Detection of Human T-Cell Lymphotropic Virus Type III-Related ...

Vol. 23, No. 6

JOURNAL OF CLINICAL MICROBIOLOGY, June 1986, p. 1072-1077 0095-1137/86/061072-06$02.00/0 Copyright © 1986, American Society for Microbiology

Detection of Human T-Cell Lymphotropic Virus Type III-Related Antigens and Anti-Human T-Cell Lymphotropic Virus Type III Antibodies by Anticomplementary Immunofluorescence RICHARD S. BLUMBERG,l* ERIC G. SANDSTROM,'t TIMOTHY J. PARADIS,' DAVID N. NEUMEYER,' M. G. SARNGADHARAN,2 KEVAN L. HARTSHORN,' ROY E. BYINGTON,' MARTIN S. HIRSCH,' AND ROBERT T. SCHOOLEY' Infectious Disease Unit, Massachusetts General Hospital, Boston, Massachusetts 02114,1 and Department of Cell Biology, Litton Bionetics Inc., Kensington, Maryland 207952 Received 23 December 1985/Accepted 7 March 1986

Techniques presently available for detection of human T-cell lymphotropic virus type III (HTLV-III) antigens and antibodies are laborious or relatively nonsensitive. We adapted anticomplementary immunofluorescence (ACIF) for these purposes. In HTLV-III-infected cells, specific ACIF was demonstrated by a diffuse speckling pattern that often resulted in a peripheral cellular rim of fluorescence. A 97% concordance was demonstrated between the ACIF assay and other sensitive tests for HTLV-III antibody detection (Western blot and membrane immunofluorescence and fixed-cell immunofluorescence tests). The ACIF assay was both more sensitive and more specific when compared with the enzyme-linked immunosorbent assay. For detection of HTLV-III antigens, the ACIF assay appeared to be as sensitive as the reverse transcriptase assay and more sensitive, with less background reactivity, than the conventional immunofluorescence assay. The ACIF assay often detected low levels of HTLV-III antigens within 3 days of infection in vitro, compared with 5 to 7 days with the indirect immunofluorescence assay, and generally paralleled the reverse transcriptase assay. The ACIF assay is a simple, sensitive, and specific assay for detection of HTLV-III-related antigens and antibodies. It should prove useful in the diagnosis of HTLV-III infection, as well as in studies of pathogenesis. The laboratory diagnosis of human T-cell lymphotropicvirus type III (HTLV-III) infection can be laborious and time consuming. Virus isolation requires prolonged lymphocyte cultures, with detection by reverse transcriptase (RT) assay or immunofluorescence assay (IFA) (17, 19). The RT assay is difficult to perform and does not distinguish among retrovirus types. In addition, it requires complete expression of the viral genome and thus does not detect abortive infection. The IFA technique uses monoclonal antibodies or alloantisera and is often characterized by large amounts of background staining or weak, specific staining, precluding the early detection of small numbers of infected cells. Other techniques, such as enzyme-linked immunosorbent assays (ELISAs) and plaque formation, are under study (8, 22). The anticomplementary immunofluorescence (ACIF) assay has been widely used in other viral systems. It is not only easy to perform but also reduces nonspecific staining and enhances sensitivity when compared with conventional IFA (7, 15, 23). Moreover, it can be used to detect both cellassociated viral antigens, as well as circulating antibodies. In the following studies, we show that the ACIF assay is both sensitive and specific in the detection of HTLV-III antigens and antibodies and can be used not only for diagnosis but also for monitoring of ongoing infections.

