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

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

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

Analysis of purified MSP-1-BBM protein by SDS-PAGE and Western blotting.

<p>A. Coomassie-stained gel of purified MSP-1-BBM protein produced in <i>T. thermophila</i>. Lane 1. Molecular weight markers. Lane 2. 0.5 µg purified MSP-1-BBM protein. B. Western blot of MSP-1-BBM protein and MSP-1 hybrid probed with with mAb 12.2, (specific for repeat sequences present in the K1 serotype of MSP-1 Block 2). Lane 1. Molecular weight markers. Lane 2. 0.5 µg of <i>Tetrahymena</i>-derived MSP-1-BBM protein. Lane 3. Negative control. Lane 4. 0.5 µg MSP-1 Block 2 hybrid protein <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087198#pone.0087198-Galamo1" target="_blank">[30]</a> (positive control).</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

A. Heat map of antibody reactivity to FVO Block 2 serotype peptides over the course of immunization. MSP-1 Block 2 specific peptide ELISA is as described in Figure 4 and in Materials and Methods. Reactivities of sera from immunized <i>Aotus</i> are shown as blue rectangles for each peptide tested, with darker colored bars indicating higher ELISA reactivity as shown in the figure key. Columns represent serum reactivity for each time point, and each panel shows reactivity for all pre-challenge samples from each animal. B. Amino acid sequence of the FVO MSP-1 Block 2 antigen. MSP-1 Block 2 flanking sequences are shown in red and internal repeat sequences in blue, matching the peptide sequences shown in Panel A.

<p>A. Heat map of antibody reactivity to FVO Block 2 serotype peptides over the course of immunization. MSP-1 Block 2 specific peptide ELISA is as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083704#pone-0083704-g004" target="_blank">Figure 4</a> and in Materials and Methods. Reactivities of sera from immunized <i>Aotus</i> are shown as blue rectangles for each peptide tested, with darker colored bars indicating higher ELISA reactivity as shown in the figure key. Columns represent serum reactivity for each time point, and each panel shows reactivity for all pre-challenge samples from each animal. B. Amino acid sequence of the FVO MSP-1 Block 2 antigen. MSP-1 Block 2 flanking sequences are shown in red and internal repeat sequences in blue, matching the peptide sequences shown in Panel A.</p

Details of <i>Aotus lemurinus griseimembra</i> used for immunization and challenge.

<p>died of non-immunization causes before challenge.</p><p>¶naïve (non-injected) animal substitute.</p><p>Removed from experiment after challenge.</p

A. Antigen specific antibody titers of sera from <i>Aotus</i> monkeys immunized with the GST-FVO Block 2 fusion protein. Serum samples collected at the time points listed in Table 2 were tested for reactivity with cleaved, purified FVO MSP-1 Block 2. Small arrows indicate immunization time points. Large arrow indicates <i>P. falciparum</i> challenge time point. Titers were calculated by interpolation from titration curves for each serum sample, with the endpoint titer defined as the dilution that gave an optical density value of 0.1. B. Parasite-reactive antibody titers of sera from four immunized <i>Aotus</i> (A53, A66, A9801, A9802) plus control animals (A9804, A9902). Sera were tested by IFA against the Wellcome <i>P. falciparum</i> strain (which has an identical MSP-1 Block 2 sequence to FVO). Hollow symbols, immunized animals; filled symbols, control (non-immunized) animals. Small arrows indicate immunization time points.

<p>A. Antigen specific antibody titers of sera from <i>Aotus</i> monkeys immunized with the GST-FVO Block 2 fusion protein. Serum samples collected at the time points listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083704#pone-0083704-t002" target="_blank">Table 2</a> were tested for reactivity with cleaved, purified FVO MSP-1 Block 2. Small arrows indicate immunization time points. Large arrow indicates <i>P. falciparum</i> challenge time point. Titers were calculated by interpolation from titration curves for each serum sample, with the endpoint titer defined as the dilution that gave an optical density value of 0.1. B. Parasite-reactive antibody titers of sera from four immunized <i>Aotus</i> (A53, A66, A9801, A9802) plus control animals (A9804, A9902). Sera were tested by IFA against the Wellcome <i>P. falciparum</i> strain (which has an identical MSP-1 Block 2 sequence to FVO). Hollow symbols, immunized animals; filled symbols, control (non-immunized) animals. Small arrows indicate immunization time points.</p

Schedule of immunization, blood sampling and parasite challenge of <i>Aotus lemurinus griseimembra</i>.

<p>The day of sampling and/or immunization is shown with the appropriate week of each time point shown in brackets.</p><p>i.v. injection (1 x 10⁵<i>P. falciparum</i> FVO parasites - ring stage).</p><p>Parasite challenge Go/No Go decision point, based on IFA titer.</p><p>Drug treatment criteria: Parasitemia ≥5% and/or haematocrit ≤20%.</p