WO1994017826A1 - Vaccins polypeptidiques - Google Patents

Vaccins polypeptidiques Download PDF

Info

Publication number
WO1994017826A1
WO1994017826A1 PCT/US1994/001149 US9401149W WO9417826A1 WO 1994017826 A1 WO1994017826 A1 WO 1994017826A1 US 9401149 W US9401149 W US 9401149W WO 9417826 A1 WO9417826 A1 WO 9417826A1
Authority
WO
WIPO (PCT)
Prior art keywords
leu
glu
gly
asp
lys
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US1994/001149
Other languages
English (en)
Inventor
Allan Shatzman
James Kane
Miller Scott
Susan Dillon
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
SmithKline Beecham Corp
Original Assignee
SmithKline Beecham Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by SmithKline Beecham Corp filed Critical SmithKline Beecham Corp
Publication of WO1994017826A1 publication Critical patent/WO1994017826A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

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 polypeptides useful in vaccine compositions and more specifically to vaccine compositions useful in providing immunity against influenza A and influenza B in an animal.
  • the present invention also relates generally to a method of enhancing expression of polypeptides and, more specifically, to a method of enhancing influenza protein expression and homogeneity in E. coli.
  • 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
  • Type A glycoproteins.
  • subtypes H1 swine flu
  • H2 asian flu
  • H3 Hong Kong flu
  • influenza A subtypes H1 and H3
  • horses, H3 and H7 atypical flu
  • 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.
  • the sequence is derived from an H3N2 subtype influenza virus.
  • 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 viruses, where the sequences are derived from other Type A influenza strains infecting animals as well as humans.
  • Such viruses 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 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.
  • 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 an HA protein from a selected Type A subtype virus.
  • the subtype virus is an H3N2.
  • the protein may be derived from the HA subunit of a Type B influenza virus.
  • Other embodiments 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 method of recombinantly producing the fusion proteins of the invention, and a method of purifying the same.
  • the invention provides a vaccine composition containing a purified protein of the invention, as described above.
  • a vaccine composition may include a fusion protein 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.
  • 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 immunogenic 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.
  • the present invention provides a method of enhancing in E. coli the expression of influenza vaccinal proteins characterized by a naturally-occurring amino acid pattern comprising Arg-Arg-Xaa-Xaa-Arg [S ⁇ Q ID NO:8].
  • Arg is arginine
  • Xaa is any amino acid
  • at least one of the arginines in the naturally-occurring sequence is encoded by the rare nucleic acid triplets AGG or AGA.
  • the method of the invention involves mutating one or more of these AGG or AGA codons to a preferred arginine codon and expressing the mutated sequence in E. coli.
  • this modification which does not result in a change in the encoded amino acid sequence, can increase the expression and homogeneity of an influenza protein in E. coli significantly.
  • the method of this invention involves increasing the expression of the above-identified proteins by inserting into the host cell tRNA molecules capable of translating the native rare arginine codons.
  • the E. coli host cells are modified such that they are capable of efficiently translating the rare, native arginine codons.
  • the present invention provides novel nucleic acid sequences of influenza proteins which contain the nucleotide sequence CGn-CGn-
  • n represents a nucleotide selected from the group consisting of T, C, A or G [S ⁇ Q ID NO:9], in place of the native nucleotide sequence AGr- AGr-Xaa-Xaa-AGr, where r represents the nucleotides A or G [S ⁇ Q ID NO: 10].
  • these sequences result in increased expression of the encoded protein as compared to the native sequence.
  • the invention provides the novel modified nucleic acid sequences described above fused in the same reading frame to another DNA sequence encoding a polypeptide or protein, i.e., a fusion partner, which may further enhance the expression of, or immunogenicity of, the encoded influenza protein. It is desirable that the fusion partner be an influenza protein sequence or fragment thereof.
  • 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
  • Fig. 2 illustrates the nucleic acid and amino acid sequences of
  • 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].
  • Fig. 5 illustrates the pOTS208NS1BLmut2 vector nucleic acid sequences [SEQ ID NO: 54] encoding the amino acid sequences [SEQ ID NO: 55] of the mutant NS (1-81) BLHA2 ( 1-223) (m et-leu) fusion protein, with the nucleic acid sequences of the coding region NS (1-81) BLHA2 (1 -223) [SEQ ID NO: 56] and native amino acid sequences [SEQ ID NO: 57], which include a Met in amino acid position 98, illustrated above the modified BLHA2 sequences.
  • Fig. 6 illustrates the nucleic acid [SEQ ID NO: 17] and amino acid [SEQ ID NO:18] sequences of the H1N1 fusion protein, NS1 ( 1-81 ) HA2 (65-222) , also known as flu D.
  • Fig. 7 illustrates the naturally-occurring nucleic acid sequence [SEQ ID NO:1] and corresponding amino acid sequence [SEQ ID NO:2] of the HA2 portion of the H3N2 virus, A/Udorn.
  • Fig. 8 illustrates the naturally-occurring nucleic acid sequence [SEQ ID NO: 1]
  • 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 characteristics of the exemplified sequences.
  • this invention provides a protein or fragment thereof characterized by an amino acid sequence derived from the HA2 subunit of an HA protein, e.g., from a H3N2 subtype virus.
  • a "fragment" of the HA2 subunit is an amino acid sequence derived from the HA2 subunit which is characterized by having an immunogenic determinant of the HA2 subunit.
  • Such a fragment is desirably at least about 8 amino acids in length.
  • H3 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 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 [University of Wisconsin].
  • Proteins according to this invention may include unfused HA2 subunits of the influenza A viruses, particularly H3N2 subtype.
  • a protein of the invention contains amino acids 1-221 of a selected H3HA2 subunit.
  • 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 protein or an HA2 subunit protein.
  • the NS1 portion of the fusion protein is derived from an HlNl subtype virus, A/PR/8/34.
  • the NS1 portion may comprise amino acid residues 1 to 42 of H1NS1.
  • 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.
  • 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.
  • the present invention provides a modified protein containing a portion of the HA2 subunit of a Type B influenza virus.
  • 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 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 to a fusion "partner" protein or protein fragment, as described above for the H3HA2 proteins of this invention, or remain unfused.
  • a linker sequence may optionally be inserted 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, collagenase, and thrombin.
  • 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.
  • 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.
  • H3 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 acids 1 to 221 of the H3HA2 subunit (amino acids 1-221).
  • NS1 ( 1-81) H3HA2 (77-221 ) [SEQ ID NO: 12] comprises the first 81 amino acids of NS1 fused to amino acids 77 to 221 of the truncated H3HA2 subunit.
  • Fig. 3 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 acids 77 to 221 of the truncated H3HA2 subunit.
  • a present preferred example of a Type B 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.
  • Another fusion protein of this invention is NS1 ( 1 -81) BHA2 (1 - 223) [SEQ ID NO: 57], which contains the first 81 amino acids of NS1 fused to amino acids 1 to 223 of the BHA2 subunit.
  • Another preferred fusion protein of the invention is NS1 ( 1-81 ) BHA2 ( 1 -223) (met-leu) SEQ ID NO: 55, which contains the same amino acid sequence as NS1 (1-81) BHA2 (1-223) , with the exception that the internal methionine residue at position 98 of the fusion protein has been changed to a leucine. (Fig. 5)
  • H3HA2 proteins 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).
  • the amino acid sequence of the NS1 ( 1 - 81 ) H3HA2 ( 1 -221 ) protein [SEQ ID NO: 10] has only approximately 50%
  • the nucleic acid sequence of the H3HA2 1-221 protein derived from A/Udorn has only approximately 60% homology with the nucleic acid sequence of the HlHA2 1-222 protein derived from strain A/PR/8/34 (nucleotides 1872-2407 from A/PR/8/34) [SEQ ID NO: 5].
  • the nucleic acid sequence of H3HA2 1-221 from A/Udorn 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] [Fiers et al, Cell, 19:683-696 (1980)].
  • Analogs of the HA2 peptides from a Type A virus, e.g., an H3, or Type 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.
  • 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.
  • 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) targeting.
  • 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.
  • 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).
  • NS1 nonstructural influenza protein 1
  • 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.
  • a DNA coding sequence for HA from the above-cited references can be prepared synthetically or can be derived from viral RNA or from available cDNA-containing plasmids by known techniques.
  • a DNA coding sequence for HA from 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,
  • influenza viruses including other strains, subtypes, and types are available from clinical specimens and from public depositories, such as the
  • 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.
  • this invention provides certain silent mutations to the coding sequences for NS1 (1 -81) H3HA2 (1-221) , which have been found to increase expression yields. See Fig. 2. Further, the NS1 ( 1-81 ) BHA2 (1 - 223) (met-leu)-encoding sequence, BC13mut2, in addition to modifying the codon encoding amino acid position 98 of the fusion protein (position 17 of the HA2 portion), contains a number of silent modifications designed to increase protein expression. See Fig. 5.
