Fig 1.
The HC mediates the off-track phenotype.
(A) Maximum likelihood phylogenetic tree, rooted on the CAP256.UCA, with 33 clonally related VH sequences of monoclonal antibodies isolated from donor CAP256. Highlighted are CAP256.04 (orange), CAP256.25 (brown), CAP256.20 (light purple) and CAP256.27 (purple). Neutralization breadth, potency, VH SHM (related to CAP256.UCA) and the number of amino acids that differ between the CDR and FR of each pair are tabulated below. Breadth and potency (mean IC50) was calculated from a panel of 46 heterologous viruses [11]. (B, left) The amino acid sequence of CAP256.04 (orange) was fitted to a structure of CAP256.25 and CAP256 34-week trimer in Swiss-PdbViewer (v4.1.0), aligned in PyMOL (v2.0.2) and displayed as cartoons. (B, right) The amino acid sequences of CAP256.27 (purple) and CAP256.20 (light purple) were fitted to CAP256.25 in Swiss-PdbViewer (v4.1.0) and aligned in PyMOL (v2.0.2). The amino acids that differ between each bNAb and matched off-track antibody are represented as yellow spheres. (C) The LCs were exchanged between each bNAb/off-track pair and neutralization (IC50, μg/mL) was tested against (n = 16*) pseudoviruses.
Fig 2.
One mutation is required to introduce breadth into CAP256.04 while mutations in CDRH1, CDRH2 and CDRH3 are required for potency.
(A) Comparison of the CDR sequences, including the length and charge of the CDRH3 of CAP256.04 (orange) and CAP256.25 (brown). The blue boxes highlights key off-track mutations. (B) Neutralization data using mutants and chimeras (indicated schematically above the table) between CAP256.25 and CAP256.04, with neutralization potency shown by color, as indicated in the key. Neutralization data represents the arithmetic mean titers (IC50, μg/mL) from at least two experiments.
Fig 3.
The structural effect and probabilities of the CAP256.04 off-track mutations S28, N30 and R100r.
(A) The amino acid sequence of CAP256.04 (orange) was fitted to a model of CAP256.25 (brown) and the V1V2 of trimeric CAP256 34-week Env (grey, [26]) in Swiss-PdbViewer (v4.1.0) and visualized in PyMOL (v2.0.2). The top inset shows the CAP256.25 CDRH1 D30 (brown) residue and a hydrogen bond between CAP256.25 CDRH1 residue R28 (brown), and the glycan at N130. The bottom inset displays CAP256.04 R100r (orange) in proximity to V2 K168. Symbols + and - denote positive and negative charge, respectively. (B) The germline (T28, S30 and Q100r) and mature amino acid sequences (CAP256.25 T28R, S30D and Q100rA, and CAP256.04 T28S, S30N and Q100rR) at codons 28, 30 and 100r are displayed with the nucleotide sequence of each codon. Blue and red text indicates AID cold-and hotspots, respectively. Green text refers to mutations and yellow to pink shading indicates low and high probability mutations, respectively. The mutability scores are shown below each nucleotide.
Fig 4.
The CDRH3 alone restricts the neutralization breadth and potency of CAP256.20.
(A) Comparison of the CDR sequences of CAP256.20 (light purple) and CAP256.27 (purple). The blue box highlights key off-track mutations. (B) Neutralization data using mutants and chimeras (indicated schematically above the table) between CAP256.27 and CAP256.20, with neutralization potency shown by color, as indicated in the key. Neutralization data represents the arithmetic mean titers (IC50, μg/mL) from at least two experiments.
Fig 5.
The probability and structural effect of the CAP256.20 off-track mutations W100dR, D100jH and G100nV.
(A) The germline (W100d, D100j and G100n) and mature nucleotide and amino acid sequences (CAP256.27 W100d, D100j and G100nE and CAP256.20 W100dR, D100jH and G100nV) at codons 100d, 100j and 100n are displayed. Blue and red text indicate AID cold- and hot-spots, respectively. Green text refers to mutations, and yellow to pink shading indicates low and high probability mutations, respectively. The mutability scores are below each nucleotide. (B) The sequence of CAP256.20 (light purple) and CAP256.27 (purple) were fitted to a structure of CAP256.25 and trimeric CAP256 34-week Env [26] in Swiss-PdbViewer (v4.1.0) and aligned and visualized in PyMOL (v2.0.2). The top panels shows the interaction between the CDRH3 YYD motif (purple/light purple) and the V2 epitope (grey). The bottom panel displays the CAP256.27 residues W100d and E100n (purple) and CAP256.20 R100d and V100n (light purple) in proximity to K169, + and—denotes positive and negative charge, respectively.
Fig 6.
The off-track antibody CAP256.20 neutralizes a globally rare 169Q immunotype that was common in donor CAP256.
(A) The K169 (orange) immunotype is common in globally circulating viruses, (B) while 169Q (green) predominates in CAP256 across most time points. (C) Slope graphs displaying the change in neutralization titer (IC50) of CAP256.20 (light purple), CAP256.27 (purple), CAP256.2027H3 (blue) and CAP256.2720H3 (cyan) against K169 and 169Q autologous viruses.