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.