RU2236256C2 - Mutant herpes virus-comprising vaccine - Google Patents

Abstract

FIELD: medicine, virology, vaccines.

SUBSTANCE: invention relates to vaccine comprising mutant herpes virus. Invention involves mutant herpes virus comprising genome wherein viral gene encoding protein that is essential for producing infectious virus is to be deleted or inactivated and therefore virus is able to infect normal cells, to replicate and to express viral genes in these cells but it can't produce normal infectious particles, except for cases when virus infects the complementary cell that has heterologous nucleotide sequence that provides expressing the essential protein by the complementary cell and this protein is encoded by deleted or inactivated viral gene. The advantage of invention involves the development of vaccine used for prophylactic or therapeutic using in producing the immune response with using live but attenuated virus.

EFFECT: valuable properties of vaccine.

18 cl, 4 tbl, 7 dwg, 1 ex

Description

The present invention relates to viral vaccines. In particular, the present invention relates to genetically engineered mutant viruses for use as vaccines; to vaccines containing mutant viruses; to recombinant cells; to methods for producing vaccines.

Viral vaccines are usually divided into two types. The first type includes the so-called "killed" vaccines, which are viral preparations that have been neutralized by treatment with appropriate chemicals, such as beta-propiolactone. The second type includes live “attenuated” vaccines, which are viruses whose pathogenicity with respect to the host has been attenuated using a specific genetic modification of the viral genome, or in a more usual way - by passing a specific type of tissue culture into the system. However, each of these two types of vaccines has its own drawbacks. Since killed vaccines are not replicated in the host, they must be administered by injection, and therefore they can produce the wrong kind of immune response. For example, the Salk vaccine, which is a killed poliovirus preparation, produces the formation of immunoglobulin (Ig) G, but does not stimulate the formation of IgA in the gut, the natural focus of the primary infection. Therefore, although this vaccine can protect against neurological complications of polymyelitis, however, it does not block the primary infection and does not report “popular immunity”. In addition, killed viruses cannot invade and replicate within host cells. Therefore, to obtain using them any desired immunological response against non-structural proteins produced during replication is not possible. These viruses also cannot contribute to the production of cytotoxic T cells directed against viral antigens. “Dead” antigens can be captured by antigen-presenting cells and presented by T cells. However, this presentation takes place through MHC class II molecules and stimulates the activation of T-herperic cells. In turn, T-herperic cells help B-cells to produce specific antibodies against antigen.

To stimulate the production of cytotoxic T cells, viral antigens must be processed through particular metabolism within the infected cell and presented as cleaved peptide fragments on MHC class 1 molecules. Obviously, this decomposition pathway works best for proteins synthesized inside the infected cell, and therefore, only a virus that is introduced into host cells and expresses an immunogenic viral protein is capable of generating virus-specific cytos toxic T cells. Therefore, killed vaccines are poor inducers of cellular immunity against viral infection. It appears that the use of live attenuated vaccines is more preferable.

To date, live attenuated viruses have been obtained by removing the non-essential gene or by partially damaging one or more essential genes (in this case, the damage is carried out in such a way that the genes remain functional, but are not so effective). However, live attenuated viruses often remain pathogenic and can adversely affect the host. In addition, in these vaccines, unless their weakening is caused by specific division, the possibility of reversion to a more virulent form is not excluded. However, the fact that a certain amount of viral protein is produced in the host means that these vaccines are more effective than killed vaccines that cannot produce this viral protein.

Live attenuated viruses, which themselves are used as vaccines, can also be used as “vaccine vectors” for other genes, that is, in other words, as carriers of genes from a second virus (or other pathogen) against which it is necessary to develop immunity. Typically, smallpox viruses, such as vaccinia, are used as vaccine vectors. If the virus is used as a vaccine vector, it is very important that this virus does not have a pathogenic effect. In other words, it may be necessary to weaken it in the same way that a simple viral vaccine is weakened. Therefore, in this case, there are disadvantages described above.

It has been found that it is possible to remove a gene from the viral genome and obtain a so-called “complementing” cell that has a virus resulting from a deletion of the gene. The indicated operation was carried out for some viruses, for example adenoviruses, herpes viruses and retroviruses. In the case of adenoviruses, the human cell line was transformed with DNA fragments of type 5 adenovirus (Graham, Smiley, Russell & Nairn, J. Gen. Virol., 36, 59-72, 1977). This cell line expressed some viral genes and has been found to be capable of supporting the development of viral mutants having delegated or inactivated genes (Harrison, Graham & Williams, Virology 77, 319-329, 1977). Although this virus developed well on this cell line (complementing cell line) and produced standard viral particles, it did not develop on normal human cells at all. Cells expressing the T-antigen-coding region of the genome of the SV40 virus (papovavirus) also have the ability to support the replication of viruses specifically delegated in this region (Gluzman, Cell, 23, 182-195, 1981). For the herpes simplex virus, cell lines expressing gB glycoprotein (Cai et al., J. Virol., 62, 714-721, 1987), gD glycoprotein gD (Ligas and Johnson, J. Virol., 62, 1486, 1988) were produced. and pre-early protein ICP4 (Deluca et al., J. Virol., 56, 558, 1985), and these cell lines have found the ability to support viral replication with specifically inactivated copies of the corresponding genes.

