RU2475264C2 - Vaccine against hiv/aids - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to field of biotechnology, virology and medicine. Vaccine against HIV/AIDS, includes compound of vaccine antigens of HIV/AIDS virus and pharmacologically acceptable carrier. As vaccine antigens of HIV/AIDS virus applied is chimeric recombinant polypeptide, copying immunoactive areas of three HIV1 proteins - fragment of transmembrane protein gp41, coded by gen env, full-size recombinant protein p24, coded by gen gag, and fragment of internal protein p17, coded by gen gag. As polymer carrier applied is derivative of heterochain polyamines with molecular weight 50000-150000D at ratio 1:10.

EFFECT: invention presence safe vaccine against HIV/AIDS, which has high immunogenic activity and antigen specificity.

4 dwg, 4 tbl, 17 ex

Description

The invention relates to medicine and biotechnology and can be used to create candidate vaccine preparations against HIV / AIDS.

The main requirements for candidate HIV / AIDS vaccines are safety, the induction of a multipotent immune response against HIV, including cellular immunity, mucosal immunity and neutralizing antibodies, as well as the use of a viral or bacterial vector capable of generating a persistent and long-lasting immune response.

Known standard approaches in creating vaccines from live attenuated virus [Hofmann-Lehmann R., Vlasak J., Williams A.L., Chenine A.L., McClure H.M., Anderson D.C., O'Neil S., Ruprecht R.M. Live attenuated, nef-deleted SIV is pathogenic in most adult macaques after prolonged observation. AIDS - 2003. - Vol.17. - P.157-166. (524)], as well as inactivated (killed) vaccines, based on recombinant proteins or based on antitoxin [Learmont JC, Geezy AF, Mills J., Ashton LJ, Raynes-Greenow CH, Garsia RJ, Dyer WB, Mclntyre L., Oelrichs RB, Rhodes DI, Deacon NJ, Sullivan JS Immunologic and virologic status after 14 to 18 years of infection with an attenuated strain of HIV-1. A report from the Sydney Blood Bank Cohort. // N. Engl. J. Med. 1999. Vol.340. - P.1715-1722], are ineffective in the development of vaccines against HIV infection / AIDS. The main reason is the unusual biology of the virus. Attenuated vaccine strains do not guarantee vaccine safety, inactivated vaccines do not induce a broad and strong protective response [Murphey-Corb M., Martin LN, Davison-Fairburn B., Montelaro RC, Miller M., West M., Ohkawa S., Baskin GB, Zhang JY, Putney SD et al. A formalin-inactivated whole SIV vaccine confers protection in macaques. // Science. - 1989 .-- Vol.246. - R.1293-1297]. Recombinant HIV envelope glycoprotein vaccines induce a moderate titer of antibodies that can neutralize a limited range of HIV isolates and do not induce the formation of cytotoxic antibodies [Johnston R. AIDSVAX results: an answer, or just more questions? // AIDS Patient Care STDS. - 2003. - Vol.17. - P. 47-51; Watanabe M.E. Skeptical scientists skewer VaxGen statistics. // Nat. Med. - 2003. - Vol. 9. - P.376].

About 40 candidate vaccines against HIV / AIDS are known, none of which has been proven to date. A significant part of the development is devoted to the creation of drugs based on recombinant technologies.

Known vaccine AIDSVAX B / B (AIDSVAX B / E) (VaxGen) [Watanabe M.E. Skeptical scientists skewer VaxGen statistics. // Nat. Med. - 2003. - Vol. 9. - P.376], including the full-size recombinant protein gp120. However, the completion of trials of the candidate vaccine gp120 (VaxGen) showed its inability to protect against HIV infection and reduce the viral load in HIV-infected patients. Perhaps this inability to protect against infection is due to the lack of the formation of antibodies that can neutralize genetically diverse primary HIV1 isolates.

A known candidate vaccine based on the live recombinant smallpox virus ALVAC-HIV (AVENTIS-PASTEUR) and the AIDSVAX B / E vaccine based on the recombinant gp120B, (VaxGen), used in the prime boost system [Belshe RB, Gorse GJ, Mulligan MJ, Evans TG , Keefer MC, Excler J.-L., Duliege A.-M., Tartaglia J., Cox WI, McNamara J., Hwang K.-L., Bradney A., Montefiori D., Weinhold KJ Induction of immune responses to HIV-1 canarypox virus (ALVAC) HIV-1 and gp120 SF-2 recombinant vaccines in uninfected volunteers. // AIDS. - 1998 .-- Vol.12. - P.2407-2415], for which phase III clinical trials of the combined action of the two above drugs are currently being conducted. It is assumed that, not individually effective, when combined, these drugs will be able to induce two types of immune response in volunteers: the induction of effector cells that kill HIV and the formation of antibodies that neutralize the virus. Researchers expect double exposure to produce a result that is higher than the sum of the responses to individual vaccines.

Adenovirus 5-based Merck candidate vaccine trials were stopped early because there were more new infections in the vaccinated group than in the placebo group [Michael Robertson, D Casimiro, S De Rosa, S Dubey, L Kierstead, and J McElrath. Immunological Characterization of Subjects from the STEP Study: A Phase IIB Test-of-Concept Trial of the MRKAd5 HIV-1 Gag / Pol / Nef Trivalent Vaccine // CROI 2008, http://www.hvtn.org/science/step_roberts. html].

