# Patent

## Title
Synbodies for detection of human norovirus

## Abstract

Synbodies specific for Norovirus and coupled with a substrate provide Norovirus binding and detection platforms (FIG. 1). A Norovirus capturing platform, comprising one or more synbodies selected from the group consisting of synbodies 6-6, 92-92, 93-93, and 94-94 coupled to a substrate, has been found to found to bind with either GII.4 Minerva or both GII.4 Minerva and GII.4 Sydney# strains of norovirus.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This Application is a 371 application of PCT/US2014/024932 filed Mar. 12, 2014, which claims benefit of U.S. Provisional Patent Application No. 61/780,102, filed Mar. 13, 2013, which are incorporated herein by reference in their entirety.


STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
This invention was made with government support under 2011-68003-30395 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
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.
Norovirus is classified as “NIAID 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 60 C 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 (48 hrs) causes vomiting, stomach pain and diarrhea. Also, it can cause chronic infections in transplant recipient.
Of the five geno-groups of Norovirus, GI, GII, 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
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).
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
FIG. 1 illustrates an overview of the identification of lead peptide candidates.
FIG. 2 depicts a heat MAP of the Optimization of lead peptide candidates for nVLP GII.4.
FIG. 3 depicts surface plasmon resonance screening of optimized peptides.
FIG. 4 depicts nVLPGII.4 synbody construction.
FIG. 5 depicts an ELISA screening of nVLPGII.4 synbodies.
FIG. 6 depicts nVLP (GII.4) synbodies with KD<50 nM.
FIG. 7 depicts the ELISA based detection of human Norovirus.
FIG. 8 depicts the enrichment of GII.4 Minerva VLP captured from a dilute solution using synbody 93-93.



DETAILED DESCRIPTION OF THE INVENTION
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.
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).
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.
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).







TABLE-1







Selected Peptides candidates for Synbody Construction















Avg




SEQ


Binding




ID
Peptide

(Spot
%
kd-1


NO.
Code
Selected Peptides
1/2)
Stability
(sec)





 1
nVLP-1WT
LLYNKTFPHGRWSPSYPGSC
 71.5
25%
7.85E-03


 


 2
nVLP-2WT
DWARSNTSRSMDFNLGWGSC
  2.5
—
2.33E-02


 


 3
Mut Peptide-81
AWARSNNSRSKAFNLGWGSC
127.9
45%
4.55E-03


 


 4
Mut Peptide-60
DWARKNNKRKMNFNLGWGSC
134.3
44%
4.87E-03


 


 5
Mut Peptide-53
VWARKNNKRKKDFNAGWGSC
188.8
51%
4.08E-03


 


 6
Mut Peptide-78
SWARSNNKRSKAFNLGWGSC
168.8
46%
4.31E-03


 


 7
nVLP-6WT
RWHRVDLRSHTELPRYIGSC
175.7
37%
5.13E-03


 


 8
Mut Peptide-92
RWHRVKLRSHTELNRYIGSC
229.5
57%
3.55E-03


 


 9
Mut Peptide-93
RWVRVKLRSHTELNRYIGSC
274.2
60%
3.32E-03


 


10
Mut Peptide-94
RWVRVKLRSHTKLRYIGSC
358.2
63%
2.94E-03









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 KD<50 nM.







TABLE-2 







nVLP (GII.4) Synbodies with KD < 50 nM


nVLP Synbodies for GII.4











SEQ ID






NO.
Synbody
Synbody Sequence
Scaffold
ELISA KD





11
nVLP6-6-1571
RWHRVDLRSHTELPRYIGSC-RWHRVDLRSHTELPRYIGSC-1571
1571
 2 nM


 


12
nVLP6-53-1571
RWHRVDLRSHTELPRYIGSC-VWARKNNKRKKDFNAGWGSC-1571
1571 
 1 nM


 


13
nVLP6-78-1571
RWHRVDLRSHTELPRYIGSC-SWARSNNKRSKAFNLGWGSC-1571
1571
 2 nM


 


14
nVLP6-92-1571
RWHRVDLRSHTELPRYIGSC-RWHRVKLRSHTELNRYIGSC-1571
1571
 2 nM


 


15
nVLP6-60-1571
RWHRVDLRSHTELPRYIGSC-DWARKNNKRKMNFNLGWGSC-1571
1571
 3 nM


 


