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Background: Latent HIV-1 is a major hurdle in obtaining HIV-1 sustained virological remission (SVR). Here we explored histone deacetylation inhibition property of nicotinamide (NAM; n=17) for the first time in comparison to a combination of methyltransferase inhibitors (MTIs; Chaetocin and BIX01294; n=25) to reactivate latent HIV ex vivo in CD8-depleted PBMCs from antiretroviral treated aviremic individuals., Results: NAM reactivated HIV-1 from 13/17 (76.4%) samples compared to 20/25 (80.0%) using MTIs with mean viral load (VLs) of 4.32 and 3.22 log 10 RNA copies/mL, respectively (p=0.004). Mean purging time after NAM and MTIs stimulation was 5.1 and 6.75 days, respectively (p=0.73). Viral purging in autologous cultures exhibited blunted HIV recovery with fluctuating VLs followed by a complete viral extinction when expanded in allogenic system. Electron microscopy from five supernatants revealed anomalous viral particles, with lack of complete viral genomes when characterized by ultradeep sequencing through metagenomics approach (n=4)., Conclusion: NAM alone was more potent HIV-1 activator than combination of MTIs, with potential of clinical use., (Copyright © 2020 Sociedade Brasileira de Infectologia. Published by Elsevier España, S.L.U. All rights reserved.)

Detailed clonal phenotypic/genotypic analyses explored viral-escape mechanisms during maraviroc-based therapy in highly treatment-experienced participants from the MOTIVATE trials. To allow real-time assessment of samples while maintaining a blind trial, the first 267 enrolled participants were selected for evaluation. At failure, plasma samples from 20/50 participants (16/20 maraviroc-treated) with CXCR4-using virus and all 38 (13 maraviroc-treated) with CCR5-tropic virus were evaluated. Of those maraviroc-treated participants with CXCR4-using virus at failure, genotypic and phenotypic clonal tropism determinations showed >90% correspondence in 14/16 at Day 1 and 14/16 at failure. Phylogenetic analysis of clonal sequences detected pre-treatment progenitor CXCR4-using virus, or on-treatment virus highly divergent from the Day 1 R5 virus, excluding possible co-receptor switch through maraviroc-mediated evolution. Re-analysis of pre-treatment samples using the enhanced-sensitivity Trofile® assay detected CXCR4-using virus pre-treatment in 16/20 participants failing with CXCR4-using virus. Post-maraviroc reversion of CXCR4-use to CCR5-tropic occurred in 7/8 participants with follow-up, suggesting selective maraviroc inhibition of CCR5-tropic variants in a mixed-tropic viral population, not emergence of de novo mutations in CCR5-tropic virus, as the main virologic escape mechanism. Maraviroc-resistant CCR5-tropic virus was observed in plasma from 5 treated participants with virus displaying reduced maximal percent inhibition (MPI) but no evidence of IC50 change. Env clones with reduced MPI showed 1-5 amino acid changes specific to each V3-loop region of env relative to Day 1. However, transferring on-treatment resistance-associated changes using site-directed mutagenesis did not always establish resistance in Day 1 virus, and key 'signature' mutation patterns associated with reduced susceptibility to maraviroc were not identified. Evolutionary divergence of the CXCR4-using viruses is confirmed, emphasizing natural selection not influenced directly by maraviroc; maraviroc simply unmasks pre-existing lineages by inhibiting the R5 virus. For R5-viral failure, resistance development through drug selection pressure was uncommon and manifested through reduced MPI and with virus strain-specific mutational patterns., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: RM is an employee of Pfizer and owns stock in the company. CC, ML, and EV are not employed but are affiliated to The Research Network, Ltd, which administers the contract with Pfizer for work on the maraviroc program, including the finalization of the analysis and preparation of the submitted work. CC, ML, and EV were previously employed by Pfizer; EV, JM, ML, MM, and MW were employed during the conduct of the trial. CC owns stock in GlaxoSmithKline. JT, WH, and JMW are employees of Monogram Biosciences. DLR reports a grant from the Medical Research Council (MRC; G1001806/1) and consultancy fees from Pfizer during the conduct of the study. JT reports a grant from Small Business Innovation Research (Grant number: 1 R21 AI114399) outside the scope of the submitted work, and a fee for service from Pfizer during the conduct of the study; in addition, JT has a patent US 9,581,595 B2 issued. Pfizer is one of the beneficiaries of the ViiV Healthcare Joint Venture along with GlaxoSmithKline and Shinogi. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Purpose of Review: Even in the era of modern HAART, antiretroviral (ARV) failure and emergence of drug resistance is still a problem worldwide. New classes with different mechanisms of action are needed to overcome this challenge. After the integrase inhibitors were launched, more than a decade ago, no new classes were added to the ARV armamentarium., Recent Findings: Fostemsavir (FTR) is an attachment inhibitor, active regardless of viral tropism, without cross-resistance to any of the existing ARV compounds. A phase 3 study showed a reduction in plasma viral RNA of 1.21-1.73 log10 copies/ml from baseline after 8 days of functional monotherapy; at 48 weeks, up to 82% of patients treated with FTR and an optimized background ARV regimen achieved virological suppression below 50 copies/ml., Summary: FTR is an investigational HIV drug with a novel mechanism of action that demonstrates virologic activity in HIV-infected treatment-experienced individuals.

