Vaccine containing mutant herpes virus

 

The invention relates to medicine and relates to a vaccine containing mutant herpes virus. The invention includes mutant herpes virus containing the gene, in which viral gene that encodes essential for production of infectious virus protein is delegated or inaktivirovannye so that the virus can infect normal cells, replicate and Express viral genes in these cells, but is unable to produce normal infectious particles, except when the virus infects complementarily cell, which has a heterologous nucleotide sequence, which allows complementary the cell to Express the essential protein encoded by delegated or inaktivirovannye the viral genome. The advantage of the invention is the development of vaccines for prophylactic or therapeutic use in the development of the immune response with the use of live, but weakened virus. 17 C.p. f-crystals., 4 tab., 7 Il.

The present invention relates to viral vaccines. In particular, the present invention relates to genetically engineered mutant virus used for the vaccine.

Viral vaccines are usually divided into two types. The first type are the so-called "dead" vaccines, which are viral drugs, neutralized by treatment with the appropriate chemicals, such as beta-propiolactone. The second type consists of live attenuated vaccines, representing viruses, the pathogenicity of which in relation to the host has been weakened through a specific genetic modification of the viral genome, or a more conventional way - by passage system tissue culture of a certain type. However, each of these two types of vaccines has its own disadvantages. As killed vaccines do not replicate in the host, they must be introduced by injection, and so they can produce the wrong type of immune response. For example, the Salk vaccine, which represents a killed preparation of poliovirus produces the formation of immunoglobulin (Ig)G, but does not stimulate the formation of IgA in the gut, natural foci of primary infection. Therefore, although the vaccine can protect against neurological complications of poliomyelitis, but it does not block the initial infection and is not reported populating immunity". Additionally, killed viruses are not mo is undesirable immunological response against non-structural proteins, produced during replication, it is not possible. These viruses also can not contribute to the production of cytotoxic T cells directed against viral antigens. "Lost" antigens can be captured by antigen-presenting cells and presented to T-cells. However, this presentation is carried by molecules of MHC class II and stimulates the activation of the T-habernig cells. In turn, T-Hibernia cells help b-cells to produce specific antibodies against the antigen.

To stimulate the production of cytotoxic T-cells, viral antigens should be processionary through the particular metabolism inside infetsirovanna cells and presented in the form of a cleaved peptide fragments on MHC molecules of class 1. Obviously, this path decomposition works most effectively for proteins synthesized within infected cells, and therefore, only the virus that is injected into the host cell and expresses immunogenic viral protein capable of generating virus-specific cytotoxic T cells. Therefore, killed vaccines are poor inducers of cell-mediated immunity against viral infection. Hence it is clear that h is e weakened viruses were obtained by removing nonessential gene or by partial damage to one or more essential genes (in this case, the damage carried out so what genes remain functional, but destouet not as effective). However, live attenuated viruses are often left pathogenic and could have a material adverse bleed to influence the owner. In addition, these vaccines, if only their extinction is not caused by a specific division, does not exclude the possibility of reversion to a more virulent form. However, the fact that the owner is producing a certain amount of viral protein means that these vaccines are more effective than killed vaccines, which cannot produce this viral protein.

Live attenuated viruses, which themselves are used as vaccines, may also be used as vaccine vectors for other genes, i.e., in other words, as carriers of genes from the second virus (or other pathogen), against which it is necessary to develop immunity. Usually as vaccine vectors using viruses group of smallpox, for example, vaccinia virus. If the virus used as a vaccine vector, it is important that this virus was not pathogenic action. In other words, it may require loosening the same way weaken simple virus is about to remove the gene from the viral genome and to obtain the so-called "complementarism" cell, who has the virus, resulting from deletion of the gene. This operation was carried out for some viruses, such as adenoviruses, herpes viruses and retroviruses. In the case of adenovirus cell line was transformed with DNA fragments of adenovirus type 5 (Graham, Smiley, Russ ell&Nairn, J. Gen. Virol., 36, 59-72, 1977). This cell line expressed some viral genes and, as has been established, it is able to support the development of viral mutants with delegated go inactivated genes (Harrison, Graham&Williams, Virology 77, 319-329, 1977). Although this virus is well developed in this cell line (lines complementary cells) and has produced a standard viral particles, however, he absolutely did not develop into normal human cells. Cell expressing T-antigen-coding region of the genome of SV40 virus (papovavirus) also have the ability to poddergivat virus replication, specifically delegated in this area (Gluzman, Cell, 23, 182-195, 1981). For herpes simplex virus were produced cell lines expressing the glycoprotein gB (Sa etc., J. Virol., 62, 714-721, 1987), glycoprotein gD (Ligas and Johnson, J. Virol., 62, 1486, 1988) and pretani protein ICP4 (Deluca and others, J. Virol., 56, 558, 1985), and these cell lines were found with the century