16
nVLP6-81-1571
RWHRVDLRSHTELPRYIGSC-RWHRVDLRSHTELPRYIGSC-1571
1571
 3 nM


 


17
nVLP6-94-1571
RWHRVDLRSHTELPRYIGSC-RWHRVDLRSHTELPRYIGSC-1571
1571
 1 nM


 


18
nVLP2-53-1571
DWARSNTSRSMDFNLGWGSC-VWARKNNKRKKDFNAGWGSC-1571
1571
20 nM


 


19
nVLP2-78-1571
DWARSNTSRSMDFNLGWGSC-SWARSNNKRSKAFNLGWGSC-1571
1571
25 nM


 


20
nVLP2-92-1571
DWARSNTSRSMDFNLGWGSC-RWHRVKLRSHTELNRYIGSC-1571
1571
15 nM


 


21
nVLP2-60-1571
DWARSNTSRSMDFNLGWGSC-DWARKNNKRKMNFNLGWGSC-1571
1571
10 nM


 


22
nVLP2-81-1571
DWARSNTSRSMDFNLGWGSC-AWARSNNSRSKAFNLGWGSC-1571
1571
40 nM


 


23
nVLP2-92-1571
DWARSNTSRSMDFNLGWGSC-RWHRVKLRSHTELNRYIGSC-1571

10 nM


 


24
nVLP2-93-1571
DWARSNTSRSMDFNLGWGSC-RWVRVKLRSHTELNRYIGSC-1571

 5 nM


 


25
nVLP2-94-1571
DWARSNTSRSMDFNLGWGSC-RWVRVKLRSHTKLNRYIGSC-1571
1571
 5 nM


 


26
nVLP1-53-1571
LLYNKTFPHGRWSPSYPGSC-VWARKNNKRKKDFNAGWGSC-1571
1571
19 nM


 


27
nVLP1-55-1571
LLYNKTFPHGRWSPSYPGSC-VWARKNNSRSKDFNAGWGSC-1571
1571
 7 nM


 


28
nVLP1-72-1571
LLYNKTFPHGRWSPSYPGSC-SWARSNNSRSMDFNLGWGSC-1571
1571
15 nM


 


29
nVLP1-93-1571
LLYNKTFPHGRWSPSYPGSC-RWVRVKLRSHTELNRYIGSC-1571
1571
 4 nM


 


30
nVLP1-94-1571
LLYNKTFPHGRWSPSYPGSC-RWVRVKLRSHTKLNRYIGSC-1571
1571
 6 nM


 


31
nVLP60-60-1571
DWARKNNKRKMNFNLGWGSC-DWARKNNKRKMNFNLGWGSC-1571
1571
 2 nM


 


32
nVLP81-81-1571
AWARSNNSRSKAFNLGWGSC-AWARSNNSRSKAFNLGWGSC-1571
1571
 3 nM


 


33
nVLP93-93-1571
RWVRVKLRSHTELNRYIGSC-RWVRVKLRSHTELNRYIGSC-1571
1571
 4 nM


 


34
nVLP78-78-1571
SWARSNNKRSKAFNLGWGSC-SWARSNNKRSKAFNLGWGSC-1571
1571
 5 nM


 


35
nVLP92-92-1571
RWHRVKLRSHTELNRYIGSC-RWHRVKLRSHTELNRYIGSC-1571
1571
 3 nM


 


36
nVLP94-94-1571
RWVRVKLRSHTKLNRYIGSC-RWVRVKLRSHTKLNRYIGSC-1571
1571
 1 nM


 


37
nVLP53-53-1571
VWARKNNKRKKDFNAGWGSC-VWARKNNKRKKDFNAGWGSC-1571
1571
 1 nM


 


38
nVLP55-55-1571
VWARKNNSRSKDFNAGWGSC-VWARKNNSRSKDFNAGWGSC-1571
1571
 9 nM


 


39
nVLP1-1-MAP2
LLYNKTFPHGRWSPSYPGSC-LLYNKTFPHGRWSPSYPGSC-MAP2
MAP-2
 4 nM


 


40
nVLP1-53-MAP2
LLYNKTFPHGRWSPSYPGSC-LLYNKTFPHGRWSPSYPGSC-MAP2
MAP-2
 2 nM


 


