MXPA01005613A - Hbv core antigen particles with multiple immunogenic components attached via peptide ligands - Google Patents

Abstract

This invention relates to hepatitis B virus ("HBV") core antigen particles that are characterized by multiple immunogen specificities. More particularly, the invention relates to HBV core antigen particles comprising immunogens, epitopes, or other related structures, crosslinked thereto by ligands which are HBV capsid-binding peptides that selectively bind to HBV core protein. Such particles may be used as delivery systems for a diverse range of immunogenic epitopes, including the HBV capsid-binding peptides, which advantageously also inhibit and interfere with HBV viral assembly by blocking the interaction between HBV core protein and HBV surface proteins. Mixtures of different immunogens and/or capsid-binding peptide ligands may be crosslinked to the same HBV core particle. Such resulting multicomponent or multivalent HBV core particles may be advantageously used in therapeutic and prophylactic vaccines and compositions, as well as in diagnostic compositions and methods using them.

Description

ANTIGEN PARTICLES OF THE HBV NUCLEUS WITH MULTIPLE IMMUNOGENIC COMPONENTS UNITED THROUGH LIGANDS OF PEPTIDE FIELD OF THE INVENTION This invention relates to antigen particles of the hepatitis B virus core ("HBV") that are characterized by multiple immunogenic specificities. More particularly, the invention relates to HBV core antigen particles comprising immunogens, epitopes or other related structures crosslinked therewith by means of ligands, which are peptides for binding to the HBV capsid, which selectively bind to the core protein of HBV. These particles can be used as delivery systems of a varied range of immunogenic epitopes, including HBV capsid binding peptides, which advantageously also inhibit and interfere with the viral assembly of HBV by blocking the interaction between the HBV capsid. HBV core protein and surface proteins of HBV. Mixtures of different immunogens, peptide ligands that bind to the HVB capsid or both can be crosslinked to the same core particle of HBV. This results in multicomponent or multivalent HBV core particles that can be used advantageously in vaccines and therapeutic and prophylactic compositions, as well as in the compositions and in the diagnostic methods that use them.

BACKGROUND OF THE INVENTION The first line of clinical immunotherapeutic regimens includes immunizations of patients against infectious pathogens and other agents that threaten health. Despite the plethora of immunization agents, inoculations can produce, at best, partial immunity, requiring frequent re-immunizations. This is the case of several conventional monovalent or polyvalent vaccines. And even among these vaccines, the number of inoculants of a single agent, capable of producing immunity against a variety of immunogens, is limited. In addition, antigenic variation among pathogens may limit the efficacy of conventional vaccines. Due to these obstacles, efforts have focused on methodologies to improve the response of the immune system to certain immunogens. To this end, immunogenic conjugates have been produced by binding immunogens to core particles of the hepatitis B virus ("HBV") (also referred to as nucleocapsid coatings), as part of the efforts to increase the immunogenicity of the ligated immunogen, by the operation of T cell-dependent determinants as independent of T cells of the HBV core antigen. See, for example, U.S. Patent 4,818,527 and R. Ulrich et al., "Core Particles of Hepatitis B Virus as Carrier for Foreign Epitopes," Adv. Virus. Res., 50, pp. 141-82 (1998). There has also been an approach to improved immunogenicity of the epitopes of interest by means of hybrid viral particle-forming proteins, comprising at least a portion of a viral particle-forming protein present in nature, for example, the HBV surface antigen. and one or more epitopic sites of interest. See U.S. Patent 5,965,140. As is evident, from these efforts, HBV proteins have been used as platforms to present the immunogens of interest to the immune system. The hepatitis B virus is a virus transported in the blood, comprising a small genome of partially double-stranded DNA, carrying four open, widely overlapping reading frames, consisting of an internal nucleocapsid, which comprises the protein of the HBV nucleus ("HBcAg"), viral polymerase and viral DNA, surrounded by a membranous envelope containing surface HBV antigens ("HBsAg"). The Viral envelope contains three different but related surface antigen proteins, the long (L), the median (M) and the short (S), which share a terminal carboxy region, but which have different amino termini, which arise from the variable use of initiation triplets at different points within a continuous open reading frame. The long polypeptide (polypeptide L) consists of the pre-Sl, pre-S2 and S regions. This is the product of the entire reading frame and comprises the pre-Sl domain of 108 amino acids (or 199, depending on the virus subtype) at its amino terminus, followed by the pre-S2 domain of 55 amino acids and the short polypeptide region (S polypeptide) of 226 amino acids. The medium length polypeptide (M polypeptide) has the pre-S2 domain at its amino terminus followed by the S region, while the S polypeptide, which is the most abundant form, consists only of the S region. Pre-S plays an important role both in the viral assembly and in the binding to the host cell. The S form is more abundant than the M and L forms of the HBsAg of the virus and occurs in both the glycosylated and non-glycosylated forms [V. Bruss and D. Ganem, "The Role of Envelope Protein in Hepatitis B Virus Assembly", Proc. Nati Acad. Sci. USA. 88, pp. 1059-63 (1991); V. Bruss et al., "Post- translational Alteration in Transmembrane Topology of Hepatitis B Virus Large Envelope Protein ", EMBO J .. 13, pp. 2273-79 (1994); AR Neurath et al.," Identification and Chemical Synthesis of a Host Cell Receptor Binding Site on Hepatitis B Virus ", Cell 46, pp. 429-36 (1986); K. Ueda et al.," Three Envelope Proteins of Hepatitis B Virus: Large S, Middle S and Major S Proteins Needed for the Formation of Dane Particles ", J. Virol .. 65, pp. 3521-29 (1991).] The specific interactions between the outer surface of the nucleus and the inner surface of the envelope will probably guide the correct assembly of the virus and stabilize the resulting particle. of HBV can be expressed efficiently in E. coli [M. Pase et al., "Hepatitis B Virus Genes and Their Expression in E. coli", Nature, 282, pp. 575-79 (1979)], where it is assembled in icosahedral shells of two sizes containing 180 (T = 4) or 240 (T = 4) subunits [RA Crowther et al., "Three- Dimensional Structure of Hepatitis B Virus Core Particles Determined by Electron Microscopy ", Cell. 77, pp. 943-50 (1994)]. The subunits are grouped as dimers and each dimer forms a peak that projects onto the surface of the shell. Using electronic cryomicroscopy and image processing, a map of the shell T = 4 was recently made at a resolution of 7.4A from the pl301 images of more than 6000 individual particles [B. Bottcher et al., "Determination of the Fold of the Core Protein of Hepatitis B Virus by Electron Cryomicroscopy", Nature, 386, p. 88-91 (1997)]. This revealed the fold of the polypeptide chain, which was mainly a-helical and very unlikely, the viral capsids previously resolved. Each diurnal peak was formed by a pair of a-helical forks, one of each dimer monomer [Bóttcher et al. (1997); J.F. Conway et al., "Visualization of a 4-Helix Bundle in the Hepatitis B Virus Capsid by Cryoelectron Microscopy", Nature. 386, pp. 91-94 (1997)]. A numbering scheme that overlapped the amino acid sequence in the fold [Bóttcher et al. (1997)] placed the major immunodominant region of the HBV core protein around amino acids 78 to 82 [J. Salfeld et al., "Antigenic Determinants and Functional Domains in Core Antigen and E Antigen from Hepatitis B Virus", J. Virol. 63, pp. 798.908 (1989); M. Sallberg et al., "Characterization of a Linear Binding Site for a Monoclonal Antibody to Hepatitis B Core Antigen", J. Med. Virol. 33, pp. 248-52 (1991)], at the tip of the peak. Agents that inhibit the viral assembly of HBV include those that bind to the HBV core antigen, thereby blocking the interaction between pl301 HBV core proteins and surface proteins of HBV. These peptides that bind to the HBV capsid are described in PCT patent application WO 98/18818 and in MR Dyson and K. Murray, "Selection of Peptide Inhibitors of Involved Involves in Complex Protein Assemblies: Association of the Core and Surface Antigens of Hepatitis B Virus ", Proc. Nati Acad. Sci. USA, 92, pp. 2194-98 (1995). One of these peptides that bind to the capsid, MHRSLLGRMKGA, was cross-linked to the HBV core antigen [B. Bóttcher et al., "Peptides that Block Hepatitis B Virus Assembly: Analysis by Cryomicroscopy, Mutagenesis and Transformation", EMBO J .. 17, pp. 6839-45 (1998)]. As will be apparent from the following discussion, peptides that bind to the HBV capsid can be advantageously used as ligands to construct antigenic particles of the HBV core, characterized by the ability to produce enhanced immune responses to single or multiple immunogens. .

