JOURNAL OF VIROLOGY, Oct. 2009, p. 9731–9742
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 83, No. 19
Identification of Ongoing Human Immunodeficiency Virus Type 1
(HIV-1) Replication in Residual Viremia during Recombinant
HIV-1 Poxvirus Immunizations in Patients with Clinically
Undetectable Viral Loads on Durable Suppressive
Highly Active Antiretroviral Therapy?
Carlum Shiu,1Coleen K. Cunningham,2Thomas Greenough,3Petronella Muresan,4
Victor Sanchez-Merino,3Vincent Carey,4J. Brooks Jackson,1Carrie Ziemniak,1
Lawrence Fox,5Marvin Belzer,6Stuart C. Ray,1Katherine Luzuriaga,3
Deborah Persaud,1* and the Pediatric AIDS Clinical Trials
Group P1059 Team
Johns Hopkins University, Baltimore, Maryland1; Duke University Medical Center, Durham, North Carolina2; University of
Massachusetts, Worcester, Massachusetts3; Statistical and Data Analysis Center, Boston, Massachusetts4; NIAID, NIH,
Bethesda, Maryland5; and Children’s Hospital Los Angeles, Los Angeles, California6
Received 19 March 2009/Accepted 2 July 2009
In most human immunodeficiency virus type 1 (HIV-1)-infected individuals who achieve viral loads of <50
copies/ml during highly active antiretroviral therapy (HAART), low levels of plasma virus remain detectable
for years by ultrasensitive methods. The relative contributions of ongoing virus replication and virus produc-
tion from HIV-1 reservoirs to persistent low-level viremia during HAART remain controversial. HIV-1 vacci-
nation of HAART-treated individuals provides a model for examining low-level viremia, as immunizations may
facilitate virus replication and sequence evolution. In a phase 1 trial of modified vaccinia virus Ankara/fowlpox
virus-based HIV-1 vaccines in 20 HIV-infected young adults receiving HAART, we assessed the prevalence of
low-level viremia and sequence evolution, using ultrasensitive viral load (<6.5 copies/ml) and genotyping
(five-copy sensitivity) assays. Viral evolution, consisting of new drug resistance mutations and novel amino acid
changes within a relevant HLA-restricted allele (e.g., methionine, isoleucine, glutamine, or arginine for leucine
at position 205 of RT), was found in 1 and 3 of 20 subjects, respectively. Sequence evolution was significantly
correlated with levels of viremia of between 6.5 and <50 copies/ml (P ? 0.03) and was more likely to occur
within epitopes presented by relevant HLA alleles (P < 0.001). These findings suggest that ongoing virus
replication contributes to low-level viremia in patients on HAART and that this ongoing replication is subject
to CD8?T-cell selective pressures.
Highly active antiretroviral therapy (HAART) inhibits hu-
man immunodeficiency virus type 1 (HIV-1) replication, de-
creasing plasma viremia below the detection limit (?50 copies/
ml) of clinically used viral load assays (32). Most patients
receiving standard HAART, however, have low levels of vire-
mia (median, 3.1 copies/ml) detectable by more sensitive assays
(11, 17, 22, 28, 31, 34), the source of which remains unclear and
controversial. Low-level viremia may arise from viral expres-
sion from cellular reservoirs established before HAART (2,
21–23, 25, 29, 31, 34, 46) or from low levels of complete cycles
of virus replication (7, 46). Evidence supporting the former
includes the stable persistence of low-level viremia during
long-term HAART (2, 11, 23, 25, 28, 31, 34), intermingling of
HIV-1 sequences from free plasma virus with persistent repli-
cation-competent viral sequences in resting memory CD4?T
cells or proviral DNA in peripheral blood mononuclear cells
(PBMC) (2, 23, 34, 46), and a lack of decay in low-level viremia
with treatment intensification of standard three-drug HAART
regimens (10, 24). The contribution of ongoing complete cycles
of virus replication during long-term suppressive HAART is
supported by findings of sequence evolution with new drug-
resistant variants in a subset of patients during seemingly ef-
fective HAART (7, 46), decreases in plasma viremia with treat-
ment intensification (22), and transient increases in episomal
cDNA following treatment intensification with an integrase
inhibitor (6). Quantitatively, the magnitude of residual viremia
is directly correlated with pretherapy, steady-state plasma viral
load and proviral DNA levels, in support of virus expression
from chronically infected cells (28, 31). Low-level viremia and
even transient increases in viral loads to above 50 copies/ml
(known as “blips”) are often not directly associated with evo-
lution of plasma virus (2, 25, 30, 33, 34) or with decreased
effectiveness of the HAART regimen (21, 26, 28, 31) but may
replenish latent reservoirs and therefore preclude viral clear-
ance with HAART (38).
Studies aimed at understanding the source(s) of residual
viremia in patients on stable HAART have examined the ef-
fects of intensification of durable HAART regimens on vire-
mia below 50 copies/ml (10, 22, 24) or on cell-associated forms
* Corresponding author. Mailing address: Johns Hopkins University
School of Medicine, Department of Pediatrics, 200 North Wolfe
Street, Room 3151, Baltimore, MD 21287. Phone: (443) 287-3735. Fax:
(410) 614-1315. E-mail: firstname.lastname@example.org.
?Published ahead of print on 15 July 2009.
of viral DNA (6). In an earlier study, the addition of the
reverse transcriptase (RT) inhibitor abacavir to a two-drug
durable HAART regimen resulted in a lowering of residual
viremia (22). More recently, a study of treatment intensifica-
tion of durable three-drug HAART regimens with protease
inhibitors or nonnucleoside RT inhibitors (NNRTI) did not
reduce residual viremia (10). However, two studies of treat-
ment intensification of three-drug HAART regimens with the
integrase inhibitor raltegravir produced different findings.
In one study, 30 days of raltegravir produced no detectable
change in residual viremia, assessed using a single-copy viral
load assay (24). In a second study, however, intensification with
raltegravir was associated with a transient increase in episomal
viral cDNA at 2 weeks, suggesting de novo infection of and
reverse transcription in susceptible target cells, although no
change in total DNA burden was observed (6). Thus, the ques-
tion remains of whether ongoing low-level viremia during
HAART is dominated by low-level virus release from latently
infected cellular reservoirs, complete cycles of virus replication
de novo in susceptible target cells, or both.
Therapeutic HIV-1 vaccines are being evaluated to boost
HIV-1-specific immunity that wanes during HAART (12). If
successful, such immune boosting carries the promise of even-
tual treatment discontinuation, although that goal was tem-
pered recently by findings of increased morbidity with treat-
ment discontinuation (1, 8, 14, 15). To our knowledge, there
are no studies to date on the effects of HIV-1 vaccines on the
frequency and evolution of low-level viremia in HAART-
We hypothesized that immune activation through HIV-1
vaccines may amplify low-level viremia and facilitate sequence
evolution through enhanced virus expression from latently in-
fected CD4?T cells (13, 19, 35) and by increasing target cell
availability for virus replication (19). We therefore examined
the prevalence and sequence evolution of low-level viremia
during a clinical trial of recombinant poxvirus vaccinations
(modified vaccinia virus Ankara/fowlpox virus [MVA/FPV]) in
infected young adults on durable suppressive HAART to in-
vestigate the dynamics of low-level viremia.
MATERIALS AND METHODS
Study cohort. The study cohort was described previously in a report detailing
the safety and immunogenicity of the vaccine products (18). Twenty HIV-1-
infected young adults from 19 to 24 years of age (median age, 23 years) who were
on HAART received up to four vaccinations while enrolled in a phase 1 clinical
trial of MVA- and FPV-based HIV-1 vaccines containing gag, env, the RT gene,
nef, tat, and rev (Pediatric AIDS Clinical Trials Group P1059) (18). The MVA
vaccinations were administered at weeks 0 and 4, and the FPV vaccine was
administered at weeks 8 and 24. Plasma samples were collected for ultrasensitive
HIV-1 load testing and genotyping at nine time points during the trial, including
screening, entry, and weeks 2, 4, 6, 24, 26, 40, and 72. Ultrasensitive HIV-1 load
and genotyping assays were part of the parent clinical trial and were included in
the consent form approved by the institutional review boards at all sites enrolling
Sample preparation. Plasmas were separated from PBMC by centrifugation
and were stored at ?70°C until use. PBMC were isolated using Ficoll-Hypaque
centrifugation, fractionated to enrich for resting CD4?T cells, and cultured at
limiting dilutions, using previously published methods (42). Individual HLA
serotyping, gamma interferon (IFN-?)-specific enzyme-linked immunospot
(ELISPOT) assays, and CD4 lymphoproliferative response assays with cryopre-
served PBMC were performed as previously described (18).
