Strategically modified hepatitis B core proteins and their derivatives

A strategically modified hepatitis B core protein is described, where an insert is provided, preferably in an immunodominant region of the nucleocapsid protein, containing a chemically reactive amino acid residue. The modified hepatitis B core protein or its aggregated nucleocapsid protein particles can be pendently linked to a hapten to form a modified nucleocapsid conjugate. Such a conjugate is useful in the preparation of vaccines or antibodies. The modified hepatitis B core protein can also be modified to include a T cell epitope.

TECHNICAL FIELD
 The present invention relates to the intersection of the fields of
 immunology and protein engineering, and particularly to carrier proteins,
 and more particularly to a hepadnavirus nucleocapsid protein strategically
 modified with an inserted chemically-reactive amino acid residue, a
 pendently-linked hapten conjugate of that hepadnavirus nucleocapsid
 protein and to an immunogenic particle comprised of those conjugates.
 BACKGROUND OF THE INVENTION
 It is known that antibodies can be raised to a small molecule by using a
 large immunogenic protein molecule as a carrier. The small molecule that
 derives enhanced immunogenicity by being conjugated to the carrier is
 called a hapten. The phenomenon of a relatively large molecule
 potentiating the immunogenicity of a small molecular entity to which it is
 attached is known in the art as the "carrier effect."
 The portion of an immunogen recognized by the helper T cell (Th cell) is
 the T cell determinant or epitope. The portion of an immunogen that is
 bound by antibody is the B cell determinant or epitope. Carrier effects
 can be defined as immunity to one determinant, the "helper" or T (T.sub.h)
 cell determinant, of a multideterminant immunogen enhancing the immune
 response to another determinant, the B cell determinant.
 It is now well established that most immunogens require T cell help to
 induce B cells to produce antibodies. Thus, T.sub.h cells, by recognizing
 helper determinants on the immunogen help B cells to make antibody against
 the immunogen.
 The antigenic determinants recognized by T helper cells (T.sub.h) and B
 cells must be associated to form a single molecular entity, but they do
 not have to be covalently linked. See, Russel et al., Nature, 280:147
 (1979), Lamb et al., J. Immunol., 129:1465 (1982), Scherle et al., J. Exp.
 Med., 164:1114 (1986) and Lake et al., Cold Spring Harbor Symp. Quant.
 Biol., 41:589 (1976). Some immunogens do not require T cell help to induce
 antibody formation, these are T-independent antigens.
 A pathogen-related protein can be immunologically mimicked by the
 production of a synthetic polypeptide whose sequence corresponds to that
 of a determinant of the pathogen. Such polypeptide immunogens are reported
 by Sutcliffe et al., Nature, 287:801 (1980), and Lerner et al., Proc.
 Natl. Acad. Sci. USA, 78:3403 (1981).
 Gerin et al., Proc. Natl. Acad. Sci. USA, 80:2365 (1983), showed limited
 protection of chimpanzees from hepatitis B virus upon immunization with
 carrier-bound synthetic polypeptides having amino acid residue sequences
 that correspond to the sequence of a determinant portion of HBsAg; in
 particular, residues 110-137 of the "S" (surface) region. However, the
 carrier protein used in those studies was keyhole limpet hemocyanin (KLH),
 a T cell-dependent carrier that is not fit for use in medical applications
 to humans because it is a source of irritation that leads to severe
 inflammation.
 T cell-stimulating carrier proteins capable of enhancing the immunogenicity
 of haptens that do not produce unacceptable side effects in human subjects
 are often immunogenic natural proteins. For example, tetanus toxoid (TT)
 has been frequently used when a carrier suitable for human administration
 was needed. However, the use of tetanus toxoid as a carrier was restricted
 due to problems with dosage limitations and risk of sensitization to the
 toxoid itself. In addition, an epitope-specific suppression of response to
 the carried hapten can occur in individuals already immunized against
 tetanus.
 The hepatitis B surface protein has been proposed as a carrier for
 heterologous epitopes. Delpeyroux et al., Science, 233:472-475 (1986),
 reported the use of the HBV surface protein (S protein) as a carrier for a
 poliovirus polypeptide hapten. Those investigators constructed a
 recombinant deoxyribonucleic acid (DNA) protein expression vehicle that
 produces a fusion protein, designated HBsPolioAg, capable of forming
 particles closely resembling authentic 22-nanometer HBsAg particles.
 HBsPolioAg consists of HBV S protein having an 11 amino acid residue
 sequence insert corresponding to amino acids 93-103 of capsid protein VPI
 of poliovirus type 1 (Mahoney strain).
 Hepadnavirus nucleocapsid proteins have been used as hapten carriers.
 Heterologous immunogenic peptide sequences inserted internally in the
 hepatitis B core, expressed as fusion particles, elicited very high immune
 responses in immunized animals in the absence of adjuvants. B. E. Clarke
 et al. Vaccines 91:313-318 (1991); F. Schodel et al. J. Virol.
 66(1):106-114 (1992). U.S. Pat. Nos. 4,818,527, 4,882,145, and 5,143,726,
 the disclosures of which are incorporated herein by reference, describe
 the use of the carrier effect with hepatitis B virus nucleocapsid protein
 to enhance the immunogenicity of an operatively linked polypeptide hapten.
 Those patents describe the linking of a polypeptide hapten to hepatitis B
 virus nucleocapsid protein through an amino acid residue side chain that
 occurs naturally in the hepatitis B nucleocapsid protein sequence.
 Hepadnavirus nucleocapsid proteins are fairly well studied. SEQ ID NOs:1
 and 2 are the DNA and amino acid sequences of the human hepatitis B core
 protein (HBc), subtype ayw, as described in U.S. Pat. Nos. 4,818,527,
 4,882,145, and 5,143,726. Other hepadnavirus nucleocapsid protein
 sequences are also known in the art, see e.g. SEQ ID NOs: 3-13.
 There are reasons to select hepadnavirus nucleocapsid proteins as a carrier
 over other carriers used in the art, such as keyhole limpet hemocyanin
 (KLH), BCG, tetanus toxoid and diphtheria toxoid. KLH, BCG, tetanus toxoid
 and diphtheria toxoid are non-particulate, whereas hepadnavirus
 nucleocapsid proteins tend to aggregate into "particles". HBc particles
 tend to have a higher immunogenicity than hepatitis B surface antigen
 (HBsAg) particles. D. R. Milich et al., Science, 234:1398-1401 (1986). HBc
 is both a T cell-independent and a T cell-dependent immunogen. Id. HBc is
 one of the most immunogenic proteins known. Almost all hepatitis
 B-infected people develop a powerful immune response to core. J. H.
 Hoofnagle, Semin. Liver Dis., 1(1):7-14 (1981). HBc can provide universal
 responsiveness, irrespective of genetic background. Id. HBc directly
 activates T cells. HBc elicits strong T.sub.h cell responses. HBc is
 efficiently processed and presented by antigen-presenting cells. Due to
 the inherently high immunogenicity of HBc, complex adjuvants are typically
 not required, for example, the common and inexpensive alum is sufficient.
 The family hepadnaviridae is a family of enveloped animal viruses with a
 core of DNA that cause hepatitis B in humans. The hepadnaviridae are not
 responsible for human hepatitis A (a single-stranded RNA enterovirus),
 human hepatitis C (Flaviridae family of single stranded RNA virus), or
 human hepatitis D (a closed circular negative-sense RNA satellite virus,
 "delta virus", that requires hepatitis B virus for replication). The
 hepadnaviridae family includes hepatitis viruses of other species, e.g.
 woodchuck, duck, ground squirrel, and heron, in addition to human and
 simian hepatitis B. Hepatitis B (HB) used hereinafter refers to the family
 hepadnaviridae, unless the discussion is referring to a specific example.
 Hepatitis B core protein monomers self-assemble into stable aggregates
 known as hepatitis B core protein particles (HBc particles). For example,
 human HBc particles are 27 nanometers (nm) in diameter. Conway et al.,
 Nature, 386:91-94 (1997), describe the structure of human HBc particles at
 9 .ANG.ngstrom resolution, as determined from cryo-electron micrographs.
 Bottcher et al., Nature, 386:88-91 (1997), describe the polypeptide fold
 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. Bottcher et al. propose a loop from about
 residues 78 to 82 of the hepatitis B core protein.
 Using synthetic peptides and monoclonoal antibodies, the immunodominant
 loop region of HBc was mapped to about amino acid residues 75 to 83. G.
 Colucci et al., J. Immunol., 141:4376-4380 (1988). Two immunodominant
 linear epitopes were reported by other workers at amino acid residues 75
 to 85 and 130 to 140. Salfeld et al. J. Virol. 63:798 (1989).
 Insertion mutants of the hepatitis B core protein still are able to form
 core particles when foreign epitopes are cloned into the immunodominant
 loop region of HBc. P. Pumpens et al., Intervirology, 38:63-74 (1995). The
 HBc fusion proteins form particles in prokaryotic and eukaryotic
 expression systems. Id.
 The ability to use a protein as a carrier for a pendently-linked hapten
 depends upon several factors that have been studied with respect to HBc.
 Chemically-reactive amino acid side chains, such as lysine (K), aspartic
 acid (D), glutamic acid (E), and reduced cysteine residues (C), provide
 functional groups that can be useful for modifying polypeptides.
 The hepatitis B core protein sequence has several chemically-reactive amino
 acid side chains in the native sequence. Core has three primary amino
 groups, one at the amino terminus, and two lysine residues (K5 and K96),
 along with four cysteine residues (C48, C61, C107 and C183). There are
 several carboxylic acid groups, D (2, 4, 22, 29, 32, 78, 83) and E (8, 14,
 40, 43, 46, 64, 77, 113, 117, 145, 179) and the carboxy terminus.
 However, the native, unmodified hepatitis B core protein particle does not
 exhibit appreciable chemical reactivity of the amino acid side chains in
 the native sequence. The chemical reactivity of an amino acid side chain
 in a protein depends upon the nature of the amino acid side chain, and its
 environment in the folded protein.