Background: In the absence of therapy, CXCR4 (X4)-tropic human immunodeficiency virus type 1 (HIV-1) increases over time, associated with accelerated disease progression. In contrast, the majority of patients receiving long-term combination antiretroviral therapy (cART) present with CCR5 (R5)-tropic HIV-1 variants. It is unclear whether cART itself mediates the reduction of X4-tropic HIV-1. The current study aimed at assessing the tropism of viral integrates in patients' blood during fully suppressive cART., Methods: The relative frequencies of X4-tropic proviral HIV-1 variants were determined by means of next-generation sequencing (False Positive Rate (FPR), 3.5%; R5- or X4 tropic variants occurring at less than 2% of the total virus population) for 35 treated patients in the Swiss HIV Cohort Study and followed longitudinally over time. Full viral suppression and a continuous CD4 T-cell recovery during cART were documented for all patients. Viral phylogenetic changes and sequence evolution were analyzed., Results: The majority of patients (80%) experienced no frequency increase in X4-tropic proviruses during therapy. Although some proviral sequence evolution was demonstrable in >50% of these patients during therapy, this growing viral diversity was in no case paralleled by the emergence or expansion of X4-tropic provirus variants. In the remaining 20% of patients, the documented expansion of X4-tropic provirus was based on the outgrowth of single viral variants from minority populations already present before therapy initiation., Conclusion: Our study demonstrates that X4-tropic HIV sharply declines in most patients during successful therapy, which indicates a preferential tropism-dependent provirus elimination in the immunocompetent host. The recently implemented World Health Organization strategies of immediate therapy initiation are fully in line with this gradual loss of X4 tropism during therapy. Moreover, the early use of coreceptor antagonists against the remaining CCR5-tropic viruses may be indicated., (© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.)

Currently, the treatment of human immunodeficiency virus involves combination therapy, as antiretroviral therapy(ART). The treatment has improved steadily since the advent of potent combination therapy in 1996. New drugs that offer new mechanisms of action, improvements in potency and activity even against multidrug-resistant viruses, dosing convenience, and tolerability have been approved. Among ART with useful drugs, there are two important examinations before starting the treatment using the two kinds of drug. CCR5 co-receptor antagonists, maraviroc, prevent HIV entry into target cells by binding to CCR5 receptors. Genotypic assays have been developed that can determine or predict the co-receptor tropism(i.e., CCR5, CXCR4, or both) of the patient's dominant virus population. The assay for HIV-1 co-receptor usage should be performed whenever the use of a CCR5 antagonist is being considered. One of the nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs), abacavir, is an important agent to develop recommended regimens for antiretroviral therapy. Serious and sometimes fatal hypersensitivity reactions have been associated with abacavir-containing products, ZIAGEN, Epzicom, and Triumeq. Patients who carry the HLA-B*5701 allele are at high-risk of a hypersensitivity reaction to abacavir. Prior to initiating therapy with abacavir, performing a screening test for the HLA-B*5701 allele is recommended. [Review].

