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We show how to preprocess a polygonal domain with a fixed starting point $s$ in order to answer efficiently the following queries: Given a point $q$, how should one move from $s$ in order to see $q$ as soon as possible? This query resembles the well-known shortest-path-to-a-point query, except that the latter asks for the fastest way to reach $q$, instead of seeing it. Our solution methods include a data structure for a different generalization of shortest-path-to-a-point queries, which may be of independent interest: to report efficiently a shortest path from $s$ to a query segment in the domain.

HIV-1 Nef mediates immune evasion and viral pathogenesis in part through downregulation of cell surface cluster of differentiation 4 (CD4) and major histocompatibility complex class I (MHC-I) on infected cells. While Nef function of circulating viral populations found early in infection has been associated with reservoir size in early-treated cohorts, there is limited research on how its activity impacts reservoir size in people initiating treatment during chronic infection. In addition, there is little research on its role in persistence of viral variants during long-term antiretroviral therapy (ART). Phylogenetically distinct nef genes (n=82) with varying estimated times of reservoir entry were selected from viral outgrowth variants stimulated from the reservoir of South African women living with HIV who initiated ART during chronic infection (n=16). These nef genes were synthesized and used in a pseudovirus infection assay that measures CD4 and MHC-I downregulation via flow cytometry. Downregulation measures were compared to the size of the replication-competent viral reservoir (RC-VR), estimated by quantitative viral outgrowth assay (QVOA) at 5 years after treatment initiation, as well as proviral survival time. Maximum Nef-mediated MHC-I downregulation was significantly associated with RC-VR size (p=0.034), but this association was not observed for CD4 downregulation. Conversely, we did not find a consistent association between intraparticipant MHC-I or CD4 downregulation and the variant timing of entry into the reservoir. These data support a role for Nef-mediated MHC-I downregulation in determining RC-VR size, but more work is needed to determine Nef's role in the survival of individual viral variants over time.

During antiretroviral therapy (ART), most people living with HIV-1 have undetectable HIV-1 RNA in their plasma. However, they occasionally present with new or progressive neurologic deficits and detectable HIV-1 RNA in the cerebrospinal fluid (CSF), a condition defined as neurosymptomatic HIV-1 CSF escape (NSE). We explored the source of neuropathogenesis and HIV-1 RNA in the CSF during NSE by characterizing HIV-1 populations and inflammatory biomarkers in CSF from 25 individuals with NSE. HIV-1 populations in the CSF were uniformly drug resistant and adapted to replication in CD4+ T cells, but differed greatly in genetic diversity, with some having low levels of diversity similar to those observed during untreated primary infection and others having high levels like those during untreated chronic infection. Higher diversity in the CSF during NSE was associated with greater CNS inflammation. Finally, optimization of ART regimen was associated with viral suppression and improvement of neurologic symptoms. These results are consistent with CNS inflammation and neurologic injury during NSE being driven by replication of partially drug-resistant virus in CNS CD4+ T cells. This is unlike nonsuppressible viremia in the plasma during ART, which typically lacks clinical consequences and is generated by virus expression without replication.

Using the Olink Explore 1536 platform, we measured 1,463 unique proteins in 303 cerebrospinal fluid (CSF) specimens from four clinical centers contributed by uninfected controls and 12 groups of people living with HIV-1 infection representing the spectrum of progressive untreated and treated chronic infection. We present three initial analyses of these measurements: an overview of the CSF protein features of the sample; correlations of the CSF proteins with CSF HIV-1 RNA and neurofilament light chain protein (NfL) concentrations; and comparison of CSF proteins in HIV-associated dementia (HAD) and neurosymptomatic CSF escape (NSE). These reveal a complex but coherent picture of CSF protein changes with highest concentrations of many proteins during CNS injury in the HAD and NSE groups and variable protein changes across the course of systemic HIV-1 progression that included two common patterns, designated as lymphoid and myeloid patterns, related to principal involvement of their underlying inflammatory cell lineages. Antiretroviral therapy reduced CSF protein perturbations, though not always to control levels. The dataset of these CSF protein measurements, along with background clinical information, is posted online. Extended studies of this unique dataset will supplement this report to provide more detailed characterization of the dynamic impact of HIV-1 infection on the CSF proteome across the spectrum of HIV-1 infection, advancing the mechanistic understanding of HIV-1-related CNS pathobiology., Competing Interests: HZ has served at scientific advisory boards and/or as a consultant for Abbvie, Acumen, Alector, Alzinova, ALZPath, Amylyx, Annexon, Apellis, Artery Therapeutics, AZTherapies, Cognito Therapeutics, CogRx, Denali, Eisai, Merry Life, Nervgen, Novo Nordisk, Optoceutics, Passage Bio, Pinteon Therapeutics, Prothena, Red Abbey Labs, reMYND, Roche, Samumed, Siemens Healthineers, Triplet Therapeutics, and Wave, has given lectures in symposia sponsored by Alzecure, Biogen, Cellectricon, Fujirebio, Lilly, Novo Nordisk, and Roche, and is a co-founder of Brain Biomarker Solutions in Gothenburg AB (BBS), which is a part of the GU Ventures Incubator Program (outside submitted work). These organizations and companies had no role in the study design, data collection, decision to publish, or publication of the manuscript. MG has received research grants from Gilead Sciences and honoraria as speaker, DSMB committee member and/or scientific advisor from Amgen, AstraZeneca, Biogen, Bristol-Myers Squibb, Gilead Sciences, GlaxoSmithKline/ViiV, Janssen-Cilag, MSD, Novocure, Novo Nordic, Pfizer and Sanofi. These organizations and companies had no role in the study design, data collection, decision to publish, or publication of the manuscript., (Copyright: © 2024 Hu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

In efforts towards eliminating malaria, a discovery program was initiated to identify a novel antimalarial using KAF156 as a starting point. Following the most recent TCP/TPP guidelines, we have identified mCMQ069 with a predicted single oral dose for treatment (∼40-106 mg) and one-month chemoprevention (∼96-216 mg). We have improved unbound MPC and predicted human clearance by 18-fold and 10-fold respectively when compared to KAF156.

