Effects of human anti-spike protein receptor binding domain antibodies on severe acute respiratory syndrome coronavirus neutralization escape and fitness

The receptor binding domain (RBD) of the spike (S) glycoprotein of severe acute respiratory syndrome coronavirus (SARS-CoV) is a major target of protective immunity in vivo. Although a large number of neutralizing antibodies (nAbs) have been developed, it remains unclear if a single RBD-targeting nAb or two in combination can prevent neutralization escape and, if not, attenuate viral virulence in vivo. In this study, we used a large panel of human nAbs against an epitope that overlaps the interface between the RBD and its receptor, angiotensin-converting enzyme 2 (ACE2), to assess their cross-neutralization activities against a panel of human and zoonotic SARS-CoVs and neutralization escape mutants. We also investigated the neutralization escape profiles of these nAbs and evaluated their effects on receptor binding and virus fitness in vitro and in mice. We found that some nAbs had great potency and breadth in neutralizing multiple viral strains, including neutralization escape viruses derived from other nAbs; however, no single nAb or combination of two blocked neutralization escape. Interestingly, in mice the neutralization escape mutant viruses showed either attenuation (Urbani background) or increased virulence (GD03 background) consistent with the different binding affinities between their RBDs and the mouse ACE2. We conclude that using either single nAbs or dual nAb combinations to target a SARS-CoV RBD epitope that shows plasticity may have limitations for preventing neutralization escape during in vivo immunotherapy. However, RBD-directed nAbs may be useful for providing broad neutralization and prevention of escape variants when combined with other nAbs that target a second conserved epitope with less plasticity and more structural constraint

A therapeutic vaccine prototype induces protective immunity and reduces cardiac fibrosis in a mouse model of chronic Trypanosoma cruzi infection.

Chagas disease, caused by the parasite Trypanosoma cruzi, develops into chronic Chagas' cardiomyopathy in ~30% of infected individuals, characterized by conduction disorders, arrhythmias, heart failure, and even sudden cardiac death. Current anti-parasitic treatments are plagued by significant side effects and poor efficacy in the chronic phase of disease; thus, there is a pressing need for new treatment options. A therapeutic vaccine could bolster the protective TH1-mediated immune response, thereby slowing or halting the progression of chronic Chagas' cardiomyopathy. Prior work in mice has demonstrated therapeutic efficacy of a Tc24 recombinant protein vaccine in the acute phase of Chagas disease. However, it is anticipated that humans will be vaccinated therapeutically when in the chronic phase of disease. This study investigates the therapeutic efficacy of a vaccine prototype containing recombinant protein Tc24, formulated with an emulsion containing the Toll-like receptor 4 agonist E6020 as an immunomodulatory adjuvant in a mouse model of chronic T. cruzi infection. Among outbred ICR mice vaccinated during chronic T. cruzi infection, there is a significant increase in the number of animals with undetectable systemic parasitemia (60% of vaccinated mice compared to 0% in the sham vaccine control group), and a two-fold reduction in cardiac fibrosis over the control group. The vaccinated mice produce a robust protective TH1-biased immune response to the vaccine, as demonstrated by a significant increase in antigen-specific IFNγ-production, the number of antigen-specific IFNγ-producing cells, and IgG2a antibody titers. Importantly, therapeutic vaccination significantly reduced cardiac fibrosis in chronically infected mice. This is a first study demonstrating therapeutic efficacy of the prototype Tc24 recombinant protein and E6020 stable emulsion vaccine against cardiac fibrosis in a mouse model of chronic T. cruzi infection

Impact of emergency medicine training implementation on mortality outcomes in Kigali, Rwanda: An interrupted time-series study

