HIGH AFFINITY VIRAL CAPTURE HUMAN DECOY BASED PROTEINS FOR DETECTION AND PROTECTION AGAINST SARS-CoV-2 AND ZOONOTIC THREATS

Abstract

Amyloid fibrils comprising pathogen binding proteins and methods of their use are provided.

Claims

1. An amyloid fibril comprising a plurality of modified β solenoid protein (mBSP) monomers, wherein the monomers are linked to a pathogen-binding protein.

2. The amyloid fibril of claim 1, wherein the mBSP monomers are derived from an antifreeze protein.

3. The amyloid fibril of claim 2, wherein the antifreeze protein is a spruce budworm antifreeze protein.

4. The amyloid fibril of claim 3, wherein the mBSP has the sequence shown in SEQ ID NO: 1 or a sequence at least 90% identical to SEQ ID NO:1.

5. The amyloid fibril of claim 1, wherein the antifreeze protein is a rye grass antifreeze protein.

6. The amyloid fibril of claim 5, wherein the mBSP has the sequence shown in SEQ ID NO: 2 or a sequence at least 90% identical to SEQ ID NO:2.

7. The amyloid fibril of claim 1, wherein the antifreeze protein is a rhagium inquisitor antifreeze protein.

8. The amyloid fibril of claim 7, wherein the mBSP has the sequence shown in SEQ ID NO: 3 or a sequence at least 90% identical to SEQ ID NO: 3.

9. The amyloid fibril of any of claim 1, wherein the mBSP is modified to remove an end cap that prevents amyloid aggregation.

10. The amyloid fibril of claim 1 that is modified to include at least one amino acid residue that promotes attachment of the fibril to a solid support, a nanoparticle, a biological molecule, or a second amyloid fibril.

11. The amyloid fibril of claim 1, attached to a solid support, a nanoparticle, a biological molecule, or a second amyloid fibril.

12. The amyloid fibril of claim 1, wherein the pathogen is a virus.

13. The amyloid fibril of claim 11, wherein the virus is SARS-COV-2.

14. The amyloid fibril of claim 1, wherein the amyloid fibril is linked to a solid support.

15. (canceled)

16. (canceled)

17. The amyloid fibril of claim 1, wherein the pathogen-binding protein comprises the N-terminal ACE2 helix-turn-helix (HTH) domain (e.g., SEQ ID NO: 4, or a sequence at least 90% identical to SEQ ID NO:4).

18. The amyloid fibril of claim 1, wherein the pathogen binding protein is linked to a detectable label.

19. (canceled)

20. A method of detecting the presence or absence of a pathogen in a biological sample, the method comprising contacting a biological sample with the amyloid fibril of claim 1 under conditions that allow the pathogen to bind to the pathogen binding protein if the pathogen is present; and detecting the presence or absence of binding of the pathogen to the amyloid fibril, wherein the detecting comprises washing unbound components of the sample from the amyloid fibril and contacting the amyloid fibril, and bound pathogen if present, with a secondary binding agent that specifically binds to the pathogen, if bound to the amyloid fibril, and wherein the secondary binding agent is a β solenoid protein linked to a pathogen binding protein.

21. (canceled)

22. (canceled)

23. (canceled)

24. The method of claim 20, wherein the secondary binding agent is linked to a detectable label.

25. The method of claim 20, wherein the β solenoid protein comprises a sequence that prevents polymerization.

26. Clothing or protective equipment coated with the amyloid fibril of claim 1.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0036] FIG. 1 Example sequence comparisons of wild type (WT) anti-freeze proteins to modified proteins that allow for self-assembly. In each case, C-terminal end cap sequences are removed to allow C-to-N-terminal amyloid assembly. Additionally, modifications are made throughout to allow for solubility, stability, and microbial expression as detailed in [0016-0018]. FIG. 1 discloses SEQ ID NOS 7, 1, 8, 2, 9, and 3, respectively, in order of appearance.

[0037] FIG. 2 Example structural comparison of wild type (WT) RiAFP to RiAFP-m9. Removed end cap region of WT is highlighted in lighter shade.

[0038] FIG. 3 Sample mBSP derived from modified Rhagium inquisitor[2] antifreeze protein (RiAFP). (Top) An engineered VEGF binding domain is shown on the right. The flat faces and the polypeptide termini can readily be modified to provide for attachment to surfaces or covalent attachment of reporter molecules such as fluorophores. (Bottom) The mBSPs are engineered to polymerize under mild conditions to provide a high degree of polyvalency and thus avidity.

[0039] FIG. 4 Model of the structure of the proposed mBSP-ACE2 HTH fusion for SARS-CoV-2 virion capture. (Top) The N-terminal HTH motif of the ACE2 structure fused to RiAFP mBSP with the SARS-CoV-2 spike protein shown bound. This structure is stable in simulations over ~10 ns at 100 C. (Bottom) Overlay of the RiAFP-ACE2 HTH fusion with the experimental structure for the ACE2-Spike protein complex, illustrating why the N-terminal HTH motif was chosen for SARS-CoV-2 capture.

