RESUMEN
Complement activation protects against infection but also contributes to pathological mechanisms in a range of clinical conditions such as autoimmune diseases and transplant rejection. Complement-inhibitory drugs, either approved or in development, usually act systemically, thereby increasing the risk for infections. We therefore envisioned a novel class of bispecific antibodies (bsAbs) which are capable of site-directed complement inhibition by bringing endogenous complement regulators in the vicinity of defined cell surface antigens. Here, we analyzed a comprehensive set of obligate bsAbs designed to crosslink a specific target with either complement regulator factor H (FH) or C4b-binding protein (C4BP). The bsAbs were assessed for their capacity to inhibit complement activation and cell lysis in an antigen-targeted manner. We observed that the bsAbs inhibited classical, lectin, and alternative pathway complement activation in which sufficient endogenous serum FH and C4BP could be recruited to achieve local inhibition. Importantly, the bsAbs effectively protected antigen-positive liposomes, erythrocytes, and human leukocytes from complement-mediated lysis. In conclusion, localized complement inhibition by bsAbs capable of recruiting endogenous human complement regulators (such as FH or C4BP) to cell surfaces potentially provides a novel therapeutic approach for the targeted treatment of complement-mediated diseases.
Asunto(s)
Anticuerpos Biespecíficos , Activación de Complemento , Proteína de Unión al Complemento C4b , Factor H de Complemento , Anticuerpos Biespecíficos/inmunología , Anticuerpos Biespecíficos/farmacología , Humanos , Activación de Complemento/inmunología , Proteína de Unión al Complemento C4b/inmunología , Proteína de Unión al Complemento C4b/metabolismo , Factor H de Complemento/inmunología , Factor H de Complemento/metabolismo , Antígenos/inmunología , Proteínas del Sistema Complemento/inmunología , Proteínas del Sistema Complemento/metabolismo , Unión ProteicaRESUMEN
All infective bacterial species need to conquer the innate immune system in order to colonize and survive in their hosts. The human respiratory pathogens Haemophilus influenzae and Moraxella catarrhalis are no exceptions and have developed sophisticated mechanisms to evade complement-mediated killing. Both bacterial species carry lipooligosaccharides preventing complement attacks and attract and utilize host complement regulators C4b binding protein and factor H to inhibit the classical and alternative pathways of complement activation, respectively. In addition, the regulator of the terminal pathway of complement activation, vitronectin, is hijacked by both bacteria. An array of different outer membrane proteins (OMP) in H. influenzae and M. catarrhalis simultaneously binds complement regulators, but also plasminogen. Several of the bacterial complement-binding proteins are important adhesins and contain highly conserved regions for interactions with the host. Thus, some of the OMP are viable targets for new therapeutics, including vaccines aimed at preventing respiratory tract diseases such as otitis media in children and exacerbations in patients suffering from chronic obstructive pulmonary disease.
Asunto(s)
Proteína de Unión al Complemento C4b/inmunología , Factor H de Complemento/inmunología , Infecciones por Haemophilus/inmunología , Haemophilus influenzae , Evasión Inmune , Moraxella catarrhalis , Infecciones por Moraxellaceae/inmunología , Infecciones del Sistema Respiratorio/inmunología , Animales , Proteínas de la Membrana Bacteriana Externa/inmunología , Infecciones por Haemophilus/patología , Haemophilus influenzae/inmunología , Haemophilus influenzae/patogenicidad , Humanos , Moraxella catarrhalis/inmunología , Moraxella catarrhalis/patogenicidad , Infecciones por Moraxellaceae/patología , Otitis Media/inmunología , Otitis Media/patología , Enfermedad Pulmonar Obstructiva Crónica/inmunología , Enfermedad Pulmonar Obstructiva Crónica/patología , Infecciones del Sistema Respiratorio/patologíaRESUMEN
C4b Binding Protein (C4BP) is a major fluid phase inhibitor of the classical and lectin pathways of the complement system. Complement inhibition is achieved by binding to and restricting the role of activated complement component C4b. C4BP functions as a co-factor for factor I in proteolytic inactivation of both soluble and cell surface-bound C4b, thus restricting the formation of the C3-convertase, C4b2a. C4BP also accelerates the natural decay/dissociation of the C3 convertase. This makes C4BP a prime target for exploitation by pathogens to escape complement attack, as seen in Streptococcus pyogenes or Flavivirus. Here, we examined whether C4BP can act on its own in a complement independent manner, against pathogens. C4BP bound H1N1 and H3N2 subtypes of Influenza A Virus (IAV) most likely via multiple sites in Complement Control Protein (CCP) 1-2, 4-5, and 7-8 domains of its α-chain. In addition, C4BP CCP1-2 bound H3N2 better than H1N1. C4BP bound three IAV envelope proteins: Haemagglutinin (~70 kDa), Neuraminidase (~55 kDa), and Matrix protein 1 (~25kDa). C4BP suppressed H1N1 subtype infection into the lung epithelial cell line, A549, while it promoted infection by H3N2 subtype. C4BP restricted viral entry for H1N1 but had the opposite effect on H3N2, as evident from experiments using pseudo-typed viral particles. C4BP downregulated mRNA levels of pro-inflammatory IFN-α, IL-12, and NFκB in the case of H1N1, while it promoted a pro-inflammatory immune response by upregulating IFN- α, TNF-α, RANTES, and IL-6 in the case of H3N2. We conclude that C4BP differentially modulates the efficacy of IAV entry, and hence, replication in a target cell in a strain-dependent manner, and acts as an entry inhibitor for H1N1. Thus, CCP containing complement proteins such as factor H and C4BP may have additional defense roles against IAV that do not rely on the regulation of complement activation.
