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Background: The Lentivirus human immunodeficiency virus (HIV) causes chronic inflammation and AIDS in humans, with variable rates of disease progression between individuals driven by both host and viral factors. Similarly, simian lentiviruses vary in their pathogenicity based on characteristics of both the host species and the virus strain, yet the immune underpinnings that drive differential Lentivirus pathogenicity remain incompletely understood., Methods: We profile immune responses in a unique model of differential lentiviral pathogenicity where pig-tailed macaques are infected with highly genetically similar variants of SIV that differ in virulence. We apply longitudinal single-cell transcriptomics to this cohort, along with single-cell resolution cell-cell communication techniques, to understand the immune mechanisms underlying lentiviral pathogenicity., Results: Compared to a minimally pathogenic lentiviral variant, infection with a highly pathogenic variant results in a more delayed, broad, and sustained activation of inflammatory pathways, including an extensive global interferon signature. Conversely, individual cells infected with highly pathogenic Lentivirus upregulated fewer interferon-stimulated genes at a lower magnitude, indicating that highly pathogenic Lentivirus has evolved to partially escape from interferon responses. Further, we identify CXCL10 and CXCL16 as important molecular drivers of inflammatory pathways specifically in response to highly pathogenic Lentivirus infection. Immune responses to highly pathogenic Lentivirus infection are characterized by amplifying regulatory circuits of pro-inflammatory cytokines with dense longitudinal connectivity., Conclusions: Our work presents a model of lentiviral pathogenicity where failures in early viral control mechanisms lead to delayed, sustained, and amplifying pro-inflammatory circuits, which in turn drives disease progression., (© 2024. The Author(s).)

Recent efforts to shift the control and leadership of health research on African issues to Africa have led to increased investments for scientific research capacity strengthening (RCS) on the continent and a greater demand for accountability, value for money and demonstration of return on investment. There is limited literature on monitoring and evaluation (M&E) of RCS systems and there is a clear need to further explore whether the M&E frameworks and approaches that are currently used are fit for purpose. The M&E approaches taken by four African RCS consortia funded under the Developing Excellence in Leadership, Training and Science in Africa (DELTAS) I initiative were assessed using several methods, including a framework comparison of the M&E approaches, semi-structured interviews and facilitated discussion sessions. The findings revealed a wide range in the number of indicators used in the M&E plans of individual consortium, which were uniformly quantitative and at the output and outcome levels. Consortia revealed that additional information could have been captured to better evaluate the success of activities and measure the ripple effects of their efforts. While it is beneficial for RCS consortia to develop and implement their own M&E plans, this could be strengthened by routine engagement with funders/programme managers to further align efforts. It is also important for M&E plans to consider qualitative data capture for assessment of RCS efforts. Efforts could be further enhanced by supporting platforms for cross-consortia sharing, particularly when trying to assess more complex effects. Consortia should make sure that processes for developmental evaluation, and capturing and using the associated learning, are in place. Sharing the learning associated with M&E of RCS efforts is vital to improve future efforts. Investing and improving this aspect of RCS will help ensure tracking of progress and impact of future efforts, and ensure accountability and the return on investment. The findings are also likely applicable well beyond health research., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Background: Polypharmacy, using five or more medications, may increase the risk of nonadherence to prescribed treatment. We aimed to identify the interrelationship between trajectories of adherence to antiretroviral therapy (ART) and polypharmacy., Methods: We included women with HIV (aged ≥ 18) enrolled in the Women's Interagency HIV Study in the United States from 2014 to 2019. We used group-based trajectory modeling (GBTM) to identify trajectories of adherence to ART and polypharmacy and the dual GBTM to identify the interrelationship between adherence and polypharmacy., Results: Overall, 1,538 were eligible (median age of 49 years). GBTM analysis revealed five latent trajectories of adherence with 42% of women grouped in the consistently moderate trajectory. GBTM identified four polypharmacy trajectories with 45% categorized in the consistently low group., Conclusions: The joint model did not reveal any interrelationship between ART adherence and polypharmacy trajectories. Future research should consider examining the interrelationship between both variables using objective measures of adherence., (© 2023. The Author(s).)

Background: The majority of studies characterizing female genital tract microbiota have focused on luminal organisms, while the presence and impact of tissue-adherent ectocervical microbiota remain incompletely understood. Studies of luminal and tissue-associated bacteria in the gastrointestinal tract suggest that these communities may have distinct roles in health and disease. Here, we performed a multi-omics characterization of paired luminal and tissue samples collected from a cohort of Kenyan female sex workers., Results: We identified a tissue-adherent bacterial microbiome, with a higher alpha diversity than the luminal microbiome, in which dominant genera overall included Gardnerella and Lactobacillus, followed by Prevotella, Atopobium, and Sneathia. About half of the L. iners-dominated luminal samples had a corresponding Gardnerella-dominated tissue microbiome. Broadly, the tissue-adherent microbiome was associated with fewer differentially expressed host genes than the luminal microbiome. Gene set enrichment analysis revealed that L. crispatus-dominated tissue-adherent communities were associated with protein translation and antimicrobial activity, whereas a highly diverse microbial community was associated with epithelial remodeling and pro-inflammatory pathways. Tissue-adherent communities dominated by L. iners and Gardnerella were associated with lower host transcriptional activity. Tissue-adherent microbiomes dominated by Lactobacillus and Gardnerella correlated with host protein profiles associated with epithelial barrier stability, although with a more pro-inflammatory profile for the Gardnerella-dominated microbiome group. Tissue samples with a highly diverse composition had a protein profile representing cell proliferation and pro-inflammatory activity., Conclusion: We identified ectocervical tissue-adherent bacterial communities in all study participants of a female sex worker cohort. These communities were distinct from cervicovaginal luminal microbiota in a significant proportion of individuals. We further revealed that bacterial communities at both sites correlated with distinct host gene expression and protein levels. The tissue-adherent bacterial community could possibly act as a reservoir that seed the lumen with less optimal, non-Lactobacillus, bacteria. Video Abstract., (© 2023. The Author(s).)

