WO1994006468A1 - Compositions de vaccin contre le virus de la griffe, obtenues par recombinaison - Google Patents

Compositions de vaccin contre le virus de la griffe, obtenues par recombinaison Download PDF

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Publication number
WO1994006468A1
WO1994006468A1 PCT/US1992/007312 US9207312W WO9406468A1 WO 1994006468 A1 WO1994006468 A1 WO 1994006468A1 US 9207312 W US9207312 W US 9207312W WO 9406468 A1 WO9406468 A1 WO 9406468A1
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Prior art keywords
polypeptide
protein
ser
thr
sequence
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PCT/US1992/007312
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English (en)
Inventor
Susan B. Dillon
Christopher S. Jones
Miller O. Scott
Allan Shatzman
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Smithkline Beecham Corporation
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Priority to AU27756/92A priority Critical patent/AU2775692A/en
Publication of WO1994006468A1 publication Critical patent/WO1994006468A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/464838—Viral antigens
    • 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
    • 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

Definitions

  • the present invention relates generally to a vaccine composition capable of conferring multi-strain immunity against influenza A in a vaccinated animal.
  • Influenza virus infection causes acute respiratory disease in man, horses and fowl, sometimes of pandemic proportions. Influenza viruses are
  • 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.
  • Type A viruses have mostly been Type A, although Type C viruses have also been isolated from swine.
  • the Type A viruses are divided into subtypes based on the antigenic properties of the hemagglutinin (HA) and neuraminidase (NA) surface glycoproteins.
  • subtypes H1 (swine flu"), H2 (“asian flu”) and H3 (“Hong Kong flu”) are predominant in human infections.
  • polypeptide is an antigen which may be administered for vaccine purposes.
  • fusion proteins include NS 1- 81 -HA2 65-222 (D) protein and NS 1-81 -HA2 1-222 (C13) protein.
  • D NS 1- 81 -HA2 65-222
  • C13 NS 1-81 -HA2 1-222
  • Other peptides or proteins disclosed in these references include the entire HA2 1-222 (C36) protein.
  • the fusion proteins were believed to influence the activity of the proteins as influenza vaccine candidates, since the individual components of the D and C13 proteins displayed no activity.
  • the C36 protein did not restimulate antiviral memory CTL in vitro [Yamada et al, J. Exp. Med., 162:163 (1985)]. These documents also describe the recombinant expression and purification of these and other proteins in detail.
  • Short synthetic peptides representing CTL epitopes are efficient for sensitizing target cells for recognition by class I restricted CTL in vitro but generally fail to induce class I restricted CTL in vivo.
  • the peptide was either injected with incomplete Freunds adjuvant [P. Aichele et al, J. EXP. Med. 171:1815 (1990)], or covalently coupled to a lipid backbone [K. Deres et al, Nature, 342:561 (1989)], CTL responses were successfully generated.
  • the present invention provides in one aspect a vaccine composition
  • a vaccine composition comprising an effective immunogenic amount of a purified recombinant influenza hemaglutinen HA2 subunit protein or fragment thereof in admixture with a selected adjuvant.
  • the presently preferred embodiment comprises H ⁇ 2 66-222 in admixture with a suitable aluminum adjuvant.
  • This vaccine composition is capable of
  • the vaccine inducing protection in mammals against challenge by more than one subtype of influenza A.
  • composition of this invention is capable of inducing protection in mammals against challenge by H1N1 subtype of influenza A.
  • the vaccine composition of this invention is capable of inducing protection in mammals against challenge by H1N1 subtype of influenza A.
  • the invention is capable of inducing protection in mammals against challenge by H2N2 subtype of influenza A.
  • the vaccine composition induces class I restricted CTL response and a proliferative T cell response in a
  • HA2 polypeptide e.g., HA2 1-222 and fragments thereof useful in the vaccine of the present invention.
  • These polypeptides are substantially isolated from association with contaminants with which they occur in nature. Additionally, natural analogs or deliberately modified polypeptide sequences which retain the
  • Another aspect of this invention is a DNA sequence encoding HA2 or a fragment thereof, substantially
  • Another aspect of the invention provides a DNA molecule comprising a coding sequence for a polypeptide of the invention, including the coding sequence for HA2 peptide or fragments thereof alone or incorporated into a larger molecule.
  • the coding sequence is operably
  • polypeptide in a selected host cell is a selected host cell.
