Journal: Brazilian Journal of Medical and Biological Research

Article Title: Recombinant 60-kDa heat shock protein from Paracoccidioides brasiliensis : is it a good antigen for serological diagnosis of paracoccidioidomycosis?

doi: 10.1590/1414-431X20175928

Figure Lengend Snippet: Western blot analysis of the recombinant proteins from P. brasiliensis recognized by sera from patients with paracoccidioidomycosis (PCM) or healthy subjects (NC). Recombinant proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. Membranes containing both proteins were incubated with sera from PCM patients ( A ) or NC subjects ( B ). After incubation with alkaline-phosphatase-conjugated antibody to human immunoglobulin, the reactions were revealed with BCIP-NBT. The arrowheads indicate the migration position of rPbHsp60 and rgp43. The identification of each patient serum is depicted by the numbers below each image. The negative reaction for both proteins is not shown. C , Relative number (%) of positive reactions with serum samples from PCM patients and NC group against rPbHsp60 and rgp43. No NC serum was reactive to rgp43.

Article Snippet: After 2 h, the plates were washed five times and the wells were incubated with 100 μL of alkaline phosphatase-conjugated goat anti-human immunoglobulin antibody (Merck Millipore) diluted to 1:2,000, at 37°C.

Techniques: Western Blot, Recombinant, SDS Page, Incubation, Migration

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