MTBVAC vaccination protects rhesus macaques against aerosol challenge with M. tuberculosis and induces immune signatures analogous to those observed in clinical studies

Abstract

A single intradermal vaccination with MTBVAC given to adult rhesus macaques was well tolerated and conferred a significant improvement in outcome following aerosol exposure to M. tuberculosis compared to that provided by a single BCG vaccination. Vaccination with MTBVAC resulted in a significant reduction in M. tuberculosis infection-induced disease pathology measured using in vivo medical imaging, in gross pathology lesion counts and pathology scores recorded at necropsy, the frequency and severity of pulmonary granulomas and the frequency of recovery of viable M. tuberculosis from extrapulmonary tissues following challenge. The immune profiles induced following immunisation with MTBVAC reflect those identified in human clinical trials of MTBVAC. Evaluation of MTBVAC- and TB peptide-pool-specific T-cell cytokine production revealed a predominantly Th1 response from poly- (IFN-γ+TNF-α+IL2+) and multi-(IFN-γ+TNF-α+) functional CD4 T cells, while only low levels of Th22, Th17 and cytokine-producing CD8 T-cell populations were detected together with low-level, but significant, increases in CFP10-specific IFN-γ secreting cells. In this report, we describe concordance between immune profiles measured in clinical trials and a macaque pre-clinical study demonstrating significantly improved outcome after M. tuberculosis challenge as evidence to support the continued development of MTBVAC as an effective prophylactic vaccine for TB vaccination campaigns.

Conflict of interest statement

E.P., E.R. and C.M. are co-inventors on a patent on MTBVAC held by the University of Zaragoza and Biofabri.

Figures

Fig. 1. Study schedule and in vivo CT imaging.

a Diagram showing the week in which clinical examinations (open circle), blood sample collections (shaded circle), CT scan collection, the aerosol challenge with M. tuberculosis and necropsy (black circle) were conducted relative to vaccination. Disease burden development quantified from CT scans reflects scores derived for total disease burden (b), pulmonary disease burden (c), pneumonia (d) and the number of TB-induced nodules in the lung (e) from CT scans collected eat weeks 3, 8, 12 and 16 after challenge with M. tuberculosis. Box plots show group median values +/− IQR with minimum and maximum values indicated by box whiskers. f Incidence of lymph node disease across the 16-week study period. Non-parametric Mann–Whitney U tests were used for comparison between groups with unadjusted results reported as: *P ≤ 0.05; **P ≤ 0.005.

Fig. 2. Tuberculosis-induced disease burden.

a Kaplan–Meier plot showing the development of progressive disease to a level that met humane endpoint criteria in vaccinated and unvaccinated macaques after challenge with M. tuberculosis. Unadjusted P values from log-rank comparisons are shown. b Change in body weight expressed as a percentage of the peak weight measured during the post-challenge study period. c Total, pulmonary and disseminated (spleen, liver, kidneys) tuberculosis-induced disease burden measured using a gross pathology score system. d The number of macroscopic lesions in the lungs following serial sectioning. e Total number of granulomas (stage I–VI combined) identified in the lung from representative H&E-stained sections. f Total number of granulomas (stage I–VI combined) identified in the lung from representative H&E-stained sections where stacked bars indicate the number of granulomas at each stage I–VI and the combined total within each experimental group. g Total number of granulomas (stage I–VI combined) identified in the extrapulmonary tissues (spleen, liver, kidneys) from representative H&E-stained sections. h The proportion of tissue samples cultured from which M. tuberculosis was isolated (>LLOD: CFU value greater than the lower limit of detection of the assay; LLOD: value recorded as the lower limit of detection of the assay). i Bacterial burden determined in lung-associated lymph nodes (LALN) and extrapulmonary tissues. The colour and symbol coding per individual is consistent throughout and between figures. Upside down triangular symbols indicate animals in which disease progressed to meet humane endpoint criteria. Non-parametric Mann–Whitney U tests were used for group-wise comparisons of pathology scores, granuloma/lesion counts and viable CFU counts recovered from tissues, with unadjusted results reported as: *P ≤ 0.05; **P ≤ 0.01. Χ2 tests were used to compare the proportion of tissues with viable M. tuberculosis CFU counts above or below the LLOD of the assay between vaccination groups and Cochran–Armitage method Χ2 tests to compare granuloma stage scores between groups, unadjusted results are reported as *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Fig. 3. Immune response to vaccination and challenge.

The frequency of M. tuberculosis antigen-specific IFN-γ-secreting cells induced following BCG vaccination and challenge measured by ELISpot (a). Top row: PPD-specific response profiles, middle row: ESAT6-specific response profiles, bottom row: CFP10-specific response profiles. Left: response profiles in the MTBVAC-vaccinated group; middle: response profiles in the BCG-vaccinated group; right: response profiles in the unvaccinated group. Vaccination with MTBVAC, or BCG indicated by the dotted line, and aerosol exposure to M. tuberculosis indicated by the dashed line at week 21. Comparison of the CFP10-specific IFN-γ response measured by ELISpot induced following vaccination with BCG or MTBVAC determined by analysis of the area under the response curve between week 2 and 20 after vaccination with group comparison made using Mann–Whitney between groups **P ≤ 0.005 (b). All line graphs show individual response (circular symbol) and Group median response (solid line). The colour and symbol coding per individual is consistent throughout and between figures. Upside down triangular symbols indicate animals in which disease progressed to meet humane endpoint criteria. The assay threshold (mean of pre-vaccination values plus 1.5× standard deviation) is indicated by a horizontal dotted line (PPD: 119 spot forming units (SFU) per million cells; ESAT6: 40 SFU per million cells; CFP10: 28 SFU per million cells).

Fig. 4. M. tuberculosis peptide-specific CD4 T-cell cytokine secretion profiles measured by whole blood intracellular cytokine staining.

Box plots show the vaccination group median frequency of CD4 T-cells producing combinations of the cytokines IFN-γ, IL-2 and TNF-α (a–d); IL-17 (e) or IL-22 (f) +/− IQR with minimum and maximum values indicated by box whiskers. Cytokine production was measured at weeks prior to (−1), and following (4, 8, 12 and 18) BCG or MTBVAC vaccination. Significant differences between pre- and post-vaccination values (Wilcoxon signed-rank) and between groups (Mann–Whitney U test) are indicated by bars and asterisks: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 (all P values are unadjusted for multiple comparisons). Frequencies measured in individual animals are represented by dots.