OBJECTIVETo investigate the effect of temperature on the stability of intermediate and final products of inactivated enterovirus 71 vaccine, which was prepared in human diploid cells.

METHODSThe different batches of harvest viral cultures, the vaccine stock solutions and the final productions of inactivated enterovirus 71 vaccine were stored at different temperatures. The samples of viral culture stored at -20°C or 4°C were harvested at 0, 6, 12 and 24 months later. The samples of vaccine stock solutions stored at -20°C were harvested at 0, 6, 12 and 24 months later, and that stored at 4°C were harvested at 0, 1, 3, 6 and 12 months later. The samples of finial products were harvested at different time points (0, 6, 12 and 24 months for storing at 4°C; 0, 7, 14, 28, 42 and 60 d for storing at 25°C; 0, 3, 7, 14 and 21 d for storing at 37°C). The viral titer, antigen content, antigen purity, endotoxin content, effectiveness, pH and appearance of samples were determined, respectively. A total of 1 800 BLAB/c mice were immunized by vaccine and 150 control mice were injected by diluents without antigen via intraperitoneal. The tail vein blood (500 µl per mouse) from 1 950 mice were harvested after 4 weeks post injected. The neutralization antibody titers of the serum were tested to calculate the half effective dose (ED50) of final products. All results were analyzed using analysis of variance to compare the differences of the above indexes.

RESULTSThe viral titers of harvest viral culture of inactivated EV71 vaccine were (6.67 ± 0.13), (6.56 ± 0.09), (6.52 ± 0.04), (6.39 ± 0.16) lgCCID50/ml (CCID50, the half cell culture infective dose) after 0, 6, 12 and 24 months storage at -20°C; and (6.67 ± 0.13), (6.41 ± 0.13), (6.19 ± 0.18), (5.97 ± 0.09) lgCCID50/ml at 4°C. The viral titers reduced with time (F = 9.81 or 44.16, P < 0.05). The antigen contents of the vaccine stock solution were maintained at (3 626.67 ± 1 382.56) EU/ml within 3 months at 4°C, but were (2 080.00 ± 876.36), (951.17 ± 346.35) EU/ml at 6 and 12 months, respectively. The ED50 of the final production were (31.00 ± 2.71), (32.93 ± 3.22), (39.37 ± 3.44) and (46.04 ± 3.25) EU/ml after 0, 6, 12 and 24 months storage at 4 °C, but were (31.00 ± 2.71), (32.23 ± 2.66), (34.70 ± 1.77), (40.04 ± 2.10), (47.78 ± 1.93) and (56.97 ± 0.50) EU/ml at 0, 7, 14, 28, 42 and 60 days at 25°C, and were (31.00 ± 0.00), (36.20 ± 0.00), (41.87 ± 0.50), (53.25 ± 0.50) and (64.84 ± 0.58) EU/ml at 0, 3, 7, 14 and 21 days at 37°C, respectively. The ED50 had increased with the time by and had significantly differences compared with the beginning level (F = 28.49, 215.15 or 156.12, P < 0.05).

CONCLUSIONThere is a good stability of the intermediate and final productions of inactivated enterovirus 71 (EV71) vaccines, within 24 months at -20°C or 6 months at 4°C storage for viral culture, 24 months at -20°C or 3 months at 4°C storage for stock solution and 24 months at 4°C or 28 d at 25°C or 7 d at 37°C storage for finial vaccine.