# US 8,771,710 B2 · Fusion proteins and their use in the diagnosis and treatment of leishmaniasis

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- Number: US 8,771,710 B2
- Document ID: US8771710B2
- Status: Granted
- Kind: B2
- Application number: 13/560,565
- Filed: 2012-07-27
- Published: 2013-03-21
- Granted: 2014-07-08
- Earliest claimed: 2008-07-03
- Assignees: Infectious Disease Research Institute
- Applicants: Ajay Bhatia; Steven G. Reed
- Inventors: Ajay Bhatia; Steven G. Reed
- Web page: https://patentgrep.com/patent/US8771710B2

## CPC

- C07K 14/44: from protozoa
- A61P 1/16: for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
- A61P 3/04: Anorexiants; Antiobesity agents
- A61P 33/02: Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
- C07K 16/20: from protozoa
- G01N 33/56905: {Protozoa}
- G01N 33/6893: {related to diseases not provided for elsewhere}
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- A61K 39/00: Medicinal preparations containing antigens or antibodies (materials for immunoassay G01N33/53)
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- Y10S 435/81: Packaged device or kit
- Y10S 435/975: Kit
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y10S: TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS

## Abstract

The present invention relates generally to a fusion protein made from a synthetic gene construct comprising of elements derived from the Leishmania antigens K26, K39, and K9. The fusion protein is particularly useful in the diagnosis of leishmaniasis, particularly visceral leishmaniasis in animals such as humans and dogs.

## Claims

### Claim 1

A diagnostic kit for detecting asymptomatic or sub-clinical visceral leishmaniasis in a biological sample selected from the group consisting of sera, blood, and saliva, comprising:
(a) an fusion polypeptide comprising Leishmania antigens K26, K39, and K9, wherein the Leishmania antigen K26 comprises a sequence having at least 98% identity to SEQ ID NO:5, the Leishmania antigen K39 comprises a sequence having at least 98% identity to SEQ ID NO:6, and the Leishmania antigen K9 comprises a sequence having at least 98% identity to SEQ ID NO:7; and
(b) a detection reagent.

### Claim 2 (depends on 1)

The kit of claim 1, wherein the detection reagent comprises a reporter group conjugated to a binding agent.

### Claim 3 (depends on 2)

The kit of claim 2, wherein the binding agent is selected from the group consisting of anti-immunoglobulin, Protein G, Protein A and lectins.

### Claim 4 (depends on 2)

The kit of claim 2, wherein the reporter group is selected from the group consisting of radioisotopes, fluorescent groups, luminescent groups, enzymes, biotin and dye particles.

### Claim 5 (depends on 1)

The kit of claim 1, wherein the Leishmania antigen K26 comprises a sequence having at least 99% identity to SEQ ID NO:5, the Leishmania antigen K39 comprises a sequence having at least 99% identity to SEQ ID NO:6, and the Leishmania antigen K9 comprises a sequence having at least 99% identity to SEQ ID NO:7.

### Claim 6 (depends on 1)

The kit of claim 1, wherein the fusion polypeptide is bound to a solid support.

### Claim 7 (depends on 6)

The kit of claim 6, wherein the solid support comprises nitrocellulose, latex or a plastic material.

### Claim 8

A diagnostic kit for identifying a patient afflicted with asymptomatic or sub-clinical visceral leishmaniasis that is likely to develop acute visceral leishmaniasis, comprising:
(a) an fusion polypeptide comprising Leishmania antigens K26, K39, and K9, wherein the Leishmania antigen K26 comprises a sequence having at least 98% identity to SEQ ID NO:5, the Leishmania antigen K39 comprises a sequence having at least 98% identity to SEQ ID NO:6, and the Leishmania antigen K9 comprises a sequence having at least 98% identity to SEQ ID NO:7;
(b) a polypeptide comprising an amino acid sequence as set forth in SEQ ID NOs:10 or 11; and
(c) a detection reagent.

### Claim 9 (depends on 8)

The kit of claim 8, wherein the detection reagent comprises a reporter group conjugated to a binding agent.

### Claim 10 (depends on 9)

The kit of claim 9, wherein the binding agent is selected from the group consisting of anti-immunoglobulin, Protein G, Protein A and lectins.

### Claim 11 (depends on 9)

The kit of claim 9, wherein the reporter group is selected from the group consisting of radioisotopes, fluorescent groups, luminescent groups, enzymes, biotin and dye particles.

### Claim 12 (depends on 8)

The kit of claim 8, wherein the Leishmania antigen K26 comprises a sequence having at least 99% identity to SEQ ID NO:5, the Leishmania antigen K39 comprises a sequence having at least 99% identity to SEQ ID NO:6, and the Leishmania antigen K9 comprises a sequence having at least 99% identity to SEQ ID NO:7.

### Claim 13 (depends on 8)

The kit of claim 8, wherein the fusion polypeptide of (a) and the polypeptide of (b) are each bound to a separate solid support.

### Claim 14 (depends on 13)

The kit of claim 13, wherein the solid supports comprise nitrocellulose, latex or a plastic material.

### Claim 15

An isolated fusion polypeptide comprising (i) a Leishmania antigen K26 comprising a sequence having at least 98% identity to SEQ ID NO:5; (ii) a Leishmania antigen K39 comprising a sequence having at least 98% identity to SEQ ID NO:6; and (iii) a Leishmania antigen K9 comprising a sequence having at least 98% identity to SEQ ID NO:7.

### Claim 16 (depends on 15)

The fusion polypeptide of claim 15, wherein the fusion polypeptide is bound to solid support.

### Claim 17 (depends on 16)

The fusion polypeptide of claim 16, wherein the solid support comprises nitrocellulose, latex or a plastic material.

### Claim 18

An isolated fusion polypeptide comprising an amino acid sequence having at least 95% identity to amino acid residues 10-262 of SEQ ID NO:8.

### Claim 19 (depends on 18)

The fusion polypeptide of claim 18, wherein the fusion polypeptide further comprises an N-terminal amino acid sequence of MHHHHHHTS (SEQ ID NO:21).

### Claim 20 (depends on 18)

The fusion polypeptide of claim 18, wherein the fusion polypeptide is bound to a solid support.

### Claim 21 (depends on 20)

The fusion polypeptide of claim 20, wherein the fusion polypeptide comprises the sequence set forth in amino acid residues 10-262 of SEQ ID NO:8.

### Claim 22 (depends on 20)

The fusion polypeptide of claim 20, wherein the solid support comprises nitrocellulose, latex or a plastic material.