JPH10503649A - Polynucleotide herpesvirus vaccine - Google Patents

Abstract

  (57) [Summary] To express the encoded protein in vivo in mammalian muscle cells, the gene encoding the herpes simplex virus type 2 (HSV-2) protein was cloned into a eukaryotic expression vector. The animals were immunized by injecting these DNA constructs, called polynucleotide vaccines or PNV, into the muscles of the animals. DNA titrations found that a single immunization of ≧ 0.5 μg of (1) PNV resulted in> 90% seroconversion at 10 weeks post immunization. The immune antiserum neutralized both HSV-2 and HSV-1 in cell culture. When animals were challenged with HSV-2, significant (p <0.001) protection from lethal infection was achieved after PNV vaccination.

Description

DETAILED DESCRIPTION OF THE INVENTION           Polynucleotide herpesvirus vaccine Background of the Invention   Viruses, especially those with multiple serotypes or high mutation rates, Key to the development of vaccines against viruses that can elicit a neutralizing and protective immune response The impairment is the diversity of viral foreign proteins between different virus isolates or strains. You. Cytotoxic T lymphocytes (CTLs) in mouse and human are preserved It is possible to recognize epitopes derived from internal viral proteins such as . W. See Yewdell et al. Proc. Natl. Acad. Sci . (USA) 82, 1785 (1985); R. M. Townsend, e t al. , Cell 44, 959 (1986); J. McMichael   et al. , J .; Gen. Virol. 67, 719 (1986); B astin et al. , J .; Exp. Med. 165, 1508 (1987 A). R. M. Townsend and H.S. See Bodmer, Annu. R ev. Immunol. 7,601 (1989)], and immunity against the virus It is considered important for the response [Y. -L. Lin and B. A. Askonas, J .; Exp. Med. 154,22 5 (1981); Gardner et al. , Eur. J. Immun ol. 4, 68 (1974); L. Yap and G. L. Ada, Na cure 273, 238 (1978); J. McMichael et a l. , New Engl. J. Med. 309, 13 (1983); M. T aylor and B.A. A. Askonas, Immunol. 58,417 (1986)] can provide heterologous protection against different virus strains Efforts are being directed at developing a novel CTL vaccine.   CTLs show that their T cell receptors have MHC class I and / or class II Kills virus-infected cells when recognizing viral peptides associated with offspring. And is known. These peptides determine the position of this protein within the virus or Can be derived from endogenously synthesized viral proteins, regardless of function. Security CTLs by recognizing epitopes derived from stored viral proteins Classes can provide heterogeneous defenses.   Many infectious agents induce themselves and this disease-inducing factor. And its by-products to kill and render harmless, or it is killed or rendered harmless. Elicit protective antibodies that can lead to Recovery from these diseases is usually Long lasting due to protective antibodies raised against the highly antigenic components of the infectious agent Immunity.   Protective antibodies are part of the natural defense mechanisms in humans and many other animals, and And in the blood in addition to body fluids. Infectious agent without causing disease and / or Or eliciting protective antibodies against their by-products is This is the first function.   Most efforts to generate CTL responses have been made using replicating vectors. Generating protein antigens in the vesicle [J. R. Benlink et al. , Supra 311,578 (1984); R. Benlink and J.M. W. Ye wdell, Curr. Top. Microbiol. Immunol. 163 , 153 (1990); K. Stover et al. , Nature3 51, 456 (1991); Aldovini and R.A. A. Youn g, Nature 351, 479 (1991); Schaffer et a l. , J. Immunol. 149, 53 (1992); S. Hahn et a l. Proc. Natl. Acad. Sci. (USA) 89, 2679 (1 992] or the introduction of peptides into the cytosol. [F. R. Carbone and M.S. J. Bevan, J .; Exp. Me d. 169, 603 (1989); Deres et al. , Natur e342, 561 (1989); Takahashi et al. , Mentioned above 344, 873 (1990); S. Collins et al. , J .; Immunol. 148, 3336 (1992); J. Newman et   al. 148, 2357 (1992)]. Both of these approaches Both have the limitation that their usefulness as vaccines may be reduced. I do. Retroviral vectors maintain the ability of the recombinant virus to replicate. Restrictions on the size and structure of polypeptides that can be expressed as fusion proteins [A. D. Miller, Curr. Top. Microbiol. Immu nol. 158, 1 (1992)], the subsequent license of vectors such as Vaccinia. Epidemiological efficacy depends on immune response to vaccinia Therefore, it cannot be fully demonstrated [E. L. Cooney et al. , Lanc et 337, 567 (1991)]. Also, viral vectors and modified pathogens Have an inherent risk that they may interfere with their use in humans [R. R. Redfield et al. , New Engl. J. Med. 316, 673 (1987); Mascola et al. Arch. Intern. Med. 149, 1596 (1989)]. Let me show you more The choice of peptide epitope to be sought depends on the structure of the individual MHC antigen, Thus, peptide vaccines are used in MHC haplotypes in crossbreeding populations. Effectiveness may be limited by the diversity of the loops.   Benvenisty, N .; And Reshef, L .; [PNAS 83,95 51-9555 (1986)] can be administered intraperitoneally (ip) or intravenously (i.p.) to mice. v. ) Or CaCl introduced by intramuscular (im) route_TwoPrecipitated DNA is expressed It was shown that it could be. Intramuscular (im) injection of DNA expression vector into mice Is the uptake of DNA by muscle cells and the proteins encoded by this DNA Has been shown to result in the expression of [J. A. Wolff et al. , Science 247, 146 5 (1990); Ascadi et al. , Nature 352, 81 5 (1991)]. These plasmids remain episomal and do not replicate It was shown that. Subsequently, the rat, fish and primate skeletal muscle, and the rat heart I. m. Sustained onset after injection has been observed [H. Lin et a l. R., Circulation 82, 2217 (1990); N. Kits is et al. Proc. Natl. Acad. Sci. (USA) 88 , 4138 (1991); Hansen et al. , FEBS Let t. 290, 73 (1991); Jiao et al. , Hum. Gen e Therapy 3, 21 (1992); A. Wolff et al. , Human Mol. Genet. 1, 365 (1992)]. Therapeutic nucleic acid The technology used as is reported in WO90 / 11092 (October 4, 1990) Where naked polynucleotides are used for vertebrate vaccination. Was.   Recently, on the surface of antigen presenting cells in the activation of CTLs to remove tumors B7 and major histocompatibility complex of epitopes The collaborative role of body (MHC) presentation was assessed [Edgington, Biot technology 11, 1171-1119, 1993]. Once the antigen MHC molecules on the surface of indicator cells (APCs) add epitopes to the T cell receptor (TCR) When passed, B7 expressed on the same APC surface binds to CTLA-4 or CD28 This acts as a second signal. The results indicate that CD4 + helper T cells It is a fast division and this CD4 + helper T cells may proliferate into CD8 + T cells. To kill the APC.   Performing the immunization intramuscularly is not essential to the success of this method. For example Tang et al. [Nature, 356, 152-154 (1992)] reported that cattle Gold coated in a microprojection with DNA encoding growth hormone (BGH) The production of anti-BGH antibodies in mice by introduction into skin. Was. Furth et al. [Analytical Biochemistry, 205 , 365-368 (1992)], on the skin, muscle, fat and milk of living animals. We have shown that jet injectors are available for transduction of tuft tissue. Various nucleic acid introduction methods have recently been evaluated [Friedman, T .; , Science, 244, 1275-1281 (1989)]. Robinso [Abstracts of Papers Presented at the 1992 meeting on Modern Approach s to New Vaccines, Included Preventi on of AIDS, Cold Spring Harbor, p92]. Teru. Here, im, ip, and avian influenza DNA to chickens It has been claimed that iv administration provides protection against lethal challenge. DNA: Intravenous injection of cationic liposome complex results in systemic expression of cloned transgene Can be generated by Zhu et al. [Science 261, 209-211 (1993) July 9); see also WO 93/24640, December 9, 1993]. Was done. Recently, Ulmer et al. [Science 259, 1747-149, ( 1993)] injects DNA encoding the influenza virus protein. Heterogeneous protection against influenza virus infection was reported.   Wang et al. N. A. S. USA 90, 4156-4160 (1993 May)] is the cloned genome (non-spliced). S) Immune response to HIV in mice by intramuscular inoculation of the HIV gene Was reported. However, the level of immune response achieved is very high Low, and this system uses the mouse mammary tumor virus (MMTV) long terminal repeat (L TR) Part of the promoter and Simian virus 40 (SV40) promoter And a part of the terminator was used. SV40 is probably the host cell D It is known that cells are transformed by incorporation into NA. others Thus, the system described by Wang et al. Is one of the objects of the present invention. It is completely unsuitable for administration to patients.   WO 93/17706 describes how to vaccinate animals against the virus Here, the carrier particles are coated with a genetic construct and the coated particles Was accelerated into animal cells.   Development of subunit vaccine against herpes simplex virus (HSV) All recent efforts have focused on the novel expression of viral antigens, especially viral glycoproteins, and The focus is on presentation. [For a review, see Burke, R .; L. , 19 93, Sem. In Virol. , 4, pp. 187-197]   Yeast (Kino., YC et al., 1989, Vaccine, 7). Pp. 155-160), insect cells (Ghiasi, H. et al., 19). 