Specific activation of B-cell clones within the central nervous system in course of herpes simplex encephalitis

Total and virus-specific immunoglobulin (Ig) G oligoclonal bands were studied in paired serum and cerebrospinal fluid (CSF) of four patients with herpes simplex virus type 1 (HSV-1) encephalitis. We used the isoelectric focusing in agarose gel, a sensitive technique for protein separation, followed by passive transfer of proteins on nitrocellulose paper and specific immunostaining. Oligoclonal bands were observed in serum and CSF of all patients. HSV-1-specific oligoclonal IgG bands were present in the CSF only during a limited period of the disease, having their counterpart in serum during the remaining periods. Our findings contribute to tackle the issue of B-cell activation within central nervous system and peripheral blood compartments in course of HSV-1 encephalitis.     

Protective mucosal immunity to ocular herpes simplex virus type 1 infection in mice by using Escherichia coli heat-labile enterotoxin B subunit as an adjuvant

The potential of nontoxic recombinant B subunits of cholera toxin (rCtxB) and its close relative Escherichia coli heat-labile enterotoxin (rEtxB) to act as mucosal adjuvants for intranasal immunization with herpes simplex virus type 1 (HSV-1) glycoproteins was assessed. Doses of 10 microg of rEtxB or above with 10 microg of HSV-1 glycoproteins elicited high serum and mucosal anti-HSV-1 titers comparable with that obtained using CtxB (10 microg) with a trace (0.5 microg) of whole toxin (Ctx-CtxB). By contrast, doses of rCtxB up to 100 microg elicited only meager anti-HSV-1 responses. As for Ctx-CtxB, rEtxB resulted in a Th2-biased immune response with high immunoglobulin G1 (IgG1)/IgG2a antibody ratios and production of interleukin 4 (IL-4) and IL-10 as well as gamma interferon by proliferating T cells. The protective efficacy of the immune response induced using rEtxB as an adjuvant was assessed following ocular challenge of immunized and mock-immunized mice. Epithelial disease was observed in both groups, but the immunized mice recovered by day 6 whereas mock-immunized mice developed more severe corneal disease leading to stromal keratitis. In addition, a significant reduction in the incidence of lid disease and zosteriform spread was observed in immunized animals and there was no encephalitis compared with 95% encephalitis in mock-immunized mice. The potential of such mucosal adjuvants for use in human vaccines against pathogens such as HSV-1 is discussed.     

Mechanism of reduced T-cell effector functions and class-switched antibody responses to herpes simplex virus type 2 in the absence of B7 costimulation

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-gamma)-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-gamma 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, na?ve 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.     

A replication-competent, neuronal spread-defective, live attenuated herpes simplex virus type 1 vaccine

Herpes simplex virus type 1 (HSV-1) produces oral lesions, encephalitis, keratitis, and severe infections in the immunocompromised host. HSV-1 is almost as common as HSV-2 in causing first episodes of genital herpes, a disease that is associated with an increased risk of human immunodeficiency virus acquisition and transmission. No approved vaccines are currently available to protect against HSV-1 or HSV-2 infection. We developed a novel HSV vaccine strategy that uses a replication-competent strain of HSV-1, NS-gEnull, which has a defect in anterograde and retrograde directional spread and cell-to-cell spread. Following scratch inoculation on the mouse flank, NS-gEnull replicated at the site of inoculation without causing disease. Importantly, the vaccine strain was not isolated from dorsal root ganglia (DRG). We used the flank model to challenge vaccinated mice and demonstrated that NS-gEnull was highly protective against wild-type HSV-1. The challenge virus replicated to low titers at the site of inoculation; therefore, the vaccine strain did not provide sterilizing immunity. Nevertheless, challenge by HSV-1 or HSV-2 resulted in less-severe disease at the inoculation site, and vaccinated mice were totally protected against zosteriform disease and death. After HSV-1 challenge, latent virus was recovered by DRG explant cocultures from <10% of vaccinated mice compared with 100% of mock-vaccinated mice. The vaccine provided protection against disease and death after intravaginal challenge and markedly lowered the titers of the challenge virus in the vagina. Therefore, the HSV-1 gEnull strain is an excellent candidate for further vaccine development.     

