# US 11,389,523 B2 · Vectors for eliciting immune responses to non-dominant epitopes in the hemagglutinin (HA) protein

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- Number: US 11,389,523 B2
- Document ID: US11389523B2
- Status: Granted
- Kind: B2
- Application number: 16/545,761
- Filed: 2019-08-20
- Published: 2020-06-18
- Granted: 2022-07-19
- Earliest claimed: 2018-08-20
- Assignees: Wisconsin Alumni Research Foundation (WARF)
- Applicants: Wisconsin Alumni Research Foundation (WARF)
- Inventors: Yoshihiro Kawaoka; Gabriele Neumann; Huihui Kong
- Web page: https://patentgrep.com/patent/US11389523B2

## CPC

- A61K 39/145: Orthomyxoviridae, e.g. influenza virus
- C07K 14/005: from viruses
- C07K 16/108: Orthomyxoviridae (F), e.g. influenza virus
- C07K 16/34: against blood group antigens
- C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00)
- C12N 15/86: Viral vectors
- A61K 2039/521: {inactivated (killed)}
- A61K 2039/5258: {Virus-like particles}
- C12N 2760/14123: Virus like particles [VLP]
- 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
- 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)
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Methods of preparing influenza viruses having altered immunodominant epitopes in HA, e.g., having one or more residues in one or more of antigenic sites A-E in HA altered, and viral vectors, e.g., influenza virus VLPs or non-influenza viruses or VLPs thereof expressing or having influenza HAs with altered immunogenicity as a result of altered immunodominant epitopes therein are provided.

## Claims

### Claim 1

A method to prepare a plurality of influenza virus nucleic acid molecules encoding a H3 or H5 hemagglutinin (HA) having a reduced number of immune dominant epitopes, comprising: introducing random mutations at a plurality of codons in one or more immune dominant epitopes in an isolated parental influenza virus nucleic acid molecule encoding an influenza virus H3 or H5 hemagglutinin having at least two immune dominant epitopes, thereby providing a library of influenza virus nucleic acid molecules encoding a mutant H3 or H5 influenza virus hemagglutinin; introducing the library into cells so as to provide a library of cells that express the mutant hemagglutinins; and identifying a mutant hemagglutinin encoded by the library with a reduced number of the immune dominant epitopes relative to the parental hemagglutinin as a result of one or more substitutions and/or deletions at residues that form the one or more immune dominant epitopes, wherein if the mutant H3 hemagglutinin has substitutions, the mutant H3 hemagglutinin has more than five substitutions relative to the parental H3 hemagglutinin.

### Claim 2 (depends on 1)

The method of claim 1 wherein at least 5, 10, 15 or 20 codons, or any integer between 5 and 20, are mutated.

### Claim 3 (depends on 1)

The method of claim 1 wherein the immune dominant epitope that is mutated corresponds to residues 121 to 146 in H3 HA (site A), residues 156 to 196 in H3 HA (site B), residues 50 to 57 or 275 to 279 in H3 HA (site C), residue 164, residue 182 or residues 208 to 217 in H3 HA (site D) or residues 62 to 83 in H3 HA (site E).

### Claim 4 (depends on 1)

The method of claim 1 wherein the cells are mammalian cells.

### Claim 5 (depends on 1)

The method of claim 1 wherein the mutant HA has a substitution at two or more of positions 121, 131, 135, 138, 140, 142, 144, 145, 155, 156, 157, 158, 171, 189, 193, 212, or 225, or a deletion at one or more of positions 121, 131, 135, 138, 140, 142, 144, 145, 155, 156, 157, 158, 171, 189, 193, 212, or 225, in H3, or a combination thereof.

### Claim 6 (depends on 1)

The method of claim 1 wherein the residue in H3 at position 121 is Q, R, I, L, V, T, S, F, Y or A, position 131 is R, K, <M, V, S, Q, C, V, Y, D, E, or L, position 135 is Y, G, R, M, K, N, V, W, S, V, or P, position 138 is S, W, K, I, R, or L, position 140 is L, M, T, S, R K, M, or P, position 142 is N, D, G, Y, Q, E, H, N, L, or P, position 144 is T, K, V, G, D, H, L or Q, position 145 is P, R, W or K, position 155 is C, I, F, H, R, A, V, S or N, position 156 is P, M, R, G, S, T, A, or C, position 157 is D, P, S, G, I, R or T, position 158 is R, V, S, A, K, C, Q, position 171 is T, F, L, E, H, C or R, position 189 is A, P, T, L, A, S, Y, or R, position 193 is Q, R, N, T, E, V, or P, position 212 is V, R, G, S, M, D or E, or position 225 is L, P, C, S, Q, G, Y, or F.

