3 bacterial expression vector. pPACIB.3 is an “in house” developed plasmid for bacterial periplasmic expression of recombinant proteins via an ompA leader

sequence. The tryptophan promoter and a terminator Duvelisib cell line sequence from the T4 phage ensure high expression levels and the vector provides expressed proteins with six-histidine tags at their C-terminus. The hrVEGF molecule was purified from bacterial periplasm using conventional IMAC procedures [16]. The recombinant P64K protein derived from Nm was supplied by the Development Department of the CIGB. P64K is produced routinely to be used as a vaccine carrier protein [17]. Clinical grade preparations (0.8 mg of protein/0.5 mL per vial) of VSSP were supplied by the Center for Molecular Immunology of Havana. The VSSP preparation is obtained by physical disorganization of outer membrane vesicles of Nm and further re-association and stabilization with the inclusion of GM3 gangliosides. VSSP induces the activation of CTL responses to peptides and proteins, and can also stimulate the humoral response to different antigens [18], [19] and [20]. The

oil-based adjuvant was obtained from Seppic (France). Emulsification was done as recommended by the supplier using two syringes, a connector, and 100 syringe passes. Animals were randomly assigned to five groups of five animals each and given: (a) six subcutaneous learn more injections of 100 μg of the recombinant protein pP64K-hVEGFKDR− mixed with 200 μg of VSSP (hereafter denominated CIGB-247), in weekly or biweekly schedules,

or (b) six intramuscular injections of CIGB-247 in a volume of 0.1 mL, mixed with 0.1 mL of montanide ISA 51, in a biweekly schedule. Control (placebo) animals received only Tris 10 mM. The rats assigned to the weekly schedule received three additional injections of CIGB-247 25 days after the sixth immunization. A week after the last booster the animals were euthanized, sera and plasma collected and their organs processed for histopathology. Platelet rich and platelet depleted plasma were obtained as described [21]. Animals (three per group) were given: (a) six subcutaneous isothipendyl injections of CIGB-247, in weekly or biweekly schedules, or (b) six intramuscular injections of CIGB-247 in a volume of 0.1 mL, mixed with 0.1 mL of montanide ISA 51, in a biweekly schedule. Control animals (two animals) received only Tris 10 mM. The animals assigned to the weekly schedule received an additional booster 21 days after the sixth immunization and were euthanized a week later. Sera were collected and organs dissected and fixed in 10% formalin for histological evaluation. Animals were screened first for antibodies to P64k and VEGF proteins and considered naive with respect to both antigens when specific antibodies were undetectable by ELISA (titer <1:50) (see methods below). Monkeys were subsequently ranked by weight and age and then randomly assigned to three groups of three animals each.