Mt10 Vaccine Protects Diversity Outbred Mice from CVB3 Infection by Producing Virus-Specific Neutralizing Antibodies and Diverse Antibody Isotypes.

Group B coxsackieviruses (CVBs) cause a wide range of diseases in humans, but no vaccines are currently available to prevent these infections. Previously, we had demonstrated that a live attenuated CVB3 vaccine virus, Mutant 10 (Mt10), offers protection against multiple CVB serotypes as evaluated in various inbred mouse strains; however, the applicability of these findings to the outbred human population remains uncertain. To address this issue, we used Diversity Outbred (DO) mice, whose genome is derived from eight inbred mouse strains that may capture the level of genetic diversity of the outbred human population. To determine the efficacy of the Mt10 vaccine, we established the CVB3 infection model in the DO mice. We noted that CVB3 infection resulted mainly in pancreatitis, although viral RNA was detected in both the pancreas and heart. Histologically, the pancreatic lesions comprised of necrosis, post-necrotic atrophy, and lymphocyte infiltration. In evaluating the efficacy of the Mt10 vaccine, both male and female DO mice were completely protected in challenge studies with CVB3, and viral RNA was not detected in the heart or pancreas. Likewise, vaccine recipients of both sexes showed significant levels of virus-neutralizing antibodies. Furthermore, by using the CVB3 viral protein 1, virus-reactive antibodies were found to be diverse in the order of IgG2c, followed by IgG2a, IgG2b/IgG3, and IgG1. Together, the data suggest that the Mt10 vaccine virus can offer protection against CVB infections that may have translational significance.

Investigation into Cardiac Myhc-α 334-352-Specific TCR Transgenic Mice Reveals a Role for Cytotoxic CD4 T Cells in the Development of Cardiac Autoimmunity.

Myocarditis is one of the major causes of heart failure in children and young adults and can lead to dilated cardiomyopathy. Lymphocytic myocarditis could result from autoreactive CD4+ and CD8+ T cells, but defining antigen specificity in disease pathogenesis is challenging. To address this issue, we generated T cell receptor (TCR) transgenic (Tg) C57BL/6J mice specific to cardiac myosin heavy chain (Myhc)-α 334-352 and found that Myhc-α-specific TCRs were expressed in both CD4+ and CD8+ T cells. To investigate if the phenotype is more pronounced in a myocarditis-susceptible genetic background, we backcrossed with A/J mice. At the fourth generation of backcrossing, we observed that Tg T cells from naïve mice responded to Myhc-α 334-352, as evaluated by proliferation assay and carboxyfluorescein succinimidyl ester staining. The T cell responses included significant production of mainly pro-inflammatory cytokines, namely interferon (IFN)-γ, interleukin-17, and granulocyte macrophage-colony stimulating factor. While the naïve Tg mice had isolated myocardial lesions, immunization with Myhc-α 334-352 led to mild myocarditis, suggesting that further backcrossing to increase the percentage of A/J genome close to 99.99% might show a more severe disease phenotype. Further investigations led us to note that CD4+ T cells displayed the phenotype of cytotoxic T cells (CTLs) akin to those of conventional CD8+ CTLs, as determined by the expression of CD107a, IFN-γ, granzyme B natural killer cell receptor (NKG)2A, NKG2D, cytotoxic and regulatory T cell molecules, and eomesodermin. Taken together, the transgenic system described in this report may be a helpful tool to distinguish the roles of cytotoxic cardiac antigen-specific CD4+ T cells vs. those of CD8+ T cells in the pathogenesis of myocarditis.

Epidemic profile of common respiratory viruses in association SARS CoV-2 among SARI and ARI-two year study.

BACKGROUND: COVID-19/SARS CoV-2 continue to pose a threat to human health and placed millions of livelihoods at risk. Surveillance for the other circulating seasonal viruses during this pandemic is necessary to understand the manifestations of the CoV-2 pandemic and their incidence. METHODS: A detailed study survey was performed on subjects with acute respiratory infections (ARI) and severe acute respiratory infection (SARI) in the King Institute of Preventive Medicine and Research, Chennai from April 2020 to March 2022. A total of 1480 patients presenting with either SARI (41.8%) or ARI (58.1%) were screened for SARS CoV-2 and other respiratory viruses. The SARS CoV-2 real-time PCR was carried out using ICMR-approved kits and other respiratory viruses were detected using the commercially available real-time kit. RESULTS: Out of the 620 SARI patients, 198 (31.9%) were positive for SARS CoV-2 RNA. Out of the 860 ARI patients, 352 (40.9%) were positive for SARS CoV-2 RNA. Among the 550 patients positive for SARS CoV-2, 7 (1.2%) were positive coexistent with other respiratory viruses. Among the 930 patients with negative SARS CoV-2, 222 (23.8%) were positive for other common respiratory viruses (p = 0.001). Influenza viruses (36.9%) predominated followed by RSV (31.9%) and Parainfluenza virus (13.5%). CONCLUSION: This study suggests that viral coinfections are significantly higher among SARS CoV-2 negative individuals (23.8 vs. 1.2%). It is possibly due to viral interference and the competitive advantage of SARS CoV-2 in modulating the host immunity. Continuous surveillance is necessary for understanding the viral co-infection mechanisms.

Transgenic Mice Expressing Functional TCRs Specific to Cardiac Myhc-α 334-352 on Both CD4 and CD8 T Cells Are Resistant to the Development of Myocarditis on C57BL/6 Genetic Background.

Myocarditis is a predominant cause of congestive heart failure and sudden death in children and young adolescents that can lead to dilated cardiomyopathy. Lymphocytic myocarditis mediated by T cells can result from the recognition of cardiac antigens that may involve CD4 or CD8 T cells or both. In this report, we describe the generation of T cell receptor (TCR) transgenic mice on a C57BL/6 genetic background specific to cardiac myosin heavy chain (Myhc)-α 334-352 and make the following observations: First, we verified that Myhc-α 334-352 was immunogenic in wild-type C57BL/6 mice and induced antigen-specific CD4 T cell responses despite being a poor binder of IAb; however, the immunized animals developed only mild myocarditis. Second, TCRs specific to Myhc-α 334-352 in transgenic mice were expressed in both CD4 and CD8 T cells, suggesting that the expression of epitope-specific TCR is common to both cell types. Third, although T cells from naïve transgenic mice did not respond to Myhc-α 334-352, both CD4 and CD8 T cells from animals immunized with Myhc-α 334-352 responded to the peptide, indicating that antigen priming is necessary to break tolerance. Fourth, although the transgenic T cells could produce significant amounts of interferon-γ and interleukin-17, the immunized animals developed only mild disease, indicating that other soluble factors might be necessary for developing severe myocarditis. Alternatively, the C57BL/6 genetic background might be a major contributing factor for resistance to the development of myocarditis. Taken together, our model permits the determination of the roles of both CD4 and CD8 T cells to understand the disease-resistance mechanisms of myocarditis in a single transgenic system antigen-specifically.

The knowns and unknowns of cardiac autoimmunity in viral myocarditis.

Myocarditis can result from various infectious and non-infectious causes that can lead to dilated cardiomyopathy (DCM) and heart failure. Among the infectious causes, viruses are commonly suspected. But the challenge is our inability to demonstrate infectious viral particles during clinical presentations, partly because by that point, the viruses would have damaged the tissues and be cleared by the immune system. Therefore, viral signatures such as viral nucleic acids and virus-reactive antibodies may be the only readouts pointing to viruses as potential primary triggers of DCM. Thus, it becomes hard to explain persistent inflammatory infiltrates that might occur in individuals affected with chronic myocarditis/DCM manifesting myocardial dysfunctions. In these circumstances, autoimmunity is suspected, and antibodies to various autoantigens have been demonstrated, suggesting that immune therapies to suppress the autoimmune responses may be necessary. From this perspective, we endeavoured to determine whether or not the known viral causes are associated with development of autoimmune responses to cardiac antigens that include both cardiotropic and non-cardiotropic viruses. If so, what their nature and significance are in developing chronic myocarditis resulting from viruses as primary triggers.