The present invention relates to a mutant virus for use as a vaccine, where the viral gene encoding the protein responsible for the production of the infectious virus is delegated or inactivated; and where the specified virus can be cultured in a cell that has a heterologous nucleotide sequence that allows the specified cell to express the desired protein encoded by the specified delegated or inactivated viral gene.

The present invention also relates to the production of a vaccine that contains the above virus in combination with one or more excipients and / or adjuvants. The viral genome may itself produce an immunogen, or it may contain a heterologous gene insert expressing the immunogenic protein.

The present invention also relates to a complementing cell transfected with the attenuated virus described above and intended to produce a vaccine.

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

The present invention also relates to a method for producing a vaccine, which comprises cultivating a cell infected with a virus having a delegated or inactivated viral gene encoding a protein responsible for producing an infectious virus, wherein said host cell has a heterologous nucleotide sequence containing a viral gene, and has the ability to express the basic protein encoded by the specified gene; the virus thus produced is collected and used for the vaccine.

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

The present invention illustrates a unique method that combines the efficacy and safety of a killed vaccine with a high immunological response induced by the in vivo production of a viral protein by an attenuated vaccine. A preferred embodiment of the present invention has two distinguishing features. First, the selected gene is inactivated in the viral genome, usually by performing a specific deletion. This gene will participate in the production of the infectious virus, preferably without interfering with the replication of the viral genome. Thus, an infected cell can produce more viral protein from replicated genetic material, and in some cases, new viral particles can be produced that should not be infectious. This means that a viral infection cannot spread from the site of inoculation.

The second distinguishing feature of the present invention is a cell that produces a virus with a deleted gene product, which makes it possible to cultivate the virus in tissue culture. Therefore, although the gene encoding an important protein will not be present in this virus when cultured in a suitable host cell, it will multiply and produce viral particles, which in their appearance will be indistinguishable from the original virus. This mutant viral preparation is inactive in the sense that since it has a defective genome and cannot produce an infectious virus in a normal host, its introduction in the amounts necessary to directly generate a humoral response in the host is absolutely safe. Thus, a mutant virus does not have to be infectious for the protected host cell, and basically it can simply act in the same way as a standard killed or attenuated viral vaccine. However, it is preferable that the immunizing virus itself is infectious in the sense that it can bind to the cell, invade it and initiate a viral replication cycle, i.e. so that he is able to initiate an infection inside the host cell belonging to the protected species and produce a certain amount of viral antigen in it. Thus, another opportunity presents itself to stimulate the cellular arsenal of the host immune system.

Preferably, the delegated or inactivated gene is involved as late as possible in the viral cycle so as to produce as much viral protein as possible in vivo to induce an immunogenic response. For example, this gene may be a gene involved in packaging or in another post-replicative stage, for example, such as the HSV glycoprotein gH gene. However, the selected gene may be a gene involved in the replication of the viral genome, and then the amount of expressed in vivo protein will depend on the stage at which this gene is usually expressed. In the case of using human cytomegalovirus (HCMV), the selected gene can be a gene (except for the early gene), which will greatly impede the replication of the viral genome, since the early gene that is produced before the viral genome is replicated (and in fact is the main one for it) is highly immunogenic.

The present invention can be applied to any virus in which one or more 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 viruses, and parvoviruses, 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-introducing the modified variant into the viral genome using the standard technique of recombination and "rescue" of the genetic marker.

However, RNA viruses can also be used in the present invention. Then any technique can be applied that allows you to manipulate complementary DNA copies of the RNA virus genome in vitro using standard genetic technology, followed by conversion to RNA by in vitro transcription. The resulting RNA can then be reintroduced into the viral genome. This technique has been used to generate specific mutations in the genome of both (+) RNA virus and (-) RNA virus, such as poliovirus (Racaniello and Baltimore, Science, 214, 916-919, 1981), and influenza virus (Lutyes et al., Cell, 59, 1107-113, 1989).

