Methods and composition for inhibition of hiv-1 multiplication

FIELD: virology.

SUBSTANCE: invention proposes different compositions able to induce production of antibodies against Tat HIV-1 that can inhibit multiplication of HIV-1. Also, invention proposes a method for induction of antibodies raised against Tat HIV-1, in vitro method for assay of the presence of antibodies and their titer values, a method for reducing HIV-1 virus levels, sequence of synthetic nucleic acid and synthetic molecule. Proposed group of inventions can be used for inhibition of multiplication of HIV-1 in infected patients and for attenuation of HIV-1 multiplication after the primary infection in early infected persons.

EFFECT: valuable methods and compositions.

39 cl, 7 dwg, 9 tbl, 5 ex

 

The scope to which the invention relates.

In General terms, the present invention relates to compositions and methods that can be used to inhibit the multiplication of human immunodeficiency virus-1 (HIV-1) infected patients with symptoms or without symptoms and to reduce the propagation of HIV-1 after primary infection in a previously uninfected individuals in order to minimize the progression of AIDS.

Background of invention

Various methods of treating infections caused by human immunodeficiency virus type 1 (HIV-1), aimed at transactivity gene (tat) of HIV-1, which produces a protein (Tat), which plays an important role in the transcription of the virus. Gene tat and its protein were sequenced and investigated the perceived possibility of their use for the treatment of HIV infections [see, for example, U.S. patent No. 5158877, 5238882 and 5110802; International patent application no WO 92/07871, WO 91/10453, WO 91/09958 and WO 87/02989, published may 14, 1992, 25 July 1991 11 July 1991 and may 21, 1987, respectively]. Protein Tat is released from the cells, which makes possible the absorption of other infected cells with increased transcription of HIV-1 in these cells and uninfected cells with changes in the activation of genes of the host cell. Tat reports these cells susceptible to infection with the indicated virus. Urbanpolicy Tat cells is very high and, as reported, is mediated by a short main sequence specified protein [S.Fawell et al., Proc. Natl. Acad. Sci., USA, 91:664-668 (1994)].

The intensive research on immunization using the Tat protein of HIV-1 as a potential AIDS vaccine. In published studies, the sequence of the Tat HIV-1 HXB/LAV was used as an immunogen or as a recombinant protein [A.Cafaro et al., Nat. Med., 5:643-650 (1999)], DNA vaccines [S.Calarota et al., Lancet. 351:1320-5 (1998)], inactivated protein (Tat-toxoid) [S.S. Cohen et al., Proc. Natl. Acad. Sci., USA, 96(19):10842-10847 (1999)], A. Gringeri et al., J. Hum. Virol. 1:293-8 (1998)] or DNA vaccines expressing inactive Tat [E.Caselli et al., J.Immunol., 162:5631-5638 (1999)]. Immunizatio full sequence Tat induced both cellular and humoral immunity. Cm. also M.C. Rhe et al., J. Acquir. Immune. Defic. Syndr. Hum. Virol. 10:408-416 (1995); C.J. Li et al., Proc. Natl. Acad. Sci. USA, 94:8116-8120 (1997) and others].

International patent application number WO 92/14755, published September 3, 1992, refers to a protein Tat and cell surface receptor - integrin, is able to connect with Tat protein. Identified two sequences of Tat binding to integrin: -Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg [SEQ ID NO: 1]and Gly-Arg-Gly-Asp-Ser-Pro [SEQ ID NO: 2]. These sequences represent the main area or domain, which is the dominant site of binding to the integrin. The op is sanija said application demonstrates the number of peptides corresponding to these sequences, Tat, and the related integrins inhibit in vitro the binding of cells with Tat-sensitized tablets as well as antibodies against the respective integrins. However, in this description is also shown that these reagents do not block the absorption of functional Tat cells (see, example 9 in WO92/14755), making ineffective the proposed mechanism of action for therapeutic treatment of HIV infection. The Tat sequence that is described in the International application different from the peptide immunogens of the present invention.

Both monoclonal and polyclonal antibodies against Tat protein were easily produced in animals and have shown that they block the uptake of Tat protein in vitro [see, for example, work D.Brake et al., J.Virol., 64:962 (1990); D.Mann et al., EMBO J. 10, 1733 (1991); J. Abraham et al.; P.Auron et al.; work M.Jaye et al.; G.Zauli et al., quoted above]. In later studies it was shown that monoclonal or polyclonal antibodies against Tat protein, was added to the medium with tissue culture, weakened HIV-1 infection in vitro [work L.Steinaa et al., Arch. Virol. 139:263 (1994); .Re et al., cited above; and G.Zauli et al., J. Acq. Imm. Def. Syndr. Hum. Retrovirol., 10:306 (1995)].

In the publications of the authors of this application [G. Goldstein, Nature Med. 2:960 (1996) and international patent application No. WO95/31999, published November 30, 1995] finds evidence that the secretion of the protein Tat of HIV-1 from infected cells and the absorption of both infected and uninfected cells plays an important role in the infectivity of HIV-1. Previous studies have also shown that antibodies against Tat protein blocked the in vitro uptake of Tat and inhibited infectivity in vitro. It has been suggested that active immunization of animals induces the formation of antibodies against the protein Tat of HIV-1 used as a potential AIDS vaccine. Cm. also G. Goldstein et al., "Minimization of chronic plasma viremia in rhesus macaques immunized with synthetic HIV-1 Tat peptides and infected with a chimeric simian/human immunodeficiency virus (SHIV33)", Vaccine, 18:2789 (2000).

In other publications of the author of the present invention (international patent application No. WO99/02185 published January 21, 1999, and U.S. patent No. 5891994, issued April 6, 1999 (both these works are introduced in the present description by reference)) proposed a new concept for the treatment and prevention of HIV-1 infection, involving the use of Tat sequences that are recognized by the immune system of rabbits as epitopes. Unlike previous works discussed above, these publications relate to therapeutic and immunogenic combinations, requiring the presence of at least two, and preferably all four peptides or polypeptides Tat, including sequence is lnasty "epitope I, covering the following amino acid residues 4 (or 5) - 10 Tat: -Asp-Pro-X7-Leu-Glu-Pro [SEQ ID NO: 3] or R1-Val-Asp-Pro-X7-Leu-Glu-Pro-R2[SEQ ID NO: 4], where X7is Arg, Lys, Ser or Asn. Such compositions induce antibodies that react with most of the protein Tat of HIV-1 and inhibit the replication of HIV-1. In accordance with this publication, this composition can be added to some other sequence Tat, which include a peptide or polypeptide "epitope II, spanning amino acid residues 41-50 Tat formula R3-Lys-X42-Leu-Gly-Ile-Ser-Tyr-Gly-Arg-Lys-R4 [SEQ ID NO: 5], where X42selected from the group consisting of Gly or Ala. Alternatively, this composition can be added to the peptide or polypeptide "epitope III, encompassing amino acid residues 56-62 Tat formula R5-Arg-Arg-X58-Z59-A60-Y61-Ser-R6 [SEQ ID NO: 6], where X58selected from the group consisting of Ala, Pro, Ser and Gln; Y61selected from the group consisting of Asp, Asn, Gly and Ser; Z59selected from the group consisting of Pro and His; and A60selected from the group consisting of Gln and Pro. In addition, the alternative, the composition can be added to the peptide or polypeptide "epitope IV", encompassing amino acid residues 62-73 Tat formula R7-Ser-Gln-X64-His-Gln-Y67-Ser-Leu-Ser-Lys-Gln-Pro-R8 [SEQ ID NO: 7], where X64selected from the group consisting of Asn and Thr; Y67selected from the group consisting of Ala and Val. the AMA this composition can be used to induce antibodies against a large number of sequences, Tat, characterizing multiple variants of HIV-1. These compositions or generated antibodies are used as vaccines or preventive treatment against these multiple options.

Despite the accumulated knowledge about the spread of diseases associated with HIV-1, the need to develop compositions and methods for prophylactic and therapeutic treatment of HIV-1 infections, which would reduce the levels of HIV-1 for the treatment and possible prevention of subsequent, mostly deadly disease AIDS, remains.

Brief description of the invention

In one of its aspects the present invention relates to compositions comprising at least two variants of a peptide or polypeptide epitope I of the formula R1-Asp-Pro-Y7-Leu-X9-Pro-Trp-Z12-R2 [SEQ ID NO: 8], where Y7selected from the group consisting of Arg, Lys, Ser and Asn; X9selected from the group consisting of Glu and Asp; Z12selected from the group consisting of Lys and Asn; R1 is selected from the group consisting of hydrogen, lower alkyl, lower alkanoyl and sequence of about 1-5 amino acids, optionally substituted lower alkyl or lower alkanoyl; R2 is selected from the group consisting of a free hydroxyl, an amide, and a sequence of about 1-5 additional is minislot, optionally substituted by amidon. In this composition, at least one of the two options should have the formula where Y7represents Arg and Z12is Lys, and at least the second of the two options should have the formula where Y7is Asn and Z12is Asn. Each peptide of this composition is recognized by the immune system of primates as the epitope I Tat of HIV-1. This formula allows us to construct and use a range of peptide combinations.

In another aspect of the present invention described above, the composition further contains one or more additional peptides or polypeptides which are other amino acid sequence corresponding to amino acid residues 5-12 Tat of HIV-1. These optional amino acid sequence described in detail below. These sequences are preferably derived from the HIV-1 strain having the variant protein Tat in the specified field.

In another aspect the present invention relates to the above compositions containing the peptides or polypeptides that contain at least two of the required peptide epitope I, recognized by the immune system of primates (and preferably, other peptides epitope I) in combination with one or more epitopes II, III and/or IV Tat of HIV-1. Epitopes II, III and IV depict ablaut a Tat peptides of HIV-1, formulas are presented in the publication of International patent application No. WO99/02185, which is introduced in the present description by reference. Such compositions can combine the corresponding peptides, Tat, HIV-1, so that these compositions induced the formation of antibodies reacting approximately more than 95% of all known protein Tat of HIV-1.

In yet another aspect the present invention relates to compositions of antibodies comprising at least one antibody, preferably generated from the primacy, which specifically binds to a peptide or polypeptide of the formula R1-Asp-Pro-Y7-Leu-X9-Pro-Trp-Z12-R2 [SEQ ID NO: 8], where Y7selected from the group consisting of Arg, Lys, Ser and Asn; X9selected from the group consisting of Glu and Asp; Z12selected from the group consisting of Lys and Asn; R1 is selected from the group consisting of hydrogen, lower alkyl, lower alkanoyl and sequence of about 1-5 amino acids, optionally substituted lower alkyl or lower alkanoyl; R2 is selected from the group consisting of a free hydroxyl, an amide, and a sequence of about 1-5 additional amino acids, optionally substituted by amidon. This antibody composition preferably contains at least two antibodies, i.e. one antibody that binds to a variant of the epitope I, where Y7is sabotaged, and Z12represents Lys, and at least a second antibody that binds with a second embodiment of the epitope I, where Y7represents Asn, and Z12represents Asn. This composition may also include other antibodies directed to these two specific variants, and other variants. Such antibodies in the specified composition are associated with sequences of epitope I, recognizable by the immune system of primates, where the specified epitope is present on many versions protein Tat of HIV-1. These antibodies include a number of structures of antibodies, such as monoclonal antibodies, which are described in more detail below.

In yet another aspect the present invention relates to an antibody, particularly a monoclonal antibody, which specifically binds with recognizable by the immune system of primates epitope of the protein Tat of HIV-1, and the specified epitope comprises the amino acid sequence-Asp-Pro-Y7-Leu-X9-Pro-Trp-Z12- [SEQ ID NO: 9], where Y7, X9and Z12defined above.

In yet another aspect the present invention relates to an antibody composition containing at least one antibody that recognizes a peptide sequence of the epitope II-Lys-X42-Leu-Gly-Ile-Ser-Tyr-Gly-Arg-Lys [SEQ ID NO: 10], where X42represents Gly or Ala, and where indicated the epitope different from the epitope-Leu-Gly-Ile-Ser-Tyr-Gly-Arg-Lys [SEQ ID NO: 11], recognized previously described antibodies. Preferably, these compositions include one antibody that recognizes a peptide, where X42represents Gly, and the peptide, where X42represents Ala. These antibodies are produced preferably in primates. These antibodies in the specified composition are associated with sequences II epitope recognized by the immune system of primates, where the specified epitope is present on many versions protein Tat of HIV-1. These antibodies include a number of structures of antibodies, which are described in more detail below.

In yet another aspect the present invention relates to the antibody, and preferably, the monoclonal antibody that recognizes a peptide sequence of the epitope II-Lys-X42-Leu-Gly-Ile-Ser-Tyr-Gly-Arg-Lys [SEQ ID NO: 10], where X42is Gly or Ala, and where specified epitope different from the epitope-Leu-Gly-Ile-Ser-Tyr-Gly-Arg-Lys [SEQ ID NO: 11], recognized the previously described antibodies.

