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WO1996003510A1 - Vaccin polynucleotidique contre le virus de l'herpes - Google Patents

Vaccin polynucleotidique contre le virus de l'herpes Download PDF

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Publication number
WO1996003510A1
WO1996003510A1 PCT/US1995/009057 US9509057W WO9603510A1 WO 1996003510 A1 WO1996003510 A1 WO 1996003510A1 US 9509057 W US9509057 W US 9509057W WO 9603510 A1 WO9603510 A1 WO 9603510A1
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WO
WIPO (PCT)
Prior art keywords
hsv
dna
polynucleotide
gene
genes
Prior art date
Application number
PCT/US1995/009057
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English (en)
Inventor
Marcy E. Armstrong
Robert D. Keys
John A. Lewis
Margaret A. Liu
William L. Mcclements
Original Assignee
Merck & Co., Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Merck & Co., Inc. filed Critical Merck & Co., Inc.
Priority to EP95926736A priority Critical patent/EP0772680A1/fr
Priority to JP8505831A priority patent/JPH10503649A/ja
Priority to AU31012/95A priority patent/AU708460B2/en
Publication of WO1996003510A1 publication Critical patent/WO1996003510A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • 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
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • 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
    • C12N2710/16622—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • CTLs cytotoxic T-lymphocytes
  • CTLs kill virally-infected cells when their T cell receptors recognize viral peptides associated with MHC class I and or class II molecules. These peptides can be derived from endogenously synthesized viral proteins, regardless of the protein's location or function within the virus. By recognition of epitopes from conserved viral proteins, CTLs may provide heterologous protection.
  • Many infectious disease causing agents can, by themselves, elicit protective antibodies which can bind to and kill, render harmless, or cause to be killed or rendered harmless, the disease causing agent and its byproducts. Recuperation from these diseases usually results in long- lasting immunity by virtue of protective antibodies generated against the highly antigenic components of the infectious agent.
  • Protective antibodies are part of the natural defense mechanism of humans and many other animals, and are found in the blood as well as in other tissues and bodily fluids. It is the primary function of most vaccines to elicit protective antibodies against infectious agents and/or their byproducts, without causing disease.
  • Retroviral vectors have restrictions on the size and structure of polypeptides that can be expressed as fusion proteins while maintaining the ability of the recombinant virus to replicate [A.D. Miller, Curr. Top. Microbiol. Immunol. 158, 1 (1992)], and the effectiveness of vectors such as vaccinia for subsequent immunizations may be compromised by immune responses against vaccinia [E.L. Cooney et al, Lancet 337, 567 (1991)]. Also, viral vectors and modified pathogens have inherent risks that may hinder their use in humans [R.R. Redfield et al, New Engl. J. Med. 316, 673 (1987); L. Mascola et al, Arch. Intern. Med.
  • peptide epitopes to be presented is dependent upon the structure of an individual's MHC antigens and, therefore, peptide vaccines may have limited effectiveness due to the diversity of MHC haplotypes in outbred populations.
  • MHC major histocompatibility complex
  • WO 93/17706 describes a method for vaccinating an animal against a virus, wherein carrier particles were coated with a gene construct and the coated particles are accelerated into cells of an animal.
  • HSV herpes simplex virus
  • Virol., 74 * pp.2 13-2817 have successfully protected animals from HSV challenge.
  • Vaccination by infection with recombinant adenovirus expressing HSV gB elicits a protective immune response in mice.
  • anti-gD antibodies can protect against HSV infection whether elicited by immunization with native protein (Long, D. et al., 1984, Infect.Immun., 43, pp.761 -764) recombinantly expressed protein (Burke, R.L., supra; Stanberry, L.R.
  • HSV-2 protein-coding DNA sequences were cloned into the eukaryotic expression vector. This DNA construction elicits an immune response when injected into animals. Immunized animals were infected with HSV to evaluate whether or not direct DNA immunization with the gD gene (or other HSV-2 genes) could protect them from disease. Nucleic acids, including DNA constructs and RNA transcripts, capable of inducing in vivo expression of human herpes simplex virus (HSV) proteins upon direct introduction into animal tissues via injection or otherwise are therefore disclosed.
  • HSV herpes simplex virus
  • nucleic acids may elicit immune responses which result in the production of cytotoxic T lymphocytes (CTLs) specific for HSV antigens, as well as the generation of HSV-specific antibodies, which are protective upon subsequent HSV challenge.
  • CTLs cytotoxic T lymphocytes
  • HSV-specific antibodies which are protective upon subsequent HSV challenge.
  • These nucleic acids are useful as vaccines for inducing immunity to HSV, which can prevent infection and/or ameliorate HSV- related disease.
  • ELISA generated group GMT data is shown for HSV PNV- immunized animals receiving a single injection of vaccine; sera were obtained at 4, 7 and 10 weeks post-immunization.
  • Fig. 4 Survival of HSV-2 challenged animals following two injections with VU;gD at 200ug; lOOug; 50 ug; 25ug; 12.5ug; 6.25ug; 3.13ug; 1.56 ug; 0.78 ug; or saline only. Since all animals in the 200ug; lOOug; 25ug; 12.5ug;
  • Fig. 8 The results of survival, mean days to death, paralysis, and vaginal virus titers in HSV-2 infected guinea pigs is shown.
