Journal of Virological Methoak 30 (1990) 301-310

Elsevier 301

VIRMET 01088

Demonstration of antigenic variation among isolates of human cytomegalovirus using

monoclonal antibodies and indirect ELISA

G.S. James and M.J. Cloonan

Virology Section, Department of Microhiolqy, Prince Henry Hospital, Little Bay, New South Wales, Australia

(Accepted 6 September 1990)

Summary

An indirect ELISA was developed for the detection of antigenic differences between isolates of human cytomegalovirus (HCMV) using a monoclonal antibody to an early (67 kDa) antigen. Antibody binding curves were analyzed using a microcomputer program (LISACRV) based on a nonlinear logistical model. The derivation of values for the average intrinsic association constant for seven isolates of HCMV and the prototype AD169 strain revealed significant differences between them. Because of the importance of HCMV as a pathogen, especially in immunosuppressed and AIDS patients, further investigation of the biological significance of differences between isolates of HCMV is clearly warranted.

Cytomegalovirus; Antigenic variation; Indirect ELISA; Monoclonal antibody

Human cytomegalovirus (HCMV) is a member of the herpesvirus group and has been associated with a wide range of diseases. These include congenital infec- tion (Yow, 1989), ~~sfusion-associated illness (Ho, I982), infection involving both high morbidity and mortality amongst immunocompromised patients espe- cially those receiving bone marrow and organ transplants (Peterson et al., 1980; Maher et al., 1985; Meyers et al., 1986; Breinig et al., 1987; Grattan et al., 1989), and severe infection in many patients with AIDS (Drew, 1988).

-_ .-..- Correspondence too: M.J. Cloonan, Virology Section, Dept. of Microbiology, Prince Henry Hospital, Little Bay, N.S.W. 2036, Australia.

016%8510/570/$03.50@ 1990 Elsevier Science Publishers B.V. (Biomedical Division)

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Antigenic heterogeneity has been recognized for some time amongst strains of CMV in serological studies with polyclonal antisera (Weller et al., 1960; Waner and Weller, 1978). A similar degree of variation has been shown using DNA restriction enzyme analysis (Huang et al., 1976). It has been demonstrated that HCMV isolates share 80-90% homology in their DNA content which might consequently allow a considerable difference in sequence homology between strains such that no classification into types or groups would be possible (Huang et al., 1976). This has been confirmed, for example, in patients with AIDS in that isolates of HCMV from different patients exhibited major differences in DNA restriction profiles whereas, in contrast, only minor differences were found when multiple isolates from the same patient were studied (Taylor et al., 1988) although some individuals may be latently infected with multiple strains of HCMV and recombination between strains may even occur (Chou, 1989).

Little systematic study has been reported on the likely biological implications of strain differences which are important for several reasons. First, antigenic variation may be a problem in the laboratory diagnosis of infection, especially with infants with congenital HCMV, both in the detection of specific antibodies using the prototype strain as antigen and in the early detection of specific antigens using monoclonal antibodies (Faix, 1985; Popow-Kraupp and Kunz, 1988; P.W. Robertson, personal communication). Second, infection with multiple strains of HCMV is relatively common in a number of patient groups including patients with AIDS (Collier et al., 1989) and the role played by strain variation in the pathogenesis of infection clearly requires detailed investigation. Third, as the use of chemotherapeutic agents such as ganciclovir in HCMV infection increases, the question of strain variation and drug resistance of variants will assume considerable relevance (Erice et al., 1989). Also important will be the ability of new HCMV vaccines to confer protection against all strains of the virus.

The use of monoclonal antibodies for studies of antigenic differences between viruses represents an important recent advance since these antibodies can not only be used to demonstrate antigenic differences between strains (Wiktor and Koprowski, 1978) but may also reveal greater antigenic heterogeneity than that shown when polyvalent antibodies are used (Wands et al., 1984). Both of these features have been noted in limited studies of differences between strains of HCMV using specific monoclonal antibodies mostly reactive with early or immediate early proteins (Rodgers et al., 1985; Popow-Kraupp and Kunz, 1988; Andreoni et al., 1989) although antigenic differences have not been demonstrated consistently (Pereira et al., 1982; Amadei et al., 1983). In conjunction with the use of monoclonal antibodies, solid phase assays such as RIA or ELISA have been shown to be of considerable value in studies of antigenic variation in a wide range of viruses including hepatitis B virus (Wands et al., 1984) and yellow fever virus (Cammack and Gould, 1986) especially when antibody binding profiles have been derived and subsequently analyzed, as with foot and mouth disease virus (Hingley and Ouldridge, 1985).

