Skin Cancer Detection in Diverse Skin Tones by Machine Learning Combining Audio and Visual Convolutional Neural Networks.

INTRODUCTION: Skin cancer (SC) is common in fair skin (FS) at a 1:5 lifetime incidence for nonmelanoma skin cancer. In order to assist clinicians' decisions, a risk intervention technology was developed, which combines a dual-mode machine learning of visual and sonified (pixel to sound) data. The addition of an audio technology enhances malignant features of lesions, increases sensitivity and was previously validated under a prospective clinical setting in FS. In dark skin (DS), although rare by a 10-30 factor, skin cancer is diagnosed at more advanced stages resulting in a delayed diagnosis and affecting life quality and expectancy. It is known as well that SC diagnostic accuracy by machine learning in DS is decreased as compared to FS. The present study tests the use of sonification aided by artificial intelligence algorithms to compare diagnostics of different skin tones. METHODOLOGY: Biopsy-validated smartphone images were diagnosed in a retrospective study by a dual audio-visual convoluted neural network. A total of 60 Fitzpatrick I-III were compared to 72 Fitzpatrick IV-VI. A dichotomous diagnostic output, either malignant or benign, was assessed for sensitivity, specificity and area under the curves (AUCs) for the receiver operating characteristic (ROC) curve. RESULTS: ROC curve analytics indicated an AUC of 0.858 (95% CI: 0.795-0.921) and 0.856 (95% CI: 0.759-0.953) for fair and DS (p = NS). Sensitivity of Fitzpatrick I-III skin and Fitzpatrick IV-VI were 84.4% (71.8-96.9) and 79.6% (63.4-93.8), respectively (p = NS). Specificity of Fitzpatrick I-III skin and Fitzpatrick IV-VI were 84.2% (72.6-95.8) and 85.3% (73.4-97.2), respectively (p = NS). The positive predictive and negative predictive values as well as accuracy (0.817 vs. 0.847) were all within the same range (p = NS). CONCLUSIONS: The results demonstrate that the dual-modality classifier identifies skin cancer of FS and DS similarly well. Sonification of malignant signs of a skin lesion demonstrates promising results, even with smartphone images, which should be considered as a tool to achieve more effective and accessible healthcare.

Mastery of assistive technology: What is it? How do we measure it?

This paper presents the results of a one-year study on mastery of assistive technology (AT). This study sought to develop a conceptual framework for talking about mastery of AT and to create an instrument for measuring individual mastery. A Delphi Study was conducted with individuals with disabilities considered to be "power users" of AT, practitioners, and researchers. Participants were asked to: identify factors that are predictors and indicators of AT mastery, determine how to measure these factors and determine criteria for each factor for the stages of AT mastery (e.g. novice, context-dependent, transitional, and power user). The resulting measure is called the Continuum of AT Mastery (CATM).

Expanded Antigen-Specific Elimination Assay to Measure Human CD8+ T Cell Cytolytic Potential.

Durable cellular immunity against pathogens is dependent upon a coordinated recall response to antigen by memory CD8+ T cells, involving their proliferation and the generation of secondary cytotoxic effector cells. Conventional assays measuring ex vivo cytotoxicity fail to capture this secondary cytolytic potential, especially in settings where cells have not been recently exposed to their cognate antigen in vivo. Here we describe the expanded antigen-specific elimination assay (EASEA), a flow cytometric endpoint assay to measure the capacity of human CD8+ T cells to expand in vitro upon antigen re-exposure and generate secondary effector cells capable of selectively eliminating autologous antigen-pulsed target cells across a range of effector-to-target ratios. Unlike alternative assays, EASEA avoids the hazards of radioactive labeling and viral infection and can be used to study responses to individual or pooled antigens of interest. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Expanded antigen-specific elimination assay.

Differences in HIV-1 reservoir size, landscape characteristics and decay dynamics in acute and chronic treated HIV-1 Clade C infection.

