JP6756950B2 - Human herpesvirus 6B antigen composition - Google Patents

Description

The present invention relates to an antigen composition of human herpesvirus 6B. Furthermore, it relates to a vaccine containing an antigen composition of human herpesvirus 6B.

This application claims the priority of Japanese application Japanese Patent Application No. 2015-069965, which is incorporated herein by reference.

Herpesvirus is a general term for viruses belonging to the herpesvirus family. Human herpes virus 6 (HHV-6) or 7 (HHV-7) is a double-stranded DNA virus belonging to the herpesvirus family β herpesvirus subfamily, and is an exanthom subitum. Is the causative virus of. HHV-6 can be classified into HHV-6A and HHV-6B as two variants. In particular, HHV-6B is considered to be the cause of exanthema subitum in infants. Infant encephalitis caused by HHV-6B is 150-200 cases per year in Japan, and it is known that 50% of them have neurological sequelae, but no effective control method has been developed so far. In recent years, it has been clarified that a specific amino acid sequence in gQ1 known as a kind of surface protein of HHV-6B induces a neutralizing antibody specific to HHV-6B, and HHV-6B using an antigen containing the sequence The development of vaccines has been expected. In particular, the dimer complex (gQ1 / gQ2), which is a complex of surface proteins gQ1 and gQ2, is known as a ligand site required for cell invasion of HHV-6B, and can be an effective candidate for HHV-6B vaccine antigen. It has been considered.

Although the primary target of HHV-6 is lymphocytes of the T cell lineage, HHV-6B is a latent infection in most adults and is the causative virus of exanthema subitum in infancy. The pathogenicity of HHV-6A has not yet been reported. In cells infected with HHV-6, gQ1 binds to gQ2 and gH / gL complexes to form gH / gL / gQ1 / gQ2 (hereinafter referred to as "quaternary complex"). This quaternary complex is found in the viral envelope. It is said that the tetrameric complex of HHV-6A binds to human CD46, and that of HHV-6B binds to human CD134 (see Non-Patent Documents 1 to 3; FIG. 8 of Non-Patent Document 2). It is also known that the ligand structure of HHV-6B, which functions functionally in binding to CD134, is a dimeric complex (gQ1 / gQ2), and the tetrameric complex structure is not an essential structure for binding to a receptor. (Non-Patent Document 4). Non-Patent Document 1 discloses that an analysis on intracellular processing of gQ1 and gQ2 was performed. Non-Patent Document 2 discloses the analysis results of the U100 gene product and the complex formation with gH and gL.

It is known that a neutralizing antibody for gQ1 was produced in HHV-6A (Patent Document 1). On the other hand, in HHV-6B, an antibody or antigen-binding fragment having a specific amino acid sequence of gQ1 as an epitope, an antigen containing a specific amino acid sequence of gQ1 or an antigen composition (gQ1 / gQ2), or an antigen composition thereof. There is disclosure about vaccines containing substances (Patent Document 1). However, it is difficult to obtain a stable peptide expression level for an antigen containing a specific amino acid sequence of gQ1. For example, the dimer complex (gQ1 / gQ2) does not yield a sufficient yield as an antigen due to the low viability of peptide-expressing cells, so that a stable and large-scale supply of vaccines is required. There is a need for an antigen suitable for this purpose.

Journal of Virology, 2004, Vol. 78 (15) pp. 7969-7983 Journal of Virology, 2003, Vol. 77 (4) pp. 2452-2458 Proc Natl Acad Sci U S A. 2013 May 28; 110 (22): 9096-9099 Journal of Virology, 2014, Vol. 88 (18) pp. 10875-10882 Cellular Microbiology, 2009, 11 (7), 1001-1006 Journal of Virology, 1993, Vol. 67 (8) pp. 4611-4620 Journal of Virology, 2004, Vol. 78 (9) pp. 4609-4616

WO2012 / 060025 (Application No. 2012-541704)

An object of the present invention is to provide an antigen composition for HHV-6B that can be stably and mass-supplied and a method for producing the same. Another object of the present invention is to provide a vaccine containing the above antigen composition.

As a result of intensive studies, the present inventors have been able to solve the above-mentioned problems by using a tetrameric complex containing gH, gL, gQ1 and gQ2 among the surface proteins of HHV-6B as an antigen composition. The present invention was completed.

The present invention comprises the following.
(1) An antigen composition of HHV-6B, which comprises a tetrameric complex containing gH, gL, gQ1 and gQ2 among the surface proteins of HHV-6B.
(2) An antigen composition consisting of a tetrameric complex, each of which contains a polypeptide consisting of any of the amino acid sequences specified in the "Form for carrying out the invention" column, in which gH, gL, gQ1 and gQ2 are each. ..
(3) An HHV-6B vaccine containing the antigen composition.
(4) A pharmaceutical composition containing the antigen composition as an active ingredient.
(5) A method for producing the antigen composition.

Among the surface proteins of HHV-6B, a tetrameric complex containing gH, gL, gQ1 and gQ2 can provide an antigen composition capable of stable and large-scale supply. As a result, it was possible to obtain an antigen composition having a more excellent neutralizing antibody-inducing ability as compared with the conventionally known antigen composition for a bimolecular complex (gQ1 / gQ2).