 As is discussed in detail hereinafter, it has now been found that the
 problem of low reactivity of the amino acid side chains in native
 hepatitis B nucleocapsid protein can be overcome by inserting a
 chemically-reactive amino acid side chain into the HBc protein sequence. A
 strategically modified hepadnavirus core protein particle of the present
 invention exhibits substantially enhanced reactivity toward derivatization
 of HBc particles with chemically linked haptens, and provides enhanced
 immunogenicity to those linked haptens.
 These modified HBc proteins and their pendently-linked hapten conjugate
 derivatives are discussed in the disclosure that follows.
 BRIEF SUMMARY OF THE INVENTION
 The present invention relates to a strategically modified hepatitis B core
 (HBc) protein that is linked to a pendent hapten through
 chemically-reactive amino acid residue inserted into the HBc sequence. The
 contemplated strategic modification of HBc is an insert mutation of the
 HBc protein. A contemplated insert is 1 to about 40 amino acid residues in
 length, preferably 1 to 10 amino acid residues in length, and includes a
 chemically-reactive amino acid residue.
 The insert is provided to the region corresponding to amino acid residues
 about 50 to about 100 of the hepatitis B core protein sequence shown in
 SEQ ID NO:2. The preferred region of insertion corresponds to the
 hepatitis B core protein immunodominant loop region at about amino acid
 residue 70 to about 90, more preferably the loop tip region at about amino
 acid 78 to about 82 and most preferably at amino acid 78 to amino acid 80.
 Such an introduced chemically-reactive amino acid residue is characterized
 in that it has a chemically-reactive side chain that provides a chemical
 group for pendently linking the strategically modified HBc to the hapten.
 Typically, the chemically-reactive amino acid residue is a lysine,
 cysteine, or histidine residue or a carboxyl-containing residue such as
 aspartic acid or glutamic acid, preferably lysine or a carboxyl-containing
 residue, and most preferably lysine.
 The hapten bonded to the chemically-reactive amino acid residue is any
 compound of interest for generating an immune response, and is typically a
 B cell determinant. Preferably, the hapten is a polypeptide hapten, a
 carbohydrate hapten, or a non-peptidal/non-saccharidal (chemical) hapten.
 In one embodiment of the invention, the hapten is a pathogen-related
 hapten, such as a B cell determinant of a pathogen.
 In another embodiment of a strategically modified hepatitis B core protein
 conjugate, the strategically modified hepatitis B core protein also has a
 T cell stimulating amino acid residue sequence operatively linked to the
 carboxy terminus of the hepatitis B core amino acid sequence. Preferably,
 both the hapten and the T cell stimulating amino acid residue sequence are
 pathogen-related, most preferably, both are related to (from) the same
 pathogen.
 In the above embodiment of the invention, the response to a B cell epitope
 is boosted by also providing the T helper (T.sub.h) cell determinant. In
 this preferred embodiment of the invention, such a T.sub.h cell
 determinant is from the same pathogen as the B cell determinant hapten
 that is pendently linked to the strategically modified hepatitis B core
 protein in order to provide pathogen-specific T cell memory in addition to
 the hepatitis B core protein antigen-specific T cell memory.
 A strategically modified hepatitis B core protein conjugate contains a
 hapten that is pendently linked to a strategically modified hepatitis B
 core protein. Looked at differently, a before-described strategically
 modified hepatitis B core protein can be considered to have three
 peptide-linked domains, I, II and III (numbered consecutively from the
 amino terminus). Domain I comprises a sequence that corresponds to
 residues numbered about 10 to 50 of the amino acid sequence of hepatitis B
 core protein of SEQ ID NO:2, and preferably corresponds to residues
 numbered 1 to 50 of that sequence. Domain II is bonded to the carboxy
 terminal residue of Domain I. Domain II corresponds to residues numbered
 50 to 100 of the amino acid sequence of hepatitis B core protein of SEQ ID
 NO:2. Domain III comprises a sequence that is bonded to the carboxy
 terminal residue of Domain II. Domain III corresponds to residues numbered
 100 to about 140 of the amino acid sequence of hepatitis B core protein,
 and preferably corresponds to residues numbered 100 to about 149 of that
 sequence.
 In an embodiment of the invention discussed before, a strategically
 modified hepatitis B core protein additionally has a Domain IV exogenous
 to HBc that is peptide-bonded to the carboxy terminal residue of Domain
 III to provide a fusion protein. Domain IV is a T cell epitope.
 A strategically modified hepatitis B core protein particle of the invention
 is made of assembled heptatitis B core protein where a plurality of the
 subunits are strategically modified hepatitis B core protein subunits.
 Also contemplated is a particle comprised of a mixture of strategically
 modified hepatitis B core protein subunits and other heptatitis B core
 protein subunits.
 A contemplated strategically modified hepatitis B core protein particle
 conjugate is comprised of assembled hepatitis B core protein subunits
 where a plurality of the subunits are strategically modified hepatitis B
 core protein subunits. In this embodiment, a hapten is pendently linked to
 a hepatitis B core protein subunit. Preferably, the hapten is
 pathogen-related. As above, a T cell stimulating amino acid residue
 sequence can be peptide-bonded to the carboxy terminal residue of the
 sequence corresponding to hepatitis B core protein. Preferably that
 pathogen-related T cell determinant is related to the same pathogen as the
 pathogen-related hapten.
 A strategically modified hepatitis B core protein particle conjugate of the
 invention has pendently-linked hapten. In a contemplated embodiment of the
 particle conjugate, the particle is made up of a mixture of strategically
 modified hepatitis B core protein subunits having pendently-linked
 haptens, and other hepatitis B core protein subunits. In one embodiment,
 about 0.1 to about 0.5 of the strategically modified hepatitis B core
 protein subunits are pendently linked to a hapten. Also contemplated is a
 particle conjugate that is made up of a mixture of strategically modified
 hepatitis B core protein subunits and other hepatitis B core protein
 subunits.
 A before-described strategically modified hepatitis B core protein of the
 invention includes a peptide insert containing a chemically-reactive amino
 acid residue. That insert can be, but is typically not, itself a separate
 B cell antigenic determinant, although some B cell immunogenicity of the
 insert can be exhibited merely because of the placement of the insert into
 the HBc protein or particle. Such an insert can be and in some embodiments
 is a T cell epitope. Placement of an insert into the HBc loop region
 greatly diminishes the HBc immunogenicity and antigenicity of the
 resulting molecule.
 An inoculum of the invention comprises an immunogenic amount of the
 strategically modified hepatitis B core protein conjugate of the
 invention. When the pendently-linked hapten is a pathogen-related hapten,
 the inoculum can be used as a vaccine to protect a mammal treated with the
 inoculum from that pathogen. Thus, in one embodiment of the invention, a
 strategically modified hepatitis B core protein conjugate is used as a
 vaccine to provide protection against the pathogen from which the hapten
 is derived. More preferably, the inoculum is comprised of strategically
 modified hepatitis B core protein particle conjugate as the immunogen.
 The present invention has several benefits and advantages.
 One benefit of a contemplated modified HBc protein is that the protein can
 be derivatized while in the aggregated form of HBc particles.
 An advantage of the invention is that the modified HBc protein displays
 appreciably enhanced chemical reactivity toward derivatization, as
 compared to use of the N-terminal primary amine, for example.
 Another benefit of a contemplated modified HBc protein is that the
 chemistry of derivatization of such side chains is well-studied,
 straightforward and relatively predictable.
 Another advantage of a contemplated modified HBc protein is that it
 enhances the immunologic response to the conjugated hapten with which it
 is derivatized.
 Yet another benefit of a contemplated modified HBc protein is that it is
 unlikely to produce undesirable immunologic side effects in humans.
 Still further benefits and advantages of the invention will be apparent to
 the skilled worker from the discussion that follows.

DETAILED DESCRIPTION OF THE INVENTION
 A. Definitions
 The term "antibody" refers to a molecule that is a member of a family of
 glycosylated proteins called immunoglobulins, which can specifically
 combine with an antigen.
 The word "antigen" has been used historically to designate an entity that
 is bound by an antibody, 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, whereas the word "immunogen"
 is used for the entity that induces antibody production. Where an entity
 discussed herein is both immunogenic and antigenic, reference to it as
 either an immunogen or antigen will typically be made according to its
 intended utility.
 The word "hapten" is used to describe molecules that are capable of
 stimulating an immune response (e.g., production of antibody) when
 chemically coupled to a protein carrier. The word is often used for small
 nonantigenic molecules in the art, but herein, it merely refers to the
 molecule that is to be pendently linked to the carrier protein, even if it
 is antigenic or not small.
 "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 noun "conjugate" as used herein refers to a molecule formed from a
 hapten pendently linked through an amino acid residue side chain to a
 carrier.
 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 "conservative
 substitution" also includes the use of a substituted amino acid in place
 of an unsubstituted parent amino acid provided that antibodies raised to
 such a polypeptide also immunoreact with the corresponding polypeptide
 having the unsubstituted amino acid.
 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. "Epitope"
 refers to that portion of a molecule that is specifically bound by a T
 cell antigen receptor or an antibody combining site.
 "Epitope" and "determinant" are used interchangeably.
 As used herein, the term "fusion protein" designates at least two amino
 acid residue sequences not normally found linked together in nature
 operatively linked together end-to-end (head-to-tail) by a peptide bond
 between their respective terminal amino acid residues.
 The phrase "hepatitis B" as used here refers in its broadest context to any
 member of the family hepadnaviridae, a family of enveloped DNA-containing
 animal viruses that can cause hepatitis B in human.
 The phrase "HBc" as used here refers to T cell stimulating proteins having
 an amino acid residue sequence that corresponds to an amino acid residue
 sequence encoded by the hepatitis B virus (HBV) nucleocapsid protein gene.
