HSV Antigens Research Articles

Antibody responses in humans to individual proteins of herpes simplex viruses.

Sera from 231 women were used to examine their frequency of precipitation of various herpes simplex virus type 1 and 2 (HSV-1 and HSV-2) proteins and to determine if there was a rank order of immune responsiveness of humans to these HSV antigens. Radiolabeled viral proteins were reacted with serum and immune complexes isolated with staphylococcal protein A. Individual antigens were resolved by polyacrylamide gel electrophoresis and visualized by fluorography. As a group, these sera precipitated 31 HSV-1 and 27 HSV-2 proteins. HSV-1 polypeptides with molecular weights of 133,000, 99,000, and 82,000, as well as HSV-2 polypeptides with molecular weights of 131,000 and 101,000, were precipitated by essentially all sera that contained antibodies to HSV-1 and HSV-2. When attempts were made to order the viral proteins by constructing precipitation profiles ranking the antigens in patterns according to their frequency of precipitation, it was observed that the antigens were generally not ordered. Demographic analysis of the sera suggested that the differences in the number of proteins precipitated were associated with differences in age, education, age at first marriage, and income, which collectively may reflect the frequency of exposure to the virus.

Herpesvirus antigens as markers for cervical cancer

A total of 393 patients representing cervical atypias, cancers, and nonneoplastic disease have been studied. Cellular specimens have been stained with antisera raised against total herpes type 2 virus (HSV-2) and subfractions labeled crude AG-e, pure AG-e, ICP 12, and ICP 14. Reproducibility, statistical significance, false positive and false negative results, specificity, and sensitivity of the various antisera have been investigated. Correlation of immunocytostaining with Papanicolaou staining has been made. Usefulness of these markers in the detection and diagnosis of premalignant and malignant cervical epithelial changes for mass screening has been discussed. Using crude AG-e, pure AG-e, HSV-2, and HSV-1 antisera in a number of blind studies, no significant differences were found in the reproducibility of results. Observations indicated that a minimum of 30 cells must be analyzed to make data amenable to statistical analysis. Exfoliated cells from 62, 75, 77, and 94% of patients respectively diagnosed as mild, moderate, and marked atypia, and cancer were studied with antisera against total HSV-2. Similar results were observed with antisera against pure AG-e. No patient reacted with anti-AG-e or anti-HSV-1 antiserum in the absence of reactivity to antisera against total HSV-2 antigens. Studies using anti-ICP 12 and -ICP 14 antigens established the wide and specific staining character of anti-HSV-2 antisera. Morphologic preservation of cells by Papanicolaou staining after the FA procedures was considered satisfactory for diagnostic purposes. Our studies indicated the potential usefulness of anti-HSV-2 and anti-AG-e antisera in mass cervical cancer screening programs and cytology automation.

Detection of nucleic acid sequences in cervical tumors

Our results suggest that HSV RNA is present in abnormal cells of a significant proportion of cervical tissue specimens containing intraepithelial neoplasia and that it is only rarely detected in sections of normal cervical epithelium. A proliferation of atypical basal-type cells and their continued persistence through the more mature upper layers is the first indication of dysplasia and CIN in cervical epithelium, and it is in these proliferating cells that evidence of HSV RNA can be found. The results of localizing an HSV-2 antigen (ICSP 11–12) closely parallel the in situ hybridization results. About 30% of patients with dysplasia (CIN I and II) have detectable antigen. It is interesting to note that the incidence of detectable antigen decreased to 12% in patients with carcinoma in situ, but was demonstrable in 20% of patients who had microinvasive or invasive squamous cell lesions.

Immunohistological studies in viral encephalitis.

Immunofluorescence (IF) techniques performed on formol-fixed, paraffin-embedded and protease-pretreated sections from autopsy brain for demonstration of rabies and Herpes simplex type 1 (HSV-1) antigens proved to be a simple and effective method to ensure the etiology of some viral encephalitides. In contrast to fresh material, the use of formol-fixed tissues rules out any risk in handling highly infectious material. Another major advantage is the possibility of comparing IF appearance with normal histology of the same section by post-staining with hematoxilineosin (H.E.) or other stains. In 2 animal and one of 2 human rabies cases, neuronal cytoplasmic inclusions were more prominent in IF than in H.E. stains. In 31 cases with a histopathological diagnosis of necrotizing encephalitis, HSV-1 antigen was demonstrated in 17 of 25 acute cases but absent in 6 cases with clinical courses longer than 4 weeks. Specific fluorescence of nuclear inclusions bodies was inconstant and less prominent than that of neuronal cytoplasm and neuronal/glial nuclear membranes. Since HSV-1 positive cells are patchily distributed, limited tissue sampling (e.g. in diagnostic biopsies) may yield false negative results. Paucity of HSV-1-positive cells in the leptomeninges renders CSF immunocytology of limited use for diagnostic purposes.

