RU2232815C2 - Self-replicating recombinant vector expressing hiv regulatory proteins, method for it preparing, dna-base vaccine containing indicated vector for immunization against hiv, method for hiv treatment or prophylaxis - Google Patents

Abstract

FIELD: molecular biology, medicine, immunology, vaccines.

SUBSTANCE: invention relates to the development of DNA-base vaccine against HIV. Invention proposes self-replicating recombinant vector expressing HIV regulatory proteins: TAT, NEF, REV, a method for it preparing, DNA-base vaccine containing indicated vector for immunization against HIV, method for HIV treatment or prophylaxis. Invention provides the development of a self-replicating vector expressing HIV regulatory proteins and a safety vaccine against HIV.

EFFECT: improved treatment and prophylaxis method, valuable medicinal properties of vaccine.

12 cl, 8 dwg, 2 tbl, 6 ex

Description

Field of 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 vector, a method for producing the vector, and a host cell containing it. The invention also relates to the use of the above vectors for the production of an HIV vaccine and to a method for treating or preventing HIV.

BACKGROUND OF THE INVENTION

The interaction of vertebrates and therefore also people with pathogenic microbes, such as bacteria, fungi and viruses, is regulated by the ability of the vertebral body to carry out an immune response against the penetrated microbe. This reaction is based on the ability of the immune system to distinguish between "one's own" and "another's"; in a normal situation, the immune response spares its own structures, cells and antigenic molecules of the body, but destroys the foreign antigens expressed by penetrated microbes. When a vertebral, such as a person, is infected with microbes, the immune response helps to get rid of the infection by killing the microbes or cells infected by the microbes or by preventing the spread of the infection as a result of neutralizing antibodies. Secondly, and more importantly, the immune response once triggered by an invaded organism has an innate memory mechanism, and therefore an individual who has once suffered an infection caused by a specific microbe is often immune and may not be infected again.

This immunological memory, caused naturally in an infection, is the basis for vaccines that mimic a natural infection in many ways. An ideal vaccine will not cause or only cause mild symptoms in vaccinated individuals, but nevertheless will lead to the induction of immunological memory and the ability to develop a strong warning the immune response if the vaccine encounters this microbe.

The development of a vaccine against a specific microbe depends on the mechanism by which the body during a natural infection can destroy this specific organism and prevent subsequent infections. There are several ways in which an immune response can fulfill its beneficial function. First, antibodies synthesized and secreted by B-lymphocytes can bind to the microbe and destroy them by complement-mediated lysis. Secondly, neutralizing antibodies can prevent the spread of infection by inhibiting the binding of the microbe to the target cell. Thirdly, antibodies in combination with complement activation can destroy infected cells, and finally, specific cytotoxic T lymphocytes (CTLs, CTLs) can kill and destroy cells infected with a microbe. It is believed that all these mechanisms are involved in the immune response caused by the virus. human immunodeficiency (HIV) type 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 a less effective or completely absent response of T-lymphocytes (Gerstott, J. et al. Scand. J. Immunol. 22 (5): 463-470, 1985; Re, MC et al. J. Clinical Pathol. 42 (5): 282-283, 1989) This may be the reason why individuals infected with HIV develop a chronic infection despite a strong antibody-mediated immune response.

The prophylactic immune response to a viral infection, in general, can be mediated by the four types of immune responses described above, but it is known that the CTL response that can kill virus-infected cells is the most effective. This is the reason why, generally speaking, live attenuated viral vaccines have been proven to be most effective. In fact, the first vaccine developed by Jenner more than 200 years ago, a live virus vaccine that can prevent smallpox virus infection, is an example of this principle. When an individual is vaccinated with a live attenuated viral vaccine, host cells become infected and viral proteins are synthesized. Some of the molecules of viral proteins are used to form viral particles, while others are proteolytically cleaved into small peptides that bind to antigens of the major histocompatibility complex (MHC) (in humans, HLA class I and II) and appear to T cells on the surface of infected cells. Subsequently, T lymphocytes carrying the corresponding T cell receptor (TCR) will recognize the foreign peptide associated with HLA and will either help B cells produce antibodies (T cells helpers / inducers; Th), or they will destroy the infected cell ( cytotoxic T lymphocytes; CTL).

Despite decades of efforts, there is still no effective vaccine against human immunodeficiency virus (HIV) infection and AIDS. The earliest attempts focused on obtaining sterilizing immunity with the help of neutralizing antibodies to the HIV outer coat glycoprotein, gp120 / gp160. Studies of phase I / II with gp160 / gp120 showed, however, the neutralization of laboratory strains, but the inability to neutralize field isolates.

In contrast, experiments with attenuated virus have been successful in the monkey immunodeficiency model (SIV). The SIV, from which the NEF or REV genes are delegated, behaves like a weakened virus and protects vaccinated animals from developing the disease, but not from being infected with a provocative wild-type virus. In the case of a REV-defective virus, the only significant immunological correlate with protection was the cellular immune (CI) response to regulatory proteins SIV NEF and TAT. An important observation made in this experiment was that vaccinated animals could even overcome the infection caused by the provocative wild-type virus. Recently, it has been reported that HIV-infected patients can sometimes overcome a clear infection. The only correlation with protection in these studies in animals and / or humans appears to be the cellular immune response to HIV.

