With the potential threat of smallpox as a bioterrorism agent, a major effort should be invested in two main areas: a) development of new effective preventive vaccines, and b) production of high titer vaccinia IGG (VIG) that can be used as a first line of defense in exposed and/or high risk populations. The current stocks of VIG are limited and outdated. Scientists at CBER and elsewhere (MPH) were encouraged to produce new lots of VIG and subject them to careful titration in validated neutralization assays. In addition, experiments are underway to compare the neutralization capacity of different VIG subclasses, in order to optimize viral neutralization in future production of VIG.The current neutralization assay for vaccinia (plaque-reduction), is labor intensive, slow (up to 7 days), and depends on scoring plaques by eye (i.e., subjective). This assay is difficult to validate and to transfer to sponsor-based laboratories. As an alternative approach, our laboratory initiated late last year the development of an alternative vaccinia neutralization assay, which is fast, quantitative, and easy to perform and transfer to other laboratories.This assay is based on the use of recombinant vaccinia virus expressing a bacterial gene coding for the enzyme b-Gal under the control of the vaccinia late promoter. Several recombinant viruses were tested and two of them, vSC8 and vSC56 are currently under investigation in our laboratory. We also tested several cell substrates for the assay: BSC1, Vero, and HeLa. Results: - We have established and standardized a novel assay to measure vaccinia neutralization. It is based on the expression of a reporter gene coding for the bacterial b-galactosidase enzyme (b-Gal) under the control of a synthetic E/L promoter. We demonstrate that the new assay is rapid (24 hr), of equal sensitivity to PRNT assays, reproducible, objective, and easy to transfer. In addition, we describe preliminary results with a second reporter gene assay based on a vaccinia-EGFP recombinant virus. The expression of the GFP is induced by IPTG. The two assays provide high throughput capabilities and may be established in clinical laboratories for the evaluation of multiple samples from clinical trials. - Thus far, it was found that the MPH VIG is somewhat better than the Baxter (4oC) VIG with ID(50) values of 10-15 ug/ml and 25 ug/ml, respectively. - We also tested Ig subclasses derived from the MPH VIG and found that the neutralization efficiency of the IgG1 subclass is superior to all other subclasses and to the total (unseparated) VIG preparation. - We tested multiple lots of IVIG from 5 different manufacturers. Several lots were found to have significant anti-vaccinia neutralization titers.