It is a conceptual diagram which shows the structure of HHV-6B. In particular, it is a conceptual diagram showing the structure of the HHV-6B antigen tetrameric complex. (Example 1-1) It is a figure which shows one example of embodiment of the HHV-6B antigen tetramer complex, and a dimer as a comparative example. (Examples 1-1 and 1-6) It is a figure which shows that HHV-6B antigen quaternary complex and gQ1 / gQ2 bind to CD134. (Example 1-1) It is a conceptual diagram which shows the production method and purification method of the HHV-6B antigen tetrameric complex. (Example 1-2) It is a figure which shows the result of having subjected | subjected to the Western blotting of the protein eluted by Ni-affinity chromatography in the manufacturing process of the HHV-6B antigen quaternary complex, and detected using the antibody against each of gH, gL, gQ1 and gQ2. (Example 1-2) It is a figure which shows the result of having performed the protein secreted in the culture supernatant of the cell which established the HHV-6B antigen quaternary complex to Western blotting, and detected using the antibody against each of gH, gL, gQ1 and gQ2. (Example 1-2) After concentrating the culture supernatant of the cells in which the HHV-6B antigen quaternary complex was established, the protease for GST fusion protein cleavage was reacted to cleave the HHV-6B antigen quaternary complex and hIgG1Fc His, and put them on a gel filtration column. It is a figure which shows the elution pattern at the time of passing. (Example 1-2) It is a figure which shows the administration schedule of the HHV-6B antigen quaternary complex to a mouse as an immune test of the HHV-6B antigen quaternary complex. (Example 1-3) It is a figure which shows the result of the cell-mediated immunity test as the immunity test of the HHV-6B antigen quaternary complex. (Example 1-5) It is a figure which shows the transition of the total viable cell number about the quaternary complex expression cell. (Example 1-6) It is a figure which shows the transition 1 of the viable cell number of the floating cell or the adherent cell about the quaternary complex expression cell. (Example 1-6) It is a figure which shows the transition 2 of the number of viable cells by floating cell or adherent cell about the quaternary complex expression cell. (Example 1-6) It is a figure which shows the administration schedule of HHV-6B antigen to a mouse as an immune test of various HHV-6B antigens of a monomer or a dimer. (Comparative Example 1-2) It is a figure which shows the ELISA antibody titer measurement result as the immunity test of various HHV-6B antigens of a monomer or a dimer. (Comparative Example 1-2) It is a figure which shows the administration schedule to a mouse as an immune test for confirming the combined effect of HHV-6B antigen quaternary complex and DPT-IPV. (Example 2) It is a figure which shows the ELISA antibody titer measurement result as an immunoassay for confirmation of the combined effect of HHV-6B antigen tetrad complex and DPT-IPV. (Example 2) It is a figure which shows the cell-mediated immunity measurement result as an immunity test for confirming the combined effect of HHV-6B antigen quaternary complex and DPT-IPV. (Example 2)

The present invention relates to an antigen composition of HHV-6B. Furthermore, it relates to a vaccine containing an antigen composition of HHV-6B.

The following is a list of definitions of terms specifically used herein. As used herein, the term "antigen composition" refers to a composition for inducing an immune response against a selected antigen in a vertebrate such as a human. The antigen composition of HHV-6B is an immunogen composition for HHV-6B. The antigen composition is described in detail below, but in the present invention it encodes a natural product, synthetic product or genetically engineered protein, peptide, or such protein purified from HHV-6B or HHV-6B. It may be a nucleic acid molecule containing a nucleic acid sequence. As used herein, the term "vaccine" refers to a factor containing the above-mentioned "antigen composition" and capable of producing an antibody, a factor capable of enhancing cell-mediated immunity, or producing such a factor when administered in vivo. A substance that can be used.

As used herein, the term "HHV-6B" includes both wild-type and mutant forms of human herpesvirus 6B. As used herein, the "wild strain type" of HHV-6B refers to the type of herpesvirus strain (HHV-6B) isolated from nature that has not undergone artificial modification. Examples of wild strains of HHV-6B include, but are not limited to, the HST strain. As used herein, the "mutant type" of HHV-6B refers to a type of HHV-6B that has been mutated into a wild type and mutated by multiple subcultures. When mutagenizing, the mutagenesis may be random mutagenesis or site-specific mutagenesis. The mutant type also includes those obtained by a method such as gene recombination.

The antigen composition of HHV-6B of the present invention refers to an antigen composition composed of an HHV-6B antigen quaternary complex containing gH, gL, gQ1 and gQ2 among the surface proteins of HHV-6B (FIGS. 1 and 2). reference). gH, gL, gQ1 and gQ2 are one molecule that forms the HHV-6B antigen tetrameric complex, and the genes for forming each protein can be found in GenBank: NC_000898.

In the present specification, the gene encoding gH among the surface proteins of HHV-6B can be specified by, for example, the nucleotide sequence shown in SEQ ID NO: 1, and gH can be specified by the amino acid sequence shown in SEQ ID NO: 2. .. Similarly, the gene encoding gL can be specified by, for example, the nucleotide sequence shown in SEQ ID NO: 3, gL can be specified by the amino acid sequence shown in SEQ ID NO: 4, and the gene encoding gQ1 can be specified, for example, by the sequence. It can be specified by the base sequence shown in No. 5, gQ1 can be specified by the amino acid sequence shown in SEQ ID NO: 6, and the gene encoding gQ2 can be specified by, for example, the base sequence shown in SEQ ID NO: 7. , GQ2 can be specified by the amino acid sequence shown in SEQ ID NO: 8. The gH, gL, gQ1 and gQ2 used in the antigen composition of the present invention are not limited to the substances specified by the above sequences. For example, each surface protein may contain a portion that can function as an antigen, and the HHV-6B antigen quaternary complex consisting of gH, gL, gQ1 and gQ2 may form a three-dimensional structure. Hereinafter, gH, gL, gQ1 and gQ2 used in the antigen composition composed of the HHV-6B antigen quaternary complex of the present invention will be described in detail.

(1) About gH
gH is one of the proteins that form the HHV-6B antigen tetrameric complex of the present invention. The gH that can be used as one of the antigen compositions is the polypeptide (10-a) specified by the amino acid sequence shown in SEQ ID NO: 10 or the polypeptide shown in any of (10-b) to (10-f). Can be included. For example, it may be a polypeptide specified by the amino acid sequence shown in SEQ ID NO: 2. The polypeptide shown in any of the following (10-a) to (10-f) may further contain the polypeptide specified by SEQ ID NO: 11 (see FIG. 2). For example, the polypeptide in which the polypeptide specified in SEQ ID NO: 11 is bound to the polypeptide specified in SEQ ID NO: 10 is the polypeptide specified in SEQ ID NO: 9. The amino acid sequence shown in SEQ ID NO: 10 is a partial sequence of the amino acid sequence shown in SEQ ID NO: 2, which is an amino acid sequence that does not contain a signal sequence and contains a portion having an antigenic function as gH. In the present specification, the polypeptide identified by SEQ ID NO: 9 is also included in the gH of the present invention.