Quantitative assessment of low-level viremia. The viral load was quantified
from 1 ml of plasma by use of a modified ultrasensitive RNA assay in which the
limit of detection was further validated to detect viremia at 6.5 copies/ml instead
of the previously reported limit of 12.5 copies/ml (36).
Genotyping of HIV-1 RT during low-level viremia. HIV genotyping of RT
from low-level viremia variants was performed using our previously published
methods with a sensitivity of 5 copies/ml (25, 34). Briefly, viral RNA was ex-
tracted from virions pelleted at 23,500 ? g for 2 h at 4°C from four 1-ml aliquots
of thawed plasma, using a QIAamp viral RNA isolation kit (Qiagen, Valencia,
CA). Following DNase treatment to ensure removal of cell-associated DNA
(Invitrogen, Carlsbad, CA), eight independent one-step RT-PCRs were set up
for the DNase-treated RNA, followed by a nested PCR with previously published
primers (49). The PCR products were purified using a QIAquick PCR purifica-
tion kit (Qiagen, Valencia, CA) and directly sequenced bidirectionally using
Analysis of HIV-1 RT sequences. Sequences were aligned using Bioedit and
cleaned using a previously published method (CleanCollapse [http://sray.med
.som.jhmi.edu/SCRoftware/CleanCollapse]) that removes sporadic changes
likely to represent PCR-induced errors (25). Evolution has been reported to be
overestimated systematically without such adjustments (44).
Nonsynonymous substitutions at known drug resistance sites (Stanford HIV
Drug Resistance Database [http://hivdb.stanford.edu]) were preserved, as were
the novel changes detected at amino acid 205 of HIV-1 RT in the sequences
amplified from episodes of low-level viremia after vaccination of four subjects.
Using this approach, only four sporadic nonsynonymous substitutions were re-
moved from the total data set of 209 sequences spanning amino acids 40 to 219
of HIV-1 RT. These sporadic changes, by definition, occurred only once in the
data set; therefore, our estimates are lower bounds for evolution occurring in this
Phylogenetic analysis parameters were estimated using ModelTest, version 3.7
(37), and these parameters were used as input to PhyML, version 3.0, to generate
maximum likelihood trees, using the SPR search option and starting from a
BioNJ tree (20). For bootstrap analysis, maximum likelihood trees were inferred
from 1,000 permutations of the original data set. Genetic diversity in each plasma
sampling from each subject was calculated by the Kimura 2 parameter distance
model, using MEGA4 (45). Nonsynonymous amino acid changes detected post-
vaccination within HIV-1 RT were also analyzed with respect to relevant HLA-
restricted CD8?T-cell epitopes in the Los Alamos Database (http://www.hiv.lanl
.gov) for subjects who maintained suppression during the trial. The two
substitutions observed at position 205 of HIV-1 RT (L205M/Q) at week 40 for
the one subject who discontinued HAART at week 24 were also included in this
analysis, since these mutations have not been reported previously for subtype B
Analysis of CD4?T cells infected with replication-competent HIV-1. The
frequencies of CD4?T cells carrying replication-competent virus and sequences
of HIV-1 RT from replication-competent isolates were assessed using previously
published methods (33). Representative HIV-1 RT sequences from replication-
competent clones recovered at the prevaccination visits were used to confirm the
patient specificity of the HIV-1 RT sequences amplified during low-level viremia.
Immunogenicity studies. HLA serotypes, HIV-1-specific immune responses,
and immune activation analyses were assessed as part of the parent trial, as
previously described (18). These measurements were used to examine potential
immunological differences among subjects experiencing sequence evolution dur-
ing the trial compared to those who did not.
Statistical analysis. Most analyses consist of descriptive summaries of the
various measurements and changes from baseline in those values. The summa-
ries are provided as medians and interquartile ranges (IQR), unless otherwise
specified. The changes from prevaccination to subsequent weeks were tested
using the Wilcoxon sign rank test. The proportion of subjects with low-level
viremia above 6.5 copies/ml at each study visit was computed and compared to
baseline by the McNemar test.
The study did not have a control group that did not receive vaccines. However,
subjects were divided into several groups based on criteria specific to each data
analysis. Exploratory data analyses were performed to compare groups with
respect to raw data or changes from baseline. No adjustments for multiple
comparisons were made. Differences in baseline levels of immune activation
(percent CD8?CD38?HLA-DR?T cells), frequencies and magnitudes of
low-level viremia, and frequencies of resting CD4?T cells with replication-
competent virus (infectious units per million) for subjects who had viremia
genotypes versus those who did not were assessed using the Wilcoxon two-
sample test. Rates of viremic episodes of between 6.5 and 50 copies/ml postvac-
cination for subjects with and without sequence evolution in RT were compared
using the Wilcoxon two-sample test. Similarly, the Wilcoxon two-sample test was
used to compare the magnitudes of low-level viremia for subjects who were
viremic above 6.5 copies/ml and of HIV-1 specific CD8?T-cell responses to Pol
9732 SHIU ET AL.J. VIROL.
in the subjects experiencing HIV-1 RT sequence evolution to the same mea-
surements for those with no evolution. The relationship between HIV-1 RT
mutation events and HLA serotypes was assessed using a logistic model for the
probability that mutations occupy epitopes recognized by HLA type (27). For
each individual, a measure of recognition capacity was computed as the number
of (nonredundant) residues occupied by epitopes associated with the individual’s
HLA type, gathered from the Los Alamos HIV Immunology Database (http:
Nucleotide sequence accession numbers. The nucleotide sequences deter-
mined in this study have been submitted to GenBank (accession numbers
GQ424058 to GQ424103 and GQ428780 to GQ428988).
Study population. The demographics, antiretroviral treat-
ment histories, and virologic data for the patients are summa-
rized in Table 1. The subjects received durable successful
HAART for a median of 3.3 years (range, 0.6 to 6.4 years)
before receipt of HIV-1 vaccines, and 11 of the 20 (55%)
subjects controlled viremia on their first HAART regimen. As
previously reported, 75% of the 20 participants acquired
HIV-1 infection through high-risk behaviors, and the remain-
der acquired infection perinatally (18). Three participants (pa-
tients 5, 11, and 15) developed rebound viremia during the
trial; patients 5 and 15 rebounded due to discontinuation of
their HAART regimens by the week 24 visit, and the third
participant (patient 11) rebounded at week 60 while on
Prevalence of low-level viremia before and after recombi-
nant HIV-1 poxvirus vaccination. We assessed the prevalence
of low-level viremia pre- and post-HIV-1 vaccination in young
adults with durable control of virus replication via HAART.
Viral loads measured in the three subjects experiencing re-
bound viremia (subjects 5, 11, and 15) were excluded from
analysis at the time of rebound and at subsequent time points.
Nineteen of the 20 subjects had plasma samples analyzed at the
two visits prior to HIV-1 vaccination. Low-level viremia at
levels greater than 6.5 copies/ml was detected in 21% (4 of 19
samples) and 25% (5 of 20 samples) of the samples at the
screening and entry visits, respectively. The nine episodes of
detectable low-level viremia prevaccination were in seven par-
ticipants, with two of the seven subjects having viremia detect-
able at both prevaccine visits (Table 2).
Following vaccination, the prevalence of low-level viremia
ranged from 12% (2/17 samples) at week 2 postvaccination to
as high as 44% (8/18 samples) at the week 40 visit but did not
differ significantly from that at study entry for each time point
tested (P ? 0.51) (Table 2). The median viral load at study
entry for the five subjects with detectable viremia of ?6.5
copies/ml was 11 copies/ml (IQR, 7 to 15 copies/ml), and this
value was 19 copies/ml for the time points with the highest
median low-level viremia postvaccination (weeks 24 [IQR, 17
to 37 copies/ml], 26 [IQR, 11 to 27.5 copies/ml], and 72 [IQR,
11 to 42.5 copies/ml]).
