HK1100812B - Composition for the prophylaxis/treatment of hbv infections and hbv-mediated diseases - Google Patents

Description

Composition for preventing/treating HBV infection and HBV-mediated disease

The present invention relates to a composition comprising at least two hepatitis b virus surface antigens (HBsAg), fragments thereof and/or nucleotides encoding the same, wherein the HBsAg differs in the Hepatitis B Virus (HBV) genotype of the HBsAg S region and the pre-S1 region and the composition does not comprise HBV core antigen (HBcAg) and nucleic acids encoding the antigens; the present invention relates to pharmaceutical compositions, in particular vaccines, comprising these compositions and their use in the prevention and treatment of HBV infection and HBV mediated diseases. The invention also relates to a method for preparing a patient-specific medicament for the therapeutic treatment of hepatitis and a kit for diagnosing the HBV genotype.

More than 2.5 million people worldwide infect Hepatitis B Virus (HBV). Most of these infected individuals exhibit pathological consequences, including chronic hepatic insufficiency, cirrhosis, and hepatocellular carcinoma (HCC). The reasons why some people develop acute HBV infection and others do not. Clinical data and similarities to other chronic viral infections underscore the importance of cell-mediated immune responses in controlling viral infections, particularly including cytotoxic T lymphocytes. Induction of cytotoxic T cell responses is a key factor in the elimination of acute HBV infection and in the prevention of chronic HBV infection. The viral genome also encodes the envelope proteins pre-S1, pre-S2 and S antigen (HBsAg), polymerase and core protein (HBcAg).

Chronic hepatitis b is a progressive inflammation of the liver with chronic persistent and chronic active processes. Chronic persistent hepatitis is manifested by an enlarged portal zone infiltration defined in the liver with increased fibrous tissue formation; clinically, the signs of persistent hepatitis persist for years (up to 10 years), with HBsAg positivity in about 80% of cases. The pathogenesis may be based on cellular immune system dysfunction and persistent viral infection.

The small surface antigen of hepatitis B (HBsAg) is a 226 amino acid protein (p24/gp27 or S protein), which is an important HBV antigen, that assembles itself into lipoprotein particles of 20-30nm, in which 100-150 subunits are cross-linked by multiple intermolecular and intramolecular disulfide bonds. The variability of the S protein from HBV strains of different subtypes and genotypes is limited. The four stable HBsAg subtypes adw, ayw, adr and ayr involve a single amino acid interchange at positions 122 and 160 adjacent to the immunodominant "a-determinant" (hydrophilic region containing residues 124-147). These subtypes have not previously been identified as having any biological or pathogenic differences in HBV infection.

The vaccine obtained from plasma of chronic HBsAg carriers was first approved in 1982 in federal germany. Since that time, vaccines can be produced by genetic techniques and used for active immunization of at risk populations. 95% of the seronegative population before vaccination showed an immune response after one year. All hepatitis b vaccines used contained high concentrations of purified HBsAg protein (corresponding to the non-infectious coat of hepatitis b virus) and contained no viral DNA or were formalin inactivated.

The prior art has the following disadvantages: at least 5% of the population immunized are "non-responders" who do not exhibit an immune response. Furthermore, none of the vaccines known to date are capable of treating chronic persistent hepatitis.

WO 01/40279 and WO 01/38498 describe vaccines based on the genotype G of the hepatitis B virus, but the specifications of the two patents do not mention the combination of different genotypes.

Michel et al, PNAS 92(1995), 5307-. The document does not mention combinations of HBsAg of different HBV genotypes.

The present invention is therefore based on the problem of providing improved means for the prevention/treatment of HBV infection or HBV mediated diseases. The present invention is also based on the problem of providing a patient-specific drug for the therapeutic treatment of hepatitis. It is a further object of the present invention to provide an improved kit for diagnosing HBV infection.

The problem underlying the present invention is solved by providing a composition comprising at least two hepatitis b virus surface antigens (HBsAg), fragments thereof and/or nucleic acids encoding them, wherein the HBsAg differs in the Hepatitis B Virus (HBV) genotype of the HBsAg S region and/or pre-S region and the composition does not comprise HBV core antigen (HBcAg) or nucleic acids encoding this antigen.

The present invention is based on the following surprising findings: transgenic mice constitutively expressing HBsAg subtype ayw in the liver can be used as a latent phase model to evaluate the efficacy of specific immunotherapy regimens against chronic HBV infection. Such mice produce large amounts of HBsAg due to persistent antigenemia, and they are substantially resistant to HBsAg. On the one hand, the inventors have now immunized HBsAg transgenic mice with a vaccine whose HBsAg genotype is exactly the transgenic mouse genotype (ayw), on the other hand, with a vaccine comprising an HBsAg genotype different from the transgenic mouse genotype. Although the application corresponds to itSelf-bodyHBsAg antigen of HBsAg transgenic mice were repeatedly immunized, but no cytotoxic T cell response was observed. On the contrary, useImmunization of transgenic mice with an HBsAg genotype different from its own genotype results in the generation of genotype specific sums for HBsAgCross reaction(iii) a cytotoxic T cell response. This suggests that naturally occurring HBsAg variants can break "tolerance" by eliciting cross-reactive T cell immunity. Activation of cytotoxic T cell immunity results in a reduction of the HBsAg ayw antigen and thus results in the production of liver-specific disorders and syndromes equivalent to acute hepatitis with HBV effectively controlled. The immune response observed was particularly pronounced since the amino acid sequence of the HBsAg ayw antigen differed from the amino acid sequence of the HBsAg adw2 antigen only in a few positions. The present inventors have demonstrated that even small conservative exchanges of amino acids within a T cell epitope result in changes in the T cell response to that epitope.

The specificity and effectiveness of T cell responses to protein antigens are controlled at various levels, with particular determinants: (i) proteolytic release of the epitope (or antigenic peptide); (ii) affinity of antigenic peptides to Major Histocompatibility Complex (MHC) presenting glycoproteins; and (iii) negative interference with competitively developed T cell responses to different epitopes of the same antigen. Natural variants of protein antigens (either by single amino acid exchanges of epitopes or key sequences in the flanking epitopes or by generation of neo-epitopes) induce T cell responses in four ways:

(i) more efficient proteolytic processing (release) of antigenic peptides from the protein;

(ii) high affinity binding of antigenic peptides to presented MHC molecules;

(iii) (iii) elimination of immunodominant epitopes capable of reducing the immunogenicity of the epitopes by a process similar to that mentioned in (i) and/or (ii) (the immunodominant epitopes suppress the response to different epitopes of the same protein antigen);

(iv) a new epitope is generated.

In the context of the present invention it has been demonstrated that natural variants of HBsAg (expressed by genotype) have a relatively broad specificity for the T cell response they stimulate.

The term "HBV genotype" in connection with the present invention means the entirety of the hepatitis B virus genome. The HBV genotype is preferably determined by total sequencing and phylogenetic analysis. Currently 8 standard genotypes are known. The nucleotide changes of the 8 genotypes to each other were 8%; see Bartholomeusz, rev. med. virol.13(2003), 1-14. Preferably, HBV genotype A has the reference nucleic acid sequence Genbank X02763 or HBV genotype A_afrHaving the reference nucleic acid sequence genbanka af 297621. For HBV genotype Ba, the reference nucleic acid sequence is Genbank D00330 and for genotype Bj the reference nucleic acid sequence is AB 073858. For HBV genotype C, the reference nucleic acid sequence is Genbank AY206389, and for genotype C_ausThe reference nucleic acid sequence is Genbank AB 048704. For genotype D, the reference nucleic acid sequence is Genbank X02496. The reference nucleic acid sequence for genotype E was X75657. The reference nucleic acid sequence for genotype F was X69798. The reference nucleic acid sequence for genotype G was AF160501 and the reference nucleic acid sequence for genotype H was AY 090454.

