VACCINE COMPOSITION FOR HERPES SIMPLEX VIRUS AND METHODS OF USING

Abstract: The present invention discloses a new vaccine composition for Herpes Simplex comprising a whole live HSV-2 virus having the oncogene deleted. Methods of using the vaccine composition are also included.

Description: TITLE OF THE INVENTION VACCINE COMPOSITION FOR HERPES SIMPLEX VIRUS AND METHODS OF L SING RACKGROI NT) OF THF INVENTION Heφes Simplex Virus (HSV) is a well-studied virus. Both distinguishable serotypes of Heφes Simplex Virus (HSV-1 and HSV-2) cause infection and disease ranging from relatively minor fever blisters on lips to severe genital infections, and generalized infections of newborns. HSV-1 and HSY-2 are 50% homologous at the DNA lev el, and polyclonal antibodies and MAbs to shared epitopes for one are cross-reactive to the other. HSV-1 and HSV-2 ha\ e RR1 proteins (respectively designated ICP6 and ICP10) that contain a unique amino terminal domain. The HSV-2 unique domain codes for a ser/thr- specific PK which has auto- and transphosphorylating activity and has a transmembrane (TM) domain. Sequences which code for the PK domain cause neoplastic transformation and are associated with cervical cancer (HSV-2 oncogene). The unique terminal domain of the HSV-1 RR1 protein (ICP6) also has PK activity but it is different from that of the HSV-2 oncoprotein, both structurally and functionally. Original studies, using enzymatic assay conditions similar to those employed for ICP 10 PK, concluded that ICP6 does not have PK activity, although the unique domain is retained (Chung et al.. J Virol 63 3389-3398, 1989) This was not unexpected since the sequence of the unique PK domains showed only 38% homology (Nikas et al.. Proteins: Structure, function and genetics 1.376-384, 1986). Further studies indicated that ICP6 has PK activity but only under different conditions. There are conflicting results about its ability to transphosphorylate other proteins (see Peng et al., Virology 216:184-196, 1996 for a review of the problem; particularly Table 1 ) The reason for the different PK activities of the ICP6 and ICP 10 proteins is likely to be that the ICP6 PK ATP binding sites are located distantly from the rest of the catalytic motifs (Cooper et al., J. Virol 69'4979-4985. 1995). ICP6 also does not have a functional TM domain and it does not localize to the cell surface (Conner et al.. Virology 213:615. 1995) The PK ac ity of the native ICP6 is \ er w eak e\ en under ideal conditions, such that its K is 10-fold higher than that of ICP 10 PK (Peng et al., Virology 216: 184, 1996. Lee and Aurehan. in preparation) The transtorming activ ity of ICP6 is located w ithin a genome fragment that is distant from that at which the HSV2 oncogene is located Transformation in this system is moφhologic (focus forming abihty) It has pre\ lously been shown that DNA sequences which encode for the amino- terminal one-third of ICP10 (amino acids 1 -417) hav e oncogenic potential Cells transfected with these DNA sequences evidence anchorage independent growth and cause tumors in animals Transformation is seen in both rodent and human cells (Jaπwalla et al , PNAS 77 2279-2283, 1980. Hayashi et al PNAS 82 8493-8497, 1985, Smith et ai Virology 200 598-612. 1994, Hunter et al Virology 210 345-360, 1995) There are three functional domains within ICP10 amino acids 1-41 1 (I) an intracellular domain, at amino acids 106-41 1 , which encompasses the PK catalytic domain with eight conserv ed catalvtic motifs (amino acids 106-41 1 ), (n) a TM, at amino acids 88- 105, and (in) an extracellular domain at amino acids 1-88 (Chung, et al , J Virol 63 3389- 3395, 1989, Virology 179 168- 178, 1990) The minimal size required for PK activity is amino acids 1-283 (pp29 ) (Luo et ai J Biol Chem 266 20976-20983, 1991 ) However, the PK activity of pp29 Ui has some properties different from the authentic ICP10 PK, presumably because it lacks part of the PK catalytic domain VI (Luo et al , J Biol Chem. 266. 20976-20983. 1991) The TM domain is also required (but insufficient) for PK activity (Luo and Aurelian. J Biol Chem 267 9645-9653, 1992) Therefore, it can be concluded that the PK acti ity is localized within amino acids 88-41 1 with an essential core at amino acids 88-283 The unique Hpal site ithin the ICP10 coding region represents the 3' end of the transforming region (Jaπwalla et al , Proc Nat Acad Sci 77 2279-2283, 1980) and cuts the gene after the codon for amino acid residue 417 It is not known whether pp29 ial has transforming activity Howev er, PK activity is required for neoplastic potential PK negative mutants do not transform cells This includes a mutant deleted in the TM domain and site directed mutants in the ATP binding sites (Lvs 1 '' and/or Lys 2M) ) or the ion-binding site (Glu 209 ) (Smith et al Virology 200 598-612. 1994. Aurelian, L Frontiers in Biology, in press) Because a PK mutant deleted only in the TM domain does not ha e transforming activity (Smith et al Virology 200 598-612. 1994) DNA sequences that code for ICP10 amino acids 106-41 1 but lack PK activ itv are not intrinsicallv neoplastic This demonstrates that (i) the HSV-2 oncoprotein is located w ithin ICP10 amino acids 1-41 1. and (u) neoplastic potential requires a functional PK activ itv The function of ICP 10 PK in v irus growth/pathogenesis is unknown The HSV-2 ICP 10 protein has intrinsic PK activ ity This as shown by demonstrating that ICP 10 PK activity is lost through site-directed mutagenesis The oncogene also has SH3- binding sites at positions 140, 149 and 396 w hich are required for interaction with signaling proteins This interaction is required for transforming activity Site directed mutagenesis was used to identify amino acids required for kinase activity and interaction with signaling proteins Mutation of Lys' 6 or Lys 2 " reduced PK activity (5-8 fold) and binding of the l4 C- labeled ATP analog p-fluorosulfonylbenzoyl 5'-adenosιne (FSBA), but did not abrogate them. Enzymatic activity and FSBA binding w ere abrogated by mutation of both Lys residues, suggesting that either one can bind ATP Mutation of Glu 209 (PK catalytic motif III) virtually abrogated kinase activity in the presence of Mg " or Mn 2 ions, suggesting that Glu 209 functions in ion-dependent PK activity ICP10PK functions as a growth factor receptor involved in signaling and it binds the adaptor protein Grb 2 in vitro The SH3-bιndιng sites ithin the ICP 10 PK domain (at positions 140, 149 and 396) are required for interaction with signaling proteins and, thereby transformation (Nelson et al , J Biol Chem 271 17021 -17027. 1996) Mutation of the ICP10 prohne-πch motifs at position 396 and 149 reduced Grb : binding 20- and 2-fold respectively Binding was abrogated by mutation of both motifs Grb 2 binding to wild type ICP 10 was competed bv a peptide for the Grb 2 C-terminal SH3 motif indicating that it involv es the Grb -terminal SH3 (Nelson et al , J Biol Chem 271 17021 -17027, 1996) The ICP 10 PK catalytic domain also contains amino acids at position 106-178 that are responsible for binding a down-regulator of PK activity (ras-GAP) Deletion of amino acids 106-178 reduces, but does not abrogate. PK activity (Luo and Aurelian, J Biol Chem 267 9645-9653, 1992) However, it abrogates ras-GAP binding, thereby increasing transforming potential (Nelson et al manuscript in preparation) The construction of the ICP 10 PK virus is described by Peng et al (Virology 216, 184-196. 1996) Briefly, the wild type sequences in a plasmid (TP101 ) that contains the HSV- 2 BamHI E and T fragments were replaced ith the 1 8kb Sail Belli fragment from pJHL9 pJHL9 is a plasmid containing the ICP 10 mutant deleted in the PK catalytic domain (Luo and Aurelian. J Biol Chem 267 9645-9653, 1992) The resulting plasmid, TP9, contains sequences hich code for ICP 10 deleted in the PK catalytic domain flanked by 4 and 2.8 kb of HSV-2 DNA sequences at the 5' and 3' ends, respectiv ely The lOkb HindllL EcoRl fragment from TP9 was introduced by marker transfer into a v lrus (ICP10ΔRR) in which the RR domain of ICP 10 had been replaced with the LacZ gene The resulting recombinant irus, designated ICP10ΔPK, was obtained by selecting white plaques on a background of blue plaques after staining ith X-gal A few white plaques were picked and purified Two were grown in Vero cells with 10% serum (exponentially growing) into individual stocks respecti ely designated RF and CS There are several known HSV vaccines in the prior art US patents 4,347,127, 4,452,734, 5,219,567, and 5.171 ,568 each teach subunit vaccines which provide some protection against HSV-2 infection These vaccines are infeπor to one in which a live, attenuated virus is used The immunity induced by a subunit vaccine is restπcted to the particular protein represented