R848 VacciGrade™

TLR7/TLR8 Agonist - Imidazoquinoline compound | Th1 response

TLR7/TLR8 activation with R848

R848 (Resiquimod), an imidazoquinoline, is a dual TLR7 and TLR8 guanosine derivative with potent antiviral activity [1-3]. It induces differential TLR7 and/or TLR8 responses in human and murine immune cells. TLR7 and TLR8 are endosomal pattern recognition receptors that play an important role in the antiviral immune response.

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Mode of action

R848 (Resiquimod), a low molecular weight synthetic molecule, acts as a selective activating ligand for both TLR7 and TLR8 in humans but only TLR7 in mice. It activates immune cells via the TLR7/TLR8 MyD88-dependent signaling pathway with the subsequent activation of the transcription factors NF-κB and interferon regulatory factor (IRF) [2, 3]. This ultimately leads to the production of pro-inflammatory cytokines and type I interferons. 

Unlike other commercially available R848 preparations, InvivoGen's R848 VacciGrade™ is water-soluble, controlled for TLR7/8 potency and TLR4/TLR2 contamination, and available in a high-quality, pre-clinical grade. 

In addition, InvivoGen offers R848 (Resiquimod) in a standard grade for in vitro experiments. 

R848 VacciGrade™ is a high-quality pre-clinical grade. 

Imiquimod VacciGrade™​ is for research use only, and not for human or veterinary use.

Key features of R848

• Agonist of hTLR7 and hTLR8
• Agonist of mTLR7
• Each lot is highly pure (≥95%) and functionally tested
• Standard R848 is also available for in vitro assays

Read our review about TLR7 and TLR8.

References:

1. Vanwalscappel B. et al., 2018. Toll-like receptor agonist R848 blocks Zika virus replication by inducing the antiviral protein viperin. Virology 522:199-208.
2. Hemmi H. et al., 2002. Small anti-viral compounds activate immune cells via the TLR7 MyD88-dependent signaling pathway. Nat Immunol. 3(2):196-200.
3. Jurk M. et al. 2002. Human TLR7 or TLR8 independently confer responsiveness to the antiviral compound R848. Nat Immunol. 3(6):499.

Figures

NF-κB response of HEK-Blue™-derived cells to R848. HEK-Blue™ cells expressing hTLR7, mTLR7, hTLR8, or mTLR8 cultured in HEK-Blue™ Detection reagent and stimulated with increasing concentrations of R848. After 24h incubation, the NF-κB-induced SEAP activity was assessed by measuring the SEAP level in the supernatant. Data are shown as optical density (OD) at 650 nm (mean ± SEM). Of note, HEK-Blue™ Null* comprises data from parental cell lines HEK-Blue Null1, HEK-Blue Null1-v, HEK-Blue Null2-k.

NF-κB and IRF responses of THP1-Dual™-derived cells to R848. THP1-Dual™, THP1-Dual™ hTLR7 cells, and THP1-Dual™ hTLR8 cells were incubated for 24 hours with increasing concentrations of R848. After 24h incubation, the (A) NF-κB-induced SEAP activity was assessed using QUANTI-Blue™. Data are shown as optical density (OD) at 650 nm (mean ± SEM). (B) The IRF response was assessed by measuring the activity of Lucia luciferase in the supernatant using QUANTI-Luc™. Data are shown in fold response over non-induced cells (mean ± SEM).

Specifications

Description: TLR7/8 agonist VacciGrade™

Polarization of adaptive immune response: Th1 response

CAS number: 144875-48-9 (free base)

Working concentration: 10 - 100 μg/mouse

Solubility: 1 mg/ml in physiological water

Quality control:

• Sterility guaranteed
• The absence of bacterial contamination (lipoproteins & endotoxins) has been confirmed using HEK-Blue™ TLR2 and HEK-Blue™ TLR4 cells
• Endotoxin level < 1 EU/mg (measurement by kinetic chromogenic LAL assay)

Contents

R848 VacciGrade™ is provided lyophilized:

• 5 mg of sterile lyophilized R848 VacciGrade™.
• 10 ml sterile endotoxin-free physiological water (NaCl 0.9%).

R848 VacciGrade™ is shipped at room temperature

R848 VacciGrade™ should be stored at 4°C or -20°C.

Lyophilized product is stable 1 year when properly stored.

Upon resuspension, prepare aliquots of R848 VacciGrade™ and store at -20°C for long term storage.

Resuspended product is stable 6 months 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.

Details

R848 (resiquimod), a small molecular weight imidazoquinoline compound, is an immune response modifier with potent antiviral and antitumor activities [1].

R848 is being evaluated as an adjuvant in FDA-approved clinical vaccine trials. R848 immune properties result from its ability to induce the production of pro-inflammatory cytokines through the activation of Toll-like receptor (TLR)-7 and TLR8 [2].

In vitro and in vivo studies have shown that R848 promotes the secretion of Th1 cytokines, including IFN-γ, IFN-α, IL-12 and TNF-α [3-7].

R848 is capable of skewing antibody responses toward a Th1 IgG2a response and away from a Th2 IgE response, a feature mediated in part by IFN-α and IL-12.

Unlike most adjuvants, R848 can be administered by a different route than the antigen, suggesting that it does not produce a depot effect.

Preclinical studies in mice have shown that R848 is able to promote adaptive immune responses to codelivered antigens and provide protection against live infection challenges [4, 6, 8, 9].

1. Stanley MA., 2002. Imiquimod and the imidazoquinolines: mechanism of action and therapeutic potential. Clin Dermatol 27:571–7.
2. Hemmi H. et al., 2002. Small anti-viralcompounds activate immune cells via the TLR7 MyD88-dependent signaling pathway. Nat. Immunol. 3:196-200.
3. Wagner tL. et al., 1999. Modulation of TH1 and TH2 cytokine production with the immune response modifiers, R-848 and imiquimod. Cell. Immunol. 191, 10, 1999.
4. Vasilakos Jp. et al., 2000. Adjuvant activities of immune response modifier R-848: comparison with CpG ODN. Cell. Immunol. 204:64-74.
5.Thomsen L. et al., 2004. Imiquimod and resiquimod in a mouse model: adjuvants for DNA vaccination by particle-mediated immunotherapeutic delivery. Vaccine 22:1799-1809.
6. Baldwin sL. et al., 2009. Intradermal immunization improves protective efficacy of a novel TB vaccine candidate. Vaccine 27:3063-3071.
7. Ma Y. et al., 2010. Assessing the immunopotency of Toll-like receptor agonists in an in vitro tissue engineered immunological model. Immunology 130:374-387.
8. tomai MA. et al., 2000. The immune response modifiers imiquimod and R-848 are potent activators of B lymphocytes. Cell. Immunol. 203:55-65.
9. Zhang WW. & G. Matlashewski. 2008.Immunization with a Toll-like receptor 7 and/or 8 agonist vaccine adjuvant increases protective immunity against Leishmania major in BALB/c mice. Infect. Immun. 76:3777-3783.