Researchers develop novel proteolysis-targeting (PROTAR) live attenuated vaccine strategies
image: PROTAR viruses efficiently reproduce owing to TEVp-mediated viral protein stabilization in TEVp-expressing cells and are attenuated by proteasome-mediated viral protein degradation in conventional cells. VP, viral protein; Ub, ubiquitin.
Credit: SIAT
Recently, Prof. SI Longlong's team from the Shenzhen Institutes of Advanced Technology of the Chinese Academy of Sciences constructed a library of live attenuated influenza A vaccines that utilize diverse E3 ubiquitin ligases to degrade viral proteins and achieve virus attenuation, and developed the next-generation proteolysis-targeting (PROTAR) strategy, PROTAR 2.0.
The studies were published in Nature Microbiology and Nature Chemical Biology, respectively, and expand on the PROTAR live attenuated vaccine technology that was initially introduced by the team's study published in Nature Biotechnology in 2022.
To prevent influenza, vaccination is widely considered as the most effective way. Currently, the majority of licensed influenza vaccines are inactivated influenza vaccine (IIV) and cold-adapted live attenuated influenza vaccine (CAIV). However, traditional vaccine strategies can result in the loss or incomplete matching of natural antigens from circulating influenza strains, potentially leading to reduced vaccine efficacy.
Converting the whole circulating virus into an attenuated, live vaccine could ensure a better antigen match and induce sufficient efficacy, offering a promising method for more effective vaccines. Using the cellular ubiquitin-proteasome system (UPS) to alter viral protein stability is a promising strategy for developing live attenuated vaccines.
Researchers first engineered PROTAR influenza viruses to be attenuated by the ubiquitin–proteasome system, which degrades viral protein in conventional host cells but allows efficient replication in engineered cell lines for large-scale manufacturing. They then constructed a PROTAR vaccine library by incorporating 22 distinct proteasome-targeting degrons (PTDs) into the C-terminus of the viral protein M1 via a conditionally cleavable linker, with each PTD being recognized by a different E3 ligases.
Depending on the PTD–E3 ligase pairs, PROTAR influenza viruses show varying levels of attenuation in vitro. In animal models, PROTAR vaccine candidates exhibit significant attenuated, induced broad-spectrum immune responses with varying intensity. They also provide robust cross-reactive protection against lethal infection by both homologous and heterologous viruses in a range of animal models, including adult mice, aged mice, mice with pre-existing flu immunity and ferrets.
Although the PROTAR vaccine approach shows potential for virus attenuation and vaccine design, the PTDs could only be incorporated at the terminal ends of viral proteins thus limiting its broad application. Therefore, researchers developed the next-generation PROTAR vaccine approach, PROTAR vaccine 2.0, aiming to optimize and enhance the versatility of the PROTAR vaccine strategy.
PROTAR 2.0 enables the incorporation of PTDs at multiple sites within viral proteins, including the N-terminal, C-terminal and internal regions. The genome-wide investigation revealed that PROTAR 2.0 viruses, with two PTD-modified viral proteins, exhibit efficient replication in E3 ubiquitin ligase-knockout cells but are attenuated in conventional cells due to PTD-mediated proteasomal degradation.
In animal models (mice and ferrets), PROTAR 2.0 viruses exhibit excellent safety characteristics. A single intranasal dose of PROTAR 2.0 virus vaccine induces robust humoral, mucosal and T cell immune responses, offering complete cross-reactive protection against both homologous and heterologous viral challenges.
Overall, these studies provide new approaches for developing safe and effective live attenuated vaccines.