Cervical antibodies in patients with oral herpes simplex virus type 1 (HSV-1) infection: local anamnestic responses after genital HSV-2 infection.

Herpes simplex virus (HSV)-specific immunoglobulin A, immunoglobulin G, and secretory-component-containing immunoglobulins were identified in cervical and salivary secretions from six subjects with oral HSV type 1 (HSV-1) infections. Anamnestic cervical and salivary antibody responses were detected in two HSV-1-seropositive women with newly acquired genital HSV-2 infections. These data implicate the common mucosal immune system in antibody responses to HSV.     

T cell immunity to herpes simplex viruses in seronegative subjects: silent infection or acquired immunity?

During the course of investigating T cell responses to HSV among volunteers entering trials of investigational genital herpes vaccines, 6 of the 24 immunocompetent subjects with no prior history of oral/labial or genital herpes possessed HSV-specific T cell immunity but, by multiple determinants of even the most sensitive serological assays, remained seronegative to HSV-1 and -2. Of these six immune seronegative (IS; HSV-seronegative with HSV-specific T cell responses) subjects, two had transient HSV-specific T cell responses, while four had CD4(+) and CD8(+) T cell responses directed at HSV that persisted for up to 4 years. CD4(+) T cell clones were isolated that recognized and had high binding affinities to epitopes in HSV-2 tegument proteins. All six IS subjects had potential sexual exposure to an HSV-2-infected sexual partner. Oral and genital mucosal secretions were sampled and tested for the presence of infectious HSV and HSV DNA. No evidence of HSV was detected in >1500 samples obtained from these IS subjects. The identification of persistent T cell responses to HSV in seronegative subjects is a novel finding in the herpesvirus field and suggests either undetected infection or acquired immunity in the absence of infection. Understanding the basis of these acquired immune responses may be critical in developing effective vaccines for genital herpes.     

Immunogenicity,Protective Efficacy,and Non-Replicative Status of the HSV-2 Vaccine Candidate HSV529 in Mice and Guinea Pigs

HSV-2 vaccine is needed to prevent genital disease, latent infection, and virus transmission. A replication-deficient mutant virus (dl5-29) has demonstrated promising efficacy in animal models of genital herpes. However, the immunogenicity, protective efficacy, and non-replicative status of the highly purified clinical vaccine candidate (HSV529) derived from dl5-29 have not been evaluated. Humoral and cellular immune responses were measured in mice and guinea pigs immunized with HSV529. Protection against acute and recurrent genital herpes, mortality, latent infection, and viral shedding after vaginal HSV-2 infection was determined in mice or in naïve and HSV-1 seropositive guinea pigs. HSV529 replication and pathogenicity were investigated in three sensitive models of virus replication: severe combined immunodeficient (SCID/Beige) mice inoculated by the intramuscular route, suckling mice inoculated by the intracranial route, and vaginally-inoculated guinea pigs. HSV529 immunization induced HSV-2-neutralizing antibody production in mice and guinea pigs. In mice, it induced production of specific HSV-2 antibodies and splenocytes secreting IFNγ or IL-5. Immunization effectively prevented HSV-2 infection in all three animal models by reducing mortality, acute genital disease severity and frequency, and viral shedding. It also reduced ganglionic viral latency and recurrent disease in naïve and HSV-1 seropositive guinea pigs. HSV529 replication/propagation was not detected in the muscles of SCID/Beige mice, in the brains of suckling mice, or in vaginal secretions of inoculated guinea pigs. These results confirm the non-replicative status, as well as its immunogenicity and efficacy in mice and guinea pigs, including HSV-1 seropositive guinea pigs. In mice, HSV529 produced Th1/Th2 characteristic immune response thought to be necessary for an effective vaccine. These results further support the clinical investigation of HSV529 in human subjects as a prophylactic vaccine.     

Human newborn natural killer cell responses to activation by monoclonal antibodies. Effect of culture with herpes simplex virus

Deficient responses by NK cells may contribute to the susceptibility of human newborns to severe HSV infections. Furthermore, HSV disseminates widely during neonatal infection and may also infect and interfere with the function of blood mononuclear cells (MNC). To determine whether the function of newborn NK cells would be affected by contact with HSV, and whether newborn NK cells might be permissive for HSV replication, newborn MNC were cultured with HSV in vitro for 3 days. Before culture, the intracellular calcium mobilization in newborn NK cells induced by mAb to CD2 and CD16 did not differ from that of adult NK cells. This result argues against immaturity of an early NK cell activation event in human newborns. After culture with HSV the Ca2+ flux response was unaffected by lysis of K562 targets by newborn NK cells and NK-dependent suppression of HSV replication in fibroblasts were preserved or increased. Incorporation of thymidine by NKH-1 cells following stimulation with PHA and IL-2 was not suppressed. NK cells recovered from these cultures did not contain infectious HSV and synthesis of HSV-specific proteins was not detected by immunoprecipitation although HSV genome was detected by DNA hybridization. Our results extend the in vitro model stimulation systems in which newborn NK cells respond positively to include triggering through the CD2 Ag, cross-linking of cell surface CD 16 Ag and response to a pathogen, HSV.     