  • Still another aspect is a cell transformed with this DNA molecule.
  • This polypeptide may be produced by culturing the transformed cell under suitable conditions and isolating the polypeptide from the culture medium or, if expressed intracellularly, from the cell.
  • a further aspect of this invention is a method for inducing in an animal protection against multiple strains of influenza A which comprises internally
  • Fig. 1 is a graph of the results of cytotoxic T-lymphocyte (CTL) induction in CB6F 1 (H-2 dxb ) mice
  • Target cells immunized with highly purified HA2 66-222 .
  • Fig. 2 in two graphs indicates CTL activity against cells either uninfected or coincubated with a peptide representing a H1N1/H2N2 virus-subtype cross-reactive HA2 epitope of amino acid sequence IleTyrSerThrValAlaSerSerLeuValLeu [SEQ ID NO: 7].
  • the vaccines used in this experiment included an adjuvant control (O), a fusion protein NS1 1-81 HA2 65-222 (106160) in CFA ( ⁇ ), or the partially purified HA2 66-222 polypeptide of this invention (•). Results are reported as % cytotoxicity.
  • Fig. 3 in four graphs illustrates the induction of H1N1 cross-reactive CTL by highly purified HA2 66-222 , as described in Example 4.
  • FIG. 4 illustrates the utility of the highly purified HA2 66-222 protein/adjuvant formulation with other antigens.
  • Figure 4A illustrates the cytotoxicity of HA2 66- 222 /Al +3 (-•-); D protein/Al +3 (- ⁇ -), and Al +3 (— - o— -) against A/PR/8/34 infected target cells at the
  • Figure 4C illustrates the cytotoxicity of HA2 66-222 /A
  • Fig. 5 illustrates by two graphs the proliferative response of HA2 66-222 -immune cells to a fusion protein NA1 1-81 HA1 65-222 (106160) (batch 8904) verses partially purified HA2 66-222 protein in vitro.
  • the present invention provides DNA sequences, polypeptides and pharmaceutical vaccine compositions including them, which are useful in conferring protection in vaccinated mammals against multiple strains of
  • influenza A particularly H1N1 subtype.
  • These vaccine compositions demonstrate the ability to stimulate or produce a protective CTL response in the mammal in the absence of neutralizing antibodies.
  • the vaccine compositions of this invention comprise a DNA sequence encoding a polypeptide having an immunogenic determinant of the hemagglutinin subunit 2 (HA2) of influenza A, subtype H1N1, in admixture with a suitable adjuvant.
  • a suitable adjuvant for use in the formulation of the invention includes the aluminum adjuvants of which aluminum hydroxide and aluminum phosphate are examples.
  • hemagglutinin subunit 2 (HA2) of influenza A subtype H1N1
  • H1N1 hemagglutinin subunit 2
  • this invention is not limited by the subtype described in the example below.
  • HA2 of influenza A, subtype H1N1 are expected to be particularly useful in the vaccine of the invention: HA2 1-222 , HA2 1-70 , HA 265-196 , HA2 65-200 , HA2 65-222 , HA2 66-222 , HA2 69-222 , HA2 81-222 and HA2 150- 222 . It is expected that any CTL epitope within the HA2 genome which is not known in man may be similarly useful in the vaccine composition of the present invention.
  • HA2 1-222 protein was tested in an in vitro assay for CTL which is less sensitive than the in vivo CTL assay now employed by the present invention to screen the various proteins.
  • the presently preferred fragment of HA2 for use in the vaccine composition of the invention is the fragment which encodes amino acid residues 66 to 222 of HA2.
  • the HA2 66-222 protein fragment is characterized by a calculated molecular weight of 18.9 kD, a calculated pI of 5.89. While DNA sequences and amino acid sequences for the HA2 protein are known, it is anticipated that other DNA and amino acid sequences of this invention will include sequences which are substantially similar to presently described sequences but which may be 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 HA2 protein sequences. Other analogs or derivatives of HA2 are also included.
  • DNA sequences which code for HA2 protein but which differ in codon sequence due to the degeneracies of the genetic code or variations in the DNA sequence encoding the HA2 peptide or fragments thereof are included within the scope of this invention. Additionally, DNA sequences which are capable of
  • the gene may be manipulated by varying individual nucleotides, while retaining the correct amino acid(s), or varying the nucleotides, so as to modify the amino acids, without loss of the necessary HA2
  • Nucleotides may be substituted, inserted, or deleted by known techniques, including, for example, in vitro mutagenesis and primer repair.
