FIG. 4.

Ability of CatL-cleaved trimeric EBOV-GP ZEBOV-GP to elicit neutralizing antibodies and bind KZ52. (A) The neutralization by antisera from groups of five mice immunized and boosted at weeks 0, 4, and 8 with 20 μg of either CatL-cleaved or uncleaved ZEBOV-GP in 50 μl PBS and an equal volume of Ribi adjuvant was analyzed by incubating the mouse sera (collected 10 days after each vaccination) with an ZEBOV-GP pseudotyped lentiviral reporter vector expressing luciferase. The neutralization activity was calculated based on the luciferase activity relative to values reported for preimmune mouse sera. CatL-cleaved ZEBOV-GP induced the highest titer of neutralizing antibody response, while the uncleaved ZEBOV-GP was 3-fold less potent. (B) Mouse sera from CatL-cleaved ZEBOV-GP mice were analyzed for neutralizing activity against all five EBOV-GP strains individually pseudotyped or a VSV control with lentiviral reporter vectors expressing luciferase. Matched Zaire GP and Ivory Coast GP pseudotyped viruses showed the highest neutralization, while Sudan GP, Bundibugyo GP, and Reston GP pseudotyped viruses showed limited neutralization. The VSV control showed no neutralization. (C) Surface plasmon resonance analysis of CatL-cleaved ZEBOV-GP (upper panel) and SEBOV-GP (lower panel) showed that KZ52 binds both CatL-cleaved EBOV GP strains with a binding affinity of 1.5 nM and 63 nM, respectively.