### Claim 7 (depends on 1)

The method of claim 1 wherein the residue in H5 at position 119 is L, K, R, S, G, T, E, A, V, F or N; position 123 is L, Y, I, M, N, S, V, K, G, T or R; position 125 is L, D, N, G, W, M, I, R, K, F, A, or S; position 126 is S, R, I, G, N, Q, A, N or R; position 127 is V, A, S, M, L, K, F or Y; position 129 is D, S, G, K, W, R, E, V, Q, A, I, or F; position 138 is G, D, E, L, A, M, V, F, R or S; position 140 is T, G, S, R, D, K, Q, E, C, or V; position 141 is R, P, W, K, E, A, M, D, L, or Q; position 151 is T, S, L, Y, K, N, or Q; position 152 is A, P, T, Y, H, E, S, I, F, or D; position 153 is R, Q, T, N, S, F, P, V, or K; position 154 L, T, D, R, P, S, or H; position 155 is N, G, K, H, T, L, S, I, P, or Q; position 156 is T, F, R, S, D, P, H, G, A, or N; position 185 is L, D, N, G, E, F, S, L, Q, P, V, M, R, A, or S; or position 189 is Y, S, L, R, K, G, E, F, D, V, E, I, or H.

### Claim 8

A method to prepare an influenza virus encoding a mutant hemagglutinin that has fewer immune dominant epitopes relative to a parental influenza virus, comprising: introducing a mutation in a parental H3 HA nucleic acid molecule at two or more codons for residue 121, 131, 135, 138, 140, 142, 144, 145, 155, 156, 157, 158, 171, 189, 193, 212, or 225, wherein the mutation encodes a residue that is not an immune dominant epitope residue in the parent H3 HA or introducing a mutation in a parental H5 HA nucleic acid molecule at two or more codons for residue 119, 123, 125, 126, 127, 129, 138, 140, 141, 151, 152, 153, 154, 155, 156, 185, or 189, wherein the mutation encodes a residue that is not an immune dominant epitope residue in the parent H5 HA; and isolating or preparing one or more influenza viruses with the mutated H3 or H5 HA, wherein the mutated H3 nucleic acid has more than five of the codons mutated, and wherein numbering for H3 is H3 numbering.

### Claim 9 (depends on 8)

The method of claim 8 wherein the residue in the mutated H3 HA at position 121 is Q, R, I, L, V, S, F, Y, or A, position 131 is R, V, S, Q, C, V, Y, D, E, or L, position 135 is Y, K, N, V, W, G, S, V, or P, position 138 is W, K, I, F, R, or L, position 140 is L, M, T, S, R K, M, Y, or P, position 142 is N, G, Y, Q, E, H, N, or Q, position 144 is T, V, G, D, P, H, L, K, or Q, position 145 is P, D, R, W or N, position 155 is C, I, R, A, V, S, or Q, position 156 is P, G, S, T, A, or C, position 157 is D, P, S, G, I, Q, R or T, position 158 is R, V, S, A K, C, Q, or G, position 171 is T, F, L, E, H, V, or R, position 189 is A, P, T, L, S, Y or R, position 193 is Q, R, N, T, E, V or P, position 212 is V, R, G, S, M, D or E, or position 225 is L, P, C, S, Q, G, Y, or F.

### Claim 10 (depends on 8)

The method of claim 8 wherein the residue in H5 at position 119 is L, K, R, S, G, T, E, A, V, F or N; position 123 is L, Y, I, M, N, S, V, K, G, T or R; position 125 is L, D, N, G, W, M, I, R, K, F, A, or S; position 126 is S, R, I, G, N, Q, A, N or R; position 127 is V, A, S, M, L, K, F or Y; position 129 is D, S, G, K, W, R, E, V, Q, A, I, or F; position 138 is G, D, E, L, A, M, V, F, R or S; position 140 is T, G, S, R, D, K, Q, E, C, or V; position 141 is R, P, W, K, E, A, M, D, L, or Q; position 151 is T, S, L, Y, K, N, or Q; position 152 is A, P, T, Y, H, E, S, I, F, or D; position 153 is R, Q, T, N, S, F, P, V, or K: position 154 L, T, D, R, P, S, or H; position 155 is N, G, K, H, T, L, S, I, P, or Q; position 156 is T, F, R, S, D, P, H, G, A, or N; position 185 is L, D, N, G, E, F, S, L, Q, P, V, M, R, A, or S; or position 189 is Y, S, L, R, K, G, E, F, D, V, E, I, or H.

### Claim 11

A composition comprising a plurality of distinct isolated recombinant influenza viruses each encoding a H3 or H5 hemagglutinin comprising a plurality of antigenically distinct residues relative to residues that form an immune dominant epitope in a parent virus, wherein each of the plurality of influenza viruses comprises substitutions at two or more of positions 121, 131, 135, 138, 140, 142, 144, 145, 155, 156, 157, 158, 171, 189, 193, 212, or 225, or one or more deletions of positions 121, 131, 135, 138, 140, 142, 144, 145, 155, 156, 157, 158, 171, 189, 193, 212, or 225, in H3, or any combination thereof, relative to the parent virus H3 hemagglutinin; or wherein each of the plurality of influenza viruses comprises a substitution at two or more of positions 119, 123, 125, 126, 127, 129, 138, 140, 141, 151, 152, 153, 154, 155, 156, 185, or 189, or a deletion in one or more of positions 119, 123, 125, 126, 127, 129, 138, 140, 141, 151, 152, 153, 154, 155, 156, 185, or 189, in H5, or any combination thereof, relative to the parent virus H5 hemagglutinin vims, wherein at least one of the plurality of distinct recombinant H3 viruses has a H3 hemagglutinin with more than five substitutions relative to the parental virus H3 hemagglutinin.

