WO1993015763A1 - Polypeptides de vaccination - Google Patents

Polypeptides de vaccination 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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PCT/US1993/001451
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English (en)
Inventor
Allan Shatzman
Miller Scott
Susan B. Dillon
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Smithkline Beecham Corporation
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Publication of WO1993015763A1 publication Critical patent/WO1993015763A1/fr

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    • 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

L'invention concerne des compositions de vaccin capables de conférer une immunité multisouche contre la grippe de type A et B.
PCT/US1993/001451 1992-02-18 1993-02-18 Polypeptides de vaccination WO1993015763A1 (fr)

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994022917A1 (fr) * 1993-04-05 1994-10-13 University Of Massachusetts Medical Center Immunisation a reaction croisee contre le virus de la grippe a
US5674502A (en) * 1990-08-08 1997-10-07 University Of Massachusetts Medical Center Cross-reactive influenza a immunization
WO2001062778A2 (fr) * 2000-02-23 2001-08-30 Smithkline Beecham Biologicals S.A. Nouveaux composes
WO2002024734A2 (fr) * 2000-09-19 2002-03-28 Chiron S.P.A. Sous-type h16 du virus grippal a
JP2006316072A (ja) * 1994-01-27 2006-11-24 Univ Of Massachusetts Medical Center Ｄｎａ転写ユニットの接種による免疫化
WO2008036146A2 (fr) 2006-07-14 2008-03-27 Sanofi Pasteur Biologics Co. Construction de vaccins antiviraux de recombinaison par insertion directe à médiation par transposon de déterminants immunologiques étrangers dans des protéines de virus vecteur
WO2008100290A2 (fr) 2006-09-29 2008-08-21 Sanofi Pasteur Biologics Co Vecteurs rhinoviraux recombinants
US7811574B2 (en) 2000-02-23 2010-10-12 Glaxosmithkline Biologicals S.A. Tumour-specific animal proteins
US20100291128A1 (en) * 2005-11-18 2010-11-18 Montelione Gaetano T Novel compositions and vaccines against influenza a and influenza b infections
CN1840178B (zh) * 2000-02-23 2014-05-28 史密丝克莱恩比彻姆生物有限公司 肿瘤特异性动物蛋白
US8916514B2 (en) 2009-05-27 2014-12-23 Glaxosmithkline Biologicals, S.A. CASB7439 constructs
CN110003314A (zh) * 2019-04-11 2019-07-12 上海市计划生育科学研究所 H1n1流感病毒血凝素可诱发广谱保护性抗体的表位肽及其应用
US10555998B2 (en) 2014-11-24 2020-02-11 Intervet Inc. Inactivated equine influenza virus vaccines

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
FEDERATION OF AMERICAN SOCIETIES FOR EXPERIMENTAL BIOLOGY, 75th Anual Meeting, Volume 5, No. 5, issued 21-25 April 1991, DILLON et al., "Activity of CD8+ CTL in Mice Immunized with Recombinant Influenza NS1-HA2 Fusion Protein or a CTL Epitope Peptide (HA2 189-199)", Abstract 5748, page A1362. *
JOURNAL OF EXPERIMENTAL MEDICINE, Volume 162, issued August 1985, YAMADA et al., "Influenza Virus Hemagglutinin-Specific Cytotoxic T Cell Response Induced by Polypeptide Produced in Escherichia Coli", pages 663-674. *
JOURNAL OF EXPERIMENTAL MEDICINE, Volume 162, issued November 1985, YAMADA et al., "Influenza Virus Subtype-Specific Cytotoxic T Lymphocytes Lyse Targer Cells Coated with a Protein Produced in E. Coli", pages 1720-1725. *
JOURNAL OF IMMUNOLOGY, Volume 140, No. 4, issued 15 February 1988, KUWANO et al., "HA2 Subunit of Influenza A H1 and H2 Subtype Viruses Induces a Protective Cross-Reactive Cytotoxic T Lymphocyte Response", pages 1264-1268. *

Cited By (27)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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 (fr) * 1993-04-05 1994-10-13 University Of Massachusetts Medical Center Immunisation a reaction croisee contre le virus de la grippe a
JP2006316072A (ja) * 1994-01-27 2006-11-24 Univ Of Massachusetts Medical Center Ｄｎａ転写ユニットの接種による免疫化
KR100848973B1 (ko) * 2000-02-23 2008-07-30 글락소스미스클라인 바이오로지칼즈 에스.에이. 종양 특이적 동물 단백질
CN1840178B (zh) * 2000-02-23 2014-05-28 史密丝克莱恩比彻姆生物有限公司 肿瘤特异性动物蛋白
WO2001062778A3 (fr) * 2000-02-23 2002-04-04 Smithkline Beecham Biolog Nouveaux composes
AU2001256156B2 (en) * 2000-02-23 2006-01-05 Smithkline Beecham Biologicals S.A. Novel compounds
CZ303468B6 (cs) * 2000-02-23 2012-10-03 Smithkline Beecham Biologicals S. A. Imunogenní smes a farmaceutická smes
EP1650221A3 (fr) * 2000-02-23 2006-12-20 GlaxoSmithKline Biologicals SA Nouveaux composés
US8207123B2 (en) 2000-02-23 2012-06-26 Glaxosmithkline Biologicals S.A. Tumour-specific animal proteins
WO2001062778A2 (fr) * 2000-02-23 2001-08-30 Smithkline Beecham Biologicals S.A. Nouveaux composes
US7811574B2 (en) 2000-02-23 2010-10-12 Glaxosmithkline Biologicals S.A. Tumour-specific animal proteins
KR100919916B1 (ko) 2000-02-23 2009-10-07 글락소스미스클라인 바이오로지칼즈 에스.에이. 종양 특이적 동물 단백질
AU2006201042B2 (en) * 2000-02-23 2009-10-08 Smithkline Beecham Biologicals S.A. Novel compounds
US7803379B2 (en) 2000-02-23 2010-09-28 Glaxosmithkline Biologicals S.A. Tumour-specific animal proteins
WO2002024734A2 (fr) * 2000-09-19 2002-03-28 Chiron S.P.A. Sous-type h16 du virus grippal a
WO2002024734A3 (fr) * 2000-09-19 2002-08-15 Chiron Spa Sous-type h16 du virus grippal a
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 (fr) 2006-07-14 2008-03-27 Sanofi Pasteur Biologics Co. Construction de vaccins antiviraux de recombinaison par insertion directe à médiation par transposon de déterminants immunologiques étrangers dans des protéines de virus vecteur
WO2008100290A2 (fr) 2006-09-29 2008-08-21 Sanofi Pasteur Biologics Co Vecteurs rhinoviraux recombinants
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 (zh) * 2019-04-11 2019-07-12 上海市计划生育科学研究所 H1n1流感病毒血凝素可诱发广谱保护性抗体的表位肽及其应用
CN110003314B (zh) * 2019-04-11 2023-06-09 上海市计划生育科学研究所 H1n1流感病毒血凝素可诱发广谱保护性抗体的表位肽及其应用

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