[0040] FIG. 5. Specific designs of mBSP-HTH constructs used to obtain affinity data for this patent. A) mBSP-HTH.1 Design - HTH is fused at a bend of modified Rhagium inquisitor antifreeze protein [SEQ ID NO: 5]. B) mBSP-HTH.2 Design - HTH with added spacing peptide sequences are fused at a bend of modified Rhagium inquisitor antifreeze protein [SEQ ID NO: 6].

[0041] FIG. 6. Table of measured dissociation constant values for primary bound protein RBD at tip and ligand proteins with HTH. Comparison with two other ACE2 N-terminal mimics from literature shown.

[0042] FIG. 7 Schematic mBSP assay for SARS-CoV-2 detection.

[0043] FIG. 8. Schematic for fluorescent assay demonstrating polyvalent binding. A) Designed viral mimic nanoparticle. Spike RBD proteins are attached to the surface of fluorescent nanoparticles to assay binding to polymeric mBSP-HTH constructs. B) Polymeric mBSP-HTH constructs are fixed to nitrocellulose followed by dried milk to coat empty space on nitrocellulose.

[0044] FIG. 9. Fluorescent Assay to demonstrate polyvalent binding of SARS-CoV-2 Spike RBDs by mBSP-HTH constructs. A) Control: BSA protein on nitrocellulose shows no fluorescence, indicating no binding of fluorescent nanoparticles, B) Image 24 hours after deposition shows fluorescence on two separate patches where mBSP-HTH.2 proteins have been deposited. C) Image 24 hours later shows little decrease in fluorescent intensity consistent with polyvalent binding.

[0045] FIG. 10. Summary of dimeric RBD capture constructs based upon ACE2 or decoys, including results from two other groups.

[0046] FIG. 11 Proof of principle VEGF binding experiment with engineered mBSP RiAFP fibrils.

DETAILED DESCRIPTION OF THE INVENTION

[0047] The inventors have discovered that amyloid fibrils comprised of monomers linked to pathogen binding proteins can be used to effectively detect pathogens. Amyloid fibrils are of particular use because they are stable under many conditions and can present a high density of binding molecules allowing for high affinity binding to pathogens. Amyloid fibrils can be formed from a number of β solenoid proteins that can be modified to remove a naturally-occurring motif that blocks polymerization. By fusing a pathogen binding protein to one, some or all of the β solenoid protein monomers, and allowing the monomers to polymerize, one can form a polymer with numerous pathogen binding sites. The polymer can be optionally linked to a solid support and can be used to detect pathogens in samples.

[0048] Naturally occurring β-solenoid proteins (BSPs) can be modified to form amyloid fibrils. These proteins have backbones that turn helically in either a left- or right-handed sense from the N-terminus to form β-sheets, and have regular geometric structures (triangles, rectangles, etc.) with 1.5-2 nm sides. The naturally occurring proteins are inhibited from amyloid aggregation (end-to-end polymerization to give cross β-fibrils) by natural capping features and/or structural distortions on one or both ends. Modification for making linear polymers (amyloids) from these proteins, molecular simulations used to assess structural stability and geometric properties for comparison to measurements, and the protocol for expressing and folding of the engineered proteins are described in, e.g., U.S. Pat. No. 10,287,332, which is incorporated by reference[5]. The correct monomeric structures can be obtained after purification and folding, amyloid fibrils can be produced by incubation at elevated temperatures, and the kinetics of fibril formation are consistent with, though slightly faster than, other amyloid polymerization reactions.

[0049] The modified BSPs (mBSPs) offer excellent platforms for functionalization with pathogen binding proteins without interfering with the native β-sheet structure. This allows for presentation of a number of the same or different pathogen binding proteins while maintaining the beta-sheet.

[0050] In one embodiment, the BSP is modified to enable one-dimensional growth through cross-beta strand (amyloid) pairing mBSPs. The exteriors and interiors of the proteins can also be modified to enable more efficient production. In some embodiments, the protein units are allowed to self-assemble in one dimension after expressing proteins (for example but not limited to, in E. coli), followed by, for example, subsequent cell lysis, purification, denaturation, refolding, and polymerization of the proteins to create the one dimensional scaffolds.

[0051] In some embodiments, at least two different mBSP monomers are designed to self-assemble in a predetermined order. This can be achieved by modifying the ends of the monomers such that, for example, the N-terminus of a first monomer interfaces with the C-terminus of a second monomer, but not with the C-terminus of another copy of the first monomer. The resulting fibril comprises the two different monomers in predetermined order (e.g., A-B-A-B-A- B, or A-B-C-A-B-C). For example, A, B, and C can represent monomers fused to different pathogen binding proteins, where different pathogen binding proteins have different sequences but bind to the same pathogen (optionally to different locations of the same pathogen) or bind to different pathogens.