  • a polynucleotide is a nucleic acid which contains essential regulatory elements such that upon introduction into a living vertebrate cell, is able to direct the cellular machinery to produce translation products encoded by the genes comprising the polynucleotide.
  • the polynucleotide is a polydeoxyribonucleic acid comprising HSV genes operatively linked to a transcriptional promoter.
  • the polynucleotide vaccine comprises polyribonucleic acid encoding HSV genes which are amenable to translation by the eukaryotic cellular machinery (ribosomes, tRNAs, and other translation factors).
  • the protein encoded by the polynucleotide is one which does not normally occur in that animal except in pathological conditions, (i.e. an heterologous protein) such as proteins associated with HSV
  • the animals' immune system is activated to launch a protective immune response. Because these exogenous proteins are produced by the animals' own tissues, the expressed proteins are processed by the major histocompatibility system (MHC) in a fashion analogous to when an actual HSV infection occurs.
  • MHC major histocompatibility system
  • the result is induction of immune responses against HSV.
  • Polynucleotides for the purpose of generating immune responses to an encoded protein are referred to herein as polynucleotide vaccines or PNV.
  • the instant invention provides a method for using a polynucleotide which, upon introduction into mammalian tissue, induces the expression, in vivo, of the polynucleotide thereby producing the encoded protein. It is readily apparent to those skilled in the art that variations or derivatives of the nucleotide sequence encoding a protein can be produced which alter the amino acid sequence of the encoded protein. The altered expressed protein may have an altered amino acid sequence, yet still elicits antibodies which react with the viral protein, and are considered functional equivalents. In addition, fragments of the full length genes which encode portions of the full length protein may also be constructed. These fragments may encode a protein or peptide which elicits antibodies which react with the viral protein, and are considered functional equivalents.
  • a gene encoding an HSV gene product is incorporated in an expression vector.
  • the vector contains a transeriptional promoter recognized by eukaryotic RNA polymerase, and a transeriptional terminator at the end of the HSV gene coding sequence.
  • the promoter is the cytomegalovirus promoter with the intron A sequence (CMV-intA), although those skilled in the art will recognize that any of a number of other known promoters such as the strong immunoglobulin, or other eukaryotic gene promoters may be used.
  • CMV-intA cytomegalovirus promoter with the intron A sequence
  • an antibiotic resistance marker is also optionally included in the expression vector under transeriptional control of a suitable prokaryotic promoter. Ampicillin resistance genes, neomycin resistance genes or any other suitable antibiotic resistance marker may be used. In a preferred embodiment of this invention, the antibiotic resistance gene encodes a gene product for neomycin resistance. Further, to aid in the high level production of the polynucleotide by growth in prokaryotic organisms, it is advantageous for the vector to contain a prokaryotic origin of replication and be of high copy number.
  • any of a number of commercially available prokaryotic cloning vectors provide these elements.
  • these functionalities are provided by the commercially available vectors known as the pUC series. It may be desirable, however, to remove non-essential DNA sequences. Thus, the lacZ and lad coding sequences of pUC may be removed. It is also desirable that the vectors are not able to replicate in eukaryotic cells. This minimizes the risk of integration of polynucleotide vaccine sequences into the recipients' genome.
  • the expression vector pnRSV is used, wherein the rous sarcoma virus (RSV) long terminal repeat (LTR) is used as the promoter.
  • RSV rous sarcoma virus
  • LTR long terminal repeat
  • VI a mutated pBR322 vector into which the CMV promoter and the BGH transeriptional terminator were cloned is used.
  • the elements of V 1 and pUC19 have been been combined to produce an expression vector named V1J.
  • VI J or another desirable expression vector is cloned an HSV gene, such as gD, or any other HSV gene which can induce anti-HSV immune responses (antibody and/or CTLs) such as gB, gC, gL, gH and ICP27.
  • the ampicillin resistance gene is removed from VI J and replaced with a neomycin resistance gene, to generate VU-neo, into which any of a number of different HSV genes may be cloned for use according to this invention.
  • the vector is VUns, which is the same as VUneo except that a unique Sfil restriction site has been engineered into the single Kpnl site at position 21 14 of VU-neo.
  • this vector allows careful monitoring for expression vector integration into host DNA, simply by Sfil digestion of extracted genomic DNA.
  • the vector is V1R.
  • this vector as much non-essential DNA as possible is "trimmed" to produce a highly compact vector.
  • This vector allows larger inserts to be used, with less concern that undesirable sequences are encoded and optimizes uptake by cells when the construct encoding specific virus genes is introduced into surrounding tissue.
  • the methods used in producing the foregoing vector modifications and development procedures may be accomplished according to methods known by those skilled in the art.
  • one of the utilities of the instant invention is to provide a system for in vivo as well as in vitro testing and analysis so that a correlation of HSV sequence diversity with serology of HSV neutralization, as well as other parameters can be made.
  • the isolation and cloning of these various genes may be accomplished according to methods known to those skilled in the art.
