JP2001501101A - Polynucleotide herpesvirus vaccine - Google Patents

Abstract

  (57) [Summary] The gene encoding herpes simplex virus type 2 (HSV-2) is cloned into a eukaryotic expression vector to express the encoded protein in mammalian muscle cells in vivo. Animals are immunized by intramuscular injection of these DNA constructs, called polynucleotide vaccines or PNV. DNA titration showed that a single immunization with 0.5 μg (one) of PNV resulted in> 90% seropositivity by 10 weeks post-immunization. The immune antiserum neutralizes both HSV-2 and HSV-1 in the cell culture medium. When animals are exposed to HSV-2, significant (p <0.001) protection from lethal infection is achieved after PNV inoculation. The DNA constructs are full-length, truncated and / or mutated, and can be immunized together or in combination to optimize immunity and protection from HSV infection.

Description

DETAILED DESCRIPTION OF THE INVENTION                 Polynucleotide herpesvirus vaccine Interaction with related applications   This application is a continuation of US application Ser. No. 08 / 279,459, filed Jul. 22, 1994. Part of a continuation-in-part application Ser. No. 08 / 715,309, filed Sep. 18, 1996. It is a continuation application. Statement on Federal Indemnity R & D   Applied addition Reference to microfish attachment   Applied addition Field of the invention   Applied additionBackground of the Invention   Viruses, especially multiple serotypes, in which neutralizing and eliciting a protective immune response are desired The main obstacle to vaccine development against high mutation rate viruses The diversity of the viral exogenous protein among the isolates or strains. Mouse Cytotoxic T lymphocytes (CTL) in both human and human It can recognize epitopes derived from viral proteins [J. W. Yellowde II, et al., Proc. Natl. Acad. Sci. (USA) Vol. 82, 17 85 (1985); R. M. Authors: Townsend et al., Cell, Volume 44 959 (1986); J. McMichael et al. Gen. V irol. 67, 719 (1986); Bastin et al. E xp. Med. 165, 1508 (1987); R. M. Town send and H.S. Bodmer, Annu. Rev .. Immunol. Seventh Vol. 601 (1989)], which is important in the immune response to the virus. [Y. -L. Lin and B.A. A. Askonas, J.A. E xp. Med. 154, 225 (1981); By Gardner et al. , Eur. J. Immunol. 4, 68 (1974); L. Ya p and G. L. Ada, Nature 273, 238 (1978) A. J. McMichael et al., New Engl. J. Med. No. 309 Volume, 13 (1983); M. Taylor and B.C. A. Askona s, Immunol. 58, 417 (1986)] Development of CTL vaccines that can provide heterologous protection against different virus strains Efforts have been made.   CTLs show that their T cell receptors are MHC class I and / or class II Kills virus-infected cells when it recognizes a viral peptide associated with the molecule It is known that These peptides include the location of the protein in the virus. Irrespective of their function or function, they can be derived from endogenously synthesized viral proteins. Wear. By recognizing epitopes from conserved viral proteins, CTLs can Protection can be provided.   Many infectious disease inducers themselves bind and injure themselves and are harmless Can induce protective antibodies, and can also induce disease inducers and their by-products Things are injured and rendered harmless. Recovery from these diseases usually results in infectious agents Sustained immunity results because of the protective antibodies generated against the high quality antigenic components.   Protective antibodies are part of the natural defense mechanism of humans and many other animals, In addition, it is found in other tissues and body fluids. Feeling without causing illness Inducing mostly protective antibodies against dyes and / or their by-products A minute Chin's main function.   Most efforts to prime a CTL response generate intracellular protein antigens For this purpose, a replicated solid is used [J. R. The same place by Benlink et al. Thirty first 1, 578 (1984); R. Benlink and J.M. W. New Dell, Curr. Top. Microbiol. Immunol. Sixteenth 3, 153 (1990); K. Stover et al., Nature 3 51, 456 (1991); Aldovini and R.A. A. You ng, Nature 351, 479 (1991); Schafe r., et al. Immunol. 149, 53 (1992); S. H ahn et al., Proc. Natl. Acad. Sci. (USA) Vol. 89, 2 679 (1992)], or the introduction of peptides into the cytosol. [F. R. Carbone and M.S. J. Bevan, J. et al. Exp. Med . 169, 603 (1989); Deres et al., Nature 342, 561 (1989); Takahashi et al., Same place, 344, 873 (1990); S. Collins et al. Im munol. 148, 3336 (1992); J. Newman et al., Same place 148, 2357 (1992)]. None of these techniques Has the limitation that its usefulness as a component can be reduced. Retro virus vector Can be expressed as a fusion protein while maintaining the ability of the recombinant virus to replicate There are restrictions on the size and structure of the polypeptide [A. D. By Miller , Curr. Top. Microbiol. Immunol. Volume 158, 1 page (1992)], Effect of Vaccinia-like Vectors for Subsequent Immunization May be impaired by an immune response to vaccinia [E. L. Coo ney et al., Lancet 337, 567 (1991)]. Also, Lus vectors and denatured pathogens can be an inherent hazard that can hinder their use in humans [R. R. Redfield et al., New Engl. J. Med. 316, 673 (1987); Mascola et al., Arch. I ntern. Med. 149, 1569 (1989)]. Furthermore, expression The choice of peptide epitope to be taken depends on the structure of the individual's MHC antigen, Peptide vaccines show diversity of MHC haplotypes in outbred populations Effects may be limited due to gender.   Benvenisty, N .; And Reshef, L .; [PNAS Vol. 83, 9551-9555 (1986)], intraperitoneal, intravenous or muscle of mice. CaCl introduced into_TwoIt was shown that the precipitated DNA could be expressed. Mouse muscle Injection of the DNA expression vector into the meat involves uptake of the DNA by muscle cells and And expression of proteins encoded by DNA and DNA [J. A. Wolff et al. G., Science, 247, 1465 (1990); Ascadi et al. Author, Nature, 352, 815 (1991)]. Plasmids are episodes And was not replicated. Followed by rats, fish and primates Following intramuscular injection into skeletal and rat myocardium, sustained expression was observed [H . Lin et al., Circulation 82: 2217 (1990); R. N. Kitsis et al., Proc. Natl. Acad. Sci. (USA 88, 4138 (1991); Hansen et al., FEBS L ett. 290, 73 (1991); Jiao et al., Hum. Ge ne Therapy, vol. 3, p. 21 (1992); A. Wolff et al. , Human Mol. Genet. 1, 363 (1992)]. Remedies Report using WO90 / 11092 (October 4, 1990) Where a bare polynucleus was used to vaccinate vertebrates. Reotide has been used.   Recently, the surface of antigen presenting cells in the activation of CTL for tumor removal has been reduced. The coordination function of pitope B7 and primary histocompatibility complex (MHC) expression was studied [Edgington, Biotechnology, Vol. 11, 1117- 1119, 1993]. MHC molecules on the surface of antigen presenting cells (APC) When an epitope is expressed on the T-cell receptor (TCR), Expressed B7 binds to CTLA-4 or CD28 to produce a second signal. Acts as a signal. As a result, CD8 + T cells are proliferated and killed by APCs. Cell division of signaling CD4 + helper T cells is accelerated.   Intramuscular immunization is not required for the success of the method. That is, Tang [Nature, 356, 152-154, (1992)] Gold microspheres coated with DNA coding for growth hormone (BGH) were Led to the skin On entry into mice produced anti-BGH antibodies. Furt H et al. [Analytical Biochemistry, Vol. 205, 36] 5-368, (1992)], skin, muscle, fat, and breast of living animals. It has been shown that a jet injector can be used to transfect tissues. Nuclear Various methods for introducing acids have recently been investigated [Friedman, T. et al. Author , Science, Vol. 244, pp. 1275-1128, (1989)]. R Obinson et al. [Abstracts of Papers Presen] ted at the 1992 meeting on Modern Ap approaches to New Vaccines, Included P Revision of AIDS, Cold Spring Harbor , P. 92]. Here, the bird flu DNA is used to Intra-, intraperitoneal and intravenous administration provide protection against lethal doses of antigen It is stated that. DNA in mice: Cationic liposome complex Intravenous injection is described by Zhu et al. [Science Vol. 261, pp. 209-211, ( Jul. 9, 1993); see also WO 93/24640, 1993. 9/9] caused systemic expression of the cloned transgene Was shown. Recently, Ulmer et al. [Science 259, 174] 5-1749, (1993)] describes the influenza virus protein Protection against influenza virus infection by injection of coding DNA And reported.   Wang et al. [P. N. A. S. USA, 90, 4156-4160, ( May 1993)] is the cloned genome (unspliced). I) Induction of an immune response in mice against HIV by intramuscular inoculation of the HIV gene We report about departure. However, the level of immune response achieved is very low The system uses a mouse mammary tumor virus (MMTV) terminal repeat (LTR) promoter. Part of the promoter and the simian virus 40 (SV40) promoter and promoter I used the part of the minator. SV40 is probably assembled into host cell DNA. It is known to transform cells by incorporation. In other words, Wang et al. The more described system is completely unsuitable for human administration, which is an object of the present invention. One.   WO 93/17706 vaccinates animals against the virus A method for immunization is described, wherein the carrier particles are coated with a genetic construct. Overlaid, the coated particles are facilitated to enter the cells of the animal.   Recent development of subunit vaccines for herpes simplex virus (HSV) Efforts are focused on novel expression of viral antigens, especially viral glycoproteins [For review, Burke, R .; L. Author, 1993, Sem. In Viro l. , Vol. 4, pp. 187-197]. Yeast (Kino., YC) . Authors, 1989, Vaccine, Vol. 7, pp. 155-160), insect cells (Ghiasi, H. et al., 1991, Arch. Virol., Vol. 121) 163-178) and mammalian cells (Burke, RL, 199). One year, Rev. Infect. Dis. , Volume 13, S906-S911; L Asky, L .; A. Author, 1990; Med. Virol. , Vol. 31, 5 9-61), recombinant HSV glycoproteins expressed by various systems Elicited protective immunity in an animal model. Chinese hamster Clinical trial of recombinant HSV-2 glycoprotein D (gD) produced in ovarian cells The test showed that the vaccine was Elicit a response, and in HSV-1 and HSV-2 seropositive individuals, It has been shown to stimulate pre-existing responses (Straus, SE et al., 19 1993; Infect. Dis. 167, 1045-1052).   Another approach to subunit vaccination is to use live virus for delivery of HSV antigens. Using a vector. gD (Aurelian, L. et al., 1991) Year, Rev. Infect. Dis. , Vol. 13, S924-S930; Ro oney, J .; F. Et al., 1991, Rev. Infect. Dis. , First 3, S898-S903; Wachsman, M .; Authors, 1992, Va ccine, vol. 10, pages 447-454), gB (Rooney, JF, et al.). GL and gH (Browne, H. et al., 1993, J. Ge. n. Virol. , Vol. 74, pp. 2813-2817). The HSV recombinant protected animals well from HSV exposure. Express HSV gB Immunization by infection with a recombinant adenovirus that protects against Induces an epidemic response (Ghiasi, H., supra; McDermott, MR). . Author, 1989, Virology, 169, 244 247). Natural protein (Long, D. et al., 1984, Infect . Immun. 43, 761-764), recombinantly expressed proteins (Bur ke, R .; L. Authors, supra; Stanbury, L .; R. Et al., 1987, J . Infect. Dis. 155, 914-920; Straus, S. . E. FIG. Authors, supra), peptides derived from gD (Eisenberg, RJ et al., 1985; Virol. , Vol. 56, pp. 1014-1027). (Dix, RD et al., 1981, In fact. Immun. 34, 192-199; Ritchie, M .; H. Authors, 1993, Investigative Ophthalmolo. gy and Visual Sciences, Vol. 34, 2460-246 8) that the anti-gD antibody can protect against HSV infection Have been widely reported.   The work by Wolff et al. (Supra) initially encodes a reporter gene. Intramuscular injection of plasmid DNA allows muscle cells at and near the site of injection The gene is expressed in. A recent paper encodes influenza A hemagglutinin Plus Midgomery, DL, et al., 1993, Cell Biol. . , Vol. 12, p. 777-783) or nucleoprotein (Montgom). ery, D .; L. Ulmer, J. et al., Supra. B. Author, 1993, Sci ence, vol. 259, 1745-1749). Excellent immunization of the mice was reported. DNA immunization against herpes virus The first use of chemicalization was reported (Cox et al., 1993, J. Virol. 67, 5664-5567). Bovine herpesvirus 1 (BHV-1) sugar Injection of the plasmid encoding the protein gIV was performed in mice and calves. To generate anti-gIV antibodies. Upon intranasal exposure to BHV-1, immunized calves Showed less symptoms and produced substantially less virus than the control. HSV Glycoprotein D is expressed in mice (Long, D. et al., Supra) and in guinea pigs (S tanbury, L .; R. Authors, supra; Stanbury, L .; R. Et al. 1989, Antiviral. Res. , Vol. 11, pp. 203-214) The ability to elicit a protective immune response in the art has been well described in the literature.Summary of the Invention   To test the effect of DNA immunization in preventing HSV disease, HSV-2 Cloning the DNA sequence encoding the protein into a eukaryotic expression vector; Was. This DNA construct elicits an immune response when injected into animals. Immunization Infected with HSV and directly with the gD gene (or other HSV-2 gene). It was evaluated whether DNA immunization could protect animals from disease. That is, injection When introduced directly into animal tissues, human herpes simplex virus (HSV) DNA structures and RNA transcripts that can induce in vivo expression of proteins Containing nucleic acids are disclosed. Injection of these nucleic acids will result in cells specific for the HSV antigen Immune response leading to the production of damaging T lymphocytes (CTLs) and subsequent HS V exposure can induce the production of protective HSV-specific antibodies. these The nucleic acid can prevent infection and / or ameliorate an HSV-related disease, It is useful as a vaccine for inducing immunity to HSV.   In a particular embodiment of the invention, the herpes simplex virus type 2 (HSV- 2) expressing full-length glycoprotein D (gD); Or using a DNA vaccine cleaved from HSV-2 glycoprotein B (gB) To provide a protective effect against HSV-2 infection.   The invention also provides for the ability to immunize a host against herpes infection. Plus a herpes simplex virus type 2 (HSV-2) protein It also relates to the use of a mid expression vector. Protein elicits an immune response to HSV Expressed together or in combination with one or more additional proteins be able to.   In a preferred embodiment of the present invention, the full length glycoprotein D (gD), and HS Combination vaccine preparation cleaved from V-2 glycoprotein B (gB) Used at low doses.   In a preferred embodiment of the invention, the amino-terminal 707 amino acids of gB (aa ) Using immunization with a plasmid expressing Induces a humoral immune response that is more detectable.   