JOURNAL OF VIROLOGY, Jan. 2004, p. 968–979
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 78, No. 2
Continued Production of Drug-Sensitive Human Immunodeficiency
Virus Type 1 in Children on Combination Antiretroviral Therapy
Who Have Undetectable Viral Loads
Deborah Persaud,1* George K. Siberry,1Aima Ahonkhai,2Joleen Kajdas,1Daphne Monie,2
Nancy Hutton,1Douglas C. Watson,3Thomas C. Quinn,2,4Stuart C. Ray,2
and Robert F. Siliciano2,5
Department of Pediatrics1and Department of Medicine,2Johns Hopkins University School of Medicine, Department of Pediatrics,
University of Maryland School of Medicine,3and Howard Hughes Medical Institute,5Baltimore, and National Institute
of Allergy and Infectious Diseases, National Institutes of Health, Bethesda,4Maryland
Received 11 July 2003/Accepted 3 October 2003
Highly active antiretroviral therapy (HAART) can suppress plasma human immunodeficiency virus type 1
(HIV-1) levels to below the detection limit of ultrasensitive clinical assays. However, HIV-1 persists in cellular
reservoirs, and in adults, persistent low-level viremia is detected with more sensitive assays. The nature of this
viremia is poorly understood, and it is unclear whether viremia persists in children on HAART, particularly
those who start therapy shortly after birth. We therefore developed a reverse transcriptase PCR (RT-PCR)
assay that allows genotyping of HIV-1 protease even when viremia is present at levels as low as 5 copies of
HIV-1 RNA/ml. We demonstrated that viremia persists in children with plasma virus levels below the limit of
detection of clinical assays. Viremia was detected even in children who began HAART in early infancy and
maintained such strong suppression of viremia that HIV-1-specific antibody responses were absent or minimal.
The low-level plasma virus lacked protease inhibitor resistance mutations despite the frequent use of nelfi-
navir, which has a low mutational barrier to resistance. Protease sequences resembled those of viruses in the
latent reservoir in resting CD4?T cells. Thus, in most children on HAART with clinically undetectable viremia,
there is continued virus production without evolution of resistance in the protease gene.
The treatment of human immunodeficiency virus type 1
(HIV-1) infection with highly active antiretroviral therapy
(HAART) significantly reduces the levels of viral RNA in
plasma and lymphoid tissue (3, 15, 17, 26, 27, 31). In many
treated adults and children, free virus becomes undetectable in
the plasma when measured by ultrasensitive clinical viral load
assays that have a detection limit of 50 copies of HIV-1 RNA/
ml. Despite the absence of clinically detectable viremia, how-
ever, ongoing viremia remains detectable when more-sensitive
reverse transcriptase PCR (RT-PCR) assays are used (8, 20,
28). The nature and clinical significance of ongoing virus pro-
duction during effective HAART remain elusive.
Several mechanisms may contribute to the persistence of
viremia during effective HAART. These include the inability
of HAART to completely suppress virus replication because of
inadequate potency (14), intermittent nonadherence resulting
in suboptimal drug concentrations (30), and the emergence of
drug-resistant variants (19). Ongoing cycles of replication in
the setting of HAART could lead to the accumulation of drug
resistance mutations and treatment failure. Another possible
explanation for ongoing low-level viremia is the continued
production of HIV-1 by infected cells harbored in viral reser-
voirs or drug sanctuary sites (1, 20, 34). One such reservoir that
is established during acute infection is a small pool of latently
infected resting memory CD4?T cells (5, 6). This latent res-
ervoir has been shown to retain HIV-1 in a replication-com-
petent form despite many years of suppression of viremia to
?50 copies/ml (6, 11, 12, 32, 38, 40, 45).
Ongoing viremia is a particular concern with HIV-1-infected
children who potentially face a full lifetime of treatment. The
extent to which low-level viremia continues in children treated
with HAART, particularly those treated from infancy, and the
clinical significance of this viremia have not been defined. In an
initial cross-sectional study, we detected and sequenced plasma
viruses in four of seven children on HAART who had plasma
virus levels below 50 copies/ml. These viruses were archival
wild-type or pre-HAART drug-resistant variants rather than
recently derived drug-resistant mutants (20). To more fully
characterize ongoing viremia in children, we have developed a
more sensitive RT-PCR assay which we have used to better
define the frequency of ongoing viremia and the evolution of
drug resistance mutations in the protease gene in children ini-
tiating suppressive HAART during acute and chronic HIV-1
infection. Our results provide insight into the nature and clin-
ical significance of low-level viremia in patients on HAART.
MATERIALS AND METHODS
Patients. We studied low-level viremia in acutely and chronically infected
children who had durable suppression of HIV-1 replication on HAART for 1 to
5 years. Study participants were recruited from the pediatric specialty clinics at
Johns Hopkins University and the University of Maryland. Written informed
consent approved by the institutional review boards was obtained from the
parents or guardians of the children. A total of 15 HIV-1-infected children were
eligible for study during the period from April 2002 to February 2003.