In yet another aspect the present invention relates to recombinant or synthetic gene, which sequentially encodes a peptide or polypeptide containing at least two variants of a peptide or polypeptide epitope I of the formula R1-Asp-Pro-Y7-Leu-X9-Pro-Trp-Z12-R2 [SEQ ID NO: 8], defined above. This synthetic gene at least Odie is of two options must have the formula, where Y7represents Arg and Z12represents Lys, and at least the second of the two options should have the formula where Y7represents Asn, and Z12represents Asn. This synthetic gene contains optional carboxy-terminal peptide epitope II, recognized by the immune system of primates. Alternatively, recombinant or synthetic gene contains seven or eight preferred amino acid sequence of the epitope I, recognized by the immune system of primates and identified below. The synthetic gene can contain every amino acid sequence, separated spacer elements of the sequence, or it can Express each peptide/polypeptide read together with protein carrier with preservation of the open reading frame. The specified synthetic gene may be separated from the protein carrier by the spacer in that case, if the spacer is attached to the sequence of the epitope I, recognized by the immune system of primates, resulting in the sequence of the epitope II will be located at the carboxy-end of the recombinant protein. Other embodiments of the invention include a variety of peptides epitope I of the above formulas attached to each other and to the protein of the host.

In still another aspect of the present invention is worn by a synthetic molecule, for example, the vector containing the above-described synthetic gene is functionally attached to a regulatory nucleic acid sequences that direct and regulate the expression product of the synthetic gene in a cell host.

In another aspect the present invention relates to the recombinant microorganism such as a virus or bacteria-commensal, which contain the above-described synthetic gene or a synthetic molecule. The specified microorganism is able to Express in a host of many copies of a gene product of the specified or the specified molecule.

In another aspect the present invention relates to pharmaceutical compositions used for the induction of the production of antibodies that react with a large number of known protein Tat of HIV-1, for example, with more than 95%and preferably more than 99% of the known protein Tat. These induced antibodies can inhibit the replication of HIV-1. Specified pharmaceutical composition includes at least one of the compositions containing recombinant or synthetic peptides/polypeptides described above; or a synthetic gene/synthetic molecule described above; or the recombinant microorganism described above, in a pharmaceutically acceptable carrier.

In another aspect of the present invention relative is seeking to pharmaceutical compositions, which can be used to inhibit the multiplication of HIV-1, where the specified composition contains the above-described composition of an antibody or composition of monoclonal antibodies.

In yet another aspect the present invention relates to a method of reducing levels of HIV-1 virus, introducing a human or other Primate antibody-inducing pharmaceutical compositions described above and actively inducing the production of antibodies that react with most of the protein Tat of HIV-1 and inhibiting the reproduction of the indicated virus in vivo. This method can be applied to HIV-1-infected individual with a competent immune system or to uninfected or chronically infected individual, but do not have symptoms. This method allows to induce the production of antibodies that react with the protein Tat of HIV-1 and reduce the replication of the virus during any initial acute infection with HIV-1 virus and who, in addition, minimize chronic viremia leading to AIDS.

In another aspect the present invention relates to a method of reducing levels of HIV-1 virus by introducing a pharmaceutical composition containing the above-described compositions of the antibodies, the person is not able to develop an effective or rapid immune response to HIV-1 infection, This method may provide for the continuous introduction of specified composition.

In other aspects the present invention relates to methods of producing the compositions described above and also to the cells of the host, transfected with the indicated compositions.

In yet another aspect the present invention relates to a kit for the measurement and detection of titles and specificdate antibodies induced by immunization compositions described above. The kit of the present invention, preferably, includes two required peptide epitope I, described above, as well as additional peptides epitope I, recognized by the immune system of primates, and possibly additional peptides epitopes II-IV, and sensitized solid carriers, labeled reagent for detection of antibody binding to these peptides, a variety of substrates and device for stimulation or detection signals generated by the labels, as well as the standard device for sampling blood, appropriate vessels and other components of the diagnostic analysis.

In another aspect the present invention relates to a method of detection of titles and evaluation patterns of antibody reactivity in individuals immunogenic compositions of the present invention. This method includes the stage of incubation of dilutions of a biological fluid of an individual, for example, serum, with planes or spheres, which is nazyvautsa one or more peptide sequences of the epitope I of the present invention, and optional epitopes II-IV, washing unbound biological materials, and measuring the level of binding of any antibodies with peptides using a labeled reagent, such as anti-human immunoglobulin, which is associated with this enzyme. Depending on the type label of the signal produced by this label, can be induced by subsequent addition of the substrate, which reacts with the enzyme, for example, can be induced discoloration. In this analytic set can be entered and other standard labels.

Other aspects and advantages of the present invention are presented below in a more detailed description of the preferred variants of its implementation.

Brief description of the graphical material

On figa shows a graph of ELISA-titer rabbit antisera against larger linear peptide epitope I truncated the detector peptides, where these titers are expressed as the percentage of maximum binding to larger peptides. N - or C-terminal amino acids of the respective detector peptides are indicated below each column one-letter code.

On FIGU shows a graph of ELISA-titer antisera primates against a larger linear peptide epitope I truncated the detector peptides, where these titles is erogeny as a percentage of the maximum binding to larger peptides. N - or C-terminal amino acids of the respective detector peptides are indicated below each column one-letter code.

On figa shows a graph of ELISA-titer rabbit antisera against a linear peptide epitope II truncated detector peptides, where these titers are expressed as the percentage of maximum binding to larger peptides. N - or C-terminal amino acids of the respective detector peptides are indicated below each column one-letter code.

On FIGU shows a graph of ELISA-titer antisera primates against a linear peptide epitope II truncated detector peptides, where these titers are expressed as the percentage of maximum binding to larger peptides. N - or C-terminal amino acids of the respective detector peptides are indicated below each column one-letter code.

On figa illustrated construction pentavalent immunogenic structure of the epitope I/II epitope. Tat HIV-1, where amino acids are denoted by the three-letter code [SEQ ID NO: 12].

On FIGU illustrated construction osmislennogo universal epitope I, where amino acids are denoted by the three-letter code [SEQ ID NO: 13].

On figs illustrated construction monovalent immunogenic universal design of epitope II, where amino acids are marked trenbolone the code [SEQ ID NO:14].

Detailed description of the invention

The present invention relates to solving the above problems by more compositions that induce the production of antibodies in uninfected individuals or individuals with early HIV-1 infection who are still capable of producing an immune response to the immunogen, and these antibodies react with a large number (i.e. more than 95%, and preferably more than 99%) of the known variants of the Tat protein of HIV-1. The term "variant sequence (or protein) Tat" means a polypeptide or peptide containing amino acid residues Tat protein or the sequence of another protein Tat of HIV-1 strain, which is basically similar to the consensus sequence shown in table I [SEQ ID NO: 15]. Each variant may differ from the consensus sequence and/or from another variant, at least one amino acid substitution at residues that are important for epitope I-IV. When introduced into the composition of the present invention, this replacement can give the same or a different antigenic specificity for a particular epitope Tat.

The antibodies induced by the compositions of the present invention, can inhibit the replication of HIV-1, and, thereby, to prevent the subsequent development of the disease AIDS. The antibody-containing compositions the AI is also intended for the introduction of infected or uninfected individuals are not able to develop an effective or rapid immune response to HIV-1 infection. These compositions have the ability to react with a large number of proteins Tat, thereby leading to lower levels of HIV-1 virus. These antibodies can be used in therapeutic and prophylactic purposes to prevent the development of AIDS in a large number of people exposed to strains of HIV-1 infected strains of HIV-1 after infection cells produce immunologically distinct proteins Tat.

Compositions of the present invention include peptides or proteins consisting of peptides comprising an epitope of the protein Tat of HIV-1, against which the Primate antibodies, or nucleic acid sequences that encode the peptides and polypeptides that induce the production of primates antibodies against Tat. These induced antibodies, in turn, inhibit the replication of HIV-1.

Protein Tat of HIV-1 is produced by two exons: exon 1, encoding a protein of 72 amino acids (AA), which can be expressed without splicing or which may be playserver with approximately 15-32 amino acids encoded by exon 2. The sequence of exon I Tat HIV-1 shown in table I and represents a consensus sequence based on the sequence of the protein at 31 known strains of HIV-1, common subtype In [database NIH Los Alamos]. The provisions of amino acids, in which there are variations, denoted by lowercase letters. In table I [SEQ ID NO: 15] amino acid residue at position 73 is the first Pro exon 2 Tat of HIV-1. Because the product of exon I of the 72 amino acid itself can be absorbed by the cell and activate, it is important that the antibodies reacted with the indicated peptide of 72 amino acids and thereby prevented the intercellular transport of the peptide. Tat of HIV-1 contains rich in cysteine region between the provisions of amino acids 22 and 37 of exon I [SEQ ID NO: 15] one covalent bond between Cys21and Cys37forming, thereby, a complex tertiary structure. The research literature shows that this area is probably not immunogenic. Prevailing antibodies against Tat are antibodies to linear N-terminal Pro-rich region (AA1-21) and to the linear core area (AE-65), it was reported on additional antibody against the region AA-73.

The authors of the present invention previously identified epitopes, i.e. the area of linking recognized by rabbit antibodies (antigenic sequence) in the N-terminal linear sequence 1-21 (22 AA) exon I [SEQ ID NO: 15] Tat variants and identified four b-cell epitope in the HIV Tat-1. As was previously described in the publication M the international patent application no WO 99/02185, immunogenic region of this larger sequence is recognized by the immune system of rabbits. These areas were identified in the consensus sequence of exon 1 in table I below. The epitope I was identified rabbit antibodies as a sequence of nine amino acids in positions 2-10 of exon 1. Epitope II was identified as a sequence of eight amino acids in positions 43-50 exon 1. Epitope III has been identified as a sequence of 7 amino acids in positions 56-62 exon 1. Epitope IV was identified as a sequence of twelve amino acids in positions 62-73 Tat, including the first Pro (A) exon 2 and overlapping with Ser62 epitope III.

However, the author of the present invention unexpectedly discovered a shift in amino acid sequences, in particular epitope I, recognized by b-cells of primates. In table I sequences of epitopes I and II, recognized by the cells of primates, are underlined. Recognized by cells of primates epitope I encompasses amino acid residues 5-12 Tat. Sequence II epitope recognized by b-cells of primates, encompasses amino acids 41-50. Epitopes III and IV are the same epitopes that are recognized by the immune system of rabbits and are described in WO99/02185, input the ima in the present description by reference.

A. Immunogenic compositions containing the epitope I, which is recognized by the immune system of primates

In one variation of its implementation of the present invention relates to compositions containing at least two variants of a peptide or polypeptide that can be recognized by the immune system of primates and stimulating specific humoral immune response (corresponding to the purposes of the present invention) in primates subjected to in vivo specified sequences. These are recognized by the immune system of primates amino acid sequence of the epitope I correspond to amino acid residues 5-12 consensus sequences of Tat [SEQ ID NO: 15], shown in table I, originating from a number of "variants of the sequence Tat". Recognized by the immune system of primates I identifies the epitope peptides of General formula R1-Asp-Pro-Y7-Leu-X9-Pro-Trp-Z12-R2 [SEQ ID NO: 8], where Y7represents Arg, Lys, Ser or Asn; X9represents Glu or Asp, and Z12represents Lys or Asn. This formula allows to determine the set of variant peptides epitope I of mammals. The composition of the present invention must contain at least one peptide variant, where Y7represents Arg and Z12represents Lys, and at least a second peptide variant, where Y7p is ecstasy an Asn and Z 12represents Asn.

Specific amino acids present in the formula epitope I, recognized by the immune system of primates and described above represent the minimum reactive sequence of the epitope I, recognized by the immune system of primates. Each immunogen, certain specified formula and used in the methods of the present invention for the production of antibodies against the minimum sequence of the epitope I, may represent a larger amino acid sequence. For example, the minimum amino acid sequence of the epitope I planiruetsja other amino acid sequences, so that all immunogenic epitope sequence I has a length of from about 8 to 25 amino acids. Type of flanking amino acids does not play an important role in the biological function of the immunogen epitope I. In particular, additional amino acids at the N-end sequences of the epitope I, recognized by the immune system of primates, do not affect immunogenicity. Thus, for each peptide epitope I, recognized by the immune system of primates, N-terminal R1 can be free hydrogen on an unmodified N-terminal amino acid or the lower alkyl (i.e. C1-C10-alkyl) or lower C1-C10-alcoolica group, such as acetyl GRU is PA. R1 may also include a sequence from about 1 to about 5 amino acids, optionally substituted lower alkyl or lower alkanoyl. Preferably, R1 is a 2 amino acids. In one of the embodiments of the invention R1 represents Val, resulting in a sequence Val-Asp-Pro-Y7-Leu-X9-Pro-Trp-Z12[SEQ ID NO: 37], where Y7, X9and Z12defined above. In another embodiment of the invention R1 represents X2-Pro-Val-, resulting in a sequence of X2-Pro-Val-Asp-Pro-Y7-Leu-X9-Pro-Trp-Z12[SEQ ID NO: 38], where X2represents Glu or Asp, and where Y7, X9and Z12defined above. Preferably, R1 is a 3 amino acids.

