WO2003072722A2

WO2003072722A2 - IMMUNOGENIC AVIAN HBc CHIMER PARTICLES HAVING ENHANCED STABILITY

Info

Publication number: WO2003072722A2
Application number: PCT/US2003/005315
Authority: WO
Inventors: Ashley J. Birkett; Birgit Peck
Original assignee: Apovia, Inc.
Priority date: 2002-02-21
Filing date: 2003-02-21
Publication date: 2003-09-04
Also published as: AU2003213194A1; AU2003213194A8; WO2003072722A3

Abstract

A chimeric, carboxy-terminal truncated avian hepatitis B virus nucleocapsid protein (AHBc) is disclosed that is engineered for both enhanced stability of self-assembled particles and the display of an immunogenic epitope. The display of the immunogenic epitope is displayed in the immunogenic loop of AHBc, whereas the enhanced stability of selfassembled particles is obtained by the presence of at least one heterologous cysteine residue near the carboxy-terminus of the chimer molecule. Methods of making and using the chimers are also disclosed.

Description

IMMUNOGENIC AVIAN HBc CHIMER PARTICLES HAVING ENHANCED STABILITY

CROSS REFERENCE TO RELATED APPLICATION

This application claims benefit of U.S. Provisional Application No. 60/359,129, filed February 21, 2002.

TECHNICAL FIELD

The present invention relates to the intersection of the fields of immunology and protein engineering, and particularly to a chimeric avian hepatitis B virus (AHBV) nucleocapsid protein that is engineered for enhanced stability of self-assembled particles and the display of an immunogenic epitope, as well as for decreased cross-reactivity.

BACKGROUND OF THE INVENTION

The family hepadnaviridae are enveloped DNA-containing animal viruses that can cause hepatitis B in humans (HBV) . Human hepatitis B core protein (HBc) sequences include subtype ayw (SEQ ID NO:l), adw (SEQ ID NO:2), adw2 (SEQ ID N0:3), and adyw (SEQ ID NO: 4) . The hepadnavirus family includes hepatitis B viruses of other mammals, e.g., woodchuck (WHV; woodchuck HBc SEQ ID NO: 5), and ground squirrel (GSHV; ground squirrel HBc SEQ ID NO: 6), as well as avian viruses (AHBV; avian HBc SEQ ID NO: 7, AHBc) found in ducks (DHV; duck HBc SEQ ID NO: 8) and herons (HeHV; heron HBc SEQ ID NO: 9) . Hepatitis B virus (HBV) used herein refers to a member of the family hepadnaviridae, unless the discussion is referring to a specific example. The sub-family of avian hepatitis B viruses are referred to as AHBV herein. The nucleocapsid or core of the mammalian hepatitis B virus (HBV or hepadnavirus) 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 nucleocapsid or viral core protein monomers of the several hepadnaviridae self-assemble in infected cells into stable aggregates known as hepatitis B core protein particles (HBc or HBcAg 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 disulfide-linked dimers or 180 individual monomeric proteins, and a second, major population that contains 120 copies of the HBc subunit protein as disulfide-linked dimers or 240 individual monomeric proteins. These particles are referred to as T = 4 or T = 3 particles, respectively, wherein "T" is the triangulation number. These HBc particles of the human-infecting virus (human virus) are about are about 30 or 34 nm in diameter, respectively. Pumpens et al . (1995) Intervirology, 38:63-74; and Metzger et al . (1998) J. Gen . Viol . , 79:587-590.

Conway et al . , (1997) Nature, 386:91-94, describe the structure of human HBc particles at 9 Angstrom resolution, as determined from cryo-electron micrographs. Bottcher et al . (1997), Nature, 386:88- 91, describe the polypeptide folding for the human HBc monomers, and provide an approximate numbering scheme for the amino acid residues at which alpha- helical regions and their linking loop regions form. Zheng et al . (1992), J. Biol . Chem. , 267 (13) : 9422- 9429 report that core particle formation is not dependent upon the arginine-rich C-terminal domain, the binding of nucleic acids or the formation of disulfide bonds based on their study of mutant proteins lacking one or more cysteines and others ' work with C-terminal-truncated proteins [Birnbaum et al., (1990) J. Virol . 64, 3319-3330].

The hepatitis B viral core protein has been disclosed as an immunogenic carrier moiety that stimulates the T cell response of an immunized host animal. See, for example, U.S. Patents No. 4,818,527, No. 4,882,145 and No . 5,143,726, the disclosures of which are incorporated herein by reference. 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 mammalian protein. Clarke et al .

(1991) F. Brown et al . eds., Vaccines 91 , Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp.313-318.

During viral replication, HBV nucleocapsids associate with the viral RNA pre-genome, the viral reverse transcriptase (Pol) , and the terminal protein

(derived from Pol) to form replication competent cores. The association between the nucleocapsid and the viral RNA pre-genome is mediated by an arginine- rich domain at the carboxyl-terminus (C-terminus) . When expressed in heterologous expression systems, such as E. coli where viral RNA pre-genome is absent, the protamine-like C-terminus; i.e., residues at positions 150 through 183, can bind E. coli RNA. Zhang et al . (1992) J. Biol . Chem. , 267 (13) : 9422-29. HbcAg is a particulate protein derived from the hepatitis B virus that has been proposed as a carrier for heterologous epitopes. The relative immunogenicity of the hepatitis B surface antigen

(HBsAg also HBs) has been compared with HbcAg, and the ability of each to evoke immune responses in different genetic backgrounds [Milich et al . , Science, (1986) 234(4782): p. 1398-1401]. These data emphasize the higher immunogenicity of HBc relative to HBs, and the universal responsiveness to HBc, irrespective of genetic background.

For example, mammalian 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. These results re-emphasize the suitability of HBc as a vaccine carrier and specifically, its superiority over HBs, hence the selection of HBc as opposed to HBs to carry heterologous epitopes. These facets of HBc are thought to be important in influenza vaccine development, because they address issues of genetic restriction and inadequate antibody titers.

Another advantage of the mammalian HBc carrier is the fact that it does not require complex adjuvants for efficacy. This is due to the high inherent immunogenicity of the particle. A comparison of the immunogenicity of HBc-P. berghei particles showed that alum, which is approved for human use, was more effective than either IFA or CFA

[Schodel et al . , J. Exp . Med. , (1994) 180(3): p. 1037-46] . The importance of this observation is highlighted by toxicity problems associated with newer, more complex adjuvants as was recently noted in clinical trials of Smith Kline Beecham' s candidate malaria vaccine [Stoute et al . , N. Engl . J. Med. , [1997] 336 (2) : p. 86-91] .

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, which is thought to bind nucleic acid, could be deleted without interfering with the protein's ability to assemble into virus-like particles. It is thus reported that proteins truncated to about position 144; i.e., containing the HBc sequence from position one through about 144, can self-assemble, whereas deletions beyond residue 139 abrogate capsid assembly [F. Birnbaum & M. Nassal (1990) J. Virol . , 64: 3319-30; Seifer et al . , (1995) Intervirology, 38:47-62].

Zlotnick et al . , (1997) Proc . Natl . Acad . Sci . , USA, 94:9556-9561 studied the assembly of full length and truncated human HBc proteins in to particles. In addition to discussing full length molecules, those authors reported the preparation of a truncated protein that contained the human HBc sequence from position 1 through 149 in which the cysteines at positions 48, 61 and 107 were each replaced by alanines and in which a cysteine residue was added at the C-terminus (position 150) . That C- terminal mercaptan was used for linkage to a gold atom cluster for labeling in electron microscopy.

More recently, Metzger et al . , (1998) J. Gen . Viol . , 79:587-590, reported that the proline at position 138 (Pro-138 or P138) of the human viral sequence is required for particle formation. Those authors also reported that assembly capability of human particles truncated at the carboxy-terminus to lengths of 142 and 140 residues was affected, with assembly capability being completely lost with truncations resulting in lengths of 139 and 137 residues .

Several groups have shown that truncated mammalian particles exhibit reduced stability relative to standard mammalian hepatitis B core particles [Galena et al . (1989) J. Virol . , 63:4645- 4652; Inada, et al . (1989) Virus Res . , 14:27-48], evident by variability in particle sizes and the presence of particle fragments in purified preparations [Maassen et al . , (1994) Arch . Virol . , 135:131-142]. Thus, prior to the report of Metzger et al . , above, Pumpens et al . , (1995) Intervirology, 38:63-74 summarized the literature reports by stating that the carboxy-terminal border for HBc sequences required for self-assembly was located between mammalian consensus amino acid residues 139 and 144, and that the first two or three amino-terminal residues could be replaced by other sequences, but elimination of four or eleven amino-terminal residues resulted in the complete disappearance of chimeric protein in transformed E. coli cells.

Such mammalian 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] . In some cases the insertion of heterologous epitopes into C-terminally truncated HBc particles has such a dramatic destabilizing effect that hybrid particles cannot be recovered following heterologous expression [Schodel et al . (1994) Infect . Immunol . , 62 : 1669 - 1616] . Thus, 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 .

Recombinantly-produced hybrid HBc particles bearing internal insertions (referred to in the art as HBc chimeric particles or HBc chimers) containing various inserted polypeptide sequences have been prepared by heterologous expression in a wide variety of organisms, including E. coli , B . subtilis, Vaccinia, Salmonella typhimurium, Saccharomyces cerevisiae . See, for example Pumpens et al . (1995) Intervirology, 38:63-74 , and the citations therein that note the work of several research groups .

The above Pumpens et al . , (1995) Intervirology, 38:63-74 , report lists particle- forming chimers in which the inserted polypeptide sequence is at the N-terminus, the C-terminus and between the termini of the mammalian HBc. Insert lengths reported in that article are 24 to 50 residues at the N-terminus, 7 to 43 residues internally, and 11 to 741 residues at the C-terminus.

Kratz et al . , (1999) Proc . Natl . Acad. Sci . , U. S.A. , 96:1915-1920 recently described the E. coli expression of chimeric HBc particles comprised of a truncated HBc sequence internally fused to the 238-residue green fluorescent protein (GFP) . This chimer contained the inserted GFP sequence flanked by a pair of glycine-rich flexible linker arms replacing amino acid residues 79 and 80 of HBc. Those particles were said to effectively elicit antibodies against native GFP in rabbits as host animals. 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.

Recently published WO 01/27281 teaches that the immune response to mammalian 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 mammalian 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 mammalian HBc sequence was said to include a C-terminal cysteine and an optional sequence that defines an epitope from other than HBc . Published PCT application WO 01/98333 teaches the deletion of one or more of the four arginine repeats present at the C-terminus of native mammalian HBc, while maintaining the C-terminal cysteine residue. That application also teaches that the deleted region can be replaced by an epitope from a protein other than HBc so that the HBc portion of the molecule so formed acts as a carrier for the added epitope .

Published PCT applications corresponding to PCT/US01/25625 and PCT/USOl/41759 of the present inventor teach that stabilization of C-terminally truncated mammalian HBc particles can be achieved through the use of one or more added cysteine residues in the chimer proteins from which the particles are assembled. Those added cysteine residues are taught to be at or near the C- terminus of the chimeric protein.

A structural feature whereby the stability of full-length mammalian HBc particles could be retained, while abrogating the nucleic acid binding ability of full-length HBc particles, would be highly beneficial in vaccine development using the hepadnaviral nucleocapsid delivery system. Indeed, Ulrich et al . in their recent review of the use of HBc chimers as carriers for foreign epitopes [Adv. Virus Res . , 50: 141-182 (1998) Academic Press] note three potential problems to be solved for use of those chimers in human vaccines. A first potential problem is the inadvertent transfer of nucleic acids in a chimer vaccine to an immunized host. A second potential problem is interference from preexisting immunity to HBc. A third possible problem relates to the requirement of reproducible preparation of intact chimer particles that can also withstand long-term storage .

The above four published PCT applications appear to contain teachings that can be used to overcome over come the potential problems disclosed by Ulrich et al . As disclosed hereinafter, the present invention provides an avian HBc chimer that provides a solution to the problems of HBc chimer stability as well as the substantial absence of nucleic acid binding ability of the construct. In addition, a contemplated recombinant chimer exhibits minimal, if any, antigenicity toward preexisting anti-HBc antibodies.

The above particle instability findings related to N-terminally truncated mammalian HBc chimer molecules notwithstanding, Neirynck et al . , (October 1999) Nature Med. , 5 (10) : 1157-1163 reported that particle formation occurred upon E. coli expression of a HBc chimer that contained the N- terminal 24-residue portion of the influenza M2 protein fused at residue 5 to full length mammalian HBc.

The previously discussed use of hybrid mammalian HBc proteins with truncated C-termini for vaccine applications offers several advantages over their full-length counterparts, including enhanced expression levels and lack of bound E. coli RNA. However, C-terminally truncated particles engineered to display heterologous epitopes are often unstable, resulting in particles that either fail to associate into stable particulate structures following expression, or that readily dissociate into non- particulate structures during and/or following purification. Such a lack of stability is exhibited by particles comprised of chimeric HBc molecules that are C-terminally truncated to HBc position 149 and also contain the above residues 1-24 of the influenza A M2 protein.

Others have reported that in wild type hepadnaviral core antigens a cysteine residue upstream of the HBcAg start codon is directly involved in the prevention of particle formation [Schodel et al . (Jan. 15, 1993) J. Biol . Chem. , 268(2) -1332-1337; Wasenauer et al . (Mar. 1993) J. Virol . , 67(3) .1315-1322; and Nassal et al . (Jul . 1993) J^". Virol . , 67 (7) .4307-4315] . All three groups reported that in wild type HBeAg, the 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, is responsible for preventing particle formation and therefore facilitating the transition from particulate HBcAg to secreted, non-particulate HBeAg.

Based upon the above three publications, one would expect the inclusion of one or more cysteine residues at a position prior to that of the initiator methionine of HBc to actually destabilize hybrid particles rather than stabilize them. As will be seen from the discussion that follows, the present invention provides teachings that are contrary to those expectations.

The core proteins of the hepadnaviridae class of viruses are considered excellent carriers of heterologous epitopes for vaccine applications. A modified version of the nucleocapsid or core of the mammalian hepatitis B virus (HBV or hepadnavirus) has proven useful in the development of new vaccines as an antigenic carrier, U.S. Patent No. 6,231,864, May 15, 2001. However, concerns have been raised over the use of such mammalian HBV-derived vaccines in providing false positive results in tests for exposure to the human disease, hepatitis B. In addition, the efficacy of vaccines derived from mammalian HBV core may be decreased if the person vaccinated already has antibodies to HBV core, from exposure due to HBV infection or an earlier- administered vaccine using HBV core.

The duck hepatitis B virus core protein is considered to be especially suited to human vaccine applications because it is distinct from the highly homologous human and woodchuck core proteins . Published PCT Application No. WO 00/46365, August 10, 2000, discloses the use of duck hepatitis B virus core protein as a carrier that does not result in false positives for exposure to human hepatitis B using the standard tests. The decreased cross- reactivity is a result of the sequence variation between mammalian and avian hepatitis B core protein. Immune responses directed to an avian core carrier are less likely to interfere with hepatitis B serological markers that are currently used to diagnose hepatitis B virus infection or exposure. Those workers disclose the attachment of antigens to the N- or C-terminus, or truncated C-terminus of the avian hepatitis B core protein.

Based upon a homology match by Standring (Chapter 7, The Molecular Biology of the Hepatitis B Virus Core Protein) , where the first 39 amino acids of the human HBV (starting with the C ORF methionine) were aligned with analogous residues of the duck and heron viruses, 41% show identity and a further 23% of the residues represent conservative substitutions. One potential drawback to the use of duck hepatitis B core protein as a vaccine carrier is its lack of stability. The introduction of N- or C-terminal antigens, as disclosed in WO 00/46365 (discussed above) tends to destabilize the formation of particles of the avian hepatitis B core protein. Even in the absence of additions on the N- or C-terminus, particles of the avian HB core are less stable than particles of mammalian HB core. In that international application, Peterson and Colement teach that exposure to metal ions (divalent cations) , inter alia, cause particles of avian HB core to dissociate.

One possible reason for the observed decreased stability of avian hepatitis B core particles is the lack of extensive disulfide cross- linking in the core protein. Unlike the human and woodchuck hepatitis B core proteins, which contain 4 conserved cysteine residues (at positions corresponding to 48, 61, 107 and 183 in SEQ ID NO:l), the duck core protein only has the cysteine that is possibly analogous to Cysl07 in the human sequence. Cysl07 is not believed to be involved in the formation of stabilizing disulfide bonds in the human core protein. Rather, cysteine residues 61 and 183 are believed to be the most important cysteine residues for imparting stability to the human core protein particles.

A structural feature whereby the stability of full-length avian HBc particles could be retained, while abrogating the nucleic acid binding ability of full-length avian HBc particles, would be highly beneficial in vaccine development using the hepadnaviral nucleocapsid delivery system. Indeed, Ulrich et al . in their recent review of the use of HBc chimers as carriers for foreign epitopes [Adv. Virus Res . , vol. 50 (1998) Academic Press pages 141- 182] note three potential problems to be solved for use of those chimers in human vaccines. A first potential problem is the inadvertent transfer of nucleic acids in a chimer vaccine to an immunized host . A second potential problem is interference from preexisting immunity to HBc. A third possible problem relates to the requirement of reproducible preparation of intact chimer particles that can also withstand long-term storage.

As disclosed hereinafter, the present invention provides one solution to the problems of HBc chimer stability as well as the substantial absence of nucleic acid binding ability of the construct, while providing powerfully immunogenic materials.

BRIEF SUMMARY OF THE INVENTION

The present invention contemplates a recombinant avian hepadnavirus nucleocapsid protein; i.e., an avian hepatitis B core (AHBc) chimeric protein [or chimer avian hepatitis B core protein molecule or AHBc chimer molecule or just chimer] that self-assembles into particles after expression in a host cell.

In a preferred embodiment, a recombinant chimer avian hepatitis B core (AHBc) protein molecule up to about 635 amino acid residues in length is contemplated.

In a preferred embodiment, a chimer molecule contains one to three cysteine residues from either an N-terminal cysteine residue, a C-terminal cysteine residue or both the N- and C-termini, and also optionally in the range of residues 55 to 65. An N-terminal cysteine residue is a cysteine residue within about 30 amino acid residues of an amino acid position of the chimer molecule corresponding the N- terminus. A C-terminal cysteine residue is a cysteine residue within about 30 amino acid residues of an amino acid position of the chimer molecule corresponding to the C-terminus. If such a cysteine residue is at the N-terminus, it is other than that of a pre-core sequence.

One or both of chimeric protein dimers and particles formed from such a chimer with cysteine residues are more stable than are corresponding dimers or particles formed from an otherwise identical AHBc chimer that lacks said cysteine (s) or in which said cysteine present in the chimer molecule is replaced by another residue. One preferred embodiment of the invention is a cysteine-stabilized avian HBc that has two heterologous cysteine residues to enable both monomer-monomer cross-linking to form dimers, and to enable dimer-dimer cross-linking to form a disulfide-linked lattice in the formed particles .

In some embodiments, the chimer molecule is free of more than one sequence of five amino acid residues that includes three arginine, lysine or both residues. The chimeric protein is thus sufficiently free of arginine residues so that the self-assembled avian core particles are substantially free of nucleic acid binding. Such a chimer self-assembles into particles that are substantially free of binding to nucleic acids upon expression in a host cell. The chimeric avian protein contains a peptide-bonded heterologous epitope or a heterologous linker residue for a conjugated epitope sequence. Preferably, the chimeric protein displays one or more immunogenic epitopes at the N-terminus, AHBc loop region or C-terminus, or has a heterologous linker residue for a conjugated epitope in the immunogenic loop. The AHBc loop region is from amino acid residue position number about 86 to about 130, preferably 90 to 110, as numbered in Fig. 1 for the AHBc consensus sequence (SEQ ID NO: 7) . In some embodiments of the AHBc chimer, some or all of the region corresponding to the loop regionfrom amino acid residue position number 86 to 130 is deleted.

In some embodiments, the heterologous epitope is a B cell epitope. In some embodiments the heterologous epitope defines a T cell epitope. In some embodiments, the peptide-bonded heterologous epitope is peptide-bonded within about 5 residues of the portion of the chimer that corresponds to the N- terminus of the AHBc sequence. In some embodiments, the peptide-bonded heterologous epitope is peptide- bonded within about 5 residues of the portion of the chimer that corresponds to the C-terminus (even if truncated) of the AHBc sequence .

Preferably, for the portions of the chimer molecule that align with segments of the consensus avian hepatitis B core protein sequence depicted in SEQ ID NO: 7, the chimeric avian protein contains no more than 20 percent, more preferably no more than 10 percent, conservatively substituted amino acid residues relative to the consensus avian hepatitis B core protein sequence (SEQ ID NO: 7), the duck HBc sequence (SEQ ID NO: 8) or the heron HBc sequence (SEQ ID NO: 9) . Exemplary preferred conserved and variable HBc amino acid residues are illustrated in the consensus avian HBc sequence in SEQ ID NO: 7.

In one embodiment , the recombinant chimer AHBc protein molecule includes the amino acid residue sequence of the avian core protein sequence (SEQ ID NO: 7, preferably the duck hepatitis B core protein (SEQ ID NO:8), from about position 5 to about position 205, although the avian portion of the chimeric sequence in some embodiments will not be consecutive due to introduction of a peptide bonded heterologous protein sequence, for example an epitope inserted within the sequence after an amino acid residue located between position 86 and position 130 of SEQ ID NO: 7 or 8.

The present invention also contemplates an immunogenic particle comprised of recombinant avian hepatitis B core (AHBc) chimeric protein molecules. The chimeric protein making up the particles are as described above .

One embodiment of the invention contemplates a recombinant chimer avian hepatitis B core (AHBc) protein molecule up to about 635 amino acid residues in length that

(a) contains (i) a sequence of at least about 150 of the N-terminal 230 amino acid residues of the AHBc molecule (SEQ ID NO: 7) and further including a covalently linked peptide-bonded heterologous epitope or a heterologous linker residue for a conjugated epitope present in the avian loop sequence) from about residue 75 to about residue 130, or (ii) a sequence of at least about 150 residues of the N-terminal 205 avian HBc amino acid residues (SEQ ID NO: 7) , and (b) contains one to three cysteine residues toward the C-terminus of the portion of the AHBc chimer molecule that corresponds to a sequence from the C-terminal residue of the AHBc sequence present and within about 30 residues from the C-terminus of the chimer molecule, referred to herein as the"C- terminal cysteine residue (s) " . Preferably, the arginine rich region following residue 214 is deleted, but at least one cysteine residue is introduced thereafter.

The contemplated chimer molecules (i) contain no more than 20 percent substituted amino acid residues in the AHBc sequence, and (ii) self- assemble on expression in a host cell into particles that are substantially free of binding to nucleic acids. In a preferred embodiment, those particles bind less nucleic acid than the corresponding full- length particle, and most preferably are substantially free of binding to nucleic acids. One or both of the chimer molecule dimers and particles are more stable than are dimers or particles formed from an otherwise identical AHBc chimer that lacks the above C-terminal cysteine residue (s) or where a C-terminal cysteine residue is present in the chimer and is replaced in the molecule by another residue such as an alanine residue.

In one aspect of this embodiment, a contemplated AHBc chimer has a sequence of about 175 to about 635 amino acid residues and contains four serially peptide-linked domains that are denominated Domains I, II, III and IV. From the N-terminus, Domain I comprises about 71 to about 100 amino acid residues whose sequence includes at least the sequence of the residues corresponding to about position 5 through position 75 of the avian HBc shown in Fig. 1 (SEQ ID NO:7), and optionally includes a heterologous epitope containing up to about 30 amino acid residues peptide-bonded to one of HBc residues 1-4. Domain II comprises 5 to about 250 amino acid residues peptide-bonded to avian HBc residue 75 of Domain I in which (i) zero to all, and preferably at least 4, residues in a sequence of avian HBc corresponding to positions 76 to 85 of SEQ ID NO: 7 are present peptide-bonded to one to about 245 amino acid residues that are heterologous (foreign) to AHBc and constitute a heterologous epitope such as a B cell epitope or a heterologous linker residue for an epitope such as a B cell epitope or (ii) the sequence of AHBc at positions 76 to 85 is present free from heterologous residues. Domain III corresponds tothe avian HBc sequence of SEQ ID NO: 7 from position 86 through position 135 peptide-bonded to residue 85 of Domain II. Domain IV comprises (i) zero through fourteen residues of an avian HBc amino acid residue sequence corresponding to SEQ ID NO: 7 from position 136 through 149 peptide-bonded to the residue of position 135 of Domain III, (ii) one to ten, and more preferably one to three, cysteine residues peptide- bonded C-terminal to that AHBc sequence [C-terminal cysteine residue(s)] and (iii) zero to about 100, more preferably zero to about 50, and most preferably about 25 amino acid residues in a sequence heterologous to avian HBc corresponding to from position 150 of SEQ ID NO : 7 to the C-terminus, with the proviso that Domain IV contain at least 6 amino acid residues including the above one to ten cysteine residues of (ii) . In a preferred embodiment, a contemplated recombinant chimer protein forms particles that are associated with signficantly less, and preferably substantially free of binding to, nucleic acids, and form more stable dimers an/or particles than those formed from an avian HBc chimer containing the same peptide-linked Domain I, II and III sequences and a Domain IV sequence that is otherwise same but lacks any cysteine residues or in which a cysteine residue is replaced by another residue such as an alanine residue. The particles formed are believed to be of the T = 4 structure, containing 240 monomeric AHBc chimers or 120 dimer AHBc chimers.

More broadly, a contemplated chimer particle comprises a C-terminal truncated AHBc protein (to at least residue 205) that contains a heterologous epitope or a heterologous linker residue for an epitope in the immunodominant loop, or an uninterrupted immunodominant loop, and regardless of the amino acid residue sequence of the immunodominant loop, one to three C-terminal cysteine residues heterologous to the AHBc sequence. Such a particle exhibits a 280/260 absorbance ratio of about 1.2 to about 1.7 and is more stable than a particle formed from an otherwise identical AHBc chimer that lacks the above C-terminal cysteine residue (s) or where a single C-terminal cysteine residue is present in the chimer and is replaced by another residue.

Another embodiment comprises an inoculum or vaccine that comprises an above AHBc chimer particle or a conjugate of a hapten with an above AHBc chimer particle that is dissolved or dispersed in a pharmaceutically acceptable diluent composition that typically also contains water. When administered in an immunogenic effective amount to an animal such as a mammal or bird, an inoculum (i) induces antibodies that immunoreact specifically with the chimer particle or the conjugated (pendently-linked) hapten or (ii) activates T cells , or (iii) both. The antibodies so induced also preferably immunoreact specifically with (bind to) an antigen containing the hapten, such as a protein where the hapten is a peptide or a saccharide where the hapten is an oligosaccharide .

The present invention has several benefits and advantages .

One advantage of the invention is that chimer AHBc particles used in mammals, for example in a vaccine, elicits lower cross-reactivity with mammalian HBc.

One benefit of the invention is that chimer AHBc particles are formed that are more stable on storage in aqueous compositions than are particles of similar sequence that lack any N-terminal (other than pre-core) or C-terminal cysteine residues.

An advantage of the invention is that chimer molecules are prepared that exhibit the self- assembly characteristics of native AHBc particles, while not exhibiting the nucleic acid binding of those native particles.

Another benefit of the present invention is that chimer particles are formed that exhibit excellent B cell and T cell immunogenicities.

