EP1517702A2 - Particules chimeriques hbc immunogenes stabilisees avec une cysteine d'extremite n-termnale - Google Patents

Particules chimeriques hbc immunogenes stabilisees avec une cysteine d'extremite n-termnale

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Publication number
EP1517702A2
EP1517702A2 EP03709214A EP03709214A EP1517702A2 EP 1517702 A2 EP1517702 A2 EP 1517702A2 EP 03709214 A EP03709214 A EP 03709214A EP 03709214 A EP03709214 A EP 03709214A EP 1517702 A2 EP1517702 A2 EP 1517702A2
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Prior art keywords
hbc
sequence
residues
chimer
residue
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EP03709214A
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German (de)
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EP1517702A4 (fr
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Ashley J. Birkett
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Apovia Inc
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Apovia Inc
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Priority claimed from US10/082,014 external-priority patent/US20030185858A1/en
Priority claimed from US10/080,299 external-priority patent/US20030175863A1/en
Priority claimed from US10/274,616 external-priority patent/US20030202982A1/en
Application filed by Apovia Inc filed Critical Apovia Inc
Publication of EP1517702A2 publication Critical patent/EP1517702A2/fr
Publication of EP1517702A4 publication Critical patent/EP1517702A4/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
    • A61K39/002—Protozoa antigens
    • A61K39/015—Hemosporidia antigens, e.g. Plasmodium 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
    • A61K39/12—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
    • A61K39/12—Viral antigens
    • A61K39/145—Orthomyxoviridae, e.g. influenza virus
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5258—Virus-like particles
    • 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/543—Mucosal route intranasal
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55572—Lipopolysaccharides; Lipid A; Monophosphoryl lipid A
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • 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
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10122—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
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • 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
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the intersection of the fields of immunology and protein engineering, and particularly to a chimeric hepatitis B virus (HBV) nucleocapsid protein that is engineered for both enhanced stability of self-assembled particles via an N-terminal cysteine residue and the display of an immunogenic epitope, and more particularly to an immunogen and vaccine useful in prevention of influenza infection by influenza A virus .
  • HBV hepatitis B virus
  • the family hepadnaviridae are enveloped DNA-containing animal viruses that can cause hepatitis B in humans (HBV) .
  • the hepadnavirus family includes hepatitis B viruses of other mammals, e.g., woodchuck (WHV) , and ground squirrel (GSHV) , and avian viruses found in ducks (DHV) and herons (HeHV) .
  • Hepatitis B virus (HBV) used herein refers to a member of the family hepadnaviridae that infects mammals, as compared to a virus that infects an avian host, unless the discussion refers to a specific example of a non-mammalian virus.
  • the nucleocapsid or core of the mammalian hepatitis B virus contains a sequence of 183 or 185 amino acid residues, depending on viral subtype, whereas the duck virus capsid contains 262 amino acid residues.
  • Hepatitis B core protein monomers of the several hepadnaviridae self-assemble in infected cells into stable aggregates known as hepatitis B core protein particles (HBc particles) . Two three-dimensional structures are reported for C-terminally truncated HBc particles.
  • These HBc particles of the human-infecting virus (human virus) are about are about 30 or 34 nm in diameter, respectively.
  • hepatitis B nucleocapsid or viral core protein has been disclosed as an immunogenic carrier moiety that stimulates the T cell response of an immunized host animal. See, for example, U.S. Patents No. 4,818,527, No 4,882,145 and No. 5,143,726.
  • a particularly useful application of this carrier is its ability to present foreign or heterologous B cell epitopes at the site of the immunodominant loop that is present at about residue positions 70-90, and more usually recited as about positions 75 through 85 from the amino-terminus (N- terminus) of the protein. Clarke et al . (1991) F. Brown et al . eds., Vaccines 91, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp.313-318.
  • HBV nucleocapsids associate with the viral RNA pre-genome, the viral reverse transcriptase (Pol) , and the terminal protein (derived from Pol) to form replication competent cores.
  • the association between the nucleocapsid and the viral RNA pre-genome is mediated by an arginine- rich domain at the carboxyl-terminus (C-terminus) .
  • C-terminus carboxyl-terminus
  • HBcAg is a particulate protein derived from the hepatitis B virus that has been proposed as a carrier for heterologous epitopes.
  • the relative immunogenicity of HBsAg (HBs) has been compared with HBcAg (HBc) , and the ability of each to evoke immune responses in different genetic backgrounds [Milich et al . , Science, (1986) 234 (4782) :1398-1401] .
  • HBc is more than 300 times more immunogenic than HBs in BALB/c mice; and, although both B10.S and B10.M mice are non-responders to HBs, every strain tested is responsive to HBc.
  • HBc carrier Another advantage of the HBc carrier is the fact that it does not require complex adjuvants for efficacy. This is due to the high inherent immunogenicity of the particle. A comparison of the immunogenicity of HBc-P. berghei particles showed that alum, which is approved for human use, was more effective than either IFA or CFA [Sch ⁇ del et al . , J. Exp . Med . , (1994) 180 (3) : 1037-46] . The importance of this observation is highlighted by toxicity problems associated with newer, more complex adjuvants as was recently noted in clinical trials of SKB's candidate malaria vaccine [Stoute et al . , N. Engl . J. Med. , [1997] 336 (2) :86-91] .
  • the HBV nucleocapsids not bind nucleic acid derived from the host. Birnbaum et al . (1990) J. Virol . , 64:3319-3330 showed that the protamine-like C-terminal domain of HBV nucleocapsids could be deleted without interfering with the protein's ability to assemble into virus-like particles. It is thus reported that proteins truncated to about position 144; i.e., containing the HBc sequence from position one through about 144, can self-assemble, whereas deletions beyond residue 139 abrogate capsid assembly [Birnbaum et al., (1990) J. Virl . , 64:3319-30].
  • HBc chimeric particles or HBc chimers Recombinantly-produced hybrid HBc particles bearing internal insertions (referred to in the art as HBc chimeric particles or HBc chimers) containing various inserted polypeptide sequences have been prepared by heterologous expression in a wide variety of organisms, including E. coli , B . subtilis , Vaccinia, Salmonella typhimurium, Saccharomyces cerevisiae . See, for example Pumpens et al . (1995) Intervirology, 38:63-74, and the citations therein that note the work of several research groups . Native HBc particles have also been produced in plants [Tsuda et al., (1998) Vox Sang, 74 (3) : 148-155] .
  • HBc chimers often appear to have a less ordered structure, when analyzed by electron microscopy, compared to particles that lack heterologous epitopes [Sch ⁇ del et al . , (1994) J. Exp . Med. , 180:1037-1046].
  • the insertion of heterologous epitopes into C-terminally truncated HBc particles has such a dramatic destabilizing affect that hybrid particles cannot be recovered following heterologous expression [Schodel et al. (1994) Infect . Immunol . , 62:1669-1676].
  • many chimeric HBc particles are so unstable that they fall apart during purification to such an extent that they are unrecoverable or they show very poor stability characteristics, making them problematic for vaccine development .
  • U.S. Patent No. 5,990,085 describes two fusion proteins formed from an antigenic bovine inhibin peptide fused into (i) the immunogenic loop between residues 78 and 79 and (ii) after residue 144 of carboxy-terminal truncated HBc. Expressed fusion proteins were said to induce the production of anti- inhibin antibodies when administered in a host animal. The titers thirty days after immunization reported in that patent are relatively low, being 1:3000-15,000 for the fusion protein with the loop insertion and 1:100-125 for the insertion after residue 144.
  • U.S. Patent No. 6,231,864 teaches the preparation and use of a strategically modified hepatitis B core protein that is linked to a hapten.
  • the modified core protein contains an insert of one to about 40 residues in length that contains a chemically-reactive amino acid residue to which the hapten is pendently linked.
  • WO 01/27281 teaches that the immune response to HBc can be changed from a Thl response to a Th2 response by the presence or absence, respectively, of the C-terminal cysteine- containing sequence of the native molecule. That disclosure also opines that disulfide formation by C-terminal cysteines could help to stabilize the particles. The presence of several residues the native HBc sequence immediately upstream of the C-terminal cysteine was said to be preferred, but not required.
  • One such alternative that might be used to replace a truncated C-terminal HBc sequence was said to include a C-terminal cysteine and an optional sequence that defines an epitope from other than HBc.
  • the present invention provides another HBc chimer that provides unexpectedly high titers of antibodies against influenza, and in one aspect also provides a solution to the problems of HBc chimer stability as well as the substantial absence of nucleic acid binding ability of the construct.
  • a contemplated recombinant chimer exhibits reducedantigenicity toward preexisting anti-HBc antibodies .
  • the present invention contemplates a recombinant hepadnavirus nucleocapsid protein; i.e., a hepatitis B core (HBc) chimeric protein [or chimer hepatitis B core protein molecule or HBc chimer molecule or just chimer] that self-assembles into particles after expression in a host cell.
  • a recombinant chimer hepatitis B core (HBc) protein molecule up to about 515 amino acid residues in length is contemplated. That chimer molecule
  • (a) contains an HBc sequence of at least about 125 of the N-terminal 150 amino acid residues of the HBc molecule that includes (i) the HBc sequence of residue positions 5 through about 75 and about 85 through about 140, (ii) a peptide-bonded heterologous immunogenic epitope at one or more of the N-terminus, in the HBc immunodominant loop or the C-terminus of the chimer, or (iii) a heterologous linker residue for a conjugated epitope present in the HBc immunodominant loop,
  • (b) contains one to three cysteine residues at an amino acid position of the chimer molecule corresponding to amino acid position -20 to about +1 from the N-terminus of the HBc sequence of SEQ ID NO:l [N-terminal cysteine residue (s) ] in a sequence other than that of the HBc precore sequence and zero to about three cysteine residues toward the C- terminus of the molecule from the C-terminal residue of the HBc sequence and within about 30 residues from the C-terminus of the chimer molecule [C-terminal cysteine residue (s) ] .
  • That chimer molecule (i) contains no more than about 20 percent conservatively substituted amino acid residues in the HBc sequence, (ii) self- assembles into particles that are substantially free of binding to nucleic acids on expression in a host cell .
  • the particles are more stable than are particles formed from otherwise identical HBc chimer molecules that are free of any above-mentioned C-terminal cysteine residue (s) and (i) lack the N-terminal cysteine residue (s) or (ii) in which an N-terminal cysteine residue (s) present in a contemplated chimer molecule is (are) replaced by another residue.
  • a preferred recombinant hepatitis B virus core (HBc) protein chimer molecule has a length of about 135 to about 515 amino acid residues that contains four peptide-linked amino acid residue sequence domains from the N-terminus that are denominated Domains I, II, III and IV.
  • Domain I of that chimer molecule comprises about 71 to about 110 amino acid residues whose sequence includes (i) at least the sequence of the residues of position 5 through position 75 of HBc, (ii) one to three cysteine residues at an amino acid position of the chimer molecule corresponding to amino acid position -20 to about +1 from the N- terminus of the HBc sequence of SEQ ID N0:1 [N- terminal cysteine residue (s) ] in a sequence other than that of the HBc precore sequence, and (iii) an optional heterologous immunogenic epitope containing up to about 30 amino acid residues peptide-bonded to one of HBc residues 2-4.
  • Domain II of that chimer molecule comprises about 5 to about 250 amino acid residues peptide- bonded to HBc residue 75 of Domain I in which (i) zero to all residues in the sequence of HBc positions 76 to 85 are present peptide-bonded to (ii) an optionally present sequence of one to about 245 amino acid residues that are heterologous to HBc and constitute a heterologous immunogenic epitope or a heterologous linker residue for a conjugated epitope.
  • Chimer Domain III is an HBc sequence from position 86 through position 135 peptide-bonded to residue 85 of Domain II.
  • Chimer molecule Domain IV comprises (i) five through fourteen residues of an HBc amino acid residue sequence from position 136 through 149 peptide-bonded to the residue of position 135 of Domain III, (ii) zero to three cysteine residues [C- terminal cysteine residue (s)] within about 30 residues from the C-terminus of the chimer molecule, and (iii) zero to about 100 amino acid residues in an immunogenic sequence heterologous to HBc from position 150 to the C-terminus.
  • the chimer molecules (i) have an amino acid residue sequence in which no more than about 10 percent of the amino acid residues are substituted in the HBc sequence of the chimer and (ii) self-assemble into particles on expression in a host cell.
  • the particles are substantially free of binding to nucleic acids and are more stable than are particles formed from otherwise identical HBc chimer molecules that are free of any above-mentioned C-terminal cysteine residue (s) and (i) lack the N-terminal cysteine residue (s) or (ii) in which an N-terminal cysteine residue (s) present in a contemplated chimer molecule is (are) replaced by another residue.
  • the HBc sequence of Domain I include the residues of position 5 through position 75 along plus at least an N-terminal cysteine residue. It is further preferred that a contemplated immunogen contain not only an N-terminal cysteine residue, but also contain one cysteine residue within Domain IV as noted above that is alone or in an amino acid residue sequence heterologous to that of HBc from position 150 to the C-terminus.
  • the before-mentioned self-assembled chimer molecule particles are a particularly contemplated embodiment of this invention.
  • a particularly preferred embodiment of the present invention contemplates an immunogen for inducing antibodies to influenza A that is a self-assembled particle comprised of recombinant hepatitis B virus core (HBc) chimer protein molecules.
  • HBc hepatitis B virus core
  • Each of those molecules has a length of about 150 to about 325 amino acid residues and contains four peptide-linked amino acid residue sequence domains from the N-terminus that are denominated Domains I, II, III and IV.
  • the first domain, Domain I comprises about 75 to about 110 amino acid residues.
  • the sequence of this Domain includes at least the sequence of the residues of position 4 through position 75 of HBc.
  • One to three cysteine residues are also present at a position in the chimer molecule of about one to about -20 relative to the N-terminus of HBc of SEQ ID NO:l [N-terminal cysteine residue (s) ] .
  • the one or more N- terminal cysteine residues are present within a sequence other than that of the pre-core sequence of HBc .
  • Domain I can further include a sequence of about 6 to about 24 residues of an influenza A M2 polypeptide X 1 X 2 X3X4X5X 6 X7X8TX ⁇ o ⁇ nR 13 x 14 x 15 x 16- x 17 x 18 x 19 x 20 x 21 x 22 x 23 x 24 of SE0 - ID NO : 9 that are peptide-bonded to or within about 15 residues of the N-terminus of the HBc sequence, and whose subscripted X residues are defined hereinafter, as well as one or more or HBc residues 1-4.
  • the second domain, Domain II comprises about 10 to about 60 amino acid residues peptide- bonded to residue 75 of Domain I of which (i) zero to all residues in the sequence of HBc positions 76 through 85 are present peptide-bonded to (ii) an optional sequence of about 6 to about 48 residues that constitute one or more repeats of the above influenza A M2 polypeptide of SEQ ID NO: 9.
  • the third domain, Domain III is an HBc sequence from position 86 through position 135 peptide-bonded to residue 85.
  • the fourth domain, Domain IV comprises (i) the residues of positions 136-140 plus up to nine residues of an HBc amino acid residue sequence from position 141 through 149 peptide-bonded to the residue of position 135 of Domain III, (ii) zero to three cysteine residues, (iii) fewer than three arginine or lysine residues, or mixtures thereof adjacent to each other, and (iv) up to about 100 amino acid residues in a sequence heterologous to HBc from position 164 to the C-terminus.
  • a contemplated chimer molecule (i) contains no more than 10 percent conservatively substituted amino acid residues in the HBc sequence, (ii) self- assembles into particles that are substantially free of binding to nucleic acids on expression in a host cell, and those particles are more stable on formation than are particles formed from an otherwise identical HBc chimer that lacks said N-terminal cysteine residue (s) or in which an N-terminal cysteine residue present in the chimer molecule is replaced by another residue. It is preferred that the HBc sequence of Domain I include the residues of position 4 through position 75 along plus at least an N-terminal cysteine residue.