by the subunit. which may not hav e sufficient protective potential Additionally it is non-replicating and there is, therefore, no amplification of the protein which would further reduce immunogenicity These problems occur in any subunit vaccine regardless of whether the method of preparation is via a recombinant protein or the puπfication of an antigen from the virus A cross recombinant vaccine, such as disclosed in US 4,554,159, does not suffer from the problems of the subunit vaccines, but contains the oncogene present in HSV-2 Unless care is taken to define and delete the oncogene, the cross recombinant vaccine would induce cancer in the vaccinee The cross recombinant of '159 is temperature sensitive Av irulence may be obtained by selecting temperature resistance, but the temperature of the mouse is 39°C while that of humans is 37°C This temperature sensitivity could well render such a cross problematic in a vaccine A superior method of selection of avirulence is by the remov al of genes coding for virulence without respect to the temperature at which the virus replicates Also, the use of prototypical crosses would preclude the use of mutants with deleted or inserted genes Due to the many type-common epitopes on HSV-1 and HSV-2, the antibodies in human serum are cross-reactiv e (Aurelian, et al , J Natl Cancer Inst 45 455-464, 1970 ) It has also been previously shown that cell-mediated immunity cross-reacts (Jacobs et al , J Immunol 116 1520-1525, 1976) A live vaccine is superior to a dead v accine because the li e v accine induces herd immunity and it also induces different tv pes of immunity, such as mucosal. cell mediated and humoral immunity. A higher level of immunity is normally obtained because the virus titers are increased through replication within the vaccinee. Finally a live vaccine is of longer duration, thus obviating boosters and lowering initial dosage. However, an absolute necessity for a live heφes vaccine is the removal of the gene responsible for causing transformation, as in the present invention. Known vaccines are not virus type-specific. All known vaccines for HSV-1 or HSV-2 are cross-reactive and provide immunity to the other virus type. Most developed vaccines (viz. those in neurovirulence genes) are in HSV-1. However, HSV-1 is not as desirable a vaccine candidate against heφes, because the major clinical problem is the sexually transmitted HSV-2, which is also associated with cancer induction. Recent studies indicate that the age-adjusted prevalence of HSV-2 in the US is now 20.8%, an increase of approximately 30% over the past 13 years (Fleming et al.. New Engl. J. Med. 337:1105-1111, 1997). The increasing rate of HSV-2 acquisition among young adults increases the likelihood that infants will be exposed to HSV-2 at delivery, resulting in an infection that, despite antiviral therapy, is still life-threatening (Whitley, et al.. New Engl. J. Med. 327:782-799, 1992 [Erratum, N.Engl. J. Med. 328:671 , 1993]). A new concern about HSV-2 infection is that it may facilitate the spread of HIV and increase the severity of the disease (Aurelian, L. Editor. Heφes viruses, the Immune System and AIDS. Kluwer Academic Publishers, Boston, Mass. 1990). Because HSV-1 has only a 50%o homology to HSV-2, this may lower the response rate against the heterologous strain in the vaccinated population. Another absolute requirement for a live vaccine is the absence of lesions upon immunization. A desirable trait in the live vaccine would be its ability to cause a reduction in the frequency of recurrent lesions in a person already infected. There is a substantial population already infected with HSV who may have intercourse with uninfected individuals who would benefit from such a vaccine. The present invention solves all the problems recited above providing a whole live attenuated HSV-2 in which the HSV-2 has a deletion of the oncogene, and is formulated in a vaccine composition. The present invention provides a method of immunizing a subject against HSV-1 or HSV-2 with said vaccine composition, providing a superior method of conferring immunity upon the subject. SI IMMAR Y OF THE INVENTION It is an object of the present invention to provide a v accine composition which when administered to an animal, including a human, provides protection from challenge with HSV- 2 or HSV- 1 infection. It is a further object of the invention to provide a vaccine composition comprising whole, liv e, attenuated HSV-2 wherein the oncogene or any portion thereof that causes transformation and does not attenuate the virus has been deleted. It is a further object of the invention to provide a method of immunizing a subject against HSV-2 or HSV-1 comprising administering a novel vaccine composition It is even a further object of the present invention to reduce or prevent clinical symptoms associated with HSV-2 or HSV-1 infections comprising administeπng a novel vaccine composition. It is yet a further object of the present invention to reduce recurrent disease associated with HSV-2 or HSV-1 in previously infected subjects composing administering a novel vaccine composition BRIEF DESCRIPTION OF THE DRAWINGS Fig. l A. Schematic representation of ICP 10ΔPK DNA Ohgoprobe AU26 should recognize the 7 6kb BamHI E fragment from HSV-2 or HSV-2(R) DNA and a 2.2kb BamHI fragment from ICP10ΔPK DNA. B. Southern blot hybridization of BamHI digested ICP10ΔPK (lane 1), HSV-2 (lane 2), or HSV-2 (R ) (lane 3) DNA with the digoxige n-labeled AU26 ohgoprobe Size markers are shown in the right margin Similar results were obtained for ICP10ΔPK stocks RF and CS. Fig. 2 Expression and PK activity of the p95 protein from ICP10ΔPK infected cells. A. Vero cells w ere infected with HSV-2 (lanes 1 ,4), ICP 1 OΔPK (lane 2), or HSV-2 (R) (lane 3), labeled with [^S]-methιonιne from 6-16 hrs p.i. and extracts were immunoprecipitated w ith anti LA-1 antibody hich is specific for ICP 10 (lanes 1 -3) or preimmune serum (lane 4) B. Immunocomplex PK assay w ith anti LA-1 antibody (lanes 1-3) or preimmune serum (lane 4) of extracts from Vero cells infected for 16 hrs with HSV-2 (lane 1 ,4). ICP10ΔPK (lane 2) or HSV-2(R) (lane 3) C. The immunoprecipitates in Panel B were immunoblotted w ith anti LA-1 antibody Similar results were obtained for ICPI OΔPK stocks RF and CS. Fig. 3 Virus growth under exponential and grow th restncted conditions. Vero cells grown in 10% serum (•) or 0 5% serum (Δ) w ere infected with HSV-2, ICPIOΔPK, or HSV-2(R) at an moi of 2. Virus titers were assayed at 2 to 36 hrs. p l Results are expressed as PFU cell (burst size). Fig 4 Extracellular and intracellular v irus titers in Vero cells infected at a high moi. Exponential Vero cells were infected w ith HSV-2 (A) or ICPIOΔPK (B) at an moi of 200 and intracellular (Δ) and extracellular ( • ) virus titers were determined at 2 to 36 hrs. p.i Fig. 5 Virus growth in dividing cells infected w ith the two ICPIOΔPK stocks. Vero cells were grown in medium with 10% serum and infected with ICPIOΔPK (RF) ( • ) or ICPIOΔPK (CS) ( ▲ ). Infection was at moi of 2 (panel A) or 200 (panel B). Virus titers w ere determined at 2-35 hrs. p.i and results are expressed as PFU/cell (burst size). Fig. 6 ICPI OΔPK virus growth in cells that constituti ely express ICP 10. JHLal cells, that constitutively express ICP 10 (Panel A) or 293 cells, that were used to establish the JHLal line (Panel B), were infected with HSV-2 (Δ) or ICPIOΔPK stock RF (•) at an moi of 200. Virus titers were assayed at 2 to 20 hrs p.i. Results are expressed as PFU cell (burst size). Similar results were obtained for ICP 10ΔPK stock C in JHLal cells. Fig. 7 Adsoφtion/penetration kinetics of ICPI OΔPK Vero cells were exposed to 200 pfu of HSV-2 (•) ICPIOΔPK (o) or HSV-2) (R)(< ) for 0, 10, 30, 60, 90, 120 min., overlaid with MEM 10% serum and 0.3% IgG , reincubated at 37°C for 48 hrs and scored for plaque foπnation. Data are presented as the % of the onginal inoculum. Similar results w ere obtained for ICPIOΔPK stocks RF and CS Fig. 8 Protein profiles of HSV-2 and ICPIOΔPK infected cells. A. Vero cells (lanes 1-6 , 8) were mock infected (lane 1) or infected with HSV-2 (lanes 2, 4), ICPIOΔPK (RF) ( lanes 3. 5. 6.) or HSV-2 (R) (lane 8) and labeled with from 2-3 hrs p ι., (lanes 1 -3, 8), 7-8 hrs p.i. (lanes 4. 