J Antimicrob Chemother 2013; 68: 2875 – 2881
doi:10.1093/jac/dkt290 Advance Access publication 18 July 2013
Characterization of CXCR4-using HIV-1 during primary infection
by ultra-deep pyrosequencing
Stéphanie Raymond1–3*, Adrien Saliou1, Florence Nicot1,3, Pierre Delobel1,2,4, Martine Dubois1,3, Romain Carcenac3,
Karine Sauné1–3, Bruno Marchou4, Patrice Massip4 and Jacques Izopet1–3
1
INSERM, UMR1043, Toulouse, France; 2Université Toulouse III Paul-Sabatier, Faculté de Médecine Toulouse-Purpan, Toulouse, France;
3
CHU de Toulouse, Hôpital Purpan, Laboratoire de Virologie, Toulouse, France; 4CHU de Toulouse, Hôpital Purpan,
Service des Maladies Infectieuses et Tropicales, Toulouse, France
*Corresponding author. CHU de Toulouse, Hôpital Purpan, Laboratoire de Virologie, Toulouse, France. Tel: +33-5-67-69-04-24;
Fax: +33-5-67-69-04-25; E-mail: [email protected]
Received 22 February 2013; returned 20 May 2013; revised 14 June 2013; accepted 16 June 2013
Objectives: R5 viruses have long been thought to account for almost all strains present in primary HIV-1 infections
(PHIs), but recent studies using sensitive phenotypic assays have revealed that 3%–6.4% of subjects also harbour
CXCR4-using viruses. Phenotypic assays provide only qualitative results: the presence or absence of CXCR4-using
viruses. We have therefore used ultra-deep pyrosequencing (UDS) to determine the frequency of CXCR4-using
viruses among HIV-1 quasispecies.
Methods: We first screened 200 patients for HIV-1 tropism using a sensitive phenotypic assay during PHI and identified 11 infected with an R5X4 dual/mixed (D/M) virus population. We then used UDS of the V3 env region with the
geno2pheno algorithm (false positive rate¼ 5.75) to identify the HIV-1 quasispecies.
Results: CXCR4-using viruses were detected in all but 1 of the 11 patients by UDS, and accounted for 0.2% –100% of
the virus populations. The frequency of CXCR4-using viruses was ,20% in six subjects and 100% in four subjects.
Virus populations containing 100% CXCR4-using variants during PHI persisted for at least 1–2 years after the acute
phase. The CCR5 D32 heterozygous genotype was similarly prevalent in patients infected with D/M (27%) and R5
(15%) viruses.
Conclusions: UDS and the phenotype were concordant for determining HIV-1 coreceptor usage. UDS analysis indicated large differences in the percentage of CXCR4-using viruses in the HIV-1 quasispecies during PHI. Further
studies should examine the impact of the proportion of CXCR4-using viruses on disease prognosis.
Keywords: HIV quasispecies, V3 genotype, chemokine receptor, HIV tropism, HIV envelope protein gp120
Introduction
HIV-1 enters CD4-expressing cells using one or both of the chemokine receptors CCR5 and CXCR4. Virus strains can be classified as R5,
X4 or R5X4 variants depending on their use of one or both coreceptors.1 But the complexity of the virus quasispecies makes it difficult
to determine virus coreceptor usage in a given HIV-infected individual. Population-based assessments of HIVcoreceptor usage do not
determine whether HIV isolates that use both CCR5 and CXCR4 coreceptors are mixtures of pure R5 and X4 monotropic variants
or contain truly R5X4 dual-tropic virus clones. Neither do these
approaches estimate the proportion of each type of variant in a
mixture. Phenotypic and genotypic methods have been developed
to assess HIV-1 coreceptor use.2 Bulk phenotyping can discriminate between pure R5 populations, pure X4 populations and R5X4
dual/mixed (D/M) populations.3 – 5 Bulk genotyping of the V3 env
region can predict the presence of viruses that use CXCR4 for
entry and is easier to perform in clinical practice.6,7 But the poor
sensitivity of bulk genotyping for detecting minor variants among
the quasispecies may lead to the virological failure of treatment
with a CCR5 antagonist.8,9 Genotyping can be improved by ultradeep pyrosequencing (UDS), which provides a new method for
studying the clones within an HIV-1 population.8,10
R5 viruses predominate during the early stage of HIV-1 infection,
probably by selective transmission or because R5 virus infections are
more readily established.11 CXCR4-using viruses emerge later and
are associated with the accelerated decline of CD4+ T lymphocytes
and progression to AIDS.1,12 – 14 However, CXCR4-using viruses have
been found even during primary infection, with a prevalence estimated at 3%–6.4% by bulk phenotyping and up to 17.2% by bulk
# The Author 2013. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.
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Raymond et al.