  • a contemplated immunogen contain one cysteine residue within Domain IV alone or in an amino acid residue sequence heterologous to that of HBc from position 164 to the C-terminus. It is particularly preferred that that heterologous sequence comprise a T cell epitope of influenza A.
  • Another embodiment comprises an inoculum or vaccine that comprises an above HBc chimer particle that is dissolved or dispersed in a pharmaceutically acceptable diluent composition that typically also contains water.
  • an inoculum When administered in an immunogenic effective amount to an animal such as a mammal or bird, an inoculum induces antibodies that immunoreact specifically with the chimer particle. The antibodies so induced also immunoreact specifically with (bind to) the N-terminal portion of the M2 protein, when such a M2-related sequence is present in the chimer .
  • the present invention has several benefits and advantages.
  • a particular benefit of the invention is that its use as a vaccine provides extraordinary antibody titers against influenza A.
  • An advantage of the invention is that those very high antibody titers have been produced with the aid of an adjuvant approved for use in humans.
  • the recombinant immunogen can be prepared easily and in large quantities using well-known cell culture techniques to grow transformed host cells.
  • Another advantage of the invention is that the immunogen is easily prepared using well-known recombinant techniques .
  • a preferred immunogen exhibits greater stability at elevated temperatures than to other HBc chimers .
  • a contemplated immunogen is substantially free of nucleic acids.
  • Fig.l shown in two panels as Fig. IA and Fig. IB, provides an alignment of six published sequences for mammalian HBc proteins from six viruses.
  • the first (SEQ ID NO:l), human viral sequence is of the ayw subtype and was published in Galibert et al . (1983) Nature, 281:646-650;
  • the second human viral sequence (SEQ ID NO: 2) , of the adw subtype, was published by Ono et al . (1983) Nucleic Acids Res . , 11(6): 1747-1757;
  • the third human viral ' sequence (SEQ ID NO:3), is of the adw2 subtype and was published by Valenzuela et al .
  • the fourth human viral sequence (SEQ ID NO: 4) is of the adyw subtype that was published by Pasek et al . (1979) Nature, 282:575-579;
  • the fifth sequence (SEQ ID NO: 5), is that of the woodchuck virus that was published by Galibert et al . (1982) J " . Virol . , 41:51-65;
  • the sixth mammalian sequence, (SEQ ID NO:6) is that of the ground squirrel that was published by Seeger et al . (1984) J. Virol . ,51:367-375.
  • Fig. 2 shows the modifications made to commercial plasmid vector pKK223-3 in the preparation of plasmid vector pKK223-3N used herein for preparation of recombinant HBc chimers.
  • the modified sequence (SEQ ID NO: 7) is shown below the sequence of the commercially available vector (SEQ ID NO:8).
  • the bases of the added Ncol site are shown in lower case letters and the added bases are shown with double underlines, whereas the deleted bases are shown as dashes.
  • the two restriction sites present in this segment of the sequence (Ncol and Hindlll) are indicated.
  • Fig. 3 is an analytical size exclusion chromatography elution profile for ICC-1603 particles in which absorbance at 280 nm is shown on the ordinate and time in seconds is shown on the abscissa.
  • Fig. 4 is an analytical size exclusion chromatography elution profile for ICC-1590 particles as discussed for Fig. 3.
  • Fig. 5 is an analytical size exclusion chromatography elution profile for ICC-1560 particles as discussed for Fig. 3.
  • Fig. 6 is an analytical size exclusion chromatography elution profile for ICC-1605 particles as discussed for Fig. 3.
  • Fig. 7 is an analytical size exclusion chromatography elution profile for ICC-1604 particles as discussed for Fig. 3.
  • Fig. 8 is an analytical size exclusion chromatography elution profile for ICC-1438 particles as discussed for Fig. 3.
  • Fig. 9 is an analytical size exclusion chromatography elution profile for ICC-1492 particles as discussed for Fig. 3.
  • Fig 10 is a photograph of an SDS-PAGE analysis under reducing conditions following particle preparation that shows the ICC-1438 monomer construct was unstable after aging (Lane 2) as compared to the ICC-1492 construct (Lane 3) , with HBc-149 (Lane 1) , ICC-1475 (Lane 4) and ICC-1473 (Lane 5) serving as additional molecular weight controls.
  • Fig. 11, taken from PCT/US01/25625 (ICC- 102.2) illustrates a reaction scheme (Scheme 1) that shows two reaction sequences for (I) forming an activated carrier for pendently linking a hapten to a chimeric hepatitis B core protein (sm-HBc) particle using sulpho-succinimidyl 4- (N-maleimidomethyl) - cyclohexane 1-carboxylate (sulpho-SMCC) , and then (II) linking a sulfhydryl-terminated (cysteine- terminated) hapten to the activated carrier to form a conjugate particle.
  • Scheme 1 shows two reaction sequences for (I) forming an activated carrier for pendently linking a hapten to a chimeric hepatitis B core protein (sm-HBc) particle using sulpho-succinimidyl 4- (N-maleimidomethyl) - cyclohexan
  • the sm-HBc particle is depicted as a box having a single pendent amino group (for purposes of clarity of the figure) , whereas the sulfhydryl-terminated hapten is depicted as a line terminated with an SH group .
  • HBcl49 indicates that the chimer ends at residue 149
  • HBcl49 + C150 indicates that that same chimer contains a cysteine ' residue at HBc position 150 relative to the sequence numbers of SEQ ID NO:l.
  • antibody refers to a molecule that is a member of a family of glycosylated proteins called immunoglobulins, which can specifically bind to an antigen.
  • antigen has been used historically to designate an entity that is bound by an antibody or receptor, and also to designate the entity that induces the production of the antibody. More current usage limits the meaning of antigen to that entity bound by an antibody or receptor, whereas the word “immunogen” is used for the entity that induces antibody production or binds to the receptor. Where an entity discussed herein is both immunogenic and antigenic, reference to it as either an immunogen or antigen is typically made according to its intended utility.
  • Antigenic determinant refers to the actual structural portion of the antigen that is immunologically bound by an antibody combining site or T-cell receptor. The term is also used interchangeably with “epitope” .
  • conjugate refers to a hapten operatively linked to a carrier protein, as through an amino acid residue side chain.
  • conservative substitution denotes that one amino acid residue has been replaced by another, biologically similar residue.
  • conservative substitutions include the substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another such as between arginine and lysine, between glutamic and aspartic acids or between glutamine and asparagine and the like.
  • Domain is used herein to mean a portion of a recombinant HBc chimer molecule that is identified by (i) residue position numbering relative to the position numbers of HBcAg subtype ayw as reported by Galibert et al . , (1979) Nature, 281:646- 650 (SEQ ID N0:1).
  • the polypeptide portions of at least chimer Domains I, II and III are believed to exist in a similar tertiary form to the corresponding sequences of naturally occurring HBcAg.
  • fusion protein designates a polypeptide that contains at least two amino acid residue sequences not normally found linked together in nature that are operatively linked together end-to-end (head-to-tail) by a peptide bond between their respective carboxy- and amino-terminal amino acid residues.
  • the fusion proteins of the present invention are HBc chimer molecules that induce the production of antibodies that immunoreact with a polypeptide that corresponds in amino acid residue sequence to the polypeptide portion of the fusion protein.
  • hepatitis B refers in its broadest context to any member of the family of mammalian hepadnaviridae, as discussed before.
  • polypeptide and “peptide” are used interchangeably throughout the specification and designate a linear series of amino acid residues connected one to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent amino acids.
  • Polypeptides can be a variety of lengths, either in their neutral (uncharged) forms or in forms that are salts. It is well understood in the art that amino acid residue sequences contain acidic and basic groups, and that the particular ionization state exhibited by the peptide is dependent on the pH value of the surrounding medium when the peptide is in solution, or that of the medium from which it was obtained if the peptide is in solid form. Thus, "polypeptide” or its equivalent terms is intended to include the appropriate amino acid residue sequence referenced. A peptide or polypeptide is always shown herein from left to right and in the direction from amino-terminus (N-terminus) to carboxy-terminus (C- terminus) .
  • amino acid residue is used interchangeably with the phrase amino acid residue. All amino acid residues identified herein are in the natural or L- configuration. In keeping with standard polypeptide nomenclature, [J. Biol . Chem. , 243, 3557-59 (1969)], abbreviations for amino acid residues are as shown in the following Table of Correspondence. TABLE OF CORRESPONDENCE
  • the present invention contemplates a chimeric hepadnavirus nucleocapsid protein; i.e., a recombinant hepatitis B core (HBc) protein, that is engineered to (a) display an immunogenic B cell or T cell epitope, or a linker for attachment of an immunogenic B cell or T cell epitope, (b) exhibit enhanced stability on formation when present in a self-assembled particle via an added cysteine residue near the N-terminus, as well as exhibit (c) a substantial absence of nucleic acid binding as a self-assembled particle.
  • HBc chimer is truncated at the C-terminus of the molecule relative to a native HBc molecule.
  • the chimeric protein displays one or more heterologous immunogenic epitopes at the N-terminus, in the HBc immunogenic (immunodominant) loop or C-terminus, or a linker for such a B cell or T cell epitope in the immunogenic loop.
  • the chimeric protein contains a cysteine residue at or near the N-terminus that confers enhanced stability on formation to the self-assembled particles.
  • a preferred chimeric protein is sufficiently free of arginine and or lysine residues downstream of (toward the carboxy-terminus from) HBc residue position 149 so that the self-assembled particles are substantially free of nucleic acid binding.
  • contemplated chimer sequences and sequence position numbers referred to herein are based on the sequence and position numbering of the human hepatitis B core protein of subtype ayw [Galibert et al . , (1979) Nature, 281:646- 650] that is shown in SEQ ID N0:1. It is to be understood, however, that in view of the great similarity between the mammalian hepadnavirus capsid protein sequences and similar particle formation exhibited by those proteins, which are well-known to skilled workers, a discussion regarding human HBc subtype ayw is also applicable to subtype adw, as well as the woodchuck and ground squirrel proteins . As a consequence of those great similarities, HBc sequences are recited generally herein as a "HBc" sequence, unless otherwise stated. In one embodiment, a contemplated HBc chimer is up to about 515 residues in length and contains
  • an HBc sequence of at least about 125 of the N-terminal 150 amino acid residues of the HBc molecule that includes (i) the HBc sequence of residue positions 5 through about 75 and about 85 through about 140, (ii) a peptide-bonded heterologous immunogenic epitope at one or more of the N-terminus, in the HBc immunodominant loop or the C-terminus of the chimer, or (iii) a heterologous linker residue for a conjugated epitope present in the HBc immunodominant loop, and
  • That chimer molecule (i) contains no more than about 20 percent conservatively substituted amino acid residues in the HBc sequence, (ii) self- assembles into particles that are substantially free of binding to nucleic acids on expression in a host cell.
  • the particles are more stable on formation than are particles formed from otherwise identical HBc chimer molecules that are free of any above- mentioned C-terminal cysteine residue (s) and (i) lack the N-terminal cysteine residue (s) or (ii) in which an N-terminal cysteine residue (s) present in a contemplated chimer molecule is (are) replaced by another residue .
  • a contemplated chimer molecule contains a cysteine residue at a position of about -20 to about +1 relative to the N-terminus of HBc as is illustrated in Fig. 1 and SEQ ID NO:l.
  • the concept of a negative amino acid position is usually associated with a leader sequence such as the precore sequence of HBc. That concept is used similarly here in that one can simply align a given chimer molecule sequence with that of SEQ ID N0:1 to determine the position of the chimer that corresponds to that of the starting methionine residue of position +1 of HBc.
  • any aligned chimer molecule residue to the left of the position occupied by, the HBc start methionine has a negative position.
  • a contemplated cysteine residue can occur at a position about twenty residues to the left of the aligned start methionine of HBc to the position corresponding to that start methionine .
  • a preferred HBc chimer has a sequence of about 135 to about 515 L- ⁇ -amino acid residues and contains four serially peptide-linked domains; i.e., Domains I, II, III and IV. Those four domains are linked together in the same manner as are native proteins; i.e., they are peptide-bonded to each other, as compared to polypeptides that contain residues of other than ⁇ -amino acids and therefore cannot form peptide bonds, those that contain D-amino acid residues, or oligopeptide conjugates in which two or more polypeptides are operatively linked through an amino acid residue side chain.
  • a contemplated chimeric HBc protein can therefore be prepared by expression using the usual methods of recombinant technology.
  • Domain I of that chimer molecule comprises about 71 to about 110 amino acid residues whose sequence includes (i) at least the sequence of the residues of position 5 through position 75 of HBc, (ii) one to three cysteine residues at an amino acid position of the chimer molecule corresponding to amino acid position -20 to about +1, and preferably amino acid position -14 to about +1, from the N-terminus of the HBc sequence of SEQ ID NO:l [N-terminal cysteine residue (s) ] in a sequence other than that of the HBc precore sequence, and (iii) an optional heterologous immunogenic epitope containing up to about 30 amino acid residues peptide-bonded to one of HBc residues 2-4. That heterologous immunogenic sequence, when present, is typically an epitope used to induce an immune response for a vaccine or inoculum.
  • Domain II of that chimer molecule comprises about 5 to about 250 amino acid residues peptide- bonded to HBc residue 75 of Domain I in which (i) zero to all residues in the sequence of HBc positions 76 to 85, and preferably at least four HBc residues, are present peptide-bonded to (ii) an optionally present sequence of one to about 245 amino acid residues that are heterologous to HBc and constitute a heterologous immunogenic epitope or a heterologous linker residue for a conjugated epitope. It is particularly preferred that the sequence of 10 residues of positions 76 trough 85 (position 76-85 sequence) be present, but interrupted by one to about 245 residues of the heterologous linker or heterologous epitope .
  • Domain III is an HBc sequence from position 86 through position 135 peptide-bonded to residue 85 of Domain II.
  • Chimer molecule Domain IV comprises (i) five through fourteen residues of an HBc amino acid residue sequence from position 136 through 149 peptide-bonded to the residue of position 135 of Domain III, (ii) zero to three cysteine residues [C-terminal cysteine residue (s) ] within about 30 residues from the C-terminus of the chimer molecule, and (iii) zero to about 100 amino acid residues in an immunogenic sequence heterologous to HBc from position 150 to the C-terminus.
  • Domain IV contains a sequence of zero to about 50 amino acid residues in a sequence heterologous to HBc, and more preferably that sequence is zero to about 25 residues. Domain IV also preferably contains one C-terminal cysteine residue.
  • the chimer molecules (i) have an amino acid residue sequence in which no more than about 10 percent of the amino acid residues are substituted in the HBc sequence of the chimer and (ii) self-assemble into particles on expression in a host cell.
  • the particles are substantially free of binding to nucleic acids and are more stable than are particles formed from otherwise identical HBc chimer molecules that are free of any above-mentioned C-terminal cysteine residue (s) and (i) lack the N-terminal cysteine residue (s) or (ii) in which an N-terminal cysteine residue (s) present in a contemplated chimer molecule is (are) replaced by another residue.
  • a contemplated chimer molecule contains a heterologous epitope at the N-terminus peptide-bonded to one of HBc residues 2-5.
  • a contemplated chimer molecule contains a heterologous epitope or a heterologous linker residue for an epitope peptide-bonded near the middle of the molecule located between HBc residues 76 and 85 in the immunodominant loop.
  • a heterologous epitope is located at the C-terminal portion of the chimer molecule peptide- bonded to one of HBc residues 136-149.
  • two or three heterologous epitopes are present at the above locations, or one or two heterologous epitopes are present along with a heterologous linker residue for an epitope.
