WO2003102165A2 - IMMUNOGENIC HBc CHIMER PARTICLES STABILIZED WITH AN N-TERMINAL CYSTEINE - Google Patents

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 B cell or T cell epitope, or both, such as a B cell epitope polypeptide of the influenza M2 protein and a T cell epitope of the influenza NP protein. An immunogenic epitope is peptide-bonded to one or more of the N-terminus, in the immunogenic loop or at the C-terminus of HBc, whereas the enhanced stability of self-assembled particles is obtained by the presence of at least one heterologous cysteine residue near the amino-terminus of the chimer molecule. Methods of making and using the chimers are also disclosed.

Description

IMMUNOGENIC HBc CHIMER PARTICLES STABILIZED WITH AN N-TERMINAL CYSTEINE

Description

CROSS-REFERENCE TO RELATED APPLICATION

This is a continuation-in-part of application Serial No.10/274, 616 filed October 21, 2002 as a continuation-in-part of application Serial No.10/080, 299 filed February 21, 2002, and as a continuation-in-part of application Serial No.10/082, 014 filed February 22, 2002, that were each a continuation-in-part of application Serial No. 09/930,915, filed August 15, 2001.

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 via an N-terminal cysteine residue and the display of an immunogenic epitope, and more particularly to an immunogen and vaccine useful in prevention of influenza infection by influenza A virus .

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 that infects mammals, as compared to a virus that infects an avian host, unless the discussion refers to a specific example of a non-mammalian virus.

The nucleocapsid or core of the mammalian hepatitis B virus (HBV or hepadnavirus) contains a sequence of 183 or 185 amino acid residues, depending on viral subtype, whereas the duck virus capsid contains 262 amino acid residues. Hepatitis B core protein monomers of the several hepadnaviridae self- assemble in infected cells into stable aggregates known as hepatitis B core protein particles (HBc particles) . Two three-dimensional structures are reported for C-terminally truncated HBc particles. 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 low resolution structure of HBc particles reported by Conway et al . , (1997) and Bottcher et al . , (1997) has been confirmed by a 3.3 A resolution crystal structure of the T=4 particles reported by Wynne at al., (1999) Mol . Cell , 3 (6) : 70-80.

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, for example, U.S. Patents No. 4,818,527, No 4,882,145 and No. 5,143,726. A particularly useful application of this carrier is its ability to present foreign or heterologous B cell epitopes at the site of the immunodominant loop that is present at about residue positions 70-90, and more usually recited as about positions 75 through 85 from the amino-terminus (N- terminus) of the protein. Clarke et al . (1991) F. Brown et al . eds., Vaccines 91, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp.313-318.

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.

HBcAg is a particulate protein derived from the hepatitis B virus that has been proposed as a carrier for heterologous epitopes. The relative immunogenicity of HBsAg (HBs) has been compared with HBcAg (HBc) , and the ability of each to evoke immune responses in different genetic backgrounds [Milich et al . , Science, (1986) 234 (4782) :1398-1401] . These data emphasize the higher immunogenicity of HBc relative to HBs, and the universal responsiveness to HBc, irrespective of genetic background.

For example, HBc is more than 300 times more immunogenic than HBs in BALB/c mice; and, although both B10.S and B10.M mice are non-responders to HBs, every strain tested is responsive to HBc. These results re- emphasize the suitability of HBc as a vaccine carrier and specifically, its superiority over HBs, hence the selection of HBc as opposed to HBs to carry heterologous epitopes. These facets of HBc are thought to be important in influenza vaccine development, because they address issues of genetic restriction and inadequate antibody titers .

Another advantage of the HBc carrier is the fact that it does not require complex adjuvants for efficacy. This is due to the high inherent immunogenicity of the particle. A comparison of the immunogenicity of HBc-P. berghei particles showed that alum, which is approved for human use, was more effective than either IFA or CFA [Schόdel et al . , J. Exp . Med . , (1994) 180 (3) : 1037-46] . The importance of this observation is highlighted by toxicity problems associated with newer, more complex adjuvants as was recently noted in clinical trials of SKB's candidate malaria vaccine [Stoute et al . , N. Engl . J. Med. , [1997] 336 (2) :86-91] .

In an application as a vaccine carrier moiety, it may be 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 144, can self-assemble, whereas deletions beyond residue 139 abrogate capsid assembly [Birnbaum et al., (1990) J. Virl . , 64:3319-30].

Zlotnick et al . , (1997) Proc. Natl . 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 position 1 through 149 in which the cysteines at positions 48, 61 and 107 were each replaced by alanines and in which a cysteine residue was added at the C-terminus (position 150) . That C-terminal mercaptan was used for linkage to a gold atom cluster for labeling in electron microscopy.

More recently, Metzger et al . (1998) J^". Gen . Viol . , 79:587-590 reported that the proline at position 138 (Pro-138 or P138) of the human viral sequence is required for particle formation. Those authors also reported that assembly capability of 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 [Gallina et al . (1989) J. Virol . , 63:4645-4652; Inada, et al . (1989) Virus Res . , 14:27-48], evident by variability in particle sizes and the presence of particle fragments in purified preparations [Maassen et al . , (1994) Arch . Virol . , 135:131-142]. Thus, prior to the report of Metzger et al . , above, Pumpens et al . , (1995) Intervirology, 38:63-74 summarized the literature reports by stating that the carboxy- terminal border for HBc sequences required for self- assembly was located between 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.

Recombinantly-produced hybrid HBc particles bearing internal insertions (referred to in the art as HBc chimeric particles or HBc chimers) containing various inserted polypeptide sequences have been prepared by heterologous expression in a wide variety of organisms, including E. coli , B . subtilis , Vaccinia, Salmonella typhimurium, Saccharomyces cerevisiae . See, for example Pumpens et al . (1995) Intervirology, 38:63-74, and the citations therein that note the work of several research groups . Native HBc particles have also been produced in plants [Tsuda et al., (1998) Vox Sang, 74 (3) : 148-155] .

Such HBc chimers often appear to have a less ordered structure, when analyzed by electron microscopy, compared to particles that lack heterologous epitopes [Schδdel 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 they fall apart during purification to such an extent that they are unrecoverable or they show very poor stability characteristics, making them problematic for vaccine development .

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

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

U.S. Patent No. 5,990,085 describes two fusion proteins formed from an antigenic bovine inhibin peptide fused into (i) the immunogenic loop between residues 78 and 79 and (ii) after residue 144 of carboxy-terminal truncated HBc. Expressed fusion proteins were said to induce the production of anti- inhibin antibodies when administered in a host animal. The titers thirty days after immunization reported in that patent are relatively low, being 1:3000-15,000 for the fusion protein with the loop insertion and 1:100-125 for the insertion after residue 144.

U.S. Patent No. 6,231,864 teaches the preparation and use of a strategically modified hepatitis B core protein that is linked to a hapten. The modified core protein contains an insert of one to about 40 residues in length that contains a chemically-reactive amino acid residue to which the hapten is pendently linked.

Recently published WO 01/27281 teaches that the immune response to HBc can be changed from a Thl response to a Th2 response by the presence or absence, respectively, of the C-terminal cysteine- containing sequence of the native molecule. That disclosure also opines that disulfide formation by C-terminal cysteines could help to stabilize the particles. The presence of several residues the native HBc sequence immediately upstream of the C-terminal cysteine was said to be preferred, but not required. One such alternative that might be used to replace a truncated C-terminal HBc sequence was said to include a C-terminal cysteine and an optional sequence that defines an epitope from other than HBc.

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

Published PCT applications corresponding to PCT/US01/25625 (WO 02/13765 A2 published February 21, 2002) and PCT/USOl/41759 (WO 02/14478 A2 published February 21, 2002) of the present inventor teach that stabilization of C-terminally truncated HBc particles can be achieved through the use of one or more added cysteine residues in the chimer proteins from which the particles are assembled. Those added cysteine residues are taught to be at or near the C-terminus of the chimeric protein.

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

The above four published PCT applications appear to contain teachings that can be used to overcome over come the potential problems disclosed by Ulrich et al . As disclosed hereinafter, the present invention provides another HBc chimer that provides unexpectedly high titers of antibodies against influenza, and in one aspect also provides a solution to the problems of HBc chimer stability as well as the substantial absence of nucleic acid binding ability of the construct. In addition, a contemplated recombinant chimer exhibits reducedantigenicity toward preexisting anti-HBc antibodies .

The above particle instability findings related to N-terminal truncated HBc chimer molecules notwithstanding, Neirynck et al . , (October 1999) 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 at residue 5 to full length HBc.

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

Others have reported that in wild type hepadnaviral core antigens a cysteine residue upstream of the HBcAg start codon is directly involved in the prevention of particle formation [Schodel et al . (Jan. 15, 1993) J^". Biol . Chem. , 268(2) :1332-1337; Wasenauer et al . (Mar. 1993) J^". Virol . , 67 (3) : 1315-1322; and Nassal et al . (Jul . 1993) J^". Virol . , 67 (7) :4307-4315] . All three groups reported that in wild type HBeAg, the cysteine residue at position -7 of the pre-core sequence, which is present when the core gene is translated from an upstream initiator methionine at position - 30, is responsible for preventing particle formation and therefore facilitating the transition from particulate HBcAg to secreted, non-particulate HBeAg.

Based upon the above three publications, one would expect the inclusion of one or more cysteine residues at a position prior to the initiator methionine of HBc; i.e., at a residue position of less than one relative to the N-terminus of the sequence of SEQ ID N0:1, to actually destabilize hybrid particles rather than stabilize them. As will be seen from the discussion that follows, the present invention provides results that are contrary to those expectations. BRIEF SUMMARY OF 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. A recombinant chimer hepatitis B core (HBc) protein molecule up to about 515 amino acid residues in length is contemplated. That chimer molecule

(a) contains an HBc sequence of at least about 125 of the N-terminal 150 amino acid residues of the HBc molecule that includes (i) the HBc sequence of residue positions 5 through about 75 and about 85 through about 140, (ii) a peptide-bonded heterologous immunogenic epitope at one or more of the N-terminus, in the HBc immunodominant loop or the C-terminus of the chimer, or (iii) a heterologous linker residue for a conjugated epitope present in the HBc immunodominant loop,

(b) contains one to three cysteine residues at an amino acid position of the chimer molecule corresponding to amino acid position -20 to about +1 from the N-terminus of the HBc sequence of SEQ ID NO:l [N-terminal cysteine residue (s) ] in a sequence other than that of the HBc precore sequence and zero to about three cysteine residues toward the C- terminus of the molecule from the C-terminal residue of the HBc sequence and within about 30 residues from the C-terminus of the chimer molecule [C-terminal cysteine residue (s) ] .

That chimer molecule (i) contains no more than about 20 percent conservatively substituted amino acid residues in the HBc sequence, (ii) self- assembles into particles that are substantially free of binding to nucleic acids on expression in a host cell . The particles are more stable than are particles formed from otherwise identical HBc chimer molecules that are free of any above-mentioned C-terminal cysteine residue (s) and (i) lack the N-terminal cysteine residue (s) or (ii) in which an N-terminal cysteine residue (s) present in a contemplated chimer molecule is (are) replaced by another residue.

A preferred recombinant hepatitis B virus core (HBc) protein chimer molecule has a length of about 135 to about 515 amino acid residues that contains four peptide-linked amino acid residue sequence domains from the N-terminus that are denominated Domains I, II, III and IV.

Domain I of that chimer molecule comprises about 71 to about 110 amino acid residues whose sequence includes (i) at least the sequence of the residues of position 5 through position 75 of HBc, (ii) one to three cysteine residues at an amino acid position of the chimer molecule corresponding to amino acid position -20 to about +1 from the N- terminus of the HBc sequence of SEQ ID N0:1 [N- terminal cysteine residue (s) ] in a sequence other than that of the HBc precore sequence, and (iii) an optional heterologous immunogenic epitope containing up to about 30 amino acid residues peptide-bonded to one of HBc residues 2-4.

Domain II of that chimer molecule comprises about 5 to about 250 amino acid residues peptide- bonded to HBc residue 75 of Domain I in which (i) zero to all residues in the sequence of HBc positions 76 to 85 are present peptide-bonded to (ii) an optionally present sequence of one to about 245 amino acid residues that are heterologous to HBc and constitute a heterologous immunogenic epitope or a heterologous linker residue for a conjugated epitope.

Chimer Domain III is an HBc sequence from position 86 through position 135 peptide-bonded to residue 85 of Domain II.

Chimer molecule Domain IV comprises (i) five through fourteen residues of an HBc amino acid residue sequence from position 136 through 149 peptide-bonded to the residue of position 135 of Domain III, (ii) zero to three cysteine residues [C- terminal cysteine residue (s)] within about 30 residues from the C-terminus of the chimer molecule, and (iii) zero to about 100 amino acid residues in an immunogenic sequence heterologous to HBc from position 150 to the C-terminus.

The chimer molecules (i) have an amino acid residue sequence in which no more than about 10 percent of the amino acid residues are substituted in the HBc sequence of the chimer and (ii) self-assemble into particles on expression in a host cell. The particles are substantially free of binding to nucleic acids and are more stable than are particles formed from otherwise identical HBc chimer molecules that are free of any above-mentioned C-terminal cysteine residue (s) and (i) lack the N-terminal cysteine residue (s) or (ii) in which an N-terminal cysteine residue (s) present in a contemplated chimer molecule is (are) replaced by another residue.

It is preferred that the HBc sequence of Domain I include the residues of position 5 through position 75 along plus at least an N-terminal cysteine residue. It is further preferred that a contemplated immunogen contain not only an N-terminal cysteine residue, but also contain one cysteine residue within Domain IV as noted above that is alone or in an amino acid residue sequence heterologous to that of HBc from position 150 to the C-terminus.

The before-mentioned self-assembled chimer molecule particles are a particularly contemplated embodiment of this invention. A particularly preferred embodiment of the present invention contemplates an immunogen for inducing antibodies to influenza A that is a self-assembled particle comprised of recombinant hepatitis B virus core (HBc) chimer protein molecules. Each of those molecules has a length of about 150 to about 325 amino acid residues and contains four peptide-linked amino acid residue sequence domains from the N-terminus that are denominated Domains I, II, III and IV.

The first domain, Domain I, comprises about 75 to about 110 amino acid residues. The sequence of this Domain includes at least the sequence of the residues of position 4 through position 75 of HBc. One to three cysteine residues are also present at a position in the chimer molecule of about one to about -20 relative to the N-terminus of HBc of SEQ ID NO:l [N-terminal cysteine residue (s) ] . The one or more N- terminal cysteine residues are present within a sequence other than that of the pre-core sequence of HBc . Domain I can further include a sequence of about 6 to about 24 residues of an influenza A M2 polypeptide X₁X₂X3X4X5X₆X7X8TXιo^χnR 13^x14^x15^x16- ^x17^x18^x19^x20^x21^x22^x23^x24 ^{of SE0}- ^ID NO : 9 that are peptide-bonded to or within about 15 residues of the N-terminus of the HBc sequence, and whose subscripted X residues are defined hereinafter, as well as one or more or HBc residues 1-4.

The second domain, Domain II, comprises about 10 to about 60 amino acid residues peptide- bonded to residue 75 of Domain I of which (i) zero to all residues in the sequence of HBc positions 76 through 85 are present peptide-bonded to (ii) an optional sequence of about 6 to about 48 residues that constitute one or more repeats of the above influenza A M2 polypeptide of SEQ ID NO: 9.

The third domain, Domain III, is an HBc sequence from position 86 through position 135 peptide-bonded to residue 85.

The fourth domain, Domain IV, comprises (i) the residues of positions 136-140 plus up to nine residues of an HBc amino acid residue sequence from position 141 through 149 peptide-bonded to the residue of position 135 of Domain III, (ii) zero to three cysteine residues, (iii) fewer than three arginine or lysine residues, or mixtures thereof adjacent to each other, and (iv) up to about 100 amino acid residues in a sequence heterologous to HBc from position 164 to the C-terminus.

A contemplated chimer molecule (i) contains no more than 10 percent conservatively substituted amino acid residues in the HBc sequence, (ii) self- assembles into particles that are substantially free of binding to nucleic acids on expression in a host cell, and those particles are more stable on formation than are particles formed from an otherwise identical HBc chimer that lacks said N-terminal cysteine residue (s) or in which an N-terminal cysteine residue present in the chimer molecule is replaced by another residue. It is preferred that the HBc sequence of Domain I include the residues of position 4 through position 75 along plus at least an N-terminal cysteine residue. It is further preferred that a contemplated immunogen contain one cysteine residue within Domain IV alone or in an amino acid residue sequence heterologous to that of HBc from position 164 to the C-terminus. It is particularly preferred that that heterologous sequence comprise a T cell epitope of influenza A.

Another embodiment comprises an inoculum or vaccine that comprises an above HBc chimer particle that is dissolved or dispersed in a pharmaceutically acceptable diluent composition that typically also contains water. When administered in an immunogenic effective amount to an animal such as a mammal or bird, an inoculum induces antibodies that immunoreact specifically with the chimer particle. The antibodies so induced also immunoreact specifically with (bind to) the N-terminal portion of the M2 protein, when such a M2-related sequence is present in the chimer .

The present invention has several benefits and advantages.

A particular benefit of the invention is that its use as a vaccine provides extraordinary antibody titers against influenza A.

An advantage of the invention is that those very high antibody titers have been produced with the aid of an adjuvant approved for use in humans.

Another benefit of the invention is that the recombinant immunogen can be prepared easily and in large quantities using well-known cell culture techniques to grow transformed host cells. Another advantage of the invention is that the immunogen is easily prepared using well-known recombinant techniques .

Yet another benefit of the invention is that a preferred immunogen exhibits greater stability at elevated temperatures than to other HBc chimers .

Yet another advantage of the invention is that a contemplated immunogen is substantially free of nucleic acids.

Still further benefits and advantages will be apparent to the worker of ordinary skill from the disclosure that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings forming a portion of this disclosure :

Fig.l, shown in two panels as Fig. IA and Fig. IB, provides an alignment of six published sequences for mammalian HBc proteins from six viruses. The first (SEQ ID NO:l), human viral sequence is of the ayw subtype and was published in Galibert et al . (1983) Nature, 281:646-650; the second human viral sequence (SEQ ID NO: 2) , of the adw subtype, was published by Ono et al . (1983) Nucleic Acids Res . , 11(6): 1747-1757; the third human viral^' sequence (SEQ ID NO:3), is of the adw2 subtype and was published by Valenzuela et al . , Animal Virus Genetics, Field et al . eds., Academic Press, New York (1980) pages 57-70; the fourth human viral sequence (SEQ ID NO: 4) , is of the adyw subtype that was published by Pasek et al . (1979) Nature, 282:575-579; the fifth sequence (SEQ ID NO: 5), is that of the woodchuck virus that was published by Galibert et al . (1982) J^". Virol . , 41:51-65; and the sixth mammalian sequence, (SEQ ID NO:6), is that of the ground squirrel that was published by Seeger et al . (1984) J. Virol . ,51:367-375.

Fig. 2 shows the modifications made to commercial plasmid vector pKK223-3 in the preparation of plasmid vector pKK223-3N used herein for preparation of recombinant HBc chimers. The modified sequence (SEQ ID NO: 7) is shown below the sequence of the commercially available vector (SEQ ID NO:8). The bases of the added Ncol site are shown in lower case letters and the added bases are shown with double underlines, whereas the deleted bases are shown as dashes. The two restriction sites present in this segment of the sequence (Ncol and Hindlll) are indicated.

Fig. 3 is an analytical size exclusion chromatography elution profile for ICC-1603 particles in which absorbance at 280 nm is shown on the ordinate and time in seconds is shown on the abscissa.

Fig. 4 is an analytical size exclusion chromatography elution profile for ICC-1590 particles as discussed for Fig. 3.

Fig. 5 is an analytical size exclusion chromatography elution profile for ICC-1560 particles as discussed for Fig. 3.

Fig. 6 is an analytical size exclusion chromatography elution profile for ICC-1605 particles as discussed for Fig. 3.

Fig. 7 is an analytical size exclusion chromatography elution profile for ICC-1604 particles as discussed for Fig. 3. Fig. 8 is an analytical size exclusion chromatography elution profile for ICC-1438 particles as discussed for Fig. 3.

Fig. 9 is an analytical size exclusion chromatography elution profile for ICC-1492 particles as discussed for Fig. 3.

Fig 10 is a photograph of an SDS-PAGE analysis under reducing conditions following particle preparation that shows the ICC-1438 monomer construct was unstable after aging (Lane 2) as compared to the ICC-1492 construct (Lane 3) , with HBc-149 (Lane 1) , ICC-1475 (Lane 4) and ICC-1473 (Lane 5) serving as additional molecular weight controls.

Fig. 11, taken from PCT/US01/25625 (ICC- 102.2) illustrates a reaction scheme (Scheme 1) that shows two reaction sequences for (I) forming an activated carrier for pendently linking a hapten to a chimeric hepatitis B core protein (sm-HBc) particle using sulpho-succinimidyl 4- (N-maleimidomethyl) - cyclohexane 1-carboxylate (sulpho-SMCC) , and then (II) linking a sulfhydryl-terminated (cysteine- terminated) hapten to the activated carrier to form a conjugate particle. 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 . Definitions

Numerals utilized in conjunction with HBc chimers indicate the position in the HBc ayw amino acid residue sequence of SEQ ID NO : 1 at which one or more residues has been added to or deleted from 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 relative to the sequence numbers of SEQ ID NO:l.

