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WO1993015763A1 - Vaccinal polypeptides - Google Patents

Vaccinal polypeptides Download PDF

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Publication number
WO1993015763A1
WO1993015763A1 PCT/US1993/001451 US9301451W WO9315763A1 WO 1993015763 A1 WO1993015763 A1 WO 1993015763A1 US 9301451 W US9301451 W US 9301451W WO 9315763 A1 WO9315763 A1 WO 9315763A1
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WIPO (PCT)
Prior art keywords
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PCT/US1993/001451
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French (fr)
Inventor
Allan Shatzman
Miller Scott
Susan B. Dillon
Original Assignee
Smithkline Beecham Corporation
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Publication of WO1993015763A1 publication Critical patent/WO1993015763A1/en

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16211—Influenzavirus B, i.e. influenza B virus
    • C12N2760/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates generally to a polypeptide useful in a composition for providing
  • Influenza virus infection causes acute respiratory disease in man, horses, swine and fowl, sometimes of pandemic proportions. Influenza viruses are orthomyxoviruses and, as such, have envelope virions of 80 to 120 nanometers in diameter, with two different glycoprotein spikes. Three types, A, B and C, infect humans. Type A viruses have been responsible for the majority of human epidemics in modern history, although there are also sporadic outbreaks of Type B infections. Known swine, equine and avian viruses have mostly been Type A, although Type C viruses have also been isolated from swine.
  • Type A viruses are divided into subtypes based on the antigenic properties of the hemagglutinin (HA) and neuraminidase (NA) surface glycoproteins.
  • HA hemagglutinin
  • NA neuraminidase
  • subtypes H1 swine flu
  • H2 asian flu
  • H3 Hong Kong flu
  • swine flu the predominant influenza A subtypes are H1 and H3; in horses, H3 and H7; and in avians, H5 and H7.
  • avians H5 and H7.
  • Type B virus Presently only one Type B virus has been identified, with no subtypes.
  • the present invention provides compositions containing, and methods for use of, a protein which is capable of inducing protection in animals and avians against challenge with more than one strain of influenza type A and influenza type B.
  • one aspect of the invention provides a DNA sequence encoding a modified purified recombinant protein.
  • the DNA sequence of the invention encodes a modified protein sequence derived from the HA2 subunit of a selected hemagglutinin (HA) protein.
  • HA hemagglutinin
  • the sequence is derived from an H3N2 subtype influenza virus. These H3N2 fusion proteins are capable of inducing T cell responses in the absence of neutralizing antibodies.
  • a DNA sequence of this invention encodes a modified protein sequence derived from the HA2 subunit from a type B influenza virus. Still further embodiments include DNA sequences obtained as described for the two above virus, where the sequences are derived from other Type A
  • influenza strains infecting animals as well as humans include, without limitation, Type A subtypes of H1, H2, H3, H4, H5, H6 and H7.
  • the invention provides a DNA sequence encoding a recombinant fusion protein, in which the desired Type A subtype HA2 subunit sequence or a portion thereof, is fused in frame to another protein or protein fragment capable of enhancing expression of the fusion protein.
  • One embodiment includes the H3N2 subtype HA2 subunit sequence described above fused in frame to another protein or fragment capable of enhancing
  • a fusion protein comprises a type B HA2 sequence, described above, or a portion thereof, fused in frame to another protein or protein fragment capable of enhancing expression of the fusion protein. Still other Type A subtype HA2 sequences can be similarly used. It is desirable that this fusion partner protein be an influenza protein sequence or fragment thereof.
  • a protein encoded by a DNA sequence of the invention is provided.
  • the protein may be a protein sequence derived from the HA2 subunit of a hemagglutinin (HA) protein from a selected Type A subtype virus. Desirably the subtype virus is an H3N2.
  • the protein may be derived from the HA subunit from a type B influenza virus.
  • H5 or H7 subtypes include H5 or H7 subtypes.
  • preferred embodiments include fusion proteins comprising a protein sequence derived from the HA2 subunit of an HA protein from a Type A virus, e.g., an H3N2 subtype, or from a type B virus fused in frame to a selected
  • influenza sequence The proteins of this invention are particularly useful in inducing protection in mammals, especially humans, against challenge by type B or an H3N2 subtype of influenza A.
  • the proteins employing other Type A subtypes, e.g., H5 and H7, are useful in inducing protection in animals against influenza viruses.
  • the invention provides a vaccine composition containing a purified protein of the invention, as described above.
  • a vaccine composition containing a purified protein of the invention, as described above.
  • composition may include a fusion protein of the
  • the vaccine compositions contain an H3HA2 protein of the invention and other influenza antigens; a type B HA2 protein of the invention and other influenza antigens; or both an H3HA2 protein, a BHA2 protein and other influenza antigens.
  • a combination vaccine of the invention will contain an H3HA2 and a BHA2 protein of the invention in combination with influenza antigens derived from the other type A influenza virus subtypes, H1 and H2.
  • An embodiment for use in animals may contain an H5HA2 or H7HA2 protein, among others.
  • a further aspect of this invention is a method for inducing in an animal protection against influenza type A, influenza type B, influenza type C, or
  • Still a further aspect of this invention is a method for inducing in an animal protection against multiple strains of influenza types A and B which
  • Fig. 1 illustrates the nucleic acid sequences of the HA2 portions of (a) A/Udorn [SEQ ID NO: 1], (b) A/Victoria [SEQ ID NO: 3], (c) A/PR/8/34 [SEQ ID NO: 5], and (d) a consensus sequence [SEQ ID NO: 7]. Dashes indicate the same nucleotide as the consensus sequence. Different nucleotides from that of the consensus sequence are reported in lower case letters. Dots indicate no corresponding nucleotide when compared to the consensus sequence.
  • Fig. 2 illustrates the nucleic acid and amino acid sequences of NS1 (1-81) H3HA2 (1-221) fusion protein [SEQ ID NO: 9 & 10].
  • Fig. 3 illustrates the nucleic acid and amino acid sequences of the NS1 (1-81) H3HA2 (77-221) fusion protein [SEQ ID NO: 11 & 12].
  • Fig. 4 illustrates the nucleic acid and amino acid sequences of the type B fusion protein, NS1 1-42 HA2 41-223 . [SEQ ID NO: 13 & 14]. Detailed Description of the Invention
  • the present invention provides novel proteins, DNA sequences, pharmaceutical vaccine compositions and methods of use thereof for conferring protection in vaccinated mammals against one strain, or desirably multiple strains, of influenza viruses.
  • the proteins and vaccine compositions of the present invention demonstrate the ability to stimulate or produce a protective immune response which is capable of recognizing an influenza virus or influenza virus-infected cells and protecting the vaccinated mammal against disease caused thereby.
  • This protective response is desirably a T cell response, produced in the substantial absence of vaccine-induced neutralizing antibody.
  • H3HA2 and BHA2 sequences originating from viral strains to which humans are susceptible
  • similar sequences and molecules can be prepared for veterinary applications.
  • selected HA2 sequences obtained from type A viral strains e.g., H5HA2, H7HA2 and other strains of interest may be obtained following the teachings described herein for the exemplified H3HA2 and BHA2 sequences.
  • H5HA2, H7HA2 and other strains of interest may be obtained following the teachings described herein for the exemplified H3HA2 and BHA2 sequences.
  • this invention is not limited to the exemplified protein and DNA sequences, even though the following disclosure is limited to the two latter sequences for simplicity.
  • Such additional viral HA2 subunits are expected to share the biological
  • this invention provides a protein or fragment thereof characterized by an amino acid sequence derived from the HA2 subunit of a hemagglutinin (HA) protein, e.g., from a H3N2 subtype virus.
  • HA hemagglutinin
  • proteins of the invention are capable of inducing T helper cells, particularly cytotoxic T lymphocytes, in the absence of neutralizing antibodies.
  • H3N2 subtype strains of influenza A include A/Udorn and
  • influenza A may also produce HA proteins for use in vaccine compositions according to this invention.
  • Fig. 1 compares the nucleic acid sequences of the HA2 portions of the A/Udorn [SEQ ID NO: 1] and A/Victoria [SEQ ID NO: 3] strains with the nucleic acid sequence of an H1N1 subtype virus, A/PR/8/34 [SEQ ID NO: 5].
  • a consensus sequence [SEQ ID NO: 7] was computer generated, and may likewise be useful in producing proteins according to this invention. This consensus sequence [SEQ ID NO: 7] can be constructed by a commercially available
  • Proteins according to this invention may include unfused HA2 subunits of the influenza A viruses, particularly H3N2 subtype.
  • H3N2 subtype For example, in one
  • a protein of the invention contains amino acids 1-221 of a selected H3HA2 subunit. In another embodiment, a protein of the invention contains amino acids 77-221 of the H3HA2 subunit. Other fragments of this HA2 amino acid sequence characterized by the ability to stimulate similar immunological activity in an
  • immunized animal are also encompassed by this invention.
  • Proteins of this invention also include fusion proteins comprising a protein sequence derived from the HA2 subunit of an HA protein from a Type A virus, e.g., an H3N2 subtype virus, fused in frame to another protein or protein fragment capable of enhancing expression of the fusion protein.
  • this fusion "partner" protein be an influenza protein sequence or fragment thereof derived from the same or another strain of influenza virus as the HA protein or protein fragment.
  • this fusion partner protein is all or a portion of the influenza virus NS1 gene or an HA2
  • the NS1 portion of the fusion protein is derived from an H1N1 subtype virus, A/PR/8/34.
  • H1N1 subtype virus A/PR/8/34.
  • the NS1 portion may comprise amino acid residues 1 to 42 of H1NS1. In another embodiment the NS1 portion may comprise amino acid residues 1 to 81 of the selected virus.
  • the HA2 fragment may alternatively be fused to a portion of the NS1 peptide derived from a selected Type A virus, e.g., an H3 subtype virus (H3HA2), or a type B (BHA2) virus.
  • H3HA2 H3 subtype virus
  • BHA2 type B virus
  • non-influenza fusion proteins may also produce desirable fusion proteins with the H3N2, or other Type A, or type B protein or portion thereof.
  • the HA2 fragment may be fused to any peptide capable of enhancing its expression in the host cell selected.
  • a fusion "partner" protein or fragment taking into account the desired host cell and utilizing the teachings herein.
  • the fusion proteins of the present invention are not limited by the selection of the "partner" protein or fragment to which the HA2 fragment is fused.
  • the present invention provides a modified protein containing a portion of the HA2 subunit of a type B influenza virus.
  • a type B influenza virus Currently, the preferred human virus strain is B/Lee/40.
  • the vaccinal proteins of this invention are not limited to this type B strain, and other strains
  • HA2 protein infecting other species, or other as yet unidentified type B virus strains, may be used to produce the HA2 protein.
  • type B HA2 proteins may be fused, as described above for the H3HA2 proteins of this invention, or remain unfused. In the construction of a fusion protein
  • a linker sequence may be inserted optionally between the two fused sequences, i.e., between the NS1 portion and the HA2 portion.
  • This optional linker may provide space between the two linked sequences.
  • this linker sequence may encode, if desired, a polypeptide which is selectively cleavable or digestible by conventional chemical or enzymatic methods.
  • the selected cleavage site may be an enzymatic cleavage site, including sites for cleavage by a proteolytic enzyme, such as
  • enterokinase factor Xa
  • trypsin trypsin
  • collagenase and
  • the cleavage site in the linker may be a site capable of being cleaved upon exposure to a selected chemical, e.g., cyanogen bromide or
  • cleavage site if inserted into a linker useful in the fusion sequences of this invention, does not limit this invention. Any desired cleavage site, of which many are known in the art, may be used for this purpose.
  • a presently preferred example of a fusion protein of this invention is NS1 (1-81) H3HA2 (1-221) [SEQ ID NO: 10], which comprises the first 81 amino acids of NS1 fused to amino acid 1 to 221 of the H3HA2 subunit (amino acids 1-221).
  • Another exemplary fusion protein, NS1 (1 - 81) H3HA2 (77-221) [SEQ ID NO: 12] comprises the first 81 amino acids of NS1 fused to amino acid 77 to 221 of the
  • H3HA2 proteins Yet another preferred example of a fusion protein of this invention is NS1 1-42 BHA2 41-223 [SEQ ID NO: 14], which comprises the first 42 amino acids of NS1 fused to amino acids 41 to 223 of the truncated BHA2 subunit.
  • SEQ ID NO: 14 comprises the first 42 amino acids of NS1 fused to amino acids 41 to 223 of the truncated BHA2 subunit.
  • the NS1 (1-81) H3HA2 (1-221) protein [SEQ ID NO: 10] of the invention has a three-dimensional structure which is substantially similar to that of the NS1 (1-81) HA2 (1-222) protein [SEQ ID NO: 16] derived from the H1N1 subtype virus
  • the amino acid sequence of the NS1 (1- 81) H3HA2 (1-221) protein [SEQ ID NO: 10] has only approximately 50% homology with the amino acid sequence of C13 protein [SEQ ID NO: 16].
  • the nucleic acid sequence of the H3HA2 1-221 fragment derived from A/Udorn (nucleotides 25-560 from that virus) [SEQ ID NO: 1] has only approximately 60% homology with the nucleic acid sequence of the H1HA2 1-222 protein derived from strain A/PR/8/34 (nucleotides 1872-2407 from A/PR/8/34) [SEQ ID NO: 5].
  • nucleic acid sequence of H3HA2 1-221 from A/Udorn (nucleotides 1-499 of A/Udorn) [SEQ ID NO: 1] has approximately 99% homology with the nucleic acid sequence of H3HA2 1-221 from A/Victoria/H3/75 (nucleotides 1226-1725 of A/Victoria) [SEQ ID NO: 3]
  • Analogs of the HA2 peptides from a Type A virus, e.g., an H3, or B viruses, included within the definition of this invention, include truncated
  • polypeptides including fragments
  • HA2 polypeptides e.g. mutants that retain the epitopes and thus the biological activity of HA2. It is anticipated that, because the NS1 portion of the fusion peptide provides a means of expressing the protein at high levels and does not appear to play as significant a role in the
  • analogs of the HA2 peptides and/or the fusion partner differ by only 1 to about 4 codon changes.
  • Other examples of analogs include
  • polypeptides with minor amino acid variations from the natural amino acid sequence of HA2 in particular, conservative amino acid replacements.
  • Conservative replacements are those that take place within a family of amino acids that are related in their side chains.
  • isoleucine or valine an aspartate with a glutamate, a threonine with a serine, or a similar conservative replacement of an amino acid with a structurally related amino acid will not have a significant effect on its activity, especially if the replacement does not involve an amino acid at an epitope of the HA2 polypeptide.
  • the HA2 portion of the fusion peptide e.g., H3HA2 1-221 , H3HA2 77-221 and
  • BHA2 41-223 confers the majority of the necessary epitopes for antibody binding or T cell (particularly CTL)
  • the present invention also encompasses DNA sequences of this invention encoding the above-described proteins and fusion proteins, the sequences characterized by having an immunogenic determinant of a modified HA2 subunit of an HA protein, derived from a Type A virus, e.g., an H3 subtype, or type B virus.
  • a Type A virus e.g., an H3 subtype, or type B virus.
  • sequences of this invention encode such HA2 subunits, optionally fused to a DNA sequence encoding a protein or peptide which is capable of enhancing expression of the protein in a selected host cell.
  • the consensus sequence illustrated in Fig. 1(d) may provide a source of HA2 DNA.
  • the currently preferred embodiment provides a DNA sequence encoding a Type A virus, e.g., an H3 or type B HA2 protein or fragment thereof fused in frame to a DNA sequence encoding a portion of the
  • N nonstructural influenza protein 1
  • Coding sequences for the HA2, NS1 and other viral proteins of influenza virus can be prepared
  • influenza viruses including other strains, subtypes and types, are
  • DNA sequences encoding the H3HA2 or BHA2 protein sequences are also included in the present invention, as well as analogs or derivatives thereof.
  • DNA sequences which code for H3 or other Type A or type B HA2 proteins of the invention but which differ in codon sequence due to the degeneracies of the genetic code or variations in the DNA sequence encoding H3HA2, other Type A or BHA2 proteins which are caused by point mutations or by induced modifications to enhance the activity, half-life or production of the peptide encoded thereby are also encompassed in the invention.
  • DNA sequences which hybridize under stringent conditions with the DNA sequences encoding the HA2 subunit proteins e.g., H3HA2 or BHA2 proteins
  • DNA sequences which hybridize under non-stringent conditions with the disclosed sequences, but which encode proteins or fragments retaining the biological activities of the H3HA2 or BHA2 proteins are also included in this
  • the fusion proteins of the invention may be prepared by conventional genetic engineering and
  • proteins may be purified from expression in host cell or vector systems by conventional means.
  • microorganisms and cells including, for example, E.
  • E. coli Bacillus, Streptomyces, Saccharomyces, mammalian and insect cells, are known and available from private and public laboratories and depositories and from commercial vendors.
  • the preferred host is E. coli
  • polypeptide employed in the presently preferred embodiment is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • a preferred method of production employs an alternative expression system in which the ⁇ -lactamase coding sequence is wholly or partially replaced by a coding sequence for an alternative selectable marker such as, for example, kanamycin or chloramphenicol.