The present invention relates to mutant virus for use as a vaccine, where viral gene that encodes a protein responsible for the production of infectious virus is delegated or inaktivirovannye; and where the virus can be cultured in the cell, which has a heterologous nucleotide sequence, allowing the specified cell to Express the necessary protein encoded specified or delegated inaktivirovannye viral genome.

The present invention also relates to the production of the vaccine, which contains the above virus in combination with one or more excipients and/or adjuvants. The viral genome itself may or may not produce the immunogen, or, it can contain the insertion of a heterologous gene, expressing the immunogenic protein.

The present invention also relates to complementary cell transfected with a weakened virus described above and intended for receiving the vaccine.

The present invention also relates to a method, which consists in using the above described virus to obtain a vaccine intended for use in therapeutic or prophylactic purposes.

tivirus cell infected with a virus that has been delegated, or an inactivated viral gene that encodes a protein responsible for the production of infectious virus, where the specified host cell is heterologous nucleotide sequence containing the viral gene, and has the ability to Express the core protein encoded by the specified genome; collect biogas produced thus the virus and use it to vaccines.

This virus can be derived from herpes simplex virus (HSV), in which, for example, delegated or inactivated the gene encoding glycoprotein H(DN). The mutant virus may also contain a heterologous sequence encoding the immunogen derived from the pathogen. The host cell must be recombinant eukaryotic cell line containing the gene encoding glycoprotein H HSV. As another example, you can use the virus derived from an orthopoxvirus such as vaccinia virus, which can also contain a heterologous sequence encoding the immunogen derived from the pathogen.

The present invention illustrates a unique way that combines the effectiveness and safety of the murdered HAC is nerovnoi vaccine. The preferred implementation of the present invention has two distinctive features. First, the selected inactivate gene in the viral genome usually through the implementation of specific deletions. This gene is involved in the production of infectious virus, preferably without interfering with the replication of the viral genome. Thus, the infected cell can produce a greater amount of viral protein from the replicated genetic material, and in some cases can be produced new viral particles, which should not be infectious. This means that the viral infection cannot spread from the inoculation.

The second distinguishing feature of the present invention is a cell, which provides the virus with the product deletirovanie gene that makes it possible to cultivate the virus in tissue culture. Therefore, although this virus will be missing a gene that encodes an important protein under cultivation in a suitable host cell, however, it will multiply and produce viral particles, which in appearance will be indistinguishable from the original virus. This mutant virus, the drug is inactive in the sense of h is about introduction in quantities necessary for the immediate generation of a humoral response in the host, is absolutely secure. Thus, the mutant virus does not have to be infectious for proektiruemoi the host cell, and basically he can just act the same way as standard dead or weakened virus vaccine. However, preferably, the immunizing virus itself was infectious in the sense that he could contact the cell to penetrate it and to initiate a cycle of viral replication, i.e. that he was able to initiate an infection inside the host cell, which belongs to proektiruemy species, and produce in it a certain amount of viral antigen. Thus, it is another opportunity to stimulate cellular Arsenal immune system of the host.

Preferably, delegated, or an inactivated gene was involved in the later phase of the viral cycle, in order to produce as much as possible the amount of viral protein in vivo to induce immunogenic response. For example, the gene may be a gene involved in the packaging or in other postreplicative stage, such as a gene, and then the number expressed in vivo protein will depend on the stage which typically is expressed this gene. In the case of human cytomegalovirus (V), the selected gene may be a gene (except pretannage gene), which will strongly inhibit replication of the viral genome, since pretani gene, which is produced to replicate the viral genome (and in fact is important), is highly immunogenic.

