Reactivation of Herpes Simplex Virus Type 1 in Cells of Latently Infected Mouse Trigeminal Ganglia Cultured in Serum-Free Medium

Herpes simplex virus type 1 (HSV-1) was reactivated more rapidly in cells of latently infected mouse trigeminal ganglia which were cultured in serum-free medium (after 3.7 days of cultivation) than in those cultured in serum-containing Dulbecco's modified Eagle's medium (after 8.5 days of cultivation). The concentration of calcium ion (Ca²⁺) in the medium affected HSV-1 reactivation in ganglionic cultures, and 0.9 mM was the optimum concentration for the reactivation. Reactivation was delayed significantly in ganglia put into culture 4 months or more after infection compared with those cultured 1 month after infection.     

Herpes Simplex Virus Type 1 Glycoprotein gC Mediates Immune Evasion In Vivo

Many microorganisms encode proteins that interact with molecules involved in host immunity; however, few of these molecules have been proven to promote immune evasion in vivo. Herpes simplex virus type 1 (HSV-1) glycoprotein C (gC) binds complement component C3 and inhibits complement-mediated virus neutralization and lysis of infected cells in vitro. To investigate the importance of the interaction between gC and C3 in vivo, we studied the virulence of a gC-null strain in complement-intact and C3-deficient animals. Using a vaginal infection model in complement-intact guinea pigs, we showed that gC-null virus grows to lower titers and produces less severe vaginitis than wild-type or gC rescued virus, indicating a role for gC in virulence. To determine the importance of complement, studies were performed with C3-deficient guinea pigs; the results demonstrated significant increases in vaginal titers of gC-null virus, while wild-type and gC rescued viruses showed nonsignificant changes in titers. Similar findings were observed for mice where gC null virus produced significantly less disease than gC rescued virus at the skin inoculation site. Proof that C3 is important was provided by studies of C3 knockout mice, where disease scores of gC-null virus were significantly higher than in complement-intact mice. The results indicate that gC-null virus is approximately 100-fold (2 log₁₀) less virulent that wild-type virus in animals and that gC-C3 interactions are involved in pathogenesis.     

The Probability of In Vivo Reactivation of Herpes Simplex Virus Type 1 Increases with the Number of Latently Infected Neurons in the Ganglia

The purpose of this study was to define the relationship between herpes simplex virus (HSV) latency and in vivo ganglionic reactivation. Groups of mice with numbers of latently infected neurons ranging from 1.9 to 24% were generated by varying the input titer of wild-type HSV type 1 strain 17syn+. Reactivation of the virus in mice from each group was induced by hyperthermic stress. The number of animals that exhibited virus reactivation was positively correlated with the number of latently infected neurons in the ganglia over the entire range examined (r = 0.9852, P < 0.0001 [Pearson correlation]).     

Effects of Nerve Growth Factor and Phorbol Derivative on Reactivation of Herpes Simplex Virus Type 1 in Cultured Cells of Latently Infected Adult Mouse Trigeminal Ganglia

Reactivation of herpes simplex virus type 1 (HSV-1) occurred rapidly in cells of latently infected adult mouse trigeminal ganglia which were cultured in serum-free medium in the presence of sufficient nerve growth factor (NGF). However, HSV-1 reactivation was delayed significantly in ganglionic cultures in the absence of exogenous NGF or in cultures treated with 2-aminopurine in the presence of NGF. The delayed viral reactivation in ganglionic cultures without NGF was accelerated by treatment with phorbol myristate acetate or dibutyryl cyclic AMP. Culture conditions which affected HSV-1 reactivation did not affect replication of HSV-1 in normal ganglionic cultures.     

Preparation of Type 2 Herpes Simplex Virus Complement-Fixing Antigen

Comparable complement-fixing antigens of type 1 and type 2 herpes simplex virus were produced by extraction of infected African green monkey cells with 0.85% NaCl which was buffered at pH 9.0 with 0.05 m glycine-NaOH. The optimal antigen dilutions were higher in titrations against hyperimmune animal sera than in titrations against human sera. Complement-fixing antibody to type 2 herpes antigen was detected in 5 of 17 sera from healthy humans.     