  • H3 or other Type A subunit or type B HA2 peptides or fusion protein To aid in expression of the H3 or other Type A subunit or type B HA2 peptides or fusion protein
  • these protein sequences or fragments thereof may also be fused to a polypeptide capable of enhancing expression of these fragments in the selected host system.
  • a polypeptide capable of enhancing expression of these fragments in the selected host system.
  • a peptide would contain a leader sequence fragment that provides for secretion of the Type A subunit fragment, e.g., the H3HA2 fragment, or type B HA2 fragment in the host cell.
  • the leader sequence fragment that provides for secretion of the Type A subunit fragment, e.g., the H3HA2 fragment, or type B HA2 fragment in the host cell.
  • sequence fragment typically encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell.
  • a promoter sequence may be linked directly with the DNA molecule encoding the HA2 fragment.
  • Such polypeptides, promoter and leader sequences are known to those of skill in the art and may be readily selected for expression in the selected host.
  • the present invention is therefore not limited to any particular expression system or vector, nor to any particular purification process from cell lysates or cell medium.
  • proteins and fusion proteins of this invention may be employed in vaccine compositions.
  • compositions of this invention therefore, contain an effective immunogenic amount of a selected HA2 protein, e.g., H3HA2 or BHA2 protein, of the invention in admixture with a suitable adjuvant in a nontoxic and sterile pharmaceutically acceptable carrier.
  • a selected HA2 protein e.g., H3HA2 or BHA2 protein
  • Suitable carriers for vaccine use are well known to those of skill in the art.
  • exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextrin, agar, pectin, peanut oil, olive oil, sesame oil, squalene and water.
  • the carrier or diluent may include a time delay material, such as glyceryl monostearate or glyceryl distearate alone or with a wax.
  • suitable chemical stabilizers may be used to improve the stability of the pharmaceutical preparation. Suitable chemical stabilizers are well known to those of skill in the art and include, for example, citric acid and other agents to adjust pH, chelating or sequestering agents, and
  • Vaccine compositions of this invention may employ an immunogenic amount of a purified recombinant protein as described above.
  • a preferred embodiment of the vaccine of the invention is composed of an aqueous suspension or solution containing the recombinant HA2 protein molecule, e.g., H3HA2 or BHA2, together with an adjuvant, preferably an aluminum, most preferably
  • a preferred protein for use in these vaccine compositions includes a protein comprising amino acid residues 1 to 81 from NS1 fused to C-terminal amino acid residues 1-221 from the hemagglutinin subunit 2 (HA2) from influenza A, subtype H3N2.
  • HA2 hemagglutinin subunit 2
  • preferred vaccine composition of this invention employs a purified recombinant protein made up of amino acid residues 1 to 81 from NS1 fused to amino acid residues
  • Still another preferred vaccine composition of this invention employs a purified recombinant protein made up of amino acid residues 1 to 42 fused to amino acid residues 41-223 of the HA2 from influenza B.
  • Vaccine compositions of the invention may also employ an immunogenic amount of a recombinant protein of the invention in combination with other influenza
  • Suitable influenza antigens for combination in a vaccine composition with the proteins of this invention may be derived from type A, H1 subtype viruses and may include the recombinant fusion proteins described in detail in copending U. S. Patent Application Ser. No.
  • suitable H1 subtype immunogenic proteins include C13 (NS1 (1-81) -D-L-S-R-HA2 (1-222) ) [SEQ ID NO: 15 & 16], D (NS1 (1-81) -Q-I-P-HA2 (65-222) ) [SEQ ID NO: 17 & 18], C13 short (NS1 (1-42) -M-D-L-S-R-HA2 (1-222) ) [SEQ ID NO: 19 & 20], D short (NS1 (1-42) -M-D-H-M-L-T-S-T-R-S-HA2 (66-222) )
  • H1 proteins consist of unfused polypeptides, such as H1HA2 66-222 [SEQ ID NO: 33 & 34] which is disclosed in copending U. S. Patent Application Ser. No. 07/751,898, incorporated herein by reference.
  • one desirable combination vaccine to provide protection against Type A influenza contains NS1 (1-81) H3HA2 (1-221) protein [SEQ ID NO: 9 & 10] of the invention, one or more proteins derived from subtype H1N1 as described above, and an aluminum
  • a combination vaccine of the invention will contain an immunogenic amount of the H3 fusion protein of the invention in combination with immunogenic amounts of influenza antigens derived from the other type A influenza virus subtypes, including among others, H1, H2, H3, H4, H5, H6 and H7 as well as a type B fusion protein of the invention.
  • other preferred combination vaccines would include the NS1 (1- 81) H3HA2 (77-221) protein [SEQ ID NO: 11 & 12] in combination with one or more additional influenza antigens derived from the type or subtype influenza viruses described above.
  • the combination vaccine will protect against influenza infections caused by both type A and type B influenza viruses.
  • Still other combination vaccine compositions will employ other proteins described herein.
  • compositions of the present invention are advantageously made up in a dose unit form adapted for the desired mode of administration.
  • Each unit will contain, at a minimum, a predetermined quantity of the selected HA2 subunit protein, e.g., H3HA2 protein and/or BHA2 protein, and adjuvant calculated to produce the desired therapeutic effect in optional association with a pharmaceutical diluent, carrier, or vehicle.
  • Dosage protocol can be optimized in accordance with standard vaccination practices.
  • the vaccine will be administered intramuscularly, although other routes of administration may be used, such as intradermal. It is expected that an effective
  • immunogenic amount of a protein, fusion protein or combination of proteins of this invention for average adult humans is in the range of 1 to 1000 micrograms.
  • Another desirable immunogenic amount ranges between 50 to 500 micrograms.
  • the proteins of the invention are in admixture with the same amount or more adjuvant to form a vaccine composition.
  • While the proteins described herein have been particularly developed for use in humans (e.g., the H3HA2 and BHA2 sequences), it is expected that due to species cross-reactivity, these vaccines will be useful in other animals, particularly swine. Additionally, similar molecules can be prepared for equine and avian veterinary applications utilizing the HA2 proteins from other strains to which animals are susceptible. Combination vaccines for use in swine would preferably include protections against both H1 and H3 viruses. Combination vaccines for use in equine would preferably include protection against H3 and H7 viruses. Combination vaccines for use in avian species would preferably confer protection against H5 and H7 viruses. Appropriate dosages can be determined by one skilled in veterinary medicine.
  • the specific effective immunogenic amount for any particular patient will depend upon a variety of factors including the age, general health, sex, and diet of the vaccinee; the species of the vaccinee; the time of administration; the route of administration; interactions with any other drugs being administered; and the degree of protection being sought.
  • the vaccine can be administered initially in late summer or early fall and can be readministered two to six weeks later, if desirable, or periodically as immunity wanes, for example, every two to five years.
  • the administration can be repeated at suitable intervals if necessary or desirable.
  • Plasmid pFV88 contains the entire 221 amino acid length HA from A/Udorn, an H3 subtype virus [C. J. Lai et al, Proc. Natl. Acad. Sci. USA. 77:210-214
  • HA nucleic acid sequence is illustrated in Fig. 1 [SEQ ID NO: 1].
  • This plasmid was cut with Pst I.
  • the resulting plasmid is termed pMS3 or pMS3H3HA.
  • Plasmid pAPR801 is a pBR322-derived cloning vector which carries the NS1 coding region (A/PR/8/34). It is described by Young et al, in The Origin of Pandemic Influenza Viruses, ed. by W. G. Laver, Elsevier Science Publishing Co. (1983).
  • Plasmid pAS1 is a pBR322-derived expression vector which contains the P L promoter, an N utilization site (to relieve transcriptional polarity effects in the presence of N protein) and the ell ribosome binding site including the ell translation initiation codon followed immediately by a BamHI site. It is described by
  • Plasmid pAS1 ⁇ EH was prepared by deleting a non-essential EcoRI-HindIII region of pBR322 origin from pAS1.
  • the resulting plasmid, pAS1 ⁇ EH/801 expresses authentic NS1 (230 amino acids).
  • the plasmid has an NcoI site between the codons for amino acids 81 and 82 and an NruI site 3' to the NS sequences.
  • the BamHI site between amino acids 1 and 2 is retained.
  • Plasmid pMG27N a pAS1 derivative [ Mol . Cell. Biol., 5:1015-1024 (1985)] was cut with BamHI and SacI and ligated to a BamHI/NcoI fragment encoding the first 81 amino acids of NS1 from pAS1 ⁇ EH801 and a synthetic DNA NcoI/SacI fragment of the following sequence:
  • Synthetic oligonucleotides were annealed to generate an NcoI 5' overhang sequence (at the 5' end) and a HhaI 3' overhang sequence (at the 3' end).
  • SEQ ID NO: 37 5' -CATGGGCGCCCATATGGGCATATTCGGCG-3'
  • SEQ ID NO: 38 3'- CCGCGGGTATACCCGTATAAGCC -5'
  • the annealing reaction was performed as follows.
  • the annealing mixture was made up of 2.5 ⁇ L each of 5' oligo (1.3 ⁇ g/ ⁇ L), the 3' oligo (1.2 ⁇ g/ ⁇ L), and added water (15 ⁇ L) to a final volume of 20 ⁇ L.
  • the reaction tubes were then placed in 4 mL culture tubes containing water which had been heated to 65°C for 10 minutes and allowed to cool down slowly. The tubes were then put on ice and used immediately for ligation.
  • This three part ligation generates pMG1H3HA2 (1-221) [SEQ ID NO: 9] which codes for the first 81 amino acids of NS1 fused to four amino acids donated from the linker and amino acids 1-221 of the HA2 subunit. This sequence is illustrated in Fig. 2 [SEQ ID NO: 9 & 10]. This molecule is also designated NS1 (1-81) H3HA2 (1-221) [SEQ ID NO: 9 & 10]. EXAMPLE 4 - NS1 (1-81) H3HA2 (77-221) [SEQ ID NO: 11 & 12]
  • pMS3H3HA described in Example 1 above, was digested with EcoRI and end-filled (Klenow).
  • the vector was digested with XbaI.
  • a 487 bp fragment which contains the coding sequence for amino acids 77-221 of the HA2 subunit, was isolated and ligated to the HpaI and XbaI sites of pMG1.
  • the resulting vector codes for a fusion polypeptide containing amino acids 1- 81 of NS1 fused to amino acids 77-221 of the HA2 subunit. This molecule has been termed NS1 (1-81) H3HA2 ⁇ 77-221) and is illustrated in Fig. 3 [SEQ ID NO: 11 & 12].
  • pMG1 was digested with BamHI and NcoI and ligated to the BamHI/NcoI fragment encoding amino acids 2 to 42 of NS1 from pNS1 42 TGF ⁇ .
  • pNS1 42 TGF ⁇ is derived when pASl ⁇ EH801 is cut with NcoI and SalI and ligated to a synthetic DNA encoding human TGF ⁇ as an NcoI/SalI fragment.
  • pNS1 42 TGF ⁇ encodes a protein
  • NS1 comprised of the first 42 amino acids of NS1 and the mature TGF ⁇ sequence.
  • the NS1 portion of pNS1 42 TGF ⁇ contains an amino acid change from Cys to Ser at amino acid #13.
  • pMG 42 A The resulting plasmid, termed pMG 42 A, was then modified to contain an alternative synthetic linker after the NS1 42 sequence with a different set of restriction enzyme sites within which to insert foreign DNA fragments into the three reading frames after the NS1 42 .
  • This linker has the following sequence:
  • pMG 42 B The resulting plasmid is called pMG 42 B.
  • This vector is needed to contain the neomycin phosphotransferase-1 (NPT- 1) gene which confers kanamycin resistance.
  • pOTS207 is a pAS derived cloning vector which carries the kanamycin resistance gene from Tn903 [Berg et al, Microbiology, ed. D.
  • the pOTS207 was digested with EcoRI and PstI, and the 1467 bp fragment containing the kanamycin
  • SEQ ID NO: 41 5' AATTCGTACCTA 3'
  • pMG 42 B was digested with BglII and PstI.
  • the EcoRI/PstI NPT-1 gene fragment and the synthetic oligo linker were ligated to the digested pMG 42 B.
  • the resulting plasmid, pMG 47 Kn allows fusions, in three different reading frames, to the NS 1-42 gene, while allowing antibiotic selection with kanamycin.
  • Plasmid pBHA is a pBR322-derived vector, containing the complete nucleotide sequence of the hemagglutinin (HA) gene of a type B influenza virus (B/Lee/40). It is described by Krystal et al, Proc.
  • pBHA was digested with Rsal and a 813 bp fragment containing the HA subunit was isolated. This fragment was ligated into plasmid pMG 42 Kn (described above) that had been digested with ScaI. During the cloning, a base (T) was deleted from the ScaI recognition site shifting the gene out of the reading frame. The vector was digested with NcoI, and filled-in using Klenow, putting the gene back into the reading frame.
  • the resulting construct expresses a fusion polypeptide containing amino acids 1-42 of NS1 and 41-233 of the HA2 subunit.
  • This construct contains the Cys to Ser change at amino acid #13 of the NS1 portion of the fusion peptide.
  • the seed virus, A/Udorn was prepared according to the procedures described in P. Palese and J. Schulman, Virol., 57:227-237 (1974). Briefly, this technique is as follows. Influenza virus strain A/Udorn was inoculated in 10-day old embryonated hen's eggs into the allantoic cavity. The eggs were incubated for 24-48 hours at 35°C then chilled at 4°C overnight. A portion of the eggshell over the airsac was removed and the allantoic fluid was aseptically removed using a 10-ml syringe. The fluid was centrifuged at low speed (3,000 ⁇ g) to remove
  • Antisera was prepared as follows. 100-200 micrograms of purified virus in complete Freund's
  • the plasmid pMG1H3HA2 (1-221) [SEQ ID NO: 9] was transfected into E. coli strain AR58 [SmithKline Beecham Pharmaceuticals]. Cultures were grown at 32°C to mid-log phase at which time cultures were shifted to 39.5°C for 2 hours. The E. coli cell pellets containing the
  • the plasmid encoding the NS1 (1-81) H3HA2 (77-221) peptide [SEQ ID NO: 11 & 12] was expressed as described in part A above. Production of this peptide was confirmed by
  • the pellet was resuspended by sonication in 50 mM glycine pH 10.0, 5% glycerol, 2 mM EDTA and then the suspension was treated with 1% Triton X-100 [J.T. Baker Chemicals Co.] at 4°C for 60 minutes and
  • the resulting pellet was solubilized in 50 mM Tris, 8 M urea, pH 8.0 and centrifuged to remove any insoluble material. This solubilized material is dialyzed against 10 mM Tris, 1 mM EDTA, pH 8.0 followed, again, by centrifugation of insoluble material.
  • the solubilized material is designated as "crude” material and is used in in vitro and in vivo mouse assays. At this point, the material is approximately 40 - 50% pure.
  • the "crude” material was electrophoresed through an SDS-PAGE and the appropriate H3HA2 protein bands were visualized by KCl staining according to D. Hager et al, Anal. Biochem. 109:76-86 (1980). The band was cut-out and eluted electrophoretically by the "S&S Elutrap Electro-Separation System” [Schleicher &
  • the electro-eluting buffer was the Tris-glycine.
  • a concentrated and eluted sample was obtained and exhaustively dialyzed against 0.01 M NH 4 HCO 3 and 0.02% SDS [M. Hunkapiller et al, Method. Enzymol., 91:227-236 (1983)]. This sample was frozen quickly by dry ice and lyophilized to complete dryness. The lyophilized
  • the protein is usually greater than 75% pure.
  • mice (NIH/Swiss; 15 per group) were vaccinated subcutaneously with 50 or 10 ⁇ g NS1 (1-81) H3HA2 (1-221) [SEQ ID NO: 9 & 10] in aluminum hydroxide on days 0 and 21. The mice were boosted intraperitoneally on day 42 with the protein without adjuvant. On day 47, mice were challenged intranasally with 2 - 3 LD 50 doses of either A/PR/8/34 (H1N1) or A/HK/68 (H3N2) virus, and survival was monitored through day 21.
  • A/PR/8/34 H1N1
  • H3N2 A/HK/68 virus
  • mice vaccinated with NS1 (1-81) H3HA2 (1-221) [SEQ ID NO: 10] and challenged with A/HK/68 (80-93%) was significantly higher than in control mice which were injected with adjuvant only (26% survival).
  • vaccination with NS1 ⁇ 1- 81) H3HA2 (1-221) [SEQ ID NO: 10] did not confer protection against challenge with A/PR/8/34, an H1N1 strain (0-26% survival).
  • protection elicited by NS1 (1-81) H3HA2 (1 . 221) [SEQ ID NO: 10] is selective for antigenically diverse virus strains within the H3 subtype.
  • NS1 (1-81) HA2 (65-222) [SEQ ID NO: 18], derived from the H1N1 subtype) elicits protection from heterosubtypic challenge with H1N1, but not the H3N2 subtype [S Dillon et al,
  • mice 5:A1362 (abs. 5749 and Table 1].
  • mice were challenged with A/HK/68 (H3N2) on day 47, four weeks after the second injection.
  • Control mice were immunized as described above for Table 1, where an ip injection was given at week 6 (5 days prior to challenge).
  • the results in Table 2 show that CB6F 1 mice (15 per group) were significantly protected when challenged with the A/HK/68 heterologous H3 virus strain 5-28 days after the last injection.
  • mice CB6F 1 were divided randomly into six groups, with fifteen in each group. The mice were injected subcutaneously with proteins in Al +3 (100 ⁇ g) on days 0 and 21, and then were challenged with 2-3 LD 50 doses of virus on day 49. Survival was monitored through day 21. The results of this study are illustrated in
  • H3C13 NS1 1-81 H3HA2 1-221 is referred to as H3C13 in the table below.
  • mice immunized with a mixture of the D protein and H3C13 protein in aluminum adjuvant were protected against challenge with either
  • mice immunized with the D protein were protected against H1 but not H3 challenge. Likewise, mice immunized with the
  • MOLECULE TYPE DNA (genomic)
  • AGC ACT CAA GCA GCC ATC GAC CAA
  • ATC 144 Gly Gln Ala Ala Asp Leu Lys Ser Thr Gln Ala Ala Ile Asp Gln Ile
  • GAG CTT CTT GTC GCT CTG GAG AAC CAA CAT ACA ATT GAT CTG ACT GAC 336 Glu Leu Leu Val Ala Leu Glu Asn Gln His Thr Ile Asp Leu Thr Asp
  • MOLECULE TYPE DNA (genomic)
  • AGC ACT CAA GCA GCC ATC GAC CAA
  • ATC 144 Gly Gln Ala Ala Asp Leu Lys Ser Thr Gln Ala Ala Ile Asp Gln Ile
  • GAG CTT CTT GTC GCT CTG GAG AAC CAA CAT ACA ATT GAT CTG ACT GAC 336 Glu Leu Leu Val Ala Leu Glu Asn Gln His Thr Ile Asp Leu Thr Asp
  • MOLECULE TYPE DNA (genomic)
  • GGT CTA TTT GGA GCC ATT GCC
  • GGG GGA TGG ACT GGA 48 Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • xi SEQUENCE DESCRIPTION: SEQ ID NO: 8:
  • MOLECULE TYPE DNA (genomic)
  • ATC AGA AAT GGG ACT TAT GAC CAT GAT GTA TAC AGA GAC GAA GCA TTA 528 Ile Arg Asn Gly Thr Tyr Asp His Asp Val Tyr Arg Asp Glu Ala Leu
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • ATC TAC TCA ACT GTC GCC AGT TCA CTG GTG CTT TTG GTC TCC CTG GGG 672 Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Leu Val Ser Leu Gly
  • MOLECULE TYPE DNA (genomic)

