WO1993018791A1 - Vaccine for preventing hiv-infected disease and production thereof - Google Patents

Vaccine for preventing hiv-infected disease and production thereof Download PDF

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Publication number
WO1993018791A1
WO1993018791A1 PCT/JP1993/000327 JP9300327W WO9318791A1 WO 1993018791 A1 WO1993018791 A1 WO 1993018791A1 JP 9300327 W JP9300327 W JP 9300327W WO 9318791 A1 WO9318791 A1 WO 9318791A1
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Prior art keywords
peptide
vaccine
peptides
protein
hiv
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PCT/JP1993/000327
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French (fr)
Japanese (ja)
Inventor
Kenji Okuda
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Inmel Co., Ltd.
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Publication of WO1993018791A1 publication Critical patent/WO1993018791A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16211—Human Immunodeficiency Virus, HIV concerning HIV gagpol
    • C12N2740/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to a vaccine for preventing human immunodeficiency virus (Human Immunodeficiency Virus, hereinafter referred to as "HIV”) infection, and more particularly to a vaccine for preventing HIV infection comprising a plurality of peptides as immunogens.
  • HIV Human Immunodeficiency virus
  • An object of the present invention is to provide a vaccine for preventing HIV infection which has high safety and high preventive and therapeutic effects.
  • the present inventor has conducted intensive studies and found that, unlike vaccines using synthetic peptides of As a vaccine license, a plurality of peptides containing at least a peptide corresponding to a specific region of the HIV envelope (env) protein gp120 and an invariant site of gp120 or the internal structure protein gag ⁇ tide are used as vaccine licenses. It is possible to efficiently produce antibodies without the need for ⁇ ⁇ ! Genetic manipulation, and it is highly safe, and the antibodies can affect each other. It has been found that it is extremely effective as an infectious disease preventive vaccine.
  • the vaccine for the prevention of HIV infection of the present invention is based on the above findings, and more specifically, the V3 region of the HIV (human immunodeficiency virus) envelope gp120 peptide (amino acid position 30 in the envelope protein). 3-322) and a plurality of peptides consisting of the constant site peptide of gpl20 or the constant site peptide of the internal structural protein gag.
  • the V3 region peptide of the HIV envelope gp120 (amino acid positions 303 to 322 in the envelope protein) and the constant site of gpl20 ⁇ the constant site of the peptide or the internal structural protein gag ⁇
  • the present invention provides a vaccine for preventing HIV infection, which comprises a complex comprising a peptide consisting of a peptide and a carrier protein.
  • the V3 region peptide of the HIV envelope gp120 (amino acid positions 303 to 322 in the envelope protein) and the invariant site of gpl20 peptide or the invariant site of the internal
  • a method for the manufacture of a vaccine to prevent HIV infection which includes a step of mixing an adjuvant with a peptide containing a peptide that can be withdrawn11.
  • the V3 region peptide of HIV envelope gp120 (amino acid positions 303 to 322 in the envelope protein) and the constant site peptide of gpl20 or the constant site peptide of internal structural protein gag
  • the present invention provides a method for producing a vaccine for preventing HIV infection, which comprises the step of binding a peptide consisting of the following to a carrier protein.
  • a vaccine for preventing HIV infection of the present invention will be described in detail with reference to the drawings as necessary.
  • amino acid sequence in the present specification, the sequence is described from (left) to C-terminal (right) (for example, in the following amino acid sequence (1), Asn is the N-terminal, Cys is the C-terminal).
  • the “V3 region peptide” corresponds to (all or part of) the sequence of amino acid positions 303 to 322 of the glycoprotein gp120 having a molecular weight of 120,000 that constitutes the envelope protein (env) of HIV. Refers to a peptide (the amino acid number may be different depending on various strains).
  • the amino acid number may be different depending on various strains.
  • Rusche et al. Proc. Nat. Acad. Sci. USA, 85, 3198-320. 2 (1988)).
  • a peptide (24 amino acids) having the following amino acid sequence is preferably used.
  • V3 region peptides (1) to (6) as described above can be used alone as V3 region peptides.
  • a plurality of types of V3 region peptides are mixed. It is preferable to use it because it is effective against a number of mutations in HIV and enhances the preventive effect against multiple types of HIV (and corresponds to a high degree of HIV isomerism).
  • Such preferred combinations include, for example, the following two or three combinations.
  • each peptide having the largest number of moles among them is 1, the mole of the peptide having the smallest number of moles is used.
  • the number is preferably l / 3tU :, more preferably 1/2 or more (particularly 4/5 or more).
  • the “invariant site peptide” includes a peptide (Ho or a part thereof) corresponding to the sequence of amino acid positions 252 to 274 of gp120 constituting the envelope protein (env) of HIV ( ⁇ or part thereof). Et al., Science, J, 1021-1023, 1985); and a constant site peptide having a sequence similar to a part of a protein (pi 7) having a molecular weight of 17,000 that constitutes the internal structural protein gag of Z or HIV ( (About 30 amino acids) (Achour et al., Proc. Nat. Acad. Sci., USA, 87, 7045-704, 1990).
  • a peptide having a sequence similar to pi 7 of gag a peptide having a structure represented by the following formula (7) (a peptide similar to HGP-30) is preferably used.
  • the constant site corresponding to the sequence of gp120 is more specific. Specifically, for example, a peptide having an amino acid sequence as shown below (number of amino acids 2
  • helper T cell activation site peptide constitutes the envelope protein (env) of HIV.
  • a peptide corresponding to! 1 ( ⁇ or part of) amino acid positions 430 to 445 of gpl20 (Cease et al., Proc. Nat. Acad. Sci, USA, 84, 424-4253, 1987). More specifically, for example, in the present invention, a peptide having an amino acid sequence as shown below (15 to 16 ms of amino acids) is preferably used.
  • cysteine (Cys) in parentheses is a melamine that is suitable for binding to a peptide or carrier (carrier) protein having a relatively low cysteine content other than KLH or HSA. Is shown.
  • the above-mentioned various peptides may be obtained from the HIV virus itself, but from the viewpoint of safety and safety, it is preferable to use a synthesis method (particularly, a synthesis method using automatic peptide synthesis).
  • the above-mentioned plural kinds of peptides are usually It can also be used as a vaccine (usually with adjuvant) without using this carrier protein (see J. Immunol., ⁇ 8_, 914-920 (1992)).
  • the carrier protein used for binding to the habutene can be used without any particular limitation.
  • proteins containing a cysteine group are particularly preferably used.
  • Such a cysteine-containing protein is preferably a protein containing 10 or more, preferably 20 or more (more preferably 30 or more) cysteine groups in one molecule (on average).
  • the molecular weight of such a protein is preferably about 100,000 to 500,000 (more preferably, about 300,000 to 400,000).
  • albumin such as human serum albumin (HSA), horseshoe hemocyanin (KLH; molecular weight of about 360 ⁇ ), and the like are preferably used.
  • the method for preparing the complex by binding the carrier protein to a plurality of peptides is not particularly limited, but the binding can be carried out using a suitable acylating agent such as N-hydroxysuccinimide ester. It is.
  • a suitable acylating agent such as N-hydroxysuccinimide ester.
  • an N-hydroxysuccinimide ester such as m-maleimidobenzoyl N-hydroxysuccinimide ester (MBS) is preferably used as the acylating agent.
  • the carrier protein for example, KLH or HSA.
  • the plurality of peptides may be preliminarily mixed and then bound to the carrier protein, or each of the peptides may be bound after binding the carrier protein.
  • One carrier protein complex may be mixed, Further, these two methods may be used in an appropriate combination.
  • the present invention comprises a multifaceted peptide comprising at least the gp120 and / or gag invariant site peptide and at least a V3 region peptide (if necessary, a carrier protein).
  • the molar ratio of the V3 region peptide to the invariant site peptide is 1: 3 to 3: l®g, and even 2: 1
  • the mole number is the same as the mole number of the smallest mole peptide. It is preferable that the peptide having a large amount is 3 mol or less, more preferably 2 mol or less (especially 1.1 to 1.0 mol ⁇ S).
  • the vaccine of the present invention containing the above-mentioned plural kinds of peptides (and, if necessary, a carrier protein) is usually used together with an appropriate adjuvant.
  • adjuvant used with the vaccine of the present invention known ones can be used without particular limitation.
  • adjuvants include aluminum (aluminum sulphide or aluminum adjuvant), Iscom (ISCOM), MDP-PE (oil-containing muramyl tripeptide), and ribosome.
  • Iscom Iscom
  • MDP-PE oil-containing muramyl tripeptide
  • ribosome aluminum sulphide or aluminum adjuvant
  • an antibacterial agent in view of the regulation.
  • the adjuvant is preferably used in an amount of 1.5 to 5 times @g (more preferably 1.5 to 2 times mS.) Of the vaccine of the present invention.
  • the above-mentioned aram is preferably mixed at a rate of about 300 yag / ml.
  • the dose of the vaccine is preferably about 5 ⁇ g / kg per 1 kg of body weight.
  • the vaccine of the present invention As an immunization method using the vaccine of the present invention, it is preferable to administer the vaccine at least four times a day.
  • the interval between the administration days is not particularly limited, but it is preferable to administer the administration on, for example, 0 days, 30 days, 60 days, and 120 days.
  • the above dose is the first dose, and the dose after the second day may be reduced to about 1/3.
  • the vaccine of the present invention consisting of a complex of a plurality of peptides and a carrier protein is administered (with an adjuvant if necessary) at the first time
  • the vaccine of the present invention consisting of a plurality of secreted peptides is administered at the second and subsequent times.
  • the vaccine may be given (with an adjuvant if necessary).
  • Figure 1 is a copy of a photograph showing the results of western blotting of HIV «cells using ⁇ 3 ⁇ 4 & Qing.
  • the above three peptides were synthesized using an automatic peptide synthesizer (43 OA, manufactured by Applied Biosystems, USA). These three types of peptides were first used in a molar ratio of 10: 10: 10: 1 using MBS (m-maleimidobenzoyl-N-hydroxysuccinimidestere) and KLH (keyhole limpet hemocyanin) or HSA (human serum albumin). After conjugation with the product, purification was carried out using a Sephadex G25 column. The above individual peptides (1) to (3) are bound to KLH: ⁇ , the molar ratio was 30: 1.
  • the following peptides were used as gp120 constant site peptides for neutralizing antibody production.
  • the cysteine in the cocoon indicates a sequence synthesized when the cysteine is combined with another peptide or carrier carrier protein).
  • Equal amounts (equal weight) of the two components obtained above ie, the V3 peptide carrier conjugate and the invariant site peptide Z helper T cell-derived peptide-one carrier conjugate
  • the heron was immunized by administering 2 Ozg / kg of the carrier-conjugated vaccine, and the immunization was performed four times at one week intervals.
  • the measurement by the ELISA method was carried out by the method described in the report (AIDS,, 765-766 (1989) and AIDS, Mutual, 1140-1141 (1991)). That is, the peptide or that of the carrier-binding peptide was coated on a 96-well microplate. Here, a mixture of three peptides from different sites was used. Other antigens were used with each of the peptides. (The C 21 E peptide corresponds to the constant site of gp120, and the HGP-30 peptide corresponds to the protein at the central gag site of the virus).
  • HIV IIIB, III RF and IIIMN strains
  • CD4 + peripheral blood T cells (2X10 5 CELLz ⁇ E Le) along with the plate of 24-well 37, in the presence of Qingqing.
