MAJOR ARTICLE
Increased In Vitro Cytopathicity of CC Chemokine Receptor 5–Restricted Human Immunodeficiency Virus Type 1 Primary Isolates Correlates with a Progressive Clinical Course of Infection David Kwa, Jose Vingerhoed, Brigitte Boeser, and Hanneke Schuitemaker Sanquin Research and Landsteiner Laboratory of the Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
The presence of only non–syncytium-inducing b-chemokine receptor 5–restricted (R5/NSI) human immunodeficiency virus type 1 (HIV-1) in an infected individual has been associated with long-term asymptomatic survival. However, the majority of R5/NSI HIV-1–infected individuals do progress to AIDS. Here, we compared the replicative capacity and cytopathicity of R5/NSI HIV-1 variants that were isolated early and late in the clinical course from 7 long-term asymptomatic individuals and 7 individuals with progressive HIV-1 infection. R5/NSI HIV-1 cytopathicity in vitro directly correlated with in vitro replication. HIV-1 variants obtained early and late during long-term asymptomatic HIV infection from the same individual were equally cytopathic. In contrast, HIV-1 variants obtained during late-stage progressive HIV infection were more cytopathic than viruses obtained early in infection from the same individuals. Our data indicate that the cytopathicity of HIV1 variants may increase with progression to disease. The asymptomatic phase of infection with human immunodeficiency virus type 1 (HIV-1) is dominated by macrophage-tropic non–syncytium-inducing (NSI) HIV1 variants that use CD4 and chemokine receptor CCR5 for entry in their target cells [1–5]. In 50% of HIV1–infected individuals, disease progression is associated with the emergence of syncytium-inducing (SI) HIV-1
Received 26 August 2002; accepted 16 December 2002; electronically published 9 April 2003. Financial support: Netherlands Council for Scientific Research (grant 901-02-214). The Amsterdam Cohort Studies are financially supported by the Netherlands Council for Scientific Research and the Netherlands AIDS Fund. Written informed consent was obtained from all participants of the Amsterdam Cohort Studies on HIV infection and AIDS, a collaboration between the Academic Medical Center, the Municipal Health Service, and Sanquin Research. Reprints or correspondence: Dr. Hanneke Schuitemaker, Sanquin Research, Dept. of Clinical Viro Immunology, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands (
[email protected]). The Journal of Infectious Diseases 2003; 187:1397–403 2003 by the Infectious Diseases Society of America. All rights reserved. 0022-1899/2003/18709-0006$15.00
variants [6, 7], which at least use the chemokine receptor CXCR4 in addition to CD4 as entry receptor [6, 8–10]. SI conversion is followed by a more rapid decrease in CD4 cell counts and an accelerated progression to AIDS [6, 10, 11]. Naive CD4⫹ T cells express high levels of CXCR4 and low levels of CCR5 and are targets for SI HIV-1 in vivo [12, 13]. Infection and death of naive CD4⫹ T cells may directly interfere with T cell renewal and maintenance of the T cell pool [12]. In addition, CXCR4, in comparison with CCR5, is more broadly expressed on memory T cells [14, 15], which provides SI HIV-1 variants with a much larger target cell population than that of R5/NSI HIV variants. A rapid loss of memory and naive CXCR4⫹CD4⫹ T cells in various cell systems in vitro has been observed after inoculation with X4/SI HIV-1 but not with R5/NSI HIV-1 [16–18]. However, the majority of individuals who never develop infection with X4/SI HIV-1 variants do progress to AIDS, some of them even rapidly [6, 19, 20]. We previously R5 HIV-1 Cytopathicity • JID 2003:187 (1 May) • 1397
demonstrated that NSI HIV-1 variants isolated from individuals with a progressive disease course are more rapidly replicating in vitro and are associated with a higher virus load in vivo, compared with NSI HIV-1 from asymptomatic individuals [20]. In the present study, we analyzed, in a system of phytohemagglutinin (PHA)–stimulated peripheral blood mononuclear cells (PBMCs), the in vitro cytopathic properties of R5/NSI HIV-1 variants that were obtained early and late in infection from long-term asymptomatic individuals (LTAs) and from individuals with a progressive disease course (“progressors”).
