A method of treating chronic hepatitis B is disclosed that comprises administering a T cell-stimulating amount of a vaccine to a patient. The vaccine comprises an immunogenic amount of chimeric, carboxy-terminal truncated hepatitis B virus nucleocapsid (core) protein (HBc) that is engineered for both enhanced stability of self-assembled particles and the substantial absence of nucleic acid binding by those particles. The chimeric protein molecule can include one or more immunogenic epitopes peptide-bonded to one or more of the N-terminus, the immunogenic loop or the C-terminus of HBc. The enhanced stability of self-assembled particles is obtained by the presence of at least one heterologous cysteine residue near one or both of the amino-terminus and carboxy-terminus of the chimer molecule.

Description of the Invention

BRIEF SUMMARY OF THE INVENTION

The present invention contemplates a method of treating an individual chronically infected with the hepatitis B virus, by administering to that patient a vaccine comprised of recombinant truncated and stabilized hepadnaviral nucleocapsid protein particles dissolved or dispersed in a pharmaceutically acceptable diluent in an amount sufficient to enhance the immune response against the virus to a patient having a chronic hepatitis B virus infection. Such enhancement of the immune response against the virus, alone or in combination with other therapies, can permit the individual to clear the virus from the body and to no longer be infectious. It is preferred that the recombinant truncated and stabilized hepadnaviral nucleocapsid protein be substantially free of host-nucleic acid.

The method utilizes a vaccine comprised of a recombinant hepadnavirus nucleocapsid protein; i.e., a hepatitis B core (HBc) chimeric protein [also referred to herein as a chimer hepatitis B core protein molecule, a HBc chimer molecule or just a chimer] that self-assembles into particles after expression in a host cell and is dissolved or dispersed in a pharmaceutically acceptable diluent. A contemplated chimer molecule is truncated at least at the C-terminus relative to a native core molecule whose C-terminus is usually at about residue position 183. Particles containing a contemplated chimer molecule are preferably stabilized by a cysteine residue at or near one or both of the N- and C-termini. A contemplated chimer molecule contains about 125 to all of the N-terminal 165 amino acid residues of HBc and can include one or more other amino acid residues or residue sequences that are typically B or T cell epitopes of HBV, another pathogen or another protein such as bovine inhibin.

In one aspect of the invention, a contemplated method of treating chronic hepatitis comprises the steps of administering an anti-HBc T cell-stimulating amount of a vaccine comprised of immunogenic particles dissolved or dispersed in a pharmaceutically acceptable diluent to a patient having a chronic hepatitis B virus infection. The immunogenic particles are preferably administered in conjunction with an immunostimulatory adjuvant. Preferred immunostimulatory adjuvants include lipid-A analogues such as monophosphoryl lipid A or synthetic aminoalkyl glucosamide phosphates. The immunostimulatory molecules are preferably associated with a microparticulate carrier such as oil-in-water emulsions or microparticulate mineral salts such as aluminium hydroxide gel. The immunogenic particles are themselves comprised of recombinant hepatitis B core (HBc) chimeric protein molecules, with the chimeric protein molecules being up to about 550 amino acid residues in length. Those chimeric protein molecules (a) contain an HBc sequence of about 125 to all of the N-terminal 165 amino acid residues of the HBc molecule and contains the HBc sequence of residue positions 4 through about 75 and about 85 through about 140. The HBc chimer molecule sequence optionally includes (a') a peptide-bonded amino acid sequence containing an immunogenic epitope at one or more of the N-terminus, in the HBc immunodominant loop (i.e., between residue positions about 76 through about 85) and the C-terminus of the chimer, or (b') an insert in the HBc immunodominant loop having a length of one to about 40 amino acid residues and containing a chemically-reactive linker residue for a conjugated hapten, or (c') zero to all of the residues of the sequence of positions 76 through 85.

The chimeric protein molecule also contains one or both of (a') 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:1 [N-terminal cysteine residue(s)] in a sequence other than that of the HBc precore sequence and (b') one to about three cysteine residues toward the C-terminus of the molecule from the C-terminal residue of the HBc sequence and within about 30 residues from the C-terminus of the chimer molecule [C-terminal cysteine residue(s)].

