Purpose To review the changing epidemiology of herpes simplex virus infection, emerging patterns of herpetic ocular disease, and the challenges and promise of herpes simplex virus vaccine therapy. Design Perspective. Methods Literature review. Results An epidemic increase in genital herpes simplex type 2 infection is reflected in a 30% increase in HSV-2 antibodies in the United States since 1976. Approximately one in four people in the United States over age 30 is infected with HSV-2. Primary acquisition of herpes simplex type 1 is becoming progressively delayed in many industrialized countries, in contrast to developing nations where the virus is acquired early in life and is ubiquitous. Changes in sexual behavior among young adults have been associated with a recent increase in genital HSV-1 infection, resulting from oral-genital rather than genital-genital contact. Clinical trials of HSV vaccines using selected herpes simplex virus type 2 proteins mixed in adjuvant have shown limited efficacy in seronegative women, but not in men. Conclusions The recent epidemic of genital herpes simplex type 2 infection is likely to result in an increase in neonatal ocular herpes and in delayed cases of acute retinal necrosis syndrome. The increase in genital HSV-1 may lead to industry production of vaccines that contain components of both HSV-1 and HSV-2 targeted toward limiting genital disease and transmission. As newer herpes simplex vaccines become available, ophthalmologists must be vigilant that a boost in immunity against HSV does not have a paradoxical effect in exacerbating break-through cases that develop immune-mediated herpes simplex stromal keratitis.

The role that T cell subsets play in herpetic stromal keratitis (HSK) has been the subject of intense research efforts. While most studies implicate CD4(+) T cells as the principal cell type mediating primary corneal disease, recent reports using knockout mice have suggested that both CD4(+) and CD8(+) T cell subsets may play integral roles in modulating the disease. Furthermore, recent studies suggest that CD8(+) T cells are directly involved in maintaining virus latency in infected trigeminal ganglia. This work has addressed these discrepancies by infecting the corneas of mice lacking CD4(+) and CD8(+) T cells with herpes simplex virus type 1 (HSV-1) and monitoring both corneal disease and latent infection of trigeminal ganglia. Results indicated that mice lacking CD8(+) T cells had more severe corneal disease than either BALB/c or B6 parental strains. In contrast, mice lacking CD4(+) T cells had a milder disease than parental strains. When mice were evaluated for persistence of infectious virus, only transient differences were observed in periocular tissue and corneas. No significant differences were found in persistence of virus in trigeminal ganglia or virus reactivation from explanted ganglia. These data support the following conclusions. CD4(+) T cells are not required for resistance to infection with HSV-1 and probably mediate HSK. Mice lacking CD8(+) T cells do not display differences in viral loads or reactivation and thus CD8(+) T cells are not absolutely required to maintain latency. Finally, CD8(+) T cells probably play a protective role by regulating the immunopathological response that mediates HSK.

Phosphorylation of the herpes simplex virus (HSV) VP22 protein is regulated by cellular kinases and the UL13 viral kinase, but the sites at which these enzymes induce phosphorylation of HSV-2 VP22 are not known. Using serine-to-alanine mutants to map phosphorylation sites on HSV-2 VP22 in cells, we made three major observations. First, phosphorylation by a cellular kinase mapped to serines 70, 71, and/or 72 within CKII consensus sites analogous to previously identified phosphorylation sites in HSV-1 VP22. Second, we mapped UL13-mediated phosphorylation of HSV-2 VP22 to serines 28 and 34, describing for the first time UL13-dependent phosphorylation sites on VP22. Third, previously identified VP22-associated cellular kinase sites in HSV-1 VP22 (serines 292 and 294) were not phosphorylated in HSV-2 VP22 (serines 291 and 293). VP22 expressed alone accumulated in the cytoplasm and to a lesser extent in the nucleus. Phosphorylation by endogenous cellular kinase(s) did not alter the localization of VP22. Co-expression of HSV-2 VP22 with active UL13, but not with enzymatically inactive UL13, resulted in nuclear accumulation of VP22 and altered nuclear morphology. Surprisingly, redistribution of VP22 to the nucleus occurred independently of UL13-induced phosphorylation of VP22. The altered nuclear morphology of UL13-expressing cells was not due to apoptosis. These results demonstrate that phosphorylation of HSV-2 VP22 at multiple serine residues is induced by UL13 and cellular kinase(s), and that the nuclear/cytoplasmic distribution of VP22 is independent of its phosphorylation status but is controlled indirectly by UL13 kinase activity.