In Vivo Immune Evasion Mediated by the Herpes Simplex Virus Type 1 Immunoglobulin G Fc Receptor

Herpes simplex virus (HSV) glycoproteins gE and gI form an immunoglobulin G (IgG) Fc receptor (FcγR) that binds the Fc domain of human anti-HSV IgG and inhibits Fc-mediated immune functions in vitro. gE or gI deletion mutant viruses are avirulent, probably because gE and gI are also involved in cell-to-cell spread. In an effort to modify FcγR activity without affecting other gE functions, we constructed a mutant virus, NS-gE₃₃₉, that has four amino acids inserted into gE within the domain homologous to mammalian IgG FcγRs. NS-gE₃₃₉ expresses gE and gI, is FcγR⁻, and does not participate in antibody bipolar bridging since it does not block activities mediated by the Fc domain of anti-HSV IgG. In vivo studies were performed with mice because the HSV-1 FcγR does not bind murine IgG; therefore, the absence of an FcγR should not affect virulence in mice. NS-gE₃₃₉ causes disease at the skin inoculation site comparably to wild-type and rescued viruses, indicating that the FcγR⁻ mutant virus is pathogenic in animals. Mice were passively immunized with human anti-HSV IgG and then infected with mutant or wild-type virus. We postulated that the HSV-1 FcγR should protect wild-type virus from antibody attack. Human anti-HSV IgG greatly reduced viral titers and disease severity in NS-gE₃₃₉-infected animals while having little effect on wild-type or rescued virus. We conclude that the HSV-1 FcγR enables the virus to evade antibody attack in vivo, which likely explains why antibodies are relatively ineffective against HSV infection.     

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes during initial entry and establishes a primary infection in the epithelium, which is followed by latent infection of neurons. After reactivation, viruses can become evident at mucocutaneous sites that appear as skin vesicles or mucosal ulcers. How HSV-1 invades skin or mucosa and reaches its receptors is poorly understood. To investigate the invasion route of HSV-1 into epidermal tissue at the cellular level, we established an ex vivo infection model of murine epidermis, which represents the site of primary and recurrent infection in skin. The assay includes the preparation of murine skin. The epidermis is separated from the dermis by dispase II treatment. After floating the epidermal sheets on virus-containing medium, the tissue is fixed and infection can be visualized at various times postinfection by staining infected cells with an antibody against the HSV-1 immediate early protein ICP0. ICP0-expressing cells can be observed in the basal keratinocyte layer already at 1.5 hr postinfection. With longer infection times, infected cells are detected in suprabasal layers, indicating that infection is not restricted to the basal keratinocytes, but the virus spreads to other layers in the tissue. Using epidermal sheets of various mouse models, the infection protocol allows determining the involvement of cellular components that contribute to HSV-1 invasion into tissue. In addition, the assay is suitable to test inhibitors in tissue that interfere with the initial entry steps, cell-to-cell spread and virus production. Here, we describe the ex vivo infection protocol in detail and present our results using nectin-1- or HVEM-deficient mice.     

Inhibition of H3K27me3-Specific Histone Demethylases JMJD3 and UTX Blocks Reactivation of Herpes Simplex Virus 1 in Trigeminal Ganglion Neurons

Herpes simplex virus 1 (HSV-1) genomes are associated with the repressive heterochromatic marks H3K9me2/me3 and H3K27me3 during latency. Previous studies have demonstrated that inhibitors of H3K9me2/me3 histone demethylases reduce the ability of HSV-1 to reactivate from latency. Here we demonstrate that GSK-J4, a specific inhibitor of the H3K27me3 histone demethylases UTX and JMJD3, inhibits HSV-1 reactivation from sensory neurons in vitro. These results indicate that removal of the H3K27me3 mark plays a key role in HSV-1 reactivation.     

