Vol. 28, No. 4
JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1990, p. 734-737 0095-1137/90/040734-04$02.00/0 Copyright © 1990, American Society for Microbiology
Microculture Assay for Isolation of Human Immunodeficiency Virus Type 1 and for Titration of Infected Peripheral Blood Mononuclear Cells DIMITRE H. DIMITROV,t JOSEPH L. MELNICK, AND F. BLAINE HOLLINGER* Division of Molecular Virology, Baylor College of Medicine, Houston, Texas 77030 Received 18 September 1989/Accepted 4 January 1990
To define the optimal conditions for human immunodeficiency virus (HIV) detection in microcultures, experiments were conducted with different ratios of patient and donor peripheral blood mononuclear cells (PBMCs). Donor/patient PBMC ratios ranged from 1:1 to 1:125. Optimal results were obtained when 1,500,000 donor cells were cocultured with equal or smaller quantities of patient PBMCs. Thus, virologie endpoints could be achieved by diluting patient cells. Smaller numbers of donor cells, with or without larger numbers of patient cells, resulted in lower rates of HIV isolation. Similarly, the direct stimulation of patient PBMCs with phytohemagglutinin without the addition of normal donor cells lowered the sensitivity of the assay significantly. We suggest that a microculture procedure using a fixed quantity of donor cells with different dilutions of patient cells may be useful for monitoring changing HIV levels during antiviral therapy.
tories, Detroit, Mich.) per ml, 5% interleukin-2 (Cellular Products, Buffalo, N.Y.) and antibiotics (100 U of penicillin per ml and 100 ,ug of streptomycin per ml). The PHAP-stimulated donor cells were incubated at 37°C for 1 to 3 days before cocultivation (6, 14). Standard HIV coculture procedure. Our standard coculturing procedure required 10 x 106 patient PBMCs to be cultured with an equal number of PHA-P-stimulated normal donor PBMCs in 10 ml of the medium described above (but without PHA-P) in a 25-cm2 tissue culture flask. Every 3 to 4 days, 5 ml of medium was removed for HIV p24 antigen analysis by enzyme immunoassay (DuPont Co., Wilmington, Del.) and replaced with 5 ml of fresh medium. In addition, PHA-P-stimulated PBMCs (107 cells) from a known seronegative donor were added to the cocultures at weekly intervals. The p24 antigen enzyme immunoassay was used, because it has been found to be 100 times more sensitive than the standard reverse transcriptase assay (5, 16). A "days-tofirst-positive" outcome was determined for the first culture specimen that yielded a value of at least 30 pg of p24 antigen per ml when the next sample was out of range or showed a fourfold or greater rise in HIV p24 antigen concentration. To estimate the concentration of HIV p24 antigen in the supernatant, a standard curve was prepared using a known concentration of HIV p24 antigen in a viral lysate. A sample was diluted until the enzyme immunoassay optical density fell within the range of the standard curve. The value obtained from the standard curve was multiplied by the reciprocal of the dilution to obtain the estimated concentration of the undiluted specimen. Microculture procedure. Eight participants volunteered for the initial pilot experiments. They included three individuals classified in Centers for Disease Control (CDC) group 11, one person who was in CDC group III, and four subjects who were in CDC group IV (3, 4). Initially, serial fivefold dilutions of cells in culture were carried out while a donor-topatient cell ratio of 1:1 was maintained. The initial total cell count was 5 x 106 cells (2.5 x 106 patient PBMCs plus 2.5 x 106 PHA-P-stimulated donor PBMCs) suspended in 2 ml of standard culture medium. Fivefold dilutions of these cells were prepared in volumes of 2 ml and added to appropriate
