Memory B cell repertoire for recognition of evolving SARS-CoV-2 spike.

Memory B cell reserves can generate protective antibodies against repeated SARS-CoV-2 infections, but with unknown reach from original infection to antigenically drifted variants. We charted memory B cell receptor-encoded antibodies from 19 COVID-19 convalescent subjects against SARS-CoV-2 spike (S) and found seven major antibody competition groups against epitopes recurrently targeted across individuals. Inclusion of published and newly determined structures of antibody-S complexes identified corresponding epitopic regions. Group assignment correlated with cross-CoV-reactivity breadth, neutralization potency, and convergent antibody signatures. Although emerging SARS-CoV-2 variants of concern escaped binding by many members of the groups associated with the most potent neutralizing activity, some antibodies in each of those groups retained affinity-suggesting that otherwise redundant components of a primary immune response are important for durable protection from evolving pathogens. Our results furnish a global atlas of S-specific memory B cell repertoires and illustrate properties driving viral escape and conferring robustness against emerging variants.

Antibodies to a Conserved Influenza Head Interface Epitope Protect by an IgG Subtype-Dependent Mechanism.

Vaccines to generate durable humoral immunity against antigenically evolving pathogens such as the influenza virus must elicit antibodies that recognize conserved epitopes. Analysis of single memory B cells from immunized human donors has led us to characterize a previously unrecognized epitope of influenza hemagglutinin (HA) that is immunogenic in humans and conserved among influenza subtypes. Structures show that an unrelated antibody from a participant in an experimental infection protocol recognized the epitope as well. IgGs specific for this antigenic determinant do not block viral infection in vitro, but passive administration to mice affords robust IgG subtype-dependent protection against influenza infection. The epitope, occluded in the pre-fusion form of HA, is at the contact surface between HA head domains; reversible molecular "breathing" of the HA trimer can expose the interface to antibody and B cells. Antigens that present this broadly immunogenic HA epitope may be good candidates for inclusion in "universal" flu vaccines.

Memory B Cells that Cross-React with Group 1 and Group 2 Influenza A Viruses Are Abundant in Adult Human Repertoires.

Human B cell antigen-receptor (BCR) repertoires reflect repeated exposures to evolving influenza viruses; new exposures update the previously generated B cell memory (Bmem) population. Despite structural similarity of hemagglutinins (HAs) from the two groups of influenza A viruses, cross-reacting antibodies (Abs) are uncommon. We analyzed Bmem compartments in three unrelated, adult donors and found frequent cross-group BCRs, both HA-head directed and non-head directed. Members of a clonal lineage from one donor had a BCR structure similar to that of a previously described Ab, encoded by different gene segments. Comparison showed that both Abs contacted the HA receptor-binding site through long heavy-chain third complementarity determining regions. Affinities of the clonal-lineage BCRs for historical influenza-virus HAs from both group 1 and group 2 viruses suggested that serial responses to seasonal influenza exposures had elicited the lineage and driven affinity maturation. We propose that appropriate immunization regimens might elicit a comparably broad response.

Protective human antibodies against a conserved epitope in pre- and postfusion influenza hemagglutinin.

Phylogenetically and antigenically distinct influenza A and B viruses (IAV and IBV) circulate in human populations, causing widespread morbidity. Antibodies (Abs) that bind epitopes conserved in both IAV and IBV hemagglutinins (HAs) could protect against disease by diverse virus subtypes. Only one reported HA Ab, isolated from a combinatorial display library, protects against both IAV and IBV. Thus, there has been so far no information on the likelihood of finding naturally occurring human Abs that bind HAs of diverse IAV subtypes and IBV lineages. We have now recovered from several unrelated human donors five clonal Abs that bind a conserved epitope preferentially exposed in the postfusion conformation of IAV and IVB HA2. These Abs lack neutralizing activity in vitro but in mice provide strong, IgG subtype-dependent protection against lethal IAV and IBV infections. Strategies to elicit similar Abs routinely might contribute to more effective influenza vaccines.

A new class of antibodies that overcomes a steric barrier to cross-group neutralization of influenza viruses.

