Mosaic influenza virus hemagglutinin polypeptides and uses thereof

A mosaic influenza virus hemagglutinin (HA) polypeptide comprising an HA ectodomain of a first group 1 influenza A virus strain HA, wherein the HA ectodomain comprises an HA stem domain of the first group 1 influenza A virus strain HA and an HA globular head domain of the first group 1 influenza A virus strain HA, wherein the HA globular head domain of the first group 1 influenza A virus strain has been engineered to comprise four or all of the following:

• a. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more amino acid substitutions within the Sa antigenic site or a corresponding hypervariable antigenic site of the globular head domain of the first group 1 influenza A virus strain HA, wherein the amino acid substitutions substitute 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more amino acid residues within the Sa antigenic site or the corresponding hypervariable antigenic site of the globular head domain of the first group 1 influenza A virus strain HA with amino acid residues found in a corresponding region of an HA globular head domain of:
• (1) a group 2 influenza A virus HA, (2) a group 1 influenza A virus HA of a different subtype or a different strain than the first group 1 influenza A virus strain, or (3) a combination of group 1 influenza A virus HAs of different subtypes or different strains than the first group 1 influenza A virus strain;
• b. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or more amino acid substitutions within the Sb antigenic site or a corresponding hypervariable antigenic site of the globular head domain of the first group 1 influenza A virus strain HA, wherein the amino acid substitutions substitute 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or more amino acid residues within the Sb antigenic site or the corresponding hypervariable antigenic site of the globular head domain of the first group 1 influenza A virus strain HA with amino acid residues found in a corresponding region of:
• (1) a group 2 influenza A virus HA, (2) a group 1 influenza A virus HA of a different subtype or a different strain than the first group 1 influenza A virus strain, or (3) a combination of group 1 influenza A virus HAs of different subtypes or different strains than the first group 1 influenza A virus strain;
• c. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions within the Ca1 antigenic site or a corresponding hypervariable antigenic site of the globular head domain of the first group 1 influenza A virus, strain HA, wherein the amino acid substitutions substitute 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residues within the Ca1 antigenic site or the corresponding hypervariable antigenic site of the globular head domain of the first group 1 influenza A virus strain HA with amino acid residues found in a corresponding region of an HA globular head domain of:
• (1) a group 2 influenza A virus HA, (2) a group 1 influenza A virus HA of a different subtype or a different strain than the first group 1 influenza A virus strain, or (3) a combination of group 1 influenza A virus HAs of different subtypes or different strains than the first group 1 influenza A virus strain;
• d. 1, 2, 3, 4, 5, 6, 7 or more amino acid substitutions within the Ca2 antigenic site or a corresponding hypervariable antigenic site of the globular head domain of the first group 1 influenza A virus strain HA, wherein the amino acid substitutions substitute 1, 2, 3, 4, 5, 6, 7 or more amino acid residues within the Ca2 antigenic site or the corresponding hypervariable antigenic site of the globular head domain of the first group 1 influenza A virus strain HA with amino acid residues found in a corresponding region of:
• (1) a group 2 influenza A virus HA, (2) a group 1 influenza A virus HA of a different subtype or a different strain than the first group 1 influenza A virus strain, or (3) a combination of group 1 influenza A virus HAs of different subtypes or different strains than the first group 1 influenza A virus strain;
• and
• e. 1, 2, 3, 4, 5 or more amino acid substitutions within the Cb antigenic site or a corresponding hypervariable antigenic site of the globular head domain of the first group 1 influenza A virus strain, wherein the amino acid substitutions substitute 1, 2, 3, 4, 5 or more amino acid residues within the Cb antigenic site or the corresponding hypervariable antigenic site of the globular head domain of the first group 1 influenza A virus strain with amino acid residues found in a corresponding region of an HA globular head domain of:
• (1) a group 2 influenza A virus HA, (2) a group 1 influenza A virus HA of a different subtype or a different strain than the first group 1 influenza A virus strain, or (3) a combination of group 1 influenza A virus HAs of different subtypes or different strains than the first group 1 influenza A virus strain.

