Herpes simplex virus type-specific antibody determination by enzyme-linked immunosorbent assay in human sera

Serodiugnosis

and Immunotherapy

in Infectious Disease (1988)2, 415422

Herpes simplex virus type-specific antibody determination enzyme-linked immunosorbent assay in human sera

by

Maria Grazia Revello and Giuseppe Gerna* Virus Laboratory,

Institute of Infectious Diseases, University of Pavia and IRCCS Policlinico S. Matteo, 27100 Pavia, ItaIy

Herpessimplexvirus (HSV) type-specific1gGantibody wasdeterminedby an indirect enzyme-linkedimmunosorbentassay(ELBA) which requiresoptimal concentrations of HSV-1 and HSV-2 crude antigensabsorbedto the solid phaseand the calculation of the ratio (r) betweenabsorbancevaluesobtainedwith HSV-1 and HSV-2 antigens. Referencepools of human seracontaining HSV-1 or HSV-2 type-specificantibody, identified by microneutralization, wereusedto calibrate the optimal concentrations of HSV-1 and HSV-2 antigens, which were selectedon the basisof the highest differential reactivity with the homotypic as compared to the heterotypic viral antigen. r value rangesfor HSV-l- (> 2.00) and HSV-Z (~0.70) specificantibody weredefinedby testing sequentialserafrom patientswith primary HSV-1 or HSV-2 infection. Intermediate r values (0.7sl.50) for dual HSV-specific antibody were determinedby testing serummixtures containing different amounts of HSV-I and HSV-2 type-specificantibody. When serumsamplesfrom 105patientswith culturepositive recurrent HSV-1 (No. 55) or HSV-2 (No. 50) infection wereexaminedfor HSV type-specificantibody by ELBA, 96.36% of serafrom HSV-1 patientsshowed r values > 2.00 (mean2.9H) whereasin the HSV-2 group of patients, 21 (42%) were shownto have HSV-2 type-specificantibody only (r < 0.70, mean0.55). Twenty-two patients (44%) fell in the r range of 0.7gl.50 (mean 1.13) typical of dual HSV antibody, and amongthe remainingseven(14%), six gave r, between1.50and 2.00 and one > 2.00.Anamnesticdata availablefrom five of thesesevenpatientsindicated a relatively recent (l-6 months) primary HSV-2 infection in the presenceof remote HSV- 1 antibody. Keywords:

HSV antibody typing, HSV strain typing, microneutralization, ELBA. Introduction

The high degree of antigenic cross-reactivity between herpes simplex virus type 1 (HSVI) and type 2 (HSV-2) is reflected in the production of broadly cross-reactive antibody after infection with either HSV serotype. Many tests have been used for typing HSV antibody in human sera, i.e. microneutralizatiorrm3, inhibition of indirect hemagglutinationhh, enzyme-linked immunosorbent assay (ELISA)‘, ‘. and radioimmunoassays’. “. The development of HSV type-specific monoclonal antibodies has recently allowed a high purification of HSV type-specific glycoproteins, and thus a more specific approach *To whom correspondence should be addressed. 0888-0786/88/060415

+ 08 $03.00/O

0 1988Academic PressLimited

416

M. G. Revello and G. Cema

to the problem of HSV antibody typing”. I’. However, all the above mentioned procedures are too cumbersome for general use in a diagnostic laboratory. We have recently developed an indirect ELISA system which, with whole, crude HSV antigens, allows differentiation between HSV-1, HSV-2 and dual HSV antibody in human sera when the two HSV antigens are used at optimal concentrations. The specificity of HSV antibody response in human sera is expressed as a ratio (r), which is calculated by dividing serum absorbance values obtained with HSV-1 and HSV-2 antigens. Materials ELBA