Antibody titers that inhibit the influenza virus hemagglutinin (HA) from engaging its receptor are the accepted correlate of protection from infection. Many potent antibodies with broad, intra-subtype specificity bind HA at the receptor binding site (RBS). One barrier to broad H1-H3 cross-subtype neutralization is an insertion (133a) between positions 133 and 134 on the rim of the H1 HA RBS. We describe here a class of antibodies that overcomes this barrier. These genetically unrestricted antibodies are abundant in the human B cell memory compartment. Analysis of the affinities of selected members of this class for historical H1 and H3 isolates suggest that they were elicited by H3 exposure and broadened or diverted by later exposure(s) to H1 HA. RBS mutations in egg-adapted vaccine strains cause the new H1 specificity of these antibodies to depend on the egg adaptation. The results suggest that suitable immunogens might elicit 133a-independent, H1-H3 cross neutralization by RBS-directed antibodies.

Transient inhibition of lysosomal functions potentiates nucleic acid vaccines.

Nucleic acid vaccines have shown promising results in the clinic against infectious diseases and cancers. To robustly improve the vaccine efficacy and safety, we developed an approach to increase the intracellular stability of nucleic acids by transiently inhibiting lysosomal function in targeted tissues using sucrose. To achieve efficient and localized delivery of sucrose in animals, we designed a biomimetic lipid nanoparticle (LNP) to target the delivery of sucrose into mouse muscle cells. Using this approach, viral antigen expression in mouse muscle after DNA vaccination was substantially increased and prolonged without inducing local or systemic inflammation or toxicity. The same change in antigen expression would be achieved if the vaccine dose could be increased by 3,000 folds, which is experimentally and clinically impractical due to material restrictions and severe toxicity that will be induced by such a high dose of nucleic acids. The increase in antigen expression augmented the infiltration and activation of antigen-presenting cells, significantly improved vaccine-elicited humoral and T cell responses, and fully protected mice against the viral challenge at a low dose of vaccine. Based on these observations, we conclude that transient inhibition of lysosome function in target tissue by sucrose LNPs is a safe and potent approach to substantially improve nucleic acid-based vaccines.

Differential immune imprinting by influenza virus vaccination and infection in nonhuman primates.

Immune memory of a first infection with influenza virus establishes a lasting imprint. Recall of that memory dominates the response to later infections or vaccinations by antigenically drifted strains. Early childhood immunization before infection may leave an imprint with different characteristics. We report here a comparison of imprinting by vaccination and infection in a small cohort of nonhuman primates (NHPs). We assayed serum antibody responses for binding with hemaglutinnins (HAs) both from the infecting or immunizing strain (H3 A/Aichi 02/1968) and from strains representing later H3 antigenic clusters ("forward breadth") and examined the effects of defined HA mutations on serum titers. Initial exposure by infection elicited strong HA-binding and neutralizing serum antibody responses but with little forward breadth; initial vaccination with HA from the same strain elicited a weaker response with little neutralizing activity but considerable breadth of binding, not only for later H3 HAs but also for HA of the 2009 H1 new pandemic virus. Memory imprinted by infection, reflected in the response to two immunizing boosts, was largely restricted (as in humans) to the outward-facing HA surface, the principal region of historical variation. Memory imprinted by immunization showed exposure to more widely distributed epitopes, including sites that have not varied during evolution of the H3 HA but that yield nonneutralizing responses. The mode of initial exposure thus affects both the strength of the response and the breadth of the imprint; design of next-generation vaccines will need to take the differences into account.

Cost analysis and clinical outcomes of anatomic pre-contoured locking versus conventional plates for distal fibula ankle fractures.

PURPOSE: To analyze differences in union, complication rates and cost from surgical fixation of distal fibula fractures with fibular plating implants. METHODS: In total, 380 adult patients from 2012 to 2015 treated with 12 fibular plates from 4 different manufacturers utilized by 9 surgeons were retrospectively reviewed. They were stratified into a conventional one-third tubular fibular plate group, pre-contoured anatomic locking plate group, or a heterogeneous group including 3.5-mm reconstruction, one-third tubular locking, composite, and limited compression plates. The outcomes included failure of fixation, deep infection requiring debridement, time to union, anatomic reduction, superficial infection, hardware removal, and post-traumatic arthritis. Plate and screw costs were calculated from hospital billing records. RESULTS: Pre-contoured locking plates were used in older, female patients with a greater number of comorbidities. Open injuries and OTA 44B fractures were more likely to be an indication for pre-contoured plates. There was no difference noted in time to union between the different plating groups. Risk factors for deep infection requiring debridement included a history of tobacco use, open fractures, and pre-contoured locking plates relative to the conventional plating group. The pre-contoured plating group was on average $586 more expensive compared to the conventional group. CONCLUSION: Pre-contoured locking plates achieved similar radiographic outcomes compared to conventional plates with an increased risk of complications and higher cost. Surgeons should consider their choice of implant based on the patient's fracture pattern, underlying comorbidities, and risk for infection.