A mosaic influenza virus HA polypeptide comprising an HA ectodomain of influenza A/Michigan/45/2015 virus HA, wherein the HA ectodomain comprises the influenza A/Michigan/45/2015 virus HA stem domain and HA globular head domain, wherein the globular head domain has been engineered to comprises four or all of the following amino acid sequence substitutions:

• a. the amino acid sequences PN, KKGNS (SEQ ID NO:
• 1), and PKLNQS (SEQ ID NO:
• 2) in the HA globular head domain Sa antigenic site of influenza A/Michigan/45/2015 virus HA have been substituted with the following amino acid sequences PS, KKNST (SEQ ID NO:
• 3), and PTIKRS (SEQ ID NO:
• 4), respectively;
• b. the amino acid sequence TTADQQSLYQNA (SEQ ID NO:
• 5) in the HA globular head domain Sb antigenic site of influenza A/Michigan/45/2015 virus HA has been substituted with the following amino acid sequence DAAEQTKLYQNP (SEQ ID NO:
• 6);
• c. the amino acid sequences INDKG (SEQ ID NO:
• 7), TSR, and EPG in the HA globular head domain Ca1 antigenic site of influenza A/Michigan/45/2015 virus HA have been substituted with the following amino acid sequences NNTTG (SEQ ID NO:
• 8), TSS, and HPG, respectively;
• d. the amino acid sequences PHAGAK (SEQ ID NO:
• 9) and RD in the HA globular head domain Ca2 antigenic site of influenza A/Michigan/45/2015 virus HA have been substituted with the following amino acid sequences PYQGKS (SEQ ID NO:10) and ND, respectively;
• and
• e. the amino acid sequence LSTASS (SEQ ID NO:
• 11) in the HA globular head domain Cb antigenic site of influenza A/Michigan/45/2015 virus HA have been substituted with the following amino acid sequence LNVPE (SEQ ID NO:
• 12).

A mosaic influenza virus 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 has been engineered to comprise four or all of the following:

• a. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or more amino acid substitutions within the A antigenic site or a corresponding hypervariable antigenic site of the globular head domain of the first group 2 influenza A virus strain HA;
• b. 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more amino acid substitutions within the B antigenic site or a corresponding hypervariable antigenic site of the globular head domain of the first group 2 influenza A virus strain HA;
• c. 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or more amino acid substitutions within the C antigenic site or a corresponding hypervariable antigenic site of the globular head domain of the first group 2 influenza A virus strain HA;
• d. 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or more amino acid substitutions within the D antigenic site or a corresponding hypervariable antigenic site of the globular head domain of the first group 2 influenza A virus strain HA;
• and
• e. 1, 2, 3, 4, 5, 6, 7 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or more amino acid substitutions within the E antigenic site or a corresponding hypervariable antigenic site of the globular head domain of the first group 2 influenza A virus strain HA.

A mosaic influenza virus 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 HA strain HA, wherein the HA globular head domain of the first group 2 influenza A virus strain HA has been engineered to comprise four or all of the following:

