Application of High Hydrostatic Pressure (HHP) for the production of whole virus inactivated vaccines: development of SARS-CoV-2 and West Nile virus vaccine candidates

Vaccination remains the most effective means to prevent infectious diseases; however, the COVID-19 pandemic has highlighted key limitations of vaccine technologies (production scalability, cold-chain dependence, antigen stability, and adaptability. Concurrently, arboviral infections such as West Nile virus (WNV) are becoming endemic in Europe and other regions due to ecological and climatic changes, despite the absence of licensed human vaccines. In this context, High Hydrostatic Pressure (HHP) processing, a chemical-free technology, has emerged as a promising approach for viral inactivation. This study aimed to develop and validate an HHP-based platform to produce whole-virus inactivated vaccines against SARS-CoV-2 and WNV. The objectives were to evaluate inactivation efficacy across structurally distinct viruses, characterize pressure-induced morphological and antigenic changes, assess immunogenicity in a murine model, and investigate thermostability under storage conditions relevant to low-resource settings. Viral stocks were subjected to pressures ranging from 400 to 600 MPa for 5-10 minutes. Residual infectivity was assessed in Vero E6 cells, while antigenic and structural integrity were analyzed by electron microscopy and Western blotting. Immunogenicity of SARS-CoV-2 vaccine candidates was evaluated in CD-1 mice following a prime-boost regimen, measuring humoral and cellular immune responses. Thermostability was assessed during storage at 4 °C and 25 °C. HHP effectively inactivated both viruses while preserving key antigenic determinants. For SARS-CoV-2, treatment at 500 MPa abolished infectivity while maintaining virion morphology and inducing robust, durable humoral and cellular immune responses, including strong neutralizing activity and broader epitope recognition compared with heat-inactivated controls. Importantly, HHP-treated preparations showed improved thermostability. For WNV, longer pressure exposure was required for complete inactivation, but major structural proteins retained immunoreactivity. Overall, these findings demonstrate that HHP is a versatile and effective viral inactivation strategy, offering a scalable and distribution-friendly platform for whole-virus vaccine development applicable to both pandemic preparedness and control of emerging or endemic arboviruses.