 An exemplary well-known naturally occurring protein encoded by the human
 HBV nucleocapsid gene is the "core" protein, subtype ayw, having the
 biological sequences of SEQ ID NOs: 1 and 2. Galibert, et al., Nature
 281:646 (1979). HBeAg protein, the precursor to HBc, includes the sequence
 of the hepatitis B core protein and a "pre-core" sequence at the amino
 terminus thereof, as shown in SEQ ID NOs: 8 and 9 in the case of a ground
 squirrel hepatitis B nucleocapsid gene. The core protein sequence begins
 at amino acid position 31 therein, thus corresponding to amino acid
 residue number 1 of SEQ ID NO:2. The sequences for other hepatitis B core
 proteins are known in the art. Human hepatitis B virus core protein
 subtype adr is provided in SEQ ID NOs: 3 and 4, and subtype adw is
 provided in SEQ ID NOs: 5 and 6. Ono et al., Nucl. Acids Res. 11:1747
 (1983). Sequences are also provided for woodchuck hepatitis B core protein
 at SEQ ID NO:7 [Schodel et al., Adv. Viral Oncol. 8:73-102 (1989)], ground
 squirrel at SEQ ID NOs:8 and 9, heron at SEQ ID NOs:10 and 11, and duck at
 SEQ ID NOs:12 and 13. For clarity, the amino acid numbering system shown
 in SEQ ID NOs:1 and 2 with respect to human hepatitis B core protein
 subtype ayw is used as a benchmark herein. Other HBc sequences can be
 aligned with that sequence using standard biological sequence alignment
 programs and protocols to determine the amino acid residues that
 "correspond to the hepatitis B core protein sequence of SEQ ID NO:2", see
 e.g. Schodel et al., Adv. Viral Oncol. 8:73-102 (1989).
 If reference is made to a polypeptide portion of any of the above described
 naturally occurring HBV nucleocapsid gene encoded proteins, that reference
 is explicit, either by stating, for example, that a T cell stimulating
 portion of the particular protein is referred to or by explicitly
 designating the particular portion of the sequence, as by indication of
 the included amino acid residue positions.
 The term "immunoreact" in its various forms means specific binding between
 an antigen as a ligand and a molecule containing an antibody combining
 site such as a Fab portion of a whole antibody.
 The phrase "operatively linked" as used herein means that the linkage does
 not interfere with the ability of either of the linked groups to function
 as described; e.g., to function as a T or B cell determinant.
 The phrase "pendently linked" refers to a single linkage, either direct or
 via a bridge, from a HBc protein to another molecule at other than the
 amino or carboxy termini. The phrase is used herein to describe the
 linkage between a hapten and a chemically-reactive amino acid side chain
 of a strategically modified hepatitis B core protein.
 "Macromolecular assembly" refers to a non-covalently bonded aggregate of
 protein subunits. Typically in this invention, the protein subunit is a
 strategically modified hepatitis B core protein monomer. As described in
 more detail hereinafter, those core protein monomers usually self-assemble
 into spherical "core particles" having either 90 or 120 core protein
 dimers (a total of 180 or 240 core protein subunits). A spherical core
 particle is an example of a macromolecular assembly.
 The phrase "pathogen-related" as used herein designates a B cell or T cell
 immunogen that is capable of inducing the production of antibodies that
 immunoreact with a pathogen in native form. Exemplary pathogen-related B
 cell and T cell immunogens are illustrated hereinafter.
 The words "polypeptide" and "peptide" as 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 variety of
 lengths, either in their neutral (uncharged) forms or in forms which are
 salts, and either free of modifications such as glycosylation, side chain
 oxidation, or phosphorylation or containing these modifications. 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 of the surrounding medium
 when the protein is in solution, or that of the medium from which it was
 obtained if the protein is in solid form. Also included in the definition
 are proteins modified by additional substituents attached to the amino
 acid side chains, such as glycosyl units, lipids, or inorganic ions such
 as phosphates, as well as modifications relating to chemical conversions
 of the chains, such as oxidation of sulfhydryl groups. Thus, "polypeptide"
 or its equivalent terms is intended to include the appropriate amino acid
 residue sequence referenced, subject to those of the foregoing
 modifications which do not destroy its functionality. A peptide or
 polypeptide used as a hapten typically contains fewer than 70 amino acid
 residues, and more typically contains a linear chain of about 5 to about
 40 amino acid residues, and more preferably about 10 to about 25 residues.
 It is noted that a contemplated polypeptide hapten can be longer than 70
 residues, but such a polypeptide is shorter than the naturally occurring
 protein that shares its sequence.
 The word "protein" designates a polypeptide having about 70 or more amino
 acid residues, and is a naturally occurring entity.
 The words "secrete" and "produce" are often used interchangeably in the art
 as to cells from which antibody molecules are obtained. Cells that produce
 antibodies may, however, not secrete those molecules into their
 environment. Herein, the antibody molecules are secreted and are obtained
 from the blood stream (humoral antibody). Nevertheless, antibodies are
 generally referred to as being "produced" in keeping with the phrase
 utilized in the art.
 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 1.
 TABLE 1
 TABLE OF CORRESPONDENCE
 SYMBOL
 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 Ile 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 Gln L-glutamine
 E Glu L-glutamic acid
 Z Glx L-qlutamic acid
 or
 L-glutamine
 W Trp L-tryptophan
 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
 B. Strategically Modified Hepatitis B Core Protein
 The present invention contemplates a strategically modified hepadnaviridae
 core ("HBc") protein that has an inserted chemically reactive amino acid
 residue for pendently linking with haptens such as polypeptides and
 carbohydrates. The strategic modification of the invention is the
 insertion of 1 to about 40 amino acid residues including a
 chemically-reactive amino acid residue into the hepatitis B core protein
 sequence in the region corresponding to amino acid residues 50 to 100 of
 the HBc sequence of SEQ ID NO:2. Such an introduced chemically-reactive
 amino acid residue has a side-chain that provides a functional group for
 pendently linking a hapten to the strategically modified carrier.
 Hepadnaviridae have a nucleocapsid, or core, surrounded by a lipid envelope
 containing surface proteins. The nucleocapsid is a generally spherical
 aggregate of core proteins ("core antigen", HBcAg) dimers. In vitro, the
 hepatitis B core protein self-assembles into "particles", spherical shells
 of icosahedral symmetry made up of 90 or 120 hepatitis B core protein
 dimers, thus 180 or 240 protein subunits. The particles are about 280 or
 310 .ANG.ngstroms in diameter, respectively. B. Bottcher et al., Nature,
 386:88-91 (1997); J. F. Conway et al. Nature 386:91-94 (1997).
 A contemplated strategically modified hepatitis B core protein also forms a
 macromolecular assembly. Such a particle can be present in the form of 180
 or 240 protein subunits, although it does not have to be such a 90 or 120
 dimer.
 Hybrid core proteins with exogenous amino acid residues inserted in the
 region near amino acid residue 80 are reported to assemble into regular
 shells, even with inserts as large as 46 amino acids in length. A. I.
 Brown et al., Vaccine, 9:595-601 (1991); F. Schodel et al., J. Virol.,
 66:106-114 (1992).
 The hepadnaviridae core protein sequence used as a benchmark sequence
 herein is that of the human hepatitis B core protein, subtype ayw, shown
 in SEQ ID NOs:1 and 2. Other subtypes of the human hepatitis B virus are
 known. SEQ ID Nos: 3 and 4 are human HBc, subtype adr, and SEQ ID NOs:5
 and 6 are HBc subtype adw. The sequences of various animal hepatitis core
 proteins are also published. The biological sequence of duck hepatitis
 core protein is disclosed herein as SEQ ID NO:12 and 13; a portion of the
 ground squirrel hepatitis nucleocapsid gene is at SEQ ID NO:8 and 9;
 woodchuck hepatitis core is at SEQ ID NO:7 and heron hepatitis core at SEQ
 ID NOs:10 and 11. Exemplary animal hepatitis B core proteins are aligned
 with human hepatitis B core protein by F. Schodel et al., Adv. Viral
 Oncology 8:73-102 (1989).
 i. Strategic Modification of the Core Protein
 The present invention contemplates a strategically modified hepatitis B
 core protein conjugate that comprises a hapten that is pendently linked to
 a strategically modified hepatitis B core protein (HBc). The strategically
 modified hepatitis B core protein itself comprises an amino acid sequence
 corresponding to the hepatitis B core protein amino acid sequence of SEQ
 ID NO:2 including the amino acid residues numbered about 10 to about 140
 of that sequence. That HBc amino acid residue sequence additionally
 contains an exogenous amino acid residue insert in the region
 corresponding to amino acid residues numbered about 50 to about 100 from
 the HBc amino terminus, wherein the exogenous insert (i) is 1 to about 40
 amino acid residues in length, and (ii) contains a chemically-reactive
 amino acid residue. The hapten is pendently linked to the strategically
 modified HBc protein by means of a chemically-reactive amino acid residue
 present in the insert.
 It is preferred that residues 1 through 10 of SEQ ID NO:2 be present in the
 strategically modified HBc protein molecule. It is further preferred when
 any residue is absent or deleted from position 1 to 10 that those residues
 be deleted in sequence and that the remaining residues be present in
 sequence. Thus, if a five residue deletion were contemplated, the deleted
 residues would be numbered 1-5, leaving residues 6 through the desired HBc
 carboxy terminus present, plus the insert.
 It is similarly preferred that residues numbered about position 140 through
 149 of SEQ ID NO:2 be present in a strategically modified HBc protein
 molecule. As noted elsewhere herein, the region of HBc numbered 150
 through the carboxy terminus contains a plurality of arginine residues.
 Those residues bind nucleic acids on purification of HBc particles after
 expression, and the sequence containing those residues is preferably
 omitted from a strategically modified HBc protein molecule. As was the
 case with the residues of positions 1 through 10, it is preferred that
 residues between about position 140 and 149 be present and correspondingly
 absent in a sequential manner. Thus, where the carboxy terminal residue
 corresponds to the residue of position 146, the residues of positions
 141-145 are also present and those of 147-149 are absent. Most preferably,
 a contemplated HBc sequence is that shown in SEQ ID NO:2 from position 1
 through position 149, plus the sequence of the insert.
 The insert can be placed within the HBc sequence in the region of positions
 numbered about 50 through about 100, as already noted. Preferably, the
 insert is present in the region corresponding to amino acid residues
 numbered about 70 to about 90. More preferably, the insert is present in
 the region corresponding to amino acid residues numbered 78 to 82. Most
 preferably, the insert is located in the region corresponding to residues
 numbered 78 through 80.