Background: Alterations to the gut microbiome have been linked to multiple chronic diseases. However, the drivers of such changes remain largely unknown. The oral cavity acts as a major route of exposure to exogenous factors including pathogens, and processes therein may affect the communities in the subsequent compartments of the gastrointestinal tract. Here, we perform strain-resolved, integrated meta-genomic, transcriptomic, and proteomic analyses of paired saliva and stool samples collected from 35 individuals from eight families with multiple cases of type 1 diabetes mellitus (T1DM)., Results: We identified distinct oral microbiota mostly reflecting competition between streptococcal species. More specifically, we found a decreased abundance of the commensal Streptococcus salivarius in the oral cavity of T1DM individuals, which is linked to its apparent competition with the pathobiont Streptococcus mutans. The decrease in S. salivarius in the oral cavity was also associated with its decrease in the gut as well as higher abundances in facultative anaerobes including Enterobacteria. In addition, we found evidence of gut inflammation in T1DM as reflected in the expression profiles of the Enterobacteria as well as in the human gut proteome. Finally, we were able to follow transmitted strain-variants from the oral cavity to the gut at the individual omic levels, highlighting not only the transfer, but also the activity of the transmitted taxa along the gastrointestinal tract., Conclusions: Alterations of the oral microbiome in the context of T1DM impact the microbial communities in the lower gut, in particular through the reduction of "mouth-to-gut" transfer of Streptococcus salivarius. Our results indicate that the observed oral-cavity-driven gut microbiome changes may contribute towards the inflammatory processes involved in T1DM. Through the integration of multi-omic analyses, we resolve strain-variant "mouth-to-gut" transfer in a disease context. Video Abstract., (© 2022. The Author(s).)

Background: A dominance of non-iners Lactobacillus species in the vaginal microbiome is optimal and strongly associated with gynecological and obstetric health, while the presence of diverse obligate or facultative anaerobic bacteria and a paucity in Lactobacillus species, similar to communities found in bacterial vaginosis (BV), is considered non-optimal and associated with adverse health outcomes. Various therapeutic strategies are being explored to modulate the composition of the vaginal microbiome; however, there is no human model that faithfully reproduces the vaginal epithelial microenvironment for preclinical validation of potential therapeutics or testing hypotheses about vaginal epithelium-microbiome interactions., Results: Here, we describe an organ-on-a-chip (organ chip) microfluidic culture model of the human vaginal mucosa (vagina chip) that is lined by hormone-sensitive, primary vaginal epithelium interfaced with underlying stromal fibroblasts, which sustains a low physiological oxygen concentration in the epithelial lumen. We show that the Vagina Chip can be used to assess colonization by optimal L. crispatus consortia as well as non-optimal Gardnerella vaginalis-containing consortia, and to measure associated host innate immune responses. Co-culture and growth of the L. crispatus consortia on-chip was accompanied by maintenance of epithelial cell viability, accumulation of D- and L-lactic acid, maintenance of a physiologically relevant low pH, and down regulation of proinflammatory cytokines. In contrast, co-culture of G. vaginalis-containing consortia in the vagina chip resulted in epithelial cell injury, a rise in pH, and upregulation of proinflammatory cytokines., Conclusion: This study demonstrates the potential of applying human organ chip technology to create a preclinical model of the human vaginal mucosa that can be used to better understand interactions between the vaginal microbiome and host tissues, as well as to evaluate the safety and efficacy of live biotherapeutics products. Video Abstract., (© 2022. The Author(s).)

Background: Cynomolgus macaque (Macaca fascicularis) is an attractive animal model for the study of human disease and is extensively used in biomedical research. Cynomolgus macaques share behavioral, physiological, and genomic traits with humans and recapitulate human disease manifestations not observed in other animal species. To improve the use of the cynomolgus macaque model to investigate immune responses, we defined and characterized the T cell receptor (TCR) repertoire., Result: We identified and analyzed the alpha (TRA), beta (TRB), gamma (TRG), and delta (TRD) TCR loci of the cynomolgus macaque. The expressed repertoire was determined using 22 unique lung samples from Mycobacterium tuberculosis infected cynomolgus macaques by single cell RNA sequencing. Expressed TCR alpha (TRAV) and beta (TRBV) variable region genes were enriched and identified using gene specific primers, which allowed their functional status to be determined. Analysis of the primers used for cynomolgus macaque TCR variable region gene enrichment showed they could also be used to amplify rhesus macaque (M. mulatta) variable region genes., Conclusion: The genomic organization of the cynomolgus macaque has great similarity with the rhesus macaque and they shared > 90% sequence similarity with the human TCR repertoire. The identification of the TCR repertoire facilitates analysis of T cell immunity in cynomolgus macaques., (© 2022. The Author(s).)

Background: The generation of over 69 spliced HIV-1 mRNAs from one primary transcript by alternative RNA splicing emphasizes the central role that RNA processing plays in HIV-1 replication. Control is mediated in part through the action of host SR proteins whose activity is regulated by multiple SR kinases (CLK1-4, SRPKs)., Methods: Both shRNA depletion and small molecule inhibitors of host SR kinases were used in T cell lines and primary cells to evaluate the role of these factors in the regulation of HIV-1 gene expression. Effects on virus expression were assessed using western blotting, RT-qPCR, and immunofluorescence., Results: The studies demonstrate that SR kinases play distinct roles; depletion of CLK1 enhanced HIV-1 gene expression, reduction of CLK2 or SRPK1 suppressed it, whereas CLK3 depletion had a modest impact. The opposing effects of CLK1 vs. CLK2 depletion were due to action at distinct steps; reduction of CLK1 increased HIV-1 promoter activity while depletion of CLK2 affected steps after transcript initiation. Reduced CLK1 expression also enhanced the response to several latency reversing agents, in part, by increasing the frequency of responding cells, consistent with a role in regulating provirus latency. To determine whether small molecule modulation of SR kinase function could be used to control HIV-1 replication, we screened a GSK library of protein kinase inhibitors (PKIS) and identified several pyrazolo[1,5-b] pyridazine derivatives that suppress HIV-1 gene expression/replication with an EC 50 ~ 50 nM. The compounds suppressed HIV-1 protein and viral RNA accumulation with minimal impact on cell viability, inhibiting CLK1 and CLK2 but not CLK3 function, thereby selectively altering the abundance of individual CLK and SR proteins in cells., Conclusions: These findings demonstrate the unique roles played by individual SR kinases in regulating HIV-1 gene expression, validating the targeting of these functions to either enhance latency reversal, essential for "Kick-and-Kill" strategies, or to silence HIV protein expression for "Block-and-Lock" strategies., (© 2022. The Author(s).)

