HRP950412A2 - A polynucleotide herpes virus vaccine - Google Patents

Description

The field of technology

The main obstacle in the development of vaccines against viruses, especially those with multiple serotypes or high mutability, against which it is desirable to induce neutralizing and protective immune reactions, is the diversity of external viral proteins in different virus isolates or strains. Because cytotoxic T-lymphocytes ( CTLi ) are able to recognize epitopes derived from conserved internal viral proteins in both humans and mice /J.W. Yevdell et al., Proc Natl Acad Sci (USA) 82, 1785 (1985); A.R.M. Townsend et al., Cell 44, 959 (1986); A.J. McMichael et al., J. Gen. Virol., 67, 719 (1986); J. Bastin et al., J. Exp. Med. 165, 1508, (1987); A.R.M. Townsend and H. Bodmer, Annu. Rev. Immun. 7, 601 (1989)/, and are considered important for the immune response against viruses / Y.-L. Lin and B.A. Askonas, J. Exp. Honey. 154, 225 (1981); I. Gardner et al., Eur. J. Immunol. 4, 68 (1974); K.L. Yap and G.J. Ada, Nature 273, 238 (1978); A.J. McMichael et al., New. English J. Med. 309, 13 (1983); PM Taylor and B.A. Askonas, Immunol. 58, 417 (1986)/, efforts have been made to develop a CTL vaccine that enables heterologous protection against different virus strains.

CTLi are known to kill virus-infected cells if their T receptors recognize viral peptides bound to class I or class II MHC molecules. These peptides can arise from endogenously synthesized viral proteins regardless of the location or function of that protein in the virus. By recognizing epitopes on conserved viral proteins, CTLi can provide heterologous protection.

Many infectious disease agents can, by themselves, induce the production of protective antibodies that can bind to and kill, incapacitate, or cause to be destroyed or incapacitated the disease agent or its by-products. Recovery from these diseases usually results in long-term immunity conditioned by the production of protective antibodies created against highly antigenic components of the infectious agent.

Protective antibodies are part of the natural defense mechanism of humans and many other animals and are found in the blood, as well as in other body fluids and tissues. The primary function of most vaccines is to stimulate the formation of protective antibodies against infectious agents and/or their by-products, without causing disease.

Most attempts to induce CTL reactions have used replicating vectors in order not to produce a protein antigen in the cell / J.R. Bennink et al., ibid., 311, 578, (1984); J.R. Bennink and Yewdell, Curr Top Microbiol Immunol 163, 153 (1990); C.K. Stover et al., Nature 351, 456 (1991); A. Aldovini and R.A. Young, Nature 351, 479 (1991); R. Schafer et al., J Immunol 149, 53 (1992); C.S. Hahn et al., Proc Natl Acad Sci (USA) 89, 2679 (1992) / or focused on the introduction of peptides into the cytosol / F.R. Carbone and M.J. Bevan, J Exp Med 1169, 603 (1989); K. Deres et al., Nature 342, 561 (1989); H. Takahashi et al., ibid. 344, 873 (1990); D.S. Collins et al., J Immunol 148, 2357 (1992) /. Both approaches have limitations that may reduce their applicability as vaccines. Retroviral vectors have limitations according to the size and structure of fusion polypeptides that can be expressed as fusion proteins, while maintaining the ability of the virus to replicate / A.D. Miller, Top. Microbiol. Immunol. 158, 1 (1992)/ and the effectiveness of vectors such as vaccination for subsequent immunization can be compromised by an immune response against vaccination / E.L. Cooney et al.. Lancet 337, 567 (1991) /. Also, viral vectors and modified pathogens carry an inevitable risk that can prevent their use in humans /R.R. Redfield et al., New Engl J Med 316, 673 (1987); L. Mascola et al., Archiv Intern Med 149, 1569 (1989)/ . In addition, the selection of peptide epitopes to be presented depends on the structure of the individual MHC antigen and therefore peptide vaccines may have limited efficacy due to the diversity of MHC haplotypes in cultured populations.

Benvenisty, N., and Reshef, L. / PNAS 83, 9551-9555, (1986)/ showed that CaCl2-precipitated DNA introduced into mice intraperitoneally (i.p.), intravenously (i.v.), or intramuscularly (i.m.) could be expressed . Intramuscular injection (i.m.) of DNA expression vectors in mice has been shown to lead to uptake of DNA by muscle cells and expression of the protein encoded by that DNA /J.A. Wolff et al., Science 247,1465 (1990); G. Ascadi et al., Nature 352, 815 (1991)/. Plasmids were retained episomally and did not replicate. Subsequently, persistent expression was observed after i.m. injections into fish and primate rat muscle and rat heart muscle. / H. Lin et al. , Circulation 82,2217, (1990) R.N. Kitsis et al., Proc Natl Acad Sci (USA) 88, 4138 (1991); E. Hansen et al., FEBS Lett. 290, 73 (1990); S. Jiao et al., Hum Gene Therapy 3, 21, (1992); I. Wolff et al., Human Mol Genet 1, 363 (1992)/.

The technique of using nucleic acid as a therapeutic agent is described in WO90/11092 (September 4, 1990), where naked polynucleotides were used for vaccination of vertebrates.

Recently, the coordinated role of B7 and the major histocompatibility complex in the presentation of antigen epitopes on the surface of antigen-presenting cells during CTLa activation for tumor elimination was described / Edington, Biotechnology 11, 1117-1119, 1993/. When the MHC molecule on the antigen-presenting cell (APC) presents an epitope to the T-cell receptor (TCR), B7 expressed on the surface of the same APC acts as a second signal by binding to CTLA-4 or CD-28. The result is rapid division of CD-4+ helper T-cells, which signal CD8+ T-cells to proliferate and destroy APCs.

For the method to be successful, it is not necessary for the immunization to be intramuscular.

Thus, Tang et al. / Nature 356,152-15419 (1992) describes that the introduction of gold microprojectiles coated with bovine growth hormone into mouse skin leads to the formation of anti-BGH antibodies in mice. Furth et al.,/Analytical Biochemistry, 205, 365-8, (1992)/ show that a jet injector can be used to introduce skin, muscle, fat, and mammalian tissue of living animals. Recently, various methods for introducing nucleic acids have been described / Friedman, T., Science, 244, 1275-1281 (1989)/. See also Robinson et al. / Abstracts of articles presented at the meeting held in 1992 "Modern approaches to new vaccines, including prevention of AIDS, Cold spring harbor, p.92/, where it was shown that i.m., i.p., or i.v. administration of bird flu virus DNA provides protection against the challenge of a deadly disease . Intravenous injection of a DNA: cationic liposomal complex in the mouse as demonstrated by Zhu et al.,/ Science 261,209-211(July 1993); see also WO93/24640, December 9, 1993/ leads to systemic expression of the cloned transgene. Recently, Ulmer et al./ Science 259, 1745-1749, 1993 / reported heterologous protection against influenza virus infection after injection of DNA encoding influenza virus proteins.

Wang et al./ P.N.A.S.USA 90, 4156-4160 (May 1993)/ reported the induction of an immune response against the HIV virus in mice by intramuscular administration of a cloned genomic (unsplit) HIVa gene. However, the level of immune response achieved was very low and the system used parts of the mouse mammary tumor virus (MMTV), long terminal repeat (LTR) promoter and parts of the simian virus 40 (SV40) promoter and terminator. SV40 is known to transform cells, possibly by integrating into host DNA.

Thus, the system described by Wang et al. completely inapplicable for use in humans, which is one of the goals of this invention.

WO 93/17706 describes a procedure for vaccinating an animal against a virus, where parts of the carrier are coated with a gene construct and, dividing by acceleration, are introduced into the cells of the animal.

Recent efforts to develop a subunit vaccine for herpes simplex virus (HSV) have focused on novel expression and presentation of viral antigens, particularly viral glycoproteins. / for review see Burke, R.L., 1993, Sem. In Virol. 4., p. 187-197/. Recombinant HSV glycoproteins expressed by various systems including yeast (Kino, Z.C. et al., 1989, Vaccine, 7, pp. 155-160), insect cells (Ghiasi, H. et al. 1991, Arch Virol, 121, p. 163 -178) and mammalian cells (Burke, R.L., 1991, Rev Infect Dis, 13, S906-S911; Lasky, L.A., 1990, J. Med. Virol induce protective immunity in an animal model Clinical trials of recombinant HSV-2 glycoprotein D (gD ) produced in Chinese hamster ovary cells showed that the vaccine elicited an antibody response in seronegative and stimulated a preexisting response in both HSV-I and HSV-2 seropositive individuals. (Straus, S.E. et al., 1993, J Infect Dis 167, pp. 1045-1052).

