, substituted, or added in the amino acid sequence represented by TGTGGRTGYIPLGGRPNT (SEQ ID NO: 7), and Variants of the L2 epitope having an amino acid sequence conferring HPV39 type antigenicity to the capsid can be included.

  Similarly, the 64-81 L2 epitope group consists of amino acids in which one or several amino acids are deleted, substituted, or added in the amino acid sequence represented by SGSGGRTGYVPLGGRSNT (SEQ ID NO: 8). Variants of the L2 epitope having an amino acid sequence conferring HPV45 type antigenicity to the capsid can be included.

  Similarly, the 64-81 L2 epitope group consists of amino acids in which one or several amino acids are deleted, substituted, or added in the amino acid sequence represented by SGSGGRTGYIPLGGGGRP (SEQ ID NO: 9). Variants of L2 epitopes having amino acid sequences that confer HPV51 type antigenicity to the capsid can be included.

  Similarly, the 64-81 L2 epitope group consists of amino acids in which one or several amino acids are deleted, substituted, or added in the amino acid sequence represented by AGSGGRAGYVPLSTRPPT (SEQ ID NO: 10), and Variants of the L2 epitope having an amino acid sequence conferring HPV52 type antigenicity to the capsid can be included.

  Similarly, the 64-81 L2 epitope group consists of amino acids in which one or several amino acids are deleted, substituted, or added in the amino acid sequence represented by TGSGGRAGYVPLGSRPST (SEQ ID NO: 11), and Variants of the L2 epitope having an amino acid sequence conferring HPV56 type antigenicity to the capsid can be included.

  Similarly, the 64-81 L2 epitope group consists of amino acids in which one or several amino acids are deleted, substituted, or added in the amino acid sequence represented by SGTGGRTGYVPLGSTPPS (SEQ ID NO: 12), and Variants of the L2 epitope having an amino acid sequence conferring HPV58 type antigenicity to the capsid can be included.

  Similarly, the 64-81 L2 epitope group consists of amino acids in which one or several amino acids are deleted, substituted, or added in the amino acid sequence represented by SGTGGRTGYIPLGGKPNT (SEQ ID NO: 13), and Variants of the L2 epitope having an amino acid sequence conferring HPV68 type antigenicity to the capsid can be included.

  In the chimeric protein of the present invention, 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. In particular, the loop site of the L1 protein into which the 64-81 L2 epitope is inserted is at least one position 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 The amino acid sequence of HPV16 type L1 protein is shown in SEQ ID NO: 1.

  From the viewpoints of structural analysis of HPV16L1 protein, distribution of neutralizing antibody epitopes, and position of cysteine residues essential for encapsidation, the region outside L1 protein and capable of being recognized as an antigen is Amino acids 52-62, 85-97, 133-152, 171-183, 266-294, 315-325, 346-360, and 426-446 regions.

  The present inventors compared the amino acid sequences of HPV16 type, 18 type, and 6 type L1 proteins side by side, and considered that the common part between the types is likely to be related to the structure, which is inappropriate for insertion of the L2 epitope. did. Regions with greatly different amino acid sequences depending on the type were predicted not to be essential for capsid formation itself, and were selected as candidate insertion regions for the L2 epitope. Furthermore, structural analysis of L1-small particles (small icosahedral particles smaller than capsids) formed by the assembly of proteins excluding 10 amino acids from the amino terminus of HPV16 L1 protein, and neutralizing antibody With reference to the distribution of the epitope (Cited document 3) and the position of the cysteine residue essential for encapsidation (Cited document 4), the L2 epitope can be inserted and recognized as an antigen outside the L1 protein. The possible regions 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), 266-294 (FG Loop), 315-325 (GH loop), 346-360 (HI loop), and 426-446 (carboxy terminus) regions. The cited documents are listed at the end of the examples.

  Furthermore, the insertion site was narrowed down for the following reasons, and a chimeric protein into which the L2 epitope was inserted was produced to examine the formation of capsid.