119 sera, 72 were from healthy homosexual men, 29 were from patients with the AIDS-related complex, and 18 were from patients with AIDS. Control sera included 15 from patients with autoimmune disease with high anti-nuclearantibody titers (512 to 4,096; homogeneous or speckled or both), obtained from Edward Paluch, Massachusetts General Hospital; 25 from patients attending an infectious disease clinic (including several patients with active cytomegalovirus [CMV] or Epstein-Barr virus [EBV] infection); and 7 from healthy laboratory workers. In addition, four ELISApositive, Western blot (WB)-negative sera, which, in three of four cases, were reactive with the HLA-Dr4 antigen, were also evaluated (kindly provided by David D. Ho). For antigen detection studies, three human sera that were positive in the ACIF and IFA reactions were used. Heparinized peripheral blood was obtained from these subjects, and peripheral blood mononuclear cell (PBMC) preparations were prepared on a Ficoll-Hypaque gradient as previously described (2). Antigen for indirect IFA and ACIF tests. HTLV-IIIBinfected and uninfected H9 cell lines were used for the serologic work (25). These cell lines were obtained from R. C. Gallo, National Cancer Institute, and were grown in RPMI 1640 medium (GIBCO Laboratories, Grand Island, N.Y.) supplemented with 20% heat-inactivated fetal calf serum, 250 U of penicillin per ml, 250 U of streptomycin per ml, 0.29 mg of L-glutamine per ml, and 0.01 M N-2hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) (Sigma Chemical Co., St. Louis, Mo.). These cells were harvested at the log phase of growth (90% viability, washed with phosphate-buffered saline (PBS), and resuspended in PBS without serum at 2 x 106 cells per ml. Ten microliters of this suspension was applied to the wells of cleaned 18-well fluorescence slides (number 10-441; Carlson Scientific, Inc., Peotone, 111.), air dried for 2

MATERIALS AND METHODS

Sera and PBMC. Sera were obtained from homosexual men enrolled in a prospective study of risk factors for the acquired immune deficiency syndrome (AIDS) at our institution. Of the *

Corresponding author.

t Present address: Department of Dermatology, Karolinska Institute, Sodersjukhuset, S-100 64, Stockholm, Sweden.

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were compared with those obtained with these techniques, by use of a previously characterized serum bank (29). ACIF was considered positive when a characteristic immunofluorescent pattern of at least 2+ intensity on a scale of 0 to 4+ was observed. The IFA technique was also compared with

rut. 1. n iLv-111-inieCLu nH meiis. NoLte I immunofluorescence. Shifting the focus reveals h, and fixed in a 50:50 mixture of methanol and acetone for 10 min at -20°C for use in the ACIF test. The slides were stored at -20°C until use. PBMC were obtained from HTLV-III viremic patients or from normal donors and infected in vitro with HTLV-III. Cells were processed as described above, and slides were fixed for 10 min in acetone for staining by the indirect IFA or in methanol-acetone as outlined above for the ACIF assay. Uninfected PBMC were used as controls. ACIF assay. The ACIF assay was performed by an adaptation of the procedure outlined by Henle et al. (11). Sera were heat inactivated at 56°C for 30 to 45 min. Ten microliters of a 1:10 dilution of these sera, diluted in PBS, was added to each well and incubated for 45 min at 37°C in a humid chamber. The slides were washed twice for 5 min in PBS, washed once in double-distilled water, and air dried. Ten microliters of non-heat-inactivated human serum stored at -70°C and thawed only once was added as a source of human complement at a 1:30 dilution in cold Hanks balanced salt solution (GIBCO) for 30 min at 37°C in a humid chamber. A single EBV-, CMV-, and HTLV-III-negative donor was used as our source of complement. Fresh serum was obtained from this individual every month. These procedures, as well as adding the serum before the complement, minimized the nonspecific reactivity of the complement. The slides were washed and air dried, and 10 ,uI of a 1:40 dilution of fluorescein isothiocyanate-labeled, F(ab')2 goat antihuman complement (C3) (Cooper Biomedical, Inc., West Chester, Pa.) in PBS was applied and incubated for 30 min at 37°C. The slides were washed, counterstained in 0.01% Evans Blue, air dried, mounted, and stored at 4°C in darkness until use. IFA, WB, MAF assay, and ELISA. IFA, WB, and the membrane antibody fluorescence (MAF) assay were performed as outlined previously (16, 29, 32, 33). ACIF results

