C3 Binding Research Articles

143 A targeted binding and activation of native C3 without proteolytic cleavage induced by contact with biosurfaces

143 A targeted binding and activation of native C3 without proteolytic cleavage induced by contact with biosurfaces

Synthesis and anti-tumor activity of piperonal substituted chalcone

Objectives: Chalcones have been identified as potential antitumor agents with a novel target, the tubulin. The aim of the study was to synthesize a piperonal substituted chalcone and evaluate its in vivo antitumor activity. Materials and Methods: Piperonal substituted chalcone was synthesized using Claisen-Schmidt condensation and characterized using various spectroscopic techniques. The lethal dose (LD50) of the synthesized compound was estimated using OECD-425 guidelines in rats. Antitumor activity of the synthesized compound was evaluated on 1-methyl nitrosourea (MNU)-induced mammary tumor in female Wistar rats. Histological evaluation was used to confirm tumor induction and assess treatment with the synthesized compound. The possible mechanism of action of the synthesized compound was elucidated in silico using molecular docking. Results: The compound was synthesized and named C2. C2 was found to be relatively safe with LD50 >2000 mg/kg orally. Moreover, C2 exhibited remarkable antitumor activity, at all the tested doses in a dose dependent manner. Histological evaluation of the MNU-induced mammary tumor rats treated with C2 displayed fewer signs of hyperplasia and small numbers of connective tissue with larger lobules when compared with the untreated group. In silico tubulin-binding interactions revealed that the kinetics of C2 binding to tubulin was like that of colchicine. Comparison of crystal structures of tubulin-C2 and tubulin-colchicine complexes showed that the binding mode of C2 to tubulin was like that of colchicine to tubulin and produced the same conformational changes on the tubulin structure as colchicine. Conclusion: The synthesized chalcone demonstrated remarkable antitumor activities in MNU-induced mammary tumors in rats possibly through inhibition of tubulin polymerization.

Discovery and biological evaluation of an adamantyl-amide derivative with likely MmpL3 inhibitory activity

A series of molecules containing bulky lipophilic scaffolds was screened for activity against Mycobacterium tuberculosis and a number of compounds with antimycobacterial activity were identified. The most active compound, (2E)-N-(adamantan-1-yl)-3-phenylprop-2-enamide (C1), has a low micromolar minimum inhibitory concentration, low cytotoxicity (therapeutic index = 32.26), low mutation frequency and is active against intracellular Mycobacterium tuberculosis. Whole genome sequencing of mutants resistant to C1 showed a mutation in mmpL3 which may point to the involvement of MmpL3 in the antimycobacterial activity of the compound. In silico mutagenesis and molecular modelling studies were performed to better understand the binding of C1 within MmpL3 and the role that the specific mutation may play in the interaction at protein level. These analyses revealed that the mutation increases the energy required for binding of C1 within the protein translocation channel of MmpL3. The mutation also decreases the solvation energy of the protein, suggesting that the mutant protein might be more solvent-accessible, thereby restricting its interaction with other molecules. The results reported here describe a new molecule that may interact with the MmpL3 protein, providing insights into the effect of mutations on protein-ligand interactions and enhancing our understanding of this essential protein as a priority drug target.

Fusion partners matter: Factor VIII light chain binding enables CD16-mediated recombinant Factor VIII-Fc fusion protein natural killer cell activation.

Abstract Recombinant Factor VIII-Fc fusion protein (rFVIIIFc) is an enhanced half-life therapeutic protein product used for the management of hemophilia A. Recent studies have demonstrated that rFVIIIFc interacts with multiple Fc gamma receptors (FcγR) resulting in the activation or inhibition of a wide variety of FcγR expressing immune cells. We demonstrated that rFVIIIFc activates natural killer (NK) cells via Fc mediated interactions with FcγRIIIA or CD16. Here, we used human NK cell lines and primary NK cells enriched from peripheral blood leukocytes to study rFVIIIFc-mediated NK cell activation. Following overnight incubation of NK cells with rFVIIIFc, we assessed cellular activation by measuring inflammatory cytokine secretion (IFNγ by ELISA) or cellular degranulation (flow cytometry-based surface LAMP1/CD107a). In some cases, we introduced specific blocking molecules (anti-FVIII, anti-Fc, or anti-CD16) into our assays to act as inhibitors of NK cell activation. We identified the FVIII light chain as playing a key role in the activation of CD16 +NK cells by rFVIIIFc. Using FVIII domain-specific inhibitors, we demonstrate that blocking C1 or C2 domain-mediated membrane binding potently inhibits rFVIIIFc-mediated CD16 +NK cell activation, while targeting FVIII heavy chain domains does not. Our results suggest FVIII light chain-mediated membrane binding results in tethering of the fusion protein to the cell surface and allows for Fc-CD16 interactions to proceed, resulting in NK cellular activation. This working model may help explain our previous results where we observed recombinant Factor IX-Fc fusion protein and rFVIIIFc exhibited different CD16-signaling properties despite having identical IgG1 Fc domains.

