Figure 6. Mapping of putative epitopes recognized by LASV mAbs by site-directed mutagenesis.

Wild-type recombinant LASV GPC was engineered with altered amino-acid sequences to map putative B-cell epitopes on LASV glycoproteins. (a) Location of mutations in engineered LASV glycoproteins are mapped to a structural model of GP1. (b) Binding of mAbs to GPC constructs in which the indicated sets of three amino acids in the amino terminus of GP1 were mutated to three alanines. ConA-capture ELISA performed as in Fig. 5; results expressed as per cent of binding to unmutated control. An expanded panel of mAbs is presented in Supplementary Fig. 8d. (c) 19.7E fails to neutralize LASVpp expressing GPC from lineage III LASV. The sequence of lineage IV GP1 was partially altered to the sequence in lineage III GPC by changing the sequence IIN to LLN. Binding of mAbs was quantified by ConA ELISA as described above. Mutations that reduced binding to 10% or less of control or 11–24% of control are highlighted. (d) Binding of mAbs to GP1 constructs in which the indicated single amino acids in GP1 were mutated to alanine and expressed as D7-tagged proteins. Binding of mAbs to mutant and control GP1-D7 constructs captured in wells coated with mAb JR-52. A mixture of anti-GP1 mAbs was included as a control. Values indicate per cent binding of mutants compared with unmutated GP1. The results shown are representative of multiple (n>4) independent experiments.