Non-neutralizing antibodies against SARS-CoV-2 nucleocapsid protein mediate variant transcendent antibody-dependent cellular cytotoxicity

Vaccination strategies and correlates of protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have predominantly focused on the spike (S) protein and neutralizing antibodies. However, the rapid emergence of SARS-CoV-2 variants has reduced the effectiveness of spike-based vaccines and monoclonal antibodies. It remains unclear how non-neutralizing antibodies that target the nucleocapsid (N) protein contribute to protection against SARS-CoV-2 variants, especially their ability to trigger antibody effector functions. These antibodies may function by binding to infected cells and initiating antibody-dependent cellular cytotoxicity (ADCC), eliminating infected cells. In this study, we demonstrate that antibodies from individuals who recovered from coronavirus disease 2019 (COVID-19) infection and/or were vaccinated with the S protein vaccine recognize viral proteins on the surface of infected cells and mediate ADCC-mediated NK cell killing of infected cells. Notably, non-neutralizing antibodies induced in COVID-19 infection recognized non-spike proteins on the surface of SARS-CoV-2 variant-infected cells, and these non-neutralizing antibodies cleared SARS-CoV-2 infected cells following depletion of spike antibodies. We identified N and minimal membrane (M) proteins as the targets of non-neutralizing antibodies on the surface of these variant-infected cells. We show that enriched N-specific antibodies from individuals who recovered from COVID-19 infection more consistently killed SARS-CoV-2 variant-infected cells than antibodies to the spike protein. The observed cross-reactivity and robust ADCC activity mediated by N-specific antibodies across various SARS-CoV-2 variant-infected cells highlight the N protein as an important vaccine target in addition to the S protein. Targeting N may provide more comprehensive and durable immunity against SARS-CoV-2 and its evolving variants.