CA2195099A1 - A polynucleotide herpes virus vaccine - Google Patents

Abstract

Genes encoding herpes simplex virus type 2 (HSV-2) proteins were cloned into eukaryotic expression vectors to express the encoded proteins in mammalian muscle cells in vivo. Animals were immunized by injection of these DNA constructs, termed polynucleotide vaccines or PNV, into their muscles. In a DNA titration, it was found that a single immunization of >= 0.5 .mu.g of (one) PNV, gave > 90 % seroconversion by ten weeks post immunization. Immune antisera neutralized both HSV-2 and HSV-1 in cell culture. When animals were challenged with HSV-2, significant (p < .001) protection from lethal infection was achieved following PNV vaccination.

Description

~ W0 96/03510 r~
21 q509q TITLE OF THE INVENTION
A POLYNllCLEOTIDE HERPES VIRUS VACCINE

BACKGROUND OF THE INVENTION
S A major obstacle to the development of vaccines against viruses, particularly those with multiple serotypes or a high rate of mutation, against which elicitation of neutralizing and protective immune responses is desirable, is the diversity of the viral external proteins among different viral isolates or strains. Since cytotoxic T-lymphocytes (CTLs) 10 in both mice and humans are capable of recognizing epitopes derived from conserved internal viral proteins [J.W. Yewdell 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. ~en. Virol. 67, 719 (1986); J. Bastin et - al., J. I~xp. Med. 165, 1508 (1987); A.R.M. Townsend and H. Bodrner, 15 Annu. Re1~. Immunol. 7, 601 (1989)], and are thought to be important in the immune response against viruses [Y.-L. Lin and B.A. Askonas, J.
~xp. Med. 154, 225 (1981); I. Gardner et al., Eur. J. Immunol. 4, 68 (1974); K.L. Yap and G.L. Ada, Nature 273, 238 (1978); A.J. McMichael et al ., New Engl . J. Med . 309, 13 (1983); P.M. Taylor and B.A. Askonas, 20 Immunol. 58, 417 (1986)], efforts have been directed towards the development of CTL vaccines capable of providing heterologous protection against different viral strains.
It is known that CTLs kill virally-infected cells when their T
cell receptors recognize viral peptides associated with MHC class I and 25 or class II molecules. These peptides can be derived from endogenously synthesized viral proteins, regardless of the protein's location or function within the virus. By recognition of epitopes from conserved viral proteins, CTLs may provide heterologous protection.
Many infectious disease causing agents can, by themselves, 30 elicit protective antibodies which can bind to and kill, render harmless, or cause to be killed or rendered harmless, the disease causing agent and its byproducts. Recuperation from these diseases usually results in long-lasting irnmunity by virtue of protective antibodies generated against the highly antigenic CU~ of the infectious agent.

2~ 95o9~

Protective antibodies are part of the natural defense mechanism of hurnans and many other animals, and are found in the blood a.s well as in other tissues and bodily fluids. It is the prirnary function of most vaccines to elicit protective antibodies against infectiou.s 5 agents and/or their byproducts, without causing disease.
Most efforts to generate CTL responses have either used replicating vectors to produce the protein antigen within the cell [J.R.
Bennink et al., ib~d. 311, 578 (1984); J.R. Bennink and J.W. Yewdell, CUrT . Top. Microbiol. Immunol. 163, 153 (1990); C.K. Stover et al., I O Nature 3~1, 456 (1991); A. Aldovini and R.A. Young, Nature 351, 479 (1991); R. Schafer et al ., J. Immunol . 149, 53 (1992); C.S . Hahn ef al., Proc. Natl. Acad. Sci. (USA) 89, 2679 (1992)], or they have focused upon the introduction of peptides into the cytosol [F.R. Carbone and MJ.
Bevan, J. Ex~. Med. 169, 603 (1989); K. Deres et al., Nature 342, 561 15 (1989); H. Takahashi etal., ibid. 344, 873 (1990); D.S. Collins etal., J.
Iml7lunol. 148, 3336 (1992); M.J. Newman et al., ibid. 148, 2357 (1992)].
Both of these ~ aches have limitations that may reduce their utility as vaccimes. Retroviral vectors have restrictions on the si~e and structure of polypeptides that can be expressed as fusion proteins while l l l,dl l~ l i l lg20 the ability of the re~;u-llbi--~ll virus to replicate [A.D. Miller, Curr. Top.
Microbiol. Irnmunol. I58, 1 (1992)], and the effectiveness of vectors such as vaccinia for subsequent i~"""~ lions may be ~.o~ ,l-lised by immune responses against vaccinia [E.L. Cooney et al., Lancet 337, 567 (1991)]. Also, viral vectors and modified pathogens have inherent risks 25 that may hinder their use in hurnans [R.R. Redfield et al., Ne~ Engl. J.
Med . 316, 673 (1987); L. Mascola et al., Arcl~. Intern. Med. 149, 1569 (1989)] . Furtherrnore, the selection of peptide epitopes to be presented i.s dependent upon the structure of an individual's MHC antigens and, therefore, peptide vaccines may have limited effectiveness due to the 30 diversity of MHC haplotypes in outbred 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 intr~m~ rly (i.m.) could be expressed. The intramllsc~ r (i.m.) injection of DNA expression ~ WO96/03510 1~,11L_ ~'C5.-I
2 l '~ 509q vectors in mice has been demonstrated to result in the uptake of DNA by the muscle cells and expression of the protein encoded by the DNA [J.A.
Wolff et al., Science 247, 1465 (1990); G. Ascadi et al., Nature 352, 815 (1991)]. The plasmids were shown to be lllAi~ ed episomally and did 5 not replicate. Snhse~ ntly, persistent expression has been observed after i.m. injection in skeletal muscle of rats, fish and primates, and cardiac muscle of rats [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 10 (1992); J.A. Wolff et al., ~luman Mol. Genet. 1, 363 (1992)] . The technique of using nucleic acids as ll"~ ic agents was reported in WO90/11092 (4 October 1990), in which naked polynucleotides were used to vaccinate vertebrates.
Recently, the coordinate roles of B7 and the major 15 histocompatibility complex (MHC) ~sGIl~lion of epitopes on the surface of antigen presenting cells in activating CTLs for the elimination of tumors was reviewed [Edgington, Biotechnology 11, 1117-1119, 1993]. Once the MHC molecule on the surface of an antigen presenting cell (APC) presents an epitope to a T-cell receptor (TCR), B7 expressed 20 on the surface of the same APC acts as a second signal by binding to CTLA-4 or CD28. The result is rapid division of CD4+ helper T-cells which signal CD8+ T-cells to proliferate and kill the APC.
It is not necessary for the success of the method that i"""""i,~rion be intr~rnll~cnl~r. Thus, Tang et al., [Nature, 356, 152-154 25 (1992)] disclosed that introduction of gold microprojectiles coated with DNA encoding bovine growth hormone (BGH) into the skin of mice resulted in production of anti-BGH antibodies in the mice. Furth et al., [Analytical Biochemistry, 205, 365-368, (1992)] showed that a jet imjector could be used to transfect skin, muscle, fat, and mammary tissues of 30 living animals. Various methods for introducing nucleic acids was recently reviewed [Friedman, T., Science, 244, 1275-1281 (1989)]. See also Robinson et al., [Abstracts of Papers Presented at the 1992 meeting on Modern Approaches to New Vaccines, Including Prevention of AIDS, Cold Spring Harbor, p92], where the im, ip, and iv ~(iminis~r~lion of 3~10 r~

avian influenza DNA into chickens was alleged to have provided protection against lethal challenge. Intravenous injection of a DNA:cationic liposome complex in mice was shown by Zhu et al., [Science 261, 209-211 (9 July 1993); see also W093/24640, 9 Dec. 1993]
5 to result in systemic expression of a cloned transgene. Recently, Ulmer et al., [Science 259, 1745- 1749, (1993)] reported on the heterologous protection against influenza virus infection by injection of DNA encoding influenza virus proteins.
Wang et al ., [P.N.A .S. USA 90, 4156-4160 (May, 1993)]
10 reported on elicitation of immune responses in mice against HIV by intramuscular inoculation with a cloned, genomic (unspliced) HTV gene.
However, the level of immune responses achieved was very low, and the system utilized portions of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) promoter and portions of the sirnian virus 40 15 (SV40) promoter and t~ lol. SV40 is known to transform cells, possibly through integration into host cellular DNA. Thus, the system described by Wang et al., is wholly illa~ l iate for administration to humans, which is one of the objects of the instant invention.
WO 93/17706 describes a method for vaccinating an animal 20 against a virus, wherein carrier particles were coated with a gene construct and the coated particles are accelerated into cells of an animal.
Recent efforts to develop subunit vaccines for herpes simplex virus (HSV) have focused on novel expression and presentation of viral antigens; especially the viral glycoproteins. [for review see 25 Burke, R.L., 1993, Sem.ln Virol., 4, pp.l87-197] Recombinant HSV
glycoproteins expressed by a variety of systems including yeast (Kino., Y.C. et ~., 1989~ Vaccine, Z, pp.l55-160), insect cells (Ghiasi, H. et al..
1991, Arch.Virol., 121, pp.l63-178), and m~mm~ n cells (Burke, R.L., 1991, Rev.Infect.lDis., 13, S906-S911; Lasky, L.A., 1990, J.Med.Virol., 30 31, pp.59-61) have been shown to elicit protective immunity in animal models. Clinical trials of a recullll;hldlll HSV-2 glycoprotein D (gD) produced in Chinese hamster ovary cells have shown that the vaccine induces an antibody response in naive individuals and stimulates the pre-WO96/03510 21 950~ t9C~/

