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.

Protection against Lethal Vaccinia Virus Challenge in HLA-A2 Transgenic Mice by Immunization with a Single CD8+T-Cell Peptide Epitope of Vaccinia and Variola Viruses

“…Inasmuch as smallpox vaccination was discontinued more than 30 years ago, one possible use for this epitope would be to detect a long-term memory response, or the response to the epitope could be used as a criterion of effective immunization. This could be utilized in addition to other methods for detection of effective poxvirus immunity or vaccine "take," such as the recently developed vaccinia virus neutralization assay (35), or it could be used to look for long-term immunity to poxviruses in human subjects (25).…”

Differential Antigen Requirements for Protection against Systemic and Intranasal Vaccinia Virus Challenges in Mice

“…NAb responses against vaccinia virus were measured using a luciferase-based assay in HeLa cells essentially as previously described for a ␤-galactosidase neutralization assay (20). We measured the reduction in luciferase reporter gene expression in target cells after a single round of virus infection.…”

“…Subtyping studies demonstrated that these antibodies were predominantly IgG2a and IgG1 (data not shown). We next assessed vaccinia virus IMV-specific NAb titers by using a luciferase-based virus neutralization assay (20). As shown in Fig.…”

Correlations Between Vaccinia-Specific Immune Responses Within a Cohort of Armed Forces Members

“…We adapted a vaccinia-specific neutralization assay originally developed at the Food and Drug Administration (FDA) to measure neutralizing antibody titer (15,19). Briefly, heat-inactivated serum samples were serially diluted and then mixed with a known quantity of a recombinant b-galactosidase (b-gal) expressing VACV for 1 h. Serum samples were then incubated with HeLa cells overnight.…”