In a particularly preferred embodiment of the present invention, the cleaved gB plasmid is In combination with a plasmid that expresses this The DNA structure of the combination may be in the state of another DNA vector or each antigenic protein. It may be a single vector expressing a protein or protein fragment . Again, these nucleic acid constructs prevent infection and / or HSV-related Useful as a vaccine to induce immunity to HSV, which can improve disease is there.BRIEF DESCRIPTION OF THE FIGURES   FIG.Panels A, B and C   A. V1J: Western blot of HSV gD expression by gD-transfected cells Shows blot analysis;     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 transfected with gD;     5. Mock transfected RD cells.   B. V1JNS: Waste of HSV gB expression by gB transfected cells Figure 4 shows a tan blot analysis.   C. V1J: HSV with ICP27 transfected cells 4 shows a Western blot analysis of ICP27 expression.   FIG.Panels A, B and C   A. HSV PNV-immunization (B-V1J: 6.25ug administration of gD, CV 1J: 50 ug of gD) and mock-immunized (A) Stern blot analysis is shown for lysates of BHK cells infected with: :     1. HSV-1 KOS;     2. HSV-2 186;     3. HSV-2Curtis;     4. False infection.   B. Western blot using immunization from HSV PNV gB immunized animals Show the analysis.   FIG. ELISA on HSV PNV immunized animals injected once with vaccine Shows the occurrence group GMT data. Serum was obtained at 4, 7 and 10 weeks after immunization.   Figure 4. 200 ug; 100 ug; 50 ug; 25 ug; 12.5 ug, 6.2 5ug; 3.13ug; 1.56ug; 0.78ug V1J: gD or physiology Of HSV-2 exposed animals after two injections of saline alone. 200ug; 10 0ug; 25 ug; 12.5 ug, 6.25 ug; and 3.13 ug group of animals all survived. Therefore, they are all represented by a single code.   Figure 5.50ug; 16.7ug; 5.0ug; 1.67ug; 0.5ug; 0 . 167ug; 0.05ug; 0.017ug; 0.005ug of V1J: gD Or the survival rate of HSV-2 exposed animals after a single injection of saline alone.   FIG. V1JNS: Survival of animals exposed to HSV after immunization with gB.   FIG. V1J: Survival of animals exposed to HSV after immunization with gC.   FIG. Survival, average days of death, paralysis and vagina in guinea pigs infected with HSV-2 The results of viral load are shown.   FIG. The guinea pig vaginal lesion value is shown.   FIG. Mice infected by intraperitoneal injection of HSV-2 in Example 11 2 shows the effect of DNA immunization on the survival of E. coli. Mice were twice as rare as gD-2 (A) Diluent, half log dilution of gB-2 (B) or twice with saline It has become epidemic. The dose (μg) is shown in the figure. Table 9 shows the number of mice in each group. Mice were clarified with the HSV-2 strain Curtis clarified material 0.2. 5 mL (10^5.7pfu) by antigen injection by intraperitoneal injection Observed for signs of survival for 3 weeks.   FIG. G for the survival of mice infected by intraperitoneal injection with HSV-2 D-2 Effect of immunization with DNA or vector DNA. The mouse is 12.5μ g (o), 1.6 μg gD-2 (·) or 12.5 μg (o) Vector V1 Immunized with J. Virus exposure is described in FIG.   FIG. GD-2 and primary HSV-2-induced disease in guinea pigs Example 11 shows the effect of immunization by the combination of gB-2 DNA and gB-2 DNA. Immunization And mock immunized guinea pigs and HSV-2 strain MS applied to vaginal and external genital skin Infected by One hour prior to infection, the vaginal closure membrane was washed with saline-moistened cotton. The bar was ruptured and the vagina and external genitalia were wiped with 0.1 N NaOH. 1 in tissue culture medium 0^6.7In clarified HSV-2 MS infected Vero cell lysate diluted to pfu / mL The virus was introduced using a cotton swab soaked in. Insert cotton swab into vagina and rotate Later, it was turned five times, removed, and the entire external genitalia was wiped. To ensure infection, The irs application was repeated after 1 hour. For mock-infected groups, mock-infected Vero cells Was used to prepare an inoculum. Movement Items were randomly caged and evaluated daily by observers unaware of the test group. 3 On the day, the virus-carrying medium (Carr-Scarborough Microb) iologicals Inc. Manufactured by Stone Mt. , GA) dissolved in 2 mL The vagina was wiped with a cotton swab moistened with the released calcium alginate. Virus re-isolation, HERPCHEK^TMKit (Dupont, North Billerica) , MA), and the appearance of positive reactions or symptomatic diseases, and non-structural HSV Infection was confirmed by the generation of antibodies on the virus. The severity of the external illness, Stanberr (1982, J. Infect. Dis. Vol. 146: 397-404) ) Was quantified using a visual evaluation system applied from those described in (1). Numerical rating Values were fitted to indicate a particular disease using the following criteria: 0; no disease, 1 Redness or swelling; 2; some (≦ 3) small blisters; 3; some (≦ 3) large Kind blisters, 4; large ulcer with soft immersion. 0.5, 1.5, 2.5, and 3. A rating of 5 applies to intermediate-grade illness. The average daily lesion rating is Calculated by dividing the sum of group lesion ratings by the number of observations. During the observation period In case of death, the last rating assigned to the animal is The final value in between. Immunizing the animals with 3 μg gD-2 + 10 μg gB-2, Exposure with HSV-2 (o) n = 7; mock immunization with saline, HSV-2 (o) n = Exposure at 8; or mock-immunized and mock-infected (t) n = 6. *: 3 animals cheap Euthanasia †: Two animals euthanized   Figures 13A and B. V1Jns for HSV-induced disease in guinea pigs Example 12 shows the effect of immunization with gB. The immunized animal was used to Antigen exposure and evaluation were performed essentially as described by ry et al. (supra). One hour after rupture of the vaginal obturator membrane with a damp swab, 10 minutes with a gavage needle^5.7 The pfu HSV-2 strain MS was placed in the vagina. HERPCHEK^TMKit (D by re-isolating the virus from the vagina on day 2 or 4 Or by evaluating serum after exposure. Animals cage randomly And evaluated daily by an observer unaware of the test group. The numerical rating is based on the following criteria Was used to indicate a particular disease: 0; no disease, 1; redness or swelling 2; some (≦ 3) small blisters, 3; some (≦ 3) large blisters, 4; Large ulcer with soft immersion. 0.5, 1.5, 2.5, and 3.5 Ratings apply to moderate severity illness. The average daily lesion rating is Calculated by dividing the sum of the lesion ratings of the group by the number of observations. Died during observation period In the case of, the last rating value applied to the animal was taken as the final value for the observation period. (A) Primary disease: 100 μg V1Jns: HA (n = 10); n, 10 μg V1Jns: gB (n = 9); 1,100 μg of V1Jns: gB (n = 9). (B) Recurrent disease: 100 μg of V1Jns: HA (n = 6); n, 10 μg of V 1Jns: gB (n = 7); 1,100 μg of V1Jns: gB (n = 9).   Figures 14A and B. Example 12 Against HSV-Induced Disease in Guinea Pigs , V1Jns: gB + V1Jns: gD. Virus exposure And the evaluation of the disease is as shown in FIG. (A) Primary disease: 100 μg of V1 Jns: HA (n = 8); 1,100 μg of V1 Jns: gB + 100 μg of V 1Jns: gD (n = 8). (B) Recurrent disease: 100 μg of V1Jns: HA ( n = 8); 1,100 μg of V1Jns: gB + 100 μg of V1Jns: gD (N = 8).   Figure 15. Group of 6 African green monkeys, gD and gB DNA DNA mixture containing 100 or 10 μg (real (See Examples 11 and 12) at weeks 0 and 4 and additional at week 24 did. The sera obtained at 4 week intervals were purified using anti-g using an antigen-specific ELISAS. Analyzed for D and anti-gB antibodies. For neutralizing activity, The plaque reduction% was determined for each serum dilution compared to the same dilution (twice), A reduction of 50% or more in the number was positive. 1:10 to 1: 10,000 10 times continuous The dilution was assayed; the amount at the end was calculated by linear regression analysis (the lowest tested The serum negative at the highest dilution was assigned an endpoint activity value of 1). Arrows indicate immunization. (A ) ELISA log_TenGMT +/− standard error: anti-gD response: 100 μg administration Amount group; ▲ 104 μdose group: anti-gB response: q 100 μg dose Group; n 104 μg dose group: (B) GMT +/− SE HSV -2 neutralizing antibody: m 100 μg dose group; 110 μg dose group.Detailed description of the invention   The present invention is directed to direct introduction into a living body of a vertebrate including a mammal such as a human. And a polynucleotide that expresses the protein encoded in the animal. here Polynucleotides used in When introduced into living vertebrate cells, tides convert into genes containing polynucleotides. Can work on cellular machinery to produce more encoded translation products It is a nucleic acid containing essential control elements. In one embodiment of the invention, the polynucleotide Otides contain plasmids containing the HSV gene operably linked to the translation promoter. Oxyribonucleic acid. In another embodiment of the present invention, a polynucleotide Vaccine is an HSV gene (ribosome, t RNA and other translation factors). Polynucleoti If the protein encoded by the disease is ill, like a protein associated with HSV, Proteins that do not normally occur in animals except in If so, the animal's immune system is activated to initiate a protective immune response. It is made. Because these exogenous proteins are produced by the animal's own tissues, The expressed protein is applied to major tissues in the same manner as when actual HSV infection occurs. Processed by a compatibility system (MHC). The results shown in this disclosure show that HSV Eliciting an immune response to Generates an immune response to the encoded protein A polynucleotide vaccine or a polynucleotide vaccine Is called PNV.   There are many embodiments of the present invention that one skilled in the art can understand from the specification. You That is, different transcription promoters, terminators, carrier Specific gene sequences can be used successfully.   The present invention induces in vivo expression of a polynucleotide upon introduction into a mammalian tissue. Provided is a method using a polynucleotide that produces the encoded protein thereby I do. One of skill in the art would be able to modify the amino acid sequence of the encoded protein Can produce variants or derivatives of the nucleotide sequence encoding Is. The modified expression protein may have a modified amino acid sequence. Can elicit additional antibodies that react with the viral proteins and are functionally equivalent. Conceivable. In addition, the fragment of the full-length gene encoding the full-length protein Can also be formed. These fragments are antibodies that react with viral proteins. Protein or peptide that induces It is thought that.   In one embodiment of the present invention, the gene encoding the HSV gene product is Incorporated into the current vector. Vector is true Transcription promoter recognized by nuclear RNA polymerase, and HSV gene A transcription terminator is included at the end of the coding sequence. In a preferred embodiment And the promoter is a site having an intron A sequence (CMV-intA). Although it is a megalovirus promoter, those skilled in the art Use many other promoters, or other eukaryotic gene promoters Understand what you can do. A preferred transcription terminator is bovine growth hormone terminator. Minator. A combination of CMVintA-BGH terminators is preferred New In addition, it is necessary to assist in the preparation of polynucleotides in eukaryotic cells. If the antibody resistance marker is also used as an expression vector under the transcriptional control of a suitable prokaryotic promoter Included. Ampicillin resistance gene, neomycin resistance gene or any Other suitable antibiotic resistance markers can be used. Preferred practice of the invention In some embodiments, the antibiotic resistance gene encodes a neomycin resistance gene product You. In addition, growth in prokaryotes assists in high-level production of polynucleotides Advantageously, the vector contains a prokaryotic origin of replication and has a high copy number to . All of the many commercially available prokaryotic cloning vectors Provide these elements. In a preferred embodiment of the present invention, these functional substances Is provided by a commercial vector known as the pUC series. But Thus, it is desirable to remove non-essential DNA sequences. That is, pUC la The sequences encoding cZ and lacI are removed. Vectors replicate in eukaryotic cells It is also desirable that they cannot be manufactured. This allows the polynucleotide vaccine sequence to be accepted The risk of integration into the human genome is minimized.   In another embodiment, the expression vector pnRSV is used, wherein the vector Rous sarcoma virus (RSV) long terminal repeat (LTR) used as promoter Can be In a further embodiment, V1, the CMV promoter and Mutant pBR322 vector into which BGH transcription terminator has been cloned Is used. In a preferred embodiment of the present invention, V1 and pUC19 are required. The elements are combined to produce an expression vector called V1J. V1J or In another desirable expression vector, an HSV gene such as gD, or gB , GC, gL, gH and anti-HSV immune responses such as ICP27 (antibodies and / or Or other HSV genes that can induce CTL) Is cloned. In another embodiment, the ampicillin resistance gene Is removed from V1J and replaced with the neomycin resistance gene, Any of the many different HSV genes as used may contain clonin V1J-neo that can be plugged. In yet another embodiment, the base Is unique to a single Kpn1 site at position 2114 of V1J-neo. Is the same as V1Jneo except that the Sfi1 restriction site of 1 Jns. Very low incidence of Sfi1 sites in human genomic DNA (About 1 per 100,000 bases). That is, this vector contains the extracted genotype. Of expression vector into host DNA by Sfi1 digestion of DNA Monitor carefully. In a further embodiment, the vector is V1R . In this vector, as much non-essential DNA as possible is "cut". A smaller vector is produced. This vector contains unwanted sequences. Large inserts can be used regardless of the specific virus When a gene-encoding structure is introduced into surrounding tissue, it is taken up by cells. Only optimization. Following the vector denaturation and expression procedures described above The methods used can be achieved by methods known to those skilled in the art. .   From this work, one of skill in the art will recognize that one of the utilities of the present invention is that HSV sequence diversity and HSV To be able to form relationships with serology and other parameters of V neutralization To provide systems for in vivo and in vitro testing and analysis to understand. The isolation and cloning of these various genes is known to those of skill in the art. Can be achieved by the following methods. The invention further provides for the production of vaccines. And methods for systematically recognizing HSV strains and sequences. Primary single HSV strain Integration of genes from isolation triggers an immune response against clinical isolates of the virus To provide an immunogen that meets the needs that have not been met in the art. In addition, toxic If the isolate changes, the immunogen can be denatured to reflect the new sequence if necessary. You.   