KR100348183B1 - Tandem Synthetic HIV-1 Peptides - Google Patents

Abstract

New synthetic peptides have been provided that provide vaccines against HIV-1, which are very useful for diagnostic applications. These peptides comprise the amino acid sequence of the T-cell epitope of the gag protein of HIV-1, in particular p24E directly linked to the amino acid sequence of the B-cell epitope of the V3 100P protein of the HIV-1 isolate, the GPGR sequence and / or the ELKDWA sequence It contains gp41 containing. Multimeric forms of tandem synthetic peptides are provided.

Description

Tandem Synthetic HIV-1 Peptides

Reference to related application

The present invention is a continuation-in-part of US patent application Ser. No. 08 / 073,378 filed June 9, 1993.

FIELD OF THE INVENTION The present invention relates to the field of immunology and, in particular, to synthetic peptides containing T- and B-cell epitopes from human immunodeficiency virus proteins.

Background of the present invention

AIDS is the end result of infection with human immunodeficiency virus (HIV). Currently, there is no effective vaccine that can protect the human population from HIV infection, so urgent development of effective HIV-vaccines is required. Already, HIV-1 microparticles that are completely inactivated by chemical treatment, vaccine vectors encoding the entire envelope protein (GP160) of HIV-1 and purified recombinant gp120 are evaluated as candidate HIV vaccines. Inactivated HIV-1 virus production induces T-cell-mediated-delayed hypersensitivity (DTH) in the human body, and although vaccine / gP160 and gp120 recombinant vaccine candidates induced viral neutralizing antibodies, none of these immunogens Neither has provided an effective human HIV vaccine (Ref. 1—the references are listed at the end of the specification).

The inventors' interest in HIV vaccology develops synthetic HIV-1 peptides for binding to vaccines, and the vaccine HIV-1-recombinant subunits used in conjunction with this HIV-1 peptide vaccine are directed to HIV-1. To induce a more effective immune response. To design synthetic HIV vaccine candidates, viral B-cell immunization epitopes (BE) of immunogens with high conserved sequences between viral isolates are strong and linked to functional T-helper cell determinants (THD). Induces a long lasting cross-protective antibody response. In addition, HIV-specific cytotoxic T-lymphocyte (CTL) epitopes may be included in the synthesis to induce cell-mediated immunity to HIV infection.

Specific and preferential spatial relationships between certain T-cell epitopes and B-cell epitopes are required for and are thus immunogens for tandem epitopes to be effectively processed. Therefore, it is important to identify the appropriate T- and B-cell epitope sequences in the HIV-1 protein and combine them in the best configuration so that T-cell memory and B-cell memory are effectively induced and produce the desired specific antibodies. THD has not been found to be universal and functions immunologically only when presented in conjunction with the appropriate major histocompatibility complex (MHC) class II antigen. There is a characteristic classification system for T-cell epitope dominance. In order to develop effective synthetic AIDS vaccines, it is important to utilize the most potent THD among various HIV-1 gp160, gag, pol proteins and other gene products. Recent studies indicate that gag gene products play an important role in inducing an immune response to HIV infection. Therefore, clinical progress of AIDS is associated with a decrease in circulating antibodies against gag p24 protein, and antibodies raised against immunodominant gag p17 peptides can inhibit HIV-1 infection in vitro (Refs. 2, 3). ).

Published International Patent Application WO 90/13564 discloses the distinction and characterization of T-cell epitopes of core proteins, p24E of HIV-1 and T-cells linked to amino acid sequences of envelope proteins of HIV-1 or B-cell epitopes of coa proteins. Disclosed is the construction of a synthetic chimeric peptide comprising the amino acid sequence of an epitope. Linking the B-cell epitope with the T-cell epitope induces an immune response to the B-cell epitope while no such response is observed when the B-cell epitope is not so linked. Data for all epitopes derived from p24 T-cell epitopes, BE3 epitopes, ENV epitopes and V3A epitopes, and HIV-1 / LAV isolates, with or without linker sequences between epitopes, are presented in the publication. do.

Specific constructs tested in the published WO specification are BE3 linked to the C-terminus of p24E by direct coupling or to the N-terminus of p24E by two proline residues or by direct coupling, In both cases by two proline residues, the ENV is linked to the N-terminus and the C-terminus of p24E, and V3A is linked to the N-terminus of p24E by two proline residues.

The V3A sequence tested in the publication of the variant loop of HIV-1 gp120 from HIV-1 / LAV isolates (residues 308-327) is made immunogen by linking the molecule to the N-terminus of p24E with a proline-proline linker. U.S. Patent No. 4,925,784 (Crowl) discloses 15 to 512 amino acids in gag protein and env proteins of LAV isolates of HIV-1 (HTLV-III) by recombinant means, ie 1093 amino acids longer than any synthetic peptide. It has been known to provide fusion proteins comprising between 44 and 140 amino acids of a polypeptide or protein containing, which do not exceed 150 amino acids in length and are generally no longer than 50 amino acids. Such large molecular fusion proteins are described as useful for diagnostic applications and vaccine materials.

Envelope glycoproteins (env) of human immunodeficiency virus (HIV) are highly variable between isolates and also successive isolates from individuals infected alone. Amino acid variability in the envelope glycoprotein (env) is concentrated with other regions of low variability into specific variability regions (most at the surface portion gp120 caused by proteolytic maturation of the primary gp160 gene product). However, most variant regions often contain neutralizing epitopes so that the virus partially escapes the host's immune response and the infection persists. This variability presents problems for diagnostic techniques based on specific interactions with separation or mixing reagents commonly used in HIV-1 test samples. This variability also presents problems for possible vaccines or immunotherapy because suitable agents must provide a response to many lines of HIV-1.

Therefore, there are two problems in generating an immune response in the host against a number of immunologically specific HIV isolates. First, any particular host in a crosscrossed population will have a particular HLA haplotype and therefore will have to respond differentially to a specific T-cell epitope. Second, the antibody does not recognize or neutralize a number of immunologically specific HIV isolates and, in particular, HIV isolates obtained from patients as primary field isolates.

For diagnosis, production of immunological reactants, treatment and vaccination against HIV, B-cell epitopes from proteins of other HIV isolates, including T-cell epitopes and primary field isolates, to which multiple hosts will respond. It is beneficial to provide synthetic peptides.

Summary of the Invention

The present invention includes synthetic peptides, particularly synthetic HIV-1 peptides, at least one such synthetic HIV-1 peptide and composition useful for undertaking an immune response to infection by human immunodeficiency virus (HIV) and for detecting HIV infection. A vaccine against AIDS, a method for detecting HIV antigens using such HIV-1 peptides, and the provision of a diagnostic kit. Here, the synthetic HIV-1 peptide is a T-helper determinant of HIV-1 coa protein (T-cell epitope), in particular the amino acid sequence GPKEPFRDYVDRFYK (SEQ ID NO: 2) of p24E and HIV-1 proteins, in particular gp160, gag and pol. Amino acid sequences corresponding to B-cell epitopes from proteins.

By the term “synthetic peptide” as used herein, for example, a T-cell epitope comprising an amino acid sequence is linked to a B-cell epitope comprising an amino acid sequence using the synthetic peptide process described in Example 1 below. By means of forming a synthetic TB or BT configuration.

The major HIV-1 strain found in people with AIDS in North America and Western Europe is an isolate of HIV-1 (MN). Therefore, synthetic HIV peptides capable of protecting against this serotype contain neutralizing epitopes of p24E as THD and HIV-1 (MN) protein as B-cell epitopes. HIV-1 (MN) envelope protein. Two regions or epitope clusters in the extracellular component of gp120 have been shown to induce neutralizing antibodies to the virus. One of these regions is the third excessively mutant (V3) loop that surrounds 301-335 amino acid residues of gp120 (MN) (Ref. 4). Lineage and group-specific monoclonal antibodies isolated from individuals infected with MN isolates are shown to recognize different core amino acid sequences in the crown region of the V3 loop (Ref. 5).