With respect to the above-mentioned genotypes, there is a certain correlation between the genotypes and the subtypes, as follows: genotype a is associated with subtypes adw2, ayw 1; genotype B is associated with adw2, ayw 1; genotype C is associated with adw2, adrq +, adrq-, ayr, adr; genotype D is associated with ayw2, ayw3, ayw 4; genotype E is associated with ayw 4; genotype F is associated with adw4q-, adw2, and ayw 4; genotype G is associated with adw2 and genotype H is associated with adw 4.

HBV subtypes of infected patients are determined by serological methods with the aid of monospecific antibodies, e.g.anti-d, anti-y, anti-r, anti-a (w). The determination can be achieved in the form of an agar gel diffusion test or in the form of a radioimmunoassay; ("HBs Antigen Subtypes", published by Coouuc, A.M., Holland, P.V., Muller, J.Y., and Soulier, J.P., Biblotheca Haematologica No.42, S.Karger, Basel, 1976).

The subtype can also be determined by sequencing DNA encoding HBsAg from the patient's serum. The amino acid sequence of HBsAg is then obtained from the determined nucleic acid sequence. The subtype is then determined by amino acids at positions 122 and 160 as described by Magnius, L.O. and Norder, H. ("Subtypes, Genotypes and molecular dynamics of the hepatitis B virus as reflected by sequence variability, for example, the S-gene" Intervirology, 38 (1-2): 24-34).

The expression "hepatitis B virus surface antigen" (HBsAg) in connection with the present invention refers to the small surface antigen or S protein of HBV (p24/gp 27). HBsAg may also include the pre-S1 protein domain. Preferably, the HBsAg consists of the S protein and/or the pre-S1 protein domain.

For the numbering of HBsAg, a system according to Kidd-Ljunggren et al J.Gen.Virol.83(2002), 1267-.

The term "fragment" according to the invention encompasses fragments of HBsAg. Fragments preferably comprise at least 5 amino acids and comprise a T cell epitope, preferably at least 10, in particular at least 20, more in particular at least 50 amino acids. According to a preferred embodiment, the composition comprises at least 2 HBsAg or 2 fragments thereof. Such compositions are particularly suitable for use as polypeptide-based vaccines. In particular in case the composition comprises 2 fragments from different HBV genotypes HBsAg, the first and second fragments have in common at least 10 amino acids, preferably 20 amino acids but differ from each other by at least one amino acid.

As mentioned above, the present invention is based on the recognition that even very small differences in the antigens produced by different genotypes (HBsAg) lead to the production of modified T cell epitopes which differ only slightly from one another but which can lead to a very variable T cell response. Thus two fragments differing from each other by at least one amino acid can be easily detected by comparison with simple sequences of known HBsAg genotypes. Suitable fragments differing from each other by at least one amino acid may be used in the composition of the invention. The fragments preferably comprise at least one T cell epitope, in particular a human T cell epitope. Methods for determining T cell epitopes are known, for example, Lauer et al, J.Virol.76(2002), 6104-6113.

According to a preferred embodiment, the composition comprises at least 2 hbsags and/or at least 2 fragments thereof.

Preferably, there is also provided a composition comprising at least a first HBsAg or fragment thereof and a nucleic acid encoding a second HBsAg or fragment thereof, wherein the HBV genotypes of the first and second hbsags are different.

According to a further preferred embodiment, the composition comprises at least 2 nucleic acids encoding 2 hbsags, wherein the HBV genotypes of the hbsags are different. The nucleic acid may also be a nucleic acid encoding a fragment as defined above. The nucleic acid may be viral DNA or synthetic DNA, which is understood to include those DNA sequences containing modified internucleoside linkages. The nucleic acid can also be an RNA molecule which is necessary for expression by means of a recombinant vector system. Furthermore, mixed DNA/RNA molecules are also contemplated as nucleic acids according to the invention.

According to a preferred embodiment, the genotype is selected from the known genotypes A, B, C, D, E, F, G and H. Reference may be made to the above definitions for each reference nucleic acid sequence. Generally, a genotype is determined by means of an 8% nucleotide variation from a reference nucleic acid sequence, that is to say a nucleic acid having at least 92% identity to a reference nucleic acid sequence is also understood as a genotype consistent with the definition. It is particularly preferred that there is at least 95%, in particular 98%, identity with respect to the reference nucleic acid sequence. The "identity" with respect to a reference nucleic acid sequence can be determined here by means of known methods. A special computer program with an algorithm taking into account the special requirements is generally used.

First, the preferred method of determining identity is used to produce the greatest agreement between the sequences being compared. Computer programs for determining identity include, but are not limited to, the GCG program package including GAP (Deveroy, J et al, Nucleic Acid Research 12(1984), 387; Genetics computer group University of Wisconsin, medicine (WI)); and BLASTP, BLASTN, and FASTA (Altschul, S. et al, J.mol.biol.215(1990), 403-. The BLASTX program is available from the National Center for Biotechnology Information (NCBI) or other sources (BLAST handbook, Altschul, S. et al, NCBI NLM NIH Bethesda ND 22894; Altschul, S. et al; supra). The identity may likewise be determined using the known Smith-Waterman algorithm.

Preferred parameters for nucleic acid comparison include the following:

needleman and Wunsch algorithm, J.mol.biol.48(1970), 443-

Comparing the matrixes:

match +10

Mismatch of 0

Opening penalty: 50

Opening length penalty: 3

The GAP program also suitably uses the above parameters. The above parameters are default parameters in nucleic acid sequence comparisons. Further examples of algorithms, opening penalty, opening extension penalty and comparison matrices include those in the program manual Wisconsin software package (version 9, 9 months 1997). The choice of which depends on the comparison being made and also on whether the comparison is made between sequence pairs (when using GAP or Best Fit) or between sequences and a large sequence data bank (when using FASTA or BLAST).

A92% identity according to the algorithm above represents a 92% identity to the sequence of the invention. The same applies to higher identities.

Preferably, the composition according to the invention is characterized in that: the variant encodes a polymerase having an activity substantially equivalent to the activity of the polymerase encoded by the reference nucleic acid sequence and/or the variant encodes an HBsAg having an immunoreactivity substantially equivalent to the immunoreactivity of the HBsAg encoded by the reference nucleic acid sequence.

Polymerase activity can be measured here in accordance with Kim et al biochem. mol. biol. int.1999; 47(2): 301-308 determination. HBsAg immunoreactivity can be determined by commercially available antigen ELISA. By "immunoreactivity substantially by the HBsAg encoded by the reference nucleic acid" is meant that the binding of the antibody to the reference HBsAg is substantially the same as the binding affinity of the antibody to the HBsAg encoded by the isotype.

According to a preferred embodiment, the composition comprises at least 3, preferably at least 5 different hbsags, fragments thereof and/or nucleic acids encoding them.

Particularly preferably, the composition comprises hbsags of all known HBV genotypes, fragments thereof and/or nucleic acids encoding them.

According to a further preferred embodiment of the composition of the invention, the nucleic acid encoding the HBsAg or a fragment thereof is present in a vector under the control of a promoter suitable for expression of the HBsAg in mammalian cells, preferably human cells. If the composition comprises at least 2 nucleic acids encoding HBsAg or fragments thereof, these nucleic acids may be present in the same vector (binary vector) or in separate vectors. Suitable vectors are, for example, plasmids, adenoviruses, vaccinia viruses, baculoviruses, measles viruses and retroviruses. Vectors generally include a source of replication that affects replication of the vector in the transfected mammalian cell.

Suitable promoters may be constitutive promoters and inducible promoters. Preferred promoters are those from CMV and SC-40.