This type of immune response is usually not obtained with protein immunization. In the case of HIV, live attenuated vaccines may be effective in preventing infection, but they are theoretically dangerous for several reasons. The recently described genetic immunization method (synonyms for nucleic acid immunization, DNA immunization) has several advantages over live attenuated vaccines, but does not have their potential harmful side effects. A DNA-based vaccine in the form of a eukaryotic expression vector that carries the gene of one or more viral proteins is transfected into a host cell. The viral proteins synthesized in the target cell will then be cleaved by proteolytic enzymes, the resulting peptides will bind to MHC / HLA molecules and appear on the surface of transfected cells. This will cause CTL-mediated immunological memory, which, if the individual is subsequently infected with a wild-type virulent virus, will effectively kill the virus-infected cells immediately after infection and thereby prevent infection. Direct intramuscular or intracutaneous administration of cDNA in a eukaryotic expression plasmid has been shown to induce an immune response (Wolff et al. Science 246: 1465-1468, 1990). The expression of foreign antigens in this way leads mainly to the immune responses of T helper cells of subclass 1 (Th1) with a strong response of cytotoxic T lymphocytes (CTLs) and sometimes also with a high antibody titer (Wang, B. 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, the use of antigens that are the nucleoproteins of influenza A virus have been described for the appearance of antigen-specific CTLs and protection (Ulmer et al. Science 259, 1745-1749, 1993). Moreover, protection against infection using DNA immunization 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) and in the case of human papillomavirus in model experiments on rabbits (Donnelly et al. J. Inf. Dis. 173 (2): 314-320, 1996). DNA immunization has also been used to induce antitumor immunity mediated by cytotoxic lymphocytes (Bohm et al. Cancer Immunol. Immunother. 44 (4): 230-238, 1997).

DNA immunization has several advantages over immunization with live attenuated viral vaccines. Since no infectious virus is formed, the viral genes introduced into the recipient organism remain only in those cells that were initially transfected, and there are no symptoms of a viral infection. In the case of HIV, the main theoretically possible detrimental effect of a live attenuated virus may be reversal by mutation into a wild-type virulent virus. In addition, when immunizing with DNA, you can use only those viral genes, or parts thereof, for which it is known that they effectively induce a preventive immune response.

For an effective CTL response, it is important that cytotoxic T cells destroy infected cells before the formation of structural proteins and before the release of mature viral particles. Therefore, an immune response to the early proteins of the virus life cycle is preferred. HIV replication is regulated by its own regulatory genes and proteins. The HIV genome encodes three non-structural regulatory proteins (NEF, TAT, REV), which are necessary for in vivo replication of the virus. REV is a protein that transports genomic RNA into the cytoplasm, TAT regulates the transcription of the virus, and NEF provides replication in resting cells. SIV experiments have shown that viruses in which one of these regulatory genes does not function may be unable to induce disease due to insufficient replication of the virus. These three proteins are expressed for a short time and in small amounts during the first hours of the infection cycle of the virus (Ranki et al. Arch. Virol. 139: 365-378, 1994). Only a small fraction of HIV-infected individuals show humoral and / or cellular responses to these proteins, and this response correlates with a favorable clinical course.

CTL responses against NEF, TAT, and REV have been extensively studied. NEF-specific responses of CTLs and Th (T helper cells) correlate with a favorable clinical prognosis. When using a REV-defective SIV vaccine, the immune responses to NEF and TAT were protective. Th and CTL epitopes have been identified in the TAT and REV proteins that are detected in HIV-infected individuals and which 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).

A joint review of these results suggests that moderate disease replication (reduced reproduction) in combination with specific immune responses against regulatory proteins involved in virus replication may be necessary for disease prevention.

For DNA immunization, several eukaryotic expression vectors can be used, but their effectiveness is different. Some parameters governing the effectiveness of a particular expression vector in inducing an immune response are unknown, but a high level of expression of the antigenic protein should be clearly preferred. The time period when a vector introduced into the cell can express a foreign antigenic viral protein can also be important. Finally, expression vectors that cause a certain level of cell damage may also have advantages, since it is known that tissue destruction will enhance the immune response through several biologically active molecules, such as cytokines, lymphokines and chemokines, secreted by a cell expressing antigenic protein. Apparently, this is another reason why a live attenuated virus, which causes a certain level of destruction of tissue and cells, induces immunity so effectively, and therefore a DNA vector that mimics a live attenuated vaccine in this regard can have advantages.

Non-specific factors, such as cytokines and lymphokines, can also regulate viral replication and immune responses in HIV infection. The role of the balance of Th1 / Th2 helper cells, which is reflected in the production of lymphokines specific for these two populations of T-helper cells, was demonstrated by Clerici and Shearer (Immunology Today 14 (3): 107-111, 1993) and other authors. Soluble factors produced by CDS cells and capable of suppressing virus production by HIV-infected CD4 cells have recently been identified as RANTES, MIP1-α and MIP1-β (Cocchi et al. Science 270 (5243): 1811-1815, 1995). It is possible that cytokines, whose production either increases or decreases with HIV infection, regulate the transcription of the virus.

