B7 costimulation molecules expressed from the herpes simplex virus 2 genome rescue immune induction in B7-deficient mice

The interaction between B7 costimulation molecules on antigen-presenting cells and CD28 on antigen-responsive T cells is essential for T-cell activation and maturation of immune responses to herpes simplex virus (HSV) infection. Vaccine-induced immune responses also depend upon adequate upregulation of B7 costimulation molecules, but this signal may be limiting for replication-defective virus vaccines. We investigated whether expression of B7 costimulation molecules by a prototypical replication-defective antiviral vaccine could enhance immune responses to the vaccine and whether B7-1 and B7-2 would be similarly effective. We altered an ICP8(-) replication-defective strain of HSV type 2 (HSV-2), 5BlacZ, to encode either murine B7-1 or B7-2. B7 molecule expression was detected on the surface of cells infected in vitro and at the RNA level in tissue of immunized mice. Immunization of B7-1/B7-2 knockout mice with B7-encoding virus modestly expanded the number of gamma interferon-producing T cells and significantly augmented class-switched HSV-specific antibody responses compared with the parental virus. Mice immunized with either B7-expressing virus showed less replication of challenge virus in the genital mucosa than mice immunized with 5BlacZ, markedly fewer signs of genital and neurological disease, and little weight loss. Virtually all mice immunized with B7-encoding virus survived challenge with a large dose of HSV-2, whereas most 5BlacZ-immunized mice succumbed to infection. These results indicate that protective immune responses can be enhanced by the inclusion of host B7 costimulation molecules in a prototypical replication-defective HSV vaccine against HSV-2 genital infection and that B7-1 and B7-2 induce immune responses with similar capacities to fight HSV-2 infection.     

Virus-encoded b7-2 costimulation molecules enhance the protective capacity of a replication-defective herpes simplex virus type 2 vaccine in immunocompetent mice

Herpes simplex virus 2 (HSV-2) and, to a lesser extent, HSV-1 cause the majority of sexually transmitted genital ulcerative disease. No effective prophylactic vaccine is currently available. Replication-defective HSV stimulates immune responses in animals but produces no progeny virus, making it potentially useful as a safe form of live vaccine against HSV. Because it does not replicate and spread in the host, however, replication-defective virus may have relatively limited capacity to solicit professional antigen presentation. We previously demonstrated that in mice devoid of B7-1 and B7-2 costimulation molecules, replication-defective HSV-2 encoding B7-1 or B7-2 induces stronger immune responses and protection against HSV-2 challenge than immunization with replication-defective virus alone. Here, we vaccinated wild-type mice fully competent to express endogenous B7 costimulation molecules with replication-defective HSV-2 or replication-defective virus encoding B7-2 and compared their capacities to protect against vaginal HSV-2 infection and disease. Replication-defective virus encoding B7-2 induced more IFN-γ-producing CD4 T cells than did replication-defective virus alone. Immunization with B7-2-expressing virus decreased challenge virus replication in the vaginal mucosa, genital and neurological disease, and mortality more effectively than did immunization with the parental replication-defective virus. Prior immunization with B7-expressing, replication-defective virus also effectively suppressed infection of the nervous system compared to immunization with the parental virus. Thus, B7 costimulation molecules expressed at the site of HSV infection can enhance vaccine efficacy even in a fully immunocompetent host.     

Disruption of virion host shutoff activity improves the immunogenicity and protective capacity of a replication-incompetent herpes simplex virus type 1 vaccine strain

