Orf virus-based vectors induce potent germinal center B cell, Tfh cell, and CD8⁺ T cell responses

Recombinant Orf virus (rORFV) vectors have proven safe and immunogenic in human clinical trials but the cellular correlates of antibody induction by this parapoxviral platform remain ill-defined. To characterize antigen-specific B cell responses at the cellular level, we performed adoptive transfer experiments with monoclonal B cells specific for the glycoproteins of either vesicular stomatitis virus or lymphocytic choriomeningitis virus. Immunizations of mice with rORFV delivering these glycoproteins stimulated antigen-specific B cells to engage in germinal center (GC) reactions and to differentiate into antibody-secreting cells and memory B cells. These responses could be recalled upon homologous rORFV boost. Moreover, rORFV induced antigen-specific CD8⁺ and CD4⁺ T cell responses including T follicular helper (Tfh) cells. These responses contracted over time but re-expanded upon vaccine re-administration. Pre-existing rORFV-specific anti-vector immunity interfered with CD8⁺ T cell responses to rORFV-vectored antigen, whereas CD4⁺ T cell and B cell responses were unaffected. rORFV-induced immune responses were comparable with those elicited by recombinant adenovirus- and modified vaccinia Ankara virus-based vectors, and they conferred protection against viral challenge. This study characterizes rORFV as a versatile inducer of protective GC B cell, Tfh cell, and CD8⁺ T cell responses, which can be augmented by homologous booster vaccination.