WO2006080420A1

WO2006080420A1 - Antigen-specific t cell inducer, antibody production inducer, immune enhancer and vaccine

Info

Publication number: WO2006080420A1
Application number: PCT/JP2006/301283
Authority: WO
Inventors: Masahiro Toda; Tomoko Muraki; Kozo Tsukada; Raita Fukaya
Original assignee: Keio University; Institute Of Gene And Brain Science
Priority date: 2005-01-27
Filing date: 2006-01-27
Publication date: 2006-08-03
Also published as: JPWO2006080420A1

Abstract

[PROBLEMS] To provide a novel antigen-specific T cell inducer, a novel antibody production inducer, a novel immune enhancer and a novel vaccine, as well as a novel method of inducing antigen-specific T cells, a method of inducing antibody production, a novel method of immune enhancement, a novel therapeutic method for tumor and a novel therapeutic method against a pathogen. [MEANS FOR SOLVING PROBLEMS] A complete adjuvant, which is prepared by inactivating herpes simplex virus (HSV) and adding it to an incomplete adjuvant, is used as an antigen-specific T cell inducer, an antibody production inducer or an immune enhancer. Such an antigen-specific T cell inducer, an antibody production inducer or an immune enhancer is used together with an antigen to give a vaccine. In this case, a cancer-specific antigen or a pathogen-specific antigen is used as the antigen to thereby establish a therapeutic method for tumor or a therapeutic method for pathogen infection.

Description

Specification

Antigen-specific T cell inducer, antibody production inducer, immune enhancer, and vaccine

[0001] The present invention relates to an antigen-specific T cell inducer, an antibody production inducer, an immunopotentiator, and a vaccine containing herpes simplex virus (HSV).

Background art

[0002] When inoculating a substance (antigen) that causes an immune response in a living body, in order to enhance the immune response, it has been practiced to inoculate the antigen with an immunopotentiator called an adjuvant. . Adjuvants such as Freund Complete Adjuvant and Freund Incom plete Adjuvant that are often used in animal experiments (Broderson, J.A "Lab. Anim. Sci. 39 (1989) 400-406), for example, Freund's complete adjuvant is an emulsion containing mycobacteria heat-sterilized in mineral oil, and Freund's incomplete adjuvant contains mycobacteria in Freund's complete adjuvant. An emulsion that is not added Mycobacteria are active ingredients that have immunopotentiating activity in Freund's complete adjuvant.

[0003] When a vaccine is administered to an animal, an adjuvant is added to enhance the effect of the vaccine. Vaccines include live vaccines, inactivated vaccines, component vaccines, etc. Adjuvants are added to inactivated vaccines and component vaccines in particular, and antibodies are effectively produced even when antigens with weak immunogenicity are used. Will be able to

[0004] In recent years, new vaccines such as component vaccines and cancer vaccines have been developed, and new types of adjuvants have been developed (Jin H, et al., Vaccine 22 ( 2004), 2925-2935; Flarend, R et al., Vaccine 15 (1997), 1314-1318; Villacres- Eriksson M, et al., Cytokine 9 (1997), 73-82; Weeratna, R, et al " Vaccine 18 (2000), 1755-1762) ₀

Disclosure of the invention

Problems to be solved by the invention [0005] With the background of the above-described technology, the present invention was made for the purpose of developing a novel antigen-specific T cell inducer, a novel immune enhancer, a novel antibody production inducer, and a novel vaccine.

Means for solving the problem

[0006] The antigen-specific T cell inducer that is effective in the present invention contains a herpes simple x virus (HSV) as an active ingredient, and induces antigen-specific T cells against antigens other than HSV. It is an agent. Therefore, a method for inducing the antigen-specific T cell against an antigen other than HSV administered to humans and non-human vertebrates, wherein the antigen-specific T cell is induced in combination with the antigen. A method characterized by administering an agent also belongs to the technical scope of the present invention. This antigen-specific T cell inducer is preferably an antigen-specific cytotoxic T cell inducer. The herpes simplex virus is preferably inactivated and may be inactivated by, for example, heating, UV irradiation, radiation irradiation, fixation, or gene recombination. Further, the antigen-specific T cell inducer may contain incomplete Freund adjuvant (IFA).

[0007] The antibody production inducer according to the present invention is a herpes simplex virus;

It is an antibody production inducer against an antigen other than HSV, containing HSV) as an active ingredient. Therefore, a method for inducing antibody production against an antigen other than HSV administered to humans and non-human vertebrates, wherein the antibody production inducer is administered in combination with the antigen. These methods also belong to the technical scope of the present invention. In addition, it is preferable that the herpes simplex virus force is inactivated. For example, it may be inactivated by heating, UV irradiation, radiation irradiation, fixation, or genetic recombination. Furthermore, the antibody production-inducing agent may contain an incomplete Freund adjuvant (IF A).

[0008] The immunopotentiator according to the present invention is an immunopotentiator for antigens other than HSV, which contains herpes simplex virus (HSV) as an active ingredient. Accordingly, a method for enhancing immunity against antigens other than HSV in humans and non-human vertebrates, which comprises administering the above-described immunity enhancing agent, also belongs to the technical scope of the present invention. The simple herpes virus is preferably inactivated. For example, it may be inactivated by heating, UV irradiation, radiation irradiation, fixation, or genetic recombination. Furthermore, the immunopotentiator may contain incomplete Freund's adjuvant. Here, the immune enhancer generally refers to a drug that enhances an immune response in a living body. This immune reaction includes antigen-specific τ cell induction, antibody production induction, natural killer cell activation induction, dendritic cell activation induction, and inhibitory T cell induction inhibition.

