UV-inactivated Herpes Simplex Virus Research Articles

Classical Type 1 Dendritic Cells Dominate Priming of Th1 Responses to Herpes Simplex Virus Type 1 Skin Infection.

CD4+ T cell responses are crucial for the control of many intracellular pathogens, yet the requirements for their induction are not fully understood. To better understand the role that various dendritic cell (DC) subtypes play in CD4+ T cell priming, we compared in vivo T cell responses to skin inoculation of mice with infectious or UV-inactivated HSV type 1. Localized infection elicited a Th1 response that was primed in skin-draining lymph nodes involving Ag presentation by migratory dermal and lymph node-resident DC. However, expansion and Th1 differentiation was impaired in response to UV-inactivated virus (UV-HSV), and this defect correlated with a restriction of Ag presentation to migratory CD103- dermal DC. A similar differentiation defect was seen in infected mice lacking CD8α+ and CD103+ classical type 1 DC (cDC1). Finally, Th1 differentiation after UV-HSV inoculation was rescued by targeted Ag delivery to CD8α+ and CD103+ cDC1 using an anti-Clec9A Ab construct. This suggests that Ag presentation by cDC1 is crucial for optimal Th1 immunity to HSV type 1 infection and potentially other pathogens of the skin.

Induction of Transcription Factor Early Growth Response Protein 1 during HSV-1 Infection Promotes Viral Replication in Corneal Cells.

To understand the mechanisms of Early Growth Response Protein 1 (Egr-1) induction upon HSV-1 lytic infection and its roles in regulating viral gene expression and replication. Rabbit corneal cell line SIRC and other cell lines were infected by HSV-1 to investigate the Egr-1 induction and its occupancy on the viral genome in different conditions. UV-inactivated HSV-1 and a recombinant virus over-expressing Egr-1 were generated to evaluate the regulatory effects on viral gene expression and replication during the infection. Egr-1 induction triggered by viral infection was determined by Western Blot analyses and immune-fluorescent microscopy. Real-time RT-PCR and a novel Cignal™ Reporter Assay were used for quantitative measurement of Egr-1 expression. Chromatin Immuno-precipitation (ChIP) was performed to address the Egr-1 occupancy to the viral regulatory sequences and the influence on viral replication was assessed by plaque assays. Our results indicated that Egr-1 expression requires viral gene expression since the UV-inactivated HSV-1 failed to produce Egr-1 protein. Blockade of viral replication did not block the Egr-1 protein synthesis, supporting the hypothesis that HSV-1 replication was not essential for Egr-1 production. Chromatin immune-precipitation (ChIP) and RT-PCR assays demonstrated that induced Egr-1 was able to interact with key regulatory elements near HSV-1 immediate-early (IE) genes and promote viral gene expression. Recombinant virus overexpressing Egr-1 revealed that Egr-1 enhanced the viral replication and the release of infectious virus. Together these results concluded that HSV-1 triggers the expression of an important host transcription factor Egr-1 via a unique mechanism and benefit the viral gene expression and replication.

Induction of Transcription Factor Early Growth Response Protein 1 during HSV-1 Infection Promotes Viral Replication in Corneal Cells

AimsTo understand the mechanisms of Early Growth Response Protein 1 (Egr-1) induction upon HSV-1 lytic infection and its roles in regulating viral gene expression and replication.Study DesignRabbit corneal cell line SIRC and other cell lines were infected by HSV-1 to investigate the Egr-1 induction and its occupancy on the viral genome in different conditions. UV-inactivated HSV-1 and a recombinant virus over-expressing Egr-1 were generated to evaluate the regulatory effects on viral gene expression and replication during the infection.MethodologyEgr-1 induction triggered by viral infection was determined by Western Blot analyses and immune-fluorescent microscopy. Real-time RT-PCR and a novel Cignal™ Reporter Assay were used for quantitative measurement of Egr-1 expression. Chromatin Immuno-precipitation (ChIP) was performed to address the Egr-1 occupancy to the viral regulatory sequences and the influence on viral replication was assessed by plaque assays.ResultsOur results indicated that Egr-1 expression requires viral gene expression since the UV-inactivated HSV-1 failed to produce Egr-1 protein. Blockade of viral replication did not block the Egr-1 protein synthesis, supporting the hypothesis that HSV-1 replication was not essential for Egr-1 production. Chromatin immune-precipitation (ChIP) and RT-PCR assays demonstrated that induced Egr-1 was able to interact with key regulatory elements near HSV-1 immediate-early (IE) genes and promote viral gene expression. Recombinant virus overexpressing Egr-1 revealed that Egr-1 enhanced the viral replication and the release of infectious virus.ConclusionTogether these results concluded that HSV-1 triggers the expression of an important host transcription factor Egr-1 via a unique mechanism and benefit the viral gene expression and replication.

