Self-replicating recombinant vector expressing regulatory proteins of hiv, its production method, a vaccine for immunization against hiv-based dna containing the specified vector, a method for the treatment or prevention of hiv

 

The invention relates to immunology, and more specifically to the creation of an HIV vaccine based on DNA. Proposed self-replicating recombinant vector expressing regulatory HIV proteins TAT, NEF, REV, method thereof, a vaccine for immunization against HIV-based DNA containing the specified vector, a method for the treatment or prevention of HIV. The technical result is to create a self-replicating recombinant vector expressing regulatory proteins of HIV and safe HIV vaccine. 4 N. and 8 C.p. f-crystals, 8 ill., table 2.

The scope of the invention

The invention is directed to a self replicating recombinant vector suitable for immunization against HIV using DNA. The invention is also directed to a vaccine containing the above-mentioned vector, a method of obtaining a vector and a cell host containing it. The invention also relates to the use of these vectors for the production of vaccines against HIV and to a method of treatment or prevention of HIV.

Background of invention

The interaction between vertebrates and therefore also people with pathogenic microbes, such as bacteria, fungi and viruses, is governed by way of the and the ability of the immune system to distinguish between "own" and "alien"; under normal circumstances, the immune response protects its own structure, cells and antigenic molecules of the body, but destroys the alien antigens expressed penetrated by microbes. When vertebrate, such as people infected with microbes, the immune response helps to get rid of the infection by killing microbes or cells infected by microbes or by preventing the spread of infection as a result of neutralizing antibodies. Secondly, and more importantly, the immune response, once called penetrated the body has an innate mechanism of memory, and therefore the individual who once had an infection caused by a specific microbe is often immune and may not be infected again.

This immunological memory that is called in a natural situation of the infection, is the basis for vaccines that mimic natural infection in different ways the Ideal vaccine will not cause or cause only mild symptoms in vaccinated individuals, but would nonetheless lead to the induction of immunological memory and ability to develop strong warning of the immune response, if the vaccine is faced with this germ.

Development of vacchi can destroy this particular organism and prevent subsequent infection. There are several ways in which the immune response can perform its useful function. First, antibodies, synthesized and secreted by b lymphocytes can contact the microbe and destroy them using mediated complement lysis. Secondly, neutralizing antibodies can prevent the spread of infection by inhibiting the binding of germ cells to target. Third, antibodies in combination with activation of complement can destroy infected cells, and, finally, specific cytotoxic T-lymphocytes (CTL, CTL) can kill and destroy cells infected with the microbe Believe that all of these mechanisms are involved in the immune response caused by the human immunodeficiency virus (HIV) types 1 and 2 (HIV-1 and HIV-2). However, the immune response in HIV-infected individuals is usually characterized by a strong response with the formation of antibodies and less effective or non-existent response of T-lymphocytes (Gerstott, J. et al. Scand. J. Immunol. 22(5): 463-470, 1985; Re, M. C. et al. J. Clinical Pathol. 42(5): 282-283, 1989) This may be the reason why individuals infected with HIV will develop chronic infection, despite a strong antibody-mediated immune response.

Prophylactic immune about the but know, what is the most effective CTL response capable of killing virus-infected cells. This is the reason why, generally speaking, live attenuated viral vaccines are proven to be the most effective. In fact, the first vaccine developed by Jenner more than 200 years ago, live virus vaccine that can prevent infection with variola virus, is an example of this principle. When an individual vaccinated with a live attenuated viral vaccine, infected cells of the host and are synthesized viral proteins. Some of the molecules of viral proteins are used for the formation of viral particles, while others proteoliticeski broken down into small peptides that bind with the antigens of the major histocompatibility complex (MHC) (human HLA class I and II) and presented to T lymphocytes on the surface of infected cells. Subsequently, T cells bearing the appropriate T-cell receptor (TCR), will recognize foreign peptide bound to HLA, and either will help b-cells to produce antibodies (T-cell helpers/inductors; Th), or will destroy the infected cell (cytotoxic T lymphocytes, CTL).

Despite making) and AIDS is still missing. Most early efforts were focused on obtaining sterilizing the immunity using neutralizing antibodies to the glycoprotein of the outer shell HIV gp120/gp160. Research phase I/II with gp160/gp120 showed, However, neutralization of laboratory strains, but the inability to neutralize the field isolates.

In contrast, experiments with a weakened virus was successful on the model of immunodeficiency monkeys (SIV). SIV, which delegated NEF genes or REV, behaves like a weak virus and protects the vaccinated animals against development of the disease but not against infection provoking wild-type virus. In the case of REV-defective virus, the only significant immunological correlate of protection was cellular immune (CI) response to regulatory proteins of SIV NEF and TAT. An important observation made in this experiment was that vaccinated animals could even overcome the infection caused by provoking wild-type virus. It was recently reported that HIV-infected patients can sometimes overcome the apparent infection. The only correlation with protection in these studies on animals and/or humans seems to be the cellular immune response to HIV.

