S. L. Keith1, N. Graham2, E. Sendra2, B. Kirkpatrick2, J. E. Boyson1 2University Of Vermont College Of Medicine / Fletcher Allen Health Care,Department Of Medicine,Burlington, VT, USA 1University Of Vermont College Of Medicine / Fletcher Allen Health Care,Department Of General Surgery,Burlington, VT, USA
Introduction: Over 40% of the world’s population lives in dengue virus (DENV) endemic areas. Therefore, the development of a safe and efficacious dengue virus vaccine is a global health priority. Althought the first dengue vaccine was recently licensed in 3 countries, it has only a 30% efficacy against some DENV serotypes in DENV-endemic areas. Interestingly, this low efficacy was not predicted during pre-clinical testing. The reasons underlying these discrepant results are unclear. One of the primary correlates of vaccine efficacy is the ability to elicit neutralizing antibodies. The standard assay in the field is a plaque reduction neutralizing titer 50% (PRNT50) assay that assesses the ability of a vaccinee’s serum to neutralize virus infection of Vero cells, which do not have the same attachment factors and receptors as human cells. This raises the question of whether the currently used PRNT50 assay is the best method for neutralizing antibody determination.
Methods: The purpose of this study was to develop a neutralization assay using primary human cells and to determine whether this assay would yield comparable results to the standard PRNT50 assay. Human CD14+ monocytes were enriched from peripheral blood mononuclear cells. Monocytes were then differentiated into immature dendritic cells (immDC) using GM-CSF and IL-4 for use in a neutralization assay. Antisera with known PRNT50 values for DENV1 were obtained from volunteers vaccinated with the NIH DENV TV005 vaccine (UVM Vaccine Testing Center) and incubated with DENV1 prior to adding to human immDCs. Cells were then harvested and DENV infection was assessed using flow cytometry. An inhibition curve (IC50) was calculated and compared to the PRNT50 value.
Results: Preliminary data indicate patient to patient variability in the level of DENV infection of monocytes. A comparison of Vero cell PRNT50 and human immDC IC50 values using DENV serotypes 1 and 4 suggests no significant difference in neutralizing antibody titers.
Conclusion: Our data indicate that the standard PRNT50 assay is suitable for testing DENV vaccinine response, at least for serotypes 1 and 4. Serotypes 2 and 3 have yet to be tested. This assay may be important in evaluating DENV candidate vaccines in the future.?