Differentiation of Cytomegalovirus Antigens by Their Reactivity with Various Classes of Human Antibodies in the Indirect Fluorescent Antibody Test

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1980, p /80/ /06$02.00/0 Vol. 11, No. 1 Differentiation of Cytomegalovirus Antigens by Their Reactivity with Various Classes of Human Antibodies in the Indirect Fluorescent Antibody Test JOHN L. RIGGS* AND NATALIE E. CREMER Viral and Rickettsial Disease Laboratory, California State Department of Health Services, Berkeley, California Human sera containing immunoglobulin G (IgG), IgA, and IgM antibodies were tested by immunofluorescence for reactivity with cytomegalovirus-infected cell cultures and with early antigens of cytomegalovirus produced by treating the infected cultures with either bromodeoxyuridine or cytosine arabinoside. IgG antibody but not IgM antibody reacted with early antigens produced in bromodeoxyuridine-treated infected cultures. This observation on a small sample of sera suggested that a positive IgM reaction with an infected, nontreated culture and a negative reaction with a bromodeoxyuridine-treated infected culture may indicate a positive specific IgM reaction for cytomegalovirus, even in the presence of IgM rheumatoid factor. The hypothesis requires further testing. The different classes of antibody did not all react or did not react to the same extent with early antigens produced in infected cells blocked with cytosine arabinoside or bromodeoxyuridine. This observation indicates that different antigens were being produced as a result of the two treatments. Problems in the detection of immunoglobulin M (IgM) antibody to viral antigens in human serum by the indirect fluorescent-antibody (IFA) technique have been reported. These include false-positive reactions due to the reaction of IgM rheumatoid factor (RF) with viral IgG antibody in the serum (5, 12) and also falsenegative reactions due to high concentrations of viral IgG antibody which successfully blocks IgM antibody through competition for viral antigen binding sites (2, 11). To investigate these problems, the antibody response in cytomegalovirus (CMV) infection was chosen for study. The immunological response in CMV infection is of particular interest in that a number of aberrant reactions occur that include formation of rheumatoid factor, induction of Fc receptors in the cytoplasm of the infected cells, production of cryoglobulins, cold agglutinins, and antinuclear antibody (1, 6, 8, 9, 15). In the course of the study, it was observed that not all classes of antibodies reacted equally well with infected cells at different stages of cell infection. The' present report gives data on the IFA test in detection of different classes of antibodies reactive with early and late antigens produced in chemically treated infected cultures and at various times after cell infection. MATERIALS AND METHODS Virus. The AD-169 strain of human CMV was used in this study. Seed virus preparations were made by harvesting infected cultures at 9 days postinfection. The cultures were frozen and thawed three times, clarified by slow-speed centrifugation, and concentrated 10 times by ultracentrifugation (78,000 x g). The concentrated virus had a titer of 2.4 x 108 plaqueforming units per ml as determined by a plaque procedure described previously (10). Cell cultures. A human fetal diploid lung (HL) strain, L645, of cells established by J. H. Schieble of this laboratory was used in these studies. The cells were routinely propagated in Eagle minimal essential medium in Hanks balanced salt solution supplemented with 10% fetal bovine serum and contained 200 U of penicillin and streptomycin per ml. Time study (arac and BrdU). A confluent monolayer of the HFDL cells in a 4-ounce (ca. 120-ml) prescription bottle (approximately 1.5 x 106 cells per bottle) was infected at a multiplicity of infection of 1 to 2 plaque-forming units per cell, and the virus was allowed to adsorb for a period of 1 h at 37 C. The inoculum was removed, and the bottles were fed with 10 ml of Dulbecco's modification of Eagle's medium fortified with 10% fetal bovine serum and containing antibiotics. After viral adsorption some of the cultures were treated with either bromodeoxyuridine (BrdU) or cytosine arabinoside (arac) for production of early CMV antigens (EA) by addition of the drug to the medium (20,ig/ml). EA in BrdU-treated infected cultures are designated as BrdU-EA, EA in arac-treated infected cultures are designated as arac-ea, and antigens in infected, nontreated cultures are designated as CMV antigens. Uninfected chemically treated and untreated cell cultures were included as controls. Sample cultures of each of the above treatments 88

