Abstract
Bacillus Calmette-Guérin (BCG), an attenuated strain of
Mycobacterium bovis (M. bovis)
, is the lead candidate vaccine for control of bovine tuberculosis (TB) in cattle. However, BCG ...vaccination sensitises cattle to bovine tuberculin, thus compromising the use of the current bovine TB surveillance tests. To address this, we have developed a diagnostic skin test that is not compromised by BCG vaccination and is able to detect BCG vaccinated animals that subsequently develop bovine TB following exposure to
M. bovis
. Building on previous work using ‘in house’ formulated protein cocktail reagents, we herein present test performance data for a single fusion protein (DST-F) containing the mycobacterial antigens ESAT-6, CFP-10 and Rv3615c formulated as a ‘ready to use’ reagent by a commercial manufacturer. Our results demonstrate that, unlike tuberculin reagents, a diagnostic skin test using DST-F maintained high specificity in BCG vaccinated animals. Furthermore, the DST-F skin test demonstrated a high relative sensitivity in identifying
M. bovis
infected animals, including those where BCG vaccination failed to prevent bovine TB pathology following experimental exposure to
M. bovis
. The DST-F is currently undergoing field trials in Great Britain to support its licensure and commercialisation.
•BCG-primed, Ad5-85A boosted cattle present Ag85A specific mycobacteriocidal T cells.•Along with mycobacteriocidal activity Ad5-85A boosting induces IL-10 transcription.•Ad5-85A boosting does not ...alter the transcription pattern of pro-inflammatory cytokines.
There is a need to improve the efficacy of the BCG vaccine against human and bovine tuberculosis. Previous data showed that boosting bacilli Calmette-Guerin (BCG)-vaccinated cattle with a recombinant attenuated human type 5 adenovirally vectored subunit vaccine (Ad5-85A) increased BCG protection and was associated with increased frequency of Ag85A-specific CD4+ T cells post-boosting. Here, the capacity of Ag85A-specific CD4+ T cell lines – derived before and after viral boosting – to interact with BCG-infected macrophages was evaluated. No difference before and after boosting was found in the capacity of these Ag85A-specific CD4+ T cell lines to restrict mycobacterial growth, but the secretion of IL-10 in vitro post-boost increased significantly. Furthermore, cell lines derived post-boost had no statistically significant difference in the secretion of pro-inflammatory cytokines (IL-1β, IL-12, IFNγ or TNFα) compared to pre-boost lines. In conclusion, the protection associated with the increased number of Ag85A-specific CD4+ T cells restricting mycobacterial growth may be associated with anti-inflammatory properties to limit immune-pathology.
Gene transcription studies have identified dual roles for the cytokines IL-17A and IL-22 in bovine tuberculosis, where they show potential as both predictors of vaccine success and correlates of ...infection. To allow for a detailed investigation of the cell populations responsible for production of these cytokines, we have utilised a novel bovine IL-22 specific recombinant antibody for flow cytometry. Bovine tuberculin (PPDB) induced greater IL-22 and IL-17A production in Mycobacterium bovis (M. bovis)-infected cattle compared to non-infected controls, while PWM-induced cytokine levels were similar between the two groups. In M. bovis-infected animals, PPDB specific IL-22 and IL-17A responses were observed in both CD4+ T cell and γδ T cell populations. Although both cytokines were detected in both cell types, IL-22/IL-17A double producers were rare and confined mainly to the γδ T cell population. These results support previous gene transcription studies and extend the observation of increased IL-22 and IL-17A responses in M. bovis-infected animals to the level of protein production. We were also able to characterise the cell populations responsible for these disease-related cytokine responses. The data generated can be used to further our understanding of the immunopathology of bovine tuberculosis and to produce more sensitive and specific immune-diagnostic reagents.
Bovine tuberculosis is an infectious disease of global significance that remains endemic in many countries. Mycobacterium bovis infection in cattle is characterized by a cell-mediated immune response ...(CMI) that precedes humoral responses, however the timing and trajectories of CMI and antibody responses determined by newer generation assays remain undefined. Here we used defined-antigen interferon-gamma release assays (IGRA) and an eleven-antigen multiplex ELISA (Enferplex TB test) alongside traditional tuberculin-based IGRA and IDEXX M. bovis antibody tests to assess immune trajectories following experimental M. bovis infection of cattle. The results show CMI responses developed as early as two-weeks post-infection, with all infected cattle testing positive three weeks post-infection. Interestingly, 6 of 8 infected animals were serologically positive with the Enferplex TB assay as early as 4 weeks post-infection. As expected, application of the tuberculin skin test enhanced subsequent serological reactivity. Infrequent M. bovis faecal shedding was observed but was uncorrelated with observed immune trajectories. Together, the results show that early antibody responses to M. bovis infection are detectable in some individuals and highlight an urgent need to identify biomarkers that better predict infection outcomes, particularly for application in low-and-middle income countries where test-and-slaughter based control methods are largely unfeasible.
In the present study, we applied microarray technology to define biosignatures by microarray transcriptome analysis in lung and spleen samples after BCG vaccination and M. bovis infection of BALB/c ...mice. The aims were two-fold, namely to define biosignatures that could predict vaccine success before challenge, and biomarker patterns that correlated with anamnestic protective responses following exposure to virulent M. bovis. Further, these biosignatures should be detectable without in vitro antigenic challenge.
After BCG vaccination, we defined a specific pulmonary gene expression signature related to the connective tissue development and function network that predicted vaccine success before M. bovis challenge. In addition, a Th17-related cytokine profile was found that correlated with vaccine-induced protective immunity following infection with virulent M. bovis in the lung as well as additional genes that were up-regulated in the spleens of vaccinated animals post-infection related to neutrophil biology and inflammation.
