CA2381056A1 - Recombinant hsv-1 and live viral vaccines - Google Patents

Abstract

The invention concerns a recombinant herpes simplex virus which genome has been altered by mutations or deletions in the unique small (US) 8 and 12 genes. The invention further concerns vaccines comprising said recombinant herpes simplex virus.

Description

FIELD OF THE INVENTION
The present invention is in the field of live viral vaccines, in particular Herpes Simplex Virus (HSV) vaccine. The present invention also concerns viral vectors carriers of heterologous or therapeutic genes for infection purposes.
BACKGROUND OF THE INVENTION
Herpes viruses are a large group of intranuclear, double-stranded DNA
viruses that are remarkably capable of establishing a latent infection for many years after primary infection. The herpes virus group is responsible for such human i o diseases as fever blisters and keratoconjunctivitis (Herpes Simplex Virus Type 1 ) venereal diseases (Herpes Simplex Virus Type l and 2), Chickenpox (Varicella) and Shingles (Herpes Zoster) cytomegalic inclusion disease (Cytomegalovirus) infectious mononucleosis (Epstein-Barr virus), Exanthem subitum (roseola) (Human Herpes virus 6 and 7) and Kaposi's sarcoma (HHVB).
HSV-1 and HSV2 are related immunologically, but most of their proteins carry distinguishing characteristics which allow them to be differentiated.
HSV is characterized by its ability to establish latent infection in the central nervous system (CNS) of its host specifically in the neural ganglia. The infection or reactivation may result in encephalitis.
2o There have been several attempts to develop a live HSV-I vaccine.
U.S. 5,328,688 discloses a herpes simplex virus which has been rendered avirulent by prevention of the expression of an active product of a gene which is designated as y,34.5.

U.S. 4,89,587 discloses a recombinant herpes virus for use as a vaccine, both against a virulent HSV-1 and against HSV-2. The virus was prepared by taking an HSV-1 like virus recombinant from which a portion of the genome, responsible for neurovirulence, was deleted, and a gene from HSV-2 genome, s responsible for coding an immunity-inducing glycoprotein, was inserted into the mutated virus genome.
U.S. 4,554,159 discloses a live viral vaccine against herpes simplex virus types 1 and 2 which includes at least one vaccinal intertypic (HSV-1 x HSV-2) recombinant virus strain prepared by crossing two prototypic HSV-1 and HSV2 parental virus strain in order to obtain a recombinant progeny. The parental strains have distinguishing genetic markers so that it is possible to distinguish the recombinant progeny from the parenteral strains on the basis of these markers, and at least one of the parenteral strains is also temperature sensitive.
U.S. 5,641,651 discloses a synthetic herpes simplex virus promoter which comprises the herpes simplex virus oc gene promoter fragment operatively 5'-linked to the herpes simplex virus Y gene promoter fragment.
U.S. 5,837,532 discloses a viral expression vector which comprises a herpes simplex virus type 1 with a DNA sequence which has an alteration in the gene coding for the Vmw65 protein, the alteration being a transition or transversion 2o alteration of 1 to 72 base pairs or a deletion of 3 to 72 base pairs, or alternatively, an insertion of an oligonucleotide sequence, wherein the alteration is carried out in the position in the gene coding for the probe in between the amino acid 289 and 412 of the protein. The viral expression vector further comprises a heterologous gene which is inserted in the region of the HSV-1 genome which is a region non 25 essential for culture of the virus, together with a suitable promoter.
Typically, the heterologous gene is a therapeutic gene which is inserted in the viral TK gene (UL23).
U.S. 5,066,492 discloses a method for treating or preventing infection of hwnan HSV-1 infections using inoculation by a live bovine mammillitis virus.

U.S. 4.024,836 discloses lyophilized live herpes virus vaccine that comprises from about 0.5% to about 8% moisture.
The above HSV-1 virus vaccines are either mutants in one pathogenecity gene (such as the y,34.5 gene in U.S. 5,328,688 and the VMW 64 in U.S.
5,837,532) or the intertypic recombinant between HSV-I and HSV-2 (U.S.

4,554,159). However, none of these publications led to the development of a single anti-HSV-1 vaccine for human use. One of the main obstacles is the property of the live HSV-1 vaccine viruses of these publications to enter into the nervous system of the immunized animals and to establish a latent infection therein. To date, the only i o live herpes virus vaccine that is being used to immunize humans is the OKA
strain of Varicells-Zoster virus (VZV) which is an attenuated virus obtained from a VZV
patient.
SUMMARY OF THE INVENTION
The present invention is based on a development of a unique HSV-1 recombinant virus termed hereinafter "HSY 1 RI S ". This recombinant virus was prepared by obtaining a parent virus, being HSV-1 HFEM originally isolated from a patient with a mild HSV-1 infection. HSV-1 HFEM was shown to have a deletion in the internal repeat of UL (IRL) so that this virus was known to be apathogenic to adult mice. However this virus was still unsuitable for human immunization 2o purposes since it retained its pathogenecity to suckling mice~~~. The HSV-1 recombinant has the HFEM DNA genome in which a BamHI-B DNA fragment from a pathogenic virus HSV was inserted by recombination~~~.
The HSV-1 R15 recombinant of the present invention was found to have several advantageous properties as follows:
25 ( 1 ) it was found to be unpathogenic to adults and suckling mice;
(2) the replication of the apathogenic HSV-1 R15 recombinant of the invention in the skin or nose epithelium of the immunized subject (mouse) is self limited and consequently the viral DNA disappears from the site of inoculation within 3-4 days post infection;

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(3) the recoxinbinant of the invention is incapable of penetrating into the peripheral and central nervous system and thus in , fact overcomes one of the main obstacles of prior art proposed live-viral based vaccines;
(4) inoculation with the recombinant of the invention prevents, in immunized s mice, the entry of other pathogenic HS'V 1 into the nervous system;

(5) the I3SV 1 recombinant of the invention is highly immunoge~oi~c and is capable of inducing, in immunized mice, an antivizal huutoral i~unune response {G) the recombinant of the invention is not pathogenic as a result of a stereotaxic injection;
(7) the recombinant of the invention, .after being injected into ~oauuse and rat brains did not activate the hypothalmie pituary adrenocortical axis (FAA), - did not increase the production of prostaglandin E2, did not induce Ii,-I
gene expression outside of the hypothalamus in the injection site, in contrast is to the condition caused by pathogenic HSV-1F;
{8) infection, of mouse astrocytes under W~ vitro, conditions with the recombinant of the iavention did not induce the expression of the IZ,-1 gene, conlarary to ' the situation where unfection. was caused with pathogenic HSV-1 Synl7 that induced the IL-1 genes within three hours post infection;
20 (9) the recombinant HSV 1 Rl s of the invention, has an active Uf,.23 gene coding for thymidine kinase (IK) anal thus, the virus is highly sensitive to treatment with anti-herpetic drug acyclvvir, so that if desired, its infection may be controlled.
The HSV-1 R1.5 recombinant carries a number of deleted o~genes, genomic zs rearrangements anal changes in gene expressioza, which contribute to its lack of pathogenicity and high innmunogEnicity in use. as a vaccine. The fact that several gex~.e deletions and mutations occur simultaneously, prevents spontax~.~ous revertants mutations, thus eliminating the possibility of the recombinant of the invention.
mutatix~,g back to produce a virulent virus.

