RU2600162C1 - Method of treating and preventing hiv infection of type 1 subtype a - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine and concerns a method of treating and preventing HIV infection of type 1 subtype A (HIV-1A), in which HIV-1A genome structure is extracted from infected patient, primary structure of 14 N-terminal amino acids of Tat-protein in extracted HIV-1A genome is determined, personalized drug is created in the form of 6-peptidyl substituted with aminopenicillanic acid derivatives (6-peptidil - APA) peptide, having amino acid sequence, coinciding with the first 14 N-terminal amino acids of Tat-protein in extracted HIV-1A genome of infected patient, and obtained personalized preparation is administrated to patient.

EFFECT: invention provides effective treatment and prevention of HIV-1A, minimized side effect of vaccination process.

3 cl, 9 ex, 7 dwg, 1 tbl

Description

Technical field

The invention relates to the field of immunology and medicine and is intended for the treatment and prevention of HIV infection of type 1 subtype "A" (HIV-1A).

Currently, the problem of combating HIV infection (human immunodeficiency virus) has become global due to the spread of HIV infection in almost all countries, including the Russian Federation, where the number of HIV-infected is officially estimated to be 1 million. The human immunodeficiency virus is characterized by a high frequency of genetic changes that occur during self-reproduction. The frequency of errors in the HIV genome is several orders of magnitude greater than in eukaryotes. From this it follows that almost every viral particle in the sequence of genomic RNA has at least one or more nucleotides by which it differs from its predecessor. Currently, 4 types of HIV are distinguished, which differ in the most characteristic features, in particular, in the different genome structure: HIV-1, HIV-2, HIV-3, HIV-4. The most common type of virus is HIV-1, discovered in 1983. Type 1 human immunodeficiency virus (HIV-1) has high genetic variability, and its strains are divided into three main groups: the main (M), which is the cause of most HIV -1 infections worldwide, a special group (O) and a new group (N), the strains of which do not belong to either M or O. Within group M, nine subtypes are identified that received the letter designation “AD”, “FH” , "J" and "K". Recombinant forms of the virus have also been identified (Grant R Campbell, Erwann Ρ Loret. What Does the HIV1 Tat Protein Teach us About Developing an AIDS Vaccine? Retrovirology 2009; 6: 50-56). The most frequently isolated strains from HIV-1 infected people in industrialized countries are viruses belonging to the subtypes "B", "C", "D". In the Russian Federation, Belarus and Ukraine, viruses belonging to the subtype “A” and its recombinant forms are more often isolated from HIV-1 infected people.

State of the art

At present, quite a lot of drugs have been developed that are used for the treatment and prevention of HIV-1, which differ in their mechanisms of exposure, route of administration and dosage forms. A known composition having immunogenic activity against HIV, containing a fusion polypeptide that includes Nef, or its immunogenic fragment, or derivative, and p17 Gag, or its immunogenic fragment, or derivative, and p24 Gag, or its immunogenic fragment, or derivative (RU 2441878, priority from 08/05/2004). The specified composition allows you to activate immune responses to various types of HIV and can be used in the form of a vaccine administered by injection.

A known method (and composition) for the prevention and treatment of HIV, comprising the introduction of one or more antiretroviral drugs, which simultaneously and simultaneously enter the activated-potentiated forms of antibodies to a protein or peptide of the immune system that interacts with HIV or the content and / or functional activity of which changes due to HIV infection (RU 2516931, priority from 08/06/2010). The proposed invention improves the effectiveness of the prevention and treatment of HIV. The composition can be obtained in the form of a solution or tablets intended for oral administration.

A known method of treating HIV based on the use of a composition comprising at least one HIV protease inhibitor selected from the group of nelfinavir, saquinavir, tipranavir and others, and pharmaceutically acceptable excipients, including water-soluble polymers, with a certain quantitative ratio of components (RU 2505286, priority from 12.29.2012). The composition is made in the form of a solid dosage form and can improve the dissolution kinetics and bioavailability of the drug, which reduces the time before the onset of the therapeutic effect.