The major tests used currently for the diagnosis of acquired immune deficiency syndrome, i.e., anti-human immunodeficiency virus (HIV) enzyme immunoassay, Western blot (immunoblot), and immunofluorescence, determine the presence of antibody directed against the virus. Confirmed positive results indicate prior exposure to the virus (10, 18). However, the definitive test for an active HIV infection is the recovery of HIV or provirus DNA from the infected individual (1, 7, 11, 12, 17, 19). The methods for HIV cultivation have improved steadily (2, 9), with coculture being the most sensitive for the isolation of the virus from patients. For determination of quantitative changes in virus concentration after antiviral therapy, endpoint virologic assays are essential. Some studies (20) have already been done with limiting dilution cultures of peripheral blood mononuclear cells (PBMCs). We report here on experiments to help to define the optimal conditions for isolation and quantification of HIV type 1 in microculture assays. MATERIALS AND METHODS Patient PBMCs. Coded samples from 16 HIV-infected patients were studied. Blood samples (7 to 10 ml) drawn from each patient were processed on the same day. The plasma was separated by centrifugation at 200 x g for 10 min. The remaining cell fraction was diluted 1:2 with phosphate-buffered saline, and 4 parts of the diluted sample were layered over 3 parts of lymphocyte separation medium. This was followed by centrifugation at room temperature for 30 min at 400 x g. The PBMCs were removed and washed twice with phosphate-buffered saline. Donor PBMCs. Donor PBMCs were isolated as described above from healthy persons who were negative for HIV antibody. The donor PBMCs were suspended (2 x 106 PBMCs per ml) in a stimulation medium containing RPMI 1640 with glutamine, 20% heat-inactivated fetal bovine serum, 3 ,ug of phytohemagglutinin P (PHA-P; Difco LaboraCorresponding author. t Present address: Department of Virology, Institute of Infectious and Parasitic Diseases, Sofia, Bulgaria. *
734
HIV TYPE 1 MICROCULTURE ASSAY
VOL. 28, 1990 1 x 16 P
2 x 105 P
1 x 106 D
1
1 x 106 P
2 x 105 P
4 x 104 P
8 x 103 P
2 x 105 D
2 x 105 D
2 x 105 D
2 x 105 D
x 106 D
4 x 104 P 1
x 106 D
8x 103 P 1
x 106 D
1 x 106 P
2 x 105 P
4 x 104 P
8sx 103 P
4 x 104 D
4 x 104 D
4 x 104 D
4 x 104 D
1 x 106 P
2 x 105 P
4 x 104 P
8 x 103 P
8 x 103 D
8 x 103 D
8 x
103 D
8 x 103 D
FIG. 1. Box titration of patient (P) and donor (D) cells.
wells in a 24-well microculture plate (Corning Glass Works, Corning, N.Y.). On day 7 of coculturing, 0.5 ml of medium was removed for HIV p24 antigen analysis and replaced with 0.5 ml of fresh medium. The culture was terminated on day 14, and the medium was tested again for HIV p24 antigen concentration. As a control, the patient cells were cultured by the standard 10-ml coculture technique described above. A positive reaction was defined as any value of at least 30 pg of p24 antigen per ml. To ascertain the optimal mixture of patient and donor cells, a box titration was designed, employing PBMCs from the same subjects used in the pilot study. For this experiment, 16 combinations of patient and donor PBMCs were examined, with PBMCs at four different cell levels: 1 x 106, 2 x 105, 4 x 104, and 8 x 103 (Fig. 1). The total culture volume was 2 ml per well. In this set of experiments, fresh medium was not added on day 7, nor were the cultures tested for p24 antigen on that day. Instead, the cultures were terminated on day 14 and the medium was reexamined for HIV p24 antigen concentration. A final set of experiments was designed to determine endpoint dilutions of PBMCs obtained from HIV-infected patients. Serial twofold dilutions of PBMCs were prepared, beginning at a cell count of 1.5 x 106 and ending at a cell count of 3 x 103. Each quantity of patient cells was cocultured with 1.5 x 106 seronegative donor PBMCs in 24-well microculture plates. The cells in each well were suspended in 2 ml of medium. The cultures were terminated on day 14, and the medium was tested for HIV p24 antigen concentration. RESULTS Decreasing quantities of PBMCs (2.5 x 106 to 4 x 103) from eight HIV-infected patients were cocultured in 24well microculture plates with equivalent quantities of donor PBMCs. While the standard cocultures were maintained for 31 days, the microcultures were terminated on day 14. Each of the eight patients was positive by the standard 10-ml coculturing procedure; cocultures from seven of the eight patients became positive by day 14. Samples from six of the eight patients also were positive by day 14 in the microculture procedure at the highest PBMC level (2.5 x 106 cells). All four patients classified in CDC group IV (3, 4) were positive by the microculture technique, and isolation of HIV
