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.

Ϫ/Ϫ , fB Ϫ/Ϫ , or fD Ϫ/Ϫ ) or antibody-deficient (RAG1 Ϫ/Ϫ ) mice prior to addition to a monolayer of BHK21-15 cells. Infectivity was determined 4 days later by plaque assay. Data are presented as the percent neutralization by a given complement-deficient or antibody-deficient serum compared to wild-type C57BL/6 serum. Error bars indicate standard errors of the means (SEM) for up to 6 independent experiments with each condition performed in duplicate. Values that are significantly different from the value for wild-type C57BL/6 serum are indicated by asterisks as follows: *, P Ͻ 0.05; **, P Ͻ 0.01; ***, P Ͻ 0.001. (B and D) Serum neutralization of C6/36 cell-derived (B) or Vero cell-derived (D) DENV is abolished in the presence of mannan. DENV was preincubated with an increasing percentage of wild-type serum in the presence or absence of mannan (100 g/ml). The percentage of neutralization was calculated based on reduction of the number of plaques for a given condition compared to heat-inactivated (HI) wild-type serum. Error bars indicate SEM from 3 independent experiments performed in duplicate. Values that are significantly different from the value for the wild type are indicated by asterisks and brackets as follows: *, P Ͻ 0.05; **, P Ͻ 0.01; ***, P Ͻ 0.001. (E) Model of DENV neutralization by complement. MBL binding to the virion surface initiates lectin pathway activation resulting in deposition of C4b and C3b. Binding of MBL also activates MASPs that directly cleave C3 without activation of C4 and C2 (the C4 and C2 bypass pathway). The alternative pathway amplification loop serves to generate more C3b deposition on the virus. DENV-2 in the absence of any other complement components neutralized infection up to 65% (Fig. 2D) . Nonetheless, the efficiency of neutralization increased significantly (2-to 6-fold [P Ͻ 0.001]) in the presence of other complement components in serum from MBL-A/C Ϫ/Ϫ mice, especially at low concentrations of purified MBL (e.g., 0.03 g/ml) (Fig. 2E ). As expected, soluble mannan competed with binding of purified MBL to DENV and inhibited both complement-independent (Fig. 2D ) and complement-dependent ( Fig. 2E ) neutralization of insect cellderived DENV-2. In contrast, purified MBL failed to directly neutralize DENV-2 propagated in several mammalian cell types, including Vero cells, primary human monocyte-derived dendritic cells (DC), and primary human peripheral blood monocytes, or WNV that was produced in insect and mammalian cells, even with concentrations of MBL as high as 30 g/ml (~10-fold above the physiological level) ( Fig. 2F ; see Fig. S1 in the supplemental material). In contrast to human MBL, purified mouse MBL inhibited insect cell-derived DENV-2 poorly without complement activation, although neutralizing activity was greatly enhanced in the presence of other complement components (Fig. S2 ). In combination with the 2-fold-lower blood levels of MBL in MASP-2 Ϫ/Ϫ mice (data not shown), these results begin to explain the lack of neutralizing activity in serum from MASP-2 Ϫ/Ϫ mice (Fig. 1A) .