Rocío Bellido

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

The interferon (IFN)‐γ component of the immune response plays an essential role in combating infectious and non‐infectious diseases. Induction of IFN‐γ 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‐γ 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‐γ when further stimulated with a combination of IL‐12 and IL‐18. In addition, naturally activated alveolar macrophages immediately secreted IFN‐γ upon treatment with IL‐12 and IL‐18. Therefore, human macrophages in addition to lymphoid cells contribute to the IFN‐γ response, providing another link between the innate and acquired immune responses.

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.

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.

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.

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.

The Magnitude of Interferon-γ 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-γ (IFN-γ) 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-γ 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-γ responses to HCMV may be employed as a predictive useful marker for the evolution of HIV-1 infection.

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 Coxs 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.

RECall for Automated Genotypic Tropism Testing

ABSTRACT 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.

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.