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.

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.

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.

Freund’s adjuvants have been used in vaccine and autoimmune settings, and their effects can be overlapping or unique to each. While both incomplete Freund’s adjuvants (IFA) and complete Freund’s adjuvants (CFA) influence antibody and T cell responses, the robust T helper 1 cytokines induced by the mycobacterial components make CFA the powerful immunostimulating adjuvant. In these studies, the adjuvant effects are investigated in a select population of cells, and the changes, if any, with the metabolic alterations in the systemic compartment are unclear. We investigated whether the effects of IFA and CFA can be influenced by the metabolic shifts in mice immunized with saline, IFA, or CFA using Mycobacterium tuberculosis var. bovis Bacillus Calmette–Guérin (BCG) as a positive control. After seven days of immunization, we analyzed the serum metabolite profiles using liquid chromatography coupled with high-resolution mass spectrometry and multivariate statistical analysis to identify metabolic features between the groups. The data revealed that, in the scores space, the CFA and BCG groups were more closely aligned compared to the saline group, while the IFA group displayed an intermediate profile. Furthermore, comparisons between the CFA and BCG groups showed more significant perturbations in lipid and amino acid metabolism, particularly involving glycerophospholipids, cysteine, and aromatic amino acids. In contrast, comparisons between the BCG and IFA groups indicated a more pronounced disruption in central energy metabolism pathways, such as the citric acid cycle and pyruvate metabolism. Together, the data suggest that the serum metabolite profiles in response to IFA and CFA might play a role in modulating the immune responses.

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 IA^b; 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.

Background/Objectives: Freund’s adjuvants induce different immunomodulatory effects, but their underlying molecular mechanisms are unclear. In this study, we investigated whether the immune-stimulating effects of the complete Freund’s adjuvant (CFA) involve the mechanisms of trained immunity (TI). Methods: We examined bone marrow cells (BMCs) isolated from CFA-immunized A/J mice to address this question. Incomplete Freund’s adjuvant (IFA) and Mycobacterium tuberculosis var. bovis Bacillus Calmette-Guérin (BCG) served as negative and positive controls, respectively. We evaluated cytokine profiles, metabolic, and epigenetic changes. Results: First, BMCs from all groups except saline showed varied levels of IL-1β, IL-6, and TNF-α. But expression of CCL5 and CXCL10 was significantly elevated only in the CFA and BCG groups. Transcriptionally, significant elevations were noted for TNF-α and IL-1β in the CFA and BCG groups, whereas CXCL10, IL-6, and IL-10 were upregulated in the CFA and BCG groups, respectively. Second, while BMCs from the BCG group expressed the markers of both the M1 and M2 macrophages, no clear trends were noted in the CFA and IFA groups. Third, cell lysates from the CFA group revealed metabolic reprogramming in the BMCs. Specifically, we observed an increased level of lactate, indicative of aerobic glycolysis, which is implicated in TI, and this was also detected in the IFA group. Fourth, epigenetic analysis revealed histone enrichment in the promoter region of TNF-α, in the CFA group, but to a lesser degree than the BCG group. However, no epigenetic changes were observed in the IFA group. Conclusions: Our data provide new insights into the mechanisms of Freund’s adjuvants and the immunomodulatory effects of CFA could involve the features of TI.