Background: Persisting HIV reservoir viruses in resting CD4 T cells and other cellular subsets are the main barrier to cure efforts. Antiretroviral therapy (ART) intensification by early initiation has been shown to enable post-treatment viral control in some cases but the underlying mechanisms are not fully understood. We hypothesized that ART initiated during the hyperacute phase of infection before peak will affect the size, decay dynamics and landscape characteristics of HIV-1 subtype C viral reservoirs. Methods: We studied 35 women at high risk of infection from Durban, South Africa identified with hyperacute HIV infection by twice weekly testing for plasma HIV-1 RNA. Study participants included 11 who started ART at a median of 456 (297-1203) days post onset of viremia (DPOV), and 24 who started ART at a median of 1 (1-3) DPOV. We used peripheral blood mononuclear cells (PBMC) to measure total HIV-1 DNA by ddPCR and to sequence reservoir viral genomes by full length individual proviral sequencing (FLIP-seq) from onset of detection of HIV up to 1 year post treatment initiation. Results: Whereas ART in hyperacute infection blunted peak viremia compared to untreated individuals (p<0.0001), there was no difference in total HIV-1 DNA measured contemporaneously (p=0.104). There was a steady decline of total HIV DNA in early treated persons over 1 year of ART (p=0.0004), with no significant change observed in the late treated group. Total HIV-1 DNA after one year of treatment was lower in the early treated compared to the late treated group (p=0.02). Generation of 697 single viral genome sequences revealed a difference in the longitudinal proviral genetic landscape over one year between untreated, late treated, and early treated infection: the relative contribution of intact genomes to the total pool of HIV-1 DNA after 1 year was higher in untreated infection (31%) compared to late treated (14%) and early treated infection (0%). Treatment initiated in both late and early infection resulted in a more rapid decay of intact (13% and 51% per month) versus defective (2% and 35% per month) viral genomes. However, intact genomes were still observed one year post chronic treatment initiation in contrast to early treatment where intact genomes were no longer detectable. Moreover, early ART reduced phylogenetic diversity of intact genomes and limited the seeding and persistence of cytotoxic T lymphocyte immune escape variants in the reservoir. Conclusions: Overall, our results show that whereas ART initiated in hyperacute HIV-1 subtype C infection did not impact reservoir seeding, it was nevertheless associated with more rapid decay of intact viral genomes, decreased genetic complexity and immune escape in reservoirs, which could accelerate reservoir clearance when combined with other interventional strategies.

Prediction of differential Gag versus Env responses to a mosaic HIV-1 vaccine regimen by HLA class I alleles.

HLA class I variation has the strongest effect genome-wide on outcome after HIV infection, and as such, an understanding of the impact of HLA polymorphism on response to HIV vaccination may inform vaccine design. We sought HLA associations with HIV-directed immunogenicity in the phase 1/2a APPROACH vaccine trial, which tested vaccine regimens containing mosaic inserts in Ad26 and MVA vectors, with or without a trimeric gp140 protein. While there were no HLA allelic associations with the overall cellular immune response to the vaccine assessed by ELISpot (Gag, Pol, and Env combined), significant associations with differential response to Gag compared to Env antigens were observed. Notably, HLA class I alleles known to associate with disease susceptibility in HIV natural history cohorts are associated with stronger Env-directed responses, whereas protective alleles are associated with stronger Gag-directed responses. Mean viral loads determined for each HLA allele in untreated individuals correlated negatively with the strength of the Gag response minus the Env response in Black vaccinees based on both ELISpot and CD8+ T cell ICS responses. As the association of T cell responses to conserved Gag epitopes with lower viral load in untreated individuals is well established, our data raise the possibility that the Ad26.Mos.HIV vaccine may induce more effective cellular responses in those with HLA alleles that confer improved virologic control in untreated HIV infection.IMPORTANCENo vaccine tested to date has shown sufficient efficacy against HIV infection. A vaccine that induces robust responses in one individual may fail to do so in another individual due to variation in HLA class I genes, loci central to the immune response. Extensive data have shown the strong effect of HLA variation on outcome after HIV infection, but very little is known about the effect of such variation on HIV vaccine success. Here, we identify a link between the effect of HLA variation on HIV disease outcome and immune responses to an HIV vaccine. HLA variants associated with better HIV control after infection also induce stronger responses against the HIV Gag protein relative to the Env protein after vaccination. Given the virologic control conferred by responses to Gag in natural history of HIV infection, these data suggest that HLA alleles conferring protection after HIV infection may also support a more effective cellular response to HIV vaccination.