The term "antibody" refers to a molecule that is a member of a family of glycosylated proteins called immunoglobulins, which can specifically bind to an antigen.

The word "antigen" has been used historically to designate an entity that is bound by an antibody or receptor, and also to designate the entity that induces the production of the antibody. More current usage limits the meaning of antigen to that entity bound by an antibody or receptor, whereas the word "immunogen" is used for the entity that induces antibody production or binds to the receptor. Where an entity discussed herein is both immunogenic and antigenic, reference to it as either an immunogen or antigen is typically made according to its intended utility.

"Antigenic determinant" refers to the actual structural portion of the antigen that is immunologically bound by an antibody combining site or T-cell receptor. The term is also used interchangeably with "epitope" .

The word "conjugate" as used herein refers to a hapten operatively linked to a carrier protein, as through an amino acid residue side chain.

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 N0:1). The polypeptide portions of at least chimer Domains I, II and III are believed to exist in a similar tertiary form to the corresponding sequences of naturally occurring HBcAg.

As used herein, the term "fusion protein" designates a polypeptide that contains at least two amino acid residue sequences not normally found linked together in nature that are operatively linked together end-to-end (head-to-tail) by a peptide bond between their respective carboxy- and amino-terminal amino acid residues. The fusion proteins of the present invention are HBc chimer molecules that induce the production of antibodies that immunoreact with a polypeptide that corresponds in amino acid residue sequence to the polypeptide portion of the fusion protein. The phrase "hepatitis B" as used here refers in its broadest context to any member of the family of mammalian hepadnaviridae, as discussed before.

The words "polypeptide" and "peptide" are used interchangeably throughout the specification and designate a linear series of amino acid residues connected one to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent amino acids. Polypeptides can be a variety of lengths, either in their neutral (uncharged) forms or in forms that are salts. It is well understood in the art that amino acid residue sequences contain acidic and basic groups, and that the particular ionization state exhibited by the peptide is dependent on the pH value of the surrounding medium when the peptide is in solution, or that of the medium from which it was obtained if the peptide is in solid form. Thus, "polypeptide" or its equivalent terms is intended to include the appropriate amino acid residue sequence referenced. A peptide or polypeptide is always shown herein from left to right and in the direction from amino-terminus (N-terminus) to carboxy-terminus (C- terminus) .

The term "residue" is used interchangeably with the phrase amino acid residue. All amino acid residues identified herein are in the natural or L- configuration. In keeping with standard polypeptide nomenclature, [J. Biol . Chem. , 243, 3557-59 (1969)], abbreviations for amino acid residues are as shown in the following Table of Correspondence. TABLE OF CORRESPONDENCE

1-Letter 3 -Letter AMINO ACID

Y Tyr L-tyrosine

G Gly glycine

F Phe L-phenylalanine

M Met L-methionine

A Ala L-alanine

S Ser L-serine

I lie 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

Z Glx L-glutamic acid or

L-glutamine

W Trp L- ryptophan

R Arg L-arginine

D Asp L-aspartic acid

N Asn L-asparagine

B Asx L-aspartic acid or

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, or a linker for attachment of an immunogenic B cell or T cell epitope, (b) exhibit enhanced stability on formation when present in a self-assembled particle via an added cysteine residue near the N-terminus, as well as exhibit (c) a substantial absence of nucleic acid binding as a self-assembled particle. A contemplated HBc chimer is truncated at the C-terminus of the molecule relative to a native HBc molecule.

Thus, the chimeric protein displays one or more heterologous immunogenic epitopes at the N-terminus, in the HBc immunogenic (immunodominant) loop or C-terminus, or a linker for such a B cell or T cell epitope in the immunogenic loop. The chimeric protein contains a cysteine residue at or near the N-terminus that confers enhanced stability on formation to the self-assembled particles. A preferred chimeric protein is sufficiently free of arginine and or lysine residues downstream of (toward the carboxy-terminus from) HBc residue position 149 so that the self-assembled particles are substantially free of nucleic acid binding.

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, 281:646- 650] that is shown in SEQ ID N0:1. It is to be understood, however, that in view of the great similarity between the mammalian hepadnavirus capsid protein sequences and similar particle formation exhibited by those proteins, which are well-known to skilled workers, a discussion regarding human HBc subtype ayw is also applicable to subtype adw, as well as the woodchuck and ground squirrel proteins . As a consequence of those great similarities, HBc sequences are recited generally herein as a "HBc" sequence, unless otherwise stated. In one embodiment, a contemplated HBc chimer is up to about 515 residues in length and contains

(a) an HBc sequence of at least about 125 of the N-terminal 150 amino acid residues of the HBc molecule that includes (i) the HBc sequence of residue positions 5 through about 75 and about 85 through about 140, (ii) a peptide-bonded heterologous immunogenic epitope at one or more of the N-terminus, in the HBc immunodominant loop or the C-terminus of the chimer, or (iii) a heterologous linker residue for a conjugated epitope present in the HBc immunodominant loop, and

(b) one to three cysteine residues at an amino acid position of the chimer molecule corresponding to amino acid position -20 to about +1 from the N-terminus of the HBc sequence of SEQ ID NO:l [N-terminal cysteine residue (s) ] in a sequence other than that of the HBc precore sequence and zero to about three cysteine residues toward the C-terminus of the molecule from the C-terminal residue of the HBc sequence and within about 30 residues from the C-terminus of the chimer molecule [C-terminal cysteine residue (s) ] .

That chimer molecule (i) contains no more than about 20 percent conservatively substituted amino acid residues in the HBc sequence, (ii) self- assembles into particles that are substantially free of binding to nucleic acids on expression in a host cell. The particles are more stable on formation than are particles formed from otherwise identical HBc chimer molecules that are free of any above- mentioned C-terminal cysteine residue (s) and (i) lack the N-terminal cysteine residue (s) or (ii) in which an N-terminal cysteine residue (s) present in a contemplated chimer molecule is (are) replaced by another residue .

A contemplated chimer molecule contains a cysteine residue at a position of about -20 to about +1 relative to the N-terminus of HBc as is illustrated in Fig. 1 and SEQ ID NO:l. The concept of a negative amino acid position is usually associated with a leader sequence such as the precore sequence of HBc. That concept is used similarly here in that one can simply align a given chimer molecule sequence with that of SEQ ID N0:1 to determine the position of the chimer that corresponds to that of the starting methionine residue of position +1 of HBc. Inasmuch as amino acid residue sequences are normally shown from left to right and in the direction from N-terminus to C-terminus, any aligned chimer molecule residue to the left of the position occupied by, the HBc start methionine has a negative position. A contemplated cysteine residue can occur at a position about twenty residues to the left of the aligned start methionine of HBc to the position corresponding to that start methionine .

In one aspect, a preferred HBc chimer has a sequence of about 135 to about 515 L-α-amino acid residues and contains four serially peptide-linked domains; i.e., Domains I, II, III and IV. Those four domains are linked together in the same manner as are native proteins; i.e., they are peptide-bonded to each other, as compared to polypeptides that contain residues of other than α-amino acids and therefore cannot form peptide bonds, those that contain D-amino acid residues, or oligopeptide conjugates in which two or more polypeptides are operatively linked through an amino acid residue side chain. A contemplated chimeric HBc protein can therefore be prepared by expression using the usual methods of recombinant technology.

Domain I of that chimer molecule comprises about 71 to about 110 amino acid residues whose sequence includes (i) at least the sequence of the residues of position 5 through position 75 of HBc, (ii) one to three cysteine residues at an amino acid position of the chimer molecule corresponding to amino acid position -20 to about +1, and preferably amino acid position -14 to about +1, from the N-terminus of the HBc sequence of SEQ ID NO:l [N-terminal cysteine residue (s) ] in a sequence other than that of the HBc precore sequence, and (iii) an optional heterologous immunogenic epitope containing up to about 30 amino acid residues peptide-bonded to one of HBc residues 2-4. That heterologous immunogenic sequence, when present, is typically an epitope used to induce an immune response for a vaccine or inoculum.

Domain II of that chimer molecule comprises about 5 to about 250 amino acid residues peptide- bonded to HBc residue 75 of Domain I in which (i) zero to all residues in the sequence of HBc positions 76 to 85, and preferably at least four HBc residues, are present peptide-bonded to (ii) an optionally present sequence of one to about 245 amino acid residues that are heterologous to HBc and constitute a heterologous immunogenic epitope or a heterologous linker residue for a conjugated epitope. It is particularly preferred that the sequence of 10 residues of positions 76 trough 85 (position 76-85 sequence) be present, but interrupted by one to about 245 residues of the heterologous linker or heterologous epitope .

Domain III is an HBc sequence from position 86 through position 135 peptide-bonded to residue 85 of Domain II.

Chimer molecule Domain IV comprises (i) five through fourteen residues of an HBc amino acid residue sequence from position 136 through 149 peptide-bonded to the residue of position 135 of Domain III, (ii) zero to three cysteine residues [C-terminal cysteine residue (s) ] within about 30 residues from the C-terminus of the chimer molecule, and (iii) zero to about 100 amino acid residues in an immunogenic sequence heterologous to HBc from position 150 to the C-terminus. 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. Domain IV also preferably contains one C-terminal cysteine residue.

The chimer molecules (i) have an amino acid residue sequence in which no more than about 10 percent of the amino acid residues are substituted in the HBc sequence of the chimer and (ii) self-assemble into particles on expression in a host cell. The particles are substantially free of binding to nucleic acids and are more stable than are particles formed from otherwise identical HBc chimer molecules that are free of any above-mentioned C-terminal cysteine residue (s) and (i) lack the N-terminal cysteine residue (s) or (ii) in which an N-terminal cysteine residue (s) present in a contemplated chimer molecule is (are) replaced by another residue. In one aspect, a contemplated chimer molecule contains a heterologous epitope at the N-terminus peptide-bonded to one of HBc residues 2-5. In another 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 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 an N-terminal cysteine residue (s), as discussed before. Specific examples of several of these chimer molecules and their self-assembled particles are discussed hereinafter.

As already noted, a contemplated HBc chimer molecule can contain about 135 to about 515 amino acid residues. In some preferred embodiments, HBc residue 4 is present, whereas residues 2-5 are present in other preferred embodiments, so that Domain I can begin at HBc residue 4 or 2 and continue through residue 75; i.e., the HBc residue at HBc position 75. Residue 1 is methionine, the amino acid of the DNA start codon. It is preferred that the native methionine that is normally present at position 1 of HBc be absent so that only one start signal is present in the encoding DNA or NA.

The heterologous immunogenic epitope that can be present in Domain I or in the immunodominant loop of Domain II preferably contains about 15 to about 50 residues, although an epitope as short as about 6 amino acid residues can induce and be recognized by antibodies and T cell receptors and is therefore useful . It is preferred that all of the residues of Domain II from position 76 through position 85 are present, although interrupted by one or more other residues. Domain II must contain at least four residues, that can have any sequence that does not interfere with expression or use, but those residues are preferably part of the sequence between the residues of positions 75 and 85.

Domain III contains HBc residues 86 through 135 peptide-bonded to residue 85.

Domain IV contains a sequence of at least five residues that are comprised of (i) a sequence of the residues of HBc positions 136 through 140, and preferably through 149 on up to 156, peptide-bonded to residue 135, (ii) zero to three cysteines residues and (iii) optionally can contain a sequence of a heterologous epitope of up to about 100 residues, particularly when the HBc sequence ends at residue 140, although a shorter sequence of up to about 25 residues is more preferred. That Domain IV heterologous sequence is heterologous to the sequence of HBc and is other than a sequence of HBc from position 150 to the HBc

C-terminus. The heterologous sequence, when present in Domain IV, is preferably a T cell epitope, but can also be a B cell epitope as are usually present in one or the other of Domains I and II.

Domain IV can also contain zero to three cysteine residues and those Cys residues are present within about 30 residues of the carboxy-terminus (C-terminus) of the chimer molecule. Preferably, one cysteine (Cys) residue is present, and that Cys is preferably present as the carboxy-terminal (C-terminal) residue, unless a T cell epitope is present as part of Domain IV. When such a T cell epitope is present, the preferred Cys is preferably within the C-terminal last five residues of the HBc chimer .

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 the sequence contain one or more B cell epitopes, that the HBc sequence between amino acid residues 76 and 85 be present, but interrupted by the heterologous epitope (s), and that the chimer further include one or more T cell epitopes in Domain IV peptide-bonded to one of HBc residues 140-149.

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 linker residue, with a T cell epitope being present peptide-bonded to one of HBc residues 140-149. The particles formed from such chimer molecules typically contain a ratio of conjugated epitope to C-terminal peptide-bonded T cell epitope of about 1:4 to 1:1, with a ratio of about 1:2 being common.

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.

One preferred contemplated HBc chimer molecule contains a sequence of about 135 to about 515 residues. A preferred HBc chimer molecule containing two heterologous epitopes of preferred lengths of about 15 to about 50 residues each and a preferred HBc portion length of about 140 to about 149 residues has a sequence length of about 170 to about 250 amino acid residues. Particularly preferred chimer molecules continuing two heterologous epitopes have a length of about 180 to about 210 residues. It is to be understood that a wide range of chimer molecule lengths is contemplated in view of the variations in length of the N- and C-terminal HBc portions and differing lengths of the several contemplated epitopes that can be inserted in the immunogenic loop. A contemplated recombinant protein, after expression in a host cell, self-assembles to form particles that are substantially free of binding to nucleic acids. The contemplated HBc, chimer particles are generally spherical in shape and are usually homogeneous in size for a given preparation. These chimeric particles thus resemble native HBc particles that have a similar shape and size and can be recovered from infected persons .

A contemplated chimer particle comprises previously discussed chimer molecules. More broadly, such a chimer particle comprises a chimeric C-terminal truncated HBc protein that has a sequence of at least about 125 of the N-terminal 150 residues and contains (i) a heterologous epitope peptide- bonded to one or more of the N-terminus, C-terminus or the immunodominant loop, or a heterologous linker residue for an epitope in the immunodominant loop, and (ii) one to three N-terminal cysteine residues and zero to three C-terminal cysteine residues as previously described, and at least a 5 HBc residue sequence from position 135.

A contemplated particle is sufficiently free of arginine and/or lysine residues in Domain IV so that the self-assembled particles are substantially free of nucleic acid binding and exhibits a 280:260 absorbance ratio of about 1.2 to about 1.7, as discussed herein after. Thus, a contemplated chimeric protein is free of the HBc sequence between positions 150 and 183, and preferably between residue positions 149 through 183.

A particularly preferred immunogen is a particle comprised of recombinant hepatitis B virus core (HBc) protein chimer molecules with a length of about 150 to about 325 and preferably about 155 to 225 amino acid residues that contains four peptide- linked amino acid residue sequence domains from the N-terminus that are denominated Domains I, II, III and IV. At least one polypeptide containing 6 to about 24 residues of the influenza A M2 polypeptide of SEQ ID NO: 9, as defined hereinbelow, is present peptide-bonded to the chimer molecule.

(a) Domain I comprises about 71 to about 110 amino acid residues whose sequence includes at least the sequence of the residues of position 5 through position 75 of HBc. One to three cysteine residues is (are) also present at a position in the chimer molecule of about one to about -20 relative to the N-terminus of HBc of SEQ ID NO : 1 [N-terminal cysteine residue (s) ] . The one or more N-terminal cysteine residues is (are) present within a sequence other than that of the pre-core sequence of HBc.

Domain I can, and preferably does, further include a (i) sequence of 6 to about 24 residues of an above-noted influenza A M2 polypeptide

X₁X₂X₃ ^x4^x5^x6^x7^x8T^χ10^xllR 13^x14^x15^x16^x17^x18^x19^x20^x21- ^x22^x23^x24 °f ^SE0- ^ID NO: 9 that are peptide-bonded to or within about 15 residues of the N-terminus of the HBc sequence, as well as (ii) one or more or HBc residues 1-4. In that influenza A M2 polypeptide sequence, residues X_]_ through X_Q are absent or present, and when present are the residues naturally present in the M2 protein sequence that are methionine, serine, leucine, leucine, threonine or proline, glutamic acid, valine, and glutamic acid, respectively, with the proviso that when one subscripted X residue is present, any remaining subscripted X with a higher subscript number up to 8 is also present,

Xio is present and is proline, leucine or histidine,

X]_l is present and is isoleucine or threonine,

Xχ3 is present and is asparagine or serine,

X]_ is present and is glutamic acid or glycine, residues X^5 and X_]_g are present or absent, and when present are tryptophan and glycine or glutamic acid, respectively, residues X17 and X^g are present or absent, and when present are independently cysteine, serine, or alanine, residue X]_g is present or absent, and when present is arginine or lysine, and residues X_2Q through X₂4 are present or absent, and when present are the residues naturally present in the M2 protein sequence that are asparagine or serine, aspartic acid or glycine, serine, serine and aspartic acid respectively, with the proviso that when one subscripted X residue is present, any remaining subscripted X residue with a lower subscript number down to 15 is also present.

(b) Domain II comprises about 10 to about 60 amino acid residues peptide-bonded to residue 75. This sequence includes (i) zero to all of the residues of a sequence of HBc from HBc position 76 through 85 peptide-bonded to (ii) an optional sequence of about 6 to about 48 residues that constitute one or more repeats of the above influenza A M2 polypeptide of SEQ ID NO: 9. (c) Domain III is an HBc sequence from position 86 through position 135 that is peptide- bonded to residue 85. d) Domain IV comprises (i) the residues of positions 136-140 plus up to nine residues of an HBc amino acid residue sequence from position 141 through 149 peptide-bonded to the residue of position 135 of Domain III, (ii) zero to three cysteine residues, (iii) fewer than three arginine or lysine residues, or mixtures thereof adjacent to each other, and (iv) up to about 100 amino acid residues in a sequence heterologous to HBc from position 164 to the C-terminus. Thus, Domain IV contains at least the 5 residues of positions 136-140.

A contemplated chimer molecule (i) contains up to about 10 percent conservatively substituted amino acid residues in the HBc sequence, (ii) self- assembles into particles that are substantially free of binding to nucleic acids on expression in a host cell, and those particles are more stable on formation than are particles formed from an otherwise identical HBc chimer that lacks said N-terminal cysteine residue (s) or in which an N-terminal cysteine residue present in the chimer molecule is replaced by another residue.

In examining the length of a particularly preferred HBc chimer, such a recombinant protein can have a length of about 150 to about 325 amino acid residues. Preferably, that length is about 155 to about 225 residues. More preferably, the length is about 155 to about 170 residues. These differences in length arise from changes in the length of Domains I, II and IV. Returning to the more broadly defined chimeric molecules and the particles assembled therefrom, 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 the other hand, a recent study in which an immunogenic 23-mer polypeptide from the influenza M2 protein was fused to the HBc N- terminal sequence reported that the resultant fusion protein formed particles when residues 1-4 of the native HBc sequence were replaced. Neirynck et al . (October 1999) Nature Med . , 5 (10) :1157-1163. Thus, the art teaches that particles can form when an added amino acid sequence is present peptide-bonded to one of residues 2-4 of HBc, whereas particles do not form if no additional sequence is present and more than residues 1-3 are deleted from the N-terminus of HBc.

An N-terminal epitope sequence peptide- bonded to one of the first five N-terminal residues of HBc can contain a single cysteine residue or a sequence of up to about 30 residues that are heterologous to HBc . The one to three cysteine residues can be present at a convenient location in the sequence, but are typically near the C-terminus of the added sequence so that the added N-terminal cysteine residue (s) are at a position of about -20 to about +1, and more preferably at a position of about -14 to about +1, relative to the HBc N-terminus as shown in SEQ ID NO:l. Exemplary sequences include a B cell or T cell epitope such as those discussed and illustrated hereinafter (Tables A and B, respectively) , the 23-mer polypeptide from the influenza M2 protein of Neirynck et al . , above, that includes two cysteine residues, and variants of that sequence containing at least about 6 residues, a sequence of another (heterologous) protein such as β-galactosidase as can occur in fusion proteins as a result of the expression system used, or another hepatitis B-related sequence such as that from the Pre-Sl or Pre-S2 regions or the major hepatitis B surface antigen (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, constitute portions of the HBc sequence at positions 76 to 85, and one to about 245 residues, and preferably one to about 50 residues are heterologous (foreign) to HBc. Thus, at least HBc residues 75 and 85 are present in Domains I and II, respectively. Those heterologous residues constitute

(i) a heterologous linker residue for a epitope such as a B cell or T cell epitope or (ii) a heterologous B or T cell epitope that preferably contains 6 to about 50, more preferably about 15 to about 50, and most preferably about 20 to about 30 amino acid residues, and are positioned so that they are peptide-bonded between zero, or more preferably at least 4, to all of the residues of positions 76 to 85 of the HBc sequence. Heterologous B cell epitopes are preferably linked at this position by the linker residue or are peptide-bonded into the HBc sequence, and use of a B cell epitope is discussed illustratively hereinafter.

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 to 85 are present in the chimer.

The one to about 245 residues added to the HBc loop sequence is (are) heterologous to a HBc sequence. A single added heterologous residue is a heterologous linker residue for a B cell epitope as discussed before. The longer sequences, typically at least 6 amino acid residues long to about 50 amino acid residues long and more preferably about 15 to about 50 residues in length, as noted before, are in a sequence that comprises a heterologous immunogen such as a B cell or T cell epitope, except for heterologous residues encoded by restriction sites. A particularly preferred heterologous sequence contains 6 to about 24 residues of the N-terminal extracellular portion of the influenza A M2 protein. It is to be understood that an above heterologous immunogenic sequence can be present within a longer heterologous, non-HBc sequence or can be repeated in such a sequence .

