Periodic Reporting for period 4 - SNUGly (Systems-level novel understanding of anti-glycan immunity)
Okres sprawozdawczy: 2025-01-01 do 2025-06-30
Many protective intestinal antibodies target bacterial glycans: long, repetitive sugar polymers that coat the surface of all bacterial cells. As these are highly flexible hydrophilic structures that interact with antibodies in a very different way to a globular protein or hapten: typical structures of model antigens such as ovalbumin or nitrophenol. In this project we have aimed to understand the nature of antibody-glycan interactions and to understand how efficient anti-glycan antibody responses are induced in vivo. This knowledge is essential for the rational design of better bacteria-targeting vaccines.
Our major findings include the observation that glycan-binding antibodies can be subdivided into “tip binders” i.e. embedding a single repeat of the glycan polymer into the binding groove, or “lateral binders”, which contact multiple repeats of the glycan and can bind in multiple places along a single glycan polymer. Lateral binders have particularly strong avidity-driven interactions. We have mapped extensive evidence for effective somatic hypermutation and affinity maturation in Salmonella glycan-binding antibodies in both mouse and human, which breaks down the dogma of low-affinity T cell-independent antibody responses against these molecules. Consistent with classical affinity maturation, short glycan structures which are more rigid and more efficiently sampled via the B cell receptor, seem to be immunodominant to long structures during vaccination in vivo.
In a final part of the project, glycan-binding antibodies were developed that can manipulate the evolutionary trajectory of Salmonella in vivo and that can drive sterilizing immunity in the gut lumen via the process of vaccine-enhanced competition.
Our major findings include the observation that glycan-binding antibodies can be subdivided into “tip binders” i.e. embedding a single repeat of the glycan polymer into the binding groove, or “lateral binders”, which contact multiple repeats of the glycan and can bind in multiple places along a single glycan polymer. Lateral binders have particularly strong avidity-driven interactions. We have mapped extensive evidence for effective somatic hypermutation and affinity maturation in Salmonella glycan-binding antibodies in both mouse and human, which breaks down the dogma of low-affinity T cell-independent antibody responses against these molecules. Consistent with classical affinity maturation, short glycan structures which are more rigid and more efficiently sampled via the B cell receptor, seem to be immunodominant to long structures during vaccination in vivo.
In a final part of the project, glycan-binding antibodies were developed that can manipulate the evolutionary trajectory of Salmonella in vivo and that can drive sterilizing immunity in the gut lumen via the process of vaccine-enhanced competition.
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.
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.
This project has generated several outcomes with major impact beyond state-of-the art:
1) By categorically demonstrating high-affinity glycan-binding and affinity maturation of O-antigen glycan binding antibodies, we are forcing a change in the textbook immunology concept that all anti-glycan responses are low-affinity and T-independent.
2) The development of evolutionary trap vaccines and vaccine-enhanced competition is game-changing in how we design vaccines and treatments to target bacteria that colonize mucosal surfaces.
3) Tools developed within the project, including novel crosslinkers, novel approaches to SPR and atomic force microscopy and panels of glycan-specific monoclonal antibodies allow the field of immunology and microbial glycobiology to interact and grow.
1) By categorically demonstrating high-affinity glycan-binding and affinity maturation of O-antigen glycan binding antibodies, we are forcing a change in the textbook immunology concept that all anti-glycan responses are low-affinity and T-independent.
2) The development of evolutionary trap vaccines and vaccine-enhanced competition is game-changing in how we design vaccines and treatments to target bacteria that colonize mucosal surfaces.
3) Tools developed within the project, including novel crosslinkers, novel approaches to SPR and atomic force microscopy and panels of glycan-specific monoclonal antibodies allow the field of immunology and microbial glycobiology to interact and grow.