Journal of Medical Virology 251-10 (1988)

Assessment of Human Immunodeficiency Virus Expression in Cocultures of Peripheral Blood Mononuclear Cells From Healthy Seropositive Subjects Paul P. Ulrich, Michael P. Busch, Tarek El-Beik, Jean Shiota, Joann Vennari, Kathy Shriver, and Girish N. Vyas

Department of Laboratory Medicine, University of California, San Francisco, California (P. P. U., T. E. - B., J. V., G. N. V.); Irwin Memorial Blood Bank, San Francisco, California (M. P. B., J. S.); Genetic Systems, Inc., Seattle, Washington (K. S.)

We have evaluated a number of methodological variables effecting the expression of human immunodeficiency virus (HIV) in cultured peripheral blood mononuclear cells (PBMCs) of 93 healthy anti-HIV-positive and 72 healthy seronegative subjects. For optimal HIV recovery, PBMCs had to be freshly separated from whole blood. Short-term freezing of purified PBMCs was practically advantageous and actually resulted in more rapid virus recovery. The minimal number of PBMCs necessary for virus expression was determined by dilutional cultures and varied from 10' to lo6 cells. HIV expression was demonstrated initially at the cellular level by immunocytochemi- cal detection of HIV core and envelope proteins using a mixture of monoclonal antibodies, subsequently confirmed by detection of viral antigens and reverse transcriptase (RT) in the culture supernatants. HIV recovery was not improved following induction with 5-iodo-2'-deoxyuridine (IUDR) and only marginally improved following depletion of the CD8'-suppressor cell population in the PBMC specimens. The overall frequency of HIV detection in cultures was 84% in healthy seropositive subjects, whereas none of the PBMCs from 72 seronegative persons yielded HIV expression.

Key words: virus cultivation, lymphocytes, AIDS, HIV cultivation, anti-HIV testing

Accepted for publication October 30, 1987.

Address reprint requests to Girish N. Vyas, PhD, University of California, San Francisco, CA 94143-0100.

0 1988 Alan R. Liss. Inc.

2 Ulrich et al.

INTRODUCTION

Exposure to human immunodeficiency virus (HIV), the causative agent of the acquired immune deficiency syndrome (AIDS), is serologically demonstrated by enzyme immunoassays for anti-HIV and is confirmed by Western blot analyses [Institute of Medicine Committee Report, 19861. Isolation of HIV from human peripheral blood mononuclear cells (PBMCs) provides definitive evidence of persistent infection and can be useful in assessing disease prognosis and in monitoring the efficacy of therapy [Levy and Shimabukuro, 1985; McDougal et al., 1985; Mitsuya and Broder, 19871. Variation in the efficiency of virus isolation from seropositive individuals has ranged from 50% to over 8096, with higher rates generally reported from symptomatic patients than from asymptomatic, healthy individuals [Levy and Shimabukuro, 1985; Salahuddin et al., 19851.

The methodology of HIV cultivation has improved significantly over the past several years in terms of both practicality and viral yield. For example, investigators have shown that cocultivation of a patient’s PBMCs with susceptible, normal PBMCs results in more frequent and rapid virus recovery than cultivation of phytohemagglutinin (PHA)- stimulated patient PBMCs alone or with various T-cell lines [Levy and Shimabukuro, 1985; Gallo et al., 19871. In one study, HIV isolation from asymptomatic individuals was dramatically improved by depletion of CD8+-suppressor cells prior to culture [Walker et al., 19861. In another study, expression of latent HIV was induced with the halogenated pyrimidine 5-iod0-2’-deoxyuridine [Folks et al., 19861.

However, the practical utility of these as well as several other aspects of HIV cultivation has not been systematically investigated. Comparative data regarding various methods of preparation and storage of PBMCs and the requisite number of inoculated PBMCs have not been previously reported. Although replication of HIV was initially established by detecting virus-associated reverse transcriptase (RT) activity in superna- tants of cultured cells, enzyme-linked antigen capture (AC) assays have been reported to detect readily HIV in culture supernatants with equal success [McDougal et al., 1985; Wittek et al., 19871. We have previously reported the rapid and efficient detection of HIV in cultured PBMCs at the cellular level by in situ hybridization (ISH) and immunocyto- chemical (IC) assays [Busch et al., 19871. We describe here our rigorous evaluation of these methods for rapid detection of HIV in cultured cells from healthy subjects positive and negative for anti-HIV.

