JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1990, p. 724-733

Vol. 28, No. 4

0095-1137/90/040724-10$02.00/0 Copyright © 1990, American Society for Microbiology

Detection and Quantitation of Human Immunodeficiency VirusInfected Peripheral Blood Mononuclear Cells by Flow Cytometry JAMES J. McSHARRY,1* ROBERT COSTANTINO,' ELLEN ROBBIANO,2 ROGER ECHOLS,2T ROY STEVENS,3 AND JOHN M. LEHMAN'

Departments of Microbiology and Immunologyl and Medicine,2 Albany Medical College, Albany, New York 12208, and The Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, New York 122013 Received 11 May 1989/Accepted 27 December 1989

A flow cytometric assay has been developed to detect and quantitate human immunodeficiency virus (HIV)infected peripheral blood mononuclear cells obtained from HIV-seropositive patients. Peripheral blood was obtained from patients attending an acquired immune deficiency syndrome clinic, and mononuclear cells were separated by centrifugation onto Ficoll-Hypaque. The cell layer at the interface was removed, washed in phosphate-buffered saline without Ca2+ and Mg2+, and fixed with 90% methanol, and intracellular HIV antigens were detected by indirect immunofluorescence with monoclonal antibodies to 1IV antigens as the primary antibody and fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin G F(ab')2 antibody as the secondary antibody. DNA content was determined by propidium diiodide staining after RNase treatment. These fluorochrome-treated cells were analyzed for two-color fluorescence by flow cytometry. The results showed that HIV-infected cells in peripheral blood that have been treated with monoclonal antibodies to the p24 or nef antigens of HIV can be detected and quantitated by flow cytometry. The percentage of p24 antigen-positive mononuclear cells had a significant correlation (P = 0.0001) with the clinical status of the patient, i.e., those with a high percentage of p24 antigen-positive cells had a poorer prognosis than those with a lower percentage of p24 antigen-positive mononuclear cells. In addition, for those in Centers for Disease Control groups III and IV, there was an inverse correlation between the percentage of p24 antigen-positive mononuclear cells and the number of T4 cells. However, cell-associated antigen detection by flow cytometry did not correlate with detection of antigen in sera of HIV-seropositive patients by the standard antigen capture enzyme-linked immunosorbent assay. This lack of correlation was probably due to the presence of immune complexes in the sera of HIV-seropositive patients. These results suggest that flow cytometry can be used as a rapid, sensitive, and quantitative assay system for the determination of the antigen status of HIV-seropositive patients and that it may be more useful as an indicator of disease progression than the currently used antigen detection methods.

Acquired immune deficiency syndrome (AIDS) is caused by infection with the human immunodeficiency virus (HIV) (2, 13). Infection is determined by detecting the presence of antibodies to specific viral proteins in sera of HIV-infected individuals by an enzyme-linked immunosorbent assay (ELISA), and those sera that are repeatedly ELISA positive are confirmed by the Western blot (immunoblot) test (27, 29). This information is useful for epidemiological purposes and may be useful for counseling HIV-seropositive individuals to modify their social behavior. However, given the long latency of HIV infection, the presence of antibodies to HIV has little clinical relevance. Therefore, more specific and reliable indicators of the clinical status of an HIVseropositive individual are needed to aid in the treatment and counseling of these individuals. Recently, a number of parameters which may be useful in predicting disease progression in HIV-infected patients have been defined. These include the presence of HIV antigenemia as measured by the presence of soluble p24 antigen, the decline in antibody to p24 antigen as measured by ELISA or Western blot assays, the decline in the total number of CD4positive cells, an increase in the level of beta 2 microglobulin in serum, and a decline in the ratio of the number of CD4positive cells to the serum neopterin level (1, 8-11, 20, 23, *

25, 30, 32, 35). Of these predictors of disease progression, the presence of HIV antigenemia may be the most important indicator of the clinical status of an infected individual (1, 10, 23, 27, 32). Two methods for detecting HIV in peripheral blood are cell culture assays for infectious virus and antigen capture ELISAs (4, 15). However, antigen detection by the commercially available p24 antigen detection assays may underestimate the presence of HIV antigens in the sera of HIV-seropositive individuals because of the presence of HIV antigen-antibody complexes (23, 24, 34). Growth of HIV in mitogen-stimulated lymphocytes has been used to detect infectious virus in peripheral blood obtained from HIV-infected individuals (4). However, this procedure is both time-consuming and expensive and has seldom detected infectious virus in all patients with pre-AIDS and AIDS. Since the currently available HIV antigen detection systems may underestimate the prevalence of HIV antigenemia, there is a need to develop more accurate assays for detecting the presence of HIV antigens in body fluids and cells of HIVseropositive individuals. Analysis of immunofluorescently labeled antigens in virus-infected cells by flow cytometry has been used to detect and quantitate infected cells in tissue culture and in clinical specimens. We have used this technique to detect and quantitate simian virus 40 (SV40)- and cytomegalovirus (CMV)-infected tissue culture cells and CMV-infected cells in bronchoalveolar lavage specimens (12, 17, 21). Cory and colleagues have used indirect immu-

Corresponding author.

t Present address: Miles Pharmaceuticals Inc., West Haven, CT 06516.

