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.

Keywords: TLR3 agonist; Th1 response; dsRNA; in vitro transcription; vaccine adjuvant.

Figure 1

Schematic illustration of NexaVant (NVT) synthesis. The partial nucleotide segment (1701-2112; 412 nucleotides) from the CSBV genome was cloned into the vector. Forward and reverse PCR primers containing the T7 RNA promoter sequence were designed for the DNA template. After in vitro transcription (IVT) on the DNA template, complementary RNA strands were annealed to form dsRNA. Template DNAs and non-specific ssRNAs were removed with the treatment of DNase I and RNase T1 to generate a dsRNA structure having UAUAG-3′ at both ends and the final product was purified through column purification. nt, nucleotide.

Figure 2

Stability and biological characteristics of NVT (A) NVT was stored at 25 ± 2°C for up to 6 months. NVT bands at various concentrations were identified by electrophoresis on 1% agarose gel in each month indicated. (B) Homogeneity of NVT samples was analyzed by RP-HPLC using a two-eluent buffer system. Peak quantification was performed by recording chromatograms at 260 nm and integrating peak areas. (C) Human TLR3-expressing HEK293 cells were stimulated with 10, 50, or 100 μg/ml of NVT, or 100 μg/ml of CpG (negative control) or poly(I:C) (positive control). After 24 h, the supernatant and Quanti-Blue solution were mixed and reacted at 37°C for 1-3 h. The change in absorbance was measured at 655 nm and the TLR3 activity of the samples was normalized relative to non-treated (NT) control. (D) Rats (n=6 per group) were subcutaneously injected with 375 μg of NVT. Blood was collected at each indicated time point, total RNA was extracted, and RT-qPCR for NVT was performed to determine in vivo pharmacokinetics of NVT. *, P < 0.05; ns: not significant.

Figure 3

NVT prompts innate immune responses. (A) Experimental scheme. C57BL/6 female mice (n=4 per group) were injected into the thigh muscles of the right hind leg with OVA (2 μg) either alone or together with NVT (10 μg) or poly(I:C) (10 μg). At 0, 6 and 24 h, draining inguinal lymph node (iLN) on the right hind leg was collected, dissociated into single cells, and whole cells were counted. (B-D) Macrophages (CD11b⁺F4/80⁺), neutrophils (CD11b⁺Ly6G⁺) and DCs (CD11c^hiMHC-II^hi) were analyzed by flow cytometry, and the absolute numbers were calculated by multiplying the total iLN cells by their percentage. (E-H) The mean fluorescence intensity of CD40, CD80, CD86, and MHC-II expression on CD11c^hiMHC-II^hi DCs was determined by flow cytometric analysis. (I) The concentration of IFN-β in serum was measured by ELISA. All data were expressed as the mean values ± S.D. *, P < 0.05; N.D., not detected.

Figure 4

NVT potently enhances Th1-skewed antibody and T cell response. (A) C57BL/6 mice (n=5 per group) were primed and boosted via an intramuscular route with OVA (2 μg) either alone or adjuvanted with NVT (10 μg) or poly(I:C) (10 μg). (B-D) The levels of OVA-specific total IgG, IgG1, and IgG2c antibodies in the serum were measured at 2 weeks post-boosting. (E, F) Spleen cells were obtained from mice 2 weeks after boosting and restimulated with OVA peptides (OVA_257-264 and OVA_323-339). After blocking cytokine secretion with protein transport inhibitor, surface staining was performed with anti-CD4, anti-CD8, and anti-CD44 antibodies, followed by intracellular staining with anti-IFN-γ and anti-IL-4 antibodies. Flow cytometric analysis showed the proportion of IFN-γ⁺-expressing CD4 and CD8 T cells in the draining iLN. Data are presented as the mean values ± S.D. *, P < 0.05; ns, not significant; N.D., not detected.