A vaccine preparation is known [RF patent No. 2179980, IPC С07К 7/00, publ. 02/27/2002], which offers, as an active vaccination component, mimetic antigens that mimic conserved fragments of the HIV1 gp41 protein epitope. The use of a known drug involves a strong immune response of the body to the introduction of this protein with the formation of virus-neutralizing antibodies. The use of peptides of the conserved epitope of the gp41 protein is intended to provide protection against many new constantly emerging HIV subtypes, however, the known vaccine preparation provides the formation of antibodies only.

A vaccine against AIDS is also known [RF patent 2014845, IPC A61K 39/12, publ. 06/30/1994], with the ability to cause the formation of antibodies only against the key epitope of the protein gp41.

The vaccines described in the above patents can cause either the induction of only neutralizing antibodies or only the lymphoproliferative response, while for the prevention and treatment of HIV infection, a multifunctional immune response, including both the humoral and cellular components, is important.

Described recombinant protein [RF patent 2214274, IPC A61K 39/21, publ. 2003], consisting of an internal p24 protein and an immunogenic fragment of gp41. It has been claimed that this recombinant polypeptide is capable of inducing a strong immune response in mammals, however, it has not yet been used in the composition of the vaccine.

Known vaccine composition against human immunodeficiency virus [RF patent No. 2223784, IPC C 12N 15/09, op. 2004], including two recombinant plasmids containing the Lpp-OmpA genes necessary for surface exposure, the genes behind them encoding HIV-1 proteins, namely, reverse transcriptase protein or transactivating protein (Tat). The known vaccine was created using an attenuated Salmonella strain designed to express specific HIV proteins on its surface. The vaccine is offered to be administered orally. The described vaccine also cannot cause a multipotent immune response of the body to the introduction of the human immunodeficiency virus. In addition, it seems that this vaccine may not have a good protective effect, because the transactivating Tat protein and reverse transcriptase are formed in the cells after the introduction of the virus, i.e. at the beginning of virus replication. Nuclear localization of the transactivating protein Tat generally makes it inaccessible to antibodies formed as a result of vaccination.

Closest to the present invention is a vaccine against HIV infection and AIDS [RF patent 2306950, IPC A61K 39/21, publ. 04/10/2007], including a peptide, which is a peptide fragment of the structural protein p17 / 18/55 HIV-1/2 (gag gene products) associated with the CD4 cell receptor. The presence of a CD4 cell receptor in the vaccine, which is a 58 kDa monomeric glycoprotein that is found on the surface of approximately 60% T-lymphocytes, precursors of T-lymphocytes in the bone marrow and thymus, as well as monocytes, macrophages, eosinophils, dendritic cells and CNS microglia cells , suggests the main purpose of the known vaccine is therapeutic, because the formation of antibodies to CD4 in a healthy person with the introduction of a vaccine can cause undesirable consequences. In addition, it has been proven that there are CD4-independent strains of HIV for which binding to CD4 is not required for entry into the cell.

Thus, the technical result obtained by the implementation of the described invention is to create a safe vaccine against HIV / AIDS with a multi-potent immune response, to increase its prophylactic effectiveness by using unique genes / fragments of genes encoding optimal antigenic to produce a chimeric recombinant HIV protein determinants capable of inducing the formation of virus-neutralizing antibodies, cellular and lymphoproliferative response with a significant reduction in vaccination her dose of the drug.

The specified technical result is achieved in that the HIV / AIDS vaccine, comprising a combination of vaccine antigens of the HIV / AIDS virus and a pharmacologically acceptable carrier, contains as a vaccine antigens of the HIV / AIDS virus a chimeric recombinant polypeptide (consisting of sequentially located fragment of p17 and full-sized protein p24, encoded by the gag gene and a fragment of the gp41 protein encoded by the env gene region) expressed in E. coli cells based on the plasmid vector pET15b, into which the Bam H1 restriction site is inserted gment DNA encoding a chimeric recombinant polypeptide and the restriction sites Hind III - Eco R1 inserted par-locus, and as the carrier polymer derivative is used heterochain polyamines having a molecular weight of 50000-150000 Yes (polioksidony) at a ratio protein: polioksidony 1:10.

Moreover, the chimeric recombinant HIV1 polypeptide is a sterile purified protein that copies the amino acid sequences of the p17 fragment, the full-sized p24 protein and the gp41 protein fragment, in which the p17 fragment has the following amino acid sequence:

SEQ ID NO: 1

full-sized protein p24 has the following amino acid sequence

SEQ ID NO: 2

The gp41 fragment has the following amino acid sequence

SEQ ID NO: 3

SEQ ID NO: 4 is a series-connected SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3.

As a result of the analysis of databases of HIV sequences, genes / gene fragments were selected that encode the optimal antigenic determinants for inclusion in the candidate vaccine.

The selection of the full-sized p24 protein, as well as protein fragments from the antigenic determinants gp41 and p17, is carried out from the following premises.