Because of the importance of strain variation in HCMV infection and of uncertainty as to the extent and significance of differences between strains, we

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have developed a method for antigenic analysis of HCMV which is based on monoclonal antibody to an early antigen and indirect ELISA. The results have been analyzed using a new microcomputer program developed for the analysis of antibody binding curves by means of a nonlinear logistical model. Response profiles have been derived from seven isolates of HCMV recovered from six patients and from the prototype strain AD169 and significant differences between strains have been demonstrated.

Material& and Methods

Antigens

All HCMV isolates were propagated in human embryonic lung fibroblast (MRCS) cells. In addition to the AD169 prototype laboratory strain of HCMV (American Type Culture Collection, Rockville, MD), seven isolates were also used as antigens and these are described in Table 1.

Antigens for indirect ELISA were prepared from inoculated MRCS cells. When approximately 90% of the cells showed a cytopathic effect, they were removed by trypsinization and centrifuged (800 x g 10 min); each pellet was resuspended in viral maintenance medium containing 2% foetal calf serum and was stored at -8OOC. Each preparation was thawed quickly prior to testing and sonicated (Branson 250, Sonic Power Co., Danbury, CO.). The suspension was then centrifuged (800 x g 10 min) and the supematant tested by indirect ELISA. A control antigen was prepared from uninfected MRCS cells in a similar manner.

Monoclonal antibodies

The commercially-available HCMV mouse monoclonal antibodies used in the study were (a) against an early antigen (approximately 67 kDa) (Whittaker M.A. Bioproducts, Walkersville, MD, cat. no. 30-552L), (b) against an early antigen

TABLE 1 Isolates of HCMV studied by indirect ELISA

Code Suecimena Date of Patient Clinical details isolation age/sex

WON7383 Urine (3) 23/10/84 15M/F Pneumonitis. Acute lymphoblastic leukaemia

WIT2053 Urine (3) 1 l/ 4185 53/M Renal transplant, febrile FIE601 Urine (6) l/ 4187 30/F Renal transplant, febrile RAH054 Urine (3) 519187 lOM/M Recurrent fits CAM5712 Buffy coat (2) 6110187 44/M AIDS, febrile CAM5860 Liver biopsy (3) 9/10/87 44/M same patient as above BUR3342 Myocardial tissue (3) 8/ 7/87 25/F Cardiomyopathy, cardiac arrest

aFigure in parentheses indicates the number of passages in human embryonic lung fibroblast (MRCS) cell culture.

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(68 kDa) (Gull Laboratories, Salt Lake City, Utah, cat. no. CEAOlO), (c) against early and late antigens (43 kDa) (Dakopatts, Glostrup, Denmark, cat. no. M757), (d) against early and late nuclear antigens (Du Pont, Billerica, MA, cat. nos. NEA9221 and NEA9220 respectively), and (e) against early antigen (67 kDa) and immediate early antigen (68 kDa) (Mallinckrodt, Chemicon International, El Segundo, CA, cat. nos. MAB8101 and MAB810).

Indirect ELISA

The wells of flat-bottomed microtitre plates (Immulon 2, Dynatech Laborato- ries, Alexandria, VA) were coated with 100 ~1 of HCMV or control antigen by dilution in 0.05 M sodium carbonate buffer pH 9.6 and incubated overnight at 4°C in a humid container. The wells were washed four times with 300 ~1 of PBS pH 7.4, containing 0.05% Tween-20 (washing buffer). After incubation at 37OC for 1 h with 150 ~1 of blocking buffer (carbonate buffer containing 4% skim milk powder, Diploma Milk Co., Dandenong, Vie.), each well was washed four times with washing buffer. Twofold serial dilutions of monoclonal antibody were made in washing buffer containing 3% bovine serum albumin (Armour Pharmaceutical Co., U.K.) and 80 ~1 was added to each well. Plates were in- cubated in a 37OC waterbath for 2 h and washed four times. Rabbit anti-mouse immunoglobulin conjugated to horseradish peroxidase (Dakopatts, Denmark, cat. no. P161) was diluted in washing buffer containing 10% foetal calf serum (Com- monwealth Serum Laboratories, Parkville, Vie.) and added to each well. Plates were incubated in a 37OC waterbath for 1 h and then washed five times. Tetrame- thylbenzidine substrate (100 ~1) (Bio-Rad Laboratories, Richmond, CA, cat. no. 172- 1066) was added to each well and the reaction stopped after 30 min at room temperature by the addition of 100 ~1 of 1 N sulphuric acid to each well. Absorbance values were read at 450 nm (Dynatech MR700 Microplate Reader). Optimal dilutions of reagents were determined by block titrations and each isolate of HCMV was tested in triplicate. The panel of eight strains was tested in nine separate assays. To permit a comparison between assays, one strain (WON7383) was used as an internal control and tolerance limits for the two parameters under study were set at the 95% confidence interval; any departure from these limits indicated that the results from a particular assay were unacceptable.