existing response in both HSV- I and HSV-2 seropositive individuals.
(Straus, S .E.et al., 1993, J.lnfect.Dis., 167,pp.1045-1052) An alternate approach to subunit vaccination has been the use of live virus vectors for delivery of HSV antigens. Vaccinia-HSV
recombinants expressing gD (Aurelian, L. el al., 1991, Rev.Infect.Dis., 13,S924-S930; Rooney, J.F. et al., 1991, Rev.lnfect.Dis., 13,S898-S903;
Wachsman, M. et ak, 1992, Vaccine, 10, pp.447-454) gB (Rooney, J.F. et al., supra), gL and gH (Browne, H. et al., 1993, J.Gen.Virol., ~
pp.2813-2817) have successfully protected animals from HSV challenge.
10 Vaccination by infection with reco.l.ll)illall~ adenovirus expressing HSV
gB elicits a protective immune response in mice. (Ghiasi, H., supra;
McDermott, M.R., 1989, Virology, 169,pp.244-247) It is well ~locllmPnt~d that anti-gD antibodies can protect against HSV infection whether elicited by i" " """i,~l ion with native protein (Long, D. et al., 1984, Infect Immlln, 43,pp.761-764)-~co---b~ lly expressed protein (Burke, R.L., supra; Stanberry, L.R. et ak, 1987, J.Infect.Dis., 155, pp.914-920; Straus, S.E., supra) peptides derived from gD (Eisenberg, RJ. I al., 1985, J.Virol., 56,pp.1014-1027) or l,~l~r~ d passively (Dix, R.D. et al., 1981,Infecf Tmmnn 34,pp.192-199; Ritchie, M.H. et al., 1993, Investigative Ophthalmology and Visual Sciences, 34,pp.2460-2468).
Studies by Wolff et al. (supra) originally demonstrated that intr~mllsrnl:~r injection of plasmid DNA encodimg a reporter gene results in the expression of that gene in myocytes, at and near the sight of injection. Recent reports demonstrated the successful i"."~ lion of mice against influenza by the injection of plasmids encoding influenza A
hPm~ggllltinin (Montgomery, D.L. et al., 1993, Cell Biol., 12,pp.777-783), or nucleoprotein (Montgomery, D.L. et ak, supra; Ulmer, J.B. et al., 1993, Science, 259,pp.1745-1749). The first use of DNA i~ l inn for a herpes virus has been reported (Cox et al., 1993, J.Virol., 67, pp.5664-5667). Injection of a plasmid encoding bovine herpesvirus I
(BHV- I ) glycoprotein g IV gave rise to anti-g IV antibodies in mice and calves. Upon intranasal challenge with BHV-I, il"""lni,~d calves showed reduced symptoms and shed sllhst~nti~lly less virus than controls.

WO96103~10 2 ~ 950 99 ~ S~

The ability of HSV glycoprotein D to elicit a protective immune response in mice (Long, D. et al., supra) and guinea pigs (Stanberry, L.R. et al., supra; Stanberry, L.R. et al., 1989, Antiviral.Res., I l, pp.203-214) is well docllmPntp~l SU~MARY OF THE INVENTION
To test the efficacy of DNA i"""~",;",lion in the prevention of HSV disease, HSV-2 protein-coding DNA sequences were cloned into the eukaryotic expression vector. This DNA construction elicits an 10 immune response when injected into animals. lmnnnni7~d animals were infected with HSV to evaluate whether or not direct DNA i" " "~l"i ".1 ion with the gD gene (or other HSV-2 genes) could protect them from disease. Nucleic acids, including DNA constructs and RNA Llal,s~ L~, capable of inducing in vivo expression of human herpes simplex virus 15 (HSV) proteins upon direct introduction into animal tissues via injection or otherwise are therefore disclosed. Injection of these nucleic acids may elicit immune responses which result in the production of cytotoxic T
Iymphocytes (CTLs) specific for HSV antigens, as well as the generation of HSV-specific antibodies, which are protective upon s--hscf~ nt HSV
20 challenge. These nucleic acids are useful as vaccines for inducing immunity to HSV, which can prevent infection and/or ameliorate HSV-related disease.

BRIEF DESCRTPTION OF THE DRAWINGS
Fig. 1. Panels A~ B. and C
A. Western blot analysis of HSV gD expression by VlJ:gD-transfected cells is shown, 1. mock infected Vero cells;
2. HSV-2 IR6 infected Vero cells (moi=l);
3. HSV-2 Curtis infected Vero cells (moi=l);

4. RD cells transfected with VlJ:gD;

5. mock transfected RD cells.

~ WO96/03510 p ~1 5/,)5 ~, 21 95a99 B. Western blot analysis of HSV gB expression by VlJNS:gB-transfected cells is shown.
C. Western blot analysis of HSV ICP27 expression by VlJ:lCP27-transfected cells is shown.

Fig. 2. Panels A. B. and C
A. Western blot analysis using sera from HSV PNV-j"""lllli,~d (B - 6.25 ug dose of VlJ:gD, C - 50 ug dose of VlJ:gD) and sham i""lllllli,,~d (A) animals is shown on Iysates of BHK cells infected with:
1. HSV-I KOS;
2. HSV-2 186;
3. HSV-2 Curtis;
4. mock infected.
B. Western blot analysis using sera from HSV PNV gB
rd animals is shown.

Fig. 3. ELISA generated group GMT data is shown for HSV PNV-rd animals receiving a single injection of vaccine;
sera were obtained at 4, 7 and 10 weeks post-ill"",ll~ n.

Fig. 4. Survival of HSV-2 rh ~ ngPd animals following two injections with VlJ:gD at 200ug; 100ug; 50 ug; ~5ug;
12.5ug; 6.~5ug; 3.13ug; 1.56 ug; 0.78 ug; or saline ~~ only. Since all animals in the 200ug; 100ug; ~5ug; 12.5ug;

6.25ug; and 3.13ug groups survived, they are all d with a single symbol.

WO96103510 2 ~ 95399 ~ c ~, ~

Fig. 5. Survival of HSV-2 challenged animals following one injection with VlJ:gD at 50 ug; 16.7 ug; 5.0 ug; 1.67 ug;
0.5 ug; 0.167 ug; 0.05 ug; 0.017 ug; 0.005 ug; or saline only.

Fig. 6. Survival of animals i"""""i,~.l with VlJNS:gB following HSV challenge is shown.

Fig. 7 Survival of animals illlllllllli~rd with VlJ:gC following HSV
challenge is shown.

Fig. R The results of survival, mean days to death, paralysis, and vaginal virus titers in HSV-2 infected guinea pigs is shown.
Fig. 9 Guinea pig vaginal lesion scores are shown.

DETAILED DESCRIPTION OF THE INVENTION
This invention provides polynucleotides which, when 20 directly introduced into a vertebrate in vivo, including mammals such as humans, induces the expression of encoded proteins within the animal.
As u.sed herein, a polynucleotide is a nucleic acid which contains essential regulatory elements such that upon introduction into a living vertebrate cell, is able to direct the cellular machinery to produce 25 translation products encoded by the genes l,Olllplisillg the polynucleotide.
In one embodiment of the invention, the polynucleotide is a polydeoxyribonucleic acid comprising HSV genes operatively linked to a Ll dns~ ional promoter. In another embodiment of the invention the polynucleotide vaccine comprises polyribonucleic acid encoding HSV
30 genes which are amenable to translation by the eukaryotic cellular machinery (ribosomes, tRNAs, and other translation factors). Where the protein encoded by the polynucleotide is one which does not normally occur in that animal except in pathological conditions, (i.e. an heterologous protein) such as proteins associated with HSV, the animals' ~ WO 96/03510 2 ~ 9 5 0 9 q . ~

g immune system is activated to launch a protective immune response.
Because these exogenous proteins are produced by the animals' own tissues, the expressed proteins are processed by the major histocu,llpdtibility system (MHC) in a fashion analogous to when an 5 actual HSV infection occurs. The result, as shown in this disclosure, is induction of immune responses against HSV. Polynucleotides for the purpose of generating immune responses to an encoded protein are referred to herein as polynucleotide vaccines or PNV.
There are many embodiments of the instant invention which 10 those skilled in the art can a~ ;idl~ from the specification. Thus, different transcriptional promoters, Ir~ , carrier vectors or specific gene seq~Pn~es may be used successfully.
The instant invention provides a method for using a polynucleotide which, upon introduction into m~mm~ n tissue, induces 15 the expression, in vivo, of the polynucleotide thereby producing the encoded protein. It is readily apparent to those skilled in the art that variations or derivatives of the nllc!.ootide sequence encoding a protein can be produced which alter the amino acid sequence of the encoded protein. The altered expressed protein may have an altered amino acid 20 sequence, yet still elicits antibodies which react with the viral protein, and are considered functional equivalents. In addition, fragments of the full length genes which encode portions of the full length protein may also be constructed. These fragments may encode a protein or peptide which elicits antibodies which react with the viral protein, and are 25 considered functional equivalents.
In one embodiment of this invention, a gene encoding an HSV gene product is incorporated in an expression vector. The vector COntdinS a L.dl~s~ lional promoter recognized by eukaryotic RNA
polymerase, and a l-d-ls~ )lional terminator at the end of the HSV gene 30 coding sequence. In a preferred embodiment, the promoter is the cytomegalovirus promoter with the intron A sequence (CMV-intA), although those skilled in the art will recognize that any of a number of other known promoters such as the strong immunoglobulin, or other eukaryotic gene promoters may be used. A preferred L.a~ Lional WO96/0351i~ 2 ~ 9 50 99 1~ .'U!~