Additional amino acids at the C-end of the minimum sequence of the epitope I, recognized by the immune system of primates, can increase the titer of antibodies. Although the C-terminal R2 can be a simple free hydroxyl group at the C-terminal amino acid, however, it can also be a C-terminal amide. However, to increase the titer R2 preferably represents a sequence of from about 1 to 14, preferably about 4 additional amino acids, amidarone at the carboxy-end. In predpochtitel the nom embodiment of the invention R2 represents His-Pro-Gly-Ser-amide, resulting in the sequence Asp-Pro-Y7-Leu-X9-Pro-Trp-Z12-His-Pro-Gly-Ser [SEQ ID NO: 16], where Y7, X9and Z12defined above.

Preferably, the composition of the present invention, in addition to the two desired peptides identified above, includes at least five or six other amino acid sequences of the epitope I, recognized by the immune system of primates. More preferably, the composition includes about seven or eight variant amino acid sequences identified directly below. This composition may also contain other peptide or polypeptide sequence, each of which includes various combinations of X9,Y7and Z12. As demonstrated in negativeone examples, which explains recognized by the immune system of primates epitope I, with three sites of antigenic variability, the preferred composition of the present invention may include a sufficient number of peptides epitope I, recognized by the immune system of primates, so that this composition contains 95% of all known cases In Cladocera and not-In-Cladonia Tat HIV-1 due to the inclusion of two "necessary" peptides:

R1-Asp-Pro-Arg-Leu-Glu-Pro-Trp-Lys-R2 [SEQ ID NO: 17] and

R1-Asp-Pro-Asn-Leu-Glu-Pro-Trp-Asn-R2 [SEQ ID NO: 18],

and one of the five trail is among the additional epitope peptides I:

R1-Asp-Pro-Lys-Leu-Glu-Pro-Trp-Lys-R2 [SEQ ID NO: 19],

R1-Asp-Pro-Ser-Leu-Glu-Pro-Trp-Lys-R2 [SEQ ID NO: 20],

R1-Asp-Pro-Asn-Leu-Glu-Pro-Trp-Lys-R2 [SEQ ID NO: 21],

R1-Asp-Pro-Lys-Leu-Glu-Pro-Trp-Asn-R2 [SEQ ID NO: 22] and

R1-Asp-Pro-Asn-Leu-Asp-Pro-Trp-Asn-R2 [SEQ ID NO:23],

and not necessarily rare variant

R1-Asp-Pro-Ser-Leu-Glu-Pro-Trp-Asn-R2 [SEQ ID NO: 24].

The composition of the epitope I, recognized by the immune system of primates, may include a number of additional peptides or polypeptides that contain other sequence corresponding to amino acid residues AA-AA SEQ ID NO: 15, but originating from other Tat variants that are not capable of cross-reacting with antibodies against compositions of epitope I, recognized by the immune system of primates. Composition of epitope I of the present invention may contain multiple copies of five or more different peptides epitope I, in any order. In one of the embodiments of the invention in the composition is at least one copy of seven or all eight of the amino acid sequences described above [SEQ ID NO: 17-24].

These peptides or polypeptides of the present invention produce synthetic or recombinantly ways. Binding of these peptides to each other or to the carrier at the ends of these peptides can be included optional amino acids (for example, -Gly-Ser-) or other amino acids or chemical compounds on - Spey is a career. This composition may be in the form of one or several of the above peptides, expressed in the form of a synthetic peptide that is attached to a protein-carrier. Alternatively, the composition may contain a variety of peptides epitope I, each of which is expressed in the form of a multiple antigenic peptide, optionally attached to a protein-carrier. Alternatively, the selected peptides can be sequentially attached and expressed in recombinante produced protein. In one of the embodiments of the invention eight sequences specifically identified above, are connected to each other directly or through a spacer elements amino acids with the formation of larger recombinant protein. Alternatively, the recombinant protein can be fused with preservation of the reading frame with the protein carrier. Such compositions epitope I, recognized by the immune system of primates, are designed to induce the production of antibodies that react with more than 95% of the known variants of the Tat protein of HIV-1, including protein Tat of HIV-1 In and not-In-clamation.

The composition of the epitope I, recognized by the immune system of primates, detect biological activity, aimed at inducing immunising immunocompetent primacy, i.e. uninfected the first man or infected persons without symptoms active humoral immune response (i.e. the formation of antibodies)that is directed against more than 95%, and preferably more than 99% of the known variants of the Tat protein of HIV-1. The end result of this treatment is the inhibition of the propagation of the HIV-1 after acute infection. This inhibition prevents high levels of HIV-1 in plasma after seroconversion associated with the development of AIDS. Active induction of antibody production in asymptotic phase of HIV infection may lead to reduced viral replication, reduce viral load in the plasma and to reduce the likelihood of developing AIDS. The composition, which contains at least two necessary immunogen epitope I, recognized by the immune system of primates, and preferably seven or eight sequences of epitope I [e.g., SEQ ID NO: 17-24], can stimulate the immune response by about 95% of the 294 well-known sequences of Tat In General-subtype HIV-1 protein Tat for all 56-b subtypes of HIV-1 that have been sequenced [with permission from Dr. Esther Guzman, database NIAID HIV Los Alamos, database Genbank].

Century Immunogenic compositions containing additional epitopes

In another embodiment, its implementation of the present invention relates to the other songs that use two or more sequences of apito is and I recognized by the immune system of primates, combined with at least one sequence of the epitope II and, optionally, with one or more peptides epitope III or IV. These epitopes II, III and IV Tat of HIV-1 that are recognized by the immune system of the rabbit, is described in detail in International patent application No. WO99/02185, which is introduced in the present description by reference.

Briefly, the sequence of the epitope II stimulates specific humoral immune response in the Primate subjected to in vivo sequence of the epitope II. The II epitope recognized by the immune system of primates, determines the peptides of the formula R3-Lys-X42-Leu-Gly-Ile-Ser-Tyr-Gly-Arg-Lys-R4 [SEQ ID NO: 5], where X42represents Gly or Ala. The minimal epitope recognized by the immune system of primates is the epitope with specifically identified amino acids of this formula ie-Lys-Gly-Leu-Gly-IIe-Ser-Tyr-Gly-Arg-Lys (amino acids 41-50 SEQ ID NO: 15). This sequence is also a sequence of preferred immunogen of the present invention to epitope II. Immunogen in which X42represents Gly, induces the production of antibodies cross-reacting with the sequence in which X42represents Ala. This sequence must respond/cross-react with more than 95% of f the x protein Tat of HIV-1. The sequence of the epitope II is not antigenic variability of the large number of known variants Tat of HIV-1. N-terminal R3 may represent a hydrogen on an unmodified N-terminal amino acid Lys, or R3 may be a lower alkyl or lower alkanoyloxy group such as acetyl group, which is a surrogate for Lys. R3 may also represent a sequence of about 1-5 amino acids, optionally substituted lower alkyl or lower alkanoyl. C-terminal R4 can be a free hydroxyl at the C-terminal amino acid, or R4 may represent an amide on this C-terminal amino acid. R4 may further include non-polar amino acids, such as spacer. Can be used illustrative spacer gly-ser-gly-ser obtaining sequence-Lys-X42-Leu-Gly-Ile-Ser-Tyr-Gly-Arg-Lys-Gly-Ser-Gly-Ser [SEQ ID NO: 25], where X42represents Gly or Ala. However, R4 cannot represent the sequence of the basic amino acids Lys-Arg-Arg, which is usually present in the Tat sequence after the last amino acid in the formula epitope II. This sequence of epitope II, found in 294 Tat variants In Cladocera, recognized by the immune system of primates (as reported in WO 99/02185, the immune system of rabbits recognizes an epitope of amino acids 43-50 SEQ ID NO: 15).

Epitope II, if it is present in other sequences, it has low immunogenicity. Thus, for optimal immunogenicity this sequence receive in the form of a synthetic peptide, fused with protein carrier or attached to it, either in the form of a multiple antigenic peptide, optionally attached to a protein-carrier. Alternatively, the epitope II can be expressed as C-terminal sequence of the recombinant protein, which is not necessarily attached in the same reading frame for the protein-carrier at its amino-terminal sequence. In the composition of the present invention the peptide epitope II is preferably separately or in combination with one or more peptides epitope I, recognized by the immune system of primates.

Epitope III briefly described and identified in WO 99/02185 determines the peptides of the formula R5-Arg-Arg-X58-Z59-A60-Y61-Ser-R6 [SEQ ID NO: 6], where X58can represent Ala, Pro, Ser or Gln; Y61can be an Asp, Asn, Gly or Ser; Z59can be a Pro, or His; and A60can be a Gln or Pro. Epitope IV defines the peptides of the formula R7-Ser-Gln-X64-His-Gln-Y67-Ser-Leu-Ser-Lys-Gln-Pro-R8 [SEQ ID NO: 7], where X64may be an Asn or Thr; and Y67may represent the Wallpaper Ala or Val.

Thus, the compositions of the present invention, i.e. peptides/polypeptide containing identified above amino acid sequence, intended for insertion person, can be used for immunological surveillance of the production of extracellular Tat protein of most strains of HIV-1. The action of these compositions is directed to a sharp decline in the continuous reproduction of the virus and allows efficient immune control the spread of this virus.

Immunogen for each epitope were constructed, preferably, to induce antibodies that react with most natural variants of each epitope. As for the epitope, such as recognized by the immune system of primates epitope I, to enhance the immunogenicity and production of antibodies with higher titers in synthetic or recombinant immunogen may be included multiple copies of the immunogen. In addition, immunogen for two or more epitopes can be combined to extend the range, because changes in the sequence of each epitope occur independently from each other. Thus, as one example is the composition of the present invention, which contains two peptide epitope I, recognizable immune si is theme of primates, and four or five other specifically identified above peptide epitope I with Cys at the end, which is linked to the protein carrier. Alternatively, it may be received multiple antigenic peptides, optionally attached to a protein-carrier and combined together to obtain the composition of the present invention. Alternatively, it may be used a mixture of two or more immunogenic.

Can be obtained immunogen recognized by the immune system of primates epitope I of the present invention together with any epitope II, III or IV or without them, or with other optional immunogenum, and these immunogen can be used in immunogenic compositions in various forms, for example in the form of chemically synthesized or recombinant peptides, polypeptides, proteins, hybrid proteins or hybrid peptides.

1. Recombinant or synthetic peptides/proteins attached to the media

In one of the embodiments of the invention, the composition of the present invention may be a synthetic or recombinante produced peptide containing at least two necessary immunogenic amino acid sequence of the epitope I, recognized by the immune system of primates (as well as other additional sequence of the epitope I), and, in addition, the containing a series of one or more immunogenic amino acid sequence of the epitope II/III/IV, attached to the selected protein-media. In this embodiment, the compositions of the present invention many of the above-mentioned amino acid sequence of the epitope I, recognized by the immune system of primates, with flanking sequences or without them, can be consistently combined in a polypeptide attached to the same protein-media. Alternatively, immunogenic epitope I, II, III or IV can individually be in the form of peptides attached to the same protein-media or other proteins, the media, and the resulting construction "immunogen-carrier" is mixed together with the formation of a single composition. Such sequences can be obtained synthetically by standard methods of chemical synthesis or recombinante by expression in the selected cell as the host, also carried out by standard methods.

In this embodiment of the invention the protein carrier is preferably a protein or other molecule that can enhance the immunogenicity of the selected immunogen. Such media can be a larger molecule, which acts as an adjuvant. Examples of standard proteins carriers include, but are not limited to, a protein of E.coli DnaK, galactokinase (galK, which catalyzes the first stage of galactose metabolism in bacteria), ubicacin, α factor crosses, β-galactosidase and protein NS-1 flu. As carriers can also be used toxoid (i.e. sequences that encode natural toxin with sufficient modifications to eliminate its toxic activity), such as diphtheria toxoid and tetanus toxoid. Likewise, there may be used a number of bacterial heat shock proteins, such as mycobacterial hsp-70. Another suitable carrier is a glutathione-reductase (GST). Each specialist can easily choose suitable media.

In the specific design of the immunogen - protein-media" two necessary immunogen epitope I and from three to six additional immunogenic epitope I, recognized by the immune system of primates, as well as optional immunogenic peptides/polypeptides can be covalently associated with mycobacterial heat shock protein 70 gene (hsp70) [K.Suzue et al., J. Immunol.156:873 (1996)]. In another preferred embodiment of the present invention the specified composition is obtained by covalent binding immunogen-containing peptide or polypeptide sequences with diphtheria toxoids.

