Correlating physicochemical and biological properties to define critical quality attributes of a rAAV vaccine...

Recombinant adeno-associated viruses (rAAVs) are a preferred vector system in clinical gene transfer. A fundamental challenge to formulate and deliver rAAVs as stable and efficacious vaccines is to elucidate interrelationships between the vector's physicochemical properties and biological potency. To this end, we evaluated an rAAV-based coronavirus disease 2019 (COVID-19) vaccine candidate that encodes the Spike antigen (AC3) and is produced by a commercially viable process. First, state-of-the-art analytical techniques were employed to determine key structural attributes of AC3, including primary and higher-order structures, particle size, empty/full capsid ratios, aggregates, and multi-step thermal degradation pathway analysis. Next, several quantitative potency measures for AC3 were implemented, and data were correlated with the physicochemical analyses on thermally stressed and control samples. Results demonstrate links between decreasing AC3 physical stability profiles, in vitro transduction efficiency in a cell-based assay, and, importantly, in vivo immunogenicity in a mouse model. These findings are discussed in the general context of future development of rAAV-based vaccine candidates as well as specifically for the rAAV vaccine application under study.