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

Abstract

The presence of only non-syncytium-inducing β-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 HIV-1 variants may increase with
progression to disease

Full text

R5 HIV-1 Cytopathicity •JID 2003:187 (1 May) •1397
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 immuno-
deficiency 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 HIV-
1 variants may increase with progression to disease.
The asymptomatic phase of infection with human im-
munodeficiency virus type 1 (HIV-1) is dominated by
macrophage-tropic non–syncytium-inducing (NSI) HIV-
1 variants that use CD4 and chemokine receptor CCR5
for entry in their target cells [1–5]. In 50% of HIV-
1–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
(h.schuitemaker@sanquin.nl).
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 ex-
pressed 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 mem-
ory and naive CXCR4
⫹
CD4
⫹
T cells in various cell sys-
tems in vitro has been observed after inoculation with
X4/SI HIV-1 but not with R5/NSI HIV-1 [16–18]. How-
ever, the majority of individuals who never develop in-
fection with X4/SI HIV-1 variants do progress to AIDS,
some of them even rapidly [6, 19, 20]. We previously

1398 •JID 2003:187 (1 May) •Kwa et al.
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 fromasymptomatic individuals[20].
In the present study, we analyzed, in a system of phytohemag-
glutinin (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 individ-
uals 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 isola-
tion, 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 (Am-
sterdam 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/
mm
3
) in the absence of antiretroviral therapy. Of the 7 progres-
sors, 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 dif-
ferent 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 di-
agnosis (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
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 ⫺70C.
For mock infections, we collected and pooled the supernatant
of uninfected PBMC cultures. Determination of virus titers in
stock preparations (TCID
50
) 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 mMEDTA
and 0.5% bovine serum albumin [BSA]) and were incubated
with microbeads labeled with CD8-directed antibodies, at 4C
for 15 min at a concentration of 20 mL/10
7
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 resus-
pended to a cell concentration of 10
6
cells/mL in IMDM sup-
plemented with 20 U/mL rIL-2, 5 mg/mL polybrene, 10% FCS,
and P/S. Titer of the stocks (TCID
50
) was determined on CD8-
depleted PBMCs from the same cell pool as the one used for
further experiments.
Replication kinetics and cytopathicity. Inoculation of
CD8-depleted PHA-stimulated peripheral blood lym-
6
6⫻10
phocytes with 2500 TCID
50
of each virus clone was performed
in a 15-mL tube in a total volume of 1 mL for 2.5 h at 37C.
Cells were subsequently washed with IMDM and were cultured
in 25-mL culture flasks at 37C 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
⫹
lym-
phocytes 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 non-
parametric tests, using SPSS version 10.0. The Mann-Whitney
Utest was used for comparison of unpaired samples. For com-
parison of longitudinal paired samples, the Wilcoxon-signedrank
test was used. Spearman’s correlation coefficient (r
s
)was used
for determination of correlations between studied parameters.