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nofluorescence and flow cytometry to detect and quantitate HIV-infected H9 cells, for which they were able to detect 1 HIV-infected H9 cell in 10,000 uninfected H9 cells (6, 7). In preliminary reports, Ohlsson-Wilhelm et al. and Cory et al. reported that they could detect HIV-infected mononuclear cells obtained from HIV-infected hemophiliacs by flow cytometry (J. M. Cory, B. M. Ohlsson-Wilhelm, F. Rapp, and M. E. Eyster, Abstr. 5th Int. Conf. AIDS, 1989; B. M. Ohlsson-Wilhelm, J. M. Cory, H. A. Kessler, M. E. Eyster, F. Rapp, and A. Landay, Abstr. 5th Int. Conf. AIDS, 1989; B. M. Ohlsson-Wilhelm, J. M. Cory, M. E. Steck, M. Rozday, M. Smithgall, N. Shaeffer, F. Rapp, and M. E. Eyster, Abstr. 13th Int. Meet. Soc. Anal. Cytometry, abstr. no. 399D, 1988). This technique is particularly useful for detecting viral antigens in cells in clinical samples because it detects viral antigens in virus-infected cells which have been separated from other body fluids that may contain antibodies that interfere with the currently available antigen detection assays. In this report, we show that indirect immunofluorescence combined with flow cytometry can be used to detect and quantitate HIV antigens in mononuclear cells obtained from the peripheral blood of HIV-seropositive patients. Furthermore, there is a highly significant correlation between the percentage of p24 antigen-positive cells detected in the peripheral blood of HIV-seropositive individuals and the clinical stage of the disease and an inverse relationship between the percentage of p24 antigen-positive cells and the total number ofT4 cells. Thus, these two parameters may be useful for determining disease progression in HIV-seropositive individuals. MATERIALS AND METHODS Cells. Uninfected H9 cells and H9 cells persistently infected with HIV were obtained from the National Institutes of Health AIDS Research and Reference Reagent Program and the American Type Culture Collection, respectively. The cells were grown in 75-cm2 Corning plastic flasks in suspension in RPMI 1640 medium (for H9 cells) or Iscove Dulbecco modified medium (for HIV-infected H9 cells) supplemented with 20% fetal bovine serum, streptomycin (100 ,ug/ml), and penicillin (100 U/ml) under an atmosphere of 5% C02. Blood was obtained by venipuncture and collected in a tube containing EDTA. To obtain mononuclear cells, the blood was diluted 1/2 with phosphate-buffered saline (PBS) without Ca2+ and Mg2+, layered over 3 ml of Ficoll-Hypaque, and centrifuged at room temperature at 400 x g for 30 min. The plasma was removed from the top of the tube and the band of mononuclear cells at the interface was aspirated, washed twice in PBS without Ca2+ and Mg2+, and suspended in PBS without Ca2+ and Mg2+; methanol was then added to a final concentration of 90% (17, 21). Monoclonal and polyclonal antibodies. HIV-specific monoclonal antibodies were obtained as follows. Monoclonal antibodies to p24, p17, and gpl20 antigens were obtained from Chemicon International, Inc., El Segundo, Calif.; monoclonal antibodies to 3'ORF (nef), gpl20, and gp4l antigens were obtained from NEN-Dupont Co., Wilmington, Del.; and monoclonal antibodies to p24, p17, and gpl6O/41 antigens were obtained from Cellular Products Inc., Buffalo, N.Y. Murine monoclonal antibodies to herpes simplex virus type 1 and cytomegalovirus antigens were obtained from NEN-Dupont. Murine monoclonal antibody to the SV40 T antigen was obtained from a hybridoma cell line supplied by the American Type Culture Collection. Known HIV-seropositive and HIV-seronegative polyclonal human sera were

725

obtained from Roy Stevens, New York State Department of Health, Albany, N.Y. The HIV-seropositive polyclonal human serum reacted with bands 24, 31, 41, 51, and 66 on a Western blot, suggesting that it contained antibodies to these HIV antigens. F(ab')2 goat anti-mouse immunoglobulin G (IgG) (heavy and light chain)-fluorescein isothiocyanate (FITC) conjugate was obtained from Boehringer Mannheim Biochemicals, Indianapolis, Ind. FITC-conjugated goat antihuman IgG was obtained from Litton Bionetics, Kensington, Md. Fixation and staining of cells for flow cytometry. The procedures for fixing and staining cells for flow cytometry have been published elsewhere (12, 17, 21). Briefly, uninfected and HIV-infected H9 cells were removed from culture flasks, washed twice in cold PBS without Ca2+ and Mg2+ by being pelleted at 400 x g for 10 min at 4°C, and suspended in PBS without Ca2+ and Mg2+ at 0°C. The cells were fixed by the addition of cold methanol to a final concentration of 90%. Mononuclear ceils obtained from peripheral blood were prepared as described above. This procedure served to fix the cells and to inactivate any HIV that was present without destroying antigenicity. The fixed cells were counted in a hemacytometer and stored at -70°C at a concentration of 106 cells per ml in 90% methanol until it was time for staining and analysis by flow cytometry. Fixed cells have been stored in this manner for several months without detectable loss of antigen (21). After removal of the fixative, the cells were washed once in PBS without Ca2" and Mg2+ and suspended in unconjugated primary antibody diluted 1:10 (unless otherwise noted) in PBS-normal goat serum (NGS) (PBS plus 145 mM NaCI, 10 mM phosphate [pH 7.4] and an equal volume of heatinactivated NGS with 0.002% Triton X-100 and 0. 1% sodium azide). For all reagents, 0.5 ml per sample was used. The antibody was in excess. The cells were incubated with the primary antibody for 2 h at 37°C, pelleted, and washed twice in PBS-NGS at 4°C for 30 min. After being pelleted, the washed cells were suspended in FITC-conjugated goat antimouse IgG F(ab')2 diluted 1:80 in PBS-NGS, incubated at 37°C for 2 h, pelleted, and washed twice in PBS-NGS at 37°C for 30 min. After the final wash, the cells were treated with RNase at 37°C for 30 min to remove any RNA which may interfere with binding of propidium diiodide to DNA and then with an equal volume of propidium diiodide to a final concentration of 50 ,ug/ml. Propidium diiodide was used in this system to determine the DNA content of a cell. Only cells with diploid DNA content were analyzed for fluorescence. The samples were stored at 4°C overnight. Analysis of data obtained by flow cytometry. Data acquisition was done with a Cytofluorografs IIs model H-H and a 2151 data analysis system (Ortho Diagnostics, Inc., Westwood, Mass.) using a 5-W argon ion laser (Coherent, Palo Alto, Calif.). The instrument was aligned immediately before use with FITC-labeled microspheres (no. 9847; Polysciences, Warrington, Pa.) for green fluorescence and with lymphocytes stained with prodidium diiodide for red fluorescence and assayed as previously described (17, 21). The coefficient of variation was typically about 1.0 for low-angle light scatter and 0.8 for both red and green fluorescence. For analysis, a 535-nm bandpass filter (40-nm width) was used for green and a 640-nm longpass filter was used for red. No filter was used for light scattering. The data were collected first into a two-parameter histogram displaying light scattering versus red fluorescence. This was gated to eliminate any particles which were not the correct size or of sufficient DNA content to be a cell. Cells passing through this gate

