Virus-specific antibodies in sera from patients with genital herpes simplex virus infection.

Virus-specific antibodies against a number of herpes simplex virus type 2 antigens were determined by radioimmunoprecipitation assays in sequential serum samples obtained from 12 patients with initial genital herpes simplex virus infection. The progressive appearance of antibodies to virus-specific antigens was observed; antibodies against a 130,000-molecular-weight glycoprotein complex appeared first, followed by antibodies against the major nucleocapsid polypeptide and then antibodies against a number of other viral antigens, including a polypeptide with a molecular weight of 62,000. Patients who developed a wide variety of antibodies to viral polypeptides shortly after resolution of their initial episode seemed to experience more severe initial infections and more recurrences than did those who reacted poorly with these virus-specific antigens. This was most apparent with respect to antibodies to virus-specific polypeptides with molecular weights between 30,000 and 43,000. Antibody specificity did not change during the course of follow-up regardless of whether serum samples were taken shortly before, during, or after recurrent episodes. Glycoprotein-specific antibodies were quantitated with the purified 130,000-molecular-weight glycoprotein material. No significant fluctuations in these antibody titers were observed before or after recurrences of the disease.     

Antibodies against varicella-zoster virus and herpes simplex virus deoxythymidine kinase in heterologous infections

Antibody responses to varicella-zoster virus (VZV) deoxythymidine kinase (dTK) and herpes simplex virus (HSV) dTK in homologous and heterologous infections were studied. Antibodies blocking the enzymatic activity of VZV-dTK appeared late after varicella and decreased more or less in parallel with the decreasing complement fixing [CF] titre. In herpes zoster, on the other hand, antibodies to VZV-dTK appeared soon after infection. Antibodies against HSV dTKs appeared long after primary infection, but they were subsequently present in all other HSV-CF positive sera. In recurrent HSV, all acute sera were already HSV-dTK antibody positive, and three of nine persons showed an increase in titer between their acute and convalescent sera. Blocking antibodies to VZV-dTK appeared rapidly in specimens from three of 18 individuals positive by an immunofluorescence VZV-immunity test during HSV infection, whereas all other specimens remained devoid of blocking antibodies against VZV-dTK. A rise in antibody titre against HSV-dTK during VZV infections was observed in serum specimens from three of 13 HSV-CF positive patients, whereas an antibody response against HSV-dTK was not found in HSV-CF negative individuals in connection with VZV infections. The relevance of the sporadic increase in the titres of antibodies against heterologous viral dTKs is discussed.     

Immune responses in mice against herpes simplex virus: mechanisms of protection against facial and ganglionic infections.

We performed experiments with mice to determine the nature of the immune response(s) that prevents primary infections of the skin and the trigeminal ganglia with herpes simplex virus. Immunization with infectious herpes simplex virus, inactivated virus, or material enriched for viral glycoproteins protected hairless mice against primary facial and ganglionic infections. Live and inactivated viruses induced neutralizing antibodies, whereas glycoprotein material did not. Instead, glycoprotein material induced antibodies that were largely directed against two glycopolypeptides with molecular weights of 120,000 to 130,000. Hairless mice immunized with glycoprotein material responded faster than control mice in the synthesis of neutralizing antibodies after challenge with infectious virus. Congenital athymic BALB/c (nu/nu) mice were protected against primary facial infections after immunization with glycoprotein material, but glycoprotein-specific antibodies were not induced.     

Multiple Sclerosis: Local Synthesis of Electrophoretically Restricted Measles, Rubella, Mumps and Herpes Simplex Virus Antibodies in the Central Nervous System

An imprint electroimmunofixation (IEIF) technique was used to study measles, rubella, mumps and herpes simplex virus antibodies in serum and concentrated cerebrospinal fluid (CSF) from ten patients with multiple sclerosis (MS). Electrophoretically restricted virus-specific antibodies were detected in sera or CSF from nine of the ten patients. Comparison of the antibody patterns in matching serum and CSF samples indicated that electrophoretically restricted populations of antibody against one or more of the four viruses were produced locally in the central nervous system of nine patients. No association between the locally produced antibody populations the oligoclonal IgG of the CSF could be demonstrated. The virus-specific antibodies studied thus seem to constitute only a minor fraction of the total IgG of the CSF from MS patients.     

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.     

Comparison of Western blot (immunoblot) and glycoprotein G-specific immunodot enzyme assay for detecting antibodies to herpes simplex virus types 1 and 2 in human sera.

