The patterns of in vitro cell-death and inflammatory cytokines induced by distinct BCG vaccine strains are differentially induced in human mononuclear cells

Abstract

Tuberculosis (TB) remains among the world's leading cause of mortality. For its control, studies of TB vaccines are needed. Since live-attenuated Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the only TB vaccine currently in use, studies on the protective role of BCG are required. In this study, we analyzed host cells purified directly from whole blood of human immunodeficiency virus (HIV)-negative volunteers, comprising adult healthy donors (HD) and neonates (umbilical cord bloods, UCB), with the aim to directly compare in vitro immune responses with distinct BCG strains in human mononuclear cells. The Moreau, Pasteur, and Danish BCG strains were used to infect mononuclear cells in vitro for 48 h; bacilli viability and cell-death were subsequently detected by flow cytometry. In addition, cell culture supernatants were used in cytokine detection assays. Overall, the Moreau BCG strain induced higher levels of apoptosis than the Pasteur and Danish BCG strains in both the HD and UCB groups (p-value < 0.05), and a human monocytic cell-line mirrored those cell-death patterns after BCG infection. The Moreau BCG strain, exclusively, induced Th1 cytokines at the highest levels in cells from adults (p-value < 0.05) when compared with both Pasteur and Danish BCG strains, whereas TGF-β1 levels were reduced significantly (p-value < 0.01) in the HD group when cells were infected with the Moreau BCG vaccine. As expected, eight out of 22 pro-inflammatory cytokines were secreted at significant levels (p-value < 0.05) above the baseline rates in all BCG-infected cell cultures, in the HD group only. When analyzing these results, we excluded confounding factors related to storage and viability of the BCG strains used. These findings suggest that Moreau BCG is a more potent immunostimulating agent than the Pasteur and Danish BCG strains. Clinical trials will be needed to confirm these findings.

Licensed Bacille Calmette-Guérin (BCG) formulations differ markedly in bacterial viability, RNA content and innate immune activation

“…It is not currently known which BCG sub-strain/formulation offers the best protection from TB disease or heterologous infections in human populations [ 15 ]. Differences in BCG-induced innate immune activation may contribute to distinct clinical effects of BCG in populations of different ages [ 16 ].…”

The in vitro Mycobacterium bovis BCG Moreau infection of human monocytes that induces Caspase-1 expression, release and dependent cell death is mostly reliant upon cell integrity

“…On the other hand, our previous study of circulating biomarkers found that active IL-18 plasma levels positively correlated with inflammatory IL-23 in cord blood, but not in adult blood [19]. Thus, IL-18 released during 48 h-BCG infection of the HD and UCB groups were previously assayed [11], and the results were found to be differentially induced (Fig. 3).…”

“…In TB, the specific cell-death pattern plays a critical role in the ensuing immune response [15]. Previously, we have found that healthy donor adults (HD) vaccinated with BCG-Moreau during childhood (BCG vaccination in Brazil is mandatory after birth) display a distinct in vitro cell-death pattern when compared with umbilical vein cells from naïve individuals (UCB) who had never been exposed to the mycobacterium before [11, 13, 16]. In order to investigate these data at the molecular level, cells were 1) infected with the BCG-Moreau vaccine for 16 h (yielding 87% live bacilli on average after reconstitution) or 2) cells were left resting (baseline) uninfected.…”

“…Data points denote individual donors and horizontal bars represent median values in each condition. * p -value ≤0.05, according to the Dunn’s paired test [11]…”

“…Both Peripheral Blood (PBMC) and Cord Blood Mononuclear Cells (CBMC) were separated within no more than 24 h (average 5 h) after obtaining and then culturing blood specimens from all study participants, as described elsewhere [11]. In vitro infections (approximately 1 × 10 7 viable bacilli per vial) with the Moreau BCG vaccine (Ataulpho de Paiva Foundation-FAP, Rio de Janeiro) of freshly-isolated PBMCs and CBMCs (5 × 10 6 cells each) were performed for 16 h (exclusively for gene expression) and then for 24, 48, and 72 h, at a multiplicity of infection (MOI) of 2:1 (bacilli:host cell).…”