Publications

Mt10-CVB3 Vaccine Virus Protects against CVB4 Infection by Inducing Cross-Reactive, Antigen-Specific Immune Responses

Published in Microorganisms, 2021

Abstract: Group B coxsackieviruses (CVB) containing six serotypes, B1–B6, affect various organs, and multiple serotypes can induce similar diseases such as myocarditis and pancreatitis. Yet, no vaccines are currently available to prevent these infections. Translationally, the derivation of vaccines that offer protection against multiple serotypes is highly desired. In that direction, we recently reported the generation of an attenuated strain of CVB3, termed Mt10, which completely protects against both myocarditis and pancreatitis induced by the homologous wild-type CVB3 strain. Here, we report that the Mt10 vaccine can induce cross-protection against multiple CVB serotypes as demonstrated with CVB4. We note that the Mt10 vaccine could induce cross-reactive neutralizing antibodies (nABs) against both CVB1 and CVB4. In challenge studies with CVB4, the efficacy of the Mt10 vaccine was found to be 92%, as determined by histological evaluation of the heart and pancreas. Antibody responses induced in Mt10/CVB4 challenged animals indicated the persistence of cross-reactive nABs against CVB1, CVB3, and CVB4. Evaluation of antigen-specific immune responses revealed viral protein 1 (VP1)-reactive antibodies, predominantly IgG2a, IgG2b, IgG3, and IgG1. Similarly, by using major histocompatibility complex class II tetramers, we noted induction of VP1-specific CD4 T cells capable of producing multiple T cell cytokines, with interferon-γ being predominant. Finally, none of the vaccine recipients challenged with CVB4 revealed the presence of viral nucleic acid in the heart or pancreas. Taken together, our data suggest that the Mt10 vaccine can prevent infections caused by multiple CVB serotypes, paving the way for the development of monovalent CVB vaccines to prevent heart and pancreatic diseases of enteroviral origin.

Recommended citation: Lasrado, N., Arumugam, R., Rasquinha, M., Sur, M., Steffen, D., Reddy, J., 2021. Mt10-CVB3 Vaccine Virus Protects against CVB4 Infection by Inducing Cross-Reactive, Antigen-Specific Immune Responses. Microorganisms, 2021; 9(11):2323.

IL-10 as a Th2 Cytokine: Differences Between Mice and Humans

Published in The Journal of Immunology, 2021

Abstract: ‘The discovery of IL-10 more than 30 years ago marked the beginning of our understanding of how cytokines regulate immune responses, based on cross-regulation between Th1 and Th2 cytokines. Although multiple cell types were shown to produce IL-10, its identity as a Th2 cytokine remained strong because it was rigidly associated with Th2 clones in mice, whereas both Th1 and Th2 clones could secrete IL-10 in humans. However, as new Th1/Th2 cell functionalities emerged, anti-inflammatory action of IL-10 gained more attention than its inhibitory effect on Th1 cells, which may occur as an indirect consequence of suppression of APCs. This notion is also supported by the discovery of regulatory T cells, whose suppressor functions involve the mediation of IL-10, among other molecules. From this perspective, we discuss the functionalities of IL-10 by highlighting important differences between mice and humans with an emphasis on the Th1 and Th2 paradigm.’

Recommended citation: Rasquinha, M.* , Sur, M.*, Lasrado, N.*, Reddy, J., 2021. IL-10 as a Th2 Cytokine: Differences Between Mice and Humans. The Journal of Immunology, 2021, 207 (9) 2205-2215. * equal first authors

Attenuated strain of CVB3 with a mutation in the CAR-interacting region protects against both myocarditis and pancreatitis

Published in Scientific Reports, 2021

Abstract: ‘Coxsackievirus B3 (CVB3), is commonly implicated in myocarditis, which can lead to dilated cardiomyopathy, in addition to causing acute pancreatitis and meningitis. Yet, no vaccines are currently available to prevent this infection. Here, we describe the derivation of a live attenuated vaccine virus, termed mutant (Mt) 10, encoding a single amino acid substitution H790A within the viral protein 1, that prevents CVB3 infection in mice and protects from both myocarditis and pancreatitis in challenge studies. We noted that animals vaccinated with Mt 10 developed virus-neutralizing antibodies, predominantly containing IgG2a and IgG2b, and to a lesser extent IgG3 and IgG1. Furthermore, by using major histocompatibility complex class II dextramers and tetramers, we demonstrated that Mt 10 induces antigen-specific T cell responses that preferentially produce interferon-γ. Finally, neither vaccine recipients nor those challenged with the wild-type virus revealed evidence of autoimmunity or cardiac injury as determined by T cell response to cardiac myosin and measurement of circulating cardiac troponin I levels, respectively. Together, our data suggest that Mt 10 is a vaccine candidate that prevents CVB3 infection through the induction of neutralizing antibodies and antigen-specific T cell responses, the two critical components needed for complete protection against virus infections in vaccine studies.’

