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

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

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

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

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

Oligonucleotides used for amplification of <i>ama1</i> gene sequences.

<p>Oligonucleotides used for amplification of <i>ama1</i> gene sequences.</p

The importance of polymorphisms in the C1-L region of W2mef for vaccine escape.

<p><b>A</b> Alignment of amino acids (aa) 196–206 of AMA1 across the different strains. W2Mef, 3D7, HB3, FVO, XIE, Pf2006 and <i>P. reichenowi</i> (Pr) AMA1 C1-L 196 to 206 region have polymorphisms at 6 out of the 11 amino acid locations within the defined loop region, located at positions 196, 197, 200, 201, 204 and 206. <b>B.</b> Plasmid design and integration. Six hybrid W2Mef-AMA1 alleles containing the aa196–206 C1-L domain from 3D7, HB3, FVO, XIE, Pf2006 or Pr were transfected into W2Mef parental parasites. The single-cross over event for allelic replacement of the wild type (WT) AMA1 with W2Mef-C1-L hybrid AMA1 alleles is illustrated. <b>C.</b> Southern blot. Genomic DNA from parental W2Mef and transfected parasite populations was digested with restriction enzymes as indicated and hybridised with an AMA1 probe. Expected sizes for WT, non-integrated plasmid and for integrated W2Mef C1-L hybrids are shown in kilobases (kb). <b>D</b> Differential growth inhibition of transgenic parasite lines by polyclonal rabbit antibodies to AMA1. The four W2Mef C1-L hybrid transgenic lines generated, and a control W2–W2 transgenic line expressing WT W2Mef AMA1, were tested in GIAs against different polyclonal rabbit AMA1 antibodies. Anti-W2Mef #1 was tested at a final dilution of 1∶10, and all other antibodies listed were tested at a final concentration of 2 mg/ml of IgG. Columns represent the mean parasite growth inhibition achieved in two separate assays tested in triplicate wells. <b>*</b> indicates a significant difference in inhibition when compared to the W2–W2 reference line, P&lt;0.05 by t-test.</p

Prevalence of parasitic infections in pregnancy and association of moderate/severe maternal anemia (Hg<9) at first ANC visit, among a cohort of pregnant women in coastal Kenya, 2006–2009.

<p>*Numbers differ due to missing values;</p>†<p>Adjusted for primagravid status, gestational age at ANC, and low maternal BMI;</p>‡<p>None of the participants had ≥50 eggs/mL; MS = Microscopy diagnosed malaria.</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

Maternal socio-demographic factors by maternal anemia status at first antenatal care visit, cohort of pregnant women in coastal Kenya, 2006–2009.

<p>*Numbers less than total enrolled reflect missing data.</p>†<p>Unadjusted Risk ratio (RR) and 95% confidence intervals (CI).</p>‡<p>Adjusted for gestational age.</p

Factors associated with moderate/severe maternal anemia at first ANC among a cohort of pregnant women, coastal Kenya 2006–2009.

<p>*Regression model adjusted for all variables listed and gestational age.</p><p>ANC = Antenatal care; MS = microscopy.</p