Adjuvanted Vaccine Induces Functional Antibodies against Pseudomonas aeruginosa Filamentous Bacteriophages.

Pseudomonas aeruginosa (Pa), a WHO priority 1 pathogen, resulted in approximately 559,000 deaths globally in 2019. Pa has a multitude of host-immune evasion strategies that enhance Pa virulence. Most clinical isolates of Pa are infected by a phage called Pf that has the ability to misdirect the host-immune response and provide structural integrity to biofilms. Previous studies demonstrate that vaccination against the coat protein (CoaB) of Pf4 virions can assist in the clearance of Pa from the dorsal wound model in mice. Here, a consensus peptide was derived from CoaB and conjugated to cross-reacting material 197 (CRM197). This conjugate was adjuvanted with a novel synthetic Toll-like receptor agonist (TLR) 4 agonist, INI-2002, and used to vaccinate mice. Mice vaccinated with CoaB-CRM conjugate and INI-2002 developed high anti-CoaB peptide-specific IgG antibody titers. Direct binding of the peptide-specific antibodies to whole-phage virus particles was demonstrated by ELISA. Furthermore, a functional assay demonstrated that antibodies generated from vaccinated mice disrupted the replicative cycle of Pf phages. The use of an adjuvanted phage vaccine targeting Pa is an innovative vaccine strategy with the potential to become a new tool targeting multi-drug-resistant Pa infections in high-risk populations.

A Novel Synthetic Dual Agonistic Liposomal TLR4/7 Adjuvant Promotes Broad Immune Responses in an Influenza Vaccine With Minimal Reactogenicity.

The limited efficacy of seasonal influenza vaccines is usually attributed to ongoing variation in the major antigenic targets for protective antibody responses including hemagglutinin (HA) and neuraminidase (NA). Hence, vaccine development has largely focused on broadening antigenic epitopes to generate cross-reactive protection. However, the vaccine adjuvant components which can accelerate, enhance and prolong antigenic immune responses, can also increase the breadth of these responses. We previously demonstrated that the combination of synthetic small-molecule Toll-like receptor 4 (TLR4) and TLR7 ligands is a potent adjuvant for recombinant influenza virus HA, inducing rapid, and sustained antibody responses that are protective against influenza viruses in homologous and heterologous murine challenge models. To further enhance adjuvant efficacy, we performed a structure-activity relationship study for the TLR4 ligand, N-cyclohexyl-2-((5-methyl-4-oxo-3-phenyl-4,5-dihydro-3H-pyrimido[5,4-b]indol-2-yl)thio)acetamide (C25H26N4O2S; 1Z105), and identified the 8-(furan-2-yl) substituted pyrimido[5,4-b]indole analog (C29H28N4O3S; 2B182C) as a derivative with higher potency in activating both human and mouse TLR4-NF-κB reporter cells and primary cells. In a prime-boost immunization model using inactivated influenza A virus [IIAV; A/California/04/2009 (H1N1)pdm09], 2B182C used as adjuvant induced higher serum anti-HA and anti-NA IgG1 levels compared to 1Z105, and also increased the anti-NA IgG2a responses. In combination with a TLR7 ligand, 1V270, 2B182C induced equivalent levels of anti-NA and anti-HA IgG1 to 1V270+1Z105. However, the combination of 1V270+2B182C induced 10-fold higher anti-HA and anti-NA IgG2a levels compared to 1V270+1Z105. A stable liposomal formulation of 1V270+2B182C was developed, which synergistically enhanced anti-HA and anti-NA IgG1 and IgG2a responses without demonstrable reactogenicity after intramuscular injection. Notably, vaccination with IIAV plus the liposomal formulation of 1V270+2B182C protected mice against lethal homologous influenza virus (H1N1)pdm09 challenge and reduced lung viral titers and cytokine levels. The combination adjuvant induced a greater diversity in B cell clonotypes of immunoglobulin heavy chain (IGH) genes in the draining lymph nodes and antibodies against a broad spectrum of HA epitopes encompassing HA head and stalk domains and with cross-reactivity against different subtypes of HA and NA. This novel combination liposomal adjuvant contributes to a more broadly protective vaccine while demonstrating an attractive safety profile.

Species-Specific Structural Requirements of Alpha-Branched Trehalose Diester Mincle Agonists.

Despite the ever present need for an effective Mycobacterium tuberculosis (Mtb) vaccine, efforts for development have been largely unsuccessful. Correlates of immune protection against Mtb are not wholly defined, but Th1 and likely Th17 adaptive immune responses have been demonstrated to be necessary for vaccine-mediated protection. Unfortunately, no approved adjuvants are able to drive a Th17 response, though recent clinical trials with CAF01 have demonstrated proof of concept. Herein we present the discovery and characterization of a new class of potential Th17-inducing vaccine adjuvants, alpha-branched trehalose diester molecules (αTDE). Based off the Mtb immunostimulatory component trehalose dimycolate (TDM), we synthesized and evaluated the immunostimulatory capacity of a library of structural derivatives. We evaluated the structure activity relationship of the compounds in relation to chain length and engagement of the Mincle receptor, production of innate cytokines from human and murine cells, and a pro-Th17 cytokine profile from primary human peripheral blood mononuclear cells. Murine cells displayed more structural tolerance, engaging and responding to a wide array of compound chain lengths. Interestingly, human cells displayed a unique specificity for ester chains between 5 and 14 carbons for maximal immune stimulating activity. Evaluation of two distinct αTDEs, B16 and B42, in concert with a recombinant Mtb antigen demonstrated their ability to augment a Th17 immune response against a Mtb antigen in vivo. Collectively this data describes the species-specific structural requirements for maximal human activity of alpha-branched trehalose diester compounds and demonstrates their capacity to serve as potent Th17-inducing adjuvants.