CGRP inhibits SARS-CoV-2 infection of bronchial epithelial cells and its pulmonary levels correlate with viral clearance in critical COVID-19 patients (preprint)

Upon infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), patients with critical coronavirus disease 2019 (COVID-19) present with life-threatening respiratory distress, pulmonary damage and cytokine storm. One unexplored hub in COVID-19 is the neuropeptide calcitonin gene-related peptide (CGRP), which is highly abundant in the airways and could converge in multiple aspects of COVID-19-related pulmonary pathophysiology. Whether CGRP affects SARS-CoV-2 infection directly remains elusive. We show that in critical COVID-19 patients, CGRP is increased in both plasma and lungs. Importantly, CGRP pulmonary levels are elevated in early SARS-CoV-2-positive patients, and restore to baseline upon subsequent viral clearance in SARS-CoV-2-negative patients. We further show that CGRP and its stable analogue SAX directly inhibit infection of bronchial Calu-3 epithelial cells with SARS-CoV-2 Omicron and Alpha variants in a dose-dependent manner. Both pre- and post-infection treatment with GRRP and/or SAX is enough to block SARS-CoV-2 productive infection of Calu3 cells. CGRP-mediated inhibition occurs via activation of the CGRP receptor and involves down-regulation of SARS-CoV-2 entry receptors at the surface of Calu-3 cells. Together, we propose that increased pulmonary CGRP mediates beneficial viral clearance in critical COVID-19 patients, by directly inhibiting SARS-CoV-2 infection. Hence, CGRP-based interventions could be harnessed for management of COVID-19. Brief summaryPulmonary levels of the neuropeptide CGRP are increased in critical COVID-19 patients, and could clear virus by directly inhibiting SRAS-CoV-2 infection of bronchial epithelia cells.

Biosynthetic proteins targeting the SARS-CoV-2 spike as anti-virals (preprint)

The binding of the SARS-CoV-2 spike to angiotensin-converting enzyme 2 (ACE2) promotes virus entry into the cell. Targeting this interaction represents a promising strategy to generate antivirals. By screening a phage-display library of biosynthetic protein sequences build on a rigid alpha-helicoidal HEAT-like scaffold (named alphaReps), we selected candidates recognizing the spike receptor binding domain (RBD). Two of them (F9 and C2) bind the RBD with affinities in the nM range, displaying neutralisation activity in vitro and recognizing distinct sites, F9 overlapping the ACE2 binding motif. The F9-C2 fusion protein and a trivalent alphaRep form (C2-foldon) display 0.1 nM affinities and EC50 of 8-18 nM for neutralization of SARS-CoV-2. In hamsters, F9-C2 instillation in the nasal cavity before or during infections effectively reduced the replication of a SARS-CoV-2 strain harbouring the D614G mutation in the nasal epithelium. Furthermore, F9-C2 and/or C2-foldon effectively neutralized SARS-CoV-2 variants (including delta and omicron variants) with EC50 values ranging from 13 to 32 nM. With their high stability and their high potency against SARS-CoV-2 variants, alphaReps provide a promising tool for SARS-CoV-2 therapeutics to target the nasal cavity and mitigate virus dissemination in the proximal environment.

Bidirectional genome-wide CRISPR screens reveal host factors regulating SARS-CoV-2, MERS-CoV and seasonal HCoVs (preprint)

Several genome-wide CRISPR knockout screens have been conducted to identify host factors regulating SARS-CoV-2 replication, but the models used have often relied on overexpression of ACE2 receptor. Additionally, such screens have yet to identify the protease TMPRSS2, known to be important for viral entry at the plasma membrane. Here, we conducted a meta-analysis of these screens and showed a high level of cell-type specificity of the identified hits, arguing for the necessity of additional models to uncover the full landscape of SARS-CoV-2 host factors. We performed genome-wide knockout and activation CRISPR screens in Calu-3 lung epithelial cells, as well as knockout screens in Caco-2 intestinal cells. In addition to identifying ACE2 and TMPRSS2 as top hits, our study reveals a series of so far unidentified and critical host-dependency factors, including the Adaptins AP1G1 and AP1B1 and the flippase ATP8B1. Moreover, new anti-SARS-CoV-2 proteins with potent activity, including several membrane-associated Mucins, IL6R, and CD44 were identified. We further observed that these genes mostly acted at the critical step of viral entry, with the notable exception of ATP8B1, the knockout of which prevented late stages of viral replication. Exploring the pro- and anti-viral breadth of these genes using highly pathogenic MERS-CoV, seasonal HCoV-NL63 and -229E and influenza A orthomyxovirus, we reveal that some genes such as AP1G1 and ATP8B1 are general coronavirus cofactors. In contrast, Mucins recapitulated their known role as a general antiviral defense mechanism. These results demonstrate the value of considering multiple cell models and perturbational modalities for understanding SARS-CoV-2 replication and provide a list of potential new targets for therapeutic interventions.

