Title of Invention

"RECOMBINANT CHIMER HEPATITIS B CORE (HBC) PROTEIN MOLECULE AND IMMUNOGENIC PARTICLES FORMED BY SAID HBC CHIMER PROTEIN MOLECULE"

Abstract A chimeric, carboxy-terminal truncated hepatitis B virus nucleocapsid protein (HBc) 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 HBc, whereas the enhanced stability of self-assembled 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.'
Full Text TECHNICAL FIELD
The present invention relates to the
intersection of the fields of immunology and protein engineering, and particularly to a chimeric hepatitis B virus (HBV) nucleocapsid protein that is engineered for both enhanced stability of self-assembled particles and the display of an immunogenic epitope.
BACKGROUND OF THE INVENTION
The family hepadnaviridae are enveloped DNA-containing animal viruses that can cause hepatitis B in humans (HBV). The hepadnavirus family includes hepatitis B viruses of other mammals, e.g., woodchuck (WHV), and ground squirrel (GSHV), and avian viruses found in ducks (DHV) and herons (HeHV). Hepatitis B virus (HBV) used herein refers to a member of the family hepadnaviridae, unless the discussion is referring to a specific example.
The nucleocapsid or core of the mammalian hepatitis B virus (HBV or hepadnavirus) contains a sequence of 183 or 135 amino acid residues, depending on viral subtype, whereas the duck virus capsid contains 262 amino acid residues. Hepatitis B core protein monomers of the several hepadnaviridae self-

assemble in infected cells into stable aggregates known as hepatitis B core protein particles (HBc particles) . Two three-dimensional structures are reported for HBc particles. A first that comprises a minor population contains 90 copies of the HBc subunit protein as dimers or 180 individual monomeric proteins, and a second, major population that contains 120 copies of the HBc subunit protein as dimers or 240 individual monomeric proteins. These 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 nucleocapsid or viral core protein (HBc) has been disclosed as an immunogenic

carrier moiety that stimulates the T cell response of an immunized host animal. See, Cor example, U.S. Patents No. 4,813,527, No 4,882,145 and No. 5,143,726. A particularly useful application of this carrier is its ability to present foreign or heterologous B cell epitopes at the site of the immunodominant loop that is present at about residue positions 70-90, and more usually recited as about positions 75 through 85 from the amino-terminus (N-terminus) of the protein. Clarke et al. (1991) F. Brown et al. eds., Vaccines 91, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp.313-318.
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) JBC, 267(13) 9422-29.'
In an application as a vaccine carrier moiety, it is preferable that the HBV nucleocapsids not bind nucleic acid derived from the host. Birnbaum et al. (1990) J.Virol., 64:3319-3330 showed that the protamine-like C-terminal domain of HBV nucleocapsids could be deleted without interfering with the protein's ability to assemble into 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 ]44, can

self-assemble, whereas deletions beyond residue 139 abrogate capsid assembly [ F. Birnijaum & M. Nassal (1990) J.Virl., 64: 3319-30].
Zlotnick et al. , '(1997) Proc. Wati. Acad. Sci., USA, 94:9556-9561 studied the assembly of full length and truncated HBc proteins in to particles. In addition to discussing full length molecules, those authors reported the preparation of a truncated protein that contained the HBc sequence from•posit ion 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-terrainal 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 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 particles exhibit reduced stability relative to standard 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. Viral., 135:131-142]. Thus, prior to the report of Metzger et al. , above, Pumpens et al.. ,

coJi expression of chimeric HBc particle'-s 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.
Chimeric hepatitis B core particles bearing internal insertions 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 affect that hybrid particles cannot be recovered following heterologous expression [Schodel et al. (1994) Infect. Immunol., 62:1669-1676]. Thus, many chimeric HBc particles are so unstable that thsy fall apart during purification to such an extent that they are unrecoverable or they show very poor

(1995) Intervirology, 38:63-74 summarized che literature reports by stating that the carboxy-terminal border for HBc sequences required for self-assembly was located between 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. Neirynck et al . , (October 1.999) Nature Med., 5 (10) :1157-1163 reported that particle formation occurred on E. coli expression of a HBc chimer that contained the N-terminal 24-residue portion of the influenza M2 protein fused to HBc at residue 5.
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. report lists particle-forming chimers in which the inserted polypeptide sequence is at the N-terminus, the C-terminus and between the termini. 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 tne E.

stability characteristics, making them problematic for vaccine development.
A structural feature whereby the stability of full-length 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., 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_gpssible__Brgblem relates to^
the requirement of reproducible preparation of intact chimer particles that can also withst^and_lojign:_t^rrnL_. s_torage_.
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 OP THE INVENTION
The present invention contemplates a
recombinant hepadnavirus nucleocapsid protein; i.e., a hepatitis B core (HBc) chimeric protein [or chimer hepatitis B core protein molecule or HBc chimer molecule or just chimer] that self -assembles into particles after expression in a host cell. The

chimeric pro.tein (i) displays one or more immunogenic epitopes at the N-terminus, HBc imrv.unogenic loop or C-terminus, or has a heterologous linker residue for a conjugated epitope in the immunogenic loop, arid contains a cysteine residue__a_t or near the C-terminus that confers enhanced stability to the particles. The chimeric protein is sufficiently free of arginine residues so that the self-assembled particles are substantially free of nucleic acid binding.
The present invention also contemplates an immunogenic particle comprised of recombinant hepatitis B core (HBc) chimeric protein molecules. The chimeric protein (i) displays one or more immunogenic epitopes at the N-terminus, HBc immunogenic loop or C-terminus, or (ii) has a heterologous linker residue for a conjugated epitope in the HBc immunogenic loop. That recombinant protein contains a cysteine residue at or near the C-terminus. The particles are substantially free of nucleic acid binding and exhibit enhanced stability relative to particles comprised of otherwise identical proteins that are free of the cysteine residue.
One embodiment of the invention
contemplates a recombinant chimer hepatitis B core (HBc) protein molecule up to about 515 amino acid residues in length that
(a) contains (i) a sequence of at least about 130 of the N-terminal 150 amino acid residues of the HBc molecule including a covalently linked peptide-bonded heterologous epitope or a heterologous linker residue for a conjugated epitope present in the HBc immunodominant loop, or (ii) a sequence of at

lease about 135 residues of the N-terminal 150 HBc arnirio acid residues,
(b) contains one to ten, and mere
preferably, one to three cysteine residues toward the C-terminus of the molecule from the C-terminal residue of the HBc sequence present and within about 30 residues from the C-terminus of the chimer molecule [C-terminal cysteine residue(s)], and
(c) contains a sequence of at least five
amino acid residues from HBc residue position 135 to
the HBc C-terminus.
The contemplated chimer molecules (i) contain no more than 20 percent substituted ammo acid residues in the HBc sequence, and (ii) self-assemble on expression in a host cell into particles that are substantially free of binding to nucleic acids. Those particles are substantially free of binding to nucleic acids and -are more stable than are particles formed from an otherwise identical HBc 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 HBc chimer has a sequence of about 135 to about 515 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 abouc 100 amino acid residues whose sequence includes at least the sequence of the residues of about position 5 through position 75 of HBc, and optionally includes a he'^erologous 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 ammo acid residues peptide-bonded to HBc residue 75 of Domain I in which (i) zero to all, and preferably at least 4, residues in a sequence of HBc positions 76 to 85 are present peptide-bonded to one to about 245 amino acid residues that are heterologous (foreign) to HBc 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 (li) the sequence of HBc at positions 76 to 85 is present free from heterologous residues. Domain III is an HEc sequence from position 86 through position 135 peptide-bonded to residue 85 of Domain II. Domain IV comprises (i) zero through fourteen residues of a HBc amino acid residue sequence 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 HBc 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 HBc from position 150 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) .
A contemplated recombinant chimer protein forms particles that are substantially free of binding to nucleic acids and are more stable than are particles formed from a 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. When chimer molecules are assembled into particles, those particles exhibit an absorbance ratio at 280 nm to 260 nm (280/260 absorbance ratio) of about 1.2 to about 1.7. The particles formed are believed to be of the T = 4 structure, containing 240 monomeric HBc chimers or 120 dimer HBc chirners.
More broadly, a contemplated chimer
particle comprises a C-terminal truncated HBc protein (to at least residue 149) 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 HBc 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 HBc 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 HBc chimer particle or a conjugate of a hapten with an above HBc 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 benefit of the invention is that chimer HBc particJLes are formed that are more stable on storage in aqueous compositions than are particles of similar sequence that lack any C-terminal cysceine residues.
An advantage of the invention is that chimer molecules are prepared that exhibit the self-assembly characteristics of native HBc particles, while not exhibiting the nucleic acid binding of those native particles.
Another benefit of the piesent 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 a 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 C-terminal cysteine residue.
A further advantage is that
immunogenicities of particles assembled from chimer molecules containing at least one 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 lorm the disclosure that follows.
BRIEF DESCRIPTION OF THE DRAWINGS
In the drawings forming a portion of this disclosure
Fig. 1 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 HBc chimers . The modified sequence (SEQ ID NO: 285) is shown below the sequence of the commercially available vector (SEQ ID NO: 286) . The bases of the added Ncol 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 Hindlll) are indicated.
Fig. 2, shown in three panels as Figs. 2A, 2B and 2C, schematically illustrates a preferred cloning strategy in which a malarial B cell epitope such as (NANP)4 (SEQ ID N0:l) is cloned into the
EcoRI and SacI sites of an engineered HBc gene (Fig. 2A) between positions 78 and 79, which destroys the EcoRI site, while preserving the SacI site. Fig. 2B shows DNA that encodes a T cell epitope such as that referred to as Pf/CS-UTC and a stop codon (SEQ ID NO-.120) cloned into the EcoRI and Hindlll sites at the C-terminus of an engineered, truncated HBc gene containing the first 149 HBc residues (HBcl49). PCR amplification of the construct of Fig. 2B using a primer having a 5'-terminal SacI restriction site adjacent to a HBc-encoding sequence beginning at

residue position 79 digestion of the amplified sequence and the construct of Fig. 2A with SacI, followed by ligation of the appropriate portions is shown in Fig. 2C to form a single gene construct referred to hereinafter as V12 that encodes B cell-and T cell-containing epitopes of an immunogen for a vaccine against P. falciparum.
Fig. 3 is a photograph of an SDS-PAGE analysis under reducing conditions to show the stabilizing•effects on expressed particles of a codon for a single cysteine residue inserted in frame between the C-terminal codon (V149) and the termination codon of HBc in a chimer that also contains (NANP)4 inserted between the amino acids of
positions 78 and 79 (V2.Pfl+C), and a similar construct whose C-terminus is residue V149 (V2.Pfl)
at day zero and after 15 days.at 37°C. [Lane 1,
V2.Pfl - day 0; Lane 2, V2.Pfl - day 15 at 37°C; Lane 3, V2.Pfl+C, day 0; Lane 4, V2.Pfl+C - day 15 at
37°C.]
Fig. 4 is a photograph of an SDS-PAGE analysis under reducing conditions that illustrates the stabilizing effects on chimer HBcl49 particles containing (NANP)4 inserted between amino acids 78 and 79 and the cysteine-containing T cell epitope fused to the C-terminus [V2.Pf1+Pf/CS-UTC also referred to as V12.Pfl] as compared to a similar particle in which the C-terminal Cys was replaced by an Ala residue [V2.Pfl+ Pf/CS-UTC(C17A) also referred to as V12.Pfl(C17A)] at day zero and after 28 days at 37°C. [Lane 1, V2.Pfl+Pf/CS-UTC - day zero; Lane 2, V2.Pfl+ Pf/CS-UTC - day 28 at 37°C; Lane 3,

V2 .Pfl+Pf/CS-UTC(C17A) - day zero; Lane 4, V2.Pfl+
Pf/CS-UTC(C17A) - day 28 at 37°C.]
Fig. 5 is a graph showing the results of an indirect immunofluorescence assay (IFA) carried out using glutaraldehyde-fixed P. falciparum sporozoites and FITC-labeled anti-mouse IgG (gamma-chain specific) to detect bound antibody titers (log of I/dilution; ordinate) over time in weeks (abscissa) for three chimeric immunogens after immunization in mice. Data for the prior art chimer immunogen, CS-2, are shown as squares, those for the recombinant HBc chimer V12.Pf1 are shown as diamonds, whereas those for the recombinant HBc chimer Vl2.Pf3.1 are shown as triangles.
Fig. 6 illustrates a reaction scheme (Scheme 1) that shows two reaction sequences for (I) forming an activated carrier for pendently linking a hapten to a chimeric hepatitis B core protein (sm-HBc) particle using sulpho-succinimidyl 4-(N-maleimidomethyl)cyclohexane 1-carboxylate (sulpho-SMCC), and then (II) linking a sulfhydryl-terminated (cysteine-terminated) hapten to the activated carrier to form a conjugate particle. The sm-HBc particle is depicted as a box having a single pendent amino group (for purposes of clarity of the figure), whereas the sulfhydryl-terminated hapten is depicted as a line terminated with an SH group.
Fig.7, shown in two panels as Fig. 7A and Fig. 7B, provides an alignment of six published amino acid residue sequences for mammalian HBc proteins from six viruses. The first (SEQ ID NO:247), human viral sequence is of the ayw subtype and was published in Galibert et al. (1983) Nature, 281:646-650; the second human viral sequence (SEQ ID N0:248),

of the adiv subtype, was published by Ono e'^ al . (1983) Nucleic Acids Res., 11(6): 1M7-1757; the third human viral sequence (SEQ ID NO: 24 9) , is or che adw2 subtype and was published by Valen-uela et al. , Animal Virus Genetics, Field et al. eds., Academic Press, New York (1980)pages 57-70; the fourth human viral sequence (SEQ ID NO:250), is of the adyw subtype that was published by Pasek et al. (1979) Nature, 282:575-579; the fifth sequence iSEQ ID NO:251), is that of the woodchuck virus that was published by Galibert et al . (1982) J. Virol., 41:51-65; and the sixth mammalian sequence, (SEQ ID N0:246), is that of the ground squirrel that was published by Seeger et al. (1984) J. Virol. ,51:367-375.
Figure 8 is a photograph of an SDS-PAGE analysis under reducing conditions following
incubations at 37°C for 0, 1 and 2 days that illustrates the stabilizing effects on (1) chimer HBcl49 particles containing the P. falciparum (NANP)4
immunogenic sequence inserted between HBc amino acid residues 78 and 79 that also contain a carboxy-terminal universal P. falciparum malarial T cell epitope peptide-bonded to HBc position 149 [UTC; V12.Pfl = V2.Pfl + Pf/CS-UTC], and (2) similar particles in which the cysteine at position 17 of the UTC was mutated to be an alanine residue and a cysteine residue was added at residue position 150, between the HBc residue at position 149 and the beginning of the UTC [V12.Pf1(C17A)+C150] .
Figure 9 is a photograph of an SDS-PAGE analysis under reducing conditions following particle preparation that shows the ICC-143C monomer construct was unstable (Lane 2) as compared to the ICC-1492

construct (Lane 3), with HBc-149 (Lane 1), ICC-1475 (Lane 4) and ICC-1473 (Lane 5) serving as additional molecular weight controls.
DEFINITIONS
Numerals utilized in conjunction with HBc chimers indicate the position in the HBc ayw amino acid residue sequence of SEQ ID NO: 247 at which one or more residues has been added to the sequence, regardless of whether additions or deletions to the amino acid residue sequence are present. Thus, HBcl49 indicates that the chimer ends at residue 149, whereas HBcl49 + C150 indicates that that same chimer contains a cysteine residue at HBc position 150. 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 ani 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 imraunologically 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 HBc 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:1 and 21 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 HBc chimer molecule that is identified by (i) residue position numbering relative to the position numbers of HBcAg subtype ayw as reported by Galibert et al. , (1979) Nature, 281:646-650 (SEQ ID N0:246) . The polypeptide portions ofjat_ least chimer Domains I, II and III are believed to exist in a similar tertiary form to the corresponding sequences of naturally occurring 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- ^nd amino-terminal amino acid residues. The fusion proteins of the present invention are HBc 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 nudeofeide 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. In keeping with

standard polypeptide nomenclature, J. B.iol. Chem. , 243:3557-59, (1969), abbreviations for amino acid residues are as shown in the following Table of Correspondence:
TABLE OF CORRESPONDENCE
SYMBOL 1-Letter 3-Letter AMINO ACID

Y Try L-tyrosine
G Gly glycine
F Phe L-phenylalanine
M Met L-methionine
A Ala L-alanine
S Ser L-serine
I He L-isoleucine
L Leu L-leucine
T Thr L-threonine
V Val L-valine
P Pro L-proline
K Lys L-lysine
H His L-histidine
Q Gin L-glutamine
E Glu L-glutamic acid
W Trp L-tryptophan
R Arg L-arginine
D Asp L-aspartic acid
N Asn L-asparagine
C Cys L-cysteine
DETAILED DESCRIPTION OF THE INVENTION
The present invention contemplates a chimeric hepadnavirus nucleocapsid protein; i.e., a

recombinant hepatitis B core (Hbc) protein, that is engineered to (a) display an immunogenic B cell or T cell epitope, a linker for attachment of an immunogenic B cell or T cell epitope or a truncated HBc protein, (b) exhibit enhanced stability when present in a self-assembled particle, as well as exhibit (c) a substantial absence of nucleic acid binding as a self-assembled particle. A contemplated HBc chiraer is truncated at the C-terminus of the molecule relative to a native HBc molecule.
Thus, the chimeric protein displays one or more immunogenic epitopes at the N-terminus, in the ! HBc immunogenic loop or C-terminus, or a linker for > such an epitope in the immunogenic loop. The / chimeric protein contains a cysteine residue at or near the C-terminus that confers enhanced stability to the self-assembled particles. The chimeric protein is sufficiently free of arginine residues downstream of (toward the carboxy-terminus from) HBc residue position 149 so that the self-assembled particles are substantially free of nucleic acid ^-^ binding.
For ease of discussion, contemplated chimer sequences and sequence position numbers referred to herein are based on the sequence and position numbering of the human hepatitis B core protein of subtype ayw [Galibert et al.(1979) Nature, 281:64:650]. It is to be understood, however, that in view of the great similarity between the mammalian hepadnavirus capsid protein sequences and similar particle formation exhibited by those proteins, which are well-known to skilled workers, a discussion regarding human HBc subtype ayw is also applicable to subtype adw, as well as the woodchuck and ground

squirrel proteins. As a conseque ice of tho.'ie great similarities, HBc seauences are recited generally herein as a "HBc" sequence, unless otherwise stated.
In one embodiment, a contemplated HBc chimer is up to about 515 residues in length and
(a) contains (i) a sequence of at least
about 130 of the N-terminal 150 amino acid residues
of the HBc molecule including a covalently linked
heterologous epitope or a heterologous linker residue
for a conjugated epitope present peptide-bonded in
the HBc immunodominant loop/ or (ii) a sequence of at
least about 135 residues of the N-terminal 150 HBc
amino acid residues,
(b) contains one to ten, and more
preferably one to three, cysteine residues toward the C-terminus of the molecule from the C-terminal residue of the HBc sequence present and within about 30 residues from the C-terminus of the chimer molecule [C-terminal cysteine residue(s)], and
(c) contains a sequence of at lease five
amino acid residues from HBc residue position 135 to
the HBc C-terminus. Five of those six residues are
preferably of the HBc sequence from positions 136-
140, with the sixth being the required cysteine.
The contemplated chimer self-assembles into particles when the chimer protein molecules are expressed in a host cell, and those particles are substantially free of binding to nucleic acids and are more stable (1) than are particles formed from an otherwise identical HBc chimer that lacks the above one to ten cysteine residues [C-terminal cysteine residue (s)] or (2) where a single C-terminal cysteine residue is present in the chimer and is replaced by another residue such as an alanine residue.

In cne aspect, a preferred HBc chimer has a sequence of about 135 to about 515 L-a-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 a-amino acids and therefore cannot form peptide bonds, those that contain D-arnino acid residues, or oligopeptide conjugates in which two or more polypeptides are operatively linked through an amino acid residue side chain. A contemplated chimeric HBc protein can therefore be prepared by expression using the usual methods of recombinant technology.
From the amino-terminus, Domain I comprises about 71 to about 100 amino acid residues whose sequence includes at least the sequence of the residues of position 5 through position 75 of HBc. Preferably, the sequence of residues 1 through 75 of the HBc sequence is present as part of Domain I. Most preferably, Domain I is comprised only of the HBc sequence from position 1 through position 75.
Domain II comprises 5 to about 250 amino" acid residues peptide-bonded to HBc residue 75 of Domain I of which (i) zero to all of the residues, and preferably at least 4 residues, and more preferably at least 8 residues, in a sequence of HBc at positions 76 through 85 are present peptide-bonded to one to about 245 residues that are heterologous (foreign) to HBc 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) the sequence of HBc at positions 76 through 85 is present free from heterologous residues.

