Resiquimod  (Synonyms: R848; S28463)

For luciferase assay, FG-9307 cells are transfected with the firefly NF-κB-specific luciferase reporter vector pNFκB-Met-Luc2. Transfection efficiency is monitored by co-transfection with the pSEAP2 control vector, which constitutively expresses the human secreted enhanced alkaline phosphatase (SEAP). Then the cells are treated with Resiquimod (R848, 1 µg/mL), CQ (10 µM), CQ plus R848 or PBS and incubated at 22°C for 24 h. The culture medium of the transfectants is then analyzed for luciferase activity and SEAP activity using Luciferase Assay Kit and the Great EscAPe™ SEAP Chemiluminescence Detection Kit, respectively. The assay is performed three times.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

For inhibition of lysosomal acidification, cells are incubated with 10 µM CQ for 1 h before Resiquimod (R848) treatment. After treatment, 20 µL of 5 mg/mL MTT is added to the plate. The plate is incubated at 22°C for 4 h, and 200 µL dimethyl sulfoxide is added to the plate to dissolve the reduced formazan. The plate is then read at 490 nm with a microplate reader. To determine the effect of Myd88 inhibition on R848-induced cell proliferation, the Myd88 inhibitor Pepinh-MYD and the control peptide Pepinh-Control are added to PBL at the concentration of 50 µM, and the plate is incubated at 22°C for 6 h. After incubation, the cells are treated with R848 and subjected to MTT assay as above. To determine the effect of NF-κB inactivation on R848-induced cell proliferation, BAY-11-7082, an irreversible inhibitor of IκB-α phosphorylation, is added to the cells at the concentration of 1 µM, and the plate is incubated at 22°C for 1 h. After incubation, the cells are treated with R848 and subjected to MTT assay as earlier. All experiments are performed three times.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

A total of 40 SPF chickens of two-week old are allotted to one of the following four experimental groups (n=10/group): Group A: PBS control; Group B: inactivated NDV vaccine; Group C: commercial oil adjuvanted inactivated NDV vaccine prepared from lentogenic strain and Group D: combination of inactivated NDV vaccine and R-848 (50 μg/bird). Vaccine or PBS is administered by intramuscular route in the thigh muscle. A booster dose is given 14-day post immunization (d.p.i). Two weeks post-booster, experimental SPF birds are challenged with velogenic strain of NDV (10⁵ ELD₅₀ per bird) intramuscularly. Clinical signs and mortality are observed daily till 14 day post-challenge (d.p.c). Cloacal swabs (n=6/group) are collected from the birds on day 0, 4, 7 and 14 post-challenge and inoculated into 10-day old embryonated chicken eggs (n=3 eggs/sample) through intra-allantoic route. Three day post-inoculation, the allantoic fluid is checked for the NDV growth by spot haemagglutination using 10% chicken RBC.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Sachan S, et al. Adjuvant potential of resiquimod with inactivated Newcastle disease vaccine and its mechanism of action in chicken. Vaccine. 2015 Aug 26;33(36):4526-32.  [Content Brief]

  [2]. Brugnolo F, et al. The novel synthetic immune response modifier R-848 (Resiquimod) shifts human allergen-specific CD4+ TH2 lymphocytes into IFN-gamma-producing cells. J Allergy Clin Immunol. 2003 Feb;111(2):380-8.  [Content Brief]

  [3]. Zhou ZX, et al. Immune effects of R848: evidences that suggest an essential role of TLR7/8-induced, Myd88- and NF-κB-dependent signaling in the antiviral immunity of Japanese flounder (Paralichthys olivaceus). Dev Comp Immunol. 2015 Mar;49(1):113-20.  [Content Brief]