Host and pathogen genetic diversity shape vaccine-mediated protection to Mycobacterium tuberculosis

Abstract

To investigate how host and pathogen diversity govern immunity against Mycobacterium tuberculosis (Mtb), we performed a large-scale screen of vaccine-mediated protection against aerosol Mtb infection using three inbred mouse strains [C57BL/6 (B6), C3HeB/FeJ (C3H), Balb/c x 129/SvJ (C129F1)] and three Mtb strains (H37Rv, CDC1551, SA161) representing two lineages and distinct virulence properties. We compared three protective modalities, all of which involve inoculation with live mycobacteria: Bacillus Calmette-Guérin (BCG), the only approved TB vaccine, delivered either subcutaneously or intravenously, and concomitant Mtb infection (CoMtb), a model of pre-existing immunity in which a low-level Mtb infection is established in the cervical lymph node following intradermal inoculation. We examined lung bacterial burdens at early (Day 28) and late (Day 98) time points after aerosol Mtb challenge and histopathology at Day 98. We observed substantial heterogeneity in the reduction of bacterial load afforded by these modalities at Day 28 across the combinations and noted a strong positive correlation between bacterial burden in unvaccinated mice and the degree of protection afforded by vaccination. Although we observed variation in the degree of reduction in bacterial burdens across the nine mouse/bacterium strain combinations, virtually all protective modalities performed similarly for a given strain-strain combination. We also noted dramatic variation in histopathology changes driven by both host and bacterial genetic backgrounds. Vaccination improved pathology scores for all infections except CDC1551. However, the most dramatic impact of vaccination on lesion development occurred for the C3H-SA161 combination, where vaccination entirely abrogated the development of the large necrotic lesions that arise in unvaccinated mice. In conclusion, we find that substantial TB heterogeneity can be recapitulated by introducing variability in both host and bacterial genetics, resulting in changes in vaccine-mediated protection as measured both by bacterial burden as well as histopathology. These differences can be harnessed in future studies to identify immune correlates of vaccine efficacy.

Keywords: genetics; heterogeneity; protection; tuberculosis; vaccine.

Figure 1

Lung bacterial burdens following vaccination across strain combinations. Right lungs were excised from C57BL/6 (B6), C3HeB/FeJ (C3H), and 129/SvJxBalb/c (C129F1) mice administered BCG [subcutaneous (SC) or intravenous (IV)] or intradermal concomitant Mtb (CoMtb) for 8 weeks and then aerosol-challenged with H37Rv, SA161, or CDC1551 Mtb for either (A–C) 28 days or (D–F) 98 days. Lungs were homogenized and plated for CFU enumeration. (G) Cumulative CFU data are plotted as a log change over unvaccinated for each host-bacterial strain combination over time. Statistical comparisons were performed by non-parametric Wilcoxon ranked test, with *, p<0.05; **, p<0.01; ns, not significant.

Figure 2

The magnitude of vaccine-induced reduction in bacterial load correlates with initial burden in unvaccinated mice. The average log reduction in CFU for each mouse strain/protective modality/bacterial strain combination at Day 28 (A) and Day 98 (B) is plotted against the bacterial burden for each mouse strain/bacterial strain combination in unvaccinated mice at Day 28. Each point represents the average of 5 mice/condition. Outline colors represent mouse strain, fill colors represent bacterial strain, and shapes represent protective modality.

Figure 3

Early vaccine-mediated reductions in bacterial load do not predict late outcomes. The strain-strain combinations were ranked in order of highest vaccine-mediated reduction in bacterial load at (A) Day 28 post-infection. Keeping the same order, these combinations were then plotted to include the Day 98 log reductions in bacterial burden (B).

Figure 4

Mouse strains present a range of histopathological features upon infection with different Mtb strains. The left lung of mice was preserved in formalin at Day 98 post-Mtb infection and subjected to hematoxylin and eosin (H&E) staining to observe 14 histopathological features, which were graded in a blinded fashion by a licensed pathologist. (A–D) Representative sections from each strain of mouse is shown to illustrate the varying histological presentations. (E) Cumulative scores were used to generate a principal component analysis (PCA) encompassing all unvaccinated mice infected with each Mtb strain, with (F) different regions representing a predominance of certain pathological features.

Figure 5

Distinct strain-strain combinations result in unique vaccine-mediated histological changes. (A) The same PCA plot shown in Figure 4 is now displayed separately for each mouse strain and includes vaccinated cohorts, with color depicting bacterial strain and shape depicting protective modality. (B–E) Shown are representative Day 98 H&E images for select strain-strain combinations of interest, alongside their associated PCA plots. The arrows indicate the change in direction of samples within the PCA plot following vaccination.

Figure 6

Pathology shows minimal correlation with bacterial burden late during infection. The PCA plots of histology scores separated by mouse genotype, as shown in Figure 5 , were placed alongside the same plots now overlaid with the matched lung CFU value for each sample from Day 98, with dark colors representing lower bacterial CFU and bright colors representing higher bacterial CFU.

Figure 7

Late pathological features correlate more strongly with early than late bacterial burdens. Spearman correlation analysis was performed between histology scores for each pathology feature and lung bacterial burdens at (A) Day 28 and (B) Day 98 for each mouse genotype. For Day 98, the pathology scores and CFU values are paired, whereas for Day 28, we used the geometric mean of CFU from 5 mice per group to generate correlation values, since we did not have matched CFU values at this time point. Statistically significant correlations are shown in red (p<0.05).