  • 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 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.
  • the recombinantly-produced fusion proteins of the invention are purified as described herein.
  • method of purification involves (step 1) the isolation of the proteins, (step 2) enzymatic digestion and extraction, (step 3) urea extraction, (step 4) solubilization, reduction, and DEAE chromatography, (step 5) reverse phase chromatography, (step 6) precipitation, and (step 7) desalting and preparation of the final product.
  • the host cells containing the fusion proteins are disrupted, either chemically or by mechanical means.
  • the cells are lysed by osmotic shock.
  • the resulting pellet (P1) is subjected to nuclease digestion extraction and centrifuged to yield pellet 2 (P2).
  • a second extraction step is then performed using urea (pH 6) and the mixture centrifuged to yield pellet 3 (P3).
  • P3 is then solubilized and reduced.
  • solubilization is performed using urea at pH 12.5 and reduction is via DTT DEAE chromatography followed by SDS elution.
  • the resulting DEAE product is further reduced, preferably using DTT, and subjected to reverse phase chromatography.
  • the reverse phase product is then precipitated by adjusting to pH 6 and centrifuged.
  • the precipitated product is resolubilized, preferably with urea at pH 12.5, and subjected to G25
  • 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.
  • a desirable 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.
  • polypeptide employed in the presently preferred
  • a suitable strain, LW14 has the following genotype: galE::Tnl0 ⁇ CI857 bio- uvrB-; phenotypically, strain LW 14 requires biotin for growth, is sensitive to UV light and DNA damaging agents, and cannot use galactose as a carbon source. Construction of this strain is described in the examples below.
  • 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 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 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.
  • 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 antioxidants.
  • compositions of this invention two desirable adjuvants are available commercially, i.e., REHSORPTARTM adjuvant [Armour Pharmaceuticals, Kankakee, IL] and REHYDRAGELTM adjuvant [Reheis Chemical Co., Berkeley Heights, NJ].
  • REHSORPTARTM adjuvant Armour Pharmaceuticals, Kankakee, IL
  • REHYDRAGELTM adjuvant Reheis Chemical Co., Berkeley Heights, NJ.
  • These products are aluminum hydroxide gels which contain approximately 2% w/v AI 2 O 3 , which is equivalent to approximately 10.6 mg/ml Al .
  • 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.
  • 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
  • HA2 hemagglutinin subunit 2
  • 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 (1-81) HA2 (65-222) ) [SEQ ID NO: 17 & 18] and other fusion proteins derived from the HlNl 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.
  • suitable H1 subtype immunogenic proteins include
  • HlNl 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 HI proteins consist of unfused polypeptides, such as H1HA266-222 [SEQ ID NO: 33 & 34] which is disclosed in co-pending 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 adjuvant.
  • 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.
  • a currently preferred combination vaccine of the invention contains the H3 subtype fusion protein NS1 ( 1 -81 ) H3HA2 ( 1 -221 ) [SEQ ID NO: 10], the B subtype fusion protein NS1 (1 -81 ) BHA2 ( 1 -223) (met-leu) [SEQ ID NO: 55], and the H1 subtype fusion protein NS1 ( 1 -81 ) HA2(65-222) [SEQ ID NO: 18].
  • Studies have shown that such a combination vaccine is protective against challenge with H1, H3 and Type B influenza viruses in mice.
  • combination vaccines would include the NS1 (1 - 81 ) H3HA2 (77-221 ) protein [SEQ ID NO: 12] or the NS1 (1 -81 ) BHA2 (1-223) [SEQ ID NO: 57] in combination with one or more additional influenza antigens derived from the type or subtype influenza viruses described above. These combination vaccines 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.
  • 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.
  • 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.
  • 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 present invention provides methods for producing enhanced expression and improved homogeneity of influenza viral proteins and polypeptides in E. coli. Also provided are novel modified nucleotide sequences which encode these influenza proteins and are useful in the methods of production.
  • influenza proteins or polypeptides produced according to the invention include the complete HA2 protein of the hemagglutinin antigen (HA) of a selected H3N2 influenza virus, a complete HA protein of an H3HA2 virus, fragments thereof, and fusion proteins containing the complete H3HA2 protein or desired fragments thereof fused in the same reading frame with a selected fusion partner polypeptide or protein.
  • HA hemagglutinin antigen
  • fragment is meant a subunit of HA, or a span of contiguous amino acids from the complete protein capable of stimulating an antigenic or protective immunogenic response in an animal.
  • a fragment may contain at least about 8 amino acids from the selected influenza protein, and can contain up to the number of amino acids which make up the entire protein.
  • fragment' refers to nucleotide sequences which encode the above-defined amino acid fragments.
  • Arg represents arginine and Xaa represents any amino acid in this formula.
  • this five amino acid sequence is referred to as Formula I.
  • Formula I sequences are typically encoded by native nucleotide sequences of the formula of codons AGr-AGr-Xaa-Xaa-AGr, where r represents the nucleotides A or G and Xaa represent any codon [SEQ ID NO:63].
  • this five codon nucleotide sequence is referred to as Formula II.
  • the native nucleic acid sequence encoding a subtype H3N2 influenza virus protein, fusion protein, or a fragment or subunit thereof, specifically the HA2 portions of H3N2 virus strains is characterized by a Formula II sequence.
  • H3N2 subtype strains of influenza A characterized by this nucleotide fragment Formula II include the A/Udom and A/Victoria viruses.
  • Figs. 7 and 8 provide the native nucleic acid sequences of the HA2 portions of the A/Udorn [SEQ ID NO: 1] and A/Victoria [SEQ ID NO: 3] strains.
  • Other H3N2 virus strains of influenza A may also provide native nucleotide sequences containing Formula II, which sequences are susceptible to the modifications described herein.
  • native nucleotide sequences encoding proteins whose expression may be enhanced according to this invention are those native sequences which encode certain fragments of influenza proteins including the fragment spanning amino acids 1 to about amino acids 221 of H3HA2 [Fig. 7 SEQ ID NO:2 and Fig. 8 SEQ ID NO:3]; the fragment spanning from about amino acid 77 to about amino acid 221 [Fig. 7 SEQ ID NO:69 and Fig. 8 SEQ ID NO:70], or other desirable fragments.
  • Other desirable fragments of this H3HA2 amino acid sequence include those characterized by the ability to stimulate immunological activity in an immunized animal similar to that stimulated by use of the entire 221 amino acid sequence of H3HA2.
  • Nucleotide sequences encoding fusion proteins which contain fragments of the native nucleotide sequences encoding these influenza proteins or subunits can also be characterized by the Formula II nucleotide sequence.
  • these fusion proteins are also desirable for enhanced expression according to the method of this invention.
  • the inventors have discovered that when native nucleotide sequences of influenza proteins, which sequences comprise Formula II, are expressed in E. coli, a frame shift of one nucleotide after the third triplet in Formula II in the native sequence occurs, resulting in the increased translation of truncated proteins. It has been surprisingly found that by application of a method of the present invention, the expression and homogeneity of the influenza protein is increased significantly.
  • the methods of this invention involve enhancing the expression of proteins characterized by the amino acid pattern of Formula I, which proteins have a native nucleotide sequence of Formula II.
  • a native nucleotide sequence encoding a selected influenza protein or fragment, which sequence comprises Formula II is modified by mutating one or more of the rare AGG or AGA arginine codons of Formula II to a preferred Arg codon.
  • a preferred arginine codon for use in replacing a native AGA or AGG codon according to this invention is defined herein by the codons CGT, CGG, CGA and CGC. Of these codons, CGT and CGC are currently the most preferred.
  • the modified influenza protein-encoding nucleotide sequence is then expressed in an E. coli expression system, resulting in enhanced expression in comparison to that obtained by expression of the native nucleotide sequence encoding the same protein in the same expression system.
  • the enhanced protein expression occurs even though the mutation does not result in a change in the encoded amino acid sequence of the protein.
  • 'enhanced expression' or 'enhanced protein expression' is meant an expression level of at least 40% higher than the expression level of the protein encoded by the native, non-mutated nucleotide sequence comprising Formula II, when expressed in E. coli.
  • the inventors believe that the enhanced expression levels are obtained because the silent mutation of the AGA or AGG to a preferred arginine codon in Formula II eliminates the frame shift mutation found in the unmutated nucleotides encoding these proteins, thus substantially reducing the production of truncated messages (proteins). It is believed that the resulting influenza proteins are more homogeneous when expressed in an E. coli expression system according to this invention.
  • the expression of the proteins containing arginines encoded by the rare codons AGG and AGA can be increased by inserting into the host in which expression is desired one or more genes for tRNA molecules which are capable of properly translating the AGG and AGG arginine codons.
