Structural Basis for Broad HIV-1 Neutralization by the MPER-Specific Human Broadly Neutralizing Antibody LN01

Abstract

Potent and broadly neutralizing antibodies (bnAbs) are the hallmark of HIV-1 protection by vaccination. The membrane-proximal external region (MPER) of the HIV-1 gp41 fusion protein is targeted by the most broadly reactive HIV-1 neutralizing antibodies. Here, we examine the structural and molecular mechansims of neutralization by anti-MPER bnAb, LN01, which was isolated from lymph-node-derived germinal center B cells of an elite controller and exhibits broad neutralization breadth. LN01 engages both MPER and the transmembrane (TM) region, which together form a continuous helix in complex with LN01. The tilted TM orientation allows LN01 to interact simultaneously with the peptidic component of the MPER epitope and membrane via two specific lipid binding sites of the antibody paratope. Although LN01 carries a high load of somatic mutations, most key residues interacting with the MPER epitope and lipids are germline encoded, lending support for the LN01 epitope as a candidate for lineage-based vaccine development.

Keywords: 10E8; 4E10; Env; HIV-1; LN01; MPER; broadly neutralizing antibody; gp41; membrane interaction.

Graphical abstract

Figure 1

Analysis of LN01 mAb Sequence and Neutralization (A) Analysis of LN01 sequences showing the inferred germline genes and alleles encoding the variable region of the heavy and light chains, the amino acid length of the CDR3 regions and the mutation frequency of the variable regions of the light and heavy chains (aa, amino acid). (B) Alignment of the amino acid sequences of the variable regions of LN01 wild-type and LN01 UCA. The CDRs regions are highlighted in gray. (C) LN01 IgG1 activity was tested in vitro in neutralization assay using TZM-bl cells. Different concentrations of the antibody were tested against nine pseudoviruses (PVs) of the Global Panel plus a control PV (SVA-MLV). Shown on y axes is the % of neutralization and the standard deviation (SD) calculated on quadruplicates. (D) Schematic of LN01 unmutated common ancestor (UCA) and variants created for investigation of the neutralization requirements of LN01 germlined variants. Light gray areas represent sequence from UCA; dark gray regions are from the somatic, mature antibody. Wild-type, somatically mutated heavy (sH), or somatically mutated light (sL) chains; gH or gL, germline V-gene revertants of sH or sL in which HCDR3 or LCDR3 are mature; gH-FR or gL-FR, germline V-gene revertants of sH or sL in which HCDRs or LCDRs are mature. (E) Box-and-whisker plots showing the neutralizing activity of LN01 germline variants against a panel of eight PVs of the Global Panel as measured using a neutralization assay on TZM-bl cells.

Figure 2

Analysis of LN01 Autoreactivity (A) Immunofluorescence on Hep-2 cells. BnAbs LN01, 4E10, and 10E8 as well as positive and negative controls provided by the diagnostic kit were tested at 50 μg/mL. (B) ELISA to measure the binding to the self-antigen cardiolipin. Assay performed according to manufacturer instructions. Shown are OD values of duplicates at 405 nm. (C) Pharmacokinetic analysis performed in huFcRn transgenic mice (Tg276, Jackson Laboratory). LN01, palivizumab and a control mAb specific for an irrelevant antigen were administered i.v. at 10 mg/kg (n = 5). The concentration of human mAbs in plasma ± standard deviation (SD) was determined at multiple time points using a total human IgG ELISA, as described in STAR Methods.

Figure 3

LN01 Neutralization Breadth, Potency, and Effector Function Killing of HIV-1-Infected Lymphocytes (A) The neutralizing activity of LN01 IgG1 tested against a cross-clade panel of 118 HIV-1 PVs. The IC50 (top panel) and IC80 (bottom panel) expressed in mg/ml were determined in TZM-bl-cell-based micro-neutralization assay as described in STAR Methods. (B) Neutralization breadth-potency curves for LN01 and 10E8, with breadth shown as percentage of PVs neutralized at each IC50 (top panel) or IC80 (bottom panel) cutoff (25 μg/mL for LN01 and 10 μg/mL for 10E8). (C) ADCC killing of HIV-1 infected lymphocytes performed with bnAbs at 15 μg/mL on CEM-NKR-CCR5 cells infected with NLAD8, YU2, CH058, or CH077 HIV-1 strains. ADCC was calculated as the disappearance of Gag+ cells with or without antibodies (n = 6–10), with each dot representing an individual donor of primary NK cell. ADCC responses of each tested antibody were compared to that of the isotype control mGO53 in the Wilcoxon test (^∗p < 0.05). (D) CDC-mediated cell killing performed with bnAbs at 15 μg/mL incubated with a Raji-YU2 Env cell line in the presence of normal human serum from six individual donors or heat-inactivated human serum. The mGO53 antibody was used as a negative control in (C) and (D).

