Human Anti-ZIKV NS1 Recombinant Antibody (clone AC10) (CAT#: FAMAB-0749WJ)

Figure 1 ELISAs were performed against MR766 ZIKV supernatant 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).

Bailey, M. J., Broecker, F., Freyn, A. W., Choi, A., Brown, J. A., Fedorova, N., ... & Tan, G. S. (2019). Human monoclonal antibodies potently neutralize Zika virus and select for escape mutations on the lateral ridge of the envelope protein. Journal of virology, 93(14), e00405-19.

Figure 2 ELISAs were performed against recombinant MR766 envelope 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).

Bailey, M. J., Broecker, F., Freyn, A. W., Choi, A., Brown, J. A., Fedorova, N., ... & Tan, G. S. (2019). Human monoclonal antibodies potently neutralize Zika virus and select for escape mutations on the lateral ridge of the envelope protein. Journal of virology, 93(14), e00405-19.

Figure 3 ELISAs were performed against 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).

Bailey, M. J., Broecker, F., Freyn, A. W., Choi, A., Brown, J. A., Fedorova, N., ... & Tan, G. S. (2019). Human monoclonal antibodies potently neutralize Zika virus and select for escape mutations on the lateral ridge of the envelope protein. Journal of virology, 93(14), e00405-19.

Figure 4 Neutralization activity of 12 antibodies against MR766 ZIKV.

Nonneutralizing antibody AA12 is designated n.n.

Bailey, M. J., Broecker, F., Freyn, A. W., Choi, A., Brown, J. A., Fedorova, N., ... & Tan, G. S. (2019). Human monoclonal antibodies potently neutralize Zika virus and select for escape mutations on the lateral ridge of the envelope protein. Journal of virology, 93(14), e00405-19.

Figure 5 Neutralization activity shown as IC50 values.

Bailey, M. J., Broecker, F., Freyn, A. W., Choi, A., Brown, J. A., Fedorova, N., ... & Tan, G. S. (2019). Human monoclonal antibodies potently neutralize Zika virus and select for escape mutations on the lateral ridge of the envelope protein. Journal of virology, 93(14), e00405-19.

Figure 6 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).

Bailey, M. J., Broecker, F., Freyn, A. W., Choi, A., Brown, J. A., Fedorova, N., ... & Tan, G. S. (2019). Human monoclonal antibodies potently neutralize Zika virus and select for escape mutations on the lateral ridge of the envelope protein. Journal of virology, 93(14), e00405-19.

Figure 7 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).

Bailey, M. J., Broecker, F., Freyn, A. W., Choi, A., Brown, J. A., Fedorova, N., ... & Tan, G. S. (2019). Human monoclonal antibodies potently neutralize Zika virus and select for escape mutations on the lateral ridge of the envelope protein. Journal of virology, 93(14), e00405-19.

Figure 8 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.

Bailey, M. J., Broecker, F., Freyn, A. W., Choi, A., Brown, J. A., Fedorova, N., ... & Tan, G. S. (2019). Human monoclonal antibodies potently neutralize Zika virus and select for escape mutations on the lateral ridge of the envelope protein. Journal of virology, 93(14), e00405-19.

Figure 9 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.

Bailey, M. J., Broecker, F., Freyn, A. W., Choi, A., Brown, J. A., Fedorova, N., ... & Tan, G. S. (2019). Human monoclonal antibodies potently neutralize Zika virus and select for escape mutations on the lateral ridge of the envelope protein. Journal of virology, 93(14), e00405-19.