Background: Insertion of T4 lysozyme (T4L) into the GPCR successfully enhanced GPCR protein stability and solubilization. However, the biological functions of the recombinant GPCR protein have not been analyzed., Methods: We engineered the CCR5-T4L mutant and expressed and purified the soluble recombinant protein using an E.coli expression system. The antiviral effects of this recombinant protein in THP-1 cell lines, primary human macrophages, and PBMCs from different donors were investigated. We also explored the possible mechanisms underlying the observed antiviral effects., Results: Our data showed the biphasic inhibitory and promotion effects of different concentrations of soluble recombinant CCR5-T4L protein on R5 tropic human immunodeficiency virus-1 (HIV-1) infection in THP-1 cell lines, human macrophages, and PBMCs from clinical isolates. We demonstrated that soluble recombinant CCR5-T4L acts as a HIV-1 co-receptor, interacts with wild type CCR5, down-regulates the surface CCR5 expression in human macrophages, and interacts with CCL5 to inhibit macrophage migration. Using binding assays, we further determined that recombinant CCR5-T4L and [125I]-CCL5 compete for the same binding site on wild type CCR5., Conclusions: Our results suggest that recombinant CCR5-T4L protein marginally promotes HIV-1 infection at low concentrations and markedly inhibits infection at higher concentrations. This recombinant protein may be helpful in the future development of anti-HIV-1 therapeutic agents.

A review of a large number of HIV-1 tropism test requests (n = 1148) performed at a London tertiary referral centre was carried out. The aim was to establish whether these were being performed in line with recommendations from published guidelines and whether this represented the most cost-effective use of these tests in informing prescribing decisions of the CCR5 antagonist drug, maraviroc. The cost of these assays within the UK was covered by commercial funding until April 2013 which has subsequently been withdrawn. Furthermore, all healthcare settings are under increasing cost constraints and hence establishing the real utility and appropriate use of these tests is of vital importance., (© The Author(s) 2014.)

Assays to identify infectious organisms are critical for diagnosis and enabling the development of therapeutic agents. The demonstration that individuals with a 32-bp deletion within the CCR5 locus were resistant to human immunodeficiency virus (HIV) infection, while those heterozygous for the mutation progressed more slowly, led to the discovery of maraviroc (MVC), a CCR5 antagonist. As MVC is only active against CCR5-tropic strains of HIV, it was critical to develop a diagnostic assay to identify appropriate patients. Trofile™, a novel phenotypic tropism assay, was used to identify patients with CCR5-tropic virus for the MVC development program. Results of these clinical studies demonstrated that the assay correctly identified patients likely to respond to MVC. Over time, the performance characteristics of the phenotypic assay were enhanced, necessitating retesting of study samples. Genotypic tropism tests that have the potential to allow for local use and more rapid turnaround times are also being developed., (© 2015 New York Academy of Sciences.)

Purpose of Review: Current genotypic resistance tests fail to amplify drug-resistant minority variants when they are present below 20% of the total virus population. Next-generation sequencing (NGS) addresses this issue and is being introduced into diagnostic laboratories. This review gives an overview of the resistance tests currently used and explores the opportunities and challenges that NGS genotypic resistance tests will bring., Recent Findings: The technical challenges of NGS, such as PCR and sequence-related errors, are being addressed and various assays are currently undergoing technical validation for clinical use. Although not conclusive, the data seem to suggest that NGS will be valuable for low genetic barrier drugs and certain types of tests such as the HIV-1 tropism test. Clinical validation of the reporting and interpretation of minority variant results are essential when laboratories start reporting these results., Summary: The first wave of NGS technology is being rolled out in diagnostic laboratories. Antiviral test benefits include increased sensitivity and eventually cheaper antiviral resistance tests. There is a risk that low percentage minority variants may be over interpreted. This could result in antiviral drugs, which may have been effective, being possibly denied to patients if proper clinical validation studies are not performed.