[0052] The correct molecular mass of the amyloid monomer can be verified through standard techniques, such as mass spectroscopy. The correct beta content can be determined through techniques such as circular dichroism. Amyloid aggregation can be confirmed by observing the growth of thioflavin T (ThT) fluorescence at 480 nm.

[0053] The length of the fibrils can be controlled, for example through a variety of approaches including varying of the temperature (e.g., between 5° C. to 45° C.), by following the incubation with sonication, by the addition of inhibitors of polymerization, or by modifying the buffer solution. For example, fibrils of several microns can be routinely produced. Alternatively, shorter fibrils (e.g., 100-200 nm) can be produced upon sonication.

[0054] Exemplary naturally-occurring BSPs include but are not limited to:

[0055] 1. Chain A, Crystal Structure Of A Lumenal Pentapeptide Repeat Protein From Cyanothece Sp 51142 At 2.3 Angstrom Resolution. Tetragonal Crystal Form

[0056] PDB: 2G0Y A

TABLE-US-00005   1 mhhhhhhssg lvprgsgmke taakferqhm dspdlgtddd dkamamvtgs sasyedvkli  61 gedfsgkslt yaqftnadlt dsnfseadlr gavfngsali gadlhgadlt nglayltsfk 121 gadltnavlt eaimmrtkfd dakitgadfs lavldvyevd klcdradgvn pktgvstres 181 lrcq (SEQ ID NO: 14)

[0057] 2. Chain X, The 2.0 Angstrom Resolution Crystal Structure Of Hetl, A Pentapeptide Repeat Protein Involved In Heterocyst Differentiation Regulation From The Cyanobacterium Nostoc Sp. Strain Pcc 7120

[0058] PDB: 3DU1_X

TABLE-US-00006   1 mgsshhhhhh ssglvprgsh mnvgeilrhy aagkrnfqhi nlqeieltna sltgadlsya  61 dlrqtrlgks nfshtclrea dlseailwgi dlseadlyra ilreadltga klvktrleea 121 nlikaslcga nlnsanlsrc llfqadlrps snqrtdlgyv lltgadlsya dlraaslhha 181 nldgaklcra nfgrtiqwgn laadlsgasl qgadlsyanl esailrkanl qgadltgail 241 kdaelkgaim pdgsihd (SEQ ID NO: 15)

[0059] 3. Chain A, Crystal Structure Of Recombinant Human Alpha Lactalbumin

[0060] PDB: 3B0I_A

TABLE-US-00007   1 mkqftkcels qllkdidgyg gialpelict mfhtsgydtq aivenneste yglfqisnkl  61 wckssqvpqs rnicdiscdk flddditddi mcakkildik gidywlahka lctekleqwl 121 cekl (SEQ ID NO: 16)

[0061] 4. Chain B, Crystal Structure Of An Ice-Binding Protein From The Perennial Ryegrass, Lolium Perenne

[0062] PDB: 3ULT .sub.-B

TABLE-US-00008  1 mdeqpntisg snntvrsgsk nvlagndntv isgdnnsvsg snntvvsgnd ntvtgsnhvv  61 sgtnhivtdn nnnvsgndnn vsgsfhtvsg ghntvsgsnn tvsgsnhvvs gsnkvvtdaa 121 klaaalehhh hhh (SEQ ID NO: 8)

[0063] 5. Chain A, Crystal Structure Of An Ice-Binding Protein From The Perennial Ryegrass, Lolium Perenne

[0064] PDB: 3ULT_A

TABLE-US-00009   1 mdeqpntisg snntvrsgsk nvlagndntv isgdnnsvsg snntvvsgnd ntvtgsnhvv  61 sgtnhivtdn nnnvsgndnn vsgsfhtvsg ghntvsgsnn tvsgsnhvvs gsnkvvtdaa 121 klaaalehhh hhh (SEQ ID NO: 8)

[0065] 6. Chain B, Crystal Structure Of Ydck From Salmonella Cholerae At 2.38 A Resolution. Northeast Structural Genomics Target Scr6

[0066] PDB: 2PIG_B

TABLE-US-00010   1 xtkyrlsegp raftyqvdge kksvllrqvi avtdfndvka gtsggwvdad nvlsqqgdcw  61 iydenaxafa gteitgnari tqpctlynnv rigdnvwidr adisdgaris dnvtiqsssv 121 reecaiygda rvlnqseila iqglthehaq ilqiydratv nhsrivhqvq lygnatitha 181 fiehraevfd faliegdkdn nvwicdcakv ygharviagt eedaiptlry ssqvaehali 241 egncvlkhhv lvgghaevrg gpillddrvl ieghaciqge ilierqveis graaviafdd 301 ntihlrgpkv ingedritrt plvgsllehh hhhh (SEQ ID NO: 17)

[0067] 7. Chain A, Crystal Structure Of Ydck From Salmonella Cholerae At 2.38 A Resolution. Northeast Structural Genomics Target Scr6