These methods and compositions according to the descriptions of the inventions can improve the effectiveness of the treatment and prevention of HIV-1, but nowhere is it said about the complete sterilization of the body from the HIV virus. The absence of such sterilization according to the available data regarding the clinical course of HIV-1 leads to various negative consequences, including neurodegenerative ones, which worsen over time (HIV-1-associated dementia). The disorder is caused by the presence in the body of a small amount of residual viral genomes that, over time, produce increasing amounts of Tat protein, which irreversibly damages neurons (Ghafouri M., S. Amini, K. Khalili, BE Sawaya. HIV-1 associated dementia: symptoms and causes // Retrovirology 2006, 3 (1): 28-38).

One of the most effective methods for the prevention and treatment of HIV-1 are methods based on the use of immunization of patients with Tat protein. A study of cases of the absence of the onset of AIDS in HIV-1-infected allowed to establish the leading role of the HIV-1 Tat protein in the mechanism of the development of the syndrome.

It is known that seropositive patients with long-term non-progressive HIV-1 infection (LTNP) have a higher level of antibodies to Tat than seropositive patients with rapid infection progression (US Patent Application 20120121632, PCT 2009/54846). Another reason for the development of a Tat vaccine was the example of LTNP from a patient from Gabon, who was seropositive and was infected with the HIV-1 strain of subtype B, which forms a specific Tat protein (WO 95/31999). Tat-protein isolated from this patient and called “Oyi” proved to be a powerful protective antigen in which subcutaneous and intramuscular immunization is effective in cases of HIV-1 subtypes “B”, “C”, “D” (US Patents 7087377 U.S. Patent Applications 20110165191). The ability to restore CD4 ⁺ cell numbers is realized using a Tat vaccine based on a biologically active protein (US Patent Applications 0110319593).

The use of Tat immunization is known to enhance the effectiveness of HAART and restore immune homeostasis (Ensoli B. et al. Therapeutic Irnmunization with HIV-1 Tat Reduces Immune Activation and Loss of Regulatory T-Cells and Improves Immune Function in Subjects on HAART // PLoS ONE 2010 5 (11): e13540, pp. 1-29).

However, immunization with the Tat protein subtype “B” (Tat protein Oyi _Cys22 ) does not stimulate an effective response against the Tat species from the “A” and HIV “C1” subtypes, which cause 75% of infections in the world (PCT / EP 09/54846). The reasons for the low efficiency of Tat protein immunization in this case were established by studying the structure of neutralizing monoclonal antibodies (7G12, 6E7, 27A8), which reacted only with the conformational epitope of the Tat protein Oyi (the 14 first N-terminal amino acids, US Patents 7087377).

A number of attempts are known to induce the formation of neutralizing antibodies in humans with a variable chain structure close to the above monoclonal antibodies. To this end, “haptenization” technology is used (US Patent Applications 0060210588, US Patent Applications 0070207952), as well as various “hapten” carriers, adjuvants (enhancers of the immune response) and vaccine administration methods. Peptides corresponding to the Oyi Tat protein fragments and, first of all, the fragment forming the conformational epitope (the 15 first N-terminal amino acids, US Patents 7087377) are used as a “hapten". The technology of “haptenization” aims to obtain an immune response to a molecule with a small molecular weight (“hapten”), which suggests the formation of a covalent bond between the “carrier” and “hapten” so that the “carrier” modified by the “hapten” would serve as a target for an immunological reaction which is enhanced by adjuvants and methods of administration. However, the experimental efforts to combine the technology of "haptenization" with the determination of conformational epitopes of proteins are in the nature of a successful combination of circumstances, which, despite all attempts, cannot be systematized and regulated.

A known method for the prevention and treatment of HIV, based on the use of vaccines containing the V3 loop gp120 and Tat HIV-1 (RU 2432356, priority from 11.03. 2005). Tat interacts with the V3 gp120 loop, thus simulating the CCR5 coreceptor both at the molecular (structural) and functional levels and thereby giving the CCR5 strains of HIV the ability to infect target cells expressing only very small amounts of CCR5 that could not be infected by the same virus in the absence of immobilized Tat. Due to the presence of molecular mimicry between CCR5 and Tat, the complexes and antibodies obtained against these complexes will also partially imitate the effect or directly act against epitopes present in the CCR5 or CCR5 / V3-loop complex, thereby increasing the effectiveness of the vaccine or antibodies used for passive immunization, however, this invention also does not solve the problem of obtaining an effective immune response against HIV-1A and its Tat protein.

