FI105105B - A self-replicating DNA vector for immunization against HIV - Google Patents

Description

! 105105

A self-replicating DNA vector for immunization against HIV

Field of the Invention

The invention relates to a self-replicating recombinant vector useful in DNA immunization against HIV. The invention also relates to a vaccine comprising said vector, a method for making the vector and a host cell comprising it. The invention further relates to the use of said vector for the preparation of a vaccine against HIV and to a method of treating or preventing HIV.

Background of the Invention

The interaction of vertebrates, and thus humans, with pathogenic microbes such as bacteria, fungi and viruses is regulated by the ability of the vertebrate body to form an immune response against an invading microbe. This reaction is based on the ability of the immune system to distinguish between self and foreign; under normal circumstances, the immune response tolerates the body's own structures, cells and antigenic molecules, while attacking foreign antigens in the microbes that invade the body. When a vertebrate, such as a human, receives a microbial infection, the immune response helps to overcome the infection by killing microbes or microbially infected cells or preventing the infection from spreading through the action of antibodies that make it harmless. On the other hand, and more importantly, a once-born immune response to an invading organism has an innate memory mechanism, and thus an individual who has once undergone a particular microbial infection is often immune and cannot obtain the infection a second time.

25 This immunological memory, which is caused by infection in the natural state, is the basis for vaccines that mimic, in many ways, the natural infection. The ideal vaccine does not induce symptoms or causes only mild symptoms in vaccinated individuals, but still results in the induction of an immunological memory with the ability to form a strong suppressive immune response if the vaccine is confronted with the microbe.

The design of a vaccine against a particular microbe depends on the mechanism by which the body, in the case of a natural infection, can survive this particular organism and prevent subsequent infections. There are a few ways in which the immune response can trigger this beneficial action. First, antibodies synthesized and secreted by B lymphocytes can bind to a microbe and destroy it through complement-mediated lysis.

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Second, the antibodies can prevent the spread of the infection by inhibiting the binding of the microbe to its target cell. Third, antibodies can kill infected cells upon complement activation, and specific cytotoxic T lymphocytes (CTLs) can also kill and destroy microbial infected cells.

All of these mechanisms are thought to be involved in the immune response elicited by human immunodeficiency virus (HIV) types 1 and 2 (HIV-1 and HIV-2). However, the immune response of HIV-infected individuals is usually characterized by a strong antibody response and a less effective T lymphocyte response or even lack thereof [J. Gerstott et al., J. Immunol. 22, 10 No. 5 (1985) 463-470; Re C. M. et al., J. Clinical Pathol. 42, No. 5, 282-283 (1989)]. This may be the reason why individuals once infected with HIV have developed a chronic infection despite a strong antibody-mediated immune response.

Generally, the preventive immune response to viral infection may be mediated by the four types of immune response described above, but it is known that a CTL response with the ability to kill virus-infected cells is most effective. For this reason, live attenuated viral vaccines have generally proven to be most effective. In fact, the first vaccine developed by Jenner over 200 years ago, a live cowpox virus that can prevent smallpox virus-20 infection, is an example of this principle. When an individual is vaccinated with a live attenuated viral vaccine, the host cells become infected and synthesize viral proteins. Some of the viral protein molecules are used to produce viral particles, while some are proteolytically cleaved into small peptides that bind to major histocompatibility (MHC) antigens (HLA Class 25 I and II in humans) and are available on T-lymphocytes on infected cells. Thereafter, T lymphocytes with the appropriate T cell receptor (TCR) recognize the foreign peptide associated with HLA and either aid in B cell production of the antibody (helper / inducer T cells; Th) or destroy the infected cell (cytotoxic). T-lymphocyte; CTL).

30 Despite decades of work, an effective vaccine against human immunodeficiency virus (HIV) infection and AIDS is still lacking. Previous attempts have been made to provide sterilizing immunity through the use of harmless antibodies against the HIV outer envelope glycoprotein, gp120 / gp160. However, Phase I / II gp160 / gp120 studies 35 only indicate that laboratory strains are harmless, but fail to render field isolates harmless.