  • the cells were observed at C for 5 days, and the appearance of fused cells was observed.
  • Vn / Vo (number of cells fused in ⁇ ⁇ / ⁇ ) /
  • Neutmalizeing activity was determined by the number of reciprocal dilutions corresponding to the neutralizing activity. As a result, fusion inhibition of 70% or more was observed. At this time, the antibody ability was examined using the serum dilution number.
  • the inhibition of HIV replication by antisera was examined by another method, namely, the activity of inhibiting the ability to synthesize viral proteins. This examination was performed according to the method described in the literature (J. Virol, 6 ⁇ ., 2622-2628 (1988), Proc. Natl Acad. "Sic. USA, 88., 2249-2253 (1991)). .
  • the CEM cells were combined with the HIV-1 virus37. After incubating with C for 4 hours and washing well, the infected CEM drops were cultured in a medium containing a final mg of 2.5% serum. After 5 days incubation, the cell-free supernatant of the medium was collected and filtered through a 0.45 m filter. Virus concentration was determined using a commercially available p24 capture reagent (Coulter Co.).
  • IL-2 interleukin-12
  • peripheral blood lymphocytes were cultured in a 96-well microplate well with each of the final 2 ⁇ 10 lO zg / m 1 antigens.
  • Recombinant gp120 obtained from the virus ( ⁇ ⁇ ⁇ ) and the Ills virus gene (rgpl20, which was provided by San-Danisei Co., Ltd. of Yokohama) was used for heat-sterilized purified HIV-1. Represents the HIV envelope prepared by protein and fly)
  • HIV-infected Mo It-4 cells were solubilized with sodium dodecyl sulfate (SDS), and each solubilized sample was electrotransferred and transferred to nitrocellulose filter paper. Next, each filter paper was stained with a peroxynase-labeled anti-Peacock (or anti-human) IgG antibody.
  • SDS sodium dodecyl sulfate
  • Table 1 above shows the antibody titers of the vaccine immune sera obtained from the egrets against the respective synthetic peptides.
  • “KLH—mixed” indicates the multivalent actin of the present invention, and other licenses are KLH and its V3 region peptide (or C21E and HGP-30 peptide). This shows the conclusion. ⁇ " ⁇ " means not tested.
  • Peptides Mean reciprocal titer of antibody ( ⁇ ')
  • Table 2 above shows the antibody titers of JfiL purified from humans immunized with the vaccine. As shown in Table 2, a high antibody titer can be obtained with ELISA when immunized with the vaccine of the present invention.
  • Table 3 above shows the HIV neutralizing antibody titers using Pergum antiserum. ⁇
  • the sera obtained from the heron are the same as those shown in Table 1.
  • Table 4 above shows the HIV neutralizing antibody titers using human antisera. 3 ⁇ 43 ⁇ 4 & QI obtained from humans are the same as those shown in Table 2.
  • Virus growth inhibition was performed by detecting free ⁇ 24 protein in the culture supernatant. Also in this case, the results induced by the vaccine of the present invention (combination of multiple peptides) were observed (see Table 5 below).
  • IB # 5 shows the activity of inhibiting the production of HIV-1 p24 protein by rabbit heron serum. ⁇ Serum from herons is the same as in Table 1. "NTj stands for not tested.
  • Table 6 above shows the activity of HIV-1p24 protein production inhibition by human serum. Serum from humans is the same as in Table 2. “NT” means not tested.
  • the vaccine of the present invention It was found that the sera obtained by immunization had strong ability to suppress virus production. From the above results, it was found that the sera obtained by using the vaccine of the present invention certainly inhibited the growth of the virus.
  • Table 7 above shows the production of antigen-specific IL-12 by peripheral leukocytes from humans (individuals) who received the vaccine.
  • the values in the table are the average soil standard deviation (SD) of 4 to 6 samples.
  • FIG. 2 shows the results of Western blotting (copy of the photo).
  • FIG. 2 above shows Western blotting of HIV-infected cells using antiserum.
  • FIG. 2 shows the antibody produced in the egret, and the meaning of each lane is as follows.
  • FIG. 2 shows an antibody produced in humans, and the meaning of each sample (each lane) used here is the same as in (A) above.
  • the results of this western-producing show that the antibody against the gp120 constant site peptide bound to an antigen consisting of a molecule having a molecular weight of 120 K daltons. That is, it was confirmed that the antibody produced by the vaccine of the present invention reacts with the protein of HIV envelope.
  • H ⁇ As described above, according to the present invention, it is an effective vaccine not only for animals but also for humans. Since it does not use the HIV virus itself, it has high safety, high prevention efficiency, and humoral immunity. At the same time, a multivalent actin capable of activating cell-mediated immunity is produced.

Abstract

A vaccine which can cope with a variation in the antigenicity of various HIVs is produced from three V3 loop peptides, two constant-region peptides and one helper peptide which activates cell-mediated immunological competence each by utilizing a method by which the immunogenicity is enhanced. It is possible to prepare a vaccine by determining the V3 peptides of, for example, Thailand, Japan and other areas by the PCR method. The use of this vaccine makes possible for an antibody against the V3 region, an antibody against the e^_n^_v^_ constant region and an antibody against the g^_a^_g^_ region to control the growth of HIV from a wide variety of aspects.

Description

H I V感染症予防ワクチンおよびその製造法 分野  Preventive vaccine for HIV infection and its production
本発明は、 ヒト免疫不全ウィルス （Human Immunodeficiency Virus、 以下「H IV」という）感染症予防ワクチンに関し、 より詳しくは、 複 のペプチドを 免疫原として含む H I V感染症予防ワクチンに関する。  The present invention relates to a vaccine for preventing human immunodeficiency virus (Human Immunodeficiency Virus, hereinafter referred to as "HIV") infection, and more particularly to a vaccine for preventing HIV infection comprising a plurality of peptides as immunogens.
棚  Shelf
背景技術  Background art
H I V感染症、 いわゆるエイズ（A I D S： Acquired Immunodeficiency Synd rome、 後天的免疫不全症候群）のまん延は社会的にも極めて重大な問題となって 来ており、 その予防ないし治療薬の開発が切望されている。  The spread of HIV infection, the so-called AIDS (Acquired Immunodeficiency Syndrome), has become a very serious problem in society, and the development of preventive or therapeutic drugs is eagerly awaited. .
δέ*、 H I V感染症予防ワクチンとしてはいくつかの提案がされており、 また、 そのうち数種のワクチン （例えば g a gの 1部に対応した合成ぺプチド HGP— 30を用いたワクチン） については、 臨床試験が開始されており、 また、 ヘルパ 一ペプチド、 HGP— 30， CE 2 1又は V 3ペプチドをそれそれ別々に免疫す ることにより、 感染防御能を得るか又はそれを補助することに関しては、 それぞ れの報告がある。 しかしながら、 H I V感染症予防ワクチンとして安全性が高く、 しかも予防効果の高い^的 フクチンは未だ^^されていない。 発明の開示  δέ *, several proposals have been made as vaccines to prevent HIV infection, and several vaccines (for example, vaccines using synthetic peptide HGP-30 corresponding to a part of gag) Testing has begun, and immunization with the helper peptide, HGP-30, CE21, or V3 peptide, separately, will provide protection against infection or support it. There are reports for each. However, there is still no effective fuctin that is highly safe as a vaccine for preventing HIV infection and has a high protective effect. Disclosure of the invention
本発明の目的は、 安全性が高く、 しかも予防効果及び治療効果が高い H I V感 染症予防ワクチンを提供することにある。  An object of the present invention is to provide a vaccine for preventing HIV infection which has high safety and high preventive and therapeutic effects.
本発明者は鋭意研究の結果、 の合成ぺプチドを用いたワクチンと異なり、 H I Vエンベロープ (env) タンパク質 gp 1 20の特定の領域に対応するぺ プチドと、 gp 1 20又は内部構造夕ンパク g a gの不変部位ぺブチドとを少く とも含む複数種のぺブチドをワクチン用免 ¾ として用いることが、 ¾¾!な遺伝 子操作を必要とせずに効率よく抗体を産生させることを可能とし、 安全性が高く、 しかも された抗体がお互いに影響し合う相 «I果の高 Lヽ H I V感染症予防ヮ クチンとして極めて有効性が強いことを見出した。 The present inventor has conducted intensive studies and found that, unlike vaccines using synthetic peptides of As a vaccine license, a plurality of peptides containing at least a peptide corresponding to a specific region of the HIV envelope (env) protein gp120 and an invariant site of gp120 or the internal structure protein gag ぺ tide are used as vaccine licenses. It is possible to efficiently produce antibodies without the need for 遺 伝! Genetic manipulation, and it is highly safe, and the antibodies can affect each other. It has been found that it is extremely effective as an infectious disease preventive vaccine.
本発明の H I V感染症予防ワクチンは、上記知見に基くものであり、 より詳し くは、 H IV (ヒト免疫不全ウィルス）エンベロープ gp 1 2 0の V3領域ぺブ チド （エンベロープタンパク質内のアミノ酸位置 30 3〜322) と、 gp l 2 0の不変部位べプチド又は内部構造タンパク質 gagの不変部位べプチドとから なる複数のぺプチドとを免 ¾Μとして含むことを とするものである。  The vaccine for the prevention of HIV infection of the present invention is based on the above findings, and more specifically, the V3 region of the HIV (human immunodeficiency virus) envelope gp120 peptide (amino acid position 30 in the envelope protein). 3-322) and a plurality of peptides consisting of the constant site peptide of gpl20 or the constant site peptide of the internal structural protein gag.
更に、本発明によれば、 H I Vエンベロープ gp 1 2 0の V3領域べプチド (エンベロープタンパク質内のアミノ酸位置 303〜3 22) と、 gp l 20の 不変部位ぺプチド又は内部構造タンパク質 gagの不変部位ぺプチドとからなる ¾ ^のぺプチドと、担体夕ンパク質とを含む複合体からなることを とする H IV感染症予防ワクチンが提供される。  Further, according to the present invention, the V3 region peptide of the HIV envelope gp120 (amino acid positions 303 to 322 in the envelope protein) and the constant site of gpl20 {the constant site of the peptide or the internal structural protein gag} The present invention provides a vaccine for preventing HIV infection, which comprises a complex comprising a peptide consisting of a peptide and a carrier protein.
更に、 本発明によれば、 H I Vエンベロープ gp 1 2 0の V3領域ペプチド (エンベロープタンパク質内のアミノ酸位置 303〜322) と、 gp l 2 0の 不変部位ぺプチド又は内 Si^造タンパク質 gagの不変部位ぺプチドとからなる 撤のぺプチドを含む免 ¾11と、 アジュバントとを混合する段階を含むことを特 徴とする H I V感染症予防ワクチンの製造法が される。  Further, according to the present invention, the V3 region peptide of the HIV envelope gp120 (amino acid positions 303 to 322 in the envelope protein) and the invariant site of gpl20 peptide or the invariant site of the internal A method for the manufacture of a vaccine to prevent HIV infection, which includes a step of mixing an adjuvant with a peptide containing a peptide that can be withdrawn11.
