Insights in AAV-mediated antigen-specific immunity and a strategy for AAV vaccine dose reduction through AAV-extracellular vesicle association

We previously showed therapeutic advantages of using a capsid-modified and encoded antigen-optimized AAV-based cancer vaccine to initiate strong antigen-specific immune responses and increase survival in a syngeneic mouse model of melanoma. In this study, we further explore AAV vaccine dose reduction and possible mechanisms of the immune response. Immunization with extracellular vesicle (EV)-associated AAV6-S663V encoded ovalbumin (OVA) or tyrosinase-related protein 1 (TRP-1) induced significantly higher levels of antigen-specific CD8⁺ T cells compared with standard AAV in mice. Importantly, a higher number of specific CD8⁺ T cells was achieved with EV-AAV several logs lower than optAAV-based doses. EV-optAAV-OVA was used in a dose 100 times lower, and EV-optTRP-1 10 times lower than optOVA and optTRP-1 correspondingly. Our data suggest that significant dose reduction for optimized AAV-based vaccines is possible without sacrificing efficiency. In addition, we studied the role of conventional type 1 dendritic cells (cDC1) in optimized AAV-based immunization using a C57BL/6-Irf8em1Kmm (Irf8 ^<i>+</i> 32^-/-) mouse model lacking cDC1. Interestingly, we found that cDC1 are not essential for conveying effector T cell responses to AAV-encoded tumor antigens.