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WO1998014586A1 - Vaccins de polynucleotides contre le virus de l'herpes - Google Patents

Vaccins de polynucleotides contre le virus de l'herpes Download PDF

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Publication number
WO1998014586A1
WO1998014586A1 PCT/US1997/017262 US9717262W WO9814586A1 WO 1998014586 A1 WO1998014586 A1 WO 1998014586A1 US 9717262 W US9717262 W US 9717262W WO 9814586 A1 WO9814586 A1 WO 9814586A1
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WO
WIPO (PCT)
Prior art keywords
hsv
dna
animals
polynucleotide
immunized
Prior art date
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PCT/US1997/017262
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English (en)
Inventor
Marcy E. Armstrong
Robert D. Keys
John A. Lewis
Margaret A. Liu
William L. Mcclements
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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 AU45955/97A priority Critical patent/AU738835B2/en
Priority to JP10516673A priority patent/JP2001501101A/ja
Priority to CA002267645A priority patent/CA2267645A1/fr
Priority to EP97944467A priority patent/EP0956352A1/fr
Publication of WO1998014586A1 publication Critical patent/WO1998014586A1/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
    • 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.
  • Retro viral vectors have restrictions on the size and structure of polypeptides that can be expressed as fusion proteins
  • WO 93/17706 describes a method for vaccinating an animal against a vims, wherein carrier particles were coated with a gene constmct and the coated particles are accelerated into cells of an animal.
  • HSV herpes simplex vims
  • Recent efforts to develop subunit vaccines for herpes simplex vims (HSV) have focused on novel expression and presentation of viral antigens; especially the viral glycoproteins.
  • Virol., 7 ⁇ pp.2813-2817 have successfully protected animals from HSV challenge.
  • Vaccination by infection with recombinant adenovims expressing HSV gB elicits a protective immune response in mice.
  • McDermott, M.R., 1989, Virology, 169, pp.244-247 It is well documented that 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) recombmantly expressed protein (Burke, R.L., supra; Stanberry, L.R.
  • HSV-2 protein-coding DNA sequences were cloned into the eukaryotic expression vector. This DNA constmction 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 constmcts and RNA transcripts, capable of inducing in vivo expression of human herpes simplex vims (HSV) proteins upon direct introduction into animal tissues via injection or otherwise are therefore disclosed.
  • HSV herpes simplex vims
  • 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
  • These nucleic acids are useful as vaccines for inducing immunity to HSV, which can prevent infection and/or ameliorate HSV-related disease.
  • DNA vaccines expressing herpes simplex vims type 2 (HSV-2) full-length glycoprotein D (gD), or a truncated form of HSV-2 glycoprotein B (gB) are used to provide for protective efficacy against HSV-2 infection.
  • the present invention also relates to use of plasmid expression vectors encoding herpes simplex vims type 2 (HSV-2) proteins for their ability to immunize a host against a herpes infection.
  • the protein may be expressed along or in combination with one or more additional proteins which will induce an immune response to HSV.
  • full-length glycoprotein D (gD) and a tmncated form of HSV-2 glycoprotein B (gB) are used in a low-dosage form as a combination vaccine preparation.
  • immunization with a plasmid expressing the amino-terminal 707 amino acids (aa) of gB is used to induce a humoral immune response detected by ELISA and vims neutralization.
  • a truncated gB plasmid is combined with a plasmid expressing full-length gD.
  • This combinantion of DNA constmcts may be in the form of distinct DNA vectors, or a single vector which expresses each antigenic protein or protein fragment.
  • these nucleic acid constmcts 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. ug; 1.56 ug; 0.78 ug; or saline only. Since all animals in the 200ug; lOOug; 25ug; 12.5ug; 6.25ug; and 3.13ug groups survived, they are all represented with a single symbol.
  • FIG. 5 Survival of HSV-2 challenged animals following one injection with VU:gD at 50 ug; 16.7 ug; 5.0 ug; 1.67 ug; 0.5 ug; 0.167 ug; 0.05 ug; 0.017 ug; 0.005 ug; or saline only.
  • Fig. 8 The results of survival, mean days to death, paralysis, and vaginal vims titers in HSV-2 infected guinea pigs is shown.
  • Fig. 10 The effect of DNA immunization on the survival of mice infected by i.p. injection with HSV-2 in Example 11 is shown.
  • Mice were immunized twice with gD-2 (A) in a two-fold dilution series, or with gB-2 (B) in a half-log dilution series, or with saline. The doses ( ⁇ g) are indicated on the figure.
  • the numbers of mice in each group are noted in Table 9.
  • Mice were challenged by i.p. injection with 0.25 mL (l ⁇ 5- pfu) of a clarified stock of HSV-2 strain Curtis, and were observed for three weeks for signs of disease and survival.
  • mice were immunized with 12.5 ⁇ g (o), 1.6 ⁇ g gD-2 (•), or with 12.5 ⁇ g (o) vector V1J. Viral challenge was as described in Figure 10.
  • Fig. 12 The effect of immunization with gD-2 and gB-2 DNA in combination on primary HSV-2-induced genital disease in guinea pigs is shown for Example 11.
  • Immunized and sham-immunized guinea pigs were infected by application of HSV-2 strain MS to the vagina and external genital skin.
  • HSV-2 strain MS HSV-2 strain MS
  • the vaginal closure membrane was ruptured with a saline moistened cotton swab; the vagina and external skin were then swabbed with 0.1 N NaOH.
  • Vims was introduced using a cotton swap dipped into a clarified HSV-2 MS -infected Vero cell lysate diluted in tissue culture medium to 10"- ' pfu / mL.
  • the swab was inserted into the vagina, twisted back and forth five times then removed and wiped over the external genitalia. To ensure infection, vims application was repeated one hour later.
  • the inoculum was prepared from mock-infected Vero cells. Animals were caged randomly and evaluated daily by observers blinded to the study groups.
