Therapeutic and vaccine-induced cross-reactive antibodies with effector function against emerging Omicron variants (preprint)

Currently circulating SARS-CoV-2 variants acquired convergent mutations at receptor-binding domain (RBD) hot spots. Their impact on viral infection, transmission, and efficacy of vaccines and therapeutics remains poorly understood. Here, we demonstrate that recently emerged BQ.1.1. and XBB.1 variants bind ACE2 with high affinity and promote membrane fusion more efficiently than earlier Omicron variants. Structures of the BQ.1.1 and XBB.1 RBDs bound to human ACE2 and S309 Fab (sotrovimab parent) explain the altered ACE2 recognition and preserved antibody binding through conformational selection. We show that sotrovimab binds avidly to all Omicron variants, promotes Fc-dependent effector functions and protects mice challenged with BQ.1.1, the variant displaying the greatest loss of neutralization. Moreover, in several donors vaccine-elicited plasma antibodies cross-react with and trigger effector functions against Omicron variants despite reduced neutralizing activity. Cross-reactive RBD-directed human memory B cells remained dominant even after two exposures to Omicron spikes, underscoring persistent immune imprinting. Our findings suggest that this previously overlooked class of cross-reactive antibodies, exemplified by S309, may contribute to protection against disease caused by emerging variants through elicitation of effector functions.

Human neutralizing antibodies to cold linear epitopes and to subdomain 1 of SARS-CoV-2 (preprint)

Emergence of SARS-CoV-2 variants diminishes the efficacy of vaccines and antiviral monoclonal antibodies. Continued development of immunotherapies and vaccine immunogens resilient to viral evolution is therefore necessary. Using coldspot-guided antibody discovery, a screening approach that focuses on portions of the virus spike that are both functionally relevant and averse to change, we identified human neutralizing antibodies to highly conserved viral epitopes. Antibody fp.006 binds the fusion peptide and cross-reacts against coronaviruses of the four genera, including the nine human coronaviruses, through recognition of a conserved motif that includes the S2' site of proteolytic cleavage. Antibody hr2.016 targets the stem helix and neutralizes SARS-CoV-2 variants. Antibody sd1.040 binds to subdomain 1, synergizes with antibody rbd.042 for neutralization and, like fp.006 and hr2.016, protects mice when present as bispecific antibody. Thus, coldspot-guided antibody discovery reveals donor-derived neutralizing antibodies that are cross-reactive with Orthocoronavirinae, including SARS-CoV-2 variants.

Maturation of SARS-CoV-2 Spike-specific memory B cells drives resilience to viral escape (preprint)

Memory B cells (MBCs) generate rapid antibody responses upon secondary encounter with a pathogen. Here, we investigated the kinetics, avidity and cross-reactivity of serum antibodies and MBCs in 155 SARS-CoV-2 infected and vaccinated individuals over a 16-month timeframe. SARS-CoV-2-specific MBCs and serum antibodies reached steady-state titers with comparable kinetics in infected and vaccinated individuals. Whereas MBCs of infected individuals targeted both pre- and postfusion Spike (S), most vaccine-elicited MBCs were specific for prefusion S, consistent with the use of prefusion-stabilized S in mRNA vaccines. Furthermore, a large fraction of MBCs recognizing postfusion S cross-reacted with human betacoronaviruses. The avidity of MBC-derived and serum antibodies increased over time resulting in enhanced resilience to viral escape by SARS-CoV-2 variants, including Omicron BA.1 and BA.2 sub-lineages, albeit only partially for BA.4 and BA.5 sublineages. Overall, the maturation of high-affinity and broadly-reactive MBCs provides the basis for effective recall responses to future SARS-CoV-2 variants.

