Mechanisms of Abrupt Loss of Virus Control in a Cohort of Previous HIV Controllers.

Elite and viremic HIV controllers are able to control their HIV infection and maintain undetectable or low-level viremia in the absence of antiretroviral treatment. Despite extensive studies, the immune factors responsible for such exclusive control remain poorly defined. We identified a cohort of 14 HIV controllers that suffered an abrupt loss of HIV control (LoC) to investigate possible mechanisms and virological and immunological events related to the sudden loss of control. The in-depth analysis of these subjects involved the study of cell tropism of circulating virus, evidence for HIV superinfection, cellular immune responses to HIV, as well as an examination of viral adaptation to host immunity by Gag sequencing. Our data demonstrate that a poor capacity of T cells to mediate in vitro viral suppression, even in the context of protective HLA alleles, predicts a loss of viral control. In addition, the data suggest that inefficient viral control may be explained by an increase of CD8 T-cell activation and exhaustion before LoC. Furthermore, we detected a switch from C5- to X4-tropic viruses in 4 individuals after loss of control, suggesting that tropism shift might also contribute to disease progression in HIV controllers. The significantly reduced inhibition of in vitro viral replication and increased expression of activation and exhaustion markers preceding the abrupt loss of viral control may help identify untreated HIV controllers that are at risk of losing control and may offer a useful tool for monitoring individuals during treatment interruption phases in therapeutic vaccine trials.IMPORTANCE A few individuals can control HIV infection without the need for antiretroviral treatment and are referred to as HIV controllers. We have studied HIV controllers who suddenly lose this ability and present with high in vivo viral replication and decays in their CD4+ T-cell counts to identify potential immune and virological factors that were responsible for initial virus control. We identify in vitro-determined reductions in the ability of CD8 T cells to suppress viral control and the presence of PD-1-expressing CD8+ T cells with a naive immune phenotype as potential predictors of in vivo loss of virus control. The findings could be important for the clinical management of HIV controller individuals, and it may offer an important tool to anticipate viral rebound in individuals in clinical studies that include combination antiretroviral therapy (cART) treatment interruptions and which, if not treated quickly, could pose a significant risk to the trial participants.

RECall for automated genotypic tropism testing.

Standardization of sequence chromatogram analysis is required for consistent genotypic tropism determination across laboratories. A freely available, fast, and automated chromatogram analysis tool (RECall) provided tropism interpretations equivalent to those of manual sequence editing of 521 V3 loop HIV-1 population sequences, suggesting that RECall can be useful in standardizing genotypic tropism testing across laboratories.

Switching the third drug of antiretroviral therapy to maraviroc in aviraemic subjects: a pilot, prospective, randomized clinical trial.

OBJECTIVES: To evaluate the safety and efficacy of switching the third drug of antiretroviral treatment to maraviroc in aviraemic subjects infected with R5 HIV. PATIENTS AND METHODS: This is a pilot, prospective, randomized clinical trial (ClinicalTrials ID: NCT00966329). Eighty HIV-1-infected aviraemic adults on stable antiretroviral treatment for ≥1 year and no antiretroviral drug resistance were screened for the presence of non-R5 HIV by triplicate proviral V3 population sequencing. From them, 30 subjects with R5 HIV-1 were randomized 1 : 1 to switch the non-nucleoside reverse transcriptase inhibitor or ritonavir-boosted protease inhibitor to maraviroc (n = 15) or to continue the same antiretroviral treatment (controls, n = 15). The principal endpoint was the proportion of subjects with HIV-1 RNA <50 copies/mL at week 48. Ultrasensitive proviral HIV-1 tropism testing (454 sequencing) was performed retrospectively at weeks 0, 4, 12, 24, 36 and 48. RESULTS: One subject in the maraviroc arm and one control had non-R5 HIV in proviral DNA by retrospective 454 sequencing. The subject receiving maraviroc was the only individual to develop virological failure. However, plasma HIV at failure was R5. Switching to maraviroc was well tolerated and associated with small, but statistically significant, declines in total, high-density lipoprotein and low-density lipoprotein cholesterol. Median (IQR) triglyceride [1 (0.67-1.22) versus 1.6 (1.4-3.1) mmol/L, P = 0.003] and total cholesterol [4.3 (4.1-4.72) versus 5.4 (4-5.7) mmol/L, P = 0.059] values were lower in the maraviroc arm than in controls at week 48. CONCLUSIONS: In this pilot, prospective, randomized clinical trial, switching the third drug to maraviroc was safe, efficacious and improved lipid parameters.

