Binding of native and MA-activated α₂M to HB3VAR06.

<p>(A) Titration of binding of native α₂M (black circles) and α₂M-MA (white circles) to recombinant full-length HB3VAR06 measured by ELISA. Means and SD are indicated. (B) Titration of binding of native α₂M (black circles) and α₂M-MA (white circles) to HB3VAR06⁺ IEs measured by flow cytometry. Means and SD are indicated. (C) Activation of α₂M measured by SDS gel electrophoresis of soluble and immobilized α₂M in the presence of mPEG: soluble α₂M alone (lane 1), soluble α₂M and MA (lane 2), soluble α₂M and FV6 (lane 3), bead-immobilized α₂M-FV6 complexes alone (lane 4), and bead-immobilized α₂M-FV6 complexes and MA (lane 5). While native α₂M was detectable in all lanes, activated α₂M having a higher molecular weight than native α₂M due to incorporation of mPEG was only detected in the presence of MA (lanes 2 and 5).</p

Identification of α₂M as the soluble serum factor binding HB3VAR06.

<p>(A) 2D gel electrophoresis of serum components pulled down with recombinant full-length HB3VAR06 (FV6; left) or PFD1235w-DBLβ3_D5 (right). Spots subsequently identified as α₂M and IgM in the left panel are boxed. Molecular weight (kDa) markers are shown along the left margins. (B) Binding of α₂M to recombinant full-length HB3VAR06 (FV6; left) and IT4VAR04 (FV2; right), measured by ELISA. Means and SD are indicated. (C) Binding of α₂M to HB3VAR06⁺ IEs (left) and IT4VAR04⁺ IEs (right), measured by flow cytometry. Control sample labeling (no α₂M added) is indicated by gray shading. (D) Fluorescence micrographs of DAPI-labeled HB3VAR06⁺ IEs in the presence (top) and absence (bottom) of fluorescein isothiocyanate-labeled α₂M at low (scale bar: 20 μm; left) and high (scale bar: 5 μm; right) magnification are shown.</p

Identification and characterization of the α₂M-binding domain in HB3VAR06.

<p>(A) Schematic representation of the domain structure of HB3VAR06. Domain nomenclature as described by Rask <i>et al</i>. [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005022#ppat.1005022.ref037" target="_blank">37</a>], as well as the first and last amino acid in each domain are indicated. (B) Binding of α₂M to recombinant HB3VAR06 single-, double-, and triple-domain constructs (labeled as in panel A) as well as to full-length HB3VAR06 (FV6) measured by ELISA. Means and SD are indicated. (C) Inhibition of α₂M binding to HB3VAR06⁺ IEs by anti-sera raised against the N-terminal head structure (D1–D3), DBLξ2 (D8), and full-length HB3VAR06 (FV6), respectively, measured by flow cytometry. Means and SD relative to binding without anti-serum are indicated. (D) Simultaneous labeling of HB3VAR06⁺ IEs by α₂M and IgM (left), measured by flow cytometry. A control experiment with all detecting antibodies present but without α₂M and IgM is shown to the right. (E) Inhibition of IgM binding to HB3VAR06⁺ IEs by increasing concentrations of α₂M, measured by flow cytometry. Means and SD relative to binding in the absence of α₂M are indicated. (F) Inhibition of α₂M binding to HB3VAR06⁺ IEs by increasing concentrations of IgM, measured by flow cytometry. Means and SD relative to binding in the absence of IgM are indicated.</p

Schematic diagram of the MSP-1-BBM construct which encodes the N-terminal MSP-1 Block 1 region, the K1 Block 2 synthetic sequence, the RO33 Block 2 sequence, the MAD20 Block 2 synthetic sequence [31] and a C-terminal fragment encoding residues 1571 to 1702 of MSP-1 precursor protein of <i>P. falciparum</i>.

<p>The Block 2 region is arranged similar to natural alleles. Synthetic Block 2 repeat sequences of the K1 and MAD20 serotypes are indicated by vertical and diagonal hatched markings, respectively.</p

Epitope mapping of sera from MSP-1-BBM immunized mice by recognition of peptide epitopes within the MSP-1 Block 2 region of the MSP-1-BBM construct.

<p>A series of 133-terminally biotinylated dodecapeptides, representing the sequence diversity of all three Block 2 serotypes were used in ELISA to map the antibody specificities present in the sera of immunized animals. Reactivity with individual peptides is shown as shaded boxes, with the depth of shading of each box representing the strength of reactivity of a 1∶500 dilution of sera with each peptide. The sequences and Block 2 serotype (K1, MAD20 and RO33) of each peptide are indicated down the right hand side of the diagram.</p

Indirect immunofluorescence assay (IFA) of sera from MSP-1-BBM immunized mice against three strains of <i>P. falciparum</i>.

<p>A. Representative micrograph of IFA assay with sera from MSP-1-BBM immunized mice. DAPI staining of parasite nuclei is shown in blue and fluorescence from the FITC-conjugated secondary antibody is shown in green. B. IFA titers of sera from mice immunized with MSP-1-BBM protein. Sera were tested by IFA against the 3D7 (K1 serotype), MAD20 and RO33 strains of <i>P. falciparum</i>, as described in materials and methods. IFA endpoint data is shown on a log₁₀ scale on the Y axis. Each symbol represents the serum reactivity for an individual animal, with the geometric mean of Ab reactivity against each parasite strain indicated by the solid line. C. Western blot of MSP-1 Block 2 hybrid and MSP1₁₉ proteins probed with pooled serum from mice immunized with MSP-1-BBM protein. Lane 1: Molecular weight markers, Lane 2: MSP-1 block 2 hybrid protein, Lane 3: MSP1₁₉-GST fusion protein.</p

Immunogenicity in mice of recombinant MSP-1-BBM protein from <i>T. thermophila</i>[49].

<p>A group of five MF1 mice were immunized s.c. three times, at 2 week intervals with MSP-1-BBM protein formulated in CoVaccine HT as described. Twelve days after the last immunization (d40), serum samples from each animal were tested by ELISA for antibody responses against the MSP-1 Block 2 hybrid protein <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087198#pone.0087198-Cowan1" target="_blank">[31]</a> K1-type Block 2 protein <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087198#pone.0087198-Cavanagh2" target="_blank">[54]</a>, MAD20-type Block 2 protein <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087198#pone.0087198-Cavanagh2" target="_blank">[54]</a>, RO33-type Block 2 protein <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087198#pone.0087198-Cavanagh2" target="_blank">[54]</a> and MSP-1₁₉ protein <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087198#pone.0087198-Burghaus1" target="_blank">[53]</a>. Titers were calculated as outlined in materials and methods and expressed as arbitrary units (AU). Data is shown on a natural logarithmic scale as dotplots of serum reactivity for individual animals with the median level of Ab reactivity indicated by the solid horizontal line.</p

Reactivity of <i>Aotus</i> sera with parasite proteins.

<p>Schizont extracts from the Wellcome (W) and 3D7 (3) isolates were probed by Western blotting with sera from all four immunized animals. Serum samples from day 97 (pre-challenge) and day 120 (post challenge) from each animal were tested in parallel on contiguous parts of the same membrane. Immunized animal code numbers are shown on the left of each panel. Arrowheads indicate reactivity with the N-terminal p83 proteolytic fragment of MSP-1. The dominant 50 kDa band in all blots is the heavy chain of human IgG, recognized by the secondary reagent (HRP conjugated anti-human IgG heavy chain).</p