Polyinosinic polycytidylic acid poly 1 c

The anti-hMR antibody B11 was generated by immunizing human immunoglobulin transgenic mice with human mannose receptor. The monoclonal antibody (mAb) B11 binds human mannose receptor, but not mouse mannose receptor.^{11 (link)} The B11-hCGβ fusion protein was generated by genetically coupling hCGβ to the carboxyl terminus of the B11 heavy chain, and clinical grade material was manufactured from transfected Chinese hamster ovary cells.^{22 (link),23 (link)} The labeling of B11-hCGβ with Alexa-647 was performed according to the manufacturer's protocol (Invitrogen, Carlsbad, CA, USA). Antibodies for staining of CD3ε (145-2C11), CD4 (H129.19), CD8α (53-6.7), CD11c (HL3), MHC class II I-A/I-E (M5/114.15.2), F4/80 (BM8) and CD103 (2E7) were purchased from BD Biosciences (San Jose, CA, USA) or eBioscience (San Diego, CA, USA). hMR was stained with either B11 or 19.2 (BD Biosciences), mouse MR (mMR) with MR5D3, and DEC-205 with NLDC-145 (AbD Serotec, Raleigh, NC, USA and BMA Biomedicals, Augst, Switzerland). Mouse GM-CSF was from Peprotech (Rocky Hill, NJ, USA). Complete Freund's adjuvant (CFA) was from Sigma-Aldrich (St. Louis, MO, USA). CpG (ODN1826) and polyinosinic-polycytidylic acid (poly-IC) were from InvivoGen (San Diego, CA, USA). Poly-ICLC (poly-IC stabilized with poly-lysine and carboxymethylcellulose) was supplied by Oncovir, Inc (Washington, DC, USA).

A RISO™ RNA isolation reagent (Biomics Biotech, China) was used to extract total RNA from cells with different treatments. The mRNA levels of target genes (VEGF, NET-1 and TLR3) were determined by RT-qPCR using EzQuick^TM one-step qPCR kit (Biomics Biotech, China) according to the manufacturer's instructions. To exclude the influence of interferon (IFN) on gene silencing effects of teRNA, the mRNA levels of 2'-5'-oligoadenylate synthetase 1 (OAS1) and interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) genes were measured. For a positive control, cells were transfected with 20 ng/mL polyinosinic-polycytidylic acid (polyI:C) (InvivoGen, USA). Specific primers for each gene were presented in Table 2. Samples were tested in triplicate. GAPDH expression was used as an internal control.

Synthetic peptides were purchased from Genecust (Dudelange, Luxembourg) and dissolved in 10% dimethyl sulfoxide in phosphate-buffered saline (PBS). The purity of the peptides was >80%. Montanide was provided by SEPPIC and Berna-Biotech (Bern). Polyinosinic‐polycytidylic acid (Poly IC) was purchased from InvivoGen (San Diego, California, USA) and was diluted in PBS before injection. Poly-ICLC (Hiltonol, a synthetic poly-IC, stabilized with polylysine and carboxymethylcellulose) was kindly provided by Dr Andrés Salazar (Oncovir, NW Washington, District of Columbia, USA). Lipopolysaccharide (LPS) from Escherichia coli 055:B5 was purchased from Sigma (Madrid, Spain).
The hEDA-HPVE7-16 and hEDA-HPVE7-16/18 were produced in E. coli BL21 using a T7 expression vector and purified from inclusion bodies by size exclusion chromatography (Biotecnol, Oeiras, Portugal). The mEDA-HPVE7-16 protein was produced at CIMA as previously described.15 (link) The levels of endotoxin were below 0.1 EU/μg protein as tested by Quantitative Chromogenic Limulus Amebocyte Lysate assay (Cambrex, Walkersville, Maryland, USA).