Complement alone drives efficacy of a chimeric antigonococcal monoclonal antibody.

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.

Human IgG Increases Virulence of Streptococcus pyogenes through Complement Evasion.

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.

Species Specificity of Vaccinia Virus Complement Control Protein for the Bovine Classical Pathway Is Governed Primarily by Direct Interaction of Its Acidic Residues with Factor I.

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 ∼43-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.

The Complement Inhibitor Factor H Generates an Anti-Inflammatory and Tolerogenic State in Monocyte-Derived Dendritic Cells.

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.

Dual Plug-and-Display Synthetic Assembly Using Orthogonal Reactive Proteins for Twin Antigen Immunization.

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.

Binding of Streptococcus pneumoniae endopeptidase O (PepO) to complement component C1q modulates the complement attack and promotes host cell adherence.

The Gram-positive species Streptococcus pneumoniae is a human pathogen causing severe local and life-threatening invasive diseases associated with high mortality rates and death. We demonstrated recently that pneumococcal endopeptidase O (PepO) is a ubiquitously expressed, multifunctional plasminogen and fibronectin-binding protein facilitating host cell invasion and evasion of innate immunity. In this study, we found that PepO interacts directly with the complement C1q protein, thereby attenuating the classical complement pathway and facilitating pneumococcal complement escape. PepO binds both free C1q and C1 complex in a dose-dependent manner based on ionic interactions. Our results indicate that recombinant PepO specifically inhibits the classical pathway of complement activation in both hemolytic and complement deposition assays. This inhibition is due to direct interaction of PepO with C1q, leading to a strong activation of the classical complement pathway, and results in consumption of complement components. In addition, PepO binds the classical complement pathway inhibitor C4BP, thereby regulating downstream complement activation. Importantly, pneumococcal surface-exposed PepO-C1q interaction mediates bacterial adherence to host epithelial cells. Taken together, PepO facilitates C1q-mediated bacterial adherence, whereas its localized release consumes complement as a result of its activation following binding of C1q, thus representing an additional mechanism of human complement escape by this versatile pathogen.

Pathogenic Leptospira species acquire factor H and vitronectin via the surface protein LcpA.

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.

Yersinia pestis Ail recruitment of C4b-binding protein leads to factor I-mediated inactivation of covalently and noncovalently bound C4b.

The outer membrane protein Ail of Yersinia pestis mediates several virulence functions, including serum resistance. Here, we demonstrate that Ail binds C4b-binding protein (C4BP), the primary fluid-phase regulator of the classical and lectin pathways. Non-covalent binding of C4 and C4b to Ail was also observed. C4BP bound to Ail can act as a cofactor to the serine protease factor I (fI) in the cleavage of fluid-phase C4b. Employing a panel of C4BP alpha-chain mutants, we observed that the absence of complement control protein domain 6 and 8 reduced binding to Ail. Immunoblot analysis of normal human serum (NHS)-treated bacteria revealed minimal C4b alpha'-chain complexes with bacterial outer membrane targets. Addition of the anti-C4BP monoclonal antibody MK104 to NHS restored C4b-alpha' chain target complexes, suggesting that C4b binds covalently to targets on the Y. pestis surface. C4b bound to Ail noncovalently was also cleaved in a C4BP and fI-dependent manner, leaving the C4c fragment bound to Ail. MK104 also prevented the cleavage of noncovalently bound C4b. Collectively, these data suggest that when C4BP is bound to Ail, fI can cleave and inactivate C4b that has bound covalently to bacterial surface structures as well as C4b bound noncovalently to Ail.

Truncated and full-length thioredoxin-1 have opposing activating and inhibitory properties for human complement with relevance to endothelial surfaces.

