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The objective of this systematic review was to identify the health impact of COVID-19 on mortality, morbidity, hospital admission, and hospital readmission rates in the Black population across Canada.A comprehensive search strategy consisting of relevant subject headings and keywords was executed in five databases: OVID Medline, OVID Embase, EBSCO CINAHL Plus, Web of Science, and Scopus. Additional searches were conducted for gray literature in ProQuest Dissertations and Theses Global, Google Scholar, and an advanced customized Google search for Canadian government documents. All eligible studies included in this review underwent quality assessment.Clinical health outcomes identified included mortality, morbidity, and hospital admission rates; none of the studies reported hospital readmission rates. The search identified 616 citations, and following the removal of duplicates and screening according to our inclusion/exclusion criteria, four articles were eligible for inclusion in the review. All of these studies were conducted in Canada. Study dates ranged from 2020 to 2021.A systematic review of studies on the impact of COVID-19 on the Black population in Canada highlights two key points. First, the collection and availability of race-based data are necessary to clarify the impact of COVID-19 and other diseases on Black populations in Canada. Second, with the limited available data, studies suggest that COVID-19 disproportionately impacts Black populations in Canada, making up high shares of cases, deaths, and hospitalizations compared to most of the population.RéSUMé: OBJECTIF: L’objectif de cette revue systématique était d’identifier l’impact de la COVID-19 sur les taux de mortalité, de morbidité, d’admission à l’hôpital et de réadmission à l’hôpital dans la population noire au Canada. MéTHODES: Une stratégie de recherche complète composée des vedettes-matières et des mots-clés pertinents a été exécutée dans cinq bases de données : OVID Medline, OVID Embase, EBSCO CINAHL Plus, Web of Science et Scopus. Des recherches supplémentaires ont été effectuées pour la littérature grise dans ProQuest Dissertations and Theses Global, Google Scholar et une recherche Google personnalisée pour les documents du gouvernement canadien. Toutes les études éligibles incluses dans cette revue ont fait l’objet d’une évaluation de la qualité. RéSULTATS: Les résultats de santé cliniques identifiés comprenaient les taux de mortalité, de morbidité et d’admission à l’hôpital; aucune des études n’a rapporté de taux de réadmission à l’hôpital. La recherche a identifié 616 citations et à la suite de la suppression des doublons et de la sélection selon nos critères d’inclusion/exclusion, quatre articles étaient éligibles pour l’inclusion dans la revue. Toutes ces études ont été menées au Canada. Les dates des études allaient de 2020 à 2021. CONCLUSION: Une revue systématique des études sur l’impact de la COVID-19 sur la population noire au Canada met en évidence deux points clés. Premièrement, la collection et la disponibilité de données fondées sur la race sont nécessaires pour mieux comprendre l’impact de la COVID-19 et d’autres maladies sur les populations noires au Canada. Deuxièmement, avec les données disponibles, les études suggèrent que la COVID-19 a un impact disproportionné sur les populations noires au Canada, représentant des proportions élevées de cas, de décès et d’hospitalisations par rapport à la plupart de la population.

Background The Meningitis Vaccine Project (MVP) was an international partnership that began in 2010 and focused on the development, testing, licensure, and widespread introduction of a conjugate vaccine with the promise of protecting millions of lives from group A meningococcal meningitis. To bridge gaps upstream of vaccine administration and in understanding the project’s planning, delivery and outcomes, the primary objective of this study was to delineate the barriers to and facilitators of sustainable implementation in an upstream context of the delivery of the MenAfriVac vaccine and to draw critical lessons for other vaccination programs. Methods We conducted a qualitative study with key project partners and leaders who worked on the MVP project. The interviewees were initially identified through a literature review and a search of publications and evaluations of the project. Eighteen key stakeholders were interviewed. The interviewees included individuals in the roles of implementation oversight, champion, partner, funder, and frontline implementer. The Consolidated Framework for Implementation Research (CFIR) informed the identification of a priori codes for the directed content analysis. Results There were multiple barriers and facilitators to implementing the vaccine under all five domains of the CFIR framework, including intervention characteristics, outer setting, inner setting, characteristics of individuals, and process. The most common domains mentioned by stakeholders that facilitated the success of the MVP were inner setting and process; specifically, three constructs were most representative—Tension for Change, Formally Appointed Implementation Leaders, and External Change Agents. Conclusions The tension for change—that is, the degree to which stakeholders perceive the current situation as intolerable or needing change— advocated by African leaders served as a crucial foundation for solid leadership for the MPV. The leadership came in the form of the project director and other formally appointed leaders, who could plan, engage and execute the project’s goals effectively. This leadership translated into consistent messaging, information sharing, and multiple levels of engagement with external change agents (including technical experts), which promoted a shared sense of readiness contributing to significant change investments in implementing MVP.