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In contrast, experiments with attenuated virus have been successful in the Monkey Immunodeficiency (SIV) model. SIV with NEF or REV gene deletions behaves as a attenuated virus and protects vaccinated animals against disease development but not infection by wild-type challenger virus. In the case of the REV-defective virus, the only immunologic factor correlating with protection was the cell-mediated immune response (CMI response) to SIV regulatory proteins NEF and TAT. One important finding in this experiment was that vaccinated animals were even able to survive infection by wild-type challenger virus. Recently, it has been reported that HIV infection patients may occasionally survive a visible infection. The only significant factor correlating with protection in these animal and / or human studies appears to be the cell-mediated immune response to HIV.

This type of immune response is generally not elicited by protein-15 immunization. In the case of HIV, live attenuated vaccines could be effective in preventing infection, but they are theoretically dangerous for a number of reasons. One method described shortly ago, genetic immunization (synonyms: nucleic acid immunization, DNA immunization), has several advantages but not potentially harmful side effects of live attenuated vaccines. A DNA vaccine in the form of a eukaryotic expression vector containing a gene corresponding to one or a few viral proteins can induce viral protein synthesis when the host cell is transfected with the DNA vector. The viral proteins synthesized in the target cell then undergo processing by proteolytic enzymes; form-25 peptides bind to MHC / HLA molecules and occur on the surface of transfected cells. This results in a CTL-mediated immunological memory which, in the event that an individual subsequently becomes infected with a virulent wild-type virus, effectively kills the virus-infected cells immediately after infection and thus prevents infection. It has been shown that cDNA. 30 direct intramuscular or intradermal injection in a eukaryotic expression plasmid induces an immune response (Wolff et al., Science 246: 1465-1468 (1990)). Expression of foreign antigens by such means results primarily in helper T cell subgroup 1 (TH1) type immune responses accompanied by a strong cytotoxic T lymphocyte (CTL) response and sometimes also by a high titer antibody response [B. Wang et al., PNAS 90: 4156-4 (1993); Wang et al., NY Acad. Sci. 772, 186-197 (1995); Haynes et al., AIDS Res. Hum. Retro-4,705,705 to Viruses 10, No. 2 (1994) 43-45]. In addition, antigen-specific CTL and protection using influenza A virus nucleoprotein antigen have been reported [Ulmer et al., Science 259 (1993) 1745-1749J. In addition, protection against mycoplasma infection in mice has been achieved using DNA-5 immunization [Barry et al., Nature 377, No. 6550 (1995) 632-635; Lai et al., DNA Cell. Biol. 14, No. 7 (1995) 643-651] and against human papilloma virus in a rabbit model [Donnelly et al., J. Inf. Dis. 173, No. 2 (1996) 314-320]. DNA immunization has also been used to induce cytotoxic lymphocyte-mediated anti-tumor immunity [Bohm et al., Cancer Immunol.

10 Immunother. 44, No. 4, 230-238 (1997)].

DNA immunization has several advantages over live attenuated viral vaccines. Since no infectious virus is produced, viral genes induced in the host organism remain only in the cells initially transfected and no symptoms of viral infection occur. In the case of HIV, the major theoretical adverse effect of live attenuated virus would be the return of mutations to virulent wild-type virus. In addition, only viral genes or portions thereof known to be effective in inducing a suppressive immune response may be used in connection with DNA immunization.

For an effective CTL response, it would be important for the cytotoxic T-20 cells to destroy the infected cells prior to the formation of structural proteins and release of mature viral particles. An immune response against early proteins in the viral cycle would therefore be favorable. HIV replication is regulated by its own regulatory genes and proteins. The HIV genome encodes three regulatory non-structural proteins (NEF, TAT, REV) which are indispensable for viral replication in vivo. REV transports genomic RNA into the cytoplasm, TAT promotes viral transcription, and NEF induces replication in dormant cells. SIV experiments indicate that viruses lacking the function of one of these regulatory genes may not be able to induce disease due to insufficient virus replication. These three proteins are transiently expressed in 30 and small amounts during the first hours of the viral infection cycle [Ranki et al., Arch. Virol. 139: 365-378 (1994)]. Only a small proportion of individuals infected with HIV show a humoral and / or cellular response to these proteins, and this response correlates favorably with clinical course of the disease.

CTL responses to NEF, TAT and REV have been extensively studied.

