Peptides that induce antibodies which neutralize genetically divergent HIV-1 isolates

Abstract: This invention refers to peptides binding to antibodies that show neutralizing activity against different strains and clinical isolates of HIV-1 and that inhibit the fusion of cells caused by HIV-1. These peptides are applied with an adjuvant, as recombinant fusion proteins, chemically coupled to carrier molecules, as recombinant chimeric viruses or as recombinant antibodies.

Description: This invention refers to peptides that induce antibodies which neutralize genetically divergent HIV-1 isolates. These peptides are applied with an adjuvant, as recombinant fusion proteins, chemically coupled to carrier molecules, as recombinant chimerical viruses or as recombinant antibodies. In addition, the stage of infection can be determined and the progression of the infection can be predicted with these peptides. INTRODUCTION The acquired immunodeficiency syndrome (AIDS) is the late stage clinical manifestation of long term persistent infection with human immunodeficiency virus type 1 (HIV-1). Immune responses directed against the virus and against virus-infected cells during the persistent infection usually fail to mediate resolution to the infection. A possibility to elicit an immune response that can prevent the establishment of a persistent infection or that can prevent the progression to AIDS are vaccines. Most vaccine strategies against HIV-1 are directed against the surface glycoprotein gp160 which is made up of gp120 and gp41 and is responsible for virus binding to the cellular receptor CD4 and fusion activity. However, in context with gp160 several phenomenons that argue against the use of whole gp160 or gp120 as an immunogen were observed. In vitro experiments showed, that synergism between HIV-1 gp120 and gp120-specific antibodies block human T cell activation (1). This result supports the hypothesis, that also in vivo the humoral immune response against gp120 of HIV-1 suppresses T-cell activation and might be one reason for immunodeficiency. The proposed mechanism for this phenomenon is cross-linking and modulation of CD4 molecules through gp120 and anti-gp120. Experiments from Kion et al. (2) suggest that sequence homologies between gp160 and class II MHC molecules lead to immunodeficiency. In addition, a number of antigenic domains on gp160 are known to induce antibodies that enhance HIV-1 infection (3). Such effects known in context with gp160 could be avoided by using synthetic peptides or other subunit vaccines that only contain immunogenic and neutralizing epitopes as immunogens. Immunogenic peptides corresponding to parts of different viral proteins were already used for successful immunization (4,5,6). The use of synthetic peptides as immunogens offers a number of advantages. The antibodies produced have a predetermined specificity, and in the case of viruses, they can be selected to represent structures on the surface of virions. The synthetic polypeptides also are interesting in that they can induce antibody responses not seen under normal conditions. For example, it was found that in the hemagglutinin of influenza virus there are five major antigenic regions and that under conditions of natural infection the immune response includes antibodies only to these regions. With synthetic polypeptides, an immune response against other regions of the hemagglutinin polypeptide can be generated, and these antibodies have been found to be capable of neutralizing the virus. Therefore it is possible to induce neutralizing antibodies that have a broader reactivity than antibodies induced by whole proteins (4). In addition, immunizations with peptides derived from the nucleotide sequence of foot and mouth disease virus (FMDV) are described. In contrast to immunizations with the corresponding whole protein of FMDV, immunizations with these peptides lead to neutralizing antibodies which were also protective (5). Furthermore, a peptide containing part of the V3 loop of gp120 from the HIV-1 isolate HIV-1 IIIB was shown to induce a protective immune response against virus challenge with the same HIV-1 isolate (7,8). Because synthetic peptides themselves have poor immunogenicity, they have to be coupled to molecules that provide an adjuvant effect such as tetanus toxoid or keyhole limpet hemocyan (5). Another possibility is to clone small peptides as fusion peptides with glutathione S-transferase of Schistosoma japonicum (9,10). In addition, attenuated viruses such as vaccinia, polio Sabin type 1 or influenza NA/B-NS can be used as vectors for immunogens. Vaccinia virus is used frequently as a vector of foreign genes of multiple pathogens. For example rabbits inoculated with recombinant vaccinia virus containing sequences from hepatitis B surface antigen (HBsAg), herpes simplex virus glycoprotein D, and influenza virus hemagglutinin produced antibodies to all three foreign antigens (11). Furthermore, a chimerical polio virus that expressed an epitope from gp41 of HIV-1 induced neutralizing antibodies against gp41 in rabbits (12). Since recently it is also possible to change the genome of influenza virus by in vitro mutagenesis (13). By means of this technique, it was possible to engineer a stable attenuated influenza A virus (14). In addition, by using this technique it was also possible to construct an intertypic chimerical virus, in which a six-amino-acid loop contained in the antigenic site B of the hemagglutinin of an H1 subtype was replaced by the corresponding structures of subtypes H2 and H3 (15). An advantage of influenza virus in this context is the availability of many variants so that repeated vaccination may be possible. Furthermore, influenza virus induces strong secretory and cellular immune responses, which may be advantageous for an anti-HIV-1 vaccine approach. In addition, it is unlikely that influenza virus is associated with the development of malignancies. There is no DNA phase involved in the replication of influenza viruses, which excludes the possibility of chromosomal integration of viral influenza genes. The use of antiidiotypic antibodies is another possibility to achieve a specific immune reaction. Antiidiotypic antibodies are antibodies that specifically recognize and bind the antigen binding site of another antibody. As the combining sites of antibodies can be structurally looked at as a mirror image of the epitope that is bound, an antiidiotypic antibody corresponds to the mirror image of this primary mirror image, which means that an antiidiotypic antibody displays the internal image of the epitope that is bound by the idiotypic antibody. Although one cannot always expect to find complete identity between the structure or the amino acid sequence respectively of the antiidiotypic antibody with that of the epitope, one can however see effects in practice that allow the conclusion that there is a structural, sequential or functional similarity between antiidiotypic antibodies and the respective epitopes. The use of antiidiotypic antibodies as a vaccine was initially proposed by Nisonoff and Lamoyi (16). In the case of African Sleeping Disease, it was first shown that a protective immune response against the causative agents, Trypanosoma brucei