Six of children (C2, C7, C8, C10, C11, and C22) were from a previously
characterized cohort and had participated in longitudinal studies of the latent
* Corresponding author. Mailing address: Department of Pediatrics,
Johns Hopkins University School of Medicine, Park 256, 600 North
Wolfe St., Baltimore, MD 21205. Phone: (410) 614-3917. Fax: (410)
614-1491. E-mail: firstname.lastname@example.org.
reservoir in resting CD4?T cells (20, 32, 38). The inclusion of this well-studied
group of children allowed for validation of the novel methodologies used for this
study and for the evaluation of the phylogenetic relatedness of plasma virus to
replication-competent latent HIV-1 retained in resting CD4?T cells during
years of effective therapy.
HIV-1 RNA isolation, amplification, and sequencing from small blood vol-
umes. Virus particles were pelleted from 3 to 4 ml of plasma by ultracentrifu-
gation at 17,000 ? g for 2 h at 4°C in a Heraeus centrifuge. Virus particles were
then lysed, and the RNA was isolated using a Qiagen column purification
method according to the manufacturer’s directions. Isolated RNA was treated
with DNase (Invitrogen Corp, Carlsbad, Calif.) and divided into a total of seven
to nine reaction tubes. For the first step, the RNA was reverse transcribed and
amplified by PCR using a one-step RT-PCR protocol with primers Prot 3?out
(nucleotides 2620 to 2647; 5?GCTTTTATTTTCTCTTCTGTCAATGGCC3?)
and 5? outer pol (nucleotides 2008 to 2031; 5?GCCCCTAGGAAAAAGGGCT
GTTGG3?). A nested PCR was then carried out with a high-fidelity proof-
reading polymerase on 10 ?l of the first round product (diluted 1:40) with the
following primers: 5? inner pol (nucleotides 2057 to 2080; 5?TGAAAGATTGT
ACTGAGAGACAGG3?) and Prot3 in (nucleotides 2569 to 2593; 5?CCTGGC
TTTA-ATTTTACTGGTACAG3?). The positions of the oligonucleotide prim-
ers are numbered according to the pol gene of the HXB2 isolate (18). In each
case, two control RT-PCRs were set up without the RT to exclude contaminating
DNA as a source for the amplified sequences. PCR products were cloned into
PCR-BluntII-TOPO vector (Invitrogen Corp, Carlsbad, Calif.) and sequenced
using a fluorescent dideoxy termination method of cycle sequencing on a 373A
automated DNA sequencer (Applied Biosystems, Foster City, Calif.), following
Applied Biosystems protocols.
HIV-1 serology. HIV-1 immunoglobulin G levels were determined using a
commercial enzyme-linked immunosorbent assay (Vironostika HIV-1 Micorelisa
system; Organon-Tek, Durham, N.C.). Antibody specificity was confirmed by
Western blotting (Calyptebiomedical, Rockville, Md.).
Sequence validation and statistical considerations. Sequence validation was
carried out according to the methods recommended by Learn et al. (24). Algo-
rithms were used to distinguish PCR errors from polymorphisms and resistance
mutations and to establish the independence of HIV-1 variants obtained from
the same patient. Using an estimation procedure for the frequency of artifactual
misincorporations (41) combined with the manufacturer’s statement of polymer-
ase fidelity, the expected frequency of sporadic misincorporations was calculated
at 1 per 104residues. To avoid overestimation of diversity, substitutions that
occurred only once in this data set were removed prior to analysis. With sporadic
substitutions removed, identical sequences derived from the same PCR were
considered redundant and likely to have been generated by resampling (25).
These “sanitized” sequences were used for the remainder of the analysis. Clones
obtained from different PCRs were also considered independent. Clones ob-
tained from the same PCR were only considered independent when they differed
by drug resistance mutations or by a number of mutations that exceeded the
estimated rates of artifactual misincorporation described above. Basic local
alignment search tool (BLAST) searches of GenBank (http://www.ncbi.nlm.nih
.gov/GenBank/GenbankOverview.html) revealed that none of the sequences
matched those of laboratory strains or other patient isolates.
Phylogenetic trees were inferred from nucleotide sequences through the use of
PAUP* version 4.0 (Sinauer Associates Inc., Sunderland, Mass) (43). The
HKY-85 model of evolution was suggested by MODELTEST analysis (35). Trees
were initially inferred using minimum evolution with 100 random addition se-
quence replicates and tree bisection-reconnection branch swapping, and the
shortest trees were used as input for a maximum likelihood estimation using the
HKY85?G model. Most-recent common ancestor sequences were obtained dur-
ing maximum likelihood analysis as the sequence inferred for the node ancestral
to each patient-specific clade. Larger data sets were examined, using the highly
efficient neighbor-joining method (39) with testing of internal node support using
the bootstrap method (10) with 1,000 replicates, for concordant clustering (e.g.,
analysis of all 221 plasma and cell sequences plus reference strains).