An alternative approach to subunit vaccination has been the use of live viral vectors to deliver HSV antigen. HSV vaccine recombinants expressing gD (Aurelijan, L. et al., 1991, Rev Infect Dis, 13, S924-S930; Rooney, J.F. et al. 1991, Rev Infect Dis, 13, S898-S903; Wachsman M. et al. .,1992, Vaccine, 10, pp. 447-454) gB( Rooney, J.F. et al., supra) gL and gH (Browne, H. et al., 1993, J Gen Virol, 74, pp. 2813-2817 ) successfully protected animals from HSV infection. Vaccination by infection with a recombinant adenovirus expressing HSVgB elicits a protective immune response in mice (Ghiasi, H. supra; McDermott, M.R. 1989, Virology, 169, pp. 244-247) The protective role of anti IgD against HSV infection is well documented whether induced by immunization with native protein (Long, D. et al. 1984, Infect Immun., 43, p.761-764) recombinantly expressed protein (Burke, L.R., supra; Stanberry, L.R. et al., 1987, J Infect Dis, 155 , pp. 914-920; Straus, S.E. supra) peptides derived from gD (Eisenberg, R.J., et al., 1985, J Virol, 56, 1014-1027) or transferred passively (Dix, R.D., et al. 1981, Infect Immun , 34, pp. 192-199; Ritchie, M.H. et al., 1993, Investigative Ophthalmology and Visual Sciences, 34, pp. 2460-2468).

Studies by Wolf et al. (supra) initially showed that intramuscular injection of a plasmid encoding a transfer gene results in the expression of that gene in myocytes, near the injection site. Recent reports have shown successful immunization of mice against influenza with the injection of a plasmid encoding influenza-A hemagglutinin (Montgomery, D.L., et al. 1993, Cell Biol., 12, p.777-783) or nucleoprotein (Montgomery, D.L., et al. , supra; UlmerJB et al., 1993, Science, 259, pp. 1745-1749). The first use of DNA immunization for herpes virus was reported (Cox et al., 1993, J Virol, 67, pp. 5664-5667). Injection of a plasmid coding for the gIV glycoprotein of bovine herpesvirus I (BHV-I) led to the generation of anti-g-IV antibodies in mice and calves. After intranasal administration of BHV-I, immunized calves showed fewer symptoms and shed significantly less virus than controls. The ability of HSV glycoprotein D to elicit a protective immune response in the mouse (Long, D. et al., supra) and in the guinea pig (Stanbery, L.R. et al., supra; Stanbery, L.R. ii al., 1989, Antiviral Res., 11, s 203-214) is well documented.

Presentation of the essence of the invention

To test the effectiveness of DNA vaccines in preventing HSV disease, DNA sequences encoding HSV-2 proteins were cloned into a eukaryotic expression vector. This DNA construct elicits an immune response when injected into animals. HSV-infected animals were immunized to assess whether or not direct DNA immunization with the gD gene (or another HSV-2 gene) could provide the animals with protection against the disease. Therefore, described nucleic acids, including DNA constructs and RNA transcripts, are capable of inducing in vivo expression of herpes simplex virus (HSV) proteins after direct introduction into animal tissues by injection or otherwise. The injection of these nucleic acids can stimulate an immune response that results in the formation of cytotoxic T lymphocytes (CTLa) specific for HSV antigens, as well as the formation of HSV-specific antibodies, which protect against subsequent HSV infection. These nucleic acids are useful as a vaccine to induce immunity to HSV, which can prevent infection and/or lead to amelioration of HSV-related diseases.

Short description of the pictures

Sl. 1 Lists A, B, C

A. Western blot analysis of HSVgD expression in VIJ:gD-transfected cells is shown;

1. mock-infected Vero cells;:

2. HSV-2 186 infected Vero cells (moi = 1);

3. HSV-2 Curtis infected Vero cells (moi = 1);

4. RD cells transfected with VIJ:gD

5. Mock-transfected RD cells

B. Western blot analysis of HSV gB expression in VIJNS: gB-transfected cells is shown

C. Western blot analysis of HSV ICP27 expression in VIJ:ICP27-transfected cells is shown.

Fig. 2 Lists A, B, C

A. Western blot analysis using serum antibodies from animals immunized with HSV PNV (B-6.25 µg dose of V1J:gD, C-50 µg dose of V1J:gD) and mock-immunized animals (A) is shown on lysates of BHK cells infected with:

1. HSV-1 KOS;

2. HSV-2 186;

3. HSV-2 Curtis;

4. Falsely infected

B. Western blot analysis using sera from animals immunized with HSV PNV gB is shown.

Sl. 3 ELISA-derived GMT data set is shown for animals immunized with HSV-PNV that received a single dose of vaccine; sera were taken 4, 7 and 10 weeks after immunization.

Fig.4 Survival of animals infected with HSV-2 after two injections of V1J:gD of 200 µg;

100 µg, 50 µg, 25 µg, 12.5 µg, 6.25 µg, 3.13 µg, 1.56 µg, 0.78 µg, or salt solutions. Since all animals in the 200µg, 100µg, 50µg, 25µg, 12.5µg, 6.25µg and 3.13µg groups survived, they are all represented by the same symbol.

Fig. 5. Survival of HSV-2-infected animals after a single injection of 50 µg V1J:gD; 16.7 µg; 5.0 µg; 1.67 µg; 0.5 µg; 0.167 µg; 0.05 µg; 0.017 µg; 0.005 µg; or only physiological solutions.

Fig. 6. Survival of animals immunized with V1JNS:gB after HSV challenge is shown.

Fig. 7. Survival of animals immunized with V1J:gC after HSV challenge is shown.

Fig. 8. Results of survival, median day of death, paralysis, and vaginal viral titer are presented in guinea pigs infected with HSV-2.

Fig.9 The evaluation of vaginal lesions of guinea pigs infected with HSV-2 is shown.

Detailed description of the invention

The present invention provides polynucleotides which, when introduced directly into vertebrates in vivo, including mammals such as humans, induce expression of the encoded proteins in the animal. As used herein, a polynucleotide is a nucleic acid that contains basic regulatory elements such that, upon introduction into a living mammalian cell, it can direct the cell to produce the translation products encoded by the genes that make up the polynucleotide. In one form of embodiment of the invention, the polynucleotide is polydeoxyribonucleic acid that contains HSVa genes operatively linked to a transcriptional promoter. In another embodiment of the invention, the polynucleotide vaccine contains polyribonucleic acid, which encodes HSV genes that are subject to translation on the eukaryotic cellular system (ribosomes, tRNA, and other translation factors). When the protein encoded by the polynucleotide is one that does not normally occur in that animal's cell except under pathological conditions, (eg, a heterologous protein) such as HSV-related proteins, the animal's immune system is activated to mount a protective immune response. Because these exogenous proteins are produced in the animal's own tissue, the expressed proteins are processed by the major histocompatibility complex (MHC) in a manner analogous to that of a true HSV infection. The result, as shown in this invention, is the induction of an immune response against HSV. Polynucleotides with the purpose of generating immune responses to the encoded protein are referred to herein as polynucleotide vaccines or PNVs.

There are many forms of implementation of this invention, as those skilled in the art will see from the specification. Various transcriptional promoters, terminators, vector carriers or specific gene sequences can thus be successfully used.

This invention provides a method for the use of polynucleotides which, after being introduced into mammalian tissue, stimulate the in vivo expression of the polynucleotide, thereby producing the encoded one. protein. Those skilled in the art will appreciate that variations can be made in the nucleotide sequence that encodes a protein, which changes the amino acid sequence of the encoded protein. An altered expressed protein can have an altered amino acid sequence and still elicit antibodies that react with the viral protein and are considered functional equivalents. In addition, full-length gene fragments can be constructed that encode parts of the full-length protein. These fragments may encode a protein or peptide that elicits antibodies that react with the viral protein and are considered functional equivalents.

In one embodiment of the present invention, the gene encoding the HSV gene product is inserted into an expression vector. The vector contains a transcriptional promoter recognized by eukaryotic RNA polymerase and a transcriptional terminator at the end of the coding sequence of the HSV gene. In a preferred embodiment of the invention, the promoter is the cytomegalovirus promoter with an intron-aA sequence (CMV-intA), although those skilled in the art will recognize that any of a number of other promoters can be used such as strong immunoglobulin or other eukaryotic promoters.

