PhIP-Seq Reveals Autoantibodies for Ubiquitously Expressed Antigens in Viral Myocarditis

Abstract

Enteroviruses such as group B coxsackieviruses (CVB) are commonly suspected as causes of myocarditis that can lead to dilated cardiomyopathy (DCM), and the mouse model of CVB3 myocarditis is routinely used to understand DCM pathogenesis. Mechanistically, autoimmunity is suspected due to the presence of autoantibodies for select antigens. However, their role continues to be enigmatic, which also raises the question of whether the breadth of autoantibodies is sufficiently characterized. Here, we attempted to comprehensively analyze the autoantibody repertoire using Phage ImmunoPrecipitation Sequencing (PhIP-Seq), a versatile and high-throughput platform, in the mouse model of CVB3 myocarditis. First, PhIP-Seq analysis using the VirScan library revealed antibody reactivity only to CVB3 in the infected group but not in controls, thus validating the technique in this model. Second, using the mouse peptide library, we detected autoantibodies to 32 peptides from 25 proteins in infected animals that are ubiquitously expressed and have not been previously reported. Third, by using ELISA as a secondary assay, we confirmed antibody reactivity in sera from CVB3-infected animals to cytochrome c oxidase assembly factor 4 homolog (COA4) and phosphoinositide-3-kinase adaptor protein 1 (PIK3AP1), indicating the specificity of antibody detection by PhIP-Seq technology. Fourth, we noted similar antibody reactivity patterns in CVB3 and CVB4 infections, suggesting that the COA4- and PIK3AP1-reactive antibodies could be common to multiple CVB infections. The specificity of the autoantibodies was affirmed with influenza-infected animals that showed no reactivity to any of the antigens tested. Taken together, our data suggest that the autoantibodies identified by PhIP-Seq may have relevance to CVB pathogenesis, with a possibility that similar reactivity could be expected in human DCM patients.

The knowns and unknowns of cardiac autoimmunity in viral myocarditis

“…We encountered such an issue in our recent investigations with the PhIP‐Seq analyses in the CVB3 infection model. Using the pathogenic Nancy strain of CVB3 that induces severe myocarditis and pancreatitis, and the E2 strain of CVB4 that primarily causes pancreatitis/insulitis, we noted similar antibody reactivity for select antigens in sera obtained from both infected groups 110 . Additionally, tissue destruction resulting from systemic infection, as might occur in the hyperinflammatory syndrome associated with SARS CoV‐2 infection, may lead to the development of autoantibodies to ubiquitously expressed antigens 233 …”

“…In that direction, we made efforts to use Phage ImmunoPrecipitation Sequencing (PhIP‐Seq) to comprehensively analyse the autoantibody repertoire in the CVB3 myocarditis model in A/J mice, leading us to detect antibodies to 26 proteins that were not previously reported (Table 1). 110 Furthermore, antibody reactivity patterns were similar in both CVB3 and CVB4 infected groups, but not in Influenza virus infection, indicating that multiple CVB infections can lead to the formation of similar autoantibodies. Ironically, however, the PhIP‐Seq analysis did not consistently reveal antibodies for some of the commonly reported antigens, such as Myhc, and Troponins, previously reported by conventional methods in various virus infections (Table 1).…”

“…Using the pathogenic Nancy strain of CVB3 that induces severe myocarditis and pancreatitis, and the E2 strain of CVB4 that primarily causes pancreatitis/insulitis, we noted similar antibody reactivity for select antigens in sera obtained from both infected groups. 110 Additionally, tissue destruction resulting from systemic infection, as might occur in the hyperinflammatory syndrome associated with SARS CoV-2 infection, may lead to the development of autoantibodies to ubiquitously expressed antigens. 233 Third, unlike liver, which has a remarkable capacity to regenerate, heart tissue, especially in adults, lacks regeneration capacity due to limited renewal of cardiomyocytes after injury, although reports suggest that cardiomyocytes can acquire regenerative potential by reentering the cell cycle.…”

Validation of a murine proteome-wide phage display library for the identification of autoantibody specificities

“…Here, we describe the construction and validation of a proteome wide murine PhIP-seq library, composed of greater than 480,000 peptides, each 62 amino acids in length, spanning over 76,000 protein sequences. While similar in design, this library includes approximately 27,000 additional protein sequences (including variants) than a previously published library (45). For any newly designed PhIP-seq library, it is essential to quantify the performance characteristics.…”

Validation of a murine proteome-wide phage display library for identification of autoantibody specificities

“…The design of short peptides of equal length across the proteome was chosen to capture antibody-epitope interactions and our bioinformatics analyses, performed on the peptide level, considers each peptide independently. While similar in design, this library includes approximately 27,000 additional protein sequences (including variants) compared with a previously published library ( 47 ). For any newly designed PhIP-seq library, it is essential to quantify the performance characteristics.…”