Extent of recovery and sequence evolution of low-level vire-
mia genotypes before and after recombinant HIV-1 poxvirus
vaccination. Longitudinal analyses of low-level viremia geno-
types were carried out to examine sequence evolution in
plasma virus. Genotyping was performed on all available
plasma samples (n ? 158 [88%]) from the 180 scheduled study
visits. Eighty-nine percent of the 158 samples genotyped were
from the 18 subjects who remained on HAART during the
trial, 97% (137/141 samples) of whom had viral loads of ?50
copies/ml at the time of genotyping. Low-level viremia geno-
types were recovered from a median of 56% (range, 20 to
100%) of the time points analyzed, for 60% (12/20 subjects) of
the study participants (Tables 1 and 2). For 8 of the 20 study
subjects, 98% of the 61 plasma samples analyzed were nega-
tive. For one subject (subject 1), the plasma sample obtained at
the last study visit at week 72 yielded genotypes (Tables 1 and
2). We were unable to identify any factors that may have
contributed to the selective sequence amplification for 12 study
subjects. The 8 subjects whose plasma samples were negative
did not differ significantly at baseline from the 12 subjects with
amplifiable genotypes with respect to median viral loads below
50 copies/ml, baseline frequencies of CD4?T cells harboring
replication-competent HIV-1, levels of immune activation, or
sequence variation at the primer binding site. Prior to vacci-
nation, 3 of 15 (20%) plasma samples tested for the eight
subjects with no amplifiable genotypes were positive for viral
loads of ?6.5 copies/ml, compared to 6 of 24 (25%) samples
from the 12 subjects with amplifiable genotypes. The median
viral load at study entry was ?6.5 copies/ml for both groups
(IQR, ?6.5 to ?6.5 copies/ml and ?6.5 to 7.0 copies/ml, re-
spectively [P ? 0.39]). The median frequencies of latently
infected CD4?T cells were 0.2 (IQR, 0.1 to 0.2) and 0.3 (IQR,
0.1 to 1.1), respectively (P ? 0.33), and the median percentages
of CD8?CD38?HLA-DR?T cells were 18% (IQR, 14 to
25%) and 29% (IQR, 18 to 33%) (P ? 0.19) for the subjects
with and without amplifiable low-level viremia genotypes, re-
spectively. Analysis of HIV-1 RT sequences derived from rep-
lication-competent viral clones cultured at the prevaccine time
points showed that only one subject (subject 2) (Table 2) had
a substitution at the 3? end that may have affected binding of
the primers used for reverse transcription (data not shown).
Together, these data argue against differences in baseline lev-
els of cell-associated, replication-competent infection, immune
activation, or residual viremia that may influence the level of
viral expression from persistently infected cells or the potency
of inhibition below 50 copies/ml. Alternatively, this selective
amplification may represent the stochastic nature of amplifi-
cation near the limit of detection.
Eleven of the 12 study participants with amplifiable geno-
types at more than one time point postvaccination also had
genotypes recovered prevaccination. Phylogenetic analysis
demonstrated patient-specific clustering of plasma genotypes
and commingling with corresponding replication-competent
clones recovered prevaccination. (This was also the case for
the one subject [subject 1] who had genotypes amplified only at
the last study visit, at week 72 [Fig. 1].) For 6 of these 11
(54.5%) subjects (subjects 4, 7, 8, 9, 12, and 17), postvaccina-
tion HIV RT sequences were identical to the prevaccine se-
quences, which persisted for a median of 28 weeks (range, 8 to
74 weeks), consistent with long-term persistence of a dominant
plasma sequence in low-level viremia during HAART (Fig. 1).
In three of these six subjects (subjects 7, 8, and 12), the
persistent plasma sequence shared sequence identity with
replication-competent virus cultured from resting CD4?T
cells at study entry (Fig. 1), in contrast to one other report
that persistent plasma sequences in low-level viremia are
genetically distinct from proviral DNA sequences in resting
CD4?T cells (2).
VOL. 83, 2009ONGOING HIV-1 REPLICATION IN RESIDUAL VIREMIA 9733
TABLE 1. Patient demographics, HAART regimens, and extent of sampling of low-level viremia genotypesa
Group and subjectb
Age at study
HAART regimen at
study entry (yr)
No. of visits with
Subjects with amplifiable low-level viremia
genotypes experiencing sequence
evolution following vaccination
AZT, 3TC, NFV (0.9)
AZT, 3TC, NVP (5.7)
AZT, 3TC, DDI, ABC, IDV, EFV
D4T, TDF, LPV/r (2.3)
Wild type, D67G K70R
V018I K219E mutant
AZT, DDI, 3TC, D4T, DDC,
NVP, ABC, RTV, AMP, LPV/r
TDF, ATZ/r, EFV (1.7)
M41L, D67N, T69D, M184V,
Subjects with amplifiable low-level viremia
genotypes with no sequence evolution
AZT, 3TC, NVP (4.3)
AZT, 3TC, EFV (2.6)
AZT, 3TC, EFV (6.4)
AZT, 3TC, ABC, LPV/r
ABC, 3TC, ATV (0.6)
AZT, DDI, D4T, ADF, 3TC,
IDV, APV, NFV, SQV, DDC,
AZT, TDF, FTC, ATZ/r (1.1)
V108I, M184V, T215S/D
DDI, FTC, EFV (3.8)
DDI, FTC, EFV (4.2)
DDI, EFV, NFV (4.5)
Wild type and M41L, M184V,
and T215F mutants
Subjects without amplifiable low-viremia
DDI, 3TC, TDF, EFV
TDF, FTC, LPV/r (0.9)
AZT, 3TC, NFV (5.1)
AZT, 3TC, EFV (4.7)
AZT, 3TC, EFV (5.3)
AZT, 3TC, LPV/r
TDF, FTC, EFV (2.8)
AZT, 3TC, NFV
TDF, FTC, ATV/r(1.4)
DDI, FTC, EFV (4.3)
aF, female; M, male; AZT, zidovudine; 3TC, lamivudine; NFV, nelfinavir; EFV, efavirenz; DDI, didanosine; FTC, emtracitabine; TDF, tenofovir; LPV/r, lopinavir boosted with ritonavir; ATV, atazanavir; NVP,
nevirapine; ABC, abacavir; IDV, indinavir; D4T, stavudine; ADF, adefovir; APV, amprenavir; SQV, saquinavir; DDC, zalcitibine; ATZ/r, atazanavir boosted with ritonavir; RTV, ritonavir; AMP, amprenavir; NA, not
b†, perinatally infected subject; *, patient developed rebound viremia while on HAART; #, self-discontinued HAART.
cReceived 30 days during pregnancy for prevention of mother-to-child transmission.
dOnly the plasma sample at the week 72 visit yielded sequences; all three variants comingled with replication-competent viral clones cultured at the prevaccine visits.
9734 SHIU ET AL.J. VIROL.
Evolution of the plasma sequence was observed during the
study for four subjects. New drug resistance mutations occur-
ring in low-level viremia during the nevirapine-based HAART
regimen were observed in one subject (subject 8). Multiple
unique amino acid substitutions in HIV-1 RT position 205
were seen at week 40 for three subjects (subjects 7, 11, and 17).
Viral variants substituted at HIV-1 RT position 205 were also
transiently detected at week 40 during rebound viremia in a
fourth subject who discontinued HAART (subject 15) (Fig. 1
and Fig. 2).