A preferred transcriptional terminator is a bovine growth hormone terminator. A CMV-intA-BHG terminator combination is preferred. Additionally, to aid in the preparation of polynucleotides in prokaryotes, an antibiotic resistance marker is optionally incorporated into the expression vector under the transcriptional control of a suitable prokaryotic promoter. Genes for resistance to ampicillin, neomycin or other suitable antibiotic resistance markers can be used. In a preferred embodiment of the invention, the antibiotic resistance gene encodes a neomycin resistance gene product. Furthermore, to support the intensive production of polynucleotides in prokaryotic cells, it is good that the vector has replication of prokaryotic origin and is made of a large number of copies. Any of the commercially available prokaryotic cloning vectors possess these elements. In a preferred embodiment of the present invention, this functionality is provided by commercially available vectors known as the pUC series. However, it may be desirable to remove non-essential sequences. Thus, pUC sequences encoding 1acY and 1acI can be removed. It is also preferred that the vector cannot replicate in a eukaryotic cell. This minimizes the risk of polynucleotide vaccine sequences being incorporated into the recipient's genome.

In a further embodiment, the expression vector pnRVS is used, where the long terminal repeat (LTR) of the russo sarcoma virus (RSV) is used as a promoter. In the following implementation of the invention, V1, a mutated pBR322 vector in which the CMV promoter and the BGH transcriptional terminator are cloned, is used. In a preferred embodiment of the present invention, the V1 and pUC19 elements are combined to produce an expression vector called V1J. An HSV gene such as gD or any other HSV gene capable of inducing an anti-HSV immune response (antibodies or CTL) such as gB, gC, gL, gH and ICP27 is cloned into the V1J or other desired expression vector. In a further step, the ampicillin resistance gene is removed and replaced with a neomycin resistance gene to produce V1J-neo, in which any of the various HSV genes can be cloned according to the present invention. In the following embodiment, the vector is V1Jns, which is the same as V1J except that a unique Sfi1 restriction site is incorporated into a single Kpn site at position 2114 of V1J-neo. The occurrence of the Sfi1 site in the human genome is very low (about 1:100,000 bases). Thus, this vector enables careful monitoring of the integration of the expression vector into the host's DNA, simply by digesting the extracted genomic DNA. In the following implementation, the vector is V1R. In this vector, as much non-essential DNA as possible is excised to produce a highly compact vector. This vector allows the use of larger inserts, with less opportunity to encode unwanted sequences and optimizes uptake by cells when a construct encoding specific viral genes is introduced into the surrounding tissue. The methods used in the modification of the preceding vectors and in the development procedures can be achieved by methods known to those skilled in the art.

Those skilled in the art will recognize from this work that one of the applications of this invention is to provide an in vivo and in vitro testing and analysis system to correlate HSV sequence diversity with HSV neutralization serology, as well as other parameters. Isolation and cloning of these various genes can be accomplished by methods known to those skilled in the art. The present invention further provides a method for systematically identifying strains and sequences for vaccine production. Insertion of genes from primary isolates of HSV strains provides an immunogen that induces an immune response against clinical isolates of the virus and thus meets an unmet need in this field. Additionally, if the viral isolate changes, the immunogen can be modified to reflect the required sequences.

In one embodiment of the present invention, the gene encoding the HSV protein is directly bound to a transcriptional promoter.

The use of tissue-specific promoters or enhancers, eg, the muscle creatine kinase (MCK) enhancer element, can be useful to restrict polynucleotide expression to a specific tissue type. For example, myocytes are terminally differentiated cells that do not divide. Integration of foreign DNA into chromosomes seems to require both cell division and protein synthesis. Thus, it may be desirable to limit protein expression to non-dividing cells, such as myocytes. However, the use of the CMV promoter is adequate to achieve expression in many tissues into which PNV is introduced.

Summary of the PNV construction

HSV and other genes are preferentially bound to an expression vector that has been adapted specifically for polynucleotide vaccines. Parts include a transcriptional promoter, immunogenic epitopes, and additional cistrons encoding immunostimulatory or immunomodulatory genes, with their promoters, transcriptional terminators, bacterial origin replication, and antibiotic resistance genes, as described. Optionally, the vector may contain internal ribosome entry sites (IRES) for the expression of polycistronic mRNA. It will be clear to those skilled in the art that within the scope of this invention lies RNA transcribed in vitro to obtain polycistronic mRNAs encoded by the corresponding DNA. For this purpose, it is preferable to use strong RNA polymerase promoters such as T7 or SP6 and perform "run on" transcription with a linearized DNA template. These methods are well known in the art.

The protective efficacy of polynucleotide HSV immunogens against the next challenge of viral infection was demonstrated by immunization with the DNA of this invention. This is prestigious because no infectious factor is used, no set of viral particles is required, and the selection of determinants is enabled. Furthermore, since the sequences of the viral gene products can be conserved in various HSV strains, a consequent protection against infection with other HSV strains is achieved.

Injection of a DNA expression vector encoding gD can lead to significant protective immunity against subsequent exposure to the virus. In particular, gD-specific antibodies and CTLi can be generated. An immune response directed against conserved proteins can be effective despite shifting antigens and derivation of variable proteins. Since each of the HSV gene products shows some degree of conservation among different HSV strains and the subsequent immune response can be generated in response to intracellular expression and processing of MHC, it is expected that many different HSVgD PNV constructs could lead to cross-reactivity of immune responses.

The invention provides a method of inducing heterologous protective immunity without the need for self-propagating agents or adjuvants. The generation of high titer antibodies against the expressed proteins after injection of viral protein and human growth hormone DNA, /Tang et al., Nature 356, 152, 1992/, shows that this is an easy and highly effective way to obtain antibody-based vaccines, either separately or in combined with cytotoxic T-lymphocyte vaccines directed against conserved antigens.

The ease of making and purifying DNA constructs compares favorably with protein purification, encouraging the development of combination vaccines. So they can be made, mixed and given together. In addition, protein expression was maintained after DNA injection /H.Lin et al., Circulation 82,2217 (1990); R.N. Kitsis et al., Proc Natl Acad Sci (USA) 88, 4138 (1991); E. Hansen et al., FEBS Lett. 290, 73, (1991); S. Jiao et al., Hum Gene Therapy 3, 21, (1992); I. Wolff et al., Human Mol Genet 1, 363, (1992)/ the durability of B and T memory states can be extended / D. Gray and P. Matzinger, J Exp Med 174, 969, (1991); S. Oehen et al., ibidem 176, 1273(1992)7, thereby inducing long-term humoral and cellular immunity.

The amount of expressible DNA or transcribed RNA that should be introduced into a vaccine recipient will depend on the strength of the transcriptional and translational promoters used. The strength of the immune response may depend on the level of protein expression and on the immunogenicity of the expressed gene product. A generally effective dose of about 1 ng to 5 mg, preferably about 10 µg to 300 µg, is administered into muscle tissue. It is also suitable for subcutaneous, intradermal, injection, pressing through the skin and other forms of administration such as intraperitoneal, intravenous or inhalation administration. It is thought that a booster vaccination can also be provided. Booster revaccination with HSV protein immunogens such as g D, gB, gC, gG and gH gene products after vaccination with HSV polynucleotide immunogen has also been considered. Parenteral administration such as intravenous, intramuscular, subcutaneous or otherwise, interleukin-12-protein, compared with or after parenteral administration of the PNV of this invention may have advantages.

The polynucleotide can be naked, ie unbound with any protein, adjuvant or other agent that could act on the recipient's immune system. In this case, it is preferred for the polynucleotide to be in a physiologically acceptable solution, such as, but not limited to, sterile saline or sterile buffered saline. Alternatively, the DNA may be bound to liposomes such as lecithin liposomes or others known in the art, such as a DNA-liposome mixture, or the DNA may be bound with an adjuvant known in the art to enhance the immune response, such as a protein or other carrier. Substances that participate in the entry of DNA into the cell can also be used, such as, but not only, calcium ions. These substances are generally referred to herein as transfection promoting substances and pharmacologically acceptable carriers. Techniques for coating microprojectiles with polynucleotide are known in the art and also useful in conjunction with the present invention. For DNA intended for use in humans, it may be useful for the final DNA product to be in a pharmacologically acceptable carrier or buffer solution. Pharmacologically acceptable carriers are known in the art as well as buffer solutions and include those described in numerous texts such as those in Remington with Pharmaceutical Sciences.

