RU2768032C1 - GENETIC CONSTRUCT FOR GENE EXPRESSION mNG_CD4-CXCR4, RECOMBINANT PLASMID rVSV_MNG_CD4-CXCR4 AND A RECOMBINANT VESICULAR STOMATITIS VIRUS STRAIN rVSV_mNG_CD4-CXCR4, WHICH PROVIDES TARGETED VIROLYSIS OF CELLS EXPOSING HIV-1 GP120/GP41 PROTEINS TROPISM X4 ON THEIR SURFACE - Google Patents

Abstract

FIELD: genetics.

SUBSTANCE: group of inventions relates to a genetic construct for mNG_CD4-CXCR4 gene expression, a recombinant plasmid coding a cDNA copy of the genome of a replication-defective vesicular stomatitis virus, and recombinant vesicular stomatitis virus exhibiting human CD4 and CXCR4 receptors on its surface. Disclosed is a genetic construct for mNG_CD4 and CXCR4 gene expression, formed by sequences of fluorescent protein mNeonGreen and a human CD4 receptor and CXCR4 coreceptor fused through a P2A peptide and having the nucleotide sequence SEQ ID NO: 1. Disclosed is a recombinant plasmid rVSV_mNeonGreen [CD4-CXCR4], which codes human CD4 and CXCR4 receptors as part of a cDNA copy of the genome of the recombinant vesicular stomatitis virus (VSV), having size of 15260 bp, and containing said genetic construct. Also disclosed is a recombinant strain rVSV_mNG_CD4-CXCR4, containing said recombinant plasmid and providing targeted virolysis of cells, exposing HIV-1 tropism X4 gp120/gp41 proteins on their surface. Strain is deposited in the State collection of agents of viral infections, rickettsial infections of the Federal State Budgetary Institution State Science Center Vector of Federal Service for Supervision of Consumer Rights and Human Welfare, No. V-1108.

EFFECT: group of inventions provides a wider range of genetic structures for mNG_CD4-CXCR4 gene expression, recombinant plasmids and recombinant replication-defective viruses providing virolysis of cells exhibiting gp120/gp41 HIV-1 tropism X4 on their surface.

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Description

The invention relates to a genetic construct for the expression of mNG_CD4-CXCR4 genes, a recombinant plasmid encoding a cDNA copy of the genome of a replication-defective vesicular stomatitis virus, and a recombinant vesicular stomatitis virus exhibiting human CD4 and CXCR4 receptors on its surface, which makes it possible to create a candidate drug for virolysis of HIV- infected cells, and can also be a component of a model for studying targeted virotherapy of HIV infection and can be used in molecular virology, biotechnology, molecular biology, genetic engineering and medicine.

The virus is able to specifically bind to HIV-1 gp120 and reproduce in cells displaying the indicated HIV-1 X4 tropism fusion proteins on their surface, followed by virolysis of such cells.

As a result of the HIV-1 life cycle, its glycoprotein gp161 (gp120/gp41) is exposed on the surface of infected cells. The resulting recombinant virus can bind to HIV-1 infected cells through interaction with the exposed gp161 protein and enter the cytoplasm through the interaction of gp161-coreceptor (CXCR4). Infection of HIV-negative cells of the body should not occur due to the absence of fusion proteins in the resulting recombinant virus.

The need to obtain different viruses with different coreceptors (CXCR4 in this case, CCR5 in alternative cases) is explained by the existence of two major variants of HIV-1 circulating in the population [1]. Binding and fusion between the HIV virion membrane and the plasma membrane of the target cell is initiated by sequential binding of the glycoprotein gp120 (gp161 subunit) to CD4 on the cell surface and then to the specific chemokine receptor CXCR4 or CCR5 [2]. It is known that R5-tropic viruses are detected in the early stages of infection, while X4 variants are characteristic of the later stages, as a result of virus adaptation to a decreasing number of cells that have CCR5 receptors on the surface.

In 2019, the number of HIV-infected people in the world approached 38 million people, of which 67% received antiretroviral therapy (ARVT) [3]. The WHO-recommended treatment regimen significantly reduces the viral load and the likelihood of virus transmission, however, ART has side effects and contraindications, and can lead to the emergence of drug-resistant forms of HIV [4]. In this regard, the development of additional and alternative strategies for the complex treatment of HIV infection is being actively pursued. One of the promising approaches is the development and application of recombinant viruses targeting HIV-infected cells [9, 10, 15].

The human immunodeficiency virus (HIV) belongs to the genus Lentivirus, family Retroviridae, subfamily Orthoretrovirinae [5]. Mature HIV particle has a spherical shape and a diameter of 100-120 nm, has an outer lipid shell. The envelope contains 72 glycoprotein complexes consisting of gp161 protein trimers (gp120/gp41). Trimers of the surface protein gp120 are attached to the membrane by trimers of the transmembrane protein gp41.

Based on genetic characteristics and differences in viral antigens, HIV is classified into two types: HIV-1 and HIV-2. Since its inception, HIV-1 has spread throughout the world, while the distribution of HIV-2 is limited to West Africa, with isolated cases recorded in Europe, India and the United States of America [6]. HIV-2 infected people often do not clinically progress in the long term, while in most HIV-1 infected people, the development of infection leads to the acquisition of AIDS [7]. Currently, the lower transmission efficiency of HIV-2 compared to HIV-1 leads to a significant decrease in the prevalence of type 2 virus [8]. Thus, the most priority direction is the development of therapy for HIV-1 infection, also due to the fact that the receptor specificity and mechanisms of replication of HIV-1 and HIV-2 are largely similar, and HIV-1 is a more urgent problem.

A solution is known according to US patent 9610346 B2, which describes the developed recombinant vesicular stomatitis virus, which is used as the basis of a prophylactic and therapeutic agent against HIV infection. The present invention relates to recombinant vesicular stomatitis virus (VSV) vectors in which the gene encoding the VSV G surface glycoprotein is replaced by an HIV Env or a modified form of VSV G that carries epitopes from the proximal outer region of the HIV Env or is truncated at the N-terminus. a VSV G form that contains a cytoplasmic tail and G transmembrane domains, a proximal extracellular polypeptide to which HIV Env epitopes are attached. Thus, in the patent, recombinant viruses are obtained for potential use as vaccine preparations, while our invention involves the development of a therapeutic agent. The use of vesicular stomatitis virus as a vector indicates the expediency of its use in the development of virotherapy. A viral vector similar to our work is used, but the approach is fundamentally different - the G protein in this patent is replaced by various modifications of HIV-1 Env, while in our work the functional activity of the synthetic virus is provided by CD4 and CXCR4 receptors on the surface of virions and their interaction with HIV -infected cells or directly with the HIV-1 virus.