[0009] The vaccine according to the present invention is a vaccine against an antigen other than a herpes simplex virus (HSV), the immunopotentiator comprising the simple herpes virus as an active ingredient, and the antigen Is a vaccine containing The vaccine may be a cancer vaccine or a vaccine against pathogen infection. Therefore, a method for treating tumors in humans and non-human vertebrates or a method for treating pathogen infections, which is characterized by vaccination, also belongs to the technical scope of the present invention. It is preferable that the herpes simplex virus is inactivated, for example, it may be inactivated by heating, UV irradiation, irradiation, fixation, or genetic recombination. Furthermore, you may contain the said vaccine power incomplete Freund's adjuvant. The treatment method referred to herein includes not only a disease treatment method but also a pre-illness prevention method.

[0010] — Cross reference with related literature

In addition, this application claims the benefit of the priority of Japanese application No. 2005-20393 filed on Jan. 27, 2005, and is incorporated herein by reference.

Brief Description of Drawings

[0011] [FIG. 1] In one embodiment of the present invention, GARC-1 peptide (mutant) of T cells obtained from a mouse administered with a vaccine containing GARC-1 mutant peptide and inactivated HSV. 2 is a graph showing the reactivity with respect to cells presenting (and wild type) in terms of the amount of IFN-γ produced.

[FIG. 2] In one example of the present invention, the reactivity of sputum cells obtained from a mouse administered with a vaccine containing GARC-1 mutant peptide and inactivated HSV to GL261 cells is shown as IFN-γ. FIG. [Fig. 3] In one example of the present invention, the reactivity of CD8-positive T cells obtained from a mouse administered with a vaccine containing GARC-1 mutant peptide and inactivated HSV to GL261 cells is shown. FIG. 3 is a graph showing the production amount of IFN-y.

[FIG. 4] In one embodiment of the present invention, in mice to which tumor cells were transplanted and administered a vaccine containing AH1 peptide and inactivated HSV, (A) and (B) are tumors transplanted into mice. It is the figure which evaluated the antitumor effect of cancer vaccine by the change of the volume of the cell, (C) is the figure which evaluated the cytotoxic activity with respect to CT26 cell of the CD8 positive T cell obtained from the mouse, (D) shows the amount of IFN-γ produced in response to the AH1 antigen of CD4 positive T cells and CD8 positive T cells obtained from mice administered a vaccine containing AH1 peptide and inactivated HSV. It is the graph showing.

[FIG. 5] In one embodiment of the present invention, in a mouse administered with a vaccine containing influenza virus ΗΑ protein and inactivated Η SV, (Α) is a CD8-positive Τ cell obtained from the mouse. , K ^d- restricted ΗΑ Peptide (MHC class I) is recognized for its cytotoxic activity, and (B) shows I—E ^d of CD4 positive T cells obtained from the mouse. FIG. 6 is a diagram showing an evaluation of proliferation activity that recognizes and reacts to a restricted HA peptide (MHC class II).

BEST MODE FOR CARRYING OUT THE INVENTION

[0012] Unless otherwise stated in the embodiments and examples, J. Sambrook, EF Fritsch & ι '· Maniatis (Ed.), Molecular cloning, a laboratory manual (3rd edition, Cold spring Harbor Press, Cold Spring Harbor, New York (2001); FM Ausubel, R. Brent, RE Kingston, DD Moore, JG Seidman, JA Smith, K. Struhl (Ed.), Current Protocols in Molecular Biology, John Wiley & Sons Ltd. Use the methods described in the standard protocol collection such as, or modified or modified methods, etc. In addition, when using commercially available reagent kits and measuring devices, unless otherwise specified, Use the attached protocol.

[0013] The objects, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description of the present specification, and those skilled in the art can easily read the description from the description of the present specification. The invention can be reproduced. The embodiments and specific examples of the invention described below are described in this document. It is intended to illustrate preferred embodiments of the invention and is presented for purposes of illustration or description and should not be construed as limiting the invention thereto. It will be apparent to those skilled in the art that various modifications and variations can be made based on the description of the present specification within the spirit and scope of the present invention disclosed herein.

[0014] = = Production of antigen-specific T cell inducer, antibody production inducer, immune enhancer and antigen mixture = =

The type of HSV contained in the antigen-specific T cell inducer, antibody production inducer, immunopotentiator or actin according to the present invention is not particularly limited, but in the examples, the KOS strain, which is a type I wild strain, is used. It was. HSV can be propagated using Vero cells, etc., according to a conventional method.

