PEPTIDES DERIVED FROM VIRAL PROTEINS FOR USE AS IMMUNOGENS AND DOSAGE REACTANTS

Abstract
The present invention relates to novel peptides and methods for treatment, diagnosis and prognosis of virus infections including infections with HCV, HIV, HPV, CMV and Influenza. The invention further relates to methods for identifying and providing peptides useful for the treatment and diagnosis.
Description
FIELD OF THE INVENTION

The present invention relates to novel peptides and methods for treatment, diagnosis and prognosis of virus infections and various cancer and inflamatory diseases including infections with HCV, HIV, HPV, CMV and Influenza. The invention further relates to methods for identifying and providing peptides useful for the treatment and diagnosis.


BACKGROUND OF THE INVENTION

Conventional approaches to vaccine development have implemented either whole replication competent virus which has been attenuated (e.g. Sabin polio vaccine, measles, mumps, rubella (MMR)) or inactivated virions that are not replication competent. On occasions, the inactivated virus vaccines may include split vaccines where the virus particles have been disrupted. Molecular techniques have also been used to develop the subunit vaccine (e.g. hepatitis B vaccine) that consists only of the surface glycoproteins of hepatitis B virus. The inactivated virus vaccines tend to induce primarily antibody responses to the viruses in question, whereas the live attenuated vaccines induce both cell-mediated immunity as well as an antibody response since the vaccine induces a transient infection.


The only disease which has been eliminated by virtue of a successful vaccination campaign is smallpox. A campaign is currently in progress to eradicate polio. Features of virus infections that can be eliminated by vaccination are infections caused by viruses with stable virus antigens (i.e. very low mutation frequency, few subtypes), that lack a reservoir in other animal species, viruses that do not persist in the body once the infection is over and where vaccination leads to long lasting immunity. Viruses such as polio and measles fulfill these criteria whereas viruses such as influenza virus (Flu), HCV, and HIV that vary their protein sequences do not. It is for this reason that new and alternate approaches are required to develop vaccines for these diseases.


Vaccination aims to stimulate the immune response to a specific pathogen in advance of infection. When an individual is exposed to that pathogen, a memory response is triggered which prevents the establishment of infection. Vaccines therefore stimulate the adaptive immune response which unlike innate immunity, is long lived and has memory. There are two major arms to the adaptive immune system. Humoral immunity which involves the development of antibodies that can bind virus particles and certain antibodies that can neutralize infection. Cell mediated immunity that leads to the development of cytotoxic T-cells that kill infected cells exposing viral epitopes in the context of human leukocyte antigen (HLA) class I, in this way eliminating infected cells.


The challenge of providing vaccines suitable for stimulation of the adaptive immune system is that peptide epitopes need to be taken up by the antigen presenting cells.


Several peptides have been demonstrated to translocate across the plasma membrane of eukaryotic cells by a seemingly energy-independent pathway. These peptides are defined as cell-penetrating peptides (CPPs). Cellular delivery using these cell-penetrating peptides offers several advantages over conventional techniques. It is non-invasive, energy-independent, is efficient for a broad range of cell types and can be applied to cells en masse.


For humoral responses and development of antibodies it may not be needed to obtain cell-penetrating properties since stimulation of B-cells are also done by extracellular peptide antigens. Hepatitis means inflammation of the liver which can be caused by a variety of factors including toxins, certain drugs, some diseases, heavy alcohol use, and bacterial and viral infections. Hepatitis is also the name of a family of viral infections that affect the liver; the most common types in the developed world are hepatitis A, hepatitis B, and hepatitis C.


Hepatitis C is a liver disease that results from infection with the hepatitis C virus (HCV). It can range in severity from a mild illness lasting a few weeks to a serious, lifelong illness. Hepatitis C is spread via blood; the most common form of transmission is through sharing needles or other equipment used to inject drugs. The infection can be either “acute” or “chronic”. Acute HCV infection is an asymptomatic, short-term illness that occurs within the first 6 months after someone is exposed to the hepatitis C virus. For most people, acute infection leads to chronic infection, which can result in long-term complications and even death.


HCV is an enveloped positive stranded ribonucleic acid (RNA) virus with a diameter of about 50 nm, belonging to the genus Hepacivirus in the family Flaviviridae that replicate in the cytoplasm of infected cells. The only known reservoir for HCV is humans, although the virus has experimentally been transmitted to chimpanzees. The natural targets of HCV are hepatocytes and possibly B-lymphocytes. As of 2008, six different genotypes and more than 100 subtypes of the virus are known. Replication occurs through an RNA-dependent RNA polymerase that lacks a proofreading function, which results in a very high rate of mutations. Rapid mutations in a hypervariable region of the HCV genome coding for the envelope proteins enable the virus to escape immune surveillance by the host. As a consequence, most HCV-infected people proceed to chronic infection.


It is estimated that 170 million people are infected with HCV worldwide, equating to approximately 3% of the global population. There are also approximately 3-4 million people who are infected every year; with an estimated 80% of these newly infected patients progressing to chronic infection.


The 6 genotypes of HCV have different geographical spread. The disease in the early stages is generally asymptomatic; the majority of patients with chronic infection eventually progress to complications such as liver fibrosis and cirrhosis, and, in 1-5% of cases, hepatocellular carcinoma.


HCV is the major cause of non-A, non-B hepatitis worldwide. Acute infection with HCV frequently leads to chronic hepatitis and end-stage cirrhosis. It is estimated that up to 20% of HCV chronic carriers may develop cirrhosis over a time period of about 20 years and that of those with cirrhosis between 1 to 4% is at risk to develop liver carcinoma.


The about 9.6 kb single-stranded RNA genome of the HCV virus comprises a 5′- and 3′-noncoding region (NCRs) and, in between these NCRs a single long open reading frame of about 9 kb encoding an HCV polyprotein of about 3000 amino acids.


HCV polypeptides are produced by translation from the open reading frame and cotranslational proteolytic processing. Structural proteins are derived from the amino-terminal one-fourth of the coding region and include the capsid or Core protein (about 21 kDa), the E1 envelope glycoprotein (about 35 kDa) and the E2 envelope glycoprotein (about 70 kDa, previously called NS1), and p7 (about 7 kDa). The E2 protein can occur with or without a C-terminal fusion of the p7 protein (Shimotohno et al. 1995). An alternative open reading frame in the Core-region has been found which is encoding and expressing a protein of about 17 kDa called F (Frameshift) protein (Xu et al. 2001; Ou & Xu in US Patent Application Publication No. US2002/0076415). In the same region, ORFs for other 14-17 kDa ARFPs (Alternative Reading Frame Proteins), A1 to A4, were discovered and antibodies to at least A1, A2 and A3 were detected in sera of chronically infected patients (Walewski et al. 2001). From the remainder of the HCV coding region, the non-structural HCV proteins are derived which include NS2 (about 23 kDa), NS3 (about 70 kDa), NS4A (about 8 kDa), NS4B (about 27 kDa), NS5A (about 58 kDa) and NS5B (about 68 kDa) (Grakoui et al. 1993).


Influenza remains a significant cause of mortality and morbidity worldwide. The World Health Organisation (WHO) estimates that seasonal epidemics affect 3-5 million people with severe illness annually and result in 250,000-500,000 mortalities. Influenza is caused by viruses in the family Orthomyxoviridae which are negative stranded RNA viruses. The influenza virus exists as three types, A, B and C of which only A is associated with pandemics. Types A viruses are found in both humans and animals, particularly birds but also other mammals such as pigs. Type A viruses are further typed into subtypes according to different kinds and combinations of virus surface proteins. Among many subtypes in 2009 influenza A (H1N1) and A (H3N2) subtypes were circulating among humans. Influenza A and B are included in the seasonal vaccine, whereas influenza C occurs only rarely, and so it is not included in the seasonal vaccine. Type B viruses are human specific and Type C viruses cause a very mild disease. The genomes of Orthomyxoviruses are segmented. Influenza viruses Types A and B have 8 segments whereas type C has seven. Pandemics may arise as a result of re-assortment of gene segments when two different type A viruses infect the same cell. There is no immunity in the population to this novel re-assorted virus. Three pandemics occurred in the twentieth century: “Spanish influenza” in 1918, “Asian influenza” in 1957, and “Hong Kong influenza” in 1968. The 1918 pandemic killed an estimated 40-50 million people worldwide. Subsequent pandemics were much milder, with an estimated 2 million deaths in 1957 and 1 million deaths in 1968. In June 2009 the WHO declared a pandemic from influenza virus H1N1 (swine Influenza) which was declared over in August 2010.


Human papillomaviruses are made up of a group of DNA viruses in the family Papillomaviridae which infect the skin and mucous membranes. Two groups which are derived from more than 100 different identified subtypes are the main cause for clinical concern: those causing warts (both benign and genital warts), and a group of 12 “high risk” subtypes that can result in cervical cancer. This latter group has been attributed as a contributory factor in the development of nearly all types of cervical cancer. Worldwide, cervical cancer remains the second most common malignancy in women, and is a leading cause of cancer-related death for females in developing countries. HPV 16 and 18 have been mainly associated with cervical cancer, however, the virus is also a cause of throat cancer in both men and women. HPV is transmitted through contact and enters the skin through abrasions. An abortive infection, where only the early proteins are expressed is associated with cancer development.


OBJECT OF THE INVENTION

It is an object of embodiments of the invention to provide peptides that may be used as immunogens to stimulate an adaptive immune response in a subject.


It is a further object of embodiments of the invention to provide peptides, including multimeric, such as dimeric peptides, that may be used as immunogens to stimulate the humoral immunity in a subject.


In particular, it is an object of embodiments of the invention to provide peptides that may be taken up by antigen presenting cells (macrophages and dendritic cells) such that epitopes within the peptides are correctly processed and presented to T-lymphocytes in order to stimulate an effective immune response.


It is a further object of object of embodiments of the invention to provide peptides including multimeric, such as dimeric peptides comprising epitopes of an antigen that stimulates cells of the B lymphocyte lineage (B-cells) to secrete antibodies against this antigen.


The B-cell activation provided by the peptides according to the present invention may be both T cell-independent and T cell-dependent. Accordingly, the peptides according to the present invention or parts thereof may interact with B-cell receptors to activate the B-cells either through a T helper cell dependent or independent manner leading to the production of specific antibodies. Furthermore, the peptides may be taken up by antigen presenting cells (macrophages and/or dendritic cells) such that epitopes within the peptides are correctly processed and presented to T-lymphocytes, such as a helper T cell, which in turn helps to activate the B cells in order to stimulate an effective immune response. The peptides may also be taken up by activated B-cells which can also act as antigen presenting cells. Peptides interact with the B-cells through the B-cell receptor and are then internalised into the cell. The epitopes within the peptides will be processed and presented to T-lymphocytes such as helper cells.


However, in some important aspects of the present invention, the peptides according to the present invention are designed to not effectively penetrate and be taken up by antigen presenting cells. Accordingly, in these aspects of the invention, the peptides according to the present invention may provide B-cell activation through interaction at the cell surface via the B-cell receptor. It is to be understood that in order to provide sustained B-cell stimulation, it is preferred that the peptides according to the present invention are designed to comprise a helper epitope that may be taken up by antigen presenting cells in order to stimulate CD4+ T-helper cells that can sustain effective humoral immunity in a subject.


Further, it is an object of embodiments of the invention to provide peptides that may be used as antigens, to provide immunogenic compositions and methods for inducing an immune response in a subject against an antigen.


Further, it is an object of embodiments of the invention to provide peptides that may be used as antigens that can serve as targets in diagnostic assays.


SUMMARY OF THE INVENTION

The present invention pertains to a peptide design promoting efficient activation of a humoral immune response against antigens contained within this peptide design as well as to a peptide design promoting uptake of peptide epitopes by antigen presenting cells (macrophages and dendritic cells) such that the epitopes can be correctly processed and presented in the context of HLA class I and II to stimulate both CD4+ and CD8+ T-lymphocytes. CD8+ T-lymphocytes with cytotoxic capacity will kill infected cells bearing the epitope of interest. CD4+ T-lymphocyte provide ‘help’ to sustain effective CD8+ T-lymphocyte responses.


It has been found by the present inventor(s) that peptide constructs—amino acid sequences with a particular pattern or scaffold design, and in particular multimeric, such as dimeric peptides of this design—have the ability to effectively elicit a humoral immune response in a subject in response to the administration of these peptides.


The peptide constructs according to the present invention may be designed to be able to attach or bind to the cell surface. The peptide constructs or parts thereof may then be taken up by the antigen presenting cells (such as macrophages and dendritic cells) and stimulate helper T-cells in order to elicit efficient and long lasting T-cell dependent B-cell activation. Alternatively the B-cells themselves may provide for the induction of help to activate the B-cells.


Accordingly the peptides according to the present invention may penetrate the cells and may be used to load cells with an immunogenically effective amount of a peptide or fragments of this peptide that can be presented by macrophages and dendritic cells. Accordingly these peptide constructs may elicit both a Cytotoxic T-lymphocyte immune (CTL) response and/or a humoral immune response.


It has been found by the present inventor(s) that peptide constructs—amino acid sequences with a particular pattern or scaffold design—have the ability to effectively penetrate the cell membrane. Accordingly, the peptide constructs according to the present invention may be used to load cells with an immunogenically effective amount of a peptide or fragments of this peptide that can be presented by macrophages and dendritic cells. Accordingly these peptide constructs may elicit a Cytotoxic T-lymphocyte immune (CTL) response and/or a Humoral Immune Response.


So, in a first aspect the present invention relates to an isolated monomeric peptide comprising the following structure





(Z1-Z2)1-Z3-(Z4-Z5)2-Z6-(Z7-Z8)3-Z9-(Z10-Z11)4-Z12  (Formula I)


wherein Z1, Z4, and optional Z2 and Z10 defines a linear sequence of one, two, or three arginine residues or derivatives thereof optionally followed by a glycine (G) or an alanine (A); Z2, Z5, Z8 and Z11 defines an optional amino acid selected from cysteine (C), lysine (K), aspartic acid (D), asparagine (N), glutamic acid (E), glutamine (Q), 2,3-Diaminopropionic acid (Dpr), tryptophan (W), or tyrosine (Y) or a derivative thereof; Z3, and optional Z6, Z9 and Z12 defines any chemical moiety, such as a linear amino acid sequence.


It is to be understood that the amino acid sequence of formula I unless otherwise indicated refers to a peptide sequence in a standard N- to C-terminal direction, wherein the first amino acid mentioned is the N-terminal amino acid that may have an amino (—NH2) group or alternatively an —NH3+ group. The last amino acid mentioned is the C-terminal that may have a free carboxyl group (—COOH) or a carboxylate group. In some embodiments the N- and/or C-terminal amino acid is modified, such as by N-terminal acetylation or C-terminal amidation. The symbol “-” used in formula I refers to a standard peptide bond, such as a standard peptide bond between Z1 and Z2 in “Z1-Z2”.


It is further to be understood that the peptides according to the invention primarily are intended for synthetic peptide synthesis, which is preferred for peptides shorter than 60 amino acids. However, the peptides may be longer than 60 amino acids, if the peptides are produced by recombinant means.


In a further aspect the peptides of the present invention is not an isolated peptide consisting of X1-X5 of formula II as defined in any one of table 1 or table 2.


In a further aspect the peptides of the present invention is not an isolated peptide consisting of X1-X6 of formula III as defined in table 8.


In a further aspect the peptides of the present invention is not an isolated multimeric, such as dimeric peptide as defined in table 8.


In a further aspect the present invention relates to a dimer peptide comprising two peptide monomers, wherein each peptide monomer is according to the invention.


In a further aspect the present invention relates to a composition comprising two or more compounds selected from a monomeric peptide according to the present invention, and an isolated multimeric peptide according to the present invention.


In a further aspect the present invention relates to an isolated nucleic acid or polynucleotide encoding a peptide according to the invention.


In a further aspect the present invention relates to a vector comprising the nucleic acid or polynucleotide encoding a peptide according to the invention.


In a further aspect the present invention relates to a host cell comprising the vector comprising the nucleic acid or polynucleotide encoding a peptide according to the invention.


In a further aspect the present invention relates to an immunogenic composition comprising at least one monomeric peptide, an isolated multimeric peptide according to the invention, a peptide composition, the nucleic acid or polynucleotide, or the vector according the invention; in combination with a pharmaceutically acceptable diluent or vehicle and optionally an immunological adjuvant. In some embodiments this immunogenic composition is in the form of a vaccine composition.


In a further aspect the present invention relates to a method for inducing an immune response in a subject against an antigen which comprises administration of at least one monomeric peptide, an isolated multimeric peptide, a peptide composition, the nucleic acid or polynucleotide, or the vector, or the composition of the invention.


In a further aspect the present invention relates to a method for reducing and/or delaying the pathological effects of a disease antigen, such as an infectious agent in a subject infected with said agent or having said disease caused by said antigen, the method comprising administering an effective amount of at least one monomeric peptide, an isolated multimeric peptide, a peptide composition, the nucleic acid or polynucleotide, or the vector, or the composition according to the invention.


In a further aspect the present invention relates to a peptide according to the invention for use as a medicament.


In a further aspect the present invention relates to a peptide according to the invention for treating the pathological effects of a virus in a subject infected with said virus.


In a further aspect the present invention relates to the use of a peptide selected from a monomeric peptide according to the present invention, and an isolated multimeric peptide according to the present invention for inducing a humoral immune response in a subject.


In a further aspect the present invention relates to a peptide according to the invention for use as a medicament, or for treating the pathological effects of a disease antigen, such as an infectious agent in a subject infected with said agent or having said disease caused by said antigen.


In a further aspect the present invention relates to a peptide according to the invention for use in a diagnostic assay. In a further aspect the present invention relates to a peptide according to the invention for use in an in vitro assay.





LEGENDS TO THE FIGURES


FIG. 1. Intracellular uptake of influenza scaffold peptides. Median and intequartile range of readouts from buffy coats from ten donors and three concentrations of peptide each, normalized by value for N-biotin for each donor.



FIG. 2. Extracellular uptake of influenza scaffold peptides. Median and intequartile range of readouts from buffy coats from ten donors and three concentrations of peptide each, normalized by value for N-biotin for each donor.



FIG. 3. Intracellular uptake of HCV scaffold peptides. Median and intequartile range of readouts from buffy coats from five donors at four different concentrations of peptide each, normalized by value for N-biotin for each donor.



FIG. 4. Extracellular uptake of HCV scaffold peptides. Median and intequartile range of readouts from buffy coats from five donors at four different concentrations of peptide each, normalized by value for N-biotin for each donor.



FIG. 5. Median loss of weight by treatment group after challenge. The median weight by treatment groups; ISA5: peptides and ISA51, Provax: peptides and Provax, PR8: inactivated influenza A/PR8 (H1N1) virus, Naïve: no treatment before challenge. For animals lost to humane endpoints a last observation carried forward method was employed for the weights





DETAILED DISCLOSURE OF THE INVENTION
Definitions

When terms such as “one”, “a” or “an” are used in this disclosure they mean “at least one”, or “one or more” unless otherwise indicated. Further, the term “comprising” is intended to mean “including” and thus allows for the presence of other constituents, features, conditions, or steps than those explicitly recited.


As used herein a “multimeric peptide” or “oligomeric peptide” refers to an assembly of two or more different or identical linear peptide sequences or subunits, preferably interconnected or assembled by one or more chemical bond of a linker. Preferably the peptide sequences are interconnected by one or more, such as one covalent bond, such as an intermolecular disulfide (S—S) bond between two Cys residues, a methylated peptide bond between a N-ε-methylated Lys side-chain and the side-chain of an Asp or Glu residue, an oxime bond, a thioether bond, or a non-covalent bond, such as in a π-stacking of rings wherein a W residue in Z2 of the first Z1-Z2 peptide repeat is linked to an Y residue in Z2 of the second Z1-Z2 peptide repeat. The term includes a dimeric (or dimer) peptide suitably formed by a chemical linking of two linear peptide sequences. The term “multimeric peptide” further includes an assembly of 2, 3, 4, 5, 6, 7, 8, 9 or 10 different or identical peptide sequences. In some embodiments, the multimeric peptide is a dimeric peptide.


As used herein a “linker” refers to any compound suitable for assembly of the two or more different or identical linear peptide sequences or subunits into a multimeric peptide, or to any other therapeutically active compound, such as an immunomodulating compound. The term includes any linker found useful in peptide chemistry. Since the multimeric peptide may be assembled or connected by standard peptide bonds in a linear way, the term linker also includes a “peptide spacer”, also referred to as a “spacer”.


In some embodiments, the linker is not a peptide sequence. In some embodiments, the linker is not a branched peptide sequence.


In some embodiments, the linker does not itself contain a peptide sequence derived from or identical to a natural antigen.


In some embodiments, the linker has a molecular weight of less than 10 kDa, such as less than 9 kDa, such as less than 8 kDa, such as less than 7 kDa, such as less than 6 kDa, such as less than 5 kDa, such as less than 4 kDa, such as less than 3 kDa, such as less than 2 kDa, such as less than 1.5 kDa, such as less than 1 kDa, such as less than 0.5 kDa, such as less than 0.2 kDa. In some embodiments, wherein the multimeric peptide is a dimeric peptide, the linker is not linking the two peptide sequences from one terminal cysteine in the first peptide to a second terminal cysteine in the second peptide.


In some embodiments, the linker is not linking the two or more peptide sequences through a terminal cysteine in any one of the peptides.


In some embodiments, the linker is not linking from a cysteine residue.


“HIV” generally denotes human immunodeficiency virus I.


“HIV disease” is composed of several stages including the acute HIV infection which often manifests itself as an influenza-like infection and the early and medium stage symptomatic disease, which has several non-characteristic symptoms such as skin rashes, fatigue, night sweats, slight weight loss, mouth ulcers, and fungal skin and nail infections. Most HIV infected will experience mild symptoms such as these before developing more serious illnesses. It is generally believed that it takes five to seven years for the first mild symptoms to appear. As HIV disease progresses, some individuals may become quite ill even if they have not yet been diagnosed with AIDS (see below), the late stage of HIV disease. Typical problems include chronic oral or vaginal thrush (a fungal rash or spots), recurrent herpes blisters on the mouth (cold sores) or genitals, ongoing fevers, persistent diarrhea, and significant weight loss. “AIDS” is the late stage HIV disease and is a condition which progressively reduces the effectiveness of the immune system and leaves individuals susceptible to opportunistic infections and tumors.


The term “cell-penetrating peptide” as used herein refers to any peptide with the capability to translocate across the plasma membrane into either cytoplasmic and/or nuclear compartments of eukaryotic and/or prokaryotic cells, such as into cytoplasm, nucleus, lysosome, endoplasmatic reticulum, golgi apparatus, mitocondria and/or chloroplast, seemingly energy-independently. This capability to translocate across the plasma membrane of a “cell-penetrating peptide” according to the invention may be non-invasive, energy-independent, non-saturable, and/or receptor independent. In one embodiment the term “cell-penetrating peptide” refers to a peptide, which is demonstrated to translocate across a plasma membrane as determined by the assay in example 5. It is to be understood that a cell-penetrating peptide according to the present invention may be translocated across the membrane with the sequence complete and intact, or alternatively partly degraded, but in a form where the antigens contained within this peptide is able to be presented within the cell to stimulate an immune response. Accordingly, a cell-penetrating peptide according to the present invention is a peptide that may be demonstrated to translocate across a plasma membrane as determined by the assay in example 5 and be demonstrated to stimulate an effective immune response.


The monomeric peptide according to the present invention may be provided in any pharmaceutically acceptable salt, such as in a salt of acetat or HCl.


The term “derived from an antigen” when in reference to a peptide derived from a source (such as a virus etc.) as used herein is intended to refer to a peptide which has been obtained (e.g., isolated, purified, etc.) from the source. Preferably, the peptide may be genetically engineered and/or chemically synthesized to be essentially identical to the native peptide of the source. The term includes the use of variants of known native peptide sequences, such as peptide sequences, where 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of the native peptide sequence have been substituted with any other amino acid, such as conservative substitutions. Alternatively, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids have been removed or added to the native peptide sequence. Accordingly, in some embodiments, the peptides according to the present invention comprises an amino acid sequence Z3, and optional Z6, Z9 and Z12, that is defined as a sequence of 8-30 amino acids, such as 8-20 amino acids derived from an antigen, wherein the peptide sequence of the antigen comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, additions or deletions relative to the antigen, such as the addition of an arginine in the N- or C-terminal of the amino acid sequence of Z3, and optional Z6, Z9 and Z12. In some embodiments, the peptides according to the present invention comprises an amino acid sequence Z3, and optional Z6, Z9 and Z12, that is defined as a sequence of 8-30 amino acids, such as 8-20 amino acids identical in sequence to a native antigen. In some embodiments, the peptides according to the present invention comprises an amino acid sequence Z3, and optional Z6, Z9 and Z12, that is defined as a sequence of 8-30 amino acids, such as 8-20 amino acids that is not identical in sequence to a native antigen.


It is to be understood that “derived from an antigen” does not exclude that an amino acid sequence defined by Z3, and optional Z6, Z9 and Z12 may be derived from more than one antigenic peptide sequence, such as from two or three different proteins or peptide sources or different sequences within the same proteins or peptide of the same virus, any different virus, or any disease antigen. However, in one embodiment Z3, and optional Z6, Z9 and Z12 are derived from one specific continuous peptide sequence. In one embodiment Z3, and optional Z6, Z9 and Z12 are derived from two different specific continuous peptide sequences of the same or different protein derived from the same virus, any different virus, or any disease antigen.


The amino acids used in the amino acid sequences according to the invention may be in both L- and/or D-form. It is to be understood that both L- and D-forms may be used for different amino acids within the same peptide sequence. In some embodiments the amino acids within the peptide sequence are in L-form, such as natural amino acids. It is to be understood that any known antigen may be used in the constructs according to the present invention.


