The replication-competent HIV reservoir is a genetically restricted, younger subset of the overall pool of HIV proviruses persisting during therapy, which is highly genetically stable over time.

Within-host HIV populations continually diversify during untreated infection, and this diversity persists within infected cell reservoirs during antiretroviral therapy (ART). Achieving a better understanding of on-ART proviral evolutionary dynamics, and a better appreciation of how the overall persisting pool of (largely genetically defective) proviruses differs from the much smaller replication-competent HIV reservoir, is critical to HIV cure efforts. We reconstructed within-host HIV evolutionary histories in blood from seven participants of the Women's Interagency HIV Study who experienced HIV seroconversion, and used these data to characterize the diversity, lineage origins, and ages of proviral env-gp120 sequences sampled longitudinally up to 12 years on ART. We also studied HIV sequences emerging from the reservoir in two participants. We observed that proviral clonality generally increased over time on ART, with clones frequently persisting long term. While on-ART proviral integration dates generally spanned the duration of untreated infection, HIV emerging in plasma was exclusively younger (i.e., dated to the years immediately pre-ART). The genetic and age distributions of distinct proviral sequences remained stable during ART in all but one participant, in whom there was evidence that younger proviruses had been preferentially eliminated after 12 years on ART. Analysis of the gag region in three participants corroborated our env-gp120-based observations, indicating that our observations are not influenced by the HIV region studied. Our results underscore the remarkable genetic stability of the distinct proviral sequences that persist in blood during ART. Our results also suggest that the replication-competent HIV reservoir is a genetically restricted, younger subset of this overall proviral pool.IMPORTANCECharacterizing the genetically diverse HIV sequences that persist in the reservoir despite antiretroviral therapy (ART) is critical to cure efforts. Our observations confirm that proviruses persisting in blood on ART, which are largely genetically defective, broadly reflect the extent of within-host HIV evolution pre-ART. Moreover, on-ART clonal expansion is not appreciably accompanied by the loss of distinct proviral lineages. In fact, on-ART proviral genetic composition remained stable in all but one participant, in whom, after 12 years on ART, proviruses dating to around near ART initiation had been preferentially eliminated. We also identified recombinant proviruses between parental sequence fragments of different ages. Though rare, such sequences suggest that reservoir cells can be superinfected with HIV from another infection era. Overall, our finding that the replication-competent reservoir in blood is a genetically restricted, younger subset of all persisting proviruses suggests that HIV cure strategies will need to eliminate a reservoir that differs in key respects from the overall proviral pool.

Differential Vpu-Mediated CD4 and Tetherin Downregulation Functions among Major HIV-1 Group M Subtypes.

Downregulation of BST-2/tetherin and CD4 by HIV-1 viral protein U (Vpu) promotes viral egress and allows infected cells to evade host immunity. Little is known however about the natural variability in these Vpu functions among the genetically diverse viral subtypes that contribute to the HIV-1 pandemic. We collected Vpu isolates from 332 treatment-naive individuals living with chronic HIV-1 infection in Uganda, Rwanda, South Africa, and Canada. Together, these Vpu isolates represent four major HIV-1 group M subtypes (A [n = 63], B [n = 84], C [n = 94], and D [n = 59]) plus intersubtype recombinants and uncommon strains (n = 32). The ability of each Vpu clone to downregulate endogenous CD4 and tetherin was quantified using flow cytometry following transfection into an immortalized T-cell line and compared to that of a reference Vpu clone derived from HIV-1 subtype B NL4.3. Overall, the median CD4 downregulation function of natural Vpu isolates was similar to that of NL4.3 (1.01 [interquartile range {IQR}, 0.86 to 1.18]), while the median tetherin downregulation function was moderately lower than that of NL4.3 (0.90 [0.79 to 0.97]). Both Vpu functions varied significantly among HIV-1 subtypes (Kruskal-Wallis P < 0.0001). Specifically, subtype C clones exhibited the lowest CD4 and tetherin downregulation activities, while subtype D and B clones were most functional for both activities. We also identified Vpu polymorphisms associated with CD4 or tetherin downregulation function and validated six of these using site-directed mutagenesis. Our results highlight the marked extent to which Vpu function varies among global HIV-1 strains, raising the possibility that natural variation in this accessory protein may contribute to viral pathogenesis and/or spread.IMPORTANCE The HIV-1 accessory protein Vpu enhances viral spread by downregulating CD4 and BST-2/tetherin on the surface of infected cells. Natural variability in these Vpu functions may contribute to HIV-1 pathogenesis, but this has not been investigated among the diverse viral subtypes that contribute to the HIV-1 pandemic. In this study, we found that Vpu function differs significantly among HIV-1 subtypes A, B, C, and D. On average, subtype C clones displayed the lowest ability to downregulate both CD4 and tetherin, while subtype B and D clones were more functional. We also identified Vpu polymorphisms that associate with functional differences among HIV-1 isolates and subtypes. Our study suggests that genetic diversity in Vpu may play an important role in the differential pathogenesis and/or spread of HIV-1.