 A strategically modified hepatitis B core protein of the invention has from
 1 to about 40 amino acid residues inserted. Preferably, the insert is 1 to
 10 amino acid residues in length. The insert contains a
 chemically-reactive amino acid residue. The insertion of more than one
 chemically-reactive amino acid residue is also contemplated.
 It is contemplated that restriction endonuclease sites be provided in the
 gene construct for the strategically modified hepatitis B protein near the
 desired insert region. The nucleotides of the restriction endonuclease
 site will be translated into amino acids upon expression, and that effect
 has some bearing on the choice of endonuclease. Several restriction
 endonucleases are commercially available (e.g. from Promega Corp.,
 Madison, Wis.) and their recognition site sequences and cleavage sites
 well known in the art. Example 1 describes such a construct for a
 strategically modified hepatitis B core protein.
 In one preferred embodiment, the insert is a single residue that is added
 as the chemically-reactive residue. In other preferred embodiments, the
 use of restriction enzymes and their recognition sequences causes about
 three to about five residues to be inserted, including the desired
 chemically reactive residue.
 An insert containing a chemically-reactive amino acid residue is inserted
 into the native hepatitis B core protein at a position corresponding to an
 amino acid residue position from about 50 to about 100. The preferred
 region of insertion into the hepatitis B core protein is in the
 immunodominant loop region (about amino acid residue 70 to about 90), more
 preferably in the loop region that corresponds to the native hepatitis B
 core protein position from about amino acid 78 to about 82. Most
 preferably, the insert is placed at residues numbered 78 to 80 of SEQ ID
 NO:2.
 As used herein when it is said that the insert is "at a position" it is
 meant that the amino terminus of the insert is peptide bonded to the
 carboxy terminus of the corresponding amino acid residue of the hepatitis
 B core protein sequence having that amino acid residue number. In other
 words, the insert immediately follows the residue at that stated position.
 Insertion can be effected by generally utilized methods in the art. Genetic
 manipulation, by a PCR-based method is illustrated in Examples 1 and 5. In
 addition, oligonucleotide-mediated site directed mutagenesis as discussed
 in J. Sambrook et al. Molecular Cloning: a laboratory manual, 2.sup.nd
 ed., Cold Spring Harbor Laboratory Press, 15.51-ff. (1989) can be used to
 add one codon by hybridizing a desired DNA sequence with a template that
 adds a codon for single residue, followed by filling in the remaining
 nucleic acid sequence. Dawson et al., Science 266:776-779 (1994), describe
 a method of linking polypeptide chains at their peptide backbone, so that
 a fusion could be built up of peptide fragments.
 The chemically-reactive amino acid residue can be at any position within
 the insert. Preferably, the chemically-reactive amino acid residue is in a
 position that corresponds to amino acid residue numbered 70 to 90 of the
 native (wild type) core, and most preferably at residue position 78 to 82.
 For example, when a 10 amino acid residue long insert is inserted at a
 position corresponding to native core protein residue 73, then the
 chemically-reactive amino acid residue is preferably at position 5 to 9 of
 the insert. When a 30 amino acid residue long insert is inserted a
 position corresponding to native core protein residue 58, then the
 chemically-reactive amino acid residue is preferably at position 22 to 24
 of the insert.
 An introduced chemically-reactive amino acid residue has a
 chemically-reactive side chain that provides a functional group for
 derivatizing the strategically modified HBc (i.e. conjugating a hapten to
 the modified HBc). Useful side chain functional groups include
 epsilon-amino groups, beta-or gamma-carboxyl groups, thiol (-SH) groups
 and aromatic rings (e.g. tyrosine and histidine). The chemically-reactive
 amino acid residue is typically a lysine, cysteine, or histidine residue
 or a carboxyl-containing residue such as aspartic acid or glutamic acid.
 Lysine is a particularly preferred chemically-reactive amino acid residue.
 It is noted that the amino acid residue sequence of the hepatitis B core
 protein encoded by and shown in SEQ ID NOs:1 and 2, respectively, has two
 consecutive endogenous carboxyl-containing residues, existing glutamic
 (glu, E) and aspartic (asp, D) acids, at positions 77 and 78. However, the
 present invention contemplates the introduction of at least one
 additional, exogenous chemically-reactive amino acid residue. European
 Patent No. 385610 reports that unsatisfactory results were achieved in
 attempts to chemically couple polypeptide haptens to HBc particles. It is
 noted that those coupling attempts were directed toward amino groups and
 not carboxyl groups of the amino acid side chains.
 In addition of the use of an individual chemically-reactive amino acid
 residue in the insert such as aspartic acid or lysine, substantially any
 sequence of the desired length that contains a chemically-reactive amino
 acid residue can be used. Exemplary inserts of greater than a single
 residue include the B cell HRV-2 VP2 epitope discussed in B. E. Clarke et
 al. Vaccines 91:313-318 (1991), the HBsAg Pre-S(1)27-53 sequence discussed
 in F. Schodel et al. J. Virol. 66(1):106-114 (1992) and the HBsAg
 Pre-S(2)133-143 sequence discussed in F. Schodel et al. Vaccines
 90:193-198 (1990). An appropriate T cell epitope discussed hereinafter as
 a hapten can also be used as an insert. Exemplary sequences include those
 of SEQ ID NOs: 28, 32, 33, 34, 47, 48, 49, 50 and 55.
 ii. Additional Modification of the Core Protein
 It is also contemplated that a hepatitis B core protein strategically
 modified as described above has other modifications. The contemplated
 modifications of the strategically modified core include the nature of the
 insert containing the chemically-reactive amino acid residue, truncation
 of the amino terminus, truncation of the carboxy terminus, fusion at the
 carboxy terminus, pendent linking to the carboxy-terminal region.
 The insert containing the chemically-reactive amino acid residue to which
 the hapten is conjugated can have a use in addition to providing the
 chemically-reactive amino acid residue. It is contemplated that an insert
 containing a chemically-reactive amino acid residue is a T cell
 stimulating amino acid sequence. Such a T cell stimulating amino acid
 sequence is preferably a T cell epitope from the same source as the B cell
 epitope that will be the conjugated antigen, e.g. both from Mycobacterium
 tuberculosis. Exemplary epitopes are discussed hereinafter.
 An insert containing a chemically-reactive amino acid residue can also be
 chosen in order to confer additional desirable properties, such as
 stability-enhancing or solubility-enhancing properties.
 A strategically modified hepatitis B core protein can be chemically
 modified by methods well known in the art. Numerous such techniques are
 disclosed in Roger L. Lundblad, Techniques in Protein Modification, CRC
 Press (Ann Arbor, Mich.: 1994). Such chemical modifications are made to
 enhance or diminish properties, for example, a lysine amino group can be
 blocked to change the isoelectric point of the protein, causing it to
 separate differently on a chromatographic ion exchange resin.
 It is also contemplated that the carboxy terminus of the core protein
 sequence be truncated, preferably down to about amino acid residue
 position 140. The arginine-rich sequence present beginning at residue 150
 of SEQ ID NO:2 binds to nucleic acids and can hinder the purification and
 handling of the expressed core protein. In SEQ ID NO:2, the arginine-rich
 stretches begin at position 150. A preferred strategically modified HBc
 protein has a carboxy terminal valine (V) residue of residue 149.
 iii. Making Strategically-Modified Core Protein
 The strategic modification of the hepatitis B core protein is typically
 made by known processes in the art on the DNA level, for example by
 inserting the codons corresponding to the amino acids to be inserted. The
 engineered gene is then expressed in a convenient system known in the art,
 for example in a viral culture in infected immortalized cells.
 Methods for producing HBcAg proteins in general and the pre-core, core and
 HBeAg proteins in particular, are well known in the art. The same methods
 readily adapted to the isolation of the modified core protein particles of
 the invention. In addition, HBcAg and HBeAg can be produced by a variety
 of well known recombinant DNA techniques. See, for example, U.S. Pat. No.
 4,356,270 to Itakura and U.S. Pat. No. 4,563,423 to Murray et al.,
 respectively. Those recombinant DNA techniques can be easily adapted to
 produce modified core particles of the invention. The modified core
 proteins can be conjugated with hapten before or after particle formation,
 preferably after core particle formation and purification.
 C. Modified Hepatitis B Core Protein Conjugate
 Any hapten against which antibody production is desired can be linked to a
 strategically modified hepatitis B core protein to form an immunogenic
 strategically modified hepatitis B core protein conjugate of this
 invention. The hapten of interest typically is a B cell determinant. The
 hapten can be a polypeptide, a carbohydrate (saccharide), or a
 non-polypeptide, non-carbohydrate chemical such as 2,4-dinitrobenzene.
 Methods for operatively linking individual haptens to a protein or
 polypeptide through an amino acid residue side chain of the protein or
 polypeptide to form a pendently-linked immunogenic conjugate, e.g., a
 branched-chain polypeptide polymer, are well known in the art. Those
 methods include linking through one or more types of functional groups on
 various side chains and result in the carrier protein polypeptide backbone
 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, Method of Enzymology, 70:85
 (1980), Aurameas, et al., Scand. J. Immunol., Vol. 8, Suppl. 7, 7-23
 (1978) and U.S. Pat. No. 4,493,795 to Nestor et al. In addition, a
 site-directed coupling reaction, as described in Rodwell et al., Biotech.,
 3, 889-894 (1985) 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 HBcAg 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
 functions with 2-iminothiolane or the N-hydroxysuccinimide ester of
 3-(3-dithiopyridyl)propionate.
 The HBc protein or hapten can also be modified to incorporate a spacer arm,
 such as hexamethylene diamine or other bifunctional molecules of similar
 size, to facilitate the pendent linking.
 Polypeptide hasten. 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. Pat. No. 4,818,527,
 m-maleimidobenzoyl-N-hydroxysuccinimde ester (ICN Biochemicals, Inc.) or
 succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC, Pierce)
 is reacted with a strategically modified hepatitis B core protein to form
 an activated carrier. That activated carrier is then reacted with 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 strategically modified hepatitis B core protein
 conjugate. As an alternative example, the amino group of a polypeptide
 hapten can be first reacted with N-succinimidyl
 3-(2-pyridylthio)propionate (SPDP, Pharmacia), 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., J. Biochem., 79:233 (1976) and
 in Lachmann et al., in 1986 Synthetic Peptides as Antigens, (Ciba
 Foundation Symposium 119), pp. 25-40 (Wiley, Chichester: 1986).