US 7,736,649 B2 proposes a method for suppressing human immunodeficiency virus infection by administering synergistic combinations of anti-CCR5 monoclonal antibodies, fusion inhibitors, and inhibitors of CD4-gp120 binding. This is a variant of the therapeutic approach to curb the development of HIV-1 infection, however, it implies a fundamentally different way, which does not include the use of viral vectors and targeted mechanisms for binding to HIV-1 gp120 on the surface of infected cells.

The invention JP 2005520482 A relates to a replication vector based on the HIV-1 genome and the use of this vector in the prevention and therapeutic treatment of viral infections (in particular HIV infections). The proposed method of use involves the use of the genome "conditionally replicating" HIV (conditionally replicating HIV-1 - crHIV) with selective advantages for packaging by providing the crHIV genome with one or more ribozymes that can cleave the wild-type HIV genome. In this invention, a modified lentiviral vector is used, which is fundamentally different from the vector based on the vesicular stomatitis virus.

The closest designs to the claimed invention were described in the international application WO 1999002657 A1 and US patent No. 7081243 B1. These documents published a generalized strategy for replacing the native fusion protein in the viral platform with the cellular receptor required for pathogenic virus to bind to the target cell. Thus, binding of the therapeutic construct to infected cells exhibiting surface proteins of the pathogenic virus on their surface is ensured. The examples provide recombinant vesicular stomatitis viruses carrying the CD4 receptor and the HIV co-receptor CXCR4 instead of the native VSV G protein on the surface of the virion.

The closest analogue (prototype) is recombinant rhabdoviruses competent for replication (US patent No. 7081243, IPC A61K 39/21, publ. 25.07.2006), which lack the functional G gene and exhibit at least one foreign polypeptide on the surface of their virions , which is a cellular receptor for another virus. Potentially can be used in the treatment of viral infections. In one embodiment, a recombinant vesicular stomatitis virus (VVS) lacking its fusion glycoprotein (G) gene and expressing instead the HIV receptor and co-receptor is used to reduce HIV titers in the blood of model subjects. The recombinant virus cannot penetrate into healthy cells, while it is able to control HIV infection in a population of T-lymphocytes by specifically infecting cells that exhibit the X4 tropism HIV-1 fusion protein on the surface, replicating and subsequent lysis of these cells.

The data presented in the patent demonstrates that rVSV specifically infects cells that transiently express HIV-1 X4 envelope glycoproteins, but does not infect non-permissive cell cultures. After primary cultures infected with X4 HIV-1 were inoculated with G-complementary VSVAG-CC4, rVSV significantly reduced the number of HIV-1-infected cells, probably through VSV-mediated cytolysis and/or through syncytium formation or through dependent from cell fusion apoptosis and markedly inhibited the production of HIV-1. These results indicate that cytolytic rVSV targeting X4 HIV-1-infected cells eliminates them and potentially has therapeutic value for the control of X4 HIV-1 infection, which was also shown later in articles [9].

Significant differences from this prototype, ensuring the achievement of a technical result, are the following points:

1. A genetic construct for gene expression was constructed, including a marker fluorescent protein and an open reading frame of the CD4 gene fused to it through the P2A peptide, and then the CXCR4 gene, which ensures the detection of penetration into the cell and reproduction of recombinant viruses due to the fluorescence of the mNeonGreen protein. The P2A peptide in the construct provides for the cleavage of the mNG protein at the translation stage, followed by independent synthesis and folding of the CD4 and CXCR4 proteins;

2. In the prototype, the production of the produced rVSV was carried out in a BHK-G cell culture expressing protein G. In our case, the cultivation of the recombinant virus was carried out on transgenic cell cultures that stably express the Env gene of Russian HIV-1 isolates of X4 tropism, which ensures greater functional stability of the resulting virus . Also, it may be necessary for practical use with circulating epidemiologically significant HIV-1 variants;

3. Known patents-analogues describe the production of viruses in cell cultures previously infected with a strain of vaccinia virus Western Reserve, which encodes bacteriophage T7 RNA polymerase. In the claimed invention, the production of recombinant viruses is provided using a plasmid construct that provides the expression of DNA-dependent RNA polymerase. This approach excludes contamination of the preparation with vaccinia during the first cultivation cycles;

4. Our invention provides for the possibility of replacing the fluorescent protein gene with open reading frames for therapeutic antibody genes or a CRISPR/Cas9 system targeting the HIV-1 genome.

Disclosure of invention

The technical result of the claimed invention is the expansion of the range of genetic constructs for the expression of the mNG_CD4-CXCR4 genes, recombinant plasmids and recombinant replication-defective viruses that provide virolysis of cells exhibiting on their surface gp120/gp41 HIV-1 tropism X4.

The specified technical result is achieved by obtaining a genetic construct for the expression of the mNG_CD4 and CXCR4 genes, formed by the sequences of the mNeonGreen fluorescent protein and fused through the P2A peptide of the CD4 receptor and the human CXCR4 co-receptor and having a nucleotide sequence (SEQ ID NO 1).

The specified technical result is also achieved by creating a recombinant plasmid rVSV_mNeonGreen [CD4-CXCR4], encoding the human CD4 and CXCR4 receptors in the cDNA of a copy of the genome of the recombinant vesicular stomatitis virus (VVS), having a size of 15260 bp, containing, in accordance with the physical and genetic map shown in FIG. 1, a target genetic construct for the expression of the mNG_CD4-CXCR4 genes according to claim 1 and consisting of the following fragments:

• T7 promoter - nucleotide sequence of the bacteriophage T7 promoter, necessary for in vitro transcription of the antigenome sequence of RNA of the recombinant vesicular stomatitis virus;

• N - open reading frame of the N gene encoding the nucleoprotein;

• P - open reading frame of the P gene encoding a phosphoprotein;

• M - open reading frame of the M gene encoding the matrix protein;

• mNeonGreen - bright monomeric yellow-green fluorescent protein derived from LanYFP and having the amino acid sequence of SEQ ID NO 2;

• P2A - 2A peptide from the polyprotein of teshovirus-1, which provides cleavage of the polyprotein at the stage of translation;

• CD4 - gene encoding CD4 glycoprotein and having the amino acid sequence of SEQ ID NO 3;

• CXCR4 - gene encoding chemokine receptor C-X-C type 4 and having the amino acid sequence of SEQ ID NO 4;

• L - open reading frame of the L gene encoding RNA-dependent RNA polymerase;

• T7 terminator - terminator of bacteriophage T7 bacteriophage RNA polymerase transcription;

• β-lactamase gene under the control of a promoter, a genetic marker that determines resistance to β-lactam antibiotics in E. coli bacteria cells;

• Origin of replication ori of the plasmid vector ColE1/pMB1/pBR322/pUC with high copy number.

