Mechanisms of mannose-binding lectin-associated serine proteases-1/3 activation of the alternative pathway of complement

Mannose-binding lectin-associated serine proteases-1/3 (MASP-1/3) are essential in activating the alternative pathway (AP) of complement through cleaving pro-factor D (pro-Df) into mature Df. MASP are believed to require binding to mannose binding lectins (MBL) or ficolins (FCN) to carry out their biological activities. Murine sera have been reported to contain MBL-A, MBL-C, and FCN-A, but not FCN-B that exists endogenously in monocytes and is thought not to bind MASP-1. We examined some possible mechanisms whereby MASP-1/3 might activate the AP. Collagen antibody-induced arthritis, a murine model of inflammatory arthritis dependent on the AP, was unchanged in mice lacking MBL-A, MBL-C, and FCN-A (MBL(-/-)/FCN A(-/-) mice) in comparison to wild-type mice. The in vitro induction of the AP by adherent mAb to collagen II was intact using sera from MBL(-/-)/FCN A(-/-) mice. Furthermore, sera from MBL(-/-)/FCN A(-/-) mice lacked pro-Df and possessed only mature Df. Gel filtration of sera from MBL(-/-)/FCN A(-/-) mice showed the presence of MASP-1 protein in fractions containing proteins smaller than the migration of MBL-A and MBL-C in sera from C4(-/-) mice, suggesting possible binding of MASP-1 to an unknown protein. Lastly, we show that FCN-B was present in the sera of MBL(-/-)/FCN A(-/-) mice and that it was bound to MASP-1. We conclude that MASP-1 does not require binding to MBL-A, MBL-C, or FCN-A to activate the AP. MASP-1 may cleave pro-Df into mature Df through binding to FCN-B or to an unknown protein, or may function as an unbound soluble protein.     

A truncated form of mannose-binding lectin-associated serine protease (MASP)-2 expressed by alternative polyadenylation is a component of the lectin complement pathway.

The lectin complement pathway is initiated by binding of mannose-binding lectin (MBL) and MBL-associated serine protease (MASP) to carbohydrates. In the human lectin pathway, MASP-1 and MASP-2 are involved in the proteolysis of C4, C2 and C3. Here we report that the human MBL-MASP complex contains a new 22 kDa protein [small MBL-associated protein (sMAP)] bound to MASP-1. Analysis of the nucleotide sequence of sMAP cDNA revealed that it is a truncated form of MASP-2, consisting of the first two domains (i.e. the first internal repeat and the epidermal growth factor-like domain) with four different C-terminal amino acids. sMAP mRNAs are expressed in liver by alternative polyadenylation of the MASP-2 gene, in which a sMAP-specific exon containing an in-frame stop codon and a polyadenylation signal is used. The involvement of sMAP in the MBL-MASP complex suggests that the activation mechanism of the lectin pathway is more complicated than that of the classical pathway.     

Murine monoclonal antibodies to type Ib polysaccharide of group B streptococci bind to human milk oligosaccharides.

The chemical structures of the repeating units of the type Ib polysaccharide of group B streptococci and of the desialylated form of this antigen are almost identical to those of some oligosaccharides in human milk and certain fetal antigens. The structural similarities suggested that the molecules may be immunologically cross-reactive. Mouse monoclonal antibodies to the sialylated and nonsialylated forms of the type Ib polysaccharide were produced and tested for their ability to bind to immobilized human milk oligosaccharides. One antibody, SMB19, reacted specifically with the sialylated form of the type Ib polysaccharide and was also bound by an affinity column containing immobilized sialyllacto-N-tetraose a. The antibody was eluted from the affinity column with EDTA, since its binding to the antigen was calcium dependent. A second monoclonal antibody. SIbD2, bound specifically to the nonsialylated form of the type Ib polysaccharide and also to immobilized lacto-N-tetraose. The antibody was eluted from the affinity column at an acidic pH and retained immunologic activity. These results further extend our previous observations that certain antibodies raised against group B streptococci can also react with normal human glycoconjugates.     

Isolation of group-specific polysaccharide complexes from group B streptococci by phenol-water extraction

The group specific polysaccharide of Group B streptococci was isolated either by means of phenol-water extraction or by Lancefield extraction procedures. Two groups of rabbits were immunized with these substances to induce polyclonal antibodies. The phenol-water extract induced antisera had a significantly higher titer against B streptococci than against A-, D- and G streptococci in the agglutination reaction. The B-specific titers in rabbits immunized with phenol-water extract were significantly higher than those obtained in the rabbits immunized with Lancefield extract. In counter immunoelectrophoresis, only the antisera prepared by immunization with phenol-water extract reacted with standard antigen from group B streptococci. The serological specificity of the phenol-water extract antigen isolated from B streptococci was confirmed by counterimmunoelectrophoresis. The antigen was a heterologous fraction with a molecular mass of 8 x 10(4)-2 x 10(6) Dalton.     