Significance A complete picture of HIV antigenicity during early replication is needed to elucidate the full range of options for controlling infection through humoral immunity. The HIV envelope protein, gp120, experiences key structural rearrangements during host cell attachment, leading to exposure of highly conserved epitopes on the virion surface. These epitopes enable Fc-mediated antiviral effector functions that may be relevant to HIV prevention. Here, we used 3D superresolution microscopy to show how gp120 epitopes are rapidly exposed distal to cell–virus interfaces, introducing the opportunity for unconstrained antibody binding. These previously unrecognized facets of HIV antigenicity further define relationships between retroviral infection and immunity and should facilitate the development of antibody-based approaches for HIV prevention., A complete picture of HIV antigenicity during early replication is needed to elucidate the full range of options for controlling infection. Such information is frequently gained through analyses of isolated viral envelope antigens, host CD4 receptors, and cognate antibodies. However, direct examination of viral particles and virus–cell interactions is now possible via advanced microscopy techniques and reagents. Using such methods, we recently determined that CD4-induced (CD4i) transition state epitopes in the HIV surface antigen, gp120, while not exposed on free particles, rapidly become immunoreactive upon virus–cell binding. Here, we use 3D direct stochastic optical reconstruction microscopy (dSTORM) to show that certain CD4i epitopes specific to transition state structures are exposed across the surface of cell-bound virions, thus explaining their immunoreactivity. Moreover, such structures and their marker epitopes are dispersed to regions of virions distal to CD4 contact. We further show that the appearance and positioning of distal CD4i exposures is partially dependent on Gag maturation and intact matrix–gp41 interactions within the virion. Collectively, these observations provide a unique perspective of HIV during early replication. These features may define unique insights for understanding how humoral responses target virions and for developing related antiviral countermeasures.

Cancer treatment decisions are increasingly guided by which specific genes are mutated within each patient’s tumor. For example, agents inhibiting the epidermal growth factor receptor (EGFR) benefit many colorectal cancer (CRC) patients, with the general exception of those whose tumor includes a KRAS mutation. However, among the various KRAS mutations, that which encodes the G13D mutant protein (KRAS(G13D)) behaves differently; for unknown reasons, KRAS(G13D) CRC patients benefit from the EGFR-blocking antibody cetuximab. Controversy surrounds this observation, because it contradicts the well-established mechanisms of EGFR signaling with regard to RAS mutations. Here, we identified a systems-level, mechanistic explanation for why KRAS(G13D) cancers respond to EGFR inhibition. A computational model of RAS signaling revealed that the biophysical differences between the three most common KRAS mutants was sufficient to generate different sensitivities to EGFR inhibition. Integrated computation with experimentation then revealed a non-intuitive, mutant-specific dependency of wild-type RAS activation by EGFR that is determined by the interaction strength between KRAS and the tumor suppressor neurofibromin (NF1). KRAS mutants that strongly interacted with and competitively inhibited NF1 drove wild-type RAS activation in an EGFR-independent manner, whereas KRAS(G13D) weakly interacted with and could not competitively inhibit NF1 and, thus, KRAS(G13D) cells remained dependent upon EGFR for wild-type RAS activity. Overall, our work demonstrates how systems approaches enable mechanism-based inference in genomic medicine and can help identify patients for selective therapeutic strategies.