Previously performed DNA immunization studies using a gene encoding the HIV regulatory protein NEF have shown proliferative T cell responses (Hinkula et al. Vaccine 15 (8): 874-878, 1997 and Hinkula et al. J. Virol. Jul., 71 (7): 5528-5539, 1997). However, the positive effect of CTLs correlates with a favorable clinical course.

Therefore, one of the main objectives of the present invention is to provide a vaccine for immunization using DNA encoding a regulatory HIV protein, a vaccine capable of eliciting a CTL response against HIV-infected cells in the early phase of the infection cycle, before isolation of new mature viral particles.

Another object of the invention is to provide a vaccine that additionally elicits a humoral response against HIV.

Another objective of the invention is to provide a vaccine against HIV, which is safe to use, since it does not expose the recipient to structural genes or HIV proteins.

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

Another objective of the invention is to create a self-replicating recombinant vector expressing HIV regulatory proteins, and this vector persists for a long time and stably and gives a large number of copies in transfected cells, including mammalian cells.

Another objective of the invention is to create a host cell containing the above vector.

Another objective of the invention is to provide a method for producing the above self-replicating vector.

The present invention further provides a method of treating or preventing HIV.

Another objective of the invention is the use of the above vector to obtain a vaccine against HIV for immunization with DNA.

SUMMARY OF THE INVENTION

The objectives of the present invention can be solved by including a heterologous nucleotide sequence encoding an HIV regulatory protein NEF, REV or TAT or an immunologically active fragment thereof, in a vector containing the E1 gene or E2 gene of the papilloma virus, a minimal source of replication of the papilloma virus and the mini-chromosomal papilloma virus support element .

In other words, the invention is directed to a self-replicating recombinant vector containing the nucleotide sequences of the papilloma virus, necessarily consisting of

(I) the E1 gene and the E2 gene of papilloma,

(Ii) a minimal source of papilloma virus replication,

(Iii) a mini-chromosomal papilloma virus support element,

a heterologous nucleotide sequence encoding an HIV regulatory protein NEF, REV or TAT or an immunologically active fragment thereof.

The invention further allows the development of a vaccine for immunization against HIV using DNA, comprising the above vector, using the above vector to produce an HIV vaccine, and a method for treating or preventing HIV, comprising administering to a person in need thereof an effective amount of a self-replicating vector and expression of an NEF, REV or TAT protein or an immunologically active fragment thereof in the aforementioned person.

The invention also provides a method for producing a self-replicating recombinant vector, the above method includes

A) insertion of a heterologous nucleotide sequence encoding an HIV regulatory protein NEF, REV or TAT, or an immunologically active fragment thereof, into a vector containing papilloma virus nucleotide sequences essentially consisting of

(1) the E1 gene and the Papilloma E2 gene,

(2) a minimal source of papilloma virus replication,

(3) the minichromosomal papilloma virus support element,

and

B) transformation of the host cell by a self-replicating recombinant vector,

B) culturing the host cells,

(D) extracting the above vector.

The invention also allows the creation of a host cell containing the above vector.

A brief description of the graphic materials

Figa depicts the shuttle vector pUE83.

Figv depicts the shuttle vector pNP177.

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

Figure 3 depicts the plasmid pBNsrαTAT according to the present invention.

Figure 4 depicts the plasmid pBNsrαNEF according to the present invention.

Figure 5 depicts the expression of NEF in COS-7 cells transfected with pBNsrαNEF. Samples for Western blotting were taken 72 hours after transfection and visualized using ECL.

Figure 6 shows anti-NEF antibodies in the serum of mice immunized with pBNsrαNEF 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.

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

Figure 8 shows the subclass distribution of immunoglobulins in three mice immunized with pBN-NEF.

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention, an heterologous sequence of HIV nucleotides is inserted into a vector containing the E1 gene and the papilloma virus E2 gene, a minimal papilloma virus (MO) replication source, and a papilloma virus minichromosome support element (MME). This vector, including E1 / E2 / MO / MME, will be referred to as pBM hereinafter, and is described in detail in the publication of international application WO 97/24451, incorporated herein by reference. The aforementioned patent publication is based on the discovery that DNA replication in papillomaviruses, provided only by MO, is insufficient for stable long-term persistence, and another MME viral sequence is needed, and that the best results occur if the vector further contains papillomavirus E1 and E2 genes.

"Papilloma virus" when used here means any member of the papilloma virus family. The preferred papilloma virus used in the invention is bovine papilloma virus (BPV) or human papillomavirus (HPV).

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

A “minimal replication source” (MO) is the minimum nucleotide sequence of the papilloma virus that is necessary to initiate DNA synthesis.