rhodesiense, could be elicited in BALB/c mice by vaccinating the mice with antiidiotypic antibody (17). In the case of viral antigens, the formation of antiidiotypic antibodies to a neutralizing epitope on the hemagglutinin molecule of Reovirus Type III was investigated. These antiidiotypic antibodies recognized the cellular receptor of Reovirus-hemagglutinin on both, cytolytic T-cells and neuronal cells, and were able to induce in mice a humoral as well as a cellular immune response specific to Reovirus-hemagglutinin (18, 19, 20). DETAILED DESCRIPTION OF THE INVENTION Peptides comprising 6 amino acid residues (aa) that bind specifically to the monoclonal antibody 2F5 were used as immunogens to induce neutralizing antibodies against HIV-1. For identification of these peptides overlapping fragments of gp41 (HIV-1 isolate BH10) were cloned as fusion peptides with glutathione transferase. The different fusion peptides were obtained through hybridization of gp41 corresponding oligonucleotides which were cloned between the Bam HI and the Eco RI site of the plasmid pGEX-2T (Pharmacia). The recombinant plasmids were transformed into the E. coli strain DH5α and expression of the fusion proteins was induced with isopropylthiogalactoside (IPTG). The E. coli extract was then purified with glutathione-sepharose 4B columns, loaded on sodiumdodecylsulfate-polyacrylamide gels, separated by electrophoresis and protein expression was analyzed by silver staining. Fusion peptides that were reactive with the monoclonal antibody 2F5 were identified by immunoblotting. Using this method peptides which bind to the monoclonal antibody 2F5 were identified: Figure 1 shows Western blots of fusion peptides with overlapping fragments of gp160 of HIV-1 (isolate BH10). In contrast to constructs that comprise aa 597 to 677, 634 to 677 and 648 to 677 (the numbering of amino acid residues corresponds to gp160 of HIV-1 isolate BH10, as described in the Swissprot database entry ENV$HIV10) which were reactive with the antibody 2F5, a fusion peptide comprising aa 667 to 677 did not show a positive reaction. This was the first indication that the epitope of the monoclonal antibody 2F5 is formed by aa within the sequence from position 648 to 667 of gp160. Based on these results, overlapping 6-mer peptides of this region were fused with the glutathione S-transferase. As shown in figure lb the peptide containing the amino acid sequence GLU LEU ASP LYS TRP ALA (aa 662-667) was highly reactive with the antibody 2F5 whereas for peptides containing the amino acid sequence LEU ASP LYS TRP ALA SER (aa 663-668) or ASP LYS TRP ALA SER LEU (aa 664-669) reactivity with the monoclonal antibody was significantly lower. A peptide containing amino acid sequence LEU GLU LEU ASP LYS TRP (aa 661-666) showed no reactivity at all. These data suggest that the epitope of the monoclonal antibody comprises the amino acid sequence GLU LEU ASP LYS TRP ALA that correspond to aa 662-667 on gp160 of the HIV-1 BH10 isolate. In this context both, a synthetic peptide corresponding to this epitope sequence and a fusion protein containing this sequence were able to inhibit neutralization mediated by the 2F5 antibody (Fig. 4). Sequence comparison of that region revealed that the corresponding amino acid sequence is highly conserved between otherwise genetically highly divergent HIV-1 isolates (Table 2a). We also were able to show that fusion peptides with amino acid substitutions-according to different HIV-1 isolates- in this region were also reactive with the 2F5 antibody (Fig. 1c). The presence of antigenic domains around this region has been reported previously (21,22). Teeuwsen et al. reported of a monoclonal antibody, that reacted with a peptide corresponding to aa 643 to 692 of gp160. In addition Broliden et al. reported that HIV-1 antibody-positive human sera were reactive with a peptide corresponding to region 657-671. However, in both cases a specific epitope was not identified. The monoclonal antibody reported by Teeuwsen et al. had no neutralizing activity. Also the sera reactive with the peptide 657-671 of Broliden et al. showed just partial neutralizing activity. In different neutralization assays this group was able to show neutralizing activity against HIV-1 isolate IIIB but not against SF2 and RF. In contrast to this result, the monoclonal antibody 2F5 neutralizes a variety of different HIV-1 isolates including SF2 and RF (table 1). These data suggest that the antibodies of the sera reported by Broliden et al. as well as the monoclonal antibody reported by Teeuwsen et al. have a different specificity and recognize a different epitope than the antibody 2F5. The application of the peptides described in the present invention as immunogen has several advantages. They comprise just 6 aa. Thus, other gp160 peptide sequences which induce antibodies that enhance HIV-1 infection or lead to immunosuppression can be avoided (2,3). Furthermore an effective HIV-1 vaccine should induce an immune response against HIV-1 isolates that vary considerably in their genomic sequences. In this context sequence comparison in the region of the 2F5 epitope revealed that the epitope of the 2F5 antibody is highly conserved between different HIV-1 isolates (Table 2a). Since peptides with aa substitutions- corresponding to genetically different HIV-1 isolates- were reactive with the 2F5 antibody (Fig. 1c), it is likely that antibodies induced by peptides described in the present invention are directed against a variety of divergent HIV-1 isolates. In addition, the 2F5 antibody showed neutralizing activity against a wide variety of genetically different HIV-1 isolates which proves that peptides described in the present invention are presented as neutralizing epitopes (Table 1). In order to know which variations of the epitope sequence are binding to the monoclonal antibody 2F5 we undertook a peptide mapping with a random hexapeptide library displayed on protein III of a filamentous phage (22a). The hexapeptide sequences of the eluted phage particles were compiled (Table 2b). There is a wide range of variation in the progression of HIV-1 related disease in different HIV-1 infected persons. In many cases HIV-1 infection ends up in AIDS-related complex (ARC) and AIDS within some years, while some HIV-1 positive persons remain asymptomatic. It has been shown that antibody-titers against certain peptide epitopes are much lower in AIDS-patients compared to asymptomatic states (23). We found a significant correlation between the antibody-titers to the peptides described in the present invention and HIV-1 related disease progression (Fig. 2). Patients number 20,25,29,35,41,44 and 46 who have a high antibody-titer to peptides described in this invention (Fig. 2), did not show any progression in disease within the last five years so far. This means that generation of antibodies induced by peptides described in the present invention can inhibit or at least reduce the progression of HIV-1 related disease. The fact that there are rarely high antibody-titers to peptides described in this invention found in sera of HIV-1 positive patients indicates that these epitopes on gp160 are not recognized readily by the human immune system, resulting in low HIV-1 