De novo protein design enables precise induction of functional antibodies in vivo

Abstract

De novo protein design has been increasingly successful in expanding beyond nature's sequence and structural space. However, most de novo designed proteins lack biological function, in part due to the structural complexity required for functional purposes. An important domain where protein design has raised expectations was on the induction of precise antibody responses that may lead to improved vaccines. Here, we showcase two computational design approaches to stabilize irregular and discontinuous binding motifs in de novo designed immunogens and tested them for the induction of respiratory syncytial virus neutralizing antibodies in vivo. The designs mimic the native conformations of the neutralization epitopes with sub-angstrom accuracy. In vivo, cocktail formulations of the immunogens induce robust neutralizing serum responses targeting three epitopes, and re-focus pre-existing antibody responses towards bona fide neutralization epitopes. Our work provides a blueprint for epitope-centric vaccine design for pathogens that have frustrated traditional vaccine development efforts, and a general methodological pipeline to create novel proteins with functional sites within tailored protein topologies.Recently, Correia and colleagues (12) have shown that computationally designed immunogens could elicit epitope-specific responses. The RSVF antigenic site II, a linear helixturn-helix motif, was transplanted onto a heterologous protein scaffold, which elicited nAbs in non-human-primates (NHPs) after repeated boosting immunizations. Despite being a proofof-principle for the induction of functional antibodies using a computationally designed immunogen, several major caveats emerged; namely, the lack of applicability of the computational approach to structurally complex epitopes, and the inconsistent neutralization titers observed in the immunogenicity studies.

Enlightening Allostery: Designing Switchable Proteins by Photoreceptor Fusion

“…Nonetheless, even variants that show modest photo‐switching can be sufficient to mediate strong cellular phenotypes, [ 68,78 ] hence enabling interesting biological insights. Looking into the future, we anticipate that generalizable strategies for engineering allostery effector proteins will emerge from the increasing understanding of structure‐function relationships in proteins, advances in computational modeling and machine learning [ 106–108 ] as well as increased cloning and high‐throughput screening capabilities and complementary reduction in costs. We foresee that engineered, allosteric switches will continue to benefit a plethora of applications in basic research, synthetic biology, and beyond.…”

AlphaDesign: A de novo protein design framework based on AlphaFold

“…The general strategy of Rosetta-based protein design has been successfully applied to a variety of design problems. These applications range from the first de novo designed proteins [48] to synthetic vaccines [49,50], complex assemblies [51,52] and enzymes [53,54]. However, Markov-Chain Monte Carlo in structure space can be time-consuming and computationally demanding.…”

A bottom-up approach for thede novodesign of functional proteins

“…The design of novel functional proteins is a fundamental test to our understanding of protein structure and function, and has enormous potential in multiple arenas in basic biology and biotechnology (7)(8)(9)(10)(11)(12)(13)(14)(15)17). Nevertheless, the majority of studies where function was the main objective, existing proteins were repurposed and used as templates to install functional sites (7,(17)(18)(19)(20).…”

“…Expression plasmids for heavy and light chains were mixed in a 1:1 stochiometric ratio before transient transfection of HEK293 cells as described previously (8). NMR NMR experiments were performed as described previously (9). Briefly, proteins enriched in 15 N were purified and buffer-exchanged to 10 mM sodium phosphate buffer containing 50 mM sodium chloride at pH 7, with 10% D2O.…”

“…1a) strategy could consider the local structural and global topological requirements of the functional motif, irrespective of its complexity, and build supporting secondary structural elements (SSEs) to stabilize the functional site in its native conformation (24). A few studies have employed such a functioncentric design strategy (9,10,13,25,26), but a general approach that allows the systematic construction of de novo proteins with embedded functional motifs, defined connectivity and…”