[0015] The HSV used in the present invention is preferably inactivated by heating, UV irradiation, radiation irradiation, fixation, gene recombination or the like so as not to grow in vivo. The following are typical conditions for each operation. When heating HSV, for example, it is treated at 56 ° C. for about 30 minutes. In the case of UV irradiation, UV lamps (wavelength, 253.7 nm) are irradiated at a distance of 10 to 15 cm for a total of 120 to 650 J / m ² (De Roda Husman AM,. Et al., Appl En viron Microbiol. 70 ( 2004): 5089-5093.). In the case of irradiation, γ rays are applied at 0.96 Gy / s for a total of 200-800 Gy (De Roda Husman AM,. Et al., Appl Environ Microbio 1. 70 (2004): 5089-5093). When doing so, a known tissue fixing agent such as formaldehyde solution, formalin or dartal aldehyde may be used. Inactivation by genetic recombination can be achieved, for example, by controlling the replication of the virus by means of genetic manipulation (such as T recombination so that the viral genome replicates only in tumor cells and not in normal cells). ) (Mmeta T, et al. Attenuated multi-mutated herpes simplex virus- 丄 for the treatment of malignant gliomas. Nat Med. 9 (1995), 938-943.), Or to prevent the virus from replicating by genetic engineering. (Lim F, et al. Biotechniques. 20 (199 6): 460-469.). The above condition is a typical condition example, and the specific operation may be other than the above condition, and may not be deactivated in some cases.

[0016] Such HSV can be used as an antigen-specific T cell inducer, antibody production inducer, or immune enhancer. It is used as an antigen mixture by mixing with an antigen that is the target of antigen-specific T cell induction, antibody production induction, or immune enhancement. The dosage forms of these antigen-specific τ cell inducer, antibody production inducer, immunopotentiator and antigen mixture are not particularly limited, and may be any of liquid, emulsion, gel, solid, dry powder, etc. . In addition to HSV, pharmacologically acceptable carriers and additives such as emulsifiers, surfactants, preservatives, and incomplete adjuvants may be contained. For example, HSV may be mixed with an incomplete Freund's adjuvant in advance to form an antigen-specific sputum cell inducer, antibody production inducer, or immune enhancer, and then mixed with an antigen to form an antigen mixture. Further, a plurality of types of antigens in the antigen mixture may be contained.

[0017] The antigen of interest in the present invention is not particularly limited as long as it is other than HSV (including antigens derived from HSV), and any antigen can be used as long as it finally has antigenicity in vivo. Originally, cancer antigens (such as cancer-specific proteins and peptides that are part of them), mycoplasma, viruses (such as AIDS virus, influenza virus, SARS (severe acute respiratory syndrome) coronavirus), Inactivated for use in preventing infection of pathogens such as Itoda (pathogenic enterococcus, green bacterium, tuberculosis, etc.), protozoa (malaria, toxoplasma, etc.), parasites (nematodes, etc.) Pathogen antigens, such as pathogens and some of them (cell wall, polysaccharides, bacterial cell components such as proteins, and proteins constituting viruses), Chin can be made. That is, the antigenicity of a vaccine can be enhanced by incorporating the antigen-specific T cell inducer, the antibody production inducer, or the immune enhancer in a vaccine and using it together with a cancer antigen or a pathogen antigen. Here, vaccines include live vaccines (attenuated vaccines), killed bacteria vaccines, inactivated vaccines, toxoids, component vaccines (component vaccines), genetically modified vaccines (including peptide vaccines), cancer vaccines, etc. A vaccine with relatively weak antigenicity, such as an inactivated vaccine, a component vaccine, a genetically modified vaccine, or a cancer vaccine, is preferable. Moreover, it is good also as a mixed vaccine containing multiple types of antigens in a vaccine.

[0018] = = Inoculation method of antigen mixture = =

Antigen-specific T cell inducers, antibody production inducers, or immune enhancers produced in this way A living body is inoculated with an antigen mixture of a strong agent and an antigen. The method for inoculating the antigen mixture is not particularly limited, and it may be administered orally or parenterally. Parenteral administration includes subcutaneous, intramuscular, intraperitoneal, intravenous injection, rectal administration, mucosal administration, transdermal administration, or mucosal administration, but is usually inoculated by subcutaneous or intraperitoneal injection There are many cases. Even if the antigen is a cancer antigen, it is not necessary to directly inoculate the tumor with the cancer antigen.

[0019] The number of inoculations and the place of inoculation are not particularly limited, and may be used in combination with other pharmaceuticals at the same time. In addition, different antigen mixtures may be inoculated simultaneously.

[0020] = = Disease treatment methods (including prevention methods) = =

As described above, when the living body is inoculated with the antigen mixture according to the present invention, differentiation-proliferation of antigen-specific T cells added to the antigen mixture and production of antigen-specific antibodies are induced. When the antigen is a cancer antigen or some of its peptides, typically antigen-specifically induced T cells attack cancer cells that present the antigen with histocompatibility antigen on the cell surface. Tumors can be treated with antigen mixtures to shoot. In addition, when the antigen is a pathogen antigen, typically, the antigen-specific production-induced antibody attacks the pathogen. Therefore, by inoculating the living body with the antigen mixture in advance of infection, Prevention of pathogen infection is possible. In addition, it is possible to treat pathogen infections and prevent recurrence of pathogen infections by inoculating a living body with an antigen mixture even after infection.

[0021] The principle of the disease treatment method for the antigen to be inoculated is not limited to the case described here. For example, when the method of the present invention is used to prevent tumors or the cancer antigen is a cell surface antigen. For example, a tumor may be treated with an antibody whose production has been induced.