neutralizing antibody titers specific to these epitopes. An objective of the present invention is also to present the peptides described in the invention in a proper form and to induce a sufficient neutralizing immune response. Example 1: The cloning and expression of peptides described in the invention as fusion proteins with glutathione S-transferase (GST) and immunizations of mice with these peptides is described. All cloning methods were done according to standard procedures (24). Oligonucleotides corresponding to the peptides described in the invention were hybridized and cloned between the Bam HI and Eco RI site of the plasmid pGEX-2T (Pharmacia). By this the NH 2 -terminal ends of these peptides were fused with the COOH-terminal ends of the GST. In addition, a stop codon was added to the COOH-terminal ends of the gp41 peptide sequences. These constructs were transformed into E. coli DH5α and expression of the fusion proteins was induced with isopropylthiogalactoside (IPTG). After three hours of induction bacteria were harvested by centrifugation, suspended in phosphate buffered saline (PBS, pH 7.2) containing 1 % Triton-X-100 and sonicated. Bacterial debris were spun down by centrifugation and the supernatant was loaded on glutathione-sepharose 4B columns (Pharmacia). Elution of the fusion proteins was done with 20mM glutathione and 120mM NaCl in 100mM Tris HCl (pH 8.0). Purified fusion proteins obtained with this procedure were used for immunizing mice according to standard procedures. As a control, mice were immunized with GST prepared in the same way as the fusion proteins. Sera from mice taken one week after the last immunization showed high neutralizing titers against peptides described in the invention and inhibited HIV-1 replication in vitro (Fig. 3b and 3c). Example 2: Example 2 describes the expression of peptide sequences described in the invention as part of the hemagglutinin of influenza A virus. In vitro mutagenesis was used to introduce this peptide sequence into the antigenic sites A,B,C,D and E of the hemagglutinin of influenza A virus (26, 27). These chimerical DNA-constructs were then "RNP-transfected" into influenza HK/WSN virus (13). These chimerical influenza/HIV viruses had the antigenic properties of said peptide. In antibody-adsorption experiments these chimerical viruses inhibited HIV-1 neutralization through the antibody 2F5 (Fig. 3a). Antisera of mice immunized with the chimerical viruses were reactive with said peptides (Fig. 3b). Furthermore, in vitro these antisera neutralized different HIV-1 isolates (Fig. 3c). Example 3: Example 3 describes the expression of peptides described in the invention as part of a so called "immunological supermolecule", in where the peptide sequence is inserted into the linker which connects the variable regions of the heavy and light chain of an immunoglobulin molecule. Specifically a single chain Fv construct of a neutralizing anti-HIV-gp120 antibody was made according to standard procedures (27) . In this construct peptide sequences described in the invention were inserted into the linker which connects the variable region of the light chain with the variable region of the heavy chain. This recombinant protein was expressed in E. coli and purified according to standard procedures. Two functions were observed with this construct. First this construct showed the antigen binding properties of the original antibody and in addition this construct induced, when injected into mice, antibodies that neutralized different HIV-1 isolates (Fig. 3c). This "immunological supermolecule" provides the possibility to obtain an active and passive immunization at the same time. Basically, in such a construct the antigen binding neutralizing properties of an antibody and the presentation of a neutralizing epitope are combined. In already HIV-1 infected persons the progression of infection could be slowed down with the first application by the antigen binding neutralizing properties, before the effective onset of the immune system is triggered by the neutralizing epitopes of this molecule. Thus the usual observed "time lag" between immunization and effective immune response of a typical active immunization could be overcome. In addition, it is most likely that during neutralization of already present HIV-1 virions the presentation of the epitope is very efficient. Example 4: Example 4 describes the formation of antiidiotypic antibodies to antibody 2F5 as well as the production of an antiidiotypic antibody by means of in vitro recombination techniques. Antibody 2F5 was used to immunize mice in order to induce the formation of antiidiotypic antibody. The immunization scheme used was according to standard procedures in order to enhance the frequency at which antiidiotypic antibodies are developed in the animal. The polyclonal sera such obtained were tested for their immunoreactivity, whereby it was determined by means of antigen-competition ELISA that a part of the humoral immune response was indeed directed against the combining site of the antibody 2F5. Thus it was proven that in those sera antiidiotypic antibodies were present. In order to test the concept of vaccination by means of antiidiotypic antibodies, the sera containing antiidiotypic antibodies were subsequently used to immunize another group of mice. After completion of this immunization procedure, it was possible to detect an immune response to the antiidiotypic sera that was qualitatively comparable to the above described immune reaction against the HIV-1 peptide-part of the glutathione-S-transferase fusion protein described in example 1. Since it was now proven, that the described peptide has the quality necessary to act as an immunogen, and since furthermore it had been shown that by using antiidiotypic antibodies with internal image quality of the described peptide it was possible to induce a HIV-1 neutralizing immune response, an antiidiotypic antibody was constructed by means of in vitro recombination techniques. In order to achieve this goal, one or more hypervariable regions (or parts thereof) of an existing, molecularly cloned antibody were substituted by peptide sequences described in the invention. The respective constructs where expressed as single chain Fv fragments in E. coli, and the recombinant proteins where purified according to standard methods. Immunization of mice with the antiidiotypic proteins such produced lead to a HIV-1 neutralizing immune response (Fig. 3c). Example 5: Example 5 describes the peptide mapping with a random hexapeptide library and immunizations of mice with phages containing peptides according to SEQ ID NO: 10 through SEQ ID NO: 25. The monoclonal antibody 2F5 was coated onto polystyrol tubes (Maxiscorp, Nunc, Denmark) at a concentration of 5µg/ml in coating buffer (0.1M Na-Carbonate buffer, pH 9.6) overnight at 4°C. After washing with PBS, the surface was blocked with PBS containing 5%w/v skimmed milk powder at 36°C for 2 hours. Washing with PBS was followed by incubation of a hexapeptide phage display library (10 11 transduction units in TPBS (PBS including 0.5% v/v Tween 20) overnight at 5°C. Extensive washing with TPBS was followed by elution of phage with elution buffer (0.1N HCl/Glycine pH 2.2, 1mg BSA/ml). The