Defining the Antigenic Diversity of Plasmodium falciparum Apical Membrane Antigen 1 and the Requirements for a Multi-Allele Vaccine against Malaria
Figure 5
The importance of polymorphisms in the C1-L region of 3D7 for vaccine escape. A.
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. 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). C. 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). D. 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. * indicate a significant difference in inhibition when compared to the W2-3D7 reference line, P<0.05 by t-test.