Vaccinia Virus Vectors Targeting Peptides for MHC Class II Presentation to CD4⁺ T Cells

CD4⁺ helper T cells play important roles in providing help to B cells, macrophages, and cytotoxic CD8⁺ T cells, but also exhibit direct effector functions against a variety of different pathogens. In contrast to CD8⁺ T cells, CD4⁺ T cells typically exhibit broader specificities and undergo less clonal expansion during many types of viral infections, which often makes the identification of virus-specific CD4⁺ T cells technically challenging. In this study, we have generated recombinant vaccinia virus (VacV) vectors that target I-A^b-restricted peptides for MHC class II (MHC-II) presentation to activate CD4⁺ T cells in mice. Conjugating the lymphocytic choriomeningitis virus immunodominant epitope GP_61-80 to either LAMP1 to facilitate lysosomal targeting or to the MHC-II invariant chain (Ii) significantly increased the activation of Ag-specific CD4⁺ T cells in vivo. Immunization with VacV-Ii-GP_61-80 activated endogenous Ag-specific CD4⁺ T cells that formed memory and rapidly re-expanded following heterologous challenge. Notably, immunization of mice with VacV expressing an MHC-II-restricted peptide from Leishmania species (PEPCK_335-351) conjugated to either LAMP1 or Ii also generated Ag-specific memory CD4⁺ T cells that underwent robust secondary expansion following a visceral leishmaniasis infection, suggesting this approach could be used to generate Ag-specific memory CD4⁺ T cells against a variety of different pathogens. Overall, our data show that VacV vectors targeting peptides for MHC-II presentation is an effective strategy to activate Ag-specific CD4⁺ T cells in vivo and could be used to study Ag-specific effector and memory CD4⁺ T cell responses against a variety of viral, bacterial, or parasitic infections.