In some specific embodiments, the first 1, 2, or 3 amino acids in the N-terminal of the amino acid sequences according to the invention are in the D-form. It is assumed that the N-terminal trimming and thereby degradation of the peptides are somewhat delayed by having amino acids of the D-form in the N-terminal of these peptides according to the present invention. Alternatively and in some embodiments, the first 1, 2, or 3 amino acids in the N-terminal of the amino acid sequences according to the invention are amino acids in beta or gamma forms. Beta amino acids have their amino group bonded to the beta carbon rather than the alpha carbon as in the 20 standard natural amino acids.


Alternatively the first 1, 2, or 3 amino acids in the N-terminal of the amino acid sequences according to the invention may be modified by incorporation of fluorine, or alternatively cyclic amino acids or other suitable non-natural amino acids are used.


A “variant” or “analogue” of a peptide refers to a peptide having an amino acid sequence that is substantially identical to a reference peptide, typically a native or “parent” polypeptide. The peptide variant may possess one or more amino acid substitutions, deletions, and/or insertions at certain positions within the native amino acid sequence.


“Conservative” amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a side chain with similar physicochemical properties. Families of amino acid residues having similar side chains are known in the art, and include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). A particular form of conservative amino acid substitutions include those with amino acids, which are not among the normal 20 amino acids encoded by the genetic code. Since preferred embodiments of the present invention entail use of synthetic peptides, it is unproblematic to provide such “non-naturally occurring” amino acid residues in the peptides disclosed herein, and thereby it is possible to exchange the natural saturated carbon chains in the side chains of amino acid residues with shorter or longer saturated carbon chains—for instance, lysine may be substituted with an amino acid having an the side chain—(CH2)nNH3, where n is different from 4, and arginine may be substituted with an amino acid having the side chain —(CH2)nNHC(═NH2)NH2, where n is different from 3, etc. Similarly, the acidic amino acids aspartic acid and glutamic acid may be substituted with amino acid residues having the side chains —(CH2)nCOOH, where n>2.


The term “substantially identical” in the context of two amino acid sequences means that the sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 95, at least about 98, or at least about 99 percent sequence identity. In one embodiment, residue positions that are not identical differ by conservative amino acid substitutions. Sequence identity is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, the publicly available GCG software contains programs such as “Gap” and “BestFit” which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild-type protein and a mutein thereof. See, e.g., GCG Version 6.1. Polypeptide sequences can also be compared using FASTA or ClustalW, applying default or recommended parameters. A program in GCG Version 6.1., FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson, Methods Enzymol. 1990; 183:63-98; Pearson, Methods Mol. Biol. 2000; 132:185-219). Another preferred algorithm when comparing a sequence to a database containing a large number of sequences from various organisms, or when deducing the is the computer program BLAST, especially blastp, using default parameters. See, e.g., Altschul et al., J. Mol. Biol. 1990; 215:403-410; Altschul et al., Nucleic Acids Res. 1997; 25:3389-402 (1997); each herein incorporated by reference. “Corresponding” amino acid positions in two substantially identical amino acid sequences are those aligned by any of the protein analysis software mentioned herein, typically using default parameters.


An “isolated” molecule is a molecule that is the predominant species in the composition wherein it is found with respect to the class of molecules to which it belongs (i.e., it makes up at least about 50% of the type of molecule in the composition and typically will make up at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more of the species of molecule, e.g., peptide, in the composition). Commonly, a composition of a peptide molecule will exhibit 98%-99% homogeneity for peptide molecules in the context of all present peptide species in the composition or at least with respect to substantially active peptide species in the context of proposed use.


The term “linear sequence” as used herein refers to the specific sequence of amino acids connected by standard peptide bonds in standard N- to C-terminal direction. The peptide may contain only peptide bonds. However the term does not exclude that an amino acid within a sequence, such as within Z3, may be connected, such as through the side chains, with another amino acid at a distant location within the peptide sequence, such as a distant location within Z3.


In the context of the present invention, “treatment” or “treating” refers to preventing, alleviating, managing, curing or reducing one or more symptoms or clinically relevant manifestations of a disease or disorder, unless contradicted by context. For example, “treatment” of a patient in whom no symptoms or clinically relevant manifestations of a disease or disorder have been identified is preventive or prophylactic therapy, whereas “treatment” of a patient in whom symptoms or clinically relevant manifestations of a disease or disorder have been identified generally does not constitute preventive or prophylactic therapy.


The term “antigen” denotes a substance of matter which is recognized by the immune system's specifically recognizing components (antibodies, T-cells).


The term “immunogen” is in the present context intended to denote a substance of matter, which is capable of inducing an adaptive immune response in an individual, where said adaptive immune response targets the immunogen. In relation to the present invention, an immunogen will induce a humoral and/or cell-mediated immune response. In other words, an immunogen is an antigen, which is capable of inducing immunity.


The terms “epitope”, “antigenic determinant” and “antigenic site” are used interchangeably herein and denotes the region in an antigen or immunogen which is recognized by antibodies (in the case of antibody binding epitopes, also known as “B-cell epitopes”) or by T-cell receptors when the epitope is complexed to a Major histocompatibility complex (MHC) molecule (in the case of T-cell receptor binding epitopes, i.e. “T-cell epitopes”).


“B cell antigen” means any antigen that naturally is or could be engineered to be recognized by a B cell, and that triggers an immune response in a B cell (e.g., an antigen that is specifically recognized by a B cell receptor on a B cell).


The term “immunogenically effective amount” has its usual meaning in the art, i.e. an amount of an immunogen, which is capable of inducing an immune response, which significantly engages pathogenic agents, which share immunological features with the immunogen.


The term “vaccine” is used for a composition comprising an immunogen and which is capable of inducing an immune response which is either capable of reducing the risk of developing a pathological condition or capable of inducing a therapeutically effective immune response which may aid in the cure of (or at least alleviate the symptoms of) a pathological condition.


The term “pharmaceutically acceptable” has its usual meaning in the art, i.e. it is used for a substance that can be accepted as part of a medicament for human use when treating the disease in question and thus the term effectively excludes the use of highly toxic substances that would worsen rather than improve the treated subject's condition.


A “T helper lymphocyte epitope” (a TH epitope) is peptide, which binds an MHC Class II molecule and can be presented on the surface of an antigen presenting cell (APC) bound to the MHC Class II molecule. An “immunological carrier” is generally a substance of matter which includes one or many TH epitopes, and which increase the immune response against an antigen to which it is coupled by ensuring that T-helper lymphocytes are activated and proliferate. Examples of known immunological carriers are the tetanus and diphtheria toxoids and keyhole limpet hemocyanin (KLH).


In the scaffold design according to the present invention, Z3, and optional Z6, Z9 and Z12 may define a sequence of amino acids, such as 8-30 amino acids, such as 8-20 amino acids derived from the antigen. This sequence of amino acids derived from an antigen may herein be referred to as an epitope.


The peptides according to the present invention may be a helper T lymphocyte (HTL) inducing peptide comprising HTL epitopes. A “HTL inducing peptide” is a HLA Class II binding peptide that is capable of inducing a HTL response. Also the peptides according to the present invention may in other embodiments be CTL inducing peptides comprising CTL epitopes in addition to or as an alternative to being a HTL inducing peptide. A “CTL inducing peptide” is a HLA Class I binding peptide that is capable of inducing a CTL response.


In some embodiments the epitopes used in the scaffold according to the present invention are CTL epitopes. A “CTL inducing peptide” is a HLA Class I binding peptide that is capable of inducing a CTL response. In other embodiments the epitopes used in the scaffold design according to the present invention are HTL inducing peptides. A “HTL inducing peptide” is a HLA Class II binding peptide that is capable of inducing a HTL response.


In other alternative embodiments, tryptophan or tryptophan derivatives are used in the sequence defined by Z2, Z5, Z8 and Z11. Any suitable tryptophan derivatives may be used. As used herein “tryptophan derivatives” means an unnatural modified tryptophan amino acid residue including those disclosed in U.S. Pat. No. 7,232,803, such as tri tert.-butyltryptophan, di-tert-butyl tryptophan, 7-benzyloxytryptophan, homotryptophan, 5′-aminoethyltryptophan (available as side chain Boc and N-alpha FMOC derivative from RSP Amino Acids Analogues Inc, Boston, Mass., USA), N-Acetylhomotryptophan (Toronto Research), 7-Benzyloxytryptophan (Toronto Research), Homotryptophan (Toronto Research), and tryptophan residues which have been substituted at the 1-, 2-, 5- and/or 7-position of the indole ring, positions 1- or 2- being preferred e.g. 5′ hydroxy tryptophan.


The term “amino acid derivative”, sometimes used in the context of a “derivative thereof” referring to a specific amino acid, means an amino acid compound, wherein one or more chemical groups has been modified, added or removed as compared to the amino acid to which the amino acid compound is a derivative of, while still having an amine group and a carboxylic acid group, as well as a side chain of an amino acid and still being able to form peptide bonds. In some embodiments an amino acid derivative is a standard amino acid that has only been modified in the side chain of the amino acid. In some embodiments an amino acid derivative is a non-natural amino acid such as Dpr. In some embodiments an amino acid is a modified moiety which is incorporated into the chemically synthesized peptide or polypeptide and that comprises an activatable group that is linkable, after activation, to another peptide, such as Dpr(Ser), Lys(Ser), or Ornithine(Ser).


The term “basic amino acid” as used herein refers to any amino acid including both natural and non-natural amino acids that has an isoelectric point above 6.3 (such as above 7.4) as measured according to Kice & Marvell “Modern Principles of organic Chemistry” (Macmillan, 1974) or Matthews and van Holde “Biochemistry” Cummings Publishing Company, 1996. Included within this definition are Arginine, Lysine, Homoarginine (Har), and Histidine as well as derivatives thereof. Suitable non-natural basic amino acids are e.g. as described in U.S. Pat. No. 6,858,396. Suitable positively charged amino acids includes non-natural alpha amino acids available from Bachem AG and includes alpha-amino-glycine, alpha,gamma-diaminobutyric acid, ornithine, alpha, beta-diaminoproprionic acid, alpha-difluoromethyl-ornithine, 4-amino-piperidine-4-carboxylic acid, 2,6-diamino-4-hexynoic acid, beta-(1-piperazinyl)-alanine, 4,5-dehydro-lysine, delta-hydroxy-lysine, omega-hydroxy-norarginine, homoarginine, omega-amino-arginine, omega-methyl-arginine, alpha-methyl-histidine, 2,5-diiodo-histidine, 1-methyl-histidine, 3-methyl-histidine, beta-(2-pyridyl)-alanine, beta-(3-pyridyl)-alanine, beta-(2-quinolyl)-alanine, 3-amino-tyrosine, 4-amino-phenylalanine, and spinacine. Furthermore, any mono or dicarboxylic amino acid is a suitable positively charged amino acid.


The term “neutral amino acid” as used herein refers to an amino acid that has an isoelectric point above between 4.8 and 6.3 as measured according to Kice & Marvell “Modern Principles of organic Chemistry” (Macmillan, 1974). The term “acidic amino acid” as used herein refers to an amino acid that has an isoelectric point below 4.8 as measured according to Kice & Marvell “Modern Principles of organic Chemistry” (Macmillan, 1974).


Unless otherwise indicated amino acids are abbreviated and mentioned by their standard nomenclature known to the person skilled in the art, such as with reference to “nomenclature and symbolism for amino acids and peptides” by the international union of pure and applied chemistry (IUPAC) (www.iupac.org).


The term “antibody response” refers to the production of antibodies (e.g., IgM, IgA, IgG) which bind to an antigen of interest, this response is measured for instance by assaying sera by antigen ELISA.


The term “adjuvant” as used herein refers to any compound which, when delivered together or simultaneously with an antigen, non-specifically enhances the immune response to that antigen. Exemplary adjuvants include but are not limited to oil in water and water in oil adjuvants, aluminum-based adjuvants (e.g., AIOH, AIPO4, etc), and Montanide ISA 720.


The terms “patient” and “subject” refer to a mammal that may be treated using the methods of the present invention.


As used herein, the term “immune response” refers to the reactivity of an organism's immune system in response to an antigen. In vertebrates, this may involve antibody production, induction of cell-mediated immunity, and/or complement activation (e.g., phenomena associated with the vertebrate immune system's prevention and resolution of infection by microorganisms). In preferred embodiments, the term immune response encompasses but is not limited to one or more of a “lymphocyte proliferative response,” a “cytokine response,” and an “antibody response.”


The term “net charge” as used herein with reference to a peptide sequence refers to the total electric charge of the peptide sequence represented by the sum of charges of each individual amino acid in the peptide sequence, wherein each basic amino acid are given a charge of +1, each acidic amino acid a charge of −1, and each neutral amino acid a charge of 0. Accordingly, the net charge will depend on the number and identities of charged amino acids.


Table 1—Specific peptides not part of the present invention


Table 1 and 2 represent peptides not part of the present invention comprising the structure





X1-X2-X3-X4-X5  (formula II),


wherein X′ and X3 independently defines a linear sequence of any 1, 2, 3 or 4 amino acid independently selected from any basic amino acid, citrulline, tryptophan, or a derivative thereof; X2 defines a linear sequence of 8-30 amino acids derived from an antigen; X4 defines a linear sequence of 8-30 amino acids derived from said antigen, said sequence X4 being different from X2; and wherein X5 is any one optional amino acid selected from a basic amino acid, citrulline, tryptophan, or a derivative thereof. Citrulline is in this document referred to with the one-letter symbol “B”.

















TABLE 1















Placement










with










reference to










positions in










SEQ ID NO: 3;










SEQ ID NO: 6;










SEQ ID NO: 7,










SEQ ID










NO: 11, SEQ










ID NO: 12,










SEQ ID










NO: 46, and










SEQ ID










NO: 126.

















Reference





Modified
X2-
x4-


Antigen
ID
x1
x2
x3
x4
x5
(m)
seq
seq






P-

R

QIKIWFQN

RR

MKWKK







biotin














N-

PVVHLTL

R

QAGDDFSR







Biotin













HCV
SP_2

RR

GYIPLVGAPLG

BGR

VARALAHGVRV


135-
147-










145
157





HCV
SP_3

R

GYIPLVGAPLG

RR

VARALAHGVRV


135-
147-










145
157





HCV
SP_4

R

GYIPLVGAPLG

RRR

VARALAHGVRV

R


135-
147-










145
157





HCV
SP_5

RR

GYIPLVGAPLG

RR

VARALAHGVRV


135-
147-










145
157





HCV
SP_6

RR

GYIPLVGAPLG

RRR

VARALAHGVRV


135-
147-










145
157





HCV
SP_7

BR

GYIPLVGAPLG

RR

VARALAHGVRV


135-
147-










145
157





HCV
SP_8

RRR

GYIPLVGAPLG

BR

VARALAHGVRV


135-
147-










145
157





HCV
SP_9

R

GYIPLVGAPLG

KKK

VARALAHGVRV


135-
147-










145
157





HCV
SP_10

R

GYIPLVGAPLG

RRR

VARALAHGVRV


135-
147-










145
157





HCV
SP_11

KK

GYIPLVGAPLG

KK

VARALAHGVRV


135-
147-










145
157





HCV
SP_12
W
GYIPLVGAPLG

RR

VARALAHGVRV


135-
147-










145
157





HCV
SP_13
WW
GYIPLVGAPLG

RR

VARALAHGVRV


135-
147-










145
157





HCV
SP_14

EE

GYIPLVGAPLG

EE

VARALAHGVRV


135-
147-










145
157





HCV
SP_15
GG
GYIPLVGAPLG
GG
VARALAHGVRV


135-
147-










145
157





HCV
SP_16

EE

GYIPLVGAPLG

RR

VARALAHGVRV


135-
147-










145
157





HCV
SP_17

RR

GYIPLVGAPLG
LRR
VARALAHGVRV


135-
147-










145
157





HCV
SP21:
WW
GYIPLVGAPLG

RR

VARALAHGVRV


135-
147-










145
157





HCV
SP22:
WW
GYIPLVGAPLG

RRR

VARALAHGVRV


135-
147-










145
157





HCV
SP23:
WW
GYIPLVGAPLG

R

VARALAHGVRV


135-
147-










145
157





HCV
SP24:

R

GYIPLVGAPLG

RR

VARALAHGVRV


135-
147-










145
157





HCV
51_
RR
GYLPAVGAPIG
BR
VIRVIAHGLRL

m
135-
147-



BIotin






144
157





HCV
51b_
RR
GYIPLVGAPLG
BR
VARALAHGVRV


135-
147-



BIotin






145
157





HCV
51_n

GYIPLVGAPLG

G

VARALAHGVRV


135-
147-










145
157





HCV
SP51_1:
WW
GYLPAVGAPI

RR

VIRVIAHGLRL

m
135-
147-










144
157





HCV
SP1_C*

GYIPLVGAPLG

G

VARALAHGVRV


135-
147-










145
157





HCV
SP2_c

RR

GYIPLVGAPLG

BGR

VARALAHGVRV


135-
147-










145
157





HCV
SP3_c

R

GYIPLVGAPLG

RR

VARALAHGVRV


135-
147-










145
157





HCV
SP4_c

R

GYIPLVGAPLG

RRR

VARALAHGVRV


135-
147-










145
157





HCV
SP5_c

RR

GYIPLVGAPLG

RR

VARALAHGVRV


135-
147-










145
157





HCV
SP6_c

RR

GYIPLVGAPLG

RRR

VARALAHGVRV


135-
147-










145
157





HCV
SP7_c

BR

GYIPLVGAPLG

RR

VARALAHGVRV


135-
147-










145
157





HCV
SP8_c

RRR

GYIPLVGAPLG

BR

VARALAHGVRV


135-
147-










145
157





HCV
SP9_c

R

GYIPLVGAPLG

KKK

VARALAHGVRV


135-
147-










145
157





HCV
SP10_c

R

GYIPLVGAPLG

RRR

VARALAHGVRV


135-
147-










145
157





HCV
SP11_c

KK

GYIPLVGAPLG

KK

VARALAHGVRV


135-
147-










145
157





HCV
SP12_c
W
GYIPLVGAPLG

RR

VARALAHGVRV


135-
147-










145
157





HCV
SP13_c
WW
GYIPLVGAPLG

RR

VARALAHGVRV


135-
147-










145
157





HCV
SP17_c

RR

GYIPLVGAPLG
LRR
VARALAHGVRV


135-
147-










145
157





HCV
SP61_2_
RR
NYVTGNIPG
BR
GITFSIFLIVS


163-
171-










171
181





HCV
SP61b_
WW
NYATGNLPG
RR
CSFSIFLLAL

m
163-
171-



2_






171
181





HCV
SP61_3_
WW
NYVTGNIPG
BR
GITFSIFLIVS


163-
171-










171
181





HCV
SP61_4_
WW
NYVTGNIPG
RR
GITFSIFLIVS


163-
171-










171
181





HCV
61b_
RR
NYATGNLPG
RR
GCSFSIFLLAL


163-
171-



BIotin






171
181





HCV
SP25
RR
VTGNIPGSTYS
GBR
GITFSIYLIVS

m
165-
171-










175
181





HCV
42_
RR
IRNLGRVIETLTG
BR
LNleGYIPLIGA

m
116-
133-



BIotin






128
142





HCV
42b_
RR
SRNLGKVIDTLT
BR
LMGYIPLVGA


116-
133-



BIotin

C




128
142





HCV
42n-

SRNLGKVIDTLT
GFAD
LMGYIPLVGA


116-
133-



BIOTIN

C




129
142





HCV
SP42_1_
WW
IRNLGRVIETLT
RR
LNleGYIPLIGA

m
116-
133-










128
142





HCV
SP42b_
WW
SRNLGKVIDTLT
RR
LMGYIPLVGA


116-
133-



1_

C




129
142





HCV
BI310-

RR

GGGQIIGGNYLI

custom-character

PBIGVRATB


 26-
 42-



11_

P




 38
 50



Biotin













HCV
BI310-

GGGQIVGGVYLL

custom-character

GPRLGVRATR


 26-
 42-



11n_

P




 38
 50



Biotin













HCV
BI310-

RR

GGGQIVGGVYLL

custom-character

GPRLGVRATR


 26-
 42-



11n_sc

P




 38
 50



_Biotin













HCV
SP11b-
WW
GGGQIVGGVYLL

RR

GPRLGVRAT


 26-
 42-



1-

P




 38
 50





FLU
BI100-
BR
LIFLARSALIV

RGSVAHKS


256-
267-



12






266
274





FLU
BI100-
ED
LIFLARSALIL

RGSVAHKS


255-
267-



22b






266
274





FLU
120b_
BR
LIFLARSALIL
BGR
SALILRGSVAHK


255-
267-



BIotin






266
274





FLU
BI100-

SAYERMCNIL
KGK
FQTAAQRAMM


217-
230-



18b






226
239





FLU
BI100-

SAYERNleVNIL
KGK
FQTAAQRAVNle


217-
230-



19






226
239





FLU
190_
BR
TAYERNleCNIL
BRGR
FQTVVQBA


217-
230-



BIotin






226
237





FLU
190b_
BR
IAYERMCNIL
LBRGK
FQTAAQRA


217-
230-



BIotin






226
237





FLU
190n-

IAYERMCNIL
KGK
FQTAAQRA


217-
230-



BIOTIN






226
237





FLU
BI100-

LFFKCIYRLFKHG
KR
GPSTEGVPESM


 46-
 62-



24b

L




 59
 72





FLU
BI100-
BRR
LFFKTITRLFBHG
RR
LLSTEGVPNSNle


 46-
 62-



26

L




 59
 72





FLU
260_
BR
GLEPLVIAGILA
RR
GSLVGLLHIVL


 23-
 30-



Biotin






 33
 40





FLU
260b_
BR
GSDPLVVAASIV
RR
ASIVGILHLIL


 23-
 30-



Biotin






 33
 40





CMV
BI 050-

R

NLVPMVATV

RR

NLVPMVATV

B


485-
485-



sc1






493
493





CMV
BI 050-

R

NLVPMVATV

BRR

NLVPMVATV

B


485-
485-



sc2






493
493





CMV
BI 050-

R

NIVPNleVVTA

RR

NIVPNleVVTA

B

m
485-
485-



sc5






493
493





HIV
N10

PEVIPMFSALS

EGA

TPQDLNTMLN









HIV
V10

R

FIIPXFTALSG
GRR
ALLYGATPYAIG









HIV
N13

K

ALGPAATL

EE

MMTACQGVG









Neg
SP_18

RR

GPVVHLTL

RR
custom-character

GQAGDDFS






c











mod














Neg
SP_19

RR

GPVVHLTL

RRR

GQAGDDFS






c











mod














Neg
SP_20

RR

GPVVHLTL

RGRR

GQAGDDFS






c











mod














HPV

RR
LECVYCKQQLL
RR
EVYDFAFRDLC


 35-
 48-










 45
 58





HPV

RR

GVYDFAFRDLC


RR


GFAFRDLCIVY


R


 49-
 52-










 58
 61





HPV

RR

GVFDYAFRDIN


RR


GFAYRDINLAY


R


 49-
 52-










 58
 61





CMV


RR


GATPVDLLGA


RR

GALNLCLPM

R


498-
505-










506
514





CMV


RR


GVTPAGLIGV


RR

GALQIBLPL

R


498-
505-










506
514





HPV


RR

VDIRTLEDLL

RR

GTLGIVCPIG

R


 74-
 84-










 83
 93









As used herein the one-letter-code ‘Nle’ refers to the non-natural amino acid norleucine.









TABLE 2







Specific peptides not part of the present invention












Antigen
X1
X2
X3
X4
X5





HCV
R
GYIPLVGAPLG
RRR
VARALAHGVRV
R





HCV
R
GYLPAVGAPIG
RRR
VIRVIAHGLRL
R





HCV
RR
GYIPLVGAPLG
RR
VARALAHGVRV






HCV
RR
GYIPLVGAPLG
RRR
VARALAHGVRV






HCV
RR
SRNLGKVIDTLTC
RR
LMGYIPLVGA






HCV
RR
GGGQIVGGVYLLP
RR
GPRLGVRATR






HCV
W
GYIPLVGAPLG
RR
VARALAHGVRV






HCV
RR
IRNLGRVIETLTLNleGYIPLIGA
RR
IRNLGRVIETLTLNleGYIPLIGA
R





Flu
BR
TAYERNleCNIL
BRGR
FQTVVQBA






cmv
R
NLVPMVATV
BRR
NLVPMVATV
B









As used herein the one-letter-code Z or ‘Nle’ refers to the non-natural amino acid norleucine.


Antigens

The specific natural antigen used in the peptide constructs according to the present invention may be a protein or peptide sequence derived from any B cell antigen, such as from any disease antigen, such as an infectious agent. Suitable antigens to be used according to the present invention include antigens derived from a bacteria, a mycobacterium, a virus, a parasite such as protozoa, a fungus, a cancer antigen, such as an oncogene, such as a thelomerase, a prion, an atopic disease antigen, an addictive or abused substance or a toxin or an antigen of an autoimmune disease, such as rheumatoid arthritis, insulin dependent diabetes, multiple sclerosis and the like.


As used herein a “disease antigen” refers to any antigen confirmed or suspected to be involved in a specific disease.


In some embodiments, the antigen is an abused or addictive substance or a portion thereof, including, but are not limited to, nicotine, a narcotic, a cough suppressant, a tranquilizer, and a sedative. In some embodiments, the antigen is a toxin, such as a toxin from a chemical weapon or natural sources, or a pollutant.


Examples of bacteria for which antigens may be provided include, but are not limited to, M. tuberculosis, Mycobacterium, mycoplasma, neisseria and legionella. Examples of parasites include, but are not limited to, rickettsia and chlamydia.


Examples of an infectious disease antigen is TbH9 (also known as Mtb 39A), a tuberculosis antigen. Other tuberculosis antigens include, but are not limited to DPV (also known as Mtb8.4), 381, Mtb41, Mtb40, Mtb32A, MΛ9.9A, Mtb9.8, Mtbló, Mtb72f, Mtb59f, Mtb88f, Mtb71f, Mtb46f and Mtb31f (“f’ indicates that it is a fusion or two or more proteins).


