Strain a1ru09ru2225 of human immunodeficiency virus type 1 subtype a used for diagnosing and studying efficacy of therapeutic and vaccine preparations

FIELD: medicine.

SUBSTANCE: presented strain of human immunodeficiency virus HIV-1 A1.RU.09RU2225 is deposited in the Collection of Microorganisms of Federal State Research Institution State Science Centre Vector of Federal Service for Supervision of Consumer Rights and Human Welfare, No. V-391.

EFFECT: strain can be used for developing and improving HIV diagnostic techniques, studying the efficacy of therapeutic and preventive chemotherapeutic and vaccine preparations, and for creating a national panel of HIV-1 strains.

4 dwg, 3 ex

 

The invention relates to a strain of human immunodeficiency virus belonging to subtype a, and can be used in Virology, medicine and biotechnology. Strain is a convenient natural model and can be used to develop and improve methods of diagnosis of HIV infection, study of the effectiveness of therapeutic and preventive chemotherapy and vaccines, as well as to create a national panel of strains of HIV-1.

A high degree of genetic heterogeneity of the human immunodeficiency virus is a major challenge faced by researchers. The variability of the nucleotide structure of the virus contributes to the rapid emergence of strains resistant to neutralizing antibodies, cytotoxic T lymphocytes and antiviral drugs. For the development of various vaccines need to take into account the high variability of the viral genome. Currently in Russia there is no representative of the Bank is characterized strains of HIV-1. Therefore, we need to work on the selection, molecular biological characterization of strains of HIV-1 and create a collection of HIV-1, which is necessary to study the properties of the new vaccine and therapeutic drugs. This collection should include epidemiologically significant viral variants, reflecting heterogenes the ü HIV-1, the most typical for certain regions or at-risk groups (gay men, nosocomial infections in the hearth, drug addicts etc). According to the latest data the greatest distribution in the Russian Federation acquire viruses belonging to subtype a, which is found in most infected people. However, the domestic isolates and strains of subtype And are not described in literature.

A known strain hominis immunodeficit virus HIV-1/Russia GM-12-95 (EN-1295) subtype In subgroups lentiviridae family Retroviridae for the preparation of diagnostic and vaccine preparations (patent RF №2121502, IPC C12N 7/00, publ. 10.11.1998,) containing the following amino acid sequence region V3: CTRPNNNTRKRIHIGPGRAYFTGRIIGD IRQAYC.

However, since most prevalent in the Russian Federation acquiring the human immunodeficiency viruses type 1, belonging to subtype a, which is found in most infected people, the strain unsuitable for diagnostic purposes and to study the effectiveness of therapeutic and preventive vaccines used in Russia.

The closest analogue (prototype) are isolates of human immunodeficiency virus type 1, including a group of isolates belonging to subtypes a, obtained in Thailand (Biologic and Genetic Characterization of a Panel of 60 Human Immunodeficiency Virus Type 1 Isolate, Reprezenting Clades A, B, C, D, CRF02_AG, for the Development and assessment of Candidate Vaccines.//Jornal of virology, Mai 2005, p.6089-6101).

However, these isolates were not suitable for diagnostic purposes and to study the effectiveness of therapeutic and preventive vaccines used on the territory of the Russian Federation.

The technical result of the invention is to obtain such a strain of HIV-1, which would be suitable for diagnostic purposes and to study the effectiveness of therapeutic and preventive vaccines used on the territory of the Russian Federation.

This technical result is achieved by obtaining a new strain A1.RU.09RU2225 of human immunodeficiency virus HIV-1 subtype a, which is allocated on the territory of Russia from the patient 29 years, injecting drug users diagnosed with acute HIV infection. The strain intended for inclusion in the national panel of strains, and can also be used to study the effectiveness of therapeutic and preventive chemotherapy and vaccines, improved methods of diagnosis of HIV infection.

The inventive strain of human immunodeficiency virus HIV-1 A1.RU.09RU2225 deposited in the Collection of microorganisms fsri SRC VB "Vector" of Rospotrebnadzor number V-391 from 15.06.09,

Figure 1 shows the nucleotide sequence of the pol gene of HIV-1 strain A1.RU.09U2225, and figure 2 is the nucleotide sequence of the env gene of HIV-1 strain A1.RU.09RU2225. Figure 3 shows the dynamics of accumulation virousspecificakih protein P24 in the mixed culture of the IPC, infected with strain A1.RU.09RU2225, and figure 4 - accumulation dynamics virusspecific protein P24 in the infected strain A1.RU.09RU2225 culture cells MT-4 and U-937.