In a further embodiment, the invention is a polynucleotide comprising continuous nucleic acid sequences that can be expressed and give a gene product after introduction of the respective polynucleotide into eukaryotic tissue in vivo. The encoded gene product acts primarily as an immunostimulant or antigen capable of stimulating an immune response. Thus, the nucleic acid sequences in this embodiment encode an epitope of the human herpes simplex virus and optionally a cytokine or T-cell costimulatory element such as a member of the B7 family of proteins.

There are several advantages of gene vaccination over the same with a gene product. The first is the relative ease with which a natural or near-natural antigen can be presented to the immune system. Mammalian proteins recombinantly expressed on bacteria, yeast, or even mammalian cells must often be extensively treated to ensure adequate antigenicity. Another advantage of DNA immunization is the antigen's ability to enter the MHC class I pathway and elicit a cytotoxic T-cell response. Immunization of mice with DNA encoding influenza A nucleoprotein (NP) elicited a CD8+ response to the NP that protected the mouse when challenged with heterologous influenza virus strains. (Montgomerv, D.L., et al., supra; Ulmer, J., et al., above).

There is evidence that cellular immunity is important in controlling HSV infection / for review see Nash, A.A. et al., 1985, Herpesviruses, Vol. 4, Plenum, New York, and Schmidt, D.S. et al. 1992, in Rouse(ed,) Current topics in Microbiology and Immunology, Vol.179, Herpes Simplex Virus; Pathogenesis Immunobiology and Control, Springer Verlag Berlin/. While the majority of HSVCTLa isolated from seropositive patients were of the CD4+ type ( Schmidt, D.S. et al., 1988, J Immunol , 140, 3610-3616; Tsutsumi, T. et al., 1986, Clin Exp Immunol, 66, pp. 507- 515) were isolated and CD8+ clones included one specific for gD. (Torpez, D.J. et al., 1989, J Immunol, 142, 1325-1332; Yasukawa, M. et al., 1989, J Immunol, 143, p. 2051-2057; ZarlingJ.M. et al., 1986, J Immunol 1986, J Immunol, 136, 4669-4673) In mice, cell transfer and depletion experiments suggest that some CD8+ protect against infection (Bonneua, R.H. et al., 1989, J Virol, 63, 1480-1484; Nash, AA et al., 1987, Gen Virol, 68, 825-833) Immunization with gD by infection with recombinant viral vectors (Paoletti, E. et al., 1984, Proc Natl Acad Sci, USA, 81, 193-197; Wachsman, M.L. et al., 1987, J Infect Dis , 155, 1188-1197; Zheng, B. et al., 1993, Vaccine, 11, 1191-1198) protects mouse from HSV infection. Live viral vectors such as DNA have the ability to present antigens in addition to MHC class I. However, a recent study using HSV gD vaccine recombinant infection found that protection from infection was dependent on a delayed-type hypersensitivity reaction, the function of L3T4+ cells. (Wachsman, M. et al, 1992, Vaccine, 10, 447-454) Although gD-specific CD8+ cells have been isolated from HSV-infected mice, their role in limiting infection is unknown. (Johnson, R.M., et al., 1990, J Immunol, 145, 702-710) Work by Koelle et al. suggests that HSV infection can render human fibroblasts and keratinocytes unrecognizable to CD8 + CTL (Koelle D.M., et al, 1993, J Clin Invest., 91, 961-968). In natural infection with HSV, the role of CD8 + cells in general and the role of CD8 + cells in the response to gD is not resolved.

Since DNA immunization can stimulate both cellular and humoral immunity, its greatest advantage may be that it provides a relatively simple method for testing the vaccination potential of a large number of viral genes. Plasmids expressing HSV-2 glycoproteins B and C also raise the level of neutralizing antibodies and thereby protect mice from lethal infection. However, ICP27, which is known to generate a CTL response and provide some degree of protection in mice immunized with recombinant ICP27-vaccinia virus (Banks, T.A. et al., 1991, J Virol, 65, pp. 3185-3191) did not provide protection against lethal infection. with HSV when mice were vaccinated only with PNV ICP27. However, the DNA encoding ICP 27 may be useful as a component of multigene-HSV PNV and the present invention is intended to encompass ICP 27 as a component of multivalent HSV PNV.

Immunization with DNA injection also allows, as mentioned, easy assembly of multicomponent vaccines. Simultaneous vaccination with multiple influenza genes has recently been reported (Donnelly, J. et al., 1994, Vaccines, in press). The inclusion of genes whose products activate certain parts of the immune system in the HSV vaccine can also ensure thorough protection against subsequent viral infection.

The following examples are provided to illustrate the present invention, but are not limited thereto.

EXAMPLE 1

Vectors for vaccine production

A) V1

The V1 expression vector was constructed from pCMVIE-AKI-DHFR / Y. VVhang et al., J Virol, 61, 1796 (1987)/. AKI and DHFR genes were removed by cutting the vector with Eco RI and self-ligating. This vector does not contain intron A in the CMV promoter, so it was added as a PCR fragment that had a deletion of the internal Sac I site / at 1855 as counted by B.S. Chapman et al., Nuc Acids Res 19,3979 (1991)/ The matrix used for PCR reactions was bilapCMVintA-Lux, created by ligation of Hind III and Nhe I fragments from pCMV6a120 / see B.S. Chapman et al., ibid./ which includes the hCMV-IE1 enhancer/promoter and intron A, in the Hind III and Xba I sites of ppBL3 to give pCMVIntBL. A fragment of 1881 base pairs of the luciferase gene fragment (HindIII-Sma I loaded according to Klenovv) from RSV-Lux /J.R. de Wet et al. Mol Cell Biol 7, 725, 1987/ was cloned into the Sal I site of pCMVIntBL, which was filled according to Klenow and treated with phosphatase.

Examples that include intron A are:

5' example, SEQ.ID:1:

5'-CTATATAAGCAGAG CTCGTTTAG-3'; 3'-example, SEQ ID:2:

5'-GTAGCAAAGATCTAAGGACGGTGA CTGCAG-3'

Examples used to remove the Sac I site are:

sense example, SEQ.ID:3:

5'-GTATGTGTCTGAAAATGAGCGTGGAGATTGGGCTCGCAC-3'

antisense example, SEQ ID:4:

5'-GTGCGAGCCCAATCTCCACGCTCATTTTCAGACACA TAC-3

The PCR fragment was cut with Sac I and Bgl II and inserted into a vector that had been cut with the same enzymes.

B) V1J Expression Vector

The purpose of creating V1J is to remove promoter and transcription termination elements from the V1 vector with the intention of placing them in a more specific context, creating a more compact vector, and improving plasmid purification.

V1J was derived from the V1 vector and pUC18, a commercially available plasmid. V1 is digested with SspI and EcoRI restriction enzymes, yielding two DNA fragments. The smaller one, which contains the CMVintA promoter and Bovine Growth Hormone (BGH) transcription termination elements that control heterologous gene expression, is purified from agarose gel electrophoresis. The ends of these DNA fragments are then "blunted" using the T4 DNA polymerase enzyme with the intention of promoting its binding to the other "blunted" end of the DNA fragment.

PUC18 was chosen to provide the "backbone" for the expression vector. It is known to produce large amounts of plasmid, has well-defined sequence and function, and is small. The entire lac operon was removed from this vector by partial digestion with HaeII restriction enzyme. The remaining plasmid was purified from agarose gel for electrophoresis, its ends were blunted with T4DNA polymerase treated with calf intestinal alkaline phosphatase and ligated to the CMVintA/BGH element described above. Plasmids with one of two possible orientations of the promoter element within the pUC base were obtained. One of these plasmids produced much more DNA in E. coli and was designated V1J. This vector structure was confirmed by sequence analysis of the junction region and was subsequently confirmed to give comparable or higher expression of heterologous genes than V1.