Thioredoxin (Trx)-1 is a small, ubiquitously expressed redox-active protein with known important cytosolic functions. However, Trx1 is also upregulated in response to various stress stimuli, is found both at the cell surface and secreted into plasma, and has known anti-inflammatory and antiapoptotic properties. Previous animal studies have demonstrated that exogenous Trx1 delivery can have therapeutic effects in a number of disease models and have implicated an interaction of Trx1 with the complement system. We found that Trx1 is expressed in a redox-active form at the surface of HUVEC and acts as an inhibitor of complement deposition in a manner dependent on its Cys-Gly-Pro-Cys active site. Inhibition occurred at the point of the C5 convertase of complement, regulating production of C5a and the membrane attack complex. A truncated form of Trx1 also exists in vivo, Trx80, which has separate nonoverlapping functions compared with the full-length Trx1. We found that Trx80 activates the classical and alternative pathways of complement activation, leading to C5a production, but the inflammatory potential of this was also limited by the binding of inhibitors C4b-binding protein and factor H. This study adds a further role to the known anti-inflammatory properties of Trx1 and highlights the difference in function between the full-length and truncated forms.

Targeted complement inhibition using bispecific antibodies that bind local antigens and endogenous complement regulators.

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.

Biofilm formation avoids complement immunity and phagocytosis of Streptococcus pneumoniae.

Streptococcus pneumoniae is a frequent member of the microbiota of the human nasopharynx. Colonization of the nasopharyngeal tract is a first and necessary step in the infectious process and often involves the formation of sessile microbial communities by this human pathogen. The ability to grow and persist as biofilms is an advantage for many microorganisms, because biofilm-grown bacteria show reduced susceptibility to antimicrobial agents and hinder recognition by the immune system. The extent of host protection against biofilm-related pneumococcal disease has not been determined yet. Using pneumococcal strains growing as planktonic cultures or as biofilms, we have investigated the recognition of S. pneumoniae by the complement system and its interactions with human neutrophils. Deposition of C3b, the key complement component, was impaired on S. pneumoniae biofilms. In addition, binding of C-reactive protein and the complement component C1q to the pneumococcal surface was reduced in biofilm bacteria, demonstrating that pneumococcal biofilms avoid the activation of the classical complement pathway. In addition, recruitment of factor H, the downregulator of the alternative pathway, was enhanced by S. pneumoniae growing as biofilms. Our results also show that biofilm formation diverts the alternative complement pathway activation by a PspC-mediated mechanism. Furthermore, phagocytosis of pneumococcal biofilms was also impaired. The present study confirms that biofilm formation in S. pneumoniae is an efficient means of evading both the classical and the PspC-dependent alternative complement pathways the host immune system.

Phosphoethanolamine residues on the lipid A moiety of Neisseria gonorrhoeae lipooligosaccharide modulate binding of complement inhibitors and resistance to complement killing.

Loss of phosphoethanolamine (PEA) from the lipid A of gonococcal strain FA19 results in increased sensitivity to killing by the classical pathway of complement. Here we demonstrate that loss of PEA from lipid A diminishes binding of the complement regulatory protein C4b binding protein (C4BP) to the FA19 porin B (PorB), providing a molecular basis to explain the susceptibility of an lptA null strain of FA19 to killing by normal human serum (NHS). Loss of PEA from lipid A in three additional gonococcal strains that expressed diverse PorB molecules also resulted in decreased C4BP binding, increased deposition of C4b, and increased susceptibility to killing by NHS. Complementation of lptA null strains with lptA restored C4BP binding, decreased C4b deposition, and increased resistance to killing by NHS. These effects of lipid A PEA on C4BP binding to gonococcal PorB and serum resistance were simulated when gonococcal PorB was expressed in a meningococcal background. Loss of PEA from lipid A also affected binding of the alternative pathway regulator factor H (fH) to PorB of some strains. For instance, PorB molecules of lptA null mutants of strains 252 and 1291 bound less fH than those of their parent strains when lipooligosaccharide (LOS) was sialylated, whereas PorB molecules of lptA null mutants of strains FA1090 and 273 retained the ability to bind fH when LOS was sialylated. These data indicate that replacement of lipid A with PEA alters binding of C4BP and fH to PorB and contributes to the ability of gonococci to resist complement-mediated killing.

Clinical isolates of Streptococcus pneumoniae bind the complement inhibitor C4b-binding protein in a PspC allele-dependent fashion.

