Abstract
Currently approved vaccines for the prevention of malaria provide only partial protection against disease, due to high variability in the quality of induced antibodies. These vaccines present the unstructured central repeat region, as well as the C-terminal domain, of the circumsporozoite protein ( Pf CSP) of the malaria parasite, Plasmodium falciparum (1). A recently discovered protective monoclonal antibody, L9, recognizes three structured copies of the Pf CSP minor repeat. Similarly to other highly potent anti-malarial antibodies, L9 relies on critical homotypic interactions between antibodies for its high protective efficacy (2, 3). Here, we report the design of immunogens scaffolding one copy of Pf CSP's minor repeat capable of binding L9. To design immunogens capable of presenting multiple, structure-based epitopes in one scaffold, we developed a machine-learning-driven structural immunogen design pipeline, MESODID, tailored to focus on multi-epitope vaccine targets. We use this pipeline to design multiple scaffolds that present three copies of the Pf CSP minor repeat. A 3.6 Å cryo-EM structure of our top design, minor repeat targeting immunogen (M-TIM), demonstrates that M-TIM successfully orients three copies of L9, effectively recapitulating its critical homotypic interactions. The wide prevalence of repeated epitopes in key vaccine targets, such as HIV-1 Envelope, SARS-CoV-2 spike, and Influenza Hemagglutinin, suggests that MESODID will have broad utility in creating immunogens that incorporate such epitopes, offering a new powerful approach to developing vaccines against a range of challenging infections, including malaria. SIGNIFICANCE STATEMENT: In this study, we present a machine learning driven, structure-guided protein design pipeline built specifically for the design of multi-epitope vaccine immunogens. We employ the pipeline here to design and solve the cryo-EM structure of a de novo immunogen that binds and properly orients three copies of the anti-malarial monoclonal antibody L9, producing a promising next-generation malaria vaccine immunogen. This design pipeline could be employed to design any number of structurally constrained multi-epitope immunogens, as well as other proteins designed to bind multiple targets.