JPWO2007102326A1 - Powerful immune induction by combined use of adenovirus type 5/35 vector and vaccinia virus MVA vector - Google Patents

Abstract

Provide effective and inexpensive anti-HIV drugs. A recombinant virus vector in which the HIV structural gene is inserted into a chimeric virus in which the fiber of adenovirus type 5 is replaced with that derived from adenovirus type 35, and a group in which the HIV structural gene is inserted into a modified vaccinia virus Ankara An anti-HIV drug characterized by comprising a combination with a recombinant virus vector.

Description

  The present invention relates to an anti-HIV drug characterized by combining a recombinant viral vector vaccine using an adenochimeric virus type 5/35 vector and a recombinant viral vector vaccine using a vaccinia virus MVA vector.

More than 20 years have passed since the identification of human immunodeficiency virus (HIV), but no complete treatment has yet been established.
After the development of azidothymidine (AZT) as the first anti-HIV drug, new anti-HIV drugs were developed one after another, and in 1995, HAART (Highly Active Anti-retrovial Therapy), which combines multiple anti-HIV drugs, was started. In developed countries, AIDS deaths have fallen dramatically.

However, with this treatment, when the administration of the anti-HIV drug is discontinued, HIV will regrow after 2-3 weeks. In addition, drug-resistant viruses are appearing one after another, and new anti-HIV drugs are needed to cope with them. In addition, patients must continue to administer drugs throughout their lives, and the medical costs associated with them are enormous.
Therefore, there is a need for an effective and inexpensive treatment.

  On the other hand, vaccines against AIDS have been developed. Currently, vaccines using the adenovirus type 5 vector among the vaccines known in the world are highly immunogenic, but there are many patients with neutralizing antibodies against adenovirus type 5, In such patients, there was a problem that the ability to induce immunity was not so strong, and adenovirus type 5 was very strong in liver damage, so that it could not be administered in a large amount (Non-patent Document 1). By replacing the fiber of adenovirus type 5 with one derived from adenovirus type 35, the present inventors have developed a vaccine that is effective for patients with neutralizing antibodies against adenovirus type 5 and has low liver damage. It succeeded in producing (Patent Document 1, Non-Patent Document 2).

As other viral vectors, the effectiveness of vaccines using modified vaccinia virus Ankara (MVA) has been reported (Non-Patent Documents 3 to 5).
However, there are no reports on the combined use of different recombinant viral vector vaccines.

Vogels R et al. Replication-deficient human adenovirus type 35 vectors for gene transfer and vaccination: efficient human cell infection and bypass of preexisting adenovirus immunity.J Virol 2003; 77: 8263-8271. K-Q Xin et al., Gene Therapy (2005) 12, 1769-1777 Robinson, H. et al., Nat. Med. 5: 526-534, 1999 Amara, R.R.et al., Science 292: 69-74, 2001 Amara, R.R. et al., Virology 343 (2005) 246-255 International Publication No. WO 2005/052165 Pamphlet

  An object of the present invention is to provide an effective and inexpensive anti-HIV drug.

  The present inventors have prepared a recombinant viral vector in which an HIV structural gene is inserted into a chimeric virus in which an adenovirus (Ad) type 5 fiber is replaced with an Ad35 type derived fiber, and a HIV structure in a modified vaccinia virus ankara. It has been found that an unprecedented powerful immune response can be induced by immunization with a recombinant viral vector into which a gene has been inserted. By using different virus vectors, it is possible to reduce the production of antibodies against each virus, produce a vaccine with very little immunoreactivity and few side reactions.

The gist of the present invention is as follows.
(1) A recombinant viral vector in which the HIV structural gene has been inserted into a chimeric virus in which the fiber of adenovirus type 5 has been replaced with that derived from adenovirus type 35, and an HIV structural gene has been inserted into the modified vaccinia virus Ankara. An anti-HIV drug comprising a combination with a recombinant virus vector.
(2) The anti-HIV drug according to (1), wherein the adenovirus type 5 and the modified vaccinia virus ankara are non-proliferating.
(3) The anti-HIV drug according to claim 1 or 2, wherein the structural gene of HIV is an env gene and / or a gag gene.

(4) The anti-HIV drug according to any one of (1) to (3), wherein an HIV replication control gene is further inserted into at least one of the recombinant viral vectors.
(5) The anti-HIV drug according to (4), wherein the HIV replication control gene is a rev gene.
(6) A recombinant viral vector in which the HIV structural gene is inserted into a chimeric virus in which the fiber of adenovirus type 5 is replaced with that derived from adenovirus type 35, and an HIV structural gene is inserted into the modified vaccinia virus Ankara. The anti-HIV drug according to any one of claims 1 to 5, which is a kit comprising a recombinant viral vector.

