In Vivo Expression and Immunological Studies of the 42-Kilodalton Carboxyl-Terminal Processing Fragment ofPlasmodium falciparumMerozoite Surface Protein 1 in the Baculovirus-Silkworm System

Abstract

The 42-kDa carboxyl-terminal processing fragment of Plasmodium falciparum merozoite surface protein 1 (MSP-1 42 ) is an anti-erythrocytic stage malaria vaccine candidate. In this study, MSP-1 42 was expressed by using the Bombyx mori nuclear polyhedrosis virus-silkworm expression system, and the antigenicity and immmunogenicity of the recombinant protein, Bmp42, were evaluated. The average yield of Bmp42, as determined by a sandwich enzyme-linked immunosorbent assay (ELISA), was 379 g/ml of infected silkworm hemolymph, which was >100-fold higher than the level attainable in cell culture medium. N-terminal amino acid sequencing revealed that Bmp42 was correctly processed in silkworm cells. Data from immunoblotting, as well as from the inhibition ELISA, suggested that the conformational B-cell epitopes of MSP-1 42 were recreated in Bmp42. Immunization of rabbits with Bmp42 in complete Freund's adjuvant generated high-titer antibody responses against the immunogen. Specificity analyses of the anti-Bmp42 antibodies using several recombinant MSP-1 19 proteins expressing variant and conserved B-cell epitopes suggested that the anti-Bmp42 antibodies recognized primarily conserved epitopes on MSP-1 19 . Furthermore, the anti-Bmp42 antibodies were highly effective in inhibiting the in vitro growth of parasites carrying homologous or heterologous MSP-1 42 . Our results demonstrated that the baculovirus-silkworm expression system could be employed to express biologically and immunologically active recombinant MSP-1 42 at elevated levels; thus, it is an attractive alternative for producing a protective MSP-1 42 vaccine for human use.

Expression and biochemical characterization of the Plasmodium falciparum DNA repair enzyme, flap endonuclease-1 (PfFEN-1)

“…The type (CBP/FLAG or polyhistidine(HIS) 6 ), and the location (N-or C-terminus) of the tags used on these recombinant proteins was empirically determined to produce an acceptable amount of soluble recombinant protein and were thus utilized in the above investigation. This situation is not uncommon, and the widespread problems and difficulties associated with the expression and purification of recombinant Plasmodial proteins has been well documented [47,48,[61][62][63][64][65]. We could not find precedence in the literature where a protein tag inhibited activity to an extent that could account for the orders of magnitude difference observed between PfFEN-1ΔC and Pf-or PyFEN-1.…”

Blood stage merozoite surface protein conjugated to nanoparticles induce potent parasite inhibitory antibodies

“…The ability of mouse sera generated from SW mice immunized with different rMSP1 formulations to inhibit parasite growth was determined using an in vitro assay [5, 45, 49, 50]. Immunoglobulins from pooled mouse serum samples from each group were purified as previously described [47] with modifications.…”

“…Parasitemias of the parasite cultures were determined microscopically by Giemsa staining of thin blood smears. The degree of parasite growth inhibition was determined by comparing the parasitemias of cultures incubated in pre-immune antibodies as previously described [45, 49, 50]. …”

Production of recombinant proteins from protozoan parasites

“…Viruses represent one of the most frequently used systems for developing vaccines, and have proved suitable both as delivery systems and as adjuvants. These include, recombinant poxvirus FP9 and a modified vaccinia virus Ankara [47, 48], the malaria protein B in yellow fever 17D virus [49], the Plasmodium merozoite surface protein (MSP) -1(42) in Bombyx mori nuclear polyhedrosis virus-silkworm [50], and the malaria MSP-1 in a replication-defective virus [51]. Toxoplasma surface antigens expressed in replication-deficient recombinant adenoviruses induce a protective immune response in mice [52].…”