Extrafollicular B cell responses correlate with neutralizing antibodies and morbidity in COVID-19.

A wide spectrum of clinical manifestations has become a hallmark of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) COVID-19 pandemic, although the immunological underpinnings of diverse disease outcomes remain to be defined. We performed detailed characterization of B cell responses through high-dimensional flow cytometry to reveal substantial heterogeneity in both effector and immature populations. More notably, critically ill patients displayed hallmarks of extrafollicular B cell activation and shared B cell repertoire features previously described in autoimmune settings. Extrafollicular activation correlated strongly with large antibody-secreting cell expansion and early production of high concentrations of SARS-CoV-2-specific neutralizing antibodies. Yet, these patients had severe disease with elevated inflammatory biomarkers, multiorgan failure and death. Overall, these findings strongly suggest a pathogenic role for immune activation in subsets of patients with COVID-19. Our study provides further evidence that targeted immunomodulatory therapy may be beneficial in specific patient subpopulations and can be informed by careful immune profiling.

Dysregulated naive B cells and de novo autoreactivity in severe COVID-19.

Severe SARS-CoV-2 infection1 has been associated with highly inflammatory immune activation since the earliest days of the COVID-19 pandemic2-5. More recently, these responses have been associated with the emergence of self-reactive antibodies with pathologic potential6-10, although their origins and resolution have remained unclear11. Previously, we and others have identified extrafollicular B cell activation, a pathway associated with the formation of new autoreactive antibodies in chronic autoimmunity12,13, as a dominant feature of severe and critical COVID-19 (refs. 14-18). Here, using single-cell B cell repertoire analysis of patients with mild and severe disease, we identify the expansion of a naive-derived, low-mutation IgG1 population of antibody-secreting cells (ASCs) reflecting features of low selective pressure. These features correlate with progressive, broad, clinically relevant autoreactivity, particularly directed against nuclear antigens and carbamylated proteins, emerging 10-15 days after the onset of symptoms. Detailed analysis of the low-selection compartment shows a high frequency of clonotypes specific for both SARS-CoV-2 and autoantigens, including pathogenic autoantibodies against the glomerular basement membrane. We further identify the contraction of this pathway on recovery, re-establishment of tolerance standards and concomitant loss of acute-derived ASCs irrespective of antigen specificity. However, serological autoreactivity persists in a subset of patients with postacute sequelae, raising important questions as to the contribution of emerging autoreactivity to continuing symptomology on recovery. In summary, this study demonstrates the origins, breadth and resolution of autoreactivity in severe COVID-19, with implications for early intervention and the treatment of patients with post-COVID sequelae.

Patients taking benralizumab, dupilumab, or mepolizumab have lower postvaccination SARS-CoV-2 immunity.

BACKGROUND: Biologic therapies inhibiting the IL-4 or IL-5 pathways are very effective in the treatment of asthma and other related conditions. However, the cytokines IL-4 and IL-5 also play a role in the generation of adaptive immune responses. Although these biologics do not cause overt immunosuppression, their effect in primary severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunization has not been studied completely. OBJECTIVE: Our aim was to evaluate the antibody and cellular immunity after SARS-CoV-2 mRNA vaccination in patients on biologics (PoBs). METHODS: Patients with severe asthma or atopic dermatitis who were taking benralizumab, dupilumab, or mepolizumab and had received the initial dose of the 2-dose adult SARS-CoV-2 mRNA vaccine were enrolled in a prospective, observational study. As our control group, we used a cohort of immunologically healthy subjects (with no significant immunosuppression) who were not taking biologics (NBs). We used a multiplexed immunoassay to measure antibody levels, neutralization assays to assess antibody function, and flow cytometry to quantitate Spike-specific lymphocytes. RESULTS: We analyzed blood from 57 patients in the PoB group and 46 control subjects from the NB group. The patients in the PoB group had lower levels of SARS-CoV-2 antibodies, pseudovirus neutralization, live virus neutralization, and frequencies of Spike-specific B and CD8 T cells at 6 months after vaccination. In subgroup analyses, patients with asthma who were taking biologics had significantly lower pseudovirus neutralization than did subjects with asthma who were not taking biologics. CONCLUSION: The patients in the PoB group had reduced SARS-CoV-2-specific antibody titers, neutralizing activity, and virus-specific B- and CD8 T-cell counts. These results have implications when considering development of a more individualized immunization strategy in patients who receive biologic medications blocking IL-4 or IL-5 pathways.

