Peptide (options) and the method of its production, pharmaceutical agent, the antibody and the method of its production
(57) Abstract:The invention relates to biotechnology and immunology, and can be used to generate neutralizing antibodies against different strains and clinical isolates of HIV-1. The peptide has the amino acid sequence ELDKWAS, LELDKWAS, ELDNWAS, ELNKWAS, LELDNWAS or LELNKWAS and promotes the formation of antibodies. The peptide can be associated with a carrier that constitutes a virus or a viral protein. The peptide associated with the carrier, receive recombinant means. Antibody with HIV-1 neutralizing activity and Sekretareva the mucosal surface, obtained by immunization of a human or animal. The invention allows to develop a pharmaceutical agent, contributing to the formation of antibodies to strains of HIV-1 and/or clinical isolates. 6 C. and 11 C.p. f-crystals, 9 Il., table 1. The invention relates to peptides, contributing to the formation of neutralizing antibodies against different strains and clinical isolates of HIV-1, and generated peptides to antibodies and, in addition, combinations of peptide - carrier, effectively transferring these peptides in the immune system. More specifically, this invention relates to a vaccine against HIV-1.C is Oh resistant infections by human immunodeficiency virus type 1 (HIV-1). Focused on virus and virus-infected cells of the immune reaction in the course of resistant infections usually are not able to stop the infection. Vaccines can create such an immune response that can prevent the emergence of resistant infections or even prevent it from developing into AIDS. Most of the strategies of vaccination against HIV-1 is directed to the surface glycoprotein gp160, consisting of gp120 and gp41 and is responsible for binding the virus to the cellular receptor CD 4 and at a subsequent startup focused on merger activity.However, as gp160, some of the observed phenomena indicate the unacceptability as immunogen full gp160 or gp120. In vitro experiments show that the synergy between gp120 and gp120-specific antibodies blocks the activity of T-cells (Mittler and others, Science (1989) 245, 1380). In addition, it is known that the number of antigenic domains in gp160 induce antibodies that enhance the HIV-1 (Jiang and others, J, Exp. Med. (1991) 174, 1557).Use as immunogenic synthetic peptides have several advantages. Created by synthetic peptides antibodies have given specificity in the case of viruses can be selected peptides forming ability to induce an antibody response, not observed under normal conditions. For example, it is possible to induce neutralizing antibodies with a wider than integral proteins, reactivity (Green other), Cell (1982) 28, 477).In addition, the peptide containing part of the V3 loop of gp120 from HIV-1 isolate (HIV-1 IIIb), as shown, induces antibodies that protect chimpanzees against infection by artificial infection of the animal with the same HIV-1 isolate (Emini and other , Nature (1992) 355, 728).Since synthetic peptides themselves have a low immunogenicity, their need to connect with molecules, creating a synergistic effect, such as a tetanus toxoid or hemocyanin lymph snails (Bittle and other , Nature (1982) 298, 30). Another possibility is to clone small peptides in the form of a fused protein with glutathione-S-transferase from Schitosoma japonicum (Fikrig and others, Science (1990) 250, 553).As vectors for immunogens can also be used and viruses, for example: the cow pox, polio or influenza. Rabbits infected with recombinant vaccinia virus containing the sequence of the surface antigen of hepatitis b virus (HBSAg), a glycoprotein of herpes simplex virus and hemagglutinin of influenza virus, produce antibodies to all three h is C gp41 of HIV-1, successfully in rabbits induces neutralizing antibodies to HIV-1 (Evans and others, Nature (1989) 339, 385).Recently it has become possible to modify in vitro mutagenesis of the genome of influenza virus (Enami and others, Proc. Natl. Acad. Sci. USA (1990) 87, 3802). Using similar technologies by means of genetic engineering has managed to create a sustainable weakened influenza virus A (Muster and others, Proc. Natl. d. Sci. USA (1991) 88, 5177).Benefits of influenza virus in this context is the availability of many options, which allows for re-vaccination. In addition, the influenza virus induces a strong secretory and cellular immune response, which can have success with the establishment of an anti-HIV-1 vaccine.The purpose of the present invention to provide peptide sequences, which are