Examples of cancer antigens may be a tumor associated antigen such as HER2, HER3 or HER4 receptor or one or more tumor-associated antigens or cell-surface receptors disclosed in US Publication No. 20080171040 or US Publication No. 20080305044 and are incorporated in their entirety by reference.


Other suitable cancer antigens that may be used by the present invention include CD proteins such as CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD14, CD18, CD19, CD22, CD21, CD22, CD25, CD26, CD27, CD28, CD30, CD33, CD36, CD37, CD38, CD40, CD44, CD52, CD55, CD56, CD70, CD79, CD80, CD81, CD103, CD105, CD134, CD137, CD138, and CD152; members of the ErbB receptor family such as the EGF receptor, HER2, HER3 or HER4 receptor; cell adhesion molecules such as LFA-I, Mac1, pi 50.95, VLA-4, ICAM-1, VCAM, EpCAM, alpha4/beta7 integrin, and alpha v/beta3 integrin including either alpha or beta subunits thereof (e.g. anti-CD11a, anti-CD18 or anti-CD11b antibodies); growth factors such as VEGF; tissue factor (TF); TGF-β.; alpha interferon (alpha-IFN); an interleukin, such as IL-8; IgE; blood group antigens Apo2, death receptor; flk2/flt3 receptor; obesity (OB) receptor; mpl receptor; CTLA-4; protein C etc. In some embodiment the antigen is selected from IGF-IR, CanAg, EphA2, MUC1, MUC16, VEGF, TF, CD19, CD20, CD22, CD27, CD33, CD37, CD38, CD40, CD44, CD56, CD138, CA6, Her2/neu, EpCAM, CRIPTO (a protein produced at elevated levels in a majority of human breast cancer cells), darpins, alphav/beta3 integrin, alphav/beta5 integrin, alpha y/beta integrin, TGF-β, CD11a, CD18, Apo2 and C242. In some embodiment the antigen is selected from a CD proteins such as CD3, CD4, CD8, CD19, CD20, CD27, CD34, CD37, CD38, CD46, CD56, CD70 and CD138; members of the ErbB receptor family such as the EGF receptor, HER2, HER3 or HER4 receptor; cell adhesion molecules such as LFA-I, Mac1, p150.95, VLA-4, ICAM-1, VCAM, EpCAM, alpha4/beta7 integrin, and alpha v/beta3 integrin including either alpha or beta subunits thereof (e.g. anti-CD11a, anti-CD18 or anti-CD11b antibodies); growth factors such as VEGF; tissue factor (TF); TGF-β.; alpha interferon (alpha-IFN); an interleukin, such as IL-8; IgE; blood group antigens Apo2, death receptor; flk2/flt3 receptor; obesity (OB) receptor; mpl receptor; CTLA-4; protein C, etc. The most preferred targets herein are IGF-IR, CanAg, EGF-R, EGF-RvIII, EphA2, MUC1, MUC16, VEGF, TF, CD19, CD20, CD22, CD27, CD33, CD37, CD38, CD40, CD44, CD56, CD70, CD138, CA6, Her2/neu, CRIPTO (a protein produced at elevated levels in a majority of human breast cancer cells), alphav/beta3 integrin, alphav/beta5 integrin, TGF-β, CD11a, CD18, Apo2, EpCAM and C242. In some embodiment the antigen is selected from a cellular oncogene, such as ras or myc.


Examples of viral antigens for use with the present invention include, but are not limited to, e.g., HIV, HCV, CMV, HPV, Influenza, adenoviruses, retroviruses, picornaviruses, etc. Non-limiting example of retroviral antigens such as retroviral antigens from the human immunodeficiency virus (HIV) antigens such as gene products of the gag, pol, and env genes, the Nef protein, reverse transcriptase, and other HIV components; hepatitis viral antigens such as the S, M, and L proteins of hepatitis B virus, the pre-S antigen of hepatitis B virus, and other hepatitis, e.g., hepatitis A, B, and C, viral components such as hepatitis C viral RNA; influenza viral antigens such as hemagglutinin and neuraminidase and other influenza viral components; measles viral antigens such as the measles virus fusion protein and other measles virus components; rubella viral antigens such as proteins E1 and E2 and other rubella virus components; rotaviral antigens such as VP7sc and other rotaviral components; cytomegaloviral antigens such as envelope glycoprotein B and other cytomegaloviral antigen components; respiratory syncytial viral antigens such as the RSV fusion protein, the M2 protein and other respiratory syncytial viral antigen components; herpes simplex viral antigens such as immediate early proteins, glycoprotein D, and other herpes simplex viral antigen components; varicella zoster viral antigens such as gpl, gpll, and other varicella zoster viral antigen components; Japanese encephalitis viral antigens such as proteins E, M-E, M-E-NSI, NSI, NS1-NS2A, 80% E, and other Japanese encephalitis viral antigen components; rabies viral antigens such as rabies glycoprotein, rabies nucleoprotein and other rabies viral antigen components. See Fundamental Virology, Second Edition, eds. Fields, B. N. and Knipe, D. M. (Raven Press, New York, 1991) for additional examples of viral antigens.


The epitopes to be incorporated into the scaffold design according to the present invention may be derived from an adenovirus, retrovirus, picornavirus, herpesvirus, rotavirus, hantavirus, coronavirus, togavirus, flavirvirus, rhabdovirus, paramyxovirus, orthomyxovirus, bunyavirus, arenavirus, reovirus, papilomavirus, parvovirus, poxvirus, hepadnavirus, degngue virus, or spongiform virus. In certain specific, non-limiting examples, the viral antigen are peptides obtained from at least one of HIV, CMV, hepatitis A, B, and C, influenza, measles, polio, smallpox, rubella; respiratory syncytial, herpes simplex, varicella zoster, Epstein-Barr, Japanese encephalitis, rabies, Influenza, and/or cold viruses.


HCV:

Peptides according to the present invention may comprise a known antigen. For antigens derived from HCV these antigens may be derived from the Core, E1, E2, P7, NS2, NS3, NS4 (NS4A and NS4B) and NS5 (NS5A and NS5B) protein of the Hepatitis C Virus (HCV). The epitopes are those which elicit a HLA class I and/or class II restricted T lymphocyte response in an immunized host. More specific, the HLA class I restricted peptides of the present invention may bind to at least one HLA molecule of the following HLA class I groups: HLA-A*01, HLA-A*02, HLA-A*03, HLA-A*11, HLA-A*24, HLA-B*07, HLA-B*08, HLA-B*35, HLA-B*40, HLA-B*44, HLA-Cw3, HLA-Cw4, HLA-Cw6 or HLA-Cw7. The HLA class II restricted peptides of the present invention may bind to at least one HLA molecule of the following HLA class II groups: HLA-DRB1, -DRB2, -DRB3, -DRB4, -DRB5, -DRB6, -DRB7, -DRB8 or -DRB9.


MHC binding HCV peptides that may be used according to the present invention as epitopes are disclosed in e.g. WO02/34770 (Imperial College Innovations Ltd), WO01/21189 and WO02/20035 (Epimmune), WO04/024182 (Intercell), WO95/25122 (The Scripps Research Institute), WO95/27733 (Government of the USA, Department of Health and Human Services), EP 0935662 (Chiron), WO02/26785 (Immusystems GmbH), WO95/12677 (Innogenetics N.V), WO97/34621 (Cytel Corp), and EP 1652858 (Innogenetics N.V.).


In other embodiments, the scaffold design according to the present invention comprises a PADRE peptide, such as the universal T cell epitope called PADRE as disclosed in WO95/07707 (Epimmune) the content of which are enclosed herein by reference. A ‘PanDR binding peptide or PADRE peptide” is a member of a family of molecules that binds more that one HLA class II DR molecule. PADRE binds to most HLA-DR molecules and stimulates in vitro and in vivo human helper T lymphocyte (HTL) responses. Alternatively T-help epitopes can be used from universally used vaccines such as tetanos toxoid.


In a further embodiment, the peptides in the composition or polyepitopic peptide are characterized in that they are derived from a HCV protein, or more specifically from at least one of the following HCV regions selected from the group consisting of Core, E1, E2/NS1, NS2, NS3, NS4A, NS4B, NS5A and NS5B. Even more preferred is that peptides are characterized in that they are present in the HCV consensus sequence of genotype 1a, 1b and/or 3 a.


Other HLA class I and II binding peptides that may be used according to the invention may be identified by the method as described in WO03/105058-Algonomics, by the method as described by Epimmune in WO01/21189 and/or by three public database prediction servers, respectively Syfpeithi, BIMAS and nHLAPred. It is also an aspect of this present invention that each peptide may be used within the scaffold design of the invention in combination with the same peptide as multiple repeats, or with any other peptide(s) or epitope(s).









TABLE 3







Specific HCV peptides in their complete length according


to the invention:



















Series
Ep.nr
Ver.
Scaf.
Z1
Z2
Z3
Z4
Z5
Z6
Z7
Z8
Z9





BI330
 72


RR

GGQLIGGIYLIPG
RR

VITFSIYLIVS








BI330
 72

b
RRR

GGQLIGGIYLIPG
RR

VITFSIYLIVS








BI330
 72

c
RR

GGQLIGGIYLIPG
RRR

VITFSIYLIVS








BI330
 72

d
RR

GGQLIGGIYLIPG
RR

VITFSIYLIVS
R







BI330
 72

e
RR

GGQLIGGIYLIPG
RR

VITFSIYLIVS
RR 







BI330
 72
2


RR


VITYSIFLIVS

RR


GGNVIGGIYZIPR








BI330
 72
2
b

RRR


VITYSIFLIVS

RR


GGNVIGGIYZIPR








BI330
 72
2
c

RR


VITYSIFLIVS

RRR


GGNVIGGIYZIPR








BI330
 72
2
d
RRR

VITYSIFLIVS
RRR

GGNVIGGIYZIPR














Z = Nle








BI330
 83


RRG

TANWARVIS
R

ANWAKVIL
R

NWAKVI





BI330
 83

b
RG

TANWARVIS
RR

ANWAKVIL
R

NWAKVI





BI330
 83

c
RG

TANWARVIS
R

ANWAKVIL
R

NWAKVI





BI330
 83

d
RG

TANWARVIS
RG

ANWAKVIL
R

NWAKVI





BI330
 83
2


RRG


TANWARVIS

R


ANWARVIL
R

NWAKVI





BI330
 83
2
b

RG


TANWARVIS

RR


ANWARVIL
R

NWAKVI





BI330
 83
2
c

RG


TANWARVIS

R


ANWARVIL
R

NWAKVI





BI330
 83
2
d

RG


TANWARVIS

RG


ANWARVIL
R

NWAKVI





BI310
511



R



GYLPAVGAPI


RRR



VIRVIAHGLRL


R








BI310
511

b
RR


GYLPAVGAPI

RR


VIRVIAHGLRL

R







BI310
511

c
RR


GYLPAVGAPI

RRR


VIRVIAHGLRL









BI310
511

d
RR


GYLPAVGAPI

RR


VIRVIAHGLRL









BI310
511

e
R


GYLPAVGAPI

RR


VIRVIAHGLRL

R







BI310
511

f
R


GYLPAVGAPI

R


VIRVIAHGLRL

R







BI310
511

g
R


GYLPAVGAPI

RR


VIRVIAHGLRL









“—” = no amino acid; B = Cit; Z = Nle; X = Har






CMV:

The epitopes to be incorporated into the scaffold design according to the present invention may be derived from cytomegalovirus (CMV) including CMV glycoproteins gB and gH.









TABLE 4







Specific CMV peptides in their complete length according


to the invention:


















Series
Nr
Ver.
Scaf.
Z1
Z2
Z3
Z4
Z5
Z6
Z7
Z8





BI050
4


RG

NIVPZVVTA
RR

IGDLIVAQV







BI050
4

b
RR

NIVPZVVTA
RR

IGDLIVAQV







BI050
4

c
RRR

NIVPZVVTA
RR

IGDLIVAQV







BI050
4

d
RR

NIVPZVVTA
RRR

IGDLIVAQV







BI050
4
2

RG

NIVPZVVTA
RR

IGDLIVQAV







BI050
4
2
b
RR

NIVPZVVTA
RR

IGDLIVQAV







BI050
4
2
c
RRR

NIVPZVVTA
RR

IGDLIVQAV







BI050
4
2
d
RR

NIVPZVVTA
RRR

IGDLIVQAV







BI050
5


RG

VTPADLIGA
RR

QYNPVAVZF







BI050
5

b
RR

VTPADLIGA
RR

QYNPVAVZF







BI050
5

c
RRR

VTPADLIGA
RR

QYNPVAVZF







BI050
5

d
RR

VTPADLIGA
RRR

QYNPVAVZF







BI050
6


RRG

PRPEGYTLFF
R

GYTLFFTS
R






BI050
6

b
RG

PRPEGYTLFF
RR

GYTLFFTS
R






BI050
6

c
RRG

PRPEGYTLFF
RR

GYTLFFTS
R






BI050
6

d
RRG

PRPEGYTLFF
RRR

GYTLFFTS
R






BI050
6

e
RRRG

PRPEGYTLFF
RR

GYTLFFTS
R






BI050
7


RG

LPYPRGYTLFV
RR

GYTLFVSD
R






BI050
7

b
RRG

LPYPRGYTLFV
RR

GYTLFVSD
R






BI050
7

c
RRG

LPYPRGYTLFV
RRR

GYTLFVSD
R






BI050
7

d
RRRG

LPYPRGYTLFV
RR

GYTLFVSD
R






BI050
7

e
RRG

LPYPRGYTLFV
RR

GYTLFVSD
R






BI050
8


RRG

ETILTPRDV
R

NTLZTPRDV
R






BI050
8

b
RG

ETILTPRDV
RR

NTLZTPRDV
R






BI050
8

c
RG

ETILTPRDV
R

NTLZTPRDV
R






BI050
8

d
RG

ETILTPRDV
RG

NTLZTPRDV
R






BI050
9


RR

SSTSPVYDL
RR

SSTSPVYNL
R






BI050
9

b
RR

SSTSPVYDL
RRR

SSTSPVYNL
R






BI050
9

c
RRR

SSTSPVYDL
RR

SSTSPVYNL
R






BI050
9

d
RRR

SSTSPVYDL
RRR

SSTSPVYNL
R






“—” = no amino acid; B = Cit; Z = Nle; X = Har






Influenza:

The epitopes to be incorporated into the scaffold design according to the present invention may be derived from fragments or portions of Influenza hemagglutinin (HA) or Influenza neuraminidase (NA), nucleoprotein (NP), M1, M2, NS1, NEP, PA, PB1, PB1-F2, PB2 for each of the subgroups, such as H1N1, H2N2 og H3N2.


Suitable epitopes may be derived from an HA protein of one, or more than one subtype, including H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 or H16 or fragment or portion thereof. Examples of subtypes comprising such HA proteins include A/New Calcdonia/20/99 (H1N1) A/Indonesia/5/2006 (H5N1), A/chicken/New York/1995, A/herring gull/DE/677/88 (H2N8), A/Texas/32/2003, A/mallard/MN/33/00, A/duck/Shanghai/1/2000, A/northern pintail/TX/828189/02, A/Turkey/Ontario/6118/68 (H8N4), A/shoveler/Iran/G54/03, A/chicken/Germany/N/1949 (H10N7), A/duck/England/56 (H11N6), A/duck/Alberta/60/76 (H12N5), A/Gull/Maryland/704/77 (H13N6), A/Mallard/Gurjev/263/82, A/duck/Australia/341/83 (H15N8), A/black-headed gull/Sweden/5/99 (H16N3), B/Lee/40, C/Johannesburg/66, A/PuertoRico/8/34 (H1N1), A/Brisbane/59/2007 (H1N1), A/Solomon Islands 3/2006 (H1N1), A/Brisbane 10/2007 (H3N2), A/Wisconsin/67/2005 (H3N2), B/Malaysia/2506/2004, B/Florida/4/2006, A/Singapore/1/57 (H2N2), A/Anhui/1/2005 (H5N1), A/Vietnam/1194/2004 (H5N1), A/Teal/HongKong/W312/97 (H6N1), A/Equine/Prague/56 (H7N7), A/HongKong/1073/99 (H9N2)).


In some embodiments of the invention, the HA protein may be an H1, H2, H3, H5, H6, H7 or H9 subtype. In other embodiments, the H1 protein may be from the A/New Calcdonia/20/99 (H1N1), A/PuertoRico/8/34 (H1N1), A/Brisbane/59/2007 (H1N1), or A/Solomon Islands 3/2006 (H1N1) strain. The H3 protein may also be from the A/Brisbane 10/2007 (H3N2) or A/Wisconsin/67/2005 (H3N2) strain. In other embodiments, the H2 protein may be from the A/Singapore/1/57 (H2N2) strain. The H5 protein may be from the A/Anhui/1/2005 (H5N1), A/Vietnam/1194/2004 (H5N1), or A/Indonesia/5/2005 strain. In other embodiments, the H6 protein may be from the A/Teal/HongKong/W312/97 (H6N1) strain. The H7 protein may be from the A/Equine/Prague/56 (H7N7) strain. In other embodiments, the H9 protein is from the A/HongKong/1073/99 (H9N2) strain. In other embodiments, the HA protein may be from an influenza virus may be a type B virus, including B/Malaysia/2506/2004 or B/Florida/4/2006. The influenza virus HA protein may be H5 Indonesia.









TABLE 5







Specific Influenza peptides according to the invention in their


complete length (Z or Nle denotes Norleucine, X or Har denotes


homoarginine):

















Series
Ep.nr
version
scaffold
Z1
Z2
Z3
Z4
Z5
Z6
Z7





BI100
330


RR

TAYERZCNIL
RR

GLEPLVIAGILA






BI100
330

b
RRR

TAYERZCNIL
RR

GLEPLVIAGILA






BI100
330

c
RR

TAYERZCNIL
RRR

GLEPLVIAGILA






BI100
330

d
RR

TAYERZCNIL
RR

GLEPLVIAGILA
R





BI100
330

e
RR

TAYERZCNIL
RR

GLEPLVIAGILA
RR








Z = Nle









BI100
270


RR

TVIGASZIPLL
RG

TPIXQDWENRAN






BI100
270

b
RRR

TVIGASZIPLL
RG

TPIXQDWENRAN






BI100
270

c
RR

TVIGASZIPLL
RRG

TPIXQDWENRAN






BI100
270

d
RRR

TVIGASZIPLL
RRG

TPIXQDWENRAN






BI100
270

e
RRR

TVIGASZIPLL
RRG

TPIXQDWENRAN
R








Z = Nle


X = Har






BI100
130


RR

AAFEEZXITS
RR

VAFEDLXZZSFI






BI100
130

b
RRR

AAFEEZXITS
RR

VAFEDLXZZSFI






BI100
130

c
RRR

AAFEEZXITS
RRG

VAFEDLXZZSFI






BI100
130

d
RRR

AAFEEZXITS
RRR

VAFEDLXZZSFI






BI100
130

e
RRR

AAFEEZXITS
RRR

VAFEDLXZZSFI
GR





BI100
190

e
RR

TAYERZCNIL
RRG

RFQTVVQBA






BI100
190

f
RR

TAYERZCNIL
RRG

RFQTVVQBA
R





BI100
190

g
R

TAYERZCNIL
RG

RFQTVVQBA
R





BI100
190

h
RR

TAYERZCNIL
RG

RFQTVVQBA






BI100
260

b
BR

GLEPLVIAGILA
RR

GSLVGLLHIVL






BI100
260

c
RR

GLEPLVIAGILA
RR

GSLVGLLHIVL






BI100
260

d
RR

GLEPLVIAGILA
RR

GSLVGLLHIVL
R





BI100
260

e
RR

GLEPLVIAGILA
RRR

GSLVGLLHIVL






BI100
260

f
RR

GLEPLVIAGILA
RRR

GSLVGLLHIVL
R





BI100
120
 −3
a
R

TAFLVRNVA
R

SIARSVTIZXASVVH






BI100
120
 −3
b
R

TAFLVRNVA
RR

SIARSVTIZXASVVH






BI100
120
 −3
c
RR

TAFLVRNVA
R

SIARSVTIZXASVVH






BI100
120
 −3
d
RR

TAFLVRNVA
RR

SIARSVTIZXASVVH






BI100
120
 −3
e
RR

TAFLVRNVA
RR

SIARSVTIZXASVVH
R





BI100
120
 −3
f
RR

TAFLVRNVA
RR

SIARSVTIZXASVVH
RR





































BI100
220


RG
Dpr
TPI(Har)QDWGN
RG

TPTRQEWDCRIS








(Aoa)
RAN









BI100
220
 −2

RG
Dpr
TPI(Har)QDWGN
RG

TPTRQEWDARIS








(Aoa)
RAN









BI100
220
 −3

RG

TPI(Har)QDWGN
RG

TPTRQEWDCRIS









RAN









BI100
220
 −4

RG

TPI(Har)QDWGN
RG

TPTRQEWDARIS









RAN









BI100
220
 −5

RG
C
TPI(Har)QDWGN
RG

TPTRQEWDCRIS









RAN









BI100
220
 −6

RG
C
TPI(Har)QDWGN
RG

TPTRQEWDARIS









RAN









BI100
220
 −7

RG
K
TPI(Har)QDWGN
RG

TPTRQEWDCRIS









RAN









BI100
220
 −8

RG
K
TPI(Har)QDWGN
RG

TPTRQEWDARIS









RAN









BI100
220
 −9

RG
Lys
TPI(Har)QDWGN
RG

TPTRQEWDCRIS








(Me)
RAN









BI100
220
−10

RG
Lys
TPI(Har)QDWGN
RG

TPTRQEWDARIS








(Me)
RAN









BI100
220
−11

RG
D
TPI(Har)QDWGN
RG

TPTRQEWDCRIS









RAN









BI100
220
−12

RG
D
TPI(Har)QDWGN
RG

TPTRQEWDARIS









RAN









BI100
220
−13

RG
E
TPI(Har)QDWGN
RG

TPTRQEWDCRIS









RAN









BI100
220
−14

RG
E
TPI(Har)QDWGN
RG

TPTRQEWDARIS









RAN









BI100
240


RG
Dpr
TPT(Har)NGWDV
RG

TPI(Har)QEW(Har)SL








(Ser)
KLS


(Nle)NQEW






BI100
240
 −3

RG

TPT(Har)NGWDV
RG

TPI(Har)QEW(Har)SL









KLS


(Nle)NQEW






BI100
240
 −4

RG
K
TPT(Har)NGWDV
RG

TPI(Har)QEW(Har)SL









KLS


(Nle)NQEW






BI100
240
 −5

RG
C
TPT(Har)NGWDV
RG

TPI(Har)QEW(Har)SL









KLS


(Nle)NQEW






BI100
240
 −6

RG
Lys
TPT(Har)NGWDV
RG

TPI(Har)QEW(Har)SL








(Me)
KLS


(Nle)NQEW






BI100
240
 −7

RG
D
TPT(Har)NGWDV
RG

TPI(Har)QEW(Har)SL









KLS


(Nle)NQEW






BI100
240 
 −8

RG
E
TPT(Har)NGWDV
RG

TPI(Har)QEW(Har)SL









KLS


(Nle)NQEW





“—” = no amino acid; B = Cit; Z = Nle; X = Har













TABLE 6







Specific dimeric Influenza peptides according to the invention


in their complete length (Z or Nle denotes Norleucine, X or


Har denotes homoarginine, residues linking A and B monomer


peptides to dimers are underlined):