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Furthermore, the positions where genomic deletions in the HSV-1 R15 recombinant of the invention took place may be used for insertion of heterologous genes into these DNA sequences. The heterologous genes may be genes which code for immunogenic proteins from other human herpes viruses (i.e. HSV-2 to HHV-8) for example genes coding for HSV-2 to HHV-8 glycoproteins immediate early genes.
Alternatively, the heterologous genes may be obtained from other pathogenic human RNA and DNA viruses, such as genes coding for HIV-1 gag and envelope genes and minigenes; Hepatitis C virus genes or structural proteins;
t o influenza virus genes and Dengue virus genes for structural proteins.
In the above two examples the recombinant of the invention is used as an effective carrier of genes coding for immunogenic proteins of a desired virus for the purpose of vaccination against said virus.
The HSV-1 R15 recombinant of the invention may also be used as a carrier ~ 5 for various heterologous genes, for purposes other than immunization, for example for therapeutic purposes utilizing gene therapy techniques. In that case the recombinant of the invention is used as a carrier for genes which expressions brings about beneficial therapeutic effect, for example, the genes may be human genes coding for cytokines and chemokines, genes coding for a missing enzyme, a 2o missing metabolite and the like, as well as genes involved with cell death.
An example of the latter heterologous genes is apoptosis and tumor suppressor genes such as p53 to be inserted into cancer cells By another option, the property of HSV-1 R15 recombinant of the invention to be able to replicate in the brain astrocyte cell cultures, while being incapable of 2s affecting nerve cells, can be used to selectively transfect only tumor astrocytes (astrocytoma) while leaving nerve cells uninfected. This property may be used to cure astrocytoma brain tumors, either by simply injecting HSV-1 R15 of the invention stereotactically to the tumor so its natural replication will destroy the tumor, or inserting in the genome of the recombinant virus of the invention various heterologous cytotoxic genes, which then can selectively destroy only the astrocytoma tumor while leaving the nerve cells undamaged.
The present invention thus concerns a recombinant herpes simplex virus, the genome of which comprises a mutant of the genome of HSV-1 with the following alterations:
a deletion or mutation in the unique small (US) 8 gene region resulting either in expression of a non-functional gE protein or in no expression of the gE protein; and a deletion or mutation in the US 12 gene region resulting either in expression of a non-functional ICP47 protein or in no expression of the ICP47 protein.
The term "non functional " refers to a protein which is incapable of carrying out this nonnal physiological activity. In the case of US 8 gene, the normal expression product of the gene is a protein termed "truncated gE" and an unfunctional product is a protein which cannot form a dimer with gI protein.
In the case of US 12 gene the nonnal expression product of the protein ICP47, which binds to and inhibits transport of nonapeptides by TAPI/TAP2 diner to HLA
(MHC) Class I polypeptides in endoplasmic reticulum (ER) of infected cells. A
"non functional " US 12 expression product is a product which cannot carry out 2o said inhibition of transport.
Within the scope of the invention are any mutations or deletions in the genes which results in an unfunctional protein or results in no expression of viral proteins altogether. The mutations or deletions may be in the coding region of the gene or in the control regions thereof, resulting in the above non-expression or expression of 25 non-functional proteins.
The term "deletion " can refer to partial deletion or complete deletion of the gene sequence. Where partial deletion occurs it should be of a length sufficient to avoid expression of the protein or of a length required to produce a non-functional protein. The term "mutation " refers to an addition, replacement or rearrangement ;o of at least one nucleic acid, as compared to the native HSV-1, resulting either in _7_ production of a non-functional expression product or in complete lack of production of expression product as explained above. Again, mutation and alteration may be in the coding or non-coding regions of the gene.
Preferably, the recombinant virus of the invention should contain, in addition to the above two alternatives, at least one additional deletion or mutation in a gene selected from the group consisting of: US 9; US 10; and US 11 tegument proteins, said deletion or mutation either eliminating production of the expression product of these genes or alternatively producing non- functional expression products as explained above, i.e. a product which is unable to carry out the normal i o physiological activites of its unmutated counterparts.
Most preferably, the recombinant of the invention should have said deletion or mutation simultaneously in US 9 and US 10 and US 11 so that either none of the expression products of these genes are produced, or the expression products produced are non-functional expression products as explained above or a i 5 combination of non-expression and non-functional expression.
US 9 codes for the synthesis of tegument phosphorylated protein, US 10 codes for another tegument protein. US 11 codes for a tegument protein that binds the 60 S ribosomal subunits in infected cells and also binds to mRNA
transcripts of the gene UL 34 (membrane-associated phsophorylated virion protein).
2o Non-functional products of the above are products which cannot carry out their usual physiological activity of the unmutated counterpart.
In accordance with the preferred embodiment of the invention, the recombinant of the invention completely lacks the US 12 region, the US 9 region, the US 10 region and the US 11 region, by deletion of the full sequence of these 25 genes; and the US 8 region is mutated by reverse splicing and recombination so that it codes for a shorter non-functional product of gE having 188aa (as compared to a functional gE having SSOaa in HSV-1 Synl7), which protein is identical to the N-terminus 170aa of gE protein of HSV-1 Synl7. The product of the mutated US 8 gene, i.e. the truncated gE protein, does not form a heterodimer with the gI
coded _g_ by US 7, and without said heterodimer HSV-1 R15 is incapable of penetrating and infecting nerve cells.
By another preferred embodiment, the recombinant herpes virus of the invention contains a duplicate of at least one of the following genes: the US
1 gene s coding for ICP 22 (IE4) and US2 gene coding for ORF 291 aa. The presence of two US genes is a special feature of HSV-1 R15. The function of ICP22 is regulation of y2 gene expression and viral gene transcription that allows the virus to produce high titers of infectious virions.
Most preferably, the duplication should be so that at least one and preferably ~ o both of these genes appear both in the Internal Repeat of S (IRS) and Terminal Repeat of S (TRS).
By an additional preferred embodiment, the recombinant of the invention does not express UL54, and the gene products ICP27 and y134.5 gene product ICP34.5, which is a neuro-virulence factor. It was reported that deletions or t 5 mutations near the US 12 gene may affect the expression of the above mentioned genes by an unknown mechanism. However, it may be that non-expression of these products is an event independent of the deletion in the US 12.
By a yet further additional embodiment the recombinant of the invention carries an active UL23 gene coding for tyrosine kinase (TK). This results in a 2o recombinant virus that is sensitive to treatment with the anti-herpetic drug acyclovir. Thus, where it is desired to control replication of the recombinant of the invention, for example where the vaccinated individual develops deficiency syndrome, control may be achieved by administration of acyclovir,.
By the most preferred embodiment the present invention concerns a 2s recombinant as shown in Figs. 10, 11 and 12 under R15.
The present invention further concerns an anti-HSV-1 vaccine comprising as an active ingredient the recombinant R15 herpes simplex virus of the invention, optionally together with an immunologically acceptable carrier. By another alternative, the invention concerns DNA vaccine expressing a viral glycoprotein B gene. Immunization with HSV DNA vaccines before or together with HSV-1 R1 ~ will increase the protection of the vaccinees.
By another embodiment the present invention concerns an anti-herpes 2, herpes 3, herpes 4, herpes 5, herpes 6, herpes 7 or herpes 8 vaccine, comprising as an active ingredient the recombinant virus of the invention having in its genome a heterologous sequence obtained from herpes virus 2 to herpes virus 8, respectively, said heterologous sequence coding an immunogenic protein of said virus.
Preferably, the immunogenic protein should be the appropriate herpes virus glycoprotein obtained from the relevant herpes sepecies.
i o The present invention also concerns a method for immunizing a subject against herpes simplex 1, or any one of herpes viruses 2-8 as described above by administering to said subject an immunologically effective amount recombinant herpes simplex virus as described above. For example, the immunization may be by infecting a superficial scratch in the skin of the upper arm of the immunized ~ s subject.
Another embodiment of the invention is based on the realization that the recombinant HSV-1 RIS of the invention is capable of replicating selectively in brain astrocytes while not infecting nerve cells. Thus, the present invention further concerns a pharmaceutical composition for the treatment of astrocytoma brain ?o tumor comprising as an active ingredient the recombinant virus of the invention.
The HSV-1 RlSmay reach the brain by stereotactic injection, into the brain tumor.
Whilst within the brain, the recombinant herpes virus of the invention will replicate only in the astrocytoma tumor cells, and thus destroy them by its natural replication, while maintaining nerve cells undamaged.
25 By another alternative, the HSV-1 R15 of the invention may comprise also heterlogous sequences which are known to be cytotoxic, such as genes that induce apoptosis.
Then, the pharmaceutical composition of the invention would contain said cytotoxic-containing recombinant viruses. These sequences responsible for ;o cytotoxicity will be expressed only in cells infected with the recombinant virus of the invention, i.e. only astrocytes while other brain cells which were unaffected by the virus will not be damaged. In addition, the pharmaceutical composition of the invention can be used for the treatment of solid tumors in the skin and internal organs, either utilizing recombination of the invention without heterologous sequences, or such recombinants having also cytotoxic heterologous sequences.
By yet another embodiment, the herpes simplex recombinant of the invention may be used to prepare other pharmaceutical compositions for treatment of diseases, disorders or pathological conditions wherein a beneficial effect may be evident by expression of a desired gene in cells, for example, genes that will inhibit ~ o tumor cell-cycle by expressing the human PML gene in-situ in gene therapy.
The present invention also concerns such recombinant virus having heterologous sequences inserted therein which upon expression, for example, in gene therapy, provide beneficial therapeutic effects such as inhibition of tumor growth.
The heterologous sequences, either for preparing vaccines for immunizing i 5 against herpes virus or other DNA or RNA viruses inserted for the purpose of treatment of astrocytoma or for any other gene therapy purposes, should preferably be inserted into HSV R15 genome at the site of the deletion of the genes (such as in sites of deletion of the US 9, US 10, US 11 or US 12 genes or that are not essential for the virus replication. The expression of the heterologous sequence can be under 2o the control of any one of known control elements, such as HSV-1 or CMV
promoters.
BRIEF DESCRIPTION OF THE TABLES
Table 1A Pathogenicity of different HSV-1 strains to adult mice;
Table 1B Pathogenicity of different HSV-1 strains to suckling mice;
25 Table 2 Distribution of HSV-1 R15 DNA in brain tissues of adult mice after intranasal immunization in comparison with the apathogenic strain HSV-1 vhs (UL41);
Table 3 the penetration of HSV-1 R15 into the spinal cord and adrenal glands after infection in the mouse footpad skin;

Table 4 Pathogenicity of HSV-1 in mouse strain A/J which were inoculated intracerebrally (50 ~.l/animal) or intraperitoneally; and Table 5 Inhibition of HSV-1 R15 and HFEM plaque formation by acyclovir.
s Table 6 ICV inoculation with R-15 and monitoring of aggressive behavior, fever, PGE2 production and the challenge with strain Syn 17+.
BRIEF DESCRIPTION OF THE DRAWINGS
In order to understand the invention and to see how it may be carried out in practice, a preferred embodiment will now be described, by way of non-limiting i o example only, with reference to the accompanying drawings, in which:
Fig. 1A shows survival of adult Sabra mice immunized with apathogenic HSV-1 strains after challenge with the pathogenic HSV-1 F;
Fig 1B shows survival of suckling Sabra mice immunized with apathogenic HSV-1 strains after challenge with the pathogenic HSV-1 F;
is Fig 2 shows PCR test to detect the presence of HSV-1 F DNA in the olfactory bulbs, amygdala and trigeminal ganglia of intranasally infected mice that were immunized with HSV-1 R15 by the footpad route;
Fig 3 shows neutralizing anti-HSV-1 antibodies in sera of mice, immunized with HSV-1 R15, prior to and after challenge with the pathogenic 2o HSV-1 F;
Fig 4 shows a scheme of the recombination experiment in which BamHI B
DNA fragment from the pathogenic HSV-1 F was recombined with HSV-1 HFEM genomic DNA. From this experiment HSV-1 R15 was isolated;
Fig 5 shows a map of the genes and their RNA transcripts in the 2s IRS-US-TRS DNA of HSV-1 Synl7 and HSV-1 R15;
Fig 6 shows Northern blot analyses of RNA transcripts of genes in the IRS-US-TRS DNA of HSV-1 R15;
Fig 7 shows Southern blot analyses of HSV-1 Synl7, HFEM and R15 IRS-US-TRS DNA with OriS and US 12 probes;

Fig 8 shows Southern blot analyses of the BamHl DNA fragments from HSV-1 Syn 17, R15 and HFEM DNA;
Fig 9 shows nucleotide sequence analysis of the 4182 by of HSV-1 R15 DNA fragment Hpal-EcoRl (coordinates 141611-146693);
Fig 10 shows rearrangements in HSV-1 R15 IRS-US-TRS DNA;
Fig 11 shows comparative maps of IRS-US-TRS regions of HSV-1 Synl7 and HSV-1 R15 US DNA;
Fig 12 shows rearrangements of the genes in the HSV-1 R15 IRS-US-TRS
DNA compared to gene arrangement in HSV-1 Synl7 DNA;
Fig 13 shows a PCR test to differentiate HSV-1 R15 DNA from DNA of pathogenic HSV-1 strains;
Fig 14 shows FACS analysis of the HLA class I molecules on the cellular membrane of human fibroblasts after infection with HSV-1 Synl7 and R15;
Fig 15 shows a map of part of UL-IRL-IRS of HSV-1 Synl7 and the ~5 deletion in HFEM BamHI-B DNA;
Fig 16 shows a western blot analysis to detect ICP 34.5 protein in cells infected with HSV-1 strains;
Fig. 17A, 17B and 17C shows survival of mice challenged with HSF-1 w.t. F and immunized by ocular route ( 17A); skin route ( 17B) or lung 2o route ( 17C);
Fig. 18 shows ICP27 protein detection in HEK 293 cells infected with HSF-1 F; R15 and Moch;
Fig. 19 shows the replication rates of R15, P-17, R-19 in astrocytes; and Fig. 20 shows induction of IL-IB gene expression (as determined by 2s RT-PCT induction) in astrocytes by HSV-1 strain Synl7+ and by R-15.

DETAILED DESCRIPTION OF THE INVENTION
EXPERIMENTAL PROCEDURES
1. Mice Mice of the Sabra strain (outbred) were supplied by the breeding facility of s the Hebrew University. Ten mice inhabited each cage. They received standard nutritional and water supplies in the SPF of the Animal Containment Facility of HUJ.
2. Cells and medium io The cell lines Vero (kidney epithelium cells of the green monkey) and HeLa (Human) were grown in a medium of DMEM (GIBCO) enriched with 10%
fetal calf serum (Befit HaEmek) and supplemented with 40 units/ml penicillin and 160mg/ml streptomycin. The cells were grown in culture dishes (NUNC, Denmark) at 37°C with 5% C02tand in 100% relative humidity. The cells were t s transferred from the dishes by immersing them in a 1:5 solution of 0.25%
trypsin (w/v) and 0.2% EDTA, and inoculated under the same conditions described above and grown to confluent monolayers.
Primary astrocyte cultures were prepared from newborn rat cerebral hemispheres. Following dissection and removal of meninges, the tissue was 2o enzymatically (trypsin) and mechanically dispersed, centrifuged through 4%
BSA
layer, and 3-3.Sx10~tcells were seeded onto poly-D-lysine coated T-75 flasks.
After 1-week growth, microglia and oligodendrocyte lineage cells were separated by the shaking method and the remaining astrocytes were passaged once, resulting in 95% pure astrocyte cultures.
3. Viruses 3.1 Virus strains and recombinants.
The HSV-1 virus strains used in this study were KOS, HFEM, F (kindly provided by Prof. Rapp, Pennsylvania, USA), the mutant vhs-1 (kindly provided by Dr. Nitza Frenkel, Tel Aviv University), and Synl7 (provided by Prof.
Subalc-Sharpe, Glasgow, Scotland).
The recombinants HSV-1 R15 and R19 were prepared by Dr. A.
Rosen-Wolf and Prof. G. Darai, Heidelberg University, Heidelberg, Germany.
They were produced by recombining the BamHI-B DNA fragment (coordinates 113.322-123,464) from HSV-1 F strain with the HSV-1 HFEM DNA genome.
The recombinant HSV-M-lacZ (F blue) was also prepared by Dr. A.
Rosen-Wolf in the laboratory of Prof. G. Darai. The DNA fragment coding for the UL56 gene (coordinates 116,030-121,753) of the HSV-1 F strain was i o exchanged with a DNA fragment containing the bacterial gene for (3-galactosidase under the control of the RSV promoter.
The pathogenic recombinant viral strains P42 and P71 were prepared by Dr. T. Ben-Hur in the laboratory of Prof. Y. Becker, Jerusalem, Israel. These strains were the result of the recombination between the NruI-BamHI DNA
fragment (coordinates 111,290-113,322) from the virulent HSV-1 R19 recombinant with the avirulent HSV-1 R15 recombinant DNA genome.
TK mutants were produced in the laboratory of Pro~ Becker using BURR
(F TK, F blue TK, R15 TK, KOS TK Syn-17 TK).
The viruses were grown in Vero cells until a full cytopathic effect was 20 obtained. The virus titer was examined using standard plaque assay and the virus stocks were kept at -70°C. Sonicated suspensions of uninfected cells (Mock) served as a negative control in the mice infection trials.
3.2 Determination of the virus titer.
2s Vero cells were infected with 10 fold dilutions of the newly prepared virus stock. After one-hour adsorption, the cells were coated with an overlayer containing 0.7% agar in DMEM and incubated in a COz incubator 37°C.
After 3 days the cells were fixed with 25% fonnalin solution and stained with gentian violet. Virus plaques were counted and titer determined in plaque forming units (pfu/ml).