Another epitope cluster of gp120 that induces neutralizing antibodies is the CD4 junction. Studies according to monoclonal antibodies isolated from HIV-1 infected individuals and chimpanzees showed that neutralizing epitopes at CD4 junctions are formed by intermittent amino acid residues from several sites of gp120.

Moreover, the results for both types of neutralizing antibodies were determined by the concentration of V3-specific neutralizing monoclonal antibodies in which a given amount of in vitro neutralization of HIV-1 virus was much lower than the concentration of one response to the CD4 junction. It is done.

In the construction of the synthetic peptides of the present invention, the inventors conserved very well in the crown region of the V3 (MN) loop linked to the amino acid (N-) or carboxy (C-) terminus of THD, p24E (CPKEPFRDYVDRFYK-SEQ ID NO: 2). Panel of linear synthetic HIV-1 (MN) peptides (indicated in Table 1 below, shown at the end of the details) comprising other flanking sequences adjacent to the isolated sequence (GPGR-SEQ ID NO: 1) Was chemically synthesized.

In addition, p24E-V3MN (MAP) comprising five tetrameric peptides, ie, the linear p24E-V3MN sequence, as shown in FIG. 1, indicating that the B-cell epitope comprising sequences is linked to the C-terminus of p24E. ); CLTB34 (MAP) comprising a linear CLTB-34 sequence; CLTB36 (MAP) comprising a linear CLTB-36 sequence; CLTB91 (MAP) comprising a linear CLTB-91 sequence; And VP-T-B (MAP) comprising a linear VP sequence (see also FIG. 1); Prepare and study each VP sequence comprising hybrid V3 sequences of residues 307-316 and 315-325 of the respective HIV-1 (MN) and HIV-1 (BRU) isolates, linked to the C-terminus of p24E, respectively. It was.

According to one aspect of the invention, human immunodeficiency virus (HIV) isolation linked to at least one amino acid sequence comprising a B-cell epitope of the V3 loop of the envelope protein of an HIV isolate at the N-terminus or C-terminus thereof Provided is a synthetic peptide comprising at least one amino acid sequence comprising a T-cell epitope of a gag protein of water. Here, when located at the N-terminal end, the B-cell epitope comprising the sequence and the T-cell epitope comprising the sequence are directly linked. Such synthetic peptides are novel and are not disclosed in WO 90/13564 described above.

According to another aspect of the invention, the sequence X ₁ LKDWX ₂ , wherein X ₁ is E, A, G or Q and X ₂ is A or T at the N-terminus or C-terminus thereof, in particular ELKDWA ( Human immunodeficiency linked to at least one amino acid sequence comprising the B-cell epitope of the gp41 protein of an HIV isolate comprising a sequence capable of inducing specific antiserum of HIV and recognizing sequence X ₁ LKDWX ₂ A synthetic peptide comprising at least one amino acid sequence comprising a T-cell epitope of a gag protein of a virus (HIV) isolate is provided.

Another aspect of the invention provides a synthetic peptide molecule comprising a plurality of individual chimeric synthetic peptides linked to form a multimeric molecule, wherein the synthetic peptide of each individual is a gag protein of a human immunodeficiency virus (HIV) isolate. Or a gag of an HIV isolate linked to an amino acid sequence comprising a B-cell epitope of an envelope protein or an amino acid comprising a T-cell epitope of an envelope protein. Such multimer molecules are novel and are not disclosed in WO 90/13564 described above.

The invention also provides antibodies specific for any of the synthetic peptides provided herein and nucleic acid sequences for encoding the synthetic peptides provided herein, wherein the nucleic acid sequence is linked to an expression vector.

HIV isolates associated with the present invention are generally HIV-1 isolates. The amino acid sequences of the synthetic peptides comprising the sequences of T-cell epitopes and B-cell epitopes comprising the sequences include LAV, BRU, MN, SF2, RF, PRI, 1714, 2054, HXB2, Z6, BX08, IIIB and SC. It can be a sequence of various HIV-1 isolates, including. Match sequences of other isolates may also be used.

In an embodiment of the invention where the B-cell epitope comprising the amino acid sequence is by a V3 loop protein, the amino acid sequence is preferably of the sequence GX ₁ GX ₂ (X ₁ is P or Z and X ₂ is P, K or Q) or HIV-specific antiserum and include a sequence capable of recognizing the sequence GX ₁ GX ₂ , in particular GFGR. A B-cell epitope comprising a sequence may comprise a B-cell epitope containing a V3 loop sequence from at least two different HIV-1 isolates, and V3 of at least two HIV-1 primary isolates It can contain a matching sequence of loops.

In various embodiments of the invention, the T-cell epitope comprising the amino acid sequence preferably comprises a sequence of p24 protein such as, for example, P24E, P24N, P24L, P24M and P24H, in particular P24E. The sequences of these peptides, which are well conserved among HIV-1 isolates, are shown in Tables 1 and 9.

Such sequences also include portions, modifications or mutations of one of the selected sequences that retain the T-cell properties of the protein or the selected sequence.

The amino acid sequence comprising the B-cell epitope may be directly linked to the C-terminal amino acid sequence of the amino acid sequence comprising the T-cell epitope.

The B-cell epitope comprising the sequence is additionally linked to a further amino acid sequence comprising an HIV T-cell epitope, which may be an amino acid sequence of HIV gag or envelope protein. B-cell epitopes comprising the sequence may also be linked to further amino acid sequences comprising the B-cell epitopes of HIV. The B-cell epitopes of the gp41 protein comprising the X ₁ LKDWX ₂ sequence are linked to each other or to the amino acid sequence comprising the B cell epitope of the V3 loop.

The multimeric molecules provided herein comprise a plurality of identical individual chimeric synthetic peptides and preferably comprise the aforementioned synthetic peptides.

The present invention provides an immunogenic composition comprising an immunologically effective amount of at least one synthetic peptide or at least one nucleic acid molecule encoding any of the peptides provided by the present invention and a pharmacologically acceptable carrier therefor. do. In addition, the present invention provides a method for immunizing a host, preferably a human host, which comprises administering an immunogenic composition as indicated.

Immunogenic compositions are selected to provide an immune response against a plurality of immunologically specific HIV-1 isolates and are preferably selected to provide an immune response in a plurality of hosts that respond differently to any T-cell epitope. Synthetic peptides.

Particularly useful “cocktails” of peptides useful in immunogenic compositions include the peptides specified in CTLB-36, CTLB-91 and BX08 in the tables below. The composition further comprises a peptide specified on MPK-2 in Table 11 below.

Immunogenic compositions are formulated for mucosal or parenteral administration. The immunogenic composition may further comprise at least one other immunogenic or immunostimulatory substance, in particular an adjuvant such as aluminum phosphate or aluminum hydroxide.

The invention also extends to diagnostic kits useful for the detection of HIV specific antibodies in test samples.

(a) a surface;

(b) at least one peptide having an amino acid sequence epitope specific for HIV-specific antibodies immobilized on a surface as set forth herein;

(c) means for contacting the antibody with at least one immobilized peptide to form a complex; And

(d) means for detecting the complex.

In another aspect of the invention, there is provided a diagnostic tool for detecting HIV antigen in a test sample, which

(a) a surface;

(b) antibodies that are epitope specific and non-cross react with specific epitopes of HIV antigens cultured with the peptides set forth herein and immobilized on the surface;

(c) means for contacting the HIV antigen with the antibody to form a complex; And

(d) means for detecting the complex.

1 shows the composition of tetrameric peptides capable of inducing polyclonal antibody responses in murine and / or guinea pigs against HIV-1;

FIG. 2 graphically depicts the antibody response in guinea pigs immunized with particulates, such as non-infectious, non-repeatable HIV-1 (IIIB) and boosted with an HIV-1 peptide cocktail, as shown in the Examples. Represented by; And

FIG. 3 graphically shows the reactivity of guinea pig antisera raised and then immunized with HIV-1 peptide cocktail after being detonated with particulates such as non-infectious, non-repeatable HIV-1 (IIIB), as shown in the Examples. .