We assessed the immunovirological response to antiretroviral regimens containing maraviroc in HIV-infected viremic patients with viral tropism predicted by different assays. We selected antiretroviral treatment-experienced HIV-1-infected patients initiating regimens containing maraviroc after different phenotypic or genotypic viral tropism assays, with at least one HIV-1 RNA determination during follow-up. Survival analysis was employed to assess the virological response as time to HIV-1 RNA <50 copies/ml and immunological response as time to a CD4 cell count increase of ≥ 100/μl from baseline. Predictors of these outcomes were analyzed by multivariate Cox regression models. In 191 treatments with maraviroc, virological response was achieved in 65.4% and the response was modestly influenced by the baseline viral load and concomitant drug activity but not influenced by the type of tropism assay employed. Immunological response was achieved in 58.1%; independent predictors were baseline HIV-1 RNA (per log10 higher: HR 1.29, 95% CI 1.05-1.60) and concomitant therapy with enfuvirtide (HR 2.05, 0.96-4.39) but not tropism assay results. Of 17 patients with baseline R5-tropic virus and available tropism results while viremic during follow-up on maraviroc, seven (41%) showed a tropism switch to non-R5 virus. A significant proportion of experienced patients treated with regimens containing maraviroc achieved virological response. The tropism test type used was not associated with immunovirological response and concomitant treatment with enfuvirtide increased the chance of immunological response. More than half of virological failures with maraviroc were not accompanied by tropism switch.

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.

Objectives: The genotypic method is reliable enough for the determination of tropism and largely preferred in Europe. However, careful interpretation is essential when assessing HIV genotypic resistance during treatment interruption (TI) due to the possible disappearance of resistant strains. The results of HIV genotypic tropism testing in such a context remain unknown., Methods: First, we studied changes in tropism in patients included in a structured TI assay: the Reverse study. Second, we investigated the unexpected tropism switches from X4 to R5 recorded in our routine database., Results: Tropism determination was possible in 21 patients of the Reverse study, 9 of whom had an X4 virus (43%) at baseline. Two patients displayed a change of tropism during TI, both switching from X4 to R5. Regarding the database investigation, 7 of the 222 patients with at least two plasma tropism determinations recorded in the database displayed a switch from X4 to R5. TI due to non-compliance at the time of the tropism change was reported for five of these seven patients., Conclusions: We have shown that the redistribution of the HIV population caused by TI could potentially result in X4 viruses becoming undetected and inappropriate prescription of a CCR5 receptor antagonist. Therefore, genotypic tropism results should be interpreted with caution in such a context.

Genotypic tropism testing methods are emerging as the first step before prescription of the CCR5 antagonist maraviroc (MVC) to HIV-infected patients in Europe. Studies validating genotypic tests have included other active drugs that could have potentially convoluted the effects of MVC. The maraviroc clinical test (MCT) is an in vivo drug sensitivity test based on the virological response to a short-term exposure to MVC monotherapy. Thus, our aim was to compare the results of genotypic tropism testing methods with the short-term virological response to MVC monotherapy. A virological response in the MCT was defined as a ≥ 1-log(10) decrease in HIV RNA or undetectability after 8 days of drug exposure. Seventy-three patients undergoing the MCT were included in this study. We used both standard genotypic methods (n = 73) and deep sequencing (n = 27) on MCT samples at baseline. For the standard methods, the most widely used genotypic algorithms for analyzing the V3 loop sequence, geno2pheno and PSSM, were used. For deep sequencing, the geno2pheno algorithm was used with a false-positive rate cutoff of 3.5. The discordance rates between the standard genotypic methods and the virological response were approximately 20% (including mostly patients without a virological response). Interestingly, these discordance rates were similar to that obtained from deep sequencing (18.5%). The discordance rates between the genotypic methods (tropism assays predictive of the use of the CCR5 coreceptor) and the MCT (in vivo MVC sensitivity assay) indicate that the algorithms used by genotypic methods are still not sufficiently optimized.

