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

Asimenia Angelidou, Maria-Giulia Conti, Joann Diray-Arce, Christine S Benn, Frank Shann, Mihai G Netea, Mark Liu, Lakshmi Prasad Potluri, Guzman Sanchez-Schmitz, Robert Husson, Al Ozonoff, Beate Kampmann, Simon Daniël van Haren, Ofer Levy, Asimenia Angelidou, Maria-Giulia Conti, Joann Diray-Arce, Christine S Benn, Frank Shann, Mihai G Netea, Mark Liu, Lakshmi Prasad Potluri, Guzman Sanchez-Schmitz, Robert Husson, Al Ozonoff, Beate Kampmann, Simon Daniël van Haren, Ofer Levy

Abstract

Background: Bacille Calmette-Guérin (BCG), the live attenuated tuberculosis vaccine, is manufactured under different conditions across the globe generating formulations that may differ in clinical efficacy. Innate immune recognition of live BCG contributes to immunogenicity suggesting that differences in BCG viability may contribute to divergent activity of licensed formulations.

Methods: We compared BCG-Denmark (DEN), -Japan (JPN), -India (IND), -Bulgaria (BUL) and -USA in vitro with respect to a) viability as measured by colony-forming units (CFU), mycobacterial membrane integrity, and RNA content, and b) cytokine/chemokine production in newborn cord and adult peripheral blood.

Results: Upon culture, relative growth was BCG-USA > JPN ≫ DEN > BUL = IND. BCG-IND and -BUL demonstrated >1000-fold lower growth than BCG-JPN in 7H9 medium and >10-fold lower growth in commercial Middlebrook 7H11 medium. BCG-IND demonstrated significantly decreased membrane integrity, lower RNA content, and weaker IFN-γ inducing activity in whole blood compared to other BCGs. BCG-induced whole blood cytokines differed significantly by age, vaccine formulation and concentration. BCG-induced cytokine production correlated with CFU, suggesting that mycobacterial viability may contribute to BCG-induced immune responses.

Conclusions: Licensed BCG vaccines differ markedly in their content of viable mycobacteria possibly contributing to formulation-dependent activation of innate and adaptive immunity and distinct protective effects.

Keywords: BCG vaccine formulation; Chemokine; Colony forming units; Cord blood; Cytokine; Viability.

Conflict of interest statement

Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: OL is a named inventor on several vaccine adjuvant formulation patent applications. The other authors do not have a commercial or other association that might pose a conflict of interest.

Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.

Figures

Fig. 1.
Fig. 1.
BCG formulations exhibit differences in mycobacterial cell membrane integrity and RNA content as assessed by flow cytometry. (A) Representative dot-plot and (B) average percentage of live cells as a proportion out of all events captured. N = 5 experiments testing at least 2 different lots of each BCG formulation. (C) Histogram showing an overlay of BCG cytometric analysis using SYTO® RNA SelectTM Green Fluorescent Cell Stain. (D) Bar graph showing the % of RNA + cells per equal number of CFU. Unstained BCG USA was used as control. N = 4. Data are presented as mean +/ SD. Statistical significance denoted by one-way non-repeated measures ANOVA with Fisher’s LSD test. **p < 0.01, ***p < 0.01, ****p < 0.001.
Fig. 2.
Fig. 2.
Licensed BCG formulations differ markedly from one another in viability as measured by colony forming units (CFU) in diverse culture media. Six serial 10-fold dilutions of each BCG formulation were plated in (A) solid enriched 7H9 medium (e7H9) and (B) commercial Middlebrook 7H11 medium (M7H11) in triplicate, and colonies counted weekly for 6 weeks. In both media types, BCG-IND and -BUL demonstrated significantly lower viability (CFU) compared to the other BCG formulations. Moreover, BCG-BUL viability (CFU) was >100-fold lower than anticipated per label. (C) Growth of BCG-IND and -BUL was significantly lower (>100-fold) in e7H9 compared to M7H11 medium. Mean CFU per ampule of each candidate was calculated from the average colonies counted in independent experiments (N = 2–6) and also plotted against the anticipated mycobacterial growth based on the vaccine label. Data are presented as mean +/ SD. Statistical comparisons with ANOVA. *p < 0.05, **p < 0.01, ***p < 0.01.
Fig. 3.
Fig. 3.
(A) BCG-induced whole blood cytokine/chemokine pattern at 18 h differs significantly by age. Heat map depicts cytokines/chemokines after 18 h stimulation with equal CFU concentrations of BCG, calculated from the vaccine inserts. Repeated-measures one-way ANOVA with Sidak’s post hoc test. N = 4–9 for newborn and 8–13 for adult whole blood. (B) In NB, BCG-IND induced the weakest IFN-c responses compared to the other formulations. Box plots display medians and min-max values. Grey stars indicate comparisons against RPMI control (Kruskal-Wallis test). (C) BCG-induced hematopoietic factors and cytokines at concentrations reflecting human equivalent doses differ significantly by BCG formulation. Radar plots representing the BCG effect as a log-fold change over RPMI control. N = 6–10 for newborn and N = 7–11 for adult whole blood. NB, newborn; AD, adult. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Fig. 4.
Fig. 4.
(A) BCG formulations tested at equal CFU concentrations differ in induction of cytokines/chemokines. Radar plots represent the BCG effect as a log-fold change over RPMI control. (B) BCG-DEN induced significantly higher log-fold changes in CCL7, G-CSF, PDGF AB/BB, IL-1α and IL-1β compared to equal calculated CFU of the other BCG formulations. ANOVA with Tukey correction. N = 6–9 for NB; 10–11 for AD. NB, newborn; AD, adult.
Fig. 5.
Fig. 5.
BCG cytokine-inducing activity significantly correlates with BCG viability. (A) Spearman correlations between CFU–cytokines produced in NB after 18 h BCG stimulation with concentrations reflecting human equivalent doses. (B) CFU-cytokine correlations for all cytokines/chemokines that were significantly changed over RPMI. NB = newborn; AD = adult.

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