Objective: To characterize and address the opioid crisis disproportionately impacting rural U.S. regions., Methods: The Rural Opioid Initiative (ROI) is a two-phase project to collect and harmonize quantitative and qualitative data and develop tailored interventions to address rural opioid use. The baseline quantitative survey data from people who use drugs (PWUD) characterizes the current opioid epidemic (2018-2020) in eight geographically diverse regions., Results: Among 3,084 PWUD, 92% reported ever injecting drugs, 86% reported using opioids (most often heroin) and 74% reported using methamphetamine to get high in the past 30 days; 53% experienced homelessness in the prior 6 months; and 49% had ever overdosed. Syringe service program use varied by region and 53% had ever received an overdose kit or naloxone prescription. Less than half (48%) ever received medication for opioid use disorder (MOUD)., Conclusions: The ROI combines data across eight rural regions to better understand drug use including drivers and potential interventions in rural areas with limited resources. Baseline ROI data demonstrate extensive overlap between opioid and methamphetamine use, high homelessness rates, inadequate access to MOUD, and other unmet needs among PWUD in the rural U.S. By combining data across studies, the ROI provides much greater statistical power to address research questions and better understand the syndemic of infectious diseases and drug use in rural settings including unmet treatment needs., (© 2022. The Author(s).)

Background: We previously identified differentially expressed genes on the basis of false discovery rate adjusted P value using empirical Bayes moderated tests. However, that approach yielded a subset of differentially expressed genes without accounting for redundancy between the selected genes., Methods: This study is a secondary analysis of a case-control study of the effect of antiretroviral therapy on apoptosis pathway genes comprising of 16 cases (HIV infected with mitochondrial toxicity) and 16 controls (uninfected). We applied the maximum relevance minimum redundancy (mRMR) algorithm on the genes that were differentially expressed between the cases and controls. The mRMR algorithm iteratively selects features (genes) that are maximally relevant for class prediction and minimally redundant. We implemented several machine learning classifiers and tested the prediction accuracy of the two mRMR genes. We next used network analysis to estimate and visualize the association among the differentially expressed genes. We employed Markov Random Field or undirected network models to identify gene networks related to mitochondrial toxicity. The Spinglass model was used to identify clusters of gene communities., Results: The mRMR algorithm ranked DFFA and TNFRSF1A, two of the upregulated proapoptotic genes, on the top. The overall prediction accuracy was 86%, the two mRMR genes correctly classified 86% of the participants into their respective groups. The estimated network models showed different patterns of gene networks. In the network of the cases, FASLG was the most central gene. However, instead of FASLG, ABL1 and LTBR had the highest centrality in controls., Conclusion: The mRMR algorithm and network analysis revealed a new correlation of genes associated with mitochondrial toxicity., (© 2021. The Author(s).)

Background: The critical role of antibody Fc-mediated effector functions in immune defense has been widely reported in various viral infections. These effector functions confer cellular responses through engagement with innate immune cells. The precise mechanism(s) by which immunoglobulin G (IgG) Fc domain and cognate receptors may afford protection are poorly understood, however, in the context of HIV/SHIV infections. Many different in vitro assays have been developed and utilized to measure effector functions, but the extent to which these assays capture distinct antibody activities has not been fully elucidated., Results: In this study, six Fc-mediated effector function assays and two biophysical antibody profiling assays were performed on a common set of samples from HIV-1 infected and vaccinated subjects. Biophysical antibody profiles supported robust prediction of diverse IgG effector functions across distinct Fc-mediated effector function assays. While a number of assays showed correlated activities, supervised machine learning models indicated unique antibody features as primary contributing factors to the associated effector functions. Additional experiments established the mechanistic relevance of relationships discovered using this unbiased approach., Conclusions: In sum, this study provides better resolution on the diversity and complexity of effector function assays, offering a clearer perspective into this family of antibody mechanisms of action to inform future HIV-1 treatment and vaccination strategies., (© 2021. The Author(s).)

Background: Cervicovaginal bacterial communities composed of diverse anaerobes with low Lactobacillus abundance are associated with poor reproductive outcomes such as preterm birth, infertility, cervicitis, and risk of sexually transmitted infections (STIs), including human immunodeficiency virus (HIV). Women in sub-Saharan Africa have a higher prevalence of these high-risk bacterial communities when compared to Western populations. However, the transition of cervicovaginal communities between high- and low-risk community states over time is not well described in African populations., Results: We profiled the bacterial composition of 316 cervicovaginal swabs collected at 3-month intervals from 88 healthy young Black South African women with a median follow-up of 9 months per participant and developed a Markov-based model of transition dynamics that accurately predicted bacterial composition within a broader cross-sectional cohort. We found that Lactobacillus iners-dominant, but not Lactobacillus crispatus-dominant, communities have a high probability of transitioning to high-risk states. Simulating clinical interventions by manipulating the underlying transition probabilities, our model predicts that the population prevalence of low-risk microbial communities could most effectively be increased by manipulating the movement between L. iners- and L. crispatus-dominant communities., Conclusions: The Markov model we present here indicates that L. iners-dominant communities have a high probability of transitioning to higher-risk states. We additionally identify transitions to target to increase the prevalence of L. crispatus-dominant communities. These findings may help guide future intervention strategies targeted at reducing bacteria-associated adverse reproductive outcomes among women living in sub-Saharan Africa. Video Abstract., (© 2021. The Author(s).)