Exemplary peptide B cell epitopes useful for both linkage to the linker residue after expression of a contemplated chimer and for expression within a HBc chimer at one or more of the N-terminus, within the immunogenic loop or at the C- terminus of the chimer are illustrated in Table A, below, along with the common name given to the gene from which the sequence is obtained, the literature or patent citation for published epitopes, and SEQ ID NO.

Table A

B Cell Epitopes

SEQ ID Organism Gene Sequence Citation* NO

Streptococcus pneumoniae

PspAl

K EΞLSDKIDELDAE 1 10

QKKYDΞDQKKTEE- PSP2 KAALEKAASEEM-

DKAVAAVQQA 1 11

Crypto sporidium parvum

P23

QDKPA-DAPAAEAPA-

AEPAAQQDKP DA 2 12

HIV GP120

RKRIHIGPGR-

AFYITKN 3 13 Foot-and-mouth virus VP1

YNGECRYNRNA- VPNLRGDLQVL- AQKVART P 14

Influenza Virus A8/PR8 HA

YRN L LTEK 15

Type A M2**

(A8/PR8/34) SLLTEVETPIR-

NEWGCRCNGSSD 29 16 SLLTEVETPIR-

NEWGCRCNDSSD 29 17 SLLTEVETPIR-

NEWGARANDSSD 18 EQQSAVDADDS-

HFVSIELE 35 19 SLLTEVETPIR-

NΞWGSRSNDSSD 20 S TEVETPIR-

NE GSRCNDSSD 21 SL TEVETPIR-

NEWGCRSNDSSD 22 SLLTEVETPIR-

NEWGCRANDSSD 23 SLLTΞVETPIR-

NE GARCNDSSD 24 MSL TEVETPIR-

NEWGCRCNDSSD 25 MS LTEVETPIR-

NE GSRSNDSSD 26 MGISLLTEVETPIR-

NEWGCRCNDSSD-

ELLG L GI 27 MSLLTEVETPIR-

NEWGARANDSSD 28 MSLLTEVETPIR-

NEWGCRANDSSD 29 MSLLTEVETPIR-

NEWGARCNDSSD 30 MSLLTEVΞTPIR-

NEWGCRSNDSSD 31 SLLTEVETPIR-

NE GSRCNDSSD 32

^X1^X2^X3^X4^X5^X6^X7^X8^{T_ X}10^X11^RX13^X14^X15^X16^X17^X18^" 19^x20^x21-^x22^x23^x24

Type B NB NNATFNYTNVNPISHIR 33

Yersinia

pestis V Ag

DILKVIVDSMNHH- GDARSKLREELAE- LTAELKIYSVIQA- EINKHLSSSGTIN- IHDKSINLMDKNL- YGYTDEEIFKASA- EYKILEKMPQTTI- QVDGSEKKIVSIK- DF GSENKRTGAL- GN KNSYSYNKDN- NELSHFATTCSD 34

Haemophilus influenza pBOMP

CSSSNNDAA-

GNGAAQFGGY 10 35

NKLGTVSYGEE 36

NDEAAYSKN-

RRAVLAY 37

Moraxella catarrhalis copB DIE DKKK- RTDEQLQAE- LDDKYAGKGY 11 38 DIEKNKKK- RTEAELQAE- LDDKYAGKGY 39 IDIEKKGKI- RTEAELLAE- LN DYPGQGY 40

Porphyromona s gingivalis HA

GVSPKVCKDVTV- EGSNEFAPVQNLT 12 41 RIQST RQKTV- DLPAGTKYV 42

Try pan osoma cruzi KAAIAPAKAAA- APAKAATAPA 14 43

Plasmodium fal ciparum CS

(NANP) ₄ 24 44

NANPNVDP- (NANP) 3NVDP 45

NANPNVDP- (NANP) ₃ 46

(NANP) 3NVDPNANP 47

NANPNVDP- (NANP) 3NVDPNANP 48

NPNVDP (NANP) 3NV 49

NPNVD - (NANP) 3NVDP 50

NPNVD (NANP) 3-

NVDPNA 51

NVDP (NANP) ₃NV 52

NVDP(NANP)₃NVDP 53

NVDP (NANP) 3 _

NVDPNA 54

DP (NANP) 3NV 55

DP (NANP) 3NVDP 56 DP (NANP) 3 ^■ NVDPNA 57

vivax CS

GDRADGQPAG-

DRADGQPAG 20 58

RADDRAAGQP-

AGDGQPAG 59

ANGAGNQPG-

ANGAGDQPG 60

ANGADNQPG-

ANGADDQPG 27 61

ANGAGNQPG-

ANGADNQPG 62

ANGAGNQPG-

ANGADDQPG 63

APGANQEGGAA-

APGANQEGGAA 28 64

ANGAGNQPGAN-

GAGDQPGANGA-

DNQPGANGADD-

QPG 65

herghi CS

DPPPPNPN- DPPPPNPN 66

yoelli CS

(QGPGAP) ₄ 67

Streptococcus sobrinus Agl/II

KPRPIYEA- KLAQNQK 16 68 AKADYEAK- LAQYEKDL 69

Shigella flexneri Invasin

KDRTLIEQK 18 70 Respiratory syncitia virus (RSV) G CSICSNNPT- CWAICK 19 71

Enta oeba histolytica lectin

VECASTVCQNDN- SCPIIADVEKCNQ 21 72

Schistosoma japonicum para

DLQSEISLSLE- NGE IRRAKSA- ES ASELQRRVD 22 73

Schistosoma mansoni para DLQSEISLSLE- NSE IRRAKAA- ESLASDLQRRVD 22 74

Bovine Inhibin α_c subuni t STPPLPWP -

SPAA RLLQ- RPPEEPAA 30 75

Ebola Virus membrane -anchored glycoprotein ATQVEQHHRR- TDNDSTA 31 76

HNTPVYK D-

ISEATQVE 31 77

GKLGLITNTI- AGVAVLI 31 78

Escherichia coli

ST CCELCCYPACAGCN 33 79 NTFYCCELCC- YPACAGCN 33 80

SSNYCCE CC- YPACAGCN 33 81

Alzheimer's disease β-Amyloid DAEFRHDSGYE- 34 82

VHHQKLVFFAE-

DVGSNKGAIIG-

LMVGGWIA

DAEFRHDSGYE- 83

VHHQKL

EDVGSNKGAII 84

DAEFRHDSGYE- 85

VHHQKLVFFAE-

DVGSNKGAIIG

Neisseria meningitidis PorA YVAVENGVAKKVA 86

HFVQQTPKSQPTLVP 87

HVWNNKVATHVP 88

PLQNIQPQVTKR 89

AQAANGGAASGQVKVTKVTKA 90

YVDEQSKYHA 91

HFVQNKQNQPPTLVP 92

KPSSTNAKTGNKVEVTKA 93

Y TTVNTGSATTTTFVP 94

YVDEKKKMVHA 95

HYTRQNNADVFVP 96

YYTKDTNNN TLVP 97

PPQKNQSQPWTKA 98

PPSKGQTGNKVTKG 99

PPSKSQPQVKVTKA 100

QPQTANTQQGGKVKVTKA 101

QPQVTNGVQGNQVKVTKA 102

QPSKAQGQTNNQVKVTKA 103

PPSSNQGKNQAQTGNTVTKA 104

PPSKSQGKTGNQVKVTKA 105

PPSKSQGTNNNQVKVTKA 106

PPSKSQPGQVKVTKVTKA 107

QLQLTEQPSSTNGQTGNQVKVT-KA 108

QLQLTEAPSKSQGAASNQVKVT-KA. 109

SAYTPAHVYVDNKVAKHVA 110

SAYTPAHFVQNKQNNNPTLVP 111

VEGRNYQLQLTE 112

PAQNSKSAYTPA 113

QLQLTEPPSKNQAQTQNKVTKA 114 GRDAFELFLLGSGSDE 115

RHANVGRDAFELFLLGSGSDEA-

KGTDPLKNH 116

GRDAFNLFLLGRIGDDDE 117

GRNAFΞLFLIGSATSDQ 118

QVKVTKAKSRIRTKI 119

TLVPAWGKPGSD 120

NspA HAKASSSLGSAKGFSPR 121

TRYKNYKAPSTDFKL 122

SLNRASVDLGGSDSFSQT 123

GKVNTVKNVRSGELSAGVRVK 124

GKVNTVKNVRSGELSVGVRVK 125

Immunoglobulin E

APEWPGSRDKRTL 126

EDGQVMDVD 127

STTQEGEL 128

GHTFEDSTKK 129

GGGHFPPT 130

PGTINI 131

FTPPT 132

INHRGY V 133

GEFCINHRGY VCGDPA 134

MAPE PGSRDKRTL 135

MEDGQVMDVD 136

MSTTQEGEL 137

MGHTFEDSTKK 138

MGGGHFPPT 139

MPGTINI 140

MFTPPT 141

MINHRGY V 142

MGEFCINHRGY VCGDPA 143

*Citations to published epitopes are provided following Table B . * * articularly preferred sequences related to the inf luenza A M2 protein are discussed in greater detail in relation to Table C .

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 example, 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 2 through 78, followed by restriction site-encoded residues, the heterologous immunogenic sequence, further restriction site-encoded residues and HBc sequence 79 through 85. The sequence of other contemplated chimers through Domains I and II should be apparent from these illustrations and those that follow and need not be enumerated.

It has been found that a short hydrophilic peptide containing a plurality of glycine residues and having a length' of about 5 to about 9 residues peptide-bonded at the C-terminus of an above-noted Neisseria meningi tidis B cell epitope sequence can assist in the expression of a chimeric particle containing that sequence. One useful short peptide is that disclosed in Karpenko et al . , Amino Acids (2000) 18:329-337, having the sequence GSGDEGG of SEQ ID NO: 144.

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 to 85 is preferably present, but interrupted by the heterologous linker for a conjugated epitope. This chimer preferably includes the HBc sequence of position 4 through at least position 140, plus a cysteine residue near the N-terminus of the chimer protein. More preferably, the HBc sequence of positions 1 through 149 are present, but interrupted between residues 76 and 85 by the heterologous linker for a conjugated epitope, and the chimer molecule contains a C-terminal cysteine.

The heterologous linker for a conjugated epitope is most preferably a lysine (K) residue. Glutamic or aspartic acid, tyrosine and cysteine residues can also be used as linker residues, as can tyrosine and cysteine residues. It is noted that more than one linker can be present such as a sequence of three lysines, but such use is not preferred because heterogeneous conjugates can be formed from such use in which the conjugated hapten is bonded to one linker in a first chimer and to a different linker in a second chimer molecule. U.S. Patent No. 6,231,864 Bl discloses HBc chimer molecules containing one or more linking residues, but lacking a stabilizing N-terminal cysteine residue .

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: 145

GGGGSGGGG SEQ ID NO: 146

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 140, and the second-discussed chimer has the sequence of HBc from position 79 through position 140.

Domain IV is a sequence that (i) includes a HBc sequence from position 136 through 140 and optionally through position 149, (ii) contains zero up to three cysteine residues, and (iii) up to about 100 amino acid residues in a sequence heterologous to HBc at position 150 to the C-terminus, with the proviso that Domain IV contain at least 5 amino acid residues of the HBc sequence from position 136 through 140. The Domain IV sequence heterologous to HBc more preferably contains up to about 50 amino acid residues, and most preferably contains up to about 25 residues. The Domain IV sequence can thus be substantially any sequence, except the C-terminal HBc sequence from position 150 to the C-terminus.

The length of the Domain IV sequence can be five residues; i.e., the residue of position 136 through 140, up to about 100 amino acid residues including up to a total of three cysteines, with the length being sufficient so that a contemplated chimeric protein has a total length of about 135 to about 515 residues, and more preferably up to about 460 residues, and most preferably up to about 435 amino acid residues. 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 can include zero up to three Cys residues. When present, it is preferred that the one or more Cys residues be at or within about five amino acid residues of the C-terminus of the chimeric protein molecule. In addition, when more than one Cys residue is present in a Domain IV sequence, it is preferred that those Cys residues be adjacent to each other.

It is 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, with illustrative added C-terminal cysteine residues underlined. Table B T Cell Epitopes

SEQ

Organism Gene Sequence¹¹ Citation ID NO

HIV P24 GPKEPFRDY- VDRFYKC 147

Coryn ebacterium diptheriae toxin

FQWHNSYN- RPAYSPGC 148

Borrelia burgdorferi ospA

VΞIKΞGTVTLKRE- IDKNGKVTVSLC 149 TLSKNISKSG- EVSVELNDC 150

Influenza Virus A8/PR8 HA SSVSSFERFEC 8 151 LIDALLGDPC 32 152 TLIDALLGC 32 153

NP FWRGENGRKTRS- 36 154 AYERMCNILKGK LRVLSFIRGTKV- 36 155 SPRGKLSTRG SLVGIDPFKLLQ- 36 156 NSQVYSLIRP AVKGVGTMVME - 36 157 IRMIKRGINDRN

Trypanosoma cruzi

SHNFTLVASVII- EΞAPSGNTC 13 158 Plasmodium falciparum MSP1

SVQIPKVPYPNGIVYC 15 159 DFNHYYTLKTGLEADC 160

PSDKHIEQYKKI- 23 KNSISC 161

EYLNKIQNSLST- 26 E SPCSVT 162 v

-ivax

YLDKVRATVGTΞ- TPCSVT 163

P. yoelii

EFVKQISSQLTE- E SQCSVT 164

Streptococcus sobrinus Agl/ll

KPRPIYEAKL- AQNQKC 16 165 AKADYEAKLA- QYEKDLC 166

LCMV (lymphocytic choriomeningitis virus) NP RPQASGVYM- GNLTAQC 17 167

Clostridium tetani tox

QYIKANSKFIG- ITELC 20 168

Neisseria meningi tidis PorB AI QVEQKASIAGTDSG C 169

NYKNGGFFVQYGGAYKRHC 170

HNSQTEVAATLAYRFGNVC 171

PorB TPRVSYAHGFKGLVDDADC 172

RFGNAVPRISYAHGFDFIC 173

AFKYARHANVGRNAFΞLFC 174

SGAWLKRNTGIGNYTQINAC 175

AGΞFGTLRAGRVANQC 176 IGNYTQINAASVGLRC 177

GRNYQLQLTEQPSRTC 178

SGSVQFVPAQNSKSAC 179

HANVGRDAFNLFLLGC 180

LGRIGDDDEAKGTDPC 181

SVQFVPAQNSKSAYKC 182

NYAFKYAKHANVGRDC 183

AHGFDFIERGKKGENC 184

GVDYDFSKRTSAIVSC 185

HDDMPVSVRYDSPDFC 186

RFGNAVPRISYAHGFDFIERGKKGENC 187

NYAFKYAKHANVGRDAFNLFLLGC 188

SGAWLKRNTGIGNYTQINAASVGLRC 189

SGSVQFVPAQNSKSAYTPAC 190

OpaB TGANNTSTVSDYFRNRITC 191

IYDFKLNDKFDKFKPYIGC 192

Opa-5d LSAIYDFKLNDKFKPYIGC 193

Opac NGWYINPWSEVKFDLNSRC 194

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

Citations for Tables A and B:

1. EPO 786 521A.

2. WO 98/07320.

3. US No. 5,639,854.

4. US No. 4,544,500.

5. EPO 399001 Bl .

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

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

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

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

10. EPO 432 220 Bl .

11. WO 98/06851.

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

13. Kahn et al. (1997) J. Immunol., 159, 9:4444.

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

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

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

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

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

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

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

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

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

30. Thompson et al. (1994) Eur. J. Biochem. , 226 (3) :751-76 .

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.

36. Brett et al., (1991) J. Immunol . , 147 (3) : 984-991.

The amino acid sequence of HBc from residue position 4 through at least position 140 is preferably present in a contemplated chimer molecule and particle. The sequence from position 2 through position 149 is more preferably present. A B cell epitope is preferably present between residues 76 and 85 and at least a single cysteine residue at or near the N-terminus in Domain I as already noted, a T cell epitope that can include a cysteine residue can also be present as a C-terminal addition to the HBc sequence. A contemplated recombinant HBc chimer is substantially free of bound nucleic acid. A contemplated chimer particle that contains an added Cys residue at or near the N-terminus of the molecule is also more stable after formation than is a similar particle that does not contain that added Cys.

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

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

These particles are similar to the particles observed in patients infected with HBV, but these particles are non-infectious . Upon expression in various prokaryotic and eukaryotic hosts, the individual recombinant HBc chimer molecules assemble in the host into particles that can be readily harvested from the host cells, and purified, if desired.

As noted before, the HBc immunodominant loop is usually recited as being located at about positions 75 to 85 from the amino-terminus (N-terminus) of the intact protein. The heterologous B cell epitope-containing sequence of Domain II is placed into that immunodominant loop sequence . That placement substantially eliminates the HBc immunogenicity of the HBc loop sequence, while presenting the heterologous sequence or linker residue in an extremely immunogenic position in the assembled chimer particles.

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. An illustrative conservative substitution is seen in the replacement of residues at positions 2 and 3 (aspartic acid and isoleucine; DI) by glutamic acid and leucine (EL) residues that are encoded by an EcoRI restriction site used to add nucleic acids that code for a desired N-terminal epitope, including an N-terminal cysteine residue.

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

The HBc portion of a chimer molecule of the present invention; i.e., the portion having the HBc sequence that has other than a sequence or residue of an added epitope, linker, flexible linker arm or heterologous residue (s) that are a restriction enzyme artifact, most preferably has the amino acid residue sequence at positions 2 through 149, or 2 through 156, of subtype ayw that is shown in Fig. 1 (SEQ ID NO:l) , less any portion or portions of the subtype ayw sequence that are absent because of truncation at one or both termini. Somewhat less preferred are the corresponding amino acid residue sequences of subtypes adw, adw2 and adyw that are also shown in Fig. 1 (SEQ ID NOs:2, 3 and 4). Less preferred still are the sequences of woodchuck and ground squirrel at aligned positions 2 through 156 that are the last two sequences of Fig 1 (SEQ ID NOs : 5 and 6) . As noted elsewhere, portions of different sequences from different mammalian HBc proteins can be used together in a single chimer.

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 2 through 156, no more than about 20 percent of the amino acid residues are substituted as compared to SEQ ID NO : 1 from position 2 through 156. It is preferred that no more than about 10 percent, and more preferably no more than about 5 percent, and most preferably no more than about 3 percent of the amino acid residues are substituted as compared to SEQ ID NO : 1 from position 2 through 156. Where an HBc sequence is truncated further at one or both termini, the number of substituted residues is proportionally different. Deletions elsewhere in the molecule are considered conservative substitutions for purposes of calculation.

A contemplated chimer of 156 HBc residues can therefore contain up to about 30 residues that are different from those of SEQ ID NO : 1 at positions 2 through 156, and preferably about 15 residues. More preferably, about 7 or 8 residues are different from the ayw sequence (SEQ ID NO:l) at residue positions 2-156, and most preferably about 4 or 5 residues are different. Substitutions, other than in the immunodominant loop of Domain II or at the termini, are preferably in the non-helical portions of the chimer molecule and are typically between residues 2 to about 15 and residues 24 to about 50 to help assure particle formation. See, Koschel et al . , J. Virol . , 73 (3) :2153-2160 (Mar. 1999).

Domain I of a particularly preferred embodiment that includes an influenza A M2 immunogen preferably has the sequence of residues of positions 2-, 3- or 4- through 75 of HBc. Domain I also contains one to three, preferably one, added cysteine residue (s) and also preferably includes about 6 to about 24 residues of the sequence of the extracellular region of the influenza A M2 protein peptide-bonded at the amino-terminus as discussed herein below. Domain I therefore contains a deletion of at least the methionine residue of position 1 of HBc and can include deletions of the residues at positions 2, 3 and 4.

The one or more cysteine residues present in Domain I is (are) located at a position in the chimer molecule of about one to about -20 relative to the N-terminus of HBc of SEQ ID NO : 1 [N-terminal cysteine residue (s) ] . Thus, using the sequence of SEQ ID NO:l as a reference point, the N-terminal cysteine residue (s) is located in the chimer molecule at a position that corresponds to the methionine at position 1 of SEQ ID NO : 1 (Fig. 1), or at a position up to about 20 residues downstream from that position. More preferably, an N-terminal cysteine is located at a position of about one to about minus 14 relative to position 1 of SEQ ID NO:l.

The one or more N-terminal cysteine residues are present within a sequence other than that of the pre-core sequence of HBc. As was noted previously, the HBeAg molecule contains the pre-core sequence that includes a cysteine residue. That molecule does not form particles, whereas particles are desired herein. Thus, although an N-terminal cysteine residue can be adjacent to a pre-core sequence, such a residue is not present within a precore sequence or a contemplated chimer molecule .

Domain I of a particularly preferred chimer molecule can have a length of about 110 residues. Preferably, Domain I has a length of about 95 to about 100 amino acid residues, and includes an influenza A M2 polypeptide epitope sequence of SEQ ID NO: 9, that preferably includes the C-terminal 23 residues .

