Cpg2006

The lymphocytes were stimulated in 48-well plates with 1 × 10⁶ cells in 1 mL of mitogen stimulation medium per well. One milliliter of mitogen stimulation medium constitutes 0.1 µL of Staphylococcus aureus Cowan (Sigma), 6 µL of CpG-2006 (Invivogen), 1 µL of beta-mercaptoethanol, 1 µL of pokeweed mitogen (MP Biomedical), and complete RPMI medium. Following 5 days of stimulation in 5% CO₂ at 37°C, the ELISPOT assay was performed as described above to determine the frequency of antigen-specific memory B cells.

All experiments were performed in accordance with IRB guidelines and regulation. B cells were isolated from pre-screened healthy donors (following informed consent and under IRB approved protocols, IRB number: 2013-04-034B #1, Taipei Veterans General Hospital) with high anti-H1N1 virus titer using the combination of Ficoll density gradient centrifugation (GE Healthcare Life Sciences), negative B cell isolation kit (Miltenyi Biotec) and CD27-positive selection kit (Miltenyi Biotec), following the manufacturer’s protocol^{32 (link),33 (link)}. Memory B cells were then infected with EBV virus according to the protocol^{34 (link)}. Briefly, memory B cells were seeded in 6-well plates with feeder cells (cobalt-60 gamma-irradiated allogeneic mononuclear cells) in complete medium containing 2.5 µg/mL CpG 2006 (InvivoGen), 400 ng/mL cyclosporine A (Sigma-Aldrich) and 30% EBV supernatant. After 28 days, the culture supernatants were screened for antibodies specific to H1N1 HA from the 2009 pandemic. The full-length cDNA of the desired IgGs were cloned from identified B cell clones for further antibody expression.

PBMCs (1 × 10⁶/well) from patients with SLE or RA, or healthy subjects in 48-well plates or pDCs (2.5 × 10⁴/well) from healthy subjects in 96-well flat-bottom plates were stimulated with TLR7 agonist, loxoribine (1 mmol/L) or R837 (5 μg/mL), or TLR9 agonist, CpG2216 (2 μmol/L) or CpG2006 (2 μmol/L, all from InvivoGen) for 5 h, and brefeldin A (2.5 μg/mL: SIGMA-Aldrich) was added during the final 3 h of stimulation to block cytokine secretion. In the case of pre-treatment experiments, PBMCs or pDCs were treated with IFN-α (IFN-α1: Abcam), IFN-β (Peprotech), IFN-γ (R&D systems), TNF-α (Peprotech), IL-6 (Miltenyi Biotech)/soluble IL-6R (R&D systems) or IL-10 (R&D systems) for 2, 12, and 24 h. Cells were washed three times by complete medium to remove cytokines, thereafter, stimulated with TLR agonist as mentioned above (Supplementary Figure S1). Cell number and viability of PBMCs after pre-treatment with each cytokine were calculated under microscope using trypan blue dye.

Female, 6–8-week-old BALB/c and CD-1 mice were purchased from the Charles River Laboratories (Frederick, MD). Mice were vaccinated once or twice (three weeks apart), depending on the study, subcutaneously (s.c.) with rF1-V, in the presence or absence of TLR Agonists (Table S1), and 250 - 500 µg of Alhydrogel (Brenntag Biosector, Denmark) in a total volume of 0.1 ml. PolyICLC (Hiltonol™) was provided by Oncovir, Inc., whereas Pam3CSK4, MPLA, CpG2006, Imiquimod and R848 were purchased from Invivogen and reconstituted per manufacturer’s recommendation. The number of mice, the amount of rF1-V, and TLR Agonists for each study are stated in the figure legends or tables. After 25 to 28 days post vaccination, depending on the study, blood was obtained by intracardiac or axillary vessel collection for serum isolation and antibody titer determination from deeply anesthetized mice. In addition, spleens were harvested for cytokine profiling from select studies. After 26-43 days, depending on the study, mice were challenged via s.c. or aerosol routes. The calculated colony-forming units (CFU) doses for each study are stated in the figure legends.