Recombinant Subolesin-BM95 Vaccine Production

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.