In one embodiment, the present invention corresponds to antigenic determinants of a V3 loop linked to the N- or C-terminus of HIV-1 core protein p24, a highly conserved T-cell epitope, P24E (as shown in Table 1). Peptides having an amino acid sequence. Such peptides are, for example, shown in bold linked to the N- and C-terminus of p24E (HIV-1 (MN) core protein, amino acids 291-305 of p24) as T-cell epitopes each containing amino acid sequences. Unless stated otherwise in the specification, the bolded sequence is a B-cell epitope containing amino acid sequences) and the sequence RIHIGPGRAFYTTKNGPKEPFRDYVDRFYK (V3MN-p24E-SEQ ID NO: 10) corresponding to 311-325 amino acids of the V3 (MN) loop. It may have the sequence GPKEPFRDYVDRFYKRIHIGPGRAFYTTKN (p24E-V3MN-SEQ ID NO: 9). Such peptides include, for example, the sequence RKRIHIGPGRAFGPKEPFRDYVDRFYK (CLTB-32-SEQ ID NO: 13) and the sequence GPKEPFRDYVDRFYKPKRIHIGPGRAF corresponding to amino acid sequences 309-320 of the bolded V3 (MN) loop, respectively, linked to the N- and C-terminus of p24E, respectively. (CLTB-28-SEQ ID NO: 12). These peptides also have the sequence RKRIHIGPGRAFYTTKNGPKEPFRDYVDRFYK (CLTB-35-SEQ ID NO: 7) and the sequence GPKETFRDYVDRFYKRKRIHIGPGRAFYTTKN corresponding to amino acid sequences 309-325 of the bolded V3 (MN) loop linked to the N- and C-terminus of p24E, respectively 34-SEQ ID NO: 6). These peptides also had the sequence NKRKRIHIGPGRAFYTTKNGPKEPFRDYVDRFYK (CLTB-37-SEQ ID NO: 4) and the sequence GPKEPFRDYVDRFYKNKRKRIHIGPGRAFTB36TTKN (SEQ ID NO: 4) corresponding to amino acid sequences 307-325 of the bolded V3 (MN) loop linked to the N- and C-terminus of p24E, respectively -SEQ ID NO: 3).

These peptides can elicit polyclonal HIV-specific antibody responses in rats, guinea pigs, and monkeys (Tables 3 and 4).

In another embodiment, the present invention is directed to quaternary peptides (as shown in Figure 1) capable of inducing polyclonal antibody responses against HIV-1 in rats or guinea pigs (Table 13). It contains multimerized molecules. Such multimeric molecules may have four linear p24E-V3MN sequences (SEQ ID NO: 9), tetramerized using lysine branching peptide synthesis technology, for example p24E-V3MN (MAP-multimer- Antigenic peptide). Such tetramers may also contain, for example, four lysine-branched CLTB-34 sequences (SEQ ID NO: 6), thus sequences represented by CLTB-34 (MAP). Such tetramers may also contain, for example, four lysine-branched CLTB-36 sequences (SEQ ID NO: 3), thus sequences designated CLTB-36 (MAP). Such tetramers may contain, for example, four lysine-branched CLTB-91 sequences (SEQ ID NO: 20), thus sequences designated CLTB-91 (MAP). These tetramers also have four lysine-branched VP-TB linear sequences linked to the C-terminus (SEQ ID NO: 2) of p24E, including, for example, the hybrid V3 sequence, VP (NTRKSIRIQRGPGRAFYTTKN-SEQ ID NO: 16). , GPKEPFRDYVDRFYKNTRKSIRIQRGPGRAFYTTKN (SEQ ID NO: 15). The hybrid V3 sequence, VP, itself is bold V3 (BRU) linked through its C-terminal end to the N-terminus of amino acid sequence 315-325 (GPGRAFYTTKN-SEQ ID NO: 18) of the bold V3 (MN) loop Amino acids 307-315 of the loop (NTRKSIRIQR-SEQ ID NO: 17).

The novel immunogenic compositions of the present invention contain T- and B-cell epitopes of HIV prepared as peptides linking the extracellular envelope region of HIV-1, specific antigenic determinants from gp120, gp41 and core protein P24. Peptides. Such compositions are useful for immunization to induce HIV-specific human immune responses, as shown in the data presented in the examples below when administered to mammals such as mice, guinea pigs, monkeys.

Synthesis of Peptides

To design synthetic peptide-based HIV immunogens, as described in Example 1, containing sequences from gp-41 linked to the N- or C-terminus of peptides containing V3 loops and T-cell epitopes Linear peptides were chemically synthesized using an automated ABI 430A solid-phase peptide synthesizer. Different combinations were formulated in Freund's adjuvant (FA) or aluminum phosphate (alum) to compare their ability to induce HIV-specific immune responses in mammals.

P24E-V37MN (MAP), CLTB34 (4) formed by tetramerization using respective lysine tandem epitopes, lysine branching peptide synthesis techniques of p24E-V3MN, CLTB-34, CLTB-36, CLTB-91, VP-TB Five multimeric molecules were prepared, labeled MAP), CLTB-36 (MAP), CLTB-91 (MAP), and TB-VP (MAP). When administered in alum or FA, their ability to induce HIV-specific immune responses in mammals was investigated.

Immunogenicity of Linear HIV Peptides in Mammals

The ability of linear HIV peptides to induce antigen responses in mammals was examined by immunizing rats, guinea pigs, or monkeys with respective peptides that were emulsified or absorbed into alum in FA. After four injections of 100 μg each by the subcutaneous route, IgG antibody responses were determined by peptide-specific EIA and in vitro synthium-blocking assays (Tables 2, 3, 4, 5, 7).

Four other V3 (MN) peptides, ie V3MN, CLTB-29, CLTB-55, and CLTB- containing other sequences flanked by the crown portion of the V3 (MN) loop but lacking the p24E sequence 56 were either non-immunogenic or immunogenic, regardless of whether injected in FA or aluminum phosphate (Table 3). The carrier function of P24E to increase the immunogenicity of these peptides was found by studies performed with each synthetic HIV-1 peptide, including T-cell epitopes and B-cell epitopes. Thus, synthetic HIV-1 in T-B orientation elicited much more V3 (MN) -specific antibody responses compared to V3 (MN) peptides or B-T corresponding ones, each without (Table 3). Therefore, these results show that the orientation of V3 (MN) peptides to p24E affects the immunogenicity of the synthetic HIV-1 peptides of the invention. According to a comparison of the titer of each anti-V3 peptide antibody measured in mouse and guinea pig antiserum generated for each synthetic HIV-1 peptide administered in FA or aluminum phosphate, CLTB-36 was a guinea pig. The highest immunogenicity was found in both rats and rats (Table 3).

The immunogenicity of CLTB-36 was further demonstrated by its ability to induce strong peptide-specific antibody responses in virus neutralizing primates when formulated in adjuvant ISA 51 (Table 4). Importantly, the antiserum of rats and guinea pigs, raised against CLTB-34 and CLTB-36, can both cross react with V3 peptides of various HIV-1 serotypes (Table 5).

