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Journal of Virology, November 1999, p. 9404-9412, Vol. 73, No. 11
University of California,
Received 17 March 1999/Accepted 9 July 1999
In human immunodeficiency virus (HIV)-infected patients treated
with potent antiretroviral therapy, the persistence of latently infected cells may reflect the long decay half-life of this cellular reservoir or ongoing viral replication at low levels with continuous replenishment of the population or both. To address these
possibilities, sequences encompassing the C2 and V3 domains of HIV-1
env were analyzed from virus present in baseline plasma and
from viral isolates obtained after 2 years of suppressive therapy in
six patients. The presence of sequence changes consistent with
evolution was demonstrated for three subjects and correlated with less
complete suppression of viral replication, as indicated by the rapidity of the initial virus load decline or the intermittent reappearance of
even low levels of detectable viremia. Together, these results provide
evidence for ongoing replication. In the remaining three patients,
virus recovered after 2 years of therapy was either genotypically
contemporary with or ancestral to virus present in plasma 2 years
before, indicating that virus recovery had indeed resulted from
activation of latently infected cells.
Treatment with combination
antiretroviral agents can reduce plasma and lymph node RNA levels below
the level of detection of sensitive assays for nucleic acid (2,
12, 31, 46). The persistence of cells harboring
replication-competent virus despite periods of up to 3 years of potent
suppressive therapy has raised questions about the feasibility of
eradication of infection with currently recommended treatments (3,
9, 10, 47, 49). Despite the apparent lack of development of drug
resistance in patients with undetectable virus (9, 47), the
inferred presence of unintegrated viral DNA in peripheral blood
mononuclear cells (PBMC) in one study (3) has raised
questions about the certainty that viral replication is controlled even
in this setting. Extrapolating from such observations, it can be argued
that the persistence of this reservoir of virus reflects ongoing viral replication at low levels and continuous replenishment rather than the
existence of long-lived, latently infected cells.
To address this question, we studied sequences of the human
immunodeficiency virus type 1 (HIV-1) envelope corresponding to the
relatively conserved C2 and the hypervariable V3 domains of env from HIV-1 present in baseline plasma and in virus
recovered from in vitro culture of PBMC following 2 years of therapy.
The V3 region is an important determinant of cell tropism and is a target of host immune responses. Consequently, it is among the most
variable regions in the HIV genome (15, 19, 21, 36). Sequence differences in this region have been used to compare the
relatedness of virus populations (20, 33, 42) and to study
viral evolution within individual hosts (1a, 6, 37-39, 45),
among patients (1a, 6, 25, 26, 37, 38, 45), and across
patient populations (21, 22, 27).
Samples were studied from six individuals from whom, despite prolonged
suppression of plasma viremia, virus had been previously recovered by
coculture of PBMC (47). We hypothesized that ongoing replication should be accompanied by changes in viral envelope sequence
and that phylogenetic analysis would permit the distinction of virus
produced by activation of latently infected cells from that produced as
a consequence of continued viral propagation during the course of therapy.
(Presented in part at the 2nd International Workshop on HIV Drug
Resistance and Treatment Strategies, Lake Maggiore, Italy, June 1998.)
Patient selection.
Subjects were chosen from a
well-characterized cohort of HIV-infected patients undergoing treatment
with indinavir, lamivudine, and zidovudine (12, 47). Each
had apparent suppression of plasma and serum viral RNA to below the
50-copy/ml level with the exception of patient M, who had 50 copies of
HIV RNA/ml on the day of PBMC sampling for virus isolation (Fig.
1). Sequence comparisons were made between virus present in baseline plasma and
virus isolated while on drug therapy at year 2.
Viral isolates.
The materials and methods for viral
isolation have been previously published (47). Early culture
supernatants were chosen to minimize ex vivo culture effects on the
composition of the viral isolates (typically at day 14 of original
cultures and before peak p24 production).
RNA extraction.
RNA was extracted from cell-free viral
culture supernatants with the Qiagen HCV RNA extraction kit (Qiagen,
Chatsworth, Calif.) and from plasma by the AMPLICOR extraction
procedure (Roche Diagnostics, Branchburg, N.J.) according to the
manufacturers' protocols.