[0068] PDB: 2PIG_A

TABLE-US-00011   1 xtkyrlsegp raftyqvdge kksvllrqvi avtdfndvka gtsggwvdad nvlsqqgdcw  61 iydenaxafa gteitgnari tqpctlynnv rigdnvwidr adisdgaris dnvtiqsssv 121 reecaiygda rvlnqseila iqglthehaq ilqiydratv nhsrivhqvq lygnatitha 181 fiehraevfd faliegdkdn nvwicdcakv ygharviagt eedaiptlry ssqvaehali 241 egncvlkhhv lvgghaevrg gpillddrvl ieghaciqge ilierqveis graaviafdd 301 ntihlrgpkv ingedritrt plvgsllehh hhhh (SEQ ID NO: 17)

[0069] 8. Chain A, Choristoneura Fumiferana (Spruce Budworm) Antifreeze Protein Isoform 501

[0070] PDB: 1M8N_A

TABLE-US-00012   1 dgtcvntnsq itansqcvks tatncyidns qlvdtsictr sqysdanvkk svttdcnidk  61 sqvylttctg sqyngiyirs stttgtsisg pgcsistcti trgvatpaaa ckisgcslsa 121 m (SEQ ID NO: 7)

[0071] 9. Chain B, Choristoneura Fumiferana (Spruce Budworm) Antifreeze Protein Isoform 501

[0072] PDB: 1M8N_B

TABLE-US-00013   1 dgtcvntnsq itansqcvks tatncyidns qlvdtsictr sqysdanvkk svttdcnidk  61 sqvylttctg sqyngiyirs stttgtsisg pgcsistcti trgvatpaaa ckisgcslsa 121 m (SEQ ID NO: 7)

[0073] 10. Chain C, Choristoneura Fumiferana (Spruce Budworm) Antifreeze Protein Isoform 501

[0074] PDB: 1M8N_C

TABLE-US-00014   1 dgtcvntnsq itansqcvks tatncyidns qlvdtsictr sqysdanvkk svttdcnidk  61 sqvylttctg sqyngiyirs stttgtsisg pgcsistcti trgvatpaaa ckisgcslsa 121 m (SEQ ID NO: 7)

[0075] 11. Chain D, Choristoneura Fumiferana (Spruce Budworm) Antifreeze Protein Isoform 501

[0076] PDB: 1M8N_D

TABLE-US-00015   1 dgtcvntnsq itansqcvks tatncyidns qlvdtsictr sqysdanvkk svttdcnidk  61 sqvylttctg sqyngiyirs stttgtsisg pgcsistcti trgvatpaaa ckisgcslsa 121 m (SEQ ID NO: 7)

[0077] Exemplary mBSPs include but are not limited to:

[0078] SEQ ID NO:1 synthetic protein SBAFP-m9

TABLE-US-00016 Ala Ser Arg Ile Thr Asn Ser Gln Ile Val Lys Ser Glu Ala Thr Asn 1               5                   10                  15 Ser Asp Ile Asn Asn Ser Gln Leu Val Asp Ser Ile Ser Thr Arg Ser             20              25                      30 Gln Tyr Ser Asp Ala Asn Val Lys Lys Ser Val Thr Thr Asp Ser Asn         35                  40                  45 Ile Asp Lys Ser Gln Val Tyr Leu Thr Thr Ser Thr Gly Ser Gln Tyr     50                  55                  60 Asn Gly Ile Tyr Ile Arg Ser Ser Asp Thr Thr Gly Ser Glu Ile Ser 65                  70                  75                  80 Gly Ser Ser Ile Ser Thr Ser Arg Ile Thr Ile                 85                  90

[0079] SEQ ID NO:2 synthetic protein RGAEP-m1

TABLE-US-00017 Ala Asn Asp Ile Asp Gly Thr Asn Asn Glu Val Asp Gly Ser Glu Asn 1               5                   10                  15 Val Leu Ala Gly Asn Asp Asn Thr Val Ser Gly Asp Asn Asn Ser Val             20                  25                  30 Ser Gly Ser Asn Asn Thr Val Ser Gly Asn Asp Asn Thr Val Thr Gly         35                  40                  45 Ser Asn Met Val Val Ser Gly Thr Asn Met Ile Val Thr Asp Asn Asn     50                  55                  60 Asn Asn Val Ser Gly Asn Asp Asn Asn Val Ser Gly Ser Phe Met Thr 65                  70                  75                  80 Val Ser Gly Gly Met Asn Thr Val Ser Gly Ser Asn Asn Thr Val Ser                 85                  90                  95 Gly Lys Arg Met Arg Val Gln Gly Thr Asn Asn Arg Val Thr Asp             100                 105                 110