Results

Analysis of the indirect ELISA results

Although data derived from ELISA have been analyzed by a number of dif- ferent methods, no single approach has been universally adopted. In the present study, the results from indirect ELISA were analyzed in two ways. First, the linear portion of the response curve (between 20-80% maximum absorbance) was analyzed by determining the line of best fit by standard regression anal-

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0 1 2 2 4 5

RECIPROCAL SERUM DILUTION (log,o)

Fig. 1. Antibody binding profile of a HCMV specific monoclonal antibody (Whittaker MA clone lC6) and an isolate of HCMV (WON7383) using indirect ELBA. Vertical bars indicate the 95% confidence

intervals.

ysis and then calculating the serum dilution co~es~nding to 50% m~imum absorbance (150 value). This value was similar but not identical to the serological differentiation index, a comparison between the ~tise~rn dilutions which lead to the same absorbance value, when serological cross-reactivity between homol- ogous and heterologous plant viruses were tested by indirect ELBA (Jaegle and Van Regenmortel, 1985). However, the data generated by ELISA dose response curves are basically sigmoidal rather than linear (Fig. 1) and consequently a more appropriate method of analysis may be one which accounts for this sig- moidicity. Accordingly the second method of analysis of the results was that of a three parameter logistic model (Ouldridge et al., 1984; Hingley and Ouldridge, 1985) using a new microcomputer program (LISACRV) developed by Chalquest (1988). Three parameters were described by the model: (i) the average intrinsic association constant (K) which is the reciprocal of the amount of substrate at the half saturation point and is an indication of the stench of binding between anti- gen and antibody, (ii) the plateau value of the saturation curve which is a measure of the number of reactive sites at fixed concentration and (iii) the slope of the curve which is a measure of the heterogeneity of the antigen-antibody reaction.

Choice of monoclonal antibody

Prior to a detailed examination of differences between the HCMV isolates, a comparison was made between the seven monoclonal antibodies for reactivity in the indirect ELISA. Three antibodies (Dako M757, Du Pont 9221 and Mallinck-

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TABLE 2 Serological differentiation between eight isolates of HCMV using indirect ELISA and values corre- sponding to 50% of maximum absorbance (Iso)

Isolate of HCMV Mean 150 value (loglo) 95% Confidence interval

WON7383 3.46 3.37-3.5s WIT2053 3.34 3.29-3.39 FIE60 1 3.23 3.10-3.36 AD169 3.18 3.10-3.27 CAM5860 3.18 3.09-3.27 BUR3342 3.17 3.10-3.24 RAH054 3.07 3.04-3.1 I CAM5712 3.07 2.97-3.17

rodt 8 10) reacted equally well with control and viral antigens and were not studied further. Of the remaining four, two antibodies (Whittaker 30-552L, Mallinckrodt 8101) reacted with all isolates, but not control antigen, and clearly revealed dif- ferences between some of them, and the remaining two (Gull CEAOlO, Du Pont 9220) also reacted specifically with all of the isolates but did not demonstrate significant differences when the results were analyzed. As one anti~dy (Whit- taker 30-552L) provided the greatest differentiation between the panel of eight viruses, it was chosen for all subsequent assays at dilutions of 1: lOO--151200. All eight isolates were tested in the same test and each test was performed nine times. The binding profile of one HCMV isolate (WON7383) with the chosen antibody is shown in Fig. 1.

Strain di~ere~~~~ revealed by indirect ELBA

From the analysis of the linear portion of the response curve, 150 values (corre- sponding to 50% maximum absorbance) showed differences between isolates (Ta- ble 2) although there was some overlap between them. Two isolates (WON’7383, WIT2053) were similar but different from a group of four (AD169, BUR3342, CAM5860, CAM57 12) which in turn differed from RAH054, and the remaining isolate (FIE601) occupied an intermediate position.

When the same data were analyzed by the logistical model, the values of ?? showed greater divergences between isolates (Table 3). In this instance, two strains which showed no difference when 150 values were used (WON7383, WIT2053) now appeared different, with the eight strains clearly falling into three distinct groups consisting of (i) WON7383, (ii) AD169, WIT2053, FIE601, and (iii) RAH054, CAM57 12, CAM5860, BUR3342.

