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WO1999036087A1 - Novel vaccine compositions for herpes simplex virus - Google Patents

Novel vaccine compositions for herpes simplex virus Download PDF

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Publication number
WO1999036087A1
WO1999036087A1 PCT/US1999/000922 US9900922W WO9936087A1 WO 1999036087 A1 WO1999036087 A1 WO 1999036087A1 US 9900922 W US9900922 W US 9900922W WO 9936087 A1 WO9936087 A1 WO 9936087A1
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Prior art keywords
hsv
virus
vaccine composition
subject
amino acids
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PCT/US1999/000922
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French (fr)
Inventor
Laure Aurelian
Michael Kulka
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Aurx, Inc.
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Application filed by Aurx, Inc. filed Critical Aurx, Inc.
Priority to AU22314/99A priority Critical patent/AU2231499A/en
Publication of WO1999036087A1 publication Critical patent/WO1999036087A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/245—Herpetoviridae, e.g. herpes simplex virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16634—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention discloses new vaccine compositions for He ⁇ es Simplex comp ⁇ sing a whole live HSV-2 virus having vanous ammo acids deleted. Methods of using the vaccine compositions are also included.
  • HSV- 1 and HSV-2 Both distinguishable serotypes of He ⁇ es Simplex V irus (HSV- 1 and HSV-2) cause infection and disease ranging from relatively minor fever blisters on lips to severe genital infections, and generalized infections of newborns HSV- 1 and HSV-2 are 50% homologous at the DNA level, and polyclonal antibodies and MAbs to shared epitopes are cross-reactive.
  • HSV-1 and HSV-2 have ⁇ bonucleotide reductase 1 (RR1 ) proteins (an alloste ⁇ c subunit of the ⁇ bonucleotide reductase (RR) enzyme) (respectively designated ICP6 and ICP10) that contain a unique ammo terminal domain
  • RR1 ⁇ bonucleotide reductase 1
  • ICP6 and ICP10 ⁇ bonucleotide reductase 1
  • ICP6 and ICP10 ⁇ bonucleotide reductase 1
  • the HSV-2 unique domain codes for a se ⁇ ne/threonine-specific protein kinase (PK) which has auto- and transphosphorylating activity and has a transmembrane (TM) domain Sequences which code for the PK domain cause neoplastic transformation and are associated with cervical cancer ( HSV-2 oncogene)
  • PK se ⁇ ne/threonine-specific protein kinase
  • ICP6 also does not have a functional TM domain and it does not localize to the cell surface (Conner, J. Murray, J.,
  • PK activity of pp29 lal has some properties different from the authentic ICP10 PK, presumably because it lacks pan of PK catalytic domain VI (Luo '91 ).
  • the TM domain is also required (but insufficient) for PK activity (Luo, J. H. and Aurelian. L.
  • the transmembrane helical segment but not the invariant lysine is required for the kinase activity of the large subunit of Herpes simplex virus type 2 ribonucleolide reductase. Journal of Biological Chemistry, Vol.
  • the unique Hpal site within the ICP10 coding region represents the 3' end of the transforming region (Jariwalla '80) and Hpal cuts the gene after the codon for amino acid residue 417. It is not known whether pp29 lal has transforming activity. However, PK activity is required for neoplastic potential. PK negative mutants do not transform cells. This includes a mutant deleted in the TM domain and site directed mutants in the ATP binding sites ( Lys 1 6 and/or Lys" 59 ) or the ion-binding site (Glu 209 ) (Smith C. C, Luo, J. H., Hunter, J. C. R.. Ordonez, J. V., and Aurelian, L.
  • the transmembrane domain of the large subunit of HSV-2 ribonucleolide reductase (ICP10) is required for protein kinase activity and transformation-related signaling pathways that results in ras activation.
  • a PK ' mutant deleted only in the TM domain does not have transforming activity (Smith '94)
  • DNA sequences that code for ICP10 amino acids 106-41 1 but lack PK activity are not intrinsically neoplastic. This demonstrates that: (i) the HSV-2 oncoprotein is located within ICP10 amino acids 1 -41 1 , and (ii) neoplastic potential requires a functional PK activity.
  • ICP10 PK in virus growth/pathogenesis The function of ICP10 PK in virus growth/pathogenesis is unknown.
  • the HSV-2 ICP10 protein has int ⁇ nsic PK activity. This was shown by demonstrating that ICP10 PK activity is lost through site-directed mutagenesis.
  • the oncoprotein also has SH3-binding motifs at positions 140, 149 and 396, which are required for interaction with signaling proteins. This interaction is required for transforming activity. Site directed mutagenesis was used to identify amino acids required for kinase activity and interaction with signaling proteins.
  • ICP10PK functions as a growth factor receptor involved in signaling and it binds the adaptor protein Grb, in vitro It has also been shown that there are SH3-bind ⁇ ng sites within the ICP10 PK domain (at positions 140. 149 and 396) and they are required for interaction with signaling proteins and, thereby transformation (Nelson , J W., Zhu, J., Smith, C. C, Kulka. M. and Aure an, L ATP and SH3 binding sites in the protein kinase of the large subunit of herpes simplex virus tvpe 2 of ribonucleolide reductase (ICP10). Journal of Biological Chemistn 271, pp 17021-17027, 1996) [Nelson '96]. Mutation of the ICP10 pro ne-nch motifs at position 396 and 149 reduced Grb : binding 20- and 2-fold respectively.
  • the ICP 10 PK catalytic domain also contains amino acids that are responsible for binding a down-regulator of PK activity (ras-GAP) They are located at position 106-178.
  • ICP10 ⁇ PK The construction of a virus deleted in ICP10 amino acids 106-446 (ICP10 ⁇ PK) is described by Peng '96 Briefly, the wild type sequences in a plasmid (TP101 ) that contains the HSV-2 BamHI E and T fragments were replaced w ith the 1 8kb Sall/Bglll fragment from pJHL9 pJHL9 is a plasmid containing an ICP10 mutant deleted in the PK catalytic domain '92). The resulting plasmid, TP9, contains sequences w hich code for ICP10 deleted in the PK catalytic domain flanked by 4 and 2.8 kb of HSV-2 DNA sequences at the 5' and 3' ends, respectively.
  • the lOkb Hindlll/EcoRI fragment from TP9 was introduced by marker transfer into a virus (ICP 10 ⁇ RR) in which the RR domain of ICP 10 had been replaced with the LacZ gene
  • ICP10 ⁇ PK The resulting recombinant virus, designated ICP10 ⁇ PK. w as obtained by selecting white plaques on a background of blue plaques after staining ith X-gal. A few white plaques w ere picked, purified and grown in Vero cells w ith 10% serum.
  • the ICP10 ⁇ PK v irus is deleted in ICP 10 amino acids 106-446 It lacks ICP 10 PK activity and retains RR activitv (Peng '96) and is attenuated for growth in culture and in infected animals
  • the v irus induces HSV -specific T cell immunitv and protects mice from challenge w ⁇ h w I Id type HSV-2 99/36087
  • a cross recombinant vaccine such as disclosed in US 4,554,159 (' 159), does not suffer from the problems of the subunit vaccines, but contains the oncogene present in HSV- 2. Unless care is taken to define and delete the oncogene. the cross recombinant vaccine could induce cancer in the vaccinee.
  • the cross recombinant of '159 is temperature sensitive. Avirulence may be obtained by selecting temperature resistance, but the temperature of the mouse is 39°C while that of humans is 37°C. This temperature sensitivity could well render such a cross problematic in a vaccine.
  • a superior method of selection of avirulence is by the removal of genes coding for virulence without respect to the temperature at which the virus replicates. Also, the use of prototypical crosses would preclude the use of gene deleted or inserted mutants. Due to the many type-common epitopes on HSV- 1 and HSV-2, cell-mediated immunity cross-reacts (Jacobs, R. P., Aurelian. L.. and Cole, G. A.
  • a live vaccine is supe ⁇ or to a dead vaccine because the liv e vaccine induces herd immunity; it induces different types of immunity, such as mucosal. cell mediated and humoral immunity.
  • a higher le el of immunity is normally obtained because the virus titers are increased through replication vv ithin the vaccinee.
  • a live vaccine is of longer duration, thus obviating boosters and lowering initial dosage.
  • the ICP I O ⁇ PK virus is deleted in ICP 10 ammo acids 106-446 It lacks ICP 10 PK activity and retains RR activity (Peng '96) and is attenuated for grow th in culture and in infected animals.
  • the virus induces HSV- specific T cell immunity and protects mice from challenge w ith ild type HSV-2
  • a patent 99/36087
  • HSV-1 Most developed vaccines (viz. those in neurovirulence genes) are in HSV-1.
  • Known vaccines are not virus type-specific. All known vaccines for HSV-1 or HSV-2 are cross- reactive and provide immunity to the other virus type.
  • HSV-1 is not as desirable a vaccine candidate against he ⁇ es, because the major clinical problem is the sexually transmitted HSV-2, which is also associated with cancer induction.
  • Recent studies indicate that the age-adjusted prevalence of HSV-2 in the US is now 20.8%, an increase of approximately 30% over the past 13 years (Fleming, D.T.. McQuillan. G. M.. Johnsons, R. E., Nahmias. A. J., Aral. S. 0. Lee, F. K..
  • HSV-2 infection A new concern about HSV-2 infection is that it may facilitate the spread of HIV and increase the severity of the disease.
  • HSV-1 has only a 50%) homology to HSV-2, this may lower the response rate against the heterologous strain in the vaccinated population.
  • Another absolute requirement for a live vaccine is the absence of lesions upon immunization.