  In the region A: amino acid 52-62 region, a report (cited document 3) in which an epitope of hepatitis B virus was inserted between amino acids 56 and 57 was helpful. Amino acid 50 is phenylalanine, 51 is proline, 52 is isoleucine and hydrophobic amino acids, 53 and 54 are lysine, 56, 57 and 58 are asparagine, 59 is lysine and almost all hydrophilic amino acids, 60 is Since it becomes hydrophobic with isoleucine, it was assumed that the L2 epitope inserted between 56 and 57 appeared on the surface, and was selected as the insertion site.

  In the region B: amino acid 133-152 region, a report (cited document 3) in which an epitope of hepatitis B virus was inserted between amino acids 140 and 141 was helpful. Since this region contains many hydrophilic amino acid residues, it was assumed that the inserted L2 epitope appeared on the surface. The insertion was between amino acids 140 and 141, avoiding the immediate vicinity of the 146th cysteine residue.

  Site C: In the region of amino acids 266-294, 267 valine, 269 glutamine coincided with the amino terminal valine of the L2 epitope, the third glutamine, and was inserted between 266 and 267. This is because it was thought that capsids could be easily formed if they matched the amino acid sequence of L1 protein. At the same time, a report (cited document 3) in which an epitope of hepatitis B virus was inserted was also referred to.

  Site D: In the region of amino acids 426 to 446, it is known that 428 cysteine residues are indispensable for capsid formation (Cited document 4), so 428 was avoided. The amino acids 431 and 432 are HPV16, 18 and 6 type L1 proteins, and the amino acids vary, and insertion of the L2 epitope is considered to have little effect on capsid formation, and was inserted between amino acids 430 and 433.

  And when I actually tried to form a capsid, it was an L2 epitope other than the 64-81 L2 epitope, but the chimeric L1 protein (16L1-266VA) with the L2 epitope inserted into site 3 and the L2 epitope inserted into site 4 The chimeric L1 protein (16L1-430VA) formed a capsid. The chimeric L1 protein (16L1-140 / 266VA) in which the L2 epitope was simultaneously inserted into site 2 and site 3 formed a capsid. The chimeric L1 protein (16L1-56 / 140 / 266VA) in which the L2 epitope was simultaneously inserted into site 1, site 2 and site 3 formed a capsid.

  64-81 L2 epitopes inserted into L1 protein can be one in one L1 protein, but two or more identical 64-81 L2 epitopes are inserted into different loop sites of one L1 protein Alternatively, at least two different types of 64-81 L2 epitopes can be inserted into different loop sites of one L1 protein.

  Furthermore, the capsid of the present invention can further comprise an L2 epitope other than the 64-81 L2 epitope. For example, the amino acid sequence represented by VEETSFIDA (SEQ ID NO: 14), VEESGIVDV (SEQ ID NO: 15), IEETTFIES (SEQ ID NO: 16), IEESSFIDA (SEQ ID NO: 17), IEDSSVVTS (SEQ ID NO: 18), or IEESAIINA (SEQ ID NO: 19) In addition to the 64-81 L2 epitope, at least one L2 epitope (109-117 L2 epitope) of the L2 epitope group consisting of the amino acid group having a capsid is also a capsid of the present invention. VEETSFIDA (SEQ ID NO: 14) is a type 16 L2 epitope of the HPV group and is particularly preferred. Also, 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 L2 IEDSSVVTS (SEQ ID NO: 18) is the HPV group 18 type L2 epitope and IEESAIINA (SEQ ID NO: 19) is the 6 and 11 type L2 epitope.

  The 109-117 L2 epitope can be inserted into the loop site of the L1 protein in the same manner as the 64-81 L2 epitope. Specifically, the inserted loop site is between amino acids 56 and 57, amino acids 140 and 141. Between amino acids 266 and 267 and at least one between amino acids 430 and 433 and where the 64-81 L2 epitope is not inserted.