The above composition can be obtained by simply mixing the respective ingredients, and thus can be prepared very simply. Suitable solvents and carriers depend on the nature of the composition (polypeptide and/or nucleic acid). In principle, systems comprising water are preferred. HBsAg or a fragment thereof may be prepared synthetically or by recombinant means. The prepared polypeptide can be purified by chromatographic methods.

Alternatively, the composition may be obtained by co-expression in a recombinant expression system of at least 2 nucleic acids encoding HBsAg or a fragment thereof. Various expression systems and methods are well known to those skilled in the art; yeast is preferably used as the host cell, and Hansenula polymorpha (Hansenula polymorpha), Saccharomyces cerevisiae (Saccharomyces cerevisiae) and Pichia pastoris (Pichia pastoris) are particularly preferably used. The nucleic acid may be present in one vector or in two vectors separated from each other. Suitable vectors and promoters are described above.

According to another aspect of the invention, a pharmaceutical composition comprising a composition of the invention and a pharmaceutically acceptable carrier is prepared. Pharmaceutically acceptable carriers are known to those skilled in the art. Examples are aluminium salts, calcium phosphate, HBsAg lyophilisate with or without added polysaccharide, oil-in-water emulsions, polylactide co-glycolates. If such carriers do not have an adjuvant effect per se, they may be mixed with additional adjuvants such as lipid A mimetics, immunostimulatory nucleotides or bacterial toxins.

The pharmaceutical composition according to the invention is preferably a vaccine. According to the present invention, the pharmaceutical composition, in particular the vaccine, is suitable for the therapeutic treatment of HBV infection or HBV mediated diseases. The pharmaceutical compositions, especially vaccines, are also suitable for prophylactic treatment of HBV infection or HBV mediated diseases. HBV infection is especially chronic persistent hepatitis B infection. The HBV mediated disease may be acute or chronic hepatitis B infection. Additional HBV mediated diseases are cirrhosis and primary hepatocellular carcinoma. The vaccine is suitable for administration to clinically insignificant HBV carriers, i.e. carriers that do not have a real disease but are at high risk of developing HBV mediated disease in the future.

The pharmaceutical composition may be administered intramuscularly, subcutaneously, intradermally, intravenously, mucosally or orally, but such administration is only preferred and is not limiting as to the mode of administration.

The pharmaceutical composition comprises at least 2 HBsAg or fragments thereof in a dose range of 0.1 to 1000 μ g HBsAg or fragments thereof, preferably 2.5 to 40 μ g HBsAg or fragments thereof.

When the pharmaceutical composition comprises nucleic acid encoding HBsAg or a fragment thereof, they are present in a dose range of 10-1000 μ g nucleic acid encoding HBsAg or a fragment thereof.

Another aspect of the present invention provides a method for the preparation of a medicament for the therapeutic treatment of hepatitis b, comprising the steps of:

a) determining the genotype of HBV infection of the patient; and

b) providing a medicament comprising at least one HBsAg of an HBV genotype, an HBsAg fragment or a nucleic acid encoding HBsAg or a fragment thereof, wherein the HBV genotype is different from according to a)

The genotype of HBV in the patient determined.

As mentioned above, important recognition of the present invention is: in the latent-phase model of chronic persistent hepatitis, therapeutic effects have been achieved by treating transgenic animals with HBsAg from an HBV genotype different from that of the transgenic animals.

The genotype can be determined by the following method: sequencing the whole HBV genome or at least the part encoding HBsAg, as well as phylogenetic analysis, Restriction Fragment Length Polymorphism (RFLP), multiplex PCR.

The drug supply is effected in a manner known per se by formulating at least one HBsAg, a fragment thereof or a nucleic acid encoding HBsAg or a fragment thereof.

According to another aspect, the present invention provides a kit for diagnosing HBV infection genotype. The kit comprises at least 2 HBsAg-specific binders, characterized in that the 2 HBsAg-specific binders are specific for different HBV genotypes. The at least 2 HBsAg-specific binders may be HBsAg genotype-specific primers and/or specific antibodies. The primers are 10-30 nucleotides in length and are complementary to the known HBsAg sequence of each genotype. The antibody may be an antibody obtained by, for example, immunizing an experimental animal (e.g., a mouse having each HBsAg corresponding to the HBsAg genotype of interest), preparing a hybridoma in a manner known per se, and screening for a subtype-specific monoclonal antibody.

Brief Description of Drawings

FIG. 1: HBsAg variants. (A) The amino acid sequences of hepatitis B small surface antigen (HBsAg) ayw (1) (corresponding to genotype D) and adw2(2) (corresponding to genotype A) are shown. (B) K from HBsAg ayw and adw2^bLimitingA sex epitope sequence. Epitope 1 (S)_208-215) Presented only by cells processing exogenous HBsAg, whereas epitope 2 (S)_190-197) Presented only by cells processing endogenous HBsAg.

FIG. 2: transfer of epitope 1 or epitope 2 specific cytotoxic T cell line (CTLL) into HBs transgenic (HBs-tg) hosts results in transient liver injury. From pCI/S_aywDNA-immunized B6 mice were splenocytes removed and restimulated in vitro with syngeneic RBL5 cells, in which RBL5 cells had been treated with K^b/S_208-215Binding peptides 1(ILSPFLPL) or K^b/S_190-197Binding peptide 2 (vwlsvivm) was loaded or stimulated with ConA. At 5X 10 per mouse⁶An individual CD8⁺Amounts of CTLL were injected intravenously (i.v.) into HBs-tg mice and mean serum alanine Aminotransferase (ALT) levels were determined.

FIG. 3: HBsAg specific CD8 present in the liver and spleen of immunized mice⁺Ex vivo demonstration of T cells. 100 ug pCI/S by single injection_aywDNA intramuscular immunization of C57BL/6 mice. Specificity CD8 was demonstrated 12 days after immunization⁺The presence of T cells. By K^b/S_208-215Binding peptides 1(ILSPFLPL) or K^b/S_190-197Binding peptide 2 (vwlsvivm) was restimulated in vitro to isolate liver mononuclear cells (MNC) and splenocytes for 4 hours (in the presence of brefeldin a). CD8 is shown for 4-6 mice per group⁺IFNγ⁺T cell/10⁵CD8⁺Mean frequency of T cells ± standard deviation (from two experiments independent of each other).

FIG. 4: reaction of HBsAg-specific CD8T cells to epitope 1 in HBs-tg mice. By encoding HBsAg subtype ayw (pCI/S)_ayw) Or adw2 (pCI/S)_adw2) The DNA vaccine of (1) or the intramuscular immunization with a negative control vector pCI (vector without insert) expressing HbsAg in its liver_aywThree HBs-tg mice (4 weeks between immunizations). Splenocytes were removed from immunized mice 12 days after the last immunization and restimulated with RBL5 cells in vitro for 4 hours (in the presence of brefeldin A), where RBL5 cells were used with ayw (RBL 5/S)^P _ayw) Or adw2(RBL 5/S)^P _adw2) Restimulation of subtype HBsAg particles or use of HBsAg_ayw(ILSPFLPL) or HBsAg_adw2K of (IVSPFIPL)^b/S_208-215Binding peptide 1 restimulation. Showing 4-6 mice spleen IFN γ per group⁺CD8⁺T cell/10⁵CD8⁺Mean number of T cells ± standard deviation (from two experiments independent of each other).

FIG. 5: reaction of HBsAg-specific CD8T cells to epitope 1 in HBs-tg mice. Spleen cells were removed from immunized mice as described in the legend of FIG. 4 and used with the syngeneic RBL5/S_aywOr RBL5/S_adw2Transfectants or HBsAg_ayw(VWLSVAVIV) or HBsAg_adw2K of (VWLAIWM)^b/S_190-197Binding peptide 2 restimulation. Spleen IFN γ of 4 mice per group is shown⁺CD8⁺T cell/10⁵CD8⁺Mean number of T cells ± standard deviation.