In another embodiment of the present invention, the gene encoding the HSV protein is Binds directly to a transcription promoter. Tissue-specific promoter or enhancer For example, the use of the muscle creatine kinase (MCK) enhancer element has been identified It is desirable to limit the expression of the polynucleotide to a given tissue type. For example, Muscle cells are terminally differentiated cells that do not divide. Integrate heterologous DNA into chromosome To do so, it appears that both cell division and protein synthesis are necessary. That is, tongue It may be preferable to limit park expression to non-dividing cells, such as muscle cells. However, the use of the CMV promoter is in many tissues where PNV is introduced. Suitable for achieving expression in E. coli.Outline of PNV structure   HSV and other genes are preferably used for polynucleotide vaccination. Ligation into the adapted expression vector. The element is its own Motor, transcription terminator, bacterial origin of replication and antibiotic resistance genes Both transcription promoters, immunogenic epitopes and immunity-enhancing or immune-modulating Includes an additional cistron encoding the gene. If necessary, the vector can be Includes an internal ribosome uptake site (IRES) for expression of tron mRNA I can see. One skilled in the art will recognize that multiple cis-cells transcribed in vitro and encoded by DNA pairs It is understood that RNA that has produced a tronic mRNA is included in the scope of the present invention. For this purpose, the transcription promoter may be a T7 or SP6 promoter. Powerful RNA polymer like Performing inflow transcription using a linear DNA template with an enzyme promoter Is desirable. These methods are well-known in the art.   The protective effect of the polynucleotide HSV immunogen against subsequent virus exposure is Demonstrated by immunization with the DNA of the present invention. It contains no infectious agents, Advantageously, there is no need to collect loose particles, and crucial selections are allowed. further Since the sequence of the viral gene product is conserved in various strains of HSV, other HSV Protection against subsequent exposure by the strain of V is obtained.   Injection of the gD-encoding DNA expression vector allows for subsequent viral exposure A strong protective immunity is generated. In particular, production of gD-specific antibodies and CTL Can be achieved. The immune response elicited against the stored protein is variable It may be effective despite antigen shifts and variations in the protein. HSV gene product Each show some conservation in different strains of HSV, and Because many immune responses can be generated in response to endogenous expression and MHC processing, many different It is expected that the HSV gD PNV construct will generate a cross-reactive immune response.   The present invention requires a self-replicating substance or adjuvant. Providing a means to elicit alloprotective immunity. Viral proteins and humans Generating large amounts of antibodies to expressed proteins after injecting growth hormone DNA [Tang et al., Nature, 356, 152, 1992] Are combined separately or with cytotoxic T lymphocyte vaccines targeting conserved antigens This shows that it is an easy and highly effective means for making antibody vaccines.   The ease of producing and purifying DNA structures is desirable for conventional protein purification. Comparable and facilitates the generation of combination vaccines. That is, for example, gD The composite structure to be loaded, and other HSVs that also contain the non-HSV genes are prepared and mixed. Can be administered together. In addition, protein expression was maintained following DNA injection. [H. Lin et al., Circulation, Vol. 82, p. 2217, ( 1990); N. Kitsis et al., Proc. Natl. Acad. S ci. (USA), 88, 4138 (1991); Hansen Et al., FEBS Lett. 290, 73, (1991); Ji ao et al., Hum. Gene Therapy, vol. 3, p. 21, (1992 ); A. Wolff et al., Human Mol. Genet. 1, 363, (1992)], B- And the maintenance of T-cell records has been improved [D. Gray and P.M. Matzing er, J .; Exp. Med. 174, 969, (1991); Oe hen, et al., 176, 1273 (1992)]. Continuous humoral and cell-mediated immunity develops.   The amount of expressed DNA or transcribed RNA introduced into the vaccine receptor depends on the Depends on the strength of the transcript and translation promoter. The magnitude of the immune response It may depend on the current level and the immunogenicity of the expressed gene product. Usually about 1 ng to 5 mg, preferably about 10 μg to 300 μg, is administered directly to the muscle tissue. You. Subcutaneous injection, intradermal introduction, pressurization through the skin, and other methods of administration, such as abdominal Intracavitary, intravenous, or inhalation delivery is also applicable. Consider providing booster vaccinations available. Following inoculation of the HSV polynucleotide immunogen, gD, gB, gC, Addition of HSV protein immunogens such as gG and gH gene products is also contemplated. . Simultaneously with or subsequent to the parenteral introduction of the PNV of the invention, the interleukin Parenteral administration of -12 protein may be by intravenous, intramuscular, subcutaneous or other means of administration. Throw by It can be advantageous to provide.   The polynucleotide may be exposed, i.e., affect the immune system of the receptor Not bound to any proteins, adjuvants or other reagents. This place If the polynucleotide is, but is not limited to, sterile saline or sterile buffered saline, It is desirable to be contained in such a physiologically acceptable solution. Also, DN A is similar to lecithin liposomes or other liposomes known in the art. Liposomes and DNA-liposome mixtures, or DNA is used to stimulate an immune response in the art, such as a protein or other carrier. It may be combined with an adjuvant known to promote. Not limited However, reagents such as calcium ions that assist in cellular uptake of DNA It can also be used. These reagents are usually easily transfected here. It is called a chemical reagent and a pharmaceutically acceptable carrier. Coated with polynucleotide Techniques for coating coated microspheres are known in the art and It is also useful. Pharmaceutically acceptable carriers for DNA for human use It is useful to include the final DNA product in a buffer solution. Pharmaceutically acceptable Possible carriers or buffer solutions are known in the art, various such as ton's Pharmaceutical Sciences Includes those described in the literature.   In another embodiment, the present invention is directed to in vivo eukaryotic tissue. Polynucleotides containing contiguous nucleic acid sequences that can be expressed to produce a gene product It is a tide. The encoded gene product is preferably as an immunostimulant and Acts as an antigen capable of generating an immune response. That is, this implementation The nucleic acid sequence in the embodiment comprises a human herpes simplex virus immunogenic epitope, and Optionally, cytokines such as the B7 family of proteins or T-cell costing Code a gigantic element.   There are several advantages to immunizing with a gene over a gene product. The first is natural Or relatively simple so that it can provide near-natural antigens to the immune system That is. Mammalian cells recombinantly expressed in bacteria, yeast, or mammalian cells Product proteins may require careful processing to ensure adequate immunogenicity. Many. The second advantage of DNA immunization is that the immunogen enters the MHC class I pathway and Ability to trigger a damaging T cell response It is. DNA encoding influenza A nucleoprotein (NP) Immunization of mice protects mice against exposure to heterologous strains of influenza N Elicited a CD8 + response to P (Montgomery, DL et al., Supra; Ulmer, J.A. Et al., Supra).   There is evidence that cell-mediated immunity is important in controlling HSV infection [reviewed For example, Nash, A .; A. Authors, 1985, In: The Herpesvi. ruses, Vol. 4, Plenum, New York, and Schm idt, D.E. S. Authors, 1992, In: Rouse (eds.), Current   Topics In Microbiology And Immunolo gy, Vol. 179, Herpes Simplex Virus; Path genesis, Immunobiology and Control, S springer-Verlag, Berlin;]. HSV serum Most HSV CTLs isolated from type 1 patients are of the CD4 + type (Sch midt, D .; S. Et al., 1988; Immunol. , Vol. 140, 3 610-3616; Tsutsumi, T .; 1986, Clin. Exp. Immunol. 66, 507-515) are specific for gD. Some CD8 + clones have been isolated (Torpey, DJ et al., 1989; Immunol. 142, 1325-1332; Ya sukawa, M .; Et al., 1989; Immunol. , Vol. 143, 2 051-2057; Zarling, J .; M. 1986, J. Imm unol. 136, 4669-4673). Cell metastasis in mice And removal experiments show that some CD8 + CTLs protect against infection (Bonneua, RH, et al., 1989, J. Virol., 63. Vol., 1480-1484; Nash, A .; A. Et al., 1987; Gen . Virol. 68, 825-833). With recombinant viral vectors Immunization with gD by infection (Paoletti, E. et al., 1984, Pro c. Natl. Acad. Sci. USA, Vol. 81, pages 193-197; Wa chsman, M .; L. Et al., 1987; Infect. Dis. , First 55, 1188-1197; Zheng, B .; Author, 1993, Vacc ine, Vol. 11, pp. 1191-1198. Protect from dyeing. Live viral vectors, such as DNA, contain the immunogen MH It has the performance of C class I expression. However, to immunize mice, HS Recent studies using infection with VgD-vaccinia recombinants have shown that Protection was found to be dependent on the delayed hypersensitivity function of L3T4 + cells (Wachs man, M .; Authors, 1992, Vaccine, Vol. 10, pages 447-454. ). gD-specific CD8 + cells were isolated from HSV infected mice, Its limiting role is not known (Johnson, RM et al., 1990). Year, J. Immunol. 145, 702-710). Koelle et al. Have shown that HSV infection of human fibroblasts and epidermal cells caused them to express CD8 + CT. L. (Koelle, DM, et al., 19) 1993; Clin. Invest. 91, 961-968). Natural In HSV infection, the role of general CD8 + cells and CD8 to specific gD + The role of the reaction has not been elucidated.   DNA immunization provokes both humoral and cell-mediated immune responses The biggest advantage is that the ratio of examining vaccine performance of large amounts of viral genes Providing a relatively simple way That is. Plasmids expressing HSV-2 glycoproteins B and C are antibodies Also protects mice from lethal exposure. However, CTL A reaction occurs and the ICP27-vaccinia recombinant virus (Banks, TA) . Authors, 1991; Virol. 65, 3185-3191). I which is known to provide protection in mice immunized by infection with CP27 showed lethal HS when mice were inoculated with PNV ICP27 alone. It did not provide protection from V exposure. However, ICP27-coded DN A can be useful as one component of a multiplex HSV gene-containing PNV, It is contemplated that the present invention includes ICP27 as a component of a multivalent HSV PNV. .   Immunization by DNA infection involves multi-component subunit vaccines, as described above. It can be easily manufactured. Simultaneous immunization with multiple influenza genes recently reported (Donnelly, J. et al., 1994, Vaccines, published. During). Include an HSV vaccine whose gene activates different parts of the immune system This also provides protection from subsequent virus exposure.   In a particular embodiment of the invention, the herpes simplex virus type 2 (HSV- 2) DNA vaccine expressing full-length glycoprotein D (gD), or HSV-2 Two experiments of HSV-2 infection were performed using the truncated state of glycoprotein B (gB). Provides a protective effect in the model. This particular embodiment of the invention is a mouse Intramuscular injection, followed by induction of neutralizing serum antibodies and lethal HSV-2 infection Discloses the protection of mice from Dose studies show low levels of any DNA constructs. Dose (1 μg) also elicits protective immunity and one immunization with gD construct Is also effective.   In a preferred embodiment of the invention, the full length glycoprotein D (gD) and HS Truncated forms of V-2 glycoprotein B (gB) are used in combination at low doses. Immune sera from DNA-injected animals contain antibodies to both gD and gB, and It had a ruth neutralizing activity. When exposed by vaginal infection with HSV-2, DN A-Immunized animals were well protected from primary illness.   Thus, the present invention simply provides for the ability to immunize a host against herpes infection. Plasmid coding for pure herpesvirus type 2 (HSV-2) protein Using the current vector Related. The protein is one or more additional proteins that elicit an immune response to HSV. Expressed with or in combination with other proteins. Immunogenicity and neutralizing activity One or more species that may be wild-type, full-length or mutant The choice of such a protein would occur to those skilled in the art after reviewing this specification. It is to be. DNA vaccine dosages useful in generating an immune response to HSV The person skilled in the art can immediately think of For example, a combination expressing ΔgB and gD The preferred dosage ratio of the combined polynucleotide patatin is the guinea pig model. And 2.0 μg gD and 0.6 μg ΔgB, respectively. Those skilled in the art Adjusting these amounts and proportions may provide optimal immune response in other hosts, such as humans. Response can be generated. One skilled in the art will appreciate that the examples of this specification Or HSV proteins and antigen fragments, such as truncated or carboxy-terminal truncations Mutations, including but not limited to amino acid insertions, deletions and point mutations Use variants. These examples show only glycoprotein B (gB), full-length glycotan Only protein C (gC), only full-length glycoprotein D, truncated form of gB, and gD , A combined immunization of a DNA vaccine comprising truncated forms of gB DNA-mediated delivery of a gene fragment to be expressed. With these teachings, other species Each mutant (eg truncated gD or truncated gC) alone or other disclosures Of DNA vaccine using combination with DNA vaccine structure prepared One skilled in the art would come up with providing protection against HSV via   In a preferred embodiment of the invention, the amino-terminal 707 amino acids of gB (aa Immunization with plasmids expressing Neutralization triggers a humoral immune response that is detected. Places exposed by vaginal infection Immunized animals are particularly protected from genital herpes and have significantly reduced primary and FIG.   