A376S in the connection subdomain of HIV-1 reverse transcriptase confers increased risk of virological failure to nevirapine therapy.

BACKGROUND: The clinical relevance of mutations in the connection subdomain and the ribonuclease (RNase) H domain of HIV-1 reverse transcriptase (RT) is uncertain. METHODS: The risk of virological failure to nonnucleoside RT inhibitor (NNRTI)-based antiretroviral therapy (ART) was evaluated in NNRTI-naive patients who started NNRTIs in the EuroSIDA study after July 1997 according to preexisting substitutions in the connection subdomain and the RNase H domain of HIV-1 RT. An observed association between A376S and virological failure was further investigated by testing in vitro NNRTI susceptibility of single site-directed mutants and patient-derived recombinant viruses. Enzymatic assays also determined the effects of A376S on nevirapine and template-primer binding to HIV-1 RT. RESULTS: Virological failure occurred in 142 of 287 (49%) individuals: 77 receiving nevirapine (67%) and 65 receiving efavirenz (38%) (P < .001). Preexisting A376S was associated with an increased risk of virological failure to nevirapine (relative hazard [RH] = 10.4; 95% confidence interval [CI], 2.0-54.7), but it did not affect efavirenz outcome the same way (RH = 0.5; 95% CI, 0.1-2.2) (P value for interaction = .013). A376S conferred selective low-level nevirapine resistance in vitro, and led to greater affinity for double-stranded DNA. CONCLUSIONS: The A376S substitution in the connection subdomain of HIV-1 RT causes selective nevirapine resistance and confers an increased risk of virological failure to nevirapine-based ART.

Secretion of interferon-gamma by human macrophages demonstrated at the single-cell level after costimulation with interleukin (IL)-12 plus IL-18.

The interferon (IFN)-gamma component of the immune response plays an essential role in combating infectious and non-infectious diseases. Induction of IFN-gamma secretion by human T and natural killer (NK) cells through synergistic costimulation with interleukin (IL)-12 and IL-18 in the adaptive immune responses against pathogens is well established, but induction of similar activity in macrophages is still controversial, with doubts largely focusing on contamination of macrophages with NK or T cells in the relevant experiments. The possible contribution of macrophages to the IFN response is, however, an important factor relevant to the pathogenesis of many diseases. To resolve this issue, we analysed the production of IFN-gamma at the single-cell level by immunohistochemistry and by enzyme-linked immunosorbent spot (ELISPOT) analysis and unequivocally demonstrated that human macrophages derived from monocytes in vitro through stimulation with a combination of IL-12 and IL-18 or with macrophage colony-stimulating factor (M-CSF) were able to produce IFN-gamma when further stimulated with a combination of IL-12 and IL-18. In addition, naturally activated alveolar macrophages immediately secreted IFN-gamma upon treatment with IL-12 and IL-18. Therefore, human macrophages in addition to lymphoid cells contribute to the IFN-gamma response, providing another link between the innate and acquired immune responses.

Variability in the plasma concentration of efavirenz and nevirapine is associated with genotypic resistance after treatment interruption.

