USES, METHODS AND PRODUCTS RELATING TO OLIGOMERIC LIPOPOLYSACCHARIDE BINDING PROTEINS

Abstract

Provided and described herein is the use of an oligomeric protein as a binding agent for binding to lipopolysaccharide (LPS), the oligomeric protein having a coiled coil structure comprising at least two monomer peptides, wherein each monomer peptide, which may be the same or different, is capable of forming an α-helix and comprises at least one core sequence having at least 60% sequence identity to the heptad repeat sequence of SEQ ID NO. 1. Also provide and described herein are methods of binding, detecting and removing LPS, and products comprising the oligomeric protein.

Claims

1. Use of an oligomeric protein as a binding agent for binding to lipopolysaccharide (LPS), the oligomeric protein having a coiled coil structure comprising at least two monomer peptides, wherein each monomer peptide, which may be the same or different, is capable of forming an α-helix and comprises at least one core sequence having at least 60% sequence identity to the heptad repeat sequence of SEQ ID NO. 1.

2. The use of claim 1, wherein the core sequence comprises at least 3 heptad motifs a-b-c-d-e-f-g, or variants thereof, each variant comprising no more than 1 insertion or deletion to the heptad motif.

3. The use of claim 1 or claim 2, wherein at least 50% of the amino acid residues corresponding to positions a and d of the heptad motifs or variants thereof are hydrophobic residues.

4. The use of any one of claims 1 to 3, wherein the core sequence is flanked on one or both sides by a flanking amino acid sequence.

5. The use of claim 4, wherein the flanking sequence comprises one or more heptad motifs, and/or one or more parts thereof, preferably wherein the heptad motif in the flanking sequence corresponds to a heptad motif as found in SEQ ID NO. 1, or in a sequence having at least 80% sequence identity thereto, with the proviso that at least one of the amino acid residues a and d in the heptad motif is a hydrophobic residue.

6. The use of claim 4 or 5, wherein the flanking sequence comprises SEQ ID NO. 1, or a part thereof, or a sequence having at least 50% sequence identity thereto, wherein at least 50% of the amino acid residues corresponding to positions a and d of the heptad motifs of SEQ ID NO. 1, or variants thereof, are hydrophobic residues.

7. The use of any one of claims 3 to 6, wherein the flanking sequence comprises one or more linker sequences.

8. The use of any one of claims 1 to 7, wherein the monomer peptides each comprise two or more core sequences, wherein said core sequences may be the same or different.

9. The use of any one of claims 1 to 8, wherein the oligomeric protein is a dimer, trimer, or tetramer.

10. The use of any one of claims 1 to 9, wherein the oligomeric protein is a trimer.

11. The use of any one of claims 1 to 10, wherein the monomer peptides are provided as separate chains.

12. The use of any one of claims 1 to 10, wherein the monomer peptides are linked together.

13. The use of claim 12, wherein the monomer peptides are linked into a single chain or wherein the monomer peptides are linked by one or more chemical cross-links.

14. The use of any one of claims 1 to 13, wherein each hydrophobic residue in the heptad motifs or variants thereof is independently selected from the group consisting of leucine, isoleucine, valine, alanine, methionine, and chemical derivatives thereof.

15. The use of claim 14, wherein each hydrophobic residue is independently selected from leucine and isoleucine, or chemical derivatives thereof.

16. The use of claim 15, wherein the chemical derivatives are fluoroleucine or fluoroisoleucine.

17. The use of any one of claims 1 to 16, wherein at least 50% of the hydrophobic residues are isoleucine or fluoroisoleucine.

18. The use of any one of claims 1 to 17, wherein: (i) at least 50% of the amino acid residues corresponding to positions b, c, e, f and g in the heptad repeats or variants thereof are polar residues; and/or (ii) at least 5% of the amino acid residues corresponding to positions b, c, e, f and g in the heptad repeats or variants thereof are aliphatic residues.

19. The use of any one of claims 1 to 18, wherein each monomer peptide comprises 18 to 40 amino acids.

20. The use of any one of claims 1 to 19, wherein each monomer peptide comprises at least 4 cationic amino acids within the core sequence.

21. The use of any one of claims 1 to 20, wherein the oligomeric protein binds to LPS with a K.sub.D in the nanomolar or lower size range.

22. The use of any one of claims 1 to 21, wherein the oligomeric protein is: (i) in the form of a conjugate or fusion with one or more additional components; (ii) immobilised on a solid substrate; or (iii) conjugated to a directly detectable detection moiety.

23. The use of claim 22, wherein: (i) the protein is conjugated with a detection moiety, an oligomerisation moiety or an immobilising moiety, or is in the form of a fusion protein with a fusion partner; (ii) the protein is immobilised on a bead or resin, or in or on a well or vessel, or a column or filter material, or on a surface of a detection device; or (iii) the detection moiety is a spectrophotometrically or spectroscopically detectable label.

24. The use of any one of claim 23, wherein the use of the oligomeric protein comprises detection and/or removal of LPS in or from a sample.

25. A method of binding LPS, the method comprising contacting the LPS, or a sample containing LPS, with an oligomeric protein as defined in any one of claims 1 to 23, to allow the protein to bind to the LPS to form a protein-lipopolysaccharide complex.