Immunochemistry of capsular type polysaccharide and virulence properties of type VI Streptococcus agalactiae (group B streptococci).

The immunochemistry of capsular type polysaccharide and virulence characteristics of group B streptococci (GBS), type VI, were studied. By high-pressure anion-exchange chromatography and pulsed amperometric detection, as well as by 13C nuclear magnetic resonance analysis, both extracellular and cell-bound polysaccharides were found to contain glucose, galactose, and N-acetylneuraminic acid in the molar ratio of 2:2:1, respectively. At variance with all other GBS serotypes described to date (Ia, Ib, II, III, IV, and V), no N-acetylglucosamine was present, whatever the source of the material (secreted or cell bound; reference or clinical isolate). Sialic acid was probably involved in the immunodeterminant structure of this new serotype since cleavage of this sugar from the polysaccharide gave rise to an antigen which reacted very weakly with type VI antiserum and to a precipitation line in immunodiffusion with no identity with the native type VI polysaccharide. By using type VI antiserum and the protein A-gold technique, a large capsule was observed in the type VI GBS reference strain by electron microscopy. All type VI strains examined were lethal for CD-1 mice, the 50% lethal dose after intraperitoneal challenge ranging from 1.0 (+/- 0.9, standard deviation) x 10(5) to 2.5 (+/- 1.5, standard deviation) x 10(5) CFU per mouse. A rabbit antiserum against capsular type polysaccharide exhibited both protective activity for mice injected intraperitoneally with type VI reference strain or with clinical isolates and opsonic activity in a phagocytosis assay.     

Specificity and protective activity of murine monoclonal antibodies directed against the capsular polysaccharide of type III group B streptococci.

We have obtained 41 monoclonal antibodies directed against type III group B streptococci by immunizing Balb/c mice with formalin-killed bacteria. All of these antibodies reacted with purified type-specific carbohydrate by enzyme-linked immunosorbent assay and immunoprecipitation tests. The epitope recognized by all of these antibodies was associated with terminal sialic acid residues, as indicated by abrogation of immune reactions by treatment of the type-specific carbohydrate with neuraminidase. Two purified monoclonal antibodies (the IgM P9D8 and the IgG3 P4F12) were further characterized for their protective activity in a neonatal rat model of infection. P9D8 and P4F12 antibodies were significantly protective when administered in a dose of 0.5 and 2.5 mg/kg, respectively, at the same time as 3 x 10(5) colony forming units of type III streptococci. Protection was still observed when the antibodies were given up to 9 h after challenge. No protection was afforded against infections with type Ia/c and II streptococci. Similarly, both antibodies effectively opsonized type III, but not Ia, Ib or II bacteria, in an in vitro assay. These and similar, previously described, monoclonal antibodies may be useful, possibly after "humanization" by genetic engineering, for the therapy of neonatal group B streptococcal infections.     

Prevention of C3 deposition by capsular polysaccharide is a virulence mechanism of type III group B streptococci.

Strains of type III group B streptococci isolated from patients with neonatal sepsis are generally resistant to complement-mediated phagocytic killing in the absence of specific antibody. It has been suggested that the resistance of type III group B streptococci to phagocytosis results from inhibition of alternative-complement-pathway activation by sialic acid residues of the type III polysaccharide. To better define the relationship between structural features of the type III capsule and resistance of type III group B streptococci to complement-mediated phagocytic killing, we measured deposition of human C3 on group B streptococcal strains with altered capsule phenotypes. C3 binding was quantified by incubating bacteria with purified human 125I-C3 in 10% serum. Wild-type group B Streptococcus sp. strain COH1 bound eightfold fewer C3 molecules than did either of two isogenic mutant strains, one expressing a sialic acid-deficient capsule and the other lacking capsule completely. Similar results were obtained when the incubation with 125I-C3 was performed in serum chelated with Mg-ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'- tetraacetic acid (MgEGTA), suggesting that the majority of C3 deposition occurred via the alternative pathway. In contrast to the wild-type strain, which was relatively resistant, both mutant strains were killed by human leukocytes in 10% serum with or without MgEGTA. We also measured C3 binding to 14 wild-type strains of type III group B streptococci expressing various amounts of capsule. Comparison of degree of encapsulation with C3 binding revealed a significant inverse correlation (r = -0.72; P less than 0.01). C3 fragments released by methylamine treatment of wild-type strain COH1 were predominantly in the form of C3bi, while those released from the acapsular mutant were predominantly C3b and those from the asialo mutant represented approximately equal amounts of C3b and C3bi. We conclude from these studies that the sialylated type III capsular polysaccharide inhibits alternative-pathway activation, prevents C3 deposition on group B streptococci, and protects the organisms from phagocytic killing.     