MATERIALS AND METHODS Cells and viruses. PBMCs from buffy coats of 10 healthy blood donors selected for the absence of the CCR5D32 allele were isolated using ficoll-hypaque density centrifugation. After isolation, cells were pooled and stored in liquid nitrogen until further use. Virus isolates from 14 participants of the Amsterdam Cohort Studies were obtained by coculture of limiting diluted patient PBMCs with PHA-stimulated healthy donor PBMCs. These 14 participants have been described elsewhere [21] and never had detectable SI variants. Of these 14 participants, 7 were classified as LTAs, and 7 were classified as progressors. The LTAs (Amsterdam Cohort homosexual men [ACH] 16, 68, 78, 337, 434, 441, and 583) had an asymptomatic follow-up time of at least 9 years (mean follow-up, 143 months after seroconversion; range, 124–152 months), with stable CD4⫹ T cell counts (1400 cells/ mm3) in the absence of antiretroviral therapy. Of the 7 progressors, 4 (ACH 53, 172, 424, and 638) progressed very rapidly to AIDS (AIDS diagnosis at 25–76 months after seroconversion), 2 (ACH 38 and 142) were classified as typical progressors (AIDS diagnosis at 99–109 months after seroconversion), and 1 (ACH 617) was classified as a slow progressor (AIDS diagnosis at 136 months after seroconversion after 10-year period with stable CD4⫹ T cell counts). From each individual, 3 virus isolates were obtained at 2 different time points: at a relatively early time point in the course of infection (mean, 21 and 18 months after seroconversion for LTAs and progressors, respectively) and at a relatively late time point in the course of HIV-1 infection. For LTAs, this time point was as late as possible (mean, 113 months after seroconversion), and for progressors, the late time point was close to AIDS diagnosis (mean, 75 months after seroconversion). Virus isolates were randomly picked and only passaged on primary PBMCs with a maximum of 4 passages/virus. Virus isolates were screened previously for coreceptor use with the U87 astroglioma cell lines stable transfected with CD4 and CCR3, CXCR4 or CCR5, and PBMCs homozygous for CCR5 D32 [21]. SI phenotype was determined by coculture of infected PBMCs with the MT2 cell line [22]. Virus stocks were grown on PBMCs, which were cultured 1398 • JID 2003:187 (1 May) • Kwa et al.
for 2–3 days in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 1 mg/mL PHA, 10% fetal calf serum (FCS), and 100 U/mL penicillin and 100 mg/mL streptomycin (P/S) before inoculation. After inoculation, PBMCs were cultured in the same medium without PHA but with 20 U/mL recombinant interleukin-2 (rIL-2; Proleukin; Chiron Benelux BV). Cell-free supernatant with HIV was preserved at ⫺70C. For mock infections, we collected and pooled the supernatant of uninfected PBMC cultures. Determination of virus titers in stock preparations (TCID50) was performed on PHA-stimulated PBMCs, which were depleted for CD8 cells using the magnetic cell sorter (MACS) system (Miltenyi Biotec), according to the manufacturer’s instructions. In brief, stimulated cells were washed in MACS buffer (PBS supplemented with 2 mM EDTA and 0.5% bovine serum albumin [BSA]) and were incubated with microbeads labeled with CD8-directed antibodies, at 4C for 15 min at a concentration of 20 mL/107 cells. Thereafter, cells were run over a LS⫹ separation column attached to a MidiMACS magnet (Miltenyi Biotec). The fraction