HIV reservoirs are dominated by genetically younger and clonally enriched proviruses.

IMPORTANCE: Characterizing the human immunodeficiency virus (HIV) reservoir that endures despite antiretroviral therapy (ART) is critical to cure efforts. We observed that the oldest proviruses persisting during ART were exclusively defective, while intact proviruses (and rebound HIV) dated to nearer ART initiation. This helps explain why studies that sampled sub-genomic proviruses on-ART (which are largely defective) routinely found sequences dating to early infection, whereas those that sampled replication-competent HIV found almost none. Together with our findings that intact proviruses were more likely to be clonal, and that on-ART low-level/isolated viremia originated from proviruses of varying ages (including possibly defective ones), our observations indicate that (i) on-ART and rebound viremia can have distinct within-host origins, (ii) intact proviruses have shorter lifespans than grossly defective ones and thus depend more heavily on clonal expansion for persistence, and (iii) an HIV reservoir predominantly "dating" to near ART initiation will be substantially adapted to within-host pressures, complicating immune-based cure strategies.

HIV reservoirs are dominated by genetically younger and clonally enriched proviruses.

In order to cure HIV, we need to better understand the within-host evolutionary origins of the small reservoir of genome-intact proviruses that persists within infected cells during antiretroviral therapy (ART). Most prior studies on reservoir evolutionary dynamics however did not discriminate genome-intact proviruses from the vast background of defective ones. We reconstructed within-host pre-ART HIV evolutionary histories in six individuals and leveraged this information to infer the ages of intact and defective proviruses sampled after an average >9 years on ART, along with the ages of rebound and low-level/isolated viremia occurring during this time. We observed that the longest-lived proviruses persisting on ART were exclusively defective, usually due to large deletions. In contrast, intact proviruses and rebound HIV exclusively dated to the years immediately preceding ART. These observations are consistent with genome-intact proviruses having shorter lifespans, likely due to the cumulative risk of elimination following viral reactivation and protein production. Consistent with this, intact proviruses (and those with packaging signal defects) were three times more likely to be genetically identical compared to other proviral types, highlighting clonal expansion as particularly important in ensuring their survival. By contrast, low-level/isolated viremia sequences were genetically heterogeneous and sometimes ancestral, where viremia may have originated from defective proviruses. Results reveal that the HIV reservoir is dominated by clonally-enriched and genetically younger sequences that date to the untreated infection period when viral populations had been under within-host selection pressures for the longest duration. Knowledge of these qualities may help focus strategies for reservoir elimination.

Serial infection with SARS-CoV-2 Omicron BA.1 and BA.2 following three-dose COVID-19 vaccination.

SARS-CoV-2 Omicron infections are common among individuals who are vaccinated or have recovered from prior variant infection, but few reports have immunologically assessed serial Omicron infections. We characterized SARS-CoV-2 humoral responses in an individual who acquired laboratory-confirmed Omicron BA.1.15 ten weeks after a third dose of BNT162b2, and BA.2 thirteen weeks later. Responses were compared to 124 COVID-19-naive vaccinees. One month post-second and -third vaccine doses, the participant's wild-type and BA.1-specific IgG, ACE2-displacement and virus neutralization activities were average for a COVID-19-naive triple-vaccinated individual. BA.1 infection boosted the participant's responses to the cohort ≥95th percentile, but even this strong "hybrid" immunity failed to protect against BA.2. Reinfection increased BA.1 and BA.2-specific responses only modestly. Though vaccines clearly protect against severe disease, results highlight the continued importance of maintaining additional protective measures to counteract the immune-evasive Omicron variant, particularly as vaccine-induced immune responses naturally decline over time.

Molecular Epidemiology of HIV-1 in Ghana: Subtype Distribution, Drug Resistance and Coreceptor Usage.

The greatest HIV-1 genetic diversity is found in West/Central Africa due to the pandemic's origins in this region, but this diversity remains understudied. We characterized HIV-1 subtype diversity (from both sub-genomic and full-genome viral sequences), drug resistance and coreceptor usage in 103 predominantly (90%) antiretroviral-naive individuals living with HIV-1 in Ghana. Full-genome HIV-1 subtyping confirmed the circulating recombinant form CRF02_AG as the dominant (53.9%) subtype in the region, with the complex recombinant 06_cpx (4%) present as well. Unique recombinants, most of which were mosaics containing CRF02_AG and/or 06_cpx, made up 37% of sequences, while "pure" subtypes were rare (<6%). Pretreatment resistance to at least one drug class was observed in 17% of the cohort, with NNRTI resistance being the most common (12%) and INSTI resistance being relatively rare (2%). CXCR4-using HIV-1 sequences were identified in 23% of participants. Overall, our findings advance our understanding of HIV-1 molecular epidemiology in Ghana. Extensive HIV-1 genetic diversity in the region appears to be fueling the ongoing creation of novel recombinants, the majority CRF02_AG-containing, in the region. The relatively high prevalence of pretreatment NNRTI resistance but low prevalence of INSTI resistance supports the use of INSTI-based first-line regimens in Ghana.

