Alternating Arenavirus Vector Immunization Generates Robust Polyfunctional Genotype Cross-Reactive Hepatitis B Virus-Specific CD8 T-Cell Responses and High Anti-Hepatitis B Surface Antigen Titers.

Hepatitis B Virus (HBV) is a major driver of infectious disease mortality. Curative therapies are needed and ideally should induce CD8 T cell-mediated clearance of infected hepatocytes plus anti-hepatitis B surface antigen (HBsAg) antibodies (anti-HBs) to neutralize residual virus. We developed a novel therapeutic vaccine using non-replicating arenavirus vectors. Antigens were screened for genotype conservation and magnitude and genotype reactivity of T cell response, then cloned into Pichinde virus (PICV) vectors (recombinant PICV, GS-2829) and lymphocytic choriomeningitis virus (LCMV) vectors (replication-incompetent, GS-6779). Alternating immunizations with GS-2829 and GS-6779 induced high-magnitude HBV T cell responses, and high anti-HBs titers. Dose schedule optimization in macaques achieved strong polyfunctional CD8 T cell responses against core, HBsAg, and polymerase and high titer anti-HBs. In AAV-HBV mice, GS-2829 and GS-6779 were efficacious in animals with low pre-treatment serum HBsAg. Based on these results, GS-2829 and GS-6779 could become a central component of cure regimens.

What is the health impact of COVID-19 among Black communities in Canada? A systematic review.

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

What are the barriers and facilitators to polio vaccination and eradication programs? A systematic review.

Global efforts to eradicate polio by the Global Polio Eradication Initiative agency partners and country-level stakeholders have led to the implementation of global polio vaccination programs. This study presents the findings of existing studies regarding the barriers and facilitators that countries face when implementing polio interventions. A comprehensive search was conducted in OVID Medline, OVID Embase, EBSCO CINAHL Plus, and Web of Science. Eligible studies underwent quality assessment. A qualitative evidence synthesis approach was conducted and aligned to the Consolidated Framework for Implementation Research (CFIR). The search identified 4147 citations, and following the removal of duplicates and screening according to our inclusion/exclusion criteria, 20 articles were eligible for inclusion in the review. Twelve countries were represented in this review, with India, Nigeria, Pakistan, Ethiopia, and Afghanistan having the most representation of available studies. We identified 36 barriers and 16 facilitators. Seven themes emerged from these barriers and facilitators: fear, community trust, infrastructure, beliefs about the intervention, influential opinions, intervention design, and geo-politics. The most frequently cited CFIR constructs for the facilitators and barriers were knowledge and beliefs about the intervention, followed by available resources. This study identified a wide range of barriers and facilitators to polio vaccination implementation across the globe, adding to the scarce body of literature on these barriers and facilitators from an implementation perspective and using a determinant framework. The diversity of factors among different groups of people or countries highlights the relevance of contexts. Implementers should be conversant with the contexts within which polio eradication programs boost intervention coverage and capacity. This study provides policymakers, practitioners, and researchers with a tool for planning and designing polio immunization programs. Trial registration: A protocol for this systematic review was developed and uploaded onto the PROSPERO international prospective register of systematic reviews database (Registration number: CRD42020222115).

A systems mechanism for KRAS mutant allele-specific responses to targeted therapy.

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 (KRASG13D) behaves differently; for unknown reasons, KRASG13D 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 KRASG13D cancers respond to EGFR inhibition. A computational model of RAS signaling revealed that the biophysical differences between the three most common KRAS mutants were sufficient to generate different sensitivities to EGFR inhibition. Integrated computation with experimentation then revealed a nonintuitive, 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 KRASG13D weakly interacted with and could not competitively inhibit NF1 and, thus, KRASG13D cells remained dependent on 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.

Concurrent Exposure of Neutralizing and Non-neutralizing Epitopes on a Single HIV-1 Envelope Structure.

The trimeric envelope spikes on the HIV-1 virus surface initiate infection and comprise key targets for antiviral humoral responses. Circulating virions variably present intact envelope spikes, which react with neutralizing antibodies; and altered envelope structures, which bind non-neutralizing antibodies. Once bound, either type of antibody can enable humoral effector mechanisms with the potential to control HIV-1 infection in vivo. However, it is not clear how the presentation of neutralizing vs. non-neutralizing epitopes defines distinct virus populations and/or envelope structures on single particles. Here we used single-virion fluorescence correlation spectroscopy (FCS), fluorescence resonance energy transfer (FRET), and two-color coincidence FCS approaches to examine whether neutralizing and non-neutralizing antibodies are presented by the same envelope structure. Given the spatial requirements for donor-acceptor energy transfer (≤10 nm), FRET signals generated by paired neutralizing and non-neutralizing fluorescent Fabs should occur via proximal binding to the same target antigen. Fluorescent-labeled Fabs of the neutralizing anti-gp120 antibodies 2G12 and b12 were combined with Fabs of the non-neutralizing anti-gp41 antibody F240, previously thought to mainly bind gp41 "stumps." We find that both 2G12-F240 and/or b12-F240 Fab combinations generate FRET signals on multiple types of virions in solution. FRET efficiencies position the neutralizing and non-neutralizing epitopes between 7.1 and 7.8 nm apart; potentially fitting within the spatial dimensions of a single trimer-derived structure. Further, the frequency of FRET detection suggests that at least one of such structures occurs on the majority of particles in a virus population. Thus, there is frequent, overlapping presentation of non-neutralizing and neutralizing epitope on freely circulating HIV-1 surfaces. Such information provides a broader perspective of how anti-HIV humoral immunity interfaces with circulating virions.

Kinase domain dimerization drives RIPK3-dependent necroptosis.

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 noncatalytic 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.

Patterns of conserved gp120 epitope presentation on attached HIV-1 virions.

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.

Antigenic properties of the human immunodeficiency virus envelope glycoprotein gp120 on virions bound to target 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.

Antigenic properties of the HIV envelope on virions in solution.

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.

Fluid shear stress-induced JNK activity leads to actin remodeling for cell alignment.

Fluid shear stress (FSS) exerted on endothelial cell (EC) 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 ECs exposed to different FSS conditions, and to evaluate the effects of active JNK on actin realignment. Exposure to 15 and 20 dyn/cm(2) FSS induced higher activity levels of JNK than the lower 2 and 4 dyn/cm(2) flow conditions. At the higher FSS treatments, JNK activity increased with increasing exposure time, peaking 30 min after flow onset with an eightfold 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 ECs.