FIG. 6: s_208-215Specific CD8⁺Demonstration of the presence of T cells in the liver of immunized HBs-tg mice. By encoding HBsAg_adw2DNA vaccine (pCI/S) of (4)_adw2) Transgenic HBs-tg mice were immunized three times (immunization interval 4 weeks). Liver and spleen cells were removed from immunized mice 12 days after the last injection and treated with K^b/S_208-215The binding peptide ILSPFLPL was restimulated in vitro. Spleen IFN γ of 4 mice per group is shown⁺CD8⁺T cell/10⁵CD8⁺Mean number of T cells ± standard deviation.

FIG. 7: using pCI/S_adw2HBs-tg mice immunized with DNA vaccine have histopathology of liver. No pathological liver histology was observed in B6 mice (a, B). HBs-tg mice (C, D) exhibited moderate cell enlargement and cytoplasm exhibited the appearance of ground glass (D). The nuclei of hepatocytes exhibit moderate polymorphism. Infiltration around the portal canal is rare. Using pCI/S_adw2Repeated DNA immunizations caused severe morphological changes in liver tissue (E-I), consistent with changes caused by acute viral hepatitis. Located in the lobular parenchyma (F) andinflammatory cell infiltration in the periportal region (G) includes Kupfer cells, lymphocytes and a small number of polymorphonuclear granulocytes. Hepatocytes develop edema and often have pycnotic nuclei, which are signs of early stages of apoptosis (F, arrows). The eosinophils (H, arrows), i.e. apoptotic hepatocytes, are commonly or often surrounded by focal inflammatory cell infiltrates. Many hepatocytes showed marked vacuolization (I, arrow). Formalin-fixed and paraffin-embedded tissues were stained with H E. Original magnification: A. c and E are 10 times; B. d and F are 40 times; G-I is 63 times.

FIG. 8: induction of HBsAg-specific serum antibody response in HBs-tg mice. By encoding HBsAg_adw2(pCI/S_adw2) Or HBsAg_ayw(pCI/S_ayw) The DNA vaccine of (3) was used to immunize B6 mice and transgenic HBs-tg mice intramuscularly, 3 weeks later, and the same vaccine was used to boost the immunization. Serum samples were tested for HBsAg antigen (a) or HBsAg-specific antibody (B) 4 weeks after the last injection. Mean antibody titers (mIU/ml) and serum HBsAg levels (ng/ml) ± standard deviation are shown for 4-6 mice per group.

FIG. 9: in normal B6 and HBs_aywHBsAg specific CD8 in tg mice⁺Epitope 1 (S) by T cell_208-215) And epitope 2 (S)_190-197) The reaction of (1). With HBsAg protein particles of subtype ayw or adw2 (S)^P) Animals were immunized intramuscularly three times (21 days at immunization interval). Each protein vaccine was mixed with CpG Oligonucleotide (ODN) or RC-529 as an adjuvant. PBS was used as negative control. Spleens were removed from animals 12 days after the last immunization, and the isolated splenocytes were restimulated in vitro with RBL5 cells previously loaded with HBsAg-specific peptides for 4 hours (in the presence of brefeldin a). For this purpose, HBsAg is used_ayw(ILSPFLPL) or HBsAg_adw2K of (IVSPFIPL)^b/S_208-215Binding peptide 1 or HBsAg_ayw(VWLSVAVIV) or HBsAg_adw2K of (VWLAIWM)^b/S_190-197Binding peptide 2. Showing 4-6 mice spleen IFN γ per group⁺CD8⁺T cell/10⁵CD8⁺Mean number of T cells. + -. standard deviation (from that)Two separate experiments).

FIG. 10: HBS_aywEpitope 1 of HBsAg-specific CD8+ T cells in tg mice (S)_208-215) The reaction of (1).

A. By encoding the individual HBsAg subtype ayw (pCI/S)_ayw) Or three subtypes ayw (pCI/S)_ayw)、adw2(pCI/S_adw2) And adr (pCI/S)_adr) The DNA vaccine of (1) or the negative control vector pCI (vector without insert) is immunized intramuscularly to express HBsAg in its liver_aywHBs-tg mice 3 times (immunization interval 4 weeks). Spleens were removed from animals 12 days after the last immunization. The separated splenocytes are treated with HBsAg_ayw(ILSPFLPL) or HBsAg_adw2K of (IVSPFIPL)^b/S_208-215Peptide 1-loaded RBL 5-bound cells were restimulated in vitro for 4 hours (in the presence of brefeldin a). Showing 4-6 mice spleen IFN γ per group⁺CD8⁺T cell/10⁵CD8⁺Mean number of T cells ± standard deviation (from two experiments independent of each other).

B. With HBsAg protein particles of subtype ayw (S)^P) Or of the HBsAg protein particle mixture of subtypes ayw, adw2 and adr_aywTg mice were three times (immunization interval 21 days). Each protein vaccine was mixed with either CpG Oligonucleotide (ODN) or RC-529 (only mixing with subtype mixtures is shown) as an adjuvant. PBS was used as negative control. Spleens were removed from animals 12 days after the last immunization. The separated splenocytes are treated with HBsAg_ayw(ILSPFLPL) or HBsAg_adw2K of (IVSPFIPL)^b/S_208-215Peptide 1-loaded RBL 5-bound cells were restimulated in vitro for 4 hours (in the presence of brefeldin a). Showing 4-6 mice spleen IFN γ per group⁺CD8⁺T cell/10⁵CD8⁺Mean number of T cells ± standard deviation (from two experiments independent of each other).

FIG. 11: induction of HBsAg-specific serum antibody response in HBs-tg mice.

Using subtypes of waterHBsAg protein particle vaccine (S) of ayw or adw2^P) Or HBsAg protein particle mixture vaccines of subtypes ayw, adw2 and adr, intramuscularly immunized against B6 mice and transgenic HBs-tg mice, 3 weeks later boosted with the same vaccine. Protein vaccines contain the additive CpG Oligonucleotide (ODN) as an adjuvant. Serum samples were tested for HBsAg antigen (a) and HBsAg-specific antibody (B) 4 weeks after booster immunization injection. Mean antibody titers (mIU/ml) and serum HBsAg levels (ng/ml) ± standard deviation are shown for 4-6 mice per group.

The present invention will be described in more detail below with reference to examples. The examples are not intended to be limiting.

Examples：

Materials and methods

SUMMARY

The HBV subtype adw2 under investigation corresponds to genotype a. HBV subtype ayw corresponds to genotype D. HBV subtype adr corresponds to genotype C.

Mouse

C57BL/6Jbom (B6) mice (H-2)^b) Placed under standard pathogen-free conditions.

C57BL/6J-TgN (Alb1HBV)44Bri transgenic (HBs-tg) mouse, HBsAg_ayw(encoded by the HBV sequence with accession number V01460J 02203) was obtained from Jackson laboratories (BarHarbour, ME). Female and male mice 8-16 weeks old were used.

Cells, recombinant HBsAg particles and antigenic HBsAg peptides

H-2 used^bThe cell line RBL5 is described [10 ]]. Preparation of HBsAg expressing similar amount_aywAnd HBsAg_adw2Stable RBL5 transfectants (data not shown). Recombinant HBsAg particles of subtypes ayw, adw2 and adr were obtained from Rhein Biotech GmbH (dusseldorf,germany) was obtained. As described in [3 ]]HBsAg particles prepared in Hansenula polymorpha host strain RB10 were purified. Synthetic K^bBinding to S_208-215ILSPFLPL (ayw) or IVSPFIPL (subtype adw2) peptide and K^bBinding to S_190-197VWLDVWM (ayw) or VWLSAThe IWM (adw2) peptide was obtained from JeriniBioTools (Berlin, germany). The peptides were dissolved in DMSO solutions at a concentration of 10mg/ml and diluted with medium before use.

