WO2003072731A2 - Particules chimeres hbc stabilisees ayant des immunogenes meningoques - Google Patents

Particules chimeres hbc stabilisees ayant des immunogenes meningoques Download PDF

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WO2003072731A2
WO2003072731A2 PCT/US2003/005499 US0305499W WO03072731A2 WO 2003072731 A2 WO2003072731 A2 WO 2003072731A2 US 0305499 W US0305499 W US 0305499W WO 03072731 A2 WO03072731 A2 WO 03072731A2
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hbc
sequence
residues
chimer
residue
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WO2003072731A3 (fr
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Ashley J. Birkett
Birgit Peck
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Apovia, Inc.
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Publication of WO2003072731A3 publication Critical patent/WO2003072731A3/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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/22—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Neisseriaceae (F)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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

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 and the display of an immunogenic Neisseria meningi tidis epitope.
  • HBV hepatitis B virus
  • Meningococcal disease in its various forms is caused by Neisseria meningi tidis, an aerobic, gram-negative diplococcus . Meningococcal disease occurs as both endemic and epidemic disease, and both forms cause substantial illness, and death, as well as persistent neurological defects, mental retardation, learning disability, and particularly deafness. Even when disease is diagnosed early and adequate therapy instituted, the case fatality rate is between 5 percent and 10 percent, and may exceed 50 percent in the absence of treatment. There are two clinical forms of meningococcal disease, meningococcal meningitis and meningococcal septicemia. Meningitis is the inflammation of the tissues which cover the brain and spinal cord.
  • Meningococcal disease is the main cause of bacterial meningitis and the only form of bacterial meningitis that causes epidemics.
  • Meningococcal septicemia is a type of blood poisoning that is caused by Neisseria meningi tidis . It is the more life-threatening form of the disease compared to meningitis .
  • N. meningi tidis The sole natural habitat of N. meningi tidis is human mucosal membrane, primarily the nasopharynx, which it normally colonizes without causing disease. Virulent strains freshly isolated from blood or cerebrospinal fluid are typically encapsulated, whereas throat isolates may or may not be encapsulated. Zollinger in New Generation Vaccines, Levine et al . eds., Marcel De ker, Inc., New York (1997) pp. 469-488.
  • the capsules are composed of anionic polysaccharides, which are the basis of classification of the species into at least 13 serogroups .
  • Meningococci are serologically classified based on the immunological reactivity of their capsular polysaccharide (serogroup) , class 2 or 3 outer membrane protein, (serotype) , class 1 outer membrane protein, (serosubtype) and lipooligosaccharide (immunotype) .
  • serogroup capsular polysaccharide
  • class 2 or 3 outer membrane protein class 2 or 3 outer membrane protein
  • serotype class 1 outer membrane protein
  • lipooligosaccharide immunotype
  • meningococcal vaccines are comprised of different combinations of the purified high-molecular weight capsule polysaccharide (CP) from serogroups A, C, Y, and W-135.
  • CP purified high-molecular weight capsule polysaccharide
  • the group A and group C CP vaccines which have been quite successful, were developed in the late 1960s by Gotschlich et al . , Zollinger in New Generation Vaccines, Levine et al . eds., Marcel Dekker, Inc., New York (1997) pp. 469-488.
  • Purified polysaccharide vaccines against meningococci and other encapsulated bacteria such as Haemophilus influenzae type b (Hib) and pneumococci are poorly immunogenic in young children. Pollard et al. (2000) Pediatr. Infect . Dis . J. , 19:333-344. However, protein-polysaccharide conjugate vaccines for Hib are protective in infants and have greatly reduced the incidence of Hib disease in many countries .
  • the Meningococcal polysaccharide was conjugated to proteins such as tetanus toxoid or CRM 197 to overcome the poor immunogenicity in infants. Conjugation to a protein seems to facilitate T cell help for the immune response and permits sustained and boostable antibody responses even in infants. Pollard et al . (2000) Pediatr. Infect . Dis . J. , 19:333-344.
  • SBA titer In order for a vaccine to be acceptable for licensure, greater than 90 percent of the immunized adult subjects must have a four-fold rise (increase of 2 dilutions) in SBA titer when tested against target strain Al (serogroup A vaccine type strain) or target strain Cll (serogroup C vaccine type strain) by the specified SBA.
  • a fourfold or greater rise in SBA titer is currently used to estimate the potential efficacy of potential vaccines during phase I and phase II field trials, as well as to determine seroconversion after immunization with the currently licensed polysaccharide vaccines. Maslanka et al . (1997) Clin . Diagn . Lab Immunol . , 4:156-167.
  • OMPs bacterial outer membrane proteins
  • Pollard and Frasch (2001) Vaccine 19:1327-1346] provide the following summary of the use of OMPs: class 1 OMP so-called porins A (PorA) [Tommassen et al . (1990) Infect . Immun . , 58:1355-1359] and class 2 or 3 OMP so-called porin B (PorB) [Frasch et al . (1974) J. Exp . Med. 140:87- 104], opacity proteins (Opa and Ope) [Virj i et al . (1993) Mol . Microbiol .
  • transferrin binding proteins Tbps [Rokbi et al . (1993) FEMS Microbiol . Lett . , 110:51-57], ferric enterobactin A (FetA) [van der Ley et al . (1996) Microbiology, 142 (Pt 11) :3269-3274] , ferric binding protein (FbpA) [Gomez et al . (1998) FEMS Immunol . Med . Mi crobiol . , 20:79-86], Meningococcal surface Protein A (NspA) [Martin et al . (1997) J. Exp. Med.
  • GAA GAA
  • LOS lipooligosaccharide
  • OMV outer membrane vesicles
  • a serogroup B meningococcal OMV vaccine in widespread use in Cuba consists of serogroup B/C outer membrane proteins and serogroup C polysaccharide. This vaccine might have prevented epidemic disease in Cuba that was caused by a single strain of meningococci. Pollard et al . (2000) Pediatr. Infect . Dis . J. , 19:333-344; and Sierra et al . (1991) NIPH Ann . 14:195-210.
  • a second approach to express multiple serotype-specific epitopes in a single strain was to insert foreign PorA VR1 (loop 1) or VR2 (loop 4) peptide sequences into loop 5 and 6 of PorA, thereby elongating those loops with additional serosubtype-specific epitopes. van der Ley et al . (1993) Infect . Immun . , 63:4217-4224. When injected into mice as components of outer membrane complexes, these hybrid PorA proteins were effective in inducing bactericidal antibodies against the foreign serosubtype-specific epitopes as well as the original epitopes .
  • PorA the class 1 outer membrane protein, which bears the determinants of serosubtype specificity, has a number of characteristics that recommend it as a vaccine candidate. Zollinger in New Generation Vaccines, Levine et al . eds., Marcel Dekker, Inc., New York (1997) pp. 469-488. Among these characteristics are its capacity to induce bactericidal antibodies that are protective in animal models [Saukkonen et al . (1987) Microb . Pathog. , 3:261-267; and Saukkonen et al . (1989) Vaccine, 7:325-328], its relatively stable expression, and its moderate degree of antigenic variation [Frasch et al . (1985) .Rev. Infect . Dis . , 7:504-510].
  • the structural gene was initially cloned and sequenced by Barlow et al . (1989) Mol . Microbiol . , 3:131-139. The gene has subsequently been amplified from many different serosubtypes using the polymerase chain reaction, and the entire gene, or the variable regions, were sequenced. McGuinness et al. (1993) Mol . Microbiol . , 7:505-514. Based on comparative DNA sequences of PorA from a number of different serosubtypes, two principle variable regions, VR1 and VR2 , have been identified. A database containing these variable regions is accessible in the Internet at "outbreak. candd.ox. ac .uk/PorA-VR" .
  • PorA In an effort to develop a vaccine based on purified PorA, a system for the production of large amounts of pure, endotoxin-free PorA was developed by expressing the cloned gene as inclusion bodies in the gram-positive bacterium Bacillus subtilis . PorA could be quite easily isolated and purified, but solubilization of the inclusion bodies required denaturing conditions. The resultant purified PorA was found to be able to induce a good, relatively cross-reactive, antibody response in animals, but the antibodies were devoid of bactericidal activity. Zollinger in New Generation Vaccines, Levine et al . eds., Marcel Dekker, Inc., New York (1997) pp. 469- 488.
  • NspA was not expressed by one-third of a wide selection of meningococci.
  • Variable susceptibility to anti-sera was found in those bacteria on which it was expressed.
  • the NspA protein is a promising candidate for inclusion in a Meningococcal vaccine to increase cross-protection, but it is unlikely to be useful as a single component vaccine.
  • Humoral immunity appears to be central to natural and vaccine-induced protection against N. meningi tidis, because disease occurs in those who lack serum bactericidal antibody. In the context of induction of protective antibodies, cellular immune response to Meningococcal antigens is of interest and importance. Pollard et al . (2001) Vaccine 19:1327- 1346.
  • Adult volunteers have lymphoproliterative responses to Opa, Ope and PorA proteins of the bacterial outer membrane [Wiertz et al . (1996) Infect . Immun . , 64:298-304; Wiertz et al . (1991) J. Immunol . , 2012-2018; and Wiertz et al . (1992) J. Exp. Med.