NEF-specific CTL and Th (T-helper cell) responses correlated with a favorable clinical prognosis. In the case of the REV defective SIV vaccine, the immune responses to NEF and TAT were protective. Th and CTL epitopes in TAT and REV which have been identified by HIV-1 infected individuals and correlated clinically have been identified [Blazevic et al., 1993, J AIDS 6: 881-890; Blazevic et al., AIDS Res. Hum. Retroviruses 11 (1995) 5, 1335-1341]. Taken as a whole, these results indicate that moderate replication (attenuated growth) of the virus, combined with specific immune responses against regulatory proteins involved in supporting viral replication, may be necessary to protect against iron.

Several eukaryotic expression vectors can be used for DNA immunization, but their efficacy varies. Some of the parameters that control the efficacy of a particular expression vector in inducing an immune response are unknown, but high expression of an antigenic protein would apparently be advantageous. The time period in which the introduced vector may express a foreign antigenic viral protein may also play a role. Expression vectors that induce a certain degree of cellular damage may also be advantageous because it is known that tissue destruction enhances the immune response through a few biologically active molecules, such as cytokines, lymphokines and chemokines, which are secreted by the cell expressing the antigenic protein. This is probably one additional reason why a live attenuated virus, which causes 20 levels of tissue and cell killing, is so effective at inducing immunity, and therefore a DNA vector mimicking a live attenuated vaccine would be preferred.

Non-specific factors such as cytokines and lymphokines can also regulate viral replication and immune responses in HIV-1 infection. Clerici and Shearer (Immunology Today 14, No. 3, 107-111 (1993)) and others have demonstrated a function that is at the Th1 / Th2 balance of helper cells reflected by the production of lymphokines specific for these two T cell populations. Soluble factors produced by CD8 cells and having the ability to reduce viral production of HIV-1 infected CD4 cells have recently been identified as RANTES, MIP1-a and MIP1-β [Cocchi et al., Science 270 , 5243, 1811-3030 (1995)]. It is possible that cytokines, whose production either increases or decreases in HIV-1 infection, regulate viral transcription.

Previous DNA immunization studies using the gene encoding the HIV regulatory protein NEF have demonstrated T cell proliferation responses [Hinkula et al., Vaccine 15, No. 8 (1997) 874-878 and Hinkula et 35 al., J. Virol. . 71, No. 7 (July 1997) 5528-5539]. However, the positive efficacy relation to the favorable clinical course is precisely the CTL response.

Therefore, one of the main objects of the present invention is to provide a DNA immunization vaccine that encodes an HIV regulatory protein and has the ability to elicit a CTL response against HIV-infected cells at an early stage of the infection cycles before release of new, ready-to-infectious virus particles.

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

It is a further object of the invention to provide an HIV vaccine that is safe to use because it does not expose the recipient to structural genes or proteins of HIV.

Another object of the invention is to provide a self-replicating vector that induces long-term and high levels of HIV regulatory protein-15 expression and a degree of cellular destruction which further stimulates the immune response.

It is another object of the invention to provide a self-replicating recombinant vector expressing HIV regulatory proteins that results in long-term stable maintenance and high copy number in transfected cells, including mammalian cells.

It is a further object of the invention to provide a host cell comprising said vector.

It is a further object of the invention to provide a method for producing the above self-replicating vector.

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

It is another object of the invention to use said vector for the preparation of a DNA immunization vaccine against HIV.

SUMMARY OF THE INVENTION The objects of the present invention may be achieved by incorporating a heterologous nucleotide sequence encoding the HIV regulatory protein NEF, REV, or TAT, or an immunologically active fragment thereof, into a vector comprising the papillomavirus E1 gene and the E2 gene, papillomavirus min .

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That is, the invention relates to a self-replicating recombinant vector comprising papilloma virus nucleotide sequences consisting essentially of (i) the papillomavirus E1 gene and the E2 gene, (5) (ii) a papillomavirus minimal replicon and (iii) a papillomavirus, regulatory protein NEF, REV or TAT or an immunologically active fragment thereof.

The invention further provides a vaccine for HIV immunization against HIV comprising said vector, the use of said vector for the preparation of a vaccine against HIV and a method of treating or preventing HIV by administering to a subject in need thereof an effective amount of a self-replicating vector. and causing the NEF, REV, or TAT protein, or an immunologically active fragment thereof, to be expressed in said individual.