A chimeric protein molecule contains no more than about 20 percent conservatively substituted amino acid residues in the HBc sequence, and self-assembles into particles. Those particles are preferably substantially free of binding to nucleic acids (exhibits a ratio of absorbance at 280 nm to 260 nm of about 1.2 to about 1.7, as discussed hereinafter) on expression in a host cell (followed by collection and purification), but can also include a minimal amount of bound nucleic acid such that the ratio of absorbance at 280 nm to 260 nm is about 0.9 to about 1.15. Thus, particles that exhibit a ratio of absorbance at 280 nm to 260 nm of about 0.9 to about 1.7 can be used herein. The particles are more stable than are particles (i) formed from otherwise identical HBc chimer molecules that are free of any above-mentioned C-terminal cysteine residue(s) or N-terminal cysteine residue(s) or (ii) in which a C-terminal or an N-terminal cysteine residue(s) present in a contemplated chimer molecule is (are) replaced by another residue.

The patient is maintained for a time sufficient to induce T cells activated against HBc. In other aspects of the invention the patient is treated with an antiviral medicament such as lamivudine to reduce viral burden. The treatment with an antiviral can be concurrent with vaccination, or can precede vaccination. A contemplated aspect of the invention includes a kit comprising both antiviral medicament and HBc chimer intended for administration to patients.

In other aspects of the invention, the patient has serum that contains HbsAg, and the treatment results in decreasing the amount of that antigen in the patient's serum. In a further aspect of the invention, the patient's serum contains HBeAg, and the treatment results in decreasing the amount of the HBeAg antigen in the patient's serum.

A preferred recombinant hepatitis B virus core (HBc) protein chimer molecule has a length of about 135 to about 525 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) zero 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:1 [N-terminal cysteine residue(s)] in a sequence other than that of the HBc precore sequence, and (iii) an optional 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 up to about 255 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 through 85 are present peptide-bonded to (ii) an optionally present sequence of one to about 245 amino acid residues that constitute an immunogenic epitope or a 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 thirty residues of an HBc amino acid residue sequence from position 136 through 165 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 other than that present in HBc from position 165 to the C-terminus.

A preferred chimer molecule (i) has 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-assembles into particles on expression by 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 some embodiments, 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. In other embodiments, it is preferred that a contemplated chimer molecule 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. In yet other embodiments, a preferred chimer molecule contains only one or more C-terminal cysteine residues and Domain I is free of non-HBc cysteine residues. A cysteine residue is present at about position 61 in each of the HBc sequences of FIG. 1 (see Original Patent).

A contemplated method utilizes a vaccine that comprises before-mentioned self-assembled chimer molecule particles dissolved or dispersed in a pharmaceutically acceptable diluent composition that typically also contains water. A particularly preferred non-HBc epitope present in a contemplated chimer molecule at one or more of Domains I, II and III is an immunogenic sequence from the pres1 or preS2 regions of the hepatitis B surface protein (HBs).

The present invention has several benefits and advantages.

A particular benefit of the invention is that its use as a therapeutic vaccine provides extraordinary T cell activation.

Another benefit of the invention is that the recombinant immunogen is prepared easily and using well known cell culture techniques.

An advantage of the invention is that the immunogen is easily prepared using well known recombinant techniques.

Another advantage of the invention is that a preferred immunogen exhibits greater stability on preparation than do other HBc chimers that lack one or both of a C-terminal or N-terminal cysteine residue, while being substantially free of nucleic acids.

DETAILED DESCRIPTION OF THE INVENTION

The present invention contemplates a method for treating chronic hepatitis B infection. A contemplated method utilizes a vaccine comprising a chimeric recombinant hepadnavirus nucleocapsid protein; i.e., a hepatitis B core (HBc) chimeric protein molecule that self-assembles into particles after expression in a host cell. A contemplated chimer molecule is truncated at least at the C-terminus relative to a native core molecule whose C-terminus is normally at residue position 183 for the ayw subtype of FIG. 1. Particles containing a contemplated chimer molecule are stabilized by a cysteine residue that is located at or near one or both of the C- and N-termini, and are preferably substantially free of binding to nucleic acids as is discussed hereinafter.