This type of immune about the cult for the prevention of infection, but, theoretically, they are dangerous for several reasons. A recently described method for genetic immunization (synonyms immunization nucleic acid immunization with DNA) has several advantages compared to live attenuated vaccines, but has no potential harmful side effects. A vaccine based on DNA, in the form of a eukaryotic expression vector that carries the gene of one or more viral proteins, transfairusa cells of the host. Viral proteins synthesized in the target cell will then be broken down by proteolytic enzymes, the resulting peptides will be in contact with the molecules of the MHC/HLA and presented on the surface of transfected cells. This will cause CTL-mediated immunological memory, which, if the individual is subsequently infected with virulent wild-type virus, will effectively kill virus-infected cells immediately after infection and thus prevent infection. It was shown that direct intramuscular or intradermal injection of cDNA in eukaryotic expressing plasmid induces an immune response (Wolff et al. Science 246: 1465-1468, 1990). The expression of foreign antigens such spacechecker T-lymphocytes (CTLs) and sometimes also with a high titer of antibodies (Wang, C. Et al. PNAS 90: 4156-4160, 1993; Wang et al. Ann. NY Acad. Sci. 772: 186-197, 1995; Hayness et al. AIDS Res. Hum. Retroviruses 10 (2): 43-45, 1994). In addition, when using antigens that are nucleoproteids of the influenza a virus were described the emergence of antigen specific CTLs and protection (Ulmer et al. Science 259, 1745-1749, 1993). Moreover, protection against infection with the use of immunization with DNA was obtained in the case of Mycoplasma in mice (Barry et al., Nature 377 (6550): 632-635, 1995; Lai et al. DNA Cell Biol. 14(7): 643-651, 1995) in the case of human papilloma virus in model experiments on rabbits (Donnelly et al. J. Inf. Dis. 173(2): 314-320, 1996). Immunization with DNA was also used for the induction of antitumor immunity mediated by cytotoxic lymphocytes (Bohm et al. Cancer Immunol. Immunother. 44(4): 230-238, 1997).

Immunization with DNA has several advantages compared to immunization with live attenuated viral vaccines. Because it does not form infectious virus, viral genes introduced into the recipient organism, remain only in those cells that are initially transliterowany, and there are no symptoms of viral infection. In the case of HIV, the main theoretically possible harmful effect of live attenuated virus can be reversed by mutations in the virulent wild-type virus. the but known they efficiently induce prophylactic immune response.

For effective CTL response, it is important that cytotoxic T cells destroy infected cells before the formation of structural proteins and prior to the release of Mature viral particles. Therefore, the preferred immune response in the early proteins of the viral life cycle. The HIV replication is governed by its own regulatory genes and proteins. The HIV genome encodes three nonstructural regulatory proteins (NEF, TAT, REV), which are necessary for virus replication in vivo. REV - a protein carrying the genomic RNA in the cytoplasm, TAT regulates the transcription of the virus, and NEF provides replication in resting cells. Experiments with SIV showed that viruses, in which one of these regulatory genes is not functioning, may be unable to induce disease due to insufficient replication of the virus. These three protein in a short time and in small amounts expressed during the first h of the infectious cycle of the virus (Ranki et al. Arch. Virol. 139: 365-378, 1994). Only a small proportion of HIV-infected individuals with detectable humoral and/or cellular responses to these proteins, and this response correlates with a favorable clinical course is irout with favorable clinical prognosis. When using REV-defective SIV vaccine immune responses to NEF and TAT were protective. Were identified Th and CTL epitopes in proteins TAT and REV, which are seen in HIV-infected individuals, and they correlate with the clinical course (Blazevic et al. J AIDS 6: 881-890, 1993, Blazevic et al. AIDS Res. Hum. Retroviruses 11: 1335-1341, 1995).

When the joint consideration of these results show that for the prevention of disease may be necessary reasonable replication of the virus (reduced reproduction) in combination with specific immune responses against regulatory proteins involved in ensuring the replication of the virus.

After immunization with DNA you can use several eukaryotic expressing vector, but their effectiveness is different. Some of the parameters governing the effectiveness of a particular expressing vector in relation to the induction of an immune response is unknown, but clearly preferred must be a high level of expression of the antigenic protein. The period of time when the vector is introduced into cells, can Express the foreign antigenic viral protein may also be important. Finally, expressing the vectors that cause a certain level of cell damage, also can the biologically active molecules, such as cytokines, lymphokines, and chemokines, secreted by the cell expressing the antigenic protein. Apparently, this is another reason why a live attenuated virus that causes a certain level of destruction of tissues and cells, as effectively induces immunity, and therefore, a DNA vector, which in this respect simulates a live attenuated vaccine, may have advantages.