SUMMARY OF THE INVENTION The present invention focuses on the aforementioned problems in providing antigenic particles of the HBV core that produce improved immunogenicity to one or more immunogenic components. These pl301 Multicomponent or multivalent HBV core antigen particles comprise immunogens, epitopes or other related structures, crosslinked thereto through ligands that are peptides that selectively bind HBV core antigen particles, in addition to immunogenic or epitope domains bound to HBV core antigen polypeptides or inserted therein, by means of genetic manipulation of the coding sequence or by synthesis of the polypeptide. These particles can be used as delivery or administration systems for a varied range of immunogenic epitopes, including peptides that bind to the capsid of HBV, which inhibit and interfere with the HBV viral assembly by blocking the interaction between the protein. of HBV core and HBV surface proteins. The resulting multicomponent or multivalent HBV core particles can be used advantageously in vaccines and therapeutic and prophylactic compositions, as well as compositions and diagnostic methods that use them. The present invention advantageously allows mixtures of different immunogens, peptide ligands that bind to the HBV capsid or both, to cross-link to the same core particle of HBV. The result is pl301 single particles that are efficient stimulators of T cells and that are immunologically multivalent. In this way, a cell presenting a single antigen can stimulate the proliferation of multiple clones of the T cell of different specificity.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 depicts the structure of a core antigen particle of the HVB comprising several capsid binding immunogens. A "capsid binding immunogen" comprises at least one peptide component that binds to the HBV capsid and at least one immunogenic component Each immunogen that binds to the capsid is bound to the core antigen particle of HBV by of a peptide that binds to the HBV capsid Figure 2 is a table summarizing various HBV core antigen fusion proteins that can also serve as the HBV core antigen particle to which various immunogens can be ligated by means of the peptides that bind to the HBV capsid.

DETAILED DESCRIPTION OF THE PREFERRED MODALITIES In order that the invention herein described can be more fully understood, the following is set forth pl301 detailed description. In accordance with one embodiment of this invention, mixtures of more than one type of immunogen and one or more types of peptide ligands that bind to the HBV capsid can be crosslinked in the same core particle of HBV. Alternatively, multiple copies of the same immunogen can be ligated to a type of capsid binding peptide of the HVB and cross-linked at various positions in the core particle of HBV. HBV multicomponent or multivalent core antigen particles, in accordance with this invention, are particularly useful for inducing antibodies in all component immunogens. The use of peptides that bind to the HBV capsid to bind immunogens to the HBV core antigen particle allows for improved immunogenic presentation, without destroying the immunogenicity or stability of the immunogen by denaturation, conformational alteration or other destabilizing influences. For example, peptide binders that bind to the HBV capsid reduce the risk that the component immunogens interfere with each other to cause the loss of functional material. As a result, the HBV core antigen particle produces an enhanced immune response in its immunogens pl301 components. Therefore, it is possible to obtain the desired therapeutic or prophylactic effects with a lower number of innoculations and / or less inoculant than is necessary if each immunogen were administered as a single agent. The binding of immunogens to the HBV core antigen particle through the peptides that bind to the HBV capsid, also allows the presentation of immunogens that vary in size, conformation and nature. As a result, the present invention allows the inclusion in a vaccine or composition, of combinations of immunogens useful for producing a broad spectrum of immunity or treatment in a particular individual.

Immunogens Immunogens that can bind peptides that bind to the HBV capsid and thereby incorporate HBV core antigen particles include any molecule that contains one or more immunological, immunogenic or antigenic epitopes. These epitopes may be linear, conformational, simple or mixed in nature. More particularly, the immunogens can be selected from any agent capable of producing an immune response. These agents include, in form pl301 enunciative, antigens, antigenic determinants, proteins, glycoproteins, antibodies, antibody fragments, peptides, peptide mimotopes that mimic an antigenic determinant or antigen, polypeptides, glycopeptides, carbohydrates, oligosaccharides, polysaccharides, oligonucleotides and polynucleotides. The immunogens can also be allergens, toxins or endotoxins. These agents also include those directed to or derived from various pathogenic agents, such as viruses, parasites, bacteria, fungi, phage, protozoa and plants. These viruses include retroviruses, which include human immunodeficiency viruses type 1 and type 2 and the T cell leukemia virus; herpes viruses, such as herpes simplex type 1 and type 2 viruses, varicella-zoster virus, cytomegalovirus and Epstein-Barr virus; ortho-ioxoviruses, such as influenza A, influenza B and influenza C viruses; paramyxoviruses such as respiratory syncytial virus, measles virus, mumps virus and parainfluenza virus; epadnaviruses, such as hepatitis B viruses; flaviviruses, such as the hepatitis C virus, the hepatitis A virus, the hepatitis E virus, the yellow fever virus, the pl301 Dengue fever and tick borne encephalitis viruses; picornaviruses, such as enteroviruses, rhinoviruses, viruses of foot and mouth diseases, and poliovirus; togavir s, such as rubella virus; rhadboviruses, such as the rabies virus; adenovirus, ebolavirus; baculovirus; Hantaviruses; papoviruses, such as papillomaviruses; parvovirus; DNA virus; RNA virus; RNA tumor viruses, such as oncoviruses; and poxviruses, such as vaccinia virus. In addition, immunogens can be those that are directed to bacilli, enterobacteria, clostridium, listeria, mycobacteria, pseudomonas, staphylococci, eubacteria, mycoplasma, chlamydia, spirochetes, neisseria or salmonellas or that are derived from these. Immunogens can also be selected from the following epitopes of the human immunodeficiency virus: GELDRWEKI (gag); EDKWAS (gp 40); IGPGRAFYTTKN (V3 loop); ELDKWA (gp 41) and DRFYKTLRA (gp 41). Glycoproteins that can bind to peptides that bind to the HBV capsid and thereby incorporate into the HBV core antigen particles include, for example, antibodies, glycopeptides of, or resembling, surface components. animal cells or viruses or bacteria, such as those that cause meningitis or fragments of these entities. pl301 As will be appreciated by those skilled in the art, the size of the immunogen should not be so large as to allow a functional group thereof to interfere with the peptide binder that binds to the HBV capsid.

HBV Anti-Enoxen Proteins Due to its particulate nature, the HBV core antigen protein is an advantageous platform for the presentation of multiple immunogens, of a similar or dissimilar type, to the immune system. In accordance with the present invention, this advantage is further improved by the use of peptides that bind to the HBV capsid as ligands to bind the desired immunogens to the core particle of HBV. These particles contain either 90 or 120 binding sites with the ligand - the capsid peaks, each composed of an antigenic dimer of the HBV core (see Figure 1). Thus, multiple immunogens can physically bind to the HBV core antigen particle by the peptides that bind to the HBV capsid as ligands. The resulting particle has the ability to induce an immune response in all its component immunogens. The HBV core antigen particles can be formed with the expression of pl301 Recombinant coding of HBV core antigen peptide in an appropriate microbial, animal or plant system. See, for example, Sambrook et al., Molecular Cloninq. A Laboratory Manual. 2a. Cold Spring Harbor Press Edition, Cold Spring Harbor, New York (1989). The polypeptide to be expressed may comprise the sequence of the full-length HBV core antigen or mutations, derivatives, truncations or portions thereof, which retain or retain the ability to assemble in a particulate form in the cells of the expression system. Recombinant methods for producing these HBV core antigen particles are known in the art. See, for example, U.S. Patent 4,710,463. Alternatively, chemical synthesis methods can be used to produce the HBV core antigen polypeptide. Based on the amino acid sequence of the antigenic polypeptides of the HBV core, chemical synthesis can be performed using solid phase synthesis [R.B. Merrifield, Fed. Proced. , 21, p. 412 (1964); R.B. Merrifield Biochemistry. 3, pp. 1385-90 (1964) and D.R. Milich et al., J. Immunol .. 139, pp 1223-31 r (1987)] Those skilled in the art will appreciate that, because the antigenic sequences of the HBV core pl301 with mutations or variants, can influence the reactions with the binding or ligand peptides, the present invention also applies to natural variants or mutations introduced by the manipulation of the coding sequences or other procedures, into the antigenic subunits of the nucleus of HBV or in the corresponding binding peptides or ligands.