Sera from patients with culture-proven genital herpes infections were tested for herpes simplex virus type 1 (HSV-1)- and HSV-2-specific antibodies by both a Western blot (immunoblot) technique (WBA) and immunodot enzyme assays (IEAs) specific for HSV-1 or HSV-2 glycoprotein G (gG). Of 137 serum samples tested, none was mistyped by either WBA or IEA. Both tests were most sensitive with sera obtained at least 21 days after onset of primary HSV-2 infections or sera drawn during recurrent HSV-2 genital episodes: 75 of 76 (99%) such serum samples were positive for HSV-2 antibody by WBA and 73 of 76 (96%) were positive by IEA. Of sera drawn earlier than 21 days from onset of primary genital HSV-2, antibodies to HSV-2 were detected in 25% by WBA and 8% by IEA. In patients with culture-proven primary genital HSV-1 infection, WBA detected antibodies to HSV-1 proteins in 16 of 17 (94%) serum samples drawn at least 21 days after onset of primary genital HSV-1 infection, compared with 9 of 17 (53%) serum samples tested for gG-1 by IEA. Both WBA and IEA are accurate and sensitive tests for HSV-2 antibody in patients convalescing from a first episode or having symptomatic or asymptomatic recurrent genital herpes. WBA was more sensitive than IEA in detecting seroconversion following primary HSV-1 genital herpes, although both assays may miss persons undergoing early seroconversion to HSV-2.     

Immunological diagnosis in viral infections of the central nervous system: course of antibody titres against homo- and heterologous viruses

In clinical cases suspected for viral encephalitis or meningoencephalitis, the estimation of virus-specific antibodies especially in liquor requires high sensitivity as well as specificity. With enzyme immunoassays the sensitivity in detecting antibodies has increased compared to e.g., complement fixation tests. This report concerns the determination of virus-specific antibodies with a commercial enzyme-linked immunosorbent assay (ELISA) in paired liquor/serum samples of four patients with encephalitis or meningoencephalitis. Up to six virus-specific antibodies of the IgG and IgM classes have been determined [herpes simplex virus (HSV), varicella-zoster virus (VZV), cytomegalo virus, mumps virus, measles virus, and rubella virus]. Additionally, serum samples from several patients suffering, or recovered from, diseases caused by HSV and VZV without CNS involvement have been included as controls. The results showed that besides the virus-specific antibody development (IgG and IgM) against the leading virus, i.e., principally concerned in the disease manifestation assumed to be primarily causing the disease, virus-specific antibodies of the IgG and IgM class against a heterologous virus (e.g., VZV) could also be measured with substantial titers. Cross-reacting antibodies to both HSV and VZV with the ELISA only appeared and were present in cases where the infection mainly affected the CNS: no such immunological cross-reactivity was observed in serum of individuals in clinically silent stages of both HSV and VZV infections. The same situation with no measurable cross-reacting antibodies was found in cases of acute HSV or VZV diseases where the CNS was not involved. These findings have been discussed with respect to the findings of common antigens, especially between HSV and VZV, and with respect to an unspecific stimulation of immunocompetent cells.Abbreviations and Definitions CSF Cerebrospinal fluid - ELISA enzyme-linked immunosorbent assay - CF-test complement fixation test - NT neutralization test - IFT immunofluorescence test - vIgG virus-specific IgG-antibody - vIgM virus-specific IgM-antibody - HSV herpes simplex virus - VZV varicella zoster virus - EEG electro-encephalogram - CCT cranial computer tomogram MEM, minim essential medium     

Virus-specific polymeric immunoglobulin A antibodies in serum from patients with rubella, measles, varicella, and herpes zoster virus infections.

More than 85% of the immunoglobulin A (IgA) antibodies in normal adult serum are monomeric (m-IgA). By contrast, virus-specific IgA is mainly polymeric (p-IgA) in sera from patients with rubella, measles, and varicella. Specific m-IgA antibodies only reach quantitative significance in late convalescence. In patients with herpes zoster, on the other hand, a varying response was observed: in three of six sera, specific IgA was absent or at a very low titer, whereas in the remaining three cases, a high titer of both p-IgA and m-IgA was noted. These results suggest that in the initial response to rubella, measles, and varicella-zoster viruses, specific IgA first appears as p-IgA and only later becomes, or is replaced by, m-IgA.     

