Determination of herpes simplex virus type-specific antibodies by enzyme-linked immunosorbent assay.

We determined type-specific antibodies to herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) by an indirect enzyme-linked immunosorbent assay, using as antigens HSV-1 glycoprotein gC-1 and a HSV-2-specific polypeptide purified on affinity columns of monoclonal antibodies. All sera were initially screened for HSV antibodies by the enzyme-linked immunosorbent assay with a pool of Triton X-100-extracted antigens of HSV-1- and HSV-2-infected HEp-2 cells. The titer of HSV antibodies was predicted from a linear regression curve based on the absorbance of the initial 1:50 serum dilution. The sensitivity and specificity of the screening assay and of the assay for type-specific antibodies were established.     

Human antibody response to herpes simplex virus-specific polypeptides after primary and recurrent infection.

Human antibody responses to specific polypeptides of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2, respectively) were assessed in serial serum specimens from 18 infected patients by immunoblot technology. Nine patients had HSV-1 infections (six genital and three oral) and nine had HSV-2 genital infections. Antibodies to homologous and heterologous HSV antigens were studied and correlated with total microneutralization and enzyme-linked immunosorbent assay antibodies as well as correlated directly to purified glycoproteins. The data indicated a sequential appearance of antibodies to specific polypeptides, according to virus type and site of infection. After HSV-1 infection, the initial response was to glycoprotein B, but the same was not true for HSV-2 infection, where the initial response appeared to be to the type-specific glycoprotein G. A difference in sequential appearance of antibodies for the two viruses indicated greater reactivity to lower-molecular-weight polypeptides after genital infection, irrespective of type, in contrast to nongenital HSV-1 infections. The antibody responses for selected sera to purified glycoproteins B and D were verified by enzyme-linked immunosorbent assay antibody determinations.     

Mapping of linear antigenic determinants on glycoprotein C of herpes simplex virus type 1 and type 2 recognized by human serum immunoglobulin G antibodies

Using membrane-based dekapeptides, the reactivity of human serum antibodies with linear antigenic determinants of herpes simplex virus (HSV) type 1 and type 2 glycoprotein C (gC-1, gC-2) was studied by pep scan and immunodot assay. The entire coding sequences of gC-1 and gC-2 were screened for the presence of linear epitopes by pep scan. Peptides recognized in an HSV-1 type-specific manner were mainly identified within the N-terminal third and at the C-terminus of gC-1, whereas most type-common antibodies were directed against colinear peptides within the central parts of gC-1 and gC-2. The type-specific reaction of human sera with gC-2 peptides in pep scan was poor. Eight peptides identified as immunoreactive by pep scan were further tested in immunodot assay for their reactivity with a human serum panel. None of the eight HSV-negative sera gave positive results by immunodot assay. Positive reactions with gC peptides were found to be strongly age-dependent, i.e., the rate of positive reactions was significantly higher in HSV-positive adults than in HSV-positive children. Antibody reactivity with two type-common gC peptides was demonstrated in 17 out of 28 HSV-positive sera. A putative type-specific gC-2 peptide employed in immunodot assay was inconsistently recognized by human sera. Twenty HSV-positive sera reacted with at least 1 of 5 type-specific gC-1 peptides. Nine sera showing no reactivity with glycoprotein G of HSV-1 (gG-1) by immunoblotting recognized type-specific gC-1 peptides in immunodot assay. Thus, gC-1 peptides might allow the detection of HSV-1-specific antibodies in individuals showing no reactivity with commonly employed HSV-1-specific diagnostic antigens, i.e., purified or recombinant gG-1. J. Med. Virol. 55:281–287, 1998. © 1998 Wiley-Liss, Inc.     

