TLR4 and TLR7/8 Adjuvant Combinations Generate Different Vaccine Antigen-Specific Immune Outcomes in Minipigs when Administered via the ID or IN Routes.

The induction of high levels of systemic and mucosal humoral immunity is a key goal for many prophylactic vaccines. However, adjuvant strategies developed in mice have often performed poorly in the clinic. Due to their closer similarity to humans, minipigs may provide a more accurate picture of adjuvant performance. Based on their complementary signalling pathways, we assessed humoral immune responses to model antigens after co-administration with the toll-like receptor 4 (TLR4) stimulator glucopyranosyl lipid adjuvant (GLA-AF) or the TLR7/8 agonist resiquimod (R848) (alone and in combination) via the intradermal (ID), intranasal (IN) or combined routes in the Gottingen minipig animal model. Surprisingly, we discovered that while GLA-AF additively enhanced the adjuvant effect of R848 when injected ID, it abrogated the adjuvant activity of R848 after IN inoculation. We then performed a route comparison study using a CN54 gp140 HIV Envelope model antigen adjuvanted with R848 + GLA-AF (ID) or R848 alone (IN). Animals receiving priming inoculations via one route were then boosted by the alternate route. Although differences were observed in the priming phase (IN or ID), responses converged upon boosting by the alternative route with no observable impact resultant from the order of administration (ID/IN vs IN/ID). Specific IgG responses were measured at a distal mucosal site (vaginal), although there was no evidence of mucosal linkage as these closely reflected serum antibody levels. These data indicate that the complex in vivo cross-talk between innate pathways are likely tissue specific and cannot be predicted by simple in vitro models.

6-(4-Amino-2-butyl-imidazoquinolyl)-norleucine: Toll-like receptor 7 and 8 agonist amino acid for self-adjuvanting peptide vaccine.

Generally, small peptides by themselves are weak to induce antibody responses. Toll-like receptor (TLR) ligands are attractive candidates of vaccine adjuvants to improve their antigenicity. The covalent conjugation of TLR ligands with antigens to produce self-adjuvanting peptide vaccine is a promising approach. Based on the structure of TLR7/8 ligands, a series of synthetic amino acids 6-imidazoquinolyl-norleucines were synthesized, wherein an imidazoquinoline structure as the TLR7/8 agonistic pharmacophores was constructed on the ε-NH2 group of Lys. Of them, 6-(4-amino-2-butyl-imidazoquinolyl)-norleucine showed the most potent TLR7 and TLR8 agonistic activities with EC50 values of 8.55 and 106 µM, respectively. Subsequently, mice were immunized with the influenza A virus M2e antigen mixed with or covalently conjugated to the TLR7/8 agonist amino acid, which led to induction of M2e specific antibody productions in the absence of other adjuvant. We successfully developed a novel efficient tool for self-adjuvanting peptide vaccines targeting TLR7/8.

Administration of a dual toll-like receptor 7 and toll-like receptor 8 agonist protects against influenza in rats.

Toll-like receptors (TLR) detect conserved molecular patterns expressed by pathogens. Detection of the "molecular signature" for RNA viruses including influenza has been attributed to TLR3, TLR7, and TLR8. In the present study, compound 3M-011 was shown to be a synthetic human TLR7/8 agonist and cytokine inducer. 3M-011 was investigated as a stand-alone immune response modifier in a rat model of human influenza. Intranasal (IN) administration of 3M-011 significantly inhibited H3N2 influenza viral replication in the nasal cavity when administered from 72 h before IN viral inoculation to 6h after inoculation. Viral inhibition correlated with the ability of the TLR7/8 agonist to stimulate type I interferon (IFN) and other cytokines such as tumor necrosis factor-alpha, interleukin-12, and IFN-gamma from rat peripheral blood mononuclear cells. Prophylactic administration of TLR7/8 agonist also suppressed influenza viral titers in the lung, which corresponded with local IFN production. The activity of the TLR7/8 agonist resulted in greater inhibition of viral titers compared to rat recombinant IFN-alpha administered in a comparable dosing regimen. These studies indicate that TLR7/8 agonists may have prophylactic and therapeutic benefits in the treatment of respiratory viral infections, such as influenza, when administered prior to or shortly after viral inoculation.