Dimeric
Dimeric peptides, composed of peptides
Constituent


Peptide
A and B. Linked residues underlined
monomers





BI-155




A
RG(Dpr(Aoa))-TPI(Har)QDWGNRAN-RG-
BI-100-220



TPTRQEWDCRIS-NH2






B
RG(Dpr(Ser))-TPT(Har)NGWDVKLS-RG-
BI-100-240



TPI(Har)QEW(Har)SL(Nle)NQEW-NH2






BI-155-2




A
RG(Dpr(Aoa))-TPI(Har)QDWGNRAN-RG-
BI-100-220-2



TPTRQEWDARIS-NH2






B
RG(Dpr(Ser))-TPT(Har)NGWDVKLS-RG-
BI-100-240



TPI(Har)QEW(Har)SL(Nle)NQEW-NH2






BI-155-3




A
RGC-TPI(Har)QDWGNRAN-RG-TPTRQEWDCRIS-
BI-100-220-5



NH2






B
RGK-TPT(Har)NGWDVKLS-RG-
BI-100-240-4



TPI(Har)QEW(Har)SL(Nle)NQEW-NH2






BI-155-4




A
RGC-TPI(Har)QDWGNRAN-RG-TPTRQEWDARIS-
BI-100-220-6



NH2






B
RGK-TPT(Har)NGWDVKLS-RG-
BI-100-240-4



TPI(Har)QEW(Har)SL(Nle)NQEW-NH2






BI-155-5




A
RGK-TPI(Har)QDWGNRAN-RG-TPTRQEWDCRIS-
BI-100-220-7



NH2






B
RGC-TPT(Har)NGWDVKLS-RG-
BI-100-240-5



TPI(Har)QEW(Har)SL(Nle)NQEW-NH2






BI-155-6




A
RGK-TPI(Har)QDWGNRAN-RG-TPTRQEWDARIS-
BI-100-220-8



NH2






B
RGC-TPT(Har)NGWDVKLS-RG-
BI-100-240-5



TPI(Har)QEW(Har)SL(Nle)NQEW-NH2






BI-155-7




A
RG(Lys(Me))-TPI(Har)QDWGNRAN-RG-
BI-100-220-9



TPTRQEWDCRIS-NH2






B
RGD-TPT(Har)NGWDVKLS-RG-
BI-100-240-7



TPI(Har)QEW(Har)SL(Nle)NQEW-NH2






BI-155-8




A
RG(Lys(Me))-TPI(Har)QDWGNRAN-RG-
BI-100-220-9



TPTRQEWDCRIS-NH2






B
RGE-TPT(Har)NGWDVKLS-RG-
BI-100-240-8



TPI(Har)QEW(Har)SL(Nle)NQEW-NH2






BI-155-9




A
RG(Lys(Me))-TPI(Har)QDWGNRAN-RG-
BI-100-220-10



TPTRQEWDARIS-NH2






B
RGD-TPT(Har)NGWDVKLS-RG-
BI-100-240-7



TPI(Har)QEW(Har)SL(Nle)NQEW-NH2






BI-155-10




A
RG(Lys(Me))-TPI(Har)QDWGNRAN-RG-
BI-100-220-10



TPTRQEWDARIS-NH2






B
RGE-TPT(Har)NGWDVKLS-RG-
BI-100-240-8



TPI(Har)QEW(Har)SL(Nle)NQEW-NH2






BI-155-11




A
RGD-TPI(Har)QDWGNRAN-RG-TPTRQEWDCRIS-
BI-100-220-11



NH2






B
RG(Lys(Me))-TPT(Har)NGWDVKLS-RG-
BI-100-240-6



TPI(Har)QEW(Har)SL(Nle)NQEW-NH2






BI-155-12




A
RGD-TPI(Har)QDWGNRAN-RG-TPTRQEWDARIS-
BI-100-220-12



NH2






B
RG(Lys(Me))-TPT(Har)NGWDVKLS-RG-
BI-100-240-6



TPI(Har)QEW(Har)SL(Nle)NQEW-NH2






BI-155-13




A
RGE-TPI(Har)QDWGNRAN-RG-TPTRQEWDCRIS-
BI-100-220-13



NH2






B
RG(Lys(Me))-TPT(Har)NGWDVKLS-RG-
BI-100-240-6



TPI(Har)QEW(Har)SL(Nle)NQEW-NH2






BI-155-14




A
RGE-TPI(Har)QDWGNRAN-RG-TPTRQEWDARIS-
BI-100-220-14



NH2






B
RG(Lys(Me))-TPT(Har)NGWDVKLS-RG-
BI-100-240-6



TPI(Har)QEW(Har)SL(Nle)NQEW-NH2






BI-155-15




A
RGC-TPI(Har)QDWGNRAN-RG-TPTRQEWDCRIS-
BI-100-220-5



NH2






B
RGC-TPT(Har)NGWDVKLS-RG-
BI-100-240-5



TPI(Har)QEW(Har)SL(Nle)NQEW-NH2






BI-155-16




A
RGC-TPI(Har)QDWGNRAN-RG-TPTRQEWDARIS-
BI-100-220-6



NH2






B
RGC-TPT(Har)NGWDVKLS-RG-
BI-100-240-5



TPI(Har)QEW(Har)SL(Nle)NQEW-NH2







A-monomer peptide variants:




RG(Dpr(Aoa))-TPI(Har)QDWGNRAN-RG-
BI-100-220



TPTRQEWDCRIS-NH2







RG(Dpr(Aoa))-TPI(Har)QDWGNRAN-RG-
BI-100-220-2



TPTRQEWDARIS-NH2







RG-TPI(Har)QDWGNRAN-RG-TPTRQEWDCRIS-NH2
BI-100-220-3






RG-TPI(Har)QDWGNRAN-RG-TPTRQEWDARIS-NH2
BI-100-220-4






RGC-TPI(Har)QDWGNRAN-RG-TPTRQEWDCRIS-
BI-100-220-5



NH2







RGC-TPI(Har)QDWGNRAN-RG-TPTRQEWDARIS-
BI-100-220-6



NH2







RGK-TPI(Har)QDWGNRAN-RG-TPTRQEWDCRIS-
BI-100-220-7



NH2







RGK-TPI(Har)QDWGNRAN-RG-TPTRQEWDARIS-
BI-100-220-8



NH2







RG(Lys(Me))-TPI(Har)QDWGNRAN-RG-
BI-100-220-9



TPTRQEWDCRIS-NH2







RG(Lys(Me))-TPI(Har)QDWGNRAN-RG-
BI-100-220-10



TPTRQEWDARIS-NH2







RGD-TPI(Har)QDWGNRAN-RG-TPTRQEWDCRIS-
BI-100-220-11



NH2







RGD-TPI(Har)QDWGNRAN-RG-TPTRQEWDARIS-
BI-100-220-12



NH2







RGE-TPI(Har)QDWGNRAN-RG-TPTRQEWDCRIS-
BI-100-220-13



NH2







RGE-TPI(Har)QDWGNRAN-RG-TPTRQEWDARIS-
BI-100-220-14



NH2







B-monomer peptide variants:




RG(Dpr(Ser))-TPT(Har)NGWDVKLS-RG-
BI-100-240



TPI(Har)QEW(Har)SL(Nle)NQEW-NH2







RG-TPT(Har)NGWDVKLS-RG-
BI-100-240-3



TPI(Har)QEW(Har)SL(Nle)NQEW-NH2







RGK-TPT(Har)NGWDVKLS-RG-
BI-100-240-4



TPI(Har)QEW(Har)SL(Nle)NQEW-NH2







RGC-TPT(Har)NGWDVKLS-RG-
BI-100-240-5



TPI(Har)QEW(Har)SL(Nle)NQEW-NH2







RG(Lys(Me))-TPT(Har)NGWDVKLS-RG-
BI-100-240-6



TPI(Har)QEW(Har)SL(Nle)NQEW-NH2







RGD-TPT(Har)NGWDVKLS-RG-
BI-100-240-7



TPI(Har)QEW(Har)SL(Nle)NQEW-NH2







RGE-TPT(Har)NGWDVKLS-RG-
BI-100-240-8



TPI(Har)QEW(Har)SL(Nle)NQEW-NH2









Human Immunodeficiency Virus (HIV):

For HIV, the epitopes to be incorporated into the scaffold design according to the present invention may be derived from the group consisting of gp120, gp160, gp41, p24gag or p55gag derived from HIV, including members of the various genetic subtypes.


Human Papillomavirus (HPV):

For HPV, the epitopes to be incorporated into the scaffold design according to the present invention may be derived from the group consisting E1, E2, E3, E4, E6 and E7, L1 and L2 proteins. The epitopes may be derived from any type including types 8, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59.









TABLE 7







Specific HPV peptides in their complete length according


to the invention:


















Series
Nr
Version
Scaffold
Z1
Z2
Z3
Z4
Z5
Z6
Z7
Z8








Native:


35-45


48-58




BI500
1


RR

LECVYCKQQLL
RR

EVYDFAFRDL













C







BI500
1

b
RR

LECVYCKQQLL
RRR

EVYDFAFRDL











G

C







BI500
1

c
RRR

LECVYCKQQLL
RRG

EVYDFAFRDL













C







BI500
1

d
RRR

LECVYCKQQLL
RRR

EVYDFAFRDL











G

C







BI500
1

e
RRRG

LECVYCKQQLL
RRR

EVYDFAFRDL











G

C










Native:


49-58


52-61




BI500
2


RR

GVYDFAFRDLC
RR

GFAFRDLCIV
R












Y







BI500
2

b
RR

GVYDFAFRDLC
RRR

GFAFRDLCIV











G

Y







BI500
2

c
RRR

GVYDFAFRDLC
RRG

GFAFRDLCIV
R












Y







BI500
2

d
RRR

GVYDFAFRDLC
RRR

GFAFRDLCIV











G

Y







BI500
2

e
RRRG

GVYDFAFRDLC
RRR

GFAFRDLCIV
R










G

Y










Native:


49-58


52-61




BI500
3


RR

GVFDYAFRDIN
RR

GFAYRDINLA
R












Y







BI500
3

b
RR

GVYDFAFRDLC
RRR

GFAFRDLCIV











G

Y







BI500
3

c
RRR

GVYDFAFRDLC
RRG

GFAFRDLCIV
R












Y







BI500
3

d
RRR

GVYDFAFRDLC
RRR

GFAFRDLCIV











G

Y







BI500
3

e
RRRG

GVYDFAFRDLC
RRR

GFAFRDLCIV
R










G

Y










Native:


74-83


84-93




BI500
4


RR

VDIRTLEDLL
RR

GTLGIVCPIG
R






BI500
4

b
RR

VDIRTLEDLL
RRR

GTLGIVCPIG











G









BI500
4

c
RRR

VDIRTLEDLL
RRG

GTLGIVCPIG
R






BI500
4

d
RRR

VDIRTLEDLL
RRR

GTLGIVCPIG











G









BI500
4

e
RRRG

VDIRTLEDLL
RRR

GTLGIVCPIG
R










G









The present invention further relates to compositions comprising two or three peptides of the invention.









TABLE 8





The table represent 10 different suitable combinations of three


monomeric peptides each peptide comprising a specific natural


antigen of a protein or peptide sequence derived from HCV.
























1
BI3
BI3
BI3
RRGGQLIGGI
RRGTANWARV
RRGYLPAVG
(SEQ ID
(SEQ ID
(SEQ ID



30-
30-
10-
YLIPGRRVITF
ISRANWAKVIL
APIRRVIRVI
NO: 357)
NO: 366)
NO: 377)



72
83
511
SIYLIVS
RNWAKVI
AHGLRL








d











2
BI3
BI3
BI3
RRRGGQLIGG
RGTANWARVI
RGYLPAVGA
(SEQ ID
(SEQ ID
(SEQ ID



30-
30-
10-
IYLIPGRRVITF
SRRANWAKVI
PIRVIRVIAH
NO: 358)
NO: 367)
NO: 379)



72b
83b
511
SIYLIVS
LRNWAKVI
GLRLR








f











3
BI3
BI3
BI3
RRGGQLIGGI
RGTANWARVI
RGYLPAVGA
(SEQ ID
(SEQ ID
(SEQ ID



30-
30-
10-
YLIPGRRRVIT
SRANWAKVIL
PIRRVIRVIA
NO: 359)
NO: 368)
NO: 380)



72c
83c
511
FSIYLIVS
RNWAKVI
HGLRL








g











4
BI3
BI3
BI3
RRGGQLIGGI
RGTANWARVI
RGYLPAVGA
(SEQ ID
(SEQ ID
(SEQ ID



30-
30-
10-
YLIPGRRVITF
SRGANWAKVI
PIRRRVIRVI
NO: 360)
NO: 369)
NO: 374)



72d
83d
511
SIYLIVSR
LRNWAKVI
AHGLRLR








5
BI3
BI3
BI3
RRGGQLIGGI
RRGTANWARV
RRGYLPAVG
(SEQ ID
(SEQ ID
(SEQ ID



30-
30-
10-
YLIPGRRVITF
ISRANWARVIL
APIRRVIRVI
NO: 361)
NO: 370)
NO: 375)



72e
83-
511
SIYLIVSRR
RNWAKVI
AHGLRLR







2
b











6
BI3
BI3 
BI3
RRVITYSIFLIV
RGTANWARVI
RRGYLPAVG
(SEQ ID
(SEQ ID
(SEQ ID



30-
30-
10-
SRRGGNVIGG
SRRANWARVI
APIRRRVIRV
NO: 362)
NO: 371)
NO: 376)



72-
83-
511
IYZIPR
LRNWAKVI
IAHGLRL






2
2b
c











7
BI3
BI3
BI3
RRVITYSIFLIV
RRGTANWARV
RRGYLPAVG
(SEQ ID
(SEQ ID
(SEQ ID



30-
30-
10-
SRRGGNVIGG
ISRANWAKVIL
APIRRVIRVI
NO: 362)
NO: 366)
NO: 377)



72-
83
511
IYZIPR
RNWAKVI
AHGLRL






2

d











8
BI3
BI3
BI3
RRRVITYSIFLI
RGTANWARVI
RGYLPAVGA
(SEQ ID
(SEQ ID
(SEQ ID



30-
30-
10-
VSRRGGNVIG
SRRANWAKVI
PIRRVIRVIA
NO: 363)
NO: 367)
NO: 378)



72-
83b
511
GIYZIPR
LRNWAKVI
HGLRLR






2b

e











9
BI3
BI3
BI3
RRVITYSIFLIV
RGTANWARVI
RGYLPAVGA
(SEQ ID
(SEQ ID
(SEQ ID



30-
30-
10-
SRRRGGNVIG
SRANWARVIL
PIRVIRVIAH
NO: 364)
NO: 372)
NO: 379)



72-
83-
511
GIYZIPR
RNWAKVI
GLRLR






2c
2c
f











10
BI3
BI3
BI3
RRRVITYSIFLI
RGTANWARVI
RGYLPAVGA
(SEQ ID
(SEQ ID
(SEQ ID



30-
30-
10-
VSRRRGGNVI
SRGANWARVI
PIRRVIRVIA 
NO: 365)
NO: 373)
NO: 380)



72-
83-
511
GGIYZIPR
LRNWAKVI
HGLRL






2d
2d
g
















TABLE 9





The table represent 10 different suitable combinations of


three monomeric peptides and one dimeric peptide each


peptide comprising specific natural antigen of a protein or


peptide sequence derived from influenza.



















 1
BI-
BI100-330;
BI100-270
BI100-130



155-
RRTAYERZCNILRRGLEP
RRTVIGASZIPLLRGTPIXQD
RRAAFEEZXITSRRVAFEDL



5
LVIAGILA
WENRAN
XZZSFI




(SEQ ID NO: 407)
(SEQ ID NO: 412)
(SEQ ID NO: 417)





 2
BI-
BI100-330b
BI100-270b
BI100-130b



155-
RRRTAYERZCNILRRGLE
RRRTVIGASZIPLLRGTPIXQ
RRRAAFEEZXITSRRVAFED



4
PLVIAGILA
DWENRAN
LXZZSFI




(SEQ ID NO: 408)
(SEQ ID NO: 413)
(SEQ ID NO: 418)





 3
BI-
BI100-330c
BI100-270c
BI100-130c



155-
RRTAYERZCNILRRRGLE
RRTVIGASZIPLLRRGTPIXQ
RRRAAFEEZXITSRRGVAFE



3
PLVIAGILA
DWENRAN
DLXZZSFI




(SEQ ID NO: 409)
(SEQ ID NO: 414)
(SEQ ID NO: 419)





 4
BI-
BI100-330d
BI100-270d
BI100-130d



155-
RRTAYERZCNILRRGLEP
RRRTVIGASZIPLLRRGTPIX
RRRAAFEEZXITSRRRVAFE



2
LVIAGILAR
QDWENRAN
DLXZZSFI




(SEQ ID NO: 410)
(SEQ ID NO: 415)
(SEQ ID NO: 420)





 5
BI-
BI100-330e
BI100-270e
BI100-130e



155
RRTAYERZCNILRRGLEP
RRRTVIGASZIPLLRRGTPIX
RRRAAFEEZXITSRRRVAFE




LVIAGILARR
QDWENRANR
DLXZZSFIGR




(SEQ ID NO: 411)
(SEQ ID NO: 416)
(SEQ ID NO: 421)





 6
BI-
BI100-330e
BI100-270e
BI100-130e



155-
RRTAYERZCNILRRGLEP
RRRTVIGASZIPLLRRGTPIX
RRRAAFEEZXITSRRRVAFE



2
LVIAGILARR
QDWENRANR
DLXZZSFIGR




(SEQ ID NO: 411)
(SEQ ID NO: 416)
(SEQ ID NO: 421)





 7
BI-
BI100-330d
BI100-270c
BI100-130c



155-
RRTAYERZCNILRRGLEP
RRTVIGASZIPLLRRGTPIXQ
RRRAAFEEZXITSRRGVAFE



3
LVIAGILAR
DWENRAN
DLXZZSFI




(SEQ ID NO: 410)
(SEQ ID NO: 414)
(SEQ ID NO: 419)





10
BI-
BI100-330
BI100-270d
BI100-130b



155
RRTAYERZCNILRRGLEP
RRRTVIGASZIPLLRRGTPIX
RRRAAFEEZXITSRRVAFED




LVIAGILA
QDWENRAN
LXZZSFI




(SEQ ID NO: 407)
(SEQ ID NO: 415)
(SEQ ID NO: 418)









Carriers, Adjuvants and Vehicles—Delivery

The isolated peptides according to the invention may be delivered by various means and within various compositions, herein referred to as “compositions”, “vaccine compositions” or “pharmaceutical compositions”. The peptides of the present invention and pharmaceutical and vaccine compositions of the invention are useful for administration to mammals, particularly humans, to treat and/or prevent virus infection. Vaccine compositions containing the peptides of the invention are administered to a patient infected with the virus in question or to an individual susceptible to, or otherwise at risk for, virus infection to elicit an immune response against the specific antigens and thus enhance the patient's own immune response capabilities.


Various art-recognized delivery systems may be used to deliver the peptides, into appropriate cells. The peptides can be delivered in a pharmaceutically acceptable carrier or as colloidal suspensions, or as powders, with or without diluents. They can be “naked” or associated with delivery vehicles and delivered using delivery systems known in the art.


A “pharmaceutically acceptable carrier” or “pharmaceutically acceptable adjuvant” is any suitable excipient, diluent, carrier and/or adjuvant which, by themselves, do not induce the production of antibodies harmful to the individual receiving the composition nor do they elicit protection. Preferably, a pharmaceutically acceptable carrier or adjuvant enhances the immune response elicited by an antigen. Suitable carriers or adjuvant typically comprise one or more of the compounds included in the following non-exhaustive list: large slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles; aluminium hydroxide, aluminium phosphate (see International Patent Application Publication No. WO93/24148), alum (KAI(SO4)2.12H2O), or one of these in combination with 3-0-deacylated monophosphoryl lipid A (see International Patent Application Publication No. WO93/19780); N-acetyl-muramyl-L-threonyl-D-isoglutamine (see U.S. Pat. No. 4,606,918), N-acetyl-normuramyl-L-alanyl-D-isoglutamine, N-acetylmuramyl-L-alanyl-D-isoglutamyl-L-alanine-2-(1′,2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)ethylamine; RIBI (ImmunoChem Research Inc., Hamilton, Mont., USA) which contains monophosphoryl lipid A (i.e., a detoxified endotoxin), trehalose-6,6-dimycolate, and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion. Any of the three components MPL, TDM or CWS may also be used alone or combined 2 by 2; adjuvants such as Stimulon (Cambridge Bioscience, Worcester, Mass., USA), SAF-1 (Syntex); adjuvants such as combinations between QS21 and 3-de-O-acetylated monophosphoryl lipid A (see International Application No. WO94/00153) which may be further supplemented with an oil-in-water emulsion (see, e.g., International Application Nos. WO95/17210, WO97/01640 and WO9856414) in which the oil-in-water emulsion comprises a metabolisable oil and a saponin, or a metabolisable oil, a saponin, and a sterol, or which may be further supplemented with a cytokine (see International Application No. WO98/57659); adjuvants such as MF-59 (Chiron), or poly[di(carboxylatophenoxy) phosphazene] based adjuvants (Virus Research Institute); blockcopolymer based adjuvants such as Optivax (Vaxcel, Cytrx) or inulin-based adjuvants, such as Algammulin and Gammalnulin (Anutech); Complete or Incomplete Freund's Adjuvant (CFA or IFA, respectively) or Gerbu preparations (Gerbu Biotechnik); a saponin such as QuilA, a purified saponin such as QS21, QS7 or QS17, -escin or digitonin; immunostimulatory oligonucleotides comprising unmethylated CpG dinucleotides such as [purine-purine-CG-pyrimidine-pyrimidine] oligonucleotides. These immunostimulatory oligonucleotides include CpG class A, B, and C molecules (Coley Pharmaceuticals), ISS (Dynavax), Immunomers (Hybridon). Immunostimulatory oligonucleotides may also be combined with cationic peptides as described, e.g., by Riedl et al. (2002); Immune Stimulating Complexes comprising saponins, for example Quil A (ISCOMS); excipients and diluents, which are inherently non-toxic and non-therapeutic, such as water, saline, glycerol, ethanol, isopropyl alcohol, DMSO, wetting or emulsifying agents, pH buffering substances, preservatives, and the like; a biodegradable and/or biocompatible oil such as squalane, squalene, eicosane, tetratetracontane, glycerol, peanut oil, vegetable oil, in a concentration of, e.g., 1 to 10% or 2.5 to 5%; vitamins such as vitamin C (ascorbic acid or its salts or esters), vitamin E (tocopherol), or vitamin A; carotenoids, or natural or synthetic flavanoids; trace elements, such as selenium; any Toll-like receptor ligand as reviewed in Barton and Medzhitov (2002).


For a further enhancement of the vaccine antigenic properties, could be to combine a well known adjuvant with an oral immune modulant, such as IMID or adjuvant such as a Cox-2 inhibitor or a immunomodulating compound.


A further aspect of the invention is the use of the vaccine combined with adjuvant, with an (oral) immunemodulating agent and a reservoir purging agent.


Other suitable adjuvants includes response-selective C5a agonists, such as EP54 and EP67 described in Hung C Y et al. An agonist of human complement fragment C5a enhances vaccine immunity against Coccidioides infection. Vaccine (2012) and Kollessery G et al. Tumor-specific peptide based vaccines containing the conformationally biased, response-selective C5a agonists EP54 and EP67 protect against aggressive large B cell lymphoma in a syngeneic murine model. Vaccine (2011) 29: 5904-10.


Other suitable adjuvants include an oil-in-water emulsion containing a stabilizing detergent, a micelle-forming agent and a biodegradable oil, such as Provax described in e.g. U.S. Pat. No. 5,585,103.


Any of the afore-mentioned adjuvants comprising 3-de-O-acetylated monophosphoryl lipid A, said 3-de-O-acetylated monophosphoryl lipid A may be forming a small particle (see International Application No. WO94/21292).


In any of the aforementioned adjuvants MPL or 3-de-O-acetylated monophosphoryl lipid A can be replaced by a synthetic analogue referred to as RC-529 or by any other amino-alkyl glucosaminide 4-phosphate (Johnson et al. 1999, Persing et al. 2002). Alternatively it can be replaced by other lipid A analogues such as OM-197 (Byl et al. 2003).


A “pharmaceutically acceptable vehicle” includes vehicles such as water, saline, physiological salt solutions, glycerol, ethanol, etc. Auxiliary substances such as wetting or emulsifying agents, pH buffering substances, preservatives may be included in such vehicles. Delivery systems known in the art are e.g. lipopeptides, peptide compositions encapsulated in poly-DL-lactide-co-glycolide (“PLG”), microspheres, peptide compositions contained in immune stimulating complexes (ISCOMS), multiple antigen peptide systems (MAPs), viral delivery vectors, particles of viral or synthetic origin, adjuvants, liposomes, lipids, microparticles or microcapsules, gold particles, nanoparticles, polymers, condensing agents, polysaccharides, polyamino acids, dendrimers, saponins, QS21, adsorption enhancing materials, fatty acids or, naked or particle absorbed cDNA.


The peptides may be delivered in oils such as Endocine™ and Montanide™ (Eurocine)-Montanide™ ISA 51 VG or Montanide™ ISA 720 VG (Seppic).


The adjuvant may be stimulators of the innate immune system that can be given separately from the peptide such as Leukotriene B4 (LTB4) and granulocyte macrophage colony stimulating factor (GM-CSF), such as Sargramostim/Leukine (glycosylated GM-CSF) and Molgramostim (nonglycosylated GM-CSF).


Typically, a vaccine or vaccine composition is prepared as an injectable, either as a liquid solution or suspension. Injection may be subcutaneous, intramuscular, intravenous, intraperitoneal, intrathecal, intradermal, or intraepidermal. Other types of administration comprise electroporation, implantation, suppositories, oral ingestion, enteric application, inhalation, aerosolization or nasal spray or drops. Solid forms, suitable for dissolving in, or suspension in, liquid vehicles prior to injection may also be prepared. The preparation may also be emulsified or encapsulated in liposomes for enhancing adjuvant effect.


A liquid formulation may include oils, polymers, vitamins, carbohydrates, amino acids, salts, buffers, albumin, surfactants, or bulking agents. Preferably carbohydrates include sugar or sugar alcohols such as mono-, di-, tri-, oligo- or polysaccharides, or water-soluble glucans. The saccharides or glucans can include fructose, dextrose, lactose, glucose, mannose, sorbose, xylose, maltose, sucrose, dextran, pullulan, dextrin, alpha and beta cyclodextrin, soluble starch, hydroxethyl starch and carboxymethylcellulose, or mixtures thereof. Sucrose is most preferred. “Sugar alcohol” is defined as a C4 to C8 hydrocarbon having an —OH group and includes galactitol, inositol, mannitol, xylitol, sorbitol, glycerol, and arabitol. Mannitol is most preferred. These sugars or sugar alcohols mentioned above may be used individually or in combination. There is no fixed limit to the amount used as long as the sugar or sugar alcohol is soluble in the aqueous preparation. Preferably, the sugar or sugar alcohol concentration is between 1.0% (w/v) and 7.0% (w/v), more preferable between 2.0 and 6.0% (w/v). Preferably amino acids include levorotary (L) forms of carnitine, arginine, and betaine; however, other amino acids may be added. Preferred polymers include polyvinylpyrrolidone (PVP) with an average molecular weight between 2,000 and 3,000, or polyethylene glycol (PEG) with an average molecular weight between 3,000 and 5,000. It is also preferred to use a buffer in the composition to minimize pH changes in the solution before lyophilization or after reconstitution. Any physiological buffer may be used, but citrate, phosphate, succinate, and glutamate buffers or mixtures thereof are preferred. Most preferred is a citrate buffer. Preferably, the concentration is from 0.01 to 0.3 molar. Surfactants that can be added to the formulation are shown in EP patent applications No. EP 0 270 799 and EP 0 268 110.