2 VOL. 11, 1980 REACTIVITY OF CLASSES OF CMV ANTIBODIES 89 were removed at 24, 48, 72, and 96 h. After washing the cell monolayers with phosphate-buffered saline, ph 7.2, they were harvested with trypsin-versene solution. The cells were recovered by centrifugation and were washed once with phosphate-buffered saline containing 0.05% gelatin. The pelleted cells were suspended in a small volume (0.25 to 0.5 ml) of phosphatebuffered saline-gelatin solution and dispensed as spots, 3 to 5 mm in diameter, on microscope slides. The spots were air dried and fixed in acetone at room temperature for 5 min. After drying they were ringed with TRI CHEM embroidery paint. The slides were stored at -20 C until used in the staining procedure. IFA. Antisera prepared against human IgG (y-chain specific), IgM (,i-chain specific), and IgA (a-chain specific) were purchased from Behring Diagnostics, Somerville, N.J. Specificity of the antisera was verified by immunoelectrophoresis. They were labeled with fluorescein isothiocyanate, and the optimally labeled IgG was isolated by diethylaminoethyl chromatography as described elsewhere (9a). The serum specimens were diluted (1:10, 1:40, 1:160) and were reacted with the fixed cells on the slides. The preparations were incubated at 37 C in a moist chamber for 1 h with the exception of the preparations which were to be examined for IgM, which were reacted for a 3-h period. The preparations were washed in phosphate-buffered saline (three changes for 5 min) and were then reacted with the fluorescein-labeled antibody (anti-igg, anti IgA, or anti-igm) in a moist chamber at 37 C for 1 h. The slides were washed as before and were then rinsed with distilled water and allowed to air dry. The preparations were mounted in Elvanol mounting medium and examined with the ultraviolet microscope, with a 100-W halogen lamp and a Zeiss fluorescein isothiocyanate interference filter as an exciter filter and a Zeiss 50 filter as a barrier filter. Endpoints were determined subjectively as a 2+ staining reaction on a 1+ to 4+ scale. Serum specimens. The serum specimens tested TABLE 1. were selected from sera submitted by physicians to our diagnostic serology section for determination of antibody titers to CMV, and had been previously analyzed for RF and for class of antibody by complement fixation (CF) and indirect hemagglutination (IHA). The results of those studies and the techniques used for separation of immunoglobulins by sucrose gradient centrifugation, removal of RF from sera by absorption with insolubilized IgG, and RF, CF, and IHA assay methods have been published before (3, 4). RESULTS Classes of antibodies reactive with infected treated and nontreated cultures. Nine single samples and one paired serum sample, collected 6 to 34 days after onset of symptoms, were tested for antibody class with cultures harvested 96 h postinfection. Certain of the sera were positive for RF. Although absorption with insolubilized human IgG removed RF from positive sera, it had no significant effect on the results by IFA (Table 1). All of the samples contained CMV antibody of the IgG class, three had CMV antibody of the IgA class, and nine of the samples had CMV antibody of the IgM class. The same serum samples were tested with cultures which had been treated with either BrdU or arac (Table 2). Although all of the sera had IgG CMV antibody reactive with BrdU-EA, only seven sera had IgG antibody reactive with arac-ea. Three sera had IgA antibody reactive with BrdU-EA. The one serum tested for IgA antibody with both arac-ea and BrdU-EA reacted to the same extent with both kinds of EA. Nine of the ten sera had anti-cmv IgM antibody, but none had IgM antibody reactive with Effect of serum absorption with insolubilized IgG on titers of different classes of antibodies Serum antibody titers by IFA Bleed date Patient no. after onset RF IgG IgA IgM of symptoms Unab- Absorbed' Unab- Absorbed Unab- Absorbed sorbed sorbed sorbed > ' <10 <10 '160 '160 3-lb '160 <10 <10 '160 ' b <10 < <10 <10 <10 < ' <10 < >160 <10 < <10 < <10 <10 40 <40c a Absorbed with insolubilized IgG; after absorption, sera were negative for RF. b 3-1 and 3-2 are paired sera. c This serum could not be checked at a lower dilution because of a nonspecific reaction at higher serum concentrations.