The virion host shutoff (vhs) protein encoded by herpes simplex virus type 1 (HSV-1) destabilizes both viral and host mRNAs. An HSV-1 strain with a mutation in vhs is attenuated in virulence and induces immune responses in mice that are protective against corneal infection with virulent HSV-1, but it has the capacity to establish latency. Similarly, a replication-incompetent HSV-1 strain with a mutation in ICP8 elicits an immune response protective against corneal challenge, but it may be limited in viral antigen production. We hypothesized therefore that inactivation of vhs in an ICP8(-) virus would yield a replication-incompetent mutant with enhanced immunogenicity and protective capacity. In this study, a vhs(-)/ICP8(-) HSV-1 mutant was engineered. BALB/c mice were immunized with incremental doses of the vhs(-)/ICP8(-) double mutant or vhs(-) or ICP8(-) single mutants, or the mice were mock immunized, and protective immunity against corneal challenge with virulent HSV-1 was assessed. Mice immunized with the vhs(-)/ICP8(-) mutant showed prechallenge serum immunoglobulin G titers comparable to those immunized with replication-competent vhs(-) virus and exceed those of mice immunized with the ICP8(-) single mutant. Following corneal challenge, the degrees of protection against ocular disease, weight loss, encephalitis, and establishment of latency were similar for vhs(-)/ICP8(-) and vhs(-) virus-vaccinated mice. Moreover, the double deleted vhs(-)/ICP8(-) virus protected mice better in all respects than the single deleted ICP8(-) mutant virus. The data indicate that inactivation of vhs in a replication-incompetent virus significantly enhances its protective efficacy while retaining its safety for potential human vaccination. Possible mechanisms of enhanced immunogenicity are discussed.     

A live-attenuated HSV-2 ICP0 virus elicits 10 to 100 times greater protection against genital herpes than a glycoprotein D subunit vaccine

Glycoprotein D (gD-2) is the entry receptor of herpes simplex virus 2 (HSV-2), and is the immunogen in the pharmaceutical industry''s lead HSV-2 vaccine candidate. Efforts to prevent genital herpes using gD-2 subunit vaccines have been ongoing for 20 years at a cost in excess of $100 million. To date, gD-2 vaccines have yielded equivocal protection in clinical trials. Therefore, using a small animal model, we sought to determine if a live-attenuated HSV-2 ICP0 ⁻ virus would elicit better protection against genital herpes than a gD-2 subunit vaccine. Mice immunized with gD-2 and a potent adjuvant (alum+monophosphoryl lipid A) produced high titers of gD-2 antibody. While gD-2-immunized mice possessed significant resistance to HSV-2, only 3 of 45 gD-2-immunized mice survived an overwhelming challenge of the vagina or eyes with wild-type HSV-2 (MS strain). In contrast, 114 of 115 mice immunized with a live HSV-2 ICP0 ⁻ virus, 0ΔNLS, survived the same HSV-2 MS challenges. Likewise, 0ΔNLS-immunized mice shed an average 125-fold less HSV-2 MS challenge virus per vagina relative to gD-2-immunized mice. In vivo imaging demonstrated that a luciferase-expressing HSV-2 challenge virus failed to establish a detectable infection in 0ΔNLS-immunized mice, whereas the same virus readily infected naïve and gD-2-immunized mice. Collectively, these results suggest that a HSV-2 vaccine might be more likely to prevent genital herpes if it contained a live-attenuated HSV-2 virus rather than a single HSV-2 protein.     

Therapeutic immunization with a virion host shutoff-defective, replication-incompetent herpes simplex virus type 1 strain limits recurrent herpetic ocular infection