26. The method of claim 25, wherein the method further comprises detecting the presence of LPS in a sample, said method comprising: (a) contacting the sample with an oligomeric protein as defined in any one of claims 1 to 23, to allow the protein to bind to the LPS to form a protein-lipopolysaccharide complex; (b) detecting the presence of a protein-lipopolysaccharide complex.

27. The method of claim 25, wherein the method further comprises removing LPS from a sample, said method comprising: (a) contacting the sample with an oligomeric protein as defined in any one of claims 1 to 23, to allow the protein to bind to the LPS to form a protein-lipopolysaccharide complex; (b) separating the peptide-lipopolysaccharide complex from the sample.

28. The method of any one of claims 25 to 27, wherein the oligomeric protein is in the form of a conjugate comprising a detectable label and/or wherein the oligomeric protein is immobilised on a solid substrate.

29. The method of any one of claims 25 to 28, wherein the sample is a clinical sample derived from a patient or a sample of a product for testing for endotoxin contamination.

30. The method of claim 29, wherein the sample is a blood sample, or a sample derived from a blood sample.

31. A kit comprising; (i) an oligomeric protein as defined in any one of claims 1 to 17; and (ii) at least one non-denaturing detergent.

32. The kit of claim 31, wherein the kit is for use according to any one of claims 1 to 24, or in the method of any one of claims 25 to 30.

33. A product comprising an oligomeric protein immobilised on a solid substrate, wherein the oligomeric protein is as defined in any one of claims 1 to 21.

Description

FIGURES

[0142] The invention will now be described in more detail in the following non-limiting Examples with reference to the following drawings:

[0143] FIG. 1 shows the structure of the GCN4-pII trimer adapted from PDB-ID 2YO0 (Hartmann et al., 2012). (A) Side view. (B) Front view with core isoleucine residues in positions a and d colored green.

[0144] FIG. 2 shows a schematic version of the general structure of LPS, based on LPS from S. typhimurium. The Lipid A moiety (insert) consists of two phosphoglucosamines with four O-linked and two N-linked acyl chains embedded in the outer membrane. The core oligo saccharide (COS) is linked to Lipid A via a glycosidic bond, and the O-antigen linked to the penultimate COS sugar. The O-antigen consists of a four-sugar repeat varying between 4 and 40 repeat units, with an average of 30 repeats (Peterson and McGroarty, 1985). Lipid A and the two proximal 3-Deoxy-D-manno-oct-2-ulosonic acid (KDO) sugars are highly conserved among Gram-negative species, while the rest of COS and O-antigen are conserved among bacterial species and serotypes, respectively.

[0145] FIG. 3 shows SPR binding curves following injection of different LPS components to immobilized K9-GCN4-PII. Vertical lines indicate start and end of injection. (A) shows the injection of whole LPS. (B) shows the injection of rough LPS lacking O-antigen. (C) shows the injection of deep rough LPS, lacking all sugars except the two proximal KDOs. (D) shows the injection of LPS polysaccharide lacking Lipid A.

[0146] FIG. 4 shows a graph of SPR difference values at end of injection normalized for the molar concentrations of the ligands, for each of the LPS components tested in FIG. 3.

[0147] FIG. 5 shows ELITA binding curves of LPS to the two GCN4-containing constructs K9-His (left) and K14-His (right).

[0148] FIG. 6 shows TEM images of LPS alone (top) and LPS with GCN4-pII (bottom) at 4k (left) and 8k (right) magnification.

[0149] FIG. 7 shows a schematic overview of the constructs that were produced. Constructs derived from SadA were originally described in Alvarez et al. (Alvarez et al., 2008) and Hartman et al. (Hartmann et al., 2012). The andreinlvpas construct was originally described by Deiss et al. (Deiss et al., 2014). The GCN4 construct was synthesized by GenScript (GenScript Biotech Corp).

[0150] FIG. 8 shows SPR Fc1, Fc2, and Fc1-F2 curves for immobilized K9 with different S. typhimurium LPS components. (A) is with smooth LPS. (B) is with rough LPS. (C) is with deep rough LPS. (D) is with polysaccharide derived from LPS.

[0151] FIG. 9 shows SPR Fc1, Fc2, and Fc1-F2 curves for immobilized K14 with different S. typhimurium LPS components. (A) is with smooth LPS. (B) is with rough LPS. (C) is with deep rough LPS. (D) is with polysaccharide derived from LPS.

[0152] FIG. 10 shows SPR Fc1, Fc2, and Fc1-F2 comparison curves for immobilized K3 (Fc1 channel) and K3-His (Fc2 channel) with different S. typhimurium LPS components. (A) is with smooth LPS. (B) is with rough LPS. (C) is with deep rough LPS. (D) is with polysaccharide derived from LPS.

[0153] FIG. 11 shows the absolute values (top) and the set-up and negative controls (bottom) of the ELITA experiments with K9 and K14. SadA=Salmonella component K9 or K14. BSA=Bovine serum albumin. TSP=phage tailspike protein. ST-HRP=streptactin conjugated horseradish peroxidase.