Mutation of the core protein residues important for peptide binding The methods used to determine the fold of the core protein have been applied to localize by cryomicroscopy the binding sites in the core protein of the SLLGRMKGA, a peptide that is binds to the HBV capsid that inhibits binding with L-HBsAg. This approach has now shown the peptide attached to the tips of the peaks, both in the T = 3 and in the T = 4 [B. Bottcher et al. , "Peptides that Block Hepatitis B Virus Assembly: Analysis by Cryomicroscopy, Mutagenesis and Transfection", EMBO J., 17, p. 6839-45 (1998)]. The analysis of the image shows that the peptide binding sites are at the tip of the peaks that in the numbering scheme proposed for the peptide fold [Bóttcher et al. (1997)] corresponds to residues in the aminácidos region 78-82. There are two acid residues (glu77 and pl301 asp78) near the tip of the core protein and the selected binding peptide contained two basic residues conserved. The importance of these residues with opposite charge in the binding reaction was confirmed when the mutation of any of the acidic residues of the protein in alanine was found to greatly reduce the affinity of the peptides for the altered core shells. The change of aspartic acid 78 in alanine reduced affinity 160 fold and the change of glutamic acid 77 in alanine reduced affinity 1000 fold. This suggests that either or both acid residues in the HBV core antigen protein can provide at least part of the binding site for the peptides that bind to the HBV capsid. These results also illustrate the importance of the amino acid sequence of the HBV core antigen in the region of the tip of the peak for binding to the ligand. Those of skill in the art will appreciate that the HBV core antigen of some strains of HBV may require the mutation for the effective binding of a ligand-immunogen peptide or the selection and adaptation of ligand variants for effective binding to the antigen variant of the specific HBV nucleus. pl301 HBV core antqene fusion proteins In accordance with one embodiment of this invention, the HBV core antigen particle to which the immunogens can be linked through the peptides that bind to the HBV capsid can be one that already shows one or more immunogens, as a result of genetic fusion techniques. In one of these techniques, the relevant coding sequences are incorporated at the appropriate positions in the plasmids or in other vectors carrying that polypeptide of the HBV core antigen. Fusions with the E. coli β-galactosidase gene used to increase the expression levels of the HBV core antigen polypeptide demonstrated that the replacement of the first two amino acids of the antigen by a sequence of eleven amino acids (eight amino terminal β-galactosidase and 3 additional residues resulting from the translation of a linker sequence introduced into the gene fusion) had no adverse impact on the ease of recovery, antigenicity or morphology of the product [S. Stahl et al., "Hepatitis B Virus Core Antigen, Synthesis in Escherichia Coli and Application in Diagnosis", Proc. Nati Acad. Sci. USAf 79 pp. 1606-10 (1982); B.J. Cohen and J.E. Richmond, "Electron Microscopy of Hepatitis B Core Antigen Synthesized in El pl301 Coli ", Nature, 296, pp. 677-78 (1982).] The HBV core antigen fusion proteins useful in the present invention can be produced as exemplified in SJ Stahl and K. Murray," Immunogenicity of Peptide Fusions. Hepatitis B Virus Core Antigen ", Proc. Nati, Acad. Sci. USA, 86, pp. 6283-87 (1989) Alternatively, fusions of the polypeptide sequences with the core segment of the HBV core antigen to provide highly immunogenic particles are exemplified with several viral coding sequences, as listed in Figure 2. These include a particulate product with high immunogenicity, produced by expression of the VP1 peptide vaccinia virus vector (residues 142-160 ) fused through a heptapeptide linker sequence with the six amino acids of the pre-core sequence immediately preceding the amino terminus of the HBV core antigen polypeptide [BE Cl arke et al., "Improved Immunogenicity of a Peptide Epitope after Fusion to Hepatitis B Core Protein", Nature, 330, p. 381-84 (1987)]. See also Ulrich et al. (1998) for other useful fusion proteins with HBV core antigen. A number of other fusion proteins are characterized by the replacement of the rich region in pl301 carboxy terminal arginine of the HBV core antigen polypeptide by other alternate coding sequences. Peptides that included the immunodominant a region of the HBV surface antigen (residues 111-165), the pre-Sl and pre-S2 epitopes and various segments of the envelope protein of the human immunodeficiency virus (HIV) were linked to residue 144 of the HBV core antigen polypeptide. All were expressed efficiently in E. coli to provide particulate products that show essentially the same morphology as that of the HBV core antigen itself [Stahl and Murray, 1989]. The products showed the antigenic reactivity of the HBV core antigen and, like truncated HBV core antigen polypeptide preparations at residue 144, those that were tested also showed HBV e antigen reactivity, while the antigen of the full-length HBV core showed very little of this activity. The fusion proteins carrying residues 111-156 or 111-165 of the HBV surface antigen showed no significant reactivity of HBV surface antigen, a result that is not inconsistent with the conformational dependence of this major epitope or the probability of the sequences that will be buried inside the particles. However, immunogenic responses to pl301 the fusion proteins reflected their various component epitopes. Immune responses to the HBV surface antigen are complex, in addition to the epitopes residing in the pre-Sl and pre-S2 regions of L-HBsAg and the main immunodominant region, several determinants of variable subtype have been assigned to other regions of the short polypeptide or S of the HBV surface antigen [GL Le Bouvier, "The Heterogeneity of Australia Antigen", J. Infect. Dis .. 123, pp. 671-75 (1971); W. R. Bancroft et al., "Detection of Additional Antigenic Determinants of Hepatitis B Antigen", J. Immunol. 109, pp. 842-48 (1972); A.M. Couroucé-Pauty P.V. and Holland, "Summary of Workshop A2: HBsAg and its Subtypes", in Viral Hepatitis. G.N. Vyas, S.N. Cohen and R. Schmid, eds. (Philadelphia, USA: Franklin Institute Press), pp. 649-54 (1978)]. The HBV surface antigen coding for determined sequences in the HBV DNA cloned from the serum of different subtypes shows differences in the corresponding protein sequences. However, specific simple mutations of apparently critical residues did not affect the switching of one serological subtype (and) in another (d), although additional simple mutations induced a gradual change in the two reactivities y and d and in the immunogenicities that pl301 they will be exhibited by the same molecule [P.G. Ashton-Rickardt and K. Murray, "Mutations that Change the Immunological Subtype of Hepatitis B Virus Surface Antigen and Distinguish Between Antigenic and Immunogenic Determination", J. Med. Virol., 29, pp. 204-14 (1989)]. The mutations involved were performed within the immunodominant region sensitive to or near the conformation and all were within the segment of the HBV core antigen used in the HBV core antigen fusions described above. The impact of the mutations on the specificity of the subtype of the induced antibodies indicated the suggestion that the fusion proteins may also provide a means to change the specificity of the response to the epitopes of interest, particularly if these depend on the conformation. Mutations of glycine ^ 5 in arginine, to mimic the natural escape mutant, and in other residues with positive or negative charge (lysine and glutamic acid) were therefore carried in this residue in HBcS ??? _56 for the studies comparisons of the humoral and cellular immune responses [AL Shiau and K. Murray, "Mutated Epitopes of Hepatitis B Surface Antigen Fused to the Core Antigen of the Virus Induces Antibodies That React with the Surface Nature Antigen", J. Med.

P1301 Virol., 51, pp. 159-66 (1997)]. All were expressed efficiently in JE ?, coli, which produce the anticipated particulate products that show a strong antigenicity of the HBV core and all induced high antibody titers to HBV core antigen in rabbits. Like their parental proteins or of HBcSn origin? _i56 / the three mutants of residue 145 showed minimal interaction with the antibody in the HBV surface antigen in solid-phase radioimmunoassay (AUSTRIA; Abbott Laboratories) or antibody precipitation analysis in solution. However, all showed strong reactions with rabbit anti-HBV surface serum in immunoblot experiments after electrophoresis in acylamide gels under denaturing conditions [A.L. Shiau, "Immunological Aspects of Hepatitis B Virus Core Antigen and its Derivatives", PhD Thesis, University of Edinburgh, UK. (1993)]. At high concentrations, the original and mutant proteins also gave weakly positive reactions with antibodies on HBV surface antigen when captured on a solid phase coated with antibodies to the HBV core antigen, possibly as a result of some alteration in the particles they provide. access to anti-HBsAg molecules [Shiau pl301 and Murray. 1997]. The immunized rabbits were used to examine the T cell responses to the fusion proteins, as well as the production of antibodies. Peripheral blood mononuclear cells (PBMC) taken at various times after immunization were used for proliferation assays based on the incorporation of [3 H] -thymidine in response to exposure to the HBV core antigen or to the fusion protein used for immunization. In all cases, intense responses were found, where the fusion protein exhibits a higher stimulation index than the HBV core antigen, and the HBV surface antigen is a poor stimulant, as expected. The double antibody radioimmunoprecipitation assay [C.J. Burrell et al., "Rapid Detection of Hepatitis B Surface Antigen by Double Antibody Radioimmunoassay", J. Med. Virol. 3, pp. 1926 (1978)] with surface antigens of [125I] -HBV were used to measure anti-HBs in the serum samples and showed the anticipated positive response to HBcSi i-isß- The mutant arginine also provided a positive response in this assay , although a little lower than that of its original molecule and a weak response of the glutamic acid mutant was obtained, although none of the lysine mutant. Of this pl301 mode, the results showed that the fusion protein (designated as HBcSi45R) that carries the arginine 145 mutant was a strong stimulant of the T cells and induced antibodies with a broader specificity of reaction. An additional pool of fusions of several portions of the HBV surface antigen polypeptide, including 145 residue mutants, was prepared with the HBV core antigen polypeptide to explore the effect of the total size and the number and position of the antigens. various additional components on the immunogenicity of the products [Shiau (1993)]. These constructs were included in Figure 2 and, as with the other fusions, all provided particulate products that show the morphology of the HBV core antigen, although fusions with the HBs fragment _i56 at the amterminus of the core antigen of HBV were less satisfactory, providing products that formed insoluble aggregates. This group of products, like the previous ones with the HBcS segments? _ 56, showed little or no reaction with the antibody in the HBV surface antigen in solid phase or in solution, although when they were captured by antibodies in antigen of HBV nucleus in solid phase, showed similar reactivity pl301 with HBV surface antigen antibody and this was a bit higher (approximately double), with fusions that carry the segments pre-Sl and pre-S2 in addition to HBcS? _i56 «The stimulation indexes for the inhibition of proliferation of lymphocytes again were important for all fusion proteins and those that included pre-S segments, as well as native or mutant HBcS n_ 56 sequences, provided the strongest responses. Inclusion of the pre-Sl and pre-S2 sequences between the HBci 44 and HBcS? _56 sequences (either wild-type or mutant) provided higher levels of antibodies in the double antibody radioimmunoprecipitation assay than the missing fusions of the pre-S segments, although the introduction of a second HBcS sequence 1-156 between HBC144 and the pre-S sequences produced no further improvement in any of the responses. The longest of these sequences linked to the HBV core antigen polypeptide in the prolinai44-165 amacids obviously did not have an adverse impact on the performance or physical properties of the fusion protein. Hepatitis C virus (HCV) fusion protein (HCc) has also been fused, partially in copies and in full length multiple copies, with truncated HBV core antigen in valine polypeptide pl301 149 [A. Yoshikawa et al., "Chimeric Hepatitis B Virus Core Particles with Parts of Copies of Hepatitis C Virus Core Protein", J. Virol. 67, pp. 6064-70 (1993)]. The fusions carrying residues 39-75 of the HCc showed a negligible HCc antigenicity, although residues l-91 of the complete sequence of 180 amacids provided positive reactions and the antigenicity increased almost arithmetically with the addition of additional copies (from up to four) of the 1-180 sequence by short linkers. Electron microscopy showed that the fusion carrying a single copy of residues 1-91 of the HCc formed particles morphologically equivalent to the peptide of the HBV core antigen, although three copies of full length HCc greatly distorted this structure and the The product was very sensitive to proteolysis, providing, however, material that retained the antigenicity of the HBV core. While the larger fusion protein carried more than 720 additional amacids, the limit for an antigen-like particle of the HBV core appears to be appreciably lower. Pre-S sequences have been used in other studies of the effect of fusion position on HBcAg on immunogenicity. Borisova et al. (1989) performed fusions with segments of pre-Sl (residues 20-68, 20-69 or 69-106) or all the pre-S2 linked to the antigen of the nucleus of the pl301 Prolonged HBV 144 in proline or inserted in this position within the sequence of the full-length HBV core antigen. In these constructs and in analogous constructions with residues 56-103 of bovine leukemic virus envelope protein (BLV) or residues 78-129 of the HIV transmembrane protein (gp41), the sequences fused to the HBV core antigen they were believed to be exposed on the surfaces of the particle, they were all reported to be both antigenic and immunogenic, and the C-terminal domain rich in arginine apparently had a small adverse effect. F. Schódel et al., "The Position of Heterologous Epitopes Inserted in Hepatitis B Virus Core Particles Determines Their Immunogenicity", J. Virol .. 6, pp. 106-14 (1992) explored the impact of fusion position on antigenicity and immune response in mice with inbreeding, when pre-Sl or pre-S2 segments were joined at the amino terminal of the HBV core antigen. complete sequence (either directly or through part of the pre-core sequence) or the terminal carboxy of the truncated HBV core antigen; an additional construct carried a pre-Sl segment between residues 75 and 83 of the HBV core antigen, as well as the pre-S2 fragment in the terminal truncated carboxy (proline) pl301 156). The complete analysis showed that the pre-Sl sequence fused to the amino terminal of the HBV core antigen by the short pre-nucleus sequence was antigenic but that it fused directly at the amino terminal was not and, while both had the same immunogenicity of the HBV core antigen, the fusion by the pre-core sequence stimulated a much higher anti-pre-Sl response. The pre-S2 sequence in the truncated terminal HBV core antigen was antigenic and immunogenic to a similar degree in both contexts, although the pre-Sl sequence fused internally, so as to replace residues 76-82 (which include the main epitope of the HBV core antigen) was substantially more antigenic and very importantly, more immunogenic than in the N-terminal fusions. As anticipated, the antigenicity and immunogenicity of the HBV core were greatly reduced in the internal fusion proteins. The replacement of an internal sequence of the HBV core antigen (residues 78 to 82) with a fragment of HBV surface antigen containing the epitope to immunodominant, also provided a product that exhibits an HBV-positive core antigenicity and immunogenicity [ G. Borisova et al., "Hybrid Hepatitis B Virus Nucleocapsid Bearing an Immunodominant Region from Hepatitis B surface Antigen11, J. Virol .. 67, pp. 3696-3701 (1993)]. As another alternative or as an addition to the fusion proteins described above, the immunogenic components can be linked to the HBV core antigen by chemical cross-linking procedures. The superposition of the amino acid sequence of the HBV core antigen to the physical structure suggested by Bottcher et al. (1997) helps explain the low antigenicity of the sequences fused at or near the carboxy terminal of the nucleus antigen. HBV, since these sequences are likely to be buried within the HBV core antigen particles, whereas N-terminal fusions may benefit from more flexible linker sequences, to bring the immunogen beyond the relatively confined space at the foot of the cells. peaks The epitope location of the immunodominant HBV core antigen [residues 78-82; Salfeld et al. (1989)] at the tip of the peak, shows the attraction of this position for the insertion or binding of the immunogen-peptide that binds to the HBV capsid. In principle, all these positions can be used simultaneously to increase the number and / or diversity of epitopes presented by an antigen particle of the pl301 HBV core determined.

