Immunogenicity of an Escherichia coli-Produced Recombinant 9-Valent Human Papillomavirus Vaccine in Mice and Rats

Background: Prophylactic human papillomavirus (HPV) vaccines are crucial for preventing HPV-related cancers. This study aimed to preclinically evaluate a novel recombinant 9-valent HPV vaccine produced in Escherichia coli (E. coli), which targets HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58, and is based on virus-like particles (VLPs) of the HPV major capsid protein L1. Methods: The molecular weight and purity of HPV L1 protein bands were assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with Coomassie Brilliant Blue staining. The morphology and size distribution of VLPs were characterized using cryo-electron microscopy and DLS. The immunogenicity and durability of the recombinant 9-valent HPV vaccine were evaluated in BALB/c mice and Wistar rats. Mice received single or triple immunizations (2-week intervals) of two vaccine batches or Gardasil^®9 (MSD, USA) control at 1/20 human dose. Antibody responses were monitored via ELISA and pseudovirus neutralization assays over 24 weeks. Rats were administered single or triple immunizations (2-week intervals) of high- (1/10), medium- (1/20), or low-dose (1/40) vaccine or Gardasil^®9 control (1/20), with neutralizing antibodies tracked for 16 weeks. Results: Cryo-electron microscopy and DLS revealed that VLPs of each type appeared as uniformly distributed, spherical or ellipsoidal hollow intact particles with a diameter of approximately 45-65 nm. This vaccine demonstrated robust immunogenicity and long-lasting efficacy in BALB/c mice and Wistar rats, with effects comparable to those of the commercially available vaccine Gardasil^®9. Conclusions: The 9-valent HPV vaccine induces robust and persistent immune responses in mice and rats, strongly supporting further clinical trials. It is expected to be an alternative to marketed vaccines and ease the global supply shortage of 9-valent HPV vaccines.