Example

<Example 1> Cancer vaccine using GARC-1 peptide

(l) Preparation of HSV

The HSV type I wild type KOS strain was used as the HSV, and Vero cells were infected according to a conventional method, and the virus released in the culture supernatant was collected. The medium used at this time was a serum-free medium (DMEM without serum). After measuring the virus concentration, Completely inactivate the virus by UV treatment for 15 minutes and heat treatment for 30 minutes at 56 ° C, or treatment with 0.1% formalin for 1 week at 4 ° C, and used in the following experiments (Hereafter, the virus concentration of HSV shows the value before inactivation). The culture supernatant of Vero cells without virus was treated in the same manner as above and used as a control (Mock).

[0023] (2) Vaccine using GARC-1 antigen (tumor antigen)

In mouse glio cell line GL261 (derived from C57BLZ6 mouse), there is a mutation in GARC 1 gene that does not exist in normal cells, so a peptide with this mutation (AA LLNKLYA, SEQ ID NO: 1) (in MHC class I there is presented on H_2D ^b) is recognized to T cells as tumor-specific antigen. Therefore, using this GARC-1 mutant peptide as an antigen used in this example, and using a GAR C 1 wild-type peptide (AALLDKLYA, SEQ ID NO: 2) present in normal cells as a control, cancer A vaccine was produced.

[0024] The vaccine

(1) GARC-1 mutant peptide containing 1 Omg / ml of GARC-1 wild type peptide is 10 μl (100 / ig),

(2) 50 μ 1 of solution containing or not containing inactivated HSV (equivalent to 2 X 10 ⁸ pfli) (Mock)

(3)? 83 to 40〃1,

Prepare 4 types of the above mixture (100 μΐ) for each combination and mix with an equal volume of incomplete Freund's adjuvant (BACTO ADJUVANT INCOMPLETE FREUND, DIFCO: ² 63910) (100 μ 1). (Total 200 μ1).

[0025] A vaccine (200 μΐ) was inoculated into the skin of both hands and feet of C57BLZ6 mice, a mouse strain derived from GL261, and again the same vaccine was inoculated again 5 days later.

[0026] Two weeks after the start of vaccine administration, mixed floating cells obtained from the lymph nodes and spleen of the mice were adjusted to 2 x 10 ⁶ Zml. On the other hand, GL261 cells were treated with mitomycin C (200 zg / ml) for 60 minutes, adjusted to ³ × 10 ⁵ cells / ml, and used as stimulator cells. Mix 1 ml each of these mixed floating cells and stimulating cells into a 24-well plate (142575 N UNC) for 1 week.

[0027] Subsequently, in the following three experimental systems, analysis of antigen-specific reactivity of T cells (IF

Ν-γ release analysis).

[0028] [1. GARC — lymphocyte reactivity against cells presenting 1 peptide (mutant and wild type)]

As described above, the mixed floating cells obtained by co-culture for 1 week were adjusted to 1 × 10 ^s Zml to obtain effector cells. On the other hand, 293TK ^b D ^b cells (expressing MHC class I molecules K ^b and D ^b , 293T cells), GARC-1 mutant peptide, and GARC-1 wild-type peptide pulse treatment ( Each peptide was added to the cell culture medium at a concentration of 10 μg / ml, cultured for 1 hour, washed twice with PBS, and then adjusted to IX 10 ⁶ cells / ml to obtain target cells. Each of these effector cells and target cells was mixed at 100 μΐ and co-cultured in a 96-well plate (167008 NUNC) for 2 days. Thereafter, the amount of IFN-γ released in the culture supernatant was analyzed using ELISA kit (KM-IFNG Endogen).

As a result, as shown in FIG. 1, GARC-1 mutant peptides were obtained from lymph nodes and spleens of mice administered with a vaccine containing GARC-1 mutant peptide, which is a tumor antigen, and mock. T cells that produce IFN-y in response to the cell-specific responses presented were induced, but compared to the vaccine administration containing this GARC-1 mutant peptide and mock, the GARC-1 mutant peptide and Mice receiving a vaccine containing activated HSV had enhanced responsiveness of cell-specific lymphocytes displaying GARC-1 mutant peptides. On the other hand, in the control, the vaccine using the autoantigen GARC-1 wild-type peptide responds specifically to cells presenting GARC-1 wild-type peptide, even when inactivated HSV is supported. Lymphocytes producing y were not induced.

[0030] Based on the above, HSV as an adjuvant enhances antigen-specific immunity against tumor antigens, but does not show a clear immunity-enhancing effect against self-antigens and is useful from a safety standpoint It became clear that. Therefore, it was concluded that a composition containing HSV is useful as an immunopotentiator against an antigen administered to a living body.

[0031] [2. Lymphocyte reactivity to GL261 cells I]

Adjust mixed suspension cells obtained for 1 week of co-culture as above to 1 X 10 ^s / ml. Effector cells. On the other hand, GL261 cells and control EL4 cells were treated with mitomycin C (200 μg / ml) for 60 minutes, adjusted to 1 × 10 ⁶ Zml, and used as target cells. 100 of each of these effector cells and target cells were mixed and co-cultured in a 96-well plate (167008 NUNC) for 2 days. Thereafter, the amount of IFN-γ released in the culture supernatant was analyzed using ELISA kit (KM-IFNG Endogen).

As a result, as shown in FIG. 2, in mice administered with GARC-1 mutant peptide, which is a tumor antigen of GL261 Dario cell, only when a vaccine containing inactivated HSV was administered, Lymphocytes that specifically react with GL261 cells and produce IFN-y were induced.