Asunto(s)
Proteína de Unión al Complemento C4b/inmunología , Subtipo H1N1 del Virus de la Influenza A/inmunología , Subtipo H3N2 del Virus de la Influenza A/inmunología , Gripe Humana/inmunología , Internalización del Virus , Células A549 , Proteína de Unión al Complemento C4b/metabolismo , Humanos , Subtipo H1N1 del Virus de la Influenza A/metabolismo , Subtipo H3N2 del Virus de la Influenza A/metabolismoRESUMEN
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.
Asunto(s)
Proteína de Unión al Complemento C4b/inmunología , Interacciones Huésped-Patógeno/inmunología , Inmunoglobulina G/inmunología , Inmunomodulación , Infecciones Estreptocócicas/inmunología , Streptococcus pyogenes/inmunología , Proteína de Unión al Complemento C4b/metabolismo , Factor H de Complemento/química , Factor H de Complemento/inmunología , Factor H de Complemento/metabolismo , Susceptibilidad a Enfermedades/inmunología , Susceptibilidad a Enfermedades/metabolismo , Humanos , Cinética , Ligandos , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/ultraestructura , Unión Proteica , Multimerización de Proteína , Infecciones Estreptocócicas/metabolismo , Infecciones Estreptocócicas/microbiologíaRESUMEN
Multidrug-resistant Neisseria gonorrhoeae is a global health problem. Monoclonal antibody (mAb) 2C7 recognizes a gonococcal lipooligosaccharide epitope that is expressed by >95% of clinical isolates and hastens gonococcal vaginal clearance in mice. Chimeric mAb 2C7 (human immunoglobulin G1 [IgG1]) with an E430G Fc modification that enhances Fc:Fc interactions and hexamerization following surface-target binding and increases complement activation (HexaBody technology) showed significantly greater C1q engagement and C4 and C3 deposition compared to mAb 2C7 with wild-type Fc. Greater complement activation by 2C7-E430G Fc translated to increased bactericidal activity in vitro and, consequently, enhanced efficacy in mice, compared with "Fc-unmodified" chimeric 2C7. Gonococci bind the complement inhibitors factor H (FH) and C4b-binding protein (C4BP) in a human-specific manner, which dampens antibody (Ab)-mediated complement-dependent killing. The variant 2C7-E430G Fc overcame the barrier posed by these inhibitors in human FH/C4BP transgenic mice, for which a single 1 µg intravenous dose cleared established infection. Chlamydia frequently coexists with and exacerbates gonorrhea; 2C7-E430G Fc also proved effective against gonorrhea in gonorrhea/chlamydia-coinfected mice. Complement activation alone was necessary and sufficient for 2C7 function, evidenced by the fact that (1) "complement-inactive" Fc modifications that engaged Fc gamma receptor (FcγR) rendered 2C7 ineffective, nonetheless; (2) 2C7 was nonfunctional in C1q-/- mice, when C5 function was blocked, or in C9-/- mice; and (3) 2C7 remained effective in neutrophil-depleted mice and in mice treated with PMX205, a C5a receptor (C5aR1) inhibitor. We highlight the importance of complement activation for antigonococcal Ab function in the genital tract. Elucidating the correlates of protection against gonorrhea will inform the development of Ab-based gonococcal vaccines and immunotherapeutics.
