CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No. 61/780, 102 filed March 13, 2013; which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under 201 1-68003-30395 awarded by U.S. Department of Agriculture/National Institute of Food and Agriculture. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] Norovirus, a Calicivirus (family Caliciviridae), is colloquially known as "stomach flu" or "food poisoning". Norovirus is recognized as one of the major causes of nonbacterial outbreaks worldwide. This accounts for an estimated of 23 million infections per year in the US (the second highest cause of nonbacterial gastroenteritis (GE) morbidity) and imposes a substantial burden on healthcare.

[0004] Norovirus is classified as "MAID category B Priority bio-defense Pathogen". It is a non-enveloped, single-stranded, positive sense RNA genome which is environmentally stable due to capsid formation. It can resist freezing and heating to up to 60C and is stable at low concentrations of chlorine. An infectious dose of 10-100 viruses via fecal-oral transmission or droplet transmission can lead to infection. This is a highly contagious but short-lived illness (48hrs) causes vomiting, stomach pain and diarrhea. Also, it can cause chronic infections in transplant recipient.

[0005] Of the five geno-groups of Norovirus, GI, Gil, and GIV are known to infect humans. There is no available vaccine for human Norovirus infection, with progress being hampered by the absence of suitable animal model/cell culture for preclinical testing of the candidate vaccine. Presently, the detection of viral RNA is limited to RT-PCR in the stool samples of affected humans.

SUMMARY OF THE INVENTION

[0006] The embodiments disclosed herein generally relate to the creation of synbodies for Norovirus and to simple, practical, and broadly reactive methods to detect human Norovirus in relevant non-clinical sample matrices (e.g., food, water, and environment).

[0007] These and other aspects of the invention will be apparent upon reference to the following detailed description and figures. To that end, any patent and other documents cited herein are hereby incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 illustrates an overview of the identification of lead peptide candidates.

[0009] Figure 2 depicts a heat MAP of the Optimization of lead peptide candidates for nVLP GII.4.

[0010] Figure 3 depicts surface plasmon resonance screening of optimized peptides.

[0011] Figure 4 depicts nVLPGII.4 synbody construction.

[0012] Figure 5 depicts an ELISA screening of nVLPGII.4 synbodies.

[0013] Figure 6 depicts nVLP (GII.4) synbodies with K _D <50nM.

[0014] Figure 7 depicts the ELISA based detection of human Norovirus.

[0015] Figure 8 depicts the enrichment of GII.4 Minerva VLP captured from a dilute solution using synbody 93-93. DETAILED DESCRIPTION OF THE INVENTION

[0016] Embodiments of the invention relate to peptide affinity ligands (synbodies) for the detection of human Norovirus. As a first step to creating a synbody, a virus-like particle (VLP) was used as a Norovirus surrogate. The Norovirus surrogate VLP (nVLP) assembled from capsid structural subunits antigenically resembles native virus yet lacks viral nucleic acid, thereby rendering it non-infectious. The nVLP can be produced in a variety of known prokaryotic and eukaryotic expression systems to provide an ample sample supply.

[0017] To engineer peptide affinity ligands for nVLP, peptides specific for nVLP were identified by screening cell lysate from baculovirus expression and transgenic tobacco expression of nVLP (type GII.4 Minerva strain) against a library of 10,000 20mer peptides of random sequences in microarray format. Three lead peptides were identified. With an aim to detect very low amount of virus coat protein present in complex mixture, we focused on improving the affinity and specificity of the identified lead peptides for nVLP GII.4 (Figs. 1 and 2).

[0018] For this, nine amino acids (Y, A, D, S, K, N, V, W, E) were selected and an amino acid point variant peptide library for each selected lead peptide was designed. These peptides (408 in all) were printed on microarrays using similar sulfhydryl chemistry as used in the 10,000 peptide microarrays and screened against nVLP GII.4. After amino acid substitutions for increased binding affinity for nVLP GII.4 were identified, a library of optimized peptides (96) was created by the addition of 5-7 amino acid combinations.

[0019] These 96 peptides were synthesized and tested unpurified against GII.4 via surface plasmon resonance (SPR), a sample of which is shown in Fig. 3. Peptides candidates were analyzed and selected for improved binding and slower dissociation rate. Finally, a list of 10 peptide candidates with improved binding and slower dissociation rates were chosen to construct bivalent peptide affinity reagents (synbodies). [0020] Table-1 Selected Peptides candidates for Synbody Construction

