Structure and recognition of a novel HIV-1 gp120-gp41 interface antibody that caused MPER exposure through viral escape.

CAPRISA, 2017.Abstract available in pdf

Structure and Recognition of a Novel HIV-1 gp120-gp41 Interface Antibody that Caused MPER Exposure through Viral Escape

A comprehensive understanding of the regions on HIV-1 envelope trimers targeted by broadly neutralizing antibodies may contribute to rational design of an HIV-1 vaccine. We previously identified a participant in the CAPRISA cohort, CAP248, who developed trimer-specific antibodies capable of neutralizing 60% of heterologous viruses at three years post-infection. Here, we report the isolation by B cell culture of monoclonal antibody CAP248-2B, which targets a novel membrane proximal epitope including elements of gp120 and gp41. Despite low maximum inhibition plateaus, often below 50% inhibitory concentrations, the breadth of CAP248-2B significantly correlated with donor plasma. Site-directed mutagenesis, X-ray crystallography, and negative-stain electron microscopy 3D reconstructions revealed how CAP248-2B recognizes a cleavage-dependent epitope that includes the gp120 C terminus. While this epitope is distinct, it overlapped in parts of gp41 with the epitopes of broadly neutralizing antibodies PGT151, VRC34, 35O22, 3BC315, and 10E8. CAP248-2B has a conformationally variable paratope with an unusually long 19 amino acid light chain third complementarity determining region. Two phenylalanines at the loop apex were predicted by docking and mutagenesis data to interact with the viral membrane. Neutralization by CAP248-2B is not dependent on any single glycan proximal to its epitope, and low neutralization plateaus could not be completely explained by N- or O-linked glycosylation pathway inhibitors, furin co-transfection, or pre-incubation with soluble CD4. Viral escape from CAP248-2B involved a cluster of rare mutations in the gp120-gp41 cleavage sites. Simultaneous introduction of these mutations into heterologous viruses abrogated neutralization by CAP248-2B, but enhanced neutralization sensitivity to 35O22, 4E10, and 10E8 by 10-100-fold. Altogether, this study expands the region of the HIV-1 gp120-gp41 quaternary interface that is a target for broadly neutralizing antibodies and identifies a set of mutations in the gp120 C terminus that exposes the membrane-proximal external region of gp41, with potential utility in HIV vaccine design

Crystal structure of CAP248-2B reveals an unusually long, protruding CDR-L3, with a hydrophobic tip.

<p>(A) Sequence alignment of the CAP248-2B heavy and light chain with their predicted V- and J-gene precursors. The CDRs are shaded, labelled, and colored. The heavy chain FR3 is similarly indicated in blue. (B) Crystal structure of the CAP248-2B Fab. The light and heavy chains are colored olive and forest green respectively, while CDR loops and FR-H3 are colored according to A. Two views are shown around a ~45° axis to highlight the long CDR-L3 (yellow). Insets show the conformational differences between the CDR-H3 and CDR-L3 loops between Fab structure 1 (shown here) and Fab structure 2 (shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006074#ppat.1006074.s001" target="_blank">S1 Fig</a>). Two Fabs were present in both asymmetric units, so four loops are shown per inset, two for Fab1 CDR-H3 (red) or L3 (yellow), and two for Fab2 CDR-H3 and L3 (both grey). Asp^100B and Asp^100C in the heavy chain and Phe^95C and Phe^95D in the light chain are shown with stick representations to highlight the conformational divergence between the two structures. Due to crystal packing all downstream analyses were based on the 3.1 Å Fab1 structure.</p

Isolated antibody CAP248-2B exhibits low neutralization plateaus, but recapitulates the plasma neutralization breadth.

<p>(A) Bar graph showing percentage neutralization breadth of CAP248 plasma at ID₅₀ titers of >1:100 (y-axis) on a 45 virus panel at one, two, and three years post-infection (x-axis). The total breadth at each time point is indicated above the bars. (B) Comparison between the neutralization breadth of CAP248 plasma at three years, and CAP248-2B at IC₂₀. The maximum percentage neutralization (neutralization plateau) reached by CAP248-2B is indicated. Palivizumab, a monoclonal antibody specific for Respiratory Syncytial Virus (RSV), was used as an IC₂₀ negative control. Titers are colored yellow, orange and red by potency. A strong inverse spearman correlation with a rho value of -0.802 (p-value <0.00001) indicates good concordance between CAP248 plasma ID₅₀ and CAP248-2B monoclonal antibody IC₂₀. (C) Percentage neutralization breadth of monoclonal antibody CAP248-2B (y-axis) on the same 45 virus panel as in A, when measured at IC₈₀, IC₅₀, and IC₂₀ (x-axis). (D) Neutralization curves of CAP248-2B against four viral strains (CAP45, CNE52, CAP228, and ZM249) plotted as percentage inhibition (y-axis) versus antibody concentration (x-axis). Dotted lines indicate y-axis intersections for IC₈₀, IC₅₀, and IC₂₀. The maximum inhibitory percentage achieved against each virus is listed to the right of each curve.</p

Broadly neutralizing antibodies that target gp41 compete with CAP248-2B for binding to cell-surface Env.