(7) The HIV structural gene is inserted into a recombinant viral vector or a modified vaccinia virus ankara in which the HIV structural gene is inserted into a chimeric virus in which the fiber of adenovirus type 5 is replaced with that derived from adenovirus type 35 The anti-HIV drug according to claim 6, wherein the subject is first immunized with one of the recombinant virus vectors and boosted with the other.
(8) The anti-HIV drug according to any one of (1) to (7), further comprising a reverse transcriptase inhibitor and / or a protease inhibitor.
(9) The anti-HIV drug according to (8), wherein the reverse transcriptase inhibitor and / or protease inhibitor is administered to the subject at least once before, after or simultaneously with the administration of the recombinant viral vector.
(10) A recombinant viral vector in which the HIV structural gene is inserted into a chimeric virus in which the fiber of adenovirus type 5 is replaced with that derived from adenovirus type 35, and an HIV structural gene is inserted into the modified vaccinia virus Ankara. A method for the treatment and / or prevention of HIV infection, comprising administering to a subject an effective amount of the recombinant virus vector for treatment and / or prevention.
(11) An HIV structural gene is inserted into a recombinant viral vector or a modified vaccinia virus ankara in which the HIV structural gene is inserted into a chimeric virus in which the fiber of adenovirus type 5 is replaced with that derived from adenovirus type 35 (10) The method according to (10), wherein the subject is first immunized with one of the recombinant virus vectors and boosted with the other.
(12) The method according to (11), wherein the interval between primary immunization and booster immunization is about 1 to 3 months.
(13) The method according to (12), wherein the interval between the first immunization and the booster immunization is about 2 months.
(14) In the production of anti-HIV drugs, a recombinant viral vector in which an HIV structural gene is inserted into a chimeric virus in which the fiber of adenovirus type 5 is replaced with that derived from adenovirus type 35, and a modified vaccinia virus ankara Use in combination with a recombinant viral vector into which the HIV structural gene has been inserted.

By immunizing a combination of two types of recombinant viral vectors, it was possible to induce a significantly higher immune response than when immunized with each recombinant viral vector alone.
This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2006-061007 which is the basis of the priority of the present application.

The schedule for immunizing mice with recombinant viral vectors is shown. The result of a pentamer assay is shown. It is the result of examining the strength of cellular immunity induction ability for each vaccine schedule by measuring the ratio of CD8 ⁺ T cells having specific TCR. The result of an ICCS assay is shown. This is a result of examining the strength of the ability to induce cellular immunity for each vaccine schedule by measuring the proportion of CD8 ⁺ T cells that produce IFN-γ by stimulation with an antigenic peptide. The result of an in vivo CTL assay is shown. It is the result of examining the strength of the cellular immunity induction ability for each vaccine schedule by measuring the survival rate of the target cells presenting the transferred antigenic peptide. The schedule for immunizing monkeys with recombinant viral vectors is shown. The cellular immune response after the first immunization with MVA vaccine and the booster immunization with Ad5 / 35 vaccine is shown. Rhesus monkeys were first immunized with MVA vaccine. Two months later, the patient was boosted with the Ad5 / 35 vaccine. The IFN-γ-ELISpot assay was performed for the first time and 2 weeks after the booster immunization.

Hereinafter, embodiments of the present invention will be described in more detail.
The present invention relates to a recombinant viral vector in which the HIV structural gene is inserted into a chimeric virus in which the fiber of adenovirus type 5 is replaced with that derived from adenovirus type 35, and the HIV structural gene is inserted into a modified vaccinia virus Ankara. The present invention provides an anti-HIV drug comprising a combination with a recombinant virus vector.
Adenovirus type 5 is preferably non-proliferating. If the recombinant viral vector is non-proliferating, it will not grow in vivo after administration and is highly safe. Non-propagating adenovirus type 5 includes those from which the initial gene E1 has been removed and those from which the early genes E1 and E3 have been removed.

  In order to replace the adenovirus type 5 fiber with one derived from adenovirus type 35, a gene encoding a fiber protein derived from adenovirus type 35 may be used. The gene encoding the fiber protein derived from adenovirus type 35 and its amino acid sequence are known (Mei, YF et al., Virology 206 (1), 686-689, 1995; Dmitry M. et al., J. Virology , Mar. 2000, vol. 74, 2567-2583; NCBI U10272; NCBI AAA66361.1; Stone, D. et al., Virology, 2003, Vol. 309, 152-156). Fiber protein is composed of shaft and knob. The fiber protein derived from adenovirus type 35 may be a wild type or a mutant. Examples of such mutants include one or several (preferably 1 to 30, more preferably 1 to 10, more preferably 1) amino acid sequences of fiber protein (wild type) derived from adenovirus type 35. A protein having an amino acid sequence in which 1 to 5 amino acid residues are deleted, substituted and / or added, and having the same adhesion to a target cell as a fiber protein derived from adenovirus type 35 Can be mentioned. Also, it is encoded by DNA that hybridizes under stringent conditions with a gene encoding a fiber protein derived from adenovirus type 35 (wild type), and adheres to the same target cells as fiber protein derived from adenovirus type 35 It may be a protein having sex. Hybridization can be easily performed by those skilled in the art according to a known method, for example, a basic book such as Molecular Cloning 2nd Edt., Cold Spring Harbor Laboratory Press (1989). Stringent conditions can be determined by those skilled in the art. Examples of stringent conditions include a condition of reacting in a solution containing 6 × SSPE, 2 × Denhardt's solution, 0.5% SDS, 0.1 mg / ml salmon testis DNA at 65 ° C. for 12 hours. The gene encoding the mutant can be obtained, for example, by site-directed mutagenesis.

  A gene encoding a fiber protein derived from adenovirus type 35 (hereinafter sometimes referred to as “fiber gene”) can be obtained by a known method using a PCR method or the like based on known gene information.

  In order to replace an adenovirus type 5 fiber with one derived from adenovirus type 35, for example, DNA adenovirus type 35-derived fiber genes connected to both ends of the adenovirus type 5 fiber gene in the vicinity of adenovirus type 5 are adenovirused. When transfected into host cells (for example, 293 cells derived from human embryonic kidney cells (HEK293)) together with virus type 5, a chimeric virus in which the fiber of adenovirus type 5 is replaced with that derived from adenovirus type 35 by homologous recombination Produces. Such chimeric viruses are commercially available (pRHSP 5/35 in the Ad generation kit sold by Avior Therapeutics Inc) and are available.

