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

<p><u>Panel A:</u> Dual immunofluorescent analyses showing apical staining of mature <i>P. falciparum</i> (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). <u>Panel B:</u> 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.</p

Scatterplot showing a dose effect on <i>P. falciparum</i> FVO growth using mAb R217.

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

EBA-175 is expressed in <i>P.falciparum</i> schizonts, and late liver stages in human hepatocytes <i>in vitro</i>, but not sporozoites.

<p>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), <b>M</b>: merozoite; <b>A</b>: 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-<i>P.falciparum</i> liver stage antigen -1 (PfLSA-1) polyclonal rabbit serum (1:50 dilution) 6 days post infection with <i>P.falciparum</i> sporozoites, F) HC-O4 human hepatocytes stained with mAb R217 (used at 100 ug/mL) 6 days post infection with <i>P.falciparum</i> sporozoites. N: nucleus of the hepatocyte; P: liver stage parasite.</p

Immunoblot analysis of EBA-175 RII mAbs against <i>P. pastoris</i> expressed recombinant RII F1 or F2 domains.

<p>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 <i>P. pastoris</i> 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).</p

MAb R217 does not inhibit invasion and development of <i>P. falciparum</i> sporozoites in the human hepatocyte line, HC04 as assessed by ILSDA.

<p>*PfLSA-1 polyclonal rabbit antibody used at 1∶50 dilution.</p

Summary of EBA-175 RII specific mAbs.

<p>ND: not determined</p><p>NA: not achievable. R216 at 333 and 666 µg/ml IgG blocked EBA-175 binding by 14 and 21%, respectively.</p><p># <i>P. falciparum</i> FVO 2 cycle suspension growth inhibition assay (GIA).</p>1<p>mAbs compete against each other for binding RII by competition ELISA.</p><p>c: constrained epitope; L: linear epitope; immppt: immunoprecipitate.</p><p>*incomplete reduction; ** partial denaturation/reduction.</p

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

<p>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.</p

Expression of <i>P. falciparum</i> EBA-175 in late (6 day) but not in early (3 day) liver stages.

<p>*PfLSA-1 polyclonal rabbit antibody used at 1∶50 dilution and mAb R217 used at 100 ug/mL.</p