In the one subject (subject 8) who developed new NNRTI
resistance mutations through sequence evolution, the V108I
mutation was first detected 4 weeks after receipt of the first
MVA vaccination. Additional NNRTI resistance mutations
(K103N and V106A) were also detected in low-level viremia at
the week 6 visit following the second vaccine dose (Fig. 1 and
2). The presence of new NNRTI-resistant variants was not
associated with breakthrough viremia in this subject, despite a
regimen of nevirapine-based HAART. In fact, prevaccine wild-
type low-level viremia was maintained at all subsequent post-
vaccination visits (weeks 24, 26, 40, and 72, when the viral load
was 172 copies/ml) (Fig. 1). In this subject, another amino acid
change reported to confer decreased susceptibility to NNRTIs
(I135T) (5) was first detected in low-level viremia linked to the
V108I mutation at the week 4 visit and subsequently fixed in
the low-level viremia through the last study visit at week 72
(Fig. 2). In addition to being associated with decreased sus-
ceptibility to NNRTIs, the I135T mutation is also reported to
reside within an epitope presented by a relevant host HLA
Preexisting NNRTI drug resistance mutations in low-level
viremia were also detected in subject 4, who was on a primary
nevirapine-based HAART regimen. In this subject, all four
sequences amplified at the two prevaccine visits contained the
substitution Y188C, which confers intermediate to high-level
nevirapine resistance (Stanford HIV Drug Resistance Data-
base [http://hivdb.stanford.edu]). The Y188C variant remained
detectable at weeks 4 and 6 postvaccination but was not asso-
ciated with breakthrough viremia following HIV-1 vaccinations
(Fig. 1). This subject maintained plasma viremia at ?50 cop-
ies/ml throughout the study, and no additional plasma se-
quences were amplified at study visits from week 24 through
Unique nonsynonymous or amino acid-changing substitu-
tions which were not previously described for subtype B HIV-1
at position 205 of HIV-1 RT (methionine [M], isoleucine [I],
glutamine [Q], or arginine for leucine [L]) were detected in
four study participants (subjects 7, 11, 15, and 17) (Fig. 1 and
2; Table 2). For all four subjects, these mutations were de-
tected at the week 40 visit (Fig. 1). For three of the four
subjects, the nonsynonymous changes were detected at viral
loads of ?50 copies/ml, and for one subject (subject 15) they
were detected during rebound viremia following HAART dis-
continuation. A leucine-to-methionine (L205M) substitution
was most common and was found in all four subjects (11/15
sequences) (Fig. 2). For two subjects (subjects 7 and 15),
TABLE 2. Plasma viral levels and recovery of amplifiable genotypes before and after recombinant HIV-1 poxvirus vaccinations
Group and subject
Level of viremia (copies/ml)/genotyping success at indicated wk of studya
Subjects with amplifiable low-level
viremia genotypes experiencing
sequence evolution following
Subjects with amplifiable low-level
viremia genotypes without sequence
evolution following vaccination
Subjects without amplifiable low-level
Total proportion with genotyping successc
Total proportion with low-level viremiad
aND, not done.
cNumber of subjects with low-level viremia genotypes detected/number of subjects assessed.
dTotal number of subjects with low-level viremia between 6.5 and 50 copies/ml/number of subjects assessed.
eViral load measurement was done by standard clinical viral load assay.
VOL. 83, 2009ONGOING HIV-1 REPLICATION IN RESIDUAL VIREMIA9735
9736 SHIU ET AL.J. VIROL.
L205I/R/Q substitutions were also detected, suggesting immu-
nologic pressure at this site.
One subject developed rebound viremia while on HAART
(18). In this one perinatally infected subject (subject 11), re-
bound viremia developed at week 60, despite no reported
change in HAART adherence. A time-dependent shift in low-
level viremia genotypes with a decrease in viral diversity was
observed as early as week 6 following vaccination (Fig. 1). For
this subject, a 3.5-fold increase in HIV-1-specific CD8?T-cell
responses to HIV-1 Pol was detected at the same time (week 6
following MVA vaccinations) (see Fig. 4). These responses
were also sustained above baseline levels (sevenfold) at week
26 following the fourth vaccine dose and preceded the amino
acid substitution at position 205 of HIV-1 RT (L205M). More-
over, the L205M substitution was detected in multiple viral
lineages (Fig. 1 and Fig. 2) at week 40, suggestive of vaccine-
induced responses contributing to selection and possible
immune escape before viral breakthrough at week 60.
Association of HIV-1 sequence evolution during recombi-
nant poxvirus vaccinations with frequency of episodes of vire-
mia below 50 copies/ml and with HIV-1-specific immune re-
sponses. Given that significant increases in HIV-1-specific
CD8?T-cell responses to Pol were detected postvaccination
among the study participants (18), we analyzed the low-viremia
genotypes for nonsynonymous substitutions within relevant
CD8?T-cell epitopes following HIV-1 vaccination (http://www
FIG. 1. Maximum likelihood phylogenetic tree of HIV-1 RT sequences as a function of time postvaccination. Representative prevaccine
replication-competent viral isolates, subtype B, C, and D reference sequences for HIV-1 RT, and the vaccine sequence (vaccine) are shown.
The plasma sequences amplified during episodes of rebound viremia postvaccination are shown with a strike through the symbols. Wild-type
variants, drug-resistant variants, and variants with amino acid substitutions at position 205 of HIV-1 RT are indicated. Bootstrap values of
?80% are shown.
FIG. 2. Amino acid alignment, relative to the consensus region of HIV-1HXB2RT, showing nonsynonymous substitutions detected during
low-level viremia postvaccination within relevant CD8?T-cell epitopes. Homology to the consensus sequence is represented by dots. (a) Subject
8 developed the nonsynonymous substitutions V108I and G112S in an HLA-A*02 epitope (red box), and the I135T substitution within an
HLA-A*02/B*51 epitope (blue box) later became fixed in the free plasma virus present at low levels. The selection of substitutions at position 205
occurred within an HLA-A*02 epitope (red box) for three subjects (subject 7 [b], subject 15 [d], and subject 17 [e]). For the one subject (subject
11) (c) who showed temporal clustering of low-level viremia genotypes during the trial and subsequently failed therapy while maintained on
HAART, and who is HLA-A*6801 positive (which is also an HLA-A*03 supertype), the L205M position also lies outside the HLA-A*68 (green
box) and HLA-A*03 (red box) epitopes.
VOL. 83, 2009ONGOING HIV-1 REPLICATION IN RESIDUAL VIREMIA9737
.hiv.lanl.gov/content/hiv-db/mainpage.html). There were 45
nonsynonymous substitutions (median of 3.5 substitutions per
subject) observed in the 111 postvaccination sequences
spanning amino acid positions 40 to 219 of HIV-1 RT from 7
of the 10 subjects (70%) with amplifiable genotypes and
maintained on HAART (Table 3). Of these 45 substitutions,
56% (25/45 changes) occurred within defined CD8?T-cell RT
epitopes, and 56% of these (14/25 changes) were in HLA-A*02
epitopes. Using logistic regression modeling for correlated
mutations in HLA epitopes were significantly more likely to
occur among individuals with the restricting HLA alleles (P ?
0.001) (Table 3).
To explore whether substitutions at position 205 were
associated with decreased epitope recognition due to CD8?
T-cell escape, we compared recognition of the HIV-1 RT
HLA-A*02 KIEELRQHL wild-type epitope to that of an
autologous L205M mutant peptide in IFN-? ELISPOT as-
says. However, none of the four subjects demonstrated
postimmunization responses to wild-type or mutant peptides
above background levels with a control peptide (an HLA-
A*02-restricted epitope in human T-cell leukemia virus type
1 Tax) (data not shown).
The five subjects whose data are depicted in Fig. 2 had
evidence of either new drug resistance or unique amino acid
substitutions in the RT region not appearing in the nearly 914
subtype B sequences in the Los Alamos HIV Sequence Data-
base. Because one of these subjects (subject 15) discontinued
HAART and had levels of viremia increasing to ?1,000 copies/
ml, pre- and postvaccination viremia levels for the four remain-
ing subjects (subjects 7, 8, 11, and 17) with unexpected se-
quence evolution were compared to those for the six subjects
with no sequence evolution (subjects 4, 9, 12, 13, 16, and 19).
For this analysis, rates of quantifiable viremia pre- and post-
TABLE 3. Nonsynonymous substitutions in HIV-1 RT detected in low-level viremia postvaccination sequences relative to prevaccination
sequences and in relationship to defined CD8?T-cell epitopes restricted by HLA serotypes
Group and subject
HLA-binding residues in HIV-1 RTb
Subjects with amplifiable low-level viremia
genotypes and sequence evolutionc
I135M, E194G, E203K, E204G,
V179I, Y181C, V184M
K70R, V75S, K101E, K122E,
Subjects with amplifiable low-level viremia
genotypes without evolution following
N54T, N123D, C188Y
K43E, V118I, E203D
aHLA serotypes of study subjects with nonsynonymous changes within the relevant HLA epitopes postvaccination.
bCD8?T-cell epitopes in HIV-1 RT encompassing the observed nonsynonymous substitutions, as defined in the Los Alamos HIV Immunology Database
(http://www.hiv.lanl.gov/content/index). Substitutions at reference sites are underlined; those in the four subjects experiencing sequence evolution are also shown in
bold. To reduce redundancy, overlapping peptides were combined into one sequence. NA, not applicable; indicates a substitution in a region not likely recognized by
the subject’s HLA serotypes.
cNew drug resistance mutations or novel changes at amino acid position 205 of RT.
dPreexisting substitutions relative to consensus HIV-1 RT sequences.