Anti-chemokine antibodies after SARS-CoV-2 infection correlate with favorable disease course (preprint)

Infection by SARS-CoV-2 leads to diverse symptoms, which can persist for months. While antiviral antibodies are protective, those targeting interferons and other immune factors are associated with adverse COVID-19 outcomes. Instead, we discovered that antibodies against specific chemokines are omnipresent after COVID-19, associated with favorable disease, and predictive of lack of long COVID symptoms at one year post infection. Anti-chemokine antibodies are present also in HIV-1 and autoimmune disorders, but they target different chemokines than those in COVID-19. Finally, monoclonal antibodies derived from COVID-19 convalescents that bind to the chemokine N-loop impair cell migration. Given the role of chemokines in orchestrating immune cell trafficking, naturally arising anti-chemokine antibodies associated with favorable COVID 19 may be beneficial by modulating the inflammatory response and thus bear therapeutic potential.

DNR order in SARS-CoV-2 patients: preliminary guidelines validation and quality of palliative care in a single Swiss COVID-19 Center. (preprint)

Background: The worldwide pandemic situation forced many hospitals to improve COVID-19 management. In this scenario, the Swiss Academy of Medical Sciences (SAMW/ASSM) organized guidelines based on expert opinion to identify Do-Not-Resuscitate (DNR) patients, which often need palliative care (PC) due to a large symptoms burden. In our specialized COVID-19 Center, we investigated characteristics and mortality of DNR patients according to SAMW/ASSM guidelines, comparing to non-DNR patients. Methods: Pilot retrospective validation study, evaluating consecutive COVID-19 patients admitted to Internal Medicine Department. A sub-analysis of the deceased patients care was performed, using both Richmond Agitation-Sedation Scale-Palliative care (RASS-PAL) for sedation and agitation (+4/-5) and modified Borg Scale for dyspnea (1-10). Primary outcome was a 30-days survival of DNR patients comparing to non-DNR patients. Secondary outcomes reported quality of PC, especially on dyspnea and restlessness. Results: From March 16 to April 1 2020, 213 consecutive patients were triaged; of 40 DNR patients 25 (64%) were males with a mean age of 79.9 years. At 30-days follow-up, 9 (22.5%) of DNR patients died; 4 patients (2.2%) died in the control group. The higher mortality rate in DNR group was further confirmed by Log-Rank Mantel-Cox (23.104, p < 0.0001). PC was applied using oxygen (100%), opioids (100%) and sedatives (89%); the mean RASS-PAL improved from 2.2 to -1.8 (p < 0.0001) and Borg scale improved from 5.7 to 4.7 (p = 0.581). Conclusion: The SAMW/ASSM guidelines identified patients at higher risk of short-term death. In the end-of-life management, an early access to PC becomes crucial, both improving patients' end-of-life treatment and reducing psychological families' post-traumatic disorders.

Dysphagic disorder in a cohort of COVID-19 patients: evaluation and evolution (preprint)

ABSTRACT Background COVID-19 is a multisystem disease complicated by respiratory failure requiring sustanined mechanical ventilation (MV). Prolongued oro-tracheal intubation is associated to an increased risk of dysphagia and bronchial aspiration. Purpose of this study was to investigate swallowing disorders in critically ill COVID-19 patients. Methods This was a retrospective study analysing a consecutive cohort of COVID-19 patients admitted to the Intensive Care Unit (ICU) of our Hospital. Data concerning dysphagia were collected according to the Gugging Swallowing Screen (GUSS) and related to demographic characteristics, clinical data, ICU Length-Of-Stay (LOS) and MV parameters. Results From March 2 to April 30 2020, 31 consecutive critically ill COVID-19 patients admitted to ICU were evaluated by speech and language therapists (SLT). Twenty-five of them were on MV (61% through endotracheal tube and 19% through tracheostomy); median MV lenght was 11 days. Seventeen (54.8%) patients presented dysphagia; a correlation was found between first GUSS severity stratification and MV days (p < 0.001), ICU LOS (p < 0.001), age (p = 0.03) and tracheostomy (p = 0.042). No other correlations were found. At 16 days, 90% of patients had fully recovered; a significant improvement was registered especially during the first week (p < 0.001). Conclusion Compared to non-COVID-19 patiens, a higher rate of dysphagia was reported in COVID-19 patients, with a more rapid and complete recovery. A systematic early SLT evaluation of COVID-19 patients on MV may thus be useful to prevent dysphagia-related complications.