[0033] From the above, it was shown that immunity specific to tumor cells expressing the tumor antigen can be enhanced by using Kay HSV as an adjuvant for the tumor antigen. Therefore, it was concluded that inclusion of HSV in the vaccine is useful for enhancing tumor immunity and preventing pathogen infection.

[0034] [3. Lymphocyte reactivity to GL-261 cells II]

As described above, after purifying CD8 positive T cells (mainly cytotoxic T cells) from the mixed suspension cells obtained by co-culture for 1 week, adjust to 5 X 10 ⁵ cells / ml to obtain effector cells. It was. On the other hand, GL261 cells were heat-treated at 56 ° C. for 30 minutes and then adjusted to ⁵ × 10 ⁵ cells / ml to obtain target cells. Each of these effector cells and target cells was mixed at 100 μl and co-cultured in a 96-well plate (167008 NUNC) for 1 day. Subsequently, the amount of IFN-y released in the culture supernatant was analyzed using an ELISA kit (KM-IFNG Endogen).

As a result, as shown in FIG. 3, when a mouse was immunized with a vaccine containing GARC-1 mutant peptide, which is a tumor antigen of GL261 dalio cell, and HSV, each of them was singly used. The reactivity of CD8 positive T cells producing IFN-γ to GL261 cells was enhanced compared to mice immunized with (Fig. 3).

[0036] From the above, it was shown that the induction of CD8-positive T cells responding to tumors was enhanced by using Kay HSV as an adjuvant as a tumor antigen. Therefore, an immunopotentiator containing HSV is useful as an antigen-specific T cell inducer for the antigen combined with it. It was concluded.

[0037] <Example 2> Cancer vaccine using AH1 peptide (tumor antigen)

HSV was prepared in the same manner as in Example 1.

The antigen is a tumor antigen of mouse colon cancer cell line CT26 (derived from BALBZc mouse), and peptide AH1 (SPSYVYHQF, SEQ ID NO: 3) presented on MHC class I (H_2L ^d ) (Huang A et al, Proc. Natl. Acad. Sci. USA 93. 9730-9735, 1996), a peptide that is the tumor antigen of the mouse mastocytoma cell line P81 as a control peptide presented on the same MHC class I (H_2L ^d )! ^ :! Eight ?丫! ^^^! ^^, SEQ ID NO: 4) (Lethe B, et al, Eur. J. Immunol 22, 1992) was used.

[0038] The vaccine

(1) AH 1 peptide (100 μg) or P815 A peptide (100 μg)

(2) Inactivated HSV (equivalent to 2 X 10 ⁸ pfli) or Mock

(3) PBS

Four mixtures (100 μ1) were prepared for each combination and mixed with an equal volume of incomplete Freund's adjuvant (100 μ1) (total 200 μ1). A vaccine (200 μl) was administered into the skin of both hands and feet of BALB / c mice, the mouse strain from which CT26 was derived, and the same vaccine was administered again 7 days later. One week after the second vaccine administration, 5 x 10 ⁵ CT26 tumor cells were transplanted on the back of BALB / c mice, and the tumor diameter was measured over time to analyze the tumor volume. The results are shown in Table 1.

[0039] [Table]

Number of mice whose transplanted tumor was rejected after vaccination

[0040] In mice administered with vaccines of P815A + HSV (3 individuals) and P815A + Mock (4 individuals), all patients were confirmed to be engrafted with CT26 on the 8th day after tumor implantation, and then tumors were observed. The volume increased and was not rejected. Meanwhile, AHl + Mock vaccine was launched. In the given mice (4 mice), all cases confirmed the engraftment of CT26 tumors on the 8th day after tumor transplantation, but the tumors shrank thereafter, and on the 22nd day after tumor transplantation, 1 case was excluded. In the example, the tumor was rejected. In addition, mice (3 mice) that received the AH1 + HSV vaccine did not show tumor engraftment within the observation period, and were completely rejected in all cases.

[0041] From the above, it was shown that HSV actually enhances the antigen-specific immune response of colorectal cancer cell CT26, and its usefulness as an adjuvant to enhance the effect as a cancer vaccine was demonstrated. . Therefore, it was concluded that a vaccine containing an immunopotentiator containing HSV is effective as a cancer vaccine.

<Example 3> Treatment of tumor using AH1 peptide (tumor antigen)

HSV was purified in the same manner as in Example 1. In addition, the tumor antigen was AH as in Example 2.

1 peptide was used, CT26 (mouse colon cancer) was used as the target tumor cell, and Meth A (mouse fibrosarcoma) was used as the control tumor cell.

[0043] First, CT26 cells (5 × 10 ⁵ cells) were transplanted into the dorsal part of Balb / c mice, and the first immunization was started on the fifth day of transplantation.

[0044] For the first immunization,

(1) AH1 peptide (50 μg) or DMSO

(2) Inactivated HSV (equivalent to 1 X 10 ⁸ Pfli) or Mock

(3) PBS

3 types (AHlZHSV and AH1 ZMock and DMSO / Mock as negative controls) were prepared and mixed with an equal volume of incomplete Freund's adjuvant (IFA) (10 μμΐ) (respectively, AH1 / HSV ( IFA), and AH1ZIFA and DMSO / IFA as negative controls; 200μ1 each). As a positive control, a mixture of AH1 peptide and PBS (100 μ1) was mixed with an equal volume of complete Freund's adjuvant (CFA) (ΙΟΟμΙ) (denoted as AH1ZCFA; 200 μ1). Antigens prepared in this manner (100 μl each) were administered to both foot pads of BalbZc mice.