and methods

procedure

For HSV antigen preparations, VERO cells infected with reference HSV-1 (McIntyre) and HSV-2 (MS) strains were harvested when cytopathic effect was loo%, suspended in 0.1 M glycine-NaCI-NaOH buffer (pH 9*0), sonicated, clarified by centrifugation and supernatants stored at - 80°C until use. Uninfected VERO cells were prepared by the same protocol for control antigen. Protein content was determined according to Lowry et ~71.‘~.The ELISA test was performed as follows. Wells of polystyrene microplates (M129A Dynatech, Plochingen, West Germany) were coated overnight at room temperature with the pre-determined optimal dilutions of HSV-I, HSV-2 and relevant control antigens (at the same protein concentration) in 0.05 M sodium carbonatebicarbonate buffer (pH 9.6). After washing, a single dilution (1: 50) of test sera in phosphate-buffered saline (PBS) containing 1% foetal calf serum (FCS) and 0.1% Tween 20 was incubated for 2 h at 37°C. Plates were washed and O-1 ml of optimally diluted peroxidase-conjugated goat anti-human IgG (Cappel Laboratories, Cooper Biomedical, Malvern, PA, U.S.A.) was added to each well. Incubation was continued for 1 h at 37°C. After washing, enzyme activity was detected by adding o-phenylenediamine in H,O, citrate buffer. Absorbance values were recorded at 492nm on a microtitre plate spectrophotometer (Titertek Multiskan, Flow Laboratories, Irvine, Scotland). The cutoff value was set at 0.20 net absorbance (difference between the absorbance with HSV antigen and control antigen) for both HSV antigens. r value was calculated for each serum by dividing net absorbance with HSV-I by net absorbance with HSV-2 antigen. The calculation could be done only when both absorbance values were between > 0.20 (cut-off value) and < 2.00 (maximum absorbance measured by the spectrophotometer). When absorbance was > 2.00, serum sample was retested at a higher dilution (1:500, I :5000, and so on). Microneutralization

test

The microneutralization (MN) assay was performed on growing VERO cells by testing serial twofold dilutions of heat-inactivated serum samples mixed with equal amounts of virus suspensions containing 100 50% tissue culture infectious doses (TCID,) of either HSV-1 or HSV-2. Corrected titres for each serum were calculated, according to Stalder et a1.3, by means of the equation t, = t + 0*5(c - a - 2), where t, = log 50% end point neutralization titre corrected to 100 TCID,, virus input, t = log 50% end point neutralization titre observed in the test, and (c - a) = log actual TCID,, virus input in the test. Typing of HSV neutralizing antibody was achieved by calculating the 1, difference (At,) between titre to HSV-I and titre to HSV-2: At, = t, (HSV-I)t, (HSV2). At, > +0.60 was typical of sera from patients with primary HSV-1 infections; At, < +0.20 was restricted to sera from patients with HSV-2 primary infections; sera containing dual HSV antibody fell in the At, range between +0.20 and +0.60.

417

HSV antibody typing

Patients and HSV isolation Vesicular swabs from mucocutaneous lesions localized above or below the waistline were collected for virus isolation from the primary lesion of 20 patients with primary HSV infection and from several recurrencies of 105 patients with recurrent HSV infections during the period 198&87. HSV isolation was performed on VERO or human embryonic lung diploid cells and typing was achieved by an indirect ELBA test using HSV type-specific rabbit immune sera14. After 1985, virus isolation was performed concomitantly with antigen detection and typing by direct immunofluorescence on cells from 91 clinical specimens. Fluorescein-labelled HSV type-specific monoclonal antibodies produced in our laboratory and commercially available monoclonals (Syva MicroTrak, Syva Company, Palo Alto, CA) were used in parallel. Human

sera

The following groups of sera were tested for HSV antibody typing in the ELISA test: (i) 57 acute and convalescent-phase sequential sera from 17 patients with primary HSV-1 infection; (ii) 16 acute and convalescent-phase sequential sera from three patients with primary HSV-2 infection; (iii) 105 sera from as many patients with recurrent HSV-1 (No. 55) or HSV-2 (No. 50) infection at different intervals from primary infection. Results

Standardization

of the ELISA

test for HSV-spec@

antibody typing

Optimal concentrations of both HSV antigens were determined by testing serial twofold dilutions of HSV-1, HSV-2, and relevant control antigens (with equalized protein content) in a checkerboard titration against three pools of human sera. Two of the pools were positive for HSV-1 and HSV-2 type-specific antibody, respectively, and the third contained serum samples from subjects negative for HSV-specific IgG. The HSV-1 and HSV-2 pools were made with sera drawn from patients recovering from primary HSV- I or HSV-2 infection, and had been previously characterized, along with single sera included in each pool, by the MN test. Table 1 reports net absorbance values obtained by the three reference pools tested at a 1:50 dilution with different concentrations of

