NexaVant™ VacciGrade™

TLR3 agonist - NexaVant™ (NVT) | Homogeneous and sterile vaccine adjuvant

NexaVant™​ VacciGrade™ is a double-stranded (ds) RNA-based TLR3 agonist and potent vaccine adjuvant, provided in a high-quality pre-clinical grade.

NexaVant™ (NVT) is a synthetic dsRNA of 424 base pairs chosen from the Chinese sacbrood virus (CSBV) genome. It displays high purity, molecular homogeneity, measurable pharmacokinetics, and non-toxicity in various animals, therefore overcoming various obstacles of currently available dsRNA-based adjuvants (e.g. Poly(I:C)) [1]. It is a potent TLR3 agonist verified using HEK-Blue™ hTLR3 cells (see figures) [1]. It also upregulates specific interferon-stimulated genes (ISGs) including RIG-I, MDA-5, and TLR3 [1]. 

In vivo experiments in mice demonstrated NVT's ability to attract immune cells, activate dendritic cells, and induce strong Th1-type immune responses, such as IFN-γ+ CD4+ and IFN-γ+ CD8+ T cells, surpassing conventional adjuvants like Alum [1]. Moreover, NVT is a promising adjuvant for T cell vaccines, showing enhanced protection against influenza infections when used intranasally, promoting lung-resident memory CD4+ T cells that provide broad and long-term immunity against heterosubtypic strains [2].

InvivoGen also offers NexaVant™ in a standard grade for in vitro experiments. 

Production of NVT

A nucleotide segment (1701-2112; 412 nucleotides) from the CSBV genome, which does not match any human DNA sequence,  was cloned into a vector. After in vitro transcription using T7 RNA polymerase technology, the remaining 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. Subsequently, the final product was purified by reverse-phase HPLC [1]. NexaVant™ is available at cGMP grade from the NA Vaccine Institute.

Key features

• Potent TLR3 agonist and vaccine adjuvant
• Strong Th1 responses inducer
• Highly pure (≥ 95%) and stable
• Ready-to-use liquid
• Batch-to-batch consistency
• Each lot is functionally tested

NexaVant™  VacciGrade™ is a high-quality pre-clinical grade. It is also available in a standard grade as NexaVant™.
NexaVant™ is a trademark that belongs to the NA Vaccine Institute. 
NexaVant™ VacciGrade™​ is for research purposes only; not for human or veterinary use.

References:

1. Ko KH, et al., 2023. A novel defined TLR3 agonist as an effective vaccine adjuvant. Front Immunol. ;14:1075291.
2. Ko KH, et al. 2024. A vaccine platform targeting lung-resident memory CD4+ T-cells provides protection against heterosubtypic influenza infections in mice and ferrets. Nat Commun.15(1):10368.

Figures

Molecular weight of NexaVant™ compared to Poly(I:C) HMW.  Poly(I:C) high molecular weight (HMW) (10 µg) and NexaVant™ (100 ng) were visualized on 1% agarose gel. NexaVant™ appears as a single dsRNA band with a length of 424 bp (approximately 275 kDa), while Poly(I:C) migrated as a smear with an average size between 1 kb to 8 kb.

TLR3 response to NexaVant™. HEK-Blue™ hTLR3 cells were stimulated with 10, 50, or 100 μg/ml of NexaVant™ (NVT). 100 µg/ml of TLR3-agonist Poly(I:C) served as a positive control. After 24h incubation, the NF-κB-induced SEAP activity was assessed using QUANTI-Blue™. The change in absorbance was measured at 655 nm and the TLR3 activity of the samples was normalized relative to non-treated (NT) control.

Note: Graphs are based on literature data taken from Ko KH, et al., 2023.

Total IgG level in serum of mice after intramuscular administration. Three mice per group were treated intramuscularly with Ovalbumin (OVA, 100 µg) ± NexaVant™ (NVT; 5 µg), Poly(I:C) (50 µg), or Alum (100 µg) twice for 4 weeks. The serum level of lgG was analyzed using ELISA in duplication.
NT = non-treated

Note: Graphs are based on literature data taken from https://navaccine.org/

Antigen-specific T cell responses after NexaVant™ treatment. C57BL/6 mice (n=5 per group) were primed and boosted via an intramuscular route with Ovalbumin (OVA, 2 μg) either alone or adjuvanted with NexaVant™ (NVT; 10 μg) or Poly(I:C) (10 μg). Spleen cells were obtained from mice two weeks after boosting and restimulated with OVA peptides. After blocking cytokine secretion with a 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. Data are presented as the mean values ± S.D.

Note: Graphs are based on literature data taken from Ko KH, et al., 2023.

Specifications

Description: TLR3 agonist VacciGrade™

CAS Number: 2839526-76-8

Polarization of innate immune response: Macrophages, Neutrophils, Dendritic cells

Polarization of adaptive immune response: Th1 response

Quantity: 100 µl (vac-nvt) or 5 x 100 µl (vac-nvt-5)

Concentration: 1 mg/ml

Buffer composition: 10mM Phosphate Buffer pH 7.2

A260/A280 ratio: 1.8 ~ 2.2

Size: 424 bp

Dosing  Guidelines:

• Mice: ≥10 µg/dose (as vaccine adjuvant) and ≥ 50 µg/dose (as anti-cancer vaccine)

Quality control:

• Sterility guaranteed
• Each lot is functionally tested using cellular assays
• The absence of bacterial contamination (lipoproteins & endotoxins) has been confirmed using HEK-Blue-Lucia™ hTLR2 and HEK-Blue-Lucia™ mTLR4 cells
• Endotoxin level < 5 EU/mg (measurement by kinetic chromogenic LAL assay)

Contents

NexaVant™ VacciGrade™ is provided as a colorless transparent liquid and is available in two quantities: 

• vac-nvt: 100 µg at 1 mg/ml
• vac-nvt-5: 5 x 100 µg at 1 mg/ml

NexaVant™ VacciGrade™ is shipped at room temperature.

Upon receipt, store at 4°C. NexaVant™ is stable at RT. However, to avoid contamination, we recommend to keep aliquots at 4°C for short-term storage or -20°C for long-term storage.

 The product is stable for up to 1 year when properly stored.

 Avoid repeated freeze-thaw cycles.

VacciGrade™

VacciGrade™ is a high-quality pre-clinical grade. VacciGrade™ products are filter-sterilized (0.2 µm) and filled under strict aseptic conditions in a clean room*. The absence of bacterial contamination is assessed by a sterility test using a pharmacopeia-derived assay. The level of bacterial contaminants (endotoxins and lipoproteins) in each lot is verified using a LAL assay and/or a TLR2 and TLR4 reporter assay.
*Except for LPS VacciGrade™, which is prepared in a laminar flow hood dedicated to LPS.