Herpesvirus DNA in Peripheral Blood Mononuclear Cells of Some Patients with Meniere's Disease

Herpes simplex virus‐1 (HSV) or varicella zoster virus (VZV) DNA was detected by nested polymerase chain reaction in peripheral blood mononuclear cells of patients with Meniere's disease (one of 28 patients for HSV‐1,2 of 28 patients for VZV) during acute illness (within 5 days after onset). On the other hand, neither HSV‐1 DNA or VZV DNA was detected in PBMCs of 50 age‐ and sex‐matched healthy individuals and 50 pregnant women. These findings may imply that reactivation of HSV‐1 or VZV may be associated with the development of some cases of Meniere's disease.     

A subset of herpes simplex virus replication genes induces DNA amplification within the host cell genome.

Herpes simplex virus (HSV) induces DNA amplification of target genes within the host cell chromosome. To characterize the HSV genes that mediate the amplification effect, combinations of cloned DNA fragments covering the entire HSV genome were transiently transfected into simian virus 40 (SV40)-transformed hamster cells. This led to amplification of the integrated SV40 DNA sequences to a degree comparable to that observed after transfection of intact virion DNA. Transfection of combinations of subclones and of human cytomegalovirus immediate-early promoter-driven expression constructs for individual open reading frames led to the identification of six HSV genes which together were necessary and sufficient for the induction of DNA amplification: UL30 (DNA polymerase), UL29 (major DNA-binding protein), UL5, UL8, UL42, and UL52. All of these genes encode proteins necessary for HSV DNA replication. However, an additional gene coding for an HSV origin-binding protein (UL9) was required for origin-dependent HSV DNA replication but was dispensible for SV40 DNA amplification. Our results show that a subset of HSV replication genes is sufficient for the induction of DNA amplification. This opens the possibility that HSV expresses functions sufficient for DNA amplification but separate from those responsible for lytic viral growth. HSV infection may thereby induce DNA amplification within the host cell genome without killing the host by lytic viral growth. This may lead to persistence of a cell with a new genetic phenotype, which would have implications for the pathogenicity of the virus in vivo.     

Specific transfer factor protects mice against lethal challenge with herpes simplex virus

Bovine transfer factor (TFd) specific to herpes simplex virus (HSV)1 or to HSV2 was prepared by immunizing calves with the corresponding virus. The TFd preparations were then injected into Swiss mice in an attempt to protect them against a subsequent lethal challenge with HSV1 or HSV2 virus. It was thus shown that injection of anti-HSV TFd protects the mice against the corresponding HSV virus, whereas the injection of a nonspecific TFd (anti-CMV) fails to protect against a challenge with HSV1. Furthermore, a dose-response effect was observed, since potent TFd preparations were ineffective when they were used at one-fifth of the original concentration. It seems, therefore, that animal models may be used to assay the potency of TFd preparations specific for herpes viruses.     

Structure of the provirus within NIH 3T3 cells transfected with Harvey sarcoma virus DNA.

NIH 3T3 cells transformed with unintegrated Harvey sarcoma virus (HSV) linear DNA generally acquired a complete HSV provirus. Infection of these transformed cells with Moloney murine leukemia helper virus was followed by release of infectious particles. The HSV provirus within these transfected cells was convalently joined to nonviral DNA sequences and was termed "cell-linked" HSV DNA. The association of this cell-virus DNA sequence with the chromosomal DNA of a transfected cell was unclear. NIH 3T3 cells could also become transformed by transfection with this cell-linked HSV DNA. In this case, the recipient cells generally acquired a donor DNA fragment containing both the HSV provirus and its flanking nonviral sequences. After cells acquired either unintegrated or cell-linked HSV DNA, the newly established provirus and flanking cellular sequences underwent amplifications to between 5 and 100 copies per diploid cell. NIH 3T3 cells transfected with HSV DNA may acquire deleted proviral DNA lacking at least 1.3 kilobase pairs from the right end of full-length HSV 6-kilobase-pair DNA (corresponding to the 3'-proximal portion of wild-type HSV RNA). Cells bearing such deleted HSV genomes were transformed, indicating that the viral transformation gene lies in the middle or 5'-proximal portion of the HSV RNA genome. However, when these cells were infected with Moloney murine leukemia helper virus, only low levels of biologically active sarcoma virus particles were released. Therefore, the 3' end of full-length HSV RNA was required for efficient transmission of the viral genome.     

