Microfluidic-Chip-Based Formulation and In Vivo Evaluations of Squalene Oil Emulsion Adjuvants for Subunit Vaccines

Adjuvants play a crucial role in improving the immunogenicity of various antigens in vaccines. Squalene-in-water emulsions are clinically established vaccine adjuvants that improve immune responses, particularly during a pandemic. Current manufacturing processes for these emulsion adjuvants include microfluidizers and homogenizers and these processes have been used to produce emulsion adjuvants to meet global demands during a pandemic. These processes, however, are complex and expensive and may not meet the global needs based on the growing populations in low- and middle-income countries. At the forefront of adjuvant research, there is a pressing need to manufacture emulsion adjuvants using novel approaches that balance efficacy, scalability, speed of production, and cost-effectiveness. In this study, we explored the feasibility of a microfluidic chip platform to address these challenges and evaluated the adjuvanticity of the emulsion adjuvant prepared using the microfluidic chip process in CB6F1 mice model, and compared it with a control formulation. We developed and optimized the process parameters to produce emulsion adjuvants with characteristics similar to SEA160 (control formulation). The resulting emulsion prepared using the microfluidic chip process (MC160) when mixed with ovalbumin, maintained antigen structural integrity. Immunogenicity studies in a CB6F1 mouse model, with the Cytomegalovirus glycoprotein B (CMV gB) antigen, resulted in humoral responses that were non-inferior between MC160 and SEA160, thereby validating the microfluidic chip approach for manufacturing emulsion adjuvants. These findings demonstrate a proof of concept for using microfluidic chip platforms for formulating emulsion adjuvants, offering a simpler manufacturing platform that can be deployed to low- and middle-income countries for rapid production, improving adjuvant access and aiding in pandemic preparedness.