Using Recombinant Coxsackievirus B3 To Evaluate the Induction and Protective Efficacy of CD8⁺T Cells during Picornavirus Infection

Abstract

Coxsackievirus B3 (CVB3) is a common human pathogen that has been associated with serious diseases including myocarditis and pancreatitis. To better understand the effect of cytotoxic T-lymphocyte (CTL) responses in controlling CVB3 infection, we have inserted well-characterized CTL epitopes into the CVB3 genome. Constructs were made by placing the epitope of interest upstream of the open reading frame encoding the CVB3 polyprotein, separated by a poly-glycine linker and an artificial 3C pro /3CD pro cleavage site. This strategy results in the foreign protein being translated at the amino-terminus of the viral polyprotein, from which it is cleaved prior to viral assembly. In this study, we cloned major histocompatibility complex class I-restricted CTL epitopes from lymphocytic choriomeningitis virus (LCMV) into recombinant CVB3 (rCVB3). In vitro, rCVB3 growth kinetics showed a 1-to 2-h lag period before exponential growth was initiated, and peak titers were ϳ1 log unit lower than for wild-type virus. rCVB3 replicated to high titers in vivo and caused severe pancreatitis but minimal myocarditis. Despite the high virus titers, rCVB3 infection of naive mice failed to induce a strong CD8 ؉ T-cell response to the encoded epitope; this has implications for the proposed role of "cross-priming" during virus infection and for the utility of recombinant picornaviruses as vaccine vectors. In contrast, rCVB3 infection of LCMV-immune mice resulted in direct ex vivo cytotoxic activity against target cells coated with the epitope peptide, demonstrating that the rCVB3-encoded LCMV-specific epitope was expressed and presented in vivo. The preexisting CD8 ؉ memory T cells could limit rCVB replication; compared to naive mice, infection of LCMV-immune mice with rCVB3 resulted in ϳ50-fold-lower virus titers in the heart and ϳ6-fold-lower virus titers in the pancreas. Although the inserted CTL epitope was retained by rCVB3 through several passages in tissue culture, it was lost in an organ-specific manner in vivo; a substantial proportion of viruses from the pancreas retained the insert, compared to only 0 to 1.8% of myocardial viruses. Together, these results show that expression of heterologous viral proteins by recombinant CVB3 provides a useful model for determining the mechanisms underlying the immune response to this viral pathogen.Coxsackieviruses are members of the family Picornaviridae and lie in the Enterovirus genus, together with polioviruses, echoviruses, and unclassified enteroviruses. Coxsackieviruses are classified, according their pathogenicity in newborn mice, into groups A and B, which comprise 24 and 6 serotypes, respectively. Type B coxsackieviruses (CVB) are common human pathogens and have been implicated in acute and chronic myocarditis; there is a strong correlation between prior CVB infection and dilated cardiomyopathy, which can be effectively treated only by heart transplantation (47). In addition to cardiovascular disease, CVB has been associated with hepatitis, encephalitis, and pancreatitis, and CVB4 inf...

Amelioration of Coxsackievirus B3-Mediated Myocarditis by Inhibition of Tissue Inhibitors of Matrix Metalloproteinase-1

“…In contrast to lymphocytic choriomeningitis virus infection, CVB3 infection caused minimal activation of T-cell responses in either WT or TIMP-1KO mice ( Figure 3A). This is consistent with a previous study showing that recombinant CVB3 induced very weak CD8 ϩ T-cell responses, 25 and others have reported that the 3A protein of the closely related poliovirus can interrupt trafficking of MHC class I molecules, 26 which is required for epitope presentation to CD8 ϩ T cells. In addition, and as was observed for CD8 ϩ T cells, the CD4 ϩ T-cell responses to CVB3 infection were similar in WT and TIMP-1KO mice ( Figure 3B).…”

Protection Against CTL Escape and Clinical Disease in a Murine Model of Virus Persistence

“…Deletion of a foreign epitope is not unique to JHM because an inserted CD8 T cell epitope was also deleted from recombinant Coxsackie B virus by day 4 p.i. (39) and from recombinant polioviruses after passage through tissue culture cells (40).…”

Analysis of Translational Initiation in Coxsackievirus B3 Suggests an Alternative Explanation for the High Frequency of R +4 in the Eukaryotic Consensus Motif

“…Twelve BALB/c mice were infected intraperitoneally with 5 ϫ 10 3 PFU of the P1 stock of Koz1 virus. Three of the mice died within 2 days of infection, indicating that this mutant virus was highly pathogenic; this finding contrasts with the severe attenuation of most recombinant coxsackieviruses, in which doses as high as 10 7 PFU may be nonlethal (40). The hearts and pancreata of the nine surviving mice were harvested for analyses at 2, 5, and 9 days postinfection (four, three, and two mice, respectively).…”

“…In brief, uncapped RNA was produced by using the MAXIscript kit (Ambion). For the translation, 65% of S10 extract from HeLa cells, 1ϫ "all four" buffer (10ϫ buffer composed of 300 l of 1 M creatine phosphate; 300 l of 2 M potassium acetate; 100 l of creatine kinase [40 The translation was performed at 30°C for 4 h, after which 2ϫ Laemmli sample buffer was added, and the reactions were heated to 90°C for 5 min. Then, 5-l portions of the reactions were loaded and run on a sodium dodecyl sulfate-12.5% polyacrylamide gel.…”