  • Each of those chimer molecules also contains an N-terminal cysteine residue (s), as discussed before. Specific examples of several of these chimer molecules and their self-assembled particles are discussed hereinafter.
  • a contemplated HBc chimer molecule can contain about 135 to about 515 amino acid residues.
  • HBc residue 4 is present, whereas residues 2-5 are present in other preferred embodiments, so that Domain I can begin at HBc residue 4 or 2 and continue through residue 75; i.e., the HBc residue at HBc position 75.
  • Residue 1 is methionine, the amino acid of the DNA start codon. It is preferred that the native methionine that is normally present at position 1 of HBc be absent so that only one start signal is present in the encoding DNA or NA.
  • the heterologous immunogenic epitope that can be present in Domain I or in the immunodominant loop of Domain II preferably contains about 15 to about 50 residues, although an epitope as short as about 6 amino acid residues can induce and be recognized by antibodies and T cell receptors and is therefore useful . It is preferred that all of the residues of Domain II from position 76 through position 85 are present, although interrupted by one or more other residues. Domain II must contain at least four residues, that can have any sequence that does not interfere with expression or use, but those residues are preferably part of the sequence between the residues of positions 75 and 85.
  • Domain III contains HBc residues 86 through 135 peptide-bonded to residue 85.
  • Domain IV contains a sequence of at least five residues that are comprised of (i) a sequence of the residues of HBc positions 136 through 140, and preferably through 149 on up to 156, peptide-bonded to residue 135, (ii) zero to three cysteines residues and (iii) optionally can contain a sequence of a heterologous epitope of up to about 100 residues, particularly when the HBc sequence ends at residue 140, although a shorter sequence of up to about 25 residues is more preferred.
  • That Domain IV heterologous sequence is heterologous to the sequence of HBc and is other than a sequence of HBc from position 150 to the HBc
  • the heterologous sequence when present in Domain IV, is preferably a T cell epitope, but can also be a B cell epitope as are usually present in one or the other of Domains I and II.
  • Domain IV can also contain zero to three cysteine residues and those Cys residues are present within about 30 residues of the carboxy-terminus (C-terminus) of the chimer molecule.
  • one cysteine (Cys) residue is present, and that Cys is preferably present as the carboxy-terminal (C-terminal) residue, unless a T cell epitope is present as part of Domain IV.
  • the preferred Cys is preferably within the C-terminal last five residues of the HBc chimer .
  • a particularly preferred chimer contains two heterologous epitopes. Those two heterologous epitopes are present in Domains I and II, or II and IV, or I and IV.
  • One of the two heterologous epitopes is preferably a B cell epitope in some embodiments.
  • one of the two heterologous epitopes is a T cell epitope.
  • one of the two heterologous epitopes is a B cell epitope and the other is a T cell epitope.
  • a plurality of B cell epitopes can be present at the B cell epitope location and a plurality of T cell epitopes can be present at the T cell epitope location.
  • the sequence contain one or more B cell epitopes, that the HBc sequence between amino acid residues 76 and 85 be present, but interrupted by the heterologous epitope (s), and that the chimer further include one or more T cell epitopes in Domain IV peptide-bonded to one of HBc residues 140-149.
  • chimer molecules in which the heterologous linker residue for a conjugated epitope is present in Domain II, thereby providing one or more heterologous epitopes in Domain II, with residues 76 and 85 present, but interrupted by the heterologous linker residue, with a T cell epitope being present peptide-bonded to one of HBc residues 140-149.
  • the particles formed from such chimer molecules typically contain a ratio of conjugated epitope to C-terminal peptide-bonded T cell epitope of about 1:4 to 1:1, with a ratio of about 1:2 being common.
  • a heterologous linker residue for a conjugated epitope is present in Domain II and a T cell epitope is present in Domain IV, with no additional B cell epitope being present in Domain II.
  • Such a chimer exhibits immunogenicity of the T cell epitope, while exhibiting minimal, if any, HBc antigenicity as measured by binding of anti-loop monoclonal antibodies in an ELISA assay as discussed hereinafter.
  • One preferred contemplated HBc chimer molecule contains a sequence of about 135 to about 515 residues.
  • a preferred HBc chimer molecule containing two heterologous epitopes of preferred lengths of about 15 to about 50 residues each and a preferred HBc portion length of about 140 to about 149 residues has a sequence length of about 170 to about 250 amino acid residues.
  • Particularly preferred chimer molecules continuing two heterologous epitopes have a length of about 180 to about 210 residues.
  • chimer molecule lengths is contemplated in view of the variations in length of the N- and C-terminal HBc portions and differing lengths of the several contemplated epitopes that can be inserted in the immunogenic loop.
  • a contemplated recombinant protein after expression in a host cell, self-assembles to form particles that are substantially free of binding to nucleic acids.
  • the contemplated HBc, chimer particles are generally spherical in shape and are usually homogeneous in size for a given preparation. These chimeric particles thus resemble native HBc particles that have a similar shape and size and can be recovered from infected persons .
  • a contemplated chimer particle comprises previously discussed chimer molecules. More broadly, such a chimer particle comprises a chimeric C-terminal truncated HBc protein that has a sequence of at least about 125 of the N-terminal 150 residues and contains (i) a heterologous epitope peptide- bonded to one or more of the N-terminus, C-terminus or the immunodominant loop, or a heterologous linker residue for an epitope in the immunodominant loop, and (ii) one to three N-terminal cysteine residues and zero to three C-terminal cysteine residues as previously described, and at least a 5 HBc residue sequence from position 135.
  • a contemplated particle is sufficiently free of arginine and/or lysine residues in Domain IV so that the self-assembled particles are substantially free of nucleic acid binding and exhibits a 280:260 absorbance ratio of about 1.2 to about 1.7, as discussed herein after.
  • a contemplated chimeric protein is free of the HBc sequence between positions 150 and 183, and preferably between residue positions 149 through 183.
  • a particularly preferred immunogen is a particle comprised of recombinant hepatitis B virus core (HBc) protein chimer molecules with a length of about 150 to about 325 and preferably about 155 to 225 amino acid residues that contains four peptide- linked amino acid residue sequence domains from the N-terminus that are denominated Domains I, II, III and IV.
  • HBc hepatitis B virus core
  • (a) Domain I comprises about 71 to about 110 amino acid residues whose sequence includes at least the sequence of the residues of position 5 through position 75 of HBc.
  • One to three cysteine residues is (are) also present at a position in the chimer molecule of about one to about -20 relative to the N-terminus of HBc of SEQ ID NO : 1 [N-terminal cysteine residue (s) ] .
  • the one or more N-terminal cysteine residues is (are) present within a sequence other than that of the pre-core sequence of HBc.
  • Domain I can, and preferably does, further include a (i) sequence of 6 to about 24 residues of an above-noted influenza A M2 polypeptide
  • residues X ] _ through X Q are absent or present, and when present are the residues naturally present in the M2 protein sequence that are methionine, serine, leucine, leucine, threonine or proline, glutamic acid, valine, and glutamic acid, respectively, with the proviso that when one subscripted X residue is present, any remaining subscripted X with a higher subscript number up to 8 is also present,
  • Xio is present and is proline, leucine or histidine,
  • X]_l is present and is isoleucine or threonine
  • X ⁇ 3 is present and is asparagine or serine
  • X]_ is present and is glutamic acid or glycine
  • residues X ⁇ 5 and X ] _g are present or absent, and when present are tryptophan and glycine or glutamic acid, respectively, residues X17 and X ⁇ g are present or absent, and when present are independently cysteine, serine, or alanine
  • residue X]_g is present or absent, and when present is arginine or lysine
  • residues X 2Q through X 2 4 are present or absent, and when present are the residues naturally present in the M2 protein sequence that are asparagine or serine, aspartic acid or glycine, serine, serine and aspartic acid respectively, with the proviso that when one subscripted X residue is present, any remaining subscripted X residue with a lower subscript number down to 15 is also present.
  • Domain II comprises about 10 to about 60 amino acid residues peptide-bonded to residue 75. This sequence includes (i) zero to all of the residues of a sequence of HBc from HBc position 76 through 85 peptide-bonded to (ii) an optional sequence of about 6 to about 48 residues that constitute one or more repeats of the above influenza A M2 polypeptide of SEQ ID NO: 9.
  • Domain III is an HBc sequence from position 86 through position 135 that is peptide- bonded to residue 85.
  • Domain IV comprises (i) the residues of positions 136-140 plus up to nine residues of an HBc amino acid residue sequence from position 141 through 149 peptide-bonded to the residue of position 135 of Domain III, (ii) zero to three cysteine residues, (iii) fewer than three arginine or lysine residues, or mixtures thereof adjacent to each other, and (iv) up to about 100 amino acid residues in a sequence heterologous to HBc from position 164 to the C-terminus.
  • Domain IV contains at least the 5 residues of positions 136-140.
  • a contemplated chimer molecule (i) contains up to about 10 percent conservatively substituted amino acid residues in the HBc sequence, (ii) self- assembles into particles that are substantially free of binding to nucleic acids on expression in a host cell, and those particles are more stable on formation than are particles formed from an otherwise identical HBc chimer that lacks said N-terminal cysteine residue (s) or in which an N-terminal cysteine residue present in the chimer molecule is replaced by another residue.
  • such a recombinant protein can have a length of about 150 to about 325 amino acid residues. Preferably, that length is about 155 to about 225 residues. More preferably, the length is about 155 to about 170 residues. These differences in length arise from changes in the length of Domains I, II and IV.
  • Domain I of a contemplated chimeric HBc protein constitutes an amino acid residue sequence of HBc beginning with at least amino acid residue position 5 through position 75, and Domain III constitutes a HBc sequence from position 86 through position 137.
  • sequences from any of the mammalian hepadnaviruses can be used for either of Domains I and III, and sequences from two or more viruses can be used in one chimer.
  • sequences from two or more viruses can be used in one chimer.
  • the human ayw sequence is used through out the chimer.
  • HBc chimers having a Domain I that contains more than a deletion of the first three amino- terminal (N-terminal) residues have been reported to result in the complete disappearance of HBc chimer protein in E. coli cells. Pumpens et al . , (1995) Intervirology, 38:63-74.
  • a recent study in which an immunogenic 23-mer polypeptide from the influenza M2 protein was fused to the HBc N- terminal sequence reported that the resultant fusion protein formed particles when residues 1-4 of the native HBc sequence were replaced. Neirynck et al . (October 1999) Nature Med . , 5 (10) :1157-1163.
  • particles can form when an added amino acid sequence is present peptide-bonded to one of residues 2-4 of HBc, whereas particles do not form if no additional sequence is present and more than residues 1-3 are deleted from the N-terminus of HBc.
  • An N-terminal epitope sequence peptide- bonded to one of the first five N-terminal residues of HBc can contain a single cysteine residue or a sequence of up to about 30 residues that are heterologous to HBc .
  • the one to three cysteine residues can be present at a convenient location in the sequence, but are typically near the C-terminus of the added sequence so that the added N-terminal cysteine residue (s) are at a position of about -20 to about +1, and more preferably at a position of about -14 to about +1, relative to the HBc N-terminus as shown in SEQ ID NO:l.
  • Exemplary sequences include a B cell or T cell epitope such as those discussed and illustrated hereinafter (Tables A and B, respectively) , the 23-mer polypeptide from the influenza M2 protein of Neirynck et al . , above, that includes two cysteine residues, and variants of that sequence containing at least about 6 residues, a sequence of another (heterologous) protein such as ⁇ -galactosidase as can occur in fusion proteins as a result of the expression system used, or another hepatitis B-related sequence such as that from the Pre-Sl or Pre-S2 regions or the major hepatitis B surface antigen (HbsAg) immunogenic sequence.
  • a B cell or T cell epitope such as those discussed and illustrated hereinafter (Tables A and B, respectively)
  • the 23-mer polypeptide from the influenza M2 protein of Neirynck et al . above, that includes two cysteine residues, and variants of that sequence
  • Domain II is a sequence of about 5 to about 250 amino acid residues. Of those residues, zero
  • a heterologous linker residue for a epitope such as a B cell or T cell epitope or (ii) a heterologous B or T cell epitope that preferably contains 6 to about 50, more preferably about 15 to about 50, and most preferably about 20 to about 30 amino acid residues, and are positioned so that they are peptide-bonded between zero, or more preferably at least 4, to all of the residues of positions 76 to 85 of the HBc sequence.
  • Heterologous B cell epitopes are preferably linked at this position by the linker residue or are peptide-bonded into the HBc sequence, and use of a B cell epitope is discussed illustratively hereinafter.
  • HBc residues can be all in one sequence such as residues 82-85, or can be split on either side of (flank) the heterologous residue (s) as where residues 76-77 and 84-85 are present or where residues 76 and 83-85 are present. More preferably, Domain II contains at least 8 residues of the HBc sequence from residue 76 to 85. Most preferably, the sequence of all 10 residues of positions 76 to 85 are present in the chimer.
  • the one to about 245 residues added to the HBc loop sequence is (are) heterologous to a HBc sequence.
  • a single added heterologous residue is a heterologous linker residue for a B cell epitope as discussed before.
  • the longer sequences typically at least 6 amino acid residues long to about 50 amino acid residues long and more preferably about 15 to about 50 residues in length, as noted before, are in a sequence that comprises a heterologous immunogen such as a B cell or T cell epitope, except for heterologous residues encoded by restriction sites.
  • a particularly preferred heterologous sequence contains 6 to about 24 residues of the N-terminal extracellular portion of the influenza A M2 protein. It is to be understood that an above heterologous immunogenic sequence can be present within a longer heterologous, non-HBc sequence or can be repeated in such a sequence .
  • Exemplary peptide B cell epitopes useful for both linkage to the linker residue after expression of a contemplated chimer and for expression within a HBc chimer at one or more of the N-terminus, within the immunogenic loop or at the C- terminus of the chimer are illustrated in Table A, below, along with the common name given to the gene from which the sequence is obtained, the literature or patent citation for published epitopes, and SEQ ID NO.
  • KDRTLIEQK 18 70 Respiratory syncitia virus (RSV) G CSICSNNPT- CWAICK 19 71
  • the remaining residues of Domain II that are present on either side of the heterologous residue or sequence are the residues of HBc position 76 to position 85.
  • the chimer sequence in Domain II is 76 through 77, followed by restriction site-encoded residues, the heterologous immunogenic (epitope) sequence, further restriction site-encoded residues, and then HBc sequence 84 through 85.
  • a typical exemplary sequence of a chimer prepared by an insertion strategy between residues 78 and 79 is that of HBc from position 2 through 78, followed by restriction site-encoded residues, the heterologous immunogenic sequence, further restriction site-encoded residues and HBc sequence 79 through 85.
  • the sequence of other contemplated chimers through Domains I and II should be apparent from these illustrations and those that follow and need not be enumerated.
  • a short hydrophilic peptide containing a plurality of glycine residues and having a length' of about 5 to about 9 residues peptide-bonded at the C-terminus of an above-noted Neisseria meningi tidis B cell epitope sequence can assist in the expression of a chimeric particle containing that sequence.
  • One useful short peptide is that disclosed in Karpenko et al . , Amino Acids (2000) 18:329-337, having the sequence GSGDEGG of SEQ ID NO: 144.
  • a heterologous linker for a conjugated epitope is peptide-bonded at a position in the HBc sequence between amino acid residues 76 and 85.
  • the HBc sequence of residues 76 to 85 is preferably present, but interrupted by the heterologous linker for a conjugated epitope.
  • This chimer preferably includes the HBc sequence of position 4 through at least position 140, plus a cysteine residue near the N-terminus of the chimer protein. More preferably, the HBc sequence of positions 1 through 149 are present, but interrupted between residues 76 and 85 by the heterologous linker for a conjugated epitope, and the chimer molecule contains a C-terminal cysteine.