Asunto(s)
Activación de Complemento/inmunología , Gonorrea/inmunología , Neisseria gonorrhoeae/inmunología , Animales , Anticuerpos Antibacterianos/inmunología , Anticuerpos Monoclonales/metabolismo , Antígenos Bacterianos , Proteína de Unión al Complemento C4b/inmunología , Factor H de Complemento/inmunología , Proteínas del Sistema Complemento/inmunología , Proteínas del Sistema Complemento/metabolismo , Epítopos/inmunología , Femenino , Voluntarios Sanos , Humanos , Inmunoglobulina G/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Neisseria gonorrhoeae/patogenicidadRESUMEN
Regulating complement is an important step in the establishment of infection by microbial pathogens. Toxoplasma gondii actively resists complement-mediated killing in non-immune human serum (NHS) by inactivating C3b, however the precise molecular basis is unknown. Here, a flow cytometry-based C3b binding assay demonstrated that Type II strains had significantly higher levels of surface-bound C3b than Type I strains. However, both strains efficiently inactivated C3b and were equally resistant to serum killing, suggesting that resistance is not strain-dependent. Toxoplasma activated both the lectin (LP) and alternative (AP) pathways, and the deposition of C3b was both strain and lectin-dependent. A flow cytometry-based lectin binding assay identified strain-specific differences in the level and heterogeneity of surface glycans detected. Specifically, increased lectin-binding by Type II strains correlated with higher levels of the LP recognition receptor mannose binding lectin (MBL). Western blot analyses demonstrated that Toxoplasma recruits both classical pathway (CP) and LP regulator C4b-binding proteins (C4BP) and AP regulator Factor H (FH) to the parasite surface to inactivate bound C3b-iC3b and C3dg and limit formation of the C5b-9 attack complex. Blocking FH and C4BP contributed to increased C5b-9 formation in vitro. However, parasite susceptibility in vitro was only impacted when FH was blocked, indicating that down regulation of the alternative pathway by FH may be more critical for parasite resistance. Infection of C3 deficient mice led to uncontrolled parasite growth, acute mortality, and reduced antibody production, indicating that both the presence of C3, and the ability of the parasite to inactivate C3, was protective. Taken together, our results establish that Toxoplasma regulation of the complement system renders mice resistant to acute infection by limiting parasite proliferation in vivo, but susceptible to chronic infection, with all mice developing transmissible cysts to maintain its life cycle.
Asunto(s)
Proteína de Unión al Complemento C4b/inmunología , Factor H de Complemento/inmunología , Citotoxicidad Inmunológica , Interacciones Huésped-Parásitos/inmunología , Toxoplasma/inmunología , Toxoplasmosis/inmunología , Toxoplasmosis/parasitología , Animales , Activación de Complemento , Complemento C3/deficiencia , Convertasas de Complemento C3-C5 de la Vía Alternativa , Proteína de Unión al Complemento C4b/metabolismo , Factor H de Complemento/metabolismo , Humanos , Inmunoglobulina G/inmunología , Ratones , Toxoplasmosis/metabolismoRESUMEN
The complement system is a tightly regulated network of proteins involved in defense against pathogens, inflammatory processes, and coordination of the innate and adaptive immune responses. Dysregulation of the complement cascade is associated with many inflammatory disorders. Thus, inhibition of the complement system has emerged as an option for treatment of a range of different inflammatory diseases. MAP-1 is a pattern recognition molecule (PRM)-associated inhibitor of the lectin pathway of the complement system, whereas C4b-binding protein (C4BP) regulates both the classical and lectin pathways. In this study we generated chimeric proteins consisting of MAP-1 and the first five domains of human C4BP (C4BP1-5) in order to develop a targeted inhibitor acting at different levels of the complement cascade. Two different constructs were designed and expressed in CHO cells where MAP-1 was fused with C4BP1-5 in either the C- or N-terminus. The functionality of the chimeric proteins was assessed using different in vitro complement activation assays. Both chimeric proteins displayed the characteristic Ca2+-dependent dimerization and binding to PRMs of native MAP-1, as well as the co-factor activity of native C4BP. In ELISA-based complement activation assays they could effectively inhibit the lectin and classical pathways. Notably, MAP-1:C4BP1-5 was five times more effective than rMAP-1 and rC4BP1-5 applied at the same time, emphasizing the advantage of a single inhibitor containing both functional domains. The MAP-1/C4BP chimeras exert unique complement inhibitory properties and represent a novel therapeutic approach targeting both upstream and central complement activation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Reguladoras de la Apoptosis , Proteína de Unión al Complemento C4b , Lectina de Unión a Manosa de la Vía del Complemento/inmunología , Proteínas Recombinantes de Fusión , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/inmunología , Animales , Proteínas Reguladoras de la Apoptosis/química , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/inmunología , Células CHO , Proteína de Unión al Complemento C4b/química , Proteína de Unión al Complemento C4b/genética , Proteína de Unión al Complemento C4b/inmunología , Cricetulus , Ensayo de Inmunoadsorción Enzimática , HumanosRESUMEN
Variable lymphocyte receptors B (VLRBs) are non-immunoglobulin components of the humoral immune system in jawless vertebrates including hagfish (Eptatretus burgeri) and lamprey (Petromyzon marinus). Hagfish VLRBs consist of leucine rich repeat (LRR) modules with a superhydrophobic C-terminal tail, the latter of which leads to extremely low expression levels in recombinant protein technology. Here, we present an artificially oligomerized VLRB (arVLRB) that conjugates via the C4bp oligomerization domain derived from human C4b-binding protein (hC4bp) rather than the superhydrophobic tail. The resulting arVLRB had a tightly multimerized form with seven monomeric VLRB arms and showed high expression and secretion levels in a mammalian expression system. To isolate antigen-specific arVLRB, we constructed large VLRB libraries from hagfish immunized with the fish pathogen, viral hemorrhagic septicemia virus (VHSV). The selected arVLRBs were found to recognize various types of antigens, including the recombinant target protein, purified viruses, and progeny viruses, with high antigen binding abilities and specificities. We also performed in vitro affinity maturation of the arVLRBs through LRRCT mutagenesis, and found that this enhanced their antigen-binding properties by at least 125-fold. Our epitope mapping analysis revealed that 37DWDTPL42, which is located in a region conserved among the glycoproteins of all VHSV isolates, is the recognition epitope of the arVLRBs. Thus, our newly developed arVLRB could prove useful in the development of universal diagnostic tools and/or therapeutic agents for the virus. Together, our novel findings provide valuable insights into hagfish VLRB and its potential use as a novel alternative to conventional antibodies for biotechnological applications.
