408 - In Silico Prediction of Plasmodium falciparum PfRipr Epitopes as Vaccine Candidates

The global malarial death toll in 2020 was ~627,000, 77% of which occurred in children < 5 years of age. Plasmodium falciparum, the most lethal parasite of its genus, has evaded vaccine development due to its complex life cycle and redundant invasion mechanisms. Epitope-based vaccines hold significant promise due to their ability to target dominant regions in antigenically variable pathogens. The recently characterized P. falciparum merozoite Rh5 interacting protein (PfRipr) is highly conserved and essential for erythrocyte invasion, making it an ideal target for a bloodstage malaria vaccine.

Objective: We sought to analyze P. falciparum sequences from highly endemic areas using bioinformatic approaches to identify candidate PfRipr bloodstage vaccine epitopes for inclusion in a multiantigen vaccine.

Design/Methods: Using P. falciparum sequence datasets from Burkina Faso and Uganda, we assessed the immunogenic potential of PfRipr epitopes for immune cell recognition. T-cell receptor binding was predicted using NetMHCpan and NetMHCIIpan searching against MHC alleles with high regional frequencies. Using an in-silico 3D model of PfRipr predicted via AlphaFold, tertiary structures of all PfRipr sample sequences were predicted via SWISS-MODEL then analyzed by ElliPro to identify linear and discontinuous B-cell epitopes. Putative epitopes were filtered using binding thresholds, allele coverage, conservation, antigenicity, and allergenicity.

Results: The Burkina Faso PfRipr dataset predicted 1729 unique T-cell and 51 unique B-cell epitopes, and 28 T-cell and 4 B-cell epitopes remained post-filtration. Epitope prediction using the Ugandan dataset predicted 576 unique T-cell and 53 unique B-cell epitopes, and 57 T-cell and 7 B-cell epitopes remained post-filtration. Between the two datasets, there were 19 matching epitopes with 7 MHC I, 9 MHC II, 1 linear, and 2 discontinuous. These epitopes were used to design a multi-epitope-based vaccine construct with an adjuvant and peptide linkers to separate functional regions for immune processing.

Conclusion(s): Immunoinformatic tools employed in sequence identified candidate PfRipr epitopes used to design a bloodstage vaccine construct against P. falciparum. To validate their predicted immunogenicity, epitopes can be further investigated using in silico stabilization and docking simulations, in vitro T-cell activation assays, and in vivo transgenic mouse models. The pipeline of immunoinformatic analyses used can be applied to a database of P. falciparum sequences collected from other malaria endemic regions to develop vaccines effective against circulating strains.