MATERIAL AND METHODS Specimen Selection

A total of 165 coded and heparinized or citrated blood specimens were collected from healthy blood donors (46 anti-HIV-positive, 46 anti-HIV-negative), recipients of blood transfusions without any other risk factors (1 1 anti-HIV-positive, 16 anti-HIV- negative), and homosexual men (36 anti-HIV-positive, ten anti-HIV-negative). The anti-HIV status of each individual was established by licensed enzyme immunoassays (Abbott Laboratories, Chicago, IL; or Genetic Systems, Inc., Seattle, WA) and was confirmed by Western blot (DuPont, Wilmington, DE). The HIV antibody status of these individuals was decoded only after determination of the culture results.

In Vitro HIV Expression 3

TABLE I. Variation in HIV Recovery Resulting From Rapid vs Delayed PBMC Isolation and From Immediate Culture vs Temporary Freezing

Days to HIV detection HIV + /tested

PBMC isolation Coculture set-up (No.) Mean Range

A: <3 hr Immediate 15/17 1 1 5-22 B: 24 hr Immediate 11/13 13 1-19

- C: 12 hr' Immediate 0112 -

D: <3 hr Frozen 717 8 6-9

"Different cells from the others.

TABLE 11. DilutionaI Culture Analysis of PBMCs From Healthy HIVSeropositive Subjects

No. of sDecimen PBMCs Der culture (%)

1 o6 1 os 1 o4 1 o3 1 o2 Frequency of HIV recovery 21/23 (91) 19/23 (83) 14/23 (65) 4/23 (17) 1/23 (4)

HIV Culture PBMCs were prepared from whole blood either by ficoll-hypaque (Pharmacia,

Uppsala, Sweden) or the Sepracell (Sepratech Corp. Oklahoma, OK) centrifugation methods. The cell separation was performed within 3 hr of phlebotomy. In selected cases, a second tube of blood was reserved for PBMC isolation after a delay of 1 day or 3 days (see Table 1). PBMCs purified from fresh blood were cultured immediately and/or frozen at -70°C at a concentration of 2 4 x lo6 cells/ml in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum (FBS) and 10% dimethylsulfoxide (DMSO; Sigma, St. Louis, MO).

Specimens of I x lo6 PBMCs were cocultured in 0.5 ml culture medium containing 1 x 106 normal PBMCs derived from the buffy coat of anti-HIV- and hepatitis B core antibody-negative donors, which had been prestimulated with PHA for 3 days according to the method of Gallo et al. [1987]. After 2 hr, 2 ml of culture medium consisting of C0,-gassed RPMI (pH = 6.8), 20% FBS, 50 pg/ml gentamycin, 1 pg/ml Fungizone, 2 pg/ml polybrene (Sigma), 5% 11-2 (Cellular Products, Inc, Buffalo, NY), and 0.001% antihuman leukocyte interferon (ICN ImmunoBiologicals, Lisle, IL) was added to the culture tubes. Twice per week, 2 ml of culture fluid and part of the cell pellet were removed, and 2 ml of fresh medium was added. The culture fluid was saved for RT and AC assays, and the cells were spotted on ply-I-lysine (F.W. 5,000; Sigma)-coated ten-well microscope slides (Organon Teknika Corp., Durham, NC) for immunocytochemical analysis. Once per week, 1 x lo6 PHA-stimulated donor PBMCs were added to the cultures.

In limiting dilution studies, the PBMCs from 25 individuals (23 anti-HIV-positive, two anti-HTV-negative) were serially diluted in RPMI medium, and inocula ranging from 1 x lo6 to 1 x 102cells (see TabIe 11) were cocultured with I x lo6 PHA-stimulated donor PBMCs.