更に、 本発明によれば、 H IVエンベロープ gp 1 2 0の V3領域ペプチド (エンベロープタンパク質内のアミノ酸位置 303〜322) と、 gp l 20の 不変部位ぺプチド又は内部構造タンパク質 gagの不変部位ぺプチドとからなる のぺブチドと、担体タンパク質とを結合させる段階を含 ことを 2^とする H I V感染症予防ワクチンの製造法が提供される。 以下、 本発明の HIV感染症予防ワクチンを、 必要に応じて図面を参照しつつ 詳細に説明する。 Further, according to the present invention, the V3 region peptide of HIV envelope gp120 (amino acid positions 303 to 322 in the envelope protein) and the constant site peptide of gpl20 or the constant site peptide of internal structural protein gag The present invention provides a method for producing a vaccine for preventing HIV infection, which comprises the step of binding a peptide consisting of the following to a carrier protein. Hereinafter, the vaccine for preventing HIV infection of the present invention will be described in detail with reference to the drawings as necessary.
なお、 本明細書におけるアミノ酸配列の表記においては、 (左側） から C末端 （右側） へ記載するものとする （例えば、 以下のアミノ酸配列 （1)にお いては、 Asnが N末端、 Cysが C末端である） 。  In the description of the amino acid sequence in the present specification, the sequence is described from (left) to C-terminal (right) (for example, in the following amino acid sequence (1), Asn is the N-terminal, Cys is the C-terminal).
(V 3領域ペプチド）  (V3 region peptide)
本発明において、 「V3領域ペプチド」 とは、 H IVのエンベロープタンパク 質（env)を構成する分子量 12万の糖タンパク質 gp 120のアミノ酸位置 303〜322の配列 （の全部又は 1部） に対応するペプチドをいう （各種株に より、 そのアミノ酸番号は異なる場合がある）。 この V3領域ペプチドに関して は、 文献（Paiierら、 Proc. Nat. Acad. Sci. USA，& 5， 1932-1336 (1988) ； Ruscheら、 Proc. Nat. Acad. Sci. USA, 85, 3198-320 2 (1988) )を参照することができる。  In the present invention, the “V3 region peptide” corresponds to (all or part of) the sequence of amino acid positions 303 to 322 of the glycoprotein gp120 having a molecular weight of 120,000 that constitutes the envelope protein (env) of HIV. Refers to a peptide (the amino acid number may be different depending on various strains). For the V3 region peptide, see the literature (Paiier et al., Proc. Nat. Acad. Sci. USA, & 5, 1932-1336 (1988); Rusche et al., Proc. Nat. Acad. Sci. USA, 85, 3198-320. 2 (1988)).
より具体的には例えば、 本発明においては、 以下に示すようなアミノ酸配列を 有するペプチド （アミノ酸数 24個 ) が好ましく用いられる。  More specifically, for example, in the present invention, a peptide (24 amino acids) having the following amino acid sequence is preferably used.
Asn- Asn- Thr- Arg- Lys- Ser- lie- Arg- I丄 e- Gln- Arg- Gly- Pro- Gl - Arg- Ala- Phe- Val- Asn- Asn- Thr- Arg- Lys- Ser- lie- Arg- I 丄 e- Gln- Arg- Gly- Pro- Gl-Arg- Ala- Phe- Val-
Thr- lie- Gly- Lys- lie- Gly- Cys (1) Thr- lie- Gly- Lys- lie- Gly- Cys (1)
(—文字表記： NNTRKSIR I QRGPGRAFVT IGKIGC)  (—Letter notation: NNTRKSIR I QRGPGRAFVT IGKIGC)
(HIV- Illsないし LAIの配列に基く）  (Based on the sequence of HIV-Ills or LAI)
Asn- Asn- Thr- Arg- Lys- Ser- lie- Thr- L s- Gl y- Pro- Gl - Arg- Val- lie- Tyr- Ala- Thr- Asn- Asn- Thr- Arg- Lys- Ser- lie- Thr- L s- Gly- Pro- Gl-Arg- Val- lie- Tyr- Ayr- Thr-
Gly- Gin- lie- lie- Gly- Cys (2) Gly- Gin- lie- lie- Gly- Cys (2)
(—文字表記： NNTRKSITKGPGRVIYATGQI IGC)  (—Letter notation: NNTRKSITKGPGRVIYATGQI IGC)
(HIV- inRFの配列に基く） (Based on HIV-in RF sequence)
T r- Asn- Lys- Arg- L s- Arg- lie- His- lie- Gl - Pro- Gl - Arg- Ala- Phe- Tyr- Thr- Thr-T r- Asn- Lys- Arg- L s- Arg- lie- His- lie- Gl-Pro- Gl-Arg- Ala- Phe- Tyr- Thr- Thr-
Lys- Asn- lie- Ile- Gly- Cys (3) Lys- Asn- lie- Ile- Gly- Cys (3)
(—文字表記： YNKRKR I H I GPGRAFYTTKN IIGC  (—Letter notation: YNKRKR I H I GPGRAFYTTKN IIGC
(HIV- IIIMNの配列に基く）  (Based on the sequence of HIV-IIIMN)
(「HIV日本型」の配列に基くアミノ酸画） ··· (4) (Amino acid sequence based on "HIV Japanese type" sequence) ··· (4)
Cys- Thr- Arg- Pro- Ser- Asn- As - Thr- Arg- Thr- Ser- lie- Thr- lie- Gly- Pro- Gly- Gln- Val- Phe- Tyr- Arg- Thr- Gly- Asp- lie- 11 e-Cys- Thr- Arg- Pro- Ser- Asn- As-Thr- Arg- Thr- Ser- lie- Thr- lie- Gly- Pro- Gly- Gln- Val- Phe- Tyr- Arg- Thr- Gly- Asp- lie- 11 e-
Gl y- Asp- lie- Arg- Lys- Ala- Tyr- C s (5) Gly-Asp-lie-Arg-Lys-Ala-Tyr-Cs (5)
(—文字表記： CTRPSNNTRTSITIGPGQVFYRTGDI IG  (—Letter notation: CTRPSNNTRTSITIGPGQVFYRTGDI IG
DIRKAYC)  DIRKAYC)
(「HIVタイ A型」の IB ^に基くアミノ酸配列）  (Amino acid sequence based on IB ^ of "HIV Thailand A type")
C s- Thr- Arg- Pro- Asn- Asn- Asn- Thr- Arg- L s- Ser- lie- His- Leu- Gly- Pro- Gl - Gin- Ala- Trp- Tyr- Thr- Thr- Gly- Gin- 1,1 e- 11 e- Gl y- Asp- lie- Arg- Gin- Ala- His- Cys ··· (6) C s- Thr- Arg- Pro- Asn- Asn- Asn- Thr- Arg- L s- Ser- lie- His- Leu- Gly- Pro- Gl-Gin- Ala- Trp- Tyr- Thr- Thr- Gly- Gin- 1,1 e- 11 e- Gly-Asp-lie-Arg- Gin- Ala-His-Cys (6)
(—文字表記： CTRPN腿 TRKSIHLGPGQAWYTTGQIIG  (—Letter notation: CTRPN thigh TRKSIHLGPGQAWYTTGQIIG
DIRQAHC)  DIRQAHC)
(「HIVタイ B jの配列に基くアミノ酸配列）  ("Amino acid sequence based on the sequence of HIV Thailand Bj")
上記したような（1)〜（6)のペプチドはそれぞれ単独で V3領域ペプチド として用いることも可能であるが、 本発明者の知見によれば、 複数種類の V3領 域べプチドを混合して用いることが、 H I Vの数々の変異に対して 効果が認 められ、複数種の HI Vに対する予防効果を高める点（及び HIVの高度異性に 対応する点）から好ましい。.このような好ましい組合せとしては、 例えば以下に 示すような 2ないし 3種の組合せが挙げられる。  The peptides (1) to (6) as described above can be used alone as V3 region peptides. However, according to the findings of the present inventors, a plurality of types of V3 region peptides are mixed. It is preferable to use it because it is effective against a number of mutations in HIV and enhances the preventive effect against multiple types of HIV (and corresponds to a high degree of HIV isomerism). Such preferred combinations include, for example, the following two or three combinations.
(1) と （2) と （3) ( IIIB + IIIRF+ II (1) と （3) と （4) ( IIIB + ΠΙΜΝ+日本型） (1) and (2) and (3) (IIIB + IIIRF + II (1), (3) and (4) (IIIB + ΠΙ ΜΝ + Japanese type)
(4) と （5) (曰本型 +タイ Α型）  (4) and (5) (this type + Thailand Α type)
(1) と （5) と （6) (III B +タイ A型 +タイ B型）  (1), (5) and (6) (III B + Thai A type + Thai B type)
上記したような V 3領域べプチドを複 組合せて用 tヽる場合、 これらのうち で最もモル数の多いぺプチドのモル数を 1とした際に、 最もモル数の小さいぺブ チドのモル数は l/3tU:であることが好ましく、 1/2以上 （特に 4/5以上） であることが更に好ましい。 本発明においては、 通常、 各ペプチドをほぼ等モル (例えばほぼ 1： 1、 又はほぼ 1 : 1 : 1)のモル数で用いることが好ましい。 (不変部位ペプチド）  In the case of using a combination of two or more V3 region peptides as described above, when the number of moles of the peptide having the largest number of moles among them is 1, the mole of the peptide having the smallest number of moles is used. The number is preferably l / 3tU :, more preferably 1/2 or more (particularly 4/5 or more). In the present invention, it is usually preferable to use each peptide in a molar number of about equimolar (for example, about 1: 1 or about 1: 1: 1). (Invariant site peptide)
本発明において、 「不変部位ペプチド」 としては、 H IVのエンベロープタン パク質 （env)を構成する上記 gp 120のアミノ酸位置 252〜274の配 列 （の ^又は 1部） に対応するペプチド （Hoら、 Science, J , 1021-1023, 1985) ；および Z又は HI Vの内部構造タンパク質 gagを構成する分子量 1万 7千のタンパク質 （pi 7)の一部と類似の配列を有する不変部位ペプチド （ァ ミノ酸 30個程度) が用いられる （Achourら、 Proc. Nat. Acad. Sci, USA, 87, 7 045-704, 1990)。  In the present invention, the “invariant site peptide” includes a peptide (Ho or a part thereof) corresponding to the sequence of amino acid positions 252 to 274 of gp120 constituting the envelope protein (env) of HIV (^ or part thereof). Et al., Science, J, 1021-1023, 1985); and a constant site peptide having a sequence similar to a part of a protein (pi 7) having a molecular weight of 17,000 that constitutes the internal structural protein gag of Z or HIV ( (About 30 amino acids) (Achour et al., Proc. Nat. Acad. Sci., USA, 87, 7045-704, 1990).
後者の gagの pi 7と類似の配列を有するペプチドとしては、 下記式 (7) に示す構造を有するペプチド （HGP— 30類似のペプチド） が好ましく用いら れ *&o  As the peptide having a sequence similar to pi 7 of gag, a peptide having a structure represented by the following formula (7) (a peptide similar to HGP-30) is preferably used.