Asunto(s)
Glicoproteínas/inmunología , Anguila Babosa/inmunología , Septicemia Hemorrágica Viral/inmunología , Linfocitos/inmunología , Novirhabdovirus/inmunología , Inmunidad Adaptativa/inmunología , Animales , Anticuerpos/inmunología , Proteína de Unión al Complemento C4b/inmunología , Epítopos/inmunología , Reordenamiento Génico/inmunología , Humanos , Inmunización/métodos , Mamíferos/inmunología , Petromyzon/inmunologíaRESUMEN
Streptococcus pyogenes is an exclusively human pathogen that can provoke mild skin and throat infections but can also cause fatal septicemia. This gram-positive bacterium has developed several strategies to evade the human immune system, enabling S. pyogenes to survive in the host. These strategies include recruiting several human plasma proteins, such as the complement inhibitor, C4b-binding protein (C4BP), and human (hu)-IgG through its Fc region to the bacterial surface to evade immune recognition. We identified a novel virulence mechanism whereby IgG-enhanced binding of C4BP to five of 12 tested S. pyogenes strains expressed diverse M proteins that are important surface-expressed virulence factors. Importantly, all strains that bound C4BP in the absence of IgG bound more C4BP when IgG was present. Further studies with an M1 strain that additionally expressed protein H, also a member of the M protein family, revealed that binding of hu-IgG Fc to protein H increased the affinity of protein H for C4BP. Increased C4BP binding accentuated complement downregulation, resulting in diminished bacterial killing. Accordingly, mortality from S. pyogenes infection in hu-C4BP transgenic mice was increased when hu-IgG or its Fc portion alone was administered concomitantly. Electron microscopy analysis of human tissue samples with necrotizing fasciitis confirmed increased C4BP binding to S. pyogenes when IgG was present. Our findings provide evidence of a paradoxical function of hu-IgG bound through Fc to diverse S. pyogenes isolates that increases their virulence and may counteract the beneficial effects of IgG opsonization.
Asunto(s)
Proteínas del Sistema Complemento/inmunología , Inmunoglobulina G/inmunología , Streptococcus pyogenes/inmunología , Virulencia/inmunología , Animales , Antígenos Bacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas Portadoras/inmunología , Proteína de Unión al Complemento C4b/inmunología , Inactivadores del Complemento/inmunología , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Fagocitosis/inmunología , Unión Proteica/inmunología , Infecciones Estreptocócicas/inmunología , Factores de Virulencia/inmunologíaRESUMEN
The complement is the first line of immune defense system involved in elimination of invading pathogens and dying host cells. Its activation is mainly triggered by immune complexes or pattern recognition molecules (PRMs) upon recognition against non-self or altered self-cells, such as C1q, collectins, ficolins, and properdin. Recent findings have interestingly shown that the pentraxins (C-reactive protein, CRP; serum-amyloid P component, SAP; long pentraxin 3, PTX3) are involved in complement activation and amplification via communication with complement initiation PRMs, but also complement regulation via recruitment of complement regulators, for instance C4b binding protein (C4BP) and factor H (fH). This review addresses the potential roles of the pentraxins in the complement system during infection and inflammation, and emphasizes the underlining implications of the pentraxins in the context of complement activation and regulation both under physiological and pathological conditions.
