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 are generated by releasing the bacterial cytoplasmic contents through a channel in the cell envelope that is created by the controlled production of the bacteriophage ϕX174 lysis protein E. While ghosts possess all the immunostimulatory surface properties of the original host strain, they do not pose any of the infectious threats associated with live vaccines. Recently, we have engineered the Escherichia coli autotransporter hemoglobin protease (Hbp) into a platform for the efficient surface display of heterologous proteins in Gram-negative bacteria, HbpD. Using the Mycobacterium tuberculosis vaccine target ESAT6 (early secreted antigenic target of 6 kDa), we have explored the application of HbpD to decorate E. coli and Salmonella ghosts with antigens. The use of different promoter systems enabled the concerted production of HbpD-ESAT6 and lysis protein E. Ghost formation was monitored by determining lysis efficiency based on CFU, the localization of a set of cellular markers, fluorescence microscopy, flow cytometry, and electron microscopy. Hbp-mediated surface display of ESAT6 was monitored using a combination of a protease accessibility assay, fluorescence microscopy, flow cytometry and (immuno-)electron microscopy. Here, we show that the concerted production of HbpD and lysis protein E in E. coli and Salmonella can be used to produce ghosts that efficiently display antigens on their surface. This system holds promise for the development of safe and cost-effective vaccines with optimal intrinsic adjuvant activity and exposure of heterologous antigens to the immune system.
Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden.
Anna
A
Hjelm
Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden.
Bill
B
Söderström
Xbrane Bioscience AB, Stockholm, Sweden.
David
D
Vikström
Abera Bioscience AB, Stockholm, Sweden Section Molecular Microbiology, Department of Molecular Cell Biology, Faculty of Earth and Life Sciences, VU University, Amsterdam, Netherlands.
Wouter S P
WS
Jong
Abera Bioscience AB, Stockholm, Sweden Section Molecular Microbiology, Department of Molecular Cell Biology, Faculty of Earth and Life Sciences, VU University, Amsterdam, Netherlands.
Joen
J
Luirink
Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden degier@dbb.su.se.
Jan-Willem
JW
de Gier
D000942:Antigens, Bacterial / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D001428:Bacterial Vaccines / Q000302:isolation & purification; D061505:Cell Surface Display Techniques; D010450:Endopeptidases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D012475:Salmonella / Q000235:genetics / Q000378:metabolism*; D015164:Vaccines, Inactivated / Q000302:isolation & purification; D014764:Viral Proteins / Q000378:metabolism
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D001428:Bacterial Vaccines; C029555:E protein, bacteriophage X174; C093276:ESAT-6 protein, Mycobacterium tuberculosis; D008565:Membrane Proteins; D015164:Vaccines, Inactivated; D014764:Viral Proteins; D010450:Endopeptidases; C114676:hemoglobin protease Hbp