Journal of Virological Methoa!s, 24 (1989) 103-110

Elsevier

103

JVM 00860

Determination of rotavirus serotype-specific antibodies in sera by competitive enhanced

enzyme immunoassay

G.M. Beards and U. Desselberger Regional Virus Laboratory, East Birmingham Hospital, Birmingham B9 5ST, V. K.

(Accepted 5 December 1988)

Summary

A method is described for the specific detection of antibody to individual rota- virus serotypes in sera. A competitive enzyme immunoassay (EIA) was developed in which rotavirus serotype-specific monoclonal antibodies against VP7 compete with antibodies in test sera for rotavirus serotype-specific antigen bound to a solid phase. There was an excellent correlation between serotype-specific EIA results and serotype-specific neutralization titres (r = 0.915, P = <O.OOl). The value of this method for rotavirus epidemiology and vaccine trials is discussed.

Rotavirus serotype; Rotavirus neutralizing antibody; Viral gastroenteritis; Rota- virus epidemiology; Rotavirus vaccine

Introduction

Rotavirus is an important cause of gastroenteritis in children in all countries, and it is a major cause of childhood death in developing nations (de Zoysa and Feachem, 1985). As a result of this a number of candidate rotavirus vaccines have been developed and have been or are currently being tested (Kapikian et al., 1986; Vesikari et al., 1987). The degree of protection afforded by monovalent vaccines has varied greatly. The RIT 4237 vaccine, a candidate rotavirus vaccine derived from a bovine strain of serotype 6 (Hoshino et al., 1984), protected children against severe infection with rotavirus serotype 1 in a Finnish study (Vesikari et al., 1984,

Correspondence to: G.M. Beards, Regional Virus Laboratory, East Birmingham Hospital, Birming- ham B9 SST, U.K.

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1985); but failed to afford any protection in Rwanda (De-M01 et al., 1986) and Peru (Vesikari et al., 1987) where rotavirus serotypes other than type 1 were shown to be causing infections. Promising results have been obtained with a candidate vaccine derived from a rhesus rotavirus strain, the MMU-18006 vaccine, especially in outbreaks involving serotype 3 rotaviruses (Kapikian et al., 1985; Vesikari et al., 1986; Flares et al., 1987). The rhesus rotavirus strain has the same serotype 3 specificity as a number of human rotaviruses whereas the RIT 4237 strain is sero- typically distinct (serotype 6). Thus it would appear that homotypic immunity is important and it has been suggested that polyvalent vaccines, that is ‘cocktails’ containing more than one serotype, might be required (Kapikian et al., 1986; Flo- res et al., 1987; Beards and Brown, 1988).

We describe here an assay for the detection of antibodies directed against in- dividual rotavirus neutralisation antigens, that is those which determine the sero- type, and propose its use for epidemiological studies and vaccine research. The test is a modification of the enhanced enzyme-immunoassay recently described for se- rotyping rotaviruses in faeces (Beards, 1987).

Materials and Methods

Sera The sera tested were from children under the age of five years attending East

Birmingham Hospital. The blood samples were collected for other investigations and not specifically for this study.

Tissue culture adapted rotavirus strains representing serotypes l-4 were used as described previously (Beards, 1987).

Virus neutralisation tests The fluorescent focus reduction neutralisation assay described by Beards et al.

(1980) was used. Briefly, mixtures of 100 fluorescent focus forming units of rota- virus with serial dilutions of serum were added to monolayers of MA104 cells and residual rotavirus infectivity was measured by immunofluorescent staining using an hyperimmune rabbit anti-rotavirus serum. Endpoints were taken at the dilution giving a 60% or greater reduction in fluorescent foci.

Polyclonal antibodies These were produced in rabbits by the method of Beards (1982) and were used

as a capture antibody in the EIA tests that have been described previously (Beards et al., 1984; Beards, 1987).

~o~oclo~aL antibodies These were all specific for the rotavirus outer capsid protein designated VP7 and

were neutralising as previously described (Coulson et al., 1985, 1986, 1987). The

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monoclonal antibodies RV4:2 (specific for serotype l), RV5:3 (serotype 2), RV3:l (serotype 3) and ST3 (serotype 4) were all used at a dilution of l/10000.