In a particularly preferred embodiment of the invention, the truncated gB plasmid expresses full length gD In combination with the plasmid. The immunized host develops a humoral response to both proteins. Reveals significant protection from viral antigens.   Also, these nucleic acids may prevent infection and / or alleviate HSV-related diseases. It is useful as a vaccine that induces immunity to HSV.   The following examples illustrate the invention, but do not limit it. Absent.                                 Example 1 Vectors for vaccine production A)V1   The expression vector V1 was formed from pCMVIE-AKI-DHFR [Y. W hLang et al. Virol. 61, 1796 (1987)]. The AKI and DHFR genes were digested with EcoRI to And self-bonding. This vector contains an intro in the CMV promoter. Fragment containing no internal A and therefore having an internal SacI site deleted [B. S. Chapman et al., Nuc. Acids Res . 1955, p. 3979 (1991). Position of]. The template used for the PCR reaction was the hCMV-IE1 enhancer. / HindI from pCMV6a120 containing promoter and intron A II and Nhe I fragments [B. S. See Chapman et al., Supra.] Is ligated into the HindIII and XbaI sites of pBL3 to create pCMVIN pCMVintA-L formed by generating tBL ux. RSV-Lux [J. R. de Wet et al., Mol. Cell   Biol. , Pp. 725, 1987]. The lase gene fragment (HindIII-SmaI Klenow filled) was SalI of pCMVIntBL loaded with renown and treated with phosphatase Cloned into the site.   Primers spanning intron A are: 5 'primer, SEQ ID NO: 1 3 'primer, SEQ ID NO: 2   Primers used to remove the Sac I site were: Sense primer, SEQ ID NO: 3: And antisense primer, SEQ ID NO: 4:  The PCR fragment is cut with Sac I and Bgl II and cut with the same enzymes. It was inserted into the previously excised vector. B)V1J expression vector   The purpose of forming V1J is to create a promoter and transcription terminator from vector V1. Remover elements and place it in a more restricted area, To improve plasmid purification yield.   V1J is derived from the commercial plasmids Better V1 and pUC18. You. V1 was digested with SspI and EcoRI restriction enzymes and the two DNA fragments were digested. Generate a comment. The CMVintA promoter and the expression of the foreign gene And small fragments of these fragments containing the bovine growth hormone (BGH) transcription termination element. One was purified from an agarose electrophoresis gel. Next, this DNA fragment "End blunt" with the T4 DNA Primerase enzyme Facilitated binding to "blunt-ended" DNA fragments.   PUC18 was chosen to provide the "backbone" of the expression vector. Plasmid High yields are well characterized by sequence and function and have small dimensions It is known. This digestion was performed by partial digestion with HaeII restriction enzyme. All lac operons were removed from the reactor. Transfer the remaining plasmid to agaro T purified from a gel electrophoresis gel and treated with calf intestinal alkaline phosphatase. 4 [The end was vertically cut with DNA polymerase, and the CMVintA / BGH Bound to the element. any of the two possible orientations of the promoter element in the pUC backbone Were obtained. One of these plasmids is E. coli. D in E. coli It provided a significantly higher yield of NA and was named V1J. The structure of this vector is Confirmed by sequence analysis of the combined region and then compared to V1 Or higher expression was obtained. C)V1Jneo expression Better   Ampicillin is undesirable in large-scale fermenters, so that bacteria with V1J Amp used for antibiotic selection^rIt was necessary to remove the gene. V1 Amp from pUC backbone of J^rGenes were transformed with SspI and Eaml 105I It was removed by digestion with restriction enzymes. Remove the remaining plasmid from the agarose gel. Purified by electrophoresis, cut vertically at the end with T4 DNA polymerase, and then , Calf intestinal alkaline phosphatase. Invited from Transposon 903 Commercially available kan derived and contained in the pUC4K plasmid^rGenes are converted to pStI Limited yeast Excised, purified by agarose gel electrophoresis, and The fragments were blunt cut with Rahse. This fragment was ligated to the V1J backbone and either Kan to orientation^rA plasmid having the gene was induced, and it was transferred to V1Jneo # 's Named 1 and 3. Each of these plasmids was subjected to restriction digest analysis, ligation Confirm with the DNA sequence of the region to produce the same amount of plasmid as V1J. It has been shown. The expression of the foreign gene product was also changed to V1J for these V1Jneo. Comparable. In the following, select V1Jneo $ 3 represented as V1Jneo, This is amp in V1J as an expression construct.^rKan in the same orientation as the gene^rRemains Contains a gene. D)V1Jns expression solid   An Sfi I site was added to V1Jneo to facilitate integration studies. Commercially available Base pair Sfi I linker (New England Biolabs) ) Was added at the Kpn I site in the BGH sequence of the vector. V1Jne o was linearized with KpnI, gel purified, blunt cut with T4 DNA polymerase, , Blunt cut Sfi I linkers. Clone isolation by restriction mapping Select the product and change the sequence via the linker. More confirmed. The new vector was named V1Jns. V1Jns (Sfi I Heterogene expression in V1Jneo (with Kpn I) Comparable to the expression of the same gene in E)V1Jns-tPA   To provide a foreign leader peptide sequence for secretion and / or membrane proteins , V1Jns to denature human tissue-specific plasminogen activator (tp A) We included a leader. Annealing two synthetic complementary oligomers And then ligated into V1Jn digested with BgIII. Sense and anti The sense oligomer is SEQ ID NO: 5: and SEQ ID NO: 6. In the sense oligomer, the Kozak sequence is underlined. These ori Gomer is suitable for binding to the BglII degradation sequence. It has overhanging bases that match. After binding, destroy the upstream BglII site , Retain downstream BglII for subsequent binding. Both binding sites and total t The PA leader sequence was confirmed by DNA sequence decoding. In addition, the consensus Constructed using the optimized vector V1Jns (= V1Jneo having SfiI site) The KpnI site is blunt cut with T4 DNA polymerase, followed by fiI linker (catalog # 1138, New England Biolab) s) (in the BGH terminator region of V1Jn-tPA). A SfiI restriction site was placed at the KpnI site. This denaturation is accomplished by restriction digestion and agaro. Confirmation was performed by gel electrophoresis. F)pGEM-3-X-IRES-B7   (X = any antigen gene) Gene encoding immunogen and immunostimulator Of a dicistronic vaccine construct that provides synchronized expression of the gene encoding the protein As an example, the murine B7 gene has been cloned from the B lymphocyte cell line CH1 (obtained from the ATCC). PCR). B7 is included in major histocompatibility complexes I and II For providing essential costimulatory T cell activation by different antigens He is a member of the Npaku family. CH1 requires an excellent source of B7 mRNA I do. Because they have an essentially activated phenotype, B7 Predominantly expressed by activating antigens expressing cells such as vesicles and macrophages This is because that. These cells are further subjected to standard guanidinium thiocyanate procedures. Ex vivo using cAMP or IL-4 and mRNA prepared using Intense. GeneAmp RNA PCR Kit (Perkin-Elmer) B7 translation open reading using this mRNA Primer oligomer specific for B7 located downstream of the frame CDNA synthesis was performed using SEQ ID NO: 7). B7 has the following sense and a Amplified by PCR using antisense PCR oligomers: SEQ ID NO: 8: and SEQ ID NO: 9. These oligomers have a BglII restriction at the end of the insert. An enzyme site, and an additional NcoI site located immediately before the 3'-terminal BglII site A Kozak translation initiation sequence is provided that includes a position and an NcoI restriction site. NcoI The digestion was suitable for cloning into NcoI digested pGEM-3-IRES. Fragments were generated. The resulting vector pGEM-3-IRES-B7 is , An IRES-B7 cassette that can be easily transferred to V1Jns-X. here X represents an antigen-encoding gene. G)pGEM-3-X-IRES-GM-CSF   (X = any antigen gene) This vector is not a B7 Same as that described in item C above except that a gene for GM-CSF is used. Including cassettes. GM-CSF has potential antitumor T cell activity in vivo Stimulatory cytokines and matalophage differentiation that have been shown to induce [G . Dranoff et al., Proc. Natl. Acad. Sci. USA, ninth 0, 3539 (1993)]. H)pGEM-3-X-IRES-IL-12   (X = any antigen gene) This vector is not a B7 Except that the gene for IL-12 was used. Including cassette. IL-1 2 shifts the immune response, in contrast to the humoral response, to a pathway where cellular T cells predominate Has been shown to affect the role of [L. Alfonso et al., Sci ence, 263, 235, 1994].                                 Example 2 Vector V1R preparation   In continuing work to optimize the basic immunization vector, V1R and A derivative of V1Jns was prepared. The purpose of this vector construct is Heterogene expression features and high plasmids provided by V1J and V1Jns Minimal size vaccine solids without unnecessary DNA sequences, still maintaining yield Was to get According to literature and experiments, (1) E. replication of E. coli Region in the pUC backbone, including the source, to affect plasmid yield from bacteria (2) Kanamycin open reading frame Kan following^rInsert the bacterial terminator into the 3'-region of the gene (3) (the first Kpn in the BGH element) 300 bp from the 3'-half of the BGH terminator (following the I restriction enzyme site) Up to It has been determined that it can be removed without affecting its control function.   V1 expressing CMVintA promoter / BGH terminator respectively Three DNA segments from Jns, origin of replication, and kanamycin resistance elements The V1R was formed using PCR to synthesize the proteins. Each segment has its own system Restriction enzymes were added to the ends of each segment using PCR oligomers. CMVint SspI and XhoI for A / BGH; kan^rEcoR for gene V and BamHI; ori^rFor BclI and SalI. These enzymes Sites selected because they directly bind each of the PCR-derived DNA segments Followed by loss of each site: EcoRV and SspI are compatible with binding. BamHI and BclI were replaced by SalI and Xh. This is because it protrudes in a sending manner like oI. By PCR these segment Each segment is digested with the appropriate restriction enzymes described above, They bound together in a single reaction mixture containing all the NA segments. ori^rOf 5 The '-end contains the T2rho independent terminator normally found in this region. Designed for Stop information for the namycin resistance gene can be provided. The bound product is Confirmation was achieved by elementary digestion (over 8 enzymes) and DNA sequencing of the bond. DNA Plasmid yield and heterologous expression using viral genes in V1R were Seems similar to s. The net reduction in vector size achieved was 1346 bp. (V1Jns = 4.86 kb; V1R = 3.52 kb).   The PCR oligomer sequence used to synthesize V1R (in the following sequence, Restriction sites are underlined and shown in parentheses): [SspI] SEQ ID NO: 10:[XhoI] SEQ ID NO: 11: (About CMVintA / BGH segment) [EcoRV] SEQ ID NO: 12: [BamHI] SEQ ID NO: 13: (About kanamycin resistance gene sequence) [BclI] SEQ ID NO: 14: [SalI] SEQ ID NO: 15: (Replication origin of E. coli)                                 Example 3 Cells, viruses and cell culture media   VERO, BHK-21, RD cells and HSV-2 strain MS from ATCC Was. Virus is routinely used in a small amount of medium without fetal bovine serum (FBS). Near confluent VERO or multiplicity of infection (moi) 0.1 at 37 ° C. Was prepared by infection of BHK cells. After 1 hour, remove the inoculated virus and add 2% heat-inactivated FBS, 2 mM L-glutamine, 25 mM HEPES, 50 High glucoside with U / ml penicillin and 50 μg / ml streptomycin Source DMEM was re-fed. Incubate until the cytotoxic effect is stronger Continued: usually 24-48 hours. Cell-associated virus was incubated at 1800 × g for 1 hour at 4 ° C. Collected by centrifugation for 0 minutes. Supernatant virus at 640 × g at 4 ° C. Centrifuge for 10 minutes at For clarification.                                 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. Authors, 1981, Gene, Vol. 15, 365-378). B glII restriction confirmation site (Midland Certified Reagent)   HSV2gB, g, from the Company; Midland, Texas) C, gD, or a sequence corresponding to the 5 'and 3' terminal flanking sequences of the The resulting oligomers were used at 20 pmol each. 1: 4 instead of dGTP Using the dGTP: dGTP ratio, buffer was 3 mM MgCl_TwoSo that PCR (Perkin Elmer Cetus) according to the manufacturer's specifications 1.1 kb flag encoding the gD gene according to La Jolla, Inc. Was amplified. 100 ng / 100 HSV-2 genomic DNA template Used in μl reaction. The PCR amplified fragment was digested with BglII and Dephosphorylated vector digested with II Ter V1J (Montgomery, DL et al., Supra). E. FIG. c oliDH5α (manufactured by BRL-Gibco, Gaithersburg, Md.) Was transformed according to the manufacturer's specifications. Ampicillin resistant colonies,³²P mark Screening was performed by hybridization with 3 'PCR primers. Restriction mapping of the plasmid to be used, and Sequenase D NA Sequencing Kit, version 2.0 (United St Sequences of vector insertion junctions using ates Biochemical) Characterized by Similarly, a 2.7 Kb fragment encoding the gB gene A 1.5 Kb fragment encoding the gC gene; and the ICP27 gene The 1.6 Kb fragment encoding was also PCR amplified. Independently induced isolation The exact DN containing either the gB, gC, gD, or ICP27 gene The presence of the A structure was confirmed and characterized.   Large-scale DNA preparation involves growing 800 ml of medium for 24-48 hours, Except that the test DNA was purified by one CsCl-EtBr equal density centrifugation. The procedure was qualitatively performed as described above (Montgomery, DL et al., Supra).   Plasmid constructs can be used for restriction mapping and sequence analysis of vector insertion junctions. More characterized. Results are from published HSV-2 strain G (Lasky, LA). . Author, 1984, DNA, Vol. 3, pp. 23-29). Start and stop codons were shown to be intact for each construct.                                 Example 5 HSV-2 gB, gC, gD and I from the V1J plasmid Expression of CP27 protein   Rhabdomyosarcoma cells (ATCC CCL136) were transferred to a 6-well tissue culture cluster. Within 9.5cm^Two1.2 × 10 per well⁶10% heat-inactivated fetal cells at cell density Serum, 2 mM L-glutamine, 25 mM HEPES, 50 U / ml penic Phosphorus and 50 μg / ml streptomycin (all from BRL-Gibco) Planted in high glucose DMEM supplemented with: chloroform extracted cesium chloride purification Rasmid DNA was transferred to each 9.5 cm RD cell.