Autophagy interaction with herpes simplex virus type-1 infection

More than 50% of the U.S. population is infected with herpes simplex virus type-I (HSV-1) and global infectious estimates are nearly 90%. HSV-1 is normally seen as a harmless virus but debilitating diseases can arise, including encephalitis and ocular diseases. HSV-1 is unique in that it can undermine host defenses and establish lifelong infection in neurons. Viral reactivation from latency may allow HSV-1 to lay siege to the brain (Herpes encephalitis). Recent advances maintain that HSV-1 proteins act to suppress and/or control the lysosome-dependent degradation pathway of macroautophagy (hereafter autophagy) and consequently, in neurons, may be coupled with the advancement of HSV-1-associated pathogenesis. Furthermore, increasing evidence suggests that HSV-1 infection may constitute a gradual risk factor for neurodegenerative disorders. The relationship between HSV-1 infection and autophagy manipulation combined with neuropathogenesis may be intimately intertwined demanding further investigation.     

Ocular avirulence of a herpes simplex virus type 1 strain is associated with heightened sensitivity to alpha/beta interferon.

BALB/c mice infected on the scarified cornea with herpes simplex virus type 1 strain 35 [HSV-1(35)] rarely developed ocular disease even at challenge doses as high as 10(7) PFU per eye. In contrast, HSV-1(RE) consistently induced stromal keratitis at an inoculum of 2 x 10(4) PFU. The goal of this study was to determine the reason for the difference in virulence between the two HSV strains. Both HSV-1 strains replicated to similar titers in excised corneal "buttons." However, after in vivo infection of the cornea, the growth of strain 35 was evident only during the first 24 h postinfection, whereas the replication of strain RE persisted for at least 4 days. In vitro tests revealed that HSV-1(35) was greater than 10 times more sensitive to alpha/beta interferon (IFN-alpha/beta) than HSV-1(RE). Both strains induced comparable serum levels of IFN after intraperitoneal inoculation. The kinetics of HSV-1(35) clearance from the eye was markedly altered by treatment with rabbit anti-IFN-alpha/beta. Virus titers exceeding 10(4) PFU per eye could be demonstrated 4 to 5 days postinfection in mice given a single inoculation of antiserum 1 h after infection. Furthermore, anti-IFN treatment in 3-week-old mice infected with HSV-1(35) led to the development of clinically apparent corneal disease which subsequently progressed to stromal keratitis in the majority of recipients. These results indicate that the striking difference in the capacity of HSV-1(35) and HSV-1(RE) to induce corneal disease was related to the inherently greater sensitivity of strain 35 to IFN-alpha/beta produced by the host in response to infection.     

Targeting apoptosis in neurological disease using the herpes simplex virus

Herpes Simplex Viruses type 1 (HSV-1) and 2 (HSV-2) cause central nervous system (CNS) disease ranging from benign aseptic meningitis to fatal encephalitis. In adults, CNS infection with HSV-2 is most often associated with aseptic meningitis while HSV-1 frequently produces severe, focal encephalitis associated with high mortality and morbidity. Recent studies suggested that the distinct neurological outcome of CNS infection with the two viruses may be due to their distinct modulation of apoptotic cell death: HSV-1 triggers neuronal apoptosis, while HSV-2 is neuroprotective. Apoptosis also occurs in the etiology of neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and Down's syndrome, and determines the loss of specific neuronal populations and the decline in cognitive functions. Notwithstanding, the therapy of these disorders may rely on the use of replication-defective HSV-1 vectors to deliver anti-apoptotic transgenes to the CNS. However, the recent discovery of a neuroprotective activity innate to the HSV-2 genome (the ICP10 PK gene) suggests that: i) ICP10 PK may constitute a novel therapeutic approach by targeting both the apoptotic cell death and the cognitive decline, and ii) HSV-2 may be more suitable than HSV-1 as a vector for targeting neuronal disease.     