Such images from the prior art known methods of anti-Tat vaccination, which showed clinical efficacy (lack of AIDS) when infected with viruses of only subtypes "B", "C", "D". However, for HIV-1A, endemic to the Russian Federation, Belarus and Ukraine, there is no anti-Tat vaccination and cases of seropositive and, at the same time, long-term non-progressive patients (LTNP) are not described.

In addition, all currently used preparations and compositions intended for influencing various types and subtypes of HIV-1 do not take into account the individual structural features of the HIV-1A genome of a specific infected patient, associated with a high frequency of HIV genetic changes, leading to the presence of in different patients with the same HIV-1A subtype, the different structures of the HIV-1A genomes are different, as well as the presence of several different structures of the HIV-1A genome in one infected patient of the same subtype. Accordingly, the methods and preparations used for the treatment and prevention of HIV-1A are not sufficiently differentiated by the selectivity of their effects and are not individualized in relation to a particular patient, which reduces their effectiveness and leads to various complications.

SUMMARY OF THE INVENTION

The present invention is the creation of a personalized method for the treatment and prevention of HIV-1A using peptide (s) 6-peptidyl substituted with derivatives of aminopenicillanic acid (6-peptidyl-APA), which allows to obtain from seropositive patients the absence of a progressive course of HIV-1A. The need to create a personalized method for the treatment and prevention of HIV-1A arises due to the very high variability of the Tat protein sequence, which was revealed by analyzing previously isolated infectious molecular clones of this virus (Desfosses Y., M. Solis, Q. Sun, N. Grandvaux , C. Van Lint, A. Burny, A. Gatignol, MA Wainberg, R. Lin, J. Hiscott. Regulation of Human Immunodeficiency Virus Type 1 Gene Expression by Clade-Specific Tat Proteins // JOURNAL OF VIROLOGY, 2005, Vol. 79, No. 14 p. 9180-9191).

The solution to this problem is provided by the fact that, in contrast to the known methods, it is new that the structure of the HIV-1A genome is isolated from the infected patient, the primary structure of the 14 N-terminal amino acids of the Tat protein is determined in the selected HIV-1A genome, and a personalized preparation is created in the form of a 6-peptidyl substituted aminopenicillanic acid derivative (6-peptidyl-APA) peptide having an amino acid sequence matching the 14th N-terminal amino acids of the Tat protein in the isolated HIV-1A genome of the infected patient and the resulting personalized preparation is administered to the patient, the preparation being administered rectally, and in the case of isolation of several different structures of the HIV-1A genome from the infected patient with differences in the composition of the 14 N-terminal amino acids of the Tat protein, for each of these structures of the HIV-1A genome, a personalized preparation is created with a structure that matches the composition of the 14 N-terminal amino acids of the Tat protein of each of the selected HIV-1A genomes, and all created drugs are administered rectally to the patient th way.

In addition, HIV-1A 6-APA modified Tat peptide (s) are administered as a suppository or as microclysters.

Determination of the primary structure of the 14 N-terminal amino acids of the Tat protein in the selected HIV-1A genome allows us to identify the structure of infectious molecular clones isolated from an infected patient and create a personalized preparation in the form of 6-peptidyl substituted with aminopenicillanic acid derivatives (6-peptidyl - APA ) a peptide having an amino acid sequence that matches the 14th N-terminal amino acids of the Tat protein in the selected HIV-1A genome of the infected patient.

The introduction of the obtained personalized drug to the patient allows ensuring the patient's immune response to HIV-1A while eliminating the risk of spontaneous infection of the patient with HIV-1A during the vaccination process, reducing the side effect of drug administration and increasing the effectiveness of its use.

The administration of several different structures of the HIV-1A genome to the patient, which differ in the composition of the 14 N-terminal amino acids of the Tat protein, if similar structures are isolated in an infected patient, allows the patient to provide the necessary immune responses to all the selected HIV-1A structures.

The introduction of the drug by the rectal method allows to increase the effectiveness of the drug by increasing the intensity of exposure, reducing its digestibility and developing an immune response to the drug, as well as reducing its dosage and reducing side effects.

The introduction of the drug in the form of a candle or microclysters increases the convenience of vaccination and expands the possibilities for its use in various conditions.

The essence of the present invention is illustrated by the following images:

FIG. 1 - The structure of the molecular clone.

FIG. 2 - Microclyster.

FIG. 3 - Identification of the pre-integration complex (PIK) in the cytoplasm of HIV-infected 106 MPCH (time after infection 8 hours, track "O").