Peptides that bind to HBV capsid used to bind immunogens to HBV core antiqeno particles As described above, immunogens of interest can be linked to a HBV core particle using a ligand that is a peptide that binds to the HBV core. the capsid of HBV. These peptides that bind to the HBV capsid are isolated and purified peptides. These peptides that bind to the HBV capsid inhibit and interfere in an advantageous manner with the HBV viral assembly by blocking the interaction between the HBV core protein and the HBV surface proteins. Preferably, the peptides that bind to the HBV capsid include peptides, fragments, analogs and homologs thereof, with an approximate length between 2 and 20 amino acids. More preferably, the peptides are approximately between 3 and 15 amino acids long. These peptides include those listed in the following tables, as well as fragments and analogs thereof. As used herein, the term "fragment" refers to an amino acid sequence that is shorter than the peptide from which it was derived, although it retains a biological activity practically similar to that of the original peptide. This fragment is at least two amino acids long. As used herein, the term "analogue" refers to variations in the amino acid sequences of the peptides, which may typically include analogs that differ only from one to about four amino acid changes. Other examples of analogs include peptides with minor amino acid variations of the peptides exemplified herein. In particular, peptides containing conservative amino acid replacements, ie, those occurring within a family of amino acids that are related in their side chains, constitute analogs. Genetically encoded amino acids are generally divided into four families: (1) acids: aspartate, glutamate; (2) basic: Usin, arginine, histidine; (3) non-polar: alanine, valine, leucine, isoleucine, prolyline, phenylalanine, methionine, tryptophan; and (4) polar no charge: glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan and tyrosine are sometimes classified together as aromatic amino acids. With respect to the peptides that bind to the HBV capsid, it may be beneficial to change one or more amino acids. The experienced pl301 in the art they can easily assess the impact of these changes. The term "homologue" includes peptide fragments that share at least 60 percent identity at the amino acid level and, preferably, 75 percent identity and, a biological activity very similar to a reference peptide. These preferred percentages reflect the small size of the peptides. Useful peptides that bind to the HBV capsid include those that are based on the peptides that are set forth in Dyson and Murray (1995). These peptides were synthesized following the random utagenesis of residues flanking the peptide LLGRMK in the fusion phage Bl and the re-selection against the antigen of the HBV nucleus in a biopane reaction to obtain derivatives that bind the antigen with an improved affinity . High-definition electronic cryomicroscopy showed that these peptides that bind to the HBV capsid bind at the tips of the protein shell peaks of the HBV core. The inhibitory effect of the peptides on the interaction between the HBV core antigen and the HBV surface antigen proteins in infected cells was examined by transfecting hepatic Hep G2 cells permeabilized with a plasmid competent to replication that carries a dimer from head to tail of the HBV genome in pl301 presence or absence of the peptide. See Bottcher et al. (1998) . HBV capsid-binding peptides carrying the LLGRMK sequence reduces the yield of HBV in cultures of transfected hepatoma cells in a dosage-dependent manner and with relative efficiencies that reflect the IC50 values of the peptides in inhibition. of their reactions between the HBV core antigen and the surface antigen of L-HBV in solution. Peptides that bind to the HBV capsid preferably have a mean maximum concentration (IC50) of less than about 10, preferably less than 5, more preferably less than about 2 and most preferably less than about 0.5 μM. Preferred peptides include, but are not limited to: SLLGRMKG (ß-A) C, RSLLGRMKGA, HRSLLGRMKGA and RSLLGRMKGA (ß-A) C or peptides that are derived therefrom. Alternatively, this peptide can be the peptide ALLGRMKG which inhibits the interaction between the surface antigen of the hepatitis B virus long (L HBsAg) and HBcAg, with a maximum average concentration (IC50) of 10.0 μM. The peptides that bind to the HBV capsid are exemplified by the following, wherein KpR (nM) represents a relative dissociation constant for the reactions between the HBV core antigen and pl301 the fd fusion phage carrying the peptide sequences in the amino-terminal region of the protein [see Dyson and Murrav (1995)]: Sequence KpRel (nM *. ADGALLGRMKGA 152 ± 5 ADGALLGRMKPA 767 ± 8 ADGSLLGRMKPA 322 ± 50 ADGALLGRMKRA 181 ± 12 ADGTLLGRMKLA 20 ± 2 ADGSLLGRMKGA 1.7 ± 0.3 ADRSLLGRMKGA 1.09 ± 0.02 ADGSRSSLLGRMKGA 1.96 ± 0.32 ADGAHSSLLGRMKGA 1.72 ± 0.17 ADGHRSSLLGRMKGA 1.40 ± 0.13 ADGPRSSLLGRMKGA 0.84 ± 0.07 ADGAHRSLLGRMKGA 0.9410.12 ADGYQRSLLGRMKGA 0.88 ± 0.08 ADGTQRSLLGRMKGA 0.84 ± 0.06 ADGMHRSLLGRMKGA 0.55 ± 0.03 These peptides, which mimic the cytoplasmic regions L HBsAg, were identified by selection of a random library of hexapeptides exposed in a filamentous phage and their affinities for the core antigen pl301 of HBV in solution were determined in the associated form of phage. The following related peptides (which are mentioned below) are examples of the HBV capsid binding peptides and the IC5Q μM values represent the concentration of the peptide that is required to inhibit the binding of L HBsAg to the HBV core antigen in a mean maximum level, N / D represents unobservable inhibition, and β-A represents beta alanine [Dyson and Murray (1995)] Sequence IC5Q IXM ALLGRMKG HO ± O.8 LLGRMKG 46.2 ± 7.4 LGRMKG 980 ± 157 GRMKG N / A LLGRM N / A CLLGRMKC 652 ± 74 ALLPRMKC N / A SLLGRMKG 6.4 ± 0.7 SLLGRMK 40.7 ± 4.8 SLLGRMKGA 2.4 ± 0.2 GSLLGRMKGA 0.79 ± 0.23 DGSLLGRMKGAA 3.0 ± 0.4 ADGSLLGRMKGAAG 4.5 ± 0.8 ACSLLGRMKG 16.2 ± 5.0 SLLGRMKG (ß-A) C 1.8 ± 0.4 pl301 Sequence IC5Q UM RSLLGRMKGA 0.29 ± 0.02 HRSLLGRMKGA 0.50 ± 0.04 MHRSLLGRMKGA O.80 ± 0.10 RSLLGRMKGA (ß-A) C 0.29 ± 0.03 MHRSLLGRMKGAG (ß-A) GC 3.80 ± 0.69 HBV capsid binding peptides, fragments, analogs and homologs thereof, which can serve as ligands for binding the immunogens to HBV core antigen particles are preferably synthesized using conventional synthesis techniques, example, chemical synthesis techniques. Alternatively, the skilled artisan can synthesize any of the peptides using an automated peptide synthesizer using standard chemical reactions, for example chemical reaction of t-BOC. See, for example L.A. Carino, J. Am. Chem. Soc., Pp. 4427 (1957). And the peptides can be prepared by chemical breakdown of a protein or by other methods. The peptides are isolated so that they are essentially free of chemical propellants or other chemical agents when chemically synthesized or obtained by chemical breakdown of a protein. Alternatively, the binding peptides to the P1301 HBV capsid can be prepared by conventional genetic engineering techniques, i.e., recombinant DNA techniques in a host cell transformed with a nucleic acid sequence encoding the peptide, by cloning and expression within a host microorganism or a cell, which they express a DNA fragment carrying a coding sequence for the selected peptide. When produced by recombinant techniques, in appropriately transformed cells, the peptides can be purified from the cell culture medium, host cells or both, using conventional methods. The recombinant peptides are isolated so that the peptide is essentially free of cellular material or culture medium when produced by recombinant DNA techniques. The coding sequences for the peptides can be prepared in synthetic form or derived from the viral RNA by known techniques, or by plasmids containing cDNA. For use in the methods of this invention, the peptides described above can be designed as alternative or conventionally known constructs, in order to enhance the replication of the peptide or its binding to the HBV core antigen. For example, the peptides may optionally be fused to a protein or a peptide fusion partner. Therefore, an expert pl301 it will be able to design the peptide in association with a selected fusion partner, for example another peptide, or other peptides or proteins imparting the desired characteristics thereto. Systems for cloning and expressing capsid binding peptides of HBV in various microorganisms and cells, including, for example, E. coli, streptomyces bacilli, saccharomyces, mammals, yeasts, insect cells and plant cells and vectors suitable for They are already known and available from public and private laboratories and from deposit agencies and commercial sellers. Regardless of whether they are produced by recombinant techniques or in synthetic form, the capsid binding peptides of HBV can be purified using conventional purification means. One skilled in the art will readily be able to determine the appropriate level of purity that is required for the desired application in which the peptides are to be used. It should be understood that the choice of HBV capsid binding peptide binder will depend, to some degree, on the nature of the HBV core antigen polypeptide that forms the core antigen particle of HBV. For example, HBV core antigen particles from different strains of viruses pl301 Originals may require different capsid binding peptide ligands of HBV, due to different amino acid sequences at or near the ligand binding sites of the antigen-specific polypeptide for the HBV core.

