title: Development of a novel vaccinia-neutralization assay based on reporter-gene expression.
abstract: In anticipation of large-scale smallpox vaccination, clinical trials of new vaccine candidates with improved safety profiles, and new vaccinia immune globulin (VIG) products, there is an immediate need to develop new assays to measure vaccinia-specific immune responses. The classical assay to measure vaccinia neutralization, the plaque-reduction neutralization test (PRNT), is slow, labor intensive, and difficult to validate and transfer. Here we describe the development of a novel vaccinia-neutralization assay based on the expression of a reporter gene, beta-galactosidase (beta-Gal). Using a previously constructed vaccinia-beta-Gal recombinant virus, vSC56, we developed a neutralization assay that is rapid, sensitive, and reproducible. The readout is automated. We show that the neutralizing titers, ID(50), for several VIG products measured by our assay were similar to those obtained by PRNTs. A new Food and Drug Administration VIG standard was established for distribution to other laboratories. The new assay will serve as an important tool both for preclinical and clinical trials of new smallpox vaccines and for evaluation of therapeutic agents to treat vaccine-associated adverse reactions.
authors.affiliation: Division of Viral Products, Center for Biologics Evaluation and Research, Food and Drugs Administration, Bethesda, Maryland 20892, USA.
authors.forename: Jody
authors.initials: J
authors.lastname: Manischewitz
authors.forename: Lisa R
authors.initials: LR
authors.lastname: King
authors.forename: Nicole A
authors.initials: NA
authors.lastname: Bleckwenn
authors.forename: Joseph
authors.initials: J
authors.lastname: Shiloach
authors.forename: Rolf
authors.initials: R
authors.lastname: Taffs
authors.forename: Michael
authors.initials: M
authors.lastname: Merchlinsky
authors.forename: Nancy
authors.initials: N
authors.lastname: Eller
authors.forename: Malgorzata G
authors.initials: MG
authors.lastname: Mikolajczyk
authors.forename: David J
authors.initials: DJ
authors.lastname: Clanton
authors.forename: Thomas
authors.initials: T
authors.lastname: Monath
authors.forename: Richard A
authors.initials: RA
authors.lastname: Weltzin
authors.forename: Dorothy E
authors.initials: DE
authors.lastname: Scott
authors.forename: Hana
authors.initials: H
authors.lastname: Golding
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000032:analysis / Q000097:blood; D002460:Cell Line; D015870:Gene Expression; D017930:Genes, Reporter; D006801:Humans; D007136:Immunoglobulins / Q000032:analysis; D009500:Neutralization Tests / Q000379:methods*; D011995:Recombination, Genetic; D012680:Sensitivity and Specificity; D014616:Vaccinia virus / Q000235:genetics / Q000276:immunology*; D001616:beta-Galactosidase / Q000235:genetics
chemical_list: D000914:Antibodies, Viral; D007136:Immunoglobulins; D001616:beta-Galactosidase