86 87  86 87
93 94  93 94
H2 N-Try -Ser -Val -His -Gin一 Arg -Leu一 Asp -Val \ H 2 N-Try -Ser -Val -His -Gin-1 Arg -Leu-1 Asp -Val \
102 Lys  102 Lys
/ lie— Lys -Glu -lie -Ala -Glu -Lys -Thr一 Asp / ··· (7) / lie— Lys -Glu -lie -Ala -Glu -Lys -Thr-One Asp / ... (7)
GlU , 115 GlU, 115
\ Glu -Glu -Gin -Asn -Lys一 Ser -Lys -Lys -Lys -Ala一 C00H —方、 上記 g p 120の配列に対応する不変部位ぺブチドとしては、 より具体 的には例えば、 以下に示すようなアミノ酸配列を有するペプチド （アミノ酸数 2\ Glu-Glu-Gin-Asn-Lys-Ser-Lys-Lys-Lys-Ala-C00H — On the other hand, the constant site corresponding to the sequence of gp120 is more specific. Specifically, for example, a peptide having an amino acid sequence as shown below (number of amino acids 2
4個 のペプチドが好ましく用いられる。 Four peptides are preferably used.
Val- Gin- Cys- Thr- His- Gly- lie- Arg- Pr o- Val- Val- Ser- Thr- Gin- Leu- Leu- Leu- Asn- Gly- Ser- Leu- Ala- Glu- Glu- Glu- (Cys) (C Val- Gin- Cys- Thr- His- Gly- lie- Arg- Pr o- Val- Val- Ser- Thr- Gin- Leu- Leu- Leu- Asn- Gly- Ser- Leu- Ala- Glu- Glu- Glu -(Cys) (C
21E) ······ (8) 21E) (8)
(—文字表記： VQCTHGIRPWSTQLLLNGSLAEEE (C) ) (ヘルパー T細胞活性化部位ぺプチド） 本発明において、 「ヘルパー T細胞活性化部位ペプチド」 とは、 H IVのェン ベロープタンパク質 （env) を構成する gpl20のアミノ酸位置 430〜4 45の !1 (の^又は 1部） に対応するペプチドをいう （Cease ら、 Proc. Na t. Acad. Sci, USA, 84, 424-4253, 1987)。 より具体的には例えば、 本発明にお いては、 以下に示すようなアミノ酸配列を有するペプチド （アミノ酸数 15個な いし 16個 ms)が好ましく用いられる。 (—Letter notation: VQCTHGIRPWSTQLLLNGSLAEEE (C)) (Helper T cell activation site peptide) In the present invention, the “helper T cell activation site peptide” constitutes the envelope protein (env) of HIV. A peptide corresponding to! 1 (^ or part of) amino acid positions 430 to 445 of gpl20 (Cease et al., Proc. Nat. Acad. Sci, USA, 84, 424-4253, 1987). More specifically, for example, in the present invention, a peptide having an amino acid sequence as shown below (15 to 16 ms of amino acids) is preferably used.
Cys) - lie- Asn- Met- Trp- Gin- Glu- Leu- Gl - Lys- Ala- Met- Tyr- Ala- Pro- Pro- (Cys)  Cys)-lie- Asn- Met- Trp- Gin- Glu- Leu- Gl-Lys- Ala- Met- Tyr- Ala- Pro- Pro- (Cys)
(9)  (9)
(—文字表記： (C) I NMWQ E L GK AM Y A P P (C) )  (—Letter notation: (C) I NMWQ E L GK AM Y A P P (C))
上記式 (8)ないし （9) において、 かっこ内のシスティン （Cys)は、 KLH又は HS A以外のシスティン含量が比較的少ないぺプチド又はキヤリァ (担体） タンパク質との結合用に好適な醇!1を示す。  In the above formulas (8) to (9), cysteine (Cys) in parentheses is a melamine that is suitable for binding to a peptide or carrier (carrier) protein having a relatively low cysteine content other than KLH or HSA. Is shown.
上記した各種ぺプチドは H I Vウイルス自身から得てもよいが、 安全性および «の点からは、 合成法（特に自動ペプチド合 «による合成法） を用いること が好ましい。  The above-mentioned various peptides may be obtained from the HIV virus itself, but from the viewpoint of safety and safety, it is preferable to use a synthesis method (particularly, a synthesis method using automatic peptide synthesis).
ズ担体タンパク質） Carrier protein)
本発明においては、 上記した複数種のペプチドは、 通常はこの担体タンパク質 とともに用いられるが、 この担体タンパク質を用いずに （通常アジュパントとと もに用いて） ワクチンとすることも可能である （J. Immunol. , Η8_, 914-920 (199 2)参照） 。 このような^、 本発明においては、 MAP (マルチブル アンティ ゲン ペプチド）法又は BLD (ブランチト' リジン オリゴマー） 法を用いる ことが好ましい。 In the present invention, the above-mentioned plural kinds of peptides are usually It can also be used as a vaccine (usually with adjuvant) without using this carrier protein (see J. Immunol., Η8_, 914-920 (1992)). In the present invention, it is preferable to use the MAP (multiple antigen peptide) method or the BLD (branched lysine oligomer) method.
本発明においては、 投にハブテンとの結合に用いられる担体タンパク質を特 に制限なく用いることが可能であるが、 なかでも、 特にシスティン基含有^ンパ ク質が好ましく用いられる。 このようなシスティン含有タンパク質は、 その 1分 子中に （平均値で） システィン基を 1 0個以上、 好ましくは 2 0個以上 （更には 30個以上）含むタンパク質が好ましい。 このようなタンパク質の分子量は 1 0 万〜 5 00万程度 （更には 3 0万〜 40 0万程度） であることが好ましい。  In the present invention, the carrier protein used for binding to the habutene can be used without any particular limitation. Among them, proteins containing a cysteine group are particularly preferably used. Such a cysteine-containing protein is preferably a protein containing 10 or more, preferably 20 or more (more preferably 30 or more) cysteine groups in one molecule (on average). The molecular weight of such a protein is preferably about 100,000 to 500,000 (more preferably, about 300,000 to 400,000).
このようなタンパク質としては、 具体的には例えば、 ヒト血清アルブミン （H SA)等のアルブミン、 カブトガ二へモシァニン （KLH ;分子量約 360^) 、 などが好ましく用いられる。  As such a protein, specifically, for example, albumin such as human serum albumin (HSA), horseshoe hemocyanin (KLH; molecular weight of about 360 ^), and the like are preferably used.
本発明において、 上記担体タンパク質を複数種のぺプチドと結合させて複合体 を調製する方法は特に限定されないが、 N—ヒドロキシサクシイミドエステル等 の適当なァシル化剤を用いて結合することが可能である。 本発明において KLH を担体タンパク質として用いる際には、 上記ァシル化剤として m—マレイミド ベンゾィルー N—ヒドロキシサクシイミドエステル （MB S) 等の N—ヒドロキ シスクシンイミドエステルが好ましく用いられる。  In the present invention, the method for preparing the complex by binding the carrier protein to a plurality of peptides is not particularly limited, but the binding can be carried out using a suitable acylating agent such as N-hydroxysuccinimide ester. It is. When KLH is used as the carrier protein in the present invention, an N-hydroxysuccinimide ester such as m-maleimidobenzoyl N-hydroxysuccinimide ester (MBS) is preferably used as the acylating agent.
担体タンパク質とペプチドとを結合させる際には、 担体タンパク質 （例えば K LH又は HSA) 1モルに対して、 ペプチドを 5モル以上髓、 更には 1 0〜4 When binding the carrier protein to the peptide, 5 mol or more of the peptide is added to 1 mol of the carrier protein (for example, KLH or HSA).
0モル as (特に ι ο〜3 οモル as) 用いることが好ましい。担体タンパク質 と複数種のぺブチドとを結合させる場合、 複数種のぺブチドをあらかじめ混合物 としてから担体夕ンパク質と結合させてもよく、 またそれぞれ担体夕ンパク質を 結合させた後にそれぞれのぺプチド一担体夕ンパク質複合体を混合してもよく、 またこれら 2通りの方法を適宜組合せて用いてもよい。 It is preferable to use 0 mol as (particularly lο to 3ο mol as). When binding a carrier protein to a plurality of peptides, the plurality of peptides may be preliminarily mixed and then bound to the carrier protein, or each of the peptides may be bound after binding the carrier protein. One carrier protein complex may be mixed, Further, these two methods may be used in an appropriate combination.
(ワクチン）  (Vaccine)
上述したように、 gp 1 2 0及び/又は gagの不変部位ペプチドと、 V3領 域ぺプチドとを少くとも含む複顏のぺプチド （必要に応じて担体夕ンパク質） とからなる本発明のワクチンにおいては、 V3領域ぺプチドと不変部位ぺプチド とのモル比（又は V3領域ペプチド一担体タンパク質結^?と、 不変部位ぺプチ ドー担体タンパク質結合物とのモル比）は、 1： 3〜3： l®g、 更には 2： 1 As described above, the present invention comprises a multifaceted peptide comprising at least the gp120 and / or gag invariant site peptide and at least a V3 region peptide (if necessary, a carrier protein). In vaccines, the molar ratio of the V3 region peptide to the invariant site peptide (or the molar ratio of the V3 region peptide-carrier protein bond to the invariant site peptide carrier protein conjugate) is 1: 3 to 3: l®g, and even 2: 1
〜ι： 2離、 特にはおよそ 1： ims (すなわち、 一方を 1とした際に、 他方 が 0. 9〜： L . であることが好ましい。 ~ Ι: 2 apart, especially about 1: ims (that is, when one is 1, the other is preferably 0.9 to: L.).
本発明のワクチンがヘルパー T細胞活性化部位べプチドを含む場合（すなわち、 3種ないし 4種のペプチドを含む場合） においても、 最もモル数の小さいぺプチ ド 1モルに対して、 最もモル数の大きいペプチドは 3モル以下、 更には 2モル以 下（特に 1 . 1〜1. 0モル §S)であることが好ましい。  Even when the vaccine of the present invention contains a helper T cell activation site peptide (ie, contains three or four peptides), the mole number is the same as the mole number of the smallest mole peptide. It is preferable that the peptide having a large amount is 3 mol or less, more preferably 2 mol or less (especially 1.1 to 1.0 mol §S).
(アジュバント）  (Adjuvant)
上記した複数種のペプチド （ 要に応じて、 更に担体タンパク質） を含む本発 明のワクチンは、通常、 適当なアジュバントとともに用いられる。  The vaccine of the present invention containing the above-mentioned plural kinds of peptides (and, if necessary, a carrier protein) is usually used together with an appropriate adjuvant.
本発明のワクチンとともに用いられるアジュバントとしては、 公知のものを特 に制限なく使用することが可能である。 このようなアジュパントの例としてはァ ラム （ス 化アルミニウムないしアルミニウム ·ァジュパント）、 イスコム （I SCOM)、 MDP-PE (合藏油性ムラミルトリペプチド）、 リボゾーム等 を用いることが可能であるが、 日本国内においてヒトに対して投与する場 には、 制等との関連からァラムを用いることが好ましい。  As the adjuvant used with the vaccine of the present invention, known ones can be used without particular limitation. Examples of such adjuvants include aluminum (aluminum sulphide or aluminum adjuvant), Iscom (ISCOM), MDP-PE (oil-containing muramyl tripeptide), and ribosome. In the case of administration to humans in Japan, it is preferable to use an antibacterial agent in view of the regulation.
