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.

ACE2 engagement exposes the fusion peptide to pan-coronavirus neutralizing antibodies (preprint)

Coronaviruses use diverse Spike (S) glycoproteins to attach to host receptors and fuse with target cells. Using a broad screening approach, we isolated from SARS-CoV-2 immune donors seven monoclonal antibodies (mAbs) that bind to all human alpha and beta coronavirus S proteins. These mAbs recognize the fusion peptide and acquire high affinity and breadth through somatic mutations. Despite targeting a conserved motif, only some mAbs show broad neutralizing activity in vitro against alpha and beta coronaviruses, including Omicron BA.1 variant and bat WIV-1, and reduce viral titers and pathology in vivo. Structural and functional analyses show that the fusion peptide-specific mAbs bind with different modalities to a cryptic epitope which is concealed by prefusion-stabilizing 2P mutations and becomes exposed upon binding of ACE2 or ACE2-mimicking mAbs. This study identifies a new class of pan-coronavirus neutralizing mAbs and reveals a receptor-induced conformational change in the S protein that exposes the fusion peptide region.

Broadly neutralizing antibodies overcome SARS-CoV-2 Omicron antigenic shift (preprint)

The recently emerged SARS-CoV-2 Omicron variant harbors 37 amino acid substitutions in the spike (S) protein, 15 of which are in the receptor-binding domain (RBD), thereby raising concerns about the effectiveness of available vaccines and antibody therapeutics. Here, we show that the Omicron RBD binds to human ACE2 with enhanced affinity relative to the Wuhan-Hu-1 RBD and acquires binding to mouse ACE2. Severe reductions of plasma neutralizing activity were observed against Omicron compared to the ancestral pseudovirus for vaccinated and convalescent individuals. Most (26 out of 29) receptor-binding motif (RBM)-directed monoclonal antibodies (mAbs) lost in vitro neutralizing activity against Omicron, with only three mAbs, including the ACE2-mimicking S2K146 mAb, retaining unaltered potency. Furthermore, a fraction of broadly neutralizing sarbecovirus mAbs recognizing antigenic sites outside the RBM, including sotrovimab, S2X259 and S2H97, neutralized Omicron. The magnitude of Omicron-mediated immune evasion and the acquisition of binding to mouse ACE2 mark a major SARS-CoV-2 mutational shift. Broadly neutralizing sarbecovirus mAbs recognizing epitopes conserved among SARS-CoV-2 variants and other sarbecoviruses may prove key to controlling the ongoing pandemic and future zoonotic spillovers.

Excessive inflammatory and metabolic responses to acute SARS-CoV-2 infection are associated with a distinct gut microbiota composition (preprint)

Protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and associated clinical sequelae requires well-coordinated metabolic and immune responses that limit viral spread and promote recovery of damaged systems. In order to understand potential mechanisms and interactions that influence coronavirus disease 2019 (COVID-19) outcomes, we performed a multi-omics analysis on hospitalised COVID-19 patients and compared those with the most severe outcome (i.e. death) to those with severe non-fatal disease, or mild/moderate disease, that recovered. A distinct subset of 8 cytokines and 140 metabolites in sera identified those with a fatal outcome to infection. In addition, elevated levels of multiple pathobionts and lower levels of protective or anti-inflammatory microbes were observed in the faecal microbiome of those with the poorest clinical outcomes. Weighted gene correlation network analysis (WGCNA) identified modules that associated severity-associated cytokines with tryptophan metabolism, coagulation-linked fibrinopeptides, and bile acids with multiple pathobionts. In contrast, less severe clinical outcomes associated with clusters of anti-inflammatory microbes such as Bifidobacterium or Ruminococcus, short chain fatty acids (SCFAs) and IL-17A. Our study uncovered distinct mechanistic modules that link host and microbiome processes with fatal outcomes to SARS-CoV-2 infection. These features may be useful to identify at risk individuals, but also highlight a role for the microbiome in modifying hyperinflammatory responses to SARS-CoV-2 and other infectious agents.

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.

SARS-CoV-2 immune evasion by variant B.1.427/B.1.429 (preprint)

SARS-CoV-2 entry is mediated by the spike (S) glycoprotein which contains the receptor-binding domain (RBD) and the N-terminal domain (NTD) as the two main targets of neutralizing antibodies (Abs). A novel variant of concern (VOC) named CAL.20C (B.1.427/B.1.429) was originally detected in California and is currently spreading throughout the US and 29 additional countries. It is unclear whether antibody responses to SARS-CoV-2 infection or to the prototypic Wuhan-1 isolate-based vaccines will be impacted by the three B.1.427/B.1.429 S mutations: S13I, W152C and L452R. Here, we assessed neutralizing Ab responses following natural infection or mRNA vaccination using pseudoviruses expressing the wildtype or the B.1.427/B.1.429 S protein. Plasma from vaccinated or convalescent individuals exhibited neutralizing titers, which were reduced 3-6 fold against the B.1.427/B.1.429 variant relative to wildtype pseudoviruses. The RBD L452R mutation reduced or abolished neutralizing activity of 14 out of 35 RBD-specific monoclonal antibodies (mAbs), including three clinical-stage mAbs. Furthermore, we observed a complete loss of B.1.427/B.1.429 neutralization for a panel of mAbs targeting the N-terminal domain due to a large structural rearrangement of the NTD antigenic supersite involving an S13I-mediated shift of the signal peptide cleavage site. These data warrant closer monitoring of signal peptide variants and their involvement in immune evasion and show that Abs directed to the NTD impose a selection pressure driving SARS-CoV-2 viral evolution through conventional and unconventional escape mechanisms.