The complement system constitutes an important component of the innate immune system. To colonize their host and/or to cause disease, many pathogens have evolved strategies to avoid complement-mediated bacterial lysis and opsonophagocytosis. In this study, using a collection of 55 clinical isolates of Streptococcus pneumoniae, we demonstrate for the first time that pneumococci bind the complement inhibitor C4b-binding protein (C4BP). C4BP binding seems to be restricted to certain serotypes such as serotype 4, 6B, 7F, and 14, of which the strains of serotype 14 are the strongest binders. We show that bacteria-bound C4BP retains its functional activity and down-regulates the activation of the classical pathway. Thus, this major respiratory pathogen may escape immune recognition and eradication by the complement system. Furthermore, we show that C4BP binding varies between strains but is dependent on the expression of pneumococcal surface protein C, PspC of group 4. The study of the distribution of group 4 pspC locus shows that most of high-binder serotype 14 isolates harbor an allelic variant of group 4 pspC. Using PspC-negative mutant strains, we identified a new allelic variant of PspC (PspC4.4) as a major ligand for C4BP, revealing a new function for this important pneumococcal virulence factor. Thus pneumococci exploit host C4BP for complement evasion in a PspC allele-dependent manner.

Binding of complement inhibitor C4b-binding protein contributes to serum resistance of Porphyromonas gingivalis.

The periodontal pathogen Porphyromonas gingivalis is highly resistant to the bactericidal activity of human complement, which is present in the gingival crevicular fluid at 70% of serum concentration. All thirteen clinical and laboratory P. gingivalis strains tested were able to capture the human complement inhibitor C4b-binding protein (C4BP), which may contribute to their serum resistance. Accordingly, in serum deficient of C4BP, it was found that significantly more terminal complement component C9 was deposited on P. gingivalis. Moreover, using purified proteins and various isogenic mutants, we found that the cysteine protease high molecular weight arginine-gingipain A (HRgpA) is a crucial C4BP ligand on the bacterial surface. Binding of C4BP to P. gingivalis appears to be localized to two binding sites: on the complement control protein 1 domain and complement control protein 6 and 7 domains of the alpha-chains. Furthermore, the bacterial binding of C4BP was found to increase with time of culture and a particularly strong binding was observed for large aggregates of bacteria that formed during culture on solid blood agar medium. Taken together, gingipains appear to be a very significant virulence factor not only destroying complement due to proteolytic degradation as we have shown previously, but was also inhibiting complement activation due to their ability to bind the complement inhibitor C4BP.

C4b Binding Protein Acts as an Innate Immune Effector Against Influenza A Virus.

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.

Complement evasion by the human respiratory tract pathogens Haemophilus influenzae and Moraxella catarrhalis.

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.

Native, amyloid fibrils and beta-oligomers of the C-terminal domain of human prion protein display differential activation of complement and bind C1q, factor H and C4b-binding protein directly.

Prion protein (PrP) is an endogenous protein involved in the pathogenesis of bovine spongiform encephalopathy and Creutzfeldt-Jakob disease. Murine PrP has been reported to bind C1q and activate the classical pathway of complement in a copper-dependent manner. Here we show that various conformational isoforms (native, amyloid fibrils, and beta-oligomers) of recombinant human PrP (90-231 and 121-231) bind C1q and activate complement. PrP binds both the globular head and collagenous stalk domains of C1q. Native, beta-oligomeric and amyloid fibrils of PrP all activate the classical and alternative pathways of complement to different extent. However, they do not trigger the lectin pathway. Of the tested PrP conformational isoforms we find that beta-oligomers bind C1q and activate complement most strongly. Membrane attack complex formation initiated by PrP is subdued in comparison to deposition of early complement components. This is most likely attributed to the interaction between human PrP and complement inhibitors factor H and C4b-binding protein. Accordingly, PrP-triggered complement activation in the terminal pathway was increased in serum lacking C4b-binding protein. Taken together the present study indicates that complement activation may be an important factor in human prion diseases, suggesting that complement induced activities may prove relevant therapeutic targets.

Toxoplasma gondii Recruits Factor H and C4b-Binding Protein to Mediate Resistance to Serum Killing and Promote Parasite Persistence in vivo.

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.

The Molecular Basis of Human IgG-Mediated Enhancement of C4b-Binding Protein Recruitment to Group A Streptococcus.

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.

Pentraxins in Complement Activation and Regulation.

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.