Figure S2.

Characterization of neutralizing FDG Ab lineage (DH898) elicited by SHIV infection in RMs, related to Figure 4

DH898 mAbs were elicited by pathogenic SHIV (CH848TF) infection in RM6163 (see STAR Methods). (A) DH898 mAbs were tested in ELISA for binding to Man₉-V3, and heat-killed yeast antigens Candida albicans or Cryptococcus neoformans, in the absence (black bars) or presence (white bars) of 1M D-mannose. Control mAbs are reactive to Env glycan (DH501 and 2G12), Env peptide (DH1013) or influenza (CH65). Binding titers were reported as Log AUC. Data shown were from a single ELISA that were in agreement with an independent experiment of DH898 mAbs binding to Man₉-V3 and yeast antigens in the absence or presence of 0.5M D-mannose. (B) DH898 mAbs were tested in ELISA for binding soluble stabilized recombinant HIV-1 trimers, CH848 10.17 DS.SOSIP wild-type and mutant trimers, in a single experiment. Mutations in CH848 10.17 DS.SOSIP trimers included deletions of potential N-linked glycan sites in the V3 (N301A_N332A) or V1 (N133D_N138T) regions. Mutations also included amino acid insertions that filled glycan holes on the trimer (D230N_H289N). Binding levels were measured at OD_450nm. CH848 10.17 SOSIP trimers were captured using PGT151. Biotinylated (B) 2G12, PGT128 (V3 glycan bnAb) and CH65 mAbs were tested as control Abs. (C) Binding levels of DH898 mAbs to CH848TF SOSIPv4.1 in one ELISA, representative of at least two independent experiments. CH848TF SOSIP trimer was captured using anti-AVI mAb. 2G12, PGT151 and CH65 were tested as control Abs. (D) NSEM class averages for DH898 mAbs with lineage member indicated at upper left of each panel. At the lower left corner, each class average was identified as Y-shaped (Y), I-shaped (I), or ambiguous (A). (E) Sequence analysis of key residues within the Fab-dimer interface shows three hydrophobic or aromatic residues in the interface, similar to 2G12. V_H interface residues were numbered according to standard Kabat numbering (Wu and Kabat, 1970). Amino acids at each position were indicated by their one-letter code and colored according to Taylor (Taylor, 1997), with polar groups orange, hydrophobic and aromatic groups in shades of green to yellow, positively charged groups blue and negatively charged groups red. Residues that are rare for a particular position, i.e., ≤ 1% in the abYsis database (Swindells et al., 2017), are indicated with an asterisk. Bottom row indicates VRC01 as a non-Fab-dimerized negative control. (F) Bar graph indicating the fraction of I-shaped Abs for wild-type (blue bars), strengthening mutants (green bars), or disrupting mutants (red bars) in DH898 mAbs. Mutations were engineered in the Ab V_H genes. The % I-shaped was estimated by the fraction of particle images that sorted into I-shaped classes as indicated in each of the NSEM 2D class averages shown in G. (G) NSEM 2D class averages from of DH898.4 wild-type (WT), strengthening double mutations T19I and T70F, and disrupting double mutations R64D and F68D. The class averages shown represented the average of ~8,000 to 20,000 individual particle images classified and averaged into ten classes, arranged from the most populated class at the top left to the least populated at the bottom right, and marked as I-shaped (I), Y-shaped (Y), or ambiguous (A). (H) DH898.4 wild-type and mutant mAbs were tested in ELISA for binding to HIV-1 CH848TF SOSIP trimer. MAbs were tested in technical replicates within a single ELISA and binding levels measured at OD_450nm; error bars represent standard error of the mean. (I) DH898.4 wild-type and mutant mAbs weres tested for neutralization against autologous (CH848TF) HIV-1 strain bearing Env with heterogeneous glycoforms as well as heterologous HIV-1 isolates bearing Envs Man₉-enriched glycans [Kif] in TZM-bl cells. These data were generated in a single neutralization assay and titers were reported as IC₅₀ in μg/ml.