CCL5 is a potential bridge between type 1 and type 2 inflammation in asthma.

BACKGROUND: Type 1 (T1) inflammation (marked by IFN-γ expression) is now consistently identified in subsets of asthma cohorts, but how it contributes to disease remains unclear. OBJECTIVE: We sought to understand the role of CCL5 in asthmatic T1 inflammation and how it interacts with both T1 and type 2 (T2) inflammation. METHODS: CCL5, CXCL9, and CXCL10 messenger RNA expression from sputum bulk RNA sequencing, as well as clinical and inflammatory data were obtained from the Severe Asthma Research Program III (SARP III). CCL5 and IFNG expression from bronchoalveolar lavage cell bulk RNA sequencing was obtained from the Immune Mechanisms in Severe Asthma (IMSA) cohort and expression related to previously identified immune cell profiles. The role of CCL5 in tissue-resident memory T-cell (TRM) reactivation was evaluated in a T1high murine severe asthma model. RESULTS: Sputum CCL5 expression strongly correlated with T1 chemokines (P < .001 for CXCL9 and CXCL10), consistent with a role in T1 inflammation. CCL5high participants had greater fractional exhaled nitric oxide (P = .009), blood eosinophils (P < .001), and sputum eosinophils (P = .001) in addition to sputum neutrophils (P = .001). Increased CCL5 bronchoalveolar lavage expression was unique to a previously described T1high/T2variable/lymphocytic patient group in the IMSA cohort, with IFNG trending with worsening lung obstruction only in this group (P = .083). In a murine model, high expression of the CCL5 receptor CCR5 was observed in TRMs and was consistent with a T1 signature. A role for CCL5 in TRM activation was supported by the ability of the CCR5 inhibitor maraviroc to blunt reactivation. CONCLUSION: CCL5 appears to contribute to TRM-related T1 neutrophilic inflammation in asthma while paradoxically also correlating with T2 inflammation and with sputum eosinophilia.

Bridging the Gap between Innate and Adaptive Immunity in the Lung: Summary of the Aspen Lung Conference 2022.

Although significant strides have been made in the understanding of pulmonary immunology, much work remains to be done to comprehensively explain coordinated immune responses in the lung. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic only served to highlight the inadequacy of current models of host-pathogen interactions and reinforced the need for current and future generations of immunologists to unravel complex biological questions. As part of that effort, the 64th Annual Thomas L. Petty Aspen Lung Conference was themed "Bridging the Gap between Innate and Adaptive Immunity in the Lung" and featured exciting work from renowned immunologists. This report summarizes the proceedings of the 2022 Aspen Lung Conference, which was convened to discuss the roles played by innate and adaptive immunity in disease pathogenesis, evaluate the interface between the innate and adaptive immune responses, assess the role of adaptive immunity in the development of autoimmunity and autoimmune lung disease, discuss lessons learned from immunologic cancer treatments and approaches, and define new paradigms to harness the immune system to prevent and treat lung diseases.

Detection of Newly Secreted Antibodies Predicts Nonrecurrence in Primary Clostridioides difficile Infection.

Within 8 weeks of primary Clostridioides difficile infection (CDI), as many as 30% of patients develop recurrent disease with the associated risks of multiple relapses, morbidity, and economic burden. There are no clear clinical correlates or validated biomarkers that can predict recurrence during primary infection. This study demonstrated the potential of a simple test for identifying hospitalized CDI patients at low risk for disease recurrence. Forty-six hospitalized CDI patients were enrolled at Emory University Hospitals. Samples of serum and a novel matrix from circulating plasmablasts called "medium-enriched for newly synthesized antibodies" (MENSA) were collected during weeks 1, 2, and 4. Antibodies specific for 10 C. difficile antigens were measured in each sample. Among the 46 C. difficile-infected patients, 9 (19.5%) experienced recurrence within 8 weeks of primary infection. Among the 37 nonrecurrent patients, 23 (62%; 23/37) had anti-C. difficile MENSA antibodies specific for any of the three toxin antigens: TcdB-CROP, TcdBvir-CROP, and/or CDTb. Positive MENSA responses occurred early (within the first 12 days post-symptom onset), including six patients who never seroconverted. A similar trend was observed in serum responses, but they peaked later and identified fewer patients (51%; 19/37). In contrast, none (0%; 0/9) of the patients who subsequently recurred after hospitalization produced antibodies specific for any of the three C. difficile toxin antigens. Thus, patients with a negative early MENSA response against all three C. difficile toxin antigens had a 19-fold greater relative risk of recurrence. MENSA and serum levels of immunoglobulin A (IgA) and/or IgG antibodies for three C. difficile toxins have prognostic potential. These immunoassays measure nascent immune responses that reduce the likelihood of recurrence thereby providing a biomarker of protection from recurrent CDI. Patients who are positive by this immunoassay are unlikely to suffer a recurrence. Early identification of patients at risk for recurrence by negative MENSA creates opportunities for targeted prophylactic strategies that can reduce the incidence, cost, and morbidity due to recurrent CDI.