Low and high molecular weight cytomegalovirus-specific immunoglobulin M antibody in renal transplant patients with cytomegalovirus infections

Sera from 27 renal transplant patients with primary and recurrent CMV infections and which were known to contain CMV-specific IgM antibodies were investigated by indirect immunofluorescence for the presence of virus-specific high molecular weight IgM (19S IgM) and low molecular weight IgM (7S IgM). After sucrose gradient fractionation of the sera, 19S IgM was found in all 27 patients, whereas 7S IgM was present in 11 out of 19 (56%) patients with primary CMV infection and in 1 out of 8 (12%) patients with recurrent CMV infection. The presence of 7S IgM was unrelated to the titre of the virus-specific IgM in whole serum. The presence of IgM rheumatoid factor was monitored by a sensitive fluorescence assay using measles virus antigen/antibody complexes. The absorption of the serum fractions with heat-aggregated gamma globulin failed to remove the specific IgM staining indicating that it was not due to IgM rheumatoid factor. On the other hand adsorption with protein A/sepharose removed the specific IgM staining from the 7S IgM fractions but not from the 19S IgM fractions. This suggests that specific 19S and 7S IgM antibodies may belong to different subclasses of IgM.     

Persistence of serum IgA antibodies to herpes simplex, varicella-zoster, cytomegalovirus, and rubella virus detected by enzyme-linked immunosorbent assays

Enzyme-linked immunosorbent assays were used to detect IgG and IgA antibodies to herpes simplex virus (HSV), varicella-zoster virus (VZV), cytomegalovirus (CMV), and rubella virus in sera from 68 adult female gynaecological patients. Of the patients who had virus-specific IgG antibodies, the proportion who also had virus-specific IgA was 98% for HSV, 75% for VZV, 73% for rubella virus, and 42% for CMV. IgA antibodies to all four viruses were only found when specific IgG antibodies were also detected in the serum. These results suggest that virus-specific IgA may persist for several years; possible explanations for this are discussed.     

Immune response to bovine herpes herpesvirus type 1 infections: virus-specific antibodies in sera from infected animals.

The virus specificity of antibodies against bovine herpes virus type 1 was determined with a radioimmunoprecipitation assay and serum collected from natural and experimentally induced infections. By using sequentially collected sera, the development of antibodies to 4 to 5 viral glycoproteins and 11 to 12 nonglycosylated proteins was followed for the first 50 days after infection. The major and most consistent responses in experimentally and naturally infected animals were to four glycoproteins with molecular weights of 102,000, 96,000, 69,000, and 55,000, as well as to a major virion 115,000-molecular-weight nonglycosylated protein. The four glycoproteins were all coprecipitated by a neutralizing monoclonal antibody and were probably involved as target antigens in virus neutralization. Another antigenically unrelated glycoprotein with a molecular weight of 82,000 and a nonglycosylated protein with a molecular weight of 91,000 were also precipitated, but the immune response to these two proteins was transient. Reactivity to gp82 was only weakly detected in serum from naturally infected animals. Contact control animals which did not contract a bovine herpes virus type 1 infection but were exposed to infected animals with signs of severe illness had antibodies which recognized gp102, gp96, gp69 and gp55 as well as p115. These antibodies were present in low amounts and, in contrast to infected animals, did not increase between acute and convalescent sampling.     

Secreted portion of glycoprotein g of herpes simplex virus type 2 is a novel antigen for type-discriminating serology

The secreted portion of glycoprotein G (sgG-2) of herpes simplex virus type 2 (HSV-2) was evaluated as a novel antigen in an enzyme-linked immunosorbent assay (ELISA) format for detection of type-specific immunoglobulin G (IgG) antibodies in HSV-2-infected patients. The results were compared with those obtained by a commercially available assay, the HerpeSelect 2 ELISA (the FOCUS2 assay). Five different panels of sera were analyzed: panel A consisted of 109 serum samples from patients with a culture-proven HSV-1 infection that were Western blotting (WB) negative for HSV-2; panel B consisted of 106 serum samples from patients with a culture-proven recurrent HSV-2 infection that were WB positive for HSV-2; panel C consisted of 100 serum samples with no detectable IgG antibodies against HSV-1 and HSV-2; panel D consisted of 70 HSV-2 negative "tricky" serum samples containing antinuclear IgG antibodies or IgM antibodies against other viruses or bacteria; and panel E consisted of consecutive serum samples from 21 patients presenting with a first episode of HSV-2-induced lesions. When sera in panels A to C were analyzed, the sgG-2 ELISA and the FOCUS2 assay both showed sensitivities and specificities of >or=98%. In total, among the samples in panel D, 13 serum samples (19%) were false positive by the FOCUS2 assay and 1 serum sample (1.4%) was false positive by the sgG-2 ELISA. When the sera in panel E were analyzed, the sgG-2 ELISA detected seroconversion somewhat later than WB or the FOCUS2 assay did. We conclude that sgG-2 induces an HSV-2 type-specific antibody response and can be used for type-discriminating serology.     

Antibody avidity following varicella-zoster virus infections.