only minimally immunogenic in comparison with the complete gp160. Such peptide sequences can be used to generate antibodies exhibiting neutralizing activity in relation to various strains and clinical isolates of HIV-1 and/or inhibit fusion of mammalian cells affected by HIV-1.Another objective of the invention is to create a strategy for the induction of secretory antibodies secreted from SL is appolagies, what is created in the tissue of the mucosal anti-HIV-1 IgA antibody will become a weapon, a particularly powerful for prevention of HIV-1 individuals at risk, since many viral infections, including HIV-1, is transmitted through the surface of the mucous membrane of the respiratory tract, gastrointestinal tract and reproductive tract.These goals are achieved by obtaining small peptides of seven or eight amino acids, obtained either by chemical synthesis or by microbiological methods. It is recommended that the peptides were derived from sequences of nucleic acids encoding Glu Leu s Liys Trp Ala Ser (=ELDKWAS in single-letter code), Leu Glu Leu Asp Lys Trp Ala Ser (LELDKWAS), Glu Leu Asn Lys Trp Ala Ser (ELNKWAS), Leu Glu Leu Asn Lys Trp Ala Ser (LELNKWAS), Glu Leu Asp Asn Trp Ala Ser (ELDNWAS) or Leu Glu Leu Asp Asn Trp Ala Ser (LELDNWAS).At least one, but preferably all six of the above amino acid sequence (hereinafter referred to as "specified six ACP") can be successfully introduced into the immune system to induce the formation and evolution of antibodies exhibiting neutralizing activity on HIV-1 strains and/or inhibit cell fusion caused by these strains.The main objective of the invention zakluychitelnoe a mix of all six of the above-mentioned peptides. The characteristics and advantages of the invention are described below.In a featured embodiment of the invention the amino acid sequence Glu Leu Asp Lys Trp Ala (ELDKWA), which determines the sequence of the epitope of the monoclonal human antibodies 2F5 (obtained from hybridoma cell line 2F5, PHLS depositor 90091704; deposited 09/17/90), connect with Ser or Leu and Ser at the same time, adjacent to the ELDKWA sequence in gp41, which determines 2F5 epitope, with the formation of the peptide sequence Glu Leu s Lys Trp Ala Ser (ELDKWAS) and Leu Glu Lu s Lys Thr Ala Ser (LELDWAS), respectively, and the introduction into the molecule of the carrier, namely, the area of the determinants of influenza virus.Modified "chimeric" protein carrying alien amino acid sequence ELDKWAS or LELDKWAS, very effectively induces antibodies directed to the specified ELDWAS or LELDKWAS sequence, with the introduction of the protein, preferably in the form of injections in the immune system of a mammal, in particular humans. Thus obtained antibodies are neutralizing activity in relation to various strains and clinical isolates of HIV-1.However, it turned out that due to a known polymorphism of the HIV gp41 activity, corresponding to amino acids 661-668 (LELDWAS) or 662-668 (ELDKWAS) of the HIV-1 isolate MN. The numbering of nucleotides and amino acids used in this description corresponds to the numbering gp160 from HIV-1 isolate MN used according to the database of Los Almås (Myers and others , Data s Nimap Retrovirus and Aids). Particularly susceptible to mutation s (D) and/or Lys (K) amino acids in the center of the sequence, the probability of neutralization of the virus by antibodies bearing 2F5 epitope. Such undesirable mechanism of escape of the virus can be successfully avoided by applying the peptides of the present invention, in which either s (D), or adjacent Lys (K) is replaced by asparagine (N).For the preparation of pharmaceutical agents designed to induce antibodies exhibiting neutralizing activity in relation to various strains and clinical isolates of HIV-1 and/or suppress the merger affected by HIV-1 cells, can be used at least one, preferably a mixture of all six peptides.In another embodiment, for manufacturing a pharmaceutical agent intended to induce anti-HIV-1 IgA antibody secreted by the mucosal surface, preferably during centuries the spine, or in a mixture with at least one other peptide.In yet another variant of the invention at least one of the following six peptides are linked with the media. Such a carrier can be, for example, the virus in his weakened and/or recombinant form. With success can be used by the influenza virus, baculovirus or vaccinia virus. Attach small peptides to the carrier, such as, for example, viral protein or virus entirely increases the efficiency of inducing antibodies after administration in the immune system such chimeric combinations