Recommended citation: Lasrado, N., Gangaplara, A., Massilamany, C., Arumugam, R., Shelbourn, A., Rasquinha, M., Basavalingappa, R., Delhon, G., Xiang, S-H., Pattnaik, A., Steffen, D., Reddy, J., 2021. Attenuated strain of CVB3 with a mutation in the CAR-interacting region protects against both myocarditis and pancreatitis. Sci Rep, 11, 12432 (2021).

Dissecting the Cellular Landscape and Transcriptome Network in Viral Myocarditis by Single-Cell RNA Sequencing

Published in bioRxiv, 2021

Abstract: Myocarditis induced with Coxsackievirus B3 (CVB3) is commonly employed to study viral pathogenesis in mice. Although infectious virus is cleared after the acute phase, affected animals chronically develop the features of dilated cardiomyopathy, which may involve the mediation of immune and non-immune cells. To dissect this complexity, we performed single-cell RNA sequencing on heart cells obtained from healthy and myocarditic mice, leading us to note that myocarditic mice had significantly higher proportions of myeloid cells, CD4 and CD8 T cells, and fibroblasts, whereas NK cells, ILCs and B cells were low. While the transcriptome profiles of myeloid cells revealed detection of monocytes and macrophages of M2 phenotype with pathways important in immune metabolism and inflammation, T cells consisted of Th17 cells, CTLs, and Treg cells with transcriptome signatures critical for cytotoxic functions. Although fibroblasts detected in myocarditic mice were phenotypically heterogeneous, their transcriptomes played roles in fibrosis and regulation of inflammation and immune responses. Additionally, analysis of intercellular communication networks revealed unique interactions and signaling pathways in the cardiac cellulome, whereas myeloid cells and T cells in myocarditic mice revealed uniquely upregulated transcription factors modulating cardiac remodeling functions. Taken together, our data suggest that M2 cells, T cells, and fibroblasts may cooperatively or independently participate in the pathogenesis of viral myocarditis.

Recommended citation: Lasrado, N., Borcherding, N., Arumugam, R., Starr, T.K., Reddy, J., 2021. Dissecting the Cellular Landscape and Transcriptome Network in Viral Myocarditis by Single-Cell RNA Sequencing

Mechanisms of sex hormones in autoimmunity: focus on EAE

Published in Biology of Sex Differences, 2020

Abstract: Sex-related differences in the occurrence of autoimmune diseases is well documented, with females showing a greater propensity to develop these diseases than their male counterparts. Sex hormones namely, dihydrotestosterone and estrogens have been shown to ameliorate the severity of inflammatory diseases. Immunologically, the beneficial effects of sex hormones have been ascribed to the suppression of effector lymphocyte responses accompanied by immune deviation from pro-inflammatory to anti-inflammatory cytokine production. In this review, we present our view of the mechanisms of sex hormones that contribute to their ability to suppress autoimmune responses with an emphasis on the pathogenesis of experimental autoimmune encephalomyelitis.

Recommended citation: Lasrado, N., Jia, T., Massilamany, C., Franco, R., Illes, Z., Reddy, J., 2020. Mechanisms of sex hormones in autoimmunity: focus on EAE. Biology of Sex Differences

Evidence for anti-viral effects of complete Freund’s adjuvant in the mouse model of enterovirus infection

Published in Vaccines, 2020

Abstract: Group B Coxsackieviruses belonging to the genus, Enterovirus, contain six serotypes that induce various diseases, whose occurrence may involve the mediation of more than one serotype. We recently identified immunogenic epitopes within CVB3 viral protein 1 that induce anti-viral T cell responses in mouse models of CVB infections. In our investigations to determine the protective responses of the viral epitopes, we unexpectedly noted that animals immunized with complete Freund’s adjuvant (CFA) alone and later challenged with CVB3 were completely protected against myocarditis. Similarly, the pancreatitis-inducing ability of CVB3 was remarkably reduced to only 10% in the CFA group as opposed to 73.3% in the control group that received no CFA. Additionally, no mortalities were noted in the CFA group, whereas 40% of control animals died during the course of 21 days post-infection with CVB3. Taken together, our data suggest that the adjuvant effects of CFA may be sufficient for protection against CVB infections. These observations may provide new insights into our understanding of the occurrence of viral infections. One example is Coronavirus disease-19 (COVID-19) as individuals suffering from COVID-19 who have been vaccinated with Bacillus Calmette–Guérin appear to have fewer morbidities and mortalities than unvaccinated individuals.’