RT-PCR.
Following denaturation of the RNA template and
primer mix at 70°C for 3 min, reverse transcription (RT) was
performed at 45°C for 1 h in the following buffer: 100 mM Tris
(pH 8.3), 10 mM MgCl, 50 mM KCl, 10 mM dithiothreitol, 250 µM
dinucleoside triphosphate, 37 U of RNAgard (Pharmacia), 2.5 U of avian
myeloblastosis virus reverse transcriptase (Gibco BRL, Gaithersburg,
Md.), 12.5 pmol of V3Bout primer (5'-ATTACAGTAGAAAAATTCCCCT-3').
Nested PCRs were then performed in quadruplicate for each sample
to minimize sampling bias. RT product (cDNA; 10 µl) was carried over
to a first-step PCR employing the primers V3Bout and V3Fout
(5'-CAAAGGTATCCTTTGAGCCAAT-3') under the following reaction
and thermocycling conditions: Tris (pH 8.3), 3 mM MgCl2,
250 µM deoxynucleoside triphosphate, 20 pmol of primers, 1 mg of
bovine serum albumin/ml, 5 U of Taq polymerase (Boehringer
Mannheim) in a 50-µl reaction volume at 95°C for 2 min, 95°C for
30 s, 55°C for 30 s, 72°C for 1 min for 35 cycles; and
72°C for 1 min. One microliter of the first-step product was then
carried over to a second-step PCR with the primers V3Fin (5'-GAACAGGACCAGGATCCAATGTCAGCACAGTACAAT-3') and V3Bin
(5'-GCGTTAAAGCTTCTGGGTCCCCTCCTGAG-3') with reaction
conditions identical to those for the first-step PCR. Ten percent of
the PCR products were resolved on 1% agarose gels and visualized by
ethidium bromide staining. We have examined the rate of
misincorporation during amplification under these nested PCR conditions
by comparing the results from sequencing of unamplified pNL4-3
(1) with those from sequencing of amplified and subsequently
cloned products and have observed a misincorporation frequency of
approximately 1 to 3 per 3,500 bases.
Cloning.
After determination that each of quadruplicate
nested PCR products yielded comparable band intensities by ethidium
staining, 5 µl of each of the quadruplicate reactions were pooled
together and gel purified by agarose gel electrophoresis. DNA was
extracted from gel slices with the Qiaex II Gel Extraction kit (Qiagen) according to the manufacturer's protocol. Two microliters of the pooled, gel-purified PCR products was ligated into the plasmid vector
pCR 2.1 and cloned according to the instructions for the TOPO Ta
Cloning system (Invitrogen, Carlsbad, Calif.) under ampicillin selection.
Sequencing.
Ten to 13 individual clones were picked based on
blue-white screening and grown in 3-ml cultures. Plasmids were purified
with the Qiaprep Turbo-8 kit (Qiagen). Approximately 300 ng of each plasmid was sequenced with the sequencing primers ForTopoEcoR1 (5'-TGGATATCTGCAGAATTCG-3') and M13 Reverse
(5'-CAGGAAACAGCTATGAC-3') with a 373A automated sequencer
and Big-dye chain terminator chemistry (Applied Biosystems, Inc.,
Foster City, Calif.). The sequences were aligned and manually edited
with Sequence Navigator 1.1 (Applied Biosystems). In total, 115 evaluable sequences were obtained for the six patients.
Phylogenetic analysis.
Tree reconstructions were performed
by a neighbor-joining method implemented with DNADIST (Kimura
distances; transition transversion ratio, 2.0) and NEIGHBOR in Phylip
version 3.54 (7) with 100 bootstrapped data sets and by a
maximum-likelihood method with fastDNAML (7, 30), applying a
transition/transversion ratio of 2.0 and a uniform substitution rate.
Graphic representation of phylogenetic trees was accomplished with
Treeview version 1.5 (30a).
Determination of pairwise distances.