  • This invention further provides a method for systematic identification of HSV strains and sequences for vaccine production. Incorporation of genes from primary isolates of HSV strains provides an immunogen which induces immune responses against clinical isolates of the virus and thus meets a need as yet unmet in the field. Furthermore, if the virulent isolates change, the immunogen may be modified to reflect new sequences as necessary.
  • a gene encoding an HSV protein is directly linked to a transeriptional promoter.
  • tissue-specific promoters or enhancers for example the muscle creatine kinase (MCK) enhancer element may be desirable to limit expression of the polynucleotide to a particular tissue type.
  • myocytes are terminally differentiated cells which do not divide. Integration of foreign DNA into chromosomes appears to require both cell division and protein synthesis. Thus, limiting protein expression to non-dividing cells such as myocytes may be preferable.
  • use of the CMV promoter is adequate for achieving expression in many tissues into which the PNV is introduced.
  • HSV and other genes are preferably ligated into an expression vector which has been specifically optimized for polynucleotide vaccinations.
  • Elements include a transeriptional promoter, immunogenic epitopes, and additional cistrons encoding immunoenhancing or immunomodulatory genes, with their own promoters, transeriptional terminator, bacterial origin of replication and antibiotic resistance gene, as described herein.
  • the vector may contain internal ribosome entry sites (IRES) for the expression of polycistronic mRNA.
  • IRS internal ribosome entry sites
  • RN A which has been transcribed in vitro to produce multi-cistronic mRNAs encoded by the DNA counterparts is within the scope of this invention.
  • it is desirable to use as the transeriptional promoter such powerful RNA polymerase promoters as the T7 or SP6 promoters, and performing run-on transcription with a linearized DNA template.
  • the protective efficacy of polynucleotide HSV immunogens against subsequent viral challenge is demonstrated by immunization with the DNA of this invention. This is advantageous since no infectious agent is involved, no assembly of virus particles is required, and determinant selection is permitted. Furthermore, because the sequence of viral gene products may be conserved among various strains of HSV, protection against subsequent challenge by another strain of HSV is obtained.
  • the injection of a DNA expression vector encoding gD may result in the generation of significant protective immunity against subsequent viral challenge. In particular, gD-specific antibodies and CTLs may be produced. Immune responses directed against conserved proteins can be effective despite the antigenic shift and drift of the variable proteins.
  • HSV gD PNV constructs may give rise to cross reactive immune responses.
  • the invention offers a means to induce heterologous protective immunity without the need for self-replicating agents or adjuvants.
  • the generation of high titer antibodies against expressed proteins after injection of viral protein and human growth hormone DNA, [Tang et al., Nature 356, 152, 1992] indicates this is a facile and highly effective means of making antibody-based vaccines, either separately or in combination with cytotoxic T-lymphocyte vaccines targeted towards conserved antigens.
  • the amount of expressible DNA or transcribed RNA to be introduced into a vaccine recipient will depend on the strength of the transeriptional and translational promoters used.
  • the magnitude of the immune response may depend on the level of protein expression and on the immunogenicity of the expressed gene product.
  • an effective dose of about 1 ng to 5 mg, and preferably about 10 ⁇ g to 300 ⁇ g is administered directly into muscle tissue.
  • Subcutaneous injection, intradermal introduction, impression through the skin, and other modes of administration such as intraperitoneal, intravenous, or inhalation delivery are also suitable. It is also contemplated that booster vaccinations may be provided.
  • HSV protein immunogens such as the gD, gB, gC, gG, and gH gene products is also contemplated.
  • Parenteral administration such as intravenous, intramuscular, subcutaneous or other means of administration of interleukin- 12 protein, concurrently with or subsequent to parenteral introduction of the PNV of this invention may be advantageous.
  • the polynucleotide may be naked, that is, unassociated with any proteins, adjuvants or other agents which affect the recipients' immune system.
  • the polycucleotide may be in a physiologically acceptable solution, such as, but not limited to, sterile saline or sterile buffered saline.
  • the DNA may be associated with liposomes, such as lecithin liposomes or other liposomes known in the art, as a DNA-liposome mixture, or the DNA may be associated with an adjuvant known in the art to boost immune responses, such as a protein or other carrier.
  • Agents which assist in the cellular uptake of DNA such as, but not limited to, calcium ions, may also be used. These agents are generally referred to herein as transfection facilitating reagents and pharmaceutically acceptable carriers.
  • Techniques for coating microprojectiles coated with polynucleotide are known in the art and are also useful in connection with this invention. For DNA intended for human use it may be useful to have the final DNA product in a pharmaceutically acceptable carrier or buffer solution.
  • the invention is a polynucleotide which comprises contiguous nucleic acid sequences capable of being expressed to produce a gene product upon introduction of said polynucleotide into eukaryotic tissues in vivo.
  • the encoded gene product preferably either acts as an immunostimulant or as an antigen capable of generating an immune response.
  • the nucleic acid sequences in this embodiment encode a human herpes simplex virus immunogenic epitope, and optionally a cytokine or a T-cell costimulatory element, such as a member of the B7 family of proteins.