It is particularly preferred that the sequence of 10 residues of positions 76 through 85 (76-85 sequence) be 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 10 residue sequence be present alone, uninterrupted by any heterologous residue.
A chimer containing only HBc residues in this Domain together with the features discussed below is useful for inducing a B and/or T cell response to HBc itself. A preferred HBc chimer molecule with an uninterrupted 76-85 sequence contains the uninterrupted HBc amino acid residue sequence of position 1 through at least position 140, and more preferably contains the uninterrupted HBc amino acid residue sequence of position 1 through position 149, plus a single cysteine residue at the C-terminus, as discussed below.
Domain III is an HBc sequence from position 86 through position 135 peptide-bonded to residue 85.
Domain IV comprises (i) zero to fourteen residues of a HBc amino acid residue sequence from position. 136 through 149 peptide-bonded to the residue of position 135 of Domain III, (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 HBc from position 150 to the C-terminus 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 135 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 HBc, 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 HBc residues 1-5. 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 HBc residues 76 and 85 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 HBc residues 136-149. 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 HBc chimer molecule can contain about 135 to about 515 amino acid residues. In preferred embodiments, HBc residues 1-5 are present, so that Domain I begins at HBc residue 1 and continues through residue 75; i.e., the HBc residue at HBc position 75. The heterologous epitope present in Domain II in the immunodominant

loop preferably contains about 15 ':o 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 HBc residues 86 through 135 peptide-bonded to residue 35. Domain IV contains a sequence of at least six residues that are comprised of (i) zero, one or a sequence of the residues of HBc positions 136 through 149 peptide-bonded to residue 135, (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 HBc sequence ends at residue 135, 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 76 and 85 "be present, but interrupted by the heterologous epitope (s), and that the chimer

further include one or more T cell epitopes in Domain IV peptide-bonded to one of HBc residues 140-149.
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 76 and 85 present, but interrupted by the heterologous linke^ residue, with a T cell epitope being present peptide-bonded to one of HBc residues 140-149. The particles formed from such chimer molecules typically contain a ratio of conjugated epitope to C-terminal peptide--bonded T cell epitope of about 1:4 to 1:1, with a ratio of about 1:2 being common.
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, HBc antigenicity as measured by binding of anti-loop monoclonal antibodies in an ELISA assay as discussed hereinafter.
A preferred contemplated HBc chimer molecule contains a sequence of about 140 to about 515 residues. A preferred HBc chimer molecule containing two heterologous epitopes of preferred lengths of about 15 to about 50 residues each and a preferred HBc portion length of about 140 to about 149 residues has a sequence length of about 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 co be understood that a wide range of chimer molecule lengths is contemplated in view of the variations in length of t .ie N- and C-terminal HBc portions and differing lengths of the several contemplated epitopes that can be inserted in the immunogenic loop.
A contemplated recombinant protein, after expression in a host cell, self-assembles to form particles that are substantially free of binding to nucleic acids. The contemplated HBc chirner particles are generally spherical in shape and are usually homogeneous in size for a given preparation. These chimeric particles thus resemble native HBc particles that have a similar shape and size and can be recovered from infected persons.
A contemplated chimer particle comprises previously discussed chimer molecules. More broadly, such a chimer particle comprises a chimeric C-terminal truncated HBc protein that has a sequence of at least about 130 of the N-terminal 150 residues and contains (i) a heterologous epitope or a heterologous linker residue for an epitope in the immunodominant loop, or at least about 130 of the N-terminal 150 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 135. Such a particle is 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 HBc sequence between positions 150 and 183 . A contemplated

particle is more stable than a particle formed from an otherwise identical HBc chimer protein that lacks the above C-terminal cysteine residue(s). Similarly, a particle whose chimer molecule contains a single C-terminal cysteine residue is more stable than a particle in which that cysteine is replaced by another residue such as an alanine residue. In some instances, particles do not iorm 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 Figs. 3, 4 and 8.
The substantial freedom of nucleic acid binding can be readily determined by a comparison of the absorbance of the particles in aqueous solution measured at both 280 and 260 nm; i.e., a 280/260 absorbance ratio. The contemplated particles do not bind substantially to nucleic acids that are oligomeric and/or polymeric DNA and RNA species originally present in the cells of the organism used to express the protein. 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 HBc particles or chimeric HBc particles that contain the arginine-rich sequence at residue positions 150-183 (or ISO-IS 5) 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 particles sufficiently free of arginine residues so that the self-assembled

particles are substantially free of nucleic acid binding such as particles that arc free of the arginine-rich nucleic acid binding region of naturally occurring HBc 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 HBc residue position 140 to position 149, exhibit a 280/260 absorbance ratio of about 1.2 to about 1.6.
Chimeric HBc particles of the present invention are substantially free of nucleic acid binding and exhibit a 280/2SO absorbance ratio of about 1.2 to about 1.6, and more typically, about 1.4 to about 1.6. This range is due in large part to the number of aromatic amino acid residues present in Domains II and IV of a given chimeric HBc particle. That range is also in part due to the presence of the Cys in Domain IV of a contemplated chimer, whose presence can diminish the observed ratio by about 0.1 for a reason that is presently unknown.
The contemplated chimer HBc particles are
more stable in aqueous buffer at 37°C over a time period of about two weeks to about one month than are particles formed from a HBc chimer containing the same pep tide-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)] are absent or a single C-terminal residue present is replaced by another residue such as an alanine residue. Stability of various chimer particles is determined as discussed hereinafter.
Thus, for example, particles containing a heterologous malarial epitope in Domain II [e.g.

(NANP)4] and a single cysteine residue C-terminal to residue valine 149 is more stable than otherwise identical particles assembled from chimetr molecules whose C-terminal residue is valine 149. Similarly, particles containing the above malarial 3 cell epitope in Domain II and the universal malarial T cell epitope that contains a single cysteine near the C-terminus are more stable than are otherwise identical particles in which that cysteine is replaced by an alanine residue. See, Figs. 3, 4 and 8 and the discussion relating thereto hereinafter.
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 and shown in Table 17.
Domain I of a contemplated chimeric HBc protein constitutes an amino acid residue sequence of HBc beginning with at least amino acid residue position 5 through position 75, and Domain III constitutes a HBc sequence from position 86 through position 137. The sequences from any of the mammalian hepadnaviruses can be used for either of Domains I and III, and sequences from two or more viruses can be used in one chimer. Preferably, and for ease of construction, the human ayw sequence is used through out the chimer.
HBc chimers having a Domain I that contains more than a deletion of the first three amino-terminal (N-terminal) residues have been reported to result in the complete disappearance of HBc chimer

protein in E. coli cells. Pumpens et al.,(1995) Intervirology, 38:63-74. On che 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 HBc can contain a sequence of up to about 15 residues that are heterologous to HBc. 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 P-galactosidase as can occur in fusion proteins as a result of the expression system used, or another hepatitis B-related sequence such as that from the Pre-Sl or Pre-32 regions or the major HbsAg immunogenic sequence.
Domain II is a sequence of about 5 to about 250 amino acid residues. Of those residues, zero
(none), and preferably at least 4 residues, and more preferably at least 8 residues, constitute portions of the HBc sequence at positions 76 to 85, and one to about 245 residue.s, and preferably one to about 50 residues are heterologous (foreign) to HBc. Those heterologous residues constitute (i) a heterologous

linker residue for a epitope such as a B ceil or T cell epitope or (ii) a heterologous B or T cell epitope that preferably contains 6 to about 50, more preferably about 15 to about 50, and most preferably about 20 to about 30 amino acid residues, and are positioned so that they are peptide-bonded between zero, or more preferably at least 4, to all of the residues of positions 76 through 85 of the HBc sequence. Heterologous B cell epitopes are preferably linked at this position by the linker residue or are peptide-bonded into the HBc sequence, and use of a B cell epitope is discussed illustratively hereinafter.
Those preferred at least 4 HBc residues can be all in one sequence such as residues 82-85, or can be split on either side of (flank) the heterologous residue(s) as where residues 76-77 and 84-85 are present or where residues 76 and 83-85 are present. More preferably, Domain II contains at least 8 residues of the HBc sequence from residue 76 to 85. Most preferably, the sequence of all 10 residues of positions 76, through 85 are present in the chimer.
The one to about 245 residues added to the HBc loop sequence is (are) heterologous to a HBc sequence. A single added heterologous residue is a heterologous linker residue for a B cell epitope as discussed before. The longer sequences, typically at least 6 amino acid residues long to about 50 amino acid residues long and more preferably about 15 to about 50 residues in length, as noted before, are in a sequence that comprises a heterologous immunogen such as a B cell epitope, except for heterologous residues encoded by restriction sites.

Exemplary peptide immune-jens useful for both linkage to the linker resicue 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 NO.
Table A B Cell Epitopes

Organism
Streptococcus
pneumoniae

Gene
PspA

Sequence Citation"
KLEELSDKIDELDAE QKKYDEDQKKTEE-KAALEKAASEEM-DKAVAAVQQA

SEQ ID NO



Cryptosporidium parvum

P23

QDKPADAPAAEAPA-AEPAAQQDKPADA



HIV

GP120

RKRIHIGPGR-AFYITKN

Foo t-and-mout h virus VP1
YKTGECRYNRNA-VPNLRGDLQVL-AQKVARTLP

Influenza Virus
A8/PR8 HA
A8/PR8/34 M2

YRNLLWLTEK
SLLTEVETPIR-NEWGCRCNGSSD SLLTEVETPIR-NEWGCRCNDSSD SLLTEVETPIR-NEWGARANDSSD EQQSAVDADDS-HFVSIELE

2 9 29

8
9
10 312 313

Yersinia
pestii

V Ag

DILKVIVDSMNHH -GDARSKLREELAE-LTAELKIYSVIQA-EINKHLSSSGTIN-IHDKSINLMDKNL-YGYTDEEIFKASA-EYKILEKMPQTTI-QVDGSEKKIVSIK-DFLGSENKRTGAL-GNLKNSYSYl^KDN-NELSHFATTCSD

11



Haemophilus influenza

pBOMP

CSSSNNDAA-
GNGAAQFGGY 10
NKLGTVSYGEE
NDEAAYSKN-
RRAVLAY

13 14



Moraxella catarrhalis
Porphyromonas gingivalis
1'rypa.nosoma cruzi
Plasmodium falciparum

copB
HA
CS

LDIEKDKKK-
RTDEQLQAE-
LDDKYAGKGY 11
LDIEKNKKK-
RTEAELQAE-
LDDKYAGKGY
IDIEKKGKI-
RTEAELLAE-
LNB03YPGQGY
GVSPKVCKDVTV-EGSNEFAPVQNLT 12 RIQSTWRQKTV-DLPAGTKYV
KAAIAPAKAAA-APAKAATAPA 14
(NANP)4
NANPNVDP-(NANP)3NVDP
NANPNVDP-(NANP)3
(NANP) 3NVDPNANP
NANPNVDP-(NAWP)3NVDPNANP
NPNVDP(NANP)3NV NPNVDP-(NANP) 3WVDP

15 15 17
18 19
20
1 21
22 23
24 25

NPNVDP(NAiJP) 3-
NVDPNA
NVDP(NANP)3NV
NVDP (NANP) 3 NVDP NVDP(NANP) 3_
NVDPNA
DP(NANP)3NV
DP(NANP)3 NVDP DP(NANP)3-NVDPNA

30 31 32



CS
vi vax
CS
berghi
CS
yoel1i
Streptococcus sobrinus Agl/II
Shigrella
flexneri Invasin
Respiratory syncitia
virus (RSV) G
Entamoeia
histolytica lectin

GDRPJ3GQPAG-
DRADGQPAG
RADDRAAGQP-
AGDGQPAG
ANGAGNQPG-
ANGAGDQPG
ANGADNQPG-
ANGADDQPG
ANGAGNQPG-
ANGADNQPG
ANGAGNQPG-
ANGADDQPG
APGANQEGGAA-
APGANQEGGAA
ANGAGNQPGAN-
GAGDQPGANGA-
DNQPGANGADD-
QPG
DPPPPNPN-DPPPPNPN
(QGPGAP)4
KPRPIYEA-KLAQNQK AKADYEAK-LAQYEKDL
KDRTLIEQK
CSICSNNPT-CWAICK
VECASTVCQNDN-SCPIIADVEKCNQ

20
27
28
IS
18
19
21

36 37 38 39 40
199
41 42
43 44
45 46
47

Schistosoma

7aoon±cum para
ScMstosoma
mansoni oara
DLQSEISLSLE-NGELIRRAKSA-ESLASELQRRVD
DLQSEISLSLE-NSELIRRAKAA-ESLASDLQRRVD
Bovine Inhibin
ac subunic STPPLPWPW-
SPAALRLLQ-RPPEEPAA
Ebola Virus
membrane-anchored glycoprotein ATQVEQHHRR-TDNDSTA HNTPVYKLD-ISEATQVE GKLGLITNTI-AGVAVLI
Escherichia coli
ST CCELCCYPACAGCN NTFYCCELCC-YPACAGCN SSNYCCELCC-YPACAGCN
Alzheimer's disease
(3-Amyloid DAEFRHDSGYE--
VHHQKLVFFAE-
DVGSNKGAIIG-
LMVGGWIA
DAEFRHDSGYE-
VHHQKL
EDVGSNKGAII
DAEFRHDSGYE-
VHHQKLVFFAE-
DVGSNKGAIIG

30
31 31 31
33 33 33
24

48 49
252
253 254 255
288 289 290
293
188
294 295



*Citations to published epitopes are provided following Table B
The remaining residues of Domain II that are present on either side of the heterologous residue or sequence are the residues of HBc position

76 to position 85. Thus, in a typical c-jxai.iple, where residues 78 through 82 have been replaced, the chimer sequence in Domain II is 76 through 77, followed by restriction site-encoded residues, the heterologous immunogenic (epitope) sequence, further restriction site-encoded residues, and then HBc sequence 84 through 85. A typical exemplary sequence of a chimer prepared by an insertion strategy between residues 78 and 79 is that of HBc from position 1 through 78, followed by restriction site-encoded residues, the heterologous immunogenic sequence, further restriction site-encoded residues and HBc sequence 79 through 85. The sequence of other contemplated chimers through Domains I and II should be apparent from these illustrations and those that follow and need not be enumerated.
As already noted, a heterologous linker for a conjugated epitope is peptide-bonded at a position in the HBc sequence between amino acid residues 76 and 85. As was the case for the heterologous epitope, the HBc sequence of residues 76 through 85 is preferably present, but interrupted by the heterologous linker for a conjugated epitope. This chimer preferably includes the HBc sequence of position 1 through at least position 140, plus a cysteine residue at the C-terminus of the chimer protein. More preferably, the HBc sequence of positions 1 through 149 are present, but interrupted between residues 76 and 85 by the heterologous linker for a conjugated epitope, and the chimer molecule contains a C-terminal cysteine. The heterologous linker for a conjugated epitope is most preferably a lysine (K) residue. Glutamic or aspartic acid, tyrosine and cysteine residues can also be used as

linker residues, as can tyrosine and cysteine residues. 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 chirner molecule. Published application PCT/US93/03055 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:256
GGGGSGGGG SEQ ID NO:257
As was noted previously, Domain III constitutes the sequence of HBc from position 86

through position 135. Consequently, the sequence of the illustrative chimers discussed above for Domains I and II, can be extended so that the first-discussed chimer has the sequence of HBc from position 84 through position 135, and the second-discussed chimer has the sequence of HBc from position 79 through position 135.
Domain IV is a sequence that (i) optionally includes a HBc sequence from position 13o through 149, (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 HBc at position 150 to the C-terminus, wich 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 HBc more preferably contains up to about 50 amino acid residues,- and most preferably contains up to about 25 residues. The Domain IV sequence can thus be substantially any cysteine-containing sequence, except the C-terminal HBc sequence from position 150 to the 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 135 to about 515 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 arnino 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 il T Cell Spl topes

I

SEQ
Organism Gene Sequence* Citation ID NO
HIV P24 GPKEPFRDY-
VDRFYKC 3 50
CorynebacCerium diptheriae toxin

Barrelia burgdorreri

ocpA

FQWHHSYH-RPAYSPGC
VEIKEGTVTLICRE-IDKNGKVTVSLC TLSKHISKSG-EVSVELNDC

51
52 53



Influenza Virus A8/PR8 HA

SSVSSFERFEC
LIDALLGDPC
TLIDALLGC

291 292

Trypa n o s oma cruzi

SHNFTLVASVII-EEAPSGNTC
Plasmodium falciparum
MSPl
SVQIPKVPYPNGIVYC
DFNHYYTLKTGLEADC
PSDKHIEQYKKI-
KNSISC
EYLNKIQNSLST-
P. vi vax
EWSPCSVT
P. yoelii
YLDKVRATVGTE-WTPCSVT
EFVKQISSQLTE-EWSQCSVT
Streptococcus sobrinus Agl/II
KPRPIYEAKL-AQNQKC AKADYEAKLA-QYEKDLC
LCMV (lymphocytic choriomeningitis virus)
NP RPQASGVYM-
GNLTAQC
Clostridium tetani tox
QYIKANSKFIG-ITELC

15 23 26
17

55
56 57
58 59
60 287
61 62
63
64



*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, 1.2:5496.
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8. Brumeanu et al. (1996) I/ranunotechnology, 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, 3:4444.
14. WO 97/18475 .
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. Letter et al. (1997) J. Exp. Med., 185, 10:1793.
22. Nara et al. (1997) Vaccine 15, 1:79.
23. U.S. Mo. 4,885,782.
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) jSur.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 HBc from residue position 1 through at least
position 140 is preferably present in a contemplated
chimer molecule and particle. The sequence from

position I through position 14j is more preferably present. A E cell epitope is preferably present between residues 76 and 85 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 HBc sequence. A contemplated recombinant HBc chimer is substantially free of bound nucleic acid. A contemplated chimer particle that contains an added Cys residue at or near the C-terminus of the molecule
is also more stable at 37°C than is a similar particle that does not contain that added Cys. This enhanced stability is illustrated in Figs. 3, 4 and 8, and is discussed hereinafter.
A contemplated recombinant HBc chimer molecule is typically present and is used as a self-assembled particle. These particles are comprised of 180 to 240 chimer molecules (90 or 120 dimer pairs) , usually 240 chimer molecules,- that separate into protein molecules in the presence of disulfide reducing agents such as 2-mercaptoethanol, and the individual molecules are therefore thought to be bound together into the particle primarily by disulfide bonds.
JQ though not wishing to be bound by theory, it is believed that the observed enhanced stability and in some cases enhanced expression for a contemplated HBc chimer is due to the formation of a further cystine disulfide bond between proteins 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 'co the
particles observed in patients infected with HBV, but these particles are non-infectious. Upon expression in various prokaryotic and eukaryotic hosts, the individual recombinant HBc chimer molecules assemble in the host into particles that can be readily harvested from the host cells, and purified, if desired. ,_.
I As noted before, the HBc immunodominant loop is usually recited as being locaced at about
positions 75 through 85 from the amino-terminus (N-
*~-i 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 HBc 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 (CNFASTAR Inc . , Madison, •Wis.)
The HBc portion of a chimer molecule of the present invention; i.e., the portion having the HBc sequence that has other than a sequence or residue of an added epitope, linker, flexible linker arm or heterologous residue(s) that are a restriction enzyme artifact, most preferably has the amino acid residue sequence at positions 1 through 149 of subtype ayw that is shown in Fig. 7 (SEQ ID NO.-247), less any portion or portions of the subtype ayw sequence that are absent because of truncation at one or both termini. Somewhat less preferred are the corresponding amino acid residue sequences of subtypes adw, adw2 and adyw that are also shown in Fig. 7 (SEQ ID N0s:248, 249 and 250). Less preferred still are the sequences of woodchuck and ground squirrel at aligned positions 1 through 149 that are the last two sequences of Fig 7 (SEQ ID N0s:251 and 246) . As noted elsewhere, portions of different sequences from different mammalian HBc proteins can be used together in a single chimer.
When the HBc portion of a chimer molecule of the present invention as above described has other than a sequence of a mammalian HBc molecule corresponding to positions 1 through 149, no more than about 20 percent of the amino acid residues are substituted as compared to SEQ ID NO:247 from position 1 through 149. 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 Z percent of the amino acid residues are substituted as compared to SEQ ID NO:247 from position 1 through 149.

