Journal: Frontiers in Immunology

Article Title: Utilization of Staphylococcal Immune Evasion Protein Sbi as a Novel Vaccine Adjuvant

doi: 10.3389/fimmu.2018.03139

Figure Lengend Snippet: Surface plasmon resonance analyses of tripartite complexes. A triply diluted concentration series (4,050 to 1.8 nM) of (A) WT or (B) K173A Sbi-III-IV were co-injected with plasma purified FH, recombinant FHR-1, FHR-2, FHR-5, or FH 19−20 . The red response curves were indicative of binding experiment in the absence of Sbi. The co-injection experiments of a fixed analyte concentration in combination with increasing Sbi concentration were depicted by increasingly dark lines. (C) Relative changes of Sbi-III-IV mediated FH (or FHR) binding to C3b. By subtracting the co-injection sensorgram (i.e., Sbi+FH) with the corresponding Sbi binding dataset (Figure ), the changes in FH (or FHRs) binding was deduced (Figure ). Changes in FH (or FHRs) binding were expressed as the relative change, derived from dividing the Sbi mediated binding by the FH (or FHR) only control, using the response-difference values at the equilibrated binding point (173.5 s ). Each sensorgram is representative of two experiments. Relative change curves were fitted using non-linear variable slope (four parameters) function in GraphPad Prism.

Article Snippet: The goat anti-human FH polyclonal serum (catalog no. 341276-1 ml) that was previously used to detect human FH and FHR-1 was purchased from Merck Millipore.

Techniques: SPR Assay, Concentration Assay, Injection, Purification, Recombinant, Binding Assay, Derivative Assay

Journal: Frontiers in Immunology

Article Title: Utilization of Staphylococcal Immune Evasion Protein Sbi as a Novel Vaccine Adjuvant

doi: 10.3389/fimmu.2018.03139

Figure Lengend Snippet: Functional characterization of tripartite complexes in complement AP regulation. NHS was incubated with Sbi-III-IV, in combination with specified reagents or just buffer, the consumption of AP activity was indicated by the protection of rabbit red blood cell from lysis. (A) Pre-incubation of recombinant FHR-1 or−2 with the presence or absence of Sbi-III-IV. (B) Pre-incubation of recombinant FHR-5 in the presence or absence of Sbi-III-IV. (C) Pre-incubation of recombinant FH 19−20 or FHR-1 1−2 in the presence or absence of Sbi-III-IV. Using an ELISA assay, the ability of FHR-1,−2,−5, FH 19−20 , or FHR-1 1−2 to modulate FH binding to a C3b coated surface was studied in the absence (D) or presence of WT Sbi-III-IV (E) , or K173A Sbi-III-IV (F) . C3 convertase formation in the absence (G) or presence of Sbi-III-IV (H) was assessed by flowing factor B (500 nM) and factor D (100 nM) in the presence of FH (2,000 nM) or FH +FHR-1 (2,000 and 200 nM) or FH+FHR-1&-5 (2,000, 200 and 20 nM) across a surface amine coupled with 500 RU C3b. To form Sbi bound C3 convertase, experiments were conducted in addition of 2,000 nM of Sbi-III-IV. Detailed experimental and data processing procedures are provided in Materials and Methods and Figure . (I) Percentage of intact C3b derived from continuous recording of ANS fluorescence changes between 465 and 475 nm spectrum. Baseline C3b breakdown curve (−) was recorded in the presence of FH and FI, interference caused by the addition of FHR-5 (+) or FHR-5 in combination of Sbi (++) was also examined. The data for FHR-1 and FHR-2 are presented in Figure 3F. Normalized data was depicted in solid lines, simulated breakdown curves were shown as dotted-lines. Each curve represents the mean value of three independent experiments. For (A–F) , the mean and standard deviation for each measurement was calculated; For (G–H) , each sensorgram is representative of two experiments. For (I) , simulated breakdown curves were fitted using one phase exponential decay function in GraphPad Prism.

Article Snippet: The goat anti-human FH polyclonal serum (catalog no. 341276-1 ml) that was previously used to detect human FH and FHR-1 was purchased from Merck Millipore.

