Mosaic influenza virus hemagglutinin polypeptides and uses thereof

A mosaic influenza virus hemagglutinin (HA) polypeptide comprising an HA ectodomain of a first group 2 influenza A virus strain HA, wherein the HA ectodomain comprises an HA stem domain of the first group 2 influenza A virus strain HA and an HA globular head domain of the first group 2 influenza A virus strain HA, wherein the HA globular head domain of the first group 2 influenza A virus strain HA comprises amino acid substitutions in antigenic sites A to E, and wherein:

• (a) antigenic site A of the globular head domain of the first group 2 influenza A virus strain HA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of the amino acid substitutions are substitutions to amino acid residues found in the antigenic site A of an HA globular head domain of a second group 2 influenza A virus of a different subtype or a different strain than the first group 2 influenza A virus strain;
• (b) antigenic site B of the globular head domain of the first group 2 influenza A virus strain HA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the amino acid substitutions are substitutions to amino acid residues found in the antigenic site B of an HA globular head domain of the second group 2 influenza A virus of a different subtype or a different strain than the first group 2 influenza A virus strain;
• (c) antigenic site C of the globular head domain of the first group 2 influenza A virus strain HA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 amino acid substitutions, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 of the amino acid substitutions are substitutions to amino acid residues found in the antigenic site C of an HA globular head domain of the second group 2 influenza A virus of a different subtype or a different strain than the first group 2 influenza A virus strain;
• (d) antigenic site D of the globular head domain of the first group 2 influenza A virus strain HA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the amino acid substitutions are substitutions to amino acid residues found in the antigenic site D of an HA globular head domain of the second group 2 influenza A virus of a different subtype or a different strain than the first group 2 influenza A virus strain;
• and
• (e) antigenic site E of the globular head domain of the first group 2 influenza A virus strain HA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the amino acid substitutions are substitutions to amino acid residues found in the antigenic site E of an HA globular head domain of the second group 2 influenza A virus of a different subtype or a different strain than the first group 2 influenza A virus strain;
• wherein antigenic sites A to E of the first group 2 influenza A virus strain HA and antigenic sites A to E of the second group 2 influenza A virus strain HA correspond to antigenic sites A to E of influenza virus A/Hong Kong/4801/2014 HA (SEQ ID NO:
• 171);
• and
• wherein the mosaic HA polypeptide comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions outside of antigenic sites A to E of the globular head domain of the first group 2 influenza A virus strain HA;
• and wherein the second group 2 influenza A virus strain is a strain of an H10 subtype or H14 subtype.

A mosaic influenza virus hemagglutinin (HA) polypeptide comprising an influenza A virus HA ectodomain of an influenza virus A/Hong Kong/4801/2014 (H3) HA, wherein the HA ectodomain comprises an influenza virus A/Hong Kong/4801/2014 (H3) HA stem domain and an influenza virus A/Hong Kong/4801/2014 HA globular head domain, wherein the influenza virus A/Hong Kong/4801/2014 HA globular head comprises amino acid substitutions in antigenic sites A to E, and wherein:

• (a) antigenic site A of the influenza virus A/Hong Kong/4801/2014 HA globular head domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of the amino acid substitutions are amino acid substitutions to amino acid residues found in the antigenic site A of an HA globular head domain of influenza virus A/Jiangxi-Donghu/346-1/2013 HA (H10) or influenza virus A/mallard/Gurjev/263/1982 HA (H14);
• (b) antigenic site B of the influenza virus A/Hong Kong/4801/2014 HA globular head domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the amino acid substitutions are substitutions to amino acid residues found in the antigenic site B of an HA globular head domain of influenza virus A/Jiangxi-Donghu/346-1/2013 HA (H10) or influenza virus A/mallard/Gurjev/263/1982 HA (H14);
• (c) antigenic site C of the influenza virus A/Hong Kong/4801/2014 HA globular head domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 amino acid substitutions, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 of the amino acid substitutions are substitutions to amino acid residues found in the antigenic site C of an HA globular head domain of influenza virus A/Jiangxi-Donghu/346-1/2013 HA (H10) or influenza virus A/mallard/Gurjev/263/1982 HA (H14);
• (d) antigenic site D of the influenza virus A/Hong Kong/4801/2014 HA globular head domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of the amino acid substitutions are substitutions to amino acid residues found in the antigenic site D of an HA globular head domain of influenza virus A/Jiangxi-Donghu/346-1/2013 HA (H10) or influenza virus A/mallard/Gurjev/263/1982 HA (H14);
• and
• (e) antigenic site E of the influenza virus A/Hong Kong/4801/2014 HA globular head domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of the amino acid substitutions are substitutions to amino acid residues found in the antigenic site E of an HA globular head domain of influenza virus A/Jiangxi-Donghu/346-1/2013 HA (H10) or influenza virus A/mallard/Gurjev/263/1982 HA (H14);
• and
• wherein the mosaic HA polypeptide comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions outside of antigenic sites A to E of the influenza virus A/Hong Kong/4801/2014 HA globular head domain.