  • the host cells are E. coli.
  • a gene for a tRNA molecule described above can be selected from among known gene sequences.
  • the genes and tRNA molecules which can translate the rare Arg codons identified above are known and readily available to one of skill in the art. See, e.g., [P. Saxena and J. Walker, J. Bacteriol., 174(6): 1956-1964 (Mar. 1992)].
  • these genes may be placed on a plasmid which will increase the copy number of these genes and therefore the tRNA molecules encoded by these genes.
  • these sequences can be genetically engineered and placed on the host cell chromosome behind an appropriate promoter element in such a manner that the effective concentration of these tRNA molecules is increased inside the cell.
  • Conventional texts describe the techniques useful in this method [See, e.g., Sambrook et al., Molecular Cloning. A Laboratory Manual. 2d edition, Cold Spring Harbor, New York (1989)].
  • this method may be used to increase expression of a protein in host cells lacking sufficient amounts of the appropriate tRNA to permit efficient expression of the protein. Use of this method obviates the need to modify the sequences encoding the selected protein, and thus provides an alternative method to the first embodiment described above.
  • novel modified nucleotide sequences are provided, which in E. coli expression systems, can be employed to produce the encoded influenza proteins, subunits, fragments and fusion proteins described above according to the first embodiment of the method of this invention.
  • the proteins encoded by these nucleotides are produced at levels of expression enhanced over that of the native sequences, by about forty percent or more.
  • novel nucleotide sequences of the invention are characterized by comprising the nucleotide sequence CGn-CGn-Xaa-Xaa-CGn, where n represents a nucleotide selected from the group consisting of T, C, A or G [S ⁇ Q ID NO:62], in place of the Formula II fragment in the native nucleotide sequence encoding the selected influenza protein or fragment.
  • the nucleotide fragment identified by the formula above is referred to herein for simplicity as Formula III.
  • a modified DNA sequence of the invention comprises the Formula El nucleotide sequence and may encode the amino acid sequences identified specifically above, e.g., Fig. 7 [SEQ ID NO:2], Fig. 8 [SEQ ID NO:3]; Fig. 7 [SEQ ID NO:69] and Fig. 8 [SEQ ED NO:70], or other fragments.
  • the nucleic acid sequence encoding the HA2 subunit protein which contains the native sequence of Formula II has been provided with three silent mutations, which have changed each of the three native arginine-encoding AGG codons each to a preferred arginine codon CGT.
  • These codons encode amino acid numbers 123, 124 and 127 of the H3HA2 subunit protein of the A/Udorn strain identified in Fig. 7.
  • the same codons (and amino acid numbers) are altered in the A/Victoria strain identified in Fig. 8 to provide another example of a modified nucleotide sequence according to this invention.
  • the native nucleotide sequences encoding the HA2 subunit proteins of the aforementioned viruses are modified according to this invention at nucleotides 367, 370, and 379.
  • the native A adenine
  • C cytosine
  • the native nucleotides at sites 369, 372 and 381 in each sequence are changed from a G (guanine) to a T (thymine), resulting in preferred Arg codons.
  • nucleotide sequences encoding the influenza vaccinal polypeptides described herein, or other such influenza proteins or subunits characterized by Formula II may be mutated into novel nucleotide sequences of this invention, i.e., by mutating Formula II into Formula III within those sequences using the first embodiment of the methods of this invention.
  • the silent mutations described herein may be inserted at analogous regions in each nucleotide sequence.
  • novel modified H3HA2 nucleotide sequences whether alone or in association with a nucleotide sequence encoding a fusion partner of a fusion protein of the invention are useful in E. coli expression systems.
  • the novel nucleotide sequences of the invention will also encode analogs of the H3HA2 peptides, such as truncated polypeptides (including fragments) and H3HA2 polypeptides, e.g. mutants that retain the epitopes and thus the biological activity of
  • the nucleotide sequence encodes a fusion protein
  • the non-HA2 fusion partner e.g., NS1 as described below
  • 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.
  • the analogs of the nucleotide sequences encoding the HA2 peptides and/or the fusion partner may differ by only 1 to about 4 codon changes, in addition to the nucleotide mutations to the above-identified fragment.
  • sequences of this invention include modified nucleotide sequences which encode polypeptides with minor amino acid variations from the natural amino acid sequence of HA2.
  • conservative amino acid replacements may be introduced by altering, deleting or replacing codons of the native sequence, in addition to altering those codons in Formula II according to one embodiment of this method.
  • Conservative replacements are those that take place within a family of amino acids that are related in their side chains and are well known in the art. For example, it is reasonable to expect that an isolated replacement of a selected amino acid with a conservative replacement of an amino acid with a structurally related amino acid will not have a significant effect on the activity of the protein, especially if the replacement does not involve an amino acid at an epitope of the HA2 polypeptide.
  • novel modified nucleotide sequences of this invention are further characterized by encoding an immunogenic determinant of a modified HA2 subunit of an HA protein, derived from an H3N2 subtype.
  • the encoded protein may contain all or a portion of the H3N2 HA2 sequence, including the Formula I amino acid sequence.
  • the currently preferred embodiment provides a novel DNA sequence encoding an H3HA2 protein or fragment thereof fused in frame to a DNA sequence encoding a portion of the nonstructural influenza protein 1 (NS1).
  • One modified fusion protein-encoding nucleotide sequence is obtained by making mutations according to this invention in the nucleotide sequence encoding the fusion protein NS1 ( 1-81 ) H3HA2 (1 -221) [SEQ ID NO:10].
  • the nucleotide sequence [SEQ ID NO:58] for this fusion protein [SEQ ID NO: 10] is referred to herein as pOTS208NS1H3mut5585.
  • the modified coding sequences for the HA2 proteins, as well as the coding sequences for 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.
  • references known to the art which disclose the nucleotide coding sequences for HA from the A/Japan/305/57 strain [Gething et al, Nature, 287:301-306 (1980)]; strain A/NT/60/68 [Sleigh et al., and Both et al., in Developments in Cell Biology, Elsevier Science Publishing Co., pages 69-79 and 81-89, respectively, (1980)]; strain A/WSN/33 [Davis et al, Gene, 10:205-218 (1980); Hiti et al., Virology, 111:113-124 (1981)]; and fowl plague virus [Porter et al.
  • 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.
  • Novel modified nucleotide sequences of this invention may also include 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 protein sequences, and the Formula III fragment [SEQ ID NO:62].
  • DNA sequences having the Formula III fragment, which sequences encode other H3N2 HA2 proteins of the invention include sequences which differ in codon sequence outside of Formula II due to degeneracies of the genetic code or variations in the DNA sequence encoding H3HA2 proteins. Such codon differences may be caused by point mutations or by induced modifications to enhance the activity, half- life or production of the peptide encoded thereby.
  • DNA sequences characterized by the above modification of Formula II into Formula III which hybridize under stringent conditions with the DNA sequences encoding the HA2 subunit proteins, e.g., H3HA2 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 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, cited above].
  • a preferred method of production which uses the modified nucleotide sequences of this invention 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, kanamycin or
  • these protein sequences or fragments thereof may also be fused to a polypeptide capable of further enhancing expression of these fragments in the selected host system.
  • such a peptide would contain a leader sequence fragment that provides for secretion of the H3HA2 subunit 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.
  • a promoter sequence may be linked directly with the DNA molecule encoding the H3HA2 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.
  • E. coli expression systems including expression vectors and transformed host cells are also within the skill of the art. See, generally, methods described in standard texts, such as Sambrook et al, cited above.
  • the present invention is therefore not limited to any particular vector, nor to any particular purification process from cell lysates or cell medium.
  • Influenza proteins encoded by the modified nucleotide sequence may be expressed in enhanced manner according to the first embodiment of the method of this invention, or the influenza proteins may be expressed in an enhanced manner by translation from their native sequences by the second embodiment of the method.
  • fusion protein which comprises a protein sequence encoded by the modified nucleotide sequence containing Formula III in place of Formula II in the native nucleotide sequence encoding an HA2 subunit of an HA protein from an H3N2 subtype virus, fused in frame to another protein or protein fragment (a "fusion partner") capable of enhancing expression of the fusion protein.
  • a fusion partner protein or fragment taking into account the desired host cell, i.e., E. coli, and utilizing the teachings herein.
  • the H3HA2 fragment or sequence encoded by a modified nucleotide sequence as described above or the native sequence used in the second embodiment of this method may be fused to any peptide capable of further enhancing its expression in the host cell selected or of increasing its immunogenicity.
