Human Adaptation of Ebola Virus during the West African Outbreak.

The 2013-2016 outbreak of Ebola virus (EBOV) in West Africa was the largest recorded. It began following the cross-species transmission of EBOV from an animal reservoir, most likely bats, into humans, with phylogenetic analysis revealing the co-circulation of several viral lineages. We hypothesized that this prolonged human circulation led to genomic changes that increased viral transmissibility in humans. We generated a synthetic glycoprotein (GP) construct based on the earliest reported isolate and introduced amino acid substitutions that defined viral lineages. Mutant GPs were used to generate a panel of pseudoviruses, which were used to infect different human and bat cell lines. These data revealed that specific amino acid substitutions in the EBOV GP have increased tropism for human cells, while reducing tropism for bat cells. Such increased infectivity may have enhanced the ability of EBOV to transmit among humans and contributed to the wide geographic distribution of some viral lineages.

Revisiting the origins of the Sobemovirus genus: A case for ancient origins of plant viruses.

The discrepancy between short- and long-term rate estimates, known as the time-dependent rate phenomenon (TDRP), poses a challenge to extrapolating evolutionary rates over time and reconstructing evolutionary history of viruses. The TDRP reveals a decline in evolutionary rate estimates with the measurement timescale, explained empirically by a power-law rate decay, notably observed in animal and human viruses. A mechanistic evolutionary model, the Prisoner of War (PoW) model, has been proposed to address TDRP in viruses. Although TDRP has been studied in animal viruses, its impact on plant virus evolutionary history remains largely unexplored. Here, we investigated the consequences of TDRP in plant viruses by applying the PoW model to reconstruct the evolutionary history of sobemoviruses, plant pathogens with significant importance due to their impact on agriculture and plant health. Our analysis showed that the Sobemovirus genus dates back over four million years, indicating an ancient origin. We found evidence that supports deep host jumps to Poaceae, Fabaceae, and Solanaceae occurring between tens to hundreds of thousand years ago, followed by specialization. Remarkably, the TDRP-corrected evolutionary history of sobemoviruses was extended far beyond previous estimates that had suggested their emergence nearly 9,000 years ago, a time coinciding with the Neolithic period in the Near East. By incorporating sequences collected through metagenomic analyses, the resulting phylogenetic tree showcases increased genetic diversity, reflecting a deep history of sobemovirus species. We identified major radiation events beginning between 4,600 to 2,000 years ago, which aligns with the Neolithic period in various regions, suggesting a period of rapid diversification from then to the present. Our findings make a case for the possibility of deep evolutionary origins of plant viruses.

Polyvalent immunization elicits a synergistic broadly neutralizing immune response to hypervariable region 1 variants of hepatitis C virus.

A hepatitis C virus (HCV) vaccine is urgently needed. Vaccine development has been hindered by HCV's genetic diversity, particularly within the immunodominant hypervariable region 1 (HVR1). Here, we developed a strategy to elicit broadly neutralizing antibodies to HVR1, which had previously been considered infeasible. We first applied a unique information theory-based measure of genetic distance to evaluate phenotypic relatedness between HVR1 variants. These distances were used to model the structure of HVR1's sequence space, which was found to have five major clusters. Variants from each cluster were used to immunize mice individually, and as a pentavalent mixture. Sera obtained following immunization neutralized every variant in a diverse HCVpp panel (n = 10), including those resistant to monovalent immunization, and at higher mean titers (1/ID50 = 435) than a glycoprotein E2 (1/ID50 = 205) vaccine. This synergistic immune response offers a unique approach to overcoming antigenic variability and may be applicable to other highly mutable viruses.

Recombinant H77C gpE1/gpE2 heterodimer elicits superior HCV cross-neutralisation than H77C gpE2 alone.

