Fig. 1. Transgene designs and antigen expression.

(A) Schematic diagrams representing the different types of constructs generated for PorA or FetA antigens. For full-length constructs, full-length genes were inserted in frame after the tPA leader, and a V5 marker peptide was added at the C-terminus (Design A). A single PorA VR loop was fused to a FliC scaffold (with or without flanking cysteines), with a V5 peptide sequence at the C-terminus (Design B). Two PorA VR loops were inserted into the FliC scaffold with a flexible polylinker sequence, SGMPGSGPAY, between the VR regions (Design C). A molecular adjuvant (IMX313) was added at the C-terminus (Design D). (B) Detection of PorA and FetA antigens expressed in mammalian cells. HeLa cells were infected with Ad-P1.7,16 and the resulting protein detected using anti-P1.16 mAb (row A), or cells were transfected with plasmids coding for full length FetA3–3 and proteins detected with anti-FetA mouse serum (row B), or FliC-VR3–3 and proteins detected with anti-FetA mouse serum (row C), or FliC-VR1.7-C and protein detected with anti-P1.7 mAb (row D). Negative controls (right image of each row) were stained identically to the test cells (left image each panel). Proteins were detected using a fluorescently labelled secondary antibody (green). Nuclei were counterstained using DAPI (blue). (C and D) Detection of PorA and FetA epitopes in supernatants of cells transfected with FliC-VR constructs. Supernatants from individual transfection supernatants were serially diluted 1:7 in blocking buffer in duplicate. Proteins were detected with anti-P1.7 mAb (C), or anti-P1.16 mAb (D). Plasmids used for transfections are indicated in the figure legends.