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Factor H-related protein 3 (FHR-3) inhibits factor H binding to pentraxins and malondialdehyde epitopes, and activates the alternative pathway via C3b binding
Abstracts / Immunobiology 221 (2016) 1131–1225
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Factor H-related protein 3 (FHR-3) inhibits factor H binding to pentraxins and malondialdehyde epitopes, and activates the alternative pathway via C3b binding
Mechanisms responsible for differential bactericidal activities of human and rabbit complement for Neisseria meningitidis
Csincsi 1,∗ ,
Pouw 2 ,
Ádám I. Richard B. Agustín Tortajada 3 , Santiago Rodríguez de Córdoba 3 , Diana Wouters 2 , Mihály Józsi 1 1
MTA-ELTE “Lendület” Complement Research Group, Department of Immunology, Eötvös Loránd University, Budapest, Hungary 2 Department of Immunopathology, Sanquin Research and Landsteiner laboratory of the Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands 3 Department of Cellular and Molecular Medicine, Centro de Investigaciones Biológicas and Ciber de Enfermedades Raras, Madrid, Spain Factor H-related 3 (FHR-3) is one of the five human FHR proteins homologous to the complement alternative pathway inhibitor factor H (FH). In contrast to FH, however, their biological function is controversial and poorly defined. Genetic and functional studies implicated FHRs, including FHR-3, in inflammatory, autoimmune and infectious diseases. The aim of the current study was to further characterize FHR-3 in terms of ligand interactions and role in the regulation or modulation of complement activation. Recombinant FHR-3 bound to the acute phase proteins pentraxin 3 (PTX3) and C-reactive protein. PTX3, but not CRP, was able to bind C1q when bound to FHR-3. Similar to FHR-5, and in contrast to FHR-1, FHR-3 enhanced C3 deposition on PTX3- and CRP-coated wells, but not on wells coated with the extracellular matrix MaxGel. Contrary to a previous report, we found that FHR-3 did not display cofactor activity for the cleavage of C3b by factor I even when added in supra-physiological, 2 M concentration. However, we found that FHR-3, similar to FHR-4 and FHR-5, was able to support the formation of the C3bBb convertase by binding C3b, and this convertase in turn could generate C3a from C3. In line with this, FHR-3 activated the alternative pathway in serum and caused enhanced C3 deposition. Importantly, FHR3 bound to malondialdehyde-BSA adduct and dose-dependently inhibited FH binding to malondialdehyde epitopes. These results identify novel pentraxin ligands of FHR-3, demonstrate complement deregulation on pentraxins by this factor H-related protein, lack of cofactor activity, but enhancement of alternative pathway activation. Thus, FHR-3 is not a complement inhibitor, but it rather promotes opsonization and complement activation. Moreover, the competitive inhibition of FH binding to malondialdehyde epitopes by FHR-3 provides an explanation for the protective role of the CFHR3-CFHR1 gene deletion in age-related macular degeneration. Conflict of interest: No relevant conflicts of interest to disclose. This work was supported by the Hungarian Scientific Research Fund (OTKAK 109055). http://dx.doi.org/10.1016/j.imbio.2016.06.118
1177
Scott Jones 1,∗ , Holly Humphries 2 , Andrew Gorringe 2 , Dominique Walters 3 , B. Paul Morgan 1 , Stephen Taylor 2 , Claire L. Harris 4 1 Division of Infection and Immunity, Cardiff University, Cardiff, UK 2 Public Health England, Salisbury, UK 3 GlaxoSmithKline (GSK), Warve, Belgium 4 Institute of Cellular Medicine, Newcastle University, Newcastle, UK
