Efficacy of Tolerizing Vaccination Strategy for Mitigating Autoimmune‐Mediated Cardiac Injury

Background Infectious agents that induce cross‐reactive myocarditic T cell responses may cause autoimmune inflammation of the heart, directly causing cardiac dysfunction, or they may produce an undetected autoimmune background that could manifest later as an exacerbating influence in the setting of future myocardial injury. Purpose This study was designed to evaluate the efficacy of GMCSF‐myosin fusion proteins as tolerogenic vaccines capable of inhibiting experimental autoimmune myocarditis (EAM). Methods The fusion proteins were comprised of an N‐terminal granulocyte macrophage‐colony stimulating factor (GM‐CSF) domain and a C‐terminal cardiac myosin antigen domain. The antigen domains Myosin 1052–1076 and myosin 614–643 were chosen based on epitopes known to cause EAM in Lewis rats and Balb/c mice respectively. Bioactivity of the cytokine domains for GMCSF‐Myo1052 and GMCSF‐Myo614 was confirmed with bone marrow proliferation assays. Enhanced antigen presentation was confirmed for GMCSF‐Myo1052. Results Both fusion proteins were found to inhibit the development of EAM when given as a pretreatment before EAM induction. Myo1052 fusion protein reduced the inducible incidence of EAM by nearly half, from 81.3% (13/16) to 46.7% (7/15), and the severity of EAM by 62%, from a histology severity score of 1.56 ± 1.41 to 0.60 ± 0.83 (mean ± SD, 5 point scale). Myo614 fusion protein reduced the inducible incidence of EAM by 67%, from 76.9% (13/16) to 25% (3/12), and the EAM severity by 85%, from a histology severity score of 2.23 ± 1.42 to 0.33 ± 0.65. Additionally GMCF‐Myo614 showed promise for inhibiting EAM when given after EAM induction. Serial assessment of myocardial performance was completed using echocardiography. Interestingly, despite the histological changes, cardiac function was preserved throughout. A transient increase in wall thickness was observed between days 14 and 17, but had resolved by day 21. Coupled with evidence of pericardial effusion, and in the context of the later histology findings, the change in wall thickness was interpreted to suggest building intramural myocardial edema over the second week that had resolved by the end of the third week. To explore the potential use of knockout mouse strain to identify mechanisms associated with fusion protein tolerizing efficacy, EAM was induced in C57BL/6 mice (the background stain for most knockout lines) using a peptide (myosin 718–736) derived from the murine cardiac myosin heavy chain and theorized to bind to IAd. Myosin 718–736 was capable of inducing EAM in Interferon Gamma receptor knockout mice on the C57BL/6 background. Additional studies revealed nitric oxide was not the primary effector molecule responsible for EAM resistance in C57BL/6 wild type mice. Summary These studies identify a novel potential means for reducing autoimmune mediated cardiac injury.