^Two5 to 15 μg per well Pharmacia Cellfect test according to the kit instructions except use Precipitated with calcium phosphate using the drug. Calcium phosphate After addition of the DNA precipitate, the medium was treated with glycerol for 6 hours; Was incubated for 2 days before harvesting.   Lysates of the transfected medium were added to 1 μM leupepsin, 1 μM 1 with the addition of pepstatin, 300 nM aprotinin and 10 μM TLCK × PIPA (0.5% SDS, 1.0% TRITON X-100, 1% na Thorium deoxycholate, 1 mM EDTA, 150 mM NaCl, 25 m M TRIS-HCl; pH 7.4) and sonicate briefly to reduce viscosity Let down. Lysate electrophoresed on 10% Tricine gel (Novex) And then transferred to a nitrocellulose membrane. Immunoblots were HSV-2 times Treated with recurrent mouse serum and developed with ECL detection kit (Amersham) .   V1J: Expression of HSV gD from gD was determined by transient transfection of RD cells. Indicated by the option. V1J: gD transfected or pseudo-transfected Cell lysates were separated by SDSPAGE and analyzed by immunoblot. FIG. 1A shows that V1J: gD transfected RD cells had an apparent molecular weight of about 55K. It shows that it expresses an immunoreactive protein. HSV-2 (Curtis), HSV-2 (186) or mock infected Vero cells Lysate from is included for comparison. From cloned gD and infected cells Equivalent migration of the reference protein to that of the reference protein indicates that the protein is full-length and And processed and glycosylated in the same manner as the gD tamper. Solid Indirect immunofluorescence of defined V1J: gD transfected cells in the cytoplasm The spread signal is shown.   V1JNS: Expression of HSV gB from gB was determined by transient transfection of RD cells. Indicated by section. V1JNS: gB transfected or pseudotran Lysates of infected cells were separated by SDS PAGE and analyzed by immunoblot. analyzed. FIG. 1B shows that V1JNS: gB transfected RD cells were This indicates that the protein expresses an immunoreactive protein with a child size of about 140K. HSV-2 (Curtis), lysis from HSV-2 (186) or mock-infected Vero cells Objects are included for comparison. Cloned gB and reference protein from infected cells Equivalent movement with protein indicates that the protein is full length. Fixed V Indirect immunofluorescence of 1JNS: gB transferato cells shows a membrane-associated punctate signal Was.   V1J: Expression of HSV gC from gC was determined by transient transfection of RD cells. Indicated by the option. Indirect immunization of fixed V1J: gC transfected cells Epifluorescence showed a signal diffused mainly in the cytoplasm.   Expression of ICP27 was determined by transient transfection of RD cells and then As indicated by Western blot analysis. Mouse monochrome specific to ICP27 The internal antibody detected a protein of about 60 kDa, which was detected in HSV-2 infected cells. In agreement with the major immunoreactive proteins in 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 HCl (A Veco, Fort Dodge, 1A) and 0.5 mg of xylazine (M obley Corp. Shawnee, KS. ) Mixture is injected intraperitoneally By anesthesia. Shade the hind legs with an electric clipper, 70% ethanol And washed with Animals were suspended in saline in 50 μl of DNA on each leg for a total of At 100 μl.   First, V1J: gD DNA demonstrates immune response to HSV gD. The triggering performance was tested by a titer test. In groups of 10 mice, 200 μg- DNA is injected intramuscularly in a dose range of 0.78 μg (8 double dilutions), and Was mock-immunized with saline. Serum obtained at 4 and 6 weeks after immunization was subjected to ELISA Analyzed by A. For ELISA, HSV-2 glycoprotein was added at pH9. 5 was diluted to 5 μg / ml in 50 mM carbonate buffer. Nunc Maxi- sorb flat bottom 96-well plate with 100 μl of HSV glycoprotein per well Coated overnight at 4 ° C. depending on quality. The plate was washed four times with PBS pH 7.2, Specific reactivity was determined using 20 mM TRIS-HCl; pH 7.5, 137 mM Na. Cl, 2.7 mM KCl, 0.5% gelatin, 0.05% Tween20. Inhibition and dilution buffer included reduced for 1 hour at room temperature. Series of mouse serum Dilutions were added and plates were incubated for 1 hour at room temperature. PB plate S 4 times, water once, then alkaline phosphatase-labeled goat anti-mouse IgG (Boehringer Mannheim, Indianapol) is, IN) and incubated at room temperature for 1 hour. Excess secondary antibody, Washed four times with PBS followed by one wash with distilled water to remove. ELISA With 10% diethanolamine at 37 ° C .; pH 9.8 100 μg / ml MgC 1.6H_Two1 mg / ml p-nitrophenyl phosphate in O An amount of 00 μl was added for development. Read absorbance at 405 nm and dilute serum , OD₄₀₅Is greater than the mean + 3-fold standard deviation of saline control serum at the same dilution Cases were rated as positive. By four weeks, animals receiving 6.25 μg of DNA Most became seropositive. At doses lower than 6.25 μg, less Animals seroconverted, but at the lowest dose, some animals It was positive. None of the saline-injected control animals were positive. In the sixth week, Most of the animals were immunopositive.   At week 7, re-immunization with the same dose of DNA (or saline) used for the first injection It has become epidemic. Serum was obtained at 10 weeks (3 weeks after the second injection) and by ELISA The end point addition amount was determined. The results are summarized in Table 1. By week 10, DNA injection 93% of the mice became seropositive. Even at a dose of 0.78 μg, 10 Eight of the animals were positive.   a-Mice were immunized at weeks 0 and 7 with the indicated amount of DNA. 10th week Serum was obtained and assayed as described herein.   For the purpose of calculating b-GMT, at the lowest dilution tested (1:30), Sex serum was assigned a value of one less dilution, ie 1:10.   To confirm that the ELISA reactivity was due to anti-gD antibodies, several high EL Reactivity of ISA titers with immunoblotting of HSV or mock infected cell lysates Characterized by FIG. 2A shows that serum from V1J: gD immunized mice was HSV   Single HSV-encoded protein with electrophoretic mobility consistent with that of gD And specifically react with. Together, this These data show that when V1J: gD DNA was administered intramuscularly to mice, gD epitope was observed. Shows that the immune response to the gD protein progresses.   To extend these results and to know the minimum effective PNV dose, V1J: gD Were further titrated in experiments in which animals were immunized only once. In a group of mice, 5 ng to 50 μg of V1J: gD DNA were injected. 4, 7 and 1 after immunization Serum collected at week 0 was assayed by ELISA; the data is summarized in FIG.   This titration indicates a threshold for a reaction of about 0.5 μg of DNA. Small amount of D A small number of animals receiving NA are seropositive by ELISA, The response was transient and occurred only at the lowest serum dilution. 1.67μ At a DNA dose of g, more than 90% of the animals were seroconverted by 4 weeks, Remained positive at 7 and 10 weeks.   The time-dependent increase in the antibody titer of the individual animals was similar to the increase in group GMT shown in FIG. Has been reflected. At a dose of 0.5 μg, GMT at 4 and 6 weeks Rose sharply between. All except one between week 7 and week 10 In the case of, the titer increased or remained constant. 0.5 μg The titers of the receiving animals at week 10 were reached in previous experiments with a second DNA immunization. Similar to what was done. The amount of PVN required to elicit an immune response is 100-fold less than published reports using similar vectors (Robin Son et al., 1993, Vaccine, Vol. 11, pp. 957-960; And Fynan et al., 1993, Proc. Natl. Acad. Sci. US A, Vol. 90, pp. 11478-11482).   The efficacy of single and double dose immunizations to achieve a standardized protocol The results were compared. Highest dose (200 μg) and lowest in two dose experiments A large difference was found in protection at lower doses (0.78 μg). Throw once If increasing in a given experiment, observe the dose response for ELISA GMT, It showed the efficiency of seroconversion and protection. The threshold for these reactions is about 0.5μ g. However, seroconversion does not occur with as little as 50 ng of DNA . Usually, the titer continues to increase throughout the 10 weeks after a single injection. Thus, there was no clear increase in titer after the second injection. Finally, the only one in both experiments With a common dose of 50 μg DNA Thus, there was no significant difference between the single and double doses.   An analysis similar to that described above was performed using the PNV construct containing the HSV genes gB and gC. I went about. Mice (10 mice per group) were treated with HSV g as described above. Immunization was performed with DNA containing the B gene or DNA containing the HSV gC gene. Serum was collected and tested for the presence of anti-gB or anti-gC antibodies in the aforementioned ELISA. Was analyzed. The ELTSA data for the gB antibody is shown in Table 2 and shows V1J Mice immunized with NS: gB were seropositive for anti-gB antibodies.  ELISA data for gC antibodies are shown in Table 3 and were immunized with V1J: gC. Mice (5 mice per group) were seropositive for anti-gC antibody. Show.   To confirm that the ELISA reactivity for gB was due to the anti-gB antibody , HSV or mock infected cell lysate reactivity to immunoblots LISA titers were characterized. FIG. 2B shows V1JNS: gB immunized mice. Of a single HSV cohort with electrophoretic mobility comparable to that of HSV gB It shows that it reacts specifically with the oxidized protein. Together, these data Data showed that HSV PNV was intramuscularly administered to mice and HSV epitopes were expressed. Shows that the immune response to these HSV proteins progresses.Example 7 HSV neutralization   Mouse serum was heat-inactivated at 56 ° C for 30 minutes, and then DMEM, 2% heat-inactivated Serially diluted in FBS-modified, then 50 μl of each dilution is added to sterile polypropylene 9 6-well plate (Marsh Biomedical, Rocheste) r, NY. ) Delivered to two wells. HSV-1 or HSV-2 Dilute the sample to 4,000 pfu / ml and then add 50 μl of virus to each sample well. 1 was added and the plate was incubated at 4 ° C. overnight. Guinea pig complement (Ca ppel) was diluted 1: 4 in DMEM, 2% heat-inactivated FBS. 50 μl was added to the wells. After incubating at 37 ° C for 1 hour, 100 µl Of medium without serum was added to each well, and each reaction mixture was used to prepare 12 wells of the medium. Confluent VERO cells were infected on a star plate (Costar). During ~ The sum virus sample was adsorbed at 37 ° C. for 1 hour. Gently aspirate adsorbate, single 1 ml of 0.5% carboxymethylcellulose in the layer, 1 × MEM, 5% heat inactivated FBS, 10 mM 1-glutamine, 25 U / ml penicillin, 25 μg / ml streptomycin, 12.5 mM HEPES was overlaid. Plates are incubated at 37 ° C for 48 hours. I did it. The overlapping layer was removed and the cell monolayer was replaced with 1% basic fuchsin, 50% methanol. And 10% phenol. Count plaques and compare with serum from mock-immunized mice Neutralization titers were determined as serum dilutions that reduced the number of plaques by 50% when compared.   Weeks 0 and 7 to determine if anti-gD antibody is biologically active Selected 10-week sera from mice immunized with HSV-2 were neutralized with HSV-2 neutralizing activity. Tested for gender. Table 4 shows the results of the plaque reduction test. V1J: gD immunized mouse Serum from HSV-2 (Curtis) as well as HSV-2 (186) Neutralized. In addition, at least a portion of the serum is in HSV-1 (KOS) infectious Includes type-common neutralizing antibodies as indicated by the sum. Neutralization titer is low in some cases However, due to these results, we believe that these anti-gD antibodies We checked whether the animals could be protected against dew.   From all animals immunized with 0.5 μg V1J: gD in a single dose experiment Week 10 sera were also tested in the HSV-2 plaque reduction assay. 49 blood tested 29 sera positive in Qing More than 50% of the plaques were reduced at a 1:10 dilution. 16.7 and 5 At the 0 μg dose level, 9 out of 10 sera from each group were neutralizing positive Met.   a-1: Neutralizes HSV-1 KOS at 100   b-1: Neutralizes HSV-2 186 at 100Example 8 HSV exposure   Infected virus material was infected with a confluent VERO monolayer with HSV-2 as described above. Prepared. Clarified supernatant virus is titrated on VERO cells , A small amount was stored at -70 ° C. Animals were injected intraperitoneally with 0.25 ml of virus material Then infected Log rank test at FETEST (McDermott et al., 1989, Virology, Vol. 169, pp. 244-247). Probability 0 . The 001 difference was determined to be highly significant.   Eleven weeks after the first DNA injection, the immunized mice received two doses of V1J: gD. The mouse is HSV-2 (Curtis) 10^5.7p. f. u. Intraperitoneal injection And exposed for 21 days. The survival data is shown in FIG. 0.78 μg Animals immunized with low V1J: gD are significantly protected from lethal infection Is evident. Of the three dead immunized animals, two were EL at week 10 Seronegative by ISA. Some surviving animals are groomed Impossible, undergrowth or song He showed signs of temporary illness, including a hurt posture. Death achieved at each dose of DNA Protection level was high (p <0.01), but these signs indicate that some Indicates that an infection has occurred. Analysis of serum obtained from convalescent animals was performed using HS V-2 infected Vero cell lysates were characterized by reactions in immunoblots. In some cases, the serum only recognizes gD and others, and the serum has many H Reacted with SV tamper. These results indicate that mice with the tested HSV infection Matches at least a part of   Animals immunized with a single DNA infection were exposed as described above. Survival day The data is shown in FIG. Statistically significant (p <0.001) protection against death was J: obtained in a group of animals receiving 1.67 μg gD. This surviving dose The response is similar to that seen in the ELISA titration (FIG. 3). For two dose experiments During the observation period, some surviving animals show a transient signal of disease, as seen in did. Surviving animals immunized with high doses (16.7 and 50 μg) of DNA A glossy and healthy appearance was maintained during the observation period.   Immunization with a PNV construct containing the HSV gB or gC gene Animals were also exposed by lethal administration of HSV as described above for gD. . Survival data for animals immunized with V1JNS: gB are shown in FIG. J: Survival data for animals immunized with gC were tested for protection against death. FIG.   These results demonstrated the performance of direct DNA immunization in preventing HSV infection. . Using glycoprotein gD as a model, a small amount of 0.5 μg V1J: gD Single intramuscular injection of DNA protects against statistically significant protection against lethal HSV exposure A neutralizing antibody response to the provided gD was elicited. 