Inhibition of mycobacterial growth in vitro following primary but not secondary vaccination with Mycobacterium bovis BCG

Abstract

Despite the widespread use of the Mycobacterium bovis BCG vaccine, there are more than 9 million new cases of tuberculosis (TB) every year, and there is an urgent need for better TB vaccines. TB vaccine candidates are selected for evaluation based in part on the detection of an antigen-specific gamma interferon (IFN-γ) response. The measurement of mycobacterial growth in blood specimens obtained from subjects immunized with investigational TB vaccines may be a better in vitro correlate of in vivo vaccine efficacy. We performed a clinical study with 30 United Kingdom adults who were followed for 6 months to evaluate the abilities of both a whole-blood- and a novel peripheral blood mononuclear cell (PBMC)-based mycobacterial growth inhibition assay to measure a response to primary vaccination and revaccination with BCG. Using cryopreserved PBMCs, we observed a significant improvement in mycobacterial growth inhibition following primary vaccination but no improvement in growth inhibition following revaccination with BCG (P < 0.05). Mycobacterial growth inhibition following primary BCG vaccination was not correlated with purified protein derivative (PPD) antigen-specific IFN-γ enzyme-linked immunospot (ELISPOT) responses. We demonstrate that a mycobacterial growth inhibition assay can detect improved capacity to control growth following primary immunization, but not revaccination, with BCG. This is the first study to demonstrate that an in vitro growth inhibition assay can identify a difference in vaccine responses by comparing both primary and secondary BCG vaccinations, suggesting that in vitro growth inhibition assays may serve as better surrogates of clinical efficacy than the assays currently used for the assessment of candidate TB vaccines.

Figures

Fig 1

Outline of the clinical study showing the schedule of blood collection and BCG vaccination.

Fig 2

T-cell responses in adults receiving primary vaccination with BCG or BCG revaccination. PPD antigen-specific T cells were detected in PBMCs from healthy, BCG-naive (A) or previously BCG-vaccinated (B) adults receiving ∼2 × 105 to 8 × 105 CFU of BCG-SSI. The T cells were stimulated overnight with PPD, and the responses were detected using an ex vivo IFN-γ ELISPOT assay. Values were determined by a Wilcoxon matched-pairs signed-rank test compared to the baseline (before BCG vaccination). SFC, spot-forming cells. Dots indicate individual data points, and the line indicates the median response for the group.

Fig 3

In vitro growth inhibition in BCG-vaccinated and BCG-naive subjects using both whole blood and cryopreserved PBMCs. (A) The abilities of whole blood from 8 BCG-naive and 10 previously BCG-vaccinated volunteers to inhibit mycobacterial growth at baseline were compared. There was a trend toward enhanced mycobacterial growth inhibition in the BCG-vaccinated group, but it was not significant. (B) The abilities of cryopreserved PBMCs from 9 BCG-naive and 10 BCG-vaccinated volunteers to inhibit mycobacterial growth were assessed. Significantly more growth inhibition was observed in the PBMC cultures from previously BCG-vaccinated subjects (P > 0.05; t test). The box plots show the lowest data point of the 25th quartile and the highest data point of the 75th quartile (Tukey).

Fig 4

Mycobacterial growth in the blood and PBMCs of adults receiving primary vaccination with BCG or following revaccination with BCG. Mycobacterial growth was measured in whole blood (A and B) and cryopreserved PBMCs (C and D) from healthy, BCG-naive (A and C) or previously BCG-vaccinated (B and D) adults receiving ∼2 × 105 to 8 × 105 CFU of BCG-SSI. Mycobacterial growth in samples collected at baseline (before BCG vaccination) was compared to growth in samples collected at 4, 8, and 24 weeks postvaccination. One-way ANOVA was used to test for significance, followed by a paired t test. The box plots show the lowest data point of the 25th quartile and the highest data point of the 75th quartile (Tukey).

Fig 5

Growth inhibition in PBMCs before and after primary vaccination with BCG. To confirm that growth inhibition can be measured in frozen PBMCs without antibiotics from subjects vaccinated with BCG, we repeated the assay using PBMCs from the same 8 subjects used in the whole-blood growth inhibition assay. Growth inhibition was measured prevaccination and 8 weeks postvaccination.

Fig 6

Correlation of mycobacterial growth with the IFN-γ ELISPOT response to PPD. (A) In cryopreserved PBMCs collected 4 weeks following immunization with BCG, there was significant correlation with the inhibition of mycobacterial growth and the number of PPD antigen-specific IFN-γ-secreting T cells. Subjects from both the primary-vaccination and revaccination groups are included. (B) In the revaccination group, a higher PPD antigen-specific IFN-γ ELISPOT response at baseline (prior to vaccination) was associated with reduced capacity to control mycobacterial growth (Spearman's correlation).