The new use of the p24E and V3 sequences for the construction of immunogenic TB peptides was followed by three different representations: p24E-SP10 (A), CLTB-91, CLTB-84, T1-SP10 (A) -MN (Table 1). This is further illustrated by studies performed with chimeric peptides. These results shown in Table 3 show the sequence GPKEPFRDYVDRFYKCTRPNYNKRKRIHIGPGRAFYTTK (SEQ ID NO: 20) comprising the N-terminal end (CTRPNYNKRKRIHIGPGRAFYTTK-SEQ ID NO: 20) of the V3 (MN) loop linked to the C-terminus of p24E when administered in FA or alum. P24E-SP10 (A) with 19) was able to induce a good titration of CLTB-56-specific antibodies in the guinea pig. p24 degrees-SP10 (A) formulated in alum similarly induced high anti-CLTB-56 antibody responses in Balb / c (H-2 ^d ). In contrast, the sequence KQIINMWQEVEKAMYACTRPNYNKRKRIHIGPGRAFYTTK-SEQ ID NO: 21, containing the same N-terminal end of the V3 (MN) loop linked to the C-terminus of the T-cell epitope, reported T1 (KQIINMWQEVEKAMYA-) T1-SP10 (A) -MN with SEQ ID NO: 22)) showed low immunogenicity in guinea pigs and Balb / c (H-2 ^d ) mice. These data suggest that p24E acts as a more effective carrier for the N-terminal sequence of the V3 (MN) loop than T1. Moreover, T1 has been found to mediate the enhancement of immunogenicity of CLTB-56. This result shows that the guinea pigs immunized with the CLTB-91 peptide of sequence KQIINMWQEVEKAMYANKRKRIHIGPGRAFYTTKN-SEQ ID NO: 23, comprising CLTB-56 linked to T-cell epitope T1 in FA or alum, and CLTB-91 in alum It is indicated by high CLTB-56-specific antibody titers measured in serum samples of injected mice. Since CLTB-91 differs only in T1-SP10 (A) -MN and V3 (MN) sequences linked to the C-terminus of T1, these data suggest that CLTB-56 was used in T1-SP10 (A) -MN. It shows a better B-cell epitope than the N-terminal end of the (MN) loop. Moreover, P24M comprising the sequence GHKARVAEMSQVT (SEQ ID NO: 30) of p24, CLTB-84 comprising the CLTB-56 sequence linked to the C-terminus of other T-cell epitopes, and also in the guinea pig when injected in alum It was found to show high immunogenicity in (Table 3).

Five different panels of HIV-1 synthetic peptides were synthesized. In the first panel of peptides shown in Table 6 (SEQ ID NOs: 38-47), two different U.S. The V3 sequences of clinical HIV isolates, 1714 (NTRKRIHMGPGRAFYATGDIIG-SEQ ID NO: 48) and 2054 (NTRKGIHIGPGRAFYTGEIVGDIRQ-SEQ ID NO: 49), were linked to the C-terminus of p24E and T1 to form p24E-1714 and T1-2054, respectively. do. In addition, the V3 consensus sequence, PRI (NTRKSIPIGPGRAFYTTG-SEQ ID NO: 50), and the consensus sequences of the New York and Amsterdam isolates were linked to p24X, T1 or p24M to form TB peptides of CLTB-PRI, T1-PRI, p24M-PRI, respectively. do. Moreover, pTB is linked to LTB (NTRKSIRIQRGPGRAFYTIG-SEQ ID NO: 51), V3 sequences of RF (NTRKSITKGPGRVIYATGQIIG-SEQ ID NO: 52), and CLTB- respectively by linking the hybrid V3 sequence of MN and RF (NKRKRIHIGPGRVIYATGQIIG-SEQ ID NO: 53). The V3B, CLTB-V3RF and CLTB-HB constructs form three different TB peptides.

The second panel of the structures is shown in Table 7 (SEQ ID NOs: 54-68). The particular gp41 sequences used in their construction share a neutralizing epitope, ELDKWA (SEQ ID NO: 69), described in Reference 6. The results in Table 8 show that each of these peptides is recognized by human neutralizing monoclonal antibody, 2F5 (cf. 6).

Table 9 shows a third panel of constructed peptides (SEQ ID NOs: 70-84) These constructs show three different T-cell epitopes from gag, ie P24N for forming BT or TB synthetic peptides, respectively. (QMREPRGSDIAGTTSTL-SEQ ID NO: 70), P24L (EEMMTACQGVGGPGHK-SEQ ID NO: 73), and the use of the CLTB-56 sequence to be linked to the N- or C-terminus of P24M (GHKARVLAEAMSQVT-SEQ ID NO: 30,76). . Furthermore, the N- or the consensus sequence PRI (NTRKSIPIGPGRAFYTTG-SEQ ID NO: 50,80) of the New York and Amsterdam isolates is also the T-cell epitope, P24H (PIVQNIQGQMVHQAI-SEQ ID NO: 79) or T5 (VKYKVVKIEPLGVAP-SEQ ID NO: 82). Linked to the C-terminus to form BT or TB peptides, respectively.

A fourth panel of peptides is shown in Table 10 (SEQ ID NOs: 85-92). Peptides CLTB-102 and CLTB-105 represent T-coupling epitopes, gp41 linked to the C- and N-terminus of p24E, respectively. Constructs containing the hybrid sequence (ELLELDKWASLWNWF-SEQ ID NO: 93) and CLTB-56. CLTB-103 and CLTB-107 are peptides containing the same gp41 sequence as CLTB-56 linked to the C- and N-terminus of P24E, respectively. For CLTB-160 and CLTB-161 constructs, the V3 sequences from the London, India and Paris isolates (LIP), the V3 sequence of Thai HIV-1 virus (THAI) and the primary isolates found in New York and Amsterdam A consensus sequence of water was used to link to the C-terminus of T-cell epitopes, p24E and T1, respectively.

The fifth panel of prepared peptides is shown in Table 11 (SEQ ID NOs: 94-97). The peptide, MPK-1, contains a copy of the gp41 neutralizing epitope (ELDKWAS-SEQ ID NO: 98) linked via a GPG linking sequence linked to T1 and p24E T-cell epitopes at their N- and C-terminus, respectively. The structure of the peptide, MPK-2, is identical to MPK-1 except that the orientations of T1 and P24E are reversed. The preparation of MPK-3 and MPK-4 involves the use of two copies of the gp41 sequence ELDKWAS to make MPK-1 in order to link the N- or C-terminus to T-1 and p24E, or p24E and T1, respectively. It includes.

Immunogenicity of the HIV-1 Peptide Cocktail

The immunogenicity of the cocktails containing the five peptides of the invention was tested against guinea pigs (Table 2). Such tandem peptides include CLTB-70 containing the V3 sequence of an HIV-1 (SF2) isolate linked to the C-terminus of p24E; CLTB-72 containing the V3 sequence of HIV-1 (IIIB) linked to the C-terminus of p24E; CLTB-74 containing the V3 sequence of HIV-1 (RF) linked to the C-terminus of p24E; CLTB-76 containing the V3 sequence of HIV-1 (Z6) linked to the C-terminus of p24E; And p24E-GP41C containing the gp41 sequence of HIV-1 (BRU) linked to the C-terminus of p24E. The results shown in Table 2 indicate that animals immunized with cocktails in FA or alum elicited high antibody responses against each of the five tandem peptides.

Therefore, the results described above are based on the V3 loops of four different HIV-1 isolates (SF2, IIIB, RF, Z6) and the gp41 sequence of HIV-1 (BRU) when peptide cocktails were absorbed into alum. It is shown that it can induce a strong antibody response.

Other HIV-1 peptide cocktails consisting of CLTB-36, CLTB-91, CLTB-84 (shown in Table 1) and CLTB-70 (shown in Table 2), and HIV-1 self-assembly, non-replicating, non- Immunization schedules using infectious HIV-like particles (described in WO 91/058564, published on 1991, 5.2) were also investigated. The results shown in FIG. 2 show that CLTB-56 is a junior pig that is first immunized with HIV-like particles emulsified in incomplete Freund's Auxiliary Solution (IFA) and further immunized with a cocktail absorbed in alum. It can be seen that a strong antibody reaction was induced against the particles. The titration of virus neutralization of antiserum against MN isolates after the second booster immunization was 1,091. Very importantly, the results shown in FIG. 3 also showed that the antisera collected from post-secondary additional immunized animals with peptide cocktails contained the V3 sequence of viruses and primary clinical isolates raised in several laboratories. It can be seen that there is a strong cross-reactivity to these.

Other peptide cocktails may comprise an immunoeffective amount of any of the disclosed peptides, including synthetic peptides, which sequence X ₁ LKDWX ₂ at the N-terminus or C-terminus, wherein X ₁ is E, A, G, Q and X ₂ is A, T) or an HIV isolate comprising a sequence capable of inducing HIV-specific antiserum and recognizing sequence X ₁ LKDWX ₂ and specifically peptide MPK-2 (SEQ ID NO: 95, Table 11) At least one amino acid sequence comprising the T-cell epitope of the gag protein of the HIV isolate, linked to at least one amino acid sequence comprising the B-cell epitope of the gp41 protein of water.