The invention further provides a method for making a self-replicating vector comprising: A) inserting a heterologous nucleotide sequence encoding the HIV regulatory protein NEF, REV, or TAT, or an immunologically active fragment thereof, into a vector comprising nucleic acid sequences consisting essentially of - a) papillomavirus E1 gene and E2 gene, 25 - (ii) papillomavirus minimal replicon and - (iii) papillomavirus minicromosomal maintenance element, and B) transforming the host cell with the resulting self-replicating recombinant vector, 30 C) cultured. vector.

The invention also provides a host cell comprising said vector.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1A shows the shuttle vector pUE83.

Figure 1B shows the shuttle vector pNP177.

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Figure 2 shows the pBNtkREV plasmid of the invention.

Figure 3 shows the pBNsraTAT plasmid of the invention.

Figure 4 shows the pBNsraNEF plasmid of the invention.

Figure 5 shows NEF expression in 5 COS-7 cells transfected with pBNsraNEF. Western blots were taken 72 hours after transfection and visualized by ECL.

Figure 6 shows anti-NEF antibodies in the serum of mice immunized with pBNsraNEF detected by Western blotting. Samples 1-4 were taken 2 weeks after the last immunization and Samples 5-8 were taken 4 to 10 weeks after the last immunization.

Figure 7 shows CTL responses of mice immunized with pBNsraNEF vector. Figure 7A shows CTL responses, expressed as percentage specific lysis of target cells, in four mice tested two weeks after the last immunization. Figure 7B shows these values four weeks after the last immunization. Specific lysis> 4% is considered a positive result.

Detailed Description of the Invention

According to the invention, the heterologous HIV nucleotide sequence is inserted into a vector comprising the papillomavirus E1 gene and the E2 gene, the papillomavirus minimal replicon (MO), and the papillomavirus minicomyosomal maintenance element (MME). This vector comprising E1 / E2 / MO / MME is hereinafter referred to as pBN and is described in detail in WO 97/24451, which is incorporated herein by reference. Said patent is based on the finding that DNA replication by MO alone is not sufficient for stable long-term survival in papillomaviruses but requires another viral sequence MME and that the best results are obtained when the vector further comprises the papillomavirus E1 and E2 genes.

"Papillomavirus" as used herein, means any member of the papillomavirus group. The papillomavirus used in the invention is preferably bovine papillomavirus (BPV) or human papillomavirus (HPV).

"E1" and "E2" are papillomavirus regulatory proteins that replicate through MO and are essential for replication.

The "minimal origin of replication" (MO) is a very small sequence of the papillomavirus necessary for the initiation of DNA synthesis.

9705705 "Minichromosomal maintenance element" (MME) refers to a region of the papillomavirus genome that binds viral or human proteins essential for papillomavirus replication. multiple binding sites for transcriptional activator protein E2.

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

10 "Heterologous" means guest. For example, a nucleotide sequence heterologous to the vectors of the invention refers to a non-papilloma sequence.

"Immunologically active fragment" means a fragment that has the ability to elicit an immunological response in a recipient.

"Papillomavirus nucleotide sequences consisting essentially of" means that the vector comprises papillomucleotide sequences which are necessary and sufficient for the long-term survival and replication of the vector. This means, for example, that extra sequences, such as all sequences encoded by papillomavirus, 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 invention comprise promoters for encoded proteins as well as regulatory sequences, polyadenylation sequences, and introns. The vectors preferably also contain a bacterial host cell replI-Kon and one or more selectable marker genes for the production of vector DNA in a bacterial host cell.

One essential feature of pBN vectors is that they are not host cell specific. This is because expression of the E1 and E2 proteins is regulated by non-native, i.e., heterologous, promoters. These promoters are either extensively active in mammalian cells or tissues or are cell or tissue specific.

In the vectors of the present invention, the E1 gene is preferably under the control of the sr-a promoter or thymidine kinase promoter (tk) and the E2 gene is preferably under the control of the LTR-gag promoter. The NEF, REV, or TAT-35 gene may be under the control of the CMV promoter or the RSV-LTR promoter. The vector may further comprise an antibiotic selection of the SV40 early promoter 10705705 (neomycin or kanamycin) to initiate gene expression.

The host cell replicon in the vectors of the invention is preferably pUC-ORI and the selectable markers used are, for example, kanamycin-5 and / or neomycin markers. The intron is preferably beta-globin IVS.

The octase sequence present in TK promoter-based plasmids is the non-coding sequence of the octameric protein. It has no functional purpose in the plasmid, but was needed to create suitable restriction sites for the preparation of the final plasmids.