of peptide - carrier or chimeric fused proteins.Particularly successful is the use of each of the above peptides in the form of a fused protein with the viral protein as a carrier, and at least one amino acid sequence according to the invention replaces at least one of the viral protein carrier or incorporated into at least one of the areas of the determinants of the viral protein. This characteristic, therefore, represents an important embodiment of the invention. The recommended option is specified viral protein is hemagglutinin () influenza virus, the neuraminidase (NA) of influenza virus or surface antigen fair) or vaccinia virus.The present invention relates also to the use of any of vysokorejtingovykh peptides, consisting of seven or eight amino acids, and/or combinations of peptide - carrier, or fused proteins for the preparation of pharmaceutical agent intended to create or induce antibodies exhibiting neutralizing activity in relation to various strains and clinical isolates of HIV-1 and/or suppress the merger affected by HIV-1 cells. If more specifically, the goal is to create an effective vaccine, based on at least one, preferably a mixture of all six of these peptides and/or six of these peptides that are connected with the carrier, for prophylactic treatment of individuals who are at risk of infection with HIV-1 and/or for the treatment of HIV-1-infected patients, especially to prevent the development of infection in AIDS.In another featured embodiment, the six peptides and/or the six peptides, United with the carrier, used for the manufacture of pharmaceutical preparations, which, after introduction into the immune system of a mammal lead to the induction of anti-HIV-1 IgA antibody secreted from the surface of the mucous membrane of the patient LM which has peptides and/or peptide combination - the carrier of the present invention can also be used for the preparation of pharmaceutical agents intended for the prophylaxis or prevention of HIV-1 infection in populations at risk for HIV-1.Apparently, it is this characteristic feature of the present invention, it is hoped that by inducing anti-HIV-1-specific secretory IgA antibodies at the mucosal surface according to the present invention provides a medical instrument, which effectively attacked viral aggression on the early stages of penetration of the virus into the body of a mammal. I believe that the ways-prototypes treatment of HIV-1 infected patients at least partially able to protect the patient because the hunt for the virus occurs in the circulatory system, i.e. the spread of the virus in the humoral system.In a featured embodiment, the specified antibody is a secretory antibody, preferably an anti-HIV-1 IgA antibody, which secretiruetsa the mucosal surface.In addition, the present invention relates also to a method for producing any of the six specified peptidome synthesized chemically by standard biochemical methods, preferably by using a peptide synthesizer model A company Applied Biosystems.In another embodiment, the above amino acid sequence receive microbiological method including the stage of incorporation of the nucleotide sequence selected from the group consisting of:
a) the nucleotide sequence corresponding to one of the following six peptides
b) a nucleotide sequence hybridizing with one of the nucleotide sequences in paragraph (a),
(C) a nucleotide sequence obtained from one of the nucleotide sequences in paragraph (a) by degeneration
in the genome of the microorganism host, leading to the expression of the genome, the use of standard technology for microbial cultivation and selection of at least one, preferably several of these peptides.Chimeric fused proteins or a combination of peptide - carrier can be obtained by a method including the connection amino acid sequence selected from the group consisting of the following six peptides with a carrier, preferably a virus or viral protein chemical or microbiological methods.
a) the nucleotide sequence corresponding to one of the amino acid sequences of these six peptides
b) the nucleotide sequence, hybridizing with one of the nucleotide sequences in paragraph (a),
(C) a nucleotide sequence obtained from one of the nucleotide sequences in paragraph (a) by degeneration
to the nucleotide sequence corresponding to the amino acid sequence of media, migration connected nucleotide sequence in a microorganism host, carrying out expression connected sequence using standard microbiological techniques and the selection of at least one, preferably several of these peptides associated with the media.