  • 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 self-assemble into stable aggregates known as hepatitis B core protein particles (HBc particles) . Two three-dimensional structures are reported for HBc particles. A first that comprises a minor population contains 90 copies of the HBc subunit protein as dimers or 180 individual monomeric proteins, and a second, major population that contains 120 copies of the HBc subunit protein as dimers or 240 individual monomeric proteins.
  • These human HBc particles are about are about 30 or 34 nm in diameter, respectively.
  • 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 via an arginine-rich domain at the carboxyl-terminus (C-terminus) .
  • C-terminus carboxyl-terminus
  • HBcAg is a particulate protein derived from the core or nucleocapsid of hepatitis B virus that has been proposed as a carrier for heterologous epitopes.
  • the protein (HBc) 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.
  • HBsAg hepatitis B surface protein
  • HbcAg HbcAg
  • 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 does not require complex adjuvants for efficacy, although such adjuvants can be used. 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 [Schodel et al . , J. Exp . Med. ,
  • HBV nucleocapsids In an application as a vaccine carrier moiety, it is preferable that 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 viruslike 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 [Seifer et al . (1995) Intervirology, 38:47-62] .
  • HBc chimeric particles or HBc chimers Recombinantly-produced hybrid particles bearing internal insertions
  • HBc chimeric particles or HBc chimers Recombinantly-produced hybrid particles bearing internal insertions
  • HBc chimers Recombinantly-produced hybrid particles bearing internal insertions
  • HBc chimers Recombinantly-produced hybrid particles bearing internal insertions
  • 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] .
  • Chimeric hepatitis B core particles 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, other than the present inventors .
  • HBc chimers often appear to have a less ordered structure, when analyzed by electron microscopy, compared to particles that lack heterologous epitopes [Schodel 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 a contemplated HBc chimer that provides high titers of antibodies against Neisseria meningi tidis serogroup B and C, 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 minimal, if any, antigenicity toward preexisting anti-HBc antibodies.
  • the present invention contemplates a chimeric immunogen for inducing antibodies to Neisseria meningitidis serogroup B, and a vaccine comprising that immunogen dispersed in a physiologically tolerable diluent.
  • a vaccine preferably contains a cross-protective immunogen that is independent of the serogroup.
  • Epitopes of the PorA protein fulfill that criterion in that that protein is present within the serosubtype and particular protective epitopes are known.
  • NspA is a conserved outer membrane protein that also fulfills the criterion of independence of serogroup and surface-exposed epitopes are characterized.
  • PorA shows some variability and at least seven different PorA B cell epitopes are preferably combined to prevent infections by serogroup B strains.
  • a consensus sequence corresponding to the relatively conserved loop 5 of Por A is the epitope expressed in chimeric HBcAg.
  • a contemplated immunogen is a recombinant chimer hepatitis B core (HBc) protein molecule up to about 230 amino acid residues in length.
  • That recombinant chimer (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 4 through about 75 and about 85 through about 140, and (ii) a peptide-bonded heterologous immunogenic sequence of about 10 to about 25 residues that constitute a B cell epitope of a variable or conserved region of the PorA protein or the NspA protein of Neisseria meningi tidis serogroup B at one or more of the N-terminus, in the HBc immunodominant loop (HBc positions about 176-185) or the C-terminus of the chimer.
  • Meningococcal B cell epitopes are discussed hereinafter.
  • That recombinant chimer also (b) contains one or both of (i) 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 (ii) one 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) ] .
  • the chimer molecule further (i) contains no more than 10 percent conservatively substituted amino acid residues in the HBc sequence, (ii) an optionally present sequence of up to about 25 amino acid residues that define a Meningococcal T cell epitope such as a sequence from the PorA protein, from the PorB protein or from a class five protein such as the OpaB protein, the Opa5d protein or the Ope protein are also preferably present peptide-bonded between HBc position 140 and the C-terminus of the recombinant chimer molecule, and (iii) self-assembles into particles that are substantially free of binding to nucleic acids on expression in a host cell .
  • particles are more stable than are particles formed from otherwise identical HBc chimer molecules that are free of one or both of the N- and C-terminal cysteine residue (s) present or in which one or both of the N- or C-terminal cysteine residue (s) present in a contemplated chimer molecule is (are) replaced by another residue.
  • the particles are substantially free of bound nucleic acids.
  • HBc hepatitis B virus core
  • the first domain, Domain I comprises about 71 to about 100 amino acid residues whose sequence includes at least the sequence of the residues of position 5 through position 75 of HBc. Domain I can optionally also include 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.
  • the second domain, Domain II comprises about 10 to about 35 amino acid residues peptide- bonded to HBc residue 75 of Domain I and residue 86 of Domain III.
  • Those about 10 to about 35 residues include a sequence of about 10 to about 25 residues that constitute a B cell epitope of a variable or conserved region of a protein of Neisseria meningi tidis such as the PorA protein or the NspA protein, and preferably also includes an HBc sequence of at least 4 residues, and more preferably at least 8 residues, and more preferably still all 10 HBc residues.
  • the Meningococcal epitope is preferably peptide-bonded between the HBc residues of positions 78 and 79.
  • 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 contains a sequence of at least five amino acid residues from HBc residue position 136 through position 140 peptide-bonded to the residue of position 135 of Domain III, as well as an optionally present one to three cysteine residues toward the C-terminus of the molecule from the C-terminal residue of the HBc sequence present and within about 30 residues from the C-terminus of the chimer molecule [C-terminal cysteine residue (s) ] .
  • HBc positions 141 through 149 can be present along with the cysteine.
  • Up to about 25 amino acid residues in a sequence heterologous to HBc from position 150 to the HBc C-terminus (e.g., HBc position 183) that define a Meningococcal T cell epitope such as from the PorA protein, from the PorB protein, from the OpaB protein, or from the Opa5d protein, are also preferably present peptide-bonded between the at least five HBc residues and the cysteine or C-terminal to the cysteine, along with fewer than three arginine or lysine residues, or mixtures thereof adjacent to each other.
  • a contemplated chimer molecule is substantially free of bound nucleic acids.
  • the immunogen is in the form of self-assembled particles. It is also preferred that the HBc sequence of Domain I include the residues of position 1 through position 75 with no additional residues at the N-terminus. It is further preferred that a contemplated immunogen contain one cysteine residue within Domain IV in an amino acid residue sequence heterologous to that of HBc from position 150 to the C-terminus. It is particularly preferred that that heterologous sequence comprise a T cell epitope from Neisseria meningi tidis , but the T cell epitope need not be from the same protein as the B cell epitope.
  • a mixture of particles be present that contain a plurality of different Meningococcal B cell epitope Domain II sequences or consensus sequences, and preferably, at least seven different Meningococcal B cell epitope sequences are present. More preferably, at least seven different particles are present, each containing a different, single Meningococcal B cell epitope sequence.
  • Another embodiment comprises an inoculum or vaccine that comprises the above HBc chimer particles dissolved or dispersed in a pharmaceutically acceptable diluent composition that typically also contains water.
  • an inoculum When administered in an immunogenic effective amount to a mammal, an inoculum induces antibodies that immunoreact specifically with the chimer particle. The antibodies so induced also preferably immunoreact specifically with (bind to) the Neisseria meningi tidis serogroup B protein of the Domain II B cell epitope protein.
  • a vaccine or inoculum preferably contains a plurality of particles that contain a single Domain II sequence or consensus sequence that differs from the other Domain II sequences, and preferably, at least seven different Meningococcal B cell epitope sequences are present . Again more preferably, at least seven different particles are present, each containing a different, single Meningococcal B cell epitope sequence
  • the present invention has several benefits and advantages .
  • a particular benefit of the invention is that its use as a vaccine provides high antibody titers against the B cell epitopes of Neisseria meningi tidis serogroup B.
  • antibodies that recognize serogroup B also recognize Neisseria meningitidis serogroup C.
  • An advantage of the invention is that those very high antibody titers can be produced with the aid of an adjuvant approved for use in humans.
  • Another benefit of the invention is that the recombinant immunogen is prepared easily and using well known cell culture techniques.
  • 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. 1 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 N0:1), 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: 8) is shown below the sequence of the commercially available vector (SEQ ID NO: 7) .
  • 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 shown in three panels as Figs. 3A, 3B and 3C, schematically illustrates a preferred cloning strategy in which a Meningococcal B cell epitope such as PorA-1 (SEQ ID NO: 9) is cloned into the EcoRI and Sad sites of an engineered HBc gene (Fig. 3A; SEQ ID NO: 149, 89 and 90) between positions 78 and 79, which can destroy the EcoRI site, while preserving the Sad site .
  • Fig. 3A Meningococcal B cell epitope
  • Fig. 3A SEQ ID NO: 149, 89 and 90
  • 3B shows DNA that encodes a T cell epitope such as that referred to as PorB-1 that includes a carboxy-terminal cysteine residue (PorB-Tl+C; SEQ ID NO: 49) and a stop codon cloned into the EcoRI and HindiII sites (SEQ ID NO: 149, 150 and 151) at the C-terminus of an engineered, truncated HBc gene containing the first 149 HBc residues (HBcl49) .
  • FIG. 3C 3A with Sad, followed by ligation of the appropriate portions is shown in Fig. 3C to form a single gene construct that encodes a B cell- and a T cell-containing immunogen for a vaccine against Neisseria meningi tidis .
  • the two engineered residues present on either side of the PorA-1 sequence are also shown in Fig. 3C.
