Search

The objective of this study was to assess the phenotypic susceptibility of HIV-1 subtype C isolates, with nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance-associated amino acid changes, to newer NNRTIs. A panel of 52 site-directed mutants and 38 clinically derived HIV-1 subtype C clones was created, and the isolates were assessed for phenotypic susceptibility to etravirine (ETR), rilpivirine (RPV), efavirenz (EFV), and nevirapine (NVP) in anin vitrosingle-cycle phenotypic assay. The amino acid substitutions E138Q/R, Y181I/V, and M230L conferred high-level resistance to ETR, while K101P and Y181I/V conferred high-level resistance to RPV. Y181C, a major NNRTI resistance-associated amino acid substitution, caused decreased susceptibility to ETR and, to a lesser extent, RPV when combined with other mutations. These included N348I and T369I, amino acid changes in the connection domain that are not generally assessed during resistance testing. However, the prevalence of these genotypes among subtype C sequences was, in most cases

Dried blood spots (DBS) are an alternative specimen type for HIV drug resistance genotyping in resource-limited settings. Data relating to the impact of DBS storage and shipment conditions on genotyping efficiency under field conditions are limited. We compared the genotyping efficiencies and resistance profiles of DBS stored and shipped at different temperatures to those of plasma specimens collected in parallel from patients receiving antiretroviral therapy in Uganda. Plasma and four DBS cards from anti-coagulated venous blood and a fifth card from finger-prick blood were prepared from 103 HIV patients with a median viral load (VL) of 57,062 copies/ml (range, 1,081 to 2,964,191). DBS were stored at ambient temperature for 2 or 4 weeks or frozen at −80°C and shipped from Uganda to the United States at ambient temperature or frozen on dry ice for genotyping using a broadly sensitive in-house method. Plasma (97.1%) and DBS (98.1%) stored and shipped frozen had similar genotyping efficiencies. DBS stored frozen (97.1%) or at ambient temperature for 2 weeks (93.2%) and shipped at ambient temperature also had similar genotyping efficiencies. Genotyping efficiency was reduced for DBS stored at ambient temperature for 4 weeks (89.3%, P = 0.03) or prepared from finger-prick blood and stored at ambient temperature for 2 weeks (77.7%, P < 0.001) compared to DBS prepared from venous blood and handled similarly. Resistance profiles were similar between plasma and DBS specimens. This report delineates the optimal DBS collection, storage, and shipping conditions and opens a new avenue for cost-saving ambient-temperature DBS specimen shipments for HIV drug resistance (HIVDR) surveillances in resource-limited settings.

Recent reports have shown that human immunodeficiency virus type 1 (HIV-1) Gag can directly affect susceptibility to protease inhibitors (PIs) in the absence of known resistance mutations in protease. Inclusion of co-evolved Gag alongside protease in phenotypic drug susceptibility assays can alter PI susceptibility in comparison with protease with a WT Gag. Using a single-replication-cycle assay encompassing full-length Gag together with protease we demonstrated significant variation in PI susceptibility between a number of PI-naïve subtype B viruses. Six publicly available subtype B molecular clones, namely HXB2, NL4-3, SF2, YU2, JRFL and 89.6, displayed up to nine-fold reduced PI susceptibility in comparison with the assay reference strain. For two molecular clones, YU2 and JRFL, Gag contributed solely to the observed reduction in susceptibility, with the N-terminal region of Gag contributing significantly. Gag and protease from treatment-naïve, patient-derived viruses also demonstrated significant variation in susceptibility, with up to a 17-fold reduction to atazanavir in comparison with the assay reference strain. In contrast to the molecular clones, protease was the main determinant of the reduced susceptibility. Common polymorphisms in protease, including I13V, L63P and A71T, were shown to contribute to this reduction in PI susceptibility, in the absence of major resistance mutations. This study demonstrated significant variation in PI susceptibility of treatment-naïve patient viruses, and provided further evidence of the independent role of Gag, the protease substrate and in particular the N-terminus of Gag in PI susceptibility. It also highlighted the importance of considering co-evolved Gag and protease when assessing PI susceptibility.

We investigated the effect of N348I alone and with M184V on nonnucleoside reverse transcriptase inhibitor (NNRTI) drug susceptibility and replicative capacity in B and non-B HIV-1 isolates. N348I reduced the susceptibility to all NNRTI drugs across subtypes. The replication capacity of all viruses in a variety of cell lines was impaired by N348I. Interestingly, the N348I and M184V double mutation compensated for the reduced NNRTI drug susceptibility observed in the N348I single mutant and marginally improved viral replicative capacity.

Other than cleavage site mutations, there is little data on specific positions within Gag that impact on HIV protease inhibitor susceptibility. We have recently shown that non-cleavage site mutations in gag , particularly within matrix protein can restore replication capacity and further reduce protease inhibitor drug susceptibility when coexpressed with a drug-resistant (mutant) protease. The matrix protein of this patient-derived virus was studied in order to identify specific changes responsible for this phenotype. Three amino acid changes in matrix (R76K, Y79F, and T81A) had an impact on replication capacity as well as drug susceptibility. Introduction of these three changes into wild-type (WT) matrix resulted in an increase in the replication capacity of the protease mutant virus to a level similar to that achieved by all the changes within the mutant matrix and part of the capsid protein. Pairs of changes to wild-type matrix led to an increased replication capacity of the protease mutant (although less than with all three changes). Having only these three changes to matrix in a wild-type virus (with wild-type protease) resulted in a 5- to 7-fold change in protease inhibitor 50% effective concentration (EC 50 ). Individual changes did not have as great an effect on replication capacity or drug susceptibility, demonstrating an interaction between these positions, also confirmed by sequence covariation analysis. Molecular modeling predicts that each of the three mutations would result in a loss of hydrogen bonds within α-helix-4 of matrix, leading to the hypothesis that more flexibility within this region or altered matrix structure would account for our findings.

Mutations can accumulate in the protease and gag genes of human immunodeficiency virus in patients who fail therapy with protease inhibitor drugs. Mutations within protease, the drug target, have been extensively studied. Mutations in gag have been less well studied, mostly concentrating on cleavage sites. A retroviral vector system has been adapted to study full-length gag , protease, and reverse transcriptase genes from patient-derived viruses. Patient plasma-derived mutant full-length gag , protease, and gag -protease from a multidrug-resistant virus were studied. Mutant protease alone led to a 95% drop in replication capacity that was completely rescued by coexpressing the full-length coevolved mutant gag gene. Cleavage site mutations have been shown to improve the replication capacity of mutated protease. Strikingly, in this study, the matrix region and part of the capsid region from the coevolved mutant gag gene were sufficient to achieve full recovery of replication capacity due to the mutant protease, without cleavage site mutations. The same region of gag from a second, unrelated, multidrug-resistant clinical isolate also rescued the replication capacity of the original mutant protease, suggesting a common mechanism that evolves with resistance to protease inhibitors. Mutant gag alone conferred reduced susceptibility to all protease inhibitors and acted synergistically when linked to mutant protease. The matrix region and partial capsid region of gag sufficient to rescue replication capacity also conferred resistance to protease inhibitors. Thus, the amino terminus of Gag has a previously unidentified and important function in protease inhibitor susceptibility and replication capacity.

HIV-1 strains use C-C-chemokine receptor 5, CCR5, as a coreceptor for host transmission. Human CCR5 chemokine ligands inhibit binding and infection, whereas CCR5 mutations also inhibit infection by preventing surface expression, resulting in delayed progression to AIDS. Here, we describe a human herpesvirus 6 (HHV-6A) chemokine, U83A, which binds CCR5 with higher affinity than human chemokines, displacing their binding and leading to inhibition of chemotaxis of human leukocytes. Similarly, U83A inhibits infection by HIV-1 strains which use CCR5, but not the CXCR4, coreceptor. Unlike human CCR5 chemokine ligands which induce rapid CCR5 internalization mediated via clathrin, treatment with U83A prevents internalization. A spliced truncated U83A isoform, U83A-N, also binds CCR5 albeit with lower affinity, and this correlates with lower HIV-1 infection inhibition, whereas further truncation abolishes binding and any inhibition. Confocal microscopy confirms CCR5 internalization inhibition by U83A treatment, whereas labeled transferrin uptake shows that endocytosis via clathrin is unaltered. Previous results show that, although U83A-N is an antagonist, U83A is an agonist for CCR1, CCR4, CCR6, and CCR8 present on immune effector and antigen-presenting cells and here also shown for CCR5. Thus, U83A could act as a novel inhibitor of HIV-1 infection while also stimulating local immunity to the virus.

Protease inhibitors (PIs) are used as a first-line regimen in HIV-1-infected children. Here we investigated the phenotypic consequences of amino acid changes in Gag and protease on lopinavir (LPV) and ritonavir (RTV) susceptibility among pediatric patients failing PI therapy. The Gag-protease from isolates from 20 HIV-1 subtype C-infected pediatric patients failing an LPV and/or RTV-based regimen was phenotyped using a nonreplicativein vitroassay. Changes in sensitivity to LPV and RTV relative to that of the matched baseline (pretherapy) sample were calculated. Gag and protease amino acid substitutions associated with PI failure were created in a reference clone by site-directed mutagenesis and assessed. Predicted phenotypes were determined using the Stanford drug resistance algorithm. Phenotypic resistance or reduced susceptibility to RTV and/or LPV was observed in isolates from 10 (50%) patients, all of whom had been treated with RTV. In most cases, this was associated with protease resistance mutations, but substitutions at Gag cleavage and noncleavage sites were also detected. Gag amino acid substitutions were also found in isolates from three patients with reduced drug susceptibilities who had wild-type protease. Site-directed mutagenesis confirmed that some amino acid changes in Gag contributed to PI resistance but only in the presence of major protease resistance-associated substitutions. The isolates from all patients who received LPV exclusively were phenotypically susceptible. Baseline isolates from the 20 patients showed a large (47-fold) range in the 50% effective concentration of LPV, which accounted for most of the discordance seen between the experimentally determined and the predicted phenotypes. Overall, the inclusion of thegaggene and the use of matched baseline samples provided a more comprehensive assessment of the effect of PI-induced amino acid changes on PI resistance. The lack of phenotypic resistance to LPV supports the continued use of this drug in pediatric patients.

Structured treatment interruption (STI) has been trialed as an alternative to lifelong antiretroviral therapy (ART). We retrospectively performed single genome sequencing of the HIV-1 pol region from three patients representing different scenarios. They were either failing on continuous therapy (CT-F), failing STI (STI-F), or suppressing on STI (STI-S). Over 460 genomes were generated from three to five different time points over a 2-year period. We found multiple-linked-resistant mutations in both treatment failures. However, the CT-F patient showed a stepwise accumulation of diverse, linked mutations whereas the STI-F patient had lineage turnover between treatment periods with recirculation of wild-type and resistant variants from reservoirs. The STI-F patient showed a 7-fold increase in the third codon position substitution rate relative to the first and second positions compared to a 2-fold increase for CT-F and increased purifying selection in the pol gene (62 vs. 22 sites, respectively). An understanding of intrapatient viral dynamics could guide the future direction of treatment interruption strategies.

OBJECTIVE To determine and compare the rates of HIV superinfection and primary HIV infection in high-risk female sex workers (FSWs) in Kampala, Uganda. DESIGN A retrospective analysis of individuals who participated in a clinical cohort study among high-risk FSWs in Kampala, Uganda. METHODS Plasma samples from HIV-infected FSWs in Kampala, Uganda were examined with next-generation sequencing of the p24 and gp41HIV genomic regions for the occurrence of superinfection. Primary HIV incidence was determined from initially HIV-uninfected FSWs from the same cohort, and incidence rate ratios were compared. RESULTS The rate of superinfection in these women (7/85; 3.4/100 person-years) was not significantly different from the rate of primary infection in the same population (3.7/100 person-years; incidence rate ratio = 0.91, P = 0.42). Seven women also entered the study dual-infected (16.5% either dual or superinfected). The women with any presence of dual infection were more likely to report sex work as their only source of income (P = 0.05), and trended to be older and more likely to be widowed (P = 0.07). CONCLUSIONS In this cohort of FSWs, HIV superinfection occurred at a high rate and was similar to that of primary HIV infection. These results differ from a similar study of high-risk female bar workers in Kenya that found the rate of superinfection to be significantly lower than the rate of primary HIV infection.