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

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.

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

“…LLC-MK2 cells or Vero cells (American type cell culture [ATCC], Manassas, VA, USA) were grown to 80-90% confluency. LLC-MK2 cells were infected with CVB1-conn5 (henceforth called CVB1), and Vero cells with wt CVB3, CVB4-E2 (henceforth called CVB4), and Mt10 as described previously [17,20]. In brief, Mt 10 vaccine virus was derived using a CVB3-infectious clone, pBRCVB3, developed previously in our laboratory [17].…”

“…LLC-MK2 cells were infected with CVB1-conn5 (henceforth called CVB1), and Vero cells with wt CVB3, CVB4-E2 (henceforth called CVB4), and Mt10 as described previously [17,20]. In brief, Mt 10 vaccine virus was derived using a CVB3-infectious clone, pBRCVB3, developed previously in our laboratory [17]. The in vitro transcribed RNA from the infectious clone was used to propagate the virus in Vero cells in EMEM containing 10% fetal bovine serum (FBS).…”

“…For challenge studies, mice were randomly assigned and administered with the vaccine virus (0.5 × 10 6 TCID 50 /mouse diluted in 200 µL of 1x PBS, i.p., n = 26) or 1x PBS for control mice (200 µL, i.p., n = 10) on day 0. After 14 days, animals were injected with saline or CVB4 [2000 TCID 50 /mouse diluted in 200 µL of 1x PBS] i.p., and body weights were recorded every two days [17]. Hearts and pancreata were collected from animals that died naturally.…”

“…We recently reported the creation of an attenuated strain of CVB3, termed Mt10, that offered complete protection against both myocarditis and pancreatitis in A/J mice, which are highly susceptible to both diseases [17,18]. Essentially, the Mt10 vaccine, hereafter termed vaccine virus, was generated by introducing an H790A mutation within the viral protein 1 (VP1) region of the CVB3 viral canyon, where the coxsackievirus-adenovirus receptor (CAR) is expected to interact with the virus [17,19]. Based on this success, we sought to determine whether the vaccine virus can also induce cross-reactive immune responses and can protect against infection caused by other serotypes of CVB.…”

Application Route and Immune Status of the Host Determine Safety and Oncolytic Activity of Oncolytic Coxsackievirus B3 Variant PD-H

“…The CVB3 serotype includes many genetic variants. These were either isolated from infected patients or represent laboratory strains which were generated by genetic engineering or emerged through in vitro or in vivo adaptation [12][13][14][15]. All CVB3 variants (Nancy, 31-1-93, H3, 2035A and PD) analyzed so far for oncolytic activity, showed potent anticancer efficiency in vivo, as shown in xenografted models of lung, colorectal, breast and endometrial cancer [3][4][5]16] and in a syngenic models of lung cancer [3].…”