genotyping methods.15 – 19 The transmission of CXCR4-using viruses
may be favoured by a defect in the expression of CCR5 at the cell
surface. Individuals who are homozygous for a 32 bp deletion in
CCR5 cannot be infected by R5 viruses but are susceptible to infection
with CXCR4-using viruses.20 Moreover, a primary HIV-1 infection
(PHI) with CXCR4-using viruses is associated with a more rapid
disease progression than is a primary infection with pure R5
viruses.18 The transmitted D/M virus populations have not been
well characterized and the proportion of CXCR4-using viruses may
influence disease progression.
We have used UDS to determine the composition of the virus
quasispecies during PHI in patients infected with D/M viruses identified using a sensitive phenotypic assay. We determined the evolution of the CXCR4-using viruses in the quasispecies in untreated
patients. Lastly, we looked for a link between the transmission of
CXCR4-using viruses and the D32 deletion in CCR5 gene of the host.
Methods
Patient selection and clinical samples
We selected 11 patients from a total of 200 patients screened for HIV-1
tropism during primary infection (Table 1) because of their D/M virus
tropism determined using a sensitive bulk phenotypic assay. These patients
were treated at the Department of Infectious Diseases of Toulouse University Hospital, France and all gave their informed consent for pathophysiological studies (ethics committee approval was not required). Recent
HIV-1 seroconversion was defined as: (i) subjects with a negative
anti-HIV-1 antibody test and positive plasma HIV-1 RNA; (ii) a positive
HIV-1 antibody test and a negative or indeterminate immunoblot, later
confirmed positive; or (iii) patients who were seronegative when tested
within the previous 6 months, but who subsequently became HIV-1 seropositive. The time since HIV infection was estimated using the Fiebig
classification (Table 1). Analysis of the env sequences using the US National
Center for Biotechnology Information genotyping tool (http://www.ncbi.
nlm.nih.gov/projects/genotyping/formpage.cgi) indicated that nine
patients were infected with subtype B HIV-1 and two with non-B subtypes
(CRF01-AE and CRF02-AG). The median age of the patients was 26 years
(IQR, 21–36) and 64% were men. Their median CD4+ T cell count was
359 cells/mm3 (IQR, 287– 618) and their median plasma HIV-1 RNA load
was 5.8 log10 copies/mL (IQR, 4.7–6.2). Plasma samples were collected
from all 11 patients and stored at 2808C. The HIV transmission routes,
virus subtypes, plasma HIV-1 RNA and CD4+ T cell counts are summarized
in Table 1. The 189 blood samples with an R5 phenotype during PHI included
125 that were available to determine the CCR5 polymorphism in the D32
deletion.
Phenotypic characterization of HIV-1 coreceptor usage
HIV-1 tropism was determined using the Toulouse Tropism Test (TTT)
recombinant phenotypic assay.4 Briefly, a fragment encompassing the
gp120 and the ectodomain of gp41 was amplified with RT–PCR using
HIV-1 RNA isolated from plasma or with PCR using HIV-1 DNA taken from
peripheral blood mononuclear cells (PBMCs). The PCR products then underwent nested PCR. The phenotype of HIV-1 coreceptor usage was determined using a recombinant virus entry assay with the pNL43-Denv-Luc2
vector and the product of the nested PCR obtained from the challenged
HIV-1-containing sample. The chimeric recombinant virus particles were
used to infect U87 indicator cells bearing CD4 and either CCR5 or CXCR4.
Virus entry was assessed by measuring the luciferase activity in lysed
cells (as relative light units). The specificity of the positive signals was
confirmed using the CXCR4 antagonist AMD-3100 obtained through the
NIH AIDS Research and Reference Reagent Program21 and the CCR5 antagonist maraviroc (Pfizer). CXCR4-using viruses were detected when they
accounted for 0.5% or more of the total population.