  • An important trait in the live vaccine is its ability to induce high levels of HSV-specific T cell immunity and prevent lesions due to infection with wild type HSV-2 and
  • a desirable trait in the live vaccine would also be its ability to cause a reduction in the frequency of recurrent lesions in a person already infected. There is a substantial population already infected with HSV who may have intercourse with uninlected individuals who would benefit from such a vaccine.
  • the present invention solves all the problems recited above providing a w hole live attenuated HSV-2 in which the HSV-2 has a deletion of the oncot-ene. has strong 99/36087
  • the present invention provides a method of immunizing a subject against HSV-1 or HSV-2 with said vaccine composition, providing a supenor method of conferring immunity upon the subject.
  • the present invention is a vaccine composition comp ⁇ sing He ⁇ es Simplex V ⁇ rus-2 recombinant selected from the group of recombinants which have deletions in the gene for ICP 10 to remove the fragments encoding for amino acids 107-351 (AuV 351) , the fragments encoding for amino acids 107-375 (AuV375) and the fragments encoding for amino acids 107- 417 (AuV417), and a pharmaceutically acceptable earner or diluent.
  • Fig. 1 Schematic representation of the construction of AuV351 DNA
  • Fig 2 Schematic representation of the construction of AuV375 DNA
  • Fig 3 Schematic representation of the construction of AuV417 DNA
  • v e w hole HS V-2 has been mutated and attenuated to prevent neoplastic transformation
  • the mutated HSV-2 can be formulated w ith immune 99/36087
  • the protein kinase (PK) domain of the large subunit of ⁇ bonucleotide reductase (ICP 10) has previously been shown to have oncogenic properties Deletion of the PK domain was shown to have delete ⁇ ous effects on the abi tv of HSV-2 to infect cells
  • the present invention consists of the construction of three viruses that have various deletions in the ICP10 PK domain These deletions encompass the minimal kinase catalytic region but do not include antigenic sites downstream thereof The mutants are, therefore, PK negative, growth attenuated, and do not have oncogenic potential Unlike the recombinant virus (ICPIO ⁇ PK) previously constructed (Peng 96), they have supenor immunogenicity Computer assisted analysis of the ICP 10 PK antigenicity profile indicates that many antigenic sites are clustered within amino acids 350-450 Because these amino acids are not required for PK activity (Luo
  • HSV-2 RR activity depends on the binding of the two RR subunits (RR1 and RR2) at RR1 sites located within amino acids 419-432 and the extreme C-terminal 145 codons (Chung, T D , Luo, J H , Wymer, J P., Smith, C C and urehan, L .
  • recombinant viruses that retain these amino acids are likely to have a higher RR activity.
  • the increased RR activity may provide an advantage in that the recombinant viruses do not have defects other than the PK which is required for the expression of the regulatory IE genes. As such they will retain attenuated growth mediated by the absence of ICP 10 PK while evidencing improved in vivo expression required for increased immunogenicity (also favored by the retention of amino acids within positions 350-450).
  • RR activity was assayed as described (Averett, D. R.. Lubbers. C, Ehon. G. B., and Spector, T Ribonucleolide reductase induced by herpes simplex virus type 1. Characterization of a distinct enzyme Journal of Biological Chemisti ⁇ 258, pp.9631-9638, 1983) in the absence or presence of 0.5 mM ohgopeptides located at various ICP 10 amino acids (aa).
  • HSV-1 and HSV-2 v iruses are very similar.
  • the DNA is 50% 0 homologous.
  • Virtually all viral proteins have both t pe-specific and type common epitopes. For all but 2 proteins (i.e.. for 82 proteins), the type-common epitopes are predominant. The exception is the HSV-
  • HSV-2 oncoprotein ICP10PK both of which elicit predominantly type-specific antibodies.
  • HSV-2 oncogene was deleted from ICPIO ⁇ PK Therefore we only have one protein that can induce tvpe specific immunity The remaining 83 proteins will induce type common immunity This incudes both antibody and cell mediated immunity.
  • v e whole HSV-2 could not be explored as a vaccine option for HSV since the oncogene had potential neoplastic implications for the patient.
  • the present invention demonstrates that by removing the oncogene, a protein kinase, from the HSV-2 genome, not only are the neoplastic properties removed, but the virus is attenuated and provides full protection against challenge for an extended pe ⁇ od of time
  • HSV-2 strain which contains the deleted oncogene is not c ⁇ tical to the present inv ention
  • examples of such strains include HSV-2(G), HSV-2(333), HSV-2(186), HSV-2(S-1), although anv strain is acceptable
  • PK oncogene
  • the construction of the mutant virus is accomplished by well known techniques.
  • the location of the oncogene (PK) is well-known (DNA Tumor Viruses Oncogenic Mechanisms, Ed C Barbanti-Brodano. et al , Plenum Press, NY, 1995, chapter 14 by L Aurelian, Transformation and Mutagenic Effects Induced by He ⁇ es Simplex Virus Types 1 and 2, pp.
  • the oncogene is located in the ICP 10 section of the HSV-2 genome It has previously been shown that the PK activity and oncogenic activity are located within the gene sequence encoding amino acids 88-41 1 Bneflv, the wild type sequences in a plasmid (TP101) that contains the HSV-2 BamHI E and T fragments are replaced with vanous fragments from pJHL2 [ICP10 mutant deleted in the PK domain (Luo '92)] The resulting plasmids contain sequences which code for ICP 10 deleted in the PK catalytic domain flanked by 4 and 2.8 kb of HSV-2 DNA sequences at the 5' and 3' ends, respectively The lOkb
  • Hindlll'EcoRI fragment from these plasmids are introduced by marker transfer into a virus (ICP10 ⁇ RR) in which the RR domain of ICP 10 had been replaced with the LacZ gene.
  • the resulting recombinant v iruses designated AuV351.
  • AuV375 and AuV 417 are obtained by selecting w hue plaques on a background of blue plaques after staining w ith X-gal A few white plaques are picked and purified
  • the hyb ⁇ dizing bands, seen in the recombinant viruses are smaller than the 7.6kb for wild type HSV-2.
  • the band seen for AuV351 DNA is 6.9kb.
  • that seen for AuV375 DNA is 2.2 kb and that seen for AuV417 DNA is 6.6 kb as compared to 7 6kb for HSV-2 or the restored viruses AuV351(R), AuV375(R) ⁇ nd AuV417(R) DNA.
  • the recombinant viruses can be differentiated from wild type HSV-2 by DNA analysis and by lmmunoprecipitation/immunoblotting w ith antibody to epitopes located at ICP 10 amino acids retained by the deleted protein, such as the ant ⁇ -LA-1 antibody (recognizes ICP10 amino acids 13-26) (Aure an, et al.. Cancer Cells 7, pp.187-191, 1989).
  • the proteins recognized by the antibody are significantly smaller than the 140kDa ICP10 protein.
  • AuV41 7 is 99 kDa
  • AuV375 is 104 kDa
  • AuV351 is 107 kDa.
  • the oncogene or any portion thereof may be deleted.
  • e mean any portion of the oncogene w hich once deleted results in attenuation of the virus and prevents neoplastic transformation of the cells. Determining if PK activitv is absent requires expression of the viral gene and subjecting the result to standard
  • PK assays (Chung '89) There is abundant guidance in the p ⁇ or art as to the sections of the ICP 10 gene hich is required for PK activity. Determining viral attenuation requires testing in animals to determine absence of lesion formation. The techniques for accomplishing this are standard and well-known in the art. The resultant mutant viruses are used in infection experiments and compared to infections ith wild-type HSV-2 and the restored viruses. The cells used in infection are not critical to the present invention. Any human or animal cell line which can be infected with ild type HSV-2 may be used in the present invention Examples of such cell lines include Vero cells, HeLa cells, 293 cells, or MRC5 cells (all av ailable from American Type Culture Collection, Rockville. Maryland). ICPIO ⁇ PK can also be grown in cells that constitutively express ICP10. for example JHLal . It is titrated by plaque assay on Vero cells with MEM- 10% FCS and 0.3% human IgG
  • Immunizing a subject indicates the standard inte ⁇ retation w ell known in the art. Upon administration w ith the v accine composition, neutralizing antibodies and cell-mediated immunity are raised in the subject and said antibodies and cell-mediated immunity confer immunity to the subject 99/36087
  • the present invention teaches immunization of a subject against HSV-2.
  • a "pfu” is a plaque forming unit and represents the quantity of virus required to form a single plaque when a cell culture is infected with the virus. It is a quantitative measure of viral infectivity used by those skilled in the art. Due to the 50% homology of HSV-1 and HSV-2 there will be a high degree of protection against HSY- 1 infection.
  • viruses AuV351 , AuV375 and AuV417 for human use is accomplished by suspension in a solution with or without stabilizing ingredients, and with or without immune stimulants and adjuvants.
  • stabilizing agents, immune stimulants, and adjuvants include alum, incomplete Freud's adjuvant, MR-59 (Chiron, Emeryville. CA), MPL (mono-phosphoryl Lipid A).
  • Such stabilizing agents, adjuvants and immune stimulants are well known in the art and can be used singly or in combination.
  • the vaccine composition of the present invention can be administered to any animal, including humans.
  • the vaccine composition may be administered via any suitable mode of administration, such as intramuscular, oral, subcutaneous, intradermal, intravaginal. rectal, or intranasal administration.
  • a prefened mode of administration is subcutaneous or intradermal administration.
  • the AuV351 , AuV375 and AuV417 viruses which provide protection against HSV-2 infection, can be administered along with a pharmaceutically acceptable earner or diluent.