735
was possible from as few as 4,000 PBMCs collected from one of these patients. In contrast, the lone individual in CDC group III and one of the three subjects in group II were negative. Samples from the same patients were tested in 24-well microculture plates with different combinations of donor and patient cells (Fig. 1). Results at 14 days from microcultures were compared to those obtained at 31 days by the standard coculture method (data not shown). By the standard method, seven of eight samples were positive by day 7, while one sample required 25 days in coculture before becoming positive. In the microculture procedure, seven of eight samples were positive when 106 donor cells and 106 patient cells were used. At this level of donor cells, culture supernatants continued to register a positive response, even at the lowest level of patient PBMCs evaluated, i.e., 8,000 cells. In the group of patient cell samples cocultured with 2 x 105 donor cells, HIV was isolated from four of the eight patient samples at the highest patient PBMC level cultured (106 cells), whereas only two of the eight patient samples were positive when 8,000 patient cells were used. No correlation with CDC classification was observed. Except for a cell sample from one individual, isolation was not successful at any patient cell level when patient cells were cocultured for 14 days with fewer than 200,000 donor cells. On the basis of these results, a final experiment was designed. In this experiment, twofold dilutions of patient PBMCs, from 1.5 x 106 to 3 x 103 cells, were cocultured with a constant quantity of donor PBMCs, i.e., 1.5 x 106 cells (Table 1). Coded samples from 10 persons were tested (two controls negative for HIV antibodies and eight patients positive for HIV antibodies). HIV was not isolated from either of the control samples that were negative for HIV antibody. All eight patients already known to be positive for HIV antibody were positive for virus when their specimens were tested by the standard coculture procedure, and seven also were positive by day 14 in the microculture assay. The lowest number of cells yielding a positive reaction in the microculture procedure could be ascertained for each of the seven positive samples. In one sample, 750,000 cells were required for a positive reaction, whereas only 6,000 cells were required in another sample. In these eight patients, the median number of cells required for a positive response was 23,000. No correlation was observed between the absolute numbers of CD4 and CD8 cells present in a specimen, or their ratios, and the level at which the virus could be detected. DISCUSSION Results obtained in this study indicate the optimum conditions needed for isolating HIV type 1 within a 14-day interval in microculture wells and for performing endpoint titrations of PBMCs. The endpoint dilution method should be considered whenever a change in the level of HIV concentration is anticipated, e.g.,.following antiviral therapy. In a few instances, a smaller number of patient PBMCs yielded more HIV p24 antigen in the supernatant than a larger number yielded (data not shown). A similar observation has been reported previously (20), suggesting that dilution of suppressor cells may enhance HIV replication. In the study of Ulrich et al. (20), using a macroculture method, HIV was isolated from dilutions of patient cells in the range of 102 to 106; these results were similar to our own. Such a variety of levels of viral concentration in infected hosts is typical of animal lentiviral infections (8, 15).
736
J. CLIN. MICROBIOL.
DIMITROV ET AL.