Regulatory T cell expression of herpes virus entry mediator (HVEM) following HSV-1 infection and its functional significance. (137.25)

Abstract In many infections especially those that are chronic, the outcome may be influenced by the activity of one or more types of regulatory T cells (Tregs), particularly those that express FOXP3. In many instances, for example the response that follows HSV infection, the majority of expanded Tregs are not antigen specific. In this report, we demonstrate one possible mechanism by which HSV-1 infection could act to signal and expand the Treg population. We show that HVEM, a tumor necrosis factor receptor superfamily member which interacts with the gD protein of HSV-1 to facilitate cell entrance, is expressed by FOXP3+ T cells and that engagement of HVEM by its ligand expands the Treg population. To demonstrate this, FOXP3-GFP knock in mice were infected with HSV kos and change in cell numbers of Tregs and their expression levels of HVEM were recorded in draining popliteal lymph node (DLN). Tregs expansion occured following HSV-1 infection, peaking at around day 8 compared to the peak numbers of CD8+T cells (day 5 p.i). Both FOXP3+ and FOXP3- CD4 T cells expressed HVEM in the resting state but Tregs upregulated HVEM reaching highest MFI at day 7 p.i. In-vitro stimulation of DLN cells with UV inactivated HSV kos, further up regulated HVEM on Tregs (possibly through gD) and down regulated on FOXP3_ cells suggesting a possibility that engaging this receptor with gD expressed by infected cells results in Treg activation and expansion which in turn modulates CD8+ effector responses.

Induction of IL-6 in Transcriptional Networks in Corneal Epithelial Cells after Herpes Simplex Virus Type 1 Infection

To determine the transcriptional responses of human corneal epithelial cells (HCECs) after herpes simplex virus type (HSV)-1 infection and to identify the critical inflammatory element(s). Immortalized HCECs were infected with HSV-1, and the global transcriptional profile determined. Molecular signaling networks were constructed from the HSV-1-induced transcriptomes. The relationships of the identified networks were confirmed by real-time-PCR and ELISA. Contributions of the critical network nodes were further evaluated by protein array analyses as candidates for inflammatory element induction. HSV-1 infection induced a global transcriptional response, with 412 genes significantly activated or suppressed compared with mock-infected HCECs (P < 0.05, 2< or 0.5> threshold). Infection by UV-inactivated HSV-1 did not induce significant transcriptional activity. Network analysis showed that the HSV-1-induced transcriptomes were associated with JUN N-terminal kinase, p38, extracellular signal-regulated kinase, and nuclear factor kappa-B signaling pathways. These findings indicate that interleukin (IL)-6 and vascular endothelial growth factor (VEGF) probably serve as critical nodes of signaling events. ELISA and protein array analyses verified the induction of the inflammatory elements by HSV infection. Blocking the induction of IL-6 significantly reduced the expression of 21 cytokines, including CCL7, CCL8, CXCL6, transforming growth factor-beta2, platelet-derived growth factor, interferon-gamma, IL-2, and VEGF, thus confirming the critical role of IL-6. HCECs respond to HSV-1 infection by initiating mitogen-activated protein kinase-related transcriptional events, and IL-6 may serve to induce expression of an array of inflammatory mediators.