Standard protocol to treat colorectal cancer (CRC) includes surgery, chemotherapy and radiotherapy as first-line treatment options. When applicable, targeting agents such as anti-EGFR therapies (i.e., cetuximab and panitumumab) have become important long-term neoadjuvant therapies that increase survival in some patients. In recent years it has become common practice to sequence patients and their tumor specimens’ DNA. This recent push in genomic medicine has led to the identification that 30-40% of CRC patient harbor an activating KRAS mutation. These mutations are thought to be an early driver in the cancer’s progression. KRAS is also considered to be the key player in conferring resistance to the anti-EGFR treatments cetuximab and panitumumab. That Ras mutations confer resistance to EGFR inhibitors is intuitive. In recent years, clinical guidelines have extended the list of contraindicated genotypes to many mutations in both the KRAS and NRAS genes. In contrast with the current guidelines for managing CRC with EGFR inhibitors, it has been shown that patients harboring a KRAS G13D mutation are sensitive to cetuximab. This has been reproducibly shown in both in vitro and in vivo model systems and is supported by retrospective analysis of initial clinical trial data. The current mutant-RAS restrictive guidelines are supported by a method that relied upon the grouping of all common Ras mutations as “equal,” which potentially washes out less common yet sensitive mutations. Whether KRAS G13D mutations are sensitive to cetuximab has remained a controversial topic for many years due to the lack of understanding of a mechanism, as it is counterintuitive to the current understanding of EGFR signaling cascade. We here identify a novel mechanism by which G13D RAS mutant is sensitive to EGFR inhibition by a nonintuitive process of reliance upon WT RAS molecules. We utilized a computational model of RAS signaling previously developed by our laboratory to explore mutant Ras signaling and thereby investigated the controversial response of KRAS G13D to anti-EGFR agents. Our computational studies of the historically reported biochemical processes that regulate Ras signals reveal a nonintuitive, mutant-specific dependency of wild-type RAS activation on EGFR. The model also reveals this dependency is determined by the interaction strength between a KRAS mutant and tumor suppressor neurofibromin. Our prospective experiments confirm this mechanism that arises from the systems-level regulation of Ras pathway signaling. Overall, our work demonstrates how systems approaches enable mechanism-based inference in genomic medicine. Citation Format: Thomas McFall, Jolene Diedrich, Stacy L. Littlechild, Laura Sisk-Hackworth, James Moresco, Meron Mengistu1, Andrey Shaw, Ed Stites. A systems biology approach to elucidate the mechanism of EGFR inhibitor sensitivity in mutant KRAS-driven colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B43.

Necroptosis, an inflammatory form of cell death, is initiated by the activation of receptor-interacting protein kinase 3 (RIPK3), which depends on its interaction with RIPK1. Although catalytically inactive, the RIPK3 mutant D161N still stimulates RIPK1-dependent apoptosis and embryonic lethality in RIPK3 D161N homozygous mice. Whereas the absence of RIPK1 rescues RIPK3 D161N homozygous mice, we report that the absence of RIPK1 leads to embryonic lethality in RIPK3 D161N heterozygous mice. This suggested that the kinase domain of RIPK3 had a non-catalytic function that was enhanced by a conformation induced by the D161N mutation. We found that the RIPK3 kinase domain homodimerized through a surface that is structurally similar to that of the RAF family members. Mutation of residues at the dimer interface impaired dimerization and necroptosis. Kinase domain dimerization stimulated the activation of RIPK3 through cis-autophosphorylation. This noncatalytic, allosteric activity was enhanced by certain kinase-deficient mutants of RIPK3, including D161N. Furthermore, apoptosis induced by certain RIPK3 inhibitors was also dependent on the kinase dimerization interface. Our studies reveal that the RIPK3 kinase domain exhibits catalytically independent function that is important for both RIPK3-dependent necroptosis and apoptosis.

Fluid shear stress (FSS) exerted on endothelial cell surfaces induces actin cytoskeleton remodeling through mechanotransduction. This study was designed to determine whether FSS activates Jun N-terminal kinase (JNK), to examine the spatial and temporal distribution of active JNK relative to the actin cytoskeleton in endothelial cells exposed to different FSS conditions, and to evaluate the effects of active JNK on actin realignment. Exposure to 15 and 20 dyn/cm2 FSS induced higher activity levels of JNK than the lower 2 and 4 dyn/cm2 flow conditions. At the higher FSS treatments, JNK activity increased with increasing exposure time, peaking 30 minutes after flow onset with an 8-fold activity increase compared to cells in static culture. FSS-induced phospho-JNK co-localized with actin filaments at cell peripheries, as well as with stress fibers. Pharmacologically blocking JNK activity altered FSS-induced actin structure and distribution as a response to FSS. Our results indicate that FSS-induced actin remodeling occurs in three phases, and that JNK plays a role in at least one, suggesting that this kinase activity is involved in mechanotransduction from the apical surface to the actin cytoskeleton in endothelial cells.

