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1.
Blood ; 136(18): 2080-2089, 2020 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-32877502

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly contagious respiratory virus that can lead to venous/arterial thrombosis, stroke, renal failure, myocardial infarction, thrombocytopenia, and other end-organ damage. Animal models demonstrating end-organ protection in C3-deficient mice and evidence of complement activation in humans have led to the hypothesis that SARS-CoV-2 triggers complement-mediated endothelial damage, but the mechanism is unclear. Here, we demonstrate that the SARS-CoV-2 spike protein (subunit 1 and 2), but not the N protein, directly activates the alternative pathway of complement (APC). Complement-dependent killing using the modified Ham test is blocked by either C5 or factor D inhibition. C3 fragments and C5b-9 are deposited on TF1PIGAnull target cells, and complement factor Bb is increased in the supernatant from spike protein-treated cells. C5 inhibition prevents the accumulation of C5b-9 on cells, but not C3c; however, factor D inhibition prevents both C3c and C5b-9 accumulation. Addition of factor H mitigates the complement attack. In conclusion, SARS-CoV-2 spike proteins convert nonactivator surfaces to activator surfaces by preventing the inactivation of the cell-surface APC convertase. APC activation may explain many of the clinical manifestations (microangiopathy, thrombocytopenia, renal injury, and thrombophilia) of COVID-19 that are also observed in other complement-driven diseases such as atypical hemolytic uremic syndrome and catastrophic antiphospholipid antibody syndrome. C5 inhibition prevents accumulation of C5b-9 in vitro but does not prevent upstream complement activation in response to SARS-CoV-2 spike proteins.


Assuntos
Betacoronavirus , Fator D do Complemento/antagonistas & inibidores , Inativadores do Complemento/farmacologia , Via Alternativa do Complemento/efeitos dos fármacos , Glicoproteína da Espícula de Coronavírus/farmacologia , Linhagem Celular , Ativação do Complemento/efeitos dos fármacos , Complemento C3/metabolismo , Complemento C5/antagonistas & inibidores , Fator H do Complemento/metabolismo , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Humanos , Glicoproteína da Espícula de Coronavírus/fisiologia
2.
Nat Commun ; 11(1): 1326, 2020 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-32165615

RESUMO

Persistent pathogens have evolved to avoid elimination by the mammalian immune system including mechanisms to evade complement. Infections with African trypanosomes can persist for years and cause human and animal disease throughout sub-Saharan Africa. It is not known how trypanosomes limit the action of the alternative complement pathway. Here we identify an African trypanosome receptor for mammalian factor H, a negative regulator of the alternative pathway. Structural studies show how the receptor binds ligand, leaving inhibitory domains of factor H free to inactivate complement C3b deposited on the trypanosome surface. Receptor expression is highest in developmental stages transmitted to the tsetse fly vector and those exposed to blood meals in the tsetse gut. Receptor gene deletion reduced tsetse infection, identifying this receptor as a virulence factor for transmission. This demonstrates how a pathogen evolved a molecular mechanism to increase transmission to an insect vector by exploitation of a mammalian complement regulator.


Assuntos
Fator H do Complemento/metabolismo , Trypanosoma/fisiologia , Moscas Tsé-Tsé/parasitologia , Animais , Anticorpos Monoclonais/metabolismo , Células CHO , Bovinos , Membrana Celular/metabolismo , Complemento C3b/metabolismo , Fator H do Complemento/química , Cricetinae , Cricetulus , Camundongos Endogâmicos BALB C , Parasitemia/sangue , Ligação Proteica , Domínios Proteicos , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Receptores de Superfície Celular/metabolismo , Regulação para Cima
3.
Nat Commun ; 11(1): 778, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32034129

RESUMO

Age-related macular degeneration (AMD) is a leading cause of blindness. Genetic variants at the chromosome 1q31.3 encompassing the complement factor H (CFH, FH) and CFH related genes (CFHR1-5) are major determinants of AMD susceptibility, but their molecular consequences remain unclear. Here we demonstrate that FHR-4 plays a prominent role in AMD pathogenesis. We show that systemic FHR-4 levels are elevated in AMD (P-value = 7.1 × 10-6), whereas no difference is seen for FH. Furthermore, FHR-4 accumulates in the choriocapillaris, Bruch's membrane and drusen, and can compete with FH/FHL-1 for C3b binding, preventing FI-mediated C3b cleavage. Critically, the protective allele of the strongest AMD-associated CFH locus variant rs10922109 has the highest association with reduced FHR-4 levels (P-value = 2.2 × 10-56), independently of the AMD-protective CFHR1-3 deletion, and even in those individuals that carry the high-risk allele of rs1061170 (Y402H). Our findings identify FHR-4 as a key molecular player contributing to complement dysregulation in AMD.