  Examples of the structural gene of HIV inserted into a chimeric virus in which the fiber of adenovirus type 5 is replaced with that derived from adenovirus type 35 include gag gene, pol gene, and env gene. Furthermore, if necessary, an HIV replication control gene may be inserted into the chimeric virus, and examples of the HIV replication control gene include rev gene and tat gene. There are two known types of HIV, HIV-1 and -2, and HIV-1 is transmitted worldwide, but HIV-2 is ubiquitous in West Africa. The HIV structural gene and the replication control gene may be those of any HIV, but are preferably those of HIV-1. The HIV structural gene inserted into the chimeric virus (if necessary, the HIV replication control gene) may be inserted so as to be expressed in the host cell.

The gag gene is a gene encoding the core protein of HIV.
The pol gene is a gene encoding HIV reverse transcriptase.
The env gene is a gene encoding an HIV envelope protein. Although HIV-1 and -2 show differences in envelope proteins, any type of HIV envelope protein may be used. Moreover, HIV-1 is classified into subtypes A to J, K and the like based on the difference in the amino acid sequence of the envelope protein, but may be any subtype of envelope protein.

The rev gene is a gene that encodes a rev protein that stabilizes unspliced viral mRNA and transports mRNA encoding viral structural proteins to the cytoplasm.
The tat gene is a gene encoding a tat protein that causes transcriptional activation of a virus as a transactivator.

  The sequences of HIV structural genes and replication control genes and the amino acid sequences encoded by them are known (Goa, F., et al., J. Virol. 70 (3), 1651-1667 (1996); Ratner, L , et al., Nature 313 (6000), 277-284 (1985); Jounai, N. et al., J. Gene Med., 5, 609-617 (2003); Kim, FM, et al., J. Virol. 69 (3), 1775-1761 (1995); Rodenburg, CM, et al., AIDS Res. Hum. Retroviruses 17 (2), 161-168 (2001); Gao, F., et al. , J. Virol. 72 (7), 5680-5698 (1998); Gao, F. et al., J. Virology 70 (10), 7013-7029 (1996)). Gene information can also be obtained from gene banks such as NCBI (U51190, K03455, U39362, AF286224, U88822, U51188).

  HIV structural genes and replication control genes may be wild-type or mutant. The mutant gene includes one or several (preferably 1 to 30, more preferably 1 to 10, more preferably 1 to 5) amino acids in the amino acid sequence of the protein encoded by the wild type gene. A protein consisting of an amino acid sequence in which residues are deleted, substituted and / or added, and which encodes a protein having the same function as the protein encoded by the wild type gene, under stringent conditions with the wild type gene And a DNA that encodes a protein that has the same function as the protein encoded by the wild-type gene. Hybridization can be easily performed by those skilled in the art according to a known method, for example, a basic book such as Molecular Cloning 2nd Edt., Cold Spring Harbor Laboratory Press (1989). Stringent conditions can be determined by those skilled in the art. Examples of stringent conditions include a condition of reacting at 65 ° C. for 12 hours in a solution containing 6 × SSPE, 2 × Denhardt's solution, 0.5% SDS, 0.1 mg / ml salmon testis DNA. A mutant gene can be obtained, for example, by site-directed mutagenesis.

The HIV structural gene and replication control gene can be obtained by a known method using PCR or the like based on known gene information.
The HIV structural gene inserted into the chimeric virus in which the fiber of adenovirus type 5 is replaced with that derived from adenovirus type 35 (optionally a replication control gene) is an anti-HIV drug when inserted into the chimeric virus. As long as the effect as described above is obtained, the entire region or a part of the region may be used.

  The HIV structural gene (optionally the HIV replication control gene) may be inserted at any site of the chimeric virus as long as it does not adversely affect the life cycle of the chimeric virus. It may be inserted into any part corresponding to 22 to 5790 parts. To insert HIV structural genes (if necessary, HIV replication control genes) into the chimeric virus, for example, expression including these genes and then poly A gene downstream of a promoter such as CMV promoter, CAG promoter, etc. Prepare a cassette, and connect the same gene to adenovirus type 5 (for example, DNA corresponding to 22-342 site of adenovirus type 5 and DNA corresponding to 3523-5790 site) to both ends of the cassette, along with the chimeric virus. When transfected into a host cell (for example, HEK293 cell), a chimeric virus into which an HIV structural gene (optionally, an HIV replication control gene) is inserted is generated by homologous recombination. The expression cassette may further include a replication origin, a drug resistance gene such as an ampicillin resistance gene (a marker indicating that the target gene has been introduced), and the like.

  A recombinant viral vector in which the structural gene of HIV is inserted into a chimeric virus in which the fiber of adenovirus type 5 is replaced with that derived from adenovirus type 35 can also be prepared using a commercially available kit. For example, in Example 1 described later, a procedure for producing a recombinant virus vector using an Ad generation kit sold by Avior Therapeutics, Inc. is described.