terminator ic the bovine growth hormone terminator. The combination of CMVintA-BCH temminator is preferred. In addition, to assist in preparation of the polynucleotides in prokaryotic cells, an antibiotic resistance marker is also optionally included in the expression vector 5 under transcriptional control of a suitable prokaryotic promoter.
Ampicillin resistance genes, neomycin resistance genes or any other suitable antibiotic resistance marker may be used. In a preferred embodiment of this invention, the antibiotic resistance gene encodes a gene product for neomycin resistance. Further, to aid in the high level 10 production of the polynucleotide by growth in prokaryotic organisms, it is advantageous for the vector to contain a prokaryotic origin of replication and be of high copy number. Any of a number of commercially available prokaryotic cloning vectors provide these elements. In a preferred embodiment of this invention, these 15 functionalities are provided by the commercially available vectors known as the pUC series. It may be desirable, however, to remove non-essential DNA seq~ n~ec Thus, the lacZ and lacl coding sequences of pUC may be removed. It is also desirable that the vectors are not able to replicate in eukaryotic cells. This lllilli",i,rs the risk of integration of 20 polynucleotide vaccine sequences into the recipients' genome.
In another embodiment, the expression vector pnRSV is used, wherein the rous sarcoma virus (RSV) long temminal repeat (LTR) is used as the promoter. In yet another embodiment, VI, a mutated pBR322 vector into which the CMV promoter and the BGH
25 transcriptional t~ atul were cloned is used. In a preferred embodiment of this invention, the elements of Vl and pUCI9 have been been combined to produce an expression vector named V IJ. Into V IJ or another desirable expression vector is cloned an HSV gene, such as gD, or any other HSV gene which can induce anti-HSV immune responses 30 (antibody and/or CTLs) such as gB, gC, gL, gH and ICP27. In another embodiment, the ampicillin resistance gene is removed from VIJ and replaced with a neomycin resistance gene, to generate V lJ-neo, into which any of a number of different HSV genes may be cloned for use according to this invention. In yet another embodiment, the vector is ~ WO96103510 r~ C~C~_5/
-- 2~ q5~9q VlJns, which is the same a.s VlJneo except that a unique Sfil restriction site has been engineered into the single Kpnl site at position 2114 of VlJ-neo. The incidence of Sfll sites in human genomic DNA is very low (approximately I site per 100,000 bases). Thus, this vector allows careful 5 monitoring for expression vector integr~tion into host DNA, simply by Sfi I digestion of extracted genomic DNA. In a further embodiment, the vector is VlR. In this vector, as much non-essential DNA as possible is "trimmed" to produce a highly compact vector. This vector allows larger inserts to be used, with less concern that undesirable sf~qu~nr~s are 10 encoded and optimizes uptake by cells when the construct encoding specific virus genes is introduced into surrounding tissue. The methods used in producing the foregoing vector modifications and development procedures may be accomplished according to methods known by those skilled in the art.
From this work those skilled in the art will recognize that one of the utilities of the instant invention is to provide a system for in vn~o as well as in vitro testing and analysis so that a correlation of HSV
sequence diversity with serology of HSV neutralization, as well as other parameters can be made. The isolation and cloning of these various 20 genes may be accomplished according to methods known to those skilled in the art. This invention further provides a method for systematic id~ntifi~tion of HSV strains and sequences for vaccine production.
Incorporation of genes from primary isolates of HSV strains provides an immunogen which induces immune responses against clinical isolates of 25 the virus and thus meets a need as yet unmet in the field. Furthermore, if the virulent isolates change, the immunogen may be modified to reflect new sequences as necessary.
In one embodiment of this invention, a gene encoding an HSV protein is directly linked to a transcriptional promoter. The use of 30 tissue-specific promoter.s or ~nh~nrers for example the muscle creatine kinase (MCK) enhancer element may be desirable to limit expression of the polynucleotide to a particular tissue type. For example, myocytes are terminally differentiated cells which do not divide. Integration of foreign DNA into chromosomes appears to require both cell division and protein WO 96/03510 2 1 9 5 0 q 9 r~

synthesis. Thus, limiting protein expression to non-dividing cells such as myocytes may be preferable. However, use of the CMV promoter is adequate for achieving expres.sion in many tissues into which the PNV is introduced.

PNV Construct Summary HSV and other genes are preferably ligated into an expression vector which has been specifically optimized for polynucleotide vaccinations. Elements include a transcriptional promoter, immunogenic epitopes, and additional cistrons encoding immunoenhancing or immunomodulatory genes, with their own promoters, L-a--s~ ional terminator, bacterial origin of replication and antibiotic resistance gene, as described herein. Optionally, the vector may contain internal ribosome entry sites (IRES) for the expression of polycistronic mRNA. Those skilled in the art will appreciate that RNA
which has been transcribed in l~itro to produce multi-cistronic mRNAs encoded by the DNA u~ul~le~d-~.~ is within the scope of this invention.
For this purpose, it is desirable to use as the transcriptional promoter such powerful RNA polymerase promoters as the 17 or SP6 promoters, and performing run-on transcription with a linearized DNA template. These methods are well known in the art.
The protective efficacy of polynucleotide HSV immunogens against snhscqll~nt viral challenge is demonstrated by i"""ll"i".lion with the DNA of this invention. This is advantageous since no infectious ~S agent is involved, no as.sembly of virus particles is required, and de~ ul~l~ selection is permitted. Furthermore, because the sequence of viral gene products may be conserved among various .strains of HSV, protection against 5nhseql~nt challenge by another strain of HSV is obtained.
The injection of a DNA expression vector encoding gD may result in the generation of .signific:~nt protective immunity against subse~uent viral challenge. In particular, gD-specific antibodies and CTLs may be produced. Immune responses directed against conserved protein,s can be effective despite the antigenic shift and drift of the ~ WO 96/03S10 2 ~ q 5 ~ ~ 9 P~ .,........................... J/

variable proteins. Because each of the HSV gene products exhibit some degree of conservation among the various strains of HSV, and because immune responses may be generated in response to intracellular expression and MHC processing, it is expected that many different HSV
S gD PNV constructs may give rise to cross reactive immune responses.
The invention offers a means to induce heterologous protective immunity without the need for self-replicating agents or adjuvants. The generation of high titer antibodies against expressed proteins after injection of viral protein and human growth hormone DNA, 10 [Tang et al., Nature 356, 152, 1992], indicates this is a facile and highly effective means of making antibody-based vaccines, either separately or in combination with cytotoxic T-lymphocyte vaccines targeted towards conserved antigens.
The ease of producing and purifying DNA constructs 15 compares favorably with traditional protein purification, facilitating the generation of combination vaccines. Thus, multiple constructs, for example encoding gD and any other HSV gene also including non-HSV
genes may be prepared, mixed and co-~lministered. Additionally, protein expression is m~inr~in~d following DNA injection [H. Lin et al..
20 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); J.A. Wolff et al., Hur~lan Mol. Genet. 1, 363 (1992)], the persistence of B- and T-cell memory may be enhanced [D. Gray and P. Matzinger, J. Exp. Med. 174, 969 (1991) S.
25 Oehen et al.. ibid. 176, 1273 (1992)], thereby engendering long-lived humoral and cell-mediated immunity.
The amount of expressible DNA or transcribed RNA to be introduced into a vaccine recipient will depend on the strength of the L~ tional and translational promoters used. The m~gnitll(ie of the 30 immune response may depend on the level of protein expression and on the immunogenicity of the expressed gene product. In general, an effective dose of about I ng to ~ mg, and preferably about 10 ~g to 300 :H
llg is administered directly into muscle tissue. Subcutaneous injection, intradermal introduction, impression through the skin, and other modes of W096/03510 2 1 95 0 9 9 P~ 3~

administration such as intraperitoneal, intravenous, or inh~l~tinn delivery are al.so suitable. It is also contemplated that booster vaccinations may be provided. Following vaccination with HSV polynucleotide immunogen, boosting with HSV protein immunogens such as the gD, gB, gC, gG, and 5 gH gene products is also contemplated. Parenteral administration, such as intravenous, intr~ml~cc~ r, s--hcut~nPous or other means of administration of iu~ lcu~ill-12 protein, concurrently with or subsequent to parenteral introduction of the PNV of this invention may be advantageous.
The polynucleotide may be naked, that is, unassociated with any proteins, adjuvants or other agents which affect the recipients' immune system. In this case, it is desirable for the polycucleotide to be in a physiologically acceptable solution, such as, but not limited to, sterile saline or sterile buffered saline. Alternatively, the DNA may be 15 associated with liposomes, such as lecithin liposomes or other liposomes known in the art, as a DNA-liposome mixture, or the DNA may be associated with an adjuvant known in the art to boost imunune responses, such as a protein or other carrier. Agents which assist in the cellular uptake of DNA, such as, but not limited to, calcium ions, may also be 20 used. These agents are generally referred to herein as transfection facilitating reagents and ph~rmS~rentin~lly ac~t~blc carriers.
Techniques for coating u~ ctiles coated with polynucleotide are known in the art and are also useful in connection with this invention.
For DNA intended for human use it may be useful to have the final DNA
25 product in a ph~rm~rellfically acceptable carrier or buffer solution.
Ph~rm~nelltically acceptable carriers or buffer solutions are known in the art and include those described in a variety of texts such as Remington's Pharmaceutical Sciences.
In another embodiment, the invention is a polynucleotide 30 which comprises contiguous nucleic acid sequences capable of being expressed to produce a gene product upon introduction of said polynucleotide into eukaryotic tissues in vivo. The encoded gene product preferably either acts as an immnnostimlll~nt or as an antigen capable of generating an immune response. Thus, the nucleic acid s~(lll/n~es in this ~ WO96/03510 r~ vJ/
~ q5~9q embodirnent encode a human herpes simplex virus immunogenic epitope, and optionally a cytokine or a T-cell costimulatory element, such as a member of the B7 family of proteins.
There are several advantages of i"""""i~"lion with a gene 5 rather than its gene product. The first is the relative simplicity with which native or nearly native antigen can be presented to the imrnune system. M~mm~ n proteins expressed recombinantly in bacteria, yeast, or even m~mm~ n cells often require extensive treatment to insure appropriate antigenicity. A second advantage of DNA imm~mi7:~tion is 10 the potential for the immunogen to enter the MHC class I pathway and evoke a cytotoxic T cell response. I"""""i,~lion of mice with DNA
encoding the influenza A nucleoprotein (NP) elicited a CD8+ response to NP that protected mice against challenge with heterologous strains of flu.
(Montgomery, D.L. et ah, supra; Ulmer, J. et ah, supra) There is evidence that cell-mediated i~ uulily is important in controlling HSV infection [for review see Nash, A.A. et al., 1985, In:
The Hel~lesvilu:,es, Vol.4, Plenum, New York, and Schrnidt, D.S. et al., 1992, In: Rouse (ed.), Current Topics In Microbiology And Irnmunology, Vol.179, Herpes Simplex Viru.s; Pathogenesis, Irnmunobiology and 20 Control, Springer-Verlag, Berlin]. While the majority of HSV CTLs isolated from HSV seropositive patients are of the CD4+ type (Schrnidt, D.S. et al., 1988, J.lmmunol., 14Q, pp.3610-3616; Tsutsurni, T. I al., 1986, Clin.Exp.lrnmunol., 66, pp.507-515) CD8+ clones, including one specific for gD, have been isolated. (Torpey, DJ. et al., 1989, 25 J.lmrnunol., 142, pp.l325-1332; Yasukawa, M. et aL, 1989,J.lrnrnunol., 143, pp.2051-2057; Zarling, J.M. et al., 1986, J.lmmunol., 136. pp.4669-4673) In mice, cell tran.sfer and depletion e~pelull~llL~ suggest that some CD8+ CTLs protect against infection. (Bonneua, R.H. et ak, 1989, J.Virol, 63, pp.l480-1484; Nash, A.A. et ah, 1987, J.Gen.Virol., 68, 30 pp.825-833) T,,,,,llllli/AIion with gD via infection with recombinant virus vectors (Paoletti, E. et al., 1984, Proc.Natl.Acad. Sci. USA, R 1, pp. l 93-197; Wachsman, M.L. et al., 1987, J.Lnfect.Dis., 155, pp. l l 88- 1197;
Zheng, B. et al., 1993, Vaccine, 11, pp. l l91 - 1198) protects mice from HSV infection. Live virus vectors, like DNA, have the potential for WO96/03510 l.~ J...................... J/~JCJl ~