  • HBcl49 indicates that the chimer ends at residue 149
  • HBcl49 + C150 indicates that that same chimer contains a cysteine residue at HBc position 150.
  • 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 NO:l) .
  • 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.
  • a contemplated immunogen is a recombinant chimer hepatitis B core (HBc) protein molecule up to about 230 amino acid residues in length. That recombinant chimer (a) contains an HBc sequence of at least about 125 of the N-terminal 150 amino acid residues of the HBc molecule that includes
  • the HBc sequence of residue positions 4 through about 75 and about 85 through about 140 and (ii) a peptide-bonded immunogenic sequence of about 10 to about 25 residues that constitute a B cell epitope of a variable or conserved region of the PorA protein or the NspA protein of Neisseria meningi tidis serogroup B at one or more of the N-terminus, in the HBc immunodominant loop or the C-terminus of the chimer.
  • That recombinant chimer also (b) contains one or both of (i) 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 (ii) one 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
  • the chimer molecule further (i) contains no more than 10 percent conservatively substituted amino acid residues in the HBc sequence, (ii) an optionally present sequence of up to about 25 amino acid residues that define a Meningococcal T cell epitope such as a sequence from the PorA protein, from the PorB protein or from a class five protein such as the OpaB protein, the Opa5d protein or the Ope protein are also preferably present peptide-bonded between HBc position 140 and the C-terminus of the recombinant chimer molecule, and (iii) self-assembles into particles that are substantially free of binding to nucleic acids on expression in a host cell.
  • an optionally present sequence of up to about 25 amino acid residues that define a Meningococcal T cell epitope such as a sequence from the PorA protein, from the PorB protein or from a class five protein such as the OpaB protein, the Opa5d protein or the Ope protein are also preferably present peptide-
  • Those particles are more stable than are particles formed from otherwise identical HBc chimer molecules that are free of one or both of the N- and C-terminal cysteine residue (s) present or in which one or both of the N- or C-terminal cysteine residue (s) present in a contemplated chimer molecule is (are) replaced by another residue.
  • the particles are substantially free of bound nucleic acids. Determination of chimer molecule stability and substantial freedom from bound nucleic acids are discussed hereinafter.
  • a contemplated chimer molecule has a length of up to about 230 peptide-bonded ⁇ -amino acid residues, and more preferably up to about 205 residues. Most preferably, a contemplated chimer contains about 180 to about 205 amino acid residues. Such a contemplated chimer molecule contains at least about 125, and more preferably at least about 135, of the N-terminal 150 amino acid residues of HBc.
  • the HBc sequence present includes the HBc sequence of residue positions 4 through about 75 and about 85 through about 140.
  • the chimer contains a peptide- bonded immunogenic sequence of about 10 to about 25 residues that constitute a B cell epitope of a variable or conserved region of the PorA protein or the NspA protein of Neisseria meningi tidis serogroup B at one or more of the N-terminus, in the HBc immunodominant loop (i.e., between residue positions 76 through 85) or the C-terminus of the chimer.
  • Preferred Meningococcal B cell epitope sequences are discussed hereinafter.
  • the immunogenic loop can contain (i) zero to all residues in a sequence of HBc positions 76 through 85 present and peptide-bonded to ten to about 25 amino acid residues that are heterologous to HBc and constitute a Meningococcal B cell epitope.
  • the Meningococcal B cell epitope sequence can be peptide- bonded to one of the first four N-terminal residues of HBc can contain a sequence of up to about 25 residues that are heterologous to HBc, with the loop region containing all or none of the residues from position 76 through 85 and the C-terminal region having a HBc sequence from position 140 through 149.
  • the Meningococcal B cell epitope sequence can be bonded to a residue of HBc position 140 through 149, with the N-terminal remaining chimer sequence containing all or none of HBc residues 1-4 and all or none of HBc residues 176- 185.
  • a contemplated chimer molecule can contain Meningococcal B cell epitopes fused (bonded) at two or more of the N-terminus, loop and C-terminus.
  • the Meningococcal B cell epitope can be present peptide-bonded in a chimer molecule at both of the N- and C-termini, in the loop and at the C-terminus or at the N-terminus and in the loop.
  • a B cell epitope can also be present at all three locations.
  • a Meningococcal B cell epitope is present at more than one location in the chimer sequence, the same or a different sequence can be used for each epitope.
  • a single Meningococcal B cell epitope is present and it is present in the HBc immunogenic loop between about HBc residues 176 and 185.
  • a contemplated recombinant chimer also (b) contains one or both of (i) 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 (ii) one 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) ] .
  • 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 upstream 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 : 1.
  • 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 at position -7 of the pre-core sequence, which is present when the core gene is translated from an upstream initiator methionine at position -30. That cysteine residue is responsible for preventing particle formation and therefore facilitating the transition from particulate HBcAg to secreted, non-particulate HBeAg.
  • the HBeAg 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 of a contemplated chimer molecule .
  • C-terminal one to three cysteine residues A discussion of location of the C-terminal one to three cysteine residues follows hereinafter in relation to another embodiment of the invention. It is preferred that only one of the N- or C-terminal one to three cysteine residues be present. It is more preferred that one rather than two or three cysteines be present and that that present cysteine residue be a C-terminal cysteine. Recombinant chimer HBc protein molecules having a single stabilizing C-terminal cysteine residue are used illustratively herein.
  • a preferred immunogen is a recombinant hepatitis B virus core (HBc) protein chimer molecule with a length of about 135 to about 215, and preferably about 145 to about 190 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
  • Domain I comprises about 71 to about 100 amino acid residues whose sequence includes at least the sequence of the residues of position 5 through position 75 of HBc. Domain I can optionally also include 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.
  • Domain II comprises a sequence of about 10 to about 35 amino acid residues peptide-bonded to residue 75.
  • This sequence preferably includes a sequence of HBc of at least 4 residues, and more preferably at least 8 residues, and more preferably still includes all ten residues, from HBc positions 76 through 85 peptide-bonded to a sequence of about 10 to about 25 residues that constitute a B cell epitope of a protein of Neisseria meningi tidis serogroup B such as the PorA protein or the NspA protein.
  • the Meningococcal epitope is preferably peptide-bonded between the HBc residues of positions 78 and 79.
  • the Meningococcal epitope is more preferably about 10 to about 25 residues in length, and most preferably about 15 to about 20 residues in length. It is to be understood that a contemplated Meningococcal epitope can also be present in a longer sequence .
  • Domain III is an HBc sequence from position 86 through position 135 that is peptide- bonded to residue 85.
  • Domain IV comprises an HBc sequence of at least five amino acid residues from HBc residue position 136 through position 140 peptide-bonded to the residue of position 135 of Domain III, as well as one to three cysteine residues toward the C-terminus of the molecule from the C-terminal residue of the HBc sequence present and within about 30 residues from the C-terminus of the chimer molecule [C-terminal cysteine residue (s) ] .
  • a contemplated chimer molecule is substantially free of bound nucleic acids. Zero to nine residues of the sequence of HBc positions 141 through 149 can also be present along with the required cysteine and the residues of positions 136 through 140.
  • a sequence heterologous to HBc from position 150 to the HBc C-terminus that define a Meningococcal T cell epitope
  • a Meningococcal T cell epitope such as a sequence from the PorA protein, from the PorB protein or from a class five protein such as the OpaB protein, the Opa5d protein or the Ope protein are also preferably present peptide- bonded between the at least five HBc residue sequence and the cysteine or C-terminal to the cysteine, along with fewer than three arginine or lysine residues, or mixtures thereof adjacent to each other.
  • such a recombinant protein can have a length of about 135 to about 215 amino acid residues. Preferably, that length is about 150 to about 195 residues. More preferably, the length is about 170 to about 190 residues. Most preferably, the length is about 175 to about 190 residues. These differences in length arise from changes in the length of Domains I, II and IV. 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 .
  • An N-terminal sequence peptide-bonded to one of the first five N-terminal residues of HBc can contain a sequence of up to about 25 residues that are heterologous to HBc .
  • Exemplary sequences include a B cell or T cell epitope such as those discussed hereinafter, 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-SI or Pre-S2 regions or the major HbsAg immunogenic sequenc .
  • the N-terminal region of a chimer molecule can also include one to three cysteine residues at a position in the HBc sequence corresponding to amino acid position -20 to about +1 from the N-terminus of the HBc sequence of SEQ ID NO : 1. When used at the N-terminus, it is preferred that only one cysteine residue be present . It is also preferred that that cysteine be located at about residue -5 to about HBc position +1.
  • Domain I preferably has the sequence of residues of positions 1 through 75 of HBc, and is free of added residues at the amino-terminus (N-terminus) . Domain I is also therefore preferably free of deletions of residues of positions 1-3.
  • Domain II which is peptide-bonded to residue 75, contains the sequence of HBc residues of positions 76 through 85, and has a Neisseria meningi tidis B cell epitope whose length is about 10 to about 25 residues peptide-bonded between residues 76 and 86.
  • This region, the immunodominant loop of HBV also preferably contains a sequence of at least 4 residues, more preferably at least eight residues, and most preferably all ten residues of the HBV core sequence from positions 76 through 85 peptide-bonded to the sequence that defines the Neisseria meningi tidis B cell epitope.
  • Those at least four residues can be on either side or both sides of the inserted Neisseria meningi tidis B cell epitope, and when present continue the HBV sequence to which the residues are bonded.
