Preparation and properties of a freeze-dried B.C.G. vaccine of increased stability.

A stable B.C.G. vaccine, prepared and distributed in the freeze-dried form, is now accepted for use in many parts of the world. Recent trials (Medical Research Council, 1958, 1960; Griffiths and Gaisford, 1956) have confirmed that the freeze-dried vaccine induces a high degree of tuberculin sensitivity without untoward local reactions. From its stability the freeze-dried vaccine has undoubted advantages over a liquid vaccine, particularly because it permits standardization and safety-testing to be completed before issue. Nevertheless, since the vaccine contains living cells, of which an optimal number is essential for the efficacy of B.C.G. vaccination, great care has to be taken to ensure that the vaccine is not exposed to elevated temperatures even for relatively short periods. It is therefore obvious that a heatresistant freeze-dried B.C.G. vaccine that could be kept at temperatures up to 370 C. for a few weeks would have advantages, since it could then be distributed under unrefrigerated conditions and transported in tropical and subtropical countries without special precautions. Numerous attempts have been made to improve the heat-stability of freeze-dried B.C.G. vaccine. Almost all of them have concentrated on the medium in which the organisms are freeze-dried, since this has usually been regarded as the most important factor affecting loss of viability during both freeze-drying and subsequent storage. Thus, for example, Miller and Goodner (1953) showed that sodium glutamate, as well as sodium aspartate and ascorbic acid, when included in the menstruum, increased the stability of the vaccine on storage. Cho and Obayashi (1956), working with the Japanese strain of B.C.G., concluded that the inclusion of I % sodium glutamate provided one of the best media for freeze-drying B.C.G. Greaves (1960), in studies with Bacterium paracoli and Nelsseria gonorrhoeae, also found that the presence of sodium glutamate in the drying medium gave subsequent enhanced stability at elevated temperatures. The rationale behind these improvements has been given by Scott (1960). In work on Salmonella newport he showed that the sucrose included in the menstruum gave better stability during storage than did glucose or arabinose, both of which contain an available carbonyl group. He advanced the theory that the action of the menstruum is one of inhibiting or preventing damage to the micro-organisms resulting from the reaction between cellular protein and available carbonyl groups. In our laboratory the actions of many different media for freeze-drying B.C.G. have been investigated: they have included that of sodium glutamate (Muggleton, 1960). Under our conditions and with Sour substrain of B.C.G., however, no outstanding improvement in survival on storage at elevated temperatures was seen with any of these media, and none showed any great merit, such as had been claimed by the Japanese workers. It was logical to assume that other factors could influence the stability of the dried vaccine, and it was decided to investigate the culture medium in which the B.C.G. was grown. In previous experimental work (Ungar et al., 1956) Sauton's medium with the addition of 1/4,000 triton WRl339 was used to grow the organisms as a uniform suspension in deep culture. Having then found that the addition of various substances to the freeze-drying menstruum had little effect