Fig 1.
Depicting the timelines of vaccination (BCG priming at wk0 and AdHu5Ag85A boosting at wk14), M.tb infectious challenge (wk20) and fixed endpoint/autopsy (wk38). Scheduled times for clinical signs, blood work for acute phase protein measurement, chest X-ray and immune monitoring are indicated. Timelines during infection phase are referred relative to the 18 weeks post-challenge timepoint (indicated within parentheses).
Table 1.
Characteristics of treatment groups.
Table 2.
Clinical outcomes post-AdHu5Ag85A boost vaccination.
Fig 2.
Antigen-specific IFN-γ+ T cell responses post-BCG vaccination or post-BCG prime-Ad boost vaccination.
Antigen-specific IFN-γ+ T cell responses in non-boosted BCG-primed animals are measured using IFN- γ ELISOPT assay at 0wk, 13wk and 17wk post BCG vaccination by stimulating fresh peripheral blood mononuclear cells (PBMC) with PPD for 24h (A). Scatter dotplot depicting spot forming cells/million PBMC representing the mean responses±standard error. P-values are relative to the time point before BCG vaccination (0wk). (B/C/D) depicts antigen-specific IFN-γ responses for each group measured using IFN-γ ELISOPT assay before vaccination (wk0) and after BCG priming (BCG) or after BCG priming and AdHu5Ag85A boosting BCG/Ad(it), BCG/Ad(aerosol), (BCG/Ad(im). Fresh PBMCs are stimulated with PPD (B) or rAg85A (C) or single pool (D) and scatter dotplot depicting mean of spot forming cells/million PBMC ± standard error. P-values above lines indicate comparison between groups. Wilcoxon signed rank test was used to compare measurements at different time points of same animal.
Fig 3.
Survival rates and mycobacterial burden in the lung and spleen.
Animals were infected with 25 colony-forming units of M.tb Erdman strain. Group coloring in the survival graph is maintained throughout the article for comparability of data. One animal in BCG/Ad(im) group died accidently and is excluded from data analysis (A). Overall log-rank test for trend yielded p = 0.0247, but no statistical differences between groups. Colony-forming units (CFU) of mycobacteria was measured per gram of randomly selected tissue from different lung lobs or spleen at necropsy. CFU per right lung (B), left lung (C) and spleen (D) were presented in logarithmic numbers. P-values above lines indicate comparison between groups. Mann-Whitney rank test is used to compare differences relative to naïve group.
Fig 4.
Evaluation of gross pathology and histopathology in M.tb-infected lungs.
Individual dots represent scores for animals in each group with mean ± standard error for gross pathology (A). (B) Representative histomicrographs (x40) of lungs of animals in each group. Arrow points to granuloma with the central caseous necrosis in the lung of non-vaccinated animal (non-v), non-necrotic granuloma in the lung of BCG vaccinated animal (BCG), scattered small size granulomas lacking necrosis in the of lung of AdHu5Ag85A boosted animals (BCG/Ad(it), BCG(aerosol) and BCG(im)). (C) Individual dots represent scores for animals in each group with mean± standard error for histopathology. Mann-Whitney rank test is used to compare differences relative to naïve group.
Fig 5.
Longitudinal kinetics of radiological changes in the lung.
Chest X-ray (CXR) was acquired by using DIGI Eye 380 by placing anesthetized animals in dorsal recumbence position. X-ray images were scored using relative scoring system. (A) Lines represent individual animals in each group and color assigned to individual animals in each group is kept consistent throughout the article for comparability. Wilcoxon ranked test is used to compare CXR scores post-infection relative to values before infection (0), showing mean. Vertical arrow lines in the graphs indicate the evidence of CXR changes 4 weeks post-infection in non-vaccinated naïve and non-boosted BCG-primed animals, but not in AdHu5Ag85A-boosted animals. (B) Representative radiographs of CXR of animals in each group 4 week post-infection. (C) Scatter dotplot depicting the mean maximum CXR scores±standard error for animals in each group. Mann-Whitney rank test is used to compare CXR scores in vaccinated groups to naïve group.
Fig 6.
Longitudinal kinetics of clinical outcomes during the course of infection.
Clinical outcomes were monitored at scheduled times as indicated in the experiment plan and as described in Methods. Line graphs depict (A) percent of weight change relative to weight at the start of the experiment, (B) appetite, (C) change in erythrocyte sedimentation rate (ESR), and (D) C-reactive protein (CRP). Lines represent individual animals in each group and color assigned to individual animal in each group is maintained to display each clinical sign evaluated for comparability. Wilcoxon ranked test is used to compare clinical signs post infection relative to values before infection.
Fig 7.
Correlation of lung histopathology with microbiological and clinical outcomes.
Bacterial burden in the lung, chest radiography (CXR) and C-reactive protein (CPR) levels in blood correlate significantly with lung histopathology. Parameters are plotted (maximum values for each of the parameters) per individual animal (color coordination consistent throughout the article) against lung histology score for right lung (A), left lung bacterial burden (B), CXR (C), and CRP (D). Spearman’s rho (Rs) correlation factors and p-values are indicated.
Fig 8.
Correlation of vaccine-activated T cell responses with lung histopathology post-vaccination and post-infection.
Maximal PPD-specific PBMC IFN-γ responses post-vaccination and maximal rAg85A-specific IFN-γ responses post-infection show significant inverse correlations with histopathology in the lung. Maximal antigen-specific PBMC responses for PPD (A), rAg85A (B) and single pool (C) post-vaccination are plotted per individual animal against histopathological scores. Maximal antigen-specific PBMC responses for PPD (D), rAg85A (E) and single pool (F) post-infection are plotted per individual animal against histopathological scores. Spearman’s rho (Rs) correlation factors and p-values are indicated.