Background: Adolescents and young adults living with HIV in sub Saharan Africa are at high risk of poor adherence to antiretroviral therapy (ART) and virologic failure (VF)., Methods: We conducted a randomized control trial among adolescents and young adults on ART with VF to assess the effectiveness of a community-based peer support intervention aimed at improving VF. Viral load (VL) levels were obtained at 12, 24 and 36 weeks. A subset of the participants had baseline HIV drug resistance (HIVDR) genotyped using Sanger sequencing., Results: The participants' median (interquartile range (IQR)) age was 18.1 (IQR: 15.1-20.0) years and half (50.5%, n = 107) were male. At week 24, the proportion of subjects with a detectable viremia was significantly lower in the intervention arm than in the standard of care (SOC) arm (76.0% (n = 79) vs. 89.0% (n = 96), p = 0.013). At Week 36, there remained a difference in the proportion of subjects with a detectable VL between the intervention arm (68.3%, n = 71) and SOC arm (79.6%, n = 86), which was trending towards statistical significance (p = 0.059). There was no difference in the probability of having a detectable VL over time between the intervention and SOC groups (adjusted odds ratio: 1.14, p = 0.439). Baseline HIVDR was observed in 44.0% of the participants in the intervention and 56.0% in the SOC group (p = 0.146)., Conclusion: A transient effect of the peer support intervention in improving VF was observed among adolescents and young people failing ART., Trial Registration: This study is registered with www.clinicaltrials.gov under the reference number: NCT02833441.

Background: Immunoglobulin G4-related disease (IgG4-RD) and systemic sclerosis (SSc) are rare autoimmune diseases characterized by the presence of CD4+ cytotoxic T cells in the blood as well as inflammation and fibrosis in various organs, but they have no established etiologies. Similar to other autoimmune diseases, the gut microbiome might encode disease-triggering or disease-sustaining factors., Methods: The gut microbiomes from IgG4-RD and SSc patients as well as healthy individuals with no recent antibiotic treatment were studied by metagenomic sequencing of stool DNA. De novo assembly-based taxonomic and functional characterization, followed by association and accessory gene set enrichment analysis, were applied to describe microbiome changes associated with both diseases., Results: Microbiomes of IgG4-RD and SSc patients distinctly separated from those of healthy controls: numerous opportunistic pathogenic Clostridium and typically oral Streptococcus species were significantly overabundant, while Alistipes, Bacteroides, and butyrate-producing species were depleted in the two diseases compared to healthy controls. Accessory gene content analysis in these species revealed an enrichment of Th17-activating Eggerthella lenta strains in IgG4-RD and SSc and a preferential colonization of a homocysteine-producing strain of Clostridium bolteae in SSc. Overabundance of the classical mevalonate pathway, hydroxyproline dehydratase, and fibronectin-binding protein in disease microbiomes reflects potential functional differences in host immune recognition and extracellular matrix utilization associated with fibrosis. Strikingly, the majority of species that were differentially abundant in IgG4-RD and SSc compared to controls showed the same directionality in both diseases. Compared with multiple sclerosis and rheumatoid arthritis, the gut microbiomes of IgG4-RD and SSc showed similar signatures; in contrast, the most differentially abundant taxa were not the facultative anaerobes consistently identified in inflammatory bowel diseases, suggesting the microbial signatures of IgG4-RD and SSc do not result from mucosal inflammation and decreased anaerobism., Conclusions: These results provide an initial characterization of gut microbiome ecology in fibrosis-prone IgG4-RD and SSc and reveal microbial functions that offer insights into the pathophysiology of these rare diseases.

Background: Africa bears a disproportionately high burden of globally significant disease but has lagged in knowledge production to address its health challenges. In this contribution, we discuss the challenges and approaches to health research capacity strengthening in sub-Saharan Africa and propose that the recent shift to an African-led approach is the most optimal., Methods and Findings: We introduce several capacity building approaches and recent achievements, explore why African-led research on the continent is a potentially paradigm-shifting and innovative approach, and discuss the advantages and challenges thereof. We reflect on the approaches used by the African Academy of Sciences (AAS)-funded Sub-Saharan African Network for TB/HIV Research Excellence (SANTHE) consortium as an example of an effective African-led science and capacity building programme. We recommend the following as crucial components of future efforts: 1. Directly empowering African-based researchers, 2. Offering quality training and career development opportunities to large numbers of junior African scientists and support staff, and 3. Effective information exchange and collaboration. Furthermore, we argue that long-term investment from international donors and increasing funding commitments from African governments and philanthropies will be needed to realise a critical mass of local capacity and to create and sustain world-class research hubs that will be conducive to address Africa's intractable health challenges., Conclusions: Our experiences so far suggest that African-led research has the potential to overcome the vicious cycle of brain-drain and may ultimately lead to improvement of health and science-led economic transformation of Africa into a prosperous continent.

An amendment to this paper has been published and can be accessed via the original article.

Introduction: Immunological damage in acute HIV infection (AHI) may predispose to detrimental clinical sequela. However, studies on the earliest HIV-induced immunological changes are limited, particularly in sub-Saharan Africa. We assessed the plasma cytokines kinetics, and their associations with virological and immunological parameters, in a well-characterized AHI cohort where participants were diagnosed before peak viremia., Methods: Blood cytokine levels were measured using Luminex and ELISA assays pre-infection, during the hyperacute infection phase (before or at peak viremia, 1-11 days after the first detection of viremia), after peak viremia (24-32 days), and during the early chronic phase (77-263 days). Gag-protease-driven replicative capacities of the transmitted/founder viruses were determined using a green fluorescent reporter T cell assay. Complete blood counts were determined before and immediately following AHI detection before ART initiation., Results: Untreated AHI was associated with a cytokine storm of 12 out of the 33 cytokines analyzed. Initiation of ART during Fiebig stages I-II abrogated the cytokine storm. In untreated AHI, virus replicative capacity correlated positively with IP-10 (rho = 0.84, P < 0.001) and IFN-alpha (rho = 0.59, P = 0.045) and inversely with nadir CD4 + T cell counts (rho = - 0.58, P = 0.048). Hyperacute HIV infection before the initiation of ART was associated with a transient increase in monocytes (P < 0.001), decreased lymphocytes (P = 0.011) and eosinophils (P = 0.003) at Fiebig stages I-II, and decreased eosinophils (P < 0.001) and basophils (P = 0.007) at Fiebig stages III-V. Levels of CXCL13 during the untreated hyperacute phase correlated inversely with blood eosinophils (rho = - 0.89, P < 0.001), basophils (rho = - 0.87, P = 0.001) and lymphocytes (rho = - 0.81, P = 0.005), suggesting their trafficking into tissues. In early treated individuals, time to viral load suppression correlated positively with plasma CXCL13 at the early chronic phase (rho = 0.83, P = 0.042)., Conclusion: While commencement of ART during Fiebig stages I-II of AHI abrogated the HIV-induced cytokine storm, significant depletions of eosinophils, basophils, and lymphocytes, as well as transient expansions of monocytes, were still observed in these individuals in the hyperacute phase before the initiation of ART, suggesting that even ART initiated during the onset of viremia does not abrogate all HIV-induced immune changes.