  • the heterologous linker for a conjugated epitope is most preferably a lysine (K) residue.
  • Glutamic or aspartic acid, tyrosine and cysteine residues can also be used as linker residues, as can tyrosine and cysteine residues.
  • more than one linker can be present such as a sequence of three lysines, but such use is not preferred because heterogeneous conjugates can be formed from such use in which the conjugated hapten is bonded to one linker in a first chimer and to a different linker in a second chimer molecule.
  • U.S. Patent No. 6,231,864 Bl discloses HBc chimer molecules containing one or more linking residues, but lacking a stabilizing N-terminal cysteine residue .
  • a heterologous epitope sequence present in a contemplated HBc chimer can also be separated from the HBc sequence residues by a "flexible linker arm" on one or both sides of (flanking) the heterologous immunogenic (epitope) sequence. This is particularly the case where the heterologous immunogenic sequence is greater than about 30 amino acid residues long.
  • Exemplary flexible linker arm sequences typically contain about 4 to about 10 glycine residues that are thought to permit the inserted sequence to "bulge" outwardly from the otherwise bulging loop sequence and add further stability to the construct.
  • Illustrative flexible linker arm sequences are disclosed in Kratz et al . ( (March 1999) Proc. Natl . Acad. Sci . , U. S.A. , 96:1915-1920 and are exemplified by the amino acid residue sequences :
  • Domain III constitutes the sequence of HBc from position 86 through position 135. Consequently, the sequence of the illustrative chimers discussed above for Domains I and II, can be extended so that the first-discussed chimer has the sequence of HBc from position 84 through position 140, and the second-discussed chimer has the sequence of HBc from position 79 through position 140.
  • Domain IV is a sequence that (i) includes a HBc sequence from position 136 through 140 and optionally through position 149, (ii) contains zero up to three cysteine residues, and (iii) up to about 100 amino acid residues in a sequence heterologous to HBc at position 150 to the C-terminus, with the proviso that Domain IV contain at least 5 amino acid residues of the HBc sequence from position 136 through 140.
  • the Domain IV sequence heterologous to HBc more preferably contains up to about 50 amino acid residues, and most preferably contains up to about 25 residues.
  • the Domain IV sequence can thus be substantially any sequence, except the C-terminal HBc sequence from position 150 to the C-terminus.
  • the length of the Domain IV sequence can be five residues; i.e., the residue of position 136 through 140, up to about 100 amino acid residues including up to a total of three cysteines, with the length being sufficient so that a contemplated chimeric protein has a total length of about 135 to about 515 residues, and more preferably up to about 460 residues, and most preferably up to about 435 amino acid residues.
  • an epitope is peptide- bonded to Domains I or II contains up to about 30 or about 50 residues, respectively, as is preferred for those epitopes, more preferred lengths of the chimer molecule, including the Domain IV epitope, are about 175 to about 240 residues.
  • Particularly preferred chimer molecules containing two heterologous epitopes have a length of about 190 to about 210 residues. Freedom of the resulting particle from nucleic acid- binding is determined by determination of the 280:260 absorbance ratio as discussed previously.
  • the Domain IV sequence can include zero up to three Cys residues. When present, it is preferred that the one or more Cys residues be at or within about five amino acid residues of the C-terminus of the chimeric protein molecule. In addition, when more than one Cys residue is present in a Domain IV sequence, it is preferred that those Cys residues be adjacent to each other.
  • the Domain IV sequence constitute a T cell epitope, a plurality of T cell epitopes that are the same or different or an additional B cell epitope for the organism against which a contemplated chimer is intended to be used as an immunogen.
  • Exemplary Domain IV T cell epitope sequences are provided in Table B, below, as in Table A, with illustrative added C-terminal cysteine residues underlined. Table B T Cell Epitopes
  • LCMV lymphocytic choriomeningitis virus
  • the amino acid sequence of HBc from residue position 4 through at least position 140 is preferably present in a contemplated chimer molecule and particle.
  • the sequence from position 2 through position 149 is more preferably present.
  • a B cell epitope is preferably present between residues 76 and 85 and at least a single cysteine residue at or near the N-terminus in Domain I as already noted, a T cell epitope that can include a cysteine residue can also be present as a C-terminal addition to the HBc sequence.
  • a contemplated recombinant HBc chimer is substantially free of bound nucleic acid.
  • a contemplated chimer particle that contains an added Cys residue at or near the N-terminus of the molecule is also more stable after formation than is a similar particle that does not contain that added Cys.
  • a contemplated recombinant HBc chimer molecule is typically present and is used as a self- assembled particle. These particles are comprised of 180 to 240 chimer molecules (90 or 120 dimer pairs) , usually 240 chimer molecules, that separate into protein molecules in the presence of disulfide reducing agents such as 2-mercaptoethanol, and the individual molecules are therefore thought to be bound together into the particle primarily by disulfide bonds.
  • the observed enhanced stability and in some cases enhanced expression for a contemplated HBc chimer is due to the formation of an N-terminal cystine disulfide bond between chimer protein molecules of the particles.
  • the N-terminal cysteine (s) residue is referred to as a cysteine inasmuch as that is the residue coded-for by the codon present in the nucleic acid from which the protein and assembled particle is expressed.
  • HBc chimer molecules assemble in the host into particles that can be readily harvested from the host cells, and purified, if desired.
  • the HBc immunodominant loop is usually recited as being located at about positions 75 to 85 from the amino-terminus (N-terminus) of the intact protein.
  • the heterologous B cell epitope-containing sequence of Domain II is placed into that immunodominant loop sequence . That placement substantially eliminates the HBc immunogenicity of the HBc loop sequence, while presenting the heterologous sequence or linker residue in an extremely immunogenic position in the assembled chimer particles.
  • a contemplated chimer molecule can also contain conservative substitutions in the amino acid residues that constitute HBc Domains I, II, III and IV. Conservative substitutions are as defined before. An illustrative conservative substitution is seen in the replacement of residues at positions 2 and 3 (aspartic acid and isoleucine; DI) by glutamic acid and leucine (EL) residues that are encoded by an EcoRI restriction site used to add nucleic acids that code for a desired N-terminal epitope, including an N-terminal cysteine residue.
  • DI amino acid and isoleucine
  • EL glutamic acid and leucine
  • a "nonconservative" change e.g., replacement of a glycine with a tryptophan is contemplated.
  • Analogous minor variations can also include amino acid deletions or insertions, or both.
  • Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological activity or particle formation can be found using computer programs well known in the art, for example LASERGENE software (DNASTAR Inc., Madison, WI)
  • the HBc portion of a chimer molecule of the present invention i.e., the portion having the HBc sequence that has other than a sequence or residue of an added epitope, linker, flexible linker arm or heterologous residue (s) that are a restriction enzyme artifact, most preferably has the amino acid residue sequence at positions 2 through 149, or 2 through 156, of subtype ayw that is shown in Fig. 1 (SEQ ID NO:l) , less any portion or portions of the subtype ayw sequence that are absent because of truncation at one or both termini. Somewhat less preferred are the corresponding amino acid residue sequences of subtypes adw, adw2 and adyw that are also shown in Fig.
  • HBc portion of a chimer molecule of the present invention as above described has other than a sequence of a mammalian HBc molecule corresponding to positions 2 through 156, no more than about 20 percent of the amino acid residues are substituted as compared to SEQ ID NO : 1 from position 2 through 156. It is preferred that no more than about 10 percent, and more preferably no more than about 5 percent, and most preferably no more than about 3 percent of the amino acid residues are substituted as compared to SEQ ID NO : 1 from position 2 through 156. Where an HBc sequence is truncated further at one or both termini, the number of substituted residues is proportionally different. Deletions elsewhere in the molecule are considered conservative substitutions for purposes of calculation.
  • a contemplated chimer of 156 HBc residues can therefore contain up to about 30 residues that are different from those of SEQ ID NO : 1 at positions 2 through 156, and preferably about 15 residues. More preferably, about 7 or 8 residues are different from the ayw sequence (SEQ ID NO:l) at residue positions 2-156, and most preferably about 4 or 5 residues are different. Substitutions, other than in the immunodominant loop of Domain II or at the termini, are preferably in the non-helical portions of the chimer molecule and are typically between residues 2 to about 15 and residues 24 to about 50 to help assure particle formation. See, Koschel et al . , J. Virol . , 73 (3) :2153-2160 (Mar. 1999).
  • Domain I of a particularly preferred embodiment that includes an influenza A M2 immunogen preferably has the sequence of residues of positions 2-, 3- or 4- through 75 of HBc. Domain I also contains one to three, preferably one, added cysteine residue (s) and also preferably includes about 6 to about 24 residues of the sequence of the extracellular region of the influenza A M2 protein peptide-bonded at the amino-terminus as discussed herein below. Domain I therefore contains a deletion of at least the methionine residue of position 1 of HBc and can include deletions of the residues at positions 2, 3 and 4.
  • the one or more cysteine residues present in Domain I is (are) located at a position in the chimer molecule of about one to about -20 relative to the N-terminus of HBc of SEQ ID NO : 1 [N-terminal cysteine residue (s) ] .
  • the N-terminal cysteine residue (s) is located in the chimer molecule at a position that corresponds to the methionine at position 1 of SEQ ID NO : 1 (Fig. 1), or at a position up to about 20 residues downstream from that position. More preferably, an N-terminal cysteine is located at a position of about one to about minus 14 relative to position 1 of SEQ ID NO:l.
  • the one or more N-terminal cysteine residues are present within a sequence other than that of the pre-core sequence of HBc.
  • the HBeAg molecule contains the pre-core sequence that includes a cysteine residue. That molecule does not form particles, whereas particles are desired herein.
  • an N-terminal cysteine residue can be adjacent to a pre-core sequence, such a residue is not present within a precore sequence or a contemplated chimer molecule .
  • Domain I of a particularly preferred chimer molecule can have a length of about 110 residues.
  • Domain I has a length of about 95 to about 100 amino acid residues, and includes an influenza A M2 polypeptide epitope sequence of SEQ ID NO: 9, that preferably includes the C-terminal 23 residues .
  • Domain II which is peptide-bonded to residue 75, contains about 10 to about 60 amino acid residues. This Domain includes zero through all of the sequence of HBc residues of positions 76 through 85. Domain II also optionally includes a sequence of about 6 to about 48 residues that constitute one or more repeats of the before-mentioned influenza A M2 polypeptide of SEQ ID NO: 9.
  • the influenza A M2 polypeptide sequence when present, is preferably peptide-bonded between HBc residues 78 and 79, and all of the HBc sequence from position 76 through 85 is present .
  • Preferred influenza A M2 polypeptide sequences for insertion into Domains I or II, or both, of a contemplated recombinant HBc chimer are enumerated in Table C, below.
  • a sequence beginning with a methionine residue (M) is designed to be N-terminal sequence for insertion into the N-terminus of Domain I, whereas a sequence free of an N-terminal M residue is designed for insertion into Domain II.
  • NNATFNYTNVNPISHIR 33 In the polypeptide of SEQ ID NO: 9, X 1 through X Q are absent or present, and when present are the residues naturally present in a reported M2 protein sequence; i.e., methionine, serine, leucine, leucine, threonine or proline, glutamic acid, valine, and glutamic acid, respectively, with the proviso that when one subscripted X is present, any remaining subscripted X residue with a higher subscript number up to 8 is also present.
  • X ⁇ each of X 2 through X Q is also present.
  • each of X4 through X Q is also present, and the like.
  • X Q can be present without any other of the remaining X residues having a lower valued subscript number being present .
  • residues of X ⁇ o , X ⁇ , X13 and X14 are present, and can be leucine or histidine for X ] _ Q , isoleucine or threonine for X t asparagine or serine for -]_ an -d glutamic acid or glycine for X]_4.
  • residues X ⁇ 5 and X ⁇ g are present or absent, and when present are tryptophan and glycine or glutamic acid, respectively.
  • Residues - ⁇ and ] _g are present or absent, and when present are independently cysteine, serine, or alanine. It is preferred that one of Xi ⁇ and X]_g be cysteine, particularly when an M2 polypeptide epitope is present at the N-terminus of the chimer molecule.
  • Residue X Q is present or absent, and when present is arginine or lysine.
  • Residues X 2Q through 4 are present or absent, and when present are the residues naturally present in the reported M2 protein sequence; i.e., asparagine or serine, aspartic acid, serine, serine and aspartic acid respectively, with the proviso that when one subscripted X is present, any remaining X residue with a lower subscript number through 15 is also present.
  • X 2 3 when X 2 3 is present, so are each of residues X ⁇ 5 through X .
  • Domain III contains the sequence of HBc position 86 through position 135 peptide-bonded at its N-terminus to residue 85.
  • the fourth domain, Domain IV comprises (i) the residues of positions 136 through 140 plus up to nine residues of an HBc amino acid residue sequence from position 141 through 149 peptide-bonded to the residue of position 135 of Domain III, (ii) zero to three cysteine residues, and preferably one cysteine residue, (iii) fewer than three arginine or lysine residues, or mixtures thereof adjacent to each other, and (iv) up to about 100 amino acid residues, preferably up to 50 amino acid residues, and more preferably up to about 25 residues, in a sequence heterologous to HBc from position 164 to the C-terminus .
  • Domain IV contain up to fourteen residues of an HBc sequence from position 136 through position 149 peptide-bonded to residue 135; i.e., an HBc sequence that begins with the residue of position 136 that can continue through position 149.
  • an HBc sequence that begins with the residue of position 136 that can continue through position 149.
  • a chimer containing a HBc sequence up to about position 156 can be used, but it is preferred to end the HBc sequence at about residue 149.
  • Domain IV can also contain zero to three cysteine residues and those Cys residues are present within about 30 residues of the carboxy-terminus (C-terminus) of the chimer molecule.
  • one cysteine (Cys) residue is present, and that Cys is preferably present as the carboxy-terminal (C-terminal) residue, unless an influenza T cell epitope is present as part of Domain IV.
  • the preferred Cys is preferably within the C-terminal last five residues of the HBc chimer.
  • HBc chimer immunogen tends to form particles that stay together upon collection and initial purification as measured by analytical size exclusion chromatography, whose details are discussed hereinafter.
  • the contemplated particles can also be more stable to decomposition at 37°C after aging than are similar chimer particles lacking that cysteine residue.
  • This latter type of enhanced stability can be measured using 15% SDS-PAGE gels with particles dispersed in sample buffer (reducing) . Gels are stained using Coomassie Blue, and then analyzed. This type of stability is believed to be exhibited against hydrolysis, whereas the stability determined by size exclusion chromatography is that of initial particle formation.
  • Particles that additionally contain one or more C-terminal cysteine residues exhibit enhanced stability in formation and also toward decomposition on aging, with some particles containing both N- and C-terminal cysteines usually exhibiting greater stability in either measure than those particles having only an added cysteine at either the N- or C-terminus .
  • Domain IV contains fewer than three arginine or lysine residues, or mixtures thereof adjacent to each other.
  • Arginine and lysines are present in the C-terminal region of HBc that extends from position 164 through the C-terminus of the native molecule. That region is sometimes referred to as the "protamine” or "arginine-rich” region of the molecule and binds nucleic acids.
  • a contemplated HBc chimer molecule and particle are substantially free of bound nucleic acids.
  • the substantial freedom of nucleic acid binding can be readily determined by a comparison of the absorbance of the particles in aqueous solution measured at both 280 and 260 nm; i.e., a 280:260 absorbance ratio.
  • the contemplated particles do not bind substantially to nucleic acids that are oligomeric and/or polymeric DNA and RNA species originally present in the cells of the organism used to express the protein.
