SUMMARYTo increase its immunostimulatory properties, BCG was genetically engineered to secrete recombinant human interferon-alpha 2B (rhIFN-a ) under control of the mycobacterial heat shock protein (hsp)60 promoter and the a antigen signal sequence. Expression of rhIFN-a was readily detectable by ELISA and on Western blotting. When compared with control BCG, rhIFN-a BCG was substantially more active in inducing the production of IFN-g and IFN-inducible protein 10 (IP-10) from human peripheral blood mononuclear cells, while IL-10 production was correspondingly decreased. These effects were reversible upon antibody neutralization of rhIFN-a . Among 10 patients tested, rhIFN-a BCG enhanced IFN-g production in all patients ranging from 1´4-to 23´7-fold with a general trend toward greatest enhancement among those with weakest baseline responses to control BCG. Correspondingly, rhIFN-a BCG decreased IL-10 production in all patients by 1´2±4´8-fold. The onset of IFN-g production induced by rhIFN-a BCG was also more rapid, occurring within 4 h after stimulation versus . 24 h with wildtype BCG. The observation that the maximum IFN-g induction depends on the simultaneous presence of both IFN-a and BCG highlights the advantages of rhIFN-a BCG. Taken together, these immunostimulatory properties of rhIFN-a BCG suggest that it may be a superior agent for immunotherapeutic protocols involving live BCG in humans.

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bThe exotoxins TcdA and TcdB are the major virulence factors of Clostridium difficile. Circulating neutralizing antitoxin antibodies are protective in C. difficile infection (CDI), as demonstrated, in part, by the protective effects of actoxumab and bezlotoxumab, which bind to and neutralize TcdA and TcdB, respectively. The question of how systemic IgG antibodies neutralize toxins in the gut lumen remains unresolved, although it has been suggested that the Fc receptor FcRn may be involved in active antibody transport across the gut epithelium. In this study, we demonstrated that genetic ablation of FcRn and excess irrelevant human IgG have no impact on actoxumab-bezlotoxumab-mediated protection in murine and hamster models of CDI, suggesting that Fc-dependent transport of antibodies across the gut wall is not required for efficacy. Tissue distribution studies in hamsters suggest, rather, that the transport of antibodies depends on toxin-induced damage to the gut lining. In an in vitro two-dimensional culture system that mimics the architecture of the intestinal mucosal epithelium, toxins on the apical side of epithelial cell monolayers are neutralized by basolateral antibodies, and antibody transport across the cell layer is dramatically increased upon addition of toxin to the apical side. Similar data were obtained with F(ab=) 2 fragments, which lack an Fc domain, consistent with FcRn-independent paracellular, rather than transcellular, transport of antibodies. Kinetic studies show that initial damage caused by apical toxin is required for efficient neutralization by basolateral antibodies. These data may represent a general mechanism of humoral response-mediated protection against enteric pathogens.

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AG. 2015. Correction for Zhang et al., Toxin-mediated paracellular transport of antitoxin antibodies facilitates protection against Clostridium difficile infection. Infect Immun 83:4899.

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