  • pharmaceutically acceptable earner or diluents include water, phosphate buffered saline or sodium bicarbonate buffer.
  • a number of other acceptable carriers or diluents are known
  • the strategy for construction of AuV351 is to create a recombinant plasmid, pAu ⁇ 351 that contains a gene cassette deleted in ICP 10 amino acids 107-351 (Fig. 1 )
  • This plasmid is used for the generation of a recombinant HSV-2 v irus deleted in ICP 10 amino acids 10"-351 through recombination w ith the appropriate v iral DNA All details of cloning methodology are based on standard procedures
  • Plasmid pJL2 (Luo '92) is digested w ith BamHI (made blunt ended) and StuI to remov e the 732bp fragment that encodes amino acids 107-351 The resulting construct is 99/36087
  • plasmid pAu ⁇ 351 contains sequences which code for ICP 10 deleted in amino acids 107-351 flanked by 4 and 2.8kb of HSV-2 DNA sequences at the 5' and 3' ends, respectively.
  • Hindlll/EcoRI fragment from pAu ⁇ 351 is introduced by marker transfer into a virus (ICP10 ⁇ RR) in which the RR domain of ICP 10 has been replaced with the LacZ gene.
  • the resulting recombinant designated AuV351 is obtained by selecting white plaques on a background of blue plaques after staining with X-gal. A few white plaques are picked, punfied. and grown in Vero cells in MEM with 10%> FCS.
  • Vero cells are co-transfected with l ⁇ g of infectious viral DNA from AuV351 and a 10-fold molar excess of the wild type BamHI E, T fragment.
  • Southern blot hyb ⁇ dization is used to confirm that the AuV351 DNA is deleted in nucleotides encoding ICP 10 amino acids 107-351
  • Viral DNA is isolated from cytoplasmic vinons as descnbed (Pignatti et al.. Virology, Vol 93, pp 260-264, 1979; Smith et al., Journal of General Virology, Vol 73. pp.1417-1428. 1992). Briefly, Vero cells are infected at a multiplicity of infection (moi ) of 5. At 48 hrs.
  • post infection (p.i.) cells are resuspended (2 x 10 7 cell/ml) in a buffer consisting of 10 mM T ⁇ s-HCl (pH 7.9), 10 mM EDTA and 0.25% Triton. Following incubation on ice ( 15 mm.), NaCl is added at a final concentration of 0.2 M and the nuclei are precipitated by centnfugation at 1 ,000 x g ( 10 min. 4°C). The supernatant, containing cytoplasmic vinons.
  • Viral DNA 15 ⁇ g is digested w ith BamH I and the fragments are separated by 1% agarose gel electrophoresis in a T ⁇ s-Acetate EDTA (TAE) buffer (40 mM T ⁇ s-acetate and 1 mM EDTA) It is transferred to Gene screen membranes ( New England Nuclear Co ⁇ ., Beverly. MA) and the membranes are incubated in a preh b ⁇ dization solution containing 5 x 99/36087
  • the hyb ⁇ dization probe is o gonucleotide AU26 (CCCCTTCATCATGTTT.AAGGA) hich represents a sequence m the ICP 10 RR coding region It is 3' tailed w ith digoxigenin-dUTP (DIG-dUTP ) by terminal transferase ( Boeh ⁇ nger Mannheim, Indianapolis, IN) in 20 ⁇ l volume with lx reaction buffer
  • the membranes are nnsed in Buffer 1(100 m.M T ⁇ s-HCl. pH 7 5.150 mM NaCl), incubated in Buffer 2 [2% (w ) casein in Buffer 1 ] for 40m ⁇ n and in Buffer 2 containing 3xl 0 U/ml of alkaline phosphatase-conjugatcd anti-digoxigenin antibody (Boeh ⁇ nger Mannheim, Indianapolis, IN) for 30 min After washing w ith Buffer 1 (twice) and soaking in Buffer 3 ( 100 mM T ⁇ s-HCl, pH 9 5, 100 mM NaCl. 50 mM MgCl, for 2 mm. the membranes are exposed to the chemiluminescent substrate Lumi-PhosTM 530 (Boeh ⁇ nger Mannheim, Indianapolis. IN) and the reaction is developed on X-ray film
  • DNA ( 15 ⁇ g) from HSV-2, AuV351 or AuV351 (R) is digested with BamHI. separated on 1 % agarose gels and transfened to nylon membranes. It is hybndized with the AU26 probe which recognizes a sequence within the ICP 10 RR coding region A hybridizing 7 6kb band which represents the BamHI E fragment is observed for HSV-2. and AuV351(R) DNA The hyb ⁇ dizing band seen for AuV351 DNA is 6.9kb.
  • Example 2 Constniction and Characterization of the AuV375 virus and AnV ⁇ 7 (R)
  • the strategy for construction of AuV375 is to create a recombinant plasmid, pAu ⁇ 375 that contains a gene cassette deleted in ICP 10 amino acids 107-375 (Fig. 1 )
  • This plasmid is used for the generation of a recombinant HSV-2 irus deleted in ICP 10 amino acids 107-375 through recombination w ith the approp ⁇ ate iral DN ⁇
  • All details of cloning methodology are based on standard procedures Plasmid pJL2 (Luo '92) is digested w ith EcoNI (partial) and BamHI to remove the
  • plasmid p ⁇ 375 The wild type sequences in a plasmid (TP 101 ) that contains the HSV-2 BamHI E and T fragments (Peng 96) are replaced with the 2 3kb Sall/Bglll fragment from p ⁇ 375
  • the resulting plasmid, pAu ⁇ 375 contains sequences which code for ICP10 deleted in amino acids 107-375 flanked by 4 and 2 8kb of HSV-2 DNA sequences at the 5' and 3' ends, respectively
  • the 10 3kb Hindlll EcoRI fragment from pAu ⁇ 375 is introduced by marker transfer into a virus (ICPIO ⁇ RR) in which the RR domain of ICP 10 has been replaced with the LacZ gene The resulting recombinant.
  • AuV375 is obtained by selecting white plaques on a background of blue plaques after staining with X-gal. A few white plaques are picked, pu ⁇ fied, and grown in Vero cells in MEM with 10% FCS
  • Vero cells are co-transfected with l ⁇ g of infectious viral DNA from AuV375 and a 10-fold molar excess of the wild type BamHI E/T fragment
  • a strategy similar to that reported for ICP6 ⁇ Goldstein and Weller, Virology, Vol 166, pp 41-51 , 1988) is used to select restored virus under growth rest ⁇ cted conditions (1 % FCS) Southern blot hybridization is used to confirm that the AuV375 DNA is deleted in nucleotides encoding ICP 10 amino acids 107-375 as descnbed in Example 1
  • DNA 15 ⁇ g from HSV-2, AuV375 or AuV375(R) is digested with BamHI, separated on 1%, agarose gels and transferred to nylon membranes It is hybndized with the AU26 probe which recognizes a sequence within the ICP 10 RR coding region A hybndizing 7 6kb band which represents the BamHI E fragment is observed for
  • Example 3 Construction and Characte ⁇ zation of the AuV417 virus and A ⁇ V417(R ⁇ ).
  • the strategy for construction of AuV417 is to create a recombinant plasmid, pAu ⁇ 417 that contains a gene casette deleted in ICP 10 amino acids 107-417 (Fig 3)
  • This plasmid is used for the generation of a recombinant HSV-2 virus deleted in ICP 10 amino acids 107-417 through recombination w ith the approp ⁇ ate v iral DNA
  • All details of cloning methodology are based on standard procedures
  • Plasmid pJL2 (Luo '92) is digested w ith BamHI (made blunt-ended) and Hpal to remov e the 936bp fragment that encodes amino acids 107-417 The resulting construct is collapsed through ligation at the BamHI EcoM sites to generate plasmid p ⁇ 41 7
  • the wild type sequences in a plasmid (TP101 ) that contains the HSV-2 BamHI E and T fragments 99/36087
  • pAu ⁇ 417 contains sequences which code for ICP 10 deleted in amino acids 107-417 flanked by 4 and 2.8kb of HSV-2 DNA sequences at the 5' and 3' ends, respectively.
  • the 10. lkb Hindlll/EcoRI fragment from pAu ⁇ 417 is introduced by marker transfer into a virus (ICPIO ⁇ RR) in which the RR domain of ICP 10 has been replaced with the LacZ gene.
  • the resulting recombinant designated AuV417 is obtained by selecting white plaques on a background of blue plaques after staining with X-gal A few white plaques are picked, pu ⁇ fied, and grown in Vero cells in MEM with 10% FCS
  • Vero cells are co-transfected with l ⁇ g of infectious viral DNA from AuV417 and a 10-fold molar excess of the wild type BamHI E/T fragment.
  • a strategy similar to that reported for ICP6 ⁇ Goldstein and Weller, Virology, Vol. 166, pp 41-51, 1988) is used to select restored virus under growth restncted conditions ( 1% FCS).
  • AuV375 and uV417 have hiaher RR activity than ICPI O ⁇ PK ICPIO ⁇ PK virus has RR activity, but it is lower than that of HSV-2 Inasmuch as amino acids 413-438 may be involved in the complexation of the two RR subunits (Table 1), viruses that retain these amino acids have an RR activity similar to that of HSV-2 (Table 2).
  • Example 5 AuV351 .
  • AuV.75 and AuV417 are attenuated for growth in infected nmmals
  • the mouse footpad model of HSV-2 infection is used to examine the growth of
  • Example 6 AnVlSI .