  • nucleic acids exhibit an absorbance at 260 nm and relatively less absorbance at 280 nm, whereas a protein such as a contemplated chimer absorbs relatively less at 260 nm and has a greater absorbance at 280 nm.
  • Chimeric HBc particles of the present invention are substantially free of nucleic acid binding and exhibit a 280:260 absorbance ratio of about 1.2 to about 1.7, and more typically, about 1.4 to about 1.7. This range is due in large part to the number of aromatic amino acid residues present in Domains II and IV of a given chimeric HBc particle. That range is also in part due to the presence of the Cys in Domain IV of a contemplated chimer, whose presence can diminish the observed ratio by about 0.1 for a reason that is presently unknown.
  • the contemplated particularly preferred chimer HBc particles are also more stable in aqueous buffer at 37°C over a time period of about two weeks to about one month than are particles formed from a HBc chimer containing the same peptide-linked Domain II, III and IV sequences and an otherwise same Domain I sequence in which the one to three cysteine residues [N-terminal cysteine residue (s) ] are absent or a single N-terminal residue present is replaced by another residue such as an alanine residue.
  • particles containing an influenza A M2 polypeptide in Domain I are more stable than otherwise identical particles [ICC- 1603 particles] assembled from chimer molecules whose N-terminal M2 variant sequence contains serine residues in place of the cysteines.
  • particles containing the above serine-containing influenza B cell epitope in Domain I and a single cysteine at the C-terminus are more stable than are otherwise identical particles in which that cysteine is absent, but are less stable than are the particles containing the two N-terminal cysteines, ICC-1590 particles, or those particles that contained both N-terminal and C-terminal cysteines [ICC-1604 particles] .
  • a contemplated particle containing a N-terminal cysteine residue is often prepared in greater yield than is a particle assembled from a chimer molecule lacking a N-terminal cysteine. This increase in yield can often be seen from the mass of particles obtained or from analytical gel filtration analysis using Superose ® 6 HR as discussed hereinafter.
  • HBc Although the T cell help afforded by HBc is highly effective in enhancing antibody responses (i.e. B cell-mediated) to 'carried' epitopes following vaccination, HBc does not activate influenza-specific T cells, except in restricted individuals for whom the B cell epitope is also a T cell epitope.
  • one or more influenza-specific T helper epitopes is preferably incorporated into a particularly preferred immunogen and is located in Domain IV of the immunogen.
  • a plurality of the above or another T cell epitopes can be present in Domain IV or another B cell epitope can be present.
  • Domain IV has up to about 50 residues in a sequence heterologous to HBc. More preferably, that sequence is up to about 25 residues and includes a T cell epitope.
  • M2 is expressed abundantly by infected cells, it has the potential to serve as a target for influenza-specific CTL activity.
  • the extracellular domain is a possible attachment site for antibody, which may function to immobilize the function of M2 , in an analogous manner to the therapeutic drug amantadine, or via the activation of antibody-dependent cellular cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) .
  • ADCC antibody-dependent cellular cytotoxicity
  • CDC complement-dependent cytotoxicity
  • the extracellular domain of M2 has been shown to contain at least two distinct human CTL epitopes, one at 7-15 [ Jameson, J. , J. Cruz, and F.A. Ennis, Human cytotoxic 1 '-lymphocyte repertoire to influenza A viruses . J Virol, 1998. 72(11): p. 8682-9] and another at 3-11 [Gianfrani, C, et al . , Human memory CTL response specific for influenza A virus is broad and mul tispecific . Hum Immunol, 2000. 61(5): p. 438-52].
  • Anti-M2e mediated lysis of influenza-infected cells via ADCC and/or CDC may be the preferred mechanisms of target cell lysis, compared with CTL, because, unlike CTL, they are not subject to MHC restriction. Therefore, an ability to evoke sufficient titers of anti-M2 antibodies displaying a required IgG subclass profdie, and M2e specificity, should evoke broad protection across diverse populations.
  • 14C2 was not virus neutralizing in vivo, but did bind infected cells and inhibited virus growth in vi tro; however, it failed to cure the infection [Palladino et al . , (1995) J. Virol . , 69(4) : 2075-2081] .
  • An immune response that can provide anti-M2e mediated lysis of influenza-infected cells via ADCC and/or CDC is thus a preferred response.
  • Th epitopes derived from the influenza nucleoprotein (NP 206-229) which is broadly reactive in humans (HLA-DR1, HLA-DR2, HLA-DRwl3) [Brett et al., (1991) J. Immunol . , 147 (3) : 984-991] and also functional in BALB/c mice are contemplated for use as T cell epitopes herein. Particles with this epitope fused to the C-terminus of HBc particles have been expressed and purified. Additional influenza Th epitopes are also considered, such as NP 341-362, NP 297-318 and NP 182-205 [Brett et al . , (1991) J. Immunol . , 147 (3) : 984-991] ; these sequences can ultimately be linked in series at the C-terminus of the M2e-expressing particle. These illustrative sequences are provided below.
  • a contemplated recombinant HBc chimer molecule is typically present and is used in an immunogen or vaccine as a self-assembled particle. These particles are comprised of 180 to 240 chimer • molecules that separate into protein molecules in the presence of disulfide reducing agents such as 2-mercaptoethanol and denaturing reagents such as SDS. The individual molecules are bound together into the particle by protein-protein interactions, and these interactions are stabilized by the presence of disulfide bonds. These particles are similar to the particles observed in patients infected with HBV, but these particles are non-infectious . Upon expression in various prokaryotic and eukaryotic hosts, the individual recombinant HBc chimer molecules assemble in the host into particles that can be readily harvested from the host cells.
  • a contemplated chimeric immunogen is prepared using the well-known techniques of recombinant DNA technology. Thus, sequences of nucleic acid that encode particular polypeptide sequences are added and deleted from the precursor sequence that encodes HBV.
  • An illustrative contemplated chimeric immunogen typically utilizes a cysteine residue present in the M2 sequence as the N-terminal cysteine. Primers for the preparation of such chimer molecules by in vi tro mutagenesis of a polynucleotide encoding an HBc molecule are discussed hereinafter.
  • the N-terminal cysteine can be provided by in vi tro mutagenesis using a primer that encodes just a cysteine- containing portion of the M2 polypeptide or a simple N-terminal start sequence such as Met-Cys- or Met- Gly-Cys- .
  • the HBc immunodominant loop is usually recited as being located at about positions 75 through 85 from the amino-terminus (N-terminus) of the intact protein.
  • the influenza A M2 B cell epitope-containing sequence can be placed into that immunodominant loop sequence of Domain II. That placement substantially eliminates the HBc immunogenicity and antigenicity of the HBc loop sequence, while presenting the influenza A M2 B cell epitope in an extremely immunogenic position in the assembled chimer particles.
  • a first, less successful strategy is referred to as replacement in which DNA that codes for a portion of the loop is excised and replaced with DNA that encodes the B cell epitope sequence.
  • the second strategy is referred to as insertion in which the B cell sequence is inserted between adjacent residues in the loop.
  • PCR polymerase chain reaction
  • a replacement approach to provide a chimeric HBc DNA sequence that encodes a pair of different restriction sites, e.g. EcoRI and Sacl, one near each end of the immunodominant loop-encoding DNA.
  • Exemplary residues replaced are 76 through 81.
  • the loop-encoding section is excised, an illustrative influenza A M2 B cell epitope-encoding sequence flanked on each side by appropriate HBc sequence residues is ligated into the restriction sites and the resulting DNA is used to express the HBc chimer. See, for example, Table 2 of Pumpens et al . , (1995) Intervirology, 38:63-74 for exemplary uses of a similar technique.
  • a single restriction site or two sites can be encoded into the region, the DNA cut with a restriction enzyme (s) to provide "sticky" or ends, and an appropriate sticky- or blunt-ended heterologous DNA segment ligated into the cut region.
  • a restriction enzyme s
  • Examples of this type of sequence replacement into HBc can be found in the work reported in Schodel et al . , (1991) F. Brown et al . eds., Vaccines 91 , Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp.319-325, Schodel et al . , Behring Inst . Mi tt . , 1997(98): p. 114-119 and Schodel et al . , J.
  • Insertion is preferred.
  • site-directed mutagenesis is used to create two restriction sites adjacent to each other and between codons encoding adjacent amino acid residues, such as those at residue positions 78 and 79. This technique adds twelve base pairs that encode four amino acid residues (two for each restriction site) between formerly adjacent residues in the HBc loop.
  • the HBc loop amino acid sequence is seen to be interrupted on its N-terminal side by the two residues encoded by the 5' restriction site, followed toward the C-terminus by the illustrative B cell epitope sequence, followed by two more heterologous, non-loop residues encoded by the 3' restriction site and then the rest of the loop sequence.
  • This same strategy is also preferably used for insertion into Domain IV of a T cell epitope or one or more cysteine residues that are not a part of a T cell epitope.
  • a DNA sequence that encodes a C-terminal truncated HBc sequence (HBcl49) is engineered to contain adjacent EcoRI and Sacl sites between residues 78 and 79. Cleavage of that DNA with both enzymes provides one fragment that encodes HBc positions 1-78 3 ' -terminated with an EcoRI sticky end, whereas the other fragment has a 5 '-terminal Sacl sticky end and encodes residues of positions 79- 149.
  • Ligation of a synthetic nucleic acid having a 5' AATT overhang followed by a sequence that encodes a desired B cell epitope and a AGCT 3 'overhang provides a HBc chimer sequence that encodes that B cell epitope flanked on each side by two heterologous residues (GI and EL, respectively) between residues 78 and 79, while destroying the EcoRI site and preserving the Sacl site.
  • GI and EL heterologous residues
  • a similar strategy can be used for insertion of a C-terminal cysteine-containing sequence.
  • EcoRI and HindiII restriction sites are engineered in to the HBc DNA sequence after amino acid residue position 149.
  • a synthetic DNA having the above AATT 5 'overhang followed by a T cell epitope-encoding sequence , a stop codon and a 3 ' AGCT overhang were ligated into the digested sequence to form a sequence that encoded HBc residues 1-149 followed by two heterologous residues (GI) , the stop codon and the HindiII site.
  • GI heterologous residues
  • PCR amplification using a forward primer having a Sacl restriction site followed by a sequence encoding HBc beginning at residue position 79, followed by digestion with Sacl and HindiII provided a sequence encoding HBc positions 79-149 plus the two added residues and the T cell epitope at the C-terminus.
  • Digestion of that construct with Sacl and ligation provides the complete gene encoding a desired recombinant HBc chimer immunogen having the sequence, from the N-terminus, of HBc positions 1-78, two added residues, the B cell epitope, two added residues, HBc positions 79-149, two added residues, and the T cell epitope.
  • a nucleic acid sequence that encodes a previously described HBc chimer molecule or a complement of that coding sequence is also contemplated herein. Such a nucleic acid segment is present in isolated and purified form in some preferred embodiments.
  • the amino acid residue sequence of a protein or polypeptide is directly related via the genetic code to the deoxyribonucleic acid (DNA) sequence of the gene that codes for the protein.
  • DNA deoxyribonucleic acid
  • additional DNAs and corresponding RNA sequences can be prepared as desired that encode the same chimer amino acid residue sequences, but are sufficiently different from a before-discussed gene sequence that the two sequences do not hybridize at high stringency, but do hybridize at moderate stringency.
  • High stringency conditions can be defined as comprising hybridization at a temperature of about 50°-55°C in 6XSSC and a final wash at a temperature of 68°C in 1-3XSSC.
  • Moderate stringency conditions comprise hybridization at a temperature of about 50°C to about 65°C in 0.2 to 0.3 M NaCl, followed by washing at about 50°C to about 55°C in 0.2X SSC, 0.1% SDS (sodium dodecyl sulfate) .
  • a nucleic sequence (DNA sequence or an RNA sequence) that (1) itself encodes, or its complement encodes, a chimer molecule whose HBc portion from residue position 1 through 136, when present, is that of SEQ ID NOs: 1, 2, 3, 4, 5 or 6 and (2) hybridizes with a DNA sequence of SEQ ID NOs: 195, 196, 197, 198, 199 and 200 at least one moderate stringency (discussed above) ; and (3) whose HBc sequence shares at least 80 percent, and more preferably at least 90 percent, and even more preferably at least 95 percent, and most preferably 100 percent identity with a DNA sequence of SEQ ID NOs: 195, 196, 197, 198, 199 and 200, is defined as a DNA variant sequence.
  • nucleic acid sequence such as a contemplated nucleic acid sequence is expressed when operatively linked to an appropriate promoter in an appropriate expression system as discussed elsewhere herein.
  • An analog or analogous nucleic acid (DNA or RNA) sequence that encodes a contemplated chimer molecule is also contemplated as part of this invention.
  • a chimer analog nucleic acid sequence or its complementary nucleic acid sequence encodes a HBc amino acid residue sequence that is at least 80 percent, and more preferably at least 90 percent, and most preferably is at least 95 percent identical to the HBc sequence portion from residue position 1 through residue position 136 shown in SEQ ID NOs : 1, 2, 3, 4, 5 and 6.
  • This DNA or RNA is referred to herein as an "analog of” or “analogous to” a sequence of a nucleic acid of SEQ ID NOs: 195, 196, 197, 198, 199 and 200, and hybridizes with the nucleic acid sequence of SEQ ID NOs : 195, 196, 197, 198, 199 and 200, or their complements herein under moderate stringency hybridization conditions.
  • a nucleic acid that encodes an analogous sequence upon suitable transfection and expression, also produces a contemplated chimer.
  • Different hosts often have preferences for a particular codon to be used for encoding a particular amino acid residue. Such codon preferences are well known and a DNA sequence encoding a desired chimer sequence can be altered, using in vi tro mutagenesis for example, so that host- preferred codons are utilized for a particular host in which the enzyme is to be expressed.
  • a useful analogous DNA sequence need not hybridize with the nucleotide sequences of SEQ ID NOs: 195, 196, 197, 198, 199 and 200 or a complement under conditions of moderate stringency, but can still provide a contemplated chimer molecule .
  • a recombinant nucleic acid molecule such as a DNA molecule, comprising a vector operatively linked to an exogenous nucleic acid segment (e.g., a DNA segment or sequence) that defines a gene that encodes a contemplated chimer, as discussed above, and a promoter suitable for driving the expression of the gene in a compatible host organism, is also contemplated in this invention.
  • an exogenous nucleic acid segment e.g., a DNA segment or sequence
  • a promoter suitable for driving the expression of the gene in a compatible host organism is also contemplated in this invention.
  • a recombinant DNA molecule that comprises a vector comprising a promoter for driving the expression of the chimer in host organism cells operatively linked to a DNA segment that defines a gene for the HBc portion of a chimer or a DNA variant that has at least 90 percent identity to the chimer gene of SEQ ID NOs: 195, 196, 197, 198, 199 and 200 and hybridizes with that gene under moderate stringency conditions.
  • a recombinant DNA molecule that comprises a vector containing a promoter for driving the expression of a chimer in host organism cells operatively linked to a DNA segment that is an analog nucleic acid sequence that encodes an amino acid residue sequence of a HBc chimer portion that is at least 80 percent identical, more preferably 90 percent identical, and most preferably 95 percent identical to the HBc portion of a sequence of SEQ ID NOs: 1, 2, 3, 4, 5 or 6. That recombinant DNA molecule, upon suitable transfection and expression in a host cell, provides a contemplated chimer molecule.
  • isolated nucleic acid segments preferably DNA sequences, variants and analogs thereof can be prepared by in vi tro mutagenesis, as is well known in the art and discussed in Current Protocols In Molecular Biology, Ausabel et al . eds., John Wiley & Sons (New York: 1987) p. 8.1.1-8.1.6, that begin at the initial ATG codon for a gene and end at or just downstream of the stop codon for each gene.