depleted for CD8⫹ cells was collected, washed once in IMDM, and resuspended to a cell concentration of 106 cells/mL in IMDM supplemented with 20 U/mL rIL-2, 5 mg/mL polybrene, 10% FCS, and P/S. Titer of the stocks (TCID50) was determined on CD8depleted PBMCs from the same cell pool as the one used for further experiments. Replication kinetics and cytopathicity. Inoculation of 6 ⫻ 10 6 CD8-depleted PHA-stimulated peripheral blood lymphocytes with 2500 TCID50 of each virus clone was performed in a 15-mL tube in a total volume of 1 mL for 2.5 h at 37C. Cells were subsequently washed with IMDM and were cultured in 25-mL culture flasks at 37C in rIL-2–supplemented medium for 14 days. At several time points, cells were harvested and washed for fluorescence-activated cell sorter analysis. CD4⫹ lymphocytes were gated on the basis of their forward and side scatter and by cell-surface expression of CD4 and CD3. Cytopathicity was determined by comparing gated CD4⫹ lymphocytes as the percentage of total cells in the HIV-1–inoculated culture, relative to the gated CD4⫹ lymphocytes as the percentage of total cells in the mock-infected control culture [17]. At several time points, culture supernatant was harvested for analysis of p24 production by an in-house p24 antigen capture ELISA. Statistical analyses. The data used for statistical analyses were obtained at day 7 after inoculation (by that time, all the viruses had replicated substantially, and no plateau was reached for cell killing). All statistical analyses were performed with nonparametric tests, using SPSS version 10.0. The Mann-Whitney U test was used for comparison of unpaired samples. For comparison of longitudinal paired samples, the Wilcoxon-signed rank test was used. Spearman’s correlation coefficient (rs) was used for determination of correlations between studied parameters.
Table 1. Characteristics of long-term asymptomatic individuals (LTAs) and individuals with a progressive disease course (progressors) and laboratory values at the time points of clonal virus isolation. Group, patient, biological clone
Genotype
Virus isolation, months after SC
CCR5
CCR2b
68.12.b5
33
WT
WT
68.39.h4
100
Clinical outcome (months after SC)
Serum RNA load, log copies/mL
CD4⫹ cell count, 103 cells/mL
3.6
1.06
4.7
0.63
3.0
1.10
3.0
0.50
3.0
0.83
3.7
0.67
3.7
0.63
3.8
0.49
LTA 68 AS (151)
441 441.6.2b11
16
441.39.2a1
111
WT
WT
AS (152)
583 583.9.2f1 583.38.1a5
24
WT
WT
AS (149)
109
16 16.10.1c3
22
16.37.2a7
114
D32/WT
WT
AS (143)
78 78.7.2g6
17
78.42.1a4
115
D32/WT
WT
AS (124)
3.0
0.75
3.7
0.38
337 337.9.1a2
24
337.43.b4
122
D32/WT
64I/WT
AS (142)
4.2
1.31
4.5
0.71
434 434.8.a3 434.43.6e12
13
D32/WT
WT
AS (140)
119
3.0
0.63
5.9
0.72
4.8
0.70
4.8
0.20
3.1
0.72
4.6
0.31
4.8
0.66
4.7
0.23
Progressors 53 53.13.d7
35
53.60.e6
77
WT
WT
PCP (76)
142 142.8.b1
21
142.32.f9
93
WT
WT
KS (109)
424 424.9.f4 424.18.a1
6
WT
WT
CO (38)
43
38 38.8.d1 38.35.e11
21
D32/WT
WT
KS (101)
102
3.8
0.81
3.9
0.17
4.5
1.58
4.5
1.30
172 172.7.f11
5
172.14.d7
25
D32/WT
WT
KS (25)
617 617.6.c7
15
617.41.e4
126
D32/WT
WT
NHL (136)
3.5
0.57
4.8
0.33
4.2
0.35
4.1
0.24
638 638.7.c10
22
638.14.g3
54
D32/WT
WT
NHL (59)
NOTE. AS, asymptomatic; CO, oesophageal candidiasis; KS, Kaposi sarcoma; mo, month; NHL, Non-Hodgkin’slymphoma; PCP, Pneumocystis carinii pneumonia; SC, seroconversion; WT, wild-type genotype; D32/WT, CCR5 D32 heterozygote; 64I/ WT, CCR2b 64I heterozygote.