Techniques: Functional Assay, Incubation, Activity Assay, Lysis, Recombinant, Enzyme-linked Immunosorbent Assay, Binding Assay, Derivative Assay, Fluorescence, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Utilization of Staphylococcal Immune Evasion Protein Sbi as a Novel Vaccine Adjuvant

doi: 10.3389/fimmu.2018.03139

Figure Lengend Snippet: Structural analysis of the Sbi-III-IV:C3d:FHR-1 tripartite complex. SAXS solution structure analysis and EOM modeling of the Sbi-III-IV:C3d:FHR-1 tripartite complex: (A) Left panel, fit of the selected ensemble of conformers to the experimental scattering. Radius of gyration (Rg, middle panel), particle maximum dimension (Dmax, right panel), and distribution histograms of the selected conformers vs. the pool. (B) Kratky plot of the tripartite complex. (C) Examples of rigid body models of the selected conformers corresponding to the histogram peaks. The volume fraction of each species is indicated. The relative positions of C3d, Sbi-III-IV, and FHR-1 in the dimeric tripartite complex are indicated, with C3d in red, Sbi-IV in dark blue, Sbi-III in turquoise and FHR-1 in orange. (D) Schematic representation of the dimeric Sbi-III-IV:C3d:FHR-1 tripartite complex. (E) Comparison of the solutions structure of wild-type Sbi-III-IV:C3d and mutated version Sbi-III-IV(K173A):C3d of the dual complex. Radius of gyration (Rg), particle maximum dimension (Dmax), and distribution histograms of the selected conformers vs. the pool are shown in Figure . (F) Ab initio shape reconstruction shown as gray spheres in comparison to the partial crystal structure Sbi-IV:C3d (2wy8). (G) Examples of rigid body models. Complete set of models as well as flexibility assessment is presented in Figure . C3d in shown red, Sbi-IV in dark blue, and Sbi-III in turquoise.

Article Snippet: The goat anti-human FH polyclonal serum (catalog no. 341276-1 ml) that was previously used to detect human FH and FHR-1 was purchased from Merck Millipore.

Techniques:

Journal: Frontiers in Immunology

Article Title: Utilization of Staphylococcal Immune Evasion Protein Sbi as a Novel Vaccine Adjuvant

doi: 10.3389/fimmu.2018.03139

Figure Lengend Snippet: Sbi-III-IV is an effective adjuvant in mice. (A) Freshly prepared CD21 −/− mouse serum was mixed with Sbi-III-IV-Ag85b or just Sbi-III-IV. The reaction was stopped at various time points (0, 30, 60, 120 min). Western blot was developed with rabbit anti-C3 at 1/1000 and goat anti-rabbit at 1/2000. C3d is shown as confirmation that C3 has been activated and broken down. (N) is Cr2 −/− serum incubated for 120 min with saline. (B) C57Bl/6 mice (groups of 6) where immunized intraperitoneally with either 2.7 μg Sbi-III-IV-Ag85b protein, 2 μg Ag85b, or 0.7 μg Sbi-III-IV plus 2 μg Ag85b in 150 mM NaCl solution, followed by weekly bleed and boosted (day 28) before terminal bleed at day 49. Serum IgG reactivity to Ag85b was measured over time by ELISA. Sera was diluted 1/50 and the mean absorbance ± SEM of each mouse group is shown. All data has been normalized to the day 0 average of all WT mice. (C) The previous experiment was repeated in C57Bl/6 mice deficient of C3 (C3 −/− )and complement receptor type I and 2 ( Cr2 −/− ). Data is representative of at least 2 repeats ( *** P < 0.001, Student's T -test, GraphPad Prism). (D) Schematic representation of the dimeric Sbi-III-IV:C3d:FHR-1 solution structure providing a nidus for AP C3 convertase generation that overwhelms local complement regulators, leading to the opsonisation of the nearby antigen surface by C3 break-down products that help facilitate the co-ligation of the B cell antigen receptor (BCR) with complement receptor 2 (CR2) thereby lowering the threshold for B cell activation.