Plasmid and DNA immunization

As described in [ 4; 5]Mixing HBsAg_ayw、HBsAg_adw2And HBsAg_adrCloned into pCI (Promega) and BMGneo vectors. As a DNA vaccine, HBsAg expression was used_ayw、HBsAg_adw2And HBsAg_adrEqually good plasmid pCI/S_ayw、pCI/S_adw2、pCI/S_adr. This was demonstrated from immunoprecipitation of HBsAg from cells transiently transfected with these plasmids (data not shown). Thus, differences in the immunogenicity of the HBsAg epitopes cannot be explained by differences in the amount of HBsAg expressed by the DNA vaccine or differences in transfectants. For intramuscular nucleic acid immunization, [4 ] as described]Mu.l PBS (phosphate buffered saline) containing 1. mu.g/. mu.l of plasmid DNA was injected into each tibialis anterior muscle. Contains 1. mu.g/. mu.l pCI/S by injection_ayw、1μg/μl pCI/S_adw2And 1. mu.g/. mu.l pCI/S_adrThe 50. mu.l PBS achieved HBsAg subtype mixture immunization.

Immunization with HBsAg protein particles

Mu.g HBsAg protein particles and 30. mu.g CpG oligonucleotide (ODN1826, MWG Biotech, Ebersberg, Germany) in 100. mu.l PBS (phosphate buffered saline) or 8. mu. gRC-529(Corixa Corp. Settle, WA, USA) were injected subcutaneously into each mouse. For immunization with a mixture of HBsAg subtypes, 5. mu.g HBsAg was injected subcutaneously in one injection_ayw、5μgHBsAg_adw2And 5. mu.g HBsAg_adrProtein particles and 30. mu.g CpG oligonucleotide adjuvant or 8. mu.g RC-529 in 100. mu.l PBS.

Specific spleen and liver CD8 ⁺ Determination of the T cell frequency

Spleen cell suspension [1]And liver NPC (parenchymal) cell preparation are described in [ 6; 7]. Spleen cells and liver NPC (1X 10)⁶Ml) in transfectants with 5. mu.g/. mu.l HBsAg-derived peptide or HBsAg expression (10)⁶/ml) or HBsAg particle-loaded cells were incubated in RPMI-1640 medium for 1 hour. Then, 5. mu.g/. mu.l of brefeldin A (BFA) (catalog No. 15870; Sigma) was added and the culture was further incubated for 4 hours. Cells were harvested and stained for their surface with anti-CD 8mAb, followed by fixation, permeabilization and cytoplasmic IFN γ staining. CD8 determination by FACS analysis⁺IFNγ⁺The frequency of the CTL. Show by 10⁵CD8 in spleen or liver T cells⁺IFNγ⁺Mean value of T cells.

Specific CD8 ⁺ Transfer of T cell lines

From using pCI/S_aywSpleen of B6 mouse immunized by DNA vaccine to obtain CD8⁺T cell line. Using warp K^b/S_208-215Binding peptides 1(ILSPFLPL) or K^b/S_190-197The splenocytes were restimulated in vitro by binding peptide 2(VWLSVIWM) -loaded syngeneic RBL5 cells. Greater than 80% of CD8 in cell lines stimulated in vitro for approximately 2 weeks⁺T cells have the expected epitope specificity as demonstrated by the specific IFN γ expression assay. Cells were washed and 5X 10 of these cell lines were used⁶The individual cells were injected intravenously. Control cells were non-specific CD8 isolated from cultures stimulated with ConA for 3 days⁺T blast (blast).

Determination of transaminase, HBsAg and anti-HBsAg antibodies in serum

Serum antibodies were obtained repeatedly from each immunized or control mouse by drawing blood from the tail vein at certain time points after injection. Using ReflotronThe assay (Cat. No. 745138; Roche diagnostics GmbH) measures serum alanine Aminotransferase (ALT) activity. The HBsAg concentration in the serum of transgenic mice was determined by the commercial ELISA AUSZYME II test (ABBOTT Laboratories, Wiesbaden, Germany). anti-HBsAg antibodies were determined using the commercial IMxAUSAB test (catalog No. 7A 39-20; Abbott, Wiesbaden, Germany).

Antibody levels were quantified using 6 standard sera. The test sera were diluted so that the OD values determined were between the values of standard sera 1 and 6. The serum dilution times the determined antibody levels (mlU/ml) gave the values shown here. The serum titers given correspond to the mean ± standard deviation of 4 individual mice.

Histology

Thin liver tissue sections (< 3mm) were fixed with 4% formalin (pH 7.0) for 24 hours and embedded in paraffin. Paraffin sections 2 μm thick were stained with hematoxylin-eosin (H & E).

HBsAg peptide and K ^b In combination with

As described in [8, 9 ]]Affinity purified MHC class I molecule K in the presence of 3 μ M human β 2M^bThe indicator peptide was incubated with increasing concentrations of the test peptide and a defined concentration (approximately 2nM) of radiolabeled VSVNP 52-59 at 18 ℃ for 48 hours. Then, gel filtration through Sephadex G50 column [8]Binding of the peptide to MHC class I molecules was determined. The radiolabeled VSV NP 52-59 peptide is located in the exclusion volume (MHC binding peptide) and inclusion volume (free peptide). This can be determined by gamma emission spectroscopy and the proportion of test peptide bound to MHC molecules relative to the total amount of test peptide is determined. The concentration of test peptide required to achieve 50% inhibition of the indicator peptide binding (IC50 value) was determined. The lower the IC50 value, the better the binding of the test peptide. To prevent ligand depletion, the MHC volume used in all binding experiments was sufficient to be realisticNow up to 15-25% bound. Under these conditions, the IC50 value was close to the dissociation constant (K)_d). All binding experiments were performed as inhibition experiments.

Example 1: epitope 1 or epitope 2 specific K^bRestrictive CD8⁺Adoptive transfer of T cell lines into HBs-tg B6 mice resulted in liver injury

From the use of pCI/S_aywSpleen of B6 mice immunized with plasmid DNA produced short-term CD8 specific for HBsAg epitope 1 or epitope 2 (FIG. 1B)⁺T cells. Of these > 95% of cells are CD8⁺And in these CD8⁺More than 80% of the T cells induced IFN γ expression. 5X 10 of these cell lines⁶Adoptive transfer of individual cells into the liver to express HBsAg from the transgene_aywCaused acute liver injury in syngeneic B6 hosts as shown by a short and dramatic increase in serum transaminase (fig. 2). At 5-6 days post-transfer, serum transaminase levels returned to normal, at which time no CD8 was detectable in the host⁺T cells. Transfer of equal amounts of polyclonal (mitogen-stimulated) CD8⁺The T blast cells showed no liver damage. Thus, it was possible to determine (i) the specificity of CD8 in HBs-tg mice⁺T cell induced liver damage (e.g. in [2 ]]The above); (ii) HBsAg epitopes produced by endogenous or exogenous HBsAg processing appear in the liver expressing the transgene; and (iii) adoptively transferred incoming CD8⁺T cells are rapidly removed from the transgenic host. Transferred CD8⁺T cells have different HBsAg specificities and therefore can enter the liver and be activated in situ, but cannot be stably attracted.

