Transplanting Supersites of HIV-1 Vulnerability

<div><p>One strategy for isolating or eliciting antibodies against a specific target region on the envelope glycoprotein trimer (Env) of the human immunodeficiency virus type 1 (HIV-1) involves the creation of site transplants, which present the target region on a heterologous protein scaffold with preserved antibody-binding properties. If the target region is a supersite of HIV-1 vulnerability, recognized by a collection of broadly neutralizing antibodies, this strategy affords the creation of “supersite transplants”, capable of binding (and potentially eliciting) antibodies similar to the template collection of effective antibodies. Here we transplant three supersites of HIV-1 vulnerability, each targeted by effective neutralizing antibodies from multiple donors. To implement our strategy, we chose a single representative antibody against each of the target supersites: antibody 10E8, which recognizes the membrane-proximal external region (MPER) on the HIV-1 gp41 glycoprotein; antibody PG9, which recognizes variable regions one and two (V1V2) on the HIV-1 gp120 glycoprotein; and antibody PGT128 which recognizes a glycopeptide supersite in variable region 3 (glycan V3) on gp120. We used a structural alignment algorithm to identify suitable acceptor proteins, and then designed, expressed, and tested antigenically over 100-supersite transplants in a 96-well microtiter-plate format. The majority of the supersite transplants failed to maintain the antigenic properties of their respective template supersite. However, seven of the glycan V3-supersite transplants exhibited nanomolar affinity to effective neutralizing antibodies from at least three donors and recapitulated the mannose₉-<i>N</i>-linked glycan requirement of the template supersite. The binding of these transplants could be further enhanced by placement into self-assembling nanoparticles. Essential elements of the glycan V3 supersite, embodied by as few as 3 <i>N</i>-linked glycans and ∼25 Env residues, can be segregated into acceptor scaffolds away from the immune-evading capabilities of the rest of HIV-1 Env, thereby providing a means to focus the immune response on the scaffolded supersite.</p></div

10E8 light chain ontogeny: lineage members and calculated intermediates.

<p>(<b>A</b>) 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>B</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., [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157409#pone.0157409.ref018" target="_blank">18</a>]. Blue circles indicate nodal points on the tree. (<b>C</b>) 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.</p

Pairing of heavy and light 10E8 intermediates.

<p>(<b>A</b>) 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 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157409#pone.0157409.ref018" target="_blank">18</a>]). (<b>B</b>) Heavy chain UCA, intermediates and mature sequences. (<b>C</b>) Light chain UCA, intermediates and mature sequences.</p

Analysis of 10E8 somatic hypermutation identifies critical K52T mutation.

<p>(<b>A</b>) 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157409#pone.0157409.s007" target="_blank">S1 Table</a> for details on HIV-1 isolate panel). (<b>B</b>) Superposition of mature 10E8 complex structure and 10E8 gHv/gLv. Coloring is the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157409#pone.0157409.g001" target="_blank">Fig 1</a>. (<b>C</b>) 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.</p

Crystallographic data collection and refinement statistics.

<p>Crystallographic data collection and refinement statistics.</p

Functional characteristics of 10E8 UCA and maturation intermediates.

<p>(<b>A</b>) Neutralization on eight diverse HIV-1 isolates (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157409#pone.0157409.s012" target="_blank">S6 Table</a> for details on HIV-1 isolate panel). (<b>B</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. (<b>C</b>) 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. (<b>D</b>) Hep2 cell assessment of autoreactivity. (<b>E</b>) 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.</p

Structures of mature10E8 antibody and its genomic revertants.

<p>(<b>A</b>) (<i>left</i>) 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. (<i>right</i>) 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>B</b>) (<i>left</i>) Ligand-free structures of germline-reverted gHv/gLv and (<i>right</i>) 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.</p