BACKGROUND: Selection and persistence of non-nucleoside reverse transcriptase inhibitor (NNRTI)-associated mutations during treatment interruptions (TIs) has been attributed to the long plasma half-life of these drugs. However, little is known about the contribution of variable NNRTI plasma levels before a TI. We evaluated the selection of NNRTI-related mutations and the coefficient of variation of NNRTI plasma concentrations during different TI periods. METHODS: The selection of NNRTI-related mutations was examined in 50 HIV type-1 (HIV-1)-infected patients on a virologically suppressive regimen who underwent TI guided by CD4+ T-cell counts and plasma viraemia. Population and clone-based sequencing of the reverse transcriptase coding region was performed using plasma HIV-1 RNA samples during TI and proviral DNA from peripheral blood mononuclear cells before TI. NNRTI plasma concentrations were determined by HPLC. RESULTS: In 7/50 treated patients, de novo and transient NNRTI-related mutations appeared when treatment was interrupted. Emergence of resistant variants (including K103N, Y181C or G190S) after interruption was associated with a higher coefficient of variation in NNRTI plasma concentrations during the treatment period. Moreover, minority HIV-1 variants containing different resistance patterns (V1061/A, K103R/E or Y188C/D/H) were detected regardless of NNRTI concentrations. CONCLUSIONS: The emergence of NNRTI-associated mutations during TI appears to be associated with the variation of NNRTI plasma concentrations during the preceding treatment period. The selection of minority HIV-1 variants with different patterns of NNRTI resistance in the absence of drug pressure should be considered for the efficacy of future NNRTI-containing antiretroviral regimens.

Partial immunological and mitochondrial recovery after reducing didanosine doses in patients on didanosine and tenofovir-based regimens.

BACKGROUND: Tenofovir disoproxil fumarate (TDF) has a safe toxicity profile; however, administration together with didanosine (ddl) increases ddl levels causing mitochondrial damage and CD4+ T-cell decline. We assessed whether a simple reduction of the ddl dose in patients receiving ddl (400 mg/day) and TDF could revert this side effect. METHODS: Immunological and mitochondrial changes were analysed in 20 patients at baseline, after 14 months of receiving ddl (400 mg/day), TDF (300 mg/day) and nevirapine (NVP; 400 mg/day) and 14 months after a ddl dose reduction to 250 mg/day. Immunological analyses measured CD4+ and CD8+ T-cell counts and mitochondrial studies in peripheral blood mononuclear cells assessed mitochondrial DNA content by quantitative real-time PCR, cytochrome c oxidase (COX) activity by spectrophotometry and mitochondrial protein synthesis (COX-II versus beta-actin or COX-IV expression) by western blot. RESULTS: Treatment with TDF, ddl (400 mg/day) and NVP for 14 months produced significant decreases in mitochondrial parameters and CD4+ T-cell counts. The reduction in ddl dose resulted in mitochondrial DNA recovery; however, the remaining mitochondrial parameters remained significantly decreased. Levels of CD4+ T-cells were partially restored in 35% of patients. Subjects presenting a significant reduction in CD4+ T-cells during the high ddl dose period showed greater mitochondrial impairment in this stage and better mitochondrial and immunological recovery after drug reduction. CONCLUSIONS: Administration of high ddl doses together with TDF was associated with mitochondrial damage, which may explain the observed CD4+ T-cell decay. A reduction of the ddl dose led to mitochondrial DNA recovery, but was not sufficient to recover baseline CD4+ T-cell counts. Other mitochondrial toxicity in addition to DNA gamma-polymerase inhibition could be responsible for CD4+ T-cell toxicity.

HIV drug resistance patterns in pregnant women using next generation sequence in Mozambique.

BACKGROUND: Few data on HIV resistance in pregnancy are available from Mozambique, one of the countries with the highest HIV toll worldwide. Understanding the patterns of HIV drug resistance in pregnant women might help in tailoring optimal regimens for prevention of mother to child transmission of HIV (pMTCT) and antenatal care. OBJECTIVES: To describe the frequency and characteristics of HIV drug resistance mutations (HIVDRM) in pregnant women with virological failure at delivery, despite pMTCT or antiretroviral therapy (ART). METHODS: Samples from HIV-infected pregnant women from a rural area in southern Mozambique were analysed. Only women with HIV-1 RNA >400c/mL at delivery were included in the analysis. HIVDRM were determined using MiSeq® (detection threshold 1%) at the first antenatal care (ANC) visit and at the time of delivery. RESULTS: Ninety and 60 samples were available at the first ANC visit and delivery, respectively. At first ANC, 97% of the women had HIV-1 RNA>400c/mL, 39% had CD4+ counts <350 c/mm3 and 30% were previously not on ART. Thirteen women (14%) had at least one HIVDRM of whom 70% were not on previous ART. Eight women (13%) had at least one HIVDRM at delivery. Out of 37 women with data available from the two time points, 8 (21%) developed at least one new HIVDRM during pMTCT or ART. Twenty seven per cent (53/191), 32% (44/138) and 100% (5/5) of the mutations that were present at enrolment, delivery and that emerged during pregnancy, respectively, were minority mutations (frequency <20%). CONCLUSIONS: Even with ultrasensitive HIV-1 genotyping, less than 20% of women with detectable viremia at delivery had HIVDRM before initiating pMTCT or ART. This suggests that factors other than pre-existing resistance, such as lack of adherence or interruptions of the ANC chain, are also relevant to explain lack of virological suppression at the time of delivery in women receiving antiretrovirals drugs during pregnancy.