The HIV-1 envelope glycoprotein, gp120, undergoes multiple molecular interactions and structural rearrangements during the course of host cell attachment and viral entry, which are being increasingly defined at the atomic level using isolated proteins. In comparison, antigenic markers of these dynamic changes are essentially unknown for single HIV-1 particles bound to target cells. Such markers should indicate how neutralizing and/or non-neutralizing antibodies might interdict infection by either blocking infection or sensitizing host cells for elimination by Fc-mediated effector function. Here we address this deficit by imaging fluorescently labeled CCR5-tropic HIV-1 pseudoviruses using confocal and superresolution microscopy to track the exposure of neutralizing and non-neutralizing epitopes as they appear on single HIV-1 particles bound to target cells. Epitope exposure was followed under conditions permissive or non-permissive for viral entry to delimit changes associated with virion binding from those associated with post-attachment events. We find that a previously unexpected array of gp120 epitopes is exposed rapidly upon target cell binding. This array comprises both neutralizing and non-neutralizing epitopes, the latter being hidden on free virions yet capable of serving as potent targets for Fc-mediated effector function. Under non-permissive conditions for viral entry, both neutralizing and non-neutralizing epitope exposures were relatively static over time for the majority of bound virions. Under entry-permissive conditions, epitope exposure patterns changed over time on subsets of virions that exhibited concurrent variations in virion contents. These studies reveal that bound virions are distinguished by a broad array of both neutralizing and non-neutralizing gp120 epitopes that potentially sensitize a freshly engaged target cell for destruction by Fc-mediated effector function and/or for direct neutralization at a post-binding step. The elucidation of these epitope exposure patterns during viral entry will help clarify antibody-mediated inhibition of HIV-1 as it is measured in vitro and in vivo., Author Summary A major strategy for blocking HIV-1 infection is to target antiviral antibodies or drugs to sites of vulnerability on the surface proteins of the virus. It is a relatively straightforward matter to explore these sites on the surfaces of free HIV-1 particles or on isolated viral envelope antigens. However, one difficulty presented by HIV-1 is that its surface proteins are flexible and change shape once the virus has attached to its host cell. To date, it has been difficult to predict how cell-bound HIV-1 exposes its sites of vulnerability. Yet the antiviral activities of certain antibodies indirectly suggest that there must be unique sites on cell-bound HIV-1 that are not found on free virus. Here, we use new techniques and tools to determine how HIV-1 exposes unique sites of vulnerability after attaching to host cells. We find that the virus exposes a remarkable array of these sites, including ones previously believed hidden. These exposure patterns explain the antiviral activities of various anti-HIV-1 antibodies and provide a new view of how HIV-1 might interact with the immune system. Our study also provides insights for how to target HIV-1 with antiviral antibodies, vaccines, or antiviral agents.

The structural flexibility found in human immunodeficiency virus (HIV) envelope glycoproteins creates a complex relationship between antigenicity and sensitivity to antiviral antibodies. The study of this issue in the context of viral particles is particularly problematic as conventional virus capture approaches can perturb antigenicity profiles. Here, we employed a unique analytical system based on fluorescence correlation spectroscopy (FCS), which measures antibody-virion binding with all reactants continuously in solution. Panels of nine anti-envelope monoclonal antibodies (MAbs) and five virus types were used to connect antibody binding profiles with neutralizing activities. Anti-gp120 MAbs against the 2G12 or b12 epitope, which marks functional envelope structures, neutralized viruses expressing CCR5-tropic envelopes and exhibited efficient virion binding in solution. MAbs against CD4-induced (CD4i) epitopes considered hidden on functional envelope structures poorly bound these viruses and were not neutralizing. Anti-gp41 MAb 2F5 was neutralizing despite limited virion binding. Similar antigenicity patterns occurred on CXCR4-tropic viruses, except that anti-CD4i MAbs 17b and 19e were neutralizing despite little or no virion binding. Notably, anti-gp120 MAb PG9 and anti-gp41 MAb F240 bound to both CCR5-tropic and CXCR4-tropic viruses without exerting neutralizing activity. Differences in the virus production system altered the binding efficiencies of some antibodies but did not enhance antigenicity of aberrant gp120 structures. Of all viruses tested, only JRFL pseudoviruses showed a direct relationship between MAb binding efficiency and neutralizing potency. Collectively, these data indicate that the antigenic profiles of free HIV particles generally favor the exposure of functional over aberrant gp120 structures. However, the efficiency of virion-antibody interactions in solution inconsistently predicts neutralizing activity in vitro .