The specified technical result is achieved by obtaining a recombinant strain of the vesicular stomatitis virus rVSV_mNG_CD4-CXCR4, obtained using the recombinant plasmid rVSV_mNeonGreen [CD4-CXCR4] according to claim 2. The virus exposes human CD4 and CXCR4 receptors on its surface, provides virolysis of cells exhibiting gp120 on its surface /gp41 HIV-1 tropism X4. The rVSV_mNG_CD4-CXCR4 strain was deposited in the State Collection of Causative Agents of Viral Infections, Rickettsiosis FBSI SRC VB "Vector" of Rospotrebnadzor under the number V-1108.

In the recombinant vesicular stomatitis virus (VSV), the gene encoding the VVS G surface glycoprotein was replaced with an artificial genetic construct for the expression of the mNG_CD4-CXCR4 genes, which includes the mNeonGreen fluorescent protein sequences and the gene for the CD4 receptor and one of the two key co-receptors of HIV-1 fused through P2A peptide CXCR4 in another viral transcription cassette (SEQ ID NO 1). In this case, the mNeonGreen gene is a marker. The P2A peptide in this construct ensures the cleavage of the mNG protein at the translation stage, followed by independent synthesis and folding of the CD4 protein. This strategy provides a higher stability of the target transgene due to the fact that the occurrence of any mutations leading to a reading frame shift excludes the possibility of synthesizing the viral proteins required for a full reproductive cycle, located after the P2A peptide. The location of the CXCR4 gene in the next viral transcription cassette from the 3' end of the genome provides the required lower level of coreceptor expression relative to CD4.

Implementation of the invention

The essence of the claimed invention is illustrated by the following drawings. Fig. 1 - genetic and physical map of the recombinant plasmid vector rVSV_mNeonGreen [CD4-CXCR4] for reverse genetics of the replication defective vesicular stomatitis virus.

Fig. 2 is a scheme for obtaining a CD4-CXCR4 product using primers that provide overlapping sequences of CD4 and CXCR4 gene fragments for subsequent PCR with overlap extension (overlap extension PCR in the scheme);

Fig. 3 is a scheme for cloning the CD4-CXCR4 OL PCR product into the VSV reverse genetics target vector pStem-rVSV-mNG;

Fig. 4 - amplicons of human CD4 and CXCR4 receptor genes (lanes 1,2 - CD4 gene, lanes 3, 4 - CXCR4 gene)

Fig. 5 - PCR products, 1411 bp long. (CD4) - bands 1.2; 1108 b.p. (CXCR4) - bands 3, 4 obtained by amplification from plasmids pJET1.2_CD4 and pJET1.2_CXCR4 using primers that provide partial overlap in the CD4 and CXCR4 sequences in order to further set up PCR with overlapping primers;

Fig. 6 - combination of CD4+CXCR4 receptor genes by PCR with overlapping primers, product size 2491 bp;

Fig. 7 - restriction analysis of the recombinant plasmid rVSV_mNeonGreen [CD4-CXCR4], M - marker;

Fig. 8 - (A, B) 24 hours after infection of HEK293_Env_X4 cells with rVSV_mNG_CD4-CXCR4 virus; (C, D) 24 hours after infection of HEK293_Env_R5 cells with rVSV_mNG_CD4-CXCR4 virus. A, C - light microscopy, B, D - fluorescence microscopy, scale scale - 300 μm.

Initially, cDNA copies of the human CD4 and CXCR4 receptor genes were obtained and subcloned into the commercial pJET1.2/blunt vector. Further, the CD4 and CXCR4 receptor gene fragments were combined, which became possible due to their amplification from plasmids using primers providing overlapping sequences for subsequent PCR with overlapping primers (overlap extension PCR in the scheme).

The resulting overlap extension PCR (OL PCR) product was digested with restriction endonucleases ApaI and SbfI and cloned into the reverse genetics vector for VVS pStem-rVSV-mNG at these sites. In the structure of the resulting recombinant plasmid rVSV_mNeonGreen [CD4-CXCR4], at this stage, a construct for the expression of the mNG_CD4 and CXCR4 genes was assembled.

Then, a replication-defective vesicular stomatitis virus rVSV_mNG_CD4-CXCR4 was obtained by transfection of a continuous cell line 4647 (collection of cell cultures of the FBSI SRC VB "Vector" of Rospotrebnadzor) and/or a transgenic HEK293 cell culture using a commercial Lipofectamine 3000 reagent (Thermo Scientific, USA) according to manufacturer's recommendations. Transfection was carried out with the obtained recombinant plasmid rVSV_mNeonGreen [CD4-CXCR4] together with helper plasmids pStem-vN, pStem-vP, pStem-vL, which ensure the expression of the protein replicative machinery of the vesicular stomatitis virus (N, P and L), as well as DNA-dependent RNA- bacteriophage T7 polymerase.

The resulting virus was used to infect transgenic monoclonal cell cultures HEK293_Env_R5 and HEK293_Env_X4, which exhibit on their surface gp161 HIV-1 tropism R5 or X4, respectively. Using light and fluorescence microscopy, the fusion of the obtained synthetic viruses with transgenic cell cultures stably expressing gp161 HIV-1 of X4 tropism on their surface and the absence of virus interaction with the HEK293_Env_R5 cell culture were functionally confirmed.

The implementation of the invention is confirmed by the following examples.

Example 1. Obtaining cDNA copies of human CD4 and CXCR4 receptor genes

The first step in obtaining a genetic construct for the expression of the mNG_CD4 and CXCR4 genes and a plasmid construct for VSV reverse genetics containing the CD4 and CXCR4 receptor genes was the amplification of the CD4 and CXCR4 receptor genes. PCR was performed with a cDNA copy of total RNA isolated from the leukocyte fraction of the blood of a healthy donor.