上記アジュバントは、通常、 本発明のワクチンに対して 1 . 5〜5倍量 @g (更には 1. 5〜2倍量 mS.)用いることが好ましい。本発明のワクチンにおい ては、例えば、 上記ァラムは 300yag/ml程度の割合で混入していることが 好ましい。 (投与量および投与方法） The adjuvant is preferably used in an amount of 1.5 to 5 times @g (more preferably 1.5 to 2 times mS.) Of the vaccine of the present invention. In the vaccine of the present invention, for example, the above-mentioned aram is preferably mixed at a rate of about 300 yag / ml. (Dosage and administration method)
本発明のワクチンをヒトに投与する場合（実験等でゥサギ等の動物に投与する 場合もほぼ同様）、 体重 lkgに対して該ワクチンの投与量は 5〃g/kg程度 であることが好ましい。  When the vaccine of the present invention is administered to humans (substantially the same when administered to animals such as egrets in experiments and the like), the dose of the vaccine is preferably about 5 μg / kg per 1 kg of body weight.
本発明のワクチンを用いる免疫法としては、 4回以上日をおいてワクチンを投 与することが好ましい。 この投与の日の間隔は特に制限されないが、 例えば、 0 日、 30日、 60日、 120日のように投与することが好ましい。 なお、 上述し た投与量は、 1回目の投与量であり、 2回日目以降の投与量は 1/3程度まで減 量してもよい。 更に、 例えば、 1回目には複数のペプチドと担体タンパク質との 複合体からなる本発明のワクチンを （必要に応じてアジュバントとともに）投与 し、 2回目以降は、複数匿のペプチドからなる本発明のワクチンを （必要に応じ てアジュバントとともに）投与してもよい。 図面の簡単な説明  As an immunization method using the vaccine of the present invention, it is preferable to administer the vaccine at least four times a day. The interval between the administration days is not particularly limited, but it is preferable to administer the administration on, for example, 0 days, 30 days, 60 days, and 120 days. The above dose is the first dose, and the dose after the second day may be reduced to about 1/3. Furthermore, for example, the vaccine of the present invention consisting of a complex of a plurality of peptides and a carrier protein is administered (with an adjuvant if necessary) at the first time, and the vaccine of the present invention consisting of a plurality of secreted peptides is administered at the second and subsequent times. The vaccine may be given (with an adjuvant if necessary). BRIEF DESCRIPTION OF THE FIGURES
図 1は ί¾&清を用いた HI V«細胞のウエスタン ·ブロッティング結果を示 す写真のコピーである。 発明を実施するための最良の形態  Figure 1 is a copy of a photograph showing the results of western blotting of HIV «cells using ί¾ & Qing. BEST MODE FOR CARRYING OUT THE INVENTION
以下、 実施例により、 本発明を更に具体的に説明する。  Hereinafter, the present invention will be described more specifically with reference to examples.
難例 1 Difficult case 1
(ぺプチド合成およびべプチド複合体の調製）  (Peptide Synthesis and Preparation of Peptide Complex)
中和抗体産生 V3領域べプチドとして、 下記 3種のぺプチドを用いた。  The following three peptides were used as V3 region peptides producing neutralizing antibodies.
As n- Asn- Thr- Arg- Ly s- S e r- lie- Ar g- 11 e- Gln- Arg- Gl - Pro- Gl y- Arg- Ala- Phe- Val- Thr- lie- Gl - Lys- lie- Gly- Cys- (1)、 As n- Asn- Thr- Arg- Lys- Ser- lie- Ar g- 11 e- Gln- Arg- Gl-Pro- Gl y- Arg- Ala- Phe- Val- Thr- lie- Gl-Lys -lie- Gly- Cys- (1),
文字表記： NNTRKSIRI QRGPGRAFVT IGKIGC1_ As n- Asn- Thr- Arg- Lys- Ser- lie- Thr- L s- Gl - Pro- Gl - Arg- Val- lie- Tyr- Ala- Thr- Gl - Gin- lie- lie- Gl - Cys- (2) Character notation: NNTRKSIRI QRGPGRAFVT IGKIGC1_ As n- Asn- Thr- Arg- Lys- Ser- lie- Thr- L s- Gl-Pro- Gl-Arg- Val- lie- Tyr- Ala- Thr- Gl-Gin- lie- lie- Gl-Cys- (2)
(—文字表記： NNTRKSITKGPGRVIYATGQI IGC)および、 T r- As n- Lys- Arg- Lys- Arg- lie- His- 11 e- Gly- Pro- Gl - Arg- Ala- Phe- Tyr- Thr- Thr- Lys- Asn- lie- lie- Gl - Cys— (3)  (—Letter notation: NNTRKSITKGPGRVIYATGQI IGC) and Tr-Asn-Lys-Arg-Lys-Arg-lie-His-11e-Gly-Pro-Gl-Arg-Ala-Phe-Tyr-Thr-Thr-Lys -Asn- lie- lie- Gl-Cys— (3)
(—文字表記： YNKRKR IHIGP GRAFYT TKNI I GO  (—Letter notation: YNKRKR IHIGP GRAFYT TKNI I GO
上記した 3種のペプチドは自動ペプチド合成機（米国 Applied Biosystems社製 43 OA)で合成した。 これら 3種のペプチドは、 まず、 モル比、 10 : 10 : 10 : 1で MBS (m—マレイミド ベンゾィルー N—ヒドロキシサクシ二イミ ドエステレ） を用いて KLH (スカシガイへモシァニン） 又は HSA (ヒト血清 アルブミン） と結合 (conjugate) させた後、 セフアデヅクス G25カラムを ¾い て精製した。上記個々のペプチド （1)〜 （3)を KLHと結合させる: ^には、 モル比は 30： 1とした。  The above three peptides were synthesized using an automatic peptide synthesizer (43 OA, manufactured by Applied Biosystems, USA). These three types of peptides were first used in a molar ratio of 10: 10: 10: 1 using MBS (m-maleimidobenzoyl-N-hydroxysuccinimidestere) and KLH (keyhole limpet hemocyanin) or HSA (human serum albumin). After conjugation with the product, purification was carried out using a Sephadex G25 column. The above individual peptides (1) to (3) are bound to KLH: ^, the molar ratio was 30: 1.
中和抗体産生用 gp 120不変部位べプチドとしては、 下記のぺプチト'を用い た。  The following peptides were used as gp120 constant site peptides for neutralizing antibody production.
Val- Gin- Cys- Thr- His- Gly- lie- Arg- Pro- Val- Val- Ser- Thr- Gin- Leu- Leu- Leu- Asn- Gly- Ser- Leu- Ala- Glu- Glu- Glu- (Cys) (C 2 IE) ··· (8)  Val- Gin- Cys- Thr- His- Gly- lie- Arg- Pro- Val- Val- Ser- Thr- Gin- Leu- Leu- Leu- Asn- Gly- Ser- Leu- Ala- Glu- Glu- Glu- (Cys) (C 2 IE) (8)
(—文字表記： VQCTHGIRPWSTQLLLNGSLAEEE (C) ) またヘルパー T細胞誘導用ペプチドとしては、 、 下記のペプチドを用いた。  (—Letter notation: VQCTHGIRPWSTQLLLNGSLAEEE (C)) The following peptides were used as peptides for inducing helper T cells.
11 e- Asn- Met- Trp- Gin- Glu- Leu- Gly- Lys- Al a- Met- Tyr- Ala- Pro- Pro- (C s) ··. (9) 11 e- Asn- Met- Trp- Gin- Glu- Leu- Gly- Lys- Ala- Met- Tyr- Ala- Pro- Pro- (C s)
(—文字表記： I NMWQE L GK AM Y AP P (C) ) 上記べプチドをそれぞれ前記と同様に自動ぺブチド合 ^を用いて合成して、(—Letter notation: I NMWQE L GK AM Y AP P (C)) Each of the above peptides was synthesized using automatic ぺ butide compound ^ in the same manner as above,
KLH又は HSAと 20： 20： 1のモル比で同様に結合させた。 C21Eぺブ チドを KL A又は HS Aと結合させる際には、 モル比は 30： 1とした。 これら 2種のペプチドは、 HIV- ΙΠΒ、 HIV— IIIBFおよび HIV— IIIMN株 (s train)に共通のものである。 It was similarly bound to KLH or HSA at a molar ratio of 20: 20: 1. When binding C21E-peptide to KLA or HSA, the molar ratio was 30: 1. These two peptides, HIV- ΙΠ Β, is common to HIV- IIIBF and HIV- III MN strain (s train).
(上記式（8) ないし （9) において、 カヅコ内のシスティンは、 他のペプチド 又はキヤリァ担体夕ンパク質との結合用に合成した際の配列を示す）。  (In the above formulas (8) to (9), the cysteine in the cocoon indicates a sequence synthesized when the cysteine is combined with another peptide or carrier carrier protein).
上記により得た 2種の成分（すなわち、 V 3ペプチドキャリア結合物、 および 不変部位ペプチド Zヘルパー T細胞誘導ペプチド一キャリア結 ） の等量 (等 重量) を混合し、 ワクチンとした。  Equal amounts (equal weight) of the two components obtained above (ie, the V3 peptide carrier conjugate and the invariant site peptide Z helper T cell-derived peptide-one carrier conjugate) were mixed to prepare a vaccine.
上記結合の後、残った MBSはセフアデヅクス G— 10カラムを用いて除去し  After the above binding, the remaining MBS was removed using a Sephadex G-10 column.
(ワクチンによる免疫化） (Immunization with vaccine)
ゥサギにキャリア結合ワクチン 2 O zg/kgを投与して免疫し、 これを 1週 間の間をおいて合計 4回行った。  ゥ The heron was immunized by administering 2 Ozg / kg of the carrier-conjugated vaccine, and the immunization was performed four times at one week intervals.
更にポランティア （健常人） には、 KLH結合ペプチドワクチンの合計量 5 g/kgをアジュバントたるァラム ワクチン +ァラム =10 S) とともに投与して 0日および 30日に免疫した。更に、 60日後および 12 0日後には、 HSA含有ワクチン 20 g/kgおよびポリマ"^匕ペプチド （キ ャリア非結合キラーペプチド） を投与した。  Further, the volunteers (healthy individuals) were immunized on days 0 and 30 with a total dose of 5 g / kg of the KLH-binding peptide vaccine administered together with the adjuvant (aram vaccine + aram = 10 S). Further, on day 60 and day 120, the vaccine containing HSA was administered at 20 g / kg and the polymer “^” (non-carrier killer peptide) was administered.
難例 3 Difficult case 3
(ェンザィムーリンクト ·ィムノソルベントァヅセィ、 ELISA)  (Enzyme Linked Immobilizer Assay, ELISA)
ELISA法による測定は.、 報文 (AIDS, ， 765〜766 (1989) および AIDS，互， 1140〜 1141 ( 1991 ) に記載された方法により 行った 0 すなわち、 ペプチド又はキヤリア結合べプチドのそれを 96穴のマイクロブレ ート上にコーティングした。 ここでは、 異なる部位由来の 3種のペプチドの混合 物を使用した。他の抗原は、 それそれのぺブチドとともに使用した。 （C 21 E ペプチドは、 gp 120の不変部位に対応し、 HGP— 30ペプチドはウィルス 中部 gag部位の蛋白に対応する）。 The measurement by the ELISA method was carried out by the method described in the report (AIDS,, 765-766 (1989) and AIDS, Mutual, 1140-1141 (1991)). That is, the peptide or that of the carrier-binding peptide was coated on a 96-well microplate. Here, a mixture of three peptides from different sites was used. Other antigens were used with each of the peptides. (The C 21 E peptide corresponds to the constant site of gp120, and the HGP-30 peptide corresponds to the protein at the central gag site of the virus).
