Highly conserved Betacoronavirus sequences are broadly recognized by human T cells.

The COVID-19 pandemic highlighted the critical need for vaccine strategies capable of addressing emerging viral threats. Betacoronaviruses, including severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome (MERS), and SARS-CoV-2, present significant pandemic risks due to their zoonotic potential and genetic diversity. T cell-mediated immunity has demonstrated durable responses and strong cross-reactivity, offering a promising avenue for achieving broad immunity within a viral family. In this study, we combined comprehensive epitope mapping with sequence conservation analyses to identify conserved T cell epitope regions (CTERs), which constitute 12% of the complete SARS-CoV-2 proteome. We showed that SARS-CoV-2 CTER-specific T cells cross-reactively recognize sequences from multiple Betacoronavirus subgenera. Importantly, incorporating CTERs from non-spike proteins significantly enhanced T cell cross-reactivity potential and human leukocyte antigen (HLA) coverage compared with T cells targeting only spike proteins. Our findings lay the groundwork for a multi-antigen vaccine strategy that includes non-spike proteins to expand cross-reactive immunity across a broader spectrum of Betacoronaviruses.