4. Infection of mice with virus Three-to-four-week-old male Sabra mice were infected with the HSV-1 strains and recombinants at different titers. Each mouse received 30-50 p,1 of diluted virus stock introduced into the nose. Suckling mice (7 days old) were s infected by the same methods with 10 ~l of the diluted virus stock. In the preliminary experiments the mice were anesthetized with Halothane vapors for 10-20 seconds before virus infection. It was found that the virus infection in these mice caused pathological changes not only in CNS, but also in the lungs.
Therefore the mice were not anaesthetized before infection. Progression of the i o disease was monitored for 4-6 weeks. Mice of the control groups (Mock) were treated by the same mode. The follow-up was done every day by two neutral investigators. The results were registered and compared.
The major stages of the developing HSV-1 viral disease (Herpetic encephalitis), in murine model, are: 1. Adynamia - Lack of movement; 2. Flexia ~ s of the body; 3. Hair-raising; 4. Conjunctivitis (eye infection); 5.
Acrocyanosis (body appendages turn blue); 6. Convulsions of the muscles; 7. Paralysis; 8.
Death.
The degree of severity of the symptoms is graded from (-) (no disease manifestation) to (++++) (most extreme expression of the disease symptom).
5. Extraction of tissue sample s from infected mice The mice were sacrificed by cervical dislocation and the tissues required for the analysis were removed by dissection. The tissue samples (nose epithelium, trigeminal ganglia, olfactory bulb, spinal cord and adrenal glands) were 2s transferred to test tubes and kept at -70°C for further analysis.
6. Preparation of the serum The HSV-1 infected mice were sacrificed by cervical dislocation and the thoraces of the mice were immediately surgically opened and the heart was 3o exposed. Blood was extracted with a Pasteur pipette, transferred to an Eppendorf test tube. incubated for two hours at room temperature and centrifuged for 10 minutes at 14000rpm. The upper part of the liquid (sera) containing the antibodies was transferred to Eppendorf test tubes and kept at -20°C
for further analysis.
s 7. Determination of the titers of neutralizing antibody in sera from infected and immunized mice 100 p1 serum (in appropriate dilution) was mixed with 100 ~l viral stock io (in a titer of 103pfu/ml) and with 50 p1 of growth medium DMEM and incubated for 2 hrs at 4"C. This mixture was added to the Vero cell monolayer incubated at 37"C with 5% CO? and standard plaque assay was performed.
The level of neutralizing antibodies was calculated according to the percentage of viral plaque inhibition.

8. Detection of infective virus in mouse tissues Tissue samples were homogenized in a Dounce homogenizer in 1 ml PBS.
After homogenization, 200 p,1 of the sample was sonicated and added to Vero cell monolayer. The cells were incubated at 37°C with 5% COZ for several days and 2o the cytopathic effect of the virus was observed.

9. Preparation of high molecular HSV-1 DNA
9.1 Preparation of purified HSV-1 virions Virus-infected cells (109) were washed in TBS buffer, scraped into a 2s minimal volume of the buffer, sonicated for 2 min and mounted onto a sucrose gradient (12%-52% w/w sucrose in TBS buffer) in nitrocellulose test tubes. The gradients were centrifuged for one hour at 25000 rpm in rotor TST-28. The band of virions was transferred to polyalomer tubes and diluted in TBS. The tubes were centrifuged again under the same conditions and the virion pellet was resuspended 3o in lml TE buffer.

9.2 Preparation of high molecular viral DNA from purified virus (9.1). from the virus-infected cells or from tissue samples.
O.SmI or l.Oml of TE buffer was added to a sample in the presence of s Proteinase K (Sigma) (final concentration of O.Smg/ml) and 1 % SDS (Sigma).
The samples were incubated for 2 hours at 55°C or at room temperature (R.T.) overnight. with gentle shaking. The sample lysate was extracted twice with phenol and then with chloroform containing 4% isoamyl alcohol. The DNA was precipitated from the aqueous phase with ethanol, dissolved in 400m1 of TE
i p buffer and kept at 4°C.

10. Indirect immunofluorescent stainings Subconfluent monolayer cell cultures that were grown on coverslides were infected with HSV-1 virus (or treated by another way). Suspension cultures were i s attached to coverslides using poly-L-Lysine solution 1:10 (Sigma). Cells were fixed with 4% formaldehyde (10', R.T.) and rehydrated with PBS (5' x 2, R.T.) with gentle shaking. Cells were washed with SOmM NH4C1 (5', R.T.) and afterwards withfPBS (5', x 2 R.T.) (At this stage the procedure may be interrupted and the slides can be frozen at -20°C).
2o For blocking of the Fc receptors on the cells, slides were incubated with S% Normal Goat Serum (20', R.T.) and then washed with PBS (5', R.T., x3).
Then cells were incubated with a primary antibody (lhr, R.T.), washed with PBS
(5', R.T. x3), incubated with secondary antibody (45', R.T., in darkness), and washed with PBS (5' R.T. in darkness). Slides were prepared using antibleaching 2s medium (45m1 glycerol pH 8.5, Sml PBS pH 7.4- pH7.5, O.Sg DABCO (Sigma) and O.OSg NaN3) and kept at +4°C in the dark.
When working with paraffin-embedded slides, the deparafinization procedure is necessary prior to fixation. Slides washed with xylene (5', R.T.
x3), then with 100% ethanol (5', R.T., x 2 (3)), then with 96% ethanol (5', R.T. x2 (3) ;o and finally with doubly distilled water (DDW) (5', R.T. x1).

In some cases the use of 0.01 % triton x 100 solution prior to incubation with a primary antibody is recommended.

11. Investigation of RNA expression in cells s 11.1 Extraction of total RNA from cells using CsCI method.
RNA was extracted from infected cells or uninfected cells by suspending the cell pellet in a solution containing 25mM sodium citrate, 4.5M guanidiurn thiocyanate, 0.5% sodium lauryl sarcosine, 0.1% antifoam, and 47mM
mercaptoethanol. Cell lysates were centrifuged for 14 hours at 36000rpm in rotor TST-55.5 of the Beckman preparative centrifuge, through a layer of CsCI, at a concentration of 5.7M. The RNA that was centrifuged to the bottom of the test tube was suspended in 0.3M NaAcetate, precipitated with ethanol, and dissolved in RNase tree DDW.
t 5 11.2 Electrophoresis of RNA in ~lyoxal gels RNA was added to a solution that contained IOmM Sodium phosphate at pH6.5, 50% DMSO, 1M de-ionized glyoxal and was incubated for 30 min at 50°C. The RNA was electrophoresed in the presence of loading buffer ( l OmM
EDTA, 0.05% Bromophenol blue and 60% glycerol) in agarose gel (1.5%) that 2o contained IOmM Sodium phosphate at pH 6.5. The gel was run in a Sodium phosphate buffer at pH 6.5. Each gel contained a sample of RNA with Ethidium bromide (EtBr), for the determination of the position of the 18S and the 28S
RNA as molecular weight markers.
25 11.3 Fixing RNA to a nylon filter (Northern blot) The RNA from the gel was transferred by the capillary blot method to a nylon filter (0.2 pm), by placing the gel on an absorption paper (Whatman 3MM) soaked in 25mM sodium phosphate at pH 6.5. A nylon filter, 8 layers of absorption paper in a phosphate buffer, 8 layers of dry absorption paper, many layers of paper towels and a one-kilogram weight were placed on the gel. After 12-18 hr the nylon filter was dried and baked in 80°C oven for one and a half hrs.
11.4 Preparation of specific DNA probes 11.4.1 Purification of DNA from a~arose Viral DNA fragments that were cloned in plasmids served as a source for molecular probe preparation. The plasmid DNA was cleaved by restriction enzymes (which were used in the cloning) and the DNA was to electrophoresed in agarose gels. The band containing the viral DNA
fragment that will be used as a probe was excised and transferred to an Eppendorf tube. To obtain an agarose-free DNA fragment, the DNA was purified by Gel extraction kit (Jetsorb, Promega), suspended in TE buffer and kept at -20°C.
IS
11.4.2 Labeling of DNA with radioactive phosphate by Nick translation Viral DNA, serving as a probe in hybridization experiments, was labeled by the Nick translation method using the BRL kit. The reaction 2o was carried out in the presence of 100p.ci of dCTP (a32P) together with the other 3 unlabeled deoxyribonucleotides. After incubation for 90 min at 14°C the reaction was stopped and the labeled DNA was separated from the free nucleotides on a G-50 sephadex column that was washed with TE
buffer. The reaction products were loaded onto the column; the fraction of 25 labeled probe was collected. The radioactivity was measured with a scintillation counter. The labeled probe was used in the Southern or Northern hybridization experiments.
11.5 Hybridization of RNA with DNA probes.
3o The nylon filter was put in a bag or hybridization bottle and incubated 3 hrs at 42°C in the presence of 50% formamide, 5 x Denhardt buffer, SOmM

Tris-HCL, pH 7.5, 0.7M NaCI 0.1% sodium pyrophosphate, 1% SDS, 10%
Dextrane sulfate. 400mg/ml of salmon sperm DNA that underwent denaturation.
The labeled DNA probe (2 x 10G cpm) was added to the bag or bottle and the filter was incubated for 16 additional hours. The filter was washed twice for min at room temperature in a solution of 2 x SSC and 0.1% sodium pyrophosphate, once for 30 min at 60°C in a solution of 2 x SSC, 0.1%
sodium pyrophosphate and 0.1% SDS, and once for 30 min at 60°C in a solution of 2 x SSC and 0.1% SDS. The filter was exposed to Agfa X-ray film in a cassette with enhancing screen and kept at -70°C for several hours.
12. Identification of specific DNA by hybridization to a DNA probe 12.1 DNA electomhoresis in a~arose gels High molecular DNA (8p,g) prepared from HSV-1-infected cells or from HSV-1 purified virions was incubated with 5 units/p,g DNA restriction enzymes in final volume of 100 p,1 overnight in 37°C. The samples of the restricted DNA
were electrophoresed in 0.8% agarose gel in TERN buffer at 25 volts (35v/150mAmp) overnight.
12.2 Denaturation and neutralization 2o The gel was placed on glass in a bowl and shaken gently with a solution of O.SN NaOH and 1.5M NaCI in room temperature for one hour. The neutralization is achieved by substitution of the alkaline solution with a neutralizing solution of 1M Tris pH8.0 and 1.5M NaCI at room temperature while gently shaking for 30 mm.
12.3 Fixation of DNA to the nylon filter (Southern blot) Same procedure as for RNA.
12.4 Hybridization of DNA with DNA probes ;o Same procedure as for RNA with the following changes:

a) Hybridization buffer contained 50% formamide, 10 x Denhardt, x SSPE, 1% SDS and 400mg/ml denaturated salmon sperm DNA.
b) The filter was washed twice for 15 minutes at R.T. in a solution of 2 x SSC and 1% SDS and once for 2 hours at 65°C in a solution of s 0.1 x SSC and 1% SDS.

13. Cloning of viral DNA and sequence analysis 13.1 Preparation of the viral DNA fragments and plasmid vector Viral DNA fragments and the plasmids cleaved with suitable restriction enzymes i o were purified from agarose gel (see also Section 11.4).
13.2 Li-;gation Plasmid and viral DNA were mixed in a molar ratio of 1:10 in the presence of the enzyme Ta DNA ligase (Promega), an appropriate buffer and i s incubated at 16°C overnight.
13.3 Bacterial transformation An E. Coli bacterial colony of the dHaS strain or the JM109 strain was grown overnight at 37°C in LB medium. The next day a culture of competent 2o bacteria was prepared by the CaCl2/RuCl2 method (Kushner 1978). SOng of plasmid DNA after ligation was added to 100p,1 competent bacterial culture for 20 minutes packed on ice. The bacteria were transferred to 42°C for one minute, then to ice for two minutes and immediately afterwards 1 ml LB was added and the bacteria were incubated for one hour at 37°C. The bacteria were seeded onto a Zs LB plate containing 1.5% agar and 100 p,g/ml of ampicillin. When necessary 40 p,1 of 2%X-Gal (sigma) solution and 40 p,1 of 2% IPTG (Sigma) was added to the bacteria during the seeding. The plate was incubated overnight at 37°C.