Humoral- and cell-mediated immune responses to herpesvirus antigens in patients with cervical carcinoma

The humoral and cellular immune responses were studied in 35 patients with cervical carcinoma (Ca Cx) and 32 healthy controls. Complement fixing (CF) and indirect hemagglutinating antibody (IHA) titers for herpes simplex type 1 (HSV-1), herpes simplex type 2 (HSV-2), and cytomegalovirus (CMV), and fluorescent antibody (FA) titers for viral capsid antigen for Epstein-Barr virus (EBV) were determined. Cellular immune response was measured by the technique of [ 3H]thymidine uptake by stimulated lymphocytes. Phytohemagglutinin (PHA), HSV-1, HSV-2, and CMV antigens were used as stimulants. CF titers (≥ 1:4) to HSV-1, HSV-2, and CMV were present in 34 ( P < 0.005), 33 ( P < 0.005), and 29 Ca Cx patients and in 22, 20, and 18 controls. IHA titers (≥ 1.8) to HSV-1 and HSV-2 were present in all 35 Ca Cx patients ( P < 0.0001) and only 20 and 19 controls, respectively. IHA titers for CMV and FA titers for EBV were not different in the two study groups. Significantly more Ca Cx patients demonstrated definite prior HSV-2 infection ( P < 0.002). Geometric means of IHA titers were higher for HSV-1 ( P < 0.01) and HSV-2 ( P < 0.005) antigens in Ca Cx patients. Lymphocyte stimulation indices of 3 or greater, indicative of cell-mediated immunity, were found in all 35 Ca Cx patients to HSV-1 ( P < 0.0005), HSV-2 ( P < 0.0002) antigens, and in only 21 and 20 controls, respectively. A significantly increased lymphocyte response to a 1 100 dilution of HSV-2 antigen with other data suggests a difference in past exposure or an altered response of lymphocytes to HSV-2 antigen in Ca Cx patients.

Expression of herpesvirus-induced antigens in human cervical cancer.

Herpes simplex virus type 2 (HSV2) was first associated with human cervical cancer by seroepidemiologic studies over 10 years ago, but there has been no clearcut evidence confirming or refuting the role of the virus in this cancer. Expression of HSV antigens in cells transformed in vitro by the virus has been reported by several groups1,2. Evidence for the association of HSV antigens with human cervical cancer has recently been reviewed3–5; general antisera prepared against HSV virions or structural proteins were used by several groups to screen cells derived from the cancer and equivocal results were obtained. The tumour-specific antigens associated with three known oncogenic DNA viruses—adenovirus type 2, SV40, and Epstein–Barr herpesvirus—are DNA-binding proteins5–8. Therefore, proteins which bind DNA were isolated9,10 from HEp-2 cells infected by HSV2, and monospecific antisera to two of these proteins (ICSP11/12 and ICSP 34/35) were prepared. These rabbit antisera react specifically with the nuclei of HSV2-infected cells11. We show here that an HSV2-specific DNA-binding antigen was expressed in 38% of tissues with pathological findings of severe dysplasia or carcinoma. This therefore constitutes further evidence for HSV2 as a factor in human cervical carcinoma.

Isolation of the major herpes simplex virus type 1 (HSV-1)-specific glycoprotein by hydroxylapatite chromatography and its use in enzyme-linked immunosorbent assay for titration of human HSV-1-specific antibodies

A 131,000 molecular weight herpes simplex virus type 1 (HSV-1) glycoprotein designated antigen number 6 (Ag-6) was previously shown to possess almost exclusively HSV-1-specific antigenic sites. Fused rocket and crossed immunoelectrophoresis of fractions obtained from hydroxylapatite chromatography of crude HSV-1 antigen (Triton X-100-solubilized, infected tissue culture cells) showed that a subfraction of Ag-6 could be separated from the other HSV antigens. Enzyme-linked immunosorbent assay with the isolated Ag-6 showed that sera from rabbits infected with HSV-1 and HSV-1 human antisera contained antibodies to Ag-6, whereas sera from HSV-2-infected rabbits and sera from patients with primary HSV-2 infections did not react with Ag-6. Enzyme-linked immunosorbent assay of 852 human sera for antibodies to HSV type-common glycoproteins, Ag-6, and HSV 2-specific antigens showed that 139 sera which reacted negatively with HSV type-common glycoproteins also did not react with Ag-6 with HSV-2 specific antigens. The 713 sera reacting positively to HSV type-common antigens either reacted with Ag-6 (328 sera) or with HSV-2-specific antigens (31 sera) or both (354 sera). This means that Ag-6 might be useful in large-scale human serology for the detection of past infection with HSV-1, irrespective of whether or not past infection with HSV-2 has occurred.