The present invention may be applied to any virus, in which one or a few major genes can be identified and delegated from the viral genome or inactivated in the viral genome. For DNA viruses, such as adenoviruses, herpes viruses, papovaviruses, papilloma virus, parvovirus, this procedure can be carried out directly by (I) in vivo modification of cloned DNA copies of the selected gene in order to obtain specific DNA mutations, and (II) re-introduction of modified variants in the viral genome using standard recombination techniques and "rescue" a genetic marker.

However, in the present invention can also be used and RNA viruses. Then can be applied to any technique that allows the manipulation of complementary DNA copies of the genome of transcriptio. The obtained RNA can then be introduced into the viral genome. This technique was used to obtain specific mutations in the genome as a (+) RNA virus, and (-) RNA virus, such as poliovirus (Racaniello and Baltimore, Science, 214, 916-919, 1981), and influenza virus (Lutyes etc., Cell, 59, 1107-113, 1989).

Theoretically, to obtain attenuirovannogo virus can be selected any gene coding for the main protein. However, in practice, the choice of such a gene should be based on the following considerations:

1. This gene is preferably a gene that is required in a later phase of infection. Thus, replication attenuirovannogo virus is not interrupted in the early phase. This means that most, and possibly the first other viral antigens are produced in the infected cell and presented to the immune system of the host together of molecules MHC class I host cell. This presentation contributes to the development of cellular immunity against viral infection through the production of cytotoxic T cells. Cytotoxic T cells can recognize these antigens and, therefore, to neutralize the virus-infected cells. It is possible that delegated gene should be the same genome, kotoroe to infect new cells. An example of such a protein is a protein Nam virus SV. In the absence of this protein virions SV will still be produced, but they will be non-infectious.

2. In the ideal case, the product selected gene should not be in itself toxic to eucharistically the cell, so that complementary cell could relatively easily produced. However, this requirement is not categorical, since this gene may be placed under the control of the inducible promoter in complementaria the cell, so that its expression could also be included, when needed.

The nature of the mutations generated in the target gene, is also a matter of choice. Any mutation that produces a non-functional gene product, is acceptable only if it does not minimized the risk of reversion to the structure of the wild type. Such mutations can be embedding in the target alien gene sequences and the creation of specific divisions. However, the preferred mutation in the manufacture of vaccines intended for use in therapeutic and/or prophylactic purposes, is a deletion that encompasses the whole posledovatelnosti entered in complementarily glue the control virus and cellular DNA in complementaria the cell.

Although currently there are several examples of specific combinations of inactivated viruses and complementary cells (see above discussion), but they were used either for fundamental research on viruses, or, in the case of retroviruses, for safe vector for producing trangenic animals. These combinations were not used for the production of vaccines and, as far as known to applicants, was not made any assumptions regarding the possibility of their use.

Besides using the specified combination of inactivated virus and complementary cells in order to produce safe vaccines against wild-type virus, the present invention also relates to the use of this system to obtain safe viral vectors for use as vaccines against foreign pathogens. As example can serve as a vector derived from HSV. Virus genome is large enough to accommodate significant additional amount of genetic information, and some examples of recombinant viruses HSV bearing and expressionwhich genetically alien who s in the main viral gene described above, as well as the host and expressing certain foreign gene can be used as a safe vector for vaccination in order to generate an immune response against the foreign protein.

A distinctive feature of the virus HSV is the fact that it can be latent in neurons infected organism, but sometimes it is again reactivated, causing focal lesion. In this way, the virus SV having a deletion in the main virus gene and expressing the foreign gene can be used for producing the desired latent infection of neurons in treated patients. Reactivation of this latent infection should not cause damage, as the viral vector is not able to fully replicate, but may initiate the initial replication cycle of the virus. During this period of time may be the expression of the foreign antigen, thereby generating an immune response. In the case when deleteriously virus HSV gene specifies a protein that is dispensable for virus Assembly, and is only required for the infectivity of precast virus, a foreign antigen can be put into teams of viral particles in order to strengthen them, what also can happen on the stage, where the mutant virus first produced in complementaria the cell, and in this case, the mutant virus used as a vaccine will immediately imagine alien protein obrabatyvaimym species.

