Multiple genetic loci influence vaccine-induced protection against Mycobacterium tuberculosis in genetically diverse mice

Abstract

Mycobacterium tuberculosis (M.tb.) infection leads to over 1.5 million deaths annually, despite widespread vaccination with BCG at birth. Causes for the ongoing tuberculosis endemic are complex and include the failure of BCG to protect many against progressive pulmonary disease. Host genetics is one of the known factors implicated in susceptibility to primary tuberculosis, but less is known about the role that host genetics plays in controlling host responses to vaccination against M.tb. Here, we addressed this gap by utilizing Diversity Outbred (DO) mice as a small animal model to query genetic drivers of vaccine-induced protection against M.tb. DO mice are a highly genetically and phenotypically diverse outbred population that is well suited for fine genetic mapping. Similar to outcomes in people, our previous studies demonstrated that DO mice have a wide range of disease outcomes following BCG vaccination and M.tb. challenge. In the current study, we used a large population of BCG-vaccinated/M.tb.-challenged mice to perform quantitative trait loci mapping of complex infection traits; these included lung and spleen M.tb. burdens, as well as lung cytokines measured at necropsy. We found sixteen chromosomal loci associated with complex infection traits and cytokine production. QTL associated with bacterial burdens included a region encoding major histocompatibility antigens that are known to affect susceptibility to tuberculosis, supporting validity of the approach. Most of the other QTL represent novel associations with immune responses to M.tb. and novel pathways of cytokine regulation. Most importantly, we discovered that protection induced by BCG is a multigenic trait, in which genetic loci harboring functionally-distinct candidate genes influence different aspects of immune responses that are crucial collectively for successful protection. These data provide exciting new avenues to explore and exploit in developing new vaccines against M.tb.

Fig 1. BCG vaccination followed by M.tb. challenge of DO mice leads to heterogenous infection outcomes by 14 weeks.

~ 871 DO mice were vaccinated with 10⁵ BCG Pasteur intradermally and challenged 8 weeks after vaccination with ~ 50 CFU M.tb. Erdman, and M.tb. burdens in organs assessed at 14 weeks after challenged (BCG). ~ 135 sham-PBS vaccinated DO mice were included as controls for M.tb. infection (naïve). At necropsy, a lung lobe was collected from each mouse, formalin-fixed, H&E stained, and analyzed by densitometry to assess the proportion of inflamed lung tissue. (A) Survival of M.tb.-challenged naïve and BCG-vaccinated DO mice through 14 weeks after challenge (*p < 0.0001; Kaplan-Meier). (B) Lung and spleen M.tb. burdens for naïve and BCG-vaccinated DO mice. (C) The ratio of the lung CFU/spleen CFU which was determined for each of the naïve-M.tb. challenged or BCG-vaccinated/M.tb.-challenged DO mice. (D) The percent of inflamed lung tissue for each mouse from naïve or BCG-vaccinated mice. Groups were compared by t-test, *p < 0.0001. (E) The lung and spleen CFU and percent lung inflammation are represented for each BCG-vaccinated/M.tb.-challenged mouse, with the percentage of lung containing inflammation depicted by color scale. (F) Body weight was tracked over time for all naïve and BCG-vaccinated mice that were challenged with M.tb. For each animal, the percent of weight loss from the peak body weight was calculated; individual animals are represented by dots. For (B), (C), (D), and (F) data are depicted as violin plots, with individual mice represented as dots. Median value for each group is represented by a solid line, with quartiles represented by dashed lines. Groups were compared by Student’s t test, *p < 0.05.

Fig 2. Lung homogenates from BCG-vaccinated/M.tb.-challenged DO mice contain variable amounts of cytokines at 14 weeks after challenge.

Lung homogenates from a subset of 300 BCG-vaccinated/M.tb.-challenged mice were analyzed for the presence of a panel of 37 chemokines and cytokines by multiplex assay or sandwich ELISA (S1 Table). Data are presented for three of the cytokines, (A) IFN-γ, (B) IL-1α, and (C) Esm1. Data are represented by violin plots, where individual mice are represented as dots, the median value for each group by a solid red line, and quartiles by dashed red lines.