Recent malaria drug discovery approaches have been extensively focused on the development of oral, smallmolecule inhibitors for disease treatment whereas parenteral routes of administration have been avoided due to limitations in deploying a shelf-stable injectable even though it could be dosed less frequently. However, an updated target candidate profile from Medicines for Malaria Venture (MMV) and stakeholders have advocated for long-acting injectable chemopreventive agents as an important interventive tool to improve malaria prevention. Here, we present strategies for the development of a long-acting, intramuscular, injectable atovaquone prophylactic therapy. We have generated three prodrug approaches that are contrasted by their differential physiochemical properties and pharmacokinetic profiles: mCBK068, a docosahexaenoic acid ester of atovaquone formulated in sesame oil, mCKX352, a heptanoic acid ester of atovaquone formulated as a solution in sesame oil, and mCBE161, an acetic acid ester of atovaquone formulated as an aqueous suspension. As a result, from a single 20 mg/kg intramuscular injection, mCKX352 and mCBE161 maintain blood plasma exposure of atovaquone above the minimal efficacious concentration for >70 days and >30 days, respectively, in cynomolgus monkeys. The differences in plasma exposure are reflective of the prodrug strategy, which imparts altered chemical properties that ultimately influence aqueous solubility and depot release kinetics. On the strength of the pharmacokinetic and safety profiles, mCBE161 is being advanced as a first-in-class clinical candidate for first-in-human trials.

Type 2 alveolar epithelial cells (AEC2s) are stem cells in the adult lung that contribute to lower airway repair. Agents that promote the selective expansion of these cells might stimulate regeneration of the compromised alveolar epithelium, an etiology-defining event in several pulmonary diseases. From a high-content imaging screen of the drug repurposing library ReFRAME, we identified that dipeptidyl peptidase 4 (DPP4) inhibitors, widely used type 2 diabetes medications, selectively expand AEC2s and are broadly efficacious in several mouse models of lung damage. Mechanism of action studies revealed that the protease DPP4, in addition to processing incretin hormones, degrades IGF-1 and IL-6, essential regulators of AEC2 expansion whose levels are increased in the luminal compartment of the lung in response to drug treatment. To selectively target DPP4 in the lung with sufficient drug exposure, we developed NZ-97, a locally delivered, lung persistent DPP4 inhibitor that broadly promotes efficacy in mouse lung damage models with minimal peripheral exposure and good tolerability. This work reveals DPP4 as a central regulator of AEC2 expansion and affords a promising therapeutic approach to broadly stimulate regenerative repair in pulmonary disease., Competing Interests: Competing interests statement:The authors declare no competing interest.

Human immunodeficiency virus type 1 typically requires a high density of CD4 for efficient entry as a mechanism to target CD4+ T cells (T-tropic), with CCR5 being used most often as the coreceptor. When target T cells are limiting, the virus can evolve to infect cells with a low density of CD4 such as macrophages (M-tropic). The entry phenotype is known to be encoded in the viral Env protein on the surface of the virus particle. Using data showing a dose response for infectivity based on CD4 surface density, we built a model consistent with T-tropic viruses requiring multiple CD4 molecules to mediate infection, whereas M-tropic viruses can infect cells using a single CD4 receptor molecule interaction. We also found that T-tropic viruses bound to the surface of cells with a low density of CD4 are released more slowly than M-tropic viruses which we modeled to be due to multiple interactions of the T-tropic virus with multiple CD4 molecules to allow the initial stable binding. Finally, we found that some M-tropic Env proteins, as the gp120 subunit, possess an enhanced affinity for CD4 compared with their T-tropic pair, indicating that the evolution of macrophage tropism can be reflected both in the closed Env trimer conformation on the virion surface and, in some cases, also in the open confirmation of gp120 Env. Collectively, these studies reveal differences in the stoichiometry of interaction of T-tropic and M-tropic viruses with CD4 and start to identify the basis of binding differences at the biochemical level., Importance: Human immunodeficiency virus type 1 normally targets CD4+ T cells for viral replication. When T cells are limiting, the virus can evolve to infect myeloid cells. The evolutionary step involves a change from requiring a high surface density of CD4 for entry to being able to infect cells with a low density of CD4, as is found on myeloid lineage cells such as macrophage and microglia. Viruses able to infect macrophages efficiently are most often found in the CNS late in the disease course, and such viruses may contribute to neurocognitive impairment. Here, we examine the CD4 binding properties of the viral Env protein to explore these two different entry phenotypes., Competing Interests: The authors declare no conflict of interest.

People with HIV-1 (PWH) on antiretroviral therapy (ART) can maintain undetectable virus levels, but a small pool of infected cells persists. This pool is largely comprised of defective proviruses that may produce HIV-1 proteins but are incapable of making infectious virus, with only a fraction (~10%) of these cells harboring intact viral genomes, some of which produce infectious virus following ex vivo stimulation (i.e. inducible intact proviruses). A majority of the inducible proviruses that persist on ART are formed near the time of therapy initiation. Here we compared proviral DNA (assessed here as 3' half genomes amplified from total cellular DNA) and inducible replication competent viruses in the pool of infected cells that persists during ART to determine if the original infection of these cells occurred at comparable times prior to therapy initiation. Overall, the average percent of proviruses that formed late (i.e. around the time of ART initiation, 60%) did not differ from the average percent of replication competent inducible viruses that formed late (69%), and this was also true for proviral DNA that was hypermutated (57%). Further, there was no evidence that entry into the long-lived infected cell pool was impeded by the ability to use the CXCR4 coreceptor, nor was the formation of long-lived infected cells enhanced during primary infection, when viral loads are exceptionally high. We observed that infection of cells that transitioned to be long-lived was enhanced among people with a lower nadir CD4+ T cell count. Together these data suggest that the timing of infection of cells that become long-lived is impacted more by biological processes associated with immunodeficiency before ART than the replication competency and/or cellular tropism of the infecting virus or the intactness of the provirus. Further research is needed to determine the mechanistic link between immunodeficiency and the timing of infected cells transitioning to the long-lived pool, particularly whether this is due to differences in infected cell clearance, turnover rates and/or homeostatic proliferation before and after ART., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Joseph et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

There remains a need to develop novel SARS-CoV-2 therapeutic options that improve upon existing therapies by an increased robustness of response, fewer safety liabilities, and global-ready accessibility. Functionally critical viral main protease (M pro , 3CL pro ) of SARS-CoV-2 is an attractive target due to its homology within the coronaviral family, and lack thereof toward human proteases. In this disclosure, we outline the advent of a novel SARS-CoV-2 3CL pro inhibitor, CMX990 , bearing an unprecedented trifluoromethoxymethyl ketone warhead. Compared with the marketed drug nirmatrelvir (combination with ritonavir = Paxlovid), CMX990 has distinctly differentiated potency (∼5× more potent in primary cells) and human in vitro clearance (>4× better microsomal clearance and >10× better hepatocyte clearance), with good in vitro -to- in vivo correlation. Based on its compelling preclinical profile and projected once or twice a day dosing supporting unboosted oral therapy in humans, CMX990 advanced to a Phase 1 clinical trial as an oral drug candidate for SARS-CoV-2.