Another advantage is that chimer particles of the present invention are typically prepared in higher yield than are similar particles that are free of an N- or C-terminal cysteine residue. A further benefit of the invention is that chimer particles are formed that are often far more immunogenic than are similar conjugates that lack a N- or C-terminal cysteine residue.

A further advantage is that immunogenicities of particles assembled from chimer molecules containing at least one N- or C-terminal cysteine residue are enhanced as compared to similar particles assembled from chimer molecules lacking at least one C-terminal cyeteine residue.

Still further benefits and advantages will be apparent to the skilled worker form the disclosure that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings forming a portion of this disclosure :

Fig. 1 provides an alignment of two published sequences for avian HBc proteins from duck (SEQ ID NO: 8) and heron (SEQ ID NO: 9), along with a consensus sequence (SEQ ID NO: 7) .

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 some recombinant avian HBc chimers. The modified sequence (SEQ ID NO: 10) is shown below the sequence of the commercially available vector (SEQ ID NO: 11) . The bases of the added JVcoI site are shown in lower case letters with all of the added bases being 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 Hindlϊl ) are indicated. Fig. 3, shown in three panels as Figs. 3A, 3B and 3C, schematically illustrates a preferred cloning strategy in which a malarial B cell epitope such as (NANP)₄ (SEQ ID NO:47) is cloned into the

.EcoRI and Sacl sites of an engineered HBc gene (Fig. 3A) between positions 95 and 96. Fig. 3B shows DNA that encodes a T cell epitope such as that referred to as Pf/CS-UTC and a stop codon (SEQ ID NO: 192) cloned into the EcoRI and HindiII sites at the C-terminus of an engineered, truncated duck HBc gene containing the first 214 HBc residues (DHc214) . PCR amplification of the construct of Fig. 2B using a primer having a 5 ' -terminal Sacl restriction site adjacent to an AHBc-encoding sequence beginning at residue position 96 digestion of the amplified sequence and the construct of Fig. 2A with Sacl, followed by ligation of the appropriate portions is shown in Fig. 2C to form a single gene construct that encodes B cell- and T cell-containing epitopes of an immunogen for a vaccine against P. falciparum. Fig. 4 illustrates a reaction scheme (Scheme 1) that shows two reaction sequences for (I) forming an activated carrier for pendently linking a hapten to a chimeric avian hepatitis B core protein (sm-AHBc) particle using sulpho-succinimidyl 4- (N-maleimidomethyl) cyclohexane 1-carboxylate (sulpho-SMCC) , and then (II) linking a sulfhydryl- terminated (cysteine-terminated) hapten to the activated carrier to form a conjugate particle. The sm-AHBc particle is depicted as a box having a single pendent amino group (for purposes of clarity of the figure) , whereas the sulfhydryl-terminated hapten is depicted as a line terminated with an SH group. Fig. 5 illustrates a synthetic version of the full-length hepatitis duck core gene (DHc gene; SEQ ID NO: 196), which encodes amino acids 1-262 of SEQ ID NO: 8. The DHc gene is chemically synthesized using codons that are most abundant in E. coli genes. In addition to using the codons favored by E. coli , the gene is also designed to avoid continuous stretches of single codons. The synthetic gene is flanked by Ncol and HindiII restriction sites (underlined) , to facilitate insertion into the pKK223-3N expression plasmid. In addition, two termination codons were introduced following amino acid K-262 to ensure efficient termination of translation.

DEFINITIONS

Numerals utilized in conjunction with AHBc chimers indicate the position as it correlates to the consensus AHBc amino acid residue sequence of SEQ ID NO: 7 at which one or more residues¹ has been added to the sequence, regardless of whether additions or deletions to the amino acid residue sequence are present. Thus, AHBc214 indicates that the chimer ends at residue 214, whereas AHBc214 + C215 indicates that the same chimer contains a cysteine residue at AHBc position 215. On the other hand, the malarial CS protein universal T cell epitope (UTC) is 20 residues long, and a replacement of the cysteine at position 17 in that sequence by an alanine is referred to as CS-UTC (C17A) .

The term "antibody" refers to a molecule that is a member of a family of glycosylated proteins called immunoglobulins, which can specifically bind to an antigen. The word "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" . The words "antigenic determinant" and "epitope" are used somewhat more broadly herein to include additional residues that are heterologous to the AHBc sequence but may not actually be bound by an antibody. Thus, for example, the malarial CS protein repeat sequences (NANP) 4 and

NANPNVDP(NANP)₃NVDP of SEQ ID Nos : 47 and 48 are each , thought to contain more than one actual epitope, but are considered herein to each constitute a single epitope. Use of both of those sequences in a single HBc chimer molecule is considered to be a use of a plurality of epitopes.

The word "conjugate" as used herein refers to a hapten operatively linked to a carrier protein, as through an amino acid residue side chain of the carrier protein such as a lysine, aspartic or glutamic acid, tyrosine or cysteine residue. The term "conservative substitution" as used herein denotes that one amino acid residue has been replaced by another, biologically similar ' residue. Examples of 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.

The term "corresponds" in its various grammatical forms as used in relation to peptide sequences means the peptide sequence described plus or minus up to three amino acid residues at either or both of the amino- and carboxy-termini and containing only conservative substitutions in particular amino acid residues along the polypeptide sequence.

The term "Domain" is used herein to mean a portion of a recombinant AHBc chimer molecule that is identified by (i) residue position numbering relative to the position numbers of the consensus AHBc sequence shown in Fig. 1 (SEQ ID NO: 7) . 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 avian and mammal HBcAg.

As used herein, the term "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 AHBc chimers that induce the production of antibodies that immunoreact with a polypeptide or pathogen-related immunogen that corresponds in amino acid residue sequence to the polypeptide or pathogen-related portion of the fusion protein.

The phrase "hepatitis B" as used here refers in its broadest context to any member of the family hepadnaviridae, as discussed before.

The term "residue" is used interchangeably with the phrase amino acid residue, and means a reacted amino acid as is present in a peptide or protein.

As used herein, the term "expression vector" means a DNA sequence that forms control elements that regulate expression of a structural gene that encodes a protein so that the protein is formed .

As used herein, the term "operatively linked" used in the context of a nucleic acid means that a gene is covalently bonded in correct reading frame to another DNA (or RNA as appropriate) segment, such as to an expression vector so that the structural gene is under the control of the expression vector. The term "operatively linked" used in the context of a protein, polypeptide or chimer means that the recited elements are covalently bonded to each other.

As used herein, the term "promoter" means a recognition site on a DNA sequence or group of DNA sequences that provide an expression control element for a gene and to which RNA polymerase specifically binds and initiates RNA synthesis (transcription) of that gene . As used herein, the term "recombinant DNA molecule" means a hybrid DNA sequence comprising at least two nucleotide sequences not normally found together in nature .

As used herein, the term "vector" means a DNA molecule capable of replication in a cell and/or to which another DNA segment can be operatively linked so as to bring about replication of the attached segment. A plasmid is an exemplary vector.

All amino acid residues identified herein are in the natural L-configuration and standard nomenclature is used, following World Intellectual Property Organization (WIPO) Handbook on Industrial Property Information and Documentation, Standard ST.25: Standard for the Presentation of Nucleotide and Amino Acid Sequence Listings in Patent Applications (1998) , incorporated herein by reference.

DETAILED DESCRIPTION OF THE INVENTION

The present invention contemplates a chimeric avian hepadnavirus nucleocapsid protein; i.e., a recombinant avian hepatitis B core (AHBc) protein, that is engineered to (a) display an immunogenic B cell or T cell epitope, or a linker for attachment of an immunogenic B cell or T cell epitope, (b) exhibit enhanced stability of one or both of the dimer and particle , as well as (c) exhibit a substantial absence of nucleic acid binding as a self-assembled particle. A contemplated AHBc chimer is typically truncated at the C-terminus of the molecule relative to a native AHBc molecule.

A chimeric protein displays one or more immunogenic epitopes at the N-terminus, in the AHBc region from about residue 85 to about residue 135 or at the C-terminus, or a linker for such a B cell or T cell epitope in the loop region from about residue 85 to about residue 135, in some embodiments, from about residue 90 to 120, more preferably 95 to 110.

For dimer and/or particle stabilization through introduction of cysteine residues, the chimeric protein contains a cysteine residue at or near the N-terminus, C-terminus or N- and C-termini, and/or in the AHBc region from about residue 55 to about residue 65. The cysteine (s) confer enhanced stability to one or both of the self-assembled dimers and particles formed by the core protein.

For minimizing impurities due to unintentionally bound nucleic acids, the chimeric protein is sufficiently free of arginine and or lysine residues downstream of (toward the carboxy- terminus from) AHBc residue position 214 so that the self-assembled particles are substantially free of nucleic acid binding.

For. ease of discussion, contemplated chimer sequences and sequence position numbers referred to herein are based on the sequence and position numbering of the consensus avian hepatitis B core protein that is shown in SEQ ID NO: 7. The consensus AHBc sequence is shown aligned with the sequences from duck and heron in Fig. 1. It is to be understood, however, that in view of the similarity between the avian hepadnavirus capsid protein sequences and similar particle formation exhibited by those proteins, which are well-known to skilled workers, a discussion regarding duck is also applicable to heron. As a consequence of those great similarities, the avian HBc sequences are recited generally herein as a "AHBc" sequences. There are significant similarities between the avian and mammalian hepatitis B core protein sequences, as well .

In one embodiment, a contemplated AHBc chimer is up to about 635 residues in length and contains

(a) an AHBc sequence of at least about of the N-terminal amino acid residues of the AHBc molecule that includes (i) the AHBc sequence of residue positions 5 through about 85 and about 130 through about 190, (ii) a peptide-bonded heterologous epitope (1 to 250 amino acid residues in length) at one or more of the N-terminus, in the region of about residue 75 to 130, or the C-terminus of the chimer, or (iii) a heterologous linker residue for a conjugated epitope present in the region of about residue 75 to 130, and

(b) one to three cysteine residues at an amino acid position of the chimer molecule corresponding to amino acid position -30 to about +1 from the N-terminus of the AHBc sequence of SEQ ID N0:7 [N-terminal cysteine residue (s) ] in a sequence other than that of the heron HBc sequence that aligns upstream of the consensus AHBc sequence and zero to about three cysteine residues toward the C-terminus of the molecule from the C-terminal residue of the AHBc sequence and within about 30 residues from the C-terminus of the chimer molecule [C-terminal cysteine residue (s) ] and/or a cysteine residue at an amino acid position of the chimer molecule corresponding to amino acid position 55 to 65 of the AHBc sequence . That chimer molecule (i) contains no more than about 20 percent conservatively substituted amino acid residues in the AHBc sequence, (ii) self- assembles into particles that are substantially free of binding to nucleic acids on expression in a host cell . One or both of the dimers and particles are more stable upon formation than are particles formed from otherwise identical AHBc chimer molecules that are free of any above-mentioned cysteine residue (s) , and lack a cysteine residue (s) present in the consensus AHBc sequence or where a consensus cysteine is (are) replaced by another residue.

A contemplated chimer molecule contains a cysteine residue at a position of about -30 to about +1 relative to the N-terminus of the consensus AHBc as is illustrated in Fig. 1 and SEQ ID NO: 7. The concept of a negative amino acid position is usually associated with a leader sequence such as the precore sequence of mammalian HBc. That concept is used similarly here in that one can simply align a given chimer molecule sequence with that of SEQ ID NO: 7 to determine the position of the chimer that corresponds to that of the starting methionine residue of position +1 of HBc. Inasmuch as amino acid residue sequences are normally shown from left to right and in the direction from N-terminus to C-terminus, any aligned chimer molecule residue to the left of the position occupied by the AHBc start methionine has a negative position. Following this convention, the heron sequence shown in Fig. 1 begins at position - 43. A contemplated cysteine residue can occur at a position about thirty residues to the left of the aligned start methionine of HBc to the position corresponding to that start methionine . In one aspect, a preferred AHBc chimer has a sequence of about 175 to about 256 L-α-amino acid residues and contains four serially peptide-linked domains; i.e., Domains I, II, III and IV. Those four domains are linked together in the same manner as are native proteins, as compared to polypeptides that contain residues of other than α-amino acids and therefore cannot form peptide bonds, those that contain D-amino acid residues, or oligopeptide conjugates in which two or more polypeptides are operatively linked through an amino acid residue side chain. A contemplated chimeric AHBc protein can therefore be prepared by expression using the usual methods of recombinant technology.

From the amino-terminus, Domain I comprises about 81 to about 110 amino acid residues whose sequence includes at least the sequence of the residues of position 5 through position 85 of HBc. Preferably, the sequence of residues 1 through 85 of the AHBc sequence is present as part of Domain I . Most preferably, Domain I is comprised only of the AHBc sequence from position 1 through position 85.

Domain II comprises 1 to 290 amino acid residues, peptide-bonded to AHBc residue 85 of Domain I of which (i) zero to all of the residues, and preferably at least 10 residues, and more preferably at least 20 residues, in a sequence of AHBc at positions 86 through 130 are present peptide-bonded to one to about 245 residues that are heterologous (foreign) to AHBc and constitute a heterologous linker residue for an epitope such as a B cell epitope or a heterologous epitope such as a B cell epitope itself or (ii) from one to all of the residues, and preferably at least 10 residues, more preferably at least 20 residues, of the sequence of AHBc at positions 86 through 130 is present free from heterologous residues .

In some embodiments, at least 10 residues, preferably at least 20 residues of the AHBc sequence from positions 86 through 130 are present in Domain II; resulting in Domain II length ranges of 11 to 255 residues or 21 to 265 residues, respectively. In some embodiments, a heterologous epitope is peptide- bonded in the included portion of the AHBc sequence, and that epitope is not longer than 50 amino acid residues, or not longer than 20 amino acid residues; resulting in Domain II length ranges of 50 to 95 residues, or 20 to 65 residues, respectively.

In a contemplated embodiment the complete AHBc sequence of 46 residues of positions 86 through 130 (86-130 sequence) is present, but interrupted by one to about 245 residues of the heterologous linker or heterologous epitope. In other instances, it is particularly preferred that that 45 residue sequence be present alone, uninterrupted by any heterologous residue .

A chimer containing only AHBc residues in this Domain II together with the features discussed below is useful for inducing a B and/or T cell response to AHBc itself. A preferred AHBc chimer molecule with an uninterrupted 86-130 sequence contains the uninterrupted AHBc amino acid residue sequence of position 1 through at least position 205, and more preferably contains the uninterrupted HBc amino acid residue sequence of position 1 through position 214, plus a single cysteine residue at the C-terminus, as discussed below. Domain III is an AHBc sequence from position 131 through position 205 peptide-bonded to the C-terminal residue of Domain II.

Domain IV comprises (i) zero to fifty-six residues, preferably five to thirty residues, more preferably eight to eighteen residues, of a AHBc amino acid residue sequence from position 206 through 262 peptide-bonded to the residue of Domain III corresponding to position 205 of the AHBc sequence, (ii) one to ten cysteine residues [C-terminal cysteine residue (s) ] , and (iii) zero to about 100 amino acid residues in a sequence heterologous to AHBc from the C-terminal residue that corresponds to AHBc to the C-terminus of the chimer that typically constitute one T cell epitope or a plurality of T cell epitopes, with the proviso that Domain IV contains at least a sequence of 6 amino acid residues from HBc residue position 206 to the C-terminus of the chimer, including the above one to ten cysteine residues of (ii) . Preferably, Domain IV contains a sequence of zero to about 50 amino acid residues in a sequence heterologous to AHBc, and more preferably that sequence is zero to about 25 residues.

In one aspect, a contemplated chimer molecule can thus be free of epitopes or residues heterologous to HBc, except for the C-terminal cysteine. In another aspect, a contemplated chimer molecule contains a heterologous epitope at the N-terminus peptide-bonded to one of AHBc residues 1 to 5. In a further aspect, a contemplated chimer molecule contains a heterologous epitope or a heterologous linker residue for an epitope peptide- bonded near the middle of the molecule located between AHBc residues 86 and 130 in the immunodominant loop. In a still further aspect, a heterologous epitope is located at the C-terminal portion of the chimer molecule peptide-bonded to one of AHBc residues 205-262. In yet other aspects, two or three heterologous epitopes are present at the above locations, or one or two heterologous epitopes are present along with a heterologous linker residue for an epitope. Each of those chimer molecules also contains a C-terminal cysteine residue (s), as discussed before. Specific examples of several of these chimer molecules and their self-assembled particles are discussed hereinafter.

As already noted, a contemplated AHBc chimer molecule can contain about 175 to about 256 amino acid residues. In preferred embodiments, AHBc residues 1-5 are present, so that Domain I begins at AHBc residue 1 and continues through residue 85; i.e., the AHBc residue at AHBc position 85. The heterologous epitope present in Domain II in the immunodominant loop preferably contains about 15 to about 50 residues, although an epitope as short as about 6 amino acid residues can induce and be recognized by antibodies and T cell receptors. Domain III contains AHBc residues 131 through 205 peptide-bonded to the C-terminal residue of Domain II. Domain IV contains a sequence of at least six residues that are comprised of (i) zero, one or a sequence of the residues of AHBc positions 206 through 262 peptide-bonded to the C-terminal residue of Domain III, (ii) at least one cysteine residue and (iii) optionally can contain a heterologous sequence of an epitope of up to about 100 residues, particularly when the AHBc sequence ends at residue 205, although a shorter sequence of up to about 25 residues is more preferred.

In one embodiment, 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. In other embodiments, one of the two heterologous epitopes is a T cell epitope. More preferably, one of the two heterologous epitopes is a B cell epitope and the other is a T cell epitope. In addition, 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.

In the embodiments in which the chimer molecule contains a heterologous epitope in Domain II, it is preferred that that epitope be one or more B cell epitopes, that the HBc sequence between amino acid residues 86 and 130 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 AHBc residues 205-262, preferably a residue closer to 205 than 262, such as 214 or 224.

This same preference holds for those chimer molecules in which the heterologous linker residue for a conjugated epitope is present in Domain II, thereby providing one or more heterologous epitopes in Domain II, with residues 86 through 130 present, but interrupted by the heterologous linker residue, with a T cell epitope being present peptide-bonded to one of AHBc residues at 205 or a later position. The particles formed from such chimer molecules typically contain a ratio of conjugated epitope to C-terminal peptide-bonded T cell epitope of about 1:4 to 1:1, with a ratio of about 1:2 being common.

In an illustrative structure of an above- described chimer molecule, a heterologous linker residue for a conjugated epitope is present in Domain II and a T cell epitope is present in Domain IV, with no additional B cell epitope being present in Domain II. Such a chimer exhibits immunogenicity of the T cell epitope, while exhibiting minimal, if any, AHBc antigenicity as measured by binding of anti-loop monoclonal antibodies in an ELISA assay as discussed hereinafter.

A preferred contemplated AHBc chimer molecule contains a sequence of about 140 to about 635 residues. A preferred AHBc chimer molecule containing two heterologous epitopes of preferred lengths of about 15 to about 50 residues each and a preferred AHBc portion length of about 140 to about 149 residues has a sequence length of about 175 to about 240 amino acid residues. Particularly preferred chimer molecules continuing two heterologous epitopes have a length of about 190 to about 210 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 AHBc portions and differing lengths of the several contemplated epitopes that can be inserted in the immunogenic loop.

A contemplated recombinant protein, after expression in a host cell, self-assembles to form particles that are substantially free of binding to nucleic acids. The contemplated AHBc chimer particles are generally spherical in shape and are usually homogeneous in size for a given preparation. , These chimeric particles thus resemble native HBc particles that have a similar shape and size and can be recovered from infected persons .

A contemplated chimer particle comprises dimers of the previously discussed chimer molecules. More broadly, such a chimer particle comprises dimers of a chimeric C-terminal truncated HBc protein that has a sequence of at least about 190 of the N-terminal 205 residues and contains (i) a heterologous epitope or a heterologous linker residue for an epitope in the immunodominant loop, or at least about 190 of the N-terminal 205 residues and an uninterrupted immunodominant loop and (ii) one to three C-terminal cysteine residues as previously described, and at least a 5 HBc residue sequence from position 190. Such a particle is preferably sufficiently free of arginine residues so that the self-assembled particles are substantially free of nucleic acid binding and exhibits a 280/260 absorbance ratio of about 1.2 to about 1.7, as discussed herein after. Thus, a contemplated chimeric protein can be free of the AHBc sequence between positions 205 and 262.

A contemplated dimer or particle is more stable than a respective dimer or particle formed from an otherwise identical HBc chimer protein that lacks the above C-terminal cysteine residue (s). Similarly, a dimer or particle whose chimer molecule contains a single C-terminal cysteine residue is more stable than a respective dimer or particle in which that cysteine is replaced by another residue such as an alanine residue. In some instances, dimers or particles do not form unless a C-terminal cysteine is present. Examples of enhanced stabilities for both types of sequences are illustrated in the Examples that follow and is particularly evident in Examples relating to the Figures.

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 preferably 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. Such 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.

Thus, recombinantly expressed AHBc particles or chimeric AHBc particles that contain the arginine-rich sequence at residue positions 205-262 (or 214-262 or 224-262) sometimes referred to in the art as the protamine region exhibit a ratio of absorbance at 280 nm to absorbance at 260 nm (280/260 absorbance ratio) of about 0.8, whereas preferred particles sufficiently free of arginine residues so that the self-assembled particles are substantially free of nucleic acid binding such as particles that are free of the arginine-rich nucleic acid binding region of naturally occurring AHBc like 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 AHBc residue position 205 to position 224, exhibit a 280/260 absorbance ratio of greater than one.

Preferably, chimeric AHBc 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 preferably, 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 dimers are more stable in aqueous buffer at 37°C over a time period of about two weeks to about one month than are dimers 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 ten cysteine residues [C-terminal cysteine residue (s) ] or [N-terminal cysteine residue) s) ] are absent or the N- or C-terminal cysteine residue present is replaced by another residue such as an alanine residue. Stability of various chimer dimers and particles can be determined by use of size- exclusion chromatography and SDS-PAGE electrophoresis under non-reducing conditions.

Solution chromatography, such as the size- exclusion chromatography discussed in the examples below, can be used to separate assembled particles from free monomers and other impurities or small molecules. As the protein degrades, for example through acid hydrolysis or oxidation, the particles disintegrate and the protein is even more susceptible to degradation.

Under non-reducing conditions, the cysteine-cysteine cross-links are intact, and the stability of the dimers can be observed directly through size exclusion gel chromatography, or gel electrophoresis, using techniques well-known in the art .

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 analytical gel filtration analysis using Superose^® 6 HR as discussed hereinafter. The increased yield also leads to an increase in purity, as the same co-purifying impurities make up respectively less of the purified particle samples.

Domain I of a contemplated chimeric AHBc protein constitutes an amino acid residue sequence of AHBc beginning with at least amino acid residue position 5 through position 85, and Domain III constitutes a HBc sequence from position 130 through position 205. The sequences from any of the avian hepadnaviruses can be used for either of Domains I and III, and sequences from two or more viruses can be used in one chimer. Preferably, and for ease of construction, the same avian sequence is used throughout the chimer .

HBc chimers having a Domain I that contains more than a deletion of the first three amino- terminal (N-terminal) residues have been reported to result in the complete disappearance of HBc chimer protein in E. coli cells. Pumpens et al . , (1995) Intervirology, 38:63-74. On the other hand, a recent study in which an immunogenic 23-mer polypeptide from the influenza M2 protein was fused to the HBc N-terminal sequence reported that the resultant fusion protein formed particles when residues 1-4 of the native HBc sequence were replaced. Neirynck et al. (October 1999) Nature Med. , 5 (10) : 1157-1163. Thus, the art teaches that particles can form when an added amino acid sequence is present peptide-bonded to one of residues 1-4 of HBc, whereas particles do not form if no additional sequence is present and more than residues 1-3 are deleted from the N- terminus of HBc .

An N-terminal sequence peptide-bonded to one of the first five N-terminal residues of AHBc can contain a sequence of up to about 25 residues that are heterologous to AHBc. Exemplary sequences include a B cell or T cell epitope such as those discussed hereinafter, the 23 -mer polypeptide from the influenza M2 protein of Neirynck et al . , above, a sequence of another (heterologous) protein such as β-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 sequence.

Domain II is a sequence of about 1 to about

290 amino acid residues. Of those residues, anywhere from zero (none) to all of the AHBc residues from position 86 to 130 are present, and preferably at least 4 residues, and more preferably at least 10 or

20 residues, constitute portions of the AHBc sequence i at positions 86 to 130, and one to about 245 residues, and preferably one to about 50 residues are heterologous (foreign) to AHBc. Those heterologous residues constitute (i) a heterologous linker residue for a epitope such as a B cell or T cell epitope or (ii) a heterologous B or T cell epitope that preferably contains 6 to about 50, more preferably about 15 to about 50, and most preferably about 20 to about 30 amino acid residues, and are positioned so that they are peptide-bonded between zero, or more preferably at least 4, to all of the residues of positions 86 through 130 of the AHBc sequence. Heterologous B cell epitopes are preferably linked at this position by the linker residue or are peptide- bonded into the HBc sequence, and use of a B cell epitope is discussed illustratively hereinafter.

The one to about 245 residues added to the AHBc loop sequence is (are) heterologous to a AHBc sequence. A single added heterologous residue is a heterologous linker residue for a B cell epitope as discussed before. The longer sequences, typically at least 6 amino acid residues long to about 50 amino acid residues long and more preferably about 15 to about 50 residues in length, as noted before, are in a sequence that comprises a heterologous immunogen such as a B cell epitope, except for heterologous residues encoded by restriction sites.