FIG. 4 - The result of electrophoresis of plasmid DNA in a 1.5% agarose gel.

FIG. 5 - Isolation of the infectious offspring of HIV-1.

FIG. 6 - Results of 2% agarose gel electrophoresis of amplification products of LTR, pol and tat sequences.

FIG. 7 - Table 1. The structure of the primers for amplification.

The implementation of the invention

The proposed method allows to obtain a personalized state of LTNP (non-progressive course of HIV-1A infection) and can be implemented on the basis of the existing level of technology with the following sequence of operations.

Initially, the necessary fragments of the 6-modified Tat peptide of HIV-1A are prepared, for which purpose the primary structure of the 14 N-terminal amino acids of the Tat protein is determined from the infectious molecular clone (s) isolated from the patient. A personalized preparation is then created: 6-peptidyl substituted 6-производ derivative, where the peptidyl is structurally similar to the 14 N-terminal amino acids of the Tat protein of the infectious molecular clone (s) in an HIV-1A infected patient. Then, rectal immunization (IR) of the patient is carried out with a personalized preparation, the introduction of which causes the patient to form three classes of IgG, IgM and IgA antibodies (immunoglobulins of classes G, M, A, respectively) in a high titer and, accordingly, an increase in the titer of immune CTL in the patient. If several different structures of the HIV-1A genome are distinguished in an infected patient, which differ in the composition of the 14 N-terminal amino acids of the Tat protein, a personalized drug is created for each of the indicated structures of the HIV-1A genome with a structure matching the composition of 14- ty of the N-terminal amino acids of the Tat protein of each of the selected HIV-1A genomes, all created drugs being administered to the patient in a rectal manner.

The invention allows to obtain personalized biologically active preparations that can form covalent bonds with soluble proteins and cell surface proteins and show the desired biological activity in vivo. The first practical objective of this invention was to determine the structure of the HIV-1 genome in an infected patient according to a scheme known to those skilled in the art (US Patent 4520113; US Patent 4647773; US Patent 4708818; EP 0178978-A / 2; WO 8601827-A / 1; EP 0185444- A2). Based on the present invention, isolation and determination of the structure of the HIV-1 genome in an infected patient allows not only to clarify the diagnosis, but also to determine the structure of the Tat protein of the infectious molecular clone (s) in the HIV-1A infected patient. During vaccination, it is possible to monitor the ratio of infectious and non-infectious molecular clones in the patient’s plasma so that the effectiveness of immunization can be evaluated. It should be noted that it is possible to control the transmission of the infectious virus to people in contact with the patient. The invention allows to obtain combined "haptens" with good pharmacodynamics due to the physiologically active half, for example, 6-ΑΠΑ, capable, after opening of the β-lactam ring, to form covalent bonds with a wide variety of protein molecules at the ε-amino groups of the amino acid residues of lysine ("penicilloyl "Configuration), or due to a disulfide bond with cysteine residues (" penicillin "or" penicillamine "configuration). In the framework of the present invention, non-toxic personalized preparations of combined “haptens” are created so that under physiological conditions they are recognized by Τ and B cells in the form of immunogenic determinants (epitopes), as is known to specialists (US Patent 6811782; US Patent 7115266; US Patent Applications 0130236478 ) Additionally, Th1 responses against combined “haptens” are achieved not only by structural polymorphism of recognition products, providing processing variation and induction of cytotoxic T-cell responses (CTL), which are normally subdominant (PCT / EP 2004/11950), but also using appropriate ODNs adjuvants known in the art (US Patent 6194388; US Patent 6,821,957). According to the scheme of this invention, a personalized preparation for rectal immunization is administered according to schemes that reduce the frequency of repeated injections. Based on the present invention, effective immunization with combined “haptens” precludes the possibility for the full-sized Tat protein to function as an adjuvant, increasing cellular immune responses against other antigens, as disclosed in WO 03/0098670.

Immunization efficiency was monitored according to well-known technological regulations (analysis of the titer of specific immunoglobulins and cytotoxic lymphocytes), and the viral load and titer of the infectious virus according to the scheme known to specialists (US Patent 4520113; US Patent 4647773; US Patent 4708818; EP 0178978-A / 2 ; WO 8601827-A / 1; EP 0185444-A2).