While human immunodeficiency virus (HIV)-1 chemokine co-receptors 5 tropism and the GWGR motif in the envelope third variable region (V3 loop) have been associated with a slower disease progression, their influence on antiretroviral response remains unclear. The impact of baseline V3 characteristics on treatment response was evaluated in a randomised, double blind, prospective cohort study with patients initiating highly active antiretroviral therapy with lopinavir or efavirenz plus azithothymidine/3TC (1:1) over 48 weeks. Similar virological and immunological responses were observed for both treatment regimens. The 43 individuals had a mean baseline CD4 T cell count of 119 cells/mm(3) [standard deviation (SD) = 99] and a mean viral load of 5.09 log(10) copies/mL (SD = 0.49). The GWGR motif was not associated with a CD4 T cell response, but predicted R5 tropism by the geno2pheno([clinical20%]) algorithm correlated with higher CD4 T cell levels at all monitoring points (p < 0.05). Moreover, higher false-positive rates (FPR) values from this analysis revealed a strong correlation with CD4 T cell recovery (p < 0.0001). Transmitted drug resistance mutations, documented in 3/41 (7.3%) cases, were unrelated to the assigned antiretroviral regimen and had no impact on patient outcomes. In conclusion, naÏve HIV-1 R5 infected patients exhibited higher CD4 T cell counts at baseline; this difference was sustained throughout therapy. The geno2pheno([clinical]) option FPR positively correlated with CD4 T cell gain and may be useful in predicting CD4 T cell recovery.

Determination of HIV-1 tropism is mandatory before using CCR5 antagonists in clinical practice. One drug of this class, maraviroc, has been approved for the treatment of HIV infection. The phenotypic assay, TrofileTM, was clinically validated in the clinical development program of maraviroc and has been widely used to select candidates for maraviroc therapy. Phenotypic tests, however, have the disadvantage of being complex, are costly and time-consuming, and their accessibility is limited, which hampers their routine use in clinical diagnosis. Genotypic assays, based on sequencing the third hypervariable (V3 loop) of the viral gene env, interpreted according to various genotypic bioinformatic tools, such as geno2pheno and PSSM, are faster and cheaper than phenotypic assays, and are also more accessible. In retrospective analyses of the maraviroc pivotal trials, genotypic methods using either conventional ("bulk") or deep-sequencing technology predicted virologic response to maraviroc similarly to phenotypic assays and are now included within several European recommendations to guide the clinical use of CCR5 antagonists., (Copyright © 2011 Elsevier España S.L. All rights reserved.)

Blocking HIV-1 cell entry has long been a major goal of anti-HIV drug development. Here, we report a successful design of two highly potent chimeric HIV entry inhibitors composed of one CCR5-targeting RANTES (regulated on activation normal T cell expressed and secreted) variant (5P12-RANTES or 5P14-RANTES (Gaertner, H., Cerini, F., Escola, J. M., Kuenzi, G., Melotti, A., Offord, R., Rossitto-Borlat, I., Nedellec, R., Salkowitz, J., Gorochov, G., Mosier, D., and Hartley, O. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 17706-17711)) linked to a gp41 fusion inhibitor, C37. Chimeric inhibitors 5P12-linker-C37 and 5P14-linker-C37 showed extremely high antiviral potency in single cycle and replication-competent viral assays against R5-tropic viruses, with IC(50) values as low as 0.004 nm. This inhibition was somewhat strain-dependent and was up to 100-fold better than the RANTES variant alone or in combination with unlinked C37. The chimeric inhibitors also fully retained the antiviral activity of C37 against X4-tropic viruses, and this inhibition can be further enhanced significantly if the target cell co-expresses CCR5 receptor. On human peripheral blood mononuclear cells, the inhibitors showed very strong inhibition against R5-tropic Ba-L strain and X4-tropic IIIB strain, with IC(50) values as low as 0.015 and 0.44 nm, which are 45- and 16-fold better than the parent inhibitors, respectively. A clear delivery mechanism requiring a covalent linkage between the two segments of the chimera was observed and characterized. Furthermore, the two chimeric inhibitors are fully recombinant and are easily produced at low cost. These attributes make them excellent candidates for anti-HIV microbicides. The results of this study also suggest a potent approach for optimizing existing HIV entry inhibitors or designing new inhibitors.