[0080] SEQ ID NO: 3 synthetic protein RiAFP-m9

TABLE-US-00018 Ser Arg Ala Glu Ala Arg Gly Glu Ala Met Ala Glu Gly His Ser Arg                 5                   10                  15 Gly Cys Ala Thr Ser His Ala Asn Ala Thr Gly His Ala Asp Ala Arg             10                  15                  20 Ser Met Ser Glu Gly Asn Ala Glu Ala Tyr Thr Glu Ala Lys Gly Thr         25                  30                  35 Ala Met Ala Thr Ser Glu Ala Ser Gly Glu Ala Arg Ala Gln Thr Asn     40                  45                  50 Ala Asp Gly Arg Ala His Ser Ser Ser Arg Thr His Gly Arg Ala Asp 55                  60                  65                  70 Ser Thr Ala Ser Ala Lys Gly Glu Ala Met Ala Glu Gly Thr Ser Asp                 75                  80                  85 Gly Asp Ala Lys Ser Tyr Ala Ser Ala Asp Gly Asn Ala Cys Ala Lys             90                  95                  100 Ser Met Ser Thr Gly His Ala Asp Ala Thr Thr Asn Ala His Gly Thr         105                 110                 115 Ala Met Ala Asp Ser Asn Ala Ile Gly Glu Ala Arg Ala Glu Thr Arg     120                 125                 130 Ala Glu Gly Arg Ala Glu Ser Ser Ser Asp Thr Asp Gly Cys 135                 140                 145

[0081] In some embodiments, binding of scaffolds to solid support (e.g., surface) can be achieved. For example, in some embodiments, binding can be achieved by: (a) sulfur chemistry of unoxidized cysteine to bind to thiols decorating a prepared surface; (b) peptide bond chemistry to link exposed lysine side chains to carboxyl groups decorating a prepared solid surface; and (c) the application of common types of bioconjugate chemistry, for example the biotin-avidin or biotin-streptavidin interacting pair.

[0082] In certain circumstances, the solid support can be mica, silicon, glass, or a transparent conducting oxide, for example, FTO or ITO. In some embodiments, the surface can be poly-L-lysine coated mica (0001) surfaces. In other embodiments, the solid support is absorbent. For example, the solid support can be paper. This will allow for collection of a liquid biological sample, for example, saliva, blood, urine, feces, waste water or other biological fluids.

[0083] As noted above, the BSP or mBSP is fused to a pathogen binding protein, which provides an affinity agent that is displayed in multiple copies in the amyloid fibril polymer. The fusion can be direct between the monomer and the pathogen binding protein or a linker can be used to link the two fusion partners. In some embodiments, the linker can be comprised of a majority or entirely of glycine, serine or alanine or combinations thereof. For example, an exemplary linker is GGG.

[0084] The pathogen binding protein can be any protein that can be formed in a translational fusion with a BSP or mBSP monomer. The pathogen binding protein will depend on the pathogen to be targeted. In some embodiments, the pathogen binding protein is a protein from a human or animal or plant cell to which the pathogen binds during infection, and thus has affinity for the pathogen. In the example of SARS-CoV-2, the virus enters cells by binding Angiotensin Converting Enzyme 2 (ACE2). Accordingly, a useful SARS-CoV-2 binding protein is the virus-binding portion of ACE2. In some embodiments, the SARS-CoV-2 binding protein comprises a 54 residue helix-turn-helix (HTH) motif from ACE2, for example

TABLE-US-00019 IEEQAKTFLDKFNHEAEDLFTQSSLASTNTNTNITEENVQNMNNAGDKTS AFLKEQSTLAQMT (SEQ ID NO:4)

or an amino acid sequence substantially identical to SEQ ID NO:4. Alternatively, the pathogen binding protein can be a peptide (e.g., identified by phage panning or other techniques), a tetrameric, single domain or single chain antibody, or other protein that selectively binds to the pathogen. Aside from SARS-CoV-2, the pathogen can be any virus, bacterium, protozoan, or fungus.

[0085] In some embodiments the combined binding protein-BSP fusion construct may be comprised from the 233 residue RiAFP and helix-turn-helix sequence (RiAFP-HTH.1)

TABLE-US-00020 ASRAEARGEAMAEGHSRGSATSHANATGHADARSMSEGNAEAYTEAKGDA MATSEASGEARAQTNADGSAHSSSRTHGRADSTASAKTNYNRECGEEQAK TFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNRNNAGDKRSAFLKEQ STLAQMYGCGSGSAMAEGTSDGDAKSYASADGNASAKSMSTGHADATTNA HGTAMADSNAIGEARAETRAEGRAESSSDTDGS (SEQ ID NO:5)

or an amino acid sequence substantially identical to SEQ. ID NO:5. In some embodiments the combined binding protein-BSP fusion construct may be comprised from the 266 residue RiAFP and helix-turn-helix sequence (RiAFP-HTH.2)

TABLE-US-00021 ASRAEARGEAMAEGHSRGSATSHANATGHADARSMSEGNAEAYTEAKGDA MATSEASGEARAQTNADGSAHSSSRTHGRADSTASAKGGKALNDKEAKNK AILNLEEIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMN NAGDKWSAFLKEQSTLAQMYQNEIKRKSEKQEDLKKEMLELEKLGGAMAE GTSDGDAKSYASADGNASAKSMSTGHADATTNAHGTAMADSNAIGEARAE TRAEGRAESSSDTDGS (SEQ ID NO:6)

or an amino acid sequence substantially identical to SEQ ID NO:6. In this embodiment the helix-turn-helix on each side is flanked by spacing linkers so it sticks out further from the mBSP scaffold.