Immunogenicity of 2 therapeutic mosaic HIV-1 vaccine strategies in individuals with HIV-1 on antiretroviral therapy.

Mosaic HIV-1 vaccines have been shown to elicit robust humoral and cellular immune responses in people living with HIV-1 (PLWH), that had started antiretroviral therapy (ART) during acute infection. We evaluated the safety and immunogenicity of 2 mosaic vaccine regimens in virologically suppressed individuals that had initiated ART during the chronic phase of infection, exemplifying the majority of PLWH. In this double-blind, placebo-controlled phase 1 trial (IPCAVD013/HTX1002) 25 ART-suppressed PLWH were randomized to receive Ad26.Mos4.HIV/MVA-Mosaic (Ad26/MVA) (n = 10) or Ad26.Mos4.HIV/Ad26.Mos4.HIV plus adjuvanted gp140 protein (Ad26/Ad26+gp140) (n = 9) or placebo (n = 6). Primary endpoints included safety and tolerability and secondary endpoints included HIV-specific binding and neutralizing antibody titers and HIV-specific T cell responses. Both vaccine regimens were well tolerated with pain/tenderness at the injection site and fatigue, myalgia/chills and headache as the most commonly reported solicited local and grade 3 systemic adverse events, respectively. In the Ad26/Ad26+gp140 group, Env-specific IFN-γ T cell responses showed a median 12-fold increase while responses to Gag and Pol increased 1.8 and 2.4-fold, respectively. The breadth of T cell responses to individual peptide subpools increased from 11.0 pre-vaccination to 26.0 in the Ad26/Ad26+gp140 group and from 10.0 to 14.5 in the Ad26/MVA group. Ad26/Ad26+gp140 vaccination increased binding antibody titers against vaccine-matched clade C Env 5.5-fold as well as augmented neutralizing antibody titers against Clade C pseudovirus by 7.2-fold. Both vaccine regimens were immunogenic, while the addition of the protein boost resulted in additional T cell and augmented binding and neutralizing antibody titers. These data suggest that the Ad26/Ad26+gp140 regimen should be tested further.

Meta-Analysis of Genome-Wide Association Studies Reveals Genetic Mechanisms of Supraventricular Arrhythmias.

BACKGROUND: Substantial data support a heritable basis for supraventricular tachycardias, but the genetic determinants and molecular mechanisms of these arrhythmias are poorly understood. We sought to identify genetic loci associated with atrioventricular nodal reentrant tachycardia (AVNRT) and atrioventricular accessory pathways or atrioventricular reciprocating tachycardia (AVAPs/AVRT). METHODS: We performed multiancestry meta-analyses of genome-wide association studies to identify genetic loci for AVNRT (4 studies) and AVAP/AVRT (7 studies). We assessed evidence supporting the potential causal effects of candidate genes by analyzing relations between associated variants and cardiac gene expression, performing transcriptome-wide analyses, and examining prior genome-wide association studies. RESULTS: Analyses comprised 2384 AVNRT cases and 106 489 referents, and 2811 AVAP/AVRT cases and 1,483 093 referents. We identified 2 significant loci for AVNRT, which implicate NKX2-5 and TTN as disease susceptibility genes. A transcriptome-wide association analysis supported an association between reduced predicted cardiac expression of NKX2-5 and AVNRT. We identified 3 significant loci for AVAP/AVRT, which implicate SCN5A, SCN10A, and TTN/CCDC141. Variant associations at several loci have been previously reported for cardiac phenotypes, including atrial fibrillation, stroke, Brugada syndrome, and electrocardiographic intervals. CONCLUSIONS: Our findings highlight gene regions associated with ion channel function (AVAP/AVRT), as well as cardiac development and the sarcomere (AVAP/AVRT and AVNRT) as important potential effectors of supraventricular tachycardia susceptibility.

