Ability of sGP to divert antibody responses against GP<sub>1,2</sub>.
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Abstract
<p>(A) Immunization study design. Female BALB/C mice were immunized IM with 50 µg of total DNA per immunization according to the schedule. Two groups of mice (n = 12) were primed and boosted as in previous experiments with either sGP Edit or GP<sub>1,2</sub> Edit in pCAGGS vector. Each group was divided in two and subgroups were boosted at week 10 with either the same construct against which they had initially been immunized, or with the opposite editing site mutant construct. (B) Comparison of antibody response against GP<sub>1,2</sub>. Sera collected at week 12 were analyzed for antibodies against GP<sub>1,2</sub> by ELISA using GP<sub>1,2</sub> as coating antigen. (C) sGP competition ELISA. The ability of sGP to compete for anti-GP<sub>1,2</sub> antibodies was determined by competition ELISA as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003065#ppat-1003065-g003" target="_blank">Figure 3B</a>. Pooled antisera were analyzed from mice immunized with sGP Edit and then boosted at week 10 with either GP<sub>1,2</sub> Edit (red), or sGP Edit (purple), and from mice immunized with GP<sub>1,2</sub> Edit and then boosted at week 10 with either GP<sub>1,2</sub>Edit (blue) or sGP Edit (green). All ELISA experiments were performed in duplicate at least three times and representative results shown. (D) Interference of EBOV GP pseudovirus neutralization by sGP. The ability of sGP to interfere with antibody-dependent neutralization was determined as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003065#ppat-1003065-g004" target="_blank">Figure 4B</a>. Pooled sGP-primed, GP<sub>1,2</sub>-boosted (red) and GP<sub>1,2</sub>-primed, sGP-boosted (green) antisera were fixed at the dilution corresponding to 50% neutralization. Antisera were co-incubated with increasing dilutions of His-tagged sGP (solid markers) or His-tagged influenza PR8 HA (open markers), and rescue of infectivity was measured as described in methods. (E) Comparison of 50% neutralization titers. Antiserum titers corresponding to 50% pseudovirus neutralization activity (NT<sub>50</sub>) were calculated for week 6 (fine checkered) and week 12 (coarse checkered) mice. Error bars correspond to 95% confidence interval as determined by Student's t-test.</p