Recombinant viral vectors in which the HIV structural gene is inserted into a chimeric virus in which the fiber of adenovirus type 5 is replaced with that derived from adenovirus type 35 can elicit very strong HIV-specific cellular immune responses. it can. In addition, toxicity to the liver is reduced, and affinity for dendritic cells is improved.
The modified vaccinia virus ankara is preferably non-proliferating. If the recombinant viral vector is non-proliferating, it will not grow in vivo after administration and is highly safe. Non-propagating vaccinia virus Ankara is a strain obtained by repeated passage and selection many times.For example, Amara, RR et al., Science 292: 69-74 (2001); Amara, RR et al. , J. Viol. 76: 7625-7631 (2002); Robinson, H. et al., Nat. Med. 5: 526-534 (1999).

  Examples of the HIV structural gene to be inserted into the modified vaccinia virus Ankara include those described above. Furthermore, if necessary, an HIV replication control gene may be inserted. Examples of the HIV replication control gene include those described above. The HIV structural gene (optionally a replication control gene) may be inserted into the modified vaccinia virus Ankara for expression in the host cell. The HIV structural gene inserted into the modified vaccinia virus Ankara (if necessary, the replication control gene) is the entire region as long as it is effective as an anti-HIV drug when inserted into the modified vaccinia virus Ankara. It may or may not be a part.

The structural gene of HIV may be inserted at any site of the modified vaccinia virus Ankara, as long as it does not adversely affect the life cycle of the modified vaccinia virus Ankara. For example, it may be inserted at the thymidine kinase (TK) gene site. .
Recombinant viral vectors in which the structural gene of HIV is inserted into a modified vaccinia virus Ankara are known, Amara, RR et al., Science 292: 69-74 (2001); Amara, RR et al., J. Viol .76: 7625-7631 (2002); Robinson, H. et al., Nat. Med. 5: 526-534 (1999).

  The recombinant viral vector is preferably dissolved in a buffer solution such as PBS, physiological saline, sterilized water or the like, and sterilized by filtration with a filter or the like as necessary, and then administered to a subject by injection. Moreover, you may add an additive (For example, an inactivation agent, a preservative, an adjuvant, an emulsifier etc.) etc. to this solution. The recombinant viral vector can be administered intravenously, intramuscularly, abdominally, subcutaneously, intradermally, or may be administered nasally or orally.

The dosage, frequency and frequency of administration of the recombinant viral vector vary depending on the subject's symptoms, age, body weight, administration method, dosage form, etc., for example, usually 10 ⁹ to 10 ¹³ virus particles per adult, preferably A recombinant viral vector in which the HIV structural gene is inserted into a chimeric virus in which the fiber of adenovirus type 5 of 10 ¹¹ to 10 ¹² virus particles is replaced with one derived from adenovirus type 35, and 10 ⁸ to 10 per adult ^The recombinant viral vector in which the HIV structural gene is inserted into ¹⁰ pfu, preferably 10 ⁹ to 10 ¹⁰ pfu of modified vaccinia virus Ankara, may be administered at least once at a frequency at which the desired effect persists.

  In one embodiment of the present invention, an HIV structural gene is inserted into a recombinant virus vector or a modified vaccinia virus Ankara in which the HIV structural gene is inserted into a chimeric virus in which the fiber of adenovirus type 5 is replaced with that derived from adenovirus type 35. The subject is first immunized with either of the recombinant viral vectors into which the is inserted and then boosted with the other. The initial immunization may be either a recombinant adenochimeric virus vector or a recombinant vaccinia virus ankara, but is preferably a recombinant adenochimeric virus vector. The interval between the initial immunization and the booster immunization is suitably about 1 to 3 months, and preferably about 2 months. Further, when a sufficient effect cannot be obtained by one booster, one or more boosters should be given at intervals of about 1 to 3 months, preferably about 2 months. . When booster immunization is performed twice or more, the same type of recombinant virus vector may be used, or different types of recombinant virus vectors may be used. The present invention is not limited to this administration method, and a recombinant viral vector in which an HIV structural gene is inserted into a chimeric virus in which the fiber of adenovirus type 5 is replaced with that derived from adenovirus type 35 and a modified vaccinia A recombinant viral vector in which the HIV structural gene is inserted into the viral ankara may be administered simultaneously.

The recombinant viral vector may further be used in combination with a reverse transcriptase inhibitor and / or a protease inhibitor. Examples of reverse transcriptase inhibitors include azidothymidine (AZT), didanosine (ddl), lambidine (3TC), nevirapine (NVP) and the like. Examples of protease inhibitors include indinavir (IDV), saquinavir (SQV), ritonavir (RTV), nelfinavir (NFV), tenofovir (PMPA) and the like. Those used in Highly Active Anti-retroviral Therapy (HAART) are preferred. Examples of reverse transcriptase inhibitors include azidothymidine (AZT), didanosine (ddl), lambidine (3TC), nevirapine (NVP) and the like. Examples of protease inhibitors include indinavir (IDV), saquinavir (SQV), ritonavir (RTV), nelfinavir (NFV) and the like. Those used in Highly Active Anti-retroviral Therapy (HAART) are preferred. A reverse transcriptase inhibitor and / or a protease inhibitor may be administered to the subject at least once before, after or simultaneously with the administration of the recombinant viral vector. For example, a reverse transcriptase inhibitor and / or a protease inhibitor is administered about 1 to 3 months, preferably about 2 months before administration of the recombinant virus vector. The dosage of reverse transcriptase inhibitors and / or protease inhibitors is the “Anti-HIV Treatment Guidelines” (FY2003, Ministry of Health, Labor and Welfare's Grant-in-Aid for Scientific Research on HIV / AIDS research project. (March) is recommended. As an example of a specific administration schedule, one month after the start of administration of a reverse transcriptase inhibitor and / or a protease inhibitor (for example, tenofovir 20 to 30 mg / kg / day once a day), adenovirus type 5 Recombinant viral vector in which HIV structural gene is inserted into a chimeric virus in which the fiber is replaced with that derived from adenovirus type 35 (for example, 10 ⁹ to 10 ¹³ viral particles once), and 2 months Later, a recombinant viral vector in which the HIV structural gene is inserted into the modified vaccinia virus Ankara is administered (for example, 10 ⁸ to 10 ¹⁰ pfu once). If it is determined that the effect is insufficient, the administration of the recombinant viral vector is repeated.