Investigation of the intraoperative cortical responses to spinal motor mapping in a patient with chronic pain.

Intraoperative neurophysiological monitoring (IONM) in spinal cord stimulation (SCS) surgery for chronic pain is shown to provide effective guidance during device placement. Electromyography (EMG) is used to determine the laterality of the paddle. In some SCS cases, laterality cannot be obtained via EMG due to patient physiology. Electroencephalography (EEG) is already used in IONM to monitor cortical responses. Here, we show proof-of-concept of assessing the responses of epidurally evoked EMGs simultaneously with EEGs to determine laterality during IONM using a high-resolution (HR) SCS paddle. An 8-column HR-SCS paddle was acutely placed at T9-T10 interspace in patients with failed back surgery syndrome. EMG signals from 18 muscle groups were recorded simultaneously with 60-channel EEG signals at various stimulation amplitudes (0-10 mA). Particular attention was paid to regions associated with pain including the somatosensory cortex (S1), prefrontal cortex (PFC), and motor cortex (M1). When left and right lateral contacts were stimulated at low amplitudes (1-2 mA), significant changes were seen in θ, α, and ß powers in the contralateral PFC but not in M1 or S1. There was a significant correlation between M1 and contralateral contacts in α power. At higher currents (7-8 mA), right-sided contacts resulted in α power change. We found significant differences in α, θ, and ß powers in PFC for contralateral stimulation of the lateral SCS contacts at low amplitudes and in α power at higher amplitudes. The changes in PFC suggest the potential of EEG for understanding a cortical mechanism of action of SCS and provide insight into the pathophysiology of chronic pain.NEW & NOTEWORTHY Here, we present proof of concept of assessing the responses of epidurally evoked electromyography simultaneously with scalp electroencephalography to determine whether both laterality and insights into pain mechanisms can be elucidated. With stimulation, significant changes were seen in θ, α, and ß band power in the contralateral prefrontal cortex and in α power in the motor cortex. We provide insight into the mechanism of action of SCS in preventing pain in this patient.

European Respiratory Society/American Thoracic Society technical statement: standardisation of the measurement of lung volumes, 2023 update.

This document updates the 2005 European Respiratory Society (ERS) and American Thoracic Society (ATS) technical standard for the measurement of lung volumes. The 2005 document integrated the recommendations of an ATS/ERS task force with those from an earlier National Heart, Lung, and Blood Institute workshop that led to the publication of background papers between 1995 and 1999 and a consensus workshop report with more in-depth descriptions and discussion. Advancements in hardware and software, new research and emerging approaches have necessitated an update to the 2005 technical standard to guide laboratory directors, physiologists, operators, pulmonologists and manufacturers. Key updates include standardisation of linked spirometry, new equipment quality control and validation recommendations, generalisation of the multiple breath washout concept beyond nitrogen, a new acceptability and grading system with addition of example tracings, and a brief review of imaging and other new techniques to measure lung volumes. Future directions and key research questions are also noted.

Development and characterization of a new monoclonal antibody against SARS-CoV-2 NSP12 (RdRp).

SARS-CoV-2 NSP12, the viral RNA-dependent RNA polymerase (RdRp), is required for viral replication and is a therapeutic target to treat COVID-19. To facilitate research on SARS-CoV-2 NSP12 protein, we developed a rat monoclonal antibody (CM12.1) against the NSP12 N-terminus that can facilitate functional studies. Immunoblotting and immunofluorescence assay (IFA) confirmed the specific detection of NSP12 protein by this antibody for cells overexpressing the protein. Although NSP12 is generated from the ORF1ab polyprotein, IFA of human autopsy COVID-19 lung samples revealed NSP12 expression in only a small fraction of lung cells including goblet, club-like, vascular endothelial cells, and a range of immune cells, despite wide-spread tissue expression of spike protein antigen. Similar studies using in vitro infection also generated scant protein detection in cells with established virus replication. These results suggest that NSP12 may have diminished steady-state expression or extensive posttranslation modifications that limit antibody reactivity during SARS-CoV-2 replication.

