12 March 2013

5 minute read

Overview

Bovine tuberculosis (bTB) is a chronic disease of cattle caused by Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex group of bacteria. Vaccination of cattle might offer a long-term solution for controlling the disease and priority has been given to the development of a cattle vaccine against bTB.

Identification of biomarkers in tuberculosis research remains elusive and the goal is to identify host correlates of protection. We hypothesized that by studying global gene expression we could identify in vitro predictors of protection that could help to facilitate vaccine development. Calves were vaccinated with BCG or with a heterologous BCG prime adenovirally vectored subunit boosting protocol. Protective efficacy was determined after M. bovis challenge.

RNA was prepared from PPD-stimulated PBMC prepared from vaccinated-protected, vaccinated-unprotected and unvaccinated control cattle prior to M. bovis challenge and global gene expression determined by RNA-seq. 668 genes were differentially expressed in vaccinated-protected cattle compared with vaccinated-unprotected and unvaccinated control cattle.

Cytokine-cytokine receptor interaction was the most significant pathway related to this dataset with IL-22 expression identified as the dominant surrogate of protection besides INF-?. Finally, the expression of these candidate genes identified by RNA-seq was evaluated by RT-qPCR in an independent set of PBMC samples from BCG vaccinated and unvaccinated calves. This experiment confirmed the importance of IL-22 as predictor of vaccine efficacy.

Discussion

The design and development of a new bTB vaccine, would be greatly facilitated by the definition of predictors and correlates of protection, which could potentially be identified by studying the vaccine-induced host responses after vaccination and both before and post M. bovis challenge.

In particular, their definition would allow rational vaccine design and testing thereby reducing the costs of large animal experiments, as well as speeding up the testing process, by selecting the most promising candidate vaccines to be challenged with M. bovis in cattle BSL3 facilities. Such an evidence-based gating strategy would allow a faster and more informed rational approach to TB vaccine development.

However, the classic reductionist scientific approach in hypothesis validation using conventional immunological methods has severe limitations when it comes to scan, identify and define thousands of potential biomarkers in parallel. Thus, systems biology, based in the integration of data generated by –omics studies, has emerged as a useful approach in order to identify gene signatures that can predict and/or correlate with protection following vaccination.

Although the immune response against TB is thought to be based primarily on the key TH1 cytokine IFN-?, its induction following vaccination does not necessarily demonstrate vaccine success. However, absence of its expression after vaccination can indicate the failure of the vaccine tested to protect against bTB.

Thus, the expression of IFN-? following vaccination alone is insufficient as a predictor of protection. In an earlier study, the transcription of IL-17A predicted protection following vaccination with BCG and correlated with protection after challenge with M. bovis. Although elevated IL-17A transcription was also found in the present study in PPD-B stimulated PBMC from vaccinated/protected compared to vaccinated/un-protected calves, the differences did not quite reach statistical significance (data not shown).

Another cytokine that is associated with a TH17 responses is IL-22 (although IL-22 can also be induced in an TH17-independent manner by TH22, for example, which is involved in barrier surface protection and healing in skin, intestine and lung.

In the present study, il-22 was the gene most up-regulated in vaccinated/protected animals and thus constitutes a surrogate of protection. To our knowledge, this is the first report describing a role for IL-22 in vaccination against bTB. Previously, it had been shown that IL-22 produced by NK cells in humans and CD4+ T cells in macaques could limit M. tuberculosis growth in macrophages by increasing phagolysosomal fusion.

However, IL-22 can play a dual role in tissue homeostasis depending on the cytokine microenvironment where it is induced. For example, Sonnenberg et al. showed that excessive pulmonary destruction after intratracheal administration of bleomycin induced IL-22, whilst in IL-17 ?/? mice, IL-22 showed tissue protective properties.

Further, a murine model of allergic asthma using OVA immunization in IL-22-deficient mice showed reduced lung inflammation during the sensitization phase but increased lung inflammation during the antigen challenge (inhibited by exogenous IL-22).

Thus, these opposing biological effects might explain in part why IL-22 in our previous study was associated with pathology in unvaccinated naturally infected cattle, whilst in this study our data suggest that BCG vaccination induced an adequate and balanced inflammatory and TH1/TH17 (or TH22) cytokine response, with the expression of IL-22 in BCG vaccinated cattle maintained at relatively stable levels after infection compared with the increased observed in unvaccinated controls at week 4 and 8 p.i., as demonstrated in our validation experiment where we measured il-22 transcription also after M. bovis challenge.

Besides the genes encoding for IL-22 and IFN-? a number of other genes were strongly and significantly regulated exclusively in vaccinated/protected calves. For example mt3, encoding the zinc metallothionein MT-3 was the second most up-regulated gene in this category after il-22. This protein is a zinc-binding protein whose role in lysosomal function and autophagy of for example neurons and astrocytes has been described.

Autophagy has been linked with innate and adaptive immune responses against intracellular pathogens including M. tuberculosis, and mt3 up-regulation could reflect IFN-? induced macrophage activation. Interestingly, the transcription of the TH2-associated cytokine il-13 is also up-regulated, which is a cytokine that inhibits autophagy alongside IL-4.

The gene encoding for the chemokine CCL3 (MIP1?) was also found to be strongly up-regulated in PBMC from protected calves. CCL3 is produced by innate cells as well as CD4+ T cells during human TB.

Its role after vaccination may be to recruit antigen-specific T cells into the airway lumen following infection with M. tuberculosis or M. bovis. Ccl3 in PPD-B stimulated PBMC may be a reflection of the strong IFN-? responses induced.

In summary, although the development of TH1 responses following vaccination is fundamental for TB vaccine success as has been confirmed in this study by the significant modulation of IFN-? expression as well as IFN-? induced genes, up-regulation of the gene encoding il-22 as the dominant gene predicting vaccine success has been the major finding of this study. Thus, induction of TH17/22 subset responses alongside TH1 responses appears to be important for the protective anti-tuberculosis response. However, this biosignature needs to be prospectively validated in future experiments to assess its full potentials for predicting protection.

Citation: Bhuju S, Aranday-Cortes E, Villarreal-Ramos B, Xing Z, Singh M, et al. (2012) Global Gene Transcriptome Analysis in Vaccinated Cattle Revealed a Dominant Role of IL-22 for Protection against Bovine Tuberculosis. PLoS Pathog 8(12): e1003077. doi:10.1371/journal.ppat.1003077

Editor: Marcel A. Behr, McGill University, Canada

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March 2013