Immunodominant T cell responses to SARS-CoV-2 nucleocapsid protein in Omicron breakthrough infection post-inactivated vaccination.

The widespread administration of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines has not eliminated breakthrough infections due to immune evasion by viral mutations. Whether individuals with breakthrough infections can elicit effective T cell responses against the circulating Omicron JN.1 variant, and the specific immunodominant characteristics of these responses, remain to be elucidated. To address this, we recruited 93 individuals who had experienced early Omicron subvariant breakthrough infections following a three-dose regimen of inactivated SARS-CoV-2 vaccines. Intracellular cytokine staining was employed to evaluate T cell responses to the JN.1 nucleocapsid (N) protein overlapping peptide pool in peripheral blood mononuclear cells. Immunodominant epitopes were identified using a three-dimensional matrix screening approach. Human leukocyte antigen (HLA) blocking assays and truncated peptides stimulation assays were conducted to define the minimal epitope and HLA restriction. We found that breakthrough infections elicited robust CD4+ and CD8+ T cell responses specific to the JN.1 N protein, characterized by Th1/Tc1-skewed phenotype and preserved polyfunctionality. The immunodominant epitope profile of the N protein has undergone a significant transformation following breakthrough infection. New epitopes targeting mutated sites have emerged, while previously dominant epitopes, such as N-4 and N-66, no longer maintained their immunodominance. Instead, highly conserved epitope N-24 has risen as the most immunodominant target for CD4+ T cells, restricted by HLA-DR, and with the minimal epitope identified as PKDHIGTRNPANNA. Our finding demonstrates that early Omicron strain breakthrough infections induce robust T cell responses targeting the circulating JN.1 subvariant and underscore the immunodominant features of these responses.