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were then displayed on a second two-parameter display of red peak versus red area to allow selection of single cells on the basis of DNA content with a second gate. Data passing through the second gate were then displayed with a third two-parameter histogram of red versus green area, which was temporarily stored on the 2151 analysis system and then transferred to an IBM PC-AT for analysis and storage. The data in all the two-dimensional histograms are presented in a smoothed form, although all of the analyses were done on the original data. The mean y value was obtained by gating on the entire population of interest and calculating the mean of those cells in the gate (17, 21). For analysis, green fluorescence (y axis, which represents HIV antigen) was correlated to both red fluorescence (x axis, representing DNA content) and the number of cells. Usually, 10,000 cells were analyzed for each sample. During each run of the flow cytometer, the staining procedure was standardized with cells stained with monoclonal antibody to the SV40 T antigen. The cells used were B-1 (negative) and A58 (positive for T antigen). Therefore, these controls provided an internal control for comparison of the mean y green fluorescence of positive and negative cell populations and allowed comparisons of data from multiple runs at different time intervals (17, 21). For each run of infected H9 cells or patient mononuclear cells, uninfected H9 cells or mononuclear cells obtained from six separate HIV-seronegative individuals were fixed, stained with the appropriate monoclonal antibodies, and analyzed by flow cytometry, and the resulting data were used to arbitrarily set a gate at 1% positive cells. The percentage of antigenpositive cells in these HIV-seronegative blood samples usually varied within ±1.0% of the gate set for cells obtained from HIV-seronegative individuals. Thus, samples containing 0 to 2% p24 antigen-positive cells were considered negative. Preparations of cells containing 2 to 4% antigenpositive cells were considered to be equivocal, whereas those containing 4% or more antigen-positive cells were considered to be HIV-positive samples. Previous studies involving mixing experiments with uninfected and SV40-infected CV-1 cells demonstrated that antigen-positive and antigen-negative cells could be resolved when analyzed by this flow cytometric technique (17). In a similar experiment, Cory et al. (6) demonstrated that uninfected and HIV-infected H9 cells treated with monoclonal antibody to the HIV-p24 antigen and FITC-conjugated goat anti-mouse IgG antibody could be distinguished by flow cytometry. Thus, this procedure is capable of distinguishing between HIV-infected and uninfected cells. Antigen detection. Soluble HIV p24 antigen in sera from HIV-seronegative and HIV-seropositive individuals was detected by using the Dupont HIV Antigen Detection Kit according to the instructions of the manufacturer. Determination of T-cell subsets. Whole blood was used to determine the number of T cells and their subsets by using monoclonal antibodies to T3, T4, and T8 cell surface antigens. Staining and analysis by flow cytometry on a Coulter EPICS V instrument was carried out according to the procedures described by Coulter Immunodiagnostics, Inc., Hialeah, Fla.

RESULTS

Detection and quantitation of HIV-infected H9 cells by flow cytometry. To determine the utility of flow cytometry for detecting HIV-infected cells, uninfected and HIV-infected H9 cells were fixed, stained by indirect immunofluorescence

TABLE 1. Flow cytometric analysis of HIV-infected H9 cells: comparison of monoclonal and polyclonal antibodies to HIV antigens Antibody

Human serum Chemicon

p24 pl7 gpl20 NEN 3'ORF (nef) gpl20 gp4l Cellular Products

p24 pl7 gpl60/41

% Positive uninfected H9 cells (mean y value)b

% Positive HIVinfected H9 cells (mean

1/200

0.81 (0.07)

81.0 (0.71)

90.1

1/10 1/10 1/10

0.34 (0.05) 0.59 (0.07) 0.59 (0.05)

81.0 (0.38) 21.1 (0.57) 2.2 (0.20)

86.8 87.7 75.0

1/10 1/10 1/10

0.48 (0.12) 0.44 (0.05) 0.44 (0.05)

78.2 (0.63) 1.3 (0.19) 12.5 (0.21)

81.0 73.6 76.2

1/10 1/10 1/10

0.22 (0.05) 0.53 (0.06) 0.71 (0.26)

16.4 (0.36) 26.6 (0.74) 24.5 (0.87)

86.1 91.9 70.1

Dilution

y

Sp actc (%)

value)

a Fluorescently tagged cells were analyzed on a logarithmic scale because the majority of HIV-infected H9 cells should be infected and each infected cell should contain significant amounts of p24 antigen yielding a high fluorescence intensity. b Mean y values were obtained by gating on the population of interest and calculating the mean fluorescence intensity of those cells in the gate (17, 21). ' Specific activity = (mean y value of infected population - mean y value of uninfected population/mean y value of infected population) x 100.

using various monoclonal or polyclonal antibodies to HIV antigens and propidium diiodide, and analyzed for fluorescence by flow cytometry. The results of this comparison between the HIV-seropositive human serum and the various commercially available monoclonal antibodies to HIV antigens are presented in Table 1. The polyclonal HIV-seropositive human serum at a 1/200 dilution detected 81% of the HIV-infected H9 cells with a specific activity of 90.1%. The Chemicon p24 and NEN 3'ORF monoclonal antibodies detected 81 and 78% of the HIV-infected H9 cells, with specific activities of 86.8 and 81.0%, respectively. The mean y value of the HIV-infected H9 cells treated with the NEN 3'ORF monoclonal antibody (0.63) was greater than that of the HIV-infected H9 cells treated with the Chemicon p24 monoclonal antibody (0.38), suggesting that the cells labeled with the NEN 3'ORF monoclonal antibody are brighter than those labeled by the Chemicon p24 monoclonal antibody. However, the mean y value of the uninfected H9 cells treated with the 3'ORF monoclonal antibody was also higher than that of the uninfected H9 cells treated with the Chemicon p24 monoclonal antibody. Thus, the Chemicon p24 antibody has greater specific activity than the NEN 3'ORF monoclonal antibody. The Chemicon p17 and the Cellular Products p17 and gpl6O/41 monoclonal antibodies detected 21, 26, and 24% of the HIV-infected H9 cells, with specific activities of 87.7, 91.9, and 70%, respectively. The Chemicon p17 monoclonal antibody and the Cellular Products pl7 monoclonal antibody had good specific activities, but the percentage of cells detected was low compared with the Chemicon p24 and NEN 3'ORF monoclonal antibodies. The remaining monoclonal antibodies tested detected less than 20% of the HIV-infected H9 cells. The inability of the Chemicon gp 120 and the NEN gp 120 and gp 41 monoclonal antibodies to detect the HIV-infected H9 cells may be due to the fixation procedure, which may disrupt or destroy antigens on the cell surface, rendering them incapable of interacting with the monoclonal antibodies to these glycopro-