The NEF, REV, or TAT genes to be inserted into plasmid pBN can be obtained from several commercial sources, such as plasmid pKP59 available from the AIDS Reagent Project MHC. Said genes are generally known and have been fully sequenced (Wain-Hobson et al., Cell 40: 9-17 (1985)). Of course, it is also possible to insert only the sequence encoding the immunologically active fragment of said HIV-15 proteins.

The NEF, REV, or TAT genes or fragments thereof are first inserted into suitable shuttle vectors. These vectors may or may not contain the MO region. The examples illustrate two shuttle vectors: pNp177, which does not contain MO, and pUE83, which contains MO. Of course, it is possible to use other shuttle vectors. Both shuttle vectors and the resultant vectors of the invention are preferably propagated in Escherichia coli. Examples of the resulting pBN-NEF, pBN-REV or pBN-TAT vectors of the present invention are shown in Figures 2, 3 and 4. The vectors of the invention are stable and self-replicating in high copy numbers. After transfection of the Euka-25 ryotic host cell, the vector (plasmid) replicates and produces 100 to 1000-fold amounts of new plasmids, each capable of expressing the desired HIV protein.

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

The expression of the resulting plasmid vectors of the present invention, HIV-NEF, -REV, and -TAT, was tested in both transfected COS-7 cells and mice immunized with said plasmids. High expression of HIV-35 proteins was detectable in COS-7 cells, and the immunized mice exhibited a significant humoral and cell-mediated (CTL) 11105105 immune response. These results indicate that the vectors of the invention have potential use as effective anti-HIV vaccines.

The present invention is further illustrated in the following examples. The examples illustrate in detail some embodiments of the invention, but should not be construed as limiting the invention as defined by the appended claims.

Example 1 Cloning of the HIV-1 genes REV and TAT into self-replicating plasmids pBNsr-α and pBNtk 10 Production of pBNtkREV and pBNsraTAT

Phase 1:

HIV-1 REV and TAT genes from the isolated strain BRU, also referred to as LAI (Wain-Hobson et al., Cell 40: 9-17 (1985)) were amplified from pcREV and pcTAT vectors [ Arya et al. Science 229 15: 69-73 (1985)] using Dynazyme Taq DNA polymerase (Finnzymes,

And the following primers containing the restriction enzyme sites for XhoI and XbaI: REV: 5'-TTTTTCTAGAACCATGGCAGGAAGAAGCGGA-3 '20 5'-TTTTCTCGAGCTATTCTTTAGTTCCTGG-3' TATTGTGTGTGTGTGTGTG CTCGAGCTAAT CGAACGGATCT GC-3 '25

The amplified genes and the shuttle vector pUE83 (Fig. 1) were digested at + 37 ° C with Xbal and Xhol (New England Biolabs, USA) to obtain overnight compatible ends. The digested DNA fragments were analyzed on 1.5% agarose gels and further purified using Band Prep 30 Kit (Pharmacia Biotech, Sweden). Each gene was ligated into the vector individually using T4 DNA ligase (New England Biolabs, USA), incubating overnight at + 16 ° C. The ligation products were transformed into competent E. coli cells (One Shot Kit; Invitrogen, The Netherlands). which were inoculated into LB plates containing kanamycin for selection. Growth clones were miniprep and analyzed for the presence of cloned genes by digestion with XhoI and XbaI. The presence of cloned genes was also confirmed by PCR with clones containing the right gene were mass cultured and plasmids were purified using Megaprep columns (Qiagen, Germany).

Step 2: 5 DNA fragment containing BPVori, RSV-LTR promoter, REV or TAT gene and b-globin IVS-poly (A) was digested from the shuttle vector by HindIII (New England Biolabs, USA) and purified using a 1% agarose gel and Band Prep Kit. The ligation with HindIII digested and dephosphorylated (alkaline phosphatase, CIP, Promega, USA) plasmid pBNsra or pBNtk, cell transformation, presence of the cloned gene, and plasmid purification was performed as in step 1.

The resulting plasmids are called pBNtkREV and pBNsraTAT and are shown in Figures 2 and 3.

