The precise elucidation of the antigen sequences for T-cell immunosurveillance greatly enhances our ability to both understand and modulate humoral responses to viral infection or active immunization. Mass spectrometry was used to identify 529 unique sequences from SARS-CoV-2 spike glycoprotein extracellular domain in a complex with human leukocyte antigen class II molecules on antigen presenting cells from a panel of healthy donors selected to represent a majority of allele usage from this highly polymorphic molecule. The identified sequences span the entire spike protein and several sequences were isolated from a majority of the donors sampled indicating promiscuous binding. Importantly, many peptides derived from the receptor binding domain used for cell entry were identified. This work represents the most precise and comprehensive immunopeptidomic investigation with SARS-CoV-2 spike glycoprotein and allows detailed analysis of features which may aid vaccine development to end the current COVID-19 pandemic. Funding: This work was supported by Eli Lilly and Company. Conflict of Interest: The authors declare no competing interests. Ethical Approval: Frozen PBMCs were obtained from 9 informed consent healthy donors (Discovery Life Sciences) according to local ethical practice.