  • AuV375 and AuV417 protect from HSV-2 challenge
  • the footpad model described in example 5 is used to examine protection by AuV351, AuV375 and AuV417
  • the expe ⁇ ment is done as previously descnbed (Wachsman '89; Wachsman '92) with mice given one or multiple immunizations with IxlO 7 pfu of virus (at 14-16 days intervals) before challenge with wild type HSV-2 Challenge is with IxlO 7 to IxlO 8 pfu of HSV-2 and it is done at 3-6 weeks after the last immunization All mice in the
  • PBS group develop skin lesions from which virus is isolated, and 50-80% die on days 8-13 after challenge Bv contrast lesions are not seen and virus is not isolated from the immunized mice
  • the AuV351 , AuV375 and AuV417 v noises have vaccine potential.
  • Example 7 AuV351 , AuV375 and AuV417 viruses induce HSV-specific immunity.
  • mice are immunized with AuV351, AuV375 and AuV417 as descnbed in example 6 Two-four weeks after the last injection spleens are removed and T cells are used in lymphocyte proliferation assays as descnbed (Wachsman et al Journal of General Virology, Vol 70, pp 2513-2520, 1989, Vaccine, Vol 10, pp 447-454, 1992) HSV-specific lymphoproliferation is seen in all animals Prohferative levels are similar to those seen for
  • AuV375 and AuV417 induce good levels of virus-specific T cell responses They also induce antibody responses as determined by neutralization assays
  • All references cited herein are inco ⁇ orated by reference in their entirety It will be apparent to those skilled in the art that the examples and embodiments descnbed herein are bv wav of illustration and not of limitation, and that other examples may be utilized w ithout departing from the spi ⁇ t and scope of the present invention, as set forth in the appended claims

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Abstract

The present invention discloses new vaccine compositions for Herpes Simplex comprising a whole live HSV-2 virus having various amino acids deleted. Methods of using the vaccine compositions are also included.

Description

TITLE OF THE INVENTION NOVEL VACCINE COMPOSITIONS FOR HERPES SIMPLEX VIRUS
BACKGROUND OF THE INVENTION The present invention discloses new vaccine compositions for Heφes Simplex compπsing a whole live HSV-2 virus having vanous ammo acids deleted. Methods of using the vaccine compositions are also included.
Both distinguishable serotypes of Heφes Simplex V irus (HSV- 1 and HSV-2) cause infection and disease ranging from relatively minor fever blisters on lips to severe genital infections, and generalized infections of newborns HSV- 1 and HSV-2 are 50% homologous at the DNA level, and polyclonal antibodies and MAbs to shared epitopes are cross-reactive.
HSV-1 and HSV-2 have πbonucleotide reductase 1 (RR1 ) proteins (an allosteπc subunit of the πbonucleotide reductase (RR) enzyme) (respectively designated ICP6 and ICP10) that contain a unique ammo terminal domain The HSV-2 unique domain codes for a seπne/threonine-specific protein kinase (PK) which has auto- and transphosphorylating activity and has a transmembrane (TM) domain Sequences which code for the PK domain cause neoplastic transformation and are associated with cervical cancer ( HSV-2 oncogene) The unique terminal domain of the HSV-1 RR1 protein (ICPό) also has PK activity but it is different from that of the HSV-2 oncogene both structurally and functionally.
Ongmal studies, using enzymatic assay conditions similar to those employed for ICP10 PK, concluded that ICP6 does not have PK activity, although the unique domain is retained (Chung, T D . Wvmer. J P., Kulka. M . Smith, C C and Aurehan. L.. Protein kinase activity associated w ith the large subunit of herpes simplex virus
Figure imgf000003_0001
2 ribonucleolide reductase (ICP 10) Journal of Virology , Vo\ 63. pp 3389-3398. 19S9).(Chung '89) This was not unexpected since the sequence of the unique PK domains sho ed only 38% homology (Nikas, I.. McLauchlan. J.. Dauson. A J.. Taylor. W R and Clements. J B.. Structural features of ribonucleolide reductase Proteins Structure Function and Genetics 1. pp 376- 384. 1986). Further studies indicated that ICP6 has PK
Figure imgf000003_0002
itv but only under different 99/36087
conditions. Also there is controversy as to whether the activity is both auto- and transphosphorylating (see Peng, T . Hunter. J R C, and Nelson, J. W. The novel protein kinase of the RR1 subunit of herpes simplex virus has autophosphonlation and transphosphon'lation activin- that differs in its A TP requirements for HSV-1 and SV-2. , Vol. 216. pp 184- 196, 1996 [Peng "96] for a review of the problem; partιcui.ar-y
Table 1 ) The reason for the different PK activities of the ICP6 and ICP10 proteins is likely to be that the ATP binding sites of ICP6 PK are located distantly from the rest of the catalytic motifs (Cooper, J ., Marsden, H., and Clements, J. B Rώonucleotide reductase of herepesviruses. Journal of Virology, Vol. 69, pp.4979-4985, 1995). ICP6 also does not have a functional TM domain and it does not localize to the cell surface (Conner, J. Murray, J.,
Cross. A , Clements. J B . and Marsden, H S Intracellular localization of herpes simplex virus t\pe 1 ribonucleotide reduciase subunits during infection of cultured cells Virology, Vol. 213, pp.615, 1995) The PK actι\ uy of native 1CP6 is \ ery weak even under ideal conditions, such that its K_, is 10-fold higher than that of ICP10 PK (Peng '96). The transforming activity of ICP6 is located within a genome fragment that is distant from that at which the HSV2 oncogene is located. Transformation in this system is based on focus formation.
It has previously been shown that DNA sequences which encode for the ammo- terminal one-third of ICP10 (amino acids 1 -41 1 ) have oncogenic potential. Cells transfected with these DNA sequences evidence anchorage independent growth and cause tumors in animals. Transformation is seen in both rodent and human cells (Jaπwalla. R J . Aurelian, L. and Ts'o. P.O P Tumorigenic transformation induced bv a specific fragment of DNA from herpes simplex virus tvpe 2 Proceedings of the National Academv of Sciences. Vol. 77, pp.2279-2283, 1980 [Jaπwalla '80]). There are three functional domains w ithin ICP 10 amino acids 1 -41 1 (ι) an intracellular domain at amino acids 106-41 1 w hich encompasses the PK catah tic domain with eight conserved catalytic motifs, (n) a TM at amino acids 88- 105 and (in ) an extracellular domain at amino acids 1 -88 (Chung '89. Virology, Vol 179. pp 168- 178, 1990). The minimal size required for PK actι\ lty is amino acids 1 -2S3 (pp29lα l) (Luo. J H., Smith, C. C. Kulka. M., and Aurehan. L A truncated pi otein kinase domain of the large subunit of herpes simplex virus
Figure imgf000004_0001
2 ribonucleolide reductase (ICP10) expressed in Eschericlua coll. Journal of Biological Chemistn , Vol 266. pp 20976-209S3. 1991 ) [Luo '91 ] However, the 99/36087
PK activity of pp29lal has some properties different from the authentic ICP10 PK, presumably because it lacks pan of PK catalytic domain VI (Luo '91 ). The TM domain is also required (but insufficient) for PK activity (Luo, J. H. and Aurelian. L. The transmembrane helical segment but not the invariant lysine is required for the kinase activity of the large subunit of Herpes simplex virus type 2 ribonucleolide reductase. Journal of Biological Chemistry, Vol.
267, pp.9645-9653, 1992) [Luo '92]. Therefore, it can be concluded that the PK activity is localized within amino acids 88-41 1 with an essential core at amino acids 88-283.
The unique Hpal site within the ICP10 coding region represents the 3' end of the transforming region (Jariwalla '80) and Hpal cuts the gene after the codon for amino acid residue 417. It is not known whether pp29lal has transforming activity. However, PK activity is required for neoplastic potential. PK negative mutants do not transform cells. This includes a mutant deleted in the TM domain and site directed mutants in the ATP binding sites ( Lys1 6 and/or Lys"59) or the ion-binding site (Glu209) (Smith C. C, Luo, J. H., Hunter, J. C. R.. Ordonez, J. V., and Aurelian, L. The transmembrane domain of the large subunit of HSV-2 ribonucleolide reductase (ICP10) is required for protein kinase activity and transformation-related signaling pathways that results in ras activation. Virology, Vol. 200, pp.598-612, 1994) [Smith '94]. Because a PK' mutant deleted only in the TM domain does not have transforming activity (Smith '94), DNA sequences that code for ICP10 amino acids 106-41 1 but lack PK activity are not intrinsically neoplastic. This demonstrates that: (i) the HSV-2 oncoprotein is located within ICP10 amino acids 1 -41 1 , and (ii) neoplastic potential requires a functional PK activity.
The function of ICP10 PK in virus growth/pathogenesis is unknown. The HSV-2 ICP10 protein has intπnsic PK activity. This was shown by demonstrating that ICP10 PK activity is lost through site-directed mutagenesis. The oncoprotein also has SH3-binding motifs at positions 140, 149 and 396, which are required for interaction with signaling proteins. This interaction is required for transforming activity. Site directed mutagenesis was used to identify amino acids required for kinase activity and interaction with signaling proteins. Mutation of Lys' '' or Lys 2>9 reduced PK activity (5-8 fold) and binding of the uC-labeled ATP analog p-fluorosulfonylbenzoyl 5'-adenosine (FSBA), but did not abrogate them. Enzymatic activity and FSBA binding were abrogated by mutation of both
Lvs residues, sussestina that either one can bind ATP. Mutation of Glu w (PK catalvtic motif 99/36087
III) virtually abrogated kinase activity in the presence of Mg2 or Mn!' ions, suggesting that Glu209 functions in ion-dependent PK activity
ICP10PK functions as a growth factor receptor involved in signaling and it binds the adaptor protein Grb, in vitro It has also been shown that there are SH3-bindιng sites within the ICP10 PK domain (at positions 140. 149 and 396) and they are required for interaction with signaling proteins and, thereby transformation (Nelson , J W., Zhu, J., Smith, C. C, Kulka. M. and Aure an, L ATP and SH3 binding sites in the protein kinase of the large subunit of herpes simplex virus tvpe 2 of ribonucleolide reductase (ICP10). Journal of Biological Chemistn 271, pp 17021-17027, 1996) [Nelson '96]. Mutation of the ICP10 pro ne-nch motifs at position 396 and 149 reduced Grb: binding 20- and 2-fold respectively.