Recommended citation: Gangaplara, A., Massilamany, C., Lasrado, N. , Steffen, D., Reddy, J., 2020. Evidence for anti-viral effects of complete Freund’s adjuvant in the mouse model of enterovirus infection. Vaccines, 2020; 8(3):364.

Viral myocarditis involves the generation of autoreactive T cells with multiple antigen specificities that localize in lymphoid and non-lymphoid organs in the mouse model of CVB3 infection

Published in Molecular Immunology, 2020

Abstract: Autoreactive T cells may contribute to post-viral myocarditis induced with Coxsackievirus B3 (CVB3), but the underlying mechanisms of their generation are unclear. Here, we have comprehensively analyzed the generation of antigen-specific, autoreactive T cells in the mouse model of CVB3 infection for antigens implicated in patients with myocarditis/dilated cardiomyopathy. First, comparative analysis of CVB3 proteome with five autoantigens led us to identify three mimicry epitopes, one each from adenine nucleotide translocator 1 (ANT), sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) and cardiac troponin I. None of these induced cross-reactive T cell responses. Next, we generated major histocompatibility complex (MHC) class II dextramers to enumerate the frequencies of antigen-specific T cells to determine whether T cells with multiple antigen specificities are generated by CVB3 infection. These analyses revealed appearance of CD4 T cells positive for SERCA2a 971-990, and cardiac myosin heavy chain-α (Myhc) 334-352 dextramers, both in the periphery and also in the hearts of CVB3-infected animals. While ANT 21-40 dextramer+ T cells were inconsistently detected, the β1-adrenergic receptor 181-200/211-230 or branched chain α-ketoacid dehydrogenase kinase 111-130 dextramer+ cells were absent. Interestingly, SERCA2a 971-990, Myhc 334-352 and ANT 21-40 dextramer+ cells were also detected in the liver indicating that they may have a pathogenic role. Finally, we demonstrate that the SERCA2a 971-990-reactive T cells generated in CVB3 infection could transfer disease to naïve mice. The data suggest that CVB3 infection can lead to the generation of autoreactive T cells for multiple antigens indicating a possibility that the autoreactive T cells localized in the liver can potentially circulate and contribute to the development of viral myocarditis.

Recommended citation: Basavalingappa R*, Arumugam R*, Lasrado N* , Yalaka B, Massilamany C, Gangaplara A, Xiang S, Steffen D and Reddy J. Viral myocarditis involves the generation of autoreactive T cells with multiple antigen specificities that compartmentalize in lymphoid and non-lymphoid organs in the mouse model of CVB3 infection, Molecular Immunology (2020). *co-first authors

An overview of the immune mechanisms of viral myocarditis

Published in Reviews in Medical Virology, 2020

Abstract: Viral myocarditis has been identified as a major cause of dilated cardiomyopathy (DCM) that can lead to heart failure. Historically, Coxsackieviruses and adenoviruses have been commonly suspected in myocarditis/DCM patients in North America and Europe. However, this notion is changing as other viruses such as Parvovirus B19 and human herpesvirus-6 are increasingly reported as causes of myocarditis in the United States, with the most recent example being the severe acute respiratory syndrome coronavirus 2, causing the Coronavirus Disease-19. The mouse model of Coxsackievirus B3 (CVB3)-induced myocarditis, which may involve mediation of autoimmunity, is routinely used in the study of immune pathogenesis of viral infections as triggers of DCM. In this review, we discuss the immune mechanisms underlying the development of viral myocarditis with an emphasis on autoimmunity in the development of post-infectious myocarditis induced with CVB3.

Recommended citation: Lasrado N, Reddy J. An overview of the immune mechanisms of viral myocarditis. Rev Med Virol. 2020;e2131. https://doi.org/10.1002/rmv.2131

Triggers of Inflammatory Heart Disease

Published in Frontiers in Cell and Developmental Biology, 2020

Abstract: Inflammatory heart disease (IHD) is a group of diseases that includes pericarditis, myocarditis, and endocarditis. Although males appear to be more commonly affected than females, IHD can be seen in any age group. While the disease can be self-limiting leading to full recovery, affected individuals can develop chronic disease, suggesting that identification of primary triggers is critical for successful therapies. Adding to this complexity, however, is the fact that IHD can be triggered by a variety of infectious and non-infectious causes that can also occur as secondary events to primary insults. In this review, we discuss the immunological insights into the development of IHD as well as a mechanistic understanding of the disease process in animal models.

Recommended citation: Lasrado N, Yalaka B and Reddy J (2020) Triggers of Inflammatory Heart Disease. Front. Cell Dev. Biol. 8:192. https://doi.org/10.3389/fcell.2020.00192.