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VOL. 28, 1990

TABLE 2. Specificity of HIV p24 monoclonal antibodies for HIV-infected H9 cells Monoclonal antibodies

CMV early Ag Herpes simplex virus type 1 Ag SV40 T Ag HIV p24 Ag

% Positive H9 cells (mean y

value)'

% Positive HIV-H9 cells (mean y value)

1.40 (0.05) 1.00 (0.05)

1.92 (0.05) 1.50 (0.05)

0.12 (0.05) 0.16 (0.05)

1.09 (0.05) 57.03 (1.05)

Sp actc

(%)

85

a Fluorescently tagged cells were analyzed on a logarithmic scale because the majority of HIV-infected H9 cells should be infected and each infected cell should contain significant amounts of p24 antigen yielding a high fluorescence intensity. Ag, Antigen. b Mean y values were obtained as described in Table 1, footnote b. C Specific activity = (mean y value of positive cells - mean y value negative cells/mean y value of positive cells) x 100.

teins. None of the monoclonal antibodies tested detected significant numbers of uninfected H9 cells. On the basis of these data, the Chemicon p24 and NEN 3'ORF monoclonal antibodies were as efficient as the HIV-seropositive human serum in detecting HIV-infected H9 cells by flow cytometry, suggesting that they would be useful for detecting HIVinfected cells obtained from HIV-seropositive patients. Specificity of HIV monoclonal antibodies for HIV-infected H9 cells. To determine the specificity of the antigen-antibody reactions shown in Table 1, uninfected and HIV-infected H9 cells were incubated with heterologous and homologous antibodies of the same isotype as those used above. The data presented in Table 2 show that there was no significant difference in the amount of green fluorescence (mean y value) and the percentage of antigen-positive cells when uninfected and HIV-infected H9 cells were treated with murine monoclonal antibodies to heterologous antigens such as CMV early nuclear antigen, herpes simplex virus type 1 antigen, and SV40 T antigen. Incubation of the HIV-infected cells with monoclonal antibodies to the p24 antigen resulted in a very specific reaction, with green fluorescence (mean y value) 20 times greater than that of the uninfected H9 cells and detection of 57% of the HIV-infected H9 cells, with a specific activity of 85%. The percentage of cells detected in this experiment was lower than that shown in Table 1 because the p24 monoclonal antibody used in this experiment was more dilute. Using a similar approach, Cory et al. (6) showed that one HIV-infected H9 cell can be detected in the presence of 10,000 uninfected H9 cells. These results demonstrate the specificity of the reaction between monoclonal antibodies to HIV antigens and HIV-infected cells and suggest that small numbers of HIV-infected cells can be detected by using this technology. Detection of HIV-infected cells in mononuclear cells of HIVseropositive patients. We attempted to determine whether the Chemicon p24 and the NEN 3'ORF monoclonal antibodies could detect HIV-infected mononuclear cells obtained from HIV-seropositive individuals but not those from HIV-seronegative people. Peripheral blood was obtained from an HIV-seropositive individual and an HIV-seronegative individual, the mononuclear cells were isolated and divided in half, and one half was treated with monoclonal antibody to the p24 antigen while the other half was treated with monoclonal antibody to the 3'ORF antigen. The samples were then analyzed by flow cytometry. The data showed that monoclonal antibodies to the p24 antigen (Fig. 1A and B) and

727

the 3'ORF (Fig. 1C and D) detected their respective antigens in mononuclear cells obtained from an HIV-seropositive individual (Fig. 1B and D), whereas they did not detect their respective antigens in mononuclear cells obtained from an HIV-seronegative individual (Fig. 1A and C). The mononuclear cells obtained from an HIV-seronegative individual treated with the monoclonal antibody to the p24 antigen had 0.18% antigen-positive cells, whereas the mononuclear cells obtained from an HIV-seropositive individual contained 4.66% p24 antigen-positive cells. Mononuclear cells obtained from the same HIV-seronegative and HIV-seropositive individuals, when treated with monoclonal antibody to the 3'ORF antigen, showed 1.61 and 15.51% positive cells, respectively. It should be noted that for studies on lymphocytes the green fluorescence was collected in the linear mode because of the lower fluorescence intensity of the infected mononuclear cells. This is in contrast to the studies using the H9 cells, which were collected in the logarithmic mode because of the greater fluorescence intensity of the persistently infected cells. Specificity of the reaction between monoclonal antibodies to HIV antigens in mononuclear cells obtained from HIV-seropositive individuals. It is clear from the data presented in Fig. 1 that we can distinguish normal mononuclear cells obtained from HIV-seronegative individuals from HIV-infected mononuclear cells obtained from HIV-seropositive individuals by flow cytometry. However, HIV-infected cells may react with these monoclonal antibodies differently than normal mononuclear cells. To determine the specificity of the reaction of these monoclonal antibodies with HIV-infected mononuclear cells, mononuclear cells obtained from an HIVseronegative individual and an HIV-seropositive individual were incubated with heterologous monoclonal antibodies such as those to SV40 T antigen, CMV early nuclear antigen, and herpes simplex virus antigens as well as homologous monoclonal antibodies to the HIV 3'ORF (nef) antigen or p24 antigen. The data are presented in Table 3. Although all three of the heterologous monoclonal antibodies reacted with mononuclear cells obtained from an HIV-seropositive individual to a greater extent than the mononuclear cells obtained from the seronegative individual, the homologous reactions between monoclonal antibody to the 3'ORF (nef) or the p24 antigen and mononuclear cells from the same HIV-seropositive individual were considerably greater than those of the heterologous reactions. The unexpectedly high reaction of the CMV-specific monoclonal antibody with the mononuclear cells of the HIV-seropositive individual suggests that this person was infected with CMV. Three months after the blood was drawn for this experiment, CMV was isolated from this patient by using standard virus culture assays. These results further verified the specificity of the reaction between the HIV monoclonal antibodies and the HIV-infected mononuclear cells. Our results suggest that monoclonal antibodies to p24 and 3'ORF antigens of HIV can be used to detect and quantitate HIV-infected mononuclear cells obtained from HIV-infected individuals. Correlation of clinical status with the percentage of p24 antigen-positive cells in peripheral blood. In the following study, the p24 monoclonal antibody was used to detect the p24 antigen in peripheral blood mononuclear cells (PBMCs) obtained from HIV-seropositive individuals by flow cytometry. We performed a prospective analysis of 85 blood samples obtained from HIV-seropositive individuals who were not on 3'-azido-3'-deoxythymidine (AZT) therapy and who consented to have blood drawn for this study. Sixty percent of these individuals were p24 antigen positive on the