  • the vagina was swabbed with a moistened calcium alginate swab which was eluted into 2 mL of vims transport medium (Carr-Scarborough Microbiologicals Inc.; Stone Mt, GA). Infection was confirmed by reisolation of vims, a positive response in the HERPCHEKTM kit (Dupont, North Billerica, MA), or appearance of symptomatic disease, and the development of antibodies to nonstmctural HSV proteins. The severity of external disease was quantified using a visual scoring system adapted from that described by Stanberry, et al. (1982, /. Infect. Dis. 146: 397-404). Numerical scores
  • /0 were assigned to specific disease signs using the following scale: 0, no disease; 1, redness or swelling; 2, several ( ⁇ 3) small vesicles; 3, several ( ⁇ 3) large vesicles; 4, large ulcers with maceration. Scores of 0.5, 1.5, 2.5, and 3.5 were assigned to disease of intermediate severity. Daily mean lesion scores were calculated by dividing the sum of a group's lesion scores by the number of observations. In the case of death during the observation period, the final score assigned to that animal was carried through to the end of the observation period.
  • 0.5, 1.5, 2.5, and 3.5 were assigned to disease of intermediate severity. Daily mean lesion scores were calculated by dividing the sum of a group's lesion scores by the number of observations. In the case of death during the ll primary phase of disease, the last score assigned to that animal was carried through to the end of the observation period.
  • FIG. 14A and B Effect of immunization with VlJns: gB + VlJns:gD on HSV-induced disease in guinea pig for Example 12 is shown. Viral challenge and disease evaluation were as described in figure 1.
  • Fig. 15 Groups of six African green monkeys were immunized with DNA mixtures containing 100 or 10 ⁇ g each, of gD and gB DNA (see Example 11 and 12) at 0 and 4 weeks, and were boosted at 24 weeks. Sera obtained at four- week intervals were analvzed for anti-gD and anti-gB antibodies with antigen-specific ELISAS. For neutralization titers, the percent plaque reduction was determined (in duplicate) for each serum dilution compared to the same dilution of preimmune semm, scoring a 50% or greater reduction in plaque number as positive. Ten-fold serial dilutions ranging from 1:10 to 1:10,000 were assayed; endpoint titers were calculated by, linear regression analysis. (Sera negative at the lowest dilution tested were assigned endpoint titers of 1.) Arrows indicate immunization. (A) ELISA logio GMT
  • anti-gD response 100 ⁇ g-dose group; A 10 4 ⁇ -dose group.
  • anti-gB response q 100 ⁇ g
  • 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
  • polynucleotide vaccines for the purpose of generating immune responses to an encoded protein are referred to herein as polynucleotide vaccines or PNV.
  • PNV polynucleotide vaccines
  • 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.
  • 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.
  • fragments of the full length genes which encode portions of the full length protein may also be constmcted. 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 transcriptional promoter recognized by eukaryotic RNA polymerase, and a transcriptional terminator at the end of the HSV gene coding sequence.
  • the promoter is the cytomegalovims promoter with the intron A sequence (CMV-intA), although those skilled in the art will recognize that any of a number of other known promoters such as the strong immunoglobulin, or other eukaryotic gene promoters may be used.
  • a preferred transcriptional terminator is the bovine growth hormone terminator. The combination of CMVintA-BGH terminator is preferred.
  • an antibiotic resistance marker is also optionally included in the expression vector under transcriptional control of a suitable prokaryotic promoter.
  • Ampicillin resistance genes, neomycin resistance genes or any other suitable antibiotic resistance marker may be used.
  • the antibiotic resistance gene encodes a gene product for neomycin resistance.
  • 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 vims (RSV) long terminal repeat (LTR) is used as the promoter.
  • RSV rous sarcoma vims
  • LTR long terminal repeat
  • VI a mutated pBR322 vector into which the CMV promoter and the BGH transcriptional terminator were cloned is used.
  • the elements of VI and pUC19 have been been combined to produce an expression vector named V1J.
  • HSV gene such as gD
  • HSV gene 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 V1J and replaced with a neomycin resistance gene, to generate VlJ-neo, into which any of a number of different HSV genes may be cloned for use according to this invention.
  • the vector is VlJns, which is the same as VUneo except that a unique Sfil restriction site has been engineered into the single Kpnl site at position 2114 of VlJ-neo.
  • the incidence of Sfil sites in human genomic DNA is very low (approximately 1 site per 100,000 bases).
  • this vector allows careful monitoring for expression vector integration into host DNA, simply by Sfil digestion of extracted genomic DNA.
  • the vector is V1R. In this vector, as much non-essential DNA as possible is "trimmed" to produce a highly compact vector.
  • This vector allows larger inserts to be used, with less concern that undesirable sequences are encoded and optimizes uptake by cells when the constmct encoding specific vims 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 vims and thus meets a need as yet unmet in the field. Furthermore, if the vimlent isolates change, the immunogen may be modified to reflect new sequences as necessary.
  • a gene encoding an HSV protein is directly linked to a transcriptional 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 transcriptional promoter, immunogenic epitopes, and additional cistrons encoding immunoenhancing or immunomodulatory genes, with their own promoters, transcriptional 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
  • RNA polymerase promoters as the T7 or SP6 promoters
  • T7 or SP6 promoters RNA polymerase promoters
  • mn-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 vims 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.
  • gD DNA expression vector encoding gD
  • 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. Because each of the HSV gene products exhibit some degree of conservation among the various strains of HSV, and because immune responses may be generated in response to intracellular expression and MHC processing, it is expected that many different HSV gD PNV constmcts 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 amount of expressible DNA or transcribed RNA to be introduced into a vaccine recipient will depend on the strength of the transcriptional 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.
  • it is desirable for the polycucleotide to be in a physiologically acceptable solution such as, but not limited to, sterile saline or sterile buffered saline.
  • 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.
  • microprojectiles coated with polynucleotide are known in the art and are also useful in connection with this invention.
  • DNA intended for human use it may be useful to have the final DNA product in a pharmaceutically acceptable carrier or buffer solution.
  • Pharmaceutically acceptable carriers or buffer solutions are known in the art and include those described in a variety of texts such as Remington's Pharmaceutical Sciences.
  • 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 vims 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.
  • Talpey DJ. et al., 1989, J.ImmunoL, 142, pp.1325-1332; Yasukawa, M. et al., 1989, J.ImmunoL, 143, pp.2051-2057; Zarling, J.M. et aL,
  • mice protects mice from HSV infection.