UDS of V3 env
UDS was performed using a 454 GS Junior. A fragment of 415 nucleotides,
encompassing the V3 env region, was generated by nested RT– PCR carried
out in duplicate as previously described.22 The nested PCR was performed
with the Expand High Fidelity Plus PCR System (Roche Diagnostics) under
the following conditions: 1 cycle of 948C for 2 min, 35 cycles of 948C for
30 s, 608C for 30 s, 728C for 3 min, followed by a final extension at 728C
for 7 min. The amplified PCR products were purified using Agencourt
AMPure PCR purification beads (Beckman Coulter, Brea, CA) and quantified
with the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen) on a LightCycler 480 (Roche). Pooled PCR products were clonally amplified on capture beads in water-in-oil emulsion microreactors. A total of 500000
Table 1. Characteristics of 11 patients infected with R5X4 D/M viruses at the primary stage of HIV-1 infection
Patient
1
2
3
4
5
6
7
8
9
10
11
CCR5
polymorphisma
Transmission
route
Fiebig
classification
HIV-1
subtype
Plasma HIV-1 RNA
(log10 copies/mL)
CD4+ T cell
count (cells/mm3)
Antiretroviral
treatment during
PHI
wt/wt
wt/wt
wt/D32
wt/wt
wt/D32
wt/wt
wt/wt
wt/wt
wt/wt
wt/wt
wt/D32
sexual
sexual
IDU
sexual
IDU
sexual
sexual
sexual
sexual
sexual
sexual
V
III
V
IV
V
V
II
V
II
IV
IV
CRF01
B
B
B
B
B
B
CRF02
B
B
B
2.69
5.9
5.58
3.83
5.78
4.34
5
5.89
6.54
6.6
4.10
796
456
940
412
240
776
359
333
164
477
759
no
yes
no
no
yes
no
no
yes
yes
no
no
IDU, intravenous drug use.
wt, wild-type allele; D32, mutant allele. wt/wt is a homozygous genotype and wt/D32 is a heterozygous genotype.
a
2876
JAC
Primary infection with CXCR4-using HIV-1
DNA-enriched beads were deposited in the wells of a full GS Junior Titanium
PicoTiterPlate device and pyrosequenced in both forward and reverse directions. The 200 nucleotide cycles were performed in a 10 h sequencing run.
Phylogenetic analyses excluded any possibility of sample contamination
(data not shown).
Determination of the D32 polymorphism in CCR5
The D32 deletion was identified in DNA from PBMCs or from plasma
by real-time PCR as described previously.24
Statistics
Genotypic prediction of HIV-1 coreceptor usage from UDS
data
The sequence reads of the V3 env regions were quantified with GS Amplicon
Variant Analyzer (AVA) software Version 2.5p1 (Roche). The AVA software
assigns each read to the proper amplicon and sample using multiplex
identifiers. The sequence reads were aligned with the R5 strain HIV-1 BaL
consensus sequence, and sequence alignments were manually edited to
correct for insertions or deletions in homopolymeric regions that would
result in a frame shift. The tropism of each virus clone was inferred from
the V3 amino acid sequence by the geno2pheno tool (false positive
rate¼5.75).22,23 The false positive rate can be adjusted for choosing how
conservative the prediction of CXCR4 usage will be. With a false positive
rate¼5.75, the specificity is expected to be around 95%. Geno2pheno is
available at http://coreceptor.bioinf.mpi-sb.mpg.de/cgi-bin/coreceptor.pl
(November 2012).
Sensitivity of UDS for detecting minor V3 env variants
The frequency of errors resulting from V3 amplification and deep sequencing was assessed by analysing the pyrosequencing data for a panel of 10
plasmid clones of env sequenced by the Sanger method. The mean error
rate of pyrosequencing was 0.031% (99% CI, 0.005%–0.056%). The
upper confidence limit of the error rate was used to calculate the sensitivity
of pyrosequencing for a given number of reads. Poisson distribution was
used to distinguish authentic variants from artefactual V3 sequences
resulting from errors arising during PCR amplification and UDS. P values
of ,0.001 were considered to be statistically significant. The detection
threshold of minor X4 variants varied according to the number of reads of
V3 for each sample.
Continuous variables were tested with the Mann– Whitney test. Categorical
variables were tested using the x2 test or Fisher’s exact test. A statistically
significant difference was defined as a P value of ,0.05.
Results
Characterization of HIV-1 quasispecies by UDS
An average of 4737 analysable reads of V3 per sample were
obtained by UDS. Nucleotide sequences were translated into
amino acid sequences and HIV-1 tropism was predicted using
the geno2pheno algorithm. The number of unique clones ranged
from 1 to 11 per sample (Table 2). UDS detected CXCR4-using
viruses in 10 samples in proportions ranging from 0.2% to 100%
of the total virus quasispecies. Four samples contained only
CXCR4-using clones. Sample 5 contained 18.4% CXCR4-using
clones and five other samples contained minor CXCR4-using variants accounting for 0.2% –2.9% of the virus quasispecies. The
last sample contained only one clone that used CCR5 for entry,
although its phenotype was R5X4. This discordance between the
results of genotyping by UDS and phenotyping by the TTT was confirmed in two independent experiments. This virus was subtype B
and the subject was heterozygous for the CCR5 D32 deletion. The
proportions and tropism of each variant in the 11 quasispecies
are shown in Figure 1.