Validation of a Genotype-Independent Hepatitis C Virus Near-Whole Genome Sequencing Assay.

Despite the effectiveness of direct-acting antiviral agents in treating hepatitis C virus (HCV), cases of treatment failure have been associated with the emergence of resistance-associated substitutions. To better guide clinical decision-making, we developed and validated a near-whole-genome HCV genotype-independent next-generation sequencing strategy. HCV genotype 1-6 samples from direct-acting antiviral agent treatment-naïve and -treated HCV-infected individuals were included. Viral RNA was extracted using a NucliSens easyMAG and amplified using nested reverse transcription-polymerase chain reaction. Libraries were prepared using Nextera XT and sequenced on the Illumina MiSeq sequencing platform. Data were processed by an in-house pipeline (MiCall). Nucleotide consensus sequences were aligned to reference strain sequences for resistance-associated substitution identification and compared to NS3, NS5a, and NS5b sequence data obtained from a validated in-house assay optimized for HCV genotype 1. Sequencing success rates (defined as achieving >100-fold read coverage) approaching 90% were observed for most genotypes in samples with a viral load >5 log10 IU/mL. This genotype-independent sequencing method resulted in >99.8% nucleotide concordance with the genotype 1-optimized method, and 100% agreement in genotype assignment with paired line probe assay-based genotypes. The assay demonstrated high intra-run repeatability and inter-run reproducibility at detecting substitutions above 2% prevalence. This study highlights the performance of a freely available laboratory and bioinformatic approach for reliable HCV genotyping and resistance-associated substitution detection regardless of genotype.

HIV-1 diversity considerations in the application of the Intact Proviral DNA Assay (IPDA).

The Intact Proviral DNA Assay (IPDA) was developed to address the critical need for a scalable method for intact HIV-1 reservoir quantification. This droplet digital PCR-based assay simultaneously targets two HIV-1 regions to distinguish genomically intact proviruses against a large background of defective ones, and its application has yielded insights into HIV-1 persistence. Reports of assay failures however, attributed to HIV-1 polymorphism, have recently emerged. Here, we describe a diverse North American cohort of people with HIV-1 subtype B, where the IPDA yielded a failure rate of 28% due to viral polymorphism. We further demonstrate that within-host HIV-1 diversity can lead the IPDA to underestimate intact reservoir size, and provide examples of how this phenomenon could lead to erroneous interpretation of clinical trial data. While the IPDA represents a major methodological advance, HIV-1 diversity should be addressed before its widespread adoption as a principal readout in HIV-1 remission trials.

Performance comparison of next generation sequencing analysis pipelines for HIV-1 drug resistance testing.

Next generation sequencing (NGS) is a trending new standard for genotypic HIV-1 drug resistance (HIVDR) testing. Many NGS HIVDR data analysis pipelines have been independently developed, each with variable outputs and data management protocols. Standardization of such analytical methods and comparison of available pipelines are lacking, yet may impact subsequent HIVDR interpretation and other downstream applications. Here we compared the performance of five NGS HIVDR pipelines using proficiency panel samples from NIAID Virology Quality Assurance (VQA) program. Ten VQA panel specimens were genotyped by each of six international laboratories using their own in-house NGS assays. Raw NGS data were then processed using each of the five different pipelines including HyDRA, MiCall, PASeq, Hivmmer and DEEPGEN. All pipelines detected amino acid variants (AAVs) at full range of frequencies (1~100%) and demonstrated good linearity as compared to the reference frequency values. While the sensitivity in detecting low abundance AAVs, with frequencies between 1~20%, is less a concern for all pipelines, their specificity dramatically decreased at AAV frequencies <2%, suggesting that 2% threshold may be a more reliable reporting threshold for ensured specificity in AAV calling and reporting. More variations were observed among the pipelines when low abundance AAVs are concerned, likely due to differences in their NGS read quality control strategies. Findings from this study highlight the need for standardized strategies for NGS HIVDR data analysis, especially for the detection of minority HIVDR variants.