4180984

Genotype-specific platform purification of norovirus VP1 virus-like particles for multivalent VP1-based vaccines | Poster Board #273

Date
March 25, 2025

Noroviruses are highly contagious non-enveloped viruses and are the leading cause of acute gastroenteritis worldwide. Norovirus particles consist of a 40 nm icosahedral capsid assembled from 90 dimers of major capsid protein VP1 and one or two copies of minor capsid protein VP2 surrounding the ~7.5 kb single-stranded RNA genome. There is currently no approved vaccine against noroviruses, but multiple recombinant protein and mRNA-based vaccines are in clinical development. Common antigens for vaccine-induced immunity are self-assembling VP1 virus-like particles (VLPs). Generation of high purity norovirus VLPs is not only important for recombinant protein-based vaccines, but also important for utilization as critical reagents in immunogenicity assays for VP1-based vaccine development. The most studied protein-based norovirus VLP vaccine candidates contain GI.1 and GII.4 genotypes and methods were established to recombinantly manufacture GI.1 and GII.4 VLPs. More recent multivalent approaches have aimed to incorporate additional VLP strains in the vaccine composition including GII.2, GII.3, and GII.6 to broaden protective immunity against circulating noroviruses. The first objective of this work was to develop an efficient purification process for GI.1 and GII.4 VLPs expressed from HEK-293 cells involving acid flocculation, cation exchange membrane chromatography, and size exclusion chromatography (SEC). The second objective was to apply this process to the less established GII.2, GII.3, and GII.6 VLPs. Unexpectedly, these additional strains were incompatible with the GI.1/GII.4 purification method due to observed differences in pH-dependent VLP solubility and aggregation propensity. A new process was developed using ammonium sulfate precipitation, anion exchange membrane chromatography, and SEC that was compatible with the three new genotypes. Five purified VLP stocks were generated from the corresponding purification process and demonstrated higher quality than commercially available VLPs. Depending on the target norovirus genotype VLP pH stability, one of the two developed methods can be implemented as a scalable platform for genotype-specific recombinant VLP production.

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