Binding w as abrogated by mutation of both motifs Grb-, binding to wild type ICP10 was competed by a peptide for the Grb: C-terminal SH3 motif indicating that it involves the Grb2 C-terminal SH3 (Nelson '96)
The ICP 10 PK catalytic domain also contains amino acids that are responsible for binding a down-regulator of PK activity (ras-GAP) They are located at position 106-178.
Deletion of these ammo acids reduces but does not abrogate PK activity (Luo '92) However, it abrogates ras-GAP binding and thereby increases transforming potential.
The construction of a virus deleted in ICP10 amino acids 106-446 (ICP10ΔPK) is described by Peng '96 Briefly, the wild type sequences in a plasmid (TP101 ) that contains the HSV-2 BamHI E and T fragments were replaced w ith the 1 8kb Sall/Bglll fragment from pJHL9 pJHL9 is a plasmid containing an ICP10 mutant deleted in the PK catalytic domain '92). The resulting plasmid, TP9, contains sequences w hich code for ICP10 deleted in the PK catalytic domain flanked by 4 and 2.8 kb of HSV-2 DNA sequences at the 5' and 3' ends, respectively. The lOkb Hindlll/EcoRI fragment from TP9 was introduced by marker transfer into a virus (ICP 10ΔRR) in which the RR domain of ICP 10 had been replaced with the LacZ gene The resulting recombinant virus, designated ICP10ΔPK. w as obtained by selecting white plaques on a background of blue plaques after staining ith X-gal. A few white plaques w ere picked, purified and grown in Vero cells w ith 10% serum.
The ICP10ΔPK v irus is deleted in ICP 10 amino acids 106-446 It lacks ICP 10 PK activity and retains RR activitv (Peng '96) and is attenuated for growth in culture and in infected animals The v irus induces HSV -specific T cell immunitv and protects mice from challenge w πh w I Id type HSV-2 99/36087
There are several known HSV vaccines in the pπor art. US patents 4,347,127; 4,452,734; 5,219.567; and 5,171 ,568 each teach subunit vaccines which provide some protection against HSV-2 infection. These vaccines are inferior to one in which a live, attenuated virus is used. The immunity induced by a subunit vaccine is restricted to the particular protein represented by the subunit, which may not have sufficient protective potential. Additionally it is non-replicating and there is therefore no amplification of the protein which would further reduce immunogenicity. These problems occur in any subunit vaccine regardless of whether the method of preparation is via a recombinant protein or a purification of antigen from a virus. A cross recombinant vaccine, such as disclosed in US 4,554,159 (' 159), does not suffer from the problems of the subunit vaccines, but contains the oncogene present in HSV- 2. Unless care is taken to define and delete the oncogene. the cross recombinant vaccine could induce cancer in the vaccinee.
The cross recombinant of '159 is temperature sensitive. Avirulence may be obtained by selecting temperature resistance, but the temperature of the mouse is 39°C while that of humans is 37°C. This temperature sensitivity could well render such a cross problematic in a vaccine. A superior method of selection of avirulence is by the removal of genes coding for virulence without respect to the temperature at which the virus replicates. Also, the use of prototypical crosses would preclude the use of gene deleted or inserted mutants. Due to the many type-common epitopes on HSV- 1 and HSV-2, cell-mediated immunity cross-reacts (Jacobs, R. P., Aurelian. L.. and Cole, G. A. Cell-mediated immune response to herpes simplex virus: Type specific Ivmphoproliferative responses in lymph nodes draining the site of primary infection Journal of Immunology, Vol. 1 16, pp.1520- 1525, 1976). A live vaccine is supeπor to a dead vaccine because the liv e vaccine induces herd immunity; it induces different types of immunity, such as mucosal. cell mediated and humoral immunity. A higher le el of immunity is normally obtained because the virus titers are increased through replication vv ithin the vaccinee. Finally a live vaccine is of longer duration, thus obviating boosters and lowering initial dosage. The ICP I OΔPK virus is deleted in ICP 10 ammo acids 106-446 It lacks ICP 10 PK activity and retains RR activity (Peng '96) and is attenuated for grow th in culture and in infected animals. The virus induces HSV- specific T cell immunity and protects mice from challenge w ith ild type HSV-2 A patent 99/36087
application for ICPI OΔPK virus was submitted. However, the levels of HSV-specific immunity induced by ICPIOΔPK virus are 3-fold lower than those induced by similar doses of HSV-2, suggesting that it has a reduced immunogenic potential. An absolute necessity for a live heφes vaccine is the removal of the gene responsible for causing transformation while retaining immunogencity. as in the present invention.
Most developed vaccines (viz. those in neurovirulence genes) are in HSV-1. Known vaccines are not virus type-specific. All known vaccines for HSV-1 or HSV-2 are cross- reactive and provide immunity to the other virus type. However, HSV-1 is not as desirable a vaccine candidate against heφes, because the major clinical problem is the sexually transmitted HSV-2, which is also associated with cancer induction. Recent studies indicate that the age-adjusted prevalence of HSV-2 in the US is now 20.8%, an increase of approximately 30% over the past 13 years (Fleming, D.T.. McQuillan. G. M.. Johnsons, R. E., Nahmias. A. J., Aral. S. 0. Lee, F. K.. St Louis, M. E. Herpes simplex virus type 2 in the United States. 1976 to 1994. New England Journal of Medicine. Vol. 337, pp.1 105- 111 1 , 1997). The increasing rate of HSV-2 acquisition among young adults increases the likelihood that infants will be exposed to HSV-2 at delivery, resulting in an infection that, despite antiviral therapy, is still life-threatening (Whitley, R. J.. and Gnann. J. W. Jr. Acyclovir: a decade later. New England Journal of Medicine, Vol. 327, pp.782-799, 1992 [Erratum, New England Journal of Medicine, Vol. 328, pp.671 , 1993]). A new concern about HSV-2 infection is that it may facilitate the spread of HIV and increase the severity of the disease.
Because HSV-1 has only a 50%) homology to HSV-2, this may lower the response rate against the heterologous strain in the vaccinated population.
Another absolute requirement for a live vaccine is the absence of lesions upon immunization. An important trait in the live vaccine is its ability to induce high levels of HSV-specific T cell immunity and prevent lesions due to infection with wild type HSV-2 and
HSV- 1.
A desirable trait in the live vaccine would also be its ability to cause a reduction in the frequency of recurrent lesions in a person already infected. There is a substantial population already infected with HSV who may have intercourse with uninlected individuals who would benefit from such a vaccine.
The present invention solves all the problems recited above providing a w hole live attenuated HSV-2 in which the HSV-2 has a deletion of the oncot-ene. has strong 99/36087
lmmunogencity, and may be formulated in a vaccine composition. The present invention provides a method of immunizing a subject against HSV-1 or HSV-2 with said vaccine composition, providing a supenor method of conferring immunity upon the subject.
BRIEF SUMMARY OF THE INVENTION
The present invention is a vaccine composition compπsing Heφes Simplex Vιrus-2 recombinant selected from the group of recombinants which have deletions in the gene for ICP 10 to remove the fragments encoding for amino acids 107-351 (AuV 351) , the fragments encoding for amino acids 107-375 (AuV375) and the fragments encoding for amino acids 107- 417 (AuV417), and a pharmaceutically acceptable earner or diluent.
It is an object of the present invention to provide a vaccine composition which when administered to an animal, including a human, provides protection from challenge with HSV- 2 or HSV-1 infection and may reduce the frequency of recurrent lesions.
It is a further object of the invention to provide a vaccine composition compπsing whole, li e, attenuated HSV-2 wherein the oncogene or any portion thereof that causes transformation and does not attenuate the virus has been deleted.