MHC class I presentation of the irnmunogen. However, a recent study using infection by an HSV gD-vaccinia reculllbill~ll to immunize mice found that protection from challenge was dependent on the delayed type hypersensitivity functions of L3T4+ cells. (Wachsman, M. _ al., 1992, S Vaccine, 10, pp.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.lmmunol., 145, pp.702-710) Work by Koelle el al., suggests that HSV infection of human fibroblasts and keratinocytes may render them unrecognizable to CD8+ CTLs 10 (Koelle, D.M. et al., 1993, J.Clin.lnvest., 91, pp.9~1-968). In natural HSV infection, the role of CD8+ cells in general, and the role of CD8+
response to gD in particular is not resolved.
Since DNA i"""""i" lion can evoke both humoral and cell-mediated immune responses, its greatest advantage may be that it 15 provides a relatively simple method to survey a large number of viral genes for their vaccine potential. Plasmids expressing HSV-2 glycoproteins B and C al.so elicit neutralizing antibodies and protect mice from lethal challenge. However, lCP27 which is known to generate a CTL response and to provide some protection in mice il"""",i,~d by 20 infection with ICP27-vaccmia recombinant virus (Banks, T.A. et al., 1991, J .Virol., 65, pp.3185-3191) did not provide protection from lethal HSV challenge when mice were vaccinated with PNV ICP27 alone.
However, ICP27-encoding DNA may be useful as one ~Ulll~)Ull~llt of a multi-HSV gene-containing PNV, and it is contemplated that the present 25 invention includes ICP27 as a component of a multivalent HSV PNV.
lmmnni7~ion by DNA injection also allows, as discussed above, the ready assembly of multicu---~unel-l subunit vaccines.
Simultaneous i,ii""",i,,,tion with multiple influenza genes has recently been reported. (Donnelly, J. et al., 1994, Vaccines, in press). The 30 inclusion in an HSV vaccine of genes whose products activate different arms of the immune system may also provide thorough protection from subse4uent virus challenge.
The following examples are provided to illustrate the present invention without, however, limiting the same thereto.

~ WO 96/03510 2 1 9 5 ~ 9 9 r~

EXAMPLE I

Vectors for Vaccine Production 5 A) Vl The expression vector Vl was constructed from pCMVlE-AKI-DHFR [Y. Whang eF Ql., J. Virol. 61, 1796 (1987)]. The AKI and DHFR genes were removed by cutting the vector with EcoR I and self-ligating. This vector does not contain intron A in the CMV promoter, so 10 it was added as a PCR fragment that had a deleted internal Sac I site [at 1855 as numbered in B.S. Chapman eF al., Nuc. Acids Res. 19, 3979 (1991)]. The template used for the PCR reactions was pCMVintA-Lux, made by ligating the Hind III and Nhe I fragment from pCMV6al20 [see B.S. Chapman ef al., ibid.,] which includes hCMV-EI
15 enhancer/promoter and intron A, into the Hind III and Xba I sites of pBL3 to generate pCMVlntBL. The 1881 base pair luciferase gene fragment (Hind m-Sma I Klenow filled-in) 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 Klenow filled-in and phosphatase treated.
The primers that spanned intron A are:
5' primer, SEQ. ID:I:
5'-CTATATAAGCAGAG CTCGmAG-3'; The 3' primer, SEQ ID:2:
5'-GTAGCAAAGATCTAAGGACGGTGA CTGCAG-3'.
The primers used to remove the Sac I site are:
sense primer, SEQ ID:3:
5-GTATGTGTCTGAAAATGAGCGTGGAGATTGGGCTCGCAC-3' and the antisense primer, SEQ ID:4:
30 5'-GTGCGAGCCCAATCTCC,~GCTCATTTTCAGACACA TAC-3'.

The PCR fragTnent was cut with Sac I and Bgl II and inserted into the vector which had been cut with the same enzymes.

WO96103510 2~ 9~09~ r~ 3c~ ~

B) VIJ Expression Vector The purpose in creating V IJ was to remove the promoter and L-dns~ ion termination elements from vector Vl in order to place them within a more defined context, create a more compact vector, and to 5 improve plasmid purification yields.
VlJ is derived from vectors Vl and pUC18, a cul~ el~,ially available plasmid. Vl was digested with Sspl and EcoRI restriction enzymes producing two fragments of DNA. The smaller of these fragments, containing the CMVintA promoter and Bovine Growth 10 Hormone (BGH) transcription termination elements which control the expression of heterologous genes, was purified from an agarose electrophoresis gel. The ends of this DNA fragment were then "blunted"
using the T4DNA polymerase enzyme in order to facilitate its ligation to another "blunt-ended" DNA fragment.
pUC18 was chosen to provide the "backbone" of the expression vector. It is known to produce high yields of plasmid, is well-characterized by sequence and function, and is of small size. The entire lac operon was removed from this vector by partial digestion with the Haen restriction enzyme. The remaining plasmid was purified from an 20 agarose electrophoresis gel, blunt-ended with the T4 DNA polymerase treated with calf intestinal aL~aline phosphatase, and ligated to the CMVintA/BGH element described above. Plasmids exhibiting either of two possible orientations of the promoter elements within the pUC
backbone were obtained. One of these plasmids gave much higher yields 2~ of DNA in ~. co7i and was d~ign~tl~d VIJ. This vector's structure was verified by sequence analysis of the junction regions and was subsequently demonstrated to give comparable or higher expression of heterologous genes compared with V 1.

~t~ WO9S/03510 21 q5099 r~ )............................... r~Jc~

C) VlJneo Expre.ssion Vector It was necessary to remove the ampr gene used for antibiotic selection of bacteria harboring VIJ because ampicillin may not be desirable in large-scale fermenters. The ampr gene from the pUC
5 backbone of VIJ was removed by digestion with Sspl and Eaml IO5I
restriction enzymes. The remaining plasmid was purihed by agarose gel ele~ ho,t;sis, blunt-ended with T4 DNA polymerase, and then treated with calf intestinal alkaline phosphatase. The commercially available kanr gene, derived from transposon 903 and contained within the pUC4K
10 plasmid, was excised using the PstI restriction enzyme, purified by agarose gel electrophoresis, and blunt-ended with T4 DNA polymerase.
This fragment was ligated with the V IJ backbone and plasmids with the kanr gene in either orientation were derived which were designated as V IJneo #'s I and 3. Each of these plasmids was confirmed by restriction 15 enzyme digestion analysis, DNA secl--~n- ing of the junction regions, and was shown to produce similar quantities of plasmid as V IJ. Expression of heterologous gene products was also comparable to VlJ for these VlJneo vectors. VlJneo#3, referred to as VlJneo hereafter, was selected which contains the kanr gene in the same orientation as the ampr gene in 20 VlJ as the expression construct.

D) VIJns Expression Vector An Sfi I site was added to VlJneo to facilitate integration studies. A co~ ,ially available 13 base pair Sfi I linker (New England 25 BioLabs) was added at the Kpn I site within the BGH sequence of the vector. VlJneo was linearized with Kpn 1, gel purified, blunted by T4 DNA polymerase, and ligated to the blunt Sfi I linker. Clonal isolates were chosen by restriction mapping and verified by sequencing through the linker. The new vector was decign~tt?d VlJns. Expression of 30 heterologous genes in VlJns (with Sfi 1) was comparable to expression of the same genes in VlJneo (with Kpn 1).

WO 96/03510 r~ c.~
21 ~5099 E) VlJns-tPA
In order to provide an heterologous leader peptide sequence to secreted and/or membrane proteins, VlJns was modified to include the human tissue-specific plasminogen activator (tPA) leader. Two synthetic complementary oligomers were annealed and then ligated into VlJn which had been Bglll digested. The sense and antisense oligomers were 5'-GATC ACC ~TG GAT GCA ATG AAG AGA GGG CTC TGC TGT
GTG CTG CTG CTG TGT GGA GCA GTC TTC GTT TCG CCC AGC
GA-3', SEQ. ID: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. The Kozak sequence is underlined in the sense oligomer. These oligomers have overh~nging bases compatible for ligation to BglII-cleaved scq~ nr~i. After ligation the upstream Bglll site is destroyed while the downstream BgllI is retained for subsequent ligations. Both the junction sites as well as the entire tPA leader sequence were verifed by DNA sequencing.
Additionally, in order to conform with the consensus optimized vector VlJns (=VlJneo with an Sfil site), an Sfil restriction site was placed at the KpnI site within the BGH terminator region of VlJn-tPA by blunting the Kpnl site with T4 DNA polymerase followed by ligation with an Sfil linker (catalogue #1138, New England Biolabs). This modification was verifed by restriction digestion and agarose gel electrophoresis.

F) pGEM-3-X-lRES-B7 (where X = any antigenic gene) As an example of a dicistronic vaccine construct which provides coordinate expression of a gene encoding an immunogen and a gene encoding an immumo-stimulatory protein, the murine B7 gene was PCR amplified from the B
Iymphoma cell line CH I (obtained from the ATCC). B7 is a member of a family of proteins which provide essential costimulation T cell activation by antigen in the context of major histocompatibility complexes I and 11. CHl cells provide a good source of B7 mRNA
because they have the phenotype of being constitutively activated and B7 is expressed primarily by activated antigen presenting cells such as B

~ WO 96/03~10 21 q 5 0 9 9 r~

cells and macrophages. These cells were further stimnl~ted in vit~-o using cAMP or IL-4 and mRNA prepared using standard gll~ni(1inillm thiocyanate procedures. cDNA synthesis was perfommed using this mRNA using the GeneAmp RNA PCR kit (Perkin -Elmer Cetus) and a 5 priming oligomer (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 PCR oligomers: 5'-GGT ACA AGA TCT
ACC ATG GCT TGC AAT TGT CAG TTG ATG C-3', SEQ. ID:8:, and 10 5'-CCA CAT AGA TCT CCA TGG GAA CTA AAG GAA GAC GGT
CTG TTC-3', SEQ. ID:9:, respectively. These oligomers provide Bglll restriction enzyme sites at the ends of the insert as well as a Kozak translation initiation sequence containing an Ncol restriction site and an additional Ncol site located imm~ t~ly prior to the 3'-terminal BglII
15 site. Ncol digestion yielded a fragment suitable for cloning into pGEM-3-lRES which had been digested with NcoI. The resulting vector, pGEM-3-IRES-B7, contains an IRES-B7 cassette which can easily be transferred to VlJns-X, where X ~ ;;S~.llS an antigen-encoding gene.