It is evident that microcultures which are terminated at day 14 are not as sensitive as those continued for 31 days. For example, although our studies indicate that less than one infected cell in a 4,000-cell sample can be detected within 14 days in HIV-infected persons, we have recently determined that even one infected cell per 100 PBMCs can be observed by extending the culture period to 21 days or longer (data not shown). Nevertheless, a 14-day period was selected for these studies as a compromise between optimal sensitivity and viability of the assay as a practical and economical procedure for determining virologic endpoints. Working with the same patients as in the pilot studies, we also examined whether direct stimulation of patient PBMCs with PHA-P would enhance the isolation of HIV in microculture without the addition of normal donor PBMCs. Under the standard culture conditions, only three patients were positive (compared with eight patients positive by coculture). With the microcultures, only one of the eight patients was positive in the absence of donor PBMCs. Thus, lymphocytes of patients with HIV infection seem to have a decreased responsiveness to PHA-P stimulation alone, and this technique, if no donor PBMCs are used, cannot be regarded as a reliable method for isolating HIV, either in microculture or in macroculture (13). Before this microculture technique, which uses coculture with donor PBMCs, can be employed to yield virologic endpoints for monitoring of antiviral therapy, reproducibility over a finite interval in untreated patients must be established with both fresh and cryopreserved samples. Should cryopreserved cells yield consistent, stable levels of infection, batch testing at the completion of a study will become feasible. This possibility is currently being evaluated.
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ACKNOWLEDGMENT This work was supported by Public Health Service grant N01AI-82517 from the National Institute of Allergy and Infectious Diseases.
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LITERATURE CITED 1. Barre-Sinoussi, F., J. C. Chermann, F. Rey, M. T. Nugeyre, S. Chamaret, J. Gruest, C. Dauguet, C. Axler-Blin, F. VezinetBrun, 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. 2. Castro, B. A., C. D. 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. 3. Centers for Disease Control. 1986. Classification system for human T-lymphotropic virus type III/lymphadenopathy-associated virus infections. Morbid. Mortal. Weekly Rep. 35:334-339. 4. Centers for Disease Control. 1987. Revision of the CDC surveillance case definition for acquired immunodeficiency syndrome. Morbid. Mortal. Weekly Rep. 36(Suppl. 1S):lS-15S. 5. Feorino, P., B. Forrester, C. Schable, D. Warfield, and G. Schochetman. 1987. Comparison of antigen assay and reverse transcriptase assay for detecting human immunodeficiency virus in culture. J. Clin. Microbiol. 25:2344-2346. 6. Folks, J. H., J. Kelly, S. Benn, A. Kinter, J. Justement, J. Gold, R. Redfield, K. W. Sell, and A. S. Fauci. 1986. Susceptibility of normal human lymphocytes to infection with HTLV-III/LAV. J. Immunol. 136:4049-4053. 7. Gallo, R. C., S. Z. Salahuddin, M. Popovic, G. M. Shearer, M. Kaplan, B. F. Haynes, T. J. Palker, R. Redfield, J. Oleske, B. Safai, G. White, P. Foster, and P. D. Markham. 1984. Frequent detection and isolation of cytopathic retroviruses (HTLV-III) from patients with AIDS and at risk for AIDS. Science 224: 500-503.
HIV TYPE 1 MICROCULTURE ASSAY
VOL. 28, 1990
8. Haase, A. T. 1986. Pathogenesis of lentivirus infections. Nature (London) 322:130-136. 9. Jackson, J. B., R. W. Coombs, K. Sannerud, F. S. Rhame, and H. H. Balfour, Jr. 1988. Rapid and sensitive viral culture method for human immunodeficiency virus type 1. J. Clin. Microbiol. 26:1416-1418. 10. Kaminsky, L. S., T. McHugh, D. P. Stites, P. Volberding, G. Henle, W. Henle, and J. A. Levy. 1985. High prevalence of antibodies to acquired immune deficiency syndrome (AIDS)associated retrovirus (ARV) in AIDS and related conditions but not in other disease states. Proc. Natl. Acad. Sci. USA 82: 5535-5539. 11. Kwok, S., D. H. Mack, K. B. Mullis, B. Poiesz, G. Ehrlich, D. Blair, A. Friedman-Kien, and J. J. Sninsky. 1987. Identification of human immunodeficiency virus sequences by using in vitro enzymatic amplification and oligomer cleavage detection. J. Virol. 61:1690-1694. 12. Levy, J. A., A. D. Hoffman, S. M. Kramer, J. A. Landis, J. M. Shimabukuro, and L. S. Oshiro. 1984. Isolation of lymphocytopathic retroviruses from San Francisco patients with AIDS. Science 225:840-842. 13. Levy, J. A., and J. Shimabukuro. 1985. Recovery of AIDSassociated retroviruses from patients with AIDS or AIDSrelated conditions and from clinically healthy individuals. J. Infect. Dis. 152:734-738. 14. McDougal, J. S., A. Mawle, S. P. Cort, J. K. A. Nicholson, G. D.