Vaccines against Group B Coxsackieviruses and Their Importance.

The group B coxsackieviruses (CVBs) exist in six serotypes (CVB1 to CVB6). Disease associations have been reported for most serotypes, and multiple serotypes can cause similar diseases. For example, CVB1, CVB3, and CVB5 are generally implicated in the causation of myocarditis, whereas CVB1 and CVB4 could accelerate the development of type 1 diabetes (T1D). Yet, no vaccines against these viruses are currently available. In this review, we have analyzed the attributes of experimentally tested vaccines and discussed their merits and demerits or limitations, as well as their impact in preventing infections, most importantly myocarditis and T1D.

A Monovalent Mt10-CVB3 Vaccine Prevents CVB4-Accelerated Type 1 Diabetes in NOD Mice.

Enteroviruses, which include Coxsackieviruses, are a common cause of virus infections in humans, and multiple serotypes of the group B Coxsackievirus (CVB) can induce similar diseases. No vaccines are currently available to prevent CVB infections because developing serotype-specific vaccines is not practical. Thus, developing a vaccine that induces protective immune responses for multiple serotypes is desired. In that direction, we created a live-attenuated CVB3 vaccine virus, designated mutant (Mt)10, that offers protection against myocarditis and pancreatitis induced by CVB3 and CVB4 in disease-susceptible A/J mice. Here, we report that the Mt10 vaccine protected against CVB4-triggered type 1 diabetes (T1D) in non-obese diabetic (NOD) mice but the expected subsequent development of spontaneous T1D in these genetically predisposed NOD mice was not altered. We noted that Mt10 vaccine induced significant amounts of neutralizing antibodies, predominantly of the IgG2c isotype, and the virus was not detected in vaccine-challenged animals. Furthermore, monitoring blood glucose levels-and to a lesser extent, insulin antibodies-was found to be helpful in predicting vaccine responses. Taken together, our data suggest that the monovalent Mt10 vaccine has the potential to prevent infections caused by multiple CVB serotypes, as we have demonstrated in various pre-clinical models.

Phylodynamic Pattern of Genetic Clusters, Paradigm Shift on Spatio-Temporal Distribution of Clades, and Impact of Spike Glycoprotein Mutations of SARS-CoV-2 Isolates from India.

INTRODUCTION: The COVID-19 pandemic is associated with high morbidity and mortality, with the emergence of numerous variants. The dynamics of SARS-CoV-2 with respect to clade distribution is uneven, unpredictable and fast changing. METHODS: Retrieving the complete genomes of SARS-CoV-2 from India and subjecting them to analysis on phylogenetic clade diversity, Spike (S) protein mutations and their functional consequences such as immune escape features and impact on infectivity. Whole genome of SARS-CoV-2 isolates (n = 4,326) deposited from India during the period from January 2020 to December 2020 is retrieved from Global Initiative on Sharing All Influenza Data (GISAID) and various analyses performed using in silico tools. RESULTS: Notable clade dynamicity is observed indicating the emergence of diverse SARS-CoV-2 variants across the country. GR clade is predominant over the other clades and the distribution pattern of clades is uneven. D614G is the commonest and predominant mutation found among the S-protein followed by L54F. Mutation score prediction analyses reveal that there are several mutations in S-protein including the RBD and NTD regions that can influence the virulence of virus. Besides, mutations having immune escape features as well as impacting the immunogenicity and virulence through changes in the glycosylation patterns are identified. CONCLUSIONS: The study has revealed emergence of variants with shifting of clade dynamics within a year in India. It is shown uneven distribution of clades across the nation requiring timely deposition of SARS-CoV-2 sequences. Functional evaluation of mutations in S-protein reveals their significance in virulence, immune escape features and disease severity besides impacting therapeutics and prophylaxis.