A mosaic influenza virus hemagglutinin (HA) polypeptide comprising an influenza A virus HA ectodomain of an influenza virus A/Hong Kong/4801/2014 (H3) HA, wherein the HA ectodomain comprises an influenza virus A/Hong Kong/4801/2014 (H3) HA stem domain and an influenza virus A/Hong Kong/4801/2014 HA globular head domain, wherein the influenza virus A/Hong Kong/4801/2014 HA globular head comprises amino acid substitutions in antigenic sites A to E, and wherein:

• (a) the amino acid sequence IRRSSSS (SEQ ID NO:
• 127) in the antigenic site A of the influenza virus A/Hong Kong/4801/2014 HA globular head domain has been substituted with the amino acid sequence MRNGGNS (SEQ ID NO:
• 128);
• (b) the amino acid sequences THLNYK (SEQ ID NO:
• 15) and TDKDQIFPYA (SEQ ID NO:
• 130) in the antigenic site B of the influenza virus A/Hong Kong/4801/2014 HA globular head domain have been substituted with the amino acid sequences THLNQK (SEQ ID NO:
• 17) and TDQDQIFPYA (SEQ ID NO:
• 131), respectively;
• (c) the amino acid sequences QNSSIGEICDS (SEQ ID NO:
• 19) and G-KCKSE (SEQ ID NO:
• 132) in the antigenic site C of the influenza virus A/Hong Kong/4801/2014 HA globular head domain have been substituted with the amino acid sequences ESTGTNRLCMK (SEQ ID NO:
• 133) and DNNCESK (SEQ ID NO:
• 134), respectively;
• (d) the amino acid sequence KRSQQA (SEQ ID NO:
• 135) in the antigenic site D of the influenza virus A/Hong Kong/4801/2014 HA globular head domain has been substituted with the amino acid sequence STYQQT (SEQ ID NO:
• 136);
• and
• (e) the amino acid sequences ENCT (SEQ ID NO:
• 124), GFQNKKWDLFVERSKAY (SEQ ID NO:
• 27) and IRSGK (SEQ ID NO:
• 137) in the antigenic site E of the influenza virus A/Hong Kong/4801/2014 HA globular head domain have been substituted with the amino acid sequences GNCH (SEQ ID NO:
• 125), GFQNKMWDLFVERSKAY (SEQ ID NO:
• 29) and LRIGR (SEQ ID NO:
• 138), respectively, and
• wherein the mosaic HA polypeptide comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions outside of antigenic sites A to E of the influenza virus A/Hong Kong/4801/2014 HA globular head domain.

A mosaic influenza virus hemagglutinin (HA) polypeptide comprising an influenza A virus HA ectodomain of an influenza virus A/Hong Kong/4801/2014 (H3) HA, wherein the HA ectodomain comprises an influenza virus A/Hong Kong/4801/2014 (H3) HA stem domain and an influenza virus A/Hong Kong/4801/2014 HA globular head domain, wherein the influenza virus A/Hong Kong/4801/2014 HA globular head comprises amino acid substitutions in antigenic sites A to E, and wherein:

• (a) the amino acid sequence NNESFNWTGVTQNGTSSACIRRSSSS (SEQ ID NO:
• 13) in the antigenic site A of the influenza virus A/Hong Kong/4801/2014 HA globular head domain has been substituted with the amino acid sequence NNESFNWTGVTQNGTSSACMRNGGNS (SEQ ID NO:
• 14);
• (b) the amino acid sequences THLNYK (SEQ ID NO:
• 15) and GTDKDQIFLYAQ (SEQ ID NO:
• 16) in the antigenic site B of the influenza virus A/Hong Kong/4801/2014 HA globular head domain have been substituted with the amino acid sequences THLNQK (SEQ ID NO:
• 17) and GTNQDQIFLYAQ (SEQ ID NO:
• 18), respectively;
• (c) the amino acid sequences QNSSIGEICDS (SEQ ID NO:
• 19) and PIGKCKSE (SEQ ID NO:
• 20) in the antigenic site C of the influenza virus A/Hong Kong/4801/2014 HA globular head domain have been substituted with the amino acid sequences ESTGTNRLCMK (SEQ ID NO:
• 133) and PIDNNCESK (SEQ ID NO:
• 22), respectively;
• (d) the amino acid sequence RITVSTKRSQQAVIPNIGS (SEQ ID NO:
• 23) in the antigenic site D of the influenza virus A/Hong Kong/4801/2014 HA globular head domain has been substituted with the amino acid sequence RITVSTSTYQQAVIPNIGS (SEQ ID NO:
• 25);
• and
• (e) the amino acid sequences ENCT (SEQ ID NO:
• 124), GFQNKKWDLFVERSKAY (SEQ ID NO:
• 27) and IRSGKS (SEQ ID NO:28) in the antigenic site E of the influenza virus A/Hong Kong/4801/2014 HA globular head domain have been substituted with the amino acid sequences GNCH (SEQ ID NO:
• 125), GFQNKMWDLFVERSKAY (SEQ ID NO:
• 29) and LRIGRS (SEQ ID NO:
• 24), respectively, and
• wherein the mosaic HA polypeptide comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions outside of antigenic sites A to E of the influenza virus A/Hong Kong/4801/2014 HA globular head domain.

A mosaic influenza virus hemagglutinin (HA) polypeptide comprising an influenza A virus HA ectodomain of an influenza virus A/Hong Kong/4801/2014 (H3) HA, wherein the HA ectodomain comprises an influenza virus A/Hong Kong/4801/2014 (H3) HA stem domain and an influenza virus A/Hong Kong/4801/2014 HA globular head domain, wherein the influenza virus A/Hong Kong/4801/2014 HA globular head comprises amino acid substitutions in antigenic sites A to E, and wherein:

• (a) the amino acid sequence IRRSSSS (SEQ ID NO:
• 127) in the antigenic site A of the influenza virus A/Hong Kong/4801/2014 HA has been substituted with the amino acid sequence LRGGRNS (SEQ ID NO:
• 148);
• (b) the amino acid sequences THLNYK (SEQ ID NO:
• 15) and TDKDQIFPYA (SEQ ID NO:
• 130) in the antigenic site B of the influenza virus A/Hong Kong/4801/2014 HA have been substituted with the amino acid sequences THLNGK (SEQ ID NO:
• 140) and TDNDQIFPYA (SEQ ID NO:
• 149), respectively;
• (c) the amino acid sequences QNSSIGEICDS (SEQ ID NO:
• 19) and G-KCKSE (SEQ ID NO:
• 132) in the antigenic site C of the influenza virus A/Hong Kong/4801/2014 HA have been substituted with the amino acid sequences ETNHTDELCPS (SEQ ID NO:
• 150) and G-SCTSP (SEQ ID NO:
• 151), respectively;
• (d) the amino acid sequence KRSQQA (SEQ ID NO:
• 135) in the antigenic site D of the influenza virus A/Hong Kong/4801/2014 HA has been substituted with the amino acid sequence RSDQQT (SEQ ID NO:
• 152);
• and
• (e) the amino acid sequences ENCT (SEQ ID NO:
• 124), K83 and IRSGK (SEQ ID NO:
• 137) in the antigenic site E of the influenza virus A/Hong Kong/4801/2014 HA have been substituted with the amino acid sequences QNCD (SEQ ID NO:
• 145), 83T, and IRKGK (SEQ ID NO:
• 153), respectively;
• and
• wherein the mosaic HA polypeptide comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions outside of antigenic sites A to E of the influenza virus A/Hong Kong/4801/2014 HA globular head domain.

A mosaic influenza virus hemagglutinin (HA) polypeptide comprising:

• (a) the amino acid sequence set forth in SEQ ID NO:
• 173;
• SEQ ID NO:
• 31;
• or SEQ ID NO:
• 175;
• or
• (b) the amino acid sequence set forth in SEQ ID NO:
• 173 or SEQ ID NO:
• 175 without the signal peptide.