A contemplated chimer of 149 HBc residues can therefore contain up to about 30 residues that are different from those of SEQ ID NO:247 at positions 1 through 149, and preferably about 15 residues. More preferably, about 7 or 8 residues are different from the ayw sequence (SEQ ID NO: 247) at residue positions 1-149, and most preferably about 4 or 5 residues are different. Substitutions, other than in the immunodominant loop of Domain II or at the termini, are preferably in the non-helical portions of the chimer molecule and are typically between residues 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) .
Where a HBc sequence is truncated at the C-" terminus beyond position 149 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 that 149 residues. Deletions elsewhere in the molecule are considered conservative substitutions for purposes of calculation.
Chimer Preparation
A contemplated chimeric HBc immunogen is typically prepared using the well-known techniques of recombinant DMA technology. Thus, sequences of nucleic acid that encode particular polypeptide sequences are added to and deleted from the precursor sequence that encodes HBc 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 HBc DNA sequence that encodes a pair of different restriction sites, e.g. EcoRI and SacI, one near each end of the immunodominant loop-encoding DNA. Exemplary residues replaced are 76 through 81. The loop-encoding section is excised, a desired sequence that encodes the heterologous B cell epitope is ligated into the restriction sites and the resulting DNA is used to express the HBc chimer. See, for example, Table 2 of Pumpens et al., (1995) Inter virology, 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. Mitt., 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 r. yoelii and P. berghei, respectively.
It has been found that the insertion position within the HBc immunogenic loop and the presence of loop residues can be of import to the activity of the immunogen. Thus, as is illustrated hereinafter, placement of a malarial B cell epitope between HBc residue positions 78 and 79 provides a particulate immunogen that is ten to one thousand times more immunogenic than placement of the same immunogen in an excised and replaced region between residues 76 and 81. In addition, placement of the same malarial immunogen between residues 73 and 79 as compared to between residues 77 and 78 provided an unexpected enhancement in immunogenicity of about 15-fold.
Insertion is therefore generally preferred. 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 78 and 79. This technique adds twelve base pairs that encode four amino acid residues (two for each restriction site) between formerly adjacent residues in the HBc loop.
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 HBc chimers, the HBc loop amino acid sequence is seen to be interrupted on its N-terminal side by the two residues encoded by the 5' restriction site, followed toward the C-terminus by the heterologous B-cell epitope sequence, followed by two more heterologous, non-loop residues encoded by the 3'

restriction site and then the re?t 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 si., (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. 2A, 2B and 2C. In Fig. 2A, a DNA sequence that encodes a C-terminal truncated HBc sequence (HBcl49) is engineered to contain adjacent EcoRI and SacI sites between residues 78 and 79. Cleavage of that DNA with both enzymes provides one fragment that encodes HBc positions 1-78 3 '-terminated with an EcoRI sticky end, whereas the other fragment has a 5'-terminal SacI sticky end and encodes residues of positions 79-149. Ligation of a synthetic nucleic acid having a 5' AATT overhang followed by a sequence that encodes a desired malarial B cell epitope and a AGCT 3'overhang provides a HBc chimer sequence that encodes that B cell epitope flanked on each side by two heterologous residues [Glylle (GI) and GluLeu (EL) , respectively] between residues 78 and 79, while usually destroying the EcoRI site and preserving the SacI site.
A similar strategy is shown in Fig. 2B 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 HBc DNA sequence after amino acid residue position 149. 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 codonr, and a 3' AGCT overhang were ligated into the digested, sequence to form a sequence that encoded HBc residues 1-149 followed by two heterologous residues (GI), the stop codon and the Hindlll site.
PCR amplification using a forward primer having a SacI restriction site followed by a sequence encoding HBc beginning at residue position 79, followed by digestion with SacI and Hindlll provided a sequence encoding HBc positions 79-149 plus the two added residues and the T cell• epitope at the C-terminus. Digestion of the construct of Fig. 2B with SacI and ligation provided the complete gene encoding a desired recombinant HBc chimer immunogen having the sequence, from the N-terminus, of HBc positions 1-78, two added residues, the malarial B cell epitope, two added residues, HBc positions 79-149, two added residues, and the T cell epitope that is shown in Fig. 2C.
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 ammo acid residue sequence shown in SEQ ID NO: 247 contains a Glu and an Asp residue at positions 11 and 78. Nonetheless, introduction of an additional, heterologous, carboxyl-containing residue is still contemplated. The chemical reactivity of the existing glutaraic and aspartic acids may be reduced by other factors. For example, it is known in the art that a neighboring proline, such as that found at position 79, can neutralize and thereby reduce the chemical reactivity of a proximal carboxyl group.
Here, using the first noted insertion strategy, 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 HBc 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 HBc sequence on either or both sides of an inserted sequence. One or both ends of the insert and HBc nucleic acid can be "chewed back" with an appropriate nuclease (e.g. SI nuclease) to provide blunt ends that can be ligated together. Added heterologous residues that are neither part of the inserted B _ell

or T cell epitopes nor a part of the HBc 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 recotnbinant HBc 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 HBc 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. 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 chimera 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 HBc chimer molecule or a complement cf 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 aminc 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 cooes 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 amir.o 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 SXSSC and a final wash at a temperature of 68°C in 1-SXSSC. 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 3SC, 0.1% SDS (sodium dodecyl sulfate) .
A nucleic sequence (DNA sequence or an RNA sequence) that (1) itself encodes, or its complement encodes, a chimer molecule whose HBc portion from residue position 1 through 136, when present, is that of SEQ ID NOs: 246, 247, 248, 249, 250 or 251 and (2) hybridizes with a DNA sequence of SEQ ID NOs: 274, 275, 276, 277, 278 or 279 at least at moderate stringency (discussed above); and (3) whose HBc sequence shares at least 80 percent, and more preferably at least 90 percent, and even more preferably at least 95 percent, and most preferably

100 percent identity with a DNA sequence of SEQ ID NOs: 274, 275, 276, 277, 273 and 27y, 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 a HBc amino acid residue sequence that is at least 80 percent, and more preferably at least 90 percent, and most preferably is at least 95 percent identical to the HBc sequence portion from residue position 1 through residue position 136 shown in SEQ ID NOs: 246, 247, 248, 249, 250 and 251. This DNA or RNA is referred to herein as an "analog of" or "analogous to" a sequence of a nucleic acid of SEQ ID NOs: 274, 275, 276, 277, 278 and 279, and hybridizes with the nucleic acid sequence of SEQ ID NOs: 274, 275, 276, 277, 278 and 279 or their complements herein under moderate stringency hybridisation 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 vitro 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 HBc portion of a sequence of SEQ ID NOs: 246, 247, 248, 249, 250 or 251 that avoids substantial identity with a DNA of SEQ ID Nos: 274, 275, 276, 277, 278 or 279, or their complements. Thus, a useful analogous DNA sequence need not hybridize with the nucleotide sequences of SEQ ID NOs: 274, 275, 276, 277, 278 or 279 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 HBc portion of a chimer or a DNA variant that has at least 90 percent identity to the chimer gene of SEQ ID NOs: 274, 275, 276, 277, 278 or 279 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 a HBc

chimer portion that is at least 80 percent identical, more preferably 90 percent identical, and most preferably 95 percent identical to the HBc portion of a sequence of SEQ ID NOs: 246, 247, 248, 249, 250 or 251. 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 30 amino acid residue N-terminal sequence of ground squirrel HBc does not align with any of the other HBc sequences, that 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 30-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 HBc-encoding bases or HBc 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 NOs: 274, 275, 2V6, 277, 278 and 279, isolated nucleic acid segments, preferably DNA sequences, variants and analogs thereof can be prepared by in vitro mutagenesis, as is well known in the art and discussed in Current Protocols In Molecular Biology, Ausabel et al . eds. , John Wiley Sc Sons (Mew York: 1987) p. 8.1.1-8.1.6, that begin at che 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 DMA 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 HBc chimer can be produced (expressed) in a number of transformed host systems, typically host cells although expression in acellular, in vitro, systems is also contemplated. These host cellular systems include, buc 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 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 a HBc chimer is referred to herein as an "expression vector" .
An expression vector contains expression control elements including the promoter. The chimer-coding gene is operatively linked to the expression vector to permit the promoter sequence to direct RNA polymerase binding and expression of the chimer-

encoding gene. Useful in expressing the polypeptide coding gene are promoters that are inducible, viral, synthetic, constitutive as described by Poszkowski et al. (1989) EMBO J. , 3:2719 and Odell et al . (1985) Nature, 313:810, as well as temporally regulated, spatially regulated, and spatiotemporally regulated as given in Chua et al. (1989) Science, 244:174-181.
One preferred promoter for use in prokaryotic cells such as E. coli is the Rec 7 promoter that is inducible by exogenously supplied nalidixic acid. A more preferred promoter is present in plasmid vector JHEX25 (available from Promega) that is inducible by exogenously supplied isopropyl-(3-D-thiogalacto-pyranoside (IPTG) . A still more preferred promoter, the tac promoter, is present in plasmid vector pKK223-3 and is also inducible by exogenously supplied IPTG. The pKK223-3 plasmid can be successfully expressed in- a number of E. coli strains, such as XL-1, TB1, BL21 and BLR, using about 25 to about 100 |iM IPTG for induction. Surprisingly, concentrations of about 25 to about 50 \M 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 Ho. 5,337,744; U.S. Patent No. 5,297,441; Ulrich et al. , (1998) Adv.

Virus Res., 50:141-182; Tacket et al. , (Aug 1991) Infecc. Immun., 65 (8) :3381-3385 ; Schodel et al., (Feb 1997) Behring Inst. Mitt., 98:114-119; Nardelli-Haefliger et al., (Dec 1996} Infect. Immun., 64 (12) :5219-5224; Londono et al., (Apr 1996) Vaccine, 14(5):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 planes 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 intc Pichia cells.

More specifically, the transformation and expression system described by Gregg et al. (1987) Biotechnology, 5:479-485; (1987) Molecular and Cellular Biology, 12:3376-3385 can be xised. 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 [Gregg 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 Gregg 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 :ntegrative transformation, which does not require the use of an ARS sequence, as described

by Gregg et al. (1987) Molecular and Cellular Biology, 12-.3376-3335 .
Production of chimer particles by
recotnbinant DMA 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 decail in Sambrook et al. , Molecular Cloning: A Laboratory Manual, 2nQ 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 NcoI/Hindlll sequence fragment that encodes the desired HBc chimer prepared as described in Example 1 is ligated into the expression plasmid, which results in the formation of a circular recombinant expression plasmid denominated pSV-Pf.
The expression plasmid pSV-Pf contains an intact E. coli ampicillin resistance gene. E. coli RR101 (Bethesda Research Laboratories, Gaithersburg, MD) , 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 HBc chimer is propagated by culturing E. coli containing the

plasmid. The plasmid DMA is isolated from E. coJi cultures as described in Sambrook et al . , above.
Expression of a chimcr is accorr.pl i shed 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 plasinids, 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 include;! in the DNA segment to which it is operatively linked.
In one preferred embodiment, the host that expresses the chimer is the prokar/ote, 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 HBc chimer gene in a host cell, such as E. coli, transformed therewith. Promoter sequences compatible with bacterial hosts are typically provided in plasmid vectors containing one or more convenient restriction sites for insertion of a contemplated DNA segment. Typical of such vector plasmids are pUC8, pUC9, and pBR329 available from Biorad Laboratories, (Richmond, CA) and pPL and pKK223-3 available from Pharmacia, Piscataway, NJ.
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-indueing (Ti) plasmid of Agrobacterium tuiaefaciens described by Rogers et al. (1987) Math, 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. (1935; Proc. Natl . Acad. Sci . USA, 82:58-24. Plasmid pCaMVCM (available

from Pharmacia, Piscataway, NJ) includes the cauliflower mosaic virus CaMV J5S 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 :.nclude 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 (1937) 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, L>copersicon esculentum. These regions and their cDKA sequences are illustrated and discussed in Deikman et al. (1988) EMBOJ., 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 ethyiene 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, p-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 mPNA. 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. A.S 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 tuniefaciens 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 polyethylene.glycol-mediated transfer 03- 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 HBc 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 homoeygous transgenic plant can

be obtained by sexually mating (selfing) sn independent segregant transge^ic 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 cwo 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 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 HBc molecule. A preferred transgenic plant is an independent segregant for the added heterologous chimeric 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 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 HBc molecule particle accumulation, preferably in the field, under a range of environmental conditions.
A transgenic plant homozygous for chimeric HBc molecule particle accumulation is crossed with a parent plant having other desired traits. The progeny, which are heterozygous or independently segregatable for chimeric HBc molecule particle accumulation, are backcrossed with one or the other parent to obtain transgenic plants that exhibit chimeric 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 a HBc chimer. For example, in one such system Autographa californica nuclear polyhediosis virus (AcNPV) o_ 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 HBc chimer can be expressed. E. Engelhard et al. (1994) Proc. Natl . .-cad. 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 Autographs, 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, 1932; 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 DMA to vectors via complementary cohesive termini or blunt ends. For instance, complementary homopolymer tracts can be added to th_-

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 incubacing 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 bacceriophage 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 vnthin a multicelled host. The particle-jontaining cells are harvested using standard procedures, and the cells are lysed using a French pressure cell, lyso~yme HBc Chimer Conjugates
Any hapten to which a B cell or T cell response is desired can be linked to a contemplated HBc chimer or chimer particle such as a chimer particle containing a heterologous linker residue such as a lysine, glutamic or aspartic acid, cysteine or tyrosine in the loop region of Domain II and an added cysteine residue in Domain IV to form a HBc chimer conjugate. The hapten of interest typicaHv 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 medicament such as cocaine or nicotine. A HBc chimer particle conjugate so formed is useful as an inoculum or vaccine, as is discussed hereinafter. Because the

chimer protein self assembles upon expression and a conjugate is formed after expression, conjugate formation is typically done using the assembled particles as compared to the free protein molecules.
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, a 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 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 prcteir or conjugate by reaction of amino functional groups with 2 -iminoth'iolane, the N-

hydroxysuccinimide ester of 3 - (3-dithiopyridyl)-propionate, or other reagents known in the art.
The HBc chimer or hapten can also be modified to incorporate a spacer arm, such as hexamethylene diamine or another bifunctional molecule, to facilitate the pendent linking. Such a procedure is discussed below.
Methods for covalent bonding oE 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 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. Biochera., 79:231 and in Lachmann et al., in 1986 S_ynthetic 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 KBc 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 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 s-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 Irmun. 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. 6 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 ucilized. For example, recombinant and PCR-based tecnniques 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. Iimunol., 154:3373-3332. The table below provides further exemplary chemical haptens.

Chemical Haptem

Chemical Hapten Citation

piperidine N-oxide U.S. Patent No. 5,304,252
phospholactone or lactamide U.S. Patent No. 5,248,611
metal ion complexes U.S. Patent No. 5,236,825
[2.2.1] or [7.2.2] bicyclic ring compounds U.S. Patent No. 5,208,152
ionically charged hydroxyl -containing compounds U.S. Patent No. 5,137,086
phosphonate analogs of carboxylate esters U.S. Patent No. 5,126,258
cocaine analogs Carrera et al . , (1995) Mature 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. rmmun., 40:245-256; Gordon, U.S. Patent No. 4,619,828 (1936); 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 Crit. 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 us~d 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.
(Figure Remove)









U.S. Patent No. 4,220,717 also discloses a polyribosyl ribitol phosphate (PRP) hapten for Haemophilias influenzae type b.
Peterson et al. (1998) Infect. Immun., 66 (8) :3848-3855, disclose a trisaccharide hapten,
aKdo(2 8)aKdo(2 4) Andersson et al., EP-0 126 043-A1, 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 GlcNAc(3l 3Gal. Andersson et al . also reported

neolactotetraosylceramide tc be useful, which is
Galpl 4GlcNAcpl BGalpl 4Glc-Cer.
McKenney et al. (1995) Science, 284:1523-1527, disclose a polysaccharide, poly-N-succinyl Pi SGlcN (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 8S3-31, 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

Danish Type Nomenclature U.S. Nomenclature 1978 ATCC Catalogue Number
1 1 6301
2 2 6302
3 3 6303
4 4 6304
5 5
6A 6 6306
6B 26 6326
7F 51 10351
8 8 6308
9N 9 6309
9V
1 68 ..

10A 34
11A 43
12F 12' 6312
14 14 6314
15B 54
17F 17
18C • 56 10356
19A 57
19F 19 6319
20 20 6320
22F 22
23F 23 6323
25 25 6325
33F 70
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-manno-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. Inmun., 66(5):1891-1897.
Group B streptococci (GBS) is a cause of sepsis, meningitis, and related neurologic disorders in humans. The Capsular pol.ysaccharide-specific antibodies arc known to protect human infants fror.i infection. Jennings et al. , U.S. Patent No.

5,795,580. The repeating unit of the GBS capsular polysaccharide type II is: 4) -ft-D-GlcpNAc- (1 3)-[p-D-Galp(l 6) ] - p-D-Galp(l 4) - (i-D-Glcp- (1 2)-(i-D-Glcp-(i 2) - [a-D-NeupNAc(2 3) ] - [3-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) - [a-D-NeupNAc- (2 3 ) -(i-D-Galp- (1 4)-p-D-GlcpNAc-(l 6) ] -cc-D-Glcp- (1 4) - [p-D-Glcp- (1 3)]-p-D-
Galp-(l 4) -(3-D-Glcp- (1 .
European patent application No. EU-0 641 568-A1, Brade, discloses the method of obtaining ladder-like banding pattern antigen from Chlamydia trachomatis, pneumonias 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 GM2 in a vaccine for treating patients with melanoma. The latter vaccine was prepared by ozone cleavage of the ceramide double bond of GM2, 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 Fuca-(l 2)-Galp(l 3) -GalNAcfi- (1 3)-

Galct-(l 4)-Galfj-(l 4)Glc, whereas the saccharide protions of gangliosides GM:, GM] and GLUa have the following structures: GalNAcp-(1 4)-[NeuAcu-(2 3)]-Gal(J- (1 4)-Glc; Galp-(l 3 )-GalNAc(3-(1 4)- [NeuAcu-(2 3)]-Galp-(l 4)-Glc; and NeuAc-(2 3)-Galp-(l 3)-GalNAcp-(l 4) - [NeuAca-(2 3)]-Galp-(l 4)-Glc, respectively.
U.S. Patent Mo. 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 p-hemolytic streptococci, Ha.emopb.ilus influenza, meningococci, pneumococci and E. coli. Rather than reductively aminating the particles, a linker arm such as that provided by an s-amino C2-C8 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, a HBc chimer particle or 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 vitro 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-y. 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 HBc chimer particles or 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 immune react with the epilope or hapten exact ivated 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 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 I 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 HBc chimer immunogen by dispersing the immunogen, preferably in oarticulate 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 dqueous composition. The diluent veiicle can also include oleaginous materials such as peanut oil, squalane or squalene as is discussed 'hereinafter.
The preparation of inocula anc'. 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 iramunogenic 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 cowards 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"'1 (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-fatty 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 s-ualane,

emulsified with the chimer protein in an aqueous phase. Well-known examples of ;-uch 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-S-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 Quill*ji

Saponaria Molina (e.g. Quil1M A) are also useful. Derivatives of Quil™ A, for example QS23. (an HPLC purified fraction derivative of QuJl'" 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-0-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/Tween13 80 emulsion. This ac.j uvant 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 \im 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) -ethylatnin (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 aci]uvant amount, which can vary with the adjuvant, mammal and recorahinant HBc chimer immunogen. Typical amounts can vary from about 1 jig 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 s.uitable 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, cablet, 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 HBc chimer or HBc chimer conjugate particles is about i fag to about 1 rnc; of active ingredient per dose, and more preferably about 5 fag to about 50 jig per dose, as noted before.
A vaccine is typically formulated for parenteral administration. Exemplary immunizations are carried out sub-cutaneously (SC) intra-muscularly (IN!) , intravenusly (IV) , intraperitor.eally (IP) or intra-dermally (ID).
The HBc chimer particles and HBc chimer particle conjugates can be formulated into the vaccine as neutral or salt forms. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the protein or hapten) and are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived form inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
In yet another embodiment, a vaccine or inoculum is contemplated in which a gene encoding a contemplated 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 5. typhi and S. typhimurium and S. typhimurium-E. coli hybrids are discussed in the citations provided before. These vaccines and inocula are particularly contemplated

