In the first half of this project we established recombinant monoclonal antibodies together with Prof. Beth Stadtmueller, Illinois, USA, as murine IgG2, dimeric IgA, secretory IgA and FAb fragments, with and without a SPI-catcher fusion to allow conjugation to SPI-tag-linked atopic force microscopy chips. Salmonella O-glycans with lengths between 1 and 200 repeats and with different chemistries (O-acetylated, glucosylated) were purified in large quantities. Combined with a newly developed bifunctional crosslinker (patent application EP24168910), we can now generate O-antigen glycoconjugates to proteins as well as to surface plasmon resonance chips and mica surfaces for atomic force spectroscopy. Whole-cell inactivated vaccines presenting glycans on between 1 and 100 repeats, and with the same varying chemistries were also generated. We additionally purified a range of E. coli capsular polysaccharides and developed a labelling technique allowing their analysis and separation by UPLC (Rutschmann et al. bioRxiv 2025).
Understanding glycan-antibody interactions
We used surface plasmon resonance(SPR) and atomic force microscopy (AFM, Fig. 1) to compare the interaction forces between monoclonal antibodies and O-antigens of different lengths and chemistries. Two antibodies were identified with single-arm binding affinities for the wildtype O-antigen in the order of 100nM, very high affinity in IgG form, and show evidence extensive somatic hypermutation (Figure 2). Interestingly, while one antibody bound equally well to both long and short O-antigens (clone STA5), while the other only bound when more than 8 O-antigen repeats were present (clone STA121). Via modelling and x-ray crystallography, we could confirm that STA5 is a “tip binder”, i.e. it embeds single repeat of the glycan polymer into its binding groove, while STA121 is a lateral binder, with groove-like interactions across multiple repeats of the glycan. Lateral binders (but not tip binders) can multivalently bind to a single glycan chain, giving very strong avidity-driven interactions. This results in particularly stable chain-like structures when Salmonella grows in the presence of the lateral-binding IgA (manuscript in preparation).
Understanding induction of antibodies against bacterial glycans
Whole live or inactivated Salmonella, as well as outer-membrane vesicles from Salmonella or virus-like particles conjugated to the Salmonella O-antigen are all capable of inducing antibody responses recognising the O-antigen. Particles can be delivered orally or parenterally, with gut IgA responses only induced by oral exposure, but serum IgG responses induced by either route. Interestingly, we find immunodominance of short O-antigen structures, and some antibody responses that bind short, but not long O-antigens which could be explained by the high entropy of long flexible glycans. Short O-antigens were also more efficiently extracted from planar lipid bilayers by B cells in vitro. Single-cell BCR sequencing from mice vaccinated with different vaccine compositions was used to identify expanded clones and to screen for O-antigen binding. By synthesizing lineages of specific B cell receptors we observe both increases in affinity and changes in specificity over time. Taken together, this demonstrates that anti-O-antigen specific B cell responses undergo affinity maturation and can be high affinity.
Understanding function of antibodies targeting bacterial glycans
We finally tested the function of oral-vaccine induced antibodies in clearing opportunistic pathogens from the gut. “Evolutionary trap vaccines” (Diard et al. Nature Microbiology 2021) can steer bacterial evolution in the gut using combinations of glycan-targeting vaccines. This ability to steer the outcome of competition in the gut can also be used to manipulate competition between a pathogen and a bacterial niche competitor. This allows a rationally designed combination of oral vaccines and niche competition to drive complete extinction of a pathogen from the gut lumen – a process we are calling vaccine-enhanced competition (Figure 3, Lentsch et al. Science 2025).
Overall this work has directly generated 4 published manuscripts and one patent, has contributed to many other published works from the lab. The work has been presented at a wide range of events worldwide. Two major manuscripts and a PhD thesis are currently in the final stages of preparation and will be submitted within the next months. The understanding of anti-glycan responses achieved here and how to apply these to generate sterilizing immunity in the gut will be of major relevance to the field, for example in controlling antibiotic-resistance bacterial colonization.