[0086] In some embodiments, the pathogen is a hepatitis virus, e.g., hepatitis A, hepatitis B (HBV) or hepatitis C (HCV). Exemplary HBV binding proteins include but are not limited to the heptapeptide

TABLE-US-00022 ETGAKPH (SEQ ID NO: 18)

(interacting with the hydrophilic loop (residues 101-159 located on the surface of the virus) with the dissociation constant of 2.9 nM [Ho, K.L., et al., Selection of high affinity ligands to hepatitis B core antigen from a phage-displayed cyclic peptide library. J Med Virol, 2003. 69(1): p. 27-32]). Exemplary HCV binding proteins include but are not limited to

TABLE-US-00023 TSQNIRS (SEQ ID NO: 19)

,_which binds to the Hepatitis C Virus Protein E2 [Hong, H.W., S.W. Lee, and H. Myung, Selection of peptides binding to HCV e2 and inhibiting viral infectivity. J Microbiol Biotechnol., 2010. 20(12): p. 1769-71] and

TABLE-US-00024  WPWHNHR (SEQ ID NO: 20)

Lu, X., et al., Identification of peptides that bind hepatitis C virus envelope protein E2 and inhibit viral cellular entry from a phage-display peptide library, in Int J Mol Med. 2014, 2011 Elsevier Inc: Greece] and

TABLE-US-00025 MARHRNWPLVMV (SEQ ID NO: 21)

[Chen, F., et al., Functional selection of hepatitis C virus envelope E2-binding Peptide ligands by using ribosome display. Antimicrob Agents Chemother., 2010. 54(8): p. 3355-64. doi: 10.1128/AAC.01357-09. Epub 2010 May 17].

[0087] In some embodiments, the amyloid fibril is composed of two or more mBSP monomer/pathogen binding protein fusions, wherein the two or more fusions differ by the pathogen binding protein. Thus, in some embodiments, the amyloid fibril comprises two or more different pathogen binding proteins that bind to the same pathogen, optionally at different targets on the pathogen. In some embodiments, the different pathogen binding proteins bind to different pathogens.

[0088] As noted above, the amyloid fibrils comprising pathogen binding proteins can be linked to a solid support, for example an absorbent solid support (e.g., paper or other cellulose or other polymer material), which can capture biological fluids. In some embodiments, the amyloid fibrils comprising pathogen binding proteins are linked to an absorbent solid support and used in detection of pathogen using lateral flow.

[0089] In other embodiments, the amyloid fibrils comprising pathogen binding proteins can be embedded in or coated on personal protective equipment (PPE). Exemplary PPE include but are not limited to clothing (e.g., lab gowns or jackets or hazmat suits), gloves, face masks, face screens or other physical barriers. In some embodiments, the amyloid fibrils comprising pathogen binding proteins can be formulated into a liquid, gel, or cream that can be applied to surfaces (e.g., countertops) or to the skin to bind and inactivate pathogen, if present, preventing the pathogens from being infectious.

[0090] In addition to binding pathogens to inactivate them, the amyloid fibrils comprising pathogen binding proteins can be used to capture and detect a pathogen. In some embodiments, the amyloid fibrils comprising pathogen binding proteins are linked to a solid support as described herein and contacted with a sample (e.g., a biological sample); pathogen specifically bound to the pathogen binding protein can be detected. In some embodiments, the bound pathogen is detected by a sandwich assay format, with a second binding agent binding the immobilized pathogen and subsequent detection of the bound second binding agent. The second binding agent can be labeled or can be contacted by a tertiary binder that comprises a detectable label. In some embodiments, the second binding agent is a mBSP comprising a cap sequence that prevents polymerization fused to a pathogen binding protein. In some embodiments, the second binding agent can be a peptide (e.g., 10-50 amino acids) or an antibody that specifically binds the pathogen. Alternatively, the amyloid fibrils comprising pathogen binding proteins can be linked to a detectable label that changes the signal depending on whether the pathogen is bound to the pathogen binding protein, thereby avoiding the need for a second binding agent.

EXAMPLES

Example 1

[0091] We propose development of direct virion capture assays based upon patented mBSP technology[5, see also 6-11] using genetically engineered, highly functionalizable beta solenoid proteins (FIGS. 1,2,3). Amino acid sequences allowing attachment to paper or nitrocellulose surfaces are well tolerated at the polypeptide termini, and the solenoid edges readily accept insertions that functionally mimic binding loops in antibodies.