Domain II, which is peptide-bonded to residue 75, contains about 10 to about 60 amino acid residues. This Domain includes zero through all of the sequence of HBc residues of positions 76 through 85. Domain II also optionally includes a sequence of about 6 to about 48 residues that constitute one or more repeats of the before-mentioned influenza A M2 polypeptide of SEQ ID NO: 9. The influenza A M2 polypeptide sequence, when present, is preferably peptide-bonded between HBc residues 78 and 79, and all of the HBc sequence from position 76 through 85 is present .

Preferred influenza A M2 polypeptide sequences for insertion into Domains I or II, or both, of a contemplated recombinant HBc chimer are enumerated in Table C, below. A sequence beginning with a methionine residue (M) is designed to be N-terminal sequence for insertion into the N-terminus of Domain I, whereas a sequence free of an N-terminal M residue is designed for insertion into Domain II.

Table C Influenza A M2 Protein B Cell Epitopes

Sequence SEQ ID NO

SLLTEVETPIRNEWGCRCNGSSD 16

SLLTΞVETPIRNEWGCRCNDSSD 17

SLLTEVETPIRNEWGARANDSSD 18

SLLTEVΞTPIRNEWGSRSNDSSD 20

SLLTEVETPIRNEWGSRCNDSSD 21

SLLTEVETPIRNEWGCRSNDSSD 22

SLLTEVETPIRNEWGCRANDSSD 23

SLLTEVETPIRNEWGARCNDSSD 24

MSLLTEVETPIRNEWGCRCNDSSD ' 25

MSLLTEVETPIRNEWGSRSNDSSD 26

MGISLLTEVETPIRNEWGCRCNDSSDELLGWLWGI 27

MSLLTEVETPIRNEWGARANDSSD 28

MSLLTEVETPIRNEWGCRANDSSD 29

MSLLTEVETPIRNEWGARCNDSSD 30

MSLLTEVETPIRNEWGCRSNDSSD 31

MSLLTEVETPIRNEWGSRCNDSSD 32

X₁X₂X₃X₄X₅X₆X₇X₈TX₁oXiiRXl3^x14- ^x15^x16^x17^x18^x19^x20^x21^x22^x23^x24 ⁹

Influenza B Protein

NNATFNYTNVNPISHIR 33 In the polypeptide of SEQ ID NO: 9, X₁ through X_Q are absent or present, and when present are the residues naturally present in a reported M2 protein sequence; i.e., methionine, serine, leucine, leucine, threonine or proline, glutamic acid, valine, and glutamic acid, respectively, with the proviso that when one subscripted X is present, any remaining subscripted X residue with a higher subscript number up to 8 is also present. Thus, when X^ is present, each of X₂ through X_Q is also present. Similarly, when X3 is present, each of X4 through X_Q is also present, and the like. On the other hand, X_Q can be present without any other of the remaining X residues having a lower valued subscript number being present .

The residues of X±o , X±χ , X13 and X14 are present, and can be leucine or histidine for X_]__Q , isoleucine or threonine for X _t asparagine or serine for -]_ ^an-d glutamic acid or glycine for X]_4.

The residues X^5 and X^g are present or absent, and when present are tryptophan and glycine or glutamic acid, respectively. Residues - η and _]_g are present or absent, and when present are independently cysteine, serine, or alanine. It is preferred that one of Xiη and X]_g be cysteine, particularly when an M2 polypeptide epitope is present at the N-terminus of the chimer molecule. Residue X _Q is present or absent, and when present is arginine or lysine.

Residues X_2Q through 4 are present or absent, and when present are the residues naturally present in the reported M2 protein sequence; i.e., asparagine or serine, aspartic acid, serine, serine and aspartic acid respectively, with the proviso that when one subscripted X is present, any remaining X residue with a lower subscript number through 15 is also present. Thus, for example, when X₂3 is present, so are each of residues X^5 through X .

Domain III contains the sequence of HBc position 86 through position 135 peptide-bonded at its N-terminus to residue 85.

The fourth domain, Domain IV, comprises (i) the residues of positions 136 through 140 plus up to nine residues of an HBc amino acid residue sequence from position 141 through 149 peptide-bonded to the residue of position 135 of Domain III, (ii) zero to three cysteine residues, and preferably one cysteine residue, (iii) fewer than three arginine or lysine residues, or mixtures thereof adjacent to each other, and (iv) up to about 100 amino acid residues, preferably up to 50 amino acid residues, and more preferably up to about 25 residues, in a sequence heterologous to HBc from position 164 to the C-terminus .

It is preferred that Domain IV contain up to fourteen residues of an HBc sequence from position 136 through position 149 peptide-bonded to residue 135; i.e., an HBc sequence that begins with the residue of position 136 that can continue through position 149. Thus, if the residue of position 148 is present, so is the sequence of residues of positions 136 through 147, or if residue 141 is present, so is the sequence of residues of positions 136 through 140. A chimer containing a HBc sequence up to about position 156 can be used, but it is preferred to end the HBc sequence at about residue 149. Domain IV can also contain zero to three cysteine residues and those Cys residues are present within about 30 residues of the carboxy-terminus (C-terminus) of the chimer molecule. Preferably, one cysteine (Cys) residue is present, and that Cys is preferably present as the carboxy-terminal (C-terminal) residue, unless an influenza T cell epitope is present as part of Domain IV. When such a T cell epitope is present, the preferred Cys is preferably within the C-terminal last five residues of the HBc chimer.

The presence of the above-discussed N-terminal cysteine residue (s) of any of the chimers discussed herein provides an unexpected enhancement of the ability of the chimer molecules to form stable immunogenic particles (discussed hereinafter) . Thus, a contemplated HBc chimer immunogen tends to form particles that stay together upon collection and initial purification as measured by analytical size exclusion chromatography, whose details are discussed hereinafter.

The contemplated particles can also be more stable to decomposition at 37°C after aging than are similar chimer particles lacking that cysteine residue. This latter type of enhanced stability can be measured using 15% SDS-PAGE gels with particles dispersed in sample buffer (reducing) . Gels are stained using Coomassie Blue, and then analyzed. This type of stability is believed to be exhibited against hydrolysis, whereas the stability determined by size exclusion chromatography is that of initial particle formation.

Particles that additionally contain one or more C-terminal cysteine residues exhibit enhanced stability in formation and also toward decomposition on aging, with some particles containing both N- and C-terminal cysteines usually exhibiting greater stability in either measure than those particles having only an added cysteine at either the N- or C-terminus .

Domain IV contains fewer than three arginine or lysine residues, or mixtures thereof adjacent to each other. Arginine and lysines are present in the C-terminal region of HBc that extends from position 164 through the C-terminus of the native molecule. That region is sometimes referred to as the "protamine" or "arginine-rich" region of the molecule and binds nucleic acids. A contemplated HBc chimer molecule and particle are substantially free of bound nucleic acids.

The substantial freedom of nucleic acid binding can be readily determined by a comparison of the absorbance of the particles in aqueous solution measured at both 280 and 260 nm; i.e., a 280:260 absorbance ratio. The contemplated particles do not bind substantially to nucleic acids that are oligomeric and/or polymeric DNA and RNA species originally present in the cells of the organism used to express the protein. 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.

Chimeric HBc particles of the present invention are substantially free of nucleic acid binding and exhibit a 280:260 absorbance ratio of about 1.2 to about 1.7, and more typically, about 1.4 to about 1.7. This range is due in large part to the number of aromatic amino acid residues present in Domains II and IV of a given chimeric HBc particle. That range is also in part due to the presence of the Cys in Domain IV of a contemplated chimer, whose presence can diminish the observed ratio by about 0.1 for a reason that is presently unknown.

The contemplated particularly preferred chimer HBc particles are also more stable in aqueous buffer at 37°C over a time period of about two weeks to about one month than are particles formed from a HBc chimer containing the same peptide-linked Domain II, III and IV sequences and an otherwise same Domain I sequence in which the one to three cysteine residues [N-terminal cysteine residue (s) ] are absent or a single N-terminal residue present is replaced by another residue such as an alanine residue.

Thus, for example, particles containing an influenza A M2 polypeptide in Domain I [e.g. ICC-1590 particles] that include two cysteine residues are more stable than otherwise identical particles [ICC- 1603 particles] assembled from chimer molecules whose N-terminal M2 variant sequence contains serine residues in place of the cysteines. Similarly, particles containing the above serine-containing influenza B cell epitope in Domain I and a single cysteine at the C-terminus [ICC-1605 particles] are more stable than are otherwise identical particles in which that cysteine is absent, but are less stable than are the particles containing the two N-terminal cysteines, ICC-1590 particles, or those particles that contained both N-terminal and C-terminal cysteines [ICC-1604 particles] .

A contemplated particle containing a N-terminal cysteine residue is often prepared in greater yield than is a particle assembled from a chimer molecule lacking a N-terminal cysteine. This increase in yield can often be seen from the mass of particles obtained or from analytical gel filtration analysis using Superose^® 6 HR as discussed hereinafter.

Although the T cell help afforded by HBc is highly effective in enhancing antibody responses (i.e. B cell-mediated) to 'carried' epitopes following vaccination, HBc does not activate influenza-specific T cells, except in restricted individuals for whom the B cell epitope is also a T cell epitope. To help ensure universal priming of influenza-specific T helper cells, in addition to B cells, one or more influenza-specific T helper epitopes is preferably incorporated into a particularly preferred immunogen and is located in Domain IV of the immunogen.

A plurality of the above or another T cell epitopes can be present in Domain IV or another B cell epitope can be present. In preferred practice, Domain IV has up to about 50 residues in a sequence heterologous to HBc. More preferably, that sequence is up to about 25 residues and includes a T cell epitope.

Because M2 is expressed abundantly by infected cells, it has the potential to serve as a target for influenza-specific CTL activity. The extracellular domain is a possible attachment site for antibody, which may function to immobilize the function of M2 , in an analogous manner to the therapeutic drug amantadine, or via the activation of antibody-dependent cellular cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) .

Indeed, the extracellular domain of M2 has been shown to contain at least two distinct human CTL epitopes, one at 7-15 [ Jameson, J. , J. Cruz, and F.A. Ennis, Human cytotoxic 1 '-lymphocyte repertoire to influenza A viruses . J Virol, 1998. 72(11): p. 8682-9] and another at 3-11 [Gianfrani, C, et al . , Human memory CTL response specific for influenza A virus is broad and mul tispecific . Hum Immunol, 2000. 61(5): p. 438-52]. Anti-M2e mediated lysis of influenza-infected cells via ADCC and/or CDC may be the preferred mechanisms of target cell lysis, compared with CTL, because, unlike CTL, they are not subject to MHC restriction. Therefore, an ability to evoke sufficient titers of anti-M2 antibodies displaying a required IgG subclass profdie, and M2e specificity, should evoke broad protection across diverse populations.

The capacity of antibodies to the extracellular domain of M2 to have a biological effect was first described by Zebedee and Lamb, who showed that the monoclonal anti-M2 antibody 14C2 dramatically slows the growth of the virus in culture [Zebedee et al, (1988) J Virol , 62 (8) : 2762-2772] . In 1990, Treanor and colleagues showed that the same monoclonal antibody successfully inhibited influenza A virus replication in mice [Treanor et al . , . (1990) J. Virol . , 64(3) :1375-1377] . Palladino et al . determined that 14C2 was not virus neutralizing in vivo, but did bind infected cells and inhibited virus growth in vi tro; however, it failed to cure the infection [Palladino et al . , (1995) J. Virol . , 69(4) : 2075-2081] . The authors of the latter paper duly noted that 14C2 is an IgGl subclass antibody, and that IgG2a and IgG2b are most effective at fixing complement; they are also superior to IgGl for binding to FcγRIII receptors on NK cells. Indeed, as is shown hereinafter, high titers of IgG2a antibodies in the mouse, which are typical of a Thl immune response, correlate with protection. An immune response that can provide anti-M2e mediated lysis of influenza-infected cells via ADCC and/or CDC is thus a preferred response.

Anamnestic anti-M2 responses have repeatedly been observed following challenge of mice previously immunized with HBC-M2 particles, which is indicative of M2e-HBc particles priming M2 -specific T cells. To investigate this, preliminary studies were conducted to investigate whether lymphocytes from immunized mice can be recalled by M2e peptide, in vi tro . Significant increases in the number of interferon-gamma secreting cells was observed following recall with peptides derived from M2 , HBc p85-100, and recombinant HBcAg, but not plOO-120 from HBc. The recall with HBcAg was most prominent, which is expected since this is a potent T cell immunogen containing many functional T cell epitopes for BALB/c mice [Saito et al . , (2001) Vaccine, 20 (1-2) : 125-33] .

Recall with all antigens appeared to be stronger for lymphocytes isolated from mice immunized with ICC-1604 particles versus ICC-1569 particles. This result indicates that ICC-1604 particles may be a superior T cell immunogen compared with to ICC-1569 particles. However, it is important to note that the M2 recall antigen for these studies was M2e (residue positions 2-24) , which contains cysteine residues at 17 and 19, and was therefore only truly homologous for ICC-1604 particles and not ICC-1569 particles, because the latter contains serine residues in place of the two cysteines residues.

The reduced level of restimulation with M2e for ICC-1569 particles versus ICC-1604 particle- immunized mice could be explained if the cysteine residues at positions 17 and/or 19 are components of one or more T cell epitope (s) . However, the presence of cysteine residue-containing T cell epitopes does not explain the reduced level of restimulation with HBc-derived antigens.

The observation of an anamnestic response, with regard to anti-M2e titers, following viral challenge of M2e-HBc-immunized mice, indicates the presence of at least one T-helper cell epitope in the M2e domain (positions 2-24) .

Th epitopes derived from the influenza nucleoprotein (NP 206-229) , which is broadly reactive in humans (HLA-DR1, HLA-DR2, HLA-DRwl3) [Brett et al., (1991) J. Immunol . , 147 (3) : 984-991] and also functional in BALB/c mice are contemplated for use as T cell epitopes herein. Particles with this epitope fused to the C-terminus of HBc particles have been expressed and purified. Additional influenza Th epitopes are also considered, such as NP 341-362, NP 297-318 and NP 182-205 [Brett et al . , (1991) J. Immunol . , 147 (3) : 984-991] ; these sequences can ultimately be linked in series at the C-terminus of the M2e-expressing particle. These illustrative sequences are provided below.

A contemplated recombinant HBc chimer molecule is typically present and is used in an immunogen or vaccine as a self-assembled particle. These particles are comprised of 180 to 240 chimer • molecules that separate into protein molecules in the presence of disulfide reducing agents such as 2-mercaptoethanol and denaturing reagents such as SDS. The individual molecules are bound together into the particle by protein-protein interactions, and these interactions are stabilized by the presence of disulfide bonds. These particles are similar to the particles observed in patients infected with HBV, but these particles are non-infectious . Upon expression in various prokaryotic and eukaryotic hosts, the individual recombinant HBc chimer molecules assemble in the host into particles that can be readily harvested from the host cells.

Chimer Preparation

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

An illustrative contemplated chimeric immunogen typically utilizes a cysteine residue present in the M2 sequence as the N-terminal cysteine. Primers for the preparation of such chimer molecules by in vi tro mutagenesis of a polynucleotide encoding an HBc molecule are discussed hereinafter. When a cysteine-containing M2 polypeptide epitope is not present at the N-terminus, the N-terminal cysteine can be provided by in vi tro mutagenesis using a primer that encodes just a cysteine- containing portion of the M2 polypeptide or a simple N-terminal start sequence such as Met-Cys- or Met- Gly-Cys- .

As was noted previously, the HBc immunodominant loop is usually recited as being located at about positions 75 through 85 from the amino-terminus (N-terminus) of the intact protein. The influenza A M2 B cell epitope-containing sequence can be placed into that immunodominant loop sequence of Domain II. That placement substantially eliminates the HBc immunogenicity and antigenicity of the HBc loop sequence, while presenting the influenza A M2 B cell epitope in an extremely immunogenic position in the assembled chimer particles.

One of two well-known strategies is particularly useful for placing an immunogenic sequence such as the influenza A M2 B cell sequence into the loop sequence at a desired location such as between residues 78 and 79. A first, less successful strategy is referred to as replacement in which DNA that codes for a portion of the loop is excised and replaced with DNA that encodes the B cell epitope sequence. The second strategy is referred to as insertion in which the B cell sequence is inserted between adjacent residues in the loop.

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 Sacl, one near each end of the immunodominant loop-encoding DNA. Exemplary residues replaced are 76 through 81. The loop-encoding section is excised, an illustrative influenza A M2 B cell epitope-encoding sequence flanked on each side by appropriate HBc sequence residues is ligated into the restriction sites and the resulting DNA is used to express the HBc chimer. See, for example, Table 2 of Pumpens et al . , (1995) Intervirology, 38:63-74 for exemplary uses of a similar technique.

Alternatively, a single restriction site or two sites can be encoded into the region, the DNA cut with a restriction enzyme (s) to provide "sticky" or ends, and an appropriate sticky- or blunt-ended heterologous DNA segment ligated into the cut region. Examples of this type of sequence replacement into HBc can be found in the work reported in Schodel et al . , (1991) F. Brown et al . eds., Vaccines 91 , Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp.319-325, Schodel et al . , Behring Inst . Mi tt . , 1997(98): p. 114-119 and Schodel et al . , J. Exp. Med . , (1994) 180(3): p. 1037-4, the latter two papers discussing the preparation of vaccines against malarial pathogens P. yoelii and P. berghei , respectively. A replacement strategy that results in a net removal of residues from the immunodominant loop is usually not used herein.

Insertion is 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 illustrative influenza A M2 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 illustrative B cell epitope sequence, followed by two more heterologous, non-loop residues encoded by the 3' restriction site and then the rest of the loop sequence. This same strategy is also preferably used for insertion into Domain IV of a T cell epitope or one or more cysteine residues that are not a part of a T cell epitope. A similar strategy using an insertion between residues 82 and 83 is reported in Schoedel et al . , (1990) F. Brown et al . eds., Vaccines 90, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp.193-198.

For example, a DNA sequence that encodes a C-terminal truncated HBc sequence (HBcl49) is engineered to contain adjacent EcoRI and Sacl sites between residues 78 and 79. Cleavage of that DNA with both enzymes provides one fragment that encodes HBc positions 1-78 3 ' -terminated with an EcoRI sticky end, whereas the other fragment has a 5 '-terminal Sacl sticky end and encodes residues of positions 79- 149. Ligation of a synthetic nucleic acid having a 5' AATT overhang followed by a sequence that encodes a desired B cell epitope and a AGCT 3 'overhang provides a HBc chimer sequence that encodes that B cell epitope flanked on each side by two heterologous residues (GI and EL, respectively) between residues 78 and 79, while destroying the EcoRI site and preserving the Sacl site.

A similar strategy can be used for insertion of a C-terminal cysteine-containing sequence. Here, EcoRI and HindiII restriction sites are engineered in to 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 , a stop codon and a 3 ' AGCT overhang were ligated into the digested sequence to form a sequence that encoded HBc residues 1-149 followed by two heterologous residues (GI) , the stop codon and the HindiII site.

PCR amplification using a forward primer having a Sacl restriction site followed by a sequence encoding HBc beginning at residue position 79, followed by digestion with Sacl and HindiII provided a sequence encoding HBc positions 79-149 plus the two added residues and the T cell epitope at the C-terminus. Digestion of that construct with Sacl and ligation provides the complete gene encoding a desired recombinant HBc chimer immunogen having the sequence, from the N-terminus, of HBc positions 1-78, two added residues, the B cell epitope, two added residues, HBc positions 79-149, two added residues, and the T cell epitope.

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 cell or T cell epitopes nor a part of the HBc sequence are not counted in the number of residues present in a recited Domain.

It is also noted that one can also synthesize all or a part of a desired recombinant 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 "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.

A nucleic acid sequence (segment) that encodes a previously described HBc chimer molecule or a complement of that coding sequence is also contemplated herein. Such a nucleic acid segment is present in isolated and purified form in some preferred embodiments.

In living organisms, the amino acid residue sequence of a protein or polypeptide is directly related via the genetic code to the deoxyribonucleic acid (DNA) sequence of the gene that codes for the protein. Thus, through the well-known degeneracy of the genetic code additional DNAs and corresponding RNA sequences (nucleic acids) can be prepared as desired that encode the same chimer amino acid residue sequences, but are sufficiently different from a before-discussed gene sequence that the two sequences do not hybridize at high stringency, but do hybridize at moderate stringency.

High stringency conditions can be defined as comprising hybridization at a temperature of about 50°-55°C in 6XSSC and a final wash at a temperature of 68°C in 1-3XSSC. Moderate stringency conditions comprise hybridization at a temperature of about 50°C to about 65°C in 0.2 to 0.3 M NaCl, followed by washing at about 50°C to about 55°C in 0.2X SSC, 0.1% SDS (sodium dodecyl sulfate) .

A nucleic sequence (DNA sequence or an RNA sequence) that (1) itself encodes, or its complement encodes, a chimer molecule whose HBc portion from residue position 1 through 136, when present, is that of SEQ ID NOs: 1, 2, 3, 4, 5 or 6 and (2) hybridizes with a DNA sequence of SEQ ID NOs: 195, 196, 197, 198, 199 and 200 at least one moderate stringency (discussed above) ; and (3) whose HBc sequence shares at least 80 percent, and more preferably at least 90 percent, and even more preferably at least 95 percent, and most preferably 100 percent identity with a DNA sequence of SEQ ID NOs: 195, 196, 197, 198, 199 and 200, is defined as a DNA variant sequence. 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 : 1, 2, 3, 4, 5 and 6. This DNA or RNA is referred to herein as an "analog of" or "analogous to" a sequence of a nucleic acid of SEQ ID NOs: 195, 196, 197, 198, 199 and 200, and hybridizes with the nucleic acid sequence of SEQ ID NOs : 195, 196, 197, 198, 199 and 200, or their complements herein under moderate stringency hybridization conditions. A nucleic acid that encodes an analogous sequence, upon suitable transfection and expression, also produces a contemplated chimer.