Cor use against diseases that infect; or are transmitted via mucosa of che 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 artive ingredient required to be administered depend on the judgment cf the practitioner and are peculiar

to each individual. However, suitable dosage ranges are of the order of tens of micrcgrams 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-sporozoite .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 ant.i gen [e.g. the P. falciparum CS protein peptide (NANP) 5 used herein] in
ar 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 immunisations 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 chroraatographic 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 HBc chimer particle or 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 plasmid vector pKK223-
3N, a modified form of pKK223-3
Plasmid vector pKK223-3 (Pharmacia) was modified by the establishment of a unique Ncol restriction site to enable insertion of HBc genes as NcoI-Hindlll restriction fragments and subsequent
expression in E.coli host cells. To modify the
pKK223-3 plasmid vector, a new SphI-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 ensymes SphI 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. 1 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. 1, 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: 65

pKK223-NcoI-mod-R
GCGAAGCTTCGGATCccatggTTTTTTCCTCCTTATGTGAAATTGTTATCCG-
CTC SEQ ID NO:66
B. Preparation of VI
and V2 Cloning Vectors
Modified HBcl49 genes, able to accept the directional insertion of synthetic dsDNA fragments into the immunodominant loop region, were constructed
/
using PCR. [The plasmid accepting inserts between amino acids E77 and D78 was named VI, whereas the plasmid accepting inserts between D78 and P79 was named V2.J The HBcl49 gene was amplified in two halves using two PCR primer pairs, one of which amplifies the amino terminus, the other amplifies the carboxyl terminus. For VI, the products of the PCR reactions (N- and C-terminus) are both 246 bp fragments; for V2, the products are a 249 bp (N-terminus) and a 243 bp fragment (C-terminus) .
The N-terminal fragments prepared were digested with Ncol and EcoRI, and the C-terminal fragments were digested with EcoRI and Hindlll. The VI and V2 fragments pairs were then ligated together at the common EcoRI overhangs. The resultant Ncol-Hindlll fragments were then ligated into the pKK223-3N vector, which had been prepared by digestion with Ncol and Hindlll.
To insert B cell epitopes into the VI and V2 plasmids, the plasmids were digested with EcoRI and SacI restriction enzymes. Synthetic dsDNA fragments containing 5' EcoRI and 3' SacI overhangs were then inserted. In both cases, VI and V2, glycine-isoleucine (EcoRI) and glnfamic acid-leucine (SacI) amino acid pairs, coded for by the restriction

sites, flank the inserted B cell epitopes. The .inserted restriction sites are underlined in the primers below.
VI
HBcl49/NcoI-F
5'-TTGGGCCATGGACATCGACCCTTA SEQ ID NO: 67
HBc-E77/EcoRI-R
5 ' -GCGGAATTCCTTCCAAATTAACACCCACC SEQ ID NO: 68
HBc-D78/EcoRI-SacI-F
5'-CGCGAATTCAAAAAGAGCTCGATCCAGCGTCTAGAGAC
SEQ ID NO:69
HBcl49/HindIII-R
5 ' -CGCAAGCTTAAACAACAGTAGTCTCgGGAAG SEQ ID NO: 70
V2
HBcl49/NcoI-F
5 ' -TTGGGCCATGGACATCGACCCTTA SEQ ID NO: 67
HBc-D78/BcoRI-R
5' -GCGGAATTCCATCTTCCAAATTAACACCCAC SEQ ID NO: 72
HBc-P79/EcoRI-SacI-F
5' -CGCGAATTCAAAAAGAGCTCCCAGCGTCTAGAGACCTAG
SEQ ID NO:73
H3cl49/HindIII-R
5 ' - CGCAAGCTTAAACAACAGTAGTCTCCGG.fi AG SEQ ID NO: 70

C. Preparation of V7 Cloning Vector To enable the fusion of T cell epitopes to the C terminus of a HBc chimer, a new vector, V7, was constructed. Unique EcoRI and Sad restriction sites were inserted between valine-149 and the Hindlll site to facilitate directional insertion of synthetic dsDNAs into EcoRI-Hindlll (or EcoRI-SacI) restriction sites. The pair of PCR primers below was used to amplify the HBc 149 gene with a Ncol restriction site at the amino-terminus and EcoRI, SacI and HindiII sites at the carboxyl-terminus. The product of the PCR reaction (479 bp) was digested with NcoI/Hindlll and cloned into pKK223-3N to form V7.
To insert T cell epitopes, the plasmid (V7) was digested EcoRI/Hindlll (or EcoRI-SacI) and synthetic dsDNA fragments having EcoRI/Hindlll {or EcoRI/SacI) overhangs, were ligated into V7. For all V7 constructs, the final amino acid of native HBc (valine-149) and the first amino acid of the inserted T cell epitope are separated by a glycine-isoleucine dipeptide sequence coded for by the nucleotides that form the EcoRI restriction site. For epitopes inserted at EcoRI/SacI, there are additional glutamic acid-leucine residues after the T cell epitope, prior to the termination codon, contributed by the SacI site. Restriction sites are again underlined in the primers shown.
HBcl49/NcoI-F
5 ' -TTGGGCCATGGACATCGACCCTTA SEQ ID NO: 67

HBcl49/SacI-EcoRI-H3-R
5 ' -CGCAAGCTTAGAGCTCTTGAATTCCAACAACAGTAGTCTCCG
SEQ ID NO: 75
D. Preparation of V12; _
Expression Constructs
V12 vectors, which contain B cell epitopes between amino acids 78 and 79, as well as T cell epitopes downstream of valine-149, were constructed from V2 and V7 vectors. The carboxyl terminus of a V7 vector containing a AT cell epitope inserted at EcoRI/Hindlll was amplified using two PCR primers (HBc-P79/SaeI-F and pKK223-2/4515-32R• to provide a dsDNA fragment corresponding to amino acids 79-149 plus the T cell epitope, flanked with SacI and HindJII restriction sites.
The PCR products were cut with Sad and Hindi!I and then cloned into the desired V2 vector prepared by cutting with the same two enzymes. The PCR primers shown are amenable for the amplification of the carboxyl terminus of all V7 genes, irrespective of the T cell epitope present after amino acid 149 of the HBc gene.
One exception to the generality of this approach was in the preparation of the V12 constructs containing the Pf-CS(C17A) mutation, which were prepared from existing V12 constructs. In this case, V12 constructs were amplified with HBcl49/NcoI-F (SEQ ID NO: 67) and the mis-match reverse PCR primer ('SEQ ID NO: 145), which facilitated the C17A mutation. The resultant PCR product was digested with Ncol and HindiII and cloned back into pKK223-3M (previously cut with the same enzymes). Restriction sites are underlined.

HBC-P7 9/SacI -F 5 ' -CC-CGAGCTCCCAGCGTCTAGAGACCTAG
SEQ ID NO: 76
pKK223-2/4515-32R 5 ' -GTATCAGGCTGAAAATC
SEQ ID NO: 77
E. P.falciparum CS-repeat B cell
Epitopes Inserted into V2
For V2 and V7 constructs, synthetic dsDNA fragments coding for the B (V2) or T cell epitope (V7) of interest were inserted into EcoRI/SacI restriction sites. Synthetic dsDNA fragments, encoding B and T cell epitopes of interest, were prepared by mixing complementary single stranded DNA oligonucleotides at equimolar concentrations, heating to 95°C for 5 minutes, and then cooling to room temperature at a rate of -1 °C per minute. This annealing reaction was performed in TE buffer. The double-stranded. DNAs are shown below with the encoded epitope sequence shown above. The pound symbol, #, is used in some of the amino acid residue sequences that follow to indicate the presence of a stop codon.
Pfl

INANPNANPNANPNA AATTAACGCTAATCCGAACGCTAATCCGAACGCTAATCCGAACGCTA TTGCGATTAGGCTTGCGATTAGGCTTGCGATTAGGCTTGCGAT
N P E L SEQ ID NO: 78
ATCCGGAGCT SEQ ID NO: 79
TAGGCC SEQ ID NO: 80

Pf3
INANPNVDPNANPNAN P AATTAACGCTAATCCGAACGTTGACCCGAACGCTAATCCGAACGCTAATCCGA TTGCGATTAGGCTTGCAACTGGGCTTGCGATTAGGCTTGCGATTAGGCT
NANPNVDPNANPEL SEQ ID NO: 81 ACGCTAATCCGAACGTTGACCCGAACGCTAATCCGGAGCT SEQ ID NO:82 TGCGATTAGGCTTGCAACTGGGCTTGCGATTAGGCCTCGAGG
SEQ ID NO:83
Pf3.1
I NANPNVDP N A N P N A N P AATTAACGCGAATCCGAACGTGGATCCGAATGCCAACCCTAACGCCAACCC TTGCGCTTAGGCTTGCACCTAGGCTTACGGTTGGGATTGCGGTTGGG
N A N P E L SEQ ID NO: 84
AAATGCGAACCCAGAGCT SEQ ID NO:85
TTTACGCTTGGGTC SEQ ID NO:86
Pf 3 . 2
INANPNANPNANPNVDP AATTAACGCGAATCCGAATGCCAACCCTAACGCCAACCCAAACGTGGATCCGA TTGCGCTTAGGCTTACGGTTGGGATTGCGGTTGGGTTTGCACCTAGGCT
N A N P E L SEQ ID NO: 87
ATGCGAACCCAGAGCT SEQ ID NO:88
TACGCTTGGGTC SEQ ID NO:8 9

Pf3.3
INANPNVDPNANPNANP AATTAACGCGAATCCGAACGTGGATCCAAATGCCAACCCTAACGCTAATCCAA TTGCGCTTAGGCTTGCACCTAGGTTTACGGTTGGGATTGCGATTAGGTT
NANPNVDPNANP EL SEQ ID NO: 90
ACGCCAACCCGAATGTTGACCCCAATGCCAATCCGGAGCT SEQ ID NO: 91
TGCGGTTGGGCTTACAACTGGGGTTACGGTTAGGCC SEQ ID NO:92
Pf3.4
INPNVDPNANPNANPNA AATTAATCCGAACGTGGATCCAAATGCCAACCCTAACGCTAATCCAAACGCCA TTAGGCTTGCACCTAGGTTTACGGTTGGGATTGCGATTAGGTTTGCGGT
N P N V E L - SEQ ID NO:93
ACCCGAATGTTGAGCT SEQ ID NO:94
TGGGCTTACAAC SEQ ID NO:95
Pf3.5
INPNVDPNANPNANPNA AATTAATCCGAACGTGGATCCAAATGCCAACCCTAACGCTAATCCAAACGCCA TTAGGCTTGCACCTAGGTTTACGGTTGGGATTGCGATTAGGTTTGCGGT
NPNVDPEL SEQ ID NO: 96
ACCCGAATGTTGACCCTGAGCT SEQ ID NO:97
TGGGCTTACAACTGGGAC SEQ ID NO:98

Pf3.6
INPNVDPNANPNANPNA AATTAATCCGAACGTGGATCCAAATGCCAACCCTAACGCTAATCCAAACGCCA TTAGGCTTGCACCTAGGTTTACGGTTGGGATTGGGATTAGGTTTGCGGT
NPNVDPNAEL SEQ ID NO: 99
ACCCGAATGTTGACCCTAATGCTGAGCT SEQ ID NO:100
TGGGCTTACAACTGGGATTACGAC SEQ ID NO:101
Pf3.7
INVDPNANPNANPNANP AATTAACGTGGATCCAAATGCCAACCCTAACGCTAATCCAAACGCCAACCCGA TTGCACCTAGGTTTACGGTTGGGATTGCGATTAGGTTTGCGGTTGGGCT
N V E L .SEQ ID NO:102
ATGTTGAGCT SEQ ID NO:103
TACAAC SEQ ID NO:104
Pf3.8
INVD PNANPNANPNANP AATTAACGTGGATCCAAATGCCAACCCTAACGCTAATCCAAACGCCAACCCGA TTGCACCTAGGTTTACGGTTGGGATTGCGATTAGGTTTGCGGTTGGGCT
N V D P E L SEQ ID NO:105
ATGTTGACCCTGAGCT SEQ ID NO-.106
TACAACTGGGAC SEQ ID NO:107

Pf3.9
I NVD PNANPNAN PNANP AATTAACGTGGATCCAAATGCCAACCCTAACGCTAATCCAAACGCCAACCCGA TTGCACCTAGGTTTACGGTTGGGATTGCGATTAGGTTTGCGGTTGGGCT
NVDPNAEL SEQ ID NO: 108
ATGTTGACCCTAATGCTGAGCT SEQ ID NO: 109
TACAACTGGGATTACGAC SEQ ID NO: 110
.Pf3.10
I DPNANPNANPNANP
AATTGATCCAAATGCCAACCCTAACGCTAATCCAAACGCCAACC
CTAGGTTTACGGTTGGGATTGCGATTAGGTTTGCGGTTGG
N V E L SEQ ID NO:111
CGAATGTTGAGCT • SEQ ID NO:112
GCTTACAAC SEQ ID NO: 113
Pf3.ll
I DPNANPNANPNANPNV AATTGATCCAAATGCCAACCCTAACGCTAATCCAAACGCCAACCCGAATGTTG CTAGGTTTACGGTTGGGATTGCGATTAGGTTTGCGGTTGGGCTTACAAC
D P E L SEQ ID NO:114
ACCCTGAGCT SEQ ID NO:115
TGGGAC SEQ ID NO:116

Pf3.12
IDPNANPNANPNANPNV AATTGATCCAAATGCCAACCCTAACGCTAATCCAAACGCCAACCCGAATGTTG CTAGGTTTACGGTTGGGATTGCGATTAGGTTTGGGGTTGGGCTTACAAC
D P N A E L SEQ ID NO:117
ACCCTAATGCCGAGCT SEQ ID NO:118
TGGGATTACGGC - SEQ ID NO:119
F. P. falciparum universal T cell epitope Pf-UTC (PF/CS326-345)
IEYLNKIQNSLSTEWSP AATTGAATATCTGAACAAAATCCAGAACTCTCTGTCCACCGAATGGTCTCCGT CTTATAGACTTGTTTTAGGTCTTGAGAGACAGGTGGCTTACCAGAGGCA
C S V T # # SEQ ID NO:120
GCTCCGTTACCTAGTA SEQ ID NO:121
CGAGGCAATGGATCATTCGA SEQ ID NO: 122
P.vivax CS-repeat B cell epitopes Pv-TIA
IP. AGDRADGQ. PAGDRAA AATTCCGGCTGGTGACCGTGCAGATGGCCAGCCAGCGGGTGACCGCGCTGCAG GGCCGACCACTGGCACGTCTACCGGTCGGTCGCCCACTGGCGCGACGTC
G Q P A G E L SEQ I'D NO: 123
GCCAGCCGGCTGGCGAGCT SEQ ID NO:124
CGGTCGGCCGACCGC SEQ ID NO:125

Pv-TIB
IDRAAGQPAGDRADGQP AATTGACAGAGCAGCCGGACAACCAGCAGGCGATCGAGCAGACGGACAGCCCG CTGTCTCGTCGGCCTGTTGGTCGTCCGCTAGCTCGTCTGCCTGTCGGGC
A G E L SEQ ID NO:126
CAGGGGAGCT SEQ ID NO:127
GTCCCC SEQ ID NO:128
PV-T2A
IANGAGNQPGANGAGDQ
AATTGCGAACGGCGCCGGTAATCAGCCGGGGGCAAACGGCGCGGGTGATCAAC CGCTTGCCGCGGCCATTAGTCGGCCCCCGTTTGCCGCGCCCACTAGTTG
P G E L SEQ ID NO:129
CAGGGGAGCT " SEQ ID NO:130
GTCGCC SEQ ID NO:131
PV-T2B
I A N G A D N Q P G A. N G A: D D Q
AATTGCGAACGGCGCCGATAATCAGCCGGGTGCAAACGGGGCGGATGACCAAC
C-/ CGCTTGCCGCGGCTATTAGTCGGCCCACGTTTGCCCCGCCTACTGGTTG
P G E L SEQ ID NO:132
CAGGCGAGCT SEQ ID NO:133
GTCCGC SEQ ID NO:134

PV-T2C
IA.NGAGNQPGANGAGDQ AATTGCGAACGGCGCCGGTAATCAGCCGGGAGCAAACGGCGCGGGGGATCAAC CGCTTGCCGCGGCCATTAGTCGGCCCTCGTTTGCCGCGCCCCCTAGTTG
PGANGADNQP GANGADD
CAGGCGCCAATGGTGCAGACAACCAGCCTGGGGCGAATGGAGCCGATGACC
GTCCGCGGTTACCACGTCTGTTGGTCGGACCCCGCTTACCTCGGCTACTGG
Q P G E L SEQ ID NO:135
AACCCGGCGAGCT SEQ ID NO:136
TTGGGCCGC SEQ ID NO:137
PV-T3
IAPGANQEGGAAA PGAN AATTGCGCCGGGCGCCAACCAGGAAGGTGGGGCTGCAGCGCCAGGAGCCAATC CGCGGCCCGCGGTTGGTCCTTCCACCCCGACGTCGCGGTCCTCGGTTAG
Q. EGGAAEL SEQ ID NO.-138
AAGAAGGCGGTGCAGCGGAGCT SEQ ID NO: 139
TTCTTCCGCCACGTCGCC SEQ ID NO: 140
O
Example 2: P.vivax universal T cell epitope
Pv-UTC
IEYLDKVRATVGTEWTP AATTGAATATCTGGATAAAGTGCGTGCGACCGTTGGCACGGAATGGACTCCGT CTTATAGACCTATTTCACGCACGCTGGCAACCGTGCCTTACCTGAGGCA

C S V T $ # SEQ ID NO:141
GCAGCGTGACCTAATA SEQ ID NO:142
CGTCGCACTGGATTATTCGA SEQ ID NO:143
A. PCR primers for site-directed mutagenesis
Pf-CS (C17A)-R FSQ ID NO:144
##TV. SAPSWETS
GCCAAGCTTACTAGGTAACGGAGGCCGGAGACCATTCGGTGG
HindiII SEQ ID NO:145
B. PCR Primers for Truncation and Cysteine Addition at C-terminus
To modify the C-terminus of HBc chimer
genes, either via the addition of cysteine residues
or varying the length of the HBe gene, PCR reactions
were performed using HBcl49 as template with the
HBc/NcoI-P primer and a reverse primer (e.g.
HBcl49+C/HindIII-R)that directed the desired
modification of the C-terminus. PCR products were
digested with Ncol and Hindi 11 arid cloned into
W pKK223-3N at the same restriction sites.
HBcl49/NcoI-F SEQ ID NO:245
M D I D P Y
5 ' -TTGGGCCATGGACATCGACCCTTA SEQ ID NO: 67

HBcl49+C/HindIII-R SEQ ID NO:147
ftCVV.TTEPL 5 ' -CGCAAGCTTACTAGCAAACAACAGTAGTCTCCGGAAG
Hindlll SEQ ID NO:148
HBc 14-4/Hindi II-R SEQ ID NO: 149
tfPLTSLIP
CGCAAGCTTACGGAAGTGTTGATAGGATAGGG SEQ ID NO: 150
H3cl42/HindIII-R SEQ ID NO:151
#TSLIPA. NP
CGCAAGCTTATGTTGATAGGATAGGGGCATTTGG SEQ ID NO: 152
HBcl40/HindIII-R SEQ ID NO:153
#LIPANPP CGCAAGCTTATAGGATAGGGGCATTTGGTGG SEQ ID NO: 154
HBcl39/HindIII-R • SEQ ID NO:155
# I P A N P P
GCGAAGCTTAGATAGGGGCATTTGGTGG SEQ ID NO: 156
HBcl38/HindIII-R SEQ ID NO:157
# P A N P P R
CGCAAGCTTAAGGGGCATTTGGTGGTCT SEQ ID NO: 158
HBcl38+C/HindIII-R SEQ ID NO:159
frCPANPPR
GCGAAGCTTAQCAAGGGGCATTTGGTGGTCT SEQ ID NO: 160
HBcl37/HindIII-R SEQ ID NO:161
#ANPPRYA
GCGAAGCTTAGGCATTTGGTGGTCTATAGC SEQ ID NO: 162

