Immunological efficacy of heat shock protein 60 peptide DiaPep277 therapy in clinical type I diabetes

Abstract

An immunogenic peptide (p277) from the 60-kDa heat shock protein (hsp60) arrested beta-cell destruction in non-obese diabetic mice. A randomized, double-blind, phase Ib/II clinical trial of DiaPep277 peptide treatment was performed in recent-onset type 1 diabetes patients with remaining insulin production. We studied the immunological efficacy of this peptide therapy and correlated this with clinical outcome. Forty-eight C-peptide-positive patients were assigned subcutaneous injections of 0.2, 1.0 or 2.5 mg p277 (n = 12 per dosage) at entry, and 1, 6 and 12 months, or four placebo injections (n = 12). T cell autoimmunity to hsp60, DiaPep277, glutamic acid decarboxylase and tetanus toxoid (recall response control) were assayed by proliferation and cytokine secretion assays (enzyme-linked immunospot) at regular intervals until 18 months after the first injection. All treated patients at each dosage of peptide demonstrated an altered immune response to DiaPep277, while the majority of placebo-treated patients remained non-responsive to treatment (P = 0.00001), indicating a 100% efficacy of immunization. Cytokine production in response to therapy was dominated by interleukin (IL)-10. IL-10 production before therapy and decreasing autoantigen-specific T cell proliferation were associated with beta-cell preservation. Third-party control immune responses were unaffected by therapy. No potentially adverse immunological side effects were noted. DiaPep277 is immunogenic in type 1 diabetic subjects and has immune modulating properties. Immunological monitoring distinguished therapy from placebo treatment and could determine immunological efficacy. Declining or temporary proliferative responses to peptide DiaPep277 treatment may serve as an immunological biomarker for clinical efficacy.

Figures

Fig. 1

Proliferative responses in peripheral blood mononuclear cells to medium (baseline response) (a), tetanus toxoid (b), glutamic acid decarboxylase 65 (c) and heat shock protein 60 (d). Arrows indicate injection time-points. Colours refer to treatment groups (black = placebo; red = 0·2 mg; green = 1·0 mg; blue = 2·5 mg). Indicated are raw counts per minute. **P-value < 0·01.

Fig. 2

Changes in immune response to heat shock protein (hsp) 60 protein, tetanus toxoid and glutamic acid decarboxylase (GAD) after treatment. Proliferative responses to antigen were calculated as fractions from the pretreatment value per patient and subsequently normalized by taking the natural logarithm (ln). Blue triangles indicate measurements of placebo-treated patients (n = 79), red dots of peptide-treated patients (n = 253). In all analyses, correlation between the measurements was highly significant (P < 0·0001 for both groups in graphs a–c). The P-value inside the graph assesses whether the differences in the slopes of the correlations are significant. The change in response after treatment was different between treated and placebo when comparing hsp with tetanus toxoid (P = 0·06) and with GAD (P = 0·01), indicating hsp-specific immune deviation by the peptide treatment.

Fig. 3

Correlation between DiaPep277 immune status before peptide therapy and changes in C-peptide production afterwards. (a) Patients showing proliferation in response to peptide DiaPep277 prior to therapy (n = 14) showed similar loss of C-peptide production as non-responsive patients (n = 26). (b) Interleukin (IL)-10 response to DiaPep277 prior to therapy (n = 10) is associated with preservation of C-peptide compared with no IL-10 response (n = 30). Horizontal lines depict medians for each group.

Fig. 4

Correlation between DiaPep277 immune status over time and changes in C-peptide production. (a) Patients showing modulated proliferation in the course of the study (n = 19) show preserved beta-cell function compared with patients who do not (n = 15). Six patients did not elicit proliferation at any time-point. (b) Interleukin (IL)-10 production after the last injection (n = 21) is not associated with C-peptide preservation when compared with no IL-10 production (n = 19). Horizontal lines depict medians for each group.