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Human Immunodeficiency Virus (HIV) maturation plays an essential role in the viral life cycle by enabling the generation of mature infectious virus particles through proteolytic processing of the viral Gag and GagPol precursor proteins. An impaired polyprotein processing results in the production of non-infectious virus particles. Consequently, particle maturation is an excellent drug target as exemplified by inhibitors specifically targeting the viral protease (protease inhibitors; PIs) and the experimental class of maturation inhibitors that target the precursor Gag and GagPol polyproteins. Considering the different target sites of the two drug classes, direct cross-resistance may seem unlikely. However, coevolution of protease and its substrate Gag during PI exposure has been observed both in vivo and in vitro . This review addresses in detail all mutations in Gag that are selected under PI pressure. We evaluate how polymorphisms and mutations in Gag affect PI therapy, an aspect of PI resistance that is currently not included in standard genotypic PI resistance testing. In addition, we consider the consequences of Gag mutations for the development and positioning of future maturation inhibitors.

Optimal regimen choice of antiretroviral therapy is essential to achieve long-term clinical success. Integrase inhibitors have swiftly been adopted as part of current antiretroviral regimens. The purpose of this study was to review the evidence for integrase inhibitor use in clinical settings. MEDLINE and Web-of-Science were screened from April 2006 until November 2012, as were hand-searched scientific meeting proceedings. Multiple reviewers independently screened 1323 citations in duplicate to identify randomized controlled trials, nonrandomized controlled trials and cohort studies on integrase inhibitor use in clinical practice. Independent, duplicate data extraction and quality assessment were conducted. 48 unique studies were included on the use of integrase inhibitors in antiretroviral therapy-naive patients and treatment-experienced patients with either virological failure or switching to integrase inhibitors while virologically suppressed. On the selected studies with comparable outcome measures and indication (n = 16), a meta-analysis was performed based on modified intention-to-treat (mITT), on-treatment (OT) and as-treated (AT) virological outcome data. In therapy-naive patients, favorable odds ratios (OR) for integrase inhibitor-based regimens were observed, (mITT OR 0.71, 95% CI 0.59-0.86). However, integrase inhibitors combined with protease inhibitors only did not result in a significant better virological outcome. Evidence further supported integrase inhibitor use following virological failure (mITT OR 0.27; 95% CI 0.11-0.66), but switching to integrase inhibitors from a high genetic barrier drug during successful treatment was not supported (mITT OR 1.43; 95% CI 0.89-2.31). Integrase inhibitor-based regimens result in similar immunological responses compared to other regimens. A low genetic barrier to drug-resistance development was observed for raltegravir and elvitegravir, but not for dolutegravir. In first-line therapy, integrase inhibitors are superior to other regimens. Integrase inhibitor use after virological failure is supported as well by the meta-analysis. Careful use is however warranted when replacing a high genetic barrier drug in treatment-experienced patients switching successful treatment.

A robust measure of the size of the latent HIV reservoir is essential to quantifying the effect of interventions designed to deplete the pool of reactivatable, replication competent proviruses. In addition to the ability to measure a biologically relevant parameter, any assay designed to be used in a clinical trial needs to be reproducible and scalable. The need to quantify the number of resting CD4+ T cells capable of releasing infectious virus has led to the development of the quantitative viral outgrowth assay (VOA). The assay as originally described has a number of features that limit its scalability for use in clinical trials; however recent developments reducing the time and manpower requirements of the assay, while importantly improving reproducibility mean that it is becoming much more practical for it to enter into more widespread use. This review describes the background to VOA development and the practical issues that they present in utilising them in clinical trials. It describes the innovations that have made their usage more practical and the limitations that still exist.

Background Emergence of resistance against integrase inhibitor raltegravir in human immunodeficiency virus type 1 (HIV-1) patients is generally associated with selection of one of three signature mutations: Y143C/R, Q148K/H/R or N155H, representing three distinct resistance pathways. The mechanisms that drive selection of a specific pathway are still poorly understood. We investigated the impact of the HIV-1 genetic background and population dynamics on the emergence of raltegravir resistance. Using deep sequencing we analyzed the integrase coding sequence (CDS) in longitudinal samples from five patients who initiated raltegravir plus optimized background therapy at viral loads > 5000 copies/ml. To investigate the role of the HIV-1 genetic background we created recombinant viruses containing the viral integrase coding region from pre-raltegravir samples from two patients in whom raltegravir resistance developed through different pathways. The in vitro selections performed with these recombinant viruses were designed to mimic natural population bottlenecks. Results Deep sequencing analysis of the viral integrase CDS revealed that the virological response to raltegravir containing therapy inversely correlated with the relative amount of unique sequence variants that emerged suggesting diversifying selection during drug pressure. In 4/5 patients multiple signature mutations representing different resistance pathways were observed. Interestingly, the resistant population can consist of a single resistant variant that completely dominates the population but also of multiple variants from different resistance pathways that coexist in the viral population. We also found evidence for increased diversification after stronger bottlenecks. In vitro selections with low viral titers, mimicking population bottlenecks, revealed that both recombinant viruses and HXB2 reference virus were able to select mutations from different resistance pathways, although typically only one resistance pathway emerged in each individual culture. Conclusions The generation of a specific raltegravir resistant variant is not predisposed in the genetic background of the viral integrase CDS. Typically, in the early phases of therapy failure the sequence space is explored and multiple resistance pathways emerge and then compete for dominance which frequently results in a switch of the dominant population over time towards the fittest variant or even multiple variants of similar fitness that can coexist in the viral population.