728

McSHARRY ET AL.

J. CILIN. MICROBIOL.

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FIG. 1. Detection of HIV-infected and uninfected mononuclear cells treated with monoclonal antibodies to p24 and 3'ORF antigens (Ag) by flow cytometry. PBMCs obtained from an HIV-seronegative individual and an HIV-seropositive individual were divided into two sets, and each population was treated with monoclonal antibody to p24 or 3'ORF antigens followed by FITC-conjugated goat anti-mouse IgG F(ab')2 antibody. After RNase treatment, the cells were treated with propidium diiodide (PI) and then analyzed for fluorescence by flow cytometry. (A and C) Contour maps of the mononuclear cells from the HIV-seronegative individual treated with the monoclonal antibodies to p24 and 3'ORF, respectively. (B and D) Contour maps of mononuclear cells obtained from the HIV-seropositive individual treated with monoclonal antibodies to p24 and 3'ORF, respectively. Data were collected in the linear mode for clinical samples because few cells were expected to be labeled and the fluorescence intensity of these cells was expected to be low. The mononuclear cells from the HIV-seropositive individual contained significant amounts of p24 or 3'ORF antigen as noted by the increased fluorescence along the y axis in both panels B and D compared with the HIV-seronegative controls in panels A and C.

basis of having >4% of analyzed mononuclear cells positive for the p24 antigen. When the percentage of p24 antigenpositive cells was compared with the clinical status of the individual as defined by the Centers for Disease Control (CDC), it was apparent that there was a significant correlation between the clinical status of the patient and the percentage of p24 antigen-positive cells in the peripheral blood of the patient. This correlation is illustrated in Fig. 2. Patients in CDC group Il had a mean of 3.8% p24 antigenpositive ceils (standard deviation, +3.7); those in CDC group III had a mean of 5.1% p24 antigen-positive cells (standard deviation, +5.2); and those in CDC group IV had a mean of 10.8% p24 antigen-positive cells (standard deviation, +6.5). The data showed that individuals with more advanced clinical disease have a greater percentage of mononuclear cells that are p24 antigen positive. A statistical analysis of variance provided very strong evidence that the percentages of p24 antigen-positive cells are different at the various clinical stages of the disease (P = 0.0001). Further analysis using the Tukey Honestly Significant Difference test showed that CDC group IV is different from CDC groups Il and III (P < 0.05). Since the percentage of p24 antigenpositive cells correlates with the CDC clinical grouping, it

TABLE 3. Specificity of HIV p24 and nef monoclonal antibodies for HIV-infected mononuclear cells Motioclonal

antibody CMV early Ag

Herpes simplex virus type 1 Ag SV40 T Ag HIV nef HIV p24

% Positive mononuclear cells from an HIV-

% Positive mononuclear cells from an HIV-

Sp actc

seronegative person (mean y value)b

seropositive

(%)

person (mean y

0.36 (1.01) 0.03 (1.02)

4.49d (1.87) 0.99 (1.10)

41 7

0.03 (1.02) 3.75 (1.38) 0.65 (1.05)

1.70 (1.35) 32.48 (11.39) 10.54 (8.58)

24 88 88

value)

a Fluorescently tagged cells were analyzed in a linear scale because few mononuclear cells were expected to be infected and the amount of antigen per cell was also expected to be low, yielding a low fluorescence intensity. Ag,

Antigen.

b Mean y values were obtained as described in Table 1, footnote b. C Specific activity = (mean y value of positive cells - mean y value of negative cells/mean y value of positive cells) x 100. d The high reactivity of these mononuclear cells with CMV antibody may be due to infection with CMV.

FLOW CYTOMETRIC ANALYSIS OF HIV-INFECTED PBMC

VOL. 28, 1990

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FIG. 3. Correlation of the percentage of p24 antigen (Ag)-positive cells with the total number of T4 cells. The percentage of p24 antigen-positive mononuclear cells was plotted against the total number of T4 cells. CDC groups Il (FI), III (+), and IV (O) are indicated. Note that patients in group IV had few T4 cells and significant percentages of p24 antigen-positive mononuclear cells. Patients in CDC group III had fewer p24 antigen-positive cells and more T4 cells, whereas for patients in group Il there was no inverse relationship between p24 antigen-positive cells and T4 cell count.