Assuntos
Apolipoproteínas/genética , Apolipoproteínas/metabolismo , Degeneração Macular/sangue , Polimorfismo de Nucleotídeo Único , Idoso , Apolipoproteínas/sangue , Capilares/metabolismo , Estudos de Casos e Controles , Ativação do Complemento , Fator H do Complemento/metabolismo , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Haplótipos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas com Domínio LIM/metabolismo , Fígado/fisiologia , Degeneração Macular/genética , Degeneração Macular/patologia , Proteínas Musculares/metabolismo , Retina/metabolismo , Retina/patologia
4.
Nephrology (Carlton) ; 25(1): 40-47, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30838755

RESUMO

BACKGROUND: Immunoglobulin A (IgA) vasculitis with nephritis (IgAVN) and IgA nephropathy (IgAN) are widely considered as related diseases. Considerable evidences support the notion of involvement of complement activation in both IgAVN and IgAN. Our previous studies identified a genetic variant in complement factor H (CFH), rs6677604, as an IgAN-susceptible variant by genome-wide association study, and further confirmed its linkage to CFHR3-1Δ and proved its influence on complement activation and thereby on IgAN susceptibility. AIM: To explore the role of rs6677604 in complement activation of IgAVN. METHODS: In this study, we enrolled 632 patients with IgAVN, 1178 patients with IgAN and 902 healthy controls. The genotype of rs6677604 was measured by TaqMan allele discrimination assays or was extracted from genome-wide association study data. RESULTS: The frequency of the rs6677604-A allele was significantly higher in IgAVN than in IgAN. However, no significant differences were observed between IgAVN and the controls. Higher complement factor H (FH) levels were observed in IgAVN than IgAN, and positive correlation between circulating FH and C3 levels was present in IgAVN. In both IgAVN and IgAN, rs6677604-A was associated with less intensity of glomerular C3 deposits. In agreement with the higher frequency of rs6677604-A in IgAVN, the glomerular C3 deposits of patients with IgAVN were less intense than those in IgAN. CONCLUSION: Our findings suggest that genetic variation in CFH (rs6677604) is involved in the phenotype of complement activation in both IgAVN and IgAN. Moreover, rs6677604 might contribute to the difference of complement activation intensity between IgAVN and IgAN.


Assuntos
Ativação do Complemento/genética , Glomerulonefrite por IGA/genética , Rim/imunologia , Polimorfismo de Nucleotídeo Único , Vasculite/genética , Adulto , Estudos de Casos e Controles , Complemento C3/metabolismo , Fator H do Complemento/genética , Fator H do Complemento/metabolismo , Feminino , Frequência do Gene , Predisposição Genética para Doença , Glomerulonefrite por IGA/sangue , Glomerulonefrite por IGA/diagnóstico , Glomerulonefrite por IGA/imunologia , Humanos , Rim/patologia , Masculino , Fenótipo , Vasculite/sangue , Vasculite/diagnóstico , Vasculite/imunologia , Adulto Jovem
5.
Front Immunol ; 10: 2573, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31824478

RESUMO

The opportunistic fungal pathogen Aspergillus fumigatus can cause severe infections, particularly in immunocompromised individuals. Upon infection, A. fumigatus faces the powerful and directly acting immune defense of the human host. The mechanisms on how A. fumigatus evades innate immune attack and complement are still poorly understood. Here, we identify A. fumigatus enolase, AfEno1, which was also characterized as fungal allergen, as a surface ligand for human plasma complement regulators. AfEno1 binds factor H, factor-H-like protein 1 (FHL-1), C4b binding protein (C4BP), and plasminogen. Factor H attaches to AfEno1 via two regions, via short conserved repeats (SCRs) 6-7 and 19-20, and FHL-1 contacts AfEno1 via SCRs 6-7. Both regulators when bound to AfEno1 retain cofactor activity and assist in C3b inactivation. Similarly, the classical pathway regulator C4BP binds to AfEno1 and bound to AfEno1; C4BP assists in C4b inactivation. Plasminogen which binds to AfEno1 via lysine residues is accessible for the tissue-type plasminogen activator (tPA), and active plasmin cleaves the chromogenic substrate S2251, degrades fibrinogen, and inactivates C3 and C3b. Plasmin attached to swollen A. fumigatus conidia damages human A549 lung epithelial cells, reduces the cellular metabolic activity, and induces cell retraction, which results in exposure of the extracellular matrix. Thus, A. fumigatus AfEno1 is a moonlighting protein and virulence factor which recruits several human regulators. The attached human regulators allow the fungal pathogen to control complement at the level of C3 and to damage endothelial cell layers and tissue components.