Genotypic and Mechanistic Characterization of Subtype-Specific HIV Adaptation to Host Cellular Immunity.

The extent to which viral genetic context influences HIV adaptation to human leukocyte antigen (HLA) class I-restricted immune pressures remains incompletely understood. The Ugandan HIV epidemic, where major pandemic group M subtypes A1 and D cocirculate in a single host population, provides an opportunity to investigate this question. We characterized plasma HIV RNA gag, pol, and nef sequences, along with host HLA genotypes, in 464 antiretroviral-naive individuals chronically infected with HIV subtype A1 or D. Using phylogenetically informed statistical approaches, we identified HLA-associated polymorphisms and formally compared their strengths of selection between viral subtypes. A substantial number (32%) of HLA-associated polymorphisms identified in subtype A1 and/or D had previously been reported in subtype B, C, and/or circulating recombinant form 01_AE (CRF01_AE), confirming the shared nature of many HLA-driven escape pathways regardless of viral genetic context. Nevertheless, 34% of the identified HLA-associated polymorphisms were significantly differentially selected between subtypes A1 and D. Experimental investigation of select examples of subtype-specific escape revealed distinct underlying mechanisms with important implications for vaccine design: whereas some were attributable to subtype-specific sequence variation that influenced epitope-HLA binding, others were attributable to differential mutational barriers to immune escape. Overall, our results confirm that HIV genetic context is a key modulator of viral adaptation to host cellular immunity and highlight the power of combined bioinformatic and mechanistic studies, paired with knowledge of epitope immunogenicity, to identify appropriate viral regions for inclusion in subtype-specific and universal HIV vaccine strategies.IMPORTANCE The identification of HIV polymorphisms reproducibly selected under pressure by specific HLA alleles and the elucidation of their impact on viral function can help identify immunogenic viral regions where immune escape incurs a fitness cost. However, our knowledge of HLA-driven escape pathways and their functional costs is largely limited to HIV subtype B and, to a lesser extent, subtype C. Our study represents the first characterization of HLA-driven adaptation pathways in HIV subtypes A1 and D, which dominate in East Africa, and the first statistically rigorous characterization of differential HLA-driven escape across viral subtypes. The results support a considerable impact of viral genetic context on HIV adaptation to host HLA, where HIV subtype-specific sequence variation influences both epitope-HLA binding and the fitness costs of escape. Integrated bioinformatic and mechanistic characterization of these and other instances of differential escape could aid rational cytotoxic T-lymphocyte-based vaccine immunogen selection for both subtype-specific and universal HIV vaccines.

Preclinical Profile of AB-423, an Inhibitor of Hepatitis B Virus Pregenomic RNA Encapsidation.