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Abstract

This invention provides vaccine compositions capable of conferring multi-strain immunity against influenza A and influenza B.

Description

VACCINAL POLYPEPTIDES
This is a continuation-in-part of pending
United States patent application Serial Number 751,896; which is a continuation-in-part of United States patent application Serial Number 387,558; which is a
continuation-in-part of United States patent application Serial Number 238,801, now abandoned; which is a
continuation-in-part of United States patent application Serial Number 645,732, now abandoned.
Field of the Invention
The present invention relates generally to a polypeptide useful in a composition for providing
immunity against influenza A and influenza B in an animal.
Background of the Invention
Influenza virus infection causes acute respiratory disease in man, horses, swine and fowl, sometimes of pandemic proportions. Influenza viruses are orthomyxoviruses and, as such, have envelope virions of 80 to 120 nanometers in diameter, with two different glycoprotein spikes. Three types, A, B and C, infect humans. Type A viruses have been responsible for the majority of human epidemics in modern history, although there are also sporadic outbreaks of Type B infections. Known swine, equine and avian viruses have mostly been Type A, although Type C viruses have also been isolated from swine.
The Type A viruses are divided into subtypes based on the antigenic properties of the hemagglutinin (HA) and neuraminidase (NA) surface glycoproteins.
Within type A, subtypes H1 ("swine flu"), H2 ("asian flu") and H3 ("Hong Kong flu") are predominant in human infections. In swine, the predominant influenza A subtypes are H1 and H3; in horses, H3 and H7; and in avians, H5 and H7. Presently only one Type B virus has been identified, with no subtypes.
Genetic "drift" or "shift", i.e., rapid and unpredictable change in the antigen, occurs at
approximately yearly intervals, and affects antigenic determinants in the HA and NA proteins. Therefore, it has not been possible to prepare a "universal" influenza virus vaccine using conventional killed or attenuated viruses, that is, a vaccine which is non-strain specific. Recently, attempts have been made to prepare such
universal, or semi-universal, vaccines from reassortant viruses prepared by crossing different strains. More recently, such attempts have involved recombinant DNA techniques focusing primarily on the HA protein.
There remains a need in the art for vaccine formulations and compositions capable of inducing
protective responses in animals against influenza
viruses. Summary of the Invention
The present invention provides compositions containing, and methods for use of, a protein which is capable of inducing protection in animals and avians against challenge with more than one strain of influenza type A and influenza type B.
Thus, one aspect of the invention provides a DNA sequence encoding a modified purified recombinant protein. The DNA sequence of the invention encodes a modified protein sequence derived from the HA2 subunit of a selected hemagglutinin (HA) protein. In one
embodiment, the sequence is derived from an H3N2 subtype influenza virus. These H3N2 fusion proteins are capable of inducing T cell responses in the absence of neutralizing antibodies. In another embodiment, a DNA sequence of this invention encodes a modified protein sequence derived from the HA2 subunit from a type B influenza virus. Still further embodiments include DNA sequences obtained as described for the two above virus, where the sequences are derived from other Type A
influenza strains infecting animals as well as humans. Such virus include, without limitation, Type A subtypes of H1, H2, H3, H4, H5, H6 and H7.
In another aspect, the invention provides a DNA sequence encoding a recombinant fusion protein, in which the desired Type A subtype HA2 subunit sequence or a portion thereof, is fused in frame to another protein or protein fragment capable of enhancing expression of the fusion protein. One embodiment includes the H3N2 subtype HA2 subunit sequence described above fused in frame to another protein or fragment capable of enhancing
expression thereof. Another embodiment of such a fusion protein comprises a type B HA2 sequence, described above, or a portion thereof, fused in frame to another protein or protein fragment capable of enhancing expression of the fusion protein. Still other Type A subtype HA2 sequences can be similarly used. It is desirable that this fusion partner protein be an influenza protein sequence or fragment thereof. In still another aspect a protein encoded by a DNA sequence of the invention is provided. The protein may be a protein sequence derived from the HA2 subunit of a hemagglutinin (HA) protein from a selected Type A subtype virus. Desirably the subtype virus is an H3N2. In another embodiment, the protein may be derived from the HA subunit from a type B influenza virus. Other embodiments include H5 or H7 subtypes. Additionally, preferred embodiments include fusion proteins comprising a protein sequence derived from the HA2 subunit of an HA protein from a Type A virus, e.g., an H3N2 subtype, or from a type B virus fused in frame to a selected
influenza sequence. The proteins of this invention are particularly useful in inducing protection in mammals, especially humans, against challenge by type B or an H3N2 subtype of influenza A. The proteins employing other Type A subtypes, e.g., H5 and H7, are useful in inducing protection in animals against influenza viruses.
In a further aspect the invention provides a vaccine composition containing a purified protein of the invention, as described above. Such a vaccine
composition may include a fusion protein of the
invention. In other embodiments of the invention, the vaccine compositions contain an H3HA2 protein of the invention and other influenza antigens; a type B HA2 protein of the invention and other influenza antigens; or both an H3HA2 protein, a BHA2 protein and other influenza antigens. In a preferred embodiment for human use, a combination vaccine of the invention will contain an H3HA2 and a BHA2 protein of the invention in combination with influenza antigens derived from the other type A influenza virus subtypes, H1 and H2. An embodiment for use in animals may contain an H5HA2 or H7HA2 protein, among others.
A further aspect of this invention is a method for inducing in an animal protection against influenza type A, influenza type B, influenza type C, or
combinations thereof, which comprises internally
administering to the animal an effective imraunogenic amount of a vaccine composition of the present invention.
Still a further aspect of this invention is a method for inducing in an animal protection against multiple strains of influenza types A and B which
comprises internally administering to the animal an effective immunogenic amount of a vaccine composition of the present invention.
Other aspects and advantages of the present invention are described further in the following detailed description of the preferred embodiments thereof. Brief Description of the Drawings
Fig. 1 illustrates the nucleic acid sequences of the HA2 portions of (a) A/Udorn [SEQ ID NO: 1], (b) A/Victoria [SEQ ID NO: 3], (c) A/PR/8/34 [SEQ ID NO: 5], and (d) a consensus sequence [SEQ ID NO: 7]. Dashes indicate the same nucleotide as the consensus sequence. Different nucleotides from that of the consensus sequence are reported in lower case letters. Dots indicate no corresponding nucleotide when compared to the consensus sequence.
Fig. 2 illustrates the nucleic acid and amino acid sequences of NS1(1-81)H3HA2(1-221) fusion protein [SEQ ID NO: 9 & 10].
Fig. 3 illustrates the nucleic acid and amino acid sequences of the NS1(1-81)H3HA2(77-221) fusion protein [SEQ ID NO: 11 & 12].
Fig. 4 illustrates the nucleic acid and amino acid sequences of the type B fusion protein, NS11-42HA241-223. [SEQ ID NO: 13 & 14]. Detailed Description of the Invention
The present invention provides novel proteins, DNA sequences, pharmaceutical vaccine compositions and methods of use thereof for conferring protection in vaccinated mammals against one strain, or desirably multiple strains, of influenza viruses. The proteins and vaccine compositions of the present invention demonstrate the ability to stimulate or produce a protective immune response which is capable of recognizing an influenza virus or influenza virus-infected cells and protecting the vaccinated mammal against disease caused thereby. This protective response is desirably a T cell response, produced in the substantial absence of vaccine-induced neutralizing antibody.
While the proteins and DNA sequences specifically described herein are directed to the H3HA2 and BHA2 sequences originating from viral strains to which humans are susceptible, it is expected that similar sequences and molecules can be prepared for veterinary applications. For example, selected HA2 sequences obtained from type A viral strains, e.g., H5HA2, H7HA2 and other strains of interest may be obtained following the teachings described herein for the exemplified H3HA2 and BHA2 sequences. One of skill in the art should understand that this invention is not limited to the exemplified protein and DNA sequences, even though the following disclosure is limited to the two latter sequences for simplicity. Such additional viral HA2 subunits are expected to share the biological
characteristics of the exemplified sequences. Thus, this invention provides a protein or fragment thereof characterized by an amino acid sequence derived from the HA2 subunit of a hemagglutinin (HA) protein, e.g., from a H3N2 subtype virus. The H3
proteins of the invention are capable of inducing T helper cells, particularly cytotoxic T lymphocytes, in the absence of neutralizing antibodies. Among H3N2 subtype strains of influenza A include A/Udorn and
A/Victoria viruses. Other H3N2 virus strains of
influenza A may also produce HA proteins for use in vaccine compositions according to this invention. Fig. 1 compares the nucleic acid sequences of the HA2 portions of the A/Udorn [SEQ ID NO: 1] and A/Victoria [SEQ ID NO: 3] strains with the nucleic acid sequence of an H1N1 subtype virus, A/PR/8/34 [SEQ ID NO: 5]. A consensus sequence [SEQ ID NO: 7] was computer generated, and may likewise be useful in producing proteins according to this invention. This consensus sequence [SEQ ID NO: 7] can be constructed by a commercially available
computerized sequence analysis program, such as Genetics Computers Group [Univeristy of Wisconsin].
Proteins according to this invention may include unfused HA2 subunits of the influenza A viruses, particularly H3N2 subtype. For example, in one
embodiment, a protein of the invention contains amino acids 1-221 of a selected H3HA2 subunit. In another embodiment, a protein of the invention contains amino acids 77-221 of the H3HA2 subunit. Other fragments of this HA2 amino acid sequence characterized by the ability to stimulate similar immunological activity in an
immunized animal are also encompassed by this invention.
Proteins of this invention also include fusion proteins comprising a protein sequence derived from the HA2 subunit of an HA protein from a Type A virus, e.g., an H3N2 subtype virus, fused in frame to another protein or protein fragment capable of enhancing expression of the fusion protein. It is desirable that this fusion "partner" protein be an influenza protein sequence or fragment thereof derived from the same or another strain of influenza virus as the HA protein or protein fragment. Preferably, this fusion partner protein is all or a portion of the influenza virus NS1 gene or an HA2
subunit.
In the embodiments exemplified herein, the NS1 portion of the fusion protein is derived from an H1N1 subtype virus, A/PR/8/34. For example, in one
embodiment, the NS1 portion may comprise amino acid residues 1 to 42 of H1NS1. In another embodiment the NS1 portion may comprise amino acid residues 1 to 81 of the selected virus. The HA2 fragment may alternatively be fused to a portion of the NS1 peptide derived from a selected Type A virus, e.g., an H3 subtype virus (H3HA2), or a type B (BHA2) virus.
However, other non-influenza fusion proteins may also produce desirable fusion proteins with the H3N2, or other Type A, or type B protein or portion thereof. Thus, in still another alternative embodiment, as
discussed below, the HA2 fragment may be fused to any peptide capable of enhancing its expression in the host cell selected. One of skill in the art may readily select a fusion "partner" protein or fragment taking into account the desired host cell and utilizing the teachings herein. The fusion proteins of the present invention are not limited by the selection of the "partner" protein or fragment to which the HA2 fragment is fused.
In yet another embodiment, the present invention provides a modified protein containing a portion of the HA2 subunit of a type B influenza virus. Currently, the preferred human virus strain is B/Lee/40. However, the vaccinal proteins of this invention are not limited to this type B strain, and other strains
infecting other species, or other as yet unidentified type B virus strains, may be used to produce the HA2 protein. These type B HA2 proteins may be fused, as described above for the H3HA2 proteins of this invention, or remain unfused. In the construction of a fusion protein
according to this invention, a linker sequence may be inserted optionally between the two fused sequences, i.e., between the NS1 portion and the HA2 portion. This optional linker may provide space between the two linked sequences. Alternatively, this linker sequence may encode, if desired, a polypeptide which is selectively cleavable or digestible by conventional chemical or enzymatic methods. For example, the selected cleavage site may be an enzymatic cleavage site, including sites for cleavage by a proteolytic enzyme, such as
enterokinase, factor Xa, trypsin, collagenase and
thrombin. Alternatively, the cleavage site in the linker may be a site capable of being cleaved upon exposure to a selected chemical, e.g., cyanogen bromide or
hydroxylamine. The cleavage site, if inserted into a linker useful in the fusion sequences of this invention, does not limit this invention. Any desired cleavage site, of which many are known in the art, may be used for this purpose.
A presently preferred example of a fusion protein of this invention is NS1(1-81)H3HA2(1-221) [SEQ ID NO: 10], which comprises the first 81 amino acids of NS1 fused to amino acid 1 to 221 of the H3HA2 subunit (amino acids 1-221). Another exemplary fusion protein, NS1(1- 81)H3HA2(77-221) [SEQ ID NO: 12], comprises the first 81 amino acids of NS1 fused to amino acid 77 to 221 of the
truncated H3HA2 subunit. Yet another preferred example of a fusion protein of this invention is NS11-42BHA241-223 [SEQ ID NO: 14], which comprises the first 42 amino acids of NS1 fused to amino acids 41 to 223 of the truncated BHA2 subunit. These proteins, fusion proteins and similar proteins encoded by the below-described DNA sequences are referred to collectively herein as H3HA2 proteins.
The NS1(1-81)H3HA2(1-221) protein [SEQ ID NO: 10] of the invention has a three-dimensional structure which is substantially similar to that of the NS1(1-81)HA2(1-222) protein [SEQ ID NO: 16] derived from the H1N1 subtype virus
(C13). However, the amino acid sequence of the NS1(1- 81)H3HA2(1-221) protein [SEQ ID NO: 10] has only approximately 50% homology with the amino acid sequence of C13 protein [SEQ ID NO: 16]. Additionally, as illustrated in Fig. 1, the nucleic acid sequence of the H3HA21-221 fragment derived from A/Udorn (nucleotides 25-560 from that virus) [SEQ ID NO: 1] has only approximately 60% homology with the nucleic acid sequence of the H1HA21-222 protein derived from strain A/PR/8/34 (nucleotides 1872-2407 from A/PR/8/34) [SEQ ID NO: 5]. However, the nucleic acid sequence of H3HA21-221 from A/Udorn (nucleotides 1-499 of A/Udorn) [SEQ ID NO: 1] has approximately 99% homology with the nucleic acid sequence of H3HA21-221 from A/Victoria/H3/75 (nucleotides 1226-1725 of A/Victoria) [SEQ ID NO: 3]
[Fiers et al, Cell, 19:683-696 (1980)].
Analogs of the HA2 peptides from a Type A virus, e.g., an H3, or B viruses, included within the definition of this invention, include truncated
polypeptides (including fragments) and HA2 polypeptides, e.g. mutants that retain the epitopes and thus the biological activity of HA2. It is anticipated that, because the NS1 portion of the fusion peptide provides a means of expressing the protein at high levels and does not appear to play as significant a role in the
immunological responses to the HA2 fusion proteins as does the HA2 portion, any number of analogs of this fusion partner can be made.
Typically, the analogs of the HA2 peptides and/or the fusion partner differ by only 1 to about 4 codon changes. Other examples of analogs include
polypeptides with minor amino acid variations from the natural amino acid sequence of HA2; in particular, conservative amino acid replacements. Conservative replacements are those that take place within a family of amino acids that are related in their side chains.
Genetically encoded amino acids are generally divided into four families: (1) acidic = aspartate, glutamate; (2) basic = lysine, arginine, histidine; (3) non-polar = alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar = glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. For example, it is reasonable to expect that an isolated replacement of a leucine with an
isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar conservative replacement of an amino acid with a structurally related amino acid will not have a significant effect on its activity, especially if the replacement does not involve an amino acid at an epitope of the HA2 polypeptide.
The construction of such analogs, given the description herein and conventional methods of protein modification known to one of skill in the art, are believed to be encompassed by this invention.
Currently, it is theorized that the HA2 portion of the fusion peptide (e.g., H3HA21-221, H3HA277-221 and
BHA241-223) confers the majority of the necessary epitopes for antibody binding or T cell (particularly CTL)
targeting. Once these epitope sequences are precisely identified, portions of the HA2 sequence which are not part of these epitopes may be altered without
significantly affecting the bioactivity of the fusion protein. The present invention also encompasses DNA sequences of this invention encoding the above-described proteins and fusion proteins, the sequences characterized by having an immunogenic determinant of a modified HA2 subunit of an HA protein, derived from a Type A virus, e.g., an H3 subtype, or type B virus. Other DNA
sequences of this invention encode such HA2 subunits, optionally fused to a DNA sequence encoding a protein or peptide which is capable of enhancing expression of the protein in a selected host cell. For example, the consensus sequence illustrated in Fig. 1(d) may provide a source of HA2 DNA. The currently preferred embodiment provides a DNA sequence encoding a Type A virus, e.g., an H3 or type B HA2 protein or fragment thereof fused in frame to a DNA sequence encoding a portion of the
nonstructural influenza protein 1 (NS1).
Coding sequences for the HA2, NS1 and other viral proteins of influenza virus can be prepared
synthetically or can be derived from viral RNA or from available cDNA-containing plasmids by known techniques.
For example, in addition to the above-cited references, a DNA coding sequence for HA from the A/Japan/305/57 strain was cloned, sequenced and reported by Gething et al,
Nature, 287: 301-306 (1980). An HA coding sequence for strain A/NT/60/68 was cloned as reported by Sleigh et al, and by Both et al, in Developments in Cell Biology, Elsevier Science Publishing Co., pages 69-79 and 81-89, respectively, (1980). An HA coding sequence for strain A/WSN/33 was cloned as reported by Davis et al, Gene.
10:205-218 (1980); and by Hiti et al, Virology. 111:113-124 (1981). An HA coding sequence for fowl plague virus was cloned as reported by Porter et al and by Emtage et al, both in Developments in Cell Biology, cited above, at pages 39-49 and 157-168. Also, influenza viruses, including other strains, subtypes and types, are
available from clinical specimens and from public
depositories, such as the American Type Culture
Collection (ATCC), Rockville, Maryland, U.S.A.
Allelic variations (naturally-occurring base changes in the species population which may or may not result in an amino acid change) of DNA sequences encoding the H3HA2 or BHA2 protein sequences are also included in the present invention, as well as analogs or derivatives thereof. Similarly, DNA sequences which code for H3 or other Type A or type B HA2 proteins of the invention but which differ in codon sequence due to the degeneracies of the genetic code or variations in the DNA sequence encoding H3HA2, other Type A or BHA2 proteins which are caused by point mutations or by induced modifications to enhance the activity, half-life or production of the peptide encoded thereby are also encompassed in the invention. Also covered by this invention are DNA sequences which hybridize under stringent conditions with the DNA sequences encoding the HA2 subunit proteins, e.g., H3HA2 or BHA2 proteins, of this invention. DNA sequences which hybridize under non-stringent conditions with the disclosed sequences, but which encode proteins or fragments retaining the biological activities of the H3HA2 or BHA2 proteins, are also included in this
invention. Typical conditions for stringent or non-stringent hybridization are known to those of skill in the art. [See, e.g., Sambrook et al, Molecular Cloning. A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, NY (1989)].
The fusion proteins of the invention may be prepared by conventional genetic engineering and
