title: Generation of recombinant vaccinia viruses expressing Puumala virus proteins and use in isolating cytotoxic T cells specific for Puumala virus.
abstract: Puumala (PUU) virus causes a form of hemorrhagic fever with renal syndrome (HFRS), called nephropathia epidemica (NE), in Europe. HFRS is characterized by an increased capillary permeability, which we hypothesize is caused by hyperactivation of the host immune system, especially cellular immune responses. To identify cytotoxic T lymphocytes (CTLs) specific for the PUU virus from NE patients, we have made recombinant vaccinia viruses expressing PUU virus proteins, the nucleocapsid (N) and two surface glycoproteins, G1 and G2. Recombinant vaccinia viruses carrying the N or the first half of the G2 cDNA under the control of a strong synthetic promoter were made. To express G1 and the second half of the G2 proteins, however, we needed to use a T7 expression system, where the T7 RNA polymerase is produced from another recombinant vaccinia virus co-infecting the same cells. These recombinant vaccinia viruses were used to detect and clone PUU virus-specific CTLs from the peripheral blood mononuclear cells of NE patients. An HLA-A24-restricted CTL line recognizing the G2 protein was isolated and its 9-mer epitope was determined.
authors.affiliation: Center for Infectious Disease and Vaccine Research, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655, USA.
authors.forename: Masanori
authors.initials: M
authors.lastname: Terajima
authors.forename: Heather L
authors.initials: HL
authors.lastname: Van Epps
authors.forename: Dexin
authors.initials: D
authors.lastname: Li
authors.forename: Anita M
authors.initials: AM
authors.lastname: Leporati
authors.forename: Sarah E
authors.initials: SE
authors.lastname: Juhlin
authors.forename: Jukka
authors.initials: J
authors.lastname: Mustonen
authors.forename: Antti
authors.initials: A
authors.lastname: Vaheri
authors.forename: Francis A
authors.initials: FA
authors.lastname: Ennis
mesh_terms: D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002522:Chlorocebus aethiops; D004279:DNA, Viral; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006480:Hemorrhagic Fever with Renal Syndrome / Q000097:blood / Q000276:immunology*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000276:immunology; D008969:Molecular Sequence Data; D019251:Nucleocapsid / Q000235:genetics / Q000276:immunology*; D019590:Nucleocapsid Proteins; D029262:Puumala virus / Q000235:genetics / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D011995:Recombination, Genetic; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D014616:Vaccinia virus / Q000235:genetics; D014759:Viral Envelope Proteins / Q000235:genetics / Q000276:immunology*
chemical_list: D004279:DNA, Viral; D019590:Nucleocapsid Proteins; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C100886:nucleocapsid protein, Puumala virus