  • the DNA sequences of this invention may also encode modified HA2 polypeptides.
  • Analogs of the HA2 peptide included within the definition of this invention, include truncated polypeptides (including fragments) and HA2-like polypeptides, e.g., mutants, that retain the epitopes of HA2.
  • Such analogs differ by only 1, 2, 3 or 4 codon changes.
  • CTL epitopes are known to be linear, it is believed to be possible to replace the cysteines of the HA2 peptide sequence with alanines and/or serines while still retaining biological activity. This can be done by using a commercially available system, such as, e.g. the Altered Sites System [Promega Corporation] among others.
  • Examples of other analogs include polypeptides with minor amino acid variations from the natural amino acid sequence of HA2 and conservative amino acid
  • the HA2 peptide or fragments thereof may optionally be fused to an additional protein or peptide at either the N- or the C- terminal end of the fragment.
  • Such an additional "fusion" peptide may contain an influenza antigen or some other antigen.
  • the fusion peptide may be highly expressed in a desired host cell system, be characterized by a high degree of secretion, or enhance the stability of the HA2 peptide in a selected host cell system.
  • the fusion peptide may be a sequence which is selectively cleavable or digestible by conventional methods.
  • the selected fusion peptide may provide an enzymatic cleavage site, including sites for cleavage by a proteolytic enzyme, such as enterokinase, factor Xa, trypsin,
  • collagenase and thrombin or a site which is 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.
  • a presently preferred vaccine composition of this invention employs a purified recombinant protein, which comprises the amino acids [SEQ ID NO:1:] Met-Leu-Thr-Ser-Thr-Arg-Ser fused to the amino acid residues 66 to 222 of HA2 from the influenza strain A/PR/8/34.
  • the above-indicated seven amino acid sequence may be eliminated from its association with the HA2 66-222 sequence for vaccine use, or eliminated from an alternative cloning strategy.
  • The-HA2 polypeptides including the HA2 66-222 polypeptide, useful in the vaccine compositions of the invention can be prepared by chemical synthesis
  • HA2 66-222 fragment generated from the HA2 gene of the influenza strain A/PR/8/34 other influenza strains may be utilized as sources of the same or homologous gene sequences.
  • a DNA coding sequence for HA from the above-cited references a DNA coding sequence for HA from the
  • Influenza viruses including other strains, subtypes and types, are available for clinical specimens and from public depositories, such as the American Type Culture Collection (ATCC), Rockville, Maryland, U.S.A.
  • ATCC American Type Culture Collection
  • Rockville Maryland
  • the polypeptide is prepared by known recombinant DNA techniques by cloning and
  • the polypeptide of this invention may be produced by transforming a selected host cell with the DNA molecule and then culturing the transformed cell under suitable conditions.
  • the polypeptide may be isolated from the culture medium or, if expressed
  • embodiment is expressed in E. coli. because it can be used to produce large amounts of desired proteins safely and cheaply.
  • one of skill in the art may use conventional techniques and known systems for cloning and expression of the vaccinal polypeptides in various other microorganisms and cells, including, for example,
  • the recombinantly-produced polypeptide may be purified from contaminants of the host cell and culture media by conventional means.
  • a presently preferred method for obtaining crude or partially purified protein is
  • Examples 4 and 5 describe the preferred method for obtaining highly purified HA2 66-222 protein.
  • the present HA2 protein fragment is capable of producing effective immunogenic responses in animals without being fused to another protein, e.g., the NS1 protein of the above-referenced European patent applications.
  • Pharmaceutical vaccine compositions of this invention contain an effective immunogenic amount of the selected HA2 protein in admixture with a suitable fusion proteins (described in these above-cited applications and by Yamada et al, cited above).
  • an adjuvant in a nontoxic and sterile pharmaceutically acceptable carrier is any aluminum adjuvant may be used in the vaccine compositions of this invention, a desirable adjuvant is commercially marketed under the trademark Rehydragel.
  • a preferred embodiment of the vaccine composition of the invention is composed of an aqueous suspension or solution containing the recombinant HA2 66-222 protein molecule, buffered at physiological pH, in a form ready for injection.