20 G) pGEM-3-X-lRES-GM-CSF
(where X = any antigenic gene) This vector contains a cas.sette analogous to that described in item C above except that the gene for the immunn~timnl:~tnry cytokine, GM-CSF, is used rather than B7.
GM-CSF is a macrophage dirre,clllialion and stimulation cytokine which 25 has been shown to elicit potent anti-tumor T cell activities in vivo [G.
Dranoff et al., P~oc. Natl. Acad. Sci. USA, 90, 3539 (1993).

H) pGEM-3-X-lRES-lL- 12 (where X = any antigenic gene) This vector contains a 30 cassette analogous to that described in item C above except that the gene for the immnnnstimlll!ltc)ry cytokine, IL-12, is used rather than B7. IL-12 has been demonstrated to have an infln~nti~l role in shifting immune responses towards cellular, T cell--lnmin~t~d pathways as opposed to humoral responses [L. Alfonso et al., Science, 263, 235, 1994].

WO 96/03510 r~
21 q5~q~ --S Vector V 11~ Pre~aration In an effort to continue to optimize the basic vaccination vector, a derivative of VlJns, d~P~ign~tPd VIR, was prepared. The purpose for this vector construction was to obtain a minimum-sized vaccine vector without unneeded DNA sequ~pnrec~ which still retained the overall optimized heterologous gene expression characteristics and high plasmid yields that VlJ and VlJns afford. It was determined from the literature as well as by ~ ,e.i~ that (I ) regions within the pUC
backbone comprising the E. coli origin of replication could be removed without affecting plasmid yield from bacteria; (2) the 3'-region of the kanr gene following the kanamycin open reading frame could be removed if a bacterial L~ illatul was inserted in its place; and, (3) ~300 bp from the 3'- half of the BGH le,ll-il-al~l could be removed without affecting its regulatory function (following the original Kpnl restriction enzyme site within the BGH element).
VlR was constructed by using PCR to synthesize three segments of DNA from VlJns representing the CMVintA promoter/BGH
terminator, origin of replication, and kanamycin resistance elements, respectively. Restriction enzymes umique for each segment were added to each segment end using the PCR oligomers: Sspl and Xhol for CMVintA/BGH; EcoRV and BamHI for the ka~l r gene; and, Bcll and Sall for the ori r These enzyme sites were chosen because they allow directional ligation of each of the PCR-derived DNA segments with sllbse(lllPnt loss of each site: EcoRV and Sspl leave blunt-ended DNAs which are compatible for ligation while BamHI and Bcll leave complementary overhangs as do Sall and Xhol. After obtaining these segments by PCR each segment was digested with the a~ )l iaL~
restriction enzymes indicated above and then ligated together in a single reaction mixture containing all three DNA segments. The 5'-end of the ori r was designed to include the T2 rho independent terminator sequence WO96/03510 21 9 5099 r~

that is normally found in this region so that it could provide termination information for the kanamycin resistance gene. The ligated product was confirrnPd by restriction enzyme digestion (>8 enzymes) as well as by DNA seqllPn( ing of the ligation junctions. DNA plasmid yields and 5 heterologous expression using viral genes within VlR appear similar to VlJns. The net reduction in vector size achieved was 1346 bp (VlJns =
4.86kb; VlR=3.52kb).

PCR oligomer sequences used to synthesize VIR (restriction enzyme 10 sites are underlined and identifled in brackets following sequence):

( I ) 5'-GGT ACA AAT ATT GG CTA TTG GCC ATT GCA TAC G-3' [Sspll, SEQ.ID:I0:, (2) 5'-CCA CAT CTC GAG GAA CCG GGT CAA TTC TTC AGC
15 ACC-3' [Xhol], SEQ.ID:Il:
(for CMVintA/BGH segment) (3) 5'-GGT ACA GAT ATC GGA AAG CCA CGT TGT GTC TCA
AAA TC-3'[EcoRV], SEQ.ID:12:
20 (4) 5'-CCA CAT GGA TCC G TAA TGC TCT GCC AGT GTT ACA
ACC-3' [BamHI], SEQ.ID:13:
(for kanamycin ~ ce gene segment) (5) 5'-GGT ACA TGA TCA CGT AGA AAA GAT CAA AGG ATC
25 TTC TTG-3'[BclI], SEQ.ID:14:, (6) 5'-CCA CAT GTC GAC CC CTA AAA AGG CCG CGT TG-C TGG- ~=
3' [Sall], SEQ.ID:15:
(for E. coli origin of replication) WO 96/03510 1 ~
~1 ~5099 EXA~IPLE 3 Cells. Viruses ~n~ Cell culture VERO, BHK-21, and RD cells were obtained from the 5 ATCC. Virus was routinely prepared by infection of nearly confluent VERO or BHK cells with a multiplicity of infection (m.o.i.) of 0.1 at 37~C in a small volume of medium without fetal bovine serum (FBS).
After one hour, virus inoculum was removed and cultures were re-fed with high glucose DMEM supplemented with 29~o heat-inactivated FBS, 10 2mM L-glllt~min,-, 25mM HEPES, 50 U/ml penicillin and 50 llg/ml streptomycin. Incubation was continued until cytopatic effect was extensive: usually 24 to 48 hours. Cell ~ssoci~d virus was collected by centrifugation at 1800 X g 10 minutes 4~C. S--pern~n~ant virus was clarified by centifugation at 640 X g for 10 minutes 4~C.
E~XAMPi F 4 Clonin.g and DNA preparation HSV-2 (Curtis) DNA for use as PCR template was prepared 20 from nucleocapsids isolated from infected VERO cells. (Denniston, K.J.
et al., 1981, Gene, 15, pp.365-378) Synthetic oligomers corresponding to 5' and 3' end flanking s.sqllrn~cs for the HSV2 gB, gC, gD, or ICP27 genes, c~-n~inins~ Bgl n restriction recognition sites (Midland Certified Reagent Company; Midland, Texas) were used at 20 pmoles each. A
25 1. Ikb fragment encoding the gD gene was amplified by PCR (Perkin Elmer Cetus, La Jolla) according to the maufacturer's specifications except that a deaza dGTP:dCTP ratio of 1 :4 was used in place of dGTP
and the buffer was supplemented to 3 mM Mg C12. HSV-2 genomic DNA template was used at 100 n~/100 ,ul reaction. The PCR amplified 30 fragments were restricted with Bgl Il and ligated to the Bgl 11 digested, dephosphorylated vector VIJ (Montgomery, D.L. et aL, supra). E. coli DH51x (BRL-Gibco, G~ilhel.,l,ul~, Md.) was transformed according to the manufacturer's specifications. Ampicillin resistant colonies were screened by hybridization with the 32p labeled 3' PCR primer. Candidate ~ W096/03510 21 95~ r~

plasmids were characterized by restriction mapping and sequencing of the vector-insert junctions using the Sequenase DNA Sequencing Kit, version 2.0 (United States Biochemical). In a similar manner, a 2.7Kb fragment encoding the gB gene; a l.SKb fragment encoding the gC gene;
5 and a 1.6Kb fragment encoding the ICP27 gene were also PCR amplified.
Independently derived isolates were identif ed and characterized for the presence of the correct DNA construct containing either the gB, gC, gD, or ICP27 gene.
Large scale DNA preparation was essentially as described 10 (Montgomery, D.L. et al., supra) except that 800 ml cultures were grown for 24 to 48 hours and for some ~l)e~ lcllL~ DNA was purified by a single CsCI-EtBr isu~yl~ ic density centifugation.
The plasmid constructions were characterized by restriction mapping and sequence analysis of the vector-insert junctions. Results 15 were cnnsistf~nt with published HSV-2 strain G (Lasky, L.A. et al., 1984, DNA, 3, pp.23-29) sequence data and showed that initiation and termination codons were intact for each construct.

Expres.sion of HSV-2gB. gC. gD and ICP27 proteins from VIJ plasmids Rhabdomyosarcoma cells (ATCC CCL136) were planted one day before use at a density of 1.2 Xl o6 cells per 9.5 cm2 well in six-well tissue culture clusters in high glucose DMEM supplemented with 25 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 25 mM
HEPES, 50 U/ml penicillin and 50 llg/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 the kit instructions except that 5 - 15 llg 30 is used for each 9.5 cm2 well of RD cells. Cultures were glycerol shocked six hours post addition of calcium phosphate-DNA precipate;
after refeeding, cultures were incubated for two days prior to harvest.
Lysates of transfected cultures were prepared in IX RIPA
(0.5% SDS, 1.0% TRITON X-100, 1 % sodium deoxycholate, ImM

... . , . . . ... . . .. . . , _ WO 96/03~10 1 ~ C.,l ~
21 q'J09q EDTA, 150mM NaCI, 2~ mM TRIS-HCI pH 7.4) supplemented with IIlM leupeptin, IIlM pepstatin, 300nM aprotinin, and IOIlM TLCK, and sonicated briefly to reduce viscosity. Lysates were resolved by electrophoresis on 10% Tricine gels (Novex) and then lldll~rell~,d to nitrocellulose membranes. Immunoblots were processed with HSV-2 convalescent mouse sera and developed with the ECL detection kit (Amersham~.
Expression of HSV gD from VlJ:gD was de-llu~ ldt~d by transient transfection of RD cells. Lysates of VlJ:gD-transfected or mock transfected cells were fractionated by SDS PAGE and analyzed by immunoblotting. Figure IA shows that VlJ:gD transfected RD cells express an immunoreactive protein with an apparent molecular weight of approximately 55 K. Ly.sates from HSV-2 (Curtis), HSV-2 (186), or mock-infected Vero cells are included for comparison. The identical migrations of cloned gD and the authentic protein from infected cells indicates that the protein is ful- length, and is processed and glycosylated similarly to that of gD in HSV-infected cells. Indirect ulullullufluorescence of fixed VlJ:gD transfected cells showed a diffuse cytoplasmic signal.
Expression of HSV gB from VlJNS:gB was demonstrated by transient transfection of RD cells. Lysates of VlJNS:gB-transfected or mock transfected cells were fractionated by SDS PAGE and analyzed by immunoblotting. Figure IB shows that VlJNS:gB transfected RD
cells express an imrnunoreactive protein with an apparent molecular 2~ weight of approximately 140 k. Lysates from HSV-2 (Curtis), HSV-2 (186), or mock infected Vero cells are included for comparison. The similar migrations of cloned gB and the authentic protein from infected cells shows that the protein is full-length. Indirect immunofluorescence of fixed VlJNS:gB transfected cells showed a membrane-associated punctate signal.

