R. G. Hewinson

Vaccination of neonatal calves with Mycobacterium bovis BCG induces protection against intranasal challenge with virulent M. bovis

Vaccination of neonates with Mycobacterium bovis bacillus Calmette–Guérin (BCG) may be a strategy that overcomes reduced vaccine efficacy associated with exposure to environmental mycobacteria in humans and cattle. Preliminary comparisons indicated that 2‐week‐old calves produced an immune response to vaccination at least as intense as that observed in adults. Subsequently, five gnotobiotic hysterotomy derived calves aged 1u2003day were inoculated with BCG and 3u2003months later were challenged intranasally with virulent M. bovis. The number of tissues with lesions and the pathological extent of these lesions was reduced significantly in vaccinates. Furthermore, lesions were evident in the lung or associated chest lymph nodes of four of five controls but none of five vaccinates. BCG vaccination reduced significantly the level of bacterial colonization. However, lesions in the head associated lymph nodes were observed in three of five BCG‐vaccinated cattle. Levels of interferon gamma (IFN‐γ) detected by enzyme‐linked immunosorbent assay (ELISA) or enzyme‐linked immunospot (ELISPOT) in individual vaccinated animals at challenge did not correlate with subsequent resistance and in general immune responses post‐challenge were lower in vaccinated calves. Low IL‐10 responses were evident but IL‐4 was not detected. Responses to ESAT‐6 and/or CFP‐10 were evident in four of four control calves that had lesions. Two of the BCG vaccinates with lesions did not produce a response to ESAT‐6 and CFP‐10, indicating that these antigens did not distinguish vaccinated immune animals from vaccinated animals with lesions. Overall, vaccination of neonatal calves with BCG induced significant protection against disease and has potential as a strategy for the reduction of the incidence of bovine tuberculosis.

Exposure to Mycobacterium avium induces low-level protection from Mycobacterium bovis infection but compromises diagnosis of disease in cattle

We assessed the effect of exposure to Mycobacterium avium on the development of immune responses and the pathogenesis of disease observed following Mycobacterium bovis challenge. A degree of protection against M. bovis was observed in calves which were pre‐exposed to M. avium as assessed by the extent of lesions and bacterial load compared to the M. bovis alone group. The immune response following M. bovis challenge in cattle previously inoculated with M. avium was biased towards antigens (PPD) present in M. avium, whereas the response following M. bovis alone was biased towards antigens present in M. bovis, indicating an imprinting of memory to avian antigens on T lymphocytes. A consequence of the memory to M. avium antigens was failure to diagnose M. bovis infection by the skin test or the IFNγ assay in some of the animals which had lesions of tuberculosis at necropsy. The use of M. bovis specific antigens ESAT‐6 and CFP‐10 increased IFNγ test specificity in animals previously exposed to M. avium but the responses to these antigens were lower than those observed in animals exposed to M. bovis alone. The implication is that responses to M. avium, although providing some immunity, may mask diagnosis of M. bovis infection, even when specific antigens are employed, potentially contributing to disease transmission in the field.

Immunogenicity of Mycobacterium tuberculosis RD1 region gene products in infected cattle

Current immuno‐diagnostic tests for the detection of Mycobacterium bovis infection in cattle rely on the use of tuberculin PPD as antigens. However, the use of a cattle vaccine is effectively prohibited because BCG, the only potentially available cattle TB vaccine, compromises the current tuberculin test. The main objective of this study was to identify specific antigens, which could increase the test sensitivity to levels achieved with tuberculin. Our approach utilized the availability of the genome sequences of Mycobactereium tuberculosis and BCG by applying principles of comparative genomics to the identification of species‐specific antigens. Eight open‐reading frames (Rv3871 to Rv3878) encoding for putative antigens in the RD1 region of the M. tuberculosis genome, which is deleted in all strains of BCG, were selected and screened in the form of pools of synthetic peptides for immunological reactivity (antigen induced proliferation and IFN‐γ secretion) with peripheral blood mononuclear cells from cattle experimentally infected with M. bovis. Our results confirm the immunodominant role of two RD1 region products, CFP‐10 (Rv3874) and ESAT‐6 (Rv3875). In addition, we were able to identify 3 more antigens (Rv3871, Rv3872 and Rv3873), which induced immunological reactivity in PBMC from more than 50%M. bovis of infected cattle.