Additionally, the peptides according to the present invention may be chemically modified by covalent conjugation to a polymer to increase their circulating half-life, for example. Preferred polymers, and methods to attach them to peptides, are shown in U.S. Pat. Nos. 4,766,106; 4,179,337; 4,495,285; and 4,609,546. Preferred polymers are polyoxyethylated polyols and polyethylene glycol (PEG). PEG is soluble in water at room temperature and has the general formula:


R(O—CH2-CH2)nO—R where R can be hydrogen, or a protective group such as an alkyl or alkanol group. Preferably, the protective group has between 1 and 8 carbons, more preferably it is methyl. The symbol n is a positive integer, preferably between 1 and 1.000, more preferably between 2 and 500. The PEG has a preferred average molecular weight between 1000 and 40.000, more preferably between 2000 and 20.000, most preferably between 3.000 and 12.000. Preferably, PEG has at least one hydroxy group, more preferably it is a terminal hydroxy group. It is this hydroxy group which is preferably activated. However, it will be understood that the type and amount of the reactive groups may be varied to achieve a covalently conjugated PEG/polypeptide of the present invention.


Water soluble polyoxyethylated polyols are also useful in the present invention. They include polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylated glycerol (POG), etc. POG is preferred. One reason is because the glycerol backbone of polyoxyethylated glycerol is the same backbone occurring naturally in, for example, animals and humans in mono-, di-, triglycerides. Therefore, this branching would not necessarily be seen as a foreign agent in the body. The POG has a preferred molecular weight in the same range as PEG. The structure for POG is shown in Knauf et al., 1988, and a discussion of POG/IL-2 conjugates is found in U.S. Pat. No. 4,766,106.


Another drug delivery system for increasing circulatory half-life is the liposome. The peptides and nucleic acids of the invention may also be administered via liposomes, which serve to target a particular tissue, such as lymphoid tissue, or to target selectively infected cells, as well as to increase the half-life of the peptide and nucleic acids composition. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. In these preparations, the peptide or nucleic acids to be delivered is incorporated as part of a liposome or embedded, alone or in conjunction with a molecule which binds to a receptor prevalent among lymphoid cells, such as monoclonal antibodies which bind to the CD45 antigen, or with other therapeutic or immunogenic compositions. Thus, liposomes either filled or decorated with a desired peptide or nucleic acids of the invention can be directed to the site of lymphoid cells, where the liposomes then deliver the peptide and nucleic acids compositions. Liposomes for use in accordance with the invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of, e.g., liposome size, acid lability and stability of the liposomes in the blood stream. A variety of methods are available for preparing liposomes, as described in, e.g., Szoka et al, 1980, and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.


For targeting cells of the immune system, a ligand to be incorporated into the liposome can include, e.g., antibodies or fragments thereof specific for cell surface determinants of the desired immune system cells. A liposome suspension containing a peptide may be administered intravenously, locally, topically, etc. in a dose which varies according to, inter alia, the manner of administration, the peptide being delivered, and the stage of the disease being treated. For example, liposomes carrying either immunogenic polypeptides are known to elicit CTL responses in vivo (Reddy et al., 1992; Collins et al., 1992; Fries et al., 1992; Nebel et al., 1992).


After the liquid pharmaceutical composition is prepared, it is preferably lyophilized to prevent degradation and to preserve sterility. Methods for lyophilizing liquid compositions are known to those of ordinary skill in the art. Just prior to use, the composition may be reconstituted with a sterile diluent (Ringer's solution, distilled water, or sterile saline, for example) which may include additional ingredients. Upon reconstitution, the composition is preferably administered to subjects using those methods that are known to those skilled in the art.


Another aspect of the present invention relates to conjugates of the isolated peptides or isolated multimeric peptides according to the present invention. Accordingly, the isolated peptides or isolated multimeric peptides according to the present invention may be an amino acid sequence conjugated at any amino acid sidechain or within the amino acid sequence with any chemical moiety, such as any therapeutic agent, such as any immunomodulating compound.


The terms “therapeutic agent”, such as “immunomodulating agent” or virus reservoir purging agent as used herein, includes but is not limited to cytokines, such as interferons, monoclonal antibodies, such as ant-PD1 antibodies, cyclophosphamide, Thalidomide, Levamisole, and Lenalidomide.


“A virus reservoir purging agent”, includes but is not limited to auranofin, IL-7, prostratin, bryostatin, HDAC inhibitors, such as vorinostat, and Disulfuram.


Use of the peptides for evaluating immune responses:


The peptides according to the present invention may be used as diagnostic reagents. For example, a peptide of the invention may be used to determine the susceptibility of a particular individual to a treatment regimen which employs the peptide or related peptides, and thus may be helpful in modifying an existing treatment protocol or in determining a prognosis for an affected individual. In addition, the peptides may also be used to predict which individuals will be at substantial risk for developing a chronic virus infection.


Accordingly, the present invention relates to a method of determining the outcome for a subject exposed to a virus, comprising the steps of determining whether the subject has an immune response to one or more peptides according to the present invention.


In a preferred embodiment of the invention, the peptides as described herein can be used as reagents to evaluate an immune response. The immune response to be evaluated can be induced by using as an immunogen any agent that may result in the production of antigen-specific CTLs or HTLs that recognize and bind to the peptide(s) to be employed as the reagent. The peptide reagent need not be used as the immunogen. Assay systems that can be used for such an analysis include relatively recent technical developments such as tetramers, staining for intracellular lymphokines and interferon release assays, or ELISPOT assays.


For example, a peptide of the invention may be used in a tetramer staining assay to assess peripheral blood mononuclear cells for the presence of antigen-specific CTLs following exposure to an antigen or an immunogen. The HLA-tetrameric complex is used to directly visualize antigen-specific CTLS (see, e.g., Ogg et al., 1998; and Altman et al., 1996) and determine the frequency of the antigen-specific CTL population in a sample of peripheral blood mononuclear cells. A tetramer reagent using a peptide of the invention may be generated as follows: a peptide that binds to an HLA molecule is refolded in the presence of the corresponding HLA heavy chain and beta2-microglobulin to generate a trimolecular complex. The complex is biotinylated at the carboxyl terminal end of the heavy chain at a site that was previously engineered into the protein. Tetramer formation is then induced by the addition of streptavidin. By means of fluorescently labeled streptavidin, the tetramer can be used to stain antigen-specific cells. The cells may then be identified, for example, by flow cytometry. Such an analysis may be used for diagnostic or prognostic purposes. Cells identified by the procedure can also be used for therapeutic purposes. As an alternative to tetramers also pentamers or dimers can be used (Current Protocols in Immunology (2000) unit 17.2 supplement 35)


Peptides of the invention may also be used as reagents to evaluate immune recall responses. (see, e.g., Bertoni et al., 1997 and Perma et al., 1991.). For example, patient PBMC samples from individuals with HCV infection may be analyzed for the presence of antigen-specific CTLs or HTLs using specific peptides. A blood sample containing mononuclear cells may be evaluated by cultivating the PBMCs and stimulating the cells with a peptide of the invention. After an appropriate cultivation period, the expanded cell population may be analyzed, for example, for cytotoxic activity (CTL) or for HTL activity.


The peptides may also be used as reagents to evaluate the efficacy of a vaccine.


PBMCs obtained from a patient vaccinated with an immunogen may be analyzed using, for example, either of the methods described above. The patient is HLA typed, and peptide epitope reagents that recognize the allele-specific molecules present in that patient are selected for the analysis. The immunogenicity of the vaccine is indicated by the presence of epitope-specific CTLs and/or HTLs in the PBMC sample.


The peptides of the invention may also be used to make antibodies, using techniques well known in the art (see, e.g. CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley/Greene, NY; and Antibodies A Laboratory Manual, Harlow and Lane, Cold Spring Harbor Laboratory Press, 1989). Such antibodies include those that recognize a peptide in the context of an HLA molecule, i.e., antibodies that bind to a peptide-MHC complex.


In certain embodiments a first monomeric peptide and the at least one second monomeric peptide are associated via a linker; the linker may comprise any peptide linker, or peptide spacer, such as a glycine, a lysine or an arginine linker/spacer, a polyhistidinyl tag, Protein G, and Protein A but it is also possible to use a bis-maleimide linker/spacer, a disulfide linker, or a polyethylene glycol (PEG) linker. In practice, any linker found useful in peptide chemistry is also useful as a linker according to the present invention. Thus, the invention contemplates the use of “simple” linear peptides which are conjugated or fused to each other, but also peptide combinations where the individual peptides derived from a natural antigen are linked via non-peptide linkers. Use of multiple linker types are also within the scope of the present invention, and it is e.g. also a part of the invention to utilise linear peptides which include intrachain disulphide linkers.


Particularly interesting peptide combinations of the invention are set forth in the preamble to the examples.


In certain embodiments, at least one of the first and at least one second peptides in the peptide combination comprises an N- or C-terminal modification, such as an amidation, acylation, or acetylation.


Since the peptide combinations are contemplated as vaccine agents or diagnostic agents, they are in certain embodiments coupled to a carrier molecule, such as an immunogenic carrier. The peptides of the peptide combinations may thus be linked to other molecules either as recombinant fusions (e.g. via CLIP technology) or through chemical linkages in an oriented (e.g. using heterobifunctional cross-linkers) or nonoriented fashion. Linking to carrier molecules such as for example diphtheria toxin, latex beads (convenient in diagnostic and prognostic embodiments), and magnetic beads (also convenient in diagnostic and prognostic embodiments), polylysine constructs etc, are all possible according to the invention.


The immunogenic carrier is conveniently selected from carrier proteins such as those conventionally used in the art (e.g. diphtheria or tetanus toxoid, KLH etc.), but it is also possible to use shorter peptides (T-helper epitopes) which can induce T-cell immunity in larger proportions of a population. Details about such T-helper epitopes can e.g. be found in WO 00/20027, which is hereby incorporated by reference herein—all immunologic carriers and “promiscuous” (i.e. universal) T-helper epitopes discussed therein are useful as immunogenic carriers in the present invention.


In certain embodiments, the carrier is a virus like particle, i.e. a particle sharing properties with virions without being infectious. Such virus-like particles may be provided chemically (e.g. Jennings and Bachmann Ann Rev Pharmacol. Toxicol. 2009. 49:303-26 Immunodrugs: Therapeutic VLP-based vaccines for chronic diseases) or using cloning techniques to generate fusion proteins (e.g. Peabody et al. J. Mol. Biol. 2008; 380: 252-63. Immunogenic display of diverse peptides on virus-like particles of RNA phage MS2). Another example is “Remune”, an HIV vaccine originally made by Immune Response Corporation, which consists of formalin inactivated HIV that has been irradiated to destroy the viral genome.


In an embodiment, a nucleic acid is encoding one or more monomeric peptide of the multimeric, such as dimeric peptide according to the invention, where the encoded first peptide and the encoded at least one second peptide of a multimeric peptide are associated via a peptide linker, including a peptide spacer, and/or a disulphide bridge. The peptide linker/spacer is typically selected from the group consisting of a glycine, an arginine, a lysine linker/spacer, or a glycine-lysine linker/spacer, but any peptide linker known in the art may be useful. The term peptide linker thus also is intended to denote coupling between the first and second peptide via a peptide bond. A peptide linker that links a first and second peptide by standard peptide bonds may also be referred to as a peptide spacer. Also, the first and second peptides may be linked via a peptide linker and a disulphide bond, as is the case when an intrachain disulphide bond is established.


In one embodiment, the nucleic acid according to the invention encodes the peptide combination, which is coupled (by fusion) to a carrier molecule, such as an immunogenic carrier; useful carriers are discussed above.


In some embodiments the linker is selected from the group consisting of a bis-maleimide linker, a disulfide linker, a polyethylene glycol (PEG) linker, a glycine linker/spacer, a lysine linker/spacer, and an arginine linker/spacer.


In some embodiments the multimeric peptide, such as a dimeric peptide contain a linker in the free amino group of the N-terminal of a monomeric peptide linking said monomeric peptide to another monomeric peptide.


In some embodiments the multimeric peptide, such as a dimeric peptide contain a linker in the free carboxyl group of the C-terminal of a monomeric peptide linking said monomeric peptide to another monomeric peptide.


At least two options for such linkers are described in A. R Jacobson et al, J. Med. Chem. 1989, 32, 1708-1717 and in D Giannotti et al, Journal of Medicinal Chemistry, 2000, Vol. 43, No. 22, the disclosures of which is hereby incorporated by reference.


Alternatively a link between the N-termini of peptides may be established by reacting with Br—(CH2)n—Br.


The length of the linker may be varied by the addition of glycine residues, for example Fmoc-NH—CH2CH2—NH-Gly-NH2 may be used.


An example of such a synthesis, wherein a dimeric peptide is prepared by conjugation through succinic acid, may be as follows:









(H-Arg-Gly-Thr-Pro-Ile-Har-Gln-Asp-Trp-Gly-Asn-





Arg-Ala-Asn-Arg-Gly-Thr-Pro-Thr-Arg-Gln-Glu-





Trp-Asp-Cys-Arg-Ile-Ser-NH2Arg-Gly-Thr-Pro-Ile-





Har-Gln-Asp-Trp-Gly-Asn-Arg-Ala-Asn-Arg-Gly-Thr-





Pro-Thr-Arg-Gln-Glu-Trp-Asp-Cys-Arg-Ile-Ser-NH2)





E(H-Arg-Gly-Thr-Pro-Thr-Har-Asn-Gly-Trp-Asp-Val-





Lys-Leu-Ser-Arg-Gly-Thr-Pro-Ile-Har-Gln-Glu-Trp-





Har-Ser-Leu-Nle-Asn-Gln-Glu-Trp-NH2)F


(Succinic acid linker between Arg1E and Arg1F)






This dimer was produced from the reaction of the following 2 monomers:









Monomer E


H-Arg-Gly-Thr-Pro-Ile-Har-Gln-Asp-Trp-Gly-Asn-Arg-





Ala-Asn-Arg-Gly-Thr-Pro-Thr-Arg-Gln-Glu-Trp-Asp-





Cys-Arg-Ile-Ser-NH2Arg-Gly-Thr-Pro-Ile-Har-Gln-





Asp-Trp-Gly-Asn-Arg-Ala-Asn-Arg-Gly-Thr-Pro-Thr-





Arg-Gln-Glu-Trp-Asp-Cys-Arg-Ile-Ser-NH2





Monomer F


H-Arg-Gly-Thr-Pro-Thr-Har-Asn-Gly-Trp-Asp-Val-Lys-





Leu-Ser-Arg-Gly-Thr-Pro-Ile-Har-Gln-Glu-Trp-Har-





Ser-Leu-Nle-Asn-Gln-Glu-Trp-NH2






The two monomers are reacted to give a heterodimer according to the reaction scheme outlined below; where the link is between N-terminal on Arg1 of on chain E and the N-terminal on Arg1 in chain F.


Monomers E and F are synthesized separately on a Sieber Amid resin. The Fmoc-groups on N-terminal Gly are removed while the peptides are still on resin. The peptides are cleaved from resin. The resulting protected peptide E is reacted with succinic acid anhydride and thereafter reacted with the protected peptide F. Protective groups are subsequently removed with 95% TFA. The formed heterodimer may be purified from un-reacted monomers by conventional purification methods known to the person skilled in the art.


An example of a synthesis, wherein a dimeric peptide is prepared by conjugation through di-amino propane, may be as follows:









(H-Gly-Gly-Ala-Lys-Arg-Arg-Val-Val-Gln-Arg-Glu-





Lys-Arg-Ala-Gly-Glu-Arg-Glu-Lys-Arg-Ala-Gly-Gly)





G(H-Gly-Gly-Ile-Glu-Glu-Glu-Gly-Gly-Arg-Asp-Arg-





Asp-Arg-Gly-Gly-Glu-Gln-Asp-Arg-Asp-Arg-Gly-Gly)H


trifluoroacetate salt (Diamino propane linker


between Gly23 and Gly23)






This dimer was produced from the reaction of the following 2 protected monomers









Monomer G


H-Arg-Gly-Thr-Pro-Ile-Har-Gln-Asp-Trp-Gly-Asn-Arg-





Ala-Asn-Arg-Gly-Thr-Pro-Thr-Arg-Gln-Glu-Trp-Asp-





Cys-Arg-Ile-Ser-COOH





Monomer H


H-Arg-Gly-Thr-Pro-Thr-Har-Asn-Gly-Trp-Asp-Val-Lys-





Leu-Ser-Arg-Gly-Thr-Pro-Ile-Har-Gln-Glu-Trp-Har-





Ser-Leu-Nle-Asn-Gln-Glu-Trp-COOH






The two monomers G and H are reacted to give a heterodimer according to the reaction scheme outlined below; where the link is between C-terminal on Ser28 of on chain G and the C-terminal on Trp31 in chain H.


Monomers G and H are synthesized separately on a 2-chlorotrityl resin. Boc-Gly-OH is coupled to the peptides on the resin before cleaving them of the resin. The resulting peptides are then Boc-protected, alternatively they may me acetylated before being cleaved of the resin. The resulting protected peptide G is reacted with Fmoc-diaminopropane, Fmoc is deprotected and G is coupled to the C-terminal of the protected peptide H via a peptide bond. Protective groups are subsequently removed with 95% TFA. The formed heterodimer may be purified from un-reacted monomers by conventional purification methods known to the person skilled in the art.


Method for Synthesis of Cys-Lys Bridge:

Exemplified with the preparation of BI-155-3 trifluoroacetate salt









(H-Arg-Gly-Cys(2-oxo-ethyl)-Thr-Pro-Ile-Har-Gln-





Asp-Trp-Gly-Asn-Arg-Ala-Asn-Arg-Gly-Thr-Pro-Thr-





Arg-Gln-Glu-Trp-Asp-Cys-Arg-Ile-Ser-NH2)A(H-Arg-





Gly-Lys-Thr-Pro-Thr-Har-Asn-Gly-Trp-Asp-Val-Lys-





Leu-Ser-Arg-Gly-Thr-Pro-Ile-Har-Gln-Glu-Trp-Har-





Ser-Leu-Nle-Asn-Gln-Glu-Trp-NH2)B


trifluoroacetate salt (Thioether bond


between Cys(2-oxo-ethyl) 3A and Lys3B)






This dimer was produced from the reaction of the following 2 protected monomers









Monomer A


H-Arg-Gly-Cys-Thr-Pro-Ile-Har-Gln-Asp-Trp-Gly-





Asn-Arg-Ala-Asn-Arg-Gly-Thr-Pro-Thr-Arg-Gln-





Glu-Trp-Asp-Cys-Arg-Ile-Ser-NH2





Monomer B


H-Arg-Gly-Lys(bromoacetyl)-Thr-Pro-Thr-Har-Asn-





Gly-Trp-Asp-Val-Lys-Leu-Ser-Arg-Gly-Thr-Pro-Ile-





Har-Gln-Glu-Trp-Har-Ser-Leu-Nle-Asn-Gln-Glu-Trp-





NH2






Or with the preparation of BI-155-4 trifluoroacetate salt









(H-Gly-Ala-Lys-Arg-Arg-Val-Val-Gly-Gly-Cys(2-oxo-





ethyl)-Gly-Gly-Ala-Lys-Arg-Arg-Val-Val-Gln-Arg-





Glu-Lys-Arg-Ala-Gly-Glu-Arg-Glu-Lys-Arg-Ala-NH2)





A(H-Gly-Lys-Gly-Gly-Ile-Glu-Glu-Glu-Gly-Gly-Arg-





Asp-Arg-Asp-Arg-Gly-Gly-Gln-Asp-Arg-Asp-Arg-NH2)B


trifluoroacetate salt (Thioether bond


between Cys(2-oxo-ethyl)9A and Lys2B)






This dimer was produced from the reaction of the following 2 protected monomers:









Monomer A


H-Arg-Gly-Cys-Thr-Pro-Ile-Har-Gln-Asp-Trp-Gly-





Asn-Arg-Ala-Asn-Arg-Gly-Thr-Pro-Thr-Arg-Gln-





Glu-Trp-Asp-Ala-Arg-Ile-Ser-NH2





Monomer B


H-Arg-Gly-Lys(bromoacetyl)-Thr-Pro-Thr-Har-Asn-





Gly-Trp-Asp-Val-Lys-Leu-Ser-Arg-Gly-Thr-Pro-Ile-





Har-Gln-Glu-Trp-Har-Ser-Leu-Nle-Asn-Gln-Glu-Trp-





NH2






The 2 monomers are reacted to give a heterodimer according to the reaction scheme outlined below; where the link is created between Lys3 (bromoacetyl) side chain on chain B and Cys in chain A.


At neutral pH and room temperature, bromoacetyl moieties in buffered aqueous solutions are very reactive towards SH-containing moieties, such as the thiol group in cysteine. Thus, if a cysteine is present on the other peptide sequence, the SH will attack the bromoacetyl to form a intermolecular thioether bridge. When the reaction is buffered with a sodium-containing buffer, such as NaHCO3, the only byproduct of the reaction is NaBr, an innocuous salt.


The formed heterodimer may be purified from un-reacted monomers by conventional purification methods known to the person skilled in the art.


Method for synthesis of oxime bond between two peptide sequences, an intermolecular bond:


Exemplified with the preparation of BI-155 trifluoroacetate salt









(H-Arg-Gly-Dpr(Ser)-Thr-Pro-Thr-Har-Asn-Gly-Trp-





Asp-Val-Lys-Leu-Ser-Arg-Gly-Thr-Pro-Ile-Har-Gln-





Glu-Trp-Har-Ser-Leu-Nle-Asn-Gln-Glu-Trp-NH2)D(H-





Arg-Gly-Dpr(Aoa)-Thr-Pro-Ile-Har-Gln-Asp-Trp-Gly-





Asn-Arg-Ala-Asn-Arg-Gly-Thr-Pro-Thr-Arg-Gln-Glu-





Trp-Asp-Cys-Arg-Ile-Ser-NH2)C


trifluoroacetate salt (oxime is created


between Dpr(Ser)3D and Dpr(Aoa)3C)






This dimer is produced from the reaction of the following two monomers:









Monomer C


H-Arg-Gly-Dpr(Aoa)-Thr-Pro-Ile-Har-Gln-Asp-Trp-





Gly-Asn-Arg-Ala-Asn-Arg-Gly-Thr-Pro-Thr-Arg-





Gln-Glu-Trp-Asp-Cys-Arg-Ile-Ser-NH2





Monomer D


H-Arg-Gly-Dpr(Ser)-Thr-Pro-Thr-Har-Asn-Gly-Trp-





Asp-Val-Lys-Leu-Ser-Arg-Gly-Thr-Pro-Ile-Har-Gln-





Glu-Trp-Har-Ser-Leu-Nle-Asn-Gln-Glu-Trp-NH2






The two monomers are reacted to give a heterodimer according to the reaction scheme outlined below; where the link is created between Dpr(Aoa)3 side chain on chain C and oxidized Dpr(Ser) in chain D.


After removal of the Mtt group from Lys and while the peptide was still attached to the resin aminooxyacetylated (AoA) monomer C was synthesized by coupling aminooxyacetic acid to Lys. The peptide was then cleaved from the solid phase support and purified by conventional purification methods. The monomer D was, after cleavage from resin and purification, created by oxidation of the serinyl diaminopropionic acid residue (Dpr(Ser)) with periodate to the aldehyde function. Equimolar amounts of monomer A and B were dissolved in acetonitrile and acetate buffer (pH 4). After reaction for 16 h at room temperature, the product C-oxime-D was isolated by conventional purification methods known to the person skilled in the art.


Dpr=diaminopropionic acid


Fmoc-Dpr (Boc-Ser(tBu))-OH Merck 04-12-1186

Method for Synthesis of Dimers with PEG-Linker:


A multimeric, such as dimeric peptide, such as a heterodimeric peptide may be synthesized by, but are not restricted to the following protocol:


To the peptidyl resin containing deblocked Asp or Glu residue (monomer 1) is added HBTU, DIPEA and Trt-amino PEG amine in DMF. The mixture is allowed to couple over night. The resin is filtered from the solution and washed by standard protocol. The Trt group is removed from the Trt-PEGylated peptide. The monomer 2 containing deblocked Asp or Glu residue is then coupled to the exposed amino group using HBTU and DIPEA. After cleavage the desired product is purified using any suitable technique to give the desired multimeric peptide.


In some embodiments the isolated monomeric peptide contain intramolecular bonds, such as in the form of intramolecular Cys-Cys bonds. It is to be understood that the “intramolecular bond”, used interchangeably with “intrachain bond”, is a bond between two different amino acids within the same peptide chain, which however is not necessarily adjacent to each other in the peptide sequence. Accordingly, in some embodiments, the isolated multimeric peptide according to the invention may contain both intramolecular bonds within one or more of the monomers, as well as an intermolecular bond between two chains of the multimeric peptide, such as a dimer. This intramolecular bond may be in the form of Cys-Cys bonds formed with cysteine residues within the same peptide sequence. In some embodiments the monomer contains an intramolecular bond derived from a Lys residue or other amino acid residue, such as a Ser, Cys, Asp or Glu that make the bond, such as a thioether bond or an oxime bond or through a PEG linker, to an amino acid residue on the other monomer peptide sequence.


Method for Synthesis of Multimeric Peptides with PolyLys or MAPS:


PolyLys or MAPS (multiple antigen peptides)—has been extensively used over the last 20 years as a carrier protein to produce strong immunogenic response. The MAP system utilizes a peptidyl core of three or more radially branched lysine core to form a backbone for which the epitope sequences of interest can be built parallel using standard solid-phase chemistry.


The MAP system is a commercial product available from several companies such as AnaSpec, Bio-synthesis Inc. and others. The product, as offered in the catalogue only allows attachment of two (identical) peptide sequence to the polyLys core. It is however possible also to link two different peptide sequences by using different protecting groups for alfa- and epsylon-amino functional groups of lysine on the two different peptide sequences.


Use of the MAP system has been described in references including: Wang, C. Y et al. “Long-term high-titer neutralizing activity induced by octameric synthetic HIV antigen” Science 254, 285-288 (1991). Posnett, D. et al. “A novel method for producing anti-peptide antibodies” J. Biol. Chem. 263, 1719-1725 (1988), and in Tam, J. P. “Synthetic peptide vaccine design: synthesis and properties of a high-density multiple antigenic peptide system” PNAS USA 85, 5409-5413 (1988).


