A high-throughput neutralizing assay for antibodies and sera evaluation against Epstein-Barr virus.

BACKGROUND: Epstein-Barr virus (EBV) is a wide-spread human herpesvirus that is highly associated with infectious mononucleosis and several malignancies. Evaluation of EBV neutralizing antibody titers is important for serological studies, vaccine development and monoclonal antibody screening. The traditional method based on antibody inhibition of EBV transformation of B cells is very time-consuming. A more practical flow cytometry-based (FCM) approach to evaluate neutralizing titers is not amenable to achieving high-throughput evaluation of large-scale samples. A high-throughput approach is urgently needed. RESULTS: Here, we present a rapid and high-throughput method based on high content imaging system (HCIS) analysis. EBV titers determined by the HCIS-based assay were similar to those obtained by the FCM-based assay. Neutralizing titers of sera and monoclonal antibodies measured by the HCIS-based assay strongly correlated with titers measured by the FCM-based assay. HCIS assays showed a strong correlation between B cell infection neutralizing titers and the anti-gp350 IgG titers in healthy EBV carriers and monkey sera. Finally, anti-gHgL IgG titers from sera of healthy EBV carriers significantly correlated with epithelial cell infection neutralizing titers. CONCLUSIONS: This HCIS-based assay is a high-throughput assay to determine viral titers and evaluate neutralizing potentials of sera and monoclonal antibodies. This HCIS-based assay will aid the development of vaccines and therapeutic monoclonal antibody against EBV.

Neutralizing antibodies against Epstein-Barr virus infection of B cells can protect from oral viral challenge in the rhesus macaque animal model.

Epstein-Barr virus (EBV) and related lymphocryptoviruses (LCVs) from nonhuman primates are transmitted through oral secretions, penetrate the mucosal epithelium, and establish persistent infection in B cells. To determine whether neutralizing antibodies against epithelial or B cell infection could block oral transmission and persistent LCV infection, we use rhesus macaques, the most accurate animal model for EBV infection by faithfully reproducing acute and persistent infection in humans. Naive animals are infused with monoclonal antibodies neutralizing epithelial cell infection or B cell infection and then challenged orally with recombinant rhesus LCV. Our data show that high-titer B cell-neutralizing antibodies alone, but not epithelial cell-neutralizing antibodies, can provide complete protection of rhesus macaques from oral LCV challenge, but not in all hosts. Thus, neutralizing antibodies against B cell infection are important targets for EBV vaccine development, but they may not be sufficient.

Molecular characterization and overexpression analyses of secologanin synthase to understand the regulation of camptothecin biosynthesis in Nothapodytes nimmoniana (Graham.) Mabb.

Camptothecin is a high-value anti-cancerous compound produced in many taxonomically unrelated species. Its biosynthesis involves a complex network of pathways and a diverse array of intermediates. Here, we report the functional characterization and regulation of secologanin synthase (NnCYP72A1), a cytochrome P450 involved in camptothecin biosynthesis from Nothapodytes nimmoniana. It comprises an open reading frame of 1566 bp in length. Heterologous expression in Saccharomyces cerevisiae and in vitro enzymatic assays using loganin as substrate confirmed the formation of secologanin. In planta transient overexpression analysis of NnCYP72A1 resulted in 4.21- and 2.73-fold increase in transcript levels of NnCYP72A1 on days 3 and 6 respectively. Phytochemical analysis of transformed tissues revealed ~ 1.13-1.43- and 2.02-2.86-fold increase in secologanin and CPT accumulation, respectively. Furthermore, promoter analysis of NnCYP72A1 resulted in the identification of several potential cis-regulatory elements corresponding to different stress-related components. Methyl jasmonate, salicylic acid, and wounding treatments resulted in considerable modulation of mRNA transcripts of NnCYP72A1 gene. Chemical analysis of elicitor-treated samples showed a significant increase in CPT content which was concordant with the mRNA transcript levels. Overall, the functional characterization and overexpression of NnCYP72A1 may plausibly enhance the pathway intermediates and serve as prognostic tool for enhancing CPT accumulation.

High Level Estradiol Induces EBV Reactivation and EBV gp350/220(+)CD138(+) Double-positive B Cell Population in Graves' Disease Patients and Healthy Controls.

Graves' disease occurs predominantly in women. Epstein-Barr virus (EBV) mainly persists in human B lymphocytes, and its reactivation stimulates antibody production. We previously suggested that the EBV reactivation-induced production of TRAb and IgM at 100 nM estradiol (pregnant level) was lower than that at 0 nM estradiol and that class switch recombination may be increased by estradiol. In this study, we examined the effect of estradiol on EBV reactivation. We identified the expression of EBV-glycoprotein 350/220 (gp350/220) in the late phase of reactivation and plasma cell differentiation of EBV-infected cells using 72A1 antibody and CD138 antibody, respectively. We found the mean ratio of gp 350/220(+) CD138(+) cells at 100 nM estradiol was higher than that at 0 nM estradiol. These results suggested that EBV-infected cells could survive with keeping the ability of antibody production in 100 nM estradiol, which is consistent with the improvement of Graves' disease during maternity and exacerbation postpartum.