13.4 Colonv hybridization The bacterial transformed colonies that were transferred to nitrocellulose filters, BA-85 0.4 ym, 80mm diameter (Schleicher and Schuell). In order to enable compatibility between the colonies on the filter that were indicated by a specific probe and the same colonies on the agar plate, the filter and the plate were designated in the same manner. Four trays are prepared upon which four layers of no. 3 Whatman paper soaked with one of the following four solutions:
Tray 1 - 0.2NNaOH, l.5NaC1 Tray 2 - 0.4MTris pH 7.6, SSC x 2 Tray 3 - 0.4MTris pH 7.0, SSC x 2 Tray4-SSCx2 In the first stage the filter is placed on the bacterial plate for one minute and then transferred to the four trays in the above numerical order in each tray for one minute. The filter was placed in each tray upon a wet Whatman paper so the colonies face up. The filter is air-dried and then baked in a vacuum oven at 80°C
for two hours. At this point the procedure is as in Southern blotting: Nick translation of probe, hybridization, and washing as specified: one half hour at room temperature in SSC x 2 + 1% SDS and twice for ten minutes at 65°C
in SSC x 0.5 + 1 %SDS.
13.5 Rapid~reparation of plasmid DNA
Bacterial colonies were grown in LB liquid culture overnight, then precipitated and suspended in 100p1 solution of SOmM glucose, lOmM EDTA, 25mMTris pH 8.0 and 4mg/ml lysosome. After 5 minutes, 200p,1 solution of 2s 0.2MNaOH and 1% SDS were added and incubated for 5 minutes on ice. 150 p,1 solution of SMKAc at pH5.6 were added for 5 additional minutes, then centrifuged and the DNA was extracted with phenol/chloroform. The aqueous phase was precipitated with ethanol and the DNA was dissolved in TE buffer.

13.6 Determination of DNA nucleotide sequence The plasmid DNA was purified using Promega's Wizard plus sv miniprep DNA purification system (Promega). Determination of the sequence is carried out on l0yg of DNA in the DNA Analysis Unit of the Life Sciences Institute of the Hebrew University of Jerusalem (ABI - PRISM, Model 377). Several of the plasmids containing cloned HSV-1 were sent for sequence determination to the laboratory of Prof. G. Darai, Heidelberg, Germany.
13.7 Computer analysis of the DNA sequences Computer analyses of amino acid sequences and proteins was carried out using the program package UWGCG of the University of Wisconsin. The nucleotide sequence of HSV-1 Synl7 DNA was obtained from Genbank.

14. Identification of viral DNA by the PCR method ~s 14.1 Identification of HSV-1 viral DNA by PCR
The primers used in the PCR experiments were synthesized according to the sequence of HSV-1 UL53 (gK) essential gene.
Direct primer 5' TCATCGTAGGCTGCGAGTTGAT 3' (22 mer) Reverse primer 5' CTCTGGATCTCCTGCTCGTAG'1~ 3' (Z2 mer) 2o Origin of the sequence: Synl7 strain of HSV-1. Size of fragment: 353bp.
The PCR reaction was carried out in a programmable thermal controller (M.J.
Research Inc. USA) in the presence of S~M of each primer, 50M of each deoxyribonucleotide, and l.SmM MgCIZ on the template of sample DNA.
Reaction was performed in a volume of 50p,1 DNA was denaturated (98°C, 10'), 2s then the temperature was lowered to 65°C for 10' and 2.5 units of Taq polymerase Diamond (Bioline) in 5~1 of Taq buffer x 10 (Bioline) was added.
The steps of the reaction were melting (95°C, 30") annealing (65°C, 30") and elongation (72°C, 1'). Total 35 cycles and a last step of elongation (72°C, 10').
The products of the PCR reaction were run on 1.8% agarose gel in the presence ;o of PCR marker (Promega).Additional primers used in the PCR experiments were P~yted '18-09 20~J1~ v~~°~ ~~ ~u~~at~r ~ ~~uuvu~.n ,z.. ~...:as~r~,~~ta.~t~'s.,;~:.:~,;~..~.:'~ ~..~. ~.=S~'~k .,a a..k~....~-:~,_,. ...,...u ~.. .".,~..~~."we,~:..

synthesized according to the seguence of HSV-I (US 6) gene that codes for glycoprotein D (gD).
Direct primer 5' TTCGGT'GTACTCCATGACCGTGAT3' (24 mer) Reverse primer 5' GCCGTGTGAC,ACTATCGTCCATAC 3' (24 mer) - Origin of the sequence: Syn 17 strain of HSY-l . Size of fragm~ot: G20 bp.
The PCR reaction was carried out ~s described above.
I4.1 ode tifica 'on of Rl5 recombinant DNA,~;r PCR
The primers were synthesized according to unique sequence of R15 in the T p right part of the LTS regi on.
Direct primer 5' ACGACCTCGGTGCTCTCCAAG 3' (2 X m.er) Reverse primer 5' TCAGCACGAACGTCTCCATC 3' (20 mer) Origin of sequence: R15 strain of HSV-1. The size of the fragment is 319bp. The PCIt reaction was performed under the same conditions (see 14.1) except that the deoxyribonucleotides were used at final concentration of 200 ~.
The steps of the reaction were: felting (94°C, 1'), annealing (65°C, I') and elongation (72°C, I'). There was a total 25 cycles ending with the last step of elongation (72°C, l0').
The products of the PCR reaction were identified by electrophoresis in 20 1.8% agarose gel ira the presence of PCR marlcer (Promega).

15. RT-PCR
RNA was purred fxorn rat brain tissue and fZOZn. astrocytes with the Qiagen kat, reverse transcribed with MuLV RT, and then PCR was done with various gene specific prina.ers.

_. , 2 AMENDED SHEET 16'=09 2401 EmPfanBStcm m .rev. m ~oo - " -16. Determination of serum corticosterone and ACTH levels and of prostaglandin synthesis Serum corticosterone and ACTH were determined by RIA, using commercial antibodies. Prostaglandin E2 (PGE2) synthesis was determined by RIA in brain slices ex-vivo.
Example 1 Screening HSV-1 isolates To determine which of the HSV-1 viruses of the HSV-1 strains shown in Table I A lacks pathogenicity to adult mice, each of the viruses were used to infect 4 week old Sabra mice by instilling 50,1 of 10' pfu/ml virus stock into the nostrils of each mouse. The infected and control mice were inspected for 2-4 weeks and the mortality of the infected mice was recorded. It was found that five virus strains (F, KOS, Syn 17, and recombinants P42, and P71 ) were highly is pathogenic to Sabra mice and killed all the infected animals. Four TK -mutants (F TK-, F blue TK-, R15 TK-, and Synl7 TK-) were devoid of pathogenicity to adult mice. Three additional HSV-1 isolates, were apathogenic to adult mice:
HFEM, HSV-1 R15 and HSV-1 vhs, a mutant with a mutation in the UL41 gene.
The TK- virus mutants cannot be used for vaccine development since they are 2o resistant to Acyclovir. It was reported that HSV-1 TK- mutants retain pathogenicity to newborn mice The results of the experiments presented in Table 1A and Table 1B led to the conclusion that HSV-1 R15 recombinant may have the properties required for development of a live HSV-1 virus vaccine.
Other HSV-1 recombinants, such as the HSV-1 R19, that were isolated from the 2s same recombination event were highly pathogenic to adult mice.
The parental strain HSV-1 (HFEM) that was used for the recombination experiment was found to be apathogenic to adult mice but was pathogenic to suckling mice as shown in Table 1B and its TK- mutant had a residual pathogenicity to suckling mice so that the parental strain was unsuitable for 3o human vaccination. HSV-1 R15, Fblue and Fblue TK- were apathogenic to the suckling mice (Table 1B).

Based on the above results, HSV-1 R15 recombinant was selected for further studies to determine its biological, immunological, and molecular properties.
Example 2 Protection by HSV-1 Rl5 inoculation from challenge with Groups of 10 adult mice were inoculated intranasally (i.n.) with each of the apathogenic viruses (HSV-1 R15, HSV-1 R15 TK , HSV-1 vhs, HSV-1 F
~ o blue) an additional group was used as a control. Fourteen days after the infection the mice were infected by the i.n. route with the pathogenic virus HSV-1 F.
The results are shown in Fig. 1A. As can be seen 90% of the unimmunized control mice died from the virus infection up to 23 day p.i. All the mice in the four groups that were immunized with the four apathogenic HSV-1 strains survived is the challenge with the pathogenic HSV-1 (F) virus (Fig. IA).
The immunization of suckling mice was performed with four apathogenic strains of HSV-1 (HSV-1 R15, Fblue, Fblue TK-, HFEM TK-). Litters of suckling mice were immunized by the i.n. route with the apathogenic virus strains and two weeks later the suckling mice were challenged with the pathogenic HSV-1 (F) 2o and were followed for two weeks. The results shown in Fig. 1B indicate that all (100%) of the suckling mice that were immunized with HSV-1 R15 recombinant survived the challenge infection with the pathogenic virus 90% of the suckling mice that were immunized with HSV-1 F blue survived the challenge infection but only 40% of the suckling mice that were immunized with Fblue TK- survived 2s the infection. All the suckling mice that were immunized with HSV-1 HFEM TK-died due to infection with the pathogenic virus as did the control mice.
It was concluded that HSV-1 R15 should be studied to define its molecular makeup and the reasons for its apathogenicity.

Example 3 Determination of penetration of HSV-1 R15 into nerve cells Adult Sabra mice (4 week old) were immunized by i.n. route with two apathogenic viruses: HSV-1 R15 and HSV-1 vhs (UL41-). At 2, 4, and 7 days p.i.
the mice were sacrificed and tissue samples from the nose epithelium (NE), olfactory bulbs (0B), brain amygdala (AM) and trigeminal ganglia (TG) were removed and pooled from ~ mice at each time point. The DNA was extracted from each tissue sample and subjected to a PCR test using primers that detect the presence of HSV-1 DNA. The results shown in Table 2A indicate that in mice that were immunized with HSV-1 R15 recombinant the viral DNA was detected io only in the nose epithelium in samples taken at days 2 and 4 p.i. Viral DNA
was not found in the nose epithelium at 7 days p.i. HSV-1 R15 recombinant DNA was not found in the olfactory bulbs (0B), amygdala (Am) and trigeminal ganglia (TG) of the immunized mice. It was concluded that HSV-1 is unable to penetrate into the nervous system of the immunized mice.
In tissue samples that were removed from mice immunized by the i.n.
route with HSV-1 UL41- (vhs) mutant viral DNA was detected in the nose epithelium at days 2 and 4 p.i. but not at day 7 p.i.. However, at day 2, 4, and 7 p.i. the viral DNA was detected in the olfactory bulbs (0B) and in the trigeminal ganglia (TG) and the virus DNA persisted in these tissues on days 4 and 7. It was 2o concluded that this virus mutant was able to penetrate into the olfactory bulbs and trigeminal ganglia but not into the amygdala in the central nervous system (CNS) of the infected mice as indicated in Table 2B.
It was concluded from these studies that HSV-1 R15 recombinant may have genetic modifications that render it incapable of penetration into the type C
25 fibers in the nose epithelium and as a result the viral DNA was unable to penetrate the trigeminal ganglia, olfactory bulbs and the amygdala in the CNS.
The HSV-1 R15 persisted in the nose epithelium for only 4 days p.i. and was not detected on day 7 p.i.

Example 4 Immunization of adult mice by injection of HSV-1 R15 recombination Three groups of 10 mice each were used: Groups A and B were immunized by injection of HSV-1 R15 into the mouse footpad and Group C was not immunized. After two weeks Groups B and C were challenged with HSV-1 F
and group A was injected with uninfected cell homogenate (Mock). Fourteen days post challenge, the mice were sacrificed and the tissues of the olfactory bulbs (0B), amygdala (Am) and trigeminal ganglia (TG) were removed at days 2 and 4 p.i., the DNA was extracted and analyzed by the PCR technique with primers that detect HSV-1 DNA.
The results shown in Fig. 2 indicate that group A mice, that were immunized with HSV-1 R15 by the footpad route and were mock challenged, did not have HSV-1 R15 DNA in their olfactory bulbs, amygdala or the trigeminal i5 ganglia (Fig 2, lanesl-3). Mice of group B that were immunized with R15 and challenged with HSV-1 F by the i.n. route, survived the challenge with the pathogenic virus but HSV-1 F DNA was not detectable in the olfactory bulbs, trigeminal ganglia and the CNS amygdala (Fig. 2, lanes 4-6).
The unimmunized mice (Group C) that were infected with HSV-1 F by the 2o i.n. route died as a result of the infection and the PCR analyses revealed the presence of viral DNA in the olfactory bulbs and the amygdala but not in the trigeminal ganglia (Fig. 2, lanes 7-9).
This study revealed that immunization of mice with HSV-1 R15 in the footpad efficiently protects against infection with a wild type HSV-1 (F) by the ?5 i.n. route and prevents the penetration of the pathogenic virus into the nervous system of the mice.

