Tools for Assessing the Protective Efficacy of TB Vaccines in Humans: in vitro Mycobacterial Growth Inhibition Predicts Outcome of in vivo Mycobacterial Infection

Abstract

Tuberculosis (TB) remains a leading global cause of morbidity and mortality and an effective new vaccine is urgently needed. A major barrier to the rational development of novel TB vaccines is the lack of a validated immune correlate or biomarker of protection. Mycobacterial Growth Inhibition Assays (MGIAs) provide an unbiased measure of ability to control mycobacterial growth in vitro, and may represent a functional correlate of protection. However, the biological relevance of any potential correlate can only be assessed by determining the association with in vivo protection from either a controlled mycobacterial infection or natural development of TB disease. Our data demonstrate that the direct MGIA using peripheral blood mononuclear cells (PBMC) is measuring a biologically relevant response that correlates with protection from in vivo human BCG infection across two independent cohorts. This is the first report of an MGIA correlating with in vivo protection in the species-of-interest, humans, and furthermore on a per-individual as well as per-group basis. Control of mycobacterial growth in the MGIA is associated with a range of immune parameters measured post-BCG infection in vivo including the IFN-γ ELISpot response, frequency of PPD-specific IFN-γ or TNF-α producing CD4+ T cells and frequency of specific sub-populations of polyfunctional CD4+ T cells. Distinct transcriptomic profiles are associated with good vs. poor mycobacterial control in the MGIA, with good controllers showing enrichment for gene sets associated with antigen processing/presentation and the IL-23 pathway, and poor controllers showing enrichment for hypoxia-related pathways. This study represents an important step toward biologically validating the direct PBMC MGIA for use in TB vaccine development and furthermore demonstrates the utility of this assay in determining relevant immune mechanisms and pathways of protection.

Trial registration: ClinicalTrials.gov NCT01194180.

Keywords: BCG; MGIA; Tuberculosis; mycobacterial growth inhibition assay; vaccine.

Copyright © 2020 Tanner, Satti, Harris, O'Shea, Cizmeci, O'Connor, Chomka, Matsumiya, Wittenberg, Minassian, Meyer, Fletcher and McShane.

Figures

Figure 1

Mycobacterial growth in the MGIA is inhibited in BCG-vaccinated compared with naïve volunteers and correlates with BCG recovered from biopsy following in vivo BCG infection (Study 1). Samples were taken from Study 1. n = 24 healthy human volunteers, half of whom were BCG-naïve and half of whom were historically BCG-vaccinated, were infected with intradermal BCG. The direct PBMC MGIA was conducted on cells and serum taken at the day of infection and the ratio of mycobacterial growth at the end of the 96 h culture relative to an inoculum control was determined (A). As previously reported, the BCG load was quantified from skin biopsy specimens at the site of challenge 2 weeks later using PCR (B). The association between mycobacterial growth in the MGIA and BCG recovered from biopsies in the BCG vaccinated group by both PCR (C) and culture CFU (D) was determined. Bars represent the median values with interquartile range (IQR). For (A,B) a Mann-Whitney U test was performed, where *p < 0.05 and ****p < 0.0001. For (C,D) a Spearman's correlation was performed. MGIA growth ratio = log10(CFU of sample/CFU of control).

Figure 2

Mycobacterial growth in the MGIA is inhibited in BCG-vaccinated compared with naïve volunteers and correlates with BCG recovered from biopsy following in vivo BCG infection (Study 2). Samples were taken from Study 2. n = 48 healthy human volunteers were assigned to groups A and B (BCG-naïve) or groups C and D (historically BCG vaccinated). Groups B and D received the candidate TB vaccine MVA85A. All volunteers were then infected with intradermal BCG. The direct PBMC MGIA was conducted on cells and plasma taken at the day of infection and the ratio of mycobacterial growth at the end of the 96 h culture relative to an inoculum control was determined (A). As previously reported, the BCG load was quantified from skin biopsy specimens at the site of challenge 2 weeks later using PCR (B). The association between mycobacterial growth in the MGIA and BCG recovered from biopsies was determined for all groups combined (C) and for the BCG-vaccinated group (group C) only (D). Bars represent the median values with IQR. For (A) a Kruskal Wallis test with Dunn's multiple comparisons was performed and for (B) a one-way ANOVA with Tukey's multiple comparisons test was performed, where *p < 0.05, **p < 0.005, and ***p < 0.0005. For (C,D) a Spearman's correlation was performed. MGIA growth ratio = log10(CFU of sample/CFU of control).

Figure 3

Frequency of cytokine-producing specific CD4+ T cells is associated with improved control of mycobacterial growth in the MGIA. Samples were taken from Study 2. n = 48 healthy human volunteers were assigned to groups A and B (BCG-naïve) or groups C and D (historically BCG vaccinated). Groups B and D received the candidate TB vaccine MVA85A. All volunteers were then infected with intradermal BCG, and frequency of PPD-specific IFN-γ producing CD4+ T cells were measured at 2 weeks post-infection using whole blood ICS (A). The direct PBMC MGIA was conducted on cells and serum taken at the day of challenge and the ratio of mycobacterial growth at the end of the 96 h culture relative to an inoculum control was determined. The association between mycobacterial growth in the MGIA and the frequency of PPD-specific IFN-γ producing CD4+ T cells was determined for all groups combined (B) and for the BCG-vaccinated group (group C) only (C). Bars represent the median values with IQR. For (A), a one-way ANOVA with Tukey's multiple comparisons was performed where *p < 0.05, ***p < 0.0005, and ****p < 0.0001. For (B,C) a Spearman's correlation was performed. MGIA growth ratio = log10(CFU of sample/CFU of control).

Figure 4

Frequency of sub-populations of cytokine-producing specific CD4+ T cells is associated with improved control of mycobacterial growth in the MGIA. Samples were taken from Study 2. n = 48 healthy human volunteers were assigned to groups A and B (BCG-naïve) or groups C and D (historically BCG vaccinated). Groups B and D received the candidate TB vaccine MVA85A. All volunteers were then infected with intradermal BCG, and the frequency of PPD-specific CD4+ T cells producing each of the possible expression profile permutations of the cytokines IFN-γ, TNF-α, IL-2, and IL-17 was determined by flow cytometry using whole blood samples taken at 2 weeks post-infection. The direct PBMC MGIA was conducted on cells and plasma taken at the day of challenge and the ratio of mycobacterial growth at the end of the 96 h culture relative to an inoculum control was determined. The association between mycobacterial growth in the MGIA at day of challenge and frequency of CD4+ T cells producing each profile was determined for all groups combined (A) or for the BCG-vaccinated group (group C) only (B) using Spearman's rank correlation, where light blue shading indicates a p < 0.05 and darker blue shading indicates a p < 0.01. Gray bars indicate the mean frequency of each sub-population of cells with the SEM.

Figure 5

Specific IgG ELISA responses are induced by BCG vaccination and are associated with improved control of mycobacterial growth in the MGIA. Samples were taken from Study 2. n = 48 healthy human volunteers were assigned to groups A and B (BCG-naïve) or groups C and D (historically BCG vaccinated). Groups B and D received the candidate TB vaccine MVA85A. All volunteers were then infected with intradermal BCG, and BCG-specific IgG responses (A) were measured at 2 weeks post-infection. The direct PBMC MGIA was conducted on cells and plasma taken at the day of challenge and the ratio of mycobacterial growth at the end of the 96 h culture relative to an inoculum control was determined. The association between mycobacterial growth in the MGIA and the BCG-specific IgG ELISA response was determined for all groups combined (B) and for the BCG-vaccinated group (group C) only (C). Bars represent the median values with IQR. For (A) a one-way ANOVA with Tukey's multiple comparisons was performed where **p < 0.005 and ***p < 0.0005. For (B,C) a Spearman's correlation was performed. MGIA growth ratio = log10(CFU of sample/CFU of control).

Figure 6

Distinct transcriptomic profiles are associated with good vs. poor mycobacterial control in the MGIA. Samples were taken from 10 volunteers assigned to group C (historically BCG vaccinated) in Study 2. Volunteers were infected with intradermal BCG and gene expression microarray analysis was conducted on PBMC taken at 2 weeks post-infection. The direct PBMC MGIA was conducted on cells and plasma taken at the day of challenge and the ratio of mycobacterial growth at the end of the 96 h culture relative to an inoculum control was determined. Volunteers were classified as ‘good' or “poor” controllers defined as having MGIA mycobacterial growth values below or above the group median, respectively. Gene expression was analyzed using a 2 × 2 factorial design with the interaction term (BCG stimulated − unstimulated PBMC for good MGIA controllers) vs. (BCG stimulated − unstimulated PBMC for poor MGIA controllers). Differentially expressed genes were defined as those with a p < 0.01 and log2 FC > 0.5 and are presented as a heatmap (A). GSEA analysis was conducted to identify biological pathways that were enriched in “good” vs. “poor” controllers and vice versa using an FDR cut-off of < 0.05. Representative top GSEA pathways for each category are shown from the top 50 gene sets ranked by normalized enrichment score (nES) (B).