AB-423 is a member of the sulfamoylbenzamide (SBA) class of hepatitis B virus (HBV) capsid inhibitors in phase 1 clinical trials. In cell culture models, AB-423 showed potent inhibition of HBV replication (50% effective concentration [EC50] = 0.08 to 0.27 µM; EC90 = 0.33 to 1.32 µM) with no significant cytotoxicity (50% cytotoxic concentration > 10 µM). Addition of 40% human serum resulted in a 5-fold increase in the EC50s. AB-423 inhibited HBV genotypes A through D and nucleos(t)ide-resistant variants in vitro Treatment of HepDES19 cells with AB-423 resulted in capsid particles devoid of encapsidated pregenomic RNA and relaxed circular DNA (rcDNA), indicating that it is a class II capsid inhibitor. In a de novo infection model, AB-423 prevented the conversion of encapsidated rcDNA to covalently closed circular DNA, presumably by interfering with the capsid uncoating process. Molecular docking of AB-423 into crystal structures of heteroaryldihydropyrimidines and an SBA and biochemical studies suggest that AB-423 likely also binds to the dimer-dimer interface of core protein. In vitro dual combination studies with AB-423 and anti-HBV agents, such as nucleos(t)ide analogs, RNA interference agents, or interferon alpha, resulted in additive to synergistic antiviral activity. Pharmacokinetic studies with AB-423 in CD-1 mice showed significant systemic exposures and higher levels of accumulation in the liver. A 7-day twice-daily administration of AB-423 in a hydrodynamic injection mouse model of HBV infection resulted in a dose-dependent reduction in serum HBV DNA levels, and combination with entecavir or ARB-1467 resulted in a trend toward antiviral activity greater than that of either agent alone, consistent with the results of the in vitro combination studies. The overall preclinical profile of AB-423 supports its further evaluation for safety, pharmacokinetics, and antiviral activity in patients with chronic hepatitis B.

Novel Acylguanidine-Based Inhibitor of HIV-1.

UNLABELLED: The emergence of transmissible HIV-1 strains with resistance to antiretroviral drugs highlights a continual need for new therapies. Here we describe a novel acylguanidine-containing compound, 1-(2-(azepan-1-yl)nicotinoyl)guanidine (or SM111), that inhibits in vitro replication of HIV-1, including strains resistant to licensed protease, reverse transcriptase, and integrase inhibitors, without major cellular toxicity. At inhibitory concentrations, intracellular p24(Gag) production was unaffected, but virion release (measured as extracellular p24(Gag)) was reduced and virion infectivity was substantially impaired, suggesting that SM111 acts at a late stage of viral replication. SM111-mediated inhibition of HIV-1 was partially overcome by a Vpu I17R mutation alone or a Vpu W22* truncation in combination with Env N136Y. These mutations enhanced virion infectivity and Env expression on the surface of infected cells in the absence and presence of SM111 but also impaired Vpu's ability to downregulate CD4 and BST2/tetherin. Taken together, our results support acylguanidines as a class of HIV-1 inhibitors with a distinct mechanism of action compared to that of licensed antiretrovirals. Further research on SM111 and similar compounds may help to elucidate knowledge gaps related to Vpu's role in promoting viral egress and infectivity. IMPORTANCE: New inhibitors of HIV-1 replication may be useful as therapeutics to counteract drug resistance and as reagents to perform more detailed studies of viral pathogenesis. SM111 is a small molecule that blocks the replication of wild-type and drug-resistant HIV-1 strains by impairing viral release and substantially reducing virion infectivity, most likely through its ability to prevent Env expression at the infected cell surface. Partial resistance to SM111 is mediated by mutations in Vpu and/or Env, suggesting that the compound affects host/viral protein interactions that are important during viral egress. Further characterization of SM111 and similar compounds may allow more detailed pharmacological studies of HIV-1 egress and provide opportunities to develop new treatments for HIV-1.

SARS-CoV-2 RNA Quantification Using Droplet Digital RT-PCR.