[0154] FIG. 12 shows the CD spectra of GCN4-pII alone and in the presence of LPS. It can be seen that there is minimal variation in the secondary structure composition of GCN4-pII before and after binding to LPS.

[0155] FIG. 13 shows a graph of the results of an LAL assay demonstrating the masking effect of GCN4-pII at concentrations ranging from 10 to 0.1 μM spiked with 0.5 EU/mL LPS. Optimal masking was observed at 1 μM GCN4-pII where the masking effect was 89% of total signal.

[0156] FIG. 14 shows the fingerprint region of a 2D .sup.1H-.sup.1H TOCSY NMR spectrum of GCN4-pII. All 29 expected spin systems were well resolved and assignable without indications of peak splitting, indicating that the sample is homogenous in solution.

[0157] FIG. 15 shows a graph of the results of a GCN4-pII based ELISA using biotinylated LPS for detection.

[0158] FIG. 16 shows a graph of the results of an LAL assay using the same samples as in the assay of FIG. 15.

[0159] FIG. 17 shows a graph comparing the results of the GCN4-pII based ELISA and the LAL assay.

[0160] FIG. 18 shows SPR binding curves for various LPS types and for PBS-E, the running buffer, as a control.

[0161] FIG. 19 shows the phylogenetic distribution of the LPS variants used in Example 5. This figure is adapted from Bern and Goldberg, 2005.

EXAMPLES

[0162] Methods

[0163] Expression and Purification of Proteins

[0164] Salmonella adhesin A (SadA) constructs (as shown in FIG. 7) flanked by GCN4 adaptors were produced as described earlier (Alvarez et al., 2008; Hartmann et al., 2012). Tranformed BL21-Gold(DE3) were grown in 2 L ZYP-5052 autoinduction medium (Studier, 2005), and overexpression induced by adding 200 ng/mL anhydrotetracycline (AHTC) at OD600=0.6 followed by expression overnight at 30° C. The cells were pelleted at 6000×g (Beckman JLA 8.1000 rotor) for 30 minutes and resuspended in 20 mL Tris/HCl pH 7.4, 40 mM NaCl, 5 mM MgCl2 containing 200 μL EDTA-free Protease Inhibitor Cocktail (Merck) and DNase I. Following resuspension, the cells were lysed by French press and the resulting lysate diluted in 50 mL equilibration buffer (20 mM Tris/HCl pH 7.9, 5 M guanidine hydrochloride, 0.5 M NaCl, 10% glycerol) and incubated at room temperature for 1 hour while stirring, followed by centrifugation at 75 000×g (Beckman Ti 70 rotor) for 1 hour to remove any undissolved particulates. The resulting solution was loaded onto a 20 mL Ni Sepharose excel columns (GE Life Sciences) pre-equilibrated with equilibration buffer. Following application of the sample, the column was washed with 4 column volumes equilibration buffer and eluted using a 0-100% gradient elution buffer (20 mM Tris/HCl, pH 7.5, 5 M guanidine hydrochloride, 0.5 M NaCl, 10% glycerol, 500 mM imidazole). The eluted fractions were analyzed by SDS-PAGE, and fractions containing the protein of interest was pooled and refolded by dialyzing twice against 2 L refolding buffer (20 mM MOPS pH 7.4, 350 mM NaCl, 10% glycerol) over night.

[0165] LPS Production and Purification

[0166] LPS was produced by inoculating a 20 mL lysogeny broth (LB) preculture from a single bacterial colony (see Table 3 below for strains used) and grown over night at 37° C.

TABLE-US-00004 TABLE 3 Strain LPS name Type Notes E. coli BL21 BL21 LPS Rough Common lab strain lacking O-antigen S. anatum S. anatum LPS Smooth Exact structure unknown S. Typhimurium Smooth LPS Smooth S. Typhimurium WaaC LPS Rough Deletion of heptosyl- ΔwaaC (ΔLPS) transferase WaaC, responsible for transfer of heptose to the Kdo-moiety of LPS-precursor S. Typhimurium WaaJ LPS Rough Deletion of ΔwaaJ glycosyltransferase WaaJ, responsible for transfer of the penultimate glucose to the core-oligo LPS S. Typhimurium Waal LPS Rough Deletion of WaaL ligase, ΔwaaL responsible for ligating O-antigen to core-oligo saccharide

[0167] 6×1 L cultures in 2 L baffled flasks were inoculated from the preculture and grown over night at 37° C. on a shaker. The bacteria were harvested by centrifuging at 6000×g (Beckman JLA 8.1000 rotor) for 30 minutes. Further purification followed two different methods depending on the type of LPS.