BACKGROUND & AIMS: An optimal HCV vaccine requires the induction of antibodies that neutralise the infectivity of many heterogenous viral isolates. In this study, we have focused on determining the optimal recombinant envelope glycoprotein component to elicit cross-neutralising antibodies against global HCV genotypes. We compared the immunoreactivity and antigenicity of the recombinant HCV genotype 1a strain H77C envelope glycoprotein heterodimer gpE1/gpE2 with that of recombinant gpE2 alone derived from an infectious molecular clone (H77C). METHODS: Characterization of the envelope glycoproteins was accomplished by determining their ability to bind to a panel of broadly cross-neutralising monoclonal antibodies (bNAbs). Immunogenicity was determined by testing the ability of vaccine antisera to neutralise the infectivity in vitro of a panel of pseudotyped HCV particles in which gpE1/gpE2 derived from representative isolates of the major global HCV genotypes were displayed. RESULTS: gpE1/gpE2 binds to more diverse bNabs than gpE2 alone and elicits a broader profile of cross-neutralising antibodies in animals, especially against more heterologous, non-1a genotypes. While not all heterologous HCV strains can be potently inhibited in vitro by gpE1/gpE2 antisera derived from a single HCV strain, the breadth of heterologous cross-neutralisation is shown to be substantial. CONCLUSIONS: Our work supports the inclusion of gpE1/gpE2 in an HCV vaccine in order to maximise the cross-neutralisation of heterogenous HCV isolates. Our data also offers future directions in formulating a cocktail of gpE1/gpE2 antigens from a small selection of HCV genotypes to further enhance cross-neutralisation of global HCV strains and hopefully, achieving global protection. IMPACT AND IMPLICATIONS: An HCV vaccine is urgently required to prevent the high global incidence of HCV infection and disease. Since HCV is a highly heterogeneous virus, it is desirable for a vaccine to elicit antibodies that neutralise the infectivity of most global strains. To this end, we have compared the immunoreactivity and antigenicity of recombinant H77C E1E2 heterodimer with that of H77C E2 alone and show that the former exhibits more cross-neutralising epitopes and demonstrates a broader cross-neutralisation profile in vitro. In addition, our data suggests a way to further broaden cross-neutralisation using a combination of E1E2 antigens derived from a few different HCV clades. Our work provides encouragement for the development of an effective global HCV vaccine.

In vitro evolution predicts emerging SARS-CoV-2 mutations with high affinity for ACE2 and cross-species binding.

Emerging SARS-CoV-2 variants are creating major challenges in the ongoing COVID-19 pandemic. Being able to predict mutations that could arise in SARS-CoV-2 leading to increased transmissibility or immune evasion would be extremely valuable in development of broad-acting therapeutics and vaccines, and prioritising viral monitoring and containment. Here we use in vitro evolution to seek mutations in SARS-CoV-2 receptor binding domain (RBD) that would substantially increase binding to ACE2. We find a double mutation, S477N and Q498H, that increases affinity of RBD for ACE2 by 6.5-fold. This affinity gain is largely driven by the Q498H mutation. We determine the structure of the mutant-RBD:ACE2 complex by cryo-electron microscopy to reveal the mechanism for increased affinity. Addition of Q498H to SARS-CoV-2 RBD variants is found to boost binding affinity of the variants for human ACE2 and confer a new ability to bind rat ACE2 with high affinity. Surprisingly however, in the presence of the common N501Y mutation, Q498H inhibits binding, due to a clash between H498 and Y501 side chains. To achieve an intermolecular bonding network, affinity gain and cross-species binding similar to Q498H alone, RBD variants with the N501Y mutation must acquire instead the related Q498R mutation. Thus, SARS-CoV-2 RBD can access large affinity gains and cross-species binding via two alternative mutational routes involving Q498, with route selection determined by whether a variant already has the N501Y mutation. These mutations are now appearing in emerging SARS-CoV-2 variants where they have the potential to influence human-to-human and cross-species transmission.

Bridging the Gap: Comparison of Outpatient Clinic and Emergency Department Patients Undergoing Cholecystectomy.

INTRODUCTION: Literature shows failure of the outpatient clinic (OC) pathway after emergency department (ED) ultrasound diagnosis of symptomatic cholelithiasis (SC). We hypothesized SC to be more prevalent on final surgical pathology (FSP) in patients who successfully completed OC pathway. METHODS: This retrospective single-institution chart review compared OC and ED patients with right upper quadrant (RUQ) pain and cholelithiasis whom underwent cholecystectomy. Clinical evaluation was considered positive if RUQ pain >4 h, or + Murphy's sign. Ultrasound was positive if two of these three were present: sonographic Murphy's, wall thickness > 4 mm, or pericholecystic fluid. Results were compared with FSP. RESULTS: Six hundred-seven patients underwent cholecystectomy, 299 OC and 308 ED. OC was more likely to SC (23% versus 4.6%) (P < 0.0001) and ED acute cholecystitis (39.3% versus 4.7%). Chronic cholecystitis was the most common FSP in both OC (72%) and ED (56%) populations, of these, 73% of OC denied pain >4 h versus only 10% of ED (P < 0.001). Median time from evaluation to cholecystectomy was 14 d versus 14 h in the OC and ED respectively (P < 0.0001). CONCLUSIONS: While chronic cholecystitis was the most common FSP in both OC and ED, the majority of OC reported RUQ pain <4 h delineating these presentations. Duration of pain should be utilized as algorithm triage. We recommend patients with pain episode <4 h should complete OC algorithm with expedited cholecystectomy within 14 d.