(10-a) Polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10;
(10-b) One or more amino acids in the amino acid sequence shown in SEQ ID NO: 10 consist of an amino acid sequence having at least one mutation selected from the group consisting of substitutions, additions and deletions, and have biological activity. Have a polypeptide;
(10-c) A polypeptide encoded by a splice or allelic variant of the nucleotide sequence encoding SEQ ID NO: 10;
(10-d) A polypeptide that is a species homologue of the amino acid sequence shown in SEQ ID NO: 10.
(10-e) A polypeptide having an amino acid sequence having at least 70% identity to any one of the polypeptides (10-a) to (10-d) and having biological activity; Alternatively, an amino acid sequence encoded by a polynucleotide encoding any one of (10-f) (10-a) to (10-d) and a polynucleotide that hybridizes under stringent hybridization conditions. It can be a polypeptide that has and has biological activity.

(2) About gL
gL is one of the proteins forming the HHV-6B antigen tetrameric complex of the present invention. The gL that can be used as one of the antigen compositions is the polypeptide (12-a) specified by the amino acid sequence shown in SEQ ID NO: 12 or the polypeptide shown in any of (12-b) to (12-f). Can be included. For example, it may be a polypeptide specified by the amino acid sequence shown in SEQ ID NO: 4. Here, the amino acid sequence shown in SEQ ID NO: 12 is a partial sequence of the amino acid sequence shown in SEQ ID NO: 4, and is an amino acid sequence that does not include a signal sequence but includes a portion having an antigenic function as gL.

(12-a) Polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 12;
(12-b) One or more amino acids in the amino acid sequence shown in SEQ ID NO: 12 consist of an amino acid sequence having at least one mutation selected from the group consisting of substitutions, additions and deletions, and have biological activity. Have a polypeptide;
(12-c) A polypeptide encoded by a splice or allelic variant of the nucleotide sequence encoding SEQ ID NO: 12;
(12-d) A polypeptide that is a species homologue of the amino acid sequence shown in SEQ ID NO: 12;
(12-e) A polypeptide having an amino acid sequence having at least 70% identity to any one of the polypeptides (12-a) to (12-d) and having biological activity; Alternatively, an amino acid sequence encoded by a polynucleotide encoding any one of the polypeptides (12-f) (12-a) to (12-d) and a polynucleotide that hybridizes under stringent hybridization conditions. It can be a polypeptide that has and has biological activity.

(3) About gQ1 “gQ” used in the present specification is a protein, and in HHV-6B, the gQ gene encodes a 37 kDa protein and is derived from an alternative splicing transcript. gQ1 is one of the proteins forming the HHV-6B antigen quaternary complex of the present invention. The gQ1 that can be used as one of the antigen compositions is the polypeptide (13-a) specified by the amino acid sequence shown in SEQ ID NO: 13 or the polypeptide shown in any of (13-b) to (13-f). Can be included. For example, it may be a polypeptide specified by the amino acid sequence shown in SEQ ID NO: 6. Here, the amino acid sequence shown in SEQ ID NO: 13 is a partial sequence of the amino acid sequence shown in SEQ ID NO: 6, and is an amino acid sequence that does not include a signal sequence but includes a portion having an antigenic function as gQ1.

(13-a) Polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 13;
(13-b) One or more amino acids in the amino acid sequence shown in SEQ ID NO: 13 consist of an amino acid sequence having at least one mutation selected from the group consisting of substitutions, additions and deletions, and have biological activity. Have a polypeptide;
(13-c) A polypeptide encoded by a splice or allelic variant of the nucleotide sequence encoding SEQ ID NO: 13;
(13-d) A polypeptide that is a species homologue of the amino acid sequence shown in SEQ ID NO: 13.
(13-e) A polypeptide having an amino acid sequence having at least 70% identity to any one of the polypeptides (13-a) to (13-d) and having biological activity; Alternatively, an amino acid sequence encoded by a polynucleotide encoding any one of the polypeptides (13-f) (13-a) to (13-d) and a polynucleotide that hybridizes under stringent hybridization conditions. It can be a polypeptide that has and has biological activity.

(4) About gQ2
gQ2 is one of the proteins forming the HHV-6B antigen tetrameric complex of the present invention. The gQ2 that can be used as one of the antigen compositions is the polypeptide (14-a) specified by the amino acid sequence shown in SEQ ID NO: 14 or the polypeptide shown in any of (14-b) to (14-f). Can be included. For example, it may be a polypeptide specified by the amino acid sequence shown in SEQ ID NO: 8. Here, the amino acid sequence shown in SEQ ID NO: 14 is a partial sequence of the amino acid sequence shown in SEQ ID NO: 8, and is an amino acid sequence that does not include a signal sequence but contains a portion having an antigenic function as gQ2.

(14-a) Polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 14;
(14-b) One or more amino acids in the amino acid sequence shown in SEQ ID NO: 14 consist of an amino acid sequence having at least one mutation selected from the group consisting of substitutions, additions and deletions, and have biological activity. Have a polypeptide;
(14-c) A polypeptide encoded by a splice or allelic variant of the nucleotide sequence encoding SEQ ID NO: 14;
(14-d) A polypeptide that is a species homologue of the amino acid sequence shown in SEQ ID NO: 14.
(14-e) A polypeptide having an amino acid sequence having at least 70% identity to any one of the polypeptides (14-a) to (14-d) and having biological activity; Alternatively, an amino acid sequence encoded by a polynucleotide encoding any one of the polypeptides (14-f) (14-a) to (14-d) and a polynucleotide that hybridizes under stringent hybridization conditions. It can be a polypeptide that has and has biological activity.

As used herein, the term "vaccine" refers to a factor containing the antigen composition and capable of producing an antibody, a factor capable of enhancing cell-mediated immunity, or a substance capable of producing such a factor when administered in vivo. Say. The present invention also extends to pharmaceutical compositions containing the antigen composition of the present invention as an active ingredient. In addition to the above active ingredients, the pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier.

The immunological effect of the antigen composition as an active ingredient in the present invention can be confirmed by using any method known in the art. Examples of such a method include, but are not limited to, the ELISA method, the neutralization method, the HI method, the RCF method, the INF-γ Elispot method, and the attack test.

In the present specification, the "effective amount" of a pharmaceutical composition containing the antigen composition of the present invention as an active ingredient means an amount in which the active ingredient can exert a desired medicinal effect.

