Role of T-lymphocyte subsets in recovery from herpes simplex virus infection.

Our investigations probed the nature of different T-lymphocyte subsets effecting clearance of herpes simplex virus after infection of the pinna. Cell populations from animals recently infected subcutaneously or intraperitoneally (acute population) or from animals infected 6 weeks previously (primed population) or the latter cells reimmunized in vitro with virus (memory population) were studied. Viral clearance was a function of the Lyt 1+2- subset in the acute population, but with the memory population both Lyt 1+ and Lyt 2+ cells affected clearance. In primed populations, viral clearance was effected only by the Lyt 2+ subset. The ability of the various cell populations to adoptively transfer delayed-type hypersensitivity was also studied. Only acute population cells from animals infected subcutaneously and memory population cells transferred delayed-type hypersensitivity. In both cases, the cell subtype was Lyt 1+2-. Our results demonstrated that the delayed-type hypersensitivity response does not always correlate with immunity to herpes simplex virus. Multiple subsets of T cells participate in viral clearance, and their respective importances vary according to the stage of the virus-host interaction.     

Role of NK cells in protection of mice against herpes simplex virus-1 infection

Natural killer (NK) cells have been implicated in the recognition and killing of a variety of virus infected target cells in vitro, yet their role in vivo remains uncertain. In these experiments, the role of NK cells in the regulation of resistance to herpes simplex virus-1 (HSV-1) was studied. Adult C57BL/6 mice are resistant to HSV-1 (HFEM strain), but are rendered highly susceptible by treatment with cyclophosphamide 24 hr prior to infection. In this model, passive transfer of 10(8) normal spleen cells or 10(7) poly I:C-treated spleen cells provided protection for 72% of the recipients. Spleen cells from NK cell-deficient beige mice similarly treated failed to engender passive protection. The phenotype of the cells responsible for transferring protection was NK1.1+, and asialo GM1+. Transfer of NK cells resulted in marked reduction of HSV titers in the livers and brains of recipients. These experiments provide direct evidence for a role for NK cells in protection against development of fatal HSV infection in mice.     

Role of suppressor T cells in herpes simplex virus-induced immune deviation.

Herpes simplex virus type 1 inoculated into the anterior chamber of the mouse eye induces suppression of anti-herpes simplex virus T-cell-mediated delayed hypersensitivity. This suppression is virus-specific, and mediated by splenic T lymphocytes, and it can be adoptively transferred to naive recipients.     

The immune response to ocular herpes simplex virus type 1 infection

Herpes simplex virus type 1 (HSV-1) is a prevalent microbial pathogen infecting 60% to 90% of the adult world population. The co-evolution of the virus with humans is due, in part, to adaptations that the virus has evolved to aid it in escaping immune surveillance, including the establishment of a latent infection in its human host. A latent infection allows the virus to remain in the host without inducing tissue pathology or eliciting an immune response. During the acute infection or reactivation of latent virus, the immune response is significant, which can ultimately result in corneal blindness or fatal sporadic encephalitis. In fact, HSV-1 is one of the leading causes of infectious corneal blindness in the world as a result of chronic episodes of viral reactivation leading to stromal keratitis and scarring. Significant inroads have been made in identifying key immune mediators that control ocular HSV-1 infection and potentially viral reactivation. Likewise, viral mechanisms associated with immune evasion have also been identified and will be discussed. Lastly, novel therapeutic strategies that are currently under development show promise and will be included in this review. Most investigators have taken full advantage of the murine host as a viable working in vivo model of HSV-1 due to the sensitivity and susceptibility to viral infection, ease of manipulation, and a multitude of developed probes to study changes at the cellular and molecular levels. Therefore, comments in this review will primarily be restricted to those observations pertaining to the mouse model and the assumption (however great) that similar events occur in the human condition.     