Mucosal Immunity to Herpes Simplex Virus Type 2 Infection in the Mouse Vagina Is Impaired by In Vivo Depletion of T Lymphocytes

Intravaginal (IVAG) inoculation of wild-type herpes simplex virus type 2 (HSV-2) in mice causes epithelial infection followed by lethal neurological illness, while IVAG inoculation of attenuated HSV-2 causes epithelial infection followed by development of protective immunity against subsequent IVAG challenge with wild-type virus. The role of T cells in this immunity was studied by in vivo depletion of these cells with monoclonal antibodies. Three groups of mice were used for each experiment: nonimmune/challenged mice, immune/challenged mice, and immune depleted mice [immune mice depleted of a T-cell subset(s) shortly before challenge with HSV-2]. Mice were assessed for epithelial infection 24 h after challenge, virus protein in the vaginal lumen 3 days after challenge, and neurological illness 8 to 14 days after challenge. Monoclonal antibodies to CD4, CD8, or Thy-1 markedly reduced T cells in blood, spleen, and vagina, but major histocompatibility complex class II antigens were still partially upregulated in the vaginal epithelium after virus challenge, indicating that virus-specific memory T-cell function was not entirely eliminated from the vagina. Nevertheless, immune mice depleted of CD4⁺ and CD8⁺ T cells, Thy-1⁺ T cells, or CD8⁺ T cells alone had greater viral infection in the vaginal epithelium than nondepleted immune mice, indicating that T cells contribute to immunity against vaginal HSV-2 infection. All immune depleted mice retained substantial immunity to epithelial infection and were immune to neurological illness, suggesting that other immune mechanisms such as virus-specific antibody may also contribute to immunity.