The functional activity of antiserum produced against synthetic HIV-1 peptides was examined by testing their ability to inhibit synthium formation induced by homologous HIV-1 (MN). Mice after immunization with four V3 (MN) peptides containing only B-cell epitopes containing sequences, V3MN, CLTB-29, CLTB-55 and CLTB-56, administered in FA or aluminum phosphate (alum) Antiserum had no synthium blocking activity (Table 12). The guinea pig antiserum produced against CLTB-56 formulated in FA rather than alum showed greater than 90% synthium inhibitory activity at a dilution of 1:10. Within FA or alum, rat and guinea pig antisera raised against BT tandem synthetic peptides, ie V3MN-p24E, CLTB-32 and CLTB-35, each contain V3MN, CLTB-29, CLTB-55 peptides. It showed a low reactive titer of antibody with respect to the B-sep epitope, but also lacked synthium-blocking activity. However, the antiserum of the guinea pigs produced for CLTB-37 administered in FA or alum containing titrations of CLTB-56-specific antibodies of 1,250 to 1 and 450 to 1, respectively, resulted in a 10 to 1 citium-blocking titration It was found to have. The results of functional studies performed with antisera raised on immunogenic TB synthetic peptides showed that, in FA or aluminum phosphate, the serum of rat and guinea pigs raised against TB synthetic peptide, CLTB-36 It has been shown to strongly inhibit synthium-forming induced by this homologous (MN) virus. In addition, antisera raised against CLTB-36 formulated in ISA 51 in cynomolgus monkeys were found to have good neutralization titers (278 and 430 in both animals) for MN isolates (Table 4). Mice and guinea pig antisera raised against p24E-V3MN and CLTB-34 in FA containing an appropriate amount of V3MN- and CLTB-55-specific antibodies in greater amounts than the respective antisera raised against peptides in It has been found to have syntium-blocking activity. It was observed that the animal species used for immunization influenced the production of V3 (MN) -specific functional antibodies. This effect is due to the fact that, for example, the TB tandem synthetic peptide CLTB-28 in FA induces the same amount of anti-CLTB-29 antibodies in rats and guinea pigs, but only high antisera raised in guinea pigs. It is explained by having the appropriate amount of synthium-blocking activity.

Immunogenicity of Multimeric Molecules in Mammals

The ability to induce antibody responses of multimer molecules CLTB-36 (MAP), CLTB-91 (MAP), CLTB-34 (MAP), p24E-V3MN (MAP), VP-TB (MAP) in mammals, And guinea pigs were examined by immunization with molecules emulsified in FA or alum. After four doses of 100 μg, IgG antibody responses were determined by peptide-specific ELISA and in vitro Cynthium-Chartan analysis. The results of immunogenicity studies conducted with multimeric molecules in rats and / or guinea pigs are shown in Table 13. CLTB-56-specific peptide antibodies were generated in rat and junior ligates immunized with tetrameric CLTB-36 (MAP) in FA or alum. CLTB-91 (MAP) formulated in FA or alum induced similarly large amounts of CLTB-56-specific antibodies in these animals. Tetrameric TB tandem synthetic peptides CLTB-34 (MAP), p24E-V3MN (MAP) or VPTB (MAP) injected in FA also have high titers of CLTB-55, V3MN and VP-specificity in the guinea pig, respectively. Antibodies could be induced. Mice and guinea pig antisera raised against CLTB-36 (MAP) in FA or alum strongly inhibited synthium formation induced by HIV-1 (MN) virus (Table 14). . The guinea pig antisera raised against branch peptides CLTB-34 (MAP) and VP-T-B (MAP) in FA showed a similar potent neo-vitamin-blocking activity.

It will be apparent to those skilled in the art that various embodiments of the present invention are possible for many applications in the fields of vaccines, diagnosis, treatment and production of immunogenic reagents for HIV infection.

Vaccine Manufacturing and Use

Peptides according to the invention can induce an immune response. One possible use of such molecules can be used as the basis of a potent vaccine for AIDS and AIDS related conditions. The present invention therefore provides a vaccine against AIDS, AIDS related conditions, comprising a molecule according to the invention.

Immunogenic compositions suitable for use as vaccines can be prepared from immunogenic peptides as disclosed herein. Immunogenic compositions induce an immune response that is opsonizing and produces antiviral antibodies. If the individual receiving the vaccine is challenged by HIV, the antibodies will bind to the virus and inactivate them.

Vaccines containing peptides are generally well known in the art. See, eg, US Pat. No. 4,601,903; 4,599,231; 4,599,230; 4,596,792. Vaccines may be prepared in a form that is easy to inject, such as liquid solutions or emulsions. Peptides may be mixed with pharmaceutically acceptable excipients that are compatible with the peptides. Excipients may include water, saline, dextrose, glycerol, ethanol and combinations thereof. The vaccine may further contain adjuvant such as wetting and emulsifying agents, pH buffers or immunoadjuvant to enhance the effectiveness of the vaccine. Methods of achieving the adjuvant effect for vaccines include the use of reagents such as aluminum hydroxide or phosphate, which are generally used as 0.05 to 0.1 percent in phosphate buffered saline. Vaccines may be administered parenterally or subcutaneously or by intramuscular injection. In addition, other forms of administration, including suppositories and oral formulations, would be desirable. In the case of suppositories, binders and carriers may be included, such as, for example, polyalkalene glycols or triglycerides. Oral formulations can usually include excipients such as, for example, pharmaceutical grades of saccharin, cellulose, magnesium carbonate. Such compositions take the form of solutions, suspensions, tablets, capsules, sustained release formulations or powders and contain 10 to 95% of peptides.

These vaccines are administered in a manner compatible with the dosage formulation, the amount of which must be therapeutically effective, protective and immunogenic. The amount to be administered depends on the individual being treated, for example, depending on the individual's ability to synthesize antibodies and generate a cell-mediated immune response. The exact amount of active ingredient to be administered is at the discretion of the practitioner. However, suitable dosage ranges can be readily determined by one skilled in the art and can be in units of micrograms of peptides. Appropriate therapies for initial and booster doses are also variable, for example self-administered after initial administration, as described in 91/058564, assigned to the assignee, who is secondarily immunized with the peptides provided herein. -Continuous administration of at least one pre-peptide with assembleable, non-infectious, non-replicating HIV-like particles. The dose of vaccine may depend on the route of administration and may vary with the size of the host.

Nucleic acid molecules encoding peptides according to the invention may be administered directly, for example by injection, or first immunized by constructing a live vector such as Salmonella, BCG, Adenovirus, Poxvirus, Vaccinia, Poliovirus and administering the vector. Can be used directly. A discussion of some live vectors used to transport heterologous antigens to the immune system is given, for example, in O'Hagau 1992, (reference 10). Procedures for injecting DNA directly into a test subject for gene immunity are described, for example, in Ulmer et al., 1993 (Ref. 11).

Since the peptides themselves do not have sufficiently long serum and / or tissue half-lives, the in vivo use of the peptides provided herein requires their modification. To this end, the molecules of the invention can be optionally linked to a carrier molecule via chemical groups of amino acids of the sequence to be conserved or additional amino acids added at the C- or N-terminus. Many suitable bonds are known and, for example, utilize side branches of the Tyr residue. Suitable carriers are, for example, kilimetpethemocyanin (KLH), serum albumin, purified protein derivatives of tubulin (PPD), ovalbumin, non-protein carriers and many others.

In addition, it may be advantageous to modify the peptides to impose morphological constraints. This is useful, for example, to mimic naturally-occurring forms of peptides in the environment within natural proteins to optimize the effector immune responses that are induced. The modified protein is referred to herein as "analog" peptides. The term "analogue" extends to functional and / or structural homogeneous peptides, characterized by increasing stability and / or efficiency in vivo or in vitro in the practice of the present invention. The term "analogue" is used to extend to any amino acid derivative of the peptides described herein.

Analogs of the peptides presented herein include, but are not limited to, modifications of the side branches, non-natural amino acids and / or derivatives thereof, non-amino acid units and coalescing with cross-linkers. Other methods for imposing morphological constraints on peptides and their analogs may also be contemplated.

