Abstract
BackgroundBCG is the most widely used vaccine of all time and remains the only licensed vaccine for use against tuberculosis in humans. BCG also protects other species such as cattle against tuberculosis, but due to its incompatibility with current tuberculin testing regimens remains unlicensed. BCG’s efficacy relates to its ability to persist in the host for weeks, months or even years after vaccination. It is unclear to what degree this ability to resist the host’s immune system is maintained by a dynamic interaction between the vaccine strain and its host as is the case for pathogenic mycobacteria.ResultsTo investigate this question, we constructed transposon mutant libraries in both BCG Pasteur and BCG Danish strains and inoculated them into bovine lymph nodes. Cattle are well suited to such an assay, as they are naturally susceptible to tuberculosis and are one of the few animal species for which a BCG vaccination program has been proposed. After three weeks, the BCG were recovered and the input and output libraries compared to identify mutants with in vivo fitness defects. Less than 10% of the mutated genes were identified as affecting in vivo fitness, they included genes encoding known mycobacterial virulence functions such as mycobactin synthesis, sugar transport, reductive sulphate assimilation, PDIM synthesis and cholesterol metabolism. Many other attenuating genes had not previously been recognised as having a virulence phenotype. To test these genes, we generated and characterised three knockout mutants that were predicted by transposon mutagenesis to be attenuating in vivo: pyruvate carboxylase, a hypothetical protein (BCG_1063), and a putative cyclopropane-fatty-acyl-phospholipid synthase. The knockout strains survived as well as wild type during in vitro culture and in bovine macrophages, yet demonstrated marked attenuation during passage in bovine lymph nodes confirming that they were indeed involved in persistence of BCG in the host.ConclusionThese data show that BCG is far from passive during its interaction with the host, rather it continues to employ its remaining virulence factors, to interact with the host’s innate immune system to allow it to persist, a property that is important for its protective efficacy.
Highlights
Mycobacterium bovis Bacillus Calmette–Guérin (BCG) is the most widely used vaccine of all time and remains the only licensed vaccine for use against tuberculosis in humans
Cattle are a particular relevant model, as they are naturally susceptible to tuberculosis and are one of the few species, other than humans, for which a BCG vaccination program has been proposed
Construction of the BCG transposon library The BCG Danish and BCG Pasteur libraries constructed by transduction with MycoMarT7 contained 1.36 × 105 cfu and 1.37 × 105 cfu respectively
Summary
BCG is the most widely used vaccine of all time and remains the only licensed vaccine for use against tuberculosis in humans. Mycobacterium bovis BCG is the only vaccine, with proven efficacy against all three major mycobacterial pathogens, M. tuberculosis [1], M. bovis [2, 3] and M. leprae [4] It is the most widely administered vaccine of all time [5] with 90% of world’s children receiving BCG and over 120 million doses used each year. In immuno-compromised mice and humans, BCG retains sufficient residual virulence to cause disseminated disease [14, 15]. This is apparent in HIV positive children [16, 17]. Is there a dynamic interplay with the host, in which its remaining virulence factors are used to resist innate killing mechanisms and to subvert the immune response in a manner similar to those observed with pathogenic mycobacteria, or are the bacilli more passive, remaining largely dormant in the host?
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