Anti-idiotypic antibodies against antibodies to the herpes simplex virus and its early antigens]

Polyclonal anti-idiotypic antibodies (anti-Id-AT or AT2) were produced to AT idiotopes of virion antigens--structural proteins of herpes simplex virus (HSV) and of early virus-induced antigens (VIA). The method of SPRIA established that anti-Id-AT seemed to be associated with AT active centers. Besides, immunization with anti-Id-AT produced anti-anti-Id-AT (AT3) similar in their specificity with antiviral AT or anti-VIA. It was shown that AT2 could be used as a diagnostic preparation instead of specific antigen for the detection of antibody to HSV or VIA in human sera by indirect RIA.     

Occurrence of antibodies against herpes simplex virus thymidine kinase in human sera

The occurrence of antibodies in human serum that block herpes simplex virus (HSV) type 1 and 2 deoxythymidine kinase (dTK) has been investigated. The antibodies were detected by means of a sensitive assay technique using [¹²⁵I]iododeoxyuridine as a substrate [Gronowitz and Källander, 1980]. A total of 213 sera was studied. They included pairs of serum from patients with an acute HSV infection, individual sera from persons not suspected of a HSV infection, as well as sera from patients from whom HSV had been isolated. The HSV complement fixing (cf) titer was determined for each serum and subsequently used as a reference. None of the HSV cf negative sera contained HSV dTK blocking antibodies, whereas all cf positive sera did, however, all excepting those that were collected in connection with primary infections. By following the serum titer after a primary infection, we have found that 12 out of 19 persons studied had detectable dTK antibodies after an average time of 179 days. The results indicate a low degree of cross-reactivity between HSV type 1 and 2 dTK blocking antibodies.     

Crossed immunoelectrophoretic analysis of herpes simplex virus type 2 proteins. Characterization of antigen-5

Summary Herpes simplex virus type 2 proteins extracted from infected cells and analysed by crossed immunoelectrophoresis identified a nonglycosylated antigen named Ag-5. The antigen contained two proteins when extracted from the agarose gel and the molecular weights were 128K and 91K. Both proteins are located in the nucleus of the infected cells and the 128K is identical to ICP-8. The 91K protein is based on the reactivity with monoclonal antibodies most likely the alkaline exonuclease mapped byPreston andCordingly (25). Our data show that although the proteins ICP-8 and 91 K coprecipitate they differ in both peptide compostion and in immunological specificity.With 7 Figures     

Enzyme immunoassay for detection of antibodies against herpes simplex virus with the use of different viral antigens

Enzyme-linked immunosorbent assays (ELISA) for detection of antibodies to herpes simplex virus type 1 (HSV-1) have been performed by using different immunosorbents prepared by passive adsorption of four HSV-1 antigen preparations to the wells of polystyrene microtitre plates in order to compare the the sensitivity, specificity and reproducibility of the tests. The following antigen preparations have been used: virus-infected native Vero cells and their lysates, membrane glycoproteins and virus nucleocapsid proteins. The optimal conditions have been established for each assay system: the concentrations of adsorbed antigen and the "threshold" values of the optical density. For each antigen tested except of the nucleocapsid (NC) proteins, comparable antibody titres were found in the sera of 6 patients with different forms of herpes infections and in 86 healthy subjects. In the sera of 6 herpetic patients the antibody titres were higher against NC antigen than against other antigen preparations.     

New antigens induced by herpes simplex virus in human tumors

Tissues of malignant tumors of the genitalia (cervix, uterus body, ovary) contain a specific antigen associated with herpes simplex virus type 2 (HSV-2) identical with the antigen of the infected cells. The virus-induced antigen was detected in tissues of cervical carcinoma in 35% of cases, in 15% of tumors of the corpus uteri and in 13% of ovary tumors. HSV-2 was isolated from pathologically altered cervical carcinoma cells in 2 out of 56 cases examined. These facts indicate the presence of HSV-2 genetic information in cells of some tumors. No virus-induced antigen was found in any of the 7 specimens of normal nontumorous tissue examined.     

Solid-phase radioimmunoassay of human immunoglobulin M and immunoglobulin G antibodies against herpes simplex virus type 1 capsid, envelope, and excreted antigens.

A solid-phase radioimmunoassay developed in our laboratory for detection of human viral immunoglobulin M (IgM) and IgG antibodies was applied to demonstrate human class-specific antibody response against capsid, envelope, and excreted antigens of herpes simplex virus type 1. In primary infections, a clear IgM and IgG antibody response was found predominantly against the envelope components, whereas the IgM and IgG antibodies to the capsid antigen appeared more slowly. Increasing IgG antibody titers to the excreted antigen were also found in primary infections, though appearing more slowly than antibodies to the other subunit antigens. The antibody response against capsid and envelope antigens was not type specific, whereas in primary infections IgG class antibodies against the excreted antigen showed distinct type specificity. In recurrent infections, no significant level of IgM class antibodies was demonstrated, but in the patients with a severe secondary herpes simplex virus infection a definite IgM class antibody response was found against the envelope antigen. In addition, during severe secondary infections the antibody response against the excreted antigen was enhanced. The host IgG antibody response in recurrent infections was directed against the envelope and excreted antigens, whereas the level of the capsid antibodies was relatively stable.     