  • the method of the present invention does not limit the nature of the "partner" protein or fragment to which the H3HA2 fragment is fused to provide the enhanced expression of the resulting fusion protein.
  • influenza protein or fragment bearing the amino acid sequence of Formula I may be fused to a number of conventionally known and used "partner" proteins [See, general texts on expression such as Current Protocols in Molecular Biology, Vol. 2, suppl. 10, publ. John Wiley and Sons, New York, NY, pp. 16.4.1-16.8.1 (1990); Smith et al, Gene, 67:31-40 (1988); U. S. Patent No. 4,801,536, among others].
  • 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 subunit.
  • a linker sequence may be inserted optionally between the two sequences, i.e., between the sequence encoding the fusion partner and the HA2 protein encoded by the modified nucleotide sequence of this invention or the native sequence for expression according to the second embodiment of the method.
  • This optional linker may provide space between the two protein sequences; and may encode a polypeptide or contain a cleavage site, which is selectively cleavable or digestible by conventional chemical or enzymatic methods.
  • An example of a fusion protein whose expression can be enhanced by a method of this invention is NS1 ( 1-81 ) H3HA2 (1 -221) illustrated in Fig.
  • SEQ ID NO: 10 which comprises the first 81 amino acids of NS1 (derived from an HlNl subtype virus, A/PR/8/34) fused to the sequences spanning amino acid 1 to 221 of the H3HA2 subunit (amino acids 1-221) via an optional four amino acid linker sequence.
  • NS1 ( 1-81 ) H3HA2 (77-221 ) SEQ ID NO:72 comprises the first 81 amino acids of NS1 fused to the sequences spanning amino acid 77 to 221 of the truncated H3HA2 subunit.
  • the NS1 portion may comprise the sequence spanning amino acid residues 1 to amino acids 42 of H1N1.
  • 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 (H3N2).
  • the host cells used to express these fusion proteins may be modified by the second embodiment of the method of this invention to contain tRNA molecules capable of translating the rare arginine codons of Formula II. See, e.g., Example 25.
  • the nucleic acid sequence encoding these and other suitable H3HA2 proteins or H3HA2-containing proteins, i.e. those comprising a native Formula II sequence [SEQ ED NO:9] may be modified by the first embodiment of the method of this invention to replace
  • the proteins and fusion proteins whose expression is enhanced by the methods of this invention may be employed in vaccine compositions.
  • Several of the specific influenza proteins or fusion proteins described herein, which are produced according to the methods of this invention, have demonstrated the ability to stimulate or produce a protective immune response 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.
  • Such H3HA2 proteins and fusion proteins are capable of inducing T helper cells, particularly cytotoxic T lymphocytes, in the absence of neutralizing antibodies.
  • compositions can contain an effective immunogenic amount of a selected H3HA2 protein produced according to this invention or encoded by a modified nucleotide sequence of this invention in admixture with a suitable adjuvant in a non toxic and sterile pharmaceutically acceptable carrier.
  • suitable carriers for vaccine use, as well as other vaccine formulation additives and adjuvants, are well known to those of skill in the art. See, e.g., European Patent Application No. 366, 238, published May 2, 1990; and
  • compositions may be effectively administered to human and animal patients to induce the appropriate immune response.
  • the details of dosage and treatment using such compositions are also described in the above-cited published patent
  • 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.
  • 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 pAS 1 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 Rosenberg et al, in Methods Enzymol., 101:123-138 (1983).
  • Plasmid pAS 1 ⁇ EH was prepared by deleting a non-essential EcoRI-
  • Hindlll region of pBR322 origin from pASl 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 pAS 1 ⁇ EH.
  • the plasmid has an Ncol site between the codons for amino acids 81 and 82 and an
  • Nrul site 3' to the NS sequences.
  • the BamHI site between amino acids 1 and 2 is retained.
  • Plasmid pMG27N a pAS1 derivative [MoI. Cell. Biol., 5:1015-1024 (1985)] was cut with BamHI and Sad and ligated to a BamHI/NcoI fragment encoding the first 81 amino acids of NS1 from pAS 1 ⁇ EH801 and a synthetic DNA Ncol/Sacl fragment of the following sequence: SEQ ID NO: 35:
  • 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.
  • Plasmid pMG1, described above in Example 2 was digested with Ncol and Xbal, releasing a 54 bp fragment, which was discarded.
  • Plasmid pMS3H3HA, described in Example 1 above was digested with Hhal and Xbal, 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 Ncol 5' overhang sequence (at the 5' end) and a Hhal 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 2.5 ⁇ 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 pMGlH3HA2 (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] or H3C13.
  • pMGl was digested with BamHI and Ncol and ligated to the BamHI/NcoI fragment encoding amino acids 2 to 42 of NS1 from pNS1 42 TGF ⁇ .
  • pNS1 4 2TGF ⁇ is derived when pAS1 ⁇ EH801 is cut with Ncol and SalI and ligated to a synthetic DNA encoding human TGF ⁇ as an Ncol/Sall fragment.
  • pNS1 42 TGFCI encodes a protein comprised of the first 42 amino acids of NS1 and the mature TGF ⁇ sequence.
  • the NS1 portion of pNS142TGF ⁇ 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 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: SEQ ID NO: 39:
  • 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. 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)].
  • 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 Pstl, and the fragment containing the kanamycin gene was inserted between the EcoRI and Pstl sites of pOTSV [Shatzman and Rosenberg, cited above].
  • the resulting plasmid is pOTS207.
  • the pOTS207 was digested with EcoRI and Pstl, and the 1467 bp fragment containing the kanamycin resistance gene was isolated. Synthetic oligonucleotides:
  • SEQ ID NO: 41 5' AATTCGTACCTA 3'
  • Plasmid pBHA is a pBR322-derived vector, containing the complete nucleotide sequence of the 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 pMG 42 -Kn (described above) that had been digested with Seal. During the cloning, a nucleotide base (T) was deleted from the Seal recognition site shifting the gene out of the reading frame. The vector was digested with Ncol, 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
  • 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.
  • NTE 1 mM EDTA
  • PBS phosphate-buffered saline
  • Antisera was prepared as follows. 100-200 micrograms of purified virus in complete Freund's adjuvant w ected 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 and antisera collected 7-10 days later. EXAMPLE 7 - EXPRESSION OF H3HA2 FUSION PROTEINS
  • the plasmid pMGlH3HA2 (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.
  • 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.
  • the plasmid encoding the NS1 ( 1-81 ) H3HA2 (77-221 ) peptide [SEQ ID NO: 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.
  • lysis buffer A 50 mM Tris-HCl, 5% glycerol, 2 mM EDTA and 0.1 mM DTT, pH 8.0
  • 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,
  • 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 KC1 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 NH 4 HCO 3 and 0.02% SDS [M. Hunkapiller et al, Method. Enzvmol., 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.
  • the protein is usually greater than 75% pure.
  • pOTSV is described in Devara et al, Cell, 36: 43-49 (1984).
  • this vector is a pAS1 derivative with t-oop inserted at the Nrul site and a synthetic oligonucleotide encoding Sad, Xhol and Xbal restriction sites inserted at the Sail site (which is destroyed).
  • pOTS208 was prepared by digesting pOTSV with EcoRI and Seal, followed by fill in reaction using Klenow.
  • Tn5 Plasmid DNA (described in R. Jorgensen et al., Mol. Gen. Genet., 177:65-72 (1979)] was digested with HindIII and Smal, followed by a fill in reaction using Klenow yielding a 1323 bp fragment encoding for neomycin phosphotransferase-2 gene (NPT-2). This fragment is described in detail in Rothstein et al., Cell, 19:795-805 (1980) and Jorgensen, cited above. This fragment and the above digested vector were ligated together to create pOTS208, which is kanamycin resistant.
  • pMGlH3HA2( 1-221) (Example 3) was digested with BamHI and Xbal, releasing two fragments: an 806 bp BamHI fragment and a 160 bp
  • BamHI/Xbal fragment BamHI/Xbal fragment. These fragments together code for NS1 (1-81) H3HA2 ( 1- 221) .
  • pOTS208NS181NS181H3HA2-26 was cut with Ncol and Sail, filled in and ligated with Linker 1041 [New England Biolabs] to insert a Kpnl site and regenerate the Ncol site. This step also deletes the H3C13 region.
  • the unique Xbal site of the parent pOTS208 vector is downstream of the deletion.
  • the resulting vector is pOTS208NS181Nco.
  • a mutant H3C13 protein was prepared by mutating the nucleotide sequences of the fusion protein prepared according to Example 3 above.
  • Site directed mutagenesis using the Altered Sites System [Promega Corporation] according to the manufacturer's directions was used to change nucleotide numbers, 622, 625, and 634 (A to C) and 624, 627, and 636 (G to T) of nucleotide sequences [SEQ ID NO:9] encoding the NS1 (1-81) H3HA2 (1 -221) fusion protein of Fig. 2 [SEQ ID NO: 10], thereby changing the codons at these regions from AGG to CGT, both encoding Arg.
  • These changes correspond to nucleotide numbers 367, 370, and 379 (A to C) and 369, 372, and 381 (G to T) of the HA2 fragment of Fig. 2 [SEQ ID NO: 58].
  • Fig. 2 illustrates the modified nucleotide sequences of the fusion protein [SEQ ID NO: 10] by contrast with the nucleotide sequence [SEQ ID NO: 9] of the "unmodified" fusion gene (nucleotide changes above sequences of unmodified gene). Mutagenesis on this sequence was carried out according to the method provided with the pSelect kit from Promega.
  • cloning for the mutagenesis was performed as follows.
