The COVID-19 pandemic has resulted in over two million deaths worldwide. Despite efforts to fight the virus, the disease continues to overwhelm hospitals with severely ill patients.

Characterization of antibody response to SARS-CoV-2 is urgently needed to predict COVID-19 disease trajectories. Ineffective antibodies or antibody-dependent enhancement (ADE) could derail patient immune responses, for example. ELISA and coronavirus antigen microarray (COVAM) analysis epitope-mapped plasma from 86 COVID-19 patients. The experiments identified antibodies to a 21-residue epitope from nucleocapsid (termed Ep9) associated with severe disease, including ICU stay, requirement for ventilators, and death. Furthermore, anti-Ep9 antibodies correlate both with various comorbidities and ADE hallmarks, including increased IL-6 levels and early IgG response. Importantly, anti-Ep9 antibodies can be detected within five days post-symptom onset and sometimes within one day. The results lay the groundwork for a new type of COVID-19 diagnostic for the early prediction of disease severity to guide more effective therapeutic interventions.

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The botulinum neurotoxin serotype A (BoNT/A) cuts a single peptide bond in SNAP25, an activity used to treat a wide range of diseases. Reengineering the substrate specificity of BoNT/A's protease domain (LC/A) could expand its therapeutic applications; however, LC/A's extended substrate recognition (approx. 60 residues) challenges conventional approaches. We report a directed evolution method for retargeting LC/A's substrate and retaining its exquisite specificity. The resultant eight-mutation LC/A (omLC/A) has improved cleavage specificity and catalytic efficiency (1300- and 120-fold, respectively) for SNAP23 versus SNAP25 compared to a previously reported LC/A variant. Importantly, the BoNT/A holotoxin equipped with omLC/A infiltrates neurons and retains its SNAP23 activity. The identification of substrate control loops outside BoNT/A's active site could guide the design of improved BoNT proteases and inhibitors.

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