  • the first is the relative simplicity with which native or nearly native antigen can be presented to the immune system. Mammalian proteins expressed recombinantly in bacteria, yeast, or even mammalian cells often require extensive treatment to insure appropriate antigenicity.
  • a second advantage of DNA immunization is the potential for the immunogen to enter the MHC class I pathway and evoke a cytotoxic T cell response. Immunization of mice with DNA encoding the influenza A nucleoprotein (NP) elicited a CD8+ response to NP that protected mice against challenge with heterologous strains of flu. (Montgomery, D.L. et al, supra; Ulmer, J.
  • CD8+ clones including one specific for gD, have been isolated.
  • mice protects mice from HSV infection.
  • Live virus vectors like DNA, have the potential for MHC class I presentation of the immunogen.
  • HSV gD-vaccinia recombinant to immunize mice found that protection from challenge was dependent on the delayed type hypersensitivity functions of L3T4+ cells.
  • DNA immunization can evoke both humoral and cell- mediated immune responses, its greatest advantage may be that it provides a relatively simple method to survey a large number of viral genes for their vaccine potential. Plasmids expressing HSV-2 glycoproteins B and C also elicit neutralizing antibodies and protect mice from lethal challenge.
  • ICP27 which is known to generate a CTL response and to provide some protection in mice immunized by infection with ICP27 -vaccinia recombinant virus (Banks, T.A. et al., 1991 , J.Virol., 65., pp.3185-3191) did not provide protection from lethal HSV challenge when mice were vaccinated with PNV ICP27 alone.
  • ICP27 -encoding DNA may be useful as one component of a multi-HSV gene-containing PNV, and it is contemplated that the present invention includes ICP27 as a component of a multivalent HSV PNV.
  • Immunization by DNA injection also allows, as discussed above, the ready assembly of multicomponent subunit vaccines. Simultaneous immunization with multiple influenza genes has recently been reported. (Donnelly, J. et ui-, 1994, Vaccines, in press). The inclusion in an HSV vaccine of genes whose products activate different arms of the immune system may also provide thorough protection from subsequent virus challenge.
  • the expression vector VI was constructed from pCMVIE- AKI-DHFR [Y. Whang et al, J. Virol 61, 1796 (1987)].
  • the AKI and DHFR genes were removed by cutting the vector with EcoR I and self- ligating. This vector does not contain intron A in the CMV promoter, so it was added as a PCR fragment that had a deleted internal Sac I site [at 1 55 as numbered in B.S. Chapman et al, Nuc. Acids Res. 19, 3979 (1991)].
  • the template used for the PCR reactions was pCMVintA-Lux, made by ligating the Hind III and Nhe I fragment from pCMV6al20 [see B.S.
  • the primers that spanned intron A are:
  • the primers used to remove the Sac I site are: sense primer, SEQ ID:3:
  • V IJ The purpose in creating V IJ was to remove the promoter and transcription termination elements from vector V 1 in order to place them within a more defined context, create a more compact vector, and to improve plasmid purification yields.
  • VIJ is derived from vectors VI and pUC18, a commercially available plasmid.
  • VI was digested with Sspl and EcoRI restriction enzymes producing two fragments of DNA. The smaller of these fragments, containing the CMVintA promoter and Bovine Growth Hormone (BGH) transcription termination elements which control the expression of heterologous genes, was purified from an agarose electrophoresis gel. The ends of this DNA fragment were then "blunted” using the T4 DNA polymerase enzyme in order to facilitate its ligation to another "blunt-ended" DNA fragment.
  • pUC18 was chosen to provide the "backbone" of the expression vector.
  • the ampr gene from the pUC backbone of VI J was removed by digestion with Sspl and Eaml 1051 restriction enzymes.
  • the remaining plasmid was purified by agarose gel electrophoresis, blunt-ended with T4 DNA polymerase, and then treated with calf intestinal alkaline phosphatase.
  • VUneo #'s 1 and 3 Each of these plasmids was confirmed by restriction enzyme digestion analysis, DNA sequencing of the junction regions, and was shown to produce similar quantities of plasmid as VI J. Expression of heterologous gene products was also comparable to VIJ for these VUneo vectors.
  • VUneo An Sfi I site was added to VUneo to facilitate integration studies.
  • a commercially available 13 base pair Sfi I linker (New England BioLabs) was added at the Kpn I site within the BGH sequence of the vector.
  • VUneo was linearized with Kpn I, gel purified, blunted by T4 DNA polymerase, and ligated to the blunt Sfi I linker. Clonal isolates were chosen by restriction mapping and verified by sequencing through the linker.
  • the new vector was designated VlJns. Expression of heterologous genes in VlJns (with Sfi I) was comparable to expression of the same genes in VUneo (with Kpn I).
  • V Uns-tPA 13 base pair Sfi I linker
  • VlJns was modified to include the human tissue-specific plasminogen activator (tPA) leader.
  • tPA tissue-specific plasminogen activator
  • Two synthetic complementary oligomers were annealed and then ligated into VUn which had been Bgi ⁇ digested.
  • the sense and antisense oligomers were 5 -GATC ACC ATG GAT GCA ATG AAG AGA GGG CTC TGC TGT GTG CTG CTG CTG TGT GGA GCA GTC TTC GTT TCG CCC AGC GA-3', SEQ.