  • a desired restriction site can be engineered at or upstream of the initiation codon, and at or downstream of the stop codon so that other genes can be prepared, excised and isolated.
  • additional base pairs can usually be present at either end of the segment and that segment can still be utilized to express the protein. This, of course, presumes the absence in the segment of an operatively linked DNA sequence that represses expression, expresses a further product that consumes the enzyme desired to be expressed, expresses a product that consumes a wanted reaction product produced by that desired enzyme, or otherwise interferes with expression of the gene of the DNA segment .
  • a DNA segment of the invention can be about 500 to about 15,000 base pairs in length.
  • the maximum size of a recombinant DNA molecule, particularly an expression vector is governed mostly by convenience and the vector size that can be accommodated by a host cell, once all of the minimal DNA sequences required for replication and expression, when desired, are present. Minimal vector sizes are well known. Such long DNA segments are not preferred, but can be used.
  • DNA segments that encode the before- described chimer can be synthesized by chemical techniques, for example, the phosphotriester method of Matteucci et al . (1981) J. Am. Chem. Soc , 103:3185. Of course, by chemically synthesizing the coding sequence, any desired modifications can be made simply by substituting the appropriate bases for those encoding the native amino acid residue sequence. However, DNA segments including sequences discussed previously are preferred.
  • a contemplated HBc chimer can be produced (expressed) in a number of transformed host systems, typically host cells although expression in acellular, in vi tro, systems is also contemplated.
  • host cellular systems include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g. baculovirus) ; plant cell systems transformed with virus expression vectors (e.g. cauliflower mosaic virus; tobacco mosaic virus) or with bacterial expression vectors (e.g., Ti plasmid); or appropriately transformed animal cell systems such as CHO or COS cells.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors
  • yeast transformed with yeast expression vectors insect cell systems infected with virus expression vectors (e.g. baculovirus)
  • plant cell systems transformed with virus expression vectors e.g. cauliflower mosaic virus; tobacco mosaic virus
  • bacterial expression vectors e.g., Ti plasmid
  • appropriately transformed animal cell systems such as CHO
  • DNA segments containing a gene encoding the HBc chimer are preferably obtained from recombinant DNA molecules (plasmid vectors) containing that gene.
  • Plasmid vectors Vectors capable of directing the expression of a chimer gene into the protein of a HBc chimer is referred to herein as an "expression vector" .
  • An expression vector contains expression control elements including the promoter.
  • the chimer- coding gene is operatively linked to the expression vector to permit the promoter sequence to direct RNA polymerase binding and expression of the chimer- encoding gene.
  • Useful in expressing the polypeptide coding gene are promoters that are inducible, viral, synthetic, constitutive as described by Poszkowski et al. (1989) EMBO J. , 3:2719 and Odell et al . (1985) Nature, 313:810, as well as temporally regulated, spatially regulated, and spatiotemporally regulated as given in Chua et al . (1989) Science, 244:174-181.
  • One preferred promoter for use in prokaryotic cells such as E. coli is the Rec 7 promoter that is inducible by exogenously supplied nalidixic acid.
  • a more preferred promoter is present in plasmid vector JHEX25 (available from Promega) that is inducible by exogenously supplied isopropyl- ⁇ -D-thiogalacto-pyranoside (IPTG) .
  • IPTG isopropyl- ⁇ -D-thiogalacto-pyranoside
  • a still more preferred promoter, the tac promoter is present in plasmid vector pKK223-3 and is also inducible by exogenously supplied IPTG.
  • the p K223-3 plasmid can be successfully expressed in a number of E.
  • coli strains such as XL-1, TB1 , BL21 and BLR, using about 25 to about 100 ⁇ M IPTG for induction.
  • concentrations of about 25 to about 50 ⁇ M IPTG have been found to provide optimal results in 2 L shaker flasks and fermentors .
  • a contemplated chimer molecule in other microbes such as Salmonella like S. typhi and S. typhimurium and S . typhimurium-E. coli hybrids, yeasts such as S. cerivisiae or Pichia pastoris, in mammalian cells such as Chinese hamster ovary (CHO) cells, in both monocot and dicot plant cells generally and particularly in dicot plant storage organs such as a root, seed or fruit as where an oral vaccine or inoculum is desired, and in insect cells such as those of S.
  • CHO Chinese hamster ovary
  • Frugiperda cells or Trichoplusia by use of Autographa calif ornica nuclear polyhedrosis virus (AcNPV) or baculovirus are discussed in detail in published before-mentioned WO 02/14478 A2. These modes of expression, although contemplated, will therefore not be discussed further herein.
  • a variety of methods have been developed to operatively link DNA to vectors via complementary cohesive termini or blunt ends. For instance, complementary homopolymer tracts can be added to the DNA segment to be inserted into the vector DNA. The vector and DNA segment are then j oined by hydrogen bonding between the complementary homopolymeric tails to form recombinant DNA molecules .
  • synthetic linkers containing one or more restriction endonuclease sites can be used to join the DNA segment to the expression vector, as noted before.
  • the synthetic linkers are attached to blunt-ended DNA segments by incubating the blunt-ended DNA segments with a large excess of synthetic linker molecules in the presence of an enzyme that is able to catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase.
  • the products of the reaction are DNA segments carrying synthetic linker sequences at their ends. These DNA segments are then cleaved with the appropriate restriction endonuclease and ligated into an expression vector that has been cleaved with an enzyme that produces termini compatible with those of the synthetic linker. Synthetic linkers containing a variety of restriction endonuclease sites are commercially available from a number of sources including New England BioLabs, Beverly, MA. A desired DNA segment can also be obtained using PCR technology in which the forward and reverse primers contain desired restriction sites that can be cut after amplification so that the gene can be inserted into the vector. Alternatively PCR products can be directly cloned into vectors containing T-overhangs (Promega Corp., A3600, Madison, WI) as is well known in the art .
  • the expressed chimeric protein self- assembles into particles within the host cells, whether in single cells or in cells within a multicelled host.
  • the particle-containing cells are harvested using standard procedures, and the cells are lysed using a French pressure cell, lysozyme, sonicator, bead beater or a microfluidizer (Microfluidics International Corp., Newton MA). After clarification of the lysate, particles are precipitated with 45% ammonium sulfate, resuspended in 20 mM sodium phosphate, pH 6.8 and dialyzed against the same buffer. The dialyzed material is clarified by brief centrifugation and the supernatant subjected to gel filtration chromatography using
  • Sepharose CL-4B Particle-containing fractions are identified, subjected to hydroxyapatite chromatography, and reprecipitated with ammonium sulfate prior to resuspension, dialysis and sterile filtration and storage at -70°C.
  • Any hapten (immunogen) to which a B cell or T cell response is desired can be linked to a contemplated HBc chimer or chimer particle such as a chimer particle containing a heterologous linker residue such as a lysine, glutamic or aspartic acid, cysteine or tyrosine in the loop region of Domain II and an added cysteine residue near the N-terminus in Domain I to form a HBc chimer conjugate.
  • the hapten of interest typically is a B cell immunogen.
  • the hapten can be a polypeptide, a protein, a carbohydrate (saccharide; i.e., oligo- or polysaccharide) , or a non-polypeptide, non- carbohydrate chemical such as 2 , 4-dinitrobenzene or a medicament such as cocaine or nicotine.
  • a carbohydrate saccharide
  • non-polypeptide non- carbohydrate chemical
  • a HBc , chimer particle conjugate so formed is useful as an inoculum or vaccine, as is discussed hereinafter. Because the chimer protein self assembles upon expression and a conjugate is formed after expression, conjugate formation is typically done using the assembled particles as compared to the free protein molecules.
  • haptens immunogenic conjugates
  • a protein or polypeptide through an amino acid residue side chain of the protein or polypeptide to form a pendently-linked immunogenic conjugate, e.g., a branched-chain polypeptide polymer
  • Those methods include linking through one or more types of functional groups on various side chains and result in the carrier protein polypeptide backbone (here, a HBc chimer) within the particle being pendently linked- -covalently linked (coupled) -- to the hapten but separated by at least one side chain.
  • both the HBc protein and a polypeptide hapten can be used in their native form or their functional group content can be modified by succinylation of lysine residues or reaction with cysteine-thiolactone.
  • a sulfhydryl group can also be incorporated into either carrier protein or conjugate by reaction of amino functional groups with 2-iminothiolane, the N- hydroxysuccinimide ester of 3- (3-dithiopyridyl) - propionate, or other reagents known in the art.
  • the HBc chimer or hapten can also be modified to incorporate a spacer arm, such as hexamethylenediamine or another bifunctional molecule, to facilitate the pendent linking. Such a procedure is discussed below.
  • That activated carrier is then reacted with a hapten such as a sulfhydryl-terminated hapten or a polypeptide that either contains a terminal cysteine or to which an additional amino- or carboxy-terminal cysteine residue has been added to form a covalently bonded HBc chimer conjugate.
  • a hapten such as a sulfhydryl-terminated hapten or a polypeptide that either contains a terminal cysteine or to which an additional amino- or carboxy-terminal cysteine residue has been added to form a covalently bonded HBc chimer conjugate.
  • the amino group of a polypeptide hapten can be first reacted with N-succinimidyl 3- (2- pyridylthio) ropionate (SPDP, Pharmacia, Piscataway, NJ) , and that thiol-containing polypeptide can be reacted with the activated carrier after reduction.
  • SPDP N-
  • U.S. Patent No. 4,767,842 teaches several modes of covalent attachment between a carrier and polypeptide that are useful here.
  • tolylene diisocyanate is reacted with the carrier in a dioxane-buffer solvent at zero degrees C to form an activated carrier.
  • a polypeptide hapten is thereafter admixed and reacted with the activated carrier to form the covalently bonded HBc chimer conjugate .
  • reagents include a cysteine residue in a polypeptide hapten and an amine on the coupling partner such as the ⁇ -amine of a lysine or other free amino group in the carrier protein.
  • a variety of such disulfide/amide forming agents are known. See for example Immun . Rev.
  • the particularly preferred coupling agent for the method of this invention is succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) obtained from Pierce Chemical Co., Rockford, IL.
  • SMCC succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate
  • Fig. 11 provides a schematic representation (Scheme 1) of the formation of a HBc activated carrier using SMCC (I) and the subsequent reaction of that activated carrier with a sulfhydryl-terminated hapten (II) .
  • a polypeptide hapten can be obtained in a number of ways well known in the art. Usual peptide synthesis techniques can be readily utilized. For example, recombinant and PCR-based techniques to produce longer peptides are useful. Because the desired sequences are usually relatively short, solid phase chemical synthesis is useful .
  • polypeptide haptens are shown in Tables A and B hereinbefore . Each of those polypeptides can be utilized via its N-terminal amino group, or by use of an additional N-terminal cysteine that is not shown in the table.
  • a before-described recombinant HBc chimer immunogen preferably in particulate form is dissolved or dispersed in an immunogenic effective amount in a pharmaceutically acceptable vehicle composition that is preferably aqueous to form an inoculum or vaccine .
  • a pharmaceutically acceptable vehicle composition that is preferably aqueous to form an inoculum or vaccine .
  • an inoculum induces antibodies that immunoreact with an added B cell epitope such as an influenza A M2 B cell epitope present in the immunogen.
  • compositions that is a vaccine in one animal can be an inoculum an inoculum for another host, as where the antibodies are induced in a second host that is not infected by influenza A.
  • the amount of recombinant HBc chimer immunogen utilized in each immunization is referred to as an immunogenic effective amount and can vary widely, depending inter alia, upon the recombinant HBc chimer immunogen, animal host immunized, and the presence of an adjuvant in the vaccine, as discussed below.
  • Immunogenic effective amounts for a vaccine and an inoculum provide the protection or antibody activity, respectively, discussed hereinbefore.
  • Vaccines or inocula typically contain a recombinant HBc chimer immunogen concentration of about 1 microgram to about 1 milligram per inoculation (unit dose) , and preferably about 10 micrograms to about 50 micrograms per unit dose. Immunizations in mice typically contain 10 or 20 ⁇ g of chimer particles.
  • unit dose refers to a physically discrete unit suitable as an unitary dosage for animals, each unit containing a predetermined quantity of active material calculated to individually or collectively produce the desired immunogenic effect in association with the required diluent; i.e., carrier, or vehicle.
  • a single unit dose or a plurality of unit doses can be used to provide an immunogenic effective amount of recombinant HBc chimer immunogen particles.
  • Vaccines or inocula are typically prepared from a recovered recombinant HBc chimer immunogen particles by dispersing the particles in a physiologically tolerable (acceptable) diluent vehicle such as water, saline phosphate-buffered saline (PBS), acetate-buffered saline (ABS), Ringer's solution or the like to form an aqueous composition.
  • a physiologically tolerable (acceptable) diluent vehicle such as water, saline phosphate-buffered saline (PBS), acetate-buffered saline (ABS), Ringer's solution or the like to form an aqueous composition.
  • PBS saline phosphate-buffered saline
  • ABS acetate-buffered saline
  • Ringer's solution or the like to form an aqueous composition.
  • the diluent vehicle can also include oleaginous materials such
  • the immunogenic active ingredient is often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient .
  • excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
  • an inoculum or vaccine can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents that enhance the immunogenic effectiveness of the composition.
  • a contemplated vaccine or inoculum advantageously also includes an adjuvant.
  • Suitable adjuvants for vaccines and inocula of the present invention comprise those adjuvants that are capable of enhancing the antibody responses against B cell epitopes of the chimer, as well as adjuvants capable of enhancing cell mediated responses towards T cell epitopes contained in the chimer, if present.
  • Adjuvants are well known in the art (see, for examp1e, Vaccine Design - The Subunit and Adjuvant Approach, 1995, Pharmaceutical Biotechnology, Volume 6, Eds. Powell, M.F., and Newman, M.J., Plenum Press, New York and London, ISBN 0-306-44867-X) .
  • Exemplary adjuvants include complete Freund' s 'adjuvant (CFA) that is not used in humans, incomplete Freund's adjuvant (IFA) , squalene, squalane and alum [e.g., AlhydrogelTM (Superfos, Denmark)], which are materials well known in the art, and are available commercially from several sources.
  • CFA complete Freund' s 'adjuvant
  • IFA incomplete Freund's adjuvant
  • squalene squalene
  • squalane e.g., AlhydrogelTM (Superfos, Denmark)
  • alum e.g., AlhydrogelTM (Superfos, Denmark)
  • Preferred adjuvants for use with immunogens of the present invention include aluminum or calcium salts (for example hydroxide or phosphate salts) .
  • a particularly preferred adjuvant for use herein is an aluminum hydroxide gel such as AlhydrogelTM.
  • AlhydrogelTM aluminum hydroxide gels
  • the chimer protein is admixed with the adjuvant so that about 50 to about 800 micrograms of aluminum are present per dose, and preferably about 400 to about 600 micrograms are present .
  • a particularly preferred adjuvant for use with an immunogen of the present invention is an emulsion.
  • a contemplated emulsion can be an oil-in- water emulsion or a water-in-oil emulsion.
  • such emulsions comprise an oil phase of squalene, squalane, peanut oil or the like as are well-known, and a dispersing agent.
  • Non-ionic dispersing agents are preferred and such materials include mono- and di-C]_2"C 2 4 _ f tty acid esters of sorbitan and mannide such as sorbitan mono-stearate, sorbitan mono-oleate and mannide mono-oleate.
  • An immunogen-containing emulsion is administered as an emulsion.
  • such emulsions are water-in-oil emulsions that comprise squalene and mannide monooleate (ArlacelTM A) , optionally with squalane, emulsified with the chimer protein particles in an aqueous phase.
  • squalene and mannide monooleate AllacelTM A
  • squalane emulsified with the chimer protein particles in an aqueous phase.