The MAP system could also be prepared by chemical (thioether, oxime, hydrazone) ligation of appropriately functionalized tetra- or octavalent polylysine constructs with the peptide antigen. By the use of this chemical ligation, the two peptide sequences being linked together would not have to be identical as they are synthesized separately.


Additionally a novel application of the MAP-based system is to synthesize on solid support a “probe” containing a poly(ethylene glycol) (PEG) chain in the dendritic arms of MAP.


Use of the MAP system will increase the size of a multimeric complex and may increase the immunogenic response.


Methods for the Synthesis of Multimeric Peptides Using PEG:

Suitable Multi-Arm Activated PEG to be used for a PEG linker are commercially available, e.g. a compound with the following structure:




embedded image


Wherein X may be ethanethiol —CH2CH2SH (could be used to form S—S bridge with the epitope or a thioether link) or propylamine —CH2CH2CH2NH2, among others. These handles preferably allows for the linking of two identical peptide sequences and may be seen as a poly-monomeric epitope presenting construct. One could, however, anchor a dimer (two epitopes linked together) to the PEG above.


Method for Synthesis of Peptide-Poly-L-Lys (PLL)-Polyethylene Glycol (PEG) Construct:

Peptide—PLL-PEG constructs, may be synthesized by, but are not restricted to the following protocol:


Fmoc-Poly-L-Lys-resin (a commercial product) is de-protected with 20% piperifine-DMF. Fmoc-NH-PEG4-COOH, in a mixed solvent of CH2Cl2-NMP is added followed by HBTU and DIPEA and the reaction is allowed to proceed for 24 h. The resultant pegylated poly-L-Lys-resin is washed and the pegylation step is repeated. The reaction is monitored by Kaiser's ninhydrin test until a negative reading is obtained. After de-protection of Fmoc group, four identical peptide chains are synthesized directly on the branched poly-L-Lys-polyethylene glycol core by a stepwise solid-phase procedure. All residues activated with HBTU and DIPEA are allowed to couple for 2 h. The coupling is monitored by Kaiser's ninhydrin test and is repeated if needed. After cleavage the desired product is purified using any suitable technique to give the desired peptide-construct.


Table 8 Specific peptides not part of the present invention. (Amino acids underlined refers to place of linker in dimeric molecules; Letter C in a large font refers to a cysteine residue optionally involved in an intramolecular bond with another cysteine residue in the same peptide sequence. Homoarginine is abbreviated Har, Norleucine is abbreviated as Nle or alternatively with the single letter “Z”, N-ε-methylated Lys is abbreviated Lys(Me), Citrulline is abbreviated with the single letter “B”, diaminopropionic acid is abbreviated with Dpr and serinyl diaminopropionic acid is abbreviated Dpr(Ser). Flu; abbreviation for Influenza).


Table 8 represent peptides not part of the present invention. These peptides relates to monomeric peptides as well as multimeric peptides comprising two or more of these monomeric peptides, each monomeric peptide independently consisting of not more than 60 amino acids with the following structure





X1-X2-X3-X4-X5-X6  (formula III),


wherein X1, X3 and optional moiety X5 independently defines a linear sequence of any 1, 2, 3, 4, or 5 amino acid independently selected from glycine, arginine, norleucine, glutamine, serine, lysine, tryptophan, cysteine, or a derivative thereof; X2, X4, and optional moiety X6 each independently defines a linear sequence of 5-17 amino acids, each having more than 50% sequence identity to a specific natural antigen, said monomeric peptides being covalently joined by one or more intermolecular bond.































Position  












with refer-












ence to












 positions   












in SEQ ID  












NO: 200,












 SEQ ID NO: 












202, and SEQ





Refer-






 ID NO: 203.





















Anti-
ence






X2-
X4-
X6-
Pro-


Chain
gen
ID
X1
X2
X3
X4
X5
X6
SEQ
SEQ
SEQ
tein






Flu
BI100_

RR

SLLTEVETP

GCG

VETPIR

G

TPIRNEWG
  2-
  7-
  9-
M2




CGn






 10
 12
 16





at
















Flu
BI100_

RR

SLZTDIETP

GCG

IDTPIR

G

TPIBQDWG
  2-
  7-
  9-
M2




CG






 10
 12
 16







Flu
BI100-

WWGC

TDIET

CG

IDTPIR

G

TPIBQDWG
  5-
  7-
  9-
M2




CGcyc






  9
 12
 16







Flu
BI100-

RRG

CSLLT

C

SLLTEVQTPIRN

GRR

SEWGSRSN
  2-
  2-
 13-
M2




Cyc_2






  5
 13
 20






A
Flu
BI150-

RRZ

C


SLLTEVQTPIRN

GRR

VETPIRN


  2-
  7-

M2




Dimer






 13
 13







B
Flu
BI150-

WWQ

C


TPIRSEWGCRSN

GRR

SNDSS

G


  9-
 19-

M2




Dimer






 20
 23







A
Flu
BI150-

WW

SLZTDIETP

G

C

G

IDTPIR

G

TPIBQDWG
  2-
  7-
  9-
M2




new






 10
 12
 16






B
Flu
BI150-

RR

IDTPIR

G

TPIBQDWG


K

G

SLZTDIETPG
  7-
  9-
  2-
M2




new

(Har)






 12
 16
 11






A
Flu
BI150-

R

SLZTDIETP


Dpr


IDTPIR
G
TPIBQDWG
  2-
  7-
  9-
M2




2mod






 10
 12
 16






B
Flu
BI150-

RR

IDTPIR

GG

TPI(Har)


Dpr


SLZTDIETPG
  7-
  9-
  2-
M2




2mod



QEW


(Ser)



 12
 15
 11






A
Flu
BI150-
RR
SLZTDIETP
GCG
IDTPIR
G
TPIBQDWG
  2-
  7-
  9-
M2




dim_2






 10
 12
 16






B
Flu
BI150-
Har
IDTPIR
G
TPIBQDWG

KG

SLZTDIETPG
  7-
  9-
  2-
M2




dim_2






 12
 16
 11







HIV
BI450-

W
D
WGC

AKRRV

CGG

AKRRVVQREKRA


501-
501-

gp120




AdjBT1






505
512








HIV
BI450-

W
D
WGC

IEEEG

CGG

IEEEGGERDR


222-
222-

gp41




AdjBT2






226









HIV


CGG

AKRRVV

GG

AKRRVV

G

QREKRAV
501-
501-
507-












506
506
513







HIV


CGGG

DQQLL

GG

AEEEIV

GG

IEEEGGERDRDR
257-
266-
221-












261
271
232







HIV


CGG

AKRRVV

GG

AKRRVV

GG

QREKR
501-
501-
507-












506
506
511







HIV


CGGG

DQQLL

GG

AEEEIV

GG

IEEEGG
257-
266-
222-












261
271
227







HIV


CGG

AEEEVV

GG

DQQLL


266-
257-













271
261








HIV


GCGG

AKRRVV

GG

AKRRVV


501-
501-













506
506







A
HIV
BI400-

G

AKRRVV

GG

C

GG

AKRRVVQREKRA

G

EREKRA
501-
501-
507-
gp120




B (a-






506
512
512





chain)















B
HIV
BI400-

G

K

G

GIEEE

GG

RDRDR

GG

EQDRDR
221-
229-
228-
gp41




B (b-






225
233
233





chain)















E
HIV



G

G

AKRRVVQREKRA

G

EREKRA


501-
507-

gp120











512
512







F
HIV



G


GIEEE

GG

RDRDR

GG

EQDRDR
221-
229-
228-
gp41











225
233
233






G
HIV



G

G

AKRRVVQREKRA

G

EREKRA

GG


501-
507-

gp120











512
512







H
HIV



G


GIEEE

GG

RDRDR

GG

EQDRDRGG
221-
229-
228-
gp41











225
233
235






A
HIV
400-

G

AKRRVV

GG

C

GG

AKRRVVQREKRA

G

EREKRA
501-
501-
507-
gp120




Seq B






506
512
512





(a-














chain)















B
HIV
400-

G

K

G

GIEEE

GG

RDRDR

GG

QDRDR
221-
229-
229-
gp41




Seq B






225
233
233





(b-














chain)















D
HIV
400- 

G

AKRRVV

GG

(Dpr


AKRRVVQREKRA

G

EREKRA
501-
501-
507-
gp120




Seq B*




(Ser))





506
512
512





(a-



GG












chain)















C
HIV
400- 

G

K

G

GIEEE

GG

RDRDR

GG

QDRDR
221-
229-
229-
gp41




Seq B*






225
233
233





(b-














chain)















A
HIV
BI400-

G

AKRRVV

GG

C

GG

AKRRVVQREKRA

G

EREKRA
501-
501-
507-
gp120




Bu1






506
512
512





(a-














chain)















B
HIV
BI400-

G

K

G

GIEEE

GG

ERDRDR

GG

QDRDR
221-
228-
229-
gp41




Bu1






225
233
233





(b-














chain)















A
HIV
BI400-

G

AKRRVV

GG

C

GG

AKRRVVEREKRA

G

QREKRA
501-
501-
507-
gp120




Bu2






506
512
512





(a-














chain)















B
HIV
BI400-

G

K

G

GIEEE

GG

QDRDR

GG

RDRDR
221-
229-
229-
gp41




Bu2






225
233
233





(b-














chain)















A
HIV
BI400-

G

AKRRVV

GG

C

GG

AKRRVVEREKRA

G

QREKRA
501-
501-
507-
gp120




Bu3






506
512
512





(a-














chain)















B
HIV
BI400-

G

K

G

GIEEE

GG

EQDRDR

GG

ERDRD
221-
228-
228-
gp41




Bu3






225
233
232





(b-














chain)















A
HIV
SEQ400_
GC
AKRRVV
CGGKG
AKRRVVQREKRA

G

EREKRA
501-
501-
507-
gp120




B






506
512
512





(Cyc)















B
HIV
SEQ400_

G

K

G

GIEEE

GG

RDRDR

GG

EQDRDR
221-
229-
228-
gp41




B






225
233
233





(Cyc)















A
HIV
SEQ400_
GC
AKRRVV

CGGKG

GAKRRVVQREKRA
G
EREKRA
501-
501-
506-
gp120




B






506
512
512





(Cyc)















B
HIV
SEQ400_
GCGG
IEEEGGRDRDR
GG
QDRDR


222-
229-

gp41




B






233
233






(Cyc)















A
HIV
BI400-
G
CAKRRVVC
GGKGG
AKRRVVQREKRA
G
EREKRA
501-
501-
507-
gp120




bu1






506
512
512





(Cyc)















B
HIV
BI400-

CGG

IEEEGGERDRDR
GG
QDRDR


222-
229-

gp41




bu1






233
233






(Cyc)















A
HIV
BI400-
G
CAKRRVVC
GGKGG
AKRRVVEREKRA
G
QREKRA
501-
501-
507-
gp120




bu2






506
512
512





(Cyc)















B
HIV
BI400-

CGG

IEEEGGQDRDR
GG
RDRDR


222-
229-

gp41




bu2






233
233






(Cyc)















A
HIV
BI400-
G
CAKRRVVC
GGKGG
AKRRVVEREKRA
G
QREKRA
501-
501-
507-
gp120




bu3






506
512
512





(Cyc)















B
HIV
BI400-

CGG

IEEEGGEQDRDR
GG
RDRDR


222-
229

gp41




bu3






233
233






(Cyc)















A
HIV
BI400-
G
CAKRRVVC
GGKGG
AKRRVVQREKRA
G
EREKRA
501-
501-
507-
gp120




rev






506
512
512





(Cyc)















B
HIV
BI400-

CGG

EEEIGGRDRD
GG
RDRDQ


222-
229-

gp41




rev






233
233






(Cyc)















A
HIV
BI450- 
GG
RLEPWKH
GC
GSQPKTA
G
HPGSQ
  7-
 15-
 13-
Tat




1 (a-






 13
 21
 17





chain)















B
HIV
BI450-
GG
FHSQV

C

FITKGLGISYGRK


 32-
 38-

Tat




1 (b-






 36
 50






chain)















A
HIV
BI450-

RLEPWKH
GC
GSQPKTA
GWK
HPGSQ
  7-
 15-
 13-
Tat




1_2






 13
 21
 17





(a-














chain)















B
HIV
BI450-

C

FITKGLGISY
G
FITKGLGISYGRK


 38-
 38-

Tat




1_2






 47
 50






(b-














chain)















A
HCV
BI350-
RR
LLADARVCS
GG
LLADARVSA


342-
342-

E2




1 (a-






350
350






chain)















B
HCV
BI350-
R
GV(Nle)

C

GVLAGIAYYS


163-
163-

E1




1 (b-

AGIAYFS




172
172






chain)















A
HCV
BI350-
RR

GNWAKVL


K


NWAKVI



366-
367-

E1




1mod1






372
372







B
HCV
BI350-
RRG

LLADARV

GCG

SGADRV

CS

342-
342-

E2




1mod1






348
348







A
HCV
BI350-
RR

GNWAKVL


Dpr


NWAKVI



366-
367-

E1




1mod2






372
372







B
HCV
BI350-
RRG

LLADARV

G(Dpr

SGADRV

CS

342-
342-

E2




1mod2



(Ser))




348
348









G












A
HCV

RR

GNWAKVL


Lys


NWAKVI



366-
367-

E1








(Me)




372
372







B
HCV

RRG

LLADARV

GEG

SGADRV

CS

342-
342-

E2











348
348







A
HCV

RR

GNWAKVL


Lys


NWAKVI



366-
367-

E1








(Me)




372
372







B
HCV

RRG

LLADARV

GDG

SGADRV

CS

342-
342-

E2











348
348







A
HCV

RR

GNWAKVL


E


NWAKVI



366-
367-

E1











372
372







B
HCV

RRG

LLADARV

G(Lys

SGADRV

CS

342-
342-

E2








(Me))




348
348









G












A
HCV

RR

GNWAKVL


D


NWAKVI



366-
367-

E1











372
372







B
HCV

RRG

LLADARV

G(Lys

SGADRV

CS

342-
342-

E2








(Me))




348
348









G









SPECIFIC EMBODIMENTS OF THE INVENTION

In some embodiments the isolated peptide according to the present invention has a total of not more than 60 amino acids.


In some embodiments the sequence of amino acids defined by (Z1-Z2)1-Z3-(Z4-Z5)2-Z6-(Z7-Z8)3-Z9-(Z10-Z11)4-Z12 is not found in any native sequence of a protein.


In some embodiments the peptide according to the present invention is demonstrated to translocate across a plasma membrane in the assay based on biotinylation of peptides as described in example 5.


In some embodiments Z3, and optional Z6, Z9 and Z12 defines an amino acid sequence identical to the native sequence of a known antigen.


In some embodiments Z3, and optional Z6, Z9 and Z12 defines an amino acid sequence not identical to the native sequence of any known antigen.


In some embodiments Z3, and optional Z6, Z9 and Z12 defines any chemical moiety, which is any therapeutical compound, such as an immunomodulating compound, such as a Cox-2 inhibitor.


In some embodiments the peptide according to the present invention is capable of inducing a T-lymphocyte response.


In some embodiments the peptide according to the present invention is capable of inducing a CD4+ and/or a CD8+ T-lymphocyte response.


In some embodiments the antigen is a viral protein, such as a capsid protein.


In some embodiments the viral protein is selected from a protein of the Hepatitis C virus, such as a core protein; protein of influenza virus, such as an M2 protein.


In some embodiments the viral protein of Hepatitis C virus is selected from HCV consensus sequence of genotype 1, such as subtypes 1a and 1b, genotype 2 such as 2a and 2b and genotype 3, such as 3a.


In some embodiments, in the peptide according to the present invention, the specific natural antigen is a protein or peptide sequence derived from a disease antigen, such as an infectious agent, such as bacteria, virus, parasite, fungus, or cancer antigens such as oncogene (lung, stomach, breast cancer) or an antigen causing an autoimmune disease such as diabetes, multiple sclerosis (MS), celiac disease, Myalgic Encephalomyelitis (ME), psoriasis, and/or Crohn's Disease.


Accordingly confirmed and suspected autoimmune diseases, where relevant antigens may be derived include Achlorhydra Autoimmune Active Chronic Hepatitis, Acute Disseminated Encephalomyelitis, Acute hemorrhagic leukoencephalitis, Addison's Disease, Agammaglobulinemia, Alopecia greata, Amyotrophic Lateral Sclerosis, Ankylosing Spondylitis, Anti-GBM/TBM Nephritis, Antiphospholipid syndrome, Antisynthetase syndrome, Arthritis, Atopic allergy, Atopic Dermatitis, Autoimmune Aplastic Anemia, Autoimmune cardiomyopathy, Autoimmune hemolytic anemia, Autoimmune hepatitis, Autoimmune inner ear disease, Autoimmune lymphoproliferative syndrome, Autoimmune peripheral neuropathy, Autoimmune pancreatitis, Autoimmune polyendocrine syndrome Types I, II, & III, Autoimmune progesterone dermatitis, Autoimmune thrombocytopenic purpura, Autoimmune uveitis, Balo disease/Balo concentric sclerosis, Bechets Syndrome, Berger's disease, Bickerstaff's encephalitis, Blau syndrome, Bullous Pemphigoid, Castleman's disease, Chagas disease, Chronic Fatigue Immune Dysfunction Syndrome, Chronic inflammatory demyelinating polyneuropathy, Chronic recurrent multifocal ostomyelitis, Chronic lyme disease, Chronic obstructive pulmonary disease, Churg-Strauss syndrome, Cicatricial Pemphigoid, Coeliac Disease, Cogan syndrome, Cold agglutinin disease, Complement component 2 deficiency, Cranial arteritis, CREST syndrome, Crohns Disease (one of two types of idiopathic inflammatory bowel disease “IBD”), Cushing's Syndrome, Cutaneous leukocytoclastic angiitis, Dego's disease, Dercum's disease, Dermatitis herpetiformis, Dermatomyositis, Diabetes mellitus type 1, Diffuse cutaneous systemic sclerosis, Dressler's syndrome, Discoid lupus erythematosus, Eczema, Endometriosis, Enthesitis-related arthritis, Eosinophilic fasciitis, Epidermolysis bullosa acquisita, Erythema nodosum, Essential mixed cryoglobulinemia, Evan's syndrome, Fibrodysplasia ossificans progressiva, Fibromyalgia, Fibromyositis, Fibrosing aveolitis, Gastritis, Gastrointestinal pemphigoid, Giant cell arteritis, Glomerulonephritis, Goodpasture's syndrome, Graves' disease, Guillain-Barré syndrome (GBS), Hashimoto's encephalitis, Hashimoto's thyroiditis, Haemolytic anaemia, Henoch-Schonlein purpura, Herpes gestationis, Hidradenitis suppurativa, Hughes syndrome (See Antiphospholipid syndrome), Hypogammaglobulinemia, Idiopathic Inflammatory Demyelinating Diseases, Idiopathic pulmonary fibrosis, Idiopathic thrombocytopenic purpura (See Autoimmune thrombocytopenic purpura), IgA nephropathy (Also Berger's disease), Inclusion body myositis, Inflammatory demyelinating polyneuopathy, Interstitial cystitis, Irritable Bowel Syndrome (IBS), Juvenile idiopathic arthritis, Juvenile rheumatoid arthritis, Kawasaki's Disease, Lambert-Eaton myasthenic syndrome, Leukocytoclastic vasculitis, Lichen planus, Lichen sclerosus, Linear IgA disease (LAD), Lou Gehrig's Disease (Also Amyotrophic lateral sclerosis), Lupoid hepatitis, Lupus erythematosus, Majeed syndrome, Ménière's disease, Microscopic polyangiitis, Miller-Fisher syndrome, Mixed Connective Tissue Disease, Morphea, Mucha-Habermann disease, Muckle-Wells syndrome, Multiple Myeloma, Multiple Sclerosis, Myasthenia gravis, Myositis, Narcolepsy, Neuromyelitis optica (Also Devic's Disease), Neuromyotonia, Occular cicatricial pemphigoid, Opsoclonus myoclonus syndrome, Ord thyroiditis, Palindromic rheumatism, PANDAS (Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus), Paraneoplastic cerebellar degeneration, Paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Parsonnage-Turner syndrome, Pars planitis, Pemphigus, Pemphigus vulgaris, Pernicious anaemia, Perivenous encephalomyelitis, POEMS syndrome, Polyarteritis nodosa, Polymyalgia rheumatica, Polymyositis, Primary biliary cirrhosis, Primary sclerosing cholangitis, Progressive inflammatory neuropathy, Psoriasis, Psoriatic Arthritis, Pyoderma gangrenosum, Pure red cell aplasia, Rasmussen's encephalitis, Raynaud phenomenon, Relapsing polychondritis, Reiter's syndrome, Restless leg syndrome, Retroperitoneal fibrosis, Rheumatoid arthritis, Rheumatoid fever, Sarcoidosis, Schizophrenia, Schmidt syndrome, Schnitzler syndrome, Scleritis, Scleroderma, Sjögren's syndrome, Spondyloarthropathy, Sticky blood syndrome, Still's Disease, Stiff person syndrome, Subacute bacterial endocarditis (SBE), Susac's syndrome, Sweet syndrome, Sydenham Chorea, Sympathetic ophthalmia, Takayasu's arteritis, Temporal arteritis (also known as “giant cell arteritis”), Tolosa-Hunt syndrome, Transverse Myelitis, Ulcerative Colitis (one of two types of idiopathic inflammatory bowel disease “IBD”), Undifferentiated connective tissue disease, Undifferentiated spondyloarthropathy, Vasculitis, Vitiligo, Wegener's granulomatosis, Wilson's syndrome, and Wiskott-Aldrich syndrome.


In some embodiments, in the peptide according to the present invention, the specific natural antigen is a viral protein, such as a structural protein, such as a capsid protein, a regulatory protein, an enzymatic protein, and a proteolytic protein.


In some embodiments, in the peptide according to the present invention, the viral protein is a protein, such as a structural protein, such as a core or envelope protein, of a virus selected from the Hepatitis C virus; influenza virus such as an M2 protein, human immunodeficiency virus (HIV), cytomegalovirus (CMV), and Human papillomavirus (HPV).


In some embodiments, in the peptide according to the present invention, the viral protein is a viral protein of Hepatitis C virus selected from any one HCV consensus sequence of a specific genotype, such as 1, such as subtypes is and 1b, genotype 2, such as 2a and 2b, genotype 3, such as 3a, genotype 4, genotype 5, and genotype 6.


In some embodiments the peptide according to the present invention is of 19-60 amino acids, such as of 20-60 amino acids, such as of 21-60 amino acids, such as of 22-60 amino acids, such as of 23-60 amino acids, such as of 24-60 amino acids, such as of 25-60 amino acids, such as of 26-60 amino acids, such as of 27-60 amino acids, such as of 28-60 amino acids, such as of 29-60 amino acids, such as of 30-60 amino acids, such as of 31-60 amino acids, such as of 32-60 amino acids, such as of 33-60 amino acids, such as of 34-60 amino acids, such as of 35-60 amino acids.


In some embodiments the peptide according to the present invention is of 18-60 amino acids, such as 18-59 amino acids, such as 18-58 amino acids, such as 18-57 amino acids, such as 18-56 amino acids, such as 18-55 amino acids, such as 18-54 amino acids, such as 18-53 amino acids, such as 18-52 amino acids, such as 18-51 amino acids, such as 18-50 amino acids, such as 18-49 amino acids, such as 18-48 amino acids, such as 18-47 amino acids, such as 18-46 amino acids, such as 18-45 amino acids, such as 18-44 amino acids, such as 18-43 amino acids, such as 18-42 amino acids, such as 18-41 amino acids, such as 18-40 amino acids, such as 18-39 amino acids, such as 18-38 amino acids, such as 18-37 amino acids, such as 18-35 amino acids, such as of 18-34 amino acids, such as of 18-33 amino acids, such as of 18-32 amino acids, such as of 18-31 amino acids, such as of 18-30 amino acids, such as of 18-29 amino acids, such as of 18-28 amino acids, such as of 18-27 amino acids, such as of 18-26 amino acids, such as of 18-25 amino acids, such as of 18-24 amino acids, such as of 18-23 amino acids, such as of 18-22 amino acids, such as of 18-21 amino acids, such as of 18-20 amino acids, such as of 18-19 amino acids.


In some embodiments in the peptide according to the present invention, the monomeric peptide contain one or more intramolecular bond, such as one or more Cys-Cys bond.


In some embodiments in the peptide according to the present invention, the monomeric peptide has delayed proteolytic degradation in the N-terminal, such as by incorporation of the first 1, 2, or 3 amino acids in the N-terminal in the D-form, or by incorporation of the first 1, 2, or 3 amino acids in the N-terminal in beta or gamma form.


In some embodiments, in the multimeric, such as a dimeric peptide according to the present invention, the two or more monomeric peptides are identical in sequence.


In some embodiments, in the multimeric, such as dimeric peptide according to the present invention, the two or more monomeric peptides are different in sequence.


In some embodiments, in the multimeric, such as dimeric peptide according to the present invention, one, two or more of the peptide strands of the multimeric, such as dimeric peptide has delayed proteolytic degradation in the N-terminal, such as by incorporation of the first 1, 2, or 3 amino acids in the N-terminal in the D-form, or by incorporation of the first 1, 2, or 3 amino acids in the N-terminal in beta or gamma form.


In some embodiments, in the multimeric, such as dimeric peptide according to the present invention, the linker is placed within any sequence selected from Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, and Z12, such as in Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, and Z12 of the first monomeric peptide to anywhere on the at least one second monomeric peptide, such as within the sequence of Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, and Z12.


In some embodiments, in the multimeric, such as dimeric peptide according to the present invention, the linker is placed at an amino acid position selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 of the first monomeric peptide to a position selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 of the at least one second monomeric peptide.