Table 1. Characteristics of long-term asymptomatic individuals (LTAs) and individuals with a pro-
gressive disease course (progressors) and laboratory values at the time points of clonal virus isolation.
Group, patient,
biological clone
Virus isolation,
months after SC
Genotype Clinical outcome
(months after SC)
Serum RNA load,
log copies/mL
CD4
⫹
cell count,
10
3
cells/mL
CCR5 CCR2b
LTA
68
68.12.b5 33 WT WT AS (151) 3.6 1.06
68.39.h4 100 4.7 0.63
441
441.6.2b11 16 WT WT AS (152) 3.0 1.10
441.39.2a1 111 3.0 0.50
583
583.9.2f1 24 WT WT AS (149) 3.0 0.83
583.38.1a5 109 3.7 0.67
16
16.10.1c3 22 D32/WT WT AS (143) 3.7 0.63
16.37.2a7 114 3.8 0.49
78
78.7.2g6 17 D32/WT WT AS (124) 3.0 0.75
78.42.1a4 115 3.7 0.38
337
337.9.1a2 24 D32/WT 64I/WT AS (142) 4.2 1.31
337.43.b4 122 4.5 0.71
434
434.8.a3 13 D32/WT WT AS (140) 3.0 0.63
434.43.6e12 119 5.9 0.72
Progressors
53
53.13.d7 35 WT WT PCP (76) 4.8 0.70
53.60.e6 77 4.8 0.20
142
142.8.b1 21 WT WT KS (109) 3.1 0.72
142.32.f9 93 4.6 0.31
424
424.9.f4 6 WT WT CO (38) 4.8 0.66
424.18.a1 43 4.7 0.23
38
38.8.d1 21 D32/WT WT KS (101) 3.8 0.81
38.35.e11 102 3.9 0.17
172
172.7.f11 5 D32/WT WT KS (25) 4.5 1.58
172.14.d7 25 4.5 1.30
617
617.6.c7 15 D32/WT WT NHL (136) 3.5 0.57
617.41.e4 126 4.8 0.33
638
638.7.c10 22 D32/WT WT NHL (59) 4.2 0.35
638.14.g3 54 4.1 0.24
NOTE. AS, asymptomatic; CO, oesophageal candidiasis; KS, Kaposi sarcoma; mo, month;NHL, Non-Hodgkin’s lymphoma;
PCP,Pneumocystis carinii pneumonia; SC, seroconversion; WT, wild-type genotype; D32/WT, CCR5 D32 heterozygote; 64I/
WT, CCR2b 64I heterozygote.

1400 •JID 2003:187 (1 May) •Kwa et al.
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 (r
s
)with Pvalue 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. PHA-
stimulated PBMCs were cell-free inoculated with HIV-1 bio-
logical clones that were isolated at relatively early and late time
points in the course of infection from 14 HIV-infected indi-
viduals who never developed infection with X4/SI HIV-1 vari-
ants. Seven individuals were classified as LTAs, which was de-
fined 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-
related the cumulative viral production in the supernatant of
inoculated PBMC cultures with the depletion of CD4
⫹
T lym-
phocytes in the same cell cultures, as measured by flow cyto-
metry. 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
( ; ). For the virus isolates that were obtained
rp0.66 Pp.011
s
relatively late in the course of infection, this correlation was
not significant, although the same trend could be observed
( ; ; data not shown). The correlation between
rp0.42 Pp.131
s
virus production and cytotoxicity was stronger for virus isolates
from progressors than from LTAs (for progressors: early time
point virus isolates, [ ] and late time pointrp0.86 Pp.014
s

R5 HIV-1 Cytopathicity •JID 2003:187 (1 May) •1401
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 Pvalue. Virus production and cytotoxicity were measured
at day 7 after inoculation. NS, not significant.
virus isolates, [ ]; for LTAs: early time pointrp0.82 Pp.023
s
virus isolates: [ ] and late time point virusrp0.57 Pp.180
s
isolates: [ ]) (figure 1). However, the coeffi-rp0.46 Pp.294
s
cients of these graphs were not significantly different from each
other (early time point, ; late time point, ).Pp.195 Pp.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 late-
stage virus isolates. We performed a pairwise analysis of the
cumulative virus production at day 7 after inoculation for early-
and 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 ( ; data not shown). This increase was not ob-Pp.01
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 pro-
duction at day 7 ( ). This confirms previous obser-Pp.028
vations 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 vi-
ruses 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 ( ; figure 2B). In conclusion, our findings sug-Pp.018
gest that increased cytopathicity of HIV may be correlated with
a progressive disease course.

1402 •JID 2003:187 (1 May) •Kwa et al.
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 vari-
ants, 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 de-
veloped 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 at-
tributed 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, dif-
ferences 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 in-
fection [27, 28].
We previously demonstrated that, with progression of dis-
ease, the in vitro replicative capacity of R5 HIV-1 variants in-
creased 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 dem-
onstrate a correlation between virus production levels and cy-
totoxicity 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 cyto-
pathicity 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 PHA-
stimulated PBMCs, the differences between early- and late-
isolated 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, late-
stage R5 isolates were more cytopathic than were R5 HIV bio-
logical 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 turn-
over rate of infected target cells. Whether this increased repli-
cation 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 tech-
nical support, and Ronald van Rij and Frank Miedema, for a
critical reading of the manuscript.
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