T cell response following anti COVID-19 BNT162b2 vaccination is maintained against the SARS-CoV-2 Omicron B.1.1.529 variant of concern

The progression of the COVID-19 pandemic leads to the emergence of variants of concern (VOC), which may compromise the efficacy of the currently administered vaccines. Antigenic drift can potentially bring about a reduced protective T cell immunity and consequently to more severe disease manifestations. To assess this possibility, the T cell responses to the wild-type, Wuhan-1 SARS-CoV-2 ancestral spike protein and Omicron B.1.1.529 spike protein were compared. Accordingly, peripheral blood mononuclear cells (PBMC) were collected from 8 healthy volunteers 4-5 months following a third vaccination with BNT162b2, and stimulated with overlapping peptide libraries representing the spike of either the ancestral or Omicron SARS-CoV- 2 virus variants. Quantification of the specific T cells was carried out by a fluorescent ELISPOT assay, monitoring interferon-gamma (IFNg), interleukin-10 (IL-10) and interleukin-4 (IL-4) secreting cells. For all the examined individuals, comparable level of reactivity to both forms of spike protein were determined. In addition, a dominant Th1 response was observed, manifested mainly by IFNg secreting cells and only limited numbers of IL-10 and IL-4 secreting cells. The data demonstrates a stable T cell activity to the emerging Omicron variant in the tested individuals, therefore the protective immunity to the variant following BNT162b2 vaccination is not significantly affected.

CRISPR screens for host factors critical for infection by SARS-CoV-2 variants of concern identify GATA6 as a central modulator of ACE2.

The global spread of SARS-CoV-2 led to the most challenging pandemic in this century, posing major economic and health challenges worldwide. Revealing host genes essential for infection by multiple variants of SASR-CoV-2 can provide insights into the virus pathogenesis, and facilitates the development of novel broad-spectrum host-directed therapeutics. Here, employing genome-scale CRISPR screens, we provide a comprehensive data-set of cellular factors that are exploited by WT-SARS-CoV-2 as well as two additional recently emerged variants of concerns (VOCs), Alpha and Beta. These screens identified known and novel host factors critical for SARS-CoV-2 infection, including various components belonging to the Clathrin-dependent transport pathway, ubiquitination and Heparan sulfate biogenesis. In addition, the host phosphatidylglycerol biosynthesis processes appeared to have major anti-viral functions. Comparative analysis of the different VOCs revealed the host factors KREMEN2 and SETDB1 as potential unique candidates required only to the Alpha variant, providing a possible explanation for the increased infectivity of this variant. Furthermore, the analysis identified GATA6, a zinc finger transcription factor, as an essential pro-viral gene for all variants inspected. We revealed that GATA6 directly regulates ACE2 transcription and accordingly, is critical for SARS-CoV-2 cell entry. Analysis of clinical samples collected from SARS-CoV-2 infected individuals showed an elevated level of GATA6, indicating the important role GATA6 may be playing in COVID-19 pathogenesis. Finally, pharmacological inhibition of GATA6 resulted in down-modulation of ACE2 and consequently to inhibition of the viral infectivity. Overall, we show GATA6 represents a target for the development of anti-SARS-CoV-2 therapeutic strategies and reaffirm the value of the CRISPR loss-of-function screens in providing a list of potential new targets for therapeutic interventions.

The neutralization potency of anti-SARS-CoV-2 therapeutic human monoclonal antibodies is retained against novel viral variants

A wide range of SARS-CoV-2 neutralizing monoclonal antibodies (mAbs) were reported to date, most of which target the spike glycoprotein and in particular its receptor binding domain (RBD) and N-terminal domain (NTD) of the S1 subunit. The therapeutic implementation of these antibodies has been recently challenged by emerging SARS-CoV-2 variants that harbor extensively mutated spike versions. Consequently, the re-assessment of mAbs, previously reported to neutralize the original early-version of the virus, is of high priority. Four previously selected mAbs targeting non-overlapping epitopes, were evaluated for their binding potency to RBD versions harboring individual mutations at spike positions 417, 439, 453, 477, 484 and 501. Mutations at these positions represent the prevailing worldwide distributed modifications of the RBD, previously reported to mediate escape from antibody neutralization. Additionally, the in vitro neutralization potencies of the four RBD-specific mAbs, as well as two NTD-specific mAbs, were evaluated against two frequent SARS-CoV-2 variants of concern (VOCs): (i) the B.1.1.7 variant, emerged in the UK and (ii) the B.1.351 variant, emerged in South Africa. Variant B.1.351 was previously suggested to escape many therapeutic mAbs, including those authorized for clinical use. The possible impact of RBD mutations on recognition by mAbs is addressed by comparative structural modelling. Finally, we demonstrate the therapeutic potential of three selected mAbs by treatment of K18-hACE2 transgenic mice two days post infection with each of the virus strains. Our results clearly indicate that despite the accumulation of spike mutations, some neutralizing mAbs preserve their potency against SARS-CoV-2. In particular, the highly potent MD65 and BL6 mAbs are shown to retain their ability to bind the prevalent novel viral mutations and to effectively protect against B.1.1.7 and B.1.351 variants of high clinical concern.