Chronic cutaneous wounds remain a persistent unmet medical need that decreases life expectancy and quality of life. Here, we report that topical application of PY-60, a small-molecule activator of the transcriptional coactivator Yes-associated protein (YAP), promotes regenerative repair of cutaneous wounds in pig and human models. Pharmacological YAP activation enacts a reversible pro-proliferative transcriptional program in keratinocytes and dermal cells that results in accelerated re-epithelization and regranulation of the wound bed. These results demonstrate that transient topical administration of a YAP activating agent may represent a generalizable therapeutic approach to treating cutaneous wounds.

Brain microglia (MG) may serve as a human immunodeficiency virus 1 (HIV) reservoir and ignite rebound viremia following cessation of antiretroviral therapy (ART), but they have yet to be proven to harbor replication-competent HIV. Here, we isolated brain myeloid cells (BrMCs) from nonhuman primates and rapid autopsy of people with HIV (PWH) on ART and sought evidence of persistent viral infection. BrMCs predominantly displayed microglial markers, in which up to 99.9% of the BrMCs were TMEM119+ MG. Total and integrated SIV or HIV DNA was detectable in the MG, with low levels of cell-associated viral RNA. Provirus in MG was highly sensitive to epigenetic inhibition. Outgrowth virus from parietal cortex MG in an individual with HIV productively infected both MG and PBMCs. This inducible, replication-competent virus and virus from basal ganglia proviral DNA were closely related but highly divergent from variants in peripheral compartments. Phenotyping studies characterized brain-derived virus as macrophage tropic based on the ability of the virus to infect cells expressing low levels of CD4. The lack of genetic diversity in virus from the brain suggests that this macrophage-tropic lineage quickly colonized brain regions. These data demonstrate that MG harbor replication-competent HIV and serve as a persistent reservoir in the brain.

Despite viral suppression with antiretroviral therapy (ART), people with human immunodeficiency virus (HIV) continue to experience central nervous system (CNS) complications, primarily in the form of mild cognitive impairment and mental health disorders (eg, depression, anxiety, other neuropsychiatric problems). The multifactorial pathogenesis and heterogeneity of mechanisms likely underlying CNS complications must be addressed in the development of preventive interventions and effective treatments. The biotyping approach has previously been useful to define phenotypes of other CNS diseases based on underlying mechanisms and could be translated to the field of neuroHIV. The purpose of the Biotype Workshop series, and the Virology, Immunology and Neuropathology Working Group in particular, is to capitalize on current and new technologies and guide future research efforts using the wealth of available immunological, virologic, and neuropathological data collected from people with HIV on and off ART., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

HIV-1 replication can be suppressed with antiretroviral therapy (ART), but individuals who stop taking ART soon become viremic again. Some people experience extended times of detectable viremia despite optimal adherence to ART. In this issue of the JCI, White, Wu, and coauthors elucidate a source of nonsuppressible viremia (NSV) in treatment-adherent patients - clonally expanded T cells harboring HIV-1 proviruses with small deletions or mutations in the 5'-leader, the UTR that includes the major splice donor site of viral RNA. These mutations altered viral RNA-splicing efficiency and RNA dimerization and packaging, yet still allowed production of detectable levels of noninfectious virus particles. These particles lacked the HIV-1 Env surface protein required for cell entry and failed to form the mature capsid cone required for infectivity. These studies improve our understanding of NSV and the regulation of viral functions in the 5'-leader with implications for rationalized care in individuals with NSV.

HIV-1 persists as a latent reservoir in people receiving suppressive antiretroviral therapy (ART). When ART is interrupted (treatment interruption/TI), rebound virus re-initiates systemic infection in the lymphoid system. During TI, HIV-1 is also detected in cerebrospinal fluid (CSF), although the source of this rebound virus is unknown. To investigate whether there is a distinct HIV-1 reservoir in the central nervous system (CNS), we compared rebound virus after TI in the blood and CSF of 11 participants. Peak rebound CSF viral loads vary and we show that high viral loads and the appearance of clonally amplified viral lineages in the CSF are correlated with the transient influx of white blood cells. We found no evidence of rebound macrophage-tropic virus in the CSF, even in one individual who had macrophage-tropic HIV-1 in the CSF pre-therapy. We propose a model in which R5 T cell-tropic virus is released from infected T cells that enter the CNS from the blood (or are resident in the CNS during therapy), with clonal amplification of infected T cells and virus replication occurring in the CNS during TI., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

HIV infection has transitioned from an acute, fatal disease to a chronic one managed by antiretroviral therapy. Thus, the aging population of people living with HIV (PLWH) continues to expand. HIV infection results in a dysregulated immune system, wherein CD4 + T cells are depleted, particularly in the gastrointestinal tract, disrupting the gut epithelial barrier. Long-term HIV infection is associated with chronic inflammation through potentially direct mechanisms caused by viral replication or exposure to viral proteins and indirect mechanisms resulting from increased translocation of microbial products from the intestine or exposure to antiretroviral therapy. Chronic inflammation (as marked by IL [interleukin]-6 and CRP [C-reactive protein]) in PLWH promotes endothelial cell dysfunction and atherosclerosis. PLWH show significantly increased rates of cardiovascular disease, such as myocardial infarction (risk ratio, 1.79 [95% CI, 1.54-2.08]) and stroke (risk ratio, 2.56 [95% CI, 1.43-4.61]). In addition, PLWH have increased levels of the coagulation biomarker D-dimer and have a two to ten-fold increased risk of venous thromboembolism compared with the general population. Several small clinical trials analyzed the effect of different antithrombotic agents on platelet activation, coagulation, inflammation, and immune cell activation. Although some markers for coagulation were reduced, most agents failed to reduce inflammatory markers in PLWH. More studies are needed to understand the underlying mechanisms driving inflammation in PLWH to create better therapies for lowering chronic inflammation in PLWH. Such therapies can potentially reduce atherosclerosis, cardiovascular disease, and thrombosis rates in PLWH and thus overall mortality in this population.