One size does not fit all: an application of stochastic modeling to estimating primary healthcare needs in Ethiopia at the sub-national level.

BACKGROUND: Primary healthcare systems require adequate staffing to meet the needs of their local population. Guidelines typically use population ratio targets for healthcare workers, such as Ethiopia's goal of two health extension workers for every five thousand people. However, fixed ratios do not reflect local demographics, fertility rates, disease burden (e.g., malaria endemicity), or trends in these values. Recognizing this, we set out to estimate the clinical workload to meet the primary healthcare needs in Ethiopia by region. METHODS: We utilize the open-source R package PACE-HRH for our analysis, which is a stochastic Monte Carlo simulation model that estimates workload for a specified service package and population. Assumptions and data inputs for region-specific fertility, mortality, disease burden were drawn from literature, DHS, and WorldPop. We project workload until 2035 for seven regions and two charted cities of Ethiopia. RESULTS: All regions and charted cities are expected to experience increased workload between 2021 and 2035 for a starting catchment of five thousand people. The expected (mean) annual clinical workload varied from 2,930 h (Addis) to 3,752 h (Gambela) and increased by 19-28% over fifteen years. This results from a decline in per capita workload (due to declines in fertility and infectious diseases), overpowered by total population growth. Pregnancy, non-communicable diseases, sick child care, and nutrition remain the largest service categories, but their priority shifts substantially in some regions by 2035. Sensitivity analysis shows that fertility assumptions have major implications for workload. We incorporate seasonality and estimate monthly variation of up to 8.9% (Somali), though most services with high variability are declining. CONCLUSIONS: Regional variation in demographics, fertility, seasonality, and disease trends all affect the workload estimates. This results in differences in expected clinical workload, the level of uncertainty in those estimates, and relative priorities between service categories. By showing these differences, we demonstrate the inadequacy of a fixed population ratio for staffing allocation. Policy-makers and regulators need to consider these factors in designing their healthcare systems, or they risk sub-optimally allocating workforce and creating inequitable access to care.

Effect of high glucose on glycosaminoglycans in cultured retinal endothelial cells and rat retina.

INTRODUCTION: The endothelial glycocalyx regulates vascular permeability, inflammation, and coagulation, and acts as a mechanosensor. The loss of glycocalyx can cause endothelial injury and contribute to several microvascular complications and, therefore, may promote diabetic retinopathy. Studies have shown a partial loss of retinal glycocalyx in diabetes, but with few molecular details of the changes in glycosaminoglycan (GAG) composition. Therefore, the purpose of our study was to investigate the effect of hyperglycemia on GAGs of the retinal endothelial glycocalyx. METHODS: GAGs were isolated from rat retinal microvascular endothelial cells (RRMECs), media, and retinas, followed by liquid chromatography-mass spectrometry assays. Quantitative real-time polymerase chain reaction was used to study mRNA transcripts of the enzymes involved in GAG biosynthesis. RESULTS AND CONCLUSIONS: Hyperglycemia significantly increased the shedding of heparan sulfate (HS), chondroitin sulfate (CS), and hyaluronic acid (HA). There were no changes to the levels of HS in RRMEC monolayers grown in high-glucose media, but the levels of CS and HA decreased dramatically. Similarly, while HA decreased in the retinas of diabetic rats, the total GAG and CS levels increased. Hyperglycemia in RRMECs caused a significant increase in the mRNA levels of the enzymes involved in GAG biosynthesis (including EXTL-1,2,3, EXT-1,2, ChSY-1,3, and HAS-2,3), with these increases potentially being compensatory responses to overall glycocalyx loss. Both RRMECs and retinas of diabetic rats exhibited glucose-induced alterations in the disaccharide compositions and sulfation of HS and CS, with the changes in sulfation including N,6-O-sulfation on HS and 4-O-sulfation on CS.

Use of Fractional Exhaled Nitric Oxide to Guide the Treatment of Asthma: An Official American Thoracic Society Clinical Practice Guideline.