Assuntos
Aspergillus fumigatus/enzimologia , Proteína de Ligação ao Complemento C4b/metabolismo , Fator H do Complemento/metabolismo , Proteínas Fúngicas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas com Domínio LIM/metabolismo , Proteínas Musculares/metabolismo , Fosfopiruvato Hidratase/metabolismo , Plasminogênio/metabolismo , Células Epiteliais Alveolares/imunologia , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/microbiologia , Aspergilose/imunologia , Aspergilose/metabolismo , Aspergilose/microbiologia , Aspergillus fumigatus/imunologia , Linhagem Celular , Fator H do Complemento/imunologia , Proteínas Fúngicas/imunologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Evasão da Resposta Imune , Cinética , Fosfopiruvato Hidratase/imunologia , Ligação Proteica
6.
Front Immunol ; 10: 2722, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31849943

RESUMO

Borrelia (B.) mayonii sp. nov. has recently been reported as a novel human pathogenic spirochete causing Lyme disease (LD) in North America. Previous data reveal a higher spirochaetemia in the blood compared to patients infected by LD spirochetes belonging to the B. burgdorferi sensu lato complex, suggesting that this novel genospecies must exploit strategies to overcome innate immunity, in particular complement. To elucidate the molecular mechanisms of immune evasion, we utilized various methodologies to phenotypically characterize B. mayonii and to identify determinants involved in the interaction with complement. Employing serum bactericidal assays, we demonstrated that B. mayonii resists complement-mediated killing. To further elucidate the role of the key regulators of the alternative pathway (AP), factor H (FH), and FH-like protein 1 (FHL-1) in immune evasion of B. mayonii, serum adsorption experiments were conducted. The data revealed that viable spirochetes recruit both regulators from human serum and FH retained its factor I-mediated C3b-inactivating activity when bound to the bacterial cells. In addition, two prominent FH-binding proteins of approximately 30 and 18 kDa were detected in B. mayonii strain MN14-1420. Bioinformatics identified a gene, exhibiting 60% identity at the DNA level to the cspA encoding gene of B. burgdorferi. Following PCR amplification, the gene product was produced as a His-tagged protein. The CspA-orthologous protein of B. mayonii interacted with FH and FHL-1, and both bound regulators promoted inactivation of C3b in the presence of factor I. Additionally, the CspA ortholog counteracted complement activation by inhibiting the alternative and terminal but not the classical and Lectin pathways, respectively. Increasing concentrations of CspA of B. mayonii also strongly affected C9 polymerization, terminating the formation of the membrane attack complex. To assess the role of CspA of B. mayonii in facilitating serum resistance, a gain-of-function strain was generated, harboring a shuttle vector allowing expression of the CspA encoding gene under its native promotor. Spirochetes producing the native protein on the cell surface overcame complement-mediated killing, indicating that CspA facilitates serum resistance of B. mayonii. In conclusion, here we describe the molecular mechanism utilized by B. mayonii to resists complement-mediated killing by capturing human immune regulators.


Assuntos
Proteínas de Bactérias/genética , Proteínas do Sistema Complemento/metabolismo , Evasão da Resposta Imune/genética , Doença de Lyme/imunologia , Infecções por Spirochaetales/imunologia , Spirochaetales/fisiologia , Proteínas de Bactérias/metabolismo , Bacteriólise , Ativação do Complemento , Proteínas Inativadoras do Complemento C3b/metabolismo , Fator H do Complemento/metabolismo , Biologia Computacional , Humanos , Imunidade Inata , Ligação Proteica
7.
J Biol Chem ; 294(52): 20148-20163, 2019 12 27.
Artigo em Inglês | MEDLINE | ID: mdl-31719147

RESUMO

Activation and suppression of the complement system compete on every serum-exposed surface, host or foreign. Potentially harmful outcomes of this competition depend on surface molecules through mechanisms that remain incompletely understood. Combining surface plasmon resonance (SPR) with atomic force microscopy (AFM), here we studied two complement system proteins at the single-molecule level: C3b, the proteolytically activated form of C3, and factor H (FH), the surface-sensing C3b-binding complement regulator. We used SPR to monitor complement initiation occurring through a positive-feedback loop wherein surface-deposited C3b participates in convertases that cleave C3, thereby depositing more C3b. Over multiple cycles of flowing factor B, factor D, and C3 over the SPR chip, we amplified C3b from ∼20 to ∼220 molecules·µm-2 AFM revealed C3b clusters of up to 20 molecules and solitary C3b molecules deposited up to 200 nm away from the clusters. A force of 0.17 ± 0.02 nanonewtons was needed to pull a single FH molecule, anchored to the AFM probe, from its complex with surface-attached C3b. The extent to which FH molecules stretched before detachment varied widely among complexes. Performing force-distance measurements with FH(D1119G), a variant lacking one of the C3b-binding sites and causing atypical hemolytic uremic syndrome, we found that it detached more uniformly and easily. In further SPR experiments, KD values between FH and C3b on a custom-made chip surface were 5-fold tighter than on commercial chips and similar to those on erythrocytes. These results suggest that the chemistry at the surface on which FH acts drives conformational adjustments that are functionally critical.