[0092] Our approach to rapidly developing inexpensive SARS-CoV-2 POC direct viral capture tests will be two-fold. First, we provide a 54 residue helix-turn-helix (HTH) motif (SEQ ID NO:4 or a substantially similar sequence) from the cell-surface bound angiotensin converting enzyme 2 (ACE2) to which the SARS-CoV-2 viral spike protein binds. Second, phage display or other established biopanning technologies including ribosome display or yeast display can be used to find novel peptides that bind tightly and specifically to the SARS-CoV-2 spike protein.

[0093] This represents a significant innovation. The cell-surface ACE2 protein [12] is the cellular entry point for SARS-CoV-2. ACE2 binds tightly to the SARS-CoV-2 spike protein, with dissociation constants in the low nanomolar range[13]. However, the single N-terminal helix of the ACE2 by itself binds weakly to the spike protein, with dissociation constants of 1.3 micromolar. [14], and a computationally designed substitute which includes a portion of the first helix only modestly improves on this with a dissociation constant of 646 nanomolar [15] We have made stable constructs of a VEGF-binding HTH fusion with RiAFP, discussed extensively in Example 2 below. Here we employ the ACE2 N-terminal HTH as a SARS-CoV-2 capture sequence (see below). This is illustrated in FIG. 4 and FIG. 5. The similarity of the HTH VEGF capture peptide with the ACE2 HTH sequence indicates the SARS-CoV-2 binding construct will function. That this use of HTH as a decoy binder is a non-obvious basis for success is seen in comparison of binding data for single RBDs to RiAFP-HTH.1 and RiAFP-.2 to single ACE2 proteins and to the single helix constructs above in FIG. 6. With the same binding motif (streptavidin-biotin) of RBD to a biolayer interferometry tip, we obtained dissociation constants in the range of 0.7-1.6 nM for the RiAFP-HTH.1 construct, and 2.1-28 nM for the RiAFP-HTH.2 construct. These are comparable to the 0.5-5.1 nM value we find for the ACE2 protein itself which are consistent with the literature (e.g., Ref. [13]).

[0094] With a mBSP SARS-CoV-2 binding fusion construct in hand, a dense mBSP polymeric array is formed (FIG. 7, schematic) on suitable paper or polymer surfaces that can then capture the virus from patient fluids. Spectroscopic readout for virion capture will come from either (i) monomeric (end-capped) versions of the same mBSP SARS-CoV-2 binding fusion attached to luminescent nanoparticles, luminescent dyes or protein, or chromogenic reactions such as those catalyzed by horseradish peroxidase (as commonly employed in ELISA assays), or (ii) an environmentally sensitive dye introduced in a location that engenders fluorescence upon virion binding with no need for secondary mBSPs. Luminescence for (i) can be confirmed by illumination with LEDs or diode lasers of the appropriate output wavelength, and in (ii) by LED fluorescence excitation in the ~400 nm region and emission in the green (with, for example, 4-dimethylaminophthalimide-type fluorophores). Inexpensive hand-held fluorometers that can be modified for this application are commercially available or could rapidly be designed in collaboration with electrical engineers.

[0095] There are several built-in advantages to this mBSP based virion capture assay. First, it would likely be less expensive to employ than immunoassays and can potentially be quickly mass produced. Second, it requires no special technology and so can be implemented in field environments like drive-through test facilities. Third, it would likely detect the presence of either live or dead virus. Fourth, it is expected to be highly stable in extreme environments of heat or humidity. Our previous studies on mBSP polymers show stability in conditions of extreme alkalinity or acidity, high protein denaturant concentration, and extreme temperature (some survive autoclaving)[7]. They are however partially protease-sensitive, so do not present the same kind of potential health problems as say mammalian prions[16]. Fifth, it can be used for environmental sampling of viruses on surfaces. Sixth, a non-obvious consequence that will benefit the sensitivity and specificity of any assay based upon the polymeric capture proteins and a divalent conjugate labeling protein is at least divalent avidity compared to the monovalent binding of antibodies. FIG. 8 shows a schematic for a fluorescent assay to test for divalent binding of the capture polymers, and FIG. 9 shows results. In this experiment, proteins are spotted on a nitrocellulose surface and after the interstitial regions between spots are filled with milk proteins. Nanoparticles that fluoresce in the red are decorated by attaching single SARS-CoV-2 RBD proteins so that the mean spacing of ~7 nm matches the periodicity of the HTH constructs on the bound polymers. This implies at best dimeric binding of the RBDs. The nanoparticles are deposited on the surface, the surface is washed to clear stray particles and measurements are taken at two 24 hour intervals after washing. No intensity is seen on the Bovine Serum Albumin (BSA) control, and on two separate RiAFP-HTH.2 coated patches, fluorescent intensity is observed with a time decay rates of 6-9× 10.sup.-7 sec.sup.-1. Using observed association rate constants of 10.sup.5 /sec-M gives dissociation constants of 6-9 pM, which we interpret as a lower bound of 10 pM. In contrast, the dimeric binding of the computational designed modified N-terminal fragments[15] and of dimeric ACE2 on an Fc dimer[17] give, respectively, binding affinities in the nM and tenths of nM. These values are summarized in FIG. 10.