Background: Genomic medicine has paved the way for identifying biomarkers and therapeutically actionable targets for complex diseases, but is complicated by the involvement of thousands of variably expressed genes across multiple cell types. Single-cell RNA-sequencing study (scRNA-seq) allows the characterization of such complex changes in whole organs., Methods: The study is based on applying network tools to organize and analyze scRNA-seq data from a mouse model of arthritis and human rheumatoid arthritis, in order to find diagnostic biomarkers and therapeutic targets. Diagnostic validation studies were performed using expression profiling data and potential protein biomarkers from prospective clinical studies of 13 diseases. A candidate drug was examined by a treatment study of a mouse model of arthritis, using phenotypic, immunohistochemical, and cellular analyses as read-outs., Results: We performed the first systematic analysis of pathways, potential biomarkers, and drug targets in scRNA-seq data from a complex disease, starting with inflamed joints and lymph nodes from a mouse model of arthritis. We found the involvement of hundreds of pathways, biomarkers, and drug targets that differed greatly between cell types. Analyses of scRNA-seq and GWAS data from human rheumatoid arthritis (RA) supported a similar dispersion of pathogenic mechanisms in different cell types. Thus, systems-level approaches to prioritize biomarkers and drugs are needed. Here, we present a prioritization strategy that is based on constructing network models of disease-associated cell types and interactions using scRNA-seq data from our mouse model of arthritis, as well as human RA, which we term multicellular disease models (MCDMs). We find that the network centrality of MCDM cell types correlates with the enrichment of genes harboring genetic variants associated with RA and thus could potentially be used to prioritize cell types and genes for diagnostics and therapeutics. We validated this hypothesis in a large-scale study of patients with 13 different autoimmune, allergic, infectious, malignant, endocrine, metabolic, and cardiovascular diseases, as well as a therapeutic study of the mouse arthritis model., Conclusions: Overall, our results support that our strategy has the potential to help prioritize diagnostic and therapeutic targets in human disease.

Vectored gene delivery of HIV-1 broadly neutralizing antibodies (bNAbs) using recombinant adeno-associated virus (rAAV) is a promising alternative to conventional vaccines for preventing new HIV-1 infections and for therapeutically suppressing established HIV-1 infections. Passive infusion of single bNAbs has already shown promise in initial clinical trials to temporarily decrease HIV-1 load in viremic patients, and to delay viral rebound from latent reservoirs in suppressed patients during analytical treatment interruptions of antiretroviral therapy. Long-term, continuous, systemic expression of such bNAbs could be achieved with a single injection of rAAV encoding antibody genes into muscle tissue, which would bypass the challenges of eliciting such bNAbs through traditional vaccination in naïve patients, and of life-long repeated passive transfers of such biologics for therapy. rAAV delivery of single bNAbs has already demonstrated protection from repeated HIV-1 vaginal challenge in humanized mouse models, and phase I clinical trials of this approach are underway. Selection of which individual, or combination of, bNAbs to deliver to counter pre-existing resistance and the rise of escape mutations in the virus remains a challenge, and such choices may differ depending on use of this technology for prevention versus therapy.

Background: Single-cell genomic methods now provide unprecedented resolution for characterizing the component cell types and states of tissues such as the epithelial subsets of the gastrointestinal tract. Nevertheless, functional studies of these subsets at scale require faithful in vitro models of identified in vivo biology. While intestinal organoids have been invaluable in providing mechanistic insights in vitro, the extent to which organoid-derived cell types recapitulate their in vivo counterparts remains formally untested, with no systematic approach for improving model fidelity., Results: Here, we present a generally applicable framework that utilizes massively parallel single-cell RNA-seq to compare cell types and states found in vivo to those of in vitro models such as organoids. Furthermore, we leverage identified discrepancies to improve model fidelity. Using the Paneth cell (PC), which supports the stem cell niche and produces the largest diversity of antimicrobials in the small intestine, as an exemplar, we uncover fundamental gene expression differences in lineage-defining genes between in vivo PCs and those of the current in vitro organoid model. With this information, we nominate a molecular intervention to rationally improve the physiological fidelity of our in vitro PCs. We then perform transcriptomic, cytometric, morphologic and proteomic characterization, and demonstrate functional (antimicrobial activity, niche support) improvements in PC physiology., Conclusions: Our systematic approach provides a simple workflow for identifying the limitations of in vitro models and enhancing their physiological fidelity. Using adult stem cell-derived PCs within intestinal organoids as a model system, we successfully benchmark organoid representation, relative to that in vivo, of a specialized cell type and use this comparison to generate a functionally improved in vitro PC population. We predict that the generation of rationally improved cellular models will facilitate mechanistic exploration of specific disease-associated genes in their respective cell types.