• a. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or more amino acid substitutions within the A antigenic site or a corresponding hypervariable antigenic site of the globular head domain of the first group 2 influenza A virus strain HA, wherein the amino acid substitutions substitute 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or more amino acid residues within the A antigenic site or the corresponding hypervariable antigenic site of the globular head domain of the first group 2 influenza A virus strain HA with amino acid residues found in a corresponding region of an HA globular head domain of:
• (1) a group 1 influenza A virus HA, (2) a group 2 influenza A virus HA of a different subtype or a different strain than the first group 2 influenza A virus strain, or (3) a combination of group 2 influenza A virus HAs of different subtypes or different strains than the first group 2 influenza A virus strain;
• b. 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more amino acid substitutions within the B antigenic site or a corresponding hypervariable antigenic site of the globular head domain of the first group 2 influenza A virus strain HA, wherein the amino acid substitutions substitute 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more amino acid residues within the B antigenic site or the corresponding hypervariable antigenic site of the globular head domain of the group 2 influenza A virus strain HA with amino acid residues found in a corresponding region of an HA globular head domain of:
• (1) a group 1 influenza A virus HA, (2) a group 2 influenza A virus HA of a different subtype or a different strain than the first group 2 influenza A virus strain, or (3) a combination of group 2 influenza A virus HAs of different subtypes or different strains than the first group 2 influenza A virus strain;
• c. 11, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or more amino acid substitutions within the C antigenic site or a corresponding hypervariable antigenic site of the globular head domain of the first group 2 influenza A virus strain HA, wherein the amino acid substitutions substitute 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or more amino acid residues within the C antigenic site or the corresponding hypervariable antigenic site of the globular head domain of the first group 2 influenza A virus strain HA with amino acid residues found in the corresponding region of an HA globular head domain of:
• (1) a group 1 influenza A virus HA, (2) a group 2 influenza A virus HA of a different subtype or a different strain than the first group 2 influenza A virus strain, or (3) a combination of group 2 influenza A virus HAs of different subtypes or different strains than the first group 2 influenza A virus strain;
• d. 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or more amino acid substitutions within the D antigenic site or a corresponding hypervariable antigenic site of the globular head domain of the first group 2 influenza A virus strain HA, wherein the amino acid substitutions substitute 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or more amino acid residues within the D antigenic site or the corresponding hypervariable antigenic site of the globular head domain of the first group 2 influenza A virus strain HA with amino acid residues found in a corresponding region of an HA globular head domain of:
• (1) a group 1 influenza A virus HA, (2) a group 2 influenza A virus HA of a different subtype or a different strain than the first group 2 influenza A virus strain, or (3) a combination of group 2 influenza A virus HAs of different subtypes or different strains than the first group 2 influenza A virus strain;
• and
• e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or more amino acid substitutions within the E antigenic site or a corresponding hypervariable antigenic site of the globular head domain of the group 2 influenza A virus strain HA, wherein the amino acid substitutions substitute 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or more amino acid residues within the E antigenic site or the corresponding hypervariable antigenic site of the globular head domain of the first group 2 influenza A virus strain HA with amino acid residues found in a corresponding region of an HA globular head domain of:
• (1) a group 1 influenza A virus HA, (2) a group 2 influenza A virus HA of a different subtype or a different strain than the first group 2 influenza A virus strain, or (3) a combination of group 2 influenza A virus HAs of different subtypes or different strains than the first group 2 influenza A virus strain.

A mosaic influenza virus HA polypeptide comprising an HA ectodomain of influenza A/Hong Kong/4801/2014 virus HA, wherein the HA ectodomain comprises the influenza A/Hong Kong/4801/2014 virus HA stem domain and HA globular head domain, wherein the globular head domain has been engineered to comprises four or all of the following amino acid sequence substitutions:

• a. the amino acid sequence NNESFNWT-GVTQNGTSSACIRRSSSS (SEQ ID NO :13) in the HA globular head domain A antigenic site of influenza A/Hong Kong/4801/2014 virus HA has been substituted with the following amino acid sequence NNESFNWT-GVTQNGTSSACMRNGGNS (SEQ ID NO:
• 14);
• b. the amino acid sequences THLNYK (SEQ ID NO:
• 15) and GTDKDQIFLYAQ (SEQ ID NO:
• 16) in the HA globular head domain B antigenic site of influenza A/Hong Kong/4801/2014 virus HA have been substituted with the following amino acid sequences THL-NQK (SEQ ID NO:
• 17) and GTNQDQIFLYAQ (SEQ ID NO:
• 18), respectively;
• c. the amino acid sequences QNSSIGEICDS (SEQ ID NO:
• 19) and PIG-KCKSE (SEQ ID NO:
• 20) in the HA globular head domain C antigenic site of influenza A/Hong Kong/4801/2014 virus HA have been substituted with the following amino acid sequences ESTGINRLCMK (SEQ ID NO:
• 21) and PIDNNCESK (SEQ ID NO:
• 22), respectively;
• d. the amino acid sequence RITVSTKRSQQAVIPNIGS (SEQ ID NO:
• 23) in the HA globular head domain D antigenic site of influenza A/Hong Kong/4801/2014 virus HA has been substituted with the following 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 HA globular head domain E antigenic site of influenza A/Hong Kong/4801/2014 virus HA have been substituted with the following amino acid sequences GNCH (SEQ ID NO:
• 125), GFQNKMWDLFVERSKAY (SEQ ID NO:
• 29) and LRIGRS (SEQ ID NO:
• 24), respectively.

