Replication of lyophilized and cultured BCG in human macrophages

Abstract

If BCG must replicate well in vivo to immunize, presumably mainly in macrophages, the potency of BCG vaccines in human subjects might be measurable as the ability of the BCG to replicate in cultured human macrophages. We used this assumption to compare 6 different batches of lyophilized BCG with freshly cultured BCG and freshly cultured virulent Erdman tubercle bacilli, and to study how tubercle bacilli infect and multiply in human macrophages. The bacilli were readily phagocytized. They replicated in macrophages in proportion to how many were alive and how virulent they were, more the former than the latter among the lyophilized bacilli. The proportion of culturable bacilli in exponentially growing suspensions of BCG cultures was unexpectedly low. For suspensions of 5 of the 6 batches of lyophilized BCG, the great majority of bacilli were nonculturable. Suspensions made from the lyophilized BCG contained many large clumps of bacteria that resisted dispersion even by ultrasonic agitation. This clumpiness, presumably imparted to the bacilli during freeze-drying, caused the lyophilized BCG to infect macrophages more heavily than freshly cultured bacilli. In these experiments, native differences among macrophages from different subjects in capacity to phagocytize BCG and to inhibit its intracellular replication also were detected. These studies thus suggest that tests measuring responses of cultured human macrophages to infection with tubercle bacilli offer relatively simple, rapid, and human-being-related means for assessing several important aspects of human immunization with BCG.