Induction of neutralizing antibodies is provided by HIV1 surface proteins encoded by the env gene; therefore, a fragment of the gene encoding HIV1 transmembrane protein gp41 was introduced into the recombinant vaccine. To induce a cellular and lymphoproliferative response, a gene encoding a p17 protein fragment and a full-length HIV1 internal protein, p24, on which numerous epitopes for T-helper cells and cytotoxic lymphocytes are located, was introduced into the plasmid. Because HIV is highly variable, the gag gene region encoding the p17 matrix protein fragment and the internal p24 protein introduced into the expression vector together with the env gene fragment encoding the 578-609 gp41 region (SEQ ID NO: 4) provide a wide range of immune response of the described candidate vaccine. Due to the conservatism of the p17 protein fragment, p24 protein and gp41 fragment that are part of the chimeric recombinant polypeptide selected for the vaccine, the latter acquires the ability to induce the formation of antibodies against HIV1 with a wide spectrum of action. The matrix protein p17, in addition to participating in the replication of the virus and the formation of the virion, acts outside of infected cells, where it disrupts the regulation of the biological activity of immune cells involved in the pathogenesis of HIV infection. In this regard, it is expected that specific antibodies induced by the vaccine will be able to recognize p17 protein, disrupt the HIV life cycle and prevent the spread of infection in humans [Fiorentini S., Giaquilli C., Caccuri F., Magiera AK, Caruso A. HIV Matrix protein p17: a candidate antigen for therapeutic vaccines against AIDS. Pharmacol Ther, 2010, Dec; 128 (3): 433-444. Epub 2010 Sep 8]. The inclusion of p17 protein in a vaccine preparation is expected to significantly increase its immunogenic and protective properties.

Polymeric carrier Polyoxidonium (PO), which belongs to the class of high molecular weight water-soluble derivatives of hetero-chain polyamines, has a pronounced intrinsic immunostimulating activity, which results in a significant increase in the immunogenicity and preventive efficacy of the vaccine.

An HIV / AIDS vaccine according to the invention is prepared as follows.

Initially, an HIV-specific nucleotide sequence is selected for inclusion in recombinant pasmids. For this, a chimeric genetic engineering construct consisting of genes and gene fragments corresponding to the p17 protein fragment, the full-size p24 protein and the gp41 protein fragment is assembled in the intermediate plasmid pBS. Primers are synthesized that correspond to the 131-136 and 365-370 amino acid sequences of the full-length cDNA of plasmid pBH10 for the p17 protein fragment, full-size p24 and 578-609 protein for the gp41 protein fragment. PCR fragments are alternately cloned into pBS. A pET15 capable of being expressed in E. coli is used as an expression vector. The expression result is a chimeric recombinant rec polypeptide (17-24-41). Cells are treated with 8M urea and ultrasound. The clarified lysate is sulfonated and purified on an affinity column with Ni2 + agarose, and then on a Q-Sepharose column (Pharmacia). The purity of the product is checked by electrophoresis in PAAG. The chimeric recombinant polypeptide thus obtained is used as an antigen to produce an HIV / AIDS vaccine.

The resulting HIV / AIDS virus antigen is combined using a conjugation reaction with a polymer carrier - Polyoxidonium (a copolymer of poly-1,4-ethylene piperazine N-oxide and N-carboxyethyl) -1,4-ethylene piperazinium bromide with a molecular weight of 50,000-150,000 Da) according to the scheme :

where PO is a copolymer of N-oxide of poly-1,4-ethylene piperazine and (N-carboxyethyl) -1,4-ethylene piperazinium bromide (Polyoxidonium),

CDI - carbodiimide,

rec (17-24-41) is a chimeric recombinant polypeptide.

In the resulting compound, the recombinant vaccination polypeptide and the PO in the vaccine are linked by strong covalent amide bonds (in addition to the hydrogen, ionic, Vander-Waals bonds and complex bonds due to NO groups).

Therefore, the combination of PO with the HIV vaccine antigen ensures high antigen stability and an increase in its immunogenicity, thereby reducing the vaccination dose of the antigen. The effect of enhancing the immune response on the recombinant HIV1 protein with Polyoxidonium reduced the dose of rec antigen (17-24-41).

An analysis of the composition of the obtained compounds and their specific properties is carried out using the following methods: SDS-polyacrylamide gel electrophoresis (PAGE), enzyme-linked immunosorbent assay (ELISA), immunoblot, MALDI-TOF, molecular hybridization method, evaluation of lymphoproliferative response.

Evaluation of the immunogenic properties of the obtained compounds is carried out by studying the reaction of the immune system and the body as a whole on experimental animals, as well as by clinical trials of a vaccine involving volunteers.

The invention is illustrated by the sequences, tables 1-4 and drawings (figures 1-4), where

figure 1 - the result of analytical chromatography of a purified chimeric recombinant polypeptide of the vaccine against HIV / AIDS on a column with Q-Sepharose;

figure 2 - the result of analytical chromatography of the purified chimeric recombinant polypeptide of the vaccine against HIV / AIDS on a column with Superdex-200;

figure 3 - the result of determining the molecular weight of three series of a chimeric recombinant polypeptide of three series of vaccines against HIV / AIDS according to the spectra of the molecular ion;

figure 4 - spectra of MALDI tryptic hydrolyzate of three series of a chimeric recombinant polypeptide of three series of vaccines against HIV / AIDS.

Below are examples of the implementation of the described invention, where examples 1-10 show the stages of the method for producing a vaccine against HIV / AIDS, and examples 11-18 provide an experimental evaluation of the effectiveness of the obtained preparations.

