Functional genomics screens reveal a role for TBC1D24 and SV2B in antibody-dependent enhancement of dengue virus infection.

Dengue virus (DENV) can hijack non-neutralizing IgG antibodies to facilitate its uptake into target cells expressing Fc gamma receptors (FcgR) - a process known as antibody-dependent enhancement (ADE) of infection. Beyond a requirement for FcgR, host dependency factors for this non-canonical infection route remain unknown. To identify cellular factors exclusively required for ADE, here, we performed CRISPR knockout screens in an in vitro system permissive to infection only in the presence of IgG antibodies. Validating our approach, a top hit was FcgRIIa, which facilitates binding and internalization of IgG-bound DENV but is not required for canonical infection. Additionally, we identified host factors with no previously described role in DENV infection, including TBC1D24 and SV2B, both of which have known functions in regulated secretion. Using genetic knockout and trans-complemented cells, we validated a functional requirement for these host factors in ADE assays performed with monoclonal antibodies and polyclonal sera in multiple cell lines and using all four DENV serotypes. We show that knockout of TBC1D24 or SV2B impaired binding of IgG-DENV complexes to cells without affecting FcgRIIa expression levels. Thus, we identify cellular factors beyond FcgR that are required for ADE of DENV infection. Our findings represent a first step towards advancing fundamental knowledge behind the biology of ADE that can ultimately be exploited to inform vaccination and therapeutic approaches.

Defining the impact of flavivirus envelope protein glycosylation site mutations on sensitivity to broadly neutralizing antibodies.

Antibodies targeting an envelope dimer epitope (EDE) cross-neutralize Zika virus (ZIKV) and dengue virus (DENV) and have thus inspired an epitope-focused vaccine design. There are two EDE antibody subclasses (EDE1, EDE2) distinguished by their dependence on viral envelope protein N-linked glycosylation at position N153 (DENV) or N154 (ZIKV) for binding. Here, we determined how envelope glycosylation site mutations affect neutralization by EDE and other broadly neutralizing antibodies. Consistent with structural studies, mutations abolishing the N153/N154 glycosylation site increased DENV and ZIKV sensitivity to neutralization by EDE1 antibodies. Surprisingly, despite their location at predicted contact sites, these mutations also increased sensitivity to EDE2 antibodies. Moreover, despite preserving the glycosylation site motif (N-X-S/T), substituting the threonine at ZIKV envelope residue 156 with a serine resulted in loss of glycan occupancy accompanied with increased neutralization sensitivity to EDE antibodies. For DENV, the presence of a serine instead of a threonine at envelope residue 155 retained glycan occupancy, but nonetheless increased sensitivity to EDE antibodies, in some cases to a similar extent as mutation at N153, which abolishes glycosylation. Envelope glycosylation site mutations also increased ZIKV and DENV sensitivity to other non-EDE broadly neutralizing antibodies, but had limited effects on ZIKV- or DENV-specific antibodies. Thus, envelope protein glycosylation is context-dependent and modulates the potency of broadly neutralizing antibodies in a manner not predicted by existing structures. Manipulating envelope protein glycosylation could be a novel strategy for engineering vaccine antigens to elicit antibodies that broadly neutralize ZIKV and DENV.IMPORTANCEAntibodies that potently cross-neutralize Zika (ZIKV) and dengue (DENV) viruses are attractive to induce via vaccination to protect against these co-circulating flaviviruses. Structural studies have shown that viral envelope protein glycosylation is important for binding by one class of these so-called broadly neutralizing antibodies, but less is known about its effect on neutralization. Here, we investigated how envelope protein glycosylation site mutations impact the potency of broadly neutralizing antibodies against ZIKV and DENV. We found that glycan occupancy was not always predicted by an intact N-X-S/T sequence motif. Moreover, envelope protein glycosylation site mutations alter the potency of broadly neutralizing antibodies in a manner unexpected from their predicted binding mechanism as determined by existing structures. We therefore highlight the complex role and determinants of envelope protein glycosylation that should be considered in the design of vaccine antigens to elicit broadly neutralizing antibodies.

The effect of single mutations in Zika virus envelope on escape from broadly neutralizing antibodies.

