Immune history confers antibody- and T cell-dependent cross-protection against highly pathogenic avian influenza H5N1 viruses.

The outbreak of highly pathogenic avian H5 influenza (HPAI) clade 2.3.4.4b in cattle has spread across the United States. Mice with pre-existing immunity to H1N1 virus or with a live-attenuated influenza vaccine showed protection against a lethal bovine-derived HPAI H5N1 viral challenge. Notably, ferrets with mixed immunity also demonstrated protection against a feline-derived H5N1 virus, independent of cross-reactive neutralization titers, but antibodies to whole virus were observed. To investigate protective factors, we conducted T cell epitope mapping using published H1N1 viral sequences and found high conservation of key T cell epitopes in the bovine HPAI H5N1 strain. Depletion of T cells in mice prior to and during primary H1N1 infection impacted cross-protective antibodies to H5N1 virus, with CD4 depletion increasing mortality and CD8 depletion mildly impacting morbidity upon H5N1 viral challenge. This underscores the need to investigate memory T cell responses alongside antibodies in assessing preexisting cross-protection to HPAI H5N1 viruses.IMPORTANCEThe rapid spread of highly pathogenic avian H5 influenza (HPAI) clade 2.3.4.4b in U.S. cattle represents an urgent and evolving public health threat. Our findings reveal that pre-existing immunity, whether from seasonal H1N1 infection or live-attenuated vaccination, can confer substantial protection against lethal bovine- and feline-derived HPAI H5N1 viruses, even in the absence of strong cross-neutralizing antibody titers. By integrating T cell epitope mapping with mechanistic depletion studies, we demonstrate that conserved CD4 and CD8 T cell epitopes across H1N1 and H5N1 strains underpin this cross-protection. Critically, loss of CD4 T cell help during primary H1N1 infection disrupts the development of cross-reactive antibody responses and markedly worsens outcomes after H5N1 challenge. These results identify memory T cell responses as important determinants of heterosubtypic immunity and highlight the need to incorporate T cell-focused metrics into risk assessment, vaccine evaluation, and preparedness strategies for emerging HPAI H5N1 viruses.