recombinant techniques known to those of skill in the art. Similarly, the proteins may be purified from expression in host cell or vector systems by conventional means.
Systems for cloning and expression of the vaccinal polypeptide of this invention in various
microorganisms and cells, including, for example, E.
coli, Bacillus, Streptomyces, Saccharomyces, mammalian and insect cells, are known and available from private and public laboratories and depositories and from commercial vendors. The preferred host is E. coli
because it can be used to produce large amounts of desired proteins safely and cheaply. The polypeptide employed in the presently preferred embodiment is
expressed in E. coli. To circumvent the requirement of ampicillin for plasmid selection in production
fermentations, a preferred method of production employs an alternative expression system in which the β-lactamase coding sequence is wholly or partially replaced by a coding sequence for an alternative selectable marker such as, for example, kanamycin or chloramphenicol.
To aid in expression of the H3 or other Type A subunit or type B HA2 peptides or fusion protein
described above, these protein sequences or fragments thereof may also be fused to a polypeptide capable of enhancing expression of these fragments in the selected host system. Ordinarily, such a peptide would contain a leader sequence fragment that provides for secretion of the Type A subunit fragment, e.g., the H3HA2 fragment, or type B HA2 fragment in the host cell. The leader
sequence fragment typically encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell. There may be processing sites encoded between the leader sequence and the Type A subtype or type B HA2 fragment that can be cleaved either in vivo or in vitro. Alternatively, a promoter sequence may be linked directly with the DNA molecule encoding the HA2 fragment. Such polypeptides, promoter and leader sequences are known to those of skill in the art and may be readily selected for expression in the selected host.
Construction of expression systems, including expression vectors and transformed host cells are thus within the art. See, generally, methods described in standard texts, such as Sambrook et al, Molecular Cloning A Laboratory Manual. 2d edit., Cold Spring Harbor
Laboratory, Cold Spring Harbor, NY (1989). The present invention is therefore not limited to any particular expression system or vector, nor to any particular purification process from cell lysates or cell medium.
The proteins and fusion proteins of this invention may be employed in vaccine compositions.
Pharmaceutical vaccine compositions of this invention, therefore, contain an effective immunogenic amount of a selected HA2 protein, e.g., H3HA2 or BHA2 protein, of the invention in admixture with a suitable adjuvant in a nontoxic and sterile pharmaceutically acceptable carrier.
Suitable carriers for vaccine use are well known to those of skill in the art. However, exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextrin, agar, pectin, peanut oil, olive oil, sesame oil, squalene and water.
Additionally, the carrier or diluent may include a time delay material, such as glyceryl monostearate or glyceryl distearate alone or with a wax. Optionally, suitable chemical stabilizers may be used to improve the stability of the pharmaceutical preparation. Suitable chemical stabilizers are well known to those of skill in the art and include, for example, citric acid and other agents to adjust pH, chelating or sequestering agents, and
antioxidants.
While any aluminum adjuvant may be used in the vaccine compositions of this invention, two desirable adjuvants are commercially marketed under the trademarks Rehsorptar [Armour Pharmaceuticals, Kankakee, IL] and Rehydragel [Reheis Chemical Co., Berkeley Heights, NJ]. These products are aluminum hydroxide gels which contain approximately 2% w/v Al2O3, which is equivalent to
approximately 10.6 mg/ml Al+3.
Vaccine compositions of this invention may employ an immunogenic amount of a purified recombinant protein as described above. A preferred embodiment of the vaccine of the invention is composed of an aqueous suspension or solution containing the recombinant HA2 protein molecule, e.g., H3HA2 or BHA2, together with an adjuvant, preferably an aluminum, most preferably
aluminum hydroxide, buffered at physiological pH, in a form ready for injection. A preferred protein for use in these vaccine compositions includes a protein comprising amino acid residues 1 to 81 from NS1 fused to C-terminal amino acid residues 1-221 from the hemagglutinin subunit 2 (HA2) from influenza A, subtype H3N2. Another
preferred vaccine composition of this invention employs a purified recombinant protein made up of amino acid residues 1 to 81 from NS1 fused to amino acid residues
77-221 of the HA2 from influenza A, subtype H3N2. Still another preferred vaccine composition of this invention employs a purified recombinant protein made up of amino acid residues 1 to 42 fused to amino acid residues 41-223 of the HA2 from influenza B.
Vaccine compositions of the invention may also employ an immunogenic amount of a recombinant protein of the invention in combination with other influenza
antigens. Suitable influenza antigens for combination in a vaccine composition with the proteins of this invention may be derived from type A, H1 subtype viruses and may include the recombinant fusion proteins described in detail in copending U. S. Patent Application Ser. No.
07/387,200, filed July 28, 1989 and its corresponding European Patent Application No. 366, 238, published May 2, 1990; and in co-pending U. S. Patent Application Ser. No. 07/387,558, filed July 28, 1989 and its corresponding European Patent Application No. 366,239, published May 2, 1990. The C13 protein (NS1(1-81)HA2(1-222,) [SEQ ID NO: 15 & 16], D protein (NS1(1-80)HA2(65-222)) [SEQ ID NO: 17 & 18] and other fusion proteins derived from the H1N1 influenza virus subtype and the recombinant expression and
purification thereof are disclosed in detail in these applications, and in the parent applications identified in this application, all of which are incorporated by reference herein.
More specifically, suitable H1 subtype immunogenic proteins include C13 (NS1(1-81)-D-L-S-R-HA2(1-222)) [SEQ ID NO: 15 & 16], D (NS1(1-81)-Q-I-P-HA2(65-222)) [SEQ ID NO: 17 & 18], C13 short (NS1(1-42)-M-D-L-S-R-HA2(1-222)) [SEQ ID NO: 19 & 20], D short (NS1(1-42)-M-D-H-M-L-T-S-T-R-S-HA2(66-222))
[SEQ ID NO: 21 & 22], A (NS1(1-81)-Q-I-P-HA2(69-222)) [SEQ ID NO: 23 & 24], C (NS1(1-81)-Q-I-P-HA2(81-222)) [SEQ ID NO: 25 & 26], ΔD (NS1(1-81)HA2(150-222)) [SEQ ID NO: 27], Δ13 (NS1(1-81)-D-L-S-R-HA2(1-70)-S-C-L-T-A-Y-H-R) [SEQ ID NO: 28], M (NS1(1-81)-Q-I-P-HA2(65-196)-G-G-S-Y-S-M-E-H-F-R-W-G-K-P-V) [SEQ ID NO: 29], ΔM (NS1(1-81)-Q-I-P-HA2(65-196)-G-G-S-Y-S-M-L-V-N) [SEQ ID NO: 30], ΔM+ (NS1(1-81)-Q-I-P-HA2(65-200)-L-V-L-L) [SEQ ID NO: 31 & 32], These H1N1 fusion proteins are described in published European Patent Application 366,238 and in copending U.S. Patent Application Ser. No. 07/751,896. Other suitable H1 proteins consist of unfused polypeptides, such as H1HA266-222 [SEQ ID NO: 33 & 34] which is disclosed in copending U. S. Patent Application Ser. No. 07/751,898, incorporated herein by reference. Thus, one desirable combination vaccine to provide protection against Type A influenza contains NS1(1-81)H3HA2(1-221) protein [SEQ ID NO: 9 & 10] of the invention, one or more proteins derived from subtype H1N1 as described above, and an aluminum
adjuvant.
Preferably, a combination vaccine of the invention will contain an immunogenic amount of the H3 fusion protein of the invention in combination with immunogenic amounts of influenza antigens derived from the other type A influenza virus subtypes, including among others, H1, H2, H3, H4, H5, H6 and H7 as well as a type B fusion protein of the invention. Therefore, other preferred combination vaccines would include the NS1(1- 81)H3HA2(77-221) protein [SEQ ID NO: 11 & 12] in combination with one or more additional influenza antigens derived from the type or subtype influenza viruses described above. Thus, the combination vaccine will protect against influenza infections caused by both type A and type B influenza viruses. Still other combination vaccine compositions will employ other proteins described herein.
The compositions of the present invention are advantageously made up in a dose unit form adapted for the desired mode of administration. Each unit will contain, at a minimum, a predetermined quantity of the selected HA2 subunit protein, e.g., H3HA2 protein and/or BHA2 protein, and adjuvant calculated to produce the desired therapeutic effect in optional association with a pharmaceutical diluent, carrier, or vehicle.
Dosage protocol can be optimized in accordance with standard vaccination practices. Typically, the vaccine will be administered intramuscularly, although other routes of administration may be used, such as intradermal. It is expected that an effective
immunogenic amount of a protein, fusion protein or combination of proteins of this invention for average adult humans is in the range of 1 to 1000 micrograms.
Another desirable immunogenic amount ranges between 50 to 500 micrograms. Most preferably, the proteins of the invention are in admixture with the same amount or more adjuvant to form a vaccine composition.
While the proteins described herein have been particularly developed for use in humans (e.g., the H3HA2 and BHA2 sequences), it is expected that due to species cross-reactivity, these vaccines will be useful in other animals, particularly swine. Additionally, similar molecules can be prepared for equine and avian veterinary applications utilizing the HA2 proteins from other strains to which animals are susceptible. Combination vaccines for use in swine would preferably include protections against both H1 and H3 viruses. Combination vaccines for use in equine would preferably include protection against H3 and H7 viruses. Combination vaccines for use in avian species would preferably confer protection against H5 and H7 viruses. Appropriate dosages can be determined by one skilled in veterinary medicine.
It will be understood, however, that the specific effective immunogenic amount for any particular patient will depend upon a variety of factors including the age, general health, sex, and diet of the vaccinee; the species of the vaccinee; the time of administration; the route of administration; interactions with any other drugs being administered; and the degree of protection being sought.
The vaccine can be administered initially in late summer or early fall and can be readministered two to six weeks later, if desirable, or periodically as immunity wanes, for example, every two to five years.
Of course, as stated above, the administration can be repeated at suitable intervals if necessary or desirable.
The following examples illustrate methods for preparing H3HA2 and BHA2 fusion proteins of the invention and demonstrate the subtype specific protection against heterologous virus induced upon vaccination with the H3HA2 proteins. These examples are illustrative only and do not limit the scope of the invention. EXAMPLE 1 - PLASMID PMS3H3HA
Plasmid pFV88 contains the entire 221 amino acid length HA from A/Udorn, an H3 subtype virus [C. J. Lai et al, Proc. Natl. Acad. Sci. USA. 77:210-214
(1980)], which HA nucleic acid sequence is illustrated in Fig. 1 [SEQ ID NO: 1]. This plasmid was cut with Pst I. The resulting 1900 bp fragment, which contains the entire HA (HA1 and HA2) fragment and some GC tailing, was then inserted into pUC18 [Bethesda Research Laboratories].
The resulting plasmid is termed pMS3 or pMS3H3HA.
EXAMPLE 2 - pPMG1
Plasmid pAPR801 is a pBR322-derived cloning vector which carries the NS1 coding region (A/PR/8/34). It is described by Young et al, in The Origin of Pandemic Influenza Viruses, ed. by W. G. Laver, Elsevier Science Publishing Co. (1983).
Plasmid pAS1 is a pBR322-derived expression vector which contains the PL promoter, an N utilization site (to relieve transcriptional polarity effects in the presence of N protein) and the ell ribosome binding site including the ell translation initiation codon followed immediately by a BamHI site. It is described by
Rosenberg et al, in Methods Enzymol., 101:123-138 (1983). Plasmid pAS1ΔEH was prepared by deleting a non-essential EcoRI-HindIII region of pBR322 origin from pAS1. A 1236 base pair BamHI fragment of pAPR801, containing the NS1 coding region in 861 base pairs of viral origin and 375 base pairs of pBR322 origin, was inserted into the BamHI site of pAS1ΔEH. The resulting plasmid, pAS1ΔEH/801 expresses authentic NS1 (230 amino acids). The plasmid has an NcoI site between the codons for amino acids 81 and 82 and an NruI site 3' to the NS sequences. The BamHI site between amino acids 1 and 2 is retained.
Plasmid pMG27N, a pAS1 derivative [ Mol . Cell. Biol., 5:1015-1024 (1985)], was cut with BamHI and SacI and ligated to a BamHI/NcoI fragment encoding the first 81 amino acids of NS1 from pAS1ΔEH801 and a synthetic DNA NcoI/SacI fragment of the following sequence:
SEQ ID NO: 35:
5'-CATGGATCATATGTTAACAGATATCAAGGCCTGACTGACTGAGAGCT-3' SEQ ID NO: 36:
3'- CTAGTATACAATTGTCTATAGTTCCGGACTGACTGACTC -5'
The resulting plasmid, pMG1, allows the
insertion of DNA fragments after the first 81 amino acids of NS1 in any of the three reading frames within the synthetic linker fragment followed by termination codons in all three reading frames. EXAMPLE 3 - PMG1H3HA
Plasmid pMG1, described above in Example 2, was digested with NcoI and XbaI, releasing a 54 bp fragment, which was discarded. pMS3H3HA, described in Example 1 above, was digested with HhaI and XbaI, and a 701 bp fragment containing the coding sequence for the HA2 subunit of influenza strain A/Udorn (H3N2) was isolated, as illustrated in Fig. 1 [SEQ ID NO: 1].
Synthetic oligonucleotides were annealed to generate an NcoI 5' overhang sequence (at the 5' end) and a HhaI 3' overhang sequence (at the 3' end). The
sequence of these oligonucleotides is as follows:
SEQ ID NO: 37: 5' -CATGGGCGCCCATATGGGCATATTCGGCG-3' SEQ ID NO: 38: 3'- CCGCGGGTATACCCGTATAAGCC -5' The annealing reaction was performed as follows. The annealing mixture was made up of 2.5μL each of 5' oligo (1.3 μg/μL), the 3' oligo (1.2 μg/μL), and added water (15 μL) to a final volume of 20 μL. The reaction tubes were then placed in 4 mL culture tubes containing water which had been heated to 65°C for 10 minutes and allowed to cool down slowly. The tubes were then put on ice and used immediately for ligation.
This three part ligation generates pMG1H3HA2(1-221) [SEQ ID NO: 9] which codes for the first 81 amino acids of NS1 fused to four amino acids donated from the linker and amino acids 1-221 of the HA2 subunit. This sequence is illustrated in Fig. 2 [SEQ ID NO: 9 & 10]. This molecule is also designated NS1(1-81)H3HA2(1-221) [SEQ ID NO: 9 & 10]. EXAMPLE 4 - NS1(1-81)H3HA2(77-221) [SEQ ID NO: 11 & 12]
pMS3H3HA, described in Example 1 above, was digested with EcoRI and end-filled (Klenow).
Subsequently, the vector was digested with XbaI. A 487 bp fragment, which contains the coding sequence for amino acids 77-221 of the HA2 subunit, was isolated and ligated to the HpaI and XbaI sites of pMG1. The resulting vector codes for a fusion polypeptide containing amino acids 1- 81 of NS1 fused to amino acids 77-221 of the HA2 subunit. This molecule has been termed NS1(1-81)H3HA2{77-221) and is illustrated in Fig. 3 [SEQ ID NO: 11 & 12].
EXAMPLE 5 - PMG42BLHA2
To derive a vector similar to pMG1 (described in Example 2), which contains the coding region for the first 42 amino acids of NS1 father than the first 81 amino acids of NS1, pMG1 was digested with BamHI and NcoI and ligated to the BamHI/NcoI fragment encoding amino acids 2 to 42 of NS1 from pNS142TGFα. pNS142TGFα is derived when pASlΔEH801 is cut with NcoI and SalI and ligated to a synthetic DNA encoding human TGFα as an NcoI/SalI fragment. pNS142TGFα encodes a protein
comprised of the first 42 amino acids of NS1 and the mature TGFα sequence. The NS1 portion of pNS142TGFα contains an amino acid change from Cys to Ser at amino acid #13.
The resulting plasmid, termed pMG42A, was then modified to contain an alternative synthetic linker after the NS142 sequence with a different set of restriction enzyme sites within which to insert foreign DNA fragments into the three reading frames after the NS142. This linker has the following sequence:
SEQ ID NO: 39:
5' -CATGGATCATATGTTAACAAGTACTCGATATCAATGAGTGACTGAAGCT-3 ' SEQ ID NO: 40:
3' - CTAGTATACAATTGTTCATGAGCTATAGTTACTCACTGACT -5'
The resulting plasmid is called pMG42B. This vector is needed to contain the neomycin phosphotransferase-1 (NPT- 1) gene which confers kanamycin resistance.
As described in Shatzman and Rosenberg, Met. Enzymol., 152:661-673 (1987), pOTS207 is a pAS derived cloning vector which carries the kanamycin resistance gene from Tn903 [Berg et al, Microbiology, ed. D.
Schlessinger, pp. 13-15, American Society for
Microbiology (Washington, DC 1978); Nomura et al, The Single-Stranded DNA Phages. ed. D. Denhardt et al, pp.467-472, Cold Spring Harbor Laboratory (New York
1978); Castellazzi et al, Molecul. Gen. Genet., 117:211-218 (1982)]. It was constructed by digesting plasmid pUC8 [Yanisch-Perron et al, Gene. 33:103-119 (1985)], with BamHI and ligated to a BcII fragment containing the kanamycin gene from Tn903. The resulting plasmid, pUC8-Kan, was digested with EcoRI and PstI, and the fragment containing the kanamycin gene was inserted between the EcoRI and PstI sites of pOTSV [Shatzman and Rosenberg, cited above]. The resulting plasmid is pOTS207.
The pOTS207 was digested with EcoRI and PstI, and the 1467 bp fragment containing the kanamycin
resistance gene was isolated. Synthetic
oligonucleotides:
SEQ ID NO: 41: 5' AATTCGTACCTA 3'
SEQ ID NO: 42: 3' GCATGGATCTAG 5'
were made to link the NPT-1 gene to pMG42B vector. pMG42B was digested with BglII and PstI. The EcoRI/PstI NPT-1 gene fragment and the synthetic oligo linker were ligated to the digested pMG42B. The resulting plasmid, pMG47Kn allows fusions, in three different reading frames, to the NS1-42 gene, while allowing antibiotic selection with kanamycin.
Plasmid pBHA is a pBR322-derived vector, containing the complete nucleotide sequence of the hemagglutinin (HA) gene of a type B influenza virus (B/Lee/40). It is described by Krystal et al, Proc.
Natl. Acad. Sci. USA. 79: 4900-4804 (1982). pBHA was digested with Rsal and a 813 bp fragment containing the HA subunit was isolated. This fragment was ligated into plasmid pMG42Kn (described above) that had been digested with ScaI. During the cloning, a base (T) was deleted from the ScaI recognition site shifting the gene out of the reading frame. The vector was digested with NcoI, and filled-in using Klenow, putting the gene back into the reading frame.
The resulting construct, pMG42BLHA2 [SEQ ID NO: 14], expresses a fusion polypeptide containing amino acids 1-42 of NS1 and 41-233 of the HA2 subunit. This construct contains the Cys to Ser change at amino acid #13 of the NS1 portion of the fusion peptide.
In preliminary studies with this construct, vaccinated laboratory mice demonstrated protection from challenge with type B influenza in the absence of
neutralizing antibody for the virus. EXAMPLE 6 - PREPARING SEED VIRUS AND RAISING ANTISERA
The seed virus, A/Udorn, was prepared according to the procedures described in P. Palese and J. Schulman, Virol., 57:227-237 (1974). Briefly, this technique is as follows. Influenza virus strain A/Udorn was inoculated in 10-day old embryonated hen's eggs into the allantoic cavity. The eggs were incubated for 24-48 hours at 35°C then chilled at 4°C overnight. A portion of the eggshell over the airsac was removed and the allantoic fluid was aseptically removed using a 10-ml syringe. The fluid was centrifuged at low speed (3,000 × g) to remove
particulates. This clarified supernatant was centrifuged at high speed using an SW28 Beckman rotor at 27,000 rpm (4°C for 90 minutes), resulting in the virus pellet. The virus was resuspended in 10 mM Tris (pH 7.5) containing 100 mM NaCl, 1 mM EDTA and repelleted as before. The virus was layered on 30-60% sucrose gradient in 1 mM EDTA (NTE) and spun for 3-5 hours at 25,000 rpm. The band in the middle of the tube was withdrawn, diluted in NTE and centrifuged at 27,000 rpm for 90 minutes. The pellet was suspended in phosphate-buffered saline (PBS). These viral particles were used as immunogens for preparation of antisera.
Antisera was prepared as follows. 100-200 micrograms of purified virus in complete Freund's
adjuvant was injected into the subscapula of a New
Zealand White rabbit. A second injection in incomplete Freund's adjuvant was done 4 weeks later, and the animals were bled 7-10 days later. EXAMPLE 7 - EXPRESSION OF H3HA2 FUSION PROTEINS
A. NS1(1-81)H3HA2(1-221) [SEQ ID NO: 9 & 10]
The plasmid pMG1H3HA2(1-221) [SEQ ID NO: 9] was transfected into E. coli strain AR58 [SmithKline Beecham Pharmaceuticals]. Cultures were grown at 32°C to mid-log phase at which time cultures were shifted to 39.5°C for 2 hours. The E. coli cell pellets containing the
recombinant polypeptide were then stored at -70°C until used.
Production of the NS1(1-81)H3HA2(1-221) protein [SEQ ID
NO: 10] was confirmed by Western blot analysis [Towbin et al, Proc. Natl. Acad. Sci. U.S.A.. 76:4350 (1979)] using antisera prepared against A/Udorn virus, as described in Example 5. A major immunoreactive species was found at a molecular weight of 35,050 daltons.
B. NS1(1-81)H3HA2(77-221) [SEQ ID NO: 11 & 12]
The plasmid encoding the NS1(1-81)H3HA2(77-221) peptide [SEQ ID NO: 11 & 12] was expressed as described in part A above. Production of this peptide was confirmed by
Western blot analysis, as described above. A major immunoreactive species was found at a molecular weight of 26,697 daltons. EXAMPLE 8 - PARTIAL PURIFICATION OF H3HA2 FUSION PROTEINS E. coli cell pellets containing the recombinant polypeptides, prepared as described in Example 6, were stored at -70°C until used. E. coli cells were thawed and resuspended in lysis buffer A (50 mM Tris-HCl, 5% glycerol, 2 mM EDTA and 0.1 mM DTT, pH 8.0) at 10