Plasma sequences that were previously published (20) and had been obtained
from three of the children (C2, C11, and C22) were included to allow a complete
assessment of the phylogenetic relatedness of samples obtained longitudinally.
Previously published (20, 32, 38) and recently determined latent reservoir se-
quences were also used to validate the patient-specific character of the plasma
sequences and to compare the HIV-1 protease of plasma variants to those
harbored in the latent reservoir in resting CD4?T cells. Reference sequences
(and their accession numbers) included strains A_SE.SE8131 (AF107771),
A1_UG.U455 (M62320), A2_CD.CDKFE4 (AF286240), B_FR.HXB2R (K03455),
C_IN.IN21068 (AF067155), C_ET.ETH2200 (U46016), and D_ZR.Z2Z6
Analysis of HIV-1 diversity in plasma. Assessment of HIV-1 diversity in the
early-treated cohort versus the late-treated group was performed using protease
sequences amplified from the first visit from which multiple viral variants were
detected. Diversity was evaluated as the average pairwise genetic distance cal-
culated using the same model and parameters as described above for the phy-
logenetic analysis. Median values for the early- and late-treated groups were
compared using a nonparametric tool (Wilcoxon rank sum test).
Nucleotide sequence accession numbers. Novel sequences have been submit-
ted to GenBank (accession numbers AY429144 to AY429259).
Patients. Two groups of children who differed by the age of
initiation of HAART were studied (Table 1). The members of
group 1, the late-treated group, initiated HAART after 1 year
of age. Group 1 was subdivided into two subgroups, groups 1A
and 1B, on the basis of the length of suppression of viremia on
HAART. Before HAART was a standard method of care, six
of these children were treated with nonsuppressive regimens
consisting of nucleoside analogue RT inhibitors. Group 2, the
early-treated group, was comprised of children initiating
HAART in the first few months of life with sustained viral
suppression resulting from their first HAART regimen. These
children were selected to evaluate the impact of early initiation
of HAART on residual HIV-1 production in children with
perinatally acquired infection.
The majority (11/15; 73%) of the children studied were on
standard three-drug HAART regimens with combinations of
nucleoside analogue RT inhibitors and protease inhibitors
(PIs). The remaining children were either on four (n ? 3) or
five (n ? 1) antiretroviral drugs, including one or more PIs. A
total of 60% (9/15) of the children were receiving the PI nelfi-
The median ages at the initiation of HAART for groups 1A
and 1B were 5.6 and 6.3 years, and the median durations of
HAART were 3.3 and 0.6 years, respectively. The median age
at the start of HAART for the early-treated group was 2.5
months (range, 1.6 to 3.8 months), and the median duration of
HAART was 3.5 years (range, 0.5 to 5.2 years). The geometric
mean plasma HIV-1 RNA levels before HAART were 257,833
copies/ml (range, 5,389 to ?750,000 copies/ml) for the late-
treated group (Table 1) and 840,000 copies/ml (range, 517,384
to ?1,500,000 copies/ml) for the early-treated group (Table 1).
Persistence of HIV-1 viremia below 50 copies/ml during
suppressive HAART in children. A novel RT-PCR assay that
detects as few as 5 copies of HIV-1 RNA in plasma (Fig. 1A)
was used to assess ongoing viremia in 30 plasma samples ob-
tained from the 15 children. With this assay, ongoing HIV-1
viremia was detectable in 26 of 30 plasma samples, including 23
samples obtained while the viral load was ?50 copies/ml (Fig.
1B; Table 2). Viremia was detected in five of six children in the
early-treated group despite the excellent response to HAART
observed in this group. In three samples from group 1, the viral
load was ?50 copies/ml at the time of analysis and then re-
turned to below 50 copies/ml. All three children with episodes
of intermittent detectable viremia of ?50 copies/ml continued
to have durable suppression of virus replication and required
no change in therapy. These episodes were therefore consid-
ered to be blips.
Seven children from the late-treated group (C2, C7, C8, C10,
C11, C22, and C40) had plasma samples analyzed at multiple
VOL. 78, 2004DRUG-SENSITIVE HIV-1 VIREMIA DURING SUPPRESSIVE HAART969
time points (Fig. 2). Despite suppression of viremia to below
the limits of detection by ultrasensitive clinical assays, HIV-1
viremia remained detectable in 9 of the 11 repeat samples
obtained from this group. All of the patients continued to have
suppression of viral replication to ?50 copies/ml for a mean of
8.1 months (range, 0.03 to 21.4 months) from the first analysis.
No patients were excluded from the original pediatric cohort
for viral rebound. Together with the results of the sequence
analysis described below, which provided definitive confirma-
tion of the patient-specific nature of the PCR products ob-
tained, these data demonstrate that HIV-1 viremia persists in
children at low levels despite durable suppressive HAART
regardless of whether treatment is initiated early and late.