9738 SHIU ET AL.J. VIROL.
vaccination were calculated for each subject by dividing the
number of viremic episodes above 6.5 but below 50 copies/ml
by the numbers of visits pre- and postvaccination, respectively,
at which viremia was measured. Since blood samples were
obtained immediately before vaccine administration, the visit
at study entry (week 0) was a prevaccine visit. For the four
subjects with sequence evolution, 26 postvaccination visits
(median, 6.5 per subject) produced quantifiable low-level vire-
mia, while 41 visits (median, 7 per subject) produced quanti-
fiable viremia for the six subjects without sequence evolution
(Table 2). The median rate of quantifiable viremia did not
change significantly for each group pre- and postvaccination
(P ? 0.1 for subjects experiencing evolution and 0.81 for those
who did not), but rates were significantly greater for subjects
experiencing sequence evolution than for subjects who did not.
The rate of quantifiable viremia postvaccination was 54%
(IQR, 39 to 62%) for those experiencing evolution versus 14%
(IQR, 14 to 29%) for those who did not (P ? 0.03) (Fig. 3).
However, this did not reflect a change from prevaccination
frequencies for these subjects. Prevaccination, 50% of the 8
samples from the subjects experiencing evolution were viremic,
compared to 17% of 12 samples from subjects not experiencing
evolution. The median change in the rate of quantifiable vire-
mia pre- and postvaccination was ?2.4% (IQR, ?38.1% to
39.3%) (P ? 1.0) for subjects experiencing evolution and 7.1%
(IQR, ?16.7% to 14.3%) (P ? 0.81) for those who did not.
When only the highest viral load postvaccination was consid-
ered for each subject, median viral loads were 21.5 copies/ml
(IQR, 13.0 to 37.5 copies/ml) and 16.0 copies/ml (IQR, 10.0 to
37.0 copies/ml) for those with evolution and those without,
respectively (P ? 0.92).
We compared HIV-1-specific CD8?T-cell responses to Pol
(defined as the sum of responses to the Pol1, Pol2, and Pol3
peptides spanning amino acids 1 to 556 of HIV-1 Pol) (18) for
the four subjects experiencing RT sequence evolution with
those for subjects with no evolution. For this analysis, which
was not adjusted for multiple comparisons, the median and
IQR for the HIV-1 Pol responses for the two groups were
compared at weeks 0, 6, and either 24 or 26. At baseline, the
median HIV-1-specific CD8?T-cell response to Pol was 547.3
spot-forming units (SFC) per million PBMC (IQR, 315.5 to
759 SFC/million PBMC) for those experiencing sequence evo-
lution, compared to 376 SFC/million PBMC (IQR, 140.5 to
692.5 SFC/million PBMC) for those with no evolution, and
these were not statistically different (P ? 0.75). At week 6, the
median changes from baseline were similar for those with and
without HIV-1 RT sequence evolution (P ? 0.75) (Fig. 4), but
at week 24 or 26, the median change was 365.5 SFC/million
FIG. 3. Frequency of episodes of low-level viremia above 6.5 cop-
ies/ml for the subjects experiencing sequence evolution (n ? 4) at viral
loads of ?50 copies/ml compared to the frequency for those who did
not (no evolution) following HIV-1 recombinant poxvirus vaccinations.
Sequence evolution was defined as either new drug resistance muta-
tions (subject 8) or novel changes at amino acid position 205 of HIV-1
RT while still being suppressed on HAART (subjects 7, 11, and 17).
The one subject (subject 15) who also developed substitutions at amino
acid position 205 of RT at week 40 in rebounding virus was excluded
from this analysis. Median values and IQR are indicated.
FIG. 4. Changes from baseline in HIV-1-specific CD8?IFN-?
ELISPOT responses to overlapping peptides spanning amino acids 1
to 556 of HIV-1 Pol at weeks 6 and 24 or 26 for the subjects experi-
encing sequence evolution in low-level viremia compared to those
without detectable sequence evolution. The one subject (subject 15)
who also developed substitutions at amino acid position 205 at week
40 in rebounding virus was excluded from this analysis. One subject
(subject 8) did not receive any additional vaccine doses after week
4 and therefore did not have immune studies performed at week 24
or 26 of the study. The colored dots indicate individual subject data.
The black symbols represent data from the one subject (subject 11)
who developed rebound viremia during the trial while still on
VOL. 83, 2009ONGOING HIV-1 REPLICATION IN RESIDUAL VIREMIA9739
PBMC (IQR, 183 to 3,400 SFC/million PBMC) for the three
subjects who experienced evolution and also received at least
one FPV booster dose and ?69.3 SFC/million PBMC (IQR,
?291 to 87.5 SFC/million PBMC) for the six subjects not
experiencing evolution (P ? 0.09) (Fig. 4). Notably, the one
subject (subject 11) who failed to maintain suppression of virus
replication while on HAART also experienced sustained in-
creases in HIV-1-specific CD8?T-cell responses to Pol pep-
tides postvaccination (Fig. 4). Furthermore, in this subject, a
decrease in viral diversity was observed during low-level vire-
mia by week 24 postvaccination and prior to viral rebound at
week 60. At screening and at entry, prevaccine low-level vire-
mia diversity levels were 0.026 and 0.027, respectively, and
remained high through week 6 of the study (0.031), after which
diversity levels decreased to 0.005, 0.01, 0.02, and 0.00 at weeks
24, 26, 40, and 72, respectively (Fig. 1).
The pathogenesis of persistent low-level viremia in patients
on HAART is unclear, but it is important to understand to
improve control of viral replication in patients on long-term
HAART. Evidence exists for both ongoing virus expression
from latent cellular reservoirs (or drug sanctuary sites) (2, 10,
21–23, 25, 26, 29, 31, 34, 46) and low-level virus replication
during HAART (6, 7, 22, 46). While these two mechanisms
may not be mutually exclusive, it is likely that interpatient
differences in the potency of suppression of virus replication
below 50 copies/ml shift the contributions of virus release
from long-lived infected cells and complete cycles of repli-
cation to sustaining residual viremia in the context of HIV-1
In this first study to examine serially the effects of immune
activation with recombinant MVA/FPV-based HIV-1 vaccina-
tions on low-level viremia in HAART-treated patients, we
found evidence for both complete cycles of virus replication,
with sequence evolution, in a subset of patients receiving du-
rable successful HAART (current clinical criterion of a plasma
viral load of ?50 copies/ml) and virus expression from a latent
reservoir. In particular, sequence evolution in free plasma virus
was significantly more likely to occur within relevant CD8?
T-cell epitopes of HIV-1 RT and consisted of novel amino acid
changes at a non-drug-resistance site, position 205 of HIV-1
RT, as well as new relevant drug resistance mutations. Fur-
thermore, we found that sequence evolution following HIV-1
vaccinations was significantly associated with more episodes of
quantifiable low-level viremia, suggesting ongoing cycles of
virus replication that may be amenable to CD8?T-cell selec-
tive pressures. At baseline, the subjects were heterogeneous
with respect to the predicted potency of their HAART regi-
mens (41) and the presence of drug resistance mutations, but
their respective HAART regimens had controlled plasma vire-
mia to clinically undetectable levels for years before study
entry. Furthermore, plasma viral loads remained low postvac-
cination even in the presence of relevant new drug resistance
mutations or evidence of sequence evolution at non-drug-re-
sistance sites during low-level viremia, except for in one sub-
ject. Importantly, for most subjects in this study, administration
of MVA/FPV HIV-1 therapeutic vaccines was not associated
with temporal shifts or sequence changes within persistent
wild-type or drug-resistant plasma viral sequences in very low
viremia or with new drug resistance mutations for up to 18
months postvaccination. The 40% of study participants without
amplifiable variants prevaccination also did not have amplifi-
able genotypes postvaccination and thus did not provide evi-
dence of either enhanced virus production or replication
following HIV-1 vaccination. Virus replication was even con-
trolled for the individuals for whom NNRTI-resistant variants
were detected postvaccination, despite their remaining on the
same nevirapine-based HAART regimen as that at study entry.
While they are important for the safety profile of these vac-
cines (18), these observations also support the clinical signifi-
cance of HAART to limit HIV-1 replication in most patients.
In particular, the current clinical criterion of ?50 copies/ml
indicates that HAART has significantly reduced the effective
virus population size, thereby slowing virus replication and
sequence evolution in most individuals (39).