A detergent-soluble extract of virus-infected cells free from infectious virus protects mice from lethal herpes simplex virus infection

Lethal infection with herpes simplex virus types 1 (HSV-1) and 2 was effectively prevented by previous immunization with a detergent-soluble extract (DSE) of virus-infected cells free from infectious virus without any adjuvant. This protective immunity seemed to last for at least one month. Neutralizing antibodies were elicited in mice immunized with DSE, but at a lower level than in animals immunized with live or killed virus. DSE did not protect athymic nude mice from death by HSV-1 infection, suggesting that a T cell-mediated immune response plays a major role in the protection.     

Vaccine-induced serum immunoglobin contributes to protection from herpes simplex virus type 2 genital infection in the presence of immune T cells

Herpes 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.     

Protease-deficient herpes simplex virus protects mice from lethal herpesvirus infection.

Null mutants and attenuated mutants of herpes simplex virus (HSV) have been shown to induce immunity against challenge from wild-type virus. Null viruses with a defect in late gene products would be expected to express more viral genes than viruses with defects in essential early gene products and thus induce a better immune response. Herpesviruses encode a late gene product (serine protease) that is autocatalytic and cleaves the capsid assembly protein during viral replication. To determine whether a virus with a mutation in this gene could induce immunity, we constructed a recombinant virus containing the gusA reporter gene in the protease domain of the HSV type 1 UL26 open reading frame (ORF). Consistent with previous results (M. Gao, L. Matusick-Kumar, W. Hurlburt, S. F. DiTusa, W. W. Newcomb, J. C. Brown, P. J. McCann, I. Deckman, and R. J. Colonno, J. Virol. 68:3702-3712, 1994), recombinant virus could be isolated only from helper cell lines expressing the product of the UL26 ORF. Mice inoculated with the recombinant virus were unaffected by doses of virus that were lethal to mice infected with wild-type virus. Mice which were previously inoculated with the recombinant virus were also protected by a subsequent challenge with wild-type virus in a dose-dependent manner. These results indicate that recombinant viruses lacking the protease gene are avirulent but render protection from subsequent challenge.     

Role of specific innate immune responses in herpes simplex virus infection of the central nervous system

Herpes simplex virus 1 (HSV-1) causes a spectrum of disease, including herpes labialis, herpes keratitis, and herpes encephalitis, which can be lethal. Viral recognition by pattern recognition receptors plays a central role in cytokine production and in the generation of antiviral immunity. The relative contributions of different Toll-like receptors (TLRs) in the innate immune response during central nervous system infection with HSV-1 have not been fully characterized. In this study, we investigate the roles of TLR2, TLR9, UNC93B1, and the type I interferon (IFN) receptor in a murine model of HSV-1 encephalitis. TLR2 is responsible for detrimental inflammatory cytokine production following intracranial infection with HSV-1, and the absence of TLR2 expression leads to increased survival in mice. We prove that inflammatory cytokine production by microglial cells, astrocytes, neutrophils, and monocytes is mediated predominantly by TLR2. We also demonstrate that type I IFNs are absolutely required for survival following intracranial HSV-1 infection, as mice lacking the type I IFN receptor succumb rapidly following infection and have high levels of HSV in the brain. However, the absence of TLR9 does not impact survival, type I IFN levels, or viral replication in the brain following infection. The absence of UNC93B1 leads to a survival disadvantage but does not impact viral replication or type I IFN levels in the brain in HSV-1-infected mice. These results illustrate the complex but important roles that innate immune receptors play in host responses to HSV-1 during infection of the central nervous system.     

Cellular proteins expressed in herpes simplex virus transformed cells also accumulate on herpes simplex virus infection.

The cell proteins expressed in rat embryo cells transformed by herpes simplex virus (HSV) have been analysed by immunoprecipitation assays to determine those polypeptides which can be identified by immunoprecipitation with the sera of tumour-bearing animals and also with antisera to herpes simplex infected cells. Cell polypeptides commonly recognised by both these sera have been further characterised using a monoclonal antibody directed against a cellular polypeptide which accumulates on HSV-2 lytic infection. This monoclonal antibody recognises in HSV-transformed cells polypeptides of mol. wts. 90 000, 40 000 and 32 000. Further studies show that the accumulation of these polypeptides in HSV-transformed cells is not HSV specific but is a common feature of transformation or of cells which have been immortalised. We suggest that cellular polypeptides accumulating as a result of HSV infection may be of importance in the initiation of transformation by HSV, i.e., at the level of immortalisation of cells.     

