Human Complement Factor Research Articles

Impaired Immunogenicity of Meningococcal Neisserial Surface Protein A in Human Complement Factor H Transgenic Mice.

Neisserial surface protein A (NspA) is a highly conserved outer membrane protein previously investigated as a meningococcal vaccine candidate. Despite eliciting serum bactericidal activity in mice, a recombinant NspA vaccine failed to elicit serum bactericidal antibodies in a phase 1 clinical trial in humans. The discordant results may be explained by the recent discovery that NspA is a human-specific ligand of the complement inhibitor factor H (FH). Therefore, in humans but not mice, NspA would be expected to form a complex with FH, which could impair human anti-NspA protective antibody responses. To investigate this question, we immunized human FH transgenic BALB/c mice with three doses of recombinant NspA expressed in Escherichia coli microvesicles, with each dose being separated by 3 weeks. Three of 12 (25%) transgenic mice and 13 of 14 wild-type mice responded with bactericidal titers of ≥1:10 in postimmunization sera (P = 0.0008, Fisher's exact test). In contrast, human FH transgenic and wild-type mice immunized with a control meningococcal native outer membrane vesicle vaccine had similar serum bactericidal antibody responses directed at PorA, which is not known to bind human FH, and a mutant factor H binding protein (FHbp) antigen with a >50-fold lower level of FH binding than wild-type FHbp antigen binding.Thus, human FH can impair anti-NspA serum bactericidal antibody responses, which may explain the poor immunogenicity of the NspA vaccine previously tested in humans. A mutant NspA vaccine engineered to have decreased binding to human FH may increase protective antibody responses in humans.

A Mutant Library Approach to Identify Improved Meningococcal Factor H Binding Protein Vaccine Antigens.

Factor H binding protein (FHbp) is a virulence factor used by meningococci to evade the host complement system. FHbp elicits bactericidal antibodies in humans and is part of two recently licensed vaccines. Using human complement Factor H (FH) transgenic mice, we previously showed that binding of FH decreased the protective antibody responses to FHbp vaccination. Therefore, in the present study we devised a library-based method to identify mutant FHbp antigens with very low binding of FH. Using an FHbp sequence variant in one of the two licensed vaccines, we displayed an error-prone PCR mutant FHbp library on the surface of Escherichia coli. We used fluorescence-activated cell sorting to isolate FHbp mutants with very low binding of human FH and preserved binding of control anti-FHbp monoclonal antibodies. We sequenced the gene encoding FHbp from selected clones and introduced the mutations into a soluble FHbp construct. Using this approach, we identified several new mutant FHbp vaccine antigens that had very low binding of FH as measured by ELISA and surface plasmon resonance. The new mutant FHbp antigens elicited protective antibody responses in human FH transgenic mice that were up to 20-fold higher than those elicited by the wild-type FHbp antigen. This approach offers the potential to discover mutant antigens that might not be predictable even with protein structural information and potentially can be applied to other microbial vaccine antigens that bind host proteins.

Identification of Inhibitors against P.Falciparum GAP50 and Human Complement Factor H Interaction in the Mosquito

Malaria is an infectious disease caused by obligate intracellular parasites of the genus Plasmodium. Severe malaria cases result in more than half a million deaths every year (World Malaria Report, 2013). Mosquitoes transmit the parasite from human to human during their blood meals. With the blood meal, mosquitoes ingest female and male gametocytes present in the blood of a Plasmodium infected human. Gametocytes develop into gametes (Aly et al, 2009) that undergo fertilization inside the mosquito's gut and later result in the formation of infectious sporozoites that are transmitted during a bite. This uptake, maturation and fertilization, represents a bottleneck in the parasite's life cycle, and a prime target for transmission blocking. In the mosquito's gut, macrogametes (female gametes) bind the human complement control regulator protein, hFactor H, via PfGAP50 to avoid complement-mediated lysis (Simon et al, 2013). A potential transmission-blocking small molecule inhibitor would disrupt the PfGAP50-hFactor H interaction, exposing the parasite to the human alternate pathway of complement for destruction.The goal of this project is to characterize the PfGAP50-hFactor H interaction in vitro, and to screen the Medicines for Malaria Venture Malaria Box library for small molecule protein-protein interaction (PPI) inhibitors using a surface plasmon resonance (SPR)-based assay. Several PPI inhibitors were identified and the inhibitory affect was further validated in a concentration-dependent SPR screen. Promising hits will be further tested in mosquitos. In vitro characterization of this interaction will provide a better understanding of pathogen-host interactions at a molecular level and allow the development of specific inhibitors of PfGAP50-hFactor H interaction in the mosquito.