COVID-19-associated pulmonary aspergillosis in mechanically ventilated patients: a prospective, multicentre UK study.

BACKGROUND: Invasive pulmonary aspergillosis is a complication of severe COVID-19, with regional variation in reported incidence and mortality. We describe the incidence, risk factors and mortality associated with COVID-19-associated pulmonary aspergillosis (CAPA) in a prospective, multicentre UK cohort. METHODS: From March 2020 to March 2021, 266 mechanically ventilated adults with COVID-19 were enrolled across 5 UK hospital intensive care units (ICUs). CAPA was defined using European Confederation for Medical Mycology and the International Society for Human and Animal Mycology criteria and fungal diagnostics performed on respiratory and serum samples. RESULTS: Twenty-nine of 266 patients (10.9%) had probable CAPA, 14 (5.2%) possible CAPA and none proven CAPA. Probable CAPA was diagnosed a median of 9 (IQR 7-16) days after ICU admission. Factors associated with probable CAPA after multivariable logistic regression were cumulative steroid dose given within 28 days prior to ICU admission (adjusted OR (aOR) 1.16; 95% CI 1.01 to 1.43 per 100 mg prednisolone-equivalent), receipt of an interleukin (IL)-6 inhibitor (aOR 2.79; 95% CI 1.22 to 6.48) and chronic obstructive pulmonary disease (COPD) (aOR 4.78; 95% CI 1.13 to 18.13). Mortality in patients with probable CAPA was 55%, vs 46% in those without. After adjustment for immortal time bias, CAPA was associated with an increased risk of 90-day mortality (HR 1.85; 95% CI 1.07 to 3.19); however, this association did not remain statistically significant after further adjustment for confounders (adjusted HR 1.57; 95% CI 0.88 to 2.80). There was no difference in mortality between patients with CAPA prescribed antifungals (9 of 17; 53%) and those who were not (7 of 12; 58%) (p=0.77). INTERPRETATION: In this first prospective UK study, probable CAPA was associated with corticosteroid use, receipt of IL-6 inhibitors and pre-existing COPD. CAPA did not impact mortality following adjustment for prognostic variables.

An Antigenically Diverse, Representative Panel of Envelope Glycoproteins for Hepatitis C Virus Vaccine Development.

BACKGROUND & AIMS: Development of a prophylactic hepatitis C virus (HCV) vaccine will require accurate and reproducible measurement of neutralizing breadth of vaccine-induced antibodies. Currently available HCV panels may not adequately represent the genetic and antigenic diversity of circulating HCV strains, and the lack of standardization of these panels makes it difficult to compare neutralization results obtained in different studies. Here, we describe the selection and validation of a genetically and antigenically diverse reference panel of 15 HCV pseudoparticles (HCVpps) for neutralization assays. METHODS: We chose 75 envelope (E1E2) clones to maximize representation of natural polymorphisms observed in circulating HCV isolates, and 65 of these clones generated functional HCVpps. Neutralization sensitivity of these HCVpps varied widely. HCVpps clustered into 15 distinct groups based on patterns of relative sensitivity to 7 broadly neutralizing monoclonal antibodies. We used these data to select a final panel of 15 antigenically representative HCVpps. RESULTS: Both the 65 and 15 HCVpp panels span 4 tiers of neutralization sensitivity, and neutralizing breadth measurements for 7 broadly neutralizing monoclonal antibodies were nearly equivalent using either panel. Differences in neutralization sensitivity between HCVpps were independent of genetic distances between E1E2 clones. CONCLUSIONS: Neutralizing breadth of HCV antibodies should be defined using viruses spanning multiple tiers of neutralization sensitivity rather than panels selected solely for genetic diversity. We propose that this multitier reference panel could be adopted as a standard for the measurement of neutralizing antibody potency and breadth, facilitating meaningful comparisons of neutralization results from vaccine studies in different laboratories.