Proteins of herpesvirus type 2. Isolation and immunologic characterization of two viral proteins in a virus-specific antigenic fraction

Previous reports from our laboratory have shown that “crude” AG-e, a type-commons HSV antigen, induces an antigen-driven cell-mediated immune response in patients with HSV cervicitis or cervix cancer. Anti-crude AG-e sera give rise in crossed immunoelectrophoresis against total viral soluble antigenic mixtures (HSV-2 (G) SAM) to one precipitin band (“pure” AG-e). Pure AG-e immunologically identical to crude AG-e as determined by immunodiffusion and it resolves into two infected cell proteins, ICP 12 (MW: 140,000) and ICP 14 (MW: 130,000) upon SDS-acrylamide gel electrophoresis. ICP 12 and ICP 14 are purified to radiochemical homogeneity by SDS-acrylamide gel electrophoresis. Antisera to ICP 12 react with HSV-2 (G) SAM in crossed immunoelectrophoresis and both anti-ICP 12 and ICP 14 sera stain HSV-2 (G) infected cells. ICP 12and ICP 14 appear to be envelope proteins as evidenced by: (i) the absorption of the reactivity of anti-ICP 12 and ICP 14 sera with HSV-2 (G) virions but not with “mock-virus” preparations, (ii) the ability of the anti-ICP 12 and ICP 14 sera to neutralize HSV-2 in presence of anti-IgG, and (iii) the resolution of a protein with the relative mobility of ICP 14 in virions the surface of which was iodinated with lactoperoxidase.

ASSOCIATION OF HERPES SIMPLEX VIRUS (HSV) WITH CERVICAL CANCER BY LYMPHOCYTE REACTIVITY WITH HSV-1 AND HSV-2 ANTIGENS

Twenty-six women with invasive cervical cancer were examined for lymphocyte responsiveness to phytohemagglutinin (PHA) herpes simplex virus (HSV). Blood specimens were obtained from each patient before radiation therapy and separated into subpopulations by Ficoll-Hypaque centrifugation. Lymphocytes were cultured in RPMI-1640 containing autologous plasma and exposed to PHA, HSV-1, HSV-2, and control antigens. The results compared with those of women having negative Papanicolaou smears and matched to the cancer patients by age, race, and socioeconomic class revealed significant differences in blastogenic response (3H-thymidine) to HSV-2 antigens. Results from groups with known HSV-1 and HSV-2 infections indicated that differences were associated with the cancer patients having a higher frequency of HSV-2 infection. Results generally agreed with the findings of previous studies in which serologic test procedures were used.

Immunoelectrophoretic identification and purification of herpes simplex virus antigens released from infected cells in tissue culture.

Proteins released from herpes simplex virus type 1 (HSV-1)-and type 2 (HSV-2)-infected HEp-2 cells have been characterized by the crossed immunoelectrophoretic technique. Both HSV type-common and type-specific antigens were found in the tissue culture medium 24 h after infection. Antigen Ag-6, an HSV-1-specific antigen, was found in high concentration in the medium as compared to other HSV antigens released from HSV-1-infected cells. The HSV-2-specific antigens, Ag-4 and Ag-1, were released in molecular modifications with altered electrophoretic mobility as compared to their cellular counterparts. Purification of HSV antigens was performed by ion-exchange chromatography, and an HSV type-common antigen, Ag-11, and an HSV-2-specific antigen, Ag-4A, were isolated.

Persistence of herpes simplex virus type 1 in rat neurotumor cells

Herpes simplex virus type 1 (HSV-1) infection of a rat central nervous system tumor cell line led to almost complete destruction of the cells. Cells that survived the infection could be isolated and shown to produce infectious HSV particles for variable lengths of time in culture ranging from 20 to 57 passages. Even though infectious virus production eventually ceased, the cell lines continued to produce herpes-specified proteins as measured by immunological techniques. These cells also showed herpesvirus-like structures in the electron microscope. The persistently infected cells that produced HSV antigens and bore HSV sequences were resistant to superinfection by HSV-1. The resistance was not due to failure of adsorption of the virus or to the production of interferon by the cells. The nature of the block in HSV replication in these neurotumor cells, which contain and partially express the HSV genome, is unknown, but may offer an interesting parallel to the known latency of HSV in neural tissues.

