# US 10,370,416 B2 · Dual purpose universal influenza vaccine confers protective immunity against anthrax

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- Number: US 10,370,416 B2
- Document ID: US10370416B2
- Status: Granted
- Kind: B2
- Application number: 15/446,315
- Filed: 2017-03-01
- Published: 2017-09-07
- Granted: 2019-08-06
- Earliest claimed: 2016-03-01
- Assignees: Texas Tech University System
- Applicants: Texas Tech University System
- Inventors: Mingtao Zeng; Maria T. Arevalo; Junwei Li
- Web page: https://patentgrep.com/patent/US10370416B2

## CPC

- C07K 14/005: from viruses
- A61K 39/07: Bacillus
- A61K 39/12: Viral antigens
- G01N 33/5023: {on expression patterns}
- G01N 33/5088: {of vertebrates}
- A61K 2039/543: {intranasal}
- A61K 2039/572: {cytotoxic response}
- A61K 2039/575: {humoral response}
- A61K 2039/70: {Multivalent vaccine}
- C07K 2319/40: containing a tag for immunodetection, or an epitope for immunisation
- C07K 2319/55: containing a fusion with a toxin, e.g. diphteria toxin
- C12N 2760/16122: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- C12N 2760/16134: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- C12N 2760/16143: viral genome or elements thereof as genetic vector
- C12N 2760/16162: by genetic engineering
- G01N 2333/11: Orthomyxoviridae, e.g. influenza virus
- G01N 2333/32: from Bacillus (G)
- 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)
- 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)
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention includes antigenic fusion proteins, nucleic acids encoding the fusion proteins and methods of making and using the same, wherein the fusion protein comprises three or more different influenza A ectodomains of Matrix Protein 2 (M2e); one or more stem regions of an influenza A hemagglutinin 2 (HA2) protein; and optionally an anthrax antigen, wherein the fusion protein is immunogenic across strains.

## Claims

### Claim 1

An antigenic fusion protein comprising:
three or more different influenza A ectodomains of matrix protein 2 (M2e) domains; one or more stem regions from a first influenza A hemagglutinin 2(HA2) protein domains; and an anthrax antigen, wherein the fusion protein is immunogenic across influenza strains.

### Claim 2 (depends on 1)

The fusion protein of claim 1, wherein the fusion protein further comprises, or is provided with, an adjuvant selected from at least one of cholera toxin B subunit, flagellin, human papillomavirus L1 or L2 protein, herpes simplex glycoprotein D (gD), complement C4binding protein, TL4 ligand, and 1L-1 beta, aluminum salt, Freund's complete or incomplete adjuvant, lysolecithin, pluronic polyols, polyanions, an oil-water emulsion, dinitrophenol, iscomatrix, and liposome polycation DNA particles.

### Claim 3 (depends on 1)

The fusion protein of claim 1, wherein influenza A ectodomains are selected from H1N1, H3N2, or H5N1.

### Claim 4 (depends on 1)

The fusion protein of claim 1, further comprises a stem region from a second influenza A hemagglutinin 2 (HA2) protein domain which is from a different strain than the first HA2 protein domain.

### Claim 5 (depends on 1)

The fusion protein of claim 1, further comprising one or more immunogenic adjuvants.

### Claim 6 (depends on 1)

The fusion protein of claim 1, further comprising one or more pharmaceutically acceptable excipients for intraperitoneal, subcutaneous, intranasal, intramuscular, oral, topical or transdermal administration.

### Claim 7 (depends on 1)

The fusion protein of claim 1, wherein the antigen further comprises at least one of a pharmaceutically acceptable diluent, excipient, carrier, solubilizing agent, emulsifying agent, or preservative.

### Claim 8 (depends on 1)

The fusion protein of claim 1, wherein the antigen comprises 4, 5, 6, 7, 8, 9, or 10 ectodomains of influenza A Matrix Protein 2 (M2e).

### Claim 9 (depends on 1)

The fusion protein of claim 1, wherein the antigen comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 stem region of an influenza A hemagglutinin 2 (HA2) protein.

### Claim 10 (depends on 1)

The fusion protein of claim 1, wherein the three or more ectodomains of influenza A Matrix Protein 2 (M2e) are separated by peptide linkers comprising one to 20 amino acids.

### Claim 11 (depends on 1)

The fusion protein of claim 1, wherein the antigen is a fusion protein that comprises amino acids encoded by nucleic acids having SEQ ID NOS.: 1, 2, 3, 4, 5 and 6.

### Claim 12 (depends on 1)

The fusion protein of claim 1, wherein the antigen is a fusion protein that comprises, in the following order, amino acids encoded by nucleic acids having SEQ ID NOS.: 1, 2, 3, 4, 5 and 6.

### Claim 13 (depends on 1)

The fusion protein of claim 1, wherein the antigen is a fusion protein that comprises amino acids encoded by nucleic acids having SEQ ID NOS.: 1, 2, 3, 4, 5 and 6 separated by one or more linkers.

### Claim 14 (depends on 1)

The fusion protein of claim 1, wherein the linkers have the sequences Ala-Ala-Ala or Gly-Ser.

### Claim 15 (depends on 1)

The fusion protein of claim 1, wherein the antigen is a fusion protein that comprises three or more amino acids peptides of SEQ ID NOS.: 11, 12, and 13, at least one peptide of amino acid sequence SEQ ID NOS.: 9 or 10, and a peptide of SEQ ID NO.: 14.

### Claim 16 (depends on 1)

The fusion protein of claim 1, wherein the fusion protein is, in order, SEQ ID NO.: 14, SEQ ID NOS.: 11, 12, and 13 in any order, and SEQ ID NOS.: 9 or 10.