C) V1Jneo Expression Vector,

It was unavoidable to remove the ampr gene used for antibiotic selection of V1J carrier bacteria strains because ampicillin can be undesirable in large fermenters. The ampr gene was removed from the pUC base V1J with the help of restriction enzymes by digestion with SspI and Eam 11051. The remaining plasmid was purified by electrophoresis on agarose gel, its ends were blunted with T4 polymerase, and then treated with alkaline phosphatase from calf intestine. The commercially available kanr gene, derived from transposon 903 and contained in the pUC4K plasmid, was excised with PstI restriction enzyme, purified on agarose gel electrophoresis and then blunted with T4 DNA polymerase. This fragment was ligated with the V1J base and plasmids with the kanr gene in both orientations were obtained, designated as V1Jneo # 1 and 3. Each of these plasmids was verified by restriction enzyme analysis, DNA sequencing of the junction regions, and was shown to produce similar amounts plasmid as V1J. Expression of heterologous gene products in these V1Jneo vectors was also comparable to V1J. V1Jneo#3, hereinafter V1Jneo, which has a kanr gene in the same orientation as the ampr gene in V1J, was selected as an expression construct.

D) V1Jns Expression vector

Added Sfi I site to V1Jneo to facilitate integration testing. A commercially available Sfi I linker of 13 base pairs was added to the Kpn I site within the BGH sequence of the vector (New England BioLabs). V1Jneo was linearized with Kpn I, gel purified, blunt-ended with T4 DNA polymerase, and ligated to a blunted Sfi I linker. Clonal isolates were selected by restriction mapping and confirmed by sequencing through the junction. The new vector is labeled V1Jns. The expression of heterologous genes in V1Jns (with Sfi I) was comparable to the expression of the same in V1Jneo (with Kpn I)

E) V1Jns-tPA

In order to obtain a heterologous peptide leader sequence for secretory or membrane proteins, V1Jns was modified to include the human tissue-specific plasminogen activator (tPA) leader sequence. The two synthetic complementary oligomers were hybridized and then ligated into BgIII-treated V1Jns. Sense and antisense oligomers were: 5'-GATC ACC ATG GAT GCA ATG AAG AGA GGG CTC TGC TGTGTG CTG CTG CTG TGT GGA GCA GTC TTC GTT TCG CCC AGC GA-3', SEQ.ID:5:, and 5'- GAT CTC GCT GGG CGA AAC GAA GAC TGC TCC ACA CAG CAG CAG CAC ACA GCA GAG CCC TCT CTT CAT TGC ATC CAT GGT-3',SEQ.ID:6. In the sense oligomer, the Kozak sequence is underlined. These oligomers have free bases suitable for binding to sequences generated by cleavage with BgIII. After binding, the upstream BgIII site is destroyed, while the downstream one is retained for further binding. The splice site as well as the complete leader sequence are verified by DNA sequencing. Additionally, with the intention of matching the consensus-optimized vector V1Jns ( = V1Jneo with an Sfil site), an SfiI restriction site is inserted into the KpnI site in the BGH terminator region of V1Jn-tPA by blunting the KpnI site with T4 DNA polymerase followed by ligation with an SfiI linker ( catalog #1138, New England Biolabs). This modification was confirmed with restriction digestion and electrophoresis on agarose gel.

F) pGEM-3-X-IRES-B7

(where X = any antigenic gene) As an example of a discistronic construct for a vaccine that provides coordinated expression of a gene encoding an immunogen and a gene encoding an immunostimulatory protein, the mouse B7 gene was amplified from the B lymphoma cell line CH 1 (obtained from ATCC) by PCR- om. B7 is a member of a family of proteins that provide essential costimulation in T cell activation by antigen within the major histocompatibility complex I and II. CH I cells are a good source of B7 mRNA because they have the phenotype of being constitutively activated and B7 is expressed primarily in activated antigen-presenting cells such as cells and macrophages. These cells are further stimulated in vitro using cAMP or IL-4 and mRNA is prepared using standard guanidine thiocyanate procedures. cDNA synthesis was performed using mRNA, using the Gene Amp RNA PCR kit (Perkin-Elmer Cetus) and primer (5'- GTA CCT CAT GAG CCA CAT AAT ACC ATG-3', SEQ.ID:7:) specific for B7 located downstream of the B7 translational open reading frame. B7 was amplified by PCR using the following sense and antisense oligomers: 5'-GGT ACA AGA TCT ACC ATG GCT TGC AAT TGT CAG TTG ATG C-3', SEQ.ID:8:, and 5'-CCA CAT AGA TCT CCA TGG GAA CTA AAG GAA GAC GGT CTG TTC-3', SEQ.ID:9:. These oligomers provide BgIII restriction enzyme sites at the ends of the insertion as the Kozak translation initiation sequence has an NcoI restriction site and an additional NcoI site located just before the 3' terminal BgIII site. NcoI digestion yielded a fragment suitable for cloning into pGEM-3-IRES-B7, which was digested with NcoI. The resulting vector, pGEM-3-IRES-B7, contains an IRES-B7 cassette that can be easily transferred to V1Jns-X, where X represents the antigen-encoding gene-

G) pGEM-3-IRES-GM-CSF

(where X is any antigenic gene) This vector contains a cassette analogous to that described in C above except that the gene for the immunostimulatory cytokine, GM-CSF, is used instead of B7. GM-CSF is a macrophage differentiation and stimulatory cytokine that has been shown to stimulate potent antitumor activities of T cells in vivo / G., Dranof et al., Proc Natl Acad Sci USA, 90, 3539 (1993).

H) pGEM-3-X-IRES-IL-12

(where X is any antigenic gene) This vector contains a cassette analogous to that described in paragraph C, except that the gene for the immunostimulatory cytokine IL-12 is used instead of B7. IL-12 has been shown to have an effect on directing the immune response towards cellular, dominated by T-cells, towards humoral./L. Alfonso et al., Science, 263, 253, 1994/.

EXAMPLE 2

Preparation of the V1R vector

In an attempt to improve the basic vaccination vector, a derivative of the V1Jns vector, designated V1R, was created. The purpose of this vector construction was to obtain the smallest possible vaccine vector, without unnecessary DNA sequences, which would still retain the optimized characteristics of heterologous gene expression and high plasmid yields achieved by V1J and V1Jns. From the literature as well as from the experiment, it was determined that: 1) the regions within the pUC base that have the base of replication of E.Coli can be removed without affecting the plasmid obtained from the bacteria (2) the 3'-region of the kanr gene that follows the open reading frame , can be removed if the bacterial terminator is returned to the site; and, (3) approx. 300 bp from the 3' end of the BGH terminator can be removed without affecting its regulatory function (it follows the original site of the restriction enzyme KpnI in the BGH element).

V1R was constructed using PCR to synthesize three segments of DNA from V1Jns representing the CMVintA promoter/BGH terminator, origin of replication, and kanamycin resistance elements. Using PCR oligomers, restriction enzymes unique to each segment were added to each end of the segment: SspI and XhoI for CMVintA/BGH; EcoRV and BamHI for the kanr gene; i, Bcll and Sall for orir. These enzyme sites were chosen because they allow directed ligation of PCR-derived segments with consequent loss of each site. EcoRV and SspI leave blunt end sites compatible for binding, while BamHI and Bcll leave complementary free bases as do SalI and XhoI. After obtaining these segments by PCR, each segment is treated with the appropriate restriction enzyme indicated above and then they are linked to each other in one common reaction mixture containing all three DNA segments. The 5'-end orir was designed to include a T2 rho-independent terminator sequence normally found in this region so that it can provide terminator information for the kanamycin resistance gene. The ligated product is checked by digestion with restriction enzymes (>8 enzymes) as well as by DNA sequencing of the binding compounds. DNA. Plasmid DNA yields and heterologous expression using viral genes in VIR are similar to those of V1Jns. The net reduction in vector size was 1346 bp (V1Jns = 4.86 kb; V1R = 3.52 kb).

