Neutralization assays on JR-FL glycan mutants indicate binding of inferred intermediate antibodies to both N301 and N332 on HIV-1 Env.

<p>(<b>A</b>) Neutralization curves of inferred intermediate antibodies on wild-type JR-FL virus (<b>B</b>) JR-FL N301A mutant virus, and (<b>C</b>) JR-FL N332A mutant virus. (<b>D</b>) Neutralization curves of 3H+3L and (<b>E</b>) PGT121 on wild-type JR-FL virus compared to single and double glycan mutant viruses. (<b>F</b>) Neutralization curves of PGT121 on wild-type 92BR020 virus compared to single and double glycan mutant viruses.</p

Antibody 3H+3L likely crosslinks between trimers.

<p>(<b>A</b>) 3H+3L binds an epitope overlapping with those of PGT121, PGT128, and 2G12 as shown by competition of biotinylated antibody 3H+3L with an antibody panel. Binding assays were performed by flow cytometry on JR-FLΔCT isolate transfected in 293T cells. (<b>B</b>) IgG and Fab fragments were tested for binding on JR-FLΔCT isolate expressed on transfected 293T cells and no substantial differences in avidity were observed. Solid lines represent IgG and dashed lines represent Fab fragments. (<b>C</b>) Purified IgGs of 3H+3L and PGT121 were digested into Fab fragments using Lys-C, purified, and tested for neutralization on a cross-clade panel. Loss of neutralization was found for 3H+3L Fab, but not for PGT121 Fab. Reported values are IC₅₀ ratios of Fab compared to IgG using the equation: (IC₅₀ Fab)∶(IC₅₀ IgG).</p

Putative germline of PGT121 does not bind monomeric gp120 or cell surface Env.

<p>(<b>A</b>) PGT121germline does not bind recombinant gp120 (92BR020). Recombinant gp120s were produced in 293F cells and purified by lectin column before use in ELISA binding assays. ELISA values are reported in optical density at 405 nm (OD405). (<b>B</b>) PGT121germline does not cell surface Env (92BR020). Cell surface Env was produced by transfecting pseudovirus in 293T cells and binding was measured by flow cytometry (reported in mean fluorescence intensity or MFI).</p

Selected heavy and light chain clones were paired and tested for neutralization breadth and potency on a cross-clade 6-virus panel.

<p>(<b>A</b>) Heavy and light chain nodes leading to mAb PGT121 and (<b>B</b>) PGT124 were paired and tested on a 74-virus panel of PGT121- or PGT124-sensitive viruses. Boxes are colored by IC₅₀ values (µg/ml) of each isolate neutralized.</p