  • Another vaccine composition may also include another vaccinal peptide, such as the fusion -proteins described in the above-incorporated European patent applications or in co-pending US patent
  • Suitable carriers 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 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.
  • the formulation may optionally be admixed or adsorbed with an additional, conventional adjuvant.
  • the adjuvant is used as a non-specific irritant to attract or enhance an immune response.
  • adjuvants include, among others, amphigen, monophosphenyl lipid A, muramyl dipeptide and saponins such as Quil A.
  • compositions of the present invention are advantageously made up in a dosage unit form adapted for the desired mode of administration.
  • Each unit will contain a predetermined quantity of HA2 protein and adjuvant calculated to produce the desired therapeutic effect in optional association with a pharmaceutical diluent, carrier, or vehicle.
  • Dosage and administration protocol can be optimized in accordance with standard vaccination
  • the vaccine will be administered intramuscularly, although other routes of administration may be used, such as intradermal administration. Based on what is known about other polypeptide vaccines, it is expected that a useful single dosage for average adult humans is in the range of 1 to 1000 micrograms of
  • recombinant protein preferably 5 to 150 micrograms, most preferably 10 to 100 micrograms in admixture with the required adjuvant.
  • 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 vaccine compositions of this invention will effectively stimulate a CTL response, or boost existing CTL in previously exposed animals.
  • the vaccine compositions of this invention will effectively stimulate a CTL response, or boost existing CTL in previously exposed animals.
  • composition could potentially limit virus replication and spread at the early stages of infection due to activation of memory CTL specific for conserved virus proteins.
  • the CTL are expected to be cross reactive against heterologous H1N1 strains representing a wide period of antigenic drift.
  • vaccination with a vaccine composition of this invention will confer broad immunity against H1N1 strains.
  • Plasmid pJZ102 is a pBR322-derived cloning vector which carries a coding region for the entire HA protein (A/PR/8/34). This plasmid is described in detail by Young et al, in The Origin of Pandemic Influenza
  • Plasmid pJZ102 was cut with HindIII to liberate the HA cDNA. This 1784 base pair fragment was isolated and ligated into the commercially available pUC18 which had been cut with HindIII. The resulting plasmid, pMS2 contains the entire A/PR/8/34 HA gene.
  • pMS2 was digested with Pvu II and Sal I to liberate a fragment encoding amino acids 66-222 of the HA2 portion of the HA gene.
  • Plasmid pAS1 ⁇ EH801 is prepared by deleting a non-essential EcoRI-HindIII region of pBR322 origin from pAS1 to make pAS1 ⁇ EH.
  • the resulting plasmid, pASl ⁇ EH801 expresses authentic NS1
  • This plasmid has an Nco I site between the codons for amino acids 81 and 82 and an Nru I site 3' to the NS sequences. The BamHI site between amino acids 1 and 2 is retained.
  • Plasmid pMG27N a pAS1 derivative (Mol. Cell Bio.,
  • the resulting plasmid, pMG1 allows the insertion of
  • pMG1 was digested with BamHI and NcoI and ligated to the
  • pNS1 1-42 TGF ⁇ is derived when pAS1 ⁇ EH801 is cut with NCoI and Sail and ligated to a synthetic DNA encoding human TGF ⁇ as an NcoI/SalI fragment.
  • pNS1 1-81 TGF ⁇ encodes a protein comprised of the first 81 amino acids of NS1 and the mature TGF ⁇ sequence.
  • the NS1 portion of pNS1 42 TGF ⁇ contains an amino acid change from Cys to Ser at amino acid #13.
  • pMG42A 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:
  • the HA2 66-222 fragment liberated by the digestion of pMS2 with Pvu II and Sal I was inserted into the E. coli expression vector, describe above, between the Eco RV and XhoI sites.
  • the resulting plasmid, pMG42H1H66-222 encodes a translational fusion between the first 42 codons of the A/PR/8/34 NS1 gene and the coding sequence for HA2 66-222 in which a decapeptide (SEQ ID NO: 6: Met-Asp-His-Met-Leu-Thr-Ser-Thr-Arg-Ser) is used to join these two gene segments.
  • the plasmid (pH1HA2 66-222 ) was transformed into several E. coli lysogenic hosts including AR58, AR13, and AR68 [SmithKline Beecham Pharmaceuticals]. Cultures were grown at 32°C to mid-log phase at which time
  • the resultant suspension was treated with 0.1% DOC for 60 minutes at 4°C, then centrifuged at 25,000 x g.