In another example, vaccinia virus, poxvirus, can have and Express genes from a variety of pathogens, and this suggests that such vaccines can be effective when used in living experimental systems. The ability to use this virus to humans is quite wide, but because of the known side effects associated with widespread use of vaccinia virus as a vaccine against smallpox, be used in a wide scale unmodified vaccinia virus in humans is undesirable. Attempts were made to weaken the cowpox virus by deletion of nonessential genes, naprimer, gene growth factor cowpox (Buller, Chakrabarti, Cooper, Twardzik & Moss, J. Virolooy, 62, 866-874, 1988). However, these weakened viruses are still able to replitsirovatsja in vivo, albeit at a lower level. But not yet received cowpox virus with a deletion in the main gene, although this virus, i.e., a virus with a deletion in glumeste described strategies of immunization against heterologous protein is that with the same viral vector can be performed multiple effective vaccination, which cannot be done using standard live viral vectors. Since the efficiency standard of live viral vaccines based, probably, on its ability to replicate in the animal host through many cycles of infection, the efficiency will be much shorter in the body, producing immunity against this virus. For example, it is highly likely that the second control infection with the same virus, regardless of whether this busterminal against the same protein, or to produce a new response against another protein, will be ineffective. However, the use of viral vector with a deletion in the main gene in the case where cyclical replication is undesirable or unnecessary, provided effective response almost immediately after immunization. The dose of the mutant virus can be relatively large, because the virus is completely safe, and it is therefore unlikely that such early exposure to mutant virus will be blocked by the immune response of the host, for mobilization which takes some time.

Although all of the above relations is about protein-pathogen however, this mutant may be defective in more than one major gene and/or contain more than one gene for an immunogenic protein of a pathogen. For example, the mutant virus can include a gene for gp 120 of HIV, resulting in a vaccine will act alleged above, as well as the gene for the gag protein of HIV, in order for this protein expressively in the vaccinated host and was presented on the surface of the host cell together on MHC-I to stimulate T-cell response in the host.

For a more visual illustration of the present invention the following examples, which, however, should not be construed as a limitation of the invention; and the following drawings:

In Fig.1 shows the production of plasmids pCHl.

In Fig.2 shows the production of plasmids GH2.

In Fig.3A shows two complementary oligonucleotide used to obtain plasmids S64; Fig.3b shows the production of plasmids S64.

In Fig.4a shows two of the oligonucleotide used to obtain plasmids MVIEP.

In Fig.4B shows a plasmid pCMVIEP.

In Fig.5 shows plasmid MVIacz.

In Fig.6 shows the plasmid pGH3.

In Fig.7 shows the strategy of constructing plasmids G-120.

A simple virus is m, which causes a wide range of pathogenic symptoms in humans, including recurrent lesions of the face and genitals, and often encephalitis fatal. Infection with these viruses can to some extent be solved by chemotherapy using drugs Acrospire, but have not yet found the appropriate vaccine that would prevent primary infection. The difficulty of vaccination against the virus SV is that this virus is mainly spread in the body by direct transfer from cell to cell. Therefore, in this case, humoral immunity is ineffective, because circulating antibodies can neutralize only the extracellular virus. To combat viral infection of this kind would be effective cellular immunity, and preferred would be such a vaccine, which would be able to generate both humoral and cellular immunity, but which would also be safe.

The target gene that is suitable for the inactivation of the genome N is the gene for glycoprotein H (DN). DN-protein is a glycoprotein present on the surface of the viral envelope. This protein apparently involved in the process sarunya mutants with a defect in this gene is not released from virus-infected cells when nopermission temperatures (desai and others, J. Gen. Vir ol., 69, 1147-1156, 1988). If this protein is expressed in the late stage of infection, and if not, there may still be a substantial synthesis of viral protein.

All genetic manipulations were carried out according to standard methods described in "Molecular Cloning", And Laboratory Manual, ed. Sambrook, Fritsch and Maniatis, Cold Spring Harbor Laboratory, 1989.