In the present specification, it is preferable to use the most effective route of administration of the pharmaceutical composition containing the antigen composition as an active ingredient in prevention or treatment, for example, intrarectal, intranasal, intradermal, subcutaneous, muscle. Parenteral administration such as intra- or intravenous administration and oral administration can be mentioned. Examples of the administration form include injections, emulsions, suppositories, patches, nasal drops and capsules, tablets, granules, powders, syrups and the like.

As used herein, the term "pharmaceutically acceptable carrier" refers to a substance used in the manufacture of a drug and which does not have a harmful effect on the active ingredient. Such pharmaceutically acceptable carriers include, for example, antioxidants, stabilizers, preservatives, antibiotics, colorants, inactivating agents, isotonic agents, pH regulators, diluents, emulsifiers, suspensions. Examples include, but are not limited to, turbinants, solvents, fillers, bulking agents, buffers, thickeners, delivery vehicles, excipients and / or pharmaceutical adjuvants.

The pharmaceutical composition containing the antigen composition of the present invention as an active ingredient can be used for the prevention and / or treatment of diseases caused by HHV-6B (for example, exanthema subitum).

The present invention also extends to the method for producing the antigen composition of the present invention. The method for producing the antigen composition as a peptide is not particularly limited, and the peptide can be produced in the same manner as a method known per se, for example, an organic synthesis method (solid phase synthesis method, liquid phase synthesis method, etc.). It can also be synthesized by a biochemical synthesis method, a gene recombination method, or the like. In the case of the gene recombination method, it can be produced by cell culture. The cells that can be used are not particularly limited as long as they can be applied as host cells to the gene recombination method. For example, animal cells, fungal cells, plant cells, insect cells, prokaryotic cells and the like may be used. The gene expression vector, host cell transformation method, cell culture method, purification method and the like are not particularly limited, and a method known per se or any method developed in the future can be applied.

In order to help understanding of the present invention, the present invention will be specifically described with reference to Examples below, but it goes without saying that the present invention is not limited to the present examples. The concept of a specific protein present in HHV-6B is shown in FIG. An example of the HHV-6B antigen quaternary complex prepared in this example for the example is shown in FIG. An example of a dimeric complex as a comparative example is also shown in FIG.

(Example 1-1) Preparation of HHV-6B antigen quaternary complex In this example, a method for producing the HHV-6B antigen quaternary complex will be described.

(1) The cDNAs of gQ1, gQ2, gH and gL of the HHV-6B HST strain with humanized codons were inserted into the pCAGGS-MCS vector to prepare each expression vector. In the gH cDNA, the signal sequence and transmembrane region were deleted, and the IL-2 signal sequence, human IgG1Fc, PreScission Protease cleavage site, and His tag were added. In this example, gH from which the signal sequence (ss) and transmembrane region (TM) have been deleted is identified by the amino acid sequence shown in SEQ ID NO: 10, and human IgG1Fc, PreScission Protease cleavage site and His tag (hIgG1Fc His) are It is specified by SEQ ID NO: 11. The gH obtained by binding the polypeptide specified by SEQ ID NO: 11 to the polypeptide specified by SEQ ID NO: 10 is the polypeptide specified by SEQ ID NO: 9, and is referred to as "gH-hIgG1Fc" in the following examples for convenience. In some cases.

(2) The gQ2 and gL expression vectors prepared in (1) above are amplified by PCR using each primer consisting of the nucleotide sequences shown in SEQ ID NOs: 15 and 16 below, and the product is treated with the restriction enzyme HindIII. , GQ2 expression cassette or gL expression cassette was prepared.
Primer 1: aca aagctt ATTGATTATTGACTAG (SEQ ID NO: 15)
Primer 2: aca aagctt GGGCTGCAGGTC (SEQ ID NO: 16)

(3) The gQ1 and gH expression vectors prepared in (1) above were treated with the restriction enzymes HindIII, respectively. The gQ2 expression cassette prepared in (2) above was ligated with a gQ1 expression vector, and the gL expression cassette was ligated with a gH expression vector. Using the pPur vector as a template, each primer consisting of the nucleotide sequences shown in SEQ ID NOs: 17 and 18 below was amplified by the PCR method, and the product was treated with the restriction enzyme XhoI to prepare a puromycin expression cassette.
Primer 3: aca ctcgag TTGTGTCAGTTAGGGTG (SEQ ID NO: 17)
Primer 4: aca ctcgagag ACATGATAAGATAC (SEQ ID NO: 18)

(4) The gQ1 and gQ2 expression vectors prepared in (3) above were treated with the restriction enzyme SalI and ligated with a puromycin expression cassette.

(5) Using the pMC1neo vector as a template, amplification was performed by PCR using each primer consisting of the nucleotide sequences shown in SEQ ID NOs: 19 and 20 below, and the product was treated with the restriction enzyme XhoI to prepare a neomycin expression cassette.
Primer 5: acactcgagCAGTGTGGTTTTCAAG (SEQ ID NO: 19)
Primer 6: acactcgagGGATCCGAACAAACGAC (SEQ ID NO: 20)

(6) The gH and gL expression vectors prepared in (3) above were treated with the restriction enzyme SalI and ligated with a neomycin expression cassette.

(7) The gQ1 and gQ2 vectors prepared in (4) above are mixed with the gH and gL expression vectors prepared in (6) above, and HEK293sGnTI-cells (ATCC: CRL-3022) are used using Lipofectamin2000 (Invitrogen). Introduced in. The introduced cells were cultured in a medium supplemented with 1 μg / μL of puromycin, and cells expressing all four genes were cloned by the limiting dilution method to establish four gene-expressing cells.

(8) The medium of the established 4 gene-expressing cells was changed to Ex-cell 293 medium (Nichirei; 24561C-10L) to which 1 μg / μL of puromycin was added, and Ex-cell 293 medium was collected every 2 days. I exchanged it.

(9) HHV-6B antigen quaternary complex in which hIgG1Fc was fused to gH bound to the surface of CD134-expressing cells by reacting the culture supernatant of 4 gene-expressing cells with CD134-expressing cells (hereinafter, "HHV-6B antigen quaternary amount"). The complex (Fc ⁺ ) ”was detected by Flow cytometry using an anti-Fc fusion protein antibody. The secreted HHV-6B antigen bound to CD134, the entry receptor for HHV-6B (Fig. 3).