  • the first two of those residues are those of HBV positions 76 and 77, whereas the other two are those of HBV positions 84 and 85.
  • the inserted Neisseria meningi tidis B cell epitope is positioned between HBV residues 78 and 79, with all ten residues of the HBV sequence from position 76 through position 85 being present .
  • Neisseria meningi tidis B cell epitopes for insertion in Domain II of a recombinant HBc chimer are enumerated in Table A, below, as consensus sequences using single letter code for amino acid residues and dashes to indicate absent residues.
  • An individual, underlying sequence other than a consensus sequence can also be used.
  • the underlying PorA sequences were obtained from the Neisseria meningi tidis PorA variable region database at "outbreak.
  • 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 Karpentko et al . , having the sequence GSGDEGG of SEQ ID NO: 152.
  • Neisseria meningi tidis B cell epitope peptide sequences that include a C-terminal short peptide are listed with a plus sign "+” and a letter “S” after the numeral designating the peptide sequence, such as PorA-4+S.
  • the immunogenic sequences of PorA-1 through PorA-22 are from the hypervariable region of the PorA protein. Data discussed hereinafter indicate that sequences from the conserved epitopes of that protein such as those of PorA-43, PorA-44, PorA-50 and PorA- 51 are similarly immunogenic. Additionally, the loop V region of the protein exemplified by the PorA-46 sequence was also shown to be immunogenic, and that immunogenicity was found to be on the order of two logs greater based on anti-peptide IgG titer studies in mice.
  • Domain III contains the sequence of HBc position 86 through position 135 peptide-bonded at its N-terminus to residue 85.
  • Domain IV comprises (i) at least five residues of a HBc amino acid residue sequence from position 136 through 140 peptide-bonded to the residue of position 135 of Domain III, (ii) zero to nine residues in the sequence of HBc from position 141 through 149, (iii) one to three cysteine residues, (iv) fewer than three arginine or lysine residues, or mixtures thereof adjacent to each other, and (v) up to about 25 amino acid residues in a sequence heterologous to HBc from position 150 to the HBc C-terminus at position 183 of the human ayw sequence shown in Fig. 1 and SEQ ID NO:l, with certain provisos.
  • a contemplated chimer molecule is substantially free of bound nucleic acids.
  • the heterologous up to about 25 amino acid residues typically constitute one or more Meningococcal T cell epitopes.
  • the inclusion of a Meningococcal T (Th) cell epitope can help ensure that, should a vaccine recipient be exposed to Neisseria meningi tidis, a more rapid and stronger anti- Meningococcal response is activated ' due to previous priming of Meningococcal - specific T-helper cells.
  • Domain IV can contain up to about 25 residues that are heterologous to HBc from position 150 through the C-terminus, this domain needs no residues in addition to those recited before (residues of positions 136-140 plus the cysteine) to provide an effective immunogen.
  • Domain IV ends at least at HBc residue 140 and contains at least 5 amino acid residues of the HBc sequence from position 136 through 140, plus at least one C-terminal cysteine residue.
  • 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 can continue through position 149.
  • residue of position 148 is present, so is the sequence of residues of positions 136 through 147, or if residue 141 is present, so is the sequence of residues of positions 136 through 140.
  • Domain IV comprises a sequence of HBc from residue 136 through 140 peptide- bonded to the residue of position 135 of Domain III.
  • the remainder of Domain IV contains (i) zero to nine residues of a HBc amino acid residue sequence from position 141 through 149 peptide-bonded to the position 136-140 sequence, (ii) one to three cysteine residues, (iii) fewer than three arginine or lysine residues, or mixtures thereof adjacent to each other, and (iv) up to about 25 residues, in a sequence that constitutes a T cell epitope of Neisseria peptide- bonded to the final HBc amino acid residue present in the chimer or a cysteine residue.
  • That Meningococcal T cell epitope can be from the same or different protein as the Meningococcal B cell epitope present in Domains I or II.
  • the T cell epitope can be bonded to the carboxy-terminal-most HBc residue such as residue 149, or to a cysteine residue that is bonded to that final HBc residue.
  • Domain IV also contains one to three cysteine residues and those Cys residues are present within about 30 residues of the carboxy-terminus (C-terminus) of the recombinant chimer molecule.
  • one cysteine (Cys) residue is present, and that Cys is preferably present as the carboxy- terminal (C-terminal) residue, unless a Neisseria (Meningococcal) T cell epitope containing a Cys residue is present as part of Domain IV.
  • the preferred Cys is preferably within the C-terminal last five residues of the HBc chimer. Preferred Meningococcal T cell epitopes are discussed hereinafter.
  • a preferred HBc chimer immunogen tends to be stable to decomposition at 37°C to a greater extent than does a similar chimer lacking the cysteine (s) residue added at one or both of the N- and C-termini.
  • Domain IV contains fewer than three arginine or lysine residues, or mixtures thereof adjacent to each other.
  • Arginines and lysines are present in the C-terminal region of HBc that extends from position 150 through the C-terminus of the native molecule (e.g. position 183 of ayw) . That region is sometimes referred to in the art as the "protamine” or "arginine-rich” region of the molecule and is thought to bind to 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.
  • particles free of the arginine- rich nucleic acid binding region of naturally occurring HBc such as those that contain fewer than three arginine or lysine residues or mixtures thereof adjacent to each other, or those having a native or chimeric sequence that ends at about HBc residue position 140 to position 149, are substantially free of bound nucleic acids and exhibit a 280:260 absorbance ratio of about 1.2 to about 1.6.
  • 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 1.6, and more typically, about 1.4 to about 1.6. 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 chimer HBc particles are 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 I, II and III sequences and an otherwise same Domain IV sequence in which the one to three cysteine residues [C-terminal cysteine residue (s) ] are absent or a single C-terminal residue present is replaced by another residue such as an alanine residue. Stability of various chimer particles is determined as discussed hereinafter.
  • particles containing a heterologous Meningococcal epitope in Domain II and a single cysteine residue C-terminal to residue valine 149 is more stable than otherwise identical particles assembled from chimer molecules whose C-terminal residue is valine 149.
  • particles containing the above Meningococcal B cell epitope in Domain II and Meningococcal T cell epitope (discussed hereinafter) that contains a naturally occurring single cysteine near the C-terminus or an added cysteine at the terminus or elsewhere are more stable than are otherwise identical particles in which that cysteine is replaced by an alanine residue.
  • a contemplated particle containing a C-terminal cysteine residue is also typically prepared in greater yield than is a particle assembled from a chimer molecule lacking a C-terminal cysteine. This increase in yield can be seen from the mass of particles obtained or from integration of traces from analytical gel filtration analysis using
  • HBc does not activate
  • Neisseria-specific T cells except in restricted individuals for whom the B cell epitope is also a T cell epitope.
  • one or more eisseria-specific T helper epitopes is preferably incorporated into a contemplated immunogen and is located in Domain IV of the immunogen .
  • Particularly preferred T cell epitopes present as a part of Domain IV are enumerated in Table B, below, with added C-terminal Cys residues indicated in bold.
  • 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 25 residues in a sequence heterologous to HBc. Most preferably, that sequence is up to about 20 residues and includes one of the T cell epitopes shown in Table B, above.
  • 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 chimer molecule contains a heterologous epitope in Domain II
  • epitope be 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.
  • a preferred contemplated HBc chimer molecule contains a sequence of about 145 to about 215 residues.
  • a preferred HBc chimer molecule containing two heterologous epitopes of preferred lengths of about 15 to about 25 residues each and a preferred HBc portion length of about 140 to about 149 residues have a sequence length of about 170 to about 190 amino acid residues.
  • Particularly preferred chimer molecules continuing two heterologous epitopes have a length of about 175 to about 190 residues. It is to be understood that a wide range of 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 .
  • 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 the individual molecules are therefore thought to be bound together into the particle primarily by 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.
  • the amino acid sequence of HBc from residue position 1 through at least position 140 is preferably present in a contemplated chimer molecule and particle.
  • the sequence from position 1 through position 149 is more preferably present.
  • a Meningococcal B cell epitope is present between residues 78 and 79 and a single cysteine residue or a Meningococcal T cell epitope containing a native or added cysteine residue is preferably present as a C- terminal addition to the HBc sequence as part of Domain IV.
  • a contemplated recombinant HBc chimer is substantially free of bound nucleic acid.
  • a preferred chimer particle contains an added Cys residue at or near the C-terminus of the molecule and is more stable at 37°C than is a similar particle that does not contain that added Cys .
  • 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 .
  • 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 can be found using computer programs well known in the art, for example LASERGENE software (DNASTAR Inc., Madison, Wis.)
  • the HBc portion of a chimer molecule of the present invention [the portion having the HBc sequence that has other than a sequence of an added epitope, or heterologous residue (s) that are a restriction enzyme artifact] most preferably has the amino acid residue sequence at positions 1 through 149 of subtype ayw that is shown in Fig. 1 (SEQ ID NO:l), when present. Somewhat less preferred are the corresponding amino acid residue sequences of subtypes adw, adw2 and adyw that are also shown in Fig. 1 (SEQ ID NOs : 2 , 3 and 4) . Less preferred still are the sequences of woodchuck and ground squirrel at aligned positions 1 through 149 that are the last two sequences of Fig. 1 (SEQ ID NOs : 5 and 6) . As noted elsewhere, portions of different sequences from different mammalian HBc proteins can be used together in a single chimer.