It is a further object of the invention to provide a method of immunizing a subject against HSV-2 or HSV-1 comprising administenng a novel vaccine composition
It is e en a further object of the present inv ention to reduce or prevent clinical symptoms associated with HSV-2 or HSV-1 infections compπsing administenng a novel vaccine composition
It is yet a further object of the present inv ention to reduce recurrent disease associated with HSV-2 or HSV-1 in previously infected subjects comprising administering a novel vaccine composition. BRIEF DESCRIPTION OF DRAWINGS
Fig. 1. Schematic representation of the construction of AuV351 DNA Fig 2 Schematic representation of the construction of AuV375 DNA Fig 3 Schematic representation of the construction of AuV417 DNA
DETAILED DESCRIPTION OF THE INVENTION
In the present inv ention. v e w hole HS V-2 has been mutated and attenuated to prevent neoplastic transformation The mutated HSV-2 can be formulated w ith immune 99/36087
stimulants or adjuvants and used to immunize a subject against HSV-1 or HSV-2. The protein kinase (PK) domain of the large subunit of πbonucleotide reductase (ICP 10) has previously been shown to have oncogenic properties Deletion of the PK domain was shown to have deleteπous effects on the abi tv of HSV-2 to infect cells The present invention consists of the construction of three viruses that have various deletions in the ICP10 PK domain These deletions encompass the minimal kinase catalytic region but do not include antigenic sites downstream thereof The mutants are, therefore, PK negative, growth attenuated, and do not have oncogenic potential Unlike the recombinant virus (ICPIOΔPK) previously constructed (Peng 96), they have supenor immunogenicity Computer assisted analysis of the ICP 10 PK antigenicity profile indicates that many antigenic sites are clustered within amino acids 350-450 Because these amino acids are not required for PK activity (Luo '91 ) the recombinant viruses descπbed in this invention were constructed to retain these antigenic sites The immunogenicity of ammo acids located at this position is also demonstrated by their ability to induce antibody that specifically stains HSV- 2 infected, but not uninfected cells Therefore, viruses that retain these antigenic sites have increased lmmunogenic potential
Previous studies documented that HSV-2 RR activity depends on the binding of the two RR subunits (RR1 and RR2) at RR1 sites located within amino acids 419-432 and the extreme C-terminal 145 codons (Chung, T D , Luo, J H , Wymer, J P., Smith, C C and urehan, L . Leucine repeats in the large subunit of herpes simplex virus type 2 iibonucleotide reductase (RR ICP 10) cue im ohed in RR actmn and subunit complex formation Journal of General Vιrolog\ , \o\ 72, pp 1 139- 1 144, 1991 ) The significance of these sites for the interaction of the two RR subunits is still controv ersial, however For example, w orking with HSV- 1 infected cells. Bonneau, A M , Kibler, P , White. P , Bousquet, C , Dansereau, N and Cordingley, M G {Resistance of herpes simplex \ ιrus type 1 to peptidomimetic ribonucleotide icductase inlubitois selection and characterization of mutant isolates Journal of l u olog , \ ol 70, pp 787-93, 1996) concluded that the interaction onlv requires the C-terminai RR 1 amino acids This inteφretation is supported for HSV-2 by the finding that ICP 10ΔPK. w Inch is deleted in ICP 10 amino acids 106-446. retains RR activ itv (Peng '96) Howev er, the RR activ ity of the ICP I OΔPK v irus is low er than that of
HS -2 Also o gopeptides that encompass ICP10 ammo acids 41 425 and 426-438 reduce HS\ -2 RR acti itv in \ uιo (58 and 31 % respectiv elv ) (Table 1 ) presumably because they 99/36087
cause subunit dissociation. Therefore, recombinant viruses that retain these amino acids, such as those descnbed in this invention, are likely to have a higher RR activity. The increased RR activity may provide an advantage in that the recombinant viruses do not have defects other than the PK which is required for the expression of the regulatory IE genes. As such they will retain attenuated growth mediated by the absence of ICP 10 PK while evidencing improved in vivo expression required for increased immunogenicity (also favored by the retention of amino acids within positions 350-450).
Therefore, novel vaccine compositions have been discovered and a novel method of immunizing a subject against HSV-2 or HSV-1.
Table 1. HSV-2 RR activity is inhibited by o gopeptides at positions 413-438
Oligopeptide Inhibition (%)
None 0 aa 165-179 0 aa 355-369 10 aa 413-425 58 aa 426-438 31
RR activity was assayed as described (Averett, D. R.. Lubbers. C, Ehon. G. B., and Spector, T Ribonucleolide reductase induced by herpes simplex virus type 1. Characterization of a distinct enzyme Journal of Biological ChemistiΥ 258, pp.9631-9638, 1983) in the absence or presence of 0.5 mM ohgopeptides located at various ICP 10 amino acids (aa).
HSV-1 and HSV-2 v iruses are very similar. The DNA is 50%0 homologous. Virtually all viral proteins have both t pe-specific and type common epitopes. For all but 2 proteins (i.e.. for 82 proteins), the type-common epitopes are predominant. The exception is the HSV-
2 gG2 (Lee. F. K , Coleman. R M . Pereira. L.. Bailey. P D . Tatsuno. M.. and Nahmias, A. J Detection of herpes simplex
Figure imgf000011_0001
enzvme-linked immunosorhent 99/36087
assay Journal of Clinical Microbiology Vol 22, pp 641 -644, 1985) and the HSV-2 oncoprotein ICP10PK, both of which elicit predominantly type-specific antibodies. In the present invention, the HSV-2 oncogene was deleted from ICPIOΔPK Therefore we only have one protein that can induce tvpe specific immunity The remaining 83 proteins will induce type common immunity This incudes both antibody and cell mediated immunity.
Previously, v e whole HSV-2 could not be explored as a vaccine option for HSV since the oncogene had potential neoplastic implications for the patient. The present invention demonstrates that by removing the oncogene, a protein kinase, from the HSV-2 genome, not only are the neoplastic properties removed, but the virus is attenuated and provides full protection against challenge for an extended peπod of time
The particular HSV-2 strain which contains the deleted oncogene is not cπtical to the present inv ention Examples of such strains include HSV-2(G), HSV-2(333), HSV-2(186), HSV-2(S-1), although anv strain is acceptable These strains are well known and readily available The construction of the mutant virus is accomplished by well known techniques. The location of the oncogene (PK) is well-known (DNA Tumor Viruses Oncogenic Mechanisms, Ed C Barbanti-Brodano. et al , Plenum Press, NY, 1995, chapter 14 by L Aurelian, Transformation and Mutagenic Effects Induced by Heφes Simplex Virus Types 1 and 2, pp. 253-280) The oncogene is located in the ICP 10 section of the HSV-2 genome It has previously been shown that the PK activity and oncogenic activity are located within the gene sequence encoding amino acids 88-41 1 Bneflv, the wild type sequences in a plasmid (TP101) that contains the HSV-2 BamHI E and T fragments are replaced with vanous fragments from pJHL2 [ICP10 mutant deleted in the PK domain (Luo '92)] The resulting plasmids contain sequences which code for ICP 10 deleted in the PK catalytic domain flanked by 4 and 2.8 kb of HSV-2 DNA sequences at the 5' and 3' ends, respectively The lOkb
Hindlll'EcoRI fragment from these plasmids are introduced by marker transfer into a virus (ICP10ΔRR) in which the RR domain of ICP 10 had been replaced with the LacZ gene. The resulting recombinant v iruses, designated AuV351. AuV375 and AuV 417 are obtained by selecting w hue plaques on a background of blue plaques after staining w ith X-gal A few white plaques are picked and purified
Southern blot hv bπdization is used to confirm that the iruses are deleted in the ICP 10 PK coding region The AL'26 (CCCCTTC ATCATGTTTAAGGA) probe is used. It 99/36087
recognizes a sequence within the ICP 10 RR coding region. The hybπdizing bands, seen in the recombinant viruses are smaller than the 7.6kb for wild type HSV-2. The band seen for AuV351 DNA is 6.9kb. that seen for AuV375 DNA is 2.2 kb and that seen for AuV417 DNA is 6.6 kb as compared to 7 6kb for HSV-2 or the restored viruses AuV351(R), AuV375(R) αnd AuV417(R) DNA.
The recombinant viruses can be differentiated from wild type HSV-2 by DNA analysis and by lmmunoprecipitation/immunoblotting w ith antibody to epitopes located at ICP 10 amino acids retained by the deleted protein, such as the antι-LA-1 antibody (recognizes ICP10 amino acids 13-26) (Aure an, et al.. Cancer Cells 7, pp.187-191, 1989). The proteins recognized by the antibody are significantly smaller than the 140kDa ICP10 protein. AuV41 7 is 99 kDa, AuV375 is 104 kDa; and AuV351 is 107 kDa.
The oncogene or any portion thereof may be deleted. By the expression "or any portion thereof"" e mean any portion of the oncogene w hich once deleted results in attenuation of the virus and prevents neoplastic transformation of the cells. Determining if PK activitv is absent requires expression of the viral gene and subjecting the result to standard
PK assays (Chung '89) There is abundant guidance in the pπor art as to the sections of the ICP 10 gene hich is required for PK activity. Determining viral attenuation requires testing in animals to determine absence of lesion formation. The techniques for accomplishing this are standard and well-known in the art. The resultant mutant viruses are used in infection experiments and compared to infections ith wild-type HSV-2 and the restored viruses. The cells used in infection are not critical to the present invention. Any human or animal cell line which can be infected with ild type HSV-2 may be used in the present invention Examples of such cell lines include Vero cells, HeLa cells, 293 cells, or MRC5 cells (all av ailable from American Type Culture Collection, Rockville. Maryland). ICPIOΔPK can also be grown in cells that constitutively express ICP10. for example JHLal . It is titrated by plaque assay on Vero cells with MEM- 10% FCS and 0.3% human IgG
Immunizing a subject indicates the standard inteφretation w ell known in the art. Upon administration w ith the v accine composition, neutralizing antibodies and cell-mediated immunity are raised in the subject and said antibodies and cell-mediated immunity confer immunity to the subject 99/36087
The present invention teaches immunization of a subject against HSV-2. A "pfu" is a plaque forming unit and represents the quantity of virus required to form a single plaque when a cell culture is infected with the virus. It is a quantitative measure of viral infectivity used by those skilled in the art. Due to the 50% homology of HSV-1 and HSV-2 there will be a high degree of protection against HSY- 1 infection.
The formulation of viruses AuV351 , AuV375 and AuV417 for human use is accomplished by suspension in a solution with or without stabilizing ingredients, and with or without immune stimulants and adjuvants. Examples of stabilizing agents, immune stimulants, and adjuvants include alum, incomplete Freud's adjuvant, MR-59 (Chiron, Emeryville. CA), MPL (mono-phosphoryl Lipid A). Such stabilizing agents, adjuvants and immune stimulants are well known in the art and can be used singly or in combination.