IMPORTANCE: The wide endemic range of mosquito-vectored flaviviruses-such as Zika virus and dengue virus serotypes 1-4-places hundreds of millions of people at risk of infection every year. Despite this, there are no widely available vaccines, and treatment of severe cases is limited to supportive care. An avenue toward development of more widely applicable vaccines and targeted therapies is the characterization of monoclonal antibodies that broadly neutralize all these viruses. Here, we measure how single amino acid mutations in viral envelope protein affect neutralizing antibodies with both broad and narrow specificities. We find that broadly neutralizing antibodies with potential as vaccine prototypes or biological therapeutics are quantifiably more difficult to escape than narrow, virus-specific neutralizing antibodies.

Single B cell transcriptomics identifies multiple isotypes of broadly neutralizing antibodies against flaviviruses.

Sequential dengue virus (DENV) infections often generate neutralizing antibodies against all four DENV serotypes and sometimes, Zika virus. Characterizing cross-flavivirus broadly neutralizing antibody (bnAb) responses can inform countermeasures that avoid enhancement of infection associated with non-neutralizing antibodies. Here, we used single cell transcriptomics to mine the bnAb repertoire following repeated DENV infections. We identified several new bnAbs with comparable or superior breadth and potency to known bnAbs, and with distinct recognition determinants. Unlike all known flavivirus bnAbs, which are IgG1, one newly identified cross-flavivirus bnAb (F25.S02) was derived from IgA1. Both IgG1 and IgA1 versions of F25.S02 and known bnAbs displayed neutralizing activity, but only IgG1 enhanced infection in monocytes expressing IgG and IgA Fc receptors. Moreover, IgG-mediated enhancement of infection was inhibited by IgA1 versions of bnAbs. We demonstrate a role for IgA in flavivirus infection and immunity with implications for vaccine and therapeutic strategies.

The effect of single mutations in Zika virus envelope on escape from broadly neutralizing antibodies.

Zika virus and dengue virus are co-circulating flaviviruses with a widespread endemic range. Eliciting broad and potent neutralizing antibodies is an attractive goal for developing a vaccine to simultaneously protect against these viruses. However, the capacity of viral mutations to confer escape from broadly neutralizing antibodies remains undescribed, due in part to limited throughput and scope of traditional approaches. Here, we use deep mutational scanning to map how all possible single amino acid mutations in Zika virus envelope protein affect neutralization by antibodies of varying breadth and potency. While all antibodies selected viral escape mutations, the mutations selected by broadly neutralizing antibodies conferred less escape relative to those selected by narrow, virus-specific antibodies. Surprisingly, even for broadly neutralizing antibodies with similar binding footprints, different single mutations led to escape, indicating distinct functional requirements for neutralization not captured by existing structures. Additionally, the antigenic effects of mutations selected by broadly neutralizing antibodies were conserved across divergent, albeit related, flaviviruses. Our approach identifies residues critical for antibody neutralization, thus comprehensively defining the as-yet-unknown functional epitopes of antibodies with clinical potential.

Single B cell transcriptomics identifies multiple isotypes of broadly neutralizing antibodies against flaviviruses.

Sequential dengue virus (DENV) infections often generate neutralizing antibodies against all four DENV serotypes and sometimes, Zika virus. Characterizing cross-flavivirus broadly neutralizing antibody (bnAb) responses can inform countermeasure strategies that avoid infection enhancement associated with non-neutralizing antibodies. Here, we used single cell transcriptomics to mine the bnAb repertoire following secondary DENV infection. We identified several new bnAbs with comparable or superior breadth and potency to known bnAbs, and with distinct recognition determinants. Unlike all known flavivirus bnAbs, which are IgG1, one newly identified cross-flavivirus bnAb (F25.S02) was derived from IgA1. Both IgG1 and IgA1 versions of F25.S02 and known bnAbs displayed neutralizing activity, but only IgG1 enhanced infection in monocytes expressing IgG and IgA Fc receptors. Moreover, IgG-mediated enhancement of infection was inhibited by IgA1 versions of bnAbs. We demonstrate a role for IgA in flavivirus infection and immunity with implications for vaccine and therapeutic strategies.

Rapid development of interprofessional in situ simulation-based training in response to the COVID-19 outbreak in a tertiary-level hospital in Ireland: initial response and lessons for future disaster preparation.

