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Abstract: Plasmodium falciparum apical membrane antigen 1 (AMA1) is an asexual blood-stage vaccine candidate against the malaria parasite. AMA1-C1/ISA720 refers to a mixture of recombinant AMA1 proteins representing the FVO and 3D7 alleles in 1:1 mass ratio, formulated with Montanide® ISA 720 as a water-in oil emulsion. In order to develop the AMA1-C1/ISA720 vaccine for human use, it was important to determine the shelf life of this formulation. Previously it was found 267mM glycine stabilized the proteins in Montanide® ISA 720 formulations for a short period of time at 2–8°C . We now test the long term stability of AMA1-C1 at 10 and 40μg/mL formulated with Montanide® ISA 720 with 50mM glycine as a stabilizer. Stability of AMA1-C1/ISA720 at different time points following formulation (0, 5, 12 or 18 months) was evaluated by determining the mean particle size (diameter of the mean droplet volume), total protein content by a Modified Lowry assay, identity and integrity using western blot and SDS–PAGE. Our results showed that the mean particle size of these emulsions increased over time, whereas protein content, as determined by an ELISA method using a monoclonal antibody against penta-his, decreased over time. For the 10μg/mL AMA1-C1/ISA720 vaccine, the protein content was 6.5±2.2μg/mL, and for the 40μg/mL AMA1-C1/ISA720 vaccine, the protein content was only 8.2±2.3μg/mL after 18 months of storage at 2–8°C. These results suggest that the integrity of the protein was affected by long-term storage. The results of the present study indicate that the AMA1-C1/ISA720 emulsion was unstable after 12 months of storage, after which AMA1-C1 proteins were partially degraded. [Copyright &y& Elsevier]

Abstract: Montanide® ISA 720 is an experimental adjuvant, formulated as water-in-oil emulsions, that induces high antibody titers in several animal species. It has been used in human vaccine trials with malaria and HIV vaccines. The heightened response is likely due, in part, to the formation of a depot at the injection site. However, post-formulation modifications were seen with seven proteins tested during storage of ISA 720 formulations at 37°C for 1 week and two proteins stored longer at 4°C. Potency studies in mice, in which the stored vaccines were diluted into placebo emulsions for appropriate dosing, indicated that this instability could lead to loss of immunogenicity in the post-injection depot, limiting the allowable storage time of preformed vaccines. We describe point-of-injection formulation for ISA 720 vaccines that meets the requirement for in vitro stability. For preformed vaccines, addition of glycine or glycylglycine prevented antigen modification on storage at 37°C, providing a potential way of stabilizing antigen/ISA 720 formulations for in vitro storage and the post-injection depot. [Copyright &y& Elsevier]