Fragment-Based Approaches Identified Tecovirimat-Competitive Novel Drug Candidate for Targeting the F13 Protein of the Monkeypox Virus.

Monkeypox is a serious public health issue in tropical and subtropical areas. Antivirals that target monkeypox proteins might lead to more effective and efficient therapy. The F13 protein is essential for the growth and maturation of the monkeypox virus. F13 inhibition might be a viable therapeutic target for monkeypox. The in silico fragment-based drug discovery method for developing antivirals may provide novel therapeutic options. In this study, we generated 800 compounds based on tecovirimat, an FDA-approved drug that is efficacious at nanomolar quantities against monkeypox. These compounds were evaluated to identify the most promising fragments based on binding affinity and pharmacological characteristics. The top hits from the chemical screening were docked into the active site of the F13 protein. Molecular dynamics simulations were performed on the top two probable new candidates from molecular docking. The ligand-enzyme interaction analysis revealed that the C2 ligand had lower binding free energy than the standard ligand tecovirimat. Water bridges, among other interactions, were shown to stabilize the C2 molecule. Conformational transitions and secondary structure changes in F13 protein upon C2 binding show more native three-dimensional folding of the protein. Prediction of pharmacological properties revealed that compound C2 may be promising as a drug candidate for monkeypox fever. However, additional in vitro and in vivo testing is required for validation.

A Novel Electrochemiluminescence Immunosensor Based on Resonance Energy Transfer between g-CN and NU-1000(Zr) for Ultrasensitive Detection of Ochratoxin A in Coffee.

In this study, an electrochemiluminescence (ECL) immunosensor based on nanobody heptamer and resonance energy transfer (RET) between g-C3N4 (g-CN) and NU-1000(Zr) was proposed for ultrasensitive ochratoxin A (OTA) detection. First, OTA heptamer fusion protein was prepared by fusing OTA-specific nanometric (Nb28) with a c-terminal fragment of C4 binding protein (C4bpα) (Nb28-C4bpα). Then, Nb28-C4bpα heptamer with the high affinity used as a molecular recognition probe, of which plenty of binding sites were provided for OTA-Apt-NU-1000(Zr) nanocomposites, thereby improving the immunosensors' sensitivity. In addition, the quantitative analysis of OTA can be achieved by using the signal quenching effect of NU-1000(Zr) on g-CN. As the concentration of OTA increases, the amount of OTA-Apt-NU-1000(Zr) fixed on the electrode surface decreases. RET between g-CN and NU-1000(Zr) is weakened leading to the increase of ECL signal. Thus, OTA content is indirectly proportional to ECL intensity. Based on the above principle, an ultra-sensitive and specific ECL immunosensor for OTA detection was constructed by using heptamer technology and RET between two nanomaterials, with a range from 0.1 pg/mL to 500 ng/mL, and the detection limit of only 33 fg/mL. The prepared ECL-RET immunosensor showed good performance and can be successfully used for the determination of OTA content in real coffee samples, suggesting that the nanobody polymerization strategy and the RET effect between NU-1000(Zr) and g-CN can provide an alternative for improving the sensitivity of important mycotoxin detection.

Novel Antibody-independent Method to Measure Complement Deposition on Bacteria.