  • an Sfil restriction site was placed at the Kpnl site within the BGH terminator region of VUn-tPA by blunting the Kpnl site with T4 DNA polymerase followed by ligation with an Sfil linker (catalogue #1 138, New England Biolabs). This modification was verified by restriction digestion and agarose gel electrophoresis.
  • X any antigenic gene
  • the murine B7 gene was PCR amplified from the B lymphoma cell line CHI (obtained from the ATCC).
  • B7 is a member of a family of proteins which provide essential costimulation T cell activation by antigen in the context of major histocompatibility complexes I and II.
  • CHI cells provide a good source of B7 mRNA because they have the phenotype of being constitutively activated and B7 is expressed primarily by activated antigen presenting cells such as B cells and macrophages. These cells were further stimulated in vitro using cAMP or IL-4 and mRNA prepared using standard guanidinium thiocyanate procedures. cDNA synthesis was performed using this mRNA using the GeneAmp RNA PCR kit (Perkin -Elmer Cetus) and a priming oligomer (5'-GTA CCT CAT GAG CCA CAT AAT ACC ATG- 3', SEQ. ID:7:) specific for B7 located downstream of the B7 translational open reading frame.
  • B7 was amplified by PCR using the following sense and antisense PCR oligomers: 5'-GGT ACA AGA TCT ACC ATG GCT TGC AAT TGT CAG TTG ATG C-3', SEQ. ID:8:, and 5 -CCA CAT AGA TCT CCA TGG GAA CTA AAG GAA GAC GGT CTG TTC-3', SEQ. 1D:9:, respectively.
  • These oligomers provide BglD restriction enzyme sites at the ends of the insert as well as a Kozak translation initiation sequence containing an Ncol restriction site and an additional Ncol site located immediately prior to the 3'-terminal Bglll site.
  • pGEM-3-IRES-B7 contains an IRES-B7 cassette which can easily be transferred to VUns-X, where X represents an antigen-encoding gene.
  • This vector contains a cassette analogous to that described in item C above except that the gene for the immunostimulatory cytokine, GM-CSF, is used rather than B7.
  • GM-CSF is a macrophage differentiation and stimulation cytokine which has been shown to elicit potent anti-tumor T cell activities in vivo [G.
  • This vector contains a cassette analogous to that described in item C above except that the gene for the immunostimulatory cytokine, IL-12, is used rather than B7.
  • IL-12 has been demonstrated to have an influential role in shifting immune responses towards cellular, T cell-dominated pathways as opposed to humoral responses [L. Alfonso et al, Science, 263, 235, 1994].
  • V1R a derivative of VlJns, designated V1R.
  • the purpose for this vector construction was to obtain a minimum-sized vaccine vector without unneeded DNA sequences, which still retained the overall optimized heterologous gene expression characteristics and high plasmid yields that VIJ and VlJns afford. It was determined from the literature as well as by experiment that (1) regions within the pUC backbone comprising the E.
  • V 1R was constructed by using PCR to synthesize three segments of DNA from VlJns representing the CMVintA promoter/BGH terminator, origin of replication, and kanamycin resistance elements, respectively.
  • Restriction enzymes unique for each segment were added to each segment end using the PCR oligomers: Sspl and Xhol for CMVintA/BGH; EcoRV and BamHI for the kan r gene; and, Bell and Sail for the ori i * . These enzyme sites were chosen because they allow directional ligation of each of the PCR-derived DNA segments with subsequent loss of each site: EcoRV and Sspl leave blunt-ended DNAs which are compatible for ligation while BamHI and Bell leave complementary overhangs as do Sail and Xhol. After obtaining these segments by PCR each segment was digested with the appropriate restriction enzymes indicated above and then ligated together in a single reaction mixture containing all three DNA segments.
  • the 5'-end of the ori r was designed to include the T2 rho independent terminator sequence that is normally found in this region so that it could provide termination information for the kanamycin resistance gene.
  • VERO, BHK-21 , and RD cells were obtained from the ATCC.
  • Virus was routinely prepared by infection of nearly confluent VERO or BHK cells with a multiplicity of infection (m.o.i.) of 0.1 at 37°C in a small volume of medium without fetal bovine serum (FBS). After one hour, virus inoculum was removed and cultures were re-fed with high glucose DMEM supplemented with 2% heat-inactivated FBS, 2mM L-glutamine, 25mM HEPES, 50 U/ml penicillin and 50 ⁇ g/ml streptomycin. Incubation was continued until cytopatic effect was extensive: usually 24 to 48 hours. Cell associated virus was collected by centrifugation at 1800 X g 10 minutes 4°C. Supernantant virus was clarified by centifugation at 640 X g for 10 minutes 4°C.
  • HSV-2 (Curtis) DNA for use as PCR template was prepared from nucleocapsids isolated from infected VERO cells. (Denniston, K.J. et ah, 1981 , Gene, 15., pp.365-378) Synthetic oligomers corresponding to 5' and 3' end flanking sequences for the HSV2 gB, gC, gD, or ICP27 genes, containing Bgl II restriction recognition sites (Midland Certified Reagent Company; Midland, Texas) were used at 20 pmoles each. A 1.