The vaccine composition of the present invention can be administered to any animal, including humans. The vaccine composition may be administered via any suitable mode of administration, such as intramuscular, oral, subcutaneous, intradermal, intravaginal. rectal, or intranasal administration. A prefened mode of administration is subcutaneous or intradermal administration.
The AuV351 , AuV375 and AuV417 viruses, which provide protection against HSV-2 infection, can be administered along with a pharmaceutically acceptable earner or diluent. Examples of such pharmaceutically acceptable earner or diluents include water, phosphate buffered saline or sodium bicarbonate buffer. A number of other acceptable carriers or diluents are known
The following examples are provided for illustrative puφoses only and are in no way intended to limit the scope of the present invention.
Example 1 Construction and Characterization of the AuV351 virus and AnV S UP )
The strategy for construction of AuV351 is to create a recombinant plasmid, pAuΔ351 that contains a gene cassette deleted in ICP 10 amino acids 107-351 (Fig. 1 ) This plasmid is used for the generation of a recombinant HSV-2 v irus deleted in ICP 10 amino acids 10"-351 through recombination w ith the appropriate v iral DNA All details of cloning methodology are based on standard procedures
Plasmid pJL2 (Luo '92) is digested w ith BamHI (made blunt ended) and StuI to remov e the 732bp fragment that encodes amino acids 107-351 The resulting construct is 99/36087
collapsed through ligation at the BamHI/StuI sites to generate plasmid pΔ351. The wild type sequences in a plasmid (TP101 ) that contains the HSV-2 BamHI E and T fragments (Peng '96) are replaced with the 2.4kb Sall/Bglll fragment from pΔ351 The resulting plasmid, pAuΔ351 , contains sequences which code for ICP 10 deleted in amino acids 107-351 flanked by 4 and 2.8kb of HSV-2 DNA sequences at the 5' and 3' ends, respectively. The 10.4kb
Hindlll/EcoRI fragment from pAuΔ351 is introduced by marker transfer into a virus (ICP10ΔRR) in which the RR domain of ICP 10 has been replaced with the LacZ gene. The resulting recombinant designated AuV351 , is obtained by selecting white plaques on a background of blue plaques after staining with X-gal. A few white plaques are picked, punfied. and grown in Vero cells in MEM with 10%> FCS. For the construction of the restored virus AuV351(R). Vero cells are co-transfected with l μg of infectious viral DNA from AuV351 and a 10-fold molar excess of the wild type BamHI E, T fragment. A strategy similar to that reported for ICP6Δ (Goldstein, D J. and Weller, S. K. F actor (s) present in the herpes simplex virus tvpe-1 infected cells can compensate for the loss of the large subunit of the viral ribonucleolide reductase- characterization of an ICP6 deletion mutant. Virology,
Vol. 166, pp.41 -51, 1988) is used to select restored virus under growth restπcted conditions
Southern blot hybπdization is used to confirm that the AuV351 DNA is deleted in nucleotides encoding ICP 10 amino acids 107-351 Viral DNA is isolated from cytoplasmic vinons as descnbed (Pignatti et al.. Virology, Vol 93, pp 260-264, 1979; Smith et al., Journal of General Virology, Vol 73. pp.1417-1428. 1992). Briefly, Vero cells are infected at a multiplicity of infection (moi ) of 5. At 48 hrs. post infection (p.i.) cells are resuspended (2 x 107 cell/ml) in a buffer consisting of 10 mM Tπs-HCl (pH 7.9), 10 mM EDTA and 0.25% Triton. Following incubation on ice ( 15 mm.), NaCl is added at a final concentration of 0.2 M and the nuclei are precipitated by centnfugation at 1 ,000 x g ( 10 min. 4°C). The supernatant, containing cytoplasmic vinons. is incubated in 200 μg/ml Proteinase K and 0 2%u SDS (4 hr at 37°C), mixed with saturated sodium iodide (Nal; final concentration 1.525 g ml) and ethidium bromide (final concentration 3 μg/ml) and centπfuged at l OO.OOOxg for 16 hrs.
Viral DNA ( 15 μg) is digested w ith BamH I and the fragments are separated by 1% agarose gel electrophoresis in a Tπs-Acetate EDTA (TAE) buffer (40 mM Tπs-acetate and 1 mM EDTA) It is transferred to Gene screen membranes ( New England Nuclear Coφ., Beverly. MA) and the membranes are incubated in a preh bπdization solution containing 5 x 99/36087
SSC [750 mM NaCl. "5 mM Sodium citrate; pH (7 0)], 2% Casein, 0.1% N-laurylsarcosine and 0 02% sodium dodecv l sulfate (SDS)] at 42° C for 2 hrs The hybπdization probe is o gonucleotide AU26 (CCCCTTCATCATGTTT.AAGGA) hich represents a sequence m the ICP 10 RR coding region It is 3' tailed w ith digoxigenin-dUTP (DIG-dUTP ) by terminal transferase ( Boehπnger Mannheim, Indianapolis, IN) in 20 μl volume with lx reaction buffer
[5 mM cobalt chlonde (CoCL), 0 05 mM DIG-dUTP, 5 nmol/ml AU26, 0 5 mM dATP and 2.5 units/μl terminal transferase] at 37°C for 15 mm diluted to a final concentration of 5 pmol/ml in prehybndization solution. Hybπdization is done at 42°C for 3 hrs. Membranes are washed once (room temperature) in a solution containing 2\SSC. 0.1 %> SDS for 5 mins and twice in 0 5xSSC, 0 1 % SDS for 15 mins. For detection of the hybndized DNA fragments, the membranes are nnsed in Buffer 1(100 m.M Tπs-HCl. pH 7 5.150 mM NaCl), incubated in Buffer 2 [2% (w ) casein in Buffer 1 ] for 40mιn and in Buffer 2 containing 3xl 0 U/ml of alkaline phosphatase-conjugatcd anti-digoxigenin antibody (Boehπnger Mannheim, Indianapolis, IN) for 30 min After washing w ith Buffer 1 (twice) and soaking in Buffer 3 ( 100 mM Tπs-HCl, pH 9 5, 100 mM NaCl. 50 mM MgCl, for 2 mm. the membranes are exposed to the chemiluminescent substrate Lumi-Phos™ 530 (Boehπnger Mannheim, Indianapolis. IN) and the reaction is developed on X-ray film
More specifically, DNA ( 15 μg) from HSV-2, AuV351 or AuV351 (R) is digested with BamHI. separated on 1 % agarose gels and transfened to nylon membranes. It is hybndized with the AU26 probe which recognizes a sequence within the ICP 10 RR coding region A hybridizing 7 6kb band which represents the BamHI E fragment is observed for HSV-2. and AuV351(R) DNA The hybπdizing band seen for AuV351 DNA is 6.9kb.
Example 2 Constniction and Characterization of the AuV375 virus and AnV^7 (R) The strategy for construction of AuV375 is to create a recombinant plasmid, pAuΔ375 that contains a gene cassette deleted in ICP 10 amino acids 107-375 (Fig. 1 ) This plasmid is used for the generation of a recombinant HSV-2 irus deleted in ICP 10 amino acids 107-375 through recombination w ith the appropπate iral DNΛ All details of cloning methodology are based on standard procedures Plasmid pJL2 (Luo '92) is digested w ith EcoNI (partial) and BamHI to remove the
804bp fragment that encodes ammo acids 107-3"5 After both sites arc made blunt-ended, the resulting construct is collapsed through ligation at the BamHI EcoNI sites to generate 99/36087
plasmid pΔ375 The wild type sequences in a plasmid (TP 101 ) that contains the HSV-2 BamHI E and T fragments (Peng 96) are replaced with the 2 3kb Sall/Bglll fragment from pΔ375 The resulting plasmid, pAuΔ375, contains sequences which code for ICP10 deleted in amino acids 107-375 flanked by 4 and 2 8kb of HSV-2 DNA sequences at the 5' and 3' ends, respectively The 10 3kb Hindlll EcoRI fragment from pAuΔ375 is introduced by marker transfer into a virus (ICPIOΔRR) in which the RR domain of ICP 10 has been replaced with the LacZ gene The resulting recombinant. designated AuV375, is obtained by selecting white plaques on a background of blue plaques after staining with X-gal. A few white plaques are picked, puπfied, and grown in Vero cells in MEM with 10% FCS For the construction of the restored virus AuV375(R), Vero cells are co-transfected with lμg of infectious viral DNA from AuV375 and a 10-fold molar excess of the wild type BamHI E/T fragment A strategy similar to that reported for ICP6Δ (Goldstein and Weller, Virology, Vol 166, pp 41-51 , 1988) is used to select restored virus under growth restπcted conditions (1 % FCS) Southern blot hybridization is used to confirm that the AuV375 DNA is deleted in nucleotides encoding ICP 10 amino acids 107-375 as descnbed in Example 1
More specifically, DNA ( 15 μg) from HSV-2, AuV375 or AuV375(R) is digested with BamHI, separated on 1%, agarose gels and transferred to nylon membranes It is hybndized with the AU26 probe which recognizes a sequence within the ICP 10 RR coding region A hybndizing 7 6kb band which represents the BamHI E fragment is observed for
HSV-2. and AuV375(R) DNA The hybπdizing band seen for AuV375 DNA is 2 2kb.