[0168] Rough LPS was purified following the protocol described by Galanos et al. (Galanos, Luderitz and Westphal, 1969), using phenol-chloroform-petroleum ether extraction. Following harvest, the bacterial pellet was washed 3 times with 40 mL ethanol and once with acetone, then left over night under an airflow. The dried out pellet was homogenized using a mortar and pestle and dissolved in a 40 ml mixture of 90% (W/V) liquid phenol, chloroform, and petroleum ether in a ratio of 2:5:8. After one hour incubation on a shaker, the undissolved material was pelleted at 4200×g for 15 minutes and the supernatant collected. Chloroform and petroleum ether was removed under an airflow for 4 hours or until the phenol started crystallizing. The solution was resuspended by heating to 40° C., and water added dropwise (3×5 drops) under stirring until the LPS precipitated. The LPS was pelleted at 4200×g for 15 minutes, and more water added to the supernatant to collect any residual LPS. The pellets were washed two times with 10 mL 80% (W/V) phenol, and taken up in 20 mL milliQ-water before centrifugation at 100 000×g (Beckman, MLA-50 rotor) for one hour. The final pellet was taken up in 50 mL MilliQ-water and lyophilized to yield pure LPS.

[0169] Smooth LPS was purified following the protocol described by Darveau et al. (Darveau and Hancock, 1983). The bacteria was washed twice and resuspended in 40 mL 10 mM Tris-HCl pH 8.0, 2 mM MgCl2, and lysed by french press followed by additional disruption by sonication. The resulting suspension was incubated with 200 μg/mL DNase I, 50 μg/mL RNase A overnight while stirring at 37° C. To 15 mL suspension, 5 mL 0.5 M EDTA in 10 mM Tris-HCL pH 8.0, 2.5 mL 20% SDS in 10 mM Tris-HCl pH 8.0, and 2.5 mL 10 mM Tris-HCl pH 8.0 were added, and the LPS micelles further disrupted by sonication. The solution was centrifuged at 39 000×g (Sorval, SS-34 rotor) for 30 minutes at 20° C. to pellet undissolved cell components, the supernatant frozen and lyophilized. The lyophilized crude extract was dissolved in a modicum of water, and the LPS precipitated with 2 volumes of ice cold ethanol and 0.375 M MgCl2 at −40° C. overnight. The precipitated LPS was centrifuged at 11 000×g (Sorvall, SLA-3000 rotor) for 15 minutes at 4° C., and the resulting pellet resuspended in the same volume 90% (W/V) phenol at 65° C. for 30 min while stirring. The mixture was centrifuged at 4000×g for 10 min to accelerate phase separation. The water phase was collected, and the phenol phase extracted once more with water. The pooled water phases were pooled, and phenol extracted using ¼ the volume of chloroform. The water phase was placed under an airflow overnight to evaporate any residual organic solvent, and dialysed against MQ-water for 3 days using a 500 MWCO dialysis membrane. The dialyzed LPS was frozen and lyophilized to yield pure LPS.

[0170] The purity of the LPS products which were isolated was controlled by tricine-SDS-polyacrylamide gel electrophoresis (Marolda et al., 2006).

[0171] Preparation of O-Antigen Polysaccharides

[0172] Polysaccharides were isolated from wild type S. typhimurium (smooth) LPS by mild acid hydrolyzation of the glycosidic bond connecting LipidA to the proximal KDO sugar (Raetz and Whitfield, 2002a). 4-5 mg/mL S. typhimurium LPS was dissolved in 10% acetic acid and incubated at 100° C. for 1 hour. The resulting Lipid A was removed from the solution by centrifugation at 10 000×g for 30 minutes at 4° C., the supernatant containing the polysaccharide was frozen and lyophilized overnight.

[0173] ELISA-Like Tailspike Adsorption (ELITA) Assay

[0174] The ELITA assay was first described by Schmidt et al (Schmidt et al., 2016) using whole bacteria. Here, we modified the assay for use with purified proteins in a Nunc MaxiSorp 96-well flat-well plate (as shown in FIG. 11). The wells were saturated by incubating with 100 μl 10 μg/mL of either K9-His or K14-His in PBS-buffer overnight. Following a 2 hour blocking step with 2% bovine serum albumin (BSA) in PBS, 100 μL dilutions of Salmonella typhimurium LPS ranging from 200 μg/mL to 0.0023 μg/mL were added as a binding partner and incubated for 1 hour. 100 μL P22 tailspike protein (P22TSP) with an N-terminal Strep-tag®II (IBA) was added for one hour, before the wells were finally incubated with 100 μL 1:10 000 StrepTactin-conjugated horse radish peroxidase (IBA, Gottingen) for one hour, and developed with 2,2′-azino-bis 3-ethylbenzothiazoline-6-sulphonic acid (ABTS, Sigma-Aldrich) for 30-60 min and read at 407 nm using a plate reader. The wells were washed 3 times with 150 μL PBS-buffer containing 0.1% BSA between each of the above steps (Tween-20 was omitted for these experiments since it interfered with the assay). The average background signal (0 μg/mL LPS) was subtracted from each average signal, propagation of error was calculated by adding the individual standard deviations for the triplicates to the baseline in quadrature (δQ=√{square root over ((δa).sup.2+(δb).sup.2+ . . . +(δz).sup.2)}) where δQ is the uncertainty of a combination of sums Q). The dose-response curve and dissociation constant K.sub.D, was calculated by curve fitting the data to the Hill equation as follows:

[00001]$Y=\frac{{Y_{\max }\left[L\right]}^n}{{\left(K_D\right)}^n+{\left[L\right]}^n}$

where Y denotes the fraction of occupied receptor binding sites, Y.sub.max the maximal binding, [L] the concentration of free ligand, and n the number of binding sites. Although each construct carried two GCN4-PII motifs, n was treated as being equal to 1, since they are localized at opposite ends of the protein, and thus are not expected to cooperate. The average molecular weight of smooth S. typhimurium LPS was calculated to 22 kDa assuming an average of 30 O-antigen repeats polysaccharide structure as reported (Peterson and McGroarty, 1985; Raetz and Whitfield, 2002b; Schmidt et al., 2016).

