Multiple Origins of Virus Persistence during Natural Control of HIV Infection.

Targeted HIV cure strategies require definition of the mechanisms that maintain the virus. Here, we tracked HIV replication and the persistence of infected CD4 T cells in individuals with natural virologic control by sequencing viruses, T cell receptor genes, HIV integration sites, and cellular transcriptomes. Our results revealed three mechanisms of HIV persistence operating within distinct anatomic and functional compartments. In lymph node, we detected viruses with genetic and transcriptional attributes of active replication in both T follicular helper (TFH) cells and non-TFH memory cells. In blood, we detected inducible proviruses of archival origin among highly differentiated, clonally expanded cells. Linking the lymph node and blood was a small population of circulating cells harboring inducible proviruses of recent origin. Thus, HIV replication in lymphoid tissue, clonal expansion of infected cells, and recirculation of recently infected cells act together to maintain the virus in HIV controllers despite effective antiviral immunity.

Structural Repertoire of HIV-1-Neutralizing Antibodies Targeting the CD4 Supersite in 14 Donors.

The site on the HIV-1 gp120 glycoprotein that binds the CD4 receptor is recognized by broadly reactive antibodies, several of which neutralize over 90% of HIV-1 strains. To understand how antibodies achieve such neutralization, we isolated CD4-binding-site (CD4bs) antibodies and analyzed 16 co-crystal structures -8 determined here- of CD4bs antibodies from 14 donors. The 16 antibodies segregated by recognition mode and developmental ontogeny into two types: CDR H3-dominated and VH-gene-restricted. Both could achieve greater than 80% neutralization breadth, and both could develop in the same donor. Although paratope chemistries differed, all 16 gp120-CD4bs antibody complexes showed geometric similarity, with antibody-neutralization breadth correlating with antibody-angle of approach relative to the most effective antibody of each type. The repertoire for effective recognition of the CD4 supersite thus comprises antibodies with distinct paratopes arrayed about two optimal geometric orientations, one achieved by CDR H3 ontogenies and the other achieved by VH-gene-restricted ontogenies.

De novo identification of VRC01 class HIV-1-neutralizing antibodies by next-generation sequencing of B-cell transcripts.

Next-generation sequencing of antibody transcripts provides a wealth of data, but the ability to identify function-specific antibodies solely on the basis of sequence has remained elusive. We previously characterized the VRC01 class of antibodies, which target the CD4-binding site on gp120, appear in multiple donors, and broadly neutralize HIV-1. Antibodies of this class have developmental commonalities, but typically share only ∼50% amino acid sequence identity among different donors. Here we apply next-generation sequencing to identify VRC01 class antibodies in a new donor, C38, directly from B cell transcript sequences. We first tested a lineage rank approach, but this was unsuccessful, likely because VRC01 class antibody sequences were not highly prevalent in this donor. We next identified VRC01 class heavy chains through a phylogenetic analysis that included thousands of sequences from C38 and a few known VRC01 class sequences from other donors. This "cross-donor analysis" yielded heavy chains with little sequence homology to previously identified VRC01 class heavy chains. Nonetheless, when reconstituted with the light chain from VRC01, half of the heavy chain chimeric antibodies showed substantial neutralization potency and breadth. We then identified VRC01 class light chains through a five-amino-acid sequence motif necessary for VRC01 light chain recognition. From over a million light chain sequences, we identified 13 candidate VRC01 class members. Pairing of these light chains with the phylogenetically identified C38 heavy chains yielded functional antibodies that effectively neutralized HIV-1. Bioinformatics analysis can thus directly identify functional HIV-1-neutralizing antibodies of the VRC01 class from a sequenced antibody repertoire.

The phenotype of the Cryptococcus-specific CD4+ memory T-cell response is associated with disease severity and outcome in HIV-associated cryptococcal meningitis.