Immunization of mice with herpes simplex virus type 1 (HSV-1) mutant viruses containing deletions in the gene for virion host shutoff (vhs) protein diminishes primary and recurrent corneal infection with wild-type HSV-1. vhs mutant viruses are severely attenuated in vivo but establish latent infections in sensory neurons. A safer HSV-1 mutant vaccine strain, Delta41Delta29, has combined vhs and replication (ICP8-) deficits and protects BALB/c mice against primary corneal infection equivalent to a vhs- strain (BGS41). Here, we tested the hypothesis that Delta41Delta29 can protect as well as BGS41 in a therapeutic setting. Because immune response induction varies with the mouse and virus strains studied, we first determined the effect of prophylactic Delta41Delta29 vaccination on primary ocular infection of NIH inbred mice with HSV-1 McKrae, a model system used to evaluate therapeutic vaccines. In a dose-dependent fashion, prophylactic Delta41Delta29 vaccination decreased postchallenge tear film virus titers and ocular disease incidence and severity while eliciting high levels of HSV-specific antibodies. Adoptive transfer studies demonstrated a dominant role for immune serum and a lesser role for immune cells in mediating prophylactic protection. Therapeutically, vaccination with Delta41Delta29 effectively reduced the incidence of UV-B-induced recurrent virus shedding in latently infected mice. Therapeutic Delta41Delta29 and BGS41 vaccination decreased corneal opacity and delayed-type hypersensitivity responses while elevating antibody titers, compared to controls. These data indicate that replication is not a prerequisite for generation of therapeutic immunity by live HSV mutant virus vaccines and raise the possibility that genetically tailored replication-defective viruses may make effective and safe therapeutic vaccines.     

Herpes Simplex Virus 2 ICP0? Mutant Viruses Are Avirulent and Immunogenic: Implications for a Genital Herpes Vaccine

Herpes simplex virus 1 (HSV-1) ICP0 ⁻ mutants are interferon-sensitive, avirulent, and elicit protective immunity against HSV-1 (Virol J, 2006, 3:44). If an ICP0 ⁻ mutant of herpes simplex virus 2 (HSV-2) exhibited similar properties, such a virus might be used to vaccinate against genital herpes. The current study was initiated to explore this possibility. Several HSV-2 ICP0 ⁻ mutant viruses were constructed and evaluated in terms of three parameters: i. interferon-sensitivity; ii. virulence in mice; and iii. capacity to elicit protective immunity against HSV-2. One ICP0 ⁻ mutant virus in particular, HSV-2 0ΔNLS, achieved an optimal balance between avirulence and immunogenicity. HSV-2 0ΔNLS was interferon-sensitive in cultured cells. HSV-2 0ΔNLS replicated to low levels in the eyes of inoculated mice, but was rapidly repressed by an innate, Stat 1-dependent host immune response. HSV-2 0ΔNLS failed to spread from sites of inoculation, and hence produced only inapparent infections. Mice inoculated with HSV-2 0ΔNLS consistently mounted an HSV-specific IgG antibody response, and were consistently protected against lethal challenge with wild-type HSV-2. Based on their avirulence and immunogenicity, we propose that HSV-2 ICP0 ⁻ mutant viruses merit consideration for their potential to prevent the spread of HSV-2 and genital herpes.     

Glutamine deprivation causes enhanced plating efficiency of a herpes simplex virus type 1 ICP0-null mutant

Isoleucine deprivation of cellular monolayers prior to infection has been reported to result in partial complementation of a herpes simplex virus type 1 (HSV-1) ICP0 null (ICP0⁻) mutant. We now report that glutamine deprivation alone is able to enhance the plating efficiency of an ICP0⁻ virus and that isoleucine deprivation has little or no effect. Because a low glutamine level is associated with stress and because stress is known to induce reactivation, low levels of glutamine may be relevant to the reactivation of HSV-1 from latency. Additionally, we demonstrate that arginine and methionine deprivation result in partial complementation of the ICP0⁻ virus.     

Antibodies Are Required for Complete Vaccine-Induced Protection against Herpes Simplex Virus 2