A contemplated chimer molecule contains at least about 125, and more preferably at least about 135, to all of the N-terminal 165 amino acid residues of HBc and can include one or more other amino acid residue sequences that are typically B or T cell epitopes of HBV, another pathogen or another protein such as bovine inhibin. Examples of B cell and T cell epitopes from non-HBV proteins that can be incorporated in the chimer molecule are illustrated hereinafter in Tables A and B (see Original Patent). An example of a T-cell epitope that is derived from the hepatitis B virus that is preferably incorporated in the chimer molecule is the surface antigen Pre-S2 sequence 144-160. An example of a B-cell epitope that is derived from the hepatitis B virus that is preferably incorporated in the chimer molecule is the surface antigen Pre-S2 sequence 130-144.

A contemplated method of treating chronic hepatitis comprises the steps of administering an anti-HBc T cell-stimulating amount of a vaccine comprised of immunogenic particles dissolved or dispersed in a pharmaceutically acceptable diluent to a patient having a chronic hepatitis B virus infection. The immunogenic particles are preferably administered in conjunction with an adjuvant.

Preferred adjuvants used herein are molecules that interact with toll-like receptors. Most preferred adjuvants are lipid-A analogues such as monophosphoryl lipid A and aminoalkyl glucosamide phosphates. Other preferred adjuvants include saponins and chemically modified alkylated saponins. The adjuvants can further comprise microparticulate carriers such as oil-in water emulsions or mineral salts.

The immunogenic particles are comprise recombinant hepatitis B core (HBc) chimeric protein molecules, with the chimeric protein molecules being up to about 550 amino acid residues in length. Those chimeric protein molecules (a) contain an HBc sequence of about 125 up to all of the N-terminal 165 amino acid residues of the HBc molecule that contains the HBC sequence of residue positions 4 through about 75 and about 85 through about 140.

The HBC chimer molecule sequence optionally includes (a') a peptide-bonded amino acid sequence containing an immunogenic epitope at one or more of the N-terminus, in the HBc immunodominant loop (i.e., between residue positions 76 through 85) and the C-terminus of the chimer, or (b') an insert in the HBc immunodominant loop having a length of one to about 40 amino acid residues that includes a chemically non-reactive residue or a chemically-reactive linker residue for a conjugated hapten, or (c') zero to all of the residues of the sequence of positions 76 through 85.

The chimeric protein molecule also contains one or both of (a') 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:1 [N-terminal cysteine residue(s)] in a sequence other than that of the HBc precore sequence and (b') one to about three cysteine residues toward the C-terminus of the molecule from the C-terminal residue of the HBc sequence and within about 30 residues from the C-terminus of the chimer molecule [C-terminal cysteine residue(s)].

A chimeric protein molecule contains no more than 20 percent conservatively substituted amino acid residues in the HBc sequence, and self-assembles into particles on expression in a host cell. In one aspect of the invention, the particles are substantially free of binding to nucleic acids and exhibit a ratio of absorbance ratio at 280 nm to 260 nm of about 1.2 to about 1.7, whereas in other aspects, more than minimal nucleic acid binding is present and the particles exhibit an absorbance ratio at 280 nm to 260 nm of about 0.9 to about 1.15. Broadly, therefore, the absorbance ratio at 280 nm to 260 nm of contemplated particles can be about 0.9 to about 1.7. Nucleic acid binding is discussed hereinafter. 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) or N-terminal cysteine residue(s) or (ii) in which a C-terminal or an N-terminal cysteine residue(s) present in a contemplated chimer molecule is (are) replaced by another residue.

The patient to whom the vaccine is administered is maintained for a time sufficient to induce T cells activated against HBc. In other embodiments, the method is carried out on patients that have HBsAg circulating in their blood stream and the patient is maintained for a time period sufficient to diminish the amount to circulating HBsAg. In a further aspect of the invention, the patient's serum contains HBeAg, and the treatment results in decreasing the amount of the HBeAg antigen in the patient's serum. Those skilled in the art are well aware of known methods for assaying for each of T cell activation against HBc/HbeAg and HBsAg.