Pairwise distances from
the most recent common ancestor (MRCA; represented by the most distal
node which gives rise to all sequences from the same patient) to clonal
sequences on the maximum-likelihood phylogenetic tree were estimated
both by summing relevant branch lengths generated with fastDNAML
version 1.0 (random input order; transition/transversion ratio, 2.0;
single rate of substitution) (7, 30) and by direct
computation with DNADIST (PHYLIP version 3.54; maximum-likelihood
estimates; transition/transversion ratio, 2.0) (7) from the
inferred MRCA sequences obtained with DNAML (PHYLIP version 3.6)
(7, 8). Branch lengths from fastDNAML were chosen over
those provided by DNAML (PHYLIP version 3.6) because the latter
presently assigns a minimum distance of 0.00005 to every segment of
inferred maximum-likelihood trees, including branches connecting
identical sequences. However, the two programs produced trees with
comparable maximum-likelihood values. Duplicate analyses were
performed, with selection of the MRCA based on all viral isolate
sequences and the nearest plasma sequences (changed the selection of
the MRCA for patients A, C, and M). Means and standard errors for
distances were calculated with JMP version 3.1.5 (SAS, Cary, N.C.).
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Evolution of Envelope Sequences of Human
Immunodeficiency Virus Type 1 in Cellular Reservoirs in the Setting of
Potent Antiviral Therapy
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
View larger version (10K):
[in a new window]
FIG. 1.
Log RNA copies/ml in serum or plasma plotted against
days under indinavir, zidovudine, and lamivudine combination therapy.
The baseline for the y axis is the limit of detection of the
Roche ultrasensitive assay (1.6 log copies/ml). The top panel
demonstrates the response of two patients (Pt.) with steep initial declines in viral load (A and B). The middle panel
shows two patients with steep initial declines followed by transient
reappearance of low levels of detectable viral RNA (K and L). The
bottom panel shows two patients with slow initial responses (C and M)
and, in one patient (M), intermittent transient reappearance of low
levels of viral RNA in the blood.
Estimation of residual replication. Residual HIV replication was approximated by calculation of the area under the curve (AUC) generated by plotting log plasma RNA against time. The observed AUC (AUCObs) represented the log RNA copy by time product which results from the decay of free virus in plasma and virus produced by cells infected prior to institution of therapy (productively infected cells; a reservoir of "long-lived" infected cells; and release of virus from the follicular dendritic network in lymphoid tissues and activation of latently infected cells) as well as from any virus produced as a consequence of ongoing viral replication during combination therapy.
The theoretical AUC for each patient was estimated according to the long-lived-infected-cell model of Perelson et al. (31). This calculation assumes decay in the absence of residual viral replication under therapy and assumes that activation of latent cells contributes relatively little to the viral RNA measured during first- or second-phase decay of plasma virus. Log10 viral RNA copy number (log10V) as a function of time (t) can be described as follows:
![<UP>log</UP><SUB>10</SUB>V(t)=<UP>log</UP><SUB>10</SUB>V<SUB>0</SUB><FENCE>[NkT<SUB>0</SUB>/(c−&dgr;)]e<SUP>−&dgr;t</SUP>+[(c−NkT<SUB>0</SUB>)/(c−&mgr;<SUB>M</SUB>)]e<SUP>−&mgr;<SUB>M</SUB>t</SUP>+{1−[NkT<SUB>0</SUB>/(c−&dgr;)]−[(c−NkT<SUB>0</SUB>)/(c−&mgr;<SUB>M</SUB>)]}e<SUP>−ct</SUP>)</FENCE>](/content/vol73/issue11/fulltext/9404/img001.gif)
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(1) |
is the decay constant of productively infected cells,
µM is the decay constant of long-lived cells,
c is the clearance constant for virions,
T0 is the CD4 count at baseline, N is
the viral burst size, and k is the infection constant
(31). Perelson et al. observed NkT0
values between 2.84 and 2.92 for a T0 of 205 to 500 cells/mm3. We utilized a mean value for
NkT0 of 2.88 and the observed virus clearance
constant, c, of 3 day
1 in our analysis
(32).