The recombinant virus vector may be used in combination with other vaccines such as a DNA vaccine. For example, another vaccine is administered to a subject at least once before, after, or concurrently with the administration of the recombinant viral vector. Other vaccines include DNA vaccines containing the rev and env genes of HIV type B HIV strain, subtype B (pCAGrev / env, Jounai N. et al., J. Gene Med. 2003; 5: 609-617) And so on. The dosage of the DNA vaccine is suitably 5 to 30 mg, preferably 10 to 20 mg. As an example of a specific administration schedule, administration of a reverse transcriptase inhibitor and / or a protease inhibitor (for example, tenofovir 20-30 mg / kg / day once a day) starts 0, 1 month, 2 months Later, the DNA vaccine is administered by intramuscular injection (for example, pCAGrev / env is 5 to 30 mg). One month after the completion of the DNA vaccine administration, the adenovirus type 5 fiber is derived from adenovirus type 35. A recombinant viral vector in which the HIV structural gene is inserted into the substituted chimeric virus is administered (for example, once in the amount of 10 ⁹ to 10 ¹³ viral particles), and one month later, the modified vaccinia virus Ankara is administered. A recombinant viral vector into which the HIV structural gene has been inserted is administered (for example, once in an amount of 10 ⁸ to 10 ¹⁰ pfu). If it is determined that the effect is insufficient, the administration of the recombinant viral vector is repeated.

  The anti-HIV drug of the present invention comprising two types of recombinant viral vectors can be expected to be effective as a vaccine for HIV treatment and further as a preventive vaccine.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example, this invention is not limited to these Examples.

Example 1
Experimental methods and materials
Recombinant vector
Ad generation of a recombinant virus in which the E1, E3-deficient non-proliferating adenovirus type 5 fiber is replaced with one derived from adenovirus type 35 and the rev and env genes of HIVIIIB, a subtype B HIV strain, are introduced. It was constructed using a kit (Avior Therapeutics Inc., Seattle, WA, USA) (Shayakhmetov DM, et al., J. Virol. 2000; 74: 2567-2583). Briefly, a 5.2 kbp SalI / PstI fragment containing the CAG promoter-HIV _IIIB rev / env gp160-polyA was isolated from pCAGrev / env (Jounai N. et al., J. Gene Med. 2003; 5: 609-617). A shuttle plasmid (pLHSP) containing adenovirus type 5 (Ad5) sites 22-342, 3523-5790, E. coli ori, and ampicillin resistance gene was obtained from Avior Therapeutics Inc. (Seattle, WA, USA). A blunt-ended 5.2 kbp fragment was subcloned into the blunt-ended EcoRI site of pLHSP to generate a pLHSP-HIV shuttle plasmid. This pLHSP-HIV shuttle plasmid was linearized with PacI and transfected into human fetal kidney (HEK293) cells by the calcium precipitation method together with the E1, E3-deficient chimeric Ad5 / 35 genome to generate recombinant virus Ad5 / 35IIIBenv.
The E1, E3-deficient chimeric Ad5 / 35 genome is a chimeric shuttle plasmid (pRHSP 5/35) in which the fiber gene of adenovirus type 5 is replaced with the fiber gene derived from adenovirus type 35, and the Ad generation kit (Avior Therapeutics Inc., Seattle, WA, USA). Recombinant virus Ad5 / 35IIIBenv was grown in HEK293 cells and purified by repeating the CsCl method (Lieber A. et al., J. Virol. 1996; 70: 8944-8960) twice. The total virus concentration was calculated from the optical density at 260 nm (OD ₂₆₀ ) using the formula 1 OD ₂₆₀ = 1 × 10 ¹² vp / ml.
Non-propagating vaccinia virus Ankara (MVAIIIBenv) expressing HIV env (Amara, RR et al., Science 292: 69-74 (2001); Amara, RR et al., J. Viol. 76: 7625-7631 ( 2002); Robinson, H. et al., Nat. Med. 5: 526-534 (1999)) was provided by Dr. Moss (Laboratory of Viral Diseases, National Institutes of Health, MD).

Animal immune female BALB / c mice (8 weeks old; H-2D ^d ) were purchased from Japan SLC Inc., Shizuoka, Shizuoka-ken, Japan. After initial immunization of mice with 5 × 10 ⁹ vp / mouse of Ad5 / 35IIIBenv (in PBS) by im administration, 5 × 10 ⁹ vp / mouse of Ad5 / 35IIIBenv (in PBS) or 1 × 10 ⁶ pfu / mouse of MVAIIIBenv at 4 weeks Booster immunization was performed by im administration (in PBS) (FIG. 1). Six weeks after the first immunization, blood was collected from the mice, and the following pentamer assay (Anjali N. et al., J. Virol. 2005, Vol. 79, No. 22, p. 14161-14168), ICCS assay (Elzbieta T. et al., The Journal of Immunology, 2002, 169: 5347-5357) and in vivo CTL assay (Michael S. et al., The Journal of Immunology, 2005, 174: 7986-7994) were performed (Fig. 1). In addition, a group in which neither primary immunization nor booster immunization was performed (Negative Control), a group immunized with Ad5 / 35IIIBenv without initial immunization, a group immunized with MVAIIIBenv without initial immunization, and a group immunized with MVAIIIBenv for the first time and booster immunization were also prepared.

