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.

Blood samples from subjects in the United States were obtained with informed consent and approval by the Washington University institutional review board. Some human samples were obtained in Denmark from anonymous blood donors with informed consent according to the Danish law of blood donation. C6/36 cell-derived DENV-1, -3, and -4 were preincubated with 2% (vol/vol) wild-type mouse serum in the presence or absence of mannan (100 g/ml). The virus was added to Vero cells, and infectivity was determined 48 hours (for DENV-1 and -4) or 60 hours (for DENV-3) later by a focus-forming assay. Neutralization was calculated based on reduction of the number of foci in a given condition compared to the value in heat-inactivated (HI) serum. Error bars indicate SEM for 5 or 6 independent experiments performed in duplicate. (B) C6/36 cell-derived DENV-1, -3, and -4 were incubated with purified human MBL (3 g/ml) in the presence or absence of mannan (100 g/ml) prior to addition to a monolayer of Vero cells. Data are presented as percent neutralization (percent reduction of the number of foci compared to the value in buffer alone) in a given condition. Error bars indicate SEM for 5 independent experiments performed in duplicate. (C) C6/36 cell-derived DENV-1, -3, and -4 were preincubated with 10% (vol/vol) naïve human serum (donor 1 or donor 2) in the absence or presence of 1 M mannose or 1.36 g/ml purified human MBL. Neutralization was calculated based on reduction of the number of foci compared to the value in heat-inactivated (HI) serum. Error bars indicate SEM for 5 independent experiments performed in duplicate. Values that are significantly different are indicated by brackets and asterisks as follows: *, P Ͻ 0.05; **, P Ͻ 0.01; ***, P Ͻ 0.001. MBL genotyping. The single-nucleotide polymorphisms in the promoter region at positions Ϫ550 (H/L variants) and Ϫ221 (X/Y variants), in the 5= noncoding region at position ϩ4 (P/Q variants) and in codons 54 (allele B), 57 (allele C), and 52 (allele D) in exon 1 of the MBL2 gene were identified as previously described (52) .