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may be a useful predictor of disease progression. When the data included the results of analysis of mononuclear cells obtained from an additional 95 HIV-seropositive patients who were on AZT therapy, the mean percentage of p24 antigen-positive cells obtained from patients in group IV dropped from 10.8 to 8.4%, suggesting that AZT lowers the number of p24 antigen-positive cells in the peripheral blood. Our results suggest that between 4 and 25% of PBMCs obtained from HIV-seropositive patients express the p24 HIV antigen. This percentage of HIV antigen-positive cells in peripheral blood is considerably higher than that reported by others (14). However, a recent report using the polymerase chain reaction to detect HIV-infected cells suggests that at least 1% of T4 cells obtained from AIDS patients are infected with HIV (33). Correlation between T4 cell number and the percentage of the p24-positive mononuclear cells in peripheral blood. Recent studies suggest that the T4 cell and cells of the monocytemacrophage lineage are the cells infected by HIV in vivo (15, 16). Figure 3 shows a plot of the percentage of p24 antigenpositive cells versus the number of T4 cells in the peripheral blood of HIV-seropositive individuals. The data clearly

show that patients with the highest percentage of p24 antigen-positive cells had the fewest T4 cells; that is, there was an inverse correlation between the percentage of p24 antigen-positive cells and the number of T4 cells. This inverse correlation between the percentage of p24 antigen-positive cells and the T4 cell number only applied to patients in groups III and IV. There was no correlation with patients in group II. Thus, the percentage of p24 antigen-positive cells obtained from patients in CDC groups III and IV correlates with an established predictor of disease progression. The p24 antigen-positive cell is a large mononuclear cell. The data presented in Fig. 3 suggest that the p24 antigen that was detected in the mononuclear cells from these patients was not in T4 cells or was present in the small number of T4 cells that remained among the mononuclear cells of these patients. To attempt to identify the p24 antigen-positive population of cells, we used the forward light-scattering properties of the flow cytometer that selects for cell size. Cells that do not have at least a diploid DNA content and cell debris were excluded. A clinical sample that had 21% p24 antigen-positive cells was analyzed by forward light scattering by using the following gates: (i) a gate set to include cells of all sizes, (ii) a gate set to include only cells the size of normal lymphocytes, and (iii) a gate set to include only cells

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McSHARRY ET AL.

J. CLIN. MICROBIOL.

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FIG. 4. Separation of p24 antigen (Ag)-positive mononuclear cells on the basis of size by light scattering. By using the light-scattering capabilities of the flow cytometer to set gates on populations of cells of different sizes, the immunofluorescence of the p24 antigen was determined for PBMCs obtained from an HIV-seronegative and an HIV-seropositive individual. (A) Contour map of the p24 antibody-treated PBMC obtained from an HIV-seronegative individual examined by light scattering with a gate set to include all cells. (B) Contour map of the p24 antibody-treated PBMC obtained from an HIV-seropositive individual examined by light scattering as described for panel A. (C) Contour map of the p24 antibody-treated PBMC shown in panel B examined by light scattering with a gate set to exclude cells larger than normal lymphocytes. (D) Contour map of the p24 antibody-treated cells from panel B examined by light scattering with a gate set to include only cells larger than normal lymphocytes. Note that only cells larger than lymphocytes had significant immunofluorescence (D). Cells the size of normal lymphocytes were devoid of p24 antigen (C). PI, Propidium diiodide.

larger than normal lymphocytes (Fig. 4). The data indicated that the p24 antigen-positive cells were larger than normal lymphocytes and that cells the size of normal lymphocytes are devoid of p24 antigen. It is possible that the large p24 antigen-positive cells are activated lymphocytes. However, there are many other possibilities, such as monocytes, NK cells, and megakaryocytes, for this large p24 antigen-positive cell (36). Further studies using panning techniques to separate monocyte-macrophages from lymphocytes (18) and cells doubly labeled for surface antigens and p24 antigen will be necessary to identify the HIV-infected cells detected by flow cytometry. Analysis of p24 antigen status in individual patients over time. During the course of this study, we monitored nine patients over a 6-month period. Two of these patients were in CDC group Il, three were in CDC group III, and four were in CDC group IV. The relevant data for three of these patients are presented in Table 4. Patient 1 was a male homosexual in CDC group Il. During the 6 months under study, his total T4 cell count remained above 400 cells per ,ul and he remained essentially antigen negative. Note that during this time period, the variation within the percentage of p24 antigen-positive cells was always below 4%, which is within our range for antigen-negative or equivocal samples.

TABLE 4. Analysis of p24 antigen-positive cells of individual patients over time Patient

1

2

3

Date blood drawn

CDC group

AZTI

T4 cells/l

os2t4ivantienlls

pstv ei

(mo/day/yr) 7/28/88 8/23/88 10/4/88 11/11/88

Il Il Il Il

N Y N N

614 714 686 992

0.29 2.82 3.07 1.43

7/26/88 8/23/88 10/4/88 11/1/88 12/8/88 3/7/89

III III III III III III

Y Y Y Y Y

151 343 74 163 262 350

2.07 8.57 11.03 3.87 2.09 2.87

7/5/88 8/9/88 10/11/88 10/20/88 11/15/88 12/10/88 2/14/89

IV IV IV IV IV IV IV

40 32 19 19 19 65 12

1.16 4.51 4.59 2.06 10.51 2.87 4.74

a N, Not on AZT; Y, on

Y Y Y Y

Y Y

AZT;-, change in AZT treatment.