Background: The fractional exhaled nitric oxide (FENO) test is a point-of-care test that is used in the assessment of asthma. Objective: To provide evidence-based clinical guidance on whether FENO testing is indicated to optimize asthma treatment in patients with asthma in whom treatment is being considered. Methods: An international, multidisciplinary panel of experts was convened to form a consensus document regarding a single question relevant to the use of FENO. The question was selected from three potential questions based on the greatest perceived impact on clinical practice and the unmet need for evidence-based answers related to this question. The panel performed systematic reviews of published randomized controlled trials between 2004 and 2019 and followed the Grading of Recommendations, Assessment, Development, and Evaluation (GRADE) evidence-to-decision framework to develop recommendations. All panel members evaluated and approved the recommendations. Main Results: After considering the overall low quality of the evidence, the panel made a conditional recommendation for FENO-based care. In patients with asthma in whom treatment is being considered, we suggest that FENO is beneficial and should be used in addition to usual care. This judgment is based on a balance of effects that probably favors the intervention; the moderate costs and availability of resources, which probably favors the intervention; and the perceived acceptability and feasibility of the intervention in daily practice. Conclusions: Clinicians should consider this recommendation to measure FENO in patients with asthma in whom treatment is being considered based on current best available evidence.

Safety and feasibility of outpatient high-dose cytarabine and intermediate-dose cytarabine for consolidation therapy in acute myeloid leukemia.

INTRODUCTION: The standard of care consolidation therapy for acute myeloid leukemia is high-dose cytarabine or intermediate-dose cytarabine, which are traditionally given inpatient. At Moffitt Cancer Center, we have moved the administration of high-dose cytarabine and intermediate-dose cytarabine to the outpatient setting through the inpatient/outpatient program. To facilitate outpatient administration, high-dose cytarabine and intermediate-dose cytarabine are given in a shorter interval of every 10 h instead of 12 h. The safety of a shorter duration interval of high-dose cytarabine and intermediate-dose cytarabine is unknown. This study aims to assess the safety and feasibility of administering high-dose cytarabine and intermediate-dose cytarabine consolidation therapy in the inpatient/outpatient setting. METHODS: This is a retrospective chart review to analyze acute myeloid leukemia patients treated with inpatient/outpatient high-dose cytarabine or intermediate-dose cytarabine consolidation therapy at Moffitt Cancer Center from January 1, 2015, through November 1, 2018. The primary objective was to determine the incidence of hospitalization during the inpatient/outpatient administration of high-dose cytarabine or intermediate-dose cytarabine. RESULTS: Two hundred fifty-three of 255 cycles of high-dose cytarabine/intermediate-dose cytarabine were delivered outpatient over the reviewed time period to 118 patients. No patients receiving outpatient high-dose cytarabine/intermediate-dose cytarabine consolidation required hospitalization during chemotherapy. Our incidence of hospitalization (24%) after chemotherapy is consistent with the reported literature. Through the inpatient/outpatient administration of high-dose cytarabine and intermediate-dose cytarabine, 1265 inpatient days were saved with an approximate revenue of $3,135,176 generated in our study period. CONCLUSION: Inpatient/outpatient administration of high-dose cytarabine and intermediate-dose cytarabine is both safe and feasible. Moving high-dose cytarabine/intermediate-dose cytarabine administration to the outpatient setting resulted in significant additional revenue vs. inpatient administration.

Decreasing measles burden by optimizing campaign timing.

Measles remains a major contributor to preventable child mortality, and bridging gaps in measles immunity is a fundamental challenge to global health. In high-burden settings, mass vaccination campaigns are conducted to increase access to vaccine and address this issue. Ensuring that campaigns are optimally effective is a crucial step toward measles elimination; however, the relationship between campaign impact and disease dynamics is poorly understood. Here, we study measles in Pakistan, and we demonstrate that campaign timing can be tuned to optimally interact with local transmission seasonality and recent incidence history. We develop a mechanistic modeling approach to optimize timing in general high-burden settings, and we find that in Pakistan, hundreds of thousands of infections can be averted with no change in campaign cost.

Affinity maturation in a human humoral response to influenza hemagglutinin.