Figure 1. Correlation between R5 human immunodeficiency virus (HIV) cytotoxicity and virus production in vitro. Correlations were calculated for R5 viruses isolated early and late in the course of infection from long-term asymptomatic individuals (LTAs) and individuals with a progressive disease course (“progressors”). Spearman’s rank correlation coefficient (rs) with P value is shown. Level of cytotoxicity was calculated as the percentage of viable cells in the infected culture relative to the uninfected control culture. Virus production is given as the amount of p24 gag antigen in the supernatant of the same culture, as measured in a p24 antigen capture ELISA.
RESULTS Virus production in vitro in PHA-stimulated PBMCs by R5 HIV-1 biological clones correlates with cytotoxicity. PHAstimulated PBMCs were cell-free inoculated with HIV-1 biological clones that were isolated at relatively early and late time points in the course of infection from 14 HIV-infected individuals who never developed infection with X4/SI HIV-1 variants. Seven individuals were classified as LTAs, which was defined as 19 years of asymptomatic follow-up with stable CD4⫹ cell counts and a CD4⫹ cell count 1400 cells/mL during the ninth year of follow-up. The other 7 individuals progressed to AIDS (table 1). To investigate whether cytotoxicity of R5 virus isolates was related to the level of virus production, we cor-
1400 • JID 2003:187 (1 May) • Kwa et al.
related the cumulative viral production in the supernatant of inoculated PBMC cultures with the depletion of CD4⫹ T lymphocytes in the same cell cultures, as measured by flow cytometry. We observed that a higher level of virus production correlated with a stronger CD4⫹ T cell depletion at day 7 after inoculation for all early virus isolates from LTAs and progressors (rs p 0.66; P p .011). For the virus isolates that were obtained relatively late in the course of infection, this correlation was not significant, although the same trend could be observed (rs p 0.42; P p .131; data not shown). The correlation between virus production and cytotoxicity was stronger for virus isolates from progressors than from LTAs (for progressors: early time point virus isolates, rs p 0.86 [P p .014] and late time point
Figure 2. Pairwise analyses of virus production (A) or cytotoxicity (B) of early- and late-stage virus isolates obtained from the same individual. Analysis was performed separately for virus isolates from long-term asymptomatic individuals (LTAs; left panel) or individuals with a progressive disease course (“progressors”) (right panel). Statistical tests were done using the Wilcoxon signed-rank test, with P value. Virus production and cytotoxicity were measured at day 7 after inoculation. NS, not significant.
virus isolates, rs p 0.82 [P p .023]; for LTAs: early time point virus isolates: rs p 0.57 [P p .180] and late time point virus isolates: rs p 0.46 [P p .294]) (figure 1). However, the coefficients of these graphs were not significantly different from each other (early time point, P p .195; late time point, P p .389). Identical results were obtained in 2 independent experiments (data not shown). The kinetics of virus replication, analyzed as cumulative p24 antigen production in the supernatant over time, were, on average almost similar for virus isolates obtained from LTAs and progressors at early and late time points (data not shown). Increased replication capacity and cytopathicity of latestage virus isolates. We performed a pairwise analysis of the cumulative virus production at day 7 after inoculation for earlyand late-stage virus isolates that were obtained from the same individual. This pairwise analysis revealed a significant increase in virus production between viruses from early and late time points (P p .01; data not shown). This increase was not ob-
served in a separate analysis of early- and late-stage virus isolates from LTAs. In contrast, early- and late-stage virus isolates from progressors were significantly different in cumulative virus production at day 7 (P p .028). This confirms previous observations that the replicative capacity of HIV-1 increases with a progressive clinical course of HIV infection [20]. A similar profile was seen when we compared cytopathicity over time. Although an increase in overall cytopathicity was observed in a pairwise comparison of early- and late-stage viruses from LTAs and progressors (data not shown), separate analysis of the LTA virus isolates revealed no differences in cytopathicity of early- and late-stage virus isolates (figure 2A). Virus isolates obtained from progressors late in infection showed a statistically increased cytopathicity, compared with the related virus isolates obtained early infection, in pairwise analysis (P p .018; figure 2B). In conclusion, our findings suggest that increased cytopathicity of HIV may be correlated with a progressive disease course.