  • the pSelect plasmid [Promega] and pMG1H3HA2 (Example 3) were each digested with HindIII. These two plasmids were ligated together and selected on tetracycline plates. The resulting vector is pSe1H3HA2.
  • Mutagenesis was performed according to Promega's kit. The following oligonucleotide was used: SEQ ID NO: 43:
  • Clones were verified by restriction endonuclease HincII.
  • the resulting plasmid, pSe1H3HA2mut5585 was digested with Ncol and Xbal, and a 748 bp fragment coding for the H3HA2mut5585 polypeptide was isolated.
  • pOTS208NS181Nco (Example 9C) was digested with NcoI and
  • polypeptide NS1 (1-81) H3HA2 (1 -221) [SEQ ID NO:10].
  • the plasmid of A was transfected into E. coli strain AR58
  • pSe1H3HA2mut2 was digested with Ncol and Xbal, and an approximately 748 bp fragment encoding for the H3HA2mut2 polypeptide was isolated.
  • pOTS208NS181Nco was digested with Ncol and Xbal.
  • the ligation of linear pOTS208NS181Nco (Example 9C) and the 748bp fragment resulted in pOTS208NS1H3mut2.
  • This vector codes for the NS1 (1-81) H3HA2 (1 -221) polypeptide [SEQ ID NO: 10].
  • Plasmid pAS1_EH/801 (described above in Example 2) was cut with Bglll, end-filled with DNA polymerase I (DNApolI; Klenow), and ligated closed, thus eliminating the Bglll site.
  • the resulting plasmid pBgl- was digested with Ncol, end-filled with DNApolI (Klenow), and ligated to a Bglll linker.
  • the resulting plasmid, pB4 contains a Bgl ⁇ site within the NS1 coding region. Plasmid pB4 was digested with Bgi ⁇ and ligated to a synthetic DNA linker of the sequence:
  • SEQ ID NO: 45 5'-GATCCCGGGTGACTGACTGA -3'
  • SEQ ID NO: 46 3'- GGCCCACTGACTGACTCTAG-5'.
  • the resulting plasmid, pB4+ permits insertion of DNA fragments within the linker following the coding region for first 81 amino acids of NS1 followed by termination codons in all three reading frames.
  • Plasmid pB4+ was digested with Xmal (cuts within linker), end-filled (Klenow), and ligated to a 520 base pair PvuII/Hindlll, end-filled fragment derived from the HA2 coding region.
  • the resulting plasmid, pD codes for a protein [SEQ ID NO: 18] comprised of the first 81 amino acids of NS1, three amino acids derived from the synthetic DNA linker (Gln-He-Pro), followed by amino acids 65-222 of the HA2.
  • Expression is obtained by transfecting pD into a desired E. coli strain, preferably LW14, using standard techniques. Purification may be by standard techniques or, preferably, as described in Example 18 below.
  • 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 LD50 doses of either A/PR/8/34 (H1N1) or A/HK/68 (H3N2) virus, and survival was monitored through day 21.
  • 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.
  • the vaccine will be effective in a majority of individuals.
  • 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.
  • 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.
  • mice CB6F1 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.
  • Table 3 For convenience, NS1 (1-81) H3HA2 (1 -221) [SEQ ID NO: 10] 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 A/PR/8/34 (H1) or A/HK/68 (H3) virus.
  • mice immunized with the D protein were protected against HI but not H3 challenge.
  • 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.
  • pMG1 (Example 2) and pMG42Kn (Example 5) were both digested with BamHI and Ncol.
  • the digested pMG42Kn and the 236 bp fragment were ligated together and transformants were selected on LB and kanamycin agar plates.
  • the resulting vector pMG181Kn(cII) maintains all regulatory elements of pMG42Kn except the NS1 (aa1-42) sequence is replaced with the NS1 (aa1-81) sequence.
  • pMG181Kn(cII) described above was digested with BstXI and BamHI.
  • the folio wing linker en coding ribosome binding site (RB S3) is cloned in the digested vector, replacing the ell RBS.
  • the linker sequence is:
  • the resulting vector is pMG181KnRBS3.
  • pEA181KnRBS3 a 1.2 kb EcoRI/Bglll fragment from similarly digested pOTSV containing the lambda rexArexB region was cloned into mpl8 [Gibco/Bethesda Research Labs] and mutagenized to create silent mutations in the two Ndel sites in this region.
  • the mutations were CATATG to CATGTG in both sites.
  • One site is in the rexA and the other in the rexB.
  • pEA181KnRBS3 has the useful properties of the pMG vectors, plus the additional attribute of nalidixic acid induction.
  • Plasmid pOTS208BLeeHA2 was created as follows. An EcoRI fragment encoding the B/Lee HA region from plasmid pBHA (Example 5) was cloned into pSelect to generate pSelectPBHAS2. Site-directed mutagenesis inserted an Ncol site at the start of HA2, resulting in an N-terminus: MET GLY PHE PHE, and a C terminus of SER ILE CYS LEU. The resulting construct is called pSelectPBHAS2-B1.
  • This plasmid was cut with Ncol and Xbal (a site in the polylinker of pSelect downstream of the HA gene), and ligated into Ncol/Xbal digested pEA181KnRBS3, described above, to generate pEA181BLeeB1-1.
  • Ncol and Xbal a site in the polylinker of pSelect downstream of the HA gene
  • a BamHI/EcoRI, filled in, fragment was cut out of pEA181BLeeB1-1 and ligated into pOTS208 (Example 9A), that had been digested with Xbal, filled in, and BamHI.
  • the EcoRI and Xbal sites were regenerated by the ligation.
  • a BamHI/Xbal fragment of about 1011 bp encoding the NS ( 1 - 81) BLHA2 (1-223) sequence from plasmid pOTS208BLeeHA2 was isolated and ligated into vector pSelect- 1 [Promega], which was also digested with BamHI and Xbal.
  • the resulting construct is called pSe1BC13.
  • This vector contains the coding sequence for NS1 (1-81) BHA2 ( 1-223) , also termed BC13 [SEQ ID NO: 57].
  • Mutagenesis was carried out on the pSelBC13 using Promega's protocol and oligonucleotide 5492, SEQ ID NO: 50
  • pSelBC13mut5492 This plasmid was then digested with Ncol and Xbal, releasing a digestion fragment encoding for HA2, and ligated into pOTS208NS 18 INco (Example 9C) that had been digested with Ncol and Xbal.
  • pOTS208NS1BLHA2mut5492 codes for the same polypeptide of pOTS208BLeeHA2, (i.e. BC13), except the internal translation start is eliminated at amino acid position 98 of the fusion protein.
  • This protein is NS1 ( 1 - 81) BHA2 (1 -223) (met-leu) [SEQ ID NO: 55].
  • the codons for GLY at positions 93, 94, 97, 187, 215, and 217 were each mutated from GGG to GGT; the codons for ILE at positions 188, 189, and 214 were each changed from ATA to ATC; the codon for ASP at position 193 was changed from GAT to GAC; and the codon for ASN at position 216 was changed from AAT to AAC.
  • pSelBC13mut2 The resulting plasmid was called pSelBC13mut2. This plasmid was then digested with Ncol and Xbal, releasing a fragment of about 775 bp encoding for HA2. This fragment was ligated into pOTS208NS181Nco (described above), that had been digested with Ncol and Xbal. The resulting construct,
  • pOTS208NS1BLmut2 (see Fig. 5 [SEQ ID NO: 54]), codes for the same polypeptide [SEQ ID NO: 55] as pOTS208NS1BLHA2mut5492, except for the silent codon changes.
  • pOTS208NS1BLmut2 [SEQ ID NO: 54] is transfected into a suitable host cell, preferably an E. coli strain and expressed essentially as described for the H3 proteins described above.
  • Strain LW14 is a derivative of E. coli K-12 strain W3110 [ATCC E. coli 27325].
  • the transducing phage PI [E. coli ATCC 25404- B1] was grown on E. coli K-12 strain AR58, described above, the genotype of which is thr-gal ⁇ ::Tn10 ⁇ CI857 bio-uvrB- rpsL.
  • strain AR58 requires threonine, biotin for growth, is sensitive to UV light and DNA damaging agents, cannot use galactose as a carbon source, and is resistant to streptomycin.
  • Strain W3110 a prototroph, is incubated with the phage and plated onto a medium containing tetracycline to select for the transduction of the Tn10 element. The P1 phage picks up the segment of DNA containing the Tn10 and brings with it the ⁇ CI857 bio- uvrB-.
  • the strain LW14 has the following genotype: galE::Tn10 ⁇ CI857 bio- uvrB-.
  • Phenotypically, strain LW14 requires biotin for growth, is sensitive to UV light and DNA damaging agents, and cannot use galactose as a carbon source.
  • E. coli whole cells transformed with the pOTS208NS1BLmut2 plasmid [SEQ ID NO: 54] as described in Example 16 above were recovered after fermentation by centrifugation or tangential flow filtration, washed to remove media, and stored at -70°C until use.
  • Step 1 Lvsis and centrifugation (Isolation)
  • E. coli cells 500 gm wet cell weight (WCW), were thawed and suspended in 4-7 volumes (2L) of buffer containing 0.025 M Tris-HCl, 0.005 M EDTA, pH 8.0.
  • Chicken egg lysozyme (Calbiochem; suspension at 100 mg/ mL) was added to a final concentration of 1 g/L and the preparation stirred with a Tekmar mixer at room temperature for 1 hour.
  • the lysate was centrifuged at 15,000 x g f or 1 hour at 4°C and the supernatant discarded.
  • the pellet (PI) was resuspended in 5 mL per gram of original wet cell weight of buffer consisting of 0.025 M Tris-HCl, 0.002 M MgCl 2 , pH 8.0 (about 2.5L).
  • the yield of this step was 90-100% by SDS-PAGE analysis, and 65- 100% by RP-HPLC for product.
  • the preparation was treated with benzonase to digest nucleic acids, then extracted with nonionic detergents to reduce the levels of E. coli contaminants in the pellet.
  • Benzon nuclease 0.2 mL per L of suspension, was added to the suspension, which was then stirred at room temperature for 1 hr.
  • the sample was diluted with one volume of cold water containing 2% w/v Triton X-100 and 0.2% deoxycholate and stirred for 30 min at or below 15°C. Centrifugation was repeated as in step 1 and the supernatant discarded.
  • the pellet (P2) was extracted with 5 mL/gm WCW of cold 0.025 M NaH 2 PO 4 , 0.025 M Tris-HCl, pH 6.0, containing 4 M urea and 10 mM
  • DTT dithiothreitol
  • the P3 pellet was solubilized and applied to anion exchange chromatography. This step removes remaining nucleic acid and major host cell proteins.
  • P3 was suspended to 5 mL per gm WCW in .01 M Tris base, 8M urea (pH not adjusted). DTT was added to 25 mM.
  • the pH was then adjusted to 12.5 using 6N NaOH, stirring for 15 min at room temperature, immediately followed by a 5- fold dilution of the same with 10 mM boric acid containing 25 mM DTT. If needed, the sample may be diluted to keep conductivity below 2mS/cm.
  • the pH was adjusted to 9.0 and the sample stirred for up to 2 hour at room temperature.
