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Published on: September 14, 2011
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.
Insights
Assessing Bacillus Calmette-Guérin (BCG) vaccine potency may involve measuring its replication in human macrophages. This study found lyophilized BCG batches had low culturability and clumpiness, impacting macrophage infection and highlighting macrophage response tests for BCG immunization assessment.
Area of Science:
- Immunology
- Microbiology
- Vaccinology
Background:
- Bacillus Calmette-Guérin (BCG) is a crucial vaccine for tuberculosis prevention.
- BCG's in vivo replication, particularly in macrophages, is presumed essential for effective immunization.
- Assessing BCG potency requires reliable methods linked to human immune responses.
Purpose of the Study:
- To evaluate the potency of different lyophilized BCG batches compared to fresh BCG and virulent strains.
- To investigate the infectivity and replication dynamics of tubercle bacilli within human macrophages.
- To explore the utility of human macrophage cultures as a model for BCG vaccine assessment.
Main Methods:
- Comparison of 6 lyophilized BCG batches, fresh BCG, and virulent Erdman strain.
- Infection and intracellular replication studies using cultured human macrophages.
- Assessment of bacillary viability, culturability, and aggregation (clumpiness).
Main Results:
- Lyophilized BCG batches showed significantly lower culturability and high bacterial clumpiness.
- Clumpiness in lyophilized BCG led to increased initial macrophage infection.
- Macrophage replication correlated with bacillary viability and virulence, with viability being more critical for lyophilized BCG.
- Inter-individual variations in macrophage phagocytosis and BCG inhibition were observed.
Conclusions:
- Lyophilized BCG potency is influenced by reduced culturability and increased clumpiness from freeze-drying.
- Cultured human macrophages provide a relevant model to assess BCG infectivity and replication.
- Macrophage-based assays offer a potentially rapid and human-relevant method for evaluating BCG vaccine potency and immunization potential.
