Inducing immune responses to influenza virus using polypeptide and nucleic acid compositions

• US 20080032921A1
• Filed: 05/18/2007
• Published: 02/07/2008
• Est. Priority Date: 05/18/2006
• Status: Active Grant

• Current Assignee: Epimmune, Inc. (Takeda Pharmaceutical Company Limited)
• Original Assignee: Pharmexa Inc.
• Inventors: Bilsel, Pamuk, Newman, Mark, Alexander, Jeffery
• Granted Patent: US 9,511,134 B2
• Time in Patent Office: Days
• US Class Current: 514/2
• CPC Class Codes: A61K 2039/53 DNA (RNA) vaccination
• CPC Class Codes: A61K 2039/545 characterised by the dose, timing or administration schedule
• CPC Class Codes: A61K 2039/55505 Inorganic adjuvants
• CPC Class Codes: A61K 2039/55566 Emulsions, e.g. Freund's adjuvant, MF59
• CPC Class Codes: A61K 2039/605 MHC molecules or ligands thereof
• CPC Class Codes: A61K 2039/627 characterised by the linker
• CPC Class Codes: A61K 39/12 Viral antigens
• CPC Class Codes: A61K 39/145 Orthomyxoviridae, e.g. influenza virus
• CPC Class Codes: A61K 39/385 Haptens or antigens, bound to carriers
• CPC Class Codes: A61K 39/39 characterised by the immunostimulating additives, e.g. chemical adjuvants
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C12N 2760/16122 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12N 2760/16134 Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Abstract

The present invention provides polynucleotides and polypeptides capable of enhancing the immune response of a human in need of protection against influenza virus infection by administering in vivo, into a tissue of the human, at least one polynucleotide comprising one or more regions of nucleic acid encoding an influenza protein or a fragment, variant, or derivative thereof, or at least one polypeptide encoded therefrom. The present invention also relates to identifying and preparing influenza virus epitopes and to polynucleotides and polypeptides comprising such influenza virus epitopes. The present invention also relates to compositions and methods of use in the prevention and treatment of influenza virus infection.

Claims

1. An isolated polynucleotide comprising:

(a) a nucleic acid encoding zero to ten HTL epitopes;

(b) a nucleic acid encoding a pan-DR binding epitope;

(c) a nucleic acid encoding a polypeptide at least 90% identical to an influenza hemagglutinin (HA) sequence, or a fragment thereof; and

(d) optionally, an influenza matrix protein 2 external (M2e) sequence, or fragment thereof;

wherein the nucleic acid of (a), (b), and (c), and optionally (d), are arranged in any order relative to one another.

2. The polynucleotide of claim 1, wherein said pan-DR-binding epitope comprises the amino acid sequence AKFVAAWTLKAAA (SEQ ID NO:

1).

3. The polynucleotide of claim 1, wherein said nucleic acid of (b) is located 5′

or 3′

of said nucleic acid of (c);

or said nucleic acid of (c) is interrupted by nucleic acid of (b).

4. The polynucleotide of claim 3, wherein said nucleic acid of (a) is located 5′

of said nucleic acid of (b) and/or nucleic acid of (c);

3′

of said nucleic acid of (b) and/or nucleic acid of (c).

5. The polynucleotide of claim 1, wherein nucleic acids of (a), (b) and (c), and optionally (d), are flanked by a spacer sequence between one and eight amino acids in length.

6. The polynucleotide of claim 1, wherein said HTL epitope is from about 6 to 30 amino acids in length.

7. The polynucleotide of claim 1, wherein said HTL epitope is flanked by a spacer sequence between one and eight amino acids in length.

8. The polynucleotide of claim 1, wherein said spacer optimizes HTL epitope processing and minimizes junctional epitopes.

9. The polynucleotide of claim 1, wherein said nucleic acids of (a), (b), and (c), and optionally (d), are operably linked.

10. The polynucleotide of claim 1, wherein said influenza HA sequence encodes a polypeptide at least 95% identical to an HA from any one of the influenza strains listed in Tables 1 and 2.

11. The polynucleotide of claim 1, wherein said influenza HA sequence is from an influenza strain selected from the group consisting of:

Human A/Viet Nam/1203/2004 (H5N1), Human A/Hong Kong/156/97 (H5N1), Human A/Hong Kong/483/97 (H5N1), Human A/Hong Kong/1073/99 (H9N2), Avian A/Chicken/HK/G9/97 (H9N2), Swine A/Swine/Hong Kong/10/98 (H9N2), Avian A/FPV/Rostock/34 (H7N1), Avian A/Turkey/Italy/4620/99 (H7N1), Avian A/FPV/Weybridge/34 (H7N7), Human A/New Caledonia/20/99 (H1N1), Human A/Hong Kong/1/68 (H3N2), Human A/Shiga/25/97 (H3N2), Human A/Singapore/1/57 (H2N2), Human A/Leningrad/134/57 (H2N2), Human A/Ann Arbor/6/60 (H2N2), Human A/Brevig Mission/1/18 (H1N1), Swine A/Swine/Wisconsin/464/98 (H1N1), Human A/Netherlands/219/03 (H7N7) and Human A/Wyoming/3/2003 (H3N2).