During infection, complement plays a critical role in inflammation, opsonisation, and destruction of microorganisms. This presents a challenge for pathogens such asStaphylococcus aureusto overcome when invading the host. Our current knowledge on the mechanisms that evolved to counteract and disable this system is limited by the molecular tools available. Present techniques utilise labelled complement-specific antibodies to detect deposition upon the bacterial surface, a method not compatible with pathogens such asS. aureus, which are equipped with immunoglobulin-binding proteins, Protein A and Sbi. This protocol uses a novel antibody-independent probe, derived from the C3 binding domain of staphylococcal protein Sbi, in combination with flow cytometry, to quantify complement deposition. Sbi-IV is biotinylated, and deposition is quantified with fluorophore-labelled streptavidin. This novel method allows observation of wild-type cells without the need to disrupt key immune modulating proteins, presenting the opportunity to analyse the complement evasion mechanism used by clinical isolates. Here, we describe a step-by-step protocol for the expression and purification of Sbi-IV protein, quantification and biotinylation of the probe, and finally, optimisation of flow cytometry to detect complement deposition using normal human serum (NHS) and bothLactococcus lactisandS. aureus.

Simultaneous heptamerization of nanobody and alkaline phosphatase by self-assembly and its application for ultrasensitive immunodetection of small molecular contaminants in agro-products

Antibody affinity and enzyme loads are two critical factors in determining the sensitivity of enzyme-linked immunosorbent assay for small molecular contaminants in food. Multimerization is considered an effective strategy to improve antibody affinity and enzyme loads. Herein, we selected ochratoxin A (OTA) as the model antigen and constructed a nanobody-alkaline phosphatase (Nb-ALP) heptamer fusion for OTA, where the Nb and ALP were simultaneously heptamerized via the self-associating peptide from the α-chains of C4 binding protein (C4bpα). The heptamer was used to develop an enhanced colorimetric enzyme immunoassay for OTA. Under the optimal experimental parameters, the proposed method has a half maximal inhibitory concentration of 0.081 ng/mL for OTA, which is 12.5-fold lower than the Nb-ALP-based method. Negligible cross-reaction was observed with OTA analogs and other common cereal mycotoxins. Moreover, good recovery rates and repeatability were obtained in the spiked cereal samples. The developed method was then applied to the analysis of OTA in real agro-products, and the results correlated well with that of the LC-MS/MS method. Overall, this work demonstrated that the simultaneous heptamerization of nanobody and alkaline phosphatase by self-assembly is an effective strategy for improving antibody affinity and enzyme loads. It also showed the great potential of Nb–C4bpα-ALP heptamer as a robust dual-functional probe for sensitive, selective, and rapid detection of mycotoxin and other small molecular contaminants in food. • Dual-functional nanobody-alkaline phosphatase heptamer (Nb–C4bpα-ALP) for OTA. • Simultaneous heptamerization of Nb and ALP by the self-assembly strategy. • Nb–C4bpα-ALP shows better binding affinity and enzyme activity than Nb-ALP. • Nb–C4bpα-ALP-based enzyme immunoassay for small molecules was first reported. • The developed method has an IC 50 of 0.081 ng/mL with the assay time of 20 min.

Association Between the C4 Binding Protein Level and White Matter Integrity in Major Depressive Disorder.

Objective Considerable evidence suggests that neuroinflammation plays an important role in the pathophysiology of major depressive disorder (MDD). However, the relationship between serum C4 binding protein (C4BP) and white matter (WM) tract integrity in MDD has not been investigated. Methods We obtained diffusion tensor images of 44 patients with MDD and 44 healthy controls and performed TRActs Constrained by UnderLying Anatomy (TRACULA) analysis to assess WM tract integrity. Serum C4-binding protein alpha chain (C4BPA) and C4- binding protein beta chain (C4BPB) levels were measured and in-between group comparisons were obtained. The correlation between serum C4BP levels and WM tract integrity was examined. Results In comparison to healthy controls, both serum C4BPA and C4BPB were higher in MDD. Also, fractional anisotropy (FA) was increased in the left cingulum-angular bundle (CAB) in MDD, but not healthy controls (HCs). A significant correlation was found between serum C4BP and FA levels in the right cingulum-cingulate gyrus bundle (CCG) in MDD. Conclusion This study is the first to investigate the correlation between serum C4BP levels and WM tract integrity in MDD. We identified an increase in WM integrity in the left CAB region in MDD. Furthermore, serum C4BP levels were higher in MDD, and this finding correlated with increased WM integrity in the right CCG region.