  • HBc portion of a chimer molecule of the present invention has other than a sequence of a mammalian HBc molecule at positions 1 through 149, when present, because one or more conservative substitutions has been made, it is preferred that no more than 10 percent, and more preferably no more than 5 percent, and most preferably no more than 3 percent of the amino acid residues are substituted as compared to SEQ ID NO : 1 from position 1 through 149.
  • a contemplated chimer of 149 HBc residues can therefore contain up to about 15 residues that are different from those of SEQ ID NO:l at positions 1 through 149, and preferably about 7 or 8 residues.
  • 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.
  • 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 Meningococcal B cell epitope-containing sequence is typically 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 Meningococcal B cell epitope in an extremely immunogenic position in the assembled chimer particles .
  • One of two well-known strategies is particularly useful for placing the Meningococcal B cell sequence into the loop sequence at the desired location.
  • 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 a Meningococcal B cell sequence.
  • the second strategy is referred to as insertion in which a Meningococcal 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 Sad, one near each end of the immunodominant loop-encoding DNA.
  • Exemplary residues replaced are 76 through 81.
  • the loop-encoding section is excised, a desired Meningococcal 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 “blunt” 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) : 114-119 and Schodel et al . (1994) J “ . Exp . Med. , 180 (3) : 1037-1044, the latter two papers discussing the preparation of vaccines against P. yoelii and P. berghei , respectively.
  • 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 Meningococcal 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 Sad 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 Sad 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 Meningococcal B cell epitope PorA-1, SEQ ID NO: 9, see Table A 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.
  • Fig. 3B A similar strategy is shown in Fig. 3B for insertion of a cysteine-containing sequence, such as a preferred T cell epitope.
  • a cysteine-containing sequence such as a preferred T cell epitope.
  • EcoRI and HindiII restriction sites are engineered in to the HBc DNA sequence after amino acid residue position 149.
  • 3B with Sad 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 Meningococcal B cell epitope, two added residues, HBc positions 79-149, two added residues, and the Meningococcal T cell epitope that is shown in Fig. 3C.
  • One or both ends of the insert and HBc nucleic acid can be "chewed back" with an appropriate nuclease (e.g. SI nuclease) to provide blunt ends that can be ligated together.
  • an appropriate nuclease e.g. SI nuclease
  • Added heterologous residues that are neither part of the inserted B cell or T cell epitopes nor a part of the HBc sequence are not counted in the number of residues present in a recited Domain.
  • 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 140, when present, is that of SEQ ID NOs:l, 2, 3, 4, 5 or 6 , and (2) hybridizes with a DNA sequence of SEQ ID NOs : 75, 76, 77, 78, 79 or 80 at least at 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: 75, 76, 77, 78, 79 and 80, is defined as a DNA variant sequence.
  • a 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.
  • 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 140 shown in SEQ ID NOs:l, 2, 3, 4, 5 or 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: 75, 76, 77, 78, 79 or 80, and hybridizes with the nucleic acid sequence of SEQ ID NOs: 75, 76, 77, 78,79 or 80 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: 75, 76, 77, 78, 79 or 80 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: 75, 76, 77, 78, 79 or 80 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:l, 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.
  • nucleic acid illustratively DNA sequence
  • 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 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.
  • any desired modifications can be made simply by substituting the appropriate bases for those encoding the native amino acid residue sequence.
  • 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 vitro, systems is also contemplated.
  • These 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.
  • the invention is not limited by the host cell employed.
  • 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 pKK223-3 plasmid can be successfully expressed in a number of E.
  • coli strains such as XL-1, TBl, 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 .
  • Expression vectors compatible with eukaryotic cells are also contemplated herein. Such expression vectors can also be used to form the recombinant DNA molecules of the present invention.
  • Vectors for use in yeasts such as S. cerivisiae or Pichia pastoris can be episomal or integrating, as is well known.
  • Eukaryotic cell expression vectors are well known in the art and are available from several commercial sources. Normally, such vectors contain one or more convenient restriction sites for insertion of the desired DNA segment and promoter sequences. Optionally, such vectors contain a selectable marker specific for use in eukaryotic cells.
  • Exemplary promoters for use in S. cerevisiae include the S.
  • PGK cerevisiae phosphoglyceric acid kinase
  • AOX1 alcohol oxidase gene
  • a gene that encodes a desired chimer is placed under the control of regulatory sequences that direct expression of structural genes in Pichia .
  • the resultant expression-competent forms of those genes are introduced into Pichia cells.
  • a gene for a chimer is placed downstream from the alcohol oxidase gene (AOX1) promoter and upstream from the transcription terminator sequence of the same AOX1 gene.
  • AOX1 alcohol oxidase gene
  • the gene and its flanking regulatory regions are then introduced into a plasmid that carries both the P. pastoris HIS4 gene and a P. pastoris ARS sequence (Autonomously Replicating Sequence) , which permit plasmid replication within P. pastoris cells [Cregg et al . (1987) Molecular and Cellular Biology, 12:3376-3385] .
  • the vector also contains appropriate portions of a plasmid such as pBR322 to permit growth of the plasmid in E. coli cells.
  • a plasmid such as pBR322
  • chimer gene can also be introduced by integrative transformation, which does not require the use of an ARS sequence, as described by Cregg et al . (1987) Molecular and Cellular Biology, 12:3376-3385.
  • chimer particles by recombinant DNA expression in mammalian cells is illustratively carried out using a recombinant DNA vector capable of expressing the chimer gene in Chinese hamster ovary (CHO) cells. This is accomplished using procedures that are well known in the art and are described in more detail in Sambrook et al . , Molecular Cloning: A Laboratory Manual, 2 nd ed., Cold Spring Harbor Laboratories (1989) .
  • the simian virus (SV40) based expression vector pKSV-10 (Pharmacia Fine Chemicals, Piscataway, NJ) , is subjected to restriction endonuclease digestion by Ncol and Hindlll.
  • a Ncol/Hindlll sequence fragment that encodes the desired HBc chimer prepared as described in Example 1 is ligated into the expression plasmid, which results in the formation of a circular recombinant expression plasmid denominated pSV-Pf .
  • the expression plasmid pSV-Pf contains an intact E. coli ampicillin resistance gene.
  • E. coli RR101 Bethesda Research Laboratories, Gaithersburg, MD
  • Plasmid- containing bacteria are then selected and the clones are subsequently screened for the proper orientation of the inserted coding gene into the expression vector.
  • the above obtained plasmid, pSV-Pf , containing the gene that encodes a desired HBc chimer is propagated by culturing E. coli containing the plasmid.
  • the plasmid DNA is isolated from E. coli cultures as described in Sambrook et al . , above.
  • a chimer is accomplished by the introduction of pSV-Pf into the mammalian cell line, e.g., CHO cells, using the calcium phosphate- mediated transfection method of Graham et al . (1973) Virol . , 52:456, or a similar technique.
  • the transfection is carried out in the presence of a second plasmid, pSV2NEO (ATCC #37149)' and the cytotoxic drug G418 (GIBCO Laboratories, Grand Island, N.Y.) as described by Southern et al . (1982) J " . Mol . Appl . Genet. , 1:327.
  • pSV2NEO ATCC #37149
  • G418 cytotoxic drug G418
  • a chimer is expressed in the resulting CHO/pSV-Pf cells and can be detected in and purified from the cytoplasm of these cells.
  • the resulting composition containing cellular protein is separated on a column as discussed elsewhere herein.
  • a vector useful in practicing the present invention can direct the replication, and preferably also the expression (for an expression vector) of the chimer gene included in . the DNA • segment to which it is operatively linked.
  • the host that expresses the chimer is the prokaryote, E. coli
  • a preferred, vector includes a prokaryotic replicon; i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extrachromosomally in a prokaryotic host cell transformed therewith.
  • a prokaryotic replicon i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extrachromosomally in a prokaryotic host cell transformed therewith.
  • Those vectors that include a prokaryotic replicon can also include a prokaryotic promoter region capable of directing the expression of a contemplated HBc chimer gene in a host cell, such as E. coli , transformed therewith.
  • Promoter sequences compatible with bacterial hosts are typically provided in plasmid vectors containing one or more convenient restriction sites for insertion of a contemplated DNA segment .
  • Typical of such vector plasmids are pUC8, pUC9, and pBR329 available from Biorad Laboratories, (Richmond, CA) and pPL and pKK223-3 available from Pharmacia, Piscataway, NJ.
  • a contemplated chimer molecule and resulting immunogenic particles can be prepared by expression in plants or insects using well-known transformation techniques for each type of organism. Illustrative vectors and techniques useful for such expressions are discussed in detail in PCT application WO 02/14478 A2 published on February 21, 2002.
  • a variety of methods has 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 joined 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.
  • 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 the Meningococcal B cell epitope present in the immunogen.
  • compositions that is an inoculum in one animal can be a vaccine for another where the Neisseria meningitidis strain against which antibodies are raised do not infect the animal inoculated, as where an inoculum against Neisseria meningi tidis is used to raise antibodies in mice .
  • 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, mammal 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.
  • Vaccines or inocula are typically prepared from a recovered recombinant HBc chimer immunogen by dispersing the immunogen 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
  • Vaccines and inocula can also include an adjuvant as part of the diluent.