91, Arch. Virol. , 121, pp. 163-178), and feeding Cell (Burke, RL, 1991, Rev. Infect. Dis., 13, S906-S911; Lasky, L .; A. , 1990, J.A. Med. V irol. , 31, pp. 59-61). Recombinant HSV glycoprotein induces protective immunity in animal models Have been. Recombinant HSV-produced in Chinese hamster ovary cells Clinical trials of diglycoprotein D (gD) have shown that this vaccine can be used in na 個体 ve individuals. Elicited an antibody response and previously appeared in both HSV-1 and HSV-2 seropositive individuals. Stimulates the response that exists from (Straus, SE et.   al. , 1993, J.A. Infect. Dis. 167 pp. 1045- 1052).   An alternative approach to subunit vaccines is to live in HSV antigen delivery Is to use a viral vector. gD (Aurelian, L. et.   al. , 1991, Re v. Infect. Dis. , 13, S924-S930; Rooney, J .; F. et al. 1991, Rev .; Infect. Dis. , 13, S89 8-S903; Wachsman, M .; et al. , 1992, Vaccin e, 10, pp. 447-454), gB (Rooney, JF, et al., Supra). , GL and gH (Browne, H. et al., 1993, J. Gen. Virol. , 74, pp. Vaccinia-HS expressing V recombinants protect animals well from HSV challenge. Expresses HSVgB Vaccination with a recombinant adenovirus infection produces a protective immune response in mice Trigger. (Ghiasi, H., supra; McDermott, MR, 19). 89, Virology, 169, pp. 244-247). Native protein (L ong, D .; et al. , 1984, INfect. Immun. , 43, p p. 761-764), and a protein expressed by recombination (Burke, R. et al., Supra). L. Stanbury, L .; R. et al. 1987; Infec t. Dis. 155 pp. 914-920; Straus, S., supra. E. FIG. ), A peptide derived from gD (Eisenberg, RJe). t al. 1985; Virol. , 56, pp. 1014-1027) Induced by immunization with E. coli or passively transmitted (Dix, RD. t al. 1981, Infect. Immun. , 34, pp. 192-1 99; Ritchie, M .; H. et al. , 1993, Investiga. five Ophthalmology and Visual Science es, 34, pp. 2460-2468) Anti-gD antibodies can protect against HSV infection Is documented.   A study by Wolff et al. (Supra) showed that plasmids encoding reporter genes Intramuscular injection of DNA into the muscle cells in and near the injection field For the first time. A recent report was on influenza A hemagglutination. Mouse against influenza by injecting a plasmid encoding (See Montgomery, DL et al, supra). . Ulmer, J .; B. et al. , 1993, Science, 259, pp. 1745-1749). First use of DNA immunization against herpes virus (Cox et al., 1993, J. V. irol. 67, pp. 5664-5667). Bovine herpesvirus 1 (BH V-1) Injection of the plasmid encoding the glycoprotein gIV was performed in mice and calves. -Generates gIV antibodies. Upon nasal challenge with BHV-1, immunized calves were It showed reduced symptoms and was substantially less virus than the control. Mouse (see above) Long, D.C. Guinea pigs (Stanbury, LR, et al., Supra); Stanbury. L. R. et al. , 1989, Antiviral. Res. , 11, pp. 203-214) elicits a protective immune response in The ability of V-glycoprotein D has been documented.Summary of the Invention   To test the efficacy of DNA immunization in preventing HSV disease, HSV-2 The DNA sequence encoding white matter was cloned into a eukaryotic expression vector. This DNA The construct elicits an immune response when injected into the animal. Immunized animals, g Direct DNA immunization using the D gene (or other HSV-2 gene) may result in disease These animals were infected with HSV to evaluate whether they were protected or not. But Thus, by injection or other methods, including DNA constructs and RNA transcripts, Introduced directly into the organization Induces in vivo expression of human herpes simplex virus (HSV) protein A possible nucleic acid is disclosed. Injection of these nucleic acids was followed by HSV antigen injection. In addition to generating HSV-specific antibodies that are protective against administration, cells specific for HSV antigens It can elicit an immune response that produces toxic T lymphocytes (CRLs). These nucleic acids HSV can prevent infection and / or ameliorate HSV-related diseases It is useful as a vaccine that induces immunity against E. coli.BRIEF DESCRIPTION OF THE FIGURES FIG.Panels A, B, and C   A. V1J: Western blot of HSV gD expression by gD-transduced cells Analysis is shown;     1. Mock infected Vero cells;     2. HSV-2 186 infected Vero cells (moi = 1);     3. HSV-2 Curtis infected Vero cells (moi = 1);     4. V1J: RD cells transduced with gD     5. Mock transduced RD cells.   B. V1JNS: HSVgB expression by gB-transduced cells The Western blot analysis of is shown.   C. V1J: ICP27-a vector of HSV ICP27 expression by transduced cells. Stern blot analysis is shown. FIG.Panels A, B, and C   A. HSV PNV immunization (B-6.25 μg dose of V1J: gD, C-50 μg dose of V1J: gD) and sham from sham immunized (A) animals Use,     1. HSV-1 KOS;     2. HSV-2 186;     3. HSV-2 Curtis;     4. Pseudo infection Shown is a Western blot analysis for lysates of BHK cells infected with BHK.   B. Western blot using serum from HSV PNVgB immunized animals Analysis is shown. FIG. ELI for HSV PNV immunized animals receiving a single injection of vaccine SA generation group GMT data is shown; sera were 4th, 7th and 10th post-immunization Got a week. Figure 4. 200 μg; 100 μg; 50 μg; 25 μg; 5.25 μg; 3.13 μg; 1.56 μg; 0.78 μg of V1J: Survival of HSV-2 challenged animals after two injections of gD or saline alone. 2 00 μg; 100 μg; 25 μg; 12.5 μg; 6.25 μg; and 3.13. Since all animals in the μg group survived, they are all represented by a single symbol. Figure 5.50 μg; 16.7 μg; 5.0 μg; 1.67 μg; 0.5 μg; 167 μg; 0.05 μg; 0.017 μg; 0.005 μg of V1J: gD or Shows survival of HSV-2 challenged animals after a single injection of saline alone. FIG. Shown is the survival of animals immunized with V1JNS: gB after HSV challenge. It is. FIG. Survival of animals immunized with V1J: gC after HSV challenge . FIG. Survival results in HSV-2 infected guinea pigs, mean days to death, hemp Paralysis and vaginal virus titers are indicated. FIG. Guinea pig vaginal lesion score is indicated.Detailed description of the invention   The present invention is directed to vertebrate organisms, including mammals such as humans, directly in vivo. Induces expression of the encoded protein in the animal when introduced A polynucleotide is provided. As used herein, a polynucleotide is a When introduced into living vertebrate cells, the cellular machinery Can be directed to the production of translation products encoded by the containing gene A nucleic acid that contains essential regulatory elements. In one embodiment of the present invention, the polynucleotide Is a polydeoxygenase containing the HSV gene operably linked to a transcription promoter. Ribonucleic acid. In another aspect of the invention, the polynucleotide vaccine is a true vaccine. Easily translated by nuclear cell mechanisms (ribosomes, tRNAs and other translation factors) A polyribonucleic acid encoding the HSV gene. This polynucleotide Is a pathological condition, such as a protein associated with HSV. Unless it normally occurs in an animal (ie, a heterologous protein) The animal's immune system is activated, triggering a protective immune response. These foreign proteins The expressed protein is produced by the animal's own tissues, and is expressed in the major histocompatibility system. Is treated in the same manner as when genuine HSV infection is caused by the system (MHC) . The results, as shown in this disclosure, Induction of an immune response. To generate an immune response to the encoded protein Are referred to herein as polynucleotide vaccines or PNVs .   There are many aspects of the present invention that can be understood by one skilled in the art from this specification. . For example, different transcription promoters, terminators, carrier vectors or specific The gene sequence can be used successfully.   The present invention provides polynucleotides in vivo when introduced into mammalian tissues. Polynucleotide that induces expression of, thereby producing an encoded protein Provides usage of Alter the amino acid sequence of an encoded protein; It is possible to generate variants or derivatives of the nucleotide sequence encoding the quality. It is easily recognized by those skilled in the art. Modified expression proteins are modified. Has the amino acid sequence obtained, but still reacts with viral proteins Can elicit antibodies and be considered functionally equivalent . In addition, constructing fragments of the full-length gene encoding a portion of the full-length protein Is also possible. These fragments are proteins that elicit antibodies that react with viral proteins. Encodes white matter or peptide And can be considered functionally equivalent.   