Nonspecific factors, such as cytokines and lymphokines, can also regulate viral replication and immune responses in HIV infection. The role of the balance of cells-helper cells Th1/Th2 reflected in the production of lymphokines, which are specific for these two populations of cells T-helper cells, was demonstrated Clerici and Shearer (Immunology Today 14(3): 107-111, 1993) and other authors. Soluble factors produced CDS-cells and can inhibit the production of HIV-infected CD4 cells, have recently been identified as RANTES, MRand MR(Cocchi et al. Science 270(5243): 1811-1815, 1995). It is possible that cytokines, products which either increases or decreases in HIV infection, regulate the transcription of the virus.

Previously performed studies on immunization using DNA using the gene code is Hinkula et al. J. Virol. Jul., 71(7): 5528-5539, 1997). However, the positive effect of CTLs correlated with a favorable clinical course.

Therefore, one of the main objectives of the present invention is the creation of a vaccine for immunization using DNA that encodes a regulatory protein of HIV, vaccines, can cause a CTL-response against HIV-infected cells during the early phase of the infectious cycle, to highlight new Mature viral particles.

Another object of the invention is to create a vaccine, which additionally causes humoral response against HIV.

Another object of the invention is to create an HIV vaccine, which is safe in use because it does not expose the recipient to the impact of the structural genes or proteins of HIV.

Another object of the invention is to create a self-replicating vector, which leads to long-term and high level of expression of regulatory proteins of HIV and a certain level of destruction of cells, which further stimulates the immune response.

Another object of the invention is to create a self-replicating recombinant vector expressing regulatory proteins of HIV, and this vector is long and steadily persists and gives a great Chi is ostoic in creating the host cell, containing the above-mentioned vector.

Another object of the invention is to provide a method for obtaining the above-mentioned self-replicating vector.

The present invention further provides a method for the treatment or prevention of HIV.

Another object of the invention is the use of the above vector to obtain an HIV vaccine for immunization using DNA.

The invention

The task of the present invention can be solved by incorporating a heterologous sequence of nucleotides that encodes a regulatory protein of HIV NEF, REV or TAT or immunologically active fragment, a vector containing the E1 gene or E2 gene of human papilloma virus, minimum source of the replication of human papilloma virus and minichromosomes the support element of the human papilloma virus.

In other words, the invention is directed to a self replicating recombinant vector containing the nucleotide sequence of the human papilloma virus, be sure to consist of

(I) gene E1 and E2 gene papilloma,

(II) the minimum of the source of replication of human papilloma virus,

(III) minichromosomes element support human papilloma virus,

heterologous nucleotide sequence that encodes an HIV-regulatory well for immunization against HIV using DNA including the above vector, using the above vector for the production of a vaccine and method for the treatment or prevention of HIV, including introduction to the needy in this person an effective amount of self-replicating vector and the expression of NEF, REV or TAT protein or immunologically active fragment of the above persons.

The invention also provides a method of obtaining self-replicating recombinant vector, the above method includes

A) the insertion of a heterologous sequence of nucleotides that encodes a regulatory protein of HIV NEF, REV or TAT or its immunologically active fragment, a vector containing the nucleotide sequence of the human papilloma virus, essentially consisting of

(1) gene E1 and E2 gene papilloma,

(2) the minimum of the source of replication of human papilloma virus,

(3) minichromosomes element support human papilloma virus,

and

B) transforming the host cell educated self-replicating recombinant vector,

B) culturing the host cells,

(G) removing the above-mentioned vector.

The invention also allows the cell host containing the above-mentioned vector.

A brief description is 177.

Fig.2 depicts the plasmid pBNtkREV according to the present invention.

Fig.3 depicts the plasmid pBNsrTAT according to the present invention.

Fig.4 depicts plasmid pBNsrNEF according to the present invention.

Fig.5 depicts the expression of NEF in cells COS-7, transfected pBNsrNEF. Samples for Western blotting were taken at 72 h after transfection and visualized using ECL.

Fig.6 shows antibodies to NEF in the serum of mice immunized pBNsrNEF, detected by Western blotting. Samples 1-4 were taken 2 weeks after the last immunization, and samples 5-8 were taken 4 weeks after the last immunization.

Fig.7 depicts the CTL responses in mice immunized with vector pBNsrNEF. Fig.7A depicts the CTL responses, expressed as % specific lysis of target cells, four mice tested two weeks after the last immunization. Fig.7B depicts the levels four weeks after the last immunization. Specific lysis, the greater of 4% is considered positive.

Fig.8 depicts the distribution of immunoglobulin subclass in three mice, immunoserological sequence of nucleotides in HIV vector, containing the gene E1 and E2 gene of human papilloma virus, minimum source of the replication of human papilloma virus (MO) and minichromosome the support element of the human papilloma virus (MIU). This vector, including E1/E2/MO/MME later in this paper will be referred to RWM, and he described in detail in a publication of the international application WO 97/24451 included in the present description by reference. The above patent publication is based on the discovery that DNA replication in viruses of the papilloma provided only MO insufficient for stable long-term persistence, and as necessary other viral sequence the MME, and that the best results occur if the vector additionally contains genes E1 and E2 of the human papilloma virus.