Locations of herpes simplex virus type 2 glycoprotein B epitopes recognized by human serum immunoglobulin G antibodies.

Herpes simplex virus type 2 (HSV-2) glycoprotein B (gB-2) gene segments were expressed as recombinant proteins in Escherichia coli. gB-2 recombinant proteins were reacted with human serum immunoglobulin G (IgG) antibodies in Western immunoblot assays. Initially, samples were tested for the presence of HSV-1-specific antibodies and HSV-2-specific antibodies by using HSV-infected cell lysates as antigen targets in Western blot assays. Serum samples that contained HSV-2-specific IgG (n = 58), HSV-1-specific IgG (n = 33), or no detectable HSV antibodies (n = 31) were tested for reactivities with the gB-2 recombinant proteins. In 58 of 58 samples that contained HSV-2-specific IgG, antibodies were present that reacted strongly with a gB-2 amino-proximal segment between amino acids (aa) 18 and 75. Three of 33 serum samples that contained HSV-1- and not HSV-2-specific IgG (as defined by the HSV lysate Western blot assay) reacted with this segment. Both HSV-2 antibodies and HSV-1 antibodies reacted strongly with a carboxy-terminal gB-2 segment between aa 819 and 904; a second minor cross-reactive region was mapped to a gB-2 segment between aa 564 and 626. The gB-2 segment from aa 18 to 75 may constitute a useful reagent for the virus type-specific serodiagnosis of HSV-2 infections. Further studies will be required to determine the relative sensitivities and specificities of the assay for gB-2 aa 18 to 75, HSV gG assays, and HSV lysate Western blot assays for detecting virus type-specific antibody responses in acute and chronic HSV-2 infections.     

Rheumatoid factors may bear the internal image of the Fc gamma-binding protein of herpes simplex virus type 1

Affinity-purified rheumatoid factors (RF) from 20 patients with rheumatoid arthritis were tested for their reactivity with the mAb II-481 against glycoprotein E (gE), the Fc gamma-binding protein of HSV-1, as well as with a panel of mAb against human Fc gamma R. All RF bound to mAb II-481 in preference to mAb IV.3 (anti-human Fc gamma RII) or MOPC 141 (control mAb) which belong to the same IgG2b subclass. Five RF showed strong reactivity with II-481. No significant reactivity was observed between RF and mAb against human Fc gamma R. Non-RF human IgM did not react with any of the mAb. Clear-cut binding to II-481 was also seen with monoclonal IgM-RF derived from MRL/1 mice (mRF-2). The reaction between RF and II-481 was completely inhibited by human IgG. It was also inhibited by BHK cell extract infected with HSV-1, and with purified gE. II-481 inhibited the binding of human IgG Fc to the infected cell extract, confirming that II-481 recognizes the Fc-binding site on gE. II-481 did not react directly with human IgG or Fc of IgG. mAb to human IgG2 showed stronger binding to II-481 than to MOPC 141, suggesting II-481 has conformational similarity to human IgG H chain. These results suggest that at least some RF bear the "internal image" of HSV-1 Fc gamma-binding protein and support the hypothesis that some RF may be generated as anti-idiotype antibodies against antiviral antibodies.     

Immunoglobulin G is the main protective antibody in mouse vaginal secretions after vaginal immunization with attenuated herpes simplex virus type 2.