  • Adjuvants such as complete Freund's adjuvant (CFA) that is not used in humans, incomplete Freund's adjuvant (IFA) and alum are materials well known in the art, and are available commercially from several sources. Alum is a preferred adjuvant. The use of small molecule adjuvants is also contemplated herein.
  • Exemplary of one group of small molecule adjuvants are the so-called muramyl dipeptide analogues described in U.S. Patent No. 4,767,842.
  • Another type of small molecule adjuvant described in U.S. Patent No. 4,787,482 that is also useful herein is a 4:1 by volume mixture of squalene or squalane and ArlacelTM A (mannide monooleate) .
  • Yet another type of small molecule adjuvant useful herein is a 7-substituted-8-oxo or 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. Of these materials, 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. of Seattle, WA (see, U.S. Patent No. 4,987,237); RC-529 is a synthetic monosaccharide analogue of MPL in a squalene emulsion as RC-529SE and in an aqueous formulation as RC-529AF also available from Corixa; CpG (also ODN; oligonucleotides containing the CpG nucleotide motif one or more times plus flanking sequences) available from Coley Pharmaceutical Group; QS21 available from Antigenics, New Yourk; SBAS2 (now AS02) available from SKB (now Glaxo-SmithKline) that contains QS21 and MPL in 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
  • 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) .
  • Adjuvants are utilized in an adjuvant amount, which can vary with the adjuvant, mammal 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) , intravenously (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.
  • 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 mammal 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 Neisseria meningitidis bacteria.
  • the maintenance time for the production of 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.
  • the vaccinated mammal is preferably maintained at or near that antibody titer by periodic booster immunizations administered at intervals of about 1 to about 5 years .
  • anti-Meningococcal antibodies is readily ascertained by obtaining a plasma or serum sample from the immunized mammal and assaying the antibodies therein for their ability to bind to a synthetic antigen [e.g. the Meningococcal peptide used herein] in an ELISA assay as described hereinafter or by another immunoassay such as a Western blot as is well known in the art. Furthermore, the antibodies are analyzed by their ability to kill Neisseria meningi tidis in a serum bactericidal assay (SBA) . In this assay homologous strains [carrying the sequence of the PorA and NspA epitopes, respectively that are expressed on the chimeric HBc particle] as well as heterologous strains are used.
  • SBA serum bactericidal assay
  • anti- Meningococcal antibodies so induced can be isolated from the blood of the host mammal 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 the Meningococcal B cell immunogen of the HBc chimer 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 mammal in which antibodies to Neisseria are desired to be induced, but in which protection from Neisseria meningi tidis is not desired.
  • a similar inoculum can be used in a horse or sheep to induce antibody production against Neisseria meningi tidis for use in a passive immunization in yet another animal such as humans .
  • PKK223-3N a modified form of pKK223-3 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 HindiII to provide a 467 bp fragment that was then ligated with a 4106 bp fragment of the pKK223-3 vector, to effectively replace the original 480 bp Sphl-HindllI fragment.
  • the resultant plasmid (pKK223-3N) 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) .
  • the final plasmid, pKK223-3N has a size of 4573 bp. Restriction sites in plasmid pKK223-3N are indicated in Fig. 2, and the nucleotide changes made to pKK223-3 to form plasmid pKK223-3N are indicated by an underline as shown below.
  • a modified HBcl49 gene able to accept the directional insertion of synthetic dsDNA fragments into the immunodominant loop region, was constructed using PCR.
  • the plasmid accepting inserts between amino acid residues D78 and P79 was named V2.
  • the HBcl49 gene was amplified in two halves using two PCR primer pairs, one of which amplifies the amino terminus, the other amplifies the carboxyl terminus.
  • the products are a 249 bp (N-terminus) and a 243 bp fragment (C-terminus) .
  • the N-terminal fragment was digested with Ncol and EcoRI, and the C-terminal fragment was digested with EcoRI and Hindlll.
  • the V2 fragment pairs were then ligated together at the common EcoRI overhangs.
  • the resultant Ncol-Hindlll fragment was then ligated into the pKK223-3N vector, which had been prepared by digestion with Ncol and HindiII .
  • the V2 plasmid was digested with EcoRI and Sa restriction enzymes to insert B cell epitopes. Synthetic dsDNA fragments containing 5 ' EcoRI and 3 ' Sad overhangs were then inserted. Glycine- isoleucine (EcoRI) and glutamic acid-leucine (Sad) amino acid pairs, coded for by the restriction sites, flank the inserted B cell epitopes. The inserted restriction sites are underlined in the primers below.
  • VI6 Cloning Vector A variant of the V2 vector was constructed having a stabilizing C-terminal cysteine residue after the terminal HBc residue (Val-149) . All of the particles prepared from recombinant chimer molecules containing inserted Meningococcal epitope sequences discussed hereinafter included a C-terminal cysteine residue unless noted to the contrary.
  • a PCR reaction was performed using V2 as the template and the oligonucleotides primers HBcl49/NcoI-F (SEQ ID NO: 83) and HBcl49+C/HindI-II-R (SEQ ID NO: 87) .
  • the resultant amplification product was cleaved with the restriction enzymes Ncol and HindiII and ligated into the plasmid pKK223-3N, which had previously been cleaved with the same enzymes, to create V16.
  • the V16 vector therefore accepts insertions in an identical manner to V2 , between amino acids D78 and P79 at EcoRI and Sac I restriction sites.
  • V7 Cloning Vector
  • EcoRI and Sad restriction sites were inserted between valine-149 and the HindiII site to facilitate directional insertion of synthetic dsDNAs into EcoRI- HindiII (or EcoRI -Sad) 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, Sad 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 EcoRI/HindHI (or EcoRI-SacI) and synthetic dsDNA fragments having EcoRI/HindHI (or EcoRI/Sad) overhangs, were ligated into V7.
  • EcoRI/HindHI or EcoRI-SacI
  • synthetic dsDNA fragments having EcoRI/HindHI or EcoRI/Sad
  • the final amino acid of native HBc valine-149
  • 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.
  • V16 vectors which contain B cell epitopes between amino acids 78 and 79, as well as as a cysteine residue at position 150, were constructed from V2 vector.
  • V16 and V7 constructs synthetic dsDNA fragments coding for the B (V16) or T cell epitope (V7) of interest were inserted into EcoRl/SacI or EcoRI/HindHI restriction sites, respectively.
  • Sequences for the double-stranded DNAs are shown below with the encoded epitope sequence shown above . Both DNA sequences are shown in the 5 ' to 3 ' direction and are referred to as "for" for the coding strand, and “rev” for the complementary strand.
  • the pound symbol, # is used in some of the amino acid residue sequences that follow to indicate the presence of a stop codon.
  • a Q A A N G G A A porA-7for AATT GCG CAG GCC GCA AAC GGC GGT GCT GCG porA-7rev: C GGC TTT CGT GAC CTT GGT AAC TTT CAC TTG S G Q V K V T K V T K A
  • K P S S T N A K T porA- 12 for: AATT AAA CCG AGC TCT ACC AAC GCG AAG ACG porA-12rev: C GGC CTT AGT AAC TTC CAC TTT ATT GCC CGT
  • GGC AAT AAA GTG GAA GTT ACT AAG GCC GAGCT SEQ ID NO: 109 CTT CGC GTT GGT AGA GCT CGG TTT SEQ ID NO: 110
  • a D porA-15for AATT CAT TAT ACC CGT CAG AAC AAT GCG GAT porA-15rev: C CGG AAC AAA CAC ATC CGC ATT GTT CTG
  • PorA-22 rev C CGC TTT GGT CAC TTT CAC CTG GTT GTT GTT
  • PorA-44for AATT CCG GCG CAG AAC AGC AAA AGC GCG TAT PorA-44rev: C CGC CGG GGT ATA CGC GCT TTT GCT
  • G K V N T V K N V NspA- for: AATT GGC AAA GTG AAC ACC GTG AAA AAC GTG NspA-4rev: C TTT CAC GCG CAC GCC CGC GCT CAG TTC GCC R S G E L S A G V R V K
  • PorB-Tl+C A I W Q V E Q K
  • a S porB-Tl+Cfor AATT GCG ATT TGG CAG GTG GAA CAA AAA GCC
  • AGC porB-Tl+Crev AG CTT TTA GCA CCA ACC AGA ATC GGT GCC TGC
  • PorB-T2+C N Y K N G G F F V Q porB-T2+Cfor: AATT AAC TAT AAA AAT GGC GGT TTT TTC GTG CAG porB-T2+Crev: AG CTT TTA GCA ATG ACG CTT ATA CGC TCC CCC
  • PorB-T3+C H N S Q T E V
  • a A T porB-T3+Cfor AATT CAT AAC AGC CAG ACC GAA GTG GCG GCC
  • ACG porB-T3+Crev AG CTT TTA GCA AAC ATT GCC AAA
  • PorB-T4+C T P R V S Y A H G F porB-T4+Cfor: AATT ACA CCA AGA GTA AGC TAC GCA CAC GGA TTC porB-T4+Crev: AG CTT TTA GCA GTC GGC ATC GTC GAC TAG GCC K G L V D D A D C # SEQ ID NO: 52
  • PorA-T5+C R F G N A V P
  • R I S porA-T5+Cfor AATT CGT TTT GGC
  • AAC GCG GTG CCG CGC ATT AGC porA-T5+Crev AG CTT TTA GCA GAT GAA ATC AAA ACC ATG GGC
  • OpaB-T6+Cfor AATT ACC GGC GCG AAC AAT ACG AGC ACT GTG TCT OpaB-T6+Crev: AG CTT TTA GCA TGT AAT ACG GTT ACG AAA ATA
  • blocking buffer 200 ⁇ L was added to each well and the plate was incubated at 20 degrees C for 1 hour, then washed 3 times with ELISA wash buffer.