Extent of HIV-1 diversity and divergence at plasma RNA
levels of <50 copies/ml. To provide definitive confirmation
that the PCR signals detected represent patient-specific se-
quences that are continuously produced in children whose
plasma virus levels are below the limit of detection of clinical
assays, we cloned and sequenced the positive PCRs. A total of
181 independent RT-PCR assays were performed on the co-
hort (median, 6 reactions per time point per patient) (Table 2).
A total of 68 amplicons (38% of total reactions) were obtained
at the limits of the serial dilution RT-PCR (median, 4 ampli-
cons per patient; range, 0 to 18) (Table 2). A total of 200 clones
derived from the PCR amplicons were sequenced and ana-
lyzed, yielding 116 distinct HIV-1 variants, as determined by
our criteria for clonal independence. As discussed below, phy-
logenetic analysis demonstrated the expected patient-specific
clustering. Nucleotide sequences obtained from the plasma of
each patient formed a distinct cluster (Fig. 3), reflecting the
patient-specific polymorphisms in the protease gene. These
results demonstrate that it is possible to obtain reliable, pa-
TABLE 1. Patient characteristics
Nonsuppressive therapySuppressive therapy
Plasma HIV RNA
at start of sup
Plasma HIV RNA
at first analysis
4.9 D4T/DDI/NFV 1.57,888
11.4M/AA 9.33TC/EFV/APV1.1 128,956
4.6 M/AANone None0.15 D4T/3TC/RTV
aF, female; M, male; AA, African-American; C, Caucasian.
bAbbreviations for drugs; ZDV, zidovudine; 3TC, lamivudine; DDI, didanosine; DDC, zalcitabine; D4T, stavudine; ABC, abacavir; EFV, efavirenz; DLV,
delavirdine; NVP, nevirapine; RTV, ritonavir; LPVr, lopinavir-ritonavir; APV, amprenavir; SQV, saquinavir; NFV, nelfinavir.
cSuppressed on first PI-based regimen.
dDid not take PI component of first two PI-based regimens.
ePrior failure on PI-based regimen.
fReceived neonatal prophylaxis (6 weeks of ZDV after birth).
970PERSAUD ET AL. J. VIROL.
tient-specific genotypic data even when the starting number of
template viral RNA molecules is extremely low.
Because the range of sequences obtained may be affected by
both diversity (variability observed within a single specimen)
and divergence (increase in genetic distance over time), we
performed a temporal analysis of the quasispecies in plasma at
viral loads of ?50 copies/ml in children. We found that HIV-1
viremia at ?50 copies/ml was comprised of diverse viral vari-
ants in the group of children who were chronically infected (?5
years of age) before HAART was initiated (Fig. 3A). The
median viral diversity in plasma in this group was 0.008 (range,
0.002 to 0.042 substitutions per nucleotide) when the viral load
was ?50 copies/ml. In the early-treated group, in contrast, the
median genetic diversity was significantly lower at 0.000 (range,
0 to 0.004; P ? 0.008) (Wilcoxon rank test). Although the
frequency of low-level viremia in the early-treated group was
similar to that observed in the late-treated group, the viremia
in the early-treated group was extremely homogeneous in char-
acter (Fig. 3B). There was minimal sequence variation de-
tected despite a mean infection time of 3.3 years (range, 0.7 to
5.1 years) and the continuous production of low levels of virus
for a mean treatment time with PIs of 3 years. These results
suggest that early effective HAART preserves the state of
HIV-1 evolution, arresting sequence variation to that which
had occurred before HAART was initiated.
Another striking feature of HIV-1 in plasma during suppres-
sive HAART was the lack of a clear relationship between the
degree of divergence (genetic distance from the root of the
tree) and the time of sampling. In the patients who were
sampled at multiple time points, no temporal structure was
identified in the phylogenetic trees. Representative trees show-
ing the relationship between divergence and sampling time for
patients C2, C8, C11, and C22 are shown in Fig. 4A. When the
distance from the most recent common ancestor (MRCA) was
calculated, no strong trend during suppressive HAART sug-
gesting divergence was seen (Fig. 4B). Even when divergence
in protease did occur due to treatment failure and the devel-
opment of resistance (C11; Fig. 4), the plasma sequences ob-
tained when viremia was suppressed by a new regimen did not
show a clear temporal pattern. Viremia at ?50 copies per
milliliter involved a mixture of HIV-1 variants. Recently gen-
erated, drug-resistant variants commingled with more ances-
tral, drug-sensitive variants (Fig. 4). Thus, we found that in
children in whom a successful HAART regimen was begun
during chronic HIV-1 infection, viremia at ?50 copies/ml
lacked temporal structure and showed degrees of heterogene-
ity and divergence that reflected prior nonsuppressive therapy.
