Cyclic peptides comprising, as a constituent chain or chains, one or two amino acid sequences selected from the groups consisting of the amino acid sequence Glu-Ala-Asp-Asp-Arg and the amino acid sequence Ser-Gln-Lys-Glu-Gly, and AIDS vaccines containing the cyclic peptide as an active ingredient. Preferably a cyclic dodecapeptide represented by the formula given below and an AIDS vaccine containing the cyclic dodecapeptide as an active ingredient. From the in vivo absorption and antibody formation viewpoint, active groups selected from among the carboxyl, amino and hydroxyl groups contained in the cyclic peptide is preferably bound to substituent groups. The cyclic dodecapeptide can neutralize the second receptors in the infection of human with HIV-1 virus -- see Original Patent.

Description of the Invention

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a National Phase Application (35 USC 371) of PCT/JP99/06174, filed Nov. 5, 1999 and claims priority of Japanese Application No. 11-32990, filed Feb. 10, 1999.

FIELD OF THE INVENTION

The present invention relates to cyclic peptides effective in preventing HIV-1 virus infection in human and to AIDS vaccines. More particularly, it relates to cyclic peptides which serve as antigens for producing a neutralizing antibody capable of neutralizing HIV-1 virus infection via the second receptors called CXCR4 and CCR5 and to AIDS vaccines which comprise the above antigens as active ingredients.

BACKGROUND OF THE INVENTION

Second receptors which the pathogenic virus causative of AIDS (HIV-1 virus) utilizes in infecting human were identified in 1996 (Yu Feng et al., Science, 272, 872-877, 1996). These two receptors are called CXCR4 and CCR5. It has been revealed that the HIV-1 virus utilizes one of the receptors for adsorption onto and entry into lymphocytes, macrophages and dendritic cells to achieve infection.

On the other hand, about 1 to 2% of Caucasians reportedly have resistance to HIV-1 virus infection and it has been revealed that this is due to a genetic defect or genetic incompleteness of the second receptors (CXCR4 and CCR5), which are chemokine receptors (Rong Liu et al., 86, 367-377, 1996).

These findings have called researchers attention to the importance of neutralization of the second receptors in the prevention of HIV-1 virus infection and, in recent years, attempts have been made to produce a neutralizing antibody capable of neutralizing the second receptors. There is no report, however, of the successful creation of such a neutralizing antibody.

Accordingly, it is an object of the present invention to provide three-dimensional antigens capable of producing, in vivo, a neutralizing antibody capable of neutralizing the second receptors from the stereoscopic viewpoint by paying attention to the loop structures of the second receptor proteins without following the conventional methods which interpret the peptides constituting the second receptors two-dimensionally. Another object is to provide AIDS vaccines which comprise such antigens as active ingredients.

DISCLOSURE OF THE INVENTION

The present inventors constructed a model of the second receptor in T cells (abbr.: CXCR4) and a model of the second receptor in macrophages (abbr.: CCR5) and observed them from a three-dimensional viewpoint. As a result, they explored the applicability of two pentapeptides constituting the second subloop (UPL) in the respective second receptor proteins, namely T cell-derived Glu.sub.179-Ala.sub.180-Asp.sub.181-Asp.sub.182-Arg.sub.183 (Seq. I.D. No. 2) and macrophage-derived Ser.sub.169-Gln.sub.170-Lys.sub.171-Glu.sub.172-Gly.sub.173 (Seq. I.D. No. 3), as constituent elements of a novel antigen for producing an HIV-1 virus infection-preventing antibody capable of neutralizing the second receptors.

Thus, the present invention provides a cyclic peptide which is a novel compound and which comprises, as a constituent chain thereof, one or two amino acid sequences selected from among amino acid sequences contained in the second subloop in the T cell second receptor protein and comprising at least five amino acid residues and amino acid sequences contained in the second subloop in the macrophage second receptor protein and comprising at least five amino acid residues. The present invention further provides AIDS vaccines comprising that compound as an active ingredient.

More specifically, the cyclic peptide of the present invention, which is a novel compound, is characterized in that it comprises one or two amino acid sequences selected from the groups consisting of the amino acid sequence Glu-Ala-Asp-Asp-Arg (Seq. I.D. No. 2) and the amino acid sequence Ser-Gln-Lys-Glu-Gly (Seq. I.D. No. 3) as a constituent chain or chains thereof, and the AIDS vaccine is characterized by comprising such compounds as active ingredients.

More particularly, the cyclic peptide of the invention is characterized in that it is a novel compound which is represented by the formula (1) (see Original Patent) given below and the AIDS vaccine of the invention is characterized in that it comprises that compound as an active ingredient.

FIG. 1 (see Original Patent) shows the configuration of a T cell-derived second receptor protein molecule on the T cell membrane (FIG. 1, top left) and the configuration of a macrophage-derived second receptor protein molecule on the macrophage membrane (FIG. 1, top right) and a cyclic dodecapeptide according to the invention as synthesized from the respective second subloop peptides of these second receptor protein molecules. In FIG. 1, the T cell-derived second receptor protein molecule (CXCR4) has a configuration comprising a first loop, a second loop, a third loop and a second subloop and the macropahge-derived second receptor protein molecule (CCR5) also has a configuration comprising a first loop, a second loop, a third loop and a second subloop.