Because recent studies of treatment intensification have
shown variable effects on low-level viremia (6, 10, 22, 24),
study-to-study variation in the baseline level of viremia must be
considered. In one study, baseline viremia was quite low, at 1
to 2 copies/ml, most likely reflecting the potency of the regi-
men, and no evidence of additional suppression was generated
by the addition of raltegravir (24). In contrast, abacavir inten-
sification of an efavirenz and indinavir two-drug regimen in
subjects with a baseline viral load of 10 copies/ml produced a
lowering of the viral load to ?2.5 copies/ml (22). Our data
demonstrate discernible sequence evolution in HAART-sup-
pressed subjects experiencing low-level viremia of ?6.5 copies/
ml. Together, these findings suggest that a threshold level of
viremia below 50 copies/ml may exist, above which productive
infection may be a greater contributor to sustained viremia
than low-level virus release from long-lived cellular reservoirs.
Under this model, the sequence evolution we observed during
therapeutic HIV-1 poxvirus vaccination, in or near epitopes
recognizable based on HLA serotypes, would be facilitated by
steady-state, low-level, productive infection. To test this will
require further study of patients receiving newer, more potent
HAART regimens than those used by the subjects in our study.
Nevertheless, the data suggest that the current therapeutic
target of control of viremia to ?50 copies/ml, while clinically
meaningful, is insufficient for distinguishing contributions of
low-level virus expression from those of productive infection.
A new therapeutic target for viremia may be necessary for
clinical trials assessing HIV-1 treatment approaches involving
newer drug combinations, therapeutic vaccinations, or treat-
ment intensification and deintensification strategies.
Among nonimmunized individuals, substitution at position
205 of HIV-1 RT is exceedingly rare, appearing in only a single
HIV-1 subtype C isolate (L205S) among 914 HIV-1 sequences
in the Los Alamos HIV Sequence Database (http://www.hiv
.lanl.gov). Amino acid position 205 of HIV-1 RT lies within an
HLA-A*02 cytotoxic T-lymphocyte epitope, KIEELRQHL
(48), and three of four subjects with L205 changes were HLA-
A*02 positive (subjects 7, 15, and 17) in our study. While the
fourth (subject 11) was HLA-A*6801 and -A*3001 positive,
and not HLA-A*02 positive, the L205M substitution was also
flanked by HLA-A*68 (FTTPDKKHQK) (9) and HLA-A*03
(DLEIGQHRTK) (48) epitopes. It is notable that HLA-
A*6801 could share binding to these regions as an A*03 su-
9740 SHIU ET AL.J. VIROL.
pertype (Fig. 2). Additionally, in the one subject (subject 8)
who developed new drug resistance mutations in low-level
viremia postvaccination, the first NNRTI resistance mutation
detected (V108I) lay within an HLA-A*02-restricted epitope
(VLDVGDAYFSV) (47) that was linked to an isoleucine-to-
threonine substitution at amino acid position 135 of HIV-1 RT
(I135T), known to confer decreased susceptibility to NNRTI
(5). This substitution also represents an amino acid change
within relevant HLA-A*02 and -B*51 epitopes (KYTAFTIPSI
and TAFTIPSI, respectively) (40, 43) for this individual, who
was indeed HLA-A*02 and -B*51 positive (Fig. 2). While the
NNRTI resistance mutations were transient, the I135T substi-
tution became fixed and persisted through week 72 of the trial,
suggesting selection due to either immune or drug pressure.
Moreover, in the one subject who developed rebound viremia
while on HAART, decreasing sequence diversity in low-level
viremia was associated with sustained CD8?T-cell responses
to HIV-1 Pol postvaccination, also suggesting specific HIV-1
immune selective pressure on residual replication during
HAART. This idea is supported by reports of selection of
immunity-escaping HIV-1 variants at clinically undetectable
viral loads in long-term nonprogressors (4, 16).
Together, these findings imply that viral populations repli-
cating at low levels may be targeted by HIV-1-specific immune
responses in HAART-treated patients. We were unable to
demonstrate that CD8?T-cell responses to the peptide con-
taining position 205 of HIV-1 RT were responsible for se-
quence evolution, though a finely detailed analysis of CD8?
T-cell recognition with overlapping peptides was not per-
formed. Finer mapping of immunological responses, including
interleukin-2-mediated responses (3), to this region of HIV-1
RT may help to elucidate the role of CD8?T-cell selective
pressures in L205 substitutions.
This study has some limitations. These include restriction of
genetic analysis to the HIV-1 RT region, the sensitivities of the
low-level viremia genotyping and viral load assays, the lack of
a placebo group to fully assess sequence evolution at viremia
levels of ?50 copies/ml within HLA-restricted CD8?T-cell
epitopes, and the small study population. Nevertheless, we
identified in vivo selection and convergent sequence evolution
associated with novel mutations within HLA epitopes in resid-
ual viremia among a subset of individuals receiving recom-
binant HIV-1 MVA/FPV vaccinations who had viral loads of
?50 copies/ml on durable successful HAART. Moreover,
this selection occurred in association with higher frequen-
cies of episodes of low-level viremia above 6.5 copies/ml.
The data support contributions of both low-level virus re-
lease (2, 10, 24, 28, 31, 34, 46) and productive infection (6,
22, 46), which suggests that a low-level threshold may exist
where the balance shifts for these two contributors. Al-
though reinforcing the benefits of HAART suppression of
viremia levels of ?50 copies/ml, this potential model sug-
gests that a new therapeutic target for viremia may be nec-
essary for clinical trials assessing newer treatment ap-
proaches for HIV-1, including vaccines. Furthermore, the
potential for virus replication and sequence evolution within
epitopes presented by relevant HLA sites observed in this
study has implications for therapeutic HIV-1 vaccines. Both
of these implications warrant further study.
This clinical trial (ClinicalTrials.gov identifier NCT00107549) was
supported by the International Maternal Pediatric Adolescent AIDS
Clinical Trials (IMPAACT) Network of the National Institute of Al-
lergy and Infectious Diseases (NIAID) (grant U01 A1068632), by the
general clinical research center units funded by the National Center
for Research Resources and the International and Domestic Pediatric
and Maternal HIV Clinical Trials Network of the National Institute of
Child Health and Human Development (grant N01-HD-3-3345), and
by the Pediatric AIDS Clinical Trials Group (PACTG) (group 1059).
The study on virus evolution in low-level viremia was supported by
NIAID grants R01 A155312 and R01 A1062446, awarded to D.
Persaud. Immunology studies were supported by an IMPAACT
Immunology Laboratory grant and NIAID grant RO1 32391 to K.
We acknowledge the contributions of the PACTG1059 study team,
clinical trials specialist Elizabeth Sheeran, and database manager Bar-
bara Heckman. We also acknowledge Linda Lambrecht from the Uni-
versity of Massachusetts, who performed the IFN-? ELISPOT assays;
Roxann Ashworth from the Institute of Genetics, Johns Hopkins
School of Medicine, for her independent analyses of the HIV-1 RT
sequence chromatograms; and Estelle Piwowar-Manning for her con-
tributions to the ultrasensitive viral load quantitation. We also express
great appreciation to the clinical trial sites and the young adults who
participated in this trial.
1. Ananworanich, J., A. Gayet-Ageron, M. Le Braz, W. Prasithsirikul, P.
Chetchotisakd, S. Kiertiburanakul, W. Munsakul, P. Raksakulkarn, S.
Tansuphasawasdikul, S. Sirivichayakul, M. Cavassini, U. Karrer, D.
Genne, R. Nuesch, P. Vernazza, E. Bernasconi, D. Leduc, C. Satchell, S.
Yerly, L. Perrin, A. Hill, T. Perneger, P. Phanuphak, H. Furrer, D.
Cooper, K. Ruxrungtham, and B. Hirschel. 2006. CD4-guided scheduled
treatment interruptions compared with continuous therapy for patients
infected with HIV-1: results of the Staccato randomised trial. Lancet
2. Bailey, J. R., A. R. Sedaghat, T. Kieffer, T. Brennan, P. K. Lee, M. Wind-
Rotolo, C. M. Haggerty, A. R. Kamireddi, Y. Liu, J. Lee, D. Persaud, J. E.
Gallant, J. Cofrancesco, Jr., T. C. Quinn, C. O. Wilke, S. C. Ray, J. D.
Siliciano, R. E. Nettles, and R. F. Siliciano. 2006. Residual human immu-
nodeficiency virus type 1 viremia in some patients on antiretroviral therapy
is dominated by a small number of invariant clones rarely found in circulat-
ing CD4?T cells. J. Virol. 80:6441–6457.