Table 1. Titration of HSV-1 and HSV-2 antigens for antibody typing by ELISA

HSV antigen concentration/well (AAg2)

Reference serum pool

2 I%

HSV-I HSV-2 Negative

HSV- I

HSV-2

> 2.00 1.62 0.10

0.85 2.00 0.06

*I, A,,, HSV-I/A,,, tNot applicable:

0.5 I%

1 I%

~-

r*

r

r

HSV- 1 HSV-2

NAT 1.42 NA NA

0.97 0.04

0.45 I .80 0.03

HSV- 1 HSV-2 3.15 0.53 NA

0.72 0.51 0.01

HSV-2.

r was not calculated when net absorbance was >

290

or

c 0.20

0.21 0.85 0.01

3.42 0.60 NA

418

M. G. Revello and G. Gerna

HSV antigens. Optimal HSV-1 and HSV-2 antigen dilutions were considered those giving the highest net absorbance values with the homotypic as compared to the heterotypic pool. In addition, absorbance had to be within the reading limits of the ELBA spectrophotometer (i.e. < 2.00) to allow the calculation of precise r values. These conditions were reached when both HSV-1 and HSV-2 antigens were diluted to 1 ug protein/well; this dilution was therefore selected for further use. r values were 3.15 for the HSV-1 and 0.53 for the HSV-2 pool, respectively. r values were similar (3.42 and O-60, respectively) when HSV antigens were used at a concentration of 0.5 ug/well, but absorbance values were much lower. HSV-spec$c antibody typing in serafrom patients with primary HSV-I or HSV-2 infection

Fifty-seven sequential sera from 17 patients with primary HSV-1 infection and 16 sera from three patients with primary HSV-2 infection were tested by the ELISA assay for HSV-specific antibody typing. In the HSV-1 group of patients, 14 out of 18 sera taken within IO days (mean 5.3 days) after the onset of clinical symptoms were still negative for HSV-specific antibody (Table 2). The remaining four, drawn seven (two sera), 8 and 10 days after onset of symptoms reacted with HSV-1 antigen only. Among the 39 serum samples collected 10 days to 13 months after the onset, 24 (mean day of sampling 45.7) resulted positive for HSV-1 only; 14 sera collected medially 92.7 days after the onset, showed r values ranging from 2.24 to 5.04 (mean 3.36). In one serum sample, drawn from a patient 130 days after onset of a primary HSV-1 infection in the nipple area, antibody to HSV fell to undetectable level. As far as primary HSV-2 infections are concerned, only two sera from two patients were available within 10 days after the onset and in both cases only HSV-2 specific antibody were detected. The remaining 14 sera collected between two weeks and 4 years after the first HSV-2 episode, gave r values ranging from 0.52 to 0.65 (mean 0.60). On the basis of these results, it was decided to preliminarily select cut-off values of < 0.70 for HSV-2, and > 2.00 for HSV-1 antibody.

Table 2. Specificity of the ELBA

Group tested

Primary HSV- I infection Primary HSV-2 infection HSV-negative *r, A,,, HSV-I/A,, tNot applicable.

No. patients

17

3 100

HSV-2.

test for HSV antibody typing

Days after onset

No.

No. HSV-

sera

negative sera (%)

< 10 >I1

18 39

< 10

2 14 100

>ll NAt

tested

No. sera with Y* value (%) < 0.70

> 2.00

14 (77.8)

0

1 (2.6)

0

4 (22.2) 38 (97.4)

0

0 100 (100)

2 (100) 14 (100) 0

0 0 0

HSV antibody typing

419

Table 3. HSV antibody typing in serum mixtures containing different proportions of HSV- I and HSV-2 antibody by microneutralization (MN) and ELISA Relative amounts of type-specific human sera*

HSV antibody type

Results

HSV-I

HSV-2

MN

ELISA

MN

ELISA

0.0 -0.2 0.4 0.6

I.0 0.8

-0.13

0.44% 0.73 0.91

2

+0.25 +0.38

1+2 1+2

7 Ii2

+ 0.50

I ,20

0.X I.0

0.2 0.0

+0.65

I,45 3.24

0.6 0.4

*The two human sera were selected tAtc according to Stalder et ~1.~ $r. Adu2 HSV- I iAlq2 HSV-2.