Theoretically, any gene encoding a major protein can be selected to produce an attenuated virus. However, in practice, the choice of such a gene should be based on the following considerations:

1. This gene should preferably be a gene that is necessary in the later phase of the infection. Thus, the replication of the attenuated virus is not interrupted in the early phase. This means that most, and possibly other, viral antigens will be produced in the infected cell and presented to the host immune system together with the MHC molecules of class I of the host cell. Such a presentation promotes the development of cellular immunity against viral infection through the production of cytotoxic T cells. Cytotoxic T cells can recognize these antigens and therefore neutralize virus infected cells. It is possible that the delegated gene should be one that is completely optional for the assembly of the virus, but which is necessary for the assembled virus to be able to infect new cells. An example of such a protein is the gH protein of the HSV virus. In the absence of this protein, HSV virions will still be produced, but they will be non-infectious.

2. Ideally, the product of the selected gene should not be toxic in itself to the eukaristic cell, so that the complementing cell can be relatively easily produced. However, this requirement is not categorical, as this gene can be placed under the control of an inducible promoter in a complementing cell, so that its expression can be switched on when necessary.

The nature of mutations created in the target gene is also a matter of choice. Any mutation that produces a non-functional gene product is perfectly acceptable, unless the risk of reversing to the wild-type structure is minimized. Such mutations can be the incorporation of foreign sequences into the target gene and the creation of specific divisions. However, the most preferred mutation in the manufacture of a vaccine intended for use in therapeutic and / or prophylactic purposes is a deletion that encompasses the entire sequence introduced into the complementing cell. This technique minimizes the risk of wild-type virus regeneration through recombination between the virus and cellular DNA in a complementing cell.

Although there are currently several examples of combinations of specifically inactivated viruses and complementing cells (see discussion above), they have been used either for basic research on viruses, or, in the case of retroviruses, to obtain a safe vector for the production of transogenic animals. The indicated combinations were not used for the production of vaccines and, to the best of the applicants' knowledge, no assumptions were made regarding the possibility of such use.

In addition to using the indicated combination of inactivated virus and complementing cell in order to produce safe vaccines against the wild-type virus, the present invention also relates to the use of said system for producing safe viral vectors for use as vaccines against foreign pathogens. An example is a vector derived from HSV. The virus genome is large enough to accommodate a significant additional amount of genetic information, and several examples of recombinant HSV viruses carrying and expressing foreign genetic material have been described in the literature (e.g., Ligas and Johnson, J. Virol. 1988, supra). So, for example, a virus having a deletion in the main viral gene described above, as well as carrying and expressing a certain foreign gene, can be used as a safe vector for vaccination in order to generate an immune response against a foreign protein.

A distinctive feature of the HSV virus is the fact that it can be latent in the neurons of an infected organism, but sometimes it reactivates again, thereby causing a focal lesion. Thus, an HSV virus having a deletion in the main gene virus and expressing a foreign gene can be used to produce the desired latent infection of neurons in the patient being treated. Reactivation of such a latent infection should not cause damage, since the viral vector is not able to fully replicate, but can initiate the initial cycle of virus replication. During this period of time, expression of a foreign antigen may occur, thereby generating an immune response. In the event that the deleted HSV virus gene determines a protein that is optional for the assembly of the virus, and is only necessary for the infectivity of the assembled virus, a foreign antigen can be introduced into the assembled viral particles in order to enhance the immunogenic effect. This expression of a foreign gene and its introduction into a viral particle by a protein can, of course, also occur at the stage where the mutant virus is first produced in a complementing cell, in which case the mutant virus, when used as a vaccine, will immediately represent the foreign protein to the species being processed.

In another example, the vaccinia virus, poxvirus, can have and express genes from various pathogens, and this suggests that such vaccines can be effective when used in living experimental systems. The possibilities for using this virus for humans are quite wide, but due to the known side effects associated with the widespread use of vaccinia virus as a vaccine against smallpox, it is undesirable to use a widespread unmodified vaccinia virus in humans. Attempts have been made to attenuate the vaccinia virus by deletion of non-essential genes, for example, the vaccinia growth factor gene (Buller, Chakrabarti, Cooper, Twardzik & Moss, J. Virolooy, 62, 866-874, 1988). However, such attenuated viruses are still able to replicate in vivo, albeit at a lower level. But so far, no vaccinia virus has been obtained with a deletion in the main gene, although it is such a virus, i.e. a deletion virus in the main gene described above would provide a safe variant of this vaccine vector.

