The emergence of drug-resistant parasites and insecticide-resistant Anopheles mosquitoes have contributed to the persistence of malaria in the world 
. New methods to control the disease are needed, and vaccination holds the promise of controlling and perhaps eventually eradicating malaria.
The asexual blood stage is responsible for malaria disease. Naturally acquired immunity develops against disease in malaria-endemic regions, where parasite density is observed to decrease with age and the clinical manifestations of malaria are generally much milder in adults than in children under 5 years of age 
. Moreover, immunoglobulin fractions from adults in these high-transmission areas have been found to passively reduce parasitemia in infected children 
.Thus an effective vaccine targeting this stage and inducing immune responses similar to that obtained by natural infection could reduce morbidity and mortality of the disease.
Several antigens thought to be targets of protective blood-stage immune responses have been identified 
. Among them, the ~200 kDa Merozoite Surface Protein 1 (MSP1) and the Apical Membrane Antigen 1 (AMA-1) of Plasmodium falciparum
are two leading asexual blood-stage vaccine candidates 
. Located on the merozoite surface, they are proposed to play a role in the process of parasitic invasion 
. A portion of MSP1 recognized by protective antibodies was mapped to the 19 kDa carboxy-terminal region (MSP1-19) which contains two epidermal growth factor (EGF)-like domains 
. Immunization with MSP1-19 of P. falciparum in monkeys and P. yoelii in mice induced protection against homologous parasite challenge 
. Data from these animal studies were supported by in vitro studies showing that MSP1-19 specific antibodies inhibited merozoite invasion 
AMA-1 is an integral membrane protein of 83 kDa. Studies in rodent and monkey models demonstrated that immunization with native or recombinant AMA-1 can provide protection against homologous parasite challenge 
. A three-domain sub-structure of the AMA1 ectodomain was recently suggested 
. The most C-terminal of the disulphide-bonded domains in AMA-1 (domain III) containing a “cysteine knot-like” structure may be carried on the surface of the invading merozoite along with MSP1-19. Studies with P. chabaudi in a mouse model have shown that antibody-mediated protection can be induced by immunizing with the AMA1 ectodomain 
. The structure of AMA-1 (III) was recently demonstrated to be the target of inhibitory antibodies isolated from humans in malaria-endemic regions 
. A recent study showed that AMA-1(III) bound to the erythrocyte membrane protein, Kx, and that the subsequent rate of invasion of Kx null erythrocytes was reduced, suggesting that AMA-1 (III) plays an important role in merozoite invasion of human erythrocytes 
Both MSP1-19 and AMA-1 (III) can be a target of inhibitory antibodies, but the small size of the antigens may limit the ability of each alone to adequately induce the high titer of antibodies that may be required to be effective in vivo. Polyvalent subunit malaria vaccines containing multiple protective domains or epitopes from different antigens may be necessary for enhanced immunogenicity and protective efficacy and may be needed to address the issue of parasite variation. Chimeric protein vaccine constructs may be an approach towards this and therefore, we have constructed a P. falciparum chimeric protein 2.9 (PfCP-2.9) by combining MSP1-19 and AMA-1 (III) sequences 
. The purified PfCP-2.9 protein, produced by Pichia pastoris in secreted form with a yield of 2,600 mg/L, was highly immunogenic in rabbits as well as in rhesus monkeys (Macaca mulatta). It induced both anti-MSP1-19 and anti-AMA-1 (III) antibodies at 11- and 18-fold higher titers, respectively, compared to individual components. Anti-PfCP-2.9 sera from rabbits and rhesus monkeys at a 6 to 7-fold dilution nearly completely inhibited in vitro growth of the P. falciparum FCC1/HN and 3D7 strains 
. The inhibition was dependent on the presence of antibodies to the chimeric protein and their disulfide bond-dependent conformations. Moreover, the activity was mediated by a combination of growth-inhibitory antibodies generated by the individual MSP1-19 and AMA-1 (III) components of PfCP-2.9.
In this study, we report the first human phase I trial of recombinant PfCP-2.9 formulated with the novel adjuvant Montanide ISA720