3.13 μg of small amount of DNA All animals are protected from death by immunization in a single dose format.                                 Example 9 Guinea pig inoculation with HSV PNV   About 200-250 g each of Hartley guinea pigs (Harlan Sp. Rugge Dawley Labs, Indianapolis, IN) , 11 and 4 weeks, 0.1 ml was inoculated intramuscularly in the right thigh, The thigh was inoculated intramuscularly with 0.1 ml and then exposed to the virus. Waku Fresh solutions of tin and placebo were obtained for each inoculation.   To determine HSV-2 antibody production in animals, 5 weeks after the first inoculation and Guinea pigs were bred for two weeks after the second inoculation. Blood (0.6-1 ml per animal) ) Was obtained by pinching the toes. Blood is transferred to a micro separation tube (Becton Dick) Inson) and centrifuged at 1000 Xg for 10 minutes to separate serum. Released.   Serum collected from guinea pigs was subjected to anti-HS using the ELISA described in Example 6. The presence of the V antibody was analyzed. Table 5 shows the results.  At the time of infection, each guinea pig weighed 600-700 g. Put them in a simple It was infected intravaginally with Lupes virus type 2 (HSV-2), strain E194. this is Achieved in a three-step process. First, the vagina of each animal was washed with 0.1N NaOH. Soaked in Wipe with cotton tip applicator for 5 seconds. This process makes the infection more likely To stimulate the vaginal area. After about 45-60 minutes, wipe each vagina dry for 5 seconds Was. Next, the virus medium (about 5 × 10 5 plaque-forming units of HSV-2 per ml) was used.⁶Pieces Each guinea pig was wiped for 20 seconds using an applicator dipped in). in the meantime, Gently and slowly swirling it back and forth.   Lesion ratings in infected animals were determined daily 2-15 days post infection. 1+ Ratings indicate that about 25% of the anal vaginal area was infected (usually immediately around the vagina). Redness); a rating of 2+ indicates that 50% of the anal vaginal area was infected; A rating of 3+ indicates that 75% was infected; a rating of 4+ indicates that 100% Indicates that it was infected. Some of the animals will die, so lesions close to the time of death Ratings were taken at the end of the 15th. If this is not done, the average lesion rating will be largely Minutes of infected animals die, so they seem to decline.   Deaths were recorded daily for 21 days. The average number of days for calculating death is the number of dead guinea pigs. Only calculated. The number of animals with hind leg paralysis was recorded during the infection. Vaginal virus drops The results were obtained by removing the vagina on days 2, 4 and 6 after virus inoculation. Ruth titration Was performed. A cotton swab was placed in a tube containing 1 ml of cell culture medium. The power of these samples Titration was performed on Vero cells in 96 well plates. Virus titer Is calculated by Reed L. J. And Muench M .; Authors, Am. J. Hyg * Vol. 27, pp. 493-498 (1938). . The virus titer is log / ml_TenExpressed as the cell culture infection dose.   Fisher exact test, mean days of death (Mann- Whitney U test, paralysis (Fisher exact test) ), Vaginal virus titration (Mann-Whitney U test), and vaginal disease The statistical interpretation of the variable rating (Mann-Whitney U test) was Performed by tailed analysis.   FIG. 8 shows survival, mean days of death, paralysis, and HSV-2-infected guinea pigs. 7 shows the results of virus and vaginal virus titrations. The high dose vaccine, compared to the placebo control, On day 4 and day 4, she protected from death and reduced vaginal viral load. High dose The vaccine prevented paralysis in these animals. Before low dose vaccines The parameters were reduced.   Table 7 shows the daily vaginal lesion ratings for the experiments. Both high and low doses Vaccination at day 3-15 of infection compared to placebo group Significantly reduced severity. The results in Table 7 are shown in the graph of FIG.   These results indicate that vaccine was protected in guinea pigs infected with HSV-2. That high dose vaccines are more effective than low dose vaccines Indicates that there is. Virus recovers from vaccine, causing low-level vaginal lesion progression Thus, high dose vaccines could not completely inhibit infection. in addition Nevertheless, the degree of protection by the vaccine at this dose is substantial. Anti The results of body studies are related to antiviral protection.   Two different injections into guinea pigs at 11 and 4 weeks before vaginal HSV-2 exposure The vaccine, administered intramuscularly at the dose, adequately protected the animals from the disease. High dose vaccines are more effective than low dose vaccines. Vaccines It seems safe in animals.  Intramuscular inoculation was performed 11 and 4 weeks before a-virus exposure.   b- After virus inoculation   ^*p <0.05,^**p <0.01,^***p <0.001                                Example 10   Mice inoculated intramuscularly with PNV HSV produce mucosal HSV-specific antibodies To determine if the mice had 12.5 or 1.56 μl of V1JNS: gD g. The vaginal fluid is collected with a swab and the antibody is dissolved from the swab with phosphate buffered saline. Released. The eluate was analyzed for the presence of IgG and IgA specific for HSV-2. ELISA was performed for HSV-specific IgG and IgA in mouse vaginal samples. To detect the presence, mouse IgG (Boehringer) and mouse As described above, except that a commercially available antibody specific to IgA (manufactured by Seralab) was used. I went. The IgG results are shown in Table 8 and IgA was detected in any animal Was not done.   a- saline injection   b-V1JNS: Inject 1.56 μg of gG   The results show that HSV-2 specific mucosal IgG was used in mice inoculated with V1J: gD. Indicates that it exists inside.Example 11   Herpes simplex virus type 2 (HSV-2) full-length glycoprotein D (gD), Or a D expressing a truncated version of HSV-2 full length glycoprotein B (gB) NA vaccine protects against two experimental models of HSV-2 infection evaluated. Intramuscular injection of mice resulted in each construct eliciting neutralizing serum antibodies and lethal H It showed that mice were protected from SV-2 infection. Dose-titer test That a low dose (1 μg) of the DNA construct elicits protective immunity, and g One immunization with the D construct has been shown to be effective. Next, the two DNAs are Tested in low dose combinations in guinea pigs. DNA-injected animals Has an antibody against both gD and gB, and has a virus neutralizing activity. I was When exposed to a vaginal infection with HSV-2, DNA-immunized animals are primary Well protected from venereal diseases.   The virus was prepared by a conventional method as described in Example 3. Briefly, Multiplicity of infection (moi) in a small volume of serum-free medium at 37 ° C. by routine methods. . 3.) Virus due to infection of nearly confluent Vero or BHK cells at 0.1 Prepare did. One hour later, the inoculated virus was removed, and the medium was supplemented with 2% heat-inactivated fetal bovine serum ( FBS), 2 mM L-glutamine, 25 mM HEPES, 50 U / ml peni High glucose DMEM with cillin and 50 μg / ml streptomycin Was resupplied. Incubation continued until cytotoxic effect was strong: normal 24-48 hours. Cell-bound virus is centrifuged at 1800 × g at 4 ° C. for 10 minutes Collected by releasing. The supernatant virus is centrifuged at 640 × g at 4 ° C. for 10 minutes. Clarified by centrifugation and stored at -70 ° C.   Female BALB / c mouse (Charles River Laboratory) es; Wilmington, MA) and male Duncan Hartley Guinea pigs (manufactured by Harlan Sprague Dawley; Indiana) poli, IN) by the Institutional Animal Care Maintain and maintain in accordance with protocols approved by the Use and Committee And used.   Template for polymerase chain reaction (PCR) as disclosed in Example 4 HSV-2 strain Curtis DNA was used. HSV-2 gD gene and And BglII system included Synthetic oligonucleotides corresponding to the 5 'and 3' flanking sequences for the restriction site Limer (Midland Certified Reagent Compa) NY (Midland, TX) using PCR (Perkin Elmer) (Cetus; La Jolla, CA) encodes the gD precursor gene A 1,182 base pair (bp) fragment was amplified. HSV-2 gB The 2,121 bp sequence encoding the amino terminal 707aa is amplified by PCR. Corresponding to the 5 'flanking sequence, nucleotides 2,110 to 2,121 (Figure 3, Stuv e, et al., 1987; Virol. 61: 326-335) A BglII restriction site adjacent to the coding sequence using the primer And a stop codon TAA immediately after nucleotide 2,121 was added. BglI I-Digest PCR-amplify fragment into vector V1J or V1Jns (See Example 1 and Example 2). E. FIG. coli DH5a (BRL-Gi bco) was transformed according to the manufacturer's specifications. Plasmi trying to use Are characterized by restriction mapping, and the Sequenase DNA Seque ncing Kit, version 2.0 (United Stat es Biochemical; Cleveland, OH) -The insertion junction was sequenced. GD encoding first cloned in V1J The sequence was subcloned into V1Jbs. For simplicity, Example 11 The final gD and cleaved gB plasmid constructs were And gB-2. Large-scale DNA preparation is essentially as described in Example 4. Met.   Pharmacia Cellfect Kit (Pharmacia B iotech Inc. Manufactured by Piscataway, NJ). Calcium phosphate according to the manufacturer's instructions except that 5 μg DNA / well was used. Plasmid D on rhabdomyosarcoma (RD) cells (ATCC CCL136) The NA was allowed to settle. After 48 hours, the cell lysate was degraded by electrophoresis and then Transferred to trocellulose membrane. Immunoblots were run against anti-HSV gD monoclonal anti- Body (manufactured by Advanced Biotechnologies Inc .; Col) umbia, MD) or sheep anti-HSV-2 antiserum (ViroStat; Portland, ME) and processed with an ECL detection kit (Amersham; Arlington He lights, IL).   With regard to immunization of animals, in all cases the "DNA dose" is Means the total amount of DNA injected per animal; half of the total is delivered to each injection site Was done. Mice were treated with 2 mg of ketamine HCl in saline (Aveco, Fort).   Dodge, IA) and 0.2 mg of xylazine (Mobley Corp.) . Anesthesia by intraperitoneal injection of a mixture of Shawnee, KS). Was. The hind legs were shaved with an electric clipper and washed with 70% ethanol. Each quadriceps Was injected with 50 μL of DNA diluted in sterile saline just before use. Control animals Mock immunizations were performed with saline or vector DNA. At the time of the first immunization, the mice had 5 ~ 6 weeks of age. 400-550 gm guinea pigs at the time of the first immunization Anesthetized by subcutaneous injection of 22 mg ketamine + 5 mg xylazine / kg Wash the hind legs with 70% ethanol and add 100 μL of DNA or Injected saline intramuscularly. Serum was lysed with HSV-2 Curtis infected BHK cells HSV glycoprotein (mouse serum) partially purified from SF21 medium infected with baculovirus gD and baculovirus gB (guinea pigs) blood HS) in ELISA using recombinant expressed gD and gB purified from Assayed for V-specific response. Recombinant virus was isolated from BacPAK Bacul Ovirus Expression System (Clontech; Pa lo Alto, CA) pBacPAK8 transcription vector and Bsu361 digestion The BacPAK6 virus, and gD-2 and gD-2 from gB-2, respectively. And gB coding sequence. HSV-2 or baculovirus Glycoproteins from the gD infection medium were essentially purified (Pachl et al., 1987, J. Virology, 61, 315-325). Lentil Lectin Sepharose Chromatograph hy (Pharmacia Biotech Inc.). Off Glycoprotein B was added to 0.1 mM MnCl_Two, Adjusted to 0.5% NP40 The batch was purified at room temperature from Lentil Lectin Seph Adsorbed to arose4B and eluted as described above. 50 for ELISA diluted to 5 μg / mL total protein in mM carbonate buffer (pH 9.5), 00 μL / well was placed in a Maxi-sorb 96-well plate (Nu nc; Naperville, IL) and adsorbed overnight at 4 ° C. all Subsequent incubations were performed for 1 hour at room temperature in a volume of 100 μL, The rate is washed four times with phosphate buffered saline (PBS, pH 7.2) and distilled between steps Wash once or not with water. Dilution buffer (920 mM Tris-HCl [pH 7 . 5], 137 mM NaCl, 2.7 mM KCl, 0.5% glutamine, 0 . 05% Tween 20) as an inhibitor and for serial dilution and And alkaline phosphatase-labeled goat anti-mouse (Boehringer Man nheim; Indianapolis, IN) or goat anti-guinea pig (A ccurate Chemical and Scientific Corp . (Westbury, NY). ELISA is 10% 1 mg / mL p-nitrophenyl phosphate in ethanolamine (pH 9 . 8), 0.5 mM MgCl.H_TwoO at 37 ° C and the optical absorption was 40 Read at 5 nm. OD₄₀₅The signal was from serum from mock-immunized mice at the same dilution. Average OD₄₀₅If the signal (6 replicates) exceeds the standard deviation by 3 times or more, or OD₄₀₅Faith The signal of the guinea pig pre-immune serum at the same dilution was 0.1 OD. A serum dilution is scored as positive if greater than one unit. The last sa that is rated positive The reciprocal of the sample dilution is the termination titer. Geometric mean titer using individual termination titers (GMT) is calculated. If, for the purpose of calculation, the dilution series is extended, Negative serum at low dilution, followed by a termination titer equal to the reciprocal of the lowest dilution Assign a fixed amount. ELISA titers were determined in serum from native or saline immunized animals. Lack of measurable ELISA titers, or adjusted from mock infected BHK cell lysates HSV-specific as initially indicated by the lack of reaction between the antigen and immune serum is there.   HSV neutralization occurs essentially as described in Example 7. That is, After heat inactivation of DNA or serum from saline immunized animals at 56 ° C. for 30 minutes, Dilute serially in DMEM, 2% heat inactivated FBS; 50 μl of each dilution is sterile Polypropylene 0.5 mL 96-well plate (Marsh Biomedic al., Rochester, NY). HSV -2 material diluted to 4,000 plaque forming units (pfu) / mL; 50 μl of irus was added and the plate was incubated at 4 ° C. overnight. Guinea pig Complement (Cappel; Durham, NC) in DMEM, 2% heat inactivated FB Dilute 1: 4 in S; 50 μl was added to each sample well. 1 hour at 37 ° C After incubation, add 100 μl serum-free medium and use each reaction mixture. 12 well cluster plate (Co confluent VERO cells. Aspirate inoculum, single layer 5% heat-inactivated FBS, 1X Basal Medium Eagle vi tamins, 10 mM L-glutamine, 25 U / mL penicillin, 25 μg / ML streptomycin, 12.5 mM HEPES, 0.5% carboxime Stack 1 mL of 1X MEM containing chilled cellulose and plate at 37 ° C for 48 hours For a while. The monolayer is treated with 1% basic fuchsin in 50% methanol, Staining with 10% phenol was performed to determine the number of plaques. Plaque counts in saline immunized control mice From a sera from the same or a pre-immune serum from the same guinea pig at the same dilution Serum dilution is neutralized if it is 50% of that obtained in the parallel control assay Consider positive. E. FIG. T. , 1980,Statistical Metho ds for survival data analysis; Lifeti me Learning Publications, Belmont, CA , Pp. 122-156). Guinea pig Of the daily lesion score of the two-tailed Student's test analyzed. To compare the overall disease within a group of guinea pigs, Kruskal- By the Wallis test and subsequent multiple comparison tests at the p <0.05 significance level The average lesion rating was analyzed.   