Competitive NADP-enhanced EIA This was a modification of the test described recently for serotyping rotaviruses

in faeces (Beards, 1987). Polyvinyl microtitre plates (Falcon 3912) were coated with a rabbit hyperimmune polyvalent antiserum to rotavirus diluted l/10000 in car- bonate-bicarbonate buffer, pH 9.8, and incubated at 37°C for 2 h. The wells were emptied and 100 l~,l of rotavirus-infected tissue culture supernatant, diluted in 0.05 TRIS-HCl buffered saline, pH 7.2, with 0.1% Tween 20 and 3% bovine serum al- bumin (= TBST/BSA), to give a optical density by EIA of around 1.0 was added to each of ten wells across the plate. This was repeated for all four serotypes. The plates were incubated at 37°C for 2 h. After washing the plates six times in TBST/BSA, 10 l~_l of serial dilutions of test serum was added to eight wells (l/20 to l/1280) for each serotype (i.e. eight dilutions per serotype). The two remaining wells were antigen controls. Without washing, 100 l.~l of homologous monoclonal antibody diluted l/10000 in TBSTBSA was added to each well and the plates were kept at 4°C overnight. Under these conditions serotype-specific antibodies present in the test serum could compete with the neutralizing, VP7 specific monoclonal antibodies for antigens. After washing the plate six times with TBST/BSA, 100 l,~l of goat anti-murine polyvalent Ig/alkaline phosphatase conjugate (Sigma) diluted l/1000 in TBST/BSA was added to each well and the plate incubated at 37°C for 2 h in order to measure the amount of bound monoclonal antibody in each well. The plates were washed and NADPH substrate (‘Ampak’, I.Q. Bio, Milton Road, Cambridge, CB4 lXG, U.K.) was added (Self, 1985; Beards, 1987). The reactions were stopped by addition of 3M sulphuric acid, and the optical densities read at 492 nM wavelength using a Flow Multiscan spectrophotometer.

The degree of inhibition caused by each serum was recorded as a percentage of the average optical densities of the control wells to which no test sera had been added. Any sample giving a >50% inhibition was recorded as positive for sero- type-specific antibody. For the comparison with the neutralization titres, the de- gree of inhibition obtained by testing l/20 screening dilutions of sera was meas- ured and expressed as a percentage inhibition by EIA.

Statistical Analysis Titres obtained by neutralization test and competitive EIA were compared by

linear regression analysis and the significance of the regression line tested by Stu- dent’s t-test.

Results

A comparison of 24 neutralisation and competitive EIA inhibition titres ob- tained with six sera (12, 15, 16, 17, 19 and 20) and four rotavirus strains of dif- ferent serotypes are shown in Fig. 1. A linear relationship was demonstrated be-

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-I

0 1 2 3 4 00 LOG.,O NEUTRALIZATION TITRE

Fig. 1. Plot of EIA results against the logarithms of neutralization titres (N=24; 6 sera each tested against 4 rotavirus serotypes). Neutralization titres were obtained by fluorescent focus reduction as- says. EIA titres are expressed as the degree of inhibition caused by a l/20 screening dilution of test

serum. The correlation is statistically highly significant.

tween the logarithms of the neutralization titres and the percentages of inhibition in EIA (measured from optical densities) given by a l/20 screening dilution of the sera. The correlation was excellent (r = 0.915) and highly significant (P = <O.OOl).

Table 1 shows the competitive EIA titres of 20 sera (1 to 20) listed in the order of the age of the child. Sera 6, 10, 11, 12, 15, 16 and 17 clearly demonstrate that the assay detects antibodies to a single epitope in a polyclonal serum (see Discus- sion). The acquisition of antibodies with increasing age against different and more serotypes is also demonstrated.

Discussion

Several assays for rotavirus antibodies in sera have been described (for review see Beards and Brown, 1988). Essentially these have detected antibody to the ro- tavirus group antigen, which, although being a marker for past infection with ro- tavirus, is not involved in protective immunity (Gaul et al:, 1982; Chiba et al.,

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TABLE 1

Serotype-specific antibody response against group A rotaviruses in human sera as determined by com-

petitive EIA

Serum No. Age of child EIA titre* vs serotype

1 2 3 4

1 6m 40 40 40

2 6m 40 40 40 40

3 6m 4 7m 40 40 40

5 7m 40 40 40

6 7m 1280 80 7 7m _

8 7m 40 _ _

9 7m 40 40

10 8m 40 1280 40

11 8m 640 40 80 40

12 8m 1280 13 9m 40 40 40 40

14 10m 40 40 15 llm 1280 _

16 lyr 80 1280 1280 17 2yr 640 320

18 3yr 320 320 320 320

19 4yr 1280 1280 1280 640

20 5yr 1280 640 640 160

*Titre is the reciprocal of the highest dilution showing an OD value of CO.5 indicating >50% inhibition

of homologous control reaction. - = a titre of less <l/40.