Toward Understanding How Staphylococcal Protein A Inhibits IgG-Mediated Phagocytosis.

IgG molecules are crucial for the human immune response against bacterial infections. IgGs can trigger phagocytosis by innate immune cells, like neutrophils. To do so, IgGs should bind to the bacterial surface via their variable Fab regions and interact with Fcγ receptors and complement C1 via the constant Fc domain. C1 binding to IgG-labeled bacteria activates the complement cascade, which results in bacterial decoration with C3-derived molecules that are recognized by complement receptors on neutrophils. Next to FcγRs and complement receptors on the membrane, neutrophils also express the intracellular neonatal Fc receptor (FcRn). We previously reported that staphylococcal protein A (SpA), a key immune-evasion protein of Staphylococcus aureus, potently blocks IgG-mediated complement activation and killing of S. aureus by interfering with IgG hexamer formation. SpA is also known to block IgG-mediated phagocytosis in absence of complement, but the mechanism behind it remains unclear. In this study, we demonstrate that SpA blocks IgG-mediated phagocytosis and killing of S. aureus and that it inhibits the interaction of IgGs with FcγRs (FcγRIIa and FcγRIIIb, but not FcγRI) and FcRn. Furthermore, our data show that multiple SpA domains are needed to effectively block IgG1-mediated phagocytosis. This provides a rationale for the fact that SpA from S. aureus contains four to five repeats. Taken together, our study elucidates the molecular mechanism by which SpA blocks IgG-mediated phagocytosis and supports the idea that in addition to FcγRs, the intracellular FcRn is also prevented from binding IgG by SpA.

Folic Acid Enables Targeting Delivery of Lipodiscs by Circumventing IgM-Mediated Opsonization.

Folic acid (FA) is one of the most widely utilized small-molecule ligands for cancer targeted drug delivery. Natural IgM was recently found to avidly absorb on the surface of FA-functionalized liposomes (FA-sLip), negatively regulating the in vivo performance by efficiently activating complement. Herein, FA-functionalized lipodiscs (FA-Disc) were constructed to successfully circumvent IgM-mediated opsonization and retained binding activity with folate receptors in vivo. The FA moiety along with the bound IgM was restricted to the highly curved rim of lipodiscs, leading to IgM incapability of presenting the membrane-bound conformation to trigger complement activation. The C1q docking, C3 binding, and C5a release were blocked and accelerated blood clearance phenomenon was mitigated of FA-Disc. FA-Disc retained folate binding activity and could effectively target folate receptor positive tumors in vivo. The present study provides a useful solution to avoid the negative regulation by IgM and achieve FA-enabled targeting by exploring disc-shaped nanocarriers.

Mapping of the Complement C9 Binding Region on Clonorchis sinensis Paramyosin.

Heliminthic paramyosin is a multifunctional protein that not only acts as a structural protein in muscle layers but as an immune-modulatory molecule interacting with the host immune system. Previously, we found that paramyosin from Clonorchis sinensis (CsPmy) is bound to human complement C9 protein (C9). To analyze the C9 binding region on CsPmy, overlapping recombinant fragments of CsPmy were produced and their binding activity to human C9 was investigated. The fragmental expression of CsPmy and C9 binding assays revealed that the C9 binding region was located at the C-terminus of CsPmy. Further analysis of the C-terminus of CsPmy to narrow the C9 binding region on CsPmy indicated that the region flanking 731Leu–780Leu was a potent C9 binding region. The CsPmy fragments corresponding to the region effectively inhibited human C9 polymerization. These results provide a precise molecular basis for CsPmy as a potent immunomodulator to evade host immune defenses by inhibiting complement attack.

Terminal complement pathway activation drives synaptic loss in Alzheimer’s disease models