Virological and immunological outcome of treatment interruption in HIV-1-infected subjects vaccinated with MVA-B.

The most relevant endpoint in therapeutic HIV vaccination is the assessment of time to viral rebound or duration of sustained control of low-level viremia upon cART treatment cessation. Structured treatment interruptions (STI) are however not without risk to the patient and reliable predictors of viral rebound/control after therapeutic HIV-1 vaccination are urgently needed to ensure patient safety and guide therapeutic vaccine development. Here, we integrated immunological and virological parameters together with viral rebound dynamics after STI in a phase I therapeutic vaccine trial of a polyvalent MVA-B vaccine candidate to define predictors of viral control. Clinical parameters, proviral DNA, host HLA genetics and measures of humoral and cellular immunity were evaluated. A sieve effect analysis was conducted comparing pre-treatment viral sequences to breakthrough viruses after STI. Our results show that a reduced proviral HIV-1 DNA at study entry was independently associated with two virological parameters, delayed HIV-1 RNA rebound (p = 0.029) and lower peak viremia after treatment cessation (p = 0.019). Reduced peak viremia was also positively correlated with a decreased number of HLA class I allele associated polymorphisms in Gag sequences in the rebounding virus population (p = 0.012). Our findings suggest that proviral DNA levels and the number of HLA-associated Gag polymorphisms may have an impact on the clinical outcome of STI. Incorporation of these parameters in future therapeutic vaccine trials may guide refined immunogen design and help conduct safer STI approaches.

Naive CD4(+) T cells and recent thymic emigrant levels in treated individuals with HIV: clinical relevance.

An increase in the levels of naive T cells after the administration of HAART is an indicator of the quality of immune reconstitution. We investigated whether levels of naive CD4 T cells (CD4(+)/CD45RA(+)) and of recent thymic emigrants (RTEs; CD4(+)/CD45RA(+)/CD31(+)) achieved in chronically treated HIV-infected patients could predict the length of time patients could interrupt antiretroviral treatment before their CD4 counts reached values < or =350 cells/mm(3) or HIV-1 RNA levels increased to > or =100,000 copies/ml (Tibet cohort). Serial measurements revealed that the level of naive CD4 T cells among patients was extremely variable (5-95%), but the values for each patient remained stable throughout the study. We then focused on those patients who showed percentages of naive CD4 T cells above 60% or below 30%. The levels of naive T cells, RTEs, mononuclear cells containing T cell receptor excision circles (TRECs), and the strength of CD4 helper responses to HIV p24 antigen during treatment failed to predict the duration of treatment interruptions. In contrast, the median survival time of patients with a CD4 nadir > or =350 cells/mm(3) was 2-fold higher than that of patients with a CD4 nadir <350. However, the probability of restarting therapy in these two groups of patients was independent of the levels of naive T cells, RTEs, or TRECs.

Gut Microbiota Linked to Sexual Preference and HIV Infection.

The precise effects of HIV-1 on the gut microbiome are unclear. Initial cross-sectional studies provided contradictory associations between microbial richness and HIV serostatus and suggested shifts from Bacteroides to Prevotella predominance following HIV-1 infection, which have not been found in animal models or in studies matched for HIV-1 transmission groups. In two independent cohorts of HIV-1-infected subjects and HIV-1-negative controls in Barcelona (n = 156) and Stockholm (n = 84), men who have sex with men (MSM) predominantly belonged to the Prevotella-rich enterotype whereas most non-MSM subjects were enriched in Bacteroides, independently of HIV-1 status, and with only a limited contribution of diet effects. Moreover, MSM had a significantly richer and more diverse fecal microbiota than non-MSM individuals. After stratifying for sexual orientation, there was no solid evidence of an HIV-specific dysbiosis. However, HIV-1 infection remained consistently associated with reduced bacterial richness, the lowest bacterial richness being observed in subjects with a virological-immune discordant response to antiretroviral therapy. Our findings indicate that HIV gut microbiome studies must control for HIV risk factors and suggest interventions on gut bacterial richness as possible novel avenues to improve HIV-1-associated immune dysfunction.

