Vectors for eliciting immune responses to non-dominant epitopes in the hemagglutinin (HA) protein

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.

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

3. 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).

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

5. 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.

6. 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.

7. 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.

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.

9. 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.

10. 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.

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.

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

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

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

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

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

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

18. 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.

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.