PEGylated PolyaspartamideNanomicelle Delivery ofSelf-amplifying Avian Influenza mRNA Vaccine

Abstract The avian influenza virus (AIV) poses a major threat to the poultry industry and public health worldwide, highlighting the need for effective vaccines. Currently, the influenza vaccine for human use is an inactivated vaccine produced from eggs, which requires time-consuming and safe production processes. Messenger RNA (mRNA) is a transcriptional product of cells that can directly produce proteins, with no safety concerns regarding DNA template residues or integrated mutations in the genomic DNA. Recently, COVID-19 vaccines developed using mRNA to express the vaccine antigen have resulted in effective humoral and cellular immune responses. Additionally, mRNA can induce a robust cellular Toll-like receptor immune response and has the potential to serve as a promising vaccine adjuvant. In this study, we developed PEGylated polyamino polyplex nanomicelles encapsulating mRNA that expresses the AIV antigen. These nanomicelles were manufactured as an AIV vaccine, and their efficacy was evaluated in vivo. We produced a highly translation-effective, self-amplifying mRNA (saRNA) that expresses the main antigen, hemagglutinin (HA) protein of AIV. Subsequently, we encapsulated the HA-expressing saRNA in self-assembled polymeric nanomicelles using a PEGylated polyaspartamide block copolymer. Then, we compared the mRNA-loaded nanomicelle administration scenarios and the vaccine-elicited humoral (antibody titer and hemagglutination inhibition capability) and cellular immune responses (interferon-gamma [IFN-γ] and interleukin-4 [IL-4] cytokines) in mice. Our results demonstrated the successful development of an avian mRNA vaccine using polyamino self-assembled nanomicelles, which significantly enhanced both the humoral and cellular immune responses against the AIV antigen, providing a valuable reference for the further improvement of avian influenza mRNA vaccines in the future.