5), or 1 1-12 hrs p.i. (lane 6) The protein profile in JHLal cells (constitutively express ICP10) infected w ith ICPIOΔPK, and labeled with [ 33 S]-methιonιne from 2-3 hrs p 1. served as control (lane 7) Proteins from cell extracts w ere resolv ed by PAGE on 8 5% SDS acrylamide gels. B Extracts of cells infected with ICPIOΔPK (RF) for 3 hrs (lanes 1,2) or 8 hrs p l. (lane 3) w ere immunoblotted w ith ICPO MAb (lane 1 ), LA-1 antibody to ICP10 (lane 2) or ICP4 MAb (lane 3) C Extracts of Vero cells mock infected (lane 1 ), infected with HSV-2 for 3 hrs (lane 2) or ICPI OΔPK for 12 hrs were immunoblotted with antibody to actm. ig 9 IE protein synthesis in HSV-2 and ICPIOΔPK infected cells. A Vero cells were mock infected (lane 1 ) or infected w ith HSV-2 (lane 2) or ICPIOΔPK (RF) (lane 3) in the presence of 50 μg/ml cycloheximide (6 hrs) and labeled with for 3 hrs in medium containing 10 μg/ml actinomycin D Proteins were resolved by PAGE on 8 5 % SDS acrylamide gels. B Immunoblotting of extracts in lanes 2,3 in panel A w ith ICPO MAb (lane 1) and antι-LA-1 antibody to ICP 10 (lane 2) ig 10 ICP4 and ICPO mRNA synthesis in cells infected with ICPIOΔPK. RNA was extracted from Vero cells infected with HSV-2 (HSV) or ICPIOΔPK (ΔPK) or HSV-2 (R)(R) for 3 hrs (panel A) 0-20 hrs (panel B) or 1-8 hrs (panel C) as listed It was hybridized with [ , P]-labeled ICP4 (4) or ICPO (0) DNA probes or GAPDH oligonucleotide (bottom panel) Molecular weight markers are indicated in the margins Fig 1 1 Indirect immunofluorescent staining of Vero cells infected with HSV-2 (Panels A,B.C) or ICPIOΔPK (RF) (Panels D,E,F) for 3 hrs (Panels A,D), 6hrs (Panels B, E) or 9 hr (Panels C,F) and stained with MAb 30 (Panels A-C ) or anti LA- 1 antibody to ICP 10 (Panels D-F) Fig 12 HSV-specific lymphoprohferative responses of spleen cells from mice immunized w ith ICP 10ΔPK (stock RF) Mice w ere given sc injection with lxlO 6 pfu of HSV-2 or ICPIOΔPK (ΔPK) Spleens were collected on day 24 p l. and assayed for [ H]-TdR incoφoration (cpm) Results are expressed as cpm for HSV-stimulated cultures - cpm for cultures stimulated w ith mock antigen Fig.13 HSV-specific lymphoprohferative responses of spleen cells from mice immunized with ICPIOΔPK (stock CS). Mice were given three sc injections with 1x10 " pfu of ICPIOΔPK (ΔPK). Spleens were collected on day 14 after the last injection and assayed for [Η]-TdR incoφoration (cpm) after culture with HSV-2 or mock antigens or the mitogen PHA. DETAILED DESCRIPTION OF THE INVENTION In the present invention, live whole HSV-2 has been mutated and attenuated to prevent neoplastic transformation. The mutated HSV-2 can be formulated with immune stimulants or adjuvants and used to immunize a subject against HSV-1 or HSV-2. The protein kinase domain of the large subunit of ribonucleotide reductase (ICP 10) has previously been shown to have oncogenic properties. Deletion of the PK domain is shown in the present invention to have deleterious effects on the ability of HSV-2 to infect cells. The present invention demonstrates for the first time that live HSV-2 lacking the ICP10PK domain, and hence the oncogene, provides immunogenic protection against challenge with live wild type HSV-2. Therefore, a novel vaccine composition has been discovered and a novel method of immunizing a subject against HSV-2 or HSV-1. HSV-1 and HSV-2 viruses are very similar. The DNA is 50% homologous. Virtually all viral proteins have both type-specific and type-common epitopes. For all but 2 proteins (i.e., for 82 proteins), the type-common epitopes are predominant. The exception is the HSV- 2 gG2 (Ashley et al . J. Clin. Invest. 90:51 1 , 1992) and the HS V-2 oncoprotein which elicit predominantly type-specific antibodies. In the present invention, the HSV-2 oncogene was deleted from ICPIOΔPK. Therefore we only have one protein that can induce type specific immunity. The remaining 83 proteins will induce type common immunity. This incudes both antibody and cell mediated immunity. Previously, live whole HSV-2 could not be explored as a vaccine option for HSV since the oncogene had potential neoplastic implications for the patient. The present invention demonstrates that by removing the oncogene, a protein kinase, from the HSV-2 genome, not only are the neoplastic properties removed, but the virus is attenuated and provides full protection against challenge for an extended period of time. The particular HSV-2 strain which contains the deleted oncogene is not critical to the present invention. Examples of such strains include HSV-2(G), HSV-2(333). HSV-2(186), HSV-2(S-1), although any strain is acceptable. These strains are well known and readily available. The constmction of the mutant virus is accomplished by well known techniques. The location of the oncogene (PK) is well-known (DNA Tumor Viruses Oncogenic Mechanisms, Ed. C. Barbanti-Brodano, et al.. Plenum Press, NY, 1995, chapter 14 by L. Aurelian, Transformation and Mutagenic Effects Induced by Heφes Simplex Virus Types 1 and 2, p253-280). The oncogene is located in the ICP 10 section of the HSV-2 genome. It has previously been shown that the PK activity and oncogenic activity are located within the gene sequence encoding ICP10 amino acids 88-41 1. Briefly, the wild type sequences in a plasmid (TP101 ) that contains the HSV-2 BamHI E and T fragments were replaced with the 1.8kb Sall/Bglll fragment from pJHL9 [ICP 10 mutant deleted in the PK domain, Luo and Aurelian, J. Biol. Chem. 267:9645-9653, 1992)]. The resulting plasmid, TP9, contains sequences which code for ICP 10 deleted in the PK catalytic domain flanked by 4 and 2.8 kb of HSV-2 DNA sequences at the 5' and 3' ends, respectively. The l Okb Hindlll/EcoRI fragment from TP9 was introduced by marker transfer into a virus (ICP10ΔRR) in which the RR domain of ICP 10 had been replaced with the LacZ gene. The resulting recombinant virus, designated ICPIOΔPK. was obtained by selecting white plaques on a background of blue plaques after staining with X-gal. A few white plaques were picked and purified. Two were grown in Vero cells with 10% serum (exponentially growing) into independent stocks, respectively designated RF and CS. Southern blot hybridization was used to confirm that the DNA in the ICPIOΔPK virus is deleted in the ICP 10 PK coding region. DNA from HSV-2 and ICPI OΔPK was digested with BamHI. separated on \ % agarose gels and transferred to nylon membranes. It was hybridized with the AU26 (CCCCTTCATCATGTTTAAGGA) probe which recognizes a sequence within the ICP 10 RR coding region. The hybridizing band seen for ICPIOΔPK DNA was 2.2kb as compared to 7.6kb for wild type HSV-2. Similar results were obtained for stocks RF and CS. ICPIOΔPK virus can be differentiated from wild type HSV-2 by DNA analysis and immunoprecipitation/irnmunoblotting with antibody to epitopes located at ICP 10 amino acids retained by the deleted protein. ICPIOΔPK was precipitated/immunoblotted w ith anti-LA-1 antibody (recognizes ICP10 amino acids 13-26) (Aurelian. et al.. Cancer Cells 7: 187-191 , 1989) and the proteins were resolved by SDS-PAGE. A 95kDa protein was recognized by the antibody in cells infected with ICPIOΔPK virus as compared to the 140 kDa protein from cells infected with the wild type virus Similar results were obtained for stocks RF and CS The oncogene or any portion thereof may be deleted Bv the expression "or any portion thereof we mean anv portion of the oncogene which once deleted results in attenuation of the virus and prev ents neoplastic transformation of the cells Determining if PK activ ity is absent requires expression of the v iral gene and subjecting the result to standard PK assays (Chung et al ,J Virol 63 3389-3398, 1989) There is abundant guidance in the prior art as to the section of the ICP 10 gene w hich is required for PK activity Determining viral attenuation requires testing in animals to determine absence of lesion formation The techniques for accomplishing this are standard and well-known in the art The resultant mutant virus. ICPIOΔPK was used in infection experiments and compared to infections w ith w ild-tvpe HSV-2 and restored HSV-2(R) virus The cells used in infection are not critical to the present inv ention Any human or animal cell line which can be infected with wild type HSV-2 av be used in the present invention Examples of such cell lines include Vero cells, HeLa cells, 293 cells, or MRC5 cells (all available from Amencan Type Culture Collection) ICPIOΔPK can also be grown in cells that constitutively express ICP10, for example JHLal It is titrated by plaque assay on Vero cells with MEM-10% FCS and 0 3%> human IgG The growth properties of the RF and CS virus stocks were independently determined The infection experiments were also conducted in animals Mice ere chosen since mice represent the standard animal model for HSV-2 (Wachsman et al , Vaccine 10 447-454, 1992) The mouse footpad model was chosen to examine the pathogemcity of the ICPIOΔPK virus in vno The RF and CS irus stocks were independently studied Severe lesions were seen in mice given HSV-2, or the restored virus designated HSV-2(R) Mice given ICPIOΔPK (RF or CS stocks) had no neurological symptoms nor skin lesions during the entire study protocol (days 1 through 21 ) Immunizing a subject indicates the standard inteφretation well