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Previous Article | Next Article 
Journal of Clinical Microbiology, December 2001, p. 4264-4268, Vol. 39, No. 12
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.12.4264-4268.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Quantification of Proviral Load of Human
Immunodeficiency Virus Type 2 Subtypes A and B Using Real-Time
PCR
Florence
Damond,1,2,*
Diane
Descamps,1,2
Isabelle
Farfara,1,2
Jean Noël
Telles,1,2,3
Sophie
Puyeo,4
Pauline
Campa,1,2
Annie
Leprêtre,1,2
Sophie
Matheron,1,2
Françoise
Brun-Vezinet,1,2 and
François
Simon1,2,5
Laboratoire de
Virologie1 et Service des Maladies
Infectieuses et Tropicales A,2 Hôpital
Bichat-Claude Bernard, Paris, Biomerieux S.A., 69280 Marcy
l'Etoile,3 INSERM, Unité de
Recherche U330, Université Victor Segalen,
Bordeaux,4 and Laboratoire de Virologie,
CHU Charles Nicolle, Rouen,5 France
Received 14 March 2001/Returned for modification 14 September
2001/Accepted 20 September 2001
 |
ABSTRACT |
We have developed and evaluated a new method to quantify human
immunodeficiency virus type 2 (HIV-2) proviral DNA based on LightCycler
real-time PCR. The assay has a detection limit of 5 copies/105 peripheral blood mononuclear cells (PBMC) and is
insensitive to HIV-2 strain variability: HIV-2 subtypes A and B are
both recognized and quantified. The intra- and interassay coefficients
of variation range from 16 to 40% for high provirus concentrations
(5 × 105 copies) and from 41 to 39% for low
concentrations (5 copies). We used this method to compare the proviral
DNA load and viral RNA load in plasma with clinical and immunological
status for 29 patients infected by HIV-2 (subtype A in 17 and subtype B
in 12). The proviral load (median, 201 copies/105
PBMC) was similar to that reported for HIV-1 infection. The median proviral loads did not correlate with the CD4+ cell
count categories and were as follows for CD4+ cell counts
of >400, 200 to 400, and <200 cells/mm3, respectively:
121 copies/105 PBMC (n = 8; range, <5 to
712 copies/105 PBMC); 114 copies/105 PBMC
(n = 9; range, <5 to 1,907 copies/105
PBMC); and 285 copies/105 PBMC (n = 12;
range, 53 to 2,524 copies/105 PBMC). Proviral load did not
correlate with plasma HIV-2 RNA positivity. As HIV-2 is considered to
replicate less efficiently than HIV-1, these high proviral loads might
be explained by the proliferation of infected cells.
| |
INTRODUCTION |
Human immunodeficiency virus
type 2 (HIV-2) was first isolated in 1986 from peripheral blood
mononuclear cells (PBMC) from patients in the Cape Verde Islands and
Guinea-Bissau (7). Heterosexual and vertical HIV-2
transmission rates are lower than those of HIV-1 (4, 21,
24). HIV-2-infected patients exhibit a longer clinical latency
period and progress more slowly toward AIDS (17, 22). The
origins of these viruses have been clearly linked to cross-species
transmission events between mangabeys (Cercocebus atys,
small West African monkeys) and humans (23). Six HIV-2 subtypes have so far been described (6, 14). Only HIV-2
subtypes A and B are prevalent, the others being considered
self-limiting infections at the epidemiological level. HIV-2 infection
is mostly confined to West Africa (12).
The CD4+ cell count correlates negatively with clinical
status in both HIV-2 and HIV-1 infections (3, 33). In
contrast, HIV-2 is more difficult to detect in plasma than HIV-1
(3, 28). This fact has led us and others to consider HIV-2
poorly replicative, at least early in the infection (3, 12, 28, 33). This factor could explain the epidemiological and clinical differences between HIV-2 and HIV-1 infections. The reasons for this
poorer fitness of HIV-2 in humans is unclear. The animal counterpart of
HIV-2, SIVsm (infecting mangabeys), is highly replicative in its
natural host, with high cellular and plasma viral loads, but is
nonpathogenic (30). The reason for this lack of
replicative robustness remains unclear.
For HIV-1 infection, it has been demonstrated that proviral load
correlates with disease progression (32). Previous
quantitative assessments of HIV-2 proviral load have proposed, as for
HIV-1, an inverse correlation with the CD4+ cell count and
clinical outcome (2, 27, 31). However, recent reports have
suggested that HIV-2 proviral load does not correlate with the
CD4+ cell count (15, 29).