In one embodiment of the present invention, the gene encoding the HSV gene product is expressed in an expression vector. To be installed in the computer. This vector is recognized by eukaryotic RNA polymerase. A transcription promoter and a transcription terminator are arranged to encode an HSV gene. Has at the end of the row. Many other known promoters, such as strong immunoglobulins, Or that any of the other eukaryotic gene promoters can be used. In a preferred embodiment, the promoter is The cytomegalovirus promoter (CMV-intA) with is there. A preferred transcription terminator is bovine growth hormone (BGH) terminator It is. Combinations of CMVintA-BGH terminators are preferred. Addition In addition, antibiotic-resistant markers are used to aid in the preparation of polynucleotides in prokaryotic cells. Carr is optionally inserted into an expression vector under the transcriptional control of a suitable prokaryotic promoter. Included. Ampicillin resistance gene, neomycin resistance gene or other suitable gene Any of the antibiotic resistance markers can be used. Preferred of the present invention In a different way Antibiotic resistance gene encodes a gene product for neomycin resistance You. In addition, it helps to produce high levels of polynucleotides by growth in prokaryotes Therefore, the vector must have a prokaryotic origin of replication and be of high copy number. This is particularly advantageous. Any of many commercially available prokaryotic cloning vectors Provide these elements. In a preferred embodiment of the invention, these functionalities are pU Provided by a commercial vector known as the C series. However, non It may be desirable to remove essential DNA sequences. For example, lac of pUC Z and lacl coding sequences may be removed. Also, these vectors Desirably, it cannot replicate in eukaryotic cells. This is a polynucleotide Minimize the risk that the Kuching sequence will be integrated into the recipient's genome.   In another embodiment, Rous sarcoma virus (RSV) long terminal repeat (LTR) Is used as an expression vector. further In another embodiment, the CMV promoter and BGH transcription terminator are V1 which is a mutated pBR322 vector that has been cloned is used. Of the present invention In a preferred embodiment, V1 and pUC19 are The elements have been combined to produce an expression vector designated V1J. V1 An HSV gene such as gD, or gB, g Anti-HSV immune responses such as C, gL, gH and ICP27 (antibodies and / or C Other HSV genes capable of inducing TLs) are cloned. In another aspect Removes ampicillin resistance gene from V1J to neomycin resistance gene Is replaced, resulting in V1J-neo. This V1J-neo includes the present invention. Cloning any of a number of different HSV genes for use Is possible. In yet another aspect, the vector is V1Jns. this Indicates that the unique Sfil restriction site is at position 2114 of V1J-neo and a single Kpnl region It is the same as V1Jneo, except that it is processed in the place. Humangeno The appearance rate of Sfil sites in DNA is very low (approximately 100,000 bases). About 1 site). For this reason, this vector simply transforms the extracted genomic DNA into Sf By digesting with il, the expression vector can be integrated into the host DNA. Enable bottom-up surveillance. In a further aspect, the vector is a V1R. Very compact in this vector As much of the non-essential DNA as possible is "trimmed" to produce I have. This vector contains unwanted sequences that are encoded and Cellular uptake is minimal when the gene-encoding construct is introduced into surrounding tissues. Larger inserts to try to use with little concern for being optimized Enable. The methods used for the vector denaturation generation and development procedures described above are This can be achieved according to methods known to those skilled in the art.   From this work, one skilled in the art will recognize that one of the applications of the present invention is that H In vitro additions were made so that a correlation between SV sequence diversity and the serology of HSV neutralization could be made. To provide a system for testing and analysis in vivo. You will know. Isolation and cloning of these viral genes is known to those of skill in the art. This can be achieved by a known method. The invention further provides for the production of vaccines. The present invention provides a method for systematically identifying HSV strains and sequences. Is it a major isolate of the HSV strain? Integration of these genes elicits an immune response against clinical isolates of the virus To fulfill the unmet needs in the field. Add In addition, if the toxic isolate changes, the immunogen may be renewed as needed. To reflect the new array Is possible.   In one embodiment of the present invention, the gene encoding the HSV protein is a transcription promoter Directly linked to Tissue-specific promoter or enhancer, eg, muscle Use of the creatine kinase (MCK) enhancer element is a specific tissue type It is desirable to limit the expression of the polynucleotide to For example, muscle cells divide Not peripheral differentiated cells. Integration of the foreign gene into the chromosome can lead to cell division and protein It appears to require both white synthesis. Therefore, non-dividing like muscle cells Preferred is expression of the restricted protein in cells. However, the use of the CMV promoter , Suitable for achieving expression in many tissues into which PNV is introduced.Outline of PNV construction   HSV and other genes are preferably specific for polynucleotide vaccination Ligated to an optimized expression vector. Element here As described, transcription promoters, immunogenic epitopes, and immune enhancing or Additional cistrons encoding immunoregulatory genes are their own promoters Includes, transcription terminator, bacterial origin of replication and antibiotic resistance gene . Optionally, the vector may be Internal ribosome entry site (IRES) for expression of tron mRNA May be provided. One skilled in the art will appreciate that multicistros encoded by DNA counterparts RNA transcribed in vitro to produce functional mRNA is within the scope of the present invention. Will understand. For this purpose, the T7 or SP6 promotion A strong RNA polymerase promoter such as a promoter as a transcription promoter It is desirable to perform run-on transcription using a linearized DNA template . These methods are well-known in the art.   Protective efficacy of polynucleotide HSV immunogen against subsequent viral challenge Is shown by immunization with the DNA of the present invention. It contains infectious agents No virions need to be assembled and the choice of determinants is acceptable This is advantageous. In addition, the sequence of the viral gene product is different for various HSV strains. To provide protection against subsequent challenge by other HSV strains. Can be   Injection of the gD-encoding DNA expression vector allows for subsequent viral challenge It can produce significant protective immunity against. In particular, gD-specific antibodies and CTL are generated obtain. Towards stored proteins The resulting immune response is despite the antigenic shift and drift of this variable protein. Can be effective. Each of the HSV gene products has some degree between the various HSV strains And the immune response is dependent on intracellular expression and MHC processing As can occur, many different HSV gD PNV constructs are cross-reactive immune responses Is expected to occur.   The present invention provides a heterologous defense license without the need for self-replicating factors or adjuvants. Provide a means of inducing epidemics. Viral proteins and human growth hormone DNA Generation of high titer antibodies against expressed proteins after injection [Tang et al. , Nature 356, 152, 1992] disclose that this could be a cell targeting a conserved antigen. Antibody-based vaccines separately from or in combination with cytotoxic T lymphocyte vaccines It has been shown to be an easy and highly effective means of producing Kuching.   The ease with which DNA constructs can be produced and purified is conveniently matched to traditional protein Comparable to purification, which facilitates the production of combination vaccines. For this reason, Encodes any of the other HSV genes, including gD and non-HSV genes Multiple conjugates can be prepared, mixed and co-administered. In addition, protein expression is maintained following DNA injection [H. Lin et al. , Circulation 82, 2217 (1990); N. Kitsis et al. Proc. Natl. Acad. Sci. (USA) 88, 41 38 (1991); Hansen et al. , FEBS Lett. 2 90, 73 (1991): S.M. Jiao et al. , Hum. Gene T herapy 3, 21 (1992); A. Wolff et al. , Hu man Mol. Genet. 1,363 (1992)], B- and T-cell Molly's persistence was enhanced [D. Gray and P.M. Matzinger, J. Exp. Med. 174,969 (1991); Oehen et a l. Supra, 176, 1273 (1992)]. This produces mon and cell-mediated immunity.   Expressable DNA or transcribed to be introduced into the vaccine recipient The amount of RNA depends on the strength of the transcription and translation promoter used. Immune response The strength of the answer can depend on the level of protein expression and the immunogenicity of the expressed gene product. Generally, about 1 ng to 5 mg, preferably about 10 μg to 300 mg An effective dose of μg is administered directly to the muscle tissue. Subcutaneous injection, introduction into skin, through skin Other modes of administration, such as intraperitoneal, intravenous, or inhalation delivery, are also suitable. is there. It is also intended to provide a booster vaccination. HSV Polynuk Following vaccination with the leotide immunogen, gD, gB, gC, gG, and And boosting with an HSV protein immunogen such as the gH gene product. ing. Simultaneous with or subsequent to parenteral administration of the PNV of the present invention, Parenteral administration of leukin-12 protein, eg, intravenous, intramuscular, subcutaneous or other Administration of the means of may also be advantageous.   