Intranasal BCG vaccination protects BALB/c mice against virulent Mycobacterium bovis and accelerates production of IFN-γ in their lungs

Local immune reactivity in the lungs of BALB/c mice was studied following (i) intranasal (i.n.) vaccination with Mycobacterium bovis BCG, (ii) intravenous (i.v.) challenge with a virulent M. bovis field isolate and (iii) i.n. vaccination with M. bovis BCG followed by i.v. challenge with an M. bovis field isolate. The results demonstrated that i.n. vaccination with BCG induced a high degree of protection against systemic M. bovis challenge, and that this protection correlated with a rapid production of IFN‐γ after M. bovis challenge by lung T cells from vaccinated mice.

Effective DNA vaccination of cattle with the mycobacterial antigens MPB83 and MPB70 does not compromise the specificity of the comparative intradermal tuberculin skin test

The current tuberculin test and slaughter strategy for the control of bovine tuberculosis in cattle has failed to prevent a sharp rise in cases over recent years, especially in the south-west of England. A recent scientific review has concluded that the development of a cattle vaccine holds the best prospect for tuberculosis control in British herds. In order to continue with test and slaughter-based control strategies, the development of TB vaccines that do not compromise the specificity of the tuberculin skin test are required. This report describes results of cattle vaccination experiments with TB DNA vaccines expressing the mycobacterial antigens MPB70, MPB83, and Ag85A and constitutes the first published vaccination study with DNA vaccines undertaken in a target host species. All calves vaccinated with the MPB83 expressing plasmid demonstrated potent cellular immune responses, characterised by CD4(+) T cells producing interferon-gamma as well as humoral immunity characterised by IgG1 biased specific antibodies. Vaccination with MPB70 was less effective with immune responses only observed in half of the vaccinated animals, while vaccination with Ag85A did not result in vaccine-induced immune responses. Intramuscular vaccination was found to stimulate stronger cellular responses than intradermal immunisation. Significantly, the specificity of tuberculin skin testing was not compromised by DNA vaccination since none of the vaccinated calves showed positive skin test reactivity.

Duration of Immunity against Mycobacterium bovis following Neonatal Vaccination with Bacillus Calmette-Guérin Danish: Significant Protection against Infection at 12, but Not 24, Months

ABSTRACT Vaccination of neonatal calves with Mycobacterium bovis bacillus Calmette-Guérin (BCG) induces a significant degree of protection against bovine tuberculosis, caused by infection with virulent M. bovis. In two independent experiments, we assessed the duration of the protective immunity induced in calves by neonatal vaccination with BCG Danish. Protection from disease was assessed at 12 and 24 months postvaccination in cattle challenged via the endotracheal route with M. bovis. We also assessed antigen-specific immune responses to assess their utility as correlates of protection. At 12 months postvaccination, significant reductions in lung and lymph node pathologies were observed compared to nonvaccinated M. bovis-challenged control cattle. At 24 months post-BCG vaccination, there was a reduction in lung and lymph node pathology scores and in bacterial burden. However, when comparing vaccinated and control groups, this did not reach statistical significance. Vaccination induced long-lived antigen (purified protein derivative [PPD])-specific gamma interferon (IFN-γ) release in whole-blood cultures, which remained above baseline levels for more than 20 months (approximately 90 weeks). The number of antigen-specific IFN-γ-secreting central memory T cells present at the time of M. bovis challenge was significantly higher in vaccinated than in control animals at 12 months postvaccination, but not at 24 months. Vaccination of neonatal calves with BCG Danish induced protective immune responses against bovine TB which were maintained for at least 12 months postvaccination. These studies provide data on the immunity induced by BCG vaccination in calves; the results could inform vaccination strategies for the control of bovine TB in United Kingdom cattle herds.