Polysaccharide conjugate vaccines are available to prevent disease caused by Neisseria meningitidis serogroups A, C, W and Y. Countering the recent escalation in serogroup W cases, two conjugate meningococcal vaccines have been introduced in the UK for individuals aged 13–25 years. Vaccine efficacy is assessed in clinical trials using serum bactericidal assays (SBAs). Rabbit complement is usually used as it lacks endogenous bactericidal activity compared to human sera. Several studies have shown that SBA activities determined with rabbit (rSBA) and human (hSBA) complement correlate poorly, possibly due to different interactions between antibody subclasses and the complement source, and species-specific interaction of N. meningitidis with complement regulators. To understand the influence of antibody subclass on SBA data, we determined the interaction of purified human IgM and IgG subclasses with human and rabbit C1q by ELISA and SPR and investigated the comparative ability of the different isotypes to activate human and rabbit complement. Using clinical trial samples, we assessed the difference in antibody subclass response to a plain and conjugated quadrivalent polysaccharide vaccine by ELISA and examined the relationship to rSBA and hSBA titres. By SPR, both rabbit and human C1q showed the same order of binding affinity with the IgG subclasses (IgG3 > IgG1 > IgG2 > IgG4). Rabbit C1q displayed lower binding affinities to IgG subclasses compared to human C1q with the greatest difference between IgG3 and IgG1. By ELISA, IgG1 and IgG3 showed significantly more binding to human C1q, whereas human IgM had significantly more binding to rabbit C1q. Conjugate vaccine induced higher titres of IgG1 and IgM antibody compared to plain vaccine. No benefit of a second dose of conjugate vaccine four months post vaccination was evident and hyporesponsiveness was exhibited for serogroup A. IgG1 titres correlated well with hSBA titres for serogroups A, W and Y whereas IgM titres correlated well with rSBA titres for all serogroups. These data show that a high IgM response to meningococcal polysaccharides positively skew SBA titres when rabbit serum is used, further questioning its use as a source of complement for SBAs. These data enhance the understanding of antibody response to meningococcal vaccines, highlight important differences in the ability of antibody subclasses to fix human and rabbit complement and support improvements in the development and testing of meningococcal vaccines. Conflict of interest: This study is a BBSRC CASE studentship with GlaxoSmithKiline plc (GSK). DW has a contract of employment with GSK and BPM is a consultant for GSK. Nimenrix® and Mencevax® are registered trademarks of GlaxoSmithKline Biologicals SA. http://dx.doi.org/10.1016/j.imbio.2016.06.119
103
104
Factor H-related protein 3 (FHR-3) inhibits factor H binding to pentraxins and malondialdehyde epitopes, and activates the alternative pathway via C3b binding
Mechanisms responsible for differential bactericidal activities of human and rabbit complement for Neisseria meningitidis
Csincsi 1,∗ ,
Pouw 2 ,
Ádám I. Richard B. Agustín Tortajada 3 , Santiago Rodríguez de Córdoba 3 , Diana Wouters 2 , Mihály Józsi 1 1
MTA-ELTE “Lendület” Complement Research Group, Department of Immunology, Eötvös Loránd University, Budapest, Hungary 2 Department of Immunopathology, Sanquin Research and Landsteiner laboratory of the Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands 3 Department of Cellular and Molecular Medicine, Centro de Investigaciones Biológicas and Ciber de Enfermedades Raras, Madrid, Spain Factor H-related 3 (FHR-3) is one of the five human FHR proteins homologous to the complement alternative pathway inhibitor factor H (FH). In contrast to FH, however, their biological function is controversial and poorly defined. Genetic and functional studies implicated FHRs, including FHR-3, in inflammatory, autoimmune and infectious diseases. The aim of the current study was to further characterize FHR-3 in terms of ligand interactions and role in the regulation or modulation of complement activation. Recombinant FHR-3 bound to the acute phase proteins pentraxin 3 (PTX3) and C-reactive protein. PTX3, but not CRP, was able to bind C1q when bound to FHR-3. Similar to FHR-5, and in contrast to FHR-1, FHR-3 enhanced C3 deposition on PTX3- and CRP-coated wells, but not on wells coated with the extracellular matrix MaxGel. Contrary to a previous report, we found that FHR-3 did not display cofactor activity for the cleavage of C3b by factor I even when added in supra-physiological, 2 M concentration. However, we found that FHR-3, similar to FHR-4 and FHR-5, was able to support the formation of the C3bBb convertase by