Involvement of gD/HVEM interaction in NF-kB-dependent inhibition of apoptosis by HSV-1 gD

In the present paper, we aimed to verify whether the interaction of the glycoprotein D (gD) of herpes simplex 1 (HSV-1) with the HSV-1 receptor HVEM is involved in NF-κB-dependent protection against apoptosis by gD. To this purpose, first we utilized MAbs that interfere with gD/HVEM interaction and U937 cells that naturally express human HVEM on their surface. Pre-incubation with these MAbs, but not with a control antibody, partially reverted the protection of infectious HSV-1 towards anti-Fas induced apoptosis in U937 cells. Similarly, pre-incubation of UV-inactivated HSV-1 (UV-HSV-1) or recombinant gD with the same MAbs, significantly reduced the inhibition of Fas-mediated apoptosis by UV-HSV-1 or gD, respectively, in U937 cells. Moreover, coculture with stable transfectants expressing at surface level wild type gD protected U937 cells against Fas-induced apoptosis, while coculture with transfectants expressing a mutated form of gD, incapable to bind HVEM, did not protect. Finally, UV-HSV-1 protected against staurosporine-induced apoptosis in U937 cells as well as in the CHO transfectants expressing human HVEM on their surface, but not in the control CHO transfectants, which did not express HVEM. These results suggest that signaling triggered by binding of gD to HVEM could represent an additional mechanism of evasion from premature apoptotic death exerted by HSV-1-gD in HVEM-expressing cells, disclosing new opportunities of cell death manipulation by using gD preparations.

Involvement of HVEM receptor in activation of nuclear factor κB by herpes simplex virus 1 glycoprotein D

The UV-inactivated herpes simplex virus 1 (HSV-1) and glycoprotein D (gD) of HSV-1 have been shown to activate nuclear factor kappaB (NF-kappaB) in U937 cells, but mechanisms involved in this activation have not been elucidated. Here we report that: (i) UV-inactivated HSV-1 induced an increased NF-kappaB activation in cells expressing human HVEM (for herpesvirus entry mediator) at surface level, naturally or following stable transfection, but not in cells in which this receptor was not detected by flow cytometry analysis, (ii) treatment with soluble gD induced a dose-dependent NF-kappaB activation in THP-1 cells naturally expressing HVEM, and a monoclonal antibody that prevents binding of gD to HVEM significantly reduced NF-kappaB activation by soluble gD in the same cells, (iii) coculture with transfectants expressing wild-type gD on their surface induced an approximately twofold increase in NF-kappaB activation in cells naturally expressing HVEM, while coculture with transfectants expressing a mutated form of gD, lacking its capability to bind HVEM, did not induce a similar effect and (iv) treatment with soluble gD induced a dose-dependent NF-kappaB activation in CHO transfectants expressing HVEM, but not in control CHO transfectants lacking any functional gD receptor. Overall, these results establish that HVEM is involved in NF-kappaB activation by HSV-1 gD.

Herpes simplex virus 1 (HSV-1) DNA and immune complex (HSV-1–human IgG) elicit vigorous interleukin 6 release from infected corneal cells via Toll-like receptors

Toll-like receptor 3 (TLR-3) and TLR-9 gene expression and interleukin 6 (IL-6) secretion were studied in corneal cells with components of herpes simplex virus (HSV). Human corneal epithelial cells (HCEs) and primary human corneal fibroblasts (HCRFs) were infected with live HSV or UV-inactivated HSV (UV-HSV), transfected with HSV DNA or treated with HSV-anti-HSV IgG immune complexes. Gene expression of TLR-3 and -9 was analysed by real-time PCR. Supernatants were assayed for IL-6 by ELISA. Incubation of HCEs and HCRFs with live HSV-1, UV-HSV and HSV DNA resulted in augmented TLR-3 and -9 gene expression and IL-6 release. Moreover, infected or transfected HCRFs released greater amounts of IL-6 than did HCEs. As virus is frequently in the form of neutralized virus immune complexes, the ability of these immune complexes to interact with TLRs and trigger IL-6 production was evaluated. Here, it is shown that HSV-anti-HSV IgG complexes were as potent as HSV DNA in their ability to induce IL-6. Treatment of HCRFs transfected with HSV DNA with the TLR-9-inhibitory oligomer iODN, anti-TLR-3 antibody or phosphatidylinositol 3-kinase inhibitor indicated that IL-6 release from HCRFs was mediated by TLR-3 and -9 gene expression. These results demonstrated that neutralized HSV immune complexes were as potent as HSV DNA in enhancing IL-6 release from corneal fibroblasts. These phenomena were mediated via augmented TLR-3 and -9 gene expression.