4 Ulrich et al.

For IUDR induction [Folks et al., 19861, specimen PBMCs from 16 individuals (ten anti-HIV-positive, six anti-HIV-negative) were exposed to 100 pg/ml IUDR (Sigma) in RPMI for 24 hr at 37OC, washed three times with RPMI, and then cocultured with PHA-stimulated donor PBMCs as described above.

For CDS+-suppressor cell depletion of PBMCs, the panning method as described by Engleman et al. [ 19811 was employed. Approximately 1 x lo6 PBMCs were incubated in 0.5 ml phosphate buffered saline (PBS; 0.01 M Na,HPO,, 0.15 M NaCI, pH 7.5) containing 10 pg/ml monoclonal antibodies to CD8 (Leu 2b; Becton Dickinson, Mountain View, CA) for 20 min at room temperature. The cells were then washed twice, resuspended in 1.5 ml 1 % FBS/PBS, and incubated on a plastic petri dish (60 x 15 mm; Falcon, Cockeyville, MD) coated with rabbit antimouse IgG (Dako Corp., Santa Barbara, CA) for 2 hr at 4OC. The nonadherent PBMC fraction was then cocultured with PHA-stimulated donor PBMCs as described above. The panning efficiency was checked by flow cytometry [Levy et al., 19851 and revealed less than 10% CDS'-cells in the nonadherent fraction.

Cultures were assessed to be HIV-positive if at least two consecutive IC assays were positive and the IC results were confirmed by RT and/or AC assay. IC- and RT- or AC-negative cultures were carried for 30 days.

lmmunocytochemistry

PBMC preparations were air dried, fixed in acetone for 5 min at 4"C, and stored at -7OOC. Slides were brought to room temperature, and the cells were rehydrated in Tris-buffered saline (TBS; 0.05 M Tris, 0.15 M NaCl, pH 7.5) for 5 min, followed by incubation with 10% normal rabbit serum in TBS (NRS/TBS) for 30 min to reduce nonspecific staining. A mixture of monoclonal antibodies (Genetic Systems, Seattle, WA) against HIV core (p18) and HIV envelope (gp41) proteins was added; preliminary titration studies established optimal final dilutions of 1 :4,000 for anti-pl8 and 1 :2,000 for anti-gp41 in NRS/TBS. After 30 min, the slides were washed three times in TBS and incubated for 30 min with rabbit antimouse IgG (1:300 in NRS/TBS; Dako Corp). Following this, the slides were washed three times and processed through three cycles of alkaline phosphatase-antialkaline phosphatase (APAAP) complex (1 :60 in NRS/TBS; Dako Corp) reagents as suggested by the supplier. The slides were immersed in Vector@ Red (Vector Laboratories, Burlingame, CA) for 20 min, rinsed in tap water, and counterstained in hematoxylin.

Each ten-well slide contained uninfected HUT 78 cells or cultured donor PBMCs as a negative control and chronic HIV-infected HUT 78 cells as a positive control. Cells were examined microscopically, and HIV-positive cells were characterized by intense red cytoplasmic staining.

RT Assay

Virus was pelleted from 1 ml cell-free culture supernatants in a 50 Ti rotor (Beckman, Palo Alto, CA) at 40,000 rpm for 1 hr. The pellets were assayed for RT activity using the conditions described by Hoffman et al. [ 19851. Twenty micro-curies 3H-dTTP (DuPont/NEN Products, North Billerica, MA) and 2.5 pg poly(rA):p(dT),,-,8 (Pharmacia Biochemicals, Piscataway, NJ) were used per reaction.

Supernatants from uninfected HUT 78 cells and from cultured donor PBMCs were used as the negative control and gave 9,000 2,000 cpm/ml supernatant. The cut-off

In Vitro HIV Expression 5

value used for RT activity was over two times the negative control. Supernatants from chronic HIV-infected HUT 78 cells were used as a positive control.