  • the pellet was resuspended by sonication in 50 mM glycine pH 10.0, 5% glycerol, 2 mM EDTA and then the suspension was treated with 1% Triton X-100 [Rohm and Haas] at 4°C for 60 minutes and centrifuged as above.
  • the resulting pellet was solubilized in 50 mM Tris, 8 M urea, pH 8.0 and centrifuged to remove any insoluble material. This solubilized material is dialyzed against 10 mM Tris, 1 mM EDTA, pH 8.0 followed, again, by centrifugation of insoluble material.
  • the solubilized material is
  • Crude material 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 HA2 66-222 protein band was visualized by KCl staining according to D. Hager et al, Anal. Biochem, 109:76-86 (1980). The band was cut-out and eluted electrophoretically by the "S&S Elutrap
  • Electro-Separation System [Schleicher & Schuell].
  • the electro-eluting buffer was the Tris-glycine.
  • the protein is usually greater than 75% pure.
  • Example 4 Highly Purified HA2 66-222 A purification procedure to enable recovery of highly purified (approximately 90%) HA2 66-222 .
  • E. coli cells (100 g) were thawed and resuspended in lysis buffer A (50 mM Tris-HCl, 5% glycerol, 2 mM EDTA and 0.1 mM DTT, pH 8.0) at 10 ml/gram. The stirred suspension was then treated with lysozyme (0.2 mg/ml) for 90 min at room temperature, passed through a Manton
  • Gaulin Homogenizer at 8,000 psi (2 passes) and the resultant suspension treated with 1% Triton X 100 [Rohm and Haas] and .1% DOC for 60 min at 4°C, then centrifuged at 25,000 x g.
  • the pellet was resuspended in 50 mM glycine, 5% glycerol, 2mM EDTA, pH 10.5 and 1% Triton X-100 [Baker (Phillipsburg, NJ)], stirred at 4°C for 60 min then centrifuged as above.
  • the resulting pellet was solubilized in 50mM Tris, 8 M urea, pH 8.0 (500 mis), and centrifuged to remove any insoluble material.
  • HA2 66-222 bound to the column, while low molecular weight (LMW) contaminating proteins were eluted in the unbound fraction.
  • LMW low molecular weight
  • Minisette tangential flow apparatus [Filtron] equipped with a 0.75 sq. ft. Omega 10 membrane and screen channel, run at 15-20 psi transmembrane pressure at a cross flow rate of 1,000 ml/min.
  • the DEAE pool was concentrated ⁇ 2.5 fold from 600 mis to ⁇ 236 mis.
  • the Superose 12 column (11.3 ⁇ 87 cm) had a plate count of 13,000 plates/m determined using acetone.
  • sample from the DEAE run was chromatographed on this Superose-12 column according to the protocol described earlier, HA2 66-222 eluted as the major peak at its
  • HA2 66-222 migrated at the correct monomer molecular weight and reacted positively on a Western blot using antibody directed against A/PR/8/34 virus.
  • the protein yield in the HA2 66-222 construct pool was 56% of the added protein, while total protein recovery off the column was 90 to 95%.
  • SDS 10 mg SDS/mg protein
  • DTT 50 mM
  • HA2 66-222 pool was comprised predominantly of a single band, Western positive for HA2 66-222 , with some very minor bands, also Western positive for HA2 66-222 .
  • the HA2 66-222 pool contained 61% of the loaded protein, with ⁇ 95% recovery of added protein observed in the combined pools.
  • the purified HA2 66-222 pool (329 ml) from the second Superose 12 column [Pharmacia, Piscataway, NJ] run was divided into 3 equal pools for processing on the G25 column. For each run 109 ml (120 mg) was loaded at 5 ml/min (20 cm/hr) onto a 1443 ml G25 fine column (4.4 ⁇ 95 cm) equilibrated with 50 mM Tris, 8 M urea, pH 8.0. The column was eluted isocratically at the same flow rate with equilibration buffer. Fractions were collected and assayed for protein and SDS levels and pooled to maximize protein recovery free of SDS contamination.
  • HA2 66-222 was eluted in the void volume fractions of this column run. HA2 66-222 fractions devoid of SDS were pooled and exhaustively dialyzed against 20 mM Tris, 1 mM EDTA pH 8.0. The SDS levels in the product were
  • the purified HA2 66-222 pool from the G25 column was exhaustively dialyzed against 20 mM Tris, 1 mM EDTA, pH 8.0, concentrated, sterile filtered and stored at 4°C used in in vitro and in vivo testing.