Dendritic Cell Numbers in the Blood of HIV-1 Infected Patients Before and After Changes in Antiretroviral Therapy

Antigen presenting dendritic cells (DCs) can serve as sites for HIV replication and as vehicles for transmission of the virus to T cells. It is known that the numbers of DCs in blood is reduced during HIV-1 infection. Here we monitored the two major subsets of blood DCs in 12 individuals undergoing a change, primarily initiation, of highly active antiretroviral therapy. The numbers of plasmacytoid DCs were reliably higher on therapy, although in the 1-3 month interval we followed, these numbers did not return to those seen in HIV uninfected controls. An increase in plasmacytoid DCs was accompanied by an increase in IFN-alpha production in response to a standard challenge in culture with UV-inactivated herpes simplex virus. The levels of myeloid DCs also demonstrated an increase while on HAART, and these numbers become comparable to the HIV uninfected controls. The numbers of plasmacytoid and myeloid DCs varied inversely with the levels of plasma HIV viremia. These longitudinal studies extend prior work showing that virus infection with HIV leads to a decrease in the number of dendritic cells in blood, and that this can be reversed at least in part by therapy.

Toll-like receptor 9-mediated recognition of Herpes simplex virus-2 by plasmacytoid dendritic cells.

Plasmacytoid dendritic cells (pDCs) have been identified as a potent secretor of the type I interferons (IFNs) in response to CpG as well as several viruses. In this study, we examined the molecular mechanism of virus recognition by pDCs. First, we demonstrated that the CD11c+Gr-1intB220+ pDCs from mouse bone marrow secreted high levels of IFN-α in response to either live or UV-inactivated Herpes simplex virus-2 (HSV-2). Next, we identified that IFN-α secretion by pDCs required the expression of the adaptor molecule MyD88, suggesting the involvement of a Toll-like receptor (TLR) in HSV-2 recognition. To test whether a TLR mediates HSV-2–induced IFN-α secretion from pDCs, various knockout mice were examined. These experiments revealed a clear requirement for TLR9 in this process. Further, we demonstrated that purified HSV-2 DNA can trigger IFN-α secretion from pDCs and that inhibitory CpG oligonucleotide treatment diminished HSV-induced IFN-α secretion by pDCs in a dose-dependent manner. The recognition of HSV-2 by TLR9 was mediated through an endocytic pathway that was inhibited by chloroquine or bafilomycin A1. The strict requirement for TLR9 in IFN-α secretion was further confirmed by the inoculation of HSV-2 in vivo. Therefore, these results demonstrate a novel mechanism whereby the genomic DNA of a virus can engage TLR9 and result in the secretion of IFN-α by pDCs.

Influence of DNA encoding cytokines on systemic and mucosal immunity following genetic vaccination against herpes simplex virus

The aim of our investigation was to improve the effectiveness of DNA vaccines against herpes simplex virus (HSV) infection. We chose coimmunization with DNA encoding cytokines known to emphasize components of immune defense that best correlate with immune protection. These include interferon-producing T and NK cells and the IgG2a isotype immunoglobulin. Our results show that the coadministration of plasmid DNA encoding IL-12 or IL-18 along with glycoprotein B (gB) DNA improves immune induction. Recipients of the coimmunization procedure had elevated humoral as well as IFN-γ-producing T cell responses and showed greater resistance to vaginal challenge with a lethal dose of HSV-1. The adjuvant effects were observed when the vaccines were administered either systemically or mucosally. By most assays, the adjuvant effect of IL-18 was superior to IL-12, although gB DNA plus IL-18 failed to induce levels of immunity achieved by UV-inactivated HSV immunization. Mucosal immunization proved as an effective means of inducing systemic immunity, but was less effective than the systemic route for inducing protection from vaginal challenge. Our results also demonstrated that protection from such challenges was mainly a property of IFN-γ. Thus, immunized IFN-γ–/– mice remained susceptible to challenges even while generating readily measurable immune responses. The approach of using DNA vaccines combined with DNA encoding cytokines holds promise and represents a potentially useful approach for vaccines.