known in the art Upon administration of the vaccine composition, neutralizing antibodies and cell-mediated immunity are raised in the subject and said antibodies and cell-mediated immunity confer immunity to the subject The present invention teaches immunization of a subject against HSV-2 A. "pfu" is a plaque forming unit and represents the quantity of virus required to form a single plaque when a cell culture is infected w ith the virus It is a quantitativ e measure of viral infectivity used by those skilled in the art A dose of 0 5-1 million pfu was used to immunize mice with ICPIOΔPK stock RF [ICPI OΔPK (RF)] A dose of 1 -10 million pfu w as used to immunize mice with ICPIOΔPK stock CS [ICPIOΔPK (CS)] The dosage range for a human is 1 to 100 million pfu A preferred range is 1000 to 75 million pfu and an especially preferred range is 10,000 to 50 million pfu Furthermore, due to the 50% homo logy of HSV-1 and HSV-2 there will be a high degree of protection against HSV-1 infection The formulation of ICPIOΔPK for human use is accomplished by suspension in a solution with or without stabilizing ingredients, and with or without immune stimulants and adjuvants Examples of stabilizing agents, immune stimulants, and adjuvants include alum, incomplete Freud's adjuvant, MR-59 (Chiron), MTPPE, MPL (mono-phosphoryl Lipid A) Such stabilizing agents, adjuvants and immune stimulants are well known in the art and can be used singly or in combination The vaccine composition of the present invention can be administered to any animal, including humans The vaccine composition may be administered v ia any suitable mode of administration, such as intramuscular, oral, subcutaneous, intradermal. intravaginal, rectal, or intranasal administration The preferred modes of administration are subcutaneous or intradermal administration The ICPIOΔPK which provides protection against HSV-2 infection can be administered along ith a pharmaceutically acceptable carrier or diluent Examples of such pharmaceutically acceptable earner or diluents include water, phosphate buffered saline or sodium bicarbonate buffer A number of other acceptable carriers or diluents are known Description of the Preferred Embodiments It has been found that the recombinant strain ICPIOΔPK(CS) is a preferred strain of Heφes Simplex type 2. which has had the gene coding for protein kinase deleted along with other unknown modifications of the genome which reduce its growth rate, for a v accme for immunizing a human host against both HSV-1 and HSV-2 This strain exhibits the desirable characteristics of immunogenicity. latency without possessing toxicitv or oncoge citv This strain has been deposited on December 18, 1997 w ith the American Culture Collection (ATCC), 12301 Parklawn Driv e. Rock ille, MD 20852. L S A . and is identified bv the ATCC accession number, VR 2592 The following examples are pro ided for illustrative puφoses only and are in no way intended to limit the scope of the present inv ention. Materials C≤lls Vero (African green monkey kidney) cells were grown in Eagle's minimum essential medium (EMEM) supplemented w ith 10% fetal calf serum (FCS) and antibiotics. JHLal cells (constituti elv expresses ICP10) were previously described (Luo and Aurelian, J. Biol Chem 267:9645-9653. 1992. Smith et al , Virology 200.598-612, 1994; Hunter et al., Virology 210 345-360. 1995) They w ere cultured in EMEM with 10% FCS, 1 mM Na pyruvate (GIBCO-BRL, Gaithersburg, MD), lx non-essential amino acids (GIBCO-BRL) and antibiotics Vero-ICPIO cells were derived by transfection of Vero cells with an ICP10 expression vector that has a SV 2 -neo cassette (pJW17N) (Luo and Aurelian, J. Biol. Chem 267:9645-9653, 1992, Smith et al., Virology 200:598-612, 1994) For serum starvation, cells grown to confluency in medium containing 10%> FCS, were washed with phosphate-buffered saline (PBS) pH 7 0. and grown for two days in medium containing 0.5% FCS. Plaque forming ability Virus titers w ere determined by plaque assay as described (Aurelian, L, Heφes Simplex Viruses In Clinical Vιrolog\> Manual, 2nd Edition. Specter, S. and Lancz, G., eds. Elsevier Science Pub pp 473-494, 1992). Vero-ICPIO cells were used under an overlay consisting of MEM supplemented with 10%. or 0.5% FCS and 0.3% IgG. Antibodies The production and specificity of the antι-LA-1 antibody specific for ICP10 amino acids 13-26 and monoclonal antibody (MAb 30) that recognizes a determinant in the ICP10 PK domain (amino acids 106-178) were previously described (Aurelian et al., Cancer Cells 7.187-191 , 1989. Chung et al , J. Gen Virol 72.1 139-1 144, 1991 ) ICP4 and ICPO MAbs were purchased from Adv anced Biotechnologies, (Columbia, MD). Antibody C- 1 1 to actin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA) Immunofluorescent Staining Vero cells grown on 22 mm 2 glass cover slips (Corning Glass Works, New York) were infected w ith HSV-2 or ICPIOΔPK and fixed in cold methanol (-70°) They ere stained (60 min, 37°C) with anti LA-1 antibody or MAb 30 followed respectively by fluorescein-conjugated goat anti-rabbit or mouse IgG (Wymer et al., J Virol 63.277-2784, 1989, Smith et al . Virology 200 598-612, 1994) Example 1 Construction and Characterization of the ICPI OΔPK and HSV-2(R) viruses The construction of the ICPI OΔPK irus has been described (Peng et al , Virology 216 184-196. 1996) Briefly, the wild type sequences in a plasmid (TP101 ) that contains the HSV-2 BamHI E and T fragments were replaced w ith the 1 8kb Sall/Bglll fragment from pJHL9 [ICP 10 mutant deleted in the PK domain (Luo and Aurelian, J Biol Chem 267 9645- 9653, 1992)] The resulting plasmid, TP9. contains sequences which code for ICP 10 deleted in the PK domain flanked by 4 and 2 8 kb of HSV-2 DNA sequences at the 5' and 3' ends, respectiv elv The lOkb Hindlll/EcoRI fragment from TP9 was introduced by marker transfer into a virus (ICP10ΔRR ) in which the RR domain of ICP 10 had been replaced with the LacZ gene The resulting recombinant virus, designated ICPIOΔPK, was obtained by selecting white plaques on a background of blue plaques after staining with X-gal A few white plaques were picked and purified Two stocks were grown in Vero cells in MEM with 10% FCS (exponential) They were respectively designated ICPIOΔPK (RF) and ICPIOΔPK (CS). For the construction of the restored virus HSV-2(R). \ ero cells were co-transfected with 1 μg of infectious v iral DNA from ICPIOΔPK and a 10-fold molar excess of the wild type BamHI E/T fragment A strategy similar to that reported for ICP6Δ (Goldstein and Weller, Virology 166 41 -51 , 1988) was used to select restored virus under growth restricted conditions (serum starved Vero cells) Southern blot hybridization was used to confirm that the ICPI OΔPK DNA is deleted in the ICP 10 PK coding region Generally, viral DNA was isolated from cytoplasmic vinons as described (Pignatti et al , Virology 93 260-264. 1979, Smith et al , J Gen Virol 73 1417- 1428, 1992) Briefly, Vero cells were infected at a multiplicity of infection (moi) of 5 At 48 hrs p l cells ere resuspended (2 x 10 7 cell/ml) in a buffer consisting of 10 mM Tπs-HCl (pH 7 9), 10 mM EDTA and 0 25% Triton Following incubation on ice (15 min ), NaCl was added at a final concentration of 0 2 M and the nuclei were precipitated by centrifugation at 1,000 x g ( 10 min, 4°C) The supernatant, containing cytoplasmic v inons, was incubated in 200 μg/ml Proteinase K and 0 2% SDS (4 hr at 37°C), mixed w ith saturated sodium iodide (Nal, final concentration 1 525 g/ml) and ethidium bromide (final concentration 3 μg/ml) and centrifuged at 100,000xg for 16 hrs Viral DNA ( 15 μg) w as digested w ith BamH I and the fragments w ere separated by 1% agarose gel electrophoresis in a Tπs- Acetate EDTA (TAE) buffer (40 mM Tπs-acetate and 1 m\l EDTA) It was transferred to Gene screen membranes (New England Nuclear Coφ ) and the membranes w ere incubated in a preh bπdization solution containing 5 x SSC [750 mM NaCl, 75 m Sodium citrate, pH (7 0)] 2% Casein 0 1 % N-lau , lsarcosine and 0 02% sodium dodecyl sulfate (SDS) at 42° C for 2 hrs The hybridization probe was ohgonucleotide AU26 (CCCCTTCATCATGTTTAAGGA) w hich represents a sequence in the ICP 10 RR coding region It was 3' tailed w ith digoxigenin-dUTP (DIG-dUTP ) by terminal transferase (Boehπnger Mannheim) in 20 μl v olume w ith lx reaction buffer [5 mM cobalt chloride (CoCIN 0 05 mM DIG-dUTP. 5 nmol/ml AU26. 