The polynucleotide is naked, ie, any protein, adjuvant or recipe It may not be bound to other factors that affect the immune system of the ent. in this case A physiologically acceptable solution, such as, but not limited to, The presence of this polynucleotide in bacterial saline or sterile buffered saline Is desirable. Alternatively, the DNA can be used in lecithin liposomes or in the art. As a DNA-liposome mixture in liposomes such as known liposomes The DNA may be associated with proteins or other proteins to enhance the immune response. May be associated with an adjuvant known in the art, such as a carrier of . Also, a factor that assists the uptake of DNA by cells, such as, but not limited to, Although not intended, calcium ions can be used. These factors are It is generally referred to herein as a transduction enhancing factor and a pharmaceutically acceptable carrier. Poly Techniques for coating microprojections coated with nucleotides are known in the art. It is well known and useful for the present invention. For DNA intended for human use Then, the final DNA product is placed in a pharmaceutically acceptable carrier or buffer solution. Is desirable. Pharmaceutically acceptable carriers or buffer solutions are known in the art. And Remington's Pharmaceutical Included are those described in various texts, such as Sciences.   In another aspect, the invention relates to a polynucleotide, wherein the polynucleotide is Expression of this polynucleotide into nuclear cells to produce the gene product A polynucleotide comprising a contiguous nucleic acid sequence that can be This coded The generated gene product is preferably capable of generating an immune stimulant or immune response Act as any of the various antigens. So this The nucleic acid sequence in an embodiment comprises a human herpes simplex virus immunogenic epitope, and Optionally, a T cell synaptic such as a member of the B7 family of cytokines or proteins Code a gigantic element.   Immunizing with that gene over the gene product has several advantages. That The first is relatively concise, thereby allowing native or nearly native antigens to be delivered to the immune system. It is possible to provide it. Even in bacteria, yeast or mammalian cells Therefore, recombinantly expressed mammalian proteins often maintain appropriate antigenicity. Requires further processing to prove. The second advantage of DNA immunization is that MHC The potential of the immunogen to enter the ras I pathway and elicit a cytotoxic T cell response. I Immunization of mice with DNA encoding the influenza A nuclear protein (NP) CD8 + response to NP protects mice against challenge with a heterologous strain of lu Elicit an answer (Montgomery, DL et al., Supra; Ulmer, supra). J. Et al.)   There is evidence that cell-mediated immunity is important in controlling HSV infection [for review In Nash, A .; A. et al. , 1985, In: The Herpe. sviruses, Vol. 4, Plenum, New York, and S chmid t, D. S. et al. , 1992, In: Rose (ed.), Curr. ent Topics In Microbiology And Immun logic, Vol. 179, Herpes Simplex Virus; P athogenesis, Immunobiology and Contro 1, Springer-Verlag, Berlin]. HSV seropositive The majority of HSV CTLs isolated from patients are of the CD4 + type (Schmid t, D. S. et al. , 1988, J.A. Immunol. , 140, pp. 3610-3616; Tsutsumi, T .; et al. , 1986, Cli n. Exp. Immunol. , 66, pp. 507-515) is specific for gD CD8 + clones, including common ones, have been isolated. (Torpey, DJ. et al. , 1989, J.A. Immunol. , 142, pP. 1325-1 332; Yaskawa, M .; et al. , 1989, J.A. Immunol . 143, pp. 2051-2057; Zarling, J .; M. et al. 1986, J. Am. Immunol. 136 pp. 4669-4673) Mau In cell transplantation and removal experiments, several CD8 + C Suggests that TL protects against infection. (Bonneua, RHE t al. , 1989, J.A. Virol. 63 pp. 1480-1484; N ash, A .; A. et al. 1987; Gen. Virol. , 68, pp. 825-833) Immunization with gD by infection with a recombinant viral vector (Paoletti, E. et al., 1984, Proc. Natl. Ac. ad. Sci. USA, 81 pp. 193-197; Wachsman, M .; L. et al. 1987; Infect. Dis. 155 pp. 1 188-1197; Zheng, B .; et al. , 1993, Vaccine , 11, pp. 1191-1198) protects mice from HSV infection. D Living viral vectors, such as NA, are capable of immunogenic MHC class I presentation. Have power. However, HSV gD-vaccinia recombinant was used to immunize mice. A recent study using infection by L.T. It has been found that it depends on delayed-type hypersensitivity function. (Wachsman, M. et. al. , 1992, Vaccine, 10, p. 447-454) HSV infection Although gD-specific CD8 + cells have been isolated from mice, Their role in limited infection is unknown. (Johnson, RM. et al. , 1990, J.A. Immunol. 145, pp. 702-71 0). Koelle et al. Studied HSV infection of human fibroblasts and keratinocytes. Make them unrecognizable to CD8 + CTL (Koell) e, D. M. et al. , 1993, J.A. Clin. Invest. , 91, pp. 961-968). The role of CD8 + cells in general in natural HSV infection, In particular, the role of the CD8 + response to gD has not been elucidated.   DNA immunization can elicit both hormonal and cell-mediated immune responses Therefore, its biggest advantage is that many viral genes can be It is to provide a relatively simple way to investigate power. HSV-2 glycoprotein Plasmids expressing B and C also elicit neutralizing antibodies, resulting in lethal challenge in mice. To protect. However, a CTL response was produced and ICP27-vaccinia May confer some protection to immunized mice by infection with the recombinant virus Known ICP27 (Banks, TA et al., 1991, J. Vi. rol. , 65, pp. 3185-3191) is a mouse Is vaccinated with PNV ICP27 alone, a lethal HSV anti- Provides no protection against the original dose. However, the DN encoding ICP27 A is useful as a component of a PNV containing multiple HSV genes, and the present invention relates to ICP2 7 is intended to be included as a component of a multivalent HSV PNV.   Also, immunization by DNA injection, as discussed above, is a multi-component subunit. Allows easy assembly of vaccines. In recent years, multiple influenza remains Co-immunization with the gene has been reported (Donnely, J. et al. . , 1994, Vaccines, printing). The products of the different immune systems Including HSV vaccines with genes that activate Complete protection against Lus challenge can be provided.   The following examples are presented to illustrate the present invention but are not intended to limit the invention thereto. It does not do.                                 Example 1 Vectors for vaccine production A)V1   Expression vector V1 was transformed into pCMVIE-AKI-DHFR [Y. Whang et al. , J .; Virol. 61, 1796 (1987)] It was built from. The AKI and DHFR genes cut this vector with EcoRI. , Self-ligated. This vector is the CMV promoter Since there is no intron A in the SacI site, an internal SacI site [B. S. Chap man et al. , Nuc. Acids Res. 19, 3979 (199 1855] in the numbering in 1) was added as a deleted PCR fragment. P The template used for the CR reaction was the hCMV-IE1 enhancer / promoter. PCMV6a120 containing the promoter and intron A [B. S. Chapman et al. HindIII and NheI fragments from Ligation into the HindIII and XbaI sites of pBL3 to give pCMVINt PCMVintA-Lux produced by generating BL. R SV-Lux [J. R. de Wet et al. , Mol. Cell Bi ol. 7,725,1987], a 188 bp luciferase gene. The child fragment (HindIII-SmaI Klenow filled) was filled with Klenow and phosphated. The SalI site of pCMVINtBL treated with It has become.   Primers that extend to intron A are: 5 'primer, SEQ ID NO: 1: 5'-CTATATAAGCAGAG CTCGTTTAG-3 '; 3 'primer, SEQ ID NO: 2 5'-GTAGCAAAGATCTAAGGACGGTGACTGCAG-3 ' .   Primers used to remove the SacI site were: Sense primer, SEQ ID NO: 3: 5'-GTATGTGTCTGAAAATGAGCGTGGAGATTGGGC TCCGAC-3 ' And an antisense primer, SEQ ID NO: 4: 5'-GTGCCGAGCCCAATCTCCACGCTCATTTTCAGAC ACA TAC-3 '.   This PCR fragment was cut with SacI and BglII and cut with the same enzymes. Into the vector. B)V1J expression vector   The purpose of producing V1Js is to make them more limited To produce a more compact vector and increase plasmid purification yield. To improve, promoter and transcription terminator elements from vector V1 Was to remove it.   V1J is derived from vector V1 and the commercially available plasmid pUC18. V1 Was digested with SspI and EcoRI restriction enzymes to generate two DNA fragments. C Bovine growth hormone (MVintA promoter and BGH) The smaller of these fragments, which have a transcription terminator element, Purified on a galose electrophoresis gel. Then another "blunt-ended" DNA fragment To facilitate ligation to T4 DNA, the ends of this DNA fragment were "Smoothing" using polymerase enzyme.   