Laboratory of Malaria Immunology and Vaccinology, National Institute of Allergy and Infectious Disease, National Institutes of Health, 5640 Fishers Lane, Rockville, MD 20852, USA.
Vaccine. 2011 May 9;29(20):3640-5. doi: 10.1016/j.vaccine.2011.03.015. Epub 2011 Apr 8.
Plasmodium falciparum apical membrane antigen 1 (AMA1) is an asexual blood-stage vaccine candidate against the malaria parasite. AMA1-C1/ISA 720 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/ISA 720 vaccine for human use, it was important to determine the shelf life of this formulation. Previously it was found 267 mM glycine stabilized the proteins in Montanide(®) ISA 720 formulations for a short period of time at 2-8°C [25]. We now test the long term stability of AMA1-C1 at 10 and 40 μg/mL formulated with Montanide(®) ISA 720 with 50mM glycine as a stabilizer. Stability of AMA1-C1/ISA 720 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 10 μg/mL AMA1-C1/ISA 720 vaccine, the protein content was 6.5±2.2 μg/mL, and for the 40 μg/mL AMA1-C1/ISA 720 vaccine, the protein content was only 8.2±2.3 μg/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/ISA 720 emulsion was unstable after 12 months of storage, after which AMA1-C1 proteins were partially degraded.
恶性疟原虫顶膜蛋白 1（AMA1）是一种抗疟寄生虫的无性血期疫苗候选物。AMA1-C1/ISA 720 是指重组 AMA1 蛋白混合物，代表 FVO 和 3D7 等位基因，以 1:1 的质量比混合，用 Montanide（®）ISA 720 制成水包油乳液。为了开发用于人类的 AMA1-C1/ISA 720 疫苗，确定该制剂的保质期非常重要。此前发现，267mM 甘氨酸可在 2-8°C 下短时间稳定 Montanide（®）ISA 720 制剂中的蛋白质[25]。我们现在以 50mM 甘氨酸作为稳定剂，测试 10 和 40μg/mL AMA1-C1 在 Montanide（®）ISA 720 中的长期稳定性。通过测定平均粒径（平均液滴体积的直径）、改良 Lowry 测定法测定的总蛋白含量、使用 Western blot 和 SDS-PAGE 测定的身份和完整性，评估制剂后不同时间点（0、5、12 或 18 个月）AMA1-C1/ISA 720 的稳定性。结果表明，这些乳液的平均粒径随时间增加而增加，而通过针对五聚体的单克隆抗体的 ELISA 方法测定的蛋白含量随时间减少。对于 10μg/mL AMA1-C1/ISA 720 疫苗，蛋白含量为 6.5±2.2μg/mL，对于 40μg/mL AMA1-C1/ISA 720 疫苗，在 2-8°C 下储存 18 个月后，蛋白含量仅为 8.2±2.3μg/mL。这些结果表明，长期储存会影响蛋白质的完整性。本研究结果表明，AMA1-C1/ISA 720 乳液在储存 12 个月后不稳定，之后 AMA1-C1 蛋白部分降解。