Exemplary peptide immunogens useful for both linkage to the linker residue after expression of a contemplated chimer and for expression within a HBc chimer are illustrated in Table A, below, along with the common name given to the gene from which the sequence is obtained, the literature or patent citation for published epitopes, and SEQ ID NOs. Table A

B Cell Epitopes

SEQ ID

Organism Gene Sequence Citation* NO

Streptococcus pneumoniae

PspAl

KLEELSDKIDELDAE 1 12

PsP2 QKKYDEDQKKTEE- KAA EKAASEE -

DKAVAAVQQA 1 13

Cryp t ospori di um parvum

P23

QDKPADAPAAEAPA-

AEPAAQQDKP DA 2 14

HIV GP120

RKRIHIGPGR-

AFYITKN 3 15

Foot-and-mouth virus VP1

YNGECRYNRNA-

VPN RGDLQVL-

AQKVARTLP 4 16

Influenza Virus Type A HA

(A8/PR8) YRNLLWLTEK 8 17

Type A M2 (A8/PR8/34) S TEVETPIR-

NE GCRCNGSSD 29 18 SLLTEVETPIR-

NE GCRCNDSSD 29 19 SLLTEVETPIR-

NE GARANDSSD 20 EQQSAVDADDS-

HFVSIELE 35 21 SLLTEVETPIR- SLLTEVETPIR-

NE GSRSNDSSD 22 SLLTEVETPIR-

NE GSRCNDSSD 23 SLLTEVETPIR-

NEWGCRSNDSSD 24 S LTEVETPIR-

NEWGCRANDSSD 25 SLLTEVETPIR-

NEWGARCNDSSD 26 MS LTEVETPIR-

NE GCRCNDSSD 27 SL TEVETPIR-

NEWGSRSNDSSD 28 MGISLLTEVETPIR-

NEWGCRCNDSSD-

ELLGW WGI 29 MS LTEVETPIR-

NE GARANDSSD 30 MSLLTEVETPIR-

NΞWGCRANDSSD 31 MSLLTEVETPIR-

NE GARCNDSSD 32 MSLLTΞVETPIR-

NEWGCRSNDSSD 33 MSLLTEVETPIR-

NE GSRCNDSSD 34

^X1^X2^X3^X4^X5^X6^X7^X8^T

X-i nX"11 13^Λ14^X15^X16^X17^X1-

^X19^x20^x21-^X22^x23^x24 35

Type B NB NNATFNYTNVNPISHIR 36

Yersinia pestis V Ag

DILKVIVDSMNHH- GDARSKLREELAE- TAELKIYSVIQA- EINKH SSSGTIN- IHDKSINLMDKNL- YGYTDEEIFKASA- EYKILΞKMPQTTI- QVDGSEKKIVSIK- DFLGSENKRTGAL- GNLKNSYSYWKDN- NELSHFATTCSD 37 Haemophilus influenza pBOMP

CSSSNNDAA-

GNGAAQFGGY 10 38

NK GTVSYGEE 39

NDEAAYSKN-

RRAVLAY 40

Moraxella catarrhalis copB DIEKDKKK- RTDEQLQAE- DDKYAGKGY 11 41 LDIEKNKKK- RTEAELQAE- LDDKYAGKGY 42 IDIEKKGKI- RTEAEL AE- LNKDYPGQGY 43

Porphyromona s gingivalis HA

GVSPKVCKDVTV- EGSNEFAPVQNLT 12 44 RIQST RQKTV- D PAGTKYV 45

Trypanosoma cruzi KAAIAPAKAAA- APAKAATAPA 14 46

Plasmodium falciparum CS

(NANP) ₄ 24 47

NANPNVDP- (NANP) ₃NVDP 48

NANPNVDP- (NANP) ₃ 49

(NANP) 3NVDPNANP 50

NANPNVDP- (NANP) 3NVDPNANP 51

NPNVDP (NANP) 3NV 52

NPNVDP- (NANP) 3NVDP 53

NPNVDP (NANP) 3-

NVDPNA 54

NVDP (NANP) 3NV 55

NVDP (NANP) 3NVDP 56

NVDP (NANP) 3 _

NVDPNA 57 DP(NANP)₃NV 58

DP(NANP)₃NVDP 59

DP (NANP) 3-

NVDPNA 60 vivax CS

GDRADGQPAG-

DRADGQPAG 20 61

RADDRAAGQP-

AGDGQPAG 62

ANGAGNQPG-

ANGAGDQPG 63

ANGADNQPG-

ANGADDQPG 27 64

ANGAGNQPG-

ANGADNQPG 65

ANGAGNQPG-

ANGADDQPG 66

APGANQEGGAA-

APGANQEGGAA 28 67

ANGAGNQPGAN-

GAGDQPGANGA-

DNQPGANGADD-

QPG 68 berghi CS

DPPPPNPN- DPPPPNPN 69 yoelli CS

(QGPGAP) ₄ 70

Streptococcus sobrinus Agl/ll

KPRPIYEA- KLAQNQK 16 71 AKADYEAK- LAQYEKDL 72

Shigella flexneri Invasin

KDRT IEQK 18 73

Respiratory syncitia virus (RSV) G CSICSNNPT- CWAICK 19 74

Entamoeba histolytica lectin

VECASTVCQNDN- SCPIIADVEKCNQ 21 75

Schistosoma japonicum para

DLQSEISLSLE- NGELIRRAKSA- ESLASELQRRVD 22 76

Schistosoma mansoni para DLQSEIS SLE- NSELIRRAKAA- ESLASDLQRRVD 22 77 Bovine Inhibin a_c subuni t STPPLP P -

SPAALRLLQ- RPPEEPAA 30 78

Ebola Virus membrane -anchored glycoprotein ATQVEQHHRR-

TDNDSTA 31 79

HNTPVYKLD-

ISEATQVE 31 80

GKLG ITNTI-

AGVAVLI 31 81

Escheri hi a col i

ST CCELCCYPACAGCN 33 82 NTFYCCELCC-

YPACAGCN 33 83

SSNYCCE CC-

YPACAGCN 33 84

Alzheimer's disease β-Amyloid DAEFRHDSGYE- 34 85

VHHQKLVFFAE-

DVGSNKGAIIG-

LMVGGWIA

DAEFRHDSGYE- 86

VHHQKL

EDVGSNKGAII 87

DAEFRHDSGYE- 88

VHHQKLVFFAE-

DVGSNKGAIIG

Neisseria meningitidis PorA YVAVΞNGVAKKVA 89

HFVQQTPKSQPTLVP 90

H WNNKVATHVP 91

PLQNIQPQVTKR 92

AQAANGGAASGQVKVTKVTKA 93

YVDEQSKYHA 94

HFVQNKQNQPPT VP 95

KPSSTNAKTGNKVEVTKA 96

YWTTVNTGSATTTTFVP 97

YVDEKKKMVHA 98

HYTRQNNADVFVP 99

YYTKDTNNN TLVP 100

PPQKNQSQPWTKA 101

PPSKGQTGNKVTKG 102

PPSKSQPQVKVTKA 103

QPQTANTQQGGKVKVTKA 104

QPQVTNGVQGNQVKVTKA 105

QPSKAQGQTNNQVKVTKA 106

PPSSNQGKNQAQTGNTVTKA 107

PPSKSQGKTGNQVKVTKA 108

PPSKSQGTNNNQVKVTKA 109

PPSKSQPGQVKVTKVTKA 110

QLQLTEQPSSTNGQTGNQVKVT-KA 111

QLQLTEAPSKSQGAASNQVKVT-KA 112

SAYTPAHVYVDNKVAKHVA 113

SAYTPAHFVQNKQNNNPTLVP 114

VEGRNYQLQLTE 115

PAQNSKSAYTPA 116

QLQLTEPPSKNQAQTQNKVTKA 117

GRDAFELFLLGSGSDE 118

RHANVGRDAFELFLLGSGSDEA-

KGTDPLKNH 119

GRDAFNLFLLGRIGDDDE 120

GRNAFELFLIGSATSDQ 121

QVKVTKAKSRIRTKI 122

TLVPAWGKPGSD 123

NspA HAKASSSLGSAKGFS R 124

TRYKNYKAPSTDFKL 125

SLNRASVDLGGSDSFSQT 126

GKVNTVKNVRSGELSAGVRVK 127

GKVNTVKNVRSGELSVGVRVK 128

Immunoglobulin E

APE PGSRDKRTL 129

EDGQVMDVD 130

STTQEGEL 131

GHTFEDSTKK 132

GGGHFPPT 133

PGTINI 134

FTPPT 135

INHRGY V 136

GEFCINHRGY VCGDPA 137

MAPE PGSRDKRTL 138

MEDGQVMDVD 139

MSTTQEGEL 140

MGHTFEDSTKK 141

MGGGHFPPT 142

MPGTINI 143

MFTPPT 144

MINHRGY V 145

MGEFCINHRGYWVCGDPA 146

*Citations to published epitopes are provided following Table B.

In the above influenza A M2 sequence of SEQ ID NO: 35, X^ through Xg are absent or present, and when present are the residues naturally present in a reported M2 protein sequence; i.e., methionine, serine, leucine, leucine, threonine or proline, glutamic acid, valine, and glutamic acid, respectively, with the proviso that when one subscripted X is present, any remaining subscripted X residue with a higher subscript number up to 8 is also present. Thus, when X]_ is present, each of X₂ through X₃ is also present. Similarly, when X₃ is present, each of X₄ through Xg is also present, and the like. On the other hand, Xg can be present without any other of the remaining X residues having a lower valued subscript number being present .

The residues of XIQ. XH. ^X ₁₃ and X₁₄ are present, and can be leucine or histidine for X]_o- isoleucine or threonine for X^, asparagine or serine for X]_₃ and glutamic acid or glycine for X₁₄. The residues X^5 and X^g are present or absent, and when present are tryptophan and glycine or glutamic acid, respectively. Residues X^7 and X^g are present or absent, and when present are independently cysteine, serine, or alanine. It is preferred that one of -^η and X^g be cysteine, particularly when an M2 polypeptide epitope is present at the N-terminus of the chimer molecule. Residue X_]_g is present or absent, and when present is arginine or lysine.

Residues X20 through X24 are present or absent, and when present are the residues naturally present in the reported M2 protein sequence; i.e., asparagine or serine, aspartic acid, serine, serine and aspartic acid respectively, with the proviso that when one subscripted X is present, any remaining X residue with a lower subscript number through 15 is also present. Thus, for example, when X23 is present, so are each of residues X^5 through X22 ■

The remaining residues of Domain II that are present on either side of the heterologous residue or sequence are the residues of AHBc position 86 to position 130. Thus, in an example, where residues 100 through 105 have been replaced, the chimer sequence in Domain II is 86 through 99, followed by restriction site-encoded residues, the heterologous immunogenic (epitope) sequence, further restriction site-encoded residues, and then AHBc sequence 106 through 130. A typical exemplary sequence of a chimer prepared by an insertion strategy between residues 95 and 96 is that of AHBc from position 1 through 95, followed by restriction site-encoded residues, the heterologous immunogenic sequence, further restriction site-encoded residues and HBc sequence 69 through 130. The sequence of other contemplated chimers through Domains I and II should be apparent from these illustrations and those that follow and need not be enumerated.

It has been found that a short hydrophilic peptide containing a plurality of glycine residues and having a length of about 5 to about 9 residues peptide-bonded at the C-terminus of an above-noted

Neisseria meningi tidis B cell epitope sequence can

/ assist in the expression of a chimeric particle containing that sequence. One useful short peptide is that disclosed in Karpenko et al . , Amino Acids

(2000) 18:329-337, having the sequence GSGDEGG of SEQ

ID NO: 147.

As already noted, a heterologous linker for a conjugated epitope is peptide-bonded at a position in the AHBc sequence between amino acid residues 85 and 130. As was the case for the heterologous epitope, the AHBc sequence of residues 85 through 130 is preferably present, but interrupted by the heterologous linker for a conjugated epitope. This chimer preferably includes the AHBc sequence of position 1 through at least position 205, plus a cysteine residue at the C-terminus of the chimer protein. More preferably, the AHBc sequence of positions 1 through 214 are present, but interrupted between residues 95 and 96 by the heterologous linker for a conjugated epitope, and the chimer molecule contains a C-terminal cysteine. The heterologous linker for a conjugated epitope is most preferably a lysine (K) residue. Glutamic or aspartic acid, tyrosine and cysteine residues can also be used as linker residues, as can tyrosine and cysteine residues. It is noted that more than one linker can be present such as a sequence of three lysines, but such use is not preferred because heterogeneous conjugates can be formed from such use in which the conjugated hapten is bonded to one linker in a first chimer and to a different linker in a second chimer molecule. Published application PCT/US99/03055 discloses HBc chimer molecules containing one or more linking residues, but lacking a stabilizing C- terminal cysteine residue.

It is also noted that a heterologous epitope sequence present in a contemplated HBc chimer can also be separated from the HBc sequence residues by a "flexible linker arm" on one or both sides of (flanking) the heterologous immunogenic (epitope) sequence. This is particularly the case where the heterologous immunogenic sequence is greater than about 30 amino acid residues long. Exemplary flexible linker arm sequences typically contain about 4 to about 10 glycine residues that are thought to permit the inserted sequence to "bulge" outwardly from the otherwise bulging loop sequence and add further stability to the construct. Illustrative flexible linker arm sequences are disclosed in Kratz et al . (March 1999) Proc . Natl . Acad. Sci . , U. S.A . , 96:1915-1920 and are exemplified by the amino acid residue sequences:

GGGGSGGGGT SEQ ID NO: 314

GGGGSGGGG SEQ ID NO: 315

As was noted previously, Domain III constitutes the sequence of AHBc from position 131 through position 214. Consequently, the sequence of the illustrative chimers discussed above for Domains I and II, can be extended so that the first -discussed chimer has the sequence of AHBc from position 96 through position up to 205, and the second-discussed chimer has the sequence of HBc from position 110 through position 205.

Domain IV is a sequence that (i) optionally includes an AHBc sequence from position 205 through 262 (preferably through 224 or 214) , (ii) contains at least one cysteine residue, up to three cysteine residues, and (iii) up to about 100 amino acid residues in a sequence heterologous to AHBc at position 205 to the avian C-terminus, with the proviso that Domain IV contain at least 6 amino acid residues, including the above one to ten cysteine residues of (ii) . The Domain IV sequence heterologous to AHBc more preferably contains up to about 50 amino acid residues, and most preferably contains up to about 25 residues. The Domain IV sequence can thus be substantially any cysteine- containing sequence, except the C-terminal AHBc sequence from position 205 to the avian C-terminus.

The length of the Domain IV sequence can be six residues; i.e., a cysteine plus any five residues containing up to a total of three cysteines, to about 100 amino acid residues, with the length being sufficient so that a contemplated chimeric protein has a total length of about 175 to about 256 residues, and more preferably up to about 460 residues, and most preferably up to about 435 amino acid residues. Where an epitope is peptide-bonded to Domains I or II contains up to about 30 or about 50 residues, respectively, as is preferred for those epitopes, more preferred lengths of the chimer molecule , including the Domain IV epitope, are about 175 to about 240 residues. Particularly preferred chimer molecules containing two heterologous epitopes have a length of about 190 to about 210 residues. Freedom of the resulting particle from nucleic acid- binding is determined by determination of the 280/260 absorbance ratio as discussed previously.

The Domain IV sequence includes at least one cysteine (Cys) residue and can contain up to three Cys residues. It is preferred that the one or more Cys residues be at or within about five amino acid residues of the C-terminus of the chimeric protein molecule. In addition, when more than one Cys residue is present in a Domain IV sequence, it is preferred that those Cys residues be adjacent to each other.

It is also preferred that the Domain IV sequence constitute a T cell epitope, a plurality of T cell epitopes that are the same or different or an additional B cell epitope for the organism against which a contemplated chimer is intended to be used as an immunogen. Exemplary Domain IV T cell epitope sequences are provided in Table B, below, as in Table A.

Table B T Cell Epitopes

SEQ

Organism Gene Sequence* Citation ID NO

HIV P24 GPKEPFRDY-

VDRFYKC 3 148

Coryn ebact eri m diptheriae toxin

FQWHNSYN-

RPAYSPGC 5 149

Borrelia burgdorferi ospA

VEIKEGTVTLKRE-

IDKNGKVTVSLC 6 150

TLSKNISKSG-

EVSVELNDC 7 151

Influenza Virus

A8/PR8 HA SSVSSFERFEC 8 152

LIDALLGDPC 32

153133

TLIDALLGC 32 154

Trypanosoma cruzi

SHNFT VASVII- EEAPSGNTC 13 155

Plasmodium falciparum MSP1

SVQIPKVPYPNGIVYC 15 156 DFNHYYTLKTGLEADC 157 PSDKHIEQYKKI- 23 KNSISC 158

EYLNKIQNSLST- 26 E SPCSVT 159

P. vivax

YLDKVRATVGTE- TPCSVT 160

P. yoelii

EFVKQISSQLTE- E SQCSVT 161

Streptococcus sobrinus Ag /ll

KPRPIYEAKL- AQNQKC 16 162 AKADYEAKLA- QYEKDLC 163

LCMV (lymphocytic choriomeningitis virus) NP RPQASGVYM- GNLTAQC 17 164

Clostridium tetani tox

QYIKANSKFIG- ITELC 20 165

Neisseria meningi tidis PorB AI QVEQKASIAGTDSG C 166

NYKNGGFFVQYGGAYKRHC 167

HNSQTEVAATLAYRFGNVC 168

PorB TPRVSYAHGFKGLVDDADC 169

RFGNAVPRISYAHGFDFIC 170

AFKYARHANVGRNAFELFC 171

SGA LKRNTGIGNYTQINAC 172

AGEFGTLRAGRVANQC 173

IGNYTQINAASVGLRC 174

GRNYQLQLTEQPSRTC 175

SGSVQFVPAQNSKSAC 176

HANVGRDAFNLFLLGC 177

LGRIGDDDEAKGTDPC 178

SVQFVPAQNSKSAYKC 179

NYAFKYAKHANVGRDC 180

AHGFDFIERGKKGENC 181

GVDYDFSKRTSAIVSC 182

HDDMPVSVRYDSPDFC 183

RFGNAVPRISYAHGFDFIERGKKGENC 184

NYAFKYAKHANVGRDAFNLFLLGC 185

SGA LKRNTGIGNYTQINAASVGLRC 186

SGSVQFVPAQNSKSAYTPAC 187

OpaB TGANNTSTVSDYFRNRITC 188

IYDFKLNDKFDKFKPYIGC 189

Opa-5d LSAIYDFKLNDKFKPYIGC 190 Opac NG YINPWSEVKFDLNSRC 191

*Underlined C (C) is not from the native sequence.

Citations :

1. EPO 786 521A.

2. WO 98/07320.

3. US No. 5,639,854.

4. US No. 4,544,500.

5. EPO 399001 Bl.

6. Bockenstedt et al . (1996) J. Immunol . , 157, 12:5496.

7. Zhong et al. (1996) Eur. J. Immunol . , 26, 11:2749.

8. Brumeanu et al. (1996) Immunotechnology, 2, 2:85.

9. Hill et al. (1997) Infect . Immun . , 65, 11:4476.

10. EPO 432 220 Bl .

11. WO 98/06851.

12. Kelly et al. (1997) Clin . Exp . Immunol . , 110, 2:285.

13. Kahn et al . (1997) J. Immunol . , 159, 9:4444. 15. Ohta et al. (1997) Int. Arch. Allergy Immunol., 114,1:15.

16. Staffileno et al . (1990) Arch. Oral Biol., 35: Suppl. 47S.

17. Saron et al . (1997) Proc. Natl. Acad. Sci. USA ,94,7:3314.

18. Corthesy et al. (1996) J^". Biol. Chem., 271, 52:33670.

19. Bastien et al . (1997) Virol., 234, 1:118.

20. Yang et al. (1997) Vaccine, 15, 4:377.

21. Lotter et al . (1997) J. Exp. Med., 185, 10:1793.

22. Nara et al. (1997) Vaccine 15, 1:79.

23. U.S. No. 4,886,782.

24. Zavala et al . (1985) Science, 228:1436.

25. Schodel et al . (1994) J^". Exper. Med., 180:1037.

26. Calvo-Calleet al. (1997) J. Immunol. 159, 3:1362.

27. Qari et al. (1992) Mol. Biochem. Parasitol. , 55 (1-2) : 105.

28. Qari et al. (1993) Lancet, 341 (8848) :780.

29. Neirynck et al . (Oct 1999) Nature Med. , 5 (10) : 1157-1163.

30. Thompson et al . (1994) Eur.J. Biochem., 226 (3) :751-764.

31. Wilson et al . (2000) Science, 287:1664-1666.

32. Brown et al. (1993) J. Virol., 67 (5) :2887-2893.

33. U.S. No. 4,886,663.

34. Schenk et al. (Jul 8, 1999) Nature, 400 (6740) : 116-117.

35. Slepushkin et al . (1995) Vaccine, 13 (15) :1399-1402.

In addition to the at least one cysteine residue present in Domain IV, the amino acid sequence of AHBc from residue position 1 through at least position 205 is preferably present in a contemplated chimer molecule and particle. The sequence from position 1 through position 214 is more preferably present. A B cell epitope is preferably present between residues 86 and 130 and at least a single cysteine residue or a T cell epitope containing a cysteine residue is present as a C-terminal addition to the AHBc sequence. A contemplated recombinant AHBc chimer is substantially free of bound nucleic acid. A contemplated chimer particle that contains an added Cys residue at or near the C-terminus of the

molecule is also more stable at 37°C than is a similar particle that does not contain that added Cys.

A contemplated recombinant AHBc chimer molecule is typically present and is used as a self- assembled particle. These particles are comprised of 180 to 240 chimer molecules (90 or 120 dimer pairs) , usually 240 chimer molecules, that separate into protein molecules in the presence of disulfide reducing agents such as 2-mercaptoethanol, and the individual molecules are therefore thought to be bound together into the particle primarily by disulfide bonds.

Although not wishing to be bound by theory, it is believed that the observed enhanced stability and in some cases enhanced expression for a contemplated AHBc chimer is due to the formation of a further cystine disulfide bond between monomeric protein chains of the chimer particles. Regardless of whether present as a cysteine or a cystine, the C-terminal cysteine (s) residue is referred to as a cysteine inasmuch as that is the residue coded-for by the codon present in the nucleic acid from which the protein and assembled particle is expressed.

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 AHBc chimer molecules assemble in the host into particles that can be readily harvested from the host cells, and purified, if desired.

As noted before, the AHBc immunodominant loop is usually recited as being located at about positions 85 through 130 from the amino-terminus (N- terminus) of the intact protein. The heterologous B cell epitope-containing sequence of Domain II is placed into that immunodominant loop sequence . That placement substantially eliminates the HBc immunogenicity of the HBc loop sequence, while presenting the heterologous sequence or linker residue in an extremely immunogenic position in the assembled chimer particles.

In addition to the before-discussed N- and C-truncations, insertion of various epitopes and spacers, a contemplated chimer molecule can also contain conservative substitutions in the amino acid residues that constitute AHBc Domains I, II, III and IV. Conservative substitutions are as defined before.

More rarely, a "nonconservative" change, e.g., replacement of a glycine with a tryptophan is contemplated. Analogous minor variations can also include amino acid deletions or insertions, or both. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological activity or particle formation can be found using computer programs well known in the art, for example LASERGENE software (DNASTAR Inc., Madison, Wis.)

The AHBc portion of a chimer molecule of the present invention; i.e., the portion having the AHBc sequence that has other than a sequence or residue of an added epitope, linker, flexible linker arm or heterologous residue (s) that are a restriction enzyme artifact, most preferably has the amino acid residue sequence at positions 1 through 205 of duck HBc (SEQ ID NO: 8) that is shown in Fig. 1, less any portion or portions of the sequences that are absent because of truncation at one or both termini or deletion of a portion between residues 75 and 130. Somewhat less preferred are the corresponding amino acid residue sequences of heron HBc (SEQ ID NO: 9) or avian HBc (SEQ ID NO: 7) that are also shown in Fig. 1. As noted elsewhere, portions of different sequences from different avian HBc proteins can be used together in a single chimer.

When the AHBc portion of a chimer molecule of the present invention as above described has other than a sequence of an avian HBc molecule corresponding to positions 1 through 205, no more than about 20 percent of the amino acid residues are substituted as compared to SEQ ID NOs : 7 , 8 or 9, most preferable SEQ ID NO: 8 from position 1 through 205 (using the numbering shown in Fig. 1 for heron) . It is preferred that no more than about 10 percent, and more preferably no more than about 5 percent, and most preferably no more than about 3 percent of the amino acid residues are substituted as compared to SEQ ID NO: 8 from position 1 through 205.

A contemplated chimer of 205 avian HBc residues can therefore contain up to about 41 residues that are different from those of SEQ ID NO: 7, 8 or 9 at positions 1 through 205, and preferably about 20 or 21 residues. More preferably, about 10 residues are different from the avian sequences (SEQ ID NOs: 7, 8 or 9) at residue positions 1-205, and most preferably about 6 residues are different. A contemplated chimer with residues 1 to 4, 86 to 130 and 215-262 deleted has 165 residues, 33 of which (and most preferably not more than 5) may differ from the residues in avian sequences. Substitutions, other than in the immunodominant loop of Domain II or at the termini, are preferably in the non-helical portions of the chimer molecule and are typically between residues 1 to about 15 and residues 24 to about 50 to help assure particle formation., See, Koschel et al . , J. Virol . , 73(3) .2153-2160 (Mar. 1999) noting this in the mammalian HBc.

As illustrated above, where a AHBc sequence is truncated at the C-terminus beyond position 205 or at the N-terminus, or contains one or more deletions in the immunogenic loop, the number of substituted residues is proportionally different because the total length of the sequence is less than 205 residues. Deletions elsewhere in the molecule are considered conservative substitutions for purposes of calculation.

Chimer Preparation

A contemplated chimeric AHBc immunogen is typically prepared using the well-known techniques of recombinant DNA technology. Thus, sequences of nucleic acid that encode particular polypeptide sequences are added to and deleted from the precursor sequence that encodes AHBc to form a nucleic acid that encodes a contemplated chimer.

Either of two strategies is preferred for placing the heterologous epitope sequence into the loop sequence. The first strategy is referred to as replacement in which DNA that codes for a portion of the immunodominant loop is excised and replaced with DNA that encodes a heterologous epitope such as a B cell sequence. The second strategy is referred to as insertion in which a heterologous epitope is inserted between adjacent residues in the loop.

Site-directed mutagenesis using the polymerase chain reaction (PCR) is used in one exemplary replacement approach to provide a chimeric AHBc DNA sequence that encodes a pair of different restriction sites, e.g. EcoRI and Sacl, one near each end of the immunodominant loop-encoding DNA. Exemplary residues replaced are 86 through 130. The loop-encoding section is excised, a desired sequence that encodes the heterologous B cell epitope is ligated into the restriction sites and the resulting DNA is used to express the AHBc chimer. See, for example, Table 2 of Pumpens et al . , (1995) Intervirology, 38:63-74 for exemplary uses of this technique .

Alternatively, a single restriction site can be encoded into the region by site-directed mutagenesis, the DNA cut with a restriction enzyme to provide "sticky" ends, the sticky ends made blunt with endonuclease and a blunt-ended heterologous DNA segment ligated into the cut region. Examples of this type of sequence replacement into HBc can be found in the work reported in Schodel et al . , (1991) F. Brown et al . eds., Vaccines 91 , Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp.319-325; Schodel et al . , Behring Inst . Mi tt . , 1997(98): p. 114-119 and Schodel et al . , J. Exp . Med. , (1994) 180(3) : p. 1037-4, the latter two papers discussing the preparation of vaccines against P. yoelii and P. berghei , respectively.

In an illustrative example of the insertion strategy, 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 95/96 and 109/110.

Upon cleavage with the restriction enzymes, ligation of the DNA coding for the heterologous B cell epitope sequence and expression of the DNA to form aHBc chimers, the AHBc 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 heterologous 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 can be used for insertion into Domain I of a N-terminal sequence as was reported in Neirynck et al . , (October 1999) Nature Med. , 5 (10) : 1157-1163 or 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 similar strategy using an insertion between residues 82 and 83 is reported in Schodel et al . , (1990) F. Brown et al . eds., Vaccines 90, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp.193-198.

More specifically, this cloning strategy is illustrated schematically in Figs. 3A, 3B and 3C. In Fig. 3A, a DNA sequence that encodes a C-terminal truncated avian HBc sequence (DHc214) is engineered to contain adjacent EcoRI and Sacl sites between residues 95 and 96. Cleavage of that DNA with both enzymes provides one fragment that encodes AHBc positions 1-95 3 ' -terminated with an EcoRI sticky end, whereas the other fragment has a 5 ' -terminal Sacl sticky end and encodes residues of positions 96- 214. Ligation of a synthetic nucleic acid having a 5 ' AATT overhang followed by a sequence that encodes a desired malarial B cell epitope and a AGCT 3 ' overhang provides a AHBc chimer sequence that encodes that B cell epitope flanked on each side by two heterologous residues [Glylle (GI) and GluLeu (EL) , respectively] between residues , while usually destroying the EcoRI site and preserving the Sad site .

A similar strategy is shown in Fig. 3B for insertion of a cysteine-containing sequence in Domain IV, such as a particularly preferred malarial T cell epitope that contains the P. falciparum CS protein sequence from position 326 through position 345 and is referred to herein as PF/CS326-345 (Pf-UTC) . Here, EcoRI and Hindlll restriction sites were engineered into the AHBc DNA sequence after amino acid residue position 214. After digestion with EcoRI and Hindlll, a synthetic DNA having the above AATT 5' overhang followed by a T cell epitope-encoding sequence, one or more stop codons and a 3' AGCT overhang were ligated into the digested sequence to form a sequence that encoded AHBc residues 1-214 followed by two heterologous residues (GI) , the stop codon and the Hindlll site.