PCR oligomer sequences used to synthesize V1R (restriction enzyme sites indicated in parentheses after the sequence are underlined):

(1) 5'-GGT ACA AAT ATT GG CTA TTG GCC ATT GCA TAC G-3'(Sspl), SEQ.ID:10:

(2) 5'-CCA GAT CTC GAG GAA CCG GGT CAA TTC TTC AGC ACC-3'(Xhol), SEQ.ID:11:

(for CMVintA/BGH segment)

(3) 5'-GGT ACA GAT ATC GGA AAG CCA AGT TGT GTC TCA AAA TC-'3 (EcoRV), SEQ.ID:12:

(4) 5'-CCA GAT GGA TCC G TAA TGC TCT GCC AGT GTT ACA ACC-3'(BamHI), SEQ.ID:13:

(for the kanamycin resistance gene segment)

(5) 5'-GGT ACA TGA TCA CGT AGA AAA GAT CAA AGG ATC TTC TTG-3' (Bcll), SEQ.ID:14:,

(6) 5'-CCA CAT GTC GAC CC GTA AAA AGG CCG CGT TGC TGG-3'(Sall), SEQ.ID:15:

(part to start the replication of E. Coli)

EXAMPLE 3

Cells, viruses and cell cultures

VERO, BHK-21 and RD cells were obtained from ATCC. Virus was routinely obtained by infection of nearly confluent VERO and BHK cells at multiplicity of infection (m.o.i.) at 37°C in a small volume of medium without fetal bovine serum (FBS). After one hour, the viral inoculum is removed and cultured again in high-glucose DMEM supplemented with 2% heat-activated FBS, 2mM L-glutamine, 25mM HEPES, 50 U/ml penicillin and 50 µg/ml streptomycin. Incubation was continued until the cytopathic effect was very pronounced: usually 24-48 hours. Viruses from the cells were collected by centrifugation at 1800 x g / 10 minutes at 4°C. The supernatant with viruses was clarified by centrifugation at 1800x g / 10 min / 4°C.

EXAMPLE 4

Cloning and preparation of DNA

HSV-2 (Curtis) DNA for use as a template for PCR was prepared from a nucleocapsid isolated from an infected VERO cell. (Denniston, K.J. et al., 1981, gen, 15, p.365-378) Synthetic oligomers corresponding to the 5' and 3' end sequences for the HSV2 gB, gC, gD, or ICP27 genes, containing BgII restriction sites for recognition (Midland Certified Reagent Companv; Midland Texas) were used at 20 pmol each. The 1.1 kb fragment encoding the gD gene was amplified by PCR (Perkin Elmer Cetus, La Jolla) according to the manufacturer's specification, except that instead of dGTP, dGTP:dGTP dease was used in a 1:4 ratio, and 3 mM MgCl2- HSV was added to the buffer. -2 genomic DNA matrix was used in the amount of 100ng/100µl reaction mixture. The PCR-amplified fragments were digested with Bgl II and then ligated into the Bgl II-treated dephosphorylated V1J vector (Montgomerv, D.L. et al., supra). E. Coli DH5α (BRL-Gibco, Gaithersburg, Md.) was transformed according to the manufacturer's specification. Colonies resistant to penicillin were screened by hybridization with a 32p-labeled 3'PCR primer. Candidate plasmids were identified by restriction mapping and sequencing of vector junctions and inserts, using the Sequenase DNA Sequencing Kit, version 2.0 (United States Biochemical). A 2.7 Kb fragment encoding the gB gene, a 1.5 Kb fragment encoding the gC gene, and a 1.6 Kb fragment encoding the ICP27 gene were similarly amplified. Independently produced isolates were identified and indicated for the presence of the correct DNA construct containing the gB, gC, gD or ICP27 gene.

Large-scale DNA production was essentially as previously described (Montgomerv, D.L. et al., supra), except that 800 ml cultures were incubated for 24 to 48 hours, and for some experiments DNA was purified by a single CsCl-EtBr isosphincic gradient centrifugation. density.

Plasmid constructs were indicated by restriction mapping and sequence analysis of vector junctions and insertions. The results were consistent with published HSV-2 strain G sequence data (Laskz, L.A. et al., 1984, DNA, 3, 23-29) and showed that the initiation and termination codons were intact in each construct.

EXAMPLE 5

Expression of HSV-2gB, gC, gD and ICP27 proteins from V1J plasmid

Rhabdomyosarcoma cells (ATCC CCL136) were grown the day before use to a density of 1.2x106 cells per 9.5 cm2 well on six-well tissue culture clusters in DMEM with high conc. glucose, supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 25 mM HEPES, 50 U/ml penicillin, and 50 µg/ml streptomycin (all from BRL Gibco). Phenol:chloroform extracted, cesium chloride purified plasmid DNA was precipitated with calcium phosphate using Pharmacia CellPhect reagents according to kit instructions except that 5-15µg was used for each 9.5 mm2 well of RD cells. Cultures were treated with glycerol six hours after the addition of calcium-phosphate-DNA precipitate; after refeeding, cultures were incubated for two days before collection.

Lysates of transfected cultures were prepared in 1 X RIPA (0.5% SDS, 1.0% TRITON X-100, 1.0% sodium deoxycholate, 1 mM EDTA, 150 mM NaCl, 25 mM TRIS-HCl pH 7.4) supplemented with 1µM leupeptin, 1µM pepstatin, 300mM aprotinin and 10(aM TLCK and briefly sonicated to reduce viscosity. Lysates were separated by electrophoresis on 10% Tricine gels (Novex) and then transferred to nitrocellulose membranes. Immunoblotting was done with HSV-2 convalescent mouse sera and developed with an ECL detection kit (Amersham).

HSVgD expression from V1J:gD was demonstrated by transient transfection of RD cells: Lysates of transfected or mock-transfected V1J:gD cells were fractionated by SDS PAGE and analyzed by immunoblot. Figure 1A shows that V1J:gD transfected RD cells express an immunoreactive protein that appears to have a molecular weight of about 55K. Lysates of HSV-2 (Curtis), HSV-"(186), or mock-infected Vero cells are shown for comparison. Identical migration of cloned gD and the authentic protein from the infected cell is the full-length protein and is processed and glycosylated similarly to gD in HSV - the infected cell.

Indirect immunofluorescence of fixed V1J:gD cells shows a diffuse cytoplasmic signal.

Expression of HSV gB in V1JNS:gB was demonstrated by transient transfection of RD cells. Lysates of V1J:g B -transfected or mock-transfected residues were fractionated by SDS PAGE and analyzed by immunoblot. Figure 1B shows that V1JNS:gb transfected RD cells express an immunoreactive protein with a molecular weight of about 140 k. Lysates from HSV-2(Curtis), HSV 2 (186), or mock-infected cells were included for comparison. Similar migrations of the cloned gB and the authentic protein from the infected cell indicate that the protein is full length. Indirect immunofluorescence of fixed V1JNS:gB transfected cells shows a punctate membrane-bound signal. Expression of HSV gC in V1JigC was demonstrated by transient transfection of RD cells. Indirect immunofluorescence of fixed V1J:gC transfected cells showed a primarily diffuse cytoplasmic signal.

ICP27 expression was demonstrated by transient transfection of RD cells, followed by Western blot analysis. Mouse monoclonal antibodies specific for ICP27 detected a protein of about 60 kDa, which is consistent with the major immunoreactive protein in cells infected with HSV 2 (Figure 1C).

EXAMPLE 6

Immunization with PNV and detection of anti-HSV antibodies

Five- to six-week-old female BALB/c mice were anesthetized with a mixture of 5 mg ketamine HCl (Aveco, Fort Dodge, IA) and 0.5 mg xylazine (Mobley Corp., Shawnee, KS.) in saline. Their hind legs were shaved with electric scissors and washed with 70% ethanol. The animals were injected with a total of 100 µl of DNA dissolved in physiological solution: 50 µl in each leg.

The ability of V1J:gD DNA to induce an immune response to HSV gD was first tested in a titration experiment. Groups of ten mice each received doses of i.m. injection of DNA ranging from 200 µg to 0.78 µg (8-fold dilutions) or were mock immunized with saline. Antibodies obtained 4-6 weeks after immunization were analyzed by ELISA. For ELISA, HSV-2 glycoprotein was diluted to 5 µg/ml in 50 mM carbonate buffer pH 9.5. Nunc Maxi-sorb flat-bottom 96-well plates were coated overnight at 4°C with 100 µl HS V-glycoprotein per well. Plates were washed four times with PBS pH 7.2 and non-specific reactivity was reduced with blocking and dilution buffer, 20 mM TRIS-HCl pH 7.5, 137 mM NaCl, 2.7 mM KCl, 0.5% gelatin, 0.05% Tween 20 for one hour at room temperature. Serial dilutions of mouse sera were added and the plates were incubated at room temperature for one hour. Plates were washed four times with PBS and once with distilled water before addition of alkaline phosphatase-labeled goat anti-mouse IgG (Boehringer Mannheim, Inedianapolis, IN) and incubated for one hour at room temperature. Excess secondary antibodies were removed by four washes with PBS followed by one with distilled water. ELISA was developed by adding 100 µl per well of 1 mg/ml p-nitrophenylphosphate in 10% diethanolamine pH 9.8 100 µg/ml MgCl·6 H2O at 37°C. Absorbance was read at 405 nm and serum dilutions were counted as positive if the OD405 was greater than the mean plus three standard deviations of the same control solution. Over four weeks, the majority of animals receiving ≥ 6.25 µg of DNA were positive. At doses lower than 6.25 µg, seroconversion occurred in a smaller number of animals, but even at the lowest doses, some animals were ELISA positive. None of the animals that received the saline solution tested positive. After six weeks, the majority of animals became seropositive.