Bidirectional genome-wide CRISPR screens reveal host factors regulating SARS-CoV-2, MERS-CoV and seasonal coronaviruses (preprint)

Several genome-wide CRISPR knockout screens have been conducted to identify host factors regulating SARS-CoV-2 replication, but the models used have often relied on overexpression of ACE2 receptor. Additionally, such screens have yet to identify the protease TMPRSS2, known to be important for viral entry at the plasma membrane. Here, we conducted a meta-analysis of these screens and showed a high level of cell-type specificity of the identified hits, arguing for the necessity of additional models to uncover the full landscape of SARS-CoV-2 host factors. We performed genome-wide knockout and activation CRISPR screens in Calu-3 lung epithelial cells, as well as knockout screens in Caco-2 intestinal cells. In addition to identifying ACE2 and TMPRSS2 as top hits, our study reveals a series of so far unidentified and critical host-dependency factors, including the Adaptins AP1G1 and AP1B1 and the flippase ATP8B1. Moreover, new anti-SARS-CoV-2 proteins with potent activity, including several membrane-associated Mucins, IL6R, and CD44 were identified. We further observed that these genes mostly acted at the critical step of viral entry, with the notable exception of ATP8B1, the knockout of which prevented late stages of viral replication. Exploring the pro- and anti-viral breadth of these genes using highly pathogenic MERS-CoV, seasonal HCoV-NL63 and -229E and influenza A orthomyxovirus, we reveal that some genes such as AP1G1 and ATP8B1 are general coronavirus cofactors. In contrast, Mucins recapitulated their known role as a general antiviral defense mechanism. These results demonstrate the value of considering multiple cell models and perturbational modalities for understanding SARS-CoV-2 replication and provide a list of potential new targets for therapeutic interventions.

Anti-Spike, anti-Nucleocapsid and neutralizing antibodies in SARS-CoV-2 hospitalized patients and asymptomatic carriers (preprint)

ObjectiveThe objective of this study was to monitor the anti-SARS-CoV-2 antibody response in infected patients. MethodsIn order to assess the time of seroconversion, we used 151 samples from 30 COVID-19 inpatients and monitored the detection kinetics of anti-S1, anti-S2, anti-RBD and anti-N antibodies with in-house ELISAs. We also monitored the presence of neutralizing antibodies in these samples as well as 25 asymptomatic carrier samples using retroviral particles pseudotyped with the spike of the SARS-CoV-2. ResultsWe observed that specific antibodies were detectable in all inpatients two weeks post-symptom onset. The detection of the SARS-CoV-2 Nucleocapsid and RBD was more sensitive than the detection of the S1 or S2 subunits. Neutralizing antibodies reached a plateau two weeks post-symptom onset and then declined in the majority of inpatients. Furthermore, neutralizing antibodies were undetectable in 56% of asymptomatic carriers. ConclusionsOur results raise questions concerning the role played by neutralizing antibodies in COVID-19 cure and protection against secondary infection. They also suggest that induction of neutralizing antibodies is not the only strategy to adopt for the development of a vaccine. Finally, they imply that anti-SARS-CoV-2 neutralizing antibodies should be titrated to optimize convalescent plasma therapy. HighlightsO_LISpecific antibodies are detectable in 100% COVID-19 inpatients two weeks post-symptom onset. C_LIO_LIThe detection of the SARS-CoV-2 Nucleocapsid and Receptor Binding Domain is more sensitive than the detection of the S1 or S2 subunits. C_LIO_LINeutralizing antibodies reach a plateau two weeks post-symptom onset and then decline in the majority of inpatients. C_LIO_LINeutralizing antibodies are undetectable in the majority of asymptomatic carriers. C_LI