Binding of HBV capsid binding peptides to immunohaenes HBV capsid binding peptides can be ligated to the immunogens of interest to form the capsid binding immunogen via a peptide bond. The immunogen is itself a peptide, and this will usually be conveniently achieved by a simple synthesis or by the expression of a corresponding coding sequence in cells transformed from a peptide comprising the capsid binding sequence of HBV linked to the sequence of immunogen, normal and preferably through two to five glycine residues (and often three), to impart a degree of flexibility between the two components of this longer peptide. Alternatively, the peptides can be cross-linked or cross-linked with the immunogens. The orientation of the binding between the binding component of the peptide and the immunogen can affect the efficiency of the final process for the cross-linking of the pl301 capsid binding immunogen to the core antigen particle of HBV. Alternatively, among several capsid-binding immunogens to be cross-linked to a core antigen particle of HBV, a specific immunogen can be placed on the amino terminus of the peptide to which it is to be linked, while another immunogen can be placed on the carboxy terminus of the peptide to be bound, in some cases, it is advantageous to place distinct or different immunogens at each end of the capsid binding peptide of HBV. This variation in the organization of the complex capsid binding peptides of the HBV-immunogen to be cross-linked with a specific particle of HBV core antigen advantageously provides superior multi-component or multivalent HBV core antigen particles. See Figure 1. Therefore, the orientation of the capsid binding immunogen to be cross-linked with the core antigen particle of HBV is important for the final immunogenicity or the multivalency of the resulting particles. Higher immunogenicity or multivalence is expected when the immunogen is oriented towards the amino terminus of the capsid binding peptide of HBV. This orientation, which provides greater flexibility, is also preferred for large immunogens. pl301 Binding of capsid binding immunohaenes to the HBV core antigen particle Capsid binding immunogens can be crosslinked to a core antigen particle of HBV using any conventional crosslinking agent. These crosslinking agents include, for example multifunctional crosslinking agents, for example, glutaldehyde, succinaldehyde, other non-dialdehyde and glyoxal. Other crosslinking agents are listed in Pierce Cataloq and Handbook Pierce Chemical Company, Rockford, Illinois (1997). Other crosslinking agents include for example l-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS), which bind to the adjacent primary amino acids and adjacent carboxyl groups to form a Amide bond. When added to a mixture of HBV core antigen particles / capsid binding immunogen, these agents cause a lychee component available in the peptide to be covalently linked with an aspartate or glutamate neighboring the HBV core antigen. It should be understood that the proportion of different immunogens bound to a specific particle of the HBV core antigen can, of course, vary in the relative proportions of the respective immunogens in the pl301 mixture used for the binding of the capsid binding immunogen to the core antigen particle of HBV.