0.5 mM dATP and 2.5 units μl terminal transferase] at 37°C for 15 min. diluted to a final concentration of 5 pmol/ml in prehybπdization solution Hybridization was done at 42°C for 3 hrs Membranes were washed once (room temperature) in a solution containing 2xSSC. 0.1% SDS for 5 mins and twice in 0.5xSSC, 0.1% SDS for 15 mins. For detection of the hybndized DNA fragments, the membranes were rinsed in Buffer 1 (100 mM Tπs-HCl, pH 7.5,150 mM NaCl), incubated in Buffer 2 [2% (w/v) casein in Buffer 1 ] for 40mιn and in Buffer 2 containing 3xl0 4 U/ml of alkaline phosphatase-conjugated anti-digoxigenin antibody (Boehπnger Mannheim) for 30 min After washing with Buffer 1 (twice) and soaking in Buffer 3 ( 100 mM Tns-HCl, pH 9.5, 100 mM NaCl, 50 mM MgCL) for 2 min. the membranes ere exposed to the chemiluminescent substrate Lumι-Phos 1 M 530 (Boehnnger Mannheim) and the reaction was developed on X-ray film. More specifically, DNA ( 15 μg) from HSV-2. ICPI OΔPK or HSV-2(R) was digested with BamHI, separated on 1 %> agarose gels and transferred to nylon membranes. It was hybridized with the AU26 probe which recognizes a sequence w ithin the ICP 10 RR coding region (Fig. 1A). A hybridizing 7.6kb band which represents the BamHI E fragment was observed for HSV-2, (Fig I B. lane 2) and HSV-2(R) (Fig IB. lane 3) DNA The hybndizing band seen for ICPIOΔPK DNA was 2.2kb (Fig. IB, lane 1 ) consistent with the expected size. Similar results were obtained for ICPIOΔPK (RF) and ICPIOΔPK (CS). The data confirm that ICPIOΔPK DNA is deleted in the PK coding region. Example 2 Expression of the 9 kDa PK deleted ICP 10 protein <p95. To determine whether ICPIOΔPK expresses an ICP 10 protein deleted in its PK domain. Vero cells were infected with ICPIOΔPK (200 pfu'cell) and labeled with [ 5 S]- methionine ( 100 μCi/ml) from 6-16 hrs p.i. Cells infected w ith HSV-2 or HSV-2(R) served as controls. Generally, cells were mock-infected with PBS (pH7 4) or infected with 200 PFU/cell of HSV-2, ICP 1 OΔPK, or HS V-2 (R). They were labeled with pS]-methιonιne ( 100 μCi/ml) (sp Act 1 120 Ci/mmol. Dupont, NEN Research Products) in EMEM containing no methionine and 10% dialyzed FCS In some experiments, infection as done in the presence of cycloheximide (50 μg/ml) for 6 hr at w hich time cycloheximide w as removed, cells were washed extensively with PBS. and incubated (3 hrs) in the presence of 10 μg / ml actinomycin D and 100 μCi/ml of [ 1s S]-methιonιne. For immunoprecipitation, cell lysates were incubated in cold RIPA buffer [ 0 01 VI Tπs-HCl (pH 8 0), 0.1 % SDS, 1 % Nomdet P40, 1% deoxycholate, 0 15 M NaCl, 1 M dithiothreitol] with ImM phenylmethylsulfonyl fluonde (PMSF), 100 Kalhkrein units ml aprotimn (Sigma) for 15 min on ice. and cleared of cell debris by centrifugation for 30 min at 20,000 x g. They were incubated with 15-20 μl of antibody (1 hr, 4°C) and lOOμ l protein A-Sepharose CL4B beads [10 mg in 0.1 M Tris-HCl (pH 8 0), 0 15 M NaCl and 0 5% No det P40] (30 mins, 4°C). Beads were washed extensively with ice-cold RIPA buffer and bound proteins were eluted by boiling (5 min) in 100 μl denatunng solution [ 150 mM Tπs hydrochloride (pH 7 0), 5 7% SDS, 14% 2- mercaptoethanol, 17% sucrose and 0 04% bromothymol blue] Proteins were resolved by SDS-PAGE on 7% or 8 5 % polyacrylamide gels and visualized by autoradiography In some experiments, cells were resuspended directly into denaturing solution, boiled for 5 min., and analyzed by SDS-PAGE More specifically, cell extracts were precipitated with antι-LA-1 antibody and the proteins were resolved by SDS- PAGE on 7% polyacrylamide gels. Antι-LA-1 antibody precipitated a 140 kDa protein from HSV-2 (Fig. 2A , lane 1 ) or HSV-2(R) (Fig. 2A, lane 3) infected cells. From ICPI OΔPK infected cells, it precipitated a 95kDa protein (p95) (Fig. 2A, lane 2) which is consistent w ith the PK deleted ICP 10 (Luo and Aurelian, J Biol Chem. 267 9645-9653, 1992) The preimmune serum was negative (Fig. 2A. lane 4) A 38 kDa protein consistent with RR2 w as co-precipitated by antι-LA-1 antibody from cells infected with all three viruses indicating that p95 can complex with RR2, presumably at the carboxy terminus previously implicated in complex formation (Chung et al , J. Gen Virol. 72.1139- 1 144, 1991). Similar results w ere obtained for ICPIOΔPK (RF) and ICPIOΔPK (CS). Example 3 p95 expressed by ICPIOΔPK lacks kinase acti ity We have previously shown that, (l) ICP 10 has kinase activity in HSV-2 infected and stably transfected cells, and ( n) PK activ ity is associated w ith the 57-60 kDa ammo terminal domain of the ICP 10 protein, but not w ith its 90-95kDa carboxy terminal domain (Chung et al.. J. Virol. 63.3389-3398, 1989, Smith et al . Virology 200 598-612. 1994) To determine whether p95 expressed by ICPI OΔPK has PK activity, extracts of cells infected with HSV-2, ICPIOΔPK or HS\ ' -2(R) (moi=200, 16 hrs p.i.) were immunoprecipitated with anti-LA-1 antibody and subjected to PK assays (Chung et al., J. Virol. 63:3389-3398, 1989). Generally, immunoprecipitates of cell extracts normalized for protein concentration by the BCA protein assay kit, (PIERCE. Rockford. IL) were washed with TS buffer containing 20 mM Tris-HCl (pH 7.4), 0.15 M NaCl, suspended in 50 μl of kinase reaction buffer consisting of 20 mM Tris-HCl (pH 7.4), 5 mM MgCL , 2 mM MnCL and 10 μCi of [ 32 P] ATP (3000 Ci/mmol, Dupont, New England Research Product), and incubated at 30°C for 15 min (Chung et al., J. Virol 63:3389-3398, 1989; Chung et al., Virology 179: 168-178, 1990; Smith et al.. J. Gen. Virol.73:1417-1428, 1992; Smith et al., Virology 200:598-612, 1994; Peng et al.. Virology 216:184-196, 1996). The beads were washed once with 1 ml TS buffer, resuspended in 100 μl of denaturing solution and boiled for 5 min. The proteins were resolved by SDS-PAGE on 7% polyacrylamide gels as described (Chung, et al., J. Virol. 63:3389-3398, 1989). Proteins were electrotransferred onto nitrocellulose membranes as previously described (Aurelian et al., Cancer Cells 7: 187-191 , 1989) and immunoblotting was performed by incubation with the respective antibodies followed by protein A-peroxidase (Sigma) for 1 hr at room temperature each. Detection was with ECL reagents (Amersham, Chicago, IL), as described (Smith et al., Virology 200:598 612, 1994). More specifically, the resolved proteins were transferred to a nitrocellulose membrane and immunoblotted with anti-LA-1 antibody to detennine the levels of protein in the precipitates. The 140kDa ICP 10 protein from HSV-2 (Fig. 2B, lane 1 ) or HSV-2(R) (Fig. 2B, lane 3) infected cells was phosphorylated. A phosphorylated 95 kDa protein was not seen in cells infected with ICPIOΔPK (Fig. 2B, lane 2). This is not due to low levels of protein in the precipitates used for PK assay, because similar protein levels were seen for all three viruses by immunoblotting with anti LA-1 antibody (Fig. 2C). A phosphorylated 38kDa protein was seen in HSV-2 (Fig. 2B, lane 1) and HSV-2(R) (Fig. 2B, lane 3) infected cells but not in cells infected with ICPI OΔPK (Fig. 2B, lane 2). Preimmune serum was negative (Fig. 2B,C, lane 4). We inteφret these data to indicate that RR2 is phosphorylated by ICP 10 PK, as previously reported by Chung et al., (J. Virol 63:3389-3398. 1989) and Peng et al. (Virology 216: 1 196, 1996). It is not phosphorylated by p95, consistent with the absence of the PK domain. Similar results were obtained for ICPIOΔPK (RF) and ICP I OΔPK (CS). The data confirm that the ICP10 PK coding region is required for kinase activity, also within the context of virus infection. Example 4 Ribonucleotide reductase activ lty of ICP 1 OΔPK It is generally believed that the RR and PK activities of the RR1 protein can be dissociated (Ingemarson et al.. Virology 156:417-422, 1987; Chung et al.. J. Virol. 63:3389- 3398, 1989). To examine the validity of this inteφretation it is important to document whether the loss of ICP10 PK activity has any effect on RR acti ity. RR assays were performed on extracts from infected cells (moι=20. 16 hrs, p.i.). RR activity was assayed as described (Smith et al., J. Gen Virol. 