PUC18 was chosen to provide the "backbone" of the expression vector. this Are known to produce high yields of plasmid, and Characterized and small in size. Partial digestion using HaeII restriction enzyme Removed the entire lac operon from this vector. Remove the remaining plasmid Purified by Loose electrophoresis gel and treated with calf intestinal alkaline phosphatase With T4 DNA polymerase It was terminated and ligated to the CMVintA / BGH element described above. pUC bag Plasmid showing one of two possible orientations of the promoter element within the bone Was obtained. One of these plasmids yields very high DNA in E. coli. And it was designated as V1J. The structure of this vector was determined by sequence analysis of the junction region. Followed by a heterologous gene comparable or higher than V1 Was obtained. C)V1Jneo expression vector   Because ampicillin cannot be desirable for large-scale fermentations, V1J Used for antibiotic selection of bacteria with^rNeed to remove genes Met. V1J amp derived from pUC backbone^rGene is SspI And by digestion with the Eam1105I restriction enzyme. Remaining plasmid Was purified by agarose gel electrophoresis and blunt-ended with T4 DNA polymerase. After that, the cells were treated with calf intestinal alkaline phosphatase. Transposon 903 From commercially available kan derived from pUC4K plasmid^rPs gene Excised using the tI restriction enzyme, purified by agarose gel electrophoresis, and The ends were blunt-ended with polymerase. This fragment Ligate to the V1J backbone and kan^rHave genes in either direction The plasmid was derived. These were called V1Jneo # 1 and # 3. these Each plasmid was confirmed by restriction enzyme digestion analysis and DNA sequencing of the junction region. And each of them was shown to produce the same amount of plasmid as V1J. these The expression of the heterologous gene product was comparable to that of V1J. V1 Amp in J^rKan in the same direction as the gene^rV1Jneo # 3 with gene Was selected as the expression construct. Hereinafter, this is referred to as V1Jneo. D)V1Jns expression vector   An SfiI site was added to V1Jneo to facilitate integration studies. this A commercially available 13 base pair SfiI linker at the KpnI site in the BGH sequence of the vector (New England BioLabs) was added. Kpn for V1Jneo I, gel purified, blunt-ended with T4 DNA polymerase, blunt-ended The ends were ligated to the SfiI linker. Select clonal isolates by restriction mapping And confirmed by sequencing in the linker. This new vector is V1J ns. Expression of heterologous genes in V1Jns (with SfiI) Comparable expression of the same gene in V1Jneo (with KpnI). E)V1Jns-tPA   To provide a heterologous leader peptide sequence for secreted and / or membrane proteins, a human V to include a tissue-specific plasminogen activator (tPA) leader 1 Jns was denatured. After annealing the two synthetic complementary oligomers, Bgl It was ligated to V1Jn that had been digested II. Sense and antisense oligo Mer is 5'-GATCACC ATG GAT GCA ATG AAG AGA GGG CTC TGC TGT GTG CTG CTG CTG TGT GGA GCA GTC TTC GTT TCG CCC AGC GA-3 ', SEQ ID NO: 5: and 5'-GAT CTC GCT GGG CGA   AAC GAA GAC TGC TCC ACA CAG CAG CAG   CAC ACA GCA GAG CCC TCT CTT CAT TGC   ATC CAT GGT-3 ', SEQ ID NO: 6. Sense oligomer smell The Kozak sequence is underlined. These oligomers Has an overhanging base that is compatible with ligation to the BglII cleavage sequence. After ligation, While retaining the downstream BglII for subsequent ligation, the upstream BglII portion Destroyed the ranks. Both junction sites in addition to all tPA leader sequences were determined by DNA sequencing. Confirmed. In addition, the consensus optimization vector V1Jns (= SfiI site KpnI site with T4 DNA polymerase to match Positions are blunt-ended and then a SfiI linker (catalog # 1138, New E ngland Biolabs) to obtain V1Jn-tP. A SfiI restriction site was located at the KpnI site in the BGH terminator region of A . This denaturation was confirmed by restriction digestion and agarose gel electrophoresis. F)pGEM-3-X-IRES-B7   (Where X = any antigenic gene) Coordinate expression of the gene encoding the immunogen and the gene encoding the immunostimulatory protein As an example of a dicistronic vaccine construct that provides PCR amplified from B lymphoma cell line CH1 (obtained from TCC). B7 is the main In the context of histocompatibility complexes I and II, the essential A member of the protein family that provides intense T cell activation is there. CH1 cells have a phenotype in which they are constitutively activated and B7 Preferentially expressed by activated antigen presenting cells such as B cells and macrophages Therefore, it provides a good source of B7 mRNA. These cells are cAMP or IL And further stimulated in vitro using the standard guanidinium thiocyanate method. Was used to prepare mRNA. Using this mRNA, GeneAmp RNA P CR kit (Perkin-Elmer Cetus) and B7 translation open Priming oligomer specific for B7 located downstream of the reading frame -(5'-GTA CCT CAT GAG CCA CAT AAT ACC   CDNA synthesis was performed using ATG-3 '(SEQ ID NO: 7). The following sense And antisense PCR oligomers: 5'-GGT ACA AGA, respectively TCT ACC ATG GCT TGC AAT TGT CAG TTG ATG C-3 ', SEQ ID NO: 8: and 5'-CCA CAT AGA TCT CCA TGG GAA CTA AAG GAA GAC GGT CTG B7 was amplified by PCR using TTC-3 ', SEQ ID NO: 9. These ori Gomer has a NcoI restriction site and a 3'-terminal BglII site. In addition to the Kozak translation initiation sequence with an additional NcoI site Provide a BglII restriction enzyme site at the end of the insert. By NcoI digestion, N Fragments suitable for cloning into pGEM-3-IRES digested with coI Occurred. The resulting vector, pGEM-3-IRES-B7, contains V1Jns- IRE that can be easily transferred to X (where X represents the gene encoding the antigen) It has an S-B7 cassette. G)pGEM-3-X-IRES-GM-CSF   (Where X = any antigenic gene) This vector has more immunostimulatory cytokine and GM-CSF genes than B7. Has a cassette similar to that described in item C above, except that it is used. I do. GM-CSF induces potent anti-tumor T cell activity in vivo Have been shown to be macrophage differentiation and stimulatory cytokines [G. Dra noff et al. Proc. Natl. Acad. Sci. USA, 9 0,3539 (1993)]. H)pGEM-3-X-IRES-IL-12   (Where X = any antigenic gene) This vector is more immunostimulatory cytokine, IL-, than B7. Similar to that described in item C above, except that 12 genes are used Cassette. IL-12 has a cellular T-cell dominant pathway in opposition to humoral responses Have been shown to play a significant role in diverting the immune response to tracts [ L. Alfonso et al. , Science, 263, 235, 199. 4].                                 Example 2 Vector V1R preparation   Named V1R in an effort to continue optimizing basic vaccination vectors A derivative of V1Jns was prepared. The purpose of the construction of this vector remains All of the optimized heterologous gene expression characteristics and the high levels provided by V1J and V1Jns Minimal size vaccine with no unwanted DNA sequences, preserving plasmid yield Was to get the vector. (1) pUC backbone containing E. coli origin of replication Region can be removed without affecting plasmid yield from bacteria (2) ka following the kanamycin open reading frame n^rThe 3'-region of the gene will be inserted if a bacterial terminator is inserted in its place. Can be removed; and (3) ~ 300 bp from the 3'-half of the BGH terminator affects its regulatory function Can be removed without effect (the original KpnI restriction in the BGH element) (Following the enzyme site) was determined from experiments and elsewhere in the literature.   V1R is a CMVintA promoter / BGH terminator, V1Jns of three DNA fragments corresponding to the origin of origin and kanamycin resistance element They were constructed by using PCR for their synthesis. Unique restriction enzymes for each fragment Was added to the end of each fragment using a PCR oligomer: CMVintA / BGH Are SspI and XhoI; kan^rThe genes include EcoRV and BamHI; and ori^rInclude BcII and SalI. These enzyme sites indicate that they Directional ligation of guided DNA fragments is possible by subsequent loss of each site EcoRV and SspI were selected to be compatible with ligation. BamHI and BclI are as well as SalI and XhoI, leaving the smooth-end DNA. Leave a complementary overhang. After obtaining these fragments by PCR, each fragment is shown above. Digested with the appropriate restriction enzymes, followed by a single digest containing all three DNA fragments. The reaction mixture was ligated together. ori^r5 ' -The terminus is such that it can provide termination information for the kanamycin resistance gene. Designed to include the T2rho independent terminator sequence normally found in the region . The ligated product was isolated by DNA sequencing at the ligation junction. And restriction enzyme digestion (> 8 enzymes). DNA plasmid yield and V1R Heterologous expression using viral genes within is likely to be similar to V1Jns . The net reduction in vector size achieved was 1346 bp (V1Jns = 4.86 kb; V1R = 3.52 kb).   