Tolerance and immunity in mice infected with herpes simplex virus: simultaneous induction of protective immunity and tolerance to delayed-type hypersensitivity.

Unresponsiveness to delayed type hypersensitivity was induced in mice following an intravenous injection of herpes simplex virus. The principal tolerogens used were thymidine kinase-deficient virus mutants which grow poorly in vivo; u.v.-inactivated and to a lesser extent formalin-inactivated virus were also tolerogenic. The tolerance induced was specific for the virus type. Despite the tolerance to delayed hypersensitivity, anti-viral immunity is present as determined by the rapid inactivation of infectious virus. The mechanism of tolerance to herpes virus and the importance of these observations for the pathogenesis of viral disease is discussed.     

Lipopolysaccharide-induced suppressor cells for delayed-type hypersensitivity to herpes simplex virus: nature of suppressor cell and effect on pathogenesis of herpes simplex.

Treatment of mice with LPS at the time of priming with herpes simplex virus type 1 (HSV1) causes the preferential activation of virus-specific T suppressor (Ts) cells. These Ts cells can transfer suppression to the efferent limb of a DTH response. Priming under these conditions is associated with enhanced cell-recruitment to the inoculation site, but had no effect on virus clearance. The induction of suppression was abrogated by pretreatment of mice with cyclophosphamide or indomethacin. LPS had no effect on the antibody response to HSV1 during acute infection, although treated mice showed a raised antibody titre one month after inoculation. Susceptible mice inoculated with HSV1 and given LPS showed protection, both from lethal herpes encephalitis and from demyelination within the CNS as reflected by ear paralysis. These results imply that, during some stages of acute infection, T cell effector mechanisms may themselves mediate tissue damage. At such times, Ts cells may perform a beneficial role leading to a reduction in pathology.     

Induction of herpes simplex virus immunity in newborn mice.

It was demonstrated that newborn mice, surviving an infection with attenuated herpes simplex virus, become resistant to challenge with a pathogenic strain of herpes simplex virus. This resistance did not seem to be mediated by antibody.     

Development of tumours in mice chronically infected with herpes simplex virus.

Tumours developed in chronic infection lasting for 150--180 days in 39 (60%) of 65 mice infected with strain L2 of herpes simplex virus (HSV) type 1 and in 12 (20%) of 60 mice infected with strain 333 of HSV type 2. Similar results were obtained in 150 immunosuppressed mice chronically infected with HSV types 1 and 2. Pathomorphologically, the neoplasias in the first group (strain L2) were similar to adenocarcinoma and malignant lymphoma and in the second (strain 333) to lymphoma and angio- or fibrosarcoma. The respective HSV strains were isolated by cocultivation of blood leukocytes from chronically infected animals and cultures of the tumour cells with human embryo fibroblasts (HEF). HSV and Gross murine leukaemia virus antigens were detected in tumour cells by immunofluorescence and radioimmunoassay, respectively, and HSV antigen by immunofluorescence also in cultures of tumour cells and in cells of the brains, spinal cords, livers and spleens of the animals. HSV antibody was demonstrable in the blood serum from chronically infected tumour-bearing mice in a titre of 32.     

Delayed hypersensitivity to herpes simplex virus: murine model.

Cell-mediated immunity has been shown to be clinically important in recovery from herpes simplex virus (HSV) infections. To investigate the role of delayed hypersensitivity (DH) in immunity and protection against HSV, we developed a murine model using the ear-swelling assay. Mice were infected subcutaneously with HSV-1 and ear-challenged, and the swelling was quantified. Significant ear swelling was detected by 3 to 4 days postinfection and peaked at 6 days. The kinetics of development of ear swelling were typical of DH: maximal swelling occurred 24 h post challenge and was diminished by 48 h, and the cellular infiltrate was predominantly mononuclear. Four-hour swelling, indicative of antibody-mediated, immediate-type hypersensitivity, was not detected until 15 days post immunization. The DH response was virus specific and could be transferred to normal recipients with lymph node T cells, but not with B cells or immune serum. This system will provide a useful model for evaluating the protective role of DH in HSV infection and for studying the specificity and interaction of T cells which mediate the response.     