  • Live vims 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 vims (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 aL, 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 vims challenge.
  • DNA vaccines expressing herpes simplex vims type 2 (HSV-2) full-length glycoprotein D (gD), or a truncated form of HSV-2 glycoprotein B (gB) are used to provide for protective efficacy in two experimental models of HSV-2 infection.
  • HSV-2 herpes simplex vims type 2
  • gD full-length glycoprotein D
  • gB truncated form of HSV-2 glycoprotein B
  • full-length glycoprotein D (gD), and a tmncated form of HSV-2 glycoprotein B (gB) are used in a low-dosage combination.
  • Immune sera from DNA-injected animals had antibodies to both gD and gB, and vims neutralizing activity.
  • the DNA-immunized animals were significantly protected from primary genital disease.
  • the present invention therefore relates to use of plasmid expression vectors encoding herpes simplex vims type 2 (HSV-2) proteins for their ability to immunize a host against a herpes infection.
  • the protein may be expressed along or in combination with one or more additional proteins which will induce an immune response to HSV. It will be within the purview of the skilled artisan, after reviewing this specification, to choose one or more such proteins which may be a wild type, full-length version or a mutant version which retains immunogenicity and neutralizing activity. The skilled artisan will also be prompted as to the DNA vaccine dosage which will be useful in generating an immune response to HSV.
  • a preferred dosage rate for a combination polynucleotide vaccine expressing ⁇ gB and gD would be 2.0ug gD and 0.6ug ⁇ gB, respectively, in a guinea pig model.
  • the skilled artisan will be able to manipulate these quantities and ratios to produce an optimal immune response in another host, such as a human.
  • the skilled artisan will also be directed by the examples of this specification to utilize various HSV proteins and antigenic fragments thereof, such as amino or carboxy terminal tmnctated forms, as well as mutated forms including but not limited to amino acid insertions, deletions and point mutations.
  • immunization with a plasmid expressing the ammo-terminal 707 amino acids (aa) of gB is used to induce a humoral immune response detected by ELISA and vims neutralization.
  • aa ammo-terminal 707 amino acids
  • gB ammo-terminal 707 amino acids
  • a truncated gB plasmid is combined with a plasmid expressing full-length gD. The immunized host develops humoral responses to both proteins and are significantly protected from viral challenge.
  • nucleic acids are useful as vaccines for inducing immunity to HSV, which can prevent infection and/or ameliorate HSV-related disease.
  • the expression vector VI was constmcted 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 1855 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 in and Nhe I fragment from pCMV6al20 [see B.S.
  • the primers that spanned intron A are: 5' primer, SEQ. ID:1 :
  • the primers used to remove the Sac I site are: sense primer, SEQ ID:3:
  • the PCR fragment was cut with Sac I and Bgl II and inserted into the vector which had been cut with the same enzymes.
  • VI J Expression Vector The purpose in creating VI J was to remove the promoter and transcription termination elements from vector VI in order to place them within a more defined context, create a more compact vector, and to improve plasmid purification yields.
  • VI J 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
  • 2 pUC18 was chosen to provide the "backbone" of the expression vector. It is known to produce high yields of plasmid, is well-characterized by sequence and function, and is of small size. The entire lac operon was removed from this vector by partial digestion with the Haell restriction enzyme. The remaining plasmid was purified from an agarose electrophoresis gel, blunt-ended with the T4 DNA polymerase treated with calf intestinal alkaline phosphatase, and ligated to the CMVintA/BGH element described above. Plasmids exhibiting either of two possible orientations of the promoter elements within the pUC backbone were obtained. One of these plasmids gave much higher yields of DNA in E. coli and was designated VI J. This vector's stmcture was verified by sequence analysis of the junction regions and was subsequently demonstrated to give comparable or higher expression of heterologous genes compared with VI.
  • ampr gene used for antibiotic selection of bacteria harboring V1J because ampicillin may not be desirable in large-scale fermenters.
  • the amp r gene from the pUC backbone of VI J was removed by digestion with Sspl and
  • the remaining plasmid was purified by agarose gel electrophoresis, blunt-ended with T4 DNA polymerase, and then treated with calf intestinal alkaline phosphatase.
  • the commercially available kanr gene derived from transposon 903 and contained within the pUC4K plasmid, was excised using the PstI restriction enzyme, purified by agarose gel electrophoresis, and blunt-ended with T4 DNA polymerase. This fragment was ligated with the VI J backbone and plasmids with the kan r gene in either orientation were derived which were designated as VlJneo #'s 1 and 3.
  • VlJneo 2 ⁇ VlJneo hereafter, was selected which contains the kanr gene in the same orientation as the ampr gene in VI J as the expression constmct.
  • VlJns Expression Vector An Sfi I site was added to VlJneo 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. VlJneo 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 VlJneo (with Kpn I).
  • 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 VlJn 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 VI Jn-tPA by blunting the Kpnl site with T4 DNA polymerase followed by ligation with an Sfil linker (catalogue #1138, New England Biolabs). This modification was verified by restriction digestion and agarose gel electrophoresis.
  • a dicistronic vaccine constmct which provides coordinate expression of a gene encoding an immunogen and a gene encoding an immuno- stimulatory protein
  • 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 H.
  • 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.
  • cAMP or IL-4 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.
  • oligomers provide Bgi ⁇ 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. Ncol digestion yielded a fragment suitable for cloning into pGEM- 3-IRES which had been digested with Ncol.
  • the resulting vector, pGEM-3-IRES-B7 contains an IRES-B7 cassette which can easily be transferred to VlJns-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. Dranoff et al, Proc. Natl Acad. Sci. USA, 90, 3539 (1993).
  • 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 constmction 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 VI J and VlJns afford. It was determined from the literature as well as by experiment that (1) regions within the pUC backbone comprising the E.
  • coli origin of replication could be removed without affecting plasmid yield from bacteria; (2) the 3 '-region of the kan* gene following the kanamycin open reading frame could be removed if a bacterial terminator was inserted in its place; and, (3) ⁇ 300 bp from the 3'- half of the BGH terminator could be removed without affecting its regulatory function (following the original Kpnl restriction enzyme site within the BGH element).