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FLOW CYTOMETRIC ANALYSIS OF HIV-INFECTED PBMC

As of 20 June 1989, this patient remained healthy and was not on AZT therapy. Patient 2 was a male homosexual and intravenous drug abuser in CDC group III. He began AZT therapy on 21 July 1988. His percentage of p24 antigenpositive cells increased over the next 3 months and then decreased over the next 5 months to a level that we would consider negative. During this period, his T4 cell number reflected his antigen course, falling when the percentage of p24 antigen-positive cells rose and rising when the percentage of p24 antigen-positive cells fell. AZT treatment eventually caused the percentage of p24 antigen-positive cells to decrease over time after the initial increase. Patient 3 was a male homosexual in CDC group IV. This patient was on AZT when we began our study. Because of complications of therapy, AZT treatment was stopped on 20 October 1988, and his percentage of p24 antigen-positive cells immediately rose. On 10 November 1988, AZT treatment was resumed, and his percentage of p24 antigen-positive cells returned to normal levels. AZT treatment had no effect on the number of T4 cells in this patient, which remained low throughout the period of study. Similar results were obtained with the six other patients monitored in this prospective study. The results of this serial prospective study suggest that this assay has some internal consistency and reflects the clinical status of the patient over time. In addition, these results suggest that this assay may be useful for monitoring patients receiving AZT for the effect of AZT on virus replication as expressed by antigen production. Comparison of p24 antigen assay by flow cytometry with standard p24 antigen assay (ELISA). To determine whether the antigen detection system using flow cytometry described above was as good as the currently available ELISA-based antigen capture assays, we obtained two EDTA-containing tubes of blood from 20 patients attending the AIDS clinics who were known to be in CDC groups Il, III, or IV and to have a significant percentage of p24 antigen-positive mononuclear cells. One tube was sent to the New York State Department of Health for detection of soluble p24 antigen in serum by the standard Dupont antigen capture ELISA, while the other tube was processed for cell-associated p24 antigen detection by flow cytometry. Only one of the samples determined to be p24 antigen positive by flow cytometry was also p24 antigen positive by the antigen capture ELISA. The results of this experiment indicated that simultaneous analysis of serum for soluble p24 antigen by ELISA and of cells for p24 antigen by flow cytometry does not correlate. In the majority (19 of 20) of cases analyzed, clinical samples that were positive for p24 antigen by flow cytometry were negative for p24 antigen by the standard ELISAs. This discrepancy is probably due to the presence of HIV-specific immune complexes present in the serum of these HIVseropositive patients, which interfere with the antigen capture detection assays (23, 24). One published report has demonstrated that immune complexes in the serum obtained from HIV-seropositive patients do interfere with the antigen capture assay (34). We have tested a number of samples of mononuclear cells obtained from patients who are both HIV seropositive and p24 antigen positive by flow cytometric analysis for the presence of HIV proviral DNA by using the polymerase chain reaction. In all cases tested, those samples that were p24 antigen positive by flow cytometry also contained HIV proviral DNA by the polymerase chain reaction (J. Conroy, personal communication). Thus, although we cannot correlate our data with the presence of free p24 antigen by the antigen capture assay, we can show that all of our p24

731

antigen-positive samples that were tested are also positive for HIV provirus. DISCUSSION Using murine monoclonal antibody to the p24 antigen of HIV and FITC-conjugated goat anti-mouse IgG F(ab')2 antibody to identify HIV-infected cells, we have demonstrated that HIV-infected H9 cells and mononuclear cells obtained from the peripheral blood of HIV-seropositive individuals can be detected and quantitated by flow cytometry. The percentage of p24 antigen-positive mononuclear cells in the peripheral blood of HIV-seropositive patients has a significant correlation (P = 0.0001) with the clinical status of the patients as defined by the CDC (5) and an inverse correlation with the total number of T4 cells, another predictor of disease progression in HIV-infected individuals. However, those who are p24 antigen positive by flow cytometric assay are seldom p24 antigen positive by the Dupont HIV antigen capture assay, indicating that the flow cytometry assay and the antigen capture assay do not correlate. When tested, those samples of mononuclear cells that are p24 antigen positive by flow cytometry contain HIV proviral DNA in their PBMC as determined by the polymerase chain reaction. We have tentatively identified the p24 antigenpositive cell in peripheral blood as a large mononuclear cell. Disease progression in AIDS is characterized by a decrease in antibody to the p24 antigen, a decrease in CD4positive T cells, an increase in serum neopterin levels, and an increase in p24 antigenemia (25). It is thought by some that an increase in p24 antigenemia may precede the decline in antibody to p24 antigen (1, 20). However, accurate detection of p24 antigenemia has always been hindered by the presence of immune complexes consisting of antibodies to p24 and p24 antigen. It has been demonstrated that these immune complexes form shortly after antibody to HIV is made, usually 4 to 12 weeks after infection (24). Therefore, over most of the course of the disease, the presence of antigenemia is masked by the presence of immune complexes, and the current mode of detection, antigen capture ELISAs, leads to an underestimate of the amount of antigen present in peripheral blood. In the technique developed for flow cytometry, we have avoided the problem of antibody by removing antibody, soluble antigen, and immune complexes from the sample and focusing our attention on the antigen present in the infected cell. We assume that if there is antigen in the plasma or serum it must have come from some infected cell in the peripheral blood. By using at least 4% p24 antigen-positive mononuclear cells as the criterion for a positive sample, we can demonstrate that approximately 67% of the samples obtained from HIV-seropositive patients attending our AIDS clinics are p24 antigen positive. The majority (16 of 25) of those samples with less than 4% p24-positive cells are in the CDC group II, whereas the majority (21 of 24) of those samples with >4% p24-positive cells are in CDC group IV. The remainder are in CDC group III. We are monitoring a number of patients in groups II, III, and IV who are receiving AZT, and we have demonstrated that this technique can be used to monitor the effect of AZT on antigenemia (Table 4). Further study of an increased number of patients in the different stages of the disease over a longer period of time and the use of other monoclonal antibodies to HIV antigens, either alone or in combination, will be necessary to determine whether this technique can be used to predict the clinical status of patients (22). We have mentioned that the data on antigenermia derived

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from flow cytometry do not agree with the data on antigenemia obtained from antigen capture ELISAs. We have suggested that this discrepancy is due to the presence of immune complexes in the serum of HIV-seropositive patients which interfere with the antigen capture assay (23, 24). In order to test this hypothesis, it is necessary to separate the immune complexes into antigen and antibody and then reassay the antigen free from antibody in the antigen capture assay. This has been performed by others, and the results confirm our assumptions (34). The p24-positive cell obtained from the peripheral blood has been tentatively identified as a large mononuclear cell. There are a number of cells that could be characterized in this manner, including NK cells, activated lymphocytes, monocyte-macrophages, and megakaryocytes. To further characterize this cell, it will be necessary to use FITCconjugated monoclonal antibodies to cell surface markers and phycoerythrin-streptavidin-biotin-labeled monoclonal antibodies to the p24 antigen. By using two-color fluorescence involving FITC-labeled monoclonal antibodies to cell surface markers on the various cells present in PBMC and biotin-labeled monoclonal antibodies to HIV antigens in conjunction with phycoerythrin-strepavidin, it will be possible to identify all of the HIV-positive cells in the peripheral blood of HIV-seropositive individuals by flow cytometry. This technique for detecting antigenemia may be particularly useful for detecting HIV infection in newborn babies who have been delivered to mothers who are HIV seropositive (3, 31). Currently available techniques detect HIV antibody but do not distinguish between maternal antibody and antibody produced by the newborn. In addition, the problem of interference due to the presence of immune complexes exists. The technique described in this communication can determine whether the newborn's blood contains p24 antigen-positive cells, immediately establishing whether the newborn is infected or not. With a modification of the technique used to obtain mononuclear cells described in this communication, only 0.1 ml of blood is required for this analysis, and a definitive answer can be available in 24 h. On the basis of this information, it would be possible to decide whether the child should be treated with antiviral chemotherapy (28). The polymerase chain reaction assay may be an useful adjunct to flow cytometry to determine the presence of latent HIV infection in these newborns who are antigen positive by the flow cytometric assay (19, 26). If both assays were positive, then it would be clear that the child is infected and expressing the HIV genome, whereas if only the polymerase chain reaction assay was positive it would suggest that the newborn is infected but not expressing the antigen in question (p24 or some other HIV antigen) at detectable levels. If neither assay was positive after several attempts over a year, then it can be assumed that the child was not infected at birth. Therefore, we believe that this new technology for detecting HIV-infected cells in clinical specimens may be useful for the rapid diagnosis of HIV infections in children and adults. Although there is an excellent correlation between the data derived from the flow cytometric analysis of p24 antigen-positive PBMC, the total number of T4 cells, and disease progression, it is clear that confirmation of the data derived from flow cytometry must be made by analyzing PBMC for the presence of HIV by other methods, such as polymerase chain reaction and coculture of infected cells with mitogen-activated lymphocytes. If the results of these studies correlate with those of flow cytometry, then the