Polymorphic residues in HLA-B that mediate HIV control distinctly modulate peptide interactions with both TCR and KIR molecules.

Immunogenetic studies have shown that specific HLA-B residues (67, 70, 97, and 156) mediate the impact of HLA class I on HIV infection, but the molecular basis is not well understood. Here we evaluate the function of these residues within the protective HLA-B∗5701 allele. While mutation of Met67, Ser70, and Leu156 disrupt CD8+ T cell recognition, substitution of Val97 had no significant impact. Thermal denaturation of HLA-B∗5701-peptide complexes revealed that Met67 and Leu156 maintain HLA-peptide stability, while Ser70 and Leu156 facilitate T cell receptor (TCR) interactions. Analyses of existing structures and structural models suggested that Val97 mediates HLA-peptide binding to inhibitory KIR3DL1 molecules, which was confirmed by experimental assays. These data thereby demonstrate that the genetic basis by which host immunity impacts HIV outcomes occurs by modulating HLA-B-peptide stability and conformation for interaction with TCR and killer immunoglobulin receptor (KIR) molecules. Moreover, they indicate a key role for epitope specificity and HLA-KIR interactions to HIV control.

Co-Designing Auditory Navigation Solutions for Traveling as a Blind Individual During the COVID-19 Pandemic.

A group co-design was held in March 2021 with six blind and low-vision individuals (BLIs) from the United States. Participants were asked to discuss problems related to travel during the COVID-19 pandemic and make recommendations for possible solutions. Two probes (prototypes) of a non-visual neighborhood travel map and a non-visual COVID-19 choropleth map (a map using colors or sounds over each state to represent different values) of COVID-19 state data were shown to participants for inspiration. The participants expressed that COVID-19 had significantly increased their apprehension and discomfort associated with activities such as venturing outside, traveling, engaging with strangers, communicating, adapting to changes in familiar environments, and wearing masks. Participants gravitated towards the need for information the probes provided, and made a number of observations and recommendations for improvement. They wanted more detailed geo-referenced COVID-19 data (including by county), information related to voting, a mobile app, and more detailed building information, such as doors on the travel map.

Impact of HLA class I functional divergence on HIV control.

Heterozygosity of Human leukocyte antigen (HLA) class I genes is linked to beneficial outcomes after HIV infection, presumably through greater breadth of HIV epitope presentation and cytotoxic T cell response. Distinct allotype pairs, however, differ in the extent to which they bind shared sets of peptides. We developed a functional divergence metric that measures pairwise complementarity of allotype-associated peptide binding profiles. Greater functional divergence for pairs of HLA-A and/or HLA-B allotypes was associated with slower AIDS progression and independently with enhanced viral load control. The metric predicts immune breadth at the peptide level rather than gene level and redefines HLA heterozygosity as a continuum differentially affecting disease outcome. Functional divergence may affect response to additional infections, vaccination, immunotherapy, and other diseases where HLA heterozygote advantage occurs.

T cell responses to SARS-CoV-2 infection and vaccination are elevated in B cell deficiency and reduce risk of severe COVID-19.

