Table 1.
Crystallographic data collection and refinement statistics.
Fig 1.
Structures of mature10E8 antibody and its genomic revertants.
(A) (left) Mature antibody-MPER complex (PDB ID: 4G6F). The MPER peptide is colored red and the mature 10E8 antibody heavy chain in pink and light chain in gray. (right) The ligand-free 10E8 Fab is structurally aligned to the 10E8-MPER complex structure with an overall Cα r.m.s.d of 0.2 Å. (B) (left) Ligand-free structures of germline-reverted gHv/gLv and (right) partially reverted gHv/mature L structurally aligned to the mature 10E8 with an overall Cα r.m.s.d of 0.2 Å. Structural differences with Cα r.m.s.d. greater than 2.0 Å are colored in red and displayed in stick representation.
Fig 2.
Analysis of 10E8 somatic hypermutation identifies critical K52T mutation.
(A) Summary of CDR H2 somatic hypermutation and 10E8 neutralization. Total mutations from germline are indicated for both heavy and light chains. The CDR H2 germline sequence is shown colored blue with somatic mutations indicated in black. Viruses neutralized with an IC50 < 50 μg/ml or < 1 μg/ml are indicated (see S1 Table for details on HIV-1 isolate panel). (B) Superposition of mature 10E8 complex structure and 10E8 gHv/gLv. Coloring is the same as in Fig 1. (C) Expanded view of CDR H2. Residues that were mutated and gave improved 10E8 gHv/gLv neutralization levels are shown in stick representation. Residues Phe 673 (MPER) and Lys 52 (10E8 gHv/gLv) overlap and are not compatible with binding due to steric clashing.
Fig 3.
10E8 heavy chain ontogeny: lineage members and calculated intermediates.
(A) Selection of 10E8 clonal variants by computational sieving. Identity/divergence plots showing the results of sieving on V gene (left), V, and J genes (panel 2), V, D, and J genes (panel 3), and CDR H3 signature (panel 4). (B) Phylogenetic tree based on CDR H3 signature-selected sequences; signature represents the overlap between the germline gene (HD3-3*01) and the mature 10E8 heavy chain sequence. Terminal branches were labeled to indicate which dataset the terminating sequence derives from. Branches ending ‘O’ indicate the sequence is derived from the published data set. Branches ending in ‘N’ indicate the sequence is derived from the new data set. And branches ending in ‘B’ indicate that the sequence can be found in both data sets. The threshold for inclusion into a data set was set at >97.5% nucleotide sequence identity and >98% alignment coverage. The coloring scheme for the ML tree was adapted from Zhu et al., [18]. Blue circles indicate nodal points on the tree. (C) Sequences of phylogenetically inferred developmental intermediates compared to mature 10E8 and constituent germline genes. Numbers appearing in parenthesis on the left side of the alignment indicate the number of identical residues to the germline V gene. The undefined residue labeled ‘X’ in I1-h was changed to a phenylalanine for pairing and functional analysis.
Fig 4.
10E8 light chain ontogeny: lineage members and calculated intermediates.
(A) Selection of 10E8 clonal variants by computational sieving. Identity/divergence plots showing the results of sieving on V gene (left), V, and J genes (panel 2), CDR L3 length between 9 and 12 residues (IMGT numbering) (panel 3), and CDR L3 signature (panel 4). (B) Phylogenetic tree based on CDR L3 length-selected sequences. To construct a phylogenetic tree from >105 sequences, representative sequences were selected randomly from bins of 0.5 divergence units (as shown in left inset). The numbers in parenthesis at the end of each branch correspond to the total number of clones that share >99% nucleotide sequence identity and 99% alignment coverage. Branches terminating without numbers indicate that the sequence has more than 10 clones with >99% nucleotide sequence identity and 99% alignment coverage. The coloring scheme for the ML tree was adapted from Zhu et al., [18]. Blue circles indicate nodal points on the tree. (C) Sequences of phylogeneticaly inferred developmental light chain intermediates compared to mature 10E8 and constituent germline genes. Numbers appearing in parenthesis on the left side of the alignment indicate the number of identical residues to the germline V gene.
Fig 5.
Pairing of heavy and light 10E8 intermediates.
(A) Heavy (top) and light (bottom) phylogenetic trees with pairing of inferred intermediates indicated by dashed lines. Intermediate pairing based on tree structure and considerations from polyreactivity (see text and [18]). (B) Heavy chain UCA, intermediates and mature sequences. (C) Light chain UCA, intermediates and mature sequences.
Fig 6.
Functional characteristics of 10E8 UCA and maturation intermediates.
(A) Neutralization on eight diverse HIV-1 isolates (see S6 Table for details on HIV-1 isolate panel). (B) Sensogram profiles shown represent two-fold serial dilutions of Fab analyte starting at top concentrations of 500 nM for UCA, pI1, and 17b, or 125 nM for pI2, pI3 and 10E8 mature, through to final concentrations of 3.9–31.25 nM. (C) ELISA assessment of MPER-liposome recognition. Shown are bare liposome (left) and MPER proteoliposome (right) competiton ELISA assays for antibody recognition of a soluble MPER peptide captured on a plate. Binding of 10E8 mature and pI3 antibodies to soluble MPER was effectively competed by MPER proteoliposomes in this assay. (D) Hep2 cell assessment of autoreactivity. (E) Structural models of 10E8 showing location and degree of somatic hypermutation by intermediate. Structural models of 10E8 intermediate antibodies shown in ribbon representation showing the location of resides mutated from the UCA as spheres and colored according to paired intermediate. The degree of somatic mutation for each 10E8 intermediate is given below each structure model with % nucleotide and % amino acid change from the germline VH gene and also for the calculated UCA gene.