2. Multiple antigenic peptide

In another embodiment, the present invention immunogenic peptide or polypeptide epitopes and any in the swear optional immunogen can take the form of a design of a multiple antigenic peptide ("MAR", also called octamer peptide with lysine core). This design can be constructed using IDA, described Tam, Proc. Natl. Acad. Sci. USA, 85:5409-5413 (1988). This system allows the use of a core matrix of lysine residues, which is synthesized multiple copies of the same recognized by the immune system of primates epitope I of the present invention, as described [D. Posnett et al., J. Biol. Chem. 263(4):1719-1725 (1988); J. Tam, "Chemically Defined Synthetic Immunogenes and Vaccine by the Multiple Antigen Peptide Approach", Vaccine Research and Developments, Vol.1 ed. W. Koff & H.Six, pp. 51-87 (Marcel Deblau, Inc. New York 1992)]. Each MAR contains multiple copies of only one peptide. Therefore, the composition containing MAR, includes at least two, and preferably, about seven MAR. One MAR is first necessary immunogen peptide or polypeptide epitope I, attached to each lysine core, and the second MAR has a second desired immunogen peptide or polypeptide epitope I, attached to each lysine core. Can be included and other MAR, each of which has different identified above amino acid sequence of the epitope I, recognized by the immune system of primates. Many different MAR can be used to obtain any desired combination of sequences of the epitope I, II, III or IV. P is edocfile, to these MAR designs were associated with other sequences that stimulate T-cells, or they were made in the form of pharmaceutical compositions, administered in combination with agents that stimulate T-cells, such as known adjuvants.

3. The spacers

In any of the above compositions, for example, are obtained in the form of structures of the peptide/polypeptide carrier or MAR, each peptide/polypeptide immunogen or each amino acid sequence in this immunogen may be optional, separated by an optional amino acid sequence, called a "spacer". The spacers are sequences having about 1 to 4 amino acids, which is embedded between two sequences that allows you to attach them without exerting any adverse influence on the three-dimensional structure of the immunogen. Optionally, the spacers may also contain sites of cleavage restricteduse the endonuclease to separate these sequences. Suitable spacers or linkers are known and can be easily designed and selected by any specialist. Preferred spacers are sequences containing amino acids Gly and/or Ser.

F. Composition of the nucleic acids of the present invention, including syntheti the ski or recombinante produced gene

In other variants of its implementation, the present invention relates to nucleic acid sequences coding for the above-described compositions of the peptides/polypeptides recognized by the immune system of primates epitope I, including peptide and polypeptide immunogen the above-described compositions comprising the peptides and polypeptides that have been attached to proteins vehicles. The nucleic acid sequences may also include sequences encoding carrier proteins.

Thus, one of the preferred embodiments of the invention refers to the "synthetic gene", which sequentially encodes at least two immunogenic peptide or polypeptide epitope I, recognized by the immune system of primates. It should be noted that although this gene is called "synthetic", but, if necessary, it can be constructed using chemical synthesis or recombinant methods. This synthetic gene preferably encodes seven or all eight specifically identified amino acid sequences of the epitope I, recognized by the immune system of primates [SEQ ID NO: 17-24]. The specified synthetic gene can also encode any selected immunogen epitope II or III, provided that the peptide epitope II or III process is dine to-end sequence of the epitope I, recognized by the immune system of primates, and is optionally modified at its C-end. The specified synthetic gene can encode multiple copies of two essential amino acid sequence of the epitope I, or additional copies of many other immunogenic or amino acid sequences or multiple copies of many other immunogenic or amino acid sequences. The specified synthetic gene can encode the selected amino acid sequence in one frame reading or being merged with the sequence of a nucleic acid encoding a carrier protein. Another feature of the synthetic gene is that it encodes a spacer located between each of the sequences encoding the immunogen, and/or between the sequence encoding the immunogen, and a sequence encoding a carrier protein.

The specified synthetic gene of the present invention may also be part synthetic or recombinant molecules. The specified synthetic molecule can be design nucleic acid, such as a vector or plasmid containing a synthetic gene that encodes a protein, a peptide, a polypeptide, a hybrid protein or hybrid peptide, and under the functional control sequence nucleotide sequence that is the new acid, coding regulatory elements such as promoters, termination signals, etc. Such synthetic molecules may be used for the recombinant production of immunogenic polypetide/peptide compositions. These synthetic gene or a synthetic molecule can be obtained by chemical synthesis or recombinant methods. So, for example, the synthetic gene or molecule can contain preferred codons for a given type specified host cell.

Synthetic gene or molecule, preferably in the form of DNA can be used in several ways. For example, these synthetic nucleic acid sequence can be used for expression of the peptides/polypeptides of the present invention in vitro in the culture of the host cell. Downregulation of immunogene after suitable purification can then be included in the pharmaceutical agent or a vaccine. Alternatively, a synthetic gene or a synthetic molecule of the present invention can be administered to a mammal, preferably a human, in the form of so-called "naked DNA" for in vivo expression of the protein/peptide immunogen of the patient. Cm. for example, the work of J. Cohen, Science 259:1691-1692 (March 19, 1993) and E. Fynan et al., Proc. Natl. Acad. Sci., USA, 90:11478-11842 (Dec. 1993) and J.A. Wolff et al., Biotehniques, 11:474-485 (1991), each of which is introduced into the present description by reference. The specified synthetic molecule, such as a vector or plasmid may be introduced directly to the mammalian host. This will lead to expression of the protein by the cells of the host and its subsequent presentation to the immune system to induce the formation of antibodies in vivo.

G. Microorganisms expressing the specified synthetic gene

In another aspect of the present invention these synthetic genes or molecules of the present invention can be included in the non-pathogenic microorganism. The obtained microorganism when it is administered to the mammal host expresses and multiplies in vivo downregulation of the composition of the present invention to induce the formation of specific antibodies. So, for example, non-pathogenic recombinant viruses or bacteria-commensal, which are compositions or synthetic genes of the present invention and which can be used to put the patient is a mammal can be obtained using standard techniques and selected from known non-pathogenic microorganisms.

Bacteria-commensals, which can be used for exogenous delivery of synthetic molecules to the patient and/or for the introduction of the synthetic gene to a patient in vivo, are, butnot limited to, various strains of Streptococcus, such as S. gordonii, or E. coli, Bacillus, Streptomyces, and Saccharomyces.

Suitable non-pathogenic viruses, which can be designed so that they gave the synthetic gene into the cells of the host are poxviruses, such as vaccinia virus, adenovirus, adeno-associated viruses, poxviruses Canaries, retroviruses and the like, the Number of such non-pathogenic viruses are typically used for human gene therapy, but also as carriers for other vaccine agents are known and can be selected by any specialist.

N. Obtaining or manufacturing the compositions of the present invention

Compositions of the present invention and a separate polypeptides/peptides containing immunogen recognized by the immune system of primates epitope I of the present invention and optionally one or more epitopes II, III or IV, synthetic genes and synthetic molecules of the present invention can be obtained by standard methods in accordance with known methods of chemical synthesis, such as described by Merrifield, J. Amer. Chem. Soc., 85:2149-2154 (1963). Alternatively, compositions of the present invention can be obtained by known methods of recombinant DNA by cloning and expression in a microorganism host or in the cell the DNA fragment carrying the sequence code is the dominant peptide/polypetide, containing at least two of the required sequence of the epitope I, recognized by the immune system of primates, optionally together with other immunogenum and optionally together with a protein-carriers. Coding sequences for epitope I and optional immunogens can be obtained by the method of synthesis [W.P.C. Stemmer et al., Gene, 164:49 (1995)], or they can be obtained from viral RNA known methods, or they can be derived from available cDNA-containing plasmids.

Can be used a combination of these methods. For example, for producing a synthetic gene can be carried out by the Assembly following each other immunogens standard methods of molecular biology, and site-directed mutagenesis can be used to obtain a desired sequence immunogen. Then perform the recombinant production of the product of the synthetic gene. All these manipulations can be carried out by standard methods.

System cloning and expression of the peptide/polypeptide compositions of the present invention with the use of synthetic genes or molecules include the use of various microorganisms and cells, are well known in the art of recombinant DNA. Such microorganisms are, for example, various strains of E. coli, Bacillus, Streptomyces and Saccharomyce, as well as mammalian cells, yeast and insects. Suitable vectors for them are known in the art and can be obtained from private and public laboratories and depositories or from commercial suppliers. Currently, the most preferred hosts are mammalian cells, such as cells of the Chinese hamster ovary (Cho) or COS cells-1. These hosts can be used in combination with vectors on the basis of poxviruses, such as vaccinia virus or variola virus of pigs. The selection of other suitable host cells and methods for transformation, culture, amplification, screening and isolation and purification of the product can be made by any person in accordance with known techniques. See, for example, Gething &Sambrook, Nature, 293:620-625 (1981), etc. Another preferred system are baculovirus expression systems and vectors.

When producing standard recombinant methods, compositions of the present invention, i.e., the polypeptides/petite containing the copies of immunogenic epitope I, recognized by the immune system of primates, and optional immunogen can be allocated either from the cellular content standard method of lysis or from the cellular environment by standard methods such as chromatography. Cm. for example, Sambrook et al., Molecular Cloning A Laboratory Manual, 2d Edit.,Cold Spring Harbor Laboratory, New York (1989). Suitable plasmid and viral vectors used for the production of peptide/polypetide components as DNA vaccines are well known in the art and do not limit the scope of the invention. See, manual Sambrook et al., as quoted above, and the above operation, in which is described the production of protein. See, International patent application PCT WO 94/01139, published 20 January 1994. Briefly, DNA encoding the selected peptide/polypeptide is inserted into a vector or plasmid that contain other optional flanking sequences, a promoter, a leader sequence of mRNA, the site of transcription initiation and other regulatory sequence capable of regulating the proliferation and the expression of the sequence in vivo and in vitro. These vectors infect cells of the patient and provide the expression of the sequence of the synthetic gene in vivo or an expression in vitro in the form of a protein/peptide or hybrid protein/peptide.

The resulting composition may be introduced into the composition of the epitope I, recognized by the immune system of primates, together with any number of optional immunogens and skanirovana on its effectiveness using in vivo analysis. Such analyses involve immunizing an animal such as monkeys, the composition and evaluation of antibody titers against the be the Cove Tat HIV-1 or against a synthetic detector peptides corresponding variants of Tat sequences (as shown in the examples below).

I. Antibody-containing compositions of the present invention

The antibody-containing composition or bind to the ligand composition of the present invention includes at least one antibody that specifically binds to the epitope I, formula-Asp-Pro-Y7-Leu-X9-Pro-Trp-Z12- [SEQ ID NO: 9], where Y7selected from the group consisting of Arg, Lys, Ser and Asn; X9selected from the group consisting of Glu and Asp; and Z12selected from the group consisting of Lys and Asn. Such antibody-containing composition preferably includes at least two or more different antibodies or ligands that specifically bind at least two defined here necessary sequences of epitope I. this produces antibodies (or other binding ligands) against two of the required sequences R1-Asp-Pro-Arg-Leu-Glu-Pro-Trp-Lys-R2 [SEQ ID NO: 17] and R1-Asp-Pro-Asn-Leu-Glu-Pro-Trp-Asn-R2 [SEQ ID NO: 18] epitope I. Thus, the antibodies of this composition is contacted with the protein Tat of HIV present in many versions protein Tat of HIV-1. Other antibodies or ligands are produced against other sequences covered by the above formulas epitope I.

In one embodiment of the invention, the selected antibody, which is bound to a peptide or polypeptide epitope I of the present invention, described above, is also an aspect of the present invention. Such compositions containing a polyclonal antibody, usually produced by immunization of a mammal, preferably a Primate, peptide/polypetides composition containing two necessary immunogen epitope I, and a set of other immunogenic epitope I, recognized by the immune system of primates, and optional immunogen described above. As immunogens especially preferred are heptavalent immunogen epitope I, which is recognized by the immune system of primates (i.e. without rare variants) or vosmiletnie immunogen, such as a synthetic gene or a hybrid protein as described below in example 3, and/or monovalent immunogen epitope II (i.e. a single peptide epitope II, not necessarily associated with the media). In addition to antibodies produced in primates, such antibodies can also be produced in transgenic animals, including so-called "humanized" transgenic mouse. However, a desirable host for production of polyclonal antibodies against the composition of the present invention is the man. Monitoring of titles such polyclonal antibodies produced by the mammal in response to the introduction of the composition of the epitope I, can be carried out by standard methods, such as terdapat the initial enzyme-linked immunosorbent assay. If desired, the antibody molecules can be isolated from a mammal, for example, from whole blood, plasma or serum, and then they can be purified from plasma or serum immunising mammal by standard methods. Standard methods of collection can be plasma exchange, chromatography on protein a and other Compositions such polyclonal antibodies can be used as pharmaceutical compositions of the present invention.