We investigated the protective role of antibodies in vaginal secretions of mice that were immune to vaginal challenge with herpes simplex virus type 2 (HSV-2). Unfractionated vaginal immunoglobulins from immune and nonimmune mice and affinity-purified immunoglobulin G (IgG) and secretory IgA (S-IgA) from immune secretions were adjusted to their concentrations in vivo. Wild-type HSV-2 was incubated in the immunoglobulin preparations for 15 min in vitro, followed by inoculation into vaginae of nonimmune mice. HSV-2 was neutralized by unfractionated antibody and purified IgG from immune secretions but not by unfractionated nonimmune antibody or by purified immune S-IgA. The protective effect of IgG in vivo was investigated by passively transferring purified serum IgG from immune and nonimmune donors to nonimmune recipients before vaginal challenge infection. Immune IgG significantly reduced the percentage of vaginal epithelium infected, concentrations of shed virus protein in the vaginal lumen, and illness scores, even though the viral antibody titers in serum and vaginal secretions of recipient mice at the time of challenge were only 29 and 8%, respectively, of those in actively immunized mice. Additionally, removal of vaginal secretions from immune mice 10 min before vaginal challenge with HSV-2 significantly increased the concentration of shed virus protein in the vaginal lumen after challenge. Collectively, the data indicate that IgG antibody in vaginal secretions of immune mice provides early protection against vaginal challenge infection, probably by neutralizing virus in the vaginal lumen. In contrast, S-IgA antibody contributed relatively little to immune protection of the vagina.     

Neutrophil-mediated suppression of virus replication after herpes simplex virus type 1 infection of the murine cornea.

Herpes simplex virus type 1 (HSV-1) infection of the murine cornea induces the rapid infiltration of neutrophils. We investigated whether these cells could influence virus replication. BALB/c mice treated with monoclonal antibody (MAb) RB6-8C5 experienced a profound depletion of neutrophils in the bloodstream, spleen, and cornea. In these animals, virus titers in the eye were significantly higher than those in the immunoglobulin G-treated controls at 3 days postinfection. By day 9, virus was no longer detectable in the controls, whereas titers of 10(3) to 10(6) PFU were still present in the neutrophil-depleted hosts. Furthermore, virus spread more readily to the skin and brains of MAb RB6-8C5-treated animals, rendering them significantly more susceptible to HSV-1-induced blepharitis and encephalitis. Only 25% of the treated animals survived, whereas all of the controls lived. Although MAb RB6-8C5 treatment did not alter the CD4+ T-cell, B-cell, natural killer cell, or macrophage populations, the CD8+ T-cell population was partially reduced. Therefore, the experiments were repeated in severe combined immunodeficiency mice, which lack CD8+ T cells. Again virus growth was found to be significantly elevated in the eyes, trigeminal ganglia, and brains of the MAb RB6-8C5-treated hosts. These results strongly indicate that in both immunocompetent and immunodeficient mice, neutrophils play a significant role in helping to control the replication and spread of HSV-1 after corneal infection.     

Suppression of annexin A1 and its receptor reduces herpes simplex virus 1 lethality in mice

Herpes simplex virus 1 (HSV-1)-induced encephalitis is the most common cause of sporadic, fatal encephalitis in humans. HSV-1 has at least 10 different envelope glycoproteins, which can promote virus infection. The ligands for most of the envelope glycoproteins and the significance of these ligands in virus-induced encephalitis remain elusive. Here, we show that glycoprotein E (gE) binds to the cellular protein, annexin A1 (Anx-A1) to enhance infection. Anx-A1 can be detected on the surface of cells permissive for HSV-1 before infection and on virions. Suppression of Anx-A1 or its receptor, formyl peptide receptor 2 (FPR2), on the cell surface and gE or Anx-A1 on HSV-1 envelopes reduced virus binding to cells. Importantly, Anx-A1 knockout, Anx-A1 knockdown, or treatments with the FPR2 antagonist reduced the mortality and tissue viral loads of infected mice. Our results show that Anx-A1 is a novel enhancing factor of HSV-1 infection. Anx-A1-deficient mice displayed no evident physiology and behavior changes. Hence, targeting Anx-A1 and FPR2 could be a promising prophylaxis or adjuvant therapy to decrease HSV-1 lethality.     