Example ~ Determination of HSV-1 R15 penetration to the spinal cord and adrenal glands One group of 10 adult Sabra mice was injected subcutaneously (s.c.) into the footpad skin with HSV-1 R15 and the second group was infected with the pathogenic HSV-1 F. At 2, and 4 days p.i. the mice were sacrificed and the footpad skin, spinal cord and adrenal glands were removed, the DNA was extracted from each pooled tissue sample and tested by PCR to detect the presence of HSV-1 DNA. Table 3 shows that HSV-1 R15 DNA was detected in i o the footpad skin tissue of the infected mice at day 2 p.i. but not at day 4 p.i. The HSV-R15 DNA was not detected in the spinal cord and the adrenal glands. It was concluded that HSV-1 R15 is unable to penetrate into the spinal cord of the mice and therefore the adrenal glands of the mice were not infected.
In contrast, the pathogenic HSV-1 (F) DNA was found to be present in the ~ 5 footpad skin tissue at day 2 and 4 p.i. and at day 4 the viral DNA was detected in the spinal cord and the adrenal glands of the infected mice (Table 3).
These experiments revealed that in the footpad skin HSV-1 R15 replicated at the site of injection for 2-3 days only and was unable to penetrate into and infect the spinal cord and the adrenal glands as did the pathogenic virus. The 2o results of this experiment are taken as an indication that HSV-1 R15 had lost the ability to penetrate into the peripheral type C fibers in the skin epidermis and therefore is incapable of penetration and infection of the spinal cord nervous system and the adrenal glands in the infected adult mice.
25 Example 6 Apathogenicity of HSV-1 R15 to strain A/J mice inoculated intracerebrally or intraperitoneally HSV-1 R15 (50 pl/animal) was inoculated intracerebrally (i.c.) into the brains of A/J mice (4 weeks old) at various concentrations ranging from 106 pfu/mouse to 10~ pfu/mouse. The results are shown in Table 4. It was found that all mice survived the infection while mice that were inoculate with HSV-1 (F) or HSV-1 HFEM at 1 x 102 pfu/ml died (Table 4).

Mice that were infected intraperitoneally with HSV-1 R15 at virus titers of 1 x'i 10~~'or 1 x 106 pfu/ml survived the infection similar to mice that were infected with HSV-1 (HFEM) at 1 x 10' pfu/ml. HSV-1 (F) at 1.0 x 106 pfu/mouse killed 9 out of 10 mice (Table 4).
s The results of the experiment revealed the apathogenicity of HSV-1 R15 recombinant (HSV-R-Fehx-C15) to A/J mice in i.c. and i.p. routes of infection.
Example 7 Determination of neurovirulance of HSV-1 K15 Inoculation of 5 x 10' pfu/ml by the nasal or corneal routes or direct io intracerebroventricular inoculation of up to 10' pfu of HSV-1 R-15 did not induce clinical signs of disease, or fever. Low virus titers were found at the site of infection at 3 days after stereotaxic injection to the hypothalamus, but no infectious virus could be isolated in other brain regions, as compared to dissemination of virulent strains. This non-virulent phenotype was stable over i s many passages of the virus. This suggests that spontaneous neurovirulent revertants are not existent, or occur in frequency less than 1.
Following ICV inoculation neurovirulent HSV-1 strains cause activation of the hypothalamic-pituitary-adrenocortical axis as measured by serum corticosterone and ACTH (by RIA). In addition, the virus induces an increase in 2o the synthesis of prostaglandin E2 in the brain, as measured by ex-vivo production in brain slices form various regions. Neurovirulent strains induce the expression of interleukin-1 gene in various brain regions. HSV-1 R-15 as a non-neurovirulent strain, did not activate the HPA axis, did not increase PGE2 production, and did not induce IL-1 gene expression outside from the 25 hypothalamus, which was the site of inoculation~2~.
In a one-step replication experiment, strain HSV-1 R-15 infected and replicated in cultured astrocytes to titers 10-100 fold lower than the neurovirulent HFEM strain. HSV-1 R-15 titers in infected astrocytes peaked at 106 pfu/ml as compared to 10'-10g pfu/ml of the pathogenic recombinants R-19 and p71.
;o Infection of the astrocytes by the neurovirulent strain Synl7 induced rapid expression of the IL-1 gene (within 3 hours postinfection), as detected by RT-PCR. HSV-1 R-15 recombinants did not induce IL-1 expression in astrocytes at the same timepoint.
s Example 8 Determination of antibodies after immunization of adult mice with HSV-1 R15 To study the immune response of adult mice to immunization with by HSV-1 R15 the mice were infected by the i.n. Route and at different time iv intervals (2, 4, 7,14 and 16 days p.i.) two mice were sacrificed, the blood was collected and the serum was prepared. The content of the antiviral neutralizing antibodies in the serum was measured. At day 14 the immunized mice were challenged with the pathogenic HSV-1 F.
Fig. 3 shows that at day 7 p.i. the HSV-1 R15 immunized mice had not yet ~ s responded with the synthesis of antiviral neutralizing antibodies, but at day 14 p.i.
neutralizing antibodies were present in the sera of immunized mice diluted up to 1:128. With a serum dilution of 1:8, 33~ inhibition of HSV-1 F plaques was found. At day 16 p.i. the titer of the neutralizing antibodies decreased.
However at day 21 post immunization (one week after the challenge infection with the 2o pathogenic HSV-1 F) 100% of the virus plaques were neutralized by the serum antiviral antibodies, at serum dilution of 1:16.
Example 9 HSV-1 R15 expresses the UL 23 gene that codes for the viral thymidine kinase (TK) and is sensitive to acyclovir The HSV-1 R15 has an active UL23 gene that codes for the viral thymidine kinase (TK) and therefore can be inhibited by the antiviral drug acyclovir.
HSV-1 R15 (100 pfu/plate) was used to infect vero cell cultures in the ;o presence and absence of different concentrations of acyclovir. It was found that acyclovir at concentrations of 10, 50 and 100 ~g/ml effectively inhibited HSV-R15 replication (Table 5).

Molecular analysis of the IRS-US-TRS DNA in HSV-1 R15 genome compared to the gene arrangements in HSV-1 Synl7 DNA
The biological properties of HSV-1 R15 indicated that changes may have s occurred in the viral DNA genome and affected some of the viral genes that are involved in the pathogenicity of this virus recombinant. Molecular analyses of the viral Unique Small (US) DNA and its flanking repeats and part of the gene in the Unique Long (UL) DNA and its interval repeat (IRL) of the HSV-1 R15 genomic DNA were undertaken.
to Example 10 Recombination events between BamHI-B DNA fragments from the pathogenic HSV-1 F with HSV-1 HFEM genomic DNA
yielded the apathogenic HSV-1 R15 recombinant t 5 HFEM DNA genome harbors a 4 Kbp deletion (coordinates 117088 -120641 ) in BamHI B sequence (coordinates 113322 - 123456) that affected exon 3 of the Immediate Early 1 (IE 1 ) gene coding for Infected Cell Protein (ICP) (IE 110) and also deleted the promoter sequence of UL56 gene.
Cells were transfected with the HFEM genomic DNA together with 2o HSV-1 F BamHI B DNA fragment and the progeny of the transfection was collected. The results are shown in Fig. 4. Many virus plaques were isolated from the virus progeny of the recombination experiment and all the plaques, except HSV-1 R15, yielded pathogenic viruses.
HSV-1 R15 recombinant (HSV-R-Fehx-C15) was found to be apathogenic 25 to adult and suckling Sabra and A/J mice while other recombinants (for example, HSV-1 R19) were highly pathogenic to mice. Recombination events that introduce BamHI B DNA fragments of HSV-1 F into the BamHI B DNA
sequence of HFEM genome may have lead to multiple changes in the HSV-1 R15 genes adjacent to the BamHI B DNA recombination sites.

Example 11 Changes in the expression of genes present in the US DNA of The organizational map of the US 1 to US 12 genes in the US DNA of s HSV-1 Syn 17 that serves as a model in the present studies is based on its published complete nucleotide sequence and is presented as Fig. 5. For each of the US genes the RNA transcripts are also shown.
To study the expression of the US genes in HSV-1 R15 infected cells, RNA was isolated from the infected cells and identified by hybridization to io restriction enzyme cleaved DNA fragments of the HSV-1 US DNA (Northern blot analysis) and the results are presented in Fig. 6. The HSV-1 DNA
fragments that were used as probes for the hybridization are as indicated in Fig 7.
It was found that the US genes US 4 (gG), US 8 (gE), and US 12 coding for the immediate early protein IE-5 were not expressed in the HSV-1 R15 ~s infected cells (Fig. 6).
These findings were taken to indicate that a major change had occurred in viral genes (marked dark in Fig. 5) near the Terminal Repeat of the US (TRS) DNA.
2o Example 12 Southern blot analyses of HSV-1 R15 US DNA.
To define the nature of the changes that occurred in the US genomic DNA
of HSV-1 R15 near the TRS, two probes were used: Us 12 gene probe (coordinates 145312-145576) and OriS probe (coordinates 146008-146592) that contains the origin of DNA replication (shown in Fig. 7A). For hybridization 2s analyses, DNA genomes of several HSV-1 strains were cleaved with BamHI
restriction enzymes.
The OriS probe detected in Synl7 DNA (Fig. 7Ba) two fragments: one of 1953 by that is the BamHI-X DNA fragment containing the OriS nucleotide present in sequence the TRS. The second DNA band of 4840 by is the BamHI-N
;o fragment containing the OriS sequence present in the internal repeat of US
(IRS).