“Microchromosomal support element (MME)” refers to the region of the papilloma virus genome that binds human or viral proteins necessary for replication of the papilloma virus. MME is necessary for stable episomal support of the papilloma virus MO in the host cell.

Preferably, the MME contains multiple binding sites for the transcription activator, E2 protein.

"Self-replicating vector" as used in this application means a vector plasmid capable of autonomous replication in a eukaryotic host cell.

“Heterologous” means alien. For example, with respect to the vectors of the present invention, a heterologous nucleotide sequence means a sequence that is not associated with papilloma.

“Immunologically active fragment” means a fragment capable of eliciting an immunological response in a recipient.

“The nucleotide sequences of the papilloma virus, necessarily consisting of” means that the vector contains the nucleotide sequences of the papilloma virus, which are necessary and sufficient for the long-term persistence of the vector and its replication. This means, for example, that extra sequences, such as all oncogenic coding sequences of papilloma, were delegated from pBN vectors used in the present invention.

In addition to the E1 and E2 genes, MO, MME and NEF, REV or TAT genes, the vectors of the present invention contain promoters for encoded proteins, as well as additional regulatory sequences, polyadenylation sequences and introns. Preferably, the vectors also include a replication source for the bacterial host cell and one or more selectable marker genes for generating vector DNA in the bacterial host cell.

An essential feature of pBN vectors is that they are not specific to the host cell. This is due to the fact that the expression of E1 and E2 proteins is controlled by promoters that are non-native, i.e. heterologous. The above promoters either function in a wide range of mammalian cells or tissues, or are cell or tissue specific.

In the vectors of the present invention, the E1 gene is preferably controlled by the srα promoter or thymidine kinase (tk) promoter, and the E2 gene is preferably controlled by the LTR gag promoter. The NEF, REV, or TAT genes can be controlled by the CMV promoter or the RSV LTR promoter. In addition, the vector may contain an early SV40 promoter for inducing gene expression for selection with an antibiotic (neomycin or kanamycin).

The source of replication in the host cell in the vectors of the present invention is preferably pUC ORI, and the selectable markers used are, for example, kanamycin and / or neomycin. Preferably, the intron is beta-globin IVS.

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

NEF, REV, or TAT genes for insertion into pBN can be obtained from several retailers, for example, plasmid pKP59, which can be purchased at the AIDS Reagent Project MHC repository. The above genes are well known and have been fully sequenced (Wain-Hobson, et al. Cell 40: 9-17, 1985). Of course, you can also insert the sequence encoding only the immunologically active fragment of the above HIV proteins.

The NEF, REV or TAT genes or fragments thereof are first inserted into the corresponding shuttle vector. These vectors may or may not contain MO regions. In the examples, two shuttle vectors are illustrated: pNp177, which does not contain MO, and pUE83, which contains MO. Of course, other shuttle vectors can also be used. Both the shuttle vectors and the final vectors according to the present invention are preferably multiplied in Eschenchia coli. Examples of final pBN-NEF, pBN-REV or pBN-TAT vectors according to the present invention are shown in FIGS. 2, 3 and 4. The vectors according to the present invention are stable and replicate to form a large number of copies. After transfection into a eukaryotic host cell, the vector (plasmid) will multiply and produce 100-1000-fold number of new plasmids, each of which is able to express the necessary HIV protein.

The host cell of the present invention can be either a eukaryotic cell transfected with a vector or a prokaryotic cell transformed with a vector. The eukaryotic cell is preferably a mammalian cell, and the prokaryotic cell is preferably a bacterial cell, especially E. coli.

The expression of NEF, REV, and TAT by HIV with the obtained plasmid vectors of the present invention was analyzed both on transfected COS-7 cells and in mice immunized with the above plasmids. High expression of HIV proteins could be demonstrated on COS-7 cells, immunized mice also showed pronounced humoral and cellular (CTL) immune responses. A pronounced CTL response was also demonstrated on monkeys. These results indicate that the vectors of the present invention could potentially be used as effective HIV vaccines.

It was further demonstrated that a mixture of pBN vectors encoding various HIV regulatory proteins enhances the immune response with several regulatory genes. The present invention therefore includes vaccines containing a mixture of vectors encoding various HIV regulatory proteins or immunologically active fragments thereof, and using the above mixture in the manufacture of a vaccine and for the treatment or prevention of HIV. The vaccine may contain a mixture of vectors encoding all three different regulatory proteins. Vaccines according to the present invention may also contain other genes or gene fragments, for example, selected from the group consisting of structural HIV genes.