Objective: Transcriptomic changes in joint tissues during the development of osteoarthritis (OA) are of interest for the discovery of biomarkers and mechanisms of disease. The objective of this study was to use the rat medial meniscus transection (MMT) model to discover stage and tissue-specific transcriptomic changes., Design: Sham or MMT surgeries were performed in mature rats. Cartilage, menisci and synovium were scored for histopathological changes at 2, 4 and 6 weeks post-surgery and processed for RNA-sequencing. Differentially expressed genes (DEG) were used to identify pathways and mechanisms. Published transcriptomic datasets from animal models and human OA were used to confirm and extend present findings., Results: The total number of DEGs was already high at 2 weeks (723 in meniscus), followed by cartilage (259) and synovium (42) and declined to varying degrees in meniscus and synovium but increased in cartilage at 6 weeks. The most upregulated genes included tenascins. The 'response to mechanical stimulus' and extracellular matrix-related pathways were enriched in both cartilage and meniscus. Pathways that were enriched in synovium at 4 weeks indicate processes related to synovial hyperplasia and fibrosis. Synovium also showed upregulation of IL-11 and several MMPs. The mechanical stimulus pathway included upregulation of the mechanoreceptors PIEZO1, PIEZO2 and TRPV4 and nerve growth factor. Analysis of data from prior RNA-sequencing studies of animal models and human OA support these findings., Conclusion: These results indicate several shared pathways that are affected during OA in cartilage and meniscus and support the role of mechanotransduction and other pathways in OA pathogenesis., (Copyright © 2022 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Background: In this work, we carried out a cross-sectional study examining HIV-1 and HCV free virus concentrations in blood and cerebrospinal fluid (CSF) to determine whether HIV-1 enters the central nervous system (CNS) passively as virus particles or in the context of migrating infected cells. If virions migrate freely across the blood-cerebrospinal fluid barrier (BCSFB) or the blood-brain barrier (BBB) then HCV and HIV-1 would be detectable in the CSF at proportions similar to that in the blood. Alternatively, virus entry as an infected cell would favor selective entry of HIV-1., Methods: We measured HIV-1 and HCV viral loads in the CSF and blood plasma of 4 co-infected participants who were not on antiviral regimens for either infection. We also generated HIV-1 env sequences and performed phylogenetic analyses to determine whether HIV-1 populations in the CSF of these participants were being maintained by local replication., Results: While CSF samples taken from all participants had detectable levels of HIV-1, HCV was not detectable in any of the CSF samples despite participants having HCV concentrations in their blood plasma, which exceeded that of HIV-1. Further, there was no evidence of compartmentalized HIV-1 replication in the CNS (Supplementary Figure 1). These results are consistent with a model where HIV-1 particles cross the BBB or the BCSFB within infected cells. In this scenario, we would expect HIV-1 to reach the CSF more readily because the blood contains a much greater number of HIV-infected cells than HCV-infected cells., Conclusions: HCV entry into the CSF is restricted, indicating that virions do not freely migrate across these barriers and supporting the concept that HIV-1 is transported across the BCSFB and/or BBB by the migration of HIV-infected cells as part of an inflammatory response or normal surveillance., Competing Interests: UNC is pursuing intellectual property protection for Primer ID. RS is listed as a co-inventor and has received nominal royalties. GS is an employee of Vir Biotechnology Inc. and may hold stock or stock options. Vir Biotechnology provided no funding for this work. The authors have no additional conflicts of interest., (Copyright © 2023 Pathogens and Immunity.)

HIV-1 infection within the central nervous system (CNS) includes evolution of the virus, damaging inflammatory cascades, and the involvement of multiple cell types; however, our understanding of how Env tropism and inflammation can influence CNS infectivity is incomplete. In this study, we utilize macrophage-tropic and T cell-tropic HIV-1 Env proteins to establish accurate infection profiles for multiple CNS cells under basal and interferon alpha (IFN-α) or lipopolysaccharide (LPS)-induced inflammatory states. We found that macrophage-tropic viruses confer entry advantages in primary myeloid cells, including monocyte-derived macrophage, microglia, and induced pluripotent stem cell (iPSC)-derived microglia. However, neither macrophage-tropic or T cell-tropic HIV-1 Env proteins could mediate infection of astrocytes or neurons, and infection was not potentiated by induction of an inflammatory state in these cells. Additionally, we found that IFN-α and LPS restricted replication in myeloid cells, and IFN-α treatment prior to infection with vesicular stomatitis virus G protein (VSV G) Envs resulted in a conserved antiviral response across all CNS cell types. Further, using RNA sequencing (RNA-seq), we found that only myeloid cells express HIV-1 entry receptor/coreceptor transcripts at a significant level and that these transcripts in select cell types responded only modestly to inflammatory signals. We profiled the transcriptional response of multiple CNS cells to inflammation and found 57 IFN-induced genes that were differentially expressed across all cell types. Taken together, these data focus attention on the cells in the CNS that are truly permissive to HIV-1, further highlight the role of HIV-1 Env evolution in mediating infection in the CNS, and point to limitations in using model cell types versus primary cells to explore features of virus-host interaction. IMPORTANCE The major feature of HIV-1 pathogenesis is the induction of an immunodeficient state in the face of an enhanced state of inflammation. However, for many of those infected, there can be an impact on the central nervous system (CNS) resulting in a wide range of neurocognitive defects. Here, we use a highly sensitive and quantitative assay for viral infectivity to explore primary and model cell types of the brain for their susceptibility to infection using viral entry proteins derived from the CNS. In addition, we examine the ability of an inflammatory state to alter infectivity of these cells. We find that myeloid cells are the only cell types in the CNS that can be infected and that induction of an inflammatory state negatively impacts viral infection across all cell types.

The simplicity, transparency, reliability, high efficiency and robust nature of PID controllers are some of the reasons for their high popularity and acceptance for control in process industries around the world today. Tuning of PID control parameters has been a field of active research and still is. The primary objectives of PID control parameters are to achieve minimal overshoot in steady state response and lesser settling time. With exception of two popular conventional tuning strategies (Ziegler Nichols closed loop oscillation and Cohen-Coon's process reaction curve) several other methods have been employed for tuning. This work accords a thorough review of state-of-the-art and classical strategies for PID controller parameters tuning using metaheuristic algorithms. Methods appraised are categorized into classical and metaheuristic optimization methods for PID parameters tuning purposes. Details of some metaheuristic algorithms, methods of application, equations and implementation flowcharts/algorithms are presented. Some open problems for future research are also presented. The major goal of this work is to proffer a comprehensive reference source for researchers and scholars working on PID controllers., Competing Interests: The authors declare no conflict of interest., (© 2022 The Author(s).)