In HFEM DNA the OriS probe detected a faint BamHI-X band and a ladder of bands (Fig 7Ba). The bands in the ladder contain the BamHI-X DNA
fragment and multiple repeats of the 472 by sequences containing the OriS from TRS (Fig. 9). The BamHI-N was detected by the OriS probe and also a ladder of bands that contain the BamHI-N sequence and multiple repeats of the 472 by OriS containing a fragment that is present in IRS.
In HSV-1 R15 DNA the OriS probe did not detect the BamHI-X DNA
band ( 1953 bp) but detected a new, wide band of about 10,000 bp. This result indicates that the OriS 472 by fragment present in the BamHI-X may be part of a io large BamHI DNA fragment. The ladder pattern of BamHI-N fragment with multiple repeats of 472 by OriS is the same as in HSV-1 HFEM (Fig. 7 Ba).
The US 12 probe (present in the US DNA) detected only the BamHI-X
band in HSV-1 F and its recombinants 601 and 602 (Fig. 7 Bb). The probe detected the ladder of bands in HFEM and recombinant R19. However, US 12 i5 gene was not detected in HSV-1 R15.
To study the molecular changes in the US and TRS DNA of HSV-1 R15 the probes Y, X, Z, J1135, and J1 in Southern blot analyses (Fig. 8 A, B). The results shown in Fig. 8B indicate the following:
(a) Probe Y detected an 1840 by fragment in HSV-1 Synl7 HFEM and 2o R15 DNAs;
(b) Probe X detected a 1953 by fragment in HSV-1 Synl7 and HFEM
but not in R15;
(c) Probe Z detected an 1841 by fragment in HSV-1 Synl7 and HFEM
but not in R15;
25 (d) Probe J1135 detected a 6400 by fragment in HSV-1 Synl7 and HFEM but detected a 10000 by fragment in HSV-1 R15;
(e) Probe J1 detected a 2055 by fragment in Synl7, HFEM and R15 DNAs.
These findings revealed that fragments BamHI-X+Z are missing from the 3o US and TRS DNA of HSV-1 R15. The HpaI-EcoRl fragment (coordinates 141611-146693) was cloned in pGEM-7 vector (Promega). The cloned DNA
fragment in the pGEM-2 was identified in bacterial extracts with the probes J1135 and OriS and the cloned viral DNA was sequenced in an automatic sequencer.
s Example 13 The nucleotide sequence of HSV-1 R15 DNA fragment Hpal-EcoRI (coordinates 141611-146693) revealed rearrangement The nucleotide sequence of the HSV-1 R15 DNA fragment of 4182 by is presented in Fig. 9. By comparison to the nucleotide sequence of HSV-1 Synl7 US and TRS DNA it was possible to identify the molecular rearrangements in HSV-1 R15 DNA as shown in Fig. 10 as follows:
(a) The sequence EcoRl (coordinates 146693) near the start of the ~s TRS sequence (145583) in Synl7 is unchanged in the HSV-1 R15 DNA fragment. This sequence is identical but in the opposite orientation to the sequence in the IRS coordinates 131534-132605.
(b) The DNA fragment from US DNA of HSV-1 Synl7 coordinates 132605-134892 was found in HSV-1 R15 DNA fragment in an 20 opposite orientation ligated to the start of the TRS coordinate 145583-142046.
(c) The HSV-1 Synl7 DNA sequence coordinates 141611-14204 is changed in HSV-1 R15 DNA.
(d) HSV-1 R15 DNA contains two tandem repeats of the 472 by 2s sequence that contains the OriS sequence.
The sequence of HpaI-EcoRl (coordinates 141611-146693) of HSV-1 Synl7 is 5082 by while the HSV-1 R15 DNA fragment HpaI-EcoRl is 3710 by the cloned DNA fragment of HSV-1 R15 is 4182 by with two repeats of 472 bp.
Since the HSV-1 Synl7 has only one 472 by sequence, the relevant size of the ;o cloned HSV-1 R15 fragment is 3710 bp. Therefore, the HSV-1 R15 US sequence is shorter than the same sequence in HSV-1 Synl7 US DNA by 1372 nucleotides (Fig 11 ).
Example 14 Rearrangements of the genes in the IRS-US-TRS DNA of The molecular changes that were identified in HSV-1 R15 DNA had changed the organization of the viral genes are shown in Fig. 12 and are as follows i o (a) US 1 gene (IE-4 gene coding for a nuclear phosphoprotein is duplicated and appears in IRS and TRS.
(b) US 2 gene (ORF 291aa) is duplicated and appears in IRS and TRS.
(c) US 8 gene (gE, SSOaa in HSV-1 Synl7) in HSV-1 R15 codes for a shorter polypeptide of 188aa, identical to the N-terminus 170aa of gE protein of HSV-1 Synl7. The truncated gE protein in HSV-1 R15 infected cells will not form a heterodimer with gI that is coded by US 7 and therefore HSV-1 R15 is incapable of infecting nerve cells~3~.
(d) US 9, 10 and 11 genes (coding for tegument proteins) are deleted.
20 (e) US 12 gene (IES) is deleted.
Example 15 PCR test To be able to distinguish between HSV-1 R15 and pathogenic viruses a PCR test was developed using primers designed according to the charged 2s nucleotide sequence in the R15 US DNA. The results presented in Fig. 13 reveal that under the conditions of the reaction the primers allow amplification of 3 l9bp DNA only from HSV-1 R15 DNA.
The biological implications of the gene rearrangements is the IRS-US- TRS
3o DNA of HSV-1 R15.

The molecular changes in the genes markedly modify the biological properties of HSV-1 R15 that lead to its apathogenicity.
Example 16 Effects of deletion of US 12 on HSV-1 R15 immunogenicity in s mice The US 12 gene codes for ICP 47 that binds to and inhibits the transport of nonapeptides by TAP 1/TAP2 diners to HLA (MHC) class I polypeptides in the endoplasmic reticulum (ER) of infected cells~4~. In the absence of ICP 47 in io HSV-1 R15 infected cells, the transport of viral nonapeptides to HLA class I
molecules is not affected and therefore the induction of the host (human) immune system will start immediately after immunization with HSV-1 R15.
Fig. 14 compares the results of FACS analyses that determine the fluorescence of cells that were stained with fluorescent anti-HLA antibodies.
The ~ s fluorescence of HLA class I molecules present on the cell surface of human fibroblasts and fibroblasts infected with HSV-1 Synl7 or HSV-1 R15 strains, determines the transport of HLA class I molecules from the cytoplasm to the cell surface during the early stage of viral infection. It was found that between 2 hr.p.i. to 4.5 hr.p.i. (the time of ICP47 activity), the fluorescence of the HLA
2o class I molecules on the outer cell membrane of fibroblasts infected with Synl7 was ~50% lower than in cells infected with HSV-1 R15. This result indicated that HSV-1 R15 from which the US 12 gene was deleted is unable to inhibit the HLA class I translocation to the cell surface. Thus, the presentation of viral nonapeptide antigens by HLA class I molecules to the immune system is 2s unaffected contrary to pathogenic HSV-1 Synl7 that inhibits translocation of HLA class I molecules to the cell membrane.
Confocal microscopy of HeLa cells that were infected with HSV-1 F, HFEM and R15, fixed at 3 and 6 hr p.i. and stained with antibodies to human HLA class I molecules revealed that in HeLa cells infected with HSV-1 F or ;o HFEM the HLA class I molecules were retained in the cell cytoplasm and were almost absent from the outer cell membrane. In R15 infected cells HLA class I
molecules were evenly distributed on the outer cell membrane (not shown).
Confocal microscopy of HeLa cells infected with HFEM, Synl7 or HSV-1 R15 and treated with rabbit antibodies prepared against a synthetic polypeptide derived from ICP47 amino acid sequence, revealed that ICP47 is absent from HSV-1 R15 infected cells. Inhibition of HLA class I translocation occurs in cells infected with HFEM or Syn 17 (not shown).
Example 17 The deletion in the US 8 gene prevents HSV-1 R15 from infecting neurons in mice In the absence of US 8 gene expression, the complex gE/gI cannot be formed, hence HSV-1 R15 is unable to penetrate into the nervous system of infected mice. It was reported~3~ that glycoprotein gE/gI heterodimer facilitates neuron to neuron spread of pathogenic HSV-1 strains. The inability of HSV-1 R15 to infect the nervous system of infected mice is one of the molecular changes that are responsible for the apathogenicity of this recombinant.
Example 18 The deletion of US 9, US 10 and US 11 genes does not affect the 2o replication of HSV-1 R15 in the mouse nose epithelium, skin and CNS
These genes code for y, tegument proteins:
(a) US 9 codes for the synthesis of tegument phosphorylated protein, 2s (b) US 10 codes for another tegument protein. US 11 codes for a tegument protein that binds the 60 S ribosomal subunits in infected cells and also binds to mRNA transcripts of the gene UL 34 (membrane-associated phosphorylated virion protein).
It was observed that HSV-1 R15 replicates in vivo cells to titers of 108 ;o pfu/ml indicating that the deletion of the three tegument genes does not affect virus replication in cell cultures in agreement with published results~s~. The ability of HSV-1 R15 to replicate to high titers in cultured cells assures the production of this virus for vaccine purposes.
Example 19 Modifications in the expression of UL54 and Y134.5 genes in the UL and Internal Repeat of UL (IRL), respectively, of HSV-1 Figure IS presents a map of the HSV-1 Synl7 genes that are located near and in the BamHI B DNA fragment: UL 53, UL54, UL 55 and UL56 latency i o genes that code for LATs mRNA, IE 110 gene and the y 34.5 gene that codes for ICP 34.5.
A 4Kbp deletion in the 5' end of the IRL was identified in the DNA
genome of HSV-1 HFEM (the parental virus of HSV-1 R15). Since the HSV-1 R15 DNA genome resulted from a recombination between HSV-1 HFEM DNA
~5 and BamHI B DNA fragment from HSV-1 F. It was decided to study two genes near the recombination sites that are important to the pathogenicity of HSV-1:
UL
54 and yi34.5 genes in HSV-R15 DNA (Fig. 15).
Example 20 Absence of detectable ICP27 (IE-2 protein) coded by UL 54 in 2o HSV-1 R15 infected cells The UL 54 codes for ICP27 (IE-2) protein of HSV-1 that shuttles between the cytoplasm and the nucleus of infected cells~6~.
Anti-ICP27 rabbit antibodies were prepared in the laboratory and used to 2s detect ICP27 protein in HSV-1 infected cells by confocal microscopy. HeLa cells infected with HSV-1 F but not with HSV-1 R15 revealed the presence of ICP27 protein cytoplasm and nucleus of the infected cell (not shown).
Since the ICP27 protein was not detected in HSV-1 R15 infected cells we cloned the UL54 gene in pCi expression vector (Promega) was cloned. The 3o pCi-UL 54 plasmid was transfected into cell-line 293 and stained with the anti-ICP27 antibodies. It was found that in the transfected cells the UL 54 gene of HSV-1 R15 was expressed and ICP27 was detected by the rabbit anti-ICP 27 antibodies. In addition, the nucleotide sequence of the UL54 gene promoter was cloned, sequenced and was found to be identical to the promoter sequence of the UL 54 gene promoter in HSV-1 Syn 17.
Since ICP27 was not detected in HSV-1 R15 infected cells while the UL54 gene was expressed when cloned in an expression vector. It may be possible that the UL54 gene expression in the infected cells is under influence of or regulated by other genes.
Example 21 The expression of the y134.5 gene in HSV-1 R15 infected cells as compared to the pathogenic viruses HSV-1 Synl7 and HFEM
The Yi34.5 gene in the IRL of HSV-1 DNA codes for two proteins: The ICP 34.5 and ORF B (Fig. 15). The ICP 34.5 is responsible for the CNS
pathogenicity of HSV-1 when injected intracerebrally~~~. To detect ICP 34.5 in ~ 5 infected cells, a rabbit antibody was prepared against a peptide derived from the amino acid sequence of the protein coded by the y, 34.5 gene. By Western blot analysis the ICP 34.5 (43 KDa) was detected in HSV-1 F infected cells while in HSV-1 Syn 17 and HFEM infected cells the ICP 34.5 has a MW of 37KDa. (Fig.
16). The HSV-1 recombinants 601 and 602 that contain inserts of the bacterial LacZ gene in the two alleles of the y~34.5 genes in the TRL and IRL did not express the y,34.5 gene.
The Western blot of ICP 34.5 in HSV-1 R15 infected cell homogenate gave an unclear result due to the unspecific staining of cellular proteins. It is suggested that y,34.5 was expressed at a lower level than in cells infected by 2s HSV-1 HFEM or Synl7.
Example 22 Survival of mice immunized with HSV-1 Rl recombinant through ocular skin or lungs route after challenge with the pathogenic HSV-1(F) Sabra mice were immunized with HSV-1 R15 recombinant (30 p,1 from a stock of 10' pfu/ml) by 3 different routes: ( 1 ) infection of the eyes ( 10 mice);

(2) subcutaneous infection in the skin (10 mice); and (3) and infection to the lungs of 10 mice that were slightly anesthetized.
Two weeks later all three groups of mice were infected with the pathogenic HSV-1(F) (30 y1 from 10' pfu/ml stock) by the nasal route. The animals were followed for three weeks and the survival of the immunized mice and the control mice (injected with 30 p,1 of an uninfected cell homogenate by the same route of infection as the mice that were immunized) was documented, the results are shown in Fig. 17A, 17B and 17C.
Results 22.1 Immunization of Sabra mice by the ocular route Fig. 17A revealed that 90% of the R1 S immunized mice survived the challenge with HSV-1 (F). About 50% of the control mice survived the challenge virus. This may be taken to indicate that the challenge virus HSV-I (F) may not have infected half of the mice since in the two additional control mice groups the survival rate was 30% (see Fig. 17C) and 20% (Fig. 17D).
22.2 Immunization of Sabra mice by the subcutaneous route 2o Fig. 17B shows that injection of R15 subcutaneously to the mouse skin fully (about 100%) protects the mice from a challenge with pathogenic HSV-1 (F). Of the control group only 30% of the mice survived.
25 22.3 Immunization of mice by the lung route Fig. 17C revealed that inhalation of R15 into the anesthasized mice fully protected the mice from intranasal challenge with HSV-1 (F) . The control group succumbed to the infection and only 20% of the mice survived.

