Inactivation of Mycoplasma gallisepticum and Mycoplasma synoviae during influenza vaccine production

Vollständiger Abstract

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INTRODUCTION. The use of chicken embryos in influenza vaccine production carries the risk of contaminating the final product with the avian mycoplasmas, Mycoplasma gallisepticum and M. synoviae , which pose a potential hazard to humans. Therefore, evaluating the effectiveness of inactivating these contaminants in influenza vaccine intermediates using inactivators (β-propiolactone, ultraviolet irradiation) and detergents is important. AIM. This study aimed to evaluate the inactivation dynamics of M. gallisepticum and M. synoviae in influenza vaccine intermediates following exposure to β-propiolactone, ultraviolet irradiation, and detergents (n-octyl-β-D-glucopyranoside, tetradecyltrimethylammonium bromide) to determine their effectiveness under vaccine production conditions. MATERIALS AND METHODS. M. synoviae strain WVU1853 (ATCC) and M. gallisepticum (VGNKI) were used. To assess inactivation dynamics, influenza vaccine intermediates containing human influenza virus were spiked with mycoplasma cultures at a concentration of at least 4 lg CFU/mL. Inactivation was performed under the following conditions: 0.09% β-propiolactone at 4 °C for 12 hours; ultraviolet irradiation (60 W) with a layer thickness of the contaminated intermediates ≤1.1 mm for 5 min; 0.98% n-octyl-β-D-glucopyranoside and 0.07% tetradecyltrimethylammonium bromide at 18 °C for 60 min. The concentrations of M. gallisepticum and M. synoviae in the test samples were determined by the colony-forming unit (CFU) assay. RESULTS. Immediately after β-propiolactone addition, the concentration of M. gallisepticum decreased by 1.88±0.20 lg CFU/mL, and that of M. synoviae decreased by 2.17±0.37 lg CFU/mL. After 4 hours of inactivation, no viable mycoplasmas of either species were detected. Under ultraviolet irradiation, viable M. gallisepticum were not detected after 1 min, and M. synoviae were not detected after 0.5 min. No viable M. gallisepticum or M. synoviae were detected immediately after the addition of detergents (n-octyl-β-D-glucopyranoside or tetradecyltrimethylammonium bromide). CONCLUSIONS. All studied mycoplasma inactivation methods used in influenza vaccine production (β-propiolactone, ultraviolet irradiation, n-octyl-β-D-glucopyranoside, and tetradecyltrimethylammonium bromide) reduce the viability of M. gallisepticum and M. synoviae by more than 4 lg CFU/mL. The described approaches for assessing the inactivation dynamics of avian mycoplasmas can be used to study the resistance of other contaminants to various inactivation methods and chemical reagents.