Detection of herpes simplex virus type-specific antibodies by an enzyme-linked immunosorbent assay based on glycoprotein G

In order to develop a simple and quantitative method to detect herpes simplex virus (HSV) type-specific antibodies, the usefulness of an enzyme-linked immunosorbent assay (ELISA) using HSV glycoprotein G (gG) captured on a plate by monoclonal antibodies as antigen was studied. The gG1- and gG2-specific IgG antibody activities were measured by the ELISA for 54 sera which had been collected from culture-proven genital herpes patients and pre-characterized by an immunodot assay using purified gG antigens. Thirty control sera without antibodies against the HSV whole antigens were also included. In comparison with the immunodot assay as standard, the sensitivities of the ELISA were 88.9% (32/36) for HSV-1 antibody and 89.2% (33/37) for HSV-2 antibody and the specificities were both 100%. Sera taken within a few months after primary infection tended to give false negative results. The HSV type-specific ELISA based on easy-to-prepare gG antigens might be useful to help improve the serological assessment of HSV infections. J. Med. Virol. 53:319–323, 1997. © 1997 Wiley-Liss, Inc.     

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%.     

Whole cell lysate enzyme immunoassays vs. recombinant glycoprotein G2-based immunoassays for HSV-2 seroprevalence studies.

Seroepidemiology studies of herpes simplex virus type 2 (HSV-2) infections have been difficult to carry out because antibodies to HSV type 1 (HSV-1) show an extensive cross-reactivity with HSV-2 antigens. Many kits available currently are not entirely type specific for serodiagnosis of HSV-2 infections and therefore do not allow reliable discrimination of past exposure to these closely related alphaherpes viruses. Attempts to develop type-specific antigens have focused on the envelope glycoproteins, particularly glycoprotein G (gG). A cross-sectional study was carried out to examine the seroprevalence of antibodies to HSV-2 among healthy university students, using different methods: a whole cell lysate enzyme-linked immunosorbent assay (ELISA), two different ELISAs, and a newly developed immunoblot assay, the last three based on recombinant gG2. HSV-2 prevalence was 24 times higher with the whole cell lysate ELISA (31%; 95% confidence interval [CI]: 27-35%) than the ELISAs and the immunoblot assay based on recombinant gG2 (1.3%; 95% CI: 0.1-2.5%), thus showing the inaccuracy of commercial tests based on whole-antigen preparations for epidemiological studies. Laboratories should be cautious and ensure that commercial tests for HSV typing are based on type-specific glycoproteins.     

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.     

ELISA for detection of IgG and IgM antibodies to HSV-1 and HSV-2 in human sera

A rapid, enzyme-linked immunoassay (ELISA) was applied to identify and measure specific IgG and IgM antibodies to herpes simplex viruses types 1 and 2 (HSV-1 and HSV-2). Detergent solubilized infected cells and mock-infected cells were used as antigens in the assay. Identification of type-specific antibodies was achieved by a competition assay in which clinical sera mixed with HSV-1 or HSV-2 antigens were assayed for reactivity to identical antigens coating wells of polystyrene microtiter plates. Reactivity and the specificity of the reactive immunoglobulin class was quantitated using biotinylated goat anti-IgG and biotinylated goat anti-IgM. Five paired sera from patients with diagnosed herpes simplex genital infections and one human anti-HSV-1 reference serum were tested with this assay and results were compared to results previously obtained using a complement fixation test and micro-SPRIA. The results indicate that the ELISA is a specific, sensitive and simple test which confirms the herpes simplex virus infection history of patients.     

A branched, synthetic oligopeptide corresponding to a region of glycoprotein G of HSV-1 reacts sensitively and specifically with HSV-1 antibodies in an ELISA

Herpes simplex viruses types 1 and 2 (HSV-1 and HSV-2), which are common worldwide, are so similar that antibodies directed against one serotype may crossreact with antigens from the other one. Methods for specific detection of antibodies against HSV-1 or HSV-2 are based upon the antigenicities of glycoproteins G. However, due to the cost, the available commercial methods may not readily be used in developing countries. A different enzyme-linked immunosorbent assay (ELISA) method, based upon a synthetic oligopeptide corresponding to an immunogenic region in glycoprotein G of HSV-2, has been used recently and successfully for detection of HSV-2 antibodies. In the present study, the sequences of a newly identified immunogenic and type-specific region in glycoprotein G of HSV-1 was used to synthesize three different, branched oligopeptides. The performances of these peptides in an ELISA were investigated by testing Scandinavian and African sera which were characterized by commercial ELISA and Western blotting methods and divided into four groups either lacking HSV antibodies, containing antibodies against one or the other virus, or against both types. The peptide which corresponded in sequence to the immunodominant region was as specific and sensitive by an ELISA as were the commercial methods. The method is inexpensive and reliable.     