Example 2 Cloning of HIV-1 NEF into Self-Replicating Plasmid pBNsraNEF Production of pBNsraNEF

Step 1: The HIV-1 NEF gene was obtained from a plasmid pcNEF vector containing 20 LAI isolates of the NEF gene inserted into the pcTAT vector lacking the TAT gene. The NEF gene used for further cloning was obtained as a 1.3ke fragment of pcNEF by SpeI and HindIII digestion. To eliminate HindIII site remodeling by ligation, the fragment was digested with Klenow after digestion with HindIII and a 1:25 mixture of nucleotides dATP, dCTP and dGTP followed by SpeI digestion. The resulting fragments were electrophoresed on a 1% agarose gel alongside standard size markers. Correct size bands were excised and DNA harvested using the Sephaglas Bandprep Kit (Pharmacia Biotech) according to the manufacturer's instructions.

The shuttle vector pNP177 of Figure 1B was first digested with Xho I, then treated with Klenow and dNTP and finally digested with Xba I. The vector was also treated with calf intestinal alkaline phosphatase (CIP).

The NEF gene containing fragment was ligated with digested pNP177 vector 35 using T4 ligase at +14 ° C overnight. A competent E. coli (One Shot; Invitrogen) was used for transformation. Positive clones were identified by restriction enzyme digestion and electrophoresis. Plasmid DNA was further amplified in E. coli and purified extensively on Qi-Agen columns. The resulting final plasmid was designated pNP177cHIVNEF.

Step 2:

The pNP177 shuttle vector is designed to contain only two Hin-dlll sites between which an insert can be cloned. Thus, digestion of Hind 11 plasmid yields a fragment that can be further cloned. The HindIII fragment of pNP177cHIVNEF was cloned into pBNsra. This vector 10 was digested with HindIII and treated with CIP. The same zone separation, ligation and transformation methods were used as in the first step and the correct orientation of the insert was confirmed by restriction analysis. The final plasmid was designated pBNsraNEF and is shown in Figure 4.

Example 3 Demonstration of HIV Nef Expression in vitro 3A. transfections

To test for the expression of the pBN constructs of Examples 1 and 2, COS-7 cells were transfected using electroporation. 10 μ9 of the comparative plasmid ρΒΝβ-Gal and 10 μ9 of pBN-NEF cotransfected with 1 μ0: η 20 were each electroporated into three million cells. Salmon mammalian carrier DNA was used. Electroporation was performed at a capacitance of 960 mF and a voltage of 260 V. Protein concentration and β-gal activity measurements were performed to control transfection efficiency and calibrate the amount of lysates in the Western blot.

25 3B. Immunohistochemistry and Western blot

Cells transfected with pBN constructs were lysed for use in Western blot. After lysis, protein samples were boiled in sample buffer, treated with 12% SDS polyacrylamide gel, and then transferred to a 2 μπν.η nitrocellulose filter, protected with a solution containing 5% milk in TBS. A mixture of murine monoclonal anti-NEFs [V. Ovod et al., AIDS 6 (1992) 25-34], each diluted 1: 1000. The secondary antibody was a biotinylated anti-mouse antibody diluted 1: 500.

After transfection of 35 COS-7 cells, the vectors induced transient transient HIV regulatory protein expression as detected by Western blot of lysed 14105TQ5 cells after 72 hours. The results obtained with plasmid pBNsraNEF are shown in Figure 5. In long-term cultures of transfected cells, NEF expression lasted for up to 7 weeks in cells transfected with the self-replicating pBN vector.

NEF-transfected cells were also used to prepare cytospin preparations, stained with hematoxylin, and used a monoclonal anti-NEF antibody, followed by a secondary biotinylated anti-mouse antibody, in an immunohistochemical study by Ovodin et al. {supra). The cytospin plates showed expression because of the high density of cells positively staining the posi-10 staining cells. Some of the cells expressing NEF showed morphological signs of cell death, indicating apoptosis. The expression level was still high, even though the cells were deteriorating.

Example 4 Demonstration of In vivo Immunogenicity of HIV-Nef, HIV-Tat or HIV-Rev Expression of pBNsra and pBNtk 4A. The gene cannon DNA was blended onto 1 µm gold particles using spermidine and CaCl2 following the procedure outlined in the Helios Gene Gun Instruction Manual (Bio-20 Rad Laboratories). Each cartridge was made with 0.5 pg of gold and 1 μg of DNA. The amount of DNA was spectrophotometrically controlled as instructed in the instruction manual. Inoculations were performed using the Helios Gene Gun System (Bio-Rad Laboratories). The helium burst pressure to introduce the DNA into the target was adjusted to 2070 kPa (300 psi). At Optimoides-25 moss bombing conditions, we found that 2070 kPa (300 psi) is enough to sweep gold particles into the skin.