Assuntos
Complemento C3b/metabolismo , Fator H do Complemento/metabolismo , Microscopia de Força Atômica , Ressonância de Plasmônio de Superfície , Ativação do Complemento , Complemento C3b/química , Complemento C3d/química , Complemento C3d/metabolismo , Fator H do Complemento/química , Humanos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/metabolismo , Cinética , Ligação Proteica
8.
Semin Immunol ; 45: 101341, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31757608

RESUMO

The complement system, while being an essential and very efficient effector component of innate immunity, may cause damage to the host and result in various inflammatory, autoimmune and infectious diseases or cancer, when it is improperly activated or regulated. Factor H is a serum glycoprotein and the main regulator of the activity of the alternative complement pathway. Factor H, together with its splice variant factor H-like protein 1 (FHL-1), inhibits complement activation at the level of the central complement component C3 and beyond. In humans, there are also five factor H-related (FHR) proteins, whose function is poorly characterized. While data indicate complement inhibiting activity for some of the FHRs, there is increasing evidence that FHRs have an opposite role compared with factor H and FHL-1, namely, they enhance complement activation directly and also by competing with the regulators FH and FHL-1. This review summarizes the current stand and recent data on the roles of factor H family proteins in health and disease, with focus on the function of FHR proteins.


Assuntos
Proteínas Sanguíneas/metabolismo , Proteínas de Transporte/metabolismo , Ativação do Complemento/imunologia , Fator H do Complemento/imunologia , Fator H do Complemento/metabolismo , Animais , Proteínas Sanguíneas/química , Proteínas Sanguíneas/genética , Proteínas de Transporte/química , Proteínas de Transporte/genética , Fator H do Complemento/química , Proteínas do Sistema Complemento/imunologia , Proteínas do Sistema Complemento/metabolismo , Suscetibilidade a Doenças , Humanos , Imunomodulação , Ligantes , Família Multigênica , Ligação Proteica , Relação Estrutura-Atividade
9.
Infect Immun ; 87(12)2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31548314

RESUMO

Leptospirosis is one of the most widespread zoonoses caused by pathogenic Leptospira spp. In this study, we report that the LIC11966/ErpY-like lipoprotein is a surface-exposed outer membrane protein exclusively present in pathogenic species of Leptospira The recombinant ErpY (rErpY)-like protein is recognized by the immunoglobulins of confirmed leptospirosis sera of diverse hosts (human, bovine, and canine), suggesting the expression of the native leptospiral surface protein during infection. Circular dichroism of pure rErpY-like protein showed the secondary structural integrity to be uncompromised during the purification process. Analysis of the rErpY-like protein by native polyacrylamide gel electrophoresis, chemical cross-linking, dynamic light scattering, and field emission transmission electron microscopy demonstrated it undergoes supramolecular assembly. The rErpY-like protein can bind to diverse host extracellular matrices, and it presented a saturable and strong binding affinity (dissociation constant [KD ] of 70.45 ± 4.13 nM) to fibrinogen, a central host plasma component involved in blood clotting. In the presence of the rErpY-like supramolecule, thrombin-catalyzed fibrin clot formation is inhibited up to 7%, implying its role in inhibiting blood coagulation during Leptospira infection. In addition, binding of the rErpY-like supramolecule to complement factors H and I suggests the protein also contributes to Leptospira evading innate host defense during infection by inactivating alternative complement pathways. This study reveals that rErpY-like protein is functionally active in the supramolecular state and performs moonlighting activity under the given in vitro conditions.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Coagulação Sanguínea/fisiologia , Fator H do Complemento/metabolismo , Fator I do Complemento/metabolismo , Leptospira/imunologia , Leptospirose/diagnóstico , Animais , Dicroísmo Circular , Via Alternativa do Complemento/imunologia , Feminino , Tempo de Lise do Coágulo de Fibrina , Lipoproteínas/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Ligação Proteica , Estrutura Secundária de Proteína , Trombina/metabolismo
10.
Clin Exp Immunol ; 198(3): 381-389, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31487400

RESUMO

Vaccination against meningococcal serogroup B is recommended for patients with a complement deficiency; however, although immunogenicity in this patient group has been shown, efficacy has not yet been established. In this study, we collected serum from children with a complement deficiency in the alternative pathway or in late terminal pathway before and after vaccination with multi-component meningococcal serogroup B (MenB)-4C. MenB-4C is a multi-component, protein-based vaccine against MenB consisting of factor H-binding protein, Neisserial heparin-binding protein, Neisserial adhesion A and outer membrane vesicles containing Porin A. We assessed the vaccine immunogenicity and vaccine-mediated protection by a whole cell enzyme-linked immunosorbent assay with Neisseria meningitidis serogroup B strains H44/76, 5/99 and NZ98/254, which shows that vaccination induced antibody titers against meningococcus. We show that the classical serum bactericidal activity assay with exogenous serum indicates the presence of vaccine-induced antibodies and capacity to activate complement-mediated pathogen lysis. However, in children with a late terminal pathway deficiency, no complement-mediated pathogen lysis was observed when autologous serum was applied in the serum bactericidal activity assay, demonstrating a lack of serum bactericidal activity in children with complement deficiencies. However, MenB-4C vaccination still induced effective complement-dependent opsonophagocytic killing against N. meningitidis serogroup B in reconstituted whole blood with autologous serum from children with an alternative pathway or late terminal pathway deficiency. These findings support the recommendation to vaccinate all complement-deficient children against MenB.