PCR amplification using a forward primer having a Sad restriction site followed by a sequence encoding AHBc beginning at residue position 96, followed by digestion with Sad and Hindlll provided a sequence encoding AHBc positions 96-214 plus the two added residues and the T cell epitope at the C- terminus . Digestion of the construct of Fig. 3B with Sad and ligation provided the complete gene encoding a desired recombinant AHBc chimer immunogen having the sequence, from the N-terminus, of AHBc positions 1-95, two added residues, the malarial B cell epitope, two added residues, AHBc positions 96-214, two added residues, and the T cell epitope that is shown in Fig. 3C.

Similar techniques can be used to place a heterologous linker residue for conjugation of a B cell epitope into the loop region sequence. Contemplated linker residues include lysine (Lys) , which is particularly preferred, aspartic acid (Asp) , glutamic acid (Glu) , cysteine (Cys) and tyrosine (Tyr) .

It is noted that the amino acid residue sequence shown in Fig. 1 contain several Glu and Asp residues in the regions of positions 75 to 130. Nonetheless, introduction of an additional, heterologous, carboxyl-containing residue is still contemplated in the region from positions 75 to 130. The chemical reactivity of the existing glutamic and aspartic acids may be reduced by other factors, such as decreased reactivity due to interaction with neighboring residues.

As an example of introduction of a chemically reactive amino acid side to act as a heterologous linker residue for conjugation of a B cell epitope onto the loop region, five heterologous residues are placed into the loop sequence; one that is the heterologous linker residue for conjugating a B cell epitope and two residues adjacent on either side of that one residue that are themselves also adjacent to loop sequence residues and are an expression product of the inserted restriction sites (restriction enzyme artifacts) . It is noted that one can also use site-directed mutagenesis to add a single codon into the AHBc loop sequence that encodes the heterologous linker residue for a B cell epitope. It is noted that the preferred use of two heterologous residues on either side of (flanking) a B cell or T cell epitope is a matter of convenience. As a consequence, one can also use zero to three or more added residues that are not part of the AHBc sequence on either or both sides of an inserted sequence . One or both ends of the insert and AHBc nucleic acid can be "chewed back" with an appropriate nuclease (e.g. SI nuclease) to provide blunt ends that can be ligated together. Added heterologous residues that are neither part of the inserted B cell or T cell epitopes nor a part of the AHBc sequence are not counted in the number of residues present in a recited Domain.

It is also noted that one can also synthesize all or a part of a desired recombinant AHBc chimer nucleic acid using well-known synthetic methods as is discussed and illustrated in U. S. Patent No. 5,656,472 for the synthesis of the 177 base pair DNA that encodes the 59 residue ribulose bis-phosphate carboxylase-oxygenase signal peptide of Nicotiana tabacum. For example, one can synthesize Domains I and II with a blunt or a "sticky end" that can be ligated to Domains III and IV to provide a construct that expresses a contemplated AHBc chimer that contains zero added residues to the N-terminal side of the B cell epitope and zero to three added residues on the C-terminal side or at the Domain II/III junction or at some other desired location.

An alternative insertion technique was reported in Clarke et al . (1991) F. Brown et al . eds., Vaccines 91 , Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp.313-318 for the mammalian HBc proteins that is equally useful for avian HBc proteins. Here, taking advantage of the degeneracy of the genetic code, those workers engineered a single restriction site corresponding to residues 80 and 81 that encoded the original residues present at those positions. Their expressed HBc chimers thereby contained no restriction site-encoded residues, and contained the residues of the HBc loop immediately adjacent to the inserted sequence.

A nucleic acid sequence (segment) that encodes a previously described avian 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.

In living organisms, 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. Thus, through the well-known degeneracy of the genetic code additional DNAs and corresponding RNA sequences (nucleic acids) 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 AHBc portion from residue position 1 through 262, when present, is that of SEQ ID NOs: 7, 8 or 9 and (2) hybridizes with a DNA sequence of SEQ ID NO: 196 with at least at moderate stringency (discussed above) ; and (3) whose AHBc 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: 7, 8 or 9, is defined as a DNA variant sequence. As is well-known, 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.

An analog or analogous nucleic acid (DNA or RNA) sequence that encodes a contemplated chimer molecule is also contemplated as part of this invention. A chimer analog nucleic acid sequence or its complementary nucleic acid sequence encodes an AHBc 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 duck HBc gene sequence shown in SEQ ID NO: 196 This DNA or RNA is referred to herein as an "analog of" or "analogous to" a sequence of a nucleic acid of SEQ ID NO: 196, and hybridizes with the nucleic acid sequence of SEQ ID NO: 196 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. In addition, one can also use the degeneracy of the genetic code to encode the avian HBc portion of a sequence of SEQ ID NO: 196 that avoids substantial identity with a DNA of SEQ ID No: 196, or their complements. Thus, a useful analogous DNA sequence need not hybridize with the nucleotide sequences of SEQ ID NO: 196 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. More particularly, also contemplated is 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 AHBc portion of a chimer or a DNA variant that has at least 90 percent identity to the chimer gene of SEQ ID NOs: 196 and hybridizes with that gene under moderate stringency conditions . Further contemplated is 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 an avian HBc chimer portion that is at least 80 percent identical, more preferably 90 percent identical, and most preferably 95 percent identical to the AHBc portion of a sequence of SEQ ID NOs: 196. That recombinant DNA molecule, upon suitable transfection and expression in a host cell, provides a contemplated chimer molecule.

It is noted that because of the 43 amino acid residue N-terminal , sequence of heron HBc does not align with the duck HBc sequence, the heron 43 amino acid N-terminal sequence and its encoding nucleic 'acid sequences and their complements are not included in the above percentages of identity, nor are the portions of nucleic acid that encode that 43 -residue sequence or its complement used in hybridization determinations. Similarly, sequences that are truncated at either or both of the HBc N- and C-termini are not included in identity calculations, nor are those sequences in which residues of the immunodominant loop are removed for insertion of a heterologous epitope. Thus, only those avian HBc-encoding bases or AHBc sequence residues that are present in a chimer molecule are included and compared to an aligned nucleic acid or amino acid residue sequence in the identity percentage calculations.

Inasmuch as the coding sequences for the gene disclosed herein is illustrated in SEQ ID NO: 196, 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. Thus, 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.

As is well known in the art, so long as the required nucleic acid, illustratively DNA sequence, is present, (including start and stop signals) , additional base pairs can usually be present at either end of the segment and that segment can still be utilized to express the protein. This, of course, presumes the absence in the segment of an operatively linked DNA sequence that represses expression, expresses a further product that consumes the enzyme desired to be expressed, expresses a product that consumes a wanted reaction product produced by that desired enzyme, or otherwise interferes with expression of the gene of the DNA segment .

Thus, so long as the DNA segment is free of such interfering DNA sequences, a DNA segment of the invention can be about 500 to about 15,000 base pairs in length. The maximum size of a recombinant DNA molecule, particularly an expression vector, is governed mostly by convenience and the vector size that can be accommodated by a host cell, once all of the minimal DNA sequences required for replication and expression, when desired, are present. Minimal vector sizes are well known. Such long DNA segments are not preferred, but can be used.

DNA segments that encode the before- described chimer can be synthesized by chemical techniques, for example, the phosphotriester method of Matteucci et al . (1981) J. Am . Chem . Soc , 103:3185. Of course, by chemically synthesizing the coding sequence, any desired modifications can be made simply by substituting the appropriate bases for those encoding the native amino acid residue sequence. However, DNA segments including sequences discussed previously are preferred.

A contemplated avian HBc chimer can be produced (expressed) in a number of transformed host systems, typically host cells although expression in acellular, in vi tro, systems is also contemplated. 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, VERO or COS cells. The invention is not limited by the host cell employed.

DNA segments containing a gene encoding the avian HBc chimer are preferably obtained from recombinant DNA molecules (plasmid vectors) containing that gene. Vectors capable of directing the expression of a chimer gene into the protein of an avian 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- β-D-thiogalacto-pyranoside (IPTG) . A still more preferred promoter, the taq 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 μM IPTG for induction. Surprisingly, concentrations of about 25 to about 50 μM IPTG have been found to provide optimal results in 2 L shaker flasks and fermentors .

Several strains of Salmonella such as S. typhi and S . typhimurium and S. typhimurium-E. coli hybrids have been used to express immunogenic transgenes including prior HBc chimer particles both as sources of the particles for use as immunogens and as live, attenuated whole cell vaccines and inocula, and those expression and vaccination systems can be used herein. See, U.S. Patent No. 6,024,961; U.S. Patent No. 5,888,799; U.S. Patent No. 5,387,744; U.S. Patent No. 5,297,441; Ulrich et al . , (1998) Adv. Virus Res . , 50:141-182; Tacket et al . , (Aug 1997) Infect . Immun . , 65 (8) .3381-3385; Schodel et al . , (Feb 1997) Behring Inst . Mi tt . , 98:114-119; Nardelli- Haefliger et al . , (Dec 1996) Infect . Immun . , 64 (12) .5219-5224; Londono et al . , (Apr 1996) Vaccine, 14 (6) .545-552 , and the citations therein.

Expression vectors compatible with eukaryotic cells, such as those compatible with yeast cells or those compatible with cells of higher plants or mammals, 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 . cerevisiae phosphoglyceric acid kinase (PGK) promoter and the divergent promoters GAL 10 and GAL 1, whereas the alcohol oxidase gene (AOX1) is a useful promoter for Pichia pastoris . For example, to produce chimers in the methylotrophic yeast, P. pastoris, 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.

More specifically, the transformation and expression system described by Cregg et al . (1987) Biotechnology, 5:479-485/ (1987) Molecular and Cellular Biology, 12:3376-3385 can be used. A gene for a chimer V12. Pf3.1 is placed downstream from the' alcohol oxidase gene (AOX1) promoter and upstream from the transcription terminator sequence of the same AOX1 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. The resultant plasmid carrying a chimer gene, as well as the various additional elements described above, is illustratively transformed into a his4 mutant of P. pastoris; i.e. cells of a strain lacking a functional histidinol dehydrogenase gene.

After selecting transformant colonies on media lacking histidine, cells are grown on media lacking histidine, but containing methanol as described Cregg et al . (1987) Molecular and Cellular Biology, 12:3376-3385, to induce the AOX1 promoters. The induced AOX1 promoters cause expression of the chimer protein and the production of chimer particles in P. pastoris .

A contemplated 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.

Production of 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) .

In one illustrative example, 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 avian 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) , when transformed with pSV-Pf , can thus be selected on the basis of ampicillin resistance for those bacteria containing the plasmid. Plasmid- containing bacteria are then cloned 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 avian 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.

Expression of 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.

To help ensure maximal efficiency in the introduction of pSV-Pf into CHO cells in culture, 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. Those CHO cells that are resistant to G418 are cultured, have acquired both plasmids, pSV2NEO and pSV-Pf , and are designated CHO/pSV-Pf cells. By virtue of the genetic architecture of the pSV-Pf expression vector, 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.

The choice of which expression vector and ultimately to which promoter a chimer-encoding gene is operatively linked depends directly on the functional properties desired, e.g. the location and timing of protein expression, and the host cell to be transformed. These are well known limitations inherent in the art of constructing recombinant DNA molecules. However, 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.

In one preferred embodiment, the host that expresses the chimer is the prokaryote, E. coli , and 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. Such replicons are well known in the art.

Those vectors that include a prokaryotic replicon can also include a prokaryotic promoter region capable of directing the expression of a contemplated AHBc 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.

Typical vectors useful for expression of genes in cells from higher plants and mammals are well known in the art and include plant vectors derived from the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens described by Rogers et al . (1987) Meth . in Enzymol . , 153:253-277 and mammalian expression vectors pKSV-10, above, and pCI-neo (Promega Corp., #E1841, Madison, WI) . However, several other expression vector systems are known to function in plants including pCaMVCN transfer control vector described by Fromm et al . (1985) Proc . Natl . Acad . Sci . USA, 82:58-24. Plasmid pCaMVCN (available from Pharmacia, Piscataway, NJ) includes the cauliflower mosaic virus CaMV 35S promoter.

The above plant expression systems typically provide systemic or constitutive expression of an inserted transgene. Systemic expression can be useful where most or all of a plant is used as the source to a contemplated chimer molecule or resultant particles or where a large part of the plant is used to provide an oral vaccine. However, it can be more efficacious to express a chimer molecule or particles in a plant storage organ such as a root, seed or fruit from which the particles can be more readily isolated or ingested.

One manner of achieving storage organ expression is to use a promoter that expresses its controlled gene in one or more preselected or predetermined non-photosynthetic plant organs. Expression in one or more preselected storage organs with little or no expression in other organs such as roots, seed or fruit versus leaves or stems is referred to herein as enhanced or preferential expression. An exemplary promoter that directs expression in one or more preselected organs as compared to another organ at a ratio 'of at least 5:1 is defined herein as an organ-enhanced promoter. Expression in substantially only one storage organ and substantially no expression in other storage organs is referred to as organ- specific expression; i.e., a ratio of expression products in a storage organ relative to another of about 100:1 or greater indicates organ specificity. Storage organ-specific promoters are thus members of the class of storage organ-enhanced promoters.

Exemplary plant storage organs include the roots of carrots, taro or manioc, potato tubers, and the meat of fruit such as red guava, passion fruit, mango, papaya, tomato, avocado, cherry, tangerine, mandarin, palm, melons such cantaloupe and watermelons and other fleshy fruits such as squash, cucumbers, mangos, apricots, peaches, as well as the seeds of maize (corn) , soybeans, rice, oil seed rape and the like.

The CaMV 35S promoter is normally deemed to be a constitutive promoter. However, recent research has shown that a 21-bp region of the CaMV 35S promoter, when operatively linked into another, heterologous usual green tissue promoter, the rbcS-3A promoter, can cause the resulting chimeric promoter to become a root-enhanced promoter. That 21-bp sequence is disclosed in U.S. Patent No. 5,023,179. The chimeric rbcS-3A promoter containing the 21-bp insert of U.S. Patent No. 5,023,179 is a useful root- enhanced promoter herein.

A similar root-enhanced promoter, that includes the above 21-bp segment is the -90 to +8 region of the CAMV 35S promoter itself. U.S. Patent No. 5,110,732 discloses that that truncated CaMV 35S promoter provides enhanced expression in roots and the radical of seed, a tissue destined to become a root. That promoter is also useful herein.

Another useful root-enhanced promoter is the -1616 to -1 promoter of the oil seed rape (Brassica napus L. ) gene disclosed in PCT/GB92/00416 (WO 91/13922 published Sep. 19, 1991) . E. coli DH5. alpha, harboring plasmid pRlambdaS4 and bacteriophage lambda. beta.1 that contain this promoter were deposited at the National Collection of Industrial and Marine Bacteria, Aberdeen, GB on Mar. 8, 1990 and have accession numbers NCIMB40265 and NCIMB40266. A useful portion of this promoter can be obtained as a 1.0 kb fragment by cleavage of the plasmid with Haelll.

A preferred root-enhanced promoter is the mannopine synthase (mas) promoter present in plasmid pKan2 described by DiRita and Gelvin (1987) Mol . Gen . Genet, 207 :233-241. This promoter is removable from its plasmid pKan2 as a Xbal-Xball fragment.

The preferred mannopine synthase root- enhanced promoter is comprised of the core mannopine synthase (mas) promoter region up to position -138 and the mannopine synthase activator from -318 to - 213, and is collectively referred to as AmasPmas . This promoter has been found to increase production in tobacco roots about 10- to about 100-fold compared to leaf expression levels.

Another root specific promoter is the about 500 bp 5' flanking sequence accompanying the hydroxyproline-rich glycopeprotein gene, HRGPnt3, expressed during lateral root initiation and reported by Keller et al . (1989) Genes Dev. , 3:1639-1646. Another preferred root-specific promoter is present in the about -636 to -1 5' flanking region of the tobacco root-specific gene ToRBF reported by Yamamoto et al . (1991) Plant Cell , 3:371-381. The cis-acting elements regulating expression are more specifically located by those authors in the region from about -636 to about -299 5' from the transcription initiation site. Yamamoto et al . reported steady state mRNA production from the ToRBF gene in roots, but not in leaves, shoot meristems or stems.

Still another useful storage organ-specific promoter are the 5 ' and 3 ' flanking regions of the fruit-ripening gene E8 of the tomato, Lycopersicon esculentum. These regions and their cDNA sequences are illustrated and¹ discussed in Deikman et al . (1988) EMBO J. , 7 (11) : 3315-3320 and (1992) Plant Physiol . , 100:2013-2017.

Three regions are located in the 2181 bp of the 5 ' flanking sequence of the gene and a 522 bp sequence 3 ' to the poly (A) addition site appeared to control expression of the E8 gene. One region from -2181 to -1088 is required for activation of E8 gene transcription in unripe fruit by ethylene and also contributes to transcription during ripening. Two further regions, -1088 to -863 and -409 to -263, are unable to confer ethylene responsiveness in unripe fruit but are sufficient for E8 gene expression during ripening .

The maize sucrose synthase-1 (Sh) promoter that in corn expresses its controlled enzyme at high levels in endosperm, at much reduced levels in roots and not in green tissues or pollen has been reported to express a chimeric reporter gene, β-glucuronidase (GUS) , specifically in tobacco phloem cells that are abundant in stems and roots. Yang et al . (1990) Proc . Natl . Acad . Sci . , U. S . A . , 87:4144-4148. This promoter is thus useful for plant organs such as fleshy fruits like melons, e.g. cantaloupe, or seeds that contain endosperm and for roots that have high levels of phloem cells.

Another exemplary tissue-specific promoter is the lectin promoter, which is specific for seed tissue. The lectin protein in soybean seeds is encoded by a single gene (Lei) that is only expressed during seed maturation and accounts for about 2 to about 5 percent of total seed mRNA. The lectin gene and seed-specific promoter have been fully characterized and used to direct seed specific expression in transgenic tobacco plants. See, e.g., Vodkin et al . (1983) Cell, 34:1023 and Lindstrom et al. (1990) Developmental Genetics, 11:160.

A particularly preferred tuber-specific expression promoter is the 5 ' flanking region of the potato patatin gene. Use of this promoter is described in Twell et al . (1987) Plant Mol . Biol . , 9:365-375. This promoter is present in an about 406 bp fragment of bacteriophage LPOTI . The LPOTI promoter has regions of over 90 percent homology with four other patatin promoters and about 95 percent homology over all 400 bases with patatin promoter PGT5. Each of these promoters is useful herein. See, also, Wenzler et al . (1989) Plant Mol . Biol . , 12:41- 50.

Still further organ-enhanced and organ- specific promoter are disclosed in Benfey et al . (1988) Science, 244:174-181.

Each of the promoter sequences utilized is substantially unaffected by the amount of chimer molecule or particles in the cell. As used herein, the term "substantially unaffected" means that the promoter is not responsive to direct feedback control (inhibition) by the chimer molecules or particles accumulated in transformed cells or transgenic plant.

Transfection of plant cells using Agrobacterium tumefaciens is typically best carried out on dicotyledonous plants . Monocots are usually most readily transformed by so-called direct gene transfer of protoplasts. Direct gene transfer is usually carried out by electroportation, by polyethyleneglycol -mediated transfer or bombardment of cells by microprojectiles carrying the needed DNA. These methods of transfection are well-known in the art and need not be further discussed herein. Methods of regenerating whole plants from transfected cells and protoplasts are also well-known, as are techniques for obtaining a desired protein from plant tissues. See, also, U.S. Patents No. 5,618,988 and 5,679,880 and the citations therein.

A transgenic plant formed using Agrobacterium transformation, electroportation or other methods typically contains a single gene on one chromosome. Such transgenic plants can be referred to as being heterozygous for the added gene. However, inasmuch as use of the word "heterozygous" usually implies the presence of a complementary gene at the same locus of the second chromosome of a pair of chromosomes, and there is no such gene in a plant containing one added gene as here, it is believed that a more accurate name for such a plant is an independent segregant, because the added, exogenous chimer molecule-encoding gene segregates independently during mitosis and meiosis. A transgenic plant containing an organ-enhanced promoter driving a single structural gene that encodes a contemplated AHBc chimeric molecule; i.e., an independent segregant, is a preferred transgenic plant .

More preferred is a transgenic plant that is homozygous for the added structural gene; i.e., a transgenic plant that contains two added genes, one gene at the same locus on each chromosome of a chromosome pair. A homozygous transgenic plant can be obtained by sexually mating (selfing) an independent segregant transgenic plant that contains a single added gene, germinating some of the seed produced and analyzing the resulting plants produced for enhanced chimer particle accumulation relative to a control (native, non-transgenic) or an independent segregant transgenic plant. A homozygous transgenic plant exhibits enhanced chimer particle accumulation as compared to both a native, non-transgenic plant and an independent segregant transgenic plant .

It is to be understood that two different transgenic plants can also be mated to produce offspring that contain two independently segregating added, exogenous (heterologous) genes. Selfing of appropriate progeny can produce plants that are homozygous for both added, exogenous genes that encode a chimeric avian HBc molecule. Back-crossing to a parental plant and out-crossing with a non- transgenic plant are also contemplated.

A transgenic plant of this invention thus has a heterologous structural gene that encodes a contemplated chimeric avian HBc molecule. A preferred transgenic plant is an independent segregant for the added heterologous chimeric avian HBc structural gene and can transmit that gene to its progeny. A more preferred transgenic plant is homozygous for the heterologous gene, and transmits that gene to all of its offspring on sexual mating.

Inasmuch as a gene that encodes a chimeric HBc molecule does not occur naturally in plants, a contemplated transgenic plant accumulates chimeric avian HBc molecule particles in a greater amount than does a non-transformed plant of the same type or strain when both plants are grown under the same conditions .

The phrase "same type" or "same strain" is used herein to mean a plant of the same cross as or a clone of the untransformed plant . Where alleic variations among siblings of a cross are small, as with extensively inbred plant, comparisons between siblings can be used or an average arrived at using several siblings. Otherwise, clones are preferred for the comparison.

Seed from a transgenic plant is grown in the field greenhouse, window sill or the like, and resulting sexually mature transgenic plants are self- pollinated to generate true breeding plants. The progeny from these plants become true breeding lines that are evaluated for chimeric avian HBc molecule particle accumulation, preferably in the field, under a range of environmental conditions.

A transgenic plant homozygous for chimeric avian HBc molecule particle accumulation is crossed with a parent plant having other desired traits. The progeny, which are heterozygous or independently segregatable for chimeric avian HBc molecule particle accumulation, are backcrossed with one or the other parent to obtain transgenic plants that exhibit chimeric avian HBc molecule particle accumulation and the other desired traits. The backcrossing of progeny with the parent may have to be repeated more than once to obtain a transgenic plant that possesses a number of desirable traits.

An insect cell system can also be used to express an avian HBc chimer. For example, in one such system Autographa calif ornica nuclear polyhedrosis virus (AcNPV) or baculovirus is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae.

The sequences encoding a chimer can be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of chimer sequence renders the polyhedrin gene inactive and produces recombinant virus lacking coat protein. The recombinant viruses can then be used to infect, for example, S. Frugiperda cells or Trichoplusia larvae in which the avian HBc chimer can be expressed. E. Engelhard et al . (1994) Proc . Natl . Acad. Sci . , USA, 91:3224-3227 ; and V. Luckow, Insect Cell Expression Technology, pp. 183-218, in Protein Engineering: Principles and Practice, J.L. Cleland et al . eds., Wiley-Liss, Inc, 1996). Heterologous genes placed under the control of the polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcNPV) are often expressed at high levels during the late stages of infection.

Recombinant baculoviruses containing the chimeric gene are constructed using the baculovirus shuttle vector system (Luckow et al . (1993) J. Virol . , 67:4566-4579], sold commercially as the

Bac-To-Bac™ baculovirus expression system (Life Technologies) . Stocks of recombinant viruses are prepared and expression of the recombinant protein is monitored by standard protocols (O'Reilly et al . , Baculovirus Expression Vectors: A Laboratory Manual, W.H. Freeman and Company, New York, 1992; and King et al . , The Baculovirus Expression System: A Laboratory Guide , Chapman & Hall, London, 1992) . A variety of methods have been developed to operatively link DNA to vectors via complementary cohesive termini or blunt ends. For instance, complementary homopolymer tracts can be added to the DNA segment to be inserted into the vector DNA. The vector and DNA segment are then joined by hydrogen bonding between the complementary homopolymeric tails to form recombinant DNA molecules.

Alternatively, 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.

Thus, 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.

Avian HBc Chimer Conjugates

Any hapten to which a B cell or T cell response is desired can be linked to a contemplated avian HBc chimer or chimer particle such as a chimer particle containing a heterologous linker residue such as a lysine, glutamic or aspartic acid, cysteine or tyrosine in the loop region of Domain II and an added cysteine residue in Domain IV to form an avian HBc chimer conjugate. The hapten of interest typically is a B cell immunogen. The hapten can be a polypeptide, a carbohydrate (saccharide; i.e., oligo- or polysaccharide) , or a non-polypeptide, non- carbohydrate chemical such as 2 , 4-dinitrobenzene or a

I medicament such as ***e or nicotine. An avian HBc chimer particle conjugate so formed is useful as an inoculum or vaccine, as is discussed hereinafter. Because the chimer protein self assembles upon expression and a conjugate is formed after expression, conjugate formation is typically done using the assembled particles as compared to the free protein molecules.

Methods for operatively linking individual haptens to a protein or polypeptide through an amino acid residue side chain of the protein or polypeptide to form a pendently-linked immunogenic conjugate, e.g., a branched-chain polypeptide polymer, are well known in the art. Those methods include linking through one or more types of functional groups on various side chains and result in the carrier protein polypeptide backbone (here, an avian HBc chimer) within the particle being pendently linked- - covalently linked (coupled) -- to the hapten but separated by at least one side chain.

Methods for linking carrier proteins to haptens using each of the above functional groups are described in Erlanger, (1980) Method of Enzymology, 70:85; Aurameas et al . , (1978) Scand. J. Immunol . , Vol. 8, Suppl. 7, 7-23 and U.S. Patent No. 4,493,795 to Nestor et al . In addition, a site-directed coupling reaction, as described in Rodwell et al . (1985) Biotech . , 3:889-894 can be carried out so that the biological activity of the polypeptides is not substantially diminished.

Furthermore, as is well known in the art, both the avian HBc protein and a polypeptide hapten can be used in their native form or their functional group content can be modified by succinylation of lysine residues or reaction with cysteine- thiolactone. A sulfhydryl group can also be incorporated into either carrier protein or conjugate by reaction of amino functional groups with 2- iminothiolane, the N-hydroxysuccinimide ester of 3- (3-dithiopyridyl) -propionate, or other reagents known in the art .

The avian HBc chimer or hapten can also be modified to incorporate a spacer arm, such as hexamethylene diamine or another bifunctional molecule, to facilitate the pendent linking. Such a procedure is discussed below.

Methods for covalent bonding of a polypeptide hapten are' extremely varied and are well known by workers skilled in the immunological arts. For example, following U.S. Patent No. 4,818,527, m- maleimidobenzoyl-N-hydroxysuccinimide ester (ICN Biochemicals, Inc., Costa Mesa, CA ) or succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC, Pierce Chemical Co., Rockford, IL) is reacted with an appropriate avian HBc chimer to form an activated carrier.

That activated carrier is then reacted with a hapten such as a sulfhydryl-terminated hapten or a polypeptide that either contains a terminal cysteine or to which an additional amino- or carboxy-terminal cysteine residue has been added to form a covalently bonded HBc chimer conjugate. As an alternative example, the amino group of a polypeptide hapten can be first reacted with N-succinimidyl 3- (2- pyridylthio) propionate (SPDP, Pharmacia, Piscataway, NJ) , and that thiol-containing polypeptide can be reacted with the activated carrier after reduction. Of course, the sulfur-containing moiety and double bond-containing Michael acceptor can be reversed. These reactions are described in the supplier's literature, and also in Kitagawa, et al . (1976) J. Biochem. , 79:233 and in Lachmann et al . , in 1986 Synthetic Peptides as Antigens, (Ciba Foundation Symposium 119) , pp. 25-40 (Wiley, Chichester: 1986) .

U.S. Patent No. 4,767,842 teaches several modes of covalent attachment between a carrier and polypeptide that are useful here. In one method, tolylene diisocyanate is reacted with the carrier in a dioxane-buffer solvent at zero degrees C to form an activated carrier. A polypeptide hapten is thereafter admixed and reacted with the activated carrier to form the covalently bonded avian HBc chimer conjugate.

Particularly useful are a large number of heterobifunctional agents that form a disulfide link at one functional group end and an amide link at the other, including N-succidimidyl-3- (2-pyridyldithio) - propionate (SPDP) , discussed before that creates a disulfide linkage between itself and a thiol in either the avian HBc chimer or the hapten. Exemplary reagents include a cysteine residue in a polypeptide hapten and an amine on the coupling partner such as the ε-amine of a lysine or other free amino group in the carrier protein. A variety of such disulfide/amide forming agents are known. See for example Immun . Rev. (1982) 62:185.

Other bifunctional coupling agents form a thioether rather than a disulfide linkage. Many of these thioether-forming agents are commercially available and include reactive esters of 6-maleimidocaproic acid, 2-bromoacetic acid, 2-iodoacetic acid, 4- (N-maleimidomethyl) cyclohexane- 1-carboxylic acid and the like. The carboxyl groups can be activated by combining them with succinimide or l-hydroxy-2-nitro-4-sulfonic acid, sodium salt. The particularly preferred coupling agent for the method of this invention is succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) obtained from Pierce Chemical Co., Rockford, IL. The foregoing list is not, meant to be exhaustive, and modifications of the named compounds can clearly be used. Fig. 4 provides a schematic representation (Scheme 1) of the formation of a HBc activated carrier using SMCC (I) and the subsequent reaction of that activated carrier with a sulfhydryl-terminated hapten (II) .

A polypeptide hapten can be obtained in a number of ways well known in the art . Usual peptide synthesis techniques can be readily utilized. For example, recombinant and PCR-based techniques to produce longer peptides are useful . Because the desired sequences are usually relatively short, solid phase chemical synthesis is useful.

Exemplary polypeptide haptens are shown in Tables A and B hereinbefore . Each of those polypeptides can be utilized via its N-terminal amino group, or by use of an additional N-terminal cysteine that is not shown in the table.

Related chemistry is used to couple what may be called "chemical compounds" to carrier proteins. Typically, an appropriate functional group for coupling is designed into the chemical compound. An exemplary chemical hapten to which induced antibodies protect against Streptococcus pneumoniae is 6-O-phosphocholine hydroxyhexanoate . Fischer et al. (1995) J^". Immunol . , 154:3373-3382. The table below provides further exemplary chemical haptens ,

Chemical Haptens

Chemical Hapten Citation piperidine N-oxide U.S. Patent No. 5,304,252 phospholactone or U.S. Patent No. 5,248,611 lactamide metal ion complexes U.S. Patent No. 5,236,825

[2.2.1] or [7.2.2] U.S. Patent No. 5,208,152 bicyclic ring compounds ionically charged U.S. Patent No. 5,187,086 hydroxyl-containing compounds phosphonate analogs U.S. Patent No. 5,126,258 of carboxylate esters ***e analogs Carrera et al . , (1995) Nature 378:725

There are many methods known in the art to couple carrier proteins to polysaccharides. Aldehyde groups can be prepared on either the reducing end [Anderson (1983) Infect . Immun . , 39:233-238; Jennings, et al . (1981) J. Immunol . , 127:1011-1018; Poren et al . (1985) Mol . Immunol . , 22:907-919] or the terminal end [Anderson et al . (1986) J. Immunol . , 137:1181-1186; Beuvery et al . (1986) Dev. Bio . Scand . , 65:197-204] of an oligosaccharide or relatively small polysaccharide, which can be linked to the carrier protein via reductive amination.

Large polysaccharides can be conjugated by either terminal activation [Anderson et al.(1986) J^". Immunol . , 137:1181-1186] or by random activation of several functional groups along the polysaccharide chain [Chu et al . (1983) Infect . Immun . , 40:245-256; Gordon, U.S. Patent No. 4,619,828 (1986); Marburg, U.S. Patent No. 4,882,317 (1989)]. Random activation of several functional groups along the polysaccharide chain can lead to a conjugate that is highly cross- linked due to random linkages along the polysaccharide chain. The optimal ratio of polysaccharide to carrier protein depend on the particular polysaccharide, the carrier protein, and the conjugate used.

Detailed reviews of methods of conjugation of saccharide to carrier proteins can be found in Dick et al . , in Contributions to Microbiology and Immunology, Vol. 10, Cruse et al . , eds., (S. Karger: 1989), pp. 48-114; Jennings et al . , in Neoglycoconjugates : Preparation and Applications, Lee et al . , eds., (Academic Press: 1994), pp. 325- 371; Aplin et al . , (1981) CRC Cri t . Rev. Biochem . , 10:259-306; and Stowell et al. (1980) Adv. Carbohydr. Chem . Biochem. , 37:225-281.

The carbohydrate itself can be synthesized by methods known in the art, for example by enzymatic glycoprotein synthesis as described by Witte et al . (1997) J. Am. Chem. Soc , 119:2114-2118.

Several oligosaccharides, synthetic and semi-synthetic, and natural, are discussed in the following paragraphs as examples of oligosaccharides that are contemplated haptens to be used in making a HBc conjugate of the present invention.

An oligosaccharide hapten suitable for preparing vaccines for the treatment of Haemophilus influenza type b (Hib) is made up of from 2 to 20 repeats of D-ribose-D-ribitol-phosphate (I, below) , D-ribitol-phosphate-D-ribose (II, below) , or phosphate-D-ribose-D-ribitol (III, below) . Eduard C. Beuvery et al . , EP-0 276 516-B1.

U.S. Patent No. 4,220,717 also discloses a polyribosyl ribitol phosphate (PRP) hapten for Haemophilus influenzae type b.

Peterson et al . (1998) Infect . Immun . , 66 (8) .3848-3855, disclose a trisaccharide hapten, αKdo(2 8)αKdo(2 4)αKdo, that provides protection from Chlamydia pneumoniae . Chlamydia pneumoniae is a cause of human respiratory infections ranging from pharyngitis to fatal pneumonia. Kdo is 3-deoxy-D- .ma.rzno-oct-2-ulosonic acid.

Andersson et al . , EP-0 126 043 -Al, disclose saccharides that can be used in the treatment, prophylaxis or diagnosis of bacterial infections caused by Streptococci pneumoniae . One class of useful saccharides is derived from the disaccharide GlcNAcβl 3Gal . Andersson et al . also reported neolactotetraosylceramide to be useful,^' which is Galβl 4GlcNAcβl 3Galβl 4Glc-Cer.

McKenney et al . (1999) Science, 284:1523- 1527, disclose a polysaccharide, poly-N-succinyl βl 6GlcN (PNSG) that provides protection from Staphylococcus aureus . S. aureus is a common cause of community-acquired infections, including endocarditis, osetemylitis, septic arthritis, pneumonia, and abscesses.

European Patent No. 0 157 899-B1, the disclosures of which are incorporated herein by reference, discloses the isolation of pneumococcal polysaccharides that are useful in the present invention. The following table lists the pneumococcal culture types that produce capsular polysaccharides useful as haptens in the present invention.

Polysaccharide Hapten Sources

Moraxella (Branhamella) catarrhalis is a reported cause of otitis media and sinusitis in children and lower respiratory tract infections in adults. The lipid A portion of the lipooligo- saccharide surface antigen (LOS) of the bacterium is cleaved at the 3-deoxy-D--τ.anno-octulosonic acid- glucosamine linkage. The cleavage product is treated with mild-alkali to remove ester-linked fatty acids, while preserving amide-linked fatty acids to yield detoxified lipopolysaccharide (dLOS) from M. catarrhalis . The dLOS is not immunogenic until it is attached to a protein carrier. Xin-Xing Gu et al . (1998) Infect . Immun . , 66 (5) : 1891-1897.

Group B streptococci (GBS) is a cause of sepsis, meningitis, and related neurologic disorders in humans. The Capsular polysaccharide-specific antibodies are known to protect human infants from infection. Jennings et al . , U.S. Patent No. 5,795,580. The repeating unit of the GBS capsular polysaccharide type II is: 4) -β-D-GlcpNAc- (1 3)-[β- D-Galp(l 6) ] - β-D-Galp(l 4) - β-D-Glcp- (1 3) - β-D-Glcp- (1 2) - [α-D-NeupNAc (2 3) ] - β-D-Galp- (1 , where the bracketed portion is a branch connected to the immediately following unbracketed subunit . The repeating unit of GBS capsular polysaccharide type V is: 4) - [α-D-NeupNAc- (2 3) -β-D-Galp- (1 4) -β-D- GlcpNAc-(l 6) ] -α-D-Glcp- (1 4) - [β-D-Glcp- (1 3) ] -β-D- Galp- (1 4) -β-D-Glcp- (1 .

European patent application No. EU-0 641 568-Al, Brade, discloses the method of obtaining ladder-like banding pattern antigen from Chlamydia trachomatis, pneumoniae and psittaci .

Slovin et al . , (1999) Proc . Natl . Acad. Sci . , U. S . A . , 96 (10) .5710-5715 report use of a synthetic oligosaccharide, globo H, linked to KLH as a carrier in the preparation of a vaccine used against prostate cancer. Similarly, Helling et al . , (July 1995) Cancer Res . , 55:2783-2788 report the use of KLH-linked G_M2 in a vaccine for treating patients with melanoma. The latter vaccine was prepared by ozone cleavage of the ceramide double bond of G_M2, introduction of an aldehyde group and reductive alkylation onto KLH. A similar procedure can be utilized with a contemplated chimer particle.

Oligosaccharidal portions of sphingolipids such as globosides and gangliosides that are present on the surface of other tumor cells as well as normal cells such as melanoma, neuroblastoma and healthy brain cells can similarly be used herein as a hapten. The oligosaccharide portion of the globoside globo H has the structure Fucα- (1 2)-Galβ(l 3) -GalNAcβ- (1 3) - Gala- (1 4) -Galβ- (1 4)Glc, whereas the saccharide protions of gangliosides G_M2/ G_Mi and G_Dι_a have the following structures: GalNAcβ- (1 4) - [NeuAcα- (2 3)]- Galβ-(1 4)-Glc; Galβ- (1 3) -GalNAcβ- (1 4)-[NeuAcα- (2 3)] -Galβ- (1 4)-Glc; and NeuAc- (2 3) -Galβ- (1 3)- GalNAcβ-(l 4) - [NeuAcα- (2 3) ] -Galβ- (1 4) -Glc, respectively.

U.S. Patent No. 4,356,170 discloses the preparation of useful polysaccharides that are reduced and then oxidized to form compounds having terminal aldehyde groups that can be reductively aminated onto free amine groups of carrier proteins such as tetanus toxoid and diphtheria toxoid with or without significant cross-linking. Exemplary useful bacterial polysaccharides include β-hemolytic streptococci, Haemophilus influenza, meningococci, pneumococci and E. coli . Rather than reductively aminating the particles, a linker arm such as that provided by an ε-amino C2~C alkylcarboxylic acid can be reductively aminated on to the polysaccharide, followed by linkage to the particles using a water- soluble carbodiimide.

Inocula and Vaccines

In yet another embodiment of the invention, an avian HBc chimer particle or avian HBc chimer particle conjugate with a hapten is used as the immunogen of an inoculum that induces a B cell or T cell response (stimulation) in an inoculated host animal such as production of antibodies that immunoreact with the heterologous epitope or hapten or T cell activation, or as a vaccine to provide protection against the pathogen from which the heterologous epitope or the hapten is derived.

T cell activation can be measured by a variety of techniques. In usual practice, a host animal is inoculated with a contemplated HBc chimer particle vaccine or inoculum, and peripheral mononuclear blood cells (PMBC) are thereafter collected. Those PMBC are then cultured in vi tro in the presence of the T cell immunogen for a period of about three to five days. The cultured PMBC are then assayed for proliferation or secretion of a cytokine such as IL-2, GM-CSF of IFN-γ. Assays for T cell activation are well known in the art. See, for example, U. S. Patent No. 5,478,726 and the art cited therein.

Using antibody formation as exemplary, a contemplated inoculum or vaccine comprises an immunogenic effective amount of avian HBc chimer particles or avian HBc chimer particle conjugates that are dissolved or dispersed in a pharmaceutically acceptable diluent composition that typically also contains water. When administered to a host animal in need of immunization or in which antibodies are desired to be induced such as a mammal (e.g., a mouse, dog, goat, sheep, horse, bovine, monkey, ape, or human) or bird (e.g., a chicken, turkey, duck or goose) , an inoculum induces antibodies that immunoreact with the conjugated (pendently-linked) hapten. Those antibodies also preferably bind to the protein or saccharide of the B cell immunogen.

A vaccine is a type of inoculum in which the heterologous B cell epitope or conjugated hapten corresponds to a portion of a protein or saccharidal structure that is related to a disease state, as is an exemplary malarial B cell sequence related to a malarial pathogen. The vaccine-induced antibodies not only immunoreact with the epitope or hapten or activated T cells respond to that heterologous epitope or hapten, but also immunoreact with the pathogen or diseased cell in vivo, and provide protection from that disease state.

The amount of recombinant avian 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. The term "unit dose" as it pertains to a vaccine or inoculum of the present invention refers to physically discrete units suitable as unitary dosages 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 .

Vaccines or inocula are typically prepared from a recovered recombinant avian HBc chimer immunogen by dispersing the immunogen, preferably in particulate form, 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. The diluent vehicle can also include oleaginous materials such as peanut oil, squalane or squalene as is discussed hereinafter.

The preparation of inocula and vaccines that contain proteinaceous materials as active ingredients is also well understood in the art. Typically, such inocula or vaccines are prepared as parenterals, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The preparation can also be emulsified, which is particularly preferred.

The immunogenic active ingredient is often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, an inoculum or vaccine can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents that enhance the immunogenic effectiveness of the composition.

A contemplated vaccine or inoculum advantageously also includes an adjuvant. Suitable adjuvants for vaccines and inocula of the present invention comprise those adjuvants that are capable of enhancing the antibody responses against B cell epitopes of the chimer, as well as adjuvants capable of enhancing cell mediated responses towards T cell epitopes contained in the chimer. Adjuvants are well known in the art (see, for example, Vaccine Design - The Subunit and Adjuvant Approach, 1995, Pharmaceutical Biotechnology, Volume 6, Eds. Powell, M.F., and Newman, M.J., Plenum Press, New York and London, ISBN 0-306-44867-X) .

Exemplary adjuvants include complete Freund's adjuvant (CFA) that is not used in humans, incomplete Freund's adjuvant (IFA), squalene, squalane and alum [e.g., Alhydrogel™ (Superfos, Denmark)], which are materials well known in the art, and are available commercially from several sources.

Preferred adjuvants for use with immunogens of the present invention include aluminum or calcium salts (for example hydroxide or phosphate salts) . A particularly preferred adjuvant for use herein is an aluminum hydroxide gel such as Alhydrogel™. For aluminum hydroxide gels, the chimer protein is admixed with the adjuvant so that between 50 to 800 micrograms of aluminum are present per dose, and preferably between 400 and 600 micrograms are present .

Another particularly preferred adjuvant for use with an immunogen of the present invention is an emulsion. A contemplated emulsion can be an oil-in- water emulsion or a water-in-oil emulsions. In addition to the immunogenic chimer protein, such emulsions comprise an oil phase of squalene, squalane, peanut oil or the like as are well-known, and a dispersing agent. Non-ionic dispersing agents are preferred and such materials include mono- and di-C]_2~C24-fstty acid esters* of sorbitan and mannide such as sorbitan mono-stearate, sorbitan mono-oleate and mannide mono-oleate. An immunogen-containing emulsion is administered as an emulsion.

Preferably, such emulsions are water-in-oil emulsions that comprise squalene and mannide mono- oleate (Arlacel™ A) , optionally with squalane, emulsified with the chimer protein in an aqueous phase. Well-known examples of such emulsions include Montanide™ ISA-720, and Montanide™ ISA 703 (Seppic, Castres, France) , each of which is understood to contain both squalene and squalane, with squalene predominating in each, but to a lesser extent in Montanide™ ISA 703. Most preferably, Montanide™ ISA- 720 is used, and a ratio of oil-to-water of 7:3 (w/w) is used. Other preferred oil-in-water emulsion adjuvants include those disclosed in WO 95/17210 and EP 0 399 843.

The use of small molecule adjuvants is also contemplated herein. One type of small molecule adjuvant useful herein is a 7-substituted-8 -oxo- or 8-sulfo-guanosine derivative described in U.S. Patents No. 4,539,205, No. 4,643,992, No. 5,011,828 and No. 5,093,318, whose disclosures are incorporated by reference. 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. (see, U.S. Patent No. 4,987,237), CPG available from Coley Pharmaceutical Group, QS21 available from Aquila Biopharmaceuticals, Inc., SBAS2 available from SKB, the so-called muramyl dipeptide analogues described in U.S. Patent No. 4,767,842, and MF59 available from Chiron Corp. (see, U.S. Patents No. 5,709,879 and No. 6,086,901).

More particularly, immunologically active saponin fractions having adjuvant activity derived from the bark of the South American tree Quillaja Saponaria Molina ( e . g. Quil™ A) are also useful. Derivatives of Quil™ A, for example QS21 (an HPLC purified fraction derivative of Quil™ A) , and the method of its production is disclosed in U.S. Patent No.5, 057, 540. In addition to QS21 (known as QA21) , 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-0-acylated monophosphoryl lipid A is in the form of an emulsion having a small particle size less than 0.2 μm in diameter (EP 0 689 454 Bl) .

The muramyl dipeptide adjuvants include N- acetyl-muramyl-L-threonyl-D-isoglutamine (thur-MDP) , N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP) , and N-acetylmuramyl- L-alanyl-D-isoglutaminyl-L-alanine-2 - (1 ' -2 ' - dipalmityol-sn-glycero-3-hydroxyphosphoryloxy) - ethylamin (CGP) 1983A, referred to as MTP-PE) .

Preferred adjuvant mixtures include combinations of 3D-MPL and QS21 (EP 0 671 948 Bl) , oil-in-water emulsions comprising 3D-MPL and QS21 (WO 95/17210, PCT/EP98/05714) , 3D-MPL formulated with other carriers (EP 0 689 454 Bl) , QS21 formulated in cholesterol-containing liposomes (WO 96/33739) , or immunostimulatory oligonucleotides (WO 96/02555) . Alternative adjuvants include those described in WO 99/52549 and non-particulate suspensions of polyoxyethylene ether (UK Patent Application No. 9807805.8) .

Adjuvants are utilized in an adjuvant amount, which can vary with the adjuvant, mammal and recombinant avian HBc chimer immunogen. Typical amounts can vary from about 1 μg 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. The use of a nasal spray for inoculation is also contemplated as discussed in Neirynck et al . (Oct. 1999) Nature Med. , 5(10) -1157- 1163. For 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 or HBc chimer conjugate, preferably as particles, as active ingredient. In a typical composition, an immunogenic effective amount of preferred avian HBc chimer or avian HBc chimer conjugate particles is about 1 μg to about 1 mg of active ingredient per dose, and more preferably about 5 μg to about 50 μg per dose, as noted before.

A vaccine is typically formulated for parenteral administration. Exemplary immunizations are carried out sub-cutaneously (SC) intra-muscularly (IM) , intravenusly (IV) , intraperitoneally (IP) or intra-dermally (ID) .

The avian HBc chimer particles and avian 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.

In yet another embodiment, a vaccine or inoculum is contemplated in which a gene encoding a contemplated avian HBc chimer is transfected into suitably attenuated enteric bacteria such as S. typhi , S. typhimurium, S. typhimurium-E. coli hybrids or E. coli . Exemplary attenuated or avirulent S. typhi and S. typhimurium and S . typhimurium-E. coli hybrids are discussed in the citations provided before. These vaccines and inocula are particularly contemplated for use against diseases that infect or i

are transmitted via mucosa of the nose, the gut and reproductive tract such as influenza, yeasts such as Aspergiullus and Candida, viruses such as polio, moot-and-mouth disease, hepatitis A, and bacteria such as Cholera, Salmonella and E. coli and where a mucosal IgA response is desired in addition to or instead of an IgG systemic response.

The enteric bacteria can be freeze dried, mixed with dry pharmaceutically acceptable diluents, made into tablets or capsules for ingestion and administered to or taken by the host animal as are usual solid phase medications. In addition, aqueous preparations of these bacterial vaccines are adapted for use in mucosal immunization as by oral, nasal, rectal or vaginal administration.

Oral immunization using plant matter containing contemplated chimeric molecule particles can be achieved by simple ingestion of the transgenic plant tissue such as a root like a carrot or seed such as rice or corn. In this case, the water of the mouth or gastrointestinal tract provides the usually used aqueous medium used for immunization and the surrounding plant tissue provides the pharmaceutically acceptable diluent.

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 tens of 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 .

Once immunized, 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 an antigen of interest such as a sporozoite for a malarial vaccine. The maintenance time for the production of illustrative anti-sporozόite antibodies typically lasts for a period of about three to about twelve weeks, and can include a booster, second immunizing administration of the vaccine. A third immunization is also contemplated, if desired, at a time 24 weeks to five years after the first immunization. It is particularly contemplated that once a protective level titer of antibodies is attained, the vaccinated mammal is preferably maintained at or near that antibody titer by periodic booster immunizations administered at intervals of about 1 to about 5 years .

The production of anti-sporozoite or other 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 an approriate antigen such as a synthetic circumsporozoite immunodominant antigen [e.g. the P. falciparum CS protein peptide (NANP) 5 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 .

It is noted that the induced antibodies such as anti-CS antibodies can be isolated from the blood of an inoculated 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. For example, 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 (NANP) 5 is utilized as the antigen immobilized on the chromatographic column. Thus, for example, an inoculum can be used in a horse or sheep to induce antibody production against a malarial species for use in a passive immunization in yet another animal such as humans .

Another embodiment of the invention is a process for inducing antibodies, activated T cells or both in an animal host comprising the steps of inoculating said animal host with an inoculum. The inoculum used in the process comprises an immunogenic amount of a before-described avian HBc chimer particle or avian HBc chimer particle conjugate dissolved or dispersed in a pharmaceutically acceptable diluent. The animal host is maintained for a time sufficient for antibodies or activated T cells to be induced, as can be assayed by well-known techniques, which typically requires a time period of weeks to months, as is again well-known. A plurality of such immunizations is contemplated during this maintenance period.

The invention is illustrated by the following non-limiting examples./

Example 1: B Cell Epitope-Containing

Chimer Preparation

A. Preparation of a Plasmid for Expression of a Modified Avian HBc in E. coli

A synthetic version of the full-length hepatitis duck core protein (DHc) gene (SEQ ID NO:196), encoding a full-length DHc amino acid residues 1-262 (SEQ ID NO: 8) was designed using codons that are most abundant in E. coli genes. In addition to using the codons favored by E. coli, the engineered duck core gene was also designed to avoid continuous stretches of single codons. The synthetic DHc gene is flanked by Ncol (CCATGG) and Hindlll (AAGCTT) restrictions sites to facilitate insertion into the pKK223-3N expression plasmid described below. In addition, two termination codons were introduced following amino acid K-262 to ensure efficient termination of translation.

CCATGGATATTAACGCGTCTCGTGCGCTGGCCAACGTTTATGATCTGCCAGAT GACTTCTTTCCGAAAATTGATGACCTGGTGCGTGATGCCAAAGACGCCCTGGA ACCGTATTGGAAATCTGATAGCATTAAGAAACATGTGCTGATTGCGACCCATT TTGTGGATCTGATTGAAGATTTCTGGCAGACCACGCAAGGCATGCATGAAATT GCGGAAAGCCTGCGTGCCGTGATTCCGCCAACGACCACGCCAGTTCCGCCAGG CTATCTGATTCAGCATGAGGAAGCGGAAGAGATTCCGTTAGGCGATCTGTTTA AACATCAGGAAGAGCGTATTGTGAGCTTTCAGCCAGATTATCCGATTACCGCG CGTATCCATGCCCACCTGAAAGCCTATGCGAAAATTAACGAAGAGAGCCTGGA TCGTGCGCGTCGCTTACTGTGGTGGCATTATAACTGCCTGTTATGGGGCGAAG CGCAGGTGACCAATTATATTAGCCGTCTGCGCACCTGGCTGTCTACCCCGGAG AAATATCGTGGCCGCGATGCGCCGACCATTGAAGCCATCACCCGTCCGATTCA GGTGGCGCAAGGCGGTCGTAAAACCACCACCGGCACGCGTAAACCGCGCGGTC TGGAΆCCGCGTCGCCGTAAAGTGAAAΆCCACCGTGGTGTATGGCCGTCGCCGT AGCAAAAGCCGTGAACGTCGCGCGCCGACCCCGCAGCGTGCGGGCAGCCCGCT GCCGCGTAGCTCTTCCAGCCATCACCGTAGCCCGAGCCCGCGTAAΆTAGTAAG CTT SEQ ID NO: 196

Plasmid vector pKK223-3 (Pharmacia) was modified by the establishment of a unique Ncol restriction site to enable insertion of HBc genes as Ncol -HindiII restriction fragments and subsequent expression in E. coli host cells. To modify the pKK223-3 plasmid vector, a new Sphl -HindiII fragment was prepared using the PCR primers pKK223 -3/433 -452 -F and pKK223-NcoI-mod-R, and pKK223-3 as the template. This PCR fragment was cut with the restriction enzymes Sphl and Hindlll to provide a 467 bp fragment that was then ligated with a 4106 bp fragment of the pKK223-3 vector, to effectively replace the original 480 bp Sphl-Hindlll 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.

pKK223-3/433-452-F GGTGCATGCAAGGAGATG SEQ ID NO: 197 pKK223-NcoI-mod-R

GCGAAGCTTCGGATCccatggTTTTTTCCTCCTTATGTGAAATTGTTATCCG- CTC SEQ ID NO: 198

The synthetic duck core protein sequence described above is inserted into the pKK223-3N plasmid cut by Ncol and Hindlll. The new plasmid, pKK223-3N DHc is transformed into E. coli using standard techniques. Expression of the DHc protein is under the control of a lacZ promoter (for example using commercially available IPTG using standard protocols) . The amino acid sequence of the DHc protein is shown below.

MDINASRALANVYDLPDDFFPKIDDLVRDAKDALEPYWKSDSIKKHVLIATHF

VDLIEDFWQTTQGMHEIAESLRAVIPPTTTPVPPGYLIQHEEAEEIPLGDLFK

HQEERIVSFQPDYPITARIHAHLKAYAKINEESLDRARRLLWWHYNCLLWGEA

QVTNYISRLRTWLSTPEKYRGRDAPTIEAITRPIQVAQGGRKTTTGTRKPRGL

EPRRRKVKTTWYGRRRSKSRERRAPTPQRAGSPLPRSSSSHHRSPSPRK

SEQ ID NO: 8

At this stage, the DHc plasmid is typically used for the preparation of avian HBc chimers according to the invention.

B. Preparation of a Modified DHc Gene Able to Accept Loop Insertions

Modified DHc genes, able to accept the directional insertion of synthetic dsDNA fragments into the immunodominant loop region between amino acid residues E95 and A96 of DHc or between amino acid residues E109 and E110 of DHc, are constructed using a series of vectors as follows. Unique inserted EcoRI and Sacl restriction sites facilitate the directional insertion of synthetic dsDNAs into EcoRI -Hindlll (or EcoRI-SacI) restriction sites.

The DHc gene is amplified in two halves (the amino terminal region to precede an insert and the carboxy terminal region to follow the insert) using two PCR primer pairs . For the amino terminal region of a modified DHc gene permitting insertion between residues E95 and A96, the forward (-F) and reverse (-R) PCR primers are DHc-JVcoI-F (SEQ ID NO: 199) and DHC-E95 (EcoRI) -R (SEQ ID NO: 200), consecutively.

DHc(NcoI)-F (SEQ ID NO:199)

GCGCCATGGATATTAACGCGTC

DHC-E95 (EcoRI) -R (SEQ ID NO:200)

CGCGAATTCCTTCCTCATGCTGAATCAGAT

For the carboxy terminal region of a modified DHc gene permitting insertion between residues E95 and A96, the forward and reverse PCR primers are DHC-A96 (EcoRl/SacI) -F (SEQ ID NO: 201) and a C-terminal reverse primer of choice, depending on how the cysteine stabilization of the particles is accomplished: DHc214(H3)-R (SEQ ID NO:202), DHc214+C(H3) -R (SEQ ID N0:203), DHc224(H3)-R (SEQ ID NO:204), DHc224+C(H3) -R (SEQ ID O:205), DHc262(H3)-R (SEQ ID NO:206), or DHc262+C (H3) -R (SEQ ID NO:207).

DHC-A96 (EcoRI/Sad) -F (SEQ ID NO: 201)

CGCGAATTCAAAAAGAGCTCGCGGAAGAGATTCCGTTA DHc214 (H3 ) -R ( SEQ ID NO : 202 )

GCGAAGCTTACTACGGTTCCAGACCGCGC

DHc214+C(H3) -R (SEQ ID NO:203)

GCGAAGCTTACTAGCACGGTTCCAGACCGCGC

DHc224(H3)-R (SEQ ID NO:204)

GCGAAGCTTACTACACCACGGTGGTTTTCAC

DHc224+C(H3) -R (SEQ ID NO:205)

GCGAAGCTTACTAGCACACCACGGTGGTTTTCAC

DHc262(H3)-R (SEQ ID NO:206)

GCGAAGCTTACTATTTACGCGGGCTCGGG

DHc262+C(H3) -R (SEQ ID NO:207)

GCGAAGCTTACTAGCATTTACGCGGGCTCGGG

For the amino terminal region of a modified DHc gene permitting insertion between residues E109 and E110, the forward (-F) and reverse (-R) PCR primers are DHc(JVcoI)-F (SEQ ID NO:199) and DHc- E109 (EcoRI) -R (SEQ ID NO:208), consecutively.

DHC-E109 (EcoRI) -R (SEQ ID NO:208)

CGCGAATTCCTTCCTGATGTTTAAACAGATC

For the carboxy terminal region of a modified DHc gene permitting insertion between residues E95 and A96, the forward and reverse PCR primers are DHc-EllO (EcoRI/Sad ) -F (SEQ ID NO:209) and a C-terminal reverse primer of choice: DHc214(H3)-R (SEQ ID NO:202), DHc214+C (H3) -R (SEQ ID NO:203), DHc224(H3)-R (SEQ ID NO:204), DHc224+C (H3) -R (SEQ ID NO:205), DHc262(H3)-R (SEQ ID NO:206), DHc262+C(H3) -R (SEQ ID NO:207).

DHc-EllO (EcoRI/Sad) -F (SEQ ID NO:209216)

CGCGAATTCAAAAAGAGCTCGAGCGTATTGTGAGCTTTC

To insert B cell epitopes into the DHc E95/A96 or DHc E109/E110 plasmids, the plasmids are digested with EcoRI and Sacl restriction endonuclease enzymes. Synthetic dsDNA fragments having B-cell epitopes and also having 5' EcoRI and 3' Sacl overhangs are then inserted. In both cases, DHc E95/A96 or DHc E109/E110, glycine-isoleucine (from the EcoRI cleavage sequence GAATTC) and glutamic acid-leucine (from the Sacl cleavage sequence GAGCTC) amino acid pairs, coded for by the restriction sites, flank the inserted B cell epitopes. The inserted restriction sites are underlined in the primers above. Various exemplary B cell epitopes are listed in Table A, hereinabove.

The complete DHc gene modified to accept inserts (such as B cell epitopes) in the loop region at either E95/A96 or E109/E110 at an EcoRI cleavage site is formed from the amplified fragments as described in the next section by digesting the N- terminal PCR product with JVcoI and EcoRI and the C- terminal PCR product with EcoRI and Hindlll. The N- terminal and C-terminal fragment pairs are then ligated together at the common EcoRI ends. The resultant Ncol-HindiII fragments are then ligated into the pKK223-3N vector, which has been prepared to accept an insert by digestion with JVcoI and Hindlll. C. Avian Chimers with B cell Epitopes in the Loop Region and Avian Loop

Region Deleted

A modified DHc gene is constructed with the avian loop insertion deleted (P86- K130) , which is able to accept insertions into the immunodominant loop region. DHc genes with residues P86 to K131 deleted that are able to accept the directional insertion of synthetic dsDNA fragments into the immunodominant loop between amino A85 and A131 are constructed using a series of vectors and PCR methods . The DHc genes are amplified in two halves using two PCR primer pairs. The amino terminus is amplified using the PCR primer pair DHc (Ncol) -F (SEQ ID NO:199) and DHC-V85 (EcoRI) -R (SEQ ID NO:210).

DHC-V85 (EcoRI) -R (SEQ ID NO:210)

CGCGAATTCCAACTGGCGTGGTCGTTGGCGG

The carboxyl terminus is amplied using a PCR primer pair that is DHC-A131 (EcoRI/Sad) -F (SEQ ID NO:211), and a C-terminal reverse primer of choice DHc214(H3)-R (SEQ ID NO:202), DHc214+C(H3) -R (SEQ ID NO : 203 ) , DHc224(H3)-R

(SEQ ID NO:204), DHc224+C (H3) -R (SEQ ID NO:205), DHc262(H3)-R (SEQ ID NO : 206) , DHC262+C (H3 ) -R

(SEQ ID NO:207) .

DHC-A131 (EcoRI/Sad) -F (SEQ ID NO:211)

CGCGAATTCAAAAAGAGCTCGCCTATGCGAAAATTAACGAAG

Epitopes are inserted into the immunodominant loop region (Domain II) by digesting the plasmids with EcoRI and Sacl restriction enzymes. Synthetic dsDNA fragments of B-cell epitopes, as in Table A, containing 5 ' EcoRI and 3 ' Sacl overhangs are then inserted. The inserts are flanked by glycine- isoleucine (GI for EcoRI) and glutamic acid-leucine (EL for Sad) amino acid pairs, coded for by those restriction sites.

D. Avian Chimers with B cell Epitopes in the Loop Region and T cell Epitopes at C-terminus

Vectors containing B cell epitopes between amino acids 95/96 or 109/110, as well as T cell epitopes downstream of 214 or 224 are constructed from combinations of. vectors described above for the N-terminal and C-terminal regions of avian HBc. The carboxyl terminus of a vector containing a T cell epitope inserted at EcoRI/HindHI is amplified using PCR primers DHc-A96 (EcoRI/Sacl) -F (SEQ ID NO:201) and an appropriate T-cell epitope-containing C-terminal reverse primer with a Hindlll restriction site for insertion into PKK223-3N to provide a dsDNA fragment corresponding to amino acids 96 to the C-terminus plus the T-cell epitope, flanked with Sad and Hindlll restriction sites. Similarly, the forward primer

DHc-EllO (EcoRI/Sad) -F (SEQ ID NO:209) is used along with the appropriate C-terminal reverse primer containing a T- cell epitope and a Hindlll restriction site to make th efragment corresponding to residues 109 to the c-terminus plus the T-cell epitope, flanked with the Sacl and Hindlll restriction sites.

The PCR products are cut with Sad and Hindlll and then cloned into the desired avian HBc vector prepared by cutting with the same two enzymes. The PCR primers shown are amenable for the amplification of the carboxyl terminus of the avian chimer HBc genes, irrespective of the T cell epitope present after C-terminal amino acid of the avian HBc gene .

E. P. falciparum CS-repeat B cell Epitopes Inserted into the Loop

For these constructs, synthetic dsDNA fragments coding for the B or T cell epitope of interest are inserted into EcoRl/SacI restriction sites. Synthetic dsDNA fragments, encoding B and T cell epitopes of interest, are prepared by mixing complementary single stranded DNA oligonucleotides at equimolar concentrations, heating to 95°C for 5 minutes, and then cooling to room temperature at a rate of -1 °C per minute. This annealing reaction is performed in TE buffer.

The double-stranded DNAs are shown below with the encoded epitope sequence shown above . The pound symbol, #, is used in some of the amino acid residue sequences that follow to indicate the presence of a stop codon.

Pfl:

I N A N P N A N P N A N P N A AATTAACGCTAATCCGAACGCTAATCCGAACGCTAATCCGAACGCTA TTGCGATTAGGCTTGCGATTAGGCTTGCGATTAGGCTTGCGAT

N P E L SEQ ID NO : 212

ATCCGGAGCT SEQ ID NO : 213 TAGGCC SEQ ID NO: 214

Pf3:

I N A N P N V D P N A N P N A N P AATTAACGCTAATCCGAACGTTGACCCGAACGCTAATCCGAACGCTAATCCGA TTGCGATTAGGCTTGCAACTGGGCTTGCGATTAGGCTTGCGATTAGGCT

N A N P N V D P N A N P E L SEQ ID NO: 215 ACGCTAATCCGAACGTTGACCCGAACGCTAATCCGGAGCT SEQ ID NO: 216 TGCGATTAGGCTTGCAACTGGGCTTGCGATTAGGCCTCGAGG SEQ ID NO: 217

Pf3.1:

I N A N P N V D P N A N P N A N P AATTAACGCGAATCCGAACGTGGATCCGAATGCCAACCCTAACGCCAACCC TTGCGCTTAGGCTTGCACCTAGGCTTACGGTTGGGATTGCGGTTGGG

N A N P E L SEQ ID NO: 218

AAATGCGAACCCAGAGCT SEQ ID NO -.219

TTTACGCTTGGGTC SEQ ID NO: 220

Pf3.2:

I N A N P N A N P N A N P N V D P AATTAACGCGAATCCGAATGCCAACCCTAACGCCAACCCAAACGTGGATCCGA TTGCGCTTAGGCTTACGGTTGGGATTGCGGTTGGGTTTGCACCTAGGCT

N A N P E L SEQ ID NO: 221

ATGCGAACCCAGAGCT SEQ ID NO: 222

TACGCTTGGGTC SEQ ID NO: 223 Pf 3.3 :

I N A N P N V D P N A N P N A N P AATTAACGCGAATCCGAACGTGGATCCAAATGCCAACCCTAACGCTAATCCAA TTGCGCTTAGGCTTGCACCTAGGTTTACGGTTGGGATTGCGATTAGGTT

N A N P N V D P N A N P E L SEQ ID NO: 224 ACGCCAACCCGAATGTTGACCCCAATGCCAATCCGGAGCT SEQ ID NO: 225 TGCGGTTGGGCTTACAACTGGGGTTACGGTTAGGCC SEQ ID NO: 226

Pf3.4:

I N P N V D P N A N P N A N P N A AATTAATCCGAACGTGGATCCAAATGCCAACCCTAACGCTAATCCAAACGCCA TTAGGCTTGCACCTAGGTTTACGGTTGGGATTGCGATTAGGTTTGCGGT

N P N V E L SEQ ID NO: 227

ACCCGAATGTTGAGCT SEQ ID NO: 228

TGGGCTTACAAC SEQ ID NO: 229

Pf3.5:

N P N V D P E L SEQ ID NO: 230 ACCCGAATGTTGACCCTGAGCT SEQ ID NO: 231 TGGGCTTACAACTGGGAC SEQ ID NO: 232 Pf3.6:

N P N V D P N A E L SEQ ID NO: 233 ACCCGAATGTTGACCCTAATGCTGAGCT SEQ ID NO: 234 TGGGCTTACAACTGGGATTACGAC SEQ ID NO: 235

Pf3.7:

I N V D P N A N P N A N P N A N P AATTAACGTGGATCCAAATGCCAACCCTAACGCTAATCCAAACGCCAACCCGA TTGCACCTAGGTTTACGGTTGGGATTGCGATTAGGTTTGCGGTTGGGCT

N V E L SEQ ID NO: 236

ATGTTGAGCT SEQ ID NO: 237

TACAAC SEQ ID NO: 238

Pf3.8:

N V D P E L SEQ ID NO: 239

ATGTTGACCCTGAGCT SEQ ID NO: 240

TACAACTGGGAC SEQ ID NO: 241 Pf3-9:

N V D P N A E L SEQ ID NO: 242 ATGTTGACCCTAATGCTGAGCT SEQ ID NO: 243 TACAACTGGGATTACGAC SEQ ID NO: 244

Pf3.10:

I D P N A N P N A N P N A N P AATTGATCCAAATGCCAACCCTAACGCTAATCCAAACGCCAACC CTAGGTTTACGGTTGGGATTGCGATTAGGTTTGCGGTTGG

N V E L SEQ ID NO: 245

> CGAATGTTGAGCT SEQ ID NO: 246

GCTTACAAC SEQ ID NO: 247

Pf3. .11:

I D P N A N P N A N P N A N P N V

AATTGATCCAAATGCCAACCCTAACGCTAATCCAAACGCCAACCCGAATGTTG CTAGGTTTACGGTTGGGATTGCGATTAGGTTTGCGGTTGGGCTTACAAC

D P E L SEQ ID NO: 248

ACCCTGAGCT SEQ ID NO: 249

TGGGAC SEQ ID NO: 250 _ _ι-

Pf3.12 :

I D P N A N P N A N P N A N P N V AATTGATCCAAATGCCAACCCTAACGCTAATCCAAACGCCAACCCGAATGTTG CTAGGTTTACGGTTGGGATTGCGATTAGGTTTGCGGTTGGGCTTACAAC

D P N A E L SEQ ID NO: 251

ACCCTAATGCCGAGCT SEQ ID NO: 252

TGGGATTACGGC SEQ ID NO: 253

F. P.falciparum universal T cell epitope

Pf-UTC (PF/CS326-345)

I E Y L N K I Q N S L S T E W S P AATTGAATATCTGAACAAAATCCAGAACTCTCTGTCCACCGAATGGTCTCCGT CTTATAGACTTGTTTTAGGTCTTGAGAGACAGGTGGCTTACCAGAGGCA

C S V T # # SEQ ID NO: 192

GCTCCGTTACCTAGTA SEQ ID NO: 193

CGAGGCAATGGATCATTCGA SEQ ID NO: 194

P.vivax CS-repeat B cell epitopes

Pv-TIA:

I P A G D R A D G Q P A G D R A A AATTCCGGCTGGTGACCGTGCAGATGGCCAGCCAGCGGGTGACCGCGCTGCAG GGCCGACCACTGGCACGTCTACCGGTCGGTCGCCCACTGGCGCGACGTC

G Q P A G E L SEQ ID NO: 254

GCCAGCCGGCTGGCGAGCT SEQ ID NO: 255

CGGTCGGCCGACCGC SEQ ID NO: 256 Pv-TIB:

I D R A A G Q P A G D R A D G Q P AATTGACAGAGCAGCCGGACAACCAGCAGGCGATCGAGCAGACGGACAGCCCG CTGTCTCGTCGGCCTGTTGGTCGTCCGCTAGCTCGTCTGCCTGTCGGGC

A G E L SEQ ID NO: 257

CAGGGGAGCT SEQ ID NO: 258

GTCCCC SEQ ID NO: 259

PV-T2A:

I A N G A G N Q P G A N G A G D Q AATTGCGAACGGCGCCGGTAATCAGCCGGGGGCAAACGGCGCGGGTGATCAAC CGCTTGCCGCGGCCATTAGTCGGCCCCCGTTTGCCGCGCCCACTAGTTG

P G E L SEQ ID NO: 260

CAGGGGAGCT SEQ ID NO: 261

GTCCCC SEQ ID NO: 262

PV-T2B:

I A N G A D N Q P G A N G A D D Q AATTGCGAACGGCGCCGATAATCAGCCGGGTGCAAACGGGGCGGATGACCAAC CGCTTGCCGCGGCTATTAGTCGGCCCACGTTTGCCCCGCCTACTGGTTG

P G E L SEQ ID NO: 263

CAGGCGAGCT SEQ ID NO: 264

GTCCGC SEQ ID NO: 265

PV-T2C:

I A N G A G N Q P G A N G A G D Q AATTGCGAACGGCGCCGGTAATCAGCCGGGAGCAAACGGCGCGGGGGATCAAC CGCTTGCCGCGGCCATTAGTCGGCCCTCGTTTGCCGCGCCCCCTAGTTG P G A N G A D N Q P G A N G A D D

CAGGCGCCAATGGTGCAGACAACCAGCCTGGGGCGAATGGAGCCGATGACC

GTCCGCGGTTACCACGTCTGTTGGTCGGACCCCGCTTACCTCGGCTACTGG

Q P G E L SEQ ID NO: 266

AACCCGGCGAGCT SEQ ID NO: 267

TTGGGCCGC SEQ ID NO: 268

PV-T3 :

I A P G A N Q E G G A A A P G A N AATTGCGCCGGGCGCCAACCAGGAAGGTGGGGCTGCAGCGCCAGGAGCCAATC CGCGGCCCGCGGTTGGTCCTTCCACCCCGACGTCGCGGTCCTCGGTTAG

Q E G G A A E L SEQ ID NO: 269

AAGAAGGCGGTGCAGCGGAGCT SEQ ID NO: 270

TTCTTCCGCCACGTCGCC SEQ ID NO: 271

G. P.vivax universal T cell epitope

Pv-UTC :

I E Y L D K V R A T V G T E W T P AATTGAATATCTGGATAAAGTGCGTGCGACCGTTGGCACGGAATGGACTCCGT CTTATAGACCTATTTCACGCACGCTGGCAACCGTGCCTTACCTGAGGCA

C S V T # # SEQ ID NO: 272

GCAGCGTGACCTAATA SEQ ID NO: 273

CGTCGCACTGGATTATTCGA SEQ ID NO: 274 H. PCR primers for site-directed mutagenesis for P. faliciparum

Pf-CS(C17A) -R: SEQ ID NO: 275

# # T V S A P S W E T S GCCAAGCTTACTAGGTAACGGAGGCCGGAGACCATTCGGTGG Hindlll SEQ ID NO: 276

Example 2 : Assay Procedures

A. Antigenicity

1. Particle ELISA

Purified particles are diluted to a concentration of 10 μg/mL in coating buffer (50 mM sodium bicarbonate, pH 9.6) and coated onto the wells of ELISA strips (50 μL/well) . The ELISA strips are incubated at room temperature overnight (about 18 hours) . Next morning the wells are washed with ELISA wash buffer [phosphate buffered saline (PBS), pH 7.4, 0.05% Tween^®-20] and blocked with 3% BSA in PBS for 1 hour (75 μL/well) . ELISA strips are stored, dry, at -20°C until needed.

To determine the antigenicity of particles, antisera are diluted using 1% BSA in PBS and 50 μL/well added to antigen-coated ELISA wells. Sera are incubated for 1 hour, washed with ELISA wash buffer and probed using an anti-mouse (IgG) -HRP (e.g. The Binding Site, San Diego, CA; HRP = horseradish peroxidase) conjugate (50 μL/well) or other appropriate antibody for 30 minutes. After washing with ELISA wash buffer, the reaction is visualized by the addition of TM blue substrate (50 μL/well) . After 10 minutes, the reaction is stopped by the addition of IN H₂S0 (100 μL/well) and read on an ELISA plate reader set at 450 nm.

2. Synthetic Peptide ELISA

A 20 amino acid residue synthetic peptide (NANP) 5 is diluted to a concentration of 2 μg/mL in coating buffer (50 mM sodium bicarbonate, pH 9.6) and coated onto the wells of ELISA strips (50 μL/well) . Peptides are dried onto the wells by incubating overnight (about 18 hours) , in a hood with the exhaust on. The next morning, the wells are washed with ELISA wash buffer (phosphate buffered saline, pH 7.4, 0.05% Tween^®-20) and blocked with 3% BSA in PBS (75 μL/well) for 1 hour. ELISA strips are stored, dry, at -20°C until needed.

To determine antibody antigenicity of particles, antisera (monoclonal or polyclonal) are diluted using 1% BSA in PBS, and 50 μL/well added to antigen-coated ELISA wells. Sera are incubated for 1 hour, washed with ELISA wash buffer, and probed using an anti-mouse (IgG) -HRP conjugate (as above at 50 μL/well) or other appropriate antibody for 30 minutes, washed again with ELISA wash buffer, and then visualized by the addition of TM blue substrate (50 μL/well) . After 10 minutes, the reaction is stopped by the addition of IN H2SO4 (100 μL/well) and read on an ELISA plate reader set at 450 nm.

B. Immunogenicity of Particles To assay the immunogenicity of particles, mice are immunized, intraperitoneally (IP) , with 20 μg of particles in Freund's complete adjuvant, and then boosted at 4 weeks with 10 μg in Freund's incomplete adjuvant. Mice are bled at 2 , 4, 6, and 8 weeks to monitor serum titer.

C. Sporozoite IFA

Indirect immunofluorescence assay (IFA) is carried out using glutaraldehyde-fixed P. falciparum sporozoites and FITC-labeled anti-mouse IgG (gamma- chain specific) (Kirkegaard and Perry, Gaithersburg, MD) to detect bound antibody [Munesinghe et al . , Eur. J. Immunol . 1991, 21, 3015-3020] . Sporozoites used are dissected from the salivary glands of Anopheles mosquitoes infected by feeding on P. falciparum (NF54 isolate) gametocytes derived from in vi tro cultures.

Example 3 : Expression of Recombinant Chimer HBc Particles

A. Effect of Insertion

Position on Immunogenicity

Antibody titers (1/reciprocal dilution) are measured for mice immunized with HBc particles containing the P. f-CS B cell epitope (NANP) ₄ inserted either between amino acids E109/E110 or E95/A96, or by using a loop replacement approach (CS- 2) [discussed in Schodel et al . , (1994) J. Exp. Med. , 180:1037-1046, using complete Freund's adjuvant]. Mice are immunized with a single 20 μg dose (IP) with adjuvant as noted before, and antibody titers determined in an ELISA using immobilized (NANP) ₅ synthetic peptide.

Another comparison is made of insertion position of the NANP CS-repeat epitope on immunogenicity using BALB/c mice. Antibody titers induced by the CS-2 particle of Schodel et al . are compared to titers achieved using the same (NANP) ₄ B cell epitope, inserted between avian HBc positions 95/96 and 109/110. Sera are analyzed 4 weeks after primary (1°) and 2 weeks after booster (2°) immunization.

A similar comparison of insertion position of the NANP CS-repeat epitope on immunogenicity is made using B10.S mice.

Example 4 : Truncation of Nucleic Acid Binding Domain

Removal of the arginine-rich nucleic acid binding domain of the hepatitis B core protein is desirable for bacterial expression of avian chimer without stray bound nucleic acid. With the mammalian hepatitis B core proteins, many groups used particles truncated to amino acid 149 because amino acid 150 represents the first arginine residue of the arginine-rich C-terminal domain.

Avian hepatitis B core particles can be truncated at the C-terminus (for example after residue number V214 or V224) and retain the ability to assemble into particulate virus-like particles.

A. Truncation to 224

An example of a truncated duck core protein is DHc224, constructed as follows. The duck gene is truncated after amino acid V224 by using the synthetic oligonucleotides DHc (VcoI) -F (SEQ ID NO:199) and DHc224 (H3) -R (SEQ ID NO:204), listed below, as PCR primers to amplify the target portion of the synthetic duck core gene optimized for expression in E. coli described herein. The resultant 693bp amplified fragment is digested with the restriction enzymes JYcoI and Hindlll and inserted into the plasmid pKK223-3N, cut with the same enzymes .

DHc (Ncol) -F (SEQ ID NO: 199)

GCGCCATGGATATTAACGCGTC

DHc224(H3)-R (SEQ ID NO:204)

GCGAAGCTTACTACACCACGGTGGTTTTCAC

B. Truncation to 214

A further example of a truncated duck core protein is DHc214, constructed as follows. The duck gene is truncated after amino acid residue P214 using the synthetic oligonucleotides DHc (Ncol) -F (SEQ ID NO:199) and DHc214(H3)-R (SEQ ID NO-202), listed below, as PCR primers to amplify the target portion of the synthetic duck core gene optimized for expression in E. coli described herein. The resultant 693bp amplified fragment is digested with the restriction enzymes JVcoI and Hindlll and inserted into the plasmid pKK223-3N, cut with the same enzymes .

DHc (Ncol) -F (SEQ ID NO: 199)

GCGCCATGGATATTAACGCGTC

DHc214(H3)-R (SEQ ID NO:202)

GCGAAGCTTACTACGGTTCCAGACCGCGC

C. Determination of Nucleic Acid Binding The determination of stray nucleic acid binding to the avian core protein is ascertained by monitoring and observance of the 280/260 absorbance ratios . Protein samples are diluted to a concentration of between 0.1 and 0.3 mg/mL using phosphate buffered saline (PBS), pH 7.4. The spectrophotometer is blanked, using PBS, and the absorbance of the protein sample measured at wavelengths of 260 nm and 280 nm. The absorbance value determined for a sample at 280 nm was then divided by the absorbance value determined for the same sample at 260 nm to achieve the 280/260 absorbance ratio for a given sample.

The ratios obtained for several samples, including native particles, avian HBc particles truncated after residue position 224, and several HBc chimers that are identified elsewhere herein, are determined.

Example 5: Stabilization of Avian HBc Particles By Adding Cysteine at the C-terminus

A. Addition of a Cysteine Residue to the C-terminus of Avian HBc Particles

Using the polymerase chain reaction (PCR) , genes expressing hybrid HBc particles are mutated to introduce a cysteine or cysteine-containing peptide to the C-terminus of avian HBc. For example, the synthetic oligonucleotides DHc (Ncol) -F (SEQ I'D NO:199) and DHC262+C (H3) -R (SEQ ID NO:207) are used as PCR primers to amplify a hybrid avian HBc gene and incorporate a cysteine codon at the C-terminus of the full-length duck core gene, after amino acid K262.

DHc(NcoI)-F (SEQ ID N0:199)

GCGCCATGGATATTAACGCGTC

DHc262+C(H3) -R (SEQ ID NO:207)

GCGAAGCTTACTAGCATTTACGCGGGCTCGGG

B. Addition of a Cysteine Residue to the C-terminus of Avian HBc Particles Truncated at V224 A cysteine residue is inserted amino acid V224 at the C-terminus of a C-terminal truncated duck core gene (DHc224+C) using the synthetic oligonucleotides DHc (Ncol) -F (SEQ ID NO:199) and DHc224+C (H3) -R (SEQ ID NO:205) as PCR amplification primers.