Comparison of a Multiplexed Herpes Simplex Virus Type-Specific Immunoglobulin G Serology Assay to Immunoblot, Western Blot, and Enzyme-Linked Immunosorbent Assays

The human herpes simplex virus (HSV) is highly pathogenic, with infections caused by two distinct antigenic types, HSV-1 and HSV-2. Differentiation of antibodies to these specific antigens can provide useful information for the diagnosis of subclinical or undiagnosed HSV-2 infections, as well as for reducing the risk of maternal transfer of HSV to the neonate. In this study, a multiplex assay capable of concurrent detection of HSV-1 and -2 immunoglobulin G (IgG) antibodies was compared to immunoblot, Western blot, and enzyme-linked immunosorbent assays. Agreement of the multiplex assay was 95% or greater (n = 332) for both HSV-1 and -2 compared to the three assays. Sensitivities for HSV-1 ranged from 94.9 to 97.9%, with specificities of 93 to 97%. For HSV-2, the sensitivity and specificity ranges were 92.6 to 98.9% and 98.3 to 98.7%, respectively. Our studies show that the multiplexed microsphere-based assay offers a sensitive and specific alternative method for the detection HSV-1 and -2 type-specific antibodies. Advantages of the multiplex assay include multiple results per assay, the inclusion of internal controls for each specimen, and higher throughput of results.     

Biotin-avidin-amplified enzyme immunoassay for detection of herpes simplex virus antigen in clinical specimens

A biotin-avidin-amplified enzyme-linked immunosorbent assay (B-A ELISA) has been developed to detect herpes simplex virus type 1 (HSV-1) and HSV-2 antigens in clinical specimens. The test was designed as a solid-phase, double-antibody, sandwich assay in which plates were coated with a polyclonal rabbit immunoglobulin G anti-HSV reagent, and the sandwich antibody was a biotin-labeled mouse immunoglobulin M monoclonal antibody that reacts with a common antigen associated with HSV-1 and HSV-2. The test can be completed in 4 h if antibody-coated plates are available. The detection limit of the B-A ELISA, determined by titration of virus stocks, was found to be approximately 90 PFU or 6 X 10(3) physical particles of either HSV-1 or HSV-2 per 50 microliter of virus stock. The following results were obtained in a study in which swabs were taken from a variety of lesions and assayed for infectivity in tissue culture and by B-A ELISA. Of 421 suspected HSV lesions tested, 69 were positive by both tests and 159 were negative by both tests. A total of 122 were positive by B-A ELISA but negative for infectivity. Seventy-one were negative by B-A ELISA but contained infectious virus. The HSV specificity of the assay was substantiated by partial blocking of reactivity with rabbit immunoglobulin G anti-HSV and by the absence of reactivity with a nonspecific biotin-labeled mouse immunoglobulin M monoclonal antibody.     

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.     

The use of peptides from glycoproteins G-2 and D-1 for detecting herpes simplex virus type 2 and type-common antibodies.