A. obtaining a cell linnie expressing the gene DN N

Gene bottoms is in the unique long region (UL) of the viral genome SV type I, between nucleotides 46382 and 43868 (cGch etc., J. Dept. Virol. 69, 1531-1574, 1988). A cloned copy of the gene present in the plasmid AF2. This plasmid was obtained by cutting out BglI-XhoI-fragment covering the full DN-coding sequence from the plasmid pTZg, and cloning in WD-site plasmids S64 described Gompels and inson (J. Virol., 63, 4744-4755, 1989). Then from plasmids SVD4 (Everett NucI. Acids Res., II, 6647-6667, 1983) cut Hindlll fragment containing the promoter sequence for a gene glycoprotein D(gD) (extending from nucleotide -392 to +11 with respect to the beginning of the gD gene), and cloned into the unique HindIII site F2 for producing pGHI (Fig.1), so that the promoter sequence was in the correct orientation required for stimulirovalis promoter gene gD of HSV type I. Then, this plasmid was purified and was transferrable in Vero cells together with a plasmid pNEO (Pharmacia LKB Biotechnology Inc.) using standard techniques of joint deposition of calcium phosphate (Graha and Ver der Eb, Virology 52, 456-467, 1973). Vero cells with acquired resistance to nemesio then selected by passage of the cells in the drug environment G418 and colonies of these cells cloned ways the final cultivation. These neomycin-resistant cells were then amplified in tissue culture, after which the samples were infected by the virus HSV type 2. An infection by the virus HSV type 2 contributes to the induction of transcription from the promoter gD type I present in the genome complementary cells, and stimulates the production of protein Nam type I in complementary cell. Lysates of infected cells were then skanirovali on the expression of DN-protein using Western blotting, using polyclonal anticigarette, which is known to be capable of specific recognition DN-protein type I (Desei and others, 1988, see above). Cells that Express a desired protein, then kept, and to prepare the frozen stock. This material represented DN + line complementary cells.

C. Obtaining virus SV type I with prervanny the mi sequences N, cut out from the plasmid pUG102 (Gompel and inson, Virology 153, 230-247, 1986) and cloned in plasmid rat (twigg and Sherrat, Nature, 283, 216, 1980), resulting in the obtained plasmid G2 (Fig.2). This plasmid was digested PvuII, and cut it only DN-coding sequences in two positions (nucleotides 44955 and 46065) according to the numbering scheme McGeoch and others, 1988, see above), and the largest of the two fragments were purified. The DNA fragment containing the complete gene for B-galactosidase from E. coli below (in forward direction) from the promoter mind gene, derived from cytomegalovirus (CMV), were then obtained using the following procedure. First of all annealed two complementary oligonucleotide (Fig.3A) and ligated with BgIII-digested and processed fosfates the plasmid S64 Krieg and Melton, NucI. Acids Res., 12, 7057-7071, 1984), resulting in the obtained plasmid SP64 shown in Fig.3V. Attached linker also contained the initiating codon and the first three codon of the gene for b-galactosidase (Iacz) E. coli. Then "Serafina" promoter pretannage gene from CMV amplified from plasmid UG-HI (Gompels and inson, 1988, see above) using polymerase chain reaction (PCR Molecular loning, ed. Sambrook and others, see above), where we used two of the oligonucleotide shown in Fig.4A, and zootech pretannage gene CMV, described Apgd etc., Virus Rsreh, 2, 107-121, 1985). These oligonucleotides also contain at their 5 ends of the restriction sites for the enzyme HindIII, and in the case of annealing of the oligonucleotide above from the promoter and even Smal site. Then PCR-amplificatory product DNA was digested HindlII, and cloned into HindIII-digested rrta, resulting in the obtained plasmid pCMVIEP (Fig.4B). And, finally, a DNA fragment that contains a complete copy of B-galactosidase, which is missing only the extreme 5'-terminal region of the coding sequence, was isolated by digesting the plasmid S8 (Chakzabarti and others, Mol. Cell. Biol., 5, 3403-3409, 1985) enzyme BamHI and cloned into the unique BoIII website pCMVIEP, resulting received pCMVlacz (Fig.5), a DNA fragment containing the gene for B-galactosidase under the control of the CMV IE promoter, was then isolated by digestion of the plasmid pCMV-Iacz enzyme Smal and ligated with purified PvuIII-fragment of plasmid G2 described above, resulting in the obtained plasmid GH3, which contained a copy of the DN-gene, interrupted funcionally gene B-galactosidase (Fig.6). The next stage was to replace the Nam-wild-type gene in the genome of HSV obtained interrupted by a mutant, and this stage osushestvljali using recombination between HSV-DNA and plasmid G3 followed was otbrosami in cells expressing DN-gene (DN+complementary cell line described in part A), together with purified HSV DNA isolated from purified HSV virions (Killington and PoweII "Technigues in Virology: A practical Appzoach" (ed. C. W. C. h, pp. 207-236, IPL Press, Oxford (1985)), using standard techniques of deposition of calcium phosphate (Grahame and Van de Eb, 1973, see above). Progeny virus HSV, resulting from the described experiment, transfection, and then sown on monolayers DN+ complementary cells, conducting the standard analysis belascoaran and using top agar layer to cover, in the presence of 5-bromochloro-3-indolyl--D-galactoside (X-Dai), a chromogenic substrate, which in the presence of-galactosidase was purchased blue color.

