Potent neutralizing monoclonal antibodies against Ebola virus infection

Abstract

Ebola virus infections cause a deadly hemorrhagic disease for which no vaccines or therapeutics has received regulatory approval. Here we show isolation of three (Q206, Q314 and Q411) neutralizing monoclonal antibodies (mAbs) against the surface glycoprotein (GP) of Ebola virus identified in West Africa in 2014 through sequential immunization of Chinese rhesus macaques and antigen-specific single B cell sorting. These mAbs demonstrated potent neutralizing activities against both pseudo and live Ebola virus independent of complement. Biochemical, single particle EM, and mutagenesis analysis suggested Q206 and Q411 recognized novel epitopes in the head while Q314 targeted the glycan cap in the GP1 subunit. Q206 and Q411 appeared to influence GP binding to its receptor NPC1. Treatment with these mAbs provided partial but significant protection against disease in a mouse model of Ebola virus infection. These novel mAbs could serve as promising candidates for prophylactic and therapeutic interventions against Ebola virus infection.

Figure 1

Binding (a) and neutralizing activities of isolated mAbs against pseudo (b) and live (c) Ebolaviruses. Pseudoviruses bearing the envelope glycoprotein from the five species of Ebolaviruses (EBOV, SUDV, BDBV, RESTV, TAFV) are presented as well as the controls from Marburg virus, human immunodeficiency virus (HIV) and vesicular stomatitis virus (VSV). Dashed line indicates 50% inhibition. Controls antibodies used here include VRC01, a human neutralizing mAb against HIV-1, c13C6 and KZ52, previously isolated neutralizing mAbs against EBOV. In live EBOV experiment (c), neutralizing activity was evaluated in the presence (dark) and absence (light) of complement in comparison with c13C6, one of the three mAbs in the ZMapp cocktail and its neutralizing activity is complement-dependent in vitro. Data presented are average values from at least two independent experiments and the error bars indicate for the standard error of the mean (SEM).

Figure 2

Binding kinetics of isolated mAbs with purified EBOV GPdM (a), sGP (b) and GPcl (c) measured by surface plasmon resonance (SPR). The purified soluble antigens GPdM, sGP and GPcl were covalently immobilized onto a CM5 sensor chip followed by injection of individual Fab of Q206, Q314, Q411 as well as control Fab c13C6 and KZ52 at five different concentrations. The black lines indicate the experimentally derived curves while the red lines represent fitted curves based on the experimental data.

Figure 3. Epitope mapping through competitive binding measured by SPR.

The sensorgrams show distinct binding patterns when pairs of testing Fabs were sequentially applied to the purified GPdM covalently immobilized onto a CM5 sensor chip. Arrows indicate the time points when Fabs were injected. Whether additional binding after injecting the second Fab is the key criteria for determining the two mAbs recognize the separate or closely situated epitopes.

Figure 4. Single-particle EM reconstructions of Q206, Q314 and Q411 Fabs bound to EBOV GPdM.

Hybrid models of negative-stain EM reconstructions fit with the EBOV GPdM crystal structures (PDB assessing number 3CSY) and a reference Marburg virus mAb MR78 Fab (PDB assessing number 3X2D). (a) Side view of individual Fab Q206 (mustard), Q314 (blue) and Q411 (magenta) bound to the EBOV GPdM. Overall as well as focused views at the binding interface between Fab and GPdM are shown in ribbon diagram. Four residues (T144, F225, E231 and W275) found to be critical for mAb binding and neutralization through mutagenesis study are highlighted and found to be located in the glycan cap (cyan) and the head regions (dark blue) within the EBOV GPdM. b) Side and top views of combined Fabs of Q206 (mustard), Q314 (blue), and Q411 (magenta) bound to the EBOV GPdM relative to the control Fabs c13C6 (cyan) and KZ52 (orange).

Figure 5. Critical residues on the EBOV envelope glycoprotein in conferring resistance to Q206, Q314 and Q411 neutralization.

Comparison of neutralization sensitivity of mutant and wild-type pseudoviruses to Q206, Q314, and Q411 in graphic (a) and numerical (b) format. Dashed line indicates 50% inhibition. Controls antibodies used here include VRC01, a human neutralizing mAb against HIV-1, c13C6 and KZ52, previously isolated neutralizing mAbs against EBOV. Data presented are average values from at least two independent experiments and the error bars indicate for the standard error of the mean (SEM).

Figure 6. Post attachment inhibition through partial interference with receptor NPC1 binding.

(a) The testing mAb were applied to pseudovirus and Vero-E6 cells before or after attachment and (b) Competitive binding of the testing mAbs with NPC1-C to immobilized GPcl on a chip. The blocking efficacy was determined as 12.9% for Q206, 3.9% for Q314, 14.9% for Q411, and 72.7% for control mAb 114, respectively.