BACKGROUND: Correlates of immune protection in patients with human immunodeficiency virus (HIV)-associated cryptococcal meningitis are poorly defined. A clearer understanding of these immune responses is essential to inform rational development of immunotherapies. METHODS: Cryptococcal-specific peripheral CD4(+) T-cell responses were measured in 44 patients with HIV-associated cryptococcal meningitis at baseline and during follow-up. Responses were assessed following ex vivo cryptococcal mannoprotein stimulation, using 13-color flow-cytometry. The relationships between cryptococcal-specific CD4(+) T-cell responses, clinical parameters at presentation, and outcome were investigated. RESULTS: Cryptococcal-specific CD4(+) T-cell responses were characterized by the production of macrophage inflammatory protein 1α, interferon γ (IFN-γ), and tumor necrosis factor α (TNF-α). Conversely, minimal interleukin 4 and interleukin 17 production was detected. Patients surviving to 2 weeks had significantly different functional CD4(+) T-cell responses as compared to those who died. Patients with a response predominantly consisting of IFN-γ or TNF-α production had a 2-week mortality of 0% (0/20), compared with 25% (6/24) in those without this response (P = .025). Such patients also had lower fungal burdens (10 400 vs 390 000 colony-forming units/mL; P < .001), higher cerebrospinal fluid lymphocyte counts (122 vs 8 cells/µL; P < .001), and a trend toward faster rates of clearance of infection. CONCLUSIONS: The phenotype of the peripheral CD4(+) T-cell response to Cryptococcus was associated with disease severity and outcome in HIV-associated cryptococcal meningitis. IFN-γ/TNF-α-predominant responses were associated with survival.

Effect of antiretroviral therapy on HIV reservoirs in elite controllers.

Elite controllers suppress human immunodeficiency virus (HIV) viremia to below the limit of detection in the absence of antiretroviral therapy (ART). However, precise frequencies of CD4(+) T cells carrying replication-competent HIV and/or the dynamics of the infectious viral reservoirs in response to initiation and discontinuation of ART in elite controllers are unknown. We show that the size of the pool of CD4(+) T cells harboring infectious HIV diminished significantly after initiation of ART and rebounded to baseline upon cessation of therapy. Our data provide compelling evidence that persistent viral replication occurs in untreated elite controllers even in the absence of detectable plasma viremia.

Therapeutic vaccination expands and improves the function of the HIV-specific memory T-cell repertoire.

BACKGROUND: The licensing of herpes zoster vaccine has demonstrated that therapeutic vaccination can help control chronic viral infection. Unfortunately, human trials of immunodeficiency virus (HIV) vaccine have shown only marginal efficacy. METHODS: In this double-blind study, 17 HIV-infected individuals with viral loads of <50 copies/mL and CD4(+) T-cell counts of >350 cells/µL were randomly assigned to the vaccine or placebo arm. Vaccine recipients received 3 intramuscular injections of HIV DNA (4 mg) coding for clade B Gag, Pol, and Nef and clade A, B, and C Env, followed by a replication-deficient adenovirus type 5 boost (10(10) particle units) encoding all DNA vaccine antigens except Nef. Humoral, total T-cell, and CD8(+) cytotoxic T-lymphocyte (CTL) responses were studied before and after vaccination. Single-copy viral loads and frequencies of latently infected CD4(+) T cells were determined. RESULTS: Vaccination was safe and well tolerated. Significantly stronger HIV-specific T-cell responses against Gag, Pol, and Env, with increased polyfunctionality and a broadened epitope-specific CTL repertoire, were observed after vaccination. No changes in single-copy viral load or the frequency of latent infection were observed. CONCLUSIONS: Vaccination of individuals with existing HIV-specific immunity improved the magnitude, breadth, and polyfunctionality of HIV-specific memory T-cell responses but did not impact markers of viral control. CLINICAL TRIALS REGISTRATION: NCT00270465.

Differential Gag-specific polyfunctional T cell maturation patterns in HIV-1 elite controllers.

A small fraction of HIV-infected individuals (<1%), referred to as elite controllers (EC), are able to maintain undetectable viral loads indefinitely without treatment. The role of the maturational phenotype of T cells in the control of HIV infection in these individuals is not well described. We compared the maturational and functional phenotypes of Gag-specific CD4 and CD8 T cells from EC, who maintain undetectable viral loads without treatment; relative controllers (RC), who maintain viral loads of <1,000 copies/ml without treatment; and noncontrollers (NC), who fail to control viral replication. EC maintained higher frequencies of HIV-specific CD4 T cells, less mature polyfunctional Gag-specific CD4 T cells (CD27(+) CD57(-) CD45RO(+)), and Gag-specific polyfunctional CD4 T cells than those observed in NC. In EC, the frequency of polyfunctional Gag-specific CD8 T cells was higher than that observed in RC and NC. RC had a similar functional phenotype to that observed in NC, despite consistently lower viral loads. Finally, we found a direct correlation between the frequency of Gag-specific CD27(+) CD57(-) CD45RO(+) CD4(+) T cells and the frequency of mature HIV-specific CD8 T cells. Altogether, our data suggest that immature Gag-specific interleukin-2 (IL-2)-producing CD4(+) T cells may play an important role in spontaneous control of HIV viremia by effectively supporting HIV-specific CD8 T lymphocytes. This difference appears to differentiate EC from RC.

