Recombinant Human Anti-ZIKV NS1 Antibody (AA12) (CAT#: HPAB-AP485-YC)

Figure 1 Human ZIKV specific-antibodies bind to both MR766 and PRVABC59 NS1 proteins.

Vero cells were infected with the indicated viruses at an MOI of 1 for 24 h. The cells were fixed with 0.5% paraformaldehyde and blocked with 5% non-fat milk. MAbs AA12, FC12, EB9, and GB5 were used at a concentration of 5 μg per mL and an anti-human antibody conjugated to Alexa Fluor 488 was used as a secondary antibody. The murine pan-flavivirus mAb 4G2 was used as a positive control and an anti-mouse antibody conjugated to Alexa Fluor 488 was used as a secondary antibody. Cells stained with mAb 4G2 were fixed and permeabilized using 80% acetone.

Bailey, M. J., Duehr, J., Dulin, H., Broecker, F., Brown, J. A., Arumemi, F. O., ... & Lim, J. K. (2018). Human antibodies targeting Zika virus NS1 provide protection against disease in a mouse model. Nature communications, 9(1), 1-11.

Figure 2 Human ZIKV specific-antibodies bind to both MR766 and PRVABC59 NS1 proteins.

ELISA assays were performed using recombinant NS1 protein from either MR766 or PRVABC59 viruses to assess the binding activity of mAbs AA12, FC12, EB9, and GB5. ELISAs were performed in duplicates. Data plotted represent mean values and the standard error of the mean (SEM); a non-linear regression line was generated using GraphPad Prism 5. Scale = 100 µm

Bailey, M. J., Duehr, J., Dulin, H., Broecker, F., Brown, J. A., Arumemi, F. O., ... & Lim, J. K. (2018). Human antibodies targeting Zika virus NS1 provide protection against disease in a mouse model. Nature communications, 9(1), 1-11.

Figure 3 NS1-specific antibodies activate Fc-FcR effector functions in vitro.

To examine the ability of NS1-specific antibodies to activate Fc-FcR mediated effector functions, a, b Vero cells were infected with MR766 and PRVABC59 Zika viruses or c, d HEK 293T cells were transfected with NS1 from MR766 and PRVABC59 Zika viruses. Infected Vero cells or transfected HEK 293T cells were used as targets for measuring antibody-mediated effector functions with a genetically modified Jurkat cell line expressing the human FcRγIIIa with an inducible luciferase reporter gene. Fold induction was measured in relative light units and calculated by subtracting background signal from wells without effector cells then dividing wells with antibody by wells with no antibody added. All mAbs were tested at a starting concentration of 10 μg per mL and were serially diluted four-fold. Assays were performed twice as technical duplicates and one of two replicates is shown. A non-linear regression best-fit curve was generated for each dataset using GraphPad Prism 5. Error bars represent SEM

Bailey, M. J., Duehr, J., Dulin, H., Broecker, F., Brown, J. A., Arumemi, F. O., ... & Lim, J. K. (2018). Human antibodies targeting Zika virus NS1 provide protection against disease in a mouse model. Nature communications, 9(1), 1-11.

Figure 4 LALAPG mutation ablates Fc-FcR mediated effector functions without affecting affinity.

Fc-FcR mediated effector functions were tested on Vero cells infected with MR766 and PRVABC59 Zika viruses. AA12 was able to elicit Fc-FcR mediated effector functions while AA12 LALAPG was not. Assays were performed twice as technical duplicates and one of two replicates is shown. A non-linear regression best-fit curve was generated for each dataset using GraphPad Prism 5. Error bars represent SEM

Bailey, M. J., Duehr, J., Dulin, H., Broecker, F., Brown, J. A., Arumemi, F. O., ... & Lim, J. K. (2018). Human antibodies targeting Zika virus NS1 provide protection against disease in a mouse model. Nature communications, 9(1), 1-11.

Figure 5 Generation of expression plasmids encoding ZIKV NS1.

HEK 293T cells were transfected with pCAGGS NS1 or pCAGGS NS1-His or not transfected (mock). At 24 h posttransfection, the cells were fixed with 0.5% paraformaldehyde, and the surface expression of NS1 was detected using an anti-ZIKV NS1 monoclonal antibody, AA12, or a polyclonal anti-histidine antibody. Secondary antibodies conjugated to Alexa Fluor 488 were used to visualize binding using a Celigo imaging cytometer. The scale bars are equal to 500 μm.

Bailey, M. J., Broecker, F., Duehr, J., Arumemi, F., Krammer, F., Palese, P., & Tan, G. S. (2019). Antibodies elicited by an NS1-based vaccine protect mice against zika virus. mBio, 10(2), e02861-18.

Figure 6 Neutralizing ZIKV-specific antibodies are induced by ZIKV-infection.

Neutralization activity of 12 antibodies against MR766 ZIKV.

Bailey, M. J., Broecker, F., Freyn, A. W., Choi, A., Brown, J. A., Fedorova, N., ... & Palese, P. (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.