3. Bailey, J. R., T. M. Williams, R. F. Siliciano, and J. N. Blankson. 2006.
Maintenance of viral suppression in HIV-1-infected HLA-B*57? elite
suppressors despite CTL escape mutations. J. Exp. Med. 203:1357–1369.
4. Bailey, J. R., H. Zhang, B. W. Wegweiser, H. C. Yang, L. Herrera, A.
Ahonkhai, T. M. Williams, R. F. Siliciano, and J. N. Blankson. 2007. Evo-
lution of HIV-1 in an HLA-B*57-positive patient during virologic escape.
J. Infect. Dis. 196:50–55.
5. Brown, A. J., H. M. Precious, J. M. Whitcomb, J. K. Wong, M. Quigg, W.
Huang, E. S. Daar, R. T. D’Aquila, P. H. Keiser, E. Connick, N. S.
Hellmann, C. J. Petropoulos, D. D. Richman, and S. J. Little. 2000.
Reduced susceptibility of human immunodeficiency virus type 1 (HIV-1)
from patients with primary HIV infection to nonnucleoside reverse trans-
criptase inhibitors is associated with variation at novel amino acid sites.
J. Virol. 74:10269–10273.
6. Buzon, M., M. Llibre, J. Gatell, P. Domingo, R. Paredes, S. Palmer, M.
Sharkey, M. Stevenson, B. Clotet, and J. Martinez-Picado. 2009. Transient
increase in episomal viral cDNA following raltegravir intensification of a
stable HAART regimen, abstr. 423a. Abstr. 16th Conf. Retrovir. Opportun.
7. Cohen Stuart, J. W., A. M. Wensing, C. Kovacs, M. Righart, D. de Jong,
S. Kaye, R. Schuurman, C. J. Visser, and C. A. Boucher. 2001. Transient
relapses (“blips”) of plasma HIV RNA levels during HAART are asso-
ciated with drug resistance. J. Acquir. Immune Defic. Syndr. 28:105–113.
8. Danel, C., R. Moh, M. L. Chaix, D. Gabillard, J. Gnokoro, C. J. Diby, T.
Toni, L. Dohoun, C. Rouzioux, E. Bissagnene, R. Salamon, and X. Anglaret.
2009. Two-months-off, four-months-on antiretroviral regimen increases the
risk of resistance, compared with continuous therapy: a randomized trial
involving West African adults. J. Infect. Dis. 199:66–76.
9. De Groot, A. S., B. Jesdale, W. Martin, A. C. Saint, H. Sbai, A. Bosma, J.
Lieberman, G. Skowron, F. Mansourati, and K. H. Mayer. 2003. Mapping
cross-clade HIV-1 vaccine epitopes using a bioinformatics approach. Vac-
10. Dinoso, J. B., S. Y. Kim, A. M. Wiegand, S. E. Palmer, S. J. Gange, L.
Cranmer, A. O’Shea, M. Callender, A. Spivak, T. Brennan, M. F. Kearney,
M. A. Proschan, J. M. Mican, C. A. Rehm, J. M. Coffin, J. W. Mellors, R. F.
VOL. 83, 2009ONGOING HIV-1 REPLICATION IN RESIDUAL VIREMIA9741
Siliciano, and F. Maldarelli. 2009. Treatment intensification does not reduce Download full-text
residual HIV-1 viremia in patients on highly active antiretroviral therapy.
Proc. Natl. Acad. Sci. USA 106:9403–9408.
11. Dornadula, G., H. Zhang, B. VanUitert, J. Stern, L. Livornese, Jr., M. J.
Ingerman, J. Witek, R. J. Kedanis, J. Natkin, J. DeSimone, and R. J.
Pomerantz. 1999. Residual HIV-1 RNA in blood plasma of patients
taking suppressive highly active antiretroviral therapy. JAMA 282:1627–
12. Dorrell, L. 2006. Therapeutic immunization for the control of HIV-1: where
are we now? Int. J. STD AIDS 17:436–441.
13. Douek, D. C., J. M. Brenchley, M. R. Betts, D. R. Ambrozak, B. J. Hill,
Y. Okamoto, J. P. Casazza, J. Kuruppu, K. Kunstman, S. Wolinsky, Z.
Grossman, M. Dybul, A. Oxenius, D. A. Price, M. Connors, and R. A.
Koup. 2002. HIV preferentially infects HIV-specific CD4? T cells. Na-
14. El-Sadr, W. M., J. D. Lundgren, J. D. Neaton, F. Gordin, D. Abrams, R. C.
Arduino, A. Babiker, W. Burman, N. Clumeck, C. J. Cohen, D. Cohn, D.
Cooper, J. Darbyshire, S. Emery, G. Fatkenheuer, B. Gazzard, B. Grund, J.
Hoy, K. Klingman, M. Losso, N. Markowitz, J. Neuhaus, A. Phillips, and C.
Rappoport. 2006. CD4? count-guided interruption of antiretroviral treat-
ment. N. Engl. J. Med. 355:2283–2296.
15. Emery, S., J. A. Neuhaus, A. N. Phillips, A. Babiker, C. J. Cohen, J. M.
Gatell, P. M. Girard, B. Grund, M. Law, M. H. Losso, A. Palfreeman, and R.
Wood. 2008. Major clinical outcomes in antiretroviral therapy (ART)-naive
participants and in those not receiving ART at baseline in the SMART study.
J. Infect. Dis. 197:1133–1144.
16. Feeney, M. E., Y. Tang, K. A. Roosevelt, A. J. Leslie, K. McIntosh, N.
Karthas, B. D. Walker, and P. J. Goulder. 2004. Immune escape precedes
breakthrough human immunodeficiency virus type 1 viremia and broadening
of the cytotoxic T-lymphocyte response in an HLA-B27-positive long-term-
nonprogressing child. J. Virol. 78:8927–8930.
17. Furtado, M. R., D. S. Callaway, J. P. Phair, K. J. Kunstman, J. L. Stanton,
C. A. Macken, A. S. Perelson, and S. M. Wolinsky. 1999. Persistence of
HIV-1 transcription in peripheral-blood mononuclear cells in patients re-
ceiving potent antiretroviral therapy. N. Engl. J. Med. 340:1614–1622.
18. Greenough, T. C., C. K. Cunningham, P. Muresan, M. McManus, D. Per-
saud, T. Fenton, P. Barker, A. Gaur, D. Panicali, J. L. Sullivan, and K.
Luzuriaga. 2008. Safety and immunogenicity of recombinant poxvirus HIV-1
vaccines in young adults on highly active antiretroviral therapy. Vaccine
19. Grossman, Z., M. Polis, M. B. Feinberg, Z. Grossman, I. Levi, S. Jankelev-
ich, R. Yarchoan, J. Boon, F. de Wolf, J. M. Lange, J. Goudsmit, D. S.
Dimitrov, and W. E. Paul. 1999. Ongoing HIV dissemination during
HAART. Nat. Med. 5:1099–1104.
20. Guindon, S., and O. Gascuel. 2003. A simple, fast, and accurate algorithm
to estimate large phylogenies by maximum likelihood. Syst. Biol. 52:696–
21. Havlir, D. V., R. Bassett, D. Levitan, P. Gilbert, P. Tebas, A. C. Collier, M. S.
Hirsch, C. Ignacio, J. Condra, H. F. Gunthard, D. D. Richman, and J. K.
Wong. 2001. Prevalence and predictive value of intermittent viremia with
combination HIV therapy. JAMA 286:171–179.
22. Havlir, D. V., M. C. Strain, M. Clerici, C. Ignacio, D. Trabattoni, P. Fer-
rante, and J. K. Wong. 2003. Productive infection maintains a dynamic
steady state of residual viremia in human immunodeficiency virus type 1-in-
fected persons treated with suppressive antiretroviral therapy for five years.
J. Virol. 77:11212–11219.
23. Hermankova, M., S. C. Ray, C. Ruff, M. Powell-Davis, R. Ingersoll, R. T.
D’Aquila, T. C. Quinn, J. D. Siliciano, R. F. Siliciano, and D. Persaud. 2001.
HIV-1 drug resistance profiles in children and adults with viral load of ?50
copies/ml receiving combination therapy. JAMA 286:196–207.