First, HIV-specific nucleotide sequences are selected for inclusion in the plasmid vector in order to obtain HIV-specific HIV / AIDS vaccine proteins.

The selection is carried out according to the analysis of an international database (Kuiken C, Foley B., Hahn B. et al. HIV Sequence Compendum 2001. // Los Alamos Nat. Lab., Los Alamos, New Mexico 87545, USA), focusing on the most relevant (immunogenic) regions of the HIV genome. The following are the nucleotide sequences encoding the p17 protein fragment, the p24 protein and the gp41 protein fragment selected for the vaccine of the invention.

The nucleotide sequence encoding a fragment of the protein p17

SEQ ID NO: 5

The nucleotide sequence encoding the protein p24

SEQ ID NO: 6

The nucleotide sequence encoding a gp41 protein fragment

SEQ ID NO: 7

SEQ ID NO: 8 is a series-connected SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7.

After selecting the nucleotide sequences, the steps are taken step by step to obtain a plasmid with selected fragments of the HIV genome, expression of the plasmid in E. coli, isolation and purification of expression products.

Example 1. Obtaining plasmids pET15par

The plasmid pET15b is hydrolyzed with Hind III restriction enzyme, the sticky ends are extruded with T4 DNA polymerase, then the plasmid is isolated with the Quiagen kit and ligated to the locus obtained by the known method [Meacock P. A., Cohen, SN, Cell, 1980, v.20 , pp. 529-542]. The obtained ligase mixture transform cells of E. coli strain Nova-Blue (Novagen) by the usual method [Maniatis T., Frig E., Sambrook J. Molecular cloning. Moscow, Mir, 1984]. Transformed cells are plated on LB agar plates containing 25 μg / ml kanamycin. The grown colonies are checked for the presence of recombinant plasmids. For this, bacterial cells lyse and secrete plasmid DNA. The isolated plasmid DNA is treated with restriction enzymes Pst I and Hind III and the hydrolyzate is examined by electrophoresis in 1% agarose.

The pET15-par plasmid DNA hydrolyzate has an 2270 base pair DNA band on the electrophoregram.

Example 2. Obtaining plasmid pET15par (17-24-41) (with an insert of DNA encoding a chimeric polypeptide)

Oligonucleotides to obtain gp41 are kinetized using DNA kinase in the usual way [Maniatis T., Frig E., Sambrook J. Molecular cloning. Moscow, Mir, 1984], are mixed in an equimolar ratio, incubated at a temperature of 95 ° C for 30 minutes, and annealed at a temperature of 15 ° C for 2 hours in a thermostat, then DNA ligase is added and incubated for 8-10 hours at 15 ° C. The target gp41 DNA fragment was isolated using a Qiagen DNA isolation kit.

The structure of oligonucleotides to obtain a copy of the gp41 fragment

DNA sections encoding a p17 protein fragment and a full-sized p24 are obtained simultaneously in a single DNA strand. For this, the plasmid rVH-10 containing the full-length HIV1 genome [Kuiken C., Foley B., Hahn B. et al. HIV Sequence Compendum 2001. // Los Alamos Nat. Lab., Los Alamos, New Mexico 87545, USA], amplified using primers N1 and N2. The selected DNA fragment was isolated using a Qiagen DNA isolation kit, digested with BamHl restriction enzyme and purified by electrophoresis in 1% agarose.

The structure of oligonucleotides to obtain a PCR fragment 17-24 and a PCR fragment 17-24-41

To obtain a full-sized DNA fragment encoding the chimeric recombinant rec (17-24-41) polypeptide, a gp41 DNA fragment with a combined DNA fragment encoding the p17 fragment and full-size p24 is mixed in a 0.5 ml tube and PCR is performed using primers N1 and N3 . The target fragment is isolated as described above, hydrolyzed by BamHl restriction enzyme, and ligated with the pET15-par vector hydrolyzed by the same restriction enzyme.

Example 3. Transformation of E. coli

The obtained ligase mixture transform cells of E. coli strain Nova-Blue (Novagen) according to the standard method [Maniatis T., Frig E., Sambrook J. Molecular cloning. Moscow, Mir, 1984]. Transformed cells are plated on LB agar plates containing 25 μg / ml kanamycin. The grown colonies are checked for the presence of recombinant plasmids. For this, bacterial clones are grown and plasmid DNA is isolated. Isolated plasmid DNA was analyzed by PCR using primers N3 and T7. The structure of the DNA fragment is confirmed by Sanger sequencing using T7 and N3 primers [Maniatis T., Frig E., Sambrook J. Molecular cloning. Moscow, Mir, 1984].

Example 4. Determination of expression of HIV-specific protein using immunoblot and selection of colonies containing the desired insert

Prepare two identical prints on nitrocellulose filters using Petri dishes with grown bacterial clones. Then one filter is kept with diluted blood serum of an HIV-infected patient, and the other with diluted blood serum of an HIV-negative donor. Next, the filters are washed with washing buffer and incubated with a peroxidase conjugate with rabbit antibodies against human IgG. The filters are again washed and treated with a buffer solution containing a peroxidase substrate - hydrogen peroxide and 4-chloro-1-naphthol chromogen. Bacterial colonies containing the HIV-specific insert express the HIV-specific rec protein (17-24-41) and interact with the anti-HIV antibodies contained in the serum of the infected person, giving a stained spot on the print. On a control filter treated with negative serum, stained spots are absent.