Another advantage of the described immunization strategy against heterologous proteins is that multiple effective vaccinations can be carried out with the same viral vector, which cannot be done using standard live viral vectors. Since the effectiveness of a standard live viral vaccine is probably based on its ability to replicate in an animal host through many infection cycles, this effectiveness will be greatly reduced in the body, which produces immunity against this virus. So, for example, it is very likely that a second control infection with the same virus, regardless of whether this booster immunization is carried out against the same protein, or in order to provoke a new response against another protein, will be ineffective. However, the use of a viral vector with a deletion in the main gene in the case where multicyclic replication is undesirable or optional gives an effective response almost immediately after immunization. The dose of the mutant virus can be relatively large, since this virus is absolutely safe, and therefore it is unlikely that such an early exposure to the mutant virus will be blocked by the host's immune response, which takes some time to mobilize.

Although all of the above refers to a mutant virus having a division in the main gene and optionally containing the gene for the immunogenic pathogen protein, this mutant may be defective in more than one main gene and / or contain more than one gene for the immunogenic pathogen protein. So, for example, a mutant virus can include a gene for HIV gp 120, as a result of which the vaccine will act in the manner proposed above, as well as a gene for HIV gag protein, so that this protein is expressed in the vaccinated host and presented on the surface of the host cells together about MHC-I in order to stimulate a T-cell response in the host.

To illustrate the present invention more clearly, examples are provided below, which, however, should not be construed as limiting the invention; as well as the following drawings:

Figure 1 shows the production of the plasmid pCHl.

Figure 2 shows the production of plasmid pGH2.

Figure 3a shows two complementary oligonucleotides used to produce plasmid pSP64Ta; Figure 3c shows the production of the plasmid pSP64TA.

Figure 4a shows two oligonucleotides used to obtain plasmid pCMVIEP.

Figure 4c shows the plasmid pCMVIEP.

Figure 5 shows the plasmid pCMVIacz.

Figure 6 shows the plasmid pGH ₃ .

7 shows a strategy for constructing plasmid pGH-120.

Herpes simplex virus deleted in the glycoprotein H gene (gH-HSV)

Herpes simplex virus (HSV) is a large DNA virus that causes a wide range of pathogenic symptoms in humans, including recurrent lesions of the face and genitals, and often fatal encephalitis. Infection with these viruses can be eliminated to some extent with chemotherapy using the drug Acyloquira, but no suitable vaccine has yet been found to prevent the primary infection. The difficulty in vaccinating against the HSV virus lies in the fact that this virus is mainly spread in the body by direct transfer from cell to cell. Therefore, in this case, humoral immunity will be ineffective, since circulating antibodies can only neutralize the extracellular virus. Cellular immunity would be effective for fighting this kind of viral infection, and a vaccine would be preferable that would be able to generate both humoral and cellular immunity, but which would also be safe.

The target gene suitable for inactivation in the H genome is the glycoprotein H (gH) gene. The gH protein is a glycoprotein present on the surface of the viral envelope. This protein is apparently involved in the process of membrane fusion during the introduction of the virus into the infected cell. Therefore, temperature-sensitive viral mutants with a defect in this gene are not ejected from virus-infected cells at non-permissive temperatures (desai et al., J. Gen. Vir ol., 69, 1147-1156, 1988). If this protein is expressed at a late stage of infection, as well as if it is absent, then significant synthesis of the viral protein may still occur.

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

A. Obtaining a cell line expressing the gene gH N

The gH gene is located in the uniquely long region (U _L ) of the HSV type I virus genome, between nucleotides 46382 and 43868 (McGeech et al., J. Gen. Virol. 69, 1531-1574, 1988). A cloned copy of this gene is found in plasmid pAF2. This plasmid was obtained by excising a BglI-XhoI fragment encompassing the complete gH coding sequence from the plasmid pTZgH and cloning it into the BgII site of the pSP64T plasmid described by Gompels and Minson (J. Virol., 63, 4744-4755, 1989 ) Then, from the plasmid pSVD4 (Everett NucI. Acids Res., II, 6647-6667, 1983), a HindIII fragment containing the promoter sequence for the glycoprotein D (gD) gene (extending from nucleotide -392 to +11 relative to the beginning of the gD gene was excised ), and cloned into the unique HindIII site of pAF2 to produce pGHI (FIG. 1), so that the promoter sequence is in the correct orientation necessary to stimulate gH gene expression. Thus, this plasmid contains the complete gH coding sequence under the control of the type I gD HSV gene promoter. This plasmid was then purified and transfected into Vero cells together with pNEO plasmid (Pharmacia LKB Biotechnology Inc.) using standard calcium phosphate co-precipitation technique (Graha and Ver der Eb, Virology 52, 456-467, 1973). The vero cells with acquired non-muscular resistance were then selected by passaging the cells into G418 drug medium, and the colonies of these cells cloned the final dilution pathways. These neomycin-resistant cells were then amplified in tissue culture, after which the samples were infected with HSV type 2 virus. Infection with HSV type 2 virus induces transcription from the gD type I promoter present in the complementing cell genome and also stimulates the production of gH type I protein in complementing cell. Lysates of infected cells were then screened for expression of the gH protein by western blotting using a polyclonal antiserum, which is known to be capable of specific recognition of the type I gH protein (Desei et al., 1988, see above). Cells that express the desired protein were then stored and a frozen stock was prepared. This material was a gH + complementing cell line.

