Complement-Mediated Neutralization of Dengue Virus Requires Mannose-Binding Lectin

Avirutnan, Panisadee; Hauhart, Richard E.; Marovich, Mary A.; Garred, Peter; Atkinson, John P.; Diamond, Michael S.

MBL directly binds and neutralizes insect cell-derived DENV-2 independent of complement activation. Whereas neutralization of WNV by MBL occurred with insect cell-derived virus, but not mammalian cell-derived virus (25) , both forms of DENV-2 were susceptible to MBL-dependent complementmediated inhibition. However, neutralization of insect cellderived DENV-2 by naïve wild-type C57BL/6 mouse serum was more efficient than mammalian cell-derived DENV-2 (100% versus 60 to 80% neutralization at 10% serum concentration, respectively; P Ͻ 0.0001) (Fig. 1A to D). This phenotype is likely due to modifications of high-mannose carbohydrate moieties on the structural proteins on the surfaces of DENV-2 virions produced in mammalian cells (26) resulting in less efficient recognition by MBL. To test for direct binding of MBL to DENV-2, we developed a capture enzyme-linked immunosorbent assay (ELISA) in which infectious virus was bound to wells of a microtiter plate coated with anti-DENV prM mutant monoclonal antibody (MAb). Purified recombinant MBL bound efficiently to immobilized insect Avirutnan et al. cell-derived DENV-2 in a dose-dependent manner, and as expected, binding was Ca 2ϩ dependent and blocked by soluble mannan ( Fig. 2A) . The specificity of the interaction was confirmed by an absence of signal when an isotype control (anti-hepatitis C virus E2 protein) MAb was used as the capture antibody. We next compared the relative binding of MBL to insect cell-and mammalian cell-derived DENV-2. Equivalent amounts of both viruses, as judged by a DENV E-protein-specific MAb that bound captured virions (Fig. 2C) , were interrogated for binding to MBL by ELISA. Notably, purified MBL preferentially bound to insect cellderived DENV-2 at all concentrations tested (P Ͻ 0.05 [ Fig. 2B] ).