  • rZg VI R was constmcted 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 r .
  • VERO, BHK-21, RD cells and HSV-2 strain MS were obtained from the ATCC.
  • Vims 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 semm (FBS). After one hour, vims 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 vims was collected by centrifugation at 1800 X g 10 minutes 4°C. Supernantant vims 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 al., 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 ⁇ restriction recognition sites (Midland Certified Reagent Company; Midland, Texas) were used at 20 pmoles each.
  • a l.lkb 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 H and ligated to the Bgl II digested, dephosphorylated vector VIJ (Montgomery, D.L. et al., supra).
  • E. coli DH5 (BRL-Gibco, Gaithersburg, Md.) was transformed according to the manufacturer's specifications. Ampicillin resistant colonies were screened by hybridization with the 32p 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).
  • 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 constmct containing either the gB, gC, gD, or ICP27 gene.
  • HSV-2gB. gC. gD and ICP27 proteins from VIJ plasmids Rhabdomyosarcoma cells 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 semm, 2 mM L-glutamine, 25 mM HEPES, 50 U/ml penicillin and 50 ⁇ g/ml streptomycin.
  • 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 RIP A
  • HSV gD from VlJ:gD was demonstrated by transient transfection of RD cells. Lysates of VlJ:gD-transfected or mock transfected cells were fractionated by SDS PAGE and analyzed by immunoblotting. Figure 1A 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
  • HSV gB from VlJNS:gB was demonstrated by transient transfection of RD cells. Lysates of VlJNS:gB-transfected or mock transfected cells were fractionated by SDS PAGE and analyzed by immunoblotting. Figure IB shows that VlJNS:gB transfected RD cells express an immunoreactive protein with an apparent molecular weight of approximately 140 k. Lysates from HSV-2 (Curtis), HSV-2
  • HSV gC from VlJ:gC was demonstrated by transient transfection of RD cells. Indirect immunofluorescence of fixed VlJ:gC transfected cells showed primarily a diffuse cytoplasmic signal.
  • 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
  • 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, I A) 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, I A
  • xylazine Mobley Corp., Shawnee, KS.
  • VU:gD DNA 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.
  • 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 H2O at 37°C. Absorbance was read at 405nm and semm 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 VlJ: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 semm 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.
  • mice 10 mice per group were immunized as described above with DNA containing the HSV gB gene or DNA containing the HSV gC gene. Serum was collected and analyzed for the presence of anti-gB or anti-gC antibodies in the ELISA described above.
  • the ELISA data for gB antibodies are shown in Table 2, and demonstrates that mice immunized with VlJNS:gB were seropositive for anti-gB antibodies.
  • FIG. 2B illustrates that sera from VlJNS: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 vims 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.
  • VU:gD in the single-dose experiment were also tested in an HSV-2 plaque reduction assay. Twenty-nine of the forty-nine sera assayed were positive: >50% plaque reduction at a 1:10 dilution. At the 16.7 and 50 ⁇ g dose level, nine of ten sera from each group were neutralization positive.
  • 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 vims stock and then observed for three weeks. Survival data were analyzed using the log-rank test (McDermott et al., 1989, Virology,
  • mice immunized with two doses of VlJ: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 VlJ: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.
  • the guinea pigs were bled 5 weeks after the first vaccination and 2 weeks after the second vaccination.
  • Blood (0.6-1 ml per animal) was obtained by toe clipping. The blood was collected in microseparation tubes (Becton Dickinson), and was later centrifuged at
  • the sera collected from the guinea pigs was analyzed for the presence of anti-HSV antibodies using the ELISA set forth in
  • the guinea pigs weighed 600-700 grams each. They were infected intravaginally with herpes simplex vims type 2 (HSV-2), E194 strain. This was accomplished in a 3-step process. First, the vagina of each animal was swabbed for 5 seconds with a cotton tip applicator dipped in 0.1 N NaOH. This treatment irritates the vaginal area so that the infection takes better. Approximately 45-60 minutes later each vagina was dry swabbed for 5 seconds. Then an applicator dipped in vims medium (about 5 x 106 plaque forming units of HSV-2 per ml) was used to swab each guinea pig for 20 seconds. The swabs were gently and slowly twisted back and forth during the time they were in place.
  • vims 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.
  • Vaginal vims titers were made by titration of vims 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 vims 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 logio 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 VlJNS: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.
  • DNA vaccines expressing herpes simplex vims type 2 (HSV-2) full-length glycoprotein D (gD), or a truncated form of HSV-2 glycoprotein B (gB) were evaluated for protective efficacy in two experimental models of HSV-2 infection.
  • Intramuscular (i.m.) injection of mice showed that each constmction induced neutralizing semm antibodies and protected the mice from lethal HSV-2 infection.
  • Dose-titration studies showed that low doses ( l ⁇ g) of either DNA constmction induced protective immunity, and that a single immunization with the gD constmction was effective.
  • the two DNAs were then tested in a low-dosage combination in guinea pigs. Immune sera from DNA-injected animals had antibodies to both gD and gB, and vims neutralizing activity. When challenged by vaginal infection with HSV-2, the DNA-immunized animals were significantly protected from primary genital disease.
  • Vims was routinely prepared as indicated in Example 3. Briefly, vims 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 cell-culture medium without semm. After one hour, vims inoculum was removed and cultures were re-fed with high glucose DMEM supplemented with 2% heat inactivated fetal bovine serum (FBS), 2mM L-glutamine, 25mM HEPES, 50 U/mL penicillin and 50 ⁇ g/mL streptomycin. Incubation was continued until cytopathic effect was extensive: usually for 24 to 48 hrs.
  • FBS heat inactivated fetal bovine serum
  • Cell-associated vims was collected by centrifugation at 1800 X g 10 min., 4°C. Supematant vims was clarified by centrifugation at 640 X g for 10 min., 4°C, and stored at -70°C.