Evolution of HIV-1 quasispecies during the early stage
of infection
After a median interval of 17 months we re-determined the HIV-1
tropism by UDS in five of the seven subjects who were not given
Table 2. HIV-1 quasispecies during PHI and their evolution in untreated patients
HIV-1 tropism at primary infection, UDS of the env V3
region using geno2pheno 5.75
Patient
1
2
3
4
5
6
7
8
9
10
11
HIV-1 tropism in untreated patients, UDS of the env V3 region using
geno2pheno 5.75
no. of reads
no. of variants
percentage of
CXCR4-using clones
timea (months)
no. of reads
no. of variants
percentage of
CXCR4-using clones
5330
4350
4703
4618
3381
6494
5938
3647
2172
6082
5395
2
5
2
5
11
5
5
10
4
2
1
100
100
100
100
18.43
2.9
1.2
1.04
0.8
0.2
,0.07
—
—
23
13
—
12
—
—
—
22
17
—
—
3445
2391
—
1296
—
—
—
5755
2814
—
—
25
14
—
10
—
—
—
4
7
—
—
98.52
100
—
0.4
—
—
—
,0.07
30
a
Time after diagnosis of PHI.
2877
Raymond et al.
Subject 1
Subject 3
Subject 2
0.3% X4
99.7% X4
0.3% X4
98.6% X4
5330 reads
4350 reads
Subject 4
Subject 5
0.3% X4
0.2% X4
92.4% X4
7.6% X4
3381 reads
12.8%
R5
0.8%
0.4%
0.3% X4
99.0% X4
0.4% X4
0.3% X4
0.3% X4
7.6%
67.5% R5
4.7%
2.4%
0.2% X4
1.7% X4
1.4%
0.5%
4703 reads
4618 reads
Subject 6
96.4% R5
0.6% R5
0.1% R5
0.3% X4
2.6% X4
2878
97.5% R5
1.2% R5
0.3% R5
1% X4
0.2% X4
Subject 11
Subject 10
0.2% R5
0.2% R5
0.8% X5
97.3% R5
3647 reads
5938 reads
Subject 9
2172 reads
Subject 8
Subject 7
6494 reads
98.8% R5
0.3%
100% R5
99.8% R5
0.2% X4
6082 reads
5395 reads
0.3%
0.3%
0.3% R5
0.2%
0.2%
0.2%
0.4%
0.4% X4
0.3%
JAC
Primary infection with CXCR4-using HIV-1
Table 3. Percentage of CXCR4-using viruses at PHI and patient characteristics
HIV-1 tropism using UDS and Geno2pheno 5.75
Characteristic at baseline
Median age, years (IQR)
CCR5 genotype: wt/D32a, n (%)
Transmission route, n (%)
sexual
IDU
HIV-1 RNA load (log copies/mL), median (IQR)
T CD4+ count (cells/mm3), median (IQR)
Antiretroviral treatment during PHI, n (%)
exclusively CXCR4-using variants (n¼4)
27 (22– 34)
1 (25)
3 (75)
1 (25)
4.7 (3.55–5.67)
626 (445–832)
1 (25)
minor CXCR4-using variants (n ¼6)
28 (19– 37)
1 (17)
5 (83)
1 (17)
5.83 (5.20–6.38)
346 (263–448)
3 (50)
P value
1
1
1
0.26
0.11
0.57
IDU, intravenous drug use.
wt/D32: heterozygous genotype with one wild-type allele (wt) and one mutant allele (D32).
a
antiretroviral treatment during PHI (Table 2). Each of these second
samples contained 4 –25 unique clones, whereas samples from
the same patients contained 1 –5 variants at the time of primary
infection. Two subjects infected with virus populations consisting
of 100% CXCR4-using variants during PHI still harboured 98.52%
and 100% CXCR4-using variants after 23 and 13 months of followup, respectively. One of the two subjects infected with ,5%
CXCR4-using variants during PHI harboured only pure CCR5-using
variants after 22 months, while the other still harboured minor
X4 variants (0.4% of the quasispecies) after 12 months. Patient
11 was infected with viruses that harboured no X4 genotypic determinants despite an R5X4 phenotype during PHI, but this genotype –phenotype discordance disappeared in the follow-up; UDS
detected 30% of viruses genotyped as CXCR4-using variants at
month 17, in agreement with the phenotype.