Discussion

Using a monoclonal antibody to an early antigen and indirect ELISA, we have been able to demonstrate differences between the prototype strain AD169 and seven recent isolates. The choice of assay is clearly important in this regard. For

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TABLE 3 Serological diffesntiation between eight isolates of HCMV using indirect ELISA and average associ- ation constant (K) values

Isolate of HCMV Mean K value (logis) 95% confidence interval

WON7383 3.43 3.36-3.50 AD169 3.14 3.08-3.19 WIT205 3 3.11 3.07-3.15 FIE60 1 3.11 3.00-3.23 RAH054 2.98 2.94-3.02 CAM57 12 2.97 2.93-3.01 CAM5860 2.96 2.92-2.99 BUR3342 2.90 2.83-2.97

example, in a study of the reactivity of a monoclonal antibody against the 86 kDa glycoprotein H of HCMV, all strains reacted to a similar degree when tested by immunofluorescence whereas differences were demonstrated between AD 169 and several low-passage clinical isolates when tested by neutralization (Baboonian et al., 1989). Comparative studies have shown that indirect immunoassay is both highly sensitive and suitable for the detection of antigenic differences between strains of viruses (Crawford et al., 1978).

Monoclonal antibodies have advantages in investigating antigenic relationships and in practice may reveal subtle differences between viral strains whereas poly- clonal antibodies in contrast may demonstrate less heterogeneity or may even fail to detect differences at all (Witkor and Koprowski, 1978; Wands et al., 1984). This applies to HCMV in that although there do not seem to be major anti- genie differences between strains, minor but variable degrees of reactivity have been shown with monoclonal antibodies (Rodgers et al., 1985; Andreoni et al., 1989; Foung et al., 1989). Some isolates of HCMV do not react with a mono- clonal antibody specific for an early (72 kDa) nuclear antigen but reacted with a monoclonal antibody specific for a late nuclear antigen and with polyclonal an- tiserum (Popow-Kraupp and Kunz, 1988). It is not surprising, therefore, that our results with seven commercially-available monoclonal antibodies were variable. The monoclonal antibody chosen for subsequent studies of antigenic differences was directed against an early (67 kDa) antigen which represents one of the major non-structural proteins of HCMV (Landini and Michelson, 1988).

The use of antibody binding curves in the analysis of antigenic differences between virus strains has been substantiated (Crawford et al., 1978; Wands et al., 1984; Ouldridge et al., 1984; Cammack and Gould, 1986) and reveals a greater degree of objectivity and sensitivity in the detection of differences than other methods. Ouldridge et al. (I 984) used this approach to differentiate between strains of foot and mouth disease virus; absorbance values were expressed as a proportion of a reference value and the resultant relationship between this value and log antibody dilution was a sigmoidal curve. Subsequent development of a method of analysis of this curve, which accounts for its nonlinear nature, has shown it to be a valuable method of differentiation between strains (Hingley and Ouldridge, 1985). The development of the computer program LISACRV

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provided a simple and rapid means of analysis of antibody saturation curves (Chalquest, 1988) which was successful in revealing differences between strains of HCMV when applied to the data obtained in the present study. Although differences between HCMV strains were shown when the linear portion of the dose response curve was analyzed, more distinct differences were revealed when the nonlinear logistical model was used; this is most likely due to the fact that the curve is essentially nonlinear and to treat it otherwise would lead to highly biased estimates of the various parameters.

The application of the nonlinear logistical model to a study of HCMV strain differences was reproducible in relation to between-assay and within-assay var& ability. Differentiation between strains was best achieved by a comparison of K values which, in a theoretical interpretation of the model, are a measure of the affinity of the particular monoclonal antibody for each strain; higher K values in- dicate a relatively greater affinity than lower ones, although the full significance of these values requires further study. The prototype strain AD169 yielded a K value similar to some of the isolates but significantly different from others. The preliminary results presented here suggest that examination of the binding pro- files enabled recognition of differences between strains and is a valid approach for study of antigenic variation in HCMV which should be carried out on a much larger number of isolates than included in the present study.

Genetic differences between isolates of HCMV have been demonstrated using restriction enzyme analysis (Huang et al., 1976; Taylor et al., 1988; Chou, 1989) and differences in the antiviral efficacy of ganciclovir against isolates of HCMV have also been demonstrated although differences in resistance to this compound do not necessarily correlate with genetic differences (Enrice et al., 1989). No detailed study with HCMV has been carried out to date to compare the results obtained by the various methods available for revealing differences between strains (serological assay, specific protein analysis, genetic analysis, antiviral drug sensitivity). However, further investigation is clearly required to define the biological significance of differences between isolates of HCMV and the role that variants may play in the pathogenesis of disease. This is particularly true with AIDS patients, in whom infection with multiple strains of HCMV occurs relatively often (Collier et al., 1989), and in transplant patients in whom infection with different strains may be associated with more severe disease (Kane&i et al., 1989).

Acknowledgements

We thank Professor R.R. Chalquest for provision of a copy of the LISACRV program, Dr L.M. deSilva for one of the HCMV isolates, Dr P.W. Robertson for helpful advice during the study, and Mr T.G. Vale for comments on the draft manuscript.

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