Functional Optimization of Broadly Neutralizing HIV-1 Antibody 10E8 by Promotion of Membrane Interactions

Abstract

The 10E8 antibody targets a helical epitope in the membrane-proximal external region (MPER) and transmembrane domain (TMD) of the envelope glycoprotein (Env) subunit gp41 and is among the broadest known neutralizing antibodies against HIV-1. Accordingly, this antibody and its mechanism of action valuably inform the design of effective vaccines and immunotherapies. 10E8 exhibits unusual adaptations to attain specific, high-affinity binding to the MPER at the viral membrane interface. Reversing the charge of the basic paratope surface (from net positive to net negative) reportedly lowered its neutralization potency. Here, we hypothesized that by increasing the net positive charge in similar polar surface patches, the neutralization potency of the antibody may be enhanced. We found that an increased positive charge at this paratope surface strengthened an electrostatic interaction between the antibody and lipid bilayers, enabling 10E8 to interact spontaneously with membranes. Notably, the modified 10E8 antibody did not gain any apparent polyreactivity and neutralized virus with a significantly greater potency. Binding analyses indicated that the optimized 10E8 antibody bound with a higher affinity to the epitope peptide anchored in lipid bilayers and to Env spikes on virions. Overall, our data provide a proof of principle for the rational optimization of 10E8 via manipulation of its interaction with the membrane element of its epitope. However, the observation that a similar mutation strategy did not affect the potency of the first-generation anti-MPER antibody 4E10 shows possible limitations of this principle. Altogether, our results emphasize the crucial role played by the viral membrane in the antigenicity of the MPER-TMD of HIV-1. The broadly neutralizing antibody 10E8 blocks infection by nearly all HIV-1 isolates, a capacity which vaccine design seeks to reproduce. Engineered versions of this antibody also represent a promising treatment for HIV infection by passive immunization. Understanding its mechanism of action is therefore important to help in developing effective vaccines and biologics to combat HIV/AIDS. 10E8 engages its helical MPER epitope where the base of the envelope spike submerges into the viral membrane. To enable this interaction, this antibody evolved an unusual property: the ability to interact with the membrane surface. Here, we provide evidence that 10E8 can be made more effective by enhancing its interactions with membranes. Our findings strengthen the idea that to elicit antibodies similar to 10E8, vaccines must reproduce the membrane environment where these antibodies perform their function.

Molecular recognition of the native HIV-1 MPER revealed by STED microscopy of single virions

“…Structural, biophysical, and biochemical data 9–11,15,43,44 suggest that the bnAb 10E8 employs three elements to facilitate access to and recognition of the membrane-inserted MPER (Fig. 5a): (1) a specificity-binding pocket, which is adapted to recognize a MPER α-helical structure, and forms a hydrophobic cleft where residues critical for binding Trp 672 and Phe 673 are buried 9,10,45 ; (2) a long heavy chain complementarity determining region 3 (HCDR3) loop, characterized by a very hydrophobic tip that submerges into the lipid matrix 9–11,43 ; and (3) the membrane-associated paratope area (MAPA) that can accommodate phospholipid polar head groups on its surface 10,11,15,43 .

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Broadly neutralizing antibodies for HIV-1: efficacies, challenges and opportunities

“…NIH45-46G54W, a single substitution in CDRH2 by structure-based design, results in increased contact with the gp120 inner domain/bridging sheet as well as enhanced potency and breadth by an order of magnitude [58]. In addition, by increasing positive charges at the paratope surface of 10E8, the S30R/N52R/S67R triple substitution enhanced electrostatic interaction between the antibody and lipid bilayer, enabling 10E8 to bind to Env spikes with a higher affinity and neutralizing the virus with greater potency [59]. More recently, an FR3-loop grafting strategy was used to construct chimeric antibodies by engrafting the extended FR3 loop of VRC03 onto different bNAbs.…”

Protein electrostatics: From computational and structural analysis to discovery of functional fingerprints and biotechnological design

“…The 10E8 antibody, targeting the membrane-proximal external region (MPER) of the gp41 subunit of the HIV-1 envelope glycoprotein (Env), shows a positively-charged patch on the Fab surface which is suggested to favor the binding to its target by engaging electrostatic interactions with negatively-charged membrane phospholipids [105] . 10E8 neutralizing activity against many HIV-1 isolates was increased by broadening its positive surface patch with the introduction of three solvent-exposed arginine residues [105] .…”