  • the pH 12.5 treatment was necessary to complete solubilization of the B/Lee protein. However since carbamylation may occur under these conditions, the time was controlled very carefully. In addition, the pH 9 adjusted sample was unstable and cannot be held.
  • the yield of this step was 85-90% by SDS-PAGE or Western blot analysis, and was estimated at 65-70% by RP-HPLC assay for product.
  • the buffer E eluate from step 4 was adjusted to no more than 1 g/L protein concentration and made 2% in SDS, 30 mM DTT, 0.1% M Tris, 5 mM EDTA, pH 9, then heated at either: 90°C for 60, 95°C for 30 min, or 100°C for 25 minutes, using a heat exchanger or water bath. This treatment was necessary to break up aggregates and prepare the sample for RP chromatography. The sample was cooled to room temperature and 2-propanol was added to 10% v/v.
  • the sample was injected on an Amberchrome reverse phase column equilibrated in 10% 2-propanol/0.2% trifluoroacetic acid (TFA)/water.
  • the gradient shown in Table 1 was used to elute the column.
  • Fractions containing product were analyzed by analytical RP-HPLC, pooled, and held at 4°C.
  • the column was 25cm in height and was run at a linear velocity of 75-80 cm/hr at ambient temperature.
  • the loading capacity of the column was 2 g/L.
  • the pH of the RP eluate was adjusted to 6.0 +/- 0.5 using 1 N NaOH. After 10-15 min of stirring at room temperature, the precipitate was collected by centrifugation at 16,000 x g for 30 min at 4°C. The precipitate was resuspended to approximately 6-8 mg/mL protein concentration in 25 mM Tris, 8 M urea. DTT was added to 25 mM, and the sample stirred for 30 min at room temperature. The pH was adjusted to 12.5 and stirring repeated for 15 min, immediately followed by pH adjustment to 9.0 using HCl.
  • the precipitate was suspended in buffer containing 0.1 M Tris-HCl, 2% SDS, 0.01 M EDTA, pH 8.0-9.0. DTT was added to 25 mM, and stirred 15-30 min until the solution was clear and all of the precipitate had dissolved. The sample was immediately taken to the next step.
  • Step 7 Desalting and preparation of final product.
  • a 7 x 10 cm column was packed with Sephadex G25M (Pharmacia) at room temperature. It was equilibrated with 3-7 column volumes of 25 mM Tris- HCl, pH 9.,0, containing 5% w/v mannitol. Sample, at 6-10 mg/mL protein concentration, is injected on the column (20-25% of total column volume, i.e. 80- 100 mL per injection). The column was developed at 150 cm/hr linear velocity and the product desalted into the column buffer. The final product can be stored at 4°C.
  • the product of the purification process was recovered at an overall yield of about 20-40%, and was over 95% pure by SDS-PAGE and RP-HPLC analysis.
  • the final yield is about 3 g/500 g well cell weight.
  • FluD (Example 10) may be purified in much the same manner as the B/Lee with the following parameter alterations.
  • the FluD column was equilibrated in 8M urea, 50 mM Tris, 25 mM borate at pH 9.0. After the sample is loaded, sequential washes are performed with the following buffers: 4M urea in Tris-borate pH 9.0, 4 M urea and 0.4 M NaCl in Tris-borate pH 9.0, and Tris-borate pH 9.0.
  • the product is eluted with a step elution of 2% SDS, 0.1 to 0.25 M NaCl, in Tris-borate pH 9.0.
  • a recombinant vaccine was formulated to contain 1 ⁇ g each of the recombinant proteins NS1 (1-81) HA2 (65-222) (Example 11), NS1 (1-81) H3HA2 (1 - 221 ) mut5255 (Example 10), and the BC13mut2 (described in Example 15 above) in Al +3 (100 ⁇ g) plus 3-o-deacylated monophosphoryl-lipid A (3D-MPL) (5 ⁇ g) [described in U.S. Patent No. 4,912,093; commercially available from Ribi
  • influenza proteins Prior to inclusion in the recombinant vaccine, the influenza proteins were purified as described in Example 15 above to remove any contaminating bacterial proteins, DNA, and endotoxin.
  • mice female, CB6F1 were divided randomly into groups with 15 mice per group. The mice w ere injected subcutaneously on days 0 and 21 with the recombinant vaccine. A group of control mice were injected with the same dose of Al/MPL without antigen according to the same schedule. Mice were challenged with 3-5 LD 50 doses of virus on day 49 and survival was monitored through day 21 post-challenge.
  • N.D. not done and under the antigens
  • H1 NS1 (1-81) HA2 (65-222)
  • H3 NS1 ( 1-81) H3HA2 ( 1 - 221) mut5855
  • B NS1 (1-81) BLHA2 (1 -221) mut2.
  • each antigen contains the NS1 (1-81) regions from
  • H1 virus protections against H1 challenge was only achieved with the D protein which contains the H1HA2 region as well.
  • the H3HA2 and Type B HA2 portions of each chimeric antigen are responsible for conferring subtype- specific protection.
  • the combined HA2 constructs provide cross-protections for all currently circulating influenza Type A (H1 and H3 subtypes) and Type B viruses.
  • Plasmid pFV88 contains the entire 221 amino acid length HA2 from A/Udorn, an H3 subtype virus [C. J. Lai et al, Proc. Natl. Acad. Sci. USA, 77:210- 214 (1980)], which HA2 nucleic acid sequence is illustrated in Fig. 7 [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.
  • 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 cll translation initiation codon followed immediately by a BamHI site. It is described by Rosenberg et al, in Methods Enzymol., 101:123-138 (1983).
  • Plasmid pAS1 ⁇ EH was prepared by deleting a non-essential EcoRI -
  • the resulting plasmid, pAS1 ⁇ EH/801 expresses authentic NS1 (230 amino acids).
  • the plasmid has an Ncol site between the codons for amino acids 81 and 82 and an Nrul site 3' to the NS sequences.
  • the BamHH site between amino acids 1 and 2 is retained.
  • Synthetic oligonucleotides were annealed to generate an Ncol 5' overhang sequence (at the 5' end) and a Hhal 3' overhang sequence (at the 3' end).
  • the sequence of these oligonucleotides is as follows:
  • SEQ ID NO: 66 5'-CATGGGCGCCCATATGGGCATATTCGGCG-3'
  • 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 pMGlH3HA2 (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 23 - 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 x 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.
  • PBS phosphate-buffered saline
  • 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.
  • the modified nucleotide sequences encoding the H3HA2 proteins were prepared by mutating the nucleotide sequences of the fusion proteins prepared according to Example 22 above. Site directed mutagenesis using the Altered Sites System [Promega Corporation] according to the manufacturer's directions was used to change nucleotide numbers, 622, 625 and 634 (A to C) and 624, 627, and 636 (G to T) of nucleotide sequences [SEQ ID NO:9] encoding the NS1 (1-81) H3HA2 ( 1- 221) fusion protein of Fig. 3 [SEQ ID NO:10], thereby changing the codons at these regions from AGG to CGT, both encoding Arg. These changes correspond to nucleotide numbers 367, 370 and 379 (A to C) and 369, 372 and 381 (G to T) of the HA2 fragment of Fig. 7 [SEQ ED NO:1].
  • Fig. 2 illustrates the modified nucleotide sequences of the fusion proteins [SEQ ID NO: 58] by contrast with the nucleotide sequence [SEQ ID NO:9] of the "unmodified" fusion proteins (nucleotide changes below and amino acid changes in above sequences of unmodified fusion protein). Mutagenesis on this sequence was carried out according to the method provided with the pSelect kit from Promega.
  • cloning for the mutagenesis was performed as follows.
  • the pSelect plasmid [Promega] and pMGlH3HA2 (Example 22) were each digested with Hindlll. These two plasmids were ligated together and selected on tetracycline plates. The resulting vector is pSelH3HA2.
  • Mutagenesis was performed according to Promega's kit. The following oligonucleotide was used: SEQ ID NO:68:
  • Clones were verified by restriction endonuclease HincII.
  • the resulting plasmid, pSe1H3HA2mut5585 was digested with Ncol and XbaI, and a 748 bp fragment coding for the H3HA2mut5585 polypeptide was isolated.
  • pOTS 208NS181 (Eco-740) was digested with Ncol and Xbal. The ligation of linear pOTS208NS181Nco and the 748 bp fragment resulted in pOTS208NS1H3mut5585 [SEQ ID NO:58].
  • This vector codes for the polypeptide, NS1 (1-81) H3HA2 (1 -221) [SEQ ID NO: 10].
  • B. Expression of mutated NS1 (1-81) H3HA2 proteins The plasmid of A was transfected into E. coli strain AR58 [SmithKline Beecham].
  • immunoreactive species is expected at a molecular weight of approximately 35,00 daltons.
  • E. coli host cells containing H3N2 fusion protein obtained as described in Example 22 above were transformed using conventional techniques. See, e.g. Sambrook et al, cited above.
  • plasmid pDC952 carries the argU gene which encodes the tRNA that recognizes the
  • the H3/AR13 overnight culture was diluted 1:50 in LB and kanamycin (50 mL total) and incubated at 37°C until it reached an O.D.650 of 0.6.
  • the culture was then transferred to a 50 mL conical tube and chilled at about 4°C. Following this, the tube was centrifuged in a TJ6 centrifuge (10 min; 2000-3000 rev/min), the pellet resuspended in 25 mL 100 mM CaCl 2 , and placed on ice for about 30 minutes. The pellet was then centrifuged as described above and resuspended in about 2.5 mL 100 mM CaCl 2 .
  • the competent cells were aliquoted (100 ⁇ l) into three separate sterile tubes.
  • the first tube was the negative control and did not receive any DNA.
  • the second tube was a positive control and 1 ⁇ l of plasmid pT 7 II was added to the cells.
  • To the third tube was added 3 ⁇ l of pDC952. These controls served to ensure that transformation occurred.
  • Each tube of cells was mixed, placed on ice for 60 min., heat shocked at 37°C in a water bath for 2 minutes, and incubated in a 32°C water bath for 60 min. after adding 1 mL LB. The tubes were then microfuged for 1 minute and the supernatants poured off until only about 200 ⁇ L were left.
  • pellets were then resuspended in the remaining supernatant and plated as follows: (1) on LB and chloramphenicol, (2) on LB and ampicillin, and (3) on LB and chloramphenicol and kanamycin. The plates were then incubated at 32°C overnight.
  • 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)
  • FEATURE FEATURE:
  • 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)
  • MOLECULE TYPE DNA (genomic)
  • 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)
  • FEATURE FEATURE
  • MOLECULE TYPE DNA (genomic)