HIV-1 tropism testing in subjects achieving undetectable HIV-1 RNA: diagnostic accuracy, viral evolution and compartmentalization.

BACKGROUND: Technically, HIV-1 tropism can be evaluated in plasma or peripheral blood mononuclear cells (PBMCs). However, only tropism testing of plasma HIV-1 has been validated as a tool to predict virological response to CCR5 antagonists in clinical trials. The preferable tropism testing strategy in subjects with undetectable HIV-1 viremia, in whom plasma tropism testing is not feasible, remains uncertain. METHODS & RESULTS: We designed a proof-of-concept study including 30 chronically HIV-1-infected individuals who achieved HIV-1 RNA <50 copies/mL during at least 2 years after first-line ART initiation. First, we determined the diagnostic accuracy of 454 and population sequencing of gp120 V3-loops in plasma and PBMCs, as well as of MT-2 assays before ART initiation. The Enhanced Sensitivity Trofile Assay (ESTA) was used as the technical reference standard. 454 sequencing of plasma viruses provided the highest agreement with ESTA. The accuracy of 454 sequencing decreased in PBMCs due to reduced specificity. Population sequencing in plasma and PBMCs was slightly less accurate than plasma 454 sequencing, being less sensitive but more specific. MT-2 assays had low sensitivity but 100% specificity. Then, we used optimized 454 sequence data to investigate viral evolution in PBMCs during viremia suppression and only found evolution of R5 viruses in one subject. No de novo CXCR4-using HIV-1 production was observed over time. Finally, Slatkin-Maddison tests suggested that plasma and cell-associated V3 forms were sometimes compartmentalized. CONCLUSIONS: The absence of tropism shifts during viremia suppression suggests that, when available, testing of stored plasma samples is generally safe and informative, provided that HIV-1 suppression is maintained. Tropism testing in PBMCs may not necessarily produce equivalent biological results to plasma, because the structure of viral populations and the diagnostic performance of tropism assays may sometimes vary between compartments. Thereby, proviral DNA tropism testing should be specifically validated in clinical trials before it can be applied to routine clinical decision-making.

Impact of CD4 T cell count on the outcome of planned treatment interruptions in early-treated human immunodeficiency virus-infected children.

Early highly active antiretroviral therapy is recommended in all vertically human immunodeficiency virus (HIV)-infected infants. We describe the long-term immunologic outcome after planned treatment interruption (PTI) in 7 children diagnosed and treated during acute HIV infection (age <12 weeks). Children had remained a median of 57 months off treatment, 3 of them indefinitely. The 2 patients with the lowest nadir CD4% reinitiated highly active antiretroviral therapy because of a CD4 cell decline of <20%; 2 children resumed treatment because of clinical progression and parents' wishes. All patients experienced a decrease in CD4% after PTI, which particularly affected the naive subpopulation. The interferon-γ response against HIV-p24 antigen directly correlated with nadir CD4%. Our results suggest that early treatment in HIV-infected infants increases their potential to safely control viral replication after PTI for long periods.

Mild improvement in mitochondrial function after a 3-year antiretroviral treatment interruption despite persistent impairment of mitochondrial DNA content.

