In vitro Mycobacterial Growth Inhibition in South Korean Adults With Latent TB Infection

Abstract

Background: It is important to understand the ability to inhibit mycobacterial growth in healthy adults who would have been Bacillus Calmette-Guérin (BCG) vaccinated in childhood as this group will be the potential target population for novel booster TB vaccine trials. In this study we investigated not only the long-term immunity induced by childhood BCG vaccination but also protective immunity in terms of the ability to inhibit mycobacterial growth in those who were BCG vaccinated in childhood, with evidence of recent or remote TB infection. Methods: We measured the baseline immune response using a functional mycobacterial growth inhibition assay (MGIA) as a novel approach and an intracellular cytokine staining (ICS) assay as a reference approach in healthy adults, with different status of Mycobacterium tuberculosis (Mtb) infection. Results: Based on MGIA responses in historically BCG-vaccinated healthy adults, demographical characteristics including age, and gender did not affect mycobacterial growth inhibition in PBMC. However, the uninfected healthy control (HC) group showed a greater ability to inhibit mycobacterial growth compared with the latent TB infection (LTBI) group (P = 0.0005). In terms of the M. tuberculosis antigen-specific T-cell immune response in diluted whole blood quantitated using an ICS assay, the LTBI group had a higher frequency of polyfunctional CD 4+ T cells compared with the HC group (P = 0.0002), although there was no correlation between ICS and the MGIA assay. Conclusion: The Mtb infection status had a significant impact on mycobacterial growth inhibition in PBMC from healthy adults in South Korea, a country with an intermediate burden of tuberculosis, with healthy controls showing the greatest mycobacterial growth inhibition.

Keywords: correlates; latent tuberculosis infection; mycobacterial growth inhibition; tuberculosis; vaccine.

Figure 1

MGIA results in comparison with general characteristics of participants. Cryopreserved PBMC prepared from blood samples from 121 healthy adults were thawed and incubated with BCG for 4 days after which remaining mycobacteria were quantified using BACTEC MGIT tubes. In vitro mycobacterial growth in the PBMC based-MGIA assay is presented in panel (A) by age group, in (B) by gender, and in (C) by presence of a BCG scar(s) in healthy adults. A Mann-Whitney U-test was used for comparison of the two groups. P-value ≤ 0.05 was taken as statistically significant. The growth of BCG Pasteur was plotted as TTP (Time to Positive) in hours.

Figure 2

Comparison of MGIA response as TTP (Time to Positive) in hours stratified by QuantiFERON-TB Gold In-Tube (QFT) IFN-γ values in LTBI or HC individuals. MGIA responses of healthy adults (n = 121) were stratified by QFT IFN-γ values at baseline. (A) Comparison of MGIA response as TTP in hours between the QFT+ (positive test results) and QFT- (negative test results) groups, according to the manufacturer's assay using < 0.35 IU/mL and > 0.35 IU/mL IFN-γ values; (B) MGIA response as TTP in hours, stratified by the three categories of IFN-γ values: > 0.7 IU/mL, between 0.2 and 0.7 IU/mL, and < 0.2 IU/mL; (C) Comparison of MGIA response as TTP in hours between individuals with latent TB infection (LTBI) defined as QFT + with known contact history with TB patients and healthy controls (HC) defined as QFT—without recent contact history with known TB patients. P-values were calculated using Mann-Whitney U-test.

Figure 3

Flow chart of study participants enrolled. A total of 121 Korean healthy adults aged more than 20 years old with normal chest x-ray results and without any tuberculosis history agreed to participate in the study at the Severance Hospital, Seoul. Sixty-four participants had had known contacts with TB patients within the past year. The other participants had had no known previous contacts with TB patients. In addition to clinical evaluation in terms of exposure to M. tuberculosis, we tested all study participants with QFT-GIT test for diagnosis of TB infection. Of the known TB contacts, 32.8% (n = 21) were QFT positive and defined as individuals with LTBI. In those without known TB contact, 75.4% (n = 43) were QFT negative, and were defined as healthy controls (HC) in this study.

Figure 4

ICS data in LTBI and healthy control groups and correlation between mycobacterial growth inhibition and ICS responses. (A) Single cytokine expression data including IFN-γ, IL-2, and/ or TNF-α for individuals with LTBI (black bars, n = 11) and healthy controls (gray bars, n = 8). The PPD-specific CD4+ T-cell populations making IFN-γ and IL-2 are dominant in the LTBI group. (B) The polyfunctionality of CD4+ T-cell populations in individuals with LTBI (black bars) and HCs (gray bars). The PPD-specific polyfunctional T helper-1 cells are the dominant cell type in the LTBI group. Whole blood samples obtained from healthy adults were stimulated with PPD overnight and CD4+ T-cells assessed for cytokine secretion by intracellular staining. All ICS data is plotted as bars showing medians and interquartile ranges. Whiskers show the range of the data. The Mann-Whitney U-test was used to determine significance. (C) Scatter plots of TTP in hours vs. the percentage of polyfunctional CD4+ T-cells were drawn for all samples where both the mycobacterial growth inhibition assay (MGIA) and the ICS assay were performed (n = 19). Inhibition of mycobacterial growth (of BCG Pasteur) is indicated by TTP in hours for the remaining mycobacteria after incubation with PBMC obtained from study participants. The Mann-Whitney U-test was used to compare growth inhibition ability between the LTBI and HC groups. Spearman's rank correlation coefficient was calculated between growth inhibition and ICS data, indicated as r.