AC Assay

Culture supernatants (0.2 ml per test) were inactivated by addition of Triton X-100 to a final concentration of 0.5%. HIV p24 antigen was detected by means of a commercially available HTLV-I11 antigen enzyme immunoassay (Abbott Laboratories). The analytical sensitivity of this assay is -50 pg/ml of purified HIV core antigen.

RESULTS Evaluation of Specimen Processing

The influences of delayed processing of whole blood for the isolation of PBMCs and the use of fresh vs DMSO-frozen PBMCs in the coculture procedure were evaluated. The impact of a 24-hr lag time in processing was practically important because of frequent late-evening collections and shipping of whole blood specimens. Freshly collected heparin- ized blood samples from 17 anti-HIV-positive subjects were processed for PBMC isolation both within 3 hr of collection and following room temperature storage for 24 hr. Another set of 12 seropositive samples were stored for 72 hr at room temperature prior to PBMC isolation. The results (Table I) show that the frequency of HIV recovery was comparable between the 3-hr- and 24-hr-processed samples. However, a 24-hr delay in PBMC isolation did result in a slight delay in HIV recovery (mean of 13 days vs 1 1 days). A delay of 72 hr was evidently unsatisfactory; none of these samples yielded HIV. For seven samples, we also compared the HIV recovery rates from freshly isolated PBMCs vs PBMCs frozen in 10% DMSO at -70°C (Table I, row A vs row D). The results demonstrated more frequent and earlier HIV recovery from frozen than from freshly cultured cells. Based on these findings, the processing method for subsequent cultures consisted of PBMC isolation within 2 4 hr of collection followed by freezing in DMSO and then convenient batch culture set-up.

Limiting Dilution Cultures of PBMCs

The number of PBMCs necessary for virus recovery in our coculture protocol was evaluated by limiting dilutions of PBMCs from 23 seropositive and two seronegative specimens. As shown in Table 11, the HIV recovery rate from seropositive subjects was dependent on the number of PBMCs inoculated and ranged from 4% to 91%. An inoculum of less than lo4 cells decreased the recovery rate dramatically (from 65% to 17%). PBMCs from six subjects were collected on two separate occasions and cultured by the limiting dilution method. In two cases, the results were comparable on both occasions; in four cases, the required inoculum varied. For example, in one case, as few as 100 cells were repeatedly sufficient for virus isolation from the initial PBMC preparation. However, a second blood sample obtained 2 months later from the same person required an inoculum of at least lo5 cells for HIV recovery. In seven cases, an inoculum of lo4 or lo5 cells yielded HIV expression 3 4 days earlier than did lo6 cells, suggesting that dilution of suppressor/ cytotoxic lymphocytes, which impede HIV replication, may have occurred. All dilutional cultures from the two seronegative persons at high risk for HIV infection were negative.

6 Ulrich et al.

TABLE III. HIV Isolation From Seropositive Healthy Individuals bv Three Different Culture Methods*

Specimen No.

Culturemethod 1 2 3 4 5 6 7 8 9 10

Standard 3 5 7 7 8 1 4 1 8 6 - - IUDR-induced 6 5 7 7 14 14 - - - -

CD8'-depleted 3 5 7 7 8 10 18 6 6 -

*The numbers in the table indicate the day when HIV was first detected by immunocytochemical analysis (IC); negative results are indicated by a minus sign.

TABLE IV. Relative Sensitivity of Immunocytochemical (IC), Antigen Capture (AC), and Reverse Transcriptase (RT) Assays for Detection of HIV From PBMCs of Ten Healthy Anti-HIV-Positive Individuals*

Specimen No

Monitorinamethod 1 2 3 4 5 6 7 8 9 10

1C 3 3 6 6 7 9 10 13 16 17 AC 6 3 9 6 3 9 1 0 1 3 1 6 1 3 RT 6 6 9 6 7 9 1 3 2 0 1 9 2 0

*The numbers in the table indicate the day when HIV was first detected.

