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Systems-level novel understanding of anti-glycan immunity

Periodic Reporting for period 1 - SNUGly (Systems-level novel understanding of anti-glycan immunity)

Reporting period: 2020-07-01 to 2021-12-31

Intestinal bacteria have an enormous influence on health, both in the form of major pathogens and as major constituents of the microbiota. We have demonstrated that high-affinity intestinal antibodies offer huge potential to protect from infection and to manipulate microbiota composition. However, designing good oral vaccines to induce these antibody responses is a compound problem. Most protective intestinal antibodies target bacterial glycans: long, repetitive sugar polymers that coat the surface of all bacterial cells. As these are typically amorphous hydrophilic structures the way that these interact with antibodies is fundamentally different to a rigid protein or small molecule. It is also currently unclear how these glycans or glycolipids are presented to B cells during germinal center responses. Starting from the well-established concept that whole-cell inactivated oral vaccines can induce high-affinity intestinal antibody responses against some glycan structures, we will dissect the critical determinants of antigen sampling, presentation and affinity maturation that are necessary for vaccine success. By combining advanced biophysical methods (e.g. atomic force spectroscopy), fluorescence and electron microscopy, and synthetic biology, we aim to:
1) Generate a "toolbox" for glycan-binding antibody research: Recombinant antibodies, BCR knock-in mice and defined glycan antigens as purified molecules, on whole bacteria or on virus-like particle will be developed for model antigens: Salmonella Typhimurium O-antigen and the E.coli K100 capsule.
2) Determine the quantitative relationship between glycan antigen sampling into gut-associated lymphoid tissues and particle size, glycan flexibility/structure, digestion resistance and natural IgA binding.
3) Determine how biophysical properties of glycan antigens affect B cell antigen uptake, T cell help and antibody affinity maturation.
4) Combine models of antigen sampling efficiency and anti-glycan antibody affinity maturation to generate a systems-level model of mucosal vaccine efficacy.
This will uncover the fundamental principles governing the induction of high-affinity anti-glycan sIgA, driving urgently required progress in mucosal vaccine design.
During the first phase of this project we have established a wide range of tools and have begun to probe the biophysics of antibody-glycan binding in detail. We have produced two recombinant antibodies with specificity for the Salmonella Typhimurium O-antigen backbone and could demonstrate using classical techniques (surface plasmon resonance) that these two antibodies have very different binding affinities (µM versus nM) and sensitivies to alterations in glycan chain length. These recombinant antibodies have been designed and produced, together with our main external collaborator Prof. Beth Stadtmueller, Illinois, USA, as murine IgG2, dimeric IgA, secretory IgA and FAb fragements. Salmonella O-glycans with different lengths (between 1 and 200 repeats) and chemistries (O-acetylated, glucosylated) have been purified in large quantities for use in all subsequent biophysical and biochemical analyses. Moreover, a Spy-catcher-FAb fragment fusion protein has been produced and use to covalently modify atomic force microscopy cantilevers for single-molecule force measurements. Preliminary atomic force microscopy data indicates that the high-affinity FAb fragment is capable of mediating glycan polymer extension before de-binding, validating our systems and confirming that the nature of antibody-glycan-binding is indeed very different to that observed with stiff protein structures.

Our other major achievement during this time-period has been the production of B-cell receptor knock-in mice. A CRISPR/Cas9 system was used to knock in the variable domain of both the heavy and light chains into their respective genomic sites. The resulting offspring were screened for successful integration. We have now established breeding lines for each of the 4 knock-in alleles, which are undergoing back-crossing to eliminate any off-target effects of the recombination process as well as inter-crossing towards establishing homozygous knock-in BCR lines in which all B cells show either a very high (line STA121) or low-moderate (line STA5) affinity for the Salmonella O-antigen. Encouragingly, KI VH and VL gene expression can be easily detected by qPCR in the B cells of knock-in mice and the first double-heterozygous animals show an elevated IgM titre specific for S.Typhimurium LPS. To our knowledge, these are the first knock-in mice to carry a bacterial glycan-specific B cell receptor and allow, for the first time, the detailed probing of bacterial-surface-specific B cell responses without the need to express heterologous protein antigens.
By combining these tools to examine antibody-glycan interactions across scales, we will dissect how protective mucosal immunity against bacterial glycans is generated, and will identify the determinants for its success. Already in the first phase of this project we have generated novel tools to probe these interactions at a level of detail never previously attempted. We have also generated the first evidence that protective antibodies bind with sufficient affinity to stretch the weak secondary structure of bacterial surface glycans: a strong hypothesis that had previously lacked direct evidence.

In the next phase of the project, we will validate the specificity of our knock-in B cell receptor models, and will use these tools to track where and how specific B cells become activated in intestine-draining lymphoid tissues, as well as the source of peptide antigen required for T cell help. These results will allow the rational design of optimal oral vaccines targeting major intestinal bacterial pathogens.
SPR analysis of glycan-binding kinetics of two recombinant antibodies
Atomic force microscopy used to probe antibody-bacterial glycan interactions