Development and characterisation of a laboratory model of post streptococcal autoimmune cardiac and neurobehavioral complications

Abstract

<p>Acute Rheumatic Fever and Rheumatic Heart Disease (ARF/RHD) are triggered by an autoimmune response against group A streptococcal (GAS) infection. In ARF/RHD and Sydenham chorea (SC) both antibodies and T-cells against immunodominant GAS virulence factors including M-protein, cross-react with the pathogen and host proteins leading to cardiac tissue damage and neurobehavioral changes. Historically, the aetiology of ARF/RHD is attributed to preceding GAS infections. However, throat carriage of <i>Streptococcus pyogenes</i> has been reported to be relatively low in some ARF/RHD endemic regions as opposed to <i>Streptococcus dysgalactiae subspecies equisimilis</i> (SDSE). Therefore, we hypothesised that streptococci other than GAS may also trigger ARF/RHD and associated neurobehavioral disorders. Post-streptococcal autoimmune complications are uniquely human conditions and modelling of these symptoms in animals is challenging and the immunopathology of these complications remain unclear. Therefore, animal models are essential to study the early mechanisms which lead to these complications. Furthermore, vaccine development against GAS infection is also hindered by the unavailability of a suitable animal model that mimics GAS infections and ensuing immune sequelae. An animal model, which exhibit characteristics of cardiac and neurobehavioral defects associated with ARF/RHD is important for such studies. In addition, an animal model is essential for the identification of disease associated common polyepitopes of GAS as a potential biomarker for the diagnosis of ARF/RHD. Therefore, the objective of this study was to characterise the Lewis rat Autoimmune Valvulitis (RAV) model to assess the mechanisms including functional, histological, immunological and neurobehavioral defects following exposure to streptococcal antigens.</p> <p>Initially we assessed two different rat strains (Lewis and Wistar) for their suitability for simultaneous investigation of cardiac and neurobehavioral abnormalities associated with poststreptococcal complications following exposure to whole-killed GAS (WK-GAS). Lewis rats injected with WK-GAS were preferred over Wistar rats when considering an animal model suitable for a longitudinal time course study, to examine the manifestation of post-streptococcal neurobehavioral and cardiac abnormalities akin to ARF/RHD. Our subsequent experiments assessed the functional, histological, immunological and neurobehavioral defects following exposure to recombinant GAS M5 protein (rM5) and SDSE M proteins (Stg480) in Lewis rats. Lewis rats injected with the M proteins of GAS and SDSE developed significant cardiac functional and neurobehavioral abnormalities comparable to human disease and demonstrated for the first time that antigen from a streptococcal species other than <i>Streptococcus pyogenes</i> can initiate and drive the autoimmune responses that cause cardiac and neurobehavioral changes. Antibodies against GAS and SDSE M proteins cross-reacted with cardiac myosin, collagen, tropomyosin, laminin, lysoganglioside, dopamine receptors 1 and 2 (DR1 and DR2) and tubulin. Furthermore, serum from rats injected with streptococcal antigens showed higher IgG binding to the striatum and cortex of the brain. Western blot analysis further confirmed the specificity of the cross-reactive anti-GAS and anti-SDSE antibody with endogenous brain antigens.</p> <p>We also extended our work in this model, to examine whether adoptive transfer of GAS rM5 specific serum into naïve rats reproduce cardiac and neurobehavioral changes observed in rats injected with GAS rM5. Adoptive transfer of GAS rM5 specific sera induced carditis and conduction abnormalities in recipient rats. Although cross-reactive antibodies against neuronal tissues proteins were elevated in recipient rats none of the rats demonstrated neurobehavioral changes. This indicates that extravasation of circulating antibodies into the brain, is a multistep process characterised by activation of many other sequential events. The potential role of immunodominant peptides from the streptococcal M proteins as serological markers for the diagnosis of ARF/RHD was also demonstrated using serum samples from patients with ARF/RHD and Lewis rats injected with GAS rM5 and SDSE Stg480 by peptide microarray technology. Combined experiments on our RAV model and clinical samples using peptide array technology has provided evidence of utilising immunodominant peptide from C-repeat regions of the M protein as potential biomarkers for ARF/RHD diagnosis. Although, these peptides elicited significant antibody response in the RAV model against host connective tissue, cardiac and neuronal proteins they did not induce any neurobehavioral changes or functional and histological changes in heart.</p> <p>Finally, the RAV model was used to evaluate the safety of a modified peptide vaccine from Crepeat region of the M protein (p*17) and an epitope of cell envelope protease (SpyCEP)(K4S2). The vaccine candidates did not induce any deleterious immune response associated with ARF/RHD. The vaccine specific antibodies generated in the RAV model were not crossreactive with cardiac, connective and neuronal tissue proteins. Subsequent histological examination also demonstrated no inflammatory cell infiltration into cardiac tissue or valvulitis or neurobehavioral changes. These results demonstrates that two vaccine candidates are safe for use in Phase I clinical trials.</p> <p>In conclusion, for the first time we have characterised a rat model that contributes to the understanding of the early events which lead to cardiac and neurobehavioral abnormalities following exposure to streptococcal antigens. Our experimentations also showed potential use of this animal model for the identification of immunodominant streptococcal epitopes as potential biomarkers for the diagnosis of ARF/RHD and to potentially evaluate the safety of GAS vaccine candidates prior to use in clinical trials.</p>