<p>(A) Comparisons of gp41 directed bNAbs bound to SOSIP trimers by EM. The CAP248-2B bound trimers are shown in solid grey surface, while 8ANC195, 3BC315, 10E8, 35O22, PGT151, and VRC34 bound trimers are shown with mesh representation. Both top views and side views are shown. (B) Binding of labelled CAP248-2B to cell-surface anchored HIV-1 Env by flow cytometry, in the presence of increasing concentrations of unlabeled competitor antibody. Median fluorescence intensity (MFI) is shown on the y-axis, and increasing concentrations of each competitor antibody is plotted on the x-axis. Decreasing MFI signals correspond to increasing competition with CAP248-2B. (C) Surface view of the envelope trimer with modelled MPER, colored to show the core epitopes for gp41 targeted bNAbs. The approximate location of the viral membrane is indicated.</p

Escape mutations from CAP248-2B accumulate in the gp120 C terminus.

<p>(A) Sequence alignment of the gp120 C-terminus (positions 500–511) from CAP248 autologous viruses at nine weeks (study enrolment), 1, 2, 3 and 3.5 years post-infection. The primary (position 511) and secondary (position 504) gp160 cleavage sites are indicated with arrows. The total number of viruses with identical amino acid sequence within this region are indicated in brackets to the right. Residues undergoing significant selection pressure are indicated with the asterisks. (B) Sequence logograms showing variation within the gp120 C-terminus for all clades, and clade C only, from the LANL HIV sequence database, as well as from CAP248-2B at 3.5 years post-infection, colored and labelled as in A. The global frequencies for each of the autologous mutations identified in CAP248 sequences were: 500 (E8.88%, K46.56%, G3.56%), 502 (K79.35%, R18.73%, Q1.37%), 505 (V98.98%, A0.55%, M0.08%, L<0.01%), 507 (E47.5%, G3.87%, A0.7%), 508 (R98.24%, K1.49%), 509 (E95.11%, G1.53%, A0.27%). (C) Neutralization by CAP248-2B of the heterologous strain CAP45, when compared to gp120 C-terminal mutant viruses with changes identified from autologous CAP248 Env sequences. Data was plotted as percent inhibition (y-axis) against antibody concentration (x-axis). The wild-type virus is shown in black. Dotted lines indicate y-axis intersections for IC₈₀, IC₅₀, and IC₂₀. (D) Neutralization by CAP248-2B of CAP45 wild-type and mutant viruses with the additional gp41 changes identified from CAP248 autologous sequences, plotted as in C. (E) Binding to cleaved (solid bars) or uncleaved (speckled bars) cell-surface expressed Env measured by flow cytometry. Median fluorescence intensity (MFI) is shown on the y-axis, and Palivizumab was used as an HIV-1 negative control. (F) An SDS-PAGE gel of a single SOSIP trimer sample that was divided into two and subsequently captured from suspension by either CAP248-2B or CAP256-VRC26.09. Samples were run in the presence or absence of dithiothreitol (DTT) to assess the level of furin cleavage.</p

Fine mapping of the CAP248-2B epitope.

<p>(A) Cartoon of the CAP248-2B paratope (shown as a mirror image of the docked model in B, and colored as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006074#ppat.1006074.g003" target="_blank">Fig 3</a>), showing amino acids that have affinity matured relative to the predicted germline genes with main chain Cα spheres. (B) A surface view schematic of gp41 (dark grey) and proximal regions in gp120 (light grey) showing the predicted location of CAP248-2B CDRs and FW3, coloured as in A. The approximate location of the viral membrane is indicated. (C) The envelope trimer is shown in cartoon view with the viral membrane estimated as in B. Regions of Env predicted to form part of the CAP248-2B epitope are coloured and labelled. Point mutants shown to significantly affect CAP248-2B neutralization are shown with black spheres. (D) Table showing neutralization IC₅₀ titers for CAP248-2B, PGT151, VRC34, 3BC315, 35O22, 10E8, and 8ANC195 against CAP45 and various mutants. The location of each mutant in either gp41 or the gp120 C terminus is shown on the left and coloured as in C. Fold effects on IC₅₀ are colored, with warmer colours to indicated increasingly negative effect.</p

The CAP248-2B CDR-L3 interacts with the viral membrane.