Role of peptidergic neurons in ocular herpes simplex infection in mice

Peptide-containing nerve fibers (peptidergic fibers) abundantly innervate the mammalian cornea. We investigated their role in ocular herpes simplex infection in mice by using capsaicin, which causes degeneration and permanent loss of peptidergic neurons in neonates and temporary peptide depletion in adult animals. The corneas of neonatally denervated BALB/c mice were observed for capsaicin-induced keratitis at 11-14 wk of age and were then infected bilaterally with herpes simplex virus 1 (HSV-1); trigeminal (TG) ganglia were cocultivated 6 wk later to establish the rate of latent infection. We also applied capsaicin eye drops to adult BALB/c mice that had been infected with HSV-1 6 wk earlier, and measured viral shedding before, and 3 days and 2 months after, administration of capsaicin drops; TG ganglia of these animals were cocultivated at 3 days and 2 months after capsaicin application. Neurotrophic keratitis was found in 81% of neonatally denervated animals; mortality rate due to HSV-1 infection was reduced from 80% in the controls to 24% in the capsaicin-treated group, and recovery of latent virus by cocultivation was reduced by 50%. Viral shedding could not be produced by capsaicin eye drops in adult animals infected with HSV-1. However, recovery of latent virus was significantly reduced in TG ganglia sampled 3 days and 2 months after capsaicin drops were instilled. Our findings suggest 1) that peptidergic fibers play a crucial role in the establishment of trigeminal HSV-1 latency and 2) that reactivation of latently infected ganglia can be inhibited by topical capsaicin.     

Intrauterine herpes simplex virus infection

Neonatal herpes simplex virus (HSV) infection usually is the consequence of intrapartum infection, with disease onset between 5 and 15 days of life. More recently, intrauterine HSV infection has been identified. Intrauterine infection is apparent within the first 24-48 hr of life and is associated with skin vesicles/scarring, chorioretinitis, and/or hydraencephaly. The recognition that babies with these findings can have disease caused by HSV should prompt enhanced physician awareness in the evaluation of newborns with suspected intrauterine infection. The frequency of intrauterine infection appears to be about 5% of all babies with neonatal HSV infection.     

Memory-type CD8+ T cells protect IL-2 receptor alpha-deficient mice from systemic infection with herpes simplex virus type 2

IL-2Ralpha-deficient (IL-2Ralpha(-/-)) mice exhibit an impaired activation-induced cell death for T cells and develop abnormal T cell activation with age. In our study, we found that IL-2Ralpha(-/-) mice at the age of 5 wk contained an increased number of CD44(+)CD69(-)CD8(+) T cells in lymph nodes, which expressed a high intensity of IL-2Rbeta and vigorously proliferated in response to a high dose of IL-15 or IL-2. The T cells produced a large amount of IFN-gamma in response to IL-15 plus IL-12 in a TCR-independent bystander manner. When IL-2Ralpha(-/-) mice were inoculated i.p. with HSV type 2 (HSV-2) 186 strain, they showed resistance to the infection accompanied by an increased level of serum IL-15. The depletion of CD8(+) T cells by in vivo administration of anti-CD8 mAb rendered IL-2Ralpha(-/-) mice susceptible to HSV-2-induced lethality. These results suggest that memory-type CD8(+) T cells play a novel role in the protection against HSV-2 infection in IL-2Ralpha(-/-) mice.     