Primer selection (Table 1) was performed using SnapGene v.3.2.1 and Oligo7 software, using Homo sapiens CD4 molecule (CD4), transcript variant 1, mRNA (GenBank sequence: NM 000616.4), Homo sapiens CC motif chemokine sequences as a reference receptor 5 (gene) (CCR5), transcript variant A, mRNA mRNA (GenBank sequence: NM_003467.2). Oligonucleotide primers were synthesized by the Evrogen biotechnology company (Moscow).

1.1. Isolation of total RNA from the leukocyte fraction of the blood of a healthy donor and setting up the reverse transcription reaction

Total RNA was isolated from the leukocyte blood fraction of a healthy donor; cells were resuspended in AVL lysis buffer. A commercial QIAamp Viral RNA Mini Kit (Qiagen, Germany) was used for isolation in accordance with the manufacturer's recommendations. The eluate was stored at minus 20°C; in the next stage of the work, it was used for reverse transcription.

To obtain the first DNA strand on the viral genomic RNA template, the RNAscribe RT kit (Biolabmix, Russia) was used. To carry out the reaction, a test tube with a mixture of necessary components was placed in a Thermal Cycler C1000 amplifier (BioRad, USA) and the temperature-time profile recommended by the manufacturer was used.

1.2. PCR amplification of cDNA fragments encoding human CD4 and CXCR4 receptor genes

The CD4 gene was amplified with primers F-hCD4_XmaI and R-hCD4_SbfI, each of which created additional restriction sites in the PCR product, which were subsequently used for targeted cloning into the target VSV reverse genetics vector. The primer annealing temperature was 60°C. The duration of the elongation is 45 seconds. Number of cycles - 25. PCR mixture was separated in 1.5% agarose gel. Product, 1400 bp long. (Fig. 4 - lanes 1, 2) was excised from the gel and eluted using the QIAquick Gel Extraction Kit (Qiagen, Germany).

The CXCR4 gene was amplified with primers F-hCXCR4_NheI and R-hCXCR4_SbfI, each of which created additional restriction sites in the PCR product, which were then used for directed cloning into the target VSV reverse genetics vector. The primer annealing temperature was 60°C. The duration of the elongation is 30 seconds. Number of cycles - 25. PCR mixture was separated in 1.5% agarose gel. Product, 1090 bp long. (Fig. 4 - lanes 3, 4) was excised from the gel and eluted using the QIAquick Gel Extraction Kit (Qiagen, Germany).

Example 2 Subcloning of PCR products of CD4 and CXCR4 in pJET1.2/bIunt vector

The PCR products of CD4 and CXCR4 were subcloned in the pJET1.2/blunt vector using the commercial CloneJET PCR Cloning Kit (Thermo Fisher Scientific, USA) according to the manufacturer's instructions. The resulting reaction mixtures were used to transform electrocompetent cells of E. coli, strain NEB Stable. The transformants were seeded on plates with agar nutrient medium containing the selective antibiotic ampicillin at a concentration of 50 mg/mL. Petri dishes were placed in a thermostat at a temperature of 30°C for 18-24 hours. Then, overnight cultures were planted from single colonies in a volume of 3.5 ml of LB medium. The liquid LB medium contained the antibiotic ampicillin at a concentration of 50 mg/mL.

Plasmids were isolated from bacteria using the QIAprep Spin Miniprep Kit (Qiagen, Germany). A restriction analysis of the clones of the pJET1.2_CD4, pJET1.2_CXCR4 vectors was carried out, on the basis of which plasmids were selected, the observed restriction pattern of which coincided with the expected one. The correctness of the nucleotide sequences of these constructs was confirmed by Sanger sequencing using primers included in the CloneJET PCR Cloning Kit (Thermo Fisher Scientific, USA) to check for the absence of significant nucleotide substitutions, insertions and deletions.

Example 3 PCR Amplification of CD4 and CXCR4 Receptor Genes from Plasmids pJET1.2_CD4 and pJET1.2_CXCR4

Amplification of the necessary receptor genes from the pJET1.2_CD4 and pJET1.2_CXCR4 plasmids was carried out using primers that provide partial overlap in the CD4 and CXCR4 sequences in order to further set up PCR with overlapping primers.

For both PCR products, the primer annealing temperature was 60°C. The duration of elongation is from 60 to 90 seconds. Number of cycles - 25. PCR mixture was separated in 1.5% agarose gel. Products, 1411 bp long (CD4), 1108 b.p. (CXCR4) (Fig. 5) were excised from the gel and eluted using a commercial QIAquick Gel Extraction Kit (Qiagen, Germany) according to the manufacturer's instructions.

Example 4 Fusion of CD4+CXCR4 Receptor Genes by Overlap PCR

The concentration of isolated DNA fragments was measured using NanoDrop One (Thermo Fisher Scientific, USA). Then the fragments were mixed in equimolar amounts and the first stage (overlap) of cDNA synthesis of copies encoding open reading frames of CD4 and CXCR4 receptors was carried out using Phusion High-Fidelity polymerase (NEB, USA), using the temperature-time profile presented in Table 4. Second stage (extension) was carried out using flanking primers containing restriction sites XmaI and SbfI (table 1) with a given temperature-time profile (table 4).

Purification of DNA amplification products by agarose gel electrophoresis was carried out as described previously. After visualization under UV radiation, DNA fragments of the required length, separated in agarose gel (Fig. 6), were excised and isolated from the gel in a similar manner using the QIAquick Gel Extraction kit (Qiagen, Germany).

Example 5 Cloning of the resulting CD4+CXCR4 receptor amplicon into a target vector for VSV reverse genetics

In FIG. Figure 3 shows a scheme for cloning transgenes, with the formation of a genetic construct for the expression of the mNG_CD4-CXCR4 genes in the structure of the rVSV_mNeonGreen [CD4-CXCR4] plasmid.

The OL PCR product CD4-CXCR4 was digested with restriction endonucleases ApaI and SbfI (NEB, USA) and cloned into the pStem-rVSV-mNG vector according to these sites (the vector was obtained earlier in the laboratory of Vector Systems Based on Viral Genomes, FBSI SRC VB Vektor, Rospotrebnadzor ). To do this, 2 µg of plasmid DNA pStem-rVSV-mNG was hydrolyzed with restriction endonuclease ApaI (NEB, USA) in CutSmart buffer at 25°C for 1 hour, after which 10 u.a. restrictase SbfI (NEB, USA) and incubated at 37°C for 1 hour, then 2 u.a. recombinant shrimp alkaline phosphatase (rSAP) (NEB, USA), necessary to prevent the "self-ligation" of the vector, was incubated at 37°C for 1 hour. Hydrolysis products were separated by electrophoresis, DNA fragments corresponding to the hydrolyzed vector (~12800 bp) were isolated from agarose gel using a commercial QIAquick Gel Extraction Kit (Qiagen, Germany) according to the manufacturer's instructions. The nucleotide insert encoding the target transgenes was prepared in a similar manner, omitting the addition of alkaline phosphatase from the protocol.