BACKGROUND: identification and discrimination of latent herpes simplex virus (HSV) infection relies on antibody identification. The inclusion of synthetic peptides with HSV glycoproteins provides means for stable and discriminatory assays for population studies. OBJECTIVE: to determine whether virus-specific synthetic peptides might identify HSV type 2 (HSV-2) antibodies in the presence of the cross-reactive and more common HSV type 1 (HSV-1) antibodies. STUDY DESIGN: the capacity of synthetic peptides as HSV antigens was analyzed in enzyme immunoassay (EIA) using well characterized human serum cohorts. The HSV peptide assays were evaluated in comparison with two commercial HSV-2 assays. RESULTS: a combination of two C-terminal HSV-1 glycoprotein D (gD-1) peptides detected type-common HSV immunoglobulin G (IgG) with high sensitivity (95%) and specificity (93%). Peptides derived from the C-terminus of HSV-2 glycoprotein G (gG-2) had a high HSV-2 type-specificity. Inclusion of both gD-1 and gG-2 peptides gave a sensitivity for human anti-HSV-2 IgG that was similar to that of assays including different amounts of native gG-2. With western blotting as a standard, the sensitivity of the peptide assay ranged between 86% for HSV-2 seropositive persons and 61% for HSV-2 seroconverters. Addition of a small amount of native gG-2 to the peptide assay tended to increase the specificity. CONCLUSION: HSV gG and gD peptides show promise as type-specific and type-common HSV antigens. These peptides are more stable and reproducibly prepared than native or recombinant glycoproteins and may be considered for inclusion in future HSV serodiagnostic assays.     

Evaluation of three glycoprotein G2-based enzyme immunoassays for detection of antibodies to herpes simplex virus type 2 in human sera

Three new glycoprotein G-based enzyme immunoassays (ETI-HSVK-G 2, Sorin Diagnostics Biomedica [assay A]; HSV Type 2 Specific IgG ELISA, Gull Laboratories, Inc. [assay B]; Cobas Core HSV-2 IgG EIA, Roche [assay C]) for the detection of herpes simplex virus (HSV) type 2 (HSV-2)-specific antibodies were evaluated. By testing sera from 25 individuals with culture-proven HSV-2 infection, the assays showed a sensitivity of 96%. The specificities, evaluated with sera from 70 HSV antibody-negative children, 75 HSV antibody-positive children, and 69 HSV antibody-negative adults, were 100% for assay A, 96.2% for assay B, and 97.8% for assay C, respectively. Discrepant results by any of the three assays, i.e., reactivity of a specimen in only one or two assays, occurred with similar frequencies for HSV-seronegative individuals as well as HSV-seropositive children and adults. For sera with discrepant results, the positive reactivity was mostly low. Thus, for determination of the prevalence of HSV-2 antibodies, only concordantly positive results were considered. On the basis of the results obtained with sera from 41 adults with culture-proven HSV-1 infection and from 173 HSV-antibody-positive pregnant women, the HSV-2 seroprevalence was 9. 8%. The results show that the new glycoprotein G2-based enzyme immunoassays are useful tools for the detection of type-specific HSV-2 antibodies. However, if only one assay is performed, careful interpretation of the results is indicated, especially if the exhibited reactivity is low, and for determination of the definitive HSV-2 serostatus, confirmatory assays may still be necessary.     

Herpes simplex virus type-2 specific glycoprotein G-2 immunomagnetically captured from HEp-2 infected tissue culture extracts

Monoclonal antibody H1206 anti-HSV-2 gG-2 bound to tosylactivated paramagnetic Dynabeads (Dynal) has been used to isolate HSV-2 type-specific gG-2 from solubilized HEp-2 HSV-2 infected cell extracts. The immunomagnetically captured type-specific glycoprotein reacted strongly with monoclonal antibody H1206 and demonstrated a single band with apparent molecular weight of 100000 (100 kDa) and a doublet band with an apparent molecular weight of 60000-64000 (60-64 kDa). We observed the same exact banding pattern when monoclonal H1206 was immunoblotted with Helix pomatia lectin purified HSV-2 gG-2. The immunomagnetically purified gG-2 was unreactive to monoclonal antibody H1379 anti-HSV-1 gG-1 and four human HSV antibody negative sera. In addition, 20 human HSV antibody positive sera obtained from the Centers for Disease Control (CDC), Atlanta, GA, were used for the evaluation of our methodology. Immunoblotting of the human HSV antibody positive samples were in agreement with the CDC HSV serological designation. Sera characterized by reactivity to the immunomagnetically purified gG-2 in conjunction with Western blot has the potential to be used as a confirmatory serological test or to determine the accuracy of clinical serological immunoassays used to determine HSV-2 seropositivity.     

