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 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 approximately 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 approximately 50-fold-lower virus titers in the heart and approximately 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.
Slifka
M K
MK
Department of Neuropharmacology, The Scripps Research Institute, La Jolla, California 92037, USA.
Pagarigan
R
R
Mena
I
I
Feuer
R
R
Whitton
J L
JL
D000595:Amino Acid Sequence; D000818:Animals; D000956:Antigens, Viral; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D003384:Coxsackievirus Infections / Q000276:immunology* / Q000821:virology; D029822:Enterovirus B, Human / Q000235:genetics / Q000254:growth & development / Q000276:immunology* / Q000472:pathogenicity; D018984:Epitopes, T-Lymphocyte / Q000235:genetics / Q000276:immunology; D006023:Glycoproteins / Q000276:immunology / Q000378:metabolism; D006801:Humans; D007156:Immunologic Memory; D008217:Lymphocytic choriomeningitis virus / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism; D011995:Recombination, Genetic; D014162:Transfection; D014764:Viral Proteins; D014779:Virus Replication
D000956:Antigens, Viral; D018984:Epitopes, T-Lymphocyte; D006023:Glycoproteins; D010446:Peptide Fragments; D014764:Viral Proteins; C094244:glycoprotein peptide 33-41, Lymphocytic choriomeningitis virus