The serially measured viral RNA concentrations and those calculated
from equation (1) were plotted against time under treatment with the
program PRISM 2.0 (Graphpad, San Diego, Calif.). AUCs were calculated
by summation of trapezoids implemented in the program PRISM 2.0. The
predicted AUC (AUCPred) was computed with a of 1.2 day1 and a µM of 0.115, based on
the best-case decay constants observed by Perelson et al.
(31). Duplicate analyses with a of 0.7 day1 and a µM of 0.066 based on
mean decay constants (31) for the calculation of
AUCPred resulted in very similar results (data not shown).
The difference between AUCObs and AUCPred,
then, represented the proportion of the RNA concentration by time
product that should be attributable to residual viral replication for each patient while under therapy (AUCNet).
In general, the use of AUC as an indicator of the difference in the
degree of ongoing viral replication between patients may be best when
their baseline RNA concentrations are similar, while the calculation of
AUCNet from AUCObs and AUCPred
becomes most necessary when baseline RNA concentrations vary greatly
among the patients being compared. In the present study, the patients had relatively uniform starting RNA levels.
Our calculations assumed negligible contribution to total viremia from
sporadic activation of latently infected cells (a simplification justified by observations of phase 1 and phase 2 decay by Perelson et
al. [31]) and may therefore tend to underestimate the
AUCPred, leading to overestimates of AUCNet.
Because of this and other assumptions made for the derivation of these
terms, it would be incorrect to treat the calculated values as
definitive for residual viral replication. Rather, they are meant to
represent a relative magnitude of residual replication (derived from
the best available kinetic data and based on prevailing theory in the
published literature) and are applied here to permit reasonable
interpatient comparison.
Statistical analyses. Correlations of AUCNet with pairwise genetic distance and Wilcoxon rank sums for comparison of pairwise distances were computed with the statistical analysis program JMP 3.1.5. (SAS) and the program Prism 2.0.
Nucleotide sequence accession numbers. All sequences reported here have been deposited in GenBank and were given accession no. AF185823 to AF185937.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Assessment of residual viral replication based on the response of
serum and plasma RNA to drug therapy permitted segregation of the
patients into three patterns (Fig. 1). Group 1 (patients A and B) had
subjects with the steepest initial declines in viral load and without
any detectable viral RNA for the duration of this study period. Group 2 patients (K and L) were subjects who had similarly rapid initial
declines in plasma virus concentrations upon initiation of therapy but
with sporadic reappearance of low but detectable levels of viremia
later. Finally, two patients (C and M) constituted a third group
exhibiting a protracted and gradual decline in plasma virus levels with
later sporadic reappearance of viremia throughout the first 2 years of
therapy. In the case of M, viral RNA was present in plasma at the same
time that samples were obtained for viral culture (47). The
amount of residual replication as reflected by AUCNet was
low to negligible for patients A, B, and L, intermediate for K and C,
and highest for patient M (Table 1).
|
Phylogenetic reconstruction. In every case, sequences from each patient clustered together and not with those from other patients or with prototypic laboratory-adapted and "primary" HIV clones, as shown by a reconstruction by maximum-likelihood analysis (Fig. 2) (24). Tree topology with a neighbor-joining reconstruction was similar: sequences from each patient formed individual clusters with highly significant bootstrap values (>80 per 100 bootstrapped data sets examined) in the consensus neighbor-joining tree (data not shown). In one case (patient B), year 2 isolates were indistinguishable from sequences present at baseline and were very homogeneous. For patients A and C, the viral isolates most closely resembled one or a subset of sequences present in baseline plasma, while for patients K, L, and M, no plasma sequences were identified which clearly clustered with sequences from year 2 viral isolates, although in the case of M, a "nearest plasma clone" could be identified. For patients M and C, branch lengths from the closest proximal common node (representing the most recent common ancestor) to the year 2 isolates appeared longer than those to the baseline plasma sequences.
