Antibody epitope identification by immunofluorescence assay.

<p>DBL domain proteins were surface expressed on HEK293 cells and probed with R217 or R218 as primary and Alexafluor-546 labeled anti-IgG₁ as secondary. Green channel shows GFP tagged expressed protein. Red channel shows Alexafluor-546 labeled proteins on HEK293 cell surface. Merged channel shows overlap between green and red channels.</p

Crystal structure of RII/R217 Fab complex.

<p>(A) Overall structure of the RII/R217 Fab complex shown in ribbon representation. The F1 domain of RII is colored in green, the F2 domain of RII is colored purple. The Fab heavy chain (VH) is in blue and the light chain (VL) in pink. The location of F2 β-finger is circled in black. (B) Ribbon representation of F2 mapping the R217 epitope. Residues contacted by the Fab are show in stick. Residues contacted by the heavy chain are colored blue, residues contacted by the Fab light chain are colored pink, and residues contacted by both chains are in beige. Residues not contacted by the antibody are in purple. (C) Surface representation of F2 mapping the R217 epitope. Color scheme as in B. (D) Surface representation of the R217 Fab, mapping heavy chain residues (blue) that contact F2 (purple). The light chain is shown in white. (E) Surface representation of the R217 Fab, mapping light chain residues (pink) that contact F2 (purple). The heavy chain is shown in white.</p

Direct antibody-inhibition of GpA binding by RII.

<p>(A) RII binds to GpA (lane 1), but not to neuraminidase treated GpA (GpA-NA – lane 2). Addition of R217 IgG or Fab prevents RII from binding GpA (lane 3 and 4). Neither R218 IgG (lane 5), R218 Fab (lane 6), control IgG (lane 7) nor control Fab (lane 8), block RII/GpA receptor binding. Binding is specific as neuraminidase treatment prevents binding in all cases (lanes 2 and 9–14). Note that an increased signal is observed with R218 IgG over R218 Fab (compare lanes 5 and 6) suggesting bivalent binding. This assay was performed with RII at 3 µM and antibody or Fab fragments at 6 µM. (B) Titration of R217 demonstrates the specificity of interaction as all concentrations around and above the available RII concentration (3 µM) prevents binding (lanes 6–10). At concentrations below the available RII binding occurs as not all the RII is bound by R217 (lanes 3–5). (C) Titration of R218 demonstrates that R218 is unable to directly prevent GpA binding at any concentration.</p

Crystal structure of F1/R218 Fab complex.

<p>(A) Overall structure of the F1/R218 Fab complex shown in ribbon representation. The F2 domain of RII is colored in green. The Fab heavy chain (VH) is in blue and the light chain (VL) in pink. (B) Ribbon representation of F1 mapping the R218 epitope. Residues contacted by the Fab are show in stick. Residues contacted by the heavy chain are colored blue, residues contacted by the Fab light chain are colored orange, and residues contacted by both chains are in beige. Residues not contacted by the antibody are in green. (C) Surface representation of F2 mapping the R217 epitope. Color scheme as in B. (D) Surface representation of the R218 Fab, mapping heavy chain residues (blue) that contact F2 (green). The light chain is shown in white. (E) Surface representation of the R218 Fab, mapping light chain residues (orange) that contact F2 (green). The heavy chain is shown in white.</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

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

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

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

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