Plasma viremia at <50 copies/ml mainly involves HIV-1
variants that are wild type for protease. To determine whether
continuous production of low levels of plasma virus during
effective HAART with PI-based regimens required or gener-
ated some level of PI resistance, we analyzed the nucleotide
sequences for amino acid substitutions known to be associated
with drug resistance to PIs. Sequence analysis revealed that
amino acid substitutions at sites of polymorphisms were unique
to each patient, confirming the patient-specific nature of the
PCR products obtained at low template concentrations (Table
2). More importantly, in patients who were PI naïve when
HAART was started, no drug resistance mutations were seen
in 92% (107/116) of the independent HIV-1 variants cloned
from plasma at a time when plasma virus levels were ?50
copies/ml or during intermittent blips. In 11 of the 13 patients
studied who had no prior history of treatment failure with PIs,
no protease mutations were seen despite a mean of 3.6 years
(range, 0.3 to 6.5 years) of continuous exposure to PIs (Table
2). In patient C10, for example, none of the 18 independent
sequences obtained during a blip to 69 copies/ml after 4.7 years
of a PI-based regimen showed PI resistance mutations, and
none of the 13 independent sequences obtained when the viral
load was below 50 copies/ml after 5 years of the PI-based
regimen had PI resistance. Thus, in at least some children
receiving potent antiretroviral therapy there can be continued
release into the plasma of viruses sensitive to the most potent
drug in the regimen for prolonged periods without the devel-
opment of resistance mutations.
Early drug-resistant variants were detected in two of the
children whose primary PI regimens were suppressive. Patient
C7 had detectable viremia (148 copies/ml) at the time of ini-
tiation of this study and a history of recent nonadherence. All
of the six clones detected had a V82I substitution associated
with low-level resistance to nelfinavir. On repeat analysis 3
months later, plasma virus levels were below 50 copies/ml, and
we were unable to amplify plasma virus using our five-copy-
sensitivity RT-PCR assay. In the second child, C40, one of the
seven clones detected at study entry had the N88S substitution,
and, at the second study visit 3 months later, two of the nine
clones had amino acid changes at position 82 (V82I) associated
with nelfinavir therapy. At 3 months later, none of these sub-
stitutions had become predominant, and the patient’s viral
load has remained undetectable at ?50 copies/ml. Thus,
plasma viremia below 50 copies/ml in patients on prolonged
FIG. 1. Nested RT-PCR assay used for genotyping the HIV-1 pro-
tease gene from the plasma in patients in whom plasma HIV-1 RNA
levels are below 50 copies/ml. (A) The sensitivity of the assay was
established using reaction mixtures containing serial dilutions of in
vitro-transcribed HIV-1 NL43 RNA in the presence (RT?) or absence
(RT?) of RT. Control PCRs for the first (1st) RT-PCR and the second
(2nd) nested PCR are also shown. (B) Representative amplifications of
protease from 4 ml of plasma from two subjects (subject C11, top
panel; subject C40, bottom panel) whose viral loads were ?50 cop-
ies/ml at the time of analysis.
VOL. 78, 2004 DRUG-SENSITIVE HIV-1 VIREMIA DURING SUPPRESSIVE HAART971
TABLE 2. HIV-1 protease sequences amplified from plasma in children with ?50 copies of HIV-1 RNA/ml on
protease-inhibitor-containing HAART regimensc
Continued on facing page
uninterrupted treatment with PI-containing regimens was
comprised largely of HIV-1 that was sensitive to the relevant
As expected, archival HIV-1 drug-resistant variants were
detected in the two children (C45 and C11) who had prior
treatment failure with a PI regimen and were known to have
developed PI-resistant HIV-1 variants (Table 1). In patient
C45, all 10 variants detected at a single time point had muta-
tions at amino acid positions 54 (I54V) and 82 (V82A) asso-
ciated with high-level drug resistance to ritonavir. This geno-
typic profile is consistent with the patient’s treatment history of
prior failure on a ritonavir regimen. Despite this finding, the
patient’s viral load remained undetectable (at ?50 copies/ml)
at a subsequent clinic visit. In this case, detection of archival
PI-resistant variants did not represent impending treatment
failure of the current suppressive regimen; rather, it likely
reflected production of archival resistant viruses selected by a
prior regimen. Similarly, for patient C11, drug-resistant vari-
ants harboring the D30N and N88D mutations that arose dur-
ing prior treatment failure with a nelfinavir-containing regimen
remained detectable in the plasma up to 6 months following
the discontinuation of nelfinavir and suppression on a lopi-
navir-containing regimen. These highly resistant, archival pro-
tease HIV-1 variants were found to coexist in plasma with the
more ancestral wild-type HIV-1 protease variants at plasma
virus levels below 50 copies/ml (clones 2.1 and 7.1, from the
second time point and clones 2, 5, and 7 from the third anal-
ysis) (Table 2 and Fig. 4A). The coexistence of wild-type,
drug-susceptible HIV-1 and more divergent drug-resistant
variants in plasma during suppressive therapy provides strong
evidence for active production of archival variants from long-
lived viral reservoirs.
aTime points with asterisks indicate previously published sequences from the same patient included to provide a complete picture of the evolutionary features
of HIV-1 in plasma during HAART.
bMay represent preexisting polymorphism or early drug resistance to nelfinavir.
cGroup 1A, late-treated (long-term suppression) group; group 1B, late-treated (short-term suppression) group; group 2, early-treated group.
