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. 2011 Dec 13;2(6):e00276-11. doi: 10.1128/mBio.00276-11

FIG 2 .

FIG 2

Human MBL neutralizes insect cell-derived DENV-2 independent of complement activation. (A and B) MBL binds to DENV-2. C6/36 cell-derived DENV-2 (A) or both C6/36 cell-derived and Vero cell-derived DENV-2 (B) virions were captured on a microtiter plate using an anti-DENV prM protein-specific MAb (2H2) or isotype control anti-hepatitis C virus (anti-HCV) E2 MAb (H77.16) and incubated with an increasing concentration of purified human MBL. MBL binding was detected with an anti-MBL specific MAb and inhibited by the presence of 10 mM EDTA or 100 µg/ml mannan. Error bars indicate SEM for up to 6 independent experiments. Black asterisks indicate statistical differences compared to EDTA-treated samples. Grey asterisks denote values of C6/36 cell-derived DENV binding to MBL that are different from those of Vero cell-derived DENV-2. O.D. 450 nm, optical density at 450 nm. (C) In parallel experiments, captured DENV virions derived from C6/36 or Vero cells were detected with an anti-DENV E-protein-specific MAb (4G2). (D and F) Human MBL restricts DENV-2 derived from insect cells but not DENV-2 derived from mammalian cells in the absence of complement activation. DENV-2 derived from C6/36 cells (D) and Vero cells (F) were incubated with increasing concentrations of purified MBL prior to addition to a monolayer of BHK21-15 cells. After 4 days, infectivity was determined by plaque assay. Data are presented as the percent neutralization (percent reduction of plaques compared to the number of plaques in buffer alone in a given condition). (E) MBL-mediated neutralization of insect-derived DENV-2 is enhanced by complement activation. C6/36 cell-derived DENV-2 was preincubated with serum from MBL-A/C−/− mice in the presence or absence of purified MBL at the indicated concentrations, with or without mannan (100 µg/ml). The percent neutralization was calculated based on the percent reduction of plaques in a given condition compared to the number of plaques in MBL-A/C−/− serum. Error bars indicate SEM from 2 to 4 independent experiments performed in duplicate. Values that are significantly different are indicated by asterisks as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001.