73: 1417-1428, 1992). Extracts from 16 hrs infected cells or mock infected cells were resuspended in HD buffer [100 mM HEPES buffer (pH 7.6), 2 mM dithiothreitol (DTT) ] at 2 x 10 7 cell equivalents/ml, incubated on ice for 15 mins, disrupted by sonication (30-60 sees at maximum setting; Ultrasonics model 220F Sonifier) and clarified of cell debris by centrifugation (100.000 x g; 1 hr, 4°C). The HSV RR activity was precipitated with crystalline ammonium sulfate [45% saturation (0.258 g/ml) ]. Following dialysis and centrifugation ( 16,000 xg; 30 min), the partially purified enzyme preparations were incubated (37°C; 10 min) with equal volumes of a 2 x standard reaction mixture containing 400 mM HEPES buffer (pH 8.0), 20 nM DTT and 0.2 mM [ H]-CDP (17.8 Ci/mmol, Amersham, IL). The reaction was terminated by the addition of 100 mM hydroxyurea with 10 mM EDTA (pH 8.0) and boiling for 3 mm. Crotalus atrox venom (Sigma, St Louis, MO) was added [0.5 mg/ml in 12 mM Tπs HCl (pH 9.0), 4 mM MgCl 2 , 1 mM deoxycytidine) and the mixture was incubated 30 min at 37°C, boiled for 3 min and applied to a 0.5 ml Dowex- 1 borate column (Sigma). The column w as washed with 2.5 ml H : O and 0.5 ml eluate fractions were mixed with Biofluor (New England Nuclear, Boston, MA) for scintillation counting. Ribonucleotide reductase activity is expressed as units/mg, where 1 unit represents the conversion of 1 nmol [Η]-CDP to dCDP'hr/mg protein. More specifically, as shown in Table 1, the ICPIOΔPK irus had a similar RR activity as that of HSV-2 and HSV-2(R) This is consistent ith the finding that p95 coprecipitates with RR2 and supports the conclusion that the PK and RR activ mes can be functionally dissociated. Table 1. Ribonucleotide reductase activity of ICPIOΔPK virus. Virus cpm RR Specific activity (Units) b HSV-2 1 1.534 10.; HSV-2(R) 10,037 8.8 ICPIOΔPK (RF) 9,540 8.4 Mock-infected 3,060 2J a cpm/270 mg protein b One RR unit = conversion of 1 nmol CDP to dCDP/h mg protein. Example 5 Growth properties of ICPIOΔPK The growth properties of ICPIOΔPK were studied under exponential ( 10% serum) and growth restricted (0.5% serum) conditions. In a first series of experiments, Vero cells were infected with HSV-2, ICPIOΔPK (RF), or HSV-2(R) at moi of 2 and virus growth examined for 36 hrs p.i. As shown in Fig. 3, HSV-2 grew equally well under exponential and growth restricted conditions. Virus replication began at 2 hrs p.i. and reached peak levels at 36 hrs p.i. (burst size 1000 pfu/cell). A similar growth pattern was evidenced by HSV-2(R). By contrast, onset of ICPIOΔPK replication was not seen until 15 hrs p.i. both in exponential and serum starved cells. At that time replication resumed, reaching titers similar to those of HSV- 2 at 36hrs p.i. in exponential cells (burst size 1000 pfu/cell), but not in serum starved cells (burst size 1 pfu/cell). In a second series of experiments, exponential Vero cells were infected with HSV-2, or ICPIOΔPK (RF) at moi of 200. HSV-2 replication began at 2 hrs p.i. and reached maximal titers at 20 hrs p.i. (Fig. 4A ). By contrast, replication of ICPIOΔPK virus was first seen at 10-12 hrs p.i., with maximal titers at 36hrs p.i. (Fig. 4B). The growth of HSV-2(R) was virtually identical to that of HSV-2 (data not shown). The titers of intracellular and extracellular virus were similar for HSV-2, ICPI OΔPK and HSV-2(R), indicating that progeny virus was released equally well (Fig 4) Virus gro th w as next compared for the two ICPIOΔPK v irus stocks Exponential \ ero cells were infected w ith ICPIOΔPK (RF) or ICPIOΔPK (CS) at moi's of 2 or 200 pfu/cell In cells infected at moi of 2 pfu/cell ICPIOΔPK (RF) replication began at 15 hrs p.i By contrast replication of ICPIOΔPK (CS) did not begin until 20hrs p l , suggesting that it was more defectiv e (Fig 5 A) For both v irus stocks, peak titers were seen at 35 hrs p.i. Those of ICPIOΔPK (RF) ere 1000 pfu/cell Those of ICPIOΔPK (CS) were 780 pfu/cell (Fig 5 A) In cells infected at moi of 200 pfu/cell, ICPIOΔPK (RF) replication began at 1 1 hrs p l By contrast replication of ICPIOΔPK (CS) did not begin until 15hrs p.i , demonstrating a delay in the onset of virus replication proportional to that seen in cells infected at low moi (Fig 5B) For both virus stocks, peak titers were seen at 35 hrs p l. They w ere 580 and 50 pfu/cell for ICPI OΔPK (RF) and ICPI OΔPK (CS) respectively (Fig. 5B) Because the ICPIOΔPK (RF) and ICPIOΔPK (CS) virus stocks were similarly constructed and both lack ICP 10 PK activ lty but retain RR activity, we conclude that their different growth patterns reflect additional differences which were presumably acquired dunng growth amplification and account for the further attenuation of ICPIOΔPK (CS) To confirm that ICP 10 PK is indeed required for virus replication, we also examined virus growth in JHLal cells that constitutively express ICP 10 293 cells which were used to establish the JHLal line were used as control (Luo and Aurelian, J Biol Chem 267 9645- 9653, 1992, Smith et al , Virology 200 598-612, 1994, Hunter et al . \ irology 210 345-360, 1995) Cells were infected at moi of 200 and overlaid with MEM-1% FCS ICPIOΔPK growth in 293 cells w as similar to that seen in Vero cells in that new ly synthesized virus was not seen before 10 hrs p I (Fig 6B) By contrast, in JHLal cells, ICPIOΔPK grew as well as HSV-2, with replication first seen at 2 hrs p l and reaching maximal lev els at 20 hrs p I (burst size, 2800 and 2500 for HS V-2 and ICP 1 OΔPK respectively) (Fig 6A) Similar results were obtained for ICPIOΔPK (RF) and ICPIOΔPK (CS) We inteφret these findings to indicate that ICP10 PK is required for v irus replication both in exponential and growth restricted cells Hovvev er a compensatory functιon(s) seen in both ero and 293 cells is responsible for the resumption of v irus growth at 10-15 hrs p I Because the growth of ICPIOΔPK virus resumed earlier in cells infected at high moi than low moi, but the burst size was significantly higher in exponential than in serum starv ed cells ( 1000 v s 1 respectively) we assume that the compensatory function is a cellular Ser/ Thr PK induced by incoming virus structural protein(s). Example 6 ICPI OΔPK and HSV-2 have similar cell adsoφtion kinetics One possible inteφretation for the growth pattern evidenced by ICPIOΔPK is that it is defective in its ability to adsorb/penetrate the cells. To address this question. Vero cells were exposed to 200 pfu of HSV-2, ICPIOΔPK. or HSV-2(R) for 0, 10, 30, 60. 90 or 120 minutes. They were extensively washed with PBS. overlayed with MEM- 10% FCS and 0.3 % IgG and re-incubated at 37°C for 48 hrs. At this time they were scored for plaque formation. As shown in FigJ, the number of plaques increased for all three viruses as a function of exposure time, reaching maximal levels at 20-30 min. and plateauing thereafter. Virus titers in the original inocula decreased in parallel, with similar patterns seen for HSV-2, ICPIOΔPK, and HSV-2(R) (data not shown). Similar results were obtained for ICPIOΔPK (RF) and ICPIOΔPK (CS). Example 7 Plaque forming ability of ICPIOΔPK virus To analyze the plaque forming ability of ICPIOΔPK we used Vero and Vero-ICPIO cells grown in 10% or 0.5% serum. Consistent with the low burst size observed in serum starved cells infected at low moi, ICPIOΔPK plaque forming ability was severely compromised in serum-starved Vero cells. Virus titers were similar to those of HSV-2 in exponentially growing Vero cells (10% serum) and in Vero-ICPIO cells (Table 2). In Vero cells (grown in 10% or 0.5% FCS), ICPI OΔPK plaques were hazy, apparently reflecting incomplete cell lysis. The extent of cell lysis differed somewhat from one experiment to the next, but it was never as complete as that seen for HSV-2. The plaques of ICPIOΔPK (CS) were smaller than those of ICPIOΔPK (RF), consistent with the conclusion that its growth was more severely compromised. The moφhology of the ICPIOΔPK plaques in Vero-ICPIO cells and that of HSV-2 (R) plaques in all cells was similar to that of HSV-2 (data not shown). Table 2 Plaquing efficiency of ICP 1 OΔPK v irus in dividing and serum starved cells Virus Cells r- 1 - serum) a Virus Titer b (wt/mutant) HSV-2 Vero (+) 5 0 x l0 7 ICPIOΔPK (RF) Vero (+) 2.8 x 10 7 (1.8) HSV-2 Vero (-) 4J x 10 7 ICPI OΔPK (RF) Vero (-) 3 5 x 10 4 (1 3 x 10 3 ) HSV-2 Vero-ICPIO (+) 4 9 x 10 7 ICPIOΔPK (RF) Vero-ICPIO (+) 2 2 x 10 7 (2 2) HSV-2 Vero-ICPI O (-) 4 6 x 10 7 ICPIOΔPK (RF) Vero-ICPIO (-) 2.0 x 10 7 (2 3) a plaque assays done in medium containing 10%> serum (+) or 0 5% serum (-) b plaque forming units/ml Example 8 IE Protein expression is inhibited in ICPI OΔPK infected cells early in infection The growth defect of ICPIOΔPK v irus may reflect its failure to initiate protein synthesis To address this possibility, Vero cells w ere mock infected or infected with HSV-2, ICPIOΔPK. or HSV-2(R) (moι=200) for 2 or 7 hrs. pulse labeled w ith S]-methιomne for an additional 60 mm , and proteins were resolved by SDS-PAGE The protein profiles in HSV-2 infected cells w ere similar to those previously described (Wilcox et al , j Virol 33 167-182, 1 1980) and included, at 3 hrs p.i. ICP4, ICPO. ICP 10, and ICP27 (Fig. 8A. lane 2). Similar protein profiles were seen in cells infected with HSV-2(R) (Fig. 8A. lane 8). By contrast, the protein profiles in cells infected with ICP IOΔPK for 3 hrs (Fig. 8 A. lane 3) resembled those in mock infected cells (Fig. 8A. lane 1 ). The exception were two bands, 1 l OkDa and 95kDa (Fig. 8 A, lane 3) which were respectively recognized by ICPO and ICP 10 antibodies in immunoblotting (Fig. 8B, lanes 1 ,2). Densitometric scanning indicated that the levels of ICPO were 4-fold lower in ICPIOΔPK than HSV-2 [or HSV-2(R)] infected cells (3130 and 782 units for HSV-2 and ICPI OΔPK respectively) and the p95 levels (in ICPI OΔPK infected cells) were 7-fold lower than the ICP 10 levels in HSV-2 and HSV-2(R) infected cells (3567 and 480 units for ICP 10 and p95 respectively). In cells infected with ICPIOΔPK for 8 hrs, the levels of ICPO and p95 were higher, and bands consistent with ICP4, ICP5, and ICP27 were detected (Fig. 8A, lane 5). The identity of the ICP4 band in 8 hrs infected cells was confirmed by immunoblotting with ICP4-specific MAb (Fig. 8B, lane 3). The protein profile in ICPIOΔPK infected cells at 12 hrs p.i. (Fig. 8A, lane 6) was similar to that of HSV-2 infected cells at 8 hrs p.i. (Fig. 8A, lane 4). These findings indicate that viral proteins other than ICPO and p95 are not expressed in cells infected with ICPIOΔPK for 3 hrs, suggesting that ICP10 PK is required for expression of IE proteins ICP4, ICP22 and ICP27. Indeed the protein profile in ICPIOΔPK infected JHLal cells (supply ICP10 PK activity) at 3 hrs p.i. is virtually identical to that of HSV-2 infected cells (Fig. 8 A, lane 7). Because these three IE proteins are responsible for the regulation of early and late viral gene expression (Sacks et al., J. Virol 55:796 05, 1985; McCarthy et al., J. Virol. 