The chimeric protein can display one or more immunogenic epitopes at the N-terminus, in the HBc immunogenic (immunodominant) loop or C-terminus, or a non-reactive (heterologous) residue or a linker residue for a B cell or T cell epitope in the immunogenic loop, or has zero to all of the residues of positions 76 through 85. In one embodiment, the chimeric protein contains one or more N-terminal cysteine residue(s) that confers enhanced stability on formation to the self-assembled particles.

In another embodiment, the chimeric protein contains one or more C-terminal cysteine residue(s) that confers enhanced stability on formation to the self-assembled particles. A contemplated chimeric protein molecule can also contain a cysteine residue at or near both of the N- and C-termini, that is a chimeric protein molecule can contain both an N-terminal cysteine residue and a C-terminal cysteine residue, as defined previously.

In some preferred embodiments, a contemplated 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. In other embodiments, the HBc sequence from position 149 through about position 163 that includes two of the arginine-rich repeat sequences is present (See, FIG. 1). In other embodiments, the HBc sequence through about position 156 that contains one arginine-rich sequence is present. In still other embodiments, the C-terminal HBc sequence ends between HBc positions 140 and 149 and the chimer molecule is free of the arginine repeats present in a native HBc sequence of FIG. 1 from position 150 through the C-terminus or a similar sequence containing lysine residues in place of one or more of the arginine residues. Substantial freedom from nucleic acid binding is discussed hereinafter and is readily determined.

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 NO: 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 550 residues in length and contains

(a) an HBc sequence of about 125 to all of the N-terminal 165 amino acid residues of the HBc molecule that includes the HBc sequence of residue positions 5 through about 75 and about 85 through about 140, (a') a peptide-bonded immunogenic epitope at one or more of the N-terminus, in the HBc immunodominant loop or the C-terminus of the chimer, or (b') an insert in the HBc immunodominant loop having a length of one to about 40 amino acid residues and containing a chemically non-reactive residue or a chemically reactive linker residue for a conjugated hapten, or (c') zero to all of the residues of the sequence of positions 76 through 85.

The chimeric protein molecule also contains one or both of (a') 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: 1 [N-terminal cysteine residue(s)] in a sequence other than that of the HBc precore sequence and (b') one to about three cysteine residues toward the C-terminus of the molecule from the C-terminal residue of the HBc sequence and within about 30 residues from the C-terminus of the chimer molecule [C-terminal cysteine residue(s)].

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

The patient is maintained for a time period sufficient to induce T cells activated against HBc. In other embodiments the patient is first treated with an antiviral drug such as lamivudine for a time sufficient to reduce viral burden, and then the patient receives one or more administrations of the contemplated chimer molecule administered in an acceptable excipient optionally with an adjuvant. In further embodiments, the method is carried out on patients that have HBsAg circulating in their blood stream and the patient is maintained for a time period sufficient to diminish the amount to circulating HBsAg. In a further aspect of the invention, the patient's serum contains HBeAg, and the treatment results in decreasing the amount of the HBeAg antigen in the patient's serum.

A contemplated chimer molecule contains at least one cysteine residue that is located at either or both of (i) 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: 1 or (ii) toward the C-terminus of the molecule from the C-terminal residue of the HBc sequence and within about 30 residues from the C-terminus of the chimer molecule. The 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 NO: 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 that can be 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 525 L-.alpha.-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 .alpha.-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: 1 [N-terminal cysteine residue(s)] in a sequence other than that of the HBc precore sequence, and (iii) an optional sequence containing up to about 30 amino acid residues peptide-bonded to one of HBc residues 2-4 that comprise an immunogenic epitope. That immunogenic sequence, when present, is typically an epitope used to induce an anti-hepatitis B immune response.

Domain II of that chimer molecule comprises up to about 255 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 through 85 are present peptide-bonded to (ii) an optionally present sequence of one to about 245 amino acid residues that constitute an immunogenic epitope, or (iii) an insert in the HBc immunodominant loop having a length of one to about 40 amino acid residues that contains a chemically non-reactive residue or a chemically-reactive linker residue for a conjugated hapten. 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 epitope- or linker-containing sequence.