  109-117 L2 epitopes inserted into L1 protein can be one in one L1 protein, but two or more identical 109-117 L2 epitopes are inserted into different loop sites of one L1 protein Alternatively, at least two different types of 109-117 L2 epitopes can be inserted into different loop sites of one L1 protein. In either case, one or more 64-81 L2 epitopes are inserted into the L1 protein. FIG. 9 shows examples of combinations of 64-81 L2 epitopes and 109-117 L2 epitopes inserted into the L1 protein and insertion positions. However, these are merely examples and are not intended to limit the combinations thereof.

(Preparation of peptide containing L2 epitope)
The L2 peptide (amino acids 64-81 or 109-117) was prepared by the Fmoc solid phase method using a 96-column automatic peptide synthesizer by a conventional method. Since this peptide is intended for antibody production, it is KLH (keyhole limpet hemocyanin) conjugated, and a cysteine residue is added to the N-terminus in order to prevent masking of the peptide region by this KLH.

  The present invention is a complex of keyhole limpet hemocyanin and human papillomavirus 64-81 L2 epitope, the L2 epitope 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), SGSGGRTGYVPLGGRSNT (SEQ ID NO: 8), SGSGGRTGYIPLGGGGRP (SEQ ID NO: 9), TGSGGRAGYVPLSTRPPT (SG No. 10), TGSGV (SEQ ID NO: 12) or a complex having an amino acid sequence represented by SGTGGRTGYIPLGGKPNT (SEQ ID NO :). Keyhole limpet hemocyanin and human papillomavirus L2 epitope can be linked via cysteine. SGTGGRTGYIPLGTRPPT (SEQ ID NO: 2) is shown in FIG.

(Preparation of L1 protein inserted with L2 epitope (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.

(A) Create a primer pair with the part you want to insert.
(B) PCR is performed by combining one of the primers having an insertion part with primer 1 or primer 2.
(C) Two DNAs with inserts on one side were synthesized. Each complementary portion is annealed and an extension reaction is performed.
(D) A chimeric gene having an inserted DNA is prepared.
(E) PCR is performed using primer 1 and primer 2 using this DNA as a template.
(F) It can amplify DNA with inserted gene.

  The present invention includes a method for producing a capsid, which comprises forming a capsid by assembling the chimeric proteins of the present invention. As described above, a chimeric L1 gene is prepared and expressed using a baculovirus vector to prepare a chimeric L1 protein. When the chimeric L1 protein is produced, a chimeric capsid (the 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.

  Hereinafter, the present invention will be described in more detail with reference to examples.

Example 1
Preparation of rabbit peptide antiserum Two Japanese white rabbits (2.5 kg to 3.0 kg) were immunized. As the antigen, a chemically synthesized peptide and KLH (keyhole limpet hemocyanin) were combined. For the first sensitization, 0.5 mg of antigen was administered subcutaneously with complete Freund's adjuvant. Two weeks after the first administration, 0.25 mg of antigen was subcutaneously administered together with complete Freund's adjuvant. Further, after 2 weeks, 4 weeks, and 6 weeks, the same sensitization (0.25 mg) was carried out for 5 times in total. One week after the final sensitization, whole blood was collected to obtain serum. (Consigned to Scrum Co., Ltd.)

Peptide ELISA measurement method Synthetic peptides were used as antigens. Antigen on ELISA plate 0.5 μg / 100 μl / well
In addition, the mixture was allowed to stand overnight at 4 ° C. to bind the antigen to the well.
2. The antigen solution was removed, washed twice with a washing solution (0.2% Teen20 in PBS), filled with the washing solution, and allowed to stand at room temperature for 2 hours.
3. After removing the washing solution from the well, 100 μl of antiserum diluted serially from 1000-fold with a diluent (0.05% Twenn20 in PBS) was added to each well, and left at room temperature for 2 hours.
4). The serum sample is removed, washed three times, and 100 μl of HRP (horse radish peroxydase) -conjugated anti-rabbit IgG antibody diluted 10,000 times is added to each well. Allow to stand at room temperature for 2 hours. After removing the solution, wash with washing solution 3 times.
5). Add 100 μl of the substrate solution (10 mg of 0-phenylenediamine dissolved in 25 ml of citrate monophosphate buffer (pH 5.0) and add 5 μl of H2O2), stop coloring with 1M H2SO4 after 20 minutes of color development, A wavelength of 490 nm was measured. The results are shown in Figure 3.

