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Journal of Virology, March 2001, p. 2377-2387, Vol. 75, No. 5
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.5.2377-2387.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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

Mark K. Slifka, Robb Pagarigan, Ignacio Mena,Dagger Ralph Feuer, and J. Lindsay Whitton*

Department of Neuropharmacology, The Scripps Research Institute, La Jolla, California

Received 15 September 2000/Accepted 29 November 2000

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 3Cpro/3CDpro 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 ~1 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 ~50-fold-lower virus titers in the heart and ~6-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.

* Corresponding author. Mailing address: Department of Neuropharmacology, CVN-9, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037. Phone: (858) 784-7090. Fax: (858) 784-7380. E-mail: lwhitton{at}scripps.edu.

dagger Manuscript 12836-NP from the Scripps Research Institute.

Dagger Present address: Institut Pasteur, 75724 Paris Cedex 15, France.

Journal of Virology, March 2001, p. 2377-2387, Vol. 75, No. 5
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.5.2377-2387.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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