Description of the proposed strain

The new strain has a number of virological characteristics, which allows to consider it as a convenient natural model for evaluating the effectiveness of new tools for the diagnosis and research of new therapeutic and preventive drugs. The strain has a high reproductive activity and can be easily reproduced in neoplastic T-lymphocytic cells MT-4 and monocytic U-937, the products of the virus can be easily judged by a pronounced cytopathic effect and accumulation virusspecific protein P24. The strains stored at - 700C. Identification of HIV-1 strain A1.RU.09RU2225 implemented method of determining the nucleotide sequences of fragments of the genome of HIV-1 coding region of the gene pol and the main envelope protein (env gene), automated sequencing machine (ABI PRISM. Subsequent phylogenetic analysis and identification of the subtype of virus isolates was performed using the program presented on the website of Stanford University http:/hivdb.stanford.edu). Determination of tropism to isolate the cellular chemokine used was performed using available on the web sites of the programs Geno2pheno (http://coreceptor.bioinf.mpi-inf.mpg.de and WebPSSM and (http://indra.mullins.microbiol.washington.edu/webpssm).

Cultural properties. The virus strain isolated from peripheral blood lymphocytes of a patient with a diagnosis of acute HIV infection. Upon receipt of the strain used method cocultivation lymphocytes infected patient blood lymphocytes of a healthy donor, stimulated by the mitogen, and the method of infection of human neoplastic suspension transplantable cell linei MT-4 and U-937.

A reproduction. The strain of the virus replicates in peripheral blood lymphocytes, lymphoblastoid cell cultures MT-4 and U-937. Virus reproduction is accompanied by a characteristic cytopathic effect of mixed type - the death of individual cells, the formation of entities of 5-10 cells and accumulation virousspecificakih protein P24. The HIV-1 strain A1.RU.09RU2225 can be maintained for a long time when passirovannye in a mixed culture of peripheral blood lymphocytes of healthy donors, in the culture of cells MT-4 or U-937. After 4-7 days after infection of the cells of the vaccinated culture fluid of the infected cell suspension is centrifuged at 1500 rpm and frozen in solution for cryopreservation (70% Siva the otka cattle, 10% glycerol) at - 70°C. After thawing is brought in fresh cell suspension at a ratio of 1:10. Reproductive characteristics of the strain are determined by enzyme-linked immunosorbent assay for the accumulation virousspecificakih protein P24 in the model above cell cultures (figure 3, figure 4).

The infectivity of the viral strain was assessed by the accumulation virousspecificakih protein P24 in the culture fluid of 7 and 15 days of cultivation mixed culture stimulated phytohemagglutinin (PHA) of the IPC from two seronegative donors after infection of these cells growth medium, taken at 7 and 15 days of cultivation allocated isolate by making the growth medium into the culture of the IPC (1:10).

Antigenic properties. HIV-1 belongs to the family retroviridae podgroup lentiviridae. Viruses of this subgroup are enveloped RNA viruses with a particle size of about 100-150 nm. In the composition of the particles find no less than 6 major structural antigens having immunogenic properties that can be detected in infected cells using various immunological and virological methods.

Analysis of the nucleotide sequence of the genome, phylogenetic analysis and identification of the subtype of virus isolates was performed using the program presented on the website of Stanford University is one University (http://hivdb.stanford.edu). Genotyping of the obtained strain of HIV-1 A1.RU.09RU2225 conducted by analyzing the nucleotide sequences of plots genes pol and env. Data selection genetic plots was determined by the fact that their analysis allows to distinguish the main variants of HIV-1 circulating in the territory of the former USSR. For the definition and analysis of the nucleotide sequence of the pol gene was used sets "Viroseq" ("Viroseq HIV-1 Genotyping System") of the company "Celera Diagnostics" (USA), and "AmpliSens HIV genotype" company "Interlabservice (Russia). Determination of tropism to isolate the cellular chemokine used was performed using available on the web sites of the programs Geno2pheno (http://coreceptor.bioinf.mpi-inf.mpg.de and WebPSSM (http://indra.mullins.microbiol.washington.edu/webpssm).

