Zika Virus Assay Systems

• US 20190317094A1
• Filed: 06/14/2019
• Published: 10/17/2019
• Est. Priority Date: 10/29/2016
• Status: Active Grant

• Current Assignee: University of Miami
• Original Assignee: University of Miami
• Inventors: Daunert, Sylvia, Deo, Sapna K., Dikici, Emre, Mascini, Marcello Mascini, Pawley, Devon
• Granted Patent: US 11,249,082 B2
• Time in Patent Office: Days
• CPC Class Codes: G01N 2333/185 Flaviviruses or Group B arboviruses, e.g. yellow fever virus, japanese encephalitis, tick-borne encephalitis, dengue
• CPC Class Codes: G01N 2469/10 Detection of antigens from microorganism in sample from host
• CPC Class Codes: G01N 33/54388 based on lateral flow
• CPC Class Codes: G01N 33/558 using diffusion or migration of antigen or antibody immunochromatographic test strips G01N33/54387
• CPC Class Codes: G01N 33/56983 Viruses

Abstract

Provided herein are Zika virus (ZIKV) binding constructs, e.g., antibodies and antigen-binding fragments thereof and antibody mimetics, as well as related conjugates, polypeptides, nucleic acids, expression vectors, host cells, kits, and assay systems. Methods detecting ZIKV infection and/or ZIKV exposure and/or ZIKV immunity are provided.

Claims

1. An assay system comprising a porous matrix comprising at least three zones, Zone A, Zone B, and Zone C, wherein

Zone A comprises a binding construct that binds to a Zika virus (ZIKV) protein and does not bind to a Dengue virus (DENV) protein, wherein the binding construct is not bound to a Zika virus,

Zone B comprises a binding construct that binds to a ZIKV protein and does not bind to a DENV protein, wherein the binding construct is bound to a Zika virus, and

Zone C comprises a secondary antibody which binds the binding construct of Zone A and Zone B.

2. The assay system of claim 1, wherein (A) the porous matrix comprises nitrocellulose of polyvinylidene fluoride (PVDF);

(B) Zones A to C are arranged along a horizontal axis, wherein each of Zones A to C is flanked by an intervening zone of the porous matrix lacking the binding construct;

(C) the method further comprises a sample application pad, a particle conjugate zone, a wick, and/or a backing;

or (D) a combination thereof.

5. The assay system of claim 2, part (C), wherein the porous matrix, the sample application pad, the particle conjugate zone, and the wick are arranged along a horizontal axis, optionally, wherein the horizontal axis is the same as the horizontal axis along which Zones A to C are arranged, optionally, arranged such that the sample application pad and the wick are located at opposite ends of the assay system along the horizontal axis, wherein the particle conjugate zone is flanked by the sample application pad and the porous matrix and the porous matrix is flanked by the particle conjugate and the wick.

7. The assay system of claim 2, part (C), wherein (A) the backing is positioned below the porous matrix, the sample application pad, the particle conjugate zone, and the wick;

(B) the particle conjugate zone is bound to a conjugate comprising a binding construct that binds to a Zika virus (ZIKV) protein and does not bind to a Dengue virus (DENV) protein, bound to an element or polymer, optionally, wherein the element is a gold particle or the polymer is polystyrene;

(C) the sample application Dad comprises cellulose or glass fiber;

(D) the wick comprises nitrocellulose;

(E) each of Zone A and Zone B is bound to a binding construct.

13. The assay system of claim 1, wherein the binding construct does not bind to a protein of any one of DENV subtype 1, DENV subtype 2, DENV subtype 3, and DENV subtype 4, optionally, wherein the binding construct does not bind to any flavivirus other than ZIKV.

15. The assay system of claim 1, wherein (A) the ZIKV protein is from a ZIKV comprising the genome of GenBank Accession No. KU926309.1 (SEQ ID NO:

4);

(B) the binding construct is an antibody mimetic, optionally, a clamp peptide;

(C) the binding construct comprises the structure A₁-B-A₂ wherein A₁ is a first peptide arm that binds to a first binding site of a target protein, A₂ is a second peptide arm that binds to a second binding site of the target protein, and B is a bridge peptide which links A₁ to A₂, optionally, wherein the bridge peptide is about 5 to about 10 amino acids in length, further optionally, wherein (a) A comprises an amino acid sequence of any one of SEQ ID NOs;

24-31, (b) A₂ comprises an amino acid sequence of any one of SEQ ID NOs;

16-23, or (c) a combination thereof.

20. The assay system of claim 15, part (C), wherein A₁ comprises an amino acid sequence of SEQ ID NO:

26 and A₂ comprises an amino acid sequence of SEQ ID NO;

18 or A comprises an amino acid sequence of SEQ ID NO;

25 and A₂ comprises an amino acid sequence of SEQ ID NO;

21 or A₁ comprises an amino acid sequence of SEQ ID NO;

26 and A₂ comprises an amino acid sequence of SEQ ID NO;

23.

23. The assay system of claim 15, part (C), wherein (A) the bridge peptide comprises the amino acid sequence of SEQ ID NO:

32, (B) the binding construct comprises an amino acid sequence of SEQ ID NO;

10, 13, or 14, or (C) a combination thereof.

37. A method of detecting a Zika virus (ZIKV) infection and ZIKV immunity in a subject, comprising adding a sample obtained from a subject to the assay system according to claim 1, wherein, when the assay system exhibits a single band in Zone C, the subject is determined as having neither a ZIKV infection nor ZIKV immunity, when the assay system exhibits a band in each of Zone A and Zone B, the subject is determined as having both a ZIKV infection and ZIKV immunity, and when the assay system exhibits a band in Zone B and a band is absent in Zone A, the subject is determined as not having a ZIKV infection but having ZIKV immunity.

3. (canceled)

4. (canceled)

6. (canceled)

8. (canceled)

9. (canceled)

10. (canceled)

11. (canceled)

12. (canceled)

14. (canceled)

16. (canceled)

17. (canceled)

18. (canceled)

19. (canceled)

21. (canceled)

22. (canceled)

24. (canceled)

25. (canceled)

26. A binding construct comprising the structure A₁-B-A₂ wherein A₁ is a first peptide arm that binds to a first binding site of a target protein, A₂ is a second peptide arm that binds to a second binding site of the target protein, and B is a bridge peptide which links A₁ to A₂, optionally, wherein the bridge peptide is about 5 to about 10 amino acids in length.

28. The binding construct of claim 26, wherein (a) A₁ comprises an amino acid sequence of any one of SEQ ID NOs:

24-31, (b) A₂ comprises an amino acid sequence of any one of SEQ ID NOs;

16-23, or (c) a combination thereof, optionally, wherein A₁ comprises an amino acid sequence of SEQ ID NO;

26 and A₂ comprises an amino acid sequence of SEQ ID NO;

18 or A₁ comprises an amino acid sequence of SEQ ID NO;

25 and A₂ comprises an amino acid sequence of SEQ ID NO;

21 or A₁ comprises an amino acid sequence of SEQ ID NO;

26 and A₂ comprises an amino acid sequence of SEQ ID NO;

23.

32. The binding construct of claim 26, wherein the bridge peptide comprises the amino acid sequence of SEQ ID NO:

32 or the binding construct comprises an amino acid sequence of SEQ ID NO;

10, 13, or 14.

27. (canceled)

29. (canceled)

30. (canceled)

31. (canceled)

33. (canceled)

34. (canceled)

35. (canceled)

36. (canceled)

38-50. -50. (canceled)