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.

Neutralization of insect cell-derived DENV-2 by human serum depends on the concentration of MBL. We next assessed the relevance of our findings with human serum. Initially, sera from two healthy adult DENV-naïve volunteers were assayed for neutralization of DENV-2. Consistent with results with wild-type 4C) were neutralized by human serum. Serum inhibition of DENV-2 infection was MBL dependent, as neutralization was abolished in the presence of soluble mannose. Mannose, a second competitor ligand for MBL, was used instead of mannan, because anti-mannan antibodies are ubiquitously present in human sera (30, 31) . MBLdependent neutralization of mammalian cell-derived DENV-2 by human serum was less effective relative to insect cell-derived DENV-2 (for donor 1, at 10% and 20% serum, P Ͻ 0.0001, at 35% serum, P ϭ 0.004; for donor 2, at 10% serum, P ϭ 0.03, at 20% serum, P ϭ 0.04, at 35% serum, P ϭ 0.001). We also noticed that serum from donor 1 neutralized DENV-2 more efficiently than that of donor 2. Blood MBL levels in humans vary considerably (up to 1,000-fold) due to at least five common genetic polymorphisms in the human MBL2 gene (15) . We speculated that the inefficient neutralization of DENV-2 by serum from donor 2 was due to a lower serum MBL concentration. Using a commercial ELISA, the concentrations of MBL in sera from donors 1 and 2 were measured as 7,950 and 1,380 ng/ml, respectively. Indeed, reconstitution of serum from donor 2 with purified MBL to the level of donor 1 resulted in neutralization of insect cell-derived DENV-2 to a degree similar to that of serum from donor 1 (Fig. 4B) . To our surprise, the addition of even high concentrations (5.4 g/ml) of purified MBL to serum from donor 2 did not neutralize mammalian cell-derived DENV-2 (Fig. 4D) . However, the addition of increasing percentages of fresh (Fig. 4E ) or heat-inactivated ( Fig. 4F ) serum from donor 1 to serum from donor 2 resulted in neutralization of mammalian cell-derived DENV-2. This result was abolished by soluble mannose, establishing that neutralization of mammalian cell-derived DENV-2 by donor 2 required MBL from the serum from donor 1 in particular ( Fig. 4E and F).