4B. Immunizations Female 6- to 8-week-old balb / c mice were used. The mice were anesthetized and depilated on the abdomen before immunization.

Inoculations were performed on the skin of 8 mice on days 1, 2, 3, 10, 11, and 12 I 'using the gene cannon described above and following the manufacturer's instructions.

A total of 6 μg of pBN-NEF per mouse was administered. Four mice from each group were sacrificed two weeks after the last immunization and the remaining four mice four weeks after the last immunization. See-35 rum samples were taken for Western blot analysis and splenocytes collected for CTL assay. All eight mice immunized with pBN-NEF with the 7 ° 5705 vector developed antibody responses after 2 and 4 weeks (Figure 6). The intensity of the reaction varied in the Western blot.

4C. Measurement of cytotoxic T cell activity in immunized mice 5 4C1. Effector cell stimulation

Spleens were aseptically removed from immunized mice two (16 mice) and four weeks (16 mice) after immunization. They were disrupted in Hanks' solution, filtered through a gauze, and erythrocytes were removed. Cells were then resuspended in 5-10® / ml medium: RPMI medium containing 10-10% fetal bovine serum (FCS; GibcoBRL), 1% glutamine, 100 units penicillin / ml, 100 µg streptomycin / ml and? 10mol / L 2-mercaptoethanol Responsive cells (5-106) were cultured in 25ml cell culture flasks in 5ml medium with 4-106 cells presenting antigen (APCs; see below) for 5 days. on day 10 10 to 15 units / ml recombinant interleukin-2 (rIL-2) [J. Hiserodt et al., J. Immunol.

135, No. 1 (July 1985) 53-59; M. Lagranderie et al., J. Virol. 71, No. 3 (March 1997) 2303-2309; T. Tsuji et al., Immunology 90, No. 1 (January 1997) 1-6; Vahlsing H.L. et al., Journal of Immunological Methods 175: 11-22 (1994); K. Varkila et al., Acta Path. Microbiol. Immunol. Scand.

20 Sect. C 95: 141-148 (1987)].

4C2. Cells expressing antigen

Synergistic P815 mastocytoma (H-2d) cells were infected with the modified chicken pox virus Ankara (MVA), which had been engineered to express the HIV-1 LAI NEF gene (MVA-HIVNEF). MVA is a highly attenuated ** 25 replication deficient vaccinia virus that can serve as an efficient vector for expression of heterologous genes and provides an exceptionally high level of biosecurity [G. Sutter et al., J. Virol 68, No. 7 (July 1994) 4109-4116; Sutter et al., Vaccine 12, No. 11 (1994) 1032-1039; I. Drexler et al., J. Gen. Virol. 79: 347-352 (1998); Sutter, G. et al., Proc.

30 Natl. Acad. Sci. USA 89: 10847-10851 (1992)]. Infections with MVA-HIVNEF 7 were performed with an infection rate (MOI) of 5 in 24-well plates (1106 cells / well). After the virus was absorbed for 1 hour at + 37 ° C, cells were incubated for 15 hours at + 37 ° C [A. Carmichael et al., 1996, Journal of Virology 70: 8468-8476]. After infection, the cells were washed twice with 35 PBS (phosphate buffered saline) containing 10% FCS and suspended in this solution at a concentration of 5 to 06 cells / ml. The cells were then gamma irradiated at 5000 rad and washed with medium before addition to the reacting cells.

4C3. Cytotoxicity Assays CTL activity was assayed by the 51 Cr release assay [J. Hiserodt et al., J. Immunol. 135, No. 1 (July 1985) 53-59; M. Lagranderie et al., J. Virol. 71, No. 3 (March 1997) 2303-2309; 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® P-815 cells were infected with MVA-HIVNEF as described above for cells expressing the anti-10 gene. After infection, the cells were washed once with serum-free medium. The target cells were then suspended in 200 μΐιββη serum-free medium and 100 μΟϊ 51Cr (Amersham) / 1106 cells were added for 1 hour at 37 ° C. The target cells were then washed four times in medium and resuspended at 5,104 cells / ml. Stimulated effector cells were washed once in culture medium prior to addition to target cells. The target cells were inoculated into a u-bottom 96-well plate at 100 μΙ (5-103) per well, and the effector cells were added in triplicate with ΙΟΟμΙ ^ βθ medium using effector-to-target ratios of 50, 25 and 12.5. For spontaneous release, the target cells were inoculated into six wells of ΙΟΟμΙιεεθ medium and for maximal release into six wells in 2.5% Triton-X-100 solution. The plates were centrifuged briefly, incubated for 4 hours at 37 ° C, and counted on supernatants in a gamma counter. Percent specific lysis of target cells was calculated by the formula (test-51 Cr release - spontaneous release) / (maximum release - spontaneous release)! 00. Percent specific lysis> 6% was considered positive.