Surface expression patterns of negative regulatory molecules identify determinants of virus-specific CD8+ T-cell exhaustion in HIV infection.

A highly complex network of coinhibitory and costimulatory receptors regulates the outcome of virus-specific CD8(+) T-cell responses. Here, we report on the expression patterns of multiple inhibitory receptors on HIV-specific, cytomegalovirus-specific, and bulk CD8(+) T-cell memory populations. In contrast to cytomegalovirus-specific CD8(+) T cells, the majority of HIV-specific CD8(+) T cells exhibited an immature phenotype and expressed Programmed Death-1, CD160 and 2B4 but not lymphocyte activation gene-3. Notably, before antiretroviral therapy, simultaneous expression of these negative regulators correlated strongly with both HIV load and impaired cytokine production. Suppression of HIV replication by antiretroviral therapy was associated with reduced surface expression of inhibitory molecules on HIV-specific CD8(+) T cells. Furthermore, in vitro manipulation of Programmed Death-1 and 2B4 inhibitory pathways increased the proliferative capacity of HIV-specific CD8(+) T cells. Thus, multiple coinhibitory receptors can affect the development of HIV-specific CD8(+) T-cell responses and, by extension, represent potential targets for new immune-based interventions in HIV-infected persons.

Trispecific antibody targeting HIV-1 and T cells activates and eliminates latently-infected cells in HIV/SHIV infections.

Agents that can simultaneously activate latent HIV, increase immune activation and enhance the killing of latently-infected cells represent promising approaches for HIV cure. Here, we develop and evaluate a trispecific antibody (Ab), N6/αCD3-αCD28, that targets three independent proteins: (1) the HIV envelope via the broadly reactive CD4-binding site Ab, N6; (2) the T cell antigen CD3; and (3) the co-stimulatory molecule CD28. We find that the trispecific significantly increases antigen-specific T-cell activation and cytokine release in both CD4+ and CD8+ T cells. Co-culturing CD4+ with autologous CD8+ T cells from ART-suppressed HIV+ donors with N6/αCD3-αCD28, results in activation of latently-infected cells and their elimination by activated CD8+ T cells. This trispecific antibody mediates CD4+ and CD8+ T-cell activation in non-human primates and is well tolerated in vivo. This HIV-directed antibody therefore merits further development as a potential intervention for the eradication of latent HIV infection.

Acquisition of direct antiviral effector functions by CMV-specific CD4+ T lymphocytes with cellular maturation.

The role of CD4+ T cells in the control of persistent viral infections beyond the provision of cognate help remains unclear. We used polychromatic flow cytometry to evaluate the production of the cytokines interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, and interleukin (IL)-2, the chemokine macrophage inflammatory protein (MIP)-1beta, and surface mobilization of the degranulation marker CD107a by CD4+ T cells in response to stimulation with cytomegalovirus (CMV)-specific major histocompatibility complex class II peptide epitopes. Surface expression of CD45RO, CD27, and CD57 on responding cells was used to classify CD4+ T cell maturation. The functional profile of virus-specific CD4+ T cells in chronic CMV infection was unique compared with that observed in other viral infections. Salient features of this profile were: (a) the simultaneous production of MIP-1beta, TNF-alpha, and IFN-gamma in the absence of IL-2; and (b) direct cytolytic activity associated with surface mobilization of CD107a and intracellular expression of perforin and granzymes. This polyfunctional profile was associated with a terminally differentiated phenotype that was not characterized by a distinct clonotypic composition. Thus, mature CMV-specific CD4+ T cells exhibit distinct functional properties reminiscent of antiviral CD8+ T lymphocytes.

PD-1 is a regulator of virus-specific CD8+ T cell survival in HIV infection.

