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. 2019 Nov 28;9(1):17791.
doi: 10.1038/s41598-019-54108-y.

Development of a diagnostic compatible BCG vaccine against Bovine tuberculosis

Aneesh Chandran  1 , Kerstin Williams  1 , Tom Mendum  1 , Graham Stewart  1 , Simon Clark  2 , Sirine Zadi  2 , Faye Lanni  2 , Neil McLeod  2 , Ann Williams  2 , Bernardo Villarreal-Ramos  3 , Martin Vordermeier  3 , Veerasamy Maroudam  4 , Aravind Prasad  5 , Neeraj Bharti  5 , Ruma Banerjee  5 , Sunitha Manjari Kasibhatla  5 , Johnjoe McFadden  6
Affiliations

Development of a diagnostic compatible BCG vaccine against Bovine tuberculosis

Aneesh Chandran et al. Sci Rep. .

Erratum in

Abstract

Bovine tuberculosis (BTB) caused by Mycobacterium bovis remains a major problem in both the developed and developing countries. Control of BTB in the UK is carried out by test and slaughter of infected animals, based primarily on the tuberculin skin test (PPD). Vaccination with the attenuated strain of the M. bovis pathogen, BCG, is not used to control bovine tuberculosis in cattle at present, due to its variable efficacy and because it interferes with the PPD test. Diagnostic tests capable of Differentiating Infected from Vaccinated Animals (DIVA) have been developed that detect immune responses to M. bovis antigens absent in BCG; but these are too expensive and insufficiently sensitive to be used for BTB control worldwide. To address these problems we aimed to generate a synergistic vaccine and diagnostic approach that would permit the vaccination of cattle without interfering with the conventional PPD-based surveillance. The approach was to widen the pool of M. bovis antigens that could be used as DIVA targets, by identifying antigenic proteins that could be deleted from BCG without affecting the persistence and protective efficacy of the vaccine in cattle. Using transposon mutagenesis we identified genes that were essential and those that were non-essential for persistence in bovine lymph nodes. We then inactivated selected immunogenic, but non-essential genes in BCG Danish to create a diagnostic-compatible triple knock-out ΔBCG TK strain. The protective efficacy of the ΔBCG TK was tested in guinea pigs experimentally infected with M. bovis by aerosol and found to be equivalent to wild-type BCG. A complementary diagnostic skin test was developed with the antigenic proteins encoded by the deleted genes which did not cross-react in vaccinated or in uninfected guinea pigs. This study demonstrates the functionality of a new and improved BCG strain which retains its protective efficacy but is diagnostically compatible with a novel DIVA skin test that could be implemented in control programmes.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
(a) Bean plot of fold changes during in vivo passage in cattle for selected gene groups and (b) Schematic representation of creating △BCG TK. (a) Black lines show the medians; white lines represent individual antigenic genes; polygons represent the estimated density of the data. The grey regions is a density plot of the data’s distribution. Values are normalised to the median value of the ‘All gene’ group. The plots were created with BoxPlotR (Spitzer, Wildenhain et al. 2014) (b) △BCG TK was created by sequential gene knock out using specialized Transduction method (Bardov et al. 2002). The Phagemid used was Phae159 and the cosmids used were p0004S, pYUB854 and pANEE001 to create △BCG 3043, ΔBCG3043/BCG2897/BCG2895 (Double knock out) and ΔBCG3043/2897/2895/3679/3680 (Triple knock out) respectively.
Figure 2
Figure 2
Competitive survival of selected mutants in vitro and ex vivo conditions. Competitive in vitro survival of selected mutants in (a) media and (b) bovine macrophages. Approximately equal number of WT BCG Danish and BCG knockout were mixed and used to inoculate media, infect PBMC derived bovine macrophages. Inoculants and recovered BCG were enumerated on selective media. Error bars represent standard errors.
Figure 3
Figure 3
Comparative analysis of body weight and survival ability of ΔBCG TK vaccinated Guinea pigs after M. bovis challenge. (a) Schematic representation of study schedule (b) Group mean body weight profiles recorded for each group of guinea pigs during the vaccination and challenge phase of the study. Solid red vertical bar on x-axis indicates the time point of vaccination and dashed-black vertical bar on x-axis indicates time point of challenge with M. bovis. (c) Survival data of unvaccinated and vaccinated (WT BCG, ΔBCG TK) guinea pigs up on M. bovis infection.
Figure 4
Figure 4
Protective efficacy of ΔBCG TK vaccination in Guinea pigs upon M. bovis challenge. (a) The bacterial load in the lungs of WT BCG (P = 0.0021) and ΔBCG TK (P = 0.0006) vaccinated guinea pigs is significantly low compared to the with the unvaccinated control group. (b) The bacterial load in spleen of WT BCG (P = 0.0015) and ΔBCG TK (P = 0.0006) vaccinated guinea pigs is significantly low compared to the unvaccinated control group. Values for each individual are shown and the horizontal bar denotes the mean for each group. *P = ≤ 0.05, P = ≤ 0.01, ***P = ≤ 0.001.
Figure 5
Figure 5
Pre and post-challenge skin test reaction in ΔBCG TK vaccinated guinea pigs. (A) Diagram to show the injection layout on animal. At least 2.5 cm between each inoculation of antigen preparations were injected on the same flank in a Latin Square arrangement. The site of injections on both sides of guinea pig’s trunk was marked “a, b, c” and “d, e, f”. (B,C) Pre-challenge skin test: The group mean size of the diameter of erythema at 24 (B) and at 48 hours (C) after injection. (D,E) Post-challenge skin test: the mean size of the diameter of erythema at 24 (D) and 48 (E) hours after injection. The red line denotes the minimum skin test response (STR) threshold (2 mm) for DIVA antigens to consider it as positive. The x-axis denotes the vaccine group. The error bars indicate standard deviation for each vaccine group.
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