B. Preparation of HSV Type I Virus with Interrupted gH Gene

A 6432 base pair fragment containing a gH coding sequence together with H flanking sequences was excised from plasmid pUG102 (Gompel and Minson, Virology 153, 230-247, 1986) and cloned into plasmid pATI53 (twigg and Sherrat, Nature, 283 , 216, 1980), as a result of which the plasmid pGH2 was obtained (FIG. 2). This plasmid was digested with PvuII, and it was cut only in the gH-coding sequence in two positions (at nucleotides 44955 and 46065) according to the numbering scheme of McGeoch et al., 1988, see above), and the largest of the two (fragments were purified. DNA- a fragment containing the full E. coli B-galactosidase gene located downstream (forward) from the promoter of the prodrug gene derived from cytomegalovirus (CMV) was then obtained using the following procedure. First, two complementary oligonucleotides were annealed (Fig. 3a) and ligated with BgIII-digested and about used by the phosphate plasmid pSP64T by Krieg and Melton, NucI. Acids Res. 12, 7057-7071, 1984), whereby the plasmid pSP64T shown in Fig. 3c was obtained. The attached linker also contained an initiating codon and the first three codons of the E. coli B-galactosidase (Iacz) gene. Then, the “core region” of the CMV early ear promoter promoter was amplified from pUG-HI plasmid (Gompels and Minson, 1988, see above) by polymerase chain reaction (PCR-Molecular Cloning, ed. Sambrook et al., Above) where two oligonucleotides were used, shown in figa, and corresponding to sequences from -302 to -288, and from -13 to -36, respectively (the numbering was relative to the beginning of the early CMV gene described by Anrigg et al., Virus Researeh 2, 107-121, 1985). These oligonucleotides also contain restriction sites for the HindIII enzyme at their 5 ends, and in the case of annealing of the oligonucleotide above the promoter, there is also a Smal site. Then, the PCR-amplified DNA product was digested with HindlII and cloned into a HindIII-digested pP64Ta, resulting in the plasmid pCMVIEP (Fig. 4c). Finally, a DNA fragment containing a complete copy of B-galactosidase, in which only the extreme 5'-terminal region of the coding sequence is missing, was isolated by digesting the plasmid pSC8 (Chakzabarti et al., Mol. Cell. Biol., 5, 3403- 3409, 1985) with the BamHI enzyme and cloned into the unique BoIII site of pCMVIEP, whereby pCMVlacz was obtained (Fig. 5), a DNA fragment containing the B-galactosidase gene under the control of the CMV IE promoter was then isolated by digesting the plasmid pCMV-Iacz Smal enzyme, and were ligated with purified PvuIII fragment of plasmid pGH2 described above, as a result of it received the plasmid pGH3, which contained a copy of the gH gene interrupted by the fungional B-galactosidase gene (Fig.6). The next stage was to replace the wild-type gH gene in the HSV genome with the obtained interrupted mutant, and this stage was carried out by recombination between HSV DNA and pGH3 plasmid, followed by selection of those viruses that acquired the functional B-gadactosidase gene. For this, pGH3 plasmid DNA was transfected into cells expressing the gH gene (gH + complementing 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. B.WCMahu, pp. 207-236, IPL Press, Oxford (1985)), using standard calcium phosphate precipitation techniques (Graham and Van de Eb, 1973, supra). Progeny of the HSV virus obtained as a result of the described transfection experiment, then were seeded on monolayers gH + complementing cells, conducting standard analysis of plaque formations and using the upper agar layer for coating, in the presence of 5-bromochloro-3-indolyl-β-D-galactoside (X-gaI), a chromogenic substrate, which in the presence of β-galactosidase turned blue.