[0096] We have proven the ability of our mBSP-HTH constructs to bind VEGF by fusing a known VEGF binding HTH motif with mBSPs. We found tight binding in a simple assay based on ultrafiltration (FIG. 11). The calculated upper limit on the dissociation constant for VEGF binding is approximately 10 nM.

[0097] Development of secondary mBSPs bound to silica coated rare earth upconversion quantum nanoparticles (REUQN) for binding to RBD domains of SARS-CoV-2 spike proteins. REUQN make use of sequential two photon transitions from the infrared part of the spectrum followed by energy transfer to induce single photon luminescence in the visible spectrum; for example, one can use nanorings made from NaYF.sub.4 doped with Er,Yb[18]. REUQN (i) can be excited by inexpensive infrared lasers[19], potentially enabling cell phone based imaging of binding, (ii) are stabilized with a silica coat to provide a shelf life of 4 years [18], (iii) can be functionalized for binding to amine or carboxyl functional groups, and (iv) are inexpensive to synthesize or purchase, and to coat. A previous antibody based FLISA assay for hepatitis B,C viruses made use of rare earth dots [20].

Example 2

[0098] Construction and testing of betabodyBSP polymer comprising VEGF-binding protein: To prove that a BSP (beta solenoid based synthetic antibody [6] can be engineered to capture proteins with high affinity, we engineered a mBSP polymer derived from the modified Ragium Inquisitor antifreeze protein (RiAFP) [4] with a helix-tum-helix (HTH) motif known to bind to the Vascular Endothelial Growth Factor (VEGF) protein with high affinity [Fedorova, A., et al., The Development of Peptide-Based Tools for the Analysis of Angiogenesis. Chemistry & Biology, 2011. 18(7): p. 839-845]. We used a flow-through assay to verify that the mBSP polymer captured the VEGF with high affinity (K.sub.d < 10 nM).

[0099] Experimental Design 1: Capture mBSPs We took a previously identified miniZ-peptide with the helix-turn-helix motif (HTH) that has approximately K.sub.d = 40 nM affinity to VEGF. We designed and synthesized a construct with the miniZ-HTH inserted at the edge (FIG. 1A) of the mBSP, and we polymerized the resulting RiAFP-miniZ-HTH proteins (FIG. 1B).

[0100] Protein expression occurred from synthetic genes in the pET28a vector in E. coli followed by purification using standard methods. Polymerization was effected by incubating the proteins at 37° C. for 48 hours.

[0101] Experimental Design 2. We tested the binding of VEGF to the RiAFP-miniZ-HTH construct with a flow-through assay. The schematic is shown in FIG. 11. A centrifugal protein concentrator (Amicon) with a 100 kDa MW cutoff filter is centrifuged, with the samples initially placed in the upper (retentate) chamber. The filter blocks the RiAFP-miniZ-HTH polymers from flowing through because of the large size of the polymer, while allowing free VEGF to pass through. The contents of the retentate and flow-through are assessed using SDS-PAGE gel electrophoresis.

[0102] The samples analyzed were as follows:

[0103] I) Control 1: VEGF. This is shown in lane 3. After centrifugation, no VEGF is found in the retentate chamber, only in the flow-through chamber, confirming that it passes through the filter.

[0104] II) Control 2: RiAFP-miniZ-HTH. This is shown in lane 2. There is no protein found in the flow-through chamber, only in the retentate. This confirms that the filter blocks the RiAFP-miniZ-HTH construct.

[0105] III) Test sample. Both RiAFP-miniZ-HTH polymer and VEGF are added to the upper chamber prior to centrifugation. Both the RiAFP-miniZ-HTH polymer and VEGF appear only in the retentate, confirming VEGF binding by the polymer.

[0106] Analysis. The binding assay with controls demonstrates qualitatively high-affinity binding of VEGF to the RiAFP-miniZ-HTH construct. The limit of protein detection on SDS-PAGE gels stained with Coomassie R250 is generally accepted as ~30 ng. The SDS-PAGE gels were loaded with 20 .Math.L of sample per lane. Thus, the test sample flow-through solution has less than 0.0015 mg/mL (~30 nM) VEGF. The concentrations of the RiAFP-miniZ-HTH and VEGF were each ~1 mg/mL (~40 .Math.M RiAFP-miniZ-HTH; ~25 .Math.M VEGF) in the initial mixture. Using the general equation for ligand binding combined with the values above allows us to estimate a Kd value of <10 nM for VEGF binding to RiAFP-miniZ-HTH.

[0107] The above examples are provided to illustrate the invention but not to limit its scope. Other variants of the invention will be readily apparent to one of ordinary skill in the art and are encompassed by the appended claims. All publications, databases, internet sources, patents, patent applications, and accession numbers cited herein are hereby incorporated by reference in their entireties for all purposes.

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