HBcl37+C/HindIII-R SEQ ID N0:163
tfCANPPRYA
GCGAAGCTTAGCAGGCATTTGGTGGTCTATAA SEQ ID NO:I64
HBcl36/HindIII-R SEQ ID NO:165
# N P P R Y A P
CGCAAGCTTAATTTGGTGGTCTATAAGCTGG SEQ ID NO:166
Example 3: Assay Procedures
A. Antigenicity
1. Particle ELISA
Purified particles were diluted to a concentration of 10 pg/mL in coating buffer (50 mM sodium bicarbonate, pH 9.6) and coated onto the wells of ELISA strips (50 jiL/well) . The ELISA strips were incubated at room temperature overnight (about 18 hours). Next morning the wells were washed with ELISA wash buffer [phosphate buffered saline (PBS), pH 7.4, 0.05% Tween®-20] and blocked with 3% BSA in PBS for 1 hour (75 p.L/well) • ELISA strips were stored, dry, at -20°C until needed.
To determine the antigenicity of particles, antisera were diluted using 1% BSA in PBS and 50 |AL/well added to antigen-coated ELISA wells. Sera were incubated for 1 hour, washed with ELISA wash buffer and probed using an anti-mouse(IgG)-HRP (The Binding Site, San Diego, CA; HRP = horseradish peroxidase) conjugate (50 |J.L/well) or other appropriate antibody for 30 minutes. After washing with ELISA wash buffer the reaction was visualized by the addition of TM blue substrate (50 (iL/well) . After 10 minutes, the reaction was stopped by the

addition of IN H2S04 (100 fiL/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 was diluted to a concentration of 2 ng/mL in coating buffer (50 mM sodium bicarbonate, pH 9.6) and coated onto the wells of ELISA strips (50 fiL/well) . Peptides were dried onto the wells by incubating overnight (about 18 hours), in a hood with the exhaust on. Next morning, the wells were washed with ELISA wash buffer (phosphate buffered saline, pH 7.4, 0.05% Tween®-20) and blocked with 3% BSA in PBS (75 |j.L/well) for 1 hour. ELISA strips were stored, dry, at -20°C until needed.
To determine antibody antigenicity of particles, antisera (monoclonal or polyclonal) were diluted using 1% BSA in PBS, and 50 joL/well added to antigen-coated ELISA wells. Sera were incubated for 1 hour, washed with ELISA wash buffer, and probed using an anti-mouse(IgG)-HRP conjugate (as above at 50 nil/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 jiL/well) . After 10 minutes, the reaction was stopped by the addition of IN H2S04 (100 jiL/well) and read on an ELISA plate reader set at 450 nm.
B. Immunogenicity of Particles To assay the itnmunogenicity of particles, mice were immunized, IP, with 20 jig of particles in Freund's complete adjuvant, and then boosted at 4

weeks with 10 p.g in Freund's incomplete adjuvant. Mice were bled at 2, 4, 6, and 8 weeks.
C. Sporozoite IFA
Indirect immunofluorescence assay (IFA) was carried out using glut araldehyde-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 were dissected from the salivary glands of Anopheles mosquitoes infected by feeding on P. fa.lcipa.rum (NF54 isolate) gametocytes derived from in vitro cultures.
Example 4: Expression of Recombinant Chimer HBc Particles
A. Effect of Insertion
Position on Immunogenicity
Antibody titers (I/reciprocal dilution) were measured for mice immunized with HBc particles containing the P. f-CS B cell epitope (NANP)^
inserted either between amino acids E77/D78 (SEQ ID NOs:260 and 261) or D78/P79 (SEQ ID NOs: 259 and 260), 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 were immunized with a single 20 ug dose, IP, with adjuvant as noted before, and antibody titers determined in an ELISA using immobilised (NANP) 5 synthetic peptide. The results of those studies are shown in Table 1, below.

Table 1

Time CS-2* E77/D7B (VI) D78/P79 (V2)
2 weeks 0 2,560 2,56'
4 weeks 640 2,560 40,960
*Schodel et al., (1994) J.Exp.Med., 180:1037-1046.
Another comparison was 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. were compared to titers achieved using the same (NANP)4 B
cell epitope, inserted between HBc positions 78 and 79, and using the above V2.Pfl particles as immunogen. Sera were analyzed 4 weeks after primary (1°) and 2 weeks after booster (2°) immunization, and the results are shown in Table 2, below.

Ghimer
CS-2
V2.Pfl

Primary
0 10,240

Table 2

Booster
640* 655,360




* Schodel et al., (1994) J.EXp.Med., ISO:1037-1046
A similar comparison of insertion position of the NANP CS-repeat epitope on immunogenici ty was made using B10.S mice, and the results are shown in Table 3 .

Table 3
Chimer Primary Booster
CS-2 640* 20,480*
V2.Pfl 163,840 655,360
* Schodel et al., (1994) J.Exp.Med., 180:1037-1046


The effect on the immunogenicity of HBc chimer particles (ELISA, Fl mice) that include the minor B cell epitope, NANPNVDP (SEQ ID N0:167), along with a repeated NANP sequence was examined. A HBc chimer was expressed that contained the sequence NANPNVDP(NANP)3NVDP (SEQ ID NO:21; V12.Pf3) inserted
between HBc positions 78 and ^9. The resulting ELISA data were compared to titers obtained using the tetrameric repeat (NANP)4 B cell epitope (V12.Pfl) or
the dimer of the minor B cell epitope at the same position (V12.Pf7). Each of these three chimers contained a Domain IV that included the HBc sequence from position 141 through 149, bonded to the P. Jfalciparum universal T cell epitope as the G-terminal sequence. The results of these studies using primary and booster immunizations as discussed before and using adjuvants , are shown below in Table 4.
Table 4
Chimer Primary Booster
V12.Pfl 163,840 655,360
V12.Pf3 2,621,440 10,485,760
V12.Pf7 2,560

The observed greater than 20-fold increase in immunogenicity by including the 'minor1 repeat epitope was quite unexpected. Because V12.Pf3 was not well expressed by E.coli, variants cf the Pf3 epitope NANPNVDP (NANP) 3NVDP (SEQ ID NO: 21) were constructed that had similar antigenicity to Pf3, but with increased expression levels, as shown below. Only constructs 3.1 and 3.2 were assayed for immunogenicity.
Relative expression levels of recombinant chimer HBc/P. falciparum particles and antigenicities for monoclonal antibodies specific for the CS epitopes (NANP)4 and (NANPNVDP) are shown in Table 5
below. Relative expression levels are as follows,-****=75-125 mg/L; ***=50-75 rag/L; **=25-50 mg/L. Antigenicity was determined by end point titer dilutions for the monoclonal antibodies [MoAb 2A10 for (NANP) 4; MoAb 2B6D8 for NANPNVDP; and P. vivax
Rpt. MoAb 2F2 provided by E. Nardin of New York University Medical Center]. The data were normalized such that the lowest titer is expressed as 1. For example, V12.Pf3 was 165 fold more antigenic than V12.Pf3.10 for the (NANP)4-specific monoclonal, and 26-fold more antigenic than V12.Pf3.2 for the NANPNVDP-specific monoclonal antibody. N.D.= no detectable antibody binding. [Note: V12.Pf3.7 was not , expressed due to a mutation in the expression vector; it was not examined further because sin,j.lar constructs were not antigenic and re-cloning was therefore not a worthwhile endeavor.]

Table 5

Name P.faJciparum B Cell Epitope Re_ative Antigenicity


Expression (NANP) 4 NANPNVDP
V12.Pfl (NANP) 4 SEQ ID N0:l **** 33 _ ND
V12.Pf3 NANPNVDP (NANP) 3NVDP SEQ ID NO: 21 ** 165 31
V12.Pf3.1 NANPNVDP (NANP) 3 SEQ ID NO -.2 2 + * **
- 33 31
V12 . Pf 3 . 2 (NANP) -jNVDPNANP SEQ ID NO: 23 *** 33 1.2
V12.Pf3.3 NANPNVDP (NANP) 3 NVDPNANP SEQ ID NO: 24 * * 5 1
V12 . PF3 . 4 NPNVDP (NANP) 3NV SEQ ID N0:25 **** 5 5
V12 . PF3 . 5 NPNVDP (NANP) 3NVDP SEQ ID N0:26 **** 5 5
V12 . PF3 . S NPNVDP (NANP) 3 NVDPNA SEQ ID NO:27 ** ** 5 5
V12 . PE3 . 7 NVDP(NRNP)3NV SEQ ID N0:28 - - -
V12 . PF3 . 8 NVDP(KANP)3NVDP SEQ ID N0:29 **** 5 1
V12 . PF3 . 9 NVDP (NANP) 3NVDPNA SEQ ID NO: 30 *** 5 ND
V12 . PF3 . 10 DP (NANP) 3NV SEQ ID NO:31 **** 1 ND
V12.PF3.il DP (NANP) 3NVDP SEQ ID NO:32 **** 5 ND
V12 . PF3 . 12 DP (NANP) 3NVDPNA SEQ ID NO:33 *** 5 ND

Itnmunogenicity of selected H3c chimer particles containing variants of the Pf3 epitope were assayed as described above. Sera were analyzed by ELISA 4 weeks after primary (1°) and 4 weeks after booster (2°) immunizations. The data obtained are shown in Table 6, below, in which the "Name" of the chimer and the corresponding sequence of the B cell immunogen are as illustrated above.
Table 6
NAME PRIMARY SECONDARY
V12.Pfl 40,960 ' 655,360
V12.Pf3 2,621,440 10,485,760
V12.Pf3.1 2,621,440 10,485,760
V12.Pf3.2 2,621,440 2,621,440
Surprisingly, a version Chat contained one copy of the NANPNVDP repeat (V12.Pf3.1) was as iramunogenic {and expressed better) as a version containing 2 copies (Vi2.Pf3), despite being 5-fold less antigenic for the NANP monoclonal antibody.
B. Expression failures
Several additional epitopes have been. attempted to be placed into the HBc loop (Domain II) between positions 78 and 79 (as in V2.Pfl), and have

failed to be expressed for reasons unknown. Table 7, below, enumerates those epitopes that have failed to express when inserted between D78 and P79 (V2) in a HBc chimer.
Table 7

Designation Source of
Epitope Epitope (single letter)
V2.FGF-KN7-K12) Human FGF-1 KYKKPK SEQ ID MO: 168
V2. FGF-1 (K118-H124) Human FGF-1 KRGPRTH SEQ ID NO: 169
V2.Arom-479 P450 Aromatase LHPDETKHMLEMIFTPRNSDR SEQ ID NO: 170
V2 . HIV3 . 1 HIV-1 (gp!20) RIKQI SEQ ID WO: 171
V2 . HIV4 . 1 HIV-1 (gpl20) RIKQIGMPGGK SEQ ID NO: 172
V2 . HIV5 . 1 HIV-1 (gp41) LCELDKWASL SEQ ID NO: 173
V2.HIV6.1 HIV-1 (gp41) • EQELLELDKWASLW SEQ ID NO -.174
V2 . HIV9 . 1 HIV-1 (gp41) VQQQNNLLRAIEAQQHLL -QLTVWGIKQ&QARIL SEQ ID NO: 17 5
V2.HIV10.1 HIV-1 (gp41) HLLQLTVWGIKQLQAR SEQ ID NO: 17 6
V2.HIV12.1 HIV-1 (gp41) YTHIiySLIEQSQNQQEK-NEQELLALDKWASLWNWF SEQ ID NO: 177
V2.HIV13.1 HIV-1 (gp41) YTHIIYSLIEQSQN-QQEKNEQEtLEL "SB'Q ID: Nd:17B
V2.1A2 (351-370) Human P450-1A2 GRERRPRLSDRPQLPYLEA
SEQ ID NO: 17 9
V2. 206(129-148) Human P450-2O6 REQRRPSVSTLRNLGLGKKS
SEQ ID HO: 180
V2.Py-Bl P. yoelii (TRAP) PNKLPRSTAWHQLKRKH SEQ ID NO: 181

V2.Py-B3 P. yoelii (TRAP) TAWHQLKRKH SEQ ID KO-.1B?.
V2.PV-T1A P . vivax PB.GDRADGQPAGDRAAAGQPAG
SEQ ID NO: 183
V2 . ALV1 . 2 ALV-J NQSWTMVSPINV .SEQ ID- NO: 1-8-4
V2.ALV1.2 ALV-J MIKNGTKRTAVTFGSV SEQ ID NO: 185
V2 . FMDV (142-160) FMDV PNLRGDLQVLAQKVARTLP SEQ ID NO: 186
V2.FMDV (135-160) FMDV RYNRNAVPNLRGDL-QVLAQKVARTLP SEQ ID NO: 187
Example 5: Determination of 280/260 Absorbance Ratios
Protein samples were diluted to a concentration of between 0.1 and 0.3 mg/mL using phosphate buffered saline (PBS), pH 7.4. The spectrophotometer was 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 (HBc 183), HBc particles truncated after residue position 149 (HBc 149), and several HBc chimers that are identified elsewhere herein, are shown below in Table 8.

Table 8
280/260
Pal-tide Absorbance Ratio
HBC183 0.84
-HBcl-49- -1.59
V2 .PF1 1.64
V2.PF1+C150 1.5
V2.PF1 + 1.54
Pf/CS-UTC
V2.PF1 + 1.42
Pf/CS-UTC(C17A)
Example 6: Cysteine at the C-terminus of Truncated HBc Particle
A. Addition of a Cysteine Residue
to the C-terminus of Hybrid HBc Particles
Using the polymerase chain reaction (PCR), genes expressing hybrid HBc particles can be easily mutated to introduce a cysteine or cysteine-containing peptide to the C-terminus of HBc. For example, a PCR oligonucleotide primer of SEQ ID NO:148 can be used, in concert with a suitable second primer, to amplify a hybrid HBc gene and incorporate a cysteine codon between codon V149 and the stop codon.
Hepatitis B core particles can be truncated from 183 (or 185, depending on viral subtype) to 140 and retain the ability to assemble into particulate virus-like particles. Many groups have used particles truncated to amino acid 149 because amino

acid 150 represents the first arginine residue of the arginine-rich C-terminal domain.
To assess the ability of a single cysteine residue to stabilize HBc particles, a codon for a cysteine residue was inserted using techniques described before between the codon for HBc amino acid residue V149 and the termination codon of a chitner HBc .molecule that contained the (NANP)4 malarial B
cell epitope inserted between residues 78 and 79 (referred to herein as V2.Pfl) to form the chimeric molecule and particle referred to as V2.Pfl+C
(HBC149C) . The thermal stabilit'y (at 37°C) of this chimer particle (V2.Pfl+C; SEQ ID'NOs: 264 and 265) as compared to a similar chimer particle lacking the inserted cysteine (V2.Pfl) was found to be dramatically increased, as is seen in Fig. 3.
It is noted that vectors and expression products that are prepared by addition of a cysteine to the C-terminus of a V2 construct are sometimes referred to herein as V16 vectors or expression products.
As can readily be seen in Pig. 3, the two particles started out similarly. 'However, after
fourteen days at 37°C, the cysteine-containing particle exhibited fewer bands on the SDS gel, indicating enhanced stability as compared to the particle lacking the added Cys residue.
B. Thermal Stability Protocol Purified particles were diluted to a concentration of 1 mg/mL using 50 mM NaPO4, -pH 6.8
and sodium azide was added to a final concentration of 0.02% to prevent bacterial growth. Particles were

incubated at 37° C and aliquots were taken at the time points indicated in the drawing description. Samples were mixed with SDS-PAGE sample buffer (reducing) and run on 15%-SOS-PAGE gels. Gels were stained using Coomassie Blue, and then analyzed.
Example 7: Cysteine at the C-terminus of a Peptide Fused, to the C-terminus of HBc
To further investigate whether terminal cysteine residues could elicit stabilizing effects at positions other than 150, a Th epitope from the hepatitis B core protein (amino acid residues 74-87) was fused to the C-terminus of HBc containing a malarial epitope in the immunodominant loop. This Th epitope does not contain a cytJteine residue, so a Cys residue was added at the C-terminus (underlined "C") . The control was the same epitope lacking the cysteine. These particles were made by combining V2.Pfl with V7.HBc74-87 (and'V7.HBc74-87+C). The V7 construct was PCR amplified with the HBc-P79/SacI-F primer (SEQ ID NO: 76) and pKK223-2/4515-32-R (SEQ ID NO: 77) . The product was cut with SacI and HindiII, and the SacI/Hindlll fragment was ligated into V2.Pfl cut with- the same enzymes.
Table 9, below, shows the amino acid sequences of C-terminal fusions HBc(74-87) and HBc (74-87) 4- C, relative to the native sequence that occurs in the wild type HBc protein, as well as the and the HBcl49 + C particle. "Cys shift" is the position of the introduced cysteine relative to its location in t^e wild type protein, where it is the last residue (position 183).

Table 9

Cys Cys
Source Sequence PI Length Position Shift
Native RRRGRSPRRRT- 12.74 34 34 Zero
PSPRRRRSQSP-
RRRRSQSRESQC
SEQ ID NO: 189
HBc (74-87) GIVNLEDPAS - 3.78 16 N/A N/A
RDLWS
SEQ ID NO: 190
HBc (74-87) +C GIVNLEDPAS - 3.78 16 16 -17
RDLWSC
SEQ ID NO: 191
HBc-149+C C N/A 1 1 -33
Example 8: Cysteine Located Within a Peptide
Fused to the C-terminus of an HBc Hybrid
Studies were conducted to determine if there were an absolute requirement for a cysteine residue to be the final amino acid of the HBc gene (as it is in wild type HBc) or if a cysteine could function internally in an introduced C-terminal sequence.
A peptide corresponding to a 20-residue universal T cell epitope, derived from the CS protein of the malarial parasite Plasmodium falciparum, which contains a cysteine at position 17 of the peptide or 342 of the CS protein, [Calvo-Calle et al., J. Iimunol., (1997) 159 (3) rp. 1362-1373], was fused to the C-terminus of a HBc chimer (V2.Pfl; SBQ ID NOs: 266 and 267) . This chimer contains the HBc sequence from position 1 through position 149, with the P. falciparum B cell epitope (NANP)4 inserted between amino acid residues 73 and 79. Domain I of this HBc

construct thus contained residues 1-75; Domain II contained residues 76-85 with the (NANP}4 epitope inserted between residues 78 and 79 (along with four residues comprising the restriction sites); Domain III .contained., residues ...86-135.;....and Domain IV contained residues 136-149 plus the 20-residue P. falciparum T cell epitope and two residues from the EcoRI cloning site (GI).
This fused C-terminal peptide is 20 amino acid residues long (12 or 14 amino acids shorter than the wild type sequence, depending on virus subtype) and has a predicted pi value more than 8 pH units lower than the wild type sequence. To ninimize potential stabilizing effects that may be contributed by amino acids other than the cysteine, a (similar) control construct was made, having an alanine instead of a cysteine at position 17 (see Table 10, below) .
To enable simple assessment of the stabilizing effects of this sequence, the peptides were fused to the C-terminus of a particle previously shown to degrade readily at 37°G (V2.Pfl) to form the HBc chimers denominated V2.Pfi+Pf/CS-UTC and V2.Pfl+Pf/CS-UTG(C17A), respectively. The results of a thermal stability study over a 28 day time period (as discussed previously) are shown in Pig. 4.
The results of this study showed that the presence of the cysteine in the T cell epitope derived from the GS protein of P. falciparum was needed for particle stability in the time period studied, and that there was no absolute requirement that that cysteine be at the C-terminus of the epitope. The table below shows the amino acid sequences of C-terminal fusions with a cysteine or

alanine at position 17, relative to the native sequence, which occurs in the wild t>pe HBc protein.
Table 10

Source

Sequence

Cys Cys
Length Position Shift

Native RRRGRSPRRRT- 12.74 34 34 zero
PSPRRRRSQSP-
RRRRSQSRESQC
SEQ ID NO.-189 Pf/CS-UTC (GI)EYLNKIQNS- 4.44 20 17 -15
LSTEWSPCSVT
SEQ ID NO:2
Pf/CS- (GI) EYLNKIQNS- 4.44 20 N/A N/A
UTC(C17A) LSTEWSPASVT
SEQ ID NO:192 (GI) = residues added from cloning site.
Example 9: P. Vivax HBc Chimers
Following the work discussed before on HBc chimers containing P. falcipa.rum B cell and T cell immunogens, similar work was carried out using sequences from the P, vivax CS protein. Exemplary constructs are illustrated below in Table LI.
Table 11

P. vivax Immunogen Type Malarial B Cell Immunogen. (Between D78/P79) CS-UTC (After V149)



Type -I Type- II (ANGA(G/D) (N/D)QPG) SEQ ID NO: 194 YLDKVRATVGTEWTPCSVT SEQ ID NO: 196
Type- III ('Vivax- like1) (APGANQEGGAA) SEQ ID NO: 195 YLDKVRATVGTEWTPCSVT SEQ ID NO: : 196

To address the variability of the repeats, the.following variant epitopes were used for insertion into HBc between amino acids 78 and 79:
1. Type-I CS-repeat
PAGDRADGQPAGDRAAGQPAG (P. vivax-type 1A)--SEQ ID NO:
197. This form of the epitope failed to make a
particle.
DRAAGQPAGDRADGQPAG {P. vivax-type IB)-- SEQ ID NO:
198. This form of the epitope, containing flanking
dipeptide cloning site remnants, successfully made a
particle and is referred to as V2.PV-TIB. An
immunogen for P. vivax-type I has been successfully
cloned, expressed, purified, and its immunogenicity
tested in mice. The results of that mouse study are
shown in Table 12, hereinafter.
2. Type-II CS-repeat
For type-II, this work is complicated by the existence of four different forms of the type-ll epitope. These forms contain either G or D at position 5, and either N or D at position 6 [Qari. et al., Afol. Bioch&n. Parasitol., (1992) 55 (1-2) :p. 105-113]. Hence, there are 4 different possible repeat sequences (GN, GD, DN, and DD) needed to maximise the possibility of success. The first, and preferred approach, is to prepare a single hybrid particle containing all four repeats, as shown below by underlines. This approach was successfully employed to address the variability in the type-I repeat.