Pentamer assay A pentamer is a pentamer of a complex of an antigenic peptide of interest and a major histocompatibility antigen (MHC). When this pentamer is reacted with leukocytes isolated from the spleen of a mouse, it binds only to T cells having antigen-specific receptors, so that antigen-specific T cells can be detected. In this experiment, HIV-specific T cell receptors on the surface of lymphocytes are stained with phycoerythrin (PE) -labeled pentamer, then stained with fluorescein isothiocyanate (FTIC) -labeled CD8 antibody, and FACS is performed. Positive and CD8 positive cells are detected, and the ratio of antigen-specific killer T cells in leukocytes is measured or estimated. The detailed procedure is as follows.
Leukocytes isolated from mouse spleen were suspended in buffer (PBS containing 3% FCS (fetal bovine serum) and 0.1% sodium azide), added with normal mouse serum (final concentration 4%), and blocked at 4 ^° C for 15 minutes. Was done. Next, a FITC-labeled anti-mouse CD8a antibody (Ly-2) (PharMingen) was added and allowed to stand at 4 ° C. for 30 minutes. After washing with buffer, PE-labeled pentamer (H-2Dd / p18 (RGPGRAFVTI) -MHC complex pentamer) (ProImmune, Oxford) was added and incubated at 37 ° C. for 15 minutes. After washing, it was suspended in a fixative (4% formaldehyde-containing PBS) and allowed to stand at 4 ° C. for 10 minutes, and then analyzed by flow cytometry.

ICCS assay Intracellular cytokine staining (ICCS) is an assay method for examining antigen-specific intracellular cytokine-producing cells. First, leukocytes are isolated from the spleen of an immunized mouse and cultured for 24 hours in the presence of an antigenic peptide (for example, p18 peptide of HIV). Thereafter, the produced cytokine is bound to a fluorescently labeled antibody and detected by FACS. In this experiment, CD8-positive and IFN-γ producing cells were detected, and the appearance rate of antigen-specific killer T cells that produced IFN-γ in response to antigenic peptides was estimated. The detailed procedure is as follows.
Leukocytes isolated from the mouse spleen were suspended in RPMI medium supplemented with 10 μg / ml HIV V3 peptide (NNTRKRIQRGPGRAFVTIGKIGN) and incubated at 37 ° C. for 24 hours. After the incubation, the cells were washed with a buffer (3% FCS (fetal bovine serum), 0.1% sodium azide-containing PBS), normal mouse serum (final concentration 4%) was added, and blocking was performed at 4 ° C. for 15 minutes. Next, a PE-labeled anti-mouse CD8 antibody (Ly-2) (PharMingen) was added and allowed to stand at 4 ° C. for 20 minutes. After washing, membrane permeation treatment was performed using Cytofix / CytoPerm Plus kit (PharMingen), and then FITC-labeled anti-mouse IFN-γ antibody (PharMingen) was added, and left at 4 ° C. for 30 minutes. After washing, analysis by flow cytometry was performed.

In Vivo CTL Assay Spleen cells of normal mice were pulsed with HIV p18 peptide and stained with CFSE (5- or 6- (N-Succinimidyloxycarbonyl) -3 ′, 6′-O, O′-diacetylfluorescein). In addition, unpulsed spleen cells were stained with 1/10 concentration of CFSE, mixed together, and injected into the tail vein of immunized mice. After 24 hours, mouse blood or spleen cells were flushed by flow cytometry to determine how much cells with p18 peptide were killed. The detailed procedure is as follows.
Leukocytes isolated from the spleen of non-immunized mice were used as CTL target cells. A part of leukocytes was incubated at 37 ° C. for 1 hour in a medium containing 10 μg / ml HIV V3 peptide (RGPGRAFVTI) to obtain peptide-pulsed target cells. In addition, some of the target cells were not treated with peptide pulses. Next, peptide-pulsed target cells were labeled with 5 μM CSFE and non-pulsed target cells with 0.5 μM CFSE. After washing with PBS, these two kinds of target cells were mixed, and 5 × 10 ⁶ cells were intravenously administered to mice immunized with the vaccine. 24 hours after target cell administration, the spleen was removed from the administered mice, and white blood cells were separated. The leukocytes were analyzed by flow cytometry, and the survival rate of the target cells was measured. The final value calculation method is
% = [1- (number of non-pulse target cells in non-immunized mouse spleen / number of peptide pulse target cells) / (number of non-pulse target cells in immunized mouse spleen / number of peptide pulse target cells)] × 100
It is.

Results The results of the pentamer assay are shown in FIG. Immunization was carried out according to the schedule shown in FIG. 1, and the strength of immunity induced in each administration schedule was analyzed by measuring the number of antigen-specific CD8 ⁺ T cells. The values in the figure indicate (HIV peptide p18-specific CD8 ⁺ T cells) / (total CD8 ⁺ T cells) ^× 100 (%).

