Peptides which can induce specific CTLs for HIV, DNAs encoding for the peptides and medicaments for preventing and/or treating HIV comprising the peptides are provided. According to the present invention, peptides carrying novel CTL epitopes derived from HIV Pol or Env proteins are provided. These peptides can bind to HLA-A11, particularly HLA-A*1101 which is frequently encountered in Japanese people and induce HIV-specific cytotoxic T cells.

Description of the Invention

TECHNICAL FIELD OF THE INVENTION

The present invention relates to peptides having an amino acid sequences of the partial region of the protein from Human Immunodeficiency Virus (referred to as "HIV" hereinafter) and which can induce an immune response against HIV, and medicaments comprising the peptide(s) for preventing and/or treating AIDS.

BACKGROUND OF THE INVENTION

Acquired immunodeficiency syndrome (referred to as "AIDS" hereinafter) is the disease caused by the infection of HIV. The investigations for medicaments to treat this disease have been actively performed, and although medicaments such as azidothymidine (referred to as "AZT" hereinafter) and dideoxyinosine (referred to as "DDI" hereinafter) have been practically used, they have some problems in their effects or side effects. Thus, medicaments which can completely treat the diseases caused by HIV infection have not been found and there is no prospect of the findings. On the other hand, a vaccine as a tool for preventing HIV infection and suppressing AIDS onset, which enhances the immune resistance against HIV, has been expected as a critical key to control the global and rapid spread of the disease and has been widely investigated. To date, various types of vaccine have been investigated and some of them are undergoing the clinical test.

The vaccines which have been reported to date include:

i) Vaccines which utilizes inactivated or attenuated virus particles: The method may be considered wherein a gene responsible for HIV pathogenicity is mutated or deleted (Proc. Natl. Acad. Sci. USA, 84, 1434, 1987) or the approach wherein related viruses such as simian viruses which shares the antigenecity of HIV are used (Science, 232, 238, 1987), but it would be difficult to practically use them due to their potential risks.

ii) Subunit vaccines utilizing partial antigen proteins from the viral antigenic proteins: This is the approach wherein only a partial antigenic protein of the virus particle is produce, for example, by genetic recombinant procedures and used as an immunogen (Proc. Natl. Acad. Sci. USA, 84, 6924, (1987), Ann. Int. Med., 114, 119, (1991), Nature, 355, 728, 1992). This approach is widely attempted and there are many clinical test cases. However, there are many problems such as poor increase in the titer of the neutralizing antibody or the persistency of the titer of the antibody. Also, although it would be expected for this approach that it would be effective in enhancing the humoral immunity such as the production of antibodies, the effect of this approach alone would not be necessary expected considering the infection manner of HIV, because this approach would not lead to the activation of cellular immunity which can kill the infected cells.

iii) Recombinant live vaccines such as vaccinia virus or BCG: This is the approach wherein a partial gene sequence from HIV is introduced into the gene of vaccinia virus (Nature, 332, 728, 1988) or BCG (Nature, 351, 479, 1991) which can propagate in human cells. Theoretically, the enhancing effect on cellular immunity would be expected but there are some problems, for example, in that at least previously produced recombinant live vaccines of vaccinia did not trigger sufficient immune response, or viruses such as vaccinia, which are usually harmless, have the possibility of severe infection (Lancet et al. 337, 1034, 1991).

iv) Anti-idiotype antibodies: This is the approach wherein an anti-idiotype antibody is used as the alternative to the virus antigen (Proc. Natl. Acad. Sci. USA, 89, 2546, 1992).

v) Synthetic peptide vaccines: Synthesized peptides are considered in this approach, wherein the sequences of the determinant region of neutralizing antibodies are chemically synthesized.

