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

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

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

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

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

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

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

Monoclonal antibodies against AMA1.

<p> GIA values are mean of 3 or more experiments against 3D7 strain.</p><p>^# 1F9 tested at 0.6 mg/ml in GIA.</p><p> Strain reactivity out of 7 allelic proteins by dot blot.</p><p> Binding location assigned by dot blot or western blot against linear and crystal domain chimeras.</p><p><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003840#ppat.1003840-Coley2" target="_blank">[29]</a><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003840#ppat.1003840-Kocken1" target="_blank">[30]</a>. previously described AMA1 mAbs </p

Biological activity of monoclonal antibodies.

<p>(<b>A</b>) Binding of 3D7 AMA1 (OD₄₅₀) to immobilized RON2 peptide inhibited by serial dilutions of the mAbs. Negative control mAb 5G8 binds to the N-terminal prosequence. (<b>B</b>) Western blot of a 3D7 parasite processing inhibition assay using 200 µg/ml mAbs. Top panel shows the merozoite bound full-length (83 kDa) 3D7 parasite AMA1 and the product of N-terminal processing (66 kDa). Bottom panel shows the co-migrating products of normal shedding (48+44 kDa) and the product of anomalous AMA1 processing (52 kDa). These fragments were captured from the culture supernatant using a sub-inhibitory concentration of polyclonal anti-3D7 AMA1 sera (1∶2500) in the processing assay <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003840#ppat.1003840-Dutta4" target="_blank">[34]</a>. (<b>C</b>) GIA against 3D7 target strain, using 1×IC₃₀ dose of individual mAbs (black), 2×IC₃₀ dose of individual mAbs (gray), 1×IC₃₀+1×IC₃₀ mixture of two 1e-loop mAbs (green) or 1e-loop+domain2-loop mAb (blue) or 1e-loop+domain-3 mAb (orange) or domain2 loop+domain-3 mAb (red). Mean+s.e.m. of 3 experiments; (*) p<0.05 comparing the mean of each group to the mean of 2×IC₃₀ dose of individual mAbs (gray bars). (<b>D</b>) GIA against the 3D7 parasite strain using increasing concentrations of mAb 1E10, with (red line) or without (blue line) the addition of 1×IC₃₀ concentration of mAb 4G2 (1.8 mg/ml, expected 30% GIA in green). Predicted inhibition for additive interaction (black line) was calculated according to “Bliss independence” as has been applied to determine synergy by Williams <i>et al. </i><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003840#ppat.1003840-Williams1" target="_blank">[55] </a><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003840#ppat.1003840-Bliss1" target="_blank">[56]</a>; data are mean+s.e.m. of triplicate wells. (<b>E</b>) Inhibition of 7 parasite strains using 2 mg/ml of the RON2 inhibitory mAb or a mixture of 1 mg/ml each of the RON2 inhibitory mAbs and processing inhibitory mAb 1E10; a representative of two experiments is shown.</p