Surface-Matrix Screening Identifies Semi-specific Interactions that Improve Potency of a Near Pan-reactive HIV-1-Neutralizing Antibody

Abstract

SUMMARY Highly effective HIV-1-neutralizing antibodies could have utility in the prevention or treatment of HIV-1 infection. To improve the potency of 10E8, an antibody capable of near pan-HIV-1 neutralization, we engineered 10E8-surface mutants and screened for improved neutralization. Variants with the largest functional enhancements involved the addition of hydrophobic or positively charged residues, which were positioned to interact with viral membrane lipids or viral glycan-sialic acids, respectively. In both cases, the site of improvement was spatially separated from the region of antibody mediating molecular contact with the protein component of the antigen, thereby improving peripheral semi-specific interactions while maintaining unmodified dominant contacts responsible for broad recognition. The optimized 10E8 antibody, with mutations to phenylalanine and arginine, retained the extraordinary breadth of 10E8 but with ~10-fold increased potency. We propose surface-matrix screening as a general method to improve antibodies, with improved semi-specific interactions between antibody and antigen enabling increased potency without compromising breadth.

HIV-1 Envelope and MPER antibody structures in lipid assemblies

“…In all tested combinations of FL Env and MPER Fab, cryo-EM analysis showed no stable, high-resolution structural features for MPER, TMD or CTD similarly to earlier cryo-EM studies ( Figure 3A) (Lee et al, 2016;Rantalainen et al, 2018;Torrents de la Pena et al, 2019). One of the tested antibodies involved a variant of 10E8, called 10E8v4_5R+100cF, which was designed to have improved membrane-interaction capacity and which showed 10-fold high neutralization potency (Kwon et al, 2018b). This antibody also failed to show stable, high-resolution structural features suggesting that stable lipid headgroup contacts are not fully recovered in this approach.…”

“…Nevertheless, the system is a step toward more native environment and open new possibilities for studying Env structure in lipid bilayers by single particle EM analysis. In addition to controlled lipid composition and the testing new MPER antibodies such as 10E8v4_5R+100cF with improved lipid contacts (Kwon et al, 2018b), these membrane embedded Envs may allow structural studies of the effect that CD4 receptor engagement has on the neutralization mechanism of the MPER bNAbs, which thus far has not been possible. As these complexes are likely heterogenous, excellent scalability and the classification power of single particle EM analysis becomes an important factor in addition to being able to yield high resolution structural data.…”

Recapitulation of HIV-1 Env-Antibody Coevolution in Macaques Leading to Neutralization Breadth

“…Variable regions of the RHA1.V2.01 antibody heavy and light chain genes were synthesized (Genscript) and subcloned into the pVRC8400 vector, in which a HRV3C cleavage site was inserted in the heavy-chain hinge region. The heavy and light chain pair was co-transfected in Expi293F cells (Thermo Fisher) using Turbo293 transfection reagent (Speed BioSystems) as described previously (113). The culture supernatant was harvested 6 days post transfection and loaded on a protein A column, the column was washed with PBS, and IgG proteins were eluted with a low pH buffer.…”

Susceptibility to Neutralization by Broadly Neutralizing Antibodies Generally Correlates with Infected Cell Binding for a Panel of Clade B HIV Reactivated from Latent Reservoirs

“…Infected cell binding of MPER-specific antibodies varied. 10E8v4-V5R-100cF (a version of 10E8 optimized for increased solubility and potency [47]) at 5 g/ml bound to 30 of 35 isolates, with high-level binding (MFI ratios of Ͼ4) observed for 13 of these. However, 10E8 and 10E8v4-V5R-100cF also showed substantial binding to uninfected bystanders (Gag Ϫ population) (see Fig.…”