At the seventh week, the animals were reimmunized with the same doses of DNA (or saline) used during the first administration. Sera were collected at the tenth week (three weeks after the secondary injection) and the final titers were determined by ELISA. The results are summarized in Table 1. At the tenth week, 93% of the injected mice were seropositive. Even at a dose of 0.78 µg, eight out of ten animals were positive.

Seroconversion of mice immunized with V1J:gD DNA

[image]

a- Mice were immunized at week seven with the indicated amount of DNA. Sera were collected at week 10 and screened as described in the text.

b- For the purpose of calculating GMT, sera negative at the lowest dilution tested were assigned values equal to one dilution less, ie, 1:10.

To confirm that the ELISA reactivity was due to anti-gD antibodies, several sera with high ELISA titers were indicated by their reactivity with an immunoblot of HSV-infected or mock-infected cell lysates. Figure 2A shows that serum from a V1J:gD immunized mouse reacts specifically with a single HSV-encoded protein whose electrophoretic mobility matches that of HSV gD. Overall, these data indicate that i.m. injection of V1J:gD DNA into a mouse results in the expression of the gD epitope and the generation of an immune response to the gD protein.

To obtain additional data and determine the minimum effective dose of PNV, V1J:gD was further titrated in an experiment where animals were immunized only once. Groups of mice were injected with V1J:gD DNA ranging from 5 ng to 50 µg. Sera collected four, seven and ten weeks after immunization were analyzed by ELISA; these data are collected in Figure 3.

This titration established a response threshold of about 0.5 µg of DNA. Although a few animals receiving the lower doses were positive by ELISA, the positive response was transient and occurred only at the lowest serum dilutions. At DNA doses of ≥ 1.67 µg, seroconversion occurred in more than 90% of animals after 4 weeks, which remained positive at weeks seven and ten.

The increase in antibody titer in individual animals is reflected by the increase in group GMTs, which can be seen in Figure 3. At doses ≥0.5 µg, GMTs increase sharply between the fourth and tenth weeks. Between the seventh and tenth week, titers rise or remain the same in all but one case. ELISA titers after ten weeks in animals receiving ≥0.5 µg are similar to those obtained in the previous experiment where a second dose of DNA was given. The amount of PNV sufficient to elicit an immune response was 100-fold less than published reports using similar vectors. (Robinson et al., 1993, Vaccine, 11, pp. 957-960; Fynan et al., 1993, Proc Natl Acad Sci USA, 90, pp. 11478-11482).

In order to establish a standardized protocol, immunizations with one and two doses were compared. In a two-dose experiment, no significant difference in protection was found between the highest (200 µg) and the lowest (0.78 µg) dose. When the titration was extended to a single-dose experiment, a dose-response was observed for the GMT ELISA, demonstrating efficacy in seroconversion and protection. The threshold of these responses was around 0.5 µg. Although seroconversion occurs with amounts as small as 50 ng of DNA. In general, titers continued to rise for ten weeks after a single injection and in the two-dose experiment there was no apparent increase in m a titers after the second injection. Finally, at a dose of 50 µg of DNA, common to both experiments, there were no significant differences between the administration of one or two doses.

Similar analyzes to those shown above were performed for PNV constructs containing the HSV genes gB and gC. Mice (10 per group) were immunized as described above with DNA containing the HSV gB or HSV gC gene. Serum was collected and analyzed for the presence of anti-gB or anti-gC antibodies by the ELISA test described above. Anti-gB ELISA results are shown in Table 2, and show that mice immunized with V1JNS:gB are seropositive for anti-gB antibodies.

[image]

ELISA results for gC antibodies are shown in Table 3, and show that mice immunized with V1J:gC are seropositive for anti-gC antibodies.

[image]

To confirm that the ELISA reactivity for gB was due to the presence of anti-gB antibodies, several sera with high ELISA titers were indicated by their immunoblot reactivity with HSV-infected or mock-infected cell lysates. Figure 2B shows that sera from mice immunized with V1JNS:gB react specifically with a single HSV-encoded protein with an electrophoretic mobility consistent with that of HSV gB. Overall, these data show that i.m. injection of HSV PNV mice leads to the expression of HSV epitopes and the development of an immune response to these HSV proteins.

EXAMPLE 7

HSV Neutralization

Mouse sera were heat-inactivated at 56°C for 30 minutes, before serial dilution in DMEM, 2% heat-inactivated FBS, after which 50 µl of each dilution was placed in duplicate wells of a sterile 96-well plate. (Marsh Biomedical, Rochester, N.Y.) . Batches of HSV1 or HSV2 were then diluted to 4000 pfu/ml, then 50 µl of virus was added to each assay well and the plate was incubated overnight at 4°C. 50 µl of guinea pig complement (Cappel) diluted in DMEM, 2% heat-inactivated FBS was added to each well. After one hour of incubation at 37°C, 100 µl of serum-free medium was added to each well and each reaction mixture was used to infect confluent VERO cells in 12-well cluster plates (Costar). Neutralized virus samples were adsorbed for one hour at 37°C. The inocula were carefully aspirated and covered in a monolayer with 1 ml of 0.5% carboxymethylcellulose 1X MEM 5% heat-inactivated FBS, 10 mM 1-glutamine, 25 U/ml penicillin, 25 µg/ml streptomycin, 12.5 mM HEPES. The plates were incubated at 37°C for 48 hours. The coverslip was removed and cell monolayers were stained with 1% basic fuchsin, 50% methanol, and 10% phenol. Plaques were counted and the neutralization titer determined as the serum dilution leading to a 50% reduction in the number of plaques compared to a mock-immunized mouse.

To determine whether anti-gD antibodies may be biologically active, the HSV-2 neutralizing activity of high-titer post-week 10 serum from mice immunized at week 0 and week 7 was examined. The results of the plaque reduction assay are shown in Table 4. Sera from mice immunized with V1J:gD neutralized not only HSV-2(Curtis) but also HSV-2(186). Moreover, at least some sera contained neutralizing antibodies of the common type, as evidenced by their neutralization of HSV-1(KOS) infectivity. Although neutralization titers were low in some cases, these results prompted us to see if these anti-gD antibodies could protect animals from lethal HSV infection.

Ten-week serum from all animals immunized with ≥0.5 µg V1J:gD in a single-dose experiment were also examined by HSV-2 plaque reduction assay. Twenty-nine out of forty-nine tested sera were positive: >50% plaque reduction at 1:10 dilution. At the dose of 16.7 and 50 µg, nine out of ten sera from each group were neutralization positive.

[image]

EXAMPLE 8

HSV exposure test

challenge virus lots were prepared by infecting a confluent VERO monolayer with HSV-2 Curtis as previously described. Clarified virus supernatant was titrated on VERO cells and aliquots were stored at -70°C. Animals were infected i.p. injection with 0.25 ml of viral batch and then observed for three weeks. Survival data were analyzed using the log-rank test (McDermot et al., 1989, Virology, 169, p.244-247) in the SAS® procedure LIFETEST. Differences in probability ≤0.001 were considered highly significant.

Eleven weeks after the initial DNA injection, mice immunized with two doses of V1J:gD were challenged i.p. injection 105.7 p.f.u. HSV-2(Curtis) and observed for 21 days. Survival data are in Figure 4. It is immediately clear that animals immunized with as little DNA as 0.78 µg V1J:gD were significantly protected from lethal infection. Of the three animals that died that were immunized, two were seronegative by ELISA at the tenth week. Several surviving animals showed signs of transient illness including inability to groom, inability to move forward, or a hunched position. While the level of protection against death at that dose of DNA was significant (p≤0.01), these symptoms indicate that there was still some outbreak of infection. Analysis of sera obtained from convalescent animals was indicated by their reaction in immunoblots of lysates of HSV-2 infected VERO cells. In some cases the serum recognized only gD, and in others it reacted with many HSV proteins. These results are consistent with at least some mice being infected with HSV.