15.
16. 17.
18. 19.
20.
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Cross, J. A. Scheppler-Campbell, D. Hicks, and J. Sligh. 1985. Cellular tropism of the human retrovirus HTLV-IIIILAV. I. Role of T cell activation and expression of the T4 antigen. J. Immunol. 135:3151-3162. Nabel, G., and D. Baltimore. 1987. An inducible transcription factor activates expression of human immunodeficiency virus in T cells. Nature (London) 326:711-713. Newell, A. L., M. Malkovsky, D. Orr, D. Taylor-Robinson, and A. G. Dalgleish. 1987. Antigen test versus reverse transcriptase assay for detecting HIV. Lancet ii:1146-1147. Ou, C. Y., S. Kwok, S. W. Mitchell, D. H. Mack, J. J. Sninsky, J. W. Krebs, P. Feorino, D. Warfield, and G. Schochetman. 1988. DNA amplification for direct detection of HIV-1 in DNA of peripheral blood mononuclear cells. Science 239:295-297. Pan, L. Z., C. Cheng-Mayer, and J. A. Levy. 1987. Patterns of antibody response in individuals infected with the human immunodeficiency virus. J. Infect. Dis. 155:626-632. Schnittman, S. M., M. C. Psallidopoulos, H. C. Lane, L. Thompson, M. Baseler, F. Massari, C. H. Fox, N. P. Salzman, and A. S. Fauci. 1989. The reservoir for HIV-1 in human peripheral blood is a T cell that maintains expression of CD4. Science 245:305-308. Ulrich, P. P., M. P. Busch, T. El-Beik, J. Shiota, J. Venari, K. Shriver, and G. N. Vyas. 1988. Assessment of human immunodeficiency virus in cocultures of peripheral blood mononuclear cells from healthy seropositive subjects. J. Med. Virol. 25:1-10.
JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1990, p. 734-737 0095-1137/90/040734-04$02.00/0 Copyright © 1990, American Society for Microbiology
Microculture Assay for Isolation of Human Immunodeficiency Virus Type 1 and for Titration of Infected Peripheral Blood Mononuclear Cells DIMITRE H. DIMITROV,t JOSEPH L. MELNICK, AND F. BLAINE HOLLINGER* Division of Molecular Virology, Baylor College of Medicine, Houston, Texas 77030 Received 18 September 1989/Accepted 4 January 1990
To define the optimal conditions for human immunodeficiency virus (HIV) detection in microcultures, experiments were conducted with different ratios of patient and donor peripheral blood mononuclear cells (PBMCs). Donor/patient PBMC ratios ranged from 1:1 to 1:125. Optimal results were obtained when 1,500,000 donor cells were cocultured with equal or smaller quantities of patient PBMCs. Thus, virologie endpoints could be achieved by diluting patient cells. Smaller numbers of donor cells, with or without larger numbers of patient cells, resulted in lower rates of HIV isolation. Similarly, the direct stimulation of patient PBMCs with phytohemagglutinin without the addition of normal donor cells lowered the sensitivity of the assay significantly. We suggest that a microculture procedure using a fixed quantity of donor cells with different dilutions of patient cells may be useful for monitoring changing HIV levels during antiviral therapy.