24. Jones, J., D. McMahon, A. Wiegand, M. Kearney, S. Palmer, S. McNulty, J.
Metcalf, J. Coffin, J. Mellors, and F. Maldarelli. 2009. No decrease in
residual viremia during raltegravir intensification in patients on standard
ART, abstr. 423b. Abstr. 16th Conf. Retrovir. Opportun. Infect.
25. Kieffer, T. L., M. M. Finucane, R. E. Nettles, T. C. Quinn, K. W. Broman,
S. C. Ray, D. Persaud, and R. F. Siliciano. 2004. Genotypic analysis of
HIV-1 drug resistance at the limit of detection: virus production without
evolution in treated adults with undetectable HIV loads. J. Infect. Dis.
26. Lee, K. J., D. Shingadia, D. Pillay, A. S. Walker, A. Riordan, E. Menson, T.
Duong, G. Tudor-Williams, and D. M. Gibb. 2007. Transient viral load
increases in HIV-infected children in the U.K. and Ireland: what do they
mean? Antivir. Ther. 12:949–956.
27. Liang, K. Y., and S. L. Zeger. 1986. Longitudinal data-analysis using gener-
alized linear-models. Biometrika 73:13–22.
28. Maldarelli, F., S. Palmer, M. S. King, A. Wiegand, M. A. Polis, J. Mican,
J. A. Kovacs, R. T. Davey, D. Rock-Kress, R. Dewar, S. Liu, J. A. Metcalf, C.
Rehm, S. C. Brun, G. J. Hanna, D. J. Kempf, J. M. Coffin, and J. W. Mellors.
2007. ART suppresses plasma HIV-1 RNA to a stable set point predicted by
pretherapy viremia. PLoS Pathog. 3:e46.
29. Nettles, R. E., T. L. Kieffer, P. Kwon, D. Monie, Y. Han, T. Parsons, J.
Cofrancesco, Jr., J. E. Gallant, T. C. Quinn, B. Jackson, C. Flexner, K.
Carson, S. Ray, D. Persaud, and R. F. Siliciano. 2005. Intermittent HIV-1
viremia (blips) and drug resistance in patients receiving HAART. JAMA
30. Nettles, R. E., T. L. Kieffer, R. P. Simmons, J. Cofrancesco, Jr., R. D.
Moore, J. E. Gallant, D. Persaud, and R. F. Siliciano. 2004. Genotypic
resistance in HIV-1-infected patients with persistently detectable low-
level viremia while receiving highly active antiretroviral therapy. Clin.
Infect. Dis. 39:1030–1037.
31. Palmer, S., F. Maldarelli, A. Wiegand, B. Bernstein, G. J. Hanna, S. C. Brun,
D. J. Kempf, J. W. Mellors, J. M. Coffin, and M. S. King. 2008. Low-level
viremia persists for at least 7 years in patients on suppressive antiretroviral
therapy. Proc. Natl. Acad. Sci. USA 105:3879–3884.
32. Perelson, A. S., P. Essunger, Y. Cao, M. Vesanen, A. Hurley, K. Saksela, M.
Markowitz, and D. D. Ho. 1997. Decay characteristics of HIV-1-infected
compartments during combination therapy. Nature 387:188–191.
33. Persaud, D., S. C. Ray, J. Kajdas, A. Ahonkhai, G. K. Siberry, K. Ferguson,
C. Ziemniak, T. C. Quinn, J. P. Casazza, S. Zeichner, S. J. Gange, and D. C.
Watson. 2007. Slow human immunodeficiency virus type 1 evolution in viral
reservoirs in infants treated with effective antiretroviral therapy. AIDS Res.
Hum. Retrovir. 23:381–390.
34. Persaud, D., G. K. Siberry, A. Ahonkhai, J. Kajdas, D. Monie, N. Hutton,
D. C. Watson, T. C. Quinn, S. C. Ray, and R. F. Siliciano. 2004. Continued
production of drug-sensitive human immunodeficiency virus type 1 in chil-
dren on combination antiretroviral therapy who have undetectable viral
loads. J. Virol. 78:968–979.
35. Persaud, D., Y. Zhou, J. M. Siliciano, and R. F. Siliciano. 2003. Latency in
human immunodeficiency virus type 1 infection: no easy answers. J. Virol.
36. Piwowar-Manning, E. M., T. A. Henderson, L. Brisbin, and J. B. Jackson.
2003. A modified ultrasensitive assay to detect quantified HIV-1 RNA of
fewer than 50 copies per milliliter. Am. J. Clin. Pathol. 120:268–270.
37. Posada, D., and K. A. Crandall. 1998. MODELTEST: testing the model of
DNA substitution. Bioinformatics 14:817–818.
38. Ramratnam, B., R. Ribeiro, T. He, C. Chung, V. Simon, J. Vanderhoeven, A.
Hurley, L. Zhang, A. S. Perelson, D. D. Ho, and M. Markowitz. 2004.
Intensification of antiretroviral therapy accelerates the decay of the HIV-1
latent reservoir and decreases, but does not eliminate, ongoing virus repli-
cation. J. Acquir. Immune Defic. Syndr. 35:33–37.
39. Rouzine, I. M., A. Rodrigo, and J. M. Coffin. 2001. Transition between
stochastic evolution and deterministic evolution in the presence of selection:
general theory and application to virology. Microbiol. Mol. Biol. Rev. 65:
40. Shankar, P., H. Sprang, and J. Lieberman. 1998. Effective lysis of HIV-1-
infected primary CD4? T cells by a cytotoxic T-lymphocyte clone directed
against a novel A2-restricted reverse-transcriptase epitope. J. Acquir. Im-
mune Defic. Syndr. Hum. Retrovirol. 19:111–120.
41. Shen, L., S. Peterson, A. R. Sedaghat, M. A. McMahon, M. Callender, H.
Zhang, Y. Zhou, E. Pitt, K. S. Anderson, E. P. Acosta, and R. F. Siliciano.
2008. Dose-response curve slope sets class-specific limits on inhibitory po-
tential of anti-HIV drugs. Nat. Med. 14:762–766.
42. Siliciano, J. D., and R. F. Siliciano. 2005. Enhanced culture assay for detec-
tion and quantitation of latently infected, resting CD4? T-cells carrying
replication-competent virus in HIV-1-infected individuals. Methods Mol.
43. Sipsas, N. V., S. A. Kalams, A. Trocha, S. He, W. A. Blattner, B. D. Walker,
and R. P. Johnson. 1997. Identification of type-specific cytotoxic T lympho-
cyte responses to homologous viral proteins in laboratory workers acciden-
tally infected with HIV-1. J. Clin. Investig. 99:752–762.
44. Smith, D. B., J. McAllister, C. Casino, and P. Simmonds. 1997. Virus
‘quasispecies’: making a mountain out of a molehill? J. Gen. Virol. 78:1511–
45. Tamura, K., J. Dudley, M. Nei, and S. Kumar. 2007. MEGA4: molecular
evolutionary genetics analysis (MEGA) software version 4.0. Mol. Biol. Evol.
46. Tobin, N. H., G. H. Learn, S. E. Holte, Y. Wang, A. J. Melvin, J. L. Mc-
Kernan, D. M. Pawluk, K. M. Mohan, P. F. Lewis, J. I. Mullins, and L. M.
Frenkel. 2005. Evidence that low-level viremias during effective highly active
antiretroviral therapy result from two processes: expression of archival virus
and replication of virus. J. Virol. 79:9625–9634.
47. van der Burg, S. H., M. R. Klein, C. J. van de Velde, W. M. Kast, F.
Miedema, and C. J. Melief. 1995. Induction of a primary human cytotoxic
T-lymphocyte response against a novel conserved epitope in a functional
sequence of HIV-1 reverse transcriptase. AIDS 9:121–127.
48. Walker, B. D., C. Flexner, K. Birch-Limberger, L. Fisher, T. J. Paradis, A.
Aldovini, R. Young, B. Moss, and R. T. Schooley. 1989. Long-term culture
and fine specificity of human cytotoxic T-lymphocyte clones reactive with
human immunodeficiency virus type 1. Proc. Natl. Acad. Sci. USA 86:9514–
49. Ziemniak, C., A. George-Agwu, W. J. Moss, S. C. Ray, and D. Persaud. 2006.
A sensitive genotyping assay for detection of drug resistance mutations in
reverse transcriptase of HIV-1 subtypes B and C in samples stored as dried
blood spots or frozen RNA extracts. J. Virol. Methods 136:238–247.
9742 SHIU ET AL.J. VIROL.