25Antibody-based interventions against SARS-CoV-2 could limit morbidity, mortality, and 26 possibly disrupt epidemic transmission. An anticipated correlate of such countermeasures is the 27 level of neutralizing antibodies against the SARS-CoV-2 spike protein, yet there is no consensus 28 as to which assay should be used for such measurements. Using an infectious molecular clone 29 of vesicular stomatitis virus (VSV) that expresses eGFP as a marker of infection, we replaced the 30 glycoprotein gene (G) with the spike protein of SARS-CoV-2 (VSV-eGFP-SARS-CoV-2) and 31 developed a high-throughput imaging-based neutralization assay at biosafety level 2. We also 32 developed a focus reduction neutralization test with a clinical isolate of SARS-CoV-2 at biosafety 33 level 3. We compared the neutralizing activities of monoclonal and polyclonal antibody 34 preparations, as well as ACE2-Fc soluble decoy protein in both assays and find an exceptionally 35 high degree of concordance. The two assays will help define correlates of protection for antibody-36 based countermeasures including therapeutic antibodies, immune γ-globulin or plasma 37 preparations, and vaccines against SARS-CoV-2. Replication-competent VSV-eGFP-SARS-38

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the global COVID-19 pandemic infecting more than 106 million people and causing 2.3 million deaths. The rapid deployment of antibody-based countermeasures has provided hope for curtailing disease and ending the pandemic1. However, the emergence of rapidly-spreading SARS-CoV-2 variants in the United Kingdom (B.1.1.7), South Africa (B.1.351), and elsewhere with mutations in the spike protein has raised concern for escape from neutralizing antibody responses and loss of vaccine efficacy based on preliminary data with pseudoviruses2-4. Here, using monoclonal antibodies (mAbs), animal immune sera, human convalescent sera, and human sera from recipients of the Pfizer-BioNTech (BNT162b2) mRNA vaccine, we report the impact on antibody neutralization of a panel of authentic SARS-CoV-2 variants including a B.1.1.7 isolate, a chimeric Washington strain with a South African spike gene (Wash SA-B.1.351), and isogenic recombinant variants with designed mutations or deletions at positions 69-70, 417, 484, 501, and/or 614 of the spike protein. Several highly neutralizing mAbs engaging the receptor binding domain (RBD) or N-terminal domain (NTD) lost inhibitory activity against Wash SA-B.1.351 or recombinant variants with an E484K spike mutation. Most convalescent sera and virtually all mRNA vaccine-induced immune sera tested showed markedly diminished neutralizing activity against the Wash SA-B.1.351 strain or recombinant viruses containing mutations at position 484 and 501. We also noted that cell line selection used for growth of virus stocks or neutralization assays can impact the potency of antibodies against different SARS-CoV-2 variants, which has implications for assay standardization and congruence of results across laboratories. As several antibodies binding specific regions of the RBD and NTD show loss-of-neutralization potency in vitro against emerging variants, updated mAb cocktails, targeting of highly conserved regions, enhancement of mAb potency, or adjustments to the spike sequences of vaccines may be needed to prevent loss of protection in vivo.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the global COVID-19 pandemic resulting in millions of deaths worldwide. Despite the development and deployment of highly effective antibody and vaccine countermeasures, rapidly-spreading SARS-CoV-2 variants with mutations at key antigenic sites in the spike protein jeopardize their efficacy. Indeed, the recent emergence of the highly-transmissible B.1.1.529 Omicron variant is especially concerning because of the number of mutations, deletions, and insertions in the spike protein. Here, using a panel of anti-receptor binding domain (RBD) monoclonal antibodies (mAbs) corresponding to those with emergency use authorization (EUA) or in advanced clinical development by Vir Biotechnology (S309, the parent mAbs of VIR-7381), AstraZeneca (COV2-2196 and COV2-2130, the parent mAbs of AZD8895 and AZD1061), Regeneron (REGN10933 and REGN10987), Lilly (LY-CoV555 and LY-CoV016), and Celltrion (CT-P59), we report the impact on neutralization of a prevailing, infectious B.1.1.529 Omicron isolate compared to a historical WA1/2020 D614G strain. Several highly neutralizing mAbs (LY-CoV555, LY-CoV016, REGN10933, REGN10987, and CT-P59) completely lost inhibitory activity against B.1.1.529 virus in both Vero-TMPRSS2 and Vero-hACE2-TMPRSS2 cells, whereas others were reduced (∼12-fold decrease, COV2-2196 and COV2-2130 combination) or minimally affected (S309). Our results suggest that several, but not all, of the antibody products in clinical use will lose efficacy against the B.1.1.529 Omicron variant and related strains.

Mucosal Associated Invariant T (MAIT) cells are an “innate” T cell population expressing an invariant TCRα chain, mVαl9 in mice and hVα7.2 in humans. Strikingly similar to CD 1d‐restricted iNKT cells, MAIT cells have a memory phenotype and rapidly release IFN‐γ, IL‐4, 5, and 10 upon TCR ligation. Interestingly, MAIT cells are also restricted by an MHC class I like molecule, MR1. But, unlike iNKT cells, MAIT cells require B cells and gut commensal flora for development and/or expansion. Although the ligand presented by MR1 is unknown, evidence strongly suggests that MAIT cell activation is ligand‐dependent. Here we demonstrate that MR1 antigen presentation is not affected by either the proteasome or the class I chaperones. However, the class II chaperone invariant chain, Ii, physically associates with MR1 and promotes its endosomal trafficking. Functionally, MAIT cell activation is enhanced by Ii overexpression but strikingly diminished by silencing endogenous Ii. Furthermore, confocal microscopy confirms localization of MR1 in late endosomes and inhibiting the acidification of these compartments reduces MR1 surface expression and ablates MAIT cell activation. These data indicate that MR1 traffics through endocytic compartments, thereby allowing MAIT cells to potentially sample both endocytosed and endogenous antigens.

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