Example 5. Expression, isolation and purification of the chimeric recombinant HIV polypeptide

The E. coli strain BL21 (DE3) containing the plasmid pET15 par (17-24-41) is grown in 1 liter of LB medium (lactobulone) containing 25 mg / l kanamycin at 37 ° C to a density of 0.5 OE (at 600 im), add isopropylthioglucopyranoside (IPTG) to a concentration of 1 mM, incubate for another 4 hours and collect cells by centrifugation at 4000 g. Cells are suspended in 50 ml of 6M urea and sonicated, potassium tetrathionate and sodium sulfite are added to the clarified lysate to a concentration of 25 mM and 100 mM, respectively, incubated for 16 hours, and then the mixture is chromatographed on an affinity column with Ni2 + agarose (Qiagen) according to the formula manufacturing company. Rechromatography was performed on a Q-Sepharose column in 6M urea and a linear NaCl gradient (0-0.2 M). The purity of the product was evaluated by electrophoresis in PAAG.

Three experimental series of a chimeric recombinant protein were obtained and analyzed by the method described above for further connection with a polymer carrier for the preparation of an HIV / AIDS vaccine in order to conduct research on laboratory animals and to conduct phase I clinical trials in order to determine the ability of the obtained vaccine to induce an immune response .

Example 6. The analysis of the purity of the chimeric recombinant HIV1 polypeptide (purity, homogeneity, molecular weight)

The purity of the resulting chimeric recombinant HIV1 polypeptide is evaluated by analytical column chromatography by elution profile from Q-Sepharose and Superdex-200 columns (FIGS. 1-2) and by PAGE. In analytical chromatography, the elution profile of the purified product from both columns should be an acute equal-arm peak. As can be seen from the drawings (FIGS. 1 and 2), the elution profiles of the purified product from Q-Sepharose and Superdex-200 columns respectively represent sharp equal-arm peaks, which indicates the purity of the product.

Example 7. Analysis of the chimeric recombinant HIV polypeptide using MALDI-TOF (confirmation of molecular weight and determination of N-terminal peptides)

Mass spectrometric testing of the chimeric recombinant HIV1 vaccination polypeptide by mass spectrometry is to confirm the primary structure of the protein, which includes confirmation of the molecular weight and determination of N-terminal peptides. In addition, the molecular weight of the protein is determined by the molecular ion method. The theoretical protein mass calculated by the amino acid sequence is 34.950 kDa (kilodaltons). The molecular weight of the samples obtained by the above method of the chimeric recombinant protein rec (17-24-41), determined on the basis of the molecular ion spectra shown in figure 3, is in the range of 34,800-35,200 for all three series of vaccines.

The determination of the molecular weight of the three experimental series of protein gave the following results. For the series 16 08 01 - 35.015 kDa, for the series 14 10 00 - 34.950 kDa and for the series 15 06 01 - 34.920 kDa. Thus, the molecular weight of the obtained proteins, determined on the basis of the spectra of the molecular ion, corresponds to the calculated one.

To confirm the amino acid composition, the recombinant protein is hydrolyzed with trypsin and the obtained hydrolyzate is analyzed by mass spectroscopy.

Figure 4 shows the MALDI spectra of the three experimental series of the protein included in the vaccine, and Table 1 shows the structure of the expected peptides resulting from exhaustive tryptic hydrolysis and their molecular weight (MH +). As can be seen from figure 4 and table 1, the spectra contain ions of all the expected peptides with a molecular mass of more than 800, the noise level on the spectra is minimal, which indicates the homogeneity and purity of the drug. Based on these spectra, it was found that the protein structure is consistent with the theoretically stated and fully confirmed, which proves the authenticity of the chimeric recombinant protein rec (17-24-41), which is part of the HIV / AIDS vaccine, i.e. protein structure is fully proven. Comparison of the mass spectra shows good reproducibility of the protein from series to series.

Example 8. The control of the purified protein preparation rec (17-24-41) on the content of impurities of bacterial DNA

The use of the chimeric recombinant rec polypeptide (17-24-41) as a substance of a candidate anti-HIV / AIDS vaccine requires monitoring for the content of bacterial DNA. The control is carried out by molecular hybridization with a radioactive breakdown. The specific radioactivity of the DNA probe is 4 × 10 ⁸ cpm / μg DNA. Dilutions of the standards of purified bacterial DNA are applied to a nitrocellulose filter in concentrations from 31.25 ng to 2 ng per point. The protein content of the vaccine dose is 50 mcg. In this regard, DNA is isolated from 50 μg of protein, which corresponds to the dose of the vaccine, and from 500 μg of protein, which is 10 times higher than the protein content in the dose of the vaccine. It has been established that bacterial DNA is not detected both in preparations corresponding to a single dose of the vaccine and in preparations corresponding to a ten-fold dose of the vaccine, i.e. the content of bacterial DNA in all series of the recombinant protein rec (17-24-41) is less than 2 pg (picograms) per ten-fold dose of the drug (500 μg), which meets WHO requirements.