Top left; The Negative Control of splenocytes, the percentage of p18-specific CD8 ⁺ T cells of total CD8 ⁺ T cells was 0.07%. Upper row; 8.64% in the group that received a single dose of Ad5 / 35IIIBenv. Upper right: 2.79% in the group that received a single dose of MVAIIIBenv. Bottom left: 5.84% in the group immunized twice with MVAIIIBenv. Lower row: 10.66% in the group immunized twice with Ad5 / 35IIIBenv. Lower right: 21.26% in the group immunized with MVAIIIBenv on week 0 and Ad5 / 35IIIBenv on week 4.

From the above, it is clear that Ad5 / 35IIIBenv has a stronger ability to induce HIV peptide-specific immunity than MVAIIIBenv, and that both Ad5 / 35IIIBenv and MVAIIIBenv induce a stronger specific immune response than single administration by two immunizations Became. In addition, the immunization schedule using Ad5 / 35IIIBenv for primary immunization and MVAIIIBenv for booster immunization yielded the highest proportion of HIV peptide p18-specific CD8 ⁺ T cells, and this schedule is the most effective vaccine. It was suggested that it was expensive.

The results of the ICCS assay are shown in FIG. Immunization with the schedule shown in Fig. 1 and the strength of immunity induced in each administration schedule was analyzed by measuring the proportion of CD8 ⁺ T cells producing IFN-γ by stimulation with HIV V3 peptide (NNTRKRIQRGPGRAFVTIGKIGN) did. The values in the figure indicate (CD8 ⁺ T cells producing IFN-γ upon stimulation with HIV V3 peptide) / (total CD8 ⁺ T cells) ^× 100.

Upper left: In the spleen cells of Negative Control, the ratio of CD8 ⁺ T cells producing IFN-γ by stimulation with HIV V3 peptide in all CD8 ⁺ T cells was 0.75 ± 0.07%. Upper row; 2.87 ± 0.52% in the group administered a single dose of Ad5 / 35IIIBenv. Upper right: 0.99 ± 0.25% in the group that received a single dose of MVAIIIBenv. Bottom left: 1.86 ± 0.14% in the group immunized twice with MVAIIIBenv. Lower row: 5.00 ± 1.46% in the group immunized twice with Ad5 / 35IIIBenv. Bottom right: 8.86 ± 1.77% in the group immunized with MVAIIIBenv at week 0 and Ad5 / 35IIIBenv at week 4.

As described above, Ad5 / 35IIIBenv has a higher ability to induce HIV peptide-specific immunity than MVAIIIBenv, and both Ad5 / 35IIIBenv and MVAIIIBenv have a stronger specific immune response than a single dose as in the case of the pentamer assay. It became clear that was induced. Among the tests performed this time, IFN-γ-producing CD8 ⁺ T cells stimulated with HIV V3 peptide appeared at the highest rate by the immunization schedule using Ad5 / 35IIIBenv for the first immunization and MVAIIIBenv for the booster immunization. It was suggested that the effectiveness is high.

  The results of the in vivo CTL assay are shown in FIG. Immunization was carried out according to the schedule shown in FIG. 1, and the strength of immunity induced in each administration schedule was analyzed by measuring the survival rate of HIV V3 peptide (RGPGRAFVTI) -presenting target cells. In the figure, M1 represents a non-pulse target cell, and M2 represents a peptide pulse target cell. The value in the figure is CTL activity = [1- (number of non-pulsed target cells in non-immunized mouse spleen / number of peptide pulse target cells) / (number of non-pulsed target cells in immunized mouse spleen / number of peptide pulse target cells)] × 100 (%) Is shown.

  Upper left: Negative control CTL activity is 0%. Upper row; CTL activity was 61.1 ± 5.4% in the group administered a single dose of Ad5 / 35IIIBenv. Upper right: 46.0 ± 3.0% in the group that received a single dose of MVAIIIBenv. Lower left: 47.7 ± 2.6% in the group immunized twice with MVAIIIBenv. Lower row: 73.1 ± 6.0% in the group immunized twice with Ad5 / 35IIIBenv. Lower right: 84.5 ± 1.4% in the group immunized with MVAIIIBenv on week 0 and Ad5 / 35IIIBenv on week 4.

  From the above, it was clarified that Ad5 / 35IIIBenv has a stronger ability to induce HIV peptide-specific immunity than MVAIIIBenv, as in the results of the pentamer assay and ICCS assay. Among the tests performed this time, the highest CTL activity was induced by the immunization schedule using Ad5 / 35IIIBenv for the first immunization and MVAIIIBenv for the booster, suggesting that this schedule is the most effective as a vaccine.

[Example 2]
Recombinant vector
MVA vaccine expressing SIVenv and SIVgag: SIVenv (GenBank No. AAA47632.1) and SIVgag gene (GenBank No.AAA47637.1) (Dr. Thomas C Friedrich, Wisconsin National Primate Research Center, Madison, WI 53706 USA (Provided by Dr. Bernard Moss, LVD, NIAID, NIH, Bethesda, MD 20892-0445 USA, PNAS 101: 6641-46, 2004). Inserted into PstI site. BHK21 cells (American Type culture Collection, The Global Bioresource Center, Manassas, VA20108, USA) infected with wild-type MVA (provided by Dr. Bernard Moss) were transferred to lipofectamin (Invitrogen, Carisbad, CA92008). , USA). Recombinant MVA virus was purified with Sucrose (Wako, Tokyo, Japan).
Method for preparing Ad5 / 35 vector expressing SIVenv and SIVgag: SIVenv (GenBank No. AAA47632.1) and SIVgag gene (GenBank No. AAA47637.1) (Dr. Thomas C Friedrich, Wisconsin National Primate Research Center, Madison, WI Ad5 / 35 vector expressing SIVenv and SIVgag genes in the same manner as in Example 1 (paragraph [0036]) was used in HEK293 cells (American Type culture Collection, The Global Bioresource Center, Manassas, VA20108, USA).
SIV is a monkey immunodeficiency virus, and its classification, nature, infection route, and onset are the same as HIV, so it is used as a monkey model for HIV.