12. The polynucleotide of claim 1, wherein said influenza HA sequence or fragment thereof is selected from the group consisting of an HA extracellular domain (ECD), an HA transmembrane domain (TM), and an HA cytoplasmic domain (CYT).

13. The polynucleotide of claim 1, wherein said HTL epitope is selected from the group consisting of the epitopes set forth in Table 3.

14. The polynucleotide of claim 1, wherein said HTL epitope is selected from the group consisting of the HTL epitopes set forth in Table 4.

15. The polynucleotide of claim 1, wherein said spacer residues are selected from the group consisting of:

G, P and N.

16. The polynucleotide of claim 1, wherein said spacer encodes an amino acid sequence selected from the group consisting of:

an amino acid sequence comprising or consisting of GPGPG (SEQ ID NO;

3), an amino acid sequence comprising or consisting of PGPGP (SEQ ID NO;

4), an amino acid sequence comprising or consisting of (GP)n, an amino acid sequence comprising or consisting of (PG)n, an amino acid sequence comprising or consisting of (GP)nG, and an amino acid sequence comprising or consisting of (PG)nP, where n is an integer between zero and eleven.

17. The polynucleotide of claim 1, wherein said M2e sequence is selected from the group consisting of the M2e sequences set forth in Table 5.

18. The polynucleotide of claim 1, wherein said M2e sequence is selected from the group consisting of the M2e sequences set forth in Table 6.

19. The polynucleotide of claim 1, wherein said M2e sequence contains amino acid substitutions at positions 10, 13, 15, 17 and/or 19.

20. The polynucleotide of claim 19, wherein an isoleucine at position 10 is substituted with a threonine at position 10, a glutamic acid at position 13 is substituted with glycine, a glycine at position 15 is substituted with glutamic acid, an arginine at position 17 is substituted with a lysine, and/or a glutamine at position 19 is substituted with a serine

21. The polynucleotide of claim 1, wherein said M2e sequence contains amino acid substitutions, and wherein said amino acid substitutions correspond to the following:

a proline at position 9 is substituted with a leucine or histidine, an aspartic acid at position 18 is substituted with a glycine, a serine at position 20 is substituted with an aspargine, a serine at position 19 is substituted with a leucine and/or a serine at position 1 is substituted with a valine.

22. The polynucleotide of claim 1, further comprising a nucleic acid encoding a targeting sequence located at the N-terminus of said construct.

23. The polynucleotide of claim 22, wherein said targeting sequence is selected from the group consisting of:

an Ig kappa signal sequence, a tissue plasminogen activator signal sequence, an insulin signal sequence, an endoplasmic reticulum signal sequence, a LAMP-1 lysosomal targeting sequence, a LAMP-2 lysosomal targeting sequence, an HLA-DM lysosomal targeting sequence, an HLA-DM-association sequence of HLA-DO, an Ig-a cytoplasmic domain, Ig-ss cytoplasmic domain, a li protein, an influenza matrix protein, an HCV antigen, a yeast Ty protein, a baculovirus signal sequence and a prokaryotic signal sequence.

24. The polynucleotide of claim 1, comprising, consisting essentially of, or consisting of an HA sequence selected from the group consisting of the nucleic acid sequence encoding the polypeptide of HA (SEQ ID NO:

5), HA ECD (SEQ ID NO;

     6), HA TM (SEQ ID NO;

7), HA CYT (SEQ ID NO;

8), and any combination thereof.

25. The polynucleotide of claim 1, comprising, consisting essentially of, or consisting of the polynucleotide sequence of PADRE®

-HA (SEQ ID NO;

13).

26. The polynucleotide of claim 1, comprising, consisting essentially of, or consisting of the polynucleotide sequence of HA-PADRE®

(SEQ ID NO;

11).

27. The polynucleotide of claim 1, wherein said nucleic acid of (c) encodes zero to three HTL epitopes.

28. The polynucleotide of claim 1, wherein the HTL epitope is from about 8 to about 20 amino acids in length.

29. The polynucleotide of claim 28, wherein the HTL epitope is from about 12 to about 18 amino acids in length.

30. A polypeptide encoded by the polynucleotide of claim 1.

35. A vector comprising the polynucleotide of claim 1.

36. The vector of claim 35, wherein said vector is an expression vector.

37. A composition comprising the polynucleotide of claim 1.

44. A cell comprising the polynucleotide of claim 1.

45. A method of inducing an immune response against influenza virus in an individual in need thereof, comprising administering to said individual the composition of claim 37.

83. The polynucleotide of claim 1, wherein the polynucleotide further comprises a signal sequence.

84. The polynucleotide of claim 83, wherein the signal sequence is a chitinase signal sequence.

85. The polynucleotide of claim 83, wherein the signal sequence is a BiP signal sequence.

86. The polynucleotide of claim 1, wherein the polynucleotide further comprises a HIS tag and optionally, a dipeptidase stop signal.