Identification of immune correlates of fatal outcomes in critically ill COVID-19 patients.

Prior studies have demonstrated that immunologic dysfunction underpins severe illness in COVID-19 patients, but have lacked an in-depth analysis of the immunologic drivers of death in the most critically ill patients. We performed immunophenotyping of viral antigen-specific and unconventional T cell responses, neutralizing antibodies, and serum proteins in critically ill patients with SARS-CoV-2 infection, using influenza infection, SARS-CoV-2-convalescent health care workers, and healthy adults as controls. We identify mucosal-associated invariant T (MAIT) cell activation as an independent and significant predictor of death in COVID-19 (HR = 5.92, 95% CI = 2.49-14.1). MAIT cell activation correlates with several other mortality-associated immunologic measures including broad activation of CD8+ T cells and non-Vδ2 γδT cells, and elevated levels of cytokines and chemokines, including GM-CSF, CXCL10, CCL2, and IL-6. MAIT cell activation is also a predictor of disease severity in influenza (ECMO/death HR = 4.43, 95% CI = 1.08-18.2). Single-cell RNA-sequencing reveals a shift from focused IFNα-driven signals in COVID-19 ICU patients who survive to broad pro-inflammatory responses in fatal COVID-19 -a feature not observed in severe influenza. We conclude that fatal COVID-19 infection is driven by uncoordinated inflammatory responses that drive a hierarchy of T cell activation, elements of which can serve as prognostic indicators and potential targets for immune intervention.

Scavenger receptor class B type I genetic variants associated with disease severity in chronic hepatitis C virus infection.

Analysis of host genetic polymorphisms is an increasingly important tool for understanding and predicting pathogenesis and treatment response of viral diseases. The gene locus of scavenger receptor class B type I (SR-BI), encoding a cell entry factor and receptor for hepatitis C virus (HCV), contains several genetic polymorphisms. We applied a probe extension assay to determine the frequency of six single nucleotide polymorphisms (SNPs) within the SR-BI gene locus in 374 individuals with history of HCV infection. In addition, SR-BI messenger RNA (mRNA) levels were analyzed in liver biopsy specimens of chronically infected HCV subjects. The rs5888 variant allele T was present at a higher frequency in subjects with advanced fibrosis (χ2 , p = 0.016) and after adjusting for age, duration of infection and alcohol intake as confounding factors. Haplotype analysis of SNP frequencies showed that a haplotype consisting of rs61932577 variant allele C and rs5888 variant allele T was associated with an increased risk of advanced liver fibrosis (defined by an Ishak score 4-6) (adjusted odds ratio 2.81; 95% confidence interval 1.06-7.46. p = 0.038). Carriers of the rs5888 variant allele T displayed reduced SR-BI mRNA expression in liver biopsy specimens. In conclusion the rs5888 polymorphism variant is associated with decreased SR-BI expression and an increased risk of development of advanced fibrosis in chronic HCV infection. These findings provide further evidence for a role of SR-BI in HCV pathogenesis and provides a genetic marker for prediction of those infected individuals at greater risk of developing severe disease.

Hepatitis C subtyping assay failure in UK patients born in sub-Saharan Africa: Implications for global treatment and elimination.

BACKGROUND AND AIMS: The newly developed direct-acting antivirals have revolutionized the treatment of chronic hepatitis C virus (HCV), with cure rates as high as 98% in some cohorts. Although genome sequencing has demonstrated that some subtypes of HCV naturally harbor drug resistance associated substitutions (RAS), these are often overlooked as "rarities." Furthermore, commercial subtyping assays and associated epidemiological findings are skewed towards Western cohorts and whole-genome sequencing can be problematic to deploy without significant infrastructure and training support. We thus aimed to develop a simple, robust and accurate HCV subtyping pipeline, to optimize and streamline molecular detection and sequence-based typing of diverse RAS-containing subtypes. METHODS: HCV serum derived from 146 individuals, whose likely source of infection was from sub-Saharan Africa (SSA) was investigated with a novel panel of single round polymerase chain reaction (PCR) assays targeting NS5B and NS5A genomic regions. Virus subtype assignments were determined by pairwise-distance analysis and compared to both diagnostic laboratory assignments and free-to-use online typing tools. RESULTS: Partial NS5A and NS5B sequences were respectively obtained from 131 to 135 HCV-positive patients born in 19 different countries from SSA but attending clinics in the UK. We determined that routine clinical diagnostic methods incorrectly subtyped 59.0% of samples, with a further 6.8% incorrectly genotyped. Of five commonly used online tools, Geno2Pheno performed most effectively in determining a subtype in agreement with pairwise distance analysis. CONCLUSION: This study provides a simple low-cost pathway to accurately subtype in SSA, guide regional therapeutic choice and assist global surveillance and elimination initiatives.