mL/gram. The stirred suspension was then treated with lysozyme (0.2 mg/mL) for 45 minutes at room temperature and sonicated 2× for 2-3 minutes each time by a
Sonicator. The resultant suspension was treated with 0.1% DOC for 60 minutes at 4°C, then centrifuged at
25,000 × g. The pellet was resuspended by sonication in 50 mM glycine pH 10.0, 5% glycerol, 2 mM EDTA and then the suspension was treated with 1% Triton X-100 [J.T. Baker Chemicals Co.] at 4°C for 60 minutes and
centrifuged as above.
The resulting pellet was solubilized in 50 mM Tris, 8 M urea, pH 8.0 and centrifuged to remove any insoluble material. This solubilized material is dialyzed against 10 mM Tris, 1 mM EDTA, pH 8.0 followed, again, by centrifugation of insoluble material. The solubilized material is designated as "crude" material and is used in in vitro and in vivo mouse assays. At this point, the material is approximately 40 - 50% pure. The "crude" material was electrophoresed through an SDS-PAGE and the appropriate H3HA2 protein bands were visualized by KCl staining according to D. Hager et al, Anal. Biochem. 109:76-86 (1980). The band was cut-out and eluted electrophoretically by the "S&S Elutrap Electro-Separation System" [Schleicher &
Schuell]. The electro-eluting buffer was the Tris-glycine. A concentrated and eluted sample was obtained and exhaustively dialyzed against 0.01 M NH4HCO3 and 0.02% SDS [M. Hunkapiller et al, Method. Enzymol., 91:227-236 (1983)]. This sample was frozen quickly by dry ice and lyophilized to complete dryness. The lyophilized
material was brought back into solution using 50 mM Tris pH 8.0 and used for in vitro and in vivo mouse assays.
Following this gel elution step, the protein is usually greater than 75% pure.
EXAMPLE 9 - H3 SUBTYPE HETEROLOGOUS PROTECTION ELICITED BY VACCINATION WITH NS1(1-81)H3HA2(1-221) [SEQ ID NO: 10]
Mice (NIH/Swiss; 15 per group) were vaccinated subcutaneously with 50 or 10 μg NS1(1-81)H3HA2(1-221) [SEQ ID NO: 9 & 10] in aluminum hydroxide on days 0 and 21. The mice were boosted intraperitoneally on day 42 with the protein without adjuvant. On day 47, mice were challenged intranasally with 2 - 3 LD50 doses of either A/PR/8/34 (H1N1) or A/HK/68 (H3N2) virus, and survival was monitored through day 21. This represents a heterologous challenge (A/PR/8/34) and an H3 heterosubtypic challenge, since the NS1(1-81)H3HA2(1-221) construct [SEQ ID NO: 9 & 10] was derived from A/Udorn/72 cDNA. The control group received adjuvant (CFA) only.
The results in Table 1 below show that survival in mice vaccinated with NS1(1-81)H3HA2(1-221) [SEQ ID NO: 10] and challenged with A/HK/68 (80-93%) was significantly higher than in control mice which were injected with adjuvant only (26% survival). In contrast, vaccination with NS1{1- 81)H3HA2(1-221) [SEQ ID NO: 10] did not confer protection against challenge with A/PR/8/34, an H1N1 strain (0-26% survival). Thus protection elicited by NS1(1-81)H3HA2(1.221) [SEQ ID NO: 10] is selective for antigenically diverse virus strains within the H3 subtype.
Likewise, vaccination with the D protein
(NS1(1-81)HA2(65-222) [SEQ ID NO: 18], derived from the H1N1 subtype) elicits protection from heterosubtypic challenge with H1N1, but not the H3N2 subtype [S Dillon et al,
Nature, in press (1992); Mbawuike et al, Faseb. J.,
5:A1362 (abs. 5749 and Table 1]. These results in outbred mice also suggest that the response to the H1 and H3 proteins will not be restricted to a limited number of individuals with certain major histocompatibility
alleles, and therefore the vaccine will be effective in a majority of individuals. Table 1
Percent Survival After Challenge:
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
Immunization HA A/PR/8/34 A/HK/68
Subtype (H1N1) (H3N2)
50 μg NS11-81H3HA21-221 H3 26 80*
10 μg NS11-81H3HA21-221 H3 0 93*
10 μg NS11-81HA244-222 H1 67* 13
A/HK/68 Virus H3 60* 100*
Control (Al+3) - 0 26
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - p ≤ 0.05 vs. control in Fishers exact probability test
Vaccination of mice with live homologous
(A/HK/68) virus provided complete or partial protection, reflecting protection mediated by neutralizing antibody
(homologous H3N2 challenge) and/or CTL (heterologous H1N1 challenge), respectively.
Duration of protective immunity was tested by immunizing mice subcutaneously with the recombinant influenza protein plus adjuvant on days 0 and 21. Some mice were also given an ip injection of the protein
(without adjuvant) on day 42. Mice were challenged with A/HK/68 (H3N2) on day 47, four weeks after the second injection. Control mice were immunized as described above for Table 1, where an ip injection was given at week 6 (5 days prior to challenge). The results in Table 2 show that CB6F1 mice (15 per group) were significantly protected when challenged with the A/HK/68 heterologous H3 virus strain 5-28 days after the last injection. Table 2
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
Dose (μg per injection) Injection Percent
of NS11-81H3HA21-221 Adjuvant Schedule Survival
50 μg CFA 0,21 86*
50 μg CFA 0,21,42 100*
0 μg CFA 0,21 6
50 μg Al+3 0,21 93*
50 μg Al+3 0,21,42 93*
0 μg Al+3 0,21 0
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
*p ≤ 0.05 v. control in Fisher's exact probability test
EXAMPLE 10 - TYPE A CROSS-PROTECTION WITH D AND H3C13 PROTEIN
Mice (CB6F1) were divided randomly into six groups, with fifteen in each group. The mice were injected subcutaneously with proteins in Al+3 (100 μg) on days 0 and 21, and then were challenged with 2-3 LD50 doses of virus on day 49. Survival was monitored through day 21. The results of this study are illustrated in
Table 3 below. For convenience, NS11-81H3HA21-221 is referred to as H3C13 in the table below.
Table 3
Percent Survival After Challenge with:
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
HA A/PR/8/34 A/HK/68
Immunization Subtype (H1N1) (H3N2}
1. 50 μg H3C13 H3 73* 73*
50 μg D H1
2. 10 μg H3C13 H3 67* 100*
10 μg D H1
3. 1 μg H3C13 H3 86* 73*
1 μg D H1
4. 50 μg H3C13 H3 7 73*
5. 50 μg D H1 47** 7
6. Al+3 control - 7 0
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
* p ≤ 0.001 vs. control group
** p ≤ 0.03 vs. control group
This data demonstrates that mice immunized with a mixture of the D protein and H3C13 protein in aluminum adjuvant were protected against challenge with either
A/PR/8/34 (H1) or A/HK/68 (H3) virus. In contrast, mice immunized with the D protein were protected against H1 but not H3 challenge. Likewise, mice immunized with the
H3C13 protein were protected against the H3 but not the H1 challenge. Therefore, the combination of the D protein and the H3C13 proteins elicited protection against the currently circulating subtypes of influenza A virus. Thus, this combination represents a subtype cross-protective vaccine. Numerous modifications and variations of the present invention are included in the above-identified specification and are expected to be obvious to one of skill in the art. Such modifications and alterations to the compositions and processes of the present invention are believed to be encompassed in the scope of the claims appended hereto.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Shatzman, Allan
Scott, Miller
Dillon, Susan B.
(ii) TITLE OF INVENTION: Vaccinal Polypeptides
(iii) NUMBER OF SEQUENCES: 42
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: SmithKline Beecham Corporation - Corporate
Patents
(B) STREET: U.S. Mailcode VW2220 - 709 Swedeland Road
(C) CITY: King of Prussia
(D ) STATE: Pennsylvania
(E) COUNTRY: USA
(F) ZIP: 19406-2799
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: US
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Canter, Carol G.
(B) REGISTRATION NUMBER: 31,151
(C) REFERENCE/DOCKET NUMBER: SBC14224-8
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 215-270-5013
(B) TELEFAX: 215-270-5090
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 666 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..663 ( xi ) SEQUENCE DESCRIPTION : SEQ ID NO: 1 :
GGC ATA TTC GGC GCA ATA GCA GGT TTC ATA GAA AAT GGT TGG GAG GGA 48
Gly Ile Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly
1 5 10 15
ATG ATA GAC GGT TGG TAC GGT TTC AGG CAT CAA AAT TCT GAG GGC ACA 96 Met Ile Asp Gly Trp Tyr Gly Phe Arg His Gln Asn Ser Glu Gly Thr
20 25 30
GGA CAA GCA GCA GAT CTT AAA AGC ACT CAA GCA GCC ATC GAC CAA ATC 144 Gly Gln Ala Ala Asp Leu Lys Ser Thr Gln Ala Ala Ile Asp Gln Ile
35 40 45
AAT GGG AAA CTG AAT AGG GTA ATC GAG AAG ACG AAC GAG AAA TTC CAT 192 Asn Gly Lys Leu Asn Arg Val Ile Glu Lys Thr Asn Glu Lys Phe His
50 55 60
CAA ATC GAA AAG GAA TTC TCA GAA GTA GAA GGG AGA ATT CAG GAC CTC 240 Gln Ile Glu Lys Glu Phe Ser Glu Val Glu Gly Arg Ile Gln Asp Leu
65 70 75 80
GAG AAA TAC GTT GAA GAC ACT AAA ATA GAT CTC TGG TCT TAC AAT GCG 288 Glu Lys Tyr Val Glu Asp Thr Lys Ile Asp Leu Trp Ser Tyr Asn Ala
85 90 95
GAG CTT CTT GTC GCT CTG GAG AAC CAA CAT ACA ATT GAT CTG ACT GAC 336 Glu Leu Leu Val Ala Leu Glu Asn Gln His Thr Ile Asp Leu Thr Asp
100 105 110
TCG GAA ATG AAC AAA CTG TTT GAA AAA ACA AGG AGG CAA CTG AGG GAA 384 Ser Glu Met Asn Lys Leu Phe Glu Lys Thr Arg Arg Gln Leu Arg Glu
115 120 125
AAT GCT GAG GAC ATG GGC AAT GGT TGC TTC AAA ATA TAC CAC AAA TGT 432 Asn Ala Glu Asp Met Gly Asn Gly Cys Phe Lys Ile Tyr His Lys Cys
130 135 140
GAC AAT GCT TGC ATA GGG TCA ATC AGA AAT GGG ACT TAT GAC CAT GAT 480 Asp Asn Ala Cys Ile Gly Ser Ile Arg Asn Gly Thr Tyr Asp His Asp
145 150 155 160
GTA TAC AGA GAC GAA GCA TTA AAC AAC CGG TTT CAG ATC AAA GGT GTT 528 Val Tyr Arg Asp Glu Ala Leu Asn Asn Arg Phe Gln Ile Lys Gly Val
165 170 175
GAA CTG AAG TCA GGA TAC AAA GAC TGG ATC CTG TGG ATT TCC TTT GCC 576 Glu Leu Lys Ser Gly Tyr Lys Asp Trp Ile Leu Trp Ile Ser Phe Ala
180 185 190
ATA TCA TGC TTT TTG CTT TGT GTT GTT TTG CTG GGG TTC ATC ATG TGG 624 Ile Ser Cys Phe Leu Leu Cys Val Val Leu Leu Gly Phe Ile Met Trp
195 200 205
GCC TGC CAG AAA GGC AAC ATT AGG TGC AAC ATT TGC ATT TGA 666
Ala Cys Gln Lys Gly Asn Ile Arg Cys Asn Ile Cys Ile
210 215 220 (2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 221 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Gly Ile Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly 1 5 10 15
Met Ile Asp Gly Trp Tyr Gly Phe Arg His Gln Asn Ser Glu Gly Thr
20 25 30
Gly Gln Ala Ala Asp Leu Lys Ser Thr Gln Ala Ala Ile Asp Gln Ile
35 40 45
Asn Gly Lys Leu Asn Arg Val Ile Glu Lys Thr Asn Glu Lys Phe His 50 55 60
Gln Ile Glu Lys Glu Phe Ser Glu Val Glu Gly Arg Ile Gln Asp Leu 65 70 75 80
Glu Lys Tyr Val Glu Asp Thr Lys Ile Asp Leu Trp Ser Tyr Asn Ala
85 90 95
Glu Leu Leu Val Ala Leu Glu Asn Gln His Thr Ile Asp Leu Thr Asp
100 105 110
Ser Glu Met Asn Lys Leu Phe Glu Lys Thr Arg Arg Gln Leu Arg Glu
115 120 125
Asn Ala Glu Asp Met Gly Asn Gly Cys Phe Lys Ile Tyr His Lys Cys 130 135 140
Asp Asn Ala Cys Ile Gly Ser Ile Arg Asn Gly Thr Tyr Asp His Asp 145 150 155 160
Val Tyr Arg Asp Glu Ala Leu Asn Asn Arg Phe Gln Ile Lys Gly Val
165 170 175
Glu Leu Lys Ser Gly Tyr Lys Asp Trp Ile Leu Trp Ile Ser Phe Ala
180 185 190
Ile Ser Cys Phe Leu Leu Cys Val Val Leu Leu Gly Phe Ile Met Trp
195 200 205
Ala Cys Gln Lys Gly Asn Ile Arg Cys Asn Ile Cys Ile
210 215 220 (2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 666 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..663
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
GGC ATA TTC GGC GCA ATA GCA GGT TTC ATA GAA AAT GGT TGG GAG GGA 48 Gly Ile Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly
1 5 10 15
ATG ATA GAC GGT TGG TAC GGT TTC AGG CAT CAA AAT TCC GAG GGC ACA 96 Met Ile Asp Gly Trp Tyr Gly Phe Arg His Gln Asn Ser Glu Gly Thr
20 25 30
GGA CAA GCA GCA GAT CTT AAA AGC ACT CAA GCA GCC ATC GAC CAA ATC 144 Gly Gln Ala Ala Asp Leu Lys Ser Thr Gln Ala Ala Ile Asp Gln Ile
35 40 45
AAT GGG AAA CTG AAT AGG GTA ATC GAG AAG ACG AAC GAG AAA TTC CAT 192 Asn Gly Lys Leu Asn Arg Val Ile Glu Lys Thr Asn Glu Lys Phe His
50 55 60
CAA ATC GAA AAG GAA TTC TCA GAA GTA GAA GGG AGA ATT CAG GAC CTC 240 Gln Ile Glu Lys Glu Phe Ser Glu Val Glu Gly Arg Ile Gln Asp Leu
65 70 75 80
GAG AAA TAC GTT GAA GAC ACT AAA ATA GAT CTC TGG TCT TAC AAT GCG 288 Glu Lys Tyr Val Glu Asp Thr Lys Ile Asp Leu Trp Ser Tyr Asn Ala
85 90 95
GAG CTT CTT GTC GCT CTG GAG AAC CAA CAT ACA ATT GAT CTG ACT GAC 336 Glu Leu Leu Val Ala Leu Glu Asn Gln His Thr Ile Asp Leu Thr Asp
100 105 110
TCG GAA ATG AAC AAA CTG TTT GAA AAA ACA AGG AGG CAA CTG AGG GAA 384 Ser Glu Met Asn Lys Leu Phe Glu Lys Thr Arg Arg Gln Leu Arg Glu
115 120 125
AAT GCT GAG GAC ATG GGC AAT GGT TGC TTC AAA ATA TAC CAC AAA TGT 432 Asn Ala Glu Asp Met Gly Asn Gly Cys Phe Lys Ile Tyr His Lys Cys
130 135 140
GAC AAT GCT TGC ATA GGG TCA ATC AGA AAT GGG ACT TAT GAC CAT GAT 480 Asp Asn Ala Cys Ile Gly Ser Ile Arg Asn Gly Thr Tyr Asp His Asp
145 150 155 160
GTA TAC AGA GAC GAA GCA TTA AAC AAC CGG TTT CAG ATC AAA GGT GTT 528 Val Tyr Arg Asp Glu Ala Leu Asn Asn Arg Phe Gln Ile Lys Gly Val
165 170 175
GAA CTG AAG TCA GGA TAC AAA GAC TGG ATC CTG TGG ATT TCC TTT GCC 576 Glu Leu Lys Ser Gly Tyr Lys Asp Trp Ile Leu Trp Ile Ser Phe Ala
180 185 190 ATA TCA TGC TTT TTG CTT TGT GTT GTT TTG CTG GGG TTC ATC ATG TGG 624 Ile Ser Cys Phe Leu Leu Cys Val Val Leu Leu Gly Phe Ile Met Trp
195 200 205
GCC TGC CAA AAA GGC AAC ATT AGG TGC AAC ATT TGC ATT TGA 666
Ala Cys Gln Lys Gly Asn Ile Arg Cys Asn Ile Cys Ile
210 215 220
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 221 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Gly Ile Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly
1 5 10 15
Met Ile Asp Gly Trp Tyr Gly Phe Arg His Gln Asn Ser Glu Gly Thr
20 25 30
Gly Gln Ala Ala Asp Leu Lys Ser Thr Gln Ala Ala Ile Asp Gln Ile
35 40 45
Asn Gly Lys Leu Asn Arg Val Ile Glu Lys Thr Asn Glu Lys Phe His
50 55 60
Gln Ile Glu Lys Glu Phe Ser Glu Val Glu Gly Arg Ile Gln Asp Leu
65 70 75 80
Glu Lys Tyr Val Glu Asp Thr Lys Ile Asp Leu Trp Ser Tyr Asn Ala
85 90 95
Glu Leu Leu Val Ala Leu Glu Asn Gln His Thr Ile Asp Leu Thr Asp
100 105 110
Ser Glu Met Asn Lys Leu Phe Glu Lys Thr Arg Arg Gln Leu Arg Glu
115 120 125
Asn Ala Glu Asp Met Gly Asn Gly Cys Phe Lys Ile Tyr His Lys Cys
130 135 140
Asp Asn Ala Cys Ile Gly Ser Ile Arg Asn Gly Thr Tyr Asp His Asp
145 150 155 160
Val Tyr Arg Asp Glu Ala Leu Asn Asn Arg Phe Gln Ile Lys Gly Val
165 170 175
Glu Leu Lys Ser Gly Tyr Lys Asp Trp Ile Leu Trp Ile Ser Phe Ala
180 185 190
Ile Ser Cys Phe Leu Leu Cys Val Val Leu Leu Gly Phe Ile Met Trp
195 200 205
Ala Cys Gln Lys Gly Asn Ile Arg Cys Asn Ile Cys Ile
210 215 220 (2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 670 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE :
(A) NAME/KEY: CDS
(B) LOCATION: 1..666
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
GGT CTA TTT GGA GCC ATT GCC GGT TTT ATT GAA GGG GGA TGG ACT GGA 48 Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly
1 5 10 15
ATG ATA GAT GGA TGG TAC GGT TAT CAT CAT CAG AAT GAA CAG GGA TCA 96 Met Ile Asp Gly Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser
20 25 30
GGC TAT GCA GCG GAT CAA AAA AGC ACA CAA AAT GCC ATT AAC GGG ATT 144 Gly Tyr Ala Ala Asp Gln Lys Ser Thr Gln Asn Ala Ile Asn Gly lie
35 40 45
ACA AAC AAG GTG AAC TCT GTT ATC GAG AAA ATG AAC ATT CAA TTC ACA 192 Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn Ile Gln Phe Thr
50 55 60
GCT GTG GGT AAA GAA TTC AAC AAA TTA GAA AAA AGG ATG GAA AAT TTA 240 Ala Val Gly Lys Glu Phe Asn Lys Leu Glu Lys Arg Met Glu Asn Leu
65 70 75 80
AAT AAA AAA GTT GAT GAT GGA TTT CTG GAC ATT TGG ACA TAT AAT GCA 288 Asn Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala
85 90 95
GAA TTG TTA GTT CTA CTG GAA AAT GAA AGG ACT CTG GAT TTC CAT GAC 336 Glu Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Phe His Asp
100 105 110
TCA AAT GTG AAG AAT CTG TAT GAG AAA GTA AAA AGC CAA TTA AAG AAT 384 Ser Asn Val Lys Asn Leu Tyr Glu Lys Val Lys Ser Gln Leu Lys Asn
115 120 125
AAT GCC AAA GAA ATC GGA AAT GGA TGT TTT GAG TTC TAC CAC AAG TGT 432 Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys
130 135 140
GAC AAT GAA TGC ATG GAA AGT GTA AGA AAT GGG ACT TAT GAT TAT CCC 480 Asp Asn Glu Cys Met Glu Ser Val Arg Asn Gly Thr Tyr Asp Tyr Pro
145 150 155 160
AAA TAT TCA GAA GAG TCA AAG TTG AAC AGG GAA AAG GTA GAT GGA GTG 528 Lys Tyr Ser Glu Glu Ser Lys Leu Asn Arg Glu Lys Val Asp Gly Val
165 170 175 AAA TTG GAA TCA ATG GGG ATC TAT CAG ATT CTG GCG ATC TAC TCA ACT 576 Lys Leu Glu Ser Met Gly Ile Tyr Gln Ile Leu Ala Ile Tyr Ser Thr
180 185 190
GTC GCC AGT TCA CTG GTG CTT TTG GTC TCC CTG GGG GCA ATC AGT TTC 624 Val Ala Ser Ser Leu Val Leu Leu Val Ser Leu Gly Ala Ile Ser Phe
195 200 205
TGG ATG TGT TCT AAT GGA TCT TTG CAG TGC AGA ATA TGC ATC 666
Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile Cys Ile
210 215 220
TGAG 670
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 222 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly
1 5 10 15
Met Ile Asp Gly Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser
20 25 30
Gly Tyr Ala Ala Asp Gln Lys Ser Thr Gln Asn Ala Ile Asn Gly Ile
35 40 45
Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn Ile Gln Phe Thr
50 55 60
Ala Val Gly Lys Glu Phe Asn Lys Leu Glu Lys Arg Met Glu Asn Leu
65 70 75 80
Asn Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala
85 90 95
Glu Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Phe His Asp
100 105 110
Ser Asn Val Lys Asn Leu Tyr Glu Lys Val Lys Ser Gln Leu Lys Asn
115 120 125
Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys
130 135 140
Asp Asn Glu Cys Met Glu Ser Val Arg Asn Gly Thr Tyr Asp Tyr Pro
145 150 155 160
Lys Tyr Ser Glu Glu Ser Lys Leu Asn Arg Glu Lys Val Asp Gly Val
165 170 175 Lys Leu Glu Ser Met Gly Ile Tyr Gln Ile Leu Ala Ile Tyr Ser Thr
180 185 190
Val Ala Ser Ser Leu Val Leu Leu Val Ser Leu Gly Ala Ile Ser Phe
195 200 205
Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile Cys Ile
210 215 220
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 670 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..670
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
GGCATATTCG GCGCAATAGC AGGTTTCATA GAAAATGGTT GGGAGGGAAT GATAGACGGT 60
TGGTACGGTT TCAGGCATCA AAATTCNGAG GGCACAGGAC AAGCAGCAGA TCTTAAAAGC 120
ACTCAAGCAG CCATCGACCA AATCAATGGG AAACTGAATA GGGTAATCGA GAAGACGAAC 180
GAGAAATTCC ATCAAATCGA AAAGGAATTC TCAGAAGTAG AAGGGAGAAT TCAGGACCTC 240
GAGAAATACG TTGAAGACAC TAAAATAGAT CTCTGGTCTT ACAATGCGGA GCTTCTTGTC 300
GCTCTGGAGA ACCAACATAC AATTGATCTG ACTGACTCGG AAATGAACAA ACTGTTTGAA 360
AAAACAAGGA GGCAACTGAG GGAAAATGCT GAGGACATGG GCAATGGTTG CTTCAAAATA 420
TACCACAAAT GTGACAATGC TTGCATAGGG TCAATCAGAA ATGGGACTTA TGACCATGAT 480
GTATACAGAG ACGAAGCATT AAACAACCGG TTTCAGATCA AAGGTGTTGA ACTGAAGTCA 540
GGATACAAAG ACTGGATCCT GTGGATTTCC TTTGCCATAT CATGCTTTTT GCTTTGTGTT 600
GTTTTGCTGG GGTTCATCAN NNTGTGGGCC TGCCANAAAG GCAACATTAG GTGCAACATT 660
TGCATTTGAN 670
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 222 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
Gly Ile Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly 1 5 10 15
Met Ile Asp Gly Trp Tyr Gly Phe Arg His Gln Asn Ser Glu Gly Thr
20 25 30
Gly Gln Ala Ala Asp Leu Lys Ser Thr Gln Ala Ala Ile Asp Gln Ile
35 40 45
Asn Gly Lys Leu Asn Arg Val Ile Glu Lys Thr Asn Glu Lys Phe His 50 55 60
Gln Ile Glu Lys Glu Phe Ser Glu Val Glu Gly Arg Ile Gln Asp Leu 65 70 75 80
Glu Lys Tyr Val Glu Asp Thr Lys Ile Asp Leu Trp Ser Tyr Asn Ala
85 90 95
Glu Leu Leu Val Ala Leu Glu Asn Gln His Thr Ile Asp Leu Thr Asp
100 105 110
Ser Glu Met Asn Lys Leu Phe Glu Lys Thr Arg Arg Gln Leu Arg Glu
115 120 125
Asn Ala Glu Asp Met Gly Asn Gly Cys Phe Lys Ile Tyr His Lys Cys 130 135 140
Asp Asn Ala Cys Ile Gly Ser Ile Arg Asn Gly Thr Tyr Asp His Asp 145 150 155 160
Val Tyr Arg Asp Glu Ala Leu Asn Asn Arg Phe Gln Ile Lys Gly Val
165 170 175
Glu Leu Lys Ser Xaa Gly Tyr Lys Asp Trp Ile Leu Trp Ile Ser Phe
180 185 190
Ala Ile Ser Cys Phe Leu Leu Cys Val Val Leu Leu Gly Phe Ile Met
195 200 205
Trp Ala Cys Gln Lys Gly Asn Ile Arg Cys Asn Ile Cys Ile
210 215 220
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 918 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic]
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..918 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
ATG GAT CCA AAC ACT GTG TCA AGC TTT CAG GTA GAT TGC TTT CTT TGG 48 Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp
1 5 10 15
CAT GTC CGC AAA CGA GTT GCA GAC CAA GAA CTA GGT GAT GCC CCA TTC 96 His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe
20 25 30
CTT GAT CGG CTT CGC CGA GAT CAG AAA TCC CTA AGA GGA AGG GGC AGC 144 Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser
35 40 45
ACT CTT GGT CTG GAC ATC GAG ACA GCC ACA CGT GCT GGA AAG CAG ATA 192 Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Arg Ala Gly Lys Gln Ile
50 55 60
GTG GAG CGG ATT CTG AAA GAA GAA TCC GAT GAG GCA CTT AAA ATG ACC 240 Val Glu Arg Ile Leu Lys Glu Glu Ser Asp Glu Ala Leu Lys Met Thr
65 70 75 80
ATG GGC GCC CAT ATG GGC ATA TTC GGC GCA ATA GCA GGT TTC ATA GAA 288 Met Gly Ala His Met Gly Ile Phe Gly Ala Ile Ala Gly Phe Ile Glu
85 90 95
AAT GGT TGG GAG GGA ATG ATA GAC GGT TGG TAC GGT TTC AGG CAT CAA 336 Asn Gly Trp Glu Gly Met Ile Asp Gly Trp Tyr Gly Phe Arg His Gln
100 105 110
AAT TCT GAG GGC ACA GGA CAA GCA GCA GAT CTT AAA AGC ACT CAA GCA 384 Asn Ser Glu Gly Thr Gly Gln Ala Ala Asp Leu Lys Ser Thr Gln Ala
115 120 125
GCC ATC GAC CAA ATC AAT GGG AAA CTG AAT AGG GTA ATC GAG AAG ACG 432 Ala Ile Asp Gln Ile Asn Gly Lys Leu Asn Arg Val Ile Glu Lys Thr
130 135 140
AAC GAG AAA TTC CAT CAA ATC GAA AAG GAA TTC TCA GAA GTA GAA GGG 480 Asn Glu Lys Phe His Gln Ile Glu Lys Glu Phe Ser Glu Val Glu Gly
145 150 155 160
AGA ATT CAG GAC CTC GAG AAA TAC GTT GAA GAC ACT AAA ATA GAT CTC 528 Arg Ile Gln Asp Leu Glu Lys Tyr Val Glu Asp Thr Lys Ile Asp Leu
165 170 175
TGG TCT TAC AAT GCG GAG CTT CTT GTC GCT CTG GAG AAC CAA CAT ACA 576 Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu Asn Gln His Thr
180 185 190
ATT GAT CTG ACT GAC TCG GAA ATG AAC AAA CTG TTT GAA AAA ACA AGG 624 Ile Asp Leu Thr Asp Ser Glu Met Asn Lys Leu Phe Glu Lys Thr Arg
195 200 205