Introduction: Although emergency medicine (EM) training programmes have begun to be introduced in low- and middle-income countries (LMICs), minimal data exist on their effects on patient-centered outcomes in such settings. This study evaluated the impact of EM training and associated systems implementation on mortality among patients treated at the University Teaching Hospital-Kigali (UTH-K). Methods: At UTH-K an EM post-graduate diploma programme was initiated in October 2013, followed by a residency-training programme in August 2015. Prior to October 2013, care was provided exclusively by general practice physicians (GPs); subsequently, care has been provided through mutually exclusive shifts allocated between GPs and EM trainees. Patients seeking Emergency Centre (EC) care during November 2012–October 2013 (pre-training) and August 2015–July 2016 (post-training) were eligible for inclusion. Data were abstracted from a random sample of records using a structured protocol. The primary outcomes were EC and overall hospital mortality. Mortality prevalence and risk differences (RD) were compared pre- and post-training. Magnitudes of effects were quantified using regression models to yield adjusted odds ratios (aOR) with 95% confidence intervals (CI). Results: From 43,213 encounters, 3609 cases were assessed. The median age was 32 years with a male predominance (60.7%). Pre-training EC mortality was 6.3% (95% CI 5.3–7.5%), while post-training EC mortality was 1.2% (95% CI 0.7–1.8%), constituting a significant decrease in adjusted analysis (aOR = 0.07, 95% CI 0.03–0.17; p < 0.001). Pre-training overall hospital mortality was 12.2% (95% CI 10.9–13.8%). Post-training overall hospital mortality was 8.2% (95% CI 6.9–9.6%), resulting in a 43% reduction in mortality likelihood (aOR = 0.57, 95% CI 0.36–0.94; p = 0.016). Discussion: In the studied population, EM training and systems implementation was associated with significant mortality reductions demonstrating the potential patient-centered benefits of EM development in resource-limited settings. Keywords: Emergency medicine, Training, Mortality, Rwanda, Afric

Derivation of the first clinical diagnostic models for dehydration severity in patients over five years with acute diarrhea.

Diarrheal diseases lead to an estimated 1.3 million deaths each year, with the majority of those deaths occurring in patients over five years of age. As the severity of diarrheal disease can vary widely, accurately assessing dehydration status remains the most critical step in acute diarrhea management. The objective of this study is to empirically derive clinical diagnostic models for assessing dehydration severity in patients over five years with acute diarrhea in low resource settings. We enrolled a random sample of patients over five years with acute diarrhea presenting to the icddr,b Dhaka Hospital. Two blinded nurses independently assessed patients for symptoms/signs of dehydration on arrival. Afterward, consecutive weights were obtained to determine the percent weight change with rehydration, our criterion standard for dehydration severity. Full and simplified ordinal logistic regression models were derived to predict the outcome of none (9%) dehydration. The reliability and accuracy of each model were assessed. Bootstrapping was used to correct for over-optimism and compare each model's performance to the current World Health Organization (WHO) algorithm. 2,172 patients were enrolled, of which 2,139 (98.5%) had complete data for analysis. The Inter-Class Correlation Coefficient (reliability) was 0.90 (95% CI = 0.87, 0.91) for the full model and 0.82 (95% CI = 0.77, 0.86) for the simplified model. The area under the Receiver-Operator Characteristic curve (accuracy) for severe dehydration was 0.79 (95% CI: 0.76-0.82) for the full model and 0.73 (95% CI: 0.70, 0.76) for the simplified model. The accuracy for both the full and simplified models were significantly better than the WHO algorithm (p<0.001). This is the first study to empirically derive clinical diagnostic models for dehydration severity in patients over five years. Once prospectively validated, the models may improve management of patients with acute diarrhea in low resource settings

Global Health and Emergency Care: Defining Clinical Research Priorities

OBJECTIVES: Despite recent strides in the development of global emergency medicine (EM), the field continues to lag in applying a scientific approach to identifying critical knowledge gaps and advancing evidence-based solutions to clinical and public health problems seen in emergency departments (EDs) worldwide. Here, progress on the global EM research agenda created at the 2013 Academic Emergency Medicine Global Health and Emergency Care Consensus Conference is evaluated and critical areas for future development in emergency care research internationally are identified. METHODS: A retrospective review of all studies compiled in the Global Emergency Medicine Literature Review (GEMLR) database from 2013 through 2015 was conducted. Articles were categorized and analyzed using descriptive quantitative measures and structured data matrices. The Global Emergency Medicine Think Tank Clinical Research Working Group at the Society for Academic Emergency Medicine 2016 Annual Meeting then further conceptualized and defined global EM research priorities utilizing consensus-based decision making. RESULTS: Research trends in global EM research published between 2013 and 2015 show a predominance of observational studies relative to interventional or descriptive studies, with the majority of research conducted in the inpatient setting in comparison to the ED or prehospital setting. Studies on communicable diseases and injury were the most prevalent, with a relative dearth of research on chronic noncommunicable diseases. The Global Emergency Medicine Think Tank Clinical Research Working Group identified conceptual frameworks to define high-impact research priorities, including the traditional approach of using global burden of disease to define priorities and the impact of EM on individual clinical care and public health opportunities. EM research is also described through a population lens approach, including gender, pediatrics, and migrant and refugee health. CONCLUSIONS: Despite recent strides in global EM research and a proliferation of scholarly output in the field, further work is required to advocate for and inform research priorities in global EM. The priorities outlined in this paper aim to guide future research in the field, with the goal of advancing the development of EM worldwide

