Autotransporter-based antigen display in bacterial ghosts.

Bacterial ghosts are empty cell envelopes of Gram-negative bacteria that can be used as vehicles for antigen delivery. Ghosts aregenerated by releasing the bacterial cytoplasmic contents through a channel in the cell envelope that is created by the controlledproduction of the bacteriophage ΦX174 lysis protein E. While ghosts possess all the immunostimulatory surface properties ofthe original host strain, they do not pose any of the infectious threats associated with live vaccines. Recently, we have engineeredthe Escherichia coli autotransporter hemoglobin protease (Hbp) into a platform for the efficient surface display of heterologousproteins in Gram-negative bacteria, HbpD. Using the Mycobacterium tuberculosis vaccine target ESAT6 (early secreted antigenictarget of 6 kDa), we have explored the application of HbpD to decorate E. coli and Salmonella ghosts with antigens. The use ofdifferent promoter systems enabled the concerted production of HbpD-ESAT6 and lysis protein E. Ghost formation was monitoredby determining lysis efficiency based on CFU, the localization of a set of cellular markers, fluorescence microscopy, flowcytometry, and electron microscopy. Hbp-mediated surface display of ESAT6 was monitored using a combination of a proteaseaccessibility assay, fluorescence microscopy, flow cytometry and (immuno-)electron microscopy. Here, we show that the concertedproduction of HbpD and lysis protein E in E. coli and Salmonella can be used to produce ghosts that efficiently displayantigens on their surface. This system holds promise for the development of safe and cost-effective vaccines with optimal intrinsicadjuvant activity and exposure of heterologous antigens to the immune system.