Conclusions It is concluded that HSV-1 R15 recombinant protects mice against challenge with HSV-1 F when immunized by the ocular, by the skin and the lung routes.
s Example 23 HSV-1 R15 UL54 gene is not expressed in infected cells The UL54 gene of pathogenic HSV-1 strains F and Synl7 is expressed early (2-4 hrs post infection) and codes for the viral Immediate Early Protein designated ICP27. This protein causes rearrangements of molecules in the ~ o nucleus of the infected cells. In the previous example it was demonstrated that in HSV-1 R15 UL54 gene is not functional.
In the present experiment HEK293 cells were treated with uninfected cell homogenate (Fig. 18 Mock) and infected HEK 293 cells treated with HSV-1 R15 (Fig 18, R15) or HSV-1 (F) (Fig. 17F). Antibodies were raised in rabbits against i s a synthetic peptide attached to KLH. The rabbits' sera was obtained and the antiICP27 antibodies were used to stain the control and the virus infected cells.
Results It can be seen in Fig. 18 in HEK cells infected with HSV-1 (F) the cells 2o were stained with the immune serum indicating that ICP27 protein was synthesized from the UL54 gene transcript. However, in the cells that were infected with HSV-1 R15 the viral protein ICP27 was missing. The control cells were negative. The results indicate that the HSV-1 R15 recombinant is unable to synthesize mRNA from Immediate Early gene UL54.

Eacample 24 Effects of apathogenic HSV-1 R15 recombinant injected by the intracerebral route on the brain functions and behavior of infected rats Behavioral studies:
Aggressive behavior was assessed 3 days post-infection by examining the responsiveness of the animal to cage opening and insertion of a gloved hand into the cage. Aggression was scored on a scale of 0-2 as follows: 0=no response;
I=overt startle response and attempt to attack the hand; 2=extreme irritability, io fierce attack and attempt to bite the hand and /or jump out of the cage.
Startle reaction was observed in response to scratching the cage.
It was previously shown that immunization with R15 fully protected both mice and rats from lethal doses of virulent strains. In this experiment it was examined whether immunization with R-15 may protect the rats from the clinical i5 signs of acute infection with virulent HSV-1 and whether acute or chronic infection with R-15 have any clinical implications. For this purpose, the following parameters were monitored: body temperature; aggressive behavior;
and brain prostaglandin E2 (PGE2) synthesis in S experimental groups as follows:
2o I. Control uninfected rats II. Rats inoculated intracerebroventricularly (ICV) with 5 x 105 pfu HSV-1 strain Syn 17+.
III. Rats inoculated ICV with 5x105 pfu R-15 IV. Rats that were immunized by two subcutaneous inoculations with 25 5x105 pfu R-15 at 2 weeks difference and examined 2 weeks after the second immunization/
V. Rats that were immunized by two subcutaneous inoculations with 5x105 pfu R-15 at 2 weeks difference and then challenged with a highly lethal dose of 106 pfu Syn 17+ administered ICV.
~o Results The results of this experiment are given in Table 6. As can be seen acute ICV inoculation with synl7+ caused significant hyperthermia and aggressive behavior as well as increased production of PGEZ in the brain. Acute ICV
inoculation with R-15 or s.c. immunization with R-15 did not induce any rise in body temperature, or PGE2 production or aggressive behavior different than that observed for control animals. Lnmunization with R-15 fully protected the animals from the Synl7+ induced aggressive behavior. These animals responded to this ultimate ICV challenge with syn 17+ by intermediate values of body temperature and PGEZ production between the control and Syn 17+ infected animals.
Conclusion Although HSV-1 R15 recombinant, when injected intracerebrally, replicates in the brain it does not affect the normal behavior of the infected rats and does not kill them. This is another marker of the apathogenicity of HSV-1 R15 recombinant since infection with the pathogenic HSV-1 (F) makes the rats very aggressive until they die of the infection.
Example 25 Defining the target cells in the rat brain in which HSV-1 R15 2o replicates One step replication of R-15 in purified primary newborn rat glial cultures:
Figure 19 shows that R-15 replicates to 1-2 log lower titers than virulent strains.
This attenuated replication in brain cells is comparable to that in other cells types.
25 The virulent HSV-1 strain Synl7+ induced interleukin-1(3 gene expression in infected astrocytes as determined by RT-PCR (Figure 20 first line on left).
In correlation, this virulent strain caused translocation of NFKB to the nucleus in the astrocytes. In comparison, strain R-15 did not induce IL-1 ~3 gene expression (Figure 20) Conclusions HSV-1 RIS recombinant and progeny from brain cells is by two logs lower than the progeny of a pathogenic HSV-1 The later induces the transcription of the gene for IL 1 ~i while HSV-2 R15 is unable to induce IL-I ~i gene.

Table 1 A. Pathogenicity of different HSV-1 strains to adult mice Virus strainGenetic modification N of exp.N of mice% of mortali F w.t 13 118 100 KOS w.t 1 9 100 Synl7 w.t 1 5 100 R19 6 53 >50 recombinant of HFEM

P42 genome with BamH-B 1 5 100 DNA from F

P71 recombinant ofRIS genome1 5 100 with UL53 DNA from F blue recombinant of RI 5 6 64 0 genome with UL53 DNA from LacZ inserted into UL56 gene F TK' of HSV-1 (F) 1 9 0 KOS TK- TK' mutant 1 9 33 Syn 17 TK- TK- mutant 1 5 0 F blue TK- ~' mutant 1 16 0 R 15 TK' TK- mutant 1 9 0 HFEM ~'lnutant 4 S 0 0 R1 S w.t., with a 4ktb deletion6 86 0 in BamHI-B fragment Vhs recombinant HFEM genome2 18 0 with BamH-B DNA from F

UL41- mutant Table 1B. Pathogenicity of different HSV-1 strains to suckling mice Virus strainGenetic modification N of exp.N of mice% of mortali HFEM w.t. with a 4 kbp 2 14 100 deletion in BamHI-B fragment HFEM TK- TK-mutant 1 10 10 R15 Recombinant of HFEM 4 67 4*
genome with BamH-B fDNA from F

F blue LacZ inserted into 2 15 6*
UL56 gene of HSV-I (F) F blue TK- 1 7 0 TK-mutant * Six and four suckling mice died 1-2 days after infection with HSV-1 F
blue and R15, respectively, possibly due to rejection by the mothers.

Table 2. Distribution of HSV-1 DNA in brain of adult mice after intranasal immunization in comparison with the apathogenic strain HSV-1 vhs (UL41-) S A. I-iSV-I R15 recombinant:
2 days 4 days 7 days p.i. p.i. p.i.

Sample Tissue Viral SampleTissue Viral Sample Tissue Viral number DNA number DNA number DNA

1 N.E. + 5 N.E. + 9 N.E. -3 Am - 7 Am - 11 Am -B. HSV-1 vhs (UL41-) 2 days 4 days 7 days p.i. p.i. p.i.

Sample Tissue Viral SampleTissue Viral Sample Tissue Viral number DNA number DNA number DNA

1 N.E. + 5 N.E. + 9 N.E. -2 OB + 6 OB + 10 OB +

3 Am - 7 Am - 11 Am -Table 3: HSV-1 R15 does not penetrate into the spinal cord and adrenal glands after infection in the mouse foodpad skin.
Time after infection 2 da s 4 da s Virus strain F R15 F R15 Tissue Foodpad + + + -Spinal cord - - + -Adrenal gland - - +

Table 4 Pathogenicity of HSV-1 in mouse strain A/Ja which were inoculated intracerebrally (50 ~1/animal) or intraperitoneally) Virus strain' PFU of No. animals Sign of Days of death dead/

inoculatedNo. of animalsillness Post infection at virus/animalinfected 15' to [No. of animals]
3'd (% survival) days post infection A: Intracerebrallv HSV-R-Fehx-C15 1.0 x 106 0/10 (100) 10/10 HSV-R-Fehx-C15 2.0 x 105 0/10 (100) 9/10 HSV-R-Fehx-C15 3.0 x l0' 1/10 (90) 9/10 6[I]

HSV-R-Fehx-C15 4.0 x 10j 0/10 (100) 6/10 HSV-R-Fehx-C15 5.0 x 10~ 0/10 (100) 4/10 HSV-R-Fehx-CIS 6.0 x 10~ 0/10 (100) 2/10 Mock (50 ~, 0/10 (100) 0/10 BME) HSV-I-F 1.0 x lOZ 10/10 (0) 10/10 5 I S ~ , 7 ~

HSV-1 HFEM 1.0x 102 10/10 (0) 10/10 6~ 1 ~,7~4~,9~3~
~, 12~1~

B: Intraneritoneally HSV-R-Fehx-C15 1.0 x 10' 0/10 (100) 2/10 HSV-R-Fehx-CIS 2.0 x 10~ 0/10 (100) 1/10 HSV-1 F 1.0 x106 9/10 (10) 4/10 7~5~,8~2~, HSV-1 HFEM 1.0 x 10' 0/10 (100) 0/10 C: Intranasal 27.10.1996 HSV-R-Fehx-CIS I.0 x 106 0/10 (100) 0/10 HSV-I F 1.0 x 106 3/10 (70) 10/10 10 ~ 1 ~ , 12 ~ 1 ~ , 16 ~
I

Mice A/J strain were infected at the age of four weeks (32 days), animals were purchased by Harlan-Winkelmann GmbH, D-33178 Borchen, Germany.
HSV-1 strains were propagated on CV-I cell cultures.

-Sl-Table 5: Inhibition of HSV-1 R15 and HFEM plaque formulation By acyclovir Virus strain Acyclovir No. of % Inhibition concentration ( plaques/plate /ml) Table 6: ICV inoculation with R-15 does not induce aggressive behavior, fever or increased PGE2 production above control levels. Immunication with R-1 S protects rats to from the behavioral changes induced by an ICV challenge with strain Syn I 7+
ImmunizationAcute AggressionMean rectalEx-vivo Ex-vivo infection index scoretemperaturePGE2 PGE2 production production Cortex) No Synl7t 1.6 0.4 39.0 0.5 249 50 220 53 immunizationICV

ImmunizationSynl7+ 0 38.2 0.4 166 19 199 56 with R-15 ICV

No R-15 0 36.90.4 10025 10427 immunizationICV

Immunization 0 36.8 0.4 94 16 123 20 Vehicle with R-15 No 0 36.4 0.4 80 35 91 28 Vehicle immunization REFERENCES
1. Rosen, A., Ernst, F., Koch, H-G., Gelderblom, H., Darai, G., Hadar, J., Tabor, E., Ben-Hur, T. and Becker, Y. Replacement of the deletion in the genome (0.762-0.789 mu) of avirulent HSV-1 HFEM using cloned MIuI DNA fragment (0.7615-0.796 mu) of virulent HSV-1 F leads to generation of virulent intratypic recombinant. Virus Res. 5:157-175 ( 1986).
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J. Neurovirol 2:279-288, ( 1996).
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facilitate neuron-to-neuron spread of Herpes simplex virus. J. Virol.
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272:484-492, ( 1997).
20 5. Nishiyama Y., Kurachi R., Daikoku T., and Umene K. The US 9, 10, 11 and 12 genes of Herpes simplex virus type 1 are of no importance for its neurovirulence and latency in mice. Virology 194:419-423, ( 1993).
6. Mears W.E. and Rice S.A. The Herpes simplex virus immediate early protein ICP 27 shuttles between nucleus and cytplasm. Virology 2s 242:128-137, (1998).
7. He B., Gross M. and Roizman B. The X34.5 protein of herpes simplex virus-1 complexes with protein phosphatase 1 a to dephosphorylate the a subunit of the eukaryotic translation initiation factor 2 and preclude the shutoff of protein synthesis by double stranded RNA-activated protein ;o kinase. Proc. Natl. Acad. Science USA 94:843-848, (1997).

