Variable contexts and levels of hypermutation in HIV-1 proviral genomes recovered from primary peripheral blood mononuclear cells

APOBEC-mediated cytidine cleamination of HIV-1 genomes during reverse transcription has been shown to be a potent mechanism of host restriction for HIV-1 infection ex vivo and in vitro. However, this defense system can be overcome by the viral protein Vif. Unlike other mechanisms of host restriction, the APOCEC-Vif interaction leaves an imprint on integrated proviruses in the form of G-A hypermutation. in the current work we systematically studied levels, contexts, and patterns of HIV-1 hypermutation in vivo. the analysis of 24 full-genome HIV-1 sequences retrieved from primary PBMCs, representing infections with several HIV-1 clades, and the inclusion of 7 cognate pairs of hypermutated/non-hypermutated sequences derived from the same patient sample, provided a comprehensive view of the characteristics of APOBEC-mediated restriction in vivo. Levels of hypermutation varied nearly 5-fold among the studied proviruses. GpG motifs were most frequently affected (22/24 proviruses). Levels of hypermutation varied across the genome. the reported twin peak pattern of hypermutation was observed in 18/24 hypermutants, but the remainder exhibited singular non-conforming patterns. These data suggest considerable complexity in the interplay of host restriction and viral defense during HIV-1 infection. (c) 2008 Elsevier Inc. All rights reserved.Henry M Jackson Fdn Advancement Mil Med, US Mil HIV Res Program, Rockville, MD 20850 USAUniversidade Federal de São Paulo, Paulista Sch Med, Div Infect Dis, BR-04039 São Paulo, BrazilWalter Reed Army Inst Res, Div Retrovirol, Rockville, MD 20850 USAUniversidade Federal de São Paulo, Paulista Sch Med, Div Infect Dis, BR-04039 São Paulo, BrazilWeb of Scienc

Analysis of SARS-CoV-2 Emergent Variants Following AZD7442 (Tixagevimab/Cilgavimab) for Early Outpatient Treatment of COVID-19 (TACKLE Trial)

Introduction: AZD7442 (tixagevimab/cilgavimab) comprises neutralising monoclonal antibodies (mAbs) that bind to distinct non-overlapping epitopes on the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. Viral evolution during mAb therapy can select for variants with reduced neutralisation susceptibility. We examined treatment-emergent SARS-CoV-2 variants during TACKLE (NCT04723394), a phase 3 study of AZD7442 for early outpatient treatment of coronavirus disease 2019 (COVID-19). // Methods: Non-hospitalised adults with mild-to-moderate COVID-19 were randomised and dosed ≤ 7 days from symptom onset with AZD7442 (n = 452) or placebo (n = 451). Next-generation sequencing of the spike gene was performed on SARS-CoV-2 reverse-transcription polymerase chain reaction-positive nasopharyngeal swabs at baseline and study days 3, 6, and 15 post dosing. SARS-CoV-2 lineages were assigned using spike nucleotide sequences. Amino acid substitutions were analysed at allele fractions (AF; % of sequence reads represented by substitution) ≥ 25% and 3% to 25%. In vitro susceptibility to tixagevimab, cilgavimab, and AZD7442 was evaluated for all identified treatment-emergent variants using a pseudotyped microneutralisation assay. // Results: Longitudinal spike sequences were available for 461 participants (AZD7442, n = 235; placebo, n = 226) and showed that treatment-emergent variants at any time were rare, with 5 (2.1%) AZD7442 participants presenting ≥ 1 substitution in tixagevimab/cilgavimab binding sites at AF ≥ 25%. At AF 3% to 25%, treatment-emergent variants were observed in 15 (6.4%) AZD7442 and 12 (5.3%) placebo participants. All treatment-emergent variants showed in vitro susceptibility to AZD7442. // Conclusion: These data indicate that AZD7442 creates a high genetic barrier for resistance and is a feasible option for COVID-19 treatment

Next-Generation Sequencing of HIV-1 Single Genome Amplicons

The analysis of HIV-1 sequences has helped understand the viral molecular epidemiology, monitor the development of antiretroviral drug resistance, and design candidate vaccines. The introduction of single genome amplification (SGA) has been a major advancement in the field, allowing for the characterization of multiple sequences per patient while preserving linkage among polymorphisms in the same viral genome copy. Sequencing of SGA amplicons is performed by capillary Sanger sequencing, which presents low throughput, requires a high amount of template, and is highly sensitive to template/primer mismatching. In order to meet the increasing demand for HIV-1 SGA amplicon sequencing, we have developed a platform based on benchtop next-generation sequencing (NGS) (IonTorrent) accompanied by a bioinformatics pipeline capable of running on computer resources commonly available at research laboratories. During assay validation, the NGS-based sequencing of 10 HIV-1 env SGA amplicons was fully concordant with Sanger sequencing. The field test was conducted on plasma samples from 10 US Navy and Marine service members with recent HIV-1 infection (sampling interval: 2005-2010; plasma viral load: 5,884-194,984 copies/ml). The NGS analysis of 101 SGA amplicons (median: 10 amplicons/individual) showed within-individual viral sequence profiles expected in individuals at this disease stage, including individuals with highly homogeneous quasispecies, individuals with two highly homogeneous viral lineages, and individuals with heterogeneous viral populations. In a scalability assessment using the Ion Chef automated system, 41/43 tested env SGA amplicons (95%) multiplexed on a single Ion 318 chip showed consistent gene-wide coverage \u3e50×. With lower sample requirements and higher throughput, this approach is suitable to support the increasing demand for high-quality and cost-effective HIV-1 sequences in fields such as molecular epidemiology, and development of preventive and therapeutic strategies