Antibody Profiles According to Mild or Severe SARS-CoV-2 Infection, Atlanta, Georgia, USA, 2020.

Among patients with coronavirus disease (COVID-19), IgM levels increased early after symptom onset for those with mild and severe disease, but IgG levels increased early only in those with severe disease. A similar pattern was observed in a separate serosurveillance cohort. Mild COVID-19 should be investigated separately from severe COVID-19.

Effect of Anti-IL5, Anti-IL5R, Anti-IL13 Therapy on Asthma Exacerbations: A Network Meta-analysis.

BACKGROUND: Several new treatments for severe asthma have become available in the last decade; yet, little data exist to guide their use in specific patient populations. OBJECTIVE: A network meta-analysis was conducted comparing the efficacy of FDA-approved monoclonal antibody therapies in preventing exacerbations in patients with severe eosinophilic asthma. METHODS: PubMed and Ovid were searched from inception until July 2019 for randomized controlled trials that studied the efficacy of benralizumab, dupilumab, mepolizumab, and reslizumab, in preventing acute exacerbations of asthma. Studies were included if they reported data for patients with severe eosinophilic asthma (defined in this meta-analysis as absolute eosinophil count ≥ 250 cells/µL). Annualized rate ratios for asthma exacerbations (during treatment) were calculated and converted to log rate ratios. Direct and indirect treatment estimates (for inter-drug differences) were analyzed using frequentist network meta-analysis methodology in R and treatments were ranked based on P-scores. RESULTS: In total, nine studies were included in the final analysis. Network meta-analysis revealed that all drugs were superior to placebo in preventing rates of asthma exacerbation in the study population and no inter-drug differences existed. Dupilumab was found to have the greatest magnitudes of effect on decreasing log rate ratio of asthma exacerbation based on P-score (0.83). CONCLUSION: Benralizumab, dupilumab, mepolizumab, and reslizumab are all associated with decreased asthma exacerbations in patients with eosinophilic asthma, with no significant inter-drug differences.

MENSA, a Media Enriched with Newly Synthesized Antibodies, to Identify SARS-CoV-2 Persistence and Latent Viral Reactivation in Long-COVID.

Post-acute sequelae of SARS-CoV-2 (SARS2) infection (PASC) is a heterogeneous condition, but the main viral drivers are unknown. Here, we use MENSA, Media Enriched with Newly Synthesized Antibodies, secreted exclusively from circulating human plasmablasts, to provide an immune snapshot that defines the underlying viral triggers. We provide proof-of-concept testing that the MENSA technology can capture the new host immune response to accurately diagnose acute primary and breakthrough infections when known SARS2 virus or proteins are present. It is also positive after vaccination when spike proteins elicit an acute immune response. Applying the same principles for long-COVID patients, MENSA is positive for SARS2 in 40% of PASC vs none of the COVID recovered (CR) patients without any sequelae demonstrating ongoing SARS2 viral inflammation only in PASC. Additionally, in PASC patients, MENSAs are also positive for Epstein-Barr Virus (EBV) in 37%, Human Cytomegalovirus (CMV) in 23%, and herpes simplex virus 2 (HSV2) in 15% compared to 17%, 4%, and 4% in CR controls respectively. Combined, a total of 60% of PASC patients have a positive MENSA for SARS2, EBV, CMV, and/or HSV2. MENSA offers a unique antibody snapshot to reveal the underlying viral drivers in long-COVID thus demonstrating the persistence of SARS2 and reactivation of viral herpes in 60% of PASC patients.