(Example 1-2) Purification of HHV-6B antigen quaternary complex In this example, purification of HHV-6B antigen quaternary complex (Fc ⁺ ) will be described. A conceptual diagram of the manufacturing method is shown in FIG. To the culture supernatant of the 4 gene-expressing cells collected in Example 1-1, add 1/20 equivalent of a 200 mM Na ₂ PO ₄ solution and 1/500 equivalent of a 50% slurry of Ni-NTA agarose resin (Qiagen), and 4 The mixture was stirred at ° C for 14 hours.

Wash with 20 equivalents of wash buffer (20 mM sodium phosphate pH 7.4, 500 mM NaCl, 20 mM imidazole) relative to the volume of Ni-NTA agarose resin and 2 equivalents of elution buffer (20 mM Tris-HCl pH 8.0, The protein was eluted by Ni-affinity chromatography with 500 mM NaCl, 120 mM imidazole). The eluted protein was subjected to Western blotting and detected using antibodies against each of gH, gL, gQ1 and gQ2 (Fig. 5). The HHV-6B antigen quaternary complex (Fc ⁺ ) was secreted into the culture supernatant of the four gene-expressing cells established in Example 1 (7) (Fig. 6).

The culture supernatant from which the HHV-6B antigen quaternary complex (Fc ⁺ ) was secreted was subjected to an absorbance of 13,500 g up to A280 = 0.2-0.5 using a centrifugal filter Amicon Ultra-0.5 (Merck Millipore) with a molecular weight cut-off of 50,000. Ultrafiltration and concentration were performed at 4 ° C. Prepare 8 units of GST fusion protein cleavage protease (GE-Healthcare) per 1 mg, add dithioslateol at a final concentration of 5 mM, and let stand on ice for 30 minutes. It was added to a sample containing the HHV-6B antigen quaternary complex (Fc ⁺ ) and reacted at 4 ° C. for 48 hours.

Proteins were purified using a liquid chromatography system (AKTA pure 25) at 4 ° C. The gel filtration column HiLoad 16/600 Superdex 200 pg (GE-Healthcare) was equilibrated with buffer A (20 mM Tris-HCl pH 8.0, 100 mM NaCl), and 2 mL of each sample was added and filtered. The elution peak was analyzed by SDS-PAGE, and the elution peak corresponding to the HHV-6B antigen quaternary complex was recovered. The HHV-6B antigen quaternary complex was eluted as a single peak and could be fractionated (Fig. 7).

The cation exchange column MonoQ 5/50 GL (GE-Healthcare) was equilibrated with the above buffer A under the condition of 4 ° C. by the liquid chromatography system (AKTA pure 25), and the HHV-6B antigen quaternary complex (Fc ^+). ) Fractions were added. After washing with 5 mL of buffer A, elution from buffer A with a concentration gradient from buffer A to buffer B (20 mM Tris-HCl pH 8.0, 1 M NaCl) corresponds to HHV-6B antigen quaternary complex (Fc ⁺ ). The peak was collected.

The HHV-6B antigen quaternary complex (Fc ⁺ ) of the present invention was prepared by ultrafiltration concentration at 13,500 g at 4 ° C. using a centrifugal filter with a molecular weight cut-off of 50,000 (Amicon Ultra-0.5 (Merck Millipore)). (Fig. 6).

(Example 1-3) Immunity test of HHV-6B antigen quaternary complex (antibody production)
Mix 100 μg of the HHV-6B antigen quaternary complex (Fc ⁺ ) prepared in Example 1-2 and an equal amount of Titer Max Gold (Funakoshi, Titer Max Gold, Inc, Cat No. CYT: G-1x5) as a solution. Then, sonication (Sonication: output = 3) was performed 4 times for 10 seconds on ice. Using a 23 G needle and a 1 mL syringe, 100 μg was subcutaneously administered to the back of mice (BALB / c, female, 4 weeks old, 3 animals in each group, Control: PBS). The administration schedule was 3 times at 2-week intervals. Blood and spleen were collected 4 weeks after the third dose (Fig. 8).

100 μL of pentobarbital (somnopentyl: 50 mg / mL) was administered to the abdominal cavity of mice, and heart blood was collected under anesthesia (23 G needle, 1 mL syringe), and the spleen was collected. Approximately 1 mL of blood was placed in a 1.5 mL tube, incubated at 37 ° C for 1 hour, and centrifuged at 14000 rpm at 4 ° C for 30 minutes. The serum (upper layer) was transferred to another 1.5 mL tube, deactivated in a block incubator at 56 ° C. for 30 minutes, and then used for antibody titer measurement. After collection, the spleen was placed in a 50 mL tube containing 20 mL of RPMI medium and transported on ice. Mononucleosis was collected by density gradient centrifugation using Ficoll-conray solution and used for measurement of cell-mediated immunity.

The antibody titer against HHV-6B antigen was measured for the above serum by the indirect fluorescent antibody method (IFA). HHV-6B HST strain-infected MT4 cells (cells on day 4 of Cell to cell, confirmed late protein expression) or quaternary complex-expressing 293T cells were used for the antigen plate. An antigen plate using 293T cells expressing the tetrameric complex was prepared by the following method. 2 × 10 ⁶ cells / 12 mL DMEM: Seed 293T cells in a 10 cm dish with 8% FBS, and the next day, transfer pCAGGSMCS-gH 3 μg, pCAGGSMCS-gL 6 μg, pCAGGSMCS-gQ1 3 μg, and pCAGGSMCS-gQ2 6 μg by calcium phosphate method. After transfection, cells were collected on the 3rd day of culture to prepare an antigen plate (expression of each protein was confirmed using each antibody of anti-gH, gL, gQ1 and gQ2).

Mouse serum serially diluted (10 to 1280 times) with PBS / 2% BSA was placed on each antigen plate and reacted by incubation at 37 ° C. for 30 minutes. Each plate was washed with PBS / 0.02% Tween 20 and then dried. A secondary antibody (using 100-fold diluted FITC conjugate rabbit anti-mouse IgG) was placed, reacted by incubation at 37 ° C for 20 minutes, washed with PBS / 0.02% Tween20, and dried. It was encapsulated and fluorescence was observed under a fluorescence microscope. The maximum dilution ratio of mouse serum in which fluorescence can be observed was defined as the antibody titer. Antibody production against the HHV-6B antigen quaternary complex (Fc ⁺ ) was confirmed in mice to which the HHV-6B antigen quaternary complex (Fc ⁺ ) was administered.