Example 2: k recognizing HBsAg epitopes 1 and 2 was observed in spleen and liver^bRestrictive CTL

Development of specific CD8 for vaccine-induced HBsAg⁺Study of whether T cells were able to enter the liver of normal or transgenic HBsAg (HBs-tg) expressing B6 mice (FIG. 3). From the previous use of pCI/S_aywVaccine immunization of 12-15 day B6 mice splenocytes and nonparenchymal hepatocytes (NPC) were isolated. From normal B6 miceSpleen and liver CD8⁺Epitope 1 or epitope 2 specific CD8 was found in T cell populations⁺T cells (fig. 3A). Albeit in liver CD8⁺HBsAg specific CD8 in T cell population⁺T cells were more frequent but their absolute values were smaller than in the spleen (data not shown). In contrast, the coding HBsAg is used_aywThe DNA vaccine of (3) immunized HBsAg_aywtgB6 none of the mice had CD8⁺T cell responses (fig. 3B). Neither three booster injections (3 weeks apart) with DNA vaccine nor repeated immunizations with HBsAg antigen particles and oligonucleotide adjuvant yielded HBsAg-specific CD8 in HBs-tg mice⁺T cell immunization (data not shown). Thus, vaccination regimens using the same HBsAg variant tolerated by mice did not elicit effective antiviral CD8⁺T cell immunization.

Implementation 3: k^bRestricted T cell vs. HBsAg_aywAnd HBsAg_adw2Reaction of variants

HBV strain HBsAg with 226 amino acids_aywAnd HBsAg_adw2Proteins differ by 16 amino acid residues (corresponding amino acid identity 93%). HBsAg used_aywThe sequence of the protein and HBsAg encoded by the transgene expressed in HBs-tg B6 mice_aywAre identical. Selected HBsAg_aywAnd HBsAg_adw2K of^bThe sequences binding epitopes 1 and 2 differ by 1 and 2 amino acid residues within the epitope, respectively, but their flanking sequences are identical (fig. 1A, 1B). HBsAg_aywAnd HBsAg_adw2S of_208-215Epitope 1 differs in 2 positions: in adw2 valine (V) at position 2 was substituted with leucine (L) and isoleucine (I) at position 6 was substituted with leucine (L) (fig. 1B). K of epitope 1^bThe binding affinity is rather low; HBsAg with epitope_aywVariant vs. HBsAg of epitope 1_adw2Variants exhibit higher K^bBinding affinity (table 1). In contrast, K of epitope 2^bThe binding affinity was high (table 1).

Table 1: immunogenic HBsAg epitope pair K^bBinding affinity of (2)

Using pCI/S_aywOr pCI/S_adw2DNA vaccine immunized B6 mice showed resistance to K^bCD8 binding epitope 1⁺T cell response, in response to already administered HBsAg_aywOr HBsAg_adw2The granule or HBsAg_aywOr HBsAg_adw2Antigenic peptide S_208-215Loaded sensitized spleen CD8⁺Observed after 5 hours ex vivo restimulation of T cells (fig. 4A, groups 2, 3). The ayw and adw2 variants of epitope 1 cross-react in that (i) is via pCI/S_aywOr pCI/S_adw2Can sensitize epitope 1-specific CTL; and (ii) has been administered HBsAg_aywOr HBsAg_adw2Granules or already coated with peptide ILSPFLPL (ayw) or peptide IVSPFIPL (adw2) loaded cells sensitized to CD8⁺T cells present epitope 1. Thus, 2 substitutions within 8-mer epitope 1 failed to exhibit efficient epitope processing, K^bBinding or presenting.

Already used pCI/S_aywDNA vaccine sensitized CD8⁺Recognition of HBsAg by T cells_aywOr HBsAg_adw2Epitope 2 (S) of (2)_190-197) (FIG. 5A; group 2). This can be achieved by using peptide-loaded cells or expressing HBsAg_aywThe transfectants of (a) were demonstrated after 5 hours of in vivo restimulation. Sensitized CD8⁺T cells do not recognize expression of endogenous HBsAg_adw2The transfectant of (1). Using pCI/S_adw2DNA vaccine immunization failed to sensitize epitope 2-specific T cells (fig. 5A, group 3). Using pCI/S_adw2(instead of pCI/S)_ayw) DNA vaccine sensitized CD8⁺T cells were able to recognize adw 2-specific epitopes of unknown restriction specificity/epitope presented by transfectants (figure 5, group 3). Thus, substitution of amino acid 5 (exchange of hydrophobic valine V for hydrophobic alanine A) inhibits epitope 2 production, but not K^bPresentation of molecules [1]。

Example 4: cross-reactivity K^bRestrictive CD8⁺T cell response to HBsAg epitope 1 sensitization in HBs-tg B6 mice

HBs-tg B6 mice express HBsAg from the transgene in the liver_ayw. HBsAg for HBs-tg mouse_ayw(pCI/S_ayw) Or HBsAg_adw2(pCI/S_adw2) Immunization (fig. 4, 5B). Using pCI/S_aywDNA vaccine repeated immunization of HBs-tg B6 mice did not result in CD8⁺T cell responses (fig. 4, 5B, group 2). In contrast, pCI/S was used_adw2Immunization of HBs-tg B6 mice with DNA vaccine resulted in CD8 for HBsAg⁺T cell responses (fig. 4B, group 3). This cross-reactive CD8⁺Identification of T cell response Using HBsAg_aywOr HBsAg_adw2Particles or cells loaded with an ayw or adw2 variant of the peptide form of epitope 1 (fig. 4B, group 3). These CDs 8⁺T cells do not recognize RBL5/S_aywTransfectants or K^bBinding epitope 2S_190-197(FIG. 5B, group 3). CD8⁺T cells showed a pattern of expression of RBL5/S_adw2Transfectants (instead of RBL5/S_aywTransfectants) presented an unidentified determinant (fig. 5B, group 3). This shows that natural variants of HBsAg are able to "break tolerance" by priming of cross-reactive T cell immunity.

For specific CD8⁺Whether T cell population is present in already used pCI/S_adw2Studies were performed in the antigen-producing liver of immunized transgenic mice. In the already used pCI/S_adw2Specific CD8 in spleen and liver NMC of immunized HBs-tg B6 mice⁺T cell reactivity may exist for a time period of months (figure 6). Thus, with adoptively transferred CD8⁺Vaccine-sensitized anti-HBV specific CD8 in comparison with T cells (FIG. 2)⁺T cells have entered and have been shown to stably attract into antigen-producing target organs for at least 3 months.

Example 5: CD8 showing specificity for HBsAg epitope 1⁺Histology of T cell reactive immunized HBs-tg mouse liver

HBsAg specific CD8⁺T cells induce an inflammatory response in the liver producing HBsAg. Untreated B6 mice exhibited normal liver histology (fig. 7A, B). Hepatocytes from HBs-tg B6 mice enlarged and exhibited fine granular, light-colored eosinophilic cytoplasm, characteristic of the "ground glass hepatocytes" observed in human HBV infection cases (fig. 7C, D). No inflammatory cell infiltration was observed.

Already used pCI/S_adw2(instead of pCI/S)_ayw) HBs-tg mice immunized with DNA vaccine showed severe liver histopathology (FIG. 7E). The inflammatory cell infiltrates found in the parenchymal (fig. 7F) and periportal (fig. 7G) areas consisted primarily of monocytes (fig. 7F). A large number of small lymphoid cells are distributed in parenchymal and periportal areas. The localized inflammatory cell population surrounded apoptotic hepatocytes (fig. 7H). The enlargement of hepatocytes and edema were more pronounced in the immunized HBs-tg mice compared to untreated HBs-tg mice. Some small nuclei showed condensed chromatin and perinuclear halo (fig. 7F, arrows), indicating early apoptosis. In addition, most of the Councilman corpuscles, apoptotic hepatocytes were observed (fig. 7H, arrows). Some hepatocytes exhibited nuclear vacuolization (fig. 7, arrows). No significant cholestasis occurred.

