Team:Slovenia/Project/Engineered flagellin vaccine

Protein flagellin is one of the most effective adjuvants. It contains a pathogen-associated molecular pattern (PAMP), which is unique in its inclusion of an antigenic hypervariable region and a conserved domain involved in TLR5-dependent systemic and mucosal proinflammatory and adjuvant activities. Flagellin has already been shown to be effective as an adjuvant in vaccines against a wide range of pathogens and some vaccines which include flagellin are already in clinical trials. In addition to mucosal response, activation of TLR5 also leads to the systemic immune response.
H.pylori flagellin fails to activate TLR5, which subverts the early innate immune response against these bacteria. TLR5 is essential for protection against most flagellated bacteria. This receptor is present on cells of the immune system and epithelial cells. Activation of TLR5 triggers epithelial expression of CCL20 (which attracts immature DCs), Th1 response and production of mucosal IgAs. Bacteria breaching the epithelial barrier of the intestine also activate TLR5 on the underlying dendritic cells and activate inflammation and strong immune response.

Amino and carboxy-terminal segment of flagellin of bacteria such as S.typhimurium, E. coli, S. marcescens… are required for activation of TLR5. Our plan was to regain activation of TLR5 by flagellin of H.pylori by replacing the N- and C-terminal segment of H.pylori flagellin with segments from TLR5-activating flagellin from E. coli.
Therefore our designed vaccine, comprising chimeric flagellin, will retain the central, most antigenic segment of H.pylori flagellin FlaA, while replacing the N- and C-terminal segment by the FliC flagellin from E. coli. We expected that such an engineered flagellin would be able to activate TLR5. Additionally, we fused an antigenic protein or multiepitope segment of virulence factors of H.pylori to the C-terminus of this engineered flagellin.

In this way, our designer vaccine should provide activation of innate immune response through activation of TLR5 (N- and C-terminal segment of FliC from E. coli) and antigenic epitopes for adaptive immune response (central segment of H.pylori FlaA and H.pylori antigens, such as UreB or designed synthetic multiepitope).
Specifically, we investigated other important H.pylori antigens and incorporated them into our chimeric flagellin chassis. The later is not only important for B-cell stimulation and humoral response leading to neutralising antibody production, but also for the enhancement of interactions between the main immune cells (CD4+T, CD8+T, B-cells). At the C-terminus of chimeric flagellin, we added urease B (UreB), one of the main H.pylori antigens. This enzyme, composed of A and B subunit neutralizes the pH around the microorganism by decomposing urea into ammonia and carbon dioxide, thus enabling H.pylori to survive in the acidic gastric environment.



Ribbon trace of the molecular model of designed chimeric flagellin vaccine fused to UreB.

We also designed synthetic multiepitope, comprising the H.pylori urease B epitope, a vacuolating cytotoxin A (Vac) epitope and an adhesin A (HpaA) epitope. B-cell epitopes were designed using available 3D models or structures, which enabled more accurate prediction. Some of them were already shown to elicit antibody production, thus our combination broadens a repertoire of activated B-cells. The multiepitope protein was also fused with the C-terminal of our chimeric flagellin.
Particularly important is the urease B epitope, including the active site of the urease beta-subunit. Application of this epitope should elicit production of neutralizing antibodies against this particular epitope, inhibiting enzymatic activity of urease, which could lead to eventual eradication of bacteria. Another important unit of the multiepitope is the vacuolating cytotoxin A epitope. VacA is a pore-forming toxin, which induces extensive vacuolation in the cytoplasm of mammalian cells. In addition it can cause depolarization of membrane potential, alteration of mitochondrial membrane permeability, apoptosis, detachment of cells from the basement membrane, activation of mitogen-activated protein kinases, inhibition of antigen presentation and inhibition of T cell activation and proliferation. VacA cytotoxic activity requires the assembly of VacA monomers into oligomeric structures. Thus we have chosen the protein segment, that is, according to the biochemical data important for its oligomerization. Neutralizing antibodies against this segment should prevent oligomerization, which should result in the inhibition of VacA cytotoxicity.



Schematic representation of the composition of multiepitope, designed based on the tertiary structure and molecular models of H.pylori proteins.

During designing a vaccine, we also took into consideration recently revealed mechanism of antigen selection in dendritic cells for a presentation by major histocompatibility complex class 2 molecules (MHC II) that is based on the origin of the antigen. The critical determinant here is whether the receptors engaged trigger activation of immune defense functions. The type of receptor–ligand interactions that occur during the early steps of phagocytosis is the essential factor in this process. Phagosomes that contain microbial cargo that engage TLRs are favored for MHC II presentation. These phagosomes mature with enhanced kinetics of fusion with the endocytic pathway, and activate hydrolytic enzymes that process li (invariant chain) making MHC II able to bind antigenic peptides. Apoptotic cells, on the other hand, do not engage TLR signaling and their phagosomes mature into terminal lysosomes where apoptotic cell proteins are completely degraded. Here, specific proteases that cleave Ii are not activated and instead MHC II bound to unprocessed Ii is targeted to lysosomes for degradation. Phagosome autonomous control by TLRs thus ensures that processing of Ii occurs only within phagosomes containing microbial cells where ligands on the cargo engage compartmentalized TLR signaling pathways. Newly formed complexes of MHC II molecules and peptides are then transported to the plasma membrane and presented to T cells, which means, that TLRs are responsible for costimulation.
To sum it all up, when designing a vaccine it is important that TLR ligands and antigens are physically linked. Thus, our idea of fusing chimeric flagellin that functions as TLR5 agonist with different antigens of Helicobacter pylori is spot on. Antigen derived peptides should be favored for presentation in MHC II molecules which would result in enhanced interactions between APCs and T-helper cells. And that is a key factor for giving rise to an effective humoral and cell immune response.