PCR oligomer sequence used for V1R synthesis (restriction enzyme sites are underlined And identified in parentheses following the sequence): (1) 5'-GGT ACAAAT ATT  GG CTA TTG GCC   ATT GCA TAC G-3 '[SspI], SEQ ID NO: 10: (2) 5'-CCA CATCTC GAG  GAA CCG GGT CA A TTC TTC AGC ACC-3 '[XhoI], SEQ ID NO: 11: (For CMVintA / BGH fragment) (3) 5'-GGT ACAGAT ATC  GGA AAG CCA CG T TGT GTC TCA AAA TC-3 '[EcoRV], SEQ ID NO: 1 2: (4) 5'-CCA CATGGA TCC  G TAA TGC TCT GCC AGT GTT ACA ACC-3 '[BamHI], SEQ ID NO: 13 : (For kanamycin resistance gene fragment) (5) 5'-GGT ACATGA TCA  CGT AGA AAA GA T CAA AGG ATC TTC TTG-3 '[BclI], SEQ ID NO: 1 4: (6) 5'-CCA CATGTC GAC  CC GTA AAA AGG   CCG CGT TGC TGG-3 '[SalI], SEQ ID NO: 15 (For E. coli replication origin)                                 Example 3 Cell, virus and cell culture   VERO, BHK-21, and RD cells were obtained from the ATCC. Virus is Infection multiplex in small volume medium without calf serum (FBS) at 37 ° C Degree (moi) 0.1 By infecting nearly confluent VERO or BHK cells with Made. After 1 hour, the virus inoculum was removed and the cultures were incubated with 2% heat-inactivated FBS, 2 mM L-glutamine, 25 mM HEPES, 50 U / ml penicillin and Resupply high glucose DMEM supplemented with 50 μg / ml streptomycin. Was. Incubation was continued until extensive cytopathy: usually 24 to 48 hours. The virus attached to the cells is centrifuged at 1800 × g for 10 minutes at 4 ° C. Collected by. Centrifuge the supernatant virus at 640 xg for 10 minutes at 4 ° C. And it became clear.                                 Example 4 Cloning and DNA preparation   HSV-2 (Curtis) DNA used as PCR template was infected Prepared from nucleocapsid isolated from VERO cells. (Dennisto n, K .; J. et al. Commun., 1981, Gene, 15, pp. 146-64. 365-378 ). HSV2gB, gC, gD, or ICP2 comprising a BglII restriction recognition site 5 'and 3' terminal flanking of 7 genes Synthetic oligomer corresponding to the sequence (Midland Certified Rea) gent Company; Midland, Texas) Used in The 1.1 kb fragment encoding the gD gene was replaced with 1: Using a deaza dGTP: dGTP ratio of 4, buffer was 3 mM MgCl_TwoSupplement PCR (Perkin Elmer) according to the manufacturer's specifications except that (Cetus, La Jolla). HSV-2 genomic DNA template Plates were used at 100 ng / 100 μg reaction. This amplified PCR fragment is vector V1J restricted with II, digested with BglII, and dephosphorylated (see above). Montgomery, D .; L. Et al.). According to the manufacturer's specifications , Escherichia coli DH5α (BRL-Gibco, Gaithersburg, Md.) Was transformed.³²Hybridization using P-labeled 3 'PCR primer Ampicillin-resistant colonies were screened by screening. A candidate plasmid, Restriction mapping and Sequenase DNA Sequencing K it, version 2.0 (United States Biochemica Sequencing of the vector-insert junction using l) Characterized by Similarly, a 2.7 Kb fragment encoding the gB gene; A 1.5 Kb fragment encoding the gene; and 1.6 encoding the ICP27 gene. The Kb fragment was also PCR amplified. Derived isolates are labeled with gB, gC, gD, or IC. Independently identified for the presence of a suitable DNA construct containing any of the P27 genes And characterized.   Large-scale DNA preparation involves growing an 800 ml culture for 24-48 hours, For some experiments, DNA was purified by single CsCl-EtBr isopycnic centrifugation. Except that it was manufactured (Montgomery, D.C., supra). L. Et al.)   These plasmid constructs are used for restriction mapping and vector-insert conjugation. Some were characterized by sequence analysis. The results are based on the published HSV-2G strain (La sky, L .; A. et al. 1984, DNA, 3, pp. 23-29) Distribution Match the column data and ensure that the start and stop codons for each construct are intact. Was shown.                                 Example 5 Expression of HSV-2 gB, gC, gD and ICP27 proteins from V1J plasmid Present   Rhabdomyosarcoma cells (ATCC CCL136) were placed 9.5 cm daily for 1 day before use.^Two 1.2 × 10 per well⁶At a cell density, in a 6-well tissue culture cluster 10% heat-inactivated calf serum, 2 mM L-glutamine, 25 mM H EPES, 50 U / ml penicillin and 50 μg / ml streptomycin (total (From BRL-Gibco). H Enol: Plasmid DNA extracted with chloroform and purified with cesium chloride 9.5 cm of D cells^TwoExcept using 5-15 μg for each of the wells Follow the kit instructions and use the Pharmacia CellPhet reagent to Precipitated with calcium phosphate. 6 hours after adding calcium phosphate-DNA precipitate After a short time, the culture was bombarded with glycerol; after refeeding, the culture was Incubated.   A lysate of the transformed culture was treated with 1 μM leupepsin, 1 μM pepstatin, 300 nM aprotinin and 10 μM 1 × RIPA (0.5% SDS) supplemented with TLCK 1.0% Triton X-10 0, 1% sodium deoxycholate, 1 mM EDTA, 150 mM NaC 1, 25 mM TRIS-HCl pH 7.4), Treated to reduce viscosity. Lysate is 10% Tricine gel (Novex) Separated by electrophoresis at, and transferred to nitrocellulose membrane . Immunoblotting using HSV-2 convalescent mouse serum, ECL detection kit (Amersham).   V1J: HSV gD expression from gD is shown by transient transduction of RD cells Was done. V1J: lysate of gD-transduced or mock-transduced cells was converted to SDS P Fractionated by AGE and analyzed by immunoblotting. FIG. 1A shows V1J: gD Transduced RD cells express immunoreactive protein with an apparent molecular weight of about 55K To do so. For comparison, HSV-2 (Curtis) and HSV-2 (18) 6) or lysates from mock-infected Vero cells. With cloned gD The same mobility as the authentic protein from infected cells indicates that this protein is full-length. , Processing as in gD in HSV-infected cells And glycosylated. Immobilized V1J: gD transformed cells Indirect immunofluorescence of the vesicles showed a diffuse cytoplasmic signal.   V1Jns: Expression of HSV gB from gB is due to transient transduction of RD cells. It was shown. V1Jns: lysate of gB-transduced or mock-transduced cells Fractionated by DS PAGE and analyzed by immunoblotting. FIG. 1B shows V1 Jns: immune response in which gB-transduced RD cells have an apparent molecular weight of about 140k 2 shows that a sex protein is expressed. For comparison, HSV-2 (Curtis), H Lysates from SV-2 (186), or mock infected Vero cells are included. K The similar mobility between the cloned gB and the authentic protein from infected cells indicates that this protein Indicates that the quality is full length. Immobilized V1Jns: indirect immunization of gB transduced cells Fluorescence showed a speckled signal associated with the membrane.   V1J: HSV gC expression from gC is shown by transient transduction of RD cells Was done. Indirect immunofluorescence of immobilized V1J: gC transduced cells was mainly due to A quality signal was indicated.   Expression of ICP27 was detected by transient transduction of RD cells followed by Western blot As indicated by analysis. With a mouse monoclonal antibody specific for ICP27, Major in HSV2 infected cells A protein of about 60 kDa was detected, consistent with the immunoreactive protein (FIG. 1C).                                 Example 6 Immunization with PNV and detection of anti-HSV antibodies   Five to six week old female BALB / c mice were injected with 5 mg of ketamine HC in saline. 1 (Aveco, Fort Dodge, IA) and 0.5 mg xylazine (Mobley Corp., Shawnee, KS.) Intraperitoneally (i. . p. ) Anesthetized by injection. Shaving the hind limbs with an electric clipper, 70% ethanol Washed with knol. Animals are injected with DNA suspended in a total volume of 100 μl saline Done: 50 μl for each limb.   First, the ability of V1J: gD DNA to elicit an immune response to HSV gD Were tested in a titer experiment. 200 μg to 0.7 μg in groups of 10 mice DNA was administered i.p. in a dose range of 8 μg (8-fold two-fold dilution). m. Injection or menstrual diet Mock immunizations were performed with saline. Serum obtained at 4 and 6 weeks after immunization was separated by ELISA. Was analyzed. For this ELISA, HSV-2 glycoprotein was added to 50 mM carbonate buffer. Diluted to 5 μg / ml in pH 9.5. Nunc Maxi-sorb 9 6-well play The plates were coated overnight at 4 ° C. with 100 μl HSV glycoprotein per well. Plates were washed four times with PBS pH 7.2, blocked and blocked for 1 hour at room temperature. Dilution buffer, 20 mM TRIS-HCl pH 7.5, 137 mM NaC 1, 2.7 mM KCl, 0.5% gelatin, 0.05% Tween 20 Reduced non-specific reactivity. Add serial dilutions of mouse serum and plate Incubated for 1 hour at room temperature. Goat anti-mouse I labeled with alkaline phosphatase gG (Boehringer Mannheim, Indianapolis, Plates were washed four times with PBS and once with distilled water prior to addition of Incubated for 1 hour. Excess secondary antibody was washed four times with PBS followed by one It was removed by washing with distilled water. ELISA was performed with 10% diethanolamine pH 9.8. , 100 μg / ml MgCl.6H_Two1 mg / ml p-nitrophenyl in O 100 μl of phosphate was added per well and developed at 37 ° C. 405 nm Read absorbance at OD⁴⁰⁵Is the mean plus of the same dilution of saline control serum Serum dilutions were evaluated as positive if greater than 3 standard deviations. ≧ 6.25 in 4 weeks Most animals receiving μg DNA are seropositive Met. Fewer animals were seroconverted at doses less than 6.25 μg, Even at the dose, some animals were ELISA positive. Saline injection control animals positive There was no sexual thing. At six weeks, most of the animals became seropositive.   At week 7, the same dose of DNA (or saline) used in the initial injection Animals were used to immunize the animals. At week 10 (3 weeks after the second injection) serum was obtained and EL Endpoint titers were determined by ISA. The results are summarized in Table 1. 93% in 10 weeks DNA-injected mice were seropositive. 9 animals, even at 0.78 μg dose 8 of them were positive.   To confirm that the ELISA reactivity was due to the anti-gD antibody, Some high ELISA titer sera were immunoblotted with HSV or mock infected cell lysates And characterized by their reactivity. FIG. 2A shows V1J: gD immunized mice. That the serum specifically reacts with a single HSV-encoded protein, Indicated by electrophoretic mobility, which corresponds to. Considering these, these data Intraperitoneal injection of V1J: gD DNA from mice showed gD epitope expression and gD 2 shows that an immune response against the protein is generated.   To extend these results and establish a minimum effective PNV dose, animals were Further titration of V1J: gD was performed in the immunization experiments. 5 in a group of mice V1J: gD ranging from ng to 50 μg was injected. 4th, 7th, after immunization And sera collected at week 10 were analyzed by ELISA; the data are shown in FIG. stop.   This titration reveals a response threshold of about 0.5 μg DNA. Fewer Some of the animals that received the DNA amount were seropositive by ELISA. The positive response was transient and occurred only at the lowest serum dilution. ≧ 1.67 μg For DNA By volume, over 90% of the animals seroconverted at 4 weeks and remained positive at 7 and 10 weeks. Up to.   The increase in antibody titer over time for individual animals is due to the increase in group GMT seen in FIG. Has been reflected. At doses of ≧ 0.5 μg, GMT rapidly increased between weeks 4 and 7. To rise. Between week 7 and week 10, all but one case had a titer increase, or Be kept constant. Week 10 ELISA titers of animals receiving ≧ 0.5 μg were: Similar to that achieved in previous experiments where a second DNA immunization was given. Immune response The amount of PNV needed to elicit is based on published reports using similar vectors. And 100 times less. (Robinson et al., 199 3, Vaccine, 11, pp. 957-960; and Fynan et a l. , 1993, Proc. Natl. Acad. Sci. USA, 90, pp . 11478-11482)   Compare the efficacy of single and double dose immunizations to establish a standardized protocol . In the two dose experiment, we have the highest (200 μg) dose and the lowest (0.78 μg). g) It was found that there was no significant difference in protection by dose. Potency in single dose experiment An ELISA showing seroconversion and protection efficacy when the assay is extended   A dose response to GMT was observed. The threshold for these responses is 0.5 μg. Was. However, seroconversion occurs with as little as 50 ng of DNA. In general The titer continued to rise for 10 weeks after a single injection, and in the two dose experiment There is no apparent increase in titer after injection 2. After all, 50 μg of DNA for both experiments And there is no significant difference between single and double doses.   An analysis similar to the above description was performed on a PNV construct containing the HSV genes gB and gC. I followed. Mice (10 mice per group) carry the HSVgB gene Immunization was performed as described above using the DNA having the HSVgC gene or . Serum was collected and tested for the presence of anti-gB or anti-gC antibodies in the ELISA described above. And analyzed. The ELISA data for the gB antibody is shown in Table 2. This is V 1JNS: that mice immunized with gB were seropositive for anti-gB antibody. And   Table 3 shows the ELISA data for the gC antibody. This is free with V1J: gC Strained mice (5 mice per group) were seropositive for anti-gC antibody. Indicates that   Confirm that ELISA reactivity to gB was due to anti-gB antibody To achieve this, several high ELISA titer sera were used with HSV or mock infected cell lysates. And were characterized by their reactivity. FIG. 2B shows V1JNS: gB immunized mouse. That HSV-specific serum reacts specifically with a single HSV-encoding protein. Indicated by electrophoretic mobility consistent with that of B. Considering these, this These data show that HSV PNV can be injected intraperitoneally into mice. Shows that expression of the cells and the development of an immune response against their HSV proteins.                                 Example 7 HSV neutralization   Mouse serum was serially diluted in DMEM, 2% heat-inactivated FBS. Heat inactivate at 56 ° C for 30 minutes prior to dilution, then add 50 μl of each dilution to a sterile pool. Lipene 96 deep well plate (Marsh Biomedical Rochester) NY. ). HSV-1 or After diluting the HSV-2 stock to 4,000 pfu / ml, 5 0 μl of virus was added and the plate was incubated at 4 ° C. overnight. Guinea pig Diluted complement (Cappel) 1: 4 in DMEM, 2% heat inactivated FBS , 50 μl was added to each sample well. After incubating at 37 ° C for 1 hour, 100 μl of serum free medium is added to each well and each reaction mixture is added to 12-well -Used for infection of confluent VERO cells in cluster plates (Costar) . The neutralized virus sample was adsorbed at 37 ° C. for 1 hour. Gently aspirate inoculum And the monolayer was mixed with 1 ml of 0.5% carboxymethylcellulose, 1 × MEM, 5% heat Inactivated FBS, 10 mM L-glutamine, 25 U / ml penicillin, 25 μ g / ml streptomycin, 12.5 mM HEPES. Plate Incubated at 37 ° C. for 48 hours. Remove coating and reconstitute cell monolayer 1% basic Stained with fuchsin, 50% methanol, 10% phenol. Step Lark was counted and 50% of the number of plaques when compared to serum from mock-immunized mice Neutralization titers were determined as the serum dilution that resulted in a decrease in   To determine if the anti-gD antibody is biologically active, HSV-2 neutralizing activity of high titer week 10 sera selected from mice immunized weekly Was tested. Table 4 shows the results of the plaque reduction test. V1J: gD immune system Serum from the mouse was not only HSV-2 (Curtis) but also HSV-12 (18 6) was also neutralized. In addition, at least some sera have HSV-1 (KOS) Includes type-common neutralizing antibodies indicated by their neutralization of infectivity. In some cases And low neutralization titers, these results indicate that these anti-gD antibodies were lethal. The animal might be able to protect the animals from HSV challenge.   All animals immunized with ≧ 0.5 μg V1J: gD in a single dose experiment Their 10 week sera were also tested in the HSV-2 plaque reduction assay. Tested 29 of the 49 sera were positive, a> 50% reduction at a 1:10 dilution. 16. At the 7 and 50 μg dose levels, 9 out of 10 sera from each group were neutralizing positive. Was.                                 Example 8 HSV antigen administration   The challenge virus stock was transferred to a confluent VERO monolayer with HSV as described above. -2 Curtis was prepared by infection. Cleared supernatant virus Titrated on ERO cells and aliquots were stored at -70 ° C. Animals are i. p. After infection with 0.25 ml virus stock by injection, observe for 3 weeks. Was. Survival data were analyzed using a log-rank test (McDermott et al., 19). 89, Virology, 169, p Was analyzed. Differences of probability of ≦ 0.001 were judged to be highly significant .   Eleven weeks after the initial DNA injection, mice immunized with two doses of V1J: gD were given i. . p. 105.7 p. f. u. HSV-2 (Curtis) as antigen Dosing and observation for 21 days. Survival data is in FIG. Only 0.78 μg of V1 J: easily recognized that animals immunized with gD were significantly protected from lethal infection Can be Two of the three animals that died were seronegative by ELISA at week 10. Met. Some of the surviving animals are hair Shows signs of transient illness, including lack of regularity, lack of growth, or hunched posture Was. The level of protection from death achieved at all doses of DNA was significant (p <0.01), but these symptoms suggest that some breakthrough infection occurred doing. Analysis of sera obtained from convalescent animals showed that HSV-2 infected Vero cells Lysates were characterized by their response in immunoblots. Some things In some cases, the serum only recognizes gD; in others, the serum contains many HSV proteins. Reacted with. These results indicate that at least some of the mice that have Matches.   Animals immunized with a single DNA injection were challenged as described above. Survival data It is presented in FIG. In the group of animals that received ≧ 1.67 μg of V1J: gD For example, a statistically significant (p <0.001) protection against death was obtained. For this survival The dose response is similar to that seen in the ELISA titers (FIG. 3). For two dose experiments Some surviving mice showed transient signs of disease during the observation period, as seen in Was. Surviving animals immunized with high doses of DNA (16.7 and 50 μg) Her coat was fine and healthy in appearance.   Animals immunized with PNV constructs containing HSV gB or gC also They were challenged with a lethal dose of HSV as described above. V1Jns: exempt with gB The survival data of the animals that were infected are shown in FIG. The data is shown in FIG. These indicate that protection from death has been obtained.   