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 not present in 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 absent from those positions of 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 sequence comprising an epitope at the N-terminus peptide-bonded to one of HBc residues 2-5. In another aspect, a contemplated chimer molecule contains an epitope- or a linker residue-containing sequence peptide-bonded near the middle of the molecule located between HBc residues 76 and 85 in the immunodominant loop. In a further aspect, an epitope-containing sequence 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 epitope-containing sequences are present at the above locations, or one or two epitope-containing sequences are present along with a linker residue for an epitope. Each of those chimer molecules also contains one or both of an N-terminal or C-terminal cysteine residue(s), as discussed before. Specific examples of several of these chimer molecules and their self-assembled particles are discussed hereinafter.

As already noted, a contemplated HBc chimer molecule of this aspect contains about 135 to about 525 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 insert as short as about 6 amino acid residues can induce and be recognized by antibodies and T cell receptors and is therefore useful.

In another embodiment of the invention, one or more chemically non-reactive (heterologous) amino acid residues is inserted in Domain II not to function as a B-epitope but to reduce the recognition of the chimeric particle by antibodies circulating in the blood of patients infected with hepatitis B virus. In a preferred aspect of the invention the chimeric molecule contains a single amino acid insertion at residue position 76, 77, 78, 79, 80, 81 or 82, and most preferably at residue position 77. That inserted chemically non-reactive residue can be an alanine, leucine or isoleucine, and is most preferably an alanine residue. It can be desirable to render the particle less antigenic than the native HBc particle; i.e., recognized less well by anti-HBc antibodies resulting from HBV infection. One skilled in the art can use any number of amino acid residues and sequences inserted into Domain II to reduce the antigenicity.

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, peptide-bonded to residue 135, (ii) zero to three cysteines residues and (iii) optionally can contain a sequence of an immunogenic 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 immunogenic sequence is preferably heterologous to the sequence of HBc and is other than a sequence of HBc from about position 165 to the HBc C-terminus. The immunogenic 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. Illustrative T cell epitopes from the HBc sequence and from the preS1 and preS2 regions of hepatitis B surface protein (HBs or HBsAg) are provided in Tables A and B, hereinafter.

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 immunogenic epitopes. Those two immunogenic epitopes are present in Domains I and II, or II and IV, or I and IV. One of the two immunogenic epitopes is preferably a B cell epitope in some embodiments. In other embodiments, one of the two immunogenic epitopes is a T cell epitope. More preferably, both of the two immunogenic epitopes are the same or different T cell epitopes. In addition, a plurality of B cell epitopes can be present at a B cell epitope location, as can a plurality of T cell epitopes be present at a T cell epitope location.

In the embodiments in which the chimer molecule contains an immunogenic 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 immunogenic epitope(s), and that the chimer further include one or more T cell epitopes in Domain IV peptide-bonded to one of HBc residues 140-165.

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 immunogenic 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 140-165. 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, HBc antigenicity as measured by binding of anti-loop monoclonal antibodies in an ELISA assay as discussed hereinafter.

A preferred contemplated HBc chimer molecule contains a sequence of about 135 to about 525 residues. A preferred HBc chimer molecule that can contain one or two immunogenic epitopes of preferred lengths of about 15 to about 50 residues each and a preferred HBc portion length of about 140 to about 165 residues has a sequence length of about 170 to about 250 amino acid residues. Particularly preferred chimer molecules that contain one or two immunogenic epitopes have a length of about 190 to about 210 residues. A particularly preferred chimer molecule that is free of added immunogenic epitopes can have a length of about 140 to about 165 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 165 residues and contains (i) an immunogenic epitope peptide-bonded to one or more of the N-terminus, C-terminus or the immunodominant loop, or a heterologous non-reactive or linker residue for an epitope in the immunodominant loop, and (ii) one or both of one to three N-terminal cysteine residues and one 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 exhibit a 280:260 absorbance ratio of about 1.2 to about 1.7, as discussed hereinafter. Thus, a contemplated chimeric protein is free of the HBc sequence between positions about 155 and 183, and is more preferably free of a HBc sequence between positions about 155 and 183.