Description of Embodiments

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

Example 1

Cloning of REV and TAT HIV-1 genes in self-replicating plasmids pBNsr-α and pBNtk

Getting pBNtkREV and pBNsraTAT

Phase 1:

HIV-1 REV and TAT genes from isolated BRU, also called LAI (Wain-Hobson et al. Cell 40: 9-17, 1985), were amplified from pcREV and pcTAT vectors (Arya et al. Science 229: 69-73, 1985) using Taq Dynazyme DNA polymerase (Finnzymes, Finland) and the following primers that have restriction sites for Xhol and Xba1 enzymes:

For REV:

5’-TTTTTCTAGAACCATGGCAGGAAGAAGCGGA-3 ’

5’-TTTTCTCGAGCTATTCTTTAGTTCCCTGG-3 ’

For TAT:

5’-TTTTTCTAGAACCATGGAGCCAGTAGATCCT-3 ’

5’-TTTTCTCGAGCTAATCGAACGGATCTGC-3 ’

The amplified genes and the pUE83 shuttle vector (FIG. 3) were enzymatically digested at + 37 ° C with Xbal and Xhol enzymes (New England Biolabs, USA) overnight to obtain compatible ends. Cleavage DNA fragments were analyzed on a 1.5% agarose gel and further purified using the Band Prep Kit (Pharmacia Biotech, Sweden). Each gene was ligated separately to the vector using T4 DNA ligase (New England Biolabs, USA) during incubation overnight at + 16 ° C. Ligation products were transformed into E. coli competent cells from the One Shot Kit (Invitrogen, Netherlands), which were applied to LB plates containing kanamycin for selection. Minipreparations were prepared from growing clones, and analysis for the presence of cloned genes was carried out by enzymatic digestion with XhoI and XbaI enzymes. The presence of cloned genes was also confirmed by polymerase chain reaction (PCR) from minipreparations using the above primers. Clones containing the desired gene were mass-cultured, and plasmids were purified using Megaprep columns (Qiagen, Germany).

Phase 2:

The DNA fragment containing BPVori, the RSV LTR promoter, the REV or TAT gene and the IVS beta-globin poly (A) was isolated from the shuttle vector by enzymatic digestion with Hind III enzyme (New England Biolabs, USA) and purified using 1% agarose gel and Band Prep Kit. Ligation of pBNsrα or pBNtk isolated with Hind III and dephosphorylated (alkaline phosphatase, CIP, Promega, USA), cell transformation, confirmation of the presence of the cloned gene, and plasmid purification were carried out in the same manner as in Phase 1.

The resulting plasmids are called pBNtkREV and pBNsrαTAT and are shown in FIG. 2 and 3.

Example 2

Cloning of HIV-1 NEF into the self-replicating plasmid pBNsrα

Obtaining pBNsrαNEF

Phase 1:

The HIV-1 NEF gene was obtained from the pcNEF plasmid vector, which contained the LAI NEF gene isolate inserted into the pcTAT vector lacking the TAT gene. The NEF gene used for further cloning was obtained as a fragment of 1.3 thousand nucleotide pairs by enzymatic digestion using Spe I and Hind III from pcNEF. To eliminate Hind III site reformation during ligation, after cleavage with Hind III, the fragment was treated with Klenov enzyme and a mixture of dATP, dCTP, dGTF nucleotides, followed by cleavage with Spe I. The obtained fragments were separated by electrophoresis in a 1% agarose gel in parallel with standard size markers. Bands of the desired size were cut out and DNA was isolated using the Sephaglas Bandprep Kit (Pharmacia Biotech) according to the manufacturer's protocol.

The shuttle vector pNP177 shown in FIG. 1 was initially digested with Xho I, then digested with Klenov enzyme and a mixture of dNTP, and finally digested with Xba I. The vector was also treated with calf intestinal alkaline phosphatase (CIF).

The fragment containing the NEF gene was ligated with the digested pNP177 vector using T4 ligase at + 14 ° C overnight. A set of competent E. coli One Shot Kit (Invitrogen) was used for transformation. Positive clones were identified using restriction enzyme digestions and electrophoresis. Plasmid DNA was further amplified in E. coli and purified in large quantities using Qiagen columns. The resulting plasmid was named pNP177cHIVNEF

Phase 2:

The shuttle vector pNP177 is designed so that it has only two sites for Hind III, between which the insert can be cloned. Therefore, cleavage of the plasmid with Hind III gives a fragment that can be cloned further. The pNP177cHIVNEF fragment obtained using Hind III was cloned into pBNsrα. The vector was digested with Hind III and treated with CIP (calf intestinal intestinal phosphatase). The same methods of band isolation, ligation and transformation were used as in the first phase, and the correct orientation of the insert was confirmed by restriction analysis. The final plasmid is called pBNsrαNEF, and it is shown in Fig.4.

Example 3

In vitro demonstration of HIV-NEF expression

3A. Transfection

To analyze the expression of pBN constructs from examples 1 and 2, they were transfected by electroporation into COS-7 cells. 10 mg of pBNβ-Gal as a control and 10 μg of pBN-NEF transfected together with 1 μg of pCMVβ-Gal were electroporated into three million cells in each case. DNA from salmon sperm was used as a carrier. Electroporation was carried out at a capacitance of 960 mF and a voltage of 260 V. Protein concentration and β-gal activity were measured in order to control the transfection efficiency and calibrate the number of lysates in the analysis by Western blotting.