the ACIF assay for the detection of HTLV-III antigens in infected PBMC. In vitro infection of PBMC with HTLV-Ill. PBMC at 4 x 105 cells per ml in RPMI 1640 medium (as outlined above) plus 20% fetal calf serum and 10% interleukin-2 (Electronucleonics, Silver Spring, Md.) were stimulated with phytohemagglutinin (25 ,ug/ml) for 3 h before the addition of cell-free HTLV-IIIB at a concentration of 100 to 125 50% tissue culture infectious doses or 26,000 to 32,500 cpm of RT activity per 2 x 106 cells. The HTLV-IIIB was harvested by clarifying the supernatant fluids of log-phase cultures of the HTLV-IIIB-infected H9 cell line; these were stored at -70°C until use. The cultures were incubated in 25- or 150-cm2 flasks (Costar, Cambridge, Mass.) at 37°C in 5% C02. Aliquots of the cell suspension of infected- and uninfected-PBMC cultures were harvested at 3, 5, 7, 10, and 12 days after infection and processed for immunofluorescence (by IFA and ACIF assay) as outlined above and for the RT assay as previously outlined (14). In our experience, negative specimens may occasionally have RT values of 1,000 cpm. Therefore, a significant RT value has been defined as one >10,000 cpm. The proportion (percentage) of fluorescent cells was determined by counting at least 500 cells; on rare occasions, less cells were counted because of the sloughing of cells during the process of staining. HTLV-III isolation from fresh PBMC. Virus isolations were performed by the method of Feorino et al. (5). Briefly, PBMC from HTLV-III-seropositive patients, prepared with a Ficoll-Hypaque gradient, were added to similarly prepared PBMC from healthy subjects that had been stimulated with phytohemagglutinin-P in the presence of 2 ,ug of Polybrene per ml and 10% interleukin-2 for 3 days. Every 3 to 4 days, aliquots of the suspension were removed for the determination of the RT activity and antigens by the ACIF technique. RESULTS Detection of antibody by ACIF. By using HTLV-IIIBinfected and uninfected H9 cells, a single pattern of immunofluorescence emerged as specific for HTLV-III antibody. The pattern was brightly fluorescent diffuse speckling that often resulted in a peripheral rim (Fig. 1). Occasionally, this rim pattern took on a capped appearance. The majority (>90%) of HTLV-IIIB-infected H9 cells displayed these patterns when reacted with a strongly positive serum. The HTLV-III-specific ACIF pattern was easily distinguished from nonspecific reactive patterns which were occasionally observed with both the uninfected and HTLV-IIIB-infected H9 cell lines. This nonspecific reactivity was manifested primarily as a perinuclear globular pattern of immunofluorescence that was most prominent when complement was applied directly to both the uninfected and HTLV-IIIBinfected H9 cell lines. Applying heat-inactivated HTLV-IIIseropositive or -seronegative sera to cells before addition of complement in the normal course of the ACIF assay generally decreased this reaction pattern to negligible levels. As the dilution of complement also affected the intensity of this nonspecific reaction, the optimal concentrations of complement and conjugate were defined with a checkerboard of varying dilutions of each. The dilutions which maximized the sensitivity and minimized the nonspecific reactivity were 1:30 and 1:40, respectively. Both cell lines also rarely

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TABLE 1. ACIF assay detection of antibody and correlation with IFA, WB, MAF assay, and ELISA No. of sera that were:

Reaction by ACIF assay

Positive Negative

MAF positive

negative

MAF

ELISA positive

negative

1

44 0

4 43

20 il

2 41

negative

WB

IFA positive

negative

23 1

1 52

67 2

54

WB

displayed a dull, mottled pattern or a diffuse, homogeneous, ground-glass type of apple-green immunofluorescence. In general, the immunofluorescence associated with the ACIF reaction was brighter than that associated with the IFA reaction. Sensitivity and specificity of the ACIF assay. The sensitivity and specificity of the ACIF assay for antibody detection were determined by comparing the ACIF assay to other tests (MAF assay, IFA, ELISA, and the WB). The ACIF assay detected 23 of 24 WB-positive sera with a concordance of 97% (Table 1). The one ACIF-negative serum was attributed to the many freeze-thawing procedures because another serum specimen of the same patient was positive by the ACIF assay. Only 1 of 53 WB-negative samples was positive by the ACIF assay. The ACIF assay results also correlated closely with those obtained by the IFA and MAF tests. There was, however, a significant discrepancy between the ACIF assay and the ELISA. Of the sera, 13 of 74 (18%) and 7 of 74 (10%) were borderline or uninterpretable by ELISA, respectively. Of these 20 sera, 9 were negative by the ACIF assay and WB, as was an additional ELISA-reactive serum. Of the others, one was ACIF negative and WB positive, one was ACIF positive and WB negative, and nine were positive by both the ACIF assay and WB. As borderline reactions in