(Example 1-4) Immunity test of HHV-6B antigen quaternary complex (neutralizing antibody titer)
Using the serum obtained in Example 1-3 as a sample, the neutralizing antibody titer of the HHV-6B antigen quaternary complex (Fc ⁺ ) produced in the serum was measured. The mouse serum obtained in Example 1-3 was serially diluted (40 to 1280 times) in RPMI medium containing 3% FBS. 100 μL of HHV-6B (HST strain) virus solution was added to 100 μL of diluted serum, and the mixture was reacted at 37 ° C. for 30 minutes (locking every 10 minutes). 3 × 10 ⁶ MT4 cells were suspended in 1 mL of RPMI medium containing FBS, and 10 μL (3 × 10 ⁴ ) of each mouse serum + virus solution was added. Infected by centrifugation at 490 x g for 40 minutes at 35 ° C. Cells were transferred to a 96-well plate at the bottom of the horn and pelleted down at 490 xg for 5 minutes at room temperature. The cell pellet was washed with 200 μL of RPMI medium containing 3% FBS. 200 μL of RPMI medium containing 3% FBS was added to the cell pellet, suspended, transferred to a 96-well plate (flat bottom), and cultured in a 37 ° C. 5% CO ₂ incubator for 4 days. After 4 days of culturing, the infected cells were transferred to a 96-well plate at the bottom of the horn and centrifuged at 490 × g ・ 5 min at room temperature, and the supernatant was aspirated leaving about 20 μL of the culture solution. All cells were collected, suspended in 30 μL of 5 × sample buffer (including 2ME), and HHV-6B protein IE1 was detected by Western blotting (anti-BIE 1 × 1000, anti-α-tubulin × 10000 as internal control). , Confirmed the presence or absence of infection. The maximum dilution ratio of mouse serum in which IE1 was not detected was defined as the neutralizing antibody titer. Neutralizing antibody production against HHV-6B was confirmed in mice treated with the HHV-6B antigen quaternary complex (Fc ⁺ ).

(Example 1-5) Immunity test of HHV-6B antigen quaternary complex (cell-mediated immunity)
Cell-mediated immunity was measured in mice treated with the HHV-6B antigen quaternary complex (Fc ⁺ ). The IFN-γ Elispot assay was performed using mouse spleen-derived mononucleosis collected by the same method as in Example 1-3. HHV-6B or varicella virus (VZV) was used as a stimulator, and the number of IFN-γ-producing cells was measured. IFN-γ was produced by HHV-6B stimulation in mice treated with the HHV-6B antigen quaternary complex (Fc ⁺ ) (Fig. 9).

(Example 1-6) Transition of cells into which HHV-6B antigen tetrameric complex has been introduced
When culturing cells into which HHV-6B antigen quaternary complex (Fc ⁺ ) has been introduced (quaternary complex-expressing cells) and cells into which gQ1-hIgG1FcQ1 / Q2 (see Fig. 2) have been introduced (gQ1 / Q2-expressing cells) The number of viable cells was compared. HEK293T cells were used as a control. The cells were seeded on a 6-well plate so that the cell concentrations were 1 × 10 ⁵ / well, 2 × 10 ⁵ / well, 3 × 10 ⁵ / well, and 5 × 10 ⁵ / well. After 3 days, the cells were replaced with serum-free Ex-cell 293 medium (Nichirei; 24561C-10L) containing 1 μg / μL puromycin, and culture was started. On the same schedule as when collecting the HHV-6B antigen quaternary complex (Fc ⁺ ) secreted in the culture supernatant, the culture supernatant was collected and the medium was exchanged, the culture was continued, and the suspended cells in the supernatant were collected. Was collected and the number of viable cells was counted over time. The number of viable cells was counted for floating cells and adherent cells in the supernatant, respectively.
As a result, the tetramer complex-expressing cells had a larger number of viable cells than the gQ1 / gQ2-expressing cells and were suitable for long-term culture (Figs. 10 to 12).

(Comparative Example 1-1) Preparation of various HHV-6B antigens In this comparative example, the HHV-6B antigen monomer (gH), for comparison with the HHV-6B antigen tetrameric complex prepared in Example 1, The immunoinducibility, etc. of each antigen of HHV-6B antigen dimer complex (gH / gL), HHV-6B antigen monomer (gQ1) and HHV-6B antigen dimer complex (gQ1 / gQ2) was confirmed. .. First, a production method for producing various HHV-6B antigens will be described.

The codon-humanized HHV-6B HST strains gH, gL, and gQ1 and the sbp (streptavidin binding peptide sequence) -tagged gQ2 sequence cDNA consisting of the streptavidin binding peptide sequence were prepared. Each cDNA containing gH or gQ1 was inserted into the pCAGGS-MCS vector to prepare each expression vector. The gL and gQ2 expression cassettes (including the promoter and polyA sequence) were amplified and inserted into plasmids incorporating gH and gQ1, respectively, using HindIII. As a result, an expression plasmid (gH-expression plasmid, gH / gL-expression plasmid, gQ1-expression plasmid, gQ1 / gQ2-expression plasmid) containing each of the four genes was prepared.

An expression plasmid containing each of the four genes was introduced into 293T cells using the calcium phosphate method and cultured. After 2 days of culture, cells were harvested and solubilized in TNE buffer (10 mM Tris-HCl [pH 7.8], 0.15 M NaCl, 1 mM EDTA, 1% NP40 [Nacalai Tesque]) containing protease inhibitors (Sigma). Streptavidin Sepharose (GE Healthcare) cross-linked with an antibody against gH or gQ1 was added to the solubilized cells, and the cells were reacted at 4 ° C. for 8 hours. Sepharose was washed with TNE buffer and HHV-6B antigens gH, gH / gL, gQ1 and gQ1 / gQ2 were eluted using 8 mM biotin solution, respectively. The eluate was replaced with PBS with Millipore ^(TM) Amicon ^(TM) 30 (Millipore), and each antigen was purified.