Immunity to herpes simplex virus type 2: cell-mediated immunity in latently infected guinea pigs.

Cell-mediated (CMI) and humoral immunity to herpes simplex virus type 2 (HSV-2) were evaluated in infected strain 13/N guinea pigs with (45%) and without a history of recurrent herpetic disease. Virus was isolated by cocultivation from active herpetic lesions (9 of 10) as well as from the footpads (17 of 38), sacral ganglia (7 of 21), and sciatic nerves (1 of 21) of asymptomatic animals. Viral isolates grew in cells of human origin and were neutralized by hyperimmune anti HSV-2 sera. Humoral immunity measured by the presence of virus-neutralizing antibody was similar in both experimental groups. The involvement of CMI in recurrent disease was assessed by comparing lymphocyte transformation (LT) and leukocyte migration inhibition factor (LIF) responses in animals with a history of recurrent disease studied while asymptomatic (quiescent) and in animals without clinical evidence of recurrent disease (seropositive controls). Spleen cells from quiescent animals evidenced significant impairment of both LIF and LT responses as reflected in the requirement of higher antigen concentrations (up to 58-fold) and longer in vitro culture periods (up to 2.5 days) to mount responses comparable in magnitude to those observed in the seropositive control groups. Peripheral blood lymphocyte cultures obtained from quiescent animals showed similar impairment of LIF responses but displayed intact LT response. The data suggest that recurrent disease is associated with an impairment in the generation of anamnestic effector functions as reflected by altered kinetics and dose response patterns in in vitro secondary responses.     

Cell-mediated immunity in mice with primary,secondary and latent herpes simplex virus infection

Summary Cell mediated immunity was studied by a cytopathic effect inhibition assay in mice infected in the ear with herpes simplex virus type 1 (HSV 1). Activity appeared rapidly, reaching a high level 6 days after primary infection. It had fallen 10 days after infection and was undetectable during latency, 3–5 weeks after infection. The activity reappeared even more rapidly and strongly after reinoculation with the virus, but stimuli designed to induce recurrent disease did not induce clinical disease in the animals and no activity was detected in them.The activity, which was specific for HSV, was shown to be mediated by T-lymphocytes.With 1 Figure     

Virus-induced antigen in cells infected with the herpes simplex virus

A new antigen differing from those of mature virion and noninfected cell was shown to appear in herpes simplex (HSV) virus-infected cells. It is virus-specified since the nonvirion antigen had a similar determinant in cells of different species origin. Nonvirion antigens induced by HSV types 1 and 2 were serologically similar. Differences in the specific activity of sera to the new nonvirion antigen and mature virus antigens were established by three different methods: complement fixation test, mixed hemadsorption, and biological neutralization. The early virus-induced antigen found in HSV-infected cells differed by many properties from the antigen previously described by Tarro and Sabin (1970).     

Clonal analysis of the T-cell response of mice to herpes simplex virus: correlation between lymphokine production in vitro and the induction of delayed-type hypersensitivity and antiviral activity in vivo

The properties of two morphologically distinct L3T4+, Lyt2- "helper" T-cell clones specific for herpes simplex virus were investigated. Both of the clones produced IL-3 and interferon, but neither produced IL-2. Clone D6.6 produced macrophage agglutinating factor, a fibronectin-like lymphokine, and also a delayed hypersensitivity (DH) response when injected locally into syngeneic mice. Despite the presence of a DH producing clone and a non-DH producing clone, both were able to reduce the local virus titre to an equivalent degree. It is suggested that this protective activity is associated with the production of interferon-gamma. The significance of these results to mechanisms of protection against herpes simplex virus in vivo is discussed.     