Herpes simplex virus 2 (HSV-2) 0ΔNLS is a live HSV-2 ICP0 ^- mutant vaccine strain that is profoundly attenuated in vivo due to its interferon-hypersensitivity. Recipients of the HSV-2 0ΔNLS vaccine are resistant to high-dose HSV-2 challenge as evidenced by profound reductions in challenge virus spread, shedding, disease and mortality. In the current study, we investigated the requirements for HSV-2 0ΔNLS vaccine-induced protection. Studies using (UV)-inactivated HSV-2 0ΔNLS revealed that self-limited replication of the attenuated virus was required for effective protection from vaginal or ocular HSV-2 challenge. Diminished antibody responses in recipients of the UV-killed HSV-2 vaccine suggested that antibodies might be playing a critical role in early protection. This hypothesis was investigated in B-cell-deficient μMT mice. Vaccination with live HSV-2 0ΔNLS induced equivalent CD8⁺ T cell responses in wild-type and μMT mice. Vaccinated μMT mice shed ~40-fold more infectious HSV-2 at 24 hours post-challenge relative to vaccinated wild-type (B-cell⁺) mice, and most vaccinated μMT mice eventually succumbed to a slowly progressing HSV-2 challenge. Importantly, passive transfer of HSV-2 antiserum restored full protection to HSV-2 0ΔNLS-vaccinated μMT mice. The results demonstrate that B cells are required for complete vaccine-induced protection against HSV-2, and indicate that virus-specific antibodies are the dominant mediators of early vaccine-induced protection against HSV-2.     

B7-H1 (CD274) inhibits the development of herpetic stromal keratitis (HSK)

The co-signaling molecule B7-H1 (CD274) functions as both a co-inhibitor through programmed death-1 (PD-1) receptor and a co-stimulator via an as-yet-unidentified receptor on T cells. We investigated the physiological role of endogenous B7-H1 in the pathogenesis of herpetic stromal keratitis (HSK) caused by herpes simplex virus type 1 (HSV-1). Following HSV-1 infection of the cornea of mice, B7-H1 expression was up-regulated in the CD11b+ macrophage population in the draining lymph nodes (dLN) and in the inflamed cornea. In addition, HSV-1 infection significantly increased PD-1 expression on CD4+ T cells in the dLN and inflamed cornea. The administration of antagonistic B7-H1 monoclonal antibody resulted in the proliferation of HSV-specific CD4+ T cells that secreted interferon (INF)-gamma, and inhibited the apoptosis of HSV-specific CD4+ T cells, which exaggerated HSK. These results strongly suggest that the B7-H1 may be involved in suppression of the development of HSK.     

Absence of CXCL10 Aggravates Herpes Stromal Keratitis with Reduced Primary Neutrophil Influx in Mice

Herpes simplex virus 1 (HSV-1) replication initiates inflammation and angiogenesis responses in the cornea to result in herpetic stromal keratitis (HSK), which is a leading cause of infection-induced vision impairment. Chemokines are secreted to modulate HSK by recruiting leukocytes, which affect virus growth, and by influencing angiogenesis. The present study used a murine infection model to investigate the significance of the chemokine CXC chemokine ligand 10 (CXCL10; gamma interferon-inducible protein 10 [IP-10]) in HSK. Here, we show that HSV-1 infection of the cornea induced CXCL10 protein expression in epithelial cells. The corneas of mice with a targeted disruption of the gene encoding CXCL10 displayed decreases in levels of neutrophil-attracting cytokine (interleukin-6), primary neutrophil influx, and viral clearance 2 or 3 days postinfection. Subsequently, absence of CXCL10 aggravated HSK with elevated levels of interleukin-6, chemokines for CD4⁺ T cells and/or neutrophils (macrophage inflammatory protein-1α and macrophage inflammatory protein-2), angiogenic factor (vascular endothelial growth factor A), and secondary neutrophil influx, as well as infiltration of CD4⁺ T cells to exacerbate opacity and angiogenesis in the cornea at 14 and up to 28 days postinfection. Our results collectively show that endogenous CXCL10 contributes to recruit the primary neutrophil influx and to affect the expression of cytokines, chemokines, and angiogenic factors as well as to reduce the viral titer and HSK severity.     