The above described vaccines are of the type of humoral immune enhancer which mainly induce the neutralizing antibodies, while it is believed that cytotoxic T cells (referred to as "CTLs" hereinafter) which can impair infected cells are more important in defending the infection than humoral immunity with neutralizing antibodies, considering that HIV spreads more easily through the cell fusion between infected cells and non-infected cells than through virus particles. Actually, there is a report where if the patients who had been exposed to the chance of HIV infection were investigated, HIV specific CTLs were detected (J. Clin. Invest., 93, 1293, (1994), Nature, Med., 1, 59, 1995). There is also a report wherein HIV carrying a mutated CTL epitope can escape from the attack of CTLs (Nature Med., 3, 205, 1997) and the appearance of HIV carrying such mutated epitope is believed to be the cause of the onset of AIDS in patients chronically infected with HIV (Nature Med., 3, 212, 1997).

Antigens such as virus-derived proteins are endogenously processed in cells to short fragments and are presented on the cell surface as a bounded form with major histocompatibility complex (referred to as "MHC" hereinafter). CTL recognizes the epitope peptide presented by Class I MHC antigen expressed on the cell surface and attack the target cells. More specifically, CTL recognizes both of the processed antigen fragment bound to the groove of MHC molecule and a part of the MHC molecule on the target cell simultaneously and attack the cell carrying them. Thus, the antigen recognition of CTL intensively depends on MHC on the cell surface. Such antigen recognition is called MHC restriction. It is known that the epitope peptides which bind to a particular MHC Class I antigen and which are presented by the antigen have about nine amino acids in length and their amino acids sequences are defined by certain rules (motif) (Nature, 351, 290, 1991, Eur. J. Immunol., 22, 2453, 1992, Nature, 353, 326, 1991, Nature, 360, 434, 1992, Immunogenetics, 38, 161, 1993).

Based on these findings, the inventors of the present invention have been searching CTL peptides which specifically impair HIV-1 infected cells and have already defined the peptides which can bind to HLA-B35, HLA-B51 and HLA-A31 among human Class I antigens, human Class I leukocyte antigens (referred to as "HLA" hereinafter), and which can induce HIV-1 specific CTLs (WO 95/11255). These peptides induced HIV-1 specific CTLs, restricted by each HLA alloantigen type.

Since HLA genes are the genes having very high diversities, the peptides which bind to HLA Class I antigens have also high diversities, although they comply with certain rules. This suggests that the peptides which are effective in the prevention and/or treatment of HIV infection may possibly differ among individuals, ethnic populations and the like. Therefore, in the prevention and/or the treatment of HIV infection wherein such peptides are used, a set of peptides is desired which can induce other HLA-restricted CTLs besides the known peptide groups.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a peptide which can induce the HIV-1 specific CTL. In particular, it is an object of the present invention to provide a peptide which can induce HIV-1 specific HLA-A*1101 restricted CTL.

It is also an object of the present invention to provide a DNA coding for the above-described peptide. It is further the object of the present invention to provide a medicament for preventing and/or treating AIDS, comprising said peptide.

It is also an object of the present invention to provide a peptide useful for developing HIV-1 vaccine, especially developing the vaccine in Asia. The major difficulty in HIV vaccine development consist in that HIV can easily mutate and escape the host immunity. Thus, the vaccine which use only one epitope as the immunogen has a great possibility of losing its effectiveness with time. It is an object of the present invention to provide multiple epitope peptides that can be simultaneously used as effective immunogens.

The peptides according to the present invention can bind to HLA-A*1101 antigen and induce HLA-A*1101 restricted CTLs targeting HIV-1 infected cells. The DNAs according to the present invention encode for said peptides.

The inventors of the present invention have defined the peptides which can bind to HLA-B*3501, HLA-B*5101 and HLA-A*3101 Class I antigen (WO 95/11255), as previously mentioned. HLA is the molecule which has high diversities among ethnics or individuals, while the peptides of the present invention are characterized in that they are the peptides derived from HIV-1 Pol or Env protein and which can bind to HLA-A11 molecule frequently found in Asian people, especially in Japanese people (HLA-A*1101) (Clayton J, Lonjon C, Whittle D: Allele and haplotype frequencies for HLA loci in various ethnic group: HLA volume I Genetic diversity of HLA functional and medical implication: Charron D. Paris Ed.: EDK Medical and Scientific International Publisher; 1997; 665-820).

