Antigenic Subversion: A Novel Mechanism of Host Immune Evasion by Ebola Virus
Figure 6
The effect of sGP on immune response when antigen exposure mimics natural infection.
(A) Immunization study design. Female BALB/C mice were immunized IM with 50 µg of total DNA per immunization according to the schedule shown. Mice were immunized with a 3∶1 ratio of sGP Edit∶GP1,2 Edit in pCAGGS. Control groups were immunized with sGP Edit or GP1,2 Edit alone plus empty pCAGGS vector to keep total amount of immunizing DNA constant. (B) Comparison of antibody response against GP1,2. Mouse sera collected at week 6 were analyzed for anti-GP1,2 antibodies by ELISA using GP1,2 as coating antigen. (C) sGP competition ELISA. The ability of sGP to compete for anti-GP antibodies was determined by competition ELISA as in Figure 3B. Pooled antisera were analyzed from mice immunized with a GP1,2 Edit (blue), sGP Edit (red), or a 3∶1 ratio of sGP Edit∶GP1,2Edit (purple), and were diluted to give roughly equivalent anti-GP1,2 signal. Competition ELISA was performed from antisera collected at both week 6 (light color) and week 12 (dark color) according to the immunization schedule. (D) Competition immunoprecipitation. Pooled antisera from sGPEdit+GP1,2Edit-immunized mice were incubated with no GP, purified sGP or GP1,2 alone, or with fixed GP1,2 and increasing concentrations of sGP to compete for anti-GP1,2 antibodies. GP1,2 was incubated with recombinant HA as a negative control, and precipitated and analyzed as in Figure 3E,F. (E) Neutralization of EBOV GP pseudovirus. Neutralizing activity of antisera was determined by incubating 500 pfu of GP1,2-pseudotyped virus with dilutions of pooled sGP+GP1,2-immunized (red), or empty pCAGGS vector-immunized (black) antisera. Neutralization was measured as decrease in luciferase expression compared to virus-only controls. (F) Interference of EBOV GP pseudovirus neutralization by sGP. The ability of sGP to interfere with antibody-dependent neutralization was determined as in Figure 4B. Pooled sGP+GP1,2-immunized antisera were fixed at the dilution corresponding to 80% neutralization. Antisera were co-incubated with increasing dilutions of purified sGP (red) or purified influenza PR8 HA (blue), and rescue of infectivity was measured as described in methods.