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.

To further investigate how the concentration of MBL impacts the ability of human serum to neutralize DENV-2, we obtained sera from fifteen additional healthy adult volunteers, including five donors with known polymorphisms (associated with changes in the level, structure, and function of the protein) in the MBL2 gene (15) ( Table 1) . We first measured the concentrations of MBL in these sera (Fig. 5A) . MBL levels varied widely, ranging from Ͻ2 ng/ml to 8 g/ml. We next tested whether native forms of MBL in human serum bound DENV-2. Similar to results with purified human MBL ( Fig. 2A and B) , MBL in serum bound to immobilized insect cell-derived DENV-2 virions (Fig. 5D ). Notably, the degree of binding to DENV-2 reflected the concentrations of MBL in different serum samples ( Fig. 5A and D) , as a positive correlation was observed (R 2 ϭ 0.868 and P Ͻ 0.0001 [ Fig. 5G] ). As expected, MBL concentrations also modulated the efficiency of serum-dependent neutralization of DENV-2; sera from individuals with higher blood MBL levels neutralized virus more effectively than those with lower MBL levels (Fig. 5E ). However, weakly neutralizing activity observed in some serum samples also could be due to deficiencies of other complement components, especially C4 whose functional level is influenced by several factors, including the number of copies of the C4A and C4B genes (32) . To evaluate this, functional C4 and total complement hemolytic activity (CH50 levels [dose of complement that lyses 50% of a red blood cell suspension]) in sera were measured. In contrast to serum MBL concentrations (Fig. 5A ), no significant difference (P Ͼ 0.05) in functional C4 (Fig. 5B) and CH50 (Fig. 5C ) levels was observed among donors. Importantly, neutralization of DENV-2 by sera from all donors except donors 8 and 11 was antibody independent and MBL specific, as the addition of excess soluble mannose abrogated the neutralizing activities (Fig. 5F ). The neutralization of DENV-2 by sera from donors 8 and 11, which later were determined to contain DENV-2 specific IgG (data not shown), was MBL independent, and likely occurred through activation of the classical complement pathway. Additionally, a high level of anti-DENV-2 specific IgG in serum from donor 8 may have interfered with the serum MBL binding to DENV-2 virions, resulting in a relatively low signal in the capture ELISA (Fig. 5D) . Overall, a positive correlation was observed between neutralization and serum MBL levels (R 2 ϭ 0.662 and P ϭ 0.0002 [ Fig. 5H]) . Notably, the sera from three individuals (donors 14, 15, and 16) carrying the structural variant MBL alleles (B, C, or D, and thus markedly reduced MBL levels) also had very low neutralizing activities (Table 1 ). Because mammalian cell-derived DENV-2 was less efficiently inhibited by human serum (Fig. 4A and C) , a higher percentage of serum (35%) was used in neutralization assays in the absence (see Fig. S3A in the supplemental material) or presence (Fig. S3B) of mannose. Despite a lower level of inhibition, a positive correlation was also present between serum MBL levels and neutralization of mammalian cell-derived DENV-2 (R 2 ϭ 0.623 and P ϭ 0.0005 [ Fig. S3C]) .