Peptides according to the invention can be modified by a variety of methods without seriously affecting their functionally important immunogenic behavior. Possible modifications to the peptide sequences include the following.

One or more individual amino acids may be compared or substituted by amino acids having similar properties. That is, V may be substituted by I; T may be substituted by S; K may be substituted by R; L may be substituted by I, V or M.

As discussed in WO 91/13909, one or more amino acids of the peptides according to the invention can be replaced by "retro-inverse" amino acids, ie bifunctional amines having functional groups corresponding to the amino acids.

One or more amino acids may be deleted.

Structural analogs that mimic the three dimensional structure of a peptide can be used in place of the peptide itself.

Examples of side branch modifications contemplated by the present invention include reductive alkylation, which reduces to NaBH ₄ after reaction with aldehydes; Amidation with methylacetimidate; Acylation with acetic anhydride; Carbamoylation of amino groups using cyanates; Trinitrobenzenelation of amino groups with 2,4,6, trinitrobenzene sulfonic acid (TNBS); Acylation of amino groups using amber anhydride and tetrahydrophthalic anhydride; And modifications of amino groups such as pyridoxylation of lysine using pyridoxal-5'-phosphate and reducing to NaBH ₄ .

Guanidino groups of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanidione, phenylglyoxal and glyoxal.

The carboxyl group can be transformed, for example, into the corresponding amides by carbodiimide activation through derivatization followed by O-acylisourea formation.

Sulfhydryl groups include carboxymethylation with ioacetic acid or ioacetamide; Perfomic acid oxidation to cysteic acid, formation of mixed disulfides with other thiol compounds; Reaction with maleimide, maleic anhydride, or other substituted maleimide; Formation of a mercury derivative using 4-chloromercurybenzoate, 4-chloromercuryphenylsulfonic acid, phenylmercury chloride, 2-chloromercury-4-nitrophenol and other mercury agents; It may be modified by a method such as carbamolation with cyanate at an alkaline pH.

Tryptophan residues can be modified, for example, by oxidation with N-bromosuccinimide, alkylation of an indole ring with 2-hydroxy-5-nitrobenzyl bromide, or sulfenhalides. Tyrosine residues, on the other hand, can be altered by tetranitromethane to form 3-nitrotyrosine derivatives.

Modification of the imidazole ring of the histidine ring can be accomplished by alkylation with iodoacetic acid derivatives or N-carbetoxylation with diethylpyrocarbonate.

Examples of binding of non-natural amino acids and derivatives during peptide synthesis include norleucine, 4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine , Norvalin, phenylglycine, orytin, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienylalanine, and / or D-isomers of amino acids.

Crosslinkers include, for example, bifunctional imido esters with (CH ₂ ) _n , spacer groups having n to 6, glutaaldehyde, N-hydroxysuccinimide esters and the like. Homo-bifunctional crosslinkers, such as maleimido or dithio (for SH) or specific-reactive moieties such as carboimide (for COOH), and amino-reactive such as N-hydroxysuccinimide Hetero-bifunctional reagents comprising moieties can be used to stabilize three-dimensional forms. In addition, peptides may be morphologically restricted, for example, by the addition of α-methylamino acids, the introduction of double bonds between neighboring C atoms of amino acids and the formation of cyclic peptides or analogs.

Peptides or analogs thereof of the present invention may appear as a single length or as multiple tandem or non-tandem repeats. A single type of peptide or analog can form repeats or the repeats can be composed of other molecules including appropriate carrier molecules.

The immunogenicity of peptides according to the invention can be regulated by coupling with fatty acid moieties to produce lipidated peptides. Suitable fatty acid moieties are glycolipide analogs, N-palmityl-S- (2RS) -2,3, -bis- (palmitoyloxy) propyl-cysteinyl-serine (PAM ₃ -Cys-Ser), N- Palmityl-S- [2,3 bis (palmitoyloxy)-(2RS) -propyl- [R] -cysteine (TPC) or dipalmityl-lysine moiety.

Peptides may also be conjugated to fatty acid amino acids, such as octadecyl esters of aromatic acids, such as tyrosine, including acetadecyl-tyrosine (OTH).

Molecules according to the invention find further use in the treatment (prophylactic or therapeutic) of AIDS and related conditions by isolating the HIV virus's binding to human or animal cells or disrupting the three-dimensional structure of the virus. Can be.

therefore. The invention also provides a method for the treatment or prevention of AIDS or related conditions, which comprises administering an effective amount of peptides according to the invention.

Immunological assay

Peptides according to the invention are useful as immunogens, as antigens in the procedures known in the art for assaying immunoassays (ELISA), RIAs and other non-enzyme linked antibody binding assays, or anti-HIV antibodies. In ELISA assays, peptides are immobilized on a selected surface, such as, for example, a peptide bindable surface, such as wells of a polystyrene microtiter plate. After washing to remove incompletely adsorbed peptides, non-specific proteins known to be antigenically neutral to the sample, such as bovine serum albumin (BSA) or casein, can be bound to the selected surface. This provides for blocking of non-specific adsorption moieties, thus reducing the background caused by non-specific binding of antiserum to the surface.

In diagnostic embodiments where it is desirable to identify antibodies that can recognize a plurality of HIV isolates, a plurality of peptides of the invention are immobilized on a selected surface. In addition, when the B-cell epitopes of the peptides of the invention are highly conserved between various HIV isolates (eg, B-cell epitopes from gag or gp41), one or a limited number of peptides can be immobilized. . In diagnostic embodiments where it is desirable to specifically identify antibodies that recognize a single HIV isolate (eg, BRU, MN or SF2), a single peptide of the invention may be immobilized. Such diagnostic embodiments are particularly useful in the medical, clinical trials, and forensic fields where it is important to determine the particular HIV isolate responsible for producing antibodies.

Typically, peptides range from about 14 to about 40 residues at about 12 residues. Mixtures of peptides can be used as immunogens or as a vaccine or diagnostic agent, for example. There may be situations where mixtures of peptides from conservative moieties and / or non-conservative moieties may be used to provide cross-isolate protection and / or diagnostics. In such cases, mixtures of peptide immunogens are generally referred to as "cocktail" preparations for use as immunogenic compositions or diagnostic samples.

The fixed surface is then contacted in a manner that aids in the formation of an immune complex (antigen / antibody) with samples such as clinical or biological substances to be tested. This involves diluting the sample with diluents such as a solution of BSA, bovine gamma globulin (BGG) and / or phosphate buffered saline (PBS) / Tween. The sample is then incubated for about 2-4 hours in the temperature range of about 25-37 ° C. After incubation, the sample-contacted surface is washed to remove non-immune complexed material. This washing procedure may include washing with a solution such as PBS / Tween or borate buffer.

After formation of the specific immunocomplexes between the peptides associated with the test sample and subsequent washing, the occurrence and amount of the immunocomplexes can also be determined by treating the immunocomplex against the second antibody having specificity to the first antibody. If the sample was human, the second antibody is an antibody that is specific for human immunoglobulin and generally IgG. In order to provide a means for detection, the second antibody may have associated activity, such as, for example, enzymatic activity, such as incubation with a suitable chromogenic substrate to give color. Quantification can then be accomplished by measuring the degree of color development, for example using a visible light spectrophotometer.

Other uses

Molecules that bind to the conserved sequences on which the present invention is based, in particular antibodies, antibody-related molecules and their structural analogs, can also be used as agents in the diagnosis and treatment of AIDS or related symptoms.

Variants of antibodies (including antigen binding sites), such as chimeric antibodies, humanized antibodies, veneded antibodies, treated antibodies bound to peptides of the invention, are within the scope of the invention. Included.

Antibodies and other molecules that bind to the peptides of the invention can be used for therapeutic (prophylactic and therapeutic) and diagnostic purposes by many different methods, including:

It is used for the purpose of passive immunization by appropriate administration of antibodies, possibly humanized antibodies, to HIV patients.

It is used for the activity, supplementation, or mediation of antibody dependent cytotoxicity (ADCC) by the use of antibodies of the appropriate subclass or isotype (obtainable by appropriate antibody treatment) capable of performing the desired function.