  • a solution was prepared of 2 ⁇ g/mL of biotinylated peptide in blocking buffer and added 100 ⁇ L to each well. The plate was incubated uncovered at room temperature on the bench for one hour, then washed 3 times with ELISA wash buffer.
  • Serial dilutions (1:100 to 1:5904900) of the test bleeds were prepared. Each dilution (100 ⁇ L) was pipetted into corresponding triplicate wells on the plate. Blocking buffer alone was used to provide the blank. The plate was incubated uncovered at room temperature on the bench for one hour.
  • the plate was washed with ELISA wash buffer 5 times.
  • the plates were coated with a 1:2000 dilution of ⁇ -mouse IgG conjugated to horseradish peroxidase (HRP) in blocking buffer.
  • the plates were washed with ELISA wash buffer 9 times, then washed with ELISA wash buffer-2 (PBS) once.
  • 100 ⁇ L TMB (3 , 3 ' , 5 , 5 ' -tetramethylbenzidine) solution was added to each well.
  • the plate was incubated at room temperature out of the light until the positive control reached a strong blue color.
  • 100 ⁇ L of IN H 2 S0 was added to each well.
  • the plate was read at 450nm using the plate reader.
  • the binding of antibodies in sera from inoculated mice to intact Neisseria meningi tidis bacteria were analyzed.
  • the samples analyzed in this assay were from mouse sera taken at sacrifice (BE2) .
  • the control sera were from mouse sera taken before the first immunization (BEO) .
  • a bacterial working suspension was prepared by inoculation of a 100 ⁇ L aliquot of a Neisseria meningi tidis working seed stock into 5 mL Tryptic-Soy broth (TSB) .
  • the culture was incubated overnight (about 18 hours) at 37 °C with 150 rpm shaking in an atmosphere containing 5% C0 2 .
  • Dilutions of sera were prepared as follows. The sera from each mouse was diluted in sterile TBS (10 mM Tris, 150 mM sodium chloride, pH 7.5) to one- half the final dilution.
  • Each serum predilution (125 ⁇ L) was pipetted into the sterile vial.
  • 125 ⁇ L of each predilution 1:2500 was pipetted into a screw cap vial, and 125 ⁇ L of sterile TBS was added to dilute the serum.
  • the Neisseria meningi tidis culture (15 mL) was centrifuged 15 minutes at 2000g, 4°C.
  • the meningococcal cell pellet was resuspended in 7.5 mL of sterile TBS.
  • the bacterial suspension (125 ⁇ L) was added to each vial with serum dilution, but not to the positive control.
  • TBS-Tween -Triton (20 mM Tris, 500 mM sodium chloride, 0.05% (v/v) Tween ® 20, 0.2% (v/v)
  • Triton X 100, pH 7.5 Triton X 100, pH 7.5
  • the prepared membrane was then washed in TBS for 5 minutes .
  • the Dot blot apparatus was assembled according to the instruction manual. TBS (100 ⁇ L) was added to each well of the apparatus. The TBS was removed from the wells of the Dot blot apparatus by vacuum before applying the samples to the membrane.
  • Tween -Triton for 5 minutes, then washed in TBS for 5 minutes. The blot procedure was continued or stored overnight (about 18 hours) at -20°C. The blot was washed 5 minutes in TBS for equilibration if it was stored at -20°C.
  • the blot was incubated for 1.5 hour with goat anti-mouse IgG alkaline phosphatase conjugate diluted 1:5000 in TBS with 3% BSA (2 ⁇ L antibody in 10 mL TBS with 3% BSA) in a plastic bag. The blot was then washed four times
  • mice immunized with chimeric core particle bearing epitopes of Neisseria meningi tidis were able to induce antibodies that recognized native epitopes on intact Neisseria meningitidis bacteria in this Dot Blot assay.
  • Serum Bactericidal Assay A bacterial working suspension was prepared by inoculation of a 100 ⁇ L aliquot of a Neisseria meningi tidis working seed into 5 mL Tryptic-Soy broth (TSB) . The culture was incubated over night (about 18 hours) at 37°C and with 150 rpm shaking in an atmosphere containing 5% C0 2 . Approximately 100-200 ⁇ L of the stock bacterial cell suspension was added to 20 mL of sterile TSB pre-equilibrated to room temperature to yield an A 60 o between 0.07 - 0.08.
  • TSB Tryptic-Soy broth
  • the culture was then incubated for approximately 2 hours at 37°C with 150 rpm shaking in an atmosphere containing 5% C0 2 until A 6 oo was between
  • the bacterial cells were diluted in PBS pH 7.2 containing 10 mM MgCl 2 , 10 mM CaCl 2 and 0.5% (w/v)
  • mice sera to be tested were heat- inactivated for 30 minutes at 56°C. Pooled sterile baby rabbit serum from 3-4 weeks old animals without bactericidal activity against the strains tested was used as exogenous complement source in this assay.
  • a 50 ⁇ L aliquot of the control samples A) , B) , C) and D) were taken and plated onto GC agar plates containing 2% (v/v) hemoglobin and 1% (v/v) supplement VX using a spiral plater.
  • the microtiter plate was incubated for 1 hour at 37°with 150 rpm shaking.
  • a 50 ⁇ L aliquot from each well was taken and plated onto GC agar plates containing hemoglobin and supplement VX using a spiral plater.
  • the plates were incubated for 18 hours at 5% C0 2 and the colonies were counted using a colony counter. Each sample was determined in triplicate. The average number of CFU at time zero was used as 100%.
  • the serum bactericidal titer was reported as the reciprocal of the serum dilution yielding greater than or equal to 50% killing of the bacteria.
  • a bacterial working suspension was prepared by inoculation of a 100 ⁇ L aliquot of JV. meningi tidis working seed stock into 5 mL Tryptic-Soy broth (TSB) .
  • the culture was incubated overnight (about 18 hours) at 37°C with 150 rpm shaking in an atmosphere containing 5% C0 2 .
  • Western blot probes were prepared as follows. The cultures were centrifuged at maximal speed for 5 minutes and resuspended in an equal volume of 0.9% sodium chloride solution. A solution of 10% SDS was added to the suspension to a final volume of 1%. An aliquot was stored for determination of protein content. The residual suspension was centrifuged at maximal speed. The pellet was resuspended with Laemmli buffer [62.5 mM Tris-HCl, pH 6.8, 25% glycerin, 2% SDS, 0,01 % bromphenol blue (Biorad article no. 161-0737) containing 5% (v/v) ⁇ -mercaptoethanol in 10% of the start volume] .
  • Laemmli buffer 62.5 mM Tris-HCl, pH 6.8, 25% glycerin, 2% SDS, 0,01 % bromphenol blue (Biorad article no. 161-0737) containing 5% (v/v) ⁇ -mercaptoethanol in 10% of the start volume
  • the probes were heated at 95°C for 10 minutes and stored at -20°C until usage.
  • the protein content was measured using BCA Protein Assay (Pierce, article no. 23225) .
  • the aliquot as well as the standard was heated for 5 minutes at 95°C. Determination of protein content was carried out by following the manufacturer's instructions.
  • the JV. meningi tidis probes were adjusted to a final concentration of 1 mg/mL with pure water .
  • the proteins were transferred to a nitrocellulose membrane (Hybond ECL; AmershaM Pharmacia, article no. RPN 203D) using a SemiDry- Blotter (Biorad) .
  • the membrane was cut to the size of the gel and incubated in pure water for 10 minutes.
  • Two filter papers were equilibrated in 1 x transfer buffer (25 (v/v) methanol, 3.03 g/L Trisbase, 14.4 g/L glycerin) .
  • the membrane was also equilibrated in 1 x transfer buffer.
  • the gel was un ipped and the slots were cut off. The gel was transferred carefully onto the membrane.
  • the filter paper was transferred onto the Blot Unit . Gel and membrane were transferred at a single blow onto the filter paper. A second filter paper was applied and the blot unit was closed.
  • the Trans-Blot Unit was plugged in to a Power Pac 200 and adjusted to the appropriate amperage (A) . The transfer was done for exactly one hour at 50 m
  • the blot was incubated for 1.5 hours with goat anti-mouse IgG alkaline phosphatase conjugate diluted 1:5000 in TBS with 3% BSA (2 ⁇ L antibody in 10 mL TBS with 3% BSA) in a plastic bag. The blot was then washed four times
  • mice per group were immunized two times by subcutaneous injection in IFA either using 10 ⁇ g of purified hepatitis B core particle (truncated particle corresponding to amino acid residues 1-149 of HBc without a C-terminal stabilizing cysteine) or chimeric hepatitis B core particles containing either the sequence corresponding to a region of PorA-16 (identified as SEQ ID NO: 20), or the sequence (SEQ ID NO: 46) corresponding to a region of NspA as well as a C-terminal stabilizing cysteine residue, or 10 ⁇ g of outer membrane vesicles (OMV) of Neisseria meningi tides strain B6707 that expresses PorA-16.
  • Boost immunization was 4 weeks after the first immunization (BE1) and sacrifice occurred two weeks later (BE2) .