  • mice Male BALB/c mice (Charles River Laboratories; Wilmington, MA) and female Duncan Hartley guinea pigs (Harlan Sprague Dawley; Indianapolis, IN) were maintained and utilized in accordance with Institutional Animal Care and Use Committee-approved protocols.
  • HSV-2 strain Curtis DNA used as template for polymerase chain reactions (PCR) as disclosed in Example 4.
  • PCR polymerase chain reactions
  • a 1,182 base pair (bp) fragment encoding the gD precursor gene was amplified by PCR (Perkin Elmer Cetus; La Jolla, CA) using synthetic oligonucleotide primers (Midland Certified Reagent Company; Midland, TX) which corresponded to 5' and 3' end flanking sequences for the HSV-2 gD gene and contained Bgl H restriction sites.
  • a 2,121 bp sequence encoding the ammo-terminal 707 aa of HSV-2 gB was amplified by PCR.
  • Candidate plasmids were characterized by restriction mapping, and the vector-insert junctions were sequenced using the Sequenase DNA Sequencing Kit, version 2.0 (United States Biochemical; Cleveland, OH).
  • the gD-coding sequence originally cloned in VIJ, was subcloned into VlJns.
  • the final gD and truncated gB plasmid constmctions were designated gD-2 and gB-2, respectively.
  • Large-scale DNA preparations were essentially as described in Example 4.
  • Plasmid DNA was precipitated onto rhabdomyosarcoma (RD) cells (ATCC CCL136) by the calcium phosphate method using Pharmacia CellPhect Kit (Pharmacia Biotech Inc.; Piscataway, NJ) reagents according to the manufacturer's instructions except that 5 or 15 ⁇ g of DNA/well were used. After 48 hrs., cell lysates were resolved by electrophoresis and then transferred to nitrocellulose membranes.
  • RD rhabdomyosarcoma
  • Immunoblots were processed with an anti-HSV gD monoclonal antibody (Advanced Biotechnologies Inc.; Columbia, MD) or sheep anti-HSV-2 antiserum (ViroStat; Portland, ME) and developed with the ECL detection kit (Amersham; Arlington Heights, EL).
  • DNA dose refers to the total amount of DNA injected per animal per round of immunization; half the total was delivered to each injection site.
  • Mice were anesthetized by intraperitoneal (i.p.) injection of a mixture of 2 mg ketamine HC1 (Aveco; Fort Dodge, IA) and 0.2 mg xylazine (Mobley Corp.; Shawnee, KS) in saline. The hind legs were shaved with electric clippers and washed with 70% ethanol. Each quadriceps muscle was injected with 50 ⁇ L of DNA diluted into sterile saline just prior to use.
  • mice were sham-immunized with saline or vector DNA. Mice were five to six weeks old at the time of the first immunization. Guinea pigs, weighing 400-550 gm at the time of the first immunization, were anesthetized by subcutaneous injection of 22 mg ketamine plus 5 mg xylazine/kg; the hind legs were washed with 70% ethanol and each quadriceps muscle was injected i. m. with 100 ⁇ L of DNA or saline.
  • Sera were assayed for HSV-specific responses in ELISAs using either HSV glycoproteins partially purified from HSV-2 Curtis-infected BHK cell lysates (mouse sera) or recombinantly-expressed gD and gB purified from recombinant baculovims-gD and baculovims-gB infected SF21 cultures (guinea pig sera).
  • Recombinant vimses were constmcted using the BacPAK Baculovims Expression System (Clontech; Palo Alto, CA) pBacPAK ⁇ transfer vector and Bsu361 digested BacPAK6 vims, and gD and gB coding sequences from gD-2 and gB-2, respectively.
  • Glycoproteins from HSV-2 or baculovims-gD infected cultures were purified by Lentil Lectin Sepharose chromatography (Pharmacia Biotech Inc) essentially as described (Pachl, et al., 1987, J. Virology 61: 315- 325).
  • Truncated glycoprotein B was purified from clarified culture medium adjusted to O.lmM MnCl2, 0.5% NP40, batch adsorbed at room temperature to Lentil Lectin Sepharose 4B and eluted as previously described.
  • glycoproteins were diluted to 5 ⁇ g/mL total protein in 50 mM carbonate buffer (pH 9.5), lOO ⁇ L/well was applied to Maxi-sorb 96-well plates (Nunc; Naperville, IL) and allowed to absorb at 4°C, overnight.
  • Dilution buffer (920 mM Tris-HCl [pH 7.5], 137 mM NaCl, 2.7 mM KC1, 0.5% gelatin, 0.05% Tween 20) was used as a blocking agent as well as for the serial dilution of immune sera and the dilution of alkaline phosphatase-labeled goat anti-mouse (Boehringer Mannheim; Indianapolis, IN), or goat anti-guinea pig (Accurate Chemical and Scientific Corp.,Westbury, NY).
  • the ELISA was developed with 1 mg/mL p-nitrophenylphosphate in 10% diethanolamine (pH 9.8), 0.5 mM MgCl-6 H2 ⁇ at 37°C and optical absorbance was read at 405nm. Serum dilutions were scored as positive if the OD405 signal exceeded by more than three standard deviations the mean OD405 signal (six replicates) of sera from sham-immunized mice at the same dilution, or if the OD405 signal exceeded by > 0.1 OD units, the signal of the guinea pig's preimmune semm at the same dilution. The reciprocal of the last sample dilution scored positive was taken as the endpoint titer.
  • ELISA titers are HSV-specific as originally shown by lack of measurable ELISA titer in sera from naive or saline-immunized animals, and by the lack of reaction of immune sera with antigen prepared from mock-infected BHK cell lysates. HSV neutalization took place essentially as described in
  • Example 7 Briefly, sera from DNA- or saline-immunized animals were heat inactivated at 56°C for 30 min. prior to serial dilution in DMEM, 2% heat-inactivated FBS; 50 ⁇ L of each dilution were delivered to duplicate wells in a sterile polypropylene 0.5mL 96 well plate (Marsh Biomedical; Rochester, NY). HSV-2 stocks were diluted to 4,000 plaque forming units (pfu)/mL; 50 ⁇ L of vims was added to sample wells and the plate incubated overnight at 4°C. Guinea pig complement (Cappel; Durham, NC) was diluted 1:4 in DMEM, 2% heat inactivated FBS; 50 ⁇ L was added to each sample well.