  • the dialyzed material was concentrated on a Filtron Omega 10k stirred cell to ⁇ 1-2 mg/ml and sterile filtered in a laminar flow hood into sterile pyrogen free containers for in vitro CTL, in vivo CTL and protection studies. This results in a highly purified HA2 66-222 .
  • HA2 66-222 final product of Example 4 migrated as a single band of correct molecular weight on reducing SDS-PAGE gels. This band reacted with antibodies directed against A/PR/8/34 virus on Western blot, as did the other minor bands observed on these gels.
  • HA2 66-222 protein described in Examples 4 and 5 was prepared for assay in a mouse model of influenza.
  • CB6F 1 mice (Charles River Laboratories or Harlan Sprague Dawley] at a minimum age of 6 weeks were immunized subcutaneously, in the footpad and at the base of the tail with the protein (100, 10 or 1 ⁇ g) emulsified in CFA.
  • lymph node cells from immunized -animals were removed and restimulated, in vitro, with A/PR/8/34 virus-infected syngeneic spleen cells for 5 days and assayed for cytotoxicity against various target cells, i.e., A/PR/8/34 infected P815 cells (H-2 d
  • mastocytoma cells available from the ATCC [the black squares on Fig. 1], A/Udorn/72 infected P815 cells [the black triangles on Fig. 1], and uninfected P815 cells [the empty squares on Fig. 1].
  • CTL activity was measured in a standard four hour Cr 51 -release assay against the target cells. An effector to target ratio of 25:1 is shown.
  • Fig. 2 using the highly purified HA2 66-222 ) HA2 66-222 -induced CTL, from mice immunized with 10 or 100 ⁇ g protein, recognized and lysed target cells infected with homologous A/PR/8/34 virus.
  • H3N2 Uninfected cells or target cells infected with A/Udorn/72 (H3N2) virus were not lysed by H ⁇ 2 66-222 -immune cells (Fig. 2).
  • Lymph node cells from mice injected with highly purified HA2 66-222 (50 or 10 ⁇ g in CFA) responded to secondary stimulation with A/PR/8/34 virus-infected stimulator cells, resulting in specific killing of
  • HA2 66-222 -immune CTL also recognized and lysed target cells infected with A/FM/1/47, a heterologous H1N1 virus. Uninfected cells or target cells infected with A/Udorn/72 (H3N2) were not lysed by HA2 66-222 -immune cells (Fig. 3).
  • HA2 66-222 -immune CTL recognized the target cells co-incubated with the CTL epitope peptide.
  • This protein was also tested for its ability to induce protective immunity from lethal virus challenge, as follows.
  • CB6F1 mice were immunized with two subcutaneous injections of protein emulsified in CFA followed by an intraperitoneal boost in the absence of adjuvant five days prior to virus challenge. Injections were given at three week intervals (week 0,3,6). Mice (15/group) were challenged intranasally under metophane anesthesia with 5 LD 50 dose of A/PR/8/34 virus. Survival was monitored for 21 days. Differences in % survival between immunized and non-immunized animals was analyzed by Fisher's Exact probability test. Differences in % survival were
  • mice immunized with 50 or 5 ⁇ g HA2 66-222 emulsified in CFA were protected against intranasal challenge with A/PR/8/34 virus (47% or 87% survival in immunized animals verses 7% survival in unimmunized controls).
  • Aluminum hydroxide is an adjuvant currently licensed for human use. This adjuvant was previously shown to have the surprising property of supporting a CTL response by SK&F 106160 (NS1 1-81 HA2 65-222 or D protein ) (see copending U.S. Patent application Ser. No. 07/664,582). Highly purified HA2 66-222 protein of Examples 4 and 5 was diluted to working concentration with 5% dextrose and then mixed with Rehsorptar [Armour Pharmaceuticals, Kankakee, IL], an aluminum hydroxide gel that contains 2% w/v Al 2 O 3 which is equivalent to 10.6 mg/ml Al +3 . Another vaccine
  • Immune responsiveness to partially purified, gel eluted HA2 66-222 was also measured with respect to the proliferative response of immune cells to antigen in vitro.