  • Well-known examples of such emulsions include MontanideTM ISA-720, and MontanideTM ISA 703 (Seppic, Castres, France) , each of which is understood to contain both squalene and squalane, with squalene predominating in each, but to a lesser extent in MontanideTM ISA 703.
  • MontanideTM ISA-720 is used, and a ratio of oil-to- water of 7:3 (w/w) is used.
  • Other preferred oil-in- water emulsion adjuvants include those disclosed in WO 95/17210 and EP 0 399 843.
  • small molecule adjuvants are also contemplated herein.
  • One type of small molecule adjuvant useful herein is a 7-substituted-8-oxo- or 8-sulfo-guanosine derivative described in U.S. Patents No. 4,539,205, No. 4,643,992, No. 5,011,828 and No. 5,093,318, whose disclosures are incorporated by reference.
  • 7-allyl-8- oxoguanosine (loxoribine) is particularly preferred. That molecule has been shown to be particularly effective in inducing an antigen- (immunogen-) specific response.
  • Still further useful adjuvants include monophosphoryl lipid A (MPL) available from Corixa Corp. (see, U.S. Patent No. 4,987,237), CpG (also ODN; oligonucleotides containing the CpG nucleotide motif one or more times plus flanking sequences) available from Coley Pharmaceutical Group, QS21 available from Aquila Biopharmaceuticals, Inc., SBAS2 (now AS02) available from SKB (now Glaxo-SmithKline) that contains QS21 and MPL ion an oil-in-water emulsion, the so-called muramyl dipeptide analogues described in U.S. Patent No. 4,767,842, and MF59 available from Chiron Corp. (see, U.S. Patents No. 5,709,879 and No. 6,086,901).
  • MPL monophosphoryl lipid A
  • CpG also ODN; oligonucleotides containing the CpG nucleotide motif one or
  • immunologically active saponin fractions having adjuvant activity derived from the bark of the South American tree Quillaja Saponaria Molina ⁇ e . g. QuilTM A are also useful.
  • Derivatives of QuilTM A for example QS21 (an HPLC purified fraction derivative of QuilTM A) , and the method of its production is disclosed in U.S. Patent No. 5,057,540.
  • QS21 an HPLC purified fraction derivative of QuilTM A
  • other fractions such as QA17 are also disclosed.
  • 3 -De-O-acylated monophosphoryl lipid A is a well-known adjuvant manufactured by Ribi Immunochem, Hamilton, Montana.
  • the adjuvant contains three components extracted from bacteria: monophosphoryl lipid (MPL) A, trehalose dimycolate (TDM) and cell wall skeleton (CWS) (MPL+TDM+CWS) in a 2% squalene/Tween ® 80 emulsion.
  • This adjuvant can be prepared by the methods taught in GB 2122204B.
  • a preferred form of 3-de-O-acylated monophosphoryl lipid A is in the form of an emulsion having a small particle size less than 0.2 ⁇ m in diameter (EP 0 689 454 Bl) .
  • the muramyl dipeptide adjuvants include N- acetyl-muramyl-L-threonyl-D-isoglutamine (thur-MDP) , N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine [CGP 11637, referred to as nor-MDP] , and N-acetylmuramyl- L-alanyl-D-isoglutaminyl-L-alanine-2- (1 ' -2 ' - dipalmityol-sn-glycero-3-hydroxyphosphoryloxy) - ethylamine [(CGP) 1983A, referred to as MTP-PE] .
  • Preferred adjuvant mixtures include combinations of 3D-MPL and QS21 (EP 0 671 948 Bl) , oil-in-water emulsions comprising 3D-MPL and QS21 (WO 95/17210, PCT/EP98/05714) , 3D-MPL formulated with other carriers (EP 0 689 454 Bl) , QS21 formulated in cholesterol-containing liposomes (WO 96/33739) , or immunostimulatory oligonucleotides (WO 96/02555) .
  • Alternative adjuvants include those described in WO 99/52549 and non-particulate suspensions of polyoxyethylene ether (UK Patent Application No. 9807805.8) .
  • Adjuvants are utilized in an adjuvant amount, which can vary with the adjuvant, host animal and recombinant HBc chimer immunogen. Typical amounts can vary from about 1 ⁇ g to about 1 mg per immunization. Those skilled in the art know that appropriate concentrations or amounts can be readily determined.
  • Inocula and vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly.
  • Additional formulations that are suitable for other modes of administration include suppositories and, in some cases, oral formulation or by nasal spray.
  • suppositories traditional binders and carriers can include, for example, polyalkalene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1-2%.
  • Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like .
  • An inoculum or vaccine composition takes the form of a solution, suspension, tablet, pill, capsule, sustained release formulation or powder, and contains an immunogenic effective amount of HBc chimer, preferably as particles, as active ingredient .
  • an immunogenic effective amount of preferred HBc chimer particles is about 1 ⁇ g to about 1 mg of active ingredient per dose, and more preferably about 5 ⁇ g to about 50 ⁇ g per dose, as noted before.
  • a vaccine or inoculum is typically formulated for parenteral administration.
  • exemplary immunizations are carried out sub-cutaneously (SC) intra-muscularly (IM) , intravenusly (IV) , intraperitoneally (IP) or intra-dermally (ID) .
  • the HBc chimer particles and HBc chimer particle conjugates can be formulated into the vaccine as neutral or salt forms.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein or hapten) and are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived form inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2- ethylamino ethanol, histidine, procaine, and the like.
  • the inocula or vaccines are administered in a manner compatible with the dosage formulation, and in such amount as are therapeutically effective and immunogenic (an antibody-inducing amount or protective amount, as is desired) .
  • the quantity to be administered depends on the subject to be treated, capacity of the subject's immune system to synthesize antibodies, and degree of protection desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual. However, suitable dosage ranges are of the order of several hundred micrograms active ingredient per individual . Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed in intervals (weeks or months) by a subsequent injection or other administration.
  • the host animal is maintained for a period of time sufficient for the recombinant HBc chimer immunogen to induce the production of a sufficient titer of antibodies that bind to the M2 protein.
  • the maintenance time for the production of anti-M2 antibodies typically lasts for a period of about three to about twelve weeks, and can include a booster, second immunizing administration of the vaccine.
  • a third immunization is also contemplated, if desired, at a time 24 weeks to five years after the first immunization. It is particularly contemplated that once a protective level titer of antibodies is attained, the vaccinated host animal is preferably maintained at or near that antibody titer by periodic booster immunizations administered at intervals of about 1 to about 5 years .
  • antibodies are readily ascertained by obtaining a plasma or serum sample from the immunized host and assaying the antibodies therein for their ability to bind to a synthetic M2 polypeptide antigen in an ELISA assay as described hereinafter or by another immunoassay such as a Western blot as is well known in the art.
  • the before-described antibodies so induced can be isolated from the blood of the host using well-known techniques, and then reconstituted into a second vaccine for passive immunization as is also well known. Similar techniques are used for gamma-globulin immunizations of humans.
  • antiserum from one or a number of immunized hosts can be precipitated in aqueous ammonium sulfate (typically at 40-50 percent of saturation) , and the precipitated antibodies purified chromatographically as by use of affinity chromatography in which an M2 polypeptide is utilized as the antigen immobilized on the chromatographic column.
  • Inocula are preparations that are substantially identical to vaccines, but are used in a host animal in which antibodies to influenza are desired to be induced, but in which protection from influenza is not desired.
  • Plasmid vector pKK223-3 (Pharmacia) was modified by the establishment of a unique Ncol restriction site to enable insertion of HBc genes as Ncol-Hindlll restriction fragments and subsequent expression in E. coli host cells.
  • pKK223-3 plasmid vector a new Sphl-HindiII fragment was prepared using the PCR primers pKK223 -3/433 -452 -F and pKK223-NcoI-mod-R, and pKK223-3 as the template.
  • This PCR fragment was cut with the restriction enzymes Sphl and Hindlll to provide a 467 bp fragment that was then ligated with a 4106 bp fragment of the pKK223-3 vector, replacing the original 480 bp Sphl-HindiII fragment.
  • the resultant plasmid (pKK223-3N; 4573 bp) is therefore 13 bp shorter than the parent plasmid and contains modified nucleotide sequence upstream of the introduced Ncol site (see Fig. 2, in which the dashes indicate the absent bases) .
  • Restriction sites in plasmid pKK223- 3N are indicated in Fig. 2 and nucleotide changes made to the pKK223-3 parent plasmid are indicated by an underline as shown below.
  • Modified HBcl49 (VI and V2) or HBcl83 (V8) genes able to accept the directional insertion of synthetic dsDNA fragments into the immunodominant loop region, were constructed using PCR.
  • VI The plasmid accepting inserts between amino acids E77 and D78 and truncated to V149 was named VI
  • the plasmid accepting inserts between D78 and P79 and truncated to V149 was named V2
  • the plasmid accepting inserts between D78 and P79 and terminating at C183 was called V8 .
  • the HBcl49 and HBcl83 genes were amplified in two halves using two PCR primer pairs, one of which amplifies the amino terminus, the other amplifies the carboxyl terminus.
  • the products of the PCR reactions are both 246 bp fragments; for V2 , the products are a 249 bp (N-terminus) and a 243 bp fragment (C-terminus) ; for V8, the products are a 249 bp (N-terminus) and a bp fragment (C-terminus) .
  • N-terminal fragments prepared were digested with Ncol and EcoRI, and the C-terminal fragments were digested with EcoRI and HindiII.
  • the VI, V2 and V8 fragment pairs were then ligated together at the common EcoRI overhangs .
  • the resultant Ncol-Hindlll fragments were then ligated into the pKK223-3N vector, which had been prepared by digestion with Ncol and Hindlll.
  • VI, V2 and V8 plasmids To insert B cell epitopes into the VI, V2 and V8 plasmids, the appropriate plasmid was digested with EcoRI and Sad restriction enzymes. Synthetic dsDNA fragments containing 5 ' EcoRI and 3 ' Sacl overhangs were then inserted. In all cases, VI, V2 , and V8, glycine-isoleucine (EcoRI) and glutamic acid- leucine (Sad) amino acid pairs, flank the inserted B cell epitopes. The inserted restriction sites are underlined in the primers below.
  • Modified HBcl49 genes able to accept the directional insertion of synthetic dsDNA fragments into the N-terminal region, 5' to the pre-core sequence LGWLWG, were constructed using PCR.
  • the plasmid that encoded an HBc sequence terminating at V149 was named V34, whereas the plasmid that encoded an HBc sequence harboring an additional cysteine, C- terminal to V149, was named V55.
  • the HBcl49 gene was amplified in two halves using two PCR primer pairs, one of which amplifies the amino terminus (for which VI was used as a template) , the other amplifies the carboxyl terminus.
  • the products of the PCR reactions were a 293 bp (N-terminus) fragment and a 484 bp (C-terminus) fragment; for V55, the same N- terminal fragment was used and a 490 bp C-terminal fragment was prepared.
  • N-terminal fragment prepared by PCR was digested with Ncol and Sad, and the C-terminal fragments were digested with Sacl and Hindlll. The V34 and V55 fragment pairs were then ligated together at the common Sad overhangs. The resultant Ncol- Hindlll fragments were then ligated into the pKK223- 3N vector, which had been prepared by digestion with Ncol and HindiII.
  • B cell epitope insertion was accomplished by a procedure identical to that outlined above for the VI cloning vector. Restriction sites are underlined in the oligonucleotides primers below.
  • Modified HBcl49 and HBcl83 genes able to accept the directional insertion of synthetic dsDNA fragments into the N-terminal region between amino acid residues 13 and D4 were constructed using PCR.
  • the plasmid encoding an HBc chimer terminating at V149 was named V47
  • the plasmid encoding an HBc chimer harboring an additional cysteine, C-terminal to V149 was named V54
  • the plasmid encoding an HBc chimer terminating at C183 was named V48
  • V47, V48 and V54 a PCR primer pairs was used to amplify the amino terminus, from the template VI, including sequence preceding the HBc gene .
  • the HBcl83 gene was amplified using a PCR primer pair, resulting in a 574 bp fragment.
  • V47, V48 and V54 plasmids were first digested with Ncol and Sad restriction enzymes. Synthetic dsDNA fragments containing 5' Afllll and 3' Sacl overhangs were then inserted (note, restriction enzymes Afllll and Ncol leave compatible overhangs) .
  • HBc residues D2 and 13 were deleted so that the sequence of the epitope directly follows residue Ml; the glutamic acid-leucine (EL) amino acid pairs, coded for by the Sad restriction site, follows the inserted epitope.
  • the inserted restriction sites are underlined in the oligonucleotide primers below.
  • V7 V7 Cloning Vector
  • Unique EcoRI and Sad restriction sites were inserted between valine-149 and the HindiII site to facilitate directional insertion of synthetic dsDNAs into EcoRI-Hindlll (or EcoRI-SacI) restriction sites.
  • the pair of PCR primers below was used to amplify the HBc 149 gene with a Ncol restriction site at the amino-terminus and EcoRI, Sa and HindiII sites at the carboxyl-terminus .
  • the product of the PCR reaction (479 bp) was digested with Ncol/Hindlll and cloned into pKK223-3N to form V7.
  • V7 The plasmid (V7) was digested EcoRl/Hindlll (or EcoRI-SacI) and synthetic dsDNA fragments having EcoRl/Hindlll (or EcoRl/SacI) overhangs, were ligated into V7.
  • the final amino acid of native HBc (valine-149) and the first amino acid of the inserted T cell epitope are separated by a glycine-isoleucine dipeptide sequence coded for by the nucleotides that form the EcoRI restriction site.
  • V12 vectors which contain B cell epitopes between amino acids 78 and 79, as well as T cell epitopes downstream of valine-149, are constructed from V2 and V7 vectors .
  • the carboxyl terminus of a V7 vector containing a T cell epitope inserted at EcoRl/Hindlll is amplified using two PCR primers (HBc-P79/SacI-F and pKK223-2/4515-32R) to provide a dsDNA fragment corresponding to amino acids 79-149 plus the T cell epitope, flanked with Sad and HindiII restriction sites.
  • the PCR products are cut with Sad and HindiII and then cloned into the desired V2 vector prepared by cutting with the same two enzymes.
  • the PCR primers are amenable for the amplification of the carboxyl terminus of all V7 genes, irrespective of the T cell epitope present after amino acid 149 of the HBc gene .
  • V12 constructs containing the Pf-CS (C17A) mutation were prepared from existing V12 constructs.
  • V12 constructs were amplified with HBcl49/NcoI-F (SEQ ID NO: 203) and the mis-match reverse PCR primer, which facilitated the C17A mutation.
  • the resultant PCR product was digested with Ncol and HindiII and cloned back into pKK223-3N (previously cut with the same enzymes) . Restriction sites are underlined.
  • Synthetic dsDNA fragments coding for the B (V2) or T cell epitope (V7) of interest are inserted into EcoRI/Sad restriction sites.
  • Synthetic dsDNA fragments, encoding B and T cell epitopes of interest are prepared by mixing complementary single stranded DNA oligonucleotides at equimolar concentrations, heating to 95°C for 5 minutes, and then cooling to room temperature at a rate of -1 °C per minute. This annealing reaction is performed in TE buffer.
  • the double-stranded DNAs are shown below with the encoded epitope sequence shown above.
  • the pound symbol, # is used in some of the amino acid residue sequences that follow to indicate the presence of a stop codon.
  • PV-T2A I A N G A G N Q P G A N G A G D Q AATTGCGAACGGCGCCGGTAATCAGCCGGGGGCAAACGGCGCGGGTGATCAAC CGCTTGCCGCGGCCATTAGTCGGCCCCCGTTTGCCGCGCCCACTAGTTG
  • V34 and V55 constructs synthetic dsDNA fragments coding for the M2 epitope (residues 1-24 of the influenza A M2 protein; SEQ ID NO: 9) were inserted into EcoRI/Sad restriction sites, whereas for V47 and V54 constructs, the same were inserted into Ncol/Sacl restriction sites.
