(413c) Single-Vial Filovirus Glycoprotein Vaccines: Oxidation Causes Lower Neutralizing Antibody Titers in Both Mice and Non-Human Primates

Zaire ebolavirus (EBOV), Sudan ebolavirus (SUDV), and Marburg marburgvirus (MARV) are the most prevalent and pathogenic species of filoviruses. Previously, we showed that vaccines comprising glycoprotein antigens from each virus could be co-lyophilized with a squalane-in-water emulsion adjuvant. Our single-vial presentation retained adjuvant properties after lyophilization and reconstitution and elicited high antibody levels against all three filovirus antigens in mice and non-human primates (NHPs). In an attempt to reduce the number of doses, we tested formulations containing increased amounts of adjuvants in NHPs. We found that the vaccines with increased adjuvant levels elicited lower neutralizing antibody titers in NHPs and therefore worse survival outcomes after challenge. We also discovered that the vaccine samples with increased concentrations of adjuvant had larger adjuvant droplet sizes after reconstitution and higher levels of peroxides due to polysorbate 80 degradation in the adjuvant. We observed that increases in emulsion droplet sizes after lyophilization and reconstitution could be mitigated by increasing cryoprotectant concentration, and oxidation could be prevented by formulating the vaccines with free methionine. Through additional exploratory mouse immunogenicity studies, we determined that oxidized protein, not emulsion particle size increases, caused lower neutralizing antibody titers in mice, confirming what was previously seen in NHPs.