S¾ f¾ iid« Selected Fspi fc A % k,

1 nVLP-lWT

LLYNKTFPHGRWSPSYPGSC 71.5 25% 7.85E-03

2 nVLP-2WT

DWARSNTSRSMDFNLGWGSC 2.5 - 2.33E-02

3 Mut Pe tide-81

v AWARSN SRS AFNLGWGSC 127.9 45% 4.55E-03

4 Mut Pept ⁱ de-60 _{DWARKmiKRKMNFNLGWGSC 1 34 44} o _/o 4.87E-03

5 Mut Peptide-53

^F VWARKNNKR KDFNAGWGSC 188.8 51% 4.08E-03

6 Mut Pept ⁱ de-78 _{SWARSNNKRSKAFNL} GWGSC 168.8 46% 4.31E-03

7 nVLP-6WT

RWHRVDLRSHTELPRYIGSC 175.7 37% 5.13E-03

8 Mut Peptide-92

RWHRV LRSHTELNRYIGSC 229.5 57% 3.55E-03

9 Mut Peptide-93

^V RWVRVKLRSHTELNRYIGSC 274.2 60% 3.32E-03

10 Mut Peptide-94

¹ RWVRVKLRSHTKLNRYIGSC 358.2 63% 2.94E-03

[0021] Construction of Peptide affinity Reagents (Synbody) for nVLP GII.4: Two scaffolds (scaffold- 1571 and scaffold-MAP-2) with maleimide functional groups were selected for synbody construction (see, for example, Fig. 4). Peptide candidates (Table 1) were constrained on two scaffolds via sulfhydryl coupling. A total of 53 synbody conjugation reactions were carried out on two different scaffold types and 98 synbodies were recovered after HPLC purification. Synbodies were characterized by a matrix- assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF) and tested against purified GII.4 nVLP binding in a direct ELISA assay (Fig. 5). Table 2 and Fig. 6 show synbodies with K _D <50 nM. 022] Table-2 nVLP (GII.4) Synbodies with K _D <50nM