 U.S. Pat. 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 strategically modified hepatitis B core protein
 conjugate.
 Particularly useful are a large number of heterobifunctional agents that
 form a disulfide link at one functional group end and a peptide link at
 the other, including N-succidimidyl-3-(2-pyridyldithio) propionate (SPDP).
 This reagent creates a disulfide linkage between itself and a thiol in
 either the strategically modified hepatitis B core protein or the hapten,
 for example a cysteine residue in a polypeptide hapten, and an amide
 linkage on the coupling partner, for example the amino on 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-maleimido-methyl)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 Company, Rockford, Ill. The foregoing list is not meant to be
 exhaustive, and modifications of the named compounds can clearly be used,
 e.g., the sulpho-SMCC depicted in the figure.
 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.
 As discussed below, DNA sequences that encode a variety of polypeptide
 haptens are known in the art. The coding sequence for peptides of the
 length contemplated herein can easily be synthesized by chemical
 techniques, for example, the phosphotriester method of Matteucci et al.,
 J. Am. Chem. Soc. 103:3185 (1981). Of course, by chemically synthesizing
 the coding sequence, any desired modification can be made simply by
 substituting the appropriate bases for those encoding the native peptide
 sequence. The coding sequence can then be provided with appropriate
 linkers and ligated into expression vectors now commonly available in the
 art, and the regulating vectors used to transform suitable hosts to
 produce the desired protein.
 A number of such vectors and suitable host systems are now available. For
 example promoter sequences compatible with bacterial hosts are provided in
 plasmids containing convenient restriction sites for insertion of the
 desired coding sequence. Typical of such vector plasmids are, for example,
 pUC8, and pUC13 available from J. Messing, at the University of Minnesota
 (see, e.g., Messing et al., Nucleic Acids Res. 9:309 (1981)) or pBR322,
 available from New England Biolabs. Suitable promoters include, for
 example, the beta-lactamase (penicillinase) and lactose (lac) promoter
 systems (Chang. et al., Nature 198:1056 (1977) and the tryptophan (trp)
 promoter system (Goeddel et al., Nucleic Acids Res. 8:4057 (1980)). The
 resulting expression vectors are transformed into suitable bacterial hosts
 using the calcium chloride method described by Cohen, et al., Proc. Natl.
 Acad. Sci. U.S.A. 69:2110 (1972). Successful transformants may produce the
 desired polypeptide fragments at higher levels than those found in strains
 normally producing the intact pili. Of course, yeast or mammalian cell
 hosts can also be used, employing suitable vectors and control sequences.
 TABLE 2
 Polypeptide haptens
 T or B
 cell SEQ
 Organism Antigen epitope Sequence ID NO
 Streptococcus PspA B KLEELSDKIDELDAE 25
 pneumoniae
 Streptococcus PspA B SQKKYDEDQKKTEEKAALEKA 26
 pneumoniae ASEEMDKAVAAVQQA
 Cryptosporidium P23 B QDKPADAPAAEAPAAEPAAQQ 27
 parvum DKPADA
 HIV P24 T GPKEPFRDYVDRFYKC 28
 HIV GP120 B RKRIHIGPGRAFYITKN 29
 Foot and Mouth VP1 B YNGECRYNRNAVPNLRGDLQV 30
 Disease Virus LAQKVARTLP
 Corynebacterium toxin T FQVVHNSYNRPAYSPGC 31
 diphtheriae
 Borrelia OspA T VEIKEGTVTLKREIDKNGKVT 32
 burgdorferi VSLC
 Borrelia OspA T TLSKNISKSGEVSVELNDC 33
 burgdorferi
 Influenza A8/PR8 HA T SSVSSFERFEC 34
 Influenza A8/PR8 HA B YRNLLWLTEK 35
 Yersinia pestis V Ag B DILKVIVDSMNHHGDARSKLR 36
 EELAELTAELKIYSVIQAEIN
 KHLSSSGTINIHDKSINLMDK
 NLYGYTDEEIFKASAEYKILE
 KMPQTTIQVDGSEKKIVSIKD
 FLGSENKRTGALGNLKNSYSY
 NKDNNELSHFATTCSD
 Haemophilus pBOMP B CSSSNNDAAGNGAAQFGGY 37
 influenzae
 Haemophilus pBOMP B NKLGTVSYGEE 38
 influenzae
 Haemophilus pBOMP B NDEAAYSKNRRAVLAY 39
 influenzae
 Moraxella copB B LDIEKDKKKRTDEQLQAELDD 40
 catarrhalis KYAGKGY
 Moraxella copB B LDIEKNKKKRTEAELQAELDD 41
 catarrhalis KYAGKGY
 Moraxella copB B IDIEKKGKIRTEAELLAELNK 42
 catarrhalis DYPGQGY
 Porphyromonas HA B GVSPKVCKDVTVEGSNEFAPV 43
 gingivalis QNLT
 Porphyromonas HA B RIQSTWRQKTVDLPAGTKYV 44
 gingivalis
 Trypanosoma T SHNFTLVASVIIEEAPSGNTC 45
 cruzi
 Trypanosoma B KAAIAPAKAAAAPAKAATAPA 46
 cruzi
 Plasmodium MSP1 T SVQIPKVPYPNGIVYC 47
 falciparum
 Plasmodium MSP1 T DFNHYYTLKTGLEADC 48
 falciparum
 Streptococcus AgI/II B & T KPRPIYEAKLAQNQKC 49
 sobrinus
 Streptococcus AgI/II B & T AKADYEAKLAQYEKDLC 50
 sobrinus
 lymphocytic NP T RPQASGVYMGNLTAQC 51
 Choriomeningitis
 virus
 Shigella Invasin B KDRTLIEQK 52
 flexneri
 respiratory G B CSICSNNPTCWAICK 53
 synctial virus
 Plasmodium vivax CS B GDRADGQPAGDRADGQPAG 54
 Clostridium tox T QYIKANSKFIGITELC 55
 tetani
 Entamoeba lectin B VECASTVCQNDNSCPIIADVE 56
 histolytica KCNQ
 Schistosoma para B DLQSEISLSLENGELIRRAKS 57
 japonicum AESLASELQRRVD
 Schistosoma para B DLQSEISLSLENSELIRRAKA 58
 mansoni AESLASDLQRRVD
 Plasmodium vivax B DRAAGQPAGDRADGQPAG 83
 Influenza virus Infl B CNNPHRIL 84
 Influenza virus Infl T CPKYVKQNTLKLATGMRNVPE 85
 KQTR
 Influenza virus Infl B SIMRSDAPIGTCSSECITPNG 14
 SIPNDKPFQNVNKITY
 Influenza virus Infl B RGIFGAIAGFIENGWEGMIDG 15
 WYGFRHQN
 Influenza virus Infl B EKQTRGIFGA 16
 Mycobacterium T AVLEDPYILLVSSKV 86
 tuberculosis
 Mycobacterium T LLVSSKVSTVKDLLP 87
 tuberculosis
 Mycobacterium T LLPLLEKVIGAGKPL 88
 tuberculosis
 Mycobacterium T AILTGGQVISEEVGL 89
 tuberculosis
 Mycobacterium T IAFNSGLEPGVVAEK 90
 tuberculosis
 Mycobacterium T ARRGLERGLNALADAVKV 91
 tuberculosis
 Mycobacterium T EKIGAELVKEVAKK 92
 tuberculosis
 Mycobacterium T GLKRGIEKAVEKVTETL 93
 tuberculosis
 Mycobacterium T IEDAVRNAKAAVEEG 94
 tuberculosis
 Feline leukemia FeLV B CDIIGNTWNPSDQEPFPGYG 95
 virus
 Feline leukemia FeLV B CIGTVPKTHQALCNETQQGHT 96
 virus
 Feline leukemia FeLV B GNYSNQTNPPPSC 97
 virus
 Feline leukemia FeLV B TDIQALEESISALEKSLTSLS 98
 virus E
 Feline leukemia FeLV AKLRERLKQRQQLF 99
 virus
 Feline leukemia FeLV DSQQGWFEGWFNKSPWFTTLI 100
 virus SS
 Feline leukemia FeLV QVMTITPPQAMGPNLVLP 101
 virus
 Feline leukemia FeLV DQKPPSRQSQIESRVTP 102
 virus
 Feline leukemia FeLV RRGLDILFLQEGGLC 103
 virus
 Feline leukemia FeLV QEGGLCAALEECQIGGLCAAL 104
 virus KEEC
 Plasmodium B NANPNANPNANP 105
 falciparum
 Circumsporozoite QAQGDGANAGQP 113
 Chemical Hapten. Related chemistry is used to couple chemical compounds to
 carrier proteins. Typically, an appropriate functional group for coupling
 is designed into the chemical compound.
 Antibodies to the compound 6-O-phosphocholine hydroxyhexanoate protect
 against Streptococcus pneumoniae. Randy T. Fischer et al. J. Immunol.,
 154:3373-3382 (1995).
 TABLE 3
 Chemical Haptens
 Chemical Hapten Citation
 piperidine N-oxide U.S. Pat. No. 5,304,252
 phospholactone or U.S. Pat. No. 5,248,611
 lactamide
 metal ion complexes U.S. Pat. No. 5,236,825
 [2.2.1] or [7.2.2] U.S. Pat. No. 5,208,152
 bicyclic ring
 compounds
 ionically charged U.S. Pat. No. 5,187,086
 hydroxyl - containing
 compounds
 phosphonate analogs U.S. Pat. No. 5,126,258
 of carboxylate
 esters
 cocaine analogs Carrera et al., Nature
 378:725 (1995)
 Carbohydrate Hapten. There are many methods known in the art to couple
 carrier proteins to polysaccharides. Aldehyde groups can be generated on
 either the reducing end [Anderson, Infect. Immun., 39:233-238 (1983);
 Jennings, et al., J. Immunol., 127:1011-1018 (1981); Poren et al., Mol.