Introduction: The first case of COVID-19 in Ireland was diagnosed on 29 February 2020. Within the same week, our Department of Anaesthesia and Critical Care at University Hospital Galway began to tackle the educational challenge by developing an  in situ interprofessional simulation programme to prepare staff for the impending outbreak. Principles and approaches used for simulation-based training: We describe principles applied to identify core educational and system engineering objectives to prepare healthcare workers (HCWs) for infection control, personal and psychological safety, technical and crisis resource management skills. We discuss application of educational theories, rationale for simulation modes and debriefing techniques. Development of the simulation programme: 3 anaesthesia (general, obstetric, paediatric) and 1 critical care silo were created. 13 simulated scenarios were developed for teaching as well as for testing workflows specific to the outbreak. To support HCWs and ensure safety, management guidelines, cognitive aids and checklists were developed using simulation. The cumulative number of HCWs trained in simulation was 750 over a 4-week period. Challenges and future directions: Due to the protracted nature of the pandemic, simulation educators should address questions related to sustainability, infection control while delivering simulation, establishment of hybrid programmes and support for psychological preparedness.

The identification of needs and development of best practice guidance for the psychological support of frontline healthcare workers during and after COVID-19: A protocol for the FLoWS project.

Recent estimates suggest that up to 34% of frontline workers in healthcare (FLWs) at the forefront of the COVID-19 pandemic response are reporting elevated symptoms of psychological distress due to resource constraints, ineffective treatments, and concerns about self-contamination. However, little systematic research has been carried out to assess the mental health needs of FLWs in Europe, or the extent of psychological suffering in FLWs within different European countries of varying outbreak severity. Accordingly, this project will employ a mixed-methods approach over three work packages to develop best-practice guidelines for alleviating psychological distress in FLWs during the different phases of the pandemic. Work package 1 will identify the point and long-term prevalence of psychological distress symptoms in a sample of Irish and Italian FLWs, and the predictors of these symptoms. Work package 2 will perform a qualitative needs assessment on a sample of Irish and Italian FLWs to identify sources of stress and resilience, barriers to psychological care, and optimal strategies for alleviating psychological distress in relation to the COVID-19 pandemic. Work package 3 will synthesise the findings from the preceding work packages to draft best practice guidelines, which will be co-created by a multidisciplinary panel of experts using the Delphi method. The guidelines will provide clinicians with a framework for alleviating psychological distress in FLWs, with particular relevance to the COVID-19 pandemic, but may also have relevance for future pandemics and other public health emergencies.

Simulation as a toolkit-understanding the perils of blood transfusion in a complex health care environment.

BACKGROUND: Administration of blood is a complex process requiring vigilance and effective teamwork. Despite strict policies and training on blood administration, errors still occur and can lead to mistransfusion with adverse patient outcomes. We used an in situ simulated scenario within an operating room (OR) to identify weaknesses in the current process and hazards that could contribute to mistransfusion. METHODS: A process checklist of critical steps of safe transfusion was developed based on a large academic centre's internal hospital policy and practice. Ten standardized operating room scenarios were conducted involving management of postoperative bleeding. Scenarios lasted 20 min or until blood transfusion was started. Debriefing followed immediately. Video recordings were reviewed, scored, and evaluated for team performance. Latent safety threats were identified. Focus groups further helped to identify rationale for decisions made. Participants completed questionnaires to evaluate the exercise. RESULTS: Forty-three experienced OR professionals participated. Of the 19 steps identified as essential for the safe administration of blood components, the median number of steps correctly completed per team was 11. The largest number of errors occurred when different team members interacted and during the immediate pre-transfusion check. We report that this type of learning immediately increased participants' self-reported ability to perform in a team (90%) and to improve clinical care (88%). CONCLUSIONS: In situ simulation is valuable in identifying common susceptibilities in blood administration error in a complex healthcare organization. Administrators and clinicians may wish to use simulation as an opportunity for system improvement in the delivery of quality care.

Permissive hypercapnia: what to remember.