Enhanced Clearance of Herpes Simplex Virus Type 1 and Reduced Herpetic Eye Disease in STAT6 Knockout Mice Is Associated with Increased IL-2

STAT6 (signal transducers and activators of transcription 6)-deficient (STAT6 −/−) mice have defects in IL-4- and IL-13-mediated functions and thus have a reduced T H2-mediated immune response. Conversely, they have elevated levels of IL-2 and thus an increased T H1-mediated immune response. To assess the relative impact of reduced T H2- and elevated T H1-dependent immune responses on HSV-1 infection, vaccinated and mock-vaccinated STAT6 −/− mice were challenged ocularly with HSV-1. Mock-vaccinated STAT6 −/− mice were as susceptible to lethal HSV-1 infection as parental BALB/c mice. Mock-vaccinated STAT6 −/− mice had reduced HSV-1 titers in their eyes compared to BALB/c mice. Furthermore, mock-vaccinated STAT6 −/− mice had significantly less corneal scarring than their BALB/c counterparts. Vaccination induced significantly higher serum-neutralizing antibody titers in STAT6 −/− mice compared to BALB/c mice, while completely protecting both types of mice against HSV-1-induced death and corneal scarring. Vaccinated STAT6 −/− mice had reduced HSV-1 titers in their eyes compared to BALB/c mice. Lymphocytes from both vaccinated and mock-vaccinated STAT6 −/− mice secreted higher amounts of IL-2 than lymphocytes from BALB/c mice, in the presence or absence of stimulation with UV-inactivated HSV-1. Finally, depletion of IL-2 increased ocular virus replication in STAT6 −/− mice to levels similar to that measured in BALB/c mice. Our results suggest that in the absence of the STAT6 pathway, IL-2-mediated immune responses are up-regulated. This, in turn, leads to faster viral clearance and, consequently, lower levels of eye disease.

Acute Effects of Purified and UV-Inactivated Herpes simplex Virus Type 1 on the Hypothalamo-Pituitary-Adrenocortical Axis

Herpes simplex virus type 1 (HSV-1) is a common cause of viral encephalitis, manifested by neuroendocrine and behavioral changes. We have previously demonstrated that HSV-1 induces marked hypothalamo-pituitary-adrenocortical (HPA) axis activation. In this study we characterized the acute effects of HSV-1 on the HPA axis occurring before viral replication and appearance of clinical signs of encephalitis. Since in previous studies we used crude virus preparations which may contain immune factors produced by the infected cells, we tested here the effects of purified HSV-1 virions. HSV-1 was propagated on Vero cells and virions were purified by centrifugation in sucrose gradients. Inactivation of viral infectivity was achieved by UV-irradiation, which caused a million-fold decrease in virus titer, as determined by plaque assay. Intracerebroventricular (ICV) inoculation of crude or purified virions induced a dose dependent increase in serum corticosterone and corticotropin (ACTH). This effect was maximal within 3.5 h postinfection and lasted for 72 h. ICV inoculation of UV-inactivated purified virions caused a marked increase in serum corticosterone and ACTH at 3.5 h, but in contrast to the effect of the active virus, the hormone levels gradually decreased at 24 h, and returned to basal levels at 72 h postinfection. HSV-1-induced HPA axis activation at 3.5 h was completely abolished by pretreatment with interleukin-1 receptor antagonist, injected ICV. Adrenalectomized rats failed to respond to ICV inoculation of purified HSV-1 by increase in ACTH. In contrast, these rats responded to ICV injection of LPS. In conclusion: (1) HSV-1 can acutely activate the HPA axis before and independently of any viral replication; (2) HSV-1-induced HPA axis activation depends on a permissive action of circulating glucocorticoids and on host derived brain interleukin-1.

Protective T-cell-based immunity induced in neonatal mice by a single replicative cycle of herpes simplex virus.

Newborns are very susceptible to infections because their immune systems are not fully developed and react to antigen exposure preferentially with unresponsiveness. UV-inactivated herpes simplex virus type 1 (HSV-1) represents such an antigen and does not induce an immune response in neonates. In contrast, protective T cells were primed in newborn mice by a single replicative cycle of DISC HSV-1 given once within 24 h of birth. Each of the HSV-1-primed CD4(+) or CD8(+) T cells induced in wild-type or interferon-deficient mice conferred resistance to naive animals exposed to a lethal virus challenge. Inactivated HSV-1, injected at variable doses up to 10(4) times that of DISC HSV-1, was ineffective in inducing any detectable immune responses in neonates. Thus, the capacity of HSV-1 to replicate once, but not the number of virus particles per se, was decisive in inducing protective T-cell-associated immunity in newborn mice.