Assuntos
Doenças da Deficiência Hereditária de Complemento/imunologia , Meningite Meningocócica/imunologia , Vacinas Meningocócicas/imunologia , Neisseria meningitidis Sorogrupo B/imunologia , Proteínas Opsonizantes/imunologia , Fagocitose/imunologia , Adolescente , Adulto , Anticorpos Antibacterianos/imunologia , Criança , Fator H do Complemento/imunologia , Fator H do Complemento/metabolismo , Feminino , Doenças da Deficiência Hereditária de Complemento/microbiologia , Doenças da Deficiência Hereditária de Complemento/terapia , Humanos , Masculino , Meningite Meningocócica/microbiologia , Meningite Meningocócica/terapia , Vacinas Meningocócicas/administração & dosagem , Neisseria meningitidis Sorogrupo B/fisiologia , Proteínas Opsonizantes/metabolismo , Vacinação
11.
Med Sci Monit ; 25: 7087-7093, 2019 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-31541546

RESUMO

BACKGROUND This study aimed to determine the diagnostic role of serum levels of complement C1q, Bb, and H in nonpregnant women, women with normal pregnancy, and women with severe pre-eclampsia. MATERIAL AND METHODS Healthy nonpregnant women (n=30), women with early, middle, and late normal pregnancy (n=30, respectively), and women with severe pre-eclampsia (n=73) were studied. The pre-eclampsia study group included early-onset cases (n=43) and late-onset cases (n=30). Serum levels of Bb were determined by enzyme-linked immunosorbent assay (ELISA), and C1q and H were tested by a turbidimetric immunoassay method. RESULTS In the pre-eclampsia study group, compared with women with normal pregnancy, serum levels of C1q remained stable throughout pregnancy, and Bb levels declined from mid-pregnancy (p=0.250). Serum levels of factor H increased in the middle and late stages of pregnancy, and C1q and H were lower in early-onset severe pre-eclampsia (p<0.001, p=0.009, respectively) and late-onset severe pre-eclampsia (p<0.001, p=0.031, respectively) compared with the early-onset control and late-onset control groups. Serum levels of Bb increased in early-onset severe pre-eclampsia (p=0.001) and late-onset severe pre-eclampsia (p=0.003) compared with early-onset control and late-onset control groups. The area under the receiver operator curve (ROC) for serum C1q, Bb, and H for the diagnosis of early-onset severe pre-eclampsia were 0.814 (95% CI, 0.712-0.917), 0.743 (95% CI, 0.638-0.859), and 0.681(95% CI, 0.556-0.806), and late-onset severe pre-eclampsia were 0.805 (95% CI, 0.694-0.913), 0.796 (95% CI, 0.680-0.911), and 0.662 (95% CI, 0.524-0.800). CONCLUSIONS The classical and alternative pathways of complement were activated in patients with severe pre-eclampsia. Serum levels of C1q, Bb, and H should be studied further as potential diagnostic markers for severe pre-eclampsia.


Assuntos
Complemento C1q/metabolismo , Fator B do Complemento/metabolismo , Fator H do Complemento/metabolismo , Pré-Eclâmpsia/sangue , Adulto , Área Sob a Curva , Estudos de Casos e Controles , Feminino , Humanos , Gravidez , Curva ROC
12.
FASEB J ; 33(11): 12099-12111, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31442074

RESUMO

The 4 component meningococcus B vaccine (4CMenB) vaccine is the first vaccine containing recombinant proteins licensed for the prevention of invasive meningococcal disease caused by meningococcal serogroup B strains. 4CMenB contains 3 main recombinant proteins, including the Neisseria meningitidis factor H binding protein (fHbp), a lipoprotein able to bind the human factor H. To date, over 1000 aa sequences of fHbp have been identified, and they can be divided into variant groups 1, 2, and 3, which are usually not crossprotective. Nevertheless, previous characterizations of a small set (n = 10) of mAbs generated in humans after 4CMenB immunization revealed 2 human Fabs (huFabs) (1A12, 1G3) with some crossreactivity for variants 1, 2, and 3. This unexpected result prompted us to examine a much larger set of human mAbs (n = 110), with the aim of better understanding the extent and nature of crossreactive anti-fHbp antibodies. In this study, we report an analysis of the human antibody response to fHbp, by the characterization of 110 huFabs collected from 3 adult vaccinees during a 6-mo study. Although the 4CMenB vaccine contains fHbp variant 1, 13 huFabs were also found to be crossreactive with variants 2 and 3. The crystal structure of the crossreactive huFab 1E6 in complex with fHbp variant 3 was determined, revealing a novel, highly conserved epitope distinct from the epitopes recognized by 1A12 or 1G3. Further, functional characterization shows that human mAb 1E6 is able to elicit rabbit, but not human, complement-mediated bactericidal activity against meningococci displaying fHbp from any of the 3 different variant groups. This functional and structural information about the human antibody response upon 4CMenB immunization contributes to further unraveling the immunogenic properties of fHbp. Knowledge gained about the epitope profile recognized by the human antibody repertoire could guide future vaccine design.-Bianchi, F., Veggi, D., Santini, L., Buricchi, F., Bartolini, E., Lo Surdo, P., Martinelli, M., Finco, O., Masignani, V., Bottomley, M. J., Maione, D., Cozzi, R. Cocrystal structure of meningococcal factor H binding protein variant 3 reveals a new crossprotective epitope recognized by human mAb 1E6.