A longitudinal study of in vitro tests for lymphocyte function in rheumatoid arthritis

In vitro tests of lymphocyte function have been performed in 61 patients with ;classical' or ;definite' rheumatoid arthritis. In vitro lymphocyte function was assessed by lymphocyte transformation responses to phytohaemagglutinin (PHA), Pokeweed mitogen (PWM), Candida antigen, and herpes simplex type I (HSV1). Follow up data were available after 6 months of treatment in 32 of these patients. Spontaneous lymphocyte transformation was assessed in all patients. Results obtained in patients with rheumatoid arthiritis were compared to those seen in a normal control population. Disease activity of patients with rheumatoid arthritis was assessed using standard clinical methods. Lymphocytes from patients with rheumatoid arthritis showed a similar degree of spontaneous transformation to that seen in normal subjects. In contrast, lymphocytes from patients with rheumatoid arthritis responded less well to PHA and Candida and HSV1 antigens when compared to normal patients. In patients with rheumatoid arthritis the response to PWM was markedly enhanced compared to normals. Clinical improvement was noted in 19 of the 32 patients seen at follow up, all of whom had received gold or penicillamine therapy. The abnormal responses of PHA and PWM seen before treatment became normal in those patients who improved clinically. The responses to Candida and HSV1 antigens not only returned to normal following treatment but were increased above those seen in normal controls. A statistically significant association was seen between clinical improvement and improvement of in vitro tests of lymphocyte function.

Possible role of Fc receptors on cells infected and transformed by herpesvirus: escape from immune cytolysis.

Receptors for the Fc portion of nonimmune immunoglobulin G were demonstrated on B103 rat brain neuroma cells infected with herpes simplex virus type 1 (HSV-1) KOS by a radioimmunoassay using 125I-labeled heat-aggregated Fc fragments. Immune F(ab')2 fragments specific for HSV antigens competed efficiently for Fc binding sites, suggesting that the binding of Fc fragments to infected cells is specific for viral cell-surface antigens. It has been suggested that the binding of immune complexes to Fc receptors on the surfaces of tumor cells in vivo plays a role in protecting these cells from immune destruction. In vitro evidence is presented for the ability of aggregated immunoglobulin G molecules bound to cell-surface Fc receptors to protect both HSV-infected and HSV-transformed cells against complement-dependent and cell-mediated immune lysis.

Conditions required for induction of murine p30 by herpes simplex virus

Mouse cells (line N cIA cl10) contain 1.2-2.5 ng murine leukaemia virus (MuLV) p30 antigen/mg of protein; this amount of antigen is measurable by competition radioimmunoassay (RIA) but is not detectable by indirect immunofluorescence (IF). Infection of N cIA cl10 cells with herpes simplex virus type 2 (HSV-2) induces expression of MuLV p30. Induction by HSV-2 does not require either cell or virus DNA synthesis and is optimal 8 h post infection when cells at 50-70% confluence are infected at a multiplicity of infection (MOI) of 5-8 PFU/cell. At an MOI of 2.5, 70-80% of the cells express HSV antigens while none of the cells express p30; at an MOI of 5.0, 70-80% of the cells express HSV antigens but 55% of the cells express p30. Using the conditions reported in this paper for preparation of competing antigen, induction of p30 by HSV-2 (strain 333) infection is not measurable by competition RIA.

778 CEREBROSPINAL FLUID AND SERUM ANTIBODY IN RABBITS WITH HERPES SIMPLEX ENCEPHALITIS

The development of effective chemotherapy for Herpes Simplex Virus Encephalitis (HSE)with adenine arabinoside has stimulated interest in pathogenesis and the need for rapid diagnosis. To evaluate pathogenesis we have characterized herpes simplex virus (HSV)antibody in CSF and serum during the course of the illness in rabbits. The rabbit model of HSE is produced by corneal inoculation with HSV-1. Seven days post inoculation they have encephalitis with inflammatory cells in CSF and HSV in brain tissue. CSF and sera were collected prior to and days 5,7,9,and 12 post inoculation. Antibody was measured with radioimmune precipitation assay. The HSV-1 and control antigens were prepared in media containing C14 amino acids. The antigen-antibody precipitate was solubilized and C14 labeled HSV antigens were characterized with SDS acrylamide gel electrophoresis and autoradiography. HSV-1 antibody appeared in CSF 5 days after inoculation and serum antibody appeared on day 7. The HSV antigens precipitated from CSF and serum collected on day 12 were the same for the predominant high molecular weight group but showed differences in lesser molecular weight ranges. In this model of primary HSV-1 encephalitis antibody is detected in CSF before serum and qualitative differences are noted. These measurements better define the pathogenesis of HSE, and clinical studies are now underway characterizing antibody in human CSF and serum.