+o.s1

on the basis of a comparable

I+? I+2 1+2 I

1+2 I I

homotypic

ELBA

and MN

titre.

Dual HSV antibodv determination Serum samplesartificially prepared in the laboratory by mixing at a different proportion two reference sera known to contain only HSV-1 or HSV-2 type-specific antibody were tested for HSV antibody typing in the ELISA system (Table 3). Ratios obtained actually covered the range of values between 0.70 and 2.00, which were preliminarily set as cutoff values for the identification of HSV-2 and HSV-1 type-specific antibody. Compared to MN, which was performed in parallel on the samemixtures, ELISA was able to detect smaller amounts of HSV-2 antibody. According to these results, it was decided to keep the r values previously set for HSV- 1 and HSV-2-specific antibody and to consider sera giving r values ranging from 0.70 to 1.50 as containing dual HSV antibody. Since no serum from HSV primary infections nor dual HSV antibody serum mixture gave r values between I.50 and 2.00, this range was considered as a grey zone. HSV-speciJic antibody typing in serafrom patients with HSV-1 or HSV-2 isolation Finally, we examined sera of patients from whom HSV-1 or HSV-2 was isolated during a recurrent HSV infection (Figure 1). Fifty-three out of 55 sera from 55 patients wih HSV1 isolation gave r values > 2.00 (mean 2.94) thus indicating presenceof HSV-l-specific antibody only. The remaining two sera gave r values of I .68 and 1.5I, respectively, and fell in the grey zone. Unfortunately, no anamnestic data were available for these two patients. In the group of 50 sera from as many patients with HSV-2 isolation, 21 (42%) showed r values < 0.70 (mean 0.55) indicating presenceof HSV-2 type-specific antibody, 22 (44%) gave intermediate r values (mean I- 13) typical for dual HSV antibody, and seven (14%) gave r values > 1.50. Among these sera, one gave an r value of 2.25, the remaining six falling in the grey zone (mean r value 1.65). When anamnestic data from these patients were examined, it turned out that for the patient whose serum fell in the HSV-1 range, the first episode of HSV-2 infection dated less than a month, for four additional patients with r values in the grey zone, lessthan 10 (mean 6) months.

M. G. Revello and G. Germ

0 00000 00000000 00000000 00000000 00000000 00000000 00000000

0.70

.a 0.70

R value

Figure isolation.

I Typing

of HSV antibody

2.00

1.50

(Absorbance

in 55 patients

HSV-11

with (0)

> 2.00

HSV-2)

HSV-1

isolation,

and 50 patients

with (0)

HSV-2

Discussion Because of the high level of antigenic relationship between HSV-1 and HSV-2, the major problem encountered in typing HSV antibody in human sera has been the unequivocal identification of type 2 antibody, usually acquired in adulthood, in serum samples from patients possessing type 1 antibody as a result of HSV-1 infection mostly acquired in childhood. This problem appears further complicated by the impairment or even the lack of type 2 antibody response in patients with remote HSV-I infection, as reported for HSV cytolytic antibody”. In addition, it is now well recognized that there is a diminution in the clinical severity of primary type 2 infections in individuals with preexisting type I antibodies ‘C ” . Among different serological assays set up in the last two decades to type HSV antibody, neutralization, in its multiple versions of kinetics of neutralization, plaque-reduction and microneutralization’*, is still the reference test for HSV antibody typing. Recently, the hybridoma technology has made available type-specific monoclonal antibodies, which have been successfully used both for direct typing of HSV strains in cell cultures and clinical specimens19 and for immunoaffinity purification of type-specific glycoproteins for HSV antibody typing “.‘O. More recently, a HSV-l-specific (gG-1) and a HSV-2-specific (gG-2) glycoprotein have been purified with monoclonal antibodies and used for HSV antibody typing I2 2’. The assay resulted highly sensitive, specific and reproducible and typing results showed a good correlation with isolation findings and antibody typing results obtained by microneutralization. However, in a small number of human sera, HSV-2 antibody detected by microneutralization was not revealed in the new assay with gG-2. Previous procedures for HSV antibody typing required absorption of sera with heterotypic HSV antigenj. 6.9.‘*, blocking of cross-reactive antigenic determinants with heterotypic antibody”, or the immunoprecipitation of HSV-specific glycoproteins with human sera followed by polyacrylamide gel electrophoresis”. All these methods were not readily applicable for routine epidemiological studies with large number of sera. On the other hand, the newly introduced immunodot assay using affinity-purified typespecific glycoproteins, even though using very small amounts of antigens, require