  • Synthetic dsDNA fragments were prepared by mixing complementary single stranded DNA oligonucleotides at equimolar concentrations, heating to 95°C for 5 minutes, and then cooling to room temperature at a rate of -1 °C per minute. This annealing reaction was performed in TE buffer. The double-stranded DNAs are shown below with the encoded epitope sequence shown above.
  • Purified particles were diluted to a concentration of 10 ⁇ g/mL in coating buffer (50 mM sodium bicarbonate, pH 9.6) and coated onto the wells of ELISA strips (50 ⁇ L/well) .
  • the ELISA strips were incubated at room temperature overnight (about 18 hours) .
  • the wells were washed with ELISA wash buffer [phosphate buffered saline (PBS) , pH 7.4, 0.05% Tween ® -20] and blocked with 3% BSA in PBS for 1 hour (75 ⁇ L/well) .
  • ELISA strips were stored, dry, at -20°C until needed.
  • IgG anti-mouse
  • HRP horseradish peroxidase conjugate
  • a 24 amino acid . residue synthetic peptide M2 is diluted to a concentration of 2 ⁇ g/mL in coating buffer (50 mM sodium bicarbonate, pH 9.6) and coated onto the wells of ELISA strips (50 ⁇ L/well) . Peptides are dried onto the wells by incubating overnight (about 18 hours) , in a hood with the exhaust on. Next morning, the wells are washed with ELISA wash buffer (phosphate buffered saline, pH 7.4, 0.05% Tween ® -20) and blocked with 3% BSA in PBS (75 ⁇ L/well) for 1 hour. ELISA strips are stored, dry, at -20°C until needed.
  • coating buffer 50 mM sodium bicarbonate, pH 9.6
  • Peptides are dried onto the wells by incubating overnight (about 18 hours) , in a hood with the exhaust on. Next morning, the wells are washed with ELISA wash buffer (phosphate buffered saline, pH 7.
  • antisera monoclonal or polyclonal
  • 1% BSA in PBS 50 ⁇ L/well added to antigen-coated ELISA wells.
  • Sera are incubated for 1 hour, washed with ELISA wash buffer, and probed using an anti-mouse (IgG) -HRP conjugate or other antibody
  • mice are immunized, IP, with 20 ⁇ g of particles in Freund's complete adjuvant, and then boosted at 4 weeks with 10 ⁇ g in Freund's incomplete adjuvant. Mice were bled at 2 , 4, 6, and 8 weeks.
  • the particles were diluted to a concentration of approximately 0.2 mg/mL in 20 mM sodium phosphate buffer, pH 6.8, and absorbance values determined at wavelengths of 260 and 280 nm. The absorbance measured at 280 nm was divided by the value at 260 nm to determine the 280:260 ratio. The ratios were obtained for several samples, including native particles (HBcl83) , HBc particles truncated after residue position 149 (HBcl49) , and several HBc chimers that are identified elsewhere herein, are shown below in Table 1. Full length particles ICC- 1559 are a preparation of the particles first reported in Neirynck et al .
  • full length particles ICC- 1607 are similar particles in which the M2 polypeptide cysteines at polypeptide positions 17 and 19, (X 17 and X_9 of SEQ ID NO: 9) were mutated to serine residues .
  • Purified particles were diluted to a concentration of 1 mg/mL using 50 mM NaP0 4 , pH 6.8 and sodium azide was added to a final concentration of 0.02% to prevent bacterial growth. Samples were mixed with SDS-PAGE sample buffer (reducing) and run on 15% SDS-PAGE gels. Gels were stained using Coomassie Blue, and then analyzed.
  • the particles to be analyzed were diluted to a concentration of 1 mg/mL using 50 mM NaP0 4 , pH
  • IM2HBc A schematic representation of that construct referred to herein as IM2HBc is shown below in which the 24- mer is linked to the N-terminus of HBc.
  • the M2 epitope was inserted into the immunodominant loop of hepatitis B core and particles referred to as ICC- 'l475 were successfully expressed and purified using techniques discussed previously for such insertions and purifications.
  • the ICC-1473 particle construct yielded approximately 7-fold more purified particles when compared with the native sequence (ICC- 1475) . It remains to be determined if the mutation of the cysteine residues alters protective potential of the particles. However, epitopes delivered on the immunodominant loops of HBc are usually significantly more immunogenic as compared to when they are fused to other regions (including the N-terminus) , and resulting particles exhibit reduced anti-HBc immunogenicity .
  • Particles have also been prepared in which the M2 N-terminal 24-mer epitope was fused to the N- terminus of C-terminal truncated hepatitis B core particles. That construct (ICC-1438) also contained the N-terminal pre-core sequence (SEQ ID NO: 27). A similar construct was prepared that contained a single cysteine residue at the end of the hybrid protein (ICC-1492) , in this case immediately after Val-149 of the HBc gene. These constructs are shown schematically below.
  • ICC-1475 (Fig.10, lane 4) and ICC-1473 (Fig.10, lane 5) were expected to have slightly lower molecular weights than ICC-1438 and ICC-1492, because the former two contain the M2 epitope inserted directly into the immunodominant loop and therefore lack the pre-core sequence (SEQ ID NO: 27) present in ICC-1438 and ICC-1498.
  • ICC-1492 was larger than ICC-1475 and ICC-1473; however, ICC-1438, which is identical to ICC-1492 save the C-terminal cysteine residue, is clearly not larger than ICC-1475 and ICC-1473 due to an apparent cleavage.
  • a construct containing a M2 N-terminal extracellular sequence as discussed above linked to the HBc N-terminus (Domain I) or loop (Domain II) and also containing a M2 protein C-terminal sequence such as that of SEQ ID NO: 12 (see Table A) linked the loop (Domain II) or at the C-terminus (Domain IV) of HBc is also contemplated.
  • Such a contemplated construct also contains at least one stabilizing C-terminal cysteine residue as discussed elsewhere herein.
  • a series of synthetic oligonucleotides are synthesized.
  • V2.Pfl(N- M2 (17-24/C17S) the oligonucleotides M2 (17-24/C17S) - Ncol-F and HBcl49/HindIII-R are used to amplify the hybrid HBc gene from vector V2.Pfl.
  • the resultant 546 bp fragment is cleaved with Ncol and HindiII and inserted into pKK-223-3N, which has been cleaved with the same two enzymes .
  • V2. Pf1 N-M2 (17-24/C19S
  • the oligonucleotides M2 (17-24/C19S) -Ncol-F and HBcl49/HindIII-R are used to amplify the hybrid HBc gene in vector V2.Pfl.
  • the resultant 540 bp fragment is cleaved with Ncol and Hindlll and inserted into pKK-223-3N, which had been cleaved with the same two enzymes .
  • HBc chimer molecule-containing particles A series of HBc chimer molecule-containing particles was prepared that contained residues 1-24 of the influenza A, M2 protein peptide-bonded at or near the N-terminus of HBc whose C-terminus was truncated at residue 149.
  • the component chimeric protein molecules contained different N-terminal sequences that included an M2 sequence or variant, and some contained a C-terminal cysteine residue.
  • Example 9 Particles With an M2 or M2 Variant
  • ICC-1603 particles were shown in Fig. 3 to rapidly disassemble following purification.
  • the HBc chimer molecules that comprise ICC-1605 particles are similar to those of ICC-1603 particles, except that the ICC-1605 component chimer molecules have a single C-terminal stabilizing cysteine.
  • a plasmid was made to direct the expression of ICC-1605 particles to investigate if the addition of a C-terminal cysteine residue to ICC-1603 particles could impart greater stability on the particle.
  • ICC-1605 particles were analyzed using analytical size exclusion chromatography (Fig. 6) .
  • ICC-1604 particles To investigate the compatibility of combined amino and carboxyl-terminal cysteine stabilization of hybrid particles, an expression plasmid was constructed to direct the expression of ICC-1604 particles.
  • the component chimer molecules of ICC-1604 particles contain both the two amino-terminal stabilizing cysteine residues present in a native M2 polypeptide sequence (as in ICC-1590) as well as a C- terminal stabilizing cysteine (as in ICC-1605 particles) .
  • Analysis of ICC-1604 particles showed that they retained a homogeneous particulate state following purification (Fig. 7) , indicating that the two stabilizing methods are complementary and can be used in concert with each other.
  • Table 3 shows an alignment that illustrates the configuration of the N-termini of HBeAg, and particles designated ICC-1590, ICC-1560, ICC-1603, ICC-1604 and ICC-1605. Sequences are aligned according to amino acid residue position 4 from the N-terminus of HBc of SEQ ID NO:l that is shared by all constructs. N-terminal cysteine residues, when present, are underlined.
  • Table 4 below, provides a tabulation of the results in which stability was assessed for particles containing an N-terminal influenza A M2 sequence or variant contemplated herein. As is seen, stable particles have been prepared from HBc chimer molecules that contain an N-terminal cysteine residue at a position of minus 14 (-14) relative to the N- terminus of the HBc sequence of SEQ ID NO : 1 to about the N-terminus itself. Table 4
  • Example 10 Yield and Nucleic Acid Binding of M2 -Containing Particles
  • Yields are expressed as milligrams of purified particles from a 500 mL culture. Presence of bound nucleic acid was determined by measuring the A280:A260 ratio of the purified particle. A ratio of more than 1.0 indicates no bound nucleic acid, and a ratio of less than 1.0 indicates the presence of bound nucleic acid.
  • the original full length IM2HBc described by Fiers and colleagues [Neirynck et al . , (1999) Nat . Med. , 5 (10) : 1157-1163] is the same as ICC- 1559. Particle Epitope Insertion C- erminus Bound Yield Site Nucleic (mg/500
  • Example 11 Antigenicity of Various M2 -Containing Particles
  • the antigenicity of the various particles to the monoclonal antibody 14C2 was examined using an ELISA.
  • ELISA plates were first coated with a polyclonal antibody (rabbit) to capture the particles, which were then probed with various dilutions of either the 14C2 monoclonal antibody, or two anti-HBc monoclonal antibodies with specificity for the immunodominant loop region of HBc particles.
  • Particles were formulated on AlhydrogelTM, and groups of 10 mice were immunized with two 10 ⁇ g doses of formulated particles on days 0 and 28. Pooled sera were analyzed 2 weeks after the second injection for anti-HBc and anti-M2e antibody responses using ELISAs. Pooled sera from 10 mice at 2 weeks post boost were analyzed in ELISAs, with M2e (1- 24) synthetic peptide and recombinant HBc (ICC- 1123) serving as the capture antigens.
  • mice IgG2a and IgG2b are the most efficient subclass for fixing complement and binding Fc ⁇ RIII receptors, which are expressed by NK cell [Ravetch et al., (1991) Annu . Rev. Immunol . , 9:457-492], the data suggests an immune mechanism involving CDC and/or ADCC.
  • mice A summary of several studies in which various M2e-HBcAconstructs (10 ⁇ g/mouse) and various adjuvants were assayed. About one-half were i.p. administration and about one-half i.n. For each group (14 mice) the sera were pooled and the titer of anti-M2e IgG subclass antibodies was determined. The results are from sera taken one week after the second boost . For mice where the IgG2a titer was more than 10 4 , the IgGl titer was 10 (*)
  • Adjuvants are increasingly being investigated for their ability to enhance the magnitude and persistence of immune responses to vaccines, as well as modulate the Thl/Th2 bias of the immune response.
  • alum remains the only adjuvant that is a component of FDA-approved vaccines in the US.
  • alum biases immune responses towards a Th2 type, which is manifested by the production of high levels of IgGl antibody in mice.
  • mice in both groups were completely protected from lethal challenge; however, there was an indication of reduced morbidity (temperature decrease and weight loss) in mice immunized with ICC-1569 formulated with AlhydrogelTM + RC529, versus AlhydrogelTM alone.
  • Example 14 Partially Truncated HBc Particles : Synthesis of Expression Vectors for Expressing Partially Truncated Particles
  • a single amino terminal oligonucleotide PCR primer (HBcl49/NcoI-F) was used in combination with a unique C-terminal primer.
  • the primers HBcl49/NcoI-F and HBcl56 (E . cR) -H3-R are used.
  • Primers HBcl49/NcoI-F and HBcl56C (E. cR) -H3-R are used to prepare the HBcl56 (E.cR) +C (ICC-1601 particles) expression plasmids.
  • the sequences of all primers used are displayed below.
  • HBcl56 genes are synthesized first (ICC-1600 and HBcl56+c ICC-1601 particles) , and then used as a template for the HBcl63 constructs (ICC- 1634 and HBcl63+C ICC-1632 particles) ; the HBcl63 constructs are thereafter used as template for the HBcl71 constructs (ICC-1642 and HBC171+C ICC-1643 particles) ; finally, the HBc 171 constructs are used as a templates for the arginine codon optimized HBcl82 and HBcl83 constructs.
  • a non-optimized HBcl82 construct (ICC-1575) is also prepared for control purposes. All PCR products are cleaved with the restriction enzymes Ncol and Hindlll and cloned into the expression vector pKK223-3N, which had been cut with the same enzymes as discussed before.

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Abstract

La présente invention concerne une protéine chimérique de capside nucléique du virus de l'hépatite B (HHc) tronquée à l'extrémité carboxy-terminale, qui est modifiée de façon à renforcer la stabilité des particules auto assemblées et à présenter un épitope de lymphocyte T ou/et de lymphocyte B, telle qu'un polypeptide épitope de lymphocyte B de la protéine M2 du virus de la grippe et qu'un épitope de protéine NP du virus de la grippe. Un épitope immunogène possède une liaison peptidique au niveau d'une ou de plusieurs extrémités N-terminale, dans la boucle immunogène ou au niveau de l'extrémité C-terminale de HBc, une stabilité renforcée des particules auto assemblées étant ainsi obtenues par la présence d'au moins un résidu de cystéine hétérologue près de l'extrémité N-terminale de la molécule chimérique. Cette invention concerne aussi des techniques de fabrication et d'utilisation de ces molécules chimériques.
EP03709214A 2002-02-21 2003-02-21 Particules chimeriques hbc immunogenes stabilisees avec une cysteine d'extremite n-termnale Withdrawn EP1517702A4 (fr)

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US10/080,299 US20030175863A1 (en) 2001-08-15 2002-02-21 Influenza immunogen and vaccine
US274616 2002-10-21
US10/274,616 US20030202982A1 (en) 2001-08-15 2002-10-21 Influenza immunogen and vaccine
PCT/US2003/005196 WO2003102165A2 (fr) 2002-02-21 2003-02-21 Particules chimeriques hbc immunogenes stabilisees avec une cysteine d'extremite n-termnale

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BRPI0714721A2 (pt) 2006-07-14 2013-04-24 Sanofi Pasteur Biologics Co construÇço de vacinas de vÍrus recombinante por inserÇço direta mediada por transpàson de determinantes imunolàgicos estranhos em proteÍnas de vÍrus vetorial
CA2664791A1 (fr) 2006-09-29 2008-08-21 Sanofi Pasteur Biologics Co. Vecteurs rhinoviraux recombinants
EP2185195A2 (fr) 2007-08-16 2010-05-19 Tripep Ab Plate-forme immunogène
CN104080475A (zh) 2008-04-18 2014-10-01 法克斯因内特公司 鞭毛蛋白的缺失突变体以及使用方法
KR101634058B1 (ko) 2008-12-09 2016-06-27 화이자 백신스 엘엘씨 ＩｇＥ ＣＨ３ 펩티드 백신
KR20130127547A (ko) 2009-07-30 2013-11-22 화이자 백신스 엘엘씨 항원성 타우 펩타이드 및 이의 용도
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