[0175] Surface Plasmon Resonance Experiments (Examples 1 to 3)

[0176] All SPR-experiments were conducted on a Reichert 2SPR system at ambient temperature using PBS-E (PBS pH 7.4+5 mM EDTA) running buffer. The proteins were diluted to 50 μg/mL in 20 mM sodium acetate buffer pH 4.5 and immobilized to a CMD200 sensor chip (Xantec Bioanalytics, Duesseldorf, Germany) using NHS-EDC amine coupling (Fischer, 2010) to a response of 2000-9 000 μRIU. Following a comparison of different reference compounds (ethanolamine, BSA, casein, and skimmed milk) (Péterfi et al., 2000), ethanolamine was chosen as the standard coating for the reference channel for all experiments.

[0177] All ligands were solubilized to 1 mg/mL in running buffer by extrusion (21 passes through a 100 μm filter at 70° C.). The experiments were performed at 50 μL/min flowrate in triplicates. Each sample was injected over both measurement and reference channel for 90 s followed by 300 s dissociation. The chip was regenerated by 2×30 s injection of regeneration buffer (0.05% (w/w) CHAPS, 0.05% (w/w) zwittergent 3-12, 0.05% (v/v) tween 80, 0.05% (v/v) tween 20, and 0.05% (v/v) triton X-100) (Andersson, Areskoug and Hardenborg, 1999). The measurement data was exported to TraceDrawer (RidgeView instruments lab) for processing, and final curves generated using Origin (OriginLab corporation). The signal for each construct was normalized to K9 using the following formula S=

[00002]$S_0\left(\frac{\frac{R}{\mathrm{MW}}}{\frac{R_{K9}}{{\mathrm{MW}}_{K9}}}\right)$

where S is the normalized signal, S0

[0178] Surface Plasmon Resonance Experiments (Examples 4 and 5)

[0179] SPR experiments were conducted on a Nicoya OpenSPR system at ambient temperature using PBS-E (PBS pH 7.4+5 mM EDTA) running buffer. SadA K9 was diluted to 50 μg/mL in 10 mM sodium acetate buffer pH 4.5 and immobilized to Carboxyl Sensor (OpenSPR) using NHS-EDC amine coupling (Fischer, 2010) to a response of 700 RU.

[0180] All ligands were solubilized to 1 mg/mL in running buffer by extrusion (21 passes through a 100 μm filter at 70° C.). The experiments were performed at 35 μL/min flowrate in triplicates. Each sample was injected over both measurement and reference channel for 125 s followed by 300 s dissociation. The chip was regenerated by 125 s injection of regeneration buffer (0.05% (w/w) CHAPS, 0.05% (w/w) Zwittergent 3-12, 0.05% (v/v) Tween 80, 0.05% (v/v) Tween 20, and 0.05% (v/v) Triton X-100) (Andersson et al., 1999). The measurement data was exported to TraceDrawer (RidgeView instruments lab) for processing, and final graphs were generated using Origin (OriginLab corporation).

[0181] Electron Microscopy

[0182] Samples were adhered to a measuring grid, stained for one minute with 1% uranyl acetate and embedded in 1.8% methylcellulose/0.4% uranyl-acetate. Images were recorded in a Philips CM100 transmission electron microscope at 80 kV using a Olympus Quemesa camera.

[0183] Limulus Amebocyte Lysate (LAL) Assay

[0184] The masking effect of GCN4-PII on LPS was tested using the LAL-assay (Pierce, Thermofisher). GCN4-PII concentrations ranging from 200 μg/mL-20 pg/mL was spiked with 0.5 endotoxin units per mL LPS (EU/mL), and developed following the provided protocol.

[0185] Circular Dichroism

[0186] Spectra were recorded using a Jasco J-810 spectropolarimeter (Jasco International Co). Measurements were done using a 1.0 cm path length quartz cuvette. Each samples was scanned five times in the range of 190 to 250 nm with a scanning rate of 50 nm/min with a bandwidth of 0.5 nm. Spectra were recorded with a GCN4-pII to LPS ratios of 0, 0.5, 1, 3, and 9 in 10 mM Tris pH 7.4 at 37° C. The approximate α-helical content of the peptide was calculated using K2D2.