"Human papilloma virus" when used here means any representative of a family of viruses of the papilloma. Preferred human papilloma virus used in the invention is the human papilloma virus bull (BPV) or human papillomavirus (HPV).

"E1" and "E2" are regulatory proteins of papilloma viruses that replicate through MO and which are necessary for replication.

"Minimum source replication” (MOE) is the minimal nucleotide sequence of the virus popes the I region of the genome of human papilloma virus, with which are associated viral proteins or proteins person required for replication of human papilloma virus. MIU necessary for stable episomal support the defense of human papilloma virus in the cell host.

Preferably, the MMA contains multiple binding sites activator of transcription protein E2.

"Self-replicating vector” as used in this application means a vector plasmid capable of Autonomous replication in eukaryotic cells is the master.

"Heterologous" means alien. For example, in relation to the vectors according to the present invention the heterologous nucleotide sequence means a sequence that is not associated with papilloma.

"Immunologically active fragment” means a fragment capable of inducing an immunological response in the recipient.

"Nucleotide sequence of the human papilloma virus, necessarily consisting of” means that the vector contains the nucleotide sequence of the human papilloma virus, which are necessary and sufficient for long-term persistence of the vector and its replication. This means, for example, that the extra sequence, such as all oncogenic coding sequences papillomas were delegated from the market in accordance with the present invention include the promoters for the encoded proteins, as well as additional regulatory sequence, a polyadenylation sequence and introns. Preferably the vectors also include the source of the replication of the bacterial host cell and one or more genes selective markers to obtain the vector DNA in a bacterial cell host.

The essential feature of pBN vectors is that they are not specific for the host cell. This is due to the fact that the expression of E1 and E2 proteins is controlled by promoters, which are negativnye, that is heterologous. The above promoters or operate in a wide range of cells, or mammalian tissue, or cell - or tissue-specific.

The vectors according to the present invention E1 gene is preferably under the control of srpromoter or promoter timedancing (tk), and E2 gene is preferably under the control of the LTR promoter dad. Genes NEF, REV or TAT can be under the control of the CMV promoter or RSV LTR promoter. In addition, the vector may contain an early promoter of SV40 for the induction of gene expression for selection with the antibiotic (neomycin or kanamycin).

Source replication in the cell-host vector-soglashenie, kanamycin and/or neomycin. Preferably the intron is beta-globin IVS.

The sequence of eight nucleotides found in the plasmids based on the TK-promoter, is a non-coding sequence Artamonova protein. It has no functional significance in the plasmid, but was required to create restriction sites suitable for the final plasmids.

Genes NEF, REV or TAT to insert a pBN can be derived from several trading firms, for example, plasmid RCR, which can be purchased in store MNF AIDS Reagent Project. The above-mentioned genes are well known and have been completely sequenced (Wain-Hobson, et al. Cell 40: 9-17, 1985). Of course, you can also perform the insert sequence that encodes only immunologically active fragment of the above proteins of HIV.

Genes NEF, REV or TAT or fragments thereof is first inserted into the appropriate Shuttle vector. These vectors can contain or not contain MO area. In the examples illustrated two Shuttle vectors: pNp177 that does not contain MO, and pUE83, which contains MO. Of course, you can use other Shuttle vectors. As Shuttle vectors and destination vectors according to the present invention preferably multiplicity and 4. The vectors according to the present invention is stable and is replicated with the formation of a large number of copies. After transfection in eukaryotic cell host vector (plasmid) will multiplicitous and give 100-1000-fold the number of new plasmids, each of which is able to Express the necessary HIV protein.

A host cell that is claimed in the present invention, may be any eukaryotic cell, transtitional vector, or a prokaryotic cell transformed by the vector. Eukaryotic cell preferably is a mammal cells, and prokaryotic cell, preferably a bacterial cell, especially E. coli.

Expression of NEF, REV and TAT HIV-derived plasmid vectors according to the present invention was analyzed as in transfected cells COS-7, and in mice immunized with the above plasmids. High expression of HIV proteins could be demonstrated in cells COS-7, immunized mice also showed a marked humoral and cellular (CTL) immune responses. Pronounced CTL-response was also demonstrated in monkeys. These results show that the vectors according to the present invention, the potential of MES pBN vectors, coding of various regulatory proteins of HIV, increases the immune response through several regulatory genes. The present invention therefore includes a vaccine containing a mixture of vectors encoding various regulatory proteins of HIV or its immunologically active fragments, and the use of the above mixture in the production of vaccines for treatment or prevention of HIV. The vaccine may contain a mixture of vectors encoding all three different regulatory protein. Vaccines according to the present invention may also contain other genes or fragments of genes, for example, selected from the group consisting of the structural genes of HIV.

