Molecular characterization of the monoclonal antibodies composing ZMAb: a protective cocktail against Ebola virus

Abstract

Ebola virus (EBOV) causes severe viral hemorrhagic fever in humans and non-human primates, with a case fatality rate of up to 88% in human outbreaks. Over the past 3 years, monoclonal antibody (mAb) cocktails have demonstrated high efficacy as treatments against EBOV infection. One such cocktail is ZMAb, which consists of three mouse antibodies, 1H3, 2G4, and 4G7. Here, we present the epitope binding properties of mAbs 1H3, 2G4, and 4G7. We showed that these antibodies have different variable region sequences, suggesting that the individual mAbs are not clonally related. All three antibodies were found to neutralize EBOV variant Mayinga. Additionally, 2G4 and 4G7 were shown to cross-inhibit each other in vitro and select for an escape mutation at the same position on the EBOV glycoprotein (GP), at amino acid 508. 1H3 selects an escape mutant at amino acid 273 on EBOV GP. Surface plasmon resonance studies showed that all three antibodies have dissociation constants on the order of 10(-7). In combination with previous studies evaluating the binding sites of other protective antibodies, our results suggest that antibodies targeting the GP1-GP2 interface and the glycan cap are often selected as efficacious antibodies for post-exposure interventions against EBOV.

Figure 1. Alignment of the amino acid sequence of the variable regions of the three monoclonal antibodies.

The antibodies were sequenced using a nested PCR protocol. The amino acid sequences were analyzed using DomainGapAlign to identify the position of the complementarity determining regions (CDRs). (A) Amino acid sequences of the heavy chain variable regions. (B) Amino acid sequences of the light chain variable regions.

Figure 2. Measurement of the affinity of each mAb for the EBOV glycoprotein (GP).

The antibodies were fixed on the BIAcore chip and the specified concentrations of EBOV GPΔTM were run. Measurements for (A) 1H3, (B) 2G4, and (C) 4G7. Fitted Langmuir model for (D) 1H3, (E) 2G4, and (F) 4G7.

Figure 3. Neutralizing activity of the three antibodies.

(A) Neutralization of of VSVΔG-EBOVGP-eGFP by fluorescent plaque counting. (B) Neutralization of EBOV/May-eGFP by fluorescent plaque counting. The results are expressed as normalized plaque count, where 100% was set to incubation of cells with virus only, i.e. in absence of mAbs.

Figure 4. Escape mutants to the monoclonal antibodies 1H3, 2G4, and 4G7.

VSVΔG-EBOVGP was passaged three times in the presence of either of the monoclonal antibodies. The EBOV GP gene was then sequenced by Sanger sequencing and aligned to that of the original virus (sequenced in parallel). (A) Alignment of the nucleotide sequences showing the three escape mutations at positions 822 (1H3), 1523 (2G4), and 1524 (4G7). (B) Alignment of the amino acid sequences showing the three escape mutations at positions 274 (1H3), and 508 (2G4 and 4G7).

Figure 5. Binding sites of the monoclonal antibodies 1H3, 2G4, and 4G7.

(A) Representation of the position of the escape mutations on the EBOV GP structure (based on PDB# 3CSY) from 5 different angles. The escape mutation to 1H3 (aa 273) is identified in blue. The escape mutation site for 2G4 and 4G7 (aa 508) is identified in red. The monomer where the two amino acids are identified is light gray and the other two monomers are light blue and light orange. The dark gray amino acids (only identified on the light gray monomer) were the amino acids with the top 20% antigenicity score calculated by the Epitopia server. (B) Representation of the position of escape mutations (arrows) and suspected binding sites (lines) on a linear map of GP. The putative binding sites are based on escape mutation data and data from previous publications.