Assuntos
Anticorpos Monoclonais/imunologia , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Fator H do Complemento/imunologia , Epitopos/imunologia , Vacinas Meningocócicas/imunologia , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Fator H do Complemento/genética , Fator H do Complemento/metabolismo , Cristalografia por Raios X , Epitopos/genética , Epitopos/metabolismo , Variação Genética , Humanos , Infecções Meningocócicas/microbiologia , Infecções Meningocócicas/prevenção & controle , Vacinas Meningocócicas/administração & dosagem , Modelos Moleculares , Neisseria meningitidis/efeitos dos fármacos , Neisseria meningitidis/imunologia , Neisseria meningitidis/fisiologia , Ligação Proteica , Conformação Proteica
13.
Commun Biol ; 2: 241, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31263785

RESUMO

Monoclonal antibody (mAb) cooperativity is a phenomenon triggered when mAbs couples promote increased bactericidal killing compared to individual partners. Cooperativity has been deeply investigated among mAbs elicited by factor H-binding protein (fHbp), a Neisseria meningitidis surface-exposed lipoprotein and one of the key antigens included in both serogroup B meningococcus vaccine Bexsero and Trumenba. Here we report the structural and functional characterization of two cooperative mAbs pairs isolated from Bexsero vaccines. The 3D electron microscopy structures of the human mAb-fHbp-mAb cooperative complexes indicate that the angle formed between the antigen binding fragments (fAbs) assume regular angle and that fHbp is able to bind simultaneously and stably the cooperative mAbs pairs and human factor H (fH) in vitro. These findings shed light on molecular basis of the antibody-based mechanism of protection driven by simultaneous recognition of the different epitopes of the fHbp and underline that cooperativity is crucial in vaccine efficacy.


Assuntos
Anticorpos Monoclonais/química , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Anticorpos Monoclonais/imunologia , Atividade Bactericida do Sangue , Fator H do Complemento/metabolismo , Mapeamento de Epitopos , Humanos , Vacinas Meningocócicas/imunologia , Microscopia Eletrônica de Transmissão , Ressonância de Plasmônio de Superfície
14.
Lupus ; 28(9): 1051-1061, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31296141

RESUMO

Pulmonary hypertension occurs in systemic lupus erythematosus (SLE) for several reasons, such as vasculopathy. Previous studies have indicated that the excessive activation of the complement alternative pathway might be involved in the pathogenesis of lupus nephritis, especially in the absence of factor H or its functional impairment. However, the clinical and pathological significance of the alternative complement activation in lupus nephritis patients with pulmonary hypertension remains elusive. The data on patients with pulmonary hypertension and non-pulmonary hypertension lupus nephritis were retrospectively analyzed in our centre. Major plasma levels of complement components were evaluated. The depositions of Bb, C3d and C5b-9 in the lung specimens of pulmonary hypertension combined with SLE patients were detected by immunofluorescence staining. Among 352 lupus nephritis cases, 24 were diagnosed with pulmonary hypertension and 328 with non-pulmonary hypertension. Higher levels of Bb and lower levels of factor H were detected in the pulmonary hypertension group in comparison with the negative group (P = 0.049, P = 0.024, respectively). Pulmonary hypertension was a risk factor for renal outcome as deduced by the log-rank and Cox test for survival analysis. C3d, C5b-9 and Bb were found to be positive in lung specimens of lupus nephritis patients with pulmonary hypertension. We concluded that activation of the complement alternative pathway may be involved in the pathogenesis of pulmonary hypertension in lupus nephritis.


Assuntos
Ativação do Complemento/fisiologia , Via Alternativa do Complemento/fisiologia , Hipertensão Pulmonar/fisiopatologia , Nefrite Lúpica/fisiopatologia , Adulto , Fator H do Complemento/metabolismo , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Adulto Jovem
15.
Stem Cell Res ; 38: 101473, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31176916

RESUMO

Age-related macular degeneration (AMD) is the leading cause of adult blindness in developed countries and is characterized by progressive degeneration of the macula, the central region of the retina. A human induced pluripotent stem cell (hiPSC) line was derived from peripheral blood mononuclear cells (PBMCs) from a patient with a clinical diagnosis of dry AMD carrying the CFH Y402H polymorphism. Sendai virus was using for reprogramming and the pluripotent and differentiation capacity of the cells were assessed by immunocytochemistry and RT-PCR.