The resultant 690bp fragment is digested with the restriction enzymes JVcoI and Hindlll and inserted into the plasmid pKK223-3N, cut with the same enzymes. The restriction endonuclease cleavage sites in the sequences below are underlined.

DHc (Ncol) -F (SEQ ID NO: 199)

GCGCCATGGATATTAACGCGTC

DHc224+C(H3) -R (SEQ ID NO:205)

GCGAAGCTTACTAGCACACCACGGTGGTTTTCAC

To assess the ability of a single cysteine residue to stabilize avian HBc particles, a codon for a cysteine residue is inserted using techniques described above between the codon for avian HBc amino acid residue 262 and the termination codon of a chimer HBc molecule that contains an (NANP) 4 malarial

B cell epitope inserted between residues 95 and 96 to form a chimeric molecule and particle referred to as

DHc224+C. The thermal stability (at 37°C) of this chimer particle is compared to a similar chimer particle lacking the inserted cysteine.

After weeks at 37°C, the cysteine- containing particle exhibits fewer bands on the SDS gel, indicating enhanced stability as compared to the particle lacking the added Cys residue. C. Thermal Stability Protocol Purified particles are diluted to a concentration of 1 mg/mL using 50 mM NaP04 , pH 6.8 and sodium azide is added to a final concentration of 0.02% to prevent bacterial growth. Particles are incubated at 37° C and aliquots were taken at the time points indicated in the drawing description. Samples are mixed with SDS-PAGE sample buffer (reducing) and run on 15% SDS-PAGE gels. Gels are stained using Coomassie Blue, and then analyzed.

D. Addition of a Cysteine Residue to the C-terminus of Avian

HBc Particles Truncated at P214 A cysteine residue is inserted after amino acid V214 at the C-terminus of a C-terminal truncated duck core gene (DHc214+C) using the synthetic oligonucleotides DHc (Ncol) -F (SEQ ID NO: 199) and DHc214+C (H3) -R (SEQ ID NO: 203) as PCR amplification primers.

The resultant 663bp fragment is digested with the restriction enzymes .Ncol and Hindlll and inserted into the plasmid pKK223-3N, cut with the same enzymes. The restriction endonuclease cleavage sites in the sequences below are underlined.

DHc (Ncol) -F (SEQ ID NO: 199)

GCGCCATGGATATTAACGCGTC

DHc214+C(H3) -R (SEQ ID NO: 203)

GCGAAGCTTACTAGCACGGTTCCAGACCGCGC Example 6: Stabilization of Avian HBc Particles

By Substituting Cysteine for F60

Mutated DHc genes are constructed with a cysteine residue in the place of the phenylalanine corresponding to residue F60 of the duck gene. A series of truncated and/or C- terminal cysteine-stabilized DHc genes are constructed with amino acid F60 mutated to a cysteine by amplifying the DHc gene in two halves. The N-terminal portion is amplified using the synthetic oligonucleotide pair DHc (Ncol) -F (SEQ ID NO: 199) and DHc207- 153/F60C(H3) -R (SEQ ID NO:277). The mutated codon 60 is designated below by a double underline .

DHc207-153/F60C(H3) -R (SEQ ID

NO : 277) GCGATGCATGCCTTGCGTGGTCTGCCAGCAATCTTCAATCA

GATCCACAAA

The C-terminal portion is amplified using the synthetic oligonucleotide primer DHc (196-219) -F (SEQ ID NO: 278) and one of the various C-terminal primers described hereinabove, DHc214(H3)-R (SEQ ID NO:202), DHc214+C(H3) -R (SEQ ID NO: 203), DHc224(H3)-R

(SEQ ID NO:204), DHc224+C (H3) -R (SEQ ID NO-.205), DHc262(H3)-R (SEQ ID NO:206), DHc262+C (H3 ) -R

(SEQ ID NO:207) .

DHc (196-219) -F (SEQ ID NO:278) GGCATGCATGAAATTGCG The resultant PCR products were cleaved with EcoT22 (ATGCA/T) , ligated, reamplified with the N- and C-terminal primers, and the resultant products cleaved with JVcoI and Hindlll and cloned into pKK223-3N which had been cleaved with the same enzymes.

Example 7 : Avian HBc Particles with

Insertions at the N-terminus

A. Stabilization of Avian HBc Particles By Adding Cysteine at the C-terminus

DHc genes able to accept the directional insertion of synthetic dsDNA fragments into the N- terminal region between amino acid residues Ml and D2 are constructed using and PCR methods. The DHc gene is amplified in two halves using two PCR primer pairs. One pair of primers amplifies the amino terminus, pKK(167-150) -F (SEQ ID NO:279) and DHc-Ml (EcoRI) -R (SEQ ID NO:280), including a sequence preceding the DHc gene . A second pair of primers amplifies the carboxyl terminus, from amino acid D2 onwards, DHc- (EcoRI/Sad) D2-F (SEQ ID NO:281) as the forward primer and the C-terminal reverse primer of choice, DHc214(H3)-R (SEQ ID NO:202), DHc214+C (H3 ) -R (SEQ ID NO:203), DHc224(H3)-R (SEQ ID NO:204), DHc224+C(H3) -R (SEQ ID NO: 205), DHc262(H3)-R (SEQ ID NO:206), DHc262+C(H3) -R (SEQ ID NO:207).

pKK(167-150) -F (SEQ ID NO:279) 5 ' -GCATAATTCGTGTCGCTC DHc-Ml (EcoRI) -R SEQ ID NO:280

5 ^• -GCGGAATTCCCATGGTTTTTTCCTCCTTA

DHc(EcoRl/SacI)D2-F SEQ ID NO: 281

5 ' -CGCGAATTCAAAAAGAGCTCGATATTAACGCGTCTCGT

B. Avian HBc Chimers with N-terminal Fusions

Avian HBc chimers with epitopes inserted at the N-terminus are constructed from AHBc plasmids digested with Ncol and Sad restriction enzymes. Synthetic dsDNA fragments containing 5' Afllll and 3' Sacl overhangs are then inserted. Restriction enzymes Afllll and JVcoI leave compatible overhangs. No DHc residues were deleted, but the glutamic acid- leucine (EL; Sacl) amino acid pairs, coded for by the Sacl restriction site, follows the inserted epitope. The inserted restriction sites are underlined in the oligonucleotide primers below, as exemplified with Influenza A M2 epitopes .

M2 (1-24) :

M S L L T E V E T P I R N E W G C R CATGTCTCTGCTGACCGAAGTTGAAACCCCTATCAGAAACGAATGGGGGTGCAGA AGAGACGACTGGCTTCAACTTTGGGGATAGTCTTTGCTTACCCCCACGTCT

C N D S S D E L SEQ ID NO: 282

TGTAACGATTCAAGTGATGAGCT SEQ ID NO: 283

ACATTGCTAAGTTCACTAC SEQ ID NO: 284 M2 (1-24 (C17S/C19S) ) :

M S L L T E V E T P I R N E W G S R CATGTCTCTGCTGACCGAAGTTGAAACCCCTATCAGAAACGAATGGGGGTCTAGA AGAGACGACTGGCTTCAACTTTGGGGATAGTCTTTGCTTACCCCCAGATCT

S N D S S D E L SEQ ID NO: 285

TCGAACGATTCAAGTGATGAGCT SEQ ID NO: 286

AGCTTGCTAAGTTCACTAC SEQ ID NO: 287

M2 (1-24 (C17S) ) :

C N D S S D E L SEQ ID NO 288 TGTAACGATTCAAGTGATGAGCT SEQ ID NO 289 ACATTGCTAAGTTCACTAC SEQ ID NO 290

M2 (1-24 (C19S) ) :

S N D S S D E L SEQ ID NO: 291

TCGAACGATTCAAGTGATGAGCT SEQ ID NO: 292

AGCTTGCTAAGTTCACTAC SEQ ID NO: 293

Example 8 : P. Vivax HBc Chimers

HBc chimers containing P. falciparum B cell and T cell immunogens are made using sequences from the P. vivax CS protein. Exemplary constructs are illustrated below in Table 11. Table 11

To address the variability of the repeats, the following variant epitopes are used for insertion into AHBc between amino acids 109 and 110 or 95 and 96.

Example 9. T cell Epitope at the

C-terminus of Avian HBc The insertion of the P. vivax Th epitope (Pv-UTC; YLDKVRATVGTEWTPCSVT; SEQ ID NO: 160) into HBc and HBc avian hybrids is performed using synthetic DNA fragments (Synthetic Genetics, San Diego CA) . However, unlike B cell epitopes, which are inserted into the loop region of the avian HBc gene, T cell epitopes are fused to the C-terminus of the HBc gene. Previously discussed cloning vectors are used for the insertion of both B and Th epitopes into HBc.

Example 10 Combining B and T cell Epitopes in a Single Particle

To combine B and Th epitopes into single HBc constructs, PCR is used to amplify N-terminal avian HBc fragments (AA 1-80, which contain the B cell epitopes) , and C-terminal HBc fragments (AA 96 to the C-terminus or AA 110 to the C-terminus, which contain the T cell epitopes) . The fragments are ligated together and amplified again by PCR. Again, clones are verified by restriction endonuclease mapping and automated DNA sequence analysis (Lark Technologies, Houston TX) . Details are essentially the same as for P. falciparum. Particles that contain each of the Type-I, -II and -III B cell epitopes and variants as well as the Pv-UTC, have been expressed and recovered.

Example 11: Relative Immunogenicities of HBc Chimers Relative immunogenicities of several avian HBc chimer immunogens are compared in mice using the IFA assay.

Table 12 Immunogen Citation

P. berghei (CS-1) A

P. yoelii (CS-3) B

P. falciparum (CS-2) A

P . fal ciparum (V12.Pf3.1)

P. vivax (V2.PV-TIB)

[A = Schodel et al . , J. Exp. Med., 1994, 180:1037-1046. B = Schodel et al . , Behring Inst . Mitt . , 1997(98): p. 114- 119.] Mice are immunized with CS-2 or V12.Pfl using 20 μg of particles on day zero and are boosted with 10 μg at four weeks. Mice immunized with particles from V12.Pf3 and V12.Pf3.1 are immunized using 20 μg of particles on day zero and are boosted with 10 μg at eight weeks using adjuvants as discussed before.

Example 12 : Construction of a Modified Hepatitis B

Core Protein Expression Vector

Using site-directed mutagenesis, a lysine codon (AAA) is introduced into the avian HBc gene, into the Sad (GAGCTC) restriction endonuclease site. The insert thus had an amino acid residue sequence of KEL, where the EL is an artifact of the Sad site.

This plasmid is used for the expression of a HBc chimer bearing a lysine as a linker group in the immunodominant loop. The expressed HBc chimer spontaneously forms particles. The linker group- containing avian HBc of this Example thus has a chemically reactive lysine linker residue, preferably at a position corresponding to the loop region.

Example 13: Chemical Coupling of Synthetic Peptides to Chimer Linker Group-containing HBc Particles as Activated Carriers Synthetic peptides (haptens) are chemically conjugated to chimer linker group-containing HBc particles using succinimidyl

4- (N-maleimidomethyl) cyclohexane 1-carboxylate (SMCC) , a water-soluble heterobifunctional cross- linking reagent used to form activated carriers. SMCC is reactive towards both sulfhydryl and primary amino groups, enabling the sequential conjugation of synthetic peptides to the activated carriers (HBc chimer particles whose primary amino groups have previously been modi ied with SMCC) . Further, the 11.6 Angstrom spacer arm afforded by SMCC helps to reduce steric hindrance between the hapten and the HBc carrier, thereby enabling higher coupling efficiencies .

Briefly, avian particles with a lysine residue inserted in the avian HBc sequence between residues 75 and 130, for example at 96, are reacted with a 5-fold excess of SMCC over total amino groups (native amino groups or native amino groups plus the one from the lysine residue of the insert) for 2 hours at room temperature in 50 mM sodium phosphate, pH 7.5, to form maleimide-activated HBc particles. Unreacted SMCC is removed by repeated dialysis against 50 mM sodium phosphate, pH 6.8. The SMCC derivitization of the HBc particles results in a minimal molecular weight increase that is not detectable by SDS-PAGE. However, the PAGE analysis does confirm the integrity of the HBc proteins prior to proceeding to the peptide conjugation step.

Synthetic peptides to be coupled to the chimer HBc particles as activated carriers are designed such that they have N-terminal cysteine residues to enable directional conjugation of peptide haptens to the primary amine on the side chain of the introduced lysine residue via the cysteine sulfhydryl of the hapten. Example 14 : Preparation of Vector for Preparation of Avian HBc Particles for Use in Humans

A. Preparation of a Vector

To manufacture an avian core chimer particle in a manner suitable for human administration, the particle is expressed using an expression system that does not require the use of ampicillin to ensure plasmid maintenance. To achieve this end, the gene coding for the particle, along with the necessary upstream regulatory sequences, is inserted into a new plasmid that utilizes kanamycin as the selectable marker. The new plasmid is synthesized using a two step cloning procedure:

Step 1: The appropriate pKK223-3N plasmid containing the desired chimer is digested with the restriction enzymes BamHI and Hindlll. The commercially available plasmid pREP4 (Qiagen) is cut with Bglll and Hindlll to yield two fragments of 320 bp and 3420 bp. The 3420 bp pREP4 fragment is ligated with the avian gene cut out of pKK223-3N to create the new plasmid. Bglll and BamHI digested DNAs can be ligated by virtue of their common 'overhang' sequences, although neither Bglll or BamHI can cut the resultant fragment .

The new plasmid, therefore, contains the desired avian HBc chimer gene, complete with EcoRI and Sad restriction sites in the immunodominant loop region to enable insertion of epitopes into the loop region of the avian HBc chimer gene .

Step 2 : The second step is to insert the desired B-cell epitope, such as the Pf CS-repeat epitope, into the loop region of the avian gene. This is achieved by digesting the plasmid with Sacl and EcoRI to provide directional insertion of the B- cell epitope. Annealed oligonucleotides encoding the desired B-cell epitope are ligated into the pREP4 plasmid. In addition to the gene that encodes the new avian HBc chimer vaccine candidate (for example the malaria vaccine) , this plasmid contains a gene for the lac repressor (lac I) to force any gene under lac promoter control to be fully repressed until induction with isopropylthiogalactoside (IPTG) . It also has a kanamycin resistance gene to permit positive selection via the addition of kanamycin to culture media. The plasmid has the replication origin of pACYC 184 and is not considered to be a high copy number plasmid.

A suitable host for the constructed avian HBc chimer plasmid is E. coli BLR, a recA derivative of E. coli BL21, and a common strain used for the production of recombinant proteins (available for purchase from Novagen) . E. coli BLR is selected as a host organism for expression because of its increased genetic stability, as well as its ability to produce assembled particles in soluble form (not in inclusion bodies) .

B. Expression of Avian HBc Chimer Particles

E. coli (Strain BLR) harboring the plasmid containing the inducible avian HBc chimer gene are streaked onto an LB agar plate supplemented with 25 μg/mL kanamycin and 10 μg/mL tetracycline, then incubated at 37°C for 16-20 hours. A single colony is then used to inoculate 3 mL of TB-Phy medium in a sterile culture tube, supplemented with 25 μg/mL kanamycin. The tube is incubated overnight (about 18 hours) on a shaker at 37°C and about 200 rpm. The following morning, 100 mL of TB-Phy medium is warmed to 37°C. One mL of the overnight culture is removed and used to inoculate the flask, which is then incubated on a shaker at 37°C at about 200 rpm for six hours.

The fermentor (Biostat™ UE20) is inoculated with 100 mL of inoculum with the fermentor conditions set as follows:

Agitation 400 rpm

Temperature 37°C

Aeration air, 10 liters per minute pH 7.0, uncontrolled

The Agoo "value is measured for the first sample, and for samples every 20-30 minutes thereafter to monitor A_{Q Q Q} . to ascertain the bacterial growth curve. An IPTG solution was prepared by dissolving 62 mg IPTG in 10-15 mL water. When the Aggo "value reaches 0.5 (approximately mid- log phase for the growth curve) , the filter- sterilized IPTG solution is aseptically added to the fermentor through a syringe. The incubation is continued until next day (e.g. about another 10-24 hours) .

At 14 hours after induction, the fermentor temperature is set to 15°C. Harvesting of cells is started by centrifugation in a Beckman^® J2-MC centrifuge with following conditions: Rotor JA10

Speed 7,500 rpm

Temperature 4°C

Time 9 minutes

The harvested cells are stored frozen in liquid nitrogen.

C. Purification of Particles Expressed by the Vector

The biomass of harvested cells are resuspended in 50 mM sodium phosphate, pH 6.8, and lysed using a French Pressure cell at 16,000 psi. The cell debris is removed by centrifugation using a Beckman^® J2-MC centrifuge and the following conditions . ι

Rotor: JA20

Speed: 15,000 rpm

Temperature: 4°C

Time: 30 minutes.

The volume of the resultant supernatant is measured and 277 g/L of solid ammonium sulfate are slowly added to the supernatant. The mixture is stirred at 4°C for 30 minutes. The solution was centrifuged in Beckman^® J2-MC centrifuge with the following conditions.

Rotor: JA20

Speed: 15,000 rpm

Temperature : 4°C

Time: 30 minutes The precipitate is then resuspended in a minimal volume of 50 mM sodium phosphate buffer and then dialyzed against the same buffer for one hour with stirring. The dialyzed solution is centrifuged in Beckman^® J2-MC centrifuge with the following conditions .

Rotor : JA20

Speed: 15,000 rpm

Temperature : 4°C

Time: 15 minutes

The supernatant is recovered and then subjected to gel filtration chromatography.

System: Pharmacia Biotech AKTA™ Explorer Buffer B (elution solvent) : 50 mM Sodium phosphate buffer (pH 6.8) . Column: Millipore Vantage™ VL44 x 1000 column (44 mm diameter, 1000 mm height, Catalog No.:

96441000) Resin: 1.5 liter Sepharose^® CL-4B manufactured by

Pharmacia Detector: UN at 210, 254 and 280 nm. Fraction: 15 mL

The column is eluted with buffer B at 2 mL per minute. Particle-containing fractions are identified using SDS-PAGE and pooled. The salt concentration of the pooled material are adjusted to 5M by adding sodium chloride . Hydrophobic Interaction Chromatography:

System: Pharmacia^® Biotech AKTA™ Explorer (System No.: 18111241 001152, University of Iowa ID No.: 540833.)

Buffer A: 50 mM sodium phosphate buffer

(pH 6.8) + 5 M NaCl. (The buffer is degassed for 30 minutes daily, before use.)

Buffer B (elution solvent) : 50 mM sodium phosphate buffer (pH 6.8) . (The buffer is degassed for 30 minutes daily, before use.)

Hydrophobic Interaction Chromatography using ToyoPearl ® ether 650 resin

Column: Millipore Vantage™ VL44 x 250 column (44 mm diameter, 250 mm height, Catalog No.: 96440250)

Resin: 200 mL Toyopearl^® ether 650 HIC resin, manufactured by Tosohaas Detector: UN at 210, 254, and 280 nm Fraction: 15 mL

The column is equilibrated with 5 column volumes (CN) of buffer A for a one hour time prior to starting puri ication, using a flow rate of 20 mL/minute. The retentate containing 5 M salt is then loaded at a rate of 20 mL/minute. The column is washed with 2 CV of buffer A, washed with 2 CV of 10% buffer B, eluted with 3 CV of 40% buffer B, and (finally eluted) with 100 % buffer B. Fractions are completely analyzed for proteins of interest by SDS PAGE analysis. Pure fractions are combined together, and a protein estimation using a Bradford assay is carried out .

Hydrophobic Interaction Chromatography using butyl resin

Column: Millipore Vantage™ VL44 x 250 column (44 mm diameter, 250 mm height, Catalog No.: 96440250) Resin: 200 mL Toyopearl^® Butyl 650-S HIC resin, manufactured by Tosohaas Detector: UN at 210, 254 and 280 nm Fraction: 15 mL

The column is equilibrated with 5 column volumes (CV) of 40% buffer B for one hour prior to starting purification, using a flow rate of 20 ml/min. The combined fractions from ether HIC are loaded at a rate of 20 mL/minute. The column is washed with 2 CV of 40% buffer B, washed with 2 CV 90% B, and eluted with 4 CV of WFI .

Fractions are analyzed for protein of interest by SDS PAGE analysis. Pure fractions are combined together. Hydroxyapatite Column Chromatography

Column: Millipore Vantage™ VL16 x 250 column (16 mm diameter, 250 mm height, Catalog No. : 96160250)

Resin: 20ml Ceramic Hydroxyapatite (Catalog No. 158-2200)

Detector: UN at 215, 254 and 280 nm

Fraction: 15 mL

The column is equilibrated with 5 column volumes (CV) of 20 mM sodium phosphate buffer, flow rate: 5 mL/min. Load combined fractions eluted from butyl HIC at 5 mL/min. The column is washed with 20 mM sodium phosphate buffer until A280 drops to baseline. Fractions are analyzed for protein of interest by SDS PAGE analysis. Pure fractions are combined together.

Desalting

Column: Prepacked desalting column, HiPrep™ 26/10, Pharmacia ^ι

Resin: 20 mL Ceramic Hydroxyapatite (Catalog No. 158-2200)

Detector: UN at 215, 254 and 280 nm

Fraction: 15 mL

The column is equilibrated with 5 CV of 15 mM Acetate Buffer, pH 6.0. The pooled fractions from the hydroxyapatite column are loaded onto the column, and then eluted with 15 mM Acetate Buffer, pH 6.0 , at a flow rate of 20 mL/min. Fractions are analyzed for protein of interest by SDS PAGE analysis. Pure fractions are combined together, and protein estimation is carried out using a Bradford assay. The pure fraction is assayed for endotoxin level, and finally passed through a 0.22-micron filter for terminal filtration.

Example 15 : Comparative Immunogenicities in Monkeys

The comparative immunogenicity of the particles expressed from the plasmids containing avian HBc chimer genes, formulated with either Seppic™ ISA-720 (Seppic Inc., Paris, France), Alhydrogel™ (Superfos, Denmark) as adjuvants, or unformulated (saline) , is studied in Cynomolgus monkeys .

The Seppic™ ISA-720 formulation is prepared according to the manufacturers directions. Briefly, the particles are mixed at the desired ratio and vortexed, using a bench top vortexer, set at maximum power, for 1 minute. The Alhydrogel™ formulation was prepared using an 8-fold excess of Alhydrogel™ (by weight) over the particles, which is physically bound to the Alhydrogel™ prior to immunization.

Groups of two monkeys are immunized with 20 μg particles as immunogen via an intramuscular route. Animals are bled on days 0, 21, 42, 56 and 70, and sera analyzed for titers of anti-NANP antibody using an ELISA.

Example 16: T Cell Activation

Mice are immunized twice with particles in Seppic™ Montanide™ ISA-720. Spleen cells are removed and stimulated in the presence of various peptides. IO⁶ cells are incubated for 3 days in the presence of peptides: UTC (universal T epitope from P. falciparum) , peptide corresponding to AHBc 110-205, NANP (B-cell epitope NANPNVDP (NANP) _{3 #} SEQ ID NO: 49) in the presence of Staphylococcal enterotoxin B (SEB) , or tissue culture medium (unstim) . Interferon gamma production after 3 days is determined by ELISA.

Immunizing with particles induces T-cells that recognize the UTC component of the protein, and drives them to a Thl type response .

Example 17 : Comparison of Various Avian Chimers A. Preparation of Expression Vectors Various nucleic acid sequences encoding the core proteins described in Examples 1 and 5 hereinabove were generated in order to make avian HBc proteins for expression in E. coli . Expression plasmid inserts having nucleic acid sequences corresponding to various avian core protein sequences were generated using forward and reverse primers upon nucleic acid amplification with the avian core plasmid described in Example 1, and the amplified fragment was digested with the restriction enzymes JVcoI and Hindlll and inserted into the plasmid pKK223-3N, cut with the same enzymes.

The expression vector pKK223-3N, called DHBc262, containing a nucleic acid encoding a full- length 262 -residue avian core protein (SEQ ID NO:295), called 1724, was prepared for expression in E. coli from an E. coli-optimized nucleic acid sequence (SEQ ID NO: 294) as described in Example 1. An expression vector, called DHBc262+C (SEQ ID NO:296), was similarly prepared, encoding a full- length avian core protein similar to 1724, but additionally having a C-terminal cysteine residue (SEQ ID NO:297), called 1728, was generated using the forward primer DHc-NcoI-F (SEQ ID NO: 136) and the cysteine-containing reverse primer DHc262+C (H3) -R (SEQ ID NO: 135) .

An expression vector, called DHBc224 (SEQ ID NO:298), encoding an avian core protein truncated after residue 224 (SEQ ID NO:299) , called 1731, was generated using the forward primer DHc-JVcoI-F (SEQ ID NO: 136) and the reverse primer introducing a termination codon after residue 224, DHc224 (H3) -R (SEQ ID NO: 204) . An expression vector, called DHBc224+C (SEQ ID NO:300), encoding an avian core protein truncated after residue 224 similar to 1731, but also having a C-terminal cysteine residue (SEQ ID NO:301) , called 1732, was generated using the forward primer DHc-JVcoI-F (SEQ ID NO: 136) and the reverse primer introducing a cysteine residue and a stop codon after residue 224, DHc224+C (H3) -R (SEQ ID NO:205) .

An expression vector, called DHBc 214 (SEQ ID NO: 302), encoding a avian core protein truncated after residue 214 (SEQ ID NO:303), called 1729, was generated using the forward primer DHc-.Ncol-F (SEQ ID NO: 136) and the reverse primer introducing a stop codon after residue 214, DHc214 (H3) -R (SEQ ID NO: 202) . An expression vector, called DHBc214+C (SEQ ID NO: 304) , encoding an avian core protein truncated at residue 214 similar to 1729, but also having a C- terminal cysteine residue (SEQ ID NO:305), called 1730, was generated using the forward primer DHc- _VcoI-F (SEQ ID NO: 136) and the reverse primer introducing a cysteine residue and a stop codon after residue 214, DHc214+C (H3) -R (SEQ ID NO:203). An expression vector, called DHBc 214(F60C)+C (SEQ ID NO:306), encoding an avian core protein truncated after residue 214 with an added C- terminal cystein residue, also having the phenylalanine residue at position 60 mutated to a cysteine residue (SEQ ID NO:307), called 1808, was generated using the forward primer DHc-JVcoI-F (SEQ ID NO: 136) and the reverse primer that has the F to C mutation, DHc207-153/F60C-R (SEQ ID NO:277) for the first portion of the protein-encoding nucleic acid together with the forward primer DHc (196-219) -F (SEQ ID NO: 278) and the reverse primer adding a C-terminal cysteine residue and a stop codon after residue 214, DHc214+C(H3) -R (SEQ ID NO:203) for the second portion of the protein-encoding nucleic acid.

B. Characterization of the Expressed Protein

The various expression vectors described in Section A of this Example were expressed in E. coli cells. The cells containing the expressed avian HBc variants were lysed via microfluidizer. The lysate was clarified via centrifugation. The clarified lysate was brought to 45% ammonium sulfate, and the protein was permitted to precipitate out of the lysate solution. The full-length (262 residue) avian HBc proteins, without the C-terminal cysteine (DHBc 262 or 1724) and with (DHBc 262+C or 1728) , did not precipitate well out of the lysate, so there is little data on these samples hereinunder.