Individuals with primary and pharmacologic B cell deficiencies have high rates of severe disease and mortality from coronavirus disease 2019 (COVID-19), but the immune responses and clinical outcomes after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and vaccination have yet to be fully defined. Here, we evaluate the cellular immune responses after both SARS-CoV-2 infection and vaccination in patients receiving the anti-CD20 therapy rituximab (RTX) and those with low B cell counts due to common variable immune deficiency (CVID) disease. Assessment of effector and memory CD4+ and CD8+ T cell responses to SARS-CoV-2 revealed elevated reactivity and proliferative capacity after both infection and vaccination in B cell-deficient individuals, particularly within the CD8+ T cell compartment, in comparison with healthy controls. Evaluation of clinical outcomes demonstrates that vaccination of RTX-treated individuals was associated with about 4.8-fold reduced odds of moderate or severe COVID-19 in the absence of vaccine-induced antibodies. Analysis of T cell differentiation demonstrates that RTX administration increases the relative frequency of naïve CD8+ T cells, potentially by depletion of CD8+CD20dim T cells, which are primarily of an effector memory or terminal effector memory (TEMRA) phenotype. However, this also leads to a reduction in preexisting antiviral T cell immunity. Collectively, these data indicate that individuals with B cell deficiencies have enhanced T cell immunity after both SARS-CoV-2 infection and vaccination that potentially accounts for reduced hospitalization and severe disease from subsequent SARS-CoV-2 infection.

Extrafollicular IgD-CD27-CXCR5-CD11c- DN3 B cells infiltrate inflamed tissues in autoimmune fibrosis and in severe COVID-19.

Although therapeutic B cell depletion dramatically resolves inflammation in many diseases in which antibodies appear not to play a central role, distinct extrafollicular pathogenic B cell subsets that accumulate in disease lesions have hitherto not been identified. The circulating immunoglobulin D (IgD)-CD27-CXCR5-CD11c+ DN2 B cell subset has been previously studied in some autoimmune diseases. A distinct IgD-CD27-CXCR5-CD11c- DN3 B cell subset accumulates in the blood both in IgG4-related disease, an autoimmune disease in which inflammation and fibrosis can be reversed by B cell depletion, and in severe COVID-19. These DN3 B cells prominently accumulate in the end organs of IgG4-related disease and in lung lesions in COVID-19, and double-negative B cells prominently cluster with CD4+ T cells in these lesions. Extrafollicular DN3 B cells may participate in tissue inflammation and fibrosis in autoimmune fibrotic diseases, as well as in COVID-19.

Molecular basis of differential HLA class I-restricted T cell recognition of a highly networked HIV peptide.

Cytotoxic-T-lymphocyte (CTL) mediated control of HIV-1 is enhanced by targeting highly networked epitopes in complex with human-leukocyte-antigen-class-I (HLA-I). However, the extent to which the presenting HLA allele contributes to this process is unknown. Here we examine the CTL response to QW9, a highly networked epitope presented by the disease-protective HLA-B57 and disease-neutral HLA-B53. Despite robust targeting of QW9 in persons expressing either allele, T cell receptor (TCR) cross-recognition of the naturally occurring variant QW9_S3T is consistently reduced when presented by HLA-B53 but not by HLA-B57. Crystal structures show substantial conformational changes from QW9-HLA to QW9_S3T-HLA by both alleles. The TCR-QW9-B53 ternary complex structure manifests how the QW9-B53 can elicit effective CTLs and suggests sterically hindered cross-recognition by QW9_S3T-B53. We observe populations of cross-reactive TCRs for B57, but not B53 and also find greater peptide-HLA stability for B57 in comparison to B53. These data demonstrate differential impacts of HLAs on TCR cross-recognition and antigen presentation of a naturally arising variant, with important implications for vaccine design.

Cytolytic CD8+ T cells infiltrate germinal centers to limit ongoing HIV replication in spontaneous controller lymph nodes.

Follicular CD8+ T cells (fCD8) mediate surveillance in lymph node (LN) germinal centers against lymphotropic infections and cancers, but the precise mechanisms by which these cells mediate immune control remain incompletely resolved. To address this, we investigated functionality, clonotypic compartmentalization, spatial localization, phenotypic characteristics, and transcriptional profiles of LN-resident virus-specific CD8+ T cells in persons who control HIV without medications. Antigen-induced proliferative and cytolytic potential consistently distinguished spontaneous controllers from noncontrollers. T cell receptor analysis revealed complete clonotypic overlap between peripheral and LN-resident HIV-specific CD8+ T cells. Transcriptional analysis of LN CD8+ T cells revealed gene signatures of inflammatory chemotaxis and antigen-induced effector function. In HIV controllers, the cytotoxic effectors perforin and granzyme B were elevated among virus-specific CXCR5+ fCD8s proximate to foci of HIV RNA within germinal centers. These results provide evidence consistent with cytolytic control of lymphotropic infection supported by inflammatory recruitment, antigen-specific proliferation, and cytotoxicity of fCD8s.