Background: The factors determining differential HIV disease outcome among individuals expressing protective HLA alleles such as HLA-B*27:05 and HLA-B*57:01 remain unknown. We here analyse two HIV-infected subjects expressing both HLA-B*27:05 and HLA-B*57:01. One subject maintained low-to-undetectable viral loads for more than a decade of follow up. The other progressed to AIDS in < 3 years., Results: The rapid progressor was the recipient within a known transmission pair, enabling virus sequences to be tracked from transmission. Progression was associated with a 12% Gag sequence change and 26% Nef sequence change at the amino acid level within 2 years. Although next generation sequencing from early timepoints indicated that multiple CD8+ cytotoxic T lymphocyte (CTL) escape mutants were being selected prior to superinfection, < 4% of the amino acid changes arising from superinfection could be ascribed to CTL escape. Analysis of an HLA-B*27:05/B*57:01 non-progressor, in contrast, demonstrated minimal virus sequence diversification (1.1% Gag amino acid sequence change over 10 years), and dominant HIV-specific CTL responses previously shown to be effective in control of viraemia were maintained. Clonal sequencing demonstrated that escape variants were generated within the non-progressor, but in many cases were not selected. In the rapid progressor, progression occurred despite substantial reductions in viral replicative capacity (VRC), and non-progression in the elite controller despite relatively high VRC., Conclusions: These data are consistent with previous studies demonstrating rapid progression in association with superinfection and that rapid disease progression can occur despite the relatively the low VRC that is typically observed in the setting of multiple CTL escape mutants.

The results of the RV144 HIV vaccine, in combination with several recent non-human primate vaccine studies continue to highlight the potentially protective role of non-neutralizing Fc functional antibodies in HIV vaccine design. For many currently licensed vaccines, assays that detect antigen-specific antibody titers or neutralization levels have been used as a correlate of protection. However, antibodies can confer protection through multiple other mechanisms beyond neutralization, or mechanisms which are not dependent on total antibody titers. Alternative strategies that allow us to further understand the precise mechanisms by which antibodies confer protection against HIV and other infectious pathogens is vitally important for the development of future vaccines. Systems serology aims to comprehensively survey a diverse array of antibody features and functions, in order to simultaneously examine the mechanisms behind and distinguish the most important antibody features required for protection, thus identifying key targets for future experimental vaccine testing. This review will focus on the technical aspects required for the application of Systems serology and summarizes the recent advances provided by application of this systemic analytical approach.

Background: Intravascular leukocyte recruitment in most vertebrate tissues is restricted to postcapillary and collecting venules, whereas capillaries and arterioles usually support little or no leukocyte adhesion. This segmental restriction is thought to be mediated by endothelial, rather than hemodynamic, differences. The underlying mechanisms are largely unknown, in part because effective tools to distinguish, isolate, and analyze venular endothelial cells (V-ECs) and non-venular endothelial cells (NV-ECs) have been unavailable. We hypothesized that the atypical chemokine receptor DARC (Duffy Antigen Receptor for Chemokines, a.k.a. ACKR1 or CD234) may distinguish V-ECs versus NV-ECs in mice., Methods: We generated a rat-anti-mouse monoclonal antibody (MAb) that specifically recognizes the erythroid and endothelial forms of native, surface-expressed DARC. Using this reagent, we characterized DARC expression and distribution in the microvasculature of murine tissues., Results: DARC was exquisitely restricted to post-capillary and small collecting venules and completely absent from arteries, arterioles, capillaries, veins, and most lymphatics in every tissue analyzed. Accordingly, intravital microscopy showed that adhesive leukocyte-endothelial interactions were restricted to DARC + venules. DARC was detectable over the entire circumference of V-ECs, but was more concentrated at cell-cell junctions. Analysis of single-cell suspensions suggested that the frequency of V-ECs among the total microvascular EC pool varies considerably between different tissues., Conclusions: Immunostaining of endothelial DARC allows the identification and isolation of intact V-ECs from multiple murine tissues. This strategy may be useful to dissect the mechanisms underlying segmental microvascular specialization in healthy and diseased tissues and to characterize the role of EC subsets in tissue-homeostasis, immune surveillance, infection, inflammation, and malignancies.

Background: Although aspirin is recommended for the prevention of colorectal cancer, the specific individuals for whom the benefits outweigh the risks are not clearly defined. Moreover, the precise mechanisms by which aspirin reduces the risk of cancer are unclear. We recently launched the ASPirin Intervention for the REDuction of colorectal cancer risk (ASPIRED) trial to address these uncertainties., Methods/design: ASPIRED is a prospective, double-blind, multidose, placebo-controlled, biomarker clinical trial of aspirin use in individuals previously diagnosed with colorectal adenoma. Individuals (n = 180) will be randomized in a 1:1:1 ratio to low-dose (81 mg/day) or standard-dose (325 mg/day) aspirin or placebo. At two study visits, participants will provide lifestyle, dietary and biometric data in addition to urine, saliva and blood specimens. Stool, grossly normal colorectal mucosal biopsies and cytology brushings will be collected during a flexible sigmoidoscopy without bowel preparation. The study will examine the effect of aspirin on urinary prostaglandin metabolites (PGE-M; primary endpoint), plasma inflammatory markers (macrophage inhibitory cytokine-1 (MIC-1)), colonic expression of transcription factor binding (transcription factor 7-like 2 (TCF7L2)), colonocyte gene expression, including hydroxyprostaglandin dehydrogenase 15-(NAD) (HPGD) and those that encode Wnt signaling proteins, colonic cellular nanocytology and oral and gut microbial composition and function., Discussion: Aspirin may prevent colorectal cancer through multiple, interrelated mechanisms. The ASPIRED trial will scrutinize these pathways and investigate putative mechanistically based risk-stratification biomarkers., Trial Registration: This protocol is registered with the U.S. National Institutes of Health trial registry, ClinicalTrials.gov, under the identifier NCT02394769 . Registered on 16 March 2015.

Background: Current HIV-1 immunogens are unable to induce antibodies that can neutralize a broad range of HIV-1 (broadly neutralizing antibodies; bNAbs). However, such antibodies are elicited in 10-30 % of HIV-1 infected individuals, and the co-evolution of the virus and the humoral immune responses in these individuals has attracted attention, because they can provide clues for vaccine design., Results: Here we characterized the NAb responses and envelope glycoprotein evolution in an HIV-1 infected "elite neutralizer" of the Amsterdam Cohort Studies on HIV-1 infection and AIDS who developed an unusually potent bNAb response rapidly after infection. The NAb response was dependent on the N332-glycan and viral resistance against the N332-glycan dependent bNAb PGT135 developed over time but viral escape did not occur at or near this glycan. In contrast, the virus likely escaped by increasing V1 length, with up to 21 amino acids, accompanied by the introduction of 1-3 additional glycans, as well as 2-4 additional cysteine residues within V1., Conclusions: In the individual studied here, HIV-1 escaped from N332-glycan directed NAb responses without changing the epitope itself, but by elongating a variable loop that shields this epitope.