Description of embodiments of the invention

The present invention is further illustrated by the following examples. The examples describe certain embodiments of the invention, but should not be interpreted as limiting the invention, which is defined by the attached claims.

Example 1

Cloning REV and TAT genes of HIV-1 in the self-replicating plasmids pBNsrand pBNtk

Getting pBNtkREV and pBNsraTAT

Phase 1:

REV and TAT genes of HIV-1 isolated from BRU, also known as LAI (Wain-Hobson et al. Cell 40: 9-17, 1985), were s is Andy) and the following primers, which are the restriction sites for enzymes Xhol and b1:

For REV:

5’-TTTTTCTAGAACCATGGCAGGAAGAAGCGGA-3’

5’-TTTTCTCGAGCTATTCTTTAGTTCCCTGG-3’

For TAT:

5’-TTTTTCTAGAACCATGGAGCCAGTAGATCCT-3’

5’-TTTTCTCGAGCTAATCGAACGGATCTGC-3’

Amplificatoare genes and Shuttle vector pUE83 (Fig.3) enzyme was digested at +37With enzymes Xbal and Xhol (New England Biolabs, USA) over night to get the compatible ends. Obtained by splitting the DNA fragments were analyzed on a 1.5% agarose gel and optionally purified using a set of Band Prep Kit (Pharmacia Biotech, Sweden). Each gene ligated to the vector separately, using T4 DNA ligase (New England Biolabs, USA), during incubation over night at +16C. the ligation Products were transformed into competent cells of E. Li from a set of One Shot Kit (Invitrogen, the Netherlands), which were applied to LB-tablets containing kanamycin for selection. Minipreparation were prepared from growing clones and analyzed for the presence of the cloned genes was performed by enzymatic cleavage with the enzymes XhoI and XbaI. The presence of the cloned genes was also confirmed using polymerase chain reaction (PCR) from minipreparation using the above primers. Clones containing nornir>The DNA fragment containing BPVori, promoter, RSV LTR, REV or TAT gene and beta-globin IVS poly(A), was isolated from the vector-dropship by enzymatic cleavage enzyme Hind III (New England Biolabs, USA) and purified using a 1% agarose gel and set Band Prep Kit. Ligation selected using Hind III and dephosphorylating (alkaline phosphatase, CIP, Promega, USA) pBNsror pBNtk, transformation of cells, confirming the presence of the cloned gene and purification of plasmids was carried out as in Phase 1.

The resulting plasmids are called pBNtkREV and pBNsrTAT and shown in Fig. 2 and 3.

Example 2

Cloning of HIV-1 NEF in self-replicating plasmid pBNsr

Getting pBNsrNEF

Phase 1:

NEF gene of HIV-1 was obtained from the plasmid vector pcNEF, which contained LAI isolate NEF gene, inserted in rstat vector not containing the TAT gene. NEF gene used for further cloning was obtained as a fragment of 1.3 thousand pairs of nucleotides by enzymatic cleavage using Spe I and Hind III from pcNEF. To resolve reform site Hind III at legirovanii, after cleavage with Hind III fragment was treated farm is Genty were separated by electrophoresis in 1% agarose gel in parallel with a standard size markers. Strips of the desired size was cut out and was isolated DNA using a set of Sephaglas Bandprep Kit (Pharmacia Biotech) according to the manufacturer's Protocol.

Shuttle vector pNP177 depicted in Fig. 1, was initially digested with h I, then processed by the enzyme maple and dNTP mixture and finally were digested using b I. the Vector also worked putting in alkaline phosphatase calves (CIF).

The fragment containing the NEF gene, ligated with the cleaved vector pNP177 using T4 ligase at +14With during the night. Recruitment of competent E. coli One Shot Kit (Invitrogen) was used for transformation. Positive clones were identified using splits restricteduse enzymes and electrophoresis. Plasmid DNA is then amplified in E. coli and purified in large quantities using Qiagen columns. The resulting plasmid was named pNP177cHIVNEF

Phase 2:

Shuttle vector pNP177 designed so that it only has two sites for Hind III, between which can be cloned insert So splitting plasmids using Hind III gives a fragment that can be cloned next. Obtained using Hind III fragment pNP177cHIVNEF cloned in pBNsr. Vector RA how the selection of bands, ligation and transformation, as in the first phase, and the correct orientation of the insert confirmed by restriction analysis. The final plasmid was named pBNsrNEF, and it is shown in Fig.4.