The development of new quantitative methods using real-time PCR
technology has led us to evaluate those methods for proviral HIV-2 DNA
quantitation in 29 HIV-2-infected patients living in France. We
also tested plasma RNA detection and compared the findings with
immunological and clinical status.
| |
MATERIALS AND METHODS |
Study population and sample collection.
The study group
consisted of 29 patients (17 males and 12 females) who had been
enrolled since 1991 in the French National HIV-2 Cohort. These patients
originated from various countries in West Africa, North Africa, and
Europe. The infecting strains had been previously sequenced in the
env region: 17 were subtype A, and 12 were subtype B
(11). The epidemiological and clinical characteristics of
the patients are summarized in Table 1.
According to Centers for Disease Control and Prevention criteria
(5), 23 patients were in stage A and 6 were in stage C. Eight patients had CD4+ cell counts above 400 × 106/liter (median, 511; range, 413 to 832), 9 had counts
between 200 × 106 and 400 × 106/liter (333; 210 to 391), and 12 had counts below
200 × 106/liter (68; 13 to 199). Fourteen
patients were receiving antiretroviral treatment at enrollment in this
study (one drug for 9, two drugs for 3, and three drugs for 2).
Sample preparation and DNA extraction.
Whole blood was
collected in EDTA-containing Vacutainer tubes, and PBMC were isolated
by Ficoll-Hypaque density gradient centrifugation. Plasma was clarified
and stored at 
80°C until viral RNA assays were done. DNA was
extracted using a QIAamp DNA mini kit (QiAgen) and quantified
spectrophotometrically. Cellular and plasma viral culturing was
performed as previously described (33).
Preparation of HIV-2 DNA and RNA assay standards.
DNA
templates were derived from a PCR-amplified region of the
gag gene of HIV-2 strain ROD. HIV-2 ROD DNA in fresh PBMC
was amplified by nested PCR with a first round of long PCR. The
LTR1-5'-LTR2-3' primer pair was used for the first amplification step
as previously described (10). Nested PCR was done using
the following primers containing enzyme restriction sequences at their
5' extremities: QC1Bam, 5'-GATTGCGGATCCGTGGGAGAATGGGCGCGAGA-3',
and GAG OG AS1 Hind,
5'-CTGCATAAGCTTGCCTTCTGAGAGTGCCTGAAATCC-3' (Genset, Paris, France). The resulting PCR product was cloned into vector pGEM3Z (Promega, Madison, Wis.). Plasmids were purified on columns with a
Qiagen kit and quantified by A260 measurements.
To control the validity of our primers, the amplified product was sequenced.
RNA transcripts were obtained from the linearized templates by using a
Riboprobe in vitro transcription system according to the
manufacturer's instructions (Promega). RNA was quantified spectrophotometrically at 260 nm. Transcripts were diluted to 106 copies/µl, divided into aliquots, and stored at
80°C. Subsequent dilutions were made in a solution containing
carrier tRNA (from Escherichia coli; 30 µg/ml; Sigma, St.
Louis, Mo.).
Plasma RNA detection.
Viral RNA was extracted from 1 ml of
plasma and ultracentrifuged for 1 h at 4°C using an HCV specimen
preparation kit (Roche Diagnostics Systems). RNA was amplified by a
one-step procedure using a Titan one-tube RT-PCR kit (Roche) with
primers located in the gag region (GAG OG S1 and GAG OG
AS1). Amplified products were subjected to nested PCR with primers GAG
OG S2 and GAG OG AS2 as previously described (16, 19). The
efficiency and sensitivity of qualitative reverse transcription-PCR for
detection of viral RNA isolated from plasma were determined by using a
transcript standard diluted in 200 µl of RPMI medium to obtain 5,000, 1,000, 500, and 250 copies (seven replicates each). The standard was extracted and processed as described above. The sensitivities of
reverse transcription-PCR were 100% at 5,000, 1,000, and 500 copies/ml
and 66% at 250 copies/ml.
PCR primers and labeled hybridization probe.
In order to
avoid major mismatches due to HIV-2 variability, the primers and probe
were designed using specific software from TibmolBiol (Berlin,
Germany), on the basis of all HIV-2 subtype A and B sequences so far
published, according to the Los Alamos National Laboratory database
(26). Primers used for amplification were located in the
highly conserved gag region: U3,
5'-GGGAGATGGGCGCGAGA-3', and L140,
5'-TCCAACAGGCTCTCTGCTAATCC-3'. Probe S65GAG2 had the sequence 5'-R-TAGGTTACGGCCCGGCGGAAAGA-Q-3', where reporter R
indicates a 6-carboxyfluorescein group and quencher Q indicates a
6-carboxytetramethylrhodamine group conjugated through a linker arm
nucleotide as described previously (35). The specificity
of the primers was evaluated with reference samples (11).