The Health Effects of 72 Hours of Simulated Wind Turbine Infrasound: A Double-Blind Randomized Crossover Study in Noise-Sensitive, Healthy Adults.

BACKGROUND: Large electricity-generating wind turbines emit both audible sound and inaudible infrasound at very low frequencies that are outside of the normal human range of hearing. Sufferers of wind turbine syndrome (WTS) have attributed their ill-health and particularly their sleep disturbance to the signature pattern of infrasound. Critics have argued that these symptoms are psychological in origin and are attributable to nocebo effects. OBJECTIVES: We aimed to test the effects of 72 h of infrasound (1.6-20 Hz at a sound level of ∼90 dB pk re 20µPa, simulating a wind turbine infrasound signature) exposure on human physiology, particularly sleep. METHODS: We conducted a randomized double-blind triple-arm crossover laboratory-based study of 72 h exposure with a >10-d washout conducted in a noise-insulated sleep laboratory in the style of a studio apartment. The exposures were infrasound (∼90 dB pk), sham infrasound (same speakers not generating infrasound), and traffic noise exposure [active control; at a sound pressure level of 40-50 dB LAeq,night and 70 dB LAFmax transient maxima, night (2200 to 0700 hours)]. The following physiological and psychological measures and systems were tested for their sensitivity to infrasound: wake after sleep onset (WASO; primary outcome) and other measures of sleep physiology, wake electroencephalography, WTS symptoms, cardiovascular physiology, and neurobehavioral performance. RESULTS: We randomized 37 noise-sensitive but otherwise healthy adults (18-72 years of age; 51% female) into the study before a COVID19-related public health order forced the study to close. WASO was not affected by infrasound compared with sham infrasound (-1.36 min; 95% CI: -6.60, 3.88, p=0.60) but was worsened by the active control traffic exposure compared with sham by 6.07 min (95% CI: 0.75, 11.39, p=0.02). Infrasound did not worsen any subjective or objective measures used. DISCUSSION: Our findings did not support the idea that infrasound causes WTS. High level, but inaudible, infrasound did not appear to perturb any physiological or psychological measure tested in these study participants. https://doi.org/10.1289/EHP10757.

Which clinical research questions are the most important? Development and preliminary validation of the Australia & New Zealand Musculoskeletal (ANZMUSC) Clinical Trials Network Research Question Importance Tool (ANZMUSC-RQIT).

BACKGROUND AND AIMS: High quality clinical research that addresses important questions requires significant resources. In resource-constrained environments, projects will therefore need to be prioritized. The Australia and New Zealand Musculoskeletal (ANZMUSC) Clinical Trials Network aimed to develop a stakeholder-based, transparent, easily implementable tool that provides a score for the 'importance' of a research question which could be used to rank research projects in order of importance. METHODS: Using a mixed-methods, multi-stage approach that included a Delphi survey, consensus workshop, inter-rater reliability testing, validity testing and calibration using a discrete-choice methodology, the Research Question Importance Tool (ANZMUSC-RQIT) was developed. The tool incorporated broad stakeholder opinion, including consumers, at each stage and is designed for scoring by committee consensus. RESULTS: The ANZMUSC-RQIT tool consists of 5 dimensions (compared to 6 dimensions for an earlier version of RQIT): (1) extent of stakeholder consensus, (2) social burden of health condition, (3) patient burden of health condition, (4) anticipated effectiveness of proposed intervention, and (5) extent to which health equity is addressed by the research. Each dimension is assessed by defining ordered levels of a relevant attribute and by assigning a score to each level. The scores for the dimensions are then summed to obtain an overall ANZMUSC-RQIT score, which represents the importance of the research question. The result is a score on an interval scale with an arbitrary unit, ranging from 0 (minimal importance) to 1000. The ANZMUSC-RQIT dimensions can be reliably ordered by committee consensus (ICC 0.73-0.93) and the overall score is positively associated with citation count (standardised regression coefficient 0.33, p<0.001) and journal impact factor group (OR 6.78, 95% CI 3.17 to 14.50 for 3rd tertile compared to 1st tertile of ANZMUSC-RQIT scores) for 200 published musculoskeletal clinical trials. CONCLUSION: We propose that the ANZMUSC-RQIT is a useful tool for prioritising the importance of a research question.