We tested the combination of a broadly neutralizing HIV antibody with the latency reversal agent vorinostat (VOR). Eight participants received 2 month-long cycles of VRC07-523LS with VOR. Low-level viremia, resting CD4+ T-cell-associated HIV RNA (rca-RNA) was measured, and intact proviral DNA assay (IPDA) and quantitative viral outgrowth assay (QVOA) were performed at baseline and posttreatment. In 3 participants, IPDA and QVOA declines were accompanied by significant declines of rca-RNA. However, no IPDA or QVOA declines clearly exceeded assay variance or natural decay. Increased resistance to VRC07-523LS was not observed. This combination therapy did not reduce viremia or the HIV reservoir. Clinical Trials Registration. NCT03803605., (© The Author(s) 2021. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Staphylococcus aureus is a serious threat to public health due to the rise of antibiotic resistance in this organism, which can prolong or exacerbate skin and soft tissue infections (SSTIs). Methicillin-resistant S. aureus is a Gram-positive bacterium and a leading cause of SSTIs. As such, many efforts are under way to develop therapies that target essential biological processes in S. aureus. Antimicrobial photodynamic therapy is an effective alternative to antibiotics; therefore we developed an approach to simultaneously expose S. aureus to intracellular and extracellular photosensitizers. A near infrared photosensitizer was conjugated to human monoclonal antibodies (MAbs) that target the S. aureus iron-regulated surface determinant (Isd) heme acquisition proteins. In addition, the compound VU0038882 was developed to increase photoactivatable porphyrins within the cell. Combinatorial photodynamic treatment of drug-resistant S. aureus exposed to VU0038882 and conjugated anti-Isd MAbs proved to be an effective antibacterial strategy in vitro and in a murine model of SSTIs.

Understanding what shapes the latent human immunodeficiency virus type 1 (HIV-1) reservoir is critical for developing strategies for cure. We measured frequency of persistent HIV-1 infection after 5 years of suppressive antiretroviral therapy initiated during chronic infection. Pretreatment CD8+ T-cell activation, nadir CD4 count, and CD4:CD8 ratio predicted reservoir size., (© The Author(s) 2021. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Despite the development of next-generation antiandrogens, metastatic castration-resistant prostate cancer (mCRPC) remains incurable. Here, we describe a unique semisynthetic bispecific antibody that uses site-specific unnatural amino acid conjugation to combine the potency of a T cell-recruiting anti-CD3 antibody with the specificity of an imaging ligand (DUPA) for prostate-specific membrane antigen. This format enabled optimization of structure and function to produce a candidate (CCW702) with specific, potent in vitro cytotoxicity and improved stability compared with a bispecific single-chain variable fragment format. In vivo, CCW702 eliminated C4-2 xenografts with as few as three weekly subcutaneous doses and prevented growth of PCSD1 patient-derived xenograft tumors in mice. In cynomolgus monkeys, CCW702 was well tolerated up to 34.1 mg/kg per dose, with near-complete subcutaneous bioavailability and a PK profile supporting testing of a weekly dosing regimen in patients. CCW702 is being evaluated in a first in-human clinical trial for men with mCRPC who had progressed on prior therapies (NCT04077021)., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

The ongoing pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), necessitates strategies to identify prophylactic and therapeutic drug candidates for rapid clinical deployment. Here, we describe a screening pipeline for the discovery of efficacious SARS-CoV-2 inhibitors. We screen a best-in-class drug repurposing library, ReFRAME, against two high-throughput, high-content imaging infection assays: one using HeLa cells expressing SARS-CoV-2 receptor ACE2 and the other using lung epithelial Calu-3 cells. From nearly 12,000 compounds, we identify 49 (in HeLa-ACE2) and 41 (in Calu-3) compounds capable of selectively inhibiting SARS-CoV-2 replication. Notably, most screen hits are cell-line specific, likely due to different virus entry mechanisms or host cell-specific sensitivities to modulators. Among these promising hits, the antivirals nelfinavir and the parent of prodrug MK-4482 possess desirable in vitro activity, pharmacokinetic and human safety profiles, and both reduce SARS-CoV-2 replication in an orthogonal human differentiated primary cell model. Furthermore, MK-4482 effectively blocks SARS-CoV-2 infection in a hamster model. Overall, we identify direct-acting antivirals as the most promising compounds for drug repurposing, additional compounds that may have value in combination therapies, and tool compounds for identification of viral host cell targets.

Loss of dopaminergic neurons along the nigrostriatal axis, neuroinflammation, and peripheral immune dysfunction are the pathobiological hallmarks of Parkinson's disease (PD). Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been successfully tested for PD treatment. GM-CSF is a known immune modulator that induces regulatory T cells (Tregs) and serves as a neuronal protectant in a broad range of neurodegenerative diseases. Due to its short half-life, limited biodistribution, and potential adverse effects, alternative long-acting treatment schemes are of immediate need. A long-acting mouse GM-CSF (mPDM608) was developed through Calibr, a Division of Scripps Research. Following mPDM608 treatment, complete hematologic and chemistry profiles and T-cell phenotypes and functions were determined. Neuroprotective and anti-inflammatory capacities of mPDM608 were assessed in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated mice that included transcriptomic immune profiles. Treatment with a single dose of mPDM608 resulted in dose-dependent spleen and white blood cell increases with parallel enhancements in Treg numbers and immunosuppressive function. A shift in CD4 + T-cell gene expression towards an anti-inflammatory phenotype corresponded with decreased microgliosis and increased dopaminergic neuronal cell survival. mPDM608 elicited a neuroprotective peripheral immune transformation. The observed phenotypic shift and neuroprotective response was greater than observed with recombinant GM-CSF (rGM-CSF) suggesting human PDM608 as a candidate for PD treatment.