tories, Detroit, Mich.) per ml, 5% interleukin-2 (Cellular Products, Buffalo, N.Y.) and antibiotics (100 U of penicillin per ml and 100 ,ug of streptomycin per ml). The PHAP-stimulated donor cells were incubated at 37°C for 1 to 3 days before cocultivation (6, 14). Standard HIV coculture procedure. Our standard coculturing procedure required 10 x 106 patient PBMCs to be cultured with an equal number of PHA-P-stimulated normal donor PBMCs in 10 ml of the medium described above (but without PHA-P) in a 25-cm2 tissue culture flask. Every 3 to 4 days, 5 ml of medium was removed for HIV p24 antigen analysis by enzyme immunoassay (DuPont Co., Wilmington, Del.) and replaced with 5 ml of fresh medium. In addition, PHA-P-stimulated PBMCs (107 cells) from a known seronegative donor were added to the cocultures at weekly intervals. The p24 antigen enzyme immunoassay was used, because it has been found to be 100 times more sensitive than the standard reverse transcriptase assay (5, 16). A "days-tofirst-positive" outcome was determined for the first culture specimen that yielded a value of at least 30 pg of p24 antigen per ml when the next sample was out of range or showed a fourfold or greater rise in HIV p24 antigen concentration. To estimate the concentration of HIV p24 antigen in the supernatant, a standard curve was prepared using a known concentration of HIV p24 antigen in a viral lysate. A sample was diluted until the enzyme immunoassay optical density fell within the range of the standard curve. The value obtained from the standard curve was multiplied by the reciprocal of the dilution to obtain the estimated concentration of the undiluted specimen. Microculture procedure. Eight participants volunteered for the initial pilot experiments. They included three individuals classified in Centers for Disease Control (CDC) group 11, one person who was in CDC group III, and four subjects who were in CDC group IV (3, 4). Initially, serial fivefold dilutions of cells in culture were carried out while a donor-topatient cell ratio of 1:1 was maintained. The initial total cell count was 5 x 106 cells (2.5 x 106 patient PBMCs plus 2.5 x 106 PHA-P-stimulated donor PBMCs) suspended in 2 ml of standard culture medium. Fivefold dilutions of these cells were prepared in volumes of 2 ml and added to appropriate
The major tests used currently for the diagnosis of acquired immune deficiency syndrome, i.e., anti-human immunodeficiency virus (HIV) enzyme immunoassay, Western blot (immunoblot), and immunofluorescence, determine the presence of antibody directed against the virus. Confirmed positive results indicate prior exposure to the virus (10, 18). However, the definitive test for an active HIV infection is the recovery of HIV or provirus DNA from the infected individual (1, 7, 11, 12, 17, 19). The methods for HIV cultivation have improved steadily (2, 9), with coculture being the most sensitive for the isolation of the virus from patients. For determination of quantitative changes in virus concentration after antiviral therapy, endpoint virologic assays are essential. Some studies (20) have already been done with limiting dilution cultures of peripheral blood mononuclear cells (PBMCs). We report here on experiments to help to define the optimal conditions for isolation and quantification of HIV type 1 in microculture assays. MATERIALS AND METHODS Patient PBMCs. Coded samples from 16 HIV-infected patients were studied. Blood samples (7 to 10 ml) drawn from each patient were processed on the same day. The plasma was separated by centrifugation at 200 x g for 10 min. The remaining cell fraction was diluted 1:2 with phosphate-buffered saline, and 4 parts of the diluted sample were layered over 3 parts of lymphocyte separation medium. This was followed by centrifugation at room temperature for 30 min at 400 x g. The PBMCs were removed and washed twice with phosphate-buffered saline. Donor PBMCs. Donor PBMCs were isolated as described above from healthy persons who were negative for HIV antibody. The donor PBMCs were suspended (2 x 106 PBMCs per ml) in a stimulation medium containing RPMI 1640 with glutamine, 20% heat-inactivated fetal bovine serum, 3 ,ug of phytohemagglutinin P (PHA-P; Difco LaboraCorresponding author. t Present address: Department of Virology, Institute of Infectious and Parasitic Diseases, Sofia, Bulgaria. *
734
HIV TYPE 1 MICROCULTURE ASSAY
VOL. 28, 1990 1 x 16 P
2 x 105 P
1 x 106 D
1
1 x 106 P
2 x 105 P
4 x 104 P
8 x 103 P
2 x 105 D
2 x 105 D
2 x 105 D
2 x 105 D
x 106 D
4 x 104 P 1
x 106 D
8x 103 P 1
x 106 D
1 x 106 P
2 x 105 P
4 x 104 P
8sx 103 P
4 x 104 D
4 x 104 D
4 x 104 D
4 x 104 D
1 x 106 P
2 x 105 P
4 x 104 P
8 x 103 P
8 x 103 D
8 x 103 D
8 x
103 D
8 x 103 D
FIG. 1. Box titration of patient (P) and donor (D) cells.