Comparison of long- and short-read metagenomic assembly for low-abundance species and resistance genes.

Recent technological and computational advances have made metagenomic assembly a viable approach to achieving high-resolution views of complex microbial communities. In previous benchmarking, short-read (SR) metagenomic assemblers had the highest accuracy, long-read (LR) assemblers generated the most contiguous sequences and hybrid (HY) assemblers balanced length and accuracy. However, no assessments have specifically compared the performance of these assemblers on low-abundance species, which include clinically relevant organisms in the gut. We generated semi-synthetic LR and SR datasets by spiking small and increasing amounts of Escherichia coli isolate reads into fecal metagenomes and, using different assemblers, examined E. coli contigs and the presence of antibiotic resistance genes (ARGs). For ARG assembly, although SR assemblers recovered more ARGs with high accuracy, even at low coverages, LR assemblies allowed for the placement of ARGs within longer, E. coli-specific contigs, thus pinpointing their taxonomic origin. HY assemblies identified resistance genes with high accuracy and had lower contiguity than LR assemblies. Each assembler type's strengths were maintained even when our isolate was spiked in with a competing strain, which fragmented and reduced the accuracy of all assemblies. For strain characterization and determining gene context, LR assembly is optimal, while for base-accurate gene identification, SR assemblers outperform other options. HY assembly offers contiguity and base accuracy, but requires generating data on multiple platforms, and may suffer high misassembly rates when strain diversity exists. Our results highlight the trade-offs associated with each approach for recovering low-abundance taxa, and that the optimal approach is goal-dependent.

Slow progression of pediatric HIV associates with early CD8+ T cell PD-1 expression and a stem-like phenotype.

HIV nonprogression despite persistent viremia is rare among adults who are naive to antiretroviral therapy (ART) but relatively common among ART-naive children. Previous studies indicate that ART-naive pediatric slow progressors (PSPs) adopt immune evasion strategies similar to those described in natural hosts of SIV. However, the mechanisms underlying this immunophenotype are not well understood. In a cohort of early-treated infants who underwent analytical treatment interruption (ATI) after 12 months of ART, expression of PD-1 on CD8+ T cells immediately before ATI was the main predictor of slow progression during ATI. PD-1+CD8+ T cell frequency was also negatively correlated with CCR5 and HLA-DR expression on CD4+ T cells and predicted stronger HIV-specific T lymphocyte responses. In the CD8+ T cell compartment of PSPs, we identified an enrichment of stem-like TCF-1+PD-1+ memory cells, whereas pediatric progressors and viremic adults had a terminally exhausted PD-1+CD39+ population. TCF-1+PD-1+ expression on CD8+ T cells was associated with higher proliferative activity and stronger Gag-specific effector functionality. These data prompted the hypothesis that the proliferative burst potential of stem-like HIV-specific cytotoxic cells could be exploited in therapeutic strategies to boost the antiviral response and facilitate remission in infants who received early ART with a preserved and nonexhausted T cell compartment.

Phenotypic signatures of immune selection in HIV-1 reservoir cells.

