BULGARIAN REFERENCE REAGENT FOR BCG VACCINE:

VIABILTY EVALUATED BY THE BIOLUMINESCENT ATP-ASSAY AND VIABLE COUNT METHOD

Elitsa L. Pavlova1, Tzvetelina R. Stefanova2 , Alexander E. Mihaylov2 , Miliana S. Chouchkova2, Varban M. Savov1, Vera I. Iordanova2, Rada M. Dimitrova2

Medical Physics, Faculty of Physics, Sofia University “St. Kliment Ohridski”1, BB-NCIPD Ltd.2, Sofia, Bulgaria

The first WHO Reference Reagents for BCG vaccines of three different substrains – Danish 1331, Tokyo 172-1 and Russian BCG-I, have been developed and adopted by the WHO ECBS in 2009. These preparations cover all substrains used for production of BCG vaccines currently prequalified by the WHO. It is investigated the BCG vaccine, Bulgarian Lot 254-2 produced from Sofia SL 222, itself originating from the Russian BCG-I substrain. All three substrains are evaluated in an international collaborative study of eleven laboratories (WHO, ECBS, K.Markey, Mei M.Ho et al., 2009) using two viability assays (cultural viable count and modified ATP assay, based on the firefly luciferase) and mPCR as an identity assay.

A study, defining the content of the Bulgarian Reference Reagent (RR) candidate in terms of viability (both ATP and CFU content) is reported.

The inclusion of a modified ATP method according to a protocol provided for the purposes of the collaborative study allowed checking the suitability of this rapid andsensitive method as an alternative method of the cultural viable count assay for viability.

For the cultural viable count assay the routine in-house method (BCG Laboratory, BulBio-NCIPD) based on the principles of the WHO recommendations (WHO)/TB/Technical Guide/77.9) is applied.

The ATP content of the RR for BCG Lot 254-2 estimated in the BulBio laboratory is mean 4.11±0.88 ng per ampoule. In the collaborative study this content is 7.52±1.48 ng ATP per ampoule (estimated from seven data sets).As the ATP conditions varied among all participating laboratories, the distribution of the mean ATP content obtained from the participants is wide (the expanded uncertainty is 95%, the confidence is 0 to 16.8).

The mean CFU determined at the Bul Bio laboratory of the Bulgarian candidate is 3.07±0.29 million per ampoule.In the collaborative study the estimated CFU (ten data sets) confirm the expected variable results due to the different culture media and methodologiesused in the laboratories. The mean CFU is 3.39±0.50 million per ampoule. The expanded uncertainty (95% confidence) varies from 0.95 to 5.83 million.

The results demonstrate the suitability of the Bulgarian Lot 254-2 for use as a Reference Reagent for the BCG vaccine and as a comparator for viability assays, residual virulence/localreactogenicity assays and protection assay in animal models.

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