Montanide isa 50v2

Immunogen was prepared as a water-in-oil emulsion of purified RABV NP and CSIRO Triple Adjuvant prepared as described previously [12 ]. For each rabbit immunized, 600 μL of immunogen was prepared by mixing 90 μL of RABV NP at 1 mg/mL with 54 μL PBS and 96 μl of 3 mg/mL QuilA (Superfos Biosector), 30 mg/mL DEAE-Dextran (Pharmacia). This aqueous phase was added to 360 μL of Montanide ISA 50 V2 (Seppic) and emulsified by repeated extrusion through an 18 gauge blunt needle.
Two New Zealand White rabbits were immunized by intramuscular injection on three occasions approximately three weeks apart. Each immunization used a total of 75 μg RABV NP in two 0.25 mL doses, one dose in each hind leg. Serum samples (~1 mL) were taken prior to immunization to test for background staining. Sera were taken after each immunization and assessed for the presence of RABV NP antibodies by immunoblotting.

The purified denatured recombinant proteins were refolded by dialysis against 1000 volumes of PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄), pH 7.4 for 12 h at 4 °C. Recombinant proteins were then concentrated using an Amicon Ultra-15 ultrafiltration device (cut off 10 kDa) (Millipore-Merck, Darmstadt, Germany), and adjusted to 0.5 mg/ml. For vaccine formulation, recombinant proteins or PBS saline control were adjuvated in Montanide ISA 50 V2 (Seppic, Paris, France) to a final protein concentration of 250 μg/ml as previously described [54 (link), 55 (link)].

The adjuvants used in this study were: Montanide ISA 50V2 (SEPPIC, Castres, France, batch 190301019150); Complete Freund (Sigma-Aldrich, St. Louis. MO, USA, batch SLCJ8308); Carbigen (MVP Adjuvants, Teaneck, NJ, USA; batch F18123); Emulsigen-D (MVP Adjuvants, batch D01449) and QH-769 (IDRI, Seattle, WA, USA, experimental batch). All adjuvants were used according to the manufacturers’ instructions.

The M. bovis strain (SB0339) used was first isolated from a naturally infected wild boar on Coletsos medium. The vaccine was prepared as described by Balseiro et al. [13 (link)]. The inactivated M. bovis suspension was adjuvated with Montanide^™ ISA 50V2 (Seppic, Paris, France) to form a water in oil emulsion in a proportion 1:1 and contained approximately 10⁷ CFU of heat-treated bacteria per dose (1 ml). Animals were injected subcutaneously, at the right axilla, or intramuscularly, at the right semitendinosus muscle.

Three groups of animals consisting of twelve white mixed-breed piglets (bought from a commercial stud) weaned 21 days after birth were used in the experiment. Pigs in the first group remained serologically negative for anti-Salmonella antibodies (determined by Pig Screen ELISA, Qiagen, Germany). Another twelve animals were orally infected one week after housing with 1 × 10⁸ CFU of Salmonella Typhimurium grown in BHI medium and blood was collected 28 days after the infection. The last group of animals was vaccinated intramuscularly into the neck with 1 ml of a vaccine prepared from 1 × 10⁹ CFU of Salmonella Typhimurium grown in BHI medium, inactivated with formaldehyde and adjuvanted with Montanide ISA50V2 (Seppic, France). The first dose was administered one week after housing and the second dose two weeks later. Blood was collected 14 days after the second dose of a vaccine.

Plasmids containing the coding Subolesin-BM95 MSP1a fusion proteins (SUB-BM95) were transformed into E. coli BL21 Star™ (DE3) One Shot^® cells (Invitrogen-Life Technologies, Inc., Grand Island, NY, USA) to express and purify SUB-BM95 as described previously [24 (link),25 (link)]. The SUB-BM95 chimeric protein is based on SUB from R. microplus Media Joya strain and conserved BM86/BM95 protective epitopes. The recombinant BM86 (from R. microplus Media Joya strain) was secreted in Pichia pastoris and purified, as reported previously [26 (link)]. Recombinant proteins were adjuvated in Montanide ISA 50 V2 (Seppic, Paris, France) in a stable water in oil (W/O) vaccine emulsion (50 parts of aqueous phase and 50 parts of oily phase in the volume) in batch-by-batch processes using a high-speed mixer Heidolph DIA× 900 (Heidolph Elektro, Kelheim, Germany) at 8000 rpm and the vaccine was filled manually under sterile conditions in pyrogen-free glass bottles of 20 mL (Wheaton, Millville, NJ, USA) at a concentration of 100 µg/2 mL dose. Calves were immunized with 2 doses (days 0 (T0) and 28 (T1)) containing 100 µg/dose [27 (link)] of purified recombinant proteins formulated as described above. Negative controls were injected with the adjuvant–saline alone (placebo). Cattle were injected intramuscularly with 2 mL/dose using a 5 mL pyrogen-free plastic syringe and an 18G needle.

Control animals (Group 1) were immunized with mock formulation (Montanide ISA50V2, SEPPIC, France) five days before viral challenge. The animals were immunized once by intramuscular injection with 2ml of formulation (25ug/mL) (50 µg of purified E2CD154 antigen). Piglets were challenged five (Group 2), three (Group 3), or one (Group 4) days post-vaccination (dpv). All of the groups were challenged Intranasally (IN) with 2 × 10³ LD₅₀ of CSFV high virulent “Margarita” strain, genotype 1.4 [14 (link)]. Clinical signs were scored daily for 28 days after challenge according to Mittelholzer et al., [15 (link)] with modifications. The modifications consist in the elimination of three parameters (body tension, body shape, and breathing) and the addition of the rectal temperature. The samples of heparinized blood and serum were taken at the moment of challenge, and at 3, 7, 14, 18, 21, and 28 days post-challenge. Blood samples were taken by ophthalmic venous sinus punctures using sterile tubes, with and without anti-coagulant (VACUATTE^® Greiner bio-one, Frickenhausen, Germany). The blood samples were placed at room temperature for 2 h and then kept overnight at 2–8 °C to allow for serum extraction.