Example 9. Refolding of the chimeric recombinant polypeptide res (17-24-41)

Proteins produced in E. coli do not undergo post-translational changes that are characteristic of HIV proteins generated during natural infection, and thus the immunological characteristics of recombinant proteins may differ slightly from the properties of viral proteins. In this connection, recombinant antigens expressed in E. coli are produced (especially encoded by the gag gene), which are soluble under physiological conditions in aqueous solutions. For this, the purified protein is refolded. For refolding, cysteine is added to the protein solution to a final concentration of 10 mM, incubated for 72 hours at 4 ° C and dialyzed stepwise against 6 M - 1 M urea, and then against 10 mM NaHCO ₃ .

Example 10. Obtaining a conjugate of the obtained recombinant HIV1 protein with a carrier - a derivative of heterochained polyamines - Polyoxidonium

The conjugation of the refolded protein rec (17-24-41) with Polyoxidonium occurs at pH 7.4 in the presence of carbodiimide (CDI) according to the scheme:

Example 11. The study of the immunogenic properties of the vaccine against HIV / AIDS in laboratory animals using enzyme-linked immunosorbent assay (ELISA)

The immunogenicity of the drug is tested in laboratory animals. They comprise 4 groups of 20 mice-hybrids (CBAxC57BL / 6) F ₁ weighing 16-18 g. Three groups are immunized with the corresponding vaccine series, and the fourth, control group are 20 non-immunized mice. The vaccine is administered to animals twice intraperitoneally with an interval of 28 days. The immunizing dose is 50 μg of antigen in the composition of 0.5 ml vaccine per animal. Blood is collected 7 days after the second immunization and serum is obtained in the usual way.

The immune response is determined by ELISA. Blood sera from mice of each group are combined and the antibody titer is determined in them using ELISA in an indirect embodiment. At the same time, the chimeric recombinant polypeptide rec (17-24-41) at a concentration of 10 μg / ml used for the HIV / AIDS vaccine is adsorbed onto the solid phase, or immunosorbent tablets of commercial ELISA systems are used to determine antibodies to HIV. The developing reagent for the detection of specific murine antibodies is a peroxidase conjugate with antibodies against mouse IgG. The results presented in table 2 show that the vaccine induces the formation of high titers of antibodies against HIV1 proteins in immunized animals. These results also indicate standard vaccine preparations.

Administration of pure recombinant protein HIV1 rec (17-24-41) to laboratory animals (mice) induces a weaker immune response, which indicates the enhancing immunogenic property of Polyoxidonium, which is part of the vaccine.

Example 12. The immunogenicity analysis of the vaccine against HIV / AIDS in laboratory animals using immunoblot

The sera of immunized animals are prepared in the same manner as described in Example 11. The specific activity of the sera of mice immunized with the HIV / AIDS vaccine is studied in an immunoblot. For this, nitrocellulose strips are used from the commercial New Lav Blot kit (BIO-RAD), onto which individual proteins of cultured HIV-1 are applied. For the detection of murine antibodies, a peroxidase conjugate with antibodies against mouse IgG is used. The positive control from the New Lav Blot kit is used to identify specific bands corresponding to HIV proteins, the HIV-negative serum from the New Lav Blot kit is used as a negative control (in both of these cases, a peroxidase conjugate with anti-IgG antibodies is used for detection person). To set up another negative control, the strip from the New Lav Blot set was treated with unimmunized mouse serum using a peroxidase conjugate with antibodies against mouse IgG as a detection reagent. The sera of mice immunized with the HIV / AIDS vaccine recognize cultural HIV-1 proteins: gp41, p24, p55 (p24 precursor) in the immunoblot, which indicates the similarity of the antigenic determinants of the chimeric recombinant rec polypeptide rec (17-24-41) to determinants of the corresponding viral proteins. Thus, the chimeric recombinant HIV1 polypeptide used in the immunization with the HIV / AIDS vaccine has the same specificity as the native HIV1 viral proteins.

Example 13. The study of the neutralizing activity of antibodies induced by the vaccine against HIV / AIDS.

Mice of the line (CBA × C57 B1 / 6) F1, females weighing 16-18 g are immunized four times with the vaccine against the HIV / AIDS virus. The dose at each immunization is 10 μg per recombinant protein in the vaccine per animal. In the first and second immunizations, the vaccine according to the invention is administered subcutaneously to the animals. The third and fourth time the drug is administered intraperitoneally. The interval between the first, second and third immunizations is 1 month, between the third and fourth - two months. Blood is collected under sterile conditions from the cervical artery after 7 days and serum is obtained in the traditional way. Control serum is obtained from non-immunized animals.

The neutralizing activity of the serum is determined in the neutralization test on the model of acute MT4 / HIV1 infection, using the HIV-1 IIIB reference strain, which is actively replicating in T-lymphoblastoid cell lines. The neutralization index is on average (for the three series of vaccines) - 54.9% with a dilution of the immune serum of 1:50. Thus, antibodies obtained in animals in response to immunization have virus-neutralizing properties.