  • the coding sequences for full-length gD and the amino-terminal 707 aa of gB were cloned from HSV-2 strain Curtis viral DNA by PCR methods into the eukaryotic expression vectors VIJ or VlJns, described at length in Examples 1 and 2, and are designated gD-2 and gB-2, respectively, for the purpose of Example 11.
  • VIJ eukaryotic expression vector
  • VlJneo and VlJns The plasmids were characterized by restriction mapping and sequence analysis of the vector-insert junctions.
  • the gD clone was identical with that published for HSV-2 strain G (Lasky and Dowbenko,1984, DNA 3: 23-29) and the gB clone sequence was identical with that published for HSV-2 strain 333 (Stuve, et al., 1987, J. Virol. 61: 326-335.).
  • the ability of gD-2, or gB-2 plasmids to express the encoded protein was demonstrated by transient transfection of RD cells. Immunoblot analysis of gD-2 DNA-transfected RD-cell lysates with an anti-HSV-2 gD monoclonal antibody detected a protein with a MW of approximately 60 K not present in mock-transfected RD cell lysates.
  • mice The biological effects of immunization with gD-2 or gB-2 DNA were investigated in separate dose-titration experiments in mice.
  • Animals were immunized by i. m. injection of DNA or were sham-immunized with saline at weeks zero and seven. Sera obtained at week ten were assayed in an HSV-specific ELISA.
  • Table 9 shows the seroconversion results and reports the geometric mean titers (GMT) ⁇ the standard error of the mean (SEM) attained for each dose group.
  • GTT geometric mean titers
  • SEM standard error of the mean
  • mice injected with doses of gD-2 DNA ranging from 3.1 to 100 ⁇ g were tested; thirteen were neutralization positive at 1 :10; of those, eleven were also positive at 1 :100; and of those, five were positive at the 1 :1,000 dilution.
  • the two negative sera (from the 50 ⁇ g dose group) also had low ELISA endpoint titers (logio 2.00 and 2.52, respectively).
  • Immunized and control mice were challenged by i.p. injection of of HSV-2 and observed daily for survival.
  • Figure 10A shows the effect of gD-2 DNA immunization on survival; significant protection from death (p ⁇ 0.001) was achieved for each dose.
  • mice were immunized with either 12.5, or 1.6 ⁇ g gD-2 DNA, or 12.5 ⁇ g of vector VIJ DNA.
  • Ten weeks after a single immunization sera were analyzed by ELISA.
  • the logio GMT ( ⁇ SEM) for the group injected with 12.5 ⁇ g gD-2 DNA was 3.89 (0.97) and that for the 1.6 ⁇ g group was 2.49 (1.20). None of the sera from mice immunized with vector DNA were seropositive; the log 10 GMT of 0.48 was background.
  • Figure 11 reports the survival data for these animals following i.p. challenge at eleven weeks. Both groups immunized with gD-2 DNA were significantly protected from compared to those immunized with the vector death (p ⁇ .001). Survival of the vector-injected animals was similar to that found for the saline-injected animals in the experiments summarized in Figure 10. These results confirmed that protection was dependent upon the gD coding sequence. Furthermore, they demonstrated that protective immunity could be established with a single injection of gD-2 DNA. Additional studies in mice and guinea pigs (see Example 12) comparing plasmids gB-2 or gD-2 with vector, or with control plasmids that expressed influenza viral proteins, also found that protection was dependent on the presence HSV protein-coding sequences.
  • the lethal infection model was useful for confirming the in vivo activity of the gD-2 and gB-2 DNA, and for establishing that low DNA doses were effective. However, to more closely mimic a human
  • a guinea pig vaginal infection model was used to assay the effects of immunization with a combination of low doses of gD-2 and gB-2 DNA.
  • Seven guinea pigs were immunized with a DNA mixture containing 3 ⁇ g gD-2 DNA and 10 ⁇ g gB-2 DNA at weeks zero and six; fourteen control guinea pigs were sham-immunized with saline.
  • Sera, obtained at nine weeks were analyzed for anti-gD and anti-gB antibodies using antigen-specific ELISAs. Results are shown in Table 10.
  • this group's scores were significantly higher than those of the DNA-immunized, or the mock-infected control groups (p ⁇ 0.01). In contrast, none of the DNA-immunized animals developed severe disease, and the scores for this group were statistically indistinguishable from those of the mock-infected group. The overall primary disease, as measured by the means of all lesion scores, was significantly lower for the DNA-immunized group compared to the infected control group (p ⁇ 0.001) but was not significantly different from the mock-infected control group (p 0.92). The scores for the mock-infected group were taken as the experimental background.
  • the DNA-immunized animals were further distinguished from the infected controls in that none of them developed signs of systemic disease.
  • six of the eight sham-immunized infected guinea pigs showed signs of severe systemic infection: five retained urine on two or more days, one developed partial paralysis of the hind limbs, and five animals became moribund during the observation period and required euthanization (Figure 12). None of the mock-infected animals showed signs of systemic disease.
  • Th challenge study of this Example indicates that immunization with low doses of DNAs which encode HSV-2 full-length gD and a tmncated form of gB protected guinea pigs from HSV-2-induced primary genital disease.
  • 5 ⁇ ⁇ gB and gD is 2.0 ⁇ g gD and 0.6 ⁇ g ⁇ gB, respectively, in a guinea pig model.
  • EXAMPLE 12 As in Example 11, plasmid expression vectors encoding herpes simplex vims type 2 (HSV-2) glycoproteins B (gB) or D (gD) were constmcted and tested for their ability to immunize guinea pigs against genital HSV infection. Immunization with a plasmid expressing the amino-terminal 707 amino acids (aa) of gB induced humoral immune responses detected by ELISA and vims neutralization. When challenged by vaginal infection, immunized animals were partially protected from genital herpes, exhibiting significantly reduced primary and subsequent recurrent disease. When the gB plasmid was combined with a plasmid expressing full-length gD, immunized guinea pigs developed humoral responses to both proteins and were also significantly protected from viral challenge.