Alternatively, the cells producing the antibody can be obtained from mammals and used to produce other forms of antibodies and ligands, such as monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, ligands, produced by screening the phage display technique, fragments of antibodies, and mixtures thereof, and synthetic antibodies, monoclonal antibodies, chimeric antibodies, "humanized" antibodies and fully human antibodies. Preparative methods for the generation of ligands of these types known in the art, and the ligands can be generated using the described amino acid sequence of the epitope I, recognized by the immune system of primates, and optional immunogens. See, for example, Kohler &Milstein (1975) Nature, 256:495-497; Kozbor et al. (1983) Immunol. Today, 4:72; Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. pp. 77-96; Harlow et al., Antibodis A Laboratory Manual, Cold Spring Harbor Laboratory (1988); Queen et al., Proc. Natl. Acad. Sci. USA, 86: 10029-10032 (1989); Hodgson et al., Bio/Technology, 9:421 (1991); International application PCT/GB91/01554, publication number WO 92/04381 and International application PCT/GB93/00725, publication number WO 93/20210].

For example, in another embodiment of the invention, the monoclonal antibody specifically binds to a minimal sequence of the epitope I, defined as-Asp-Pro-Y7-Leu-X9-Pro-Trp-Z12- [SEQ ID NO: 9] protein Tat of HIV-1 containing any larger immunogen defined by the formula epitope I, where the variable amino acids and groups R are defined above. In one of the embodiments of the invention, the monoclonal antibody specifically binds to the amino acid sequence-Asp-Pro-Asn-Leu-X9-Pro - Trp-Asn [SEQ ID NO: 26], where X9is Glu or Asp. Other monoclonal antibodies that specifically bind with minimal sequences of epitope I, defined by the above formulas are part of the present invention.

In another embodiment the invention, the monoclonal antibody specifically binds to a minimal sequence II epitope containing amino acid sequence is Lys-X42-Leu-Gly-Ile-Ser-Tyr-Gly-Arg-Lys [SEQ ID NO: 10], where X42represents Gly or Ala and where specified epitope different from the epitope-Leu-Gly-Ile-Ser-Tyr-Gly-Arg-Lys- [EQ ID NO: 11], recognized previously described antibodies. Preferably, the antibody-containing composition comprises one antibody that cross-reacts with the peptide, where X42represents Gly, and with the peptide, where X42represents Ala. These antibodies are preferably produced in primates. Other monoclonal antibodies that specifically bind with minimal sequences of epitope II, defined by the above formula, are also part of the present invention.

Other antibodies against Tat can be obtained by screening a recombinant combinatorial library of the immunoglobulin (e.g., phage display antibodies using recognized by the immune system of primates Tat epitopes of HIV-1 of the present invention, for selecting members of the library of immunoglobulins that bind to Tat of HIV-1 [W.D. Huse et al., Science, 246:1275-1281 (1988)]. Kits for generating and screening libraries of phage display are commercially available, for example, in the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; Stratagene Phage Display kits, etc. See for example, U.S. patent No. 5223409, the publication of international application no WO 92/09690, WO 90/02809, etc. Chimeric antibodies can be similarly obtained with known methods [Morrison et al. (1984) Proc. Natl. Acad. Sci. USA, 81:6851; Takeda et al., Nature, 313:452 (1984) and the like]. Chimeric antibodies are the Wallpaper of the molecule, have different parts come from different species of animals. Single-chain antibodies can also be obtained by standard methods [see U.S. patent No. 4946778 and 4704692] with use of the variable parts of polyclonal or monoclonal antibodies produced in accordance with the present invention. Antibody fragments, such as Fab fragments, F(ab')2and Fv and their libraries can also be used in various aspects of the present invention.

These antibody/ligand-containing compositions preferably associated with most known variants of the protein Tat of HIV-1 (e.g., more than 95%, and preferably more than 99% of the known variants of the Tat protein) and prevent further replication of HIV-1, which is ensured by the Tat protein. Such compositions may include a mixture of many different antibodies that bind to the epitope sequences of the protein Tat of HIV-1, derived from multiple strains of HIV-1. Thus, these antibodies can be used in the methods and pharmaceutical preparations described below.

J. Pharmaceutical compositions of the present invention

In another aspect of the present invention the pharmaceutical composition used for the production of antibodies that react with most (for example, more than 95%, and preferably more than 99%) are known Belko is Tat HIV-1 and inhibiting the replication of HIV-1, can contain as active agent, at least two of the required peptide or polypeptide recognized by the primates of the epitope I of the present invention and, preferably, additional peptides epitope I. Some preferred compositions above are the following components:

(a) peptide/polypeptide immunogen, which contains at least two are needed, and more preferably, at least seven amino acid sequence of the epitope I, recognized by the immune system of primates [SEQ ID NO: 17-24];

(b) peptide/polypeptide immunogen (a), which further contains any one of the amino acid sequence of the epitope II, III or IV, preferably a monovalent immunogen epitope II;

(C) synthetic or recombinante produced the gene encoding two of the required sequence of the epitope I, recognized by the immune system of primates, and preferably seven sequences of epitope I [SEQ ID NO: 17-24], and optionally the sequence described above;

(d) a synthetic molecule containing a synthetic gene (s);

(e) recombinant virus carrying the synthetic gene or molecule described above; and

(f) bacterium-commensal, carrying the synthetic gene or molecule described above.

Selected(e) active(e) component(s) prists who meet(s) in a pharmaceutically acceptable carrier, and the said composition may contain additional ingredients. Pharmaceutical preparations containing compositions of the present invention may include other active agents, such as agents that stimulate T-cells, for MAR, adjuvants and immunostimulatory cytokines, such as IL-12, and other well-known cytokines for protein/peptide compositions. All these pharmaceutical compositions can reduce levels of the virus in a mammal.

Upon receipt of the pharmaceutical compositions compositions comprising a peptide sequence or nucleic acid sequence of the epitope I, recognized by the immune system of primates, and an optional sequence of immunogenes, mixed with a pharmaceutically acceptable carrier suitable for injection mammal for the prevention or treatment of viral infections. These proteins/peptides can be combined in a single pharmaceutical preparation for injection. Suitable pharmaceutically acceptable carriers used in the immunogenic protein compositions of the present invention, are well known in the art. These carriers are, for example, saline, buffered saline, selected adjuvant, such as water suspension of hydroxides of aluminum and magnesium, liposomes, Amul is these type of "oil in water", etc. In protein-containing compositions of the present invention can also be used suitable adjuvants. Suitable carriers for direct delivery of DNA, plasmid nucleic acid or recombinant vector include, but are not limited to, saline or sucrose, Protamine, polybrene, polylysin, polycation, proteins, CaPO4or spermidine. See, for example, PCT application WO94/01139 and work cited above. Peptide/polypeptide compositions and synthetic genes or molecules in vivo is capable of stimulating immunising host mammal, such as humans, the immune response, contributing to the suppression of many (e.g., approximately more than 95%-99%) known extracellular variants of the Tat protein of HIV-1 and, thereby, reduce levels of the virus.

Another pharmaceutical composition that can be used to inhibit the multiplication of HIV-1, consists of a composition of antibodies containing one or more antibodies described in detail above. In the pharmaceutical compositions of these antibodies may be present in saline or other suitable medium. These antibody-containing composition capable of providing Needlenose exogenous inhibition of Tat.

The present invention is not limited to the choice of standard physiologically acceptable carriers, is Duvanov or other ingredients, which can be used in pharmaceutical preparations of the above types. Preparation, including the specified pharmaceutically acceptable composition consisting of the above components and having the appropriate pH, isotonicity, stability and other acceptable properties can be obtained by any professionals.

K. the Method of the present invention is the Inhibition of the propagation of the HIV-1

In accordance with the present invention a method of reducing levels of HIV-1 provides an introduction to the person above-described pharmaceutical compositions, which induce the production of antibodies against Tat, actively induce the production of antibodies that react with many (e.g., approximately more than 95%, and preferably more than 99%) of the known protein Tat of HIV-1 and inhibit the replication of the virus in vivo. This method is designed for HIV-1-infected individual with a competent immune system or for active immunization uninfected individual. This method allows to induce antibodies that react with the protein Tat of HIV-1 and inhibit the reproduction of the virus during the initial acute infection with HIV-1 virus and minimize chronic viremia leading to the development of AIDS. This method also aims at reducing the constant reproduction of the virus in infected Indus is videolow, as well as to minimize the progression of AIDS. The use of these methods can take control of chronic HIV-1 infections and provides a new mechanism of treatment that have not developed resistance. Antibodies against Tat inhibit replication of Quasimodo HIV-1 regardless of the type of protein Tat, which they produce, because extracellular Tat protein is not associated with can replicate part of this virus. Therefore there is no obvious mechanism by which antibodies against Tat could generate selective pressure on non-reactive and escape from immunological surveillance options Tat.

In accordance with this method, pharmaceutical composition containing the peptide/polypeptide compositions, synthetic genes or molecules, recombinant viruses and recombinant bacterium-commensal. Preferably, these compositions contain heptavalent synthetic gene or a hybrid protein (without rare variants of epitope I) or vosmiletnie synthetic gene or a hybrid protein of example 3 and optional monovalent peptide epitope II. Each of these active components in pharmaceutical compositions actively induces in humans, which was introduced this composition, the formation of antibodies against Tat, which block the transfer of Tat from infic the level of cells in other infected or piperitenone cells. This action reduces the multiplicity of infection and blocks the outbreak of the virus HIV-1 and, thus, reduces the levels of virus. In already infected patients this way to reduce levels of the virus can reduce chronic viremia and reduce the progression of AIDS. Uninfected individuals introduction the composition of the present invention can lead to the suppression of acute infection and, thereby, minimize chronic viremia leading to the development of AIDS.

In yet another aspect the present invention relates to a method of reducing levels of HIV-1 virus by introducing the person is not able to develop an effective or rapid immune response to HIV-1 infection, pharmaceutical compositions containing the compositions of the antibodies described above. This method may provide for the continuous introduction of the composition. Patients susceptible to treatment by the above method are HIV-1-infected patients with immunological reactivity, disturbed by the disease, and unable to develop a strong immune response. In the later stages of HIV-1 infection, the probability of generating effective titers of antibodies is reduced due to the weakening of the immune system associated with the specified disease. Such patients are HIV-1-infected pregnant the women, newborns from infected mothers and non-immunized patients who are assumed to have been subjected to contact with the virus (for example, a man who accidentally made "the shot" needle used HIV-1-infected person).

For these patients, the method of the present invention preferably involves the use of antibody-containing compositions of the present invention as a pharmaceutical composition. The antibody-containing composition includes a composition comprising a polyclonal antibody obtained from other mammals, and preferably from normal human or other alternative forms of the above antibodies, for example monoclonal antibodies, etc. These antibody-containing composition is administered as passive immunotherapy for inhibition of viral replication and reduce viral load. Exogenous antibodies that react with a set of known protein Tat of HIV-1, quickly prevent in this patient transfer Tat from virusinfizierovannah cells in other infected or neetilirovannye cells. In accordance with the method of this patient can continuously enter the antibody-containing composition over an extended course of treatment.

In each of the above methods, the composition of this image is to be placed impose appropriate way for example, subcutaneously, orally, intravenously, intraperitoneally, intramuscularly, intranasally or by inhalation. In accordance with the present invention the preferred route of administration is intramuscular immunization (active induction) compositions and intravenous (i.v.), subcutaneous (s.c.) or intramuscular (i.m.) introduction antibody-containing compositions (passive therapy). Recombinant viral vectors or naked DNA, preferably intramuscularly, however, some other recombinant viral vectors and/or live bacteria are commensals can be administered orally.

The number of sequences of a protein, peptide or nucleic acid of the present invention, present in each vaccine dose, choose depending on age, weight, sex, General physical condition of the patient, etc. the Number of active components that are required to induce an immune response, preferably a protective response, or for producing exogenous effect in a patient without any significant adverse side effects varies depending on the pharmaceutical compositions and the presence optional adjuvant (for protein-containing compositions).

In General, to compositions containing protein/peptide hybrid protein, MAR or wired the th protein, or for compositions containing the antibody, each dose may contain from about 50 μg to 20 mg of the peptide/polypeptide immunogen per ml of the sterile solution. A more preferred dose may be about 500 μg of the immunogen. Other dose ranges can also be determined by the specialist. After administration of the initial dose can be, but not necessarily, introduced repeated booster doses if necessary.

Composition of antibodies of the present invention can be used for continuous administration to patients with risk of acute infections caused by injection needles or infection of the mother. Frequency of doses for each acute infection can vary from injection once a day to once or twice a week, i.v., s.c. or i.m. for about 6 weeks. The antibody-containing compositions of the present invention can also be used for permanent treatment of infected patients or patients with advanced HIV infection. In infected patients, the frequency of continuous administration of doses can vary from injection once a day to once or twice a month, i.v., s.c. or i.m. and may depend on the time-life immunogen (e.g., about 7-21 days). However, it is expected that the permanent treatment of such infected patients may continue for an indefinite, but prodoljitel the aqueous period of time.