|
Protein sequence comparisons. In four patients, inferred protein sequences demonstrated little difference between baseline and year 2 isolates in the C2 region (Fig. 3). In the V3 region, baseline sequences differed from year 2 viral isolates at multiple positions in the case of patient M, while few differences were noted for the other five subjects. For patient M, an S-to-R substitution was seen at position 306 (according to HXB2 sequence by convention of the Los Alamos National Laboratories) in the V3 loop which has been associated with a switch from M- to T-cell tropism (4, 11), but cell tropism and presumably coreceptor usage are context dependent (40). In this patient there was an accompanying R-to-S replacement at residue 322. Cultivation of the year 2 virus in an MT-2 cell assay (34) showed that it was non-syncytium inducing (data not shown). In the case of patient K, multiple replacements were noted in the region downstream from the V3 loop, towards V4.
|
Pairwise genetic distances between baseline plasma virus and year 2 viral isolates.
Pairwise genetic distances (both corrected and
uncorrected for overall diversity of clones from each source)
demonstrated a continuum of small but measurable distances between
baseline plasma-derived sequences and 2-year viral isolates. Corrected distances demonstrated considerably greater variability than
uncorrected ones due to substantial variation among patients in the
level of diversity in plasma samples (Table
2). A more conservative calculation of
net divergence was also made by using the diversity of the time zero
plasma population as an estimate of the true diversity of virus in the
population of viably infected cells at year 2. The rank orders of
divergence in these analyses were similar, with M and K showing the
greatest divergence (data not shown).
|
Genetic distance to MRCA.
Based on coalescence theory
(17, 18, 35, 41), each sequence can be traced back to the
most recent common ancestral sequence (the MRCA, represented by the
most distal node common to all sequences from each patient on the
phylogenetic tree [Fig. 2]), with branch lengths proportional to the
number of elapsed generations. Here we assume that the mutation rate µ is relatively constant among individuals so that sequence
divergence is determined by gt, where g is the
number of generations (replication cycles) times time1
and t is the elapsed time. Although mutations within the
reverse transcriptase have been reported to affect the fidelity of
HIV-1 RT (43), such mutations were not observed in these
viral isolates (47).
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Correlation of evolutionary distance with residual replication represented by AUCNet. Mean pairwise distances between plasma virus and virus isolated at year 2 (both corrected and uncorrected for intracompartment diversity) correlated with AUCNet, with r2 values of 0.55 and 0.6, respectively, but P values only approached statistical significance (0.09 and 0.067, respectively). However, differences in mean distance by analysis of branch lengths from MRCA to all virus isolates and to all plasma sequences from the same patient showed a significant correlation with AUCNet: r2 = 0.889 (P = 0.0048). Using a direct calculation from the inferred ancestral sequences, the r2 value was 0.68 (P = 0.043) with AUCNet (Fig. 4). When MRCAs were selected based on all viral isolates but only the nearest plasma sequence(s) for each patient, similar correlations were noted (data not shown).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Latently infected resting T cells harboring replication-competent virus persist in treated patients despite the absence of detectable free virus in the blood (3, 9, 47). Whether this reservoir of virus is maintained as a result of a low level of ongoing viral replication or as a result of a slow decay rate carries important implications for the risk of development of drug resistance and treatment failure in such patients. This question is central to the success or failure of any eradication strategy based on currently available antiviral drugs.
By conventional measures, all subjects studied had excellent responses to therapy, with viral loads rapidly falling to below 200 copies/ml of serum or plasma upon initiation of antiviral therapy. These responses were sustained at levels below the threshold of detection of a more sensitive assay (<50 copies/ml) for periods approaching 2 years at the time virus was isolated (12, 13). Previous studies of these subjects had demonstrated the lack of development of new genotypic drug resistance over this period (47). However, a detailed examination of the serial HIV RNA levels in blood from these patients revealed varying patterns based on rapidity of decline of the viral load and in some cases on the intermittent reappearance of low-level viremia during the course of therapy. These qualitative differences were reflected quantitatively in the calculated AUCNet values representing residual viremia from ongoing viral replication (and/or, to a small extent, from activation of latently infected cells in vivo [see Materials and Methods]). It is not known why the antiviral drug effect may vary among these patients in spite of identical therapies, similar prestudy drug treatment histories, and similar incidence of preexisting drug resistance (13, 46, 47). Such differences could be attributable to individual differences in drug absorption or drug metabolism or to variable adherence.