Background Maturation inhibitors are an experimental class of antiretrovirals that inhibit Human Immunodeficiency Virus (HIV) particle maturation, the structural rearrangement required to form infectious virus particles. This rearrangement is triggered by the ordered cleavage of the precursor Gag polyproteins into their functional counterparts by the viral enzyme protease. In contrast to protease inhibitors, maturation inhibitors impede particle maturation by targeting the substrate of protease (Gag) instead of the protease enzyme itself. Direct cross-resistance between protease and maturation inhibitors may seem unlikely, but the co-evolution of protease and its substrate, Gag, during protease inhibitor therapy, could potentially affect future maturation inhibitor therapy. Previous studies showed that there might also be an effect of protease inhibitor resistance mutations on the development of maturation inhibitor resistance, but the exact mechanism remains unclear. We used wild-type and protease inhibitor resistant viruses to determine the impact of protease inhibitor resistance mutations on the development of maturation inhibitor resistance. Results Our resistance selection studies demonstrated that the resistance profiles for the maturation inhibitor bevirimat are more diverse for viruses with a mutated protease compared to viruses with a wild-type protease. Viral replication did not appear to be a major factor during emergence of bevirimat resistance. In all in vitro selections, one of four mutations was selected: Gag V362I, A364V, S368N or V370A. The impact of these mutations on maturation inhibitor resistance and viral replication was analyzed in different protease backgrounds. The data suggest that the protease background affects development of HIV-1 resistance to bevirimat and the replication profiles of bevirimat-selected HIV-1. The protease-dependent bevirimat resistance and replication levels can be explained by differences in CA/p2 cleavage processing by the different proteases. Conclusions These findings highlight the complicated interactions between the viral protease and its substrate. By providing a better understanding of these interactions, we aim to help guide the development of second generation maturation inhibitors.

Background Mutations in the substrate of HIV-1 protease, especially changes in the NC/p1 cleavage site, can directly contribute to protease inhibitor (PI) resistance and also compensate for defects in viral replicative capacity (RC) due to a drug resistant protease. These NC/p1 changes are known to enhance processing of the Gag protein. To investigate the capacity of HIV-1 to modulate Gag cleavage and its consequences for PI resistance and RC, we performed a detailed enzymatic and virological analysis using a set of PI resistant NC/p1 variants (HXB2 431V , HXB2 436E+437T , HXB2 437T and HXB2 437V ). Results Here, we demonstrate that single NC/p1 mutants, which displayed only a slight increase in PI resistance did not show an obvious change in RC. In contrast, the double NC/p1 mutant, which displayed a clear increase in processing efficiency and PI resistance, demonstrated a clear reduction in RC. Cleavage analysis showed that a tridecameric NC/p1 peptide representing the double NC/p1 mutant was cleaved in two specific ways instead of one. The observed decrease in RC for the double NC/p1 mutant (HXB2 436E+437T ) could (partially) be restored by either reversion of the 436E change or by acquisition of additional changes in the NC/p1 cleavage site at codon 435 or 438 as was revealed during in vitro evolution experiments. These changes not only restored RC but also reduced PI resistance levels. Furthermore these changes normalized Gag processing efficiency and obstructed the novel secondary cleavage site observed for the double NC/p1 mutant. Conclusions The results of this study clearly demonstrate that HIV-1 can modulate Gag processing and thereby PI resistance. Distinct increases in Gag cleavage and PI resistance result in a reduced RC that can only be restored by amino acid changes in NC/p1 which reduce Gag processing to an optimal rate.

Antiretroviral therapy (ART) cannot cure HIV infection because of a persistent reservoir of latently infected cells. Approaches that force HIV transcription from these cells, making them susceptible to killing—termed kick and kill regimens—have been explored as a strategy towards an HIV cure. RIVER is the first randomised trial to determine the effect of ART-only versus ART plus kick and kill on markers of the HIV reservoir. This phase 2, open-label, multicentre, randomised, controlled trial was undertaken at six clinical sites in the UK. Patients aged 18–60 years who were confirmed as HIV-positive within a maximum of the past 6 months and started ART within 1 month from confirmed diagnosis were randomly assigned by a computer generated randomisation list to receive ART-only (control) or ART plus the histone deacetylase inhibitor vorinostat (the kick) and replication-deficient viral vector T-cell inducing vaccines encoding conserved HIV sequences ChAdV63. HIVconsv-prime and MVA.HIVconsv-boost (the kill; ART + V + V; intervention). The primary endpoint was total HIV DNA isolated from peripheral blood CD4+ T-cells at weeks 16 and 18 after randomisation. Analysis was by intention to treat. This trial is registered with ClinicalTrials.gov, NCT02336074. Between June 14, 2015 and Jul 11, 2017, 60 men with HIV were randomly assigned to receive either an ART-only (n=30) or an ART + V + V (n=30) regimen; all 60 participants completed the study, with no loss-to-follow-up. Mean total HIV DNA at weeks 16 and 18 after randomisation was 3·02 log10 copies HIV DNA per 106 CD4+ T-cells in the ART-only group versus 3·06 log10 copies HIV DNA per 106 CD4+ T-cells in ART + V + V group, with no statistically significant difference between the two groups (mean difference of 0·04 log10 copies HIV DNA per 106 CD4+ T-cells [95% CI −0·03 to 0·11; p=0·26]). There were no intervention-related serious adverse events. This kick and kill approach conferred no significant benefit compared with ART alone on measures of the HIV reservoir. Although this does not disprove the efficacy kick and kill strategy, for future trials enhancement of both kick and kill agents will be required. Medical Research Council (MR/L00528X/1).

