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. 2011;6(6):e21371.
doi: 10.1371/journal.pone.0021371. Epub 2011 Jun 21.

Immune Responses to the Enduring Hypoxic Response Antigen Rv0188 Are Preferentially Detected in Mycobacterium Bovis Infected Cattle With Low Pathology

Free PMC article

Immune Responses to the Enduring Hypoxic Response Antigen Rv0188 Are Preferentially Detected in Mycobacterium Bovis Infected Cattle With Low Pathology

Gareth J Jones et al. PLoS One. .
Free PMC article


The DosR regulon and the Enduring Hypoxic Response (EHR) define a group of M. tuberculosis genes that are specifically induced in bacilli exposed in vitro to conditions thought to mimic the environment encountered by Mycobacteria during latent infection. Although well described in humans, latent mycobacterial infection in cattle remains poorly understood. Thus, the aim of this study was to identify antigens that may potentially disclose cattle with latent M. bovis infection. To this end, we initially screened 57 pools of overlapping peptides representing 4 DosR regulon and 29 EHR antigens for their ability to stimulate an immune response in whole blood from TB-reactor cattle using IFN-γ and IL-2 as readouts. All 4 DosR regulon proteins were poorly recognized (maximum responder frequency of 10%). For the EHR antigens, both IFN-γ and IL-2 revealed similar response hierarchies, with responder frequencies ranging from 54% down to 3% depending on the given EHR antigen. Furthermore, these results demonstrated that responses in the infected cattle were largely IFN-γ biased. To support the concept for their role in latency, we evaluated if EHR antigen responses were associated with lower pathology. The EHR antigen Rv0188 was recognised predominantly in animals presenting with low pathology scores, whereas responses to ESAT-6/CFP-10 or the other EHR antigens tested were prevalent across the pathology spectrum. However, when we determined the production of additional cytokines induced by the M. bovis antigens PPD-B or ESAT-6/CFP-10, we detected significantly greater PPD-B-induced production of the pro-inflammatory cytokine IL-1β in animals recognizing Rv0188 (i.e. those with limited or no pathology). Thus, these results are consistent with the idea that responses to Rv0188 may identify a subset of animals at early stages of infection or in which disease progression may be limited.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.


Figure 1
Figure 1. IL-2 and IFN-γ response hierarchy after stimulation with EHR antigens.
Responses were established using blood samples from at least 19 TB-reactor animals. IFN-γ responses were determined using the BOVIGAM kit, IL-2 responses using a bio-assay (see Materials and Methods). Results are shown as responder frequencies (proportion of animals tested that gave a positive response). For comparison, the responder frequencies for ESAT-6/CFP-10 using IFN-γ and IL-2 as a readout were 89% and 84% respectively.
Figure 2
Figure 2. Comparison of antigen specific IFN-γ responses with pathology scores.
Antigen induced IFN-γ responses were plotted in relation to the pathology score, with each symbol representing a single animal. Horizontal and vertical dotted lines define the cut offs for IFN-γ responders/non-responders and low pathology/high pathology respectively. Responses to ESAT-6/CFP-10 (A), Rv0188 and Rv1954c (B and E) and Rv0826, Rv1284c and Rv0990c (C, D and F) were evaluated in 38, 19 and 36 animals respectively.
Figure 3
Figure 3. IFN-γ responses to Rv0188 were observed only in animals with low pathology scores.
Graph represents the relative proportion of animals responding to either ESAT-6/CFP-10 (n = 34), Rv0188 (n = 6), Rv0826 (n = 13), Rv1284 (n = 15), Rv1954c (n = 9) or Rv0990c (n = 20) that showed either low or high pathology.
Figure 4
Figure 4. Elevated cytokine production in Rv0188-responder animals.
Graph shows the relative cytokine levels (mean ± SEM) in Rv0188 responder animals (n = 6, filled bars) and Rv0188 non-responder animals (n = 12, open bars). Data are normalized to the mean cytokine concentration for all animals. Mean PPD-B induced cytokine concentrations were: 2.56 ng/ml (IL-1β); 0.52 ng/ml (IL-4); 18.04 units/ml (IL-10); 36.85 units/ml (IL-12); 64.47 ng/ml (MIP-1β); 15.67 ng/ml (IFN-γ); 3.99 ng/ml (TNF-α); and 26.76 SI (IL-2). Mean ESAT-6/CFP-10 induced cytokine concentrations were: 0.65 ng/ml (IL-1β); 0.43 ng/ml (IL-4); 9.44 units/ml (IL-10); 17.28 units/ml (IL-12); 53.77 ng/ml (MIP-1β); 6.90 ng/ml (IFN-γ); 2.73 ng/ml (TNF-α); and 11.61 SI (IL-2). * p<0.05 on non-normalised raw data, Unpaired T Test.

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