1 μg of the hydrolyzed pStem-rVSV-mNG/ApaI+SbfI vector was mixed with the transgene DNA fragment hydrolyzed by ApaI and SbfI in an equimolar ratio, the reaction buffer and T4 DNA ligase were added. After incubation at 20° C. for 60 minutes, the ligation mixture was used to transform competent E. coli cells. Screening for transformant clones was performed using PCR methods and restriction analysis of recombinant plasmids (Fig. 7). The correctness of the nucleotide sequences of the plasmid constructs was confirmed by Sanger sequencing.

Thus, in the structure of the resulting recombinant plasmid rVSV_mNeonGreen [CD4-CXCR4], a genetic construct for the expression of the mNG_CD4-CXCR4 genes was obtained, formed by the sequences of the fluorescent protein mNeonGreen and fused through the P2A peptide of the CD4 receptor and the human CXCR4 co-receptor and having the nucleotide sequence (SEQ ID NO 1 ).

Example 6. Obtaining a replication-defective vesicular stomatitis virus rVSV_mNG_CD4-CXCR4

Replication-defective vesicular stomatitis virus was obtained by transfection of a continuous HEK293 cell line. A cell culture monolayer (90% confluence) was transfected using a commercial Lipofectamine 3000 kit (Thermo Scientific, USA) according to the manufacturer's instructions with the obtained recombinant rVSV_mNeonGreen [CD4-CXCR4] plasmid, together with the helper plasmids pStem-vN, pStem-vP, pStem-vL providing expression of the protein replicative machinery of the vesicular stomatitis virus (N, P and L), as well as DNA-dependent RNA polymerase of bacteriophage T7. The amount of each of the plasmid constructs was: pStem-N - 0.25 μg; pStem-P - 0.25 mcg; pStem-L - 1.0 µg; pStem-T7 - 1.5 mcg; rVSV_mNeonGreen [CD4-CXCR4] - 2 mcg.

One hour before transfection, DMEM/F-12 growth medium was removed in a Petri dish with HEK293 cell culture (monolayer density 90-95%) and 10.0 ml of serum-free OptiMEM medium was added. The reaction mixture was prepared, for this, 50 µl of Lipofectamine 3000 was diluted in 500 µl of OptiMEM medium, incubated for 5 min at a temperature of (22 ± 2)°C and mixed with 4 µg of total plasmid DNA, also diluted in 500 µl of OptiMEM with the addition of the recommended by the manufacturer amount of P3000 reagent. The resulting mixture was incubated for 20 minutes at a temperature of (22±2)°C.

After incubation, the resulting complexes were added dropwise to HEK293 cells evenly over the entire area of the monolayer and cultivated at (37±1)°C. A day later, the nutrient medium was changed to a maintenance medium (DMEM/F-12 with 2% fetal bovine serum), followed by cultivation in a CO2 incubator at a temperature of (37±1)°C in an atmosphere of (5.0±0.5)% CO2 for 120-168 hours.

The virus-containing culture fluid was collected in a sterile 15 ml centrifuge tube, clarified by low-speed centrifugation at 10,000 rpm for 15 minutes, aliquoted, and stored at minus 80°C until use.

The strain was identified at the FBSI SSC VB "Vector" of Rospotrebnadzor in February 2019 and deposited in the State Collection of Causative Agents of Viral Infections, Rickettsiosis of the FBSI SRC VB "Vector" of Rospotrebnadzor under the number V-1108.

The accumulation titer in the HEK293_Env_X4 cell culture of the recombinant strain of vesicular stomatitis virus rVSV_mNG_CD4-CXCR4 was up to 4.0±0.33 lg TCD50/ML.

Example 7. Infection of transgenic HEK293_Env_R5 and HEK293_Env X4 cell cultures displaying on their surface HIV-1 gp161 of one of two types of tropisms

The resulting rVSV_mNG_CD4-CXCR4 virus was used to infect transgenic cell cultures HEK293_Env_R5 and HEK293_Env_X4, which exhibit HIV-1 gp161 on their surface with tropisms for CCR5 or CXCR4 co-receptors, respectively.

The recombinant virus specifically binds to gp161 X4-tropic glycoproteins on the surface of HEK293_Env_X4 cells. Binding to receptors on the membranes of transgenic cells and penetration of the virus into the cell were detected due to the fluorescence of the mNeonGreen protein in the case of virus reproduction. Subsequent specific virolysis of transgenic HEK293_Env_X4 cells was manifested by cell fusion and destruction of the monolayer. At the same time, no changes in the monolayer were observed in the transgenic HEK293_Env_R5 cell culture upon infection with the rVSV_mNG_CD4-CXCR4 virus. The results are presented in figure 8.

The presence and functional activity of the CD4 and CXCR4 receptor genes is confirmed by the efficient reproduction of recombinant viruses in HEK293_Env_X4 transgenic cell lines mimicking HIV-1 infected X4 tropism cells (a safe model of HIV-infected cells).

When studying the cultural properties, it was found that the resulting recombinant virus exhibits tropism for gp161 HIV-1 X4-expressing cells. The recombinant virus is unable to bind and reproduce in eukaryotic cells that do not exhibit HIV-1 X4 tropism on the gp161 surface. The nature of the cytopathic action during reproduction is the fusion of cells with the formation of syncytium, followed by the destruction of the monolayer.

Example 8. Determination of the accumulation titer of the replication defective virus rVSV_mNG_CD4-CXCR4

The infectious activity of the recombinant vesicular stomatitis virus was evaluated using classical virological titration methods in sensitive HEK293_Env_X4 cell cultures expressed at 50% tissue cytopathic doses (TCD50).