Role of physiotherapy team in critically ill COVID-19 patients pronation: can a multidisciplinary management reduce the complications rate? (preprint)

Objectives: During the pandemic, critically ill COVID-19 patients' management presented an increased workload for Intensive Care Unit (ICU) nursing staff, particularly during pronation maneuvers, with high risk of complications. In this scenario, a support during pronation by the ICU Physiotherapy Team was introduced. Research methodology: Retrospective analysis. Consecutive critically ill COVID-19 patients. Setting: A COVID-19 Center in southern Switzerland, between March 16th and April 30th, 2020. Main Outcome Measures: Rates and characteristics of pronation-related complications. Results: Forty-two patients on mechanical ventilation (MV) were treated; 296 standard prone/supine positioning were performed, with 3.52 cycles/patient. One (0.3%) major complication was observed, while fourteen (33.3%) patients developed minor complications, e.g. pressure injuries. The incidence of pressure sores was related to ICU length-of-stay (LOS) (p = 0.029) and MV days (p = 0.015), while their number (n = 27) further correlated with ICU LOS (p = 0.001) and MV days (p = 0.001). The propensity matching score analysis did not show any protective factor of pronation regarding pressure injuries (p = 0.448). No other significant correlation was found. Conclusion: The specific pronation team determined a low rate of major complications in critically ill COVID19 patients. The high rate of minor complications appeared to be related to disease severity, rather than from pronation.

Admission criteria in critically ill COVID-19 patients: a physiology-based approach. (preprint)

Introduction: The COVID-19 pandemic required a careful management of intensive care unit (ICU) admissions, to reduce ICU overload while facing resources' limitations. We implemented standardized, physiology-based, ICU admission criteria and analyzed the mortality rate of patients refused from the ICU. Materials and Methods: COVID-19 patients proposed for ICU admission were consecutively analyzed; Do-not-resuscitate patients were excluded. Patients presenting a SpO2 lower than 85% and/or dyspnea and/or mental confusion resulted eligible for ICU admission; patients not presenting these criteria remained in the ward. Primary outcome was both groups' survival rate. Secondary outcome was a sub analysis correlating SpO2 cutoff with ICU admission. Results: From March 2020 to January 2021, 1623 patients were admitted to our Center; 208 DNR patients were excluded; 97 patients underwent intensivist evaluation. The ICU-admitted group mortality rate resulted 15.9% at 28 days and 27% at 40 days; the ICU-refused group mortality rate resulted 0% at both intervals (p < 0.001). With a SpO2 cut-off of 92%, the hypoxia rate distribution did not correlate with ICU admission (p = 0.26); with a SpO2 cut-off of 85%, a correlation was found (p = 0.009). A similar correlation was also found with dyspnea (p =0.0002). Conclusion: In COVID-19 patients, standardized ICU admission criteria appeared to reduce safely ICU overload. In the absence of dyspnea and/or confusion, a SpO2 cutoff up to 85% for ICU admission was not burdened by negative outcomes. In a pandemic context, the SpO2 cutoff of 92%, as a threshold for ICU admission, needs critical re-evaluation.

Low-PEEP Mechanical Ventilation and P/F Ratio Evolution in COVID-19 Patients (preprint)