Pairwise genetic distances from year 2 viral isolates to corresponding baseline plasma virus and pairwise distances from year 2 viral isolates and baseline plasma virus to the ancestral sequence inferred from a maximum-likelihood phylogenetic reconstruction were examined to estimate viral evolution in these subjects. Use of simple pairwise distances from year 2 viral isolates to baseline plasma virus would be expected to be an accurate reflection of generations elapsed between time points if year 2 viral isolates were, in every case, descendent from the virus present in plasma at baseline. However, if viral isolates at year 2 arise from latently infected cells that might have been ancestral to those present in plasma at baseline or which might have descended independently from a common ancestor, simple pairwise distances and generations elapsed need not be directly proportional. So, while those three patients (C, K, and M) having the largest AUCNet (e.g., those with slower initial decline of viremia and those with sporadic reappearance of viremia) had the highest mean pairwise distances and the three subjects (A, B, and L) with the lowest AUCNet had the lowest pairwise distances, the correlation of these parameters failed to reach statistical significance.
To quantitatively analyze genetic relationships of virus isolated at year 2 and virus present in baseline plasma (time zero) without having to assume that year 2 virus descended directly from baseline plasma virus, we examined pairwise distances from each cloned sequence to the corresponding MRCAs. The approach of summing relevant branch segments benefits from the incorporation of the structure of the inferred phylogenies into the distance measurements but does not readily yield an appropriate confidence interval. Therefore, we have also reconstructed the MRCA sequences with a maximum-likelihood approach (implemented in PHYLIP version 3.6) (7, 8) and directly estimated evolutionary distance between these and the baseline (plasma) and year 2 (virus isolate) sequences. The estimates resulting from both of these approaches demonstrated significant correlations of differences in distance to the MRCA with AUCNet (Fig. 4).
Findings consistent with sequence evolution in cultured virus from some patients but not from others could be explained by a sampling artifact (isolation of virus from few latently infected cells) or by a selection artifact from in vitro coculture conditions with some patients but not others (5, 23, 28). We have analyzed sequences of virus cultured at time zero from patient A (virus isolates from time zero were not available from the other patients). This baseline isolate clustered more closely with virus in plasma at time zero than with viral isolates from the year 2 time point (data not shown), indicating greater effects from the time of sampling than from in vitro culturing in this setting. Moreover, the strong correlation between sequence evolution (measured by pairwise distances to the MRCA) and residual replication inferred by the magnitude of AUCNet supports in vivo biological differences over random sampling artifacts or artifacts from in vitro culture to explain these differences. Additional studies of the nature of the sequence changes observed have been performed which show that in the cases of M and K, but not the other four subjects, the changes in fact reflect selective differences between year 0 and year 2 (11a).
In the case of patient M and possibly patient K, the intermittent appearance of detectable plasma viral RNA into the second year of therapy and the finding of evolution in the envelope sequences would be consistent with continuous or intermittent viral replication. Alternatively, but not mutually exclusively, in the case of patient M, where nonsynonymous substitutions in V3 were so frequent, sequences of year 2 viral isolates might reflect differences of a viral population maintained in an isolated anatomic compartment or a sanctuary site (20, 33, 48, 49). For patient C, reappearance of detectable free virus did not occur in the second year, although it was seen late into the first year, and the initial decline of plasma viremia was much more gradual than that seen for patient A, B, or L. In this case, the intermediate level of sequence evolution observed might be attributable either to viral replication occurring soon after initiation of therapy or to low levels of ongoing replication throughout the course of treatment.