Splenic marginal metallophilic macrophages and marginal zone macrophages are the major interferon-alpha/beta producers in mice upon intravenous challenge with herpes simplex virus.

The interferon (IFN)-alpha/beta-producing cells (IPCs) are localized predominantly in the spleen, in particular in the marginal zones (MZ), in C57BL/6 mice injected intravenously (i.v.) with UV-inactivated herpes simplex virus (HSV). We defined the phenotype of these murine IPCs using simultaneous immunohistochemical labelling of intracellular IFN-alpha/beta and various surface antigens. We found that the IPCs in the MZ are not dendritic cells because they did not express major histocompatibility complex (MHC) class II and CD11c molecules. Furthermore, they did not express antigenic markers typical for T cells, B cells or red pulp macrophages. In contrast, the majority of IPCs were stained by the anti-sialoadhesin monoclonal antibody (MoAb) SER-4, which is specific for marginal metallophilic macrophages. In addition, a minor part of the IPCs with a more outward localization in the MZ were stained by a MoAb specific for MZ macrophages. We conclude that the massive IFN-alpha/beta production in the MZ of the spleen upon in vivo stimulation by HSV is mainly exerted by marginal metallophilic macrophages and to a lesser extent by MZ macrophages.

Enhancement of the immune response to non-replicating herpes simplex virus type-1 preparations by mucosal administration in the presence of cholera toxin

Different immunization regimes were compared in order to enhance the immune response following mucosal administration of non-replicating HSV-1 preparations to mice. The serum anti-HSV Ig response following intragastric administration of heat or UV inactivated HSV-1 strain SC16 was compared with that elicited by an attenuated derivative of SC16 (TKDM21). The highest response followed immunization with TKDM21 and this was markedly enhanced by repeated intragastric administration, reaching ca 35% of that elicited following a cutaneous infection with live virus. Repeated doses of killed virus produced only a minimal increase in the response even when given intranasally (i.n.). However, cholera toxin and its B-subunit with UV-inactivated virus or a mixture of purified viral glycoproteins enhanced the anti-HSV response after i.n. immunization and produced antibody levels equivalent to those following intragastric delivery of live TKDM21.

CD8 + type-2 T cells enhance the severity of acute herpes virus infection in mice

The role of CD8 + suppressor T cells in acute herpes simplex virus type 1 (HSV) infection was investigated in mice. CD8 + CD11b + TCR-γ/δ + suppressor T cells (HSV-STC) were demonstrated in spleens of mice infected intraperitoneally (i.p.) with HSV. When HSV-STC from mice infected with a 10 LD 50 of HSV (donors) were adoptively transferred to mice 3 days after being infected with a 1 LD 50 dose of HSV (recipients), the morbidity and mortality of recipients were greatly increased (mean survival time in days (MSD): 9.4 days; mortality, 100%) as compared with controls that received CD4 + T cells or a whole T-cell lysate from donors (MSD, > 19.6 days or > 19.1 days; mortality, 38% or 50%). The morbidity and mortality of mice exposed to a 1 LD 50 of HSV were also increased when they were continuously treated with recombinant murine IL-4. However, the survival rate of mice exposed to a 10 LD 50 of HSV increased after multiple treatments of these mice with anti-IL-4 monoclonal antibody. IL-4-producing cells were detected in a population of HSV-STC, and IL-4 was produced when these cells were cultured in the presence of UV-inactivated HSV in vitro. These results indicate that IL-4 plays an important role in the progression of acute HSV infection and, through the production of IL-4, HSV-STC may increase the severity of the acute-phase infection of HSV in mice.

Interferon-α Generation in Mice Responding to Challenge with UV-Inactivated Herpes Simplex Virus

