1F7 binding to sensitive and resistant Envs.

<p>Binding of 1F7 and other mAbs to detergent-solubilized Env from different isolates of pseudotyped HIV-1: (A) JR-FL, (B) JR-CSF, (C) MN, and (D) 93MW959. Env A (JR-FL) is 1F7-sensitive whereas Envs B-D are 1F7-resistant. Curves were analyzed with GraphPad Prism 5.04 using a four-parameter fit with shared values for minimum binding. Data are representative of at least 3 titrations of each MAb against the respective detergent-solubilized Env.</p

Neutralization (IC₅₀s) of HIV-1 JR-CSF and JR-FL N-glycosylation mutants using 1F7 and other CD4bs mAbs.

<p>Neutralization assay was performed in the TZM-bl assay format. Boxes are color-coded as follows: white, median potency >50 µg/mL; green, median potency between 20 and 50 µg/mL; yellow, median potency between 2 and 20 µg/mL; orange, median potency between 0.2 and 2 µg/mL; red, median potency <0.2 µg/mL.</p

1F7 sequence alignment.

<p>Amino acid alignment of the 1F7 (A) heavy chain and (B) light chain variable regions along with several other CD4bs antibodies. The 1F7 germline (gl) is shown in blue and residues that differ between 1F7 and its germline sequence are underlined. (A) Antibody F105 and 1F7 share the same variable heavy chain germline family IGHV4-59*01. However, the 1F7 heavy chain sequence is less conserved than the one of F105 (73.5% versus 91.8%). (B) The variable light chain of 1F7 derives from a different germline gene (IGKV1D-33*01) than the other CD4bs antibody light chain fragments.</p

Flow cytometry analysis on cell surface expressed Env.

<p>1F7 shows increased apparent binding to HIV-1 JR-CSF NGS mutant Env spikes displayed on cells as compared to wildtype spikes as measured using flow cytometry. Dashed lines represent the mean fluorescence (MFI) of control samples treated using secondary antibody only. Curves were prepared in GraphPad Prism 5.04 using a four-parameter fit. Data are representative of at least 3 titrations of each MAb against cell surface expressed Env.</p

MAb 1F7 recognition of detergent-solubilized Env from neutralization-resistant wildtype JR-CSF and neutralization-sensitive glycovariants.

<p>1F7 shows similar EC₅₀s as well as relatively low maximum binding to gp120 relative to b12. 2G12 was used throughout the experiment to normalize the quantities of gp120. Each assay was performed in duplicate. Data are representative of at least 3 repeats.</p

MAbs and HIVIG competition with 1F7 binding to gp120.

<p>MAbs b6, 15e, b12, HIVIG, F425-b4e8, VRC03, and VRC01 were co-incubated with a constant concentration of biotinylated mAb 1F7 and allowed to bind to immobilized gp120_JR-FL. Curves were analyzed with GraphPad Prism 5.04 using a three-parameter fit with shared values for maximum and minimum binding. Data are representative of at least 3 titrations of each MAb against biotinylated mAb 1F7.</p

1F7 binding is independent of the gp120 variable loops V1/V2 and V3.

<p>Binding of 1F7 to recombinant gp120_JR-FL and its truncation mutants in a direct ELISA shows that 1F7 binding is independent of V1, V2, and V3. (A) 1F7 binding to wild type gp120_JR-FL, (B) the variable loop truncated variants gp120_JR-FL ΔV1/V2, and (C) gp120_JR-FL ΔV3. Antibodies b12, VRC01, F105, 15e, 2G12, and HIVIG served as positive controls for each gp120, and F425-b4e8 to V3 is a negative control for the ΔV3 construct. Each curve is representative of at least three independent experiments performed in duplicate.</p

V3-peptide depletion of SF162 neutralization.

<p>Midpoint neutralizing titers against SF162 at week 18 in the absence and presence of V3-peptides. Experimental conditions are similar to those in Fig. 3. The titer data are colored according to the following color scale: yellow, 50% neutralization titers between 30 and 60; orange, between 60 and 300; red, >300. ^a Values represent 50% neutralization titers in the presence of irrelevant peptide. ^b Values represent 50% neutralization titers in the presence of V3 peptides. ^c Values represent fold decreases in 50% neutralization titers caused by V3 peptides. ^d Values represent percentages of V3-specific neutralization. †Animals died of unrelated causes between week 12 and week 18. N.A., not analyzed; sera from these rabbits did not neutralize SF162 potently in earlier experiments (see Fig. 3).</p

50% neutralization titers against SF162, JR-FL and LAI.

<p>Data are from the Academic Medical Center. Midpoint neutralizing titers against SF162 at week 0, 6, 12 and 18 and midpoint neutralizing titers against LAI and JR-FL virus at week 12 and 18. The titer data are colored according to the following color scale: yellow, 50% neutralization titers between 30 and 60; orange, between 60 and 300; red, >300. † Animals died of unrelated causes between week 12 and week 18.</p

Env ΔV1V2 mutant design and expression.

<p>(<b>A</b>) Linear representation of Env and the mutants Env ΔV1V2.2, ΔV1V2.4.DNGSEK and ΔV1V2.9.VK. The clade B JR-FL gp140 (amino acids 31–681) contains several modifications that have been indicated in the schematic (see materials and methods and results sections for more details). (<b>B</b>) Schematic of the D7324-tagged JR-FL used in ELISAs. (<b>C</b>) Schematic of the His-tagged ΔV1V2.2 JR-FL gp140 used in ELISAs. (<b>D</b>) Reducing SDS-PAGE (upper panel) and Blue Native-PAGE (lower panel) analysis of full length Env, Env ΔV1V2.2, ΔV1V2.4.DNGSEK and ΔV1V2.9.VK secreted from transiently transfected HEK 293T cells.</p