3 90 RIGGS AND CREMER J. CLIN. MICROBIOL. TABLE 2. Reaction of different classes of antibodies with CMV-infected, drug-treated, and nontreated cultures Bleed Serum antibody titers by IFA Patient no. date after IgG IgA IgM symptoms CMVa BrdUb arac' CMV BrdU arac CMV BrdU arac < NDC -160 < ' <10 ND ND 2160 <10 <10 3- ld <10 <10 ND ND 2160 <10 < <10 ND ND 2160 <10 < > <10 ND ND <10 <10 < <10 ND ND 40 <10 < ND 2160 <10 < <10 ND ND 2160 <10 < <10 <10 ND ND 10 < <10 ND ND 40 < QNS' <10 QNS anontreated cultures infected with CMV. b Infected cultures treated with either BrdU or arac. 'ND, Not done. d 3-1 and 3-2 are paired sera. e QNS, Quantity not sufficient. BrdU-EA. The anti-cmv IgM antibody in only three of the nine sera reacted with arac-ea. To further investigate the lack of IgM reactivity with BrdU-EA, an additional 16 serum samples (5 paired and 6 single specimens) were tested at a 1:10 dilution for IgM and IgG antibody to CMV and to BrdU-EA. All of these serum samples possessed IgG antibody to CMV and to BrdU-EA. Anti-CMV IgM antibody was also present in five of the sera. The IgM antibody did not react with BrdU-EA. Morphology of staining pattern. The staining patterns of the three classes of antibody were similar. The antigen was usually confined to the nucleus of the infected cells and consisted of a punctate to a diffuse pattern (Fig. la and b). In the BrdU-treated or arac-treated infected cultures the stain was of a punctate to fleck-like intranuclear pattern with no diffuse staining seen (Fig. lc). The nonspecific Fc receptor staining of the infected cells was not always present in every preparation. Certain cultures did show a diffuse cytoplasmic stain when a human serum negative for CMV antibodies was used as the intermediate serum (Fig. ld). The results in this study were based only on the nuclear staining patterns. Time study on antigen formation and antibody reactivity. The infected, infected and drug treated, and noninfected cultures prepared for the time study were reacted with dilutions of three serum samples so that all the classes of antibodies (IgG, IgA, and IgM) were represented. Both the IgG and IgA classes of antibodies reacted with CMV and with arac-ea and BrdU-EA at 24 h postinfection. The IgM antibody did not react with CMV antigens until 96 h postinfection (Table 3). There was no significant reaction of the IgM antibody with BrdU- EA. The antigen reactive with IgM antibody in arac blocked cultures was first evident at 96 h. Sucrose gradient fractions. Doubling dilutions of fractions containing IgM from sucrose gradients of antisera nonabsorbed and absorbed with insolubilized human IgG were reacted with CMV antigens and with BrdU-EA. The same fractions had been previously assayed for RF and antibody by the IHA and CF procedures (4). After staining with fluorescein-labeled anti- IgM and IgG, it was evident that the fractions containing only IgM antibody to CMV (Table 4) did not react with the BrdU-EA at the lowest dilution tested (1:4). It was also evident that absorption with insolubilized human IgG, while completely removing CF activity in some specimens (4), had little or no effect on the IgM antibody reaction to CMV when determined by the IFA procedure. There was no contamination of the IgM fractions with IgG antibody as shown by the lack of staining with the anti-igg conjugate. DISCUSSION The present sample of sera had true IgM antibody to CMV, since removal of RF did not remove the IgM antibody to CMV. These were well-characterized sera that had been described previously relative to CF and IHA antibody and were known to contain IgM antibody by IHA (3,