Each of these constructs contains flanking dipeptide cloning site remnants.
ANGAGNQPGANGAGDQPGANGADNQPGANGADDQ PG (P. vivax-type II -GN/GD/DN/DD) SEQ ID NO: 199.
The above sequence has been cloned, expressed, and purified as a HBc chimer with no modification to the C-terminus.-
The second approach was to prepare two hybrid particles,, whereby each particle contained two of the variant epitopes (see below) . This approach is less preferable because it requires either the use of a more complex expression system to direct the
production of 'mixed' particles during expression, or
the mixing of type-II particles following manufacture.
ANGAGMQPGANGAGDQPG (P. vivax-type II-GN/GD)
SEQ ID NO: 200.
QANGADNQPGANGADDQPG (P. vivax-type II-DN/DD)
SEQ ID NO: 201.
CGCGAATTCAAGCGAACGGGGCCGATAATCAGCCGGCGGGTGCA (P. vivax-type IIB-ERl-wt-F) SEQ ID NO: 146.
3. Type-III ('vivax-like') CS-repeat The third P. vivax CS-epitope, which is quite different from the other two, is not associated with amino acid variation (see below) [Qari et al., Lancet, 1993. 341(8848): p. 780-783]. This sequence was cloned into the HBc expression system, and

hybrids were produced that contained flanking
dipeptide cloning site remnants.
APGANQEGGAAAPGANQEGGAA (P.vivax-type III)
...... ._ ...... _ ..... .. ................ ______________ , ____________ SEQ mJ?Q; _202, _. ...... .__. ..._
1 4. T cell Spitope at the C- terminus of HBc
The insertion of the P. vivax Th epitope (Pv-UTC; YLDKVRATVGTEWTPCSVT; SEQ ID NO: 196} into KBc and HBc hybrids was also performed using synthetic DNA fragments (Synthetic Genetics, San Diego CA) . However, unlike B cell epitopes, which are inserted into the immunodominant loop region of the HBc gene, T cell epitopes are fused to the C-terminus of the HBc gene . Previously discussed cloning vectors were used for the insertion of both B and Th epitopes into HBc. The particle expressing just the Pv-UTC at the C-terminus has also been successfully made.
5 . 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 HBc fragments (AA 1-80, which contain the B cell epitopes), and C- terminal HBc fragments (AA 81-150, 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.10: Relative Immunogenicities of HBc Chimers
Relative immunogenicities of several HBc chimer immunogens were compared in mice using the IFA assay discussed previously. The results of those studies using two dose immunization regimens as before are shown below in Table 12.

Immunogen

Table 12 IFA titer Protection Citation



P.berghei (CS-1)

40,960

95%



P.yoelii (CS-3)

12,800

95%*

B



P. falciparum (CS-2) 1,200

NT



P. falciparum (V12.Pf3.1)

5,200,000

NT



P.vivax (V2.PV-TIB) 160,000

NT

[A = Schodel et al., J. Exp. Med. , 1994, 100:1037-1046. B = Schodel et al., Behring Tnst. Mitt.,, 1997(98) : p. 114-119. NT = not tested. * = protection for greater than 3 months.]
As is seen from the above data, titers of for P. falciparum were achieved using a chimeric immunogen; this compares to titers of only 104 for P. berghei and 103-for P. falciparum using the replacement technology of Schodel et al.
Mice were immunized with CS-2 or V12.Pfl using 20 ug of particles on day zero and were boosted

with 10 fig at four weeks. Mice immunized with particles from V12.Pf3 and V12.Pf3.1 were immunized using 20 jig of particles on day zero and were boosted 'with 10 (ig at eight weeks using adjuvants as discussed^ before. Data showing the duration of the titers achieved are shown in Fig. 5, with data for use of V12.Pf3 particles being essentially identical to data with V12.Pf3.1 particles, and not shown.
Example 11: Relative HBc antigenicities
A series of studies was carried out to determine the relative antigenicities of several malarial HBc chitner particles toward two monoclonal antibodies (MoAb-3120 and MoAb-3105) as compared to native HBcAg (particle). These antibodies are specific to the loop region of HBc, and were the gracious gift of the Immunology Institute, Tokyo, Japan. Studies were carried .out using the chimers of Table 5 that contain malarial epitopes inserted into HBc particles at various positions as antigens in ELISA assays with the monoclonals as probes. The results of these studies (as end point dilutions) are shown below in Table 13A, 13B, and 13C, and illustrate the substantial lack of antigenicity of a contemplated chimer toward monoclonal antibodies that bind to the loop region, the primary immunogen, of HBc. Put differently, monoclonal antibodies that bind specifically to the loop region of HBc barely recognize a contemplated chimer, if at all.

Table 12A
Anti-MoAb-31?0- Relative
Particle End Point Dilution Antigenicity
HBcAg 625000 100
V12.Pf.3 80000 12.8
V12.Pf3.1 20000 3.2
V12.Pf3.2 10000 1.6
V12.Pf3.3 10000 1.6
V12.Pf3.4 80000 12.8
V12.Pf3.5 40000 6.4
V12.Pf3.6 80000 12 . 8
V12.Pf3.8 80000 12.8
V12.Pf3.9 160000 25.6
V12.Pf3.10 10000 1.6
V12 .Pf3.11 80000 12 . 8
V12 . Pf 3 . 12 80000 12.8
Table 13B
Anti-MoAb-3105
Particle End Point Dilution
HBcAg 1,300,000
V2.Pfl Zero
(78/79)
V12.Pfl Zero
(78/79)
V12.Pf3 Zero
(78/79)
Vl.Pfl Zero
(77/78)
V13.Pfl 1,300,000

An insertion into several sites in the immunodominant loop (including positions 77-78 or 78-79) totally eliminates binding of MoAb-3105. V13 is an insertion between residues 129 and 130, and is
used as a control because the native HBc
immunodominant loop remains intact in this construct.
Table 13 C Anti-MoAb-3120
Particle End Point Dilution 77/78 Vl.Pfl 102,400 78/79 V2.Pfl 400 HBcAg 409,600
These data show that insertion between residues 78 and 79 causes a more drastic reduction in anti-MoAb-3120 binding, as compared with insertion between residues 77 and 78.
Example 12: Construction of a Modified Hepatitis B
Core Protein Expression Vector
Using site-directed mutagenesis, a lysine codon (AAA) was introduced between amino acids E77 and P78 of the HBc gene, along a Sad (GAGCTC) restriction endonuclease site, to facilitate -the genetic insertion of other codons for producing linker group-containing HBc particles. The insert thus had an aminp acid residue sequence.. of KEL, where the EL is an artifact of the SacI site. The linker group-containing HBc protein was therefore 152 amino acid residues long. The construction of the pKK223-3-HBcl52-K78 expression plasmid is described below.
Oligonucleotide primers PIP (SEQ ID NO:203) and P1R (SEQ ID NO: 204, on the complementary strand)

were used to amplify the 5' end of the HBc gene (bases 1-234, amino acids 1-77), and simultaneously incorporate an Ncol restriction site (CCATGG) at the 5' end, a SacI restriction site (GAGCTC) at the 3'
(
end of the amplified product, and a lysine codon (AAA) preceding the SacI site Oligonucleotide primers P2F (SEQ ID NO: 205) and P2R (SEQ ID NO: 206, on the complementary strand) were used to amplify the 3' end of the HBc gene (bases 235-450, amino acids 78-149), and simultaneously incorporate a SacI restriction site (GAGCTC) at the 5' end and a Hindlll restriction site (AAGCTT) at the 3' -end of the
©
amplified product. , The two PCR products (encoding amino acids
1-77 and amino acids 78-149) were cleaved with SacI, ligated together at their common SacI overhangs, cleaved with Ncol and Hindi 11 and cloned into the expression plasmid pKK223-3 (Pharmacia), using standard techniques. The resulting plasmid was called pKK223-3-HBC152-K78.
This plasmid can be used for the expression
of a HBc chimer bearing a lysine as a linker group in
the immunodominant loop. The expressed HBc chimer
spontaneously formed particles. The linker group.-.
/«N containing HBc of this Example thus had an insert
corresponding to position 77 of the HBc of SEQ ID NO: 247, a chemically reactive lysine linker residue at a position corresponding to position 78 of the HBc of SEQ ID NO: 247, and was truncated at a position corresponding to position 149 of the HBc of SEQ ID N0:247.
A plasmid that encodes the above chimer and further includes a C-terminal cysteine residue can be prepared using the PCR techniques described in

Example II, along with the preparation described immediately above. HBc chimer particles containing a C-terminal Cys residue and a linking residue that can be conjugated to an immunogenic hapten result from expression of the plasmid following the procedures described herein.
Primer PIP
TTGGGCCATGGACATCGACCCTTA SEQ ID NO: 203
Primer P1R
GCGGAGCTCTTTTTCCAAATTAACACCCAC SEQ ID NO: 204
Primer P2F
CGCGAGCTCGATCCAGCGTCTAGAGAGACC SEQ ID NO: 205
Primer P2R
CGCAAGCTTAAACAACAGTAGTCTCCGGAAG SEQ ID NO: 206
Example 13: Modified Hepatitis B Core Particle Purification
Chimeric linker group-containing HBc particles of Example 12 were expressed in E. coll typically E. coli BLR or BL21 from Novagen (Madison, Wisconsin) or E. coli TB1 from Amersham (Arlington Heights, Illinois) . The transfected E. coli [denoted HBcl52-K78], expressed plasmid pKK223-3-HBcl52-K78 . The chimer linker group-containing HBc particles [HBcl52 (K78) particles] were purified via Sepharose'5

CL-4B - (Pharmacia) chromatography using established procedures.
In the nomenclature system used for these chimer molecules and particles, "HBc" denotes hepatitis B core protein sequence; "152" denotes the number of amino acid residues present in the chimer with lysine and two restriction site residues (glutaraic acid and leucine; EL) being added to the HBcl49 sequence from the SacI site; and " (K79)" denotes that the lysine (K) is added to the sequence after residue 78 as new residue 79. Chimer molecules and particles containing a cysteine residue as the C-terminal residue of the molecule, which are discussed hereinafter, are denoted as "+C".
Because particles purify in a predictable manner, the monitoring of particle elution using simple spectroscopy (OD2ao) * in concert with SDS-PAGE analysis to assess purity of individual fractions prior to pooling, was sufficient to enable the routine purification of electrophoretically pure particles in high yield (5-120 mg/L cell culture) . The spherical- structure of the pure chimer linker group-containing HBc particles was clearly visible in an electron micrograph.
Example 14: Chemical Coupling of Synthetic Peptides to Chimer Linker Group-containing HBc Particles as Activated Carriers The chimer linker group-containing HBc particle product of the expression plasmid pKK223-3-HBC152 (K78) from Example 13 was assayed for its chemical reactivity compared with similarly expressed and purified "wild type" truncated hepatitis B core particle (HBcl49), which is identical to HBcl52(K78)

except that it lacks the introduced lysine residue linker group and flanking five amino acids.
Synthetic peptides (haptens) were chemically conjugated to chimer linker group-
cqntaining HBc_ particles _using succinitnidyl
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 modified 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, HBcl52(K78) and HBcl49 particles were separately 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-aetivated HBc particles. Unreacted SMCC was removed by repeated dialysis against 50 mM sodium phosphate, pH 6.8. The SMCC derivitization of the HBc particles resulted in a minimal molecular weight increase that was not detectable by SDS-PAGE. However, the PAGE analysis did 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 were designed such that they had 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.
Table 14 shows the synthetic peptides derived from human cytochrome P450 enzymes that were chemically conjugated to HBc particle activated carriers to form HBc chimer particle conjugates containing pendently linked cytochrome P450 determinant haptens, or more simply, HBc chimer particle conjugates. The synthetic peptides were dissolved in 50 mM sodium phosphate, pH 6.8, to a concentration of 10 mg/ml. The synthetic peptides were then added, drop-wise, to a 5-fold excess over total amino groups in maleimide-activated, strategically modified HBcl52(K78) particles, and permitted to react at room temperature for 2 hours. Maleimide-activated HBcl43 particles were reacted with the two 2D6 peptides (2D6 and 2D6-C)as controls.

Table 14 Cytochrome P450 Haptens

Peptide Name Sequence SEQ


ID NO
1A1 (289-302) CQEKQLDENANVQL 207
1A2 (291-302) CSKKGPRASGNLI 208
2D6 (263-277) CLLTEHRMTWDPAQPPRDLTE 209
3A4 (253-273) CVKRMKESRLEDTQKHRVDFLQ 210
lAl-c CMQLRS 211
1A2-C CRFSIN 212
2D6-C CAVPR 213
2E1-C CVIPRS 214
2C-C CEIPV 215
3A3/4/7-C CTVSGA 216
3*5 -c CTLSGE 217

O

Example 15: Analysis of Chimer
HBc Particle Conjugates
HBc chimer particle conjugates containing pendently linked to cytochrome P450 determinant haptens of Example 14 were analyzed by SDS-PAGE and immunoblots to- determine if synthetic- peptides had been successfully conjugated to HBc. The denaturing conditions of the electrophoresis procedure dissemble particles into their constituent subunits: HBc monomersX Because HBc monomers have a molecular weight of approximately 17,000 Da, it was simple to resolve HBcl52(K78) particles chemically conjugated to either 1A1 (289-302), 1A2 (291-302), 2D6 (263-277) or 3A4 (253-273) peptides, as chose peptides have a relative molecular mass of approximately 2,000 Da and

therefore cause a visible increase in the molecular mass of the HBc protein monomers.
From the relative .intensities of the conjugated and non-conjugated bands on SDS-PAGE, it was determined that approximately 50 percent of the HBcl52(K78) monomers were covalently linked to hapten, whereas only about 5 percent of the "wild type" HBcl49 particles were linked to hapten. The marked increase in the observed success in pendently linking hapten to the activated carrier supports the conclusion that the observed linking occurs via the inserted lysine as opposed to a lysine residue that is also present in the "wild type" .
The shift in mobility of HBc particles conjugated to shorter C-terminal P450-derived peptides (5- and 6-mers) is not as pronounced in the SDS-PAGE as that of the longer inhibitory-peptides, but shifts of approximately 1 kDa were clearly evident in successfully coupled HBcl52(K78) monomers. The chimeric HBc 152{K78) protein exhibited markedly enhanced ability to pendently link to a hapten over the "wild type" HBcl49 particles, which showed minimal conjugation.
In. the model of core particles' propounded of icosahedral particles of either 180 or 240 associated core protein monomers [Conway et al. (1997) Nature, 386:91-94)], dimers of the relatively exposed immunodominant loop regions of the core monomers extend out from the assembled core particle into solution like spikes on a mace. The "spikes" are closely arranged spatially on the HBc particles. The strategic location of the introduced lysine residue on the tip of th
propensity for steric constraints to reactions linking haptens to assembled core particle.
A maximum of 50 percent of the strategically modified HBc monomers was successfully
conjugated- toL the synthetic pept.idea__af__Cyt._P.45..Q_.._
That amount of pendent linkage corresponds- to an average of one hapten attached per core protein dimer. This proposed distribution of hapten linkage to the strategically modified HBc particle is supported by PAGE results under semi-denaturing conditions that disassemble the particle while maintaining the dimer association.
HBc-2D6 particles prepared by peptide coupling were examined using immunoblots to confirm the presentation of the 2D6 polypeptide epitope. When probed with anti-HBc antisera, the chemically coupled particle yielded two different monomer bands representing monomers with and without the 2D6 polypeptide. Only the upper band of these blotted with anti-2D6 antisera, thereby confirming the correlation between mobility shift and attachment of the 2D6 polypeptide.
Example 16: Strategic Lysine Insertions
To construct HBc particles with inserted lysine residues at every position in the immunodominant, surface-exposed loop region (amino acids 75-85), PCR was used to amplify the 5' and 3' fragments of the HBc gene and a single lysine codon was introduced via the oligonucleotide primers. The oligonucleotide primers and the resulting amino acid sequences are shown in SEQ ID NOs: 220-241. The "wild type" sequences are SEQ ID NOs : 218-219. These HBc chimers had a length of 150 residues with an added

lysine at the postition noted by the number in each chimer and particle name.
•In order to prepare lysine inserts at
positions 75 to 84 [HBcl50(K75) through HBclBO(K84) ] , the pairs of PCR fragments were digested with the restriction endonuclease Msel, which recognizes the sequence, TTAA. The modified gene was restored by ligating the oligonucleotide primer (containing the lysine) at the convenient Msel restriction site located at nucleotides 221-224. For H3c-K85 (SEQ ID N0s:240-241) it was necessary to prepare two fragments that were ligated at a common Xhol restriction site (CTCGAG) that is not present in the wild type gene, but could be introduced at position 239-244 without altering any amino acids.
Table 15
Lysine Insertion Mutants of HBc in the Iramunodominant Loop

Name Sequence SEQ ID NO:
Wild Type HBc TWVGVNLED PASRDLWSYV 218
HBC150K75 TWVGVKNLBDPASRDLWSYV 220
,HBcl50K76 TWVGWKLEDP&SRDLWS YV 2-22
HBC150K77 TWVGVNLKBDPASRDLWSYV 224
HBC150K78 TWVGVNLEKDFASRDLWSYV 226
HBC150K79 TWVGVNLEDKPASRDLWSYV 228
HBC150K80 TWVGVNLED PKASRDLWSYV 230
HBclSOKSl TWVGVNLED P AKSRDL WSYV 232
HBC150K82 TWVGVNLEDPASKRDLWSYV 234
HBC150K83 TWVGVNLEDPASRKDLWSYV 236
HBC150K84 TWVGVNLED PAS RDKL WS YV 23,8
HBC150K85 TWVGVNLED PAS RD LKWS YV 240

To purify the linker group-containing HBc chimers, cleared cell lysates from a 1L fermentation were precipitated with 45% ammonium sulfate and the resultant pellet subjected to _gel filtration using Sepharose® (Pharmacia) CL-4B chromatography (2.5ctnx 100cm). Particulate HBc has a characteristic elution position when analyzed using this type of column, independent of the amino acid insertions made to the particle. The eleven linker group-containing HBc chimer particles prepared for this study were analyzed using this procedure and the elution profiles were measured spectrophotometrically at an absorbance of 280 nm.
Three of the linker group-containing HBc chimer particles prepared from constructs [HBcl50(K75), HBclSO (K77), and HBclSO(K79) ] were produced at levels of between 50 and 100 mg/L, which is comparable with typical yields for wild-type, unmodified HBc particles, e.g. HBcl49 particles. Linker group-containing HBc chimer particles of four of the constructs [HBclSO (K76) , HBclSO (K78), HBclSO(K81), and HBclSO(K82)] were produced at relatively low levels (between 1 and: 20' mg/L) . Finally, four of the particles [HBclSO (K804) , HBclSO(K83), HBclSO(KB4), and HBclSO(K8S) ] were produced at levels deemed to be barely detectable (less than 1 mg/L) . The yields of these expression products are shown in Table 16, below.

Table 16
Purified Lysine-Containing Chimer HBc Particles from a One L Fermentation

Particle Yield (mg/L)
HBcl50(K75) 77
• HBclSO (K76) 5
HBcl50(K77) 74
HBclSO (K78) 10
HBclSO (K79) 94
HBclSO (K80) 0
HBCISO(KSI) 17
HBclSO (K82) 1
HBclSO (K83) 0
HBclSO (K84) 0
HBclSO (K85) 0
As before, a plasmid that encodes the above chimer and further includes a C-terminal cysteine residue can be prepared using the PCR techniques described above or in Example II by insertion of a Cys codon just upstream from the termination codon, along with the preparation described immediately above.
Example 17: Chimers with HIV Sequences
Recombinant chimer particles were prepared in which the HIV-1 gp41 sequence' of positions 631-665 was present between HBc residues 78 and 79. One preparation contained a C-terminal Cys residue (SEQ ID NOs: 272 and 273), whereas the other did not and was terminated at the valine of HBc position 149 (SEQ ID NOs: 270 and 271). The particles with no terminal

Cys were expressed using the V2 vector discussed in Example IB, whereas the Cys-terminated particles were expressed from a vector prepared as discussed in Example II. Those constructs are referred to as V2.HIV11.1 and V16.HIV11.1, respectively. The yields on expression were 1.6 mg/L and 12.4 mg/L, respectively, thereby illustrating an almost 8-fold increase in yield for the particles assembled from the Cys-terminated protein.
The sequence of the HIV B cell epitope is shown below, as are the coding'and complementary DNA sequences for that epitope. The HIV sequence conveniently ends with a C-terminal EL residue and begins with added N-terminal GI residues, so that there are two added (heterologous) residues in total that are neither from the HBc sequence nor from the inserted peptide sequence.
Inserted B cell epitope sequence GIQWMEWDREINNYTSLIHSLIBESQNQQBKNEQEL
SEQ ID NO: 242
Coding sequence 5' ......
AATTTGGATGTGGGAAGATCGTGAGATCAACAATTATACCAGCCTGATACATT CTTTAATTGAAGAGTCCCAGAACCAACAGGAGAAAAATGAACAAGAGCT
SEQ ID NO: 243
Complementary sequence
5'
CTTGTTCATTTTTCTCCTGTTGGTTCTGGGACTCTTCAATTAAAGAATGTATC
AGGCTGGTATAATTGTTGATCTCACGATCTTCCCACATCCA
SEQ ID NO: 244

Example 18: Comparative Expression
,,.A similar comparative expression study was carried out using the previously described HBcl50{K77) vector that expresses a chimer molecule containing a lysine between residues 76 and 77 of HBc (along with two exogenous residues on either side of the added lysine) and a similar vector that also contained a Cys residue at the C-terminus of the protein. The latter vector was prepared by the techniques discussed before by using a C-terminal PCR primer that contained a codon for Cys between the Val-149 and stop codons. In a paired expression study, the former vector expressed particj.es in an amount of 55 mg/L, whereas the latter vector expressed particles in an amount of 60 mg/L.
Example 19: Preparation of C-Terminus Truncated
HBc Chimer Genes and Particles The HBc gene was amplified using HBc-NcoI-fwd (shown hereinafter) in concert with each of the following reverse primers: HBcl38+139C-H3-rev, HBcl39-H3-rev, and HBcl40-H3-rev (shown hereinafter) to generate the following HBc genes: HBol40, HBcl39 and HBcl38+139C. The PCR products* were cut with Mcol and Hindlll and cloned into pKK223-3N, which was prepared by cutting with same two enzymes. Plasmids were then transformed into B.coli strain TB1 and grown for 24 hours in 500 mL of TB media supplemented with 8 ml g/L glucose and 50 |o.g/mL ampicillin. Particle production was determined by analyzing crude B.coli preparations using a Sepharose® CL-4B sizing column (Pharmacia), whereby particles are associated with a characteristic elution position.

Thus, five grams of harvested cells were lysed in 25 mL of 50 mM Tris-HCl buffer, pK 8.0, 10 mM EDTA using a French press. The lysate was clarified by centrifugation at 16,000 rpm (JA-30.50 Ti rotor, Beckman) for 20 minutes. Ammonium sulfate precipi"t:ation"(45%) was" used "to "precipitate particles, and the precipitate was recovered by centrifugation at 16,000 rpm (JA-30.50 Ti rotor, Beckman) for 20 minutes. The pellet so formed was resuspended in 5 mL of 50 mM Tris-HCl, pH 8.0, 10 mm EDTA and dialyzed against the 20 mm Tris-HCl, pH 8.0 until soluble. The material was then loaded onto a Sepharose CL-4B chromatography column (2.5 x 100 cm) and allowed to run at a flow rate of 1 mL/minute for 500 minutes, by which' time all material was eluted. Elution of particles was monitored at 280 nm.
Based upon the elution profiles, HBc 140 makes particles, whereas HBc 139 does not. Particles also were not formed by the addition of a cysteine at position 139 of a particle that otherwise ended at residue 138. Vectors were constructed using DNA of SEQ ID Nos: 275, 146, 159, 160, 155, 156, 153 and 154, shown previously.
Example 20: Preparation of Vector for Preparation of HBc Particles for Use in Humans
A. Preparation of Vector Vl7Pf3.1 To manufacture the particle V12.Pf3.1 (SEQ ID NOs: 268 and 269) in a manner suitable for human administration, it was necessary to express the particle using an expression system that did not require the use of ampicillin to ensure plasmid maintenance. To achieve this, the gene coding for the particle, along with the necesrary upstream

regulatory sequences, was inserted into a new plasmid that utilizes kanamycin as the selectable marker. The new plasmid (V17.Pf3.1} was synthesized using a two step cloning procedure:
Step 1: The plasmid pKK223-3M-V12 was digested with the restriction enzymes BamHI and Hindlll to yield two DNA fragments of 801 and 4869 bp. In addition, the commercially available plasmid pREP4 (Qiagen) was cut with Bglll and HindiII to yield two fragments of 320 bp and 3420 bp. The 3420 bp and 801 bp fragments were ligated to create plasmid V17. (It is noted that 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 V17 plasmid, therefore, contains the HBcl49 gene, complete with Pf-UTC sequence fused to the C-terminus, and BcoRI and SacI restriction sites in the immunodominant loop region to enable insertion of epitopes between D78 and P79 of the HBc gene.
Step 2: The second step was to insert the Pf3.1 version of the Pf CS-repeat epitope into the immunodominant loop region of the gene. This was achieved; by digesting VI7 wi.tn.Sacr and EcoEI to yield IS bp and 4206 bp DNA fragments. Annealed oligonucleotides encoding the Pf3.1 epitope were ligated with the 4206 bp fragment to yield V17.Pf3.1, a 4275 base pair plasmid. In addition to the gene 'that encodes the 195 amino acid malaria vaccine candidate, this plasmid contains a gene for the lac represser (lac I) to force any gene under lac promoter control to be fully repressed until induced by isopropylthiogalactosi^e (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.
The locations of the genes of interest are:
Amino . Molecular Gene Start Stop Acids Weight (kDa)
Lac I 2128 3087 319 34.1 V17.Pf3.1 281 868 195 2.1.7 KrnR 4259 3465 264 29.1
A suitable host for V17.Pf3.1 is E. coll BLR, a rec A derivative of B.coli BL21, and a common strain used for the production of recombinant proteins (available for purchase from Novagen). E. cold BLR was 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 Particles Using Plasmid V17 . Pf 3 .-1
E. coli (Strain BLR) containing the
V17.Pf3 .1 plasmid were streaked onto an LB agar plate supplemented with 25 /ig/mL kanamycin and 10. /zg/mL

tetracycline, then incubated at 37°C for 16-20 hours. A single colony was then used to inoculate 3 mL of TB-Phy medium in a sterile culture tube, supplemented with 25 /ig/mL kanamycin. The tube was incubated
overnight (about 18 hours) on a shaker at 37°Cand about 200 rpm.
The following morning, 100 mL of TB-Phy medium was warmed to 37°C. One mL of the- overnight culture was removed and used to inoculate the flask,
which was then incubated on a shaker at 37°C at about 200 rpm for six hours.
The fermentor (Biostat™ UE20) was
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 ASQO value was measured for the first sample, and for samples every 20-30 minutes thereafter to monitor A60o- An ±ETG solution was prepared by dissolving 62 mg IPTG in 10-15 mL water. When the A6oo value reached 0.5, the filter-sterilised IPTG solution was aseptically added to the fermentor through a syringe. The incubation was continued until next day (e.g. about another 10-24 hours).
At 14 hours after induction, the fermentor temperature was set to 15°C. Harvesting of cells was

started by centrifugation in e. Beckman® J2-MC centrifuge with following conditions:
Rotor JA10
Speed 7,500 rpm
Temperature 4°C
Time 9 minutes
The cells were harvested by freezing into liquid nitrogen.
C. Purification of Particles
Expressed by Vector V17.Pf 3 .1/BLR
The biomass of harvested cells was resuspended in 50 mM sodium phosphate, pH 6.8, and lysed using a French Pressure cell at 16,000 psi. The cell debris was removed by centrifugation using a Beckman® J2-MC centrifuge .and the following conditions.
Rotor: JA20
Speed: 15,.000 rpm
Temperature: 4°C
Time: 30 minutes.
The volume of the resultant supernatant was measured and 277 g/L of solid ammonium sulfate were slowly added, to the supernatant. The mixture was
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 rptn
Temperature: 4°C
Time: 30 minutes
; __.. ..._ ... The precipitate ._was__tij.en._.res.uapended.. in... a_
minimal volume of 50 itiM sodium phosphate buffer and then dialyzed against the same buffer for one hour with stirring. The dialyzed solution was centrifuged in Beckman® J2-MC centrifuge with the following conditions.
Rotor: JA20
Speed: 15,000 rpm
Temperature: 4°C
Time: 15 minutes
The supernatant was 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: UV at 210, 254 and 280 nm. Fraction: 15 mL
The column was eluted with buffer B at 2 mL per minute. Particle-containing fractions were identified using SDS-PAGE and pooled. The salt

concentration of the pooled material was 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) H- 5 M NaCl. (The buffer was degassed for 30 minutes daily, before use.)
Buffer B (elution solvent): 50 mM sodium phosphate buffer (pH 6,8). (The buffer was 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.: 96C40250)
Resin: 2-00 mL Toyopearl® ether 650 HIC resin, manufactured by Tosohaas
Detector: UV at 210, 254, and 280 nm
Fraction: 15 mL
The column was equilibrated with 5 column volumes (CV) of buffer A for a one hour time prior to starting purification, using a flow rate of 20

mL/minute. -The retentate containing 5 M salt was then loaded at a rate of 20 mL/minute. The column was 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:_el.uted)__.wi.Jt.h._ 1Q.Q_...%_buf.£.er_.E._ ..Fractions, were, completely analysed for proteins of interest by SDS PAGE analysis. Pure fractions were combined together, and a protein estimation using a Bradford assay was carried out.
Hydrophobia Interaction Chromatography using butyl
resin . •
Column: Millipore Vantage™ VL44 x 250
column (44 mm diameter, 250 mm
height, Catalog No.: 96440250) Resin: 200 tnL Toyopearl® Butyl €50-S HIC
resin, manufactured by Tosohaas Detector: UV at 210, 254 and 280 nm Fraction: 15 mL
The column was equilibrated, with. 5 column volumes (CV) of 40% buffer B for one hour prior to starting purification, using a flow rate of 20 ml/man. The combined fractions from ether HIC were loaded at a rate of 20 mL/minute-. The column was washed with 2 CV of 40% buffer B, washed with 2 CV. 90% B_, and eluted with 4 CV of: WFI,
Fractions were analyzed'for protein of interest by SDS PAGE analysis. Pure fractions were combined together

Hydroxyapatite Column Chromatography
Column: Millipore Vantage™ VL16 x 250 column (16 mm diameter, 250 mm
__ height-,- Catalog- Ha- L-96160250-)
Resin: 20ml Ceramic Hydroxyapatite (Catalog No.
• 158-2200)
Detector: UV at 215, 254 and 280 nm Fraction: 15 mL
The column was 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. Wash the column with 20 mM sodium phosphate buffer until A280 drops to baseline. Fractions were analyzed for protein of interest by SDS PAGE analysis. Pure fractions were combined together.
Desalting
Column: Prepacked desalting column, HiPrep™ 26/10,
Pharmacia Resin: 20 mL Ceramic Hydroxyapatite (Catalog No.
158-2200)
Detector: UV at 215, 254 and 280 nm Fraction: 15 mL
The column was equilibrated with 5 CV of 15. mM Acetate Buffer, pH 6.0. The pooled fractions from the hydroxyapatite column were loaded onto the column, and then eluted with 15 mM Acetate Buffer, pH 6.0, at a flow rate of 20 mL/min. Fractions were

analyzed for protein of interest ty SDS PAGE analysis. Pure fractions were ccmbined together, and protein estimation was carried out using a Bradford assay. The pure fraction was assayed for endotoxin level, and finally passed through a 0.22-micron, filter for terminal filtration.
Example 21: Comparative Expression of Chimers with Cytochrome P450 sequences
Recombinant chimer particles were prepared in which the human cytochrome P450 1A1 sequence of positions 290-302 was present between HBc residues 78 and 79. One preparation contained a C-terminal Cys residue, whereas the other did not and was terminated at the valine of HBc position 149. The particles with no terminal Cys were expressed using the V2 vector discussed in Example IB, whereas the Cys-terminated particles were expressed from a vector prepared as discussed in Example II. Those vectors are referred to as V2.1A1(290-302) and V16.1A1(290-302) , respectively. The yields on expression were 2.7 mg/g cells, 36 mg/L culture and 8.8 mg/g, 144 mg/Ii,.. respectively, thereby illustrating' the- ability of the terminal • cysteine modification to stabilize chimer molecule particle production and yield.
The sequence of the P450 1A1 peptide is shown below, as are the coding and complementary DNA sequences for that epitope. The P450 1A1 sequence begins with a N-terminal GI and ends with a C-terminal EL residue sequence, so that there are only four added (heterologous) residues, in total, that are neither from the HBc ^equence, nor that of the inserted peptide sequence.

Inserted B cell epitope sequence
(GI)QEKQLDENANVQL(EL) SEQ ID NO: 230
Coding sequence
5'
CAAGAAAAACAGCTAGACGAAAACGCAAATGTACAGCTC
SEQ ID NO: 74
Complementary sequence
5'
CGAGCTGTACATTTGCGTTTTCGTCTAGCTGTTTTTCTTG
:EQ ID NO: 71
Example 22: Preparation of Vectors to Express Particles with a Cysteine Residue Prior to C-Terminal
FUsed Epitope
To prepare particles with a single cysteine after VX49 of the HBc gene, followed by a T cell epitope, a PCR primer was synthesized (SEQ ID NO: 282) . This primer, in conjunction with HBcl49/NcoI-F (SEQ ID No: 67), was used to amplify the HBc gene to produce a version of HBc having a single cysteine codon introduced directly after VI£9, as well as BcoRI and Hindlll restriction sites (after the introduced cysteine) . The 478 bp PCR product was cut with Ncol and Hindlll and cloned into pKK223-3N.
SEQ ID No. 281
C V V T T E P
5' GCAAGCTTACTATTGAATTCCQCAAACAACAGTAGTCTCCGG Hindlll EcoRI
SEQ ID No:282

The resultant plasmid was then cut with EcoRI and HindiII and the annealed oligonucleotides coding for the Pf/CS-UTC (PF/CS326-345; SEQ ID Nos: 121 and 122) ligated into the plasmid. This plasmid was then used as the template in a PCR reaction along with the primers HBc-P79/SacI-F (SEQ ID No: 73) and Pf/CS'(C17A) {SEQ ID No: 145) the resultant PCR product (307 bp) coded for amino acid residues 79 through 149 of HBc, followed by the introduced cysteine, followed by the Pf/CS-UTC sequence having the C17A mutation, and flanked by SacT (5') and Hindi!I (3') restriction sites. This fragment was cut with Sad and Hindlll and ligated with the plasmid V2.Pfl [encoding the malarial (NANP)4
epitope] that had been cut with the same two enzymes.
The resultant gene codes for a 190 amino acid residue HBc chimera having (NANP)4 inserted
between amino acids 78 and 79 of HBc, (flanked by the Gly-Ileand Glu-Leu sequences derived from the EcoRI and SacI restriction, sites respectively) and the C17A version of the Pf/CS326-345 at the C terminus. The single cysteine was therefore located between V149 of HBc and: the Gly-Ile linker sequence (derived from the EcoRI --restrict'ton site) located prior to the first amino acids of the Pf/CS326-345 (C17A) [Pf/CS-UTC(C17A)] T cell epitope (see SEQ ID No. 284).
This hybrid particle was expressed,
purified and analyzed for stability by incubating at 3.7°C for several weeks. The stability of this particle (V12.Pfl(C17A)C150) was comparedto V12.Pfl, with the only difference between the two particles being the position of the cysteine residue. For V12.Pfl the cysteine is followed by three amino acid

residues (SVT) at the C-terminus of the protein (SEQ ID No: 283), whereas'for V12.Pfl(C17A)C150 the cysteine is followed by 22 additional amino acid residues (SEQ ID No: 284).
V12.Pfl
TTW GI EYLNKIQNSLSTEWSPCSVT SEQ ID No: 283
V12.Pfl (C17A)C150
TTW G GI EYLNKIQNSLSTEWSPASVT SEQ ID No: 284
The effect of inserting the cyst.eine residue between HBc and the T cell epitope (V12.Pfl (C17A) C15.0) was to create a particle that was significantly more stable than a similar particle without the C terminal cysteine (V12.Pf1(G17A)). This was evident from the fact that unlike , V12.Pfl(C17A), V12.Pfl(C17A)C15Q could be easily purified without a significant degree of degradation of monomers (compare T=O for these particles in Figures 4 and 8); further, VX2.Pfl (C17A) C150 was significantly more stable than V12.Pfl (C17S.) following incubation at 37°C. After 14 days at 37°C, . •V12 ...Pf 1.(.-C1.7A) monomers are totally degraded (Figure 4) , whereas V12 .Pfl (C17A) C150 monomers are only partially degraded (Figure; 8) .
It was apparent thatV12.Pfl(C17A)CIS0 was not as stable V12.Pfl (Figure 8). These data indicate that the stabilizing effects of a single C-terminal cysteine residue are most effective when placed at or near, e.g., within five residues of, the C-terminus of the HBc chimer.

Example 23: Analytical Gel Filtration
Analysis of Hybrid particles
Analytical gel filtration analysis of purified hybrid HBc particles was performed using a 25 mL Superose® 6 HR 10/30 chromatographic^column (Amershara Pharmacia # 17-0537-01) and a BioCAD™ SPRINT Perfusion Chromatography System. The UV detector was set to monitor both wavelengths of 260 and 280 nm. The column was equilibrated with 3 column volumes (CV; about 75 mL) of buffer (50 mM NaP04, pH 6.8) at a flow rate of 0.75 mL/minute.
The particles to be analyzed were diluted to a concentration of 1 mg/mL using 50 mM NaPO^, pH 6.8. 200 Microliters (/iL) of the sample were then loaded onto a 200 /zL loop and injected onto the column. The sample was eluted from the column with 50 mM NaPO4, pH 6.8 at a flow rate of 0.75 mL/minute.
Several particles containing C-terminal cysteine residues or similar particles free of such cysteines were analyzed using the above procedure. Integration of the 280 nm trace was carried out using BioCAD1" software (PerSeptive™) to provide the results in Table 17, below.

Table 17

Particle Percent After Purification


Particulate _ -Non.
Particulate




V2.1A1C290 to 302) 43 57

V16 . 1A1 (290 to 302) * 96 4

V12.Pfl(C17A). 67 33

V12.Pfl (C17A) + C150 * 100 0

V12.P£1 * 98 2

HBcl50(K77) 40 . 1 59.9
^

HBcl50(K77) + C * 100 0

HBcl50(K79) 59 41

HBcl50(K79) + C * 100 0

V2.Pfl + CF/HBC74-87 + C* 97 . 8 2.2

V2.Pfl + CF/HBC74-87 80 . 7 19.3
* C-terrainal cysteine-stabilized particles
Purified particles were assayed for the percentage of particles and then incubated in aqueous solution at 37°C as discussed before. The compositions were assayed for stability after fourteen days of incubation. The results of- this analysis are shown in Table 18, below.