OBJECTIVE: The ability of a prolonged antiretroviral treatment interruption to reverse mitochondrial toxicity was evaluated in a sub-study of TIBET, a prospective trial examining antiretroviral treatment interruption guided by CD4+ cell count. PATIENTS AND METHODS: The study population was composed of patients from the TIBET study who had been followed for > or =96 weeks and whose peripheral blood mononuclear cells (PBMCs) had been collected at baseline and throughout the study period. Of the 201 patients included in the TIBET study, 38 were selected for the mitochondrial sub-study; 18 patients discontinued antiretroviral therapy for > or =96 weeks and 20 maintained therapy. Mitochondrial DNA (mtDNA) and RNA (mtRNA) were measured in PBMCs using real-time polymerase chain reaction, and mitochondrial function relative to mitochondrial content was assessed using the cytochrome c oxidase and citrate synthase ratio (COX/CS). RESULTS: Whereas mtDNA content showed a similar progressive decrease throughout the study period in both arms, the mtRNA amount remained stable in both groups and the COX/CS ratio improved significantly in patients who interrupted therapy CONCLUSIONS: Mitochondrial function improved during a prolonged antiretroviral treatment interruption despite a decrease in mtDNA levels in PBMCs, probably because of the existence of a mitochondrial transcriptional and translational upregulation mechanism or the reversion of mitochondrial toxicity by a mechanism that is independent of DNA polymerase gamma. The reduction in virus-related mitochondrial damage should be considered another relevant benefit of antiretroviral therapy.

Drug-resistance mutations number and K70R or T215Y/F substitutions predict treatment resumption during guided treatment interruptions.

The role of antiretroviral history and genotypic resistance information as predictors of the first treatment interruption (TI) length in a CD4(+) cell count and plasma viremia-guided TI study (GTI) was assessed. Drug-resistance mutations (DRMs) were monitored in chronically HIV-1-infected subjects who underwent GTI. Patients were retrospectively classified into those who received monotherapy or dual therapy prior to HAART (pre-HAART group, n = 44) or directly initiated HAART (HAART group, n = 43). DRMs were assessed by population-based sequencing of proviral DNA at baseline and plasma RNA monthly during TI up to 180 weeks. Univariate and multivariate Cox's proportional hazard models were used to determine time off therapy predictors. The emergence of viruses with DRMs during TI was 5.1-fold more likely in pre-HAART than in HAART patients. The presence of DRMs in proviral DNA or plasma RNA was associated with shorter time off therapy. An accumulation of three or more DRMs duplicated the risk of restarting therapy with respect to having one or two mutations. Regardless of the number of DRMs, the presence of K70R or T215F/Y predicted the shortest TI time. Multivariate analyses adjusted by nadir CD4(+) counts supported the presence of DRMs in plasma HIV-1 RNA, and specifically the K70R or T215F/Y, as potent predictors of time off therapy. A history of monotherapy or dual therapy, accumulation of three or more key DRMs in the HIV-1 polymerase, and/or the presence of substitutions K70R or T215F/Y were associated with shorter time off therapy during GTI. A genotypic profile could provide clinicians with a predictive tool for time off therapy when TI is required in patients with suppressed viremia in whom nadir CD4(+) count is not available.

The magnitude of interferon-gamma responses to human cytomegalovirus is predictive for HIV-1 disease progression.

BACKGROUND: Human cytomegalovirus (HCMV) infection has been strongly associated to HIV-1 progression. We have investigated whether the magnitude of the overall peripheral blood mononuclear cell responses to HCMV stimulation correlated with HIV-1 progression. METHODS: Blood samples were collected from 75 HIV-1-positive individuals on highly active antiretroviral therapy with CD4 count>500 cells per cubic millimeter and undetectable HIV RNA just before interrupting treatment. Specific interferon-gamma (IFN-gamma) HCMV cell responses were measured by an enzyme-linked immunospot (ELISPOT) assay. The results were analyzed by Kaplan-Meier survival curves, contingency tests, and the Cox proportional hazard models to evaluate the predictive value of peripheral blood responses to HCMV and the length of time that patients were off treatment. RESULTS: Patients were stratified into those with weak (<500 spot-forming units) or strong (>500 spot-forming units) IFN-gamma responses to HCMV. During the 3-year follow-up, 51% of patients with strong responses remained untreated compared with 14% of patients with weak HCMV responses (P=0.0015). Length of time without therapy was also longer in patients with stronger responses (hazard ratio=2.08; P=0.001). HCMV responses were still predictive of restarting therapy after adjusting for the CD4 nadir counts. CONCLUSION: Specific IFN-gamma responses to HCMV may be employed as a predictive useful marker for the evolution of HIV-1 infection.