The herpes simplex virus 2 (HSV-2) virion host shutoff (vhs) protein is a ribonuclease contained in the virion tegument. vhs-deficient mutants of HSV-2 are profoundly attenuated in vivo, and we have previously shown that replication and virulence of vhs-deficient HSV-2 are largely restored to levels of wild-type virus in mice lacking the interferon alpha/beta receptor (IFNalphabetaR(-/-)). This result demonstrated that HSV-2 vhs interferes with the type I IFN response, but whether vhs inhibits production of type I IFN or synthesis or function of key mediators of the IFN-induced antiviral state was not clear. Here we address these questions using primary murine embryonic fibroblasts (MEFs), which produce and respond to IFNalphabeta. The vhs-deficient HSV-2 strain 333d41 replicated similarly to wild-type virus (333 clone SB5) and vhs rescue virus (333d41(R)) after infection of MEFs at high moi, but at low moi, 333d41 replication was severely attenuated, recapitulating the attenuated phenotype of vhs-deficient HSV-2 in vivo. Replication of 333d41 at low moi was restored to levels of wild-type virus in MEFs lacking the IFNalphabeta receptor or when IFNalphabeta was neutralized, thus establishing the IFNalphabeta response as the sole mechanism attenuating vhs-deficient HSV-2 replication in MEFs. MEFs infected with 333d4l produced >50-fold more IFNalphabeta than cells infected with 333 and 333d41(R). Pretreatment of MEFs with type I IFN inhibited replication of 333d41 more than 333 and 333d41(R), indicating that vhs also interferes with activation of the IFNalpha-induced antiviral response. We therefore examined vhs interference with PKR and RNase L, two key mediators of the IFNalphabeta response. 333d41 replication was restored to wild-type levels after low moi infection of PKR-/- and RNase L-/- MEFs, and was not inhibited in PKR-/- MEFs pretreated with IFNalpha. Together, these observations indicate that HSV-2 vhs is a broad and potent countermeasure to the IFN-mediated antiviral response in IFN-naive and -sensitized MEFs. (C) 2004 Elsevier Inc. All rights reserved.

ABSTRACT T-cell costimulation molecules B7-1 and B7-2 play an important role in activation of T cells to cytolytic effector function and production of cytokines. Interaction with B7 also causes T cells to upregulate surface molecules, such as CD40L, that effectively stimulate antibody responses in conjunction with cytokines. We have shown that mice lacking both B7-1 and B7-2 (B7KO mice), when infected intravaginally with virulent herpes simplex virus type 2 (HSV-2), developed more severe disease and higher mortality than their wild-type counterparts. We have now investigated the effects of B7 costimulation deficiency on induction of immune responses to HSV-2 infection of the genital tract. Fewer gamma interferon (IFN-γ)-producing T cells were present in the genital lymph nodes of B7KO mice compared to wild-type mice, either acutely after primary infection or in recall responses. Less IFN-γ and especially interleukin-10 were produced by B7KO mice, and cytolytic T-lymphocyte activity was also attenuated. Reduced expression of CD25 on CD4+ T cells after infection of B7KO mice was consistent with deficits in T-cell activation to effector functions. Although HSV-specific immunoglobulin M (IgM) titers were comparable for both B7KO mice and wild-type mice, B7KO mice had significant deficits in HSV-specific serum IgG responses, with markedly reduced levels of IgG2a and IgG1. In addition, significantly less IgG was detected in the vaginal secretions of B7KO mice than in those from wild-type mice. CD4+ T-cell expression of CD40L was depressed in B7KO mice in vivo and in vitro. Together with reduced cytokine production, these results suggest a mechanism for decreased IgG class switching or production. Thus, in the absence of B7 costimulation, naïve T cells fail to undergo proper activation in response to HSV-2, which limits T-cell cytokine production, cytotoxic T lymphocyte activity, and provision of help for class-switched antibody responses.