Virulence properties of type VII Streptococcus agalactiae (group B streptococci) and immunochemical analysis of capsular type polysaccharide.

Strains of a new polysaccharide type of group B streptococci (GBS), type VII, have been isolated from human carriers and invasive infections. Some of these strains bear the protein antigen c or R, as do other GBS serotypes. The capsular type polysaccharide is sialylated and this residue is involved in the immunodeterminant structure. All type VII strains examined were virulent in CD-1 mice; the LD50 after intraperitoneal (i.p.) challenge was 4.57 (SD 0.12) x10(7) cfu for the reference strain and 5.49 (SD 1.5) x10(7) cfu for clinical isolates. A particular feature of this serotype was the ability to induce septic arthritis not only when injected intravenously (i.v.), but also when injected i.p. Rabbit antiserum against the capsular type VII polysaccharide exhibited opsonic activity in a phagocytosis assay and protective activity against infection.     

Expression of H-ficolin/Hakata antigen,mannose-binding lectin-associated serine protease (MASP)-1 and MASP-3 by human glioma cell line T98G

Mannose-binding lectin (MBL) is a C-type lectin involved in the first line of host defense and it requires MBL-associated serine proteases (MASP) for activation of the lectin complement pathway (LCP). Recently we reported that human ficolins, L-ficolin/P35 and H-ficolin/Hakata antigen, as well as MBL activate the LCP in association with MASP. We investigated in vitro expression of complements of the lectin complement pathway in several cell lines. Out of 17 cell lines tested using RT-PCR, a human glioma cell line, T98G, expressed high levels of H-ficolin/Hakata antigen, MASP1 and MASP3 mRNAs. Similar results were obtained in four other glioma lines. In addition, mRNAs for C1r, C1s, C2, C3, C4, C5 and C6 were also detected in T98G cells, but very low amount of mRNAs for C1q and MBL. MBL mRNA was seen in two of the other glioma cell lines. An ELISA of culture supernatants showed that T98G cells secreted a considerable amount of MASP-1 and MASP-3 proteins. SDS-PAGE and immunoblotting analyses showed the secreted H-ficolin/Hakata antigen, MASP-1 and MASP-3 to be 34, 81 and 105 kDa in size respectively, similar to their serum counterparts. Since the glioma cells used are derived from astrocytes, this suggests that human astrocytes may be a source of some components of the LCP in the brain.     

Functional activity of antibodies to the group B polysaccharide of group B streptococci elicited by a polysaccharide-protein conjugate vaccine.

Group B streptococci (GBS) are a major cause of sepsis and meningitis in infants. While antibodies directed to the type-specific GBS capsule have been shown to be protective, it is less clear whether antibodies to the group B polysaccharide, a noncapsular, cell wall-associated antigen, may play a role in immunity. To investigate the functional activity of group B polysaccharide-specific antibodies, we tested sera from rabbits vaccinated with group B polysaccharide coupled to tetanus toxoid (B-TT). Anti-B-TT was weakly opsonic in vitro for a highly encapsulated type III strain, while antiserum elicited by vaccination with type III capsular polysaccharide linked to tetanus toxoid (III-TT) was a very effective opsonin. In contrast to anti-III-TT, anti-B-TT given before or after bacterial challenge was only marginally effective in protecting newborn mice against lethal infection with type III GBS. The number of C3 molecules bound to type III GBS was augmented by anti-III-TT but not by high antibody concentrations of anti-B-TT. These results suggest that the difference in opsonic activity between anti-B-TT and anti-III-TT may be due to a difference in their ability to deposit C3. In addition, the maximum number of antibody molecules bound to the bacterial surface was greater for anti-III-TT than for anti-B-TT. That anti-B-TT binds to fewer sites than anti-III-TT may explain the differences in complement activation and in opsonic and protective efficacy of antibodies to group B polysaccharide compared with antibodies to the type-specific capsular polysaccharide.     

Sialic acid of group B Neisseria meningitidis regulates alternative complement pathway activation.

