Development of New Toll-Like Receptor 4-directed adjuvants and Clarification of their Mechanism of Action

Antimicrobial resistance microorganisms are now a permanent concern in global health systems, causing high number of infection-associated morbidity and mortality. While the pipeline for new antibiotics has been halted, investing in this strategy does not tackle the resistance problem. Vaccines have been decreasing disease burden since their creation in the 18th century. Undeniably, their contribution to public health has been dramatic, contributing to the decrease of severity and incidence of infectious diseases. Subunit vaccines are of particular interest since they can contain a desired antigen, responsible for eliciting a pathogen-specific response. While safer than whole pathogen vaccines, they are often less immunogenic. Accordingly, subunit vaccines should be formulated with an adjuvant. An adjuvant is a chemical entity capable of enhancing and/or modulating the antigen’s immune response and overall vaccine efficacy. Adjuvant development is slow and for several decades, alum remained the only approved adjuvant for human use. Thus, there is still a need for novel chemical entities. Considering the role of an adjuvant in a vaccine, clarification of its mechanism of action is essential for the formulations’ success. Toll-like receptors are important innate immune receptors that recognize microbial components and trigger a downstream of cellular events leading to a proinflammatory immune response and modulation of the adaptive memory. Particularly, TLR4, whose natural ligand is lipopolysaccharide from gram negative bacteria, has been extensively studied and several new agonists have been developed as vaccine adjuvants. MPLA, a detoxified analog of the bioactive portion of LPS, is a TLR4-agonist that has been approved as vaccine adjuvant for human use. It is now incorporated into different marketed vaccines in different presentations. The aim of this work was to design and synthesize new glycolipid TLR4-directed adjuvants and clarify their mechanism of action using different cell-biology techniques. New TLR4 agonists have been synthesized based on lipid X and its TLR4-stimulating analogs. Their biological characterization was carried out using a human macrophage- like cell line and by employing ELISA and western blot techniques to measure cytokine production and protein expression. High throughput imaging techniques were also used to follow intracellular targets using fluorescence labelling. FP20 and its derivatives have demonstrated to be active and selective TLR4 agonists with activity both in vitro and in vivo. Interestingly, its C6 functionalized derivatives, FP20Glyco and FP20Mpa, have demonstrated a dramatic increase in activity compared to the parent compound. In particular, FP20Mpa was formulated with an Enterococcus faecium antigen, in a novel vaccine formulation, and it enhanced the production of antigen-specific antibodies. Additionally, a novel FTIR screening method for proinflammatory compounds was developed using LPS and applied to the project in order to identify new proinflammatory TLR4-stimulating molecules. Overall, this project combined a multidisciplinary approach for the development of new TLR4-directed adjuvants and clarification of their mechanism of action.