Herpes simplex virus type 2 (HSV-2) is a sexually transmitted pathogen that infects the human genital tract. The prevalence of this infection is increasing worldwide, and at present over 20% of the adult U.S. population is infected with the virus (12). The virus spreads from the genital tract to the nervous system, and latent virus can persist in infected ganglia for long periods after primary infection is resolved. Activation of latent virus causes recurrent lesions in the genital tract and adjacent tissues (3). Infections are particularly severe in immunocompromised individuals and in infants who are infected during delivery through an infected birth canal. Oral treatment with acyclovir can reduce the severity of infections, but vaccination to prevent or control HSV-2 infections is highly desirable. Development of an effective vaccine to prevent genital HSV-2 infection in women is problematic at present because we do not clearly understand how to elicit strong protective immunity in the mucosa of the female genital tract. Investigations of immunity to genital HSV-2 infection in animal models are likely to play an important part in the development of a vaccine for human use. An added advantage of such investigations is that the basic information so obtained may be applicable to vaccines for other human sexually transmitted diseases.Experimental studies of host resistance to genital herpes have been carried out by using a mouse model (7–9). In this model, intravaginal (IVAG) inoculation of wild-type, thymidine kinase-expressing HSV-2 (TK⁺HSV-2) into young BALB/c mice caused epithelial infection followed by lethal neurological illness. The investigators also constructed an attenuated strain of the virus, ΔTK⁻HSV-2, that contained a partial deletion of the thymidine kinase gene (9). Unlike its wild-type counterpart, the attenuated virus inoculated IVAG caused mild inflammation in the vagina and was incapable of lethal neurological spread. Importantly, IVAG inoculation of BALB/c mice with ΔTK⁻HSV-2 induced a protective immunity to subsequent lethal challenge with TK⁺HSV-2 (9).Studies of immunity to vaginal HSV-2 infection in the young-mouse model were constrained by the relationship between vaginal infection and age (9, 21). Approximately 100% of weaned mice were susceptible to vaginal HSV-2 infection, but infection declined exponentially with increasing host age; fewer than 10% of mice were susceptible to HSV-2 at 14 to 16 weeks of age (9). However, several studies have shown that adult female mice treated with progesterone or sequentially with estradiol and Depo-Provera (E/DP-treated mice) became uniformly susceptible to vaginal HSV-2 infection (1, 13, 16). Vaginal infection of E/DP-treated mice with attenuated HSV-2 produced immunity that protected the mice against later infection by wild-type virus (16). Interestingly, 35 of 36 nonimmune mice showed immunostaining of virus proteins in the vaginal epithelium 24 h after IVAG inoculation of HSV-2, while only 1 of 9 immune mice challenged with the virus showed epithelial infection at this time (16). This indicates that virus infection or replication in the vaginal epithelium was rapidly and severely inhibited in the immune mice and suggests that local immune mechanisms in the vaginal mucosa were important in protection against challenge infection.One local immune mechanism that could prevent infection of the vaginal epithelium is neutralization of challenge virus by secreted antibody in the vaginal lumen. McDermott et al. (7) and Milligan and Bernstein (11) demonstrated immunoglobulin G (IgG) antibodies specific for HSV-2 in vaginal secretions of young immune mice; antiviral IgA either was not detected or was detected only at very low titers in vaginal fluids in these mice. More recently, Parr et al. (14) found IgG viral antibody in vaginal secretions of adult immune mice at a mean titer of 6,200, whereas the mean titer of viral secretory IgA (S-IgA) in the same secretions was only 1.9. The protective role of IgG and S-IgA in vaginal secretions of adult immune mice has also been studied (15). Unfractionated vaginal antibodies from immune and nonimmune mice and affinity-purified IgG and S-IgA from immune vaginal secretions were adjusted to their in vivo concentrations in the vagina. Neutralization of HSV-2 was studied by incubating the virus in the antibody preparations in vitro, followed by inoculation into vaginas of nonimmune test mice. Virus was neutralized by unfractionated immune antibody and by purified immune IgG but not by unfractionated nonimmune antibody or by purified immune S-IgA. To determine whether immune IgG alone could protect against vaginal HSV-2 infection in vivo, purified serum IgG from immune and nonimmune donors was passively transferred to nonimmune recipients 72 h prior to virus challenge in the vagina. Passively transferred immune IgG reduced virus infection of vaginal epithelium, shed virus protein concentrations in the vaginal lumen, and illness scores, even though the viral antibody titers in serum and vaginal secretions of recipient mice were only 29 and 8%, respectively, of those in standards prepared from actively immunized mice. Collectively, the data indicated that IgG viral antibody in vaginal secretions of immune mice provided early protection against vaginal challenge infection, probably by neutralizing virus in the vaginal lumen before it could infect the epithelium. In contrast, viral S-IgA antibody contributed relatively little to immune protection of the vagina in this model.Another immune mechanism that might reduce infection of the vaginal epithelium after viral challenge is T-cell-mediated immunity. Adoptive transfer of lymphocytes from the genital lymph nodes of immune mice protected nonimmune mice against neurological illness after vaginal challenge with wild-type HSV-2 (8). This observation indicates that virus-specific T cells, if present in sufficient numbers, can protect against neurological illness, but it remains unknown whether the T cells that are actually present in immune mice protect against either vaginal epithelial infection or neurological illness. Few T cells were present in the vaginas of normal mice (17), but the numbers of CD4⁺, CD8⁺, and Thy-1.2⁺ T cells increased markedly in the vaginas of immune mice after challenge with wild-type virus (16). Similarly, we have shown that T cells with the memory phenotype continuously recirculate through the vaginal epithelium and that the number of recirculating memory cells was markedly increased when immune mice were challenged in the vagina with HSV-2 (5). The presence of specific HSV-2 memory T cells in the vaginal epithelium of immune mice is also indicated by the rapid (less than 24 h) upregulation of major histocompatibility complex (MHC) class II antigen expression in the epithelium after vaginal challenge with HSV-2. In comparison, upregulation of MHC class II antigens was not detected in the vaginal epithelium until 3 days after a primary vaginal HSV-2 infection in nonimmune mice (16). In the present study, we used the adult mouse model to examine the effects of acute in vivo depletion of T-cell subsets in immune mice on vaginal epithelial infection and neurological illness after vaginal challenge with wild-type HSV-2.     