The effect of meningococcal cell-associated sialic acid on activation of the human alternative complement pathway was examined by using a quantitative fluorescence immunoassay to assess alternative pathway-mediated C3 binding to a group B strain of Neisseria meningitidis from which graded amounts of sialic acid had been removed with neuraminidase. Using human serum absorbed with strain B16B6 (B:2a:L2,3) and chelated with 10 mM MgCl2 and 10 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, we found an increase in the amount of C3 bound by enzymatically desialylated B16B6 organisms over the amount bound by fully sialylated organisms. This increase was proportional to the amount of sialic acid cleaved from the bacteria. Enhanced C3 binding was accompanied by an increase in factor B deposition. A sialic acid-deficient mutant of strain B16B6, designated 2T4-1, bound C3 via the alternative pathway at a level equivalent to that bound by wild-type meningococci from which 88% of the sialic acid had been removed. Strain B16B6 was resistant to the alternative pathway-mediated bactericidal activity of both absorbed and hypogammaglobulinemic human sera, whereas noncapsular variant 2T4-1 was sensitive to these sera. The addition of purified immune immunoglobulin M (IgM) and IgG significantly increased the alternative pathway-mediated killing of strain B16B6 organisms. IgM mediated increased bactericidal activity without an increase in C3 or factor B deposition. In contrast, the IgG-mediated killing was associated with increased binding of C3 and factor B to the organisms. Absorption studies showed that the IgM bound to the sialic acid capsule, whereas the IgG bound to noncapsular surface antigens. We conclude from these results that the group B meningococcal sialic acid capsule inhibits activation of the alternative pathway in the nonimmune host and that both IgM and IgG, although specific for different surface antigens, are capable of augmenting the alternative pathway-mediated killing of group B meningococci.     

Antibodies to the capsular polysaccharide of Neisseria meningitidis group B or E. coli K1 bind to the brains of infant rats in vitro but not in vivo

The binding of monoclonal and polyclonal IgG antibodies specific to the capsular polysaccharide of Neisseria meningitidis group B and E. coli K1 was tested to the cross-reacting polysialosyl structures previously shown to be present in the brain of infant rats (Lancet 1983; ii: 355-7). Strong immunofluorescence was obtained after in vitro incubation of the brains of 1 to 13 days old rats with the antibodies whereas the brains of adult rats remained negative. The number of antibody-binding structures decreased as a function of age, being highest at the age of 1 to 5 days. However, when the same antibodies were injected intraperitoneally into the infant rat, or into the mother rat 2 days before parturition, no binding of antibodies to the infant rat brain tissue was observed.     

Impaired opsonophagocytosis of serotypes Ib and II of group B streptococci as compared with serotypes Ia and III: role of the alternative pathway of complement in opsonisation of serotype III of group B streptococci.

Using the technique of phagocytic chemiluminescence, we have shown that serotypes Ib and II of group B streptococci are resistant to opsonophagocytosis. The resistant strains became susceptible to opsonophagocytosis by trypsin treatment, but neuraminidase had no effect. Several studies have failed to define a significant role for the alternative pathway of complement in opsonisation of group B streptococci. By simple chelation and heat inactivation studies, we have shown that the alternative pathway of complement is activated by serotype III of group B streptococci.     

Exon structure of the gene encoding the human mannose-binding protein-associated serine protease light chain: comparison with complement C1r and C1s genes

Mannan or mannose-binding protein (MBP) requires a novel serineprotease termed MBP-associated serine protease (MASP) for activationof the complement cascade. In this study, we analyzed MASP genomicclones and found that the light chain (catalytic domain) isencoded by six exons, whereas those of the complement C1 subunits,C1r and C1s, and the haptoglobin segment have been reportedto be encoded by a single exon. We confirmed the intron-lackingsequence of C1r by analysis of its genome. These results, inconjunction with those obtained by constructing a phylogenetictree for these proteins, suggest that the MASP gene is a prototypeand that the Intron-lacking sequences of the other serine proteaseshave a more recent history.     

L-ficolin and capsular polysaccharide-specific IgG in cord serum contribute synergistically to opsonophagocytic killing of serotype III and V group B streptococci