Figure 4

Relevant Residues on HIV-1 PVs for LN01 Binding (A) HIV-2 chimera containing HIV-1 MPER region and parental HIV-2 were used to determined the binding of recombinant LN01. In the table are shown the IC50 (mg/mL) calculated based on the neutralization assay with TZM-bl cells (middle column), and the different mutations in the sequence carried by MPER of each chimeras (right column). (B) The activities of LN01, 10E8, and 4E10 mAbs against several MPER mutants of the COT6.15 virus were assessed. Shown are the IC50 values expressed in μg/mL.

Figure 5

Structure of LN01 in Complex with gp41 MPER-TM1 and with Lipid (A) Structure of the LN01-MPER-TM1 complex. LN01 is colored in yellow (light chain) and orange (heavy chain) and the N-acetyl-β-d-glucosamine (NAG) on Asn107 of the light chain is shown with spheres. Gp41 is colored in purple (MPER) and beige (TM). The same coloring was used in all figures. (B) Close up of the interactions with gp41 MPER, Fos-Choline-12, and PS revealing two lipid-binding sites on either side of the MPER helix. Fos-Choline-12, PS, and residues involved in polar contacts and important hydrophobic contacts are indicated and represented in sticks; hydrogen bond interactions are represented as dashed lines. The upper two panels show the interactions of LN01 with gp41 MPER from two different orientations. The lower left panel shows the interaction with Fos-Choline-12, a putative lipid-binding site accommodating phosphatidylcholine, and the lower right panel shows the hydrogen bond network that coordinates 06:0 PS forming a second lipid-binding pocket.

Figure 6

Structure of LN01 in Complex with gp41 MPER-TM2 Reveals a Continuous Helix of MPER and TM (A) Structure of the LN01-MPER-TM2 complex with the TM in the straight conformation. Color coding is the same as in Figure 5. Gp41 MPER residues 650 to 670 are disordered in the LN01-MPER-TM2 structure. (B) Structure of the LN01-MPER-TM2 complex with the TM in the bent conformation. (C) Orientation of the LN01-MPER-TM2 complex in the lipid bilayer based on the MD simulation result (lipids are represented in sticks). (D) LN01-MPER-TM2 complex with modeled phosphatidylcholine and 06:0 PS (based on the LN01-MPER-TM1 structure) demonstrates that both lipids are well positioned to be part of the bilayer. Phosphatidylcholine and 06:0 PS are shown in spheres. (E) The angle of approach of MPER bnAbs. The upper panel shows the bnAbs LN01 (yellow), 10E8 (orange), 4E10 (blue), and DH511.1 (red), represented with a sphere at the center of the variable domain for each antibody. The middle panel shows their orientations upon recognition of the linear helical epitope as determined by Cα superposition of the MPER peptide of the four complexes (two side views and one top view looking down the helical axis of MPER). The lower panel shows the representation of the angle of approach of the different bnAbs on the MPER-TM domain inserted in the lipid bilayer (lipids and basic residues are shown as sticks). The trajectory between the center of each antibody and gp41 T676 is depicted by a straight line.

Figure 7

Molecular Dynamics Simulation of the Straight and Bent TM Conformations in a Lipid Bilayer The lipid bilayer is composed of POPC (turquoise), POPE (yellow-green), POPS (red), SSM (pink), and cholesterol (mauve). The lipid head groups are represented as van der Waals spheres of the corresponding colorcolour. The G residues of the α helix are highlighted as orange spheres. (A) MD simulation of the straight TM conformation reveals a tilt angle of 17°. The inset shows the complete assay, featuring water and the K⁺ (violet) and Cl⁻ (green) ions. (B) MD simulation of the bent TM conformation demonstrates that the bent form thins the membrane locally. (C) Time evolution of the bending angle, φ, formed by the long axes of the α-helical segments spanning residues I675 to M687 and I697 to N706 determined from six independent MD simulations. The inset of (C) depicts the time evolution of the tilt angle, θ, formed by the long axis of gp41 and the normal to the lipid bilayer. (D) Free-energy profile characterizing the transition between the straight (left minimum) and the bent (right minimum) conformations of gp41 in a membrane. The error bars correspond to the standard deviation measured from the eight walkers of the 11-μs MW-ABF simulation.