Citations

Immunogenicity Studies of PA83 variants in Mice

Immunogenicity studies were performed with 3 and 10 μg of plant-produced dPA83(E), dPA83(P) and commercial recombinant PA (rPA, List Biological Laboratories, Campbell, CA, used as a positive control) proteins, administered intraperitoneally in seven- to eight-week-old Balb/c mice (6 animals/group). Mice were injected with three doses of protein samples adsorbed in 0.3% Alhydrogel at three-week intervals (day 0, 21 and 42). …

• Product #171E – Anthrax Protective Antigen (PA), Recombinant from B. anthracis

… Goat anti-CTb (1:5000) List Biological Labs Cat# 703; …

• Product #703 – Anti-Cholera Toxin B Subunit (Goat)

… Corynebacterium diphtheriae Diphtheria toxoid DipTox Com. product 68,71,72 73,74,75 60 5 Native, List Biological Labs, Campbell, CA …

• Product #151 – Diphtheria Toxoid, from Corynebacterium diphtheriae

… 100 µl cholera toxin (List Biological Laboratories, 100B; …

• Product #100B – Cholera Toxin (AZIDE-FREE) from Vibrio cholerae

Systemic LPS challenge

After participants had undergone the IMQ skin challenge, an i.v. LPS challenge was performed on Day 7 after the start of treatment. All participants received 1 ng/kg purified Escherichia coli O113 LPS (List Labs, US, Lot #94332B4) reconstituted in 0.5 mL glucose 2.5%–sodium chloride (NaCl) 0.45% and administered as a 2- min infusion. To ensure that participants stayed adequately hydrated, glucose 2.5%–NaCl 0.45% was infused, starting 2 h prior to LPS administration and continuing until 6 h afterwards.18 Circulating inflammatory proteins (acute phase proteins, cytokines), leukocyte differentiation, and clinical parameters (pulse rate, blood pressure, and temperature) were evaluated over time, starting 0.5 h after the end of the infusion and ending up to 24 h after infusion.

• Product #9433 – GMP LPS Lipopolysaccharide List™ Hpt™ From Escherichia Coli Type O113

… Anthrax lethal toxin List Biological Laboratories Inc. #171B/#172B …

Treatments

See Table S1 for list of Figures/panels in each figure and corresponding dose and exposure time. General information for the methods is provided here. To induce cytokine release from macrophages, microglia and BMDM cells were primed for 4–5 h (or time indicated in Table S1)with 1 μg/mL of LPS in culture medium, and some cells were pre-treated with 1 μM of CP456773/MCC950 for 30 min before ATP stimulation. Cells were then pulsed with 5 mM ATP for 30 min. For cytokine induction by anthrax lethal toxin (LeTx), LPS-primed cells were treated with LeTx at a final concentration of 250 ng/mL (LF and PA) for 3 h. Cell supernatants were collected and frozen at −80°C until assayed.

• Product #171E – Anthrax Protective Antigen (PA), Recombinant from B. anthracis
• Prodcut #172 – Anthrax Lethal Factor (LF), Recombinant from B. anthracis

… Pertussis Toxin from B. pertussis, Lyophilized (Salt-Free) List Biological Cat#181 …

• Product #181 – Pertussis Toxin from B. pertussis, Lyophilized (Salt-Free)

EAE

EAE was induced as previously described52. In brief, 8–12-week-old female C57Bl/6J mice were immunized using 150 μg of MOG35–55 (Genemed Synthesis, 110582) mixed with freshly prepared Complete Freund’s Adjuvant (using 20 ml of Incomplete Freund’s Adjuvant (BD Biosciences, no. BD263910) mixed with 100 mg of Myobacterium tuberculosis H37Ra (BD Biosciences, no. 231141)) at a ratio of 1:1 (v/v at a concentration of 5 mg ml−1). All mice received two subcutaneous injections of 100 μl each of the MOG35-55/CFA mix. All mice then received a single intraperitoneal injection of 200 ng of pertussis toxin (List Biological Laboratories, no. 180) at a concentration in 200 μl of PBS. …

• Product #180 – Pertussis Toxin from B. pertussis, Lyophilized in Buffer

Tetanus toxin labeling

Alexa Fluor®647 was attached to the tetanus toxin (List Biologicals cat. no. 19050A2) according to the manufacturer’s protocol from Alexa Fluor®647 Labeling Kit (Microscale Protein Labeling Kit, cat. no. A30009; ThermoFisher Scientific) and stored at −20°C. Quantification of the Alexa Fluor® 647 ratio per tetanus toxin was obtained by scanning measurements using the NanoDrop One c device (Thermo Fisher Scientific) with 280 and 650 nm readings. The average dye per mole of protein obtained was 1.7 moles.

Tetanus toxin internalization inhibition assay

The TeNT entry inhibition by mAbs was initiated by the pre-incubation of 25 nM TeNT-Alexa-Fluor 647 and 25 nM or 50 nM mAbs in Neurobasal-A medium on an orbital shaker at 37°C, 5% CO2 for 1 hour. The mixtures and increased concentrations of TeNT-Alexa-Fluor 647 (25 nM, 50 nM, and 100 nM) were added to cells for 1 hour at 37°C and 5% CO2. Cells were washed twice in 2% formaldehyde solution in PBS and processed for immunofluorescence.

• Product #190 – Tetanus Toxin from Clostridium tetani

Mucosal sensitisation model

The animal study protocol was approved by the “Service Vétérinaire du Canton de Vaud”, Switzerland and the experiments were performed at Ambiotis (Toulouse, France). The model has been previously described in detail (Zuercher et al., Citation2012). Briefly, five-week-old conventional female BALB/cOlaHsd mice from Envigo (Gannat, France) were sensitised for a total of seven consecutive weeks by weekly gavage of 50 mg ovalbumin (OVA, Merck) [sensitised group (S.)] or water [non-sensitised group (N.S.)] in combination with 20 μg cholera toxin (CT) (List Biologicals, purchased from LuBioscience, Lucerne, Switzerland). On day 67 or 68, mice were challenged with an oral administration of 200 mg ovalbumin (OVA; Sigma, Switzerland) and harvested at day 68 or 69 (Supplementary Figure 1A). Two independent experiments were performed with n = 8 animals per group for the exploratory experiment and n = 13 animals per group for the confirmatory experiment.

• Product #100B – Cholera Toxin (AZIDE-FREE) from Vibrio cholerae