[0187] Nuclear Magnetic Resonance (NMR) Spectroscopy

[0188] NMR experiments for assignment were carried out in Bel-Art™ SP Scienceware™ 5 mm O.D. Thin Walled Precision NMR Tubes containing 450 μL 1.5 mM synthetic FMet-GCN4-PII (Genscript, China) in 50 mM NaCl, 7% D2O, and 0.2 mM 4,4-dimethyl-4-silapentane-sulfonic acid (DSS). Spectra were acquired at 308 K on a Bruker Avance II 600 MHz NMR spectrometer equipped with a 5 mm 1H/13C/15N-cryoprobe. DSS was used as internal chemical shift standard, and 13C and 15N was referenced using frequency ratios as described (Wishart et al., 1995). The following spectra were collected for assignment: 13C-1H-HSQC, 15N-1H-HSQC, 1H-1H COSY, 1H-1H TOCSY using a mixing time of 60 and 80 ms, and 1H-1H NOESY using a mixing time of 80 and 100 ms. All spectra were processed using Topspin 4.0 and peaks picked using CARA 1.9.1 (Keller, 2004).

[0189] Biotin-LPS (B-LPS) Based ELISA

[0190] Black 96-well Greiner microplates were coated by incubating 100 μl 10 μg/mL SadA K9 in PBS-buffer (Cold spring harbor) overnight at 4° C. Wells were blocked the next day by incubating by 150 μL 2% bovine serum albumin (BSA) in PBS. 100 μL dilutions of Biotinylated-LPS ranging from 4 ng/mL to 0.06 ng/mL were added as a binding partner and incubated for 1 hour. Plates washed 3 times with 150 μL PBS+0.1% BSA. 100 μL 1:10 000 StrepTactin-conjugated horse radish peroxidase (IBA) for one hour, and developed with QuantaRed fluorescent substrate (Thermo) for 15 min and read fluorescence at Ex: 550 nm, Em: 610 nm.

[0191] Protocol: [0192] 1. Coat 96-well black Greiner/Nunc Maxisorp plate by adding 100 μL 10 μg/mL SadA fragment solution, leave overnight at 4° C. [0193] 2. Empty wells, block with 150 μL 5% BSA in PBS for 2 hours. [0194] 3. Wash 3 times with 150 μL 0.1% BSA in PBS [0195] 4. Empty wells, add 100 μL Biotinylated-LPS dilutions. [0196] 5. Wash 3 times with 150 μL 0.1% BSA in PBS [0197] 6. Add 100 μL Strep-Tactin conjugated HRP (IBA) (1:20,000 dilution in PBS+0.35 M NaCl, 50 mM MgSO4, 0.1% BSA) for 60 min [0198] 7. Wash 4 times with 150 μL PBS+0.35 M NaCl, 50 mM MgSo4, 0.1% BSA and then once with PBS+0.1% BSA. [0199] 8. Incubate with Quantared HRP-substrate, quench after 15 min. [0200] 9. Read fluorescence at Ex: 550 nm, Em: 610 nm.

[0201] All substrates were prepared following the instructions of the vendor. Where background was subtracted from signal, propagation of error was calculated by adding the individual standard deviations for the replicates to the baseline in quadrature (δQ=√(δa.sup.2+δb.sup.2+ . . . +δz.sup.2) where δQ is the uncertainty of a combination of sums Q). Error bars represent one standard deviation.

Example 1—GCN4-PII Binds Lipid A

[0202] It was intended to investigate a putative interaction between LPS and two domains belonging to the trimeric autotransporter adhesin, SadA. Two earlier described SadA constructs (Alvarez et al., 2008; Hartmann et al., 2012), K9 and K14 were used, both stabilized by flanking GCN4-PII segments. K9 or K14 were covalently linked to a SPR-chip, and various LPS components injected. A schematic version of the structure of LPS is provided in FIG. 2 for reference.

[0203] Injection of smooth LPS immediately gave a response, which approached a steady state towards the end of injection (FIG. 3a). During the following dissociation stage, the signal remained at the plateau, indicating that there was no off-rate. Injection of the rough and deep-rough LPS variants (FIGS. 3b and 3c), showed similar binding curves, except for a slight increase in signal during the dissociation phase, while the purified polysaccharide showed no binding characteristics (FIG. 3d).

[0204] The results showed that all variants containing the Lipid A moiety bound strongly to GCN4-PII, but the pure polysaccharide did not, thus localizing the interaction to the Lipid A moiety. However, the absent off-rate and the propensity of LPS to form aggregates in solution (Sasaki and White, 2008; Richter et al., 2011), meant complicated potential biophysical characterization of the interaction, and meant that the results could only be interpreted qualitatively. It was believed that the increase in signal following injection of the rough and deep-rough variants of LPS was inversely proportional to the number of sugar residues present in each variant. Particularly, deep rough LPS has a significantly higher hydrophobic to hydrophilic ratio, adopting a larger, less fluid morphology compared to LPS with longer sugar moieties (Richter et al., 2011). The signal increase following injection was thus interpreted as being due to a slower reorganization, and breakdown of the deep-rough aggregates compared to the smooth variant.

[0205] The constructs were purified using a 6×His-tag, which has been implicated to have an endotoxin depleting effect during purification due to unspecific binding (Mack et al., 2014). To evaluate the effect of the His-tag on binding, two GCN4-pII flanked SadA constructs which were identical except for the His-tag (K3, and K3-His) were compared. These yielded almost identical curves to each other and to the previous constructs, showing that the His-tag had no effect on binding (FIG. 10).