The full length gD and the coding sequence of the amino terminal 707aa of gB were analyzed by PCR. The length was determined in Examples 1 and 2 from the HSV-2 strain Curtis via DNA. Eukaryotic expression vector V1J or gD-2 and gB-2, respectively. Cloned into V1Jns. As described in the disclosure, expression was at V1 Similar to those from J, V1Jneo and V1Jns. Control plasmid Characterization and sequencing of the vector insert junction. Arrange In the region, the gD clone was HSV-2 strain G (Lasky and Dow). Benko, 1984, DNA, vol. 3, p. 23-29) The gB clone sequence was identical to that published for HSV-2 strain 33. 3 (Stove, et al., 1987, J. Virol. 61: 326-3). 35 page). gD-2 or gB-2 plus The ability of the mid to express the encoded protein depends on the transient transfection of RD cells. Indicated by the option. gD-2 DNA transfected RD cell lysate anti- Immunoblot analysis with the HSV-2gD monoclonal antibody was performed using pseudotransfer. MW of about 60K, which is not present in the cell lysate, was detected. . Conditioned media from gB-2 DNA transfected RD cells and cell lysates Immunoblot analysis with sheep anti-HSV-2 antiserum was not present in the control. MW detected a 106K protein, indicating that most of the protein was in the medium. all right. The dimensions observed are consistent with the gB 707aa truncated form. The cleavage removes the transmembrane and cytoplasmic regions from HSV-2gB, so expression Park was not expected to be cell-related.   The biological effects of immunization with gD-2 or gB-2 DNA were measured separately in mice. Was studied in a dose-titer experiment. Animals Immunization by intramuscular injection of DNA at week 0 and 7 or mock by saline Immunized. Serum obtained at 10 weeks was tested in an HSV-specific ELISA. Specified. Table 9 shows the results of seroconversion and the geometric flatness obtained for each treatment group. The titrated volume (GMT) ± standard error of the mean (SEM) is reported. These In serum, stored sera from saline-injected control mice were used as negative controls. Conclusion The result is that each DNA construct causes protein expression in vivo, even at low doses. Elicited a substantial antibody response. 0.8, the lowest dose tested In μg of gD-2 DNA, 8 out of 9 immunized mice are detectable Evoked an antibody response.   Representative sera from gD-2 DNA- and gB-2 DNA immunized animals were HSV-2 (strain C) at dilutions of 1:10, 1: 100, and 1: 1000. (urtis) neutralizing activity. 3.1 to 100 μg of gD-2 DNA Testing 15 sera from mice injected with Yes; 11 were positive at 1: 100; 5 were 1: 100 Positive at 0 dilution. Two negative sera (from the 50 μg dose group) are low ELISA endpoint force Value (each log_Ten  2.00 and 2.52). 30 μg A more restrictive study of sera from animals immunized with gB-2 DNA showed that Of the three sera obtained, all were positive at 1:10 and two were 1:10 0 indicates positive and 1: 1000 indicates no positive Was. These results indicate that immunization with either DNA construct resulted in H It has been shown that SV-2 neutralizing antibodies can be induced.   Immunized and control (saline injected) mice were exposed to an intraperitoneal injection of HSV-2. Observed daily for survival. FIG. 10A shows gD-2 DNA immunity on survival. And sufficient protection from death for each dose (P <0.001). ) Was achieved. 82 out of 86 gD-2 DNA immunized mice were exposed. Survived. Survival results for gB-2 DNA-immunized mice are shown in FIG. 10B. Show. Sufficient protection from death was observed for each dose tested (30, 10 and 3 μg P <0.01 for loop and p = 0.027 for 1 μg group ) Was seen about. That is, low doses of gD-2 or gB-2 DNA Immunization elicits an antibody response in mice and is lethal Protect against HSV-2 infection. Some animals show temporary illness during the observation period: He showed no grooming, poor growth or bent posture. Delinquency in convalescent serum Confirmation of infection in some animals by detecting antibodies to the active HSV protein did.   Mock immunization with saline was used as a control in dose-activity measurements. Protection, To ensure that it relies on the HSV coding sequence rather than injection of the DNA itself A group of 10 mice was treated with 12.5 or 1.6 μg of gD-2 DNA or Immunization was performed with 12.5 μg of vector V1J DNA. 10 weeks after one immunization The serum was analyzed by ELISA. Inject 12.5 μg of gD-2 DNA Iog about the group_TenGMT (± SEM) is 3.89 (0.97) And 2.49 (1.20) for the 1.6 μg group. Vector -None of the sera from mice immunized with DNA were seropositive; Iog_TenGMT was the background. FIG. 11 shows the abdominal cavity at 11 weeks. We report survival data for these animals following internal exposure. exempt with gD-2 DNA Any group that had become infected had more deaths than those immunized with the vector. Protected (p <0.001). Vector injection animal raw Is similar to that seen in saline injected animals in the experiments summarized in FIG. I was similar. These results confirm that protection is dependent on the gD coding sequence. I accepted. Furthermore, they achieve protective immunity with a single injection of gD-2 DNA. Showed that you get. A vector expressing influenza virus protein or Mice comparing the control plasmid with plasmid gB-2 or gD-2 and Further studies in guinea pigs (see FIG. 12) show that protection was also due to HSV protein Found to be dependent on the presence of the doping sequence.   The lethal infection model confirms the in vivo activity of gD-2 and gB-2 DNA Was useful to show that low DNA doses were effective . However, to more closely mimic human pathologies, gD-2 and gB -2 In order to suppress the effect of immunization by combining low doses of DNA, The Mott vaginal infection model was used. 3 μg gD-2 DN at weeks 0 and 6 A and a DNA mixture containing 10 μg gB-2 DNA in 7 guinea pigs Immunized, 14 subject guinea pigs were mock-immunized with saline. Obtained in the 9th week Serum was subjected to anti-gD and anti-gB using an antigen-specific ELISA. Was analyzed. Table 10 shows the results. All DNA immunized animals have gD and ELISA titrations are performed on both gB and gB; individual endpoint titers are 300. Izu None of these mock-immunized animals were positive at the lowest dilution (1:30). Conclusion The results showed that both DNAs in the mixture were expressed. These sera are The SV-2 antibody was also assayed. Immune blood from a total of 7 DNA-immunized animals Qing is a neutralization solution at a 1:10 dilution, 6 of 7 at 1: 100 And two are positive at 1: 1,000, but at 1: 10,000 Neither is positive. 4 randomly selected representatives from mock-immunized control animals Target sera were positive at 1:10 or 1: 100.   At 10 weeks, all of the DNA immunized and mock immunized guinea pigs Eight of the animals were exposed by vaginal introduction of HSV-2 strain MS. As an effect control Thus, the treatment used in the infection procedure had an external disease rating and the remaining 6 Mock immunized animals were mock infected with inoculum prepared from mock infected Vero cells . The severity of the primary disease was tested by the lesion rating system described in the description of FIG. The course of primary disease, 3-14 post-infection FIG. 12 is summarized in FIG. 12 by reporting the daily average lesion rating on day. infection All animals in the control group showed severe external disease. From day 5, this group's reviews The limits were significantly greater than DNA-immunized or mock-infected controls (p <0 . 01). In contrast, none of the DNA-immunized animals showed severe disease, Are statistically indistinguishable from those of the mock infected group. Evaluation of all lesions The overall primary disease, as measured by the mean of the fixed values, was DNA- It was significantly lower for the immunized group (P <0.001), but the mock-infected control group Did not differ significantly from P. (P = 0.92). The score for the mock infected group is Used as experimental background.   DNA immunized animals were further exposed to infected controls that did not show any signs of systemic disease. Distinguished. In contrast, six of the eight mock-immunized guinea pigs had severe whole body 5 showed urinary illness for more than 2 days, 1 showed partial paralysis in hind legs Five animals died during the observation period and required euthanasia (FIG. 12). Mock infection None of the animals showed any signs of systemic disease. The exposure study in this example Low dose immunization of DNA encoding SV-2 full length gD and truncated form of gB But HSV-2 induction It indicates that guinea pigs were protected from primary diseases.   Data include Ds encoding full length HSV-2gD or truncated forms of HSV-2gB. Immunization with NA elicits an immune response in mice, which renders them immune to HSV-2. Protecting against lethal exposure, and combining low doses of these two DNAs It shows that the combination protects guinea pigs from primary diseases. This protection is D At low doses of NA occurred in a mouse model of single immunization.   When gD-2 DNA was titrated in mice over a 250-fold concentration range, all of the tested All doses induced serum antibodies and protected mice from lethal infection. 0.8μg The level of protection elicited by is the highest level induced by the highest test dose. Could not be distinguished statistically. In subsequent titrations, 50 ng of A single injection of a small amount of gD DNA can elicit a detectable antibody response. However, a dose of 500 ng requires obtaining a corresponding seroconversion and 1.6 μg Immunization with gD-2 DNA protected mice from lethal intraperitoneal exposure (FIG. 11). Titration of gB-2 DNA in mice was as low as 2 μg in 1 μg. Shows that one immunization leads to great protection did. Expresses, but is not limited to, ΔgB and gD in a guinea pig model The preferred range of the combination polynucleotide vaccine is 2.0 μg each.   gD and 0.6 μg △ gB.   Competitors due to competition for DNA uptake or expression, or antigen It is hypothesized that immunization with simulated DNA will reduce the response to individual components. It is. The combination of gD-2 and gB-2 DNAs does not react to any of the components. It did not seem to decrease. Furthermore, this is achieved with this low dose combination Protection was achieved with a similar exposure using 100 μg of gD-2 or gB-2 DNA alone. It was similar or better than that seen in Russian studies. gD-2 DNA and gB- Combination with 2 DNAs allows for a wide range of epitopes obtained in two separate antigens. Can be more effective than either ingredient alone Had the following performance. The result is that the combination is more effective than the individual components To conclude from this study, since no comparisons were made directly Can not. gD-2 DNA and gB-2 DNA alone and in combination Is necessary to solve the problem directly, and with minimal This has been done to achieve effective dosages.   The examples show that immunization with low doses of DNA can be used in two animal models of HSV infection. Highly effective in generating protective immunity in mice and in mice One immunization is protective. These results indicate that the intramuscular injection was D It has the ability to be effective for NA delivery, low doses and limited number of injections Supports the potential to develop DNA vaccines for human use with desirable properties are doing. The combination of gD-2 DNA and gB-2 DNA shows that both proteins And elicited an immune response to the disease and was effective in preventing HSV-2-induced mucosal disease. This result indicates that multivalent vaccines can be made simply by combining DNA. Supports concepts that can be done.*: Doses at weeks 0 and 7; serum obtained at week 10. †: Number of serum tested ‡: By conventional method, stored sera from mice injected with saline were diluted at all dilutions tested. The endpoint titer equal to the reciprocal of the next lower dilution is extrapolated. 1: 3 in these cases.*: Number of animals †: For the calculation of GMT, sera that are negative at all dilutions tested Then, an endpoint titer equal to the reciprocal of the next lower dilution is extrapolated; in this case 1: 3 is there.                                Example 12   As in Example 11, herpes simplex virus type 2 (HSV-2) glycoprotein Forming a plasmid expression vector encoding the cytoplasm B (gB) or D (gD) Was tested for its ability to immunize guinea pigs against genital HSV infection. gB Immunization with a plasmid expressing the amino terminal 707 amino acids (aa) of The humoral immune response detected by ISA and virus neutralization was elicited. Vaginal infection Immunized animals are partially protected from genital herpes when exposed to A widely reduced primary and subsequently recurring disease is indicated. The gB plasmid is Immunization when combined with a plasmid expressing full length gD Guinea pigs show a low humoral response to both proteins, again significantly protecting them from viral exposure. Was protected.   Plasmid vector expressing full-length gD or carboxy-terminal truncated form of gB Is generally described throughout this specification, and in particular in Example 11. As described above, using an expression vector V1Jns which is a derivative of the vector V1J. Was. In this vector, expression is immediately before cytomegalovirus (CMVIE). Driven by protein promoters. a sequence encoding gD or gB, PCR amplification was performed from the HSV-2 strain Curtis virus DNA. Plasmid V1 Jns: Expression of full length 393 amino acid (aa) gD from gD was determined by gD-specificity Transiently Transfected Human Human Fetal Rhabdomyosarcoma (RD) Using Noclonal Antibody ) Confirmed by immunoblot analysis of cells. Indirect immunofluorescence indicates that the expressed protein It was shown to be related to the cell membrane. 904aa-gB amino-terminal 707aa The gB plasmid: V1Jns: gB was formed for expression. This cutting tamper Has a gB-deleted transmembrane and cytoplasmic region, and thus It is expected to be secreted from cut cells. Transiently transfected RD cells Sheep anti-HSV -2- Immunoblot analysis with antiserum (ViroStat) showed the expected dimensions (106 kDa) protein, most of which are found in conditioned media Was. The in vivo activity of these DNA constructs was measured in mice, guinea pigs and humans. Inducing gD- or gB-specific serum antibodies upon injection in outside primates Confirmed by All animal studies are performed by the Institutional Animal 1. Performed using Care and Use Committee approved protocols. Was.   