Relationship Between Anxiety, Depression, and Susceptibility to Severe Acute Respiratory Syndrome Coronavirus 2 Infection: Proof of Concept.

BACKGROUND: Psychological factors can influence susceptibility to viral infections. We examined whether such influences are evident in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. METHODS: Participants (n = 102) completed measures of anxiety, depression, positive mood, and loneliness and provided a blood sample for the measurement of antibodies to the SARS-CoV-2 spike and nucleocapsid proteins. RESULTS: SARS-CoV-2 was significantly negatively associated with anxiety and depression. The model remained significant after adjustment for age and gender, although anxiety and depression were no longer significant independent predictors. CONCLUSIONS: These findings offer early support for the hypothesis that psychological factors may influence susceptibility to SARS-CoV-2 infection.

The Impact of Real-Time Whole-Genome Sequencing in Controlling Healthcare-Associated SARS-CoV-2 Outbreaks.

Nosocomial severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections have severely affected bed capacity and patient flow. We utilized whole-genome sequencing (WGS) to identify outbreaks and focus infection control resources and intervention during the United Kingdom's second pandemic wave in late 2020. Phylogenetic analysis of WGS and epidemiological data pinpointed an initial transmission event to an admission ward, with immediate prior community infection linkage documented. High incidence of asymptomatic staff infection with genetically identical viral sequences was also observed, which may have contributed to the propagation of the outbreak. WGS allowed timely nosocomial transmission intervention measures, including admissions ward point-of-care testing and introduction of portable HEPA14 filters. Conversely, WGS excluded nosocomial transmission in 2 instances with temporospatial linkage, conserving time and resources. In summary, WGS significantly enhanced understanding of SARS-CoV-2 clusters in a hospital setting, both identifying high-risk areas and conversely validating existing control measures in other units, maintaining clinical service overall.

Real-World Outcomes of Direct-Acting Antiviral Treatment and Retreatment in United Kingdom-Based Patients Infected With Hepatitis C Virus Genotypes/Subtypes Endemic in Africa.

BACKGROUND: Chronic hepatitis C virus (HCV) infection affects 71 million individuals, mostly residing in low- and middle-income countries (LMICs). Direct-acting antivirals (DAAs) give high rates of sustained virological response (SVR) in high-income countries where a restricted range of HCV genotypes/subtypes circulate. METHODS: We studied United Kingdom-resident patients born in Africa to examine DAA effectiveness in LMICs where there is far greater breadth of HCV genotypes/subtypes. Viral genome sequences were determined from 233 patients. RESULTS: Full-length viral genomic sequences for 26 known subtypes and 5 previously unidentified isolates covering 5 HCV genotypes were determined. From 149 patients who received DAA treatment/retreatment, the overall SVR was 93%. Treatment failure was associated primarily with 2 subtypes, gt1l and gt4r, using sofosbuvir/ledipasvir. These subtypes contain natural resistance-associated variants that likely contribute to poor efficacy with this drug combination. Treatment failure was also significantly associated with hepatocellular carcinoma. CONCLUSIONS: DAA combinations give high SVR rates despite the high HCV diversity across the African continent except for subtypes gt1l and gt4r, which respond poorly to sofosbuvir/ledipasvir. These subtypes are widely distributed across Western, Central, and Eastern Africa. Thus, in circumstances where accurate genotyping is absent, ledipasvir and its generic compounds should not be considered as a recommended treatment option.

Optimization of the pseudoparticle system for standardized assessments of neutralizing antibodies against hepatitis C virus.