A lipid nanoparticle RBD-hFc mRNA vaccine protects hACE2 transgenic mice against lethal SARS-CoV-2 infection

The current global COVID-19 pandemic led to an unprecedented effort to develop effective vaccines against SARS-CoV-2. mRNA vaccines were developed very rapidly during the last year, and became the leading immunization platform against the virus, with highly promising phase-3 results and remarkable efficacy data. Since most animal models are not susceptible to SARS CoV-2 infection, pre-clinical studies are often limited to infection-prone animals such as hamsters and non-human primates. In these animal models, SARS-CoV-2 infection results in viral replication and a mild disease disease. Therefore, the protective efficacy of the vaccine in these animals is commonly evaluated by its ability to elicit immunologic responses, diminish viral replication and prevent weight loss. Our lab recently reported the design of a SARS-CoV-2 human Fc-conjugated receptor-binding domain (RBD-hFc) mRNA vaccine delivered via lipid nanoparticles (LNPs). These experiments demonstrated the development of a robust and specific immunologic response in RBD-hFc mRNA-vaccinated BALB/c mice. In the current study, we evaluated the protective effect of this RBD-hFc mRNA vaccine by employing the K18-hACE2 mouse model. We report that administration of RBD-hFc mRNA vaccine to K18-hACE2 mice led to a robust humoral response comprised of both binding and neutralizing antibodies. In accordance with the recorded immunologic immune response, 70% of vaccinated mice were protected against a lethal dose (3000 plaque forming units) of SARS-CoV-2, while all control animals succumbed to infection. To the best of our knowledge, this is the first non-replicating mRNA vaccine study reporting protection of K18-hACE2 against a lethal SARS-CoV-2 infection.

Therapeutic antibodies, targeting the SARS-CoV-2 spike N-terminal domain, protect lethally infected K18-hACE2 mice

Since the onset of the current COVID-19 pandemic, high priority is given to the development of neutralizing antibodies, as a key approach for the design of therapeutic strategies to countermeasure and eradicate the disease. Previously, we reported the development of human therapeutic monoclonal antibodies (mAbs) exhibiting very high protective ability. These mAbs recognize epitopes on the spike receptor binding domain (RBD) of SARS-CoV-2 that is considered to represent the main rout of receptor engagement by the SARS-CoV-2 virus. The recent emergence of viral variants emphasizes the notion that efficient antibody treatments need to rely on mAbs against several distinct key epitopes in order to circumvent the occurrence of therapy escape-mutants. Here we report the isolation and characterization of 12 neutralizing mAbs, identified by screening a phage-display library constructed from lymphatic cells collected from severe COVID-19 patients. The antibodies target three distinct epitopes on the spike N-terminal domain (NTD) of SARS-CoV-2, one of them defining a major site of vulnerability of the virus. Extensive characterization of these mAbs suggests a neutralization mechanism which relies both on amino-acid and N-glycan recognition on the virus, and involvement of receptors other than the hACE2 on the target cell. Two of the selected mAbs, which demonstrated superior neutralization potency in vitro, were further evaluated in vivo, demonstrating their ability to fully protect K18-hACE2 transgenic mice even when administered at low doses and late after infection. The study demonstrates the high potential of the mAbs for therapy of SARS-CoV-2 infection and underlines the possible role of the NTD in mediating infection of host cells via alternative cellular portals other than the canonical ACE2 receptor.

Post-exposure protection of SARS-CoV-2 lethal infected K18-hACE2 transgenic mice by neutralizing human monoclonal antibody

Coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), exhibits high levels of mortality and morbidity and has dramatic consequences on human life, sociality and global economy. Neutralizing antibodies constitute a highly promising approach for treating and preventing infection by this novel pathogen. In the present study, we characterized and further evaluated the recently identified human monoclonal MD65 antibody for its ability to provide protection against a lethal SARS-CoV-2 infection of K18-hACE2 transgenic mice. Eighty percent of the untreated mice succumbed 6-9 days post-infection while administration of the MD65 antibody as late as 3 days after exposure, rescued all infected animals. In addition, the efficiency of the treatment is supported by prevention of morbidity and ablation of the load of infective virions in the lungs of treated animals. The data unprecedentedly demonstrate, the therapeutic value of human monoclonal antibodies as a life-saving treatment of severe COVID-19 infection.

Tiger team: a panel of human neutralizing mAbs targeting SARS-CoV-2 spike at multiple epitopes

The novel highly transmissible human coronavirus SARS-CoV-2 is the causative agent of the COVID-19 pandemic. Thus far, there is no approved therapeutic drug, specifically targeting this emerging virus. Here we report the isolation and characterization of a panel of human neutralizing monoclonal antibodies targeting the SARS-CoV-2 receptor binding domain (RBD). These antibodies were selected from a phage display library constructed using peripheral circulatory lymphocytes collected from patients at the acute phase of the disease. These neutralizing antibodies are shown to recognize distinct epitopes on the viral spike RBD, therefore they represent a promising basis for the design of efficient combined post-exposure therapy for SARS-CoV-2 infection.