Assuntos
Técnicas de Reprogramação Celular , Células-Tronco Pluripotentes Induzidas , Degeneração Macular , Polimorfismo Genético , Idoso de 80 Anos ou mais , Linhagem Celular , Fator H do Complemento/genética , Fator H do Complemento/metabolismo , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Degeneração Macular/genética , Degeneração Macular/metabolismo , Degeneração Macular/patologia
16.
Front Immunol ; 10: 1230, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31214187

RESUMO

Streptococcus pyogenes infects over 700 million people worldwide annually. Immune evasion strategies employed by the bacteria include binding of the complement inhibitors, C4b-binding protein (C4BP) and Factor H in a human-specific manner. We recently showed that human IgG increased C4BP binding to the bacterial surface, which promoted streptococcal immune evasion and increased mortality in mice. We sought to identify how IgG promotes C4BP binding to Protein H, a member of the M protein family. Dimerization of Protein H is pivotal for enhanced binding to human C4BP. First, we illustrated that Protein H, IgG, and C4BP formed a tripartite complex. Second, surface plasmon resonance revealed that Protein H binds IgG solely through Fc, but not Fab domains, and with high affinity (IgG-Protein H: KD = 0.4 nM; IgG-Fc-Protein H: KD ≤ 1.6 nM). Each IgG binds two Protein H molecules, while up to six molecules of Protein H bind one C4BP molecule. Third, interrupting Protein H dimerization either by raising temperature to 41°C or with a synthetic peptide prevented IgG-Protein H interactions. IgG-Fc fragments or monoclonal human IgG permitted maximal C4BP binding when used at concentrations from 0.1 to 10 mg/ml. In contrast, pooled human IgG enhanced C4BP binding at concentrations up to 1 mg/ml; decreased C4BP binding at 10 mg/ml occurred probably because of Fab-streptococcal interactions at these high IgG concentrations. Taken together, our data show how S. pyogenes exploits human IgG to evade complement and enhance its virulence. Elucidation of this mechanism could aid design of new therapeutics against S. pyogenes.


Assuntos
Proteína de Ligação ao Complemento C4b/imunologia , Interações Hospedeiro-Patógeno/imunologia , Imunoglobulina G/imunologia , Imunomodulação , Infecções Estreptocócicas/imunologia , Streptococcus pyogenes/imunologia , Proteína de Ligação ao Complemento C4b/metabolismo , Fator H do Complemento/química , Fator H do Complemento/imunologia , Fator H do Complemento/metabolismo , Suscetibilidade a Doenças/imunologia , Suscetibilidade a Doenças/metabolismo , Humanos , Cinética , Ligantes , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Ligação Proteica , Multimerização Proteica , Infecções Estreptocócicas/metabolismo , Infecções Estreptocócicas/microbiologia
17.
Int J Mol Sci ; 20(11)2019 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-31159152

RESUMO

Coagulation factor XIII (FXIII) is a plasma-circulating heterotetrameric pro-transglutaminase complex that is composed of two catalytic FXIII-A and two protective/regulatory FXIII-B subunits. FXIII acts by forming covalent cross-links within a preformed fibrin clots to prevent its premature fibrinolysis. The FXIII-A subunit is known to have pleiotropic roles outside coagulation, but the FXIII-B subunit is a relatively unexplored entity, both structurally as well as functionally. Its discovered roles so far are limited to that of the carrier/regulatory protein of its partner FXIII-A subunit. In the present study, we have explored the co-presence of protein excipients in commercial FXIII plasma concentrate FibrogamminP by combination of protein purification and mass spectrometry-based verification. Complement factor H was one of the co-excipients observed in this analysis. This was followed by performing pull down assays from plasma in order to detect the putative novel interacting partners for the FXIII-B subunit. Complement system proteins, like complement C3 and complement C1q, were amongst the proteins that were pulled down. The only protein that was observed in both experimental set ups was alpha-2-macroglobulin, which might therefore be a putative interacting partner of the FXIII/FXIII-B subunit. Future functional investigations will be needed to understand the physiological significance of this association.


Assuntos
Transtornos da Coagulação Sanguínea/etiologia , Transtornos da Coagulação Sanguínea/metabolismo , Proteínas de Transporte/metabolismo , Fator XIII/metabolismo , Mapeamento de Interação de Proteínas , Coagulação Sanguínea , Transtornos da Coagulação Sanguínea/sangue , Fator H do Complemento/metabolismo , Fibrinogênio/metabolismo , Humanos , Espectrometria de Massas , alfa 2-Macroglobulinas Associadas à Gravidez/metabolismo , Ligação Proteica
18.
J Cell Physiol ; 234(11): 19842-19851, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-30972735

RESUMO

Considerable advances have been made in identification of the involvement of immune modulators in diseases. There is growing evidence on the role of complement pathway in pathogenesis and course of multiple sclerosis (MS). Moreover, it has been recognized that microRNAs (miRNAs) play an essential role in modulation and development of immune response in the central nervous system. We aimed to investigate the expression profile of complement factor H (CFH) and miR-146a genes in experimental autoimmune encephalomyelitis (EAE) mouse model of MS to detect the possible roles of CFH and miR-146a as biomarkers of MS disease stats. Expression of CFH and miR-146a genes in liver and brain tissues of EAE mice was measured in acute and chronic phases of disease compared to matched controls using real-time polymerase chain reaction. In the liver, increased expression of CFH gene was observed in the chronic phase compared to the acute phase. However, no significant difference was observed between acute and chronic phase mice with normal mice, while miR-146a expression was significantly decreased in livers of EAE mice in chronic group compared to acute and control groups. The expression of CFH gene in brain had a significant decrease in acute and chronic phases compared to healthy mice. Taken together, these observations indicate probable implication of complement system and miR-146a in course of immune-related diseases and reveal more facts about the pathogenesis of MS. However, further work is needed to determine protein levels of CFH and other possible targets of miR-146a in serum and cerebrospinal fluid of MS patients.