 Immunol., 22:907-919 (1985)] or the terminal end [Anderson et al., J.
 Immunol., 137:1181-1186 (1986); Beuvery et al., Dev. Bio. Scand.,
 65:197-204 (1986)] of an oligosaccharide or relatively small
 polysaccharide, which can be linked to the carrier protein via reductive
 amination.
 Large polysaccharides can be conjugated by either terminal activation
 [Anderson et al., J. Immunol., 137:1181-1186 (1986)] or by random
 activation of several functional groups along the polysaccharide chain
 [Chu et al., Infect. Immun., 40:245-256 (1983); Gordon, U.S. Pat. No.
 4,619,828 (1986); Marburg, U.S. Pat. No. 4,882,317 (1989)]. Random
 activation of several functional groups along the polysaccharide chain can
 lead to a conjugate that is highly cross-linked due to random linkages
 along the polysaccharide chain. The optimal ratio of polysaccharide to
 carrier protein depend on the particular polysaccharide, the carrier
 protein, and the conjugate used.
 See Dick et al., in Contributions to Microbiology and Immunology, Vol. 10,
 Cruse et al., eds., (S. Karger: 1989), pp. 48-114; Jennings et al., in
 Neoglycoconjugates: Preparation and Applications, Lee et al., eds.,
 (Academic Press: 1994), pp. 325-371; Aplin et al., CRC Crit. Rev.
 Biochem., 10:259-306 (1981); and Stowell et al., Adv. Carbohydr. Chem.
 Biochem., 37:225-281 (1980) for detailed reviews of methods of conjugation
 of saccharide to carrier proteins.
 The carbohydrate itself can be synthesized by methods known in the art, for
 example by enzymatic glycoprotein synthesis as described by Witte et al.,
 J. Am. Chem. Soc., 119:2114-2118 (1997).
 Several oligosaccharides, synthetic and semi-synthetic, and natural, are
 discussed in the following paragraphs as examples of oligosaccharides that
 are contemplated haptens to be used in making a strategically modified
 core protein conjugate of the present invention.
 An oligosaccharide hapten suitable for preparing vaccines for the treatment
 of Haemophilus Influenza type b (Hib) is made up of from 2 to 20 repeats
 of D-ribose-D-ribitol-phosphate (I, below), D-ribitol-phosphate-D-ribose
 (II, below), or phosphate-D-ribose-D-ribitol (III, below). Eduard C.
 Beuvery et al., EP-0 276 516-B1.
 ##STR1##
 U.S. Pat. No. 4,220,717 also discloses a polyribosyl ribitol phosphate
 (PRP) hapten for Haemophilus influenzae type b.
 Ellena M. Peterson et al., Infection and Immunity, 66(8):3848-3855 (1998),
 disclose a trisaccharide hapten, .alpha.Kdo(2 8).alpha.Kdo(2 4).alpha.Kdo,
 that provides protection from Chlamydia pneumoniae. Chlamydia pneumoniae
 is a cause of human respiratory infections ranging from pharyngitis to
 fatal pneumonia. Kdo is 3-deoxy-D-manno-oct-2-ulosonic acid.
 Bengt Andersson et al., EP-0 126 043-A1, disclose saccharides that can be
 used in the treatment, prophylaxis or diagnosis of bacterial infections
 caused by Streptococci pneumoniae. One class of useful saccharides are
 derived from the disaccharide GlcNAc.beta.1 3 Gal. Andersson et al. also
 found neolactotetraosylceramide to be useful, which is Gal.beta.1
 4GlcNAc.beta.1 3Gal.beta.1 4Glc-Cer.
 European Patent No. 0 157 899-B1, the disclosures of which are incorporated
 herein by reference, discloses the isolation of pneumococcal
 polysaccharides that are useful in the present invention. The following
 table lists the pneumococcal culture types that produce capsular
 polysaccharides useful as haptens in the present invention.
 TABLE 4
 Polysaccharide Hapten Sources
 Danish Type U.S. 1978 ATCC Catalogue
 Nomenclature Nomenclature Number
 1 1 6301
 2 2 6302
 3 3 6303
 4 4 6304
 5 5
 6A 6 6306
 6B 26 6326
 7F 51 10351
 8 8 6308
 9N 9 6309
 9V 68
 10A 34
 11A 43
 12F 12 6312
 14 14 6314
 15B 54
 17F 17
 18C 56 10356
 19A 57
 19F 19 6319
 20 20 6320
 22F 22
 23F 23 6323
 25 25 6325
 33F 70
 Moraxella (Branhamella) catarrhalis is a know cause of otitis media and
 sinusitis in children and lower respiratory tract infections in adults.
 The lipid A portion of the lipooligosaccharide surface antigen (LOS) of
 the bacterium is cleaved at the 3-deoxy-D-manno-octulosonic
 acid-glucosamine linkage. The cleavage product is treated with mild-alkali
 to remove ester-linked fatty acids while preserving amide-linked fatty
 acids to yield detoxified lipopolysaccharide (dLOS) from M. catarrhalis.
 The dLOS is not immunogenic until it is attached to a protein carrier.
 Xin-Xing Gu, et al. Infection and Immunity 66(5):1891-1897 (1998).
 Group B streptococci (GBS) is a cause of sepsis, meningitis, and related
 neurologic disorders in humans. The Capsular polysaccharide-specific
 antibodies are known to protect human infants from infection. Jennings et
 al., U.S. Pat. No. 5,795,580. The repeating unit of the GBS capsular
 polysaccharide type II is: 4)-.beta.-D-GlcpNAc-(1 3)-[.beta.-D-Galp(1
 6)]-.beta.-D-Galp(1 4)-.beta.-D-Glcp-(1 3)-.beta.-D-Glcp-(1
 2)-[.alpha.-D-NeupNAc(2 3)]-.beta.-D-Galp-(1, where the bracketed portion
 is a branch connected to the immediately following unbracketed subunit.
 The repeating unit of GBS capsular polysaccharide type V is:
 4)-[.alpha.-D-NeupNAc-(2 3)-.beta.-D-Galp-(1 4)-.beta.-D-GlcpNAc-(1
 6)]-.alpha.-D-Glcp-(1 4)-[.beta.-D-Glcp-(1 3)]-.beta.-D-Galp-(1
 4)-.beta.-D-Glcp-(1.
 European patent application No. EU-0 641 568-A1, Dr. Helmut Brade,
 discloses the method of obtaining ladder-like banding pattern antigen from
 Chlamydia trachomatis, pneumoniae and psittaci.
 D. Pathogen-related Conjugate to the Modified HBc
 In one embodiment of the invention, the hapten that is conjugated to
 strategically modified HBc protein is a B cell determinant of a pathogen.
 B cell determinants of numerous pathogens are known in the art, and
 several were illustrated in the preceding discussions of polypeptide and
 carbohydrate haptens.
 In preferred embodiments, the hapten is a pathogen-related hapten. The use
 of a portion of a pathogen's protein sequence or carbohydrate sequence as
 a hapten has distinct advantages over the exposure to an actual pathogen,
 and even over a passivated or "killed" version of the pathogen.
 Exemplary pathogen-related haptens of particular importance are derived
 from bacteria such as B. pertussis, S. typosa, S. paratyphoid A and B, C.
 diptheriae, C. tetani, C. botulinum, C. perfringens, B. anthracis, P.
 pestis, P. multocida, V. cholerae, N. meningitides, N. gonorrhea, H.
 influenzae, T. palladium, and the like.
 Other exemplary sources of pathogen-related haptens of particular
 importance are viruses such as poliovirus, adenovirus, parainfluenza
 virus, measles, mumps, respiratory syncytical virus, influenza virus,
 equine encephalomyelitis virus, hog cholera virus, Newcastle virus, fowl
 pox virus, rabies virus, feline and canine distemper viruses, foot and
 mouth disease virus (FMDV), human and simian immunodeficiency viruses, and
 the like. Other important sources of pathogen-related haptens include
 rickettsiae, epidemic and endemic typhus, the spotted fever groups, and
 the like.
 Pathogen-related polypeptide haptens are well-known in art and are
 discussed in numerous disclosures such as U.S. Pat. Nos. 3,149,036,
 3,983,228, and 4,069,313; in Essential Immunology, 3.sup.rd Ed., by Roit,
 published by Blackwell Scientific Publications; in Fundamentals of
 Clinical Immunology, by Alexander and Good, published by W. B. Saunders;
 and in Immunology, by Bellanti, published by W. B. Saunders.
 Particularly preferred pathogen-related haptens are those described in U.S.
 Pat. Nos. 4,625,015, 4,544,500, 4,545,931, 4,663,436, 4,631,191, 4,629,783
 and in Patent Cooperation Treaty International Publication No. WO87/02775
 and No. WO87/02892, all of whose disclosures are incorporated herein by
 reference.
 Antibodies that immunoreact with the hepatitis B virus can be obtained by
 using modified HBc conjugated with a polypeptide hapten that corresponds
 to the sequence of a determinant portion of HBsAg; in particular, residues
 110-137 of the "S" (surface) region disclosed in Gerin et al., Proc. Natl.
 Acad. Sci. USA, 80:2365 (1983).
 Another conjugate corresponds to amino acids 93-103 of capsid protein VPI
 of poliovirus type 1 (PV1, Mahoney strain), analogous to the work by
 Delpeyroux et al., Science, 233:472-475 (1986). Such a modified HBc
 conjugate provides antibodies that immunoreact with polio. Other potential
 haptens from poliovirus type 1, Mahoney and Sabin strains are described in
 European Patent No. 385610.
 In preferred embodiments, the hapten is a pathogen-related hapten that
 immunoreacts with; i.e., is immunologically bound by, antibodies induced
 by the pathogen. More preferably, the pathogen-related hapten induces an
 antibody response that provides protection against infection by the
 pathogen.
 Methods for determining the presence of antibodies to an immunogen in a
 body sample from an immunized animal are well known in the art. Methods
 for determining the presence of both cross-reactive and protective
 antibodies are well known in the art.