Some structural antigens of herpes simplex virus type 1.

Several of the major structural polypeptides of herpes simplex virus were obtained in purified form by polyacrylamide gel electrophoresis of purified virus particle polypeptides. Antisera made by footpad inoculation of these polypeptides into rabbits were used to study the antigenic properties of two envelope glycoproteins and of the major capsid protein.     

Enzyme-linked immunosorbent assay for determination of antibodies against herpes simplex virus types 1 and 2 in human sera.

A rapid and reproducible enzyme-linked immunosorbent assay (ELISA) is described for determining antibodies in human sera against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2). The sera were absorbed for 30 min with heterologous virus-infected-cell extracts to remove cross-reacting antibodies and then were applied to ELISA plates containing the target antigens, immunoaffinity-purified HSV-1 glycoproteins gC and gD and HSV-2 glycoproteins gD and gF. The absorbance index, defined as the ratio of A414 generated by a serum sample absorbed with a heterologous virus-infected-cell extract versus the A414 of a serum sample absorbed with an uninfected-cell extract, was used to determine the presence or absence of antibodies to HSV-1 and HSV-2. Results of the ELISA for detecting antibodies against HSV-2, when compared with results obtained for the same sera by the microneutralization test, showed an index of overall agreement of 91%. Results of the ELISA for detecting antibodies against HSV-1, when compared with microneutralization test results for sera negative for HSV-2 antibodies but positive for HSV antibodies by ELISA, showed an index of agreement of 99%.     

Monoclonal antibodies against diagnostic Anisakis simplex antigens

Five monoclonal antibodies (UA2, UA3, UA5, UA6, and UA8) specific for Anisakis simplex are described. All are IgG1/κ monoclonal antibodies, except for UA2, which is an antibody IgM/κ. The molecular weights of the major components recognized in immunoblotting are 48 and 67 kDa (UA2); 139 kDa (UA3 and UA5; same epitope); 35, 38, and 139 kDa (UA6); and 205 kDa (UA8). UA2 was the only monoclonal antibody to recognize both components of an excretion-secretion antigen preparation and antigens in the excretory cell and esophageal glands of third-stage A.␣simplex larvae; antigens in the excretory cell were also recognized by UA3 and UA6. Cross-reactivity studies using a hyperimmune polyclonal rabbit serum reacting with various ascaridoid nematodes indicated that the antigens captured by our monoclonal antibodies were specific for A. simplex. Finally, comparative studies of our monoclonal antibodies and An2 (the only monoclonal antibody currently available for serodiagnosis of human anisakiasis), based on the calculation of multiples of normal activity for human anisakiasis sera, indicated that our monoclonal antibodies (and particularly UA3) recognized antigens that are good candidates for serodiagnostic purposes. Received: 13 February 1997 / Accepted: 16 March 1997     

Cell-mediated immunity against herpes simplex virus envelope, capsid, excreted, and crude antigens.

Cell-mediated immunity to herpes simplex virus envelope, capsid, excreted, and crude antigens was studied by in vitro lymphocyte stimulation tests during 198 recurrent attacks in 69 patients. Excreted antigen caused no blast transformation. Envelope and capsid antigen-induced lymphocyte stimulation was at the maximum 7 to 14 days postinfection, declining thereafter to a rather constant level in 1 to 2 months. The lowest levels were measured just a few days before a new attack. In persons with frequent relapses, the fluctuation was more rapid and stimulation index levels stayed higher, although no protective level seemed to exist. Cultures stimulated with the crude antigen in autologous serum showed rapid increases and declines in the stimulation index values, contrary to those grown in agamma serum, in which the stimulation level stayed rather constant up to 1 year postinfection.     

Neutralizing activity of antibodies against the major herpes simplex virus type 1 glycoproteins

The specificity and neutralizing activity of antibodies against the major herpes simplex virus type 1 (HSV-1) glycoproteins were tested in serum samples of patients with a history of HSV-1 infection. By preabsorption of sera to preparations of native and denatured HSV-1 proteins, followed by immunoblotting and microneutralization, it was shown that the majority of neutralizing antibodies are directed against denaturation-sensitive epitopes. Furthermore, preabsorption of sera to proteins of viral ts and deletion mutants revealed that antibodies specific for gB, gC, and gE had a low neutralizing activity. These results suggest a major role of anti-gD in neutralization of viral infectivity. In addition, it was shown that antibodies directed against the gB monomer were distinct from antibodies against the gB homodimers. The latter, however, did not reveal any measurable neutralizing activity.     