Example 3

Demonstration of the expression of HIV NEF in vitro

3A. Transfection

To analyze the expression of pBN structures of examples 1 and 2, using the electroporation was transfusional cells COS-7. 10 mg pBN-Gal as control and 10 µg pBN-NEF, transfitsirovannykh together with 1 μg pCMV-Gal, was electroporative three million cells in each case. As a carrier used DNA from salmon sperm. Electroporation was carried out with the capacity of 960 MT and the voltage of 260 C. was measured protein concentration and activity-gal to monitor the efficiency of transfection and calibration number lysates in the analysis by Western blotting.

3B. Immunohistochemistry and Western blotting

The accumulated cells, transfetsirovannyh pBN designs, literally for use in Western-blotting After lysis, the protein samples were boiled in sample buffer and transferred in 12% of the LTO-polyacrylamide gel, then transferred to nutraceutical used a mixture of monoclonal mouse antibodies to NEF (V. Ovod Et al. AlDS 6 25-34, 1992), diluted in the ratio 1: 1000 each. The secondary antibody was biotinylated antibody to proteins in mice dilution of 1: 500.

After transfection in COS cells vectors cause severe short-term expression of the regulatory protein of HIV that were detected by Western-blotting lysed cells after 72 hours the Results obtained with pBNsrNEF, shown in Fig.5 During prolonged cultivation of the transfected cells, the expression of NEF was maintained for up to 7 weeks in cells transfected with the self-replicating pBN vector.

NEF-transfetsirovannyh cells were also used for cooking cytospin drugs, they were stained with hematoxylin and monoclonal antibodies to NEF and then biotinylated antibodies to the mouse protein were used for immunohistochemistry as described in the work Ovod et al. (see above). Cytospin-sections revealed expression as positive staining was seen in a large number of cells in the form of granules, which occupies the cytoplasm of cells. Part of expressing NEF cells found morphological signs of cell destruction, indicating apoptosis. The expression level was high, although the conditions for the cells became .<5.gif">and pBNtk in vivo

4A. Gene gun

DNA precipitiously (besieged) on gold particle diameter of 1 μm using spermidine and l2in accordance with the procedure described in the manual gene gun Helios Gene Gun (Lab Bio Rad). Were manufactured cartridges, each of which contained 0.5 mg of gold and 1 µg DNA. The amount of DNA was monitored spectrophotometrically in accordance with the instructions in the manual. Inoculation was made using the Helios Gene Gun System (Laboratory Bio-Rad). Pressure helium discharge to deliver DNA was set equal to 300 psig (about 21 kg/cm2). During the optimization of the conditions of bombardment by the authors of the present invention, it was found that 300 pounds per square inch is sufficient to advance the particles in the dermis.

4B. Immunization

Used female mice Balb/c mice aged 6-8 weeks. Before immunization mice were anestesiology and they removed the hair on the abdomen.

Inoculation of the skin on the abdomen of 8 mice were performed on days 1, 2, 3, 10, 11 and 12 using a gene gun, described above, in accordance with the manufacturer's instructions. Each mouse was injected with a total of 6 µg pBN-NEF. Four mice from about - the via 4 weeks after the last immunization. Took serum samples for Western blotting, and splenocytes collected for analysis on CTLs. All eight mice immunized with pBN-NEF vector, showed antibody responses after 2 weeks and after 4 weeks (Fig. 6). The intensity of the reactions in Western-blotting varied

4B. Measurement of cytotoxic activity of T cells from immunized mice

W. Stimulation of the cells to effector

The immunized mice two weeks (16 mice) and four weeks (16 mice) after immunization, aseptically remove the spleen. They were crushed in Hanks buffer, filtered through cheesecloth and remove the erythrocytes. Then the cells were suspensively at a concentration of 5106/ml in culture medium RPMI 1640 containing 10% fetal serum of calves (FCS, GibcoBRL), 1% glutamine, 100 U of penicillin per ml, 100 μg streptomycin per ml, and 510-5M 2-mercaptoethanol. Reactive cells (5106) were cultured in the flask for cell culture volume of 25 ml in 5 ml culture medium together with 4106antigen presenting cells (APC; see below) within five days. On the first day to the cells was added 10 Units/ml logy Jan. 90(1): 1-6, 1997; Vahlsing, H. L. et al. Journal of Immunological Methods 175: 11-22, 1994, Varkila, K. et al. Acta path. Environ. Immunol. Scand. Sect. With 95: 141-148, 1987).