The presence of the above-discussed N-terminal cysteine residue(s) provides an unexpected enhancement of the ability of the chimer molecules to form stable immunogenic particles (discussed hereinafter). Thus, a contemplated HBc chimer particle 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.

Contemplated particles are more stable upon formation than are particles formed from otherwise identical HBc chimer molecules that (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 and are also are free of any above-mentioned C-terminal cysteine residue(s). In some instances, particles do not form unless an N-terminal cysteine is present. Examples of enhanced stabilities for both types of sequences are illustrated in the Examples that follow.

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

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

Thus, recombinantly expressed HBc particles or chimeric HBc particles that contain the arginine-and lysine-rich sequence at residue positions 150-183 (or 150-185) sometimes referred to in the art as the protamine region exhibit a ratio of absorbance at 280 nm to absorbance at 260 nm (280:260 absorbance ratio) of about 0.8. On the other hand, particles sufficiently free of arginine and lysine residues in Domain IV so that the self-assembled particles are substantially free of nucleic acid binding such as particles that are free of the arginine-rich nucleic acid binding region of naturally occurring HBc like those that contain fewer than about ten, preferably fewer than about 6, and more preferably fewer than three arginine or lysine residues or mixtures thereof adjacent to each other. Illustrative proteins have a native or chimeric sequence that ends at about HBc residue position 165, preferably at about 155 and more preferably at about position 140 to position 149, exhibit a 280:260 absorbance ratio of about 0.9 to about 1.7. A more typical 280:260 absorbance ratio is about 0.9 to about 1.0 for a sequence ending at about position 165, about 1.1 to about 1.2 for a sequence ending at about position 155, and about 1.4 to about 1.7 for a sequence ending at about position 140 to about 149. 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.

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 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 comprise an immunogenic sequence. 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: 1. 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 .beta.-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 PreS1 or PreS2 regions or the major HbsAg immunogenic sequence.

Domain II is a sequence of about 5 to about 250 amino acid residues. Of those residues, zero (none), and preferably at least 4 residues, and more preferably at least 8, constitute portions of the HBc sequence at positions 76 through 85, and one to about 245 residues, and preferably one to about 50 residues are heterologous (foreign) to HBc or correspond to an immunogenic HBc sequence such as a B or T cell epitope.

Thus, at least HBc residues 75 and 85 are present in Domains I and II, respectively. Those residues constitute (i) a heterologous linker residue for a epitope such as a B cell or T cell epitope or (ii) an immunogenic 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 preferably at least 4 and more preferably at least 8 residues, or all of the residues of positions 76 through 85 of the HBc sequence. Immunogenic 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 linker residue(s) as where residues 76-77 and 84-85 are present or where residues 76 and 83-85 are present. More preferably, Domain II contains at least 8 residues of the HBc sequence from residue 76 to 85. Most preferably, the sequence of all 10 residues of positions 76 through 85 is present in the chimer.

The one to about 245 residues added to the HBc loop sequence can be heterologous to a HBc sequence or can correspond to one or more immunogenic portions of the 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 an immunogen such as a B cell or T cell epitope, except for heterologous residues encoded by restriction sites.

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 (see Original Patent), 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.

In the above influenza A M2 sequence of SEQ ID NO: 32,

residues X.sub.1 through X.sub.8 are absent or present, and when present are the residues naturally present in the M2 protein sequence that are methionine, serine, leucine, leucine, threonine, 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,

residues X.sub.15 and X.sub.16 are present or absent, and when present are tryptophan and glycine, respectively,

residues X.sub.17 and X.sub.19 are present or absent, and when present are independently cysteine, serine, or alanine,

residue X.sub.18 is present or absent, and when present is arginine, and

residues X.sub.20 through X.sub.24 are present or absent, and when present are the residues naturally present in the M2 protein sequence that are asparagine, aspartic acid, 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.