Inhibition of Major Histocompatibility Complex Class I Antigen Presentation in Pig and Primate Cells by Herpes Simplex Virus Type 1 and 2 ICP47

Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) express an immediate-early protein, ICP47, that effectively inhibits the human transporter associated with antigen presentation (TAP), blocking major histocompatibility complex (MHC) class I antigen presentation to CD8⁺ T cells. Previous work indicated that the mouse TAP is relatively resistant to inhibition by the HSV-1 and HSV-2 ICP47 proteins (ICP47-1 and ICP47-2) and that mouse cells infected with HSV-1 are lysed by anti-HSV CD8⁺ cytotoxic T lymphocytes (CTL). Therefore, mice are apparently not suitable animals in which to study the in vivo effects of ICP47. In order to find an animal model, we introduced ICP47-1 and ICP47-2 into cells from various animal species—mice, rats, guinea pigs, rabbits, dogs, pigs, cows, monkeys, and humans—and measured TAP activity in the cells. Both proteins were unable to inhibit TAP in mouse, rat, guinea pig, and rabbit cells. In contrast, ICP47-1 and ICP47-2 inhibited TAP in pig, dog, cow, and monkey cells, and the TAP in pig and dog fibroblasts was often more sensitive to both proteins than TAP in human fibroblasts. These results were extended by measuring CD8⁺-T-cell recognition (CTL lysis) of cells from various species. Cells were infected with recombinant HSV-1 constructed to express murine MHC class I proteins so that the cells would be recognized and lysed by well-characterized murine anti-HSV CTL unless antigen presentation was blocked by ICP47. Anti-HSV CD8⁺ CTL effectively lysed pig and primate cells infected with a recombinant HSV-1 ICP47⁻ mutant but were unable to lyse pig or primate cells infected with a recombinant HSV-1 that expressed ICP47. Therefore, pigs, dogs, and monkeys may be useful animal models in which to test the effects of ICP47 on HSV pathogenesis or the use of ICP47 as a selective immunosuppressive agent.Herpes simplex virus (HSV) infection of human fibroblasts leads to inhibition of antigen presentation to CD8⁺ T cells so that the virus-infected fibroblasts are not lysed by cytotoxic T lymphocytes (CTL) (10, 12, 14). The principal reason for this resistance to CTL appears to be the expression of an HSV immediate-early protein, ICP47, which causes major histocompatibility complex (MHC) class I proteins to accumulate in infected cells in a peptide-empty form (19). ICP47 was subsequently shown to inhibit the transporter associated with antigen presentation (TAP), which functions to translocate antigenic peptides across the membrane of the endoplasmic reticulum (ER) (3, 8), and without antigenic peptides, MHC class I proteins accumulate in the ER. More recent results demonstrated that ICP47 blocks peptide binding to TAP by binding with high affinity to a domain of TAP that includes the peptide binding site (1, 15).Although HSV type 1 (HSV-1) ICP47 (ICP47-1) effectively blocks TAP in human fibroblasts, it inhibits TAP little, if at all, in a variety of mouse cells unless applied in high concentrations (1, 3, 15, 19). Similarly, HSV-2 ICP47 (ICP47-2), which has only 42% amino acid identity with ICP47-1 (4), effectively blocks human TAP but inhibits murine TAP less effectively (16). Inhibition of murine TAP with these proteins occurs at ICP47-1 and ICP47-2 concentrations 50- to 100-fold higher than those required to inhibit human TAP. ICP47-1 and ICP47-2 bind poorly to mouse TAP (15, 16), which explains their inability to block peptide transport and antigen presentation in mouse cells.We were interested in extending the study of the species specificity of ICP47 for several reasons. Firstly, we wanted to find an animal model with which to assess the effects of ICP47 in vivo, both to assess its role in virus-host interactions and to provide a model for the use of ICP47 in autoimmunity, in transplantation, and in gene therapy vectors. Secondly, we wanted to determine whether ICP47 was functional in the species currently widely used for HSV pathogenesis and vaccine studies—mice, rabbits, and guinea pigs. Thirdly, we were interested in the mechanism of the extraordinary virulence of HSV in owl monkeys (aotus), speculating that the TAP in this New World primate might be exceptionally susceptible to ICP47.In order to assess the effects of ICP47 on the TAPs of various species, cells were permeabilized, recombinant ICP47-1 and ICP47-2 were introduced into the cells, and assays of TAP activity were performed. To examine the effects of ICP47 on antigen presentation and recognition by CD8⁺ T cells, fibroblasts were infected with recombinant HSV-1 that expresses mouse class I proteins and not ICP47, and lysis of the cells by mouse anti-HSV CTL was tested. We found that ICP47-1 and ICP47-2 did not block TAP in mouse, rat, guinea pig, or rabbit skin fibroblasts but effectively inhibited TAP and antigen presentation in pig, dog, cow, and monkey fibroblasts. Therefore, pigs, dogs, and monkeys can be used to study the in vivo effects of ICP47, though for several reasons, the use of pigs might be a practical starting point.     