Human immunodeficiency virus 1 (HIV-1) reservoir cells persist lifelong despite antiretroviral treatment1,2 but may be vulnerable to host immune responses that could be exploited in strategies to cure HIV-1. Here we used a single-cell, next-generation sequencing approach for the direct ex vivo phenotypic profiling of individual HIV-1-infected memory CD4+ T cells from peripheral blood and lymph nodes of people living with HIV-1 and receiving antiretroviral treatment for approximately 10 years. We demonstrate that in peripheral blood, cells harbouring genome-intact proviruses and large clones of virally infected cells frequently express ensemble signatures of surface markers conferring increased resistance to immune-mediated killing by cytotoxic T and natural killer cells, paired with elevated levels of expression of immune checkpoint markers likely to limit proviral gene transcription; this phenotypic profile might reduce HIV-1 reservoir cell exposure to and killing by cellular host immune responses. Viral reservoir cells harbouring intact HIV-1 from lymph nodes exhibited a phenotypic signature primarily characterized by upregulation of surface markers promoting cell survival, including CD44, CD28, CD127 and the IL-21 receptor. Together, these results suggest compartmentalized phenotypic signatures of immune selection in HIV-1 reservoir cells, implying that only small subsets of infected cells with optimal adaptation to their anatomical immune microenvironment are able to survive during long-term antiretroviral treatment. The identification of phenotypic markers distinguishing viral reservoir cells may inform future approaches for strategies to cure and eradicate HIV-1.

Subtle Longitudinal Alterations in Env Sequence Potentiate Differences in Sensitivity to Broadly Neutralizing Antibodies following Acute HIV-1 Subtype C Infection.

Broadly neutralizing antibodies (bNAbs) for HIV-1 prevention or cure strategies must inhibit transmitted/founder and reservoir viruses. Establishing sensitivity of circulating viruses to bNAbs and genetic patterns affecting neutralization variability may guide rational bNAbs selection for clinical development. We analyzed 326 single env genomes from nine individuals followed longitudinally following acute HIV-1 infection, with samples collected at ~1 week after the first detection of plasma viremia; 300 to 1,709 days postinfection but prior to initiating antiretroviral therapy (ART) (median = 724 days); and ~1 year post ART initiation. Sequences were assessed for phylogenetic relatedness, potential N- and O-linked glycosylation, and variable loop lengths (V1 to V5). A total of 43 env amplicons (median = 3 per patient per time point) were cloned into an expression vector and the TZM-bl assay was used to assess the neutralization profiles of 15 bNAbs targeting the CD4 binding site, V1/V2 region, V3 supersite, MPER, gp120/gp41 interface, and fusion peptide. At 1 µg/mL, the neutralization breadths were as follows: VRC07-LS and N6.LS (100%), VRC01 (86%), PGT151 (81%), 10-1074 and PGT121 (80%), and less than 70% for 10E8, 3BNC117, CAP256.VRC26, 4E10, PGDM1400, and N123-VRC34.01. Features associated with low sensitivity to V1/V2 and V3 bNAbs were higher potential glycosylation sites and/or relatively longer V1 and V4 domains, including known "signature" mutations. The study shows significant variability in the breadth and potency of bNAbs against circulating HIV-1 subtype C envelopes. VRC07-LS, N6.LS, VRC01, PGT151, 10-1074, and PGT121 display broad activity against subtype C variants, and major determinants of sensitivity to most bNAbs were within the V1/V4 domains. IMPORTANCE Broadly neutralizing antibodies (bNAbs) have potential clinical utility in HIV-1 prevention and cure strategies. However, bNAbs target diverse epitopes on the HIV-1 envelope and the virus may evolve to evade immune responses. It is therefore important to identify antibodies with broad activity in high prevalence settings, as well as the genetic patterns that may lead to neutralization escape. We investigated 15 bNAbs with diverse biophysical properties that target six epitopes of the HIV-1 Env glycoprotein for their ability to inhibit viruses that initiated infection, viruses circulating in plasma at chronic infection before antiretroviral treatment (ART), or viruses that were archived in the reservoir during ART in subtype C infected individuals in South Africa, a high burden country. We identify the antibodies most likely to be effective for clinical use in this setting and describe mutational patterns associated with neutralization escape from these antibodies.