Assuntos
Fator H do Complemento/genética , Encefalomielite Autoimune Experimental/genética , Regulação da Expressão Gênica , MicroRNAs/metabolismo , Doença Aguda , Animais , Encéfalo/metabolismo , Doença Crônica , Fator H do Complemento/metabolismo , Regulação para Baixo/genética , Encefalomielite Autoimune Experimental/patologia , Feminino , Fígado/metabolismo , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Regulação para Cima/genética
19.
Mol Vis ; 25: 174-182, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30996586

RESUMO

Purpose: To evaluate the efficacy of using a CRISPR/Cas-mediated strategy to correct a common high-risk allele that is associated with age-related macular degeneration (AMD; rs1061170; NM_000186.3:c.1204T>C; NP_000177.2:p.His402Tyr) in the complement factor H (CFH) gene. Methods: A human embryonic kidney cell line (HEK293A) was engineered to contain the pathogenic risk variant for AMD (HEK293A-CFH). Several different base editor constructs (BE3, SaBE3, SaKKH-BE3, VQR-BE3, and Target-AID) and their respective single-guide RNA (sgRNA) expression cassettes targeting either the pathogenic risk variant allele in the CFH locus or the LacZ gene, as a negative control, were evaluated head-to-head for the incidence of a cytosine-to-thymine nucleotide correction. The base editor construct that showed appreciable editing activity was selected for further assessment in which the base-edited region was subjected to next-generation deep sequencing to quantify on-target and off-target editing efficacy. Results: The tandem use of the Target-AID base editor and its respective sgRNA demonstrated a base editing efficiency of facilitating a cytosine-to-thymine nucleotide correction in 21.5% of the total sequencing reads. Additionally, the incidence of insertions and deletions (indels) was detected in only 0.15% of the sequencing reads with virtually no off-target effects evident across the top 11 predicted off-target sites containing at least one cytosine in the activity window (n = 3, pooled amplicons). Conclusions: CRISPR-mediated base editing can be used to facilitate a permanent and stably inherited cytosine-to-thymine nucleotide correction of the rs1061170 SNP in the CFH gene with minimal off-target effects.


Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Edição de Genes/métodos , RNA Guia/genética , Sequência de Bases , Proteína 9 Associada à CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Fator H do Complemento/genética , Fator H do Complemento/metabolismo , Citosina/metabolismo , Expressão Gênica , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Óperon Lac , Degeneração Macular/genética , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Mutação , Plasmídeos/química , Plasmídeos/metabolismo , RNA Guia/metabolismo , Timina/metabolismo
20.
Microbes Infect ; 21(8-9): 377-385, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30923000

RESUMO

Several pathogens including Gram-negative bacteria hijack complement regulators to escape host's innate response. Pathogenic Leptospira species bind Factor H, C4b binding protein and vitronectin from the complement system. We evaluated the ability of low passage (LP) and culture-attenuated (CA) pathogenic strains of Leptospira, to bind Factor H. We used LOCaS46 (Leptospira interrogans sv Canicola), LOVe30 (L. interrogans sv Icterohaemorrhagiae) and MOCA45 (L. santarosai sv Tarassovi), and ten high passage strains of Leptospira [used in the microscopic agglutination test (MAT)]. Afterwards, we assessed their survival in normal human serum (NHS). Interestingly, the ability in binding Factor H was higher for LOCaS46 and LOVe30 LP strains, than for the respective CA strains suggesting that the ability of evading the alternative complement pathway is lost after culture attenuation. Accordingly, the level of mRNA expression of the Factor H binding proteins, LigA, LigB and Lsa23 was higher in these LP strains than in the corresponding CA strains. Unexpectedly, no difference in Factor H binding and surviving was observed between LP and CA MOCA45 strains. The high passage MAT-reference strains showed variation in Factor H binding ability, but, in most cases, the ability for capturing Factor H by Leptospira strains correlated with their survival in NHS.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Regulação Bacteriana da Expressão Gênica , Leptospira/imunologia , Leptospira/patogenicidade , Proteínas de Transporte/genética , Fator H do Complemento/metabolismo , Humanos , Evasão da Resposta Imune/genética , Leptospira/genética , Leptospirose/microbiologia , Viabilidade Microbiana/genética , Viabilidade Microbiana/imunologia , Ligação Proteica , RNA Mensageiro/genética
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