W0 96103510 2 1 9 5 0 9 9 F~

Expression of HSV gC from VlJ:gC was demonstrated by transient transfection of RD cells. Indirect immunofluorescence of fixed VlJ:gC transfected cells showed primarily a diffuse cytoplasmic signal.
Expression of ICP27 was demonstrated by transient 5 transfection of RD cells, followed by Western blot analysis. A mouse monoclonal antibody specific for ICP27 detected a protein of about 60 k Da, which is consistent with the major immunoreactive protein in HSV 2 infected cells (Figure IC).

I"""""i, .lion with PNV and detection of anti-HSV antibodies Five- to six-week-old female BALB/c mice were anesthetized by inlld,uelilulleal (i.p.) injection of a mixture of 5 mg 15 ketamine HCI (Aveco, Fort Dodge, IA) and 0.~ mg xylazine (Mobley Corp., Shawnee, KS.) in saline. The hind legs were shaved with electric clippers and washed with 70% ethanol. Animals were injected with a total of 100 111 of DNA suspended in saline: 50 111 each leg.
The ability of VlJ:gD DNA to elicit an immune response to 20 HSV gD was first examined in a titration e~,ueliu~ L. Groups of ten mice received i.m. injections of DNA in a dose range from 200 llg to 0.78 llg (8 two-fold dilutions) or were sham i""llll~ d with saline. Sera, obtained four and six weeks post i, ,,,, ,,ll,i,~ ion, were analyzed by ELISA. For the ELISA, HSV-2 glyuuplulei l was diluted to 5 llg/ml in 25 50 mM carbonate buffer pH 9.5. Nunc Maxi-sorb flat bottom 96-well plates were coated at 4~C, ovemight with 100 1l1 per well of HSV
glycoproteins. Plates were washed four times with PBS pH 7.2 and nonspecific reactivity was reduced with blocking and dilution buffer, 20 mM TRIS-HCI pH 7.5, 137 mM NaCI, 2.7 mM KCI, 0.5% gelatin, 0.05%
30 Tween 20 for one hour at room telll~,eldlul~. Serial dilutions of mouse sera were added, and plates were incubated one hour at room temperature. Plates were washed four times with PBS and once with distilled water prior to the addition of alkaline phosphdtase-labeled goat anti mouse IgG (Boehringer Mannheim, Indianapolis, IN) and incubated .. . .... _ . . . . .

WO 96~03510 1 ~
21 950~9 - 2~ -for one hour at room temperature. Excess secondary antibody was removed with four PBS washes followed by one distilled water wash.
The ELISA was developed with the addition of 100 111 per well of I
mg/ml p~ lupll~llylphosphate in 10% diethanolamine pH 9.~ IOO~lg/ml 5 MgCI-6 H20 at 37~C. Absorbance was read at 405nm and serum dilutions were scored as positive if the OD405 was greater than the mean plus three standard deviations of the same dilution of the saline control sera. By four weeks the majority of animals receiving > 6.25 llg of DNA
were seropositive. At doses lower than 6.25 ~lg, fewer animals had 10 seroconverted, however even at the lowest dose some animals were ELISA positive. None of the saline injected control animals were positive. At six weeks a majority of the animals had become seropositive.
At seven weeks, the animals were re-illllllllll;~d with the 15 same doses of DNA (or saline) used in the initial injections. Sera were obtained at ten weeks (three weeks after the second injection) and endpoint titers were deterrnined by ELISA. The results are ~ull~,.ali~ed in Table 1. By ten weeks, 93% of the DNA injected mice were seropositive. Even at the 0.7~ g dose, eight of the nine animals were 20 positive.

WO 96/03510 2 1 9 5 ~ ~

Table I

Seroconversion of mice imm~lni7f~d with VIJ:~D ~NAa DNA dose (u~ no. seropositive/no. ill",.,ll,;~d EL~
GMTb 200 9/9 31,R08 100 10/10 44,904 9/10 8,027 8/8 13,512 12.5 10/10 14,199 6.25 10/10 16,016 3.13 10/10 9,054 1.56 7/10 360 0.7~ 8/9 4,641 saline 0/10 10 a - Mice were il,.""ll,i,rd at weeks O and 7 with the indicated amount of DNA. Sera were obtained at 10 weeks and assayed as described herein.
b - For purposes of calculating the GMT, sera negative at the lowest dilution tested (I :30), were assigned a value equal to one dilution less, i.e.
10 1:10.
To confirm that the ELISA reactivity was due to anti-gD
antibodies, several high ELISA titer sera were characterized by their reactivity with immunoblots of HSV or mock-infected cell Iysates.
Figure 2A illustrates that sera from VlJ:gD il,l,,,~llli,lSd mice react 15 specifically with a single HSV encoded protein with an electrophoretic mobility consistent with that of HSV gD. Taken together, these data show that i.m. injection of mice with VlJ:gD DNA results in the expression of gD epitopes and the development of an immune response to - gD protein.

WO96/03510 21 950q9 ~ Jv t~

To extend these results and to establish the minimal effective PNV dose, VlJ:gD was titrated further in an experiment where animals were immllni7Pd only once. Groups of mice were injected with VlJ:gD
DNA ranging from S ng to 50 !lg Sera collected at four, seven, and ten S weeks post i""~ ;".lion were assayed by ELISA; the data are summarized in Figure 3.
This titration reveals a threshold of response of about 0.5 ,ug DNA. While a few animals receiving lower amounts oi~ DNA were seropositive by ELISA, the positive respon.se was transient and occurred 10 only at the lowest serum dilution. At DNA doses of 2 1.67 llg, more than 90% of animals seroconverted by four weeks and remained positive at seven and ten weeks.
Increases in antibody titers of individual animals with time are reflected by increases in the group GMTs seen in Figure 3. At doses 15 > 0.5 ~Lg, the GMT rises sharply between four and seven weeks. Between seven and ten weeks, the titers increase or remain constant in all but one case. The ten-week ELISA titers of ar~imals receiving ~ 0~ llg are similar to those attained in the previous ~ Jelhllellt where a second DNA
i"""""i ".1 ion was given. The amounts of PNV required to provoke an 20 immune response was as much as 100-fold less than published reports using similar vectors. (Robinson et al., 1993, Vaccine, 11, pp.957-960;
and Fynan et al., 1993, Proc.Natl.Acad.Sci. USA, 90, pp.l 147~-114~2) In order to establish a standardi~ed protocol, the effectiveness of one- and two-dose i"""""i,~t;on was compared. ln the 25 two-dose c~,e~ , we found no signifi~nt differences in protection at the highest (200 llg) dose and the lowest (0.78 llg) dose. When the titration was extended in the single-dose experiment, a dose response for ELISA CMT was observed, showing efficiency of seroconversion and protection. The threshold for these responses was about 0.5 llg.
30 However, .seroconversion does occur with as little as 50 ng of DNA. In general, titers continued to rise through ten weeks after a single WO96/03510 21 9 5099 ~ J... a3~

injection and in the two-dose experiment there was no obvious boost in titers after the second injection. Finally, at 50 llg of DNA, the only dose common to both experiments, there were no .signifir~nt differences between one and two dose3.
Similar analyses as set forth above were done for the PNV
constructions containing the HSV genes gB and gC. Mice (10 mice per group) were i~ "ll"i,rd as described above with DNA containing the HSV gB gene or DNA c~nt~ining the HSV gC gene. Serum was collected and analyzed for the presence of anti-gB or anti-gC antibodies in the ELISA described above. The ELISA data for gB antibodies are shown in Table 2, and demonstrates that mice ill""ll"i,rd with VlJNS:gB were seropositive for anti-gB antibodies.

10 WEEK SERA ELISA gB
Treatment weeks GMT SEM (range) saline 0 3 3-3 I ug 0 3 3-3 lug 0, 7 3 3-3 3ug 0 6 4-10 3ug 0, 7 24 8-68 lOug 0 48 13-170 lOug 0, 7 150 38-595 30ug 0 300 87-1034 30ug 0, 7 39 10-150 l OOug 0 378 69-2062 lOOug 0, 7 7536 1893-30,000 The ELISA data for gC antibodies are shown in Table 3, and demonstrates that mice (five mice per group) illlllllllli~rd with VlJ:gC
were .seropositive for anti-gC antibodies.

10 WEEK SERA ELISA gC
Treatment weeksGMT SEM (range) saline 0 10 10-10 S IOug 0, 74642 2154-10,000 lOOug 0, 7 3162 1440-6943 To confirm that the ELISA reactivity for gB was due to anti-gB antibodies, several high lELISA titer sera were characterized by their reactivity with immunoblots of HSV or mock infected cell Iysates.
Figure 2B illustrates that sera from VlJNS:gB immnni7~d mice reacts specifically with a single HSV encoded protein with an ele-;LIupl~ol~lic mobility consistent with that of HSV gB. Taken together, these data show that i.m. injection of mice with HSV PNV results in the expression of HSV epitopes and the development of an immune response to those HSV proteins.