[0045] For the second immunization 7 days later,

(1) AH1 peptide (for AHlZHSV (IFA), AH1 / IFA, and AHl / CFA) or DMSO (for DMSO / IFA) (2) PBS

For each combination, mix with an equal volume of incomplete Freund's adjuvant IFA (100 μ1) (total 200 μ1), and apply the antigen (100 μ 1) was administered.

[0046] Thereafter, the antigen-specific reactivity of sputum cells was analyzed in the following three experimental systems.

[0047] 1. Evaluation of antitumor effect

The volume of tumor transplanted into mice was measured every 2 days up to 14 days and every 4 days up to 21 days, and the immune effect was evaluated by the change in the tumor volume. Fig. 4 (b) shows the experimental results for each mouse, and Fig. 4 (b) shows the mean value of the tumor volume on day 21.

[0048] From the time course of tumor volume (Fig. 4 IV), the increase in tumor volume was suppressed when AH1 / CFA (positive control) was used compared to when DMSO / IFA and AH1 / IFA were used. It can be seen that the increase in tumor volume is further suppressed when the force AH1 / HSV is used. In addition, when comparing the mean tumor volume on day 21 (Fig. 4 Β), the tumor volume was not statistically significant when AH1 / CFA was used compared to when DMSO / IFA and AH1 / IFA were used. On the other hand, when AH1 / HSV was used, the increase in tumor volume was significantly suppressed (ρ 0.05).

[0049] Thus, it has been clarified that HSV exhibits an inhibitory effect on tumor growth of colon cancer cell CT26 when administered together with AH1.

[0050] 2. Evaluation of cytotoxic activity

21 days after tumor cell transplantation, mixed floating cells were isolated from the spleen and lymph nodes of each mouse, and prepared to 3 to 5 × 10 ⁶ / ml. On the other hand, as antigen-presenting cells, spleen cells prepared from normal mice were irradiated with X-rays (60 Gy) and reacted with peptide antigen AH1 (10 μM) for 90 minutes to prepare 1 × 10 ⁶ / ml. These mixed floating cells and antigen-presenting cells were mixed in lml each and co-cultured in a 24-well plate (NUNC 142475) for 5 days. Thereafter, cocultured CD8-positive T cells purified from suspended cells (E: T_ratio: 60, 30 , 15; 3 X 10 5, 1. 5 X 10 5, 0. 75 X 10 5) and ⁵¹ Cr (Amersham Corp. ) in CT26 cells (5 X 10 ³ cells were labeled) and engaged mixed, a 96-well plate (U-bottom: BD35 after 4 hours co-cultured in 3077), ⁵¹ Cr release mediation Cytotoxic activity was evaluated by SE. In addition, Meth A cells (5 × 10 ³ cells) were used as control cells. In addition, the CT26 cells, 1. added of ⁵¹ Cr 85MBq in lml of cell floating遊液(IX 10 ⁶ cells), 1-2 hours, was incorporated label by shaking at 37 ° C.

[0051] As a result, as shown in FIG. 4C, when AH1 / HSV was used, there was a significant (p 0.05) specific cytotoxicity against CT26 cells compared with DMSO / IFA and AHlZlFA. Enhanced.

[0052] Thus, HSV strongly induces antigen-specific CD8-positive T cells (mainly thought to be cytotoxic T cells) when administered together with the AH1 peptide, and cells against CT26 cells. It has been shown to enhance the disability activity.

[0053] 3. Site force in IFN—y production evaluation

Using BD Cytofix / Cytoperm kit (Pharmingen), IFN-γ producing cells in the mixed suspension cells co-cultured as described above were analyzed.

[0054] First, in order to inhibit intracellular protein transport for mixed cultured floating cells, add BD GolgiStop containing mone nsin to the well of a 24-well plate for 8 hours. Then, in order to suppress non-specific reaction, antibody Fc-Block (mAb 2.4G2: diluted 100 times) against Fc receptor was added, and FITC-labeled anti-CD4 antibody (mAb GK1.5 eB Bioscience 400 times diluted) Alternatively, it was stained with FITC-labeled anti-CD8 antibody (mAb 53-6.7 e-Bioscience 400-fold dilution). After that, add 100 μ 1 of BD Cytofix / Cytoperm solution, fix the cells at 4 ° C for 20 minutes, wash twice with BD Perm / Wash solution, PE (phycoerythrin) -labeled anti-IFN-γ antibody (mAb XMG1. 2: e-Bioscience 200-fold diluted) BD Perm / Wash solution 50 μ 1 for 30 minutes 4 Reaction with C and FACS analysis

As a result, in the AH1ZHSV immunized group, it was found that most of the IFN-producing cells increased by AH1 peptide co-culture were CD8-positive T cells but not CD4-positive T cells (FIG. 4D).

[0056] From the above, in the treatment of tumors using cancer antigens, cancer antigen-specific CD8-positive T cells are strongly induced by using HSV as an adjuvant together with cancer antigens. Was shown to be enhanced. Therefore, it was concluded that a vaccine containing an immunopotentiator containing HSV together with a cancer antigen is effective for treating a tumor using the cancer antigen.