Bacitracin and the membrane-impermeant thiol reagent 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) are agents known to inhibit protein disulfide isomerase (PDI), a cell-surface protein critical in HIV-1 entry therefore they are fusion inhibitors (FI). Here we investigated the possibility that Bacitracin and or DTNB might have other antiviral activities besides FI. By means of residual activity assays, we found that both compounds showed antiviral activity only to viruses T-tropic HIV-1 strain. Cell-based fusion assays showed inhibition on HeLa-CD4-LTR-β-gal (CD4) and HL2/3 cells treated with Bacitracin, and DTNB with the latest compound we observed fusion inhibition on both cells but strikingly in HL2/3 cells (expressing Env) indicating a possible activity on both, the cell membrane and the viral envelope. A time-of-addition experiment showed that both compounds act on HIV entry inhibition but DTNB also acts at late stages of the viral cycle. Lastly, we also found evidence of long-lasting host cell protection in vitro by DTNB, an important pharmacodynamic parameter for a topical microbicide against virus infection, hours after the extracellular drug was removed; this protection was not rendered by Bacitracin. These drugs proved to be leading compounds for further studies against HIV showing antiviral characteristics of interest.

Disease progression during human immunodeficiency virus type 1 (HIV-1) infection has been associated with a switch of viral coreceptor usage from CCR5 to CXCR4. The current study investigates the effect of anti retroviral therapy (ART) on the viral tropism in a group of patients based on the V3 loop sequence, in ART naïve patients prior to and 24 weeks after ART. Genomic DNA was extracted from the PBMCs of these patients, and the C2-V5 region of the HIV-1 env genes were cloned and sequenced. The coreceptor usage was predicated based on V3 loop amino acid sequences using Geno2pheno and PSSM programs. Our results indicate that following ART, the plasma viral loads of both CXCR4 and CCR5 viruses were significantly decreased. We observed a relatively higher ratio of R5 than X4 virus after 24 weeks of ART and both the positive charges and the net charges of the V3 regions were decreased significantly (p < 0.05) after ART. We conclude that ART significantly, reduced both X4 and R5 viruses with a preferential suppression of X4 virus. These data will help improve prognostic outcomes and help clinicians determine the course of treatment in patients who exhibit virologic failure while taking a CCR5 antagonist.

Characterization of residual plasma virus during antiretroviral therapy (ART) is a high priority to improve understanding of HIV-1 pathogenesis and therapy. To understand the evolution of HIV-1 pol and env genes in viremic patients under selective pressure of ART, we performed longitudinal analyses of plasma-derived pol and env sequences from single HIV-1 genomes. We tested the hypotheses that drug resistance in pol was unrelated to changes in coreceptor usage (tropism), and that recombination played a role in evolution of viral strains. Recombinants were identified by using Bayesian and other computational methods. High-level genotypic resistance was seen in approximately 70% of X4 and R5 strains during ART. There was no significant association between resistance and tropism. Each patient displayed at least one recombinant encompassing env and representing a change in predicted tropism. These data suggest that, in addition to mutation, recombination can play a significant role in shaping HIV-1 evolution., (Copyright 2010 Elsevier Inc. All rights reserved.)

Objectives: The use of maraviroc as part of a simplification of antiretroviral therapy (ART) is hampered by the difficulty of assessing viral tropism in patients with undetectable viraemia. In this context, information on tropism might be obtained from testing either older stored viraemic sera collected before initiation of ART or current proviral DNA in peripheral blood cells., Methods: HIV-1-infected individuals who had initiated ART and had undetectable viraemia for >2 years were identified. V3 genotyping was performed in parallel from plasma HIV-RNA and proviral DNA before starting ART and from proviral DNA while on suppressive ART. Viral tropism was interpreted using geno2pheno (false positive rate = 10%) and an optimized version of position specific scoring matrices (PSSM) with a greater sensitivity to detect X4 variants (PSSM(X4/R5-8))., Results: A total of 78 HIV-1 infected individuals were examined. Mean time under suppressive ART was 3.5 years (interquartile range: 2.3-4.4). The rate of X4 variants in plasma and proviral DNA samples at baseline was 32.8% and 34.0%, respectively. It was 33.9% after >2 years of suppressive ART in DNA samples. Paired RNA/DNA tropism results at baseline could be obtained for 38 patients, with an overall 82% concordance. After >2 years of suppressed plasma viraemia, HIV tropism was re-assessed in proviral DNA; tropism switches were uncommon, especially comparing baseline and most recent DNA longitudinal specimens (12%)., Conclusions: HIV tropism switches over time under suppressive ART are rare. There is a relatively good correlation between RNA and DNA tropism estimations using genotypic tests. Thus, HIV-1 tropism might confidently be examined either in older stored viraemic plasma specimens or in current proviral DNA samples.