AGG CAA CTG AGG GAA AAT GCT GAG GAC ATG GGC AAT GGT TGC TTC AAA 672 Arg Gln Leu Arg Glu Asn Ala Glu Asp Met Gly Asn Gly Cys Phe Lys
210 215 220
ATA TAC CAC AAA TGT GAC AAT GCT TGC ATA GGG TCA ATC AGA AAT GGG 720 Ile Tyr His Lys Cys Asp Asn Ala Cys Ile Gly Ser Ile Arg Asn Gly
225 230 235 240 ACT TAT GAC CAT GAT GTA TAC AGA GAC GAA GCA TTA AAC AAC CGG TTT 768 Thr Tyr Asp His Asp Val Tyr Arg Asp Glu Ala Leu Asn Asn Arg Phe
245 250 255
CAG ATC AAA GGT GTT GAA CTG AAG TCA GGA TAC AAA GAC TGG ATC CTG 816 Gln Ile Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys Asp Trp Ile Leu
260 265 270
TGG ATT TCC TTT GCC ATA TCA TGC TTT TTG CTT TGT GTT GTT TTG CTG 864 Trp Ile Ser Phe Ala Ile Ser Cys Phe Leu Leu Cys Val Val Leu Leu
275 280 285
GGG TTC ATC ATG TGG GCC TGC CAA AAA GGC AAC ATT AGG TGC AAC ATT 912 Gly Phe Ile Met Trp Ala Cys Gln Lys Gly Asn Ile Arg Cys Asn Ile
290 295 300
TGC ATT 918
Cys Ile
305
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 306 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp
1 5 10 15
His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe
20 25 30
Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser
35 40 45
Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Arg Ala Gly Lys Gln Ile
50 55 60
Val Glu Arg Ile Leu Lys Glu Glu Ser Asp Glu Ala Leu Lys Met Thr
65 70 75 80
Met Gly Ala His Met Gly Ile Phe Gly Ala Ile Ala Gly Phe Ile Glu
85 90 95
Asn Gly Trp Glu Gly Met Ile Asp Gly Trp Tyr Gly Phe Arg His Gln
100 105 110
Asn Ser Glu Gly Thr Gly Gln Ala Ala Asp Leu Lys Ser Thr Gln Ala
115 120 125
Ala Ile Asp Gln Ile Asn Gly Lys Leu Asn Arg Val Ile Glu Lys Thr
130 135 140
Asn Glu Lys Phe His Gln Ile Glu Lys Glu Phe Ser Glu Val Glu Gly
145 150 155 160 Arg Ile Gln Asp Leu Glu Lys Tyr Val Glu Asp Thr Lys Ile Asp Leu
165 170 175
Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu Asn Gln His Thr
180 185 190
Ile Asp Leu Thr Asp Ser Glu Met Asn Lys Leu Phe Glu Lys Thr Arg
195 200 205
Arg Gln Leu Arg Glu Asn Ala Glu Asp Met Gly Asn Gly Cys Phe Lys
210 215 220
Ile Tyr His Lys Cys Asp Asn Ala Cys Ile Gly Ser Ile Arg Asn Gly
225 230 235 240
Thr Tyr Asp His Asp Val Tyr Arg Asp Glu Ala Leu Asn Asn Arg Phe
245 250 255 Gln Ile Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys Asp Trp Ile Leu
260 265 270
Trp Ile Ser Phe Ala Ile Ser Cys Phe Leu Leu Cys Val Val Leu Leu
275 280 285
Gly Phe Ile Met Trp Ala Cys Gln Lys Gly Asn Ile Arg Cys Asn Ile
290 295 300
Cys Ile
305
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 690 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
( ix ) FEATURE :
(A) NAME/KEY: CDS
(B) LOCATION: 1..690
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
ATG GAT CCA AAC ACT GTG TCA AGC TTT CAG GTA GAT TGC TTT CTT TGG 48 Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp
1 5 10 15
CAT GTC CGC AAA CGA GTT GCA GAC CAA GAA CTA GGT GAT GCC CCA TTC 96 His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe
20 25 30
CTT GAT CGG CTT CGC CGA GAT CAG AAA TCC CTA AGA GGA AGG GGC AGC 144 Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser
35 40 45
ACT CTT GGT CTG GAC ATC GAG ACA GCC ACA CGT GCT GGA AAG CAG ATA 192 Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Arg Ala Gly Lys Gln Ile
50 55 60 GTG GAG CGG ATT CTG AAA GAA GAA TCC GAT GAG GCA CTT AAA ATG ACC 240 Val Glu Arg Ile Leu Lys Glu Glu Ser Asp Glu Ala Leu Lys Met Thr
65 70 75 80
ATG GAT CAT ATG TTA ATT CAG GAC CTC GAG AAA TAC GTT GAA GAC ACT 288 Met Asp His Met Leu Ile Gln Asp Leu Glu Lys Tyr Val Glu Asp Thr
85 90 95
AAA ATA GAT CTC TGG TCT TAC AAT GCG GAG CTT CTT GTC GCT CTG GAG 336 Lys Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu
100 105 110
AAC CAA CAT ACA ATT GAT CTG ACT GAC TCG GAA ATG AAC AAA CTG TTT 384 Asn Gln His Thr Ile Asp Leu Thr Asp Ser Glu Met Asn Lys Leu Phe
115 120 125
GAA AAA ACA AGG AGG CAA CTG AGG GAA AAT GCT GAG GAC ATG GGC AAT 432 Glu Lys Thr Arg Arg Gln Leu Arg Glu Asn Ala Glu Asp Met Gly Asn
130 135 140
GGT TGC TTC AAA ATA TAC CAC AAA TGT GAC AAT GCT TGC ATA GGG TCA 480 Gly Cys Phe Lys Ile Tyr His Lys Cys Asp Asn Ala Cys Ile Gly Ser
145 150 155 160
ATC AGA AAT GGG ACT TAT GAC CAT GAT GTA TAC AGA GAC GAA GCA TTA 528 Ile Arg Asn Gly Thr Tyr Asp His Asp Val Tyr Arg Asp Glu Ala Leu
165 170 175
AAC AAC CGG TTT CAG ATC AAA GGT GTT GAA CTG AAG TCA GGA TAC AAA 576 Asn Asn Arg Phe Gln Ile Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys
180 185 190
GAC TGG ATC CTG TGG ATT TCC TTT GCC ATA TCA TGC TTT TTG CTT TGT 624 Asp Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser Cys Phe Leu Leu Cys
195 200 205
GTT GTT TTG CTG GGG TTC ATC ATG TGG GCC TGC CAA AAA GGC AAC ATT 672 Val Val Leu Leu Gly Phe Ile Met Trp Ala Cys Gln Lys Gly Asn Ile
210 215 220
AGG TGC AAC ATT TGC ATT 690
Arg Cys Asn Ile Cys Ile
225 230
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 230 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp
1 5 10 15
His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe
20 25 30 Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser 35 40 45
Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Arg Ala Gly Lys Gln Ile 50 55 60
Val Glu Arg Ile Leu Lys Glu Glu Ser Asp Glu Ala Leu Lys Met Thr 65 70 75 80
Met Asp His Met Leu Ile Gln Asp Leu Glu Lys Tyr Val Glu Asp Thr
85 90 95 Lys Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu
100 105 110
Asn Gln His Thr Ile Asp Leu Thr Asp Ser Glu Met Asn Lys Leu Phe
115 120 125
Glu Lys Thr Arg Arg Gln Leu Arg Glu Asn Ala Glu Asp Met Gly Asn 130 135 140
Gly Cys Phe Lys Ile Tyr His Lys Cys Asp Asn Ala Cys Ile Gly Ser
145 150 155 160 Ile Arg Asn Gly Thr Tyr Asp His Asp Val Tyr Arg Asp Glu Ala Leu
165 170 175
Asn Asn Arg Phe Gln Ile Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys
180 185 190
Asp Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser Cys Phe Leu Leu Cys
195 200 205
Val Val Leu Leu Gly Phe Ile Met Trp Ala Cys Gln Lys Gly Asn Ile 210 215 220
Arg Cys Asn Ile Cys Ile
225 230
(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 699 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..699 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
ATG GAT CCA AAC ACT GTG TCA AGC TTT CAG GTA GAT TCC TTT CTT TGG 48 Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Ser Phe Leu Trp
1 5 10 15
CAT GTC CGC AAA CGA GTT GCA GAC CAA GAA CTA GGT GAT GCC CCA TTC 96 His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe
20 25 30
CTT GAT CGG CTT CGC CGA GAT CAG AAA TCC ATG CAT GGA TCA TAT GTT 144 Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Met His Gly Ser Tyr Val
35 40 45
AAC AAG ACA CAA GAA GCT ATA AAC AAG ATA ACA AAA AAT CTC AAC TAT 192 Asn Lys Thr Gln Glu Ala Ile Asn Lys Ile Thr Lys Asn Leu Asn Tyr
50 55 60
TTA AGT GAG CTA GAA GTA AAA AAC CTT CAA AGA CTA AGC GGA GCA ATG 240 Leu Ser Glu Leu Glu Val Lys Asn Leu Gln Arg Leu Ser Gly Ala Met
65 70 75 80
AAT GAG CTT CAC GAC GAA ATA CTC GAG CTA GAC GAA AAA GTG GAT GAT 288 Asn Glu Leu His Asp Glu Ile Leu Glu Leu Asp Glu Lys Val Asp Asp
85 90 95
CTA AGA GCT GAT ACA ATA AGC TCA CAA ATA GAG CTT GCA GTC TTG CTT 336 Leu Arg Ala Asp Thr Ile Ser Ser Gln Ile Glu Leu Ala Val Leu Leu
100 105 110
TCC AAC GAA GGG ATA ATA AAC AGT GAA GAT GAG CAT CTC TTG GCA CTT 384 Ser Asn Glu Gly Ile Ile Asn Ser Glu Asp Glu His Leu Leu Ala Leu
115 120 125
GAA AGA AAA CTG AAG AAA ATG CTT GGC CCC TCT GCT GTA GAA ATA GGG 432 Glu Arg Lys Leu Lys Lys Met Leu Gly Pro Ser Ala Val Glu Ile Gly
130 135 140
AAT GGG TGC TTT GAA ACC AAA CAC AAA TGC AAC CAG ACT TGC CTA GAC 480 Asn Gly Cys Phe Glu Thr Lys His Lys Cys Asn Gln Thr Cys Leu Asp
145 150 155 160
AGG ATA GCT GCT GGC ACC TTT AAT GCA GGA GAT TTT TCT CTT CCC ACT 528 Arg Ile Ala Ala Gly Thr Phe Asn Ala Gly Asp Phe Ser Leu Pro Thr
165 170 . 175
TTT GAT TCA TTA AAC ATT ACT GCT GCA TCT TTA AAT GAT GAT GGC TTG 576 Phe Asp Ser Leu Asn Ile Thr Ala Ala Ser Leu Asn Asp Asp Gly Leu
180 185 190
GAT AAT CAT ACT ATA CTG CTC TAC TAC TCA ACT GCT GCT TCT AGC TTG 624 Asp Asn His Thr Ile Leu Leu Tyr Tyr Ser Thr Ala Ala Ser Ser Leu
195 200 205
GCT GTA ACA TTA ATG ATA GCT ATC TTC ATT GTC TAC ATG GTC TCC AGA 672 Ala Val Thr Leu Met Ile Ala Ile Phe Ile Val Tyr Met Val Ser Arg
210 215 220
GAC AAT GTT TCT TGT TCC ATC TGT CTG 699
Asp Asn Val Ser Cys Ser Ile Cys Leu
225 230 (2) INFORMATION FOR SEQ ID NO: 14 :
( i ) SEQUENCE CHARACTERISTICS :
(A) LENGTH: 233 amino acids
( B ) TYPE : amino acid
(D ) TOPOLOGY: linear
( ii ) MOLECULE TYPE : protein
( xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14 :
Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Ser Phe Leu Trp
1 5 10 15
His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe
20 25 30
Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Met His Gly Ser Tyr Val
35 40 45
Asn Lys Thr Gln Glu Ala Ile Asn Lys Ile Thr Lys Asn Leu Asn Tyr 50 55 60
Leu Ser Glu Leu Glu Val Lys Asn Leu Gln Arg Leu Ser Gly Ala Met 65 70 75 80
Asn Glu Leu His Asp Glu Ile Leu Glu Leu Asp Glu Lys Val Asp Asp
85 90 95
Leu Arg Ala Asp Thr Ile Ser Ser Gln Ile Glu Leu Ala Val Leu Leu
100 105 110
Ser Asn Glu Gly Ile Ile Asn Ser Glu Asp Glu His Leu Leu Ala Leu
115 120 125
Glu Arg Lys Leu Lys Lys Met Leu Gly Pro Ser Ala Val Glu Ile Gly 130 135 140
Asn Gly Cys Phe Glu Thr Lys His Lys Cys Asn Gln Thr Cys Leu Asp 145 150 155 160
Arg Ile Ala Ala Gly Thr Phe Asn Ala Gly Asp Phe Ser Leu Pro Thr
165 170 175
Phe Asp Ser Leu Asn Ile Thr Ala Ala Ser Leu Asn Asp Asp Gly Leu
180 185 190
Asp Asn His Thr Ile Leu Leu Tyr Tyr Ser Thr Ala Ala Ser Ser Leu
195 200 205
Ala Val Thr Leu Met Ile Ala Ile Phe Ile Val Tyr Met Val Ser Arg 210 215 220
Asp Asn Val Ser Cys Ser Ile Cys Leu
225 230 (2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 924 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
( ix ) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..921
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
ATG GAT CCA AAC ACT GTG TCA AGC TTT CAG GTA GAT TGC TTT CTT TGG 48 Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp
1 5 10 15
CAT GTC CGC AAA CGA GTT GCA GAC CAA GAA CTA GGT GAT GCC CCA TTC 96 His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe
20 25 30
CTT GAT CGG CTT CGC CGA GAT CAG AAA TCC CTA AGA GGA AGG GGC AGC 144 Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser
35 40 45
ACT CTT GGT CTG GAC ATC GAG ACA GCC ACA CGT GCT GGA AAG CAG ATA 192 Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Arg Ala Gly Lys Gln Ile
50 55 60
GTG GAG CGG ATT CTG AAA GAA GAA TCC GAT GAG GCA CTT AAA ATG ACC 240 Val Glu Arg Ile Leu Lys Glu Glu Ser Asp Glu Ala Leu Lys Met Thr
65 70 75 80
ATG GAT CTG TCC AGA GGT CTA TTT GGA GCC ATT GCC GGT TTT ATT GAA 288 Met Asp Leu Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu
85 90 95
GGG GGA TGG ACT GGA ATG ATA GAT GGA TGG TAC GGT TAT CAT CAT CAG 336 Gly Gly Trp Thr Gly Met Ile Asp Gly Trp Tyr Gly Tyr His His Gln
100 105 110
AAT GAA CAG GGA TCA GGC TAT GCA GCG GAT CAA AAA AGC ACA CAA AAT 384 Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Gln Lys Ser Thr Gln Asn
115 120 125
GCC ATT AAC GGG ATT ACA AAC AAG GTG AAC TCT GTT ATC GAG AAA ATG 432 Ala Ile Asn Gly Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met
130 135 140
AAC ATT CAA TTC ACA GCT GTG GGT AAA GAA TTC AAC AAA TTA GAA AAA 480 Asn Ile Gln Phe Thr Ala Val Gly Lys Glu Phe Asn Lys Leu Glu Lys
145 150 155 160
AGG ATG GAA AAT TTA AAT AAA AAA GTT GAT GAT GGA TTT CTG GAC ATT 528 Arg Met Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe Leu Asp Ile
165 170 175 TGG ACA TAT AAT GCA GAA TTG TTA GTT CTA CTG GAA AAT GAA AGG ACT 576 Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu Arg Thr
180 185 190
CTG GAT TTC CAT GAC TCA AAT GTG AAG AAT CTG TAT GAG AAA GTA AAA 624 Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys Val Lys
195 200 205
AGC CAA TTA AAG AAT AAT GCC AAA GAA ATC GGA AAT GGA TGT TTT GAG 672 Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu
210 215 220
TTC TAC CAC AAG TGT GAC AAT GAA TGC ATG GAA AGT GTA AGA AAT GGG 720 Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser Val Arg Asn Gly
225 230 235 240
ACT TAT GAT TAT CCC AAA TAT TCA GAA GAG TCA AAG TTG AAC AGG GAA 768 Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ser Lys Leu Asn Arg Glu
245 250 255
AAG GTA GAT GGA GTG AAA TTG GAA TCA ATG GGG ATC TAT CAG ATT CTG 816 Lys Val Asp Gly Val Lys Leu Glu Ser Met Gly Ile Tyr Gln Ile Leu
260 265 270
GCG ATC TAC TCA ACT GTC GCC AGT TCA CTG GTG CTT TTG GTC TCC CTG 864 Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Leu Val Ser Leu
275 280 285
GGG GCA ATC AGT TTC TGG ATG TGT TCT AAT GGA TCT TTG CAG TGC AGA 912 Gly Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg
290 295 300
ATA TGC ATC TGA 924 Ile Cys Ile
305
(2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 307 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp
1 5 10 15
His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe
20 25 30
Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser
35 40 45
Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Arg Ala Gly Lys Gln Ile
50 55 60
Val Glu Arg Ile Leu Lys Glu Glu Ser Asp Glu Ala Leu Lys Met Thr
65 70 75 80 Met Asp Leu Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu 85 90 95
Gly Gly Trp Thr Gly Met Ile Asp Gly Trp Tyr Gly Tyr His His Gln
100 105 110
Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Gln Lys Ser Thr Gln Asn
115 120 125
Ala Ile Asn Gly Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met 130 135 140
Asn Ile Gln Phe Thr Ala Val Gly Lys Glu Phe Asn Lys Leu Glu Lys 145 150 155 160
Arg Met Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe Leu Asp Ile
165 170 175
Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu Arg Thr
180 185 190
Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys Val Lys
195 200 205
Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu 210 215 220
Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser Val Arg Asn Gly 225 230 235 240
Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ser Lys Leu Asn Arg Glu
245 250 255
Lys Val Asp Gly Val Lys Leu Glu Ser Met Gly Ile Tyr Gln Ile Leu
260 265 270
Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Leu Val Ser Leu
275 280 285
Gly Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg 290 295 300
Ile Cys Ile
305
(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 729 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..726 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
ATG GAT CCA AAC ACT GTG TCA AGC TTT CAG GTA GAT TGC TTT CTT TGG 48 Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp
1 5 10 15
CAT GTC CGC AAA CGA GTT GCA GAC CAA GAA CTA GGT GAT GCC CCA TTC 96 His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe
20 25 30
CTT GAT CGG CTT CGC CGA GAT CAG AAA TCC CTA AGA GGA AGG GGC AGC 144 Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser
35 40 45
ACT CTT GGT CTG GAC ATC GAG ACA GCC ACA CGT GCT GGA AAG CAG ATA 192 Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Arg Ala Gly Lys Gln Ile
50 55 60
GTG GAG CGG ATT CTG AAA GAA GAA TCC GAT GAG GCA CTT AAA ATG ACC 240 Val Glu Arg Ile Leu Lys Glu Glu Ser Asp Glu Ala Leu Lys Met Thr
65 70 75 80
ATG CAG ATC CCG GCT GTG GGT AAA GAA TTC AAC AAA TTA GAA AAA AGG 288 Met Gln Ile Pro Ala Val Gly Lys Glu Phe Asn Lys Leu Glu Lys Arg
85 90 95
ATG GAA AAT TTA AAT AAA AAA GTT GAT GAT GGA TTT CTG GAC ATT TGG 336 Met Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp
100 105 110
ACA TAT AAT GCA GAA TTG TTA GTT CTA CTG GAA AAT GAA AGG ACT CTG 384 Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu
115 120 125
GAT TTC CAT GAC TCA AAT GTG AAG AAT CTG TAT GAG AAA GTA AAA AGC 432 Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys Val Lys Ser
130 135 140
CAA TTA AAG AAT AAT GCC AAA GAA ATC GGA AAT GGA TGT TTT GAG TTC 480 Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe
145 150 155 160
TAC CAC AAG TGT GAC AAT GAA TGC ATG GAA AGT GTA AGA AAT GGG ACT 528 Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser Val Arg Asn Gly Thr
165 170 175
TAT GAT TAT CCC AAA TAT TCA GAA GAG TCA AAG TTG AAC AGG GAA AAG 576 Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ser Lys Leu Asn Arg Glu Lys
180 185 190
GTA GAT GGA GTG AAA TTG GAA TCA ATG GGG ATC TAT CAG ATT CTG GCG 624 Val Asp Gly Val Lys Leu Glu Ser Met Gly Ile Tyr Gln Ile Leu Ala
195 200 205
ATC TAC TCA ACT GTC GCC AGT TCA CTG GTG CTT TTG GTC TCC CTG GGG 672 Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Leu Val Ser Leu Gly
210 215 220 GCA ATC AGT TTC TGG ATG TGT TCT AAT GGA TCT TTG CAG TGC AGA ATA 720 Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile
225 230 235 240
TGC ATC TGA 729
Cys Ile
(2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 242 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp
1 5 10 15
His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe
20 25 30
Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser
35 40 45
Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Arg Ala Gly Lys Gln Ile
50 55 60
Val Glu Arg Ile Leu Lys Glu Glu Ser Asp Glu Ala Leu Lys Met Thr
65 70 75 80
Met Gln Ile Pro Ala Val Gly Lys Glu Phe Asn Lys Leu Glu Lys Arg
85 90 95
Met Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp
100 105 110
Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu
115 120 125
Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys Val Lys Ser
130 135 140
Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe
145 150 155 160
Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser Val Arg Asn Gly Thr
165 170 175
Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ser Lys Leu Asn Arg Glu Lys
180 185 190
Val Asp Gly Val Lys Leu Glu Ser Met Gly Ile Tyr Gln Ile Leu Ala
195 200 205
Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Leu Val Ser Leu Gly
210 215 220 Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile 225 230 235 240
Cys Ile
(2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 810 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..807
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:
ATG GAT CCA AAC ACT GTG TCA AGC TTT CAG GTA GAT TGC TTT CTT TGG 48 Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp
1 5 10 15
CAT GTC CGC AAA CGA GTT GCA GAC CAA GAA CTA GGT GAT GCC CCA TTC 96 His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe
20 25 30
CTT GAT CGG CTT CGC CGA GAT CAG AAA TCC ATG GAT CTG TCC AGA GGT 144 Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Met Asp Leu Ser Arg Gly
35 40 45
CTA TTT GGA GCC ATT GCC GGT TTT ATT GAA GGG GGA TGG ACT GGA ATG 192 Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met
50 55 60
ATA GAT GGA TGG TAC GGT TAT CAT CAT CAG AAT GAA CAG GGA TCA GGC 240 Ile Asp Gly Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly
65 70 75 80
TAT GCA GCG GAT CAA AAA AGC ACA CAA AAT GCC ATT AAC GGG ATT ACA 288 Tyr Ala Ala Asp Gln Lys Ser Thr Gln Asn Ala Ile Asn Gly Ile Thr
85 90 95
AAC AAG GTG AAC TCT GTT ATC GAG AAA ATG AAC ATT CAA TTC ACA GCT 336 Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn Ile Gln Phe Thr Ala
100 105 110
GTG GGT AAA GAA TTC AAC AAA TTA GAA AAA AGG ATG GAA AAT TTA AAT 384 Val Gly Lys Glu Phe Asn Lys Leu Glu Lys Arg Met Glu Asn Leu Asn
115 120 125
AAA AAA GTT GAT GAT GGA TTT CTG GAC ATT TGG ACA TAT AAT GCA GAA 432 Lvs Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu
130 135 140
TTG TTA GTT CTA CTG GAA AAT GAA AGG ACT CTG GAT TTC CAT GAC TCA 480 Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser
145 150 155 160 AAT GTG AAG AAT CTG TAT GAG AAA GTA AAA AGC CAA TTA AAG AAT AAT 528 Asn Val Lys Asn Leu Tyr Glu Lys Val Lys Ser Gln Leu Lys Asn Asn
165 170 175
GCC AAA GAA ATC GGA AAT GGA TGT TTT GAG TTC TAC CAC AAG TGT GAC 576 Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp
180 185 190
AAT GAA TGC ATG GAA AGT GTA AGA AAT GGG ACT TAT GAT TAT CCC AAA 624 Asn Glu Cys Met Glu Ser Val Arg Asn Gly Thr Tyr Asp Tyr Pro Lys
195 200 205
TAT TCA GAA GAG TCA AAG TTG AAC AGG GAA AAG GTA GAT GGA GTG AAA 672 Tyr Ser Glu Glu Ser Lys Leu Asn Arg Glu Lys Val Asp Gly Val Lys
210 215 220
TTG GAA TCA ATG GGG ATC TAT CAG ATT CTG GCG ATC TAC TCA ACT GTC 720 Leu Glu Ser Met Gly Ile Tyr Gln Ile Leu Ala Ile Tyr Ser Thr Val
225 230 235 240
GCC AGT TCA CTG GTG CTT TTG GTC TCC CTG GGG GCA ATC AGT TTC TGG 768 Ala Ser Ser Leu Val Leu Leu Val Ser Leu Gly Ala Ile Ser Phe Trp
245 250 255
ATG TGT TCT AAT GGA TCT TTG CAG TGC AGA ATA TGC ATC TGA 810
Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile Cys Ile
260 265
(2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 269 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:
Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp
1 5 10 15
His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe
20 25 30
Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Met Asp Leu Ser Arg Gly
35 40 45
Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met
50 55 60
Ile Asp Gly Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly
65 70 75 80
Tyr Ala Ala Asp Gln Lys Ser Thr Gln Asn Ala Ile Asn Gly Ile Thr
85 90 95
Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn Ile Gln Phe Thr Ala
100 105 110 Val Gly Lys Glu Phe Asn Lys Leu Glu Lys Arg Met Glu Asn Leu Asn 115 120 125
Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu
130 135 140
Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser
145 150 155 160
Asn Val Lys Asn Leu Tyr Glu Lys Val Lys Ser Gln Leu Lys Asn Asn
165 170 175
Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp
180 185 190
Asn Glu Cys Met Glu Ser Val Arg Asn Gly Thr Tyr Asp Tyr Pro Lys
195 200 205
Tyr Ser Glu Glu Ser Lys Leu Asn Arg Glu Lys Val Asp Gly Val Lys
210 215 220
Leu Glu Ser Met Gly Ile Tyr Gln Ile Leu Ala Ile Tyr Ser Thr Val
225 230 235 240
Ala Ser Ser Leu Val Leu Leu Val Ser Leu Gly Ala Ile Ser Phe Trp
245 250 255
Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile Cys Ile
260 265
(2) INFORMATION FOR SEQ ID NO: 21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 630 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE :
(A) NAME/KEY: CDS
(B) LOCATION: 1..627
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:
ATG GAT CCA AAC ACT GTG TCA AGC TTT CAG GTA GAT TGC TTT CTT TGG 48 Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp
1 5 10 15
CAT GTC CGC AAA CGA GTT GCA GAC CAA GAA CTA GGT GAT GCC CCA TTC 96 His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe
20 25 30
CTT GAT CGG CTT CGC CGA GAT CAG AAA TCC ATG GAT CAT ATG TTA ACA 144 Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Met Asp His Met Leu Thr