Here, we report on the expression of programmed death (PD)-1 on human virus-specific CD8(+) T cells and the effect of manipulating signaling through PD-1 on the survival, proliferation, and cytokine function of these cells. PD-1 expression was found to be low on naive CD8(+) T cells and increased on memory CD8(+) T cells according to antigen specificity. Memory CD8(+) T cells specific for poorly controlled chronic persistent virus (HIV) more frequently expressed PD-1 than memory CD8(+) T cells specific for well-controlled persistent virus (cytomegalovirus) or acute (vaccinia) viruses. PD-1 expression was independent of maturational markers on memory CD8(+) T cells and was not directly associated with an inability to produce cytokines. Importantly, the level of PD-1 surface expression was the primary determinant of apoptosis sensitivity of virus-specific CD8(+) T cells. Manipulation of PD-1 led to changes in the ability of the cells to survive and expand, which, over several days, affected the number of cells expressing cytokines. Therefore, PD-1 is a major regulator of apoptosis that can impact the frequency of antiviral T cells in chronic infections such as HIV, and could be manipulated to improve HIV-specific CD8(+) T cell numbers, but possibly not all functions in vivo.

A steady state of CD4+ T cell memory maturation and activation is established during primary subtype C HIV-1 infection.

The functional integrity of CD4(+) T cells is crucial for well-orchestrated immunity and control of HIV-1 infection, but their selective depletion during infection creates a paradox for understanding a protective response. We used multiparameter flow cytometry to measure activation, memory maturation, and multiple functions of total and Ag-specific CD4(+) T cells in 14 HIV-1- and CMV- coinfected individuals at 3 and 12 mo post HIV-1 infection. Primary HIV-1 infection was characterized by elevated levels of CD38, HLA-DR, and Ki67 in total memory and Gag-specific CD4(+) and CD8(+) T cells. In both HIV-infected and 15 uninfected controls, the frequency of activated cells was uniformly distributed among early differentiated (ED; CD45RO(+)CD27(+)), late differentiated (CD45RO(+)CD27(-)), and fully differentiated effector (CD45RO(-)CD27(-)) memory CD4(+) T cells. In HIV-1-infected individuals, activated CD4(+) T cells significantly correlated with viremia at 3 mo postinfection (r = 0.79, p = 0.0007) and also harbored more gag provirus DNA copies than nonactivated cells (p = 0.04). Moreover, Gag-specific ED CD4(+) T cells inversely associated with plasma viral load (r = -0.87, p < 0.0001). Overall, we show that low copy numbers of gag provirus and plasma RNA copies associated with low CD4 activation as well as accumulation of ED HIV-specific CD4(+) memory. Significant positive correlations between 3 and 12 mo activation and memory events highlighted that a steady state of CD4(+) T cell activation and memory maturation was established during primary infection and that these cells were unlikely to be involved in influencing the course of viremia in the first 12 mo of HIV-1 infection.

Application of B cell immortalization for the isolation of antibodies and B cell clones from vaccine and infection settings.

The isolation and characterization of neutralizing antibodies from infection and vaccine settings informs future vaccine design, and methodologies that streamline the isolation of antibodies and the generation of B cell clones are of great interest. Retroviral transduction to express Bcl-6 and Bcl-xL and transform primary B cells has been shown to promote long-term B cell survival and antibody secretion in vitro, and can be used to isolate antibodies from memory B cells. However, application of this methodology to B cell subsets from different tissues and B cells from chronically infected individuals has not been well characterized. Here, we characterize Bcl-6/Bcl-xL B cell immortalization across multiple tissue types and B cell subsets in healthy and HIV-1 infected individuals, as well as individuals recovering from malaria. In healthy individuals, naïve and memory B cell subsets from PBMCs and tonsil tissue transformed with similar efficiencies, and displayed similar characteristics with respect to their longevity and immunoglobulin secretion. In HIV-1-viremic individuals or in individuals with recent malaria infections, the exhausted CD27-CD21- memory B cells transformed with lower efficiency, but the transformed B cells expanded and secreted IgG with similar efficiency. Importantly, we show that this methodology can be used to isolate broadly neutralizing antibodies from HIV-infected individuals. Overall, we demonstrate that Bcl-6/Bcl-xL B cell immortalization can be used to isolate antibodies and generate B cell clones from different B cell populations, albeit with varying efficiencies.

Potent anti-viral activity of a trispecific HIV neutralizing antibody in SHIV-infected monkeys.