(a) nucleotide R> b) the nucleotide sequence, hybridizing with one of the nucleotide sequences under item (a),
(C) a nucleotide sequence obtained from one of the nucleotide sequences in paragraph (a) by degeneration
connect with nucleotide sequence corresponding to the amino acid sequence of a viral protein as a carrier, with the substitution in the at least part of the nucleotide sequence corresponding to the amino acid sequence of a viral protein or embedding in at least one plot sequence corresponding to a region determinants of viral protein.In the typical embodiment of the invention applied viral protein selected from the group comprising: the hemagglutinin of influenza virus, the neuraminidase of influenza virus and the surface antigen of hepatitis b virus, and in another typical embodiment, as the microorganism-host use influenza virus, baculovirus or vaccinia virus, preferably each of them in recombinant form.The embedding of the six sequences of these peptides in the area of determinants of B ON strains of influenza virus that is different from the WSN, and/or chimeric viruses, creating a neutralizing anti-HIV-1 antibodies and prevent directed HIV-1 cell fusion.The introduction of these sequences in other sites IN influenza virus or neuraminidase (NA) of influenza virus also causes fused proteins, the peptide-carrier and/or chimeric viruses, creating a neutralizing anti-HIV antibodies and prevent directed HIV-1 cell fusion.The present invention then further clarified and illustrated by the following examples which in no way limit the scope of the present invention.Example 1. Mapping of the epitope
The epitope of the monoclonal antibody 2F5 identified by Western blot turns according to the method described in Towbin and others, Proc. Natl. Acad. Sci. USA (1979) 76, 4850. Peptides overlapping fragments of gp41 of HIV-1 isolate MN clone in the form of peptides, merged with glutationtransferase. Various fused peptides obtained by hybridization of the corresponding gp41 oligonucleotides, cloned between the Bam H1 and ECO R1 sites of the plasmid pGEX-2T (Pharmacia). Recombinant plasmids transform E. coli strain D5, and the expression of the fused proteins induce isopropylthioxanthone (IPTG). The extract of E. Li then purified on Colo is om, and protein expression analyzed by silver staining and immunoblotting (Fig. 1). Received by immunoblotting data showed that the epitope of the monoclonal antibody 2F5 person includes amino acid sequence ELDKWA, corresponding in gp160 amino acids 662-667.Fig. 1 illustrates the specificity of the monoclonal antibodies 2F5 person (see example 1).Fig. 2 illustrates the construction of chimeric hemagglutinin, bearing a sequence of 2F5-epitope (see example 2).In Fig. 3 shows the titles in enzyme immunosorbent test (ELISA) for antisera to mouse immunized with chimeric influenza virus (see example 3).In Fig. 4 shows the titers in ELISA mouse immunized with cells infected with recombinant baculovirus (see example 5).In Fig. 1 shows the results of Western blot turns peptides, merged with overlapping fragments of gp160 of HIV-1 (isolate WN). In contrast to structures containing amino acids 597-677 (lane 2), 634-677 (lane 3) and 648-677 (lane 4) and reactive antibody 2F5, protein, containing amino acids 667-677 (lane 5), does not give a positive reaction. This is a direct indication that the epitope of the monoclonal antibody 2F5 education is ausina 6-dimensional peptides from this region is drained with glutathione-S-transferase.As can be seen from Fig. 1b, the peptide containing the amino acid sequence GLU LEU ASP LYS TRP ALA (amino acids 662-667, lane 2), high reaktsionnosposobnykh relatively antibodies 2F5, while the peptides containing the amino acid sequence LEU S LYS TRP ALA SER (LDKWAS, amino acids 663-668, lane 3) or S LYS TRP ALA SER LEU (DKWASL, amino acids 664-669, lane 4), reaktsionnosposobnykh relatively monoclonal antibodies reduced. The peptide containing the amino acid sequence LEU GLU LEU S LYS TRP (LELDKW, amino acids 661-666, lane 1), does not show significant reaktsionnosposobnykh. These data indicate that the epitope of the monoclonal antibody includes the amino acid sequence GLU LEU ASP LYS TRP ALA (ELDKWA) corresponding to amino acids 662-667 in gp160 from HIV-1 (isolate WN). In this context, as a synthetic peptide corresponding to the sequence of this epitope, or protein