Article Snippet: The goat anti-human FH polyclonal serum (catalog no. 341276-1 ml) that was previously used to detect human FH and FHR-1 was purchased from Merck Millipore.

Techniques: Western Blot, Incubation, Enzyme-linked Immunosorbent Assay, Ligation, Activation Assay

Journal: Blood

Article Title: Molecular bases for the association of FHR-1 with atypical hemolytic uremic syndrome and other diseases

doi: 10.1182/blood.2020010069

Figure Lengend Snippet: Complement deregulation and C3-binding activities of recombinant FHR-1 proteins. (A) Alignment of FH and FHR-1. Numbers in between structures indicate percentages of homology. Proposed FHR-1 dimerization topology is shown. (B) Coomassie-stained sodium dodecyl sulfate–polyacrylamide gel electrophoresis of the recombinant FHR-1 proteins purified from the culture supernatants. (C) FH deregulation assay in sheep erythrocytes (ShEs). Approximately 30% of ShEs are lysed when they are exposed to 20% of a human serum that has been depleted of 75% of the FH. Adding increasing amounts of purified wild-type FHR-1 (FHR-1 WT ) or FHR-1 A296V does not significantly increase this percentage of lysis. This is in contrast with the dose-response hemolysis of ShEs that results from the addition of FHR-1 L290S,A296V , FHR-1 L290V , and FHR-1 L290S . Data are means ± standard deviations of triplicates. (D) Binding to immobilized C3b of the different FHR-1 mutants is compared with that of FHR-1 WT using a plate assay. Although FHR-1 L290V and FHR-1 A296V bind similarly to FHR-1 WT (1 ± 0.11 and 0.9 ± 0.4 times, respectively), binding of FHR-1 L290S and FHR-1 L290S,A296V to C3b was estimated in this assay to be 3.65 ± 0.4 and 4.5 ± 1.56 times lower, respectively, than that of FHR-1 WT . (E) FHR-1 WT binds similarly to immobilized native C3 (nC3), C3b, iC3b, and C3dg. The same is also true for mutant FHR-1 L290S,A296V and all other FHR-1 mutants (data not shown). (F) Binding of FHR-1 WT and FH at 300 nM to immobilized C3b and nC3. Only FHR-1 binds to immobilized nC3, indicating that our nC3 preparations were not contaminated with significant amounts of C3(H 2 O) forms. OD, optical density.

Article Snippet: FHR-1 complementary DNA was purchased from Tebu-Bio Innovative Laboratory Services and Reagents (EX-Z0243-M02), and mutagenesis was performed using the Q5 Site-Directed Mutagenesis Kit (E0552S; New England BioLabs).

Techniques: Binding Assay, Recombinant, Staining, Polyacrylamide Gel Electrophoresis, Purification, Lysis, Mutagenesis

Journal: Blood

Article Title: Molecular bases for the association of FHR-1 with atypical hemolytic uremic syndrome and other diseases

doi: 10.1182/blood.2020010069

Figure Lengend Snippet: Structural aspects of FHR-1 interaction with C3 TED and sialic acids. (A) Superimposition of C3 (cyan; Protein Data Bank [PDB] identifier 2A73; TED domain is shown in dark blue) and FHR-1 (yellow)/C3d (pink) complex from MD simulation (100 ns). Key residues are shown in sticks. (B) Root-mean-square deviation (RMSD; calculated with backbone heavy atoms) of FHR-1 WT (red) and FHR-1 L290S,A296V (black) during 500 ns of MD simulation. (C) Root-mean-square fluctuation (RMSF; calculated by residue) of FHR-1 WT (red) and FHR-1 L290S,A296V (black) during 500 ns of MD simulation. (D) RMSD (calculated with backbone heavy atoms) of FHR-1/C3dg complex during 500 ns of MD simulation. (E) Electrostatic potential surface of FHR-1 L290S,A296V (left) and FHR-1 WT (right). The sialic acid–binding sites in SCR-5 of FHR-1 L290S,A296V and FHR-1 WT are framed by squares that are enlarged below. (F) FH/3′-SL complex (cyan/orange) from docking calculations superimposed to 3′-SL (magenta) from x-ray structure (PDB identifier 4ONT). Key residues are shown in sticks. RMSD of the FH/3′-SL complex (calculated with heavy atoms for protein and with carbons for 3′-SL) from the MD simulation is also depicted (complex in black; 3′-SL in red).