Background: HIV gp120 experiences multiple molecular interactions and structural rearrangements during host cell attachment and entry. However, the antigenic correlates of these dynamic changes are essentially unknown for single HIV particles bound to target cells. Gp120 on cell-bound virions is a target for multiple humoral effector mechanisms mediated by anti-gp120 antibodies, including direct neutralizing activity and elimination by Fc-mediated effector functions such as antibody-dependent cell cytotoxicity (ADCC). It is therefore critical to understand the antigenicity of these conserved epitopes that become exposed during productive viral replication, to provide important guidance for designing antiviral strategies such as vaccines to raise protective antibody responses.Methods: We studied the antigenicity of conserved epitopes by visualizing their exposure on single HIV particles as they interact with target TZM-bl cells using confocal microscopy. Epitope exposure was probed by visualizing the binding of cognate antibodies.We then employed direct stochastic optical reconstruction microscopy (dSTORM) to determine the number of antibodies bound to cell-attached HIV, and their location relative to the virus - cell contact site.Results: CD4-induced (CD4i) epitopes thought to be sterically occluded from the virus - cell interface, were exposed on cell-bound HIV gp120. The level of A32, 17b and C11 CD4i antibody staining was similar to neutralizing antibodies b12 and 2G12. The location of these CD4i epitopes was distal to the virus - cell contact site.Conclusion: The patterns of cell-bound gp120 epitope exposure are consistent with the ADCC activities of cognate antibodies. CD4i epitopes were unexpectedly exposed distal to the HIV - cell interface, where they can be accessed by antibodies involved in ADCC. These findings indicate that HIV-1 exhibits a diversity of epitope exposure upon attachment that may provide unique insights for understanding how humoral immunity impacts HIV infection.

A preventive vaccine is potentially the most effective way to control the HIV pandemic. Such a vaccine needs to successfully harness humoral immunity and produce cross-reactive anti-envelope antibodies that mediate direct virus neutralization and/or Fc receptor-dependent killing. For these antibodies to carry out their functions in clearing HIV infection, they must bind the virus and prevent it from infecting target CD4+ cells. The capacity of an antibody to do this is dependent on the timing, duration and extent of cognate epitope exposure before and during the attachment and entry processes. The goal of this study was (i) to quantify antibody binding to HIV, and (ii) to characterize when and for how long antibody epitopes are exposed before and during virus-cell fusion. We studied the binding properties and epitope dynamics of antibodies against HIV envelope gp120 [b12 (CD4 binding site), 2G12 (carbohydrate clusters), A32 (C1, C4, & C5 domains)], CD4-induced epitope of gp120 (17b & 19e), and the membrane-proximal external region (MPER) of gp41 (4E10). To directly quantify antibody binding to virus in solution, we developed a fluorescence correlation spectroscopy (FCS) methodology that uses fluctuations in fluorescent signals to measure diffusion and reaction kinetics of fluorescently-labeled anti-envelope Mabs as they attach to HIV-1-JRFL, HIV-1-Bal, and HIV-1-NL4-3 pseudoviruses and infectious molecular clones. We have also developed methods to visualize the temporal appearance and disappearance of cognate epitopes during virus-cell fusion using immunofluorescence and live-cell imaging techniques. In this case, viral particles were labeled with a novel SNAP-tag technology that permits tracking of particles during different stages of fusion with CD4+ target cells, and concurrent imaging of epitopes that become exposed on the HIV envelope.