The peptides according to the present invention or the peptides encoded by the DNAs according to the present invention are the fragments of HIV-1 Pol or Env protein, which contain any of the amino acid sequence of QIYAGIKVK (SEQ ID NO: 1), SVITQACPK (SEQ ID NO: 2), QIIEELIKK (SEQ ID NO: 3), QIIEKLIEK (SEQ ID NO: 4), ACQGVGGPSHK (SEQ ID NO: 5), AFDLSFFLK (SEQ ID NO: 6) and ALDLSHFLK (SEQ ID NO: 7) and carry HLA-A*1101 binding motif, and which can actually induce HLA-A*1101 restricted CTLs against HIV-1 infected cells.

DETAILED DESCRIPTION OF THE INVENTION

The whole proteins of HIV are described, for example, in Nature Vol. 313, pp. 277-283 (1985) or Proc. Natl., Acad. Sci. USA Vol. 83, 002209-2213 (1986). The peptides of the present invention are the fragments of HIV and, furthermore, they have HLA-A*1101 motif and actually bind to HLA. Exemplary HLA-A*1101 binding motif includes the peptides consisting of 8 to 12 amino acids wherein the second amino acid thereof is selected from Val, Ile, Thr, Leu, Tyr, Cys or Phe and the C-terminal amino acid is Lys. Such peptides may be synthesized using a peptide-synthesizer. The binding of the peptides of the present invention containing HLA-A*1101 binding motif may be confirm by using the cells expressing HLA-A*1101. C1R or RMA-S cells may be used as such cells.

According to the present invention, the synthesized peptides are confirmed for their ability to actually stimulate the patient's peripheral blood monocytes (PBLs) and induce cytotoxic T cells (CTLs). Using such a procedure, peptides having the amino acids sequences of QIYAGIKVK (SEQ ID NO: 1), SVITQACPK (SEQ ID NO: 2), QIIEELIKK (SEQ ID NO: 3), QIIEKLIEK (SEQ ID NO: 4), ACQGVGGPSHK (SEQ ID NO: 5). AFDLSFFLK (SEQ ID NO: 6) and ALDLSHFLK (SEQ ID NO: 7) were obtained, which are the fragments of HIV-1 Pol protein. The peptides of the present invention may be prepared by synthesizing them with peptide-synthesizer or by expressing the DNAs encoding for the peptides having any of the amino acids sequences of SEQ ID NO: 1 to SEQ ID NO: 7 with appropriate host-vector systems. Since the codon usage may vary depending on the host cell used, the preferred codon usage may be selected for each cell. DNAs which can hybridize with such DNAs under the stringent condition, and which bind to HLA-A11 antigen and induce cytotoxic T cells targeting HIV infected cells, may be used also. The stringent condition as used herein means, for example, the condition wherein annealing at 20-25.degree. C. below the melting temperature Tm (Tm=81.5.degree. C.+log.sub.10[Na.sup.+]+0.41(G+C content %)-(600/sequence length)) in 2.times.SSC (20.times.SSC: NaCl 75.3 g/Lm sodium citrate 8802 g/L, pH7.0), and washing at 12-20.degree. C. below the Tm with salt concentration of 0.1.times.SSC, regarding to the temperature and the salt concentration.

The techniques of isolation of DNA fragments, construction of vectors, transformation using recombinant DNA technology, production of peptides and measurement of CTL activity are well known to those skilled in the art.