Complement is involved in developmental synaptic pruning and pathological synapse loss in Alzheimer’s disease. It is posited that C1 binding initiates complement activation on synapses; C3 fragments then tag them for microglial phagocytosis. However, the precise mechanisms of complement-mediated synaptic loss remain unclear, and the role of the lytic membrane attack complex (MAC) is unexplored. We here address several knowledge gaps: (i) is complement activated through to MAC at the synapse? (ii) does MAC contribute to synaptic loss? (iii) can MAC inhibition prevent synaptic loss? Novel methods were developed and optimised to quantify C1q, C3 fragments and MAC in total and regional brain homogenates and synaptoneurosomes from WT and AppNL−G−F Alzheimer’s disease model mouse brains at 3, 6, 9 and 12 months of age. The impact on synapse loss of systemic treatment with a MAC blocking antibody and gene knockout of a MAC component was assessed in Alzheimer’s disease model mice. A significant increase in C1q, C3 fragments and MAC was observed in AppNL−G−F mice compared to controls, increasing with age and severity. Administration of anti-C7 antibody to AppNL−G−F mice modulated synapse loss, reflected by the density of dendritic spines in the vicinity of plaques. Constitutive knockout of C6 significantly reduced synapse loss in 3xTg-AD mice. We demonstrate that complement dysregulation occurs in Alzheimer’s disease mice involving the activation (C1q; C3b/iC3b) and terminal (MAC) pathways in brain areas associated with pathology. Inhibition or ablation of MAC formation reduced synapse loss in two Alzheimer’s disease mouse models, demonstrating that MAC formation is a driver of synapse loss. We suggest that MAC directly damages synapses, analogous to neuromuscular junction destruction in myasthenia gravis.

POS-024 COMPLEMENT FACTOR I VARIANTS IN ATYPICAL HEMOLYTIC UREMIC SYNDROME AND C3 GLOMERULOPATHY

Atypical hemolytic uremic syndrome (aHUS) and C3 glomerulopathy (C3G) are two rare diseases caused by dysregulated activity of the alternative pathway of complement secondary to the presence of genetic and/or acquired factors. Complement factor I (FI) is a serine protease that downregulates complement activity in the fluid phase and/or on cell surfaces in conjunction with one of its cofactors, factor H (FH), complement receptor 1 (CR1/CD35), C4 binding protein (C4BP) or membrane cofactor protein (MCP/CD46).

Complement Factor I Variants in Complement-Mediated Renal Diseases.

C3 glomerulopathy (C3G) and atypical hemolytic uremic syndrome (aHUS) are two rare diseases caused by dysregulated activity of the alternative pathway of complement secondary to the presence of genetic and/or acquired factors. Complement factor I (FI) is a serine protease that downregulates complement activity in the fluid phase and/or on cell surfaces in conjunction with one of its cofactors, factor H (FH), complement receptor 1 (CR1/CD35), C4 binding protein (C4BP) or membrane cofactor protein (MCP/CD46). Because altered FI activity is causally related to the pathogenesis of C3G and aHUS, we sought to test functional activity of select CFI missense variants in these two patient cohorts. We identified 65 patients (16, C3G; 48, aHUS; 1 with both) with at least one rare variant in CFI (defined as a MAF < 0.1%). Eight C3G and eleven aHUS patients also carried rare variants in either another complement gene, ADAMTS13 or THBD. We performed comprehensive complement analyses including biomarker profiling, pathway activity and autoantibody testing, and developed a novel FI functional assay, which we completed on 40 patients. Seventy-eight percent of rare CFI variants (31/40) were associated with FI protein levels below the 25th percentile; in 22 cases, FI levels were below the lower limit of normal (type 1 variants). Of the remaining nine variants, which associated with normal FI levels, two variants reduced FI activity (type 2 variants). No patients carried currently known autoantibodies (including FH autoantibodies and nephritic factors). We noted that while rare variants in CFI predispose to complement-mediated diseases, phenotypes are strongly contingent on the associated genetic background. As a general rule, in isolation, a rare CFI variant most frequently leads to aHUS, with the co-inheritance of a CD46 loss-of-function variant driving the onset of aHUS to the younger age group. In comparison, co-inheritance of a gain-of-function variant in C3 alters the phenotype to C3G. Defects in CFH (variants or fusion genes) are seen with both C3G and aHUS. This variability underscores the complexity and multifactorial nature of these two complement-mediated renal diseases.