41
nVLP1-60-MAP2
LLYNKTFPHGRWSPSYPGSC-LLYNKTFPHGRWSPSYPGSC-MAP2
MAP-2
 5 nM


 


42
nVLP1-78-MAP2
LLYNKTFPHGRWSPSYPGSC-LLYNKTFPHGRWSPSYPGSC-MAP2
MAP-2
 7 nM


 


43
nVLP1-81-MAP2
LLYNKTFPHGRWSPSYPGSC-LLYNKTFPHGRWSPSYPGSC-MAP2
MAP-2
 7 nM


 


44
nVLP1-92-MAP2
LLYNKTFPHGRWSPSYPGSC-LLYNKTFPHGRWSPSYPGSC-MAP2
MAP-2
 8 nM


 


45
nVLP1-94-MAP2
LLYNKTFPHGRWSPSYPGSC-LLYNKTFPHGRWSPSYPGSC-MAP2
MAP-2
 1 nM


 


46
nVLP2-53-MAP2
DWARSNTSRSMDFNLGWGSC-VWARKNNKRKKDFNAGWGSC-MAP2
MAP-2
 5 nM


 


47
nVLP2-92-MAP2
DWARSNTSRSMDFNLGWGSC-RWHRVKLRSHTELNRYIGSC-MAP2
MAP-2
34 nM


 


48
nVLP6-6MAP2
RWHRVDLRSHTELPRYIGSC-RWHRVDLRSHTELPRYIGSC-MAP2
MAP-2
39 nM


 


49
nVLP6-53-MAP2
RWHRVDLRSHTELPRYIGSC-VWARKNNKRKKDFNAGWGSC-MAP2
MAP-2
 0 nM


 


50
nVLP6-81-MAP2
RWHRVDLRSHTELPRYIGSC-AWARSNNSRSKAFNLGWGSC-MAP2
MAP-2
40 nM


 


51
nVLP6-92-MAP2
RWHRVDLRSHTELPRYIGSC-RWHRVKLRSHTELNRYIGSC-MAP2
MAP-2
33 nM


 


52
nVLP6-93-MAP2
RWHRVDLRSHTELPRYIGSC-RWVRVKLRSHTELNRYIGSC-MAP2
MAP-2
50 nM









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 (FIG. 7).
As shown in FIG. 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.










VP1 Protein
Enrichment Factor from 1 ng/μL solution













Identity
Syn 6-6
Syn 92-92
Syn 93-93
Syn 94-94
















GII.4
100%
16
3
10
7


Minerva


GII.4
 95%
n.e.
n.e.
3
3


Sydney#









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

## Claims

1. A norovirus detection agent selected from the group consisting of synbodies 6-6 (SEQ ID NO:11), 92-92 (SEQ ID NO:35), 93-93 (SEQ ID NO:33), and 94-94 (SEQ ID NO:36).

2. A Norovirus capturing platform, comprising one or more synbodies selected from the group consisting of synbodies 6-6 (SEQ ID NO:11), 92-92 (SEQ ID NO:35), 93-93 (SEQ ID NO:33), and 94-94 (SEQ ID NO:36) coupled to a substrate.

3. A method for binding Norovirus, comprising the steps of coupling one or more synbodies of claim 1 to a substrate and contacting said substrate with a sample.

4. The norovirus detection agent of claim 1, wherein said agent is synbody 6-6 (SEQ ID NO:11).

5. The norovirus detection agent of claim 1, wherein said agent is synbody 92-92 (SEQ ID NO:35).

6. The norovirus detection agent of claim 1, wherein said agent is synbody 93-93 (SEQ ID NO:33).

7. The norovirus detection agent of claim 1, wherein said agent is synbody 94-94 (SEQ ID NO:36).

8. The method for binding Norovirus of claim 3, wherein said agent is synbody 6-6 (SEQ ID NO:11).

9. The method for binding Norovirus of claim 3, wherein said agent is synbody 92-92 (SEQ ID NO:35).

10. The method for binding Norovirus of claim 3, wherein said agent is synbody 93-93 (SEQ ID NO:33).

11. The method for binding Norovirus of claim 3, wherein said agent synbody 94-94 (SEQ ID NO:36).