35 40 45
AGT ACT CGA TCT GTG GGT AAA GAA TTC AAC AAA TTA GAA AAA AGG ATG 192 Ser Thr Arg Ser Val Gly Lys Glu Phe Asn Lys Leu Glu Lys Arg Met
50 55 60 GAA AAT TTA AAT AAA AAA GTT GAT GAT GGA TTT CTG GAC ATT TGG ACA 240 Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr
65 70 75 80
TAT AAT GCA GAA TTG TTA GTT CTA CTG GAA AAT GAA AGG ACT CTG GAT 288 Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp
85 90 95
TTC CAT GAC TCA AAT GTG AAG AAT CTG TAT GAG AAA GTA AAA AGC CAA 336 Phe His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys Val Lys Ser Gln
100 105 110
TTA AAG AAT AAT GCC AAA GAA ATC GGA AAT GGA TGT TTT GAG TTC TAC 384 Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr
115 120 125
CAC AAG TGT GAC AAT GAA TGC ATG GAA AGT GTA AGA AAT GGG ACT TAT 432 His Lys Cys Asp Asn Glu Cys Met Glu Ser Val Arg Asn Gly Thr Tyr
130 135 140
GAT TAT CCC AAA TAT TCA GAA GAG TCA AAG TTG AAC AGG GAA AAG GTA 480 Asp Tyr Pro Lys Tyr Ser Glu Glu Ser Lys Leu Asn Arg Glu Lys Val
145 150 155 160
GAT GGA GTG AAA TTG GAA TCA ATG GGG ATC TAT CAG ATT CTG GCG ATC 528 Asp Gly Val Lys Leu Glu Ser Met Gly Ile Tyr Gln Ile Leu Ala Ile
165 170 175
TAC TCA ACT GTC GCC AGT TCA CTG GTG CTT TTG GTC TCC CTG GGG GCA 576 Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Leu Val Ser Leu Gly Ala
180 185 190
ATC AGT TTC TGG ATG TGT TCT AAT GGA TCT TTG CAG TGC AGA ATA TGC 624 Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile Cys
195 200 205
ATC TGA 630 Ile
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 209 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:
Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp
1 5 10 15
His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe
20 25 30
Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Met Asp His Met Leu Thr
35 40 45
Ser Thr Arg Ser Val Gly Lys Glu Phe Asn Lys Leu Glu Lys Arg Met
50 55 60 Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr 65 70 75 80
Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp
85 90 95
Phe His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys Val Lys Ser Gln
100 105 110
Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr
115 120 125
His Lys Cys Asp Asn Glu Cys Met Glu Ser Val Arg Asn Gly Thr Tyr
130 135 140
Asp Tyr Pro Lys Tyr Ser Glu Glu Ser Lys Leu Asn Arg Glu Lys Val
145 150 155 160
Asp Gly Val Lys Leu Glu Ser Met Gly Ile Tyr Gln Ile Leu Ala Ile
165 170 175
Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Leu Val Ser Leu Gly Ala
180 185 190
Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile Cys
195 200 205
Ile
(2) INFORMATION FOR SEQ ID NO: 23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 717 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..714
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:
ATG GAT CCA AAC ACT GTG TCA AGC TTT CAG GTA GAT TGC TTT CTT TGG 48 Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp
1 5 10 15
CAT GTC CGC AAA CGA GTT GCA GAC CAA GAA CTA GGT GAT GCC CCA TTC 96 His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe
20 25 30
CTT GAT CGG CTT CGC CGA GAT CAG AAA TCC CTA AGA GGA AGG GGC AGC 144 Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser
35 40 45
ACT CTT GGT CTG GAC ATC GAG ACA GCC ACA CGT GCT GGA AAG CAG ATA 192 Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Arg Ala Gly Lys Gln Ile
50 55 60 GTG GAG CGG ATT CTG AAA GAA GAA TCC GAT GAG GCA CTT AAA ATG ACC 240 Val Glu Arg Ile Leu Lys Glu Glu Ser Asp Glu Ala Leu Lys Met Thr
65 70 75 80
ATG CAG ATC CCG GAA TTC AAC AAA TTA GAA AAA AGG ATG GAA AAT TTA 288 Met Gln Ile Pro Glu Phe Asn Lys Leu Glu Lys Arg Met Glu Asn Leu
85 90 95
AAT AAA AAA GTT GAT GAT GGA TTT CTG GAC ATT TGG ACA TAT AAT GCA 336 Asn Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala
100 105 110
GAA TTG TTA GTT CTA CTG GAA AAT GAA AGG ACT CTG GAT TTC CAT GAC 384 Glu Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Phe His Asp
115 120 125
TCA AAT GTG AAG AAT CTG TAT GAG AAA GTA AAA AGC CAA TTA AAG AAT 432 Ser Asn Val Lys Asn Leu Tyr Glu Lys Val Lys Ser Gln Leu Lys Asn
130 135 140
AAT GCC AAA GAA ATC GGA AAT GGA TGT TTT GAG TTC TAC CAC AAG TGT 480 Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys
145 150 155 160
GAC AAT GAA TGC ATG GAA AGT GTA AGA AAT GGG ACT TAT GAT TAT CCC 528 Asp Asn Glu Cys Met Glu Ser Val Arg Asn Gly Thr Tyr Asp Tyr Pro
165 170 175
AAA TAT TCA GAA GAG TCA AAG TTG AAC AGG GAA AAG GTA GAT GGA GTG 576 Lys Tyr Ser Glu Glu Ser Lys Leu Asn Arg Glu Lys Val Asp Gly Val
180 185 190
AAA TTG GAA TCA ATG GGG ATC TAT CAG ATT CTG GCG ATC TAC TCA ACT 624 Lys Leu Glu Ser Met Gly Ile Tyr Gln Ile Leu Ala Ile Tyr Ser Thr
195 200 205
GTC GCC AGT TCA CTG GTG CTT TTG GTC TCC CTG GGG GCA ATC AGT TTC 672 Val Ala Ser Ser Leu Val Leu Leu Val Ser Leu Gly Ala Ile Ser Phe
210 215 220
TGG ATG TGT TCT AAT GGA TCT TTG CAG TGC AGA ATA TGC ATC 714
Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile Cys Ile
225 230 235
TGA 717
(2) INFORMATION FOR SEQ ID NO: 24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 238 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:
Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp
1 5 10 15
His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe
20 25 30 Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser 35 40 45
Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Arg Ala Gly Lys Gln Ile 50 55 60
Val Glu Arg Ile Leu Lys Glu Glu Ser Asp Glu Ala Leu Lys Met Thr
65 70 75 80
Met Gln Ile Pro Glu Phe Asn Lys Leu Glu Lys Arg Met Glu Asn Leu
85 90 95
Asn Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala
100 105 110
Glu Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Phe His Asp
115 120 125
Ser Asn Val Lys Asn Leu Tyr Glu Lys Val Lys Ser Gln Leu Lys Asn
130 135 140
Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys 145 150 155 160
Asp Asn Glu Cys Met Glu Ser Val Arg Asn Gly Thr Tyr Asp Tyr Pro
165 170 175
Lys Tyr Ser Glu Glu Ser Lys Leu Asn Arg Glu Lys Val Asp Gly Val
180 185 190
Lys Leu Glu Ser Met Gly Ile Tyr Gln Ile Leu Ala Ile Tyr Ser Thr
195 200 205
Val Ala Ser Ser Leu Val Leu Leu Val Ser Leu Gly Ala Ile Ser Phe 210 215 220
Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile Cys Ile
225 230 235
(2) INFORMATION FOR SEQ ID NO: 25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 681 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..678 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:
ATG GAT CCA AAC ACT GTG TCA AGC TTT CAG GTA GAT TGC TTT CTT TGG 48 Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp
1 5 10 15
CAT GTC CGC AAA CGA GTT GCA GAC CAA GAA CTA GGT GAT GCC CCA TTC 96 His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe
20 25 30
CTT GAT CGG CTT CGC CGA GAT CAG AAA TCC CTA AGA GGA AGG GGC AGC 144 Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser
35 40 45
ACT CTT GGT CTG GAC ATC GAG ACA GCC ACA CGT GCT GGA AAG CAG ATA 192 Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Arg Ala Gly Lys Gln Ile
50 55 60
GTG GAG CGG ATT CTG AAA GAA GAA TCC GAT GAG GCA CTT AAA ATG ACC 240 Val Glu Arg Ile Leu Lys Glu Glu Ser Asp Glu Ala Leu Lys Met Thr
65 70 75 80
ATG CAG ATC CCG AAT AAA AAA GTT GAT GAT GGA TTT CTG GAC ATT TGG 288 Met Gln Ile Pro Asn Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp
85 90 95
ACA TAT AAT GCA GAA TTG TTA GTT CTA CTG GAA AAT GAA AGG ACT CTG 336 Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu
100 105 110
GAT TTC CAT GAC TCA AAT GTG AAG AAT CTG TAT GAG AAA GTA AAA AGC 384 Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys Val Lys Ser
115 120 125
CAA TTA AAG AAT AAT GCC AAA GAA ATC GGA AAT GGA TGT TTT GAG TTC 432 Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe
130 135 140
TAC CAC AAG TGT GAC AAT GAA TGC ATG GAA AGT GTA AGA AAT GGG ACT 480 Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser Val Arg Asn Gly Thr
145 150 155 160
TAT GAT TAT CCC AAA TAT TCA GAA GAG TCA AAG TTG AAC AGG GAA AAG 528 Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ser Lys Leu Asn Arg Glu Lys
165 170 175
GTA GAT GGA GTG AAA TTG GAA TCA ATG GGG ATC TAT CAG ATT CTG GCG 576 Val Asp Gly Val Lys Leu Glu Ser Met Gly Ile Tyr Gln Ile Leu Ala
180 185 190
ATC TAC TCA ACT GTC GCC AGT TCA CTG GTG CTT TTG GTC TCC CTG GGG 624 Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Leu Val Ser Leu Gly
195 200 205
GCA ATC AGT TTC TGG ATG TGT TCT AAT GGA TCT TTG CAG TGC AGA ATA 672 Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile
210 215 220
TGC ATC TGA 681
Cys Ile
225 (2) INFORMATION FOR SEQ ID NO: 26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 226 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp
1 5 10 15
His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe
20 25 30
Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser
35 40 45
Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Arg Ala Gly Lys Gln Ile 50 55 60
Val Glu Arg Ile Leu Lys Glu Glu Ser Asp Glu Ala Leu Lys Met Thr 65 70 75 80
Met Gln Ile Pro Asn Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp
85 90 95
Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu
100 105 110
Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys Val Lys Ser
115 120 125
Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe 130 135 140
Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser Val Arg Asn Gly Thr
145 150 155 160
Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ser Lys Leu Asn Arg Glu Lys
165 170 175
Val Asp Gly Val Lys Leu Glu Ser Met Gly Ile Tyr Gln Ile Leu Ala
180 185 190
Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Leu Val Ser Leu Gly
195 200 205
Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile 210 215 220
Cys Ile
225 (2) INFORMATION FOR SEQ ID NO: 27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 158 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:
Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp 1 5 10 15
His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe
20 25 30
Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser
35 40 45
Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Arg Ala Gly Lys Gln Ile 50 55 60
Val Glu Arg Ile Leu Lys Glu Glu Ser Asp Glu Ala Leu Lys Met Thr 65 70 75 80
Met Gln Ile Pro Val Glu Ser Val Arg Asn Gly Thr Tyr Asp Tyr Pro
85 90 95
Lys Tyr Ser Glu Glu Ser Lys Leu Asn Arg Glu Lys Val Asp Gly Val
100 105 110
Lys Leu Glu Ser Met Gly Ile Tyr Gln Ile Leu Ala Ile Tyr Ser Thr
115 120 125
Val Ala Ser Ser Leu Val Leu Leu Val Ser Leu Gly Ala Ile Ser Phe 130 135 140
Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile Cys Ile
145 150 155
(2) INFORMATION FOR SEQ ID NO: 28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 163 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:
Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp
1 5 10 15
His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe
20 25 30
Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser
35 40 45 Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Arg Ala Gly Lys Gln Ile 50 55 60
Val Glu Arg Ile Leu Lys Glu Glu Ser Asp Glu Ala Leu Lys Met Thr 65 70 75 80
Met Asp Leu Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu
85 90 95
Gly Gly Trp Thr Gly Met Ile Asp Gly Trp Tyr Gly Tyr His His Gln
100 105 110
Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Gln Lys Ser Thr Gln Asn
115 120 125
Ala Ile Asn Gly Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met 130 135 140
Asn Ile Gln Phe Thr Ala Val Gly Lys Glu Phe Ser Cys Leu Thr Ala 145 150 155 160
Tyr His Arg
(2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 231 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:
Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp
1 5 10 15
His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe
20 25 30
Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser
35 40 45
Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Arg Ala Gly Lys Gln Ile 50 55 60
Val Glu Arg Ile Leu Lys Glu Glu Ser Asp Glu Ala Leu Lys Met Thr 65 70 75 80
Met Gln Ile Pro Ala Val Gly Lys Glu Phe Asn Lys Leu Glu Lys Arg
85 90 95
Met Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp
100 105 110
Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu
115 120 125
Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys Val Lys Ser 130 135 140 Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe 145 150 155 160
Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser Val Arg Asn Gly Thr
165 170 175
Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ser Lys Leu Asn Arg Glu Lys
180 185 190
Val Asp Gly Val Lys Leu Glu Ser Met Gly Ile Tyr Gln Ile Leu Ala
195 200 205 Ile Tyr Ser Thr Val Ala Ser Ser Gly Gly Ser Tyr Ser Met Glu His 210 215 220
Phe Arg Trp Gly Lys Pro Val
225 230
(2) INFORMATION FOR SEQ ID NO: 30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 225 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:
Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp 1 5 10 15
His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe
20 25 30
Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser
35 40 45
Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Arg Ala Gly Lys Gln Ile 50 55 60
Val Glu Arg Ile Leu Lys Glu Glu Ser Asp Glu Ala Leu Lys Met Thr 65 70 75 80
Met Gln Ile Pro Ala Val Gly Lys Glu Phe Asn Lys Leu Glu Lys Arg
85 90 95
Met Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp
100 105 110
Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu
115 120 125
Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys Val Lys Ser 130 135 140
Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe 145 150 155 160
Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser Val Arg Asn Gly Thr
165 170 175 Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ser Lys Leu Asn Arg Glu Lys
180 185 190
Val Asp Gly Val Lys Leu Glu Ser Met Gly Ile Tyr Gln Ile Leu Ala
195 200 205
Ile Tyr Ser Thr Val Ala Ser Ser Gly Gly Ser Tyr Ser Met Leu Val
210 215 220
Asn
225
(2) INFORMATION FOR SEQ ID NO: 31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 912 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE :
(A) NAME/KEY: CDS
(B) LOCATION: 1..912
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:
ATG GAT CCA AAC ACT GTG TCA AGC TTT CAG GTA GAT TGC TTT CTT TGG 48 Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp
1 5 10 15
CAT GTC CGC AAA CGA GTT GCA GAC CAA GAA CTA GGT GAT GCC CCA TTC 96 His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe
20 25 30
CTT GAT CGG CTT CGC CGA GAT CAG AAA TCC CTA AGA GGA AGG GGC AGC 144 Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser
35 40 45
ACT CTT GGT CTG GAC ATC GAG ACA GCC ACA CGT GCT GGA AAG CAG ATA 192 Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Arg Ala Gly Lys Gln Ile
50 55 60
GTG GAG CGG ATT CTG AAA GAA GAA TCC GAT GAG GCA CTT AAA ATG ACC 240 Val Glu Arg Ile Leu Lys Glu Glu Ser Asp Glu Ala Leu Lys Met Thr
65 70 75 80
ATG CAG ATC CCG GGT CTA TTT GGA GCC ATT GCC GGT TTT ATT GAA GGG 288 Met Gln Ile Pro Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly
85 90 95
GGA TGG ACT GGA ATG ATA GAT GGA TGG TAC GGT TAT CAT CAT CAG AAT 336 Gly Trp Thr Gly Met Ile Asp Gly Trp Tyr Gly Tyr His His Gln Asn
100 105 110
GAA CAG GGA TCA GGC TAT GCA GCG GAT CAA AAA AGC ACA CAA AAT GCC 384 Glu Gln Gly Ser Gly Tyr Ala Ala Asp Gln Lys Ser Thr Gln Asn Ala
115 120 125 ATT AAC GGG ATT ACA AAC AAG GTG AAC TCT GTT ATC GAG AAA ATG AAC 432 Ile Asn Gly Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn
130 135 140
ATT CAA TTC ACA GCT GTG GGT AAA GAA TTC AAC AAA TTA GAA AAA AGG 480 Ile Gln Phe Thr Ala Val Gly Lys Glu Phe Asn Lys Leu Glu Lys Arg
145 150 155 160
ATG GAA AAT TTA AAT AAA AAA GTT GAT GAT GGA TTT CTG GAC ATT TGG 528 Met Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp
165 170 175
ACA TAT AAT GCA GAA TTG TTA GTT CTA CTG GAA AAT GAA AGG ACT CTG 576 Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu
180 185 190
GAT TTC CAT GAC TCA AAT GTG AAG AAT CTG TAT GAG AAA GTA AAA AGC 624 Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys Val Lys Ser
195 200 205
CAA TTA AAG AAT AAT GCC AAA GAA ATC GGA AAT GGA TGT TTT GAG TTC 672 Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe
210 215 220
TAC CAC AAG TGT GAC AAT GAA TGC ATG GAA AGT GTA AGA AAT GGG ACT 720 Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser Val Arg Asn Gly Thr
225 230 235 240
TAT GAT TAT CCC AAA TAT TCA GAA GAG TCA AAG TTG AAC AGG GAA AAG 768 Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ser Lys Leu Asn Arg Glu Lys
245 250 255
GTA GAT GGA GTG AAA TTG GAA TCA ATG GGG ATC TAT CAG ATT CTG GCG 816 Val Asp Gly Val Lys Leu Glu Ser Met Gly Ile Tyr Gln Ile Leu Ala
260 265 270
ATC TAC TCA ACT GTC GCC AGT TCA CTG GTG CTT TTG GTC TCC CTG GGG 864 Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Leu Val Ser Leu Gly
275 280 285
GCA ATC AGT TTC TGG ATG TGT TCT AAT GGA TCT TTG CAG TGC AGA ATA 912 Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile
290 295 300
(2) INFORMATION FOR SEQ ID NO: 32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 304 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:
Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp
1 5 10 15
His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe
20 25 30 Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser
35 40 45
Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Arg Ala Gly Lys Gln Ile 50 55 60
Val Glu Arg Ile Leu Lys Glu Glu Ser Asp Glu Ala Leu Lys Met Thr 65 70 75 80
Met Gln Ile Pro Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly
85 90 95
Gly Trp Thr Gly Met Ile Asp Gly Trp Tyr Gly Tyr His His Gln Asn
100 105 110
Glu Gln Gly Ser Gly Tyr Ala Ala Asp Gln Lys Ser Thr Gln Asn Ala
115 120 125
Ile Asn Gly Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn 130 135 140
Ile Gln Phe Thr Ala Val Gly Lys Glu Phe Asn Lys Leu Glu Lys Arg 145 150 155 160
Met Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp
165 170 175
Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu
180 185 190
Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys Val Lys Ser
195 200 205
Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe 210 215 220
Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser Val Arg Asn Gly Thr 225 230 235 240
Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ser Lys Leu Asn Arg Glu Lys
245 250 255
Val Asp Gly Val Lys Leu Glu Ser Met Gly Ile Tyr Gln Ile Leu Ala
260 265 270
Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Leu Val Ser Leu Gly
275 280 285
Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile 290 295 300
(2) INFORMATION FOR SEQ ID NO: 33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 474 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D ) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) ( ix ) FEATURE :
(A) NAME/KEY: CDS
(B) LOCATION: 1..471
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:
GTG GGT AAA GAA TTC AAC AAA TTA GAA AAA AGG ATG GAA AAT TTA AAT 48 Val Gly Lys Glu Phe Asn Lys Leu Glu Lys Arg Met Glu Asn Leu Asn
1 5 10 15
AAA AAA GTT GAT GAT GGA TTT CTG GAC ATT TGG ACA TAT AAT GCA GAA 96 Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu
20 25 30
TTG TTA GTT CTA CTG GAA AAT GAA AGG ACT CTG GAT TTC CAT GAC TCA 144 Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser
35 40 45
AAT GTG AAG AAT CTG TAT GAG AAA GTA AAA AGC CAA TTA AAG AAT AAT 192 Asn Val Lys Asn Leu Tyr Glu Lys Val Lys Ser Gln Leu Lys Asn Asn
50 55 60
GCC AAA GAA ATC GGA AAT GGA TGT TTT GAG TTC TAC CAC AAG TGT GAC 240 Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp
65 70 75 80
AAT GAA TGC ATG GAA AGT GTA AGA AAT GGG ACT TAT GAT TAT CCC AAA 288 Asn Glu Cys Met Glu Ser Val Arg Asn Gly Thr Tyr Asp Tyr Pro Lys
85 90 95
TAT TCA GAA GAG TCA AAG TTG AAC AGG GAA AAG GTA GAT GGA GTG AAA 336 Tyr Ser Glu Glu Ser Lys Leu Asn Arg Glu Lys Val Asp Gly Val Lys
100 105 110
TTG GAA TCA ATG GGG ATC TAT CAG ATT CTG GCG ATC TAC TCA ACT GTC 384 Leu Glu Ser Met Gly Ile Tyr Gln Ile Leu Ala Ile Tyr Ser Thr Val
115 120 125
GCC AGT TCA CTG GTG CTT TTG GTC TCC CTG GGG GCA ATC AGT TTC TGG 432 Ala Ser Ser Leu Val Leu Leu Val Ser Leu Gly Ala Ile Ser Phe Trp
130 135 140
ATG TGT TCT AAT GGA TCT TTG CAG TGC AGA ATA TGC ATC TGA 474
Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile Cys Ile
145 150 155
(2) INFORMATION FOR SEQ ID NO: 34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 157 amino acids
(B) TYPE: amino acid
(D ) TOPOLOGY: 1inear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:
Val Gly Lys Glu Phe Asn Lys Leu Glu Lys Arg Met Glu Asn Leu Asn
1 5 10 15 Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu
20 25 30
Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser
35 40 45
Asn Val Lys Asn Leu Tyr Glu Lys Val Lys Ser Gln Leu Lys Asn Asn
50 55 60
Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp
65 70 75 80
Asn Glu Cys Met Glu Ser Val Arg Asn Gly Thr Tyr Asp Tyr Pro Lys
85 90 95
Tyr Ser Glu Glu Ser Lys Leu Asn Arg Glu Lys Val Asp Gly Val Lys
100 105 110
Leu Glu Ser Met Gly Ile Tyr Gln Ile Leu Ala Ile Tyr Ser Thr Val
115 120 125
Ala Ser Ser Leu Val Leu Leu Val Ser Leu Gly Ala Ile Ser Phe Trp
130 135 140
Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile Cys Ile
145 150 155
(2) INFORMATION FOR SEQ ID NO: 35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 47 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:
CATGGATCAT ATGTTAACAG ATATCAAGGC CTGACTGACT GAGAGCT 47
(2) INFORMATION FOR SEQ ID NO: 36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D ) TOPOLOGY : unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:
CTAGTATACA ATTGTCTATA GTTCCGGACT GACTGACTC 39 (2) INFORMATION FOR SEQ ID NO: 37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:
CATGGGCGCC CATATGGGCA TATTCGGCG 29
(2) INFORMATION FOR SEQ ID NO:38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:
CCGCGGGTAT ACCCGTATAA GCC 23
(2) INFORMATION FOR SEQ ID NO: 39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 49 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:
CATGGATCAT ATGTTAACAA GTACTCGATA TCAATGAGTG ACTGAAGCT 49
(2) INFORMATION FOR SEQ ID NO: 40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:
CTAGTATACA ATTGTTCATG AGCTATAGTT ACTCACTGAC T 41 (2) INFORMATION FOR SEQ ID NO: 41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41:
AATTCGTACC TA 12
(2) INFORMATION FOR SEQ ID NO: 42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:
GCATGGATCT AG 12