Broadly neutralizing antibodies (bNAbs) represent an alternative to drug therapy for the treatment of HIV-1 infection. Immunotherapy with single bNAbs often leads to emergence of escape variants, suggesting a potential benefit of combination bNAb therapy. Here, a trispecific bNAb reduces viremia 100- to 1000-fold in viremic SHIV-infected macaques. After treatment discontinuation, viremia rebounds transiently and returns to low levels, through CD8-mediated immune control. These viruses remain sensitive to the trispecific antibody, despite loss of sensitivity to one of the parental bNAbs. Similarly, the trispecific bNAb suppresses the emergence of resistance in viruses derived from HIV-1-infected subjects, in contrast to parental bNAbs. Trispecific HIV-1 neutralizing antibodies, therefore, mediate potent antiviral activity in vivo and may minimize the potential for immune escape.

Safety and tolerability of AAV8 delivery of a broadly neutralizing antibody in adults living with HIV: a phase 1, dose-escalation trial.

Adeno-associated viral vector-mediated transfer of DNA coding for broadly neutralizing anti-HIV antibodies (bnAbs) offers an alternative to attempting to induce protection by vaccination or by repeated infusions of bnAbs. In this study, we administered a recombinant bicistronic adeno-associated virus (AAV8) vector coding for both the light and heavy chains of the potent broadly neutralizing HIV-1 antibody VRC07 (AAV8-VRC07) to eight adults living with HIV. All participants remained on effective anti-retroviral therapy (viral load (VL) <50 copies per milliliter) throughout this phase 1, dose-escalation clinical trial ( NCT03374202 ). AAV8-VRC07 was given at doses of 5 × 1010, 5 × 1011 and 2.5 × 1012 vector genomes per kilogram by intramuscular (IM) injection. Primary endpoints of this study were to assess the safety and tolerability of AAV8-VRC07; to determine the pharmacokinetics and immunogenicity of in vivo VRC07 production; and to describe the immune response directed against AAV8-VRC07 vector and its products. Secondary endpoints were to assess the clinical effects of AAV8-VRC07 on CD4 T cell count and VL and to assess the persistence of VRC07 produced in participants. In this cohort, IM injection of AAV8-VRC07 was safe and well tolerated. No clinically significant change in CD4 T cell count or VL occurred during the 1-3 years of follow-up reported here. In participants who received AAV8-VRC07, concentrations of VRC07 were increased 6 weeks (P = 0.008) and 52 weeks (P = 0.016) after IM injection of the product. All eight individuals produced measurable amounts of serum VRC07, with maximal VRC07 concentrations >1 µg ml-1 in three individuals. In four individuals, VRC07 serum concentrations remained stable near maximal concentration for up to 3 years of follow-up. In exploratory analyses, neutralizing activity of in vivo produced VRC07 was similar to that of in vitro produced VRC07. Three of eight participants showed a non-idiotypic anti-drug antibody (ADA) response directed against the Fab portion of VRC07. This ADA response appeared to decrease the production of serum VRC07 in two of these three participants. These data represent a proof of concept that adeno-associated viral vectors can durably produce biologically active, difficult-to-induce bnAbs in vivo, which could add valuable new tools to the fight against infectious diseases.

Autocrine production of beta-chemokines protects CMV-Specific CD4 T cells from HIV infection.

Induction of a functional subset of HIV-specific CD4+ T cells that is resistant to HIV infection could enhance immune protection and decrease the rate of HIV disease progression. CMV-specific CD4+ T cells, which are less frequently infected than HIV-specific CD4+ T cells, are a model for such an effect. To determine the mechanism of this protection, we compared the functional response of HIV gag-specific and CMV pp65-specific CD4+ T cells in individuals co-infected with CMV and HIV. We found that CMV-specific CD4+ T cells rapidly up-regulated production of MIP-1alpha and MIP-1beta mRNA, resulting in a rapid increase in production of MIP-1alpha and MIP-1beta after cognate antigen stimulation. Production of beta-chemokines was associated with maturational phenotype and was rarely seen in HIV-specific CD4+ T cells. To test whether production of beta-chemokines by CD4+ T cells lowers their susceptibility to HIV infection, we measured cell-associated Gag DNA to assess the in vivo infection history of CMV-specific CD4+ T cells. We found that CMV-specific CD4+ T cells which produced MIP-1beta contained 10 times less Gag DNA than did those which failed to produce MIP-1beta. These data suggest that CD4+ T cells which produce MIP-1alpha and MIP-1beta bind these chemokines in an autocrine fashion which decreases the risk of in vivo HIV infection.

Differential association of programmed death-1 and CD57 with ex vivo survival of CD8+ T cells in HIV infection.