Expression of Human Complement Factor H Prevents Age-Related Macular Degeneration–Like Retina Damage and Kidney Abnormalities in Aged Cfh Knockout Mice

Complement factor H (CFH) is an important regulatory protein in the alternative pathway of the complement system, and CFH polymorphisms increase the genetic risk of age-related macular degeneration dramatically. These same human CFH variants have also been associated with dense deposit disease. To mechanistically study the function of CFH in the pathogenesis of these diseases, we created transgenic mouse lines using human CFH bacterial artificial chromosomes expressing full-length human CFH variants and crossed these to Cfh knockout (Cfh(-/-)) mice. Human CFH protein inhibited cleavage of mouse complement component 3 and factor B in plasma and in retinal pigment epithelium/choroid/sclera, establishing that human CFH regulates activation of the mouse alternative pathway. One of the mouse lines, which express relatively higher levels of CFH, demonstrated functional and structural protection of the retina owing to the Cfh deletion. Impaired visual function, detected as a deficit in the scotopic electroretinographic response, was improved in this transgenic mouse line compared with Cfh(-/-)mice, and transgenics had a thicker outer nuclear layer and less sub-retinal pigment epithelium deposit accumulation. In addition, expression of human CFH also completely protected the mice from developing kidney abnormalities associated with loss of CFH. These humanized CFH mice present a valuable model for study of the molecular mechanisms of age-related macular degeneration and dense deposit disease and for testing therapeutic targets.

Identification of human complement factor B as a novel biomarker candidate for pancreatic ductal adenocarcinoma.

Pancreatic cancer (PC; pancreatic ductal adenocarcinoma) is characterized by significant morbidity and mortality worldwide. Although carbohydrate antigen (CA) 19-9 has been known as a PC biomarker, it is not commonly used for general screening because of its low sensitivity and specificity. Therefore, there is an urgent need to develop a new biomarker for PC diagnosis in the earlier stage of cancer. To search for a novel serologic PC biomarker, we carried out an integrated proteomic analysis for a total of 185 pooled or individual plasma from healthy donors and patients with five disease groups including chronic pancreatitis (CP), PC, and other cancers (e.g., hepatocellular carcinoma, cholangiocarcinoma, and gastric cancer) and identified complement factor b (CFB) as a candidate serologic biomarker for PC diagnosis. Immunoblot analysis of CFB revealed more than two times higher expression in plasma samples from PC patients compared with plasma from individuals without PC. Immunoprecipitation coupled to mass spectrometry analysis confirmed both molecular identity and higher expression of CFB in PC samples. CFB showed distinctly higher specificity than CA 19-9 for PC against other types of digestive cancers and in discriminating PC patients from non-PC patients (p < 0.0001). In receiver operator characteristic curve analysis, CFB showed an area under curve of 0.958 (95% CI: 0.956 to 0.959) compared with 0.833 (95% CI: 0.829 to 0.837) for CA 19-9. Furthermore, the Y-index of CFB was much higher than that of CA 19-9 (71.0 vs 50.4), suggesting that CFB outperforms CA 19-9 in discriminating PC from CP and other gastrointestinal cancers. This was further supported by immunoprecipitation and qRT-PCR assays showing higher expression of CFB in PC cell lines than in normal cell lines. A combination of CFB and CA 19-9 showed markedly improved sensitivity (90.1 vs 73.1%) over that of CFB alone in the diagnosis of PC against non-PC, with similar specificity (97.2 vs 97.9%). Thus, our results identify CFB as a novel serologic PC biomarker candidate and warrant further investigation into a large-scale validation and its role in molecular mechanism of pancreatic carcinogenesis.