The avidity of IgG antibodies following varicella-zoster virus (VZV) infections was investigated using urea treatment of antigen-bound serum antibody by indirect radioimmunoassay (RIA) and immunoblotting techniques. Sequential sera from 16 patients with varicella and 17 patients with zoster were tested, as well as sera from 80 seropositive individuals without a recent history of VZV disease. Both types of assay showed that low-avidity antibodies predominate early after primary infection, but that antibody avidity increases markedly during convalescence. Using RIA, all sera taken up to 12 weeks after the onset of varicella showed greater than 50% reduction in antibody titre after treatment with 8 M urea but thereafter the proportion of urea resistant antibody increased with time. In contrast, after recurrent infection, high avidity antibodies were found to predominate at all times. Only 6 of 47 sera tested from zoster cases showed greater than 30% reduction after urea treatment and all these were taken within 2 weeks after onset of rash. Immunoblotting also showed that the highly immunogenic p32/p36 nucleoproteins appear to induce predominantly low avidity antibodies, even after recurrent VZV infection. The results of this study indicate that treatment with 8 M urea in RIA for IgG antibodies may be a simple and reliable method for distinguishing primary and anamnestic antibody responses against VZV.     

Detection of anti-platelet antibodies in patients with idiopathic thrombocytopenic purpura (ITP) and in patients with rubella and herpes group viral infections

A highly sensitive enzyme-linked immunosorbent assay (ELISA) technique was observed for serological detection of antibodies against platelets. A covalently bound to Sepharose CL-4B was used to enrich sera in the IgG3 subclass of antibodies. The ELISA procedure as applied to detect anti-platelet antibodies in patients with herpes or rubella viral infections and in patients with idiopathic thrombocytopenic purpura (ITP). Twenty-eight sera from 13 thrombocytopenic patients showed high levels of anti-platelet antibodies. Two splenectomized patients in remission became negative for anti-platelet antibodies. Seventy-four sera from patients with serological diagnosis of herpes group viral infections comprising 10 cases of cytomegalovirus, nine cases of varicella or zoster, six cases of herpes simplex and four cases of Epstein-Barr virus were examined for the presence of anti-platelet antibodies. Except for two patients with varicella and zoster and one patient with rubella infection, all cases examined showed positive titres of anti-platelet antibodies. Sera from a group of 51 healthy controls were evaluated for anti-platelet antibodies. Forty-nine (96%) were negative (less than 40), whereas the other two were only slightly positive.     

Specific serum IgA, IgG and IgM antibody determination by a modified indirect ELISA-technique in primary and recurrent herpes simplex virus infection

Twenty-three patients with a herpetic infection as diagnosed by a positive culture of herpes simplex virus (HSV) were studied with respect to serological responses of IgA, IgG and IgM antibodies in paired serum samples by an indirect (sandwich) enzyme linked immunosorbent assay (ELISA). Eight of the patients had a primary infection and 15 a recurrent one. In the ELISA test a detergent treated cell lysate of HSV type 1 was used as antigen. In the IgM assay all sera were pretreated with antihuman IgG with the purpose to precipitate IgG of the samples. The conjugate was a F(ab)2-fragment of antihuman-IgM. In primary infections all patients had significant titre rises of IgG and presence of high IgM titres in the convalescent serum. IgA antibodies were found in all of them, while titre rises were detected in 5/8. In recurrent infections titre rises of IgG and IgA antibodies were found in 4 and 5, respectively. Six had detectable IgM in one or both of the paired samples. The IgG titres were higher in recurrent infections than in primary, in contrast to IgM of which much higher titres were found in primary infections. It is concluded that in primary infections a conclusive serological diagnosis was established in all patients, whereas in recurrent infections this was achieved in two of three patients. The indirect ELISA method used for IgM detection was sensitive, reliable and convenient. Interfering rheumatoid factor was effectively eliminated by treatment with antihuman IgG.     

Herpes simplex virus-specific serum immunoglobulin a: detection in patients with primary or recurrent herpes infections and in healthy adults.

A sensitive radioimmunoassay was used to determine levels of herpes simplex virus (HSV)-specific immunoglobulin A (IgA) in serial serum samples drawn from patients with primary HSV infections and from persons with recurrent HSV infections, and in single samples from 90 healthy adults. Significantly rising HSV IgA titers were detected in patients with primary infections, whereas those with recurrent infections had nonfluctuating titers. Sera of IgG-seropositive healthy adults were all positive for HSV-specific IgA without special pretreatment.     

Differentiation of primary from nonprimary genital herpes infections by a herpes simplex virus-specific immunoglobulin G avidity assay.