  • Ikb fragment encoding the gD gene was amplified by PCR (Perkin Elmer Cetus, La Jolla) according to the maufacturer's specifications except that a deaza dGTP:dGTP ratio of 1 :4 was used in place of dGTP and the buffer was supplemented to 3 mM Mg Cl2» HSV-2 genomic DNA template was used at 100 ng/100 ⁇ l reaction.
  • the PCR amplified fragments were restricted with Bgl II and ligated to the Bgl II digested, dephosphorylated vector V IJ (Montgomery, D.L. et ah, supra).
  • coli DH5 ⁇ (BRL-Gibco, Gaithersburg, Md.) was transformed according to the manufacturer's specifications. Ampicillin resistant colonies were screened by hybridization with the 2p labeled 3' PCR primer.
  • Candidate plasmids were characterized by restriction mapping and sequencing of the vector-insert junctions using the Sequenase DNA Sequencing Kit, version 2.0 (United States Biochemical). In a similar manner, a 2.7Kb fragment encoding the gB gene; a 1.5Kb fragment encoding the gC gene; and a 1.6Kb fragment encoding the ICP27 gene were also PCR amplified. Independently derived isolates were identified and characterized for the presence of the correct DNA construct containing either the gB, gC, gD, or ICP27 gene.
  • the plasmid constructions were characterized by restriction mapping and sequence analysis of the vector-insert junctions. Results were consistent with published HSV-2 strain G (Lasky, L.A. et ai., 1984, DNA, 3, pp.23-29) sequence data and showed that initiation and termination codons were intact for each construct.
  • Rhabdomyosarcoma cells (ATCC CCL136) were planted one day before use at a density of 1.2 XI 06 cells per 9.5 cm2 well in six- well tissue culture clusters in high glucose DMEM supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 25 mM
  • Phenol chloroform extracted cesium chloride purified plasmid DNA was precipitated with calcium phosphate using Pharmacia CellPhect reagents according to the kit instructions except that 5 - 15 ⁇ g is used for each 9.5 cm2 well of RD cells. Cultures were glycerol shocked six hours post addition of calcium phosphate-DNA precipate; after refeeding, cultures were incubated for two days prior to harvest.
  • Lysates of transfected cultures were prepared in IX RIPA (0.5% SDS, 1.0% TRITON X-100, 1 % sodium deoxycholate, ImM EDTA, 150mM NaCl, 25 mM TRIS-HC1 pH 7.4) supplemented with l ⁇ M leupeptin, l ⁇ M pepstatin, 300nM aprotinin, and lO ⁇ M TLCK, and sonicated briefly to reduce viscosity. Lysates were resolved by electrophoresis on 10% Tricine gels (Novex) and then transferred to nitrocellulose membranes. Immunoblots were processed with HSV-2 convalescent mouse sera and developed with the ECL detection kit (Amersham).
  • HSV gD from VU:gD was demonstrated by transient transfection of RD cells. Lysates of VU:gD-transfected or mock transfected cells were fractionated by SDS PAGE and analyzed by immunob lotting. Figure 1 A shows that VU:gD transfected RD cells express an immunoreactive protein with an apparent molecular weight of approximately 55 K. Lysates from HSV-2 (Curtis), HSV-2 (186), or mock-infected Vero cells are included for comparison. The identical migrations of cloned gD and the authentic protein from infected cells indicates that the protein is ful- length, and is processed and glycosylated similarly to that of gD in HSV-infected cells.
  • Indirect immunofluorescence of fixed VUNS:gB transfected cells showed a membrane-associated punctate signal.
  • Expression of HSV gC from VU:gC was demonstrated by transient transfection of RD cells.
  • Indirect immunofluorescence of fixed V U:gC transfected cells showed primarily a diffuse cytoplasmic signal.
  • ICP27 was demonstrated by transient transfection of RD cells, followed by Western blot analysis.
  • a mouse monoclonal antibody specific for ICP27 detected a protein of about 60 k Da, which is consistent with the major immunoreactive protein in HSV 2 infected cells ( Figure 1C).
  • mice Five- to six-week-old female BALB/c mice were anesthetized by intraperitoneal (i.p.) injection of a mixture of 5 mg ketamine HC1 (Aveco, Fort Dodge, IA) and 0.5 mg xylazine (Mobley Corp., Shawnee, KS.) in saline. The hind legs were shaved with electric clippers and washed with 70% ethanol. Animals were injected with a total of 100 ⁇ l of DNA suspended in saline: 50 ⁇ l each leg.
  • ketamine HC1 Aveco, Fort Dodge, IA
  • xylazine Mobley Corp., Shawnee, KS.
  • VU:gD DNA The ability of VU:gD DNA to elicit an immune response to HSV gD was first examined in a titration experiment. Groups of ten mice received i.m. injections of DNA in a dose range from 200 ⁇ g to 0.78 ⁇ g (8 two-fold dilutions) or were sham immunized with saline. Sera, obtained four and six weeks post immunization, were analyzed by ELISA. For the ELISA, HSV-2 glycoprotein was diluted to 5 ⁇ g/ml in 50 mM carbonate buffer pH 9.5. Nunc Maxi-sorb flat bottom 96-well plates were coated at 4°C, overnight with 100 ⁇ l per well of HSV glycoproteins.