containing this sequence, was able to suppress the neutralization taking place with the participation of the 2F5 antibody.Example 2. Construction of plasmids and recombinant influenza viruses.All of the techniques of genetic engineering carried out according to standard methods described in the publication: Sambro is egovernance Leu Glu Leu s Lys Thr l Ser (LELDKWAS) or Glu Lu s Lys Thr l Ser (ELDKWAS) is injected into the determinants In ON virus WSN influenza. Plasmid pHA-ELDKWAS and RNA-1 ELDKWAS design replacement st I-Hind III fragment of plasmid RTZ/WSN-HAml described by Li and others (Proc. Natl. Acad. Sci. USA 90, 5214-5218 (1993)), PCR product obtained by using RT/WSN-HAml as a matrix, and sense and antisense primers derived from WSN virus HAml flu, and antisense primer further comprises a 21 or 24 nucleotide insertion, corresponding to the position of 1981 or 1984-2004 in gp160. Similar to the obtained plasmid pHA-ELNKWAS, pHA-LELNKWAS, pHA-ELDNWAS and RNA-1 ELDNWAS.The sequence ON WSN is given in Hiti and others (J. Virol. 41 730-734 (1982)), and the gp160 sequence contained in the database Swissport, receipt ENVSHTV10. Transfection of RNA derived from this plasmid, D cells, select, and obtain the chimeric viruses is carried out according EPAM and others (WGE. Natl. Acad. Sci. USA 87, 3802-3805 (1990)) with modifications described EPAM and Palese (J. Virol. 65. 2711-2713 (1991)).Example 3. Immunization and the reaction of antibodies in mice immunized with chimeric influenza viruses.Mice OF-1 subjected to immunization chimeric virus ELDKWAS-influenza (mouse M1, M2, M3, M4) or virus LELDKWAS-influenza (mouse M5). First, mice are subjected to immunization 102TCID50through the nose (IN) with subsequent booster immunization including 510 N.5TCID50spec booster injection of 20 µg VAT-denatured virus in incomplete Freund's adjuvant at intervals in 3 more weeks. When immunization through the nose mice under ether anesthesia. For WSN control wild-type virus (WT1, WT2) used the same technique. 12 days after the final booster injection in mice selected samples of blood, anticigarette inactivate 1 h at 56oTo determine the titers in ELISA and neutralizing activity of antisera.In Fig. 3a shows the binding of the antisera ELDKWAS-influenza peptide, fused with glutathione transferase (GST) and contains at its C-end sequence ELDKWA. The sequence determined using ELISA. Titration microplates 96-well cover GST-ELDKWA fused peptide in the amount of 4 μg/ml (100 µl/well) in carbonate buffer (pH 9,6) and incubated for 4 h at room temperature. Then the tablets washed FBSR-0.05% Tween. Anticigarette diluted FBSR-1% BSA-0.05% Tween, making tablets and incubated for 1 hour at room temperature. After washing, the antibodies detect by incubation with goat artemisinin IgG (chain) antibody conjugated to horseradish peroxidase. Tablets paint application as dihydrochloride substrate o-phenylenediamine. The reaction is stopped by adding 2.5 M H2SO4and carry out the measurement of the tablets (wavelength 492 nm, ucheniem, for detection of antibodies used goat antimurine IgA antibody conjugated with horseradish peroxidase. Neutralizing activity of antisera determined by analysis with suppression of sizinti. Equivalent dilution of antisera, which is suppressed formation of sizinti 50% (ED50) given in the table. Anticavity from mice M1-M3 will neutralize the test panel fully at various dilutions of antisera. Anticavity mice M4 and M5 will neutralize HIV-1 isolates mn and RF, but not III BC Anticavity induced WSN virus wild type, do not neutralize any of the tested HIV-1 isolates even at the lowest dilutions of serum (1:20).For analysis with the suppression of sizinti use of indicator cell line AA-2, described in the work: Chaffee and others, J. Exp. Med. (1988) 168, 605, and the source of the frozen samples viral inoculum HIV-1 strains MP, RF and IIB. All virus samples were diluted to a concentration of 910-5102TCID50on ml Mouse anticigarette diluted 2-fold environment and contribute in the traditional microplate of 96 wells (4 repeat for each dilution). To 50 µl of the diluted antisera add 50 ál of the virus and the mixture is virus - antibody incubated 2 h at 4oC. the display after 5 days as an indication of HIV-1 infection. Determination of 50% inhibitory dose (ED50) carried out by the method of Reed and Muench given in Am. J. Hyg. (1938) 27, 493. Given