V1Jns: Genital HSV of gB plasmid (i.e., gB-2 of Example 11). The effect on infection was measured using female Duncan Hartley guinea pigs (HRP Inc.). . Immunized with 100 μg or 10 μg of V1Jns: gB Studied. Control animals were isolated from plants expressing influenza A virus hemagglutinin. Immunization was performed with 100 g of the rasmid V1Jns: HA. DNA in saline 200μ L and delivered to each quadriceps to 100 μL. At week 6, all animals Was re-immunized.   Serum obtained at 10 weeks was used as a capture antigen for partial analysis and purification of HSV-infected hamster milk. Analyzed by HSV-specific ELISA using fetal kidney cell lysates. ELIS A is infected, immunized Initially, due to the different reactions of the sera from It was qualified to be HSV-specific. The endpoint titer was determined using the same dilution of control animal serum as 6 Average OD of two replicates₄₀₅OD that exceeds the signal by more than 3 times the standard deviation₄₀₅Give the signal Is also the reciprocal of the higher serum dilution. V1Jns: All gB immunized animals were seropositive. Gender. 100 μg V1Jns: geometric mean titer for gB group ± standard Error (GMT ± SE) is 120,000 ± 30,000, low (10 μg) The dose group was 147,000 ± 32,300. V1Jns: HA The GMT of the immunized control group was 3.0 ± 0 by definition. These sera are used in complement Dependent virus infection was also assayed. Dilutions of heat-inactivated serum were added to HSV-2 Incubate overnight at 4 ° C with Curtis 200 plaque forming units (pfu) Was. Add guinea pig complement for 1 hour at 37 ° C, then allow for Vero cells Virus was assayed. Percentage plaque reduction was compared to each dilution of the pre-immune serum at the same dilution. Determine the clear dilution (twice) and score a positive of 50% or more in the number of plaques. 1: At initial considerations at 10 and 1: 100 dilutions, all V1Jns: gB Formation from animals is positive at both dilutions; V1Jns: None of the sera from the HA immunized animals were positive. In the definition of endpoint titer , One serum from the 100 μg dose group, four sera from the 10 μg dose group, And one serum from the HA DNA immunization group was randomized for further titration. Selected. A total of 5 sera from V1Jns: gB immunized pigs were 1: 1, Neutralization positive at 000 dilution and negative at 1: 10,000; The control serum is negative at 1:10. These V1Jns: gB immunized guinea pigs The immune sera from the kits were also HSV-HSV-2 with the same titers seen for HSV-2 neutralization. One strain KOS infectivity was neutralized. Control guinea that survived vaginal infection with HSV-2 Convalescent sera from the kits were positive for neutralization at 1: 1,000 but higher. It was negative at high dilutions. That is, DNA expressing a truncated form of gB Immunization of antibodies in guinea pigs similar to those found in immunized animals The titer was neutralized.   Ten weeks after the initial immunization, guinea pigs were inoculated by vaccination with HSV-2. Exposure. FIG. 13A shows the daily lesion evaluation values for each test group on days 3-18. The course of primary disease is summarized by reporting the mean. By day 5, high dose And low dosing The mean lesion rating of both groups in the amount of V1Jns: gB group was reduced compared to the control. all Reducing somatic disease is greater than measuring repeat mutations : V1Jns at dose of 100 μg: p <0.01 for gB group, and dose P = 0.04 for 10 μg. The occurrence of severe disease (lesion rating ≥ 3.0) 4 (out of 10) during primary infection higher in control group than in any test group Of the HA-DNA immunized animals showed systemic disease, became moribund and euthanized, and 2 10-μg gB DNA immunized guinea pigs (out of 9) required euthanasia . A total of nine 100 μg-gB DNA immunized pigs survived.   Following a primary infection, guinea pigs, like humans, sometimes become reactive and re-disease. Indicates a latent herpes infection that develops. Live animals 4 weeks after virus inoculation For 10 weeks. Observation of 39 pairs, primary illness Disease was rated on the same scale as the illness. Recurrent disease is usually more severe than primary disease The relapse data is calculated as the cumulative average The lesion evaluation value is shown in FIG. 13B. The running display of the daily average lesion rating is The frequency of each recurrent event, Includes results by severity and duration. The figure shows that control animals were isolated with gB DNA. It shows a more frequent (P = 0.03) recurrence than any of the groups that became infected. The curve is not limited to the case where there is a quantitative difference. Medium, relapse continues, but most animals immunized with 100 μg V1Jns: gB No recurrence. These results indicate that the truncated form of HSV-2gB Immunization with the loading DNA elicits an immune response in guinea pigs, which And subsequent partial protection from recurrent HSV-induced disease are doing.   V1Jns: gB plasmid was replaced with plasmid V expressing full length HSV-2gD. Tested in combination with 1Jns: gD (i.e., gD-2 of Example 11). Guinea pigs were prepared according to the method described above by 100 μg V1Jns: gB + 100 μg Immunization twice with a mixture of V1Jns: gD or 100 μg V1Jns: HA did. At week 10, serum and vaginal secretions were obtained and gD- and gB-specific. Analyzed by ELISA. These ELISAs use clonin as a capture antigen. Using baculovirus-expressed gD or truncated gB protein, V1J Guinea pigs and mice immunized with ns: gD or V1Jns: gB only The use of these sera was first qualified as antigen-specific. V1Jns : GB + V1Jns: gD immunized guinea pigs are seropositive for both gD and gB Yes; GMT for both proteins is the same: 6,926 ± 2,945, Individual animals responded differently to the two immunogens. V1Jns: HA immunization control The GMT for the group is 3 ± 0 by definition. These results are When immunized with a mixture of DNAs encoding gD and gB, Elicited an immune response to   Vaginal secretions were obtained from 7 V1Jns: gB + V1Jns: gD immunized guinea pigs. Collected by wiping with a saline-wet calcium alginate swab and Eluted in mL. The seven animals tested had a detectable anti-gD response. The titer was 2- ≧ 8. 4 out of 7 animals have detectable anti-gB anti- The titer was ≧ 8 in all cases. HA DNA immunization mole Secretion from the mott was used as a negative control. In this same experiment, HSV Immunized with 100 μg of plasmid expressing VP16, a two-interacting protein. 3 Secretion from one animal is negative for anti-gD or -gB response when tested net Met. These vaginal anti-gD or -gB antibodies were found to be IgG and serum It may be the result of antibody leakage.   At week 10, animals were exposed to HSV-2 and primary and recurrent as described above. The disease was evaluated. The course of the primary infection is summarized in FIG. 14A. Until the 7th day, V1Jns: gB + V1Jns: The average lesion rating value for the gD group was lower than that for the control group. Was substantially lower. The reduction was significant during the entire observation period; p <0.001.8. V1Jns: gB + V1Jns: gD immunized group showed severe disease No (lesion rating ≧ 3), but 3 (out of 8) control animals were consistently diseased Had a variable rating> 3. In this exposure, all animals survive the primary infection After resolving, the disease was evaluated for recurrent disease for 5 weeks. The results are shown in FIG. 14B. V Cumulative mean lesion rating for 1Jns: gB + V1Jns: gD immunized group is Significantly lower than the control group throughout the observation period; the overall reduction was significant (p <0.001). As can be seen in the previous experiment, there was no recurrence between the control and test groups. There were qualitative and quantitative differences. Unlike the control group, V1 In the Jns: gB + V1Jns: gD group, observation started on the 8th day and there was no long-term relapse Was.   The results disclosed in this example show that the DNA encoding the HSV-2 gB sequence or H Immunization of guinea pigs with a mixture of DNA encoding SV-2 gD and 2 gB Elicits a humoral immune response to the encoded protein, and these responses are primary and And reduced HSV-2 induced disease.   Further studies in mice indicate that DNA immunization with V1Jns: gB is humoral. But also elicits a cell-mediated immune response.   Additional lymphocyte proliferation data in mice, guinea pigs and monkeys (Example Immunization with V1Jns: gB in all models Elicit a memory response.                                Example 13   Adolescent (body weight 2.2-7 kg) seronegative female African green monkeys (3 / group) One triangular and one quadriceps have 0 per site containing the indicated amount of DNA. . A 5 ml inoculation volume was injected intramuscularly. Control monkeys were licensed for influenza virus Kuching (F luzone Whole Virion or Subvirion, Conn out Laboratories Inc. SWIFTWATER, PA, USA; trivalent, prepared in 1992-93), the total amount for human use (1 for each HA antigen) 5 μg). Two immunizations are pre-exposed to influenza virus The immunization of humans was performed without the recommended clinical protocol.   Hemagglutination-inhibiting (HI) antibodies were isolated from 4 HA units of the selected virus strain and Determined using a chicken RBC. Serum was purified using receptor-destroying enzyme (RDE, Sigma).   Chemical Co. Manufactured by St. Louis, MO) overnight The mixture was baked, heated at 56 ° C. for 30 minutes, and adsorbed on chicken RBC.   Groups of 6 African green monkeys were treated with gD and gB DNA at 0 and 4 weeks. With 100 or 10 μg of each, and boosted at 24 weeks . Serum obtained at 4 week intervals was antigen specific for anti-gD and anti-gB antibodies. Was analyzed by dynamic ELISA. These ELISAs use cloni as a capture antigen. Using baculovirus-expressed gD or cleaved gB protein , Vljns: gD or Vljn s: Guinea pigs and mice immunized with gB (Examples 11 and 12) alone It was first qualified as antigen-specific using sera from Immunity from the animal An OD405 signal that exceeds the signal obtained at the same dilution of preserum by more than 0.05 OD units. The endpoint titer is obtained as the reciprocal of the highest serum dilution giving the signal. Geometric mean titer (G For the calculation of MT), sera that are negative at the lowest dilution tested are terminated. It is assumed that the point titer is 3. These sera were also tested for complement-dependent virus neutralization. Was. Dilute the heat inactivated serum with 200 plaque forming unit (pfu) HSV-2Curt Incubated overnight at 4 ° C with is. Guinea pig complement at 37 ° C for 1 hour After addition, the virus was assayed for growth on Vero cells. Same dilution Plaque reduction% was determined for each serum dilution compared to pre-immune serum (2 times) and evaluated A decrease of 50% or more in the value was regarded as positive. 1:10 to 1: 10,000 10 times continuous Dilutions were assayed; endpoint titers were calculated by linear regression analysis (the lowest tested Sera that were negative in dilution were assigned an endpoint titer of 1).   DNA from other etiologies also elicits humoral immune responses in African green monkeys I found out. Herpes simplex virus Immunization with DNA encoding (HSV) is performed using glycoprotein D (gD DNA). And the truncated form of glycoprotein B (gB DNA) is Evoked an antibody response that could neutralize gender. 10 μg small amount of each DNA structure After two immunizations, all animals had detectable neutralization titers. FIG. As shown in FIG. 5A, the antibodies measured by ELISA were 6 immunizations after two immunizations. Survived the moon and was facilitated by subsequent injections. Generates neutralizing antibodies and titers decrease However, as shown in FIG. 15B, it could be easily promoted.

────────────────────────────────────────────────── ─── Continuation of front page    (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, L U, MC, NL, PT, SE), OA (BF, BJ, CF) , CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (GH, KE, LS, MW, S D, SZ, UG, ZW), EA (AM, AZ, BY, KG) , KZ, MD, RU, TJ, TM), AL, AM, AU , AZ, BA, BB, BG, BR, BY, CA, CN, CU, CZ, EE, GE, HU, ID, IL, IS, J P, KG, KR, KZ, LC, LK, LR, LT, LV , MD, MG, MK, MN, MX, NO, NZ, PL, RO, RU, SG, SI, SK, SL, TJ, TM, T R, TT, UA, US, UZ, VN, YU (72) Inventor Kizz, Robert Day             United States, Alabama 35222, Bar             Mingham, Towelves Court Sau             Su 3847 (72) Inventor Lewis, Jiong Ai             New Jersey, United States             07065, Lowway, East Linker             N Avenue 126 (72) Inventor Liu, Margaret A             New Jersey, United States             07065, Lowway, East Linker             N Avenue 126 (72) Inventors Matsuclements, William L             New Jersey, United States             07065, Lowway, East Linker             N Avenue 126

Claims (1)

[Claims] 1. Induces anti-HSV antibodies or protective immune responses when introduced into vertebrate tissues A polynucleotide, wherein said polynucleotide comprises at least one H At least one gene encoding an SV protein or a functional equivalent thereof; Wherein the gene is operably linked to a transcription promoter. Polynucleotide. 2. The gene is gB, gC, gD, gH, gL, ICP27 and its genes. 2. The method of claim 1 which encodes an HSV protein selected from the group consisting of functional equivalents. Polynucleotides listed. 3. 2. The method of claim 1, wherein said gene encodes a carboxy-terminally truncated gB protein. Polynucleotides listed. 4. The truncated gB deletion comprises the amino terminal 707 amino acids of wild-type gB. Item 4. The polynucleotide according to Item 3, 5. The polynucleotide according to claim 4, which is V1Jns: gB. 6. 3. The method according to claim 2, wherein said gene encodes gD which is an HSV protein. Renucleotide. 7. The polynucleotide according to claim 6, which is V1Jns: gD. 8. A first polynucleotide that expresses HSV protein gD; Against HSV comprising a second polynucleotide that expresses a truncated gB protein Vaccine that induces an immune response. 9. 9. The method according to claim 8, wherein the first polynucleotide is V1Jns: gD. vaccine. 10. 9. The method according to claim 8, wherein the second polynucleotide is V1Jns: gB. Vaccine. 11. The method according to claim 10, wherein the first polynucleotide is V1Jns: gD. On the vaccine. 12. 12. Introducing the vaccine of claim 11 into a vertebrate tissue. A method of eliciting an immune response to an HSV epitope in a vertebrate. 13. 12. The polynucleotide of claim 11 and a pharmaceutically acceptable carrier. A vaccine that elicits an immune response against HSV.