W. Antigen cells

Syngeneic cells mastocytoma R (H-2d) were infected with a modified vaccinia virus Ankara (MBA), rebuilt so that it is expressed gene of HIV-1 LAI NEF (MBA-HIVNEF). MBA is strongly weakened clubreplicarolex the cowpox virus, which can serve as an effective vector for the expression of heterologous genes, providing an exceptionally high level of biological security (Sutter G. Et al. J. Virol. Jul, 68(7): 4109-4116, 1994, Sutter et al. Vaccine 12(11): 1032-1039, 1994; Drexler I. Et al. J. Gen. Virol. 79: 347-352, 1998; Sutter G. et al., Proc. Natl. Acad. Sci. USA 89: 10847-10851, 1992). Infection with MVA-HIVNEF produced when the number of infections (FS) equal to 5, 24 cell plates (1106cells on the cell). After absorption of the virus for 1 hour at +37With cells were incubated for 15 h at +37(Carmichael A. Et al. Journal of Virology 70: 8468-8476, 1996). After infection, the cells were twice washed in phosphate buffer solution (PBS) containing 10% fetal serum of calves, and suspensively this solution to a concentration of 5106cells/ml the cells are Then processed

W. Tests for cytotoxicity

The activity of CTLs was investigated using analysis of allocation51SG (Hiserodt J. Et al. J. Immunol. Jul, 135(1): 53-59, 1985; Lagrandene M. et al. J. Virol. Mar, 71(3): 2303-2309, 1997; Varkila K. et al. Acta path. Environ. Immunol. Scand. Sect. With 95: 141-148, 1987; van Baalen C. et al. AIDS 7: 781-786, 1993). In brief, 2106cells P-815 infected MBA-HIVNEF as described above for antigen presenting cells. After infection cells were once washed in culture medium containing no serum. Target cells were then suspensively in 200 µl of culture medium containing no serum, and added 100 µci51Cr (Amersham)/1106cells for 1 hour at 37C. Then the target cells four times washed in the environment and suspensively to a concentration of 5104/ml Stimulated effector cells once were washed in culture medium before adding to the target cells. Target cells were applied to 96-cell tablet in the amount of 100 μl per cell (5103) and were added to the effector cells in the form of three samples with a volume of 100 μl at a ratio of effector: target equal to 50, 25 and 12.5. For spontaneous selection of target cells nanos is quickly turned, were incubated for 4 h at 37With, and the number of decays in supernatant counted using a gamma counter. The percentage of specific lysis of target cells was calculated as (selection51IG analysis - spontaneous release)/(maximum release - spontaneous release)X100. The percentage of specific lysis greater than or equal to 6% was considered positive.

Example demonstrating the activity of CTLs in 6 of 8 mice immunized pBNsrNEF, is shown in Fig.7.

, Humoral immune response in immunized mice

To analyze the formation of antibodies to HIV-1 NEF from immunized mice, serum NEF protein were subjected to electrophoresis on a PAGE, transferred to nitrocellulose filters, and the reactivity of the antibody was determined as described above.

A summary of the results of

The results with transfection and immunization are summarized in table.1. Immune response in immunized mice was assessed by Western blot turns (Western blotting) for humoral response and analysis on cytotoxic T-lymphocytes (CTLs) for cellular immunity. As can be seen from the table.1, all eight mice immunized with expressing NEF vector, discovered and what has prodemonstrirovali, that immunization with DNA with the use of self-replicating expression vector, in accordance with the description, can cause easily detectable CTL response in mice. In addition, there was obtained the humoral immune response. In light of the above results it can be assumed that the plasmid pBN-NEF, pBN-REV and pBN-TAT can Express NEF, REV and TAT in vivo in sufficient quantity to induce both humoral and cellular immune response necessary for the prevention and treatment of HIV.

Example 5

Measurement of response n|Th-type mice immunized by intramuscular injection

Humoral immune response detected in mice immunized with pBN constructs expressing regulatory proteins of HIV, was analyzed for specificity subclass of immunoglobulins. It is well known that the response with the release of antibodies, which are dominated by lgG2a subclasses of immunoglobulins, characteristic Th1-type cellular immune response, whereas lgG1, lgG2b and lgG3 characteristic of Th2 dysbalance-cell response. Moreover, it is known that the responses of Th1-type induce and promote cellular cytotoxic immune responses (CTL-responses), whereas Th2 dysbalance-the answer is to induce antibody responses, but less active C is from example 2 in the next two weeks, the total number of DNA therefore amounted to 144 mcg, and serum was analyzed for antibodies by Western blotting. Three of the four mice had antibodies to HIV-1 Nef, and a subclass of these antibodies were determined using enzyme-linked immunosorbent assay (ELISA) as follows.

Antigen pipette was applied to plates Nunc Maxi Sorb for incubation overnight at +4C; antigen used was HIV-1 Nef protein (NIH AIDS Research and Reference Reagent Program) in phosphate buffer (50 ng/cell). Cup after incubation over night blocked with 1% serum albumin of bovine (BSA, Sigma) and then incubated with serum of mice (diluted in the ratio 1: 100 blocking solution) for 4 h at room temperature. Cups were washed in phosphate buffer/ 0,1% Tween-20 three times and then phosphate buffer twice. As secondary antibodies used conjugated with peroxidase antibodies against antigens of the mouse lgG1, lgG2a, lgG2b, lgG3 and IgM (Calbiochem), diluted in the ratio 1: 1000 with blocking solution, and the cups were incubated for 2 h at room temperature. After washing stages, performed as described above, was added to the substrate ABTS (Sigma) and N2O2in citrate buffer for 10 min and proizvoditeley in Fig.8. The highest response in the three mice was detected for lgG2 secondary antibodies (absorbance at a wavelength of 405 nm 0,117, 0,262, 0,743 respectively), the response to antibody lgG1 was significantly lower (And(405) 0,004, 0,020, 0,038). This showed that the type of response in these mice after intramuscular immunization is largely a response Th1-type, leading to cellular immune response.