Example 1. Genotyping of HIV-1 strain A1.RU.09RU2225. Genotyping of the obtained strain of HIV-1 A1.RU.09RU2225 conducted by analyzing the nucleotide sequences of plots genes pol and env. Data selection genetic plots was determined by the fact that their analysis allows to distinguish the main variants of HIV-1 circulating in the territory of the former USSR. For the definition and analysis of the nucleotide sequence of the pol gene using sets "Viroseq" ("Viroseq HIV-1 Genotyping System") of the company "Celera Diagnostics" (USA), and "AmpliSens HIV genotype" company "Interlabservice (Russia). For amplification of the sequence of the env gene using polymerase chain reaction (PCR) using the following oligonucleotide primers:

ED3 (5'-TTAGGCATCTCCTATGGCAGGAAGAAGCGG-3'),

ED51 (5'-TGGGACCAAAGCCTAAAGCCATGTG-3'),

ED13 (5'-AATTGTCTGGCCTGTACCGTCAGCGT-3'),

ED71 (5'-CTGTTAAATGGCAGTCTAGCA-3'),

ED8 (5'-CACTTCTCCAATTGTCCCTCA-3'),

ED32 (5'-CCTTTGAGCCAATTCCTATACATTATTGTG-3'),

ED34 (5'-GGAGGGGCATACATTGCTTGTCCTA-3').

Virousspecificakih RNA extracted from serum samples. Virousspecificakih DNA obtained by the method of reverse transcription of viral RNA and used for subsequent amplification using nested polymerase chain reaction. Determination of the nucleotide sequences carried out automatically on the device model 3130 XL firm "Aplaud Biosystems (USA).

The obtained genome sequences of HIV-1 include: for pol gene full - length nucleotide sequence of the gene of the protease and partial sequence of a gene reverse transcriptase - 1323 nucleotide residues (figure 1), for the env gene is the partial nucleotide sequence of the core protein of the virus envelope (gp120) - 732 nucleotide residues, including the V3 region (figure 2).

The obtained data show that the HIV-1 strain A1.RU.09RU2225 is a previously unknown strain of HIV-1 and belongs to the subtype of A.

Spent using Geno2pheno and WebPSSM analysis indicates that HIV-1 isolate A1.RU.09RU2225 has the greatest trapnest to the cellular chemokine used CCR5-type.

Conducted using jpHMM-HIV (http://jphmm.gobics.de the analysis showed that the isolate A1.RU.09RU2225 Hara is marked unique genetic organization of the locus pol.

Example 2. For selection of primary isolates of HIV-1 in peripheral blood mononuclear cells (IPC) from the patient is cultured with a mixed culture stimulated phytohemagglutinin (PHA) of the IPC from two seronegative donors. The selection of the IPC carried out by centrifugation of whole blood on a Ficoll gradient. PHA stimulation donor IPC spend 2-3 days (5 μg PHA/ml). For cocultivation mix 4-6" IPC cells of a patient with 8-12×106IPC donor cells in 10 ml growth medium (MS) of the following composition is RPMI-1640, 20% fetal bovine serum (red), 100 EA./ml IL-2, 300 mg/ml glutamine, 150 μg/ml of lincomycin and 100 μg/ml of gentamicin. Every 3-4 days carry out the replacement of the culture medium. Selected culture fluid examined for the content of P24 antigen and are cryopreserved at - 70°C. For 7 and 15 day cocultivation after replacing the culture medium to the remaining infected cells add 4-6" PHA-activated donor of the IPC. Virus replication was assessed by the accumulation of virousspecificakih protein P24. Virousspecificakih protein P24 detected in the culture fluid by the method of enzyme-linked immunosorbent assay using enzyme immunoassay system for the detection and confirmation of the presence of P24 antigen of HIV-1 "Vuckovic-1 P24-antigen-confirming test Fund 42-0117-5686-04 ", with sensitivity - 5 is g/ml for CCA risk, or set of reagents for immunoassay to detect antibodies to HIV-1,2 and antigen P24 HIV-1 "Combivent HIV-1,2 AH/at, THE 9398-098-23548172-2007".

Figure 3 presents the results of the accumulation of P24 in the culture fluid in the culture of the isolate of HIV-1 in a mixed, activated mitogen culture IPC, reflecting the dynamics of the reproduction of the virus. It is seen that the isolates are characterized by the average rate of accumulation of the viral protein P24. The protein concentration reaches the maximum value (more 649800 PCG/ml) on the 29th day of cultivation. Due to the fact that this indicator at 7 and 11 days of cultivation is much lower (401 PCG/ml and 4046 PCG/ml, respectively), then by the type of replication this isolate can be classified as Slow/low.