For example, in order to bind directly or indirectly to the target conserved sequence of gp120 or gp41, targeted delivery of toxins or other agents can be achieved, for example, by the use of immunotoxins consisting of conjugates of antibodies and cytotoxic moieties. To be used.

It is used for the intended delivery of highly immunogenic substances to the surface of HIV-infected cells that allow for the removal of the cells in the host's body fluids or by the cell's immune system.

For example, it is used to detect HIV using various immunological assay techniques.

In another diagnostic embodiment, the peptides according to the present invention (mono, respectively or as a mixture prepared in a cocktail) are useful for detecting HIV or antigens or HIV antigen specific antibodies (mono) useful for neutralizing HIV in samples including biological samples. Useful for the production of clonal antibodies).

In another diagnostic embodiment, peptides according to the invention can be used to specifically stimulate HIV specific T-cells, for example in biological samples from HIV-infected individuals.

The above disclosure generally describes the invention. With reference to the following embodiments a more complete understanding can be achieved. These examples are disclosed for illustrative purposes only and do not limit the scope of the invention. Changes in form and substitution of equivalents may be considered when presented or made convenient. Although specific terms are used herein, these terms are for illustrative purposes and are not intended to be limiting.

Examples

Dr. Duke University, which is not expressly disclosed herein. Methods of synthesizing peptides, enzyme immunoassays (EIA) and in vitro syntium-blocking assays (Ref. 7) used by Thomas Mattews group (NC, USA) have been fully reported in the scientific literature and It is well known to the skilled people.

First embodiment

This example illustrates the synthesis of shaped peptides.

The peptides set forth in Tables 1, 2, 6, 7, 9, 10, 11 below are synthesized according to the amino acid sequences reported for the various HIV-1 isolates, identified using an ABI 430 peptide synthesizer, and confirmed by the manufacturer. T-Boc chemistry was optimized as described. The initial peptide product was treated with hydrofluoric acid (HF) to remove from the resin and 15-55% acetonitrile gradient in 0.1% (v / v) acetic acid with trifluoro developed for 40 minutes at a flow rate of 2 ml / min. Purification was performed by reverse-phase high performance liquid chromatography (RP-HPLC) using the used Vydac c4 semi-prepared column (1 × 30 cm). All synthetic proteins used in the immunoassays and immunological studies (Table 1 below) were determined to be at least 95% pure by analytical HPLC. The amino acid composition analyzes performed on the Waters Pico-T-AG system were in good agreement with the theoretical composition ratio.

Second embodiment

This example illustrates the synthesis of sided peptides.

The synthetic branched HIV-1 peptides (MAPs) shown in FIG. 1 were prepared using an AIB 430A peptide synthesizer and synthesized according to the manner already described by Tam (reference 8). As such, it was purified by RP-HPLC.

Third embodiment

This example describes the protocol used for immunogenicity testing of HIV-1 chimeric peptides.

Each of Canada in Montreal, Charles River Animal Farm and the United States of America Colorado Denver Haj reton animals purchased five each from the main farm 6-12 ^{Balb / c (H-2 d} ) female mice and 6-8 in three main tree and Duncan Affinity Guinea They were each immunized with 100 μg of constant peptides as follows. Animals were injected subcutaneously with a constant dose of peptides emulsified in CFA or adsorbed to 3 mg of alum. Three weeks later, the same amount emulsified in IFA or adsorbed to 3 mg of alum was further dosed. Mice were further dosed twice with equal amounts of the same peptide prepared in each adjuvant at three week intervals. Serum from post-immunized test mice and guinea pigs, respectively, were collected and assayed for peptide-specific IgG antibodies using standard EIA on day 9 and 14 and analyzed for synthium-blocking activity.

Fourth embodiment

This example illustrates the testing of anti-peptide antibodies using EIA.

Reactive BEs containing V3 peptides were detected in EIA plates (Covalink, Nunc, Denmark) by EIA for detecting antibodies reacting with V3 peptides as shown in FIG. 1 and Table 1 at 1 mg per well according to the procedures described in Reference 9. ) Was carried out by coating. After incubation for 30 min at 4 ° C., unbound peptide was washed three times with washing buffer [phosphate-buffered saline (PBS) pH 7.0 containing 0.025% Tween 20 (Bio-rad Laboratories, Richmond, CA)] Remove them. A 3-fold diluted solution of each serum sample, starting with 1 of 50, is then made in PBS containing 0.05% skim milk, and then 100 μl of diluted serum is added to each peptide-coated wells. Is added. Each dilution of serum samples is analyzed repeatedly. Incubate the plate for 1 hour at room temperature to allow V3 peptide-specific antibodies to bind to the immobilized peptide. Unbound antibodies are removed by washing the plate three times with a wash buffer. Then, as recommended by the manufacturer, 100 microliters of goat anti-rat IgG antibody horseradish peroxidase conjugate (Jackson Lab.) Diluted 1 in 5.000 in the wash buffer as recommended to each test well was added to the target peptide. Detect specific binding of anti-V3 peptide antibody. After incubation for 1 hour at room temperature, unbound antibody-conjugates are removed by washing the plate four times with a wash buffer. 100 μl of a mixture of tetramethylbenzidine (TMB) and hydrogen peroxide (1 part TMB for 9 parts hydrogen peroxide as recommended by the manufacturer, ADI Diagnostics Inc., Willowdale, Canada) is added to investigate the amount of conjugate conjugated. The color is allowed to appear for 10-15 minutes in the dark at room temperature, and stopped by adding 100 µl of 1N sulfuric acid. The optical density of the enzymatic reaction is read by a spectrophotometer (Titert Multi Skan Spectrophotometer (MCC / 340model) at 450 nm.) The results are shown in Table 3 and expressed as mean reciprocal titration. The amount of reciprocal titration of serum was always 50 or less.

Summary of the Invention

The present invention provides amino acid sequences comprising T-cell epitopes of HIV-1 gag protein, amino acid sequence corresponding to gP41 protein comprising V3 loop and / or ELKDWA sequence of envelope protein comprising GPGR sequence, HIV-1 Provided are quaternary forms of such peptides that elicit immunity against infection and vaccine compositions comprising such tandem synthetic peptides. Variants are possible within the scope of this invention.

Claims (32)

