Human Monoclonal Antibodies Potently Neutralize Zika Virus and Select for Escape Mutations on the Lateral Ridge of the Envelope Protein

Abstract

The mosquito-borne Zika virus (ZIKV) has been causing epidemic outbreaks on a global scale. Virus infection can result in severe disease in humans, including microcephaly in newborns and Guillain-Barré syndrome in adults. Here, we characterized monoclonal antibodies isolated from a patient with an active Zika virus infection that potently neutralized virus infection in Vero cells at the nanogram-per-milliliter range. In addition, these antibodies enhanced internalization of virions into human leukemia K562 cells in vitro, indicating their possible ability to cause antibody-dependent enhancement of disease. Escape variants of the ZIKV MR766 strain to a potently neutralizing antibody, AC10, exhibited an amino acid substitution at residue S368 in the lateral ridge region of the envelope protein. Analysis of publicly availably ZIKV sequences revealed the S368 site to be conserved among the vast majority (97.6%) of circulating strains. We validated the importance of this residue by engineering a recombinant virus with an S368R point mutation that was unable to be fully neutralized by AC10. Four out of the 12 monoclonal antibodies tested were also unable to neutralize the virus with the S368R mutation, suggesting this region to be an important immunogenic epitope during human infection. Last, a time-of-addition infection assay further validated the escape variant and showed that all monoclonal antibodies inhibited virus binding to the cell surface. Thus, the present study demonstrates that the lateral ridge region of the envelope protein is likely an immunodominant, neutralizing epitope.IMPORTANCE Zika virus (ZIKV) is a global health threat causing severe disease in humans, including microcephaly in newborns and Guillain-Barré syndrome in adults. Here, we analyzed the human monoclonal antibody response to acute ZIKV infection and found that neutralizing antibodies could not elicit Fc-mediated immune effector functions but could potentiate antibody-dependent enhancement of disease. We further identified critical epitopes involved with neutralization by generating and characterizing escape variants by whole-genome sequencing. We demonstrate that the lateral ridge region, particularly the S368 amino acid site, is critical for neutralization by domain III-specific antibodies.

Keywords: Zika virus; antibody-dependent cellular cytotoxicity; monoclonal antibodies; neutralizing antibodies; prM-E; whole-genome sequencing.

FIG 1

Neutralizing ZIKV-specific antibodies are induced by ZIKV-infection. (A to C) ELISAs were performed against MR766 ZIKV supernatant, recombinant MR766 envelope protein, or recombinant MR766 NS1 protein to assess binding activities. ELISAs were performed as duplicates, and results are reported as values of the area under the concentration-time curve (AUC). Error bars represent standard errors of the means (SEM). (D) Neutralization activity of 12 antibodies against MR766 ZIKV. (E) Neutralization activity shown as IC₅₀ values. Nonneutralizing antibody AA12 is designated n.n.

FIG 2

Neutralizing antibodies cannot elicit ADCC but can elicit ADE in vitro. We examined the ability of neutralizing antibodies to elicit Fc-mediated effector functions either on the surface of infected cells or against the virion itself. (A) Vero cells were infected with MR766 ZIKV and used as targets for measuring Fc-FcγR effector functions with a genetically modified Jurkat cell line expressing human FcγRIIIa with an inducible luciferase reporter gene. Fold induction was measured in relative light units, and results were compared to infected cells with no antibody added. Error bars represent standard error of the means (SEM). (B) To examine whether enhancement of flavivirus infection in vitro is observed, monoclonal antibodies were incubated with ZIKV and added to FcγR-bearing K562 cells. All monoclonal antibodies were tested at a starting concentration of 3.3 μg per ml and serially diluted 4-fold. Both assays were run in duplicate, and fold induction was measured as the percentage of infected cells as determined by flow cytometry divided by the percentage of infected cells with no antibody added (virus alone). (C) Representative flow cytometry plots for antibody AD5 and control IgG are shown.

FIG 3

Whole-genome sequencing of escape variants. Panels A to H show sequence alignments of escape mutations generated by antibodies AC10, AC4, GD12, FA12, AC11, FC3, FC11, and GA3, respectively. The corresponding protein is labeled at the top with the relevant amino acid. Six plaque-purified viruses (numbered 1 to 6) were sequenced, and amino acids are shown in red (mutated) or black (control; CTL). Six plaque-purified wild-type viruses were sequenced in parallel after an identical number of passages in Vero cells. pr, premembrane.

FIG 4

Escape mutants are mapped to a crystal structure of the ZIKV E protein. Panels A to H show graphical representations of the envelope protein with relevant mutations found on plaque-purified viruses 1 to 6 used for the experiment shown in Fig. 3 indicated in red. PDB accession number 5JHM was used to generate the three-dimensional model using UCSF Chimera.

FIG 5

Sequence alignment of ZIKV E protein. (A) A sequence alignment and a consensus sequence were generated from 173 publicly available sequences from the Virus Pathogen Resource. Each amino acid position was then assigned a polymorphism score based on Crooks et al. (46). The polymorphism score represents the normalized entropy of an observed allele distribution. Amino acid scores can have values of 0 (no observed polymorphism) to 439 (20 alleles and an indel, ∼4.7% frequency each). The red line indicates amino acid position 368. (B) The residue at site 368 (highlighted in red) has a polymorphism score of 38, where the majority of amino acids (n = 164) are serines. Of note, Xaa indicates a missing or ambiguous amino acid.

FIG 6

A recombinant MR766 ZIKV with an S368R point mutation escapes neutralization by AC10 in vitro. Serial passaging of MAb AC10 led to the identification of critical residue S368, which lies in the lateral ridge epitope of domain III of the ZIKV envelope protein. Site-directed mutagenesis was performed to generate recombinant MR766 ZIKV with the S368R point mutation. (A) Rescue titer after 72 h posttransfection demonstrates a reduction in viral titer. The dotted line represents the limit of detection. (B) Multicycle growth curves were performed to compare viral fitness between wild-type (WT) and S368R viruses. Supernatants were collected at the indicated time points, and titers were determined by plaque assays. Data points represent the means for two biological replicates, and error bars represent standard errors of the means (SEM). (C) To confirm that the S368R mutation is sufficient for escape from neutralization by MAb AC10, a plaque reduction neutralization test was performed with equivalent amounts of wild-type or S368R MR766 ZIKV. The assay was performed in duplicate, and error bars represent SEM. (D and E) Antibodies GA3 and AA1, which induce escape mutations in different sites, were tested by the same plaque reduction neutralization test. The assay was performed in duplicate, and error bars represent SEM. *, P < 0.05.

FIG 7

Neutralization occurs at a binding step. Analysis of the timing of antibody neutralization activity was performed. The results for synchronized infections of Vero cells with wild-type or S368R MR766 virus are shown. Vero cells were equilibrated to 4°C at −3 h, and virus plus antibody or virus alone was added to the cells. At 0 h Vero cells were washed twice with PBS, warm medium was added with the relevant antibody, and cells were moved to 37°C. For each assay, results are normalized to those of infections performed without any antibody added. Antibody was added at a concentration of 10 IC₅₀s as determined by plaque reduction neutralization test. Infection was measured by 4G2 anti-envelope staining at 48 h postinfection. Assays were performed in duplicate, and error bars represent SEM. *, P < 0.05; ns, not significant.