binding C3b, and this convertase in turn could generate C3a from C3. In line with this, FHR-3 activated the alternative pathway in serum and caused enhanced C3 deposition. Importantly, FHR3 bound to malondialdehyde-BSA adduct and dose-dependently inhibited FH binding to malondialdehyde epitopes. These results identify novel pentraxin ligands of FHR-3, demonstrate complement deregulation on pentraxins by this factor H-related protein, lack of cofactor activity, but enhancement of alternative pathway activation. Thus, FHR-3 is not a complement inhibitor, but it rather promotes opsonization and complement activation. Moreover, the competitive inhibition of FH binding to malondialdehyde epitopes by FHR-3 provides an explanation for the protective role of the CFHR3-CFHR1 gene deletion in age-related macular degeneration. Conflict of interest: No relevant conflicts of interest to disclose. This work was supported by the Hungarian Scientific Research Fund (OTKAK 109055). http://dx.doi.org/10.1016/j.imbio.2016.06.118
1177
Scott Jones 1,∗ , Holly Humphries 2 , Andrew Gorringe 2 , Dominique Walters 3 , B. Paul Morgan 1 , Stephen Taylor 2 , Claire L. Harris 4 1 Division of Infection and Immunity, Cardiff University, Cardiff, UK 2 Public Health England, Salisbury, UK 3 GlaxoSmithKline (GSK), Warve, Belgium 4 Institute of Cellular Medicine, Newcastle University, Newcastle, UK
Polysaccharide conjugate vaccines are available to prevent disease caused by Neisseria meningitidis serogroups A, C, W and Y. Countering the recent escalation in serogroup W cases, two conjugate meningococcal vaccines have been introduced in the UK for individuals aged 13–25 years. Vaccine efficacy is assessed in clinical trials using serum bactericidal assays (SBAs). Rabbit complement is usually used as it lacks endogenous bactericidal activity compared to human sera. Several studies have shown that SBA activities determined with rabbit (rSBA) and human (hSBA) complement correlate poorly, possibly due to different interactions between antibody subclasses and the complement source, and species-specific interaction of N. meningitidis with complement regulators. To understand the influence of antibody subclass on SBA data, we determined the interaction of purified human IgM and IgG subclasses with human and rabbit C1q by ELISA and SPR and investigated the comparative ability of the different isotypes to activate human and rabbit complement. Using clinical trial samples, we assessed the difference in antibody subclass response to a plain and conjugated quadrivalent polysaccharide vaccine by ELISA and examined the relationship to rSBA and hSBA titres. By SPR, both rabbit and human C1q showed the same order of binding affinity with the IgG subclasses (IgG3 > IgG1 > IgG2 > IgG4). Rabbit C1q displayed lower binding affinities to IgG subclasses compared to human C1q with the greatest difference between IgG3 and IgG1. By ELISA, IgG1 and IgG3 showed significantly more binding to human C1q, whereas human IgM had significantly more binding to rabbit C1q. Conjugate vaccine induced higher titres of IgG1 and IgM antibody compared to plain vaccine. No benefit of a second dose of conjugate vaccine four months post vaccination was evident and hyporesponsiveness was exhibited for serogroup A. IgG1 titres correlated well with hSBA titres for serogroups A, W and Y whereas IgM titres correlated well with rSBA titres for all serogroups. These data show that a high IgM response to meningococcal polysaccharides positively skew SBA titres when rabbit serum is used, further questioning its use as a source of complement for SBAs. These data enhance the understanding of antibody response to meningococcal vaccines, highlight important differences in the ability of antibody subclasses to fix human and rabbit complement and support improvements in the development and testing of meningococcal vaccines. Conflict of interest: This study is a BBSRC CASE studentship with GlaxoSmithKiline plc (GSK). DW has a contract of employment with GSK and BPM is a consultant for GSK. Nimenrix® and Mencevax® are registered trademarks of GlaxoSmithKline Biologicals SA. http://dx.doi.org/10.1016/j.imbio.2016.06.119