These results demonstrate the potential of direct DNA immunization in preventing HSV infection . Using the glycoprotein gD as a model, only 0.5 μg of V1J: gD DNA Once i. m. Injection provides statistically significant protection against lethal HSV challenge Eliciting a neutralizing antibody response against gD. For two dose prescription Immunization with as little as 3.13 μg of DNA protected all animals from death.                                 Example 9 Vaccination of guinea pigs with HSV PNV   Hartley guinea pigs (Harlan S) weighing approximately 200-250 g each (Prague Dawley Labs, Indianapolis, IN) 11 and 4 weeks prior to virus challenge, 0.1 ml and left thigh in right thigh 0. Vaccinated 1 ml intramuscularly. For each vaccination, a fresh solution of the vaccine Fluid and placebo were sent to us.   To determine the production of HSV-2 antibodies in these animals, The animals were kept 5 weeks after the first vaccination and 2 weeks after the second vaccination. Was. Blood (0.6-1 ml per animal) was obtained by cutting the toes. Small blood Collected in a separation tube (Becton Dickinson) and later at 1000 × g for 10 The serum was separated by centrifugation for minutes.   Serum collected from these guinea pigs was used for the ELISA described in Example 6. Was analyzed for the presence of anti-HSV antibodies. Table 5 shows the results.   At the time of infection, guinea pigs weighed 600-700 g each. These in the vagina To infect herpes simplex virus type 2 (HSV-2) and E194 strain. this is Achieved in a three-step process. First, the vagina of each animal was soaked in 0.1 N NaOH. And wiped with a cotton tip applicator for 5 seconds. This treatment irritates the vaginal area and Good infection results. After about 45-60 minutes, each vagina was wiped dry for 5 seconds . Thereafter, each guinea pig was wiped for 20 seconds using virus medium (about 5 × 1 / ml). 0⁶An applicator dipped in plaque forming units HSV-2) was used. This wipe Or gently and slowly turn back and forth while they are in place Was something.   Lesion scores in infected animals were measured daily on days 2-15 post-infection. Score 1+ indicates that 25% of the anal-vaginal area was affected (usually redness immediately around the vagina) 2+ indicates that 50% of the anus-vagina were affected; 3+ was 75% affected And 4+ indicates that 100% was affected. Several animals on the way The lesion score near death was transferred at the end of 15 days. Do this If not, most of the affected animals will die and Scores are likely to decrease.   Deaths were recorded daily for 21 days. The calculation of the average death date includes the number of dead guinea pigs. Only consideration. The number of animals with hind limb paralysis was noted throughout the infection. Vagina ui Ruth titers were obtained by wiping the vagina 1, 4 and 6 days after virus inoculation. This was done by measuring the titer of Irus. These swabs contain 1 ml of cell culture medium Into a tube. The titration of these samples was performed using the Ver. o Performed on cells. The calculation of the virus titer is described in Reed L. et al. J. And Mue nch M. Am. J. Hyg. 50% end of 27,493-498 (1938) Performed by the endpoint dilution method. Virus titer is log 10 cell culture per ml Expressed as infectious dose.   Survival (Fisher exact test), death Average days to death (Mann-Whitney U test)   test)), paralysis (Fisher precision test), vaginal virus titer (Man- Statistical solution of Vutney U test) and vaginal lesion score (Mann-Whitney U test) The analysis was performed by two-sided analysis.   FIG. 8 shows survival, mean days to death, paralysis in guinea pigs infected with HSV-2. , And vaginal virus titer results. Higher dose vaccines will be better than placebo controls To reduce mortality, And reduced vaginal virus titers on day four. High dose vaccines are available for these animals. And significantly prevented paralysis. Low-dose vaccine also reduced these parameters .   Table 7 shows the daily vaginal lesion score for this experiment. High and low capacity wax Both chins had severe vaginal lesions from day 3 to 15 of infection compared to placebo. Caused a significant decrease in gender. The results in Table 7 are shown schematically in FIG.   These results indicate that this vaccine was protective in guinea pigs infected with HSV-2. And clearly show that the high dose vaccine is more active than the low dose. High A dose of vaccine will recover the virus from the vaccinated recipient, Infection cannot be completely blocked because of the development of abnormalities. Or maybe Nevertheless, the degree of protection obtained with this dose of vaccine was considerable. This The results of this antibody study correlate with antiviral protection.   Guinea pigs were administered at two different doses 11 and 4 weeks before vaginal HSV-2 challenge. Intramuscularly administered vaccine significantly protected animals from the disease. High dose Vaccine was more effective than the lower dose. This vaccine is safe in these animals It seems to be.                                 Example 10   Mice vaccinated intramuscularly with PNV HSV have mucosal HSV-specific anti- To determine whether or not to produce a body, the mice were exposed to 12.5 or 1.56 μg of V Vaccinated with 1Jns: gD. Collect by wiping the vaginal fluid The antibody was eluted from the slab using phosphate buffered saline. This eluate is washed with HS The presence of IgG and IgA specific for the V-2 protein was analyzed. This mouse Mouse IgG specific for detection of the presence of HSV-specific IgG and IgA in the sample A commercially available antibody (Boehringer) and a commercially available antibody specific for mouse IgA (S ELISA was performed as described above, except that eralab) was used. Ig The results for G are shown in Table 8: Is IgA not detected in any animal Was.   These results indicate that HSV- in mice vaccinated with V1J: gD. 2 shows the presence of mucosal IgG specific for 2.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI // (C12N 15/09 ZNA C12R 1:92) (81) Designated country EP (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (KE, MW, SD, SZ, UG), AM, AU, BB, BG, BR, BY, CA, CN, CZ, EE, FI, GE, HU, IS, JP, KG, KR , KZ, LK, LR, LT, LV, MD, MG, MN, MX, NO, NZ, PL, RO, RU, SG, SI, SK, TJ, TM, TT, UA, US, UZ (72 ) Inventor Keith, Robert Day USA, New Jersey 07065, Lowway, East Lincoln Avenue 126 (72) Inventor Louis, Jyon Ay United States, New Jersey 07065, Lowway, East Lincoln Avenue 126 (72) Inventor Liu, Margaret A. United States, New Jersey 07065, Lowway, East Lincoln Avenue 126 (72) Inventor McClements, William El United States, New Jersey 07065, Lowway, East Lincoln Avenue 126

Claims (1)

[Claims] 1. Induces anti-HSV antibodies or protective immune responses when introduced into vertebrate tissues A polynucleotide comprising one or more HSV proteins or It contains one or more genes encoding their functional equivalents, which genes A polynucleotide operably linked to a motor. 2. The gene is gB, gC, gD, gH, gL, ICP27, and 2. An HSV protein selected from the group consisting of: Polynucleotides listed. 3. Induces an immune response to HSV epitopes in non-human vertebrates A method according to claim 1, wherein 1 ng to 5 mg of the polynucleotide according to claim 1 is used in a vertebrate. A method comprising introducing into an organization. 4. A vaccine for eliciting an immune response against HSV, comprising the vaccine of claim 1. A vaccine containing a polynucleotide and a pharmaceutically acceptable carrier. 5. A method for eliciting an immune response to HSV, comprising the steps of: Prevent HSV infection in the tissue and / or One or more isolated and purified Hs that elicit an immune response that ameliorates HSV disease A method comprising introducing an SV gene. 6. a) eukaryotic transcription promoter;   b) coding one or more HSV epitopes operably linked to the promoter; An open reading frame and a translation termination signal;   c) optionally, one or more operably linked IRESs, one or more additional genes One or more open reading frames encoding one or more transcription tags Terminator signal, A polynucleotide comprising 7. The additional gene of c) is GM-CSF, IL-12, And a member of the B7 family of T cell costimulatory proteins. The polynucleotide according to claim 6, which is an immunomodulatory or immunostimulatory gene obtained. 8. The HSV gene of a) is gB, gC, gD, gH, gL, ICP27. And HSV proteins selected from the group consisting of functional equivalents thereof. The polynucleotide according to claim 6, wherein 9. The further gene of c) is gB, gC, gD, gH, HSV selected from the group consisting of gL, ICP27, and functional equivalents thereof. The polynucleotide according to claim 6, which is a gene. 10. Anti-HSV antibodies or protective antibodies when introduced into non-human vertebrate tissue Non-human vertebrates in need of treatment with polynucleotides that trigger an epidemic response A method of treating, wherein said polynucleotide comprises one or more HSV proteins or a combination thereof. A gene that encodes a functional equivalent of How to combine as possible. 11. The polynucleotide is gB, gC, gD, gH, gL, ICP27; And one or more HSV proteins selected from the group consisting of: 11. The method of claim 10, comprising an encoding gene.