Cure of Human Immunodeficiency Virus (HIV) infection remains elusive due to the persistence of HIV in a latent reservoir. Strategies to eradicate latent infection can only be evaluated with robust, sensitive and specific assays to quantitate reactivatable latent virus. We have taken the standard peripheral blood mononuclear cell (PBMC) based viral outgrowth methodology and from it created a logistically simpler and more highly reproducible assay to quantify replication-competent latent HIV in resting CD4 + T cells, both increasing accuracy and decreasing cost and labour. Purification of resting CD4 + T cells from whole PBMC is expedited and achieved in 3 hours, less than half the time of conventional protocols. Our indicator cell line, SupT1-CCR5 cells (a clonal cell line expressing CD4, CXCR4 and CCR5) provides a readily available standardised readout. Reproducibility compares favourably to other published assays but with reduced cost, labour and assay heterogeneity without compromising sensitivity.

The persistence of infected T cells harbouring intact HIV proviruses is the barrier to the eradication of HIV. This reservoir is stable over long periods of time despite antiretroviral therapy. There has been controversy on whether low level viral replication is occurring at sanctuary sites periodically reseeding infected cells into the latent reservoir to account its durability. To study viral evolution in a physiologically relevant population of latent viruses, we repeatedly performed virus outgrowth assays on a stably treated HIV positive patient over two years and sequenced the reactivated latent viruses. We sought evidence of increasing sequence pairwise distances with time as evidence of ongoing viral replication. 64 reactivatable latent viral sequences were obtained over 103 weeks. We did not observe an increase in genetic distance of the sequences with the time elapsed between sampling. No evolution could be discerned in these reactivatable latent viruses. Thus, in this patient, the contribution of low-level replication to the maintenance of the latent reservoir detectable in the blood compartment is limited.

Introduction Dolutegravir is a second generation integrase inhibitor with a proposed high genetic barrier to resistance. However, in clinical trials, decreased virological response was seen in a subset of patients with prior exposure to raltegravir and multiple integrase resistance mutations. Methods We describe two cases of HIV subtype B‐infected patients starting dolutegravir after previous failure on a raltegravir‐containing regimen with extensive resistance. Genotypic analysis was performed using population sequencing and 454 ultradeep sequencing of integrase at time of raltegravir exposure. Results Both patients were diagnosed in early 1990s and received mono‐ and dual therapy, followed by several cART‐regimens. Due to presence of extensive resistance, the genotypic susceptibility score of these regimens never reached a score >2 and never resulted in sustained virological suppression despite good adherence. Early 2012, the clinical condition of patient 1 worsened during persistent failure of a mega‐cART regimen despite excellent drug levels. Six major PI, six minor PI, seven NRTI, six NNRTI and two INI mutations plus DM‐virus were detected (Table 1). Ultra‐deep sequencing of integrase showed the selection of Q148R, E138K+Q148K, and N155H variants and phenotypic raltegravir resistance was demonstrated. After addition of dolutegravir and enfuvirtide to the failing regimen (zidovudine, lamivudine, tenofovir, etravirine, darunavir/ritonavir, maraviroc), viral load (VL) decreased from 244,000 to <20 cps/mL within five months, CD4‐count increased (33 to 272 mm3) and the clinical condition improved substantially. In patient 2, similar worsening of the clinical condition was observed late 2012 during persistent failure on mega‐cART. Five major PI, six minor PI, nine NRTI, seven NNRTI and one INI mutation plus DM‐virus were detected. Ultra‐deep sequencing showed selection of N155H, followed by Q95K and V151I variants and phenotypic raltegravir resistance was demonstrated. Dolutegravir was added to his failing regimen (zidovudine, lamivudine, etravirine, atazanavir/ritonavir, maraviroc) at a VL of 39,000 cps/mL. Sustained virological suppression was reached within five months with considerable increase of CD4‐count (41 to 175 mm3) and slight improvement of clinical condition. Conclusions We present the first patients with extensive integrase resistance who were treated with dolutegravir in clinical practice and who achieved excellent virological and immunological success. These cases demonstrate the high genetic barrier of dolutegravir.