Example 14. Evaluation of the lymphoproliferative response to the HIV / AIDS vaccine

Hybrid mice (CBA × C57BL / 6) F ₁ weighing 16-18 g are immunized subcutaneously with the HIV / AIDS vaccine at a dose corresponding to 50 μg of antigen (chimeric recombinant HIV1 polypeptide) per animal. After 30 days, mice were injected intraperitoneally at the rate of 500 μg of recombinant protein in the composition of the vaccine in saline. 48 and 72 hours after the second immunization, spleen and lymph node cells are isolated from animals. The proliferative activity of the cells of the lymph nodes and separately the spleen cells is determined by the inclusion of ³ H-thymidine and visually, using an inverted microscope, by the formation of blasts in cell culture. The negative control is unimmunized mice. Cells were cultured for 12 h at 37 ° C in 5% CO ₂ in three parallels in 24-well plates with a density of 5 × 10 ⁶ cells per well in growth medium (DMEM with 20% FCS) and ³ H-thymidine. At the end of the incubation, 1 × 10 ⁶ cells are transferred to the U-shaped wells of a 96-well plate, and from there to the harvester. The incorporation of ³ H-thymidine was evaluated by calculating the radioactivity on a beta counter in a toluene scintillator. The antigen-specific proliferation index (IP) averages for all three vaccine series IP = 10 for spleen cells and IP> 8 for lymph node cells. This suggests that immunized animals developed a strong lymphoproliferative response in response to the vaccine.

Example 15. The specific interaction of the chimeric recombinant polypeptide rec (17-24-41), which is part of the HIV / AIDS vaccine, with HIV antibodies contained in the blood serum of HIV-infected people (vaccine specificity study)

The determination is carried out using indirect ELISA. The vaccine is sorbed on the solid phase (polystyrene tablets Greiner, Germany) at a concentration of 10 μg / ml per antigen. As HIV-positive samples, a standard panel of human blood serum OSO 42-28-212-02 P (samples containing antibodies to HIV1) is used. As HIV-negative samples, use a standard panel of human blood sera that do not contain antibodies to HIV1 (OSO 42-28-214-02 P). The developing reagent is a horseradish peroxidase conjugate with antibodies against human IgG. The results presented in table 3 show that when using the experimental vaccine series, positive samples were identified as positive and negative as negative, which indicates the antigenic activity of the vaccine. Close results were obtained for all series, which indicates the standardization of the vaccine preparation. Thus, it was shown that the chimeric recombinant polypeptide rec (17-24-41) in the vaccine against the HIV / AIDS virus specifically interacts with antibodies to HIV1 contained in the blood serum of HIV-infected people. A comparison of these results with the data obtained in the analysis of control collections of HIV-positive and HIV-negative samples using the commercial Peptoscrin-2 HIV antibody test system also indicates the specificity and sensitivity of the recombinant antigen included in the proposed vaccine against HIV / AIDS.

Example 16. Vaccine Safety Assessment

Preclinical trials have shown that the vaccine in accordance with the invention is a pyrogen-free drug that does not cause inflammatory reactions in animals at the injection site and morphological changes in internal organs. Pyrogenicity was determined according to MUK 4.1. / 4.2.588-96 p.52-53 [Methods of control of medical immunobiological drugs administered to people, Ministry of Health of Russia, M. - 1998, p.15-25]. According to these requirements, the drug should be pyrogen-free at a test dose of 10 μg of protein per 1 kg of rabbit weight. The tests showed the pyrogen-free drug. The vaccine does not affect the state of the central nervous system (CNS), the cellular elements of the blood, the detoxifying function of the liver, the biochemical parameters of blood, reflecting the state of protein, carbohydrate, fat metabolism, liver enzymes and the level of calcium in the blood. Sterility was determined by direct inoculation of the vaccine into sugar broth with reseeding on the 5th day. The sterility, toxicity, and pyrogenicity of the series of the HIV / AIDS vaccine were monitored according to the methods set forth in the draft FSP (Pharmacopoeial Article of the Enterprise) for the preparation. It has been shown that all three vaccine series are sterile, non-toxic and non-pyrogenic.

Example 17. The study of the immunogenic properties of the vaccine in clinical trials involving healthy uninfected HIV volunteers

The study involves healthy HIV-infected non-HIV volunteers who give written informed consent to participate in clinical trials and meet the inclusion / exclusion criteria. A vaccine at a dose of 50 μg of antigen and 500 μg of polyoxidonium is administered intramuscularly 4 times. Participants (3 people in the group) are vaccinated according to the following scheme: the second immunization is carried out 1 month after the first, the third immunization 2 months after the second and the fourth 3 months after the third immunization. Blood is collected in test tubes. It is kept for 1 hour at 37 ° C, then transferred to a refrigerator at 4 ° C for 30 minutes, centrifuged for 10 minutes (1000-1500 rpm), serum is taken.

Antibodies to the vaccine antigen are determined in individual sera. The determination is carried out using indirect enzyme-linked immunosorbent assay. The rec antigen (17-24-41), which is the active substance of the vaccine, is used as a solid-phase immunosorbent.