A better understanding of the antibody response during natural infection and the effect on disease progression and reinfection is necessary for the development of a protective hepatitis C virus (HCV) vaccine. The HCV pseudoparticle (HCVpp) system enables the study of viral entry and inhibition by antibody neutralization. A robust and comparable neutralization assay is crucial for the development and evaluation of experimental vaccines.With the aim of optimizing the HCVpp-murine leukaemia virus (MLV) system, we tested the neutralization of HCVpp-harbouring E1E2 from 21 HCV isolates representing 6 different genotypes by several monoclonal antibodies (mAbs). HCVpps are generated by expressing functional envelope glycoproteins (E1E2) onto pseudoparticles derived from env-deleted MLV. Adjustments of E1E2, gag-pol and luciferase plasmid ratios resulted in increased yields for most HCVpps and recovery of one non-infectious HCVpp. We simplified and improved the protocol to achieve higher signal/noise ratios and minimized the amount of HCVpps and mAbs needed for the detection of neutralization. Using our optimized protocol, we demonstrated comparable results to previously reported data with both diluted and freeze-thawed HCVpps.In conclusion, we successfully established a simplified and reproducible HCVpp neutralization protocol for studying a wide range of HCV variants. This simplified protocol provides highly consistent results and could be easily adopted by others to evaluate precious biological material. This will contribute to a better understanding of the antibody response during natural infection and help evaluate experimental HCV vaccines.

Immunogenicity of a new gorilla adenovirus vaccine candidate for COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic caused by the emergent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) threatens global public health, and there is an urgent need to develop safe and effective vaccines. Here, we report the generation and the preclinical evaluation of a novel replication-defective gorilla adenovirus-vectored vaccine encoding the pre-fusion stabilized Spike (S) protein of SARS-CoV-2. We show that our vaccine candidate, GRAd-COV2, is highly immunogenic both in mice and macaques, eliciting both functional antibodies that neutralize SARS-CoV-2 infection and block Spike protein binding to the ACE2 receptor, and a robust, T helper (Th)1-dominated cellular response. We show here that the pre-fusion stabilized Spike antigen is superior to the wild type in inducing ACE2-interfering, SARS-CoV-2-neutralizing antibodies. To face the unprecedented need for vaccine manufacturing at a massive scale, different GRAd genome deletions were compared to select the vector backbone showing the highest productivity in stirred tank bioreactors. This preliminary dataset identified GRAd-COV2 as a potential COVID-19 vaccine candidate, supporting the translation of the GRAd-COV2 vaccine in a currently ongoing phase I clinical trial (ClinicalTrials.gov: NCT04528641).

Retrospective screening of routine respiratory samples revealed undetected community transmission and missed intervention opportunities for SARS-CoV-2 in the United Kingdom.

In the early phases of the SARS coronavirus type 2 (SARS-CoV-2) pandemic, testing focused on individuals fitting a strict case definition involving a limited set of symptoms together with an identified epidemiological risk, such as contact with an infected individual or travel to a high-risk area. To assess whether this impaired our ability to detect and control early introductions of the virus into the UK, we PCR-tested archival specimens collected on admission to a large UK teaching hospital who retrospectively were identified as having a clinical presentation compatible with COVID-19. In addition, we screened available archival specimens submitted for respiratory virus diagnosis, and dating back to early January 2020, for the presence of SARS-CoV-2 RNA. Our data provides evidence for widespread community circulation of SARS-CoV-2 in early February 2020 and into March that was undetected at the time due to restrictive case definitions informing testing policy. Genome sequence data showed that many of these early cases were infected with a distinct lineage of the virus. Sequences obtained from the first officially recorded case in Nottinghamshire - a traveller returning from Daegu, South Korea - also clustered with these early UK sequences suggesting acquisition of the virus occurred in the UK and not Daegu. Analysis of a larger sample of sequences obtained in the Nottinghamshire area revealed multiple viral introductions, mainly in late February and through March. These data highlight the importance of timely and extensive community testing to prevent future widespread transmission of the virus.

Structure-Based Design of Hepatitis C Virus E2 Glycoprotein Improves Serum Binding and Cross-Neutralization.