1時間の後、 ゥエルを洗浄し、 それそれの希釈した抗血清を 芯させた。次に ペルォキシダーゼ標識した抗マウス、抗ゥサギ又は抗ヒト I gGを させた。 夕ィ夕一法により、 最終的に検出可能な希釈度の逆数（逆タイ夕一値） を^し た。以下に示す^ S例 4以降は、上記した報文に準じて行った。  After 1 hour, the wells were washed and each diluted antiserum was cored. Then, a peroxidase-labeled anti-mouse, anti-heron or anti-human IgG was used. The reciprocal of the ultimately detectable dilution (inverse Thai value) was calculated by the Yuichi method. The following ^ S examples 4 and following were performed according to the above-mentioned report.
麵例 4 麵 Example 4
(細胞融合の抑制）  (Suppression of cell fusion)
ウィルス成長の阻害は、 文献 (Science, 239， 1021〜： L023 (198 8) ； Nature, 332, 469〜470 (1988) ； J. Virol. 62., 262 2〜2628 (1988) ； Proc. Natl. Acad. Sci. USA, 8_5, 3198〜3 202 (1988) ) I3¾の方法に準じて行った。  Inhibition of virus growth is described in the literature (Science, 239, 1021-: L023 (1988); Nature, 332, 469-470 (1988); J. Virol. 62., 2622-2628 (1988); Proc. Natl. Acad. Sci. USA, 8_5, 3198-3202 (1988)).
すなわち、 HIV ( IIIB、 IIIRFおよび IIIMN株） と希釈した血清とを 37 °Cで 30分間反応させた後、 CD4+末しょう血 T細胞 (2X105 CellZゥェ ル） とともに 24穴のプレート中で、被^ ώΐ清の存在下に 37。Cで 5日間観察し、 融合細胞の出現を観察した。 That, HIV (IIIB, III RF and IIIMN strains) After the serum diluted reacted for 30 minutes at 37 ° C, in CD4 + peripheral blood T cells (2X10 5 CELLz © E Le) along with the plate of 24-well 37, in the presence of Qingqing. The cells were observed at C for 5 days, and the appearance of fused cells was observed.
融合活性 (fused activity)数は、  The number of fused activities is
Vn/Vo= (被^ ώι清中で融合した細胞の数） /  Vn / Vo = (number of cells fused in 被 ^ / ιώ) /
(コントロール血清中で融合した細胞の数）  (Number of cells fused in control serum)
で表示した。 中和活性 (neutmalizeing activity)は、 該中和活性に対応するレシ プロカル希釈の回数で ,した。 この結果、 70%以上の融合阻害が見られた。 この際、血清希釈数を用いて抗体能の検討を行った。 Displayed with. Neutmalizeing activity was determined by the number of reciprocal dilutions corresponding to the neutralizing activity. As a result, fusion inhibition of 70% or more was observed. At this time, the antibody ability was examined using the serum dilution number.
難例 5 (p 24産生の阻害） Difficult case 5 (Inhibition of p24 production)
抗血清による HIV複製の阻害は、 もう一つの方法、 すなわちウィルスの蛋白 の合成能の阻害活性で検討した。 この検討は、 文献 (J. Virol, 6^., 2622 〜2628 (1988) ， Proc. Natl Acad." Sic. USA， 88., 2249〜22 53 (1991) )記載の方法に準じて行った。  The inhibition of HIV replication by antisera was examined by another method, namely, the activity of inhibiting the ability to synthesize viral proteins. This examination was performed according to the method described in the literature (J. Virol, 6 ^., 2622-2628 (1988), Proc. Natl Acad. "Sic. USA, 88., 2249-2253 (1991)). .
すなわち、 C EM細胞を H I V— 1ウィルスとともに 37。Cで 4時間ィンキュ ペートし、 よく洗浄した後、 感染 CEM垂を、 最終 mg2.5%の ί¾ώ清を含 有するメディウム中で培養した。 5日間インキュベーションの後、 培地の細胞を 含んでいない上清を採取し、 0. 45 mフィルターで濾過した。 ウィルス濃度 は市販の p 24捕かく測定試薬 (Coulter Co. ) を用いて行った。 That is, the CEM cells were combined with the HIV-1 virus37. After incubating with C for 4 hours and washing well, the infected CEM drops were cultured in a medium containing a final mg of 2.5% serum. After 5 days incubation, the cell-free supernatant of the medium was collected and filtered through a 0.45 m filter. Virus concentration was determined using a commercially available p24 capture reagent (Coulter Co.).
me  me
特異 I - 2産生）  Specific I-2 production)
IL-2 (インターロイキン一 2)産生のァヅセィは、 文献（Imnmnology，^： , 113〜119 (1988) )記載の方法に準じて行った。  The production of IL-2 (interleukin-12) was carried out according to the method described in the literature (Imnmnology, ^ :, 113-119 (1988)).
すなわち、 2. 5xl04個の末しょう血リンパ細胞を最終 2〜： lO zg /m 1のそれぞれ抗原とともに 96穴マイクロブレートのゥエル中で培養したThat is, 2.5xl0 4 peripheral blood lymphocytes were cultured in a 96-well microplate well with each of the final 2 ~ 10 lO zg / m 1 antigens.
(ウィルス （ ΙΠΒ )および Illsウィルス遺伝子から得られた組換え gp 12 0 (rgpl20、横浜巿の三 匕成（株）から供与されたものを用いた） は、 加熱殺菌された精製 H IV- 1タンパク質および えにより調製された H I V エンベロープを代表している） (Recombinant gp120 obtained from the virus (お よ び) and the Ills virus gene (rgpl20, which was provided by San-Danisei Co., Ltd. of Yokohama) was used for heat-sterilized purified HIV-1. Represents the HIV envelope prepared by protein and fly)
8時間後、 それぞれの培地の上清 100〃1を IL一 2依存性の T一細胞ラ イン CTLLを用いて した。後述する表の数値は、 4ないし 6試料の平均 c pm土 SD (標準偏差） を示す。  Eight hours later, 100〃1 of the supernatant of each medium was used for IL-12-dependent T-cell line CTLL. The figures in the table below show the average cpm soil SD (standard deviation) of 4 to 6 samples.
餓例 7 . Starvation 7.
(ウエスタン ·ブロッテイング）  (Western Blotting)
免疫した血清と HI V感染細胞への抗体の結合は、 文献 (Proc. Natl. Acad. Sc i. USA, 85_} 3198-3202 (1988) )記載の方法に準じたウェス夕 ン ·ブロヅティング法にて行った。 Binding of antibodies to immunized sera and HIV-infected cells was described in the literature (Proc. Natl. Acad. i. USA, 85_ } 3198-3202 (1988)), according to the Westin blotting method.
すなわち、 H IV感染 Mo It— 4細胞を、 ソディウム ドデシルサルフエ一 ト （SDS)により可溶化し、 それぞれの 溶化サンプルを電 動にかけて二 トロセルロース濾紙中に転移させた。次いで、 それぞれの濾紙をペルォキシ夕一 ゼ標識抗ゥサギ（又は抗ヒト） IgG抗体を用いて染色した。  That is, HIV-infected Mo It-4 cells were solubilized with sodium dodecyl sulfate (SDS), and each solubilized sample was electrotransferred and transferred to nitrocellulose filter paper. Next, each filter paper was stained with a peroxynase-labeled anti-Peacock (or anti-human) IgG antibody.
(ヒト血清の抗体価） (Antibody titer of human serum)
まず、 それそれの免疫 ί¾Μに対する抗体価（力価） を調べた。 ELISAによ るアツセィにおいては、 免疫された個体からの血清は 400〜51， 200の抗 体価を示した （下記表 1および表 2参照）。 First, the antibody titer (titer) for each type of immunity was examined. In the assay by ELISA, sera from immunized individuals showed titers of 400-51,200 (see Tables 1 and 2 below).
Reciprocal titer of antibody Reciprocal titer of antibody
Peptides El. EJL El.  Peptides El. EJL El.
coaled KLH-mlxed» LH-ΙΠΒ LH-ΠΙΜΝ LH»mftn KLH-C21Bcoaled KLH-mlxed »LH-ΙΠΒ LH-ΠΙΜΝ LH» mftn KLH-C21B
V3 V3
pepddes  pepddes
DIB 25.600 12,800 25.600 12,800 く 100 <100 く 100 <100 N.T N.T  DIB 25.600 12,800 25.600 12,800 c 100 <100 c 100 <100 N.T N.T
ΙΠΜΝ 12,800 6,400 く 100 く 100 25,600 12,800 <100 く 100 N.T N.T mRF 12,800 12,800 <100 NT <100 く 100 12,800 51,200 N.T N.TΙΠΜΝ 12,800 6,400 c 100 c 100 25,600 12,800 <100 c 100 N.T N.T mRF 12,800 12,800 <100 NT <100 c 100 12,800 51,200 N.T N.T
Constant Constant
peptide  peptide
C21E 12,800 6,400 <100 <100 NT NT NT NT 12,800 6,400 Killer  C21E 12,800 6,400 <100 <100 NT NT NT NT 12,800 6,400 Killer
peptides 6,400 3,200 く 100 N.T NT N.T NT NT T N.T (mixed)  peptides 6,400 3,200 ku 100 N.T NT N.T NT NT T N.T (mixed)
Helper 3,200 3,200 <100 N.T NT NT NT NT N.T N.T peptide  Helper 3,200 3,200 <100 N.T NT NT NT NT N.T N.T peptide
KLH 6.400 6,400 6,400 12.800 12,800 51,200 25,600 51,200 12,800 25.600 KLH 6.400 6,400 6,400 12.800 12,800 51,200 25,600 51,200 12,800 25.600
上記表 1は、 ゥサギから得たワクチン免疫血清のそれそれの合成ペプチドに対 する抗体価を示している。上記表 1中、 「KLH— mixed」は、 本発明の多価ヮ クチンを示し、 他の免 ¾ί¾©は、 KLHと れそれの V3領域ペプチド （又は C 2 1 E及び HGP— 30のペプチド） との結 を示している。 ·「ΝΤ」は、 試 験していない （not tested) の意味である。 Table 1 above shows the antibody titers of the vaccine immune sera obtained from the egrets against the respective synthetic peptides. In Table 1 above, “KLH—mixed” indicates the multivalent actin of the present invention, and other licenses are KLH and its V3 region peptide (or C21E and HGP-30 peptide). This shows the conclusion. · "ΝΤ" means not tested.