A nonlytic transforming mutant of herpes simplex virus type 2

A small-plaque mutant (NO.69) of herpes simplex virus type 2 (HSV-2) strain 333 has been previously isolated and characterized in this laboratory. This mutant was shown to produce a high ratio of noninfectious to infectious particles when grown at the nonpermissive temperature in hamster embryo fibroblasts [Westmoreland D. and Rapp F. (1976). Journal of Virology [8:92--102]. In this study, we have demonstrated that it is possible to obtain noninfectious stocks of this virus which retain transforming ability in a biochemical transformation assay specific for detection of the HSV gene for thymidine kinase. This mutant contains a DNA genome that has a density identical to the DNA of wild-type virus. Virus and cell DNA synthesis after infection with the mutant at both the permissive and nonpermissive temperature are similar to that observed in cultures infected with the parental virus. Clones of mouse cells biochemically transformed by this virus contain HSV antigens and are presently being examined for oncogenicity.

Thymidine kinaseless revertants of Ltk- cells transformed by herpes simplex virus type 1 are resistant to retransformation by homologous virus

Mouse L cells lacking thymidine kinase (Ltk-) that had been transformed to the thymidine kinase-positive (tk+) phenotype by herpes simplex virus type 1 (HSV-1) were cultured in medium containing tritiated thymidine. Six clonal lines of cells surviving this treatment were found to have the following properties: (i) the cells were tk- and had spontaneous back-reversion frequencies to the tk+ phenotype of 10(-5) or less, (ii) the cells contained HSV antigens, although in lesser amounts than in the parental transformed cells, and (iii) the cells were retransformable to the tk+ phenotype by HSV-1 at frequencies of about 1 to 13% of the frequency of the primary transformation of LtK- cells. HSV-1 plaqued as efficiently on monolayers of these cells and replicated in them to the same extent as it did in Ltk- cells. These results indicate that HSV-1-transformed L cells surviving selection with tritiated thymidine are unlike the parental Ltk- cells in that they are damaged in such a way that the cells are resistant to retransformation by homologous virus, although they remain fully permissive for virus replication.

Herpes Simplex-Raji(A44), a New Cell Line for Serologic Testing by Immunofluorescence

An IF technique for the detection of HSV antibodies by using a lymphoblastoid cell line, Raji(A44), which continuously produce a constant amount of HSV antigen, is described. IF and IH tests were found to be similar with respect to sensitivity and reproducibility. Sero-conversions detected by the CF test were detected with this cell line. The Raji(A44) line (cells in suspension) can be routinely used for the diagnosis of HSV infection.

Proteins of herpesvirus type 2: II. Studies demonstrating a correlation between a tumor-associated antigen (AG-4) and a virion protein

Antigen AG-4, a HSV-2 antigen associated with actively growing squamous cervical tumors, correlates with infected cell protein number 10 (ICP 10), a minor component of the HSV-2 virion. This association is based on the following evidence: (i) There is a positive correlation between the amounts of AG-4 and ICP 10 produced at various times following sequential treatment of infected cells with cycloheximide and actinomycin-D; (ii) the passage history of HSV-2 affects the synthesis of AG-4 and ICP 10 in a similar manner; their synthesis is unaffected by the cell type; (iii) ICP 10 is precipitated by AG-4 positive but not by AG-4 negative sera; and (iv) following partial biochemical purification of crude AG-4 preparations, those fractions containing the AG-4 complement-fixing activity differ from those without this activity in that they contain ICP 10.

Transformation of mouse cells after infection with ultraviolet irradiation‐inactivated herpes simplex virus type 2

A transformed mouse cell line (H238) was obtained following the infection of 238 mouse cells with ultraviolet (UV) irradiation-inactivated herpes simplex virus type 2 (HSV-2). The transformed cells produced tumors with a 100% incidence within 8 weeks in 6-week-old syngeneic BALB/c mice at an inoculum of 1 times 10(6). Indirect immunofluorescence (IF) tests revealed the presence of HSV antigens in the transformed cells. Antibodies to HSV-2 were found in the sera of tumor-bearing animals by neutralization and IF techniques. Neither HSV-2 infectious virus nor viral antigens could be detected by the transfer of transformed-cell DNA into permissive cells.