In some embodiments, in the multimeric, such as dimeric peptide according to the present invention, the multimeric, such as dimeric peptide contain a helper epitope of at least 12 amino acids, such as at least 13, 14, 15 or 17 amino acids, which helper epitope consist of a combined sequence of amino acids, which is a sequence of amino acids from a first specific continuous antigenic peptide sequences, and a sequence of amino acids from at least one second specific continuous antigenic peptide sequence of the same or different protein derived from the same virus, any different virus, or any disease antigen, such as between 2-12 amino acids from the first specific continuous antigenic peptide sequences and 2-12 amino acids from the at least one second specific continuous antigenic antigenic peptide sequence.


In some embodiments, in the isolated peptide according to the present invention, the peptide contain a helper epitope of at least 12 amino acids, such as at least 13, 14, 15 or 17 amino acids, which helper epitope consist of a combined sequence of amino acids, which is a sequence of amino acids from a first specific continuous antigenic peptide sequences, and a sequence of amino acids from at least one second specific continuous antigenic peptide sequence of the same or different protein derived from the same virus, any different virus, or any disease antigen, such as between 2-12 amino acids from the first specific continuous antigenic peptide sequences and 2-12 amino acids from the at least one second specific continuous antigenic antigenic peptide sequence.


It is to be understood that an epitope may not only be present within the sequence of the monomeric peptide. An epitope may also be present with a combination of amino acids of the first and the at least one second monomeric peptide in a multimeric, such as dimeric peptide sequence, wherein this combination of amino acids forms a sequence that span from the first to the at least one second monomeric peptide sequence. This epitope may be a continuous sequence of amino acids or it may be a three-dimensional epitope with amino acids found in both monomeric peptides.


In some embodiments, in the multimeric, such as dimeric peptide according to the present invention, the intermolecular bond is a disulfide (S—S) bond between two Cys residues.


In some embodiments, in the multimeric, such as dimeric peptide according to the present invention, the intermolecular bond is a methylated peptide bond between a N-ε-methylated Lys side-chain and the side-chain of an Asp or Glu residue.


In some embodiments, in the multimeric, such as dimeric peptide according to the present invention, the intermolecular bond is a thioether bond between a Cys residue in the first monomeric peptide and a modified Lys residue in the at least one second monomeric peptide.


In some embodiments, in the multimeric, such as dimeric peptide according to the present invention, the intermolecular bond is an oxime bond.


In some embodiments, in the multimeric, such as dimeric peptide according to the present invention, the intermolecular bond is an oxime bond between a derivatized Lys residue in the first monomeric peptide and a derivatized Ser residue in the at least one second monomeric peptide.


In some embodiments, in the multimeric, such as dimeric peptide according to the present invention, the intermolecular bond is an oxime bond between a derivatized lysine, ornitine or diaminopropionic acid residue in the first monomeric peptide and a derivatized serine moiety, such as a serine residue, such as in a serinyl diaminopropionic acid residue, such as in a serinyl lysin residue or such as in a serinyl ornitine residue, in the at least one second monomeric peptide.


In some embodiments, in the multimeric, such as dimeric peptide according to the present invention, the monomeric peptides are linked by a polyethylene glycol (PEG) linker, such as through an Asp or a Glu residue in the first monomeric peptide and an Asp or a Glu residue in the at least one second monomeric peptide.


In some embodiments, in the multimeric, such as dimeric peptide according to the present invention, any one of the monomeric peptides is independently as defined herein.


In some embodiments, the peptide according to the present invention is essentially a non-cell-penetrating peptide. In other embodiments, the peptide according to the present invention is a cell-penetrating peptide. In some embodiments, the peptide according to the present invention is able to attach to the cell membrane of an antigen presenting cell.


It is to be understood that when referring to the peptides ability to attach to and enter a cell, such as an antigen presenting cell, it may be with reference to the complete sequence of the peptide as well as a fragment thereof, such as a fragment representing an epitope. Accordingly, it may be the case that the entire sequence is essentially a non-cell-penetrating peptide, whereas a fragment of the peptide is able to efficiently enter a cell, such as an antigen presenting cell.


In some embodiments, the peptide according to the present invention is not a peptide or a dimeric peptide as specifically disclosed in International Patent Application No: PCT/DK2011/050460.


In some embodiments, the peptide according to the present invention is not a peptide or a dimeric peptide as specifically disclosed in International Patent Application No: PCT/EP2010/059513, such as one selected from:











(SEQ ID NO: 267)



CGGAKRRVVGGAKRRVVGQREKRAV







(SEQ ID NO: 268)



CGGGDQQLLGGAEEEIVGGIEEEGGERDRDR







(SEQ ID NO: 269)



CGGAKRRVVGGAKRRVVGGQREKR







(SEQ ID NO: 270)



CGGGDQQLLGGAEEEIVGGIEEEGG







(SEQ ID NO: 271)



CGGAEEEVVGGDQQLL







(SEQ ID NO: 272)



GCGGAKRRVVGGAKRRVV







(SEQ ID NO: 273)



GAKRRVVGGCGGAKRRVVQREKRAGEREKRA







(SEQ ID NO: 274)



GKGGIEEEGGRDRDRGGEQDRDR







(SEQ ID NO: 275)



GAKRRVVGGCGGAKRRVVQREKRAGEREKRA







(SEQ ID NO: 276)



GKGGIEEEGGERDRDRGGQDRDR







(SEQ ID NO: 277)



GAKRRVVGGCGGAKRRVVEREKRAGQREKRA







(SEQ ID NO: 278)



GKGGIEEEGGQDRDRGGRDRDR







(SEQ ID NO: 279)



GAKRRVVGGCGGAKRRVVEREKRAGQREKRA







(SEQ ID NO: 280)



GKGGIEEEGGEQDRDRGGERDRD






In some embodiments, the peptide according to the present invention is not a dimeric peptide selected from (The peptides are linked via the underlined amino acid):




embedded image


In some embodiments Z3, and optional Z6, Z9 and Z12 consist of a sequence selected from GYIPLVGAPLG, GYLPAVGAPIG, GYLPAVGAPI, NYVTGNIPG, NYATGNLPG, NYATGNLPG, VTGNIPGSTYS, IRNLGRVIETLTG, SRNLGKVIDTLTC, IRNLGRVIETLT, GGGQIIGGNYLIP, GGGQIVGGVYLLP, LIFLARSALIV, LIFLARSALIL, LIFLARSALIL, SAYERMCNIL, SAYERNleVNIL, TAYERNleCNIL, IAYERMCNIL, IAYERMCNIL, LFFKClYRLFKHGL, LFFKTITRLFBHGL, GLEPLVIAGILA, GSDPLVVAASIV, NLVPMVATV, NLVPMVATV, NIVPNleVVTA, PEVIPMFSALS, FIIPXFTALSG, ALGPAATL, GPVVHLTL, LECVYCKQQLL, GVYDFAFRDLC, GVFDYAFRDIN, GATPVDLLGA, GVTPAGLIGV, VARALAHGVRV, VIRVIAHGLRL, GITFSIFLIVS, CSFSIFLLAL, GCSFSIFLLAL, GITFSIYLIVS, LNleGYIPLIGA, LMGYIPLVGA, LNleGYIPLIGA, PBIGVRATB, GPRLGVRATR, GPRLGVRAT, RGSVAHKS, SALILRGSVAHK, FQTAAQRAMM, FQTAAQRAVNle, FQTVVQBA, FQTAAQRA, GPSTEGVPESM, LLSTEGVPNSNle, GSLVGLLHIVL, ASIVGILHLIL, NLVPMVATV, NIVPNleVVTA, TPQDLNTMLN, ALLYGATPYAIG, MMTACQGVG, GQAGDDFS, EVYDFAFRDLC, GFAFRDLCIVY, GFAYRDINLAY, GALNLCLPM, and GALQIBLPL, IRNLGRVIETLTLNleGYIPLIGA, or a fragment or variant thereof.


In some embodiments Z3, and optional Z6, Z9 and Z12 consist of a sequence derived from an amino acid sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:126, SEQ ID NO:198, SEQ ID NO:198, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, and SEQ ID NO:205, or a fragment or variant thereof.


In some embodiments the peptide according to the invention is not a peptide selected from RRGYIPLVGAPLGBGRVARALAHGVRV, RGYIPLVGAPLGRRVARALAHGVRV, RGYIPLVGAPLGRRRVARALAHGVRVR, RRGYIPLVGAPLGRRVARALAHGVRV, RRGYIPLVGAPLGRRRVARALAHGVRV, BRGYIPLVGAPLGRRVARALAHGVRV, RRRGYIPLVGAPLGBRVARALAHGVRV, RGYIPLVGAPLGKKKVARALAHGVRV, RGYIPLVGAPLGRRRVARALAHGVRV, KKGYIPLVGAPLGKKVARALAHGVRV, WGYIPLVGAPLGRRVARALAHGVRV, WWGYIPLVGAPLGRRVARALAHGVRV, EEGYIPLVGAPLGEEVARALAHGVRV, GGGYIPLVGAPLGGGVARALAHGVRV, EEGYIPLVGAPLGRRVARALAHGVRV, RRGYIPLVGAPLGLRRVARALAHGVRV, WWGYIPLVGAPLGRRVARALAHGVRV, WWGYIPLVGAPLGRRRVARALAHGVRV, WWGYIPLVGAPLGRVARALAHGVRV, RGYIPLVGAPLGRRVARALAHGVRV, RRGYLPAVGAPIGBRVIRVIAHGLRL, RRGYIPLVGAPLGBRVARALAHGVRV, GYIPLVGAPLGGVARALAHGVRV, WWGYLPAVGAPIRRVIRVIAHGLRL, GYIPLVGAPLGGVARALAHGVRV, RRGYIPLVGAPLGBGRVARALAHGVRV, RGYIPLVGAPLGRRVARALAHGVRV, RGYIPLVGAPLGRRRVARALAHGVRV, RRGYIPLVGAPLGRRVARALAHGVRV, RRGYIPLVGAPLGRRRVARALAHGVRV, BRGYIPLVGAPLGRRVARALAHGVRV, RRRGYIPLVGAPLGBRVARALAHGVRV, RGYIPLVGAPLGKKKVARALAHGVRV, RGYIPLVGAPLGRRRVARALAHGVRV, KKGYIPLVGAPLGKKVARALAHGVRV, WGYIPLVGAPLGRRVARALAHGVRV, WWGYIPLVGAPLGRRVARALAHGVRV, RRGYIPLVGAPLGLRRVARALAHGVRV, RRNYVTGNIPGBRGITFSIFLIVS, WWNYATGNLPGRRCSFSIFLLAL, WWNYVTGNIPGBRGITFSIFLIVS, WWNYVTGNIPGRRGITFSIFLIVS, RRNYATGNLPGRRGCSFSIFLLAL, RRVTGNIPGSTYSGBRGITFSIYLIVS, RRIRNLGRVIETLTGBRLNleGYIPLIGA, RRSRNLGKVIDTLTCBRLMGYIPLVGA, SRNLGKVIDTLTCGFADLMGYIPLVGA, WWIRNLGRVIETLTRRLNleGYIPLIGA, WWSRNLGKVIDTLTCRRLMGYIPLVGA, RRGGGQIIGGNYLIPRBPBIGVRATB, GGGQIVGGVYLLPRRGPRLGVRATR, RRGGGQIVGGVYLLPRRGPRLGVRATR, WWGGGQIVGGVYLLPRRGPRLGVRAT, BRLIFLARSALIVRGSVAHKS, EDLIFLARSALILRGSVAHKS, BRLIFLARSALILBGRSALILRGSVAHK, SAYERMCNILKGKFQTAAQRAMM, SAYERNleVNILKGKFQTAAQRAVNle, BRTAYERNleCNILBRGRFQTVVQBA, BRIAYERMCNILLBRGKFQTAAQRA, IAYERMCNILKGKFQTAAQRA, LFFKClYRLFKHGLKRGPSTEGVPESM, BRRLFFKTITRLFBHGLRRLLSTEGVPNSNle, BRGLEPLVIAGILARRGSLVGLLHIVL, BRGSDPLVVAASIVRRASIVGILHLIL, RNLVPMVATVRRNLVPMVATVB, RNLVPMVATVBRRNLVPMVATVB, RNIVPNleVVTARRNIVPNleVVTAB, PEVIPMFSALSEGATPQDLNTMLN, RFIIPXFTALSGGRRALLYGATPYAIG, KALGPAATLEEMMTACQGVG, RRGPVVHLTLRRRGQAGDDFS, RRGPVVHLTLRRRGQAGDDFS, RRGPVVHLTLRGRRGQAGDDFS, RRLECVYCKQQLLRREVYDFAFRDLC, RRGVYDFAFRDLCRRGFAFRDLCIVYR, RRGVFDYAFRDINRRGFAYRDINLAYR, RRGATPVDLLGARRGALNLCLPMR, RRGVTPAGLIGVRRGALQIBLPLR, RGYLPAVGAPIGRRRVIRVIAHGLRLR, RRSRNLGKVIDTLTCRRLMGYIPLVGA, RRIRNLGRVIETLTLNleGYIPLIGARRIRNLGRVIETLTLNleGYIPLIGAR, or a fragment or variant thereof.


In some embodiments the peptide according to the invention is not a peptide consisting of a sequence selected from X1-NYVTGNIPG-X3-GITFSIYLIVS; X1-IRNLGRVIETLT-X3-LNleGYIPLIGA; X1-GYLPAVGAP1-X3-VIRVIAHGLRL; X1-GGGQIIGGNYLIP-X3-PBIGVRATB; X1-NYATGNLPG-X3-GCSFSIFLLAL; X1-SRNLGKVIDTLTC-X3-LMGYIPLVGA; X1-GYIPLVGAPL-X3-VARALAHGVRV; X1-GGGQIVGGVYLLP-X3-PRLGVRATR; X1-LTFLVRSVLL1-X3-GSVLIVRGSLVH; X1-TAYERNleCNIL-X3-GRFQTVVQBA; X1-SDPLVVAASIV-X3-ASIVGILHLIL; X1-LIFLARSALIL-X3-SALILRGSVAH; X1-IAYERMCNIL-X3-GKFQTAAQRA; and X1-LEPLVIAGILA-X3-GSLVGLLHIVL; X1-NLVPMVATV-X3-NLVPMATV; X1-GYLPAVGAPIG-X3-VIRVIAHGLRL; X1-IRNLGRVIETLTG-X3-LNleGYIPLIGA; X1-GVYDFAFRDLC-X3-GFAFRDLCIVYR, X1-GVFDYAFRDIN-X3-GFAYRDINLAYR, X1-GATPVDLLGA-X3-GALNLCLPMR, X1-GVTPAGLIGV-X3-GALQIBLPLR, and X1-IRNLGRVIETLTLNleGYIPLIGA-X3-IRNLGRVIETLTLNleGYIPLIGA; optionally with an X5 in the C-terminal of the peptide wherein X1, X3 and X5 refers to X1, X3, and X5 of formula II.


In some embodiments the peptide according to the invention is not a peptide consisting of a sequence selected from RRGYIPLVGAPLGBGRVARALAHGVRV (SEQ ID NO:47), RGYIPLVGAPLGRRVARALAHGVRV (SEQ ID NO:48), RGYIPLVGAPLGRRRVARALAHGVRVR (SEQ ID NO:49), RRGYIPLVGAPLGRRVARALAHGVRV (SEQ ID NO:50), RRGYIPLVGAPLGRRRVARALAHGVRV (SEQ ID NO:51), BRGYIPLVGAPLGRRVARALAHGVRV (SEQ ID NO:52), RRRGYIPLVGAPLGBRVARALAHGVRV (SEQ ID NO:53), RGYIPLVGAPLGKKKVARALAHGVRV (SEQ ID NO:54), RGYIPLVGAPLGRRRVARALAHGVRV (SEQ ID NO:55), KKGYIPLVGAPLGKKVARALAHGVRV (SEQ ID NO:56), WGYIPLVGAPLGRRVARALAHGVRV (SEQ ID NO:57), WWGYIPLVGAPLGRRVARALAHGVRV (SEQ ID NO:58), EEGYIPLVGAPLGEEVARALAHGVRV (SEQ ID NO:59), GGGYIPLVGAPLGGGVARALAHGVRV (SEQ ID NO:60), EEGYIPLVGAPLGRRVARALAHGVRV (SEQ ID NO:61), RRGYIPLVGAPLGLRRVARALAHGVRV (SEQ ID NO:62), WWGYIPLVGAPLGRRVARALAHGVRV (SEQ ID NO:63), WWGYIPLVGAPLGRRRVARALAHGVRV (SEQ ID NO:64), WWGYIPLVGAPLGRVARALAHGVRV (SEQ ID NO:65), RGYIPLVGAPLGRRVARALAHGVRV (SEQ ID NO:66), RRGYLPAVGAPIGBRVIRVIAHGLRL (SEQ ID NO:67), RRGYIPLVGAPLGBRVARALAHGVRV (SEQ ID NO:68), GYIPLVGAPLGGVARALAHGVRV (SEQ ID NO:69), WWGYLPAVGAPIRRVIRVIAHGLRL (SEQ ID NO:70), GYIPLVGAPLGGVARALAHGVRV (SEQ ID NO:71), RRGYIPLVGAPLGBGRVARALAHGVRV (SEQ ID NO:72), RGYIPLVGAPLGRRVARALAHGVRV (SEQ ID NO:73), RGYIPLVGAPLGRRRVARALAHGVRV (SEQ ID NO:74), RRGYIPLVGAPLGRRVARALAHGVRV (SEQ ID NO:75), RRGYIPLVGAPLGRRRVARALAHGVRV (SEQ ID NO:76), BRGYIPLVGAPLGRRVARALAHGVRV (SEQ ID NO:77), RRRGYIPLVGAPLGBRVARALAHGVRV (SEQ ID NO:78), RGYIPLVGAPLGKKKVARALAHGVRV (SEQ ID NO:79), RGYIPLVGAPLGRRRVARALAHGVRV (SEQ ID NO:80), KKGYIPLVGAPLGKKVARALAHGVRV (SEQ ID NO:81), WGYIPLVGAPLGRRVARALAHGVRV (SEQ ID NO:82), WWGYIPLVGAPLGRRVARALAHGVRV (SEQ ID NO:83), RRGYIPLVGAPLGLRRVARALAHGVRV (SEQ ID NO:84), RRNYVTGNIPGBRGITFSIFLIVS (SEQ ID NO:85), WWNYATGNLPGRRCSFSIFLLAL (SEQ ID NO:86), WWNYVTGNIPGBRGITFSIFLIVS (SEQ ID NO:87), WWNYVTGNIPGRRGITFSIFLIVS (SEQ ID NO:88), RRNYATGNLPGRRGCSFSIFLLAL (SEQ ID NO:89), RRVTGNIPGSTYSGBRGITFSIYLIVS (SEQ ID NO:90), RRIRNLGRVIETLTGBRLNleGYIPLIGA (SEQ ID NO:91), RRSRNLGKVIDTLTCBRLMGYIPLVGA (SEQ ID NO:92), SRNLGKVIDTLTCGFADLMGYIPLVGA (SEQ ID NO:93), WWIRNLGRVIETLTRRLNleGYIPLIGA (SEQ ID NO:94), WWSRNLGKVIDTLTCRRLMGYIPLVGA (SEQ ID NO:95), RRGGGQIIGGNYLIPRBPBIGVRATB (SEQ ID NO:96), GGGQIVGGVYLLPRRGPRLGVRATR (SEQ ID NO:97), RRGGGQIVGGVYLLPRRGPRLGVRATR (SEQ ID NO:98), WWGGGQIVGGVYLLPRRGPRLGVRAT (SEQ ID NO:99), BRLIFLARSALIVRGSVAHKS (SEQ ID NO:100), EDLIFLARSALILRGSVAHKS (SEQ ID NO:101), BRLIFLARSALILBGRSALILRGSVAHK (SEQ ID NO:102), SAYERMCNILKGKFQTAAQRAMM (SEQ ID NO:103), SAYERNleVNILKGKFQTAAQRAVNle (SEQ ID NO:104), BRTAYERNleCNILBRGRFQTVVQBA (SEQ ID NO:105), BRIAYERMCNILLBRGKFQTAAQRA (SEQ ID NO:106), IAYERMCNILKGKFQTAAQRA (SEQ ID NO:107), LFFKClYRLFKHGLKRGPSTEGVPESM (SEQ ID NO:108), BRRLFFKTITRLFBHGLRRLLSTEGVPNSNle (SEQ ID NO:109), BRGLEPLVIAGILARRGSLVGLLHIVL (SEQ ID NO:110), BRGSDPLVVAASIVRRASIVGILHLIL (SEQ ID NO:111), RNLVPMVATVRRNLVPMVATVB (SEQ ID NO:112), RNLVPMVATVBRRNLVPMVATVB (SEQ ID NO:113), RNIVPNleVVTARRNIVPNleVVTAB (SEQ ID NO:114), PEVIPMFSALSEGATPQDLNTMLN (SEQ ID NO:115), RFIIPXFTALSGGRRALLYGATPYAIG (SEQ ID NO:116), KALGPAATLEEMMTACQGVG (SEQ ID NO:117), RRGPVVHLTLRRRGQAGDDFS (SEQ ID NO:118), RRGPVVHLTLRRRGQAGDDFS (SEQ ID NO:119), RRGPVVHLTLRGRRGQAGDDFS (SEQ ID NO:120), RRLECVYCKQQLLRREVYDFAFRDLC (SEQ ID NO:121), RRGVYDFAFRDLCRRGFAFRDLCIVYR (SEQ ID NO:122), RRGVFDYAFRDINRRGFAYRDINLAYR (SEQ ID NO:123), RRGATPVDLLGARRGALNLCLPMR (SEQ ID NO:124), RRGVTPAGLIGVRRGALQIBLPLR (SEQ ID NO:125), RGYLPAVGAPIGRRRVIRVIAHGLRLR (SEQ ID NO:196), RRSRNLGKVIDTLTCRRLMGYIPLVGA (SEQ ID NO:197), and RRIRNLGRVIETLTLNleGYIPLIGARRIRNLGRVIETLTLNleGYIPLIGAR (SEQ ID NO:199), or a fragment or variant thereof.


In some embodiments the peptide according to the invention is not a peptide consisting of a sequence selected from X1-NYVTGNIPG-X3-GITFSIYLIVS; X1-IRNLGRVIETLT-X3-LNleGYIPLIGA; X1-GYLPAVGAP1-X3-VIRVIAHGLRL; X1-GGGQIIGGNYLIP-X3-PBIGVRATB; X1-NYATGNLPG-X3-GCSFSIFLLAL; X1-SRNLGKVIDTLTC-X3-LMGYIPLVGA; X1-GYIPLVGAPL-X3-VARALAHGVRV; X1-GGGQIVGGVYLLP-X3-PRLGVRATR; X1-LTFLVRSVLL1-X3-GSVLIVRGSLVH; X1-TAYERNleCNIL-X3-GRFQTVVQBA; X1-SDPLVVAASIV-X3-ASIVGILHLIL; X1-LIFLARSALIL-X3-SALILRGSVAH; X1-IAYERMCNIL-X3-GKFQTAAQRA; and X1-LEPLVIAGILA-X3-GSLVGLLHIVL; X1-NLVPMVATV-X3-NLVPMATV; X1-GYLPAVGAPIG-X3-VIRVIAHGLRL; X1-IRNLGRVIETLTG-X3-LNleGYIPLIGA; X1-GVYDFAFRDLC-X3-GFAFRDLCIVYR, X1-GVFDYAFRDIN-X3-GFAYRDINLAYR, X1-GATPVDLLGA-X3-GALNLCLPMR, X1-GVTPAGLIGV-X3-GALQIBLPLR, and X1-IRNLGRVIETLTLNleGYIPLIGA-X3-IRNLGRVIETLTLNleGYIPLIGA; optionally with an X5 in the C-terminal of the peptide, wherein X1 and X3 and X5 refers to X1, X3, and X5 of formula II.


In some embodiments the peptide comprises one or more cysteine.


In some embodiments the peptide contain intramolecular bonds, such as intramolecular disulfide (S—S) bonds between two cys residues.


In other embodiments the peptide contains intramolecular bonds, such as in the form of a acylal moiety (COO—CH2-OOC. COO—CHR—OOC or COO—CR2-OOC).


In some embodiments the peptide according to the present invention is not more than 58 amino acids, such as not more than 56, 54, 52, 50, 48, 46, 44, 42, 40, 38, 36, 34, 32, 30, 28, 26, 24, 22, 20, 18 amino acid residues.


In some embodiments an isolated peptide according to the present invention is not a peptide consisting of a sequence of X2 or X4 as defined in table 1, table 2, or table 8.


In some embodiments an isolated peptide according to the present invention comprises a sequence of X2 and/or X4 as defined in table 1, table 2, table 5, or a fragment thereof.


In some embodiments the dimer peptide according to the invention consist of two identical peptide monomers.


In some embodiments the immunogenic composition according to the invention is in the form of a vaccine composition.


In some embodiments, the peptide of the invention comprises at most 60, at most 59, at most 58, at most 57, at most 56, at most 55, at most 54, at most 53, at most 52, at most 51, at most 50, at most 49, at most 48, at most 47, at most 46, at most 45, at most 44, at most 43, at most 42, at most 41, at most 40, at most 39, at most 38, at most 37, at most 36, at most 35, at most 34, at most 33, at most 32, at most 31, at most 30, at most 29, at most 28, at most 27, at most 26, at most 25, at most 24, at most 23, at most 22, at most 21, at most 20, at most 19, at most 18 amino acids.


In some embodiments, the peptide of the invention comprises at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35 at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60 amino acid residues.


In some embodiments, the peptide of the invention consists of 18 amino acid residues or 19 amino acid residues or 20 amino acid residues or 21 amino acid residues or 22 amino acid residues or 23 amino acid residues or 24 amino acid residues or 25 amino acid residues or 26 amino acid residues or 27 amino acid residues or 28 amino acid residues or 29 amino acid residues or 30 amino acid residues or 31 amino acid residues or 32 amino acid residues or 33 amino acid residues or 34 amino acid residues or 35 amino acid residues or 36 amino acid residuesor 37 amino acid residues or 38 amino acid residues or 39 amino acid residues or 40 amino acid residues or 41 amino acid residues or 42 amino acid residues or 43 amino acid residues or 44 amino acid residues or 45 amino acid residues or 46 amino acid residues or 47 amino acid residues or 48 amino acid residues or 49 amino acid residues or 50 amino acid residues or 51 amino acid residues or 52 amino acid residues or 53 amino acid residues or 54 amino acid residues or 55 amino acid residues or 56 amino acid residues or 57 amino acid residues or 58 amino acid residues or 59 amino acid residues or 60 amino acid residues.