Tegument-specific, virus-reactive CD4 T cells localize to the cornea in herpes simplex virus interstitial keratitis in humans

Herpes stromal keratitis (HSK) is a prevalent and frequently vision-threatening disease associated with herpes simplex virus type 1 (HSV-1) infection. In mice, HSK progression occurs after viral clearance and requires T cells and neutrophils. One model implicates Th1-like CD4 T cells with cross-reactivity between the HSV-1 protein UL6 and a corneal autoantigen. HSK can be prevented by establishing specific immunological tolerance. However, HSK can also occur in T-cell receptor-transgenic X SCID mice lacking HSV-specific T cells. To study the pathogenesis of HSK in the natural host species, we measured local HSV-specific T-cell responses in HSK corneas removed at transplant surgery (n = 5) or control corneas (n = 2). HSV-1 DNA was detected by PCR in two specimens. HSV-specific CD4 T cells were enriched in three of the five HSK specimens and were not detectable in the control specimens. Reactivity with peptide epitopes within the tegument proteins UL21 and UL49 was documented. Responses to HSV-1 UL6 were not detected. Diverse HLA DR and DP alleles restricted these local responses. Most clones secreted gamma interferon, but not interleukin-5, in response to antigen. HSV-specific CD8 cells were also recovered. Some clones had cytotoxic-T-lymphocyte activity. The diverse specificities and HLA-restricting alleles of local virus-specific T cells in HSK are consistent with their contribution to HSK by a proinflammatory effect.     

Characterization of strain HSZP of herpes simplex virus type 1 (HSV1)

The genetic background of HSZP virus, an HSV1 strain with extensive passage history, was analyzed by parallel comparative sequencing of four relevant genes (UL27/gB, UL41/vhs, UL44/gC and UL53/gK) of HSZP and additional three selected viruses [strains ANGpath, strains KOS(a) and KOS(b) and the prototype strain 17]. Mutation at position 858 (His for Arg) in gB of HSZP was found to be responsible for giant cell formation (syn ³gB mutation) similarly as the 855 mutation (Val for Ala) in the gB of ANGpath. Nosyn ¹gK mutations were detected in the UL53 gene either of HSZP or of ANGpath viruses. The reduced virulence of HSZP for adult mice after peripheral inoculation, similarly as that of KOS virus, seems to be related (at least in part) to numerous mutations in the gB ectodomain. Of these, two mutations located in the antigenic domain IV were the same in gB^HSZP as well as in gB^KOS (at amino acids 59 and 79), at least two (amino acids 313 and 553) were specific for gB^KOS, while one mutation (Ser for Ala at position 108) was specific for gB^HSZP. The abolished shutoff function of the HSZP virus was related to at least four out of six specific mutations seen in thevhs polypeptide (vhs ^HSZP) encoded by the UL41 gene, of which three (amino acids 374, 386, 392) were clustered in the semiconservative box A ofvhs ^HSZP (the truncation of which abrogates the inhibition provided by this protein) and one mutation (at amino acid 18) was situated in the highly conservative locus I ofvhs ^HSZP. In addition, the twovhs ^KOS specific mutations (amino acids 19 and 317) not found invhs ^HSZP, enhanced the early host shutoff function of thevhs ^KOS protein. Finally, gC^HSZP had two specific mutations (amino acids 137 and 147) located in the antigenic domain II of gC, which is responsible for binding of HSV1 virions to the glycoso-aminoglycan (GAG) receptor. When expressed in Sf21 cells using the recombinatt baculovirus system (Bac-to-Bac), gC^HSZP and gC^KOS showed no essential antigenic differences. Presented at theInternational Conference on Recent Problems in Microbiology and Immunology, Košice (Slovakia), 13–15 October 1999.     