(Comparative Example 1-2) Confirmation of immunostimulatory ability In this Comparative Example, the monomer (gH), dimer complex (gH / gL), monomer (gQ1) and dimer prepared in Comparative Example 1-1. The immunoinducible ability was confirmed for each HHV-6B antigen of the amount complex (gQ1 / gQ2). Mix each antigen solution with an equal amount of Titer Max Gold (Funakoshi, Titer Max Gold, Inc, Cat No. CYT: G-1x5) and perform sonication (Sonication: output = 3) 4 times on ice for 10 seconds. , Each antigen sample. Using a 23 G needle and a 1 mL syringe, 50 μg of each antigen sample was subcutaneously administered to the back of mice (BALB / c, female, 4 weeks old, 3 animals in each group, Control: PBS). The administration schedule was 0, 4, and 8 weeks, for a total of 3 times. Blood was collected 4 weeks after the third administration (Fig. 13).

100 μL of pentobarbital (somnopentyl: 50 mg / mL) was administered to the abdominal cavity of mice, and heart blood was collected under anesthesia (23 G needle, 1 mL syringe). Approximately 1 mL of blood was placed in a 1.5 mL tube, incubated at 37 ° C for 1 hour, and centrifuged at 14000 rpm at 4 ° C for 30 minutes. The serum (upper layer) was transferred to another 1.5 mL tube, deactivated in a block incubator at 56 ° C. for 30 minutes, and used as a test sample for confirming immunoinducibility.

(1) Confirmation of immuno-inducing ability by ELISA The immuno-inducing ability of each of the above-mentioned test samples subjected to the immunoinducing treatment was confirmed by the ELISA method. The HHV-6B antigen quaternary complex used in the ELISA method was prepared by the following method. The codon-humanized HHV-6B HST strains gH, gL, and gQ1 and the sbp (streptavidin binding peptide sequence) -tagged gQ2 sequence cDNA consisting of the streptavidin binding peptide sequence were prepared. Each cDNA containing gH or gQ1 was inserted into the pCAGGS-MCS vector to prepare each expression vector. The gL and gQ2 expression cassettes (including the promoter and polyA sequence) were amplified and inserted into plasmids incorporating gH and gQ1, respectively, using HindIII. Two expression plasmids were introduced into 293T cells using the calcium phosphate method, and 2 days after culture, the cells were harvested and TNE buffer containing protease inhibitors (Sigma) (10 mM Tris-HCl [pH 7.8], 0.15 M NaCl, It was solubilized with 1 mM EDTA, 1% NP40 [Nacalai Tesque]). Streptavidin Sepharose (GE Healthcare) was added to the solubilized cells, and the cells were reacted at 4 ° C. for 8 hours to elute the HHV-6B antigen tetrameric complex with biotin. The eluate was replaced with PBS with Millipore ^(TM) Amicon ^(TM) 30 (Millipore) to purify the HHV-6B antigen quaternary complex. An ELISA measurement plate was prepared using the purified HHV-6B antigen quaternary complex prepared above. Antigen-antibody reaction was carried out on each diluted test sample from 25 times to 51,200 times according to a conventional method on an ELISA measurement plate, and the absorbance at a wavelength of 405 nm was measured to confirm the immunoinducibility (FIGS. 14 and Table). 3). As a result, among various antigens, it was confirmed that the immunoinducible ability was higher when treated with an antigen composed of a dimer complex than with a monomer.

(2) Confirmation of immunostimulatory ability by the neutralization method The immunoinducible ability of each of the test samples subjected to the above immunoinduction treatments was confirmed by the neutralization method. Approximately 1 mL of the collected blood is placed in a 1.5 mL tube, incubated at 37 ° C for 1 hour, centrifuged at 14000 rpm at 4 ° C for 30 minutes, and the unactivated blood is immunoinducible by neutralizing antibody titer measurement. It was used as a test sample for confirmation. The test sample diluted 10, 20, 40, 80, and 160 times in stages is mixed with a virus solution (HHV-6B, HST strain) and reacted at 37 ° C for 30 minutes, and each mixture of the test sample and virus Was infected with MT4 cells and then centrifuged at 37 ° C. for 1 hour. The cells were cultured in RPMI (3% FBS), and after 1 day, the cells were collected, and the neutralizing antibody titer was measured by the indirect fluorescent antibody method (IFA) using an antibody against IE1 (Table 4). As a result, the immunoinducible ability was hardly confirmed by the treatment with the antigen consisting of the monomer and the dimer complex, and the immunoinducible ability was higher in the HHV-6B antigen tetrameric complex.

(Example 2) Confirmation of combined effect of HHV-6B antigen quaternary complex and DPT-IPV In this example, the HHV-6B antigen quaternary complex of the present invention and a known four-kind combination vaccine, DPT- We confirmed the ability to induce immunity to HHV-6B when combined with IPV. Here, DPT-IPV is a preventive vaccine against diphtheria, pertussis, tetanus and polio, and its antibody-inducing ability against tetanus and pertussis has been confirmed. In this example, the HHV-6B antigen quaternary complex of the present invention was prepared by the same method as in Example 1 except that it did not contain the hIgG1Fc portion of gH. HHV-6B antigen tetrameric complexes HHV-6B antigen tetrameric complexes containing no hIgG1Fc of gH used in this example (Fc ^-) called.

In this embodiment, HHV-6B antigen tetrameric complexes (Fc ^-) and pentathlon combination vaccine containing DPT-IPV, DPT-IPV alone, HHV-6B antigen tetrameric complexes (Fc ^-) using a mouse ( BALB / c, female, 4 weeks old) were treated with immunoinduction (7 animals in each group). HHV-6B antigen tetrameric complexes (Fc ^-) is 100 [mu] g, DPT-IPV was administered 1/8 amount of human dosage. Each vaccine was subcutaneously administered 500 μL to the back of mice (BALB / c, female, 4 weeks old, 7 animals in each group, Control: PBS). Administration was performed 4 times in total at 0, 4, 10 and 16 weeks. Blood was collected by the same method as in Example 1-3 at 2, 6, 12, and 18 weeks after the start of administration, and whole blood was collected at 20 weeks (Fig. 15). The collected blood was treated in the same manner as in Comparative Example 1 to prepare a test sample for confirming immunoinducibility.

