Memory B cells were immortalized from children experiencing primary or secondary DENV3 infections in Nicaragua. TS hmAbs were isolated that bound and neutralized DENV3.
A) DENV Immune PBMCs from children in a Nicaraguan Cohort study experienced DENV3 as either a primary infection or secondary infection after a DENV2 primary infection.
B) Fifteen hmAbs were tested against DENV1–4 by Vero-81 focus reduction neutralization test (FRNT). Positive controls were as follows, DENV1-specific hmAbs 1F4 and 14c10 for DENV1, DENV2-specific hmAb 2D22 for DENV2, DENV3-specific hmAb 5J7 for DENV3 and crossreactive hmAb EDEI C8 for DENV4. All assays were performed twice in duplicate.
C) DENV 3 neutralization by hmAbs was evaluated by FRNT using wildtype and recombinant viruses. Comparison of DENV3-specific hmAbs to 5J7. Using wild-type DENV3, EC50 values from repeat experiments performed on different days were averaged to show reproducibility. EC50 value denotes amount of hmAb needed to neutralize 50% of the virus in a Vero-81 FRNT. Error bars indicate standard deviation.
D) DENV4/3 M16 ic-PyMOL representation of DENV3 residues (blue) that capture the hmAb 5J7 epitope transplanted into a DENV4 backbone.
E) DENV3-specific hmAbs do not use 5J7 epitope. DENV4/3 M16 ic is neutralized by hmAb 5J7 but not by the panel of 15 DENV3-specific hmAbs. EC50 values in a FRNT of hmAbs of DENV4/3 M16 ic, and parental DENV3 ic and DENV4ic.