Research Article

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

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Science  15 May 2020:
Vol. 368, Issue 6492, eaay5051
DOI: 10.1126/science.aay5051

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TopoBuilding precision vaccines

To induce strong and targeted neutralizing antibody (nAb) responses against vaccine targets, one strategy has been to use computationally designed immunogens. However, the structural complexity of many known neutralization epitopes has posed a major challenge for the design of accurate epitope mimetics. Sesterhenn et al. created a protein design algorithm called TopoBuilder to design scaffolds for irregular and discontinuous neutralization epitopes. As a proof of principle, the authors generated epitope-focused immunogens based on the prefusion conformation of the respiratory syncytial virus (RSV) fusion protein. When these immunogens were used to vaccinate mice and nonhuman primates in RSV infection models, they generated targeted nAb responses to RSV and boosted site-specific nAb responses in heterologous prime-boost vaccination schemes.

Science, this issue p. eaay5051

Structured Abstract

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 naïve 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.

De novo design of a trivalent cocktail vaccine.

Structurally complex RSV neutralization epitopes were stabilized in de novo–designed proteins. The computational tool TopoBuilder builds customized protein topologies to stabilize functional structural motifs, followed by folding and sequence design using Rosetta. In vivo, a three-scaffold cocktail induced focused RSV nAbs against the target epitopes in mice and NHPs.

Abstract

De novo protein design has been successful in expanding the natural protein repertoire. However, most de novo proteins lack biological function, presenting a major methodological challenge. In vaccinology, the induction of precise antibody responses remains a cornerstone for next-generation vaccines. Here, we present a protein design algorithm called TopoBuilder, with which we engineered epitope-focused immunogens displaying complex structural motifs. In both mice and nonhuman primates, cocktails of three de novo–designed immunogens induced robust neutralizing responses against the respiratory syncytial virus. Furthermore, the immunogens refocused preexisting antibody responses toward defined neutralization epitopes. Overall, our design approach opens the possibility of targeting specific epitopes for the development of vaccines and therapeutic antibodies and, more generally, will be applicable to the design of de novo proteins displaying complex functional motifs.

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