Recent studies have revealed the critical role of programmed death-1 (PD-1) in exhaustion of HIV- and SIV-specific CD8(+) T cells. In this study, we show that high expression of PD-1 correlates with increased ex vivo spontaneous and CD95/Fas-induced apoptosis, particularly in the "effector-memory" CD8(+) T cell population from HIV(+) donors. High expression of PD-1 was linked to a proapoptotic phenotype characterized by low expression of Bcl-2 and IL7-R alpha, high expression of CD95/Fas and high mitochondrial mass. Expression of PD-1 and CD57 was differentially associated with the maturation status of CD8(+) T cells in HIV infection. CD57 was linked to higher apoptosis resistance, with cells expressing a PD-1(L)CD57(H) phenotype exhibiting lower levels of cell death. The majority of HIV-specific CD8(+) T cells were found to express a PD-1(H)CD57(L) or PD-1(H)CD57(H) phenotype. No correlation was found between PD-1 expression and ex vivo polyfunctionality of either HIV- or CMV-specific CD8(+) T cells. Contrary to CD57, high expression of PD-1 was characterized by translocation of PD-1 into the area of CD95/Fas-capping, an early necessary step of CD95/Fas-induced apoptosis. Thus, our data further support the role of PD-1 as a preapoptotic factor for CD8(+) T cells in HIV infection.

Principles Governing Establishment versus Collapse of HIV-1 Cellular Spread.

A population at low census might go extinct or instead transition into exponential growth to become firmly established. Whether this pivotal event occurs for a within-host pathogen can be the difference between health and illness. Here, we define the principles governing whether HIV-1 spread among cells fails or becomes established by coupling stochastic modeling with laboratory experiments. Following ex vivo activation of latently infected CD4 T cells without de novo infection, stochastic cell division and death contributes to high variability in the magnitude of initial virus release. Transition to exponential HIV-1 spread often fails due to release of an insufficient amount of replication-competent virus. Establishment of exponential growth occurs when virus produced from multiple infected cells exceeds a critical population size. We quantitatively define the crucial transition to exponential viral spread. Thwarting this process would prevent HIV transmission or rebound from the latent reservoir.

Optimizing peptide matrices for identifying T-cell antigens.

Mapping T-cell epitopes for a pathogen or vaccine requires a complex method for screening hundreds to thousands of peptides with a limited amount of donor sample. We describe an optimized deconvolution process by which peptides are pooled in a matrix format to minimize the number of tests required to identify peptide epitopes. Four peptide pool matrices were constructed to deconvolute the HIV-specific T-cell response in three HIV-infected individuals. ELISpot assays were used to map peptide antigens. Many HIV peptides were mapped in all three individuals. However, there were several challenges and limitations associated with the deconvolution process. Peptides that induced low-frequency responses or were masked by peptide competition within a given pool were not identified, because they did not meet the threshold criteria for a positive response. Also, amino acid sequence variation limited the ability of this method to map autologous HIV peptides. Alternative analysis strategies and revisions to the original matrix optimizations are presented that address ways to increase peptide identification. This optimized deconvolution method allows for efficient mapping of T-cell peptide epitopes. It is rapid, powerful, efficient, and unrestricted by HLA type.

Slow human immunodeficiency virus type 1 evolution in viral reservoirs in infants treated with effective antiretroviral therapy.

A longitudinal study of viral reservoirs in children initiating highly active antiretroviral therapy (HAART) in early infancy was undertaken to test the hypothesis that early effective treatment affects the persistence of replication-competent viral latency and the evolution of HIV-1 in resting CD4(+) T cells. An end point dilution culture assay was used to measure the frequencies of latently-infected resting CD4(+) T cells harboring replication-competent virus in early and late treated children. Gag, pol, and env also were sequenced and compared to pretreatment sequences. HIV-1-specific humoral and cellular immune responses were also assessed. Blood samples were obtained from 12 HIV-1-infected children who started HAART at a median of 1.9 months of age and who maintained suppression of HIV-1 replication for up to 5.5 years. Replication-competent HIV-1 was recovered from 10/12 (84%) subjects. Evolution in gag, pol, and env was restricted for years in early-treated children. HAART initiated from early infancy does not prevent the establishment of a reservoir of latent provirus, but does significantly limit the evolution of HIV-1 in viral reservoirs. The effect of early therapy on HIV-1 evolution may have implications for long-term pharmacologic control of HIV-1.