Latent HIV-1 infection of resting CD4⁺ T cells in the humanized Rag2⁻/⁻ γc⁻/⁻ mouse

Persistent human immunodeficiency virus type 1 (HIV-1) infection of resting CD4⁺ T cells, unaffected by antiretroviral therapy (ART), provides a long-lived reservoir of HIV infection. Therapies that target this viral reservoir are needed to eradicate HIV-1 infection. A small-animal model that recapitulates HIV-1 latency in resting CD4⁺ T cells may accelerate drug discovery and allow the rational design of nonhuman primate (NHP) or human studies. We report that in humanized Rag2^−/− γ_c^−/− (hu-Rag2^−/− γ_c^−/−) mice, as in humans, resting CD4⁺ T cell infection (RCI) can be quantitated in pooled samples of circulating cells and tissue reservoirs (e.g., lymph node, spleen, bone marrow) following HIV-1 infection with the CCR5-tropic JR-CSF strain and suppression of viremia by ART. Replication-competent virus was recovered from pooled resting CD4⁺ T cells in 7 of 16 mice, with a median frequency of 8 (range, 2 to 12) infected cells per million T cells, demonstrating that HIV-1 infection can persist despite ART in the resting CD4⁺ T cell reservoir of hu-Rag2^−/− γ_c^−/− mice. This model will allow rapid preliminary assessments of novel eradication approaches and combinatorial strategies that may be challenging to perform in the NHP model or in humans, as well as a rigorous analysis of the effect of these interventions in specific anatomical compartments.     

Development of a glycoprotein D-expressing dominant-negative and replication-defective herpes simplex virus 2 (HSV-2) recombinant viral vaccine against HSV-2 infection in mice

Using the T-REx (Invitrogen, California) gene switch technology and a dominant-negative mutant polypeptide of herpes simplex virus 1 (HSV-1)-origin binding protein UL9, we previously constructed a glycoprotein D-expressing replication-defective and dominant-negative HSV-1 recombinant viral vaccine, CJ9-gD, for protection against HSV infection and disease. It was demonstrated that CJ9-gD is avirulent following intracerebral inoculation in mice, cannot establish detectable latent infection following different routes of infection, and offers highly effective protective immunity against primary HSV-1 and HSV-2 infection and disease in mouse and guinea pig models of HSV infections. Given these favorable safety and immunological profiles of CJ9-gD, aiming to maximize levels of HSV-2 glycoprotein D (gD2) expression, we have constructed an ICP0 null mutant-based dominant-negative and replication-defective HSV-2 recombinant, CJ2-gD2, that contains 2 copies of the gD2 gene driven by the tetracycline operator (tetO)-bearing HSV-1 major immediate-early ICP4 promoter. CJ2-gD2 expresses gD2 as efficiently as wild-type HSV-2 infection and can lead to a 150-fold reduction in wild-type HSV-2 viral replication in cells coinfected with CJ2-gD2 and wild-type HSV-2 at the same multiplicity of infection. CJ2-gD2 is avirulent following intracerebral injection and cannot establish a detectable latent infection following subcutaneous (s.c.) immunization. CJ2-gD2 is a more effective vaccine than HSV-1 CJ9-gD and a non-gD2-expressing dominant-negative and replication-defective HSV-2 recombinant in protection against wild-type HSV-2 genital disease. Using recall response, we showed that immunization with CJ2-gD2 elicited strong HSV-2-specific memory CD4(+) and CD8(+) T-cell responses. Collectively, given the demonstrated preclinical immunogenicity and its unique safety profiles, CJ2-gD2 represents a new class of HSV-2 replication-defective recombinant viral vaccines in protection against HSV-2 genital infection and disease.     

Herpes simplex virus virion host shutoff (vhs) activity alters periocular disease in mice

During lytic infection, the virion host shutoff (vhs) protein of herpes simplex virus (HSV) mediates the rapid degradation of RNA and shutoff of host protein synthesis. In mice, HSV type 1 (HSV-1) mutants lacking vhs activity are profoundly attenuated. HSV-2 has significantly higher vhs activity than HSV-1, eliciting a faster and more complete shutoff. To examine further the role of vhs activity in pathogenesis, we generated an intertypic recombinant virus (KOSV2) in which the vhs open reading frame of HSV-1 strain KOS was replaced with that of HSV-2 strain 333. KOSV2 and a marker-rescued virus, KOSV2R, were characterized in cell culture and tested in an in vivo mouse eye model of latency and pathogenesis. The RNA degradation kinetics of KOSV2 was identical to that of HSV-2 333, and both showed vhs activity significantly higher than that of KOS. This demonstrated that the fast vhs-mediated degradation phenotype of 333 had been conferred upon KOS. The growth of KOSV2 was comparable to that of KOS, 333, and KOSV2R in cell culture, murine corneas, and trigeminal ganglia and had a reactivation frequency similar to those of KOS and KOSV2R from explanted latently infected trigeminal ganglia. There was, however, significantly reduced blepharitis and viral replication within the periocular skin of KOSV2-infected mice compared to mice infected with either KOS or KOSV2R. Taken together, these data demonstrate that heightened vhs activity, in the context of HSV-1 infection, leads to increased viral clearance from the skin of mice and that the replication of virus in the skin is a determining factor for blepharitis. These data also suggest a role for vhs in modulating host responses to HSV infection.     

The dominant-negative herpes simplex virus type 1 (HSV-1) recombinant CJ83193 can serve as an effective vaccine against wild-type HSV-1 infection in mice

By selectively regulating the expression of the trans-dominant-negative mutant polypeptide UL9-C535C, of herpes simplex virus type 1 (HSV-1) origin binding protein UL9 with the tetracycline repressor (tetR)-mediated gene switch, we recently generated a novel replication-defective and anti-HSV-specific HSV-1 recombinant, CJ83193. The UL9-C535C peptides expressed by CJ83193 can function as a potent intracellular therapy against its own replication, as well as the replication of wild-type HSV-1 and HSV-2 in coinfected cells. In this report, we demonstrate that CJ83193 cannot initiate acute productive infection in corneas of infected mice nor can it reactivate from trigeminal ganglia of mice latently infected by CJ83193 in a mouse ocular model. Given that CJ83193 is capable of expressing the viral alpha, beta, and gamma1 genes but little or no gamma2 genes, we tested the vaccine potential of CJ83193 against HSV-1 infection in a mouse ocular model. Our studies showed that immunization with CJ83193 significantly reduced the yields of challenge HSV in the eyes and trigeminal ganglia on days 3, 5, and 7 postchallenge. Like in mice immunized with the wild-type HSV-1 strain KOS, immunization of mice with CJ83193 prevents the development of keratitis and encephalitis induced by corneal challenge with wild-type HSV-1 strain mP. Delayed-type hypersensitivity (DTH) assays demonstrate that CJ83193 can elicit durable cell-mediated immunity at the same level as that of wild-type HSV-1 and is more effective than that induced by d27, an HSV-1 ICP27 deletion mutant. Moreover, mice immunized with CJ83193 developed strong, durable HSV-1-neutralizing antibodies at levels at least twofold higher than those induced by d27. The results presented in this report have shed new light on the development of effective HSV viral vaccines that encode a unique safety mechanism capable of inhibiting the mutant's own replication and that of wild-type virus.