Article Snippet: FHR-1 complementary DNA was purchased from Tebu-Bio Innovative Laboratory Services and Reagents (EX-Z0243-M02), and mutagenesis was performed using the Q5 Site-Directed Mutagenesis Kit (E0552S; New England BioLabs).

Techniques: Binding Assay

Journal: Blood

Article Title: Molecular bases for the association of FHR-1 with atypical hemolytic uremic syndrome and other diseases

doi: 10.1182/blood.2020010069

Figure Lengend Snippet: Binding of FHR-1 to glomeruli from Cfh −/− mice. (A) Illustration of the rationale for the experiments using cryostat sections of Cfh −/− mouse kidneys. Photomicrograph shows staining with a biotinylated anti–mouse C3 antibody. (B) Three different concentrations of FHR-1 proteins were added to cryostat kidney sections of Cfh −/− mice. Bound FHR-1 was detected with an in-house biotinylated anti–FHR-1 mAb (2C6). All FHR-1 proteins bound exclusively to the glomerulus. Fluorescence intensity (FI) is expressed in arbitrary units. Data are means ± standard deviations of a minimum of 20 glomeruli. FI values obtained from FHR-1 WT were used to make a reference curve. FI values of all FHR-1 mutants were interpolated in this curve to calculate the relative binding between the FHR-1 proteins. SIA, sialic acid. All photomicrographs were taken at ×40 magnification. Average diameter of mouse glomeruli is 70 μm.

Article Snippet: FHR-1 complementary DNA was purchased from Tebu-Bio Innovative Laboratory Services and Reagents (EX-Z0243-M02), and mutagenesis was performed using the Q5 Site-Directed Mutagenesis Kit (E0552S; New England BioLabs).

Techniques: Binding Assay, Staining, Fluorescence

Journal: Blood

Article Title: Molecular bases for the association of FHR-1 with atypical hemolytic uremic syndrome and other diseases

doi: 10.1182/blood.2020010069

Figure Lengend Snippet: Decreased binding of aHUS-associated FHR-1 mutants to desialylated Cfh −/− mouse glomeruli. Optimal concentrations of FHR-1 WT (1 μM), FHR-1 L290V (0.3 μM), FHR-1 296A (1 μM), FHR-1 L290S (0.3 μM), and FHR-1 L290S,A296V (0.3 μM) in PBS/BSA were added to normal and neuraminidase (NA)-treated cryostat sections of Cfh −/− mouse kidneys and incubated at 37°C for 30 minutes. Bound FHR-1 was detected with an anti–FHR-1 mAb (2C6). Images of representative glomeruli are shown to illustrate the decreased binding to desialylated glomeruli of the aHUS-associated FHR-1 mutants compared with the binding to untreated glomeruli. Loss of binding for the different FHR-1 proteins was calculated basically as described in . All FHR-1 proteins bound similarly to desialylated sections of Cfh −/− mouse kidneys when they were used at the same concentration (data not shown). All photomicrographs were taken at ×40 magnification.

Article Snippet: FHR-1 complementary DNA was purchased from Tebu-Bio Innovative Laboratory Services and Reagents (EX-Z0243-M02), and mutagenesis was performed using the Q5 Site-Directed Mutagenesis Kit (E0552S; New England BioLabs).

