High viremia and low level of transmitted drug resistance in anti-retroviral therapy-naïve perinatally-infected children and adolescents with HIV-1 subtype C infection

BACKGROUND: High plasma viremia in HIV-1 infection is associated with rapid CD4 cell decline and faster disease progression. Children with HIV infection have high viral loads, particularly in early childhood. In this study we sought to understand the relationship between duration of HIV-1 infection and viral dynamics among perinatally-infected children and adolescents in India along with transmitted drug resistance in this population. METHODS: During 2007–2011, cross-sectional samples were collected from ART-naïve children (n = 105) with perinatally-acquired HIV infection, aged 2–16 years from Bangalore, India. CD4 counts, viral load and in-house genotyping were performed and transmitted drug resistance mutations were identified using the World Health Organization recommendations for Surveillance of Drug Resistance Mutations (SDRM_2009) list. RESULTS: Among 105 children studied, 73.3% (77/105) were asymptomatic, but had a median viral load of 5.24 log copies/mL (IQR 4.62-5.66). In the adolescent age group, 54% (21/39) had high levels of viremia (median 5.14 log copies/mL) but were asymptomatic. HIV-1 subtyping identified 98% strains (103/105) as subtype C; one A1 and one unique recombinant form (URF). Transmitted NRTI resistance was 1.9% (2/105); NNRTI resistance was 4.8% (5/105) and overall prevalence of transmitted drug resistance was 5.7% (6/105). CONCLUSIONS: The high burden of plasma viremia found among untreated asymptomatic adolescents needs to be addressed both from an individual angle to halt disease progression, and from a public health perspective to arrest horizontal transmission. The low level of transmitted drug resistance among perinatally-infected children is reassuring; however with improving ART access globally, regular genotyping surveillance is indicated

Co-receptor tropism prediction among 1045 Indian HIV-1 subtype C sequences: Therapeutic implications for India

<p>Abstract</p> <p><b>Background</b></p> <p>Understanding co-receptor tropism of HIV-1 strains circulating in India will provide key analytical leverage for assessing the potential usefulness of newer antiretroviral drugs such as chemokine co-receptor antagonists among Indian HIV-infected populations. The objective of this study was to determine using <it>in silico </it>methods, HIV-1 tropism among a large number of Indian isolates both from primary clinical isolates as well as from database-derived sequences.</p> <p>Results</p> <p>R5-tropism was seen in 96.8% of a total of 1045 HIV-1 subtype C Indian sequences. Co-receptor prediction of 15 primary clinical isolates detected two X4-tropic strains using the C-PSSM matrix. R5-tropic HIV-1 subtype C V3 sequences were conserved to a greater extent than X4-tropic strains. X4-tropic strains were obtained from subjects who had a significantly longer time since HIV diagnosis (96.5 months) compared to R5-tropic strains (20.5 months).</p> <p>Conclusions</p> <p>High prevalence of R5 tropism and greater homogeneity of the V3 sequence among HIV-1 subtype C strains in India suggests the potential benefit of CCR5 antagonists as a therapeutic option in India.</p

Human APOBEC3G-mediated hypermutation is associated with antiretroviral therapy failure in HIV-1 subtype C-infected individuals

Introduction: Human APOBEC3G/F (hA3G/F) restricts retroviral replication through G-to-A hypermutations, which can generate drug-resistant progenies in vitro. The clinical relevance is still inconclusive. To bridge this gap, we aim to study the role of these hypermutations in evolution of drug resistance; we characterised hA3G/F-mediated hypermutations in the RT region of the pol gene of patients with or without antiretroviral therapy (ART). Methods: In 88 HIV-1-positive individuals, drug resistance genotyping was carried out in plasma virus and provirus by population sequencing. Hypermutations were determined by three different approaches using Hypermut 2.0 software, cluster analysis and APOBEC3G-mediated defectives indices. Clinical and demographic characteristics of these individuals were studied in relation to these hypermutations. Results: hA3G/F-mediated hypermutated sequences in proviral DNA, but not in plasma virus, were identified in 11.4% (10/88) subjects. Proviral hypermutations were observed more frequently in patients with ART failure than in ART-na&#x00EF;ve individuals (p=0.03). In therapy failure patients, proviral hypermutation were associated with greater intra-compartmental genetic diversity (p&#60;0.001). In therapy-na&#x00EF;ve individuals, hypermutated proviral DNA with M184I and M230I mutations due to the editing of hA3G, had stop codons in the open reading frames and the same mutations were absent in the plasma virus. Only a limited concordance was found between the drug resistance mutations in plasma RNA and proviral DNA. Conclusions: hA3G lethal hypermutation was significantly associated with ART failure in Indian HIV-1 subtype C patients. It is unlikely that viral variants, which exhibit hypermutated sequences and M184I and/or M230I, will mature and expand in vivo

Genetic and functional analysis of HIV-1 Rev Responsive Element (RRE) sequences from North-India

HIV-1 Rev protein regulates the expression of HIV-1 transcripts by binding to a highly structured stem loop structure called the Rev Responsive Element (RRE) present in the genomic and partially spliced RNAs. Genetic variation in this structure is likely to affect binding of Rev protein and ultimately overall gene expression and replication. We characterized RRE sequences from 13 HIV-1 infected individuals from North India which also included two mother-child pairs following vertical transmission. We observed high degree of conservation of sequences, including the 9-nt (CACUAUGGG) long sequence in stem-loop B, required for efficient binding of Rev protein. All of our 13 RRE sequences possessed G to A (position 66) mutation located in the critical branched-stem-loop B which is not present in consensus C or B sequence. We derived a consensus RRE structure which showed interesting changes in the stem-loop structures including the stem-loop B. Mother-Child RRE sequences showed conservation of unique polymorphisms as well as some new mutations in child RRE sequences. Despite these changes, the ability to form multiple essential stem-loop structures required for Rev binding was conserved. RRE RNA derived from one of the samples, VT5, retained the ability to bind Rev protein under in vitro conditions although it showed alternate secondary structure. This is the first study from India describing the structural and possible functional implications due to very unique RRE sequence heterogeneity and its possible role in vertical transmission and gene expression

Phylogenetic Characterization of Six Full-Length HIV-1 Subtype C Molecular Clones from Three Patients: Identification of Rare Subtype C Strains Containing Two NF-κB Motifs in the Long Terminal Repeat

Molecular surveillance is the backbone of HIV-1 vaccinology. Full-length HIV-1 sequences are useful tools that can provide a better understanding of the epidemiology in a given region. A limited number of full-length HIV-1 sequences are available from India, where >95% of the HIV infections are due to HIV-1 subtype C (HIV-1C), which is distinct from the prototype African HIV-1C. In this study, we sequenced six full-length clones isolated from three patients. Extensive phylogenetic analyses of the full-length viral sequences using bioinformatic tools identified a separate cluster of Indian strains, thus confirming the distinct phylogenetic identity of the Indian HIV-1C. Notably, the long terminal repeat (LTR) of two of the six molecular clones contained only two NF-κB binding sites. The sequences also displayed features characteristic of HIV-1C including a Tat dicysteine motif, a shortened Rev open reading frame, and a predicted CCR5 coreceptor tropism for gp120 of three of the proviral sequences

Characterization of Inducible Transcription and Translation-Competent HIV-1 Using the RNAscope ISH Technology at a Single-Cell Resolution

Identifying the source and dynamics of persistent HIV-1 at single-cell resolution during cART is crucial for the design of strategies to eliminate the latent HIV-1 reservoir. An assay to measure latent HIV-1 that can distinguish inducible from defective proviruses with high precision is essential to evaluate the efficacy of HIV-1 cure efforts but is presently lacking. The primary aim of this study was therefore to identify transcription and translation competent latently infected cells through detection of biomolecules that are dependent on transcriptional activation of the provirus. We investigated the applicability of two commercially available assays; PrimeFlowTM RNA Assay (RNAflow) and RNAscope® ISH (RNAscope) for evaluation of the efficacy of latency reversal agents (LRAs) to reactivate the HIV-1 latent reservoir. The J-Lat cell model (clones 6.3, 9.3, and 10.6) and four LRAs was used to evaluate the sensitivity, specificity, and lower detection limit of the RNAflow and RNAscope assays for the detection and description of the translation-competent HIV-1 reservoir. We also checked for HIV-1 subtype specificity of the RNAscope assay using patient-derived subtype A1, B, C, and CRF01_AE recombinant plasmids following transfection in 293T cells and the applicability of the method in patient-derived peripheral blood mononuclear cells (PBMCs). The lower detection limit of RNAflow was 575 HIV-1 infected cells/million and 45 cells/million for RNAscope. The RNAscope probes, designed for HIV-1B, also detected other subtypes (A1, B, C, and CRF01_AE). RNAscope was applicable for the detection of HIV-1 in patient-derived PBMCs following LRA activation. In conclusion, our study showed that RNAscope can be used to quantify the number of directly observed individual cells expressing HIV-1 mRNA following LRA activation. Therefore, it can be a useful tool for characterization of translation-competent HIV-1 in latently infected cell at single-cell resolution in the fields of HIV-1 pathogenesis and viral persistence

Effectiveness of current anti-HIV regimen in low-and middle-income countries

Nevirapine (NVP) is a first-generation non-nucleoside reverse transcriptase inhibitor (NNRTI) of human immunodeficiency virus type 1 (HIV-1). However, with the emergence of resistance mutations due to a low genetic barrier under NVP pressure, new (second generation) NNRTIs have been approved. Rilpivirine (RPV), a second generation NNRTI, is not frequently used in low- and middle- income countries (LMICs) that bear the major HIV burden. RPV has been co-formulated with tenofovir (TDF) and emtricitabine (FTC) and has been recommended for patients with viral loads <100,000 copies/mL, inhibiting viruses that are resistant to NVP. It is now being considered in many LMICs. To understand RPV efficacy in HIV-1 subtypes prevalent in LMICs, we cloned RT genes from patients infected with four different HIV-1 subtypes: subtype B (HIV-1B), subtype C (HIV-1C), and recombinant forms CRF01_AE and CRF02_AG. HIV-1B is most prevalent in western countries and accounts for only ~12% of all infections. However, HIV-1C, which accounts for ~52% of all HIV infections, is most prevalent in LMICs. In vitro inhibition assays were performed with the four patient-derived RTs. Our results show that overall, NVP binds RTs with lower affinity than RPV, suggesting that NVP has lower effectiveness than RPV. However, NV binds 02_AG RT with better affinity than RPV. Hence, NVP may still be effective for patients infected with 02_AG. Furthermore, RPV binding affinity with HIV-1C is lower than other subtypes. This result is consistent with clinical results, showing less efficacy of RPV among HIV-1C infected patients