<Example 4> Influenza virus vaccine using hemagglutinin antigen (HA)

HSV was purified in the same manner as in Example 1. The animals used here are BalbZc mice (early: _n = 4 / group).

[0058] For the first immunization, hemagglutinin antigen protein (HA) (HlNl: IFA-055-B052 2) (10 x g) was used,

(Ι) ΗΑ (ΙΟ μ g / 10 μ 1) or DMSO (10 μ 1)

(2) Inactivated HSV (low): 1 X 10 ⁷ Pill, Inactivated HSV (high): 1 X 10 ⁸ Pili, or Moc k

(3) PBS

Prepare the following 5 types of liquid mixture (100 μ1) for each combination and mix with an equal volume of incomplete Freund's adjuvant (IFA) (ΙΟΟ μΙ) (total 200 / il). c The above vaccine (100 μΐ each) was administered to the foot pads of mice.

(D DMSO / IFA

(2) DMSO / HSV

(3) HA / IFA

(4) HA / HSV (low)

(5) HA / HSV (high)

[0059] The second immunization, immune responses to CD4 Epitopu or CD8 Epitopu of Maguruchinin antigen for the purpose of enhancing the, as a peptide antigen, I one E ^d restricted HA peptidase de (SVSSFERFEIFPK SEQ ID NO: 5, amino acids No. 124-136) and ^Kd- restricted HA peptide (IYSTVASSL SEQ ID NO: 6, corresponding to amino acid Nos. 533-541) mixed peptides (50 zg each) were used. That is,

The second vaccine after 7 days

(1) I—E ^d- restricted HA peptide (50 μg) and K ^d- restricted HA peptide (50 μg) or DMS

(2) PBS (100 μΐ) was mixed with an equal volume of incomplete Freund's adjuvant (50 μ1) (100 μΐ). 50 μΐ each).

(D DMSO / IFA

(2)-(5) Peptide / IFA

[0060] Thereafter, the antigen-specific reactivity of the sputum cells was analyzed in the following two experimental systems, and the antibody titer of the induced antibody was measured.

[0061] 1. Evaluation of cytotoxic activity

One week after the second vaccination, mixed floating cells were isolated from the spleen and lymph nodes of each mouse and prepared to 3 to 5 ^× 10 ^s / ml. On the other hand, as antigen-presenting cells, spleen cells prepared from normal mice were irradiated with X-rays (60 Gy), treated with IE ^d- restricted HA peptide or K ^d- restricted HA peptide (10 μΜ) for 90 minutes, and 1 X Prepared to 10 ⁶ / ml. These mixed floating cells and antigen-presenting cells were mixed in 1 ml each, and co-cultured in a 24-well plate (NUNC 14247 5) for 5 days. After that, CD8 positive T cells (E: T-ratio: 60, 30, 15; 3 X 10 ⁵ , 1.5 X 10 ⁵ , 0.775 X 10 ⁵ ) purified from co-cultured floating cells with ⁵¹ Cr Mix with CT26 cells labeled as described above (treated with 10 μΜ ^Kd- restricted HA peptide) (5 × 10 ³ cells) and co-culture for 4 hours in 96-well plate (U-bottom: BD35 3077) Thereafter, the cytotoxic activity was evaluated by ⁵¹ Cr release assay. As control cells, CT26 cells (treated with 10 μΜ of P815 A peptide) (5 × 10 ³ cells) were used.

[0062] As a result, as shown in Fig. 5 (1), (5) HAZHSV (high), compared to (1) DMS ○ ZIFA, DMSO / IFA, (3) HA / IF A, HA peptide / IFA combination The HA peptide / IFA combination significantly (p 0.05) strongly induces antigen-specific CD8-positive T cells and has cytotoxic activity against CT26 cells (treated with 10 μΜ ^Kd- restricted HA peptide). It was shown to enhance.

[0063] 2. Evaluation of proliferative response

CD4-positive T cells purified from a mixed suspension cells were co-cultured (1 X 10 ⁵ cells), X-ray irradiation (60 Gy) after the antigen-presenting cells (spleen cells treated with I _ E ^d restricted HA peptide) ( 5 x 10 ⁴ or 1 x 10 ⁵ ) for 3 days and put [ ³ H] _th in the medium 5 hours before the end of the culture. After addition of ymidine (18.5 kBq / well), the growth response was evaluated by the amount of uptake (count value cpm of []) after completion of the culture.

[0064] As shown in FIG. 5B, the results are as follows: (4) HA4HSV (low), HA peptide / IF A combination, against CD4 positive T cells of mice immunized with DMSOZlFA, DMSO / IFA combination And (5) CD4 positive T cells of mice immunized with the combination of HA / HSV (high) and HA peptide / IFA showed a significant (p 0.05) proliferation promoting effect depending on the number of antigen presenting cells.

[0065] Thus, it was revealed that antigen-specific CD4-positive T cells can be induced by using HSV as an adjuvant together with influenza antigen when administering an influenza vaccine.

[0066] 3. Measurement of antibody titer

The antibody titer was measured in 4 mice each of (1) to (4) by collecting sera from peripheral blood one week after the second vaccine and measuring it by hemagglutination inhibition test (HI).