Thus, the plaques resulting from infection viral genomes containing and expressing the gene for b-galactosidase, was blue. This meant that these viral genomes must contain option interrupted DN-gene. Then the virus was purified from these plaques by removing agar plugs from the corresponding part of the tablets, and viral billet is produced by culturing the virus in DN+complementary cell lines. These viruses, which, is the quiet do not contain or Express endogenous functional copy of DN-gene, and to confirm this fact, the samples were analyzed for their ability to form plaques in monolayers of wild-type cells of the Vero line compared to the DN-complementarity cells. And finally, from these blanks received viral DNA and tested on the expected structure of the DNA near the bottom-about gene using southern blotting. After confirming the genetic structure to prepare large stocks of the virus with scarce DN-genome by inoculation of the virus in large-scale culture DN+ comlementary cell lines (multiplicity of infection=0.01) and after three days the cells were collected. Infected cells were destroyed on ultrasound for release associated with cells of the virus, and the entire mixture is treated with ultrasound, and kept at -70in the form of viral uterine preparation. The titer of this virus preparation was then established using analysis of belascoaran on DN+complementary cell lines. After that the samples of this viral drug used for work preparations described above, then these work drugs used is compared with the virus, neutralized the effects of heat

For analysis of the immunological response of the host to the virus have conducted experiments on the control infected mice in accordance with the scheme described below.

Then compare the protective effect of living gH--viral drug action inactivated preparation of wild-type virus (WT) (strain SCI6). Receipt of inactivated wild-type virus for vaccination

The virus HSV type I (strain SC16) were cultured at low multiplicity of infection (0,01.about.e/cell) cell line Vero. Three days later, the virus was harvested and cytoplasmic virus was isolated by using a homogenizer of the downs. Nuclei were removed by centrifugation for 15 minutes at 500g, and the virus was isolated from the supernatant by centrifugation in a sucrose gradient (40%) at 12 K for 60 min, using a rotor Beckman SW27. Stripe virus bred, besieged and purified by centrifugation in a sucrose gradient (Killington and Powell, see above). The band of the virus were collected from the gradient, the virus was isolated by centrifugation. The virus then resuspendable in phosphate-buffered saline (PBS), were analyzed for infectivity by plaque titration on cells of the kidney ditanya hamster (KSS), and the province of the CI/B. about.E. The virus was diluted to 2.51010B. about.E./ml in PBS, and iactiveaware by processing-propiolactone at 20C for 60 minutes. The resulting aliquots were then stored at -70C.

Getting the living Nam--virus for vaccination

This material was obtained in the same way as the wild-type virus, except that this virus was cultured in gH+ complementary cell lines containing and expressing DN-HSV gene wild type, and was not subjected to inactivation by processing-propionolactone. The attitude of "particle: infectivity" this drug was 150:1. The concentration of this drug brought up to 2,51010B. about.E./ml, and aliquots were stored in PBS at -70C.

Scheme vaccination

4-week-old female mice lb/S (supplied Tucksv. k. Ltd.) subjected to vaccination with different doses of inactivated wild-type virus or a live virus gH-2 μl-volume of phosphate-saline buffer by drip application and scarification needle right ear according to the following scheme:

Group And the Control was not treated with virus

Group 5104B. about.E. f">106B. about.E. viral vaccines

Group E 5107B. about.E. viral vaccines

After 14 days, all mice were subjected to control infection by a similar inoculation left ear 2106B. about.E. strain SCI6 HSV-I (wild-type virus). After 5 days, mice were killed and analyzed for viral infectivity in the left ear and left cervical ganglia S, III and IV (combined data). To study latent States, other vaccinated and subjected to the control and infected animals were scored after 1 month and suffered from a latent infection by excision of gingly cII, cIII and cIV. This tissue is incubated for about 5 days, and then homogenized and evaluated for the presence of infectious virus by standard analysis belascoaran. All the results were expressed in B. about.E./body (plaque-forming units is not the body).

the combined data for cervical ganglion cII, cIII and cIV (b.about.E. - blastobasidae unit; DN-virus with defective gH-gene).

