AUTHOR=Chesterman Chelsy , Desautels Thomas , Sierra Luz-Jeannette , Arrildt Kathryn T. , Zemla Adam , Lau Edmond Y. , Sundaram Shivshankar , Laliberte Jason , Chen Lynn , Ruby Aaron , Mednikov Mark , Bertholet Sylvie , Yu Dong , Luisi Kate , Malito Enrico , Mallett Corey P. , Bottomley Matthew J. , van den Berg Robert A. , Faissol Daniel 

TITLE=Design of cross-reactive antigens with machine learning and high-throughput experimental evaluation

JOURNAL=Frontiers in Bioinformatics

VOLUME=Volume 5 - 2025

YEAR=2025

URL=https://www.frontiersin.org/journals/bioinformatics/articles/10.3389/fbinf.2025.1580967

DOI=10.3389/fbinf.2025.1580967

ISSN=2673-7647

ABSTRACT=Selecting an optimal antigen is a crucial step in vaccine development, significantly influencing both the vaccine’s effectiveness and the breadth of protection it provides. High antigen sequence variability, as seen in pathogens like rhinovirus, HIV, influenza virus, complicates the design of a single cross-protective antigen. Consequently, vaccination with a single antigen molecule often confers protection against only a single variant. In this study, machine learning methods were applied to the design of factor H binding protein (fHbp), an antigen from the bacterial pathogen Neisseria meningitidis. The vast number of potential antigen mutants presents a significant challenge for improving fHbp antigenicity. Moreover, limited data on antigen-antibody binding in public databases constrains the training of machine learning models. To address these challenges, we used computational models to predict fHbp properties and machine learning was applied to select both the most promising and informative mutants using a Gaussian process (GP) model. These mutants were experimentally evaluated to both confirm promising leads and refine the machine learning model for future iterations. In our current model, mutants were designed that enabled the transfer of fHbp v1.1 specific conformational epitopes onto fHbp v3.28, while maintaining binding to overlapping cross-reactive epitopes. The top mutant identified underwent biophysical and x-ray crystallographic characterization to confirm that the overall structure of fHbp was maintained throughout this epitope engineering experiment. The integrated strategy presented here could form the basis of a next-generation, iterative antigen design platform, potentially accelerating the development of new broadly protective vaccines.