Most myeloid lineage cells express the receptor and coreceptors that make them susceptible to infection by primate lentiviruses (SIVs and HIVs). However, macrophages are the only myeloid lineage cell commonly infected by SIVs and/or HIVs. The frequency of infected macrophages varies greatly across specific host and virus combinations as well as disease states, with infection rates being greatest in pathogenic SIV infections of non-natural hosts (i.e., Asian nonhuman primates (Asian NHPs)) and late in untreated HIV-1 infection. In contrast, macrophages from natural SIV hosts (i.e., African NHPs) are largely resistant to infection due to entry and/or post-entry restriction mechanisms. These highly variable rates of macrophage infection may stem from differences in the host immune environment, entry and post-entry restriction mechanisms, the ability of a virus to adapt to efficiently infect macrophages, and the pleiotropic effects of macrophage-tropism including the ability to infect cells lacking CD4 and increased neutralization sensitivity. Questions remain about the relationship between rates of macrophage infection and viral pathogenesis, with some evidence suggesting that elevated levels of macrophage infection may contribute to greater pathogenesis in non-natural SIV hosts. Alternatively, extensive infection of macrophages may only emerge in the context of high viral loads and immunodeficiency, making it a symptom of highly pathogenic infections, not a primary driver of pathogenesis.

Bariatric surgery results in increased intestinal secretion of hormones GLP-1 and anorexigenic PYY, which is believed to contribute to the clinical efficacy associated with the procedure. This observation raises the question whether combination treatment with gut hormone analogs might recapitulate the efficacy and mitigate the significant risks associated with surgery. Despite PYY demonstrating excellent efficacy and safety profiles with regard to food intake reduction, weight loss, and glucose control in preclinical animal models, PYY-based therapeutic development remains challenging given a low serum stability and half-life for the native peptide. Here, combined peptide stapling and PEG-fatty acid conjugation affords potent PYY analogs with >14 h rat half-lives, which are expected to translate into a human half-life suitable for once-weekly dosing. Excellent efficacy in glucose control, food intake reduction, and weight loss for lead candidate 22 in combination with our previously reported long-acting GLP-1 analog is demonstrated in a diet-induced obesity mouse model.

Stimulator of interferon genes (STING) links innate immunity to biological processes ranging from antitumor immunity to microbiome homeostasis. Mechanistic understanding of the anticancer potential for STING receptor activation is currently limited by metabolic instability of the natural cyclic dinucleotide (CDN) ligands. From a pathway-targeted cell-based screen, we identified a non-nucleotide, small-molecule STING agonist, termed SR-717, that demonstrates broad interspecies and interallelic specificity. A 1.8-angstrom cocrystal structure revealed that SR-717 functions as a direct cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) mimetic that induces the same "closed" conformation of STING. SR-717 displayed antitumor activity; promoted the activation of CD8 + T, natural killer, and dendritic cells in relevant tissues; and facilitated antigen cross-priming. SR-717 also induced the expression of clinically relevant targets, including programmed cell death 1 ligand 1 (PD-L1), in a STING-dependent manner., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Farnesoid X receptor (FXR) agonists are emerging as important potential therapeutics for the treatment of nonalcoholic steatohepatitis (NASH) patients, as they exert positive effects on multiple aspects of the disease. FXR agonists reduce lipid accumulation in the liver, hepatocellular inflammation, hepatic injury, and fibrosis. While there are currently no approved therapies for NASH, the bile acid-derived FXR agonist obeticholic acid (OCA; 6-ethyl chenodeoxycholic acid) has shown promise in clinical studies. Previously, we described the discovery of tropifexor (LJN452), the most potent non-bile acid FXR agonist currently in clinical investigation. Here, we report the discovery of a novel chemical series of non-bile acid FXR agonists based on a tricyclic dihydrochromenopyrazole core from which emerged nidufexor (LMB763), a compound with partial FXR agonistic activity in vitro and FXR-dependent gene modulation in vivo . Nidufexor has advanced to Phase 2 human clinical trials in patients with NASH and diabetic nephropathy.

Human immunodeficiency virus type 1 (HIV-1) infection persists despite years of antiretroviral therapy (ART). To remove the stigma and burden of chronic infection, approaches to eradicate or cure HIV infection are desired. Attempts to augment ART with therapies that reverse viral latency, paired with immunotherapies to clear infection, have advanced into the clinic, but the field is still in its infancy. We review foundational studies and highlight new insights in HIV cure research. Together with advances in ART delivery and HIV prevention strategies, future therapies that clear HIV infection may relieve society of the affliction of the HIV pandemic., Competing Interests: Declaration of Interests J.J.E. reports grants and personal fees from Merck and personal fees from Gilead outside of the submitted work. D.M.M. reports personal fees from Merck and ViiV Healthcare and holds common stock in Gilead. C.L.G. reports research grants from Viiv and Gilead., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Objective: To study the contribution of γδ T cells to the persistent HIV reservoir., Design: Fifteen HIV-seropositive individuals on suppressive ART were included. We performed parallel quantitative viral outgrowth assays (QVOA) of resting CD4 T (rCD4) cells in the presence or absence of γδ T cells., Methods: Resting αβ+CD4 T cells were magnetically isolated from PBMCs using two different custom cocktails, only one kit contained antibodies to deplete γδ T cells, resulting in two populations: rCD4 cells and rCD4 cells depleted of γδ cells. Frequency of infection was analyzed by QVOA and DNA measurements., Results: Recovery of replication-competent HIV from cultures of rCD4 cells was similar in 11 individuals despite the presence of γδ T cells. In four donors, HIV recovery was lower when γδ T cells were present. Expression of the cytotoxic marker CD16 on Vδ2 cells was the only variable associated with the lower HIV recovery. Our results highlight the potency of those responses since a mean of 10 000 γδ T cells were present within 2.5 million rCD4 cells. However, despite the low frequency of γδ T cells, the presence of cytotoxic Vδ2 cells correlated with lower HIV recovery from cultures of rCD4 cells., Conclusion: Results of this study show that quantification of the contribution of γδ T cells to the reservoir is challenging because of their low numbers compared with conventional rCD4 cells and highlights the potent antiviral function of γδ T cells and the impact of their presence on the frequency of latent HIV infection.