The results show that the titer of virus infection control die indicates good performance of the schemes of vaccination virus Nam-while a higher titer indicates lower efficiency. From the obtained results show that vaccination living gH-virus KSV significantly more effective than the equivalent amount of inactivated virus wild type. In the case of inactivated preparation, to prevent replication in the ear virus infection control is required dose 5107B. about.E., and in the case of living qH-virus this virus dose required 100-1000 times less. Vaccination living gH-virus at a dose of 5105B. about.E. and above was also able to block viral replication control of infection in the cervical ganglia during acute phase of infection and, in addition, clearly showed protective activity against activation of latent infection in the cervical ganglia.

The virus HSV, which has no DN-gene and which is used as a vector for immunization against foreign antigen: introduction Dr gene of strain 142SIVmac in the genome gH-virus HSV

As described above, the viruses with divisions in major genes can be used as a safe vectors for the presentation of foreign antigens to the immune system, and as a suitable prospect is hurt any foreign antigen, but especially attractive is the ability to Express the major antigenic proteins of human immunodeficiency virus (HIV) that causes AIDS. For example, these sequences can be entered in qH--HSV genome to provide their expression during infection of normal cells (i.e., complementary cells) recombinant virus. The organism infection the virus leads to latent infection, which after some time reactivated, which contributes to a sharp increase in the production of foreign antigen and thereby stimulation of the immune response to this protein.

Because conduct research to test this method on man not possible, as the initial stage of the study, this method can be tested on monkeys using monkey AIDS virus (SIVmac, simian immunodeficiency virus, obtained from macaques). Suitable for this purpose SIV genome is a gene that encodes a protein gp120, one of the major antigenic targets for this virus. Therefore, this gene was introduced in gH--HSV genome, after which the analysis was performed on the effectiveness of this virus as a vaccine in developing a well adjacent the promoter of the cytomegalovirus IE (Gompels and Minson, 1989, see above), and then a DNA cassette containing the gp120 gene and located above the CMV-promoter cloned in plasmid G2 (Fig.2). The obtained plasmid then was co-transferrable in DN+ complementarily cell line with DNA purified from gH-SV, and recombinant virus that instead of the gene-galactosidase present in the DN-HSV-virus, acquired Geng or, was isolated by screening for the presence of the gene-galactosidase.

A. Construction of plasmids for recombination SIV-gp120 coding sequence in the genome SV.

The full scheme of this procedure is shown in Fig.7. Fragment restricteduser enzyme Sacl (corresponding to bases 5240-8721) cut from a cloned DNA copy of the genome SIV (Chakrabarti and others, Nature 328, 543 (1987) and cloned into the Sacl website plasmids V118 (Viera and Messing, Methods in Enzymology, 153, 3, 1987) to generate the plasmid pSIVI, which can then be transformed into single-stranded DNA manipulation using site-directed mutagenesis. Then this DNA region, which includes env-SIV (located between 6090-8298), modified using site-directed mutagenesis (Brierley and others, Cell, 57, 537, 1989) in shares of the injection site for restricteduser enzyme EcoRV in the provisions of the regulations 7671-7676 within the env gene SIV, which correspond to the cleavage site between gp120 and gp40-domains env gene sequences using synthetic oligonucleotide

5'AAGAAGGTGTG,

as a result, we obtained a plasmid pSlV2. Then ways digestion SIV2 enzyme EcoRV were obtained DNA fragment (1617 base pairs) corresponding to the gp120-part env-gene SIV.

Region of the core promoter mind CMV gene was obtained from plasmid VG-HI (Gompels and Minson, 1989, see above) using polymer eco-chain reaction using the following two synthetic oligonucleotides:

primer above (in the direction 53)

primer below

Then the product of this reaction is hydrolyzed by the enzymes EcoRI and HindIII to obtain a DNA fragment, which is then cloned in RI - and HindIII-digested plasmid RMS, resulting in the obtained plasmid pCMVIE2, which had a unique smal site, located directly below the promotor sequence of the CMV. EcoRV fragment containing SIVmac. gp120-encoding sequence obtained in accordance with the description above, then cloned into the specified Smal site then selected the setup portion of the sequence from the promoter. Then, this plasmid was digested by the enzyme RV, resulting in the obtained DNA fragment with blunt ends, which contains SIV sequence together with CMV-promoter, and which is then cloned into PVuII-digested plyamide pGH2 (Fig.2) to obtain plasmid GH-120.