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<141>
<160> 1 <170> PatentIn Ver. 2.1 <210> 1 <211> 4182 <212> DNA
<213> Homo Sapiens <400> 1 gaattccatt atgcacgacc ccgccccgac gccggcacgc cgggggcccg tggccgcggc 60 ccgttggtcg aacccccggc cccgcccatc cgcgccatct gccatgggcg gggcgtgagg 120 gcgggtgggc ccgcgccccg ccccgcatgg catctcatta ccgcccgatc cggcggtttc 180 cgcttccgtt ccgcatgcta acaaggaacg ggcagggggc ggggcccggg ccccgacttc 240 ccggttcggc ggtaatgaga tacgagcccc gcgcgcccgt tggccgtccc cgggcccccg 300 gtcccgcccg ccggacgccg ggaccaacgg gacggcgggc ggcccaaggg ccgcccgcct 360 tgccgccccc ccattggccg gcgggcggga ccgccccaag ggggcggggc cgccgggtaa 420 aagaagtgag aacgcgaagc gttcgcactt cgtcccaata tatatatatt attagggcga 480 agtgcgagca ctggcgccgt gcccgactcc gcgccggccc cgtggccgcg gcccgttggt 540 cgaacccccg gccccgccca tccgcgccat ctgccatggg cggggcgtga gggcgggtgg 600 gcccgcgccc cgccccgcat ggcatctcat taccgcccga tccggcggtt tccgcttccg 660 ttccgcatgc taacaaggaa cgggcagggg gcggggcccg ggccccgact tcccggttcg 720 gcggtaatga gatacgagcc ccgcgcgccc gttggccgtc cccgggcccc cggtcccgcc 780 cgccggacgc cgggaccaac gggacggcgg gcggcccaag ggccgcccgc cttgccgccc 840 ccccattggc cggcgggcgg gaccgcccca agggggcggg gccgccgggt aaaagaagtg 900 agaacgcgaa gcgttcgcac ttcgtcccaa tatatatata ttattagggc gaagtgcgag 960 cactggcgcc gtgcccgact ccgcgccggc cccgggggcg ggcccgggcg gcggggggcg 1020 ggtctctccg gcgcacataa aggcccggcg cgaccgacgc ccgcagacgg cgccggccac 1080 gaacgacggg agcggctgcg gagcacgcgg accgggagcg ggagtcgcag agggccgtcg 1140 gagcggacgg cgtcggcatc gcgacgcccc ggctcgggat cgggatcgca tcggaaaggg 1200 acacgcggac gcgggggggg ggggaagacc cgcccacccc acccacgaaa cacaggggac 1260 gcaccccggg ggcctccgac gacagaaacc caccggtccg cctttttgca cgggtaagca 1320 ccttgggtgg gcggaggagg gggggacgcg ggggcggagg agggggctca cccacgttcg 1380 tgccttcccg caggaggaac gccctcgtcg aggcgaccgg cggcgaccgt tgcgtggacc 1440 gcttcctgct cgtcggcggg ggggaagcca ctgtggtcct ccgggacgtt ttctggatgg 1500 ccgacatttc cccaggcgct tttgcgcctt gtgtaaaagc gcggcgtccc gctctccgat 1560 ccccgcccct gggcacgcgc aagcgcaagc gccctgcccg ccccctctca tcggagtctg 1620 aggtcgaaac cgatacagcc ttggagtctg aggtcgaatc cgagacagca tcggattcga 1680 ccgagtctgg ggaccaggag gaagcccccc gcatcggtgg ccgtagggcc ccccggaggc 1740 ttggggggcg gttttttctg gacatgtcgg cggaatccac cacggggacg gaaacggata 1800 cggcggtgtc ggacgacccc gacgacacgt ccgactggtc ttatgacgac attcccccac 1860 gacccaagcg ggcccgggta aacctgcggc tcacgagctc tcccgatcgg cgggatgggg 1920 ttatttttcc taagatgggg cgggtccggt ctacccggga aacgcagccc cgggccccaa 1980 ccccgtcggc cccaagccca aatgcaatgc tacggcgctc ggtgcgccag gcccagaggc 2040 ggagcagcgc acgatggacc cccgacctgg gctacatgcg ccagtgtatc aatcagctgt 2100 ttcgggtcct gcgggtcgcc cgggaccccc acggcagtgc caaccgcctg cgccacctga 2160 tacgcgactg ttacctgatg ggatactgcc gagcccgtct ggccccgcgc acgtggtgcc 2220 gcttgctgca ggtgtccggc ggaacctggg gcatgcacct gcgcaacacc atacgggagg 2280 tggaggctcg attcgacgcc accgcggaaa cccgtgtgca aacttccttg tttggaggcc 2340 agacggtacg gcccggagtg tgatcttagt aatctcgaga ttcatctcag cgcgacaagc 2400 gatgatgaaa tctccgatgc caccgatctg gaggccgccg gttcggacca cacgctcgcg 2460 tcccagtccg acacggagga tgccccctcc cccgttacgc tggaaacccc agaaccccgc 2520 gggtccctcg ctgtgcgtct ggaggatgag tttggggagt ttgactggac cccccaggag 2580 ggctcccagc cctggctgtc tgcggtcgtg gccgatacca gctccatgga acgcccgggc 2640 ccatccgatt ctggggcggg tcgcgccgca gaagaccgca agtgtctgga cggctgccgg 2700 aaaatgcgct tctccaccgc ctgcccctat ccgtgtagcg acacgtttcc ccggccgtga 2760 gtccagtcgc cccgacccct ttgtatgtca ccaaaataaa agaccaaaat caaagcgttt 2820 gtcccagcgt cttaatggcg ggaagggcgg agagaaacag accacgcgga catggggggt 2880 gtttgggggt ttattggcac cgggggctaa agggtggtaa ccggatagca gatgtgagga 2940 agtcggggcc gttcgccgcg aacggcgatc agagggtcag tttcttgcgg accacggccc 3000 ggcgatgtgg gttgctcgtc tgggacctcg ggcatgccca tacacgcaca acacggacgc 3060 cgcaccggat gggacgtcgt aagggggcct ggggtagctg ggtggggttt gtgcagagca 3120 atcagggacc gcagccagcg catacaatcg cgctcccgtc cgtttgtccc gggcagtacc 3180 acgccgtact ggtattcgta ccggctgagc agggtctcca gggggtggtt gggggccgcg 3240 gggaacgggg tccacgccac ggtccacttg ggcaaaaacc gagtcggcac ggcccacggt 3300 tctcccaccc acgcgtctgg ggtcttgatg gcgataaatc ttaccccgag ccggattttt 3360 tgggcgtatt cgagaaacgg cacacacaga tccgccgcgc ctaccaccca caagtggtag 3420 aggcgagggg ggctgggttg gtctcggtgc agcagtcgga agcacgccac ggcgtccacg 3480 acctcggtgc tctccaaggg gctgtcctcc gcaaacaggc ccgtggtggt gtttgggggg 3540 cagcaacagg acctagtgcg cacgatcggg cgggtgggtt tgggtaagtc catcagcggc 3600 tcggccaacc gtcgaagatt ggccggacga acgacgaccg gggtacccag gggttctgat 3660 gccaaaatgc ggcactgacc taagcaggaa gctccacagg gccgggcttg cgtcgacgga 3720 agtccggggc agggcgttgt tctggtccgg cacgtcgaac ctcaaccaga cgacgtccat 3780 ggcgtaggtc tggtcgtcgt gggcgatggc atggatggag acgttcgtgc tgaacgcctc 3840 cccgggggaa aacaggatag cttccggagt ctccatacgc acggtcaccc cacgcacgtg 3900 tgagacttcg ggggcgctgg gccaagacct cgggggggcg gggggaggcg ggagccgggg 3960 ggtcccgctg gcgggagtgc cggctagact ttcgtcctcg ccctcgtcat tgtcatcctc 4020 gtcgtaatcg gctggggtcg gggtggggtc ggaactgggg ccggttgcac caccaggacc 4080 accgaggcca cttggcgagc cgggtccttt atgtcgccca cagacagggt atacaggccg 9140 ctgtccgtct ctcggacccc gtaaataacc aagactccgg tt 4182

Claims (18)

CLAIMS:

1. An anti HSV-1 vaccine comprising as an active ingredient a recombinant herpes simplex virus, the genome of which comprises a mutant of the genome of HSV-1, with the following alterations:

a deletion or mutation in the unique small (US) 8 gene region resulting either in expression of a non-functional gE protein or in no expression of the gE
protein; and a deletion or mutation in the US 12 gene (IE-5) region resulting either in expression of a non-functional ICP47 protein or in no expression of the ICP 47

2. The vaccine according to Claim 1, wherein the recombinant virus further comprises a mutation or deletion in at least one of US 9, US 10 or US 11, said mutation or deletion resulting either in production of a non-functional expression product of the respective gene, or in no expression of the product.

3. The vaccine according to Claim 2, wherein the recombinant virus comprises a mutation or deletion in US 9, US 10 and US 11.

4. The vaccine according to Claim 3, wherein the recombinant virus comprises deletion of the full sequence of US 9, US 10 and US 11.

5. The vaccine according to Claim 1, wherein the recombinant virus comprises a deletion of the full sequence of US 12.

6. The vaccine according to Claim 1, wherein the US 8 region of the recombinant virus is mutated by reverse splicing and recombination resulting in an expression product of said region being a non-functional expression product identical to the N'-terminal amino acids of gE protein of HSV-1 Syn17.

7. The vaccine according to any one of the preceding claims, wherein the recombinant virus comprises a duplication of the sequence of at least one of US 1 or US2.

8. A vaccine according to Claim 7, wherein the recombinant virus comprises a duplication of both US 1 and US 2 genes.

9. The vaccine according to Claim 8, wherein the US 1 and US 2 of the virus both appear in the IRS and TRS regions,

10. The vaccine according to any one of the preceding claims, wherein the recombinant virus has a non-functional or features non-expression of at least one of the proteins: ICP27 or ICP34.5.

11. The vaccine according to Claim 10, wherein the recombinant virus features essentially no expression of ICP27 and ICP34.5

12. The vaccine according to any one of the preceding claims, wherein the recombinant virus has an UL23 gene coding for a physiologically active thymidine kinase (TK).

13. The vaccine according to Claims 1 to 12, wherein the recombinant virus comprises the genome depicted in Fig. 10.

14. The vaccine according to Claims 1 to 13, wherein the recombinant virus further comprises a heterologous sequence.

15. The vaccine according to Claim 14, wherein the heterologous sequence is inserted in the position of the genes US 9, US 10, US 11 or US 12.

16. The vaccine according to Claim 14, wherein the heterologous sequence is selected from the group consisting of:
(a) a sequence coding for the immunogenic protein glycoprotein B
(UL27gene) and/or glycoprotein M(UL 10 gene) and/or glycoprotein D
(US6 gene) or a sequence coding for the combined sequences coding for the antigenic domains from gB, gD and gM of Human Herpes virus 2 (HH-2);

(b) a sequence coding for the immogenic protein gB (UL31gene) and/or gH glycoprotein (UL37gene) or a sequence coding for the antigenic domains from each of the genes gB and gH from Human Herpes Virus 3 (Varicella Zoster virus (VZV);

(c) a sequence coding for an immunogenic glycoprotein gB (BALF4 gene) and/or gH glycoprotein (GBRF -3 gene) and/or glycoprotein gL (BKRF-4 gene), or a combined sequence coding for antigenic domains of the glycoproteins gB, gH and gL from Hunan Herpes Virus 4 (Epstein-Barr Virus (EBV));

(d) ~A sequence coding for the immunogenic glycoprotein gB (UL55 gene) and/or glycoprotein gM (UL 100 gene) or a sequence coding for the combined antigenic domains of the glycoproteins gB and gM of Human Herpes Virus 5 (HHV-5, Human cytomegalovirus (HCMV));
(e) ~a sequence coding for an immunogenic glycoprotein gB (U39 gene) and/or glycoprotein gM (U72 gene) and/or glycoprotein gH (U48 gene) or a sequence coding for the combined antigenic domains of gB, gM and gH
glycoproteins from Human Herpes-Virus -6 (HHV-6);
(f) ~a sequence coding for an immunogenic glycoprotein gB (U39 gene) and/or glycoprotein gH (U42 gene) and/or glycoprotein gM (U72 gene) or a sequence coding for the combined antigenic domains of the proteins from Human Herpes Virus 7 (HIV-7); and (g) a sequence coding for an immunogenic glycoprotein gB (ORF 8 gene) and/or glycoprotein gH (ORF22 gene) and/or glycoprotein gM (ORF 39 gene) and/or gL (ORF 47 gene) and/or the protein (LANA) coded by ORF 73 gene or a sequence coding for the combined antigenic domains of the glycoproteins gB, gH, gM, and gL, and LANA protein from Human Herpes Virus 8 (HHV-8, Kaposi sarcoma virus).

17. The vaccine according to Claim 14, wherein the recombinant virus comprises as a heterologous sequence a cytytoxic gene.

18. The vaccine according to Claim 14, wherein the recombinant virus comprises as a heterologous sequence an apoptosis gene.