Objective: Few data are available about the efficacy of maraviroc (MVC) during routine use. We characterized indications for MVC use and the efficacy of MVC in clinical practice., Methods: Thirty-two patients treated with MVC at our institution between 2006 and 2009 were included. Genotypic (n +/- 31) and phenotypic (n +/- 13) tropism analysis was performed. We determined indications for MVC use, characteristics of antiretroviral combination partners and treatment outcome., Results: Complete suppression of viral replication was achieved in 78% after 6 months. A median increase of 124 CD4+ cells/microl after 6 months was observed. Concordance between phenotypic and genotypic tropism was found in 75%. Indications for MVC treatment included treatment failure (n +/- 15), intolerance to previous antiretrovirals (n +/- 6) and add-on MVC for intensification without changing the current regimen (n +/- 11). The add-on strategy was used in patients with a relatively low viremia in order to achieve complete viral load suppression or in situations with suppressed viral load but judged as unstable due to an extensive resistance pattern. Salvage drugs most frequently combined with MVC were darunavir (n +/- 14) and raltegravir (n +/- 14). - The genotypic assay had predicted CXCR4 tropism in 5 patients, using a false positive rate (FPR) of 20%. Lowering the FPR to 5% predicted CCR5 tropism in 4 cases, still resulting in sustained complete viral response under MVC use., Conclusions: MVC containing salvage regimens achieve relevant CD4 cell increases and high viral response rates. In patients with few remaining treatment options it may be justified to lower the FPR-cutoff to 5% when predicting the coreceptor usage. Hereby, MVC could still be applied in selected patients with otherwise limited treatment options.

The influence of antiretroviral therapy on co-receptor tropism remains controversial. To verify if co-receptor tropism shift was affected by HAART, the evolution of proviral DNA V3 genotype after 12 months of a new antiretroviral regimen was compared between responder and non-responder patients. Baseline blood samples were collected from 36 patients infected with HIV-1 subtype-B (18 naïve and 18 experienced) for virus isolation and env V3 genotyping from plasma HIV-1 RNA and PBMC DNA. DNA V3 genotyping was repeated after 12 months from initiating HAART. WebPSSM was used for categorizing V3 sequences into X4 or R5; for analysis purposes, dual/mixed viruses were considered as X4. From the 10 (28%) patients changing their proviral DNA V3 genotype during therapy, six shifted from R5-to-X4 and four from X4-to-R5. The lack of reaching virological suppression was not associated with an X4-to-R5 (P = 0.25) or R5-to-X4 (P = 0.14) shift; time-to-viral suppression and CD4 increase were similar in both groups. No association was found between tropism shift and patient baseline characteristics including age, sex, CDC stage, CD4 count, viral load, exposure and length of previous HAART, enfuvirtide use in the new regimen, number of reverse transcriptase and protease resistance-associated mutations. Conversely, CD4 nadir was correlated to emergence of X4 virus in proviral DNA (mean 27.2 +/- 30.6 in R5-to-X4 shifting patients vs. 161.6 +/- 150.6 in non-shifting patients, P = 0.02). The occurrence of a tropism shift in both directions was independent of HAART use, irrespective of its efficacy. The CD4 count nadir was the only baseline characteristic able to predict an R5-to-X4 viral shift., ((c) 2009 Wiley-Liss, Inc.)