The herpes simplex virus (HSV) virion host shutoff (vhs) protein, the product of the UL41 (vhs) gene, is an important determinant of HSV virulence. vhs has been implicated in HSV interference with host antiviral immune responses, down-regulating expression of major histocompatibility complex molecules to help HSV evade host adaptive immunity. The severe attenuation of vhs-deficient viruses in vivo could reflect their inability to escape immune detection. To test this hypothesis, BALB/c or congenic SCID mice were infected intravaginally (i.vag.) with the HSV type 2 (HSV-2) vhs null mutant 333d41 or the vhs rescue virus 333d41(R). vhs-deficient virus remained severely attenuated in SCID mice compared with rescue virus, indicating that vhs regulation of adaptive immune responses does not influence HSV pathogenesis during acute infection. Innate antiviral effectors remain intact in SCID mice; prominent among these is alpha/beta interferon (IFN-alpha/beta). The attenuation of HSV-2 vhs mutants could reflect their failure to suppress IFN-alpha/beta-mediated antiviral activity. To test this hypothesis, 129 and congenic IFN-alpha/beta receptor-deficient (IFN-alpha/betaR(-/-)) mice were infected i.vag. with wild-type virus, vhs null mutants 333-vhsB or 333d41, or the vhs rescue virus 333d41(R). Whereas vhs-deficient viruses showed greatly reduced replication in the genital mucosa of 129 mice compared with wild-type or vhs rescue viruses, they were restored to nearly wild-type levels of replication in IFN-alpha/betaR(-/-) mice over the first 2 days postinfection. Only wild-type and vhs rescue viruses caused severe genital disease and hind limb paralysis in 129 mice, but infection of IFN-alpha/betaR(-/-) mice restored the virulence of vhs-deficient viruses. vhs-deficient viruses replicated as vigorously as wild-type and rescue viruses in the nervous systems of IFN-alpha/betaR(-/-) mice. Restoration was specific for the vhs mutation, because thymidine kinase-deficient HSV-2 did not regain virulence or the capacity to replicate in the nervous systems of IFN-alpha/betaR(-/-) mice. Furthermore, the defect in the IFN-alpha/beta response was required for restoration of vhs-deficient virus replication and virulence, but the IFN-alpha/beta-stimulated protein kinase R pathway was not involved. Finally, vhs of HSV-2 has a unique capacity to interfere with the IFN-alpha/beta response in vivo, because an HSV-1 vhs null mutant did not recover replication and virulence after i.vag. inoculation into IFN-alpha/betaR(-/-) mice. These results indicate that vhs plays an important role early in HSV-2 pathogenesis in vivo by interfering with the IFN-alpha/beta-mediated antiviral response.

Herpes simplex virus type 2 (HSV-2) is responsible for most cases of genital herpes and also can cause fatal disseminated disease in perinatally infected newborns. Sexually transmitted infections initiate in the skin or mucosa and quickly spread into peripheral nerves to establish latency. Innate immunity, the first line of defense during both primary and recurrent infection, is essential during this period of acute infection to limit initial viral replication and to facilitate an appropriate adaptive immune response. The innate immune response consists of a complex multilayered system of mechanical and secreted defenses, immediate chemokine and IFN responses, and rapidly recruited cellular defenses. HSV has devised equally elaborate strategies to evade or interfere with innate immunity. This review summarizes our current understanding of the innate immune responses to HSV-2 and the mechanisms by which HSV-2 can overcome these barriers. Newly emerging links between products of innate responses and the development of adaptive immune responses are also discussed.