PURPOSE OF REVIEW: Hypercapnia is a central component of diverse respiratory disorders, while 'permissive hypercapnia' is frequently used in ventilatory strategies for patients with severe respiratory failure. This review will present data from recent studies relating to hypercapnia, focusing on issues that are of importance to anesthesiologists caring for the surgical and/or critically ill patient. RECENT FINDINGS: Protective ventilatory strategies involving permissive hypercapnia are widely used in patients with severe respiratory failure, particularly in acute respiratory distress syndrome, status asthmaticus, chronic obstructive pulmonary disease and neonatal respiratory failure. The physiologic effects of hypercapnia are increasingly well understood, and important recent insights have emerged regarding the cellular and molecular mechanisms of action of hypercapnia and acidosis. Acute hypercapnic acidosis is protective in multiple models of nonseptic lung injury. These effects are mediated in part through inhibition of the NF-κB pathway. Hypercapnia-mediated NF-κB inhibition may also explain several deleterious effects, including delayed epithelial wound healing and decreased bacterial killing, which has been demonstrated to cause worse lung injury in prolonged untreated pneumonia models. SUMMARY: The mechanisms of action of hypercapnia and acidosis continue to be elucidated, and this knowledge is central to determining the safety and therapeutic utility of hypercapnia in protective lung ventilatory strategies.

Hypercapnic acidosis attenuates ventilation-induced lung injury by a nuclear factor-κB-dependent mechanism.

OBJECTIVES: Hypercapnic acidosis protects against ventilation-induced lung injury. We wished to determine whether the beneficial effects of hypercapnic acidosis in reducing stretch-induced injury were mediated via inhibition of nuclear factor-κB, a key transcriptional regulator in inflammation, injury, and repair. DESIGN: Prospective randomized animal study. SETTING: University research laboratory. SUBJECTS: Adult male Sprague-Dawley rats. INTERVENTIONS: In separate experimental series, the potential for hypercapnic acidosis to attenuate moderate and severe ventilation-induced lung injury was determined. In each series, following induction of anesthesia and tracheostomy, Sprague-Dawley rats were randomized to (normocapnia; FICO2 0.00) or (hypercapnic acidosis; FICO2 0.05), subjected to high stretch ventilation, and the severity of lung injury and indices of activation of the nuclear factor-κB pathway were assessed. Subsequent in vitro experiments examined the potential for hypercapnic acidosis to reduce pulmonary epithelial inflammation and injury induced by cyclic mechanical stretch. The role of the nuclear factor-κB pathway in hypercapnic acidosis-mediated protection from stretch injury was then determined. MEASUREMENTS AND MAIN RESULTS: Hypercapnic acidosis attenuated moderate and severe ventilation-induced lung injury, as evidenced by improved oxygenation, compliance, and reduced histologic injury compared to normocapnic conditions. Hypercapnic acidosis reduced indices of inflammation such as interleukin-6 and bronchoalveolar lavage neutrophil infiltration. Hypercapnic acidosis reduced the decrement of the nuclear factor-κB inhibitor IκBα and reduced the generation of cytokine-induced neutrophil chemoattractant-1. Hypercapnic acidosis reduced cyclic mechanical stretch-induced nuclear factor-κB activation, reduced interleukin-8 production, and decreased epithelial injury and cell death compared to normocapnia. CONCLUSIONS: Hypercapnic acidosis attenuated ventilation-induced lung injury independent of injury severity and decreased mechanical stretch-induced epithelial injury and death, via a nuclear factor-κB-dependent mechanism.

Evolution of the inflammatory and fibroproliferative responses during resolution and repair after ventilator-induced lung injury in the rat.

BACKGROUND: The time course and mechanisms of resolution and repair, and the potential for fibrosis following ventilation-induced lung injury (VILI), are unclear. We sought to examine the pattern of inflammation, injury, repair, and fibrosis following VILI. METHODS: Sixty anesthetized rats were subject to high-stretch; low-stretch, or sham ventilation, and randomly allocated to undergo periods of recovery of 6, 24, 48, and 96 h, and 7 and 14 days. Animals were then reanesthetized, and the extent of lung injury, inflammation, and repair determined. RESULTS: No injury was seen following low-stretch or sham ventilation. VILI caused severe lung injury, maximal at 24 h, but largely resolved by 96 h. Arterial oxygen tension decreased from a mean (SD) of 144.8 (4.1) mmHg to 96.2 (10.3) mmHg 6 h after VILI, before gradually recovering to 131.2 (14.3) mmHg at 96 h. VILI induced an early neutrophilic alveolitis and a later lymphocytic alveolitis, followed by a monocyte/macrophage infiltration. Alveolar tumor necrosis factor-α, interleukin-1ß, and transforming growth factor-ß1 concentrations peaked at 6 h and returned to baseline within 24 h, while interleukin-10 remained increased for 48 h. VILI generated a marked but transient fibroproliferative response, which restored normal lung architecture. There was no evidence of fibrosis at 7 and 14 days. CONCLUSIONS: High-stretch ventilation caused severe lung injury, activating a transient inflammatory and fibroproliferative repair response, which restored normal lung architecture without evidence of fibrosis.