Example 2
Method for preparing capsid antigen
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 sf9 cells (night stealing-derived cells). The 16L1 (as well as 52L1 and 58L1) gene was cloned into the pFastbac1 vector to obtain pFsatbac1 / 16L1. 16L1 / L2 (52L1 / L2, 58L1 / L2) was cloned into pFastbac dual vector to obtain pFastbac dual / 16L1 / L2. Next, each cloned pFastbac vector was introduced into DH10BAC E. coli (Max Efficiency competent cell containing baculovirus DNA and helper plasmid, GIBCO BRL) 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.

  Recombinant baculovirus was infected with sf-9, and cells were collected 72 hours later. Infected cells were suspended in a 0.5% NP40 solution, allowed to stand at room temperature for 10 minutes, and then centrifuged (9000 rpm, 15 minutes, 4 ° C.) to separate into a nuclear fraction (precipitation) and a cytoplasmic fraction. The nuclear fraction was suspended in a 1.28 g / ml cesium chloride-PBS solution, sonicated (Sonifier250, Branson), and then ultracentrifuged (34000 rpm, 20 hours) with SW50.1 roter (Beckman Coulter Inc., Fulleron, CA). , 20 ° C.). A protein collected at a specific gravity of about 1.28 g / ml in a cesium chloride gradient was collected and dialyzed against 0.5 M NaCl-PBS to obtain a capsid protein solution.

Capsid ELISA assay 1. Add 1 μg / 100 μl / well of each capsid antigen and leave at 4 ° C. overnight.
2. After removal of the antigen solution, 350 μl of blocking solution (5% skim milk in PBS) was added and allowed to stand at 37 ° C. for 2 hours.
3. After removing the blocking solution, the plate was washed 3 times with a washing solution (0.05% Teen20 / 0.05% NP40 in PBS).
4). 50 μl of antiserum diluted 500-fold with blocking solution was added to each well, and allowed to stand at room temperature for 1 hour.
5). After removing the serum sample, the plate is washed 9 times, and HRP-conjugated anti-rabbit IgG antibody diluted 2000 times is added to each well at 50 μl. Allow to stand at room temperature for 30 minutes. After removing the solution, wash with washing solution 6 times.
6). Substrate solution (24 mg of 0-phenylenediamine dissolved in 12 ml of citrate monophosphate buffer (pH 5.0) and 1.2 μl of H2O2 added) was added 50 μl, and after 15 minutes of color development, the wavelength of 450 nm was measured with an immuno reader. . The results are shown in FIG.

Example 3
Preparation of infectious pseudovirus 1. HPV16L1 protein expression plasmid, HPV16L2 protein expression plasmid and secreted alkaline phosphatase expression plasmid were transfected into 293TT cells using Lipofectamine2000. (In the case of preparation of type 18 infectious pseudovirus, 18L1 plasmid and 18L2 plasmid were used, respectively). Cells were collected 72 hours after transfection.
2. The collected cells were suspended in Detergent buffer (0.5% Briji58, 0.5% Benzonase, 1% ATP dependent plasmid safe exonuclease C in D-PBS (CaCl ₂ 1 mM, MgCl ₂ 10 mM)) and allowed to stand overnight at 37 ° C. .
3. Then, after leaving still at 4 degreeC for 10 minutes, 5M NaCl was added so that it might become final 850 mM NaCl.
4. Centrifugation was performed at 1500 g for 10 minutes, and the supernatant was collected.
5). The collected supernatant was layered on a 27%, 33%, 39% optiprep (AXIS-SHIELD PoCAS) solution (diluted with PBS), and ultracentrifuged at 43700 rpm for 3 hours at 16 ° C.
6). After ultracentrifugation, fractions of about 300 μl were collected from the bottom, and fraction 3 or 4 was used as an infectious pseudovirus fraction for infection experiments.

