Delineation of stage specific expression of Plasmodium falciparum EBA-175 by biologically functional region II monoclonal antibodies

Abstract

Background: The malaria parasite Plasmodium falciparum EBA-175 binds its receptor sialic acids on glycophorin A when invading erythrocytes. The receptor-binding region (RII) contains two cysteine-rich domains with similar cysteine motifs (F1 and F2). Functional relationships between F1 and F2 domains and characterization of EBA-175 were studied using specific monoclonal antibodies (mAbs) against these domains.

Methods and findings: Five mAbs specific for F1 or F2 were generated. Three mAbs specific for F2 potently blocked binding of EBA-175 to erythrocytes, and merozoite invasion of erythrocytes (IC(50) 10 to 100 µg/ml IgG in growth inhibition assays). A mAb specific for F1 blocked EBA-175 binding and merozoite invasion less effectively. The difference observed between the IC(50) of F1 and F2 mAbs was not due to differing association and disassociation rates as determined by surface plasmon resonance. Four of the mAbs recognized conformation-dependent epitopes within F1 or F2. Used in combination, F1 and F2 mAbs blocked the binding of native EBA-175 to erythrocytes and inhibited parasite invasion synergistically in vitro. MAb R217, the most potent, did not recognize sporozoites, 3-day hepatocyte stage parasites, nor rings, trophozoites, gametocytes, retorts, ookinetes, and oocysts but recognized 6-day hepatocyte stage parasites, and schizonts. Even though efficient at blocking binding to erythrocytes and inhibiting invasion into erythrocytes, MAb R217 did not inhibit sporozoite invasion and development in hepatocytes in vitro.

Conclusions: The role of the F1 and F2 domains in erythrocyte invasion and binding was elucidated with mAbs. These mAbs interfere with native EBA-175 binding to erythrocyte in a synergistic fashion. The stage specific expression of EBA-175 showed that the primary focus of activity was the merozoite stage. A recombinant RII protein vaccine consisting of both F1 and F2 domains that could induce synergistic activity should be optimal for induction of antibody responses that interfere with merozoite invasion of erythrocytes.

Figure 1. EBA-175 RII mAbs generated against baculovirus expressed recombinant EBA-175 RII protein recognizes native EBA-175.

Panel A: Dual immunofluorescent analyses showing apical staining of mature P. falciparum (FVO strain) schizont with EBA-175 RII specific mAb R217 used at 10 ug/mL and rabbit polyclonal sera KLS13 against baculovirus expressed EBA-175 RII (used at 1:200 dilution). Panel B: Phosphoimager detection of parasite culture supernatant containing [35S]-labeled native EBA-175 immunoprecipitated with mAbs and polyclonal sera. MAb R216, R217, R218 and KLS13 (polyclonal sera against EBA-175 RII) immunoprecipitated native EBA-175, whereas mAb 48F8 (isotype control) and polyclonal sera KLS15 raised against Freund’s adjuvant did not.

Figure 2. Immunoblot analysis of EBA-175 RII mAbs against P. pastoris expressed recombinant RII F1 or F2 domains.

MAb R216 recognized a linear epitope within the F2 domain reacting against both reduced recombinant RII and F2 domain. MAb R217 recognized an epitope within F2 that was conformationally dependent. Reduction abrogated reactivity of R217 against recombinant RII and the F2 domain. MAb R218 was conformationally dependent and specific against the F1 domain and reacted against non-reduced RII and F1. Purified recombinant baculovirus EBA-175 RII protein at 0.5 µg per lane, or 10 uL per lane of supernatant of P. pastoris cultures expressing recombinant EBA-175 RII F1 or F2 domains were separated by SDS-PAGE under reduced or non-reduced conditions and electroblotted onto nitrocellulose membranes. In analyses against R216, a small fraction of the recombinant proteins were slightly denatured or reduced. In analysis using R218, reduction of the recombinant proteins was not absolute. Membranes were probed with 10 ug/mL each of mAbs R216, R217 or R218 separately. A similar staining pattern to that of R217 was observed for mAbs R215 and R256 (data not shown).

Figure 3. MAbs against the F1 and F2 domains block native [³⁵S]-labeled EBA-175 binding to erythrocytes synergistically.

Effects of mAbs on immunoprecipitation of [³⁵S]-labeled parasite culture supernatant containing labeled native EBA-175. Panel A shows that ratios of mAb R217 (against F2) and R218 (against F1) together increased the blocking of native EBA-175 binding (a synergistic effect). In contrast, Panel B shows that different ratios of mAb R217 (against F2) and R256 (also against F2) together resulted in similar levels of blocking (an additive effect). R217 and R256 may recognize a common epitope within the F2 domain. Bars show % blocking values as assessed by a phosphoimager.

Figure 4. Scatterplot showing a dose effect on P. falciparum FVO growth using mAb R217.

The solid line shows the linear regression and the dashed lines represent 95% confidence intervals. The goodness of fit R² was 0.9017.

Figure 5. EBA-175 is expressed in P.falciparum schizonts, and late liver stages in human hepatocytes in vitro, but not sporozoites.

A) Sporozoites stained with anti-PfCSP mAb 2A10 (used at 0.136 ug/mL), B) sporozoites stained with mAb R217 (used at 300 ug/mL), C) schizont stained with mAb R217 (used at 2.34 ug/mL), M: merozoite; A: apical end of the merozoite expressing EBA175, D) schizont stained with anti-PfCSP mAb 2A10 (used at 68 ug/mL). The nuclei were stained with DAPI, E) HC-O4 human hepatocytes stained with anti-P.falciparum liver stage antigen -1 (PfLSA-1) polyclonal rabbit serum (1:50 dilution) 6 days post infection with P.falciparum sporozoites, F) HC-O4 human hepatocytes stained with mAb R217 (used at 100 ug/mL) 6 days post infection with P.falciparum sporozoites. N: nucleus of the hepatocyte; P: liver stage parasite.