<p>Two docking orientations for the CAP248-2B Fab are modelled with (A) the CDR-L3 in close proximity to the viral membrane, and (B) the CDR-L3 in close proximity to the fusion peptide. The trimer is coloured as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006074#ppat.1006074.g004" target="_blank">Fig 4</a>, and the Fab heavy and light chains shown in forest and olive green respectively, and the approximate location of the viral membrane is indicated with dotted lines. In the zoomed panel insets, the CDR-H1 (pink) and CDR-L3 (yellow) are shown in their predicted binding locations for each model. The fusion peptide is colored purple and shown with surface representation. (C) Neutralization of three heterologous viruses by CAP248-2B and related CDR-H1 and CDR-L3 mutants. Percentage inhibition was plotted on the y-axis versus antibody concentration on the x-axis. Dotted lines indicate y-axis intersections for IC₈₀, IC₅₀, and IC₂₀. (D) ELISA showing binding of CAP248-2B and related mutant antibodies to the BG505(gp120)-CAP45(gp41) chimeric SOSIP trimer. Absorbance readings are plotted on the y-axis and antibody concentration on the x-axis. CAP256-VRC26.09 and F105 are used are positive and negative control antibodies. (E) Anti-cardiolipin antibody ELISA, labelled as in D. (F) HEp-2 cell reactivity assays comparing a no antibody control to 50 μg/mL concentrations of either 4E10 (positive control), 35O22 (negative control), or CAP248-2B.</p

The CAP248-2B epitope is proximal to Env glycans, but not affected by glycan heterogeneity.

<p>Schematic of CAP248-2B bound trimer (colored as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006074#ppat.1006074.g004" target="_blank">Fig 4</a> with gp120 shown in transparent surface grey scale) including modelled (NAG)₂MAN₃ basic glycans at epitope proximal N88, N611, N616, N625, and N637 residues (colored brown, yellow, pink, purple, and blue respectively). Two views are shown: (A) Top view, and (B) Side view zoom. Relocation of the N88 glycan between 35O22 and CAP248-2B bound states is indicated. (C) Wong glycan array data for CAP248-2B. Weak binding was detected for two hybrid glycans, but significant binding of >500 a.u. was only detected for one biantennary mono-sialylated complex N-glycan (indicated by the glycan schema). (D) Neutralization of CAP45 by CAP248-2B when compared to epitope proximal glycan mutants. The S613A mutant that removed the glycan at N611 but maintains the amino acid side chain properties is shown with red dashed lines and open circles. Percentage inhibition was plotted on the y-axis versus CAP248-2B antibody concentration on the x-axis. (E) Neutralization of the CAP45 N611D single mutant, and various N611D including double glycan mutants, plotted as in D. (F) Neutralization of heterologous tier-2 strains CAP45, CNE52, CAP228, and ZM249 when grown normally (black lines), with co-transfected furin (dashed pink lines), or in the presence of kifunensine (blue lines), swainsonine (cyan lines), and an O-linked glycosylation inhibitor (dashed orange lines), or in a GnTI deficient cell line (grey lines). Neutralization was also assessed in the presence of sCD4 at predetermined IC₄₀ concentrations for each virus (dashed green lines), or after a 24 hour virus-target cell incubation period (red lines). Graphs are plotted as in D.</p

Escape mutations from CAP248-2B enhance the neutralization of broadly neutralizing antibodies that bind to gp41.

<p>(A) Neutralization of CAP45 and a mutant variant that includes the six gp120 C-terminal mutations identified in CAP248 autologous sequences (CS-Mut), by broadly neutralizing antibodies with epitopes in V2, V3, the CD4bs, the MPER, and the gp120-gp41 interface. Fold changes less than three are within the variation of the assay (no effect). Fold enhancement in neutralization sensitivity to CAP248-2B is indicated in orange (3–10 fold increased sensitivity) and red (> 10 fold increased sensitivity). Conferred resistance to the mutant virus at IC₅₀ is shown by grey shading. (B) Neutralization of five paired WT (blue) and CS-Mut (red) viruses by 35O22, 10E8, and 4E10.</p