Asunto(s)
Proteína C-Reactiva/metabolismo , Activación de Complemento/inmunología , Infecciones/inmunología , Inflamación/inmunología , Componente Amiloide P Sérico/metabolismo , Animales , Proteína C-Reactiva/inmunología , Proteína de Unión al Complemento C4b/inmunología , Proteína de Unión al Complemento C4b/metabolismo , Factor H de Complemento/inmunología , Factor H de Complemento/metabolismo , Modelos Animales de Enfermedad , Humanos , Infecciones/microbiología , Receptores de Reconocimiento de Patrones/inmunología , Receptores de Reconocimiento de Patrones/metabolismo , Componente Amiloide P Sérico/inmunologíaRESUMEN
The use of C3d, the final degradation product of complement protein C3, as a "natural" adjuvant has been widely examined since the initial documentation of its immunogenicity-enhancing properties as a consequence of binding to complement receptor 2. Subsequently it was demonstrated that these effects are most evident when oligomeric, rather than when monomeric forms of C3d, are linked to various test protein antigens. In this study, we examined the feasibility of enhancing the adjuvant properties of human C3d further by utilizing C4b-binding protein (C4BP) to provide an oligomeric arrayed scaffold fused to the model antigen, tetanus toxin C fragment (TTCF). High molecular weight, C3d-containing oligomeric vaccines were successfully expressed, purified from mammalian cells and used to immunize groups of mice. Surprisingly, anti-TTCF antibody responses measured in these mice were poor. Subsequently we established by in vitro and in vivo analysis that, in the presence of mouse C3, human C3d does not interact with either mouse or even human complement receptor 2. These data confirm the requirement to develop murine versions of C3d based adjuvant compounds to test in mice or that mice would need to be developed that express both human C3 and human CR2 to allow the testing of human C3d based adjuvants in mouse in any capacity.
Asunto(s)
Linfocitos B/fisiología , Complemento C3d/inmunología , Proteína de Unión al Complemento C4b/genética , Fragmentos de Péptidos/inmunología , Toxina Tetánica/inmunología , Vacunas Sintéticas/inmunología , Adyuvantes Inmunológicos , Animales , Anticuerpos/sangre , Línea Celular , Complemento C3d/genética , Proteína de Unión al Complemento C4b/inmunología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Animales , Fragmentos de Péptidos/genética , Multimerización de Proteína/genética , Receptores de Complemento 3d/genética , Receptores de Complemento 3d/metabolismo , Toxina Tetánica/genética , Vacunación , Vacunas Sintéticas/genéticaRESUMEN
Poxviruses display species tropism-variola virus is a human-specific virus, while vaccinia virus causes repeated outbreaks in dairy cattle. Consistent with this, variola virus complement regulator SPICE (smallpox inhibitor of complement enzymes) exhibits selectivity in inhibiting the human alternative complement pathway and vaccinia virus complement regulator VCP (vaccinia virus complement control protein) displays selectivity in inhibiting the bovine alternative complement pathway. In the present study, we examined the species specificity of VCP and SPICE for the classical pathway (CP). We observed that VCP is â¼43-fold superior to SPICE in inhibiting bovine CP. Further, functional assays revealed that increased inhibitory activity of VCP for bovine CP is solely due to its enhanced cofactor activity, with no effect on decay of bovine CP C3-convertase. To probe the structural basis of this specificity, we utilized single- and multi-amino-acid substitution mutants wherein 1 or more of the 11 variant VCP residues were substituted in the SPICE template. Examination of these mutants for their ability to inhibit bovine CP revealed that E108, E120, and E144 are primarily responsible for imparting the specificity and contribute to the enhanced cofactor activity of VCP. Binding and functional assays suggested that these residues interact with bovine factor I but not with bovine C4(H2O) (a moiety conformationally similar to C4b). Mapping of these residues onto the modeled structure of bovine C4b-VCP-bovine factor I supported the mutagenesis data. Taken together, our data help explain why the vaccine strain of vaccinia virus was able to gain a foothold in domesticated animals.IMPORTANCE Vaccinia virus was used for smallpox vaccination. The vaccine-derived virus is now circulating and causing outbreaks in dairy cattle in India and Brazil. However, the reason for this tropism is unknown. It is well recognized that the virus is susceptible to neutralization by the complement classical pathway (CP). Because the virus encodes a soluble complement regulator, VCP, we examined whether this protein displays selectivity in targeting bovine CP. Our data show that it does exhibit selectivity in inhibiting the bovine CP and that this is primarily determined by its amino acids E108, E120, and E144, which interact with bovine serine protease factor I to inactivate bovine C4b-one of the two subunits of CP C3-convertase. Of note, the variola complement regulator SPICE contains positively charged residues at these positions. Thus, these variant residues in VCP help enhance its potency against the bovine CP and thereby the fitness of the virus in cattle.