87. The polynucleotide of claim 1, wherein the HA sequence comprises an HA polybasic cleavage site.

88. The polynucleotide of claim 87 wherein the HA polybasic cleavage site is a wild-type sequence that encodes for the amino acid sequence of SEQ ID NO:

186.

89. The polynucleotide of claim 87, wherein the HA polybasic cleavage site is a mutant sequence that encodes for the amino acid sequence of SEQ ID NO:

187.

90. The polynucleotide of claim 1, wherein the HA sequence includes an HA transmembrane domain.

91. The polynucleotide of claim 1, wherein the HA sequence includes an HA cytoplasmic domain.

92. The polynucleotide of claim 1, wherein the polynucleotide further comprises a thrombin cleavage site.

93. The polynucleotide of claim 1, wherein the polynucleotide further comprises a foldon sequence.

94. The polynucleotide of claim 1 wherein the polynucleotide comprises the sequence of SEQ ID NO:

173.

95. The polynucleotide of claim 1 wherein the polynucleotide comprises the sequence of SEQ ID NO:

184.

96. The polynucleotide of claim 1 wherein the polynucleotide comprises the sequence of SEQ ID NO:

182.

97. The polynucleotide of claim 1 wherein the polynucleotide comprises the sequence of SEQ ID NO:

180.

98. The polypeptide encoded by the polynucleotide of claim 83.

99. A vector comprising the polynucleotide of claim 83.

115. A composition comprising the polynucleotide of claim 83.

116. A cell comprising the polynucleotide of claim 83.

117. A method of inducing an immune response against influenza virus in an individual in need thereof, comprising administering to said individual the composition of claim 115.

119. A composition comprising the vector of claim 35.

121. A cell comprising the vector of claim 35.

126. A composition comprising the vector of claim 99.

127. A composition comprising the polypeptide of claim 98.

128. A cell comprising the vector of claim 99.

129. A cell comprising the polypeptide of claim 98.

31. An isolated polypeptide comprising:

(a) zero to ten HTL epitopes;

(b) a pan-DR binding epitope;

(c) a polypeptide at least 90% identical to an influenza hemagglutinin (HA) sequence, or a fragment thereof; and

(d) optionally, an influenza matrix protein 2 external (M2e) sequence, or fragment thereof;

wherein the polypeptide of (a), (b), and (c), and optionally (d), are arranged in any order relative to one another; and

wherein the pan-DR binding epitope comprises the amino acid sequence a₁KXVAAWTLKAAa₂ (SEQ ID NO;

2), where “

X”

is selected from the group consisting of cyclohexylalanine, phenylalanine, and tyrosine; and

“

a₁”

is either D-alanine or L-alanine; and

“

a₂”

is either D-alanine or L-alanine.

41. The composition comprising the polypeptide of claim 31 and a virosome.

42. The composition of claim 41, wherein said virosome is an immunopotentiating reconstituted influenza virosome (IRIV).

43. A composition, comprising two or more polypeptides of claim 31, wherein said polypeptides are different polypeptides.

118. A composition comprising the polypeptide of claim 31.

120. A cell comprising the polypeptide of claim 31.

32-34. -34. (canceled)

38-40. -40. (canceled)

46. An isolated polynucleotide comprising:

(a) a nucleic acid encoding zero to ten HTL epitopes;

(b) a nucleic acid encoding a pan-DR binding epitope;

(c) a nucleic acid encoding an influenza matrix protein 2 external (M2e) sequence, or fragment thereof; and

(d) optionally, a nucleic acid sequence encoding a polypeptide at least 90% identical to an influenza hemagglutinin (HA) sequence, or fragment thereof.

69. A polypeptide encoded by the nucleic acid of claim 46.

72. A vector comprising the polynucleotide of claim 46.

74. A composition comprising the polynucleotide of claim 46.

81. A cell comprising the polynucleotide of claim 46.

82. A method of inducing an immune response against influenza virus in an individual in need thereof, comprising administering to said individual the composition of claim 74.

123. A composition comprising the vector of claim 72.

125. A cell comprising the vector of claim 72.

47-68. -68. (canceled)

70. An isolated polypeptide comprising:

(e) zero to ten HTL epitopes;

(f) a pan-DR binding epitope;

(g) and an influenza matrix protein 2 external (M2e) sequence, or fragment thereof;

(h) optionally, a polypeptide at least 90% identical to an influenza hemagglutinin (HA) sequence, or a fragment thereof;

wherein the polypeptide of (a), (b), and (c), and optionally (d), are arranged in any order relative to one another; and

wherein the pan-DR binding epitope comprises the amino acid sequence a₁KXVAAWTLKAAa₂ (SEQ ID NO;

2), where “

X”

is selected from the group consisting of cyclohexylalanine, phenylalanine, and tyrosine; and

“

a₁”

is either D-alanine or L-alanine; and

“

a₂”

is either D-alanine or L-alanine.

122. A composition comprising the polypeptide of claim 70.

124. A cell comprising the polypeptide of claim 70.

71. (canceled)

73. (canceled)

75-80. -80. (canceled)

100-114. -114. (canceled)