Different hosts often have preferences for a particular codon to be used for encoding a particular amino acid residue. Such codon preferences are well known and a DNA sequence encoding a desired chimer sequence can be altered, using in vi tro mutagenesis for example, so that host- preferred codons are utilized for a particular host in which the enzyme is to be expressed. In addition, one can also use the degeneracy of the genetic code to encode the HBc portion of a sequence of SEQ ID NOs: 1, 2, 3, 4, 5 or 6 that avoids substantial identity with a DNA of SEQ ID Nos: 195, 196, 197, 198, 199 and 200 or their complements. Thus, a useful analogous DNA sequence need not hybridize with the nucleotide sequences of SEQ ID NOs: 195, 196, 197, 198, 199 and 200 or a complement under conditions of moderate stringency, but can still provide a contemplated chimer molecule .

A recombinant nucleic acid molecule such as a DNA molecule, comprising a vector operatively linked to an exogenous nucleic acid segment (e.g., a DNA segment or sequence) that defines a gene that encodes a contemplated chimer, as discussed above, and a promoter suitable for driving the expression of the gene in a compatible host organism, is also contemplated in this invention. 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: 195, 196, 197, 198, 199 and 200 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: 1, 2, 3, 4, 5 or 6. That recombinant DNA molecule, upon suitable transfection and expression in a host cell, provides a contemplated chimer molecule. 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: 195, 196, 197, 198, 199 and 200, isolated nucleic acid segments, preferably DNA sequences, variants and analogs thereof can be prepared by in vi tro mutagenesis, as is well known in the art and discussed in Current Protocols In Molecular Biology, Ausabel et al . eds., John Wiley & Sons (New York: 1987) p. 8.1.1-8.1.6, that begin at the initial ATG codon for a gene and end at or just downstream of the stop codon for each gene. Thus, a desired restriction site can be engineered at or upstream of the initiation codon, and at or downstream of the stop codon so that other genes can be prepared, excised and isolated. As is well known in the art, so long as the required nucleic acid, illustratively a DNA sequence, is present, (including start and stop signals) , additional base pairs can usually be present at either end of the segment and that segment can still be utilized to express the protein. This, of course, presumes the absence in the segment of an operatively linked DNA sequence that represses expression, expresses a further product that consumes the enzyme desired to be expressed, expresses a product that consumes a wanted reaction product produced by that desired enzyme, or otherwise interferes with expression of the gene of the DNA segment .

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

DNA segments that encode the before- described chimer can be synthesized by chemical techniques, for example, the phosphotriester method of Matteucci et al . (1981) J. Am. Chem. Soc , 103:3185. Of course, by chemically synthesizing the coding sequence, any desired modifications can be made simply by substituting the appropriate bases for those encoding the native amino acid residue sequence. However, DNA segments including sequences discussed previously are preferred. A contemplated HBc chimer can be produced (expressed) in a number of transformed host systems, typically host cells although expression in acellular, in vi tro, systems is also contemplated. These host cellular systems include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g. baculovirus) ; plant cell systems transformed with virus expression vectors (e.g. cauliflower mosaic virus; tobacco mosaic virus) or with bacterial expression vectors (e.g., Ti plasmid); or appropriately transformed animal cell systems such as CHO 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- β-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 p K223-3 plasmid can be successfully expressed in a number of E. coli strains, such as XL-1, TB1 , BL21 and BLR, using about 25 to about 100 μM IPTG for induction. Surprisingly, concentrations of about 25 to about 50 μM IPTG have been found to provide optimal results in 2 L shaker flasks and fermentors .

Expression of a contemplated chimer molecule in other microbes such as Salmonella like S. typhi and S. typhimurium and S . typhimurium-E. coli hybrids, yeasts such as S. cerivisiae or Pichia pastoris, in mammalian cells such as Chinese hamster ovary (CHO) cells, in both monocot and dicot plant cells generally and particularly in dicot plant storage organs such as a root, seed or fruit as where an oral vaccine or inoculum is desired, and in insect cells such as those of S. Frugiperda cells or Trichoplusia by use of Autographa calif ornica nuclear polyhedrosis virus (AcNPV) or baculovirus are discussed in detail in published before-mentioned WO 02/14478 A2. These modes of expression, although contemplated, will therefore not be discussed further herein. A variety of methods have been developed to operatively link DNA to vectors via complementary cohesive termini or blunt ends. For instance, complementary homopolymer tracts can be added to the DNA segment to be inserted into the vector DNA. The vector and DNA segment are then j oined by hydrogen bonding between the complementary homopolymeric tails to form recombinant DNA molecules .

Alternatively, synthetic linkers containing one or more restriction endonuclease sites can be used to join the DNA segment to the expression vector, as noted before. The synthetic linkers are attached to blunt-ended DNA segments by incubating the blunt-ended DNA segments with a large excess of synthetic linker molecules in the presence of an enzyme that is able to catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase.

Thus, the products of the reaction are DNA segments carrying synthetic linker sequences at their ends. These DNA segments are then cleaved with the appropriate restriction endonuclease and ligated into an expression vector that has been cleaved with an enzyme that produces termini compatible with those of the synthetic linker. Synthetic linkers containing a variety of restriction endonuclease sites are commercially available from a number of sources including New England BioLabs, Beverly, MA. A desired DNA segment can also be obtained using PCR technology in which the forward and reverse primers contain desired restriction sites that can be cut after amplification so that the gene can be inserted into the vector. Alternatively PCR products can be directly cloned into vectors containing T-overhangs (Promega Corp., A3600, Madison, WI) as is well known in the art .

The expressed chimeric protein self- assembles into particles within the host cells, whether in single cells or in cells within a multicelled host. The particle-containing cells are harvested using standard procedures, and the cells are lysed using a French pressure cell, lysozyme, sonicator, bead beater or a microfluidizer (Microfluidics International Corp., Newton MA). After clarification of the lysate, particles are precipitated with 45% ammonium sulfate, resuspended in 20 mM sodium phosphate, pH 6.8 and dialyzed against the same buffer. The dialyzed material is clarified by brief centrifugation and the supernatant subjected to gel filtration chromatography using

CD

Sepharose CL-4B. Particle-containing fractions are identified, subjected to hydroxyapatite chromatography, and reprecipitated with ammonium sulfate prior to resuspension, dialysis and sterile filtration and storage at -70°C.

HBc Chimer Conjugates

Any hapten (immunogen) to which a B cell or T cell response is desired can be linked to a contemplated HBc chimer or chimer particle such as a chimer particle containing a heterologous linker residue such as a lysine, glutamic or aspartic acid, cysteine or tyrosine in the loop region of Domain II and an added cysteine residue near the N-terminus in Domain I to form a HBc chimer conjugate. The hapten of interest typically is a B cell immunogen. The hapten can be a polypeptide, a protein, a carbohydrate (saccharide; i.e., oligo- or polysaccharide) , or a non-polypeptide, non- carbohydrate chemical such as 2 , 4-dinitrobenzene or a medicament such as cocaine or nicotine. It is thus seen that the word "hatpen" is used herein somewhat more broadly that is usual to include small molecules that do not themselves induce an immune response, as well as larger molecule such as proteins that often can themselves induce an immune response . 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 (immunogens) 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 protein or conjugate by reaction of amino functional groups with 2-iminothiolane, the N- hydroxysuccinimide ester of 3- (3-dithiopyridyl) - propionate, or other reagents known in the art.

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

Methods for covalent bonding of a polypeptide hapten are extremely varied and are well known by workers skilled in the immunological arts. For example, following U.S. Patent No. 4,818,527, m- maleimidobenzoyl-N-hydroxysuccinimide ester (ICN Biochemicals, Inc., Costa Mesa, CA ) or succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC, Pierce Chemical Co., Rockford, IL) is reacted with an appropriate 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) ropionate (SPDP, Pharmacia, Piscataway, NJ) , and that thiol-containing polypeptide can be reacted with the activated carrier after reduction. Of course, the sulfur-containing moiety and double bond-containing Michael acceptor can be reversed. These reactions are described in the supplier's literature, and also in Kitagawa, et al . (1976) J. Biochem. , 79:233 and in Lachmann et al . , in 1986 Synthetic Peptides as Antigens, (Ciba Foundation Symposium 119), pp. 25-40 (Wiley, Chichester: 1986).

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

Particularly useful are a large number of heterobifunctional agents that form a disulfide link at one functional group end and an amide link at the other, including N-succidimidyl-3- (2-pyridyldithio) - propionate (SPDP) , discussed before that creates a disulfide linkage between itself and a thiol in either the HBc chimer or the hapten. Exemplary reagents include a cysteine residue in a polypeptide hapten and an amine on the coupling partner such as the ε-amine of a lysine or other free amino group in the carrier protein. A variety of such disulfide/amide forming agents are known. See for example Immun . Rev. (1982) 62:185. Other bifunctional coupling agents form a thioether rather than a disulfide linkage. Many of these thioether-forming agents are commercially available and include reactive esters of 6-maleimidocaproic acid, 2-bromoacetic acid, 2-iodoacetic acid, 4- (N-maleimidomethyl) cyclohexane- 1-carboxylic acid and the like. The carboxyl groups can be activated by combining them with succinimide or 1-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. 11 provides a schematic representation (Scheme 1) of the formation of a HBc activated carrier using SMCC (I) and the subsequent reaction of that activated carrier with a sulfhydryl-terminated hapten (II) .

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

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

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

Further specifics concerning useful haptens can be found in before-mentioned, and now published, U.S. Patent Application Serial No. 09/930,915.

Inocula and Vaccines

A before-described recombinant HBc chimer immunogen preferably in particulate form is dissolved or dispersed in an immunogenic effective amount in a pharmaceutically acceptable vehicle composition that is preferably aqueous to form an inoculum or vaccine . 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 an added B cell epitope such as an influenza A M2 B cell epitope present in the immunogen. In a vaccine, those induced antibodies also believed to immunoreact in vivo with (bind to) the virus or virally- infected cells and protect the host from influenza infection. A composition that is a vaccine in one animal can be an inoculum an inoculum for another host, as where the antibodies are induced in a second host that is not infected by influenza A.

The amount of recombinant HBc chimer immunogen utilized in each immunization is referred to as an immunogenic effective amount and can vary widely, depending inter alia, upon the recombinant HBc chimer immunogen, animal host immunized, and the presence of an adjuvant in the vaccine, as discussed below. Immunogenic effective amounts for a vaccine and an inoculum provide the protection or antibody activity, respectively, discussed hereinbefore.

Vaccines or inocula typically contain a recombinant HBc chimer immunogen concentration of about 1 microgram to about 1 milligram per inoculation (unit dose) , and preferably about 10 micrograms to about 50 micrograms per unit dose. Immunizations in mice typically contain 10 or 20 μg of chimer particles.

The term "unit dose" as it pertains to a vaccine or inoculum of the present invention refers to a physically discrete unit suitable as an unitary dosage for animals, each unit containing a predetermined quantity of active material calculated to individually or collectively produce the desired immunogenic effect in association with the required diluent; i.e., carrier, or vehicle. A single unit dose or a plurality of unit doses can be used to provide an immunogenic effective amount of recombinant HBc chimer immunogen particles.

Vaccines or inocula are typically prepared from a recovered recombinant HBc chimer immunogen particles by dispersing the particles in a physiologically tolerable (acceptable) diluent vehicle such as water, saline phosphate-buffered saline (PBS), acetate-buffered saline (ABS), Ringer's solution or the like to form an aqueous composition. The diluent vehicle can also include oleaginous materials such as peanut oil, squalane or squalene as is discussed hereinafter.

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

A contemplated vaccine or inoculum advantageously also includes an adjuvant. Suitable adjuvants for vaccines and inocula of the present invention comprise those adjuvants that are capable of enhancing the antibody responses against B cell epitopes of the chimer, as well as adjuvants capable of enhancing cell mediated responses towards T cell epitopes contained in the chimer, if present. Adjuvants are well known in the art (see, for examp1e, Vaccine Design - The Subunit and Adjuvant Approach, 1995, Pharmaceutical Biotechnology, Volume 6, Eds. Powell, M.F., and Newman, M.J., Plenum Press, New York and London, ISBN 0-306-44867-X) .

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

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

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 emulsion. In addition to the immunogenic chimer protein particles, 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^_f tty acid esters of sorbitan and mannide such as sorbitan mono-stearate, sorbitan mono-oleate and mannide mono-oleate. An immunogen-containing emulsion is administered as an emulsion.

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

The use of small molecule adjuvants is also contemplated herein. One type of small molecule adjuvant useful herein is a 7-substituted-8-oxo- or 8-sulfo-guanosine derivative described in U.S. Patents No. 4,539,205, No. 4,643,992, No. 5,011,828 and No. 5,093,318, whose disclosures are incorporated by reference. Of these materials, 7-allyl-8- oxoguanosine (loxoribine) is particularly preferred. That molecule has been shown to be particularly effective in inducing an antigen- (immunogen-) specific response.

Still further useful adjuvants include monophosphoryl lipid A (MPL) available from Corixa Corp. (see, U.S. Patent No. 4,987,237), CpG (also ODN; oligonucleotides containing the CpG nucleotide motif one or more times plus flanking sequences) available from Coley Pharmaceutical Group, QS21 available from Aquila Biopharmaceuticals, Inc., SBAS2 (now AS02) available from SKB (now Glaxo-SmithKline) that contains QS21 and MPL ion an oil-in-water emulsion, the so-called muramyl dipeptide analogues described in U.S. Patent No. 4,767,842, and MF59 available from Chiron Corp. (see, U.S. Patents No. 5,709,879 and No. 6,086,901).

More particularly, immunologically active saponin fractions having adjuvant activity derived from the bark of the South American tree Quillaja Saponaria Molina { e . g. Quil™ A) are also useful. Derivatives of Quil™ A, for example QS21 (an HPLC purified fraction derivative of Quil™ A) , and the method of its production is disclosed in U.S. Patent No. 5,057,540. In addition to QS21 (known as QA21) , other fractions such as QA17 are also disclosed.

3 -De-O-acylated monophosphoryl lipid A is a well-known adjuvant manufactured by Ribi Immunochem, Hamilton, Montana. The adjuvant contains three components extracted from bacteria: monophosphoryl lipid (MPL) A, trehalose dimycolate (TDM) and cell wall skeleton (CWS) (MPL+TDM+CWS) in a 2% squalene/Tween^® 80 emulsion. This adjuvant can be prepared by the methods taught in GB 2122204B. A preferred form of 3-de-O-acylated monophosphoryl lipid A is in the form of an emulsion having a small particle size less than 0.2 μm in diameter (EP 0 689 454 Bl) .

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

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

Adjuvants are utilized in an adjuvant amount, which can vary with the adjuvant, host animal and recombinant HBc chimer immunogen. Typical amounts can vary from about 1 μg to about 1 mg per immunization. Those skilled in the art know that appropriate concentrations or amounts can be readily determined.

Inocula and vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Additional formulations that are suitable for other modes of administration include suppositories and, in some cases, oral formulation or by nasal spray. For suppositories, traditional binders and carriers can include, for example, polyalkalene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1-2%. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like .

An inoculum or vaccine composition takes the form of a solution, suspension, tablet, pill, capsule, sustained release formulation or powder, and contains an immunogenic effective amount of HBc chimer, preferably as particles, as active ingredient . In a typical composition, an immunogenic effective amount of preferred HBc chimer particles is about 1 μg to about 1 mg of active ingredient per dose, and more preferably about 5 μg to about 50 μg per dose, as noted before.

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

The HBc chimer particles and HBc chimer particle conjugates can be formulated into the vaccine as neutral or salt forms. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the protein or hapten) and are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived form inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2- ethylamino ethanol, histidine, procaine, and the like.

The inocula or vaccines are administered in a manner compatible with the dosage formulation, and in such amount as are therapeutically effective and immunogenic (an antibody-inducing amount or protective amount, as is desired) . The quantity to be administered depends on the subject to be treated, capacity of the subject's immune system to synthesize antibodies, and degree of protection desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual. However, suitable dosage ranges are of the order of several hundred micrograms active ingredient per individual . Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed in intervals (weeks or months) by a subsequent injection or other administration.

Once immunized, the host animal is maintained for a period of time sufficient for the recombinant HBc chimer immunogen to induce the production of a sufficient titer of antibodies that bind to the M2 protein. The maintenance time for the production of anti-M2 antibodies typically lasts for a period of about three to about twelve weeks, and can include a booster, second immunizing administration of the vaccine. A third immunization is also contemplated, if desired, at a time 24 weeks to five years after the first immunization. It is particularly contemplated that once a protective level titer of antibodies is attained, the vaccinated host animal is preferably maintained at or near that antibody titer by periodic booster immunizations administered at intervals of about 1 to about 5 years .

The production of antibodies is readily ascertained by obtaining a plasma or serum sample from the immunized host and assaying the antibodies therein for their ability to bind to a synthetic M2 polypeptide antigen in an ELISA assay as described hereinafter or by another immunoassay such as a Western blot as is well known in the art.

It is noted that the before-described antibodies so induced can be isolated from the blood of the host using well-known techniques, and then reconstituted into a second vaccine for passive immunization as is also well known. Similar techniques are used for gamma-globulin immunizations of humans. 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 an M2 polypeptide is utilized as the antigen immobilized on the chromatographic column.

Inocula are preparations that are substantially identical to vaccines, but are used in a host animal in which antibodies to influenza are desired to be induced, but in which protection from influenza is not desired.

Without further elaboration, it is believed that one skilled in the art can, using the preceding description and the detailed examples below, utilize the present invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limiting of the remainder of the disclosure in any way whatsoever.

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 Ncol-Hindlll restriction fragments and subsequent expression in E. coli host cells. To modify the pKK223-3 plasmid vector, a new Sphl-HindiII fragment was prepared using the PCR primers pKK223 -3/433 -452 -F and pKK223-NcoI-mod-R, and pKK223-3 as the template.

This PCR fragment was cut with the restriction enzymes Sphl and Hindlll to provide a 467 bp fragment that was then ligated with a 4106 bp fragment of the pKK223-3 vector, replacing the original 480 bp Sphl-HindiII fragment. The resultant plasmid (pKK223-3N; 4573 bp) is therefore 13 bp shorter than the parent plasmid and contains modified nucleotide sequence upstream of the introduced Ncol site (see Fig. 2, in which the dashes indicate the absent bases) . Restriction sites in plasmid pKK223- 3N are indicated in Fig. 2 and nucleotide changes made to the pKK223-3 parent plasmid are indicated by an underline as shown below.

pKK223-3/433-452-F GGTGCATGCAAGGAGATG SEQ ID NO: 201

pKK223-NcoI-mod-R

GCGAAGCTTCGGATCccatggTTTTTTCCTCCTTATGTGAAATTGTTATCCG- CTC SEQ ID NO: 202

B. Preparation of VI, V2 and V8 Cloning Vectors

Modified HBcl49 (VI and V2) or HBcl83 (V8) genes, able to accept the directional insertion of synthetic dsDNA fragments into the immunodominant loop region, were constructed using PCR. (The plasmid accepting inserts between amino acids E77 and D78 and truncated to V149 was named VI, the plasmid accepting inserts between D78 and P79 and truncated to V149 was named V2 , and the plasmid accepting inserts between D78 and P79 and terminating at C183, was called V8) . The HBcl49 and HBcl83 genes were amplified in two halves using two PCR primer pairs, one of which amplifies the amino terminus, the other amplifies the carboxyl terminus. 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) ; for V8, the products are a 249 bp (N-terminus) and a 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 HindiII. The VI, V2 and V8 fragment pairs were then ligated together at the common EcoRI overhangs . The resultant Ncol-Hindlll fragments were then ligated into the pKK223-3N vector, which had been prepared by digestion with Ncol and Hindlll.

To insert B cell epitopes into the VI, V2 and V8 plasmids, the appropriate plasmid was digested with EcoRI and Sad restriction enzymes. Synthetic dsDNA fragments containing 5 ' EcoRI and 3 ' Sacl overhangs were then inserted. In all cases, VI, V2 , and V8, glycine-isoleucine (EcoRI) and glutamic acid- leucine (Sad) amino acid pairs, flank the inserted B cell epitopes. The inserted restriction sites are underlined in the primers below.

