Cross-strain growth inhibition by antibodies to different AMA1 alleles.

<p><b>A.</b> The growth-inhibitory activity of polyclonal rabbit antibodies raised against W2Mef, 3D7, HB3 and FVO AMA1 alleles. Anti-W2Mef whole serum was tested at a dilution of 1∶10, and anti-3D7, anti-HB3 and anti-FVO rabbit purified IgG was tested a final concentration of 2 mg/ml IgG. Columns represent the mean parasite growth inhibition achieved in two separate assays tested in triplicate wells. The 4-way pool contains 25% (v/v) of each antibody and was tested at a final dilution of 1∶10. <b>B</b> Summary of cross-strain growth-inhibitory activity of antibodies against all isolates. Results show the median (horizontal line) level of inhibitory activity against the 18 isolates tested, and the interquartile range (box) and range (whiskers) of inhibitory activity. <b>C</b> Schematic representation of PfAMA1. The positions of amino acids (aa) that define Domain I (D1), Domain II (DII) and Domain III (DIII) of the PfAMA1 ectodomain are shown. The extracellular ectodomain is composed of aa 25 to 546 and excludes the signal sequence (SS), transmembrane domain (TM) and intracellular cytoplasmic tail (CT) regions. Not to scale.</p

<i>P. falciparum</i> isolates used in this study.

<p><i>P. falciparum</i> isolates used in this study.</p

The importance of polymorphisms in the C1-L region of 3D7 for vaccine escape. A.

<p>Plasmid design and integration. The C1-L of 3D7 and FVO AMA1 differ by 5 amino acid (aa) residues located at positions 196, 197, 200, 204 and 206. The hybrid 3F3 AMA1 (3D7 allele with the FVO C1-L sequence) was transfected into W2Mef parental parasites. The single-crossover event for allelic replacement of the wild type (WT) AMA1 with 3F3 is illustrated. <b>B.</b> Southern blot. Genomic DNA from parental W2Mef and transfected parasites was digested with restriction enzymes as indicated and hybridised with an AMA1 probe. Expected sizes for WT, non-integrated plasmid and integrated 3F3-AMA1 are shown in kilobases (kb). <b>C.</b> Phenotypic analysis of transgenic parasites expressing the 3F3-AMA1 hybrid. Transgenic W2Mef parasites expressing 3D7-AMA1 (W2-3D7), FVO-AMA1 (W2-FVO) or the hybrid 3F3-AMA1 (W2-3F3) were tested for their susceptibility to growth inhibition with the R1 peptide (final concentration 100 mg/ml) or the monoclonal antibody 1F9 (final concentration 0.2 mg/ml). <b>D.</b> Differential growth inhibition of transgenic parasite lines by polyclonal rabbit antibodies to AMA1; anti-W2Mef #1 and anti-FVO#2 rabbit sera were tested at a final dilution of 1∶10, all other antibodies listed were tested at a final concentration of 2 mg/ml IgG. Columns represent the mean parasite growth inhibition achieved in two separate assays tested in triplicate wells. <b>*</b> indicate a significant difference in inhibition when compared to the W2-3D7 reference line, P&lt;0.05 by t-test.</p

Phylogenetic analysis of AMA1 sequences.

<p><b>A.</b> Phylogenetic tree of the AMA1 alleles expressed by 18 different isolates examined in this study in relation to 250 other AMA1 alleles obtained from the public database. Analysis was based on the ectodomain sequence. <b>B.</b> Phylogenetic tree of the AMA1 alleles expressed by the 18 different isolates used in this study, based on the AMA1 ectodomain sequence (amino acids 25–456). The AMA1 sequences of HCS-E5 and CSL-2 were found to be identical. <b>C.</b> Phylogenetic tree of the AMA1 alleles expressed by isolates used in this study, based on the C1-L sequence of AMA1 (amino acids 196–207). Translated ectodomain and C1-L protein sequences were aligned and phylogenetic trees constructed using ClustalW2.</p

Transgenic and wild type parasite lines expressing the same AMA1 alleles share the same phenotype. A.

<p>Plasmid design and integration. Codon optimised W2Mef, 3D7 and FVO AMA1 alleles were transfected into W2Mef parental parasites. The single-crossover event for allelic replacement of the wild type (WT) AMA1 is illustrated. <b>B.</b> Southern blot. Genomic DNA from parental W2Mef and transfected parasites was digested with restriction enzymes as indicated and hybridised with an AMA1 probe. Expected sizes for WT, non-integrated plasmid and integrated 3F3-AMA1 are shown in kilobases (kb). <b>C.</b> Differential growth inhibition of wild type and transgenic parasite lines by anti-W2Mef #1 antibodies tested at a final dilution of 1∶10. <b>*</b> indicate a significant difference in inhibition when compared to the W2Mef reference line, P&lt;0.05 by t-test. <b>D, E, F</b> Phenotypic comparisons between the parental and transgenic parasites for W2Mef, 3D7 and FVO alleles of AMA1. Each figures shows the expression of W2Mef, 3D7 and FVO AMA1 in transgenic parasites compared to the corresponding parental parasite isolate by western blot and growth inhibition of transgenic parasites compared to the corresponding parental parasite isolate with AMA1 allele-specific antibodies.</p

Vaccination with Conserved Regions of Erythrocyte-Binding Antigens Induces Neutralizing Antibodies against Multiple Strains of <i>Plasmodium falciparum</i>

<div><p>Background</p><p>A highly effective vaccine against <i>Plasmodium falciparum</i> malaria should induce potent, strain transcending immunity that broadly protects against the diverse population of parasites circulating globally. We aimed to identify vaccine candidates that fulfill the criteria.</p><p>Methods</p><p>We have measured growth inhibitory activity of antibodies raised to a range of antigens to identify those that can efficiently block merozoite invasion for geographically diverse strains of <i>P. falciparum</i>.</p><p>Results</p><p>This has shown that the conserved Region III-V, of the <i>P. falciparum</i> erythrocyte-binding antigen (EBA)-175 was able to induce antibodies that potently inhibit merozoite invasion across diverse parasite strains, including those reliant on invasion pathways independent of EBA-175 function. Additionally, the conserved RIII-V domain of EBA-140 also induced antibodies with strong <i>in vitro</i> parasite growth inhibitory activity.</p><p>Conclusion</p><p>We identify an alternative, highly conserved region (RIV-V) of EBA-175, present in all EBA proteins, that is the target of potent, strain transcending neutralizing antibodies, that represents a strong candidate for development as a component in a malaria vaccine.</p></div

IC₅₀ values for total IgG against different antigens in GIA.

#<p>IC₅₀ values were calculated by interpolation on the plot of total IgG[log₁₀] versus % GIA with measured points connected by straight lines (as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072504#pone.0072504-Williams1" target="_blank">[56]</a>, calculations were performed on % GIA values in a 1-cycle assay, with IgG titrated from 2 mg/ml.</p

Data from independent testing of rabbit IgG for GIA activity.

<p>Data points indicate mean values of triplicate wells from a single-cycle pfLDH-based assay using 3D7 parasites. % GIA was calculated relative to pre-immune IgG.</p