Animals immunized with a single dose were challenged as previously described. Survival data are shown in Figure 5. Statistically significant protection (p≤.001) from death was achieved in the group of animals receiving ≥1.67 µg V1J:gD. This dose of survival is similar to that seen in the ELISA titer (Fig.3). As in the two-dose experiment, the few surviving animals showed transient signs of disease during the observation period. Surviving animals immunized with higher doses of DNA (16.7 and 50 µg) remained trim and healthy-looking throughout the observation period.

Animals immunized with PNV constructs containing HSV gB or gC genes were also challenged with a lethal dose of HSV in the manner described earlier for gD. Survival data for animals immunized with V1JNS:gB are shown in Figure 6 and survival data for animals immunized with V1J:gC are shown in Figure 7 showing that protection from death was achieved.

These results demonstrate the potential of direct DNA immunization in the prevention of HSV infection. Using glycoprotein gD as a model, it was found that one i.m. injection of as little as 0.5 µg of V1J:gD DNA elicited a neutralizing antibody response to gD that achieved statistically significant protection against lethal HSV exposure. Immunization with only 3.13 ng of DNA in two doses protected the animals from death.

EXAMPLE 9

Vaccination of guinea pigs with HSV PNV

Hartley guinea pigs (Harlan Sprague Dawley Labs, Indianapolis, IN), each weighing about 200-250 g were vaccinated intramuscularly with 0.1 ml in the right thigh and 0.1 ml in the left thigh 11 and 4 weeks before exposure to the virus. Fresh vaccine and placebo solutions are sent to us for each vaccination.

In order to determine the production of antibodies to HSV-2 in animals, blood was taken from guinea pigs 5 weeks after the first and two weeks after the second vaccination. Blood was collected (0.6-1 ml per animal) by cutting off the toes. Blood was collected in microseparation tubes (Becton Dickinson) and later centrifuged at 1000xg for 10 min to separate the serum.

The collected guinea pig sera were analyzed by ELISA for the presence of anti-HSV antibodies as shown in Example 6. The results are shown in Table 5.

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At the time of infection, the weight of the guinea pigs was 600-700 grams. They were infected intravaginally with herpes simplex virus type 2 (HSV-2), strain E194. This was achieved through a three-stage process. First, the vagina of each animal was rubbed with a cotton applicator dipped in 0.1 N NaOH for 5 seconds. This procedure irritates the vaginal wall so that the infection is better received. Approximately 45-60 minutes later, each vagina was dry cleaned for 5 seconds. Then, an applicator dipped in viral medium (about 5x106 plaque-forming units of HSV-2 per milliliter) was used to rub each guinea pig for 20 seconds. The application was done by gently moving the applicator back and forth.

The number of lesions was determined 2-15 days after infection. A score of 1+ indicates that about 25% of the anus-vaginal area is affected. (usually redness immediately around the vagina); 2+ indicates that about 50% of the anal-vaginal area is affected; 3+ indicates 75% involvement; 4+ indicates 100% coverage. Since some animals died, the number of lesions was determined close to the day of death and observed until the 15th day. If this had not been done, the total number of lesions could have apparently decreased since the most severely affected animals died.

Deaths were recorded daily on the 21st day. In calculating the mean day of death, only dead guinea pigs were taken into account. The number of animals with limb paralysis was recorded throughout the infection. Vaginal virus titer was obtained by titration of viruses obtained from vaginal swabs, 2, 4, and 6 days after virus inoculation. Swabs were placed in test tubes with 1 ml of cell culture medium. Titration of these samples was performed on Vero cells in 96-well plates. Titers were calculated using the 50% final dilution method according to Reed L.J. and Muench M., Am J Hyg 27, 493-498 (1938). Viral titers are expressed as log10 infectious dose of cell culture per milliliter.

Statistical interpretation of survival (Fisher's exact test), median day of death (Mann-Whitney U test), paralysis (Fisher's exact test), vaginal viral titer (Mann-Whitney U test), and sum of vaginal lesions (Mann-Whitney In the test) were made by separate two analyses.

Figure 8 shows the results of survival, median day of death, paralysis, and vaginal viral titers in HSV-2-infected guinea pigs. High doses of vaccine prevented death and reduced vaginal virus titers on days 2 and 4 compared to the control group. High doses of the vaccine significantly prevented paralysis in these animals. Lower vaccine doses also reduced these indicators.

Table 7 shows daily collections of vaginal lesions from the experiment. Both lower and higher doses of the vaccine led to a significant reduction in the sum of vaginal lesions from 3 to 15 days of infection compared to the control placebo group. The results in Table 7 are graphically presented in Figure 9.

These results clearly show that the vaccine is protective for HSV-2 infected guinea pigs and that high doses are more effective than low doses. High doses of the vaccine could not completely prevent infection, since the virus was obtained from the vaccinated, and low-grade vaginal lesions also developed. Nevertheless, the degree of protection achieved by these doses was considerable. Antibody test results correlate with the degree of protection against the virus.

The vaccine given to guinea pigs intramuscularly, in two different doses 11 and 4 weeks before intravaginal exposure to HSV-2, significantly protected the animals from the disease. The high dose of the vaccine was more effective than the low dose. The vaccine appears safe in animals.

[image]

a Intramuscular vaccinations were given 11 and 4 weeks before exposure to the virus

b After viral inoculation

* P<0.05, ** P<0.01, *** P<0.001.

EXAMPLE 10

To determine whether mice vaccinated intramuscularly with PNV HSV would produce HSV-specific mucosal antibodies, mice were vaccinated with 12.5 or 1.56 µg of V1JNS:gD. Vaginal fluid was collected with a swab and antibodies were washed from the swab using phys. phosphate buffered solutions. The wash was tested for the presence of IgG and IgA specific for the HSV-2 protein. An ELISA was performed as previously described except that commercially available antibodies specific for mouse IgG (Boehringer) and specific for mouse IgA (Seralab) were used to detect the presence of HSV-specific IgG and IgA in mouse vaginal samples. The results for IgG are shown in Table 8; IgA was not found in any animal.

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The results show the presence of mucosal IgG specific for HSV-2 in mice vaccinated with V1J:gD.

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Claims (11)

1. A polynucleotide that induces the formation of anti-HSV antibodies or a protective immune response after introduction into vertebrate tissue, characterized by the fact that said polynucleotide contains one or more genes encoding one or more HSV proteins or their functional equivalents, and said genes are operatively linked to transcriptional promoter. 2. The polynucleotide of claim 1, characterized in that said gene encodes an HSV protein selected from the group consisting of gB, gC, gD, gH, gL, ICP27 and their functional equivalents. 3. A method for inducing the generation of an immune response in a vertebrate against an HSV epitope, characterized in that it comprises introducing between 1 ng and 5 mg of the polynucleotide of claim 1 into the vertebrate tissue. 4. A vaccine for stimulating immune responses, characterized in that it comprises the polynucleotide of claim 1 and a pharmacologically acceptable carrier. 5. A method for inducing immune responses against HSV, characterized in that it comprises the introduction of one or more isolated and purified HSV genes into vertebrate tissue, inducing an immune response that prevents HSV infection and/or alleviates HSV disease. 6. Polynucleotide, indicated by the fact that it includes: a) eukaryotic transcription promoter; b) an open reading frame operably linked to said promoter, which encodes one or more HSV epitopes and a translation termination signal; and c) optionally contains one or more operably linked IRES, one or more open reading frames encoding one or more additional genes, and one or more transcription termination signals. 7. The polynucleotide of claim 6, indicated by the fact that said additional genes from c) are immunomodulating or immunostimulating genes selected from the group consisting of GM-CSF, IL-12, interferon and members of the B7 family of T-cell costimulator proteins. 8. The polynucleotide of claim 6, characterized in that said HSV gene from a) encodes an HSV protein selected from the group consisting of gB, gC, gD, gH, gL, ICP27 and their functional equivalents. 9. The polynucleotide of claim 6, characterized in that said additional genes from c), HSV genes selected from the group consisting of gB, gC, gD, gH, gL, ICP27 and their functional equivalents. 10. A method for treating a patient, in need of such treatment, with a polynucleotide that promotes the formation of anti-HSV antibodies or a protective immune response after introduction into the vertebrate tissue, characterized by the fact that this polynucleotide contains a gene encoding one or more HSV proteins or their functional equivalents, and said gene is operatively linked to a transcriptional promoter. 11. The method of claim 10, characterized in that the respective polynucleotide contains a gene encoding one or more HSV proteins selected from the group consisting of gB, gC, gD, gH, gL, ICP27 and their functional equivalents.