PROJECT SUMMARY: Antibody-dependent enhancement (ADE) is a phenomenon characteristic of some flavivirus infections and best known to occur between the four DENV serotypes in humans, but may also occur between sequential DENV, then ZIKV infections (or vice versa). ADE occurs when subneutralizing levels of antibodies raised against one flavivirus infection cross-react to enhance a closely related flavivirus infection. ADE is entirely dependent on the binding of IgG antibodies to FcRs found on susceptible cells, such as monocytes/macrophages. In humans, there is a nonsynonymous polymorphism that occurs in the FcRIIA gene (rs1801274) that leads to an arginine (Arg) to histadine (His) change at position 131 that has been shown to alter the affinity of this receptor for IgG antibodies. We hypothesize that individuals homozygous for the FcRIIA encoding for the high affinity His allele are at higher risk for developing ADE of flaviviruses than individuals homozygous for the low affinity Arg allele. In this application, we will test this using K562 cells homozygous for the Arg131 or the His131 allele as well as primary human monocytes stratified based on genotype to test how this SNP impacts ADE of flaviviruses in vitro (Aim 1). Using two flavivirus-specific monoclonal antibody clones that we found to enhance flavivirus infection in K562 cells, we will generate IgG subclass switch variants (IgG1, IgG2, IgG3, or IgG4) to determine the relative contribution of each IgG subclass in mediating ADE of flaviviruses (Aim 2).