Quantitative viral load assays have transformed our understanding of viral diseases. They hold similar potential to advance COVID-19 control and prevention, but SARS-CoV-2 viral load tests are not yet widely available. SARS-CoV-2 molecular diagnostic tests, which typically employ real-time RT-PCR, yield semiquantitative results only. Droplet digital RT-PCR (RT-ddPCR) offers an attractive platform for SARS-CoV-2 RNA quantification. Eight primer/probe sets originally developed for real-time RT-PCR-based SARS-CoV-2 diagnostic tests were evaluated for use in RT-ddPCR; three were identified as the most efficient, precise, and sensitive for RT-ddPCR-based SARS-CoV-2 RNA quantification. For example, the analytical efficiency for the E-Sarbeco primer/probe set was approximately 83%, whereas assay precision, measured as the coefficient of variation, was approximately 2% at 1000 input copies/reaction. Lower limits of quantification and detection for this primer/probe set were 18.6 and 4.4 input SARS-CoV-2 RNA copies/reaction, respectively. SARS-CoV-2 RNA viral loads in a convenience panel of 48 COVID-19-positive diagnostic specimens spanned a 6.2log10 range, confirming substantial viral load variation in vivo. RT-ddPCR-derived SARS-CoV-2 E gene copy numbers were further calibrated against cycle threshold values from a commercial real-time RT-PCR diagnostic platform. This log-linear relationship can be used to mathematically derive SARS-CoV-2 RNA copy numbers from cycle threshold values, allowing the wealth of available diagnostic test data to be harnessed to address foundational questions in SARS-CoV-2 biology.

ARB-1740, a RNA Interference Therapeutic for Chronic Hepatitis B Infection.

Current approved nucleoside analogue treatments for chronic hepatitis B virus (HBV) infection are effective at controlling viral titer but are not curative and have minimal impact on the production of viral proteins such as surface antigen (HBsAg), the HBV envelope protein believed to play a role in maintaining the immune tolerant state required for viral persistence. Novel agents are needed to effect HBV cure, and reduction of HBV antigenemia may potentiate activation of effective and long-lasting host immune control. ARB-1740 is a clinical stage RNA interference agent composed of three siRNAs delivered using lipid nanoparticle technology. In a number of cell and animal models of HBV, ARB-1740 caused HBV RNA reduction, leading to inhibition of multiple elements of the viral life cycle including HBsAg, HBeAg, and HBcAg viral proteins as well as replication marker HBV DNA. ARB-1740 demonstrated pan-genotypic activity in vitro and in vivo, targeting three distinct highly conserved regions of the HBV genome, and effectively inhibited replication of nucleoside analogue-resistant HBV variants. Combination of ARB-1740 with a capsid inhibitor and pegylated interferon-alpha led to greater liver HBsAg reduction which correlated with more robust induction of innate immune responses in a human chimeric mouse model of HBV. The preclinical profile of ARB-1740 demonstrates the promise of RNA interference and HBV antigen reduction in treatment strategies driving toward a cure for HBV.

Extensive host immune adaptation in a concentrated North American HIV epidemic.

OBJECTIVE: HIV incidence in the Canadian province of Saskatchewan, where Indigenous persons make up 80% of those infected, are among the highest on the continent. Reports of accelerated HIV progression, associated with carriage of certain human leukocyte antigen (HLA) alleles (including the typically protective HLA-B*51) have also emerged from the region. Given that acquisition of HIV preadapted to host HLA negatively impacts clinical outcome, we hypothesized that HIV-host adaptation may be elevated in Saskatchewan. DESIGN: Comparative analysis of population-level HIV sequence datasets from Saskatchewan and elsewhere in Canada/USA. METHODS: We analyzed 1144 HIV subtype B Pol sequences collected in Saskatchewan between 2000 and 2016, comprising ∼65% of cumulative provincial HIV cases, for the presence of 70 HLA-associated Pol mutations. Sequences from British Columbia (N = 6525) and elsewhere in Canada/USA (N = 6517) were used for comparison. HIV adaptation levels to 34 HLA alleles were also computed. Putative HIV transmission clusters were identified, and the prevalence of HLA-associated adaptations within and outside these clusters was investigated. RESULTS: Analyses confirmed significantly elevated and temporally increasing levels of HIV adaptation to commonly expressed HLA alleles, in particular B*51. Notably, HLA-adapted HIV strains were significantly enriched among phylogenetic clusters in Saskatchewan. CONCLUSION: Extensive circulating HIV adaptation to HLA in Saskatchewan provides a plausible explanation for accelerated progression, while enrichment of adapted variants in phylogenetic clusters suggests they are being widely transmitted. Results highlight the utility of Pol sequences, routinely collected for drug resistance monitoring, for surveillance of HIV-host adaptation, and underscore the urgent need to expand HIV prevention and treatment programmes in Saskatchewan.

