Identification of Growth Promoting Effect of <i>r</i>BCG/BCG Culture Supernatant and Its Potential Applications

Abstract

Background: Although BCG is the most widely administered vaccine in the world, there have never been as many cases of TB as there are now. Globally, more than 8.8 million people developed active TB and 1.4 million-many of them-died in 2010. It is estimated that half of pulmonary TB cases arise from latent Mtb infection, making the study of latency and reactivation of utmost importance. Methods: Widely administered BCG vaccines and a gene modified recombinant BCG (rBCG) strain, AERAS-422, were used as models to investigate the growth promoting function of resuscitation-promoting factors (Rpfs) in different bacilli culture phases. Different supernatant fractions were prepared by ultrafiltration, and the promoting function of each fraction containing secreted Rpf(s) was evaluated by growth curve monitoring and colony counting on 7H10 agar plates. Results: The promoting effect of culture supernatants was mainly associated with the high molecular weight fraction (>30 kDa), which stimulated bacterial growth, but did not extend the exponential phase of stimulated culture. Anti-RpfB antibody showed significant growth restriction of the tested cultures. When comparing rBCG cultures containing 7H9 medium, the 10-30 kDa fraction, or the >30 kDa fraction, only the >30 kDa fraction was displayed with down-regulation of the secretion of RpfC, D and E. In colony counting tests, the plates containing the >30 kDa fraction had total countable colony numbers 2 to 3 fold higher than the plates with the 10-30 kDa fraction, and colonies appeared one to two weeks earlier than on the regular plates. The potential applications of the prepared supernatant fractions containing RpfA and RpfB are discussed, which may include accelerating diagnosis of Mtb infection and future TB vaccine development.

High monocyte to lymphocyte ratio is associated with impaired protection after subcutaneous administration of BCG in a mouse model of tuberculosis

“…The absolute numbers of BCG might be under-estimated in this study as we only enumerated bacilli that were readily growing on standard 7H11 agar plates. Use of PCR methods or culture with resuscitation promoting factors may increase yield and allow for detection of dormant bacilli and their contribution to protection 31 , 32 .…”

Improvements of ATP Assay as a Substitute for the CFU Method in Estimating Viable Cell Count for BCG/rBCG Vaccine Preparations

“…In this study, we reevaluated critical conditions for the ATP luminescence assay, and established a linear relationship between ATP concentration and CFU count for all stage preparations (live culture, frozen bulk, lyophilized high and low dosages, and stability samples) of three different BCG/ rBCG vaccines. Mycobacteria have a tendency to clump together in all different stages of BCG/rBCG production, mostly because of the lipid content of the cell wall [12]. Highly branched structures heavily covered by lipid cell walls with few free bacilli are common observations under microscopy using the acid fast staining procedure.…”

“…Fresh cultures of AERAS-422, Danish 1331 and Pasteur BCG were initiated from their frozen working seeds (~4.5 mL) inoculated into Middlebrook 7H9 medium plus extra nutrients, with a volume ratio of 1:100 [12]. One set of fresh cultures was grown in a 37°C incubator at a shaking rate of 120 rpm for 3 to 4 days, until the OD 600nm reached 4.0 to 5.0.…”