Antigen processing in the class II MHC pathway depends on conventional proteolytic enzymes, potentially acting on antigens in native-like conformational states. CD4+ epitope dominance arises from a competition among antigen folding, proteolysis, and MHCII binding. Protease-sensitive sites, linear antibody epitopes, and CD4+ T-cell epitopes were mapped in plague vaccine candidate F1-V to evaluate the various contributions to CD4+ epitope dominance. Using X-ray crystal structures, antigen processing likelihood (APL) predicts CD4+ epitopes with significant accuracy for F1-V without considering peptide-MHCII binding affinity. We also show that APL achieves excellent performance over two benchmark antigen sets. The profiles of conformational flexibility derived from the X-ray crystal structures of the F1-V proteins, Caf1 and LcrV, were similar to the biochemical profiles of linear antibody epitope reactivity and protease sensitivity, suggesting that the role of structure in proteolysis was captured by the analysis of the crystal structures. The patterns of CD4+ T-cell epitope dominance in C57BL/6, CBA, and BALB/c mice were compared to epitope predictions based on APL, MHCII binding, or both. For a sample of 13 diverse antigens, the accuracy of epitope prediction by the combination of APL and I-A b -MHCII-peptide affinity reached 36%. When MHCII allele specificity was also diverse, such as in human immunity, prediction of dominant epitopes by APL alone reached 42% when using a stringent scoring threshold. Because dominant CD4+ epitopes tend to occur in conformationally stable antigen domains, crystal structures typically are available for analysis by APL, and thus, the requirement for a crystal structure is not a severe limitation.

16Antigen processing in the class II MHC pathway depends on conventional proteolytic enzymes, 17 potentially acting on antigens in native-like conformational states. CD4+ epitope dominance 18 arises from a competition between antigen folding, proteolysis, and MHCII binding. Protease-19 sensitive sites, linear antibody epitopes, and CD4+ T-cell epitopes were mapped in the plague 20 vaccine candidate F1-V to evaluate the various contributions to CD4+ epitope dominance. Using 21 X-ray crystal structures, antigen processing likelihood (APL) predicts CD4+ epitopes with 22 significant accuracy without considering peptide-MHCII binding affinity. The profiles of 23 conformational flexibility derived from the X-ray crystal structures of the F1-V proteins, Caf1 and 24 LcrV, were similar to the biochemical profiles of linear antibody epitope reactivity and protease- 25 sensitivity, suggesting that the role of structure in proteolysis was captured by the analysis of the 26 crystal structures. The patterns of CD4+ T-cell epitope dominance in C57BL/6, CBA, and BALB/c 27 2 mice were compared to epitope predictions based on APL, peptide binding to MHCII proteins, or 28 both. For a sample of 13 diverse antigens larger than 200 residues, accuracy of epitope prediction 29 by the combination of APL and I-A b -MHCII-peptide affinity approached 40%. When MHCII allele 30 specificity is also diverse, such as in human immunity, prediction of dominant epitopes by APL 31 alone approached 40%. Since dominant CD4+ epitopes tend to occur in conformationally stable 32 antigen domains, crystal structures typically are available for analysis by APL; and thus, the 33 requirement for a crystal structure is not a severe limitation. 34 Introduction 35Rational vaccine design continues to be challenging, due in no small part to the multiple disparate 36 mechanisms and signals that regulate the strength and specificity of the immune response. 37 Antibodies and T cells form the core of adaptive immune responses, but they depend on each 38 other and on potent signals from the innate immune system (1). T-cells recognize polypeptide 39 fragments displayed on the cell surface by polymorphic major histocompatibility complex (MHC) 40 molecules. The two main types of MHC molecules, class I and class II, differ in their source of 41 peptides and the type of type of T cells that recognize them (2, 3). Class I MHC molecules (MHCI) 42 present mostly endogenous peptides and are recognized by CD8+ T cells. Class II MHC molecules 43 (MHCII) present mostly exogenous peptides and are recognized by CD4+ T-cells. 44 The analysis of large numbers of natural and synthetic MHC-bound peptides, combined with the 45 study of X-ray crystal structures revealed that the specificity of peptide-binding to MHCI and 46 MHCII can be explained by the shape and chemical environment of the peptide-binding cleft (4-47 6). A substantial degree of variability in peptide specificity derives from the polymorphism of 48 MHCI and MHCII molecules, wherein variant residues in...