Background Critically ill COVID-19 patients are burdened by high mortality. Evaluation to improve patients’ management was performed using strict patient criteria for Intensive Care Unit (ICU) admission, a low- positive end-expiratory pressure (PEEP) setting and standard thromboembolism treatment. Objectives of this study was to report the effects of this strategy on P/F-ratio evolution during mechanical ventilation (MV), ICU length of stay (LOS) and MV length.Methods A retrospective analysis was conducted on all consecutive patients with acute respiratory distress due to COVID-19 pneumonia admitted into ICU from March 2nd to January 15th, 2021. Patients were treated with a low-PEEP strategy (PEEP 10 cmH2O if BMI < 30 Kg m-2, PEEP 12 cmH2O if BMI 30-50 Kg m-2, PEEP 15 cmH2O if BMI > 50 Kg m-2) and therapeutic anticoagulation in case of thrombosis or D-dimer greater than 1’500 ng ml-1.Results 79 patients were on invasive MV. Average applied PEEP was 11 ± 2.9 cmH2O for BMI < 30 Kg m-2, 16 ± 3.18 cmH2O for BMI > 30 Kg m-2. After low-PEEP application, patients’ P/F ratio presented daily improvement from admission during next 72 hours (p<0.001; CI 99%) that resulted statistically significant for each single day after oro-tracheal intubation (OTI). Median ICU length of stay (LOS) was 15 days (10–28); median duration of MV was 12 days (8–26). The ICU mortality rate was 31.6%.Conclusions A combination of low-PEEP treatment resulted in P/F persistent daily ratio improvement during first 72 hours after OTI. A low-PEEP strategy could be beneficial in hemodynamic than respiratory terms.

Clonal dissection of immunodominance and cross-reactivity of the CD4+ T cell response to SARS-CoV-2 (preprint)

The identification of CD4+ T cell epitopes is essential for the design of effective vaccines capable of inducing neutralizing antibodies and long-term immunity. Here we demonstrate in COVID-19 patients a robust CD4+ T cell response to naturally processed SARS-CoV-2 Spike and Nucleoprotein, including effector, helper and memory T cells. By characterizing 2,943 Spike-reactive T cell clones, we found that 34% of the clones and 93% of the patients recognized a conserved immunodominant region encompassing residues S346-365 in the RBD and comprising three nested HLA-DR and HLA-DP restricted epitopes. By using pre- and post-COVID-19 samples and Spike proteins from alpha and beta coronaviruses, we provide in vivo evidence of cross-reactive T cell responses targeting multiple sites in the SARS-CoV-2 Spike protein. The possibility of leveraging immunodominant and cross-reactive T helper epitopes is instrumental for vaccination strategies that can be rapidly adapted to counteract emerging SARS-CoV-2 variants.

Reduced mortality and shorten ICU stay in SARS-COV-2 pneumonia: a low PEEP strategy (preprint)

Background Intensive Care Unit (ICU) management of COVID-19 patients with severe hypoxemia is associated with high mortality. We implemented a "care map", as a standardized multidisciplinary approach to improve patients monitoring using: uniform patient selection for ICU admission, a low-PEEP strategy and a pharmacologic strategic thromboembolism management. Methods A standardized protocol for managing COVID-19 patients and ICU admissions was implemented through accurate Early Warning Score (EWS) monitoring and thromboembolism prophylaxis at hospital admission. Dyspnea, mental confusion or SpO2 less than 85% were criteria for ICU admission. Ventilation approach employed low PEEP values (about 10 cmH2O in presence of lung compliance > 40 mL/cmH2O) and FiO2 as needed. In presence of lower lung compliance (< 40 mL/cmH2O) PEEP value was increased to about 14 cmH2O. Results From March 16th to April 12nd 2020, 41 COVID-19 patients were admitted to our ICU from a total of 310 patients. 83% (34) of them needed mechanical ventilation. The ventilation approach chosen employed low PEEP value based on BMI (PEEP 11+/- 3.8 (10-12) cmH2O if BMI < 30 Kg/m2; PEEP 15+/- 3.26 (12-18) cmH2O if BMI >30 Kg/m2). To date, ten patients (24%) died, four (9.7%) received mechanical ventilation, two were transferred to another hospital and 25 (60.9%) were discharged from ICU after a median of nine days. Discussion A multimodal approach for COVID-19 patients is mandatory. The knowledge of this multi-organ disease is growing rapidly, requiring improvements in the standard of care. Our approach implements an accurate pre-ICU monitoring and strict selection for ICU admission, and allows to reduce mechanical ventilation, ICU stay and mortality. Funding No funding has been required.