Sequential tenfold dilutions were prepared from vaccinated material in DMEM/F-12 nutrient medium from 10 ^-1 to 10 ^-8 . Growth medium was removed from 96-well plates (Corning Costar, USA) with a daily culture of transgenic HEK293_Env_X4 cells, and each dilution was added in 100 μl to 8 wells of a 96-well culture plate. An intact cell culture was used as a control, into which a maintenance medium (DMEM/F-12 with 2% fetal bovine serum) was added. Tablets with cell culture were incubated in CO ₂ -incubator at 37°C in an atmosphere of 5.0% CO ₂ . At the end of cultivation in each well, the presence or absence of virus reproduction was assessed by the presence or absence of mNeonGreen fluorescent protein expression. The titration results were recorded on the 4th day after the reaction was set up.

The calculation of the infectious activity of the virus was determined as the number of 50% tissue cytopathic doses (TCD50) in 1 ml according to the method of Reed and Mench. The accumulation titer in the HEK293_Env_X4 cell culture of the recombinant strain of vesicular stomatitis virus rVSV_mNG_CD4-CXCR4 was up to 4.0 lg ± 0.33 lg TCD50/ML.

Example 9 Cultivation of recombinant vesicular stomatitis virus rVSV_mNG_CD4-CXCR4

The recombinant vesicular stomatitis virus rVSV_mNG_CD4-CXCR4 was cultivated in a transplantable transgenic cell line HEK293_Env_X4. The growth medium was removed from the culture flasks with the formed cell monolayer, and the cell monolayer was washed twice with Hanks' solution. The multiplicity of infection when infecting cells was 0.1-0.00001 TCD50/cell. A supporting nutrient medium (DMEM/F-12 with 2% fetal bovine serum) was added, followed by cultivation in a CO ₂ incubator at 37°C in an atmosphere of 5.0 CO ₂ for 48-72 hours. Collection of virus-containing culture fluid was carried out at 75% destruction of the cell monolayer. Further frozen and stored until use at a temperature of -80°C.

Thus, the claimed technical result is the expansion of the range of genetic constructs for the expression of the mNG_CD4-CXCR4 genes, recombinant plasmids and recombinant replication-defective viruses that provide virolysis of cells exhibiting on their surface gp120/gp41 HIV-1 tropism X4. The implementation is confirmed by examples 1-8 presented in the description of the invention.

Sources of patent and scientific and technical information:

1. Zaitseva M.V. et al. CXCR4 and CCR5 on human thymocytes: biological function and role in HIV-1 infection // The Journal of Immunology. - 1998. - T. 161. - No. 6. - C. 3103-3113.

2. Moore J.P. et al. The CCR5 and CXCR4 coreceptors-central to understanding the transmission and pathogenesis of human immunodeficiency virus type 1 infection // AIDS research and human retroviruses. - 2004. - T. 20. - no. 1. - C. 111-126.

3. www.who.int (World Health Organization)

4. Kemnic T.R., Gulick P.G. HIV antiretroviral therapy // StatPearls [Internet]. - StatPearls Publishing, 2019.

5. Blood G.A.C. et al. Human immunodeficiency virus (HIV) // Transfusion Medicine and Hemotherapy. - 2016. - T. 43. - No. 3. - C. 203.

6. Visseaux B. et al. Hiv-2 molecular epidemiology // Infection, Genetics and Evolution. - 2016. - T. 46. - C. 233-240.

7. Nyamweya S. et al. Comparing HIV-1 and HIV-2 infection: Lessons for viral immunopathogenesis //Reviews in medical virology. - 2013. - T. 23. - no. 4. - C. 221-240.

8. Campbell-Yesufu O.T., Gandhi R.T. Update on human immunodeficiency virus (HIV)-2 infection // Clinical infectious diseases. - 2011. - T. 52. - no. 6. - C. 780-787.

9. Okuma K. et al. A recombinant vesicular stomatitis virus encoding CCR5-tropic HIV-1 receptors targets HIV-1-infected cells and controls HIV-l infection // Microbes and infection. - 2017. - T. 19. - no. 4-5. - C. 277-287.

10. Schnell M.J. et al. Construction of a novel virus that targets HIV-1-infected cells and controls HIV-l infection // Cell. - 1997. - T. 90. - no. 5. - C. 849-857.

11. Liu Y. et al. Broadly neutralizing antibodies for HIV-1: efficacies, challenges and opportunities // Emerging microbes & infections. - 2020. - T. 9. - no. 1. - C. 194-206.

12. W. Hu, R. Kaminski, F. Yang et al., "RNA-directed gene editing specifically eradicates latent and prevents new HIV-1 infection," Proceedings of the National Academy of Sciences of the United States of America, vol. . 111, no. 31, pp. 11461-11466, 2014.

13. Kaminski R. et al. Negative feedback regulation of HIV-1 by gene editing strategy // Scientific reports. - 2016. - T. 6. - no. 1. - C. 1-11.

14. Lebbink R.J. et al. A combinational CRISPR/Cas9 gene-editing approach can halt HIV replication and prevent viral escape // Scientific reports. - 2017. - T. 7. - no. 1. - C. 1-10.

15. Jabbar M.A., Nayak D.P. Intracellular interaction of human immunodeficiency virus type 1 (ARV-2) envelope glycoprotein gp160 with CD4 blocks the movement and maturation of CD4 to the plasma membrane // Journal of virology. - 1990. - T. 64. - No. 12. - C. 6297-6304.

16. US patent No. 9610346.

17. US patent No. 7736649.

18. Application of Japan No. 2005520482.

19. International application WO 1999002657.

20. US patent No. 7081243, IPC A61K 39/21, publ. 07/25/2006 (prototype).

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Sequence listing

<110> Federal budgetary institution of science "State Scientific Center

virology and biotechnology "Vector" of the Federal Service for Supervision in the

protection of consumer rights and human well-being (FBSI SSC VB "Vector"

Rospotrebnadzor)

<120> Genetic construct for expression of mNG_CD4 CXCR4 genes,

recombinant rVSV_mNG_CD4 CXCR4 plasmid and recombinant virus strain

vesicular stomatitis rVSV_mNG_CD4-CXCR4, providing targeted

virolysis of cells displaying gp120/gp41 HIV-1 proteins on their surface

tropism X4.

<160> SEQ ID NO 4

<210> SEQ ID NO:1

<211> 3339

<212> PRT

<213> Artificial Sequence

<220>

<223> Nucleotide sequence of the genetic construct for expression

mNG_CD4-CXCR4 genes.