[0067] Specifically, the antibody titer against influenza A virus HA antigen was examined using a kit of influenza virus HI reagent "Seiken" (Den Rikiseiken Co., Ltd.). Prepare 0.1 ml of RDE (Receptor Destroying Enzyme) in 0.1 ml of sample, remove non-specific inhibitors by incubating at 37 ° C and heat at 30 ° C for 30 minutes to react with RDE treatment. Stopped and added 0.6 ml of normal saline. Add 50 µl of 50% (v / v) chicken erythrocyte suspension to the RDE-treated sample, mix, leave at 4 ° C for 60 minutes, centrifuge, and start the supernatant at 1:10 for serial dilution. Had made. After adding HA antigen to each and mixing well, we further measured 0.5% (v / v) chicken erythrocyte suspension, and investigated the force at which dilution of erythrocyte aggregation was suppressed to determine the HI antibody titer. .

[0068] As a result of this hemagglutination inhibition test, (1) the combination of DMS 0 / IFA and DMSO / IFA, and (2) the combination of DMS 0 / HSV, HA peptide / IFA, the HI antibody titer was It was less than 10. On the other hand, (3) HI antibody titer was detected by the combination of HAZIFA and HA peptide ZIFA (65 ± 64. 03), but (4) the combination of HA / HSV (low) and HA peptide ZIFA resulted in an HI antibody titer Significantly (p 0.05) increased (140 ± 40). (However, the average soil standard deviation is in Katsuko) From this result, it was revealed that the serum antibody titer against influenza antigen was significantly increased by simultaneously administering HSV as an adjuvant with influenza antigen.

4. Conclusion

Based on the above, HSV was shown to enhance the antigen-specific immune response of influenza antigens, and it was shown to be useful as an adjuvant to enhance the effect as an influenza vaccine. Therefore, it was concluded that a vaccine containing an immunopotentiator containing HSV together with an influenza antigen is effective as an influenza vaccine.

According to the present invention, it has become possible to provide a novel immunopotentiator containing a simple herpes virus, a novel antibody production inducer, and a vaccine containing them.

Claims

The scope of the claims

[I] An antigen-specific T cell inducer for antigens other than HS V, which contains herpes simplex virus (HSV) as an active ingredient.

[2] The antigen-specific T cell inducer according to claim 1, which is an antigen-specific cytotoxic T cell inducer

[3] The antigen-specific T cell inducer according to claim 1 or 2, wherein the herpes simplex virus is inactivated.

[4] The antigen-specific T cell inducer according to claim 3, wherein the herpes simplex virus is inactivated by heating, UV irradiation, irradiation, fixation, or genetic recombination. .

[5] The antigen-specific T cell inducer according to any one of [5] to [4], further comprising an incomplete Freund adjuvant (IFA).

[6] HS containing herpes simplex virus (HSV) as an active ingredient

Antibody production inducer against antigens other than V.

[7] The antibody production inducer according to [6], which is an antigen-specific cytotoxic T cell inducer.

[8] The antibody production-inducing agent according to [6] or [7], wherein the simple herpesvirus is inactivated.

[9] The antibody production inducer according to claim 8, wherein the herpes simplex virus is inactivated by heating, UV irradiation, irradiation, fixation, or genetic recombination.

[10] The antibody production-inducing agent according to any one of [6] to [9], further comprising an incomplete Freund adjuvant (IFA).

[I I] An immunopotentiator against antigens other than HS V, which contains herpes simplex virus (HSV) as an active ingredient.

[12] The immunopotentiator according to [11], wherein the simple herpesvirus is inactivated.

[13] The simple herpes virus is heated, UV irradiated, irradiated, fixed, or gene 13. The immunopotentiator according to claim 12, which has been inactivated by recombination.

[14] The immunopotentiator according to any one of [11] to [13], further comprising Incomplete Freund adjuvant (IFA).

[15] A vaccine against antigens other than herpes simplex virus (HSV),

An immunopotentiator comprising a simple herpes virus as an active ingredient and a vaccine containing the antigen.

16. The vaccine according to claim 15, wherein the simple herpesvirus is inactivated.

17. The vaccine according to claim 16, wherein the herpes simplex virus is inactivated by heating, UV irradiation, irradiation, fixation, or genetic recombination.

[18] The vaccine according to any one of [15] to [17], further comprising Incomplete Freund adjuvant (IFA).

[19] The vaccine according to any one of [15] to [18], which is a cancer vaccine.

[20] The vaccine according to any one of [15] to [18], which is a preventive vaccine against infection with a pathogen.

[21] A method for inducing antigen-specific T cells to an antigen other than HSV administered to a vertebrate,

A method comprising administering the antigen-specific T cell inducer according to any one of claims: to 5 or the vaccine according to any one of claims 15 to 20 in combination with the antigen.

[22] The method of claim 21, wherein the T cell is a cytotoxic T cell.

[23] A method for inducing antigen-specific antibody production against an antigen other than HSV administered to a vertebrate,

A method comprising administering the antibody production-inducing agent according to any one of claims 6 to 10 or the vaccine according to any one of claims 15 to 20 in combination with the antigen.

[24] A method of enhancing immunity in vertebrates other than humans, A method comprising administering the immunopotentiator according to any one of claims 11 to 14 or the vaccine according to any one of claims 15 to 20.

[25] A method for treating tumors in vertebrates other than humans,

A method comprising inoculating the vaccine according to any one of claims 15 to 19.

[26] A method for treating pathogen infection in non-human vertebrates,

A method comprising inoculating the vaccine according to any one of claims 15 to 18 and 20