### Claim 12 (depends on 11)

The composition of claim 11 which has at least three, four or five distinct viruses with the substitutions.

### Claim 13 (depends on 11)

The composition of claim 11 which has five to ten distinct viruses with the substitutions.

### Claim 14 (depends on 11)

The composition of claim 11 which has ten to twenty distinct viruses with the substitutions.

### Claim 15 (depends on 11)

The composition of claim 11 wherein each distinct virus has at least one to five substitutions in antigenic site A or site B.

### Claim 16 (depends on 11)

The composition of claim 11 wherein each distinct virus has at least one to ten substitutions in antigenic sites A and B.

### Claim 17 (depends on 11)

The composition of claim 11 wherein each distinct virus has altered binding to antibodies that bind the corresponding parental hemagglutinin.

### Claim 18 (depends on 11)

The composition of claim 11 wherein one of the influenza viruses comprises a) a H5 HA where the residue at position 119 is L, K, R, S, G, T, E, A, V, F or N; position 123 is L, Y, I, M, N, S, V, K, G, T or R; position 125 is L, D, N, G, W, M, I, R, K, F, A, or S; position 126 is S, R, I, G, N, Q, A, N or R; position 127 is V, A, S, M, L, K, F or Y; position 129 is D, S, G, K, W, R, E, V, Q, A, I, or F; position 138 is G, D, E, L, A, M, V, F, R or S; position 140 is T, G, S, R, D, K, Q, E, C, or V; position 141 is R, P, W, K, E, A, M, D, L, or Q; position 151 is T, S, L, Y, K, N, or Q; position 152 is A, P, T, Y, H, E, S, I, F, or D; position 153 is R, Q, T, N, S, F, P, V, or K; position 154 L, T, D, R, P, S, or H; position 155 is N, G, K, H, T, L, S, I, P, or Q; position 156 is T, F, R, S, D, P, H, G, A, or N; position 185 is L, D, N, G, E, F, S, L, Q, P, V, M, R, A, or S; or position 189 is Y, S, L, R, K, G, E, F, D, V, E, I, or H, or any combination of those residues at those positions; or b) a H3 HA wherein the residue at position 121 is Q, R, I, L, V, S, F, Y, or A, position 131 is R, V, S, Q, C, V, Y, D, E, or L, position 135 is Y, K, N, V, W, G, S, V, or P, position 138 is W, K, I, F, R, or L, position 140 is L, M, T, S, R K, M, Y, or P, position 142 is N, G, Y, Q, E, H, N, or Q, position 144 is T, V, G, D, P, H, L, K, or Q, position 145 is P, D, R, W or N, position 155 is C, I, R, A, V, S, or Q, position 156 is P, G, S, T, A, or C, position 157 is D, P, S, G, I, Q, R or T, position 158 is R, V, S, A K, C, Q, or G, position 171 is T, F, L, E, H, V, or R, position 189 is A, P, T, L, S, Y or R, position 193 is Q, R, N, T, E, V or P, position 212 is V, R, G, S, M, D or E, or position 225 is L, P, C, S, Q, G, Y, or F, or any combination of those residues at those positions.

### Claim 19

A method to immunize an animal, comprising: administering an effective amount of a composition comprising a plurality of distinct isolated recombinant influenza viruses each encoding a H3 or H5 hemagglutinin comprising a plurality of antigenically distinct residues relative to residues that form an immune dominant epitope in a parent virus, wherein each of the plurality of influenza viruses comprises substitutions at two or more of positions 121, 131, 135, 138, 140, 142, 144, 145, 155, 156, 157, 158, 171, 189, 193, 212, or 225, or one or more deletions of positions 121, 131, 135, 138, 140, 142, 144, 145, 155, 156, 157, 158, 171, 189, 193, 212, or 225, in H3, or any combination thereof relative to the parent virus H3 hemagglutinin; or wherein each of the plurality of influenza viruses comprises a substitution at two or more of positions 119, 123, 125, 126, 127, 129, 138, 140, 141, 151, 152, 153, 154, 155, 156, 185, or 189, or a deletion in one or more of positions 119, 123, 125, 126, 127, 129, 138, 140, 141, 151, 152, 153, 154, 155, 156, 185, or 189, in H5, or any combination thereof, relative to the parent virus H5 hemagglutinin to an animal.