Proof-of-concept, randomized, controlled clinical trial of Bacillus-Calmette-Guerin for treatment of long-term type 1 diabetes

Abstract

Background: No targeted immunotherapies reverse type 1 diabetes in humans. However, in a rodent model of type 1 diabetes, Bacillus Calmette-Guerin (BCG) reverses disease by restoring insulin secretion. Specifically, it stimulates innate immunity by inducing the host to produce tumor necrosis factor (TNF), which, in turn, kills disease-causing autoimmune cells and restores pancreatic beta-cell function through regeneration.

Methodology/principal findings: Translating these findings to humans, we administered BCG, a generic vaccine, in a proof-of-principle, double-blind, placebo-controlled trial of adults with long-term type 1 diabetes (mean: 15.3 years) at one clinical center in North America. Six subjects were randomly assigned to BCG or placebo and compared to self, healthy paired controls (n = 6) or reference subjects with (n = 57) or without (n = 16) type 1 diabetes, depending upon the outcome measure. We monitored weekly blood samples for 20 weeks for insulin-autoreactive T cells, regulatory T cells (Tregs), glutamic acid decarboxylase (GAD) and other autoantibodies, and C-peptide, a marker of insulin secretion. BCG-treated patients and one placebo-treated patient who, after enrollment, unexpectedly developed acute Epstein-Barr virus infection, a known TNF inducer, exclusively showed increases in dead insulin-autoreactive T cells and induction of Tregs. C-peptide levels (pmol/L) significantly rose transiently in two BCG-treated subjects (means: 3.49 pmol/L [95% CI 2.95-3.8], 2.57 [95% CI 1.65-3.49]) and the EBV-infected subject (3.16 [95% CI 2.54-3.69]) vs.1.65 [95% CI 1.55-3.2] in reference diabetic subjects. BCG-treated subjects each had more than 50% of their C-peptide values above the 95(th) percentile of the reference subjects. The EBV-infected subject had 18% of C-peptide values above this level.

Conclusions/significance: We conclude that BCG treatment or EBV infection transiently modified the autoimmunity that underlies type 1 diabetes by stimulating the host innate immune response. This suggests that BCG or other stimulators of host innate immunity may have value in the treatment of long-term diabetes.

Trial registration: ClinicalTrials.gov NCT00607230.

Figure 1. CONSORT flow chart (A) and flow diagraph (B) with depicts of treatment concept, outcomes and subject comparison groups for the study.

Figure 2. Clinical characteristics of groups of clinical trial subjects and reference subjects.

Figure 3. Clinical laboratory studies reveal acute EBV infection in placebo-treated diabetic.

(A) Weekly course of EBV infection from serum of diabetic subject #vi (B) Positive Early Antigen D antibody versus negative values. (C) CD8 T-cell proliferative response. (D) Flow scatter plots of appearance of EBV-reactive T-cells vs. paired control, week 6 to 8. All newly appearing EBV-reactive T-cells were viable.

Figure 4. Insulin-autoreactive T-cells released into the circulation are dead after BCG treatment or EBV infection.

(A) Percentage of insulin-autoreactive T-cells of total CD8 T-cells over 12 weeks for BCG-treated (Ai, Bi), placebo-treated, (Aii, Bii) and EBV-infected clinical trial subjects (Aiii, Biii). Reference diabetics without or with insulin-autoreactive T-cells vs. reference healthy controls (Aiv,v). (B) Insulin-autoreactive T-cells stratified by viability in clinical trial subjects. Red diamonds are long-term diabetics; blue diamonds paired healthy controls. Arrows are BCG or placebo injection times.

Figure 5. Two-color flow pictures of the serial weekly blood monitoring of dead and live insulin autoreactive T cells in a control subject (left) and BCG-treated diabetic subject (right).

After the first BCG treatment, predominantly dead insulin-autoreactive T cells appear in the circulation of the diabetic compared to the simultaneously studied paired healthy control. For all recruited BCG-treated diabetic subjects, the start of the trial shows fresh blood samples with no insulin-autoreactive T-cells in these longterm diabetics, followed by dead insulin-autoreactive T-cells that persist through week 4, recurrent dead insulin-autoreactive cells released again after the second injection of BCG followed by the gradual disappearance of the dead insulin-autoreactive T-cells by week 12 of monitoring. It should be noted that the newly released insulin-autoreactive cells after BCG are unique in representing both low affinity (*) autoreactive T-cells that can be observed in the routine monitoring of positive patients and high affinity(***) autoreactive T-cells that are never observed in routine monitoring of diabetic patients. In contrast to the serial monitoring of a BCG treated subject, the serial studied fresh blood samples of the control subject reveal throughout the study the lack of either live or dead insulin-autoreactive T-cells.

Figure 6. T_REG cells and GAD-autoantibodies change in response to BCG and EBV.

(A) T_REG cell ratios in BCG-treated, placebo, and EBV-infected clinical trial subjects by week vs. paired healthy controls. (B) GAD autoantibody levels vs. own baseline in BCG-treated placebo-treated, and EBV-infected clinical trial in each subject, by week. B is baseline prior to trial. Arrows are BCG or placebo injection times.

Figure 7. IA-2A and ZnT8 autoantibodies in clinical trial subjects by study week.

Figure 8. Fasting C-peptide levels show transient increase in BCG-treated and EBV-infected clinical trial subjects.

Fasting C-peptide for (A) BCG-treated, (B) Placebo-treated, and (C) EBV-infected clinical trial subjects by week vs. (D) Reference diabetics, by visit. C-peptide levels are measured by ultrasensitive C-peptide assay in duplicate. Arrows are BCG or placebo injection times.

Figure 9. C-peptide levels remain stable and near the lower limit of an ultrasensitive assay in a longterm diabetic group (N = 17) sampled weekly for 12 weeks in a fasting state.