The peptide of the present invention is useful as a vaccine, because it can induce HIV-1 specific CTL functioning as the T cell epitope. As a vaccine, the peptide solution alone or the combination thereof with one or more physiologically acceptable adjuvants may be administrated with a syringe, or they may be administrated, for example, by aerosol, through dermal absorption from mucous membrane. The peptide may be administrated by a single dose or multiple doses with 0.1 mg-100 mg/dosage. Plural peptides may be used simultaneously, which may be effective in certain cases. The formulation is not particularly limited, and may be freeze-dried or granulated with an excipient such as sugar. There are no acute toxicity observed for thus prepared peptide formulations according to the present invention. Adjuvants which may be used and which can increase the immunogenicity of the vaccine include a composition from cells such as BCG cells, ISCOM extracted from QuillA developed by Morein et al. (Immunostimulating complex, Nature, 308, 457, 1984; Nature, 344, 873, 1990), a saponin system QS-21 (J. Immunol., 148, 1438, 1992), liposome (J. Immunol., 148, 1585, 1992), alminium hidroxyde (alum), KLH (keyhole limpet hemocyanin) J. Virol., 65, 489, 1991). Each of above indicated references and Science, 255, 333, (1992) describes that immune response such as CTL can be induced in vivo by using such methods.

By using the epitope peptides according to the present invention, the methods may be applicable wherein CTLs are effectively induced within the body of patients by administrating the cells from the patients or the cells carrying HLA Class I antigen of the same haplotype, which were previously exposed to the epitope peptide, into the vein of the patients, or wherein CTLs are induced and expanded in vitro by culturing the peripheral blood lymphocytes from patients in vitro with the peptide, followed by returning the cells to the patients. Therefore, CTLs obtained by culturing peripheral blood lymphocytes carrying HLA-A*1101 antigen in the presence of any of the peptides having the sequence according to SEQ ID NO: 1 to SEQ ID NO: 7 may be used as an AIDS vaccine.

In practice, the peptide of the present invention is added to 10.sup.7-10.sup.9 peripheral blood lymphocytes of the patient and the lymphocytes are administrated into the vein of the patient after culturing for from a few hours to one day, or alternatively, after CTLs are induced by continuously culturing the lymphocytes in the medium supplemented with 50 U/ml of recombinant IL-2 and 1 .mu.g/ml of the peptide for a few weeks, they are injected into the vein of the patient. The cells may be cultured by the methods well known to those skilled in the art, then the cultured cells are suspended, for example, in saline after the medium component is washed out, for example, by centrifugation. Such therapeutic methods using cell injection have been utilized as a cancer treatment, which are well known to those skilled in the art (New 1 ng. J. Med., 313, 4185, 1085; Science, 233, 1318, 1986).

Additionally, the identified CTL epitopes according to the present invention may be effectively employed in vaccinia viruses or BCG live recombinant vaccines. When the DNA encoding for the peptides comprising any of the amino acid sequence according to SEQ ID NO: 1 to SEQ ID NO: 7 is introduced into the genes for recombinant proteins which are intended to be expressed in the recombinant live vaccine, the peptide will be processed in cells to be presented by HLA-A*1101 antigen after being expressed as a part of the antigen protein. This will allow the induction of CTL which recognize the peptide. The methods for expressing foreign genes in recombinant BCG live vaccines are detailed in WO 88/06626. The recombinant BCG live vaccines are detailed in J. Exp. Med., 178, 197 (1993). The dose and the dosage methods may be employed according to the dose and methods for the conventional variolation or BCG vaccine. They do not differ from the conventional variolation or BCG vaccine in the acute toxicity and the like. However, vaccinia virus should be carefully used as a therapeutic vaccine, because there may be the risk of sever infection in the patients whose immunocompetence was reduced by the onset of AIDS. Regarding with BCG vaccine, such cases have not been reported yet. The possibility of inducing the immuneresponse including CTL in vivo by such methods is shown, for example, in Nature, 332, 728 (1988) and Nature, 351, 479 (1991).
 

Claim 1 of 25 Claims

1. An isolated peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, wherein said peptide can induce a cytotoxic CTL targeting to a HIV infected cell.

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