Therapeutic compositions according to this invention The present invention also provides compositions useful for the therapeutic or prophylactic treatment of individuals with multivalent or multicomponent HBV core antigen particles that are discussed herein. In addition, humans and other mammals are included, as well as any type of animals, which can be treated with the HBV core antigen particles that are set forth herein. The therapeutic compositions comprise a pharmaceutically effective amount of the core antigen particles of HBV, ie, an amount that is effective to be immunized against one or more of the infectious agents or to treat one or more conditions in an individual to whom they have been subjected. administered these in certain periods. The prophylactic compositions comprise a prophylactically effective amount of HBV core antigen particles, i.e., an amount that is effective to prevent one or more conditions in an individual being administered over a period. In cases where the HBV core antigen particles contain multiple immunogens of different types, the compositions and vaccines that the pl301 comprise can be used to produce a reinforced immune response in an individual, against each of the component immunogens. In cases where the HBV core antigen particles contain multiple immunogens of a common type, the compositions and vaccines comprising them can be used to produce a strengthened immune response in an individual against the common immunogen. These latter compositions and vaccines are characterized by enhanced immunogenicity, compared to conventional monotherapies. Compositions comprising multivalent or multicomponent HBV core antigen particles of the invention can be administered alone or as part of a pharmaceutical or prophylactic preparation, with or without adjuvants, wherein the controlled release formulations are included. They may additionally contain suitable pharmaceutically acceptable diluents or carriers for the administration and treatment of these infections. Suitable and pharmaceutically acceptable carriers are physiologically inert and / or non-toxic. Various carriers are known in the art and can be selected based on the desired application. Examples of carriers include, unrestrictedly, sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextrin, agar, alum, alumina, pl301 aluminum hydroxide, pectin, peanut oil, olive oil, sesame oil and water. Also, the carrier or diluent may include a time delay material, for example glycerol monostearate or glycerol distearate, alone or in combination with a wax. In addition, slow-release polymer formulations of the conventional type can be used, including, for example, soluble glasses. Potentially, compositions comprising multicomponent or multivalent HBV core antigen particles may contain other therapeutic or prophylactic agents. For example, these compositions may comprise a "mixture" of multiple reagents useful in the treatment or prevention of infections. One of these mixtures may include other reagents such as for example interferons, nucleoside analogs and / or N-acetyl-cysteine. Optionally, the compositions comprise HBV core antigen particles of immunogenic type and, in addition, may contain immune system modifiers, for example adjuvants or cytosine which are useful for further inducing antibodies and T cell responses in the patient. These modifiers include conventional adjuvants based on alum or muramyl dipeptides, preservatives, chemical stabilizers or other antigenic proteins. Typically, pl301 stabilizers, adjuvants and preservatives, etc., are optimized to determine the best formulation to obtain the efficacy in the desired application. Suitable preservatives may include chlorylbutinol, potassium sorbate, sorbic acid, sulfur dioxide, propyl galade, parabens, glycerin, and phenol. Suitable amounts of these compositions can be determined based on the level of response desired. In general, compositions comprising immunogenic HBV core antigen particles can contain between about 5 μg and about 200 μg of the particles. These compositions can be administered as an inoculation or a series of them, for example, three inoculations at intervals of two to six months. The appropriate doses can also be determined at the discretion of the attending physician, taking into account factors such as the patient's health status, weight or age, as well as the conventional doses of a component immunogen, when administered as monotherapy. By improving the condition of a patient or with the likelihood of increased exposure to a specific pathogen, a maintenance dose of the composition comprising HBV immunogenic core antigen particles may be administered, if necessary. Subsequently, the dose or frequency of pl301 administration, or both may be reduced to a level at which the desired effect is retained. At that point, the treatment should stop. However, it may be that some individuals require long-term intermittent treatment when recurrence of a specific unwanted condition occurs. Compositions comprising multicomponent or multivalent HBV core antigen particles can be administered by any suitable route, for example, parenteral, particularly intramuscular or subcutaneous administration, as well as oral administration. Other routes that can be used are: pulmonary, nasal, aural, anal, dermal, ocular, intravenous, intraarterial, intraperitoneal, mucosal, sublingual, subcutaneous and intracranial. The HBV core antigen particles immunogenic according to this invention can be used in the active therapy of individuals infected with HBV in order to inhibit, slow down or slow down the proliferation of the virus within the body. Therapeutic compositions comprise immunogenic type HBV core antigen particles capable of disabling, inhibiting, or preventing the assembly mechanism of the virus. These therapeutic compositions can be formulated to contain carriers or diluents and one or more particles pl301 Immunogens of the HBV core antigen of the invention. These carriers and diluents are discussed above in connection with other types of compositions and can be identified by those skilled in the art. The preparation of compositions or vaccines containing HBV core antigen immunogenic particles as active ingredients can be carried out to formulate vaccines or injectable compositions, either as suspensions or liquid solutions. Suitable solid forms for the solution or suspension in liquids prior to the invention may also be prepared. Also the preparations, for some modalities, may be emulsified or encapsulated in liposomes or in soluble glasses, for their gradual release and / or prolonged administration. Alternatively, the preparations may be in the form of an aerosol or spray. They can also be included in transdermal patches. The active ingredient can be mixed with various types of excipients that are pharmaceutically acceptable and compatible with the active ingredients. These excipients include, for example, incomplete Freund's adjuvant, bacterial lipopolysaccharides, ion exchangers, alumina, aluminum stearate, muramyl dipeptide, lecithin, buffer substances, cellulose-based substances and polyethylene glycol. pl301 Advantageously, vaccines comprising core antigen particles of HBV according to this invention can be combination vaccines comprising several different immunogens. These vaccines include, for example, combination vaccines comprising immunogens against two or more of the following diseases: diphtheria, tetanus, acellular pertussis, haemophilus influenza, polio, measles, mumps, rubella, varicella, hepatitis B virus, hepatitis virus A or pneumococcal pneumonia. Other vaccines include those that are used for the inoculation of people before making an international trip. These vaccines, include, for example, vaccines comprising immunogens against two or more of the following diseases: yellow fever, hepatitis B virus, hepatitis A virus, typhoid fever, meningococcal encephalitis or cholera. Compositions comprising particles of HBV core antigen, according to this invention can also be used in immunotherapeutic regimens for the desensitization of individuals to one or more allergens, for example animal allergens, insect allergens, plant allergens, allergens and allergens. of inhalation. According to an alternative embodiment of the present invention, the core antigen particles pl301 of HBV can be used to produce antigens against the immunogens of interest, for use in immunotherapy or diagnosis. For example, antibodies obtained in individuals inoculated with HBV core antigen particles can be isolated and used in purified form. Alternatively, these antibodies or B cells of individuals may be used to produce monoclonal antibodies, through conventional techniques.

Detection methods according to the invention The HBV core antigen particles of the present invention can also be used in several conventional assay formats, in particular, immunoassay formats for the diagnosis of infections or exposure to infectious agents. This utility is apparent when the components of the HBV capsid binding peptide of the constructs of the present invention are associated with a diagnostic tag, a chemical tag, a toxin or any other protein or peptide. For example, HBV capsid binding peptides may be associated with conventional labels that are capable, either alone or in combination with other compositions or compounds, of providing a detectable signal that could indicate the presence of an analyte. pl301 blank in a sample, during exposure of the immunogen bound to the antigen-binding peptide of the specific HBV nucleus. These detectable labels can be selected from several known compositions and are readily available to experts in diagnostic assays. The invention, therefore, is not limited to the selection of the particular assay format and is considered to encompass assay formats that are known to the experts. For convenience, the test reagents can be provided in the form of kits. These kits may include microtitre plates to which the HBV core antigen particles of this invention have been previously adsorbed, and also include various diluents and buffers, labeled conjugates for the detection of the peptide immunogens of the capsid binding , to those that bind specifically, and other reagents that generate signals, for example enzymatic substrates, cofactors and chromogens. Other compounds will be readily determined by the experts. Alternatively, the core antigen particles of HBV according to the invention can be used in currently existing immunological diagnostic tests for the detection of pathogens, i.e., radioimmunoassay or ELISA (immunosorbent binding assay). pl301 enzymes). In an embodiment of the present invention, a sample to be tested for the presence of antibodies to the various immunogens, may be contacted with a particle of HBV core antigen containing HBV capsid binding immunogens labeled to be detected, and having different immunogenic components, for a time sufficient to allow any antibody that is present in the sample to form a complex with one or more of the capsid binding immunogens of HBV. The detection means can then be used to detect the complex formed between the immunogen or capsid binding immunogens and the antibodies in the sample. A second classification or assay can be carried out on the sample based on each component immunogen, in order to identify the specificity of the antibodies in the sample. In an alternative embodiment of the invention, a sample to be tested for the presence of antibodies to a specific immunogen may be contacted with an HBV core antigen particle comprising the HBV capsid binding immunogens. , labeled, in detectable form, that have that specific immunogen as an immunogenic component, pl301 for a time sufficient to allow any antibody in the sample to form a complex with one or more HBV capsid binding immunogens. Due to the high valence of the specific immunogen demonstrated by the core antigen particle of HBV, this diagnostic assay is characterized by a higher sensitivity than conventional assays.

EXAMPLES In order that the invention described here can be more easily understood, the following examples are shown. It should be understood that these examples are for illustrative purposes only and should not be construed as limitations of the invention.

Example 1 Preparations of HBV core antigen Expression of an HBV core antigen (aa3-183) or truncated C terminal HBV core antigen (aa3-148) in E. coli and purification were carried out as described in Dyson and Murray (1995). The protein preparations were stored at 4 ° C as gradient fractions of sucrose in a buffer containing TBS, sucrose (20%) and NaN3 (0.02%). The preparations were stable with the above during pl301 at least six months.

Chemical cross-linking of HBV capsid-binding peptides with the HBV core antqene HBV capsid-binding peptide MHRSLLGRMKGA (Albachem, University of Edinburgh) reticulated with HBV core antigen particles using l-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS) (both purchased of Pierce Europe BV). These reagents bind to the adjacent amino and carboxyl primary groups to form an amide linkage [Staros et al., "Enhancement by N-Hydroxysulfosuccinimide of Water-Soluble Carbonddimide-Mediated Coupling Reactions", Analytical biochem. 156, p. 220-22 (1986)). When added to a mixture of HBV capsid binding peptide / HBV core antigen, they must covalently cross-link the lysine of the peptide with an aspartate or glutamate neighboring the HBV core antigen, causing its molecular weight to increase. More specifically, the truncated HBV core antigen (15μg) was incubated at room temperature in a buffer (30μl) containing potassium phosphate (25mM, pH7), NaCl (150mM), (EDC 1.8mM) and sulfo- NHS (1.8mM) in the presence or absence of the peptide MHRSLLGRMKGA (1M).

P1301 After 18 hours, the reaction was analyzed with SDS / PAGE (15% w / v) as described in (Sambrook et al (1989)). The addition of EDC and sulfo-NHS to the core antigen particle complex of the HBV-peptide resulted in a band shift corresponding to 1 kd, which was presented on SDS-PAGE by a fraction of the HBV core antigen . Despite making several runs of the reaction under various conditions, a protein band displaced by more than 50% was not obtained. This was consistent with a binding of the peptide with a dimer of the HBV core antigen near the double local axis and, therefore, sterically hindered the binding of another peptide to the double related site.

EXAMPLE 2 Preparations of HBV core antigen In addition to the two core HBV antigen samples prepared in example 1, samples of the HBV core antigens with the pre-Sl sequence of HBV 1-36 or the sequence of surface antigen of HBV 111-156 or 111-165 bound to the truncated polypeptide of the HBV core antigen (truncated at residue 144) by the short sequence of the binder peptide, was also prepared as described in Stahl and Murrav (1989 ). pl301 Chemical cross-linking of HBV capsid binding peptide with HBV core antqene The following capsid binding immunogens were obtained by solid phase synthesis at Albachem, University of Edinburgh: AS-151: GSLLGRMKGA GGG LDPAFRG AS -152: GSLLGRMKGA GGG EQKLISEEDL AS-163: LDPAFR GG GSLLGRMKGA AS-164: EQKLISEEDL GG GSLLGRMKGA, wherein the sequence GSLLGRMKGA is the capsid binding peptide of HBV, the sequence LDPAFR is the epitope pre-Sl HBV or the immunogen and the sequence EQKLISEEDL is the epitope of the myc oncogene or the immunogen. These peptides and HBV's basic capsid-binding peptide, GSLLGRMKGA, were bound to a core antigen particle of HBV separately or in combination, at different concentrations, and cross-linked with EDC or with sulfo-NHS, as it is described in example 1.

