Plasmodium vivax Pre-Erythrocytic–Stage Antigen Discovery: Exploiting Naturally Acquired Humoral Responses

Abstract

Abstract. The development of pre-erythrocytic Plasmodium vivax vaccines is hindered by the lack of in vitro culture systems or experimental rodent models. To help bypass these roadblocks, we exploited the fact that naturally exposed Fy− individuals who lack the Duffy blood antigen (Fy) receptor are less likely to develop blood-stage infections; therefore, they preferentially develop immune responses to pre-erythrocytic-stage parasites, whereas Fy+ individuals experience both liver-and blood-stage infections and develop immune responses to both pre-erythrocytic and erythrocytic parasites. We screened 60 endemic sera from P. vivax-exposed Fy+ or Fy− donors against a protein microarray containing 91 P. vivax proteins with P. falciparum orthologs that were up-regulated in sporozoites. Antibodies against 10 P. vivax antigens were identified in sera from P. vivax-exposed individuals but not unexposed controls. This technology has promising implications in the discovery of potential vaccine candidates against P. vivax malaria.

Predicting Antidisease Immunity Using Proteome Arrays and Sera from Children Naturally Exposed to Malaria

“…The array was fabricated as previously described (41). Briefly, coding sequences were PCR-amplified from Pv (Sal I strain [MRA-552, MR4]) or Pf (clone 3D7, [MRA-102, MR4]) genomic DNAs and cloned into the PXT7 plasmid with a T7 transcription terminator, and tagged with 5Ј polyhistidine (HIS) and 3Ј hemagglutinin (HA) epitopes.…”

“…Recombinant proteins were expressed using E. coli cell-free in vitro transcription and translation reactions (RTS 100 HY kits from 5 PRIME, Gaithersburg, MD) according to the manufacturer's instructions. Proteome arrays were printed as previously described, with each recombinant protein spotted once in each array (41). Each array contained 256 negative control spots made with an in vitro transcription translation master mix without plasmid DNA.…”

“…Each array contained 256 negative control spots made with an in vitro transcription translation master mix without plasmid DNA. Once printed, recombinant protein expression was verified using antipolyhistidine (clone His-1, Sigma) and antihemagglutinin (clone 3F10, Roche) monoclonal antibodies, as previously described (41). All signal intensities were corrected for spot-specific background.…”

“…Pf/Pv infection) by LM or PCR, as previously described (41). Sera were diluted to 1/100 in 1x Blocking Buffer containing 1 mg/ml E. coli lysate and incubated at room temperature for 30 min with constant mixing to remove E. coli-specific antibodies.…”

“…We probed the arrays with sera from the 224 PNG children, as well as sera from 18 US malaria naïve controls (Table I), and quantified the resulting signals as previously described (41). The strategy used to analyze antibody responses to the Plasmodium recombinant proteins contained in the array is illustrated in Fig.…”