<400> 1

AACAGAGATCGATCTGTTTCCTTGACGCTAGCCGCCACCATGGTGAGCAAGGGCG 55

AGGAGGATAATATGGCCTCTCTCCCAGCGACACATGAGTTACACATCTTTGGCTC 110

CATCAACGGTGTGGACTTTGACATGGTGGGTCAGGGCACCGGCAATCCTAACGAC 165

GGTTATGAGGAGTTAAACCTGAAGTCCACCAAGGGTGACCTCCAGTTCTCCCCCT 220

GGATTCTGGTCCCTCATATCGGGTATGGCTTCCATCAGTACCTGGCCCTACCCTGA 275

CGGGATGTCGCCTTTCCAGGCTGCTATGGTCGATGGCTCCGGATACCAAGTCCAT 330

CGCACAATGCAGTTTGAAGATGGTGCCTCCCTTACTGTCAACTACCGCTACACCT 385

ACGAGGGAAGCCACATCAAAGGAGAGGCCCAGGTGAAGGGGACTGGTTTCCCCTGC 440

TGACGGTCCTGTGTGATGACCAACTCGCTGACCGCTGCGGACTGGTGCAGGTCGAAG 495

AAGACTTACCCCCAACGACAAAACCATCATCAGTACCTTTAAGTGGAGTTACACCA 550

CTGGAAATGGCAAGCGGTACAGGAGCACTGCGCGGACCACCTACACCTTTGCCAA 605

GCCAATGGCGGCTAACTATCTGAAGAACCAGCCGATGTACGTGTTCCGTAAGACG 660

GAGCTGAAGCACTCCAAGACCGAGCTTAACTTCAAGGAGTGGCAAAAGGCATTCA 715

CCGATGTGATGGGCATGGACGAGCTGTACAAGGGATCCGGCTCCGGAGCTACTAA 770

CTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCCGGGCCCAACCGG 825

GGAGTCCCTTTTAGACACTTGCTTCTGGTGCTGCAACTGGCGCTCCTCCCAGCAG 880

CCACTCAGGGAAAGAAAGTGGTGCTGGGCAAAAAAGGGGATACAGTGGAACTGAC 935

CTGTACAGCTTCCCAGAAGAAGAGCATACAATTCCACTGGAAAAACTCCAACCAG 990

ATAAAGATTCTGGGAAATCAGGGCCTCCTTCTTAACTAAAGGTCCATCCAAGCTGA 1045

ATGATCGCGCTGACTCAAGAAGAAGCCTTTGGGACCAAGGAAACTTTCCCCTGAT 1100

CATCAAGAATCTTAAGATAGAAGACTCAGATACTTACATCTGTGAAGTGGAGGAC 1155

CAGAAGGAGGAGGTGCAATTGCTAGTGTTCGGATTGACTGCCAACTCTGACACCC 1210

ACCTGCTTCAGGGGCAGAGCCTGACCCTGACCTTGGAGAGCCCCCCTGGTAGTAG 1265

CCCCTCAGTGCAATGTAGGAGTCCAAGGGGTAAAAACATACAGGGGGGAAGAC 1320

CTTCCGTGTCTCAGCTGGAGCTCCAGGATAGTGGCACCTGGACATGCACTGTCT 1375

TGCAGAACCAGAAGAAGGTGGAGTTCAAAATAGACATCGTGGTGCTAGCTTTTCCA 1430

GAAGGCCTCCAGCATAGTCTATAAGAAAGAGGGGGAACAGGTGGAGTTTCTCCTTC 1485

CCACTCGCCTTTACAGTTGAAAAGCTGACGGGCAGTGGCGAGCTGTGGTGGCAGG 1540

CGGAGAGGGCTTTCCTCCTCCAAGTCTTGGATCACCTTTGACCTGAAGAACAAGGA 1595

AGTGTCTGTAAAACGGGTTACCCAGGACCCTAAGCTCCAGATGGGCAAGAAGCTC 1650

CCGCTCCACCTCACCCTGCCCCAGGCCTTGCCTCAGTATGCTGGCTCTGGAAACC 1705

TCACCCTGGCCCTTGAAGCGAAAACAGGAAAGTTGCATCAGGAAGTGAACCTGGT 1760

GGTGATGAGAGCCACTCAGCTCCAGAAAAATTTGACCTGTGAGGTGTGGGGACCC 1815

ACCTCCCCTAAGCTGATGCTGAGTTTGAAACTGGAGAACAAGGAGGCAAAGGTCT 1870

CGAAGCGGGAGAAGGCGGTGTGGGTGCTGAACCCTGAGGCGGGGATGTGGCAGTG 1925

TCTGCTAGTGACTCGGGACAGGTCCTGCTGGAATCCAACATCAAGGTTCTGCCC 1980

ACATGGTCCACCCCGGTGCAGCCAATGGCCCTGATTGTGCTGGGGGCGTCGCCG 2035

GCCTCCTGCTTTTCATTGGGCTAGGCATCTTCTTCTGTGTCAGGTGCCGGCACCG 2090

AAGGCGCCAAGCAGAGCGGATGTCTCAGATCAAGAGACTCCTCAGTGAGAAGAAG 2145

ACTGCCAGTGTCCTCACCGGTTTCAGAAGACATGTAGCCCCATTTGACTATGAA 2200

AAAAACTAACAGTAATCAGCCACCATGGAGGGGATCAGTATATACACTTCAGATA 2255

ACTACACCGAGGAAATGGGCTCAGGGGACTATGACTCCATGAAGGAACCCTGTTT 2310

CCGTGAAGAAAATGCTAATTTCAATAAAATCTTCCTGCCCACCATCTACTCCATC 2365

ATCTTCTTAACTGGCATTGTGGGCAATGGATTGGTCATCCTGGTCATGGGTTACC 2420

AGAAGAAACTGAGAAGCATGACGGACAAGTACAGGCTGCACCTGTCAGTGGCCGA 2475

CCTCCTCTTTGTCATCACGCTTCCCTTCTGGGCAGTTGATGCCGTGGCAAACTGG 2530

TACTTTGGGAACTTCCTATGCAAGGCAGTCCATGTCATCTACACAGTCAACCTCT 2585

ACAGCAGTGTCCTCATCCTGGCCTTCATCAGTCTGGACCGCTACCTGGCCATCGT 2640

CCACGCCACCAACAGTCAGAGGCCAAGGAAGCTGTTGGCTGAAAAGGTGGTCTAT 2695

GTTGGCGTCTGGATCCCTGCCCTCCTGCTGACTATTCCCGACTTCATCTTTGCCA 2750

ACGTCAGTGAGGCAGATGACAGATATATCTGTGACCGCTTCTACCCCAATGACTT 2805

GTGGGTGGTTGTGTTCCAGTTTCAGCACATCATGGTTGGCCTTATCCTGCCTGGT 2860

ATTGTCATCCTGTCCTGCTATTGCATTATCATCTCCAAGCTGTCACACTCCAAGG 2915

GCCACCAGAAGCGCAAGGCCCTCAAGACCACAGTCATCCTCATCCTGGCTTTCTT 2970

CGCCTGTTGGCTGCCTTACTACATTGGGATCAGCATCGACTCCTTCATCCTCCTG 3025

GAAATCATCAAGCAAGGGTGTGAGTTTGAGAACACTGTGCACAAGTGGATTTCCA 3080

TCACCGAGGCCCTAGCTTTCTTCCACTGTTGTCTGAACCCCATCCTCTATGCTTT 3135

CCTTGGAGCCAAATTTAAAACCTCTGCCCAGCACGCACTCACCTCTGTGTGAGCAGA 3190

GGGTCCAGCCTCAAGATCCTCTCCAAAGGAAAGCGAGGTGGACATTCATCTGTTT 3245

CCACTGAGTCTGAGTCTTCAAGTTTTCACTCCAGCTAACCCTGCAGGCCATGCTC 3300

AAAGAGGCCTCAATTATATTTGAGTTTTTAATTTTTATG 3339

<210> SEQ ID NO:2

<211> 236

<212> PRT

<213> Artificial Sequence

<220>

<223> Amino acid sequence of mNeonGreen protein

<400> 2

MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYEELNLKSTKGD 55

LQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGASLTV 110

NYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIISTF 165

KWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKE 220

WQKAFTDVMGMDELYK 236

<210> SEQ ID NO:3

<211> 457

<212> PRT

<213> Artificial Sequence

<220>

<223> Amino acid sequence of CD4 protein

<400> 3

NRGVPFRHLLLVLQLALLPAATQGKKVVLGKKGDTVELTCTASQKKSIQFHWKNS 55

NQIKILGNQGSFLTKGPSKLNDRADSRRSLWDQGNFPLIIKNLKIEDSDTYICEV 110

EDQKEEVQLLVFGLTANSDTHLLQGQSLTLTLESPPGSSPSVQCRSPRGKNIQGG 165

KTLSVSQLELQDSGTWTCTVLQNQKKVEFKIDIVVLAFQKASSIVYKKEGEQVEF 220

SFPLAFTVEKLTGSGELWWQAERASSKSWITFDLKNKEVSVKRVTQDPKLQMGK 275

KLPLHLTLPQALPQYAGSGNLTLALEAKTGKLHQEVNLVVMRATQLQKNLTCEVW 330

GPTSPKLMSLKLENKEAKVSKREKAVWVLNPEAGMWQCLLSDSGQVLLESNIKV 385

LPTWSTPVQPMALIVLGGVAGLLLFIGLGIFFCVRCRHRRRQAERMSQIKRLLSE 440

KKTCQCPHRFQKTCSPI* 457

<210> SEQ ID NO:4

<211> 352

<212> PRT

<213> Artificial Sequence

<220>

<223> Amino acid sequence of CXCR4 protein

<400> 4

MEGISIYTSDNYTEEMGSGDYDSMKEPCFREENANFNKIFLPTIYSIIFLTGIVG 55

NGLVILVMGYQKKLRSMTDKYRLHLSVADLLFVITLPFWAVDAVANWYFGNFLCK 110

AVHVIYTVNLYSSVLILAFISLDRYLAIVHATNSQRPRKLLAEKVVYVGVWIPAL 165

LLTIPDFIFANVSEADDRYICDRFYPNDLWVVVFQFQHIMVGLILPGIVILSCYC 220

IIISKLSHSKGHQKRKALKTTVILILAFFACWLPYYIGISIDSFILLEIIKQGCE 275

FENTVHKWISITEALAFFHCCLNPILYAFLGAKFKTSAQHALTSVSRGSSLKILS 330

KGKRGGHSSVSTESESSSFHSS* 352

<---

Claims (15)

1. A genetic construct for expression of the mNG_CD4-CXCR4 genes formed by the mNeonGreen fluorescent protein sequences fused through the P2A peptide of the human CD4 receptor and the human CXCR4 co-receptor and having the nucleotide sequence of SEQ ID NO: 1. 2. Recombinant plasmid rVSV_mNeonGreen [CD4-CXCR4] encoding human CD4 and CXCR4 receptors in the cDNA of a copy of the genome of the recombinant vesicular stomatitis virus (VVS), having a size of 15260 bp, containing, in accordance with the physical and genetic map shown in Fig. . 1, a genetic construct for gene expression according to claim 1 and consisting of the following fragments: • T7 promoter - nucleotide sequence of the bacteriophage T7 promoter, necessary for in vitro transcription of the antigenome sequence of RNA of the recombinant vesicular stomatitis virus; • N - open reading frame of the N gene encoding the nucleoprotein; • P - open reading frame of the P gene encoding a phosphoprotein; • M - open reading frame of the M gene encoding the matrix protein; • mNeonGreen - bright monomeric yellow-green fluorescent protein derived from LanYFP and having the amino acid sequence of SEQ ID NO: 2; • P2A - 2A peptide from the polyprotein of teshovirus-1, which provides cleavage of the polyprotein at the stage of translation; • CD4 - gene encoding CD4 glycoprotein and having the amino acid sequence of SEQ ID NO: 3; • CXCR4 - gene encoding chemokine receptor C-X-C type 4 and having the amino acid sequence of SEQ ID NO: 4; • L - open reading frame of the L gene encoding RNA-dependent RNA polymerase; • T7 terminator - terminator of bacteriophage T7 bacteriophage RNA polymerase transcription; • β-lactamase gene under the control of a promoter, a genetic marker that determines resistance to β-lactam antibiotics in E. coli bacteria cells; • Origin of replication ori of the plasmid vector ColE1/pMB1/pBR322/pUC with high copy number. 3. Recombinant strain of vesicular stomatitis virus rVSV_mNG_CD4-CXCR4, obtained using the recombinant rVSV_mNeonGreen [CD4-CXCR4] plasmid according to claim 2, exhibiting human CD4 and CXCR4 receptors on its surface, capable of providing targeted virolysis of cells exhibiting gp120 proteins on their surface. gp41 HIV-1 troprost X4 and deposited in the State Collection of Causative Agents of Viral Infections, Rickettsiosis FBSI SRC VB "Vector" of Rospotrebnadzor under the number V-1108.

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