Serological analysis of herpes simplex virus types 1 and 2 with monoclonal antibodies.

A panel of monoclonal antibodies to herpes simplex virus glycoproteins was used for serological analysis of 130 strains. Based on specific immunological determinants, strains of each serotype clustered into subgroups. Monoclonal antibodies were suitable reagents for serotyping and have potential application to epidemiology of herpes simplex virus infections.     

Cross-reacting herpes simplex virus antigens in hamster and mouse cells transformed by ultraviolet light-inactivated herpes simplex virus type 2.

Murine and hamster cell lines, each transformed with a different strain of herpes simplex virus (HSV), were examined for cross-reacting antigens by in vitro and in vivo assays. A comparative study by the indirect immunofluorescence technique detected common cross-reacting viral antigens. Cytoplasmic fluorescence patterns were observed in the 333-8-9 hamster line, the H238 murine line, and the H238 clonal lines; these patterns were identical to the fluorescence pattern of HSV -2-infected controls when reacted with HSV antiserum. Tumor rejection studies in the BALB/c host indicated that each cell line provided immunity against a tumorigenic challenge of transformed mouse cells. The H238 clone EC1 3 provided a 53% immunity against itself at an inoculum of 10(6); the 333-8-9 line supported a 26% immunity. These data demonstrate a common HSV antigenicity between the murine and hamster transformed lines and further indicate that the HSV genome is involved in transformation.     

Solid-phase radioimmunoassay of herpes simplex virus IgG and IgM antibodies.

A solid-phase radioimmunoassay for detection of herpes simplex virus-specific IgG and IgM antibodies in human serum specimens is presented. Virus antigen is adsorbed on polystyrene balls and antibodies which attach to the antigen are detected by 125I-labeled antihuman-gamma or antihuman-mu immunoglobulins. A total of 76 specimens have been tested. The appearance of virus-specific IgG and IgM antibodies in primary herpetic infections was readily demonstrated. When serum samples from patients with past exposure to herpes simplex virus were tested, endpoint titers of virus-specific IgG antibodies were found to be 8 to 2048 times higher than titers determined by a complement fixation test. Apparent cross reactivity with varicella-zoster virus was observed in the present radioimmunoassay.     

Thin-layer immunoassay for determination of antibodies to herpes simplex virus.

Thin-layer immunoassay (TIA) is a simple serological technique suitable for analysis of large numbers of samples. In this study, TIA was evaluated for determination of antibodies to herpes simplex virus. Herpes simplex virus antigen used in TIA was purified from material released from virus-infected cells. The results obtained by TIA were compared with those obtained by neutralization and complement fixation tests. TIA was found to be as sensitive as the neutralization test for demonstration of herpes simplex virus antibodies. No false-negative or -positive reactions were observed. In primary herpes simplex virus-1 infections, an antibody response was demonstrated by TIA, whereas antibodies could not be demonstrated in patients with primary herpes simplex virus-2 infections.     

Further analysis of physicochemical and immunologic properties of herpes simplex virus precipitating antigens

Summary Evidence was obtained indicating that fresh antigen preparations of herpes simplex virus contained a number of antigenic components, which formed precipitates with human anti-herpes sera. Many of these were quite labile and lost during fractionations by DEAE cellulose chromatography. Several precipitating antigens with relative stability were further analysed by density gradient centrifugation and zone electrophoresis. Most of the antigen fractions thus obtained were found to contain subunits of surface antigens of the virus, which were capable of inducing virus neutralizing antibodies by immunization of animals and combiningin vitro with neutralizing antibodies. There were some special antigens with cathodic migration in electrophoresis and stability at pH 2 which were incapable of producing neutralizing antibodies in animals or combining with themin vitro. This investigation was supported by research grant (AI-05612) from The National Institute of Allergy and Infectious Diseases, U.S. Public Health Service, Bethesda. Maryland, U.S.A.     

Production and some properties of neutralizing antigens of herpes simplex virus.

Human diploid cells (LEP) infected with herpes simplex virus (HSV) type 1 were extracted by treatment with Nonidet P-40. The content of antigens reactive with neutralizing antibody in the clarified extract was measured by the blocking test. Influence of various factors on the production of the blocking antigens (BA) was tested. Highest BA titres in LEP cells were achieved at a multiplicity of infection (m.o.i.) of 0.5 TCD50/cell, medium 199 and 22 to 29 hours incubation at 37 degrees C. The use of resting cultures or the presence of cytosine arabinoside in medium reduced the BA production. The activity of BA was reduced by repeated freezing and thawing. The antigen was stable at -20 and -70 degrees C. One hour heating at 60 degrees C resulted in a marked decrease of the antigen titre. BA was resistant to formalin; formalin treatment did not influence its thermosensitivity.