TABLE V. HIV Recovery Rates From Healthy Subjects

Positive/total no. of cultures ( W )

Seronegative subjects 0/72

Seropositive subjects 78/93 (84) Blood donor 39/46 (85) Transfusion recipients 10/11 (91) Homosexual men 29/36 (81)

Influence of IUDR Induction and CD8+-Cell Depletion

We randomly selected 10 seropositive and six seronegative subjects and cultured fresh PBMCs (1) by the standard method, (2) after exposure to IUDR, and (3) after CD8+-cell depletion. The time period to and the rate of HIV recovery from cultured PBMCs with each of the three protocols are shown in Table 111. The IUDR treatment of PBMCs actually resulted in a lower HIV recovery rate (six of ten vs eight of ten for the standard protocol) and in a longer time period to positivity in several cases. The PBMCs following depletion of CD8+ cells yielded virus from nine of ten seropositive subjects, including one case in whom HIV was isolated only after CD8+-cell depletion. Another seropositive individual and all six seronegative subjects were HIV culture-negative with each of the three culture protocols.

In Vitro H N Expression 7

Comparison of HIV Monitoring A s s a y s

To determine the relative sensitivity of HIV-detection assays, we monitored ten PBMC cultures from anti-HIV-positive and five cultures from anti-HIV-negative individ- uals by IC, AC, and RT assays. Table IV presents the resuits for the ten anti-HIV-positive individuals. Each assay detected HIV at varying periods in each of the ten cultures. However, the IC and the AC assays were positive earlier than the RT assay in eight of ten cultures. In no case was RT positive prior to IC or AC, whereas AC and iC each detected HIV replication first in two of ten cultures. All the assays were negative in cultured PBMCs from five anti-HIV-negative blood donors.

Frequency of HIV Recovery From Asymptomatic Persons

Based on the above data, we employed the optimized culture protocol in a prospective study of i 65 asymptomatic individuals. PBMCs were prepared from whole blood within 3 hr of collection and were frozen in DMSO. After thawing and washing, I x lo6 PBMCs were set into coculture with 1 x 106 PHA-stimulated cells and monitired by 1C and RT. The total HIV recovery rate from healthy seropositive subjects was 84%; similar results were obtained for blood donors, transfusion recipients, and homosexual men (Table V). We could not isolate HIV from any of the 72 seronegative subjects, including ten homosexual men at high risk for HIV infection.

DISCUSSION

Serological testing for HIV antibodies establishes whether an individual has immunologically responded to viral infection but does not distinguish whether the infectious agent was eliminated by the immune response of the host or persists as an active or latent infection. In addition, a time window exists between initial viral infection and the production of detectable levels of anti-HIV in the serum. Thus an infectious person may not be recognized by serological tests for HIV antibodies. Determination of active HIV infection requires either direct detection of viral nucleic acids or antigens, or virus culture. Although assays for direct detection of HIV DNA and RNA in PBMCs and of HIV antigens in serum have recently been reported, they identify HIV in only a proportion of the individuals who actuaIIy harbor the virus as determined by culture (Harper et al., 1986; Kwok et al., 1987; Pellegrino et al., 1987; Wittek et al., 19871. Thus, HIV culture remains a n important laboratory method in clinical studies of AIDS.

Over the past several years, HiV cultivation has been steadily improved, with progressively higher recovery rates reported. The earliest successful methods employed direct PHA stimulation of PBMCs from AIDS patients and patients with AIDS-related complex (ARC) and yielded virus recovery rates of about 50% [Gallo et a)., 19841. The coculture of PHA-stimulated normal PBMCs with the patient’s PHA-stimulated cells improved HIV isolation rates to 80% for symptomatic patients with AIDS and ARC and to 50% for healthy seropositive individuals [Levy and Shiniabukuro, 1985; Salahuddin et al., 19851. A more efficient protocol for HIV recovery from PBMCs in cocultures was recently reported by Gallo et al. 119871. This coculture protocol, which we have adapted, differs from others in that ( 1 ) the patient’s PBMCs are not directly PHA stimulated but instead are cocultured with 3 day-prestimulated normal PBMCs; this avoids any direct PHA toxicity to the patient cells; (2) propagation of HIV is promoted by the use of culture rubes rather than flasks, wiih the cell pellets left undisturbed throughout the course of

8 Ulrich et al.

culture such that high cell density promotes direct cell-to-cell viral propagation; and (3) culture media are maintained at pH 6.8 rather than the conventional pH 7.2; slightly acidic culture media are better for initial lymphocyte growth in culture [Glick, 19801 and may facilitate virus infection of cells by promoting the fusion of the viral envelope with cell membranes and the consequent release of the nucleocapsid into the cytosol [Helenius et al., 19801.