New insights into disease-specific absence of complement factor H related protein C in mouse models of spontaneous autoimmune diseases

Complement factor H (CFH) protein is an inhibitor of the alternative pathway of complement (AP) both in the fluid phase and on the surface of host cells. Mouse and human complement factor H-related (CFHR) proteins also belong to the fH family of plasma glycoproteins. The main goal of the current study was to compare the presence of mRNA for two mCFHR proteins in spontaneously developing autoimmune diseases in mice such as dense deposit disease (DDD), diabetes mellitus (DM), basal laminar deposits (BLD), collagen antibody-induced arthrits (CAIA) and systemic lupus erythematosus (SLE). Here we report for the first time that the CFHR-C mRNA was universally absent in the liver from three strains of lupus-prone mice and in a diabetic-prone mouse strain. The mRNA levels (pg/ng) for CFH and CFHR-B in MRL-lpr/lpr, at 9wks and 23wks were 707.2±44.4, 54.5±5.75 and 729±252.9, 74.04±22.76, respectively. The mRNA levels for CFH and CFHR-B in NZB/NZW mice, at 9wks and 54wks were 579.9±23.8, 58.8±1.41 and 890.3±135.2, 63.30±9.2, respectively. CFHR-C protein was absent in the circulation of MRL-lpr/lpr and NZB/NZW mice before and after the development of lupus. Similarly, mRNA and protein for CFHR-C was universally absent in liver and other organs and in the circulation of NOD mice before and after the development of DM. In contrast, the mRNAs for CFH, CFHR-B and CFHR-C were universally present in the liver from mice with and without DDD, BLD and CAIA. The levels of mRNA for CFHR-B in mice with and without BLD were ∼4 times higher than the mice with lupus. The complete absence of mRNA for CFHR-C in lupus and diabetic-prone strains indicates that polymorphic variation within the mouse CFHR family exists and raises the possibility that such variation contributes to lupus and diabetic phenotypes.

Ensemble refinement shows conformational flexibility in crystal structures of human complement factor D

Human factor D (FD) is a self-inhibited thrombin-like serine proteinase that is critical for amplification of the complement immune response. FD is activated by its substrate through interactions outside the active site. The substrate-binding, or `exosite', region displays a well defined and rigid conformation in FD. In contrast, remarkable flexibility is observed in thrombin and related proteinases, in which Na(+) and ligand binding is implied in allosteric regulation of enzymatic activity through protein dynamics. Here, ensemble refinement (ER) of FD and thrombin crystal structures is used to evaluate structure and dynamics simultaneously. A comparison with previously published NMR data for thrombin supports the ER analysis. The R202A FD variant has enhanced activity towards artificial peptides and simultaneously displays active and inactive conformations of the active site. ER revealed pronounced disorder in the exosite loops for this FD variant, reminiscent of thrombin in the absence of the stabilizing Na(+) ion. These data indicate that FD exhibits conformational dynamics like thrombin, but unlike in thrombin a mechanism has evolved in FD that locks the unbound native state into an ordered inactive conformation via the self-inhibitory loop. Thus, ensemble refinement of X-ray crystal structures may represent an approach alternative to spectroscopy to explore protein dynamics in atomic detail.

Structural Model for Covalent Adhesion of the Streptococcus pyogenes Pilus through a Thioester Bond

The human pathogen Streptococcus pyogenes produces pili that are essential for adhesion to host surface receptors. Cpa, the adhesin at the pilus tip, was recently shown to have a thioester-containing domain. The thioester bond is believed to be important in adhesion, implying a mechanism of covalent attachment analogous to that used by human complement factors. Here, we have characterized a second active thioester-containing domain on Cpa, the N-terminal domain of Cpa (CpaN). Expression of CpaN in Escherichia coli gave covalently linked dimers. These were shown by x-ray crystallography and mass spectrometry to comprise two CpaN molecules cross-linked by the polyamine spermidine following reaction with the thioester bonds. This cross-linked CpaN dimer provides a model for the covalent attachment of Cpa to target receptors and thus the streptococcal pilus to host cells. Similar thioester domains were identified in cell wall proteins of other Gram-positive pathogens, suggesting that thioester domains are more widely used and provide a mechanism of adhesion by covalent bonding to target molecules on host cells that mimics that used by the human complement system to eliminate pathogens.