J. CLIN. MICROBIOL.

relevance of flow cytometry for antigen detection will be determined. ACKNOWLEDGMENTS This work was supported in part by Public Health Service grant CA 41608 from the National Institutes of Health. We are grateful for the excellent technical assistance provided to the project by Ann C. Ogden-McDonough and Evelyn Keller, for statistical analysis provided by Paul Wing and Shelley Landon, for analysis of T-cell subsets by members of the Cellular Immunology Laboratory, and for secretarial support provided by JoAnne D'Annibale. LITERATURE CITED 1. Allain, J. P., Y. Laurian, D. A. Paul, F. Verroust, M. Leuther, C. Gazengel, D. Senn, M. J. Larrieu, and C. Bosser. 1987. Longterm evaluation of HIV antigen and antibodies to p24 and gp4l in patients with hemophilia. N. Engl. J. Med. 317:1114-1121. 2. Barré-Sinoussi, F., J. C. Chermann, F. Rey, M. T. Nugeyre, S. Chamaret, J. Gruest, C. Dauguet, C. Axler-Blin, F. VézinetBrun, C. Rouzioux, W. Rozenbaum, and L. Montagnier. 1983. Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS). Science 220:868-871. 3. Blanche, S., C. Rouzioux, M.-L. G. Moscato, F. Veber, M.-J. Mayaux, C. Jacomet, J. Tricoire, A. Deville, M. Vial, G. Firtion, A. de Crepy, D. Douard, M. Robin, C. Courpotin, N. CiraruVigneron, F. LeDeist, C. Griscelli, and the HIV Infection in Newborn French Collaborative Study Group. 1989. A prospective study of infants born to women seropositive for human immunodeficiency virus type 1. N. Engl. J. Med. 320:16431648. 4. Castro, B. A., C. A. Weiss, L. D. Wiviott, and J. A. Levy. 1988. Optimal conditions for recovery of the human immunodeficiency virus from peripheral blood mononuclear cells. J. Clin. Microbiol. 26:2371-2376. 5. Centers for Disease Control. 1986. CDC classification system for HIV infections. Morbid. Mortal. Weekly Rep. 35:334-339. 6. Cory, J. M., B. M. Ohlsson-Wilhelm, E. J. Brock, N. A. Sheaffer, M. E. Steck, M. E. Eyster, and F. Rapp. 1987. Detection of human immunodeficiency virus-infected lymphoid cells at low frequency by flow cytometry. J. Immunol. Methods 105:71-78. 7. Cory, J. M., B. M. Ohlsson-Wilhelm, M. E. Steck, M. D. Smithgall, S. V. Rozday, M. E. Eyster, and F. Rapp. 1989. Kinetics of infected cell appearance as a determinant of the number of human immunodeficiency virus 1 infectious units. AIDS Res. Hum. Retroviruses 5:97-106. 8. Creemers, P. C., M. O'Shaughnessy, and W. J. Boyko. 1988. Analysis of absolute T helper cell number and cellular immune defects in HIV antibody positive and negative homosexual men. AIDS Res. Hum. Retroviruses 4:269-278. 9. Crocchiolo, P. R., A. Lizioli, G. Bedarida, and M. P. Panzeri. 1988. CD4+: neopterin ratio significantly improves correlation with the Walter Reed staging system if compared with CD4+ and neopterin considered separately. AIDS 2:481-482. 10. DeWolf, F., J. M. A. Lange, J. T. M. Houweling, R. A. Coutinho, P. T. Schellekens, J. van der Noordaa, and J. Goudsmit. 1988. Numbers of CD4+ cells and the levels of core antigens of and antibodies to the human immunodeficiency virus as predictors of AIDS among seropositive homosexual men. J. Infect. Dis. 158:615-622. 11. DeWolf, F., M. Roos, J. M. A. Lange, J. T. M. Houweling, R. A. Coutinho, J. van der Noordaa, P. T. Schellekens, and J. Goudsmit. 1988. Decline in CD4+ cell numbers reflects increase in HIV-1 replication. AIDS Res. Hum. Retroviruses 4:433-440. 12. Elmendorf, S., J. McSharry, J. Laffin, D. Fogleman, and J. Lehman. 1988. Detection of an early cytomegalovirus antigen with two-color quantitative flow cytometry. Cytometry 9:254260. 13. Gallo, R., P. S. Sarin, E. P. Gelmann, M. Robert-Guroff, E. Richardson, V. S. Kalyanaraman, D. Mann, G. D. Sidhu, R. E.

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Detection and quantitation of human immunodeficiency virus-infected peripheral blood mononuclear cells by flow cytometry.

A flow cytometric assay has been developed to detect and quantitate human immunodeficiency virus (HIV)-infected peripheral blood mononuclear cells obt...
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