Table 18

Particle Percent Particles Following Incut ations at 37CC (Days)

Zero 14
V12.Pfl * 98 96
V12.Pfl(C17A) 67 63
V12.Pfl * See the note to Table 17.
Fig. 8 shows the results of a SDS-PAGE analysis of the particles of Table 18 at days zero, 7 and 14 following incubation at 37°C. Results of a densitometric analysis of that a SDS-PAGE analysis are shown in Table 19, below.
Table 19

Particle Percent Full Length Monomer Following Incubation at 37°C
Days
Zero 7 1-4
V12.Pfl * 100 94 93
V12.Pfl{C17A) 100 • ; li- 1
V12 .-Pf 1 (C17A) +C150 * 100 sa 63
* See the note to Table 17.
The particles of Tables 18 and 19 and control particles of Example 16 with and without a C-terminal Cys residue were analyzed for immunogenicity in BALB/c mice via intraperitoneal injection using 20 p,g of the respective particles in phosphate buffered saline (pH 7.4) in the absence'of adjuvant, contrary to the results reported in Example 4. *Sera were analyzed two weeks after immunization using an ELISA

with HBc particles (Anti-HBc) or (NANP>5 synthetic
peptide [Anti-(NANP)n] as the solid phase capture
antigen. The results of this study are shown in
Table 20, below
Table 20

Particle End Point Titer

Anti-HBc Anti- (NANP)n
V12.Pfl(C17A) 10,240 0
V12.P.fl (C17A)+C150 * 10,240 ' 2,5~60
i
V12.Pfl * 10,240 10,240
HBcl50(K77) 40,960 0
HBcl50(K77)+C* 163,840 o
* See the note to, Table 17.
The data from this 'study are interpreted to mean that the C-terminal cysteine-stabilized particles are more stable immediately on production as well as after incubation at 37°C for various time periods. The stabilized particles also exhibit enhanced immunogenicity even in the absence of adjuvant. In addition, although particulate matter is present in the non-stabilised material such as V12.££l(C17A) , there are no monotneric chimeric proteins _after fourteen days of incubation and the material present does not induce antibodies toward the initially introduced heterologous B cell epitope sequence, here a malarial immunogen.

Example 24: Chimers Containing Beta-Amyloid Protein Epitope Sequ?nces
Antibodies to the 42 amino acid beta-amyloid precursor protein fragment have been proposed
.. as.. a_therapeutic and_ prophylactic, vaccine. Lor.
treating Alzheimer's Disease (REF) [Schenk et al. (Jul 8, 1999) Nature, 400(6740) : 116-117] . The C-terminus of that fragment contains a region that is extremely hydrophobia, and therefore potentially problematic for expression at the surface of chimeric HBc particles.
Therefore, in addition to a particle ' . containing the complete 42 amino acid sequence [VI6. P-Am(.1-4-2-) ] , three other particles were constructed that contain only the relatively hydrophilic regions: amino acid residues 1-17 [V16..J3-Am(l-17)], amino acid residues 22-32 [VI6. p-Am-('22-32)], and amino acid residues.1-32 [V16. P-Am1 (I-3-2) ] . Chimeric genes coding particles VIS. p-Am(l-17) and V16. p'-Am("22-3-2 ) were constructed by annealing complimentary oligonucleotides and inserting them • into the plasmid V16 that had previously been digested with; BcoRI and: Sac I.
p-Aia('l-17)-T
5' -AATTGATGCGGAATTTCGTCATGACAGCGGCTATGAGGTGCACCATC -AGAAACTGGAGCT SEQ ID NO: "9'6
p-Am(l-17)-B
5' - CCAGTTTCTGATGGTGCACCTCATAGCCGGTGTCATGACG -AAATTCCGCATC SEQ ID NO: 297

p-Am(22-32)-T
5'-AATTGAAGATGTCGGTTCTAACAAGGGGGCAATTATCGAGCT
SEQ ID NO: 298
5'-CGATAATTGCCCCCTTGTTAGAACCGACATCTTC
SEQ ID NO: 299
For chimeric genes containing residues 1-42 [V16. p-Am(l-42)] and 1-32 [Vi:6 . p-Am(l-32) ] , the oligonucleotides p-Amtl-32/42) -T and p-Am(l-42).-B or p-Am(l-32)-B were annealed, and then filled-in to make the fragment completely double stranded using 5 cycles of melting (94°C) and filling-in (72°C) . The reactions were performed in a total volume of 100 jaL using Vent polymerase (NEB) , dNTPs (250 jiM) and the annealed fragments (250 nM). Two microliters of these reaction products were then used as templates in two PCR reactions to prepare the fragments coding for residues 1>32 and 1-42, flanked by EcoRT and Sad restriction sites. (Note: Leu codon (CTG) is introduced by the primer "p-Am(L.+l-32/42)-5'-PCR" and precedes the first j3-Am amino acid in the following two constructs to restore EcoRI site for the cloning purposes).
Oligonucleotides for preparation of p-amyloid residue
1 -3"2 and 1-4Z fragments: -
p-Am(1-32/42)-T
5' -GCGGGAATTGATGCGGAATTTCGTCATGACAGCGGCTATGAGGTG-CACCATCAGAAACTGGTTTTCTTTGCCGAAGATGTCG
SEQ ID NO: 300

p-Am(l-42)-B
S' -GCGGAGCTCCGCTATGACAACCCCACCCACCArTAAGCCGAT-
AATTGCCCCCTTGTTAGAACCGACATCTTCGGCAAAGAAAA
SEQ ID NO: 301
p-Am(l-32)-B
5' -GCGGAGCTCGATAATTGCCCCCTTGTTAGAACCGACAT -CTTCGGCAAAGAAAA
.SEQ ID XC: 202
PCR Primers for residue 1-42 amplification
P-Am(L+l-32/42)-5'-PGR
5'-GGGGGAATTCTGGATGCGGAATTTCGTCATG
SEQ ID NO: 303
p-Am(l-42)-3'PCR
5'-GCGGAGCTCCGCTATGA
• SEQ ID NO: 304
PCR Primers for residue 1-32 amplification
p-Am(L+l-32/42)-5'-PCR
5' -GCGGGAATTCTGGATGCGGAATTTCGTCATG
SEQ IDr NO:- 305
P-Am(l-32)-3'PCR
5'-GCGGAGCTCGATAATTGC
SEQ ID NO:/ 306
Example 25: Influensa M2 Constructs
Recently, Neirynck et al. , (Oct 1399)
Nature Med., 5(10):1157-1163 and Wo 99/07829 reported the fusion of the 24 amino acid extracellular domain

of M2 to the N-terminus of full-length HBc particles
(H3cl83), lacking amino acid residues 1-4. A
schematic representation of that construct referred
to herein as IM2HBc is shown below in which the 24-
rner is .linked _to_ the, N-terminus _o_f._.HE.c.,_..-.
IM2HBc
MSLLTEVETPIRNEWGCRCNDSSD-HBc(5 -183) SEQ ID NO: 307
*V
In one illustrative preparation, the M2 epitope was inserted into the immunodominant loop of hepatitis B core and particles referred to as ICC-1475 were successfully expressed and purified using techniques discussed previously for such insertions and purifications. A mutated version of the M2 epitope, in which two cysteine residues at M2 native positions 17 and 19 were substituted by alanine residues, was also expressed in the immunodominant loop (ICC-1473) and the resulating particles purified. These two particles are illustrated schematically below.
ICC-1475
HBc (1-78) -GI-SLLTEVETPIRNEWGCRCNDSSD-EL-HBc (79-149)
SEQ ID NO: .308
ICC-1473
HBc (1-78) -GI-SLLTEVETPIRNEWGAKANDSSD-SL-HBc(79-149)-C
SEQ ID NO: 309

The ICC- 14 73 construct yielded
approximately 7-fold more purified particles when compared with the native sequence (ICC- 147 5) . It remains to be determined if the mutation of the _cyj3teine_j:esijdj4e^^
particles. However, epitopes delivered on the immunodominant loops of HBc are usually significantly more immunogenic as compared to when they are fused to other regions (including the N-terminus}, and resulting particles exhibit reduced anti-HBc-immunogenic ity.
Particles have; also, been prepared 'in which the M2 N- terminal 24-mer epitope was fused to the N-terminus of C- terminal truncated hepatitis B core particles. That construct (lCC-143 8) also contained the N-terminal pre-core sequence (SEQ ID NO:3lQ) . A similar construct was prepared: that contained a single cysteine residue at the end of the hybrid protein (ICC- 1492), in this case immediately after Val-149 of the HBc gene. These constructs are shown schematically below.
ICC- 1438
MG^SELl-EVETPrEISIEWGCRCNDSSDELLGWLWGr-HBc ( 2 - 14 9 ) SEQ ID NO: 310
ICO1492
MGISLLTEVETPIRNEWGCRCNDSSDELLGWLWGI-HBc (2-149) -C
It should be noted that to guard against translation initiation from the natural HBc initiator methionine, the codon for that residue was mutated to code for' ctn -isoleucine. residue. Residues contributed

by EcoRI (GI). and SacI (EL) restriction sites are underlined. The precore sequence iu recited between the underlined EL residues and "-HBc(2-149)".
Analysis by SDS-PAGE as discussed elsewhere
-herein,- showed- that upon preparation-, the-ICC-1438
monomer construct was unstable (Lane 2) as compared to the ICC-1492 (Lane 3), with HBc-149 (Lane 1), ICC-1475 (Lane 4) and ICC-1473 (Lane 5) serving as additional molecular weight controls on the SDS-PAGE gel in Figure 9. The instability of the ICC-1438 monomers was not evident using analytical gel filtration of particles.
Both ICC-1475 (Fig. V9, lane 4) and ICC-1473 (Fig. 9, lane 5) were expected to have slightly lower molecular weights than ICC-1438 and ICC-1492, because the former two contain the M2 epitope inserted directly into the immunodominant loop and. therefore lack the precore sequence (SEQ ID NO: 310) present in ICC-1438 and ICC-1498. As expected, ICC-1492 was larger than ICC-1475 and ICC-1473; however, ICC-1438, which is identical to ICC-1492 save the C-terminal cysteine residue, is clearly not larger than ICC-1475 and ICC-1473 due to an apparent cleavage.
A construct conataining a M2 N-terminal extracellular sequence as discussed above linked to the HBc KT-terminus (Domain I) or loop (Domain II) and also containing a M2 protein C-terminal sequence such as that of SEQ ID NO: 10 (see Table A) linked the loop (Domain II) or at the C-terminus (Domain IV) of HBc is also contemplated. Such a contemplated construct also contains at least one stabilizing C-terminal cysteine residue as discussed elssewhere herein.

Example 26: Comparative Immunogenir.ities in Monkeys
The comparative immunogenicity of the particles expressed by V12.Pf3.1, formulated with
either Seppic^1 J[SA-J2_p CSepp,i_c Inc,_,__P.aris;,_FranceL,
'Alhydrogel"* (Superfos, Denmark) as adjuvants, or unformulated (saline), was studied in Cynomolgus monkeys.
The Seppic™ ISA-720 formulation was prepared according to the manufacturers directions. Briefly, the ISA-720 and V12.PfS.l particles were/ mixed at 70:30 (w/w) 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 V12. Pf3.1 particles, which was shown to be physically bound to the Alhydrogel™ prior to immunization.
Groups of two monkeys (one male and one female) were immunized with 20 |jg V12.Pf3.1 particles as immunogenvia the intramuscular route. Animals were bled on days 0, 21, 42, 56 and 70-, and sera analyzed for titers of anti-NANP antibody using an ELI'SA. the results, shown in Table 15, below, demonstrate the extremely high immunogenicity of Vl2,.Pf3.i particles when formulated with Seppic™ ISA-720 versus Alhydrogel ""-formulated or unformulated material. The kinetics of the antibody response^ were more rapid when Seppic™' ISA-72,0 was used as the adjuvant, and the end-point titers were more than 100- and 1000-fold higher than for Alhydrogel™ and saline respectively.

Table 15

Adjuvant Antibody liters at Stated T. me (Days) !
:

Zero 21 42 | 56 ,0
1
- ----- Sa-l-ine - -Zero- - 4-0 24~0 — 1-7-2-e-Q -- - 640
Anhydrogel™ Zero 2,880 1920 11, 500 6400
Seppic™ ISA-720 Zero 81, 920 348, 160 25, 000, 000 1, 520, 000
Example 27: T Cell Activation
Mice were immunized twice with Vl2.Pf3.1 particles in Seppic™ Montanide™ ISA-720. Spleen cells were removed and stimulated in the presence of various peptides. 10s cells were incubated for 3 days in the presence of peptides: UTG (universal T epitope from P. falciparum; Seq IN NO: 120), p85-100 peptide corresponding to HBc 85-100 ,'NANP (B-cell epitope from V12.Pf3.1; NANPNVDP (NANP) 3 / SEQ ID NO: 22) in the presence of Staphylococcal enterotoxin B (SEB), or tissue culture medium (unstim). Interferon gamma production after 3 days was determined by ELISA.
The results shown in 'Table Iff, below, indicate that immunizing with V12.Pf3.1 induces T-cells that recognize the UTC component of the protein, and drives them to a Thl type response.

Table 16

900
1100 i

Immunogen IFN- 7 (pg/tnl) S.D.*

UTC 1600 750
. p85-100 350 3-0
NANPNVDP (NANP) 3 SEQ ID NO: 22
I 370 50
SEE 4300 MD**
unstim

* S.D. = Standard Deviation **- ND = Not Done
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.












WE CLAIM :
1. A vaccine or inoculum comprising an immunogenic effective amount of immunogenic particles dissolved or dispersed in a pharmaceutically acceptable diluent, wherein said immunogenic particles are comprised of recombinant chimeric HBc protein molecules that have a length of about 135 to about 515 amino acid residues and contain 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 71 to about 100 amino acid residues whose sequence includes at least the sequence of the residues of position 5 through position 75 of HBc and optionally includes a heterologous epitope containing up to about 30 amino acid residues peptide-bonded to one of HBc residues 1-4; (b) Domain II comprises about 5 to about 250 amino acid residues peptide-bonded to HBc residue 75 of Domain I in which (i) at least 4 residues in a sequence of HBc positions 76 through 85 are present peptide-bonded to one to about 245 amino acid residues that are heterologous to HBc and constitute a heterologous epitope or a heterologous linker residue for a conjugated epitope or (ii) the sequence of HBc at positions 76 to 85 is present free from heterologous residues; (c) Domain III is an HBc sequence from position 86 through position 135 peptide-bonded to residue 85 of Domain II; and d) Domain IV comprises (i) zero through fourteen residues of a HBc amino acid residue sequence from position 136 through 149 peptide-bonded to the residue of position 135 of Domain III, (ii) one to ten cysteine residues [C-terminal cysteine residue (s)] within about 30 residues from the C- terminus of the chimer molecule, and (iii) zero to about 100 amino acid residues in a sequence heterologous to HBc from position 150 to the C- terminus, with the proviso that Domain IV contain at least 6 amino acid residues including said one to ten cysteine residues of (ii), said recombinant chimeric HBc protein molecules having an amino acid residue sequence in which no more than about 5 percent of the amino acid residues are substituted in the HBc sequence.

2. The vaccine or inoculum as claimed in claim 1 that contains a heterologous linker residue for a conjugated epitope in Domain II and further includes a hapten linked to said heterologous linker residue.
3. The vaccine or inoculum as claimed in claim 1 wherein said recombinant chimeric HBc protein molecules have a length of about 175 to about 240 amino acid residues and contain 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 1 through position 75 of HBc; (b) Domain II comprises about 5 to about 55 amino acid residues peptide-bonded to HBc residue 75 of Domain I in which at least 4 residues in a sequence of HBc positions 76 through 85 are present peptide-bonded to 6 to about 50 amino acid residues that are heterologous to HBc and constitute a heterologous epitope; (c) Domain III is an HBc sequence from position 86 through position 135 peptide-bonded to residue 85 of Domain II; and d) Domain IV comprises (i) 5 through fourteen residues of a HBc amino acid residue sequence from position 136 through 149 peptide-bonded to the residue of position 135 of Domain III, and (ii) zero to about 50 amino acid residues in a sequence heterologous to HBc from position 150 to the C-terminus, said particles exhibiting a ratio of absorbance at 280 nm to 260 nm of about 1.4 to about 1.6.
4. The vaccine or inoculum as claimed in claim 1 wherein said recombinant chimeric HBc protein molecule particles are present in an attenuated strain of S. typhi, S. typhimurium or a S. typhimurium-E. coli hybrid.
5. The vaccine or inoculum as claimed in claim 1 wherein said recombinant chimeric HBc protein molecule particles are present in plant tissue.
6. The vaccine or inoculum as claimed in claim 1 that further includes an adjuvant.

7. The vaccine or inoculum as claimed in claim 6 wherein said adjuvant is alum.
8. The vaccine or inoculum as claimed in claim 6 wherein said adjuvant is a small molecule selected from the group consisting of a muramyl dipeptide, 7-substituted-8-oxo-or 8-sulfo-guanosine derivative, monophosphoryl lipid A, aluminum or calcium salts.
9. The vaccine or inoculum as claimed in claim 6 wherein said adjuvant is an oil that is emulsified with said immunogenic particles and said pharmaceutically acceptable diluent.

10. The vaccine or inoculum as claimed in claim 9 wherein said emulsion is an water-in-oil emulsion having a water phase and an oil phase.
11. The vaccine or inoculum as claimed in claim 9 wherein said emulsion is an oil-in-water emulsion having a water phase and an oil phase.
12. The vaccine or inoculum as claimed in claim 9 wherein the oil phase of said emulsion comprises squalene.
13. The vaccine or inoculum as claimed in claim 9 wherein the oil phase of said emulsion comprises squalane.
14. The vaccine or inoculum as claimed in claim 9 wherein the water and oil phases of said emulsion are emulsified by an emulsifying agent that is a sorbitan or mannide C12-C24 fatty acid ester.
15. The vaccine or inoculum as claimed in claim 14 wherein said emulsifying agent is a mannide C12-C24 fatty acid ester.

16. The vaccine or inoculum as claimed in claim 15 wherein said C12-C24 fatty acid of said mannide C12-C24 fatty acid ester is oleic acid.

Documents:

6137-DELNP-2006-Abstract-(27-08-2010).pdf

6137-delnp-2006-abstract.pdf

6137-DELNP-2006-Claims-(07-02-2011).pdf

6137-DELNP-2006-Claims-(23-02-2011).pdf

6137-DELNP-2006-Claims-(27-08-2010).pdf

6137-DELNP-2006-Claims-(29-09-2010).pdf

6137-delnp-2006-claims.pdf

6137-delnp-2006-Correspondence-Others-(01-11-2010).pdf

6137-DELNP-2006-Correspondence-Others-(07-02-2011).pdf

6137-DELNP-2006-Correspondence-Others-(23-02-2011).pdf

6137-DELNP-2006-Correspondence-Others-(27-08-2010).pdf

6137-DELNP-2006-Correspondence-Others-(27-09-2010).pdf

6137-delnp-2006-correspondence-others-1.pdf

6137-delnp-2006-correspondence-others.pdf

6137-delnp-2006-description (complete).pdf

6137-delnp-2006-drawings.pdf

6137-delnp-2006-Form-1-(01-11-2010).pdf

6137-DELNP-2006-Form-1-(27-08-2010).pdf

6137-delnp-2006-form-1.pdf

6137-DELNP-2006-Form-13-(23-02-2011).pdf

6137-DELNP-2006-Form-13-(29-09-2010).pdf

6137-delnp-2006-form-18.pdf

6137-DELNP-2006-Form-2-(27-08-2010).pdf

6137-delnp-2006-form-2.pdf

6137-DELNP-2006-Form-3-(27-09-2010).pdf

6137-delnp-2006-form-3.pdf

6137-delnp-2006-form-5.pdf

6137-delnp-2006-pct-304.pdf

6137-delnp-2006-Petition 137-(01-11-2010).pdf

6137-DELNP-2006-Petition-137-(29-09-2010).pdf


Patent Number 246663
Indian Patent Application Number 6137/DELNP/2006
PG Journal Number 10/2011
Publication Date 11-Mar-2011
Grant Date 09-Mar-2011
Date of Filing 19-Oct-2006
Name of Patentee CELLDEX THERAPEUTICS LIMITED,FORMERLY KNOWN AS LORANTIS,LIMITED
Applicant Address 410 CAMBRIDGE SCIENCE PARK CAMBRIDGE CB4,OPE UNITED KINGDOM
Inventors:
# Inventor's Name Inventor's Address
1 BIRKETT,ASHLEY 210,SOUTH STREET,# 7-2,BOSTON MASSACHUSETTS 02111 UNITED STATES OF AMERICA
PCT International Classification Number C07K 14/02
PCT International Application Number PCT/US01/41759
PCT International Filing date 2001-08-16
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/226,867 2000-08-22 U.S.A.
2 60/225,843 2000-08-16 U.S.A.
3 09/930,915 2001-08-15 U.S.A.