eluate is neutralized with 1 M Tris and used for infection of E. coli K91Kan. Phage is prepared from the infected culture by the methods described (22a). The procedure is repeated 3 times. The final eluate was used to produce transduced E. coli K91Kan. DNA of these clones was sequenced and the respective phage displayed hexapeptide sequence was derived by computer translation. Clones with the SEQ ID NO:10 through 25 (Table 2) were used for phage preparation and the respective phages were injected into mice. After two booster injections the respective sera were tested for HIV-1 neutralizing activity. All of these sera were neutralizing in vitro. The control serum was produced by immunization with wild-type phage f1 and did not neutralize HIV-1. In addition, oligonucleotides coding for SEQ ID NO: 10 through 25 were introduced into gene VIII of fd-tet between aa 27 and 28 of the immature protein VIII by standard cloning techniques. The recombinant phages were produced in E. coli K91Kan, purified by standard techniques (PEG mediated precipitation followed by CsCl gradient centrifugation) and used as immunogen. The respective sera were found to be neutralizing in HIV-1 neutralizing assays whereas anti-wild type fd-tet was not. Sequence Listing : SEQ ID NO: 1 LENGTH OF SEQUENCE: 6 amino acid residues TYPE OF SEQUENCE: amino acid sequence TYPE OF FRAGMENT: inner fragment ORIGIN: GP160 of HIV-1 isolate BH10 POSITION OF THE SEQUENCE IN GP160: from residue 662 to 667 PROPERTIES: Epitope of a human monoclonal antibody directed against HIV-1 GP160 REFERENCE: translated from GenBank accession M15654, nucleotides 7563 to 7580 SEQ ID NO: 2 LENGTH OF SEQUENCE: 6 amino acid residues TYPE OF SEQUENCE: amino acid sequence TYPE OF FRAGMENT: inner fragment ORIGIN: GP160 of HIV-1 isolate JS4/26 POSITION OF THE SEQUENCE IN GP160: from residue 655 to 660 PROPERTIES: Epitope of a human monoclonal antibody directed against HIV-1 GP160 REFERENCE: translated from GenBank accession M37576, nucleotides 1963 to 1980 SEQ ID NO: 3 LENGTH OF SEQUENCE: 6 amino acid residues TYPE OF SEQUENCE: amino acid sequence TYPE OF FRAGMENT: inner fragment ORIGIN: GP160 of HIV-1 isolate (patient 3L) POSITION OF THE SEQUENCE IN GP41: from residue 164 to 169 PROPERTIES: Epitope of a human monoclonal antibody directed against HIV-1 GP160 REFERENCE: translated from GenBank accession X61352, nucleotides 490 to 507 SEQ ID NO: 4 LENGTH OF SEQUENCE: 6 amino acid residues TYPE OF SEQUENCE: amino acid sequence TYPE OF FRAGMENT: inner fragment ORIGIN: GP160 of HIV-1 isolate SF170 POSITION OF THE SEQUENCE IN GP160: from residue 667 to 672 PROPERTIES: Epitope of a human monoclonal antibody directed against HIV-1 GP160 REFERENCE: translated from GenBank accession M66533, nucleotides 1999 to 2016 SEQ ID NO: 5 LENGTH OF SEQUENCE: 6 amino acid residues TYPE OF SEQUENCE: amino acid sequence TYPE OF FRAGMENT: inner fragment ORIGIN: GP160 of HIV-1 isolate JH3 POSITION OF THE SEQUENCE IN GP160: from residue 673 to 678 PROPERTIES: Epitope of a human monoclonal antibody directed against HIV-1 GP160 REFERENCE: translated from GenBank accession M21138, nucleotides 2263 to 2280 SEQ ID NO: 6 LENGTH OF SEQUENCE: 6 amino acid residues TYPE OF SEQUENCE: amino acid sequence TYPE OF FRAGMENT: inner fragment ORIGIN: GP160 of HIV-1 isolate Z-84 POSITION OF THE SEQUENCE IN GP160: from residue 669 to 674 PROPERTIES: Epitope of a human monoclonal antibody directed against HIV-1 GP160 REFERENCE: translated from GenBank accession J03653, nucleotides 2037 to 2054 SEQ ID NO: 7 LENGTH OF SEQUENCE: 6 amino acid residues TYPE OF SEQUENCE: amino acid sequence TYPE OF FRAGMENT: inner fragment ORIGIN: GP160 of HIV-1 isolate CAM1 proviral genome POSITION OF THE SEQUENCE IN GP160: from residue 662 to 667 PROPERTIES: Epitope of a human monoclonal antibody directed against HIV-1 GP160 REFERENCE: translated from GenBank accession D10112, nucleotides 8209 to 8226 SEQ ID NO: 8 LENGTH OF SEQUENCE: 6 amino acid residues TYPE OF SEQUENCE: amino acid sequence TYPE OF FRAGMENT: inner fragment ORIGIN: GP160 of HIV-1 isolate JS4/6 POSITION OF THE SEQUENCE IN GP160: from residue 659 to 664 PROPERTIES: Epitope of a human monoclonal antibody directed against HIV-1 GP160 REFERENCE: translated from GenBank accession M37491, nucleotides 2416 to 2433 SEQ ID NO: 9 LENGTH OF SEQUENCE: 6 amino acid residues TYPE OF SEQUENCE: amino acid sequence TYPE OF FRAGMENT: inner fragment ORIGIN: GP160 of HIV-1 isolate SBB POSITION OF THE SEQUENCE IN GP160: from residue 413 to 418 (partial sequence) PROPERTIES: Epitope of a human monoclonal antibody directed against HIV-1 GP160 REFERENCE: translated from GenBank accession M77229, nucleotides 1239 to 1256 SEQ ID NO: 10 LENGTH OF SEQUENCE: 6 amino acid residues TYPE OF SEQUENCE: amino acid sequence TYPE OF FRAGMENT: inner fragment ORIGIN: p3 fusion protein of filamentous phage fUSE5 POSITION OF THE SEQUENCE IN p3: from residue 4 to 9 PROPERTIES: hexapeptide binding to monoclonal antibody 2F5 SEQ ID NO: 11 LENGTH OF SEQUENCE: 6 amino acid residues TYPE OF SEQUENCE: amino acid sequence TYPE OF FRAGMENT: inner fragment ORIGIN: p3 fusion protein of filamentous phage fUSE5 POSITION OF THE SEQUENCE IN p3: from residue 4 to 9 PROPERTIES: hexapeptide binding to monoclonal antibody 2F5 SEQ ID NO: 12 LENGTH OF SEQUENCE: 6 amino acid residues TYPE OF SEQUENCE: amino acid sequence TYPE OF FRAGMENT: inner fragment ORIGIN: p3 fusion protein of filamentous phage fUSE5 POSITION OF THE SEQUENCE IN p3: from residue 4 to 9 PROPERTIES: hexapeptide binding to monoclonal antibody 2F5 SEQ ID NO: 13 LENGTH OF SEQUENCE: 6 amino acid residues TYPE OF SEQUENCE: amino acid sequence TYPE OF FRAGMENT: inner fragment ORIGIN: p3 fusion protein of filamentous phage fUSE5 POSITION OF THE SEQUENCE IN p3: from residue 4 to 9 PROPERTIES: hexapeptide binding to monoclonal antibody 2F5 SEQ ID NO: 14 LENGTH OF SEQUENCE: 6 amino acid residues TYPE OF SEQUENCE: amino acid sequence TYPE OF FRAGMENT: inner fragment ORIGIN: p3 fusion protein of filamentous phage fUSE5 POSITION OF THE SEQUENCE IN p3: from residue 4 to 9 PROPERTIES: hexapeptide binding to monoclonal antibody 2F5 SEQ ID NO: 15 LENGTH OF SEQUENCE: 6 amino acid residues TYPE OF SEQUENCE: amino acid sequence TYPE OF FRAGMENT: inner fragment ORIGIN: p3 fusion protein of filamentous phage fUSE5 POSITION OF THE SEQUENCE IN p3: from residue 4 to 9 PROPERTIES: hexapeptide binding to monoclonal antibody 2F5 SEQ ID NO: 16 LENGTH OF SEQUENCE: 6 amino acid residues TYPE OF SEQUENCE: amino acid sequence TYPE OF FRAGMENT: inner fragment ORIGIN: p3 fusion protein of filamentous phage fUSE5 POSITION OF THE SEQUENCE IN p3: from residue 4 to 9 PROPERTIES: hexapeptide binding to monoclonal antibody 2F5 SEQ ID NO: 17 LENGTH OF SEQUENCE: 6 amino acid residues TYPE OF SEQUENCE: amino acid sequence TYPE OF FRAGMENT: inner fragment ORIGIN: p3 fusion protein of filamentous phage fUSE5 POSITION OF THE SEQUENCE IN p3: from residue 4 to 9 PROPERTIES: hexapeptide binding to monoclonal antibody 2F5 SEQ ID NO: 18 LENGTH OF SEQUENCE: 6 amino acid residues TYPE OF SEQUENCE: amino acid sequence TYPE OF FRAGMENT: inner fragment ORIGIN: p3 fusion protein of filamentous phage fUSE5 POSITION OF THE SEQUENCE IN p3: from residue 4 to 9 PROPERTIES: hexapeptide binding to monoclonal antibody 2F5 SEQ ID NO: 19 LENGTH OF SEQUENCE: 6 amino acid residues TYPE OF SEQUENCE: amino acid sequence TYPE OF FRAGMENT: inner fragment ORIGIN: p3 fusion protein of filamentous phage fUSE5 POSITION OF THE SEQUENCE IN p3: from residue 4 to 9 PROPERTIES: hexapeptide binding to monoclonal antibody 2F5 SEQ ID NO: 20 LENGTH OF SEQUENCE: 6 amino acid residues TYPE OF SEQUENCE: amino acid sequence TYPE OF FRAGMENT: inner fragment ORIGIN: p3 fusion protein of filamentous phage fUSE5 POSITION OF THE SEQUENCE IN p3: from residue 4 to 9 PROPERTIES: hexapeptide binding to monoclonal antibody 2F5 SEQ ID NO: 21 LENGTH OF SEQUENCE: 6 amino acid residues TYPE OF SEQUENCE: amino acid sequence TYPE OF FRAGMENT: inner fragment ORIGIN: p3 fusion protein of filamentous phage fUSE5 POSITION OF THE SEQUENCE IN p3: from residue 4 to 9 PROPERTIES: hexapeptide binding to monoclonal antibody 2F5 SEQ ID NO: 22 LENGTH OF SEQUENCE: 6 amino acid residues TYPE OF SEQUENCE: amino acid sequence TYPE OF FRAGMENT: inner fragment ORIGIN: p3 fusion protein of filamentous phage fUSE5 POSITION OF THE SEQUENCE IN p3: from residue 4 to 9 PROPERTIES: hexapeptide binding to monoclonal antibody 2F5 SEQ ID NO: 23 LENGTH OF SEQUENCE: 6 amino acid residues TYPE OF SEQUENCE: amino acid sequence TYPE OF FRAGMENT: inner fragment ORIGIN: p3 fusion protein of filamentous phage fUSE5 POSITION OF THE SEQUENCE IN p3: from residue 4 to 9 PROPERTIES: hexapeptide binding to monoclonal antibody 2F5 SEQ ID NO: 24 LENGTH OF SEQUENCE: 6 amino acid residues TYPE OF SEQUENCE: amino acid sequence TYPE OF FRAGMENT: inner fragment ORIGIN: p3 fusion protein of filamentous phage fUSE5 POSITION OF THE SEQUENCE IN p3: from residue 4 to 9 PROPERTIES: hexapeptide binding to monoclonal antibody 2F5 SEQ ID NO: 25 LENGTH OF SEQUENCE: 6 amino acid residues TYPE OF SEQUENCE: amino acid sequence TYPE OF FRAGMENT: inner fragment ORIGIN: p3 fusion protein of filamentous phage fUSE5 POSITION OF THE SEQUENCE IN p3: from residue 4 to 9 PROPERTIES: hexapeptide binding to monoclonal antibody 2F5 SEQ ID NO: 26 LENGTH of SEQUENCE: 18 base pairs TYPE OF SEQUENCE: nucleotide sequence TYPE OF STRAND: single strand TOPOLOGY OF SEQUENCE: linear TYPE OF SEQUENCED MOLECULE: cDNA to viral RNA TYPE OF FRAGMENT: inner fragment ORIGIN: GP160 of HIV-1 isolate BH10 REFERENCE: GenBank accession M15654 POSITION OF THE SEQUENCE IN DATABASE ENTRY: 7564-7580 SEQ ID NO: 27 LENGTH of SEQUENCE: 18 base pairs TYPE OF SEQUENCE: nucleotide sequence TYPE OF STRAND: single strand TOPOLOGY OF SEQUENCE: linear TYPE OF SEQUENCED MOLECULE: cDNA to viral RNA TYPE OF FRAGMENT: inner fragment ORIGIN:GP160 of HIV-1 isolate JS4/26 REFERENCE: GenBank accession M37576 POSITION OF THE SEQUENCE IN DATABASE ENTRY: 1963-1980 SEQ ID NO: 28 LENGTH of SEQUENCE: 18 base pairs TYPE OF SEQUENCE: nucleotide sequence TYPE OF STRAND: single strand TOPOLOGY OF SEQUENCE: linear TYPE OF SEQUENCED MOLECULE: cDNA to viral RNA TYPE OF FRAGMENT: inner fragment ORIGIN: GP160 of HIV-1 isolate (patient 3L) REFERENCE: GenBank accession X61352 POSITION OF THE SEQUENCE IN DATABASE ENTRY: 490-507 SEQ ID NO: 29 LENGTH of SEQUENCE: 18 base pairs TYPE OF SEQUENCE: nucleotide sequence TYPE OF STRAND: single strand TOPOLOGY OF SEQUENCE: linear TYPE OF SEQUENCED MOLECULE: cDNA to viral RNA TYPE OF FRAGMENT: inner fragment ORIGIN: GP160 of HIV-1 isolate SF170 REFERENCE: GenBank accession M66533 POSITION OF THE SEQUENCE IN DATABASE ENTRY: 1999-2016 SEQ ID NO: 30 LENGTH of SEQUENCE: 18 base pairs TYPE OF SEQUENCE: nucleotide sequence TYPE OF STRAND: single strand TOPOLOGY OF SEQUENCE: linear TYPE OF SEQUENCED MOLECULE: cDNA to viral RNA TYPE OF FRAGMENT: inner fragment ORIGIN: GP160 of HIV-1 isolate JH3 REFERENCE: GenBank accession M21138 POSITION OF THE SEQUENCE IN DATABASE ENTRY: 2263-2280 SEQ ID NO: 31 LENGTH of SEQUENCE: 18 base pairs TYPE OF SEQUENCE: nucleotide sequence TYPE OF STRAND: single strand TOPOLOGY OF SEQUENCE: linear TYPE OF SEQUENCED MOLECULE: cDNA to viral RNA TYPE OF FRAGMENT: inner fragment ORIGIN: GP160 of HIV-1 isolate Z-84 REFERENCE: GenBank accession J03653 POSITION OF THE SEQUENCE IN DATABASE ENTRY: 2037-2054 SEQ ID NO: 32 LENGTH of SEQUENCE: 18 base pairs TYPE OF SEQUENCE: nucleotide sequence TYPE OF STRAND: single strand TOPOLOGY OF SEQUENCE: linear TYPE OF SEQUENCED MOLECULE: cDNA to viral RNA TYPE OF FRAGMENT: inner fragment ORIGIN: GP160 of HIV-1 isolate CAM1 proviral genome REFERENCE: GenBank accession D10112 POSITION OF THE SEQUENCE IN DATABASE ENTRY: 8209-8226 SEQ ID NO: 33 LENGTH of SEQUENCE: 18 base pairs TYPE OF SEQUENCE: nucleotide sequence TYPE OF STRAND: single strand TOPOLOGY OF SEQUENCE: linear TYPE OF SEQUENCED MOLECULE: cDNA to viral RNA TYPE OF FRAGMENT: inner fragment ORIGIN: GP160 of HIV-1 isolate JS4/6 REFERENCE: GenBank accession M37491 POSITION OF THE SEQUENCE IN DATABASE ENTRY: 2416-2433 SEQ ID NO: 34 LENGTH of SEQUENCE: 18 base pairs TYPE OF SEQUENCE: nucleotide sequence TYPE OF STRAND: single strand TOPOLOGY OF SEQUENCE: linear TYPE OF SEQUENCED MOLECULE: cDNA to viral RNA TYPE OF FRAGMENT: inner fragment ORIGIN: GP160 of HIV-1 isolate SBB REFERENCE: GenBank accession M77229 POSITION OF THE SEQUENCE IN DATABASE ENTRY: 1239-1256 SEQ ID NO: 35 LENGTH of SEQUENCE: 18 base pairs TYPE OF SEQUENCE: nucleotide sequence TYPE OF STRAND: single strand TOPOLOGY OF SEQUENCE: linear TYPE OF SEQUENCED MOLECULE: phage DNA TYPE OF FRAGMENT: inner fragment ORIGIN: gene coding for p3 fusion protein of filamentous phage fUSE5 POSITION OF THE SEQUENCE IN p3: 10 to 27 SEQ ID NO: 36 LENGTH of SEQUENCE: 18 base pairs TYPE OF SEQUENCE: nucleotide sequence TYPE OF STRAND: single strand TOPOLOGY OF SEQUENCE: linear TYPE OF SEQUENCED MOLECULE: phage DNA TYPE OF FRAGMENT: inner fragment ORIGIN: gene coding for p3 fusion protein of filamentous phage fUSE5 POSITION OF THE SEQUENCE IN p3: 10 to 27 SEQ ID NO: 37 LENGTH of SEQUENCE: 18 base pairs TYPE OF SEQUENCE: nucleotide sequence TYPE OF STRAND: single strand TOPOLOGY OF SEQUENCE: linear TYPE OF SEQUENCED MOLECULE: phage DNA TYPE OF FRAGMENT: inner fragment ORIGIN: gene coding for p3 fusion protein of filamentous phage fUSE5 POSITION OF THE SEQUENCE IN p3: 10 to 27 SEQ ID NO: 38 LENGTH of SEQUENCE: 18 base pairs TYPE OF SEQUENCE: nucleotide sequence TYPE OF STRAND: single strand TOPOLOGY OF SEQUENCE: linear TYPE OF SEQUENCED MOLECULE: phage DNA TYPE OF FRAGMENT: inner fragment ORIGIN: gene coding for p3 fusion protein of filamentous phage fUSE5 POSITION OF THE SEQUENCE IN p3: 10 to 27 SEQ ID NO: 39 LENGTH of SEQUENCE: 18 base pairs TYPE OF SEQUENCE: nucleotide sequence TYPE OF STRAND: single strand TOPOLOGY OF SEQUENCE: linear TYPE OF SEQUENCED MOLECULE: phage DNA TYPE OF FRAGMENT: inner fragment ORIGIN: gene coding for p3 fusion protein of filamentous phage fUSE5 POSITION OF THE SEQUENCE IN p3: 10 to 27 SEQ ID NO: 40 LENGTH of SEQUENCE: 18 base pairs TYPE OF SEQUENCE: nucleotide sequence TYPE OF STRAND: single strand TOPOLOGY OF SEQUENCE: linear TYPE OF SEQUENCED MOLECULE: phage DNA TYPE OF FRAGMENT: inner fragment ORIGIN: gene coding for p3 fusion protein of filamentous phage fUSE5 POSITION OF THE SEQUENCE IN p3: 10 to 27 SEQ ID NO: 41 LENGTH of SEQUENCE: 18 base pairs TYPE OF SEQUENCE: nucleotide sequence