equivalent dilution of serum at which 50% inhibition of the formation of sizinti (ED50).Example 4. Expression of recombinant baculoviruses chimeric hemagglutinin carrying extra long 2F5-epitope.The chimeric hemagglutinin containing six of these peptides, receive according to the method of example 1. The sequence encoding these chimeric the hemagglutinin, flanked by site of restriction enzyme BamHI and built into the BamHI site of plasmid Bluebac III (Invitrogen, San Diego, CA). Experiments with transfection with the aim of obtaining recombinant baculoviruses containing the chimeric hemagglutinin conducted according to the methodology provided in the works: Groebe and others, Nucleic. ids Res. (1990) 18, 4033 and Felgner and others, Proc. Natl. Acad. Sci. USA (1987) 84, 7413. The obtained recombinant recombinant baculoviruses can be used to generate neutralizing HIV-1 antibodies.Example 5. Immunization and antibody responses in mice immunized with cells infected with recombinant baculoviruses.Used for immunization Sf9 cells infect recombinant baculovir the CF and re-suspended in FBSR at a concentration of 5106cells/ml of these cells react with specific monoclonal antibody 2F5 in the analysis Western blotting, which can be considered as an indication of the content in these cells hemagglutinin carrying ELDKWAS or LELDKWAS sequence. Mice of Balb/cA subjected to immunization four injections intraperitoneally 1106infected Sf9 cells with an interval in 2 weeks (Van Wyyke Coelingh and other Virology (1987) 160, 4465). Seven days after the fourth immunization in mice selected blood samples and determine the ELISA titers of the antisera. In Fig. 4 shows the binding created by recombinant baculovirus antisera with a synthetic peptide consisting of the sequence Gly Gly Gly Glu Leu s Lys Trp Ala (GGGELDKWA) using ELISA method, carried out by the method of example 2.The induction of secretory antibodies.Immunization is carried out by introduction of at least one, preferably a mixture of six of these peptides led to a significant improvement IgG ELDKWA-specific ELISA titers. Moreover, in contrast to immunization with the use of ELDKWA sequence immunization six of these peptides also leads to significant IgA response. Was surprised that this IgA immune response is triggered also urovnem flu-ELDKWAS.Mice subjected to immunization introduction through the nose 102FU and after 4 weeks again subjected to immunization introduction in the same way 105PFU. The third immunization carried out after another 4 weeks introduction through the nose (IN) or intraperitoneally (IP) of 107PFU. 8 weeks after the third immunization collect and combine products nasal lavage and ELISA method to determine the reactivity of these samples with peptide ELDKWA. As a control (sample WT) analyze the joint products washing noses of mice immunized with influenza virus wild-type (WT). Applied the same scheme immunization, and IN groups (see Fig. 5). Induced antibodies are produced predominantly mucous membranes of the nose, and we can assume that the titer of antibodies unusually high. It is evident from Fig. 5 also shows that the introduction through the nose flu virus-LDKWAS leads to a higher concentration of HIV-1 neutralizing antibodies compared to intraperitoneal injection.Example 7. TDA antibodies in secretions of the intestine lb/C mice immunized with influenza virus-ELDKWAS.Mice subjected to immunization introduction through the nose 102PFU and after 4 weeks again subjected to immunization introduction in the same way 106FU. The third immunization carried out after another 4 weeks introduction chartextarearows faeces, containing antibodies isolated from the mucosa of the intestine and the ELISA method to determine reaktsionnosposobnykh these samples relative to the peptide ELDKWA. INO, INR, IN INS and samples from mice that third immunization was performed through the nose, and samples IPO, IPR, IPRS and IS selected in mice, which is the third immunization was performed intraperitoneally.WT1 and W2 samples of control mice immunized with influenza virus wild-type (WT). 