Proviral load quantitation.
Real-time quantitative PCR was
performed using an LC Fast Start DNA master mix hybridization probe kit
(Roche Molecular Diagnostics). Each PCR mixture (20-µl total
volume) contained the following: 2 µl of DNA master mix, 2.4 µl of
25 mM MgCl2, primers U3 and L140 (0.8 µM each), TaqMan probe S65 (100 nM), and 20 µl of H2O qsp. Cycling parameters were
as follows: denaturation for 8 min at 95°C, followed by 45 cycles of
10 at 95°C and 40 at 60°C. For each run, a standard curve was
generated from purified HIV-2 gag plasmid ranging from
5 × 105 to 5 copies. Fresh dilutions of the control
were prepared with salmon sperm DNA (100 ng/ml) before each experiment
from a stock stored at 20°C. Five hundred nanograms of DNA from
each patient was analyzed. Results were expressed in copies per
105 PBMC.
Reproducibility of DNA quantitation with the LightCycler
system.
To assess the inter- and intra-assay reproducibilities of
the LightCycler system, we first analyzed two- and fivefold differences in the starting copy number using 25,000 to 50,000 and 5,000 to 25,000 copies per capillary (12 replicates each). Calculations were determined
with the cycle threshold (CT). We also tested 10-fold differences in
copy number with a broader concentration range: from 5 to 50 copies (10 replicates each), 500 to 5,000 copies (6 replicates), and 50,000 to
500,000 copies (4 replicates). The last experiment was used to assess
the interassay reproducibility (10 runs at each point from 5 to 5 × 105 copies).
| |
RESULTS |
Reproducibility of the LightCycler system for HIV-2 DNA
quantitation.
DNA concentrations of 5,000, 25,000, and 50,000 copies were distinguished with high confidence. The coefficients of
variation (CVs) ranged between 0.31 and 0.56% for the CT and between
7.5 and 12.9% for the copy number (Table
2). At a high virus concentration (mean,
534,340 copies per 105 PBMC), the intra-assay CV was 16%.
At a mean copy number of 586,692 per 105 PBMC, the
interassay CV was 40%. At the lowest concentration (five copies), the
intra-assay CV was 41% and the interassay CV was 39%. Intra- and
interassay CVs are summarized in Table 3.
Sensitivity of the LightCycler system for HIV-2 DNA
quantitation.
The sensitivities of the assay on the basis of
repeated testing (eight replicates) of the lower concentrations of our
plasmid standard were 100% at 5 copies, 62% at 2.5 copies, and 75%
at 1 copy. Consequently, we set the detection limit of the assay at
five copies.
HIV-2 proviral DNA detection according to subtype.
DNA viral
load, expressed as copies per 105 PBMC, was determined for
the 29 HIV-2-infected patients. DNA was detected in 24 (83%) of the 29 patients and was consistently below five copies in the other 5 patients. The lack of HIV-2 provirus amplification was not linked to
the subtype, three subtype A and two subtype B samples being below the
cutoff. This result indicates that the selected primers and probes
cover a wide range of HIV-2 diversity.
HIV-2 proviral load is high whatever the CD4+ cell
count.
Levels of proviral DNA ranged from 5 to 2,524 copies per
105 PBMC, with a median of 201 copies/105 PBMC.
These values are similar to those previously reported for both HIV-2
and HIV-1 (1, 2, 8, 15, 27, 29, 31). DNA viral load in
these 29 patients was then examined according to the following
CD4+ cell count categories: <200/mm3 (all
these patients being on antiretroviral therapy), 200 to 400/mm3, and >400/mm3. The respective median
proviral loads per 105 cells were 285 (12; 53 to 2,524 [n, range]), 114 (9; <5 to 1,907), and 121 (8; <5
to 712). This trend toward increasing proviral load according to
CD4+ cell count was not significant. All the samples with
negative provirus amplification were from patients with
CD4+ cell counts above 328 × 106 cells/liter.
Viral load did not differ significantly among these three
categories of CD4+ cell count (
2 = 3.071; P < 0.3; Kruskal-Wallis test) (Fig.
1).
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FIG. 1.
