Design and Development of Stable, Water-Soluble, Human Toll-like Receptor 2 Specific Monoacyl Lipopeptides as Candidate Vaccine Adjuvants

Abstract

Antigens in modern subunit vaccines are largely soluble and poorly immunogenic proteins inducing relatively short-lived immune responses. Appropriate adjuvants initiate early innate immune responses, amplifying subsequent adaptive immune responses. Agonists of TLR2 are devoid of significant pro-inflammatory activity in ex vivo human blood models, and yet potently adjuvantic, suggesting that this chemotype may be a safe and effective adjuvant. Our earlier work on the monoacyl lipopeptide class of TLR2 agonists led to the design of a highly potent lead, but with negligible aqueous solubility, necessitating the reintroduction of aqueous solubility. We explored several strategies of introducing ionizable groups on the lipopeptide, as well as the systematic evaluation of chemically stable bioisosteres of the ester-linked palmitoyl group. These studies have led to a fully optimized, chemically stable, and highly water-soluble, human TLR2-specific agonist, which was found to have an excellent safety profile and displayed prominent adjuvantic activities in rabbit models.

Structure‐Based Design of Novel Human Toll‐like Receptor 8 Agonists

“…The most potent analogue 14b was characterized further in cytokine/chemokine induction profiles in a panel of secondary screens employing human peripheral blood mononuclear cells [8a] as well as whole human blood. [22c] Consistent with its specificity for TLR8, we observed the induction of a specific set of chemokines and proinflammatory cytokines, including interleukins 12 and 18 (Fig. 6).…”

“…All analogues were counter-screened [8c, 19b, 22] in reporter cell lines specific for human TLR2, TLR3, TLR4, TLR5, TLR7, TLR9, TLR10, Nod1 and Nod2, and compounds 6 , 9 , 12 , and 14a - f were confirmed to be specific for human TLR8. The most potent analogue 14b was characterized further in cytokine/chemokine induction profiles in a panel of secondary screens employing human peripheral blood mononuclear cells [8a] as well as whole human blood.…”

Structure-Based Design of Human TLR8-Specific Agonists with Augmented Potency and Adjuvanticity

“…Pure TLR8 agonists, as discussed earlier, evoke the production of Th1-biased cytokines such as TNF-α, IL-1, IL-12, IL-18, and IFN-γ from cells of the monocytoid lineage; pure TLR7-active compounds induce the copious production of IFN-α from low-abundance plasmacytoid cells, activate natural killer (NK), 73 and induce mitogenicity in B lymphocytes (manuscript in preparation) and are much weaker in inducing TNF-α and IFN-γ; TLR2 agonists, in contrast, activate neutrophils as evidenced by rapid upregulation of CD11b and p38 MAP kinase activity. 43 , 44 The observation that all these chemotypes display adjuvantic activities may signify that the disparate outcomes in different cell types may point to different mechanisms mediating adjuvantic activities such as, as discussed earlier, enhanced antigen uptake and presentation by APCs, 23 − 30 enhanced CD4 + T helper cell activation, 31 − 33 or affinity maturation of antibodies. 38 , 39 …”

Strategies for designing synthetic immune agonists

“…Many synthetic lipopeptides have introduced polyethylene glycol (PEG) or sugar moieties at the peptide region with the aim of improving solubility and/or amphiphilic properties of these otherwise poorly soluble and lipophilic molecules . Further simplifying the structure, a series of monoacyl lipopeptides was discovered to be human‐specific TLR2 agonists . Another simplified class of lipoamino acid agonist was identified by maintaining the minimal structure requirement for TLR2 activity …”