Hybrid insulin peptides (HIPs) are unique post-translationally modified antigens whereby derivatives of proinsulin (PI) form peptide bonds with endogenous granule peptides. HIPs have been identified in mouse β-cell extracts and human islets by mass spectrometry. Diabetogenic CD4 T cell clones in NOD mice and CD4 T cells from T1D patients are stimulated by HIP ligands and imply that HIPs may also serve as epitopes for humoral immunity. To identify and characterize antibodies (Abs) targeting HIPs among T1D individuals and evaluate their utility as biomarkers, a sensitive and specific ECL-based HIP Ab assay was adapted from the hallmark ECL-IAA protocol. The key determinant for the HIP-Ab assay was design and implementation of a conformational HIP protein probe reflecting derivatives of the HIPs capable of eliciting an antigenic response in CD4 T cell lines from T1D individuals and NOD mice. Sera from a new onset T1D cohort using a PI/IAPP HIP probe showed the presence of anti-PI/IAPP Abs (32.2%, 58/180). However unexpected prevalence was measured among control (18.8%, 18/96) and T2D (25.9%, 29/112) sera samples even when the data was stratified according to HLA genotype. These HIP-Ab epitopes are both conformation and HIP-sequence specific as they are pre-absorbed only with refolded PI/IAPP, but not alternate or random HIPs, linear PI/IAPP HIPs or cyclic PI/IAPP peptides. In sera from prediabetic subjects from the DAISY cohort, 7 of 22 (31.8%) display a peak of HIP-Abs that appears prior to the peak of IAA autoAbs in most cases (87.5%, 7/8). Large number of individuals have PI/IAPP-specific Abs in their circulation, irrespective of T1D disease state or risk. Although HIP-Abs are pervasive and emerge early in the disease process in prospectively followed at risk prediabetic individuals, they are not predictive of disease. As the appearance of PI/IAPP HIP-Abs typically precedes IAAs, it is possible that Abs to other HIPs may serve as earlier biomarkers for ensuing disease.
Y. Gu: None. J.M. Wenzlau: None. A.W. Michels: Stock/Shareholder; Self; IM Therapeutics. M. Rewers: None. K. Haskins: None. L. Yu: None.