nVLP Synbodies for GII.4

S. No. Synbody Synbody Sequence i Scaffold ELISA KD

1 i nVLP6-6-1571 RWH VDLRSHTELPRYIGSC-RWH VDLRSHTELPRYIGSC-1571 1571 2nM

2 : nVLP6-53-1571 RWHRVDLRSHTELPRYIGSC-VWARKNNKRKKDFNAGWGSC-1571 1571 InM

3 : nVLP6-78-1571 RWH RVDLRSHTE LPRYIGSC-SWARSN N KRSKAFN LGWGSC-1571 1571 2nM

4 ; nVLP6-92-1571 RWHRVDLRSHTELPRYIGSC-RWHRVKLRSHTELNRYIGSC-1571 1571 2nM

5 i nVLP6-60-1571 RWHRVDLRSHTELPRYIGSC-DWARKNNKRKMNFNLGWGSC-1571 1571 3nM

6 : nVLP6-81-1571 RWH RVDLRSHTE LPRYIGSC-RWHRVDLRSHTELPRYIGSC-1571 1571 3nM

7 ; nVLP6-94-1571 RWH RVDLRSHTE LPRYIGSC-RWHRVDLRSHTELPRYIGSC-1571 1571 InM

8 i nVLP2-53-1571 i DWARSNTSRSMDFNLGWGSC-VWARKNNKRKKDFNAGWGSC-1571 1571 20nM

9 : nVLP2-78-1571 i DWARSNTSRSM DFN LGWGSC-SWARSN N KRSKAFN LGWGSC-1571 1571 25nM

10 ; nVLP2-92-1571 DWARSNTSRSMDFNLGWGSC-RWHRVKLRSHTELNRYIGSC-1571 1571 15nM

11 i nVLP2-60-1571 : DWARSNTSRSM DFN LGWGSC-DWARKNNKRKMN FN LGWGSC-1571 1571 ΙΟηΜ

12 : nVLP2-81-1571 ; DWARSNTSRSM DFN LGWGSC-AWARSNNSRSKAFN LGWGSC-1571 1571 40nM

13 : nVLP2-92-1571 DWARSNTSRSM DFN LGWGSC-RWHRVKLRSHTELNRYIGSC-1571 ΙΟηΜ

14 ; nVLP2-93-1571 DWARSNTSRSM DFN LGWGSC-RWVRVKLRSHTELNRYIGSC-1571 5nM

15 i nVLP2-94-1571 DWARSNTSRSM DFN LGWGSC-RWVRVKLRSHTKLNRYIGSC-1571 1571 5nM

16 : nVLPl-53-1571 i LLYNKTFPHGRWSPSYPGSC-VWARKNNKRKKDFNAGWGSC-1571 1571 19nM

17 ; nVLPl-55-1571 LLYNKTFPHGRWSPSYPGSC-VWARKNNSRSKDFNAGWGSC-1571 1571 7nM

18 i nVLPl-72-1571 LLYNKTFPHGRWSPSYPGSC-SWARSNNSRSM DFN LGWGSC-1571 1571 15nM

19 : nVLPl-93-1571 LLYNKTFPHGRWSPSYPGSC-RWVRVKLRSHTELNRYIGSC-1571 1571 4nM

20 ; nVLPl-94-1571 LLYNKTFPHGRWSPSYPGSC-RWVRVKLRSHTKLNRYIGSC-1571 1571 6nM

21 i nVLP60-60-1571 ; DWARKNNKRKMNFNLGWGSC-DWARKNNKRKMN FN LGWGSC-1571 1571 2nM

22 : nVLP81-81-1571 i AWARSNNSRSKAFNLGWGSC-AWARSNNSRSKAFN LGWGSC-1571 1571 3nM

23 ; nVLP93-93-1571 RWVRVKLRSHTELNRYIGSC-RWVRVKLRSHTELNRYIGSC-1571 1571 4nM

24 i nVLP78-78-1571 i SWARSNN KRSKAFN LGWGSC-SWARSN NKRSKAFN LGWGSC-1571 1571 5nM

25 : nVLP92-92-1571 RWHRVKLRSHTELNRYIGSC-RWHRVKLRSHTELNRYIGSC-1571 1571 3nM

26 : nVLP94-94-1571 RWVRVKLRSHTKLNRYIGSC-RWVRVKLRSHTKLNRYIGSC-1571 1571 InM

27 ; nVLP53-53-1571 : VWARKNNKRKKDFNAGWGSC-VWARKNNKRKKDFNAGWGSC-1571 1571 InM

28 i nVLP55-55-1571 : VWARKNNSRSKDFNAGWGSC-VWARKNNSRSKDFNAGWGSC-1571 1571 9nM

29 : nVLPl-l-MAP2 LLYNKTFPHGRWSPSYPGSC-LLYNKTFPHGRWSPSYPGSC-MAP2 : MAP 2 4nM

30 ; nVLPl-53-MAP2 LLYNKTFPHGRWSPSYPGSC-LLYNKTFPHGRWSPSYPGSC-MAP2 ; MAP 2 2nM

31 i nVLPl-60-MAP2 LLYNKTFPHGRWSPSYPGSC-LLYNKTFPHGRWSPSYPGSC-MAP2 i MAP 2 5nM

32 : nVLPl-78-MAP2 LLYNKTFPHGRWSPSYPGSC-LLYNKTFPHGRWSPSYPGSC-MAP2 i MAP 2 7nM

33 ; nVLPl-81-MAP2 LLYNKTFPHGRWSPSYPGSC-LLYNKTFPHGRWSPSYPGSC-MAP2 ; MAP 2 7nM

34 ; nVLPl-92-MAP2 LLYNKTFPHGRWSPSYPGSC-LLYNKTFPHGRWSPSYPGSC-MAP2 ; MAP 2 8nM

35 i nVLPl-94-MAP2 LLYNKTFPHGRWSPSYPGSC-LLYNKTFPHGRWSPSYPGSC-MAP2 i MAP 2 InM

36 ; nVLP2-53-MAP2 ; DWARSNTSRSMDFNLGWGSC-VWARKNNKRKKDFNAGWGSC-MAP2 i MAP 2 5nM 37 i nVLP2-92-MAP2 I DWARSNTSRSM DFN LGWGSC-RWHRVKLRSHTELNRYIGSC-MAP2 i MAP 2 34nM

38 : nVLP6-6MAP2 RWHRVDLRSHTELPRYIGSC-RWHRVDLRSHTELPRYIGSC-MAP2 : MAP 2 39nM

39 ; nVLP6-53-MAP2 i RWHRVDLRSHTELPRYIGSC-VWARKNNKRKKDFNAGWGSC-MAP2 ; MAP 2 OnM 40 i nVLP6-81-MAP2 : RWHRVDLRSHTELPRYIGSC-AWARSNNSRSKAFNLGWGSC-MAP2 i MAP 2 40nM

41 : nVLP6-92-MAP2 RWHRVDLRSHTELPRYIGSC-RWHRVKLRSHTELNRYIGSC-MAP2 : MAP 2 33nM

42 : nVLP6-93-MAP2 RWHRVDLRSHTELPRYIGSC-RWVRVKLRSHTELNRYIGSC-MAP2 : MAP 2 50nM

43 ; nVLP6-94-MAP2 RWHRVDLRSHTELPRYIGSC-RWVRVKLRSHTKLNRYIGSC-MAP2 ; MAP 2 15nM [0023] ELISA detection. A direct ELISA method for detection of HuNoV also has been developed. Stool samples containing HuNoV (GII.3, GII.4, or no NoV) were coated onto an ELISA plate and detected with a fixed concentration of the candidate synbody. Initial results clearly indicate that the synbody performs similarly to a polyclonal antibody raised against the GII.4 strain (Figure 7).

[0024] As shown in Figure 8 and Table 3, enrichment of either GII.4 Minerva or both GII.4 Minerva and GII.4 Sydney# occurs for synbodies 6-6, 92-92, 93-93, and 94-94 from Table 2.

Table 3: Enrichment of two different Norovirus VLPs captured from a dilute solution.

[0025] In view of the above, a series of affinity agents for the detection of Norovirus have been developed. These synbodies can be used for detection of Norovirus or in capture assays for Norovirus concentration or enrichment. These Norovirus detecting synbodies can be coupled with filtration procedures, which may be used to remove low levels of viruses present in naturally contaminated surfaces or samples.

[0026] The claims are not intended to be limited to the embodiments and examples described herein.