Asunto(s)
Activación de Complemento/inmunología , Vía Alternativa del Complemento/inmunología , Vía Clásica del Complemento/inmunología , Proteínas de la Matriz Viral/inmunología , Proteínas Virales/inmunología , Tropismo Viral/genética , Secuencia de Aminoácidos , Animales , Bovinos , Proteína de Unión al Complemento C4b/inmunología , Fibrinógeno/metabolismo , Humanos , Alineación de Secuencia , Especificidad de la Especie , Virus Vaccinia/inmunología , Virus Vaccinia/patogenicidad , Proteínas de la Matriz Viral/genética , Proteínas Virales/genéticaRESUMEN
Engineering modular platforms to control biomolecular architecture can advance both the understanding and the manipulation of biological systems. Icosahedral particles uniformly displaying single antigens stimulate potent immune activation and have been successful in various licensed vaccines. However, it remains challenging to display multiple antigens on a single particle and to induce broader immunity protective across strains or even against distinct diseases. Here, we design a dually addressable synthetic nanoparticle by engineering the multimerizing coiled-coil IMX313 and two orthogonally reactive split proteins. SpyCatcher protein forms an isopeptide bond with SpyTag peptide through spontaneous amidation. SnoopCatcher forms an isopeptide bond with SnoopTag peptide through transamidation. SpyCatcher-IMX-SnoopCatcher provides a modular platform, whereby SpyTag-antigen and SnoopTag-antigen can be multimerized on opposite faces of the particle simply upon mixing. We demonstrate efficient derivatization of the platform with model proteins and complex pathogen-derived antigens. SpyCatcher-IMX-SnoopCatcher was expressed in Escherichia coli and was resilient to lyophilization or extreme temperatures. For the next generation of malaria vaccines, blocking the transmission of the parasite from human to mosquito is an important goal. SpyCatcher-IMX-SnoopCatcher multimerization of the leading transmission-blocking antigens Pfs25 and Pfs28 greatly enhanced the antibody response to both antigens in comparison to the monomeric proteins. This dual plug-and-display architecture should help to accelerate vaccine development for malaria and other diseases.
Asunto(s)
Proteína de Unión al Complemento C4b/inmunología , Proteínas de Escherichia coli/inmunología , Proteínas Protozoarias/inmunología , Animales , Proteína de Unión al Complemento C4b/química , Proteínas de Escherichia coli/química , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Proteínas Protozoarias/química , Vacunación , Vacunas Sintéticas/química , Vacunas Sintéticas/inmunologíaRESUMEN
The surface protein SdrE, a microbial surface components recognizing adhesive matrix molecule (MSCRAMM) family protein expressed on the surface of Staphylococcus aureus (S. aureus), can recognize human complement regulator Factor H and C4BP, thus making it a potentially promising vaccine candidate. In this study, SdrE278-591 was found to directly affect S. aureus host cell invasion. Additionally, the crystal structure of SdrE278-591 at a resolution of 1.25 Å was established, with the three-dimensional structure revealing N2-N3 domains which fold in a manner similar to an IgG fold. Furthermore, a putative ligand binding site located at a conserved charged groove formed by the interface between N2 and N3 domains was identified, with ß2 suspected to occupy the ligand recognizing site and undergo a structural rearrangement to allow ligand binding. Overall, these findings have further contributed to the understanding of SdrE as a key factor for S. aureus invasivity and will enable a better understanding of bacterial infection processes.
Asunto(s)
Proteínas Bacterianas/química , Proteína de Unión al Complemento C4b/química , Factor H de Complemento/química , Mutación , Staphylococcus aureus/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Sitios de Unión , Línea Celular Tumoral , Clonación Molecular , Proteína de Unión al Complemento C4b/genética , Proteína de Unión al Complemento C4b/inmunología , Factor H de Complemento/genética , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Inmunoglobulina G/química , Inmunoglobulina G/genética , Ligandos , Modelos Moleculares , Osteoblastos/inmunología , Osteoblastos/microbiología , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Pliegue de Proteína , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Staphylococcus aureus/patogenicidadRESUMEN
Mycobacterium tuberculosis is an accomplished intracellular pathogen, particularly within the macrophage and this is of the utmost importance in the host-pathogen stand-off observed in the granuloma during latent tuberculosis. Contact with innate immune molecules is one of the primary interactions that can occur with the pathogen M. tuberculosis once inhaled. Complement proteins may play a role in facilitating M. tuberculosis interactions with macrophages. Here, we demonstrate that factor H, a complement regulatory protein that down-regulates complement alternative pathway activation, binds directly to the model organism M. bovis BCG. Binding of factor H reaches saturation at 5-10µg of factor H/ml, well below the plasma level. C4 binding protein (C4BP) competed with factor H for binding to mycobacteria. Factor H was also found to inhibit uptake of M. bovis BCG by THP-1 macrophage cells in a dose-dependent manner. Real-time qPCR analysis showed stark differential responses of pro- and anti-inflammatory cytokines during the early stages of phagocytosis, as evident from elevated levels of TNF-α, IL-1ß and IL-6, and a concomitant decrease in IL-10, TGF-ß and IL-12 levels, when THP-1:BCG interaction took place in the presence of factor H. Our results suggest that factor H can interfere with mycobacterial entry into macrophages and modulate inflammatory cytokine responses, particularly during the initial stages of infection, thus affecting the extracellular survival of the pathogen. Our results offer novel insights into complement activation-independent functions of factor H during the host-pathogen interaction in tuberculosis.