[0206] It was considered whether the nature of the interaction between GCN4-PII was hydrophobic, electrostatic, or a combination of both. The choice of regeneration solution helped to determine this. In the process of testing suitable regeneration buffers prior to the experiments, it was found that 1 M NaCl had no effect, whilst a mixture of non-denaturing detergents tallying to 0.3% regenerated the samples in less than 60 seconds. This indicated a strong hydrophobic factor involved in the interaction.

Example 2—GNC4-PII Binds with High Affinity

[0207] The SPR results were not suitable for determining the binding kinetics of the GCN4-pII/LPS interaction. In order to quantify the affinity, an ELISA-like tailspike adsorption (ELITA) assay described earlier (Schmidt et al., 2016) was modified by using purified proteins in lieu of whole bacteria. The assay was similar to a traditional ELISA, except that the antibody was replaced with a phage tailspike protein that recognized the O-antigen of LPS (FIG. 11). The results showed that both constructs exhibited an extremely high binding affinity in the lower pM range (FIG. 5), which is in concordance with the zero off-rates which were observed in the SPR experiments. This setup proved advantageous since it allowed for the use of LPS concentrations below the critical micelle concentration (CMC) of smooth LPS, which would otherwise have complicated the interpretation (Yu et al., 2006; Sasaki and White, 2008). However, due to the propensity of LPS to coat the microtiter wells prior to blocking, an indirect ligand-receptor interaction setup was not possible.

Example 3—GCN4-pII Dissolves LPS Aggregates

[0208] It was observed that adding GCN4-PII to LPS caused visible breakdown of the LPS aggregates. This was investigated by comparing the structures of rough LPS at different GCN4-pII ratios using transmission electron microscopy (FIG. 6). It was confirmed prior to the experiments that the synthetic GCN4-pII bound LPS and retained its α-helical structure upon binding using a LAL-masking assay (Schwarz et al., 2017), circular dichroism, and NMR. The NMR spectrum confirmed that the peptide existed in a homologous α-helical state (FIG. 14), which was retained upon LPS binding (FIG. 12), and showed at least 89% neutralizing effect (binding) on LPS at a GCN4-pII concentration of 1 μM (FIG. 13).

[0209] Rough LPS was observed with TEM to form tubular micelles with a radius of around 10 nm and lengths ranging up to hundreds of nm (FIG. 6, top), as reported earlier with cryo-EM (Richter et al., 2011; Broeker et al., 2018). Following incubation with equimolar GCN4-PII, the micellar structures completely disappeared, leaving occasional aggregates, probably caused by slight aggregation of the peptide-LPS complexes (FIG. 6, bottom).

[0210] Discussion of Results (Examples 1 to 3)

[0211] We originally set out to study a putative interaction between trimeric SadA domains and LPS. Our results however, show that the GCN4-pII adapters we used to stabilize our constructs displays an extremely high affinity for LPS. Interestingly, the affinity of GCN4-pII, with a K.sub.D in the picomolar range, is 3-5 orders of magnitude higher than the human LPS immune receptors TLR4 (141 μM), CD14 (74 nM), MD-2 (2.33 μM), and LPS binding protein (3.5 nM). The dissociation constants we obtained with GCN4-pII are also 1-6 orders of magnitude higher than for polymxin B (48 μM), and even peptide avibodies specifically designed with the aim of highest achievable affinity. Furthermore, as opposed to several of the binding partners mentioned above, we have shown that GCN4-pII is specific to LipidA. We demonstrated that this interaction is reversible using detergents and that GCN4-pII readily dissolves LPS aggregates in solution, indicating that the interaction is largely hydrophobic. As far as we are aware, this is the first report of a trimeric coiled-coil motif binding LPS. GCN4-pII containing crystal structures earlier reported (Hartmann et al., 2012) show that the γ.sub.2 and δ-carbons belonging to the core isoleucines protrude from the core, forming hydrophobic surfaces along the coiled-coil grooves. It is conceivable that one or more of the LipidA acyl chains can align along these grooves to form the extremely strong interaction, a model that also explains how GCN4-pII can break down LPS aggregates. However, GCN4-pII also has a C-terminal patch of cationic residues, and these may also contribute to the interaction.

Example 4—Sensitivity of GCN4-pII Based ELISA

[0212] The aim of this experiment was to show that in principle the binding of the oligomeric protein to LPS, as shown here with GCN4-PII, could detect LPS quantities with equal or similar sensitivity to the LAL assay. As in previous examples, SadA-based constructs were used, in particular the K9 construct described above.

[0213] To ensure full reproducibility of the assay, the sensitivity experiment was conducted in 4 replicates with the final optimized conditions. In order to counteract the edge effect, only internal, randomized wells were used. The only exception was A3:A10, which was reserved for the highest concentration sample. The same samples were subsequently measured with the LAL assay for comparison. Only one replicate has been included in the results.

[0214] In the GCN4-pII based ELISA using biotinylated LPS (B-LPS) for detection (FIG. 15), a linear signal response was observed within the 0.06-1 ng/mL LPS concentration range, meaning that the assay could consistently detect B-LPS down to the lowest dilution (0.06 ng/mL).