Single-cell analysis of human nasal mucosal IgE antibody secreting cells reveals a newly minted phenotype.

Immunoglobulin (Ig) E is central to the pathogenesis of allergic conditions, including allergic fungal rhinosinusitis. However, little is known about IgE antibody secreting cells (ASCs). We performed single-cell RNA sequencing from cluster of differentiation (CD)19+ and CD19- ASCs of nasal polyps from patients with allergic fungal rhinosinusitis (n = 3). Nasal polyps were highly enriched in CD19+ ASCs. Class-switched IgG and IgA ASCs were dominant (95.8%), whereas IgE ASCs were rare (2%) and found only in the CD19+ compartment. Through Ig gene repertoire analysis, IgE ASCs shared clones with IgD-CD27- "double-negative" B cells, IgD+CD27+ unswitched memory B cells, and IgD-CD27+ switched memory B cells, suggesting ontogeny from both IgD+ and memory B cells. Transcriptionally, mucosal IgE ASCs upregulate pathways related to antigen presentation, chemotaxis, B cell receptor stimulation, and survival compared with non-IgE ASCs. Additionally, IgE ASCs have a higher expression of genes encoding lysosomal-associated protein transmembrane 5 (LAPTM5) and CD23, as well as upregulation of CD74 (receptor for macrophage inhibitory factor), store-operated Calcium entry-associated regulatory factor (SARAF), and B cell activating factor receptor (BAFFR), which resemble an early minted ASC phenotype. Overall, these findings reinforce the paradigm that human ex vivo mucosal IgE ASCs have a more immature plasma cell phenotype than other class-switched mucosal ASCs and suggest unique functional roles for mucosal IgE ASCs in concert with Ig secretion.

Transcriptional reprogramming of infiltrating neutrophils drives lung pathology in severe COVID-19 despite low viral load.

Troubling disparities in COVID-19-associated mortality emerged early, with nearly 70% of deaths confined to Black/African American (AA) patients in some areas. However, targeted studies on this vulnerable population are scarce. Here, we applied multiomics single-cell analyses of immune profiles from matching airways and blood samples of Black/AA patients during acute SARS-CoV-2 infection. Transcriptional reprogramming of infiltrating IFITM2+/S100A12+ mature neutrophils, likely recruited via the IL-8/CXCR2 axis, leads to persistent and self-sustaining pulmonary neutrophilia with advanced features of acute respiratory distress syndrome (ARDS) despite low viral load in the airways. In addition, exacerbated neutrophil production of IL-8, IL-1ß, IL-6, and CCL3/4, along with elevated levels of neutrophil elastase and myeloperoxidase, were the hallmarks of transcriptionally active and pathogenic airway neutrophilia. Although our analysis was limited to Black/AA patients and was not designed as a comparative study across different ethnicities, we present an unprecedented in-depth analysis of the immunopathology that leads to acute respiratory distress syndrome in a well-defined patient population disproportionally affected by severe COVID-19.

Chronic inflammation, neutrophil activity, and autoreactivity splits long COVID.

While immunologic correlates of COVID-19 have been widely reported, their associations with post-acute sequelae of COVID-19 (PASC) remain less clear. Due to the wide array of PASC presentations, understanding if specific disease features associate with discrete immune processes and therapeutic opportunities is important. Here we profile patients in the recovery phase of COVID-19 via proteomics screening and machine learning to find signatures of ongoing antiviral B cell development, immune-mediated fibrosis, and markers of cell death in PASC patients but not in controls with uncomplicated recovery. Plasma and immune cell profiling further allow the stratification of PASC into inflammatory and non-inflammatory types. Inflammatory PASC, identifiable through a refined set of 12 blood markers, displays evidence of ongoing neutrophil activity, B cell memory alterations, and building autoreactivity more than a year post COVID-19. Our work thus helps refine PASC categorization to aid in both therapeutic targeting and epidemiological investigation of PASC.

Inactivation of SARS-CoV-2 and COVID-19 Patient Samples for Contemporary Immunology and Metabolomics Studies.