TYPE OF STRAND: single strand TOPOLOGY OF SEQUENCE: linear TYPE OF SEQUENCED MOLECULE: phage DNA TYPE OF FRAGMENT: inner fragment ORIGIN: gene coding for p3 fusion protein of filamentous phage fUSE5 POSITION OF THE SEQUENCE IN p3: 10 to 27 SEQ ID NO: 42 LENGTH of SEQUENCE: 18 base pairs TYPE OF SEQUENCE: nucleotide sequence TYPE OF STRAND: single strand TOPOLOGY OF SEQUENCE: linear TYPE OF SEQUENCED MOLECULE: phage DNA TYPE OF FRAGMENT: inner fragment ORIGIN: gene coding for p3 fusion protein of filamentous phage fUSE5 POSITION OF THE SEQUENCE IN p3: 10 to 27 SEQ ID NO: 43 LENGTH of SEQUENCE: 18 base pairs TYPE OF SEQUENCE: nucleotide sequence TYPE OF STRAND: single strand TOPOLOGY OF SEQUENCE: linear TYPE OF SEQUENCED MOLECULE: phage DNA TYPE OF FRAGMENT: inner fragment ORIGIN: gene coding for p3 fusion protein of filamentous phage fUSE5 POSITION OF THE SEQUENCE IN p3: 10 to 27 SEQ ID NO: 44 LENGTH of SEQUENCE: 18 base pairs TYPE OF SEQUENCE: nucleotide sequence TYPE OF STRAND: single strand TOPOLOGY OF SEQUENCE: linear TYPE OF SEQUENCED MOLECULE: phage DNA TYPE OF FRAGMENT: inner fragment ORIGIN: gene coding for p3 fusion protein of filamentous phage fUSE5 POSITION OF THE SEQUENCE IN p3: 10 to 27 SEQ ID NO: 45 LENGTH of SEQUENCE: 18 base pairs TYPE OF SEQUENCE: nucleotide sequence TYPE OF STRAND: single strand TOPOLOGY OF SEQUENCE: linear TYPE OF SEQUENCED MOLECULE: phage DNA TYPE OF FRAGMENT: inner fragment ORIGIN: gene coding for p3 fusion protein of filamentous phage fUSE5 POSITION OF THE SEQUENCE IN p3: 10 to 27 SEQ ID NO: 46 LENGTH of SEQUENCE: 18 base pairs TYPE OF SEQUENCE: nucleotide sequence TYPE OF STRAND: single strand TOPOLOGY OF SEQUENCE: linear TYPE OF SEQUENCED MOLECULE: phage DNA TYPE OF FRAGMENT: inner fragment ORIGIN: gene coding for p3 fusion protein of filamentous phage fUSE5 POSITION OF THE SEQUENCE IN p3: 10 to 27 SEQ ID NO: 47 LENGTH of SEQUENCE: 18 base pairs TYPE OF SEQUENCE: nucleotide sequence TYPE OF STRAND: single strand TOPOLOGY OF SEQUENCE: linear TYPE OF SEQUENCED MOLECULE: phage DNA TYPE OF FRAGMENT: inner fragment ORIGIN: gene coding for p3 fusion protein of filamentous phage fUSE5 POSITION OF THE SEQUENCE IN p3: 10 to 27 SEQ ID NO: 48 LENGTH of SEQUENCE: 18 base pairs TYPE OF SEQUENCE: nucleotide sequence TYPE OF STRAND: single strand TOPOLOGY OF SEQUENCE: linear TYPE OF SEQUENCED MOLECULE: phage DNA TYPE OF FRAGMENT: inner fragment ORIGIN: gene coding for p3 fusion protein of filamentous phage fUSE5 POSITION OF THE SEQUENCE IN p3: 10 to 27 SEQ ID NO: 49 LENGTH of SEQUENCE: 18 base pairs TYPE OF SEQUENCE: nucleotide sequence TYPE OF STRAND: single strand TOPOLOGY OF SEQUENCE: linear TYPE OF SEQUENCED MOLECULE: phage DNA TYPE OF FRAGMENT: inner fragment ORIGIN: gene coding for p3 fusion protein of filamentous phage fUSE5 POSITION OF THE SEQUENCE IN p3: 10 to 27 SEQ ID NO: 50 LENGTH of SEQUENCE: 18 base pairs TYPE OF SEQUENCE: nucleotide sequence TYPE OF STRAND: single strand TOPOLOGY OF SEQUENCE: linear TYPE OF SEQUENCED MOLECULE: phage DNA TYPE OF FRAGMENT: inner fragment ORIGIN: gene coding for p3 fusion protein of filamentous phage fUSE5 POSITION OF THE SEQUENCE IN p3: 10 to 27 Literature 1. 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Vaccination against ovine cysticercosis using a defined recombinant antigen, Nature 338: 585-587. 11. Perkus, M.E., A. Piccini, R.R. Lipinskas, and E. Paoletti. 1985. Recombinant vaccinia virus:Immunization against multiple pathogens, Science 229: 981-984 12. Evans J.D., J.McKeating, J.M. Meredith, K.L. Burke, K. Katrak,l A. John, M. Ferguson, P.D.Minor, R.A. Weiss, and J.W. Almond. 1989. An engineered Poliovirus chimaera elicits broadly reactive HIV-1 neutralizing antibodies, Nature 339: 385-388. 13. Enami M., W.Luytjes, M. Krystal, and P.Palese et al. 1990. Introduction of site-specific mutations into the genome of Influenza virus, Proc. Natl. Acad. Sci. USA 87: 3802-3805. 14. Muster T., E.K.Subbarao, M.Enami, B.R. Murphy, and P. Palese. 1991. An Influenza A virus containing Influenza B virus 5' and 3' noncoding regions on the neuraminidase gene is attenuated in mice, Proc.Natl. Acad. Sci. USA 88: 5177-5181. 15. Li, S, J.L. Schulmann, T. Moran, C.Bona, and P.Palese. 1992. Influenza-A virus transfectants with chimeric Hemagglutinins containing epitopes from different subtypes, J.Virol.66.: 399-404 16. Nisonoff A., E. Lamoyi. 1981. Clin. Immunol. Immunopathol. 21: 397 17. Sacks D.L., K.M. Esser and A. Sher. 1982. J. Exp. Med. 155: 1108 18. Kauffman R.S., J.H. Noseworthy, J.T. Nepom, R. Finberg, B.N. Fields, and M.I. Greene. 1983. J. Immunol. 131: 2539 19. Sharpe A.H., G.N. Gaulton, K.K. McDade, B.N. Fields, and M.I. Greene. 1984. J. Exp. Med. 160: 1195 20. Gaulton G.N., and M.I. Greene. 1986. Ann. Rev. Immunol. 4: 253 21. Broliden, P., A. Gegerfelt, P. Clapham, J. Rosen, E. Fenyö, B. Wahren, and K. Broliden. 1992. Identification of human neutralizing regions of the human immunodeficiency virus type-1 envelope glycoproteins, Proc. Natl. Acad. Sci. USA 89/2:461-465. 22. Teeuwsen V.J., K.H. Siebelnik, S. Crush-Stanton, A.D.B. Swerdlow, J.J. Schalken, J. Goudsmit, R. Van De Akker, M.J. Stukart, F.G. Uytdehaag, and A.D. Osterhaus. 1990. Production and characterization of a human monoclonal antibody, reactive with conserved epitope on gp41 of human immunodeficiency virus type 1, AIDS Res Hum Retroviruses 6: 381-392. 22a. Scott, J. K. and G. P. Smith. 1990. Searching for peptide ligands with an epitope library, Science 249: 386-390 23. Neurath, A.R., N. Strick, P.Taylor, P. Rubinstein, and C.E. Stevens. 1990. Search for epitope- specific antibody responses to the human immunodeficiency virus (HIV-1) envelope glycoproteins signifying resistance to desease development, AIDS Res Hum Retroviruses 6: 1183-1191 24. Sambrook, J., E. F. Fritsch and T. Maniatis. 1989. Molecular Cloning. A Laboratory manual. Cold Spring Harbor Laboratory Press, New York 25. Wharton, S.A., W. Weis, J.J. Shekel, and D.C. Wiley. 1989. Strucutre, function and antigenicity of the Hemagglutinin of Influenza virus, in: R.M. Krug (ed), The Influenza Viruses, Plenum Press, New York 26. Wiley, D.C., I.A. Wilson and J.J. Shekel. 1981. Structural identification of the antibody binding sites of Hong Kong Influenza Hemagglutinin and their involvement in the antigenic variation, Nature 289: 373-378 27. Kohl, J., F. Rüker, G. Himmler, E. Razazzi, and H. Katinger. 