### Claim 17 (depends on 1)

The fusion protein of claim 1, wherein the fusion protein is, in order, SEQ ID NOS.: 11, 12, and 13 in any order, and SEQ ID NOS.: 9 or 10.

### Claim 18 (depends on 1)

A nucleic acid encoding the fusion protein of claim 1.

### Claim 19

A method of making a mucosal vaccine against influenza A comprising:
combining two or more different influenza A ectodomains of Matrix Protein 2 (M2e); one or more stem regions from a first influenza A hemagglutinin 2 (HA2) protein; and an anthrax antigen into an antigen that is immunogenic across strains.

### Claim 20 (depends on 19)

The method of claim 19, wherein the vaccine further comprises, or is provided with, an adjuvant selected from at least one of anthrax protective antigen (PA), cholera toxin B subunit, flagellin, human papillomavirus L1 or L2 protein, herpes simplex glycoprotein D (gD), complement C4 binding protein, TL4 ligand, and 1L-1 beta, aluminum salt, Freund's complete or incomplete adjuvant, lysolecithin, pluronic polyols, polyanions, an oil-water emulsion, dinitrophenol, iscomatrix, and liposome polycation DNA particles.

### Claim 21 (depends on 19)

The method of claim 19, wherein the two or more different influenza ectodomains are selected from H1N1, H3N2, or H5N1.

### Claim 22 (depends on 19)

The method of claim 19, further comprising a second stem region of an influenza A hemagglutinin 2 (HA2) protein from a different influenza virus A strain than the first HA2 protein.

### Claim 23 (depends on 19)

The method of claim 19, further comprising one or more immunogenic adjuvants.

### Claim 24 (depends on 19)

The method of claim 19, further comprising one or more pharmaceutically acceptable excipients for intraperitoneal, subcutaneous, intranasal, intramuscular, oral, topical or transdermal administration.

### Claim 25 (depends on 19)

The method of claim 19, wherein the antigen further comprises at least one of a pharmaceutically acceptable diluent, excipient, carrier, solubilizing agent, emulsifying agent, or preservative.

### Claim 26 (depends on 19)

The method of claim 19, wherein the antigen comprises 3, 4, 5, 6, 7, 8, 9 or 10 ectodomains of influenza A Matrix Protein 2 (M2e).

### Claim 27 (depends on 19)

The method of claim 19, wherein the antigen comprises 3, 4, 5, 6, 7, 8, 9 or 10 stem region of an influenza A hemagglutinin 2 (HA2) protein.

### Claim 28 (depends on 19)

The method of claim 19, wherein the antigen comprises 3 ectodomains of influenza A Matrix Protein 2 (M2e) separated by peptide linkers comprising one to 20 amino acids.

### Claim 29 (depends on 19)

The method of claim 19, wherein the antigen is a fusion protein that comprises SEQ ID NOS.: 1, 2, 3, 4, 5 and 6.

### Claim 30 (depends on 19)

The method of claim 19, wherein the antigen is a fusion protein that comprises, in the following order, SEQ ID NOS.: 1, 2, 3, 4, 5 and 6.

### Claim 31 (depends on 19)

The method of claim 19, wherein the antigen is a fusion protein that comprises SEQ ID NOS.: 1, 2, 3, 4, 5 and 6 separated by one or more amino acid or peptide linkers.

### Claim 32 (depends on 19)

The method of claim 19, wherein the linkers have the sequences Ala-Ala-Ala or Gly-Ser.

### Claim 33 (depends on 19)

The method of claim 19, wherein the antigen is a fusion protein that comprises three or more amino acids peptides of SEQ ID NOS.: 11, 12, and 13, at least one peptide of amino acid sequence SEQ ID NOS.: 9 or 10, and a peptide of SEQ ID NO.: 14.

### Claim 34 (depends on 19)

The method of claim 19, wherein the fusion protein is, in order, SEQ ID NO.: 14, SEQ ID NOS.: 11, 12, and 13 in any order, and SEQ ID NOS.: 9 or 10.

### Claim 35 (depends on 19)

The method of claim 19, wherein the fusion protein is, in order, SEQ ID NOS.: 11, 12, and 13 in any order, and SEQ ID NOS.: 9 or 10.

### Claim 36

A method of evaluating a candidate drug believed to provide cross-immunity against Influenza A and anthrax, the method comprising:
(a) measuring the immune response from a set of patients suspected of having or being exposed to influenza A;
(b) administering a candidate drug to a first subset of the patients, and a placebo to a second subset of the patients, wherein the candidate drug comprises: two or more different influenza A ectodomains of Matrix Protein 2 (M2e); one or more stem regions of an influenza A hemagglutinin 2 (HA2) protein; and an anthrax antigen into a fusion protein that is immunogenic across strains;
(c) repeating step (a) after the administration of the candidate drug or the placebo; and
(d) determining if the candidate drug triggers an increase in the immune response against two or more influenza A strains and anthrax that is statistically significant as compared to any increase occurring in the second subset of patients, wherein a statistically significant increase indicates that the candidate drug is useful in treating two or more strains of influenza A and anthrax.

### Claim 37

An isolated immune response stimulating fusion protein against influenza A comprising:
two or more different influenza ectodomains of A Matrix Protein 2 (M2e); one or more stem regions of an influenza A hemagglutinin 2 (HA2) protein comprising SEQ ID NO.: 14, SEQ ID NOS.: 11, 12, and 13 in any order, and SEQ ID NOS.: 9 or 10 into a fusion protein that is immunogenic across strains.