ABSTRACT During lytic infection, the virion host shutoff (vhs) protein mediates the rapid degradation of mRNA and the shutoff of host protein synthesis. In vivo, herpes simplex virus type 1 (HSV-1) mutants lacking vhs activity are profoundly attenuated. Homologs of vhs exist in all of the neurotropic herpesviruses, and the goal of this study was to determine the virulence of HSV-2 mutants lacking vhs. Two HSV-2 recombinants were used in this study: 333-vhsB, which has a lacZ cassette inserted into the N terminus of vhs, and 333d41, which has a 939-bp deletion in vhs. As expected, both 333-vhsB and 333d41 failed to induce the cellular RNA degradation characteristic of HSV. Corneal, vaginal, and intracerebral routes of infection were used to study pathogenesis. Both viruses grew to significantly lower titers in the corneas, trigeminal ganglia, vaginas, dorsal root ganglia, spinal cords, and brains of mice than wild-type and rescue viruses, with a correspondingly reduced induction of disease. Both viruses, however, reactivated efficiently from explanted trigeminal ganglia, showing that vhs is dispensable for reactivation. The lethality of 333d41 following peripheral infection of mice, however, was significantly higher than that of 333-vhsB, suggesting that some of the attenuation of 333-vhsB may be due to the presence of a lacZ cassette in the vhs locus. Taken together, these data show that vhs represents an important determinant of HSV-2 pathogenesis and have implications for the design of HSV-2 recombinants and vaccines.

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.

Virion host shutoff (vhs)-deficient herpes simplex virus (HSV) was tested as a therapeutic vaccine in a mouse model of UV light-induced recurrent herpetic stromal keratitis. Four weeks after primary corneal infection, mice were vaccinated intraperitoneally with vhs(-) vaccine or control. Four weeks after vaccination, the eyes of latently infected mice were UV-B irradiated to induce recurrent virus shedding and disease. Post-irradiation corneal opacity in latently infected, vhs(-)-vaccinated mice was significantly reduced compared to control-vaccinated mice (P=0.007 to 0.035). The incidence and duration of recurrent virus shedding were the same in both groups. Antibody titres were increased (P=0.05) and delayed type hypersensitive responses were unaffected by vhs(-) vaccination. Combined with studies using different vaccination timing and vhs(-) genotypes, these data suggest that deletion of vhs is a useful strategy in the development of a therapeutic HSV vaccine, and that temporal and genetic factors influence vaccination outcome.

We have used mice lacking both B7-1 and B7-2 costimulation molecules (B7KO) to investigate the effects of B7 costimulation on herpes simplex virus type 2 (HSV-2) pathogenesis. B7KO mice infected intravaginally with virulent HSV-2 showed more severe genital and neurologic disease and higher mortality rates than their wild-type counterparts. These results suggest that B7 costimulation molecules play an important role in the development of primary immune responses protective against HSV-2.

Some subunit vaccines composed of herpes simplex virus (HSV) glycoproteins have been shown to protect guinea pigs against primary and recurrent genital infection by HSV-2. However, these vaccines were ineffective or only marginally effective in clinical trials. To attempt to define an animal model that would better discriminate the protective capacity of different vaccine formulations, we have examined the requirements for vaccine-induced protection against HSV-2 infection and disease in a mouse genital model. Unlike the guinea pig model where inactivated viral vaccines can protect nearly as well as live viral vaccines, inactivated viral vaccine afforded little protection in this mouse model. Using replication-defective mutant viruses as a form of live viral vaccine, we found that the extent of protection conferred by live vaccine was proportional to the amount of replication-defective mutant virus inoculated, over doses from 10(4) to 10(6) PFU. Furthermore, the mouse genital model showed quantitative differences in the degree of protection induced by various viral vaccine constructs. An HSV-2 replication-defective mutant virus protected better than an HSV-1 replication-defective mutant that expressed HSV-2 glycoprotein D, which in turn protected better than an HSV-2 replication-defective mutant virus. We conclude that this mouse genital model can rank different vaccine constructs for their capacity to induce protective immunity. Thus, genital infection of the mouse with HSV-2 may provide a stringent animal model that can predict the relative capacity of viral vaccines to stimulate protective immunity against HSV-2.

ABSTRACTHerpes simplex type virus 2 (HSV-2) is a sexually transmitted pathogen that causes genital lesions and spreads to the nervous system to establish acute and latent infections. Systemic but not mucosal cellular and humoral immune responses are elicited by immunization of mice with a replication-defective mutant of HSV-2, yet the mice are protected against disease caused by subsequent challenge of the genital mucosa with virulent HSV-2. In this study, we investigated the role of immune serum antibody generated by immunization with a replication-defective HSV-2 vaccine prototype strain in protection of the genital mucosa and the nervous system from HSV-2 infection. Passive transfer of replication-defective virus-immune serum at physiologic concentrations to SCID or B-cell-deficient mice had no effect on replication of challenge virus in the genital mucosa but did significantly reduce the incidence and severity of genital and neurologic disease. In contrast, B-cell-deficient mice immunized with replication-defective HSV-2 were able to control replication of challenge virus in the genital mucosa, but not until 3 days postchallenge, and were not completely protected against genital and neurologic disease. Passive transfer of physiologic amounts of immune serum to immunized, B-cell-deficient mice completely restored their capacity to limit replication of challenge virus in the genital mucosa and prevented signs of genital and systemic disease. In addition, the numbers of viral genomes in the lumbosacral dorsal root ganglia of immunized, B-cell-deficient mice were dramatically reduced by transfer of immune serum prior to challenge. These results suggest that there is an apparent synergism between immune serum antibody and immune T cells in achieving protection and that serum antibody induced by vaccination with replication-defective virus aids in reducing establishment of latent infection after genital infection with HSV-2.

ABSTRACT The VP22 protein of herpes simplex virus type 2 (HSV-2) is a major component of the virion tegument. Previous work with HSV-1 indicated that VP22 is phosphorylated during infection, and phosphorylation may play a role in modulating VP22 localization in infected cells. It is not clear, however, when phosphorylation occurs in infected cells or how it is regulated. Less is known about the synthesis and phosphorylation of HSV-2 VP22. To study the complete biosynthetic history of HSV-2 VP22, we generated a monoclonal antibody to the carboxy terminus of VP22. Using immunoprecipitation and Western blot analyses, we show that HSV-2 VP22 can be found in three distinct isoforms in infected cells, two of which are phosphorylated. Like HSV-1 VP22, HSV-2 VP22 is synthesized ca. 4 h after infection, and the isoform later incorporated into virions is hypophosphorylated. In addition, we demonstrate for the first time (i) that newly synthesized VP22 is phosphorylated rapidly after synthesis, (ii) that this phosphorylation occurs in a virus-dependent manner, (iii) that the HSV-2 kinase UL13 is capable of inducing phosphorylation of VP22 in the absence of other viral proteins, (iv) that phosphorylated VP22 is very stable in infected cells, (v) that phosphorylated isoforms of VP22 are gradually dephosphorylated late in infection to produce the virion tegument form, and (vi) that this dephosphorylation occurs independently of viral DNA replication or virion assembly. These results indicate that HSV-2 VP22 is a stable protein that undergoes highly regulated, virus-dependent phosphorylation events in infected cells.

ABSTRACTThe 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.

IL-18 induces IFN-gamma and NK cell cytotoxicity, making it a logical target for viral antagonism of host defense. We demonstrate that the ectromelia poxvirus p13 protein, bearing homology to the mammalian IL-18 binding protein, binds IL-18, and inhibits its activity in vitro. Binding of IL-18 to the viral p13 protein was compared with binding to the cellular IL-18R, The dissociation constant of p13 For murine IL-18 is 5 nM, compared with 0.2 nM for the cellular receptor heterodimer. Mice infected with a p13 deletion mutant of ectromelia virus had elevated cytotoxicity for YAC-1 tumor cell targets compared with control animals. Additionally, the p13 deletion mutant virus exhibited decreased levels of infectivity. Our data suggest that inactivation of IL-18, and subsequent impairment of NK cell cytotoxicity, may be one mechanism by which ectrometia evades the host immune response.