VI

HBcl49/NcoI-F

5 ' -TTGGGCCATGGACATCGACCCTTA SEQ ID NO: 203

HBc-E77/EcoRI-R

5 ' -GCGGAATTCCTTCCAAATTAACACCCACC SEQ ID NO: 204

HBc-D78/EcoRI-SacI-F

5 ' -CGCGAATTCAAAAAGAGCTCGATCCAGCGTCTAGAGAC

SEQ ID NO: 205

HBcl49/HindIII-R

5 ' -CGCAAGCTTAAACAACAGTAGTCTCCGGAAG SEQ ID NO: 206

V2

HBcl49/NcoI-F

5' -TTGGGCCATGGACATCGACCCTTA SEQ ID NO: 203 HBc-D78/EcoRI-R

5 ' -GCGGAATTCCATCTTCCAAATTAACACCCAC SEQ ID NO: 207

HBc-P79/EcoRI-SacI-F

5 ' -CGCGAATTCAAAAAGAGCTCCCAGCGTCTAGAGACCTAG

SEQ ID NO: 208

HBcl49/HindIII-R

5'-CGCAAGCTTAAACAACAGTAGTCTCCGGAAG SEQ ID NO: 206

V8

HBcl49/NcoI-F

5 ' -TTGGGCCATGGACATCGACCCTTA SEQ ID NO: 203

HBC-D78/ECORI-R

5 ' -GCGGAATTCCATCTTCCAAATTAACACCCAC SEQ ID NO: 207

HBc-P79/EcoRI-SacI-F

5 ' -CGCGAATTCAAAAAGAGCTCCCAGCGTCTAGAGACCTAG

SEQ ID NO: 208

HBcl83/HindIII-R

5'-GGAAAGCTTACTAACATTGAGATTCCCG SEQ ID NO: 209

Preparation of V34 and V55 Cloning Vectors

Modified HBcl49 genes, able to accept the directional insertion of synthetic dsDNA fragments into the N-terminal region, 5' to the pre-core sequence LGWLWG, were constructed using PCR. (The plasmid that encoded an HBc sequence terminating at V149 was named V34, whereas the plasmid that encoded an HBc sequence harboring an additional cysteine, C- terminal to V149, was named V55. ) The HBcl49 gene was amplified in two halves using two PCR primer pairs, one of which amplifies the amino terminus (for which VI was used as a template) , the other amplifies the carboxyl terminus. For V34, the products of the PCR reactions were a 293 bp (N-terminus) fragment and a 484 bp (C-terminus) fragment; for V55, the same N- terminal fragment was used and a 490 bp C-terminal fragment was prepared.

The N-terminal fragment prepared by PCR was digested with Ncol and Sad, and the C-terminal fragments were digested with Sacl and Hindlll. The V34 and V55 fragment pairs were then ligated together at the common Sad overhangs. The resultant Ncol- Hindlll fragments were then ligated into the pKK223- 3N vector, which had been prepared by digestion with Ncol and HindiII.

B cell epitope insertion was accomplished by a procedure identical to that outlined above for the VI cloning vector. Restriction sites are underlined in the oligonucleotides primers below.

V34/V55

pKK-BamHI-F

5 ' -GCGGGATCCGGAGCTTATCGA SEQ ID NO: 210

HBc-NcoI/EcoRI/Sacl-R

5 ' -GCGGAGCTCTTTTTGAATTCCCATGGTTTTTTCCTCCTTAT

SEQ ID NO: 211

PreC- SacI -HBc -F 5 ' -GCGGAGCTCCTTGGGTGGCTTTGGGGCATTGACATCGACCCTTATAAAG

SEQ ID NO: 212

V34

HBcl49/HindIII-R

5 ' -CGCAAGCTTAAACAACAGTAGTCTCCGGAAG SEQ ID NO: 206

V55

HBcl49+C/HindIII-R

5 ' -CGCAAGCTTACTAGCAAACAACAGTAGTCTCCGGAAG

SEQ ID NO: 213

D. Preparation of V47, V48 and V54 Cloning Vectors

Modified HBcl49 and HBcl83 genes, able to accept the directional insertion of synthetic dsDNA fragments into the N-terminal region between amino acid residues 13 and D4 were constructed using PCR. (The plasmid encoding an HBc chimer terminating at V149 was named V47, the plasmid encoding an HBc chimer harboring an additional cysteine, C-terminal to V149, was named V54, and the plasmid encoding an HBc chimer terminating at C183 was named V48) . For V47, V48 and V54, a PCR primer pairs was used to amplify the amino terminus, from the template VI, including sequence preceding the HBc gene . The resultant PCR fragment of 190 bp for the C-terminus of V47, the HBc gene was amplified using a PCR primer pair resulting in a 469 bp fragment; for V54, the C- terminal fragment is 475 bp . For the C-terminus of V48, the HBcl83 gene was amplified using a PCR primer pair, resulting in a 574 bp fragment.

The cloning procedure used from this point was identical to that outlined for the cloning vector VI.

To insert epitopes into the V47, V48 and V54 plasmids, the plasmids were first digested with Ncol and Sad restriction enzymes. Synthetic dsDNA fragments containing 5' Afllll and 3' Sacl overhangs were then inserted (note, restriction enzymes Afllll and Ncol leave compatible overhangs) . In all cases, V47, V48, and V54, HBc residues D2 and 13 were deleted so that the sequence of the epitope directly follows residue Ml; the glutamic acid-leucine (EL) amino acid pairs, coded for by the Sad restriction site, follows the inserted epitope. The inserted restriction sites are underlined in the oligonucleotide primers below.

pKK(167-150) -F

5' -GCATAATTCGTGTCGCTC

SEQ ID NO: 214

HBc-I3/EcoRI-R

5' -GCGGAATTCCGATGTCCATGGTTTTTTCCT

SEQ ID NO: 215

HBc-EcoRl/Sacl/D4-F

5 ' -GCGGAATTCAAAAAGAGCTCGACCCTTATAAAGAATTTGGA

SEQ ID NO: 216

V47 HBcl49/HindIII-R

5 ' -CGCAAGCTTAAACAACAGTAGTCTCCGGAAG

SEQ ID NO: 206

V54

HBcl49+C/HindIII-R

5' -CGCAAGCTTACTAGCAAACAACAGTAGTCTCCGGAAG

SEQ ID NO: 213

V48

HBcl83/HindIII-R SEQ ID NO: 209

5' -GGAAAGCTTACTAACATTGAGATTCCCG

E. 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 HindiII site to facilitate directional insertion of synthetic dsDNAs into EcoRI-Hindlll (or EcoRI-SacI) restriction sites. The pair of PCR primers below was used to amplify the HBc 149 gene with a Ncol restriction site at the amino-terminus and EcoRI, Sa and HindiII sites at the carboxyl-terminus . The product of the PCR reaction (479 bp) was digested with Ncol/Hindlll and cloned into pKK223-3N to form V7.

To insert T cell epitopes, the plasmid (V7) was digested EcoRl/Hindlll (or EcoRI-SacI) and synthetic dsDNA fragments having EcoRl/Hindlll (or EcoRl/SacI) overhangs, were ligated into V7. 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/Sad, there are additional glutamic acid-leucine residues after the T cell epitope, prior to the termination codon, contributed by the Sad site. Restriction sites are again underlined in the primers shown .

HBcl49/NcoI-F

5 ' -TTGGGCCATGGACATCGACCCTTA SEQ ID NO: 203

HBcl49/SacI-EcoRI-H3-R

5 ' -CGCAAGCTTAGAGCTCTTGAATTCCAACAACAGTAGTCTCCG

SEQ ID NO: 221

F. 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, are constructed from V2 and V7 vectors . The carboxyl terminus of a V7 vector containing a T cell epitope inserted at EcoRl/Hindlll is amplified using two PCR primers (HBc-P79/SacI-F and pKK223-2/4515-32R) to provide a dsDNA fragment corresponding to amino acids 79-149 plus the T cell epitope, flanked with Sad and HindiII restriction sites.

The PCR products are cut with Sad and HindiII and then cloned into the desired V2 vector prepared by cutting with the same two enzymes. The PCR primers are amenable for the amplification of the carboxyl terminus of all V7 genes, irrespective of the T cell epitope present after amino acid 149 of the HBc gene .

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: 203) and the mis-match reverse PCR primer, which facilitated the C17A mutation. The resultant PCR product was digested with Ncol and HindiII and cloned back into pKK223-3N (previously cut with the same enzymes) . Restriction sites are underlined.

HBc-P79/SacI-F

5 ' -CGCGAGCTCCCAGCGTCTAGAGACCTAG SEQ ID NO: 222

pKK223-2/4515-32R

5 ' -GTATCAGGCTGAAAATC SEQ ID NO: 223

G. 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 are inserted into EcoRI/Sad restriction sites. Synthetic dsDNA fragments, encoding B and T cell epitopes of interest, are prepared by mixing complementary single stranded DNA oligonucleotides at equimolar concentrations, heating to 95°C for 5 minutes, and then cooling to room temperature at a rate of -1 °C per minute. This annealing reaction is performed in TE buffer. The double-stranded DNAs are shown below with the encoded epitope sequence shown above. The pound symbol, #, is used in some of the amino acid residue sequences that follow to indicate the presence of a stop codon. Pfl

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

N P E L SEQ ID NO: 224

ATCCGGAGCT SEQ ID NO: 225

TAGGCC SEQ ID NO: 226

P-----3

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

N A N P N V D P N A N P E L SEQ ID NO: 227

ACGCTAATCCGAACGTTGACCCGAACGCTAATCCGGAGCT SEQ ID NO: 228

TGCGATTAGGCTTGCAACTGGGCTTGCGATTAGGCCTCGAGG SEQ ID NO: 229

Pf3.1

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

N A N P E L SEQ ID NO: 230

AAATGCGAACCCAGAGCT SEQ ID NO: 231

TTTACGCTTGGGTC SEQ ID NO: 232

Pf3.2

I N A N P N A N P N A N P N V D P AATTAACGCGAATCCGAATGCCAACCCTAACGCCAACCCAAACGTGGATCCGA TTGCGCTTAGGCTTACGGTTGGGATTGCGGTTGGGTTTGCACCTAGGCT N A N P E L SEQ ID NO: 233

ATGCGAACCCAGAGCT SEQ ID NO: 234

TACGCTTGGGTC SEQ ID NO: 235

Pf3.3

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

N A N P N V D P N A N P E L SEQ ID NO:236 ACGCCAACCCGAATGTTGACCCCAATGCCAATCCGGAGCT SEQ ID NO: 237 TGCGGTTGGGCTTACAACTGGGGTTACGGTTAGGCC SEQ ID NO: 238

Pf3.4

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

N P N^' V E L SEQ ID NO: 239

ACCCGAATGTTGAGCT SEQ ID NO: 240

TGGGCTTACAAC SEQ ID NO: 241

Pf3.5

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

N P N V D P E L SEQ ID NO: 242

ACCCGAATGTTGACCCTGAGCT SEQ ID NO: 243

TGGGCTTACAACTGGGAC SEQ ID NO: 244

Pf3.6 I N P N V D P N A N P N A N P N A AATTAATCCGAACGTGGATCCAAATGCCAACCCTAACGCTAATCCAAACGCCA TTAGGCTTGCACCTAGGTTTACGGTTGGGATTGCGATTAGGTTTGCGGT

N P N V D P N A E L SEQ ID NO: 245

ACCCGAATGTTGACCCTAATGCTGAGCT SEQ ID NO: 246

TGGGCTTACAACTGGGATTACGAC SEQ ID NO: 247

Pf3.7

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

N V E L SEQ ID NO: 248

ATGTTGAGCT SEQ ID NO: 249

TACAAC SEQ ID NO: 250

Pf3.8

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

N V D P E L SEQ ID NO: 251

ATGTTGACCCTGAGCT SEQ ID NO: 252

TACAACTGGGAC SEQ ID NO: 253

Pf3.9

I N V D P N A N P N A N P N A N P AATTAACGTGGATCCAAATGCCAACCCTAACGCTAATCCAAACGCCAACCCGA TTGCACCTAGGTTTACGGTTGGGATTGCGATTAGGTTTGCGGTTGGGCT N V D P N A E L SEQ ID NO: 254

ATGTTGACCCTAATGCTGAGCT SEQ ID NO:255

TACAACTGGGATTACGAC SEQ ID NO: 256

Pf3.10

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

N V E L SEQ ID NO: 257 CGAATGTTGAGCT SEQ ID NO: 258 GCTTACAAC SEQ ID NO: 259

Pf3.11

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

D P E L SEQ ID NO: 260

ACCCTGAGCT SEQ ID NO: 261

TGGGAC SEQ ID NO: 262

Pf3.12

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

D P N A E L SEQ ID NO: 263

ACCCTAATGCCGAGCT SEQ ID NO: 264

TGGGATTACGGC SEQ ID NO: 265 I . P.falciparum Universal T cell Epitope

Pf-UTC (PF/CS326-345)

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

C S V T # # SEQ ID NO: 266

GCTCCGTTACCTAGTA SEQ ID NO: 267

CGAGGCAATGGATCATTCGA SEQ ID NO: 268

P.vivax CS-repeat B cell Epitopes

Pv-TIA

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

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

GCCAGCCGGCTGGCGAGCT SEQ ID NO: 270

CGGTCGGCCGACCGC SEQ ID NO: 271

Pv-TIB

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

A G E L SEQ ID NO: 272

CAGGGGAGCT SEQ ID NO: 273

GTCCCC SEQ ID NO: 274

PV-T2A I A N G A G N Q P G A N G A G D Q AATTGCGAACGGCGCCGGTAATCAGCCGGGGGCAAACGGCGCGGGTGATCAAC CGCTTGCCGCGGCCATTAGTCGGCCCCCGTTTGCCGCGCCCACTAGTTG

P G E L SEQ ID NO: 275

CAGGGGAGCT SEQ ID NO: 276

GTCCCC SEQ ID NO: 277

PV-T2B

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

P G E L SEQ ID NO: 278

CAGGCGAGCT SEQ ID NO: 279

GTCCGC SEQ ID NO: 280

PV-T2C

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

P G A N G A D N Q P G A N G A D D

CAGGCGCCAATGGTGCAGACAACCAGCCTGGGGCGAATGGAGCCGATGACC

GTCCGCGGTTACCACGTCTGTTGGTCGGACCCCGCTTACCTCGGCTACTGG

Q P G E L SEQ ID NO: 281

AACCCGGCGAGCT SEQ ID NO: 282

TTGGGCCGC SEQ ID NO: 283

PV-T3

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

Q E G G A A E L SEQ ID NO: 284

AAGAAGGCGGTGCAGCGGAGCT SEQ ID NO: 285

TTCTTCCGCCACGTCGCC SEQ ID NO: 286

Example 2 :> Preparation of Chimers Containing

Influenza A M2 Polypeptide Sequences

A. Insertion of Influenza A M2 N-terminal Domain into V34, V47, V48, V54, and V55 Cloning Vectors

V47

M2 (1-24/C17S)

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

C N D S S D E L SEQ ID NO: 287

TGTAACGATTCAAGTGATGAGCT SEQ ID NO: 288

ACATTGCTAAGTTCACTAC SEQ ID NO: 289

M2 (1-24/C19S)

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

S N D S S D E L SEQ ID NO: 290

TCGAACGATTCAAGTGATGAGCT SEQ ID NO: 291

AGCTTGCTAAGTTCACTAC SEQ ID NO: 292 B. Insertion of a Mutated Influenza A M2 (M2 (2-24/C17A,C19A) into Expression Vectors V2, V8 and V16

V2, V8, V16

M2 (2-24/C17A,C19A)

I S L L T E V E T P I R N E W G A R AATTTCTCTGTTAACCGAAGTGGAGACGCCGATTCGTAACGAATGGGGTGCGCGC AGAGACAATTGGCTTCACCTCTGCGGCTAAGCATTGCTTACCCCACGCGCG

A N D S S D E L SEQ ID NO: 293

GCCAATGATAGCTCTGACGAGCT SEQ ID NO: 294

CGGTTACTATCGAGACTGC SEQ ID NO: 295

C. Insertion of Influenza A M2 N-terminal domain into Vectors V34, V47, V54, and V55 For V34 and V55 constructs, synthetic dsDNA fragments coding for the M2 epitope (residues 1-24 of the influenza A M2 protein; SEQ ID NO: 9) were inserted into EcoRI/Sad restriction sites, whereas for V47 and V54 constructs, the same were inserted into Ncol/Sacl restriction sites. Synthetic dsDNA fragments were prepared by mixing complementary single stranded DNA oligonucleotides at equimolar concentrations, heating to 95°C for 5 minutes, and then cooling to room temperature at a rate of -1 °C per minute. This annealing reaction was performed in TE buffer. The double-stranded DNAs are shown below with the encoded epitope sequence shown above.

V3 /V55

M2 (2-24)

I S L L T Ξ V E T P I R N E W G C R

AATTAGCCTGTTAACCGAAGTGGAGACGCCGATCCGTAACGAATGGGGCTGCCG

TCGGACAATTGGCTTCACCTCTGCGGCTAGGCATTGCTTACCCCGACGGC

C N D S S D E L SEQ ID NO: 296

CTGTAATGATTCTTCCGACGAGCT SEQ ID NO: 297

GACATTACTAAGAAGGCTGC SEQ ID NO -.298

V47/V54

M2(l-24)

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

C N D S S D E L SEQ ID NO:299

TGTAACGATTCAAGTGATGAGCT SEQ ID NO: 300

ACATTGCTAAGTTCACTAC SEQ ID NO: 301

M2 (l-24/C17S,C19S)

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

S N D S S D E L SEQ ID NO: 302

TCGAACGATTCAAGTGATGAGCT SEQ ID NO: 303

AGCTTGCTAAGTTCACTAC SEQ ID NO: 304

D. Construction of Truncated Version of Native M2-HBc

The original M2-HBc construct [Neirynck et al., (October 1999) Nature Med. , 5 (10) : 1157-1163 : WO 99/07839] that contained the 183-residue, full length HBc sequence was truncated to V149, and the entire gene was moved into the pKK223-3 expression vector. To achieve this, the plasmid 3453, which was provided by the University of Gent, was used as a template for a PCR reaction that yielded a product of 523 bp. This product was digested with restriction enzymes Afllll and HindiII, and then ligated into the pKK223- 3Ν vector, which had been prepared by digestion with Ncol and Hindlll.

AflIII-M2-F

5' -CGCGACATGTCTCTGCTGACCG SEQ ID NO: 305

HBcl49/HindIII-R 5 ' -CGCAAGCTTAAACAACAGTAGTCTCCGGAAG SEQ ID NO : 206

Example 3 : Assay Procedures

A. Antigenicity

1. Particle ELISA

Purified particles were diluted to a concentration of 10 μg/mL in coating buffer (50 mM sodium bicarbonate, pH 9.6) and coated onto the wells of ELISA strips (50 μL/well) . The ELISA strips 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 μL/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 μL/well added to antigen-coated ELISA wells. Sera were incubated for 1 hour, washed with ELISA wash buffer (above) and probed using an anti-mouse (IgG) - HRP (The Binding Site, San Diego, CA; HRP = horseradish peroxidase) conjugate (50 μL/well) or other appropriate antibody for 30 minutes. After washing with ELISA wash buffer the reaction was visualized by the addition of TM blue substrate (50 μL/well) . After 10 minutes, the reaction was stopped by the addition of IN H₂S0₄ (100 μL/well) and read on an ELISA plate reader set at 450 nm.

2. Synthetic Peptide ELISA

A 24 amino acid _.residue synthetic peptide M2 is diluted to a concentration of 2 μg/mL in coating buffer (50 mM sodium bicarbonate, pH 9.6) and coated onto the wells of ELISA strips (50 μL/well) . Peptides are dried onto the wells by incubating overnight (about 18 hours) , in a hood with the exhaust on. Next morning, the wells are washed with ELISA wash buffer (phosphate buffered saline, pH 7.4, 0.05% Tween^®-20) and blocked with 3% BSA in PBS (75 μL/well) for 1 hour. ELISA strips are stored, dry, at -20°C until needed.

To determine antibody antigenicity of particles, antisera (monoclonal or polyclonal) are diluted using 1% BSA in PBS, and 50 μL/well added to antigen-coated ELISA wells. Sera are incubated for 1 hour, washed with ELISA wash buffer, and probed using an anti-mouse (IgG) -HRP conjugate or other antibody

(as above at 50 μL/well) for 30 minutes, washed again with ELISA wash buffer, and then visualized by the addition of TM blue substrate (50 μL/well) . After 10 minutes, the reaction is stopped by the addition of IN H₂S0₄ (100 μL/well) and read on an ELISA plate reader set at 450 nm.

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

Example 4 : Determination of 280:260 Absorbance Ratios

To determine the 280:260 absorbance ratio of purified particles, the particles were diluted to a concentration of approximately 0.2 mg/mL in 20 mM sodium phosphate buffer, pH 6.8, and absorbance values determined at wavelengths of 260 and 280 nm. The absorbance measured at 280 nm was divided by the value at 260 nm to determine the 280:260 ratio. The ratios were obtained for several samples, including native particles (HBcl83) , HBc particles truncated after residue position 149 (HBcl49) , and several HBc chimers that are identified elsewhere herein, are shown below in Table 1. Full length particles ICC- 1559 are a preparation of the particles first reported in Neirynck et al . , (Oct 1999) Nature Med. , 5(10) :1157-1163, whereas full length particles ICC- 1607 are similar particles in which the M2 polypeptide cysteines at polypeptide positions 17 and 19, (X₁₇ and X_9 of SEQ ID NO: 9) were mutated to serine residues .

Table 1

Example 5 : Thermal Stability Protocol

Purified particles were diluted to a concentration of 1 mg/mL using 50 mM NaP0₄ , pH 6.8 and sodium azide was added to a final concentration of 0.02% to prevent bacterial growth. Samples were mixed with SDS-PAGE sample buffer (reducing) and run on 15% SDS-PAGE gels. Gels were stained using Coomassie Blue, and then analyzed.