In some embodiments the peptide of the invention does not consist of the following sequence RFIIP[Nle]FTALSGGRRALLYGATPYAIG, where Nle denotes a nor-leucine.


In some embodiments Z3, and optional Z6, Z9 and Z12 is not derived from HIV.


Numbered embodiments according to the invention:


1. An isolated monomeric peptide comprising the following structure





(Z1-Z2)1-Z3-(Z4-Z5)2-Z6-(Z7-Z8)3-Z9-(Z10-Z11)4-Z12


wherein Z1, Z4, and optional Z7 and Z10 defines a linear sequence of one, two, or three arginine residues or derivatives thereof optionally followed by a glycine (G) or an alanine (A); Z2, Z5, Z8 and Z11 defines an optional amino acid selected from cysteine (C), lysine (K), aspartic acid (D), asparagine (N), glutamic acid (E), glutamine (Q), 2,3-Diaminopropionic acid (Dpr), tryptophan (W), or tyrosine (Y) or a derivative thereof; Z3, and optional Z6, Z9 and Z12 defines any chemical moiety, such as a linear amino acid sequence.


2. The isolated monomeric peptide according to embodiment 1, wherein said chemical moiety of Z3, and optional Z6, Z9 and Z12 is a linear amino acid sequence of 8-30 amino acids or a compound with our without immune modulating properties.


3. The isolated monomeric peptide according to embodiments 1 or 2, wherein Z2 defines an amino acid selected from cysteine (C), lysine (K), aspartic acid (D), asparagine (N), glutamic acid (E), glutamine (Q), 2,3-Diaminopropionic acid (Dpr), tryptophan (W), or tyrosine (Y) or a derivative thereof.


4. The isolated monomeric peptide according to any one of embodiments 1-3, wherein Z5 defines an amino acid selected from cysteine (C), lysine (K), aspartic acid (D), asparagine (N), glutamic acid (E), glutamine (Q), 2,3-Diaminopropionic acid (Dpr), tryptophan (W), or tyrosine (Y) or a derivative thereof.


5. The isolated monomeric peptide according to embodiments 1-4, wherein Z8 defines an amino acid selected from cysteine (C), lysine (K), aspartic acid (D), asparagine (N), glutamic acid (E), glutamine (Q), 2,3-Diaminopropionic acid (Dpr), tryptophan (W), or tyrosine (Y) or a derivative thereof.


6. The isolated monomeric peptide according to embodiments 1-5, wherein Z11 defines an amino acid selected from cysteine (C), lysine (K), aspartic acid (D), asparagine (N), glutamic acid (E), glutamine (Q), 2,3-Diaminopropionic acid (Dpr), tryptophan (W), or tyrosine (Y) or a derivative thereof.


7. The isolated monomeric peptide according to any one of embodiments 1-6, wherein Z7 defines a linear sequence of one, two, or three arginine residues or derivatives thereof optionally followed by a glycine (G) or an alanine (A).


8. The isolated monomeric peptide according to any one of embodiments 1-7, wherein Z10 defines a linear sequence of one, two, or three arginine residues or derivatives thereof optionally followed by a glycine (G) or an alanine (A).


9. The isolated monomeric peptide according to any one of embodiments 1-8, wherein Z6 defines any chemical moiety, such as a linear amino acid sequence.


10. The isolated monomeric peptide according to any one of embodiments 1-9, wherein Z9 defines any chemical moiety, such as a linear amino acid sequence.


11. The isolated monomeric peptide according to any one of embodiments 1-10, wherein Z12 defines any chemical moiety, such as a linear amino acid sequence.


12. The isolated monomeric peptide according to any one of embodiments 1-11, wherein Z1, Z4, and optional Z7 and Z10 is followed by a glycine (G) or an alanine (A).


13. The isolated monomeric peptide according to any one of embodiments 1-12, wherein Z3, and optional Z6, Z9 and Z12 is a linear amino acid sequence of 8-30 amino acids derived from an antigen with more than 40%, such as more than 45%, such as more than 50%, such as more than 55%, such as more than 60%, such as more than 65%, such as more than 70%, such as more than 75%, such as more than 80%, such as more than 85%, such as more than 90%, such as more than 95%, such as more than 96%, such as more than 97%, such as more than 98%, such as more than 99%, such as 100% sequence identity to a specific natural antigen.


14. The isolated monomeric peptide according to any one of embodiments 1-13, wherein Z3, and optional Z6, Z9 and Z12 defines a specific natural antigen of a protein or peptide sequence derived from a disease antigen, such as an infectious agent, such as bacteria, virus, parasite, fungus, or cancer antigens such as oncogene (lung, stomach, breast cancer) or an antigen causing an autoimmune disease such as diabetes, multiple sclerosis (MS), celiac disease, Myalgic Encephalomyelitis (ME), psoriasis, and/or Crohn's Disease.


15. The isolated monomeric peptide according to embodiment 14, wherein said specific natural antigen is a viral protein, such as a structural protein, such as a capsid protein, a regulatory protein, an enzymatic protein, and a proteolytic protein.


16. The isolated monomeric peptide according to any one of embodiments 14-15, wherein said viral protein is selected from a core protein or an envelope protein, of a virus selected from the Hepatitis C virus, influenza virus, such as an M2 protein, human immunodeficiency virus (HIV), cytomegalovirus (CMV), and Human papillomavirus (HPV).


17. The isolated monomeric peptide according to embodiment 16, wherein said viral protein is a viral protein of Hepatitis C virus selected from any one HCV consensus sequence of a specific genotype, such as 1, such as subtypes 1a and 1b, genotype 2, such as 2a and 2b, genotype 3, such as 3a, genotype 4, genotype 5, and genotype 6.


18. The isolated monomeric peptide according to any one of embodiments 1-17, wherein a sequence of amino acids defined by (Z1-Z2)1-Z3-(Z4-Z5)2-Z6-(Z7-Z8)3-Z9-(Z10-Z11)4-Z12 is not found in the native sequence of a natural antigen.


19. The isolated monomeric peptide according to any one of embodiments 1-18, which monomeric peptide is of 10-60 amino acids, such as of 11-60 amino acids, such as of 12-60 amino acids, such as of 13-60 amino acids, such as of 14-60 amino acids, such as of 15-60 amino acids, such as of 16-60 amino acids, such as of 17-60 amino acids, such as of 18-60 amino acids, such as of 19-60 amino acids, such as of 20-60 amino acids, such as of 21-60 amino acids, such as of 22-60 amino acids, such as of 23-60 amino acids, such as of 24-60 amino acids, such as of 25-60 amino acids, such as of 26-60 amino acids, such as of 27-60 amino acids, such as of 28-60 amino acids, such as of 29-60 amino acids, such as of 30-60 amino acids, such as of 31-60 amino acids, such as of 32-60 amino acids, such as of 33-60 amino acids, such as of 34-60 amino acids, such as of 35-60 amino acids, such as of 36-60 amino acids, such as of 37-60 amino acids, such as of 38-60 amino acids, such as of 39-60 amino acids, such as of 40-60 amino acids, such as of 42-60 amino acids, such as of 44-60 amino acids, such as of 46-60 amino acids, such as of 48-60 amino acids, such as of 50-60 amino acids, such as of 52-60 amino acids, such as of 54-60 amino acids, such as of 56-60 amino acids, such as of 58-60 amino acids.


20. The isolated monomeric peptide according to any one of embodiments 1-19, which monomeric peptide is of 10-60 amino acids, such as 10-58 amino acids, such as 10-56 amino acids, such as 10-54 amino acids, such as 10-52 amino acids, such as 10-50 amino acids, such as 10-48 amino acids, such as 10-46 amino acids, such as 10-44 amino acids, such as 10-42 amino acids, such as 10-40 amino acids, such as 10-39 amino acids, such as 10-38 amino acids, such as 10-37 amino acids, such as 10-36 amino acids, such as 10-35 amino acids, such as 10-34 amino acids, such as 10-33 amino acids, such as 10-32 amino acids, such as 10-31 amino acids, such as 10-30 amino acids, such as 10-29 amino acids, such as 10-28 amino acids, such as 10-27 amino acids, such as 10-26 amino acids, such as 10-25 amino acids, such as 10-24 amino acids, such as 10-23 amino acids, such as 10-22 amino acids, such as 10-21 amino acids, such as 10-20 amino acids, such as 10-19 amino acids, such as 10-18 amino acids, such as 10-17 amino acids, such as 10-16 amino acids, such as 10-15 amino acids, such as 10-14 amino acids, such as 10-13 amino acids, such as 10-12 amino acids, such as 10-11 amino acids.


21. The isolated monomeric peptide according to any one of embodiments 1-20, which monomeric peptide consist of not more than about 55 amino acids, such as not more than about 50 amino acids, such as not more than about 45 amino acids, such as not more than about 40 amino acids, such as not more than about 38 amino acids, such as not more than about 36 amino acids, such as not more than about 34 amino acids, such as not more than about 32 amino acids, such as not more than about 30 amino acids, such as not more than about 28 amino acids, such as not more than about 26 amino acids, such as not more than about 24 amino acids, such as not more than about 22 amino acids, such as not more than about 20 amino acids, such as not more than about 18 amino acids, such as not more than about 16 amino acids, such as not more than about 14 amino acids, such as not more than about 12 amino acids, such as not more than about 10 amino acids.


22. The isolated monomeric peptide according to any one of embodiments 1-21, which monomeric peptide consist of at least about 10 amino acids, such as at least about 12 amino acids, such as at least about 14 amino acids, such as at least about 16 amino acids, such as at least about 18 amino acids, such as at least about 20 amino acids, such as at least about 22 amino acids, such as at least about 24 amino acids, such as at least about 26 amino acids, such as at least about 28 amino acids, such as at least about 30 amino acids, such as at least about 32 amino acids, such as at least about 34 amino acids, such as at least about 36 amino acids, such as at least about 38 amino acids, such as at least about 40 amino acids, such as at least about 45 amino acids, such as at least about 50 amino acids, such as at least about 55 amino acids, such as at least about 60.


23. The isolated monomeric peptide according to any one of embodiments 1-22, wherein the overall net charge of (Z1-Z2)1-Z3-(Z4-Z5)2-Z6-(Z7-Z8)3-Z9-(Z10-Z11)4-Z12 is equal to or above 0, such as above 1, 2, 3, 4, or 5.


24. The isolated monomeric peptide according to any one of embodiments 1-23, wherein said monomeric peptide is capable of inducing a humoral immune response.


25. The isolated monomeric peptide according to any one of embodiments 1-24, wherein said monomeric peptide comprises at least one amino acid selected from a Cys, a Lys, an Asp, and a Glu residue, or derivatives thereof.


26. The isolated monomeric peptide according to any one of embodiments 1-25, which monomeric peptide contain one or more intramolecular bond, such as one or more Cys-Cys bond.


27. The isolated monomeric peptide according to any one of embodiments 1-26, which monomeric peptide has delayed proteolytic degradation in the N-terminal, such as by incorporation of the first 1, 2, or 3 amino acids in the N-terminal in the D-form, or by incorporation of the first 1, 2, or 3 amino acids in the N-terminal in beta or gamma form.


28. The isolated peptide according to any one of embodiment 1-27, wherein said peptide is demonstrated to translocate across a plasma membrane in the assay based on biotinylation of peptides as described in example 5.


29. The isolated peptide according to any one of embodiments 1-28, wherein said peptide is capable of inducing a T lymphocyte response.


30. The isolated peptide according to any one of embodiments 1-29, wherein the net charge of Z3, and/or optional Z6, Z9 and Z12 is below or equal to 0.


31. The isolated peptide according to any one of embodiments 1-30, wherein the net charge of Z3 is below or equal to 0; and wherein the net charge of Z6 and/or optional Z9 and Z12 is above or equal to 1.


32. The isolated peptide according to any one of embodiments 1-31, wherein the net charge of Z3, and/or optional Z6, Z9 and Z12 are above or equal to 1.


33. The isolated peptide according to any one of embodiments 1-32, wherein the net charge of Z3 is above or equal to 1; and wherein the net charge of Z6 and/or optional Z9 and Z12 is below or equal to 0.


34. The isolated peptide according to any one of embodiments 1-33, wherein the peptide comprises one or more cysteine.


35. The isolated peptide according to any one of embodiments 1-34, wherein the N- and/or C-terminal amino acid in Z3, and/or optional Z6, Z9 and Z12 is a hydrophilic or polar amino acid.


36. The isolated peptide according to any one of embodiments 1-35, wherein Z3, and/or optional Z6, Z9 and Z12 defines a sequence of 8-25 amino acids, such as 8-20 amino acids, such as 8-15 amino acids.


37. The isolated peptide according to any one of embodiments 1-36, wherein Z3, and/or optional Z6, Z9 and Z12 defines a sequence of less than 25, such as less than 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7 or 6 amino acids.


38. The isolated peptide according to any one of embodiments 1-37, wherein Z3, and/or optional Z6, Z9 and Z12 defines a sequence of more than 8, such as more than 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 amino acids.


39. The isolated peptide according to any one of embodiments 1-38, which does not consist of the following sequence RFIIP[Nle]FTALSGGRRALLYGATPYAIG, where Nle denotes a nor-leucine.


40. The isolated peptide according to any one of embodiments 1-39, wherein Z3, and/or optional Z6, Z9 and Z12 is not derived from HIV.


41. The isolated peptide according to any one of embodiments 1-40, wherein Z3, and/or optional Z6, Z9 and Z12 is a linear sequence of less than 12 amino acids.


42. The isolated peptide according to any one of embodiments 1-41, wherein Z3, and/or optional Z6, Z9 and Z12 is a linear sequence of less than 12 amino acids.


43. The isolated peptide according to any one of embodiments 1-42, wherein Z3, and/or optional Z6, Z9 and Z12 do not contain nor-leucine.


44. The isolated peptide according to any one of embodiments 1-43, wherein Z3, and/or optional Z6, Z9 and Z12 do not contain nor-leucine.


45. The isolated peptide according to any one of embodiments 1-44, wherein Z3, and/or optional Z6, Z9 and Z12 only contains natural amino acids.


46. The isolated peptide according to any one of embodiments 1-45, wherein Z3, and/or optional Z6, Z9 and Z12 only contains natural amino acids.


47. The isolated peptide according to any one of embodiments 1-46, wherein Z3, and/or optional Z6, Z9 and Z12 only contains natural amino acids if derived from HIV.


48. The isolated peptide according to any one of embodiments 1-47, wherein Z3, and/or optional Z6, Z9 and Z12 is derived from HCV, CMV, HPV, Influenza, adenoviruses, herpesviruses, or picornaviruses.


49. The isolated peptide according to any one of embodiments 1-48, wherein Z1 is as defined in any one of table 3, table 4, table 5, or table 7, such as any one selected from R, RR, RRR, RG, RRG and RRRG.


50. The isolated peptide according to any one of embodiments 1-49, wherein Z2 is as defined in any one of table 3, table 4, table 5, or table 7, such as any one selected from Dpr(Aoa), C, K, Lys(Me), D, E, Dpr(Ser).


51. The isolated peptide according to any one of embodiments 1-50, wherein Z3 is as defined in any one of table 3, table 4, table 5, or table 7, such as any one selected from GGQLIGGIYLIPG (SEQ ID NO:313), VITYSIFLIVS (SEQ ID NO:314), TANWARVIS (SEQ ID NO:315), GYLPAVGAPI (SEQ ID NO:316), NIVPZVVTA (SEQ ID NO:317), VTPADLIGA (SEQ ID NO:318), PRPEGYTLFF (SEQ ID NO:319), LPYPRGYTLFV (SEQ ID NO:320), ETILTPRDV (SEQ ID NO:321), SSTSPVYDL (SEQ ID NO:322), TAYERZCNIL (SEQ ID NO:323), TVIGASZIPLL (SEQ ID NO:324), AAFEEZXITS (SEQ ID NO:325), GLEPLVIAGILA (SEQ ID NO:326), TAFLVRNVA (SEQ ID NO:327), TPI(Har)QDWGNRAN (SEQ ID NO:328), TPT(Har)NGWDVKLS (SEQ ID NO:329), LECVYCKQQLL (SEQ ID NO:330), GVYDFAFRDLC (SEQ ID NO:331), GVFDYAFRDIN (SEQ ID NO:332), and VDIRTLEDLL (SEQ ID NO:333).


52. The isolated peptide according to any one of embodiments 1-51, wherein Z4 is as defined in any one of table 3, table 4, table 5, or table 7, such as any one selected from R, RR, RRR, RG, RRG and RRRG.


53. The isolated peptide according to any one of embodiments 1-52, wherein Z5 is as defined in any one of table 3, table 4, table 5, or table 7, such as any one selected from Dpr(Aoa), C, K, Lys(Me), D, E, Dpr(Ser).


54. The isolated peptide according to any one of embodiments 1-53, wherein Z6 is as defined in any one of table 3, table 4, table 5, or table 7, such as any one selected from EVYDFAFRDLC (SEQ ID NO:334), GFAFRDLCIVY (SEQ ID NO:335), GFAYRDINLAY (SEQ ID NO:336), GTLGIVCPIG (SEQ ID NO:337), GLEPLVIAGILA (SEQ ID NO:338), TPIXQDWENRAN (SEQ ID NO:339), VAFEDLXZZSFI (SEQ ID NO:340), RFQTVVQBA (SEQ ID NO:341), GSLVGLLHIVL (SEQ ID NO:342), SIARSVTIZXASVVH (SEQ ID NO:343), TPTRQEWDCRIS (SEQ ID NO:344), TPTRQEWDARIS (SEQ ID NO:345), TPI(Har)QEW(Har)SL(Nle)NQEW (SEQ ID NO:346), IGDLIVAQV (SEQ ID NO:347), QYNPVAVZF (SEQ ID NO:348), GYTLFFTS (SEQ ID NO:349), GYTLFVSD (SEQ ID NO:350), NTLZTPRDV (SEQ ID NO:351), SSTSPVYNL (SEQ ID NO:352), VITFSIYLIVS (SEQ ID NO:353), GGNVIGGIYZIPR (SEQ ID NO:354), ANWAKVIL (SEQ ID NO:355), VIRVIAHGLRL (SEQ ID NO:356), and IGDLIVQAV (SEQ ID NO:478).


55. The isolated peptide according to any one of embodiments 1-54, wherein Z7 is as defined in any one of table 3, table 4, table 5, or table 7, such as any one selected from R, RR, RRR, RG, RRG and RRRG.


56. The isolated peptide according to any one of embodiments 1-55, wherein Z8 is as defined in any one of table 3, table 4, table 5, or table 7, such as any one selected from Dpr(Aoa), C, K, Lys(Me), D, E, Dpr(Ser).


57. The isolated peptide according to any one of embodiments 1-56, wherein Z9 is as defined in any one of table 3, table 4, table 5, or table 7, such as NWAKVI.


58. The isolated peptide according to any one of embodiments 1-57, which peptide consist of (Z1-Z2)1-Z3-(Z4-Z5)2-Z6-(Z7-Z8)3-Z9 as defined in any one of table 3, table 4, table 5, or table 7, such as any one selected from RRGGQLIGGIYLIPGRRVITFSIYLIVS (SEQ ID NO:357), RRRGGQLIGGIYLIPGRRVITFSIYLIVS (SEQ ID NO:358), RRGGQLIGGIYLIPGRRRVITFSIYLIVS (SEQ ID NO:359), RRGGQLIGGIYLIPGRRVITFSIYLIVSR (SEQ ID NO:360), RRGGQLIGGIYLIPGRRVITFSIYLIVSRR (SEQ ID NO:361), RRVITYSIFLIVSRRGGNVIGGIYZIPR (SEQ ID NO:362), RRRVITYSIFLIVSRRGGNVIGGIYZIPR (SEQ ID NO:363), RRVITYSIFLIVSRRRGGNVIGGIYZIPR (SEQ ID NO:364), RRRVITYSIFLIVSRRRGGNVIGGIYZIPR (SEQ ID NO:365), RRGTANWARVISRANWAKVILRNWAKVI (SEQ ID NO:366), RGTANWARVISRRANWAKVILRNWAKVI (SEQ ID NO:367), RGTANWARVISRANWAKVILRNWAKVI (SEQ ID NO:368), RGTANWARVISRGANWAKVILRNWAKVI (SEQ ID NO:369), RRGTANWARVISRANWARVILRNWAKVI (SEQ ID NO:370), RGTANWARVISRRANWARVILRNWAKVI (SEQ ID NO:371), RGTANWARVISRANWARVILRNWAKVI (SEQ ID NO:372), RGTANWARVISRGANWARVILRNWAKVI (SEQ ID NO:373), RGYLPAVGAPIRRRVIRVIAHGLRLR (SEQ ID NO:374), RRGYLPAVGAPIRRVIRVIAHGLRLR (SEQ ID NO:375), RRGYLPAVGAPIRRRVIRVIAHGLRL (SEQ ID NO:376), RRGYLPAVGAPIRRVIRVIAHGLRL (SEQ ID NO:377), RGYLPAVGAPIRRVIRVIAHGLRLR (SEQ ID NO:378), RGYLPAVGAPIRVIRVIAHGLRLR (SEQ ID NO:379), RGYLPAVGAPIRRVIRVIAHGLRL (SEQ ID NO:380), RGNIVPZVVTARRIGDLIVAQV (SEQ ID NO:381), RRNIVPZVVTARRIGDLIVAQV (SEQ ID NO:382), RRRNIVPZVVTARRIGDLIVAQV (SEQ ID NO:383), RRNIVPZVVTARRRIGDLIVAQV (SEQ ID NO:384), RGVTPADLIGARRQYNPVAVZF (SEQ ID NO:385), RRVTPADLIGARRQYNPVAVZF (SEQ ID NO:386), RRRVTPADLIGARRQYNPVAVZF (SEQ ID NO:387), RRVTPADLIGARRRQYNPVAVZF (SEQ ID NO:388), RRGPRPEGYTLFFRGYTLFFTSR (SEQ ID NO:389), RGPRPEGYTLFFRRGYTLFFTSR (SEQ ID NO:390), RRGPRPEGYTLFFRRGYTLFFTSR (SEQ ID NO:391), RRGPRPEGYTLFFRRRGYTLFFTSR (SEQ ID NO:392), RRRGPRPEGYTLFFRRGYTLFFTSR (SEQ ID NO:393), RGLPYPRGYTLFVRRGYTLFVSDR (SEQ ID NO:394), RRGLPYPRGYTLFVRRGYTLFVSDR (SEQ ID NO:395), RRGLPYPRGYTLFVRRRGYTLFVSDR (SEQ ID NO:396), RRRGLPYPRGYTLFVRRGYTLFVSDR (SEQ ID NO:397), RRGLPYPRGYTLFVRRGYTLFVSDR (SEQ ID NO:398), RRGETILTPRDVRNTLZTPRDVR (SEQ ID NO:399), RGETILTPRDVRRNTLZTPRDVR (SEQ ID NO:400), RGETILTPRDVRNTLZTPRDVR (SEQ ID NO:401), RGETILTPRDVRGNTLZTPRDVR (SEQ ID NO:402), RRSSTSPVYDLRRSSTSPVYNLR (SEQ ID NO:403), RRSSTSPVYDLRRRSSTSPVYNLR (SEQ ID NO:404), RRRSSTSPVYDLRRSSTSPVYNLR (SEQ ID NO:405), RRRSSTSPVYDLRRRSSTSPVYNLR (SEQ ID NO:406), RRTAYERZCNILRRGLEPLVIAGILA (SEQ ID NO:407), RRRTAYERZCNILRRGLEPLVIAGILA (SEQ ID NO:408), RRTAYERZCNILRRRGLEPLVIAGILA (SEQ ID NO:409), RRTAYERZCNILRRGLEPLVIAGILAR (SEQ ID NO:410), RRTAYERZCNILRRGLEPLVIAGILARR (SEQ ID NO:411), RRTVIGASZIPLLRGTPIXQDWENRAN (SEQ ID NO:412), RRRTVIGASZIPLLRGTPIXQDWENRAN (SEQ ID NO:413), RRTVIGASZIPLLRRGTPIXQDWENRAN (SEQ ID NO:414), RRRTVIGASZIPLLRRGTPIXQDWENRAN (SEQ ID NO:415), RRRTVIGASZIPLLRRGTPIXQDWENRANR (SEQ ID NO:416), RRAAFEEZXITSRRVAFEDLXZZSFI (SEQ ID NO:417), RRRAAFEEZXITSRRVAFEDLXZZSFI (SEQ ID NO:418), RRRAAFEEZXITSRRGVAFEDLXZZSFI (SEQ ID NO:419), RRRAAFEEZXITSRRRVAFEDLXZZSFI (SEQ ID NO:420), RRRAAFEEZXITSRRRVAFEDLXZZSFIGR (SEQ ID NO:421), RRTAYERZCNILRRGRFQTVVQBA (SEQ ID NO:422), RRTAYERZCNILRRGRFQTVVQBAR (SEQ ID NO:423), RTAYERZCNILRGRFQTVVQBAR (SEQ ID NO:424), RRTAYERZCNILRGRFQTVVQBA (SEQ ID NO:425), BRGLEPLVIAGILARRGSLVGLLHIVL (SEQ ID NO:426), RRGLEPLVIAGILARRGSLVGLLHIVL (SEQ ID NO:427), RRGLEPLVIAGILARRGSLVGLLHIVLR (SEQ ID NO:428), RRGLEPLVIAGILARRRGSLVGLLHIVL (SEQ ID NO:429), RRGLEPLVIAGILARRRGSLVGLLHIVLR (SEQ ID NