Short Report: An Automated Dengue Virus Microneutralization Plaque Assay Performed in Human Fcγ Receptor-expressing CV-1 Cells

We describe microneutralization assays that used automated 96-well enzyme-linked immunospot (ELI- SPOT) readout instrumentation to measure human anti-dengue virus (DENV) antibodies in CV-1 cells that were stably transfected to express human FcγRIIA (CD32) using conventional Vero cells as a comparator. Classic plaque reduction neutralization test (PRNT) end-point titers were determined by probit analysis. Neutralization titers against DENV measured in CV-1 transfectants were expressed in terms of both conventional 50% to 90% PRNT end-point titers and differential infectivity of antibody-treated virus in control and CD32-expressing CV-1 cells. Significantly reduced PRNT titers and strikingly heightened infectivity (up to 100-fold) of antibody-treated DENV was observed in CV-1 CD32 transfectants compared with that observed in control CV-1 or Vero cells. Because DENVs may preferentially replicate in CD32-expressing monocytes/macrophages and dendritic cells, in vivo , it is possible that CD32 intro duced into a conventional DENV neutralization assay might provide results that better correlate with protection. Dengue virus (DENV) human immune serum volumes are often limited, especially from children and infants, and DENV plaque neutralization assays, conventionally per- formed in cells of monkey kidney origin (i.e., Vero, LLC-MK 2 , CV-1), use relatively large formats (e.g., 6-well cluster plates). The DENV microneutralization assays that use such cell types have been described using an enzyme-linked immunosorbent assay (ELISA) format 1 or immunofluores- cent microscopy. 2 In this work, we describe two microneu- tralization plaque assays that adopted widely used 96-well enzyme-linked immunospot (ELISPOT) readout instrumen- tation to measure DENV antibodies in African green mon- key Vero and CV-1 cells used in DENV plaque assays. The first assay method used Vero cells, which are conventionally used in DENV neutralization tests, 3 as a comparator and to calibrate the ELISPOT instrument for DENV plaque count- ing to determine traditional DENV plaque reduction neu- tralization test (PRNT) end-point serum titers against each of the four DENV serotypes. The second method used CV-1 cells that were engineered to constitutively express func- tional human FcγRIIA (CD32), or were stably transfected with an empty vector to serve as a control. This modification was introduced with the notion that neutralization measure- ments performed in such cells would better reflect the anti- body effect on DENV infectivity for CD32-expressing cells of monocyte/macrophage (Mo/MΦ) lineage, the putative in vivo targets of DENV. 4-7