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Vaccination responses to Neisseria meningitidis and Haemophilus influenzae type b in two C2-deficient sisters
74 215- A CLUSTER OF POSITIVELY CHARGED AMINO ACIDS AT THE INTERFACE BETWEEN CIBP CCPl AND CCP2 IS CRUCIAL FOR BINDING OF C4b AND STREPTOCOCCAL PROTEINS Blom A. &I.*,Villoutreix, B. O.*, Webb, J. H.*, Be&rd K.s, Fohyn&dura A.*, Lii G.s and DablbiickB’. *LundUniversity, Dept. of Cliical Chemistry The Wallenberg Laboratorv: MAS: S-205 02 Malmd SWEDEN: Fax: +46 40 33 70 44; &roil: ‘[email protected] #Lund Universitv. Dent. of Labontorv Medicine
role of a clusterof positively &s&d amino acids at the interface between CCPl and CCP2 of the a-chain for the nhvsioloeical functionsof C4BP. We have qressed and puri6ed9;rmtsn7sof C4BP where positively charged aa were mutatedinto Glu. We found that all -mutants displayed decmamd apjmrent aftinities for C4b clearly showing the importance of the cluster for this interaction. The mutations also affected the decay accelerating activity of C4BP and the ability of the protein to inhibit assemblv of the C3-convettase. It ten tbereforc be conch&d thst these ti&tions sre related to the C4b biding ability of CQBP. Binding of CXBP to many strains of Strqtococcws uj.wgeacs is mediated by surtbce M proteins (Arp4&?2), which r& major virulence factors. Results obtained from the analvsis of our mutants indicate thst C4b and M proteins have overlapping, but not identical, binding sites on C4BP. Accordingly, six moncclonal antibodies dimcted against the a-chain interfered with the C4b-C4BP interaction, whereas only two of them efftciently inhibited binding of C4BP to Arp4/Sir22. The interaction Lxtwcen CXBPand Arp4 wss relatively insensitive to changes in salt concentration, m contrast to the C4BP-C4b binding. Our results suggest that biing between C4h and C4BP is governed mostly by electrostatic interactions, while additional non-covalent forces cause tight binding of C4BP to strrptoeoccal M proteins.
216- THE MEMBRANE ATIACK COMPLEX INHIBITOR OF STREPTOCOCCUS PYOGENES (SIC) BINDS PREFEREi’iTIALLY TO C567 Barbara A. Fen&-King. David J. ScilJy, Alexaudra Davies, PetcrJ. Lachmann Microbial Immunology Group, Centre for Veterinary Science, University of Cambridge,Madingley Road, Cambridge, UK Streptococcal Inhibitor of Complement (SIC) (1). is an extremely variable 3lkDa secreted protein with no homology to any other known proteins. The SIC gene is found in only 4 of the many M types of S. pyog., including the extremely virulent Ml strain, and is reported to bind to Clusterin and to incorporate into the membrane attack complex (MAC) and inhibit its function To investigate the mcchauism by which SIC inhibits the activity of MAC, we have generated His-tagged recombinant SIC (rSIC) in Ecoli and have purified native SIC from type Ml culture supernatauts. We find that both SIC and rSIC migrate on SDS-PAGE at -37 kDa and have hydrophobic characteristics, although from sequence analysis SIC should have a high negative charge. ELISA results show that SIC and rSIC bind to preformed tennirxd complement complexes (ICC) and C5b-7 complexes but not to C5b6. demonstrating that SIC binding is C7 dependent. Since these assays were performed using whole/deficient sera, it may be that SIC binds indirectly to the TCC via Clusterin. SIC and rSIC also weakly inhibit formation of the TCC. SIC and rSIC inhibit reactive lysis of guinea pig crythrocytes by human complement, albeit weakly, in that 2 PM SIC is required for 50% inhibition. These data suggest that inhibition of the MAC may not be the primary role of SIC. Further characterisation of SIC is ongoing. 1. Akesson, P, Sjoholm, A. G. and Bjorck, L. (1996). J. Biol. ‘&cm. 271(2), 1081.1088
217- EFFECTS ON C3 OF THE SECRETED CATHEPSIN L PROTEINASES FROM THE PARASITE FASCIOLA HEPATICA. MartIn Cigandar, Antony C. Willil, John P. Dalton3 & Alvaro Diaz’. 1. Immunology Dept., Faculty of Chemistry/Sciences, University of Uruguay. 2. MRC hnnumochemistry Unit, Dept. of Biochemistry, University of Oxford, UK. 3. School of Biotechnology, Dublin City University, Dublin, Ireland. Fasciola hepatica (liver fluke) is a trematode parasite of livestock and humans. Juvenile flukes migrate in the peritoneal cavity and liver pamnchyma, while adult worms dwell in the bile ducts. A major biological fact about F. hepotica is that it secretes strikingly high levels of proteinases. The major protein species secreted by adult flukes are two cysteine proteinases of the cathepsin L family (CL1 and CL2). Juvenile flukes also secrete cathepsin Ls. We are analysing the actions of these proteinases on host complement. Both CL1 and CL2 were observed to cleave human C3 cleanly, generating C3b-like and C3a-lie products. The C3b-like fragments formed were found to be active in a factor B activation assay. The fragments were also susceptible to inactivation by thctor I in the presence of factor H. The cleavage site on C3 was hxalised, in the case of CL2, to the bond between A“” and R748,ie one residue Nterminal from the physiological activation cleavage site. Thus the action of the proteinase on C3 generates Arg-C3b, which would be inactivated in viva by factors I and H, and C3a de&g, inactive as an inflammatory mediator. This could be the basis of an evasion mechanism by the parasite. .............................................................................................................. ,..............