Peptides Mean reciprocal titer of antibody (κΐθ') Peptides Mean reciprocal titer of antibody (κΐθ ')
HI H2 H3 H4 Ηβ HI H2 H3 H4 Ηβ
V 3peptldes  V 3peptldes
ΠΐΒ 5.311.5 β·5±3·0 4.311.5 8.5±3.0 8.513.0 6.410  ΠΐΒ 5.311.5 β5 ± 3.0 4.311.5 8.5 ± 3.0 8.513.0 6.410
HIM N 2.410.8 5.3±1.5 3.210 6.4±4.5 A.2±i.5 5.311.5 HIM N 2.410.8 5.3 ± 1.5 3.210 6.4 ± 4.5 A.2 ± i.5 5.311.5
IIlR F 8.513.0 14.4±7.0 5.3±1.5 4.211.5 3.7*2.0 14.4±7 IIlR F 8.513.0 14.4 ± 7.0 5.3 ± 1.5 4.211.5 3.7 * 2.0 14.4 ± 7
Constant peptide Constant peptide
t C21B 6.4±0 S.6±1.3 3.5H.9 5.3 1.5 12.810 8.5±3.0  t C21B 6.4 ± 0 S.6 ± 1.3 3.5H.9 5.3 1.5 12.810 8.5 ± 3.0
Killer peptides Killer peptides
(mixed) 1.3±0.4 ί.8±0.7 4.3±1.S 3.210 3.7±2.0 4.2±1.5  (mixed) 1.3 ± 0.4 ί.8 ± 0.7 4.3 ± 1.S 3.210 3.7 ± 2.0 4.2 ± 1.5
Helper peptide .2.110.8 2.1±0.8 4.3H.5 3.7±2.0 1.3*0.4 10.710.4 LH 14.416.2 8.S±3.0 6.414.5 10.613.0 6.4±4.S 10.613.0 Helper peptide 2.110.8 2.1 ± 0.8 4.3H.5 3.7 ± 2.0 1.3 * 0.4 10.710.4 LH 14.416.2 8.S ± 3.0 6.414.5 10.613.0 6.4 ± 4.S 10.613.0
BSA く 0.2 <0.2 <0.2 <0.2 く 0.2 <0.2 BSA 0.2 0.2 <0.2 <0.2 <0.2 0.2 0.2 <0.2
5 Five
上記表 2は、 ワクチン免疫したヒト 人）からの免 ¾JfiL清の抗体価を示し ている。表 2に示されているように、本発明のワクチンを用いて免疫すれば、 E L I S Aで高い抗体価を得ることができる。 Table 2 above shows the antibody titers of JfiL purified from humans immunized with the vaccine. As shown in Table 2, a high antibody titer can be obtained with ELISA when immunized with the vaccine of the present invention.
それそれの 1価ワクチンにおいては、 多価ワクチンの場合と比較して、 多少高 いレベルの抗ペプチド抗体が見られた。ォク夕ロニー法においては、 主な抗体の クラスは I gGであることが見い出された。 ゥサギ δ¾清の夕イタ一は、 ヒト抗 血清のそれよりも高かった。 これのデータから、 ヒトにおいても、 本発明のワク チンを用いた免疫法は抗体レベルを良好に上昇させることが確認された。  For each monovalent vaccine, slightly higher levels of anti-peptide antibodies were seen than for the multivalent vaccine. In the Okuno Ronny method, the major antibody class was found to be IgG. In the evening of Egret δ¾sei, it was higher than that of human antiserum. From these data, it was confirmed that the immunization method using the vaccine of the present invention satisfactorily increased antibody levels even in humans.
(ί¾ύΐ清の中和活性）  (ί¾ύΐ Neutralizing activity)
H I V感染 CD 4+ T細胞および B細胞の融合に対する阻害活性の測定結果を 下記表 3および表 4に示す。 The measurement results of the inhibitory activity against the fusion of HIV-infected CD4 + T cells and B cells are shown in Tables 3 and 4 below.
表 3 Table 3
Reciprocal HIV neotraUsdng liter Reciprocal HIV neotraUsdng liter
Sera Sera
HILV-IIIB HTLV-IIIRF HTLY-IIIMNHILV-IIIB HTLV-IIIRF HTLY-IIIMN
Rl reimmnne <4 <4 <4Rl reimmnne <4 <4 <4
Rl immune 128 128 64Rl immune 128 128 64
R2 preimmnne く 4 <4 <4R2 preimmnne c 4 <4 <4
R2 immnne 256 128 64R2 immnne 256 128 64
R3 immane 32 <4 <4R3 immane 32 <4 <4
R4 immune 64 <4 <4 iU immune <4 32 <4R4 immune 64 <4 <4 iU immune <4 32 <4
R6 immune <4 32 <4R6 immune <4 32 <4
R7 immune <4 <4 16R7 immune <4 <4 16
R8 immune <4 <4 32R8 immune <4 <4 32
R9 immune S 16 8R9 immune S 16 8
RIO immune 4 <4 16 RIO immune 4 <4 16
上記表 3は、 ゥサギ抗血清を用いた H IV中和抗体価を示している。 ゥサギか ら得た 清は、表 1に示したものと同じである。 Table 3 above shows the HIV neutralizing antibody titers using Pergum antiserum.清 The sera obtained from the heron are the same as those shown in Table 1.
表 4  Table 4
Keciprocal HIY neutralizing titer Keciprocal HIY neutralizing titer
HTLY-ΙΠΒ HILV-IIIRF HILV HTLY-ΙΠΒ HILV-IIIRF HILV
Hi preimmune く 4 <4 く 4  Hi preimmune ku 4 <4 ku 4
HI immune 64 32 w  HI immune 64 32 w
H2 preimmune <4 <4 <4  H2 preimmune <4 <4 <4
H2 immane 64 64 64  H2 immane 64 64 64
H3 preimmane <4 <4 <4  H3 preimmane <4 <4 <4
H3 immane 128 64 32  H3 immane 128 64 32
H4 preimmune <4 <4 <4  H4 preimmune <4 <4 <4
H4 immune 128 32 32  H4 immune 128 32 32
H5 preimmune <4 <4 <4  H5 preimmune <4 <4 <4
H5 immune 64 64 32  H5 immune 64 64 32
H6 preimmune <4 <4 く 4  H6 preimmune <4 <4
H6 immane 128 32 64  H6 immane 128 32 64
上記表 4は、 ヒト抗血清を用いた H I V中和抗体価を示している。 ヒトから得 た¾¾&清は、 表 2に示したものと同じである。 Table 4 above shows the HIV neutralizing antibody titers using human antisera. ¾¾ & QI obtained from humans are the same as those shown in Table 2.
ゥサギからの免疫血清は、.顕著な巨細 成阻害効果を誘起した。 しかしなが ら、 1価ワクチンで免疫された血清は、 強い阻害タイターを示さなかった。 ゥサ ギ抗血清においては本発明の多価ワクチン（すなわち、 3種の V3領域ペプチド 'および不変部位ペプチドを含むワクチン）で免疫した血清においては、 激合活 性の強い相^!果が見られた （表 3) o このような強い！^ ¾¾果は、 のワク チンを用いた場合には見出だされていなか たものである。良好なレベルの阻害 は、 免疫したヒトからの血清においても顧された （表 4)。 ヒトに関し、 この ような相^!果は現在まで報告されていなかったものである。 Immune sera from egrets induced a profound macrophage inhibitory effect. However, sera immunized with the monovalent vaccine did not show strong inhibition titers.に お い て In the heron antiserum, the polyvalent vaccine of the present invention (ie, three V3 region peptides) ) And a vaccine immunized with a peptide containing an invariant site peptide), a highly vigorous phase was observed (Table 3). ^ The results have not been found when using the vaccine. Good levels of inhibition were also noted in sera from immunized humans (Table 4). For humans, such an outcome has not been reported to date.
( ρ 24タンパク質産生に対する 清による阻害）  (Inhibition of ρ24 protein production by Qing)
ゥィルス成長阻害は、培地上清中の遊離 ρ 24夕ンパク質を検出することによ り行った。 この場合にも本発明のワクチン（複 ペプチドの組合せ） により誘 起された相^!果が見られた （下記表 5参照）。  Virus growth inhibition was performed by detecting free ρ24 protein in the culture supernatant. Also in this case, the results induced by the vaccine of the present invention (combination of multiple peptides) were observed (see Table 5 below).
表 5  Table 5
HIY-l p24 (ng/ml) HIY-l p24 (ng / ml)
Sera Sera
HTLV-ΠΙΒ HTLY-IURF HTLV-IIIM  HTLV-ΠΙΒ HTLY-IURF HTLV-IIIM
Rl preimmtine 19.0 28.7 18.6  Rl preimmtine 19.0 28.7 18.6
Rl immune 6.9 12^ 52 Rl immune 6.9 12 ^ 52
2 preimmune 18.7 26.4 16.0  2 preimmune 18.7 26.4 16.0
R2 immune 83 10.6 8.3  R2 immune 83 10.6 8.3
R3 preimmune 16.0 24.6 18.8  R3 preimmune 16.0 24.6 18.8
R3 immune 132 25.7 19.9  R3 immune 132 25.7 19.9
R5 preimmune 18.7 28.3 18.4  R5 preimmune 18.7 28.3 18.4
R5 immune 18.1 20.7 19.9  R5 immune 18.1 20.7 19.9
R7 preimmune 20.4 N T 19.7  R7 preimmune 20.4 N T 19.7
R7 immune 13.7 N T 13.4 R7 immune 13.7 N T 13.4
9 preimmune * 20.1 25.7 18.5  9 preimmune * 20.1 25.7 18.5
R9 immune 17.6 20.3 13.0 上 IB¾5は、 ゥサギ血清による H I V— 1 p 24タンパク質産生阻害活性を 示している。 ゥサギからの血清は、 ΙίϋΒ表 1と同じである。 「NTjは、 I ^し ていない （not tested) の意味である。 R9 immune 17.6 20.3 13.0 Above IB # 5 shows the activity of inhibiting the production of HIV-1 p24 protein by rabbit heron serum.血清 Serum from herons is the same as in Table 1. "NTj stands for not tested.
表 6  Table 6
t XiITXV V--i1. t XiITXV V--i1.
Sera Sera
UIB IIIRF ΏΙΜΝ UIB IIIRF ΏΙΜΝ
HI 15 7 307 200 HI 15 7 307 200
HI inunune 6.0 14.4 14 8  HI inunune 6.0 14.4 14 8
H2 preimmune 16.6 31.6 21J  H2 preimmune 16.6 31.6 21J
H2 immune 8.0 12·5 8·5  H2 immune 8.0 12
H3 preimmune 18.1 27.7 19.1  H3 preimmune 18.1 27.7 19.1
H3 immune 9ュ 10.0 11.0  H3 immune 9 10.0 10.0 11.0
H4 preimmune 18.9 24.8 19.6  H4 preimmune 18.9 24.8 19.6
H4 immune 0 6·5 8.2  H4 immune 0 6.58.2
H5 preimmune 14.0 31.1 23.9  H5 preimmune 14.0 31.1 23.9
H5 immune 7.1 Z5  H5 immune 7.1 Z5
H6 preimmune 16.6 N T N T  H6 preimmune 16.6 N T N T
H6 immune 10 N T N T  H6 immune 10 N T N T
上記表 6は、 ヒ卜血清による H IV— 1 p 24タンパク質産生阻害活性を示 している。 ヒトからの血清は、 編己表 2と同じである。 「NT」は、 試験してい ない （not tested) の意味である。 表 6に示すように、 ヒトに本発明のワクチン を免疫して得られた血清は、 強いウイルス産生抑制能があることが判明した。 以上の結果から、 本発明のワクチンを用いて得られた ί¾ώ清が、 ウィルスの増 殖を確実に阻害することが判明した。 Table 6 above shows the activity of HIV-1p24 protein production inhibition by human serum. Serum from humans is the same as in Table 2. “NT” means not tested. As shown in Table 6, the vaccine of the present invention It was found that the sera obtained by immunization had strong ability to suppress virus production. From the above results, it was found that the sera obtained by using the vaccine of the present invention certainly inhibited the growth of the virus.