The precipitated protein was collected via centrifugation, and the lysate precipitation supernatant decanted off. The precipitated protein pellets were resuspended in 20 mM sodium phosphate, pH 6.8, and dialyzed against the same buffer for several hours with a few changes of dialysis buffer. The dialyzed protein solution was clarified by a brief centrifugation step. The clarified protein supernatant was subjected to gel filtration chromatography on Sepharose^® CL-4B. The absorbance units of the peak maximum for the various proteins are reported in the table below. The fractions containing avian HBc protein were pooled.

The presence of a C-terminal cysteine residue did not affect the expression level, despite dimer formation. Truncation at the C-terminus permitted more facile purification of protein. The yield of full-length particles using the above purification procedure was negligible.

The avian HBc protein was present in the form of avian HBc particles. The avian HBc protein peaks for shortest truncated variants, DHBc214 (1729), DHBc 214+C (1730) and DHBc 214 (F60C) +C (1808) were the most intense, showing the least shoulder and tailing.

As noted above, the purified avian HBc was present in solution in the form of an assembled HBc particle. Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) of the avian HBc variants was carried out under reducing (addition of di- thiothreitol to the gel sample) or non-reducing conditions. The gels were visualized using silver stain.

Under reducing conditions, the avian HBc samples show a strong band at the molecular weight of the corresponding avian HBc monomer, in the case of the avian HBc variants that did not have an additional cysteine residue at the C-terminus (DHBc 214 or 1729, and DHBc 224 or 1731) a similar dominant monomer band was evident . In the case of the avian

HBc variants that had an additional cysteine residue at the C-terminus (DHBc 214+C or 1730, DHBc 214

(F60C+C) or 1808, and DHBc 224+C or 1732), a band was present at the molecular weight corresponding to a dimer of the avian HBc variant .

Samples of the various avian HBc variant proteins were diluted to 0.5 mg/mL and incubated at -70°C, +4°C, room temperature (about +20°C) and +37°C for a week. After one week, 200 ng of each sample were analyzed using SDS-PAGE (silver stain) under reducing and non-reducing conditions. The stability of the various protein particles was suggested in these data.

The DHBc 214 (1729) variant was very stable for a week at -70°C, +4°C, but at room temperature (about +20°C) and +37°C, the sample had degraded significantly. The DHBc 224 (1731) variant was very stable at all four temperatures for the entire week.

The DHBc 224+C (1732) variant was stable at all four temperatures for the full week, also demonstrating dimer cross-linking after a week at all storage temperatures. The DHBc 214+C (1730) variant was stable for a week at -70°C and +4°C, and also showed cross-linking; at room temperature (about +20°C) and +37°C, however, the sample had degraded significantly.

The DHBc 214 (F60C) +C (1808) sample was stable after the week at -70°C and +4°C upon reduction, but showed degradation to smaller protein sizes at room temperature and above. At room temperature and higher, there was a significant amount of cross-linking that was not dimer of the two original monomer chains .

To summarize, DHBc 214 and DHBc214+C showed degradation at room temperature and at +37°C. The monomers of DHBc 214 (F60C) +C were degraded to smaller fragments after incubation at RT and +37°C. Under non-reducing conditions monomers and dimers formed after storage at -70°C and +4°C. DHBc 224 showed partial degradation at all temperatures tested, and no dimer formation. DHBc224+C formed monomers and dimers at all storage temperatures.

As described in Example 4 hereinabove, truncation of the arginine-rich nucleic acid binding domain of the hepatitis B core protein can reduce spurious nucleic acid binding to the expressed modified hepatitis B core protein. The ratio of absorbance of 280 nm over 260 nm of the purified protein suggests whether spurious nucleic acid co- purified with the protein. The A280:A260 data for the various purified proteins is shown the table below. An ethidium bromide-stained agarose gel of the protein samples confirmed the presence of some nucleic acid in the DHBc224 sample, but the protein band seemed to show some particle disassembly at the time of the ethidium gel. The DHBc 214 samples did not contain detectable amounts of nucleic acid, even from 10 micrograms of particle. The protein bands were analyzed in the ethidium bromide-stained gel by staining with coomassie blue overnight.

The truncation of the C-terminal arginine residues (e.g. 224 or 214) did result in a decrease in the A280 to A260 ratio, demonstrating less nucleic acid contamination of the purified proteins. The truncated HBc protein variants that also had a C- terminal cysteine residue had less nucleic acid contamination in the purified protein samples.

DHBc 262 (1724) nucleic acid sequence (SEQ ID NO:294)

ATGGATATTAACGCGTCTCGTGCGCTGGCCAACGTTTATGATCTGCCAGATGA CTTCTTTCCGAAAATTGATGACCTGGTGCGTGATGCCAAAGACGCCCTGGAAC CGTATTGGAAATCTGATAGCATTAAGAAACATGTGCTGATTGCGACCCATTTT GTGGATCTGATTGAAGATTTCTGGCAGACCACGCAAGGCATGCATGAAATTGC GGAAAGCCTGCGTGCCGTGATTCCGCCAACGACCACGCCAGTTCCGCCAGGCT ATCTGATTCAGCATGAGGAAGCGGAAGAGATTCCGTTAGGCGATCTGTTTAAA CATCAGGAAGAGCGTATTGTGAGCTTTCAGCCAGATTATCCGATTACCGCGCG TATCCATGCCCACCTGAAAGCCTATGCGAAAATTAACGAAGAGAGCCTGGATC GTGCGCGTCGCTTACTGTGGTGGCATTATAACTGCCTGTTATGGGGCGAAGCG CAGGTGACCAATTATATTAGCCGTCTGCGCACCTGGCTGTCTACCCCGGAGAA ATATCGTGGCCGCGATGCGCCGACCATTGAAGCCATCACCCGTCCGATTCAGG TGGCGCAAGGCGGTCGTAAAACCACGACCGGCACGCGTAAACCGCGCGGTCTG GAACCGCGTCGCCGTAAAGTGAAAACCACGGTGGTTTATGGCCGTCGCCGTAG CAAAAGCCGTGAACGTCGCGCGCCGACCCCGCAGCGTGCGGGCAGCCCGCTGC CGCGTAGCTCTTCCAGCCATCACCGTAGCCCGAGCCCGCGTAAATAGTAA

DHBc 262 (1724) amino acid sequence (SEQ ID NO-295)

MDINASl^LANVYDLPDDFFPKIDDLVRDAKDALEPYWKSDSIKKHVLIATHF VDLIEDFWQTTQGMHEIAESLRAVIPPTTTPVPPGYLIQHEEAEEIPLGDLFK HQEERIVSFQPDYPITARIHAHLKAYAKINEESLDRARRLLWWHYNCLLWGEA QVTNYISRLRTWLSTPEKYRGRDAPTIEAITRPIQVAQGGRKTTTGTRKPRGL EPRRRKVKTTWYGRRRSKSRERRAPTPQRAGSPLPRSSSSHHRSPSPRK@#

DHBc 262+C (1728) nucleic acid sequence (SEQ ID NO-296)

ATGGATATTAACGCGTCTCGTGCGCTGGCCAACGTTTATGATCTGCCAGATGA CTTCTTTCCGAAAATTGATGACCTGGTGCGTGATGCCAAAGACGCCCTGGAAC CGTATTGGAAATCTGATAGCATTAAGAAACATGTGCTGATTGCGACCCATTTT GTGGATCTGATTGAAGATTTCTGGCAGACCACGCAAGGCATGCATGAAATTGC GGAAAGCCTGCGTGCCGTGATTCCGCCAACGACCACGCCAGTTCCGCCAGGCT ATCTGATTCAGCATGAGGAAGCGGAAGAGATTCCGTTAGGCGATCTGTTTAAA CATCAGGAAGAGCGTATTGTGAGCTTTCAGCCAGATTATCCGATTACCGCGCG TATCCATGCCCACCTGAAAGCCTATGCGAAAATTAACGAAGAGAGCCTGGATC GTGCGCGTCGCTTACTGTGGTGGCATTATAACTGCCTGTTATGGGGCGAAGCG CAGGTGACCAATTATATTAGCCGTCTGCGCACCTGGCTGTCTACCCCGGAGAA ATATCGTGGCCGCGATGCGCCGACCATTGAAGCCATCACCCGTCCGATTCAGG TGGCGCAAGGCGGTCGTAAAACCACGACCGGCACGCGTAAACCGCGCGGTCTG GAACCGCGTCGCCGTAAAGTGAAAACCACGGTGGTTTATGGCCGTCGCCGTAG CAAAAGCCGTGAACGTCGCGCGCCGACCCCGCAGCGTGCGGGCAGCCCGCTGC CGCGTAGCTCTTCCAGCCATCACCGTAGCCCGAGCCCGCGTAAATGCTAGTAA

DHBc 262+C (1728) amino acid sequence (SEQ ID NO-297)

MDINASRALANVYDLPDDFFPKIDDLVRDAKDALEPYWKSDSIKKHVLIATHF VDLIEDFWQTTQGMHEIAESLRAVIPPTTTPVPPGYLIQHEEAEEIPLGDLFK HQEERIVSFQPDYPITARIHAHLKAYAKINEESLDRARRLLWWHYNCLLWGEA QVTNYISRLRTWLSTPEKYRGRDAPTIEAITRPIQVAQGGRKTTTGTRKPRGL EPRRRKVKTTWYGRRRSKSRERRAPTPQRAGSPLPRSSSSHHRSPSPRKC@#

DHBc 224 (1731) nucleic acid sequence (SEQ ID NO-298)

ATGGATATTAACGCGTCTCGTGCGCTGGCCAACGTTTATGATCTGCCAGATGA CTTCTTTCCGAAAATTGATGACCTGGTGCGTGATGCCAAAGACGCCCTGGAAC CGTATTGGAAATCTGATAGCATTAAGAAACATGTGCTGATTGCGACCCATTTT GTGGATCTGATTGAAGATTTCTGGCAGACCACGCAAGGCATGCATGAAATTGC GGAAAGCCTGCGTGCCGTGATTCCGCCAACGACCACGCCAGTTCCGCCAGGCT ATCTGATTCAGCATGAGGAAGCGGAAGAGATTCCGTTAGGCGATCTGTTTAAA CATCAGGAAGAGCGTATTGTGAGCTTTCAGCCAGATTATCCGATTACCGCGCG TATCCATGCCCACCTGAAAGCCTATGCGAAAATTAACGAAGAGAGCCTGGATC GTGCGCGTCGCTTACTGTGGTGGCATTATAACTGCCTGTTATGGGGCGAAGCG CAGGTGACCAATTATATTAGCCGTCTGCGCACCTGGCTGTCTACCCCGGAGAA ATATCGTGGCCGCGATGCGCCGACCATTGAAGCCATCACCCGTCCGATTCAGG TGGCGCAAGGCGGTCGTAAAACCACGACCGGCACGCGTAAACCGCGCGGTCTG GAACCGCGTCGCCGTAAAGTGAAAACCACCGTGGTGTAGTAA

DHBc 224 (1731) amino acid sequence (SEQ ID NO-299)

MDINASRALANNYDLPDDFFPKIDDLVRDAKDALEPYWKSDSIKKHVLIATHF VDLIEDFWQTTQGMHEIAESLRAVIPPTTTPVPPGYLIQHEEAEEIPLGDLFK HQEERIVSFQPDYPITARIHAHLKAYAKIΝEESLDRARRLLWWHYΝCLLWGEA QVTΝYISRLRTWLSTPEKYRGRDAPTIEAITRPIQVAQGGRKTTTGTRKPRGL EPRRRKVKTTW@#

DHBc 224+C (1732) nucleic acid sequence (SEQ ID ΝO-300)

ATGGATATTAACGCGTCTCGTGCGCTGGCCAACGTTTATGATCTGCCAGATGA CTTCTTTCCGAAAATTGATGACCTGGTGCGTGATGCCAAAGACGCCCTGGAAC CGTATTGGAAATCTGATAGCATTAAGAAACATGTGCTGATTGCGACCCATTTT GTGGATCTGATTGAAGATTTCTGGCAGACCACGCAAGGCATGCATGAAATTGC GGAAAGCCTGCGTGCCGTGATTCCGCCAACGACCACGCCAGTTCCGCCAGGCT ATCTGATTCAGCATGAGGAAGCGGAAGAGATTCCGTTAGGCGATCTGTTTAAA CATCAGGAAGAGCGTATTGTGAGCTTTCAGCCAGATTATCCGATTACCGCGCG TATCCATGCCCACCTGAAAGCCTATGCGAAAATTAACGAAGAGAGCCTGGATC GTGCGCGTCGCTTACTGTGGTGGCATTATAACTGCCTGTTATGGGGCGAAGCG CAGGTGACCAATTATATTAGCCGTCTGCGCACCTGGCTGTCTACCCCGGAGAA ATATCGTGGCCGCGATGCGCCGACCATTGAAGCCATCACCCGTCCGATTCAGG TGGCGCAAGGCGGTCGTAAAACCACGACCGGCACGCGTAAACCGCGCGGTCTG GAACCGCGTCGCCGTAAAGTGAAAACCACCGTGGTGTGCTAGTAA

DHBc 224+C (1732) amino acid sequence (SEQ ID NO: 301)

MDINASRALANVYDLPDDFFPKIDDLVRDAKDALEPYWKSDSIKKHVLIATHF VDLIEDFWQTTQGMHEIAESLRANIPPTTTPVPPGYLIQHEEAEEIPLGDLFK HQEERIVSFQPDYPITARIHAHLKAYAKIΝEESLDRARRLLWWHYΝCLLWGEA QVTΝYISRLRTWLSTPEKYRGRDAPTIEAITRPIQVAQGGRKTTTGTRKPRGL EPRRRKVKTTWC@#

DHBc 214 (1729) nucleic acid sequence (SEQ ID ΝO:302)

ATGGATATTAACGCGTCTCGTGCGCTGGCCAACGTTTATGATCTGCCAGATGA CTTCTTTCCGAAAATTGATGACCTGGTGCGTGATGCCAAAGACGCCCTGGAAC CGTATTGGAAATCTGATAGCATTAAGAAACATGTGCTGATTGCGACCCATTTT GTGGATCTGATTGAAGATTTCTGGCAGACCACGCAAGGCATGCATGAAATTGC GGAAAGCCTGCGTGCCGTGATTCCGCCAACGACCACGCCAGTTCCGCCAGGCT ATCTGATTCAGCATGAGGAAGCGGAAGAGATTCCGTTAGGCGATCTGTTTAAA CATCAGGAAGAGCGTATTGTGAGCTTTCAGCCAGATTATCCGATTACCGCGCG TATCCATGCCCACCTGAAAGCCTATGCGAAAATTAACGAAGAGAGCCTGGATC GTGCGCGTCGCTTACTGTGGTGGCATTATAACTGCCTGTTATGGGGCGAAGCG CAGGTGACCAATTATATTAGCCGTCTGCGCACCTGGCTGTCTACCCCGGAGAA ATATCGTGGCCGCGATGCGCCGACCATTGAAGCCATCACCCGTCCGATTCAGG TGGCGCAAGGCGGTCGTAAAACCACGACCGGCACGCGTAAACCGCGCGGTCTG GAACCGTAGTAA

DHBc 214 (1729) amino acid sequence (SEQ ID NO: 303)

MDINASRALANVYDLPDDFFPKIDDLVRDAKDALEPYWKSDSIKKHVLIATHF VDLIEDFWQTTQGMHEIAESLRAVIPPTTTPVPPGYLIQHEEAEEIPLGDLFK HQEERIVSFQPDYPITARIHAHLKAYAKINEESLDRARRLLWWHYNCLLWGEA QVTNYISRLRTWLSTPEKYRGRDAPTIEAITRPIQVAQGGRKTTTGTRKPRGL EP@#

DHBc 214+C (1730) nucleic acid sequence (SEQ ID NO:304)

ATGGATATTAACGCGTCTCGTGCGCTGGCCAACGTTTATGATCTGCCAGATGA

CTTCTTTCCGAAAATTGATGACCTGGTGCGTGATGCCAAAGACGCCCTGGAAC

CGTATTGGAAATCTGATAGCATTAAGAAACATGTGCTGATTGCGACCCATTTT

GTGGATCTGATTGAAGATTTCTGGCAGACCACGCAAGGCATGCATGAAATTGC

GGAAAGCCTGCGTGCCGTGATTCCGCCAACGACCACGCCAGTTCCGCCAGGCT

ATCTGATTCAGCATGAGGAAGCGGAAGAGATTCCGTTAGGCGATCTGTTTAAA

CATCAGGAAGAGCGTATTGTGAGCTTTCAGCCAGATTATCCGATTACCGCGCG

TATCCATGCCCACCTGAAAGCCTATGCGAAAATTAACGAAGAGAGCCTGGATC

GTGCGCGTCGCTTACTGTGGTGGCATTATAACTGCCTGTTATGGGGCGAAGCG '

CAGGTGACCAATTATATTAGCCGTCTGCGCACCTGGCTGTCTACCCCGGAGAA

ATATCGTGGCCGCGATGCGCCGACCATTGAAGCCATCACCCGTCCGATTCAGG

TGGCGCAAGGCGGTCGTAAAACCACGACCGGCACGCGTAAACCGCGCGGTCTG

GAACCGTGCTAGTAA

DHBc 214+C (1730) amino acid sequence (SEQ ID NO:305)

MDINAS1^]--ANVYDLPDDFFPKIDDLVRDAKDALEPYWKSDSIKKHVLIATHF VDLIEDFWQTTQGMHEIAESLRAVIPPTTTPVPPGYLIQHEEAEEIPLGDLFK HQEERIVSFQPDYPITARIHAHLKAYAKINEESLDRARRLLWWHYNCLLWGEA QVTNYISRLRTWLSTPEKYRGRDAPTIEAITRPIQVAQGGRKTTTGTRKPRGL EPC@#

DHBc 214(F60C)+C (1808) nucleic acid sequence (SEQ ID NO:306)

ATGGATATTAACGCGTCTCGTGCGCTGGCCAACGTTTATGATCTGCCAGATGA CTTCTTTCCGAAAATTGATGACCTGGTGCGTGATGCCAAAGACGCCCTGGAAC CGTATTGGAAATCTGATAGCATTAAGAAACATGTGCTGATTGCGACCCATTTT GTGGATCTGATTGAAGATTGCTGGCAGACCACGCAAGGCATGCATGAAATTGC GGAAAGCCTGCGTGCCGTGATTCCGCCAACGACCACGCCAGTTCCGCCAGGCT ATCTGATTCAGCATGAGGAAGCGGAAGAGATTCCGTTAGGCGATCTGTTTAAA CATCAGGAAGAGCGTATTGTGAGCTTTCAGCCAGATTATCCGATTACCGCGCG TATCCATGCCCACCTGAAAGCCTATGCGAAAATTAACGAAGAGAGCCTGGATC GTGCGCGTCGCTTACTGTGGTGGCATTATAACTGCCTGTTATGGGGCGAAGCG CAGGTGACCAATTATATTAGCCGTCTGCGCACCTGGCTGTCTACCCCGGAGAA ATATCGTGGCCGCGATGCGCCGACCATTGAAGCCATCACCCGTCCGATTCAGG TGGCGCAAGGCGGTCGTAAAACCACGACCGGCACGCGTAAACCGCGCGGTCTG GAACCGTGCTAGTAA

DHBc 214(F60C)+C (1808) amino acid sequence (SEQ ID NO:307)

MDINASRALANVYDLPDDFFPKIDDLVRDAKDALEPYWKSDSIKKHVLIATHF VDLIEDCWQTTQGMHEIAESLRAVIPPTTTPVPPGYLIQHEEAEEIPLGDLFK HQEERIVSFQPDYPITARIHAHLKAYAKINEESLDRARRLLWWHYNCLLWGEA QVTNYISRLRTWLSTPEKYRGRDAPTIEAITRPIQVAQGGRKTTTGTRKPRGL EPC@#

Each of the patents and articles cited herein is incorporated by reference. The use of the article "a" or "an" is intended to include one or more .

The foregoing description and the examples are intended as illustrative and are not to be taken as limiting. Still other variations within the spirit and scope of this invention are possible and will readily present themselves to those skilled in the art .

Claims

WHAT IS CLAIMED IS:

1. A recombinant chimer avian hepatitis B core (AHBc) protein molecule

(i) is free more than one sequence of five amino acid residues that includes three arginine, lysine or both residues;

(ii) contains one to about three cysteine residues within about 30 amino acid residues of the N-terminus, C-terminus or both N- and C-termini in a sequence other than that of a pre-core sequence; and

(iii) contains a peptide-bonded heterologous epitope or a heterologous linker residue for a conjugated epitope sequence, said chimer molecule (a) containing no more than 20 percent conservatively substituted amino acid residues in the AHBc sequence, (b) self-assembling into particles that are substantially free of binding to nucleic acids on expression in a host cell, and (c) said dimers being more stable than are particles formed from an otherwise identical AHBc chimer that lacks said cysteine of (ii) or in which said cysteine of (ii) present in the chimer molecule is replaced by another residue .

2. The recombinant chimer AHBc protein molecule according to claim 1 that includes the amino acid residue sequence of the avian hepatitis B core protein of about position 5 to about position 205 of SEQ ID NO: 7.

3. The recombinant chimer AHBc protein molecule according to claim 2 wherein said peptide- bonded heterologous epitope is inserted within the sequence of about position 2 to about position 205 of SEQ ID NO: 7.

4. The recombinant chimer AHBc protein molecule according to claim 3 wherein said peptide- bonded heterologous epitope defines a B cell epitope.

5. The recombinant chimer AHBc protein molecule according to claim 3 wherein said peptide- bonded heterologous epitope defines a T cell epitope.

6. The recombinant chimer AHBc protein molecule according to claim 3 wherein said peptide- bonded heterologous epitope is peptide-bonded within about 5 residues of the N-terminus of the AHBc sequence .

7. The recombinant chimer AHBc protein molecule according to claim 3 wherein said peptide- bonded heterologous epitope is peptide-bonded within about 5 residues of the C-terminus of the AHBc sequence .

8. The recombinant chimer AHBc protein molecule according to claim 3 wherein said peptide- bonded heterologous epitope is peptide-bonded at a position of about 70 to about 77 of the AHBc sequence of the chimer relative to the sequence of SEQ ID

NO: 7.

9. The recombinant chimer AHBc protein molecule according to claim 2 wherein said one to about three cysteine residues are present within about 30 amino acid residues of the N-terminus.

10. The recombinant chimer AHBc protein molecule according to claim 2 wherein said one to about three cysteine residues are present within about 30 amino acid residues of the C-terminus.

11. The recombinant chimer AHBc protein molecule according to claim 2 wherein said one to about three cysteine residues are present both within about 30 amino acid residues of the N-terminus and within about 30 amino acid residues of the C- terminus .

12. The recombinant AHBc chimer protein molecule according to claim 4 wherein said B cell epitope is an amino acid sequence present in a pathogen selected from the group consisting of Streptococcus pneumonia, Cryptosporidium parvum, HIV, foot-and-mouth disease virus, influenza virus, Yersinia pestis, Haemophilus influenzae, Moraxella catarrhalis, Porphyromonas gingivalis, Trypanosoma cruzi , Plasmodium falciparum, Plasmodium vivax, Plasmodium berghi , Plasmodium yoelli , Streptococcus sobrinus, Shigella flexneri , RSV, Plasmodium Entamoeba histolytica, Schistosoma japonicum, Schistosoma mansoni , and ebola virus.

13. The recombinant chimer AHBc protein molecule according to claim 2 wherein said peptide- bonded heterologous epitope is a heterologous linker residue for a conjugated epitope sequence inserted within the sequence of about position 2 to about position 205 of SEQ ID NO: 7.

14. The recombinant HBc chimer protein molecule according to claim 13 wherein said heterologous linker residue for a conjugated epitope is selected from the group consisting of a lysine, aspartic acid, glutamic acid, cysteine and a tyrosine residue .

39. A recombinant avian hepatitis B virus core (AHBc) protein chimer molecule with a length of about 175 to about 435 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, wherein

(a) Domain I comprises about the sequence of the residues of position 4 through position 75 of AHBc, a sequence of up to about 30 residues that contains a cysteine residue at an amino acid position of the chimer molecule corresponding to amino acid position -6 to about +4 from the N-terminus of the HBc sequence of SEQ ID NO: 7 [N-terminal cysteine residue] , and up to three cysteine residues added or substituted into the AHBc sequence at position 55 to position 65;

(b) Domain II comprises about 0 to about 106 amino acid residues peptide-bonded to AHBc residue 75 of Domain I in which at least 4 residues in a sequence of AHBc positions 76 to 131 are present and peptide-bonded to zero to about 50 amino acid residues that are heterologous to AHBc and constitute a heterologous epitope;

(c) Domain III is an AHBc sequence from position 132 through position 205 peptide-bonded to the C-terminal residue of Domain II; and (d) Domain IV comprises (i) 8 through nineteen residues of an AHBc amino acid residue sequence from position 206 through 224 peptide-bonded to the residue of position 205 of Domain III, (ii) zero or one cysteine residue [C-terminal cysteine residue] within about 30 residues of the C-terminus of the chimer molecule, and (iii) zero to about 50 amino acid residues in a sequence heterologous to AHBc from the C-terminus of the portion of the AHBc sequence in (i) to the C-terminus of the chimer, said chimer self-assembling into particles on expression in a host cell, said particles being more stable than are particles formed from an otherwise identical AHBc chimer molecule that lacks said cysteine residue introduced in (a) or (d) or in which a N-terminal cysteine residue present in the chimer molecule is replaced by another residue, and having an amino acid residue sequence in which no more than about 5 percent of the amino acid residues are substituted in the AHBc sequence of the chimer.

40. The recombinant HBc chimer protein molecule according to claim 39 wherein said heterologous epitope of Domain II is a B cell epitope .

41. The recombinant HBc chimer protein molecule according to claim 40 wherein said heterologous epitope contains 15 to about 50 amino acid residues.

42. The recombinant HBc chimer protein molecule according to claim 40 wherein said heterologous epitope contains 20 to about 30 amino acid residues.

43. The recombinant HBc chimer protein molecule according to claim 40 wherein the HBc sequence between amino acid residues 76 and 109 is present, but interrupted by said heterologous epitope.

44. The recombinant HBc chimer protein molecule according to claim 40 wherein said B cell epitope is an amino acid sequence present in a pathogen selected from the group consisting of Streptococcus pneumonia, Cryptosporidium parvum, HIV, foot-and-mouth disease virus, influenza virus, Yersinia pestis, Haemophilus influenzae, Moraxella catarrhalis, Porphyromonas gingivalis, Trypanosoma cruzi, Plasmodium falciparum, Plasmodium vivax, Plasmodium berghi, Plasmodium yoelli, Streptococcus sobrinus, Shigella flexneri , RSV, Plasmodium Entamoeba histolytica, Schistosoma japonicum, Schistosoma mansoni , bovine inhibin and ebola virus.

45. The recombinant AHBc chimer protein molecule according to claim 40 wherein said sequence heterologous to AHBc in (d) is a T cell epitope peptide-bonded to the C-terminal residue of the sequence in (d) (i) or (d) (ii) .

46. The recombinant AHBc chimer protein molecule according to claim 45 wherein said T cell epitope is from the organism against which a contemplated chimer is to be used as an immunogen.

47. The recombinant AHBc chimer protein molecule according to claim 40 wherein said N- terminal cysteine residue is located within about five amino acid residues of the N-terminal of the chimer protein molecule.