Background: Broadly neutralizing antibodies (bnAbs) directed against the mannose-patch on the HIV envelope glycoprotein gp120 have several features that make them desirable targets for vaccine design. The PGT125-131 bnAb family is of particular interest due to its superior breadth and potency. The overlapping epitopes recognized by this family are intricate and neutralization requires interaction with at least two N-linked glycans (N332/N334, N295 or N301) in addition to backbone-mediated contact with the (323)IGDIR(327) motif of the V3 loop. We have recently shown that this bnAb family consists of two distinct antibody classes that can bind alternate arrangements of glycans in the mannose-patch in the absence of N332 thereby limiting viral escape. This led us to further investigate viral resistance and escape mechanisms to the PGT125-131 bnAb family., Results: Using an escape virus isolated from the PGT125-131 donor as a guide, we show that mutating both the V3 core protein epitope and repositioning critical N-linked glycosylation sites are required to restore neutralization sensitivity. Interestingly, neutralization sensitivity could be restored via different routes for the two distinct bnAb classes within the PGT125-131 family, which may have been important in generating the divergence in recognition. We demonstrate that the observed V3 mutations confer neutralization resistance in other virus strains through both gain-of-function and escape studies. Furthermore, we show that the V3 loop is important in facilitating promiscuous binding to glycans within the mannose-patch., Conclusions: These data highlight the importance of the V3 loop in the design of immunogens aimed at inducing broad and potent bnAbs that can bind promiscuously to the mannose-patch.

Background: Killer-cell Immunoglobulin-like Receptors (KIR) interact with Human Leukocyte Antigen (HLA) to modify natural killer- and T-cell function. KIR are implicated in HIV acquisition by small studies that have not been widely replicated. A role for KIR in HIV disease progression is more widely replicated and supported by functional studies., Methods: To assess the role of KIR and KIR ligands in HIV acquisition and disease course, we studied at-risk women in South Africa between 2004-2010. Logistic regression was used for nested case-control analysis of 154 women who acquired vs. 155 who did not acquire HIV, despite high exposure. Linear mixed-effects models were used for cohort analysis of 139 women followed prospectively for a median of 54 months (IQR 31-69) until 2014., Results: Neither KIR repertoires nor HLA alleles were associated with HIV acquisition. However, KIR haplotype BB was associated with lower viral loads (-0.44 log10 copies/ml; SE = 0.18; p = 0.03) and higher CD4+ T-cell counts (+80 cells/μl; SE = 42; p = 0.04). This was largely explained by the protective effect of KIR2DL2/KIR2DS2 on the B haplotype and reciprocal detrimental effect of KIR2DL3 on the A haplotype., Conclusions: Although neither KIR nor HLA appear to have a role in HIV acquisition, our data are consistent with involvement of KIR2DL2 in HIV control. Additional studies to replicate these findings are indicated.

Background: HIV-1 subtype C Nef sequences have a significantly lower ability overall to down-regulate CD4 and HLA-I than subtype B Nef sequences. Here we investigated whether Nef amino acids differing in frequency between HIV-1 subtypes B and C explain lower CD4 and HLA-I down-regulation ability of subtype C., Findings: Subtype-specific mutations were introduced into representative subtype B and C Nef sequences and the CD4 and HLA-I down-regulation ability of these mutants was measured by flow cytometry in a CD4+ T cell line. Subtype C consensus 20I and subtype B consensus 20M reduced and increased HLA-I down-regulation respectively, and the S88G immune escape mutation (which is significantly more frequent in subtype C than subtype B) reduced CD4 and HLA-I down-regulation., Conclusions: Our data suggest that these subtype-specific differences may partly contribute to inter-subtype functional differences, and identification of an immune escape mutation - S88G - that impairs Nef function is of relevance to vaccine design.

Background: The precise immune responses mediated by HLA class I molecules such as HLA-B*27:05 and HLA-B*57:01 that protect against HIV disease progression remain unclear. We studied a CRF01&#95;AE clade HIV infected donor-recipient transmission pair in which the recipient expressed both HLA-B*27:05 and HLA-B*57:01., Results: Within 4.5 years of diagnosis, the recipient had progressed to meet criteria for antiretroviral therapy initiation. We employed ultra-deep sequencing of the full-length virus genome in both donor and recipient as an unbiased approach by which to identify specific viral mutations selected in association with progression. Using a heat map method to highlight differences in the viral sequences between donor and recipient, we demonstrated that the majority of the recipient's mutations outside of Env were within epitopes restricted by HLA-B*27:05 and HLA-B*57:01, including the well-studied Gag epitopes. The donor, who also expressed HLA alleles associated with disease protection, HLA-A*32:01/B*13:02/B*14:01, showed selection of mutations in parallel with disease progression within epitopes restricted by these protective alleles., Conclusions: These studies of full-length viral sequences in a transmission pair, both of whom expressed protective HLA alleles but nevertheless failed to control viremia, are consistent with previous reports pointing to the critical role of Gag-specific CD8+ T cell responses restricted by protective HLA molecules in maintaining immune control of HIV infection. The transmission of subtype CRF01&#95;AE clade infection may have contributed to accelerated disease progression in this pair as a result of clade-specific sequence differences in immunodominant epitopes.