Example 3 Construction and Characteπzation of the AuV417 virus and AιιV417(R^ The strategy for construction of AuV417 is to create a recombinant plasmid, pAuΔ417 that contains a gene casette deleted in ICP 10 amino acids 107-417 (Fig 3) This plasmid is used for the generation of a recombinant HSV-2 virus deleted in ICP 10 amino acids 107-417 through recombination w ith the appropπate v iral DNA All details of cloning methodology are based on standard procedures
Plasmid pJL2 (Luo '92) is digested w ith BamHI (made blunt-ended) and Hpal to remov e the 936bp fragment that encodes amino acids 107-417 The resulting construct is collapsed through ligation at the BamHI EcoM sites to generate plasmid pΔ41 7 The wild type sequences in a plasmid (TP101 ) that contains the HSV-2 BamHI E and T fragments 99/36087
(Peng 96) are replaced with the 2 lkb Sall/Bglll fragment from pΔ417 The resulting plasmid. pAuΔ417. contains sequences which code for ICP 10 deleted in amino acids 107-417 flanked by 4 and 2.8kb of HSV-2 DNA sequences at the 5' and 3' ends, respectively. The 10. lkb Hindlll/EcoRI fragment from pAuΔ417 is introduced by marker transfer into a virus (ICPIOΔRR) in which the RR domain of ICP 10 has been replaced with the LacZ gene. The resulting recombinant designated AuV417, is obtained by selecting white plaques on a background of blue plaques after staining with X-gal A few white plaques are picked, puπfied, and grown in Vero cells in MEM with 10% FCS For the construction of the restored virus AuV417(R), Vero cells are co-transfected with lμg of infectious viral DNA from AuV417 and a 10-fold molar excess of the wild type BamHI E/T fragment. A strategy similar to that reported for ICP6Δ (Goldstein and Weller, Virology, Vol. 166, pp 41-51, 1988) is used to select restored virus under growth restncted conditions ( 1% FCS).
Southern blot hybπdization is used to confirm that the AuV417 DNA is deleted in nucleotides encoding ICP 10 amino acids 107-417 as described in Example 1. More specifically, DNA (15 μg) from HSV-2, AuV417 or AuV417(R) is digested with BamHI, separated on 1 % agarose gels and transfened to nylon membranes. It is hybndized with the AU26 probe which recognizes a sequence within the ICP 10 RR coding region A hybπdizing 7 6kb band which represents the BamHI E fragment is observed for HSV-2, and AuV417(R) DNA The hybridizing band seen for AuV417 DNA is 6.6kb. Example 4
AuV351. AuV375 and uV417 have hiaher RR activity than ICPI OΔPK ICPIOΔPK virus has RR activity, but it is lower than that of HSV-2 Inasmuch as amino acids 413-438 may be involved in the complexation of the two RR subunits (Table 1), viruses that retain these amino acids have an RR activity similar to that of HSV-2 (Table 2).
99/36087
Table 2. Ribonucleotide reductase activity of ICPIOΔPK virus.
Virus RR Specific activity (Units)3
HSV-2 10.2
ICPIOΔPK 8.0
AuV351 10.2 AuV375 10.2
AuV417 10.2
Mock-infected 2.7
a One RR unit = conversion of 1 nmol CDP to dCDP/h/mg protein. Data are expenmentally determined for HSV-2, ICPIOΔPK and Mock-infected. They are projected for AuV351 , AuV375 and AuV417
Example 5 AuV351 . AuV.75 and AuV417 are attenuated for growth in infected nmmals The mouse footpad model of HSV-2 infection is used to examine the growth of
AuV351. AuV375 and AuV417 in vivo. Seven groups of Swiss Webster mice are inoculated s.c. in the footpad with lxl07 pfu of HSV-2, AuV351 , AuV375, AuV417 or the restored viruses AuV351(R), AuV375(R) and AuV417(R). Neurological symptoms and severe skin lesions are seen in mice given HSV-2 or the restored viruses beginning on day 6 p.i. (Wachsman, M Luo, J H., Aurelian, L., Perkus, M E., and Paoletti, E. 3intιgen-presentιng capacity of epidermal cells infected u ith vaccinia virus recombinants containing the herpes simplex virus glycoprotein D and protective immunitx Journal of General Virology, Vol. 70, pp.2513-2520, 1989 [Wachsman '89]; (Wachsman, M Luo, J H., Aurelian, L., and Paoletti, E. Protection from herpes simplex virus npe 2 is associated with T cells involved in delayed type hvpersensitivitx that recognize gl\ cos\ lation-related epitopes on gly coprotein D
Vaccine Vol 10, pp 447-454. 1992) [Wachsman '92] Mice infected w ith AuV351 , AuV375 or AuV417 hav e no neurological svmptoms nor skin lesions HSV-2 and the restored viruses 9/36087
are isolated from the footpad and ganghonic homogenates for 7-9 days AuV351, AuV375 and AuV417 are isolated for only 3-4 days Maximum titers and the proportion of latently infected ganglia are lower than those seen for HSV-2 (1-3x10' pfu and 80-90%> latency).
Example 6 AnVlSI . AuV375 and AuV417 protect from HSV-2 challenge
The footpad model described in example 5 is used to examine protection by AuV351, AuV375 and AuV417 The expeπment is done as previously descnbed (Wachsman '89; Wachsman '92) with mice given one or multiple immunizations with IxlO7 pfu of virus (at 14-16 days intervals) before challenge with wild type HSV-2 Challenge is with IxlO7 to IxlO8 pfu of HSV-2 and it is done at 3-6 weeks after the last immunization All mice in the
PBS group develop skin lesions from which virus is isolated, and 50-80% die on days 8-13 after challenge Bv contrast lesions are not seen and virus is not isolated from the immunized mice The AuV351 , AuV375 and AuV417 v noises have vaccine potential.
Example 7 AuV351 , AuV375 and AuV417 viruses induce HSV-specific immunity.
Groups of mice are immunized with AuV351, AuV375 and AuV417 as descnbed in example 6 Two-four weeks after the last injection spleens are removed and T cells are used in lymphocyte proliferation assays as descnbed (Wachsman et al Journal of General Virology, Vol 70, pp 2513-2520, 1989, Vaccine, Vol 10, pp 447-454, 1992) HSV-specific lymphoproliferation is seen in all animals Prohferative levels are similar to those seen for
HSV-2 AuV351. AuV375 and AuV417 induce good levels of virus-specific T cell responses They also induce antibody responses as determined by neutralization assays All references cited herein are incoφorated by reference in their entirety It will be apparent to those skilled in the art that the examples and embodiments descnbed herein are bv wav of illustration and not of limitation, and that other examples may be utilized w ithout departing from the spiπt and scope of the present invention, as set forth in the appended claims

Claims

99/36087CLAIMS We claim:
1. A vaccine composition comprising a live Heφes Simplex Virus Type-2 recombinant virus having a genome with a deletion in the gene coding for the protein kinase domain of the large subunit of ribonucleotide reductase and a pharmaceutically acceptable carrier or diluent.
2. The vaccine composition of claim 1 wherein the live Heφes Simplex Virus-2 recombinant virus is selected from the group consisting of AuV 351, AuV375 and AuV417.
3. A method of immunizing a subject against Heφes Simplex virus comprising the step administering the vaccine composition of claim 1 to said subject.
4. The method of claim 3 wherein said subject is a human.
5. The method of claim 3 wherein the dosage range for said vaccine composition is 1 pfu to 100 million pfu.
6. The method of claim 3 wherein said vaccine composition is administered via an intranasal, oral, intravaginal, subcutaneous or intradermal route.
7. A method of conferring immunity against Heφes Simplex virus to a subject comprising the step administering the vaccine composition of claim 1.
8. The method of claim 7 wherein said subject is a human.
9. The method of claim 7 wherein the dosage range for said vaccine composition is 1 pfu to 100 million pfu.
10. The method of claim 7 wherein said vaccine composition is administered via an intranasal, oral, intravaginal, subcutaneous or intradermal route.
1 1. A method of preventing clinical symptoms in a subject associated with Heφes Simplex
Virus in a subject comprising the step administering the vaccine composition of claim 1 to said subject.
12. The method of claim 1 1 wherein said subject is a human.
13. The method of claim 1 1 wherein the dosage range for said vaccine composition is 1 pfu to 100 million pfu.
14. The method of claim 1 1 wherein said vaccine composition is administered via an intranasal. oral, intravaginal, subcutaneous or intradermal route.
PCT/US1999/000922 1998-01-20 1999-01-15 Novel vaccine compositions for herpes simplex virus WO1999036087A1 (en)

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MILLIGAN G N, BERNSTEIN D I: "GENERATION OF HUMORAL IMMUNE RESPONSES AGAINST HERPES SIMPLEX VIRUSTYPE 2 IN THE MURINE FEMALE GENITAL TRACT", VIROLOGY, ELSEVIER, AMSTERDAM, NL, vol. 206, 1 January 1995 (1995-01-01), AMSTERDAM, NL, pages 234 - 241, XP002917744, ISSN: 0042-6822, DOI: 10.1016/S0042-6822(95)80038-7 *
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EP1370283A2 (en) * 2000-11-16 2003-12-17 University of Maryland, Baltimore Prevention of recurrent viral disease
EP1370283A4 (en) * 2000-11-16 2004-12-15 Univ Maryland PREVENTING RECURRING VIRUS DISEASES
EP3139745A4 (en) * 2014-05-09 2017-11-08 Board of Supervisors of Louisiana State University And Agricultural and Mechanical College Vaccines against genital herpes simplex infections
US10130703B2 (en) 2014-05-09 2018-11-20 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Vaccines against genital herpes simplex infections
US10596253B2 (en) 2014-05-09 2020-03-24 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Vaccines against genital herpes simplex infections
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