1991. Cloning and expression of a HIV-1 specific single-chain Fv region fused to Escherichia coli Alkaline Phosphatase, Ann. New York Acad. Sci. 646: 106-114 Tables Neutralizing properties of human monoclonal antibody 2F5 in vitro neutralization assays: Isolate IIIB MN RF SF2 A C number of positive tests 8/8 2/2 2/2 n.t. 8/8 4/4 neutralizing concentration (Ìg/ml) 10 10 10 50 10 syncitia inhibition assay: Isolate IIIB MN RF SF2 A C number of positive tests 18/18 11/11 6/10 1/1 1/1 2/3 EC 50 (Ìg/ml) 12,8 12 13,7 1,9 27 10 Figure legends: Fig.1: Western blots of fusion peptides. Recombinant proteins expressed in E. coli were purified as described in example 1 and 100ng of each fusion peptide was fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis on a 20% polyacrylamidegel and electroblotted onto a nitrocellulose filter. The blots were blocked with 0.5% nonfat dried milk in phosphate-buffered saline containing 0.1% Tween for 1h at room temperature. After washing, blots were incubated with antibody 2F5 (500ng/ml) for 1h at room temperature. After washing, blots were incubated for 1 h at room temperature with anti-human IgG-alkaline-phosphatase-conjugat. Blots were developed with 1M diethanolaminbuffer(pH9.6) containing 350ug/ml nitro-blue tetrazolium chloride and 350 ug/ml 5-bromo-4-chloro-3-indolyl-phosphate. Fig.1a: Lane 1, glutathion S-transferase (GST); lane 2, amino acids (aa) 597-677 of gp160 fused with GST; lane 3, aa 634-677 fused with GST; lane 4, aa 648-677 fused with GST; lane 5 aa 667-677 fused with GST. Fig.1b: lane 1, GST; lane 2, GST fused with aa GLU LEU ASP LYS TRP ALA (aa 662-667); lane 3, GST with aa LEU ASP LYS TRP ALA SER (aa 663-668); lane 4, GST with ASP LYS TRP ALA SER LEU (aa 664-669); lane 5, GST with aa LEU GLU LEU ASP LYS TRP (aa 661-666) Fig.1c: Fusionpeptides with amino acid substitutions according to HIV-1 isolates with differences in the region of the 2F5 epitope. Amino acid differences are underlined. Lane 1, GST; lane 2, GLU LEU ASP LYS TRP ALA; lane 3, GLN LEU ASP LYS TRP ALA; lane 4, GLY LEU ASP LYS TRP ALA; lane 5, ALA LEU ASP LYS TRP ALA; lane 6, GLU LEU ASN LYS TRP ALA (reaction of this fusion peptide with the 2F5 antibody is not visible in this Western blot; however in ELISA competition assays this peptide was competitive to recombinant gp41 for binding to the 2F5 antibody); lane 7, GLU LEU ASP THR TRP ALA; lane 8, GLU LEU ASP LYS TRP ASP

Claims: Claims for the following Contracting States : BE, CH, DE, DK, FR, GB, IE, IT, LI, NL, PT, SE Peptides binding to antibodies that show neutralizing activity against different strains and clinical isolates of HIV-1 and that inhibit the fusion of cells caused by HIV-1, characterised in that said peptides are composed according to anyone of the SEQ ID NO: 1 through SEQ ID NO: 25. Peptides according to claim 1, characterised in that they are genetically encoded by the nucleotide sequence according to anyone of the SEQ ID NO: 26 through SEQ ID NO: 50 or by sequences that are deduced from SEQ ID NO: 26 through SEQ ID NO: 50 by degeneration. Peptides according to claim 1 or 2, which upon injection into a mammal either alone or in combination with an adjuvant cause an immune response that leads to the generation of HIV-1 neutralizing antibodies. Peptides according to anyone of claims 1 to 3 in combination with an adjuvant, wherein the adjuvant is a substance to which said peptides are bound through chemical interaction. Peptides according to claim 4 in the form of fusion peptides, characterised in that a protein or part of a protein is used as the adjuvant, to which said peptides are bound by fusion of the respective nucleotide sequences and subsequent expression of the fusion genes in a biological expression system. Fusion peptides according to claim 5, characterised in that one or more peptides according to anyone of the SEQ ID NO:1 through SEQ ID NO: 25 are used as linker or as part thereof in order to link the variable domains of a single chain Fv fragment. Fusion peptides according to claim 5, characterised in that one ore more peptides according to anyone of the SEQ ID NO:1 through SEQ ID NO: 25 substitute one or more parts of the peptide sequence of a monoclonal antibody. Fusion peptides according to claims 5 and 7, characterised in that one ore more peptides according to one of the SEQ ID NO: 1 through SEQ ID NO: 25 are expressed as part of one or more hypervariable regions of a monoclonal antibody. Fusion peptides according to anyone of claims 5, 7 or 8, characterised in that they are either expressed, or chemically or enzymatically synthesized as part of a single chain Fv fragment or as part of a Fab fragment. Fusion peptides according to claim 5, characterised in that one or more peptides according to anyone of the SEQ ID NO:1 through SEQ ID NO: 25 substitute one or more parts of the peptide sequence of a viral protein, or are inserted into antigenic sites of a viral protein. Fusion peptides according to claim 10, characterised in that they are part of a virus. Fusion peptides according to claim 10 or 11, characterised in that the viral protein is the hemagglutinin or neuraminidase of influenza virus. Fusion peptides according to claim 10 or 11, characterised in that the viral protein is the surface antigen or the core antigen of hepatitis B virus. Use of peptides as defined in anyone of claims 1 through 5 to select antibodies or antibody fragments binding to HIV-1 in vitro. Use of peptides as defined in anyone of claims 1 through 5 in an immunological test in vitro to determine the neutralization titer in complete sera of patients or experimental animals infected with HIV-1, or to determine the status of infection or to make a prognosis on the further progress of infection. Use of peptides as defined in anyone of claims 1 to 5 for the manufacture of antiidiotypic antibodies. A vaccine against HIV-1, characterised in that it comprises at least one peptide and/or fusion peptide as defined in anyone of claims 1 to 13 and/or at least one antiidiotypic antibody obtained according to claim 16. Claims for the following Contracting State : ES A method for the manufacture of peptides which bind to antibodies, that show neutralizing activity against genetically divergent strains and clinical isolates of HIV-1 and which inhibit the fusion of cells caused by HIV-1, characterized in that oligonucleotides corresponding to one of the amino acid sequences of SEQ ID NO: 1 through SEQ ID NO: 25 are cloned, transformed into and expressed in E.coli, preferably E. coli DH5 α. A method according to Claim 1, characterized in that the peptides are expressed as fusion proteins with glutathion S-transferase (GST), preferably with said oligonucleotides being hybridized and cloned between the Bam HI and Eco RI site of the plasmid pGEX-2T (Pharmacia). A method according to Claim 1 or 2, characterized in that after expression of said amino acid sequences and/or said peptides the E. coli cells are disrupted and said amino acid sequences and/or said peptides are separated from the liquid fraction and purified. A method according to Claim 3, characterized in that disruption of the E. coli cells is achieved by sonication. A method according to Claim 3 or 4, characterized in that the separation and purification of the amino acid sequences and/or peptides is carried out by affinity chromatography, preferably using a glutathion sepharose 4B column, wherefrom the amino acid sequences and/or peptides are eluted, preferably with a solution containing glutathione, NaCI and a buffer. A method according to Claim 5, characterized in that the elution medium is a solution composed of 20 mM glutathione and 120 mM NaCI in 100 mM Tris-HCI, pH 8.0 A method for the manufacture of fusion proteins which bind to antibodies that show neutralizing activity against different strains and clinical isolates of HIV-1 and which inhibit the fusion of cells caused by HIV-1, characterized in that a) at least one of the amino acid sequences of SEQ ID NO: 1 through SEQ ID NO: 25 is introduced into antigenic sites of the hemagglutinin of influenza A virus by in vitro mutagenesis, thereby leading to