The distribution of labeled neurons in the brain and spinal cord was studied after injecting the Bartha strain of pseudorabies virus (PRV) into the sciatic nerve to provide a baseline for studying neural circuitry after spinal cord injury (SCI) and regeneration. Following a single injection of viral particles into the left sciatic nerve, PRV labeling was found in the spinal cord at 2 days post-injection (p.i.). Increasing complexity in viral labeling from the spinal cord to supraspinal regions became apparent with increasing survival time. In brain regions, several neuronal groups that regulate sympathetic outflow, such as the rostroventrolateral medulla, the lateral paragigantocellular nuclei, and the A5 cells, were densely labeled. However, relatively sparse labeling was noticed in the lateral vestibular nuclei, the red nucleus and the motor cortex whose spinal projections regulate somatic motor function, although those areas were abundantly labeled with Fast blue (FB) in a double-labeling experiment in which FB was co-injected into the lumbar cord. The pattern of viral labeling became more complex beyond 5 days p.i. when increased numbers of cell groups were labeled with PRV but not FB. In addition, some infected neurons started to lyse, as evidenced by a decrease in viral labeling at 7 days p.i. Thus, the 5th day post-viral injection would appear to be an appropriate survival time to obtain maximal labeling with acceptable specificity. We suggest that transneuronal labeling using PRV should be appropriate for studying multi-neural circuitry after SCI and regeneration.

Herpes simplex virus (HSV) most frequently initiates infection at a mucosal surface; thus mucosal immune responses are likely to be important in defense against HSV infection. We have examined the effects of eliciting mucosal as well as systemic immune responses on protection against genital challenge infection with virulent HSV-2 in mice immunized with a replication-defective mutant of HSV-2. In addition, we have examined the types of immune responses elicited by immunization by the different routes under conditions known to provide protection. We observed that immunizations at parenteral and distal mucosal sites generate immune responses that have an additive effect in protection against challenge infection with virulent HSV-2. Immunization at either of these sites alone prevented paralysis and death after challenge virus infection and reduced replication of the challenge virus in the genital mucosa, although subcutaneous immunization was more effective in reducing virus replication. Simultaneous immunization at the two sites led to the greatest reduction in mucosal replication of challenge virus. The type of response generated was also affected by the route of immunization. Subcutaneous immunization results in a strong systemic immune response that is somewhat biased toward a Th1 T cell response, while intranasal immunization induces mucosal as well as systemic immunity, as evidenced by HSV-specific IgA in vaginal secretions, and a stronger bias toward a Th1 response. These results suggest that mucosal immunization may complement protective immunity against HSV-2 genital infection generated by parenteral immunization with replication-defective mutant virus.

Replication-defective mutants of herpes simplex virus 1 (HSV-1) elicit immune responses in mice that reduce acute and latent infection after corneal challenge and are protective against development of disease. To understand the basis for the protective immunity induced by this new form of immunization, we investigated the contribution of various components of the immune response to protection against corneal infection and disease. Passive transfer of sera from mice immunized with the replication-defective mutant virus,d301, its parental HSV-1 strain, or uninfected cell lysate was used to examine the role of antibody. Despite posttransfer neutralizing antibody titers equivalent to those in control mice directly immunized with mutant virus, recipients of immune serum showed no reductions in primary replication in the eye, keratitis, or latent infection of the nervous system. However, immune serum protected mice from encephalitis and death. To examine the contribution of T cell subsets to protection, mice were immunized once with mutant virus and then were depletedin vivoof CD4+or CD8+T cells prior to corneal challenge. CD4 depletion resulted in higher titers of challenge virus in the eye at 3 to 4 days after challenge compared to control mice. Latent infection of the nervous system was increased by depletion of CD4+T cells but not by depletion of CD8+T cells. Keratitis developed only in a portion of the CD8+T cell-depleted mice, suggesting that an immunopathologic potential of CD4+T cells is held in check when immune CD8+T cells are also present. Taken together, these data support a role for antibody induced by immunization with a replication-defective virus principally in protecting the central nervous system from disease, roles for CD4+T cells in reducing primary replication in the eye and protecting against latent infection of the nervous system, and a role for CD8+T cells in regulating the immunopathologic activity of CD4+T cells.