A mosaic influenza virus HA polypeptide comprising an HA ectodomain of influenza A/Hong Kong/4801/2014 virus HA, wherein the HA ectodomain comprises the influenza A/Hong Kong/4801/2014 virus HA stem domain and HA globular head domain, wherein the globular head domain has been engineered to comprise four or all of the following amino acid sequence substitutions:

• (a) the amino acid sequence IRRSSSS (SEQ ID NO:
• 127) in the HA globular head domain A antigenic site of influenza A/Hong Kong/4801/2014 virus HA has been substituted with the following amino acid sequence MRNGGNS (SEQ ID NO:
• 128);
• (b) the amino acid sequences THLNYK (SEQ ID NO:
• 15) and TDKDQIFPYA (SEQ ID NO:
• 130) the HA globular head domain B antigenic site of influenza A/Hong Kong/4801/2014 virus HA have been substituted with the following 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 HA globular head domain C antigenic site of influenza A/Hong Kong/4801/2014 virus HA have been substituted with the following 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 HA globular head domain D antigenic site of influenza A/Hong Kong/4801/2014 virus HA has been substituted with the following amino acid sequence STYQQT (SEQ ID NO:
• 136);
• and
• (e) the amino acid sequences ENCT (SEQ ID NO:
• 124), K and IRSGK (SEQ ID NO:
• 137) the HA globular head domain E antigenic site of influenza A/Hong Kong/4801/2014 virus HA have been substituted with the following amino acid sequences GNCH (SEQ ID NO:
• 125), M, and LRIGR (SEQ ID NO:
• 138), respectively.

A mosaic influenza virus HA polypeptide comprising an HA ectodomain of influenza A/Hong Kong/4801/2014 virus HA, wherein the HA ectodomain comprises the influenza A/Hong Kong/4801/2014 virus HA stem domain and HA globular head domain, wherein the globular head domain has been engineered to comprise four or all of the following amino acid sequence substitutions:

• (a) the amino acid sequence NNESFNWT-GVTQNGTSSACIRRSSSS (SEQ ID NO :13) in the HA globular head domain A antigenic site of influenza A/Hong Kong/4801/2014 virus HA has been substituted with the following amino acid sequence NNESFNWT-GVTQNGTSSACLRGGRNS (SEQ ID NO:
• 139);
• (b) the amino acid sequences THLNYK (SEQ ID NO:
• 15) and GTDKDQIFLYAQ (SEQ ID NO:
• 16) in the HA globular head domain B antigenic site of influenza A/Hong Kong/4801/2014 virus HA have been substituted with the following amino acid sequences THL-NGK (SEQ ID NO:
• 140) and GTDNDQIFLYAQ (SEQ ID NO:
• 141), respectively;
• (c) the amino acid sequences QNSSIGEICDS (SEQ ID NO:
• 19) and PIG-KCKSE (SEQ ID NO:
• 20) in the HA globular head domain C antigenic site of influenza A/Hong Kong/4801/2014 virus HA have been substituted with the following amino acid sequences ETTNHTDECPK (SEQ ID NO:
• 142) and PIGKSCTSP (SEQ ID NO:
• 143), respectively;
• (d) the amino acid sequence RITVSTKRSQQAVIPNIGS (SEQ ID NO:
• 23) in the HA globular head domain D antigenic site of influenza A/Hong Kong/4801/2014 virus HA has been substituted with the following amino acid sequence RITVSTRSDQQTVIPNIGS (SEQ ID NO:
• 144);
• and
• (e) the amino acid sequences ENCT (SEQ ID NO:
• 124), GFQNKKWDLFVERSKAY (SEQ ID NO:
• 27) and IRSGKS (SEQ ID NO:28) in the HA globular head domain E antigenic site of influenza A/Hong Kong/4801/2014 virus HA have been substituted with the following amino acid sequences QNCD (SEQ ID NO:
• 145), GFQNKTWDLFVERSKAY (SEQ ID NO:
• 146) and IRKGRS (SEQ ID NO:
• 147), respectively.