  • the IgG titers were high from 4 out of 5 mice immunized with HBc-NspA-3
  • mice per group were immunized two times intraperitoneally in IFA either with 15 ⁇ g purified particles or 30 ⁇ g inactivated Neisseria meningitidis strains.
  • Boost immunization was 6 weeks after the first immunization and sacrifice was two weeks later.
  • Immunizing truncated core particles, C-terminal stabilized chimer particles and inactivated JV. meningi tidis control strains with expressed PorA ligands shown in parentheses are provided below.
  • Anti-core endpoint titers of about 10 s were determined in all groups. These data show, that the chimeric particles are highly immunogenic The data showed that five out of six of the chimeric particles induced antibody levels at about the same magnitude as the whole killed JV. meningi tidis control strains. The sixth chimeric particle that bore the PorA-46 sequence induced a significantly greater antibody level (more than 2 log units greater) . Data from a comparison of the biotinylated peptide binding of sera from mice immunized with a variety of stabilized peptide-containing chimer particles as compared to mice immunized with corresponding phenoxyohenol-inactivated bacteria displaying those peptides are shown in Table 5, below.
  • mice were immunized with truncated stabilised HBc particles containing sequences from PorA in the immundominant (El) loop, or else with whole killed bacteria of strains expressing PorA with the defined sequences in their hypervariable domains..
  • immunization with an HBc chimer displaying a PorA peptide induced antibodies to the corresponding synthetic biotinylated PorA peptide as well as, if not significantly better (as in the case of HBc-PorA- 46) , than immunization with the whole inactivated bacteria.
  • all of these sera gave positive results in the Dot Blot assay.
  • Epitope PorA-46 is derived from loop V, indicating that loop V is immunogenic.
  • Epitope PorA- 44 is derived from a conserved region of PorA. It appears that the conserved epitopes, PorA-43, PorA- 44, PorA-50 and PorA-51, are as immunogenic as the hypervariable ones (all epitopes from PorA-1 to PorA- 22) .
  • mice immunized with the different chimeric core particles recognize their corresponding epitope on intact JV. meningi tidis . These data are shown in Table 6, below.
  • mice per group were immunized two times subcutanously in IFA either with 15 ⁇ g purified particles or 30 ⁇ g inactivated Neisseria meningi tidis strains.
  • Boost immunization was 6 weeks after the first immunization and sacrifice was two weeks later.
  • Immunizing truncated C-terminal stabilized chimer particles and inactivated JV. meningi tidis control strains with expressed PorA ligands shown in parentheses are provided below.
  • Anti-core endpoint titers of about IO 5 were determined in all groups. Therefore, the titers in this study are about 1 log unit higher (about 10-fold higher) than in study GSF #3 , but they are about 1 log unit lower than in GSF #8. These data show, that the chimeric particles are less immunogenic when administered subcutaneously compared to intraperitoneal immunization and that a 6 week interval between prime and boost immunization is superior to a 4 week interval .
  • Anti-peptide titer showed that four out of five of the chimeric particles induced antibody levels between IO 3 and 5xl0 4 which is about 2 log units (100-fold) higher the levels measured with the corresponding JV. meningi tidis control strains.
  • the fifth chimeric particle that bore the PorA-43 sequence induced a significantly lower antibody level .
  • mice immunized with a variety of stabilized peptide-containing chimer particles as compared to mice immunized with corresponding phenoxyphenol-inactivated bacteria displaying those peptides are shown in Table 7, below.
  • Mice were immunized with HBc-PorA-2+S, HBc- PorA-7+S, JV. meningi tidis strain 0028/96 (PorA-7) , HBc-PorA-43, HBc-NspA-1+S and HBc-NspA-2+S .
  • immunization with an HBc chimer displaying a PorA peptide induced antibodies to corresponding biotinylated PorA peptide, which are significantly better, than immunization with the control, inactivated bacteria alone.
  • Epitopes PorA-2 and Por-7 are derived from the hypervariable regions of loop IV and loop I, respectively. In contrast PorA-43 is derived from a conserved region of loop I . This shows that the hypervariable regions display a significantly higher immunogenicity.
  • Epitopes NspA-1 and NspA-2 are derived from the conserved Meningococcal surface protein A and located in proposed surface exposed loops of the protein. HBc-NspA-2+S elicited the highest anti-peptide titer measured in this animal experiment . Therefore both epitopes are at least as immunogenic as the hypervariable ones.

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Abstract

L'invention concerne une protéine nucléocapside du virus de l'hépatite B (HBc) tronquée, chimère et à terminaison carboxy contenant un immunogène d'induction de la production d'anticorps sur des protéines méningocoques. Un épitope méningocoque immunogène est exprimé entre les résidus 76 et 85 de la séquence en boucle immunogène HBc. Le chimère contient de préférence un épitope de lymphocyte T spécifique au méningocoque et comprend un résidu de cystéine à terminaison C assurant une meilleure stabilité des particules chimères assemblées automatiquement. Font également l'objet de cette invention, les procédés d'obtention et d'utilisation des chimères.
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US9555097B2 (en) 2006-02-23 2017-01-31 Era Biotech, S.A. Recombinant protein bodies as immunogen-specific adjuvants
US9637751B2 (en) 2010-05-28 2017-05-02 Era Biotech, S.A. Recombinant protein body-inducing polypeptides
US10617748B2 (en) 2008-02-14 2020-04-14 Life Sciences Research Partners Vzw Immunogenic control of tumours and tumour cells
US10662232B2 (en) 2006-08-11 2020-05-26 Life Sciences Research Partners Vzw Immunogenic peptides and their use in immune disorders
US10729791B2 (en) 2015-05-18 2020-08-04 Imcyse Sa Animal models for evaluating pharmaceutical compounds
US10982196B2 (en) 2008-02-14 2021-04-20 Life Sciences Research Partners Vzw Immunotherapy targeting intracellular pathogens
US11193114B2 (en) 2010-11-25 2021-12-07 Imnate Sarl Immunogenic peptides for use in the prevention and/or treatment of infectious diseases, autoimmune diseases, immune responses to allofactors, allergic diseases, tumors, graft rejection and immune responses against viral vectors used for gene therapy or gene vaccination
US11226332B2 (en) 2013-05-28 2022-01-18 Imcyse Sa Method for the detection, preparation and depletion of CD4+ t lymphocytes
US11485768B2 (en) 2016-04-19 2022-11-01 Imcyse Sa Immunogenic CD1d binding peptides
US11787849B2 (en) 2015-09-25 2023-10-17 Imcyse Sa Methods and compounds for eliminating immune responses to therapeutic agents
US12023384B2 (en) 2014-10-17 2024-07-02 Imcyse Sa Immunogenic peptides comprising an MHC class II T cell epitope and a redox motif

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US9555097B2 (en) 2006-02-23 2017-01-31 Era Biotech, S.A. Recombinant protein bodies as immunogen-specific adjuvants
US10662232B2 (en) 2006-08-11 2020-05-26 Life Sciences Research Partners Vzw Immunogenic peptides and their use in immune disorders
US11718650B2 (en) 2006-08-11 2023-08-08 Imcyse Sa Immunogenic peptides and their use in immune disorders
US10617748B2 (en) 2008-02-14 2020-04-14 Life Sciences Research Partners Vzw Immunogenic control of tumours and tumour cells
US10982196B2 (en) 2008-02-14 2021-04-20 Life Sciences Research Partners Vzw Immunotherapy targeting intracellular pathogens
US9637751B2 (en) 2010-05-28 2017-05-02 Era Biotech, S.A. Recombinant protein body-inducing polypeptides
US11193114B2 (en) 2010-11-25 2021-12-07 Imnate Sarl Immunogenic peptides for use in the prevention and/or treatment of infectious diseases, autoimmune diseases, immune responses to allofactors, allergic diseases, tumors, graft rejection and immune responses against viral vectors used for gene therapy or gene vaccination
CN104220080A (zh) * 2012-01-30 2014-12-17 鲁汶天主教大学 用于加强cd4+t细胞应答的经修饰表位
AU2013214700B2 (en) * 2012-01-30 2017-09-28 Katholieke Universiteit Leuven Modified epitopes for boosting CD4+ T-cell responses
CN104220080B (zh) * 2012-01-30 2018-10-12 鲁汶天主教大学 用于加强cd4+t细胞应答的经修饰表位
RU2724994C2 (ru) * 2012-01-30 2020-06-29 Католике Университейт Левен Модифицированные эпитопы для усиления ответов cd4+ т-клеток
US10899795B2 (en) 2012-01-30 2021-01-26 Life Sciences Research Partners Vzw Modified epitopes for boosting CD4+ T-cell responses
US11226332B2 (en) 2013-05-28 2022-01-18 Imcyse Sa Method for the detection, preparation and depletion of CD4+ t lymphocytes
US12023384B2 (en) 2014-10-17 2024-07-02 Imcyse Sa Immunogenic peptides comprising an MHC class II T cell epitope and a redox motif
US10729791B2 (en) 2015-05-18 2020-08-04 Imcyse Sa Animal models for evaluating pharmaceutical compounds
US11787849B2 (en) 2015-09-25 2023-10-17 Imcyse Sa Methods and compounds for eliminating immune responses to therapeutic agents
US11485768B2 (en) 2016-04-19 2022-11-01 Imcyse Sa Immunogenic CD1d binding peptides

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