(1) Confirmation of immunoinducible ability by ELISA method Comparative Example 1 The antibody titer in serum was confirmed by the ELISA method according to the same method (Fig. 16, Table 5). As a result, HHV-6B antigen tetrameric complexes (Fc ^-) any time higher immunity-inducing ability in HHV-6B were obtained, HHV-6B antigen tetrameric complexes ^- in combination with the DPT-IPV (Fc) Higher immunoinducibility was confirmed with the five-kind vaccine. On the other hand, in the case of DPT-IPV alone, almost no immunity was induced as in the case of PBS. HHV-6B antigen tetrameric complexes (Fc ^-) and the combined use of DPT-IPV, a synergistic effect was confirmed.

(2) Confirmation of immunoinducible ability by neutralization method The neutralizing antibody titer was measured by the indirect fluorescent antibody method (IFA) according to the same method as in Comparative Example 1 (Table 6). As a result, HHV-6B antigen tetrameric complexes (Fc ^-) even higher immune inducibility is obtained for HHV-6B, HHV-6B antigen tetrameric complexes (Fc ^-) and DPT-IPV and combination with five kinds of It was confirmed that the combination vaccine had the same or higher immunoinducing ability. On the other hand, in the case of DPT-IPV alone, almost no immunity was induced as in the case of PBS.

(3) Confirmation of immune-inducing ability by cell-mediated immunity (Elispot method)
Using mouse spleen-derived mononucleosis collected by the same method as in Example 1-3, an assay was performed using an Elispot plate (millipore). The ELISpot assay is used to determine the effectiveness of a vaccine by measuring its ability to induce a T cell response. The Elispot plate (Millipore) was blocked with RPMI containing FBS, then 1 × 10 ⁶ or 0.5 × 10 ⁶ cells / well of mouse spleen cells were added, stimulated with 100 μL of PHA, and the HHV-6B antigen tetrameter was added. complex (Fc ^-) and the like, or PBS was added, and cultured at 40 hours 37 ° C., the spots of the formed IFN-gamma-producing cells was counted using a KS ELISPOT (Carl Zeiss) (Fig. 17). As a result, HHV-6B antigen tetrameric complexes (Fc ^-) even although cellular immunity-inducing ability was obtained for HHV-6B, pentathlon mixed vaccine in combination with HHV-6B antigen tetrameric complexes and DPT-IPV Was confirmed to have higher immunoinducibility. On the other hand, in the case of DPT-IPV alone, almost no immunity was induced as in the case of PBS.

As described in detail above, among the surface proteins of HHV-6B, the HHV-6B antigen quaternary complex containing gH, gL, gQ1 and gQ2 provides an antigen composition capable of stable and large-scale supply. Can be done. Since the antigen composition can be stably and mass-supplied, a vaccine or pharmaceutical composition containing the antigen composition can be stably supplied, which is industrially advantageous.

(SEQ ID NO: 1) GenBank: Nucleotide sequence of gH encoding based on NC_000898 information (SEQ ID NO: 2) GenBank: Amino acid sequence of gH based on NC_000898 information (SEQ ID NO: 3) GenBank: Gene encoding gL based on NC_000898 information Nucleotide sequence (SEQ ID NO: 4) GenBank: amino acid sequence of gL based on NC_000898 information (SEQ ID NO: 5) GenBank: Nucleotide sequence of gene encoding gQ1 based on NC_000898 information (SEQ ID NO: 6) GenBank: gQ1 based on NC_000898 information Amino acid sequence (SEQ ID NO: 7) GenBank: Nucleotide sequence of the gene encoding gQ2 based on NC_000898 information (SEQ ID NO: 8) GenBank: Amino acid sequence of gQ2 based on NC_000898 information (SEQ ID NO: 9) Amino acid sequence shown in SEQ ID NOs: 10 and 11. Amino acid sequence (SEQ ID NO: 10) of gH in which the signal sequence and transmembrane region have been deleted from the amino acid sequence specified by SEQ ID NO: 2 (SEQ ID NO: 11) and the amino acid sequence of hIgG1Fc (SEQ ID NO: 12). Of the amino acid sequences specified by No. 4, the amino acid sequence of gL from which the signal sequence has been deleted (SEQ ID NO: 13) Of the amino acid sequences specified by SEQ ID NO: 6, the amino acid sequence of gQ1 from which the signal sequence has been deleted (SEQ ID NO: 14) ) Of the amino acid sequences specified by SEQ ID NO: 8, the amino acid sequence of gQ2 from which the signal sequence has been deleted (SEQ ID NO: 15), the sequence that specifies primer 1 (SEQ ID NO: 16), and the sequence that specifies primer 2 (SEQ ID NO: 17) Sequence that identifies 3 (SEQ ID NO: 18) Sequence that identifies primer 4 (SEQ ID NO: 19) Sequence that identifies primer 5 (SEQ ID NO: 20) Sequence that identifies primer 6

Claims (8)

A composition for inducing an immune response against human herpes virus 6B (HHV-6B), of the surface proteins of HHV-6B, gH, gL, said composition comprising a tetrameric complex comprising gQ1 and gQ2 .. The composition according to claim 1, wherein gH comprises a polypeptide identified by the amino acid sequence set forth in SEQ ID NO: 10. The composition according to claim 1 or 2, wherein gH comprises the polypeptide specified by the amino acid sequence shown in SEQ ID NO: 10 and the polypeptide specified by the amino acid sequence shown in SEQ ID NO: 11. The composition according to any one of claims 1 to 3, wherein gL comprises a polypeptide identified by the amino acid sequence set forth in SEQ ID NO: 12. The composition according to any one of claims 1 to 4, wherein gQ1 comprises a polypeptide identified by the amino acid sequence set forth in SEQ ID NO: 13. The composition according to any one of claims 1 to 5, wherein gQ2 comprises a polypeptide identified by the amino acid sequence set forth in SEQ ID NO: 14. An HHV-6B vaccine comprising the composition according to any one of claims 1-6. A pharmaceutical composition for preventing or treating a disease caused by HHV-6B, which comprises the composition according to any one of claims 1 to 6 as an active ingredient.