3B. Immunohistochemistry and Western blotting

The accumulated cells transfected with pBN constructs were lysed for use in Western blotting. After lysis, the protein samples were boiled in a sample buffer and transferred to a 12% SDS-polyacrylamide gel, then transferred to a nitrocellulose filter with a pore diameter of 2 μm, which was blocked 5 % solution of milk in TBS. As a primary antibody, a mixture of mouse monoclonal antibodies to NEF (Ovod V. Et al. AlDS 6 25-34, 1992) diluted in a ratio of 1: 1000 each was used. The secondary antibody was a biotinylated antibody to mouse proteins at a dilution of 1: 500.

After transfection into COS cells, the vectors caused strong short-term expression of the HIV regulatory protein, which was detected by Western blotting of lysed cells after 72 hours. The results obtained with pBNsrαNEF are shown in Fig. 5. For long-term cultivation of transfected cells, NEF expression remained for up to 7 weeks in cells transfected with a self-replicating pBN vector.

NEF-transfected cells were also used to prepare cytospin preparations, they were stained with hematoxylin, and monoclonal antibodies to NEF and then biotinylated antibodies to mouse proteins were used for immunohistochemistry, as described by Ovod et al. (see above). Cytospin sections revealed expression, since positive staining was visible in a large number of cells in the form of granules occupying the cell cytoplasm. A portion of NEF-expressing cells showed morphological signs of cell destruction, indicating apoptosis. The expression level was high, although the conditions for the cells were getting worse.

Example 4

Demonstration of the immunogenicity of the expression of HIV-Nef, HIV-Tat or HIV-Rev vectors pBNsrα and pBNtk in vivo

4A. Gene gun

DNA was precipitated (precipitated) on particles of gold with a diameter of 1 μm using spermidine and CaCl ₂ in accordance with the procedure described in the instruction manual for the gene gun Helios Gene Gun (Laboratory Bio Rad). Cartridges were made, each containing 0.5 mg of gold and 1 μg of DNA. The amount of DNA was controlled spectrophotometrically in accordance with the instructions in the manual. Inoculations were performed using the Helios Gene Gun System (Bio-Rad Laboratories). The discharge pressure of helium for the purpose of DNA delivery was set to 300 psi (approximately 21 kg / cm ² ). During the optimization of the bombardment conditions, the inventors found that 300 psi was sufficient to move particles into the dermis.

4B. Immunization

Used female Balb / c mice at the age of 6-8 weeks. Before immunization, mice were anesthetized and abdominal hair was removed.

Inoculation of the skin on the abdomen of 8 mice was performed on days 1, 2, 3, 10, 11 and 12 using the gene gun described above, in accordance with the manufacturer's instructions. A total of 6 μg pBN-NEF was injected into each mouse. Four mice from both groups were sacrificed painlessly two weeks after the last immunization, and the remaining four mice - 4 weeks after the last immunization. Serum samples were taken for Western blotting, and splenocytes were accumulated for analysis on CTLs. All eight mice immunized with pBN-NEF vector showed an antibody response after 2 weeks and after 4 weeks (FIG. 6). The reaction rate for Western blotting varied

4B. Measurement of the cytotoxic activity of T cells in immunized mice

4B1. Stimulation of effector cells

In immunized mice, spleen was aseptically removed two weeks after (16 mice) and four weeks (16 mice) after immunization. They were ground in Hanks buffer, filtered through cheesecloth and red blood cells removed. Then the cells were suspended at a concentration of 5 × 10 ⁶ / ml in culture medium RPMI 1640 medium containing 10% fetal calf serum (FCS, GibcoBRL), 1% glutamine, 100 units of penicillin per ml, 100 μg of streptomycin per ml and 5 × 10 ^{- 5} M 2-mercaptoethanol. The reacting cells (5 × 10 ⁶ ) were cultured in a 25 ml cell culture flask in 5 ml of culture medium together with 4 × 10 ⁶ antigen-presenting cells (APC; see below) for five days. On the first day, 10 U / ml of recombinant interleukin-2 (rlL-2) was added to the cells (Hiserodt J. Et al. J. Immunol. Jul, 135 (1): 53-59, 1985, Lagrandene M. et al. J Virol. Mar. 71 (3): 2303-2309, 1997; tsuji T. et al. Immunology Jan. 90 (1): 1-6, 1997; Vahlsing HL et al. Journal of Immunological Methods 175: 11- 22, 1994, K. Varkila et al. Acta path. Microbiol. Immunol. Scand. Sect. C 95: 141-148, 1987).

4B2. Antigen presenting cells

Syngeneic P815 mastocytoma cells (H-2d) were infected with a modified Ankara vaccinia virus (MBA), rearranged to express the HIV-1 LAI NEF gene (MBA-HIVNEF). MBA is a highly attenuated, weakly replicating vaccinia virus, which can serve as an effective vector for the expression of heterologous genes, providing an exceptionally high level of biological safety (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). MBA-HIVNEF infection was performed with the number of infections (ChZ) equal to 5 on 24-cell plates (1 × 10 ⁶ cells per cell). After the virus was absorbed for 1 hour at + 37 ° C, the cells were incubated for 15 hours at + 37 ° C (Carmichael A. Et al. Journal of Virology 70: 8468-8476, 1996). After infection, the cells were washed twice with phosphate buffered saline (PBS) containing 10% fetal calf serum and suspended in this solution to a concentration of 5 × 10 ⁶ cells / ml. Then the cells were treated with γ-radiation at a dose of 5000 rad and washed with culture medium before adding the corresponding cells.