Claims

WHAT IS CLAIMED IS:
1. A vaccine for stimulating protection in animals against infection by influenza virus which comprises a an effective amount of an immunogenic
fragment of the HA2 subunit of an HA protein selected from the group consisting of a type A subtype influenza virus or a type B influenza virus.
2. The vaccine according to claim 1 wherein said type A subunit is H3N2.
3. The vaccine according to claim 1 wherein the polypeptide is fused to a second polypeptide.
4. The vaccine according to claim 2 wherein the second polypeptide comprises the N terminal amino acids of a NS1 protein.
5. The vaccine according to claim 1 wherein the immunogenic fragment of the HA2 subunit is selected from the group consisting of a peptide comprising amino acids 1 to 221 of the H3HA2 subtype, a peptide comprising amino acids 77 to 221 of the H3HA2 subtype, a peptide comprising amino acids 1 to 223 of the BHA2 type, and a peptide comprising amino acids 41 to 223 of the BHA2 type.
6. The vaccine according to claim 5
comprising NS1(1-81)H3HA2(1-221) SEQ ID NO: 10.
7. The vaccine according to claim 5 comprising NS1(1-81)H3HA2(77-221) SEQ ID NO: 12.
8. The vaccine according to claim 5 comprising NS11-42BLHA241-223 SEQ ID NO: 14.
9. A protein comprising an immunogenic fragment of the HA2 subunit of an HA protein selected from the group consisting of Type A subtype or type B influenza virus.
10. The protein according to claim 9 wherein said type A subtype is H3N2.
11. The protein according to claim 9 wherein the peptide containing the immunogenic fragment is fused to a second peptide or protein.
12. The protein according to claim 10 wherein the second peptide comprises the N terminal amino acids of a NS1 protein.
13. The protein according to claim 10 wherein the immunogenic fragment of the HA2 subunit is selected from the group consisting of a peptide comprising amino acids 1 to 221 of the H3HA2 subunit, a peptide comprising amino acids 77 to 221 of the H3HA2 subunit, a peptide comprising amino acids 1-223 of the BHA2 subunit, and a peptide comprising amino acids 41-223 of the BHA2
subunit.
14. A polypeptide NS1(1-81)H3HA2(1-221) SEQ ID NO: 10.
15. A polypeptide NS1(1-81)H3HA2(77-221) SEQ ID NO: 12.
16. A polypeptide NS11-41BLHA241-223 SEQ ID NO: 14.
17. A DNA molecule comprising a coding
sequence for an immunogenic fragment of the HA2 subunit of an HA protein selected from the group consisting of a Type A subtype or type B influenza virus.
18. The DNA molecule according to claim 17 wherein said Type A subunit is H3N2.
19. The DNA molecule according to claim 17 comprising a coding sequence for the polypeptide NS1(1- 81)H3HA2(1-221) SEQ ID NO: 10.
20. The DNA molecule according to claim 17 comprising a coding sequence for the polypeptide NS1(1- 42)H3BLHA2(41-223) SEQ ID NO: 14.
21. The DNA molecule according to claim 17 comprising a coding sequence for the polypeptide NS1(1- 81)H3HA2(77-221) SEQ ID NO: 12.
22. Plasmid pMG13H3HA SEQ ID NO: 9.
23. Plasmid pNS11-41BLHA241-223 SEQ ID NO: 13.
24. A microorganism transformed with a DNA molecule comprising a coding sequence for an immunogenic fragment of the HA2 subunit of an HA protein selected from the group consisting of a Type A subtype or type B influenza virus.
25. The microorganism according to claim 24 wherein said Type A subunit is H3N2.
26. The microorganism according to claim 24 wherein said DNA molecule comprises a coding sequence for the polypeptide NS1(1-81)H3HA2(1-221) SEQ ID NO: 10.
27. A combination vaccine for stimulating protection in animals against infection by influenza virus which comprises a first polypeptide having an immunogenic fragment of the HA2 subunit of an influenza H3 subtype virus and a second polypeptide selected from the group consisting of a polypeptide having an
immunogenic fragment of the HA2 subunit of a type B influenza virus, and a polypeptide having an immunogenic fragment of the HA2 subunit of an H1 subtype influenza virus, and a polypeptide having an immunogenic fragment of the HA2 subunit of an H2 subtype influenza virus.
28. The combination vaccine according to claim 27 wherein the first polypeptide is selected from the group consisting of NS1(1-81)H3HA2(1-221) SEQ ID NO: 10 and NS1(1- 81)H3HA2(77-221) SEQ ID NO: 12.
29. The combination vaccine according to claim 27 wherein the second polypeptide is a polypeptide having an immunogenic fragment of the HA2 subunit of an H1 subtype influenza virus.
30. The combination vaccine according to claim 27 wherein said second polypeptide is selected from the group consisting of C13 SEQ ID NO: 16, D SEQ ID NO: 18, C13 short SEQ ID NO: 20, D short SEQ ID NO: 22, A SEQ ID NO: 24, C SEQ ID NO: 26, ΔD SEQ ID NO: 27, Δ13 SEQ ID NO: 28, M SEQ ID NO: 29, ΔM SEQ ID NO: 30, ΔM+ SEQ ID NO: 32, and H1HA266-222 SEQ ID NO: 34.
31. The combination vaccine according to claim 27 wherein said second polypeptide is NS11-42BLHA241-223 SEQ ID NO: 14.
32. A combination vaccine for stimulating protection in animals against infection by influenza virus which comprises a first polypeptide having an immunogenic fragment of the HA2 subunit of an influenza H3 subtype virus, a second polypeptide having an
immunogenic fragment of the HA2 subunit of an influenza B type virus, and a third polypeptide selected from the group consisting of a polypeptide having an immunogenic fragment of the HA2 subunit of an H1 subtype influenza virus and a polypeptide having an immunogenic fragment of the HA2 subunit of an H2 subtype influenza virus.
PCT/US1993/001451 1992-02-18 1993-02-18 Vaccinal polypeptides WO1993015763A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US83777392A 1992-02-18 1992-02-18
US07/837,773 1992-02-18

Publications (1)

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WO1993015763A1 true WO1993015763A1 (en) 1993-08-19

Family

ID=25275375

Family Applications (1)

Application Number Title Priority Date Filing Date
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Country Status (3)

Country Link
AU (1) AU3724093A (en)
MX (1) MX9300883A (en)
WO (1) WO1993015763A1 (en)

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WO1994022917A1 (en) * 1993-04-05 1994-10-13 University Of Massachusetts Medical Center Cross-reactive influenza a immunization
US5674502A (en) * 1990-08-08 1997-10-07 University Of Massachusetts Medical Center Cross-reactive influenza a immunization
WO2001062778A3 (en) * 2000-02-23 2002-04-04 Smithkline Beecham Biolog Tumour-specific animal proteins
WO2002024734A3 (en) * 2000-09-19 2002-08-15 Chiron Spa Influenza a virus haemagglutinin subtype h16 proteins and their encoding nuclei c acid
JP2006316072A (en) * 1994-01-27 2006-11-24 Univ Of Massachusetts Medical Center Immunization by inoculation with DNA transcription unit
WO2008036146A2 (en) 2006-07-14 2008-03-27 Sanofi Pasteur Biologics Co. Construction of recombinant virus vaccines by direct transposon-mediated insertion of foreign immunologic determinants into vector virus proteins
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US8916514B2 (en) 2009-05-27 2014-12-23 Glaxosmithkline Biologicals, S.A. CASB7439 constructs
CN110003314A (en) * 2019-04-11 2019-07-12 上海市计划生育科学研究所 H1N1 influenza virus hemagglutinin can induce epitope peptide and its application of broad spectrum protection antibody
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Cited By (25)

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US5674502A (en) * 1990-08-08 1997-10-07 University Of Massachusetts Medical Center Cross-reactive influenza a immunization
US5766601A (en) * 1990-08-08 1998-06-16 University Of Massachusetts Medical Center Cross-reactive influenza a immunization
US5882650A (en) * 1990-08-08 1999-03-16 University Of Massachusetts Medical Center Cross-reactive influenza A immunization
WO1994022917A1 (en) * 1993-04-05 1994-10-13 University Of Massachusetts Medical Center Cross-reactive influenza a immunization
JP2006316072A (en) * 1994-01-27 2006-11-24 Univ Of Massachusetts Medical Center Immunization by inoculation with DNA transcription unit
KR100919916B1 (en) 2000-02-23 2009-10-07 글락소스미스클라인 바이오로지칼즈 에스.에이. Tumour-specific animal proteins
US7803379B2 (en) 2000-02-23 2010-09-28 Glaxosmithkline Biologicals S.A. Tumour-specific animal proteins
CN1840178B (en) * 2000-02-23 2014-05-28 史密丝克莱恩比彻姆生物有限公司 Tumour-specific animal proteins
EP1650221A3 (en) * 2000-02-23 2006-12-20 GlaxoSmithKline Biologicals SA Novel compounds
CZ303468B6 (en) * 2000-02-23 2012-10-03 Smithkline Beecham Biologicals S. A. Immunogenic mixture and pharmaceutical mixture
KR100848973B1 (en) * 2000-02-23 2008-07-30 글락소스미스클라인 바이오로지칼즈 에스.에이. Tumour-specific animal proteins
US8207123B2 (en) 2000-02-23 2012-06-26 Glaxosmithkline Biologicals S.A. Tumour-specific animal proteins
WO2001062778A3 (en) * 2000-02-23 2002-04-04 Smithkline Beecham Biolog Tumour-specific animal proteins
AU2006201042B2 (en) * 2000-02-23 2009-10-08 Smithkline Beecham Biologicals S.A. Novel compounds
AU2001256156B2 (en) * 2000-02-23 2006-01-05 Smithkline Beecham Biologicals S.A. Novel compounds
US7811574B2 (en) 2000-02-23 2010-10-12 Glaxosmithkline Biologicals S.A. Tumour-specific animal proteins
WO2002024734A3 (en) * 2000-09-19 2002-08-15 Chiron Spa Influenza a virus haemagglutinin subtype h16 proteins and their encoding nuclei c acid
US20100291128A1 (en) * 2005-11-18 2010-11-18 Montelione Gaetano T Novel compositions and vaccines against influenza a and influenza b infections
US9119810B2 (en) * 2005-11-18 2015-09-01 Rutgers, The State University Of New Jersey Compositions and vaccines against influenza A and influenza B infections
WO2008036146A2 (en) 2006-07-14 2008-03-27 Sanofi Pasteur Biologics Co. Construction of recombinant virus vaccines by direct transposon-mediated insertion of foreign immunologic determinants into vector virus proteins
WO2008100290A2 (en) 2006-09-29 2008-08-21 Sanofi Pasteur Biologics Co Recombinant rhinovirus vectors
US8916514B2 (en) 2009-05-27 2014-12-23 Glaxosmithkline Biologicals, S.A. CASB7439 constructs
US10555998B2 (en) 2014-11-24 2020-02-11 Intervet Inc. Inactivated equine influenza virus vaccines
CN110003314A (en) * 2019-04-11 2019-07-12 上海市计划生育科学研究所 H1N1 influenza virus hemagglutinin can induce epitope peptide and its application of broad spectrum protection antibody
CN110003314B (en) * 2019-04-11 2023-06-09 上海市计划生育科学研究所 Epitope peptide capable of inducing broad-spectrum protective antibody by H1N1 influenza virus hemagglutinin and application thereof

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AU3724093A (en) 1993-09-03
MX9300883A (en) 1994-08-31

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