wells in a 24-well microculture plate (Corning Glass Works, Corning, N.Y.). On day 7 of coculturing, 0.5 ml of medium was removed for HIV p24 antigen analysis and replaced with 0.5 ml of fresh medium. The culture was terminated on day 14, and the medium was tested again for HIV p24 antigen concentration. As a control, the patient cells were cultured by the standard 10-ml coculture technique described above. A positive reaction was defined as any value of at least 30 pg of p24 antigen per ml. To ascertain the optimal mixture of patient and donor cells, a box titration was designed, employing PBMCs from the same subjects used in the pilot study. For this experiment, 16 combinations of patient and donor PBMCs were examined, with PBMCs at four different cell levels: 1 x 106, 2 x 105, 4 x 104, and 8 x 103 (Fig. 1). The total culture volume was 2 ml per well. In this set of experiments, fresh medium was not added on day 7, nor were the cultures tested for p24 antigen on that day. Instead, the cultures were terminated on day 14 and the medium was reexamined for HIV p24 antigen concentration. A final set of experiments was designed to determine endpoint dilutions of PBMCs obtained from HIV-infected patients. Serial twofold dilutions of PBMCs were prepared, beginning at a cell count of 1.5 x 106 and ending at a cell count of 3 x 103. Each quantity of patient cells was cocultured with 1.5 x 106 seronegative donor PBMCs in 24-well microculture plates. The cells in each well were suspended in 2 ml of medium. The cultures were terminated on day 14, and the medium was tested for HIV p24 antigen concentration. RESULTS Decreasing quantities of PBMCs (2.5 x 106 to 4 x 103) from eight HIV-infected patients were cocultured in 24well microculture plates with equivalent quantities of donor PBMCs. While the standard cocultures were maintained for 31 days, the microcultures were terminated on day 14. Each of the eight patients was positive by the standard 10-ml coculturing procedure; cocultures from seven of the eight patients became positive by day 14. Samples from six of the eight patients also were positive by day 14 in the microculture procedure at the highest PBMC level (2.5 x 106 cells). All four patients classified in CDC group IV (3, 4) were positive by the microculture technique, and isolation of HIV
735
was possible from as few as 4,000 PBMCs collected from one of these patients. In contrast, the lone individual in CDC group III and one of the three subjects in group II were negative. Samples from the same patients were tested in 24-well microculture plates with different combinations of donor and patient cells (Fig. 1). Results at 14 days from microcultures were compared to those obtained at 31 days by the standard coculture method (data not shown). By the standard method, seven of eight samples were positive by day 7, while one sample required 25 days in coculture before becoming positive. In the microculture procedure, seven of eight samples were positive when 106 donor cells and 106 patient cells were used. At this level of donor cells, culture supernatants continued to register a positive response, even at the lowest level of patient PBMCs evaluated, i.e., 8,000 cells. In the group of patient cell samples cocultured with 2 x 105 donor cells, HIV was isolated from four of the eight patient samples at the highest patient PBMC level cultured (106 cells), whereas only two of the eight patient samples were positive when 8,000 patient cells were used. No correlation with CDC classification was observed. Except for a cell sample from one individual, isolation was not successful at any patient cell level when patient cells were cocultured for 14 days with fewer than 200,000 donor cells. On the basis of these results, a final experiment was designed. In this experiment, twofold dilutions of patient PBMCs, from 1.5 x 106 to 3 x 103 cells, were cocultured with a constant quantity of donor PBMCs, i.e., 1.5 x 106 cells (Table 1). Coded samples from 10 persons