Finally, in the cases of A, B, and L, treatment was associated with both the rapid decay of plasma virus levels and the sustained suppression of plasma viremia to undetectable levels. In these subjects, corrected pairwise genetic distances between sequences from baseline plasma and virus isolated later in therapy were smaller. In these three cases, mean distances from sequences of viral isolates at year 2 were equivalent to or even shorter than mean distances from baseline plasma to the MRCA, findings consistent with recovery of virus from cells infected at the time of or before the baseline plasma sequences. However, the failure to demonstrate sequence evolution for these patients in this particular viral reservoir does not rule out the possibility that very low levels of replication or replication in other compartments and in sanctuary sites not accessible to drug therapy persist.
These findings have several important implications for the understanding of HIV latency and the evaluation of antiviral therapies. First, they confirm that, in subjects who have sustained suppression of plasma viremia documented by the frequent application of the most sensitive assays (such as patients A, B, and L), a population of latently infected cells likely persists into the third year of therapy and that the maintenance of this reservoir of cells appears to be largely independent of discernible viral replication. From the available data, one would surmise that in such patients, the likelihood for emergence of drug resistance should be small. Second, even among patients who have apparent "suppression" of plasma viremia based on less sensitive assays (thresholds between 50 and 200 copies/ml), a subset will not have achieved complete suppression of viral replication. In these cases, the reservoir of replication-competent virus (whether in latently or productively infected cells) may in fact be replenished by continued viral propagation, a conclusion consistent with the demonstration of spliced RNA message and, in one case, changes seen in pol sequences in subjects previously studied (14).
Frequent testing for viremia with the most sensitive assays may permit the identification of such individuals with incomplete suppression of viral replication. The magnitude of risk for development of drug resistance under these circumstances is not known. Consequently, it is also not known whether, in the absence of demonstrable drug resistance, additions to or substitutions in antiviral regimens are warranted. However, these observations suggest that a less rapid initial decline in viral load and the sporadic reappearance of low levels of detectable viremia during therapy may signify incomplete suppression of viral replication and that such assessments may be meaningful in evaluating and comparing the potency of antiviral therapies, as has been proposed by others (44). Finally, these findings indicate that any measurement of the "clearance rate" of the reservoir of latently infected T cells can only be accurately performed on the very best characterized patients, employing, at a minimum, both sensitive and frequent assays of residual RNA in blood to screen for incomplete suppression of viral replication.
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ADDENDUM |
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Following the submission of our manuscript, Zhang et al. (50) reported the demonstration of viral replication in two of eight patients with primary HIV infection treated with antiviral therapy who had achieved undetectable plasma RNA levels. In the same issue, Furtado and colleagues (11b) reported the persistence of viral transcriptional activity in PBMC from treated, chronically infected patients with undetectable viral RNA, suggesting either ongoing replication or a transcriptionally active form of latent infection.
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ACKNOWLEDGMENTS |
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We gratefully acknowledge the technical assistance of Linda Terry and Nancy Keating; the administrative assistance of Sharon Wilcox, Darica Smith, and Mark Biedermann; and the participation of the patients from the San Diego Cohort of M035. We further acknowledge helpful comments and constructive criticism from Steven Wolinsky, John Guatelli, James Mullins, Allen Rodrigo, Gerald Learn, Jon Condra, and Lisa Frenkel and the cooperation of Emilio Emini, Leslie Jonas, and Robin Isaacs.
This work was supported by NIH grants AI 01361 and AI 43752 and a VA Career Development Award (J.K.W.); NIH grants AI 27670, AI 38858, and AI 29164 (D.D.R.); NIH grant AI 36214 (Center for AIDS Research) (D.D.R. and D.J.L.); NIH grant RR 06555 (A.S.P.); Swiss National Science Foundation Grant 84AD-046176 (H.F.G.); a Medical Research Council grant (S.D.W.F.); an unrestricted educational grant from the Merck Research Laboratories; and a grant from the Research Center for AIDS and HIV Infection of the San Diego Veterans Affairs Medical Center.
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FOOTNOTES |
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* Corresponding author. Mailing address: Stein Clinical Research Building No. 326, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0679. Phone: (619) 552-8585, ext. 7193. Fax: (619) 552-7445. E-mail: j2wong{at}ucsd.edu.
Present address: University of Zürich, Zürich, Switzerland.
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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