Due to the severity of COVID-19 disease, the U.S. Centers for Disease Control and Prevention and World Health Organization recommend that manipulation of active viral cultures of SARS-CoV-2 and respiratory secretions from COVID-19 patients be performed in biosafety level (BSL)3 laboratories. Therefore, it is imperative to develop viral inactivation procedures that permit samples to be transferred to lower containment levels (BSL2), while maintaining the fidelity of complex downstream assays to expedite the development of medical countermeasures. In this study, we demonstrate optimal conditions for complete viral inactivation following fixation of infected cells with commonly used reagents for flow cytometry, UVC inactivation in sera and respiratory secretions for protein and Ab detection, heat inactivation following cDNA amplification for droplet-based single-cell mRNA sequencing, and extraction with an organic solvent for metabolomic studies. Thus, we provide a suite of viral inactivation protocols for downstream contemporary assays that facilitate sample transfer to BSL2, providing a conceptual framework for rapid initiation of high-fidelity research as the COVID-19 pandemic continues.

Checklists Improve Team Performance During Simulated Extracorporeal Membrane Oxygenation Emergencies: A Randomized Trial.

Extracorporeal membrane oxygenation-related complications are potentially catastrophic if not addressed quickly. Because complications are rare, high-fidelity simulation is recommended as part of the training regimen for extracorporeal membrane oxygenation specialists. We hypothesized that the use of standardized checklists would improve team performance during simulated extracorporeal membrane oxygenation emergencies. DESIGN: Randomized simulation-based trial. SETTING: A quaternary-care academic hospital with a regional extracorporeal membrane oxygenation referral program. SUBJECTS: Extracorporeal membrane oxygenation specialists and other healthcare providers. INTERVENTIONS: We designed six read-do checklists for use during extracorporeal membrane oxygenation emergencies using a modified Delphi process. Teams of two to three providers were randomized to receive the checklists or not. All teams then completed four simulated extracorporeal membrane oxygenation emergencies. MEASUREMENTS AND MAIN RESULTS: Simulation sessions were video-recorded, and the number of critical tasks performed and time-to-completion were compared between groups. A survey instrument was administered before and after simulations to assess participants' attitudes toward the simulations and checklists. We recruited 36 subjects from a single institution, randomly assigned to 15 groups. The groups with checklists completed more critical tasks than participants in the control groups (90% vs 75%; p < 0.001). The groups with checklists performed a higher proportion of both nontechnical tasks (71% vs 44%; p < 0.001) and extracorporeal membrane oxygenation-specific technical tasks (94% vs 86%; p < 0.001). Both groups reported an increase in reported self-efficacy after the simulations (p = 0.003). After adjusting for multiple comparisons, none of the time-to-completion measures achieved statistical significance. CONCLUSIONS: The use of checklists resulted in better team performance during simulated extracorporeal membrane oxygenation emergencies. As extracorporeal membrane oxygenation use continues to expand, checklists may be an attractive low-cost intervention for centers looking to reduce errors and improve response to crisis situations.

Longitudinal trends using a point-of-care gelatin-based model for ultrasound-guided central venous catheter insertion.

Ultrasound (US)-guided central venous catheter (CVC) insertion is a procedure that carries the risk of significant complications. Simulation provides a safe learning atmosphere, but most CVC simulators are not available outside of simulation centers. To explore longitudinal trends in US-guided CVC insertion competency in internal medicine (IM) interns, we studied the use of a low-fidelity, gelatin-based, US-guided CVC insertion simulation model combined with a simulation curriculum. This prospective observational study of IM interns was performed over the course of one academic year. Interns (n = 56) underwent model-based, US-guided procedure simulation training program and a repeated training course prior to their intensive care unit (ICU) rotation. CVC insertion competency at different timepoints was recorded. Survey data about intern experience and attitudes were also collected. Out of the 56 interns initially trained, 40 were included in the final analysis. Across all outcomes, interns experienced skill atrophy between initial training and the beginning of their ICU month. However, by the end of the month, there was a significant improvement in competency as compared to initial procedural training, which then waned by the end of the intern year. Attitudes toward the model were generally positive and self-reported confidence improved throughout the course of the year and correlated with objective measures of competency. Over the course of their intern year, which included simulation training using a gelatin-based model, interns demonstrated consistent competency trends. The use of a gelatin-based CVC insertion simulation model warrants further study as an adjunctive aid to existing simulation training.