High-Throughput High-Resolution Class I HLA Genotyping in East Africa

HLA, the most genetically diverse loci in the human genome, play a crucial role in host-pathogen interaction by mediating innate and adaptive cellular immune responses. A vast number of infectious diseases affect East Africa, including HIV/AIDS, malaria, and tuberculosis, but the HLA genetic diversity in this region remains incompletely described. This is a major obstacle for the design and evaluation of preventive vaccines. Available HLA typing techniques, that provide the 4-digit level resolution needed to interpret immune responses, lack sufficient throughput for large immunoepidemiological studies. Here we present a novel HLA typing assay bridging the gap between high resolution and high throughput. The assay is based on real-time PCR using sequence-specific primers (SSP) and can genotype carriers of the 49 most common East African class I HLA-A, -B, and -C alleles, at the 4-digit level. Using a validation panel of 175 samples from Kampala, Uganda, previously defined by sequence-based typing, the new assay performed with 100% sensitivity and specificity. The assay was also implemented to define the HLA genetic complexity of a previously uncharacterized Tanzanian population, demonstrating its inclusion in the major East African genetic cluster. The availability of genotyping tools with this capacity will be extremely useful in the identification of correlates of immune protection and the evaluation of candidate vaccine efficacy

The Role of Natural Killer (NK) Cells and NK Cell Receptor Polymorphisms in the Assessment of HIV-1 Neutralization

The importance of innate immune cells in HIV-1 pathogenesis and protection has been highlighted by the role of natural killer (NK) cells in the containment of viral replication. Use of peripheral blood mononuclear cells (PBMC) in immunologic studies provides both HIV-1 target cells (ie. CD4+ T cells), as well as anti-HIV-1 effector cells, such as NK cells. In this study, NK and other immune cell populations were analyzed in HIV-negative donor PBMC for an impact on the anti-HIV activity of polyclonal and monoclonal antibodies. NK cell percentages were significantly higher in donor PBMC that supported lower levels of viral replication. While the percentage of NK cells was not directly associated with neutralization titers, NK cell-depletion significantly diminished the antiviral antibody activity by up to three logs, and polymorphisms in NK killer immunoglobulin receptor (KIR) and FcγRIIIa alleles appear to be associated with this affect. These findings demonstrate that NK cells and NK cell receptor polymorphisms may influence assessment of traditional HIV-1 neutralization in a platform where antibody is continuously present. This format appears to simultaneously assess conventional entry inhibition (neutralization) and non-neutralizing antibody-dependent HIV inhibition, which may provide the opportunity to delineate the dominant antibody function(s) in polyclonal vaccine responses

High Affinity Allele for the Gene of FCGR3A Is Risk Factor for HIV Infection and Progression

Background: We investigated the genetics of Fc receptors, which function as activating receptors on immune cells and help to control HIV through antibody-mediated cellular cytotoxicity. Thus, Fc receptors may be important for virus immunity but might also promote immune hyperactivation that would enhance infection. Methodology/Principal Findings: We measured abundance of low and high activity alleles in two Fc receptor genes, FCGR2A and FCGR3A, for persons with HIV disease, natural virus suppressors (HIV+, without disease) and healthy controls to show whether genotypes were associated with infection and disease. Individuals homozygous for the high activity allele of FCGR3A (158VV) were predominantly found among HIV progressors and this group was also skewed toward higher allele frequencies for the V158 variant. Both of the HIV positive groups (progressors and natural virus suppressors) had significantly higher frequencies of the V158 allele compared with uninfected controls. There were no apparent associations among FCGR2A alleles and HIV status. Conclusions/Significance: Our results indicate that high activity alleles of FCGR3A may be risk factors for HIV infection or progression and we need to understand how allelic variants affect the balance between virus control and immune activation

Association of SNPs rs1801274 (FCGR2A) and rs396991 (FCGR3A) with HIV status.

<p>Significant differences related to HIV status are shown. For FCGR3A, carriage of V allele was associated with increased risk of HIV seropositivity. Among HIV sero-positive cases, there was a significant association between VV genotype (i.e., lack of allele F) and the progressor phenotype. For the FCGR2A∶FCGR3A extended haplotype, the RR∶FF genotype was associated with disease progression.</p><p>Fisher's exact test (2-tailed) p values and Odds ratios are listed where significant.</p><p>*None of the NVS donors carried the F allele, so odds ratio were not calculated.</p><p>#Include Progressors and NVS.</p><p>**Not significant.</p

Genotypic and allelic frequencies of SNP rs1801274 (FCGR2A) and rs396991 (FCGR3A) in HIV negative controls, NVS and HIV Progressors.

<p>Abbreviations: SNP, Single Nucleotide Polymorphism; NVS, Natural Virus Suppressors.</p><p>FCGR genotypic variants (FCGR2A H131R and FCGR3A F158V) were studied in HIV progressors, NVS as well as HIV negative controls. Genotypic and allelic frequencies of rs396991 and rs1801274 are shown. The genotypic frequencies of the HIV sero-negative patients are in Hardy-Weinberg equillibrium.</p