To assess immunogenicity and development of antibodies in the context of vaccination, it is critical to quantify titers of neutralizing antibodies. We have been employing the 293TT cell-based neutralization assay system to quantify anti-HPV neutralizing antibodies. In this system, human papillomavirus (HPV) pseudovirion (PsV) particles encapsidating secreted alkaline phosphatase (SEAP) gene are used to measure infection of 293TT cells in 72-hr cell-culture supernatants. SEAP has traditionally been measured by Great EscAPe™ SEAP Chemiluminescence Kit 2.0 (GE). To reduce the cost, and to potentially increase efficiency, we sought a cheaper kit with better detection capability. Performance characteristics of the newer chemiluminescence kit, ZiVa® Ultra SEAP Plus Assay (Ziva) and GE were compared using the 293TT system. Dose titration of HPV PsV 16 or 18 showed that signal-to-noise ratios at 48 and 72 hr post-infection were higher for ZiVa at nearly all doses. ZiVa was superior to GE as it was able to detect SEAP at 48 hr, as well as when lower numbers of 293TT cells were used. The ability of ZiVa to quantitate HPV-16 and -18 neutralizing antibody titers was tested using sera from Cervarix® immunized individuals. Spearman rank correlational analyses showed excellent correlations between the titers obtained with ZiVa and GE for anti-HPV16 (r = 0.9822, p < 0.0001) and anti-HPV18 (r = 0.9832, p < 0.0001) antibodies. We concluded that ZiVa is superior to GE in detecting SEAP, and the antibody titers in sera of vaccinated individuals were similar to those obtained with GE. Thus, Ziva is a suitable alternative to GE.

01 Jul 1995·Journal of VirologyQ2 · MEDICINE

YY1 binds to and regulates cis-acting negative elements in the Epstein-Barr virus BZLF1 promoter

Q2 · MEDICINE

Article

Author: Wang, Y J ; Cottam, M ; Montalvo, E A ; Hill, S

A 48-bp cis-acting negative element in the Epstein-Barr virus BZLF1 gene P1 promoter has been described previously. By DNase I footprinting experiments, two regions were identified as the protein-binding sites (previously designated site I and site II). In this report, the cellular transcription factor YY1 has been identified as a protein which binds to both of these elements, now designated ZIVA and ZIVB. Both ZIVA and ZIVB conferred cis-acting negative regulation on an enhancerless simian virus 40 promoter. In cotransfection experiments, overexpression of YY1 caused further repression of the enhancerless simian virus 40 promoter containing either the ZIVA or ZIVB element. Cotransfection of a plasmid expressing antisense to YY1 increased the expression of the heterologous promoter containing ZIVA but not ZIVB. In similar experiments carried out with the P1 promoter, overexpression of YY1 caused downregulation of P1 whereas antisense RNA to YY1 caused a slight increase in expression. Analyses of various P1 mutant constructions revealed additional YY1 sites downstream of ZIVB. Overexpression of YY1 also caused downregulation of a P1 mutant with no apparent YY1-binding sites. TPA treatment of Raji cells caused a temporal loss of YY1-binding activity but had no effect on the intracellular levels of YY1 protein. Serum induction of quiescent B cells also caused loss of YY1 binding to the ZIVB site, which was found to be a weak serum response element. In contrast, anti-immunoglobulin G treatment of Akata cells had no effect on either the YY1-binding activity or protein levels. The binding of YY1 to the cis-acting negative elements in infected B cells may play a pivotal role in the maintenance of Epstein-Barr virus latency.

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Indication	Highest Phase	Country/Location	Organization	Date
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