  • the ELISA was developed with the addition of 100 ⁇ l per well of 1 mg/ml p-nitrophenylphosphate in 10% diethanolamine pH 9.8 lOO ⁇ g/ml MgCl «6 H2 ⁇ at 37°C. Absorbance was read at 405nm and serum dilutions were scored as positive if the OD405 was greater than the mean plus three standard deviations of the same dilution of the saline control sera. By four weeks the majority of animals receiving > 6.25 ⁇ g of DNA were seropositive. At doses lower than 6.25 ⁇ g, fewer animals had seroconverted, however even at the lowest dose some animals were ELISA positive. None of the saline injected control animals were positive. At six weeks a majority of the animals had become seropositive.
  • mice were re-immunized with the same doses of DNA (or saline) used in the initial injections.
  • Sera were obtained at ten weeks (three weeks after the second injection) and endpoint titers were determined by ELISA. The results are summarized in Table 1.
  • 93% of the DNA injected mice were seropositive. Even at the 0.78 ⁇ g dose, eight of the nine animals were positive.
  • FIG. 2A illustrates that sera from VU:gD immunized mice react specifically with a single HSV encoded protein with an electrophoretic mobility consistent with that of HSV gD.
  • This titration reveals a threshold of response of about 0.5 ⁇ g DNA. While a few animals receiving lower amounts of DNA were seropositive by ELISA, the positive response was transient and occurred only at the lowest serum dilution. At DNA doses of > 1.67 ⁇ g, more than 90% of animals seroconverted by four weeks and remained positive at seven and ten weeks.
  • FIG. 2B illustrates that sera from VUNS:gB immunized mice reacts specifically with a single HSV encoded protein with an electrophoretic mobility consistent with that of HSV gB.
  • HSV-1 or HSV- 2 stocks were diluted to 4,000 pfu/ml, 50 ⁇ l of virus were then added to each sample well and the plate was incubated overnight at 4°C.
  • Guinea pig complement (Cappel) was diluted 1 :4 in DMEM, 2% heat inactivated FBS and 50 ⁇ l were added to each sample well.
  • HSV Challenge Stocks of challenge vims were prepared by infection of confluent VERO monolayers with HSV-2 Curtis as described above. Clarified supematant vims was titered on VERO cells and aliquots were stored at -70°C. Animals were infected by i.p. injection with 0.25 ml of virus stock and then observed for three weeks. Survival data were analyzed using the log-rank test (McDermott et ai., 1989, Virology, 169. ⁇ p.244-247) in the SAS® procedure LIFETEST. Differences in probability ⁇ 0.001 were judged highly significant.
  • mice immunized with two doses of VU:gD were challenged by i.p. injection of 105.7 p.f.u. of HSV-2 (Curtis) and observed for 21 days. Survival data are in Figure 4. It is readily apparent that animals immunized with as little 0.78 ⁇ g of VU:gD were significantly protected from lethal infection. Of the three immunized animals that died, two were seronegative by ELISA at ten weeks. A few of the surviving animals did show signs of transient illness including failure to groom, failure to thrive, or a hunched posture. While the level of protection from death achieved at every dose of DNA was significant (p ⁇ 0.01 ), these symptoms suggest some break-through infection occurred.
  • Blood (0.6- 1 ml per animal) was obtained by toe clipping. The blood was collected in micro separation tubes (Becton Dickinson), and was later centrifuged at 1000 x g for 10 minutes to separate the serum.
  • virus medium about 5 x 106 plaque forming units of HSV-2 per ml
  • Lesion scores in infected animals were determined daily at day 2-15 post infection. A score of 1+ indicates about 25% of the anal- vaginal area was affected (usually by redness immediately around the vagina); 2+ indicates 50% of the anal-vaginal area affected; 3+ indicates 75% affected; and 4+ indicates 100% affected. Because some of the animals went on to die, the lesion score near the time of death carried through to the end of the 15 days. If this were not done, average lesion scores would appear to go down since the most affected animals died off. Deaths were recorded daily for 21 days. The mean day of death calculation took into account only guinea pigs that die. Numbers of animals with hind limb paralysis were noted throughout the infection.
  • Vaginal vims titers were made by titration of virus obtained from vaginal swabs at 2, 4 and 6 days after vims inoculation. The swabs were placed into tubes containing 1 ml of cell culture medium. The titration of these samples was conducted in Vero cells in 96-well plates. Calculation of virus titer was made by the 50% endpoint dilution method of Reed L. J. and Muench M., Am. J. Hyg. 27, 493-498 (1938). Vims titers were expressed as log 10 cell culture infectious doses per ml.
  • FIG. 8 shows the results of survival, mean days to death, paralysis, and vaginal vims titers in HSV-2 infected guinea pigs.
  • the high dose of vaccine prevented mortality and reduced vaginal vims titers on days 2 and 4 relative to the placebo control.
  • the high dose of vaccine significantly prevented paralysis in these animals.