 In another embodiment of the invention, the immune response to the B cell
 determinant is boosted by also providing a T cell determinant.
 For example, U.S. Pat. No. 4,882,145 describes T cell stimulating
 polypeptides derived from the HBV nucleocapsid protein. Other T cell
 determinants are known in the art and can be used as an operatively linked
 determinant in a contemplated modified HBc protein or particle.
 In a particularly preferred embodiment of the invention, such a T cell
 determinant is derived from the same pathogen as the B cell determinant
 that is conjugated to the modified HBc. The T cell determinants of various
 pathogens are reported in the art.
 Although it is preferred that the B and T cell determinants are derived
 from the same pathogen, it is not necessary that they be from the same
 protein of that pathogen. For example, the B cell determinant from the VP1
 protein of the foot and mouth disease virus (FMDV) can be conjugated to a
 modified HBc particle, wherein the HBc protein is further modified by
 having a T cell determinant derived from the VP4 protein of FMDV.
 The additional T cell determinant can be introduced into a modified HBc
 protein on the genetic level using well-known methods within or at a
 terminus of the HBc protein sequence (amino or carboxy termini), including
 as part of the DNA inserted to introduce the chemically-reactive amino
 acid residue, but preferably as a fusion protein at the carboxy terminus.
 Alternatively, the additional T cell determinant can be operatively linked
 to the conjugated B cell determinant or the strategically modified HBc
 protein.
 Exemplary disclosures that describe techniques for genetically engineering
 a DNA sequence that can be used to produce a fusion protein of the present
 invention can be found in: U.S. Pat. No. 4,428,941 to Galibert et al.,
 U.S. Pat. No. 4,237,224 to Cohen et al.: U.S. Pat. No. 4,273,875 to Manis;
 U.S. Pat. No. 4,431,739 to Riggs; U.S. Pat. No. 4,363,877 to Goodman et
 al., and Rodriguez & Tait, Recombinant DNA Techniques: An Introduction,
 The Benjamin-Cummings Publishing Co., Inc. Menlo Park, Calif. (1983),
 whose disclosures are incorporated by reference.
 E. Inocula and Vaccines
 In yet another embodiment of the invention, a modified HBc protein or
 particle conjugated to a hapten (a HBc conjugate) is used as the immonogen
 of an inoculum that induces production of antibodies that immunoreact with
 the hapten or as a vaccine to provide protection against the pathogen from
 which the hapten is derived.
 A contemplated inoculum or vaccine comprises a HBcAg conjugate 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 (e.g., a mouse,
 dog, goat, sheep, horse, bovine, monkey, ape, or human) or bird (e.g., a
 chicken, turkey, duck or goose), an inoculum induces antibodies that
 immunoreact with the conjugated (pendently-linked) hapten. A vaccine is a
 type of inoculum in which the hapten is pathogen related and the induced
 antibodies not only immunoreact with the hapten, but also immunoreact with
 the pathogen or diseased cell, and neutralize the pathogen or diseased
 cell with which they immunoreact.
 The preparation of inocula and vaccines that contain proteinaceous
 materials as active ingredients is also well understood in the art.
 Typically, such inocula or vaccines are prepared as parenterals, either as
 liquid solutions or suspensions; solid forms suitable for solution in, or
 suspension in, liquid prior to injection can also be prepared. The
 preparation can also be emulsified. 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 or adjuvants which enhance the
 immunogenic effectiveness of the composition.
 Exemplary adjuvants include complete Freund's adjuvant (CFA) that is not
 used in humans, incomplete Freund's adjuvant (IFA) and alum, which are
 materials well known in the art, and are available commercially from
 several sources. The use of small molecule adjuvants is also contemplated
 herein.
 Exemplary of one group of small molecule adjuvants are the so-called
 muramyl dipeptide analogues described in U.S. Pat. No. 4,767,842. Another
 type of small molecule adjuvant described in U.S. Pat. No. 4,787,482 that
 is also useful herein is a 4:1 by volume mixture of squalene or squalane
 and Aracel.TM. A (mannide monooleate).
 Yet another type of small molecule adjuvant useful herein is a
 7-substituted-8-oxo or 8-sulfo-guanosine derivative described in U.S. Pat.
 Nos. 4,539,205, 4,643,992, 5,011,828 and 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
 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 amount of strategically modified HBc
 protein conjugate or strategically modified HBc protein particle conjugate
 as active ingredient. In a typical composition, an immunogenic amount of
 strategically modified HBc protein conjugate or strategically modified HBc
 protein particle conjugate is about 50 .mu.g to about 2 mg of active
 ingredient per dose, and more preferably about 100 .mu.g to about 1 mg per
 dose.
 The particles and protein 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 peptide) and
 which 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 may
 also be derived form inorganic bases such as, for example, sodium,
 potassium, ammonium, calcium, or ferric hydroxides, and such organic bases
 as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine,
 procaine, and the like.
 The inocula or vaccines are administered in a manner compatible with the
 dosage formulation, and in such amount as are therapeutically effective
 and immunogenic. The quantity to be administered depends on the subject to
 be treated, capacity of the subject's immune system to synthesize
 antibodies, and degree of protection desired. Precise amounts of active
 ingredient required to be administered depend on the judgment of the
 practitioner and are peculiar to each individual. However, suitable dosage
 ranges are of the order of several hundred micrograms active ingredient
 per individual. Suitable regimes for initial administration and booster
 shots are also variable, but are typified by an initial administration
 followed in intervals (weeks or months) by a subsequent injection or other
 administration.
 Another embodiment of the invention is a process for inducing antibodies in
 an animal host comprising the steps of inoculating said animal host with
 an inoculum. The inoculum used in the process comprises an immunogenic
 amount of a strategically modified hepatitis B core protein conjugate
 dissolved or dispersed in a pharmaceutically acceptable diluent. The
 strategically modified hepatitis B core protein conjugate used in the
 process comprises a hapten pendently linked to a strategically modified
 hepatitis B core protein. Preferably the strategically modified hepatitis
 B core protein is in particle form. The strategically modified hepatitis B
 core protein comprises an amino acid sequence corresponding to the
 hepatitis B core protein amino acid sequence of SEQ ID NO:2 including the
 amino acid residues numbered about 10 to about 140 and additionally having
 an insert in the region corresponding to amino acid residues numbered
 about 50 to about 100, said insert (i) being 1 to about 40 amino acid
 residues in length, and (ii) containing a chemically-reactive amino acid
 residue. The hapten is pendently linked to the strategically modified
 hepatitis B core protein through said chemically-reactive amino acid
 residue. Preferably, the hapten is pathogen-related. The animal is
 maintained for a time sufficient for antibodies to be induced.
 The invention is illustrated by the following non-limiting examples.
 EXAMPLE 1
 Construction of a Modified Hepatitis B Core Protein Expression Vector
 Using site-directed mutagenesis, a lysine codon (TTT) was introduced
 between amino acids D78 and P79 of the HBc gene, along with EcoRI (GAATTC)
 and SacI (GAGCTC) restriction endonuclease sites to facilitate the genetic
 insertion of other condons for producing strategically modified hybrid HBc
 particles. The insert thus had an amino acid residue sequence GIQKEL,
 where the GIQ is an artifact of the EcoRI site and the EL is an artifact
 of the SacI site. The strategically modified hepatitis B core protein was
 therefore 155 amino acid residues long. The construction of the
 pKK322-HBc155-K81 expression plasmid is described below.
 Oligonucleotide primers P1F (SEQ ID NO:17) and P1R (SEQ ID NO:18, on the
 complementary strand) were used to amplify the 5' end of the HBc gene
 (bases 1-234, amino acids 1-78), and simultaneously incorporate an NcoI
 restriction site (CCATGG) at the 5' end, and an EcoRI restriction site
 (GAATTC) at the 3' end of the amplified product. Oligonucleotide primers
 (SEQ ID NO:19) P2F and P2R (SEQ ID NO:20, on the complementary strand)
 were used to amplify the 3' end of the HBc gene (bases 232-450, amino
 acids 79-149), and simultaneously incorporate an EcoRI restriction site
 (GAATTC) at the 5' end, a SacI restriction site (GAGCTC) adjacent to it,
 an inserted lysine codon (AAA) between them, and a HindIII restriction
 site at (AAGCTT) the 3' end of the amplified product.
 The two PCR products (encoding amino acids 1-78 and amino acids 79-149)
 were cleaved with EcoRI, ligated together at their common EcoRI overhangs,
 cleaved with NcoI and HindIII and cloned into the expression plasmid
 pKK332 (Pharmacia), using standard techniques. The resulting plasmid was
 called pKK332-HBc-K81. This plasmid can be used for the expression of a
 strategically modified HBc protein bearing a lysine in the immunodominant
 loop. The expressed strategically modified HBc protein spontaneously
 formed particles. The strategically modified HBc of this Example thus had
 an insert corresponding to position 78 of the HBc of SEQ ID NO:2, a
 chemically reactive lysine residue at a position corresponding to position
 82 of the HBc of SEQ ID NO:2, and was truncated at a position
 corresponding to position 149 of the HBc of SEQ ID NO:2.
 EXAMPLE 2
 Modified Hepatitis B Core Particle Purification
 Strategically modified HBc particles of Example 1 were expressed in E. coli
 typically E. coli BLR or BL21 from Novagen (Madison, Wis.) or E. coli TB11
 from Amersham (Arlington Heights, Ill.). The transfected E. coli denoted
 HBc155-K81, were expressed plasmid pKK332-HBc155-K81. The strategically
 modified HBc particles were purified via Sepharose CL-4B chromatography
 using established procedures. Because particles purify in a predictable
 manner, the monitoring of particle elution using simple spectroscopy
 (OD.sub.280), in concert with SDS-PAGE analysis to assess purity of
 individual fractions prior to pooling, was sufficient to enable the
 routine purification of electrophoretically pure particles in high yield
 (5-120 mg/L cell culture). The spherical structure of the pure
 strategically modified hepatitis B core particles was clearly visible in
 an electron micrograph.