Animal immunity (Figure 5)
Five Rhesus monkeys (purchased from the Chinese Academy of Military Medical Sciences Experimental Animal Center) were immunized first with 10 ⁹ pfu of MVA vaccine expressing SIVenv and SIVgag (described above). Two months later, the animals were boosted with Ad5 / 35 vaccine (described above) expressing 10 ¹² particles of SIVenv and SIVgag. Cellular immunity (IFN-γ-ELISpot) was measured 2 weeks after immunization with MVA vaccine and Ad5 / 35 vaccine.

Measurement of Cellular Immunity (IFN-γ-ELISpot) Immunized monkey lymphocytes were stimulated with SIVenv and SIVgag pool peptide (AIDS research and Reference Reagent Program, National Institutes of Health, Rockville, MD 20850, USA) for 24 hours. The number of cells secreting IFN-γ was measured by ELISpot method (Xin et al, Gene Ther. 2005, 12: 1769-1777.).

result
The result of IFN-γ-ELISpot is shown in FIG. The immunization with MVA vaccine, whereas SIV-specific IFN-.gamma.-secreting lymphocytes was 470 cells / 10 ⁶ cells, blanking with Ad5 / 35 Vaccine - the cell numbers by strike more than three times (1708 cells / 10 ⁶ cells). Based on the above, it can be expected that the combination of MVA vaccine and Ad5 / 35 vaccine will provide a high immune effect that cannot be obtained by single vaccine administration, and preventive and therapeutic effects on SIV and HIV. .

  All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

  By using two types of recombinant viral vectors in combination, a strong immune response against HIV can be induced. The anti-HIV drug of the present invention enables effective treatment for the eradication of HIV.

Claims (14)

A recombinant virus vector in which the HIV structural gene is inserted into a chimeric virus in which the fiber of adenovirus type 5 is replaced with that derived from adenovirus type 35, and a group in which the HIV structural gene is inserted into a modified vaccinia virus Ankara An anti-HIV drug characterized by comprising a combination with a recombinant virus vector. The anti-HIV drug according to claim 1, wherein the adenovirus type 5 and the modified vaccinia virus ankara are non-proliferating. The anti-HIV drug according to claim 1 or 2, wherein the structural gene of HIV is an env gene and / or a gag gene. The anti-HIV drug according to any one of claims 1 to 3, wherein an HIV replication control gene is further inserted into at least one of the recombinant viral vectors. The anti-HIV drug according to claim 4, wherein the HIV replication control gene is a rev gene. A recombinant virus vector in which the HIV structural gene is inserted into a chimeric virus in which the fiber of adenovirus type 5 is replaced with that derived from adenovirus type 35, and a group in which the HIV structural gene is inserted into a modified vaccinia virus Ankara The anti-HIV drug according to any one of claims 1 to 5, which is a kit comprising a recombinant virus vector. Recombinant viral vector in which the structural gene of HIV is inserted into a chimeric virus in which the fiber of adenovirus type 5 is replaced with that derived from adenovirus type 35 or recombination in which the structural gene of HIV is inserted into a modified vaccinia virus Ankara The anti-HIV drug according to claim 6, wherein the subject is first immunized with one of the viral vectors and then boosted with the other. The anti-HIV drug according to any one of claims 1 to 7, further comprising a reverse transcriptase inhibitor and / or a protease inhibitor. The anti-HIV drug according to claim 8, wherein the reverse transcriptase inhibitor and / or protease inhibitor is administered to the subject at least once before, after or simultaneously with the administration of the recombinant viral vector. A recombinant virus vector in which the HIV structural gene is inserted into a chimeric virus in which the fiber of adenovirus type 5 is replaced with that derived from adenovirus type 35, and a group in which the HIV structural gene is inserted into a modified vaccinia virus Ankara A method for treating and / or preventing HIV infection, comprising administering a recombinant virus vector to a subject in an amount effective for treatment and / or prevention. Recombinant viral vector in which the structural gene of HIV is inserted into a chimeric virus in which the fiber of adenovirus type 5 is replaced with that derived from adenovirus type 35 or recombination in which the structural gene of HIV is inserted into a modified vaccinia virus Ankara The method according to claim 10, wherein the subject is first immunized with one of the viral vectors and then boosted with the other. The method according to claim 11, wherein the interval between the first immunization and the booster immunization is about 1 to 3 months. The method according to claim 12, wherein the interval between the first immunization and the booster immunization is about 2 months. In the manufacture of anti-HIV drugs, a recombinant viral vector in which the HIV structural gene is inserted into a chimeric virus in which the fiber of adenovirus type 5 is replaced with that derived from adenovirus type 35, and the structure of HIV in the modified vaccinia virus Ankara Use in combination with a recombinant viral vector into which the gene has been inserted.

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