Properties of the resulting HBV core antiqene particles The products were analyzed by electrophoresis in acrylamide gels and in the presence of SDS (SDS-PAGE), followed by staining with Coomassie blue and the analysis pl301 Western blot with monoclonal antibodies and rabbit polyclonal sera raised against HBV core antigen particles or denatured HBV surface antigen particles. The monoclonal antibodies for each pre-Sl epitope of HBV and the epitome of the myc oncogene are commercially available. Respectively they are the monoclonal antibody 18/7 [K.H. Heermann et al., J. Virol. 52, pp. 396-402 (1984)] and the monoclonal antibody 9E10 [Invitrogen, Catalog # R950-25]. These experiments demonstrate that the products of all the crosslinking reactions exhibited positive reactions with the antibodies against each of the constituent epitopes in the ligation reaction components. Positive reactions were obtained with the immunogen linked through the amino group or the carboxy terminal group of the peptide ligand. As already mentioned above, preparations of HBV core antigen particles from reactions involving cross-linking with two or more different immunogens, using a common-type HBV capsid-binding peptide ligand, react with the antibodies against all the component immunogens. In addition, the HBV core antigen particles precipitated with the specific antibody for one of the pl301 immunogens, exhibit cross-reactivity with the antibodies of other peptides included in the ligand crosslinking process. The products of the ligature were subjected to ultracentrifugation through sucrose gradients. They were precipitated with one of the antibodies, the anti-myc antibody, then analyzed by SDS-PAGE and Western blotting. The material precipitated with one of the antibodies, for example, anti-myc antibody, showed strong cross-reactivity with the anti-myc antibody and the anti-pre-Sl antibody, in the Western blot. The products precipitated with the other antibody, the antibody, the pre-Sl antibody, also showed the same. In reagents where two HBV capsid-binding peptides, which carried different immunogens, were mixed in different ratios of binding and cross-linking with the core particles, analysis by Western blotting and by SDS-PAGE showed that the relative intensities of the staining with the two monoclonal antibodies reflected the proportion of the two immunogens in the mixture used for the crosslinking. These experiments showed that at least pl301 some of the HBV core particles that resulted from the reactions had the two immunogens covalently bound to them. Since the ligand peptide binds to the tips of the HBV core antigen particles [nucleocapsids], these preparations will exhibit high immunogenic potency for the two components and will be expected to produce high antibody titers in individuals to whom they are administered. Although various embodiments of the invention have been presented so far, it will be apparent that our basic construction may be altered to provide other embodiments utilizing the process of this invention. Therefore, it will be appreciated that the scope of the invention will be defined by the appended claims and not by the specific embodiments that have been presented as examples only. pl301

Claims (50)