4 VOL. 11, 1980 REACTIVITY OF CLASSES OF CMV ANTIBODIES 91 FIG. 1. CMV-infected cells harvested at 96 h postinfection and reacted in the IFA procedure: (a) stained for human IgG (x470); (b) stained for human IgM (x470); (c) cultures treated with BrdU and stained for human IgG (x6o0); (d) negative human serum stained for IgG. The diffuse staining in the cytoplasm is the nonspecific Fc receptor site (x600). 4). The reactivity in these sera of IgG antibody, IgM RF, directed against IgG, can complex with but not IgM antibody, to BrdU-EA is of interest IgG specific for CMV (as well as with other IgG and suggests a possible method for detection of molecules), thus giving a false-positive reaction IgM antibody in the presence of IgM RF. The for anti-cmv IgM antibody (5, 13). Since spe-

5 92 RIGGS AND CREMER cific anti-cmv IgM antibody does not react with BrdU-EA, a positive IgM reaction with an infected, nontreated culture and a negative reaction with a BrdU-treated infected culture should indicate a positive specific IgM reaction for CMV. This was the case for the 15 sera with IgM antibody, as well as for isolated IgM fractions in our study. To extrapolate to CMV IgM antibody in the general population, testing of more IgM-containing serum samples is necessary. In a study by The et al. of serial serum samples from 45 acutely infected patients, a transient appearance of an antibody that reacted with arac-ea was reported (14). The antibody was present in sera by 10 to 19 days after onset of symptoms and declined within a few months. It was suggested that the presence in acute-phase serum of this antibody at a titer of -1:80 may be TABLE 3. Temporal appearance of CMV antigen(s) reactive with different classes of antibodies Result of IFA at post- Antibody Treatment of cells infection h: class IgG CMV +' CMV + BrdU CMV + Ara C IgA CMV CMV + BrdU CMV + Ara C IgM CMV CMV + BrdU CMV + Ara C Sera screened at a 1:10 serum dilution. indicative of a current primary infection. The FA reagent used was a fluorescein-conjugated anti-human immunoglobulin and therefore the class of antibody involved was not determined. A later study by The et al. (13) indicated that some immunosuppressed patients with CMV infection produced antibody to arac-ea for several years. They concluded that production of antibody to arac-ea was associated with active proliferation of the virus. Gerna et al. (7) confirmed that production of antibody to arac-ea was not a marker only for current primary CMV infection, but for active CMV replication in primary, congenital, or reactivated CMV infections. Nine of ten patients in our study (Table 2) had a current CMV infection as evidenced by a fourfold or greater rise in CF or IHA antibody titer (3, 4) in paired sera, and the tenth patient (no. 2) had a high stationary titer. We have no information on virus production in these patients, because material for viral isolation was not received. In our study the sera reacted differently to BrdU-EA and arac-ea, suggesting that different early antigens are being produced by the two treatments. Thus, not only did IgG serum antibody titers differ with the two preparations, but some patients (sera 1, 3-1, and 8) had high antibody titers to BrdU-EA and none demonstrable to arac-ea. Furthermore, three patients (sera 1, 8, and 9) had low IgM antibody titers to arac-ea and none detectable to BrdU-EA (Table 2). Patients when infected by a virus such as CMV which contains a number of structural proteins apparently respond differently with different classes of antibody to these antigens. In a test such as IFA, which is not limited to the detection of a single or just a few antigens, a TABLE 4. IgM fractions isolated by sucrose gradient centrifugation IHAa CFa Titers by IFA IgM BrdU-treated BrdU-treated Serum no. g IgM IgG Unabsorbed Ab- Unabsorbedh sorbed Absorbed Unab- Ab- Unab- Ab- Unab- Ab- sorbed sorbed sorbed sorbed sorbed sorbed 1-lC d < < < >2,048 2, < ,048 1, , < a Data from reference 4. ' Absorbed with insolubilized IgG. 1-1 and 1-2 are paired sera. d = <4. J. CLIN. MICROBIOL.