4B3. Cytotoxicity assays

CTL activity was investigated using ⁵¹ Cr analysis (Hiserodt J. Et al. J. Immunol. Jul, 135 (1): 53-59, 1985; Lagrandene M. et al. J. Virol. Mar, 71 (3 ): 2303-2309, 1997; K. Varkila et al. Acta path. Microbiol. Immunol. Scand. Sect. C 95: 141-148, 1987; van Baalen C. et al. AIDS 7: 781-786, 1993) . Briefly, 2 × 10 ⁶ P-815 cells infected with MBA-HIVNEF as described above for antigen presenting cells. After infection, the cells were washed once with serum-free culture medium. The target cells were then suspended in 200 μl of serum-free culture medium, and 100 μCi ⁵¹ Cr (Amersham) / 1 × 10 ⁶ cells was added for 1 hour at 37 ° C. Then the target cells were washed four times in the medium and suspended to a concentration of 5 × 10 ⁴ / ml. Stimulated effector cells were washed once with culture medium before being added to target cells. The target cells were applied to a 96-cell plate in an amount of 100 μl per cell (5 × 10 ³ ) and effector cells were added in the form of three samples with a volume of 100 μl at an effector: target ratio of 50, 25, and 12.5. For spontaneous isolation, target cells were applied to 6 cells in 100 μl of culture medium, and for maximum isolation, to 6 cells with 2.5% Triton-X-100. The plates were quickly rotated, incubated for 4 hours at 37 ° C, and the number of decays in the supernatants was calculated using a gamma counter. The percentage of specific lysis of target cells was calculated as ( ⁵¹ Cr excretion in the analysis — spontaneous excretion) / (maximum excretion — spontaneous excretion) x100. A specific lysis percentage greater than or equal to 6% was considered positive.

An example showing CTL activity in 6 of 8 mice immunized with pBNsrαNEF is depicted in FIG. 7.

D. Humoral immune response in immunized mice

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

Summary of Results

The results of analyzes with transfection and immunization are summarized in table 1. The immune response in immunized mice was assessed by immunoblotting (western blotting) for a humoral response and by analysis of cytotoxic T lymphocytes (CTLs) for cellular immunity. As can be seen from table 1, all eight mice immunized with an NEF expressing vector showed both a humoral and a cellular immune response.

In the present study, we demonstrated that immunization with DNA using a self-replicating expression vector, as described, can elicit a readily detectable CTL response in mice. In addition, a humoral immune response was obtained. In light of the above results, it can be assumed that plasmids pBN-NEF, pBN-REV and pBN-TAT can express NEF, REV and TAT in vivo in an amount sufficient to elicit both a humoral and cellular immune response necessary for prevention and HIV treatment.

Example 5

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

The humoral immune response found in mice immunized with pBN constructs expressing HIV regulatory proteins was analyzed for the specificity of the immunoglobulin subclass. It is well known that the response with the isolation of antibodies, among which IgG2a subclasses of immunoglobulins dominate, is characteristic of the Th1 type of the cellular immune response, while logG1, IgG2b and IgG3 are characteristic of the Th2 cell response. Moreover, it is known that Th1-type responses induce and promote cellular cytotoxic immune responses (CTL responses), while a Th2 response will induce an antibody response, but less active CTL responses.

The immunization schedule was as follows.

Four Balb / c mice were immunized six times with 24 μg of pBN-Nef from Example 2 for two weeks, the total DNA was therefore 144 μg, and the serum was analyzed for antibodies by Western blotting. Three out of four mice had antibodies to HIV-1 Nef, and a subclass of these antibodies was determined by enzyme-linked immunosorbent assay (ELISA) as follows.

The antigen was pipetted onto Nunc Maxi Sorb plates for overnight incubation at + 4 ° C; the antigen used was HIV-1 Nef protein (NIH, AIDS Research and Reference Reagent Program) in phosphate buffer (50 ng / well). Cups after incubation overnight were blocked with 1% bovine serum albumin (BSA, Sigma) and then incubated with mouse serum (diluted 1: 100 with blocking solution) for 4 hours at room temperature. The plates were washed with phosphate buffer / 0.1% Tween 20 three times and then with phosphate buffer two times. Peroxidase-conjugated antibodies against mouse antigens IgG1, IgG2a, IgG2b, IgG3 and IgM (Calbiochem) diluted 1: 1000 with a blocking solution were used as secondary antibodies, and the plates were incubated for 2 hours at room temperature. After the washing steps performed as described above, ABTS substrate (Sigma) and H ₂ O ₂ were added in citrate buffer for 10 min and photometric determination was performed on an ELISA-recording device at a wavelength of 405 nm.