Tubular structures in the cervix of mice experimentally infected with herpes simplex virus type 2

Experimental infection of the C3H/N mouse genital tract was demonstrated after intravaginal inoculation with herpes simplex virus type 2 (HSV-2). About 75% of the infected animals died by Day 7, and 75% of the surviving animals had severe vaginitis or neurological signs on Day 7. Titers of the virus recovered from vaginal secretions of infected animals reached a maximum on Day 2 and gradually decreased until Day 7. On the other hand, under the electron microscope, virus particles and tubular structures could be found in the nuclei of infected cells of the cervix in the 1st, 2nd and 4th days after infection. All cases in which virus particles could be found in the nuclei of infected cells were also positive for tubular structures and vice versa. These observations indicate that in situ diagnosis of HSV-2 infection can be made in the mouse model. The same method would be applicable for the diagnosis of human HSV-2 infection.     

Detection of herpes simplex virus in the ependyma of experimentally infected mice.

Forty-five Swiss albino mice were inoculated with HSV-1 (strain McKrae) by the corneal route. The spread of HSV to the brain stem and the ventricular ependyma was investigated. The polymerase chain reaction (PCR) was used to detect HSV-DNA in the CSF during the course of infection. The ependyma of the third and fourth ventricle and the central canal contained viral antigen at a late stage of infection in up to 60% of mice. At this stage we found that many animals gave a positive PCR in the CSF although no antigen could be detected in the ependymal cells. The presence or absence of antigen containing cells could not be related to detection of HSV-DNA in the CSF. The results show that infection of the ventricular wall is not important for the spread of HSV to the CSF.     

Viral-specific thymidine kinase in sensory ganglia of mice infected with herpes simplex virus.

Dorsal root ganglia (DRG) from herpes simplex virus (HSV)-infected mice were assayed for thymidine kinase (TK) activity at various times after infection. Polyacrylamide-gel electrophoresis revealed two peaks of activity with Rf values ranging from 0.18 to 0.24 (peak I) and 0.40 to 0.46 (peak II). In contrast, DRG from uninfected animals showed only one peak of enzyme activity with an R_f of 0.23. Incubation of infected DRG extracts with anti-HSV γ-globulin eliminated the TK activity in peak II but had no effect on the TK activity in peak I or on the TK activity found in DRG extracts from vaccinia-infected mice. The demonstration that infectious virus could be recovered from cell-free ganglionic homogenates for only 14 days, while HSV-specific TK (peak II) could be detected for up to 60 days, suggests that at least a portion of the viral genome is being continually or intermittently expressed during the chronic phase of the infection.     

T-enriched spleen cells in delayed-type hypersensitivity to influenza virus in mice

Restimulation in vitro of T-enriched spleen cells from CBA mice with influenza virus A/Bangkok 1/79/H3N2 or its hemagglutinin (HA) leads to enhancement of delayed-type hypersensitivity (DTH) to virus and HA in recipients of transfer. The enhancement of DTH measured by tail swelling is accompanied by 20-fold increase of binding affinity of transferring cells to HA measured by saturation analysis. DTH induced by HA in vivo is weaker than induced by virus in this system. However, when HA is used in vitro as a restimulating antigen of virus primed in vivo lymphocytes it leads to generation of such lymphocytes population which after transfer mediates DTH response to virus or HA to the same level as virus restimulated cells. The increase of binding affinity of restimulated T lymphocytes to HA accompanying the enhancement of DTH activity is considered in the relation to quantitative and qualitative changes of antigen binding cell populations and their role in antiviral response in this systems.     

Induction and characterization of delayed-type hypersensitivity to influenza virus in mice.

Mice infected with an aerosol of influenza virus or immunized with purified UV- inactivated whole virus or viral subunits developed delayed-type hypersensitivity (DTH) to influenza virus. The level of DTH was greatly enhanced when mice were injected intraperitoneally 2 days prior to immunization with 200 mg/kg of cyclophosphamide. The reaction peaked at 24 h after elicitation, had the classical DTH histology, and was transferable by immune cells but not by immune serum. DTH induced in cyclophosphamide-pretreated mice persisted for at least 40 days after sensitization. DTH induced by deoxycholate-treated subviral particles (DC particles) or the matrix protein of the influenza virus was type-specific, i.e. DTH induced by DC particles or matrix protein of type A virus cross-reacted only with type A virus and not with type B virus. DTH induced by purified hemagglutinin (HA) subunits, on the other hand, showed subtype specificty. Thus, DTH induced by HA of X 31 virus (H3) did not cross-react with PR 8 virus (H 0). However, DTH induced by HA of one subtype cross-reacted significantly with the variants of the same subtype. Thus, it appears that the effector T cells of DTH do not discriminate between the antigenic variants of influenza virus that are distinguishable by serology. DTH induced by purified, UV- inactivated whole virus showed extensive cross-reactivity. This nonspecific reaction was shown to be due to host-derived material, the lipid bilayer. - The theoretical and practical implications of these findings are discussed.     