Neutralization experiment On the day before the neutralization experiment, 20000 cells / well of 293TT cells are planted in a 96-well cell culture plate.
2. Serum is diluted with Neautralization Buffer (phenolic red-free DMEM medium with 10% FCS, 1% non-essential amino acid, 1% L-glutamic acid, 10 mM HEPES) and mixed with infectious pseudovirus for 1 hour at 4 ° C And added to the 293TT cells planted the day before.
3. After culturing for about 72 hours, 15 μl of the supernatant was collected, and alkaline phosphatase activity was measured with a Great ESCAPE SEAP Chemiluminescence Kit from BD clontech using a Luminometer. The results are shown in FIGS.

Example 4
In Example 3, the same operation as in Example 3 was performed except that instead of the HPV16L2 protein expression plasmid, an L2 mutant type HPV16L2 protein expression plasmid into which mutations were introduced in 64-81 shown in FIG. 6 was used. The results showing the relationship between the difference in the L2 mutant and the infectivity are shown in FIG.

Documents cited in the specification other than Non-Patent Documents 1 to 5 are as follows.
Cited Reference 1) Matsumoto. K, et al.: Antibodies to human papillomavirus 16, 18, 58, and 6b major capsid proteins among Japanese females. Jpn. J. Cancer Res., 88, 369-375, 1997.
2) Xiaojiang S. Chen, et al .: Structure of small virus-like particles assembled from the L1 protein of human papillomavirus 16. Mol. Cell., 5, 557-567, 2000.
3) Jean-Remy Sadeyen, et al .: Insertion of a foreign sequence on capsid surface loops of human papillomavirus type 16 virus-like particles reduce their capacity to induce neutralizing antibodies and delineates a conformational neutralizing epitope.Virology., 309, 32- 40, 2003.
4) Ishii. Y, et al,: Mutational analysis of human papillomavirus type 16 major capsid protein L1: the cysteines affecting the intermolecular bonding and structure of L1-capsids. Virology., 308, 128-136, 2003

Particles from chimeric proteins in which the amino acid 108-120 region of HPV16 type L2 protein is inserted into HPV16L1 protein have strong immunogenicity and the ability to induce neutralizing antibodies common to high-risk HPV groups (patent pending). By adding a 64-81 region to this chimeric protein, a vaccine antigen capable of inducing a stronger type-common neutralizing antibody can be produced.
If this vaccine can prevent high-risk HPV infection, it will help prevent cervical cancer.

Schematic diagram of a chimera capsid for which a patent application was previously filed Region of the peptide antibody produced. The peptide was combined with KLH to obtain an immunizing antigen. Reactivity of antisera and peptides obtained by immunizing 2 rabbits with each peptide (results of 1 animal were shown). ELISA antigens are peptides only. Serum contained specific antibodies. Reactivity of each peptide antibody to L1- or L1 / L2-capsid. ELISA antigens include HPV16 type L1 / L2-capsid (16L1 / L2), L1-capsid (16L1), etc. It was examined whether the antibody was bound to the region of the L2 protein particle surface. Binding by crossing with type 52 and type 58 L2 proteins was also examined. Neutralizing activity of each peptide antibody against HPV16. 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. Comparison of amino acid sequences in the 64-81 region. Representative anti-risk HPVL2 protein is shown. Neutralization of HPV18 infection with anti-64-81 antibody. The two doses of pseudovirus used were efficiently neutralized. Infectivity of HPV16 pseudovirus having a mutant with amino acid substitution in the 64-81 region. Examples of combinations and insertion positions of 64-81 L2 epitope and 109-117 L2 epitope inserted into L1 protein are shown.