HSV antibody typing

421

sophisticated methods for antigen preparation. The ELISA assayherein reported, which allows simultaneous detection and typing of HSV-specific IgG antibody, overcomes the potential drawback of using whole, crude antigens by the determination of the optimal protein concentration of the two antigens to be usedin the assay. By determining HSV- 1 and HSV-2 antigen concentrations against reference pools of sera previously determined to contain either HSV-1 or HSV-2 antibody, it was possible to select optimal concentrations of the two antigens, allowing optimal differentiation between homotypic and heterotypic reactivity. r value was introduced to normalize the results and define the ranges to be used for type-specific and dual antibody identification. The specificity of the ELISA method described was excellent, since more than IO days after onset of primary infection, 38 out of 39 HSV- 1 sera had HSV- 1 antibody, and all of 14 HSV-2 sera had HSV-2 antibody. Similarly, the sensitivity was very high, since, if we include the grey zone in the range of dual antibody, 49 out of 50 patients with HSV-2 isolation, presented HSV-2 antibody either single or dual, and only one patient with a recent HSV-2 primary infection fell in the HSV-I antibody area. On the other hand, all 55 patients with HSV- 1 isolation showed presenceof HSV- 1 antibody, either single (53) or possibly dual (two in the grey zone). In addition, the sensitivity of the ELISA assay in detecting small amounts of HSV-2 antibody, in sera containing higher levels of HSV-I antibody, was greater than that displayed by MN. Finally, the reproducibility of antibody typing results was consistently greater than 95%. In conclusion, we believe that the ELISA assay here reported is a very simple and useful test for HSV antibody typing and can be routinely employed in diagnostic virology laboratories. Results show its high sensitivity, specificity and reproducibility. We have now developed in our laboratory several type-specific monoclonal antibodies which are currently used for typing of HSV strains in clinical specimens(Gerna et ~1.. manuscript in preparation). These antibodies will possibly be usedin the near future for purification of type-specific glycoproteins to be tested in an ELISA system for HSV antibody typing. When available, this test will be used as a reference test for discriminating equivocal results given by sera with r values falling in the grey zone. Acknowledgements The technical assistance of Daniela Burroni is greatly appreciated. This work was partially supported by Minister0 Sanitd Ricerca Finalizzata “Tutela materno-infantile”, by CNR grant No. 86.01625.52, and by Minister0 Pubblica Istruzione Ricerca Scientifica 60%. References I. Pa& RP, Dowdle WR. A serologic study of herpesvirus hominis strains by microneutraliration tests. J Immunol 1966; 98: 941-7. 2. Rawls WE, Iwamoto K, Adam E, Melnick JL. Measurementof antibodiesto herpesvirus types 1 and 2 in humansera. J Immunol 1970; 104: 5999606. 3. Stalder H. Oxman MN, Hermann KL. Herpes simplex virus neutralization: a simplification of the test. J Infect Dis 1975; 131: 43G-2. 4. Schneweis KE, Nahmias AJ. Antigens of herpes simplex virus type I and 2 immunodiffusion and inhibition passive hemagglutination studies. Z lmmunitaetsforsch Immunobiol 1971; 22: 70X -24. 5. Back AF, Schmidt NJ. Typing Herpesvirus hominis antibodies and isolates by inhibition of the indirect hemagglutination reaction. Appl Microbial 1974; 28: 4OG5.

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M. G. Revelloand G. Gerna

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uccepred

10th June 1988)