Recombinant protein-based vaccines must be further evaluated for antigen stability. The PfCP-2.9 efficacy correlated with the integrity of its tertiary structure maintained by inter-molecular disulfide bonds. Accumulated evidence has Caspase inhibitor indicated that reduced and alkylated components in PfCP-2.9 lost their GIA activities [4]. Therefore, assessing the conformational nature of this protein following the emulsion process was extremely important for vaccine development. To date, there were

no available methods for the detection of intact protein once it had been emulsified. The Montanide ISA720 adjuvant has been widely utilized in HIV and malaria vaccine development and it was shown to be an effective delivery system for human vaccines [13], [14], [15] and [16]. However, Montanide ISA720 has been reported to modify the antigen after emulsification [21]. Therefore, the stability of the formulated emulsion with the adjuvant was an initial concern. We used available methods as well as new developed methods (such as the sandwich ELISA method) to assess the stability and

potency of the PfCP-2.9 vaccine formulation. This ELISA-based mTOR inhibitor method utilized two types of antibodies and demonstrated that emulsified PfCP-2.9 maintained its integrity for periods of up to 18 months suggesting that protein integrity would not easily be lost in ISA720 adjuvant formulations stored at 4 °C. Furthermore, no degradation of PfCP-2.9 was observed by SDS-PAGE for samples stored for up to 2 years. We noted that PfCP-2.9 formed aggregates (which increased over time in samples stored at warmer temperatures) in some of the emulsion preparations but these aggregates were a small percentage of total protein. However, the aggregates retained their tertiary structure as noted by the ability of mAb5.2 Calpain to bind to them in Western blot assays. Moreover, the potency of the stored emulsion containing aggregated PfCP-2.9 was not affected and the stored emulsion

induced specific antibodies that inhibited parasite growth at the same level as a freshly prepared antigen emulsions, indicating that aggregate formation did not influence the potency and function of the vaccine emulsion. Taken together, the physical and biological properties of the vaccine emulsion preparations used in the described pre-clinical studies demonstrated that PfCP-2.9 was stable for at least 1.5 years. Although some protein aggregation was observed during storage at 4 °C, the aggregated protein retained its conformational integrity and immunogenic potency. This investigation received financial support from the National Basic Research Program (973 Program) in China (2007CB513100) the National 863 Program (2006AA02A222), and Shanghai leading Academic Discipline Project (B901). “
“Bovine herpesvirus-1 (BHV-1) is a pathogen of major economic importance in the cattle industry worldwide.