Example 6 : 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

(Amersham Pharmacia # 17-0537-01) and a BioCAD™ SPRINT Perfusion Chromatography System. The UV detector was set to monitor a wavelength of 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 NaP0₄, pH

6.8. 200 Microliters (μL) of the sample were then loaded onto a 200 μL loop and injected onto the column. The sample was eluted from the column with 50 M NaP0 , pH 6.8 at a flow rate of 0.75 mL/minute.

Particles containing N-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 BioCAD™ software (PerSeptive™) to provide the results in

Example 7 : Influenza M2 Constructs

Recently, Neirynck et al . , (Oct 1999) Nature Med. , 5 (10) : 1157-1163 and WO 99/07839 reported the fusion of the 24 amino acid extracellular domain- of M2 to the N-terminus of full-length HBc particles (HBcl83) , lacking amino acid residues 1-4. A schematic representation of that construct referred to herein as IM2HBc is shown below in which the 24- mer is linked to the N-terminus of HBc.

IM2HBG

MSLLTEVETPIRNEWGCRCNDSSD-HBc (5-183) SEQ ID NO: 306

In one illustrative preparation, the M2 epitope was inserted into the immunodominant loop of hepatitis B core and particles referred to as ICC- 'l475 were successfully expressed and purified using techniques discussed previously for such insertions and purifications. 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 particles) and the resulting particles purified. These two particles are illustrated schematically below.

ICC-1475 HBc (1-78) -GI-SLLTEVETPIRNEWGCRCNDSSD-EL-HBc (79- 149)

SEQ ID NO: 307

ICC-1473

HBc (1-78) -GI-SLLTEVETPIRNEWGARANDSSD-EL-HBc (79- 149) -C

SEQ ID NO: 308

The ICC-1473 particle construct yielded approximately 7-fold more purified particles when compared with the native sequence (ICC- 1475) . It remains to be determined if the mutation of the cysteine residues alters protective potential of the particles. However, epitopes delivered on the immunodominant loops of HBc are usually significantly more immunogenic as compared to when they are fused to other regions (including the N-terminus) , and resulting particles exhibit reduced anti-HBc immunogenicity .

Particles have also been prepared in which the M2 N-terminal 24-mer epitope was fused to the N- terminus of C-terminal truncated hepatitis B core particles. That construct (ICC-1438) also contained the N-terminal pre-core sequence (SEQ ID NO: 27). A similar construct was prepared that contained a single cysteine residue at the end of the hybrid protein (ICC-1492) , in this case immediately after Val-149 of the HBc gene. These constructs are shown schematically below.

ICC-1438

MGISLLTEVETPIRNEWGCRCNDSSDELLGWLWGI -HBc (2-149) SEQ ID NO: 309 ICC- 1492

MGISLLTEVETPIRNEWGCRCNDSSDELLGWLWGI-HBc (2-149) -C SEQ ID NO: 310

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 an isoleucine residue. Residues contributed by EcoRI (GI) and Sad (EL) restriction sites are underlined. The pre-core sequence is 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 Fig. 10. The instability of the ICC-1438 monomers was not evident using analytical gel filtration of particles.

Both ICC-1475 (Fig.10, lane 4) and ICC-1473 (Fig.10, lane 5) were expected to have slightly lower molecular weights than ICC-1438 and ICC-1492, because the former two contain the M2 epitope inserted directly into the immunodominant loop and therefore lack the pre-core sequence (SEQ ID NO: 27) present in ICC-1438 and ICC-1498. 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 containing a M2 N-terminal extracellular sequence as discussed above linked to the HBc N-terminus (Domain I) or loop (Domain II) and also containing a M2 protein C-terminal sequence such as that of SEQ ID NO: 12 (see Table A) linked the loop (Domain II) or at the C-terminus (Domain IV) of HBc is also contemplated. Such a contemplated construct also contains at least one stabilizing C-terminal cysteine residue as discussed elsewhere herein.

To modify the amino-terminus of hybrid HBc particles containing immunodominant loop fusions to incorporate a cysteine residue, and minimal M2- derived sequence, a series of synthetic oligonucleotides are synthesized. To make V2.Pfl(N- M2 (17-24/C17S) , the oligonucleotides M2 (17-24/C17S) - Ncol-F and HBcl49/HindIII-R are used to amplify the hybrid HBc gene from vector V2.Pfl. The resultant 546 bp fragment is cleaved with Ncol and HindiII and inserted into pKK-223-3N, which has been cleaved with the same two enzymes .

To make V2. Pf1 (N-M2 (17-24/C19S) , the oligonucleotides M2 (17-24/C19S) -Ncol-F and HBcl49/HindIII-R are used to amplify the hybrid HBc gene in vector V2.Pfl. The resultant 540 bp fragment is cleaved with Ncol and Hindlll and inserted into pKK-223-3N, which had been cleaved with the same two enzymes .

M2 (17-24/C17S) -Ncol-F G S R C N D S S D I D P Y K E F G ■ GGCGCCATGGGGTCTAGATGTAACGATTC.AAGTGACATCGACCCTTATAAAGAATTTCG

SEQ ID NO: 311

SEQ ID NO: 312

M2 (17-24/C19S) -Ncol-F

M G C N D S S D I D P Y K E SEQ ID NO : 313 GCGCCATGGGGTGTAACGATTCAAGTGACATCGACCCTTATAAAGAATTTGG

SEQ ID NO : 314

Example 8 : HBc Chimer Molecules With and Without

Both N- and C-Terminal Cysteine Residues

A series of HBc chimer molecule-containing particles was prepared that contained residues 1-24 of the influenza A, M2 protein peptide-bonded at or near the N-terminus of HBc whose C-terminus was truncated at residue 149. The component chimeric protein molecules contained different N-terminal sequences that included an M2 sequence or variant, and some contained a C-terminal cysteine residue.

All purified particles listed in Table 2, below, were analyzed by analytical size exclusion chromatography to assess the retention of particulate structure following purification. Particles designated ICC-1603, which contain no N-terminal cysteine residues, displayed evidence of disassembly back to sub-particulate structures (Fig. 3) because the protein eluted in the 1500 second range (particles elute at approximately 1000 seconds) .

Similar analysis of particles ICC-1590, which are similar to ICC-1603 ICC-particles except for the mutation of two serine residues to cysteine residues in the N-terminal M2 sequence, revealed that that construct remained particulate following purification, with elution occurring at around 1000 seconds, which is typical for a hybrid particle (Fig. 4) . There was no evidence of disassembly for ICC- 1590 particles. Analysis of ICC-1560 particles, whose chimer protein also has two N-terminal cysteine residues, revealed that it too was particulate following purification, although it did exhibit some degree of disassembly (Fig. 5) , suggesting that the stabilization was not quite as robust as it was for ICC-1590 particles. Comparison of the N-terminal configurations of ICC-1590 and ICC-1560 particles (Table 2, hereinafter), shows that the relative position of the two cysteine residues in ICC-1560 particles is shifted by 3 amino acid residues relative to ICC-1590 particles via the deletion of three amino acid residues (DEL) , indicating that the cysteine residues may be required to be a minimal distance from the start of the core gene to enable optimal cross-linking.

Example 9 : Particles With an M2 or M2 Variant

Sequence and A C-Terminal Cysteine Residue

ICC-1603 particles were shown in Fig. 3 to rapidly disassemble following purification. The HBc chimer molecules that comprise ICC-1605 particles are similar to those of ICC-1603 particles, except that the ICC-1605 component chimer molecules have a single C-terminal stabilizing cysteine. A plasmid was made to direct the expression of ICC-1605 particles to investigate if the addition of a C-terminal cysteine residue to ICC-1603 particles could impart greater stability on the particle. Following purification, ICC-1605 particles were analyzed using analytical size exclusion chromatography (Fig. 6) .

The results of this study demonstrated that particle stabilization was more complete than for the ICC-1603 particles, but incomplete compared to ICC- 1590 particles, which contains two amino-terminal cysteine residues and no C-terminal stabilizing cysteine. Although a significant amount of ICC-1605 remained particulate, there was evidence of a heterogeneous mixture of sub-particulate structures that eluted over a broad range. These observations suggest that for this hybrid particle (ICC-1603) , C- terminal stabilization as found in ICC-1605 particles was less complete than for the N-terminal stabilization found in ICC-1590 particles.

To investigate the compatibility of combined amino and carboxyl-terminal cysteine stabilization of hybrid particles, an expression plasmid was constructed to direct the expression of ICC-1604 particles. The component chimer molecules of ICC-1604 particles contain both the two amino-terminal stabilizing cysteine residues present in a native M2 polypeptide sequence (as in ICC-1590) as well as a C- terminal stabilizing cysteine (as in ICC-1605 particles) . Analysis of ICC-1604 particles showed that they retained a homogeneous particulate state following purification (Fig. 7) , indicating that the two stabilizing methods are complementary and can be used in concert with each other.

Alternative linker sequences between the N- terminus of HBc and the N-terminal cysteine residues were investigated using particles ICC-1438 and ICC- 1492. Both of these particles contain the amino acid sequence ELLGWLWGIDI (SEQ ID NO: 319) between the M2 fusion and amino acid D4 of HBc. Amino acid residues LGWLWGIDI are derived from amino acids -6 of pre-core to amino acid 13 of HBc, with the initiator codon of HBc mutated to an isoleucine to prevent translation initiation from this position, which would compromise the study. The HB pre-core sequence includes a cysteine at position -7.

These particles differed only in the fact that the ICC-1438 component chimer molecule terminated at position 149 of HBc, whereas the ICC- 1492 component chimer molecule terminated at 149 of HBc and contained a terminal cysteine at position 150 relative to the HBc of SEQ ID NO:l. When analyzed by analytical gel filtration, using an alternative but similar method to that discussed before, whereby particles elute at approximately 10 minutes, both constructs were shown to be particulate following purification (ICC-1438 in Fig. 8 and ICC-1492 in Fig.10). This study demonstrated the compatibility of amino- and carboxyl-terminal, cysteine stabilization of truncated particles, and the tolerance of substantial variability in the amino acid sequence and distance between the N-terminal cysteine residues and start of the HBc gene.

Table 2 :

Table 3, below, shows an alignment that illustrates the configuration of the N-termini of HBeAg, and particles designated ICC-1590, ICC-1560, ICC-1603, ICC-1604 and ICC-1605. Sequences are aligned according to amino acid residue position 4 from the N-terminus of HBc of SEQ ID NO:l that is shared by all constructs. N-terminal cysteine residues, when present, are underlined.

Table 3 Construct Name Sequence SEQ ID NO

HBeAg

SKLCLGWLWGMDID 315

ICC-1590/ICC-1604

MSLLTEVETPIRNEWGCRCNDSSDELD 316

ICC-1560

MSLLTEVETPIRNEWGCRCNDSSD 25

ICC-1603/ICC-1605

MSLLTEVETPIRNEWGSRSNDSSDELD 317

ICC-1438/ICC-1492

MGISLLTEVETPIRNEWGCRCNDSSDELLGWLWGIDID 318

Table 4, below, provides a tabulation of the results in which stability was assessed for particles containing an N-terminal influenza A M2 sequence or variant contemplated herein. As is seen, stable particles have been prepared from HBc chimer molecules that contain an N-terminal cysteine residue at a position of minus 14 (-14) relative to the N- terminus of the HBc sequence of SEQ ID NO : 1 to about the N-terminus itself. Table 4

Example 10 : Yield and Nucleic Acid Binding of M2 -Containing Particles

Yields are expressed as milligrams of purified particles from a 500 mL culture. Presence of bound nucleic acid was determined by measuring the A280:A260 ratio of the purified particle. A ratio of more than 1.0 indicates no bound nucleic acid, and a ratio of less than 1.0 indicates the presence of bound nucleic acid. The original full length IM2HBc described by Fiers and colleagues [Neirynck et al . , (1999) Nat . Med. , 5 (10) : 1157-1163] , is the same as ICC- 1559. Particle Epitope Insertion C- erminus Bound Yield Site Nucleic (mg/500

Acid mL)

1123 None NA Truncated/ No 16.6

(HBC149+ Stabilized

C)

1559 M2 (1-24) N-terminus Full Length Yes 3.2

(IM2HBC)

1604 M2 (1-24) N-terminus Truncated/ No 16.7 Stabilized

1569 M2 (2-24) ImmunodomiTruncated/ No 11.2 (C17S, nant loop Stabilized C19S) (Between D78 and P79)

1475 M2 (2-24) Immunodomi- Truncated/ No 1.1 nant loop Stabilized

(Between

D78 and

P79)

Example 11 : Antigenicity of Various M2 -Containing Particles

The antigenicity of the various particles to the monoclonal antibody 14C2 was examined using an ELISA. To ensure retention of particles in their native conformation, ELISA plates were first coated with a polyclonal antibody (rabbit) to capture the particles, which were then probed with various dilutions of either the 14C2 monoclonal antibody, or two anti-HBc monoclonal antibodies with specificity for the immunodominant loop region of HBc particles. The data, presented in the table below, demonstrate that presentation of M2e in the immunodominant loop of HBc does not significantly alter the accessibility of the M2e epitope to the 14C2 monoclonal antibody, relative to presentation at the N-terminus (IM2HBc/ICC-1559 and ICC-1604) . These observations were not surprising because it has shown previously been shown that 14C2 binds to an internal region of M2e (amino acids 8,10,11, and 14 of M2 , as opposed to the N-terminus [Zebedee et al . , (1988) J. Virol . , 62(8) :2762-2772] .

In addition, all particles, with the exception of ICC-1569, retained antigenicity to anti- HBc monoclonal antibodies 3120 and 3105. The loss of recognition by 3105 is a previously observed phenomenon for particles with sequences inserted into the immunodominant loop, and this typically translates to reduced anti-HBc responses for these particles following immunization. Monoclonal antibodies 3105 and 3120 were purchased from the Tokyo Institute of Immunology, Japan.

Example 12 : Immunogenicity of Various M2 -Containing Particles

The immunogenicity of ICC-1604 and ICC-1569 particles was investigated in mice. There was little difference in anti-M2e titers^' between the two particles, whereas a significant difference in the anti-HBc titers was observed between the two particles. ICC-1604 particles, with a native immunodominant loop, elicited anti-HBc titers that, like IM2HBc, were approximately 100 -fold higher than those for ICC-1569 particles. These data re-emphasize the fact that disruption of the immunodominant loop of HBc, by epitope insertion, which results in loss of recognition by the HBc monoclonal antibody 3105, dramatically decreases anti-HBc antibody responses. Conversely, both particles elicited anti-M2e antibody responses that were similar, and comparable to those seen previously for IM2HBc/lCC-1559, with end point titers of approximately 1:100,000.

Particles were formulated on Alhydrogel™, and groups of 10 mice were immunized with two 10 μg doses of formulated particles on days 0 and 28. Pooled sera were analyzed 2 weeks after the second injection for anti-HBc and anti-M2e antibody responses using ELISAs. Pooled sera from 10 mice at 2 weeks post boost were analyzed in ELISAs, with M2e (1- 24) synthetic peptide and recombinant HBc (ICC- 1123) serving as the capture antigens.

A direct comparison of ICC-1569 and ICC- 1604 particles in the mouse lethal challenge model revealed that both particles, when formulated on Alhydrogel™, afforded complete protection from a lethal challenge dose. These results are therefore consistent with the observation that both particles elicit similar titers of anti-M2e antibodies.

Compilation of data from multiple mouse studies, using an array of different particles and adjuvants, has revealed evidence of a possible correlation between titers of anti-M2e of the IgG2a subclass and protective efficacy. Mice displaying anti-M2e IgG2a titers of more than 10⁴ are reliably protected from a lethal challenge, whereas mice that exhibit anti-M2e IgG2a titers below 10⁴, but IgGl titers of more than 10⁴, typically show less complete protection. These data have relevance to the potential mechanism of protection in that they suggest that anti-M2e antibodies do not simply block the function of M2 , otherwise the protection would be independent of IgG subclass bias. Rather, because mouse IgG2a (and IgG2b) are the most efficient subclass for fixing complement and binding FcγRIII receptors, which are expressed by NK cell [Ravetch et al., (1991) Annu . Rev. Immunol . , 9:457-492], the data suggests an immune mechanism involving CDC and/or ADCC.

Example 13 : Antibody Subclass and Protection

A summary of several studies in which various M2e-HBcAconstructs (10 μg/mouse) and various adjuvants were assayed. About one-half were i.p. administration and about one-half i.n. For each group (14 mice) the sera were pooled and the titer of anti-M2e IgG subclass antibodies was determined. The results are from sera taken one week after the second boost . For mice where the IgG2a titer was more than 10 ⁴, the IgGl titer was 10 (*)

Adjuvants are increasingly being investigated for their ability to enhance the magnitude and persistence of immune responses to vaccines, as well as modulate the Thl/Th2 bias of the immune response. Although many experimental adjuvants are under investigation, alum remains the only adjuvant that is a component of FDA-approved vaccines in the US. Typically, alum biases immune responses towards a Th2 type, which is manifested by the production of high levels of IgGl antibody in mice.

It is found that alum-formulated M2e-HBc particles do elicit a significant IgGl response; however, IgG2a and IgG2b antibodies, which are Thl indicators, are also elicited. In an attempt to enhance the production of Thl-type IgG subclasses, the immunogenicity of Alhydrogel™-formulated particles supplemented with RC529, a synthetic derivative of MPL developed by Corixa Corporation, was tested in mice . These studies revealed that inclusion of RC529 in the Alhydrogel™ formulation resulted in a dramatic enhancement of anti-M2e IgG2a titers, increasing the anti-M2e IgG2a:IgGl ratio by approximately 10-fold. All mice in both groups were completely protected from lethal challenge; however, there was an indication of reduced morbidity (temperature decrease and weight loss) in mice immunized with ICC-1569 formulated with Alhydrogel™ + RC529, versus Alhydrogel™ alone.

Example 14. Partially Truncated HBc Particles : Synthesis of Expression Vectors for Expressing Partially Truncated Particles To prepare expression plasmids for expressing partially truncated HBc particles, a single amino terminal oligonucleotide PCR primer (HBcl49/NcoI-F) was used in combination with a unique C-terminal primer. For example, to prepare the HBcl56(E.Cr; ICC-1600 particles) expression plasmid, the primers HBcl49/NcoI-F and HBcl56 (E . cR) -H3-R are used. Primers HBcl49/NcoI-F and HBcl56C (E. cR) -H3-R are used to prepare the HBcl56 (E.cR) +C (ICC-1601 particles) expression plasmids. The sequences of all primers used are displayed below.

In addition to truncating the particles - and in some cases the incorporating a C-terminal cysteine residue - codons that are optimal for expression in E. coli were also used. It is known that several arginine codons, particularly AGA and AGG are rarely used by E. coli and are believe to be problematic for efficient expression of proteins in E. coli by leading to stalling of polypeptide synthesis during translation, resulting in premature termination. Of the 16 arginine codons between 150 and 183 of HBc, 7 are encoded by the rare AGA codon and 2 are encoded by the very rare AGG codon. Therefore, in this study, all AGA and AGG codons were replaced with codons that are more frequently used by E. coli . To enable sequential replacement of the rare arginine codons, HBcl56 genes are synthesized first (ICC-1600 and HBcl56+c ICC-1601 particles) , and then used as a template for the HBcl63 constructs (ICC- 1634 and HBcl63+C ICC-1632 particles) ; the HBcl63 constructs are thereafter used as template for the HBcl71 constructs (ICC-1642 and HBC171+C ICC-1643 particles) ; finally, the HBc 171 constructs are used as a templates for the arginine codon optimized HBcl82 and HBcl83 constructs. A non-optimized HBcl82 construct (ICC-1575) is also prepared for control purposes. All PCR products are cleaved with the restriction enzymes Ncol and Hindlll and cloned into the expression vector pKK223-3N, which had been cut with the same enzymes as discussed before.

