Proof-of-Concept, Randomized, Controlled Clinical Trial of Bacillus-Calmette-Guerin for Treatment of Long-Term Type 1 Diabetes

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 95th 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 NCT0060723

Novel Automated Blood Separations Validate Whole Cell Biomarkers

Progress in clinical trials in infectious disease, autoimmunity, and cancer is stymied by a dearth of successful whole cell biomarkers for peripheral blood lymphocytes (PBLs). Successful biomarkers could help to track drug effects at early time points in clinical trials to prevent costly trial failures late in development. One major obstacle is the inaccuracy of Ficoll density centrifugation, the decades-old method of separating PBLs from the abundant red blood cells (RBCs) of fresh blood samples.To replace the Ficoll method, we developed and studied a novel blood-based magnetic separation method. The magnetic method strikingly surpassed Ficoll in viability, purity and yield of PBLs. To reduce labor, we developed an automated platform and compared two magnet configurations for cell separations. These more accurate and labor-saving magnet configurations allowed the lymphocytes to be tested in bioassays for rare antigen-specific T cells. The automated method succeeded at identifying 79% of patients with the rare PBLs of interest as compared with Ficoll's uniform failure. We validated improved upfront blood processing and show accurate detection of rare antigen-specific lymphocytes.Improving, automating and standardizing lymphocyte detections from whole blood may facilitate development of new cell-based biomarkers for human diseases. Improved upfront blood processes may lead to broad improvements in monitoring early trial outcome measurements in human clinical trials

Strategic internal covalent cross-linking of TNF produces a stable TNF trimer with improved TNFR2 signaling

Background: Soluble TNF superfamily (TNFSF) ligands are less stable and less active than their transmembrane (tm) analogues. This is a problem for the therapeutic use of recombinant TNFSF ligands in diverse diseases including cancer and autoimmunity. Creating TNFSF ligand analogues with improved targeting of their respective receptors is important for research and therapeutic purposes. Findings: Covalent internal cross-linking of TNF monomers by double mutations, S95C/G148C, results in stable trimers with improved TNFR2 function. The resulting mutein induced the selective death of autoreactive CD8 T cells in type-1 diabetic patients and demonstrates targeted proliferation and expansion of human CD4 Tregs. Conclusions: Stable TNF trimers, created by internal covalent cross-linking, show improved signaling. The high structural homology within the TNF superfamily provides an opportunity to extend internal cross-linking to other TNF superfamily proteins to produce active trimers with improved stability and receptor signaling, and with potential applications for cancer, autoimmunity, infections, and transplantation. Electronic supplementary material The online version of this article (doi:10.1186/s40591-015-0044-4) contains supplementary material, which is available to authorized users

Proof-of-Concept, Randomized, Controlled Clinical Trial of Bacillus-Calmette-Guerin for Treatment of Long-Term Type 1 Diabetes

<div><h3>Background</h3><p>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.</p> <h3>Methodology/Principal Findings</h3><p>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.</p> <h3>Conclusions/Significance</h3><p>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.</p> <h3>Trial Registration</h3><p>ClinicalTrials.gov <a href="http://clinicaltrials.gov/ct2/show/NCT00607230?term=NCT00607230&rank=1">NCT00607230</a></p> </div

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).

<p>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.</p

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

<p>(<b>A</b>) Weekly course of EBV infection from serum of diabetic subject #vi (<b>B</b>) Positive Early Antigen D antibody versus negative values. (<b>C</b>) CD8 T-cell proliferative response. (<b>D</b>) 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.</p

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

<p>IA-2A and ZnT8 autoantibodies in clinical trial subjects by study week.</p

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

<p>(<b>A</b>) 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.</p

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.

<p>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.</p