Gender influences herpes simplex virus type 1 infection in normal and gamma interferon-mutant mice

Gender influences the incidence and severity of some bacterial and viral infections and autoimmune diseases in animal models and humans. To determine a gender-based difference, comparisons were made between male and female mice inoculated with herpes simplex virus type 1 (HSV-1) by the corneal route. Mortality was higher in the male mice of the three strains tested: 129/Sv//Ev wild type, gamma interferon (IFN-gamma) knockout (GKO), and IFN-gamma receptor knockout (RGKO). Similarly, in vivo HSV-1 reactivation occurred more commonly in male mice, but the male-female difference in reactivation was restricted to the two knockout strains and was not seen in the 129/Sv//Ev control. Comparison among male mice of the three strains showed a higher mortality of the RGKO mice and a higher reactivation rate of the GKO and RGKO mice than of the 129/Sv//Ev males. In contrast, female RGKO and GKO mice did not differ from female 129/Sv//Ev controls in either mortality or reactivation. HSV-1 periocular and eyelid disease was also more severe in male and dihydrotestosterone (DHT)-treated female mice than in control female mice. These results show a consistent gender difference in HSV-1 infection, with a worse outcome in male mice. In addition, the results comparing GKO and RGKO mice to controls show differences only in male mice, suggesting that some effects of IFN-gamma, a key immunoregulatory molecule, are gender specific.     

Protection of mice from herpes simplex virus-induced retinitis by in vitro-activated immune cells.

A form of acute retinal necrosis occurred in the contralateral eyes of susceptible mice 1 week after each received a uniocular injection of live herpes simplex virus type 1 (HSV-1) in the anterior chamber. Although these mice did not develop systemic delayed hypersensitivity to virus antigens, their sera contained virus-specific antibodies at the time contralateral retinitis occurred. These findings suggest that systemic immunity might not be able to protect against contralateral retinitis. To explore this possibility further, we examined lymph nodes and spleens of intraocularly infected mice to determine whether their lymphoid tissues contained primed HSV-1-specific cytotoxic T cells. Virus-specific cytotoxic T cells were readily identified in these mice. We wondered why successful immune priming did not confer protection against HSV-1 retinitis. We examined this issue by evaluating the capacity of in vitro-generated, HSV-1-specific effector T cells to prevent retinitis by infusing these cells by various routes and at various times into mice that received an intracameral injection of HSV-1. The results revealed that virus-specific effector cells could prevent contralateral retinitis if injected intravenously or into the anterior chamber of the contralateral eye at the same time that virus was injected into one eye. However, the effector cells failed to prevent retinitis if they were injected into the same eye that received HSV-1 or if their intravenous administration was delayed until 24 h after the HSV-1 injection into the eye. We concluded that immune T cells can protect against contralateral retinal necrosis caused by uniocular injection of HSV-1 into the anterior chamber but only if they are administered during the first 24 h after virus infection. We propose that a retinitis-inducing process is set in motion during this early time interval postinfection. Once the process has been initiated and established, it is no longer susceptible to immune intervention. It would appear that mice that are susceptible to contralateral retinitis fail to mobilize a protective response quickly enough to ward off the establishment of the retinitis-inducing process and its disastrous eventuality.     

Noninvasive bioluminescence imaging of herpes simplex virus type 1 infection and therapy in living mice

Mouse models of herpes simplex virus type 1 (HSV-1) infection provide significant insights into viral and host genes that regulate disease pathogenesis, but conventional methods to determine the full extent of viral spread and replication typically require the sacrifice of infected animals. To develop a noninvasive method for detecting HSV-1 in living mice, we used a strain KOS HSV-1 recombinant that expresses firefly (Photinus pyralis) and Renilla (Renilla reniformis) luciferase reporter proteins and monitored infection with a cooled charge-coupled device camera. Viral infection in mouse footpads, peritoneal cavity, brain, and eyes could be detected by bioluminescence imaging of firefly luciferase. The activity of Renilla luciferase could be imaged after direct administration of substrate to infected eyes but not following the systemic delivery of substrate. The magnitude of bioluminescence from firefly luciferase measured in vivo correlated directly with input titers of recombinant virus used for infection. Treatment of infected mice with valacyclovir, a potent inhibitor of HSV-1 replication, produced dose-dependent decreases in firefly luciferase activity that correlated with changes in viral titers. These data demonstrate that bioluminescence imaging can be used for noninvasive, real-time monitoring of HSV-1 infection and therapy in living mice.