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Virology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne des compositions de vaccin capables de conférer une immunité multisouche contre la grippe de type A et B. L'invention porte également sur des procédés d'augmentation de l'expression et d'amélioration de l'homogénéité de la protéine H3HA2, de fragments de celle-ci et de protéines de fusion la contenant ainsi que sur de nouvelles séquences nucléotidiques codant ces protéines et fragments.
PCT/US1994/001149 1993-02-01 1994-02-01 Vaccins polypeptidiques Ceased WO1994017826A1 (fr)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US1341593A 1993-02-01 1993-02-01
US08/013,415 1993-02-01
US10891493A 1993-08-18 1993-08-18
US08/108,914 1993-08-18
US14915093A 1993-11-05 1993-11-05
US08/149,150 1993-11-05

Publications (1)

Publication Number Publication Date
WO1994017826A1 true WO1994017826A1 (fr) 1994-08-18

Family

ID=27359852

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1994/001149 Ceased WO1994017826A1 (fr) 1993-02-01 1994-02-01 Vaccins polypeptidiques

Country Status (1)

Country Link
WO (1) WO1994017826A1 (fr)

Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0621339A3 (en) * 1992-09-17 1995-11-29 Takara Shuzo Co Immunogenic human influenza a virus haemagglutinin polypeptides.
WO2005044992A2 (fr) 2003-11-04 2005-05-19 The Administrators Of The Tulane Educational Fund Methode de prevention des infections virales: fusion cellulaire par inhibition de la region de depart de fusion dans des virus a arn avec proteines d'enveloppe fusiogenes a membrane de classe i
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
US7959929B2 (en) 2005-04-21 2011-06-14 University Of Florida Research Foundation, Inc. Materials and methods for respiratory disease control in canines
US8222204B2 (en) * 2006-05-03 2012-07-17 The Administrators of the Tulane Educational Fund and Autoimmune Technologies, LLC Influenza inhibiting compositions and methods
US8604165B2 (en) 2007-06-25 2013-12-10 The Administrators Of The Tulane Educational Fund Influenza inhibiting compositions and methods
US9175069B2 (en) 2009-05-26 2015-11-03 Icahn School Of Medicine At Mount Sinai Monoclonal antibodies against influenza virus generated by cyclical administration and uses thereof
US9371366B2 (en) 2012-12-18 2016-06-21 Icahn School Of Medicine At Mount Sinai Influenza virus vaccines and uses thereof
AU2011217903B2 (en) * 2010-02-18 2017-03-02 Mount Sinai School Of Medicine Vaccines for use in the prophylaxis and treatment of influenza virus disease
US9708373B2 (en) 2010-03-30 2017-07-18 Icahn School Of Medicine At Mount Sinai Influenza virus vaccine and uses thereof
US9849172B2 (en) 2009-03-30 2017-12-26 Icahn School Of Medicine At Mount Sinai Influenza virus vaccines and uses thereof
US9908930B2 (en) 2013-03-14 2018-03-06 Icahn School Of Medicine At Mount Sinai Antibodies against influenza virus hemagglutinin and uses thereof
US10131695B2 (en) 2011-09-20 2018-11-20 Icahn School Of Medicine At Mount Sinai Influenza virus vaccines and uses thereof
US10555998B2 (en) 2014-11-24 2020-02-11 Intervet Inc. Inactivated equine influenza virus vaccines
US10736956B2 (en) 2015-01-23 2020-08-11 Icahn School Of Medicine At Mount Sinai Influenza virus vaccination regimens
US11254733B2 (en) 2017-04-07 2022-02-22 Icahn School Of Medicine At Mount Sinai Anti-influenza B virus neuraminidase antibodies and uses thereof
US11266734B2 (en) 2016-06-15 2022-03-08 Icahn School Of Medicine At Mount Sinai Influenza virus hemagglutinin proteins and uses thereof
US11865172B2 (en) 2005-04-21 2024-01-09 University Of Florida Research Foundation, Inc. Materials and methods for respiratory disease control in canines
US12364746B2 (en) 2018-06-21 2025-07-22 Icahn School Of Medicine At Mount Sinai Mosaic influenza virus hemagglutinin polypeptides and uses thereof
US12545718B2 (en) 2019-04-24 2026-02-10 Icahn School Of Medicine At Mount Sinai Anti-influenza B virus neuraminidase antibodies and uses thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0366238A2 (fr) * 1988-08-31 1990-05-02 Smithkline Beecham Corporation Polypeptides vaccinaux d'influenza

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0366238A2 (fr) * 1988-08-31 1990-05-02 Smithkline Beecham Corporation Polypeptides vaccinaux d'influenza

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
THE JOURNAL OF IMMUNOLOGY, Volume 140, Number 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. *
VIROLOGY, Volume 178, issued 1990, KUWANO et al., "Cross-Reactive Protection Against Influenza A Virus Infections by an NS1-Specific CTL Clone", pages 174-179. *

