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

<i>P. falciparum</i> parasitaemia development in all challenged <i>Aotus</i>.

<p>Percentage parasitaemia is plotted against day of sampling post challenge. Open symbols, immunized animals; filled symbols, control (naïve, non-immunized) animals.</p

Immunogenicity and adjuvant testing of MSP-1 Block 2 proteins from the FVO isolate.

<p>Effect of adjuvants on antibody responses against MSP-1 Block 2. Groups of five outbred (MF1, circles) or inbred (CBA, inverted triangles) mice were immunized s.c. three times at 4 week intervals with FVO MSP-1 Block 2 formulated with the adjuvants as indicated on the X axis. AH, Alhydrogel; AP, AdjuPhos; ISA51, Montanide ISA51; ISA720, Montanide ISA720; NISV, Non-ionic surfactant vesicles. Two weeks after the last immunization, serum samples were tested by ELISA for antibody responses against thrombin-cleaved and purified FVO MSP-1 Block 2 (Panel A) and by IFA reactivity with <i>P. falciparum</i> parasites of the Wellcome isolate, which has the same Block 2 sequence as FVO (Panel B). ELISA and IFA titers were calculated as outlined in Materials and Methods. Data is shown on a log₁₀ scale as dotplots of serum reactivity for individual animals with the median level of Ab reactivity for each group indicated by a horizontal line.</p

Recognition of peptide epitopes within MSP-1 Block 2.

<p>A panel of 38 N-terminally biotinylated dodecapeptides representing the sequence diversity of the MSP-1 MAD20 Block 2 serotype was used in ELISA to map the antibody specificities present in the sera of immunized monkeys, as described in Materials and Methods. Reactivity with individual peptides is shown in columns, with the strength of reactivity of each serum sample with each peptide shown as ELISA optical density. The sequence of each peptide used is indicated on the X-axis below each column.</p

Construction and analysis of fluorescent <i>P. cynomolgi</i> using a novel centromere construct.

<p>(A) Dot matrix analysis of a <i>P. cynomolgi</i> and <i>P. vivax</i> putative centromere (PCEN). Graphical representation of a matrix analysis of a <i>P. cynomolgi</i> PCEN aligned against itself (<i>left</i>), <i>P. cynomolgi</i> PCEN against the <i>P. vivax</i> PCEN (<i>middle</i>) and <i>P. vivax</i> PCEN aligned against itself (<i>right</i>). The analysis was performed using Dotlet <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054888#pone.0054888-Junier1" target="_blank">[46]</a> as described before <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054888#pone.0054888-Iwanaga1" target="_blank">[22]</a>. The diagonal line within each analysis represents sequence identity, and the diagonal line indicates repetitive regions within each PCEN. Note the absence of the diagonal in the repetitive regions of the <i>P. cynomolgi</i> and <i>P. vivax</i> alignment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054888#pone-0054888-g001" target="_blank">Figure 1</a>, <i>middle</i> panel). (B) Schematic representation of the pPcyC-PAC-GFP_hsp70-mCherry_ef1α plasmid. The plasmid contains the <i>Tgdhfr-ts</i> selectable marker that confers resistance against pyrimethamine and two expression cassettes for constitutive expression of GFP and mCherry. Additionally, to maintain the plasmid throughout the life cycle, a putative <i>P. cynomolgi</i> centromere (PcyCEN) is included. (C) Schematic representation of the procedure used for transfection and analysis of <i>P. cynomolgi</i>. (D) PCR amplification of <i>gfp</i> and <i>mCherry</i> in PcyC-PAC-GFP_hsp70-mCherry_ef1α (PcyC-PAC) blood stage parasites. Wild type gDNA of <i>P. cynomolgi</i> M served as negative control. For a control PCR primers for the <i>circumsporozoite protein (csp)</i> were used. For primers used, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054888#pone.0054888.s001" target="_blank">Table S1</a>. (E) GFP and mCherry expression throughout the life cycle of <i>P. cynomolgi.</i> GFP and mCherry expression in pPcyC-PAC-GFP_hsp70-mCherry_ef1α transfected <i>P. cynomolgi</i> blood stage parasites (a ring and a trophozoite or gametocyte), in oocysts 5 days post mosquito feeding and in salivary gland sporozoites 12 days post feeding. In the Brightfield panel two salivary gland lobes can be distinguished; only one lobe contains sporozoites. In the panel on the right GFP and mCherry expression is shown in Hoechst 33342 stained day 6 liver stages. Note the autofluorescence of hepatocytes in the GFP channel in contrast to the mCherry channel. A small uninucleate (arrow) and a large multinucleate liver stage are visible, confirmed by staining of fixed parasites with anti-HSP70 antibodies (<i>lower right panel</i>). White bars correspond to 10 µm (blood and mosquito stages) and 50 µm (liver stages).</p

Flow cytometry and cell sorting of <i>P. cynomolgi</i> liver stage parasites, including hypnozoite-forms.

<p>(A) Liver stage parasites used for flowcytometry as detected by anti-HSP70 antibodies 3 days and (B) 6 days post hepatocyte infection. White bars correspond to 50 µm. Note that day 3 cultures contain uniform small parasites while day 6 cultures contain both small and large liver stages (arrows). Flow cytometric plots of PcyC-PAC-GFP_hsp70-mCherry_ef1α (PcyC-PAC) <i>P. cynomolgi</i> liver stage parasites show a single GFP positive population compared to wild type parasites 3 days post hepatocyte infection (A, Gate 1) and two GFP positive populations 6 days post hepatocyte infection (B, Gates 2 and 3). The y-axis represents the PE-Texas Red Channel (for detection of autofluorescence), while the x-axis represents the GFP signal. (C) Post-sorting images of PcyC-PAC-GFP_hsp70-mCherry_ef1α<i>P. cynomolgi</i> liver stage parasites ‘GFPlow’ (Gate 2) and ‘GFPhigh’ (Gate 3) parasites sorted at day 6 post hepatocyte infection. The upper panel shows a GFP/Brightfield overlay while the lower panel shows mCherry/Brightfield overlay. The panels below show close-ups of the sorted parasites revealing the size differences between the ‘GFPlow’ and ‘GFPhigh’ populations. White bars correspond to 50 µm.</p