Human cytotoxic T cell clones directed against herpes simplex virus-infected cells. II. Bifunctional clones with cytotoxic and virus-induced proliferative activities exhibit herpes simplex virus type 1 and 2 specific or type common reactivities

Human cytotoxic T lymphocyte (CTL) clones directed against herpes simplex virus (HSV)-infected cells were generated after stimulation of peripheral blood lymphocytes (PBL) with HSV type 1 (HSV-1) and HSV type 2 (HSV-2). These CTL clones were studied with regard to HSV type specificity and with regard to whether they also express helper cell activity. Some clones, generated after stimulation with HSV-1, were cytotoxic for autologous cells infected with either HSV-1 or HSV-2 ("HSV type common clones"), whereas other clones lysed HSV-1-infected cells only ("type-specific clones"). Similarly, after HSV-2 stimulation, both HSV-2 specific and HSV type common clones were obtained, indicating the heterogeneity of human cytotoxic T cells to HSV. All CTL clones tested were found to be bifunctional in that they also proliferated in response to stimulation with HSV. The HSV type specificity of the proliferative response was identical to that of the cytotoxic activity of the clones. An HSV type common clone, when stimulated with either HSV-1 or HSV-2, and an HSV-1 specific clone, when stimulated with HSV-1 but not with HSV-2, produced a factor, presumably interleukin 2 (IL 2), which induced proliferation of CTLL, an IL 2-dependent T cell line, providing evidence that our HSV-directed CTL clones also express helper cell activity. CTL clones that we previously reported were restricted in cytotoxic activity by HLA class II DR-1 or MB-1 antigens were found, in this study, to be restricted in proliferative response to HSV by these same HLA antigens. These results suggest that our bifunctional T cell clones directed against HSV may recognize the same viral antigenic determinants and the same HLA antigens for both cytotoxic and virus-induced proliferative activities. This is the first demonstration of human HSV type specific and HSV type common T cell clones and HSV specific T cell clones with both cytotoxic and helper cell activities.     

Evidence that neomycin inhibits HSV 1 infection of BHK cells

The effect of neomycin on the Herpes Simplex Virus (HSV) type 1 and 2 infection of baby hamster kidney cells was studied. Neomycin concentrations of 3 mM or more caused a more than 90% inhibition of HSV 1 proliferation, while it had no effect on HSV 2 proliferation, measured as plaque-forming units. Furthermore, neomycin must be present at the time of infection in order to exert full effect, addition 1 hour postinfection was comparable to untreated cells. This indicates that neomycin may specifically interfere with very early stages of HSV 1 infection.     

Gene delivery to neurons: Is herpes simplex virus the right tool for the job?

Herpes simplex virus (HSV)-derived vectors are currently being developed for the introduction of foreign DNA into neurons. HSV vectors can facilitate a range of molecular studies on postmitotic neurons and may ultimately be used for somatic cell gene therapy for certain neurologic diseases. In this article, the salient features of the pathogenesis and molecular biology of HSV relevant to its use as a vector are described, along with an overview of the methods used to derive these vectors. The accomplishments which have been made to date using the HSV vector system are discussed, with emphasis on the issues of this technology which remain to be addressed. HSV has the potential to be a most useful tool for neuronal cell transgenesis and it is likely that important neurobiological questions will be answered using this vector system.     

Vaccine protection against simian immunodeficiency virus by recombinant strains of herpes simplex virus

An effective vaccine for AIDS may require development of novel vectors capable of eliciting long-lasting immune responses. Here we report the development and use of replication-competent and replication-defective strains of recombinant herpes simplex virus (HSV) that express envelope and Nef antigens of simian immunodeficiency virus (SIV). The HSV recombinants induced antienvelope antibody responses that persisted at relatively stable levels for months after the last administration. Two of seven rhesus monkeys vaccinated with recombinant HSV were solidly protected, and another showed a sustained reduction in viral load following rectal challenge with pathogenic SIVmac239 at 22 weeks following the last vaccine administration. HSV vectors thus show great promise for being able to elicit persistent immune responses and to provide durable protection against AIDS.     