Alternatively, compositions of the present invention can be obtained for the controlled introduction of synthetic genes or molecules of the present invention in the form of "naked DNA". As in the case of immunogenic protein-containing compositions, the number of components in the DNA and the vector-containing compositions and method of administration, for example, by injection or insertion through the nose, can be selected and adjusted by any specialist. Basically, each dose may contain from about 50 μg to 1 mg of immunogen-encoding DNA per ml of the sterile solution.

As for recombinant viruses containing synthetic genes or molecules, their dose can be from about 20 to 50 ml of saline solution containing from about 1×107up to 1×1010boe/ml recombinant virus of the present invention. The preferred dose for humans is about 20 ml of physiological solution with the above concentrations of the virus. However, it should be noted that each specialist may change the dose depending on specifically used recombinant virus and composition of the immunogen, which must be delivered to the owner.

The number of bacterial commensals carrying the synthetic gene or molecule to be delivered to a patient, will mainly be varied from about 10310 12cells/kg, This dose may be changed by a specialist depending on the bacteria and the specific composition containing the epitope I and optional immunogen delivered this living bacteria.

Thus, the compositions of the present invention are intended to slow or minimize infection uninfected mammal, such as a person, selected by a virus. Such compositions, therefore, are used as vaccines. Antibodies against Tat protein does not react with proteins of HIV-1 used in diagnostic assays for detection of seroconversion after infection. Thus, individuals who have entered the compositions of the present invention should not give false positive results in tests for HIV-1 infection and, thus, should remain the possibility of detection of seroconversion in that case, if the individuals who have been introduced this song become HIV-1 infected people.

Introduction to the mammal the composition of the present invention regardless of whether it is a protein/peptide-containing composition, or a new sequence of nucleic acid encoding the immunogen is a radically different strategy for vaccination against AIDS, since it allows to reduce the levels of virus by biological ing is berbania, preferably more than about 95%, and more preferably more than about 99% of the known variants of the Tat protein of HIV-1, in order to reduce the replication of HIV-1.

The use of compositions containing the immunogen Tat, is particularly desirable advantage compared with other methods of treatment and preventive methods against such viruses. Since the suppression of Tat protein extraclean leads to inhibition of reproduction of all Quasimodo or strains of HIV indiscriminately, it does not create a selective pressure on the parent virus for selection of mutant viral variants. Thus, blocking the absorption of protein Tat cells of the patient not only reduces the level of viremia, but also prevents the selection of "escaping from control" options.

In addition, the present invention relates to a method of active asymptotic treatment of HIV-1 infections in patients with viremia, because as the disease extracellular Tat protein likely plays a role in the development of chronic infection and immune pathologies that arise at this stage of HIV-1 infections. Inhibition of extracellular Tat protein by antibodies induced by immunization in accordance with the present invention may result in reduced viremia through more effective IMM is sent to the steering control and to slow or prevent the development of AIDS.

The mechanism of the present invention described above can be used to prevent the development of viral infection and achieve the desired clinical results. More specifically, compositions of the present invention is able to reduce viremia in patients already infected with the virus by blocking the subsequent absorption of the protein Tat uninfected cells. It is expected that the compositions of the present invention, used either alone or in combination with other therapeutic treatment regimens for HIV-inficirovannyh patients will contribute to the reduction of viremia and prevention of clinical deterioration of the patient's condition.

For such therapeutic use of drugs and routes of administration, basically, identical to the drugs and methods specifically described above, and these drugs can be introduced together or in conjunction with other standard therapies intended for a specific viral infection. For therapeutic or prophylactic use may be desirable repeated administration of doses of immunization compositions, such as the booster dose, administered once a year or booster doses, administered through other intervals.

L. Diagnostic kits of the present invention

The peptides and polypeptides described above, can be t is the train used as reagents in the kit, used for measuring and determining the titer and specificity of antibodies induced by vaccination compositions described above. The kit of the present invention may include at least two of the required peptide epitope I, defined above, and preferably two or more I epitopes that are recognized by the immune system of primates, and optional immunogen. In one of the embodiments of the invention, each peptide has at its N-end protein Biotin and the spacer, for example-Ser-Gly-Ser-Gly- [SEQ ID NO: 27]. Alternatively, the specified peptide can have at its C-end of the spacer, for example,- Gly-Ser-Gly-Ser- [SEQ ID NO: 39], and protein - biocytin. These options provide the binding of these peptides to a solid carrier, sensitized by Avidya, for example, a flat surface or spheres. For these purposes may also be used and other binding agents known to experts in the field of diagnostic analysis. In this set also includes labeled reagents that detect the binding of an antibody to the immobilized peptide epitopes, such as goat antibody against human immunoglobulin or similar Mark for the reagent can be selected from many known diagnostic labels, such as radioactive compounds, fluorescent compounds and proteins, colorimetric enzymes, etc. Thus the m this set also contains a variety of reagents and device for reading labels, for example, some substrates that interact with the enzyme label for producing color signals and the like, a device for sampling blood, as well as the appropriate vessels and other components for diagnostic analysis. Each technician can easily select other common diagnostic features for this set.

These kits and reagents can be used in the method of detection of titles and evaluation patterns of antibody reactivity in patients vaccinated compositions of the present invention. The method of determining the presence and/or titer of antibodies induced by immunization against the immunogen Tat involves the stages of contacting a biological sample taken from immunising of the individual, for example, physiological fluid, preferably blood, serum or plasma, but also urine, saliva and other fluids or tissues with one or more binding sequences of the epitope I, recognized by the immune system of primates, and with optional immunogenum, preferably immobilized on a solid medium such as a flat surface or sphere. The epitope I, which is recognized by the immune system of primates, and an optional connecting the follower of the spine, used in this way, can be an unmodified minimum region of the binding epitope.

After processing a biological sample immobilized peptides for a sufficient period of time the carrier is washed to remove from a given biological sample of any material that is not associated with these peptides. Such stage of leaching are traditional diagnostic tests and are performed using saline solution. If antibodies against the epitope I and optional immunogens or their combinations were induced in a patient by means of the above processing, the immobilized peptides bind with the antibody from the biological sample. Then for the detection of binding between the peptides on a solid carrier and an antibody in the specified biological sample material on the media type labeled reagent. This reagent is preferably anti-human immunoglobulin, such as goat anti-human immunoglobulin. The specified label is chosen from a wide range of commonly used diagnostic labels discussed above. In one of the embodiments of the invention, a label may be a colorimetric enzyme, which, after its contact with the substrate, produces the detected color signal is al. The presence and/or intensity of the color signal is an indicator of the induction of antibodies in the treated individual. This analysis can be used to determine the effectiveness of immunization, as well as for monitoring the immune status of the patient.

The choice of specific stages of the analysis, as well as various systems of detectable labels can be successfully carried out by the specialist. Such a selection is a routine procedure and does not limit the scope of the invention.

M. Advantages of the present invention

One of the advantages of the compositions of the present invention is a small number of immunogen required for inclusion in the composition of the present invention to cross-reactions with more than 95-99% of the known variants of the Tat protein of HIV-1 is most common In-subtype. As illustrated in the following examples, the immunogenic composition is recognized by the primates of the epitope I, which contains two necessary amino acid sequence recognized by the primates of the epitope I, as well as six additional sequences of epitope I cross-react with 95% protein Tat of HIV-1 is most common In-subtype, and all 56 sequences of protein Tat more rare not-b subtypes of HIV-1. Thus, a composition can be successfully used to protect against infect and or to treat infection, caused by most common strains of HIV-1.

In addition, when the accurate identification of epitopes Tat, with whom it would be desirable binding (i.e. AA-12 SEQ ID NO: 15), the new desired peptide immunogen Tat originating from newly emerging strains of HIV-1 or newly discovered strains can be easily identified by the methods described here and included in the composition. This versatility allows the use of compositions of the present invention for prevention in order to protect from any new strain or strains of HIV-1, which can be identified in the future. It is assumed that based on the descriptions given here, any expert can easily introduce new combinations of immunogens Tat (and structures of nucleic acids encoding these immunogen) in the composition.

So, for example, using standard methods, such as PCR and oligonucleotide matrix high-density [M.J. Kozal et al., Nature Med., 2:753 (1996)], allows the technician to obtain amino acid sequences for a large variety of protein Tat of HIV-1, representing variants of clinical isolates strains and subtypes of HIV-1. This technique allows you to define other options In-subtype HIV-1, as well as other subtypes of this virus, occurring in less developed countries, which were hitherto not been the residual studied. The identification of new sequences Tat will make it easy to include the corresponding peptides as immunogens in the composition of the present invention and, thereby, to induce a humoral response against other rare protein Tat of HIV-1.

Studies of cross-reactivity with antibodies produced against synthetic peptides corresponding to each variant Tat, can be used to eliminate the need to immunize such variants Tat, in which the replacement sequence is immunologically silent, in which these peptides strongly associated with antibodies against the consensus sequence or other options.

The following examples illustrate the preferred methods of obtaining compositions of the present invention and use of these compositions for the induction of antibodies against the protein Tat of the indicated virus in immunogenum the owner. These examples are only for illustrative purposes and do not limit the scope of the present invention.

Example 1 - Immunological research in order to determine the minimum amino acid sequence of the protein Tat required for binding with the antibody against recognized primates epitope I in the protein Tat of HIV-1 variants of the sequence and immunological cross-reactivity and is of thisisarto against these sequences

A. Synthetic peptides and conjugates

Synthetic peptides were synthesized by solid-phase synthesis on derivatizing plastic carriers [R.M.Valerio et al., Int. J. Peptide Res., 44:158-165 (in Russian) (1994)]. Were synthesized immunizing peptides with amino-terminal Cys introduced to facilitate accession to the protein-carrier and to aminirovanie To-end. There were also synthesized detector peptides with amino-terminal sequence of the Biotin-Ser-Gly-Ser-Gly- [SEQ ID NO: 27] and the free acid functional group at the C-end for use in ELISA assays for detection of reactivity and cross-reactivity. Immunizing peptides, covalently conjugated to protein carrier of diphtheria toxoid (DT) by cysteineless side-chain peptide:carrier =5:8 [A.C.J. Lee et al., Molec. Immunol. 17:749 (1980)]were purified using liquid chromatography high pressure (ghvd) with a purity of more than 95%, as it was shown by analytical ghvd and mass spectrometry, and use a detector peptides had a purity of more than 50%.

C. Immunization

Peptide conjugates were dissolved in purified water and emulsiable 1:1 complete adjuvant's adjuvant (CFA) or incomplete adjuvant's adjuvant (IFA) [ANTIBODIES - A LABORATORY MANUAL, Eds. E. Harlow & P.Lane, Cold Spring Harbor Laboratory (1998)]. The total amount for the primacy was 1 ml and the volume of content the elk 100 µg peptide, associated with DT.

Rhesus monkeys who participated in the study on viral infection, were immunized with antigen in IFA/CFA as follows. Several primates used for immunization peptide by initial intramuscular (i.m.) injection of the conjugate in CFA, and then, two weeks later, a booster injection i.m. conjugate in IFA. Before the first injection took preliminary blood samples, and after 3 and 5 weeks after the booster injection was taking a larger sample.

C. ELISA-titles

ELISA assays were conducted as described N.M. Geysen et al., Proc. Natl. Acad. Sci. USA, 81:3998 (1983). The titer of antibodies was represented by the reciprocal of the serum dilution that gave an optical density of 1.0 OD units above background levels. For each response was calculated geometric mean titer (GMT) for 2-3 serum samples, or only a single serum were available for immunization of some monkeys.

The results of the comparative analysis of ELISA conducted on rabbits and monkeys presented on figa and 1B, respectively. The results of ELISA showed that antibodies primates against the immunogen-containing epitope I react with the sequence-Asp-Pro-Arg7-Leu-Glu9-Pro-Trp-Lys12- [AA-12 SEQ ID NO: 15]. As discussed below, the provisions of 7, 9 and 12 are the most common options specified peptide epitope I recognize is imago primates.

D. Analysis of the diversity of amino acid sequences of the epitopes

The sequence of the first exon of Tat in HIV-1 were taken from the Bank Genbank database and retroviruses and AIDS person in Los Alamos [HUMAN RETROVIRUSES and AIDS 1996, published by the group "Theoretical biology and Biophysics National laboratory Los Alamos, Los Alamos, NM, and additional sequences, courtesy of Genbank Dr. Esther Guzman, laboratory Los Alamos]. Incomplete sequences and sequences with stop codons or deletions of bases, leading to a shift in the reading frame, were deleted because they were obviously identical repetitive sequences from the same isolate. Variants of amino acids in these positions within epitopes were registered and presented in tables.

That is Antigenic cross-reactivity between variants

Antisera against the consensus sequence of the epitope was titrated by ELISA on consensus sequences and the sequences with the most common variants of amino acids to determine the effect of amino acid polymorphism on antigenicity.

F. Differences in the sequences

Consensus sequences recognized by the primates of the epitope I, evaluated at the maximum frequency and recognize what avanie antibodies primates. For these epitopes was evaluated antigenic conservatism and conservatism sequences in the protein Tat of HIV-1 to 294 protein Tat of HIV-1 originating from 294 virus-Cladonia (I) and 56 virus not-In-Cladonia (II), and the results are presented in tables II-VI.