(細胞性免疫の活性化）  (Activation of cell-mediated immunity)
本発明のワクチンによる細胞性免疫の活性化も、 i¾i特異的な I L一 2が本発 明の多価ワクチンにより誘起されるか否かにより評価した。 結果を下記表 7に示 す。 Activation of cell-mediated immunity by the vaccine of the present invention was also evaluated based on whether i¾i-specific IL-12 was induced by the multivalent vaccine of the present invention. The results are shown in Table 7 below.
Peripheral -2 production (cpm》 Peripheral -2 production (cpm)
Peptide lor helper Peptide for KillerPeptide lor helper Peptide for Killer
Blood Virus K3P120 Vaccine (IN ····· APP) (RIQ-"IGK) Blood Virus K3P120 Vaccine (IN ... APP) (RIQ- "IGK)
Vaccinated group Vaccinated group
H1 2853±S37 3120±279 420±15 300±5 259183 H1 2853 ± S37 3120 ± 279 420 ± 15 300 ± 5 259183
H2 2390±206 2920±279 390±32 215±40 211 ±83H2 2390 ± 206 2920 ± 279 390 ± 32 215 ± 40 211 ± 83
H3 402S±315 3562±663 711±63 377±24 257±42H3 402S ± 315 3562 ± 663 711 ± 63 377 ± 24 257 ± 42
H4 3102±6S7 2866±299 452±68 150±13 239±27H4 3102 ± 6S7 2866 ± 299 452 ± 68 150 ± 13 239 ± 27
Control group Control group
C1 30θ±24 220±20 259±10 NT 261 ±30 C1 30θ ± 24 220 ± 20 259 ± 10 NT 261 ± 30
C2 219± 234±85 213±39 NT 238146 C2 219 ± 234 ± 85 213 ± 39 NT 238146
上記表 7は、 ワクチンを投与したヒト （各個人） からの末梢白血球による抗源 特異的 I L一 2の産生を示している。表中の数値は、 4ないし 6サンプルの平均 土標準偏差（SD)である。 Table 7 above shows the production of antigen-specific IL-12 by peripheral leukocytes from humans (individuals) who received the vaccine. The values in the table are the average soil standard deviation (SD) of 4 to 6 samples.
上記表 7に示したように、 H I V全体又 ismえ g p 1 2 0夕ンパク質を培養 系に添化した場合、 I L一 2産生の実質的なレベルが観察された。 この結果は、 本発明のワクチンが H I V抗原に特異的な I L— 2を産生する T細胞を誘起可能 なこと、 すなわち細胞性免疫能が活性化されていることを示している。  As shown in Table 7 above, a substantial level of IL-12 production was observed when whole HIV or isp120 protein was added to the culture system. This result indicates that the vaccine of the present invention can induce T cells that produce IL-2 specific to HIV antigen, that is, that cell-mediated immunity is activated.
(ウエスタン ·ブロッテイング）  (Western Blotting)
図 2にウエスタン ·ブロヅティングの結果（写真のコピー） を示す。上記図 2 は、 抗血清を用いた H I V感染細胞のウエスタン ·ブロッテイングである。 図 2 中、 （A) は、 ゥサギ中に産生された抗体であり、 各レーンの意味は、 以下の通 りである。  Figure 2 shows the results of Western blotting (copy of the photo). FIG. 2 above shows Western blotting of HIV-infected cells using antiserum. In FIG. 2, (A) shows the antibody produced in the egret, and the meaning of each lane is as follows.
1 · · ·可溶化した Mo I t— 4細胞  1 · · · Solubilized Mo It— 4 cells
2 · · · III B株によって感染した Mo I t細胞の可溶化物  2 · · · III Lysate of Mo It cells infected by strain B
3 · · · III MN株によって感染した Mo 1 細胞の可溶化物  3 · · · Lysate of Mo 1 cells infected by III MN strain
4 · · · III RF株によって感染した Mo I t細胞の可溶化物 4 · · · III Lysate of Mo It cells infected by RF strain
また、 図 2中 （B) は、 ヒト中に産生された抗体であり、 ここで用いた各サン プル （各レーン）の意味は、 上記 (A) におけると同様である。  In addition, (B) in FIG. 2 shows an antibody produced in humans, and the meaning of each sample (each lane) used here is the same as in (A) above.
このウエスタン ·プロヅティングの結果は、 gp 1 2 0不変部位べプチドに対す る抗体が、 分子量 1 2 0 Kダルトンサイズの分子からなる抗原と結合したことを 示している。 すなわち、 本発明のワクチンにより産生された抗体が、 H I Vェン ベローブのタンパク質と反応することが ¾ された。 The results of this western-producing show that the antibody against the gp120 constant site peptide bound to an antigen consisting of a molecule having a molecular weight of 120 K daltons. That is, it was confirmed that the antibody produced by the vaccine of the present invention reacts with the protein of HIV envelope.
また上記した各実施例においては、 いかなる副作用も観察されなかった。  Also, in each of the above Examples, no side effects were observed.
産 H±の利用可能性 上述したように本発明によれば、 動物のみならずヒトにも効果的なワクチンで あって、 H IVウィルス自体を使用していないために安全性が高く、 予防効率が 高く、 しかも体液性免疫とともに細胞性免疫を活性化させることが可能な多価ヮ クチンが される。 Availability of H ± As described above, according to the present invention, it is an effective vaccine not only for animals but also for humans. Since it does not use the HIV virus itself, it has high safety, high prevention efficiency, and humoral immunity. At the same time, a multivalent actin capable of activating cell-mediated immunity is produced.

Claims

請求の範囲 The scope of the claims
1. HIV (ヒト免疫不全ウィルス） エンベロープ gp 120の V3領域ぺ プチド （エンベロープタンパク質内のアミノ酸位置 303〜322) と、 gpl 20又は内部構造夕ンパク質 gagの不変部位ぺプチドとからなる複数のぺプチ ドとを免 源として含むことを とする HI V感染症予防ワクチン。  1. V3 region of HIV (human immunodeficiency virus) envelope gp120 複数 Multiple peptides consisting of peptide (amino acid positions 303 to 322 in the envelope protein) and gpl 20 or invariant site peptide of internal structure protein gag. A vaccine for the prevention of HIV infection, which contains peptide as an exemption.
2. HIV (ヒト免疫不全ウィルス） エンベロープ gp 120の V3領域ぺ プチド （エンベロープタンパク質内のアミノ酸位置 303〜322) と、 gpl 20の不変部位べプチド又は内部構造夕ンパク質 g a gの不変部位ぺプチドとか らなる複数のペプチドと、 担体タンパク質とを少なくとも含む複合体からなるこ とを とする HI V感染症予防ワクチン。  2. The V3 region of the HIV (human immunodeficiency virus) envelope gp120 peptide (amino acid positions 303-322 in the envelope protein) and the constant site peptide of gpl 20 or the constant site peptide of the internal structure protein gag. A preventive vaccine for HIV infection, comprising a complex comprising at least a plurality of peptides and a carrier protein.
3. アジュバントを更に含む請求項 1又は 2記載の HI V感染症予防ワクチ ン。  3. The vaccine for preventing HIV infection according to claim 1 or 2, further comprising an adjuvant.
4. 複数のぺプチドとしてヘルパー T細胞活性化部位ぺプチドを更に含 む請求項 1又は 2記載の H I V感染症予防ワクチン。  4. The preventive vaccine for HIV infection according to claim 1 or 2, further comprising a helper T cell activation site peptide as the plurality of peptides.
5. 担体タンパク質がシスティン基含有タンパク質からなる請求項 2記 載の H I V感染症予防ヮクチン。  5. The HIV infection preventive pectin according to claim 2, wherein the carrier protein comprises a cysteine group-containing protein.
6. 前記担体タンパク質がヒト血清アルブミン （HSA)、 カブトガ二へモ シァニン （KLH)又はマルチプル アンティゲン ペプチド （multiple antig en peptide) からなる請求項 1記載の H IV感染症予防ワクチン。  6. The vaccine for preventing HIV infection according to claim 1, wherein the carrier protein comprises human serum albumin (HSA), horseshoe hemocyanin (KLH), or multiple antigen peptide (multiple antigen peptide).
7. 前記複数のぺプチドが合成べプチドからなる請求項 1ないし 6のいずれ かに記載の H I V感染症予防ワクチン。  7. The vaccine for preventing HIV infection according to any one of claims 1 to 6, wherein the plurality of peptides are composed of synthetic peptides.
8. HIV (ヒト免疫不全ウィルス） エンベロープ gp 120の V3領域ぺ プチド （エンベロープタンパク質内のアミノ酸位置 303〜322) と、 gpl 20の不変部位べプチド又 {ま内 造夕ンパク質 ga gの不変部位ぺプチドとか らなる複数のぺブチドを含む免疫原と、 アジュバントとを混合する段階を含むこ とを とする H I 症予防ワクチンの製造法。 8. V3 region of HIV (human immunodeficiency virus) envelope gp120 peptide (amino acid positions 303 to 322 in the envelope protein) and invariant site of gpl 20 peptide or invariant site of ga g protein Admixing an immunogen comprising a plurality of peptides comprising the peptide and an adjuvant. A method for producing an HI disease prevention vaccine comprising:
9. H I V (ヒト免疫不全ウィルス）エンベロープ gp 1 20の V3領域ぺ ブチド （エンベロープタンパク質内のアミノ酸位置 3 03〜322 ) と、 gp l 20の不変部位ぺブチド又は内部構造夕ンパク質 g a gの不変部位ぺブチドとか らなる撤のペプチドと、 担体タンパク質とを結合させる段階を含むことを特徴 とする H I 感«予防ヮクチンの製造法。  9. V3 region of HIV (human immunodeficiency virus) envelope gp120 ぺ butid (amino acid positions 303-322 in the envelope protein) and invariant site of gpl20 不 invariant site of butide or internal structure protein gag A method for producing an HI-sensitive preventive pectin, comprising a step of binding a peptide consisting of ぺ butide and a carrier protein.
1 0. |?13 のぺブチドが合成ぺブチドからなる請求項 8又は 9 の H I V感染症予防ワクチンの製造法。  10. The method for producing a vaccine for preventing HIV infection according to claim 8 or 9, wherein the peptide of | ?? 13 is a synthetic peptide.
PCT/JP1993/000327 1992-03-26 1993-03-19 Vaccine for preventing hiv-infected disease and production thereof WO1993018791A1 (en)

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JPH01502119A (en) * 1987-01-16 1989-07-27 アンステイテユ・パストウール Peptides that can be recognized by antibodies raised against the human immunodeficiency retrovirus (virus HIV) and the use of said peptides in the diagnosis of infections caused by certain species of said virus and optionally in vaccination against AIDS.
JPH02160800A (en) * 1988-04-26 1990-06-20 E I Du Pont De Nemours & Co Human immunodeficiency virus (hiv)
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JPH01502119A (en) * 1987-01-16 1989-07-27 アンステイテユ・パストウール Peptides that can be recognized by antibodies raised against the human immunodeficiency retrovirus (virus HIV) and the use of said peptides in the diagnosis of infections caused by certain species of said virus and optionally in vaccination against AIDS.
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EP4276106A3 (en) * 2015-05-13 2024-01-24 The United States of America as represented by the Secretary of the Department of Health and Human Services Methods and compositions for inducing an immune response using conserved element constructs

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