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Abstract
Class I fusion proteins are critical mediators of viral entry in a range of pathogenic viruses, making them prime candidates for vaccine development. Despite their potential, the design of
effective vaccines based on these proteins is hindered by two challenges: structural instability and
antigenic variability. To address these limitations, we developed three distinct computational
strategies aimed at optimizing the vaccine potential of class I fusion proteins. Our initial two
methodologies targeted the enhancement of structural stability. The first approach sought to
optimize electrostatic contacts within the protein structure, while the second aimed at the strategic
introduction of disulfide bonds. Notably, the electrostatic optimization method was implemented
across three different viral families and demonstrated significant effectiveness, requiring only a
handful of protein variants for successful stabilization. The structural integrity of the leading
candidates was subsequently validated through crystallography and cryo-electron microscopy
analyses. In a vaccine application focusing on the respiratory syncytial virus (RSV), our designed
protein exhibited protective efficacy in a mouse model comparable to that of a recently approved
RSV vaccine. Further stabilization was achieved through the introduction of a single disulfide
bond, resulting in a thermal stability increase of approximately 12°C without compromising
immunogenicity. Our third computational strategy addressed the issue of antigenic variability by
targeting the hemagglutinin (HA) protein of the H1 influenza virus. Utilizing a hybrid approach
that combines consensus design with the presentation of a diverse array of immunogenic epitopes,
we engineered a HA variant with broad reactivity. This variant induced a diverse antibody response
in a mouse model and demonstrated protective capabilities against heterologous viral challenge.
Collectively, our computational approaches offer a comprehensive toolkit for the optimization of
class I fusion proteins as vaccine candidates. They present substantial promise for the development
of effective preventive measures against a wide spectrum of viral infections.