In the top row in tables II and III shows the consensus sequence having the maximum occurrence. In the middle rows specified percentage of occurrence of the amino acids detected in more than 5% of sequences at each position, if there are several of them. In the bottom row of each table specified percentage of the total occurrence, including amino acids, present in more than 5% of sequences at each position, if there are several of them. All of these data sets in table II represent different antigenic epitopes (cross-reactivity <25%) and this applies to all specified data sets in table III, except for the graphs under amino acid 4.

Table II
The epitope I - 294 sequence In Cladonia
Val4Asp5Pro6Arg7Leu8Glu9Pro10 Trp11Lys12
Arg(73)Lys(96)
Lys(12)Asn(2)
Ser(11)
Asn(4)
100%98%100%100%100%100% 100%100%100%
Table III
The epitope I - 56 sequences not-In-Cladonia
Val4Asp5Pro6Asn7Leu8Glu9Pro10Trp11Asn12
Val(89)Asn(79)Glu(86)Asn(87)
Ile(11)Lys(14)Asp(14)Lys(13)
Ser(5)
100%100%100%98%96%100%98%100%100%

As shown in tables II and III, the epitope I, recognized by primates, has the potential 16x antigenic polymorphism, but one major antigen present in In-Cladonia, and the other major antigen has not-In-Cladonia. Five other variants responsible for more than 95% of the known variants of the Tat. Cm. tables IV and V; the sequence indicated by the asterisks that appear in both In - and not-In-Cladonia.

Table VI illustrates the weak antigenic cross-reactivity variants of the epitope I, in position 7, antigenic difference between these variants at position 9 and an almost complete absence of cross-reactivity of antisera against GluProValAspProArg7LeuGlu9ProTrpAsn12[A-235 SEQ ID NO: 13] sequence GluProValAspProArg7LeuGlu9ProTrpLys12[AA2-12 SEQ ID NO: 15], containing modifications in both positions 7 and 12. It is also shown antigenic difference Glu9 and Asp9-options.

Example 2 - Immunological research in order to determine the minimum the amino acid sequence of the protein Tat, necessary for binding with antibody against recognized primates epitope II in the protein Tat of HIV-1 variants of the sequence and immunological cross-reactivity of antisera against these sequences

Using the procedures described in Example 1, was determined by the occurrence of variants of amino acid sequences for 294 sequences Tat HIV-1-Cladonia and 56 sequences not-In-Cladonia within the boundaries of epitope II for binding antibodies present in monkeys. The results are presented in tables VII and VIII. The top row of the tables contain data for the consensus sequence. In the middle row specified percentage of occurrence of the amino acids detected in more than 5% of sequences at each position, if there are several of them. In the bottom row of each table shows the total number of cases of occurrence, including amino acids, present in more than 5% of sequences at each position, if there are several of them. Amino acid variants at position 42 Tat possessed antigenic cross-reactivity.

As shown in tables VII and VIII, II epitope detects almost full antigenic conservatism.

Was measured by ELISA-reactivity monkey antisera against the immunogen is epitope II detector peptides with Gly 42or Ala42Tat (options) in the detector sequences and the results are presented below in table IX. See, for example, figa and 2B, where graphically presents comparative results for rabbits and monkeys, respectively.

Example 3: Production of human monoclonal antibodies. The asymptotic treatment of HIV-1 infections

Commercially available mouse with "humanized" antibodies were immunized with a suitable amount of immunogen epitope II: Cys-Gly-Ser-Lys-Gly-Leu-Gly-Ile-Ser-Tyr-Gly-Arg-Lys-amide [SEQ ID NO: 34]attached to the protein-carrier of diphtheria toxoid. Hybridoma was skanirovali on the sequence Biotin-Ser-Gly-Ser-Gly-Leu-Gly-Ile-Ser-Tyr-Gly-Arg-Lys [SEQ ID NO: 35] immobilized with streptavidin tablets and selected monoclonal antibody IgG with subnanomolar the affinity of the binding, which was not associated with complementary receptors. Were confirmed specificity against recombinant Tat protein of HIV-1.

Because this monoclonal antibody is directed not to the antigen, it is necessary to conduct traditional procedures for its preclinical production, purification and safety tests. Human monoclonal antibodies have a half-life of 20 days in a person, then, as the half-life of OCT mouse monoclonal antibodies, which in Encino is absorbed on the inner CD3, was 18 hours. Daily dose in 5 mg ACT contributed to maintaining minimum levels of antibodies to about 1 micrograms/ml in humans. Thus, it was expected that the dose of anti-Tat monoclonal antibodies in 5 mg, administered once every two weeks is sufficient to maintain the same minimum levels that molar ratio is more than 50 times the estimated maximum circulating levels in infected individuals, up to 1 ng/ml for protein Tat of HIV-1.

Currently, the control of viral load in plasma is generally accepted criterion for the effectiveness of treatment of HIV-1 infections. The effectiveness of monoclonal antibodies against Tat can be quickly identified in HIV-1-infected individuals without symptoms of the disease in the first four weeks of treatment. This Protocol can be used for patients who have not undergone treatment; for patients who for various reasons have proved impervious to treatment in accordance with the protocols of HAART; or for patients undergoing HAART therapy with the cessation of this therapy after 4 weeks (viral load was rapidly restored upon termination of HAART-terapii). 2-3 log magnitude reduction in viral load in the plasma to values below LOD (50 copies of viral RNA/ml) argue in favor of monotherapy mo is oclonal antibody which was evaluated over a longer period of time. The reduction above one log (90%), but not below the LOD, suggests usage of the specified monoclonal antibodies as a component of therapy.

Example 4 - Development of a universal vaccine to prevent the development of AIDS in individuals

A. Design of synthetic gene

Figure 3 illustrates the synthetic gene that encodes four copies of each of the four polymorphs of epitope I, can be detected by the humoral response in rabbits, and four copies of the epitope II, expressed as a linear hybrid protein in a strain of E. coli DnaK (HSP70). When tested in ELISA using epitope-specific rabbit antisera, this downregulation of protein contained all antigenic epitopes. However, when used to immunize rabbits or monkeys all variants of the epitope I were immunogenic, in addition to epitope II. Thus, the epitope II is best used in the form of a synthetic peptide conjugate is attached to a suitable protein-bearer.

On FIGU illustrated new vosmiletnie synthetic gene, which was constructed on the basis of polymorphism within the borders recognized by the primates of the epitope I for the introduction, with preservation of the reading frame, eight polymorphs recognized primates epitope I:

R1-Asp-Pro-Arg-Lu-Glu-Pro-Trp-Lys-R2 [SEQ ID NO: 17]

R1-Asp-Pro-Lys-Leu-Glu-Pro-Trp-Lys-R2 [SEQ ID NO: 19]

R1-Asp-Pro-Lys-Leu-Glu-Pro-Trp-Asn-R2 [SEQ ID NO: 22]

R1-Asp-Pro-Ser-Leu-Glu-Pro-Trp-Lys-R2 [SEQ ID NO: 20]

R1-Asp-Pro-Ser-Leu-Glu-Pro-Trp-Asn-R2 [SEQ ID NO: 24]

R1-Asp-Pro-Asn-Leu-Glu-Pro-Trp-Lys-R2 [SEQ ID NO: 21]

R1-Asp-Pro-Asn-Leu-Glu-Pro-Trp-Asn-R2 [SEQ ID NO: 18] and

R1-Asp-Pro-Asn-Leu-Asp-Pro-Trp-Asn-R2 [SEQ ID NO: 23].

The sequence of the epitope I divided the dipeptide spacer containing residues Gly and/or Ser. The Assembly of this gene was performed as described W.P.C. Stemmer et al., Gene, 164:49 (1995). Briefly, along with dvukhkontsevaya 50-measures, synthesized 60-dimensional oligonucleotides upper chain (oligos) and oligonucleotides bottom of chain overlap of 20 nucleotides (nt). These 60-measures were incubated together under conditions of hybridization was carried out by polymerase chain reaction (PCR) for completing the sequence and its amplification. Then added a limit of 50 measures and carried out the Assembly by PCR with the release of a full-sized gene on agarose gel. This gene is sequenced and it was found that he has the "right" sequence in the desired epitopes. Similar heptavalent gene can be constructed by excluding rare variant R1-Asp-Pro-Ser-Leu-Glu-Pro-Trp-Asn-R2 [SEQ ID NO: 24].

C. Expression of the hybrid protein

Then the above gene cut restricteduse enzymes and was built in suitable expressing a vector containing, in the same reading frame, the sequence defteri the tion toxoid (HSP 70). E.coli was transferrable and colonies expressing this protein was identified. The selected colonies were cultured and induced expression. Then identified protein from the colonies expressing the hybrid protein. The obtained protein was purified by standard methods.

On figs illustrated polyvalent immunogen epitope II received, but not necessarily, in the form of a conjugate of synthetic peptide to a protein carrier, such as diphtheria toxoid, using standard techniques.

C. Analyses to estimate the "correct" expression of epitopes in hybrid protein and the efficiency of induction of antibodies against Tat

Four variants of the epitope I, where Y7represents Arg, Asn, Lys or Ser, X9represents Glu, and Z12represents Lys, and both epitope II was designed in the synthetic gene and expressed as a hybrid protein as described above in paragraphs a and B. in order to determine whether each is expressed epitope hybrid protein in a form that can be recognized by anticorodal primates, antisera primates generated against synthetic peptides corresponding to sequences of the epitope I, were tested using ELISA using standard techniques. Tablets first directly senzibilizirani hybrid protein, and then amrabat the Wali 100 μg/ml of antisera (for example, rabbit antisera), which are known to react with epitopes I and II. Sequence variants of these epitopes expressed in conformation recognized by antibodies against the corresponding synthetic peptides, indicating the title, amounting to more than 32000 for each epitope.

To assess the immunogenicity of multivalent immunogen monkeys were immunized hybrid protein in IFA. Monkey antisera were analyzed on synthetic peptides epitopes I and II. Against variants of the epitope I developed high titers, i.e. titer 28000 to epitope I, where Y7represents Arg and Z12represents Lys; title 16000 to epitope I, where Y7represents Asn, and Z12represents Lys; title 37000 to epitope I, where Y7represents Lys, and Z12represents Lys, and the titer of 4000 to epitope I, where Y7represents Ser, and Z12represents Lys. The title for epitope II was 700, indicating that immunization with this epitope is best used in the form of a synthetic peptide linked to a carrier.

Example 5: the Method and kits for the detection of titles and specificdate antibodies induced by vaccination

To assess the titer and specificdate antibodies induced after immunization with vaccines of the present invention, may be the used analytical method. In one embodiment, such analysis of peptides containing the sequence recognized by the primates of the epitope I, described in Example 1 (depending on the composition of immunizing vaccine)was used in the development of kits to measure titles and evaluation pattern of reactivity of antibodies in vaccinated individuals.

These peptides were synthesized using the sequence Biotin-Ser-Gly-Ser-Gly- [SEQ ID NO: 36] the N-end. Each peptide was applied to a separate sensitized by Avidya tablets, where the sequence-Ser-Gly-Ser-Gly- [SEQ ID NO: 27] served as a spacer to ensure that the relevant peptide sequence will remain external to the Biotin-binding pocket avidin. Then the plates were incubated with dilutions of test serum, washed and evaluated for binding antibodies using reagent against human immunoglobulin, such as goat anti-human immunoglobulin, directly labeled with an enzyme. This reagent used for the reaction with the enzyme, resulting in producyrovtsa colorimetric signal (liners set R&D).

In vicepresidencia description of the invention can be made of numerous modifications and changes which are obvious to every specialist. Such modifications and changes, VNO which are in the compositions and methods of the present invention, included in the scope of the attached claims.

1. A composition comprising at least two variants of a peptide or polypeptide of the formula

R1-Asp-Pro-Y7-Leu-X9-Pro-Trp-Z12-R2 SEQ ID NO:8,

where Y7selected from the group consisting of Arg, Lys, Ser and Asn;

X9selected from the group consisting of Glu and Asp;

Z12selected from the group consisting of Lys and Asn;

R1 is selected from the group consisting of hydrogen, lower alkyl, lower alkanoyl and sequence of about 1-5 amino acids, optionally substituted lower alkyl or lower alkanoyl;

R2 is selected from the group consisting of a free hydroxyl, an amide, and a sequence of about 1-5 additional amino acids, optionally substituted by Amida, and

where at least one of these two options Y7represents Arg and Z12represents Lys; and at least a second of the two above options Y7represents Asn, and Z12represents Asn,

this composition is able to induce the production of antibodies against Tat of HIV-1 that can inhibit the replication of HIV-1.

2. The composition according to claim 1, where R1 represents Val, resulting in a sequence

Val-Asp-Pro-Y7-Leu-X9-Pro-Trp-Z12SEQ ID NO:37.