  • HSV-2 herpes simplex vims type 2
  • gD glycoproteins B
  • gD glycoproteins B
  • gD glycoprotein
  • Plasmid vectors expressing full-length gD, or a carboxy- terminal deleted form of gB were constmcted using the expression vector VlJns, a derivative of the vector VIJ, as described throughout this specification in general and Example 11 in particular. Expression, in this vector, is driven by the cytomegalovims immediate early
  • CMVIE human embryonal rhabdomyosarcoma
  • This truncated protein has had the transmembrane and cytoplasmic domains of gB deleted and therefore, was expected to be secreted from transfected cells.
  • Immunoblot analysis of transiently-transfected RD cells with sheep anti-HSV-2 antisemm (ViroStat) detected a protein of the expected size (106 kDa), the majority of which was found in the conditioned medium.
  • the in vivo activities of these DNA constmctions were confirmed by induction of gD-or gB-specific serum antibodies in mice, guinea pigs, and nonhuman primates upon im injection. All animal studies were carried out in accordance with Institutional Animal Care and Use Committee-approved protocols.
  • VlJns gB plasmid (i.e., gB-2 of Example 11) on genital HSV infection was studied by immunizing female Duncan Hartley guinea pigs (HRP Inc.) with 100 ⁇ g or 10 ⁇ g of VlJns: gB.
  • Control animals were immunized with 100 ⁇ g of VlJns:HA, a plasmid expressing influenza A vims hemagglutinin.
  • the DNA was delivered in 200 ⁇ L of saline; 100 ⁇ L in each quadriceps muscle. At six weeks, all animals were reimmunized.
  • HSV-specific ELISA used partially purified HSV-infected baby hamster kidney cell lysate as capture antigen.
  • the ELISA was originally qualified as HSV specific by the differential responses of sera from infected, immunized, or control mice and guinea pigs. Endpoint titers were taken as the reciprocal of the highest serum dilution which gave an OD405 signal greater than three standard deviations above the mean OD405 signal of six replicates of control-animal sera at the same dilution. All of the VlJns: gB-immunized animals were seropositive.
  • the geometric mean titer ⁇ the standard error (GMT ⁇ SE) for the 100 ⁇ g VlJns: gB group was 120,000 ⁇ 30,000; for the low (lO ⁇ g) dose group it was 147,000 ⁇ 32,300.
  • the GMT of the VlJns:HA-immunized control group was 3.0 ⁇ 0 by definition.
  • the percent plaque reduction was determined (in duplicate) for each serum dilution compared to the same dilution of pre-immune serum, scoring a 50% or greater reduction in plaque number as positive.
  • sera from all VlJns: gB animals were positive at both dilutions; none of the sera from VlJns:HA-immunized animals were positive.
  • endpoint titers one semm from the 100 ⁇ g-dose group, four sera from the 10 ⁇ g-dose group, and one semm from the HA DNA-immunized group were chosen randomly for further titration.
  • Figure 13A summarizes the course of primary disease by reporting the mean of the
  • guinea pigs like humans, develop latent herpes infections which occasionally reactivate to cause recurrent disease.
  • the surviving animals were observed for recurrent disease over a ten week period beginning four weeks after vims inoculation. Thirty-nine sets of observations were made, and disease was scored by the same scale used for primary disease. Because recurrent disease is usually less severe than the primary disease and because it can occur infrequently, recurrence data are presented in
  • Figure 13B as cumulative mean lesion scores for each observation day. This miming tally of the daily mean lesion scores includes contributions from the frequency, severity, and duration of each recurrent episode.
  • VlJns: gB plasmid was tested in combination with the plasmid VlJns:gD (i.e., gD-2 of Example 11), which expresses full- length HSV-2 gD.
  • Guinea pigs were immunized twice with either a mixture of 100 ⁇ g VlJns: gB + 100 ⁇ g VUns:gD, or with 100 ⁇ g VUns:HA by the regimen described above.
  • sera and vaginal secretions were obtained and analyzed by gD- and gB-specific ELISAs.
  • Vaginal secretions were collected from seven of theVUns: gB + VlJns:gD-immunized guinea pigs by swabbing with saline-moistened calcium alginate swabs which were eluted into 1 mL of saline. All seven animals tested had detectable anti-gD responses; titers ranged from 2 to > 8. Four of the seven animals had a detectable anti- gB response; titers were > 8 in all cases. Secretions from the HA DNA immunized pigs were used as negative controls.
  • VlJns: gB + VlJns:gD group experienced an extended period free of recurrence beginning at observation day eight.
  • the results disclosed within this Example indicate that immunization of guinea pigs with DNA encoding HSV-2 gB sequences, or a mixture of DNAs encoding HS V-2 gD and gB sequences induces humoral immune responses to the encoded proteins, and those responses are associated with the reduction of primary and recurrent HSV-2- induced disease.
  • mice guinea pigs and monkeys also, see Example 13 for primate data
  • immunization with VlJns: gB induces a memory response in all three models.
  • EXAMPLE 13 Young adult (2.2-7 kg body mass) seronegative male African green monkeys (3/group) were injected intramuscularly in one deltoid and one quadriceps with 0.5 ml of inoculum per site containing the indicated dosages of DNA. Control monkeys received a full human dose (15 ⁇ g of each HA antigen) of licensed influenza vaccine (Fluzone Whole Virion or Subvirion, Connaught Laboratories Inc., Swiftwater,
  • Hemagglutination inhibiting (HI) antibodies were determined using 4 HA units of selected vims strain and chicken RBCs. Sera were incubated overnight with receptor-destroying enzyme (RDE, Sigma Chemical Co, St. Louis, MO), heated for 30 min at 56°C, and absorbed with chicken RBC.
  • RDE receptor-destroying enzyme
  • Groups of six African green monkeys were immunized with DNA mixtures containing 100 or 10 ⁇ g each, of gD and gB DNA at 0 and 4 weeks, and were boosted at 24 weeks. Sera obtained at four-week intervals were analyzed for anti-gD and anti-gB antibodies with antigen- specific ELISAS. These ELISAs used cloned, baculovims-expressed gD or tmncated gB proteins as capture antigens and were originally qualified as antigen specific using sera from guinea pigs and mice immunized singly with Vljns:gD or Vljns: gB (Example 11 and 12).
