A single-dose inhalable rAAV-vectored vaccine against mpox virus.

In response to the global mpox outbreak, an inhalable vaccine platform based on rAAV/DJ vectors is developed. This platform represents a needle-free, single-dose, highly immunogenic defense against monkeypox virus (MPXV) and related orthopoxviruses (OPXVs). Two multivalent vaccines, MV-A (encoding M1R/A35R) and MV-B (encoding A29L/B6R), are engineered, and MV-A, MV-B, and their combination (MV-C) are assessed preclinically. The vector integrates the mucosal adjuvant cholera toxin subunit B (CTB) at the 3' terminus of antigen genes, enabling the sustained production of self-assembled antigen-adjuvant nanoparticles. A single dose induces potent activation of antigen-presenting cells and pulmonary T cell responses, generating lung tissue-resident memory T cells. MV-C induces strong, durable MPXV-specific antibodies in mice and non-human primates, with sera and bronchoalveolar lavage fluid showing high neutralizing activity. Single pulmonary immunization with MV-C establishes a protective mucosal barrier in MPXV-susceptible CAST/EiJ mice and confers protection against OPXVs via respiratory or genital routes. These vaccines hold promise for combating future MPXV variants.