  • the low dose of vaccine also reduced the above parameters.
  • Table 7 shows daily vaginal lesion scores for the experiment. Both the high and low doses of the vaccine caused significant reductions in vaginal lesion severity from days 3 through 15 of the infection compared to the placebo group. The results in Table 7 are presented graphically in Figure 9.
  • mice were vaccinated with 12.5 or 1.56 ⁇ g of VUNS:gD.
  • Vaginal fluid was collected by swab and the antibodies were eluted from the swab using phosphate buffered saline.
  • the eluant was analyzed for the presence of IgG and IgA, specific for HSV-2 protein.
  • the ELISA was performed as described above except that commercially available antibodies specific for mouse IgG (Boehringer) and specific for mouse IgA (Seralab) were used to detect the presence of HSV-specific IgG and IgA in the mouse vaginal samples.
  • the results for IgG are shown in Table 8; IgA was not detected in any animal.
  • MOLECULE TYPE DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 : CTATATAAGC AGAGCTCGTT TAG 23
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)

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Abstract

On a cloné des gènes codant les protéines du virus du type 2 de l'herpès (VHS-2) dans des vecteurs d'expression eucaryotiques, pour exprimer les protéines codées dans des cellules musculaires de mammifères in vivo. Des animaux ont été immunisés par l'injection dans leurs muscles de ces produits de recombinaison d'ADN, dénommés vaccins polynucléotidiques (VPN). Un titrage d'ADN a montré qu'une seule immunisation avec » 0,5 νg de (un) VPN aboutit à une séroconversion supérieure à 90 %, dix semaines après l'immunisation. Des immun-antisérums ont neutralisé aussi bien le VHS-2 que le VHS-1 en culture cellulaire. Lorsqu'on a administré un VHS-2 à des animaux, on a observé une protection significative (p < 0,001) contre une infection létale, après la vaccination avec le VPN.
PCT/US1995/009057 1994-07-22 1995-07-18 Vaccin polynucleotidique contre le virus de l'herpes WO1996003510A1 (fr)

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WO1998014210A1 (fr) * 1996-10-04 1998-04-09 The Regents Of The University Of California Methode de traitement d'une maladie pulmonaire allergique
WO1998014586A1 (fr) * 1996-10-01 1998-04-09 Merck & Co., Inc. Vaccins de polynucleotides contre le virus de l'herpes
WO1998017689A3 (fr) * 1996-10-18 1998-08-20 Genemedicine Inc Expression du gene il-12, systemes d'apport et utilisations
WO1998017814A3 (fr) * 1996-10-18 1998-08-27 Genemedicine Inc Expression de genes, systemes d'apport et utilisations
WO1999016892A1 (fr) * 1997-09-29 1999-04-08 University Of Bristol Vecteur a base d'herpes-virus 2 bovin (bhv-2) et utilisations de celui-ci
US6488936B1 (en) * 1998-02-12 2002-12-03 Wyeth Immunogenic composition of interleukin-12 (IL-12), alum, herpes simplex viral (HSV) antigen, and method thereof
US6867000B2 (en) * 2000-12-07 2005-03-15 Wyeth Holdings Corporation Method of enhancing immune responses to herpes
US7094767B2 (en) 1994-07-22 2006-08-22 Merck & Co., Inc. Polynucleotide herpes virus vaccine
US9012349B1 (en) 2013-11-01 2015-04-21 Ut-Battelle Llc Method of synthesizing bulk transition metal carbide, nitride and phosphide catalysts

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7094767B2 (en) 1994-07-22 2006-08-22 Merck & Co., Inc. Polynucleotide herpes virus vaccine
WO1998014586A1 (fr) * 1996-10-01 1998-04-09 Merck & Co., Inc. Vaccins de polynucleotides contre le virus de l'herpes
WO1998014210A1 (fr) * 1996-10-04 1998-04-09 The Regents Of The University Of California Methode de traitement d'une maladie pulmonaire allergique
US6174872B1 (en) 1996-10-04 2001-01-16 The Regents Of The University Of California Method for treating allergic lung disease
US6426336B1 (en) 1996-10-04 2002-07-30 The Regents Of The University Of California Method for treating allergic lung disease
WO1998017689A3 (fr) * 1996-10-18 1998-08-20 Genemedicine Inc Expression du gene il-12, systemes d'apport et utilisations
WO1998017814A3 (fr) * 1996-10-18 1998-08-27 Genemedicine Inc Expression de genes, systemes d'apport et utilisations
WO1999016892A1 (fr) * 1997-09-29 1999-04-08 University Of Bristol Vecteur a base d'herpes-virus 2 bovin (bhv-2) et utilisations de celui-ci
US6488936B1 (en) * 1998-02-12 2002-12-03 Wyeth Immunogenic composition of interleukin-12 (IL-12), alum, herpes simplex viral (HSV) antigen, and method thereof
US6867000B2 (en) * 2000-12-07 2005-03-15 Wyeth Holdings Corporation Method of enhancing immune responses to herpes
US9012349B1 (en) 2013-11-01 2015-04-21 Ut-Battelle Llc Method of synthesizing bulk transition metal carbide, nitride and phosphide catalysts

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