Group B streptococci (GBS; Streptococcus agalactiae) are the most common cause of neonatal sepsis and meningitis. Serotype-specific IgG antibody is known to protect neonates against GBS infections by promoting opsonophagocytosis. The L-ficolin-mediated lectin pathway of the complement is also a potential mechanism for opsonization of GBS, because L-ficolin activates the complement after binding to serotype Ib, III, V, VI, and VIII GBS. In the present study, we investigated how L-ficolin and serotype-specific IgG in cord sera contribute to opsonophagocytic killing of GBS. Neither L-ficolin nor serotype-specific IgG concentrations correlated with C3b deposition on serotype Ib and VI GBS, suggesting L-ficolin- and serotype-specific IgG-independent mechanisms of complement activation. The percentage of serotype VIII GBS killed was high regardless of the concentration of L-ficolin and IgG. In contrast, L-ficolin and serotype-specific IgG can each initiate C3b deposition on serotype III and V GBS and promote phagocytosis by polymorphonuclear leukocytes, but L-ficolin and serotype-specific IgG together promote opsonophagocytic killing to a greater extent than does either alone in vitro. This synergy was observed when serotype III-specific IgG concentrations were between 1 and 6 μg/ml and when serotype V-specific IgG concentrations were between 2 and 5 μg/ml. Concentrations of serotype III-specific IgG in cord blood above 7 μg/ml are considered protective for neonates colonized with GBS, but most neonates with IgG levels of less than 7 μg/ml do not develop GBS infections. The data presented here suggest that L-ficolin enhances opsonophagocytosis of serotype III and V GBS when serotype-specific IgG alone is suboptimal for protection.     

脑膜炎奈瑟菌A群荚膜多糖与B群外膜蛋白复合物偶联物的制备和免疫原性

目的　脑膜炎奈瑟菌 (Nm)A群荚膜多糖与B群菌株 34 0 7或 5 42 85 2外膜蛋白复合物偶联 ,希望获得 1种对A和B群Nm感染皆有预防效果 ,并可提高A群荚膜多糖对婴幼儿免疫力的偶联物。方法　用饱和硫酸铵沉淀细菌培养上清液 ,沉淀物再经SephacrylS 30 0纯化获得外膜蛋白复合物。通过碳化二亚铵 (EDC)介导的缩合反应将B群外膜蛋白复合物与A群荚膜多糖偶联。偶联物、未偶联的荚膜多糖、B群外膜蛋白复合物及A群荚膜多糖和B群外膜蛋白复合物简单混合物按相同程序免疫小鼠获得抗体 ,经ELISA、杀菌力试验和Westernblotting测定了偶联物的免疫原性。结果　偶联物比未偶联的荚膜多糖或者荚膜多糖同B群外膜蛋白复合物简单混合抗原的免疫原性增强 2 1～32 0倍 ;其中以B群 34 0 7菌株的外膜蛋白复合物与A群荚膜多糖偶联的效果更好。偶联物免疫血清不仅对A群代表菌株 (2 90 19)和B群菌株 (34 0 7,5 42 85 2和 2 90 2 1)均有较强的杀菌活性 ,而且还对所试的具有不同菌型特征的其它 8株B群菌株仍有较强的交叉反应性。Westernblotting初步分析发现上述两种偶联物免疫的血清与各自的外膜蛋白复合物在相对分子质量约 42× 10 3 、39× 10 3 和 2 6× 10 3 处有相同的反应带 ,其中 42× 10 3 左右的条带为 1类外膜蛋白。结论     

Synthesis and immunological properties of conjugates composed of group B streptococcus type III capsular polysaccharide covalently bound to tetanus toxoid

A synthetic scheme for covalently binding group B streptococcus type III to tetanus toxoid (TT), using adipic acid dihydrazide as a spacer, is described. Type III alone or as a conjugate with TT was injected subcutaneously into laboratory mice, and the type-specific and TT antibody responses elicited by these immunogens were assayed. Type III-TT elicited significantly higher levels of type-specific antibodies after each immunization than did the type III alone. These levels were related to the dosage of the conjugate, enhanced by Freund adjuvant, and exhibited booster responses. Type III alone elicited only immunoglobulin M (IgM) antibodies in Swiss albino mice and mostly IgM and low levels of IgG antibodies of the IgG3 subclass in BALB/c mice. Type III-TT conjugates, in contrast, elicited mostly IgG antibodies in both strains of mice. IgA type III antibodies were not detected. The first two immunizations with the conjugates elicited type III antibodies in the IgG1 and in the IgG3 subclasses. Low levels of IgG2a type III antibodies were detected after a third injection of type III-TT. Conjugate-induced antibodies facilitated opsonization of group B streptococcus type III organisms and did not react with the structurally related pneumococcus type 14. TT alone or as a component of type III-TT induced mostly antibodies of the IgG class: IgG1 levels were the highest of the four subclasses. No IgA TT antibodies were detected. The conjugation procedure, therefore, enhanced the immunogenicity of and conferred T-cell dependent properties to the type III while preserving the immunogenicity of the TT component. The T-cell dependent properties of the conjugates were responsible for stimulating IgG type III antibodies which could be boosted. Evaluation of type III-TT conjugates in antibody-negative women of child-bearing age is planned.