Overexpression of pulmonary extracellular superoxide dismutase attenuates endotoxin-induced acute lung injury.

PURPOSE: Superoxide is produced by activated neutrophils during the inflammatory response to stimuli such as endotoxin, can directly or indirectly injure host cells, and has been implicated in the pathogenesis of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). We wished to determine the potential for pulmonary overexpression of the extracellular isoform of superoxide dismutase (EC-SOD) to reduce the severity of endotoxin-induced lung injury. METHODS: Animals were randomly allocated to undergo intratracheal instillation of (1) surfactant alone (vehicle); (2) adeno-associated virus (AAV) vectors containing a null transgene (AAV-null); and (3) adeno-associated virus vectors containing the EC-SOD transgene (AAV-EC-SOD) and endotoxin was subsequently administered intratracheally. Two additional groups were randomized to receive (1) vehicle or (2) AAV-EC-SOD, and to undergo sham (vehicle) injury. The severity of the lung injury was assessed in all animals 24 h later. RESULTS: Endotoxin produced a severe lung injury compared to sham injury. The AAV vector encoding EC-SOD increased lung EC-SOD concentrations, and enhanced the antioxidant capacity of the lung. EC-SOD overexpression decreased the severity of endotoxin-induced ALI, reducing the decrement in systemic oxygenation and lung compliance, decreasing lung permeability and decreasing histologic injury. EC-SOD attenuated pulmonary inflammation, decreased bronchoalveolar lavage neutrophil counts, and reduced interleukin-6 and CINC-1 concentrations. The AAV vector itself did not contribute to inflammation or to lung injury. CONCLUSIONS: Pulmonary overexpression of EC-SOD protects the lung against endotoxin-induced ALI.

Clinical review: gene-based therapies for ALI/ARDS: where are we now?

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) confer substantial morbidity and mortality, and have no specific therapy. The accessibility of the distal lung epithelium via the airway route, and the relatively transient nature of ALI/ARDS, suggest that the disease may be amenable to gene-based therapies. Ongoing advances in our understanding of the pathophysiology of ALI/ARDS have revealed multiple therapeutic targets for gene-based approaches. Strategies to enhance or restore lung epithelial and/or endothelial cell function, to strengthen lung defense mechanisms against injury, to speed clearance of infection and to enhance the repair process following ALI/ARDS have all demonstrated promise in preclinical models. Despite three decades of gene therapy research, however, the clinical potential for gene-based approaches to lung diseases including ALI/ARDS remains to be realized. Multiple barriers to effective pulmonary gene therapy exist, including the pulmonary architecture, pulmonary defense mechanisms against inhaled particles, the immunogenicity of viral vectors and the poor transfection efficiency of nonviral delivery methods. Deficits remain in our knowledge regarding the optimal molecular targets for gene-based approaches. Encouragingly, recent progress in overcoming these barriers offers hope for the successful translation of gene-based approaches for ALI/ARDS to the clinical setting.

Hypercapnia and acidosis in sepsis: a double-edged sword?

Acute respiratory distress syndrome is a devastating disease that causes substantial morbidity and mortality. Mechanical ventilation can worsen lung injury, whereas ventilatory strategies that reduce lung stretch, resulting in a "permissive" hypercapnic acidosis (HCA), improve outcome. HCA directly reduces nonsepsis-induced lung injury in preclinical models and, therefore, has therapeutic potential in these patients. These beneficial effects are mediated via inhibition of the host immune response, particularly cytokine signaling, phagocyte function, and the adaptive immune response. Of concern, these immunosuppressive effects of HCA may hinder the host response to microbial infection. Recent studies suggest that HCA is protective in the earlier phases of bacterial pneumonia-induced sepsis but may worsen injury in the setting of prolonged lung sepsis. In contrast, HCA is protective in preclinical models of early and prolonged systemic sepsis. Buffering of the HCA is not beneficial and may worsen pneumonia-induced injury.