HSV Neutrali7ation Mouse sera were heat-inactivated at 56~C 30 minutes prior to serial dilution in DMEM, 2% heat inactivated FBS and then 50 ~11 of 25 each dilution was delivered to duplicate wells in a sterile polypropylene 96 deep well plate (Marsh Biomedical, Rochester, NY.). HSV-I or HSV-2 stocl;.s were diiuted to 4,000 pfu/ml, 50 111 of virus were then added to each sample well and the plate was incubated overnight at 4~C. Guinea pig complement (Cappel) was diluted I :4 in DMEM, 2% heat inactivated 30 FBS and 50 1ll were added to each sample well. After a one hour incubation at 37~C, 100 111 of serum free medium was added to each well and each reaction mixture wa.s used to infect confluent VERO cells in 12-well cluster plates. (Costar) Neutralized virus samples were adsorbed for one hour at 37"C. Inocula were gently aspirated and monolayers were -~ WO96/03510 2 ~ q 50q9 r~ J. r~ ~3, overlaid with I ml 0.5% carboxymethylcellulose IX MEM 5% heat inactivated FBS 10 mM l-~lu~ hle, 25U/ml penicillin, 25 llg/ml streptomycin, 12.5 mM HEPES. Plates were incubated at 37~C for4~
hours. Overlays were removed and cell monolayers were stained with 5 1% basic fuchsin, 50% methanol 10% phenol. Plaques were counted and the neutralization titer was determined as the serum dilution which yielded a 50% reduction in plaque number when compared to sera from sham-h,-...u--i~ed mice.
To determine whether the anti-gD antibodies might be 10 biologically active, selected high-titer ten-week sera from mice il"""l"i,ed at zero and seven weeks were assayed for HSV-2 neutralizing activity. The results of plaque reduction assays are in Table 4. Sera from VlJ:gD immnni7Pd mice neutralized not only HSV-2 (Curtis) but also HSV-2 (1~6). Furthermore, at least some of the sera contain type 15 common neutralizing antibodies as shown by theirneutralization of HSV-I (KOS) infectivity. Although the neutralizing titers were low in some cases, these results encouraged us to see if these anti-gD antibodies could protect the animals against a lethal HSV f h~ nge Ten week sera from all animals il"".,llli,ed with 20.5 llg 20 VlJ:gD in the single-dose experiment were also tested in an HSV-2 plaque reduction assay. Twenty-nine of the forty-nine sera assayed were positive: >50% plaque reduction at a 1:10 dilution. At the 16.7 and 50 ',lg dose level, nine of ten sera from each group were neutralization positive.

WO 96/03510 2 1 9 5 0 9 9 r~

HSV-2 Curtis neutralization animal no.(serum dilution) DNA dose (llg) ELISA titer 1:10 1:100 1:1000 4353 a + + - 3.13 2 33,333 4354 + + - 3.13 33,333 4362 + + - 6.25 > 33,333 4363 b + - - 6.25 2 33,333 4371 + + - 12.5 > 33,333 4391 + + + 50 1000 4392 a + + + 50 10,000 4395 - - - 50 < 100 4396 b + + - 50 233,333 4397 + - - 50 2 33,333 4398 + + + 50 2 33,333 4399 + + + 50 2 33,333 4400 + + - 50 3,333 4405 b + + + 100 100,000 a - neutralizes HSV-I KOS at 1:100.
5 b - neutrali~ed HSV-2 186 at 1: 100.

HSV Ch~llen~e Stocks of challenge virus were prepared by infection of confluent VERO monolayers with HSV-2 Curtis as described above.
Clarified supematant virus was titered on VERO cells and ali~uots were stored at -70~C Anirnals were infected by i.p. injection with 0.25 ml of virus stock and then observed for three weeks. Survival data were 15 analy~ed using the log-rank test (McDemmott et ak, 1989, Virology, 169, WO96/03510 2 l 9 509~ P._l/u~

pp.244-247) in the SAS~) procedure LIFETEST. Differences in probability < 0.001 were judged highly significant.
Eleven weeks after the initial DNA injection, mice immnni7ed with two doses of VlJ:gD were challenged by i.p. injection of 5 105.7 p.f.u. of HSV-2 (Curtis) and observed for 21 days. Survival data are in Figure 4. It is readily apparent that animals i~ ed with as little 0.7~ llg of VlJ:gD were .cignific~ntly protected from lethal infection. Of the three il,,,llll,,i,~d animals that died, two were .seronegative by ELISA at ten weeks. A few of the surviving animals did 10 show signs of tran.sient illness including failure to groom, failure to thrive, or a hunched posture. While the level of protection from death achieved at every dose of DNA was sip;nific~nt (p< 0.01), these symptoms suggest some break-through infection occurred. Analysis of sera obtained from convalescent animals were characterized by their 15 reaction in immunoblots of HSV-2 infected Vero cell Iysates. In some cases, a serum recognized only gD and in others, the serum reacted with many HSV proteins. These results are consistent with at least some of the mice having experienced HSV infection.
Animals immllni7ed with a single DNA injection were 20 challenged as described above. Survival data are p~esented in Figure 5.
Statistically .cignific~nt (p < .001) protection against death was obtained in groups of animals receiving 21.67 ,ug VlJ:gD. This survival dose response is similar to that seen for ELISA titer (Fig. 3). As was seen in the two-dose experiment, a few surviving animals displayed transient 25 signs of illness during the observation period. Surviving animals ill,lllll"i,.ed with higher doses of DNA (16.7 and 50 llg) remained sleek and healthy-looking throughout the observation period.
Animals il~""l~ ed with PNV constructs cnnt~ining HSV
gB or gC genes were also eh~ nged with a lethal dose of HSV as 30 described above for gD. Survival data for animals illllllllll;,rd with VlJNS:gB are shown in Figure 6, and survival data for animals i" " "", li,Pd with V IJ:gC are shown in Figure 7 demonstrating that protection from death was obtained.

WO 96/03510 2 ~ 9 5 ~ 9 9 These results demonstrate the potential for direct DNA
ion in the prevention of HSV infection. Using glycoprotein gD as a model, it was found that a single i.m. injection of as little as 0.5 llg V lJ:gD DNA elicited a neutralizing antibody response to gD that 5 afforded statistically significant protection against lethal HSV challenge.
lllllllllrli~lion with as little as 3.13 ~lg of the DNA in a two-dose regimen protected all animalc from death.

E~XAMPLE 9 Vaccination of Guinea Pigs with HSV PNV
Hartley strain guinea pigs (Harlan Sprague Dawley Labs, Indianapolis, IN) weighing d~ ately 200-250 grarns each were vaccinated intr~mllce~ rly 0.1 ml in the right thigh and 15 0.1 ml in the left thigh at 11 and 4 weeks prior to virus challenge. Fresh solutions of the vaccine and placebo were sent to us for each vaccination.
In order to determine HSV-2 antibody production in the animals, the guinea pigs were bled 5 weeks after the first vaccination and 2 weeks after the second vaccination. Blood (0.6-1 ml per animal) was 20 obtained by toe clipping. The blood was collected in mi~ s~àlalion tubes (Becton Dickinson), and was later centrifuged at 1000 x g for 10 minutes to separate the serurn.
The sera collected from the guinea pigs was analyzed for the presence of anti-HSV antibodies using the ELISA set forth in Exarnple 6.
25 The results are shown in Table 5.

10 WEEK GUINEA PIG SERA ELISA ~D
TreatmentGMT SEM (range) saline 3 3 3 lOug DNA 19 ~-43 lOOug DNA 599 277-1295 ~ W096/03510 21 q50qq J~ 3.~1 At the time of infection, the guinea pigs weighed 600-700 grams each. They were infected intravaginally with herpes simplex virus type 2 (HSV-2), E194 strain. This was accomplished in a 3-step process.
First, the vagina of each animal was swabbed for 5 seconds with a cotton tip applicator dipped in 0.1 N NaOH. This treatment irritates the vaginal area so that the infection takes better. Approximately 45-60 minutes later each vagina was dry swabbed for 5 seconds. Then an applicator dipped in virus medium (about 5 x 106 plaque forming units of HSV-2 per ml) was used to swab each guinea pig for 20 seconds. The swabs were gently and slowly twisted back and forth during the time they were in place.
Lesion scores in infected animals were determined daily at day 2-15 post infection. A score of l+ indicates about 25% of the anal-vaginal area was affected (usually by redness imm~ t~ly around the vagina); 2+ indicates 50% of the anal-vaginal area affected; 3+ indicates 759~O affected; and 4+ indicates 100% affected. Because some of the animals went on to die, the lesion score near the time of death carried through to the end of the 15 days. If this were not done, average lesion scores would appear to go down since the most affected animals died off.
Deaths were recorded daily for 21 days. The mean day of death calculation took into account only guinea pigs that die. Numbers of animals with hind limb paralysis were noted throughout the infection.
Vaginal virus titers were made by titration of virus obtained from vaginal swabs at 2, 4 and 6 days after virus inoculation. The swabs were placed into tubes c~-nt~ining I ml of celi culture medium. The titration of these samples was c--n~llnt~d in Vero cells in 96-well plates. Calculation of virus titer was made by the 50% endpoint dilution method of Reed L. J.
and Muench M., A~1. J. Hyg~. 27, 493~98 (1938). Virus titers were expressed as loglo cell culture infectious doses per ml.
Statistical interpretations of survival (Fisher exact test), mean days to death (Mann-Whitney U test), paralysis (Fisher exact test), vaginal virus titers (Mann-Whitney U test), and vaginal lesion scores (Mann-Whitney U test) were made by two-tailed analyses.

WO 96/03~;10 I ~
21 9~099 Figure 8 shows the results of survival, mean days to death, paralysis, and vaginal viru.s titers in HSV-2 infected guinea pigs. The high dose of vaccine prevented mortality and reduced vaginal virus titers on days 2 and 4 relative to the placebo control. The high dose of vaccine significantly prevented paralysis in these animals. The low dose of vaccine also reduced the above parameters.
Table 7 shows daily vaginal lesion scores for the ~e~ L Both the high and low doses of the vaccine caused signific~ni reductions in vaginal lesion severity from days 3 through 15 of the infection compared to the placebo group. The results in Table 7 are presented graphically in Figure 9.
These results clearly indicate that the vaccine was protective in guinea pigs infected with HSV 2, and that the high dose of vaccine was more active than the low dose. The high dose of vaccine was not able to completely block the infection, since virus was recovered from vaccinees and a low grade of vaginal lesion development occurred.
Nevertheless, the degree of protection afforded by the vaccine at this dose was substantial. The results of the antibody studies correlate with antiviral protection.
The vaccine :~d~ t;d intr~mnsc~ rly at two different doses to guinea pigs 11 and 4 weeks prior to intravaginal HSV-2 challenge ~ignifir~ntly protected animals from the disease. The high dose of vaccine was more effective than the low dose. The vaccine appears to be safe in the animals.

