Isolation of HIV-1 gp120 CD4bs-specific vaccine-induced NHP memory B cells, NHP MAbs and their binding specificities.

(A) Structural representation of the TriMut and TriMut368/370 probes that were used for sorting CD4bs-specific memory B cells. The key mutations for TriMut (I423M, N425K and G431E) are highlighted in hot pink. The additional TriMut368/370 mutations (D368R and E370F) used for negative selection are highlighted in green and the CD4 footprint is in yellow. The inserted table shows the binding profile of the probes with a panel of CD4bs antibodies and CD4-Ig. Relative binding to the probes was based on the OD450 values at the highest concentration of protein (10 µg/ml) and EC50 values: OD450≥3.0 and EC50≤0.10 = ++++; OD450≥3.0 and EC50>0.10 = +++; 2.0≤OD450<3.0 = ++; 0.2≤OD450<2.0 = +; OD450<0.2 = −. (B) Details describing the flow cytometric sorting of CD4bs-directed memory B cells using the TriMut and TriMut368/370 probes. Gating strategy for sorting IgG+ and CD4bs-specific memory B cells; CD20+, IgG+, CD27+, CD3−, CD8−, CD14−, IgM−, TriMut and TriMut-368/370 were sorted at single cell density. (C) Four VH1.23-using MAbs (GE331, GE356, GE443 and GE453) were characterized for their capacity to bind trimeric gp140-F+/−D368R and TriMut+/−368/370 by ELISA. Titration curves are shown as Log10 dilutions (µg/ml). All antibodies were isolated from the post 2 immunization time point.