In humans with advanced human immunodeficiency virus (HIV) infection, an interferon-alpha (IFN-alpha) response by a specialized blood mononuclear cell to herpes simplex virus (HSV) in vitro is associated with resistance to opportunistic infections. A cell type of unknown lineage, designated the natural IFN-producing cell (NIPC), has been identified preliminarily as the source of these IFNs and may have a role in other host defense functions. Earlier studies suggested the existence of analogous HSV-responsive cell populations in mice. The role specifically of IFN-alpha in the murine system, however, has not been characterized. Using IFN bioassay and neutralization with antisera against Type I IFNs and IFN-beta, we have defined the types and sources of IFNs produced by mice in response to in vivo and in vitro challenge with UV-inactivated HSV. After intraperitoneal inoculation with HSV, BALB/c and C57Bl/6 strains produced characteristically different levels of serum IFNs that appeared principally to be IFN-alpha. The response of mononuclear cells from these mice differed from that of the intact mouse. Isolated cells from bone marrow and spleen released detectable IFNs much later than did whole animals, and the IFNs produced by marrow, spleen, and peritoneal cells were usually neutralized by the anti-IFN-beta. Only bone marrow cells produced detectable amounts of IFN-alpha. Both intact mice and their cells became refractory to restimulation with similar kinetics.

Suppression of immune responses by herpes virus type 2-transformed murine tumor cells.

Work done in our laboratories, using a murine model, indicates that suppression of host immune responses might be due to secretion of soluble factors by tumor cells. The H238 cells (BALB/c embryonic fibroblasts transformed by UV-inactivated herpes simplex virus Type 2) exhibit progressive tumor growth with subsequent decrease in lymphoproliferation. To further study the suppressive effects of a tumor, H238 conditioned medium (CM) was tested for its ability to block murine and human mitogenic and allogeneic lymphocyte responses. PHA, Con A and LPS were used as mitogens. Lymphoproliferation, in the presence of increasing amounts of H238 CM, resulted in a greater degree of suppression of [3H]thymidine ([3H]Tdr) uptake, in both human and mouse systems. The kinetics of proliferation in the presence of concentrated H238 CM (cCM) showed that depression was evident regardless of the time of cCM addition, thereby affecting it at any stage of the cell cycle. Treatment of H238 cCM using acid (pH 2.3), base (pH 9.6), trypsin (100 micrograms/ml), heat (56 degrees C, 100 degrees C) and freeze-thawing, restored PHA-stimulated lymphoproliferation. Dialysis of H238 cCM showed that the molecular weight of the suppressor lies between 15 and 25 kDa. Northern blot analysis demonstrated the presence of a TGF-beta transcript in H238 cells. Neutralization of the H238 cCM with monoclonal antibody to TGF-beta resulted in complete abrogation of suppressive activity in spleen cell lymphoblastogenesis. These results suggest that TGF-beta appears to be the main inhibitor of immune responses found in this HSV-2-induced murine tumor cell line. Such tumor-induced modulations may contribute to the outcome of immunotherapy in the tumor-bearing host.

Immediate-early gene expression is sufficient for induction of natural killer cell-mediated lysis of herpes simplex virus type 1-infected fibroblasts

Herpes simplex virus type 1 (HSV-1)-infected human fibroblast (HSV-FS) targets are susceptible to lysis by natural killer (NK) cells, whereas uninfected FS are resistant to lysis. Studies were undertaken to determine the mechanism of this preferential susceptibility. HSV-FS were not intrinsically less stable than FS, as determined by a 51Cr release assay under hypotonic shock in the presence of rat granule cytolysin and by sensitivity to anti-human leukocyte antigen class I antibody plus complement. Single-cell assays in agarose demonstrated that although similar numbers of large granular lymphocytes bound to the HSV-FS and FS targets, the conjugates with HSV-FS were lysed at a much higher frequency than those with FS. These results suggested that both targets are bound by the NK cells but only the HSV-FS were able to trigger lysis. The requirement for active virus expression was demonstrated by failure of emetine-treated HSV-FS targets or targets infected with UV-inactivated HSV to be lysed by NK effectors. To evaluate the role of viral glycoproteins in conferring susceptibility to lysis, Fab were prepared from HSV-1-seropositive sera; these Fab were unable to block lysis of the HSV-FS. Furthermore, incubation in phosphonoacetic acid failed to reduce NK(HSV-FS) activity despite sharp reductions in viral glycoprotein synthesis. Finally, targets infected with tsLB2 at the nonpermissive temperature were lysed as well as or better than targets infected with wild-type virus, indicating that HSV immediate-early gene product expression is sufficient for conferring susceptibility to lysis. We conclude that expression of nonstructural viral proteins or virally induced cellular gene products early in the course of infection rather than structural glycoproteins is required for NK lysis of HSV-FS targets.