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 through 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 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 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 meningitidis 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 chemically non-reactive residue or linker for a conjugated epitope can be peptide-bonded at a position in the HBc sequence between amino acid residues 76 and 85. As was the case for the immunogenic epitope, the HBc sequence of residues 76 through 85 is preferably present, but interrupted by the added residue or residues. This chimer preferably includes the HBc sequence of position 4 through at least position 140, plus a cysteine residue near the N-terminus or the C-terminus of the chimer protein. More preferably, the HBc sequence of positions 1 through 149 are present, but interrupted between residues 76 and 85 by the heterologous linker for a conjugated epitope, and the chimer molecule contains a C-terminal cysteine.

A chemically non-reactive residue was discussed previously. 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. Pat. No. 6,231,864 B1 discloses HBc chimer molecules containing one or more linking residues, but lacking a stabilizing N-terminal cysteine residue.

It is also noted that an inserted chemically non-reactive residue, linker residue or immunogenic epitope-containing 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 immunogenic (epitope) sequence. This is particularly the case where the 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. These flexible linker arms are similar to those discussed before in relation to a Neisseria meningitidis B cell epitope sequence such as the peptide of SEQ ID NO: 125. Illustrative other 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:

The sequence immediately below is utilized at the C-terminus of an inserted epitope-containing sequence, whereas the sequences thereafter are used at each of the N- and C-termini of inserted immunogenic sequences

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 an immunogenic sequence that is preferably heterologous to HBc at position 165 to the C-terminus, with the proviso that Domain IV contains at least 5 amino acid residues of the HBc sequence from position 136 through 140. The Domain IV immunogenic sequence 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 165 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 125 amino acid residues (up to about HBc position 165 plus up to about 100 immunogenic residues of an immunogenic sequence) 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 525 residues. Where an epitope peptide-bonded to one or both of 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 170 to about 250 residues. Particularly preferred chimer molecules containing two immunogenic 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 (see Original Patent), as in Table A (see Original Patent), with illustrative added C-terminal cysteine residues underlined.
 

Claim 1 of 12 Claims

1. A method of enhancing the production of one or more of gamma-producing CD 8+, CD 4+ T cells and cytotoxic T lymphocytes against hepatitis B virus that comprises; (a) administering to a patient chronically infected with hepatitis B virus a T cell-stimulating amount of a composition comprising immunogenic particles dispersed in a squalene oil-in-water emulsion that includes an adjuvant comprised of a 2-[(R)-3-tetradecanoyloxytetra-decanoylamino]-ethyl-2-deoxy-4-O-phosphono- -3-O-[(R)-3-tetradecanoyloxytetra-decanoyl]-2-[(R)-3-tetra-decanoyloxytetr- adecanoyl-amino]-p-D-glucopyranoside triethylammonium salt, said immunogenic particles comprising recombinant hepatitis B core (HBc) chimeric protein molecules, said chimeric protein molecules being up to about 550 amino acid residues in length and containing (i) an HBc sequence of at least about 125 of the N-terminal 165 amino acid residues of the HBc molecule that includes the HBc sequence of residue positions 4 through about 75 and about 85 through about 140, and includes an insert in the HBc immunodominant loop, said insert having a length of one to about 40 amino acid residues containing one or more chemically non-reactive heterologous amino acid residues that render the immunogenic particles less antigenic than the native HBc particles, (ii) one or both of (a') 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:1 [N-terminal cysteine residue(s)] in a sequence other than that of the HBc precore sequence and (b') one to about three cysteine residues toward the C-terminus of the molecule from the C-terminal residue of the HBc sequence and within about 30 residues from the C-terminus of the chimer molecule [C-terminal cysteine residue(s)], said chimer molecule (a') containing no more than about 5 percent conservatively substituted amino acid residues in the HBc sequence relative to one of SEQ ID NO:1-6 from position 2 through 165, (b') having one or both HBc cysteine residues at positions 48 and 107 replaced by another residue, while the HBc cysteine at residue at position 61 is present, and (c') self-assembling into particles that upon expression in a host cell are substantially free of binding to nucleic acids, 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) or N-terminal cysteine residue(s) or in which a C-terminal or an N-terminal cysteine residue(s) present in a contemplated chimer molecule is(are) replaced by another residue; and (b) maintaining said patient for a time sufficient to induce T cells activated against HBc.
 

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