VOL. 78, 2004 DRUG-SENSITIVE HIV-1 VIREMIA DURING SUPPRESSIVE HAART973
Phylogenetic relatedness of plasma virus to replication-
competent HIV-1 in the latent reservoir in resting CD4?T-
cells. Because six of the children (C2, C7, C8, C10, C11, and
C22) included in this study were previous participants in a
5-year longitudinal study of HIV-1 latency in resting CD4?T
cells, we compared the protease sequences of the persistent
HIV-1 in plasma during effective HAART to those of the
replication-competent HIV-1 recovered from the resting
CD4?T-cell reservoir. As previously reported (20), in all pa-
tients there was extensive comingling of the viral variants am-
plified from plasma at ?50 copies/ml and HIV-1 recovered
from the resting CD4?T-cell compartment (Fig. 4A). Overall,
there was no distinct clustering of plasma-derived sequences
compared with that of the latent reservoir sequences.
Impact of early HAART on low-level viremia, HIV-1 diver-
sity in plasma, and the HIV-1 antibody responses. As dis-
cussed above, there was no apparent difference in the fre-
quency of ongoing low-level viremia in children initiating
HAART during acute, perinatally acquired HIV-1 infection
compared to that seen with those treated during chronic in-
fection (Table 2). Five of the six children in the early-treated
group and all of the children in the late-treated group had
viremia that was detectable using our sensitive RT-PCR assay.
The one child for whom ongoing viremia was not detected was
from the early-treated group. We were unable to amplify
plasma virus obtained on two separate occasions 3 months
Despite the prevalence of viremia in both the early- and
late-treated groups, phylogenetic analysis revealed differences
in the pace and character of sequence variation between the
children with early suppressive therapy and those with late
suppressive therapy. In the children initiating HAART during
the first 4 months of life, there was little sequence variation
(median genetic diversity, 0.00) between plasma virus clones
amplified at below 50 copies/ml. In addition, there were no
sequence variations in the protease gene between plasma viral
variants and replication-competent HIV-1 in resting CD4?T
cells (Fig. 3B).
Importantly, and consistent with previous reports (26), all of
the children tested in the early-treated group had limited or
absent HIV-1-specific antibody responses (Fig. 5). While the
absence of HIV-1-specific antibody and cellular immune re-
sponses has been used as an indicator of complete control of
virus replication in children treated from early infancy, our
results show that as determined by Western blot analysis, on-
going low-level viremia continues even in children who have
negative or highly restricted HIV-1-specific antibody re-
sponses. Together, these results show that in early-treated chil-
dren there is a continuous release into the plasma of low levels
of HIV-1 virions that do not show evidence of genetic diver-
FIG. 2. Viral load data from study subjects who were monitored longitudinally. Plasma HIV-1 RNA levels are indicated by closed symbols.
Open symbols indicate that the level of viremia was below the limit of detection of the assay used (400, 200, or 50 copies/ml); the plotted values
serve to indicate the limit of detection. Solid arrows indicate times of sampling. Dashed arrows indicated previously reported time points (20).
974 PERSAUD ET AL.J. VIROL.
sification in pol and that do not trigger a normal HIV-1-specific
Our study of 15 children with durable suppression of HIV-1
replication treated with HAART for up to 6 years demon-
strates that HIV-1 viremia persists at plasma virus levels below
50 copies/ml in most if not all infected children on HAART.
Viremia was even detected in a unique subset of children who
started HAART in early infancy, some of whom reverted to
HIV-1 seronegativity. We were able not only to detect viremia
but also to clone and characterize the protease gene from the
rare plasma virions that constitute viremia at this level. Am-
plification from low numbers of template molecules raises con-
cerns about contamination, but phylogenetic analysis con-
firmed the patient-specific nature of the plasma viral sequences
obtained. Most importantly, despite the finding of continued
virus production in the setting of PI-based HAART regimens,
HIV-1 viremia in children achieving durable suppression with
their first PI-HAART regimen consisted primarily of HIV-1
variants that were wild type in protease. For the six subjects
monitored longitudinally (C2, C7, C8, C10, C22, and C40), the
median duration of continuous exposure to PIs was 3.2 years
(range, 0.59 to 5.12). The median duration of follow-up was 21
months (range, 5 to 37 months). Despite the length of the
observation period, accumulation of mutations in protease was
not observed. These findings suggest that persistent low-level
viremia is characteristic of HIV-1 infection during effective
HAART and is comprised largely of PI-susceptible HIV-1
variants rather than protease variants with some degree of PI
Two potential processes might be involved in the stable
persistence of HIV-1 viremia in the setting of potent antiret-
roviral therapy. One possibility is that viremia at plasma RNA
levels of ?50 copies/ml represents a new steady state, reflect-
ing ongoing cycles of HIV-1 replication. Under these condi-
tions, HIV-1 replication might be continuous but might occur
only at low levels because of efficacy of the HAART regimen.