  • Endpoint titers were taken as the reciprocal of the highest serum dilution giving an OD405 signal > 0.05 OD units above the signal obtained with the same dilution of preimmune semm from that animal.
  • GMT geometric mean titers
  • sera negative at the lowest dilution tested were assigned an endpoint titer of 3.
  • endpoint titer of 3.
  • Dilutions of heat inactivated serum were incubated with 200 plaque forming units (pfu) HSV-2 Curtis overnight at 4°C.
  • Guinea pig complement was added for one hour at 37 S C before assaying for viable vims on Vero cells.
  • the percent plaque reduction was determined (in duplicate) for each semm dilution compared to the same dilution of preimmune semm, scoring a 50% or greater reduction in plaque number as positive.
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • SEQUENCE DESCRIPTION SEQ ID NO : 3 : GTATGTGTCT GAAAATGAGC GTGGAGATTG GGCTCGCAC 39
  • 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)
  • xi SEQUENCE DESCRIPTION: SEQ ID NO: 11: CCACATCTCG AGGAACCGGG TCAATTCTTC AGCACC 36
  • MOLECULE TYPE DNA (genomic)
  • SEQUENCE DESCRIPTION SEQ ID NO : 12 : GGTACAGATA TCGGAAAGCC ACGTTGTGTC TCAAAATC 38
  • MOLECULE TYPE DNA (genomic)
  • xi SEQUENCE DESCRIPTION: SEQ ID NO: 14: GGTACATGAT CACGTAGAAA AGATCAAAGG ATCTTCTTG 39
  • MOLECULE TYPE DNA (genomic)

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Abstract

On a cloné des gènes codant des protéines de type 2 du virus d'herpès simplex (HSV-2) dans des vecteurs d'expression eucaryote afin d'exprimer les protéines codées dans des cellules de muscles de mammifères in vivo. On a immunisé ces animaux par injection de ces produits de recombinaison d'ADN, désignés vaccins de polynucléotides ou PNV, dans leurs muscles. On a découvert, dans un titrage d'ADN, qu'une seule immunisation de 0,5 νg d'un PNV, produisait ⊃90 % de séroconversion sur dix semaines de post-immunisation. Des antisérums immuns ont neutralisé à la fois HSV-2 et HSV-1 dans des cultures de cellules. Quand on a testé HSV-2 sur des animaux, on a obtenu une protection importante (p∫0.001) contre l'infection mortelle en utilisant la vaccination de PNV. Ces produits de recombinaison d'ADN peuvent être des formes de longueur totale, tronquées ou ayant subi une mutation et peuvent être administrés seuls ou combinés, de manière à optimiser l'immunisation et la protection contre l'infection par HSV.
PCT/US1997/017262 1994-07-22 1997-09-26 Vaccins de polynucleotides contre le virus de l'herpes WO1998014586A1 (fr)

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AU45955/97A AU738835B2 (en) 1994-07-22 1997-09-26 A polynucleotide herpes virus vaccine
JP10516673A JP2001501101A (ja) 1996-10-01 1997-09-26 ポリヌクレオチドヘルペスウイルスワクチン
CA002267645A CA2267645A1 (fr) 1996-10-01 1997-09-26 Vaccins de polynucleotides contre le virus de l'herpes
EP97944467A EP0956352A1 (fr) 1996-10-01 1997-09-26 Vaccins de polynucleotides contre le virus de l'herpes

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0958373A1 (fr) * 1996-10-23 1999-11-24 American Home Products Corporation Vaccins
WO2023107999A3 (fr) * 2021-12-08 2023-07-13 Modernatx, Inc. Vaccins à arnm contre le virus de l'herpès simplex

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3050897A1 (fr) 2015-01-28 2016-08-03 Polichem S.A. Anticorps monoclonaux humains dotés d'une activité neutralisante contre de fortes HSV-2 et HSV-1

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JPS62115288A (ja) * 1985-11-14 1987-05-26 Chemo Sero Therapeut Res Inst 単純ヘルペスウイルス遺伝子を組込んだ組換えｄｎａ、形質転換動物細胞および単純ヘルペスウイルス蛋白質の製法
US4891135A (en) * 1988-01-25 1990-01-02 Hoechst Aktiengesellschaft Macroporous, asymmetric, hydrophilic polyaramide membrane
US5244792A (en) * 1984-04-06 1993-09-14 Chiron Corporation Expression of recombinant glyoprotein B from herpes simplex virus
WO1996003510A1 (fr) * 1994-07-22 1996-02-08 Merck & Co., Inc. Vaccin polynucleotidique contre le virus de l'herpes

Patent Citations (4)

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Publication number Priority date Publication date Assignee Title
US5244792A (en) * 1984-04-06 1993-09-14 Chiron Corporation Expression of recombinant glyoprotein B from herpes simplex virus
JPS62115288A (ja) * 1985-11-14 1987-05-26 Chemo Sero Therapeut Res Inst 単純ヘルペスウイルス遺伝子を組込んだ組換えｄｎａ、形質転換動物細胞および単純ヘルペスウイルス蛋白質の製法
US4891135A (en) * 1988-01-25 1990-01-02 Hoechst Aktiengesellschaft Macroporous, asymmetric, hydrophilic polyaramide membrane
WO1996003510A1 (fr) * 1994-07-22 1996-02-08 Merck & Co., Inc. Vaccin polynucleotidique contre le virus de l'herpes

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Title
PATENT ABSTRACTS OF JAPAN vol. 011, no. 332 (C - 455) 29 October 1987 (1987-10-29) *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0958373A1 (fr) * 1996-10-23 1999-11-24 American Home Products Corporation Vaccins
EP0958373A4 (fr) * 1996-10-23 2001-11-28 American Home Prod Vaccins
WO2023107999A3 (fr) * 2021-12-08 2023-07-13 Modernatx, Inc. Vaccins à arnm contre le virus de l'herpès simplex

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