63: 18-27, 1989; Samaniego et al.. J. Virol. 69:5705-5715, 1995; Dixon and Schaffer, J. Virol 36: 189-203, 1980; Rice et al., J. Virol. 69:5550-5559, 1995; Leopardi and Roizman. Proc. Natl. Acad. Sci. USA 93:4562 576, 1996), their absence from ICPIOΔPK infected cells results in complete inhibition of viral protein synthesis and infectious virus production. To further examine the synthesis of IE proteins in ICPIOΔPK infected cells, infection was done in the presence of 50 μg/ml cycloheximide (6 hr) and cells were labeled with [ 35 S]- methionine for 3 hrs in medium containing lOμg/ml of actinomycin D, conditions that allow IE gene expression but not expression of other viral genes (Honess and Roizman, J. Virol. 14:8-19. 1974; Strnad and Aurelian. Virology 73:244-258, 1976). Proteins consistent with ICP4, ICP10. ICPO, ICP22. and ICP27 were seen in HSV-2 infected cells (Fig. 9A, lane 2). By contrast. ICP4, ICP 10. ICP22 and ICP27 were not seen in ICPI OΔPK infected cells (Fig. 9A, lane 3). A 1 lOkDa protein, consistent with ICPO, and a 95kDa protein, consistent with p95, were seen in ICPIOΔK infected cells (Fig. 9A, lane 3), but their levels were respectively 2-fold and 3-fold lower than in HSV-2 infected cells (Fig. 9A, lane 2) (densitometric integration units 1760 and 3520 for ICPO; 733 and 2200 for p95 and ICP10, in ICPIOΔPK and HSV-2 infected cells, respectively). Immunoblotting confirmed that the 1 lOkDa and 95 kDa proteins were ICPO and p95 respectively (Fig. 9B. lanes 1,2). The protein profiles for HSV-2(R) were similar to those for HSV-2 (data not shown). These data support the conclusion that ICP 10 PK is required for expression of ICP4, ICP22. and ICP27, but not ICPO and p95. Example 9 ICPIOΔPK virus is defective for ICP4 transcription. Northern hybridization was used to examine whether the failure to detect ICP4 in ICPIOΔPK infected cells is due to a transcriptional defect. RNA was obtained from Vero cells infected with HSV-2, ICPIOΔPK (RF) or HSV-2(R). ICP4 or ICPO DNA were used as probes. GΛPDH served as control transcript. The guanidinium isothiocyanate/cesium chloride gradient method was used to isolate and purify RNA from Vero cells infected with HSV-2, ICPIOΔPK or HSV-2(R) (moi = 200). Northern blot hybridization was done as described (Feng et al., Antisense Nucleic Acid Development 6:25-35, 1996). Hybridization was for 16 hrs at 42°C with [ 32 P]-labeled ICP4, ICPO or GAPDH probes in a solution containing 40% formamide, 6X SSPE, 2X Denhardt's, 0.1 % SDS, and 250 μg/ml salmon sperm DNA. The ICP4 probe was a 1.9kb BamHI DNA fragment derived from pXhoI-C. The ICPO probe was a l Jkb Nrul-Sall fragment derived from pIGA15 (O'Hare and Hayward, j. Virol. 53:751 -760, 1985). The human GAPDH probe was a 40-mer oligonucleotide purchased from Oncogene Science (Cat No. ON407). The probes were [ 3:> P]dCTP labeled by the random priming method using an oligonucleotide kit (Pharmacia, Uppsala. Sweden) according to manufacturer's instructions. Blots were washed twice in 2X SSC-0.1% SDS and twice in 0.1X SSC-0.01 % SDS for 10 min. each at ambient temperature followed by one wash in 0.1X SSC-0.1% at 50°C and visualized by autoradiography. The relative abundance of ICP4 and ICPO mRNA was estimated by first normalizing to the value of GAPDH mRNA in each sample. Both ICP4 and ICPO mRNA w ere seen in Vero cells infected with HSV-2 (Fig. 10A, lanes 1,2) or HSV-2(R) (Fig. 10A, lanes 5.6) for 3 hrs. The kinetics of ICP4 expression in HSV-2 infected cells ere similar to those previously described for HSV-1 infected cells. Optimal lev els were seen at 3 hrs p 1 (Fig I OC, lane 2) and the transcript was no longer detectable at 8 hrs p l (Fig I OC. lane 4) By contrast, ICP4 mRNA w as not seen in cells infected w ith ICPI OΔPK for 3 hrs (Fig 10A. lane 3) However, by 8 hrs p i. (Fig. 10B, lane 3). its levels were similar to those seen earlier (at 3hrs p I ) in HSV-2 infected cells (Fig. IOC, lane 2) The transcript was no longer seen at 20 hrs p.i. (Fig. 10B, lane 6) Cells infected w ith ICPI OΔPK for 3 hrs were positive for ICPO mRNA (Fig 10, lane 4), but its relative abundance (expressed as ICPO/GIPDH mRNA) was 3-fold lower than in HSV-2 infected cells (0 32 and 1 0 respectively) The data indicate that ICP 10 PK is required for early transcription of ICP4 and contributes to optimal transcnption of ICPO. Example 10 ICPI O PK plays a role in inhibition of host cell gene expression and cell lysis. The moφhology of the ICPIOΔPK plaques is consistent with incomplete cell lysis. Because ICP27 plays a role in the shut-off of host protein synthesis (Hardw icke et al., J. Virol 68 4797-4810, 1994) and it is not expressed in cells infected with ICPI OΔPK for 8-12 hrs p ι., we also examined the expression of a host cell gene (actin) in cells infected with HSV-2 or ICPIOΔPK. Vero cells were mock infected or infected with HSV-2 or ICPI OΔPK at moi of 200 and assayed for actin expression by immunoblotting with anti-actin antibody. Actin was not seen in HSV-2 infected cells as early as 3 hrs p.i (Fig 8C, lane 2). By contrast, actm levels in cells infected with ICPI OΔPK (Fig. 8C, lane 3) were similar to those in mock infected cells (Fig. 8C, lane 1 ) as late as 12 hrs p I. These findings are consistent with the observation that cytopathogenic effect (CPE) is not seen in ICPIOΔPK infected cells until 15- 20 hrs p.i. when the compensatory functιon(s) come into play. Example 1 1 Intracellular localization of ICP10 and p95 proteins Previous studies of cells infected with HSV-2 for 8 and 12 hrs had shown that ICP10 localizes in the cytoplasm and is also associated w ith the cytoskeleton (Chung et al, J. Virol 63:3389-3398, 1989). However cells infected for less than 8 hrs were not studied. Because ICP10 PK is required for IE gene expression before 8 hrs, the question arises whether at that time it is also present in the nucleus Vero cells infected with HSV-2 or HSV-2(R), for 3,6, or 9 hrs were stained in lmmunofluorescence vv ith MAb30 (recognizes ICP10 amino acids 106- 178). Cells similarly infected w ith ICPIOΔPK were stained ith anti LA- 1 antibody (recognizes ICP 10 amino acids 13-26) Strong intranuclear staining was seen in cells infected with HSV-2 for 3 hrs (Fig 1 1 A) It had a punctate appearance consisting of discrete sphencal structures (granules) similar to those previously described for viral replication compartments (Rice et al . J Virol 68 988-1001 , 1994, Mullen et al . J Virol 68 3250-3266, 1994) At later times p l , staining took the characteristic peπnuclear and diffuse cytoplasmic pattern prev iously described for ICP 10 (Fig 1 1B,C) Similar staining patterns were seen for HSV- 2(R) (data not shown) By contrast, in ICPIOΔPK infected cells, staining was not seen before 9 hrs p l (Fig 1 ID, E), at which time it was localized only in the cytoplasm (Fig 1 IF) Nuclear staining was also not seen in ICPIOΔPK infected cells at 12 or 15 hrs p I (data not sho n) These findings suggest that the PK domain of ICP 10 is required for nuclear localization early in infection (before 6 hrs) Example 12 ICPIOΔPK virus is attenuated for growth in infected animals We used the mouse footpad model of HSV-2 infection, in order to examine the role of ICPIOΔPK in virus growth in vivo Swiss- Webster mice w ere inoculated s c in the footpad with 5xl 0 6 pfu of HSV-2, ICPIOΔPK(RF) or a restored virus designated HSV-2(R) Neurological symptoms and severe skin lesions ere seen in mice given HSV-2 or HSV- 2(R). beginning on day 6 p I ICPIOΔPK infected mice had no neurological symptoms nor skin lesions HSV-2 and HSV-2(R) were isolated from the footpad and ganghonic homogenates for 7-9 days p l ICPIOΔPK w as only isolated for 4 days p I Maximum titers