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Journal of Virology, March 2001, p. 2377-2387, Vol. 75, No. 5
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.
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
*
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.
Manuscript 12836-NP from the Scripps Research Institute.
Present address: Institut Pasteur, 75724 Paris Cedex 15, France.
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