Amino Terminal Primer Sequence (Ncol restriction site is underlined) :

HBcl49/NcoI-F

5 ' -TTGGGCCATGGACATCGACCCTTA SEQ ID NO: 203

Carboxyl -Terminal Primer Sequences (Hindlll restriction sites are underlined) :

HBcl56(E.cR) -H3-R

5 ' -GCGAAGCTTACTAAGGGGAGCGGCCTCGTCGACGAACAACAGTAGTCTCCGG

SEQ ID NO -.217

HBcl56C(E.cR) -H3-R

5 ' -GCGAAGCTTACTAACAAGGGGAGCGGCCTCGTCGACGAACAACAGTAGT- CTCCGG SEQ ID NO: 218

HBcl63 (E.cR) -H3-R

5 ' -GCGAAGCTTACTAAGGCGAGGGAGTGCGCCGACGAGGGGAGCGGCCTCG

SEQ ID NO: 219

HBcl63C(E.cR) -H3-R

5 ' -GCGAAGCTTACTAACAAGGCGAGGGAGTGCGCCGACGAGGGGAGCGGCCTCG

SEQ ID NO: 220

HBcl71(E.cR) -H3-R

5 ' -GCGAAGCTTACTACGGCGATTGAGAGCGTCGACGGCGAGGCGAGGGAGT

SEQ ID NO: 320

HBcl71C(E.cR) -H3-R

5 ' -GCGAAGCTTACTAACACGGCGATTGAGAGCGTCGACGGCGAGGCGAGGGAGT

SEQ ID NO: 321

HBcl83 (E.cR) -H3-R

5 ' -GCGAAGCTTACTAACATTGAGATTCCCGAGATTGAGATCGCCGGCGACGCGG-

CGATTGAGAGCGTC SEQ ID NO: 322 HBcl82 -H3 -R

5 ' -GCGAAGCTTACTATTGAGATTCCCGAGATTGA

SEQ ID NO: 323

HBC183-H3-R

5' -GGAA GCTTACTAACATTGAGATTCCCG

SEQ ID NO: 324

HBc1 9/HindiII-R

5 ' -CGCAAGCTTAAACAACAGTAGTCTCCGGAAG SEQ ID NO: 206

HBcl49+C/HindIII-R

5' -CGCAAGCTTACTAGCAAACAACAGTAGTCTCCGGAAG SEQ ID NO: 213

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

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

Claims

What is Claimed:

1. A recombinant chimer hepatitis B core (HBc) protein molecule up to about 515 amino acid residues in length that

(a) contains an HBc sequence of at least about 125 of the N-terminal 150 amino acid residues of the HBc molecule that includes (i) the HBc sequence of residue positions 4 through about 75 and about 85 through about 140, (ii) a peptide-bonded heterologous immunogenic epitope at one or more of the N-terminus, in the HBc immunodominant loop or the C-terminus of the chimer, or (iii) a heterologous linker residue for a conjugated epitope present in the HBc immunodominant loop,

(b) contains one to three cysteine residues at an amino acid position of the chimer molecule corresponding to amino acid position -20 to about +1 from the N-terminus of the HBc sequence of SEQ ID N0:1 [N-terminal cysteine residue (s) ] in a sequence other than that of the HBc precore sequence and zero to about three cysteine residues toward the C-terminus of the molecule from the C-terminal residue of the HBc sequence and within about 30 residues from the C-terminus of the chimer molecule [C-terminal cysteine residue (s)], said chimer molecule (i) containing no more than 20 percent conservatively substituted amino acid residues in the HBc sequence, (ii) self-assembling into particles that are substantially free of binding to nucleic acids on expression in a host cell, and said particles being more stable than are particles formed from otherwise identical HBc chimer molecules that are free of any above-mentioned C-terminal cysteine residue (s) and (i) lack the N-terminal cysteine residue (s) or (ii) in which an N-terminal cysteine residue (s) present in a contemplated chimer molecule is (are) replaced by another residue.

2. The recombinant HBc chimer protein molecule according to claim 1 wherein said peptide- bonded heterologous immunogenic epitope or a heterologous linker residue for a conjugated epitope is a heterologous immunogenic epitope.

3. The recombinant HBc chimer protein molecule according to claim 2 wherein said heterologous immunogenic epitope is a B cell epitope.

4. The recombinant HBc chimer protein molecule according to claim 3 that contains a second heterologous immunogenic epitope peptide-bonded to the N-terminus, in the HBc immunodominant loop or to the C-terminus of the chimer at a position different from that to which the first-named immunogenic epitope was bonded.

5. The recombinant HBc chimer protein molecule according to claim 3 wherein said B cell epitope is peptide-bonded in the immunodominant loop at a position between amino acid residues 76 and 85, and at least 5 residues of the HBc sequence of positions 76 to 85 are present.

6. The recombinant HBc chimer protein molecule according to claim 5 wherein the HBc sequence between amino acid residues 76 and 85 is present, but interrupted by said B cell epitope.

7. The recombinant HBc chimer protein molecule according to claim 2 further including a peptide-bonded immunogenic heterologous T cell epitope peptide-bonded to the N-terminus, in the HBc immunodominant loop or to the C-terminus of the chimer at a position different from that to which the first-named immunogenic epitope was bonded.

8. The recombinant HBc chimer protein molecule according to claim 7 wherein said T cell immunogenic epitope is peptide-bonded to the C-terminal HBc amino acid residue.

9. The recombinant HBc chimer protein molecule according to claim 8 wherein at least one of said C-terminal cysteine residue (s) is present.

10. The recombinant HBc chimer protein molecule according to claim 1 wherein said chimer contains the uninterrupted HBc amino acid residue sequence of position 4 through at least position 140, plus a cysteine residue at the N-terminus of the HBc chimer protein molecule.

11. The recombinant HBc chimer protein molecule according to claim 10 wherein said chimer contains the uninterrupted HBc amino acid residue sequence of position 4 through position 149.

12. The recombinant HBc chimer protein molecule according to claim 1 wherein said chimer contains a heterologous linker residue for a conjugated epitope present in the HBc immunodominant loop.

13. The recombinant HBc chimer protein molecule according to claim 12 wherein said heterologous linker residue for a conjugated epitope is peptide-bonded in the immunodominant loop at a position between amino acid residues 76 and 85, and at least 4 residues of the HBc sequence of positions 76 to 85 are present.

14. The recombinant HBc chimer protein molecule according to claim 13 wherein the HBc sequence between amino acid residues 76 and 85 is present, but interrupted by said heterologous linker residue for a conjugated epitope.

15. The recombinant HBc chimer protein molecule according to claim 14 that contains the HBc amino acid residue sequence of position 4 through at least position 140.

16., The recombinant HBc chimer protein molecule according to claim 15 wherein said chimer contains the HBc amino acid residue sequence of position 4 through position 149.

17. The recombinant HBc chimer protein molecule according to claim 16 wherein said heterologous linker residue for a conjugated epitope is selected from the group consisting of a lysine, aspartic acid, glutamic acid, cysteine and a tyrosine residue .

18. A recombinant hepatitis B virus core (HBc) protein chimer molecule with a length of about 135 to about 515 amino acid residues that contains four peptide-linked amino acid residue sequence domains from the N-terminus that are denominated Domains I, II, III and IV, wherein

(a) Domain I comprises about 71 to about 110 amino acid residues whose sequence includes (i) at least the sequence of the residues of position 5 through position 75 of HBc, (ii) one to three cysteine residues at an amino acid position of the chimer molecule corresponding to amino acid position -20 to about +1 from the N-terminus of the HBc sequence of SEQ ID NO:l [N-terminal cysteine residue (s) ] in a sequence other than that of the HBc precore sequence, and (iii) an optional heterologous immunogenic epitope containing up to about 30 amino acid residues peptide-bonded to one of HBc residues 2-4;

(b) Domain II comprises about 5 to about 250 amino acid residues peptide-bonded to HBc residue 75 of Domain I in which (i) zero to all residues in the sequence of HBc positions 76 to 85 are present peptide-bonded to (ii) an optionally present sequence of one to about 245 amino acid residues that are heterologous to HBc and constitute a heterologous immunogenic epitope or a heterologous linker residue for a conjugated epitope;

(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) five through fourteen residues of an HBc amino acid residue sequence from position 136 through 149 peptide-bonded to the residue of position 135 of Domain III, (ii) zero to three cysteine residues [C-terminal cysteine residue (s) ] within about 30 residues from the C- terminus of the chimer molecule, and (iii) zero to about 100 amino acid residues in an immunogenic sequence heterologous to HBc from position 150 to the C-terminus, said chimer molecule (i) having an amino acid residue sequence in which no more than about 10 percent of the amino acid residues are substituted in the HBc sequence of the chimer and (ii) self- assembling into particles on expression in a host cell, said particles being substantially free of binding to nucleic acids and being more stable than are particles formed from otherwise identical HBc chimer molecules that are free of any above-mentioned C-terminal cysteine residue (s) and (i) lack the N- terminal cysteine residue (s) or (ii) in which an N- terminal cysteine residue (s) present in a contemplated chimer molecule is (are) replaced by another residue.

19. The recombinant HBc chimer protein molecule according to claim 18 that contains two heterologous immunogenic epitopes.

20. The recombinant HBc chimer protein molecule according to claim 19 wherein said two heterologous immunogenic epitopes are present in Domains I and II, II and IV or I and IV.

21. The recombinant HBc chimer protein molecule according to claim 19 wherein one of said two heterologous immunogenic epitopes is a B cell epitope .

22. The recombinant HBc chimer protein molecule according to claim 19 wherein one of said two heterologous immunogenic epitopes is a T cell epitope .

23. The recombinant HBc chimer protein molecule according to claim 19 wherein one of said two heterologous epitopes is a B cell epitope and the other is a T cell epitope.

24. The recombinant HBc chimer protein molecule according to claim 18 wherein said Domain I includes a heterologous immunogenic epitope peptide- bonded to one of HBc residues 2-4 and said heterologous epitope is a B cell epitope.

25. The recombinant HBc chimer protein molecule according to claim 18 wherein Domain II contains a heterologous immunogenic epitope and said heterologous epitope is a B cell epitope. '

26. The recombinant HBc chimer protein molecule according to claim 18 wherein said sequence heterologous to HBc from position 150 to the C-terminus is an immunogenic T cell epitope peptide- bonded to one of HBc residues 140-149.

27. The recombinant HBc chimer protein molecule according to claim 18 wherein Domain II contains a heterologous linker residue for a conjugated epitope.

28. The recombinant HBc chimer protein molecule according to claim 24 that contains one to one C-terminal cysteine residue (s) within about 30 residues of the C-terminus of the chimer molecule.

29. The recombinant HBc chimer protein molecule according to claim 28 that contains a heterologous immunogenic epitope present in Domain II that is a B cell epitope.

30. The recombinant HBc chimer protein molecule according to claim 29 wherein said B cell epitope contains 6 to about 50 amino acid residues.

31. The recombinant HBc chimer protein molecule according to claim 28 wherein the HBc sequence between amino acid residues 76 and 85 is present, but interrupted by said heterologous immunogenic epitope.

32. The recombinant HBc chimer protein molecule according to claim 28 wherein said cysteine residue is located within about five amino acid residues of the C-terminus of the chimer protein molecule .

33. The recombinant HBc chimer protein molecule according to claim 18 wherein said sequence heterologous to HBc from position 150 to the C- terminus is an immunogenic T cell epitope peptide- bonded to one of HBc residues 140-149.

34. The recombinant HBc chimer protein molecule according to claim 18 wherein said heterologous linker residue for a conjugated epitope or a heterologous epitope is a heterologous linker residue for a conjugated epitope, and that heterologous linker residue for a conjugated epitope is selected from the group consisting of a lysine, aspartic acid, glutamic acid, cysteine and a tyrosine residue .

35. The recombinant HBc chimer protein molecule according to claim 34 that contains a single cysteine residue at the C-terminus of the HBc chimer protein molecule.

36. The recombinant HBc chimer protein molecule according to claim 29 wherein said B cell epitope is an amino acid sequence present in a pathogen selected from the group consisting of Streptococcus pneumonia, Cryptosporidium parvum, HIV, foot-and-mouth disease virus, influenza virus, Yersinia pestis, Haemophilus influenzae, Moraxella catarrhalis , Porphyromonas gingivalis, Trypanosoma cruzi , Plasmodium falciparum, Plasmodium vivax, Plasmodium berghi , Plasmodium yoelli , Streptococcus sobrinus, Shigella flexneri , RSV, Plasmodium Entamoeba histolytica, Schistosoma japonicum, Schistosoma mansoni , and ebola virus.

37. The recombinant HBc chimer protein molecule according claim 1 that is present as immunogenic particles, said particles being substantially free of bound nucleic acids, and being more stable than are particles formed from an otherwise identical HBc chimer substantially free of bound nucleic acids, and being more stable than are particles formed from an otherwise identical HBc chimer that lacks one or both of said N- or C- terminal cysteine residue (s) or in which one or both of said N- or C-terminal cysteine residues present in the chimer molecule are replaced by another residue.

38. The immunogenic particles according to claim 37 that exhibits a 280:260 absorbance ratio of about 1.2 to about 1.7.

39. The recombinant HBc chimer protein molecule according claim 18 that is present as immunogenic particles, said particles being substantially free of bound nucleic acids, and being more stable than are particles formed from an otherwise identical HBc chimer substantially free of bound nucleic acids, and being more stable than are particles formed from an otherwise identical HBc chimer that lacks one or both of said N- or C- terminal cysteine residue (s) or in which one or both of said N- or C-terminal cysteine residues present in the chimer molecule are replaced by another residue.

40. The immunogenic particles according to claim 39 that exhibits a 280:260 absorbance ratio of about 1.2 to about 1.7.

41. A vaccine or inoculum comprising an immunogenic effective amount of immunogenic particles in accordance with claim 37 dissolved or dispersed in a pharmaceutically acceptable diluent.

42. The vaccine or inoculum according to claim 41 that is adapted for parenteral administration or mucosal immunization.

43. The vaccine or inoculum according to claim 41 wherein said recombinant chimeric HBc protein molecule particles are present in plant tissue .

44. The vaccine or inoculum according to claim 41 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.

45. The vaccine or inoculum according to claim 41 that further includes an adjuvant.

46. The vaccine or inoculum according to claim 45 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.

47. The vaccine or inoculum according to claim 45 wherein said adjuvant is an oil that is emulsified with said immunogenic particles and said pharmaceutically acceptable diluent to provide an emulsion having a water phase and an oil phase.

48. The vaccine or inoculum according to claim 47 wherein the oil phase of said emulsion comprises squalene or squalane.

49. The vaccine or inoculum according to claim 47 wherein the water and oil phases of said emulsion are emulsified by an emulsifying agent that is a sorbitan C12-C24 fatty acid ester or a mannide ^c12~^c24 fatty acid ester.

50. A recombinant hepatitis B virus core (HBc) protein chimer molecule with a length of about 150 to about 325 amino acid residues that contains four peptide-linked amino acid residue sequence domains from the N-terminus that are denominated Domains I, II, III and IV, and includes at least one peptide-bonded polypeptide of about 6 to about 24 residues of the influenza A M2 polypeptide of SEQ ID NO : 9 wherein

(a) Domain I comprises (i) about 75 to about 110 amino acid residues whose sequence includes at least the sequence of the residues of position 4 through position 75 of HBc, (ii) one to three cysteine residues present at a position in the chimer molecule of about one to about -20 relative to the N- terminus of HBc of SEQ ID NO:l [N-terminal cysteine residue (s) ] , said one or more N-terminal cysteine residues being present within a sequence other than that of the pre-core sequence of HBc, and optionally includes said sequence of about 6 to about 24 residues of the influenza A M2 polypeptide of SEQ ID NO: 9 that, when present, is peptide-bonded to or within about 15 residues of the N-terminus of the HBc sequence,

(b) Domain II comprises about 10 to about 60 amino acid residues peptide-bonded to residue 75 of which (i) zero to all of the sequence of HBc is present from position 76 through 85 and (ii) an optional sequence of 6 to about 48 residues that constitute one or more repeats of the influenza A M2 polypeptide of SEQ ID NO: 9;

(c) Domain III is an HBc sequence from position 86 through position 135 peptide-bonded to residue 85; and d) Domain IV comprises (i) the residues of positions 136-140 plus up to nine residues of an HBc amino acid residue sequence from position 141 through 149 peptide-bonded to the residue of position 135 of Domain III, (ii) zero to three cysteine residues, and (iii) up to about 100 amino acid residues in a sequence heterologous to HBc from position 164 to the HBc C-terminus; said chimer molecule (i) containing no more than 10 percent conservatively substituted amino acid residues in the HBc sequence, (ii) self-assembling into particles that are substantially free of binding to nucleic acids on expression in a host cell, and said particles being more stable on formation than are particles formed from an otherwise identical HBc chimer that lacks said N-terminal cysteine residue (s) or in which an N-terminal cysteine residue present in the chimer molecule is replaced by another residue.

51. The recombinant HBc chimer protein molecule according to claim 50 wherein one of said ' residues Xiη and X]_o, of said M2 polypeptide of SEQ ID

NO: 9 is cysteine.

52. The recombinant HBc chimer protein molecule according to claim 50 wherein said M2 polypeptide of SEQ ID NO : 9 includes residues X2 through X24.

53. The recombinant HBc chimer protein molecule according to claim 50 wherein said M2 polypeptide of SEQ ID NO : 9 is present in Domain I.

54. The recombinant HBc chimer protein molecule according to claim 50 wherein said M2 polypeptide of SEQ ID NO: 9 is present in Domain II.

55. The recombinant HBc chimer protein molecule according to claim 50 wherein Domain I consists essentially of the HBc sequence from position 2 through position 75.

56. The recombinant HBc chimer protein molecule according to claim 50 wherein Domain IV contains zero cysteine residues .

57. The recombinant HBc chimer protein molecule according to claim 50 wherein Domain IV is free of said sequence heterologous to HBc at position 164 to the C-terminus.

58. A recombinant hepatitis B virus core (HBc) protein chimer molecule with a sequence of about 155 to about 225 amino acid residues that contains four peptide-linked domains from the N-terminus that are denominated Domains I, II, III and IV, and includes at least one peptide-bonded polypeptide of about 6 to about 24 residues of the influenza A M2 polypeptide of SEQ ID NO : 9 wherein (a) Domain I comprises (i) about 95 to about 100 amino acid residues whose sequence includes at least the sequence of the residues of position 4 through position 75 of HBc, (ii) one to three cysteine residues present at a position in the chimer molecule of about one to about -14 relative to the N-terminus of HBc of SEQ ID N0:1 [N-terminal cysteine residue (s) ] , said one or more N-terminal cysteine residues being present within a sequence other than that of the pre-core sequence of HBc, and optionally includes said sequence of about 6 to about 24 residues of the influenza A M2 polypeptide of SEQ ID NO: 9 that, when present, is peptide-bonded to or within about 15 residues of the N-terminus of the HBc sequence,

(b) Domain II comprises about 10 to about 60 amino acid residues peptide-bonded to residue 75 of which (i) zero to all of the sequence of HBc is present from position 76 through 85 and (ii) an optional sequence of 6 to about 48 residues that constitute one or more repeats of the influenza A M2 polypeptide of SEQ ID NO: 9;

(c) Domain III consists essentially of the HBc sequence from position 86 through position 135; and d) Domain IV comprises (i) the residues of positions 136-140 plus up to nine residues of an HBc amino acid residue sequence from position 141 through 149 peptide-bonded to the residue of position 135 of Domain III, (ii) zero or one cysteine residue, and (iii) up to about 50 amino acid residues in a sequence heterologous to HBc from position 164 to the HBc C-terminus ; said chimer molecule (i) containing no more than 5 percent conservatively substituted amino acid residues in the HBc sequence, (ii) self-assembling into particles that are substantially free of binding to nucleic acids on expression in a host cell, and said particles being more stable on formation than are particles formed from an otherwise identical HBc chimer that lacks said N-terminal cysteine residue (s) or in which an N-terminal cysteine residue present in the chimer molecule is replaced by another residue.

59. The recombinant HBc chimer protein molecule according to claim 58 wherein Domain IV includes a sequence of about nine amino acid residues of the HBc sequence from residue position 141 through about position 149 peptide-bonded to residue 140.

60. The recombinant HBc chimer protein molecule according to claim 58 wherein Domain I includes one N-terminal cysteine residue.

61. The recombinant HBc chimer protein molecule according to claim 58 wherein Domain IV includes one C-terminal cysteine residue.

62. The recombinant HBc chimer protein molecule according to claim 58 wherein Domain I includes one N-terminal cysteine residue and Domain IV includes one C-terminal cysteine residue.

63. The recombinant HBc chimer protein molecule according to claim 58 wherein one of said residues Xχ7 and X^g of said M2 polypeptide of SEQ ID NO: 9 is cysteine.

64. The recombinant HBc chimer protein molecule according to claim 58 wherein said M2 polypeptide of SEQ ID NO: 9 includes residues X2 through X2 .

65. The recombinant HBc chimer protein molecule according to claim 58 wherein Domain II comprises the amino acid residues of the sequence of HBc from position 76 through 85.

66. The recombinant HBc chimer protein molecule according to claim 58 wherein Domain II comprises the amino acid residues of the sequence of HBc from position 76 through 85 that further includes 6 to about 23 residues of the influenza A M2 polypeptide of SEQ ID NO: 9.

67. The recombinant HBc chimer protein molecule according to claim 58 wherein Domain I includes 6 to about 23 residues of the influenza A M2 polypeptide of SEQ ID NO : 9.

68. The recombinant HBc chimer protein molecule according to claim 58 that has a length of about 155 to about 170 residues.

69. Particles comprised of recombinant hepatitis B virus core (HBc) protein chimer molecules according to claim 50.

70. Particles comprised of recombinant hepatitis B virus core (HBc) protein chimer molecules according to claim 58.

71. A vaccine or inoculum comprising an immunogenic effective amount immunogenic particles according to claim 69 dissolved or dispersed in a pharmaceutically acceptable diluent .

72. A vaccine or inoculum comprising an immunogenic effective amount immunogenic particles according to claim 70 dissolved or dispersed in a pharmaceutically acceptable diluent.

73. A nucleic acid that encodes a recombinant HBc protein molecule according to claim 1, or a variant, analog or complement thereof.

74. A nucleic acid that encodes a recombinant HBc protein molecule according to claim 18, or a variant, analog or complement thereof.

75. A recombinant nucleic acid molecule that comprises a vector operatively linked to a nucleic acid segment defining a gene that encodes a recombinant HBc protein molecule according to claim 1, or a varient, analog or complement thereof, and a promoter suitable for driving the expression of the gene in a compatible host organism.

76. A recombinant nucleic acid molecule that comprises a vector operatively linked to a nucleic acid segment defining a gene that encodes a recombinant HBc protein molecule according to claim 58, or a varient, analog or complement thereof, and a promoter suitable for driving the expression of the gene in a compatible host organism.

77. A host cell transformed with a recombinant nucleic acid molecule according to claim 75.

78. The transformed host cell according to claim 77 wherein said host cell is selected from the group consisting of E. coli , S . typhi , S. typhimurium and a S. typhimurium-E . coli hybrid.