The Influence of Stress Factors on the Reactivation of Latent Herpes Simplex Virus Type 1 in Infected Mice

This study shows that the influence of different stress factors impacts the reactivation of latent herpes simplex virus type 1 (HSV-1) specifically in the trigeminal ganglion of infected mice. Different stress factors including hyperthermia, hypothermia, fatigue, and immunosuppression were exerted on mice infected with HSV-1. These viral antigens were then detected in the trigeminal ganglion region of infected mice under the influence of each stress factor, with hyperthermia having the most influence on reactivation. Interestingly, an increase in IL-6 was also detected in mice subjected to hyperthermia. These studies therefore suggest that stress can induce the reactivation of latent HSV-1, possibly through the induction of IL-6, in the trigeminal ganglion region of infected mice. This reveals a new insight on the pathogenesis of relapse infection of HSV-1.     

In Vivo Modulation of Vaccine-Induced Immune Responses toward a Th1 Phenotype Increases Potency and Vaccine Effectiveness in a Herpes Simplex Virus Type 2 Mouse Model

Several vaccines have been investigated experimentally in the herpes simplex virus type 2 (HSV-2) model system. While it is believed that CD4⁺-T-cell responses are important for protection in general, the correlates of protection from HSV-2 infection are still under investigation. Recently, the use of molecular adjuvants to drive vaccine responses induced by DNA vaccines has been reported in a number of experimental systems. We sought to take advantage of this immunization model to gain insight into the correlates of immune protection in the HSV-2 mouse model system and to further explore DNA vaccine technology. To investigate whether the Th1- or Th2-type immune responses are more important for protection from HSV-2 infection, we codelivered the DNA expression construct encoding the HSV-2 gD protein with the gene plasmids encoding the Th1-type (interleukin-2 [IL-2], IL-12, IL-15, and IL-18) and Th2-type (IL-4 and IL-10) cytokines in an effort to drive immunity induced by vaccination. We then analyzed the modulatory effects of the vaccine on the resulting immune phenotype and on the mortality and the morbidity of the immunized animals following a lethal challenge with HSV-2. We observed that Th1 cytokine gene coadministration not only enhanced the survival rate but also reduced the frequency and severity of herpetic lesions following intravaginal HSV challenge. On the other hand, coinjection with Th2 cytokine genes increased the rate of mortality and morbidity of the challenged mice. Moreover, of the Th1-type cytokine genes tested, IL-12 was a particularly potent adjuvant for the gD DNA vaccination.     

PKR-Dependent Xenophagic Degradation of Herpes Simplex Virus Type 1

The lysosomal pathway of autophagy is the major catabolic mechanism for degrading long-lived cellular proteins and cytoplasmic organelles. Recent studies have also shown that autophagy (xenophagy) may be used to degrade bacterial pathogens that invade intracellularly. However, it is not yet known whether xenophagy is a mechanism for degrading viruses. Previously, we showed that autophagy induction requires the antiviral eIF2alpha kinase signaling pathway (including PKR and eIF2alpha) and that this function ofeIF2alpha kinase signaling is antagonized by the herpes simplex virus (HSV-1) neurovirulence gene product, ICP34.5. Here, we show quantitative morphologic evidence of PKR-dependent xenophagic degradation of herpes simplex virions and biochemical evidence of PKR and eIF2alpha-dependent degradation of HSV-1 proteins, both of which are blocked by ICP34.5. Together, these findings indicate that xenophagy degrades HSV-1 and that this cellular function is antagonized by the HSV-1 neurovirulence gene product, ICP34.5. Thus, autophagy-related pathways are involved in degrading not only cellular constituents and intracellular bacteria, but also viruses.