 EXAMPLE 3
 Chemical Coupling of Synthetic Peptides and Modified Hepatitis B Core
 Particles
 The strategically modified heptatitis B core particle product of the
 expression plasmid pKK332-HBc155-K81 from Example 1 was assayed for its
 chemical reactivity compared with similarly expressed and purified "wild
 type" truncated hepatitis B core particle HBc149, which is identical to
 HBc155-K81 except that it lacks the introduced lysine residue and flanking
 five amino acids.
 Synthetic peptides (haptens) were chemically conjugated to HBc particles
 using succinimidyl 4-(N-maleimido-methyl) cyclohexane 1-carboxylate
 (SMCC), a water-soluble heterobifunctional cross-linking reagent. SMCC is
 reactive towards both sulfhydryl and primary amino groups, enabling the
 sequential conjugation of synthetic peptides to HBc particles whose
 primary amino groups have previously been modified with SMCC. Further, the
 11.6 angstrom spacer arm afforded by SMCC helps to reduce steric hindrance
 between the hapten and the HBc carrier, thereby enabling higher coupling
 efficiencies.
 Briefly, HBc155-K81 and HBc149 particles were separately reacted with a
 3-fold excess of SMCC over total amino groups (native amino groups or
 native amino groups plus the one from the lysine residue of the insert)
 for 2 hours at room temperature in 50 AM sodium phosphate, pH 7.5, to form
 maleimide-activated HBc particles. Unreacted SMCC was removed by repeated
 dialysis against 50 mM sodium phosphate, pH 7.0. The SMCC derivatization
 of the HBc particles resulted in a minimal molecular weight increase which
 was not detectable by SDS-PAGE. However, the PAGE analysis did confirm the
 integrity of the HBc proteins prior to proceeding to the peptide
 conjugation step.
 Synthetic peptides to be coupled to the HBc particles were designed such
 that they had N-terminal cysteine residues to enable directional
 conjugation of peptide haptens to the primary amine on the side chain of
 the introduced lysine residue via the cysteine sulfhydryl of the hapten.
 Table 2 shows the synthetic peptides derived from human cytochrome P450
 enzymes that were chemically conjugated to HBc particles. The synthetic
 peptides were dissolved in 50 mM sodium phosphate, pH 7.0, to a
 concentration of 10 mg/ml. The synthetic peptides were then added,
 dropwise, to a 5-fold excess over total amino groups in
 maleimide-activated strategically modified HBc155-K81 particles, and
 permitted to react at room temperature for 2 hours. Maleimide-activated
 HBc149 particles were reacted with the two 2D6 peptides (206 and 206-C)as
 controls.
 TABLE 5
 Cytochrome P-450 Haptens
 Peptide Name Sequence SEQ ID NO.:
 1A1 (289-302) CQEKQLDENANVQL 21
 1A2 (291-302) CSKKGPRASGNLI 22
 2D6 (263-277) CLTEHRMTWDPAQPPRDLT 23
 3A4 (253-273) CVKRMKESRLEDTQKHRVDFLQ 24
 1A1-c CMQLRS 106
 1A2-c CRFSIN 107
 2D6-c CAVPR 108
 2E1-c CIPRS 109
 2C-c CFIPV 110
 3A3/4/7-c CTVSGA 111
 3A5-c CTLSGE 112
 EXAMPLE 4
 Analysis of Modified Core Particles Conjugates
 Strategically modified HBc particles pendently linked to cytochrome P-450
 determinant haptens of Example 3 were analyzed by SDS-PAGE and immunoblots
 to determine if synthetic peptides had been successfully conjugated to
 HBc. The denaturing conditions of the electrophoresis dissemble of
 particles into their constituent subunits, HBc monomers. Because HBc
 monomers have a molecular weight of approximately 16,000 Da, it was simple
 to resolve HBc155-K81 particles chemically conjugated to either 1A1
 (289-302), 1A2 (291-302), 2D6 (263-277) or 3A4 (253-273) peptides, as
 those peptides have a relative molecular mass of approximately 2,000 Da
 and therefore cause a visible increase in the molecular mass of the HBc
 protein monomers. From the relative intensities of the conjugated and
 non-conjugated bands on SDS-PAGE, it was revealed that approximately 50
 percent of the HBc155-K81 monomers were operatively linked to hapten,
 whereas only about 5 percent of the "wild type" HBc149 particles were
 linked to hapten. The marked increase in the observed success in pendently
 linking hapten to the strategically modified hepatitis B core protein
 supports the conclusion that the observed linking occurs via the inserted
 lysine as opposed to a lysine residue that is also present in the "wild
 type."
 The shift in mobility of HBc particles conjugated to shorter C-terminal
 P450 derived peptides (5 and 6-mers) is not as pronounced in the SDS-PAGE
 as that of the longer inhibitory peptides, but shifts of approximately 700
 Da were clearly evident in successfully coupled HBc155-K81 monomers. The
 strategically modified HBc 155-K81 protein exhibited markedly enhanced
 ability to pendently link to a hapten over the "wild type" HBc149
 particles, which showed minimal conjugation.
 In the model of core particles propounded by Conway et al. of icosahedral
 particles of either 180 or 240 associated core protein monomers [Nature,
 386:91-94 (1997)], dimers of the relatively exposed immunodominant loop
 regions of the core monomers extend out from the assembled core particle
 into solution like spikes on a mace. The "spikes" are closely arranged
 spatially on the HBc particles. The strategic location of the introduced
 lysine residue on the tip of the spike minimizes the propensity for steric
 constraints to reactions to link haptens to the assembled core particle. A
 maximum of 50 percent of the strategically modified HBc monomers were
 successfully conjugated to the synthetic peptides of Cyt P-450. That
 amount of pendent linkage corresponds to an average of one hapten attached
 per core particle spike. This proposed distribution of hapten linkage to
 the strategically modified HBc particle is supported by PAGE results under
 semi-denaturing conditions that dissemble the particle while maintaining
 the dimer association.
 HBc-2D6 particles, prepared by peptide coupling, were examined using
 immunoblots to confirm the presentation of the 2D6 epitope. When probed
 with anti-HBc antisera, the chemically coupled particle yielded two
 different monomers representing particles with and without the 2D6
 peptide. only the upper band of which blotted with anti-2D6 antisera,
 thereby confirming the correlation between mobility shift and attachment
 of the 2D6 peptide.
 EXAMPLE 6
 Strategic Lysine Insertions
 To construct HBc particles with inserted lysine residues at every position
 in the immunodominant, surface-exposed loop region (amino acids 75-85),
 PCR was used to amplify the 5' and 3' fragments of the HBc gene and a
 single lysine codon was introduced via the oligonucleotide primers. The
 oligonucleotide primers and the resulting amino acid sequences are shown
 in SEQ ID NOs: 61-82. The "wild type" sequences are SEQ ID NOs:59-60.
 In order to generate lysine inserts at positions 75 to 84 (HBc-K75 through
 HBc-K84), the pairs of PCR fragments were digested with the restriction
 endonuclease MseI, which recognizes the sequence, AATT. The modified gene
 was restored by ligating the oligonucleotide primer (containing the
 lysine) at the convenient MseI restriction site located at nucleotides
 221-224. For HBc-K85 (SEQ ID NOs:85-86) it was necessary to generate two
 fragments that were ligated at a common XhoI restriction site (CTCGAG)
 that is not present in the wild type gene, but could be introduced at
 position 239-244 without altering any amino acids.
 TABLE 6
 Lysine insertion mutants of HBc
 in the immunodominant loop
 Name Sequence SEQ ID NO:
 Wild Type HBc TWVGVNLEDPASRDLVVSYV 60
 K75 TWVGVKNLEDPASRDLVVSYV 62
 K76 TWVGVNKLEDPASRDLVVSYV 64
 K77 TWVGVNLKEDPASRDLVVSYV 66
 K78 TWVGVNLEKDPASRDLVVSYV 68
 K79 TWVGVNLEDKPASRDLVVSYV 70
 K80 TWVGVNLEDPKASRDLVVSYV 72
 K81 TWVGVNLEDPAKSRDLVVSYV 74
 K82 TWVGVNLEDPASKRDLVVSYV 76
 K83 TWVGVNLEDPASRKDLVVSYV 78
 K84 TWVGVNLEDPASRDKLVVSYV 80
 K85 TWVGVNLEDPASRDLKVVSYV 82
 To purify the strategically modified HBc proteins, cleared cell lysates
 from a 1L fermentation were precipitated with 45% ammonium sulfate and the
 resultant pellet subjected to gel filtration using Sepharose CL-4B
 chromatography (2.5cm.times.100cm). Particulate HBc has a characteristic
 elution position when analyzed using this type of column, which was
 independent of the amino acid insertions made to the particle. The eleven
 strategically modified HBc particles generated for this study were
 analyzed using this procedure, and the elution profiles were measured
 spectrophotometrically at an absorbance of 280 nm.
 Three of the constructs (HBc-K75, HBc-K77, and HBc-K79) were produced at
 levels of between 50 and 100 mg/L, which is comparable with typical yields
 for wild-type, unmodified HBc particles, e.g. HBc149 particles. Four of
 the constructs (HBc-K76, HBc-K78, HBc-K81, and HBc-K82) were produced at
 relatively low levels of between 1 and 20 mg/L. Finally, four of the
 particles (HBc-K80, HBc-K83, HBc-K84, and HBc-K85) were produced at levels
 deemed to be barely detectable (&lt;1 mg/L).
 TABLE 7
 Yields of purified lysine-containing
 HBc particles from a 1L fermentation
 Particle Yield (mg/L)
 HBc-150 (K75) 77
 HBc-150 (K76) 5
 HBc-150 (K77) 74
 HBc-150 (K78) 10
 HBc-150 (K79) 94
 HBc-150 (K80) 0
 HBc-150 (K81) 17
 HBc-150 (K82) 1
 HBc-150 (K83) 0
 HBc-150 (K84) 0
 HBc-150 (K85) 0
 The foregoing description of the invention, including the specific
 embodiments and examples, is intended to be illustrative of the present
 invention and is not to be taken as limiting. Numerous other variations
 and modifications can be effected without departing from the true spirit
 and scope of the present invention.