Therapeutic efficiency of vector-expressed soluble virus receptor proteins in cardiac enterovirus infections

柯萨奇病毒是引起病毒性心肌炎的最常见的感染性病原体。然而，目前还没有一种特定的CV疗法。因此，开发新的治疗策略具有很高的相关性，并代表了本项目提案的主要目标。在第一个资助期，我们已经证明了从病毒载体表达的可溶性CV受体蛋白(SCAR-Fc)可以在体内抑制急性CVB3心肌炎和心功能不全的发展。此外，应用SCAR-FC足以治愈新建立的持续感染CVB3的心肌细胞系。在进一步的研究中，我们已经证明，同时给予抗CVB3 siRNA可以提高SCAR-FC治疗的效率。最后，我们首次证明了用间充质干细胞和心脏贴壁增殖细胞进行细胞治疗可以改善CVB3引起的心肌炎。第二个资助期的项目提案包括五个复合体，目标如下。(I)在证明了SCAR-FC治疗急性CVB3所致心肌炎的有效性之后，我们现在想要研究SCAR-FC在慢性心肌炎中的治疗潜力。(2)病毒诱导和反应免疫机制是CVB3慢性心肌炎发生的重要机制。我们将确定抗病毒治疗与SCAR-FC、免疫抑制药物治疗和免疫调节干细胞治疗相结合在多大程度上提高了治疗效率。(3)微小核糖核酸病毒，其中包括CVB3，在药物治疗后迅速产生逃逸突变。然而，到目前为止，还没有研究抗SCAR-FC的CVB3突变体的发生。我们打算分析来自经SCAR-FC处理的CVB3感染小鼠的CVB3分离株，以寻找SCAR-FC耐药突变。此外，还将在体外特意产生SCAR-Fc逃逸突变体，并研究其产生抗药性的机制。(Iv)我们已经证明了CAR的D2结构域对于SCAR-FC的CVB3中和性能是重要的，它不直接参与与CVB3的结合。我们将进一步阐明D2结构域的功能及其相关机制。(V)最后，由于到目前为止还没有可用的数据，我们打算调查患者的CVB3分离株对SCAR-FC的敏感性。这一预期结果可能与SCAR-FC潜在的临床治疗应用密切相关。

Coxsackieviruses (CV) are the most common infectious agents causing virus-induced myocarditis. A specific CV therapy, however, is currently not yet available. The development of new therapeutic strategies is therefore of high relevance and represents the main objective of this project proposal. In the first funding period we have shown that a soluble CV receptor protein (sCAR-Fc) expressed from a viral vector inhibits acute CVB3 myocarditis and the development of cardiac dysfunction in vivo. Moreover, application of sCAR-Fc was sufficient to cure a newly established persistently CVB3-infected myocardial cell line. In a further study we have demonstrated that the efficiency of sCAR-Fc therapy can be improved by concomitant administration of anti-CVB3 siRNAs. Finally, we have shown for the first time that CVB3-induced myocarditis can be improved by cell therapy with mesenchymal stem cells and cardiac adherent proliferating cells.The project proposal for the second funding period involves five complexes with the following aims. (I) After having demonstrated the effectiveness of sCAR-Fc therapy in acute CVB3-induced myocarditis, we now want to investigate the therapeutic potential of sCAR-Fc in chronic myocarditis. (II) Virus-induced as well as reactive immunologic mechanisms are important mechanisms for the development of chronic CVB3-induced myocarditis. We will determine the extent to which the combination of antiviral therapy with sCAR-Fc and immunosuppressive pharmacotherapy and immunomodulatory stem cell therapy results in an improvement of the therapeutic efficiency. (III) Picornaviruses, among them CVB3, rapidly develop escape mutants following pharmacotherapy. However, there are thus far no studies investigating the occurrence of sCAR-Fc-resistant CVB3 mutants. We intend to analyze CVB3 isolates from sCAR-Fc-treated, CVB3-infected mice for sCAR-Fc-resistant mutants. Furthermore, sCAR-Fc escape mutants will be deliberately generated in vitro and the mechanisms for the development of resistance will be investigated. (IV) We have shown that the D2 domain of CAR, which is not directly involved in binding to CVB3, is important for the CVB3 neutralizing properties of sCAR-Fc. We will further elucidate the function of the D2 domain and the involved mechanisms. (V) Finally, since there are no data available to date, we intend to investigate CVB3 isolates from patients for susceptibility to sCAR-Fc. The expected results may be highly relevant for a potential clinical therapeutic application of sCAR-Fc.