FIG. 2. HTLV-III-infected PBMC. Note that the morphology of the immunofluorescence is similar to that of the HTLV-III-infected H9 cell in Fig. 1.

ELISA

IFA

positive

the ELISA may be partly related to the presence of HLADr4-reactive antibodies (18), we tested four such sera with the ACIF assay. All four sera were negative by the ACIF assay and WB yet reactive by ELISA. Of the control sera studied, 3 of 15 from patients with autoimmune disease and speckled anti-nuclear antibodies reacted nonspecifically in the ACIF assay. However, these sera also reacted with the uninfected H9 cell line and had a pattern of staining that was distinctly different from the usual ACIF speckling. One of the sera from a patient attending an infectious disease clinic was from a homosexual male with AIDS-related complex and was positive by both IFA and ACIF techniques. The remainder of the control sera were negative by the ACIF assay. Antigen detection in infected PBMC. It had been our experience that the IFA, although an excellent test of serologic status, is not sensitive in antigen detection during HTLV-III infection of PBMC primarily because the relatively high level of background staining makes detection of a low number of positive cells difficult. Therefore, we evaluated the ACIF technique in the early detection of HTLV-III antigens. Phytohemagglutinin-stimulated PBMC were infected with cell-free HTLV-IIIB in media containing 10% interleukin-2 and observed every 3 to 4 days for evidence of viral antigen by IFA and ACIF assay and for viral RT activity. With the ACIF technique, infected PBMC expressed a pattern of immunofluorescence that was similar to that observed in HTLV-III-infected H9 cells, i.e., diffuse speckling or rim pattern or both (Fig. 2). The background reactivity was negligible and thus allowed a great deal of sensitivity in detecting small numbers of virus-infected cells. A nonspecific homogeneous pattern was occasionally seen in infected and uninfected cells but was easily distinguished from the specific HTLV-III staining. Whereas virus was detected by RT assay on day 7, the ACIF assay was able to detect low amounts of viral infection on day 3 in 4 of 5 donors tested and on day 5 in the other (Table 2). Significant amounts of virus were detected by the ACIF assay by day 5 in all 5 donors. These observations were confirmed by using three different alloantisera. When the RT assay became positive (>104 cpm), 10 to 15% of the cells displayed specific immunofluorescence with the ACIF assay. RT and ACIF activity appeared to parallel one another and peak at approximately 10 days after infection. With IFA, viral antigen was variably detected on day 5 and often not detected until day 7 of in vitro infection. Even on day 7, the levels of viral antigen detected were less than that defined by the ACIF test. Subsequently, the two techniques paralleled one another. However, the IFA staining was consistently weaker in intensity and was marked by distinctly more background reactivity. Detection of HTLV-III infection in PBMC isolated from peripheral blood. The ACIF technique was applied to the identification of HTLV-III in cultured, fresh PBMC. PBMC obtained from men seropositive for HTLV-III were cultured with 10% interleukin-2 and phytohemagglutinin-stimulated

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TABLE 2. In vitro infection of PBMC Course of infection as detected by the following test on day: 3 Donor

1 3 4 5 a

7

10

12

ACIF IFA (% cells (% cells

RT (cpm,

ACIF IFA (% cells (% cells

RT (cpm,

ACIF IFA (% cells (% cells

RT (cpm,

ACIF IFA (% cells (% cells

RT (cpm,

104)a

ACIF IFA (% cells (% cells

positive) positive)

104)a

positive) positive)

104)a

positive) positive)

104)a

positive) positive)

104)a

positive) positive)

0 0 0 0 0

2

5

RT (cpm,

0