Experimental studies of the proposed method were carried out by CJSC YurVeles. The involvement of patients in the research was carried out taking into account the provisions of the Helsinki Declaration of the World Medical Association (WMA) “Ethical Principles for Medical Research with a Person”, paragraph 22 of part “B” of which reads: “In any study in humans, every potential participant in the experiment should be adequately informed ... After the participant of the experiment is provided with an understanding of the information, the doctor must obtain the voluntary informed consent of this participant in the experiment, preferably in writing, which ... must be legally registered and attested. ”

All manipulations with materials suspicious of HIV-1 infection were carried out taking into account the rules of conduct in a laboratory working with material infected with HIV-1. The research results are illustrated by graphic materials (Figs. 1, 7), and in Figs. 2-6 are presented in the form of photographs.

The molecular clone structure is shown in FIG. 1. Plots of sequences of ID duplications of plasmid pBR-322 (K00005, GenBank) are indicated by nucleotide numbers; the nucleotide sequences of the joints between the LTR of HIV-1 and pBR-322 are shown, the ID sequence is underlined. Designations:

. В обоих случаях на картине разделения выделяются интенсивные полосы, принадлежащие рибосомальным РНК (около 5 и 2 т.п.о.).The result of the identification of a pre-integration complex (PIK) in the cytoplasm of HIV-1 infected MPPC (human peripheral blood mononuclear cells) is shown in FIG. 3 (10 ⁶ MCPC, time after infection 8 hours, lane “O." The virus was obtained after transfection of MCPC-activated infectious viral cDNA). PIKs were isolated from the cytoplasmic fraction. The PIK band was excised from the gel and the presence of LTR sequences and gag, pol, env genes were determined in PCR (polymerase chain reaction). Lane “K” - control: uninfected cells. Gel staining - propidium iodide, laser irradiation with

. In both cases, intense bands belonging to ribosomal RNA (about 5 and 2 kb) are highlighted in the separation picture.

The result of plasmid DNA electrophoresis on a 1.5% agarose gel is shown in FIG. 4. Tracks: “A” - linear DNA fragments with sizes from 4-0.5 kbp (thousands of base pairs). “B-D” - plasmid DNA isolated from the material of transformed clones, the presence of a hybrid plasmid (shown by an arrow) with a molecular mass many times greater than that of pBR322 (4.36 kbp, lane “D”) is evident.

The isolation of HIV-1 infectious progeny was determined by the characteristic change in the cell monolayer obtained by deposition of PEI on the plastic surface of the well. In FIG. 5 halves of the holes are brought together, on one of which (on the left) characteristic violations of the cell monolayer (“plaque”) are visible.

In FIG. Figure 6 presents the results of electrophoresis in a 2% agarose gel of amplification products of LTR, pol, and tat sequences. Lanes: 1 - LTR, 2 - pol, 3 - tat from infectious cDNA, 4 - tat from non-infectious cDNA.

The structure of the amplification primers is shown in Table 1, FIG. 7.

The invention is illustrated by the following examples.

Example 1. Isolation and cloning of PIC owls

From the blood of a healthy donor, the fraction containing MPCC was isolated by centrifugation on a step gradient of Ficol 400. Cells were cultured for 2 days at 37 ° C in RPMI1640 medium with 10% fetal bovine serum, recombinant human IL-2 (50 units / ml), as well as PHA (up to 0.5%). 2 × 10 ⁷ MPHCs activated with PHA (phytohemagglutinin) were added to the serum of AIDS patients (acquired immune deficiency syndrome) with a sufficiently high titer of p24 viral protein and the mixture was incubated for 6-8 hours at 37 ° C. Pre-integration complexes (PIKs) were precipitated by centrifugation (8000 rpm, 15 minutes) from the clarified lysate. The presence of HIV-1 PICs in the cell lysates was monitored by agarose gel electrophoresis (see Fig. 3). The pellet was mixed in 100 μl of buffer A (10 mm MgCl ₂ , 20 mm HEPES (N-hydroxyethylpiperazine-N′-2-ethanesulfonic acid) [pH = 7.2], 10% PEG8000, 5 mM aprotinin, 1 mm DTT (dithiothreitol )) with plasmid DNA pBR322 (50 ng) and the mixture was incubated at 25 ° C for 30 minutes. The mixture was used to transform competent cells of strain χ6019. Transformed cells were plated on a medium with undekilviologen (20 μg / ml). Crops were incubated at 37 ° C for 18 hours. The frequency of colony formation containing hybrid plasmids with HIV-1A molecular clones was at least 15%.

Example 2. The determination of the infectivity of the molecular clones of HIV-1A and the determination of their structure (sequencing)

Clones whose progeny contained hybrid plasmids were determined by agarose gel electrophoresis as previously described (Promega Technical Manual No. 012). In FIG. 3 shows the result of electrophoresis of a DNA clone containing a hybrid plasmid with very low mobility. The cell mass of the progeny of colonies containing low mobility hybrid plasmids was harvested and destroyed to isolate plasmid DNA using the technology described in US Pat. No. 6,242,220 and subsequent electrophoresis on a 1.5% agarose gel (see FIG. 4). DNA of hybrid plasmids with a molecular weight many times greater than that of pBR322 (4.36 kb) isolated from agarose was used to transform Jurkat cells (using the Promega Transfection Guide technology, see US Patent 5824812; US Patent 5869715) deposited on the surface of PEI cups. The formation of HIV-1 infectious progeny was determined by the characteristic change in the cell monolayer (see Fig. 5). DNA of hybrid plasmids, which during the transformation of Jurkat cells caused a characteristic change in the monolayer of cells, was used for amplification in PCR (the technology is given according to US Patent 4,683,202) to identify LTR sequences, as well as pol and tat genes. Table 1 shows the structures of the amplification primers, and FIG. 6 is the result of a PCR experiment.

Example 3. Determination of the Tat structure of the coding exon and determination of the amino acid sequence that matches that at the N-terminal positions of the 2-15 Tat protein

The DNA fragment of hybrid plasmids with infectious molecular clones of HIV-1A and encoding Tat protein was amplified in Poland. The amplification product was automatically sequenced on a 373A Automatic Sequencer (Applied Biosysthems USA) using the Dye Deoxy Terminator ABI sequencing Kit with Taq-polymerase FS (Perkin-Elmer, USA) reagent kit, as is known to those skilled in the art. The primary structure of the HIV-1A Tat protein was determined using the Vector NTI 11.5 program, as described in US Patent 5801056.

The structures of Tat peptides found in the genomes of a group of HIV-1A-infected patients involved in trials can be represented as XPXDPXXEPWXHPG. As can be seen from the presented sequence in five positions there can be replacements. Thus, the discussion of the possibility of creating an industrial anti-Tat vaccine against HIV-1A should be postponed until more statistics of the infectious molecular clones of the virus of this subtype are obtained.

Example 4. Synthesis of oligopeptides

Obtained on an ABI 488A peptide synthesizer (Perkin Elmer, Norwalk, Conn.) By the F-moc method (solid-phase synthesis of peptides with florenylmethoxycarbonyl / tert-butyl derivatives of amino acids), the peptides required a deprotection step with 15 mM trifluoroacetic acid (TFA), and the desired product was isolated by high pressure liquid chromatography (HPLC) according to the attached instructions.

Example 5. Synthesis of 6-APA modified Tat-peptide HIV-1A (combined "hapten") and determination of its ability to covalently attach to human serum albumin

Lyophilically dried 14-membered peptides with the structure indicated below were used to create conjugates with sodium salt of bromoacetylated 6-APA in 100 mM buffer (pH = 8.3). Conjugates of peptides with 6-APA were again isolated using HPLC and lyophilized. A portion of the combined hapten preparation was used to determine its ability to covalently attach to human serum albumin (the technology is generally described in US Patent 4,596,768).

Example 6. Immunization Protocol

The introduction of at least a combined "hapten" of the same type, through the mucous membrane of the gastrointestinal tract of the subject

The value of a single dose containing a therapeutically effective amount of the active principle in the preparation was determined empirically and was equal to an average of 200 μg. Moreover, since the method proposes a means for immunization, individualized for a particular patient, the choice of doses of the drug was adjusted based on the principle of individual selection of the active active substance, taking into account the results of evaluating the results of a skin test after each injection of the drug. Rectal immunization of volunteers was performed two or three times with an interval of two weeks. The number of immunizations was also determined based on the results of the evaluation of the skin test.

According to studies, immunization preparations had Limulus toxicity levels for amoebic lysate <0.1 u / μg. Other toxicological characteristics of the drugs were studied at the Laboratory of Drug Toxicology of the Federal State Institution “Russian Cardiological Research and Production Complex” in accordance with the regulations (“Guidelines for the experimental (preclinical) study of new pharmacological drugs” (Ministry of Health of the Russian Federation, Moscow, 2000). in mice and rats of Wistar showed that the doses of acute and chronic toxicity of the drug used exceeded the daily therapeutic dose for eloveka 1400 and 20 times.

Example 7. ELISA

Microtiter plates were coated with 200 ng of N-terminal peptides or 1 μg of beige dissolved in 50 mM carbonate-bicarbonate buffer (Sigma-Aldrich). After the blocking step of non-specific binding, dilutions of plasma samples (obtained from HIV-1A infected patients) or control materials (healthy patients, Trastozumab) in 25% DMSO (dimethyl sulfoxide) were added to the wells for 18 hours at + 5 ° C. Binding of antibodies and their classes was detected using rabbit anti-human IgG labeled with TAMPA fluorogenic dye (tetramethylrodamine fluorescent dye) (US Patent Applications 20080274488).

Plasma IgE levels were determined using a commercial IgE ELISA kit (Mabtech Inc., Sweden) and in accordance with the attached instructions.

IFA production. Antibodies against Tat protein in a patient were detected by standard ELISA with U96 plates (Nunc). The bottom of the well was preliminarily coated with 0.5 μg / well of each Tat species in 100 mM phosphate buffer pH = 4.5 and incubated for 16 h at 4 ° С followed by incubation with 2% milk (to reduce non-specific binding). Serums (one serum for one cup) were added in various dilutions of PBS with 0.2% milk and gently pumped on a shaker for 1 hour in RT. The secondary antibody (rabbit anti-human IgG labeled with TAMRA) was diluted 1: 1000 in PBS with 0.2% milk; it was added to each well and gently pumped on a shaker for 1 hour in RT. The dishes were washed three times with 200 μl of a single PBS / 0.05% Tween 20 (Genaxis) between each subsequent step. The discovery was performed with 100 μl ABTS (2,2′-azino-bis-3-ethylbenz-thiazoline-6-sulfonic acid from Sigma), added well to each, and the light absorption measure was read in a universal EL800 microspectrophotometer for reading tablets (BIO -TECH INSTRUMENT, INC) at 405 nm.

Example 8. Determination of CTL activity

CTL (cytotoxic lymphocyte) activity was performed by intracutaneous assay with a synthetic syngeneic peptide conjugate with human serum albumin.

Example 9. Determination of the effectiveness of immunization

CD4 counting technique (“helper” cell receptor marker of lymphocytes): isolation on magnetic beads with monoclonal antibodies against CD4 receptors (Becton Dickinson, USA).

Viral load assessment: quantitative PCR performed by Invitro LLC.

QoL assessment (Quality of Life Index): a survey conducted by the attending physician on the WHOQJ-100 Questionnaire (World Health Organization Questionnaire).

Industrial applicability

The proposed personalized method allows to provide effective treatment and prevention of HIV-1A, minimize side effects from the vaccination process and can be implemented on an outpatient basis on the basis of existing equipment.

Claims (3)

1. The method of treatment and prevention of HIV infection of type 1 of subtype A (HIV-1A), in which the structure of the HIV-1A genome is isolated from the infected patient, the primary structure of the 14 N-terminal amino acids of the Tat protein is determined in the selected HIV-1A genome create a personalized preparation in the form of a 6-peptidyl substituted aminopenicillanic acid derivative (6-peptidyl-APA) peptide having an amino acid sequence that matches the 14th N-terminal amino acids of the Tat protein in the isolated HIV-1A genome of the infected patient, and the resulting P a personalized preparation to the patient, and the administration of the drug is carried out in a rectal manner, and in the case of isolation of several different structures of the HIV-1A genome from the infected patient with differences in the composition of the 14 N-terminal amino acids of the Tat protein, for each of these structures of the HIV 1A, a personalized preparation is created with a structure that matches the composition of the 14 N-terminal amino acids of the Tat protein of each of the selected HIV-1A genomes, all created drugs being administered to the patient in a rectal manner. 2. The method according to p. 1, characterized in that the 6-APA modified Tat-peptide HIV-1A is administered in the form of a candle. 3. The method of claim 1, wherein the 6-APA modified HIV-1A Tat peptide is administered as microclysters.

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