A Modified Bacillus Calmette-Guérin (BCG) Vaccine with Reduced Activity of Antioxidants and Glutamine Synthetase Exhibits Enhanced Protection of Mice despite Diminished in Vivo Persistence

Abstract

Early attempts to improve BCG have focused on increasing the expression of prominent antigens and adding recombinant toxins or cytokines to influence antigen presentation. One such modified BCG vaccine candidate has been withdrawn from human clinical trials due to adverse effects. BCG was derived from virulent Mycobacterium bovis and retains much of its capacity for suppressing host immune responses. Accordingly, we have used a different strategy for improving BCG based on reducing its immune suppressive capacity. We made four modifications to BCG Tice to produce 4dBCG and compared it to the parent vaccine in C57Bl/6 mice. The modifications included elimination of the oxidative stress sigma factor SigH, elimination of the SecA2 secretion channel, and reductions in the activity of iron co-factored superoxide dismutase and glutamine synthetase. After IV inoculation of 4dBCG, 95% of vaccine bacilli were eradicated from the spleens of mice within 60 days whereas the titer of BCG Tice was not significantly reduced. Subcutaneous vaccination with 4dBCG produced greater protection than vaccination with BCG against dissemination of an aerosolized challenge of M. tuberculosis to the spleen at 8 weeks post-challenge. At this time, 4dBCG-vaccinated mice also exhibited altered lung histopathology compared to BCG-vaccinated mice and control mice with less well-developed lymphohistiocytic nodules in the lung parenchyma. At 26 weeks post-challenge, 4dBCG-vaccinated mice but not BCG-vaccinated mice had significantly fewer challenge bacilli in the lungs than control mice. In conclusion, despite reduced persistence in mice a modified BCG vaccine with diminished antioxidants and glutamine synthetase is superior to the parent vaccine in conferring protection against M. tuberculosis. The targeting of multiple immune suppressive factors produced by BCG is a promising strategy for simultaneously improving vaccine safety and effectiveness.

Keywords: Bacillus Calmette-Guérin (BCG); antioxidants; glutamine synthetase; immune suppression; immunity; sigma factor; superoxide dismutase; tuberculosis; vaccine.

Figure 1

Construction of 4dBCG. (A) Hexameric ring representing half of the enzymatically-active dodecameric form of GlnA1 (left) and enlargement (right) of the area within the rectangle to show the deleted amino acids in relationship to the active site manganese atom, represented by the black dot, with amino acids numbered according to the glutamine synthetase of Salmonella [30]. As the active site comprises residues from adjacent monomers, insertion of one ∆D50∆E327 dnGlnA1 monomer into the ring is predicted to inactivate two active sites. (B) SDS-PAGE (upper) and immunoblot (lower) for GlnA1 of lysates of Bacillus Calmette-Guérin (BCG), 3dBCG and 4dBCG. Lanes are labeled on the figures as L (lysate) and AS (ammonium sulfate-treated lysate) for each strain, kDaM = Kilodalton markers. (C) Graph of a representative experiment comparing the glutamine synthetase activity of undiluted and diluted AS preparations from 3dBCG and 4dBCG as determined by monitoring A₅₄₀ over time.

Figure 2

Spleen titers of BCG and 4dBCG in C57Bl/6 mice. Mean ± SEM spleen titers on day 2, day 30, and day 60 after IV inoculation of mice with 2 × 10⁷ cfu of BCG or 4dBCG. Each value represents four to six mice. The P value represents the comparison of the BCG and 4dBCG groups at 60 days. The day 0 value is estimated as 10% of the original IV inoculum.