Differential effects of buffered hypercapnia versus hypercapnic acidosis on shock and lung injury induced by systemic sepsis.

BACKGROUND: Acute hypercapnic acidosis protects against lung injury caused by nonseptic insults and after both pulmonary and systemic sepsis. The authors wished to dissect the contribution of the acidosis versus hypercapnia per se to the effects of hypercapnic acidosis on the hemodynamic profile and severity of lung injury induced by systemic sepsis. METHODS: In the hypercapnic acidosis series, adult male Sprague-Dawley rats were randomized to normocapnia or hypercapnic acidosis-produced by adding 5% carbon dioxide to the inspired gas-and cecal ligation and puncture performed. In the buffered hypercapnia series, animals were first randomized to housing under conditions of environmental normocapnia or hypercapnia-produced by exposure to 8% carbon dioxide-to allow renal buffering. After 96 h, cecal ligation and puncture was performed. In both series, the animals were ventilated for 6 h, and the severity of the lung injury and hemodynamic deterioration were assessed. RESULTS: Both hypercapnic acidosis and buffered hypercapnia attenuated the development and severity of hypotension and reduced lactate accumulation compared to normocapnia. Hypercapnic acidosis reduced lung injury and inflammation, decreased mean (+ or - SD) bronchoalveolar lavage protein concentration (232 + or - 50 versus 279 + or - 27 microg x ml(-1)) and median neutrophil counts (3,370 versus 9,120 cells x ml(-1)), and reduced histologic lung injury. In contrast, buffered hypercapnia did not reduce the severity of systemic sepsis induced lung injury. CONCLUSIONS: Both hypercapnic acidosis and buffered hypercapnia attenuate the hemodynamic consequences of systemic sepsis. In contrast, hypercapnic acidosis, but not buffered hypercapnia, reduced the severity of sepsis-induced lung injury.

Hypercapnic acidosis attenuates shock and lung injury in early and prolonged systemic sepsis.

OBJECTIVE: To investigate whether acute hypercapnic acidosis--induced by adding CO2 to inspired gas--would protect against severe systemic sepsis-induced lung and systemic organ injury resulting from cecal ligation and puncture. Acute hypercapnic acidosis protects against lung injury after both nonseptic and early pneumonia-induced lung injury. In contrast, prolonged hypercapnia worsens pneumonia-induced lung injury. The effects of hypercapnia and acidosis in the setting of systemic sepsis remain to be determined. DESIGN: Prospective randomized animal study. SETTING: University research laboratory. SUBJECTS: Adult male Sprague-Dawley rats. INTERVENTIONS: In the early systemic sepsis series, post induction of anesthesia and tracheostomy placement, animals were randomized to normocapnia (Fico2 = 0.00, n = 12) or hypercapnic acidosis (Fico2 = 0.05, n = 12). Cecal ligation and puncture were performed and the animals were ventilated for 3 hrs. In the prolonged systemic sepsis series, rats were anesthetized, cecal ligation and puncture were performed, and the animals were allowed to recover. The animals were then randomized to housing under conditions of environmental normocapnia (Fico2 = 0.00, n = 20) or hypercapnia (Fico2 = 0.08, n = 20). After 96 hrs, the animals were reanesthetized, and the severity of lung and hemodynamic injury was assessed. RESULTS: In early systemic sepsis, hypercapnic acidosis attenuated the development and severity of hypotension, and reduced lactate accumulation and the decrement in central venous oxyhemoglobin levels, compared with normocapnia. Hypercapnic acidosis reduced bronchoalveolar lavage neutrophil infiltration, and lung wet/dry weight ratios. In prolonged systemic sepsis, hypercapnic acidosis reduced histologic indices of lung injury. There was no evidence that hypercapnia worsened prolonged systemic sepsis-induced lung injury. Hypercapnic acidosis did not alter lung or systemic bacterial loads in early or prolonged systemic sepsis. CONCLUSION: Hypercapnic acidosis exerts beneficial effects in early and prolonged cecal ligation and puncture-induced polymicrobial systemic sepsis.