Neisseria meningitidis NalP cleaves human complement C3, facilitating degradation of C3b and survival in human serum

The complement system is a crucial component of the innate immune response against invading bacterial pathogens. The human pathogen Neisseria meningitidis (Nm) is known to possess several mechanisms to evade the complement system, including binding to complement inhibitors. In this study, we describe an additional mechanism used by Nm to evade the complement system and survive in human blood. Using an isogenic NalP deletion mutant and NalP complementing strains, we show that the autotransporter protease NalP cleaves C3, the central component of the complement cascade. The cleavage occurs 4 aa upstream from the natural C3 cleavage site and produces shorter C3a-like and longer C3b-like fragments. The C3b-like fragment is degraded in the presence of the complement regulators (factors H and I), and this degradation results in lower deposition of C3b on the bacterial surface. We conclude that NalP is an important factor to increase the survival of Nm in human serum.

Human Complement Factor H and Factor H-Like Protein 1 Are Expressed in Human Retinal Pigment Epithelial Cells

Background: A common haplotype in the gene for the regulator of the alternative pathway of complement activation factor H has been linked to individual predisposition to age- related macular degeneration (AMD). Methods: In this study, retinal pigment epithelial (RPE) cells, i.e. immortalized ARPE-19 as well as primary human RPE cells, were investigated for expression of factor H and FHL-1 by immunohistochemistry and in situ hybridization analysis. Results: Factor H and the alternative spliced product FHL-1 are expressed in RPE cells, i.e. in immortalized ARPE-19 and primary human RPE cells. Factor H and FHL-1 expression was induced in a dose-dependent manner in ARPE-19 cells upon treatment with the inflammatory marker interleukin-6 (IL-6). In situ hybridization experiments confirmed an elevated expression rate of the factor H gene in IL-6-treated ARPE-19 cells. AMD is characterized by complement-associated inflammatory processes in the retina. Thus, local synthesis of complement regulators affects the protection of retinal cells and may be involved in the pathogenesis at the RPE-choroid interface.

Meningococcal factor H-binding protein vaccines with decreased binding to human complement factor H have enhanced immunogenicity in human factor H transgenic mice

Factor H-binding protein (fHbp) is a component of a meningococcal vaccine recently licensed in Europe for prevention of serogroup B disease, and a second vaccine in clinical development. The protein specifically binds human factor H (fH), which down-regulates complement activation and enhances resistance to bactericidal activity. There are conflicting data from studies in human fH transgenic mice on whether binding of human fH to fHbp vaccines decreases immunogenicity, and whether mutant fHbp vaccines with decreased fH binding have enhanced immunogenicity. fHbp can be classified into two sub-families based on sequence divergence and immunologic cross-reactivity. Previous studies of mutant fHbp vaccines with low fH binding were from sub-family B, which account for approximately 60% of serogroup B case isolates. In the present study, we evaluated the immunogenicity of two mutant sub-family A fHbp vaccines containing single substitutions, T221A or D211A, which resulted in 15- or 30-fold lower affinity for human fH, respectively, than the corresponding control wild-type fHbp vaccine. In transgenic mice with high serum concentrations of human fH, both mutant vaccines elicited significantly higher IgG titers and higher serum bactericidal antibody responses than the control fHbp vaccine that bound human fH. Thus, mutations introduced into a sub-family A fHbp antigen to decrease fH binding can increase protective antibody responses in human fH transgenic mice. Collectively the data suggest that mutant fHbp antigens with decreased fH binding will result in superior vaccines in humans.