The second subloop in the T cell-derived second receptor protein molecule (CXCR4) contains the amino acid sequence Glu.sub.179-Ala.sub.180-Asp.sub.181-Asp.sub.182-Arg.sub.183 (Seq. I.D. No. 2) and the second subloop in the macrophage-derived second receptor protein molecule (CCR5) contains the amino acid sequence Ser.sub.169-Gln.sub.170-Lys.sub.171-Glu.sub.172-Gly.sub.173 (Seq. I.D. No. 3).

A novel compound cyclic dodecapeptide of the present invention as represented by the formula (1) shown above (cyclic peptide shown in FIG. 1, bottom) can be obtained by causing both the peptides respectively having the above-identified amino acid sequences of both the second subloops of CXCR4 and CCR5 to form a ring via -Gly-Asp- as a spacer arm dipeptide.

Preferably, an active group selected from among the carboxyl, amino and hydroxyl groups contained in the cyclic dodecapeptide represented by the above formula (1) is bonded to a substituent group so that the absorption into the living body and antibody formation may be facilitated. Such a substituent can be selected from among the residues of fatty acids CH.sub.3(CH.sub.2).sub.n--COOH (n: 0 to 20), the residues of alcohols CH.sub.3(CH.sub.2).sub.n--OH (n: 0 to 20) and the unsaturated compound residues corresponding to such compound residues and preferably has biocompatibility. As appropriate examples of the fatty acid, there may be mentioned lauric acid, myristic acid, palmitic acid, stearic acid, arachidonic acid, and unsaturated fatty acids corresponding thereto. As appropriate higher alcohols, there may be mentioned lauryl alcohol, myristyl alcohol, palmityl alcohol, stearyl alcohol, eicosanol, and unsaturated alcohols corresponding thereto.

The cyclic dodecapeptide represented by the above formula (1) can be utilized as an immunogen for producing a second receptor neutralizing antibody capable of inhibiting HIV-1 virus infection.

An assaying antigen for antibody screening is prepared by binding the cyclic dodecapeptide to a solid phase resin. Separately, mice were immunized with an immunogen, for example a cyclic dodecapeptide-multiple antigen peptide (abbr.: CDP-MAP), and monoclonal antibodies are prepared by the conventional hybridoma technique. For confirming the anti-infective activity against HIV-1 virus infection, several hybridomas (cells produced by fusion of antibody-producing B cells and myeloma cells (cancer cells)) are prepared by the above method and anti-HIV-1 virus activity assaying is carried out in the conventional manner using the hybridoma culture supernatants, to show the culture supernatants prevent HIV-1 virus infection.

Thus, the cyclic dodecapeptide represented by the formula (1) can be used as an immunogen for producing antibodies having inhibitory effects against HIV-1 virus infection and therefore is useful as an active agent in AIDS vaccines.

The AIDS vaccines according to the invention contain as an active agent, a cyclic peptide comprising, as a constituent chain or chains thereof, one or two amino acid sequences selected from the amino acid sequence Glu-Ala-Asp-Asp-Arg (Seq. I.D. No. 2) and the amino acid sequence Ser-Gln-Lys-Glu-Gly (Seq. I.D. No. 3).

The AIDS vaccines according to the invention may comprise the above cyclic peptides as active agents or the active agents may be modifications of the cyclic peptides by substitution and/or addition or may be in the form of a pharmacologically acceptable salt. Pharmacologically acceptable salts include salts with hydrochloric acid, sulfuric acid, nitric acid, nitrous acid, hydrobromic acid, hydroiodic acid, phosphoric acid and organic acids.

An example of a suitable derivative (modification) of the compound of the above formula (1) is that in which the substituent group is a higher fatty acid group as shown (see Original Patent).

Five equivalents of 9-fluorenylmethoxycarbonyl-dimethylsulfonium methyl sulfate (Fmoc-DSP; tradename, product of Novabiochem) are added to 1 equivalent of the cyclic dodecapeptide-MAP represented by the formula (2) to thereby block the E-amino group of K.sub.4 of the cyclic dodecapeptide-MAP and then the carboxyl groups (E.sub.5, E.sub.7, D.sub.9, D.sub.10) are activated with EDC, DCC, BOP or the like, and a higher alcohol [CH.sub.3(CH.sub.2).sub.n--OH] is added in excess to thereby effect esterification. Alternatively, the hydroxyl group of Ser of the cyclic dodecapeptide-MAP represented by the above formula (2) is esterified by the acid chloride [CH.sub.3(CH.sub.2).sub.nCOCl] method and, after elimination of Fmoc, the ester is used as a base material of the peptide vaccine. When the vaccine is administered to a living body, it is delivered to lymphoid tissues where the ester is hydrolyzed. The thus-recovered original cyclic peptide-MAP represented by formula (2) activates the immune system, whereby antibodies are produced and the AIDS virus infection is neutralized.

The AIDS vaccines according to the invention can be used as pharmaceutical compositions in the form or oral or nonoral preparations. The oral dosage form includes tablets, powders, granules, capsules, microcapsules, solutions and the like. The nonoral or parenteral dosage form includes solutions, mainly injectable solutions, and suppositories, among others. Generally, these preparations may contain one or more pharmaceutical preparation auxiliaries such as carriers, excipients, binders, disintegrants, lubricants, stabilizers, flavors, and the like.

The dose may vary according to the symptom and/or age. In the case of oral administration, a daily dose of 0.1 to 1000 mg/kg body weight can be administered to normal adults.
 

Claim 1 of 3 Claims

1. A cyclic peptide represented by the formula -- see Original Patent.

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