Thus, plaques obtained by infection with viral genomes containing and expressing the B-galactosidase gene became blue. This meant that these viral genomes should contain a variant of the disrupted gH gene. The virus was then purified from these plaques by removing agar plugs from the corresponding portion of the plate, and the viral preforms were obtained by culturing the virus in a gH + complementing cell line. These viruses, since they carry non-functional variants of the gH gene, must be unable to form plaques on cells that do not contain and do not express an ecdogenic functional copy of the gH gene, and to confirm this fact, the samples were analyzed for their ability to form plaques on monolayers of wild-type cells Vero lines compared to gH-complementing cells. And finally, viral DNA was obtained from these blanks and tested for the expected DNA structure near the gH gene using Southern blotting. After confirming the correctness of the genetic structure, large stocks of the virus with a deficient gH gene were prepared by inoculating a virus sample in a large-scale gH + culture of a complementing cell line (infection multiplicity = 0.01), and after three days the cells were harvested. The infected cells were destroyed by ultrasound to release the virus associated with the cells, and the entire mixture treated with ultrasound was stored at -70 ° in the form of a viral uterine preparation. The titer of this viral preparation was then established using plaque analysis on gH + complementing cell lines. After that, samples of this viral preparation were used to obtain the working preparations described above, after which these working preparations were used to infuse laboratory animals, as described below.

C. Investigation of the protective effect of gH-HSV compared to heat-neutralized virus

To analyze the host immunological response to this virus, experiments were carried out to control the infection of mice in accordance with the scheme described below.

Then, the protective effect of a live gH ^- virus preparation was compared with the effect of an inactivated preparation of wild-type virus (WT) (strain SCI6). Obtaining inactivated wild-type virus for vaccination

Type I HSV virus (strain SC16) was cultured with low infection multiplicity (0.01 bfu / cell) of Vero cells. Three days later, the virus was collected, and the cytoplasmic virus was isolated using a Downes homogenizer. Nuclei were removed by centrifugation for 15 minutes at 500 × g, and the virus was isolated from the supernatant by centrifugation in a sucrose gradient (up to 40%) at 12 K for 60 min using a Beckman SW27 rotor. The virus bands were diluted, besieged and purified by sucrose gradient centrifugation (Killington and Powell, see above). The virus band was collected from the gradient, and the virus was isolated by centrifugation. Then the virus was resuspended in phosphate-buffered saline (PBS), analyzed for infectivity by titration of plaques on the cells of a hamster baby kidney (BHK), and the particles were counted on an electron microcope. The particle: inactivity ratio of the preparation was 110 particles / b.p.u. The virus was diluted to 2.5 × 10 ¹⁰ bfu / ml in PBS, and inactivated by treatment with β-propiolactone at 20 ° C for 60 minutes. The resulting aliquots were then stored at -70 ° C.

Obtaining live gH ^- virus for vaccination

This material was obtained in the same way as the wild-type virus, except that the virus was cultured in a gH + complementing cell line containing and expressing the wild-type g HSV gene and was not inactivated by treatment with β-propionolactone. The particle: infectivity ratio of this drug was 150: 1. The concentration of this preparation was adjusted to 2.5 × 10 ¹⁰ b.p.e. / ml, and aliquots were stored in PBS at -70 ° C.

Vaccination schedule

4-week-old female mice balb / C (.. Supplied Tucksv k Ltd.) were vaccinated with various doses of inactivated wild type virus or live virus gH ^- 2-.mu.l volumes of phosphate-buffered saline by droplet application and needle scarification of the right ear as follows :

Group A Control - not treated with virus

Group B 5 × 10 ⁴ b.p. viral vaccine

Group C 5 × 10 ⁵ b.p.u. viral vaccine

Group D 5 × 10 ⁶ b.p. viral vaccine

Group E 5 × 10 ⁷ b.p. viral vaccine

After 14 days, all mice were challenged by similar inoculation of the left ear with 2 × 10 ⁶ b.p. strain SCI6 HSV-I (wild-type virus). After 5 days, the mice were sacrificed and analyzed for viral infectivity in the left ear and in the left cervical ganglia CII, CIII and CIV (combined data). To study the latent state, other vaccinated and challenged animals were killed after 1 month and tested from latent infection by excision of ginglia cII, cIII and cIV. This tissue was incubated for an average of 5 days, and then homogenized and assessed for the presence of an infectious virus using a standard plaque assay. All results were expressed in bpu / organ (plaque-forming units are not an organ).

combined data for cervical ganglia cII, cIII and cIV (b.p.u. - plaque ^- forming units; gH ^- virus with a defective gH gene).