In this study, we assessed several additional practical aspects of HIV cultures. We have demonstrated that a 24-hr delay in processing whole blood stored at room temperature had no adverse influence on the overall HIV recovery rate, although immediate PBMC isolation resulted in the most rapid recovery of HIV. In contrast, a prolonged delay in PBMC isolation was severely detrimental to viral and cellular propagation. Following early isolation, the PBMCs could be suspended in 10% DMSO and stored at -7OOC without impeding the ability to recover HIV subsequently. This permitted convenient, batched set-up of HIV cultures and also allowed for shipping of PBMCs on dry ice to another laboratory for culture. Moreover, the HIV recovery rate was actually higher and the average time period to HIV detection was often shorter with frozen cells. This could be due to the fact that even optimal freezing and thawing of cells in cryoprotectants like DMSO caused considerable cell membrane damage, which in turn may have facilitated virus release from in vivo infected cells [Griffiths, 1985; Gallo et al., 19871.

In the limiting dilutionnl studies, the minimum number of cells in the inoculum for HIV recovery varied widely from lo2 to lo6 cells, suggesting that the number of HIV-infected cells varies from one seropositive subject to another and also within a single person over the course of the infection. Such a fluctuating level of viral replication is typical of animal lentiviral infections and likely reflects the intermittent activation of HIV expression in vivo by antigenic stimulation or other viral infections [Haase, 1986; Nabel and Baltimore, 19871. Although it is possible that recovery rates could have been increased even further with larger inocula, studies using more than lo6 PBMCs per culture have not been performed by us for lack of adequately large specimens. Whether larger inocula would have increased the rate of HIV recovery is uncertain, since the ratio between unstimulated specimen PBMCs and PHA-stimulated donor PBMCs may also be important. The failure to recover HIV from occasional asymptomatic seropositive subjects possibly indicates very low-level infection in circulating PBMCs, although virus may persist in other cells such as reticuloendothelial or brain cells [Koenig et al., 19861.

Our results differ from previous reports in that IUDR induction [Folks et al., 19861 did not lead to improved HIV recovery, and depletion of CD8+ cells [Walker et al., 19861 resulted in only a marginal improvement in HIV recovery rates. The failure to obtain significantly improved virus yield with these modifications probably reflects the near- optimal recovery obtained with our standard culture protocol. In critical cases, CD8+-cell depletion provides a useful but labor-intensive adjunct for virus expression.

We did confirm previous reports regarding the use and sensitivity of immunocyto- chemical and AC assays for detecting HIV replication. These assays offer many advantages over the more tedious and expensive RT assay. In particular, they can be readily performed in most microbiology and immunology laboratories and hence will allow for a wider availability of HIV cultures.

Our results of an 84% HIV recovery rate from PBMCs of healthy anti-HIV-positive persons validate the sensitivity and efficiency of our culture protocol. This recovery rate was obtained after only a single PBMC isolation attempt, and repeated PBMC cultures

In Vitro HIV Expression 9

would likely have yielded a higher rate. Our data indicate that virtually all seropositive persons, irrespective of clinical status, must be considered to be persistently infected with HIV.

ACKNOWLEDGMENTS

This study was supported in part by research grant PO1 HL-36589 and contracts HB-6-7024 and AI-32519 (San Francisco Men’s Health Study) from the National Institutes of Health. We thank Dana Gallo of California State Department of Health Services, Berkeley for sharing her method for HIV cocultures.

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