Direct Immunofluorescence Test for the Diagnosis of Genital Herpesvirus Infections

Herpetic lesions of the genitalia may be confused clinically with other ulcerative, genital lesions. Direct immunofluorescence (FA) provides a rapid method of diagnosis, and the utility of this method for the diagnosis of genital ulcers was examined. One hundred and ten patients with genital lesions were examined by darkfield for syphilis and by FA and culture for herpes simplex virus (HSV) infections. Satisfactory samples were obtained from 102 patients, of which 81 were clinically suspected cases of HSV. Acetone-fixed slides of scrapings of ulcerative lesions were stained with conjugated antiserum prepared in rabbits against HSV type 2. HSV was isolated from 73% of specimens of suspected herpetic lesions, and 77% of these specimens were positive by FA. Nine percent were positive by FA only and these were not thought to represent false positives. Five percent were positive by culture only. A comparison of clinical diagnoses with laboratory findings revealed that 4% of the cases were misdiagnosed when only the clinical evaluation was considered. The data suggest that the inclusion of a diagnostic FA test for HSV along with the darkfield examination may be useful for differentiating the etiological agents of ulcerative, genital lesions.     

Herpes simplex virus in postradiation cervical smears. A morphologic and immunocytochemical study.

From January 1987 to August 1988, cytomorphologic criteria of both herpes simplex virus (HSV) and radiation effects were observed in Papanicolaou smears from 3 of 1,340 patients who had received radiotherapy for squamous cell carcinoma of the cervix. Avidin-biotin immunoperoxidase staining, using a rabbit IgG polyclonal HSV antibody, confirmed the presence of HSV antigen in those three postradiation smears. Both multinucleated molded cells and epithelial cells that lacked cytopathic effects were positive for HSV. Three other postradiation smears from these cases were similarly positive for HSV antigen; the one preradiation smear was negative. In situ hybridization and immunoperoxidase studies on sections from the preradiation biopsies were negative: severely altered neoplastic cells showed no reactivity. The absence of HSV markers in the preradiation specimens suggests that the HSV infections were secondary to the radiotherapy; further studies are needed to prove this association and to assess the possible mechanisms. These cases clearly indicate that the overlapping features of radiation and viral effects (such as multinucleation) may be present simultaneously.     

Survival of 60Co-irradiated herpes simplex virus in 15 human diploid fibroblast cell strains

The present study was designed to determine the extent to which herpes simplex virus (HSV) may be utilized to study the repair of DNA damaged by ionizing radiation. We investigated the survival of 60Co-irradiated HSV in cell strains derived from 2 normal controls and 13 patients with a broad range of diseases associated with possible DNA repair deficiencies. Irradiation was performed under two conditions to vary the type of damage incurred by the virus. HSV survival was greatly enhanced when the virus was irradiated in such a way that the indirect effects of ionizing radiation were minimized. We found no correlation between cellular hypersensitivity to ionizing radiation and survival of irradiated HSV. Reduced levels of virus survival were found in only 1 cell strain. When cells were treated with ionizing radiation or UV light prior to infection, no enhancement of virus survival was observed.     

HSV neutralization by the microbicidal candidate C5A

Genital herpes is a major risk factor in acquiring human immunodeficiency virus type-1 (HIV-1) infection and is caused by both Herpes Simplex virus type 1 (HSV-1) and HSV-2. The amphipathic peptide C5A, derived from the non-structural hepatitis C virus (HCV) protein 5A, was shown to prevent HIV-1 infection but neither influenza nor vesicular stomatitis virus infections. Here we investigated the antiviral function of C5A on HSV infections. C5A efficiently inhibited both HSV-1 and HSV-2 infection in epithelial cells in vitro as well as in an ex vivo epidermal infection model. C5A destabilized the integrity of the viral HSV membrane. Furthermore, drug resistant HSV strains were inhibited by this peptide. Notably, C5A-mediated neutralization of HSV-1 prevented HIV-1 transmission. An in vitro HIV-1 transmigration assay was developed using primary genital epithelial cells and HSV infection increased HIV-1 transmigration. Treatment with C5A abolished HIV-1 transmigration by preventing HSV infection and by preserving the integrity of the genital epithelium that was severely compromised by HSV infection. In conclusion, this study demonstrates that C5A represents a multipurpose microbicide candidate, which neutralizes both HIV-1 and HSV, and which may interfere with HIV-1 transmission through the genital epithelium.