  • In vitro proliferation in response to influenza derived proteins is an established property of polyclonal T cell cultures with helper activity, and T helper cell clones which can augment or support influenza-specific antibody-production by B cells (Scherle and Gerhard, J. EXP. Med., 164:1114-1128 (1986). Production of
  • neutralizing antibodies can also be facilitated by helper T cells in vivo [Scherle and Gerhard, 1986, supra and 1988, PNAS, 4446-4450; Tite et al, J. Immunol.,
  • HA2 purified from A/USSR/77, (H1N1, Type A influenza) provided help for secondary anti-hapten IgG antibody responses [Garcon and Six, J. Immunol., 140:3697-3702 (1991)] Therefore, proteins which induce cells with potential T helper cell activity are also considered candidates for human vaccine immunogens.
  • the proliferative response of HA2-immune cells was comparable to the response of SKF 106160-immune cells when SKF 106160 was used to restimulate the cells in vitro (Fig. 5; upper graph). Stimulation, in vitro, with
  • HA2 66-222 was found to be mitogenic to non-immune cells (CFA control group) and therefore could not be used to measure antigen-specific proliferation (Fig. 5; lower graph).
  • the non-specific stimulation observed upon culture with the HA2 protein may be attributed to residual
  • Neutralization titers are expressed as the reciprocal of the highest serum dilution that neutralized 100 tissue culture infectious doses 50 (TCID 50 ) of the A/PR/8 virus.
  • Titers of antibody reactive with the D protein were determined by an ELISA assay in which microtiter wells were coated with 0.5 ⁇ g purified D protein, and specific antibody was detected with horseradish peroxidase-conjugated rabbit anti mouse IgG (H+L chain specific)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)

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Abstract

Nouvelle composition de vaccin capable de conférer une immunité contre des souches multiples de virus de la grippe A.
PCT/US1992/007312 1991-08-30 1992-09-17 Compositions de vaccin contre le virus de la griffe, obtenues par recombinaison WO1994006468A1 (fr)

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AU27756/92A AU2775692A (en) 1991-08-30 1992-09-17 Recombinant influenza virus vaccine compositions

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6306404B1 (en) 1998-07-14 2001-10-23 American Cyanamid Company Adjuvant and vaccine compositions containing monophosphoryl lipid A
WO2002024734A2 (fr) * 2000-09-19 2002-03-28 Chiron S.P.A. Sous-type h16 du virus grippal a
US6635261B2 (en) 1999-07-13 2003-10-21 Wyeth Holdings Corporation Adjuvant and vaccine compositions containing monophosphoryl lipid A
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

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0336238A2 (fr) * 1988-03-24 1989-10-11 Canon Kabushiki Kaisha Procédé de formation d'images et appareil

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0336238A2 (fr) * 1988-03-24 1989-10-11 Canon Kabushiki Kaisha Procédé de formation d'images et appareil

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
GENE, Volume 21, issued 1983, A.R. DAVIS et al., "Immune Response to Human Influenza Virus Hemagglutinin Expressed in E. Coli", pp. 273-284. *
JOURNAL OF IMMUNOLOGY, Volume 140, No. 4, issued 15 February 1988, K. KUWANO et al., "HA2 Subunit of Influenza A H1 and H2 Suntype Viruses Induces a Protective Cross-Reactive Cytotoxic T Lymphocyte Response", pp. 1264-1268. *
METHODS IN ENZYMOLOGY, Volume 152, issued 1987, A.R. SHATZMAN et al., "Expression, Identification, and Characterization of Recombinant Gene Products in Escherichia Coli", pp. 661-6673. *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES U.S.A., Volume 80, issued October 1983, J.F. YOUNG et al., "Efficient Expression of Influenza Virus NS1 Nonstructural Proteins in Escherichia Coli", pp. 6105-6109. *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6306404B1 (en) 1998-07-14 2001-10-23 American Cyanamid Company Adjuvant and vaccine compositions containing monophosphoryl lipid A
US6635261B2 (en) 1999-07-13 2003-10-21 Wyeth Holdings Corporation Adjuvant and vaccine compositions containing monophosphoryl lipid A
WO2002024734A2 (fr) * 2000-09-19 2002-03-28 Chiron S.P.A. Sous-type h16 du virus grippal a
WO2002024734A3 (fr) * 2000-09-19 2002-08-15 Chiron Spa Sous-type h16 du virus grippal a
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

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