One example showing CTL activity in six of the eight mice immunized with pBNsrcxNEF is shown in Figure 7.

D. Humoral immune response in immunized mice

To test for the presence of HIV-1 anti-NEF antibodies in serum from immunized mice, the NEF protein was electrophoresed on PAGE, transferred to nitrocellulose filters, and antibody reactivity detected as described above.

Summary of results

The results of transfection and immunization assays are summarized in Table 1. 35 Immune response in immunized mice was assessed by immunoblot assay for humoral and cytotoxic T lymphocytes (CTL) in 10 5 cell-mediated immunity. As shown in the table, all eight mice immunized with NEF-expressing vectors exhibited both a humoral and a cell-mediated immune response.

Table 15 Demonstration of HIV-1 NEF, TAT, and REV Protein Expression in COS-7 Cells Transfected with Vectors by Western Blotting (WB) or Immunohistochemical and Human Induced Immunoassay, pBNsraNEF, Immunized Mice 10 ___ __Transfection__Immunization

__WB__Immuunihistokemia__WB__CTL

pBNsraTAT ND __ ++ _ 6/8 6/8 pBNtkREV + __ ++ __ 7 / 8__7 / 8 pBNsraNEF ++ __ ND_ 6/8 6/8

In this study, we demonstrate that DNA immunization using a self-replicating expression vector as described can induce a clearly detectable CTL response in mice. In addition, a humoral immune response was achieved. In light of the above results, it is possible to assume that pBN-NEF, pBN-REV, and pBN-TAT plasmids express NEF, REV, and TAT in vivo in amounts sufficient to induce both a humoral and a cell-mediated immune response. To prevent or treat HIV.

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Claims (11)

A self-replicating recombinant vector, characterized in that it contains papillomavirus nucleotide sequences which essentially comprise (i) a papilloma virus E1 gene and E2 gene, (ii) a minimal replicon of papillomavirus and (iii) a minichromosomal papillomavirus retrieval element, and a heterologous nucleotide sequence encoding the regulatory protein NEF, REV or TAT or immunologically active fragments thereof. Self-replicating vector according to claim 1, characterized in that the papillomavirus is a bovine papillomavirus (BPV). The self-replicating vector of claim 1 or 2, characterized in that the heterologous nucleotide sequence encodes the NEF protein of HIV-1. 4. Self-replicating vector according to which, as claimed in the preceding claims, characterized by controlled by the sra promoter or thymidine kinase promoter. C) the host cell is grown, and D) the vector is utilized. Self-replicating vector according to claim 4, characterized in that it is the pBNt requirement, pBNsrATAT or pBNsraNEF specified in figures 2, 3 or 4. Vaccine for DNA immunization against HIV, characterized in that it contains a self-replicating vector as claimed in claims 1-5. A method for producing a self-replicating vector according to any one of claims 1-5, characterized in that the method comprises the following: A) a heterologous nucleotide sequence encoding the regulatory protein NEF, REV or TAT, or immunologically active fragments thereof, is inserted into a vector containing papillomavirus nucleotide sequences essentially comprising - (i) a papilloma virus E1 gene and E2 gene, - (ii) a minimal papillomavirus replicon and (iii) a minichromosomal papillomavirus retention element, and B) a host cell is transformed with the resulting self-replicating recombinant vector; 8. A method according to claim 7, characterized in that the host cell is an E.co./ cell. Use of a self-replicating vector according to claim 10 of claims 1 to 5 for the preparation of a DNA immunization vaccine against HIV. A host cell, characterized in that it contains a self-replicating vector as claimed in claims 1-5. Host cell according to claim 10, characterized in that it is a bacterial cell or a mammalian cell. • ·