The results show that the wild-type SCI6 challenge virus titer was present in the ears and cervical ganglia during the acute phase of infection. For example, a low titer indicates a good efficacy of the gH ^- vaccination schedule, while a higher titer indicates a lower efficacy. The results show that vaccination with live gH ^- virus KSV is significantly more effective than the equivalent amount of inactivated wild-type virus. In the case of an inactivated preparation, a dose of 5 × 10 ⁷ pfu is required to prevent replication in the ear of the control virus, and in the case of live qH ^- virus, this dose of the necessary virus is 100-1000 times smaller. Vaccination with live gH ^- virus at a dose of 5 × 10 ⁵ p.u. and above, it was also able to block the replication of the control virus in the cervical ganglia during the acute phase of infection and, in addition, clearly showed a protective effect against the activation of latent infection in the cervical ganglia.

HSV virus in which there is no gH ^- gene and which is used as a vector for immunization against a foreign antigen: introduction of the gp120 gene of strain 142SIVmac into the genome of the gH virus of HSV

As described above, viruses with division in the main genes can be used as safe vectors for the presentation of foreign antigens to the immune system, and the gH virus (HIV) described above was used as a suitable example of such a vector. This virus can be used to express any desired foreign antigen, but the ability to express the main antigenic proteins of the human immunodeficiency virus (HIV) causing AIDS is especially attractive. For example, these sequences can be introduced into the qH ^- -HSV genome so as to ensure their expression during the infection of normal cells (i.e., non-complementing cells) with a recombinant virus. Infection of the body with such a virus leads to a latent infection, which, after some time, reactivates, which contributes to a sharp increase in the production of foreign antigen and thereby stimulation of the immune response to this protein.

Since it is not possible to conduct studies to test this method directly in humans, this method can be tested on monkeys using the monkey AIDS virus (SIV _mac , monkey immunodeficiency virus obtained from macaques) as the initial stage of the study. A suitable SIV gene is the gene encoding the gp120 protein, one of the main antigenic targets for this virus. Therefore, this gene was introduced into the HSV gH ^- gene, after which an analysis was carried out on the effectiveness of this virus as a vaccine in the development of immunity in monkeys against the control of infection with virus S IV.

First of all, the gp120 SIV gene was cloned into the middle of the neighboring promoter of the cytomegalovirus IE (Gompels and Minson, 1989, see above), and then the DNA cassette containing the gp120 gene and the CMV promoter located above was cloned into the pGH2 plasmid (Fig. 2 ) The resulting plasmid was then co-transfected into a gH + complementing cell line together with DNA purified from gH-HSV, and the recombinant virus, which instead of the β-galactosidase gene present in the gH-HSV virus, acquired the gene op120, was isolated by screening for the gene β-galactosidase.

A. Construction of a plasmid for recombination of the SIV-gp120 coding sequence in the HSV genome.

A complete diagram of this procedure is shown in FIG. A Sacl restriction enzyme fragment (corresponding to bases 5240-8721) was excised from a cloned DNA copy of the SIV genome (Chakrabarti et al., Nature 328, 543 (1987) and cloned into the Sacl site of pVC118 plasmid (Viera and Messing, Methods in Enzymology, 153 3, 1987) in order to generate the plasmid pSIVI, which can then be transformed into single-stranded DNA for manipulation using site-directed mutagenesis.Then this DNA region, which includes the SIV env gene (located between 6090-8298), was modified using site-directed mutagenesis (Brierley et al., Cell, 57, 537, 1989) in the shares of input Site tions for restriction enzyme EcoRV at positions 6053-6058, using this synthetic oligonucleotide

5'GААГАAGGCТАТАГСТАААТАТАТ

The second EcoRV site was then introduced at positions 7671-7676 inside the SIV env gene, which correspond to the cleavage site between the gp120 and gp40 domains of the env gene sequence, using a synthetic oligonucleotide

5'СAAGAААТАААСТАТАГGTСТТТТGTGС,

resulting in the obtained plasmid pSlV2. Then, the SIV2 digestion pathway with the EcoRV enzyme produced a DNA fragment (1617 base pairs) corresponding to the gp120 portion of the SIV env gene.

The core region of the promoter of the extended CMV gene was obtained from plasmid pVG-HI (Gompels and Minson, 1989, see above) using an eco-chain reaction polymer using the following two synthetic oligonucleotides:

primer located above (in the direction 5 → 3)

primer located below

The product of this reaction was then hydrolyzed with the EcoRI and HindIII enzymes to produce a DNA fragment, which was then cloned into the ECORI and HindIII digested plasmid pMS118, resulting in the plasmid pCMVIE2, which had a unique smal site located immediately below the CMV promoter sequence. EcoRV fragment containing SIV _mac . The gp120 coding sequence obtained in accordance with the above description was then cloned into the indicated Smal site, after which the plasmid pSIV8 was selected with the SIV coding region correctly oriented, i.e. so that expression of the coding sequence from the promoter takes place. This plasmid was then digested with the EcoRV enzyme, resulting in a blunt-ended DNA fragment that contains the SIV sequence together with the CMV promoter, and which was then cloned into the PVuII-digested plasmid pGH2 (FIG. 2) to obtain plasmid pGH- 120.