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DISCUSSION HIV isolates obtained during the late stages in the course of progressive HIV-1 infection replicate more efficiently in tissue culture than isolates that are obtained during the earlier stages [20, 23]. This has been best documented for X4/SI HIV-1 variants, which emerge in 50% of HIV-infected individuals. In the present study, we demonstrated that R5/NSI-restricted HIV-1 clones obtained from patients with AIDS who had never developed infection with X4/SI virus variants are more cytopathic in vitro in PHA-stimulated PBMCs, compared with pre-AIDS R5/NSI HIV-1 clones or R5/NSI isolates obtained from LTAs. The development of increased pathogenicity of HIV during the course of natural infection has long been known [8,24]. It has been previously suggested that cytopathicity of primary HIV isolates may solely depend on the coreceptor usage of the virus and not on the patient’s clinical status at the moment of virus isolation [25]. The differences in CD4⫹ T cell depletion in vitro after R5/NSI infection or X4/SI infection could indeed be attributed to the capacity of X4 HIV to infect more target cells [16, 17, 25]. Naive T cells express CXCR4 but not CCR5 and, consequently, are targets for X4/SI infection [12, 13]. X4/SI HIV infection of naive T cells is considered to directly interfere with T cell renewal [12], and, after the emergence of X4/SI HIV variants, a dramatic acceleration in CD4 cell loss in vivo can indeed be observed [6, 10,26]. However, within the R5/NSI HIV-infected population, differences in cytopathicity can be expected and appear to not be related to differences in coreceptor usage [21]. Transmission of an R5 SIV isolate that was isolated early in infection to a new rhesus macaque resulted in a relatively mild clinical course of infection in the recipient. In contrast, transmission of an R5 SIV isolate that was obtained relatively late in infection resulted in rapid disease progression in the recipient animal, indeed pointing to increasing viral pathogenicity in the course of infection [27, 28]. We previously demonstrated that, with progression of disease, the in vitro replicative capacity of R5 HIV-1 variants increased and that this increase correlated with increased virus load in vivo [1, 20, 29]. In our present study, we found no significant correlation between virus production level in vitro and RNA virus load in vivo (data not shown). We could demonstrate a correlation between virus production levels and cytotoxicity in vitro. Although the level of cytotoxicity did not correlate with CD4 cell loss in vivo (data not shown), our in vitro data point to a virus-mediated cell killing that also could be relevant in vivo. In the course of R5 HIV infection, CD4⫹ T cell loss is relatively constant [6]. This indicates that the increased replicative capacity and the coinciding increased cytopathicity of R5 HIV-1 in the course of infection have much less dramatic effect on CD4⫹ T cell loss than does cytopathicity induced by X4 HIV-1. Similarly, in our in vitro system of PHA1402 • JID 2003:187 (1 May) • Kwa et al.
stimulated PBMCs, the differences between early- and lateisolated virus in their capacity to induce CD4⫹ cell killing were much more subtle than the differences in cytopathicity seen between X4 and R5 HIV-1 variants in general (data not shown). Furthermore, in collaborative studies with Scoggins et al. [30], we also observed that, in Thy/Liv SCID-hu mouse system, latestage R5 isolates were more cytopathic than were R5 HIV biological clones obtained during early asymptomatic infection, but, in this system, late-stage R5 HIV-1 variants were never as cytopathic as X4 HIV-1 variants [31]. The underlying mechanism for the increased cytopathicity of AIDS-associated R5 HIV-1 variants remains to be established. An increased replicative capacity may simply increase the turnover rate of infected target cells. Whether this increased replication capacity is due to enhanced affinity for CD4 or CCR5, or whether other mechanisms are involved, requires further study.
Acknowledgments
We wish to thank Fransje Koning and Jos Dekker, for technical support, and Ronald van Rij and Frank Miedema, for a critical reading of the manuscript.
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