In this study, we investigated the contribution of L-ficolin and serotype-specific IgG in cord sera to the opsonophagocytic killing of strains of various serotypes of GBS. The contributions of these factors varied between serotypes. L-ficolin and serotype-specific IgG appeared to contribute synergistically in cord sera to opsonophagocytic killing of bacteria when the concentration of serotype III-specific IgG was 1 to 6 μg/ml and when the concentration of serotype V-specific IgG was 2 to 5 μg/ml. Serotype III-specific IgG is mostly IgG2 (9
), a subclass that is less effective in both activating the classical pathway and binding to PMN Fc receptors (12
). Using adult serum, Aoyagi et al. (2
) showed that an increase in L-ficolin-mediated opsonophagocytic killing of serotype III GBS is observed when bacteria are preincubated with serotype-specific IgG2 before the alternative pathway is activated but that no increase in killing is observed when bacteria are incubated with the IgG2 anti-serotype III antibody after the alternative pathway is activated, indicating that the increase in killing is due not to Fc receptor-mediated binding to PMNs but to IgG2-mediated alternative-pathway activation. By assessing the inhibitory effects of monoclonal antibodies to FcRII and FcRIII, Noya et al. (31
) showed that FcRII is the primary PMN receptor in the phagocytosis of serotype III GBS opsonized with antibody alone whereas FcRII is not necessary in the presence of both antibody and complement when adult serum is used as the source of complement. Two experiments, in the present study and in that by Aoyagi et al. (2
), show that C3b deposition is markedly lower on GBS incubated in cord sera than on GBS incubated in adult serum. Deposition of C3b on a serotype III GBS strain that had been opsonized with cord sera was less than 60% of that of the same bacteria opsonized with an adult serum, with the exception of a single cord serum (). This low level of C3b deposition in cord serum compared with that in adult serum is probably a consequence of the low complement activity in cord serum compared to that in adult serum, a result which we demonstrated by measurement of CH50. It is possible that bacteria cannot be opsonized with C3b in cord serum to a level at which the interaction of IgG with FcRII is not needed for opsonophagocytosis. For serotype III GBS, and perhaps for serotype V GBS, opsonization in cord blood sera presumably involves L-ficolin and/or serotype-specific IgG initiating complement activation, leading to C3b deposition, and then both C3b and IgG, bound to bacteria, acting as opsonins.
For serotype VIII bacteria, it seems that L-ficolin initiates C3b deposition by activating the lectin pathway and then both C3b and serotype-specific IgG, bound to the bacteria, act as opsonins. The bactericidal index was high, however, and serum opsonized the bacteria effectively, independent of L-ficolin and serotype-specific IgG concentrations. This observation may help explain the discrepancy between the high rate of maternal colonization and the low incidence of early-onset disease associated with serotype VIII in Japan (10
The contribution of L-ficolin and serotype-specific IgG to C3b deposition in cord serum was not clear for serotype Ib and VI bacteria, since neither L-ficolin nor IgG concentrations correlated with C3b deposition despite experimental evidence that L-ficolin can bind to these serotypes and initiate complement activation (1
). It is possible that there is another molecule that contributes to complement activation and/or opsonization on serotype Ib and VI bacteria. Mannose-binding lectin, which, like L-ficolin, also initiates the lectin pathway activation, cannot bind to GBS (1
), but antibodies directed against GBS cell wall components other than serotype-specific IgG, such as the α and β components of the C protein complex (17
), the surface immunogenic protein (Sip) (22
), and the surface-bound C5a peptidase (7
), may potentially contribute to complement activation and/or opsonization in these circumstances.
The present study is the first report to determine the contribution of L-ficolin and serotype-specific IgG in cord sera to the opsonophagocytic killing of various serotypes of GBS. It is particularly noteworthy that L-ficolin and serotype-specific IgG appear to contribute synergistically to the opsonophagocytic killing of serotype III GBS, because this serotype causes a significant percentage of early-onset diseases, most late-onset diseases in neonates, and the majority of neonatal meningitis cases (33
). Lin et al. (20
) showed that risk for the early-onset disease caused by serotype III GBS is reduced with increasing serotype III-specific IgG concentrations in cord serum, but both neonates with early-onset disease and neonates colonized by the bacteria at birth who did not develop disease had serotype-specific IgG concentrations that were less than 7 μg/ml. This range of serotype-specific IgG concentrations corresponds to that in some cord sera from neonates whose mothers were immunized with a serotype-specific CPS-tetanus toxoid conjugate vaccine (geometric mean concentration is 7.5 μg/ml) (3
). Since the range of serotype-specific IgG concentrations in which synergy between L-ficolin and IgG was observed is similar to that in cord sera from infected and uninfected neonates, relative L-ficolin deficiency may be associated with an increased risk of early-onset disease caused by serotype III bacteria. The present set of experiments, however, did not address this question, which may be difficult to study because the incidence of early-onset disease has been markedly reduced following the widespread introduction of intrapartum antibiotic prophylaxis.
Three single nucleotide polymorphisms (SNPs) in the promoter region and two SNPs in exon 8 of a L-ficolin gene (FCN2
) that are associated with marked alterations in L-ficolin concentrations and with ligand binding, respectively, have been described (11
). Variations in ligand binding due to this polymorphism may explain the observation that L-ficolin concentrations from some sera bound to bacteria are markedly less than what would be expected from the regression line between concentration and amount of binding to bacteria, but we did not perform SNP analysis on the FCN2
genes in the cord blood samples. The variations in L-ficolin levels and ligand binding associated with these SNPs may also play a role in susceptibility to late-onset neonatal disease for which there are no obvious risk factors except for low levels of serotype-specific IgG (33
). Another factor to be considered is that some clones of GBS may be more highly associated with neonatal infections. In particular, one serotype III clone, previously identified as multilocus enzyme electrophoresis type 1 (ET1) (29
) or restriction digestion pattern type III-3 (RDP III-3) (30
) and now designated CC17 by multilocus sequence typing (MLST) (13
), seems to have a particular propensity to be associated with both early- and late-onset disease in neonates (5
). For the other serotypes, there appear to be one or more clones that are associated with disease and a number of rarer clones that colonize mothers and their babies but that are not usually associated with disease (5
). Clinical investigations into the role of L-ficolin in the prevention of neonatal GBS infection should include analysis of the SNPs of the FCN2
gene and MLST of the isolate.