Cited By (40)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0621339A3 (en) * 1992-09-17 1995-11-29 Takara Shuzo Co Immunogenic human influenza a virus haemagglutinin polypeptides.
US8598116B2 (en) 2003-11-04 2013-12-03 The Administrators Of The Tulane Treatment of influenza virus infection
WO2005044992A2 (fr) 2003-11-04 2005-05-19 The Administrators Of The Tulane Educational Fund Methode de prevention des infections virales: fusion cellulaire par inhibition de la region de depart de fusion dans des virus a arn avec proteines d'enveloppe fusiogenes a membrane de classe i
JP2007514408A (ja) * 2003-11-04 2007-06-07 ザ アドミニストレイターズ オブ ザ テューレイン エデュケイショナル ファンド クラスｉ膜融合誘発性エンベロープタンパク質をもつｒｎａウイルスの融合開始領域の機能を阻害することによってウイルス：細胞融合を妨げる方法
US9725487B2 (en) 2003-11-04 2017-08-08 The Administrators Of The Tulane Educational Fund Compositions and methods for measles virus inhibition
US7491793B2 (en) * 2003-11-04 2009-02-17 The Administrators Of The Tulane Educational Fund Influenza virus inhibiting peptides
AU2004288218B2 (en) * 2003-11-04 2009-12-24 Autoimmune Technologies, Llc Method of preventing virus: cell fusion by inhibiting the function of the fusion initiation region in RNA viruses having Class I membrane fusogenic envelope proteins
US9913892B2 (en) 2005-04-21 2018-03-13 University Of Florida Research Foundation, Inc. Materials and methods for respiratory disease control in canines
US7959929B2 (en) 2005-04-21 2011-06-14 University Of Florida Research Foundation, Inc. Materials and methods for respiratory disease control in canines
US11160859B2 (en) 2005-04-21 2021-11-02 University Of Florida Research Foundation, Inc. Materials and methods for respiratory disease control in canines
US11865172B2 (en) 2005-04-21 2024-01-09 University Of Florida Research Foundation, Inc. Materials and methods for respiratory disease control in canines
US9345758B2 (en) 2005-04-21 2016-05-24 University Of Florida Research Foundation, Inc. Materials and methods for respiratory disease control in canines
US10258686B2 (en) 2005-04-21 2019-04-16 University Of Florida Research Foundation, Inc. Materials and methods for respiratory disease control in canines
US8222204B2 (en) * 2006-05-03 2012-07-17 The Administrators of the Tulane Educational Fund and Autoimmune Technologies, LLC Influenza inhibiting compositions and methods
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
US9353157B2 (en) 2007-06-25 2016-05-31 The Administrators Of The Tulane Educational Fund Influenza inhibiting compositions and methods
US8604165B2 (en) 2007-06-25 2013-12-10 The Administrators Of The Tulane Educational Fund Influenza inhibiting compositions and methods
US9849172B2 (en) 2009-03-30 2017-12-26 Icahn School Of Medicine At Mount Sinai Influenza virus vaccines and uses thereof
US9175069B2 (en) 2009-05-26 2015-11-03 Icahn School Of Medicine At Mount Sinai Monoclonal antibodies against influenza virus generated by cyclical administration and uses thereof
US9701723B2 (en) 2010-02-18 2017-07-11 Icahn School Of Medicine At Mount Sinai Vaccines for use in the prophylaxis and treatment of influenza virus disease
AU2011217903B2 (en) * 2010-02-18 2017-03-02 Mount Sinai School Of Medicine Vaccines for use in the prophylaxis and treatment of influenza virus disease
US9708373B2 (en) 2010-03-30 2017-07-18 Icahn School Of Medicine At Mount Sinai Influenza virus vaccine and uses thereof
US10179806B2 (en) 2010-03-30 2019-01-15 Icahn School Of Medicine At Mount Sinai Influenza virus vaccines and uses thereof
US10131695B2 (en) 2011-09-20 2018-11-20 Icahn School Of Medicine At Mount Sinai Influenza virus vaccines and uses thereof
US10583188B2 (en) 2012-12-18 2020-03-10 Icahn School Of Medicine At Mount Sinai Influenza virus vaccines and uses thereof
US9968670B2 (en) 2012-12-18 2018-05-15 Icahn School Of Medicine At Mount Sinai Influenza virus vaccines and uses thereof
US10137189B2 (en) 2012-12-18 2018-11-27 Icahn School Of Medicine At Mount Sinai Influenza virus vaccines and uses thereof
US9371366B2 (en) 2012-12-18 2016-06-21 Icahn School Of Medicine At Mount Sinai Influenza virus vaccines and uses thereof
US9908930B2 (en) 2013-03-14 2018-03-06 Icahn School Of Medicine At Mount Sinai Antibodies against influenza virus hemagglutinin and uses thereof
US10544207B2 (en) 2013-03-14 2020-01-28 Icahn School Of Medicine At Mount Sinai Antibodies against influenza virus hemagglutinin and uses thereof
US10555998B2 (en) 2014-11-24 2020-02-11 Intervet Inc. Inactivated equine influenza virus vaccines
US10736956B2 (en) 2015-01-23 2020-08-11 Icahn School Of Medicine At Mount Sinai Influenza virus vaccination regimens
US11266734B2 (en) 2016-06-15 2022-03-08 Icahn School Of Medicine At Mount Sinai Influenza virus hemagglutinin proteins and uses thereof
US11865173B2 (en) 2016-06-15 2024-01-09 Icahn School Of Medicine At Mount Sinai Influenza virus hemagglutinin proteins and uses thereof
US12233123B2 (en) 2016-06-15 2025-02-25 Icahn School Of Medicine At Mount Sinai Influenza virus hemagglutinin proteins and uses thereof
US11254733B2 (en) 2017-04-07 2022-02-22 Icahn School Of Medicine At Mount Sinai Anti-influenza B virus neuraminidase antibodies and uses thereof
US12030928B2 (en) 2017-04-07 2024-07-09 Icahn School Of Medicine At Mount Sinai Anti-influenza B virus neuraminidase antibodies and uses thereof
US12364746B2 (en) 2018-06-21 2025-07-22 Icahn School Of Medicine At Mount Sinai Mosaic influenza virus hemagglutinin polypeptides and uses thereof
US12545718B2 (en) 2019-04-24 2026-02-10 Icahn School Of Medicine At Mount Sinai Anti-influenza B virus neuraminidase antibodies and uses thereof

Similar Documents

Publication Publication Date Title
WO1994017826A1 (fr) Vaccins polypeptidiques
RU2761631C2 (ru) Белки rsv в предшествующей слиянию конформации и их применение
CA1341643C (fr) Virus non transmissibles
JP3955315B2 (ja) フラビウイルス組換えポックスウイルスワクチン
JP2001269185A (ja) Ｃ型肝炎ウイルスエピトープ
AU621358B2 (en) Mutant human angiogenin (angiogenesis factor with superior angiogenin activity) genes therefor and methods of expression
CA1334387C (fr) Vaccin contre le virus de la leucemie feline
AU7182991A (en) Method of delivering molecules into eukaryotic cells using tat protein conjugate
EP0662133B1 (fr) Proteines recombinees d'une souche pakistanaise du virus de l'hepatite e et leur utilisation dans des procedes diagnostiques et des vaccins
US5229293A (en) Recombinant baculovirus
KR20110088526A (ko) 구제역 백신 조성물 및 이의 제조 방법
CA2257137A1 (fr) Genes synthetiques de l'hepatite c
CZ369696A3 (en) Nucleotide sequences pestviral strains, polypetides encoded by those sequences and their use in diagnostic and prevention of pestviral infections
KR940005586B1 (ko) 플라비 비루스 항원
DE69133552T2 (de) Nicht-A, nicht-B Hepatitis Virus Antigen, und diagnostische Verfahren.
CN112552413B (zh) 新型冠状病毒重组蛋白亚单位疫苗
EP0237546A1 (fr) Mutants de deletion du virus pseudo-rabique et vaccins les contenant
US20230405109A1 (en) Nucleic acids, proteins, and vaccines of sars-cov-2
CA2295980A1 (fr) Utilisation de l'asparaginase wolinella succinogenes pour traiter des maladies associees a la dependance de l'asparagine
JP2008543301A (ja) フィブロネクチンのｅｄａドメインの使用を基にした剤および方法
PL150654B1 (en) Production of herpes simplex viral proteins
EP0117657A1 (fr) Polypeptides immunogènes contre la rage et vaccin utilisant ces peptides, séquences d'ADN, vecteurs d'expression, cellules recombinantes, cultures et procédés pour leur production
WO1993015763A1 (fr) Polypeptides de vaccination
Ogrina et al. Bacterial expression systems based on Tymovirus-like particles for the presentation of vaccine antigens
CA2363493C (fr) Virus de la diarrhee virale bovine de formes attenuees

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): JP US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
122 Ep: pct application non-entry in european phase