1
21 S- VACCINATION RESPONSES TO NEISSERIA AND HAEMOPHIL US INFL UENZAE TYPE b IN TWO CZ-DEFICIENT SISTERS. Barbro Selander*, Helena Kayhty”,Elisabeth Wedege5, EvsL Hoimstrom*, Lennart Truedsson’, Claes Soderstr6m5s and Anders G. Sjoholm*. *Institute of Laboratory Medicine, and @Departmentof Infectious Diseases, University Hospital of Lund, Sweden. %ational Public Health Institute, Helsinki, Finland. *Department of Vaccinology, National Institute of Public Health, Oslo, Norway. The CZ-deficient sisters were vaccinated with tetravalent meningococcal vaccine and a conjugate Haemophilus infruemae type b vaccine, Three main points emerged from the analysis. First, vaccination resulted in serum bactericidal responses demonstrating anticapsular antibodymediated recruitment of the alternative pathway. Second, addition of C2 to prevaccination sera produced bactericidal activity in the absence of anticapsular antibodies, which suggested that the bactericidal action of antibodies to subcapsular antigens detected in the sera require an intact classical pathway. A third point concerned a previously unrecognized type of blocking activity. Thus, the postvaccination sers of one sister contained IgG that inhibited killing of serogroup W-135 in C2-deficient serum, and the deposition of C3 on ELISA plates coated with purified W-135 polysaccharide. Our findings suggested that the blocking was serogroup-specific and dependent on early classical pathway components. The functional vaccination responses should encourage further trial of capsular vaccines in classical pathway deficiency states. J MENINGITIDIS
role of a clusterof positively &s&d amino acids at the interface between CCPl and CCP2 of the a-chain for the nhvsioloeical functionsof C4BP. We have qressed and puri6ed9;rmtsn7sof C4BP where positively charged aa were mutatedinto Glu. We found that all -mutants displayed decmamd apjmrent aftinities for C4b clearly showing the importance of the cluster for this interaction. The mutations also affected the decay accelerating activity of C4BP and the ability of the protein to inhibit assemblv of the C3-convettase. It ten tbereforc be conch&d thst these ti&tions sre related to the C4b biding ability of CQBP. Binding of CXBP to many strains of Strqtococcws uj.wgeacs is mediated by surtbce M proteins (Arp4&?2), which r& major virulence factors. Results obtained from the analvsis of our mutants indicate thst C4b and M proteins have overlapping, but not identical, binding sites on C4BP. Accordingly, six moncclonal antibodies dimcted against the a-chain interfered with the C4b-C4BP interaction, whereas only two of them efftciently inhibited binding of C4BP to Arp4/Sir22. The interaction Lxtwcen CXBPand Arp4 wss relatively insensitive to changes in salt concentration, m contrast to the C4BP-C4b binding. Our results suggest that biing between C4h and C4BP is governed mostly by electrostatic interactions, while additional non-covalent forces cause tight binding of C4BP to strrptoeoccal M proteins.