Techniques: Binding Assay, Incubation, Concentration Assay

Journal: Blood

Article Title: Molecular bases for the association of FHR-1 with atypical hemolytic uremic syndrome and other diseases

doi: 10.1182/blood.2020010069

Figure Lengend Snippet: NMR data for the interaction of FHR-1 proteins with 3′-SL and 6′-SL. (A) Structures of 3′-SL (α-Neu5Ac-[2-3]-β-D-Gal-[1-4]-D-Glc) and 6′-SL (α-Neu5Ac-[2-6]-β-D-Gal-[1-4]-D-Glc). (B) STD-NMR spectra with 3′-SL. Lower panel shows reference spectra of 3′-SL corresponding to the off-resonance spectra acquired in presence of FHR-1 WT . Above this are panels showing the STD spectra in presence of FHR-1 WT at 9 µM and FHR-1 L290S, A296V at 16 µM; the acetate of the 5-NAc group of the sialic residue received the highest saturation. In both cases, 3′-SL concentration was adjusted to 50:1 ligand/protein ratio. (C) STD-NMR spectra of FHR-1 L290V mutant with 3′-SL and 6′-SL. From bottom to top, panels show reference spectra of 3′-SL corresponding to the off-resonance spectra in presence of FHR-1 L290V , STD-NMR spectra of 3′-SL in presence of FHR-1 L290V at 15 µM, reference spectra of 6′-SL corresponding to the off-resonance spectra in presence of FHR-1 L290V , and STD-NMR spectra of 6′-SL in presence of FHR-1 L290V at 16 µM; the acetate of the 5-NAc group of the sialic residue received the highest saturation. In both cases, 3′-SL and 6′-SL concentrations were adjusted to 50:1 ligand/protein ratio. All STD-NMR spectra were acquired with 3-s saturation on −0.14 ppm. Background signal of protein was subtracted in each case from a spectrum of the protein acquired under the same conditions but in absence of ligand. Signals of selected protons are labeled.

Article Snippet: FHR-1 complementary DNA was purchased from Tebu-Bio Innovative Laboratory Services and Reagents (EX-Z0243-M02), and mutagenesis was performed using the Q5 Site-Directed Mutagenesis Kit (E0552S; New England BioLabs).

Techniques: Concentration Assay, Mutagenesis, Labeling

Journal: Blood

Article Title: Molecular bases for the association of FHR-1 with atypical hemolytic uremic syndrome and other diseases

doi: 10.1182/blood.2020010069

Figure Lengend Snippet: Surface-bound FHR-1 promotes complement activation. (A) Cryostat kidney sections from Cfh −/− ; Cfhr −/− mice were incubated with 10% NHSΔFHR in presence or absence of 0.5 µM of the different FHR-1 proteins. Bound FHR-1 was detected using the mAb 2C6, and deposits of hC3-activated fragments were detected with the mAb 12.17. (B) Cryostat kidney sections from Cfh −/− ; Cfhr −/− mice were incubated with 10% NHSΔFHR and 0.25 µM of FHR-1 L290S,A296V in the presence or absence of FH (1 µM) or EDTA (20 mM). (C) Increasing concentrations of FHR-1 WT or FHR-1 L290S,A296V were added to 10% NHSΔFHR and then incubated with cryostat kidney sections from Cfh −/− ; Cfhr −/− mice. Fluorescence intensity is expressed in arbitrary units. Data are means ± standard deviations of a minimum of 40 glomeruli. All photomicrographs were taken at ×40 magnification.

Article Snippet: FHR-1 complementary DNA was purchased from Tebu-Bio Innovative Laboratory Services and Reagents (EX-Z0243-M02), and mutagenesis was performed using the Q5 Site-Directed Mutagenesis Kit (E0552S; New England BioLabs).

Techniques: Activation Assay, Incubation, Fluorescence

Journal: Blood

Article Title: Molecular bases for the association of FHR-1 with atypical hemolytic uremic syndrome and other diseases

doi: 10.1182/blood.2020010069

Figure Lengend Snippet: Interaction of FHR-1 WT and FHR-1 mutants with normal host cells and altered host surfaces and pathogens and their consequences in complement regulation.

Article Snippet: FHR-1 complementary DNA was purchased from Tebu-Bio Innovative Laboratory Services and Reagents (EX-Z0243-M02), and mutagenesis was performed using the Q5 Site-Directed Mutagenesis Kit (E0552S; New England BioLabs).

Techniques: