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

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-2D^b, with an IC₅₀ of 7.83 nM, and exhibits higher thermal stability (T_m = 59.16 °C) than the immunodominant LCMV gp33 epitope (T_m = 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-2D^b 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.