216- THE MEMBRANE ATIACK COMPLEX INHIBITOR OF STREPTOCOCCUS PYOGENES (SIC) BINDS PREFEREi’iTIALLY TO C567 Barbara A. Fen&-King. David J. ScilJy, Alexaudra Davies, PetcrJ. Lachmann Microbial Immunology Group, Centre for Veterinary Science, University of Cambridge,Madingley Road, Cambridge, UK Streptococcal Inhibitor of Complement (SIC) (1). is an extremely variable 3lkDa secreted protein with no homology to any other known proteins. The SIC gene is found in only 4 of the many M types of S. pyog., including the extremely virulent Ml strain, and is reported to bind to Clusterin and to incorporate into the membrane attack complex (MAC) and inhibit its function To investigate the mcchauism by which SIC inhibits the activity of MAC, we have generated His-tagged recombinant SIC (rSIC) in Ecoli and have purified native SIC from type Ml culture supernatauts. We find that both SIC and rSIC migrate on SDS-PAGE at -37 kDa and have hydrophobic characteristics, although from sequence analysis SIC should have a high negative charge. ELISA results show that SIC and rSIC bind to preformed tennirxd complement complexes (ICC) and C5b-7 complexes but not to C5b6. demonstrating that SIC binding is C7 dependent. Since these assays were performed using whole/deficient sera, it may be that SIC binds indirectly to the TCC via Clusterin. SIC and rSIC also weakly inhibit formation of the TCC. SIC and rSIC inhibit reactive lysis of guinea pig crythrocytes by human complement, albeit weakly, in that 2 PM SIC is required for 50% inhibition. These data suggest that inhibition of the MAC may not be the primary role of SIC. Further characterisation of SIC is ongoing. 1. Akesson, P, Sjoholm, A. G. and Bjorck, L. (1996). J. Biol. ‘&cm. 271(2), 1081.1088
217- EFFECTS ON C3 OF THE SECRETED CATHEPSIN L PROTEINASES FROM THE PARASITE FASCIOLA HEPATICA. MartIn Cigandar, Antony C. Willil, John P. Dalton3 & Alvaro Diaz’. 1. Immunology Dept., Faculty of Chemistry/Sciences, University of Uruguay. 2. MRC hnnumochemistry Unit, Dept. of Biochemistry, University of Oxford, UK. 3. School of Biotechnology, Dublin City University, Dublin, Ireland. Fasciola hepatica (liver fluke) is a trematode parasite of livestock and humans. Juvenile flukes migrate in the peritoneal cavity and liver pamnchyma, while adult worms dwell in the bile ducts. A major biological fact about F. hepotica is that it secretes strikingly high levels of proteinases. The major protein species secreted by adult flukes are two cysteine proteinases of the cathepsin L family (CL1 and CL2). Juvenile flukes also secrete cathepsin Ls. We are analysing the actions of these proteinases on host complement. Both CL1 and CL2 were observed to cleave human C3 cleanly, generating C3b-like and C3a-lie products. The C3b-like fragments formed were found to be active in a factor B activation assay. The fragments were also susceptible to inactivation by thctor I in the presence of factor H. The cleavage site on C3 was hxalised, in the case of CL2, to the bond between A“” and R748,ie one residue Nterminal from the physiological activation cleavage site. Thus the action of the proteinase on C3 generates Arg-C3b, which would be inactivated in viva by factors I and H, and C3a de&g, inactive as an inflammatory mediator. This could be the basis of an evasion mechanism by the parasite. .............................................................................................................. ,..............
1
21 S- VACCINATION RESPONSES TO NEISSERIA AND HAEMOPHIL US INFL UENZAE TYPE b IN TWO CZ-DEFICIENT SISTERS. Barbro Selander*, Helena Kayhty”,Elisabeth Wedege5, EvsL Hoimstrom*, Lennart Truedsson’, Claes Soderstr6m5s and Anders G. Sjoholm*. *Institute of Laboratory Medicine, and @Departmentof Infectious Diseases, University Hospital of Lund, Sweden. %ational Public Health Institute, Helsinki, Finland. *Department of Vaccinology, National Institute of Public Health, Oslo, Norway. The CZ-deficient sisters were vaccinated with tetravalent meningococcal vaccine and a conjugate Haemophilus infruemae type b vaccine, Three main points emerged from the analysis. First, vaccination resulted in serum bactericidal responses demonstrating anticapsular antibodymediated recruitment of the alternative pathway. Second, addition of C2 to prevaccination sera produced bactericidal activity in the absence of anticapsular antibodies, which suggested that the bactericidal action of antibodies to subcapsular antigens detected in the sera require an intact classical pathway. A third point concerned a previously unrecognized type of blocking activity. Thus, the postvaccination sers of one sister contained IgG that inhibited killing of serogroup W-135 in C2-deficient serum, and the deposition of C3 on ELISA plates coated with purified W-135 polysaccharide. Our findings suggested that the blocking was serogroup-specific and dependent on early classical pathway components. The functional vaccination responses should encourage further trial of capsular vaccines in classical pathway deficiency states. J MENINGITIDIS