C2 by-pass: Cross-talk between the complement classical and alternative pathways

Several disorders associated with the total or partial absence of components of the human complement system are known. Deficiencies of classical pathway (CP) components are generally linked to systemic lupus erythematosus (SLE) or SLE-like syndromes. However, only approximately one-third of patients who lack C2 show mild symptoms of SLE. The relatively high frequency of homozygous C2 deficiency without or with minor disease manifestation suggests that there might be a compensatory mechanism which allows the activation of the CP of complement without the absolute requirement of C2. In this study we show that factor B (FB), the C2 homologue of the alternative pathway (AP) of complement, can substitute for C2. This was confirmed by using C4b as immobilised ligand and FB as analyte in Surface Plasmon Resonance (BIACORE). C2 binding to the immobilised C3b-like molecule C3(CH3NH2) was not seen. The estimated binding constant for C4bB complex formation was 2.00 * 10−5 [M]. We were further able to demonstrate that C4b supports the cleavage of Factor B by Factor D. Finally, cleavage of 125I-C3 by C4bBb was evaluated and gave strong evidence that the “hybrid” convertase C4bBb can cleave and activate C3 in vitro. Cleavage activity is very low, but consistent with some of the “C2-bypass” observations of others.

Screening of Surface-Exposed Lipoproteins of Leptospira Involved in Modulation of Host Innate Immune Response.

Leptospira, a zoonotic pathogen, is capable of causing both chronic and acute infection in a susceptible host. Surface-exposed lipoproteins play a major role in modulating the host immune response by activating the innate cells like macrophages and dendritic cells or evading complement attack and killing by phagocytes like neutrophils to favor pathogenesis and establish infection. In this study, we screened some surface-exposed lipoproteins known to be involved in pathogenesis to assess their possible role in immune modulation (innate immune activation or evasion). Surface proteins of the Len family (LenB, LenD, and LenE), Lsa30, Loa22, and Lipl21 were purified in recombinant form and then tested for their ability to activate macrophages of the different host (mouse, human, and bovine). These proteins were tested for binding with complement regulators like Factor H (FH), C4 Binding Protein (C4BP), and host protease Plasminogen (PLG) and also as nucleases to access their possible role in innate immune evasion. Our results show that, of various proteins tested, Loa22 induced strong innate activation and Lsa30 was least stimulatory, as evident from the production of pro-inflammatory cytokines (interleukin-6 and tumor necrosis factor–α) and expression of surface markers [CD80, CD86, and major histocompatibility complex class II (MHCII)]. All the tested proteins were able to bind to FH, C4BP, and PLG; however, Loa22 showed strong binding to PLG correlating to plasmin activity. All the proteins except Loa22 showed nuclease activity, albeit with a requirement of different metal ions. The nuclease activity of these proteins correlated to in vitro degradation of neutrophil extracellular trap (NET). In conclusion, our results indicate that these surface proteins are involved in innate immune modulation and may play a critical role in assisting the bacteria in invading and colonizing the host tissue for persistent infection.

Activity of Protein S-C4b Binding Protein and Total TFPI Levels in Egyptian SLE Patients: A Cross-Sectional Study

BACKGROUND: Systemic lupus erythematosus (SLE) is an immune disorder with alternating active and remission phases. Cardiovascular diseases and thrombosis are the major causes of mortality in SLE. The anticoagulant activity of Protein S (PS) is complemented by C4 binding protein (C4BP) and tissue factor pathway inhibitor (TFPI). AIM: This study aims to determine the extent of change in the levels of PS activity, C4BP, and total TFPI in active SLE in comparison to the SLE remission phase and their association with thrombosis during SLE flare. METHODS: The study included 180 Egyptian SLE patients who were classified into two groups: 100 SLE cases as the active group and 80 SLE cases as the remission group. The PS activity levels were processed on automated coagulation analyzers, whereas the C4BP and total TFPI levels were measured via enzyme-linked immunosorbent assay. RESULTS: The PS activity and C4BP levels were lower in the active SLE cases than in the remitted ones (p &lt; 0.05). The levels of PS activity and C4BP were revealed to be independent predictors of SELENA-SLEDAI flare scores. In active SLE cases, the PS activity and C4BP levels were rated as excellent and fair classifiers of thrombotic risk in SLE flare, respectively. The total TFPI levels showed no association with SLE activity or its thrombotic consequences. CONCLUSIONS: The levels of PS activity and C4BP act as important biomarkers for SLE activity. Both can be implanted as predictive tools for thrombosis during activity.