Example 6: HBsAg specific CD8 in HBs-tg mice⁺Sensitization of T cells is associated with a reduction in antigenicity

Untreated HBs-tg mice showed HBsAg serum levels of 30-50ng/ml (FIG. 8A). In HBsAg_adw2Development of CD with Cross-reactivity to epitope 1 after immunization⁺T cell mice show reduced antigenicity (antigen levels ranging from 5 to 15ng/ml) compared to HBsAg_aywNo HBsAg-specific CD8 developed after immunization⁺There was no change in the level of antigenicity in T cell immunized animals (fig. 8A). Thus, partial control of antigenicity in immunized transgenic mice and specificity of CD8⁺The appearance of T cells is relevant.

Example 7: after having used HBsAg_adw2The appearance of the immunized HBs-tg miceanti-HBsAg serum antibody

In addition to T cell immunity, anti-HBsAg humoral immunity also plays a role in monitoring antigenemia. The presence of serum antibodies against HBsAg was observed in normal and transgenic mice immunized with the vaccine. Using pCL/S_aywOr pCL/S_adw2The DNA vaccine immunized normal (non-transgenic) B6 mice twice with syngeneic HBs-tg B6 mice. After 2 weeks of the last immunization, serum antibody titers specific for HBsAg were determined using the ImxAUSAB test (Abbott) capable of determining different subtypes of HBsAg. In the already used pCL/S_aywOr pCL/S_adw2High levels of anti-HBsAg serum antibodies were developed in non-transgenic mice immunized with plasmid DNA, whereas HBs-tg mice were only developed with pCL/S_adw2(instead of pCL/S)_ayw) The plasmid DNA showed a serum antibody response against HBsAg after immunization (FIG. 8B). In use of HBsAg_aywOr HBsAg_adw2Similar antibody responses were observed in particle-immunized mice (data not shown). Subtype specific ELISA (Using HBsAg)_aywOr HBsAg_adw2Particle coated panels) show that: in normal mice, > 95% of the antibody responses generated by all vaccines are directed against the HBsAg "a" determinant; in HBs-tg mice, > 90% of the antibody responses were directed against adw 2-specific determinants (data not shown).

Example 8: cross-reactivity K against epitope 1 of HBsAg in HBs-tg B6 mice immunized with HBsAg protein particles^bRestrictive CD8⁺Efficient sensitization of T cell responses

Immunization of Normal B6 mice with HBsAg protein particles of subtype ayw or adw2 resulted in targeting K^bBinding epitope 1 (S)_208-215) CD8 (1)⁺T cell mediated immune response (fig. 9A). It can thus be seen that epitopes with different sequences are able to elicit cross-reactive T cell responses regardless of the nature of the vaccine (protein particles or DNA). Similar to immunization with DNA vaccine (FIG. 5), vaccination of B6 mice with HBsAg protein particles of subtype ayw elicited protection against HBsAg K^bRestriction epitope 2 (S)_190-197) CD8 (1)⁺T cell response using subtype adw2Vaccination with HBsAg protein particles failed to elicit this response (fig. 9A).

Immunization of HBs with particulate vaccine of HBsAg protein corresponding to subtype ayw or subtype adw2_ayw-tg mice. Using HBsAg_aywNo CD8 was produced after repeated immunizations with protein vaccines⁺T cell response, with heterologous HBsAg_aywImmunization with a protein antigen resulted in HBsAg-specific CD8 directed against epitope 1⁺T cell responses (fig. 9B). Thus, it was shown that natural variants of HBsAg are able to break existing tolerance by priming of cross-reactive T cell responses and by vaccination with protein subunits.

Example 9: cross-reactivity K against epitope 1 of HBsAg in HBs-tg B6 mice immunized with a mixture of natural variants of HBsAg^bRestrictive CD8⁺Efficient sensitization of T cell responses

The gene encoding three HBsAg subtypes ayw (pCI/S) was used_ayw)、adw2(pCI/S_adw2) And adr (pCI/S)_adr) The DNA vaccine of (1) (FIG. 10A) and HBsAg protein particles containing a mixture of subtypes ayw, adw2 and adr (FIG. 10B) immunize HBs_ayw-tg mice. After immunization with DNA and protein particles, the HBsAg natural variant mixture elicits a cross-reactivity K against epitope 1^bRestrictive CD⁺T cell responses.

Example 10: reduction of antigen blood disease in HBs-tg mice after immunization with HBsAg natural variant mixture

In untreated HBs-tg mice, serum antigen levels of 30-50ng/ml were observed. In the use of heterologous HBsAg vaccine (HBsAg)_adw2) Or a mixture of natural HBsAg variants (HBsAg)_ayw+HBsAg_adw2+HBsAg_adr) Development of cross-reactive CD8 against epitope 1 following immunization⁺In T cell-responsive animals, the antigenicity was reduced (HBsAg levels were 5-17 ng/ml). In the case of using homologous HBsAg alone_aywThere was no change in the number of serum antigens in animals immunized and therefore unable to produce HBsAg-specific T cell immunity. Immunization with a mixture of natural variants of HBsAg correspondingly results inThe antigenemia is reduced.

Example 11: induction of serum antibodies against HBsAg in HBs-tg mice after immunization with a mixture of natural variants of HBsAg

In use of HBsAg_ayw、HBsAg_adw2、HBsAg_adrAnd a mixture of the three subtypes, a significant antibody response was seen in normal B6 mice (not shown).

The formation of HBsAg-specific serum antibodies in HBs-tg mice after immunization was investigated. HBs-tg mice develop serum antibody responses only after immunization with the native HBsAg variant mixture or with the heterosubtype adw 2. No anti-HBsAg response was induced after immunization with the homologous subtype ayw. Subtype specific ELISA (Using HBsAg)_aywAnd HBsAg_adw2Protein particle coated microtiter plates) showed that > 90% of HBsAg-specific antibody responses in HBs-tg mice were directed against adw 2-specific determinants (data not shown).

Reference to the literature

Schirmbeck, R., Boehm, W., Fissolo, N., Melber, K., and Reimann, J., Difference informality of H-2K^b-restricted epitopes in natural variantsof the hepatitis B surface antigen.Eur.J.lmmunol.2003.in press：xx-yy.

Ando, k. -I., Guidotti, l.g., Wirth, s., Ishikawa, t., missal, g., Moriyama, t., Schreiber, r.d., Schicht, h.j., Huang, s.n., and Chisari, f.v., Class I-corrected cytotoxin T lymphocyte area direct cyclopathic cytopathic for the target cell in vivo.j. immune.1994.152: 3245-3253.

S. Schaefer, M.Pionek, S.J.Ahn, A.Papendineck, Z.Janouwicz, I.Timmermans and G.Gellissen.2002.Recommendant hepatitis Bvacines-dis-ease characteristics and vaccine production.in Hansenula polymorpha-Biology and applications.G.Gellissen (eds.) 175-210 pages, Wiley-VCH, Weinheim, Germany.

4.Schirmbeck, r., Boehm, w., Ando, k. — i., Chisari, f.v., and Reimann, j., Nucleic acid catalysis purposes B surface antigens-specific cytology T lymphocytes in non-responsive mice, 1995.69: 5929-5934.

5.Boehm，W.，A, Paier, T, Mertens, T, Reimann, J, and Schirmeck, R, DNA vector constructs which primer primers B surface-specific primer T simple and antibody responses in microbial vectors in methods 1996.193: 29-40.

6.Trobonjaca，Z.，F, Muller, P, Schirmbeck, R, and Reimann, J, Activating immunity in the lift.I. Liverdent cells (but hotspots) area potential activators of IFN γ release by lift NKT-cells J.Immunol.2001.167: 1413-1422.

7.Trobonjaca，Z.，Kroger，A.，Stober，D.，F, Moller, P, Hauser, H, Schirmbeck, R, and Reimann, J, Activating mm units in the lift.II. IFN-. beta.attributes NK cell-dependent lift tertiary by lift NKT-cell activation.J.Immunol.2002.168: 3763-3770.