1. Peptide, contributing to the formation of antibodies exhibiting neutralizing activity against different strains and clinical isolates of HIV-1 and/or suppress cell fusion of HIV-1, wherein the peptide consists of an amino acid sequence selected from the group consisting of ELDKWAS, LELDKWAS, ELDNWAS, ELNKWAS, LELDNWAS and LELNKWAS.2. The peptide associated with the media, promoting the formation of antibodies exhibiting neutralizing activity against different strains and/or clinical isolates of HIV-1 and/or inhibit fusion of HIV-1 cells, wherein the peptide consists of an amino acid sequence selected from the group consisting of ELDKWAS, LELDKWAS, ELDNWAS, LELDNWAS, ELNKWAS, LELNKWAS, and associated with the media, which is the virus or viral protein.4. The peptide under item 2 or 3, characterized in that at least one of the amino acid sequences described in paragraph 1, replace at least part of the amino acid sequence of a viral protein or embedded in at least one antigenic site of a viral protein.5. The peptide under item 2, wherein the peptide is linked to the viral protein, the affected cells, optional Sf9 cells pre-infected with a virus containing the above-mentioned peptide associated with said viral protein.6. Pharmaceutical agent, contributing to the formation of antibodies that are neutralizing activity against different strains and clinical isolates of HIV-1 and/or inhibit fusion of HIV-1 cells, characterized in that it comprises at least one peptide selected from the group consisting of ELDKWAS, LELDKWAS, ELDNWAS, ELNKWAS, LELDNWAS and LELNKWAS, and/or at least one of the aforementioned peptides associated with noiselevels cowpox, or is part of a virus, preferably a viral protein selected from the group consisting of hemagglutinin (ON) of influenza virus, neuraminidase (NA) of influenza virus and the surface antigen of hepatitis C.7. Pharmaceutical facility under item 6, characterized in that it induces the secretion of anti-HIV-1-IgA antibodies, preferably the mucosal surface of the mammal.8. Pharmaceutical facility under item 6 or 7 to prevent HIV-1 infection in individuals at risk.9. Method for production of a peptide associated with the media, contributing to the formation of antibodies exhibiting neutralizing activity against different strains and clinical isolates of HIV-1 and/or inhibit fusion of HIV-1 cells, which includes the preparation of the nucleotide sequence corresponding to the amino acid sequence of a peptide associated with viral protein, where the peptide is selected from the group consisting of ELDKWAS, LELDKWAS, ELDNWAS, ELNKWAS, LELDNWAS and LELNKWAS, with the transfer of the mentioned nucleotide sequence in the host organism to obtain chimeric virus by expression in suitable conditions, and selecting peptides, associated with the carrier in Vieste amino acid sequence of a viral protein or embedded in antigenic site of viral protein.11. The method according to p. 9 or 10, where viral protein selected from the group consisting of hemagglutinin of influenza virus, the neuraminidase of influenza virus and the surface antigen of hepatitis C.12. The method according to PP.9, 10 or 11, where the virus group consisting of preferably a recombinant influenza virus, baculovirus and vaccinia virus is used as the host body.13. Method for the production of antibodies exhibiting HIV-1 neutralizing activity and is able to prevent the fusion of HIV-1 cells, wherein the peptide is selected from the group consisting of ELDKWAS, LELDKWAS, ELDNWAS, ELNKWAS, LELDNWAS and LELNKWAS, is introduced into the immune system of a human or animal, and the peptide is preferably linked to a carrier, which is a virus, preferably selected from the group consisting of influenza virus, baculovirus and vaccinia virus, or part of a virus, preferably a viral protein selected from the group consisting of hemagglutinin () influenza, neuraminidase (NA) of influenza virus and the surface antigen of hepatitis b virus, producing as a result of these antibodies.14. The method according to p. 13, characterized in that said peptide is administered in the immune system across the eat, what antibody is produced by secretions of the mucosal surface.16. The method according to p. 13, characterized in that the peptides associated with the media used the virus, preferably influenza virus, or part of a virus, preferably the hemagglutinin of influenza virus, as a carrier.17. Antibody expressing HIV-1 neutralizing activity and is able to prevent the fusion of HIV-1 cells, characterized by the following properties: refers to the type of IgA; directed to the epitope ELDKWA; get method according to p. 13; is mainly secreted by the mucosal surface.