Relationships between the percentage of CXCR4-using
viruses and the patients’ characteristics
We determined whether the patients harbouring minor
CXCR4-using variants (0.2% –18.4%, n¼ 6) and a majority of R5
viruses differed from those harbouring exclusively CXCR4-using
viruses (n¼ 4) and no R5 viruses (Table 3). The HIV-1 transmission
route, HIV-1 virus load and the CD4+ T cell count at the time of PHI
were similar in both groups. The four patients given antiretroviral
treatment during acute HIV infection included one who harboured
pure CXCR4-using variants and three others who harboured only
minor proportions of CXCR4-using viruses.
Impact of the D32 deletion in CCR5 on HIV-1 tropism
during PHI
We determined the CCR5 polymorphism for the D32 deletion of the
11 D/M-infected patients (Table 1) and of 125 consecutive
R5-infected patients screened during PHI. The D/M group was
made up of eight patients with homozygous wild-type CCR5 and
three that were heterozygous for D32. The 125 R5-infected
patients included 19 who were heterozygous for D32. None of
our patients was D32 homozygous. The prevalence of the D32 deletion was thus 27% in the D/M group and 15% in the R5 group
(Fisher’s exact test, P ¼ 0.38).
Discussion
Primary infection is a crucial stage for understanding the pathogenesis of HIV-1 infection. Many studies have shown that
CCR5-using viruses predominate during early infection, but that
CXCR4-using viruses can also be transmitted.1,15,17,18 Populationbased methods have been used to infer HIV-1 tropism, but the
viruses making up the quasispecies were not well characterized.15 – 19 As CXCR4-using viruses have a negative impact on
the clinical evolution of an HIV-1 infection13,18 and a primary infection with D/M viruses implies the presence of both CCR5-using
and CXCR4-using viruses, it is important to know the percentage
of CXCR4-using viruses in the quasispecies. In a previous study,
we accurately detected CCR5-using and CXCR4-using viruses
using UDS during PHI and showed a CXCR4-using virus frequency
similar to that found using TTT.23 Thus we used UDS to analyse
the components of D/M virus quasispecies during PHI and hence
the composition of the HIV-1 population.
We used a phenotypic assay to screen 200 patients for HIV-1
tropism during PHI and selected 11 who were infected with D/M
viruses. Their HIV quasispecies were then explored by UDS. The
HIV-1 tropism determined using UDS and the phenotypic assay
were concordant for 10 of the 11 patients, as previously shown
in other populations.10,22 UDS revealed that four patients
(36%) were infected with virus populations containing exclusively
CXCR4-using variants, with no pure R5 variants. The genotypic
algorithms cannot distinguish X4 from R5X4 clones because the
determinants in their V3 env regions are similar. All are thus
Figure 1. Proportion of V3 env variants among the quasispecies and prediction of HIV-1 coreceptor use during PHI. V3 sequences were generated by UDS
and coreceptor usage was predicted using geno2pheno 5.75. Each pie chart represents the virus quasispecies in an individual subject. The sectors are
proportional to the frequencies of the V3 amino acid variants whose tropism is indicated to be R5 (light grey) or X4 (dark grey). The number of reads
obtained with UDS is indicated beneath each pie chart. The quasispecies harboured by subjects 1– 10 contained CXCR4-using variants, whereas the
quasispecies infecting subject 11 were pure CCR5-using viruses.
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Raymond et al.
classified as CXCR4-using viruses by genotyping.25 The susceptibility to treatment with CCR5 antagonists of virus populations containing minor CXCR4-using variants during PHI (0.2%– 18.4%)
and a majority of R5 viruses could vary, but the clinically relevant
cut-off of CXCR4-using variants above which a CCR5 antagonistbased treatment leads to virological failure is still unknown. It
may depend on whether CXCR4-using variants are pure X4-tropic
clones, which seems quite unusual, or R5X4 clones. Moreover, the
replication of R5X4 clones could be partly inhibited by CCR5 antagonists, depending on their affinities for CCR5 and CXCR4 receptors.