1986). Methods for the detection of serotype-specific antibody have been reported previously (Hoshino et al., 1984), but involved neutralisation testing in vitro and were time consuming, expensive and laborious when carried out on a large scale. Thus only a few data are available for convalescent human sera (Linhares et al., 1981).

The assay described here exploits the exquisite specificity of VP7 specific, neu- tralizing monoclonal antibodies for the detection of naturally occurring antibody of the same specificity in polyclonal test sera. The results indicate that the mono- clonal antibodies are directly competing for epitopes with serum antibodies.

The in vitro neutralisation titres of all sera correlated very well with the results obtained by competitive EIA. Although relatively few sera have been tested to date, the results of the statistical analysis indicate that the competitive EIA pro- vides a reliable alternative to serum neutralization tests.

There is a second rotavirus surface protein, designated VP4 (McCrae and McCorquodale, 1982; Liu et al., 1988) which is important in protective immunity (Offit and Blavat, 1986). It could be argued whether blocking of serotype (VP7)- specific monoclonal antibody by a polyclonal serum occurs by true competition of antibody specific for the same epitope or by steric hindrance exerted by antibody directed against other surface components.

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In the test presented here the following points support the first of the two pos- sibilities: 1. The monoclonal antibodies used have been shown previously to be serotype

(VP7)-specific and have been selected for their capacity to neutralize (Coulson et al., 198.5, 1986, 1987).

2. If the inhibition of VP7-specific monoclonal antibody were exerted by steric hindrance, e.g. due to binding of VPCspecific antibody, it would be unlikely that monotypic serum antibody responses could be measured with polyclonal sera against one or more serotypes in different combinations (Table 1). Several neutralizing VP4-specific monoclonal antibodies which have been described by Taniguchi et al. (1987) and more recently by Mackow et al. (1988) do not dis- tinguish between serotypes 3, 3 and 4 of symptomatic strains. If these antibod- ies acted by steric hindrance it would be impossible to selectively measure monotypic antibody against VP7 of serotypes 1, 3 and 4. Other neutralizing monoclonal antibodies specific for the VP8 portion of the VP4 molecules hardly cross-neutralized (Mackow et al., 1988). It is unlikely that VP8-specific neu- tralizing monoclonal antibodies act by steric hindrance on VP7. The conclu- sions by Taniguchi et al. (1987) and Mackow et al. (1988) have been further corroborated by data of Gorziglia et al. (1988).

3. If steric hindrance of VP7 sites by VP4-specific antibody occurs, it would be dif- ficult to understand why there is an excellent correlation between the antibody response in a polyclonal serum as measured in neutralization tests against dif- ferent serotypes and the response as measured by competitive EIA (Fig. 1).

4. Finally, recent studies on the three-dimensional structure of rotaviruses have shown that the outer capsid of rotavirus particles is probably composed of 780 molecules of VP7 and only 60 molecules of VP4 which protrudes as 60 spikes (Prasad et al., 1988). Given these differences in copy number and architecture it is not very likely that antibodies directed against VP4 do sterically interfere with the major mass of VP7 antigen, though the converse might apply.

Thus we conclude that the competitive EIA test measures indeed a VP7 di- rected serotype-specific neutralizing immune response.

Antibodies to VP4 in polyclonal sera have not been assayed yet in a competitive EIA test as monoclonal antibodies to VP4 were not available at the time; but it should be possible to incorporate testing for such antibodies into the assay.

There is a need for a simple and rapid assay for serotype-specific antibodies. Testing for rotavirus serotype-specific antibody will establish the prevalence of in- fections by different serotypes with regard to different geographical areas, seasons and ages of children. A titre of l/l28 as measured in neutralization tests has been reported to be protective (Chiba et al., 1986) and this roughly corresponds to 50% inhibition in the EIA test using a l/20 screening dilution of test serum. Thus the level of protective antibody in serum can be deduced directly from the assay (Fig.

1). Such a test will also be useful in determining the efficacy of candidate vaccines

as the homotypic and heterotypic immune responses (and pre-existing antibody) can be directly measured. In a recent vaccine study (Flores et al., 1987), the ef-

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ficacy of the vaccine was deduced from serotyping of isolates obtained from chil- dren under investigation during the post-vaccination period. Such findings should be complemented by and correlated with data encompassing the serotype-specific (homotypic and heterotypic) host immune responses. Further studies are under- way to ascertain the usefulness of this technique in larger epidemiological surveys.

Acknowledgements

We thank Dr. B. Coulson for the generous gift of the monoclonal antibodies used in this study. The use of monoclonal antibodies in a competitive assay was suggested to us by Dr. H.B. Greenberg. Thanks are due to Dr. T.H. Flewett for critical reading of the manuscript.

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