Claims (12)

Capsid is a collection of chimeric proteins in which at least one L2 epitope of the human papillomavirus L2 epitope group (hereinafter referred to as 64-81 L2 epitope) is inserted into the loop site of human papillomavirus (HPV) type 16 L1 protein. And
The L2 epitope group, SGTGGRTGYIPLGTRPPT, SGTGGRTGYIPLGGRSNT, SGTGGRTGYVPLSTRPST, SGSGGRTGYVPIGTDPPT, SGTGGRSGYVPLGTTPPT, TGTGGRTGYIPLGGRPNT, SGSGGRTGYVPLGGRSNT, SGSGGRTGYIPLGGGGRP, AGSGGRAGYVPLSTRPPT, TGSGGRAGYVPLGSRPST, SGTGGRTGYVPLGSTPPS or capsid consisting of a group of amino acids having the amino acid sequence represented by SGTGGRTGYIPLGGKPNT,. Loop site for inserting the 64-81 L2 epitope between amino acids 56 and 57, between amino acids 140 and 141, between amino acids 266 and 267, and claim 1 is at least one site between amino acids 430 and 433 Capsid as described in. Request for further insertion of at least one L2 epitope (hereinafter referred to as 109-117 L2 epitope) of L2 epitope group consisting of amino acid group having amino acid sequence represented by VEETSFIDA, VEESGIVDV, IEETTFIES, IEESSFIDA, IEDSSVVTS, or IEESAIINA Item 3. The capsid according to any one of Items 1 and 2 . The 109-117 L2 epitope insertion loop site is at least one 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, and The capsid according to claim 3 , wherein the capsid is not inserted with the 64-81 L2 epitope. The capsid according to any one of claims 1 to 4, which is used for producing an L1-capsid vaccine. A chimeric protein in which at least one L2 epitope of the human papillomavirus L2 epitope group (hereinafter referred to as 64-81 L2 epitope) is inserted into the loop site of human papillomavirus (HPV) type 16 L1 protein,
The L2 epitope group, SGTGGRTGYIPLGTRPPT, SGTGGRTGYIPLGGRSNT, SGTGGRTGYVPLSTRPST, SGSGGRTGYVPIGTDPPT, SGTGGRSGYVPLGTTPPT, TGTGGRTGYIPLGGRPNT, SGSGGRTGYVPLGGRSNT, SGSGGRTGYIPLGGGGRP, AGSGGRAGYVPLSTRPPT, TGSGGRAGYVPLGSRPST, SGTGGRTGYVPLGSTPPS, or chimeric protein comprising the amino acid group having an amino acid sequence represented by SGTGGRTGYIPLGGKPNT. Loop site for inserting the 64-81L2 epitope between amino acids 56 and 57, between amino acids 140 and 141, between amino acids 266 and 267, and to claim 6 which is at least one site between amino acids 430 and 433 The chimeric protein described. Request for further insertion of at least one L2 epitope (hereinafter referred to as 109-117 L2 epitope) of L2 epitope group consisting of amino acid group having amino acid sequence represented by VEETSFIDA, VEESGIVDV, IEETTFIES, IEESSFIDA, IEDSSVVTS, or IEESAIINA Item 8. The chimeric protein according to any one of Items 6 to 7 . The 109-117 L2 epitope insertion loop site is at least one 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, and The chimeric protein according to claim 8 , wherein the chimeric protein is a portion into which the 64-81 L2 epitope is not inserted. The method for producing a capsid according to any one of claims 1 to 4 , further comprising forming a capsid by assembling the chimeric protein according to any one of claims 6 to 9. It is a complex of keyhole limpet hemocyanin and human papillomavirus L2 epitope (hereinafter referred to as 64-81 L2 epitope). A complex having an amino acid sequence represented by AGSGGRAGYVPLSTRPPT, TGSGGRAGYVPLGSRPST, SGTGGRTGYVPLGSTPPS, or SGTGGRTGYIPLGGKPNT. The complex according to claim 11 , wherein the keyhole limpet hemocyanin and the 64-81 L2 epitope of human papillomavirus are bound via cysteine.