Example 3. The study of the accumulation of the virus in various cell cultures. The suspension of cells MT-4 or U-937 infect the culture fluid from an infected mixed culture IPC method joint incubation in the ratio of 5 cells per 1 ml of culture fluid for 1 h at 37°C. Centrifuged at 1000 rpm for 5 min, remove supernatant, cells resuspended in a nutrient medium RPMI-1640 with the addition of 10% fetal serum, previously inactivated by heating at 56°C for 30 minutes, 300 mg/ml Ŀ-glutamine, 80 μg/ml gentamicin and 30 mg/ml of lincomycin to a concentration of 0.5" cells in mil is litre. Vials with cell culture incubated in a thermostat at 37°C. Control of the cytopathic effect of the virus and sensitivearea carried out by the method of inverted microscopy of cell suspension in the culture bottles. Every 3-4 days carry out the replacement of the culture medium. Selected culture fluid examined for the content of P24 antigen and are cryopreserved at - 70°C. On day 10 of cultivation after replacing the culture medium to the remaining infected cells add a suspension of uninfected cell cultures in growth medium with a concentration of 1 cells/ml of virus Replication was assessed by the accumulation of virusspecific protein P24. Virusspecific protein P24 detected in the culture fluid by the method of enzyme-linked immunosorbent assay using enzyme immunoassay system for the detection and confirmation of the presence of P24 antigen of HIV-1 "Vuckovic-1 P24-antigen-confirming test Fund 42-0117-5686-04 ", with a sensitivity of 5 PG/ml for CCA risk., or set of reagents for immunoassay to detect antibodies to HIV-1,2 and antigen P24 HIV-1 "Combivent HIV-1,2 AH/at, THE 9398-098-23548172-2007".

Figure 4 presents the results of the accumulation of P24 in the culture fluid in the culture of the isolate of HIV-1 in neoplastic T-lymphocytic cells MT-4 and monocytic U-937, reflecting the dynamics rap is oductio virus. It is seen that the isolate is characterized by different speed and dynamics of the accumulation of viral protein P24 in different cell cultures. Observed 4-fold excess concentration over 3 days of cultivation on neoplastic T-lymphocytic cells MT-4 compared to the macrophage culture U-937, but further to 10 days concentration are aligned and registered with 2-fold excess concentration of the protein P24 in the culture supernatant U-937. This is probably due to different tropism strain to cell cultures of different pool of origin.

The inventive strain of HIV-1 A1.RU.09RU2225 used in the development of a national panel of genetically and biologically characterized strains of HIV-1 that have epidemiological significance on the territory of Russia (subtype And accounts for more than 90%) for standardization of the evaluation of the effectiveness of the developed vaccine and antiviral drugs. Panel to assess the effectiveness of the vaccine is created from strains isolated from patients in the acute stage of HIV infection. In addition, the panel should include genetically and biologically heterogeneous strains belonging to the same subtype of HIV-1, representing the maximum diversity possible circulating on the territory of viral variants that would allow a more adequate and correct evaluation of the antiviral effect ol the preparations on the model as close as possible to the real processes occurring in the body of an infected HIV-1 person.

The inventive strain of HIV-1 A1.RU.09RU2225 supposed to be used also in the panel to assess the effectiveness of antiviral drugs. Panel to assess the effectiveness of antiviral drugs includes genetically and biologically different HIV strains isolated from HIV-1-infected patients at different stages of the disease and including patients taking antiviral drugs. The presence of such panels in Russia will allow standardized adequate assessment of promising antiviral drugs to treat HIV, which is a topical problem for Russia, because our country has the scientific potential for drug development, but not certified tool for screening drugs for their effectiveness against HIV-1 on the model of the live virus.

In addition, the claimed strain of HIV-1 can be used as a producer of antigens which are diagnostic kits and test systems, in particular antigens of HIV-1, required for the production of immunoassay diagnostic test kits for detection of HIV infection and kits for immunoblot confirming HIV infection in newly diagnosed patients.

Strain A1.RU.09RU2225 virus immunodefi the ITA human type 1 subtype a, used for the diagnosis and study of the efficiency of therapeutic and preventive vaccines, deposited in the Collection of microorganisms fsri SRC VB "Vector" of Rospotrebnadzor number V-391 and having the nucleotide sequence of the gene l shown in figure 1 and the nucleotide sequence of the env gene, is shown in figure 2.



 

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