A synthetic peptide comprising an amino acid sequence comprising a gag of an HIV isolate or a T-cell epitope of an envelope protein linked to an amino acid sequence comprising a gag of an HIV isolate or a B-cell epitope of an envelope protein, The synthetic peptide, (A) a synthetic peptide, wherein a plurality of such respective synthetic peptides are joined to form a multimeric molecule; (B) the T-cell epitope comprises the T-cell epitope of the gag protein of the HIV isolate and the T-cell epitope of the gag protein comprises P24N, P24L, having amino acid sequences of SEQ ID NOs: 70, 73, 76, 79, respectively, Is a portion, modification or mutation of any of the selected sequences selected from P24M and P24H or retaining the T-cell properties of the selected sequence, wherein the B-cell epitope is the HIV isolate linked to the C-terminal end of the T-cell epitope Synthetic peptides comprising the B-cell epitope of the V3 loop of the envelope protein of water; (C) the T-cell epitope comprises a T-cell epitope of the gag protein of the HIV isolate and the B-cell epitope from at least two other HIV-1 isolates linked to the C-terminal chisel of the T-cell epitope Synthetic peptides comprising B-cell epitopes comprising a hybrid V3 loop sequence; (D) the consensus sequence of the V2 loop of at least two primary isolates comprising a T-cell epitope of the gag protein of said T-cell epitope HIV isolate, said B-cell epitope being linked to the C-terminal end of the T-cell epitope Synthetic peptides comprising B-cell epitopes comprising; (E) said T-cell epitope comprises a T-cell epitope of a gag protein of an HIV isolate and said B-cell epitope each comprises a V3 loop sequence from another HIV-1 isolate or HIV-isolate match sequence Synthetic peptides comprising two amino acid sequences; And (F) said T-cell epitope comprises a T-cell epitope of a gag protein of an HIV isolate and said B-cell epitope has an amino acid sequence X wherein X ₁ is E, A, G, or Q and X ₂ is A or T A synthetic peptide selected from the group consisting of ₁ LKDWX ₂ or a synthetic peptide comprising sequences capable of inducing HIV-specific antiserum and recognizing sequence X ₁ LKDWX ₂ . The synthetic peptide of claim 1, wherein the HIV isolate is an HIV-1 isolate. The synthetic peptide of claim 2, wherein the V3 loop is a V3 loop of an HIV-1 isolate selected from LAV, BRU, MN, SF2, RF, PRI, 1714, 2054, HXB2, Z6, BX08, IIIB and SC. The method according to any one of claims 1 to 3, wherein the multimeric molecule (A) has the following amino acid sequence: Or a synthetic peptide comprising a portion, modification or mutation of this sequence that retains the T- and B-cell properties of the sequence. The sequence GX ₁ GX according to any one of claims 1 to 3, wherein the B-cell epitope containing the amino acid sequence in (B) is X ₁ is P or L and X ₂ is R, K, Q. _2. A synthetic peptide comprising ₂ or comprising a sequence capable of inducing HIV specific antiserum and recognizing sequence GX ₁ GX ₂ . The synthetic peptide of claim 5, wherein said B-cell epitope containing an amino acid sequence comprises a sequence GPGR or comprises a sequence capable of inducing HIV-specific antiserum and recognizing sequence GPGR. 6. The synthetic peptide of claim 5, wherein said B-cell epitope containing an amino acid sequence binds directly to the C-terminus of said T-cell containing an amino acid sequence. The method of claim 5, wherein the B-cell containing an amino acid sequence is a sequence Or a synthetic peptide selected from parts, modifications or mutations thereof that retain the B-cell properties of this sequence. The method of claim 5; B-cell epitopes containing sequences are synthetic peptides additionally linked to additional amino acid sequences containing gag proteins of HIV or T-cell epitopes of envelope proteins. The method according to any one of claims 1 to 3, wherein in (C), the B-cell epitope containing an amino acid sequence is a sequence. Or a synthetic peptide comprising a portion, modification or mutation of a sequence that retains the B-cell nature of that sequence. The method according to any one of claims 1 to 3, wherein the amino acid sequence of the B-cell epitope in (D) is sequence NTRKSIPIGPGRAFYTTG (PRI), NTRKSIHIGPGRAFYTTGEIIGC (FRE), KSIHIGPGKTLYAT (LIP), RKSIPIGPGRAFYTSG (NYA), Or a synthetic peptide comprising a portion, modification or mutation of a sequence that retains the B-cell properties of the sequence. The amino acid sequence of any one of claims 1 to 3, wherein in (E) the amino acid sequence comprising a B-cell epitope from another V3 loop sequence from an HIV-isolate or from another HIV-isolation sequence is A synthetic peptide linked to at least two inoic acid sequences. The method of claim 12, wherein the synthetic peptide is a CTLB-160 or CTLB-161 having an amino acid sequence of SEQ ID NO: 91 or 92, or a portion, modification, or mutation of a sequence that retains the T- and B-cell properties of the peptides. Synthetic peptides. The T-cell epitope according to any one of claims 1 to 3, wherein the T-cell epitope containing the amino acid sequence is P24E having the amino acid sequences of SEQ ID NOs: 2, 70, 73, 76 and 79, A synthetic peptide comprising a portion selected from P24N, P24L, P24M and P24H or a portion, modification or mutation of any one of the selected sequences which retains the T-cell properties of the selected sequence. 15. The synthetic peptide of claim 14, wherein said B-cell epitope containing a sequence is additionally linked to a further amino acid sequence containing a gag protein of HIV or a T-cell epitope of an envelope protein. The synthetic peptide of claim 15, wherein said T-cell epitope has been selected to provide an immune response that responds differently to T-cell epitopes in a plurality of hosts. 16. The synthetic peptide of claim 15, wherein said additional T-cell epitope containing sequence is additionally linked to at least one additional amino acid sequence containing gp41 or V3 loop envelope protein of an HIV isolate. 18. The synthetic peptide of any one of claims 15-17, wherein the synthetic peptide is one of the amino acid sequences of SEQ ID NOs: 94-97 or a portion, modification or mutation of this sequence that retains the T- and B-cell properties of the peptide. Synthetic peptides. The T-cell epitope according to any one of claims 1 to 3, wherein the T-cell epitope containing the amino acid sequence in (E) comprises p24E or a portion thereof, modification or mutation that maintains the T-cell properties of the sequence. Wherein said B-cell epitope comprising an amino acid comprises a sequence ELKDWA or comprises a sequence capable of inducing HIV specific antiserum and recognizing sequence ELKDWA. The synthetic peptide of claim 1, wherein said B-cell epitope containing an amino acid sequence is directly bound to the C-terminus of said T-cell containing an amino acid sequence. The B-cell epitope according to claim 1, wherein the B-cell epitope containing the amino acid sequence is of the sequence EQELLELDKWASLWNWFDIT (CLTB-92A), ELLELDKWASLWNWFDIT (CLTB-94), ELDKWASLWNWFDIT (CLTB-96), EQELLELDKWASLWNWF, CTLBLDWAS, WBNWF A synthetic peptide selected from EQELLELDKWA, ELLELDKWA, ELDKWAS and GPGELLELDKWASL, or a portion, modification or mutation thereof that maintains the B-cell nature of the sequence. The amino acid sequence of any one of claims 1 to 3, wherein the B-cell epitope containing the sequence in (E) comprises at least one B-cell epitope of the V3 loop of the envelope protein of an HIV isolate. Synthetic peptides additionally linked to. The method of claim 22, wherein the synthetic peptide retains the T- and B-cell properties of CTLB-102, CTLB-103, CTLB-105, CTLB-107, p24E-KAT4, or a peptide of SEQ ID NOs: 85-88, 90 Synthetic peptides that are part, modification or mutations thereof. An immunoeffective amount of at least one synthetic peptide claimed in any one of claims 1 to 3 or at least one nucleic acid molecule encoding any of said synthetic peptides, and a pharmacologically acceptable carrier for it Immunogenic composition comprising a. The method of claim 24, wherein the immunogenic composition is selected to provide an immune response against a plurality of immunogenicly-separated HIV-1 isolates and responds differently to T-cell epitopes in the plurality of hosts. An immunogenic composition comprising a plurality of synthetic peptides further selected to provide said immune response. The method of claim 25, wherein the plurality of synthetic peptides comprises a mixture of CTLB-36 and / or CTLB-84, CTLB-91 and CTLB-70 having amino acid sequences of SEQ ID NOs: 3, 29, 23 and 24, respectively. Immunogenic Compositions. 27. The immunogenic composition of claim 26, wherein the plurality of synthetic peptides further comprises peptide MPK-2 having the amino acid sequence of SEQ ID NO: 95. The composition as claimed in claim 24 formulated as an HIV vaccine for use in humans. In a diagnostic kit useful for detecting HIV specific antibodies in a test sample, (a) a surface; (b) having an amino acid sequence epitope specific for HIV-specific antibodies immobilized on a surface thereof, and comprising: 1 to 3, 6 to 9, 14 and 15 to 17. At least one peptide as claimed in claim 20; (c) means for contacting the antibodies with at least one immobilized peptide to form a complex; And (d) means for detecting said complex. In a diagnostic tool for detecting HIV antigens in a test sample, (a) a surface; (b) Claims 1 to 3, 6 to 9, 14 and 15 to 15 which are comparatively reactive and epitope specific for different epitopes of the HIV antigen immobilized on the surface. 23. An antibody raised against said peptide as claimed in any one of claims 17, 20 and 21; (c) means for contacting the antibodies with the HIV antigens to form a complex; And (d) a diagnostic tool comprising means for detecting the complex. A nucleic acid molecule comprising a synthetic peptide as claimed in any one of claims 1 to 3, 6 to 9, 13, 15 to 17, 20 and 21. Nucleic acid molecule encoding. An antibody comprising the synthetic peptides claimed in any one of claims 1 to 3, 6 to 9, 13, 15 to 17, 20 and 21. Antibodies having specificity for either.