Graphs were generated from an experiment performed in triplicate for both DENV serotypes and are representative of three similar experiments. IgG engagement by the FcR transfectants was assessed by a competition binding assay (Fig. manner, likely through effects on virion and FcR binding. Thus, the IgG antibody subclass profile generated by DENV contamination or vaccination may independently influence the magnitude of the neutralizing response. Keywords: Dengue computer virus, computer virus neutralization, humanized monoclonal antibody, Fc receptor Introduction Current experimental evidence supports a multi-hit occupancy model of flavivirus neutralization where antibodies directed at a virion envelope protein interfere with attachment or fusion if covering levels are high enough (examined D77 in: (Pierson et al., 2008)). This notion also suggests an explanation for the paradox of enhanced flavivirus contamination by neutralizing antibodies in Fc receptor (FcR)-bearing cells; at lesser levels of antibody occupancy, which are sub-neutralizing in nature, virus-immune IgG complexes can enhance dengue computer virus (DENV) attachment by engaging FcRs and contamination of monocyte/macrophages (Mo/M?). This in vitro obtaining provides the basis for any hypothesis of antibody-dependent enhanced infection (ADE) to explain more severe contamination in individuals with pre-existing DENV antibodies acquired by maternal transfer or main infection (examined in: (Halstead, 2003)). DENVs exist as four serologically unique but antigenically related computer virus types (DEN1 through 4), NSD2 which can cause sequential human infection. Homotypic antibodies confer durable DENV serotype-specific immunity whereas cross-reactive neutralizing antibodies D77 are generally weaker and less protective, and presumably can more readily form DENV immune complexes that remain infectious upon FcR engagement. FcRs comprise a multi-gene family of type 1 integral membrane glycoproteins (examined in: (Nimmerjahn and Ravetch, 2008)). Among these, IgG receptors of two activation FcR classes displayed on DENV-permissive human Mo/M? and dendritic cells (DCs) have specifically been shown to modulate DENV immune complex infectivity (Boonnak et al., 2008; Kou D77 et al., 2008; Littaua, Kurane, and Ennis, 1990; Mady et al., 1991; Rodrigo et al., 2006). The first, FcRIA (CD64), has high affinity for both monomeric and complexed IgG and is found exclusively on antigen-presenting macrophages and DCs. The second, FcRIIA (CD32), exists in two allotypic forms (H/R131) that preferentially bind multimeric IgG complexes with at least 100-fold lower affinity than FcRIA. FcRIA acquires signaling function by association with the common -chain subunit, whereas the FcRIIA ligand binding chain is usually intrinsically signaling-competent. The DENV virion E protein ectodomain is usually comprised of three domains (DI, DII, DIII), each of which is usually targeted by neutralizing antibodies. However, the IgG subclass and antigenic distribution of naturally stimulated neutralizing antibodies remains to be fully elucidated and molecular dissection of DENV immune complex conversation with FcRs is just beginning. Here, we use epitope-matched humanized mAb Fc variants to evaluate the effect of IgG subclass on DENV virion binding, and neutralization in African green monkey kidney Vero and CV-1 cells which lack Fc receptors, and in CV-1 cells which were stably transfected to express human FcRIA or FcRIIA. Our results demonstrate that DENV neutralization is usually modulated in an IgG subclass manner, possibly through impartial Fc effects on virion and FcR binding. Results Functional human FcRs displayed on DENV-permissive CV-1 cells We evaluated DENV immune complex interactions with FcRIA and FcRIIA transfected individually into Fc receptor-negative CV-1 cells, which like Vero cells are used in standard DENV plaque reduction neutralization (PRNT) assays. Coding sequences of human chain and FcRIA arranged in a bicistronic expression cassette to ensure optimal and comparative protein expression (Rodrigo et al., 2006), and those of both human FcRIIA allotypes were respectively integrated into the identical single chromosomal site in CV-1/FRT cells. A CV-1 vacant vector-transfected cell collection served as the FcR-negative CV-1 cell control. The three FcR-expressing CV-1 cell lines (Fig. 1A) displayed the respective FcR type in comparable large quantity (~300,000 FcR molecules per cell) that was essentially unchanged during continuous culture.