Buus, S.A., Stryhn, A.A., Winther, K.A., Kirkby, N.and Pedersen, L.O., Receptor-ligand interactions measured by an enhanced piston column chromatography technique. A high efficiency and high throughput sizing method. Biochim.Biophys.acta 1995.1243: 453-460.

Olsen, a.c., Pedersen, l.o., Hansen, a.s., Nissen, m.h., Olsen, m.m., Hansen, p.r., Holm, a. and Buus, s., a qualitative assessment to a measuremetic interaction between genetic peptides and a simplified class I major compatibility, eur.j.lmmunol.1994.24: 385-392.

T.van-Hall, J.van-Bergen, P.A.van-Veelen, M.Kraakman, L.C.Heukamp, F.Koning, C.J.Melief, F.Ossendorp and R.Offringa.2000.identification of a novel tumor-specific CTL epitope detected by RMA, EL-4, and MLB-2lymphoma derivatives of the same common origin.J.Immunol.165: 869-877.

Sequence listing

<110> Leyin Biotechnology Process and products Ltd

<120> composition for preventing/treating HBV infection and HBV-mediated diseases

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Ile Ile Phe Leu Phe Ile Leu Leu Leu Cys Leu Ile Phe Leu leu Val

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Ser Thr Thr Thr Ser Thr Gly Pro Cys Lys Thr Cys Thr Thr Pro Ala

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Gln Gly Asn Ser Met Phe Pro Ser Cys Cys Cys Thr Lys Pro Thr Asp

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Gly Asn Cys Thr Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Ala Lys

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Ser Ala Ile Trp Met Met Trp Tyr Trp Gly Pro Ser Leu Tyr Ser Ile

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Asp Ser Trp Trp Thr Ser Leu Asn Phe Leu Gly Gly Thr Thr Val Cys

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Leu Gly Gln Asn Ser Gln Ser Pro Thr Ser Asn His Ser Pro Thr Ser

50 55 60

Cys Pro Pro Thr Cys Pro Gly Tyr Arg Trp Met Cys Leu Arg Arg Phe

65 70 75 80

Ile Ile Phe Leu Phe Ile Leu Leu Leu Cys Leu Ile Phe Leu leu Val

85 90 95

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100 105 110

Ser Ser Thr Thr Ser Thr Gly Pro Cys Arg Thr Cys Met Thr Thr Ala

115 120 125

Gln Gly Thr Ser Met Tyr Pro Ser Cys Cys Cys Thr Lys Pro Ser Asp

130 135 140

Gly Asn Cys Thr Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Gly Lys

145 150 155 160

Phe Leu Trp Glu Trp Ala Ser Ala Arg Phe Ser Trp Leu Ser Leu Leu

165 170 175

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180 185 190

Ser Val Ile Trp Met Met Trp Tyr Trp Gly Pro Ser Leu Tyr Ser Ile

195 200 205

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<223> HBsAg polypeptide of HBV subtype adr/genotype C

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Met Glu Asn Thr Thr Ser Gly Phe Leu Gly Pro Leu Leu Val Leu Gln

1 5 10 15

Ala Gly Phe Phe Leu Leu Thr Arg Ile Leu Thr Ile Pro Gln Ser Leu

20 25 30

Asp Ser Trp Trp Thr Ser Leu Asn Phe Leu Gly Gly Ala Pro Ala Cys

35 40 45

Pro Gly Gln Asn Ser Gln Ser Pro Thr Ser Asn His Ser Leu Thr Ser

50 55 60

Cys Pro Pro Ile Cys Pro Gly Tyr Arg Trp Met Cys Leu Arg Arg Phe

65 70 75 80

Ile Ile Phe Leu Phe Thr Leu Leu Leu Cys Leu Ile Phe Leu leu Val

85 90 95

Leu Leu Asp Tyr Gln Gly Met Leu Pro Val Cys Pro Leu Leu Pro Gly

100 105 110

Thr Ser Thr Thr Ser Thr Gly Pro Cys Lys Thr Cys Thr Ile Pro Ala

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Gln Gly Thr Ser Met Phe Pro Ser Cys Cys Cys Thr Lys Pro Ser Asp

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Gly Asn Cys Thr Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Ala Arg

145 150 155 160

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165 170 175

Val Pro Phe Val Gln Trp Phe Val Gly Leu Ser Pro Thr Val Trp Leu

180 185 190

Ser Val Ile Trp Met Met Trp Tyr Trp Gly Pro Ser Leu Tyr Asn Ile

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225

Claims (19)

1. Use of a composition comprising at least 2 hepatitis b virus surface antigens (hbsags), fragments thereof containing T cell epitopes and/or nucleic acids encoding them, wherein each HBsAg is in the form of particles wherein all hbsags have the same genotype, and each HBsAg has a different Hepatitis B Virus (HBV) genotype in the HBsAg S region and/or the pre-S1 region, and the composition does not comprise HBV core antigens (hbcags) or nucleic acids encoding such antigens, for the preparation of a medicament for the therapeutic or prophylactic treatment of HBV infections or HBV-mediated diseasesOf at least 2 HBsAgThe segments have at least10 amino acids in common, but at least one amino acid different from each other,and the fragment of the at least 2 HBsAg comprises the at least 2 HBsAg Modified T cell epitopes from different HBV genotypes。

2. Use according to claim 1, characterized in that the composition comprises at least 2 hbsags and/or at least 2 fragments thereof.

3. Use according to claim 1, characterized in that the fragment of HBsAg comprises at least 20 amino acids.

4. Use according to claim 1, characterized in that the fragment of HBsAg comprises at least 50 amino acids.

5. Use according to claim 1, characterized in that the fragment comprises the "A determinant" of HBsAg.

6. Use according to claim 1, characterized in that the first and second fragments have at least 20 amino acids in common, but differ from each other by at least one amino acid.

7. Use according to claim 1, characterized in that the composition comprises at least 2 nucleic acids encoding HBsAg or a fragment thereof.

8. Use according to any one of claims 1 to 7, characterized in that the genotype is selected from: A. b, C, D, E, F, G or H.

9. Use according to any one of claims 1 to 8, characterized in that the composition comprises at least 3 different HBsAgs, fragments thereof and/or nucleic acids encoding them.

10. Use according to claim 9, characterized in that the composition comprises at least 5 different hbsags, fragments thereof and/or nucleic acids encoding them.

11. Use according to any one of claims 1 to 10, characterized in that the composition comprises hbsags of all known HBV genotypes, fragments thereof and/or nucleic acids encoding them.

12. Use according to any one of claims 1 to 11, characterized in that the nucleic acid encoding the HBsAg or a fragment thereof is present in a vector and is under the control of a promoter suitable for expression of the HBsAg in mammalian cells.

13. Use according to claim 12, characterized in that the vector is selected from: plasmids, adenoviruses, vaccinia viruses, baculoviruses, measles viruses, and retroviruses.

14. Use according to claim 13, characterized in that the promoter is selected from the group consisting of constitutive promoters and inducible promoters.

15. Use according to any one of claims 1 to 14, characterized in that the HBV infection is chronic persistent hepatitis b.

16. Use according to any one of claims 1 to 14, characterized in that the HBV mediated disease is acute or chronic hepatitis b infection, cirrhosis or primary hepatocellular carcinoma.

17. Use according to any one of claims 1 to 16, characterized in that the composition is administered intramuscularly, subcutaneously, intradermally, intravenously, mucosally or orally.

18. Use of at least one HBsAg, a fragment thereof comprising at least 10 amino acids and containing a T cell epitope, or a nucleic acid encoding HBsAg, for the preparation of a medicament for the therapeutic treatment of a patient with hepatitis b, wherein the HBV genotype of the HBsAg is different from the HBV genotype with which the patient is infected.

19. Use according to claim 18, characterized in that the genotype is determined by means of a PCR method.