were tested (two controls negative for HIV antibodies and eight patients positive for HIV antibodies). HIV was not isolated from either of the control samples that were negative for HIV antibody. All eight patients already known to be positive for HIV antibody were positive for virus when their specimens were tested by the standard coculture procedure, and seven also were positive by day 14 in the microculture assay. The lowest number of cells yielding a positive reaction in the microculture procedure could be ascertained for each of the seven positive samples. In one sample, 750,000 cells were required for a positive reaction, whereas only 6,000 cells were required in another sample. In these eight patients, the median number of cells required for a positive response was 23,000. No correlation was observed between the absolute numbers of CD4 and CD8 cells present in a specimen, or their ratios, and the level at which the virus could be detected. DISCUSSION Results obtained in this study indicate the optimum conditions needed for isolating HIV type 1 within a 14-day interval in microculture wells and for performing endpoint titrations of PBMCs. The endpoint dilution method should be considered whenever a change in the level of HIV concentration is anticipated, e.g.,.following antiviral therapy. In a few instances, a smaller number of patient PBMCs yielded more HIV p24 antigen in the supernatant than a larger number yielded (data not shown). A similar observation has been reported previously (20), suggesting that dilution of suppressor cells may enhance HIV replication. In the study of Ulrich et al. (20), using a macroculture method, HIV was isolated from dilutions of patient cells in the range of 102 to 106; these results were similar to our own. Such a variety of levels of viral concentration in infected hosts is typical of animal lentiviral infections (8, 15).
736
J. CLIN. MICROBIOL.
DIMITROV ET AL.
It is evident that microcultures which are terminated at day 14 are not as sensitive as those continued for 31 days. For example, although our studies indicate that less than one infected cell in a 4,000-cell sample can be detected within 14 days in HIV-infected persons, we have recently determined that even one infected cell per 100 PBMCs can be observed by extending the culture period to 21 days or longer (data not shown). Nevertheless, a 14-day period was selected for these studies as a compromise between optimal sensitivity and viability of the assay as a practical and economical procedure for determining virologic endpoints. Working with the same patients as in the pilot studies, we also examined whether direct stimulation of patient PBMCs with PHA-P would enhance the isolation of HIV in microculture without the addition of normal donor PBMCs. Under the standard culture conditions, only three patients were positive (compared with eight patients positive by coculture). With the microcultures, only one of the eight patients was positive in the absence of donor PBMCs. Thus, lymphocytes of patients with HIV infection seem to have a decreased responsiveness to PHA-P stimulation alone, and this technique, if no donor PBMCs are used, cannot be regarded as a reliable method for isolating HIV, either in microculture or in macroculture (13). Before this microculture technique, which uses coculture with donor PBMCs, can be employed to yield virologic endpoints for monitoring of antiviral therapy, reproducibility over a finite interval in untreated patients must be established with both fresh and cryopreserved samples. Should cryopreserved cells yield consistent, stable levels of infection, batch testing at the completion of a study will become feasible. This possibility is currently being evaluated.
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ACKNOWLEDGMENT This work was supported by Public Health Service grant N01AI-82517 from the National Institute of Allergy and Infectious Diseases.
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