The effects of oxytocin on food intake and body weight reduction have been demonstrated in both animal models and human clinical studies. Despite being efficacious, oxytocin is enzymatically unstable and thus considered to be unsuitable for long-term use in patients with obesity. Herein, a series of oxytocin derivatives were engineered through conjugation with fatty acid moieties that are known to exhibit high binding affinities to serum albumin. One analog ( OT-12 ) in particular was shown to be a potent full agonist at the oxytocin receptor (OTR) in vitro with good selectivity and long half-life (24 h) in mice. Furthermore, OT-12 is peripherally restricted, with very limited brain exposure (1/190 of the plasma level). In a diet-induced obesity mouse model, daily subcutaneous administration of OT-12 exhibited more potent anorexigenic and body weight reducing effects than carbetocin. Thus, our results suggest that the long-acting, peripherally restricted OTR agonist may offer potential therapeutic benefits for obesity.

Objective: To review the evidence that CSF (cerebrospinal fluid) escape populations are produced by viral reservoirs in the central nervous system (CNS)., Design: CSF escape is a rare phenomenon in which individuals on suppressive ART have well controlled systemic infections with elevated levels of HIV-1 RNA in their CSF. However, the rarity of CSF escape coupled with relatively low CSF viral loads has impeded detailed analyses of these populations. Here, and in a previous study, we performed genetic and phenotypic assessments of CSF escape populations to determine whether CSF escape is produced by CNS reservoirs or by cells trafficking through the CNS., Methods: We report HIV-1 viral loads in the CSF and blood plasma of four individuals with CSF escape (one new example and three previously described examples). We performed phylogenetic analyses of the viral env gene to evaluate diversity within the CSF escape populations and performed entry analyses to determine whether Env proteins were adapted to entering macrophage/microglia., Results: Two individuals had CSF escape produced by CNS reservoirs. In contrast, the remaining two cases were likely because of transient viral production from cells migrating into the CNS and releasing virus., Conclusion: Together our analyses indicate that replication-competent HIV-1 can persist in the CNS during ART, but that not all cases of CSF escape are produced by CNS reservoirs. Our results also suggest that both CD4 T cells and macrophage/microglia can serve as persistent viral reservoirs in the CNS.

Although antiretroviral therapy (ART) is highly effective at suppressing HIV-1 replication, the virus persists as a latent reservoir in resting CD4 + T cells during therapy. This reservoir forms even when ART is initiated early after infection, but the dynamics of its formation are largely unknown. The viral reservoirs of individuals who initiate ART during chronic infection are generally larger and genetically more diverse than those of individuals who initiate therapy during acute infection, consistent with the hypothesis that the reservoir is formed continuously throughout untreated infection. To determine when viruses enter the latent reservoir, we compared sequences of replication-competent viruses from resting peripheral CD4 + T cells from nine HIV-positive women on therapy to viral sequences circulating in blood collected longitudinally before therapy. We found that, on average, 71% of the unique viruses induced from the post-therapy latent reservoir were most genetically similar to viruses replicating just before ART initiation. This proportion is far greater than would be expected if the reservoir formed continuously and was always long lived. We conclude that ART alters the host environment in a way that allows the formation or stabilization of most of the long-lived latent HIV-1 reservoir, which points to new strategies targeted at limiting the formation of the reservoir around the time of therapy initiation., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Background: Human immunodeficiency virus type 1 (HIV-1) populations are detected in cerebrospinal fluid (CSF) of some people on suppressive antiretroviral therapy (ART). Detailed analysis of these populations may reveal whether they are produced by central nervous system (CNS) reservoirs., Methods: We performed a study of 101 asymptomatic participants on stable ART. HIV-1 RNA concentrations were cross-sectionally measured in CSF and plasma. In participants with CSF HIV-1 RNA concentrations sufficient for analysis, viral populations were genetically and phenotypically characterized over multiple time points., Results: For 6% of participants (6 of 101), the concentration of HIV-1 RNA in their CSF was ≥0.5 log copies/mL above that of plasma (ie, CSF escape). We generated viral envelope sequences from CSF of 3 participants. One had a persistent CSF escape population that was macrophage-tropic, partially drug resistant, genetically diverse, and closely related to a minor macrophage-tropic lineage present in the blood prior to viral suppression and enriched for after ART. Two participants (1 suppressed and 1 not) had transient CSF escape populations that were R5 T cell-tropic with little genetic diversity., Conclusions: Extensive analysis of viral populations in 1 participant revealed that CSF escape was from a persistently replicating population, likely in macrophages/microglia, present in the CNS over 3 years of ART. CSF escape in 2 other participants was likely produced by trafficking and transient expansion of infected T cells in the CNS. Our results show that CNS reservoirs can persist during ART and that CSF escape is not exclusively produced by replicating CNS reservoirs., (© The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Recent studies demonstrate that the stable HIV-1 reservoir in resting CD4 + T cells is mostly formed from viruses circulating when combination antiretroviral therapy (ART) is initiated. Here we explore the immunological basis for these observations. Untreated HIV-1 infection is characterized by a progressive depletion of memory CD4 + T cells which mostly express CD127, the α chain of the IL-7 receptor (IL-7R). Depletion results from both direct infection and bystander loss of memory CD4 + T cells in part attributed to dysregulated IL-7/IL-7R signaling. While IL-7/IL7R signaling is not essential for the generation of effector CD4 + T cells from naïve cells, it is essential for the further transition of effectors to memory CD4 + T cells and their subsequent homeostatic maintenance. HIV-1 infection therefore limits the transition of CD4 + T cells from an effector to long-lived memory state. With the onset of ART, virus load (VL) levels rapidly decrease and the frequency of CD127 + CD4 + memory T cells increases, indicating restoration of effector to memory transition in CD4 + T cells. Collectively these data suggest that following ART initiation, HIV-1 infected effector CD4 + T cells transition to long-lived, CD127 + CD4 + T cells forming the majority of the stable HIV-1 reservoir. We propose that combining ART initiation with inhibition of IL-7/IL-7R signaling to block CD4 + T cell memory formation will limit the generation of long-lived HIV-infected CD4 + T cells and reduce the overall size of the stable HIV-1 reservoir.