Indeed, studies in HIV-1-infected adults have shown that the
decay kinetics of plasma HIV-1 RNA during initial HAART
can be accelerated when four- or five-drug HAART regimens
are used (14, 29). This finding suggests that during standard
HAART with three antiretroviral agents, newly infected cells
are generated, albeit at a low level. If the stable persistence of
HIV-1 viremia observed during HAART is in fact due to
continuous cycles of HIV-1 replication, one might expect the
genetic composition to show progressive divergence and the
eventual emergence of drug-resistant variants (13). However,
neither drug-resistant HIV-1 nor viral divergence was observed
in the children who were fully compliant, as evidenced by
durable suppression with their first PI regimen. It remains
possible that the pace of evolution of drug resistance in pro-
tease is too slow for detection over the treatment period (36,
37). The pol gene is more stable than other regions (e.g., env)
of the HIV-1 genome; therefore, it is possible that analysis of
changes in env sequences over time might provide some evi-
dence for evolution (16, 47). Nevertheless, the evolutionary
changes that are likely to be of clinical significance in these
patients are the changes affecting drug susceptibility, and these
were not apparent in our study.
The persistence of low-level viremia in children on HAART
FIG. 3. Maximum-likelihood phylogenetic analysis of HIV-1 pro-
tease gene sequences from plasma (red circles) of study subjects
treated with HAART either later during childhood (A) or from early
infancy (B). Sequence information obtained from latently infected
CD4?T cells from the early-treated group (black circles) are included
in panel B to confirm the patient-specific nature of the protease se-
quences amplified at low levels and to emphasize the homogeneity of
HIV-1 protease sequences in these individuals. Previously published
plasma virus sequences are indicated in open circles (A) and are
included to provide a complete picture of the evolutionary profile of
HIV-1 in plasma at low levels during prolonged suppressive
HAART. Reference sequences from clades A and C are labeled as
follows: A*, A.SE.SE8131; A1, A.UG.U455; A2, A2.CD.CDKFE4;
C*, C.IN.IN21068; C**, C.ET.ETH2220. Strain B.F.R.HXB2R was
used as a reference sequence for clade B.
VOL. 78, 2004DRUG-SENSITIVE HIV-1 VIREMIA DURING SUPPRESSIVE HAART 975
can also be understood in the context of the latent reservoir for
HIV-1. Although resting CD4?T cells harboring replication-
competent HIV-1 are present at a low frequency (1 per million
resting CD4?T cells), they can be readily detected using cel-
lular activation methods in all infected individuals (6, 12, 33,
40, 45). It is therefore plausible that resting CD4?T cells
harboring latent replication-competent HIV-1 are continu-
ously activated for virus production during HAART. In our
phylogenetic analysis, we found extensive commingling of pro-
tease sequences from plasma virus and replication-competent
HIV-1 recovered from resting CD4?T cells during HAART.
Several genetic studies in HIV-1-infected adults have impli-
cated viral reservoirs, including the resting CD4?T-cell reser-
voir, as sources of rebound viremia following treatment dis-
continuation (4, 9, 22, 46). In patients failing antiretroviral
therapy with multidrug-resistant HIV-1, wild-type HIV-1 be-
FIG. 4. Maximum-likelihood phylogenetic analysis of HIV-1 protease gene sequences from individual study subjects. (A) Phylogenetic trees
showing sequences obtained from plasma virus (diamonds) and from latently infected resting CD4?T cells (circles). Time of sampling is indicated
by the color scale. The red bracket used with the patient C11 data indicates sequences bearing drug resistance mutations. (B) Plots of genetic
distance versus time on HAART for subjects who provided multiple specimens during HAART. An MRCA sequence was reconstructed for each
subject, and distances from the MRCA to each sequence were calculated using the HKY85?G model. Sequences obtained from plasma
(diamonds) and latently infected CD4?T cells (circles) are indicated.
976 PERSAUD ET AL.J. VIROL.
comes predominant when the drugs are discontinued (7). The
only documented site where archival, wild-type, replication-
competent HIV-1 can coexist for long periods of time with
drug-resistant variants generated during treatment failure is
the latent reservoir in resting CD4?T cells (38). Therefore, the
reemergence of wild-type HIV-1 in the plasma of patients who
clearly have had viral divergence with progressive high-level
drug-resistant virus provides further evidence for the active
contribution of latent viral reservoirs to plasma virus. The
continued production of drug-sensitive HIV-1 that we ob-
served in children on suppressive HAART can be explained by
the release of virus from stable reservoirs.
The failure to detect PI-resistant variants in most patients
was not due to any inherent bias in the assay. Possible early
PI-resistant variants were detected in two children (C7 and
C40) who had no history of failure on a PI regimen, one with
suboptimal adherence and the other in the setting of fully
suppressive HAART. In patient C7, a V82I substitution was
present in all clones amplified during a blip to 148 copies/ml
following recent nonadherence. This substitution, while not
typically observed with nelfinavir failure, could represent a
preexisting polymorphism or an uncommon early resistance
mutation (23). At 3 months later, the viral load was ?50
copies/ml and we were unable to detect persistent low-level
viremia with our assay. In patient C40, changes at two sites, a
N88S mutation at the first analysis and a V82I substitution at
the second analysis, were seen in individual clones. Neither was
detected at a subsequent time point. While these amino acid
substitutions may represent polymorphisms, they have also
been observed early in the course of nelfinavir treatment fail-
ure. Again, the detection of early drug resistance mutations did
not represent impending treatment failure. Therefore, while
PI-resistant variants may arise at plasma virus levels below 50
copies/ml in the presence of drug-selective pressure, drug-
resistant variants do not become predominant or accumulate
additional mutations when plasma virus levels are maintained
below 50 copies/ml.
On the basis of the results of this study, we propose that
every day a small subset of the cells within the latent reservoir
become activated through an encounter with an antigen or
another activating stimuli and that the virus released by these
cells contributes to the low-level viremia. These viruses may
undergo some additional rounds of replication. However, our
results suggest that this additional replication is limited suffi-
ciently that resistance mutations do not accumulate. This
model explains why the reservoir for HIV-1 in resting CD4?T
cells does not decay with prolonged HAART even though
there is continuous activation of cells in the reservoir. When
additional rounds of replication are occurring, the number of
latently infected cells that must be activated to fuel this repli-
cation may be such a small fraction of the reservoir that decay
will not be significant even over a time scale of years (40).
Using an exponential decay model (31), we calculate that the
number of productively infected cells required to give rise to
plasma virus levels of between 5 and 50 copies per ml in a child
weighing 30 kg (estimated total extracellular fluid volume, 6
liters) ranges between 1,000 and 10,000 cells, respectively. This
calculation assumes that N, the number of virions produced
per productively infected cell, is 1,000 virions (21), and that the
clearance rate constants c, for plasma virions, and ?, for pro-
ductively infected CD4?T cells, are 23 day?1and 0.7 day?1,
respectively (29). If all of these productively infected cells were
to be generated by the activation of cells in the latent reservoir
and if all of the activated cells were to die, then decay of the
reservoir would be expected. Because the reservoir is stable, it
is necessary to postulate that not all of the productively in-
fected cells come from the reservoir. Our model suggests that
virus released by cells reactivated from latency may infect some
other cells secondarily. The stability of the reservoir could
also reflect the process of proliferative renewal that maintains
the memory T-cell compartment at roughly constant levels
throughout life (42). In this case, proviral DNA is preserved by
cell division and should show genetic stability consistent with
the previous observations reported by Ruff et al. on the stabil-
ity of this reservoir in children (38). It is also possible that the
stability of the reservoir reflects survival of some latently in-
fected cells after a cytokine-induced state of partial activation
in which the cells become permissive for some level of virus
FIG. 5. Representative HIV-1 Western blots of samples from chil-
dren who were treated with HAART from early infancy (C101, C103,
and C108) or who initiated HAART at later times (C10). Patient C102
had no viremia detectable using our RT-PCR assay. Ages at the time
of HIV-1 antibody testing are indicated in parentheses. y, year.
VOL. 78, 2004DRUG-SENSITIVE HIV-1 VIREMIA DURING SUPPRESSIVE HAART977
production but do not die as quickly as antigen-stimulated
cells. Indeed, in vitro studies have shown resting CD4?T cells
can be partially activated by cytokine signals to result in HIV-1
production (44). Infected cells may be more likely to persist
during antiretroviral treatment due to waning cytotoxic T lym-
phocyte responses (2). Finally, if the burst size is significantly
greater (for example, 50,000), then the activation of fewer than
200 latently infected cells per day would give rise to a plasma
virus level of 50 copies/ml. At this rate of activation, only
limited decay of the latent reservoir would be expected. Thus,
there are several possible mechanisms that would allow the
pool of latently infected cells to contribute to ongoing viremia
without showing substantial decay.
In summary, the data presented above provide evidence that
during effective HAART, HIV-1 infection is dynamic, with the
persistent production of virus that is sensitive to the PIs and
that is related to latent HIV-1 in resting CD4?T cells. This
dynamic model of HIV-1 infection during suppressive
HAART is consistent with recent studies of the suppressed
state in HIV-1-infected adults (8, 28). Understanding the con-
tribution of viral reservoirs to the fueling of virus replication
during effective HAART in children is important for targeting
future therapeutic strategies for HIV-1 infection. Further-
more, knowledge of the clinical implications of low levels of
plasma virus during HAART is of paramount importance as
more sensitive plasma HIV-1 RNA assays are incorporated
into care and are used to guide therapeutic decisions.
Funding and support: this work was supported by an Elizabeth
Glaser Pediatric AIDS Foundation Award (D. Persaud), Doris Duke
Charitable Foundation Awards (D. Persaud and R. F. Siliciano), and
NIH grants AI43222 and AI51178 (R. F. Siliciano).
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