HIV-2 proviral load according to CD4+ cell
count category. Horizontal lines represent median values in each
category. Samples below the limit of detection were given a value of
four copies/105 PBMC. The number of samples in each
category is shown. There is no correlation among the three groups
(2 = 3.071; P < 0.3;
Kruskal-Wallis test).
|
|
HIV-2 proviral load does not influence plasma RNA detection.
There was no significant relationship between the level of HIV-2
proviral load and the rate of plasma RNA detection (U = 61; P < 0.07, Mann-Whitney test). All but one of
the patients with a CD4+ cell count below 200 × 106/liter were positive for plasma RNA; the remaining
patient was receiving antiretroviral tritherapy (Table
4). A correlation was found between
positive cell culture and viral DNA load (U = 26;
P < 0.02; Mann-Whitney test).
View this table:
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|
TABLE 4.
Relationships among the CD4+ cell
count, HIV-2 proviral load, cell culture positivity, and plasma RNA
positivity
|
|
| |
DISCUSSION |
We evaluated a real-time PCR assay with the LightCycler system for
HIV-2 proviral DNA quantitation in clinical specimens. The LightCycler
system is simple and rapid and offers a standardized approach to HIV-2
DNA quantitation. It also avoids false-positive results due to PCR contamination.
In our experiments, the reproducibility of the system was similar to
that of commercial viral load kits (9, 25). The high
sensitivity of our assay (detection limit, 5 DNA copies/105
cells) must be emphasized, as it provides ample opportunities for
further clinical studies. Given the importance of HIV-2 genetic diversity, we first tried to select conserved and efficient segments of
the viral genome to use as primers. During a previous evaluation (data
not shown) with different gag primers (U5,
5'-GAGAATGGGCGCGAGAAACT-3', and L130,
5'-AATTCATTCGCTGCCCACAC-3'), we successfully amplified all
the A subtypes but failed to amplify any B subtypes. This result
convinced us of the underlying importance of primer and probe design in
genome-based detection assays. Previous studies of HIV-2 quantitation
were performed in West Africa, where only subtype A circulates
(1, 2, 31). In contrast, subtype B is frequent in
HIV-2-infected patients living in France (11) and probably
in other Western countries as well (18), creating the need
to assess a large panel of representative HIV-2 strains.
The levels of HIV-2 proviral DNA found in the 29 patients tested here
were high and similar to those observed in studies of HIV-2 subtype A
infections (1, 2, 15). We found no significant differences
in the amount of proviral DNA according to subtype. Likewise, we found
no correlation between proviral DNA level and CD4+ cell
count or viral RNA positivity. These results for DNA quantitation are
similar to those recently reported by Popper et al. (29) and Gomes et al. (15), although other studies have shown a
correlation between these parameters (2, 27, 31). Popper
et al. also reported a lack of correlation between levels of viral RNA
and proviral DNA (29).
Because of the low pathogenicity of HIV-2 compared to that of
HIV-1, we were intrigued in find low HIV-2 proviral loads but relatively large amounts of HIV-2 DNA in our patients. A selection bias
can be ruled out, as the majority of the patients were symptom free and
the CD4+ cell count distribution was normal. In a
community-based study, Ariyoshi et al. found similar high levels of
HIV-2 DNA, regardless of the percentage of CD4+ cells
(1). Although not statistically significant, we tended to
find higher levels of HIV-2 DNA and a trend toward lower
CD4+ cell counts with more advanced stages of infection.
The effect of antiretroviral treatment in patients with
CD4+ cell counts of <200/µl could explain the lack of
correlation between proviral loads and CD4+ cell counts.
Alternatively, our results could be due to a high rate of defective
integrated virus. Low cellular pathogenicity of HIV-2 in the early
stages of infection, together with the long duration of the
symptom-free period, could explain the high proviral loads. As with
human T-cell leukemia virus type 1 infection, where high proviral loads
have been reported (13, 20, 34), normal proliferation of
infected cells could explain the large number of PBMC harboring
proviral DNA. This discordance between high proviral loads and
inefficient replication of HIV-2 might help to explain the roles of
viral replication and immune responses in HIV-2 disease.
| |
ACKNOWLEDGMENT |
This study was supported by French National Agency on AIDS
Research (A.N.R.S.) grant 98015.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratoire de
Virologie, Hôpital Bichat-Claude Bernard, 46 Rue Henri Huchard,
75877 Paris Cedex 18, France. Phone: 00 33 1 40 25 88 96. Fax: 00 33 1 40 25 67 69. E-mail:
florence.damond{at}bch.ap-hop-paris.fr.
| |
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Journal of Clinical Microbiology, December 2001, p. 4264-4268, Vol. 39, No. 12
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.12.4264-4268.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
