Using recombinant coxsackievirus B3 to evaluate the induction and protective efficacy of CD8(+) T cells during picornavirus infection

Coxsackievirus B3 (CVB3) is a common human pathogen that has been associate d with serious diseases including myocarditis and pancreatitis. To better u nderstand the effect of cytotoxic T-lymphocyte (CTL) responses in controlli ng CVB3 infection, we have inserted well-characterized CTL epitopes into th e CVB3 genome. Constructs were made by placing the epitope of interest upst ream of the open reading frame encoding the CVB3 polyprotein, separated by a poly-glycine linker and an artificial 3C(pro)/3CD(pro) cleavage site. Thi s strategy results in the foreign protein being translated at the amino- te rminus of the viral polyprotein, from which it is cleaved prior to viral as sembly. In this study, we cloned major histocompatibility complex class I-r estricted 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 similar to1 log unit lower than for wild-type virus. rCVB3 replicated to hi gh titers in vivo and caused severe pancreatitis but minimal myocarditis. D espite the high virus titers, rCVB3 infection of naive mice failed to induc e a strong CD8(+) T-cell response tea the encoded epitope; this has implica tions for the proposed role of "cross-priming" during virus infection and f or the utility of recombinant picornaviruses as vaccine vectors. In contras t, rCVB3 infection of LCMV-immune mice resulted in direct ex vivo cytotoxic activity against target cells coated with the epitope peptide, demonstrati ng 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 similar to 50-fold-lower virus titers in the heart and similar to6-fold- lower virus titers in the pancreas. Although the inserted CTL epitope was r etained 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 vira l proteins by recombinant CVB3 provides a useful model for determining the mechanisms underlying the immune response to this viral pathogen.