An immunoglobulin G (IgG) antibody avidity assay which uses protein-denaturing agents and a modification of an enzyme-linked immunosorbent assay have been investigated for their usefulness in distinguishing primary genital herpes simplex virus (HSV) infections from nonprimary infections. Forty-nine serum specimens from patients with primary, recurrent, and nonprimary first-episode genital herpes were studied. The clearest separation was obtained with 6 M urea treatment, giving mean avidity indices of 0.398 for sera < or = 100 days after the infection and 0.879 for sera > 100 days after the infection (P < 0.001). No significant difference in avidity indices was observed between the recurrent and nonprimary first-episode infections. Determination of the avidity of HSV-specific IgG will improve the diagnostic potential of HSV serology.     

Quality control assessment for the serological diagnosis of tick borne encephalitis virus infections.

BACKGROUND: The diagnosis of tick borne encephalitis (TBE) is mainly based on the demonstration of specific antibodies in serum when neurological disease is manifested. Improving diagnostics is the most important step in detecting and dealing with these pathogens. Quality control measures are essential for TBE diagnosis. OBJECTIVE: To assess an external quality assurance (EQA) program for the serologic diagnosis of TBE infections. STUDY DESIGN: A panel of 12 serum samples was sent out to be tested for the presence of TBE virus-specific IgM and IgG. This panel contained seven TBE-positive samples for IgM and/or IgG; three negative samples; two samples positive either for West Nile virus (WNV) or Dengue virus (DENV). RESULTS: Fourty-two laboratories from 25 European and 2 non-European countries participated in this EQA. The correct answer by each laboratory for all samples ranked between 58 and 96% and sera with IgM antibody positive for TBE were correctly recognized by 46-88% of the laboratories. Sera with IgG antibody positive for TBE were correctly recognized by 83-95% of the laboratories. False TBE-positive results were obtained with DENV, WNV or negative sera only for IgG-based assays. CONCLUSION: Correct results for at least 90% of the samples were obtained by 33 of 40 participating laboratories for IgM and for 16 of 42 laboratories for IgG.     

Neutralizing serum IgM antibodies in infections withHerpes simplex virus hominis

Forty-two patients with herpes simplex virus infections were tested for neutralizing serum IgM antibodies. A technique in which serum antibody fluorescein staining was combined with sucrose gradient centrifugation facilitated the isolation of the serum IgM fraction for the use in neutralization tests. In nearly all cases with primary infection, especially those presenting heavy clinical signs (encephalitis/meningitis) the IgM tests were positive. In one case we could detect the IgM antibodies for 11 weeks after the onset of the illness, in another case in cerebrospinal fluid samples for 6 weeks. In localized herpes infections, which were mostly due to reactivations, serum IgM antibodies could only rarely be demonstrated. Among the serologic tests used in this study (NT, CFT, IFT, ACIFT), only the CFT beside the NT (with certain reservations) can be applied for subtyping HSV serum antibodies.The work was supported by the Deutsche Forschungsgemeinschaft, SFB 31.     

Antibody responses in patients with staphylococcal septicemia against two Staphylococcus aureus fibrinogen binding proteins: clumping factor and an extracellular fibrinogen binding protein

We analyzed the serum antibody responses against two Staphylococcus aureus fibrinogen binding proteins, the cell-bound clumping factor (Clf) and an extracellular fibrinogen binding protein (Efb). The material consisted of 105 consecutive serum samples from 41 patients suffering from S. aureus septicemia and 72 serum samples from healthy individuals. An enzyme-linked immunosorbent assay (ELISA) was developed. Healthy individuals showed variable levels of antibodies against the studied antigens, and cutoff levels (upper 95th percentile) against these antigens were determined. No correlation was seen between serum antibody levels against Clf and Efb. In acute-phase samples 27% of patients showed positive antibody levels against Clf and 10% showed positive levels against Efb, while in convalescent-phase samples 63% (26 of 41) showed a positive serology against Clf and 49% (20 of 41) showed a positive serology against Efb. Antibody levels against Efb were significantly lower in the acute-phase sera than in sera from healthy individuals (P = 0. 002). An antibody response against Clf was most frequent in patients suffering from osteitis plus septic arthritis and from endocarditis (80% positive). The antibody response against Efb appeared to develop later in the course of disease. A possible biological effect of measured antibodies was demonstrated with the help of an inhibition ELISA, in which both high-titer and low-titer sera inhibited the binding of bacteria to fibrinogen. In conclusion, we have demonstrated in vivo production of S. aureus fibrinogen binding proteins during deep S. aureus infections and a possible diagnostic and prophylactic role of the corresponding serum antibodies in such infections.