Complement-requiring neutralizing antibody in guinea pigs with primary and recurrent genital herpes

Guinea pigs inoculated intravaginally with herpes simplex virus type 2 (HSV-2) strain 1868 produced a serum complement-requiring neutralizing (CRN) antibody during primary acute infection, i.e., 10 days postinoculation. The CRN antibody titers in the guinea pig sera decreased to less than 1:10 after heating at 56 degrees C for 30 min. It was found that 32 units of complement were necessary to obtain a satisfactory HSV-2 neutralizing antibody titer. Nonheated sera significantly reduced virus infectivity titers when mixed with 3.5 log10 PFU of HSV-2 and incubated at 37 degrees C for 20 to 60 min (P less than 0.001), whereas the same sera after heating at 56 degrees C for 30 min showed no inhibitory effect. Only 27.3% of infected guinea pigs had low serum non-CRN antibody titers ranging from 1:20 to 1:40. In addition, no evidence of increase in CRN antibody titers was noted during spontaneous recurrent genital herpes infection.     

Ketogenic diet restrains herpes simplex encephalitis via gut microbes

Herpes simplex virus type 1 (HSV-1) infection-associated herpes simplex encephalitis (HSE) is an occasionally but severe neuronal disease that causes behavioral disorder and impairs cognition. Herein, we demonstrate that the consumption of ketogenic diet (KD), a low-carbohydrate high-fat diet, restricts the neurotropic infection of HSV-1 and HSE progression in mice. KD reduced weight loss, neurodegenerative symptoms, virus production and neuroinflammation, resulting in the enhanced survival rate of HSE mice. Notably, depletion of gut microbes by antibiotics attenuated the protective function of KD on HSV-1-related neuroinflammation and HSE development. Therefore, KD represents as an alternative therapeutic strategy to alleviate or prevent HSE via gut microbiota.     

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.     

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.     

The contrasting IgG-binding interactions of human and herpes simplex virus Fc receptors

A virally encoded, high-affinity Fc receptor (FcR) is found on herpes simplex virus type 1 (HSV-1) particles and infected cells where its binding of non-immune IgG protects cells from host-mediated lysis. Whilst mutation or aglycosylation of the IgG CH2 domain reduced binding to human FcR, the interaction with HSV-1 FcR was not affected. However, the HSV-1 FcR, unlike human FcR, discriminates between human IgG1 allotypes, being sensitive to changes at positions 214 (CH1) and 356/358 (CH3), away from its proposed binding site at the CH2-CH3 interface. The biological consequences are not known but this is the first evidence of a major functional difference between IgG1 allotypes.     

Re-evaluating natural resistance to herpes simplex virus type 1

It is often stated that individuals of a species can differ significantly in their innate resistance to infection with herpes simplex virus type 1 (HSV-1). Three decades ago Lopez reported that C57BL/6 mice could survive a 5,000-fold-higher inoculum of HSV-1 given intraperitoneally than mice of the A or BALB/c strain (Nature 258:152-153, 1975). Susceptible strains of mice died of encephalitis-like symptoms, suggesting that viral spread to the central nervous system was the cause of death. Although Lopez's study documented that C57BL/6 mice were resistant to the development of HSV-1 encephalitis and mortality, the resistance of C57BL/6 mice to other steps of the HSV-1 infection process was not assessed. The results of the present study extend these observations to clarify the difference between resistance to (i) HSV-1 pathogenesis, (ii) HSV-1 replication, (iii) HSV-1 spread, and (iv) the establishment of latent HSV-1 infection. Although C57BL/6 mice are more resistant to HSV-1 pathogenesis than BALB/c mice, the results of the present study establish that HSV-1 enters, replicates, spreads, and establishes latent infections with virtually identical efficiencies in C57BL/6 and BALB/c mice. These observations raise questions about the validity of the inference that differences in natural resistance are relevant in explaining what differentiates humans with recurrent herpetic disease from the vast majority of asymptomatic carriers of HSV-1 and HSV-2.