An effective vaccine for hepatitis C virus (HCV) is a major unmet need, and it requires an antigen that elicits immune responses to key conserved epitopes. Based on structures of antibodies targeting HCV envelope glycoprotein E2, we designed immunogens to modulate the structure and dynamics of E2 and favor induction of broadly neutralizing antibodies (bNAbs) in the context of a vaccine. These designs include a point mutation in a key conserved antigenic site to stabilize its conformation, as well as redesigns of an immunogenic region to add a new N-glycosylation site and mask it from antibody binding. Designs were experimentally characterized for binding to a panel of human monoclonal antibodies (HMAbs) and the coreceptor CD81 to confirm preservation of epitope structure and preferred antigenicity profile. Selected E2 designs were tested for immunogenicity in mice, with and without hypervariable region 1, which is an immunogenic region associated with viral escape. One of these designs showed improvement in polyclonal immune serum binding to HCV pseudoparticles and neutralization of isolates associated with antibody resistance. These results indicate that antigen optimization through structure-based design of the envelope glycoproteins is a promising route to an effective vaccine for HCV.IMPORTANCE Hepatitis C virus infects approximately 1% of the world's population, and no vaccine is currently available. Due to the high variability of HCV and its ability to actively escape the immune response, a goal of HCV vaccine design is to induce neutralizing antibodies that target conserved epitopes. Here, we performed structure-based design of several epitopes of the HCV E2 envelope glycoprotein to engineer its antigenic properties. Designs were tested in vitro and in vivo, demonstrating alteration of the E2 antigenic profile in several cases, and one design led to improvement of cross-neutralization of heterologous viruses. This represents a proof of concept that rational engineering of HCV envelope glycoproteins can be used to modulate E2 antigenicity and optimize a vaccine for this challenging viral target.

Tracheostomy care and decannulation during the COVID-19 pandemic. A multidisciplinary clinical practice guideline.

PURPOSE: Traditional critical care dogma regarding the benefits of early tracheostomy during invasive ventilation has had to be revisited due to the risk of COVID-19 to patients and healthcare staff. Standard practises that have evolved to minimise the risks associated with tracheostomy must be comprehensively reviewed in light of the numerous potential episodes for aerosol generating procedures. We meet the urgent need for safe practise standards by presenting the experience of two major London teaching hospitals, and synthesise our findings into an evidence-based guideline for multidisciplinary care of the tracheostomy patient. METHODS: This is a narrative review presenting the extensive experience of over 120 patients with tracheostomy, with a pragmatic analysis of currently available evidence for safe tracheostomy care in COVID-19 patients. RESULTS: Tracheostomy care involves many potentially aerosol generating procedures which may pose a risk of viral transmission to staff and patients. We make a series of recommendations to ameliorate this risk through infection control strategies, equipment modification, and individualised decannulation protocols. In addition, we discuss the multidisciplinary collaboration that is absolutely fundamental to safe and effective practise. CONCLUSION: COVID-19 requires a radical rethink of many tenets of tracheostomy care, and controversy continues to exist regarding the optimal techniques to minimise risk to patients and healthcare workers. Safe practise requires a coordinated multidisciplinary team approach to infection control, weaning and decannulation, with integrated processes for continuous prospective data collection and audit.

Open versus percutaneous tracheostomy in COVID-19: a multicentre comparison and recommendation for future resource utilisation.

PURPOSE: The COVID-19 pandemic placed an unprecedented demand on critical care services for the provision of mechanical ventilation. Tracheostomy formation facilitates liberation from mechanical ventilation with advantages for both the patient and wider critical care resource, and can be performed using both percutaneous dilatational and surgical techniques. We compared outcomes in those patients undergoing percutaneous dilatational tracheostomy to those undergoing surgical tracheostomy and make recommendations for provision of tracheostomy services in any future surge. METHODS: Multicentre multidisciplinary retrospective observational cohort study including 201 patients with COVID-19 pneumonitis admitted to an ICU in one of five NHS Trusts within the South London Adult Critical Care Network who required mechanical ventilation and subsequent tracheostomy. RESULTS: Percutaneous dilatational tracheostomy was performed in 124 (62%) of patients, and surgical tracheostomy in 77 (38%) of patients. There was no difference between percutaneous dilatational tracheostomy and surgical tracheostomy in either the rate of peri-operative complications (16.9 vs. 22.1%, p = 0.46), median [IQR(range)] time to decannulation [19.0 (15.0-30.2 (5.0-65.0)] vs. 21.0 [15.5-36.0 (5.0-70.0) days] or mortality (13.7% vs. 15.6%, p = 0.84). Of the 172 patients that were alive at follow-up, two remained ventilated and 163 were decannulated. CONCLUSION: In patients with COVID-19 pneumonitis that require tracheostomy to facilitate weaning from mechanical ventilation, there was no difference in outcomes between those patients that had percutaneous dilatational tracheostomy compared with those that had surgical tracheostomy. Planning for future surges in COVID-19-related critical care demands should utilise all available resource and expertise.