Human Skin Mast Cells Express Complement Factors C3 and C5

We examine whether complement factor C3 or C5 is synthesized by human skin-derived mast cells and whether their synthesis is regulated by cytokines. C3 and C5 mRNAs were assessed by RT-PCR, and proteins by flow cytometry, confocal microscopy, Western blotting, and ELISA. C3 and C5 mRNAs were each expressed, and baseline protein levels/10(6) cultured mast cells were 0.9 and 0.8 ng, respectively, and located in the cytoplasm outside of secretory granules. C3 accumulated in mast cell culture medium over time and by 3 d reached a concentration of 9.4 ± 8.0 ng/ml, whereas C5 levels were not detectable (<0.15 ng/ml). Three-day incubations of mast cells with IL-1α, IL-1β, IL-17, IFN-γ, IL-6, or anti-FcεRI did not affect C3 protein levels in culture medium, whereas incubations with PMA, TNF-α, IL-13, or IL-4 enhanced levels of C3 1.7- to 3.3-fold. In contrast with C3, levels of C5 remained undetectable. Importantly, treatment with TNF-α together with either IL-4 or IL-13 synergistically enhanced C3 (but not C5) production in culture medium by 9.8- or 7.1-fold, respectively. This synergy was blocked by attenuating the TNF-α pathway with neutralizing anti-TNF-α Ab, soluble TNFR, or an inhibitor of NF-κB, or by attenuating the IL-4/13 pathway with Jak family or Erk antagonists. Inhibitors of PI3K, Jnk, and p38 MAPK did not affect this synergy. Thus, human mast cells can produce and secrete C3, whereas β-tryptase can act on C3 to generate C3a and C3b, raising the likelihood that mast cells engage complement to modulate immunity and inflammation in vivo.

Elongation Factor Tu (Tuf) is a new virulence factor of Streptococcus pneumoniae that binds human complement factors, aids in immune evasion and host tissue invasion

Elongation Factor Tu (Tuf) is a new virulence factor of Streptococcus pneumoniae that binds human complement factors, aids in immune evasion and host tissue invasion

Multiplex Protein Signature for the Detection of Bladder Cancer in Voided Urine Samples

Accurate urine assays for bladder cancer detection would benefit patients and health care systems. Through extensive genomic and proteomic profiling of urine components we previously identified a panel of 8 biomarkers that can facilitate the detection of bladder cancer in voided urine samples. In this study we confirmed this diagnostic molecular signature in a diverse multicenter cohort. We performed a case-control, phase II study in which we analyzed voided urine from 102 subjects with bladder cancer and 206 with varying urological disorders. The urinary concentration of 8 biomarkers (IL-8, MMP-9 and 10, PAI-1, VEGF, ANG, CA9 and APOE) was assessed by enzyme-linked immunosorbent assay. Diagnostic performance of the panel of tested biomarkers was evaluated using ROCs and descriptive statistical values, eg sensitivity and specificity. Seven of the 8 urine biomarkers were increased in subjects with bladder cancer relative to those without bladder cancer. The 7 biomarkers were assessed in a new model, which had an AUROC of 0.88 (95% CI 0.84-0.93), and 74% sensitivity and 90% specificity. In contrast, the sensitivity of voided urine cytology and the UroVysion® cytogenetic test in this cohort was 39% and 54%, respectively. Study limitations include analysis performed on banked urine samples and the lack of voided urine cytology and cytogenetic test data on controls. The study provides further evidence that the reported panel of diagnostic biomarkers can reliably achieve the noninvasive detection of bladder cancer with higher sensitivity than currently available urine based assays.

Correction: Expression and Subcellular Targeting of Human Complement Factor C5a in Nicotiana species

The second author's name is misspelled. The correct spelling is: Heike Mikschofsky The correct citation is: Nausch H, Mikschofsky H, Koslowski R, Meyer U, Broer I, et al. (2012) Expression and Subcellular Targeting of Human Complement Factor C5a in Nicotiana species. PLoS ONE 7(12): e53023. doi:10.1371/journal.pone.0053023

A hypervariable STR polymorphism in the CFI gene: Southern origin of East Asian-specific group H alleles

Previous studies of four populations revealed that a hypervariable short tandem repeat (iSTR) in intron 7 of the human complement factor I (CFI) gene on chromosome 4q was unique, with 17 possible East Asian-specific group H alleles observed at relatively high frequencies. To develop a deeper anthropological and forensic understanding of iSTR, 1161 additional individuals from 11 Asian populations were investigated. Group H alleles of iSTR and c.1217A allele of a SNP in exon 11 of the CFI gene were associated with each other and were almost entirely confined to East Asian populations. Han Chinese in Changsha, southern China, showed the highest frequency for East Asian-specific group H alleles (0.201) among 15 populations. Group H alleles were observed to decrease gradually from south to north in 11 East Asian populations. This expansion of group H alleles provides evidence that southern China and Southeast Asia are a hotspot of Asian diversity and a genetic reservoir of Asians after they entered East Asia. The expected heterozygosity values of iSTR ranged from 0.927 in Thais to 0.874 in Oroqens, higher than those of an STR in the fibrinogen alpha chain (FGA) gene on chromosome 4q. Thus, iSTR is a useful marker for anthropological and forensic genetics.

