Fig. 7: The citrOgen platform as a versatile, quick and cost-effective method of Ab production and multiple downstream applications.

A citrOgen strain generation. Selected Ag sequences are incorporated into the appropriate gene editing vectors designed for optimal chromosomal expression of heterologous O-Ag, capsule or complex protein Ags, the latter under the map promoter to ensure timely expression during mucosal infection. pespO is then transformed to finalise the new citrOgen strain. QC checks include assessment of heterologous expression and pedestal formation in vitro (as shown in Supp Figs. 1A, 4A and 5B) before proceeding to in vivo Ab production. B In vivo Ab production. The CR infection cycle and immune responses are co-opted by the citrOgen platform: (i) EspO-mediated boosting of CR attachment to intestinal epithelial cells, (ii) continuous exposure of immune cells to the heterologous Ags, (iii) B cell affinity maturation results in Abs, primarily IgG, which mediate CR clearance by 28 dpi and can be obtained as pAb sera from blood. C Downstream applications. Following citrOgen infection/immunisation, the functionality of Abs can be assessed in vitro or by in vivo challenge studies, the pAb sera can be used for serotyping and the B-cells can be used for mAb production, either by using hybridoma technology to immortalise single-cell cloned B-cells or by expressing heterologous Ag-specific Ab sequences in cell lines to obtain recombinant mAb. Ab Antibodies, mAb monoclonal antibodies, pAb polyclonal antibodies, Ag Antigen, CR Citrobacter rodentium. Created using BioRender. Frankel, G. (2025) https://BioRender.com/h0xs2kn.