Figure 3

Titer of M. tuberculosis challenge bacilli in the spleens and left lungs of C57Bl/6 mice. Mice received aerosol challenge with 100 cfu of M. tuberculosis strain Erdman S-1. The whole spleens and left lungs were assessed for cfu titer while the right lungs were processed for histopathology as shown in Figure 4, Figure 5, Figure 6 and Figure 7. The individual data points (circles) and median (bar) for each vaccination arm are shown at 8 weeks (top, six mice per vaccination arm) and 26 weeks (bottom, eight or nine mice per vaccination arm) post-challenge. The dotted lines extend the bar representing the median value of the PBS-vaccinated and BCG-vaccinated groups and are marked P* and P**, respectively. Analysis of spleen and lung results at 8 weeks and lung results at 26 weeks by 1 way ANOVA demonstrated that the median values of the groups varied significantly by Kruskal-Wallis test. The groups were also compared using the Mann-Whitney test and values in the row labeled P* above the panel indicate the comparison of each vaccinated group against the phosphate-buffered saline (PBS) group. The values in the row labeled P** indicate the comparison of the 4dBCG and BCG groups.

Figure 4

Low-power photomicrographs of sections of lung tissue. (Upper left panel, labeled “Slides”) Photographs of the microscope slides containing H&E-stained lung sections are shown from right lungs of three mice from each of the PBS, BCG, and 4dBCG vaccination arms at 8 weeks post-aerosol challenge with 100 cfu of M. tuberculosis strain Erdman S-1. Three rectangles are labeled a, b, and c and cover approximately 70–80% of the lung tissue on each slide. (Panels labeled “PBS”, “BCG”, and “4dBCG”) Three low-power photomicrographs of the lung tissue were taken through the 2× microscope objective and correspond to the three rectangles in each lung labeled a, b, and c in the “slides” panel. Within these photomicrographs, the black rectangles highlight lymphohistiocytic nodules in relatively consolidated regions of lung tissue, which are enlarged further and displayed in Figure 5. The white rectangles in the 4dBCG group of mice outline lung tissue containing bronchus-associated lymphoid tissue (BALT) and are displayed in Figure 7.

Figure 5

Lymphohistiocytic nodules in the lung parenchyma. Mid-power (×10 microscope objective) photomicrographs of regions of lung consolidation within the tissue sections in Figure 4 are displayed at a greater magnification. Most of the areas that stain dark blue in the PBS and BCG groups represent dense collections of lymphocytes within the lung parenchyma that are accompanied by lymphohistiocytic infiltration and consolidation of the alveolar spaces. Examples of these regions are indicated with arrows (black arrowheads) and the white rectangles outline regions of lung tissue that are enlarged further in Figure 6. Lymphoid aggregates were also observed in the lung parenchyma in the 4dBCG group of mice however they were typically smaller with less dense alveolar infiltrate compared to the PBS and BCG groups. The arrows with white arrowheads indicate BALT, which is distinguished from lymphohistiocytic nodules in the lung parenchyma by their association with blood vessels and bronchi.

Figure 6

High-power views of lymphohistiocytic nodules. Consolidated lung tissue with many epithelioid cells containing pale oval nuclei and eosinophilic cytoplasm are observed in the PBS-vaccinated mice and to a lesser degree in the BCG-vaccinated mice. The alveolar spaces adjacent to the smaller lymphoid nodules in the 4dBCG-vaccinated mice are edematous, contain relatively fewer epithelioid cells, and exhibit greater preservation of alveolar wall architecture than in the PBS- and BCG-vaccinated mice.

Figure 7

Bronchus-associated lymphoid tissue (BALT). The panels display ×10 enlargements of the lung sections from mice vaccinated with 4dBCG as represented by the white rectangles in Figure 4. BALT was prominent along the central bronchovascular structures in all three groups of mice (see Figure 5) and its association with blood vessels and bronchi distinguishes BALT from collections of lymphocytes in the lung parenchyma.

Figure 8

Inverse correlation between protection effectiveness in man and spleen titer in mice of three BCG daughter strains. The protection effectiveness values were taken from a cohort study in Kazakhstan [80] and involved BCG Russia (Microgen), Pasteur 1173P2 (Serbian “Torlak” vaccine), and BCG Tokyo 172 (Japan BCG Laboratory). The log10 spleen titer values were taken from a comparison of 5 BCG strains in Balb/C mice [37] and represent the values at 12 weeks after intravenous inoculation of mice with 10⁶ cfu of the vaccine strain.