were lower for ICPI OΔPK than HSV-2 (4 3xl0 and 3x10 pfu for ICPIOΔPK and HSV-2 respectiv ely), and the proportion of latently infected ganglia yielding virus was 90% and 80% for HSV-2 and HSV-2(R) as compared to 10% for ICPIOΔPK (Table 3) These data suggest that ICP 10 PK is involved in acute infection and directly or indirectly, in latency reactiv ation/establishment Table 3. Growth and latency reactivation of ICPIOΔPK virus Mean virus titer in footpads'' Latency' (%) (pfu/ml) Virus Day 2 Day 3 Day 4 Day 7 HSV-2 1.5xl0 3 (+) h 2xl0 4 (+) 3xl0 7 (+) lxl0 7 (+) 9/10 (90) HSV-2(R) ND 2x l 0 (+) ND 6.1x10" 8/10 (80) ICPIOΔPK (RF) 6.3x10 3 2.5xl0 3 (+ 4.3xl0 4 (+) - 1/10 (10) a Mice (n=10) were infected with 5xl 0 6 pfu in the footpad. Virus titers were determined by plaque assay. Lesions were seen beginning on day 7 in HSV-2 and HSV-2(R) infected mice. b Ganglia were obtained on days 3 and 5 p.i., homogenized and assayed for virus. (+) means virus was isolated; (-) means virus was not isolated c Latency is no. of explanted ganglia positive for virus at 30 days p.i./no. tested ND = not done Example 13 ICPI OΔPK virus protects from HSV-2 challenge Two groups of 10 mice each were respectively mock-infected with phosphate buffered saline (PBS) or ICPIOΔPK(RF) (5xl0 5 to 1 x 10 6 pfu) by one sc injection in the footpad. They did not develop any visible symptoms. On day 16 p.i. they were challenged with 1x10' pfu of HSV-2 All mice in the PBS group developed lesions consisting of swelling and redness, first visible on day 5 p.i. and virus (HSV-2) was isolated from the footpads of 10/10 infected mice. On day 15 p.i., 5/10 (50%) of the mice in the PBS group developed paralysis. Mice immunized with ICPIOΔPK virus did not develop visible lesions after HSV-2 challenge. Virus was isolated from the footpad in 3/10 (30%) animals. Virus was not isolated from the footpads of 7/10 (70%) of the ICPI OΔPK immunized mice Table 4 Protection mediated bv ICP IOΔPK Immunization Lesions Challenge Lesions Virus isolation PBS None HSV-2 ( lxl0 7 ) 10/10 10/10 ICPIOΔPK (RF) None HSV-2 ( lxl0 7 ) 0 10 3/10 Mice (groups of 10) were immunized with PBS or ICPIOΔPK (one sc injection) and challenged with HSV-2 in the footpad These findings indicate that immunization with relatively low doses of ICPIOΔPK protects from challenge with high doses of HSV-2 There is absolute protection in terms of lesion development, in that skin lesions w ere seen in all unimmunized mice as compared to no lesions in immunized mice Non-immunized mice were not protected from virus replication, with virus being isolated from all animals on day 5 post challenge. Immunized mice were protected, with virus being isolated from only 3/10 (30%) of the animals The absence of detectable lesions in the 3 animals from which virus was isolated, presumably refiect relatively low titers, suggesting that even though immunization did not afford a 100% level of protection, it reduced the titers of the challenge virus Example 14 ICPI OΔPK virus induces HSV specific immunity Two groups of 4 mice each w ere immunized with HSV-2 or ICPI OΔPK(RF) ( lxlO 6 pfu) by sc injection in the footpad On day 24 p ι., spleens were remov ed and T cells were used in lymphocyte proliferation assay ith HSV-2 antigen as we prev lously described (Wachsman, et al., Bioscience Reports, 8 323 334, 1985; Wachsman. et al , J Inf Dis 159:625 34, 1989; Wachsman, et al . Vaccine 10 447454, 1992) This assay measures the development of HSV-specific memory L ninfected cell extracts prepared in parallel to the v irus antigen (mock antigen) were used as specificity control As show n in Fig 12. HSV specific immunity w as induced by ICP 1 OΔPK v irus The response w as only 2-3 fold lower than that seen for HSY-2 under the same conditions. Example 15 ICPIOΔPK (CS) is more attenuated than ICPIOΔPK (RF) Because ICPI OΔPK (CS) appears to be more attenuated than ICPIOΔPK (RF) in cultured cells, e asked whether the same is also true in infected animals. We used the same mouse footpad model as used for ICPIOΔPK (RF). In order to at least partially compensate for the reduced gro th of the ICPIOΔPK (CS) virus [relative to ICPIOΔPK (RF)] mice were injected with 1x10 pfu of virus and a proportional dose of HSV-2 was used as control. The titers of HSV-2 isolated from the footpads were not significantly higher than those seen in mice given 5xl 0 6 pfu (Example 12). By contrast, the titers of isolated ICPIOΔPK (CS) were lower than those of ICPIOΔPK (RF) and virus w as only isolated for 3, as compared to 4, days p.i. The proportion of latently infected ganglia was 100% and 0% for HSV-2 as compared to ICPIOΔPK (CS) infected animals (Table 5). Table 5. Growth and latencv reactivation of ICPIOΔPK virus Mean virus titer in footpads a (pfu/ml) Latency c (%) Virus Day 2 Day 3 Day 4 HSV-2 5xl0 3 (+) b 4.1 xl 0 (+) 3.5xl0 7 (+) 10/10 (100) ICPIOΔPK (CS) l .OxlO 3 2.8xl0 (+) 0/10 (0) a Mice (n=10) were infected with lxlO 7 pfu in the footpad. Virus titers were determined by plaque assay. Lesions were seen beginning on day 7 p.i. in HSV-2 infected mice. b Ganglia were obtained on days 3 and 5 p.i., homogenized and assayed for virus. (+) means virus was isolated. c Latency is no. of explanted ganglia positive for virus at 30 days p.i./no. tested ND = not done Example 16 ICPIOΔPK (CS) protects from HSV-2 challenge better than ICP 1 OΔPK (RF) We used the footpad model to examine protection by ICPI OΔPK (CS). The experiment was done as previously described for ICPI OΔPK (RF) except that the mice were immunized with lxl 0 7 pfu of virus and they were given three immunizations (at 14-16 days intervals) before challenge with wild type HSV-2. Challenge was with 1 x 10 s pfu of HSV-2 and it was done 3 weeks after the last immunization. All mice in the PBS group developed skin lesions from which virus was isolated, and 8/10 died on days 8- 13 after challenge. By contrast, lesions were not seen and virus was not isolated from anyone of the immunized mice (Table 6). These findings indicate that the ICPIOΔPK (CS) virus has superior vaccine potential than the ICPIOΔPK (RF) virus in that it is somewhat more attenuated while providing superior protection. The latter is evidenced by the finding that v irus was not isolated from any of the animals although the challenge was done with 10-fold higher titers of HSV-2 than those used for animals immunized with ICPI OΔPK (RF) where virus was isolated from 3 10 mice (Example 13) Table 6 Protection mediated bv ICPI OΔPK Immunization Lesions Challenge Lesions (death) Virus isolation PBS None HSV-2 ( 1 \10 ) 10/10 (8 10) 10/10 CPIOΔPK (CS) None HSV-2 ( l xlO y ) 0/10 (0/10) 0/10 Mice (groups of 10) were immunized w ith PBS or ICPIOΔPK (3 sc injections) and challenged with HSV-2 in the footpad Example 17 ICPI OΔPK (CS) virus induces HSV-specific immunity A group of 3 mice w as immunized with ICPIOΔPK (CS) ( 1x10 pfu, 3 injections) as described in example 16 Two weeks after the last injection spleens w ere removed and T cells were used in lymphocyte proliferation assays done as in example 14 HSV-specific lymphoproliferauon was seen in all animals Proliferative levels (Fig 13) were significantly higher than those seen before (Fig 12), and approximately three-fold higher than those seen with the mitogen PHA These findings indicate that ICPIOΔPK (CS) induces good levels of virus-specific T cell responses All references cited herein are incoφorated by reference in their entirety While the invention has been described in detail, and w ith reference to specific embodiments thereof, it will be apparent to one ith ordinary skill in the art that various changes and modifications can be made therein w ithout departing from the spirit and scope thereof

Claims: We claim: 1. A vaccine composition comprising Heφes Simplex Virus-2 ICPIOΔPK (CS) and a pharmaceutically acceptable carrier or diluent. 2. A method of immunizing a subject against Heφes Simplex virus administering to said subject the vaccine composition of claim 1. 3. A method of conferring immunity against Heφes Simplex virus to a subject comprising administering the vaccine composition of claim 1. 4. A method of preventing clinical symptoms in a subject associated with Heφes Simplex Virus comprising administering the vaccine composition of claim 1. 5. The method of claim 2, 3, or 4 wherein said subject is a human. 6. The method of claim 5 wherein the dosage range for said vaccine is 1-100 million pfu. 7. The method of claim 5 wherein said vaccine composition is administered via an intranasal, oral, intravaginal, subcutaneous or intradermal route.

Inventors: AURELIAN LAURE, AURELIAN, LAURE
Applicants: UNIV MARYLAND, UNIVERSITY OF MARYLAND, BALTIMORE
Application year: 1999
Main classification: A61K39
IPC codes: A61K39, A61P31, C07K14, C12N7, C12N15