The results show that almost all mice had a strong response lgG2 type against recombinant NEF, whereas the concentration of antibodies representing other IgG subclasses was very low Results further showed that pBN NEF design is able to provoke a response Th1-type and as a consequence, a strong cellular immune response that can destroy HIV-infected cells during the early phase of the viral infectious cycle.

Example 6

The generation of cellular immune response in monkeys of Massa fasciculans pBN constructs expressing regulatory proteins of HIV-1

Experiments with mice have clearly shown that pBN NEF, pBN REV and pBN TAT designs can cause a CTL response in immunized mice. There was conducted an additional experiment with nonhuman primates to ensure that designs can be used as pricelinemortgage primates, are genetically closer to humans and which can be infected with the respective retrovirus Primate SIV, which is closely related to HIV-1 and HIV-2, infecting people. Therefore, there was conducted an experiment in which monkeys of Massa fascicularis were immunized pBN constructs expressing regulatory proteins of HIV-1 NEF, REV and TAT. These proteins were obtained as described in examples 1 and 2. Three monkeys of Massa fascicularis were immunized with a mixture of pBN NEF, pBN REV and pBN TAT. Three monkeys served as controls. Immunization schedule was as follows.

Monkeys were immunized with a total number equal to 300 μg, pBN-NEF, pBN-REV and pBN-TAT (100 μg of each design) twice. The first immunization was produced in the deltoid muscle, and the second (after 2 weeks) produced intradermal Analysis of cytotoxic T-lymphocytes were performed two months after the last immunization, as described in example S. The results were as follows.

One of the three monkeys was pronounced CTL-response against autologous b-cells expressing NEF and TAT of HIV-1. The results demonstrate that not only mouse and primates can be immunized pBN constructs expressing regulatory proteins of HIV, and immunosera utstein cytotoxic T-lymphocytes, which is able to destroy HIV-infected cells during the early phase of the viral infectious cycle. Moreover, the results show that the design can be entered simultaneously in the form of a mixture and that the presence of one design in a mixture interferes with the immune response generated by other construction.

Claims

1. Self-replicating recombinant vector expressing regulatory HIV proteins containing nucleotide sequence of the human papilloma virus, essentially consisting of (I) gene E1 and E2 gene papilloma, (II) the minimum of the source of replication of human papilloma virus, (III) minichromosomes element support human papilloma virus and the heterologous sequence of nucleotides that encodes a regulatory protein of HIV NEF, REV or TAT or its immunologically active fragment.

2. Self-replicating vector for p. 1, wherein the human papilloma virus is a virus of papilloma of the bull (big).

3. Self-replicating vector according to any one of paragraphs.1 and 2, characterized in that the heterologous nucleotide sequence encodes a protein of HIV-1 NEF.

4. Self-replicating vector according to any one of paragraphs.1-3, wherein E1 is under the control of promotora fact, he is a pBNtkREV, pBNsrTAT or pBNsrNEF, is shown in Fig.2, 3, or 4.

6. Vaccine for immunization against HIV-based DNA containing at least one self-replicating vector as defined in any of paragraphs.1-5.

7. The vaccine under item 6, characterized in that it contains a mixture of vectors encoding various regulatory HIV proteins selected from NEF, REV or TAT, or immunologically active fragments.

8. A method of obtaining self-replicating recombinant vector as defined in any of paragraphs.1-5, includes the following steps: A) inserting a heterologous sequence of nucleotides that encodes a regulatory protein of HIV NEF, REV or TAT, or immunologically active fragment, a vector containing the nucleotide sequence of the human papilloma virus, comprising (I) a gene E1 and E2 gene papilloma, (II) the minimum of the source of replication of human papilloma virus, (III) minichromosomes element support human papilloma virus, (B) transforming the host cell obtained self-replicating vector, B) cultivating the host cell, and G) removing the above-mentioned vector.

9. The method according to p. 8, characterized in that a host cell is an E. coli cell.

CTB vaccine for immunization against HIV DNA-based.

11. Method for the treatment or prevention of HIV, including introduction to the needy in this person an effective amount of at least one self-replicating vector as defined in any of paragraphs.1-5, and the expression of protein NEF, REV or TAT or its immunologically active fragment of the above persons.

12. The method according to p. 11, including the introduction of a mixture of vectors encoding various regulatory HIV proteins selected from NEF, REV or TAT, or immunologically active fragments.

 

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