Vaccines Incorporating Toll-Like Receptor Ligands - Attaching the HA antigen to a TLR can produce a strong immunogenic response and can be produced quickly and easily in E. coli. - BioPharm


Vaccines Incorporating Toll-Like Receptor Ligands
Attaching the HA antigen to a TLR can produce a strong immunogenic response and can be produced quickly and easily in E. coli.

BioPharm International Supplements

How does this relate to vaccines? First, it explains why FCA works so well. Mycobacteria, the active ingredient of FCA, are also pathogens, and therefore, contain numerous PAMPs. More importantly, it suggests that using a TLR as a portal of entry for an antigen into an APC might result in more efficient antigen processing and presentation. This, in turn, should elicit a more robust immune response.

Multiple PAMP–TLR pairs are available for use as a portal of entry (Figure 1).6 However, only one of these pairs, flagellin-TLR5, involves a purely protein ligand and a cell- surface TLR. Coupling the DNA sequence for flagellin in frame with the DNA sequence for a protein antigen and expressing this chimeric assembly in E. coli yields a protein that contains the cell entry and immunostimulatory activity of both a TLR ligand and an antigen in a single molecule. This ensures that both components enter not only the same cell but also the same endocytic vesicle within that cell. Thus, the target antigen is present within an endosome that contains an activated TLR, ensuring that both components are trafficked through the same endosome-lysosome-processing and presentation cycle in close proximity. Elegant confocal microscopy studies using other PAMPs, including LPS, have shown that this is the case at the cellular level.4

Co-Delivering Antigens and TLR Ligands to APCs

We have built our platform technology on the concept of co-delivering vaccine antigens and TLR ligands to APCs. Using this technology, we have demonstrated that coupling flagellin to a model antigen, ovalbumin, can elicit a rapid IgG antibody response in mice one week after a single dose; ovalbumin alone on aluminum hydroxide takes three weeks to reach the same level of antibody.7 The same flagellin–ovalbumin fusion elicits a classical T-helper 1 antibody and CD8+ T-cell response, while ovalbumin alone, or mixed with flagellin, yields a classical T-helper 2 response.1,7 Similar results have been obtained by a fusion of flagellin to antigens from Listeria monocytogenes.1

The same strategy has recently been applied for developing a West Nile virus vaccine that is based on the conformation-dependent structure of a subunit of the envelope of the virus, EIII. EIII contains a disulfide bond that is essential for proper folding of the domain and presentation of epitopes that are recognized by virus-neutralizing antibodies.8 Genetic fusion of the sequence for EIII to the sequence for flagellin yields a protein that retains TLR5 activity and properly folds the EIII domain such that it is recognized by neutralizing monoclonal antibodies and induces protective immune response in mice.8 The ability to express such a conformation-dependent protein in E. coli suggested that this strategy could be applied to other vaccine targets.7

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