3. The composition according to claim 1, g is e R1 represents X 2-Pro-Val, where X2selected from the group comprising Glu and Asp, resulting in a sequence of X2-Pro-Val-Asp-Pro-Y7-Leu-X9-Pro-Trp-Z12(SEQ ID NO:38), optionally substituted lower alkyl or lower alkanoyl.

4. The composition according to claim 1, where R2 represents the sequence-His-Pro-Gly-Ser-(SEQ ID NO:16), optionally substituted by amidon.

5. The composition according to claim 1, where the specified variant of a sequence selected from the group consisting of:

R1-Asp-Pro-Arg-Leu-Glu-Pro-Trp-Lys-R2SEQ ID NO:17,
R1-Asp-Pro-Arg-Leu-Asp-Pro-Trp-Lys-R2
R1-Asp-Pro-Arg-Leu-Glu-Pro-Trp-Asn-R2
R1-Asp-Pro-Arg-Leu-Asp-Pro-Trp-Asn-R2
R1-Asp-Pro-Lys-Leu-Glu-Pro-Trp-Lys-R2SEQ ID NO:19
R1-Asp-Pro-Lys-Leu-Asp-Pro-Trp-Lys-R2
R1-Asp-Pro-Lys-Leu-Glu-Pro-Trp-Asn-R2SEQ ID NO:22
R1-Asp-Pro-Lys-Leu-Asp-Pro-Trp-Asn-R2
R1-Asp-Pro-Ser-Leu-Glu-Pro-Trp-Lys-R2SEQ ID NO:20
R1-Asp-Pro-Ser-Leu-Asp-Pro-Trp-Lys-R2
R1-Asp-Pro-Ser-Leu-Glu-Pro-Trp-Asn-R2SEQ ID NO:24
R1-Asp-Pro-Ser-Leu-Asp-Pro-Trp-Asn-R2
R1-Asp-Pro-Asn-Leu-Glu-Pro-Trp-Lys-R2SEQ ID NO:21

R1-Asp-Pro-Asn-Leu-Asp-Pro-Trp-Lys-R2
R1-Asp-Pro-Asn-Leu-Glu-Pro-Trp-Asn-R2SEQ ID NO:18
and
R1-Asp-Pro-Asn-Leu-Asp-Pro-Trp-Asn-R2SEQ ID NO:23.

6. The composition according to claim 1, containing

R1-Asp-Pro-Arg-Leu-Glu-Pro-Trp-Lys-R2SEQ ID NO:17
and
R1-Asp-Pro-Asn-Leu-Glu-Pro-Trp-Asn-R2SEQ ID NO:18

and one or more sequences selected from the group including:

R1-Asp-Pro-Lys-Leu-Glu-Pro-Trp-Lys-R2SEQ ID NO:19
R1-Asp-Pro-Lys-Leu-Glu-Pro-Trp-Asn-R2SEQ ID NO:22
R1-Asp-Pro-Ser-Leu-Glu-Pro-Trp-Lys-R2SEQ ID NO:20
R1-Asp-Pro-Ser-Leu-Glu-Pro-Trp-Asn-R2SEQ ID NO:24
R1-Asp-Pro-Asn-Leu-Glu-Pro-Trp-Lys-R2SEQ ID NO:21
and
R1-Asp-Pro-Asn-Leu-Asp-Pro-Trp-Asn-R2SEQ ID NO:23.

7. The composition according to claim 1, containing at least seven of these variants amino acid sequence SEQ ID No:8.

8. The composition according to claim 6, containing at least eight of these sequences.

9. The composition according to claim 1, which further contains at least one shall epted or polypeptide of the formula R3-Lys-X 42-Leu-Gly-Ile-Ser-Tyr-Gly-Arg-Lys-R4 SEQ ID No:5, where X42selected from the group comprising Gly or Ala; R3 is selected from the group comprising hydrogen, lower alkyl, lower alkanoyl and sequence of 1-5 amino acids, optionally substituted lower alkyl or lower alkanoyl; R4 is selected from the group comprising free hydroxyl, an amide, and a sequence of one or up to 5 additional amino acids, excluding the basic amino acids Lys-Arg-Arg-, optionally substituted amidon.

10. Composition according to any one of claims 1 to 9, where these peptides or polypeptides derived recombinant or synthetic means.

11. Composition according to any one of claims 1 to 9, where one or more of these peptides are expressed as synthetic peptides associated with protein carrier.

12. Composition according to any one of claims 1 to 9, where one or more of these peptides are expressed as a multiple antigenic peptide, optionally linked to a protein carrier.

13. Composition according to any one of claims 1 to 9, where one or more of these peptides are expressed in the protein produced by recombinant technologies, optionally fused with a protein carrier in the same reading frame.

14. The composition according to claim 11, where specified a carrier protein selected from the group comprising protein of E. coli DnaK protein GST, mycobacterial heat shock protein 70, diphtheria anatox is h, tetanus toxoid, galactokinase, ubiquitin; α-factor crosses, β-galactosidase and protein NS-1 flu.

15. The composition according to item 12, where specified a carrier protein selected from the group comprising protein of E. coli DnaK protein GST, mycobacterial heat shock protein 70, diphtheria toxoid, tetanus toxoid, galactokinase, ubiquitin; α-factor crosses, β-galactosidase and protein NS-1 flu.

16. The composition according to item 13, where specified a carrier protein selected from the group comprising protein of E. coli DnaK protein GST, mycobacterial heat shock protein 70, diphtheria toxoid, tetanus toxoid, galactokinase, ubiquitin; α-factor crosses, β-galactosidase and protein NS-1 flu.

17. A pharmaceutical composition comprising a composition according to any one of claims 1 to 9, pharmaceutically acceptable carrier and optional adjuvant, the composition is able to induce the production of antibodies against Tat of HIV-1 that can inhibit the replication of HIV-1.

18. The pharmaceutical composition according to 17, where one or more of these peptides are associated with protein carrier.

19. Method of induction of antibodies against the HIV Tat-1, where the method includes a step

introduction to the individual an effective amount of a composition according to claim 1, which actively induces the production of antibodies that interact with the protein Tat of HIV-1 of the x strains or subtypes of HIV-1.

20. In vitro method for determining the presence and titer of antibodies induced by immunization with an immunogen Tat, where the method includes:

(A) contacting a biological sample taken from an immunized individual, with a peptide or polypeptide composition according to claim 1, associated with a solid carrier;

(B) washing the specified media to remove from the specified biological sample of any material that is not associated with the specified sequences;

(C) contacting the specified media reagent associated with a detectable label, where the specified reagent detects binding between these sequences on the specified solid carrier and an antibody in the specified biological sample and where the specific label that produces a detectable signal.

21. The composition of the antibody containing multiple antibodies, each antibody linked to a peptide or polypeptide composition according to any one of claims 1 to 9, where the composition interacts with the protein Tat of HIV-1 strains and subtypes of HIV-1 and inhibits the replication of HIV-1.

22. The composition of the antibody according to item 21, where each antibody selected from the group including the selected polyclonal antibody, monoclonal antibody, chimeric antibody, humanitariannet antibody, a human antibody, an antibody obtained by screening the GA phage display, single-chain antibody and the antibody fragment.

23. The composition of the antibody according to item 21, containing the antibody, which is associated with the epitope R1-Asp-Pro-Asn-Leu-Glu-Pro-Trp-Asp-R2 (SEQ ID NO:18) or R1-Asp-Pro-Asn-Leu-Asp-Pro-Trp-Asn-R2 (SEQ ID NO:23).

24. The composition of the antibody according to item 21, containing an antibody that specifically binds to an epitope with the amino acid sequence Lys-X42-Leu-Gly-Ile-Ser-Tyr-Gly-Arg-Lys SEQ ID No:10, where X42selected from the group comprising Gly and Ala.

25. A pharmaceutical composition comprising the antibody or composition of the antibody according to item 21, and a pharmaceutically acceptable carrier and, optionally, an adjuvant, where this composition inhibits the replication of HIV-1.

26. A pharmaceutical composition comprising the antibody or composition of the antibody according to item 22, and a pharmaceutically acceptable carrier and, optionally, an adjuvant, where this composition inhibits the replication of HIV-1.

27. A pharmaceutical composition comprising the antibody or composition of the antibody according to item 23, and a pharmaceutically acceptable carrier and, optionally, an adjuvant, where this composition inhibits the replication of HIV-1.

28. A pharmaceutical composition comprising the antibody or composition of the antibody according to paragraph 24, and a pharmaceutically acceptable carrier and, optionally, an adjuvant, where this composition inhibits the replication of HIV-1.

29. The pharmaceutical composition containing the anti-Christ. ate or composition antibodies on A.25, and a pharmaceutically acceptable carrier and, optionally, an adjuvant, where this composition inhibits the replication of HIV-1.

30. The method of reducing levels of HIV-1, where the method includes the stage of introduction of human antibodies or compositions of the antibody according to item 21.

31. The method of reducing levels of HIV-1, where the method includes the stage of introduction of human antibodies or compositions of the antibodies on p.22.

32. The method of reducing levels of HIV-1, where the method includes the stage of introduction of human antibodies or compositions of the antibody according to item 23.

33. The method of reducing levels of HIV-1, where the method includes the stage of introduction of human antibodies or compositions of the antibodies according to point 24.

34. The method of reducing levels of HIV-1, where the method includes the stage of introduction of human antibodies or compositions of the antibodies on A.25.

35. The method of reducing levels of HIV-1, where the method includes the stage of introduction of human antibodies or compositions of the antibodies on p.

36. The synthetic nucleic acid sequence encoding a peptide or polypeptide compositions according to claims 1-9, where the specified peptide or polypeptide induces production of antibodies inhibiting the replication of HIV-1.

37. Synthetic sequence of the nucleic acid p, which is introduced into the cell by the host mammal.

38. Formats viteska composition, containing synthetic nucleic acid sequence according p, pharmaceutically acceptable carrier and, optionally, an adjuvant, where this composition is able to induce the production of antibodies against Tat of HIV-1 that can inhibit the replication of HIV-1.

39. A synthetic molecule expressing peptides and polypeptides that are able to induce the production of antibodies against Tat of HIV-1 that can inhibit the replication of HIV-1 containing synthetic sequence of nucleic acid according p, effectively linked with regulatory sequences of nucleic acids, which carry out and control the expression product such as a synthetic gene in a cell host.



 

Same patents:

FIELD: biochemistry, molecular biology.

SUBSTANCE: invention proposes two variants for analysis of binding a target comprising a polynucleotide sequence. In accordance with given variants a target is hybridized with a probe comprising a nucleic acid sequence or its analogue in the presence of intercalating fluorophore. A sample obtained in hybridization is irradiated by exciting radiation. As result of such treatment a fluorophore containing in a sample emits the fluorescent radiation. The degree of erroneous pairing a probe with a target or degree of the correspondence of probe and target are assayed by intensity if indicated radiation. Using the invention allows simplifying analysis of interaction between nucleic acids and/or analogues of nucleic acids and enhancing sensitivity and rate of the indicated process. Invention can be used for homogenous analysis of nucleic acids hybridization.

EFFECT: improved method of analysis.

29 cl, 13 dwg, 8 ex

FIELD: biotechnology, microbiology.

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EFFECT: improved preparing method, improved and valuable properties of medium.

1 ex

FIELD: biochemistry, molecular biology.

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EFFECT: improved method of analysis.

64 cl, 17 dwg, 6 ex

FIELD: virology, biotechnology, molecular biology.

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EFFECT: improved methods for detection, diagnosis and selection.

7 tbl, 1 dwg, 8 ex

FIELD: genetic engineering, biotechnology, biochemistry, medicine.

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EFFECT: valuable medicinal and biochemical properties of polypeptide.

27 cl, 3 tbl, 11 ex

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EFFECT: improved marking method.

3 dwg, 1 tbl

FIELD: biochemistry, in particular method for determination of antioxidant enzyme superoxide dismutase (SOD) activity.

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EFFECT: method of increased accuracy and reliability.

FIELD: medicine, therapy.

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EFFECT: improved prognosis method of treatment.

3 cl, 4 dwg, 3 ex

FIELD: biotechnology.

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EFFECT: improved preparing method.

5 cl, 1 dwg, 1 tbl

FIELD: medicine, infectious diseases.

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EFFECT: improved detecting method.

2 ex

FIELD: genetic engineering and medicine genetic.

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EFFECT: method for diagnosis of congenital gene resistance to infection by HIV1.

2 dwg, 1 tbl, 2 ex

FIELD: biotechnology, genetic engineering, molecular biology.

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EFFECT: valuable biological and medicinal properties of strain and vaccine.

2 cl, 3 dwg, 3 tbl, 5 ex

The invention relates to the field of biotechnology and medicine, in particular to genetic engineering and immunology

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FIELD: biotechnology, medicine, oncology.

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EFFECT: valuable medicinal properties of peptides.

2 dwg, 2 ex

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