Human Leukocyte Antigens (HLA) are immunoreceptors that present peptide antigens at the cell surface to T cells as a primary mechanism of immune surveillance. Malaria, a disease associated with the Plasmodium parasite, claims >
600,000 lives per year globally with most deaths occurring in Africa. Development of efficacious prophylactic vaccines or therapeutic treatments for malaria has been hindered by the lack of a basic understanding of the role of HLA-mediated peptide antigen presentation during Plasmodium infection. In particular, there is (i) little understanding of which peptide antigens are presented by HLAs in the context of malaria, and (ii) a lack of structural insights into Plasmodium peptide antigen presentation by HLAs, which underpins peptide/HLA stability, specificity, cross-presentation across HLA alleles, and recognition by T cell receptors. To begin to address these knowledge gaps, we identify and characterize candidate peptide antigens derived from Plasmodium falciparum with potential for presentation by common class I HLA alleles. We computationally screen nine proteins from the P. falciparum proteome to predict eight peptides with potential for cross-presentation by common alleles in African populations, HLA-A*02:01 and HLA-B*08:01. We then validate the predictions by producing recombinant HLAs in complex with the eight identified peptides by in vitro refolding. We evaluate the folding and thermal stability of the resulting sixteen peptide/HLA complexes by CD spectroscopy and nanoDSF. In silico modeling of peptide/HLA complexes informs a plausible structural basis for mechanisms for cross-presentation of P. falciparum peptides across HLA-A*02:01 and HLA-B*08:01 alleles. Finally, we expand our identified P. falciparum peptides to cover a broader range of HLA alleles in malaria endemic populations with experimental validation provided for HLA-C*07:01 and HLA-E*01:03. Together, our results are a step forward towards a deeper understanding of the potential for multi-allele cross-presentation of peptides in malaria. These results further inform future development of multivalent vaccine strategies targeting HLA profiles in malaria endemic populations.