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Display of Antigens on Polyester Inclusions Lowers the Antigen Concentration Required for a Bovine Tuberculosis Skin Test.

ABSTRACT: The tuberculin skin test is the primary screening test for the diagnosis of bovine tuberculosis (TB), and use of this test has been very valuable in the control of this disease in many countries. However, the test lacks specificity when cattle have been exposed to environmental mycobacteria or vaccinated with Mycobacterium bovis bacille Calmette-Guérin (BCG). Recent studies showed that the use of three or four recombinant mycobacterial proteins, including 6-kDa early secretory antigenic target (ESAT6), 10-kDa culture filtrate protein (CFP10), Rv3615c, and Rv3020c, or a peptide cocktail derived from those proteins, in the skin test greatly enhanced test specificity, with minimal loss of test sensitivity. The proteins are present in members of the pathogenic Mycobacterium tuberculosis complex but are absent in or not expressed by the majority of environmental mycobacteria and the BCG vaccine strain. To produce a low-cost skin test reagent, the proteins were displayed at high density on polyester beads through translational fusion to a polyhydroxyalkanoate synthase that mediates the formation of antigen-displaying inclusions in recombinant Escherichia coli. Display of the proteins on the polyester beads greatly increased their immunogenicity, allowing for the use of very low concentrations of proteins (0.1 to 3 ?g of mycobacterial protein/inoculum) in the skin test. Polyester beads simultaneously displaying all four proteins were produced in a single fermentation process. The polyester beads displaying three or four mycobacterial proteins were shown to have high sensitivity for detection of M. bovis-infected cattle and induced minimal responses in animals exposed to environmental mycobacteria or vaccinated with BCG.

SUBMITTER: Parlane NA

PROVIDER: S-EPMC4711088 | biostudies-other | 2015 Oct

REPOSITORIES: biostudies-other

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Display of Antigens on Polyester Inclusions Lowers the Antigen Concentration Required for a Bovine Tuberculosis Skin Test.

Parlane Natalie A NA Chen Shuxiong S Jones Gareth J GJ Vordermeier H Martin HM Wedlock D Neil DN Rehm Bernd H A BH Buddle Bryce M BM

Clinical and vaccine immunology : CVI 20151028 1

The tuberculin skin test is the primary screening test for the diagnosis of bovine tuberculosis (TB), and use of this test has been very valuable in the control of this disease in many countries. However, the test lacks specificity when cattle have been exposed to environmental mycobacteria or vaccinated with Mycobacterium bovis bacille Calmette-Guérin (BCG). Recent studies showed that the use of three or four recombinant mycobacterial proteins, including 6-kDa early secretory antigenic target ( ...[more]

PMID: 26512049

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Project description:Bovine tuberculosis (bTB) remains endemic in domestic water buffaloes (Bubalus bubalis) in India and elsewhere, with limited options for control other than testing and slaughter. The prescribed tuberculin skin tests with purified protein derivative (PPD) for diagnosis of bTB preclude the use of Bacille Calmette-Guérin (BCG)-based vaccination because of the antigenic cross-reactivity of vaccine strains with Mycobacterium bovis and related pathogenic members of the M. tuberculosis complex (MTBC). For the diagnosis of bTB in domestic water buffaloes, we here assessed a recently described defined-antigen skin test (DST) that comprises overlapping peptides representing the ESAT-6, CFP-10 and Rv3615c antigens, present in disease-causing members of the MTBC but missing in BCG strains. The performance characteristics of three doses (5, 10 or 20 μg/peptide) of the DST were assessed in natural tuberculin skin test reactor (n = 11) and non-reactor (n = 35) water buffaloes at an organized dairy farm in Hisar, India, and results were compared with the single intradermal skin test (SIT) using standard bovine tuberculin (PPD-B). The results showed a dose-dependent response of DST in natural reactor water buffaloes, although the SIT induced a significantly greater (P < 0.001) skin test response than the highest dose of DST used. However, using a cut-off of 2 mm or greater, the 5, 10, and 20 μg DST cocktail correctly classified eight, 10 and all 11 of the SIT-positive reactors, respectively, suggesting that the 20 μg DST cocktail has a diagnostic sensitivity (Se) of 1.0 (95% CI: 0.72-1.0) identical to that of the SIT. Importantly, none of the tested DST doses induced any measurable skin induration responses in the 35 SIT-negative animals, suggesting a specificity point estimate of 1.0 (95% CI: 0.9-1.0), also identical to that of the SIT and compares favorably with that of the comparative cervical test (Se = 0.85; 95% CI: 0.55-0.98). Overall, the results suggest that similar to tuberculin, the DST enables sensitive and specific diagnosis of bTB in water buffaloes. Future field trials to explore the utility of DST as a defined antigen replacement for tuberculin in routine surveillance programs and to enable BCG vaccination of water buffaloes are warranted.

Project description:Mycobacterium avium subspecies paratuberculosis (MAP) causes chronic progressive granulomatous enteritis leading to diarrhea, weight-loss, and eventual death in ruminants. Commercially available vaccine provides only partial protection against MAP infection and can interfere with the use of current diagnostic tests for bovine tuberculosis in cattle. Here, we characterized immune responses in calves to vaccines containing four truncated MAP antigens as a fusion (Ag85A202-347-SOD1-72-Ag85B173-330-74F1-148+669-786), either displayed on protein particles, or expressed as a soluble recombinant MAP (rMAP) fusion protein as well as to commercially available Silirum® vaccine. The rMAP fusion protein elicited the strongest antigen-specific antibody responses to both PPDA and recombinant antigen and strong and long-lasting T-cell immune responses to these antigens, as indicated by increased production of IFN-γ and IL-17A in antigen-stimulated whole blood cultures. The MAP fusion protein particle vaccine induced minimal antibody responses and weak IFN-γ responses but stimulated IL-17A responses to recombinant antigen. The immune response profile of Silirum® vaccine was characterized by weak antibodies and strong IFN-γ and IL-17A responses to PPDA. Transcription analysis on antigen-stimulated leukocytes from cattle vaccinated with rMAP fusion protein showed differential expression of several immune response genes and genes involved in costimulatory signaling, TLR4, TLR2, PTX3, PTGS2, PD-L1, IL1B, IL2, IL6, IL12B, IL17A, IL22, IFNG, CD40, and CD86. Moreover, the expression of several genes of immune pathways correlated with cellular immune responses in the rMAP fusion protein vaccinated group. These genes have key roles in pathways of mycobacterial immunity, including autophagy, manipulation of macrophage-mediated killing, Th17- and regulatory T cells- (Treg) mediated responses. Calves vaccinated with either the rMAP fusion protein or MAP fusion protein particle vaccine did not induce reactivity to PPDA and PPDB in a comparative cervical skin test, whereas Silirum® induced reactivity to these tuberculins in most of the vaccinated animals. Overall, our results suggest that a combination of recombinant MAP antigens in the form of a soluble fusion protein vaccine are capable of inducing strong antigen-specific humoral and a balanced Th1/Th17-cell immune response. These findings, together with the absence of reactivity to tuberculin, suggest this subunit vaccine could provide protective immunity against intracellular MAP infection in cattle without compromising the use of current bovine tuberculosis surveillance test.

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Display of Antigens on Polyester Inclusions Lowers the Antigen Concentration Required for a Bovine Tuberculosis Skin Test.

Publications

Display of Antigens on Polyester Inclusions Lowers the Antigen Concentration Required for a Bovine Tuberculosis Skin Test.

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