Rec antigen (17-24-41) at a concentration of 8 μg / ml was adsorbed on Greiner polystyrene plates (Germany) in a 0.05 M solution of carbonate-bicarbonant buffer pH 9.5, adding 100 μl of antigen solution to the well. It is kept for 24 hours at room temperature in a humid chamber. After incubation, the contents of the wells are shaken off. A series of serial binary dilutions of sera are prepared in 0.01 M phosphate-buffered saline containing 0.05% Tween-20 solution (FSB-T) and 0.02% bovine serum albumin (FSB-AT). Samples are added to the well in three parallels for each dilution, and incubated for 60 min at 37 ° C. At the end of the incubation, the wells are washed five times with PBS-T to remove unbound antibodies. The horseradish peroxidase conjugate with mouse monoclonal antibodies against human IgG is used as a detecting reagent. Conjugate in working dilution on FSB-AT contribute to the hole. Incubated for 60 minutes at 37 ° C. After this, the wells are again washed five times with FSB-T. The reaction is shown by adding to each well a 0.2% solution of orthophenylenediamine in a substrate buffer. The development of a color enzymatic reaction is stopped after a 15-minute incubation in the dark, adding 50 μl of a 10% sulfuric acid solution to each well. The reaction is recorded using a spectrophotometer reader at a wavelength of 492 nm.

The result is considered positive if the OD (optical density) in the analyzed well exceeds the OD crit., Calculated by the formula:

OP crit. = cf. value OD K (-) + 0.2,

where 0.2 is the coefficient determined by the method of statistical processing of the results of the ELISA, OP K (-) is the optical density of the HIV-negative sample from the commercial Bio-Rad New LAV Blot I kit (USA) at a 1:10 dilution.

Immunization with a vaccine causes the formation of antibodies in all subjects (table 4). Specific antibodies are determined in all volunteers after the third administration of the antigen. The maximum titer of antibodies is achieved after the fourth injection of antigen. Those. the vaccine is a good immunogen capable of inducing antibodies in vaccinated people.

Example 18. The definition of antibodies against HIV1 antigens in the serum of immunized volunteers using immunoblot

The study is carried out using the commercial test system New LAV Blot I Assay (HIV-1 Ab confirmation, Western Blot) (Bio-Rad, USA) according to the manufacturer's instructions attached to the kit. Antigenic basis of the test system are the proteins of the culture virus HIV-1, adsorbed on nitrocellulose.

Before the reaction, the strips of nitrocellulose with the individual HIV-1 proteins (strips) adsorbed on them were incubated for 5 min in a solution for diluting samples at room temperature. Next, test sera of volunteers immunized with the HIV / AIDS vaccine are added (final dilution of serum 1: 100). A negative control sample (blood serum of an HIV-negative person) and a positive control sample (blood serum of an HIV-positive person) were added to control wells at a 1: 100 dilution. Strips are kept with serum for 2 hours at room temperature. At the end of the incubation, the strips are washed 3 times with a sample dilution solution, keeping the interval between each washing for 5 minutes. After this, the conjugate solution is added and left at room temperature for 1 hour. The washing is repeated, the substrate solution is added, and incubated for 15 minutes at room temperature. The reaction is stopped by washing the strips with distilled water.

The reaction is taken into account by comparing the location of the strips on the strip incubated with the test sample, with the strip incubated with the positive sample attached to the kit.

Studies have shown that already after the second immunization, antibodies that specifically recognize cultural HIV antigens are detected in serum samples. The number of antibodies increases after the third and fourth immunization.

Studies have shown that the vaccine obtained in accordance with the described invention against HIV / AIDS as a complex preparation has the following advantages:

- the drug is completely safe to use, since it uses only highly purified recombinant poly polypeptide res (17-24-41);

- in animal experiments, the HIV / AIDS vaccine showed the ability to induce high titers of specific antibodies to HIV1 antigens;

- antibodies to the vaccine are able to neutralize HIV in cell culture;

- preclinical trials showed immunogenicity, safety and standardization of the drug;

- polymer carriers based on high molecular weight water-soluble derivatives of hetero-chain polyamines have a pronounced intrinsic immunostimulating activity, thereby increasing the immunogenicity of the vaccine;

- The first phase of clinical trials showed the safety, immunogenicity and antigenic activity of the presented vaccine.

The invention is the first domestic candidate vaccine against HIV / AIDS, which is included in clinical trials. Clinical trials of the vaccine are registered in the International AIDS Vaccine Initiative (IAVI) registry of clinical trials of anti / HIV / AIDS vaccines, and the vaccine itself has been included in the list of promising Global HIV Vaccine Enterprise candidate vaccines. To date, the vaccine has passed the first phase of clinical trials, the purpose of which was to study safety and reactogenicity and as a result of which its safety, immunogenicity and antigenic specificity were proved.

The previous descriptions and examples are given by way of illustration and do not exhaust the scope of this invention.

Claims (1)

An HIV / AIDS vaccine comprising a combination of HIV / AIDS vaccine antigens and a pharmacologically acceptable carrier, characterized in that it contains a chimeric recombinant polypeptide as a vaccine antigen of the HIV / AIDS virus, consisting of a sequentially arranged p17 fragment and a full-sized p24 protein encoded by the gene gag, and a gp41 protein fragment encoded by the env gene expressed in E. coli cells based on the plasmid vector pET15b into which a chimeric DNA fragment is inserted at the Bam Hl restriction site the recombinant polypeptide is inserted at the Hind III – Eco R1 restriction sites and the par locus is inserted, and a derivative of heterochain polyamines with a molecular weight of 50000-150000D (Polyoxidonium) in a ratio of 1:10 is used as a polymer carrier, while the chimeric recombinant polypeptide has the sequence SEQ ID NO : 4 and is a purified protein copying the amino acid sequences of HIV1 proteins: a p17 fragment, a full-sized p24 protein, and a gp41 protein fragment.

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