The airway epithelium is the primary target of respiratory syncytial virus infection. It is an important component of the antiviral immune response. It contributes to the recruitment and activation of innate immune cells from the periphery through the secretion of cytokines and chemokines. This paper provides a broad review of the cytokines and chemokines secreted from human airway epithelial cell models during respiratory syncytial virus (RSV) infection based on a comprehensive literature review. Epithelium-derived chemokines constitute most inflammatory mediators secreted from the epithelium during RSV infection. This suggests chemo-attraction of peripheral immune cells, such as monocytes, neutrophils, eosinophils, and natural killer cells as a key function of the epithelium. The reports of epithelium-derived cytokines are limited. Recent research has started to identify novel cytokines, the functions of which remain largely unknown in the wider context of the RSV immune response. It is argued that the correct choice of in vitro models used for investigations of epithelial immune functions during RSV infection could facilitate greater progress in this field.

We recently described a protein O-glycosylation pathway conserved in all species of the Burkholderia genus that results in the synthesis and incorporation of a trisaccharide glycan to membrane-exported proteins. Here, we exploited this system to construct and evaluate a diagnostic tool for glanders. Burkholderia mallei, the causative agent of glanders, is a highly infectious and fatal zoonotic pathogen that infects horses, mules, donkeys, and occasionally humans. A highly sensitive and specific diagnostic tool is crucial for the control, elimination, and eradication of B. mallei infections. We constructed plasmids carrying synthetic genes encoding a modified, previously unannotated Burkholderia glycoprotein containing three glycosylation sequons fused to the cholera toxin B-subunit. The resulting proteins were glycosylated in the B. cenocepacia K56-2 parental strain, but not in glycosylationdeficient mutants, as determined by SDS-PAGE and fluorescent lectin blots. One of these glycoproteins was used as an antigen in ELISA and western blots to screen a panel of serum samples collected from glanders-infected and healthy horses, which were previously investigated by complement fixation test and indirect ELISA based on a semi-purified fraction of B. mallei. We show that ELISA and western blot assays based on our glycoprotein antigen provide 100% specificity, with a sensitivity greater than 88%. The glycoprotein antigen was recognized by serum samples collected from patients infected with B. pseudomallei, B. mallei, B. multivorans, and B. cenocepacia. Our results indicate that protein O-glycosylation in Burkholderia can be exploited as a biomarker for diagnosis of Burkholderiaassociated infections.

Previously, we discovered a protein O-glycosylation (ogc) cluster conserved in all Burkholderia species, which glycosylates proteins with a trisaccharide glycan. Sera from Burkholderia-infected patients produce anti-glycan antibodies, suggesting that the Burkholderia protein glycosylation pathway can be exploited for potential vaccine development. Here, we successfully produced two prototypes of anti-Burkholderia vaccines: a recombinant glycoprotein-based vaccine and an E. coli LPS-display vaccine. To generate the former, we constructed a plasmid carrying a chimeric gene encoding three glycosylation sequons fused to the cholera toxin B subunit. The presence of the glycan was observed in recombinant proteins expressed in B. cenocepacia parental strain, but not in proteins expressed by the glycosyltransferase-deficient ΔpglLstrain, as determined by SDS-PAGE and fluorescent lectin blots. For the development of an E. coli LPS-display vaccine, we constructed a plasmid expressing the ogc cluster, which was introduced into an E. coli strain unable to synthesize O-antigen but carrying the O-antigen ligase WaaL. Our results show that the LPS of this strain contained an additional moiety consistent with the B. cenocepacia trisaccharide glycan, as demonstrated by silver-stained LPS gels and lectin blot. This extra moiety was not detected in aΔwaaL mutant. These results suggest that the plasmid was able to provide the necessary functions for the synthesis and membrane translocation of the lipid-linked trisaccharide, which became a substrate for the WaaL ligase and incorporation into the E. coli LPS. Therefore, we demonstrate that the O-glycosylation pathway can be manipulated for the construction of potential anti-Burkholderia vaccines.

We recently described a protein O-glycosylation pathway conserved in all species of the Burkholderia genus that results in synthesis and incorporation of a trisaccharide glycan to membrane-exported proteins. Here, we exploited this system to construct and evaluate a diagnostic tool for glanders. Burkholderia mallei, the causative agent of glanders, is a highly infectious and fatal zoonotic pathogen that mainly infects horses, mules, donkeys and occasionally humans. A highly sensitive and specific diagnostic tool is crucial for the control, elimination and eradication of B. mallei infections. We constructed plasmids carrying synthetic genes encoding a modified, previously unannotated Burkholderia glycoprotein containing three glycosylation sequons fused to the cholera toxin B-subunit. The resulting proteins were glycosylated in the B. cenocepacia K56-2 parental strain, but not in glycosylation-deficient mutants, as determined by SDS-PAGE and fluorescent lectin blots. One of these glycoproteins was used as an antigen in ELISA and western blots to screen a panel of serum samples collected from glanders-infected and healthy horses previously investigated by complement fixation test and indirect ELISA based on a semi-purified fraction of B. mallei. We show that ELISA and western blot assays based on our glycoprotein antigen provide 100 % specificity, with a sensitivity greater than 88%. The glycoprotein antigen was recognized by serum samples collected from patients infected with B. pseudomallei, B. mallei, B. multivorans and B. cenocepacia. Our results indicate that protein O-glycosylation in Burkholderia can be exploited as a biomarker for diagnosis of Burkholderia-associated infections.IMPORTANCEGlanders is a severe zoonotic disease caused by the Gram-negative bacterium Burkholderia mallei, which affects horses, mules and donkeys, as well as humans. B. mallei is also considered a category B biothreat agent. Due to insufficient pathognomonic symptoms in the early stages of glanders, diagnosis can be difficult. Complement fixation is the most accurate and reliable serological test prescribed by the World Organization for Animal Health; however, this test has a considerable number of false-positive results. We have recently described a conserved protein O-glycosylation pathway present in all species of the Burkholderia genus; we also demonstrated that Burkholderia-infected humans develop anti-glycan antibodies. Here, we exploited this system to construct and evaluate a synthetic glycoengineered protein antigen as a diagnostic tool for glanders. Our results show 100 % specificity in the detection of antibodies from infected horses, indicating that protein O-glycosylation in Burkholderia can be exploited as a biomarker for diagnosis of Burkholderia-associated infections.