chimerical DNA constructs, b) said chimerical DNA constructs thereafter being RNP-transfected into influenza HK/WSN virus, whereby chimerical influenza/HIV viruses are created which exhibit antigenic properties of said fusion proteins, said chimerical influenza/HIV viruses preferably being capable of inducing a neutralizing immune response against genetically divergent HIV-1 strains. A method for the manufacture of fusion proteins which bind to antibodies that show neutralizing activity against different strains and clinical isolates of HIV-1 and which inhibit the fusion of cells caused by HIV-1, characterized in that a) a single chain Fv construct of a neutralizing anti-HIV-gp120 antibody is prepared, b) at least one of the amino acid sequences of SEQ ID NO: 1 through SEQ ID NO: 25 is inserted into the linker which connects the variable regions of the heavy and light chain of an immunoglobulin molecule, c) said fusion proteins are then expressed as part of said immunoglobulin molecule containing the inserted amino acid sequence. A method for the manufacture of fusion proteins which bind to antibodies that show neutralizing activity against different strains and clinical isolates of HIV-1 and which inhibit the fusion of cells caused by HIV-1, characterized in that one or more hypervariable regions or parts thereof of a monoclonal antibody are substituted by at least one of the amino acid sequences of the SEQ ID NO: 1 through SEQ ID NO: 25, by using standard methods. A method according to Claim 9, characterized in that said fusion proteins are expressed as single chain Fv fragments in E.coli, thereafter purified and injected into mice, which thereupon leads to the formation of antiidiotypic antibodies that are capable of inducing a neutralizing immune response against genetically divergent HIV-1 strains. A method for the selection of antibodies and/or antibody fragments binding to HIV-1 in vitro, characterized in that at least one of the amino acid sequences of the SEQ ID NO: 1 through SEQ ID NO: 25 is used to bind to said antibodies and/or antibody fragments, whereafter the resulting molecules are separated and purified according to standard techniques. A method for the determination of the status of infection of HIV-1 infected humans and/or animals in vitro, characterized in that at least one of the amino acid sequences of the SEQ ID NO: 1 through SEQ ID NO: 25 is added to a serum of an infected patient and/or experimental animal, whereupon an HIV-1 neutralization titer is determined following state of the art methods. Use of peptides or fusion proteins manufactured according to anyone of Claims 1 to 10 to select antibodies and/or antibody fragments binding to HIV-1 in vitro and/or to determine neutralization titers in sera of humans or animals. Use of peptides or fusion proteins manufactured according to anyone of Claims 1 to 10 for the manufacture of a vaccine to elicit HIV-1 neutralizing antibodies in humans. Use according to claim 14 to prevent progression of HIV-1 infection to AIDS Use of peptides or fusion proteins manufactured according to anyone of claims 1 to 10 for the manufacture of antiidiotypic antibodies. Peptides binding to antibodies that show neutralizing activity against different strains and clinical isolates of HIV-1 and that inhibit the fusion of cells caused by HIV-1, characterised in that said peptides are composed according to anyone of the SEQ ID NO: 1 through SEQ ID NO: 25. Peptides according to claim 17, characterised in that they are genetically encoded by the nucleotide sequence according to anyone of the SEQ ID NO: 26 through SEQ ID NO: 50 or by sequences that are deduced from SEQ ID NO: 26 through SEQ ID NO: 50 by degeneration. Peptides according to claim 17 or 18, which upon injection into a mammal either alone or in combination with an adjuvant cause an immune response that leads to the generation of HIV-1 neutralizing antibodies. Peptides according to anyone of claims 17 to 19 in combination with an adjuvant, wherein the adjuvant is a substance to which said peptides are bound through chemical interaction. Peptides according to claim 20 in the form of fusion peptides, characterised in that a protein or part of a protein is used as the adjuvant, to which said peptides are bound by fusion of the respective nucleotide sequences and subsequent expression of the fusion genes in a biological expression system. Fusion peptides according to claim 21, charadterised in that one or more peptides according to anyone of the SEQ ID NO: through SEQ ID NO: 25 are used as linker or as part thereof in order to link the variable domains of a single chain Fv fragment. Fusion peptides according to claim 21, characterised in that one ore more peptides according to anyone of the SEQ ID NO: 1 through SEQ ID NO: 25 substitute one or more parts of the peptide sequence of a monoclonal antibody. Fusion peptides according to claims 21 and 23, characterised in that one ore more peptides according to one of the SEQ ID NO: 1 through SEQ ID NO: 25 are expressed as part of one or more hypervariable regions of a monoclonal antibody. Fusion peptides according to anyone of claims 21, 23 or 24, characterised in that they are either expressed, or chemically or enzymatically synthesized as part of a single chain Fv fragment or as part of a Fab fragment. Fusion peptides according to claim 21, characterised in that one or more peptides according to anyone of the SEQ ID NO: through SEQ ID NO: 25 substitute one or more parts of the peptide sequence of a viral protein, or are inserted into antigenic sites of a viral protein. Fusion peptides according to claim 26, characterised in that they are part of a virus. Fusion peptides according to claim 26 or 27, characterised in that the viral protein is the hemagglutinin or neuraminidase of influenza virus. Fusion peptides according to claim 26 or 27, characterised in that the viral protein is the surface antigen or the core antigen of hepatitis B virus. Use of peptides as defined in anyone of claims 17 through 21 to select antibodies or antibody fragments binding to HIV-1 in vitro. Use of peptides as defined in anyone of claims 17 through 21 in an immunological test in vitro to determine the neutralization titer in complete sera of patients or experimental animals infected with HIV-1, or to determine the status of infection or to make a prognosis on the further progress of infection. Use of peptides as defined in anyone of claims 17 to 21 for the manufacture of antiidiotypic antibodies. A vaccine against HIV-1, characterised in that it comprises at least one peptide and/or fusion peptide as defined in anyone of claims 17 to 29 and/or at least one antiidiotypic antibody obtained according to claim 16.

Inventors: HIMMLER GOTTFRIED, KATINGER HERMANN, MAIWALD GEORG, MUSTER THOMAS, PURTSCHER MARTIN, RUEKER FLORIAN, STEINDL FRANZ, TRKOLA ALEXANDRA, HIMMLER, GOTTFRIED, KATINGER, HERMANN, MAIWALD, GEORG, MUSTER, THOMAS, PURTSCHER, MARTIN, RUEKER, FLORIAN, STEINDL, FRANZ, TRKOLA, ALEXANDRA, RÜKER, Florian
Applicants: POLYMUN SCIENT IMMUNBIO FORSCH, POLYMUN SCIENTIFIC IMMUNBIOLOGISCHE FORSCHUNG GMBH
Application year: 1993
Main classification: C12N15
IPC codes: A61K39, C07K16, C12N15, C07K19, C12P21, A61P31, C07K14, A61K38, C12R1, C07K7