Identification of Surrogates and Correlates of Protection in Protective Immunity against Mycobacterium bovis Infection Induced in Neonatal Calves by Vaccination with M. bovis BCG Pasteur and M. bovis BCG Danish

ABSTRACT Vaccination of neonatal calves with Mycobacterium bovis bacillus Calmette-Guérin (BCG) induces a significant degree of protection against infection with virulent M. bovis, the causative agent of bovine tuberculosis (bTB). We compared two strains of BCG, Pasteur and Danish, in order to confirm that the current European human vaccine strain (BCG Danish) induced protective immunity in calves, and we assessed immune responses to determine correlates of protection that could assist future vaccine evaluation in cattle. Both vaccine strains induced antigen (purified protein derivate [PPD])-specific gamma interferon (IFN-γ) in whole-blood cultures. These responses were not significantly different for BCG Pasteur and BCG Danish and peaked at week 2 to 4 postvaccination. Vaccination with either BCG Danish or BCG Pasteur induced significant protection against bTB, with reductions in both lesion score and bacteriological burden evident in both groups of vaccinated calves compared with nonvaccinated control calves. Measurement of IFN-γ-expressing T lymphocytes postvaccination and postchallenge revealed both correlates and surrogates of protective efficacy. The frequency of central memory T lymphocytes present at 12 weeks postvaccination (at the time of M. bovis challenge) correlated significantly with protection. Conversely, the number of IFN-γ-expressing effector T cells present after M. bovis challenge was correlated with disease. These results demonstrate that vaccination of neonatal calves with either BCG Pasteur or BCG Danish induces protective immune responses against TB. In addition, we show that measurement of antigen-specific T lymphocyte populations may provide a reliable means for identifying protective vaccine candidates.

A lymphocyte transformation assay for the detection of Mycobacterium bovis infection in the Eurasian badger (Meles meles).

The Eurasian badger (Meles meles) is a significant wildlife reservoir of Mycobacterium bovis in Great Britain. Improved control strategies against the disease in badgers require the development of diagnostic tests and vaccines. Here, we report the development of a comparative lymphocyte transformation assay (LTA) using bovine and avian tuberculin as antigen to detect cell-mediated responses in M. bovis-infected badgers. In a pilot study, the performance of this assay was compared with the existing indirect ELISA assay for the detection of tuberculous badgers. The sensitivity of the Comparative LTA was 87.5% compared with 62.5% for the indirect ELISA whereas the ELISA test gave a greater specificity (100% compared with 84.6% for the comparative LTA). Preliminary evidence suggests that for the comparative LTA, the blood may be stored overnight prior to testing and that this procedure might improve the specificity of the assay without compromising the sensitivity.

Vaccination of mice and cattle with plasmid DNA encoding the Mycobacterium bovis antigen MPB83.

A scientific review of bovine tuberculosis in Great Britain has concluded that the development of a cattle vaccine holds the best prospect for long-term disease control. Recent reports of successful DNA vaccination against Mycobacterium tuberculosis in small animal models have raised the possibility of using a similar strategy to produce vaccines against Mycobacterium bovis infection in cattle. To test this possibility, BALB/c mice were immunized with DNA encoding the M. bovis antigen MPB83. The mice responded to vaccination with a mixed IgG1/IgG2a response to the antigen and were protected from intravenous challenge with virulent M. bovis to a similar extent as those vaccinated with bacille Calmette-Guérin. The immunogenicity of the DNA vaccine in cattle was tested, after having established that DNA encoding MPB83 was immunogenic and elicited protective immunity in mice. In these studies, vaccinated animals had strong proliferative responses to MPB83.

Maturation of bovine dendritic cells by lipopeptides

The response of DC, and the subsequent stimulation of T cells, is an essential part of the initiation of immune responses following microbial challenge. The response of human DC to bacterial lipopeptides is mediated by toll-like receptor 2, and is characterised by DC maturation and the enhanced capacity to stimulate of T cells. We report here that bovine DC are also induced to mature following lipopeptide stimulation. Exposure of DC to the model lipopeptide Pam3CSK4 was associated with increased expression of MHC, costimulatory molecules, and enhanced secretion of IL-12 and TNFalpha. Lipopeptide-matured DC were superior in their ability to induce T cell activation and IFNgamma secretion. In contrast, exposure of MPhi to lipopeptides induced down-regulation of MHC expression and much lower increases in IL-12 secretion. A lipopeptide derived from the sequence of a relevant mycobacterial lipoprotein, MPB83, also influenced bovine DC by stimulating increases in IL-12 and TNFalpha secretion. These different changes in bovine DC and MPhi may have important implications for immune responses induced following bacterial infection with uptake of microbes by DC resulting in potentiation of their immunostimulatory capacity and uptake by MPhi having a much less marked effect on immune responses.