Serological evidence for variation in the incidence of herpesvirus infections in different species of apes.

Sera from captive lowland gorillas, chimpanzees, orangutans, and gibbons were screened by enzyme-linked immunosorbent assay (ELISA) for antibody to herpesviruses serologically related to human herpes simplex virus types 1 and 2 (HSV-1, HSV-2), a baboon virus (SA8), and a macaque herpesvirus (B virus). The incidence of herpesvirus antibodies varied considerably among the different species, gorillas having the highest incidence of seropositivity (65.4%) and orangutans the lowest. The virus specificity of positive sera was further analyzed by examining the kinetics of virus neutralization, competition of reactivity in ELISAs, and immunoblotting against HSV-1, HSV-2, SA8, and B virus antigens. Using these assays, the majority of positive gorilla sera (49 of 53, 92%) were determined to react in a manner identical to human HSV-1 immune sera. The remaining four positive gorilla sera reacted as HSV-2-positive sera. In contrast, the majority of positive chimpanzee sera (5 of 7, 71%) reacted as HSV-2 immune rather than HSV-1 immune. All positive sera from gibbon apes reacted as HSV-1 positive. No orangutan sera were identified which gave positive reactions by ELISAs to any of the four primate herpesviruses tested. Although four orangutan sera gave equivocal results against HSV-1 antigen, further analysis by immunoblotting could not confirm any specific reactivity with any of the primate herpesvirus antigens. Varied reactivity among individual animals with both SA8 and B virus proteins was observed, but none of the seropositive primates detected appeared to be infected with either of these simian viruses. Three gorilla sera had antigen recognition patterns slightly different from those of HSV-2-positive human and chimpanzee sera and another HSV-2-positive gorilla serum, raising the possibility that these animals harbor an indigenous virus related to HSV-2.     

Use of a fragment of glycoprotein G-2 produced in the baculovirus expression system for detecting herpes simplex virus type 2-specific antibodies

Fragments of glycoprotein G (gG-2(281-594His)), comprising residues 281 to 594 of herpes simplex virus type 2 (HSV-2), glycoprotein G of HSV-1 (gG-1(t26-189His)), and glycoprotein D of HSV-1 (gD-1(1-313)), were expressed in the baculovirus expression system to develop an assay for the detection of HSV-1 and HSV-2 type-specific antibodies. The expression of the gG-1(t26-189His) and gG-2(281-594His) fragments was analyzed by Western blotting using monoclonal antibodies LP10 and AP1, respectively. The molecular masses of the major products of gG-1(t26-189His) and the fragment of gG-2(281-594His) were 36 to 39 kDa and 64 to 72 kDa, respectively. Human sera positive for HSV-1 reacted with gG-1(t26-189His), sera positive for HSV-2 reacted with the gG-2(281-594His) fragment, and sera positive for both types reacted with gG-1(t26-189His) and gG-2(281-594His) in Western blotting. The human sera recognized polypeptides of gG-2(281-594His) with molecular masses of 57 to 67 and 120 to 150 kDa and additional faint bands of 21, 29, and 45 kDa. The recombinant gG-1(t26-189His) and the recombinant gG-2(281-594His) fragment were used as type-specific antigens for the detection of HSV-1- and HSV-2-specific antibody responses in human sera, respectively. As type-common antigens, an extract of HSV-1-infected Vero cells and recombinant gD-1(1-313) were used. An enzyme-linked immunosorbent assay to detect type-specific antibodies was developed, and the sensitivity and specificity were evaluated by comparison with commercial tests by using sera obtained from different sources. The sensitivity and specificity were 91.5 and 95.5%, respectively, compared to the Gull assay. The gG-2(281-594His) fragment can be obtained in relatively large quantities at low cost.     

单克隆抗体间接荧光法分型检测单纯疱疹病毒的实验研究

本文选择３株抗单纯疱疹病毒型共同性和型特异性单克隆抗体，建立了分型检测单纯疱疹病毒的单克隆抗体间接荧光法（ＭｃＡｂ－１ＦＡ）．特异性试验和重复性试验证实，ＭｃＡｂ－ＩＦＡ特异性强、重复性好．用此法检测了不同部位来源的５１份临床分离株，分型检测结果与ＥＬＩＳＡ法检测结果一致．提示ＭｃＡｂ－ＩＦＡ有可能成为实验室分型检测单纯疱疹病毒可靠的方法．     

Detection of human serum antibodies against type-specifically reactive peptides from the N-terminus of glycoprotein B of herpes simplex virus type 1 and type 2 by surface plasmon resonance

A single-step surface plasmon resonance protocol for the detection of antibodies against herpes simplex virus type 1 and type 2 (HSV-1, HSV-2) in human sera was established using the BIAcore system. Two peptides from corresponding segments of the N-terminus of HSV-1 and HSV-2 glycoprotein B (gB), i.e. peptide gB-1 (60-73) (GAAPTGDPKPKKNK) and peptide gB-2 (55-68) (SPATTKARKRKTKK), were identified as immunogenic. Employing both peptides as diagnostic antigens in the surface plasmon resonance assay, a sensitivity for the detection of HSV-1 and HSV-2 type-specific antibodies of 83 and 86%, respectively, was achieved as compared with immunoblotting as a reference method. Peptide gB-1 (60-73) allowed the discrimination between HSV-1 and HSV-2 type-specific antibodies with a specificity of 67%, whereas peptide gB-2 (55-68) reacted in a strictly HSV-2 type-specific manner. It is concluded that peptides from the N-terminus of gB-1 and gB-2 are recognized predominantly by human sera in an HSV-specific manner. Peptide gB-2 (55-68) can be employed successfully for the determination of type-specific antibodies against HSV-2.     

Seroprevalence of herpes simplex virus types 1 and 2 in HIV-infected and uninfected homosexual men in a primary care setting.

BACKGROUND: Genital herpes is usually caused by herpes simplex virus type 2 (HSV-2), with infections often being unrecognised by patients and/or clinicians. HSV-2 infections may be a risk factor for the transmission of human immunodeficiency virus (HIV) infection. Reliable tests for type-specific HSV antibodies are now readily available. OBJECTIVES: To determine the seroprevalence of HSV-1 and -2 in HIV-seronegative gay men in a primary care setting in Melbourne, Australia, and to compare it with the rate in HIV-infected gay men. To assess the utility in a clinical setting of a type-specific HSV enzyme linked immunosorbent assay (ELISA) as compared with western blot. STUDY DESIGN: We recruited a total of 300 HIV-seronegative homosexual men attending for HIV antibody testing, and HIV-infected men attending for CD4 lymphocyte count and viral load estimation. The subjects completed a questionnaire, and sera were sent for total IgG HSV testing and testing by Gull type-specific HSV ELISA assay. Selected serum samples were retested by western blotting and the results analysed. RESULTS: In total, 168 HIV-antibody negative men and 132 HIV-antibody positive men were recruited. Of all subjects, 73.3% had HSV-1 antibodies. This proportion did not differ between HIV-seronegative and seropositive men (P=0.48). About twenty percent of HIV-seronegative men and 61% of HIV-seropositive men had antibodies to HSV-2 (P<0.0001); 75.6% of HIV-seronegative men with antibodies to HSV-2 gave no history of genital herpes, as did 66.7% of HIV-seropositive men. Overall, in using the type-specific ELISA (Gull) assay, false negative, false positive or equivocal results were obtained in 33/300 (11%) of samples tested compared with western blot. CONCLUSIONS: High rates of HSV-2 infection were found in homosexual males, with the rate for HIV-seropositive men being over twice that for HIV uninfected men. Most subjects were not aware of their infection with HSV-2. HIV-infected individuals were also older and had higher numbers of sexual partners, but we were unable to unambiguously establish that these variables contributed to the difference in HSV-2 seroprevalence rates. The Gull type-specific assay for HSV antibodies has significant problems with sensitivity and specificity at a discrepancy rate of 11%. Caution is advised in using this type-specific commercial assay for clinical purposes.