Expression and Subcellular Targeting of Human Complement Factor C5a in Nicotiana species

We evaluated transgenic tobacco plants as an alternative to Escherichia coli for the production of recombinant human complement factor 5a (C5a). C5a has not been expressed in plants before and is highly unstable in vivo in its native form, so it was necessary to establish the most suitable subcellular targeting strategy. We used the strong and constitutive CaMV 35S promoter to drive transgene expression and compared three different subcellular compartments. The yields of C5a in the T0 transgenic plants were low in terms of the proportion of total soluble protein (TSP) when targeted to the apoplast (0.0002% TSP) or endoplasmic reticulum (0.0003% TSP) but was one order of magnitude higher when targeted to the vacuole (0.001% TSP). The yields could be increased by conventional breeding (up to 0.014% TSP in the T2 generation). C5a accumulated to the same level in seeds and leaves when targeted to the apoplast but was up to 1.7-fold more abundant in the seeds when targeted to the ER or vacuole, although this difference was less striking in the better-performing lines. When yields were calculated as an amount per gram fresh weight of transgenic plant tissue, the vacuole targeting strategy was clearly more efficient in seeds, reaching 35.8 µg C5a per gram of fresh seed weight compared to 10.62 µg C5a per gram fresh weight of leaves. Transient expression of C5aER and C5aVac in N. benthamiana, using MagnICON vectors, reached up to 0.2% and 0.7% of TSP, respectively, but was accompanied by cytotoxic effects and induced leaf senescence. Western blot of the plant extracts revealed a band matching the corresponding glycosylated native protein and the bioassay demonstrated that recombinant C5a was biologically active.

Intravitreal human complement factor H in a rat model of laser-induced choroidal neovascularisation

PurposeTo investigate the inhibitory effect of intravitreally administered human complement factor H (CFH) in a rat model of laser-induced choroidal neovascularisation (CNV).MethodsAnalysis of alternative pathway inhibition by human plasma-purified CFH...

A hypervariable STR polymorphism in the CFI gene: Mutation rate and no linkage disequilibrium with FGA

A hypervariable short tandem repeat (STR) polymorphism in intron 7 of the human complement factor I gene (CFI) was investigated to estimate the mutation rate in Japanese samples and to test linkage disequilibrium (LD) with an STR in the fibrinogen alpha chain gene (FGA). The expected heterozygosity and the mutation rate of CFI were estimated to be 0.917 and 0.002, respectively. No LD was observed between CFI and FGA. CFI is a useful supplementary marker for forensic science.

Factor H-related Protein 4 Activates Complement by Serving as a Platform for the Assembly of Alternative Pathway C3 Convertase via Its Interaction with C3b Protein

Human complement factor H-related protein (CFHR) 4 belongs to the factor H family of plasma glycoproteins that are composed of short consensus repeat (SCR) domains. Although factor H is a well known inhibitor of the alternative complement pathway, the functions of the CFHR proteins are poorly understood. CFHR4 lacks SCRs homologous to the complement inhibitory domains of factor H and, accordingly, has no significant complement regulatory activities. We have previously shown that CFHR4 binds C-reactive protein via its most N-terminal SCR, which leads to classical complement pathway activation. CFHR4 binds C3b via its C terminus, but the significance of this interaction is unclear. Therefore, we set out to clarify the functional relevance of C3b binding by CFHR4. Here, we report a novel role for CFHR4 in the complement system. CFHR4 serves as a platform for the assembly of an alternative pathway C3 convertase by binding C3b. This is based on the sustained ability of CFHR4-bound C3b to bind factor B and properdin, leading to an active convertase that generates C3a and C3b from C3. The CFHR4-C3bBb convertase is less sensitive to the factor H-mediated decay compared with the C3bBb convertase. CFHR4 mutants containing exchanges of conserved residues within the C-terminal C3b-binding site showed significantly reduced C3b binding and alternative pathway complement activation. In conclusion, our results suggest that, in contrast to the complement inhibitor factor H, CFHR4 acts as an enhancer of opsonization by promoting complement activation.