Fig. 4. Antibodies are sensitive to naturally occurring mutations.

The binding of these Gn-specific antibodies to Gn proteins with previously identified naturally occurring mutations²⁷ was tested next. Gene constructs encoding Gn with WT or variant sequence were synthesized and transiently expressed in suspension cells. Cells were stained for viability and fixed and then subsequently permeabilized for staining with an individual antibody and then with anti-human Fc PE-conjugated antibodies. The variants tested were: T173L, E175G, and K294E-D230N. Cells were assessed on the high-throughput IntelliCyt iQue flow cytometric screener. Cells were gated for viability and values are expressed as a percentage of positive cells (signal over 10^5 on the BL2-H channel) over the total count of viable cells. The data shown represents technical triplicate values over three independent experiments. RVFV-296 was not sensitive to any mutation, revealing that the antigens were expressed at similar levels. CCHF-245 was used as a negative control. Data are presented as mean values +/− SD and statistical test was done by using an ordinary one-way ANOVA test corrected for multiple comparisons. Source data are provided as a Source data file.