Asunto(s)
Citocinas/inmunología , Interacciones Huésped-Patógeno , Macrófagos/microbiología , Mycobacterium bovis/fisiología , Línea Celular Tumoral , Proteína de Unión al Complemento C4b/inmunología , Factor H de Complemento/inmunología , Citocinas/genética , Humanos , Macrófagos/inmunología , FagocitosisRESUMEN
The activation of the complement system is a key initiating step in the protective innate immune-inflammatory response against injury, although it may also cause harm if left unchecked. The structurally related soluble complement inhibitors C4b-binding protein (C4BP) and factor H (FH) exert a tight regulation of the classical/lectin and alternative pathways of complement activation, respectively, attenuating the activity of the C3/C5 convertases and, consequently, avoiding serious damage to host tissues. We recently reported that the acute-phase C4BP isoform C4BP lacking the ß-chain plays a pivotal role in the modulation of the adaptive immune responses. In this study, we demonstrate that FH acts in the early stages of monocyte to dendritic cell (DC) differentiation and is able to promote a distinctive tolerogenic and anti-inflammatory profile on monocyte-derived DCs (MoDCs) challenged by a proinflammatory stimulus. Accordingly, FH-treated and LPS-matured MoDCs are characterized by altered cytoarchitecture, resembling immature MoDCs, lower expression of the maturation marker CD83 and the costimulatory molecules CD40, CD80, and CD86, decreased production of key proinflammatory Th1-cytokines (IL-12, TNF-α, IFN-γ, IL-6, and IL-8), and preferential production of immunomodulatory mediators (IL-10 and TGF-ß). Moreover, FH-treated MoDCs show low Ag uptake and, when challenged with LPS, display reduced CCR7 expression and chemotactic migration, impaired CD4(+) T cell alloproliferation, inhibition of IFN-γ secretion by the allostimulated T cells, and, conversely, induction of CD4(+)CD127(low/negative)CD25(high)Foxp3(+) regulatory T cells. Thus, this novel noncanonical role of FH as an immunological brake able to directly affect the function of MoDCs in an inflammatory environment may exhibit therapeutic potential in hypersensitivity, transplantation, and autoimmunity.
Asunto(s)
Células Dendríticas/inmunología , Tolerancia Inmunológica , Inflamación/inmunología , Monocitos/inmunología , Antígenos CD/metabolismo , Diferenciación Celular , Células Cultivadas , Quimiotaxis , Proteína de Unión al Complemento C4b/inmunología , Factor H de Complemento/inmunología , Citocinas/inmunología , Endocitosis , Humanos , Linfocitos T Reguladores/inmunologíaRESUMEN
C4b-binding protein (C4BP) is best known as a potent soluble inhibitor of the classical and lectin pathways of the complement system. This large 500 kDa multimeric plasma glycoprotein is expressed mainly in the liver but also in lung and pancreas. It consists of several identical 75 kDa α-chains and often also one 40 kDa ß-chain, both of which are mainly composed of complement control protein (CCP) domains. Structure-function studies revealed that one crucial binding site responsible for inhibition of complement is located to CCP1-3 of the α-chain. Binding of anticoagulant protein S to the CCP1 of the ß-chain provides C4BP with the ability to strongly bind apoptotic and necrotic cells in order to prevent inflammation arising from activation of complement by these cells. Further, C4BP interacts strongly with various types of amyloid and enhances fibrillation of islet amyloid polypeptide secreted from pancreatic beta cells, which may attenuate pro-inflammatory and cytotoxic effects of this amyloid. Full deficiency of C4BP has not been identified but non-synonymous alterations in its sequence have been found in haemolytic uremic syndrome and recurrent pregnancy loss. Furthermore, C4BP is bound by several bacterial pathogens, notably Streptococcus pyogenes, which due to inhibition of complement and enhancement of bacterial adhesion to endothelial cells provides these bacteria with a survival advantage in the host. Thus, depending on the context, C4BP has a protective or detrimental role in the organism.