[0215] An LAL assay was also conducted for comparison. The concentration range of LPS used in the LAL assay was 0.01-0.1 EU/mL. The lowest dilution that gave a clear signal was 0.13 ng/mL (FIG. 16), meaning that the GNC4-pII based assay has comparable sensitivity to the LAL assay. A comparison of the results of the two assays is shown in FIG. 17.

Example 5—Robustness of Binding Between GCN4-pII and LPS

[0216] To investigate the robustness of binding between GCN4-pII and different LPS types, SPR was used to check a broad selection of LPS-variants collected from various pathogens and proteobacteria (Table 4). In short, K9 was immobilized to a carboxyl matrix on the SPR-chip using EDC-NHS based amine coupling. Different LPS types were injected at 0.5 mg/mL in triplicates, to observe the signal.

TABLE-US-00005 TABLE 4 Type Candidate Type Note α- B. henselae Rough B. henselae LPS is one of few proteobacteria known non-pyrogenic LPS types (Chenoweth et al., 2004). β- Neisseria Rough Neisseria spp. Carry LPS-like proteobacteria lactamica molecules referred to as lipooligosaccharides (LOS). LOS are structurally very similar to classical LPS, but seldomly comes with O- antigen (Moran et al., 1996). γ- E. coli BL21, — Binding shown for several proteobacteria S. enterica, species earlier. S. anatum Bacteroidetes Porphyromonas Smooth Exact structure unknown, gingivalis probably smooth variant. Unusual LPS V. cholerae Rough V. cholerae LPS have unusual acylation-patterns of LipidA, and absence of phosphate group on the Kdo2-sugar (Reidl and Klose, 2002).

[0217] In earlier work, we compared the binding of LPS sourced from γ-proteobacteria, namely S. enterica, S. anatum, and E. coli BL21. Injection of LPS immediately gave a response that approached a steady state towards the end of injection. During the following dissociation stage, the signal remained at the plateau, indicating that there was no measurable off-rate. Although the different curves had very similar shape, the final response (pRIU) varied between them with an inverse correlation to the amount of sugar moieties per LPS molecule. This means that rough LPS types (lacking the O-antigen) typically gave a significantly stronger signal compared to their smooth counterparts (with 0-antigen repeats). Since all LPS types were injected at the same gravimetric concentration (mg/mL), the difference in response probably reflected the lower molarity of the high molecular weight variants.

[0218] The binding curves of the LPS types we checked in our current work (FIG. 18) all share similar binding curves with no off-rate, indicating strong binding. S. typhimurium WaaL LPS is a rough type, expected to give a moderately high response upon injection. N. lactamica and B. henselae are both large rough variants, expected to give a response in the same range as WaaL, however, N. lactamica gave a response which was almost twice as large, which could be explained by a large amount of sialic acid modifications to the core-sugars. V. cholerae has a mixture of rough and low molecular weight smooth LPS. A response around ⅓ of the WaaL signal was therefore not unexpected. The P. gingivalis LPS had a low response, suggesting a medium-to-large smooth LPS type. The documentation coming with the commercially obtained P. gingivalis LPS did not name the strain or variant, so the weight will have to be confirmed by SDS-PAGE.

[0219] In conclusion, GCN4-pII bound to all LPS types that were tested.

[0220] Discussion of Results (Examples 4 and 5)

[0221] The LAL assay uses an enzymatic cascade in the blood of the horseshoe crab (Lee, 2007) that is highly sensitive to low amounts of LPS. Using the LAL assay in direct comparison, we were able to show that the GCN4-pII peptide can bind biotinylated LPS in concentrations that are barely detectable with the LAL assay, and that this binding still results in a visible signal when using routine detection methods for biotin coupled to a fluorescent enzyme substrate. Importantly, this detection method worked in different buffer backgrounds and also in an injectable drug background. We have achieved a sensitivity of 0.01 EU/mL LPS, which is comparable to the LAL assay, and our data suggests that even higher sensitivities can be achieved with our ELISA-like assay, e.g. by fine-tuning wash buffer conditions.

[0222] We used LPS variants from different clades of the proteobacteria, ranging from alpha- to gamma-proteobacteria, and from the Bacteroidetes (FIG. 19). Our selection covers Enteropathogens (Vibrio cholerae, Salmonella spp., Escherichia coli), intracellular pathogens (Bartonella henselae), and oral pathogens (Porphyromonas gingivalis), as well as commensal bacteria (Neisseria lactamica). One of the species used is known to have an LPS variant that do not elicit a strong immune response (Bartonella henselae) (Zahringer et al., 2004).

[0223] Salmonella spp. are located with Escherichia, and not displayed separately in the tree shown in FIG. 19. It is also noted that Bartonella spp. are closest to the displayed Brucella, and that Porphyromonas gingivalis is part of the Bacteroides for the purpose of this figure. Furthermore, the large groups of the Firmicutes, Actinobacteria, and Spirochaetes do not have LPS as part of their membrane components.

[0224] Overall, using SPR, we were able to show that all LPS variants used bound strongly to the GCN4-pII peptide. While there were visible differences in the “on rate” of binding, there was no detectable “off rate” for any of the LPS variants, suggesting that the peptide can detect all of these variants in a similar fashion (albeit with slight differences in binding kinetics).

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