Intravenous BCG Vaccination of Diversity Outbred Mice Results in Moderately Enhanced Protection against Challenge with Mycobacterium tuberculosis Compared to Intradermal Vaccination

Abstract

Tuberculosis is still the leading cause of death globally from any infectious disease, despite the widespread use of the live attenuated vaccine Bacille Calmette Guerin (BCG). While BCG has some efficacy against disseminated TB disease in children, protection wanes into adulthood resulting in over 1.8 million TB deaths per year. This has led to efforts to develop novel vaccine candidates that either replace or boost BCG, as well as to test novel delivery mechanisms to enhance BCG's efficacy. Traditional BCG vaccination is performed as an intradermal (ID) injection but delivering BCG by an alternate route may enhance the depth and breadth of protection. Previously, we demonstrated that phenotypically and genotypically disparate Diversity Outbred (DO) mice have heterogenous responses to M. tuberculosis challenge following intradermal BCG vaccination. Here, we utilize DO mice to examine BCG-induced protection when BCG is delivered systemically via intravenous (IV) administration. We find that DO mice vaccinated with IV BCG had a greater distribution of BCG throughout their organs compared to ID-vaccinated animals. However, compared to ID-vaccinated mice, M. tuberculosis burdens in lungs and spleens were not significantly reduced in animals vaccinated with BCG IV, nor was lung inflammation significantly altered. Nonetheless, DO mice that received BCG IV had increased survival over those vaccinated by the traditional ID route. Thus, our results suggest that delivering BCG by the alternate IV route enhances protection as detected in this diverse small animal model.

Keywords: BCG; Mycobacterium tuberculosis; diversity outbred; intravenous; mycobacterium; route; tuberculosis; vaccine.

FIG 1

Delivering BCG intravenously increases the deposition of BCG in organs compared to parenteral vaccination. Groups of 10 DO mice were vaccinated intradermally (ID) or intravenously (IV) with 10⁵ BCG Pasteur. Animals were euthanized 8 weeks after vaccination for BCG enumeration in A) lungs, B) spleens, C) livers, and D) bone marrow. Bone marrow comprises eluate from both femurs of each mouse. Data from two independent experiments are combined and are represented as log₁₀ BCG burden from individual mice with bars representing the group mean. The limit of detection for CFU enumeration is denoted with a dotted line (L.O.D.). Results were analyzed Student's t test; **, P < 0.0001, *, P < 0.05.

FIG 2

Intravenous BCG provides enhanced M.tb. control compared to intradermal BCG. Groups of 15–20 female DO mice were vaccinated with 10⁵ BCG IV or ID, with heat-killed BCG (HK) or treated with PBS (naive). Mice were aerogenically challenged with ~50 CFU of M.tb. 8 weeks after vaccination and euthanized 7 weeks after challenge for M.tb. CFU enumeration. Data from four experiments of similar design and outcome were combined and presented as individual mice with bars depicting the mean log₁₀ M.tb. burden in A) lungs and B) spleens. The limit of detection for CFU enumeration is denoted with a dotted line (L.O.D.). Results were analyzed by one-way ANOVA with Tukey's posttest; *, P < 0.01.

FIG 3

Naïve and BCG-vaccinated DO mice challenged with M.tb. exhibit a range of lung inflammation outcomes. Groups of 15–20 female DO mice were vaccinated with 10⁵ BCG ID or IV, HK-BCG, or treated with PBS (naive). Mice were challenged by aerosol with ~50 CFU M.tb. Animals were euthanized 7 weeks after infection, and lung sections isolated and prepared for slides with H&E staining. Animals that became moribund before 7 weeks were euthanized and lung samples similarly isolated. H&E-stained slides were scanned, and images were analyzed to assess the percentage of lung tissue with disease involvement compared to the total lung area. Dots represent the percentage of each lung that was inflamed and/or had disease involvement in individual animals, and lines represent the median of the group. Data represent the combined results from four independent experiments of similar design. Differences between the groups indicated by the lines were significant by one-way ANOVA with Tukey's posttest; *, P < 0.01.

FIG 4

Representative images of lung sections prepared for H&E staining demonstrate qualitatively different disease pathologies. Lung tissues were collected from naive or BCG-vaccinated DO mice 7 weeks after M.tb. challenge. Tissues were formalin-fixed and processed for H&E staining. Slides were scanned at 20× on an Olympus VS200 slide scanner. Sections were scored by a veterinary pathologist who categorized the lesion distribution as focal (A), multifocal (B), coalesced (C, D), or diffuse (E, F). The pathologist also categorized the predominant cell type within lesions as monocytes (M) or monocytes/neutrophils (MN). Images presented are representative of the various types of lung pathology found throughout all groups of mice.

FIG 5

Weight gain, but not overall weight loss, is impacted by BCG vaccination. Groups of 15–20 DO mice were vaccinated with 10⁵ BCG ID or IV, HK-BCG, or treated with PBS (naive). Mice were aerogenically challenged with ~50 CFU of M.tb. 8 weeks after vaccination and followed for survival. Animals were weighed weekly throughout the experiment, and daily if they demonstrated signs of illness, such as ruffled fur or cachexia. Animals were euthanized when they met established humane endpoint criteria. Animal weights were used to determine several weight related traits. A) The weight at euthanasia was divided by peak body weight to generate the “percentage of peak body weight” at euthanasia. B) The peak body weight was divided by the initial body weight to determine “percentage weight gain at peak.”

FIG 6

Intravenous BCG extends survival of DO mice following M.tb. challenge. Groups of 15–20 DO mice were vaccinated with 10⁵ BCG ID or IV, HK-BCG, or treated with PBS (naive). Mice were aerogenically challenged with ~50 CFU of M.tb. 8 weeks after vaccination and followed for survival. Animals were euthanized when they met established humane endpoint criteria. DO IV-BCG mice survived longer than any of the other three vaccine groups; P < 0.05 by the Log-rank Mantel-Cox test.