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

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... alization assay based on the expression of a reporter gene, b-galactosidase (b-Gal). Using a previously constructed vaccinia-b-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. With the renewed threat of smallpox as a bioterrorism agent [1-4], major efforts are underway in the following areas: (1) development of additional stocks of smallpox vaccines in alternative cell substrates (e.g., diploid cell lines and Vero cells), (2) development of more-attenuated strains of vaccinia (as vaccine candidates for immunodeficient individuals), and (3) production of hightiter vaccinia immune globulin (VIG) to treat or prevent severe vaccinia-associated adverse reactions [5, 6] . In parallel, it is essential to develop laboratory-based assays to evaluate the potency of the new vaccines and VIG products. One of the key assays to assess both humoral immune responses in vaccines and the potency of VIG