6 VOL. 11, 1980 REACTIVITY OF CLASSES OF CMV ANTIBODIES 93 variety of antibody-combining site specificities will be observed. ACKNOWLEDGMENTS We thank Beatrice O'Keefe for excellent technical assistance. This investigation was supported by Public Health Service grant Al from the National Institute of Allergy and Infectious Diseases. LITERATURE CITED 1. Andersen, P., and H. K. Andersen Smooth-muscle antibodies and other tissue antibodies in cytomegalovirus infection. Clin. Exp. Immunol. 22: Ankerst, J., P. Christensen, L. Kjellen, and G. Kronvall A routine diagnostic test for IgA and IgM antibodies to rubella virus: absorption of IgG with Staphylococcus aureus. J. Infec. Dis. 130: Cremer, N. E., M. Hoffman, and E. H. Lennette Analysis of antibody assay methods and classes of viral antibodies in serodiagnosis of cytomegalovirus infection. J. Clin. Microbiol. 8: Cremer, N. E., M. Hoffman, and E. H. Lennette Role of rheumatoid factor in complement fixation and indirect hemagglutination tests for immunoglobulin M antibody to cytomegalovirus. J. Clin. Microbiol. 8: Fraser, K. B., P. V. Shirodaria, and C. F. Stanford Fluorescent staining and human IgM. Br. Med. J. 3: Furukawa, T., E. Hornberger, S. Sakuma, and S. A. Plotkin Demonstration of immunoglobulin G receptors induced by human cytomegalovirus. J. Clin. Microbiol. 2: Gerna, G., P. M. Cereda, E. Cattaneo, G. Achilli, and M. G. Revello Immunoglobulin G to virus-specific early antigens in congenital, primary, and reactivated human cytomegalovirus infections. Infect. Immun. 22: Kantor, G. L., L. S. Goldberg, B. L. Johnson, M. M. Derechin, and E. V. Barnett Immunologic abnormalities induced by postperfusion cytomegalovirus infection. Ann. Intern. Med. 73: Pien, F. D., T. F. Smith, H. F. Taswell, and K. A. Hable Cold reactive antibodies in a case of congenital cytomegalovirus infection. Am. J. Clin. Pathol. 61: a.Riggs, J. L Immunofluorescent staining, p In E. H. Lennette and N. J. Schmidt (ed.), Diagnostic procedures for viral, rickettsial, and chlamydial infection. American Public Health Association, Inc., New York. 10. Schmidt, N. J., J. Dennis, and E. H. Lennette Plaque reduction neutralization test for human cytomegalovirus based upon enhanced uptake of neutral red by virus-infected cells. J. Clin. Microbiol. 4: Schmitz, H., and R. Haas Determination of different cytomegalovirus immunoglobulins (IgG, IgA, IgM) by immunofluorescence. Arch. Ges. Virusforsch. 37: Shirodaria, P. V., K. B. Fraser, and F. Stanford Secondary fluorescent staining of viral antigens by rheumatoid factor and fluorescein-conjugated anti IgM. Ann. Rheum. Dis. 32: The, T. H., H. K. Andersen, E. S. Spencer, and G. Klein Antibodies against cytomegalovirus-induced early antigens (CMV-EA) in immunosuppressed renal-allograft recipients. Clin. Exp. Immunol. 28: The, T. H., G. Klein, and M. M. A. C. Langenhuysen Antibody reaction to virus-specific early antigens (EA) in patients with cytomegalovirus (CMV) infection. Clin. Exp. Immunol. 16: Wager, O., J. Rasanen, A. Hagman, and E. Klemola Mixed cryoimmunoglobulinaemia in infectious mononucleosis and cytomegalovirus mononucleosis. Int. Arch. Allergy 34: