Journal of Virological Methods

ELSEVIER Journal of Virological Methods 55 (1995) 27-35

Monoclonal antibody blocking tests for the detection of HSV-l- and HSV-2-specific

humoral responses: comparison with Western blot assay

M.J. Slomka a, R.L. Ashley b, F.M. Cowan ‘, A. Cross d, D.W.G. Brown a, *

a Enteric and Respiratory Virus Laboratory, Central Public Health Laboratory, 61 Colindale Avenue,

London hW9 5HT, UK

b Children’s Hospital and Medical Center, University of Washington, Seattle, WA 98105, USA

‘Academic Department of Genito-Urinary Medicine, University College London Medical School, London, UK

’ MRC Virology Unit, Institute of Virology, Glasgow, UK

Accepted 30 March 1995

Abstract

Monoclonal antibody blocking radioimmunoassays (MAb block RIAsI which detect specific humoral responses to each of the two human herpes simplex virus (HSV) types are described. RIAs were compared with Western blot assay (WBA) in a blind study of 64 sera obtained from clinically well-documented cases of genital herpes. WBA and MAb block RIA each detected HSV-1 antibodies in 16/17 (944) D sera from confirmed HSV-1 genital infections (first episodes and recurrent infections). Detection of HSV-2 antibody in 21 sera from HSV-2 first episodes was more effective by WBA which identified homologous antibody in 19 (96%), whereas MAb block RIA detected HSV-2 antibody in 16 (76%). HSV-2 antibody was detected in 24/25 (96%) sera from recurrent HSV-2 infections by WBA and by MAb block RIA, the highest degree of concordance for both methods. In addition, the MAb block RIA may be more effective in detecting the presence of HSV-1 antibody in sera from recurrent HSV-2 cases. Prevalence of HSV-1 and HSV-2 antibody was measured by the MAb block RIAs in 3 UK human study populations which consisted of 100 children/young adolescents, 104 adult blood donors and 80 genito-urinary medicine clinic attenders.

* Corresponding author. Fax: +44 (181) 200 1569/7874.

0166-0934/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0166.0934(95)00042-9

28 M.J. Slomka et al. /Journal of Virological Methods 55 (1995) 27-35

Keywords: Herpes simplex virus (HSV); Type-specific antibody; Monoclonal antibody-blocking radioim-

munoassay (MAb block RIA)

1. Introduction

The two human herpes simplex viruses (HSV-1 and HSV-2) cause both primary and recurrent infections (Whitley and Gnann, 1993). HSV-1 infects both the oral cavity and the genital tract while HSV-2 generally causes a genital infection, and most infections with either HSV type are asymptomatic (Whitley and Gnann, 1993). First episodes of genital herpes include true primary infections, i.e. infection in patients who were previously HSV seronegative, and non-primary first episodes where clinical infection occurs in an individual who already possesses antibodies to the heterologous HSV type

(Corey, 1994). It is well known that HSV-1 and HSV-2 share common epitopes which elicit a

predominantly cross-reactive antibody response in infected individuals (Corey, 1994). This makes accurate identification of type-specific antibody difficult (Ashley, 1993). Commercial ELISAs (Ashley et al., 1991), complement fixation (Field et al., 1992) and neutralization (Ashley et al., 1993) are all unreliable for determination of type-specific

antibody. However, HSV glycoproteins C (gC) and glycoproteins G (gG) have been shown to be predominantly type-specific antigens (Marsden, 1987). Purified gG from HSV-1 (gG-1) and from HSV-2 (gG-2) have been used to identify type-specific antibody responses (Lee et al., 1985, 1986; Ho et al., 1992). The most exhaustively validated method for identifying type-specific antibody is the Western blot assay (WBA) (Ashley et al., 1988b; Ho et al., 1993). While effective, WBA is cumbersome to perform and is not widely available.

The present study describes a novel method for identifying type-specific HSV serological responses. Two monoclonal antibody-blocking radioimmunoassays (MAb block RIAs) are presented, one for each HSV type. These tests were compared with WBA using sera from culture-documented cases of genital HSV-1 or genital HSV-2 to assess their accuracy in detecting antibodies to the two HSV types. The potential use of the MAb block RIAs in seroepidemiological studies is also described.

2. Materials and methods

2.1. Serum specimens from clinical cases of genital herpes

Sixty-four sera were collected from individuals with genital herpes who had attended the Genito-Urinary Medicine (GUM) Clinic at University College Hospital Medical School, London, UK, and the Virological Research Clinic, University of Washington, Seattle, USA. The group included 7 patients from whom paired sera were obtained. HSV was isolated from all subjects, and typing was by indirect fluorescent monoclonal antibody testing (University of Washington patients) or restriction endonuclease analysis of viral DNA (University College patients) (Peterson et al., 1983). Serum specimens

M.J. Slomka et al. /Journal of Virological Methods 55 (1995) 27-35 29

included: (1) 8 from HSV-1 first episodes; (2) 9 from HSV-1 recurrences; (3) 21 from HSV-2 first episodes; and (4) 25 from HSV-2 recurrences. Sera from first episodes were drawn within 2 months of symptoms, while for the recurrent cases sera were drawn at least 4 months after the corresponding first episode. All first episodes received acyclovir (ACV) therapy, while recurrences did not. Ten sera from individuals with no history of HSV infection who were confirmed HSV antibody negative by the sensitive enhanced chemiluminescence (ECL)-WBA (Dalessio and Ashley, 1992) were also included in the evaluation.

2.2. Sera from 3 study groups

Single sera were obtained in 1991 from 100 children/young adolescents (age 2-15 years) who had been admitted to hospital in the UK with a variety of conditions (collected by the Central Public Health Laboratory (CPHL), London, UK). Single sera from 104 healthy adult blood donors (North London Blood Transfusion Service, collected in 1988) and 80 GUM clinic attenders originally referred for HIV serology at the CPHL in 1993 were also tested. None of these 3 groups included clinical data regarding HSV history.

2.3. Western blot assay (WBA)

This was carried out as previously described (Ashley et al., 1988b).

2.4. Antigen preparation

This was adapted from the protocol of Norcott and Brown (1993). Confluent monolayers of Vero E6 cells (Centre for Applied Microbiology and Research, Porton Down, Salisbury, UK) were infected at a multiplicity of infection of approximately l-2 with either HSV-1 strain SC16 or HSV-2 strain 186, and incubated at 37°C in maintenance medium (20 ml per flask) until advanced cytopathic effect was observed. The flask contents were frozen and thawed ( X 3), ultrasonicated, clarified and stored at - 70°C (Norcott and Brown, 1993).

2.5. Monoclonal antibodies (MAbs)

Two HSV type-specific MAbs (murine ascitic fluids) were selected for each blocking RIA: (1) MAb 2454, HSV-l-specific, raised to glycoprotein C (gC-1) (Palfreyman et al., 1983); and (2) MAb APl, HSV-2-specific, raised to glycoprotein G (gG-2) (Marsden et al., 1984). The latter was a gift from Professor Tony Minson (Division of Virology, University of Cambridge, UK). Each MAb was titrated in a two-fold dilution series in a binding assay with the homologous antigen (Norcott and Brown, 1993), and the end point determined as the highest dilution which gave maximal MAb/antigen binding: This was 1 : 160,000 and 1 : 20,000 for MAb 2454 and MAb APl, respectively. Diluent was 1% BSA in 0.05% Tween 20 PBS (PBS-T).

30 M.J. Slomka et al. /Journal of Virological Methods 55 (1995) 27-35

2.6. Blocking RLA

Etched polystyrene beads were coated overnight at 37°C with sonicated antigen diluted 1 : 25 in 0.1 M carbonate-bicarbonate (pH 9.6) buffer, and washed the next day (Norcott and Brown, 1993). Two hundred microlitres of diluted test serum (1: 20 in 1% BSA PBS-T) was added to each well of a 20-well plate (Abbott), followed by an antigen-coated bead. After incubation for 1 h at 37°C (blocking), 20 ~1 homologous MAb (10 X end-point concentration) was added and incubation continued overnight. The

beads were washed and incubated at 37°C for 2 h with 200 ~1 ‘251-labelled sheep anti-mouse antibody (Amersham, IM131) diluted in 1% BSA PBS-T to give 95-100 X lo3 counts per minute. After washing, beads were counted in a gamma counter for 5 min. Washes were performed (X 4) with PBS-T. Antigen and test serum dilutions were

optimised by chessboard titration. Results were quantified by calculating percentage inhibition of MAb binding to its

homologous antigen as follows:

CC&l - ct) Resu1t = loo x (Cdi, - cpos)

Cdi, is the average count of 4 beads incubated with 200 ~1 diluent (no test serum

included), C,, is the average count of 4 beads incubated with 200 ~1 diluted strong positive control sera to the homologous antigen, and C, is the count given by the test serum sample.

2.7. Polyclonal competitive radioimmunoassay

As previously described (Norcott and Brown, 1993), this assay detects antibodies to HSV and related primate viruses, herpes B virus and SA8, but cannot discriminate between HSV-l- and HSV-2-specific antibodies.

3. Results

3.1. Determination of cut-off values

One hundred and fifteen sera negative for HSV antibodies by polyclonal RIA were selected from the children/young adolescent and adult blood donor groups, and were tested by the two MAb block RIAs to determine the mean negative inhibition for each assay for the purpose of this study. The cut-off was set 3 standard deviations above the mean negative result, giving cut-offs for the HSV-1 and HSV-2 MAb block RIAs at 50 and 56%, respectively.

,3.2. Comparison of WBA and MAb block RL4 results

The panel of 74 sera gave 4 patterns of antibody response by WBA: (1) HSV negative; (2) HSV-l-positive; (3) HSV-2-positive; and (4) dual positive. The panel was

M.J. Slomka et al. /Journal of Virological Methods 55 (1995) 27-35 31

Table 1

Comparison of HSV-l- and HSV-2-specific antibody responses detected by MAb block RIA and WBA in sera

collected from 4 categories of culture-typed genital herpes cases

WBA MAb block RIA

HSV-1 first episodes (8 sera)

HSV-1 recurrences (9 sera)

All HSV-1 genital cases (17 sera)

HSV-2 first episodes (21 sera)

HSV-2 recurrences (25 sera)

All HSV-2 genital cases (46 sera)

7/8 (88%) 8/8 (100%) 9/9 (100%) 8/9 (89%)

16/17 (94%) 16/17 (94%)

19/21(91%) 16/21(76%) 24/25 (96%) 24/25 (96%)

43/46 (94%) 40/46 (87%)

tested blind in the MAb block RIAs. In 58 out of 74 sera (78%) there was complete agreement between the two tests, including the identification of 10 HSV-negative sera. For 64 sera from individuals with clinically proven genital herpes, typing of isolated HSV indicated which type-specific antibodies would be anticipated in a given patient’s serum, and the comparative results for WBA and MAb block RIAs are shown in Table 1. Among the 8 HSV-1 first episodes, absence of HSV-2 antibodies by both methods confirmed these to be true primary infections. Five sera collected from the HSV-2 first episodes failed to give positive blocking in the HSV-2 RIA (Table l), two of these were collected 16 and 60 days after onset of symptoms, subsequently seroconverting to HSV-2 in paired sera drawn 60 days and > 1 year later, respectively. The HSV-2 recurrences included 12 sera with dual antibody status by MAb block RIA, whereas WBA revealed only 6 which possessed dual antibody.

A fifth clinical category, represented by a single serum, was from a recurrent genital herpes case where both HSV types were isolated from the genital lesions. This serum contained only HSV-2 antibodies by WBA but dual antibodies by MAb block RIA.

3.3. Reactivity of sera from 3 human study groups in the two A4Ab block RkAs

The distributions of reactivities of the 284 human sera, collected from 3 study groups, are shown for the specific HSV-1 and HSV-2 RIAs in Figs. 1 and 2, respectively. None of the children/ young adolescents possessed HSV-2 antibodies, but these were detected in 3 (2.9%) adult blood donors (2 of whom were dual positive), and in 20 (25%) GUM clinic attenders (17 dual positives). These 3 groups included 36 (36%), 46 (44.2%) and 44 (55%) individuals, respectively, who possessed antibodies to HSV-1 only.

4. Discussion

WBA and MAb block RIA appeared to be equally effective for detecting HSV-1 antibody in sera from culture-proven HSV-1 genital herpes cases (first episodes and recurrences). One serum indicated HSV-1 antibody presence by WBA and not by MAb block RIA, and another vice versa. Such discrepancies may represent sensitivity differences between the two tests for these two specimens.

M.J. Slomka et al./Journal of Virological Methods 55 (1995) 27-35

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represented are: children/young adolescents (n = 1001, adult blood donors fn = 104) and GUM clinic

attenders (n = 80). Filled circles correspond to serum obtained from one individual. Setting of the cut-off

(50%) is described in the text.

WBA demonstrated greater sensitivity for detecting HSV-2 antibody (19/21 (91%)) following HSV-2 first episodes than MAb block RIA (16/21 (76%)). In paired sera from 2 patients following HSV-2 first episodes, a late seroconversion to HSV-2 was detected by MAb block RIA. WBA detected HSV-2 antibodies in all 4 sera, indicating that it can detect HSV-2 antibody sooner after infection than MAb block RIA. HSV-2-specific IgG appears more slowly by WBA than type-common IgG following primary HSV-2 infections (Ho et al., 1993) and type-specific antibody to gG-2 may require 6-8 weeks to develop after infection (Ashley, 1993). It has also been reported that 5-10% of infected individuals may not possess antibodies to gG-2 (Ashley, 1993). Because the HSV-2 MAb block RIA relies upon a clear humoral response to the APl targetted epitope on gG-2, delay or even failure in this response following primary infection may account for the decreased sensitivity of the HSV-2 MAb block RIA compared to WBA. ACV therapy during first episodes can blunt the humoral response (Bernstein et al., 1984; Ashley et al., 1988a); and this may have suppressed type-specific antibody in some of the HSV-2 first episodes.

M.J. Slomka et al. /Journal of Virological Methods 55 (1995) 27-35 33

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Fig. 2. Distribution of percentage inhibition (blocking) values for the HSV-2 MAb block RL4. The 3 study

populations represented are as in Fig. 1. Setting of the cut-off (56%) is described in the text. Filled circles

correspond to serum from one individual.

MAb block RIA appeared more sensitive for detection of HSV-2 type-specific humoral responses following recurrent infection than first episodes. All recurrences except 4 were drawn at least 1 year after the corresponding first episode, so a more complete antibody profile to both type-common and type-specific epitopes would be anticipated. The one specimen from recurrent HSV-2 infection which was negative by MAb block RIA gave a ‘high negative’ (52%) blocking value, and was drawn 8 months after the first episode. This, together with a retrospective examination of other discrepant sera in the comparison with WBA, suggested that the cut-off for the HSV-2 RIA may be altered from 56 to 50%.

MAb block RIAs for the recurrent HSV-2 cases revealed a greater number of individuals (12) who possessed dual HSV antibody compared to WBA (6). It is possible that some dual infections contain strong type-common responses which made it difficult to identify HSV-l-specific responses by WBA (Ashley, 1993). The one totally contra- dictory serotyping result among all of the recurrent genital herpes cases in the compari- son was a serum drawn from a patient with recurrent genital HSV-1. This possessed HSV-l-specific antibodies by WBA, but MAb block RIA revealed HSV-2-specific antibodies.

34 M.J. Slomka et al. /Journal of Vimlogical Methods 55 (1995) 27-35

Application of the two MAb block RIAs in a small seroepidemiological survey

revealed separate clustering of specimens into high and low percentage blockers, corresponding to positive and negative sera, respectively (Figs. 1 and 2). The survey results reflect the accepted view that many oral HSV-1 infections are acquired in childhood, as well as the sexual acquisition of HSV-2 (Whitley and Gnann, 1993). Ten out of 80 sera in the GUM clinic group were HIV-positive, two of these cases had overt AIDS, but both were HSV dual positive by the MAb block RIAs. In view of these small numbers, it is not possible to conclude whether HIV status/immune suppression may effect the sensitivity of the RIAs. A separate study of London GUM clinic attenders by WBA revealed a total HSV-2 seroprevalence of 22.6% (Cowan et al., 1994), a similar value to that obtained in the present study. This, together with the clear discrimination between positive and negative sera, suggest that the assays will be useful for seroepi- demiological studies.

These assays are a useful alternative reference test to WBA since the MAb block RIAs have the advantage of utilising a cheap and easy to prepare antigen, the test itself being free of labour-intensive procedures. Future adaptation of the blocking assays to an ELISA format has the attraction of non-radioactive reagents and will make the assays more suitable for screening large numbers of sera.

Acknowledgements

The authors wish to express their gratitude to Professor A. Mindel for supplying clinical information relating to the London genital herpes cases, and to Mrs. Inger Seth for typing the manuscript.

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