The EP has been utilized for over 20 years as a means of introducing macromolecules, including DNA into cellsin vitro[21] and for transfection of plasmids into different tissuesin vivo[22].In vivoEP is believed to increases the immunogenicity of DNA vaccines via recruitment of immune cells such as dendritic cells, T and B lymphocytes to the site of immunization [25,26]. method to elicit potent immunogenicity of malaria DNA vaccines in nonhuman primates and provides the basis for further evaluation in human volunteers. Keywords:Pfs25, Malaria, Transmission blocking vaccine, DNA vaccine, Electroporation, Prime boost == 1. Introduction == Malaria remains one of Cardiolipin the major causes of mortality and morbidity, with 3.2 billion people Rabbit Polyclonal to ERCC5 at risk, 300500 million clinical cases and more than 1 million deaths annually [1]. The malaria parasite has Cardiolipin a complex life cycle and various stages provide potential targets for the development of vaccines. In particular, vaccines that target antigens found on sexual and mosquito midgut stages of the parasite are uniquely capable of interrupting malaria transmission [24]. Transmission blocking vaccines (TBV) elicit antibodies which, upon ingestion, block parasite development in the mosquito midgut thus rendering them transmission incompetent. TBV candidates in various stages of development forP. falciparuminclude proteins Pfs48/45, Pfs230 and Pfs25 [58]. Among them, Pfs25 expressed on the surface of gametes, zygotes and ookinetes, is one of the most investigated targets for TBV development. Pfs25 has been hard to produce in reproducibly functional conformation in recombinant expression systems [10,11]. Despite these difficulties, considerable progress has been made in the expression and enhancement of immunogenicity along with formulation in various adjuvants [1214]. A phase I clinical trial of Pfs25 expressedin Pichia pastorisformulated with Montanide ISA 51 revealed moderate immunogenicity [15] emphasizing the need for improved vaccine design and alternate methods. Cardiolipin We have been investigating Pfs25 in the form of DNA vaccine plasmids [1619] as an alternate approach. The rationale for DNA based vaccine development has been to exploit the mammalian hosts cellular machinery to produce the protein antigen for presentation to the host immune system [20]. In previous studies in mice, a DNA vaccine expressing Pfs25 elicited strong antibody responses [16], while delivery byin vivoelectroporation (EP) significantly enhanced immunogenicity [19]. The EP has been utilized for over 20 years as a means of introducing macromolecules, including DNA into cellsin vitro[21] and for transfection of plasmids into different tissuesin vivo[22].In vivoEP is believed to increases the immunogenicity of DNA vaccines via recruitment of immune cells such as dendritic cells, T and B lymphocytes to the site of immunization [25,26]. Motivated by enhanced immunogenicity of Pfs25 DNA vaccine by EP in mice, we evaluated EP delivery of Pfs25 DNA vaccine in nonhuman primates (Olive baboons) for the development of a potential transmission blocking vaccine againstP. falciparum. == 2. Materials & Methods == == 2.1. DNA vaccine plasmids == Pfs25 sequence optimized for expression in mammalian cells [18], lacking signal and anchor sequences was cloned in VR1020 (Vical, Inc., San Diego, CA). Additionally, all the three putative N-glycosylation sites were mutated to replace N residues with Q. Plasmid DNA (<30 EU per mg endotoxin) was purified by Aldevron (ND, USA) and supplied at a Cardiolipin concentration of 2.5 mg/ml. == 2.2. Immunizations of Olive baboons (Papio anubis) == The immunization studies were approved by the scientific and ethics review committee of the Institute Cardiolipin of Primate Research, (Karen) Kenya (#10-10-2007). The baboons were trapped from their wild habitats, managed in quarantine for 3 months, screened for the presence of contamination with worms and protozoan parasites and successfully treated using ivermectin and/or metronidazole as appropriate months before the start of immunization. All the animals (7.0 to 12.4 kg) were free of mycobacterial infection. Detailed hematological tests qualified all 20 animals during their quarantine period, just prior to and at the termination.