Farnesoid X receptor (FXR) agonism is emerging as an important potential therapeutic mechanism of action for multiple chronic liver diseases. The bile acid-derived FXR agonist obeticholic acid (OCA) has shown promise in a phase 2 study in patients with nonalcoholic steatohepatitis (NASH). Here, we report efficacy of the novel nonbile acid FXR agonist tropifexor (LJN452) in two distinct preclinical models of NASH. The efficacy of tropifexor at <1 mg/kg doses was superior to that of OCA at 25 mg/kg in the liver in both NASH models. In a chemical and dietary model of NASH (Stelic animal model [STAM]), tropifexor reversed established fibrosis and reduced the nonalcoholic fatty liver disease activity score and hepatic triglycerides. In an insulin-resistant obese NASH model (amylin liver NASH model [AMLN]), tropifexor markedly reduced steatohepatitis, fibrosis, and profibrogenic gene expression. Transcriptome analysis of livers from AMLN mice revealed 461 differentially expressed genes following tropifexor treatment that included a combination of signatures associated with reduction of oxidative stress, fibrogenesis, and inflammation. Conclusion : Based on preclinical validation in animal models, tropifexor is a promising investigational therapy that is currently under phase 2 development for NASH.

Parasitic filarial nematodes cause debilitating infections in people in resource-limited countries. A clinically validated approach to eliminating worms uses a 4- to 6-week course of doxycycline that targets Wolbachia , a bacterial endosymbiont required for worm viability and reproduction. However, the prolonged length of therapy and contraindication in children and pregnant women have slowed adoption of this treatment. Here, we describe discovery and optimization of quinazolines CBR417 and CBR490 that, with a single dose, achieve >99% elimination of Wolbachia in the in vivo Litomosoides sigmodontis filarial infection model. The efficacious quinazoline series was identified by pairing a primary cell-based high-content imaging screen with an orthogonal ex vivo validation assay to rapidly quantify Wolbachia elimination in Brugia pahangi filarial ovaries. We screened 300,368 small molecules in the primary assay and identified 288 potent and selective hits. Of 134 primary hits tested, only 23.9% were active in the worm-based validation assay, 8 of which contained a quinazoline heterocycle core. Medicinal chemistry optimization generated quinazolines with excellent pharmacokinetic profiles in mice. Potent antiwolbachial activity was confirmed in L. sigmodontis , Brugia malayi , and Onchocerca ochengi in vivo preclinical models of filarial disease and in vitro selectivity against Loa loa (a safety concern in endemic areas). The favorable efficacy and in vitro safety profiles of CBR490 and CBR417 further support these as clinical candidates for treatment of filarial infections., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Sparse data exist on the penetration of antiretrovirals into brain tissue. In this work, we present a framework to use efavirenz (EFV) pharmacokinetic (PK) data in plasma, cerebrospinal fluid (CSF), and brain tissue of eight rhesus macaques to predict brain tissue concentrations in HIV-infected individuals. We then perform exposure-response analysis with the model-predicted EFV area under the concentration-time curve (AUC) and neurocognitive scores collected from a group of 24 HIV-infected participants. Adult rhesus macaques were dosed daily with 200 mg EFV (as part of a four-drug regimen) for 10 days. Plasma was collected at 8 time points over 10 days and at necropsy, whereas CSF and brain tissue were collected at necropsy. In the clinical study, data were obtained from one paired plasma and CSF sample of participants prescribed EFV, and neuropsychological test evaluations were administered across 15 domains. PK modeling was performed using ADAPT version 5.0 Biomedical Simulation Resource, Los Angeles, CA) with the iterative two-stage estimation method. An eight-compartment model best described EFV distribution across the plasma, CSF, and brain tissue of rhesus macaques and humans. Model-predicted median brain tissue concentrations in humans were 31 and 8,000 ng/mL, respectively. Model-predicted brain tissue AUC was highly correlated with plasma AUC (γ = 0.99, P < 0.001) but not CSF AUC (γ = 0.34, P = 0.1) and did not show any relationship with neurocognitive scores (γ < 0.05, P > 0.05). This analysis provides an approach to estimate PK the brain tissue in order to perform PK/pharmacodynamic analyses at the target site., (© 2019 The Authors. Clinical and Translational Science published by Wiley Periodicals, Inc. on behalf of the American Society for Clinical Pharmacology and Therapeutics.)

Objective: HIV infection chronically affects the central nervous system (CNS). Olfactory mucosa is a unique site in the respiratory tract that is directly connected to the CNS; thus we wanted to evaluate olfactory mucosa as a surrogate of CNS sampling., Design: We conducted a preliminary study examining HIV populations and susceptible cells in the olfactory mucosa., Methods: Olfactory mucosa was sampled by minimally invasive brushing. Cerebrospinal fluid (CSF) analyses were performed as per routine clinical procedures. Olfactory marker protein, CD4+, CD8+, and trans-activator of transcription (TAT) expressions were assessed by immunohistochemistry. Plasma, CSF, and olfactory mucosa HIV-RNA were quantified using the Cobas AmpliPrep/Cobas TaqMan assay, whereas HIV proviral DNA was evaluated on peripheral blood mononuclear cell and olfactory mucosa. HIV-1 env deep sequencing was performed for phylogenetic analysis., Results: Among ART-naive participants, 88.2% (15/17), and among ART-treated participants, 21.4% (6/28) had detectable HIV-RNA in samples from their olfactory mucosa; CSF escape was more common in patients with olfactory mucosa escape (50 vs. 7.9%; P = 0.010). Olfactory mucosa samples contained few cells positive for CD4, CD8, or HIV-DNA, and no HIV TAT-positive cells, indicating that this approach efficiently samples virions in the olfactory mucosa, but not HIV-infected cells. Yet, using a deep sequencing approach to phylogenetically compare partial HIV env genes in five untreated participants, we identified distinct viral lineages in the OM., Conclusions: The results of this study suggest that nasal brushing is a well tolerated and useful technique for sampling the olfactory mucosa. HIV-RNA was detected in most naïve and in some treated patients, warranting larger longitudinal studies.

After initiating antiretroviral therapy (ART), a rapid decline in HIV viral load is followed by a long period of undetectable viremia. Viral outgrowth assay suggests the reservoir continues to decline slowly. Here, we use full-length sequencing to longitudinally study the proviral landscape of four subjects on ART to investigate the selective pressures influencing the dynamics of the treatment-resistant HIV reservoir. We find intact and defective proviruses that contain genetic elements favoring efficient protein expression decrease over time. Moreover, proviruses that lack these genetic elements, yet contain strong donor splice sequences, increase relatively to other defective proviruses, especially among clones. Our work suggests that HIV expression occurs to a significant extent during ART and results in HIV clearance, but this is obscured by the expansion of proviral clones. Paradoxically, clonal expansion may also be enhanced by HIV expression that leads to splicing between HIV donor splice sites and downstream human exons.