WO 96/03510 r~ c~ -~1 95~)99 DaYb Vaccinea~Q~ Vaccinea~ 100 llg Placebo 3 0.2 _ 0.2 0. 1 _ 0.20.3 _ 0.3 4 0.2 _ 0.3* 0.3 i 0-4* 0.7 _ 0.3 0.4 + 0.4** 0.4 + 0.4** 1.6_ 1.1 6 1.0 _ 1.0 0.5 _ 0.6* 1.9 I .4 7 1.5 + 1.2* 0.6 _ 0.7*** 3.0 + 1.3 ~s 1.0 _ 1.0** 0.5 i 0.5*** 3.2 + 1.3 9 0.6 _ 0.7*** 0.6 _ 0.7*** 2.7 1.4 0.5 i 0.5*** 0.4 _ 0.5*** 2.5 1.1 Il 0.5+0.5*** 0.3+0.4*** 2.3+0.8 12 0.5+0.4*** 0.3~ 0.4*** 2.4_0.7 13 0.5 _ 0.5*** 0.5 + 0.6*** 2.3 _ 0.8 14 0.5 _ 0.6*** 0.6 _ 0.6*** 2.3 _ 0.6 0.5 + 0.6*** 0.7 + 0.7*** 2.0 + 0.7 Grand Avg. 0.6 + 0.4*** 0.4 + 0.2*** 2.1 + 0.8 (Days 3-15) a Intr~mnsc~ r vaccinations were given 11 and 4 weeks before virus 5 ~h~llPngP.
b A.fter virus inoculation.
* P<0.05, **P<O.OI, ***P<O.OOI.

EXA~PLE 10 -~
To ~IPtPrminP whether mice vaccinated intr~mnsc~ rly with PNV HSV would produce mucosal HSV-specific antibodies, mice were vaccinated with 12.5 or 1.56 llg of VlJNS:gD. Vaginal fluid was collected by swab and the antibodies were eluted from the swab using . .

WO96/03510 2 1 9 ~09~ C~l ~

phosphate buffered saline. The eluant was analyzed for the presence of IgG and IgA, specific for HSV-2 protein. The ELISA was performed as described above except that commercially available antibodies specific for mouse IgG (Boehringer) and specific ~or mouse IgA (Seralab) were 5 used to detect the presence of HSV-specific IgG and IgA in the mouse vaginal samples. The results for IgG are shown in Table ~s; IgA was not detected in any animal.

TABLE
Animal ELISA Development Time (minutes) No. 30 60 1031 <0.1 <0.1 1032 0.1 0.1 1033 0.01 0.01 1035 _ <0.1 0.1 1037 <0.1 <0.1 1038 <0.1 <0.1 1039 <0.1 <0.1 1040 <0.1 <0.1 a injected with saline 25 b - injected with 1.56 ~g VlJNS:gD

The results demonstrate the presence of mucosal IgC
specific for ~ISV-2 in mice vaccinated with V lJ:gD.

~ W 096/03510 21 95099 r~

- 4l -SEQUENCE LISTING

(1) GENERAL INFORMATION:
(i) APPLICANT: Armstrong, Marcy E.
Keys, Robert D.
Lewis, John A.
Liu, Margaret A.
McClements, William L.
(ii) TITLE OF INT~ENTION: A POLYNUCLEOTIDE HERPES VIRUS VACCINE
(iii) NUMBER OF SEQUENCES: 15 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: John W. Wallen III
(B) STREET: 126 E. Lincoln Avenue (C) CITY: Rahway (D) STATE: New Jersey (E) COUNTRY: USA
(F) ZIP: 07056-0900 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/279,459 (B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Wallen III, Uohn W.
(B) REGISTRATION NUMBER: 35,403 (C) FEFERENCE/DOC ~ T N~MBER: 19258 (ix) TT~TE~MMTTNICATION INFORMATION:
(A) TELEPHONE: (908) 594-3905 (B) TELEFAX: (908) 594-4720 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHP~RACTERISTICS:
(A) LENGT~: 23 base pairs (B) TYPE: nucleic acid (C) STRAT~T~TTTEcc single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA ~genomic) W 096/03510 2 1 9 5 0 9 9 P~

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
CTATATAAGC AqAGCTCGTT TAG 23 (2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (B) TYPE: nucleic aci-d (C) sTR~NnEnN~ss single (D) TOPOLOqY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPT~ON: SEQ ID NO:2:
GTAGCAAAGA TCTA~GGACG GTGACTGCAG 30 (2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) STR~NnEnN~CC: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
GTATGTqTcT qAAAATqAGC GTqGAqATTq qqCTCGCAC 39 (2) INFORMATION FOR SEQ ID No 4 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) sTR~NnEnN~cc: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (~i) SEQ~ENCE DESCRIPTION: SEQ ID NO:4:
GTGCGAGCCC AATCTCCACG'CTCATTTTCA qACACATAC 39 (2) INFORMATlON FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 78 base p~irs ~ W 096103510 2~ 95099 .~ a~ J/

(B) TYPE: nucleic ~cid (C) sTRANnFn~Ecs single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi~ SEQUENCE DESCRIPTION: SEQ ID NO:5:
GATCACCATG GATGCAATGA AGAGAGGGCT ~ ' GTGGAGCAGT 60 (2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS: :
(A) LENGTH: 78 base pairs ,(B) TYPE: nucleic acid ~C) sTR~Nn~nNEc~ single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
GATCTCGCTG rGrr~Arr~ AGACTGCTCC ACACAGCAGC AGCACACAGC AGAGCCCTCT 60 . E~

(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STR~NDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi~ SEQUENCE DESCRIPTION: SEQ ID NO:7:
GTACCTCATG Ar-rrAr~TA~ TACCATG 27 (2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 40 base pairs (B) TYPE: nucleic ~cid (C) sTRANn~nN~cc single (D) TOPOLOGY: linear WO96/03510 r~ . J5~
2l 95~9q ~ii) MOLECULE TYPE: DNA ~genomic) ~xi~ SEQUENCE DESCRIPTION: SEQ ID NO:8:

~2) INFORMATION FOR SEQ ID NO:~:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 ~ase pairs ~Bl TYPE: nucleic acid ~Cl sTRANnFnNE~c: single ~D) TOPOLOG~ linear ~ii) MOLECULE TYPE: DNA (genomicl ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
r~AoATAr.AT CTCCATGGGA ACTA~AGGAA GACGGTCTGT TC 42 ~2) INFORMATION FOR SEQ ID NO:l0:
(i) SEQUENCE CHAP~ACTERISTICS:
(A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) sTRANn~nNRcc: single ::
~D) TOPOLOG~: linear (ii~ MOLECULE TYPE: DNA (genomic) (xi~ SEQUENCE DESCRIPTION: SEQ ID NO:l0:

~2) INFORMATION FOR SEQ ID NO:ll:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 36 base pairs ::
~B) TYPE: nucleic acid ~C) STRAN~Fn~ : single (D) TOPOLOGY: linear (ii) MOLECULE ~YPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll:
CCACATCTCG Ar.~AArrr~r.-TcAATTcTTc AGCACC 36 ~ W096/035l0 21 950~9 .~

(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS: -(A) LENGTH: 38 bAse pairs ~ (B) TYPE: nucleic acid (C) 5~R~NDEnNES~: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:

(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE r~R~TERTcTIcs:
(A) LENGTH: 37 base pairs (B) TYPE: nucleic acid (C) sTR~NDEnN~s~ single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) s~R~NnEnNE~ single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) sTR~Nn~nN~ single (D) TOPOLOGY: linear WO 96/03510 P~
21 95~99 (ii) MOLECULE TYPE: DNA (genomic) (xi) sEQuENcE DESCRIPTION: SEQ ID NO:15:
CCACATGTCG ACCCGTAAAA"AGGCCGCGTT GCTGG _. 3

Claims (11)

WHAT IS CLAIMED IS:

1. A polynucleotide which induces anti-HSV antibodies or protective immune responses upon introduction into vertebrate tissue, wherein said polynucleotide comprises one or more genes encoding one or more HSV proteins or functional equivalents thereof, said genes being operably linked to a transcription promoter.

2. The polynucleotide of Claim 1, wherein said gene encodes an HSV protein selected from a group consisting of gB, gC, gD, gH, gL, ICP27, and functional equivalents thereof.

3. A method for inducing immune responses in a vertebrate against HSV epitopes which comprises introducing between 1 ng and 5 mg of a polynucleotide according to Claim 1 into a tissue of a vertebrate.

4. A vaccine for inducing immune responses against HSV which comprises the polynucleotide of Claim 1 and a pharmaceutically acceptable carrier.

5. A method for inducing immune responses against HSV which comprises introducing into a tissue of a vertebrate one or more isolated and purified HSV genes eliciting an immune response which prevents HSV infection and/or ameliorates HSV disease.

6. A polynucleotide comprising:

a) a eukaryotic transcription promoter;
b) an open reading frame operably linked to said promoter encoding one or more HSV epitopes, and a translation termination signal; and c) optionally containing 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 wherein said additional genes of c) are immunomodulatory or immunostimulatory genes selected from a group consisting of GM-CSF, IL-12, interferon, and a member of the B7 family of T-cell costimulatory proteins.

8. The polynucleotide of Claim 6 wherein said HSV
gene of a) encodes an HSV protein selected from a group consisting of gB, gC, gD, gH, gL, ICP27, and functional equivalent thereof.

9. The polynucleotide of Claim 6 wherein said additional genes of c) are HSV genes selected from a group consisting of gB, gC, gD, gH, gL, ICP27, and functional equivalent thereof.

10. A method of treating a patient in need of such treatment with a ploynucleotide which induces anti-HSV antibodies or protective immune responses upon introduction into vertebrate tissue, wherein said polynucleotide comprises a gene encoding one or more HSV proteins or functional equivalents thereof, said gene being operably linked to a transcription promoter.

11. The method of claim 10 wherein said polynucleotide comprises a gene encoding one or more HSV proteins selected from a group consisting of gB, gC, gD, gH, gL, ICP27, and functional equivalents thereof.