Plasmodium falciparum merozoite surface and rhoptry proteins as malaria vaccine candidates

Locher, Christopher P.
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Plasmodium falciparum merozoite surface proteins were isolated from in vitro cultured parasites using monoclonal antibody (mAb) 5.2. Serum samples from rabbits immunized with the native merozoite surface precursor glycoprotein (gp195) had a mean EUSA titer of 1/560,000 against native gp195 but only 1/890 to denatured, reduced and alkylated (dR/A) gp195, while rabbits immunized with dR/A gp195 had mean ELISA titers of 1/23,100 and 1/14,650 to native gpI95 and dR/A gp195, respectively. Serum samples from rabbits immunized with native gp195 had a mean parasite growth inhibition of 87% and recognized the 42 and 19 kDa C-terminal processing fragments. Serum samples from rabbits immunized with dR/A gp195 did not inhibit parasite growth in vitro and poorly recognized the native C-terminal processing fragment. This study suggests that recombinant or synthetic peptide gp195 subunit vaccines must contain the native secondary and/or tertiary protein structures. Forty-five gp195-specific mAbs were produced in BALB/c, C57/B1.10, or Swiss-Webster mice using Freund's complete adjuvant (FCA) or Lipid A-15 PH. Of 26 mAbs tested for inhibition of parasite growth in vitro, two mAbs (CE2 and EB2) inhibited parasite growth partially (59% and 52%, respectively) at high concentrations (500 µg/ml). These antibodies recognized linear, group specific epitope(s) on the 83 kDa N-terminal processing fragment. All other mAb, including mAb 5.2 and two others to the C-terminal 19 kDa processing fragment, were weakly or non-inhibitory. Combinations of N-terminal or C-terminal mAb were also not inhibitory. The inability to produce a mAb which inhibits parasite growth at low concentration (1-10 µg/ml) is discussed. MAb AC9 was produced and used to isolate the 80, 70 and 40 kDa rhoptry associated protein-1 (RAP-1) complex. The isolated RAP-1 proteins reacted with mAb 30c13, previously used by Perrin to isolate a protective 42 kDa protein and by Braun-Breton to isolate a rhoptry-associated serine protease, as well as mAb 2.13, previously used by Ridley to demonstrate induction of protective immunity by RAP-1. In addition, mAb 219.5 was used to isolate the 140, 130, and 105 kDa RAP-3 complex. Both RAP-1 and RAP-3, along with gpI95 as a control, were used to immunize rabbits. Serum samples from rabbits immunized with gp195, RAP-1, and RAP-3 inhibited parasite growth in vitro 87%, 89%, and 89%, respectively. These results suggest that RAP-3 as well as RAP-I, should be investigated as a possible blood-stage malaria vaccine candidates. Three matrix metalloproteinases (MMP's) having relative molecular weight (Mr) 220, 95 and 75K in gelatin zymograms were found to be associated with RAP-1, but not RAP-3 or gp195. The MMP activity was inhibited by EDTA but not PMSF and was restored by addition of calcium. Of eight divalent metal cations tested, 0.1 mM cobalt optimized gelatinolytic activity when combined with 1.0 mM calcium. Gelatinases having the same M, were also precipitated by antiserums to normal human macrophage and fibroblast MMP's. Therefore, these MMP's are believed to be host enzymes non-covalently associated with RAP-1 proteins.
Thesis (Ph. D.)--University of Hawaii at Manoa, 1992.
Includes bibliographical references (leaves 126-137)
x, 137 leaves, bound ill. (some col.) 29 cm
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Theses for the degree of Doctor of Philosophy (University of Hawaii at Manoa). Biomedical Sciences (Tropical Medicine); no. 2724
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