Generation of a nanobody-alkaline phosphatase heptamer fusion for ratiometric fluorescence immunodetection of trace alpha fetoprotein in serum

Alpha fetoprotein (AFP) is an important biomarker for diagnosis of hepatocellular carcinoma (HCC). Whereas, it is always a challenge to detect trace AFP in serum. In this work, a ratiometric fluorescence enzyme immunoassay (RFEIA) was developed using nanobody-alkaline phosphatase (Nb-AP) heptamer and MnFe layered double hydroxides nanoflakes (MnFe LDH) for ultrasensitive detection of AFP. The Nb-AP heptamer (Nb-C4bpα-AP) was constructed by fusion expression of Nb, AP, and α-chain of C4 binding protein (C4bpα), where the C4bpα contributed to multimerization through self-assembly. The dual functional Nb-C4bpα-AP can recognize AFP, dephosphorylate ascorbic acid-2-phosphate (AAP) into ascorbic acid (AA), and thus tune the MnFe LDH-mediated ratiometric fluorescence, which was generated from the oxidization of MnFe LDH on o-phenylenediamine (OPD) and the catalyzation of MnFe LDH on the cyclization reaction between AA and OPD. By integration of Nb-C4bpα-AP, MnFe LDH, AAP, and OPD, the RFEIA showed a limit of detection of 0.013 ng/mL with good selectivity, accuracy and precision. Furthermore, results of clinical serum samples tested by the RFEIA were well confirmed by the automated chemiluminescence immunoassay analyzer. Thus, this work demonstrated that the Nb-C4bpα-AP is a robust immunoreagent and the developed RFEIA could be a very promising tool for diagnosis of HCC.

1000. Serotype 3 pneumococci evade activation of the classical complement pathway

BackgroundComplement classical pathway (CCP) activation is the major mechanism leading to opsonophagocytic pneumococcal killing. Following immunization with 13-valent pneumococcal conjugate vaccine (PCV13), opsonophagocytic titers are lowest against serotype 3 among the 13 vaccine serotypes. Post licensure surveillance indicated early declines in serotype 3 invasive pneumococcal disease (IPD) were not sustained over time MethodsUsing flow cytometry, we measured C3 and C4 deposition on serotype 3 strains from children with IPD or nasopharyngeal [NP] carriage, and analyzed by clade. C4 deposition is an indicator of CCP, while C3 deposition is common to all complement pathways. We measured C3/C4 deposition on serotype 3 pneumococcal strains incubated with antibody depleted complement alone or with complement and the following antibodies: mouse monoclonal anti-capsular IgG or IgM, rabbit polyclonal serotype 3 antisera (IgG + IgM) [RPS3A] and RPS3A combined with anti-rabbit IgM, which blocks IgM function, leaving only polyclonal IgGResultsSerotype 3 strains demonstrated high variability in C3 binding when incubated with complement alone. RPS3A (containing both IgM+IgG) and monoclonal IgM activated CCP in all strains. Anti- serotype 3 monoclonal IgG and polyclonal IgG demonstrated absent or limited CCP activation; but activated alternative pathway in some strains. When analyzing complement deposition by clade, a lower proportion of clade II NP serotype 3 strains bound C3 when incubated with complement or monoclonal IgG, compared to clade Ia NP strains. Differences between clade Ia and II IPD strains were not apparent. ConclusionSerotype 3 strains did not demonstrate activation of the CCP in the presence IgG and varied in C3 deposition. Pneumococcal strains that evade CCP activation may be less sensitive to opsonophagocytosis. Our findings suggest a mechanism by which serotype 3 carriage and disease may persist despite immunization with conjugate vaccine containing serotype 3 polysaccharide.Disclosures Rotem Lapidot, MD MSCI, Pfizer (Consultant, Grant/Research Support, Advisor or Review Panel member) Mario Ramirez, PhD, GlaxoSmithKline (Advisor or Review Panel member)Merck Sharp & Dohme (Advisor or Review Panel member)Pfizer (Speaker’s Bureau) Ingrid L. Scully, PhD, Pfizer (Employee, Shareholder) Bradford D. Gessner, MD, MPH, Pfizer Inc. (Employee) stephen pelton, MD, Merck Vaccines (Advisor or Review Panel member, Research Grant or Support)Pfizer, Inc. (Consultant, Advisor or Review Panel member, Research Grant or Support)Sanofi pasteur (Advisor or Review Panel member, Research Grant or Support, DSMB)Seqirus (Consultant)