Affinity maturation of the B cell antigen receptor (BCR) is a conserved and crucial component of the adaptive immune response. BCR lineages, inferred from paired heavy- and light-chain sequences of rearranged Ig genes from multiple descendants of the same naive B cell precursor (the lineages' unmutated common ancestor, "UCA"), make it possible to reconstruct the underlying somatic evolutionary history. We present here an extensive structural and biophysical analysis of a lineage of BCRs directed against the receptor binding site (RBS) of subtype H1 influenza virus hemagglutinin (HA). The lineage includes 8 antibodies detected directly by sequencing, 3 in 1 principal branch and 5 in the other. When bound to HA, the heavy-chain third complementarity determining region (HCDR3) fits with an invariant pose into the RBS, but in each of the 2 branches, the rest of the Fab reorients specifically, from its position in the HA-bound UCA, about a hinge at the base of HCDR3. New contacts generated by the reorientation compensate for contacts lost as the H1 HA mutated during the time between the donor's initial exposure and the vaccination that preceded sampling. Our data indicate that a "pluripotent" naive response differentiated, in each branch, into 1 of its possible alternatives. This property of naive BCRs and persistence of multiple branches of their progeny lineages can offer broader protection from evolving pathogens than can a single, linear pathway of somatic mutation.

How does hostile resistance interfere with the benefits of cognitive-behavioral therapy for panic disorder? The role of therapist adherence and working alliance.

Objective Although clients' hostile behavior directed at therapists (hostile resistance) predicts worse outcomes in cognitive-behavioral therapy (CBT) for panic disorder, the process by which this happens remains unknown. This study examines two putative mechanisms: working alliance and therapist adherence. Method: Seventy-one adults with primary panic disorder received CBT in a larger trial. Hostile resistance and adherence in Sessions 2 and 10 were reliably coded using observer-rated measures; client- and therapist-rated questionnaires assessed working alliance. Outcome measures were attrition and symptomatic improvement, assessed at multiple timepoints with the Panic Disorder Severity Scale. Results: Hostile resistance was significantly related to both preexisting (r = -.36, p = .04) and subsequent declines (r = -.58, p < .0001) in the working alliance. Nevertheless, hierarchical linear modeling revealed that neither a declining alliance nor therapist adherence (whether treated as linear or curvilinear) was independently predictive of symptom change, nor did these factors mediate hostile resistance's association with worse symptomatic improvement. Exploratory logistic regressions similarly indicated that neither adherence nor alliance moderated whether hostilely resistant clients dropped out. Conclusion: This is the first study to establish a bidirectional association between hostile resistance and a declining working alliance. Findings also add to a mixed literature on the adherence-outcome relationship.

Assessing the stability of polio eradication after the withdrawal of oral polio vaccine.

The oral polio vaccine (OPV) contains live-attenuated polioviruses that induce immunity by causing low virulence infections in vaccine recipients and their close contacts. Widespread immunization with OPV has reduced the annual global burden of paralytic poliomyelitis by a factor of 10,000 or more and has driven wild poliovirus (WPV) to the brink of eradication. However, in instances that have so far been rare, OPV can paralyze vaccine recipients and generate vaccine-derived polio outbreaks. To complete polio eradication, OPV use should eventually cease, but doing so will leave a growing population fully susceptible to infection. If poliovirus is reintroduced after OPV cessation, under what conditions will OPV vaccination be required to interrupt transmission? Can conditions exist in which OPV and WPV reintroduction present similar risks of transmission? To answer these questions, we built a multi-scale mathematical model of infection and transmission calibrated to data from clinical trials and field epidemiology studies. At the within-host level, the model describes the effects of vaccination and waning immunity on shedding and oral susceptibility to infection. At the between-host level, the model emulates the interaction of shedding and oral susceptibility with sanitation and person-to-person contact patterns to determine the transmission rate in communities. Our results show that inactivated polio vaccine (IPV) is sufficient to prevent outbreaks in low transmission rate settings and that OPV can be reintroduced and withdrawn as needed in moderate transmission rate settings. However, in high transmission rate settings, the conditions that support vaccine-derived outbreaks have only been rare because population immunity has been high. Absent population immunity, the Sabin strains from OPV will be nearly as capable of causing outbreaks as WPV. If post-cessation outbreak responses are followed by new vaccine-derived outbreaks, strategies to restore population immunity will be required to ensure the stability of polio eradication.