This study has therefore prioritized both biomarkers predicting vaccination success before challenge and bio-signatures that are potentially associated with protective immune responses that will be useful to evaluate future vaccine candidates.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Memory B cells and tuberculosis Lyashchenko, Konstantin P.; Vordermeier, H. Martin; Waters, W. Ray
Veterinary immunology and immunopathology,
March 2020, 2020-Mar, 2020-03-00, 20200301, Letnik:
221
Journal Article
Recenzirano
Odprti dostop
•Heterogeneous populations of memory B cells are generated in tuberculosis.•Memory B cells in tuberculosis can regulate T cell-mediated immunity.•Atypical memory B cells may have adverse effects on ...disease pathogenesis.•Memory B cells are reactivated after tuberculin injection in animal tuberculosis.
Immunological memory is a central feature of adaptive immunity. Memory B cells are generated upon stimulation with antigen presented by follicular dendritic cells in the peripheral lymphoid tissues. This process typically involves class-switch recombination and somatic hypermutation and it can be dependent or independent on germinal centers or T cell help. The mature B cell memory pool is generally characterized by remarkable heterogeneity of functionally and phenotypically distinct sub-populations supporting multi-layer immune plasticity. Memory B cells found in human patients infected with Mycobacterium tuberculosis include IgD+ CD27+ and IgM+ CD27+ subsets. In addition, expansion of atypical memory B cells characterized by the lack of CD27 expression and by inability to respond to antigen-induced re-activation is documented in human tuberculosis. These functionally impaired memory B cells are believed to have adverse effects on host immunity. Human and animal studies demonstrate recruitment of antigen-activated B cells to the infection sites and their presence in lung granulomas where proliferating B cells are organized into discrete clusters resembling germinal centers of secondary lymphoid organs. Cattle studies show development of IgM+, IgG+, and IgA+ memory B cells in M. bovis infection with the ability to rapidly differentiate into antibody-producing plasma cells upon antigen re-exposure. This review discusses recent advances in research on generation, re-activation, heterogeneity, and immunobiological functions of memory B cells in tuberculosis. The role of memory B cells in post-skin test recall antibody responses in bovine tuberculosis and implications for development of improved immunodiagnostics are also reviewed.
•A dual colour IFN-γ/IL-2 FluoroSpot assay has been developed for enumeration of bovine T cell responses.•Dual IL-2+IFN-γ+ T cells were identified as the largest PPD-B responsive T cell subset.•The ...frequency of cytokine producing cells and the proportion of IL-2 single producers were significantly higher in animals with visible lesions.•The greater levels of single IL-2 producing T cells associated with the presence of pathology could be a potential biomarker for active TB in cattle.
Human studies have identified the potential of measuring Mycobacterium tuberculosis specific IFN-γ and/or IL-2 secreting T cell subsets to distinguish different clinical stages of human tuberculosis (TB). To assess these functional T cell subsets in different states of bovine TB we have established a bovine dual IFN-γ/IL-2 fluorescence-immunospot (FluoroSpot) assay and analysed the frequencies of Mycobacterium bovis (M. bovis) specific IL-2 and/or IFN-γ producing cells in PBMC from 30 cattle naturally infected with M. bovis. Depending on their post mortem results the animals were grouped in 22 cattle with visible lesions (VL) and 8 cattle without visible lesions (NVL). In response to bovine tuberculin purified protein derivative (PPD-B) the frequencies of cytokine producing cells and proportions of IL-2 single producers were significantly higher in VL compared to NVL while PWM-induced cytokine responses were similar between the two groups. Dual IL-2+IFN-γ+ T cells could be identified as the largest PPD-B responsive T cell subset in both cattle groups. In conclusion, our FluoroSpot is a valid method to enumerate individual antigen-specific IFN-γ+ and IL-2+ T cell subsets ex vivo. The greater levels of single IL-2 producing T cells associated with the presence of pathology could be a potential biomarker for active TB in cattle.
Bovine tuberculosis (bTb) remains a major and economically important disease of livestock. Improved ante-mortem diagnostic tools would help to underpin novel control strategies. The definition of ...biomarkers correlating with disease progression could have impact on the rational design of novel diagnostic approaches for bTb. We have used a murine bTb model to identify promising candidates in the host transcriptome post-infection. RNA from in vitro-stimulated splenocytes and lung cells from BALB/c mice infected aerogenically with Mycobacterium bovis were probed with high-density microarrays to identify possible biomarkers of disease. In antigen-stimulated splenocytes we found statistically significant differential regulation of 1109 genes early (3 days) after infection and 1134 at a later time-point post-infection (14 days). 618 of these genes were modulated at both time points. In lung cells, 282 genes were significantly modulated post-infection. Amongst the most strongly up-regulated genes were: granzyme A, granzyme B, cxcl9, interleukin-22, and ccr6. The expression of 14 out of the most up-regulated genes identified in the murine studies was evaluated using in vitro with antigen-stimulated PBMC from uninfected and naturally infected cattle. We show that the expression of cxcl9, cxcl10, granzyme A and interleukin-22 was significantly increased in PBMC from infected cattle compared to naïve animals following PPD stimulation in vitro. Thus, murine transcriptome analysis can be used to predict immunological responses in cattle allowing the prioritisation of CXCLl9, CXCL10, Granzyme A and IL-22 as potential additional readout systems for the ante-mortem diagnosis of bovine tuberculosis.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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