Deciphering the influence of paraimmunobiotic bifidobacteria on the innate antiviral immune response of bovine intestinal epitheliocytes by transcriptomic analysis.

Previously, we reported that the non-viable immunomodulatory Bifidobacterium infantis MCC12 and Bifidobacterium breve MCC1274 strains (paraimmunobiotic bifidobacteria) were able to increase the protection against rotavirus infection in bovine intestinal epithelial (BIE) cells. In order to gain insight into the influence of paraimmunobiotic bifidobacteria on the innate antiviral immune response of BIE cells, their effect on the transcriptomic response triggered by Toll-like receptor 3 (TLR3) activation was investigated. By using microarray technology and qPCR analysis, we obtained a global overview of the immune genes involved in the innate antiviral immune response in BIE cells. Activation of TLR3 by poly(I:C) in BIE cells significantly increased the expression of interferon (IFN)-α and IFN-ß, several interferon-stimulated genes, cytokines, and chemokines. It was also observed that both paraimmunobiotic bifidobacteria differently modulated immune genes expression in poly(I:C)-challenged BIE cells. Most notable changes were found in genes involved in antiviral defence (IFN-ß, MX1, OAS1X, MDA5, TLR3, STAT2, STAT3), cytokines (interleukin (IL)-6), and chemokines (CCL2, CXCL2, CXCL6) that were significantly increased in bifidobacteria-treated BIE cells. B. infantis MCC12 and B. breve MCC1274 showed quantitative and qualitative differences in their capacities to modulate the innate antiviral immune response in BIE cells. B. breve MCC1274 was more efficient than the MCC12 strain to improve the production of type I IFNs and antiviral factors, an effect that could be related to its higher ability to protect against rotavirus replication in BIE cells. Interestingly, B. infantis MCC12 showed a remarkable anti-inflammatory effect. The MCC12 strain was more efficient to reduce the expression of inflammatory cytokines and chemokines (IL-16, IL-20, CX3CL1) when compared with B. breve MCC1274. These results provided valuable information for the deeper understanding of the antiviral immune response of intestinal epithelial cells as well as the host-paraimmunobiotic interaction in the bovine host.

Expression and diagnostic value of proteins in Mycobacterium tuberculosis.

We constructed a prokaryotic expression vector expressing the Mycobacterium tuberculosis protein TB16.3, as well as 3 other proteins, including TB15.3, CFP-10, and Rv2626C, which were purified and analyzed for their effectiveness as detection antibodies. The TB16.3 genes of M. tuberculosis H37Rv genomic DNA were amplified by polymerase chain reaction, inserted into the expression vector pET-30a, and expressed in Escherichia coli. An enzyme-linked immunosorbent assay was used to detect the 4 M. tuberculosis antibodies. Engineered E. coli bacteria expressing TB16.3 and the 3 other proteins were constructed and found mainly to be soluble. For recombinant TB16.3 proteins, serum samples of 118 tuberculosis (TB) patients and 96 healthy controls were analyzed. Sensitivity, specificity, and adjusted concordance rate for the TB16.3 antibody were 72.9, 86.5, and 79.6%, respectively. The positive rate of Rv2626C antibody in TB patients (44.1%) was significantly lower than that in normal controls (75.0%, χ(2) = 20.8, P < 0.01). TB15.3 and TB16.3 were used for simultaneous detection and showed sensitivity, specificity, and repeatability rates of 69.4, 96.9, and 83.7%. The antibody positive rate and specificity for patients with lung disease was 9.6 and 90.4%, respectively. TB15.3 and TB16.3 were mixed and detected simultaneously. Combined with the results for TB15.3, the sensitivity, specificity, and concordance rates were 82.2, 95.9, and 88.9%, respectively. The concordance rate was the highest value observed. Target genes were cloned into a host strain and expressed successfully. The TB16.3 recombinant protein may be used as a new serological antigen for tuberculosis diagnosis.

Expression and serological diagnosis of Mycobacterium tuberculosis CFP-10 and Rv2626c proteins.

We constructed a Mycobacterium tuberculosis vector expressing CFP-10 and Rv2626c to examine the expression of these proteins in Escherichia coli as well as their immunoreactivity. The CFP-10 and Rv2626c genes were amplified from tuberculosis H37Rv genomic DNA using polymerase chain reaction. They were ligated into the expression vector PET30a and expressed in E. coli. Histidine tag nickel column chromatography was used to purify the recombinant protein. An enzyme-linked immunosorbent assay (ELISA) was used for detection. In our E. coli-engineered bacteria containing a CFP10 and Rv2626c plasmid, the target protein was found mainly to be in the soluble form. We formed mixed antigens of the recombinant CFP10 and Rv2626c proteins. ELISA results showed that in 214 blood samples, the positive rate was 77.1%. The target gene was successfully expressed in the host strain. Mixed antigens of the recombinant CFP-10 and Rv2626c proteins can be used as a combination antigen in the serological diagnosis of tuberculosis.