Isa 206

The purified VLPs (100 and 25 μg/dose per 2 mL) were emulsified with adjuvant ISA 206 (SEPPIC, France) using the R30A Electric (FLUKO, German) under sterile conditions and were stored at 2–8°C until use. A total of 20 pigs were divided into four groups (Table 1) and immunized by intramuscular injection. Group 1 was designed for the evaluation of a commercial inactivated serotype O vaccine. Groups 2 and 3 were used to assess the effect of VLPs single immunization with the doses of 100 and 25 μg, respectively. Group 4 with phosphate-buffered saline (PBS) inoculation was set as the control. Blood samples were collected preimmune and weekly up to 10 weeks postimmunization, and sera anti-FMDV antibody was analyzed by a commercial LPB-ELISA kit (Lanzhou Veterinary Research Institute, China). Peripheral blood mononuclear cells (PBMCs) were isolated from all the pigs in the four groups at 28 dpi by centrifugation in Ficoll-Paque Plus (GE Healthcare, USA) at RT for 30 min.

Calves were randomly separated into four groups and vaccinated according to the following scheme: non-vaccinated control (n = 4): PBS; Group 3Ag (n = 6): IntiminC₂₈₀ + EspB + BLS-Stx2B; Group 2Ag (n = 6): IntiminC₂₈₀ + EspB; Group Stx (n = 2): BLS-Stx2B. The immunization protocol consisted on two doses 15 days apart, with 100 μg of each antigen by intramuscular route. The antigens were diluted in 1 mL of PBS and emulsified in 1 mL of mineral oil-based adjuvant (Montanide ISA206, Seppic, France). The control group was vaccinated only with PBS emulsified in the adjuvant.

The cloning and construction of the expression vector for rPMT-NC and rSly were performed as described previously [11 (link)]. Escherichia coli strain BL21 (DE3) (Invitrogen, CA, USA) harbouring the PMT-NC and Sly recombinant plasmids were cultured in LB broth (Luria–Bertani, Difco, MD, USA) medium and incubated at 37 °C overnight, and the procedures for purification were as previously described.
The construction of a CpG plasmid containing 12 copies of the GACGTT motif is described in the Taiwan patent entitled “DNA adjuvant for waterfowl and live-stock vaccines” (Patent No. I425091), which is available for viewing online through the Intellectual Property Office, Taiwan. Plasmids were amplified in E. coli, purified using a QIAGEN-tip 500 (Qiagen, Hilden, Germany) and dissolved in sterile PBS for formulation. The purified rPMT-NC (200 μg/mL) as an antigen was emulsified with 50% water-in-oil-in-water (w/o/w) adjuvant (ISA206, Seppic, France), CpG plasmid (200 μg/mL) or rSly protein (100 μg/mL) and stored at 4 °C. The sterility of the vaccines was confirmed by culture with trypticase soy agar (Difco, MD, USA) (TSA, 37 °C), thioglycollate agar (Difco) (TGC, 37 °C) and Sabouraud dextrose agar (Difco) (SDA, 25 °C). All vaccine dosages had a final volume of 2 mL.

The purified capsid protein and adjuvant ISA 206 (SEPPIC, France) was emulsified using a RW 20 homogenizer (IKA, German) under sterile conditions at 300 rpm for 5 min, in a ratio of 46:54 (antigen:adjuvant) in volume. The preparations were stored at 4 °C until use.

The safety of the FMD vaccine containing D-galacto-D-mannan was evaluated in mice as previously described (29 (link)). The vaccine compositions for the usual vaccination in mice were as follows: purified antigens obtained via antigen purification from FMDV type O (O PA2) and type A (A YC) (0.375 μg + 0.375 μg/dose), ISA 206 (Seppic, Paris, France; 50% w/w), 10% aluminum hydroxide [Al(OH)₃], and 15 μg/mouse Quil-A (InvivoGen, CA, USA), with the addition of 100 μg of D-galacto-D-mannan/dose/mouse in a total volume of 100 μL. Mice were administered with a vaccine equivalent to fivefold (500 μL) the volume of the usual vaccination dose (100 μL). All mice (n = 5/group) were vaccinated with IP injection into the peritoneum (0 dpi). To evaluate the safety of the vaccines, survival rates and body weight changes were evaluated up to 7 dpi.