1. CLAIMS. A particle of HBV core antigen having multiple immunogen specificities, wherein the particle comprises at least one capsid binding immunogen, and this immunogen comprises at least one component of the HBV capsid binding peptide. and at least one immunogenic component.
2. 2. An HBV core antigen particle having multiple immunogen specificities according to claim 1, wherein the capsid binding immunogen is oriented on the particle, so that it allows the immunogenic component to produce an immune response when the particle is administered to an individual.
3. 3. An HBV core antigen particle having multiple immunogen specificities according to claim 1, wherein the capsid binding immunogen is bound to the particle through any amino acid residue of the peptide binding component of the peptide. HBV capsid.
4. 4. A core antigen particle of HBV having multiple immunogen specificities according to claim 1, wherein the capsid binding immunogen is bound to the particle through any amino acid residue or any other residue of the component pl301 immunogenic
5. 5. A core antigen particle of HBV having multiple immunogen specificities according to claim 4, wherein the other residue of the immunogenic component is a carbohydrate.
6. 6. An HBV core antigen particle having multiple immunogen specificities according to claim 1, wherein the capsid binding immunogen is bound to the particle through the amino terminus of the capsid binding peptide component of the capsid. HBV.
7. 7. An HBV core antigen particle having multiple immunogen specificities according to claim 1, wherein the capsid binding immunogen is bound to the particle to the particle through the terminal caboxy of the binding partner component of the peptide. the capsid of HBV.
8. 8. A core HBV antigen particle having multiple immunogen specificities according to claim 1, wherein the capsid binding immunogen is crosslinked with the particle by a crosslinker.
9. 9. An HBV core antigen particle having multiple immunogen specificities according to claim 1, wherein the immunogenic component binds to the HBV capsid binding peptide component. pl301 directly or through a binding sequence.
10. 10. A core HBV antigen particle having multiple immunogen specificities according to claim 1, wherein the immunogenic component binds to the amino terminus of the HBV capsid binding peptide component directly or through a binding sequence.
11. 11. A core HBV antigen particle having multiple immunogen specificities according to claim 1, wherein the immunogenic component binds to the carboxy terminus of the HBV capsid binding peptide component directly or through a binding sequence.
12. 12. A core HBV antigen particle having multiple immunogen specificities according to any of claims 9-11, wherein the immunogenic component binds to the HBV capsid binding peptide component via a crosslinker.
13. 13. A core antigen particle of HBV having multiple immunogen specificities according to claim 8, wherein the crosslinker is a multifunctional crosslinker.
14. 14. A particle of HBV core antigen having multiple immunogen specificities according to claim 12, wherein the crosslinker is a pl301 multifunctional crosslinker.
15. 15. A core HBV antigen particle having multiple immunogen specificities according to claim 14, wherein the multifunctional crosslinker is selected from the group consisting of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and N- hydroxysulfosuccinimide.
16. 16. A particle of HBV core antigen having multiple immunogen specificities according to claim 1, wherein the immunogenic component comprises one or more epitopes selected from the group consisting of in unological epitopes, immunogenic epitopes and antigenic epitopes.
17. 17. A core HBV antigen particle having multiple immunogen specificities according to claim 16, wherein the epitopes are selected from the group consisting of linear epitopes, conformational epitopes, single epitopes and mixed epitopes.
18. 18. A core HBV antigen particle having multiple immunogenic specificities according to claim 1, wherein the immunogenic component is selected from the group consisting of: antigens, allergens, antigenic determinants, proteins, glycoproteins, antibodies, antibody fragments , peptides, peptide mimotopes that mimic an antigenic determinant or pl301 antigen, to polypeptides, glycopeptides, carbohydrates, oligosaccharides, polysaccharides, oligonucleotides and polynucleotides.
19. 19. A core HBV antigen particle having multiple immunogenic specificities according to claim 1, wherein the immunogenic component is directed to or derives from a pathogenic agent, which is selected from the group consisting of virus, parasites, mycobacteria, bacteria, bacilli, fungi, protozoa, plants, phages, animal cells and plant cells.
20. 20. An HBV core antigen particle having multiple immunogen specificities according to claim 19, wherein the virus is selected from the group consisting of retroviruses, herpes viruses, orthomyxoviruses, paramyxoviruses, hepadnaviruses, flaviviruses, picornaviruses, papoviruses, adenovirus, baculovirus, hantavirus, parvovirus, enterovirus, rhinovirus, tumor virus, DNA virus, RNA virus, togavirus, rhabdovirus and poxvirus.
21. 21. A core HBV antigen particle having multiple immunogen specificities according to claim 20, wherein the virus is selected from the group consisting of human immunodeficiency virus type 1, human immunodeficiency virus type 2, human pl301 T cell leukemia, herpes simplex virus type 1, herpes simplex virus type 2, varicella zoster virus, cytomegalovirus, Epstein-Barr virus, influenza A virus, influenza B virus, influenza C virus, respiratory syncytial virus , measles-like virus, mumps virus, paraenfluenza virus, hepatitis B virus, hepatitis C virus, hepatitis A virus, hepatitis E virus, yellow fever virus, malaria, dengue virus, virus transmitted by tick, oncovirus, poliomyelitis virus, papillomavirus, rubella virus, rabies virus and vaccinia virus.
22. 22. A particle of HBV core antigen having multiple immunogen specificities according to claim 19, wherein the immunogenic component is directed towards bacilli, enterobacteria, clostridium, listeria, mycobacteria, pseudomonas, staphylococcus, eubacteria, mycoplasma, chlamydia, spirochetes , neisseria or salmonella, or is derived from them.
23. 23. A particle of HBV core antigen having multiple immunogen specificities according to claim 19, wherein the immunogenic component is directed towards diphtheria, tetanus, acellular pertussis, haemofilus influenza, polio, measles, mumps, rubella, varicella, virus of hepatitis B, hepatitis A virus, pneumococcal pneumonia, yellow fever, malaria, pl301 hepatitis B, hepatitis A virus, typhoid fever, meningococcal encephalitis or cholera.
24. 24. A core HBV antigen particle having multiple immunogenic specificities according to claim 18, wherein the immunogenic component is selected from the group consisting of animal allergens, insect allergens, plant allergens, atmospheric allergens and inhalant allergens.
25. 25. An HBV core antigen particle having multiple immunogen specificities according to claim 1, wherein the HBV core antigen is a fusion protein of the HBV core antigen.
26. 26. A core HBV antigen particle having multiple immunogen specificities according to claim 25, wherein the core antigen fusion protein of HBV comprises an immunological epitope, an immunogenic epitope or an antigenic epitope.
27. 27. A core HBV antigen particle having multiple immunogen specificities according to claim 26, wherein the core antigen fusion protein of HBV comprises an immunological epitope, an immunogenic epitope or an antigenic epitope fused to the core antigen. of HBV directly through the binding sequence. pl301
28. 28. A particle of HBV core antigen having multiple immunogen specificities according to claim 26, wherein the HBV core antigen fusion protein comprises an immunological epitope, an immunogenic epitope or an antigenic epitope fused to the carboxy terminus of the HBV core antigen either directly or through the binding sequence.
29. 29. A core HBV antigen particle having multiple immunogen specificities according to claim 26, wherein the core antigen fusion protein of HBV comprises an immunological epitope, an immunogenic epitope or an antigenic epitope fused to the amino terminus of the HBV core antigen. HBV core antigen either directly or through the binding sequence.
30. 30. A core HBV antigen particle having multiple immunogen specificities according to claim 25, wherein the HBV core antigen fusion protein comprises the truncated HBV core antigen.
31. 31. A HBV core antigen particle having multiple immunogen specificities according to claim 25, wherein the core antigen fusion protein of HBV comprises an antigen of pl301 HBV surface or a portion thereof.
32. 32. A core HBV antigen particle having multiple immunogen specificities according to claim 31, wherein the core antigen fusion protein of HBV comprises the sequence selected from the group consisting of the pre-Sl region of the antigen of the HBV core antigen. HBV surface, the pre-S2 region and the HBV surface antigen, the immunodominant region a of the HBV surface antigen and portions thereof.
33. 33. A core HBV antigen particle having multiple immunogen specificities according to claim 1, wherein the HBV core antigen is a full-length polypeptide of the HBV core antigen, or portions, truncated sections, mutations or derivatives thereof that are capable of assembling in particulate form.
34. 34. A particle of HBV core antigen having multiple immunogen specificities according to claim 1, wherein the component of the capsid binding peptide of HBV is selected from the group consisting of: SLLGRMKGA, GSLLGRMKGA, DGSLLGRMKGAA, ADGSLLGRMKGAAG, SLLGRMKG (ß-A) C, RSLLGRMKGA, HRSLLGRMKGA, ALLGRMKG, MHRSLLGRMKGA, RSLLGRMKGA (ß-A) C and MHRSLLGRMKGAG (ß-A) GC.
35. 35. A vaccine comprising an amount pl301 Prophylactically effective of an HBV core antigen particle having multiple immunogen specificities according to claim 1.
36. 36. A pharmaceutical composition comprising a therapeutically effective amount of an HBV core antigen particle having multiple immunogen specificities. according to claim 1.
37. 37. A method for producing an immune response in an individual, comprising the step of administering to the individual a core antigen particle of HBV having multiple immunogen specificities according to claim 1, in an effective amount to produce an immune response.
38. 38. The method according to claim 37, wherein the core antigen particle of HBV is administered to the individual parenterally.
39. 39. The method for enhancing the immunogenicity of an immunogen by linking the immunogen to an HBV core antigen particle through a capsid binding peptide of HBV.
40. 40. The HBV core antigen particle having multiple immunogen specificities according to claim 1, wherein the capsid binding immunogen comprises a diagnostic tag or a pl301 chemical marker
41. 41. A method for detecting the presence of antibodies to an immunogen in a sample comprising the steps of: (a) contacting the sample with an HBV core antigen particle having multiple immunogen specificities according to the claim 40, for a time sufficient to allow any antibody in the sample to form a complex with the capsid binding immunogen and (b) use a detection means to detect the complex formed between the capsid binding immunogen and the antibodies in the sample.
42. 42. An HBV capsid-binding peptide immunogen comprising at least one capsid-binding peptide component and at least one immunogenic component.
43. 43. An HBV capsid-binding peptide immunogen according to claim 42, wherein the immunogenic component is linked to the capsid binding peptide of HBV directly or through a binding sequence.
44. 44. An HBV capsid-binding peptide immunogen according to claim 42, wherein the immunogenic component is linked to the amino terminal of the HBV capsid. pl301 component of HBV capsid binding peptide directly or through a binding sequence.
45. 45. An HBV capsid binding peptide immunogen according to claim 42, wherein the immunogenic component is linked to the carboxy terminus of the direct HBV capsid binding peptide component or through a binding sequence.
46. 46. An HBV capsid binding peptide immunogen according to any of claims 42 to 44, wherein the immunogenic component is crosslinked with the capsid binding peptide component of HBV by a crosslinker.
47. 47. An HBV capsid binding peptide immunogen according to claim 46, wherein the crosslinker is a multifunctional crosslinker.
48. 48. An HBV capsid binding peptide immunogen according to claim 47, wherein the multifunctional crosslinker is selected from the group consisting of l-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysulfosuccinimide.
49. 49. An HBV capsid-binding peptide immunogen according to claim 42, wherein the immunogenic component comprises one or more epitopes selected from the group consisting of immunological epitopes, immunogenic epitopes and epitopes. pl301 antigenic
50. 50. An HBV capsid-binding peptide immunogen according to claim 49, wherein the epitopes are selected from the group consisting of linear epitopes, conformational epitopes, simple epitopes and mixed epitopes. A capsid binding peptide immunogen of HBV according to claim 42, wherein the immunogenic component is selected from the group consisting of: antigens, allergens, antigenic determinants, proteins, glycoproteins, antibodies, antibody fragments, peptides, mimotopes peptides that mimic an antigenic determinant or antigen, polypeptides, glycopeptides, carbohydrates, oligosaccharides, polysaccharides, oligonucleotides and polynucleotides. 52. An HBV capsid binding peptide immunogen according to claim 42, wherein the immunogenic component is directed to or derived from pathogenic agents, and these are selected from the group consisting of viruses, parasites, mycobacteria, bacteria, bacilli, fungi, protozoa, plants, phages, animal cells and plant cells. 53. An HBV capsid-binding peptide immunogen according to claim 52, wherein the virus is selected from the group consisting of retroviruses, pl301 Herpes viruses, orthomyoxoviruses, paramyxoviruses, hepadnaviruses, flaviviruses, picornaviruses, papoviruses, adenoviruses, baculoviruses, hantaviruses, parvoviruses, enteroviruses, rhinoviruses, tumor viruses, DNA viruses, RNA viruses, togaviruses, rhabdoviruses and poxviruses. 54. A capsid binding peptide immunogen of HBV according to claim 53, wherein the virus is selected from the group consisting of human immunodeficiency virus type 1, human immunodeficiency virus type 2, T cell leukemia virus, herpes simplex virus type 1, herpes simplex virus type 2, varicella-zoster virus, cytomegalovirus, Epstein-Barr virus, influenza A virus, influenza B virus and influenza C virus, respiratory syncytial virus, virus similar to measles, mumps virus, parainfluenza virus, hepatitis B virus, hepatitis C virus, hepatitis A virus, hepatitis E virus, yellow fever virus, dengue virus, malaria, tick-borne virus, polio, rubella virus, rabies virus and vaccinia virus. 55. An HBV capsid binding peptide immunogen according to claim 42, wherein the immunogenic component is directed to bacilli, enterobacteria, clostridium, listeria, mycobacteria, pseudomonas, staphylococcus, eubacteria, mycoplasma, pl301 Chlamydia, spirochetes, neisseria or salmonella, or is derived from these. 56. An HBV capsid binding peptide immunogen according to claim 42, wherein the immunogenic component is directed towards diphtheria, tetanus, acellular pertussis, haemofilus influenza, polio, measles, mumps, rubella, varicella, hepatitis B virus. , hepatitis A virus, pneumococcal pneumonia, yellow fever, malaria, hepatitis B virus, hepatitis A virus, typhoid fever, meningococcal encephalitis or cholera. 57. An HBV capsid-binding peptide immunogen according to claim 42, wherein the immunogenic component is selected from the group consisting of animal allergens, insect allergens, plant allergens, atmospheric allergens and inhalant allergens. 58. An HBV capsid binding peptide immunogen according to claim 42, wherein the capsid binding peptide component of HBV is selected from the group consisting of: SLLGRMKGA, GSLLGRMKGA, DGSLLGRMKGAA, ADGSLLGRMKGAAG, SLLGRMKG (ß -A) C, RSLLGRMKGA, HRSLLGRMKGA, ALLGRMKG, MHRSLLGRMKGA, RSLLGRMKGA (ß-A) C and MHRSLLGRMKGAG (ß-A) GC. 59. An HBV capsid binding peptide immunogen according to claim 42, wherein the pl301 HBV capsid binding peptide component is a fragment or analogue of: SLLGRMKGA, GSLLGRMKGA, DGSLLGRMKGAA, ADGSLLGRMKGAAG, SLLGRMKG (ß-A) C, RSLLGRMKGA, HRSLLGRMKGA, ALLGRMKG, MHRSLLGRMKGA, RSLLGRMKGA (ß-A) C and MHRSLLGRMKGAG (ß-A) GC. 60. An HBV capsid-binding peptide immunogen according to claim 1, wherein the capsid-binding peptide component of HBV is a fragment or analogue of: SLLGRMKGA, GSLLGRMKGA, DGSLLGRMKGAA, ADGSLLGRMKGAAG, SLLGRMKG (ß- A) C, RSLLGRMKGA, HRSLLGRMKGA, ALLGRMKG, MHRSLLGRMKGA, RSLLGRMKGA (ß-A) C and MHRSLLGRMKGAG (ß-A) GC. pl301