Role for cell-mediated immunity in the resistance of mice to subcutaneous herpes simplex virus infection.

The role of cell-mediated immunity in the resistance of young adult mice to subcutaneous herpes simplex virus (HSV) type I infection was studied in mice receiving immunosuppressive doses of antilymphocyte sera (ALS) or antithymocyte sera (ATS). The effectiveness of these treatments to reduce cell-mediated responses was measured by their ability to prolong the life of allografts transplanted to ALS- or ATS-treated mice. It was found that subcutaneous infection of these mice with HSV resulted in spread of virus from the site of inoculation to the central nervous system. Neutralizing antibody could not be detected in the sera of ALS- or ATS-treated mice after HSV inoculation. Passive transfer of neutralizing antibody to ATS-treated mice did not restore resistance to subcutaneous HSV infection. However, adoptive transfer of HSV-sensitized spleen cells did provide significant protection against infection unless the spleen cells were treated with ATS prior to transfer. These experiments suggest that lymphocytes are involved in a cell-mediated response to subcutaneous HSV infection and demonstrate the importance of a noncompromised immune response in controlling spread of HSV from localized areas of infection.     

Heterotypic and homotypic re-inoculation of mice already latently infected with herpes simplex virus type 1

Summary Mice were infected at 4 weeks of age with a type 1 strain of herpes simplex virus (HSV) and re-infected 4 weeks later with either a type 1 or a type 2 strain of HSV. The virus used for first infection could be distinguished from that used later since it was resistant to phosphonoformic acid and formed syncytial plaques. Sites used for the second inoculation were as follows: at the site of primary infection, at a different site within the same dermatome or in the equivalent dermatome on the opposite side (also called remote site).Re-infection caused no detectable reactivation of the latent PFA resistant virus. After re-infection with a homotypic virus replication of the re-infecting virus was limited to the inoculation site. However after heterotypic re-infection the type 2 strain was occasionally isolated from the ganglia. Previous infection with the PFA resistant type 1 strain clearly reduced the ability of the homotypic or heterotypic strains to establish a latent infection. However, in a few animals ganglia were found to be latently infected with virus from both the first and second inoculations. Analysis of the results suggests that resistance to the establishment of a second latent infection in a ganglion is determined by the general immunity of the animal rather than immunity of the latently infected ganglion itself.     

Split T-cell tolerance in herpes simplex virus-infected mice and its implication for anti-viral immunity.

Mice simultaneously injected intravenously and subcutaneously with herpes simplex virus fail to adoptively transfer delayed hypersensitivity (DH) to syngeneic recipients. The transferred lymph node cells also failed to rapidly eliminate infectious herpes from the pinna, despite the presence of cytotoxic T cells in the transferred suspension. Both primary and secondary cytotoxic cell responses in the draining lymph node were unaffected by the inhibition of DH. The lymph nodes from DH tolerized mice also contain lymphocytes capable of undergoing a proliferative response in vitro to herpes antigens. In addition, a neutralizing antibody response with IgG antibodies against herpes are also present in DH tolerized mice. These data suggest a form of split T-cell tolerance in which only DH responses are directly compromised. The implication of these findings for the pathogenesis of herpes simplex virus is discussed.     

Polyamine metabolism in cells infected with herpes simplex virus.

Ifection of LS cells with HSV-1 resulted in an inhibition of spermidine and spermine synthesis from putrescine, possibly through inhibition of host cell protein synthesis. The rate of putrescine uptake increased soon after infection, and later, polyamines were lost from the cells. Inhibition of spermidine and spermine synthesis by methylglyoxal bis (amidinohydrazone) did not affect virus replication.