C. Construction of recombinant DN-HSV carrying the gene gp120S1

From DN-virus SV, skontrolovane in cootvetstvii with a detailed description, given above, was isolated DNA, which was transferrable in DN+complementaria cells together with purified DNA G-120. Virus progeny resulting from this transfection was passively on monolayers DN+complementary cell lines using standard analysis belascoaran, as described above, and using top agar in the presence of X-gal. Parent gH-virus contained functional gene-galactosidase, located inside the residual DN-coding sequences, and formed in the presence of X-gal blue plaques. However, recombinant viruses, which, instead of the gene-galactosidase acquired SIV-gp120-encoding sequence, produce white plaques. Then the virus was isolated from these white plaques by removing agar tubes, and prepared virusnyye received DNA which was tested for the presence of the correct DNA structure near the bottom-gene through southern blotting using appropriate probes derived from the coding sequence of SIV. And, finally, received viral preparations in accordance with the above description, which can then be used for studies on vaccination of animals.

Vaccine containing attenyerevan virus, can be obtained and used in accordance with standard techniques, well known to specialists. For example, the vaccine may also contain one or more fillers and/or advantiv. Effective dose attenuating virus contained in accine may be determined by conventional methods well known in the art.

Claims

1. Vaccine containing mutant herpes virus, for prophylactic or therapeutic use in the development of immune response to the herpes virus wild type or another alien pathogen, characterized in that the mutant herpes virus contains a gene in which a viral gene that encodes essential for production of infectious virus protein is deletionism or ironnie genes in these cells, but unable to produce normal infectious particles, except when the virus infects complementarily cell, which has a heterologous nucleotide sequence, which allows complementary the cell to Express the essential protein encoded by the specified deletionism or inactivant the viral genome, and these normal cells are other than specified complementary cell.

2. The vaccine under item 1, characterized in that, when mutant virus infects normal cells, are produced by non-infectious viral particles.

3. The vaccine under item 1 or 2, characterized in that further comprises a pharmaceutically acceptable filler.

4. The vaccine according to any one of paragraphs.1-3, characterized in that the specified deleteriously or inactivated gene is functionally late in the viral cycle.

5. Vaccine for p. 4, characterized in that the gene encodes a protein that is involved in postreplicative event.

6. Vaccine for p. 5, characterized in that the specified deleteriously or inactivated gene is involved in the packaging.

7. The vaccine according to any one of paragraphs.1-3, characterized in that the gene encodes a protein that is not the TCI.

8. The vaccine under item 1, characterized in that it consists essentially of a pharmaceutically acceptable excipient and an effective immunizing amount of the indicated mutant herpes virus.

9. The vaccine according to any one of paragraphs.1-8, characterized in that the mutant herpes virus has the ability to establish latent infection with periodic reactivation.

10. The vaccine according to any one of paragraphs.1-3, characterized in that the specified deleteriously or inactivated gene is a gene glycoprotein.

11. The vaccine according to any one of paragraphs.1-7, characterized in that the herpes virus is a herpes simplex virus.

12. The vaccine under item 7, characterized in that the specified deleteriously or inactivated gene is a gene gH.

13. The vaccine under item 12, characterized in that it includes a dose containing from about 5 to104to approximately 5107The BATTLE of the indicated mutant virus.

14. Vaccine for p. 13, characterized in that it includes a dose containing from about 5 to104to approximately 5106The BATTLE of the indicated mutant virus.

15. The vaccine under item 14, characterized in that it includes a dose containing from these mutant virus.

16. The vaccine according to any one of paragraphs.1-15, characterized in that the mutant herpes virus is defective in more than one gene essential for production of infectious virus.

17. The vaccine according to any one of paragraphs.1-16, characterized in that the mutant herpes virus carries the genetic material encoding the immunogen of the pathogen, exogenous with respect to the specified virus and mutant herpes virus during infection with specified normal cells in which the mutant virus is not able to cause the production of new infectious virus particles, is able to Express the specified genetic material.

18. The vaccine under item 17, characterized in that the mutant herpes virus causes the immune system to a specific pathogen in susceptible species immunized with them.

 

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