Identification of immunogenic epitopes that permit the detection of antigen-specific T cell responses in multiple serotypes of group B coxsackievirus infections

Published in Viruses, 2020

Abstract: Coxsackievirus group B (CVB) contains six serotypes that can affect various organs. Some of these organ-specific diseases such as myocarditis and pancreatitis can be caused by more than one serotype. Thus, development of immunological tools common to multiple serotypes is desired. This is especially critical for analyzing antigen-specific T cell responses at a single cell level. To this end, we made efforts to identify the immunogenic epitopes of CVB3 leading us to localize three T cell epitopes within the viral protein 1 (VP1) namely, VP1 681–700, VP1 721–740 and VP1 771–790. First, we confirmed their immunogenicity in the immunization settings. Second, we sought to verify the ability of VP1 epitopes to bind major histocompatibility complex (MHC) class II (IAk) molecules. Third, we created MHC class II (IAk) dextramers and tetramers and ascertained the T cell responses to be antigen-specific. Fourth, we analyzed the T cell responses in animals infected with CVB3 and noted the magnitude of antigen-specific T cell responses occurring in the order of VP1 721–740 and VP1 681–700 followed by VP1 771–790 as verified by proliferation assay and IAk tetramer staining. All epitopes induced interferon (IFN)-γ as a major cytokine. Finally, we investigated whether the VP1 tools generated for CVB3 can also be used to verify T cell responses in infections caused by other serotypes. To this end, we established the CVB4 infection model in A/J mice and found that the CVB4 infection led to the induction of IFN-γ-producing T cell responses primarily for VP1 721–740 and VP1 681–700. Thus, the VP1-specific tools, particularly IAk tetramers can be used to monitor anti-viral T cell responses in multiple CVB serotypes.

Recommended citation: Lasrado, N., Gangaplara, A., Arumugam, R., Massilamany, C., Pokal, S., Zhou, Y., Xiang, S.-H., Steffen, D., Reddy, J. Identification of Immunogenic Epitopes That Permit the Detection of Antigen-Specific T Cell Responses in Multiple Serotypes of Group B Coxsackievirus Infections. Viruses 2020, 12, 347.

An evidence for surface expression of an immunogenic epitope of sarcoplasmic/endoplasmic reticulum calcium-ATPase2a on antigen-presenting cells from naive mice in the mediation of autoimmune myocarditis

Published in Immunobiology, 2019

Abstract: We recently reported identification of sarcoplasmic/endoplasmic reticulum calcium-ATPase2a (SERCA2a) 971-990, which induces atrial myocarditis by generating autoreactive T cells in A/J mice. However, it was unknown how antigen-sensitized T cells could recognize SERCA2a 971-990, since SERCA2a-expression is confined to an intracellular compartment. In this report, we present evidence that antigen-presenting cells (APCs) from lymphoid and non-lymphoid organs in naïve animals present SERCA2a 971-990 and stimulate antigen-specific T cells. Using major histocompatibility complex (MHC) class II dextramers for SERCA2a 971-990, we created a panel of T cell hybridomas and demonstrated that splenocytes from naïve A/J mice stimulated the hybridoma cells without exogenous supplementation of SERCA2a 971-990. We then recapitulated this phenomenon by using SERCA2a 971-990 -specific primary T cells, verifying that the T cell responses were MHC-restricted. Furthermore, SERCA2a 971-990 -sensitzed T cells exposed to APCs from naïve mice were found to produce the inflammatory cytokines interferon-γ, granulocyte macrophage colony stimulating factor, and interleukin-17A, which are implicated in the induction of myocarditis. Finally, while T cells exposed to mononuclear cells (MNCs) obtained from heart and liver also responded similarly to splenocytes, endothelial cells (ECs) generated from the corresponding organs displayed opposing effects, in that the proliferative responses were suppressed with the heart ECs, but not with the liver ECs. Taken together, our data suggest that the surface expression of SERCA2a 971-990 by naïve APCs can potentially trigger pathogenic autoreactive T cell responses under conditions of autoimmunity, which may have implications in endothelial dysfunction.

Recommended citation: Arumugam, R., Yalaka, B., Massilamany, C., Haider Ali, M., Lasrado, N., Jayaraja, S., Riethoven, J. J., Sun, X., & Reddy, J. (2020). An evidence for surface expression of an immunogenic epitope of sarcoplasmic/endoplasmic reticulum calcium-ATPase2a on antigen-presenting cells from naive mice in the mediation of autoimmune myocarditis. Immunobiology, 225(2), 151896.

Ninaad Lasrado

Publications