The phenotypic assay detected very low percentages (0.2% or
1%) of CXCR4-using variants. We reanalysed the virus populations
of two of the five patients with ,5% CXCR4-using clones after the
PHI. The patient who harboured 2.9% X4 variants during PHI still
harboured minor CXCR4-using variants (0.4%) after 12 months,
while the patient with 0.2% CXCR4-using variants harboured exclusively R5 variants after 22 months. Perhaps a very low frequency of
CXCR4-using variants during PHI indicates that these clones have
an overall disadvantage that prevents their further expansion
in competition with more-fit R5 viruses. If so, determining the
percentage of CXCR4-using variants by ultrasensitive sequencing
methods at PHI would provide substantial data for predicting
the evolution of virus tropism in subsequent years.
Patient 11 was infected with an R5X4 virus according to the
phenotypic assay, but with an R5 genotype according to UDS.
The discordance could be explained by analysis of the short V3
env region for the genotypic prediction, whatever the prediction algorithm used, while determinants outside V3 could influence
HIV-1 coreceptor use.26 – 28 The sensitivity of UDS for detecting
minor variants (0.07% –0.2% in our dataset) and the sensitivity
of the phenotypic assay (0.5%) were similar; hence this cannot
explain the failure to detect CXCR4-using variants.4 Patient 11 unexpectedly harboured 30% CXCR4-using variants 17 months after
PHI without any selective pressure from antiretroviral drugs. The
detection of X4 variants shortly after PHI, plus a high CD4+ T cell
count (550 cells/mm3), tends to confirm that these variants had
been transmitted rather than emerged.
The proportion of patients with D/M HIV-1 infection during PHI
has previously been estimated to be between 3 and 17%.15 – 17
The selective advantage of CCR5-tropic viruses in establishing an
HIV-1 infection may be reduced if the host has a polymorphism
in CCR5 that lowers the amount of CCR5 at the cell surface. The
most frequent polymorphism in CCR5 is a 32 bp deletion whose
heterozygous prevalence is estimated to be about 16% in the
French Caucasian population.29,30 A few cases of PHI with
CXCR4-using viruses have been reported in subjects homozygous
for the CCR5 D32 deletion. The consequences of heterozygous carriage of this genotype on the transmission rate of CXCR4-using variants are less clear. We determined the prevalence of the D32
deletion in the group of D/M-infected patients and in 125 consecutive R5-infected patients at the early stage of infection. The prevalence of the D32 deletion in the D/M-infected patients (27%) and in
the R5-infected patients (15%) was not different. However, the
small number of D/M-infected subjects during PHI is a limiting
factor in these studies. The lack of relationship between HIV-1
tropism and the D32 deletion should be confirmed in larger
cohorts of patients at the stage of PHI. Another limitation is
the potential influence of the route of transmission. Two of our
D/M-infected patients were infected via the parenteral route and
were D32 heterozygous. Previous studies have shown a correlation
2880
between infection with CXCR4-using viruses and the parenteral
transmission of HIV-1,15,18,31,32 suggesting that R5 viruses cross
the mucosal barrier most readily, while other variants can enter
via the parenteral route.11 The D32 deletion in CCR5 may be an additional factor favouring the transmission of CXCR4-using viruses via
the parenteral route, but further studies are needed. By contrast,
the abundance of CCR5-expressing target cells in genital and
rectal mucosae, where the CXCR4 ligand SDF-1 is highly concentrated, favours the sexual transmission of R5 viruses.
We monitored five patients given no antiretroviral treatment for
about 17 months after PHI. One of the two patients harbouring
,5% CXCR4-using variants during PHI was still infected with
CXCR4-using minor variants, whereas the two patients infected exclusively with CXCR4-using variants still had these variants as a
dominant population after the acute phase of the infection, as
shown previously in patients infected with CXCR4-tropic viruses
harbouring mutations associated with antiretroviral resistance
during PHI.33 The dominance of CXCR4-using viruses from PHI
could negatively influence the rate at which an HIV-1 infection
evolves, as already reported.18,34
In conclusion, we have determined the composition of 11 virus
quasispecies with D/M tropism during PHI and observed various
proportions of CXCR4-using variants that may influence subsequent disease progression. We found that a virus population containing only CXCR4-using viruses can persist long after PHI with
no link to CCR5 D32 deletion. Further studies are needed to determine the impact of the proportion of CXCR4-using variants on
disease progression and susceptibility to CCR5 antagonist-based
therapy. Knowledge of the viruses present very early in an infection
may lead to a better understanding of HIV pathophysiology
and the design of antiretroviral therapies.
Funding
This work was supported by the Institut National de la Santé et de la Recherche Médicale U1043.
Transparency declarations
None to declare.
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