A novel defined TLR3 agonist as an effective vaccine adjuvant

Abstract

Synthetic double-stranded RNA analogs recognized by Toll-like receptor 3 (TLR3) are an attractive adjuvant candidate for vaccines, especially against intracellular pathogens or tumors, because of their ability to enhance T cell and antibody responses. Although poly(I:C) is a representative dsRNA with potent adjuvanticity, its clinical application has been limited due to heterogeneous molecular size, inconsistent activity, poor stability, and toxicity. To overcome these limitations, we developed a novel dsRNA-based TLR3 agonist named NexaVant (NVT) by using PCR-coupled bidirectional in vitro transcription. Agarose gel electrophoresis and reverse phase-HPLC analysis demonstrated that NVT is a single 275-kDa homogeneous molecule. NVT appears to be stable since its appearance, concentration, and molecular size were unaffected under 6 months of accelerated storage conditions. Moreover, preclinical evaluation of toxicity under good laboratory practices showed that NVT is a safe substance without any signs of serious toxicity. NVT stimulated TLR3 and increased the expression of viral nucleic acid sensors TLR3, MDA-5, and RIG-1. When intramuscularly injected into C57BL/6 mice, ovalbumin (OVA) plus NVT highly increased the migration of dendritic cells (DCs), macrophages, and neutrophils into inguinal lymph node (iLN) compared with OVA alone. In addition, NVT substantially induced the phenotypic markers of DC maturation and activation including MHC-II, CD40, CD80, and CD86 together with IFN-β production. Furthermore, NVT exhibited an appropriate adjuvanticity because it elevated OVA-specific IgG, in particular, higher levels of IgG2c (Th1-type) but lower IgG1 (Th2-type). Concomitantly, NVT increased the levels of Th1-type T cells such as IFN-γ+CD4+ and IFN-γ+CD8+ cells in response to OVA stimulation. Collectively, we suggest that NVT with appropriate safety and effectiveness is a novel and promising adjuvant for vaccines, especially those requiring T cell mediated immunity such as viral and cancer vaccines.

Amphiphilic Block Copolymer Nanostructures as a Tunable Delivery Platform: Perspective and Framework for the Future Drug Product Development

“…Synthetic nucleic acid analogues that bind and activate these receptors have long been recognized for their potential as immune stimulants and vaccine adjuvants. These include poly­(I:C) (TLR3 agonist), imidazoquinolines, and oxoadenines (TLR7/8 agonists). , Unlike the limited distribution of TLR9 in humans, TLR3 is expressed in various tissues and organs, whereas TLR7 and TLR8 are expressed by all types of APCs (B cells, pDCs, mDCs, monocytes, and macrophages) as well as neutrophils . The broad distribution profile of these receptors is expected to facilitate efficient induction of an immune response to an appropriate ligand.…”

De novo design of protein minibinder agonists of TLR3

“…Upon ligand binding, TLR3 forms highly organized and cooperative complexes of dimers that cooperatively assemble along linear dsRNA 11 . However, dsRNA is not an ideal adjuvant due to its instability, structural heterogeneity, and promiscuity 16,36 . Stabilized formulations exist (e.g., poly-ICLC) but are still heterogeneous and activate other immune receptors (MDA-5, RIG-1) [37][38][39][40] .…”

The defined TLR3 agonist, Nexavant, exhibits anti-cancer efficacy and potentiates anti-PD-1 antibody therapy by enhancing immune cell infiltration

“…In this regard, It is attracting attention as vaccine adjuvants and in situ vaccines. Poly(I:C) is synthesized poly-inosine and poly-cytidine conjugates with dsRNAs’ structure but have unordered complementarities, resulting in high heterogeneity 16, 17 . The nature of Poly(I:C) significantly challenges stability, quality control, and monitoring in vivo PK.…”

“…Unlike Poly(I:C), Nexavant can be produced up to 99% purity with a scale-up and cost-effective synthesis. Moreover, it provides accurate pharmacokinetic and pharmacodynamic data, offering significant regulatory advantages 17 .…”

“…We recently presented Nexavant as a well-defined TLR3 agonist 17 . Nexavant offers several advantages over Poly(I:C) regarding quality control and clinical applicability.…”