Asunto(s)
Aborto Espontáneo/inmunología , Síndrome Hemolítico Urémico Atípico/inmunología , Proteína de Unión al Complemento C4b/metabolismo , Complicaciones Hematológicas del Embarazo/inmunología , Aborto Espontáneo/genética , Animales , Síndrome Hemolítico Urémico Atípico/genética , Activación de Complemento/genética , Proteína de Unión al Complemento C4b/genética , Proteína de Unión al Complemento C4b/inmunología , Femenino , Predisposición Genética a la Enfermedad , Homeostasis , Humanos , Mutación/genética , Polimorfismo Genético , Embarazo , Complicaciones Hematológicas del Embarazo/genéticaRESUMEN
The complement system consists of more than 40 proteins that participate in the inflammatory response and in pathogen killing. Complement inhibitors are necessary to avoid the excessive consumption and activation of this system on host cells. Leptospirosis is a worldwide zoonosis caused by spirochetes from the genus Leptospira. Pathogenic leptospires are able to escape from complement activation by binding to host complement inhibitors Factor H [FH] and C4b-binding protein (C4BP) while non-pathogenic leptospires are rapidly killed in the presence of fresh serum. In this study, we demonstrate that complement control protein domains (CCP) 7 and 8 of C4BP α-chain interact with the outer membrane proteins LcpA, LigA and LigB from the pathogenic leptospire L. interrogans. The interaction between C4BP and LcpA, LigA and LigB is sensitive to ionic strength and inhibited by heparin. We fine mapped the LigA and LigB domains involved in its binding to C4BP and heparin and found that both interactions are mediated through the bacterial immunoglobulin-like (Big) domains 7 and 8 (LigA7-8 and LigB7-8) of both LigA and LigB and also through LigB9-10. Therefore, C4BP and heparin may share the same binding sites on Lig proteins.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteína de Unión al Complemento C4b/metabolismo , Interacciones Huésped-Patógeno , Leptospira interrogans/metabolismo , Mapeo de Interacción de Proteínas , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteína de Unión al Complemento C4b/inmunología , Humanos , Leptospira interrogans/inmunologíaRESUMEN
Upon infection, pathogenic Leptospira species bind several complement regulators in order to overcome host innate immunity. We previously characterized a 20-kDa leptospiral surface protein which interacts with C4b binding protein (C4BP): leptospiral complement regulator-acquiring protein A (LcpA). Here we show that LcpA also interacts with human factor H (FH), which remains functionally active once bound to the protein. Antibodies directed against short consensus repeat 20 (SCR20) inhibited binding of FH to LcpA by approximately 90%, thus confirming that this particular domain is involved in the interaction. We have also shown for the first time that leptospires bind human vitronectin and that the interaction is mediated by LcpA. Coincubation with heparin blocked LcpA-vitronectin interaction in a dose-dependent manner, strongly suggesting that binding may occur through the heparin binding domains of vitronectin. LcpA also bound to the terminal pathway component C9 and inhibited Zn(2+)-induced polymerization and membrane attack complex (MAC) formation. Competitive binding assays indicated that LcpA interacts with C4BP, FH, and vitronectin through distinct sites. Taken together, our findings indicate that LcpA may play a role in leptospiral immune evasion.
Asunto(s)
Proteínas Bacterianas/química , Leptospira interrogans/química , Leptospira/química , Fragmentos de Péptidos/química , Vitronectina/química , Anticuerpos Monoclonales/química , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/inmunología , Sitios de Unión , Unión Competitiva , Activación de Complemento , Proteína de Unión al Complemento C4b/química , Proteína de Unión al Complemento C4b/inmunología , Complemento C9/química , Complemento C9/inmunología , Factor H de Complemento/química , Factor H de Complemento/inmunología , Complejo de Ataque a Membrana del Sistema Complemento/química , Heparina/química , Humanos , Evasión Inmune , Leptospira/inmunología , Leptospira/patogenicidad , Leptospira interrogans/inmunología , Leptospira interrogans/patogenicidad , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/inmunología , Unión Proteica , Vitronectina/inmunología , Zinc/químicaRESUMEN
Inappropriate complement activation is often responsible for incompatibility reactions that occur when biomaterials are used. Complement activation is therefore a criterion included in legislation regarding biomaterials testing. However, no consensus is yet available regarding appropriate complement-activation-related test parameters. We examined protein adsorption in plasma and complement activation/cytokine release in whole blood incubated with well-characterized polymers. Strong correlations were found between the ratio of C4 to its inhibitor C4BP and generation of 10 (mainly pro-inflammatory) cytokines, including IL-17, IFN-γ, and IL-6. The levels of complement activation products correlated weakly (C3a) or not at all (C5a, sC5b-9), confirming their poor predictive values. We have demonstrated a direct correlation between downstream biological effects and the proteins initially adhering to an artificial surface after contact with blood. Consequently, we propose the C4/C4BP ratio as a robust, predictor of biocompatibility with superior specificity and sensitivity over the current gold standard.