The results are presented in FIG. The highest response in three mice was found for IgG2 secondary antibodies (absorbance at a wavelength of 405 nm 0.117, 0.262, 0.743, respectively), the response for IgG1 antibodies was significantly lower (A (405) 0.004, 0.020, 0.038). This indicated that the type of response in these mice after intramuscular immunization is mainly a Th1-type response leading to a cellular immune response.

The results demonstrate that almost all mice had a strong IgG2 type response against recombinant NEF, while the concentration of antibodies representing other IgG subclasses was very low. The results further showed that pBN NEF constructs are able to elicit a Th1 type response and therefore a strong cellular immune a response that can destroy HIV-infected cells in the early phase of the viral infection cycle.

Example 6

Generation of a cellular immune response in Masas monkeys by fasciculans with pBN constructs expressing HIV-1 regulatory proteins

Experiments with mice clearly showed that pBN NEF, pBN REV, and pBN TAT constructs are capable of eliciting a CTL response in immunized mice. An additional experiment was conducted with non-human monkeys to confirm that the constructs can be used as prophylactic vaccines in humans. It is important to demonstrate that an immune response can also be generated in nonhuman primates that are genetically closer to humans and that can be infected with the corresponding SIV primitive retrovirus, which is closely related to HIV-1 and HIV-2, infecting humans. Therefore, an experiment was conducted in which the Masas monkeys fascicularis were immunized with pBN constructs expressing the HIV-1 regulatory proteins NEF, REV, and TAT. These proteins were prepared as described in Examples 1 and 2. Three Masas fascicularis monkeys were immunized with a mixture of pBN NEF, pBN REV and pBN TAT. Three monkeys served as control. The immunization schedule was as follows.

Monkeys were immunized with a total amount of 300 μg, pBN-NEF, pBN-REV and pBN-TAT (100 μg of each construct) twice. The first immunization was performed in the deltoid muscle, and the second (2 weeks later) was performed intradermally. Cytotoxic T-lymphocyte assays were performed two months after the last immunization, as described in Example 4C3. The results were as follows.

One of the three monkeys had a pronounced CTL response against autologous B cells expressing HIV-1 NEF and TAT. The results demonstrate that not only mice, but also primates can be immunized with pBN constructs expressing HIV regulatory proteins, and immunized animals will develop a T-cell mediated immune response characterized by the presence of cytotoxic T lymphocytes that can destroy HIV-infected cells in the early phase of the viral infection cycle. Moreover, the results show that the constructs can be administered simultaneously as a mixture and that the presence of one construct in the mixture does not interfere with the immune response generated by the other construct.

Claims (12)

1. A self-replicating recombinant vector expressing HIV regulatory proteins containing the nucleotide sequences of the papilloma virus, essentially consisting of (I) the E1 gene and the E2 gene of the papilloma virus, (II) the minimal source of replication of the papilloma virus, (III) the minichromosomal papilloma virus support element, and a heterologous nucleotide sequence encoding an HIV regulatory protein NEF, REV or TAT, or an immunologically active fragment thereof. 2. The self-replicating vector according to claim 1, characterized in that the papilloma virus is a bovine papilloma virus (VPB). 3. A self-replicating vector according to any one of claims 1 and 2, characterized in that the heterologous nucleotide sequence encodes an HIV-1 NEF protein. 4. A self-replicating vector according to any one of claims 1 to 3, characterized in that E1 is under the control of the srα promoter or the thymidine kinase promoter. 5. The self-replicating vector according to claim 4, characterized in that it is pBNtkREV, pBNsrαTAT or pBNsrαNEF, depicted in figure 2, 3 or 4. 6. A vaccine for immunization against HIV based on DNA, containing at least one self-replicating vector, as defined in any one of claims 1 to 5. 7. The vaccine according to claim 6, characterized in that it contains a mixture of vectors encoding various HIV regulatory proteins selected from NEF, REV or TAT, or immunologically active fragments thereof. 8. A method for producing a self-replicating recombinant vector, as defined in any one of claims 1 to 5, comprising the following steps: A) inserting a heterologous nucleotide sequence encoding an HIV regulatory protein NEF, REV or TAT, or an immunologically active fragment thereof, into a vector, containing the nucleotide sequences of the papilloma virus, consisting of (I) the E1 gene and the E2 gene of the papilloma, (II) the minimal source of replication of the papilloma virus, (III) the minichromosomal element of support for the papilloma virus, B) transformation of the host cell a self-replicating vector, B) culturing a host cell, and D) extracting the above vector. 9. The method of claim 8, wherein the host cell is an E. coli cell. 10. A self-replicating recombinant vector, as defined in any one of claims 1 to 5, used to manufacture a DNA-based vaccine for immunization against HIV. 11. A method of treating or preventing HIV, comprising administering to a person in need of an effective amount of at least one self-replicating vector as defined in any one of claims 1-5, and expressing an NEF, REV, or TAT protein or immunologically active fragment thereof at the above person. 12. The method according to claim 11, comprising administering a mixture of vectors encoding various HIV regulatory proteins selected from NEF, REV or TAT, or immunologically active fragments thereof.

Images