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.

Impact of SARS-CoV-2 B.1.1.7 Spike variant on neutralisation potency of sera from individuals vaccinated with Pfizer vaccine BNT162b2 (preprint)

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) transmission is uncontrolled in many parts of the world, compounded in some areas by higher transmission potential of the B1.1.7 variant now seen in 50 countries. It is unclear whether responses to SARS-CoV-2 vaccines based on the prototypic strain will be impacted by mutations found in B.1.1.7. Here we assessed immune responses following vaccination with mRNA-based vaccine BNT162b2. We measured neutralising antibody responses following a single immunization using pseudoviruses expressing the wild-type Spike protein or the 8 amino acid mutations found in the B.1.1.7 spike protein. The vaccine sera exhibited a broad range of neutralising titres against the wild-type pseudoviruses that were modestly reduced against B.1.1.7 variant. This reduction was also evident in sera from some convalescent patients. Decreased B.1.1.7 neutralisation was also observed with monoclonal antibodies targeting the N-terminal domain (9 out of 10), the Receptor Binding Motif (RBM) (5 out of 31), but not in neutralising mAbs binding outside the RBM. Introduction of the E484K mutation in a B.1.1.7 background to reflect newly emerging viruses in the UK led to a more substantial loss of neutralising activity by vaccine-elicited antibodies and mAbs (19 out of 31) over that conferred by the B.1.1.7 mutations alone. E484K emergence on a B.1.1.7 background represents a threat to the vaccine BNT162b.

Human Identical Sequences of SARS-CoV-2 Promote Clinical Progression of COVID-19 by Upregulating Hyaluronan via NamiRNA-Enhancer Network (preprint)

The COVID-19 pandemic is a widespread and deadly public health crisis. The pathogen SARS-CoV-2 replicates in the lower respiratory tract and causes fatal pneumonia. Although tremendous efforts have been put into investigating the pathogeny of SARS-CoV-2, the underlying mechanism of how SARS-CoV-2 interacts with its host is largely unexplored. Here, by comparing the genomic sequences of SARS-CoV-2 and human, we identified five fully conserved elements in SARS-CoV-2 genome, which were termed as "human identical sequences (HIS)". HIS are also recognized in both SARS-CoV and MERS-CoV genome. Meanwhile, HIS-SARS-CoV-2 are highly conserved in the primate. Mechanically, HIS-SARS-CoV-2 RNA directly binds to the targeted loci in human genome and further interacts with host enhancers to activate the expression of adjacent and distant genes, including cytokines gene and angiotensin converting enzyme II (ACE2), a well-known cell entry receptor of SARS-CoV-2, and hyaluronan synthase 2 (HAS2), which further increases hyaluronan formation. Noteworthily, hyaluronan level in plasma of COVID-19 patients is tightly correlated with severity and high risk for acute respiratory distress syndrome (ARDS) and may act as a predictor for the progression of COVID-19. HIS antagomirs, which downregulate hyaluronan level effectively, and 4-Methylumbelliferone (MU), an inhibitor of hyaluronan synthesis, are potential drugs to relieve the ARDS related ground-glass pattern in lung for COVID-19 treatment. Our results revealed that unprecedented HIS elements of SARS-CoV-2 contribute to the cytokine storm and ARDS in COVID-19 patients. Thus, blocking HIS-involved activating processes or hyaluronan synthesis directly by 4-MU may be effective strategies to alleviate COVID-19 progression.

The circulating SARS-CoV-2 spike variant N439K maintains fitness while evading antibody-mediated immunity (preprint)

SARS-CoV-2 can mutate to evade immunity, with consequences for the efficacy of emerging vaccines and antibody therapeutics. Herein we demonstrate that the immunodominant SARS-CoV-2 spike (S) receptor binding motif (RBM) is the most divergent region of S, and provide epidemiological, clinical, and molecular characterization of a prevalent RBM variant, N439K. We demonstrate that N439K S protein has enhanced binding affinity to the hACE2 receptor, and that N439K virus has similar clinical outcomes and in vitro replication fitness as compared to wild- type. We observed that the N439K mutation resulted in immune escape from a panel of neutralizing monoclonal antibodies, including one in clinical trials, as well as from polyclonal sera from a sizeable fraction of persons recovered from infection. Immune evasion mutations that maintain virulence and fitness such as N439K can emerge within SARS-CoV-2 S, highlighting the need for ongoing molecular surveillance to guide development and usage of vaccines and therapeutics.

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.