De novo protein design enables the precise induction of RSV-neutralizing antibodies.

INTRODUCTION The ultimate goal of de novo protein design is to create proteins endowed with new biological functions. From a structural perspective, this remains a challenge because most biological functions in natural proteins are mediated by irregular and discontinuous structural motifs. By contrast, state-of-the art techniques for de novo protein design excel at designing highly regular structures. Thus, most de novo proteins designed so far are either functionless or present functions that are encoded by regular, continuous secondary structures. A promising application for de novo proteins is in vaccine design, more specifically the design of proteins that mimic a viral epitope outside the context of the native protein. These proteins, when used as immunogens, have shown promise in inducing targeted virus-neutralizing antibodies (nAbs) in vivo. To date, epitope-focused immunogens have been limited to single epitopes that are regular and continuous, greatly limiting their potential in the field of vaccine design. RATIONALE A major bottleneck for the design of proteins endowed with complex functional motifs is the lack of appropriate design templates in the known structural repertoire. Here, we propose a strategy to assemble protein topologies tailored to the functional motif with the ultimate aim of enabling the design of de novo proteins endowed with complex structural motifs. We sought to apply this approach to develop an immunogen cocktail presenting three major antigenic sites of the respiratory syncytial virus (RSV) fusion protein (RSVF), aiming to induce nAbs acting through precisely defined epitopes. RESULTS We developed a novel computational design strategy, TopoBuilder, to build de novo proteins presenting complex structural motifs. TopoBuilder enabled us to define and build protein topologies to stabilize functional motifs, followed by in silico folding and sequence design using Rosetta. In vitro, the computationally designed proteins bound with high affinity to a panel of human, site-specific RSV nAbs. High-resolution crystal structures of the designs confirmed the atomic-level accuracy of the models and the presented neutralization epitopes. In vivo, cocktail formulations of the immunogens (“Trivax”) induced a balanced antibody response targeting three defined epitopes, yielding neutralizing serum levels in mice and nonhuman primates (NHPs) after a single boost. Trivax elicited a remarkably focused immune response toward the target antigenic sites. Moreover, when used as a boosting immunogen after prefusion RSVF administration, Trivax profoundly reshaped the serum composition, leading to a higher fraction of epitope-specific antibodies and an increased quality of the antibody response compared with prefusion RSVF boosting immunizations. At the molecular level, monoclonal antibodies isolated from Trivax-immunized NHPs were epitope specific, and in one instance resembled those induced by viral infection in humans. CONCLUSION Our work provides a new route to functionalizing de novo proteins and presents a blueprint for epitope-centric vaccine design, offering an unprecedented level of control over induced antibody specificities in both naive and primed antibody repertoires. Beyond immunogens, the ability to design de novo proteins presenting functional sites with high structural complexity will be broadly applicable to expanding the structural and sequence repertoires, but above all, the functional landscape of natural proteins.