Ribosome-Templated Azide–Alkyne Cycloadditions: Synthesis of Potent Macrolide Antibiotics by In Situ Click Chemistry

Over half of all antibiotics target the bacterial ribosomenature’s complex, 2.5 MDa nanomachine responsible for decoding mRNA and synthesizing proteins. Macrolide antibiotics, exemplified by erythromycin, bind the 50S subunit with nM affinity and inhibit protein synthesis by blocking the passage of nascent oligopeptides. Solithromycin (<b>1</b>), a third-generation semisynthetic macrolide discovered by combinatorial copper-catalyzed click chemistry, was synthesized in situ by incubating either <i>E. coli</i> 70S ribosomes or 50S subunits with macrolide-functionalized azide <b>2</b> and 3-ethynylaniline (<b>3</b>) precursors. The ribosome-templated in situ click method was expanded from a binary reaction (i.e., one azide and one alkyne) to a six-component reaction (i.e., azide <b>2</b> and five alkynes) and ultimately to a 16-component reaction (i.e., azide <b>2</b> and 15 alkynes). The extent of triazole formation correlated with ribosome affinity for the <i>anti</i> (1,4)-regioisomers as revealed by measured <i>K</i>_d values. Computational analysis using the site-identification by ligand competitive saturation (SILCS) approach indicated that the relative affinity of the ligands was associated with the alteration of macrolactone+desosamine-ribosome interactions caused by the different alkynes. Protein synthesis inhibition experiments confirmed the mechanism of action. Evaluation of the minimal inhibitory concentrations (MIC) quantified the potency of the in situ click products and demonstrated the efficacy of this method in the triaging and prioritization of potent antibiotics that target the bacterial ribosome. Cell viability assays in human fibroblasts confirmed <b>2</b> and four analogues with therapeutic indices for bactericidal activity over in vitro mammalian cytotoxicity as essentially identical to solithromycin (<b>1</b>)

Munc18a Scaffolds SNARE Assembly to Promote Membrane Fusion

Munc18a is an SM protein required for SNARE-mediated fusion. The molecular details of how Munc18a acts to enhance neurosecretion have remained elusive. Here, we use in vitro fusion assays to characterize how specific interactions between Munc18a and the neuronal SNAREs enhance the rate and extent of fusion. We show that Munc18a interacts directly and functionally with the preassembled t-SNARE complex. Analysis of Munc18a point mutations indicates that Munc18a interacts with helix C of the Syntaxin1a NRD in the t-SNARE complex. Replacement of the t-SNARE SNAP25b with yeast Sec9c had little effect, suggesting that Munc18a has minimal contact with SNAP25b within the t-SNARE complex. A chimeric Syntaxin built of the Syntaxin1a NRD and the H3 domain of yeast Sso1p and paired with Sec9c eliminated stimulation of fusion, suggesting that Munc18a/Syntaxin1a H3 domain contacts are important. Additionally, a Syntaxin1A mutant lacking a flexible linker region that allows NRD movement abolished stimulation of fusion. These experiments suggest that Munc18a binds to the Syntaxin1a NRD and H3 domain within the assembled t-SNARE complex, positioning them for productive VAMP2 binding. In this capacity, Munc18a serves as a platform for trans-SNARE complex assembly that facilitates efficient SNARE-mediated membrane fusion