Background: HLA-B alleles are associated with viral control in chronic HIV-1 infection, however, their role in primary HIV-1 disease is unclear. This study sought to determine the role of HLA-B alleles in viral control during the acute phase of HIV-1 infection and establishment of the early viral load set point (VLSP)., Findings: Individuals identified during primary HIV-1 infection were HLA class I typed and followed longitudinally. Associations between HLA-B alleles and HIV-1 viral replication during acute infection and VLSP were analyzed in untreated subjects. The results showed that neither HLA-B*57 nor HLA-B*27 were significantly associated with viral control during acute HIV-1 infection (Fiebig stage I-IV, n=171). HLA-B*57 was however significantly associated with a subsequent lower VLSP (p<0.001, n=135) with nearly 1 log10 less median viral load. Analysis of a known polymorphism at position 97 of HLA-B showed significant associations with both lower initial viral load (p<0.01) and lower VLSP (p<0.05). However, this association was dependent on different amino acids at this position for each endpoint., Conclusions: The effect of HLA-B*57 on viral control is more pronounced during the later stages of primary HIV-1 infection, which suggests the underlying mechanism of control occurs at a critical period in the first several months after HIV-1 acquisition. The risk profile of polymorphisms at position 97 of HLA-B are more broadly associated with HIV-1 viral load during primary infection and may serve as a focal point in further studies of HLA-B function.

Background: Endolysosomes play a key role in maintaining the homeostasis of the cell. They are made of a complex set of proteins that degrade lipids, proteins and sugars. Studies involving endolysosome contribution to cellular functions such as MHC class I and II epitope production have used recombinant endolysosomal proteins, knockout mice that lack one of the enzymes or purified organelles from human tissue. Each of these approaches has some caveats in analyzing endolysosomal enzyme functions., Results: In this study, we have developed a simple methodology to assess endolysosomal protease activity. By varying the pH in crude lysate from human peripheral blood mononuclear cells (PBMCs), we documented increased endolysosomal cathepsin activity in acidic conditions. Using this new method, we showed that the degradation of HIV peptides in low pH extracts analyzed by mass spectrometry followed similar kinetics and degradation patterns as those performed with purified endolysosomes., Conclusion: By using crude lysate in the place of purified organelles this method will be a quick and useful tool to assess endolysosomal protease activities in primary cells of limited availability. This quick method will especially be useful to screen peptide susceptibility to degradation in endolysosomal compartments for antigen processing studies, following which detailed analysis using purified organelles may be used to study specific peptides.

Background: Natural killer (NK) cells constitutively express high levels of Tim-3, an immunoregulatory molecule recently proposed to be a marker for mature and functional NK cells. Whether HIV-1 infection modulates the expression of Tim-3 on NK cells, or the levels of its ligand Galectin-9 (Gal-9), and how signaling through these molecules affects the NK cell response to HIV-1 remains inadequately understood., Results: We analyzed Tim-3 and Gal-9 expression in a cohort of 85 individuals with early and chronic HIV-1 infection, and in 13 HIV-1 seronegative control subjects. HIV-1 infection was associated with reduced expression of Tim-3 on NK cells, which was normalized by HAART. Plasma concentrations of Gal-9 were higher in HIV-1-infected individuals than in healthy individuals. Interestingly, Gal-9 expression in immune cells was significantly elevated in early infection, with monocytes and dendritic cells displaying the highest expression levels, which correlated with HIV-1 viral loads. In vitro, Gal-9 triggered Tim-3 downregulation on NK cells as well as NK cell activation., Conclusions: Our data suggest that high expression levels of Gal-9 during early HIV-1 infection can lead to enhanced NK cell activity, possibly allowing for improved early control of HIV-1. In contrast, persistent Gal-9 production might impair Tim-3 activity and contribute to NK cell dysfunction in chronic HIV-1 infection.

Background: Herpes Simplex Virus Type 2 (HSV-2) is highly prevalent in regions disproportionately affected by the human immunodeficiency virus (HIV-1) epidemic. The objective of our study was to identify the risk factors of HSV-2 and HIV-1 infections and to examine the association between the two infections., Methods: The study participants were recruited through a community based cross-sectional study that was conducted from November 2002 to March 2003 in the Moshi urban district of Northern Tanzania. A two-stage sampling design was used in recruiting the study participants. Information on socio-demographics, alcohol use, sexual behaviors, and STIs symptoms were obtained. Blood and urine samples were drawn for testing of HIV-1, HSV-2 and other STIs., Results: The prevalence of HSV-2 infection among all study participants was 43%. The prevalence rate of HSV-2 among the HIV-negative and HIV-positive women was 40% and 65%, respectively. We found 2.72 times odds of having HIV-1 in an HSV-2 positive woman than in an HSV-2 negative woman. Furthermore, HIV-1 and HSV-2 shared common high-risk sexual behavior factors such as early onset of sexual debut, and testing positive for other STIs., Conclusions: Our findings suggest that HSV-2 may be both a biological and risk-associated cofactor for HIV-1 acquisition. In resource-limited countries, where both infections are prevalent efforts at symptomatic and diagnostic screening and treatment of HSV-2 should be part of HIV-1 prevention programs.

Background: HIV-1 infection is associated with profound dysfunction of myeloid dendritic cells, for reasons that remain ill-defined. Soluble HLA class I molecules can have important inhibitory effects on T cells and NK cells, but may also contribute to reduced functional properties of professional antigen-presenting cells. Here, we investigated the expression of soluble HLA class I isoforms during HIV-1 infection and assessed their functional impact on antigen-presenting characteristics of dendritic cells., Results: Soluble HLA class I molecules were highly upregulated in progressive HIV-1 infection as determined by quantitative Western blots. This was associated with strong increases of intracellular expression of HLA class I isoforms in dendritic cells and monocytes. Using mixed lymphocyte reactions, we found that soluble HLA class I molecules effectively inhibited the antigen-presenting properties of dendritic cells, however, there was no significant influence of HLA class I molecules on the cytokine-secretion properties of these cells. The immunomodulatory effects of soluble HLA class I molecules were mediated by interactions with inhibitory myelomonocytic MHC class I receptors from the Leukocyte Immunoglobulin Like Receptor (LILR) family., Conclusions: During progressive HIV-1 infection, soluble HLA class I molecules can contribute to systemic immune dysfunction by inhibiting the antigen-presenting properties of myeloid dendritic cells through interactions with inhibitory myelomonocytic HLA class I receptors.