Structural Basis for the Immunological Paradox of a High-Affinity Yet Non-Immunogenic MHC-I Epitope from Cryptosporidium parvum

Cryptosporidium parvum is an important apicomplexan parasite that causes severe diarrheal disease in children and immunocompromised individuals. However, the structural basis for the limited immunogenicity of its T-cell epitopes remains poorly understood. This study integrates structural biology and immunological approaches to elucidate the molecular basis underlying the non-immunogenicity of KAV9, a Cp23-derived epitope with the sequence KAVKNPAPI. Biophysical analyses demonstrated that KAV9 forms a high-affinity complex with H-2Db, with an IC50 of 7.83 nM, and exhibits higher thermal stability (Tm = 59.16 °C) than the immunodominant LCMV gp33 epitope (Tm = 51.15 °C). Despite strong pMHC binding and high pMHC stability, in vivo peptide immunization failed to elicit a detectable KAV9-specific CD8+ T-cell response. Crystal structure analysis revealed that KAV9 is tightly accommodated within the H-2Db binding groove through an extensive hydrogen-bond network. However, its distinct peptide conformation, particularly involving P4-Lys and the proline residues at P6 and P8, markedly reshapes the TCR-exposed surface compared to gp33. AlphaFold3 (AF3) modeling further suggested that these structural deviations disrupt critical hydrogen-bond interactions with the T-cell receptor (TCR) CDR3 loops, thereby eliminating contacts required for TCRβ engagement. Sequence analysis revealed that KAV9 is highly conserved across multiple Cryptosporidium species, suggesting a conserved structural feature associated with limited T-cell recognition. Together, these findings demonstrate that strong MHC binding and pMHC stability are insufficient to ensure CD8+ T-cell immunogenicity. Instead, the topology of the TCR-accessible peptide surface represents a critical determinant of epitope immunogenicity, with significant implications for epitope selection and vaccine design against cryptosporidiosis.