B. Construction of recombinant gH-HSV carrying the gp120S1 gene

DNA was constructed from the HSV gH virus constructed in response to the detailed description above, which was transfected into gH + complementing cells together with purified pGH-120 DNA. The progeny virus resulting from this transfection was passaged onto gH + monolayers complementing the cell line using a standard plaque assay as described above and using top agar in the presence of X-gal. The parent gH virus contained a functional galactosidase gene located inside the residual gH coding sequences and formed blue plaques in the presence of X-gal. However, recombinant viruses that instead of the β-galactosidase gene have acquired the SIV-gp120 coding sequence produce white plaques. The virus was then isolated from these white plaques by removing agar plugs, and viral preparations were prepared by culturing the virus in a gH + complementing cell line. From these preparations, DNA was obtained which was tested for the presence of the correct DNA structure near the gH gene by Southern blotting using appropriate probes derived from the SIV coding sequence. And finally, viral preparations were obtained in accordance with the above description, which can then be used for animal vaccination studies.

An attenuated virus containing vaccine can be prepared and used in accordance with standard techniques well known in the art. For example, this vaccine may also contain one or more excipients and / or adjuvants. The effective dose of the attenuated virus contained in the acin can be determined by traditional methods well known in the art.

Claims (18)

1. A vaccine containing a mutant herpes virus for prophylactic or therapeutic use in generating an immune response to a wild-type herpes virus or other foreign pathogen, characterized in that said mutant herpes virus contains a genome in which a viral gene encoding essential for the production of an infectious virus the protein is deleted or inactivated so that the virus is able to infect normal cells, replicate and express viral genes in these cells, but not Jet produce normal infectious particles except when the virus infects a complementing cell which has a heterologous nucleotide sequence which allows a complementing cell expressing an essential protein encoded by said deleted or inaktirovannym viral gene, said normal cells are other than said complementing cell. 2. The vaccine according to claim 1, characterized in that when a mutant virus infects normal cells, non-infectious viral particles are produced. 3. The vaccine according to claim 1 or 2, characterized in that it further comprises a pharmaceutically acceptable excipient. 4. The vaccine according to any one of claims 1 to 3, characterized in that said deleted or inactivated gene is functionally late in the viral cycle. 5. The vaccine according to claim 4, characterized in that said gene encodes a protein that is involved in a post-replicative event. 6. The vaccine according to claim 5, characterized in that said deleted or inactivated gene is involved in packaging. 7. The vaccine according to any one of claims 1 to 3, characterized in that said gene encodes a protein that is not required for the assembly of the virus, but is necessary for the collected virus to be able to infect new cells. 8. The vaccine according to claim 1, characterized in that it consists essentially of a pharmaceutically acceptable excipient and an effective immunizing amount of said mutant herpes virus. 9. The vaccine according to any one of claims 1 to 8, characterized in that the mutant herpes virus has the ability to establish a latent infection with periodic reactivation. 10. A vaccine according to any one of claims 1 to 3, characterized in that said deleted or inactivated gene is a glycoprotein gene. 11. The vaccine according to any one of claims 1 to 7, characterized in that the herpes virus is a herpes simplex virus. 12. The vaccine according to claim 7, characterized in that the deleted or inactivated gene is a gH gene. 13. The vaccine according to item 12, characterized in that it includes a dose containing from about 5 · 10 ⁴ to about 5 · 10 ⁷ PFU of the indicated mutant virus. 14. The vaccine according to item 13, characterized in that it includes a dose containing from about 5 · 10 ⁴ to about 5 · 10 ⁶ PFU of the indicated mutant virus. 15. The vaccine according to 14, characterized in that it includes a dose containing from about 5 · 10 ⁴ to about 5 · 10 ⁵ PFU of the specified mutant virus. 16. A vaccine according to any one of claims 1 to 15, characterized in that the mutant herpes virus is defective in more than one gene essential for the production of an infectious virus. 17. A vaccine according to any one of claims 1 to 16, characterized in that the mutant herpes virus carries genetic material encoding the immunogen of a pathogen exogenous to the specified virus, and the mutant herpes virus upon infection of the specified normal cell in which the mutant virus is not capable cause the production of new infectious viral particles, capable of expressing the specified genetic material. 18. The vaccine according to claim 17, wherein the mutant herpes virus causes immunity to said pathogen in susceptible species immunized with it.

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