Fig 3. Pearson’s correlations reveal associations between complex disease traits and lung cytokine content.

Data from all complex and cytokine traits for BCG-vaccinated/M.tb. challenged DO mice collected at 14 weeks after challenge of vaccinated mice were used to determine Pearson’s correlations between each trait. Data were analyzed using R studio, ggplot package. Color scale represents the strength of the Pearson’s r coefficient from -1 (red; negative correlation) to 1 (blue, positive correlation). Asterisks represent those with significant associations; p-values are represented by *p < 0.05, ** p < 0.01, ***p < 0.001.

Fig 4. QTL mapping reveals novel QTL associated with complex outcomes following BCG vaccination and M.tb. challenge.

Genome wide QTL scans were performed for the complex traits A) Lung CFU, B) Spleen CFU, and C) the Lung/Spleen CFU ratio. QTL plots represent the region of the chromosome (x-axis) and the LOD score, interpreted as the strength of the association between the trait and a particular region of the chromosome (y-axis). Dashed and dotted lines indicate P value thresholds of 0.05 and 0.2, respectively.

Fig 5. Genotype analyses infer that the Vip15 QTL driving control of lung CFU is biallelic and dominant.

Lung CFU data for each animal in the mapping study were Z-scale transformed, and founder alleles were determined for each mouse at chromosome 17 position 44.3093, the QTL peak. Alleles contributed by each of the eight founder strains are designated A: AJ, B: C57BL/6, C: 129, D: NOD, E: NZO, F: CAST, G: PWK, H: WSB. A) Individual mice are represented by dots, plotted according to lung CFU and the founder allele genotype at the QTL peak. B) Animals were separated in two populations that were either homozygous for the low allele (BB or CC) or the high alleles (Other Homozygotes: AA/DD/EE/FF/GG/HH). The two groups were compared by Welch’s two sample t-test and were found to be significantly different, * p < 0.05. C) Animals were separated into three populations based on whether they possessed a high (H) allele (A/D/E/F/G/H) or low allele (L) (B/C) at the peak position. Populations representing mice homozygous for a high allele (HH), homozygous for a low allele (LL), or heterozygous with a mix of high and low alleles (HL) were compared for differences in variance by an F-test. Variance was significantly different, and therefore groups were further compared in a pairwise fashion by Welch’s two sample t-tests, * p < 0.05, or NS = p > 0.05. Animals that were homozygous for a given founder allele are represented by red dots; heterozygous animals by black dots.

Fig 6. QTL mapping reveals novel QTL associated with lung cytokines following BCG vaccination and M.tb. challenge.

Data from 37 cytokines and chemokines derived from 300 BCG-vaccinated/M.tb.-challenged DO mice were used to perform QTL mapping. Nine cytokines had significant or suggestive QTL, of which three are presented here: A) RANTES/CCL5, B) MIP-2, C) CXCL1. A threshold of p < 0.05 for significant (dashed line) or p < 0.2 for suggestive (dotted line) traits was set for the analyses.

Fig 7. Founder allele effects for each locus demonstrate the relative genetic contributions of each founder strain to the QTL.

Allele effect plots were generated using the plotting functions of the qtl2 package. Each colored line represents the allelic contribution of a given founder strain as depicted in the legend. The allele effects were determined for A) Lung CFU/Vip15, B) Spleen CFU/Vip15, C) Spleen CFU/Vip13, D) Mip-2/Vip16, E) KC/CXCL1/Vip12, F) IL-1α/Vip12, and G) RANTES/CCL5/Vip11.

Fig 8. Individual single nucleotide polymorphisms (SNP) are associated with complex infection and cytokine traits.

Allele probabilities were converted to SNP probabilities over selected intervals of interest and scanned for trait association. SNPs within 1.5 LOD of the maximum are plotted in pink, with SNPs below this threshold plotted in blue. Annotated coding regions for genes are listed across the chromosomal region of interest, and the chromosomal position (Mbp). SNPs are presented for A) the overlapping region for Vip12, and for Vip15 the SNPs associated with B) Lung CFU and C) Spleen CFU.