High Levels of Dual-Class Drug Resistance in HIV-Infected Children Failing First-Line Antiretroviral Therapy in Southern Ethiopia.

Clinical monitoring of pediatric HIV treatment remains a major challenge in settings where drug resistance genotyping is not routinely available. As a result, our understanding of drug resistance, and its impact on subsequent therapeutic regimens available in these settings, remains limited. We investigate the prevalence and correlates of HIV-1 drug resistance among 94 participants of the Ethiopia Pediatric HIV Cohort failing first-line combination antiretroviral therapy (cART) using dried blood spot-based genotyping. Overall, 81% (73/90) of successfully genotyped participants harbored resistance mutations, including 69% (62/90) who harbored resistance to both Nucleoside Reverse Transcriptase Inhibitors (NRTIs) and Non-nucleoside Reverse Transcriptase Inhibitors (NNRTIs). Strikingly, 42% of resistant participants harbored resistance to all four NRTIs recommended for second-line use in this setting, meaning that there are effectively no remaining cART options for these children. Longer cART duration and prior regimen changes were significantly associated with detection of drug resistance mutations. Replicate genotyping increased the breadth of drug resistance detected in 34% of cases, and thus is recommended for consideration when typing from blood spots. Implementation of timely drug resistance testing and access to newer antiretrovirals and drug classes are urgently needed to guide clinical decision-making and improve outcomes for HIV-infected children on first-line cART in Ethiopia.

A strong association of human leukocyte antigen-associated Pol and Gag mutations with clinical parameters in HIV-1 subtype A/E infection.

OBJECTIVES: Identification of human leukocyte antigen-associated HIV-1 polymorphisms (HLA-APs) in different global populations furthers our understanding of HIV-1 pathogenesis and may help identify candidate immunogens for HIV vaccines targeted to these populations. Although numerous population-based studies identifying HLA-APs have been conducted in HIV-1 subtype B- and subtype C-infected cohorts, few have focused on subtype A/E. DESIGN: We investigated HLA-APs in a cohort of chronically HIV-1 subtype A/E-infected Vietnamese individuals. METHODS: HLA-APs in HIV-1 Gag, Pol, and Nef regions from 388 treatment-naive individuals chronically infected with HIV-1 subtype A/E were analyzed using phylogenetically informed approaches. RESULTS: A total of 303 HLA-APs were identified. HLA-APs occurring at six positions in Gag and six positions in Pol were significantly associated with higher plasma viral load (pVL), whereas HLA-APs occurring at two positions in Gag and 13 positions in Pol were significantly associated with lower CD4 T-cell counts. Furthermore, the proportion of Pol codons harboring an HLA-AP specific to the host's HLA correlated positively with HIV-1 pVL (R = 0.22; P < 0.0001) and inversely with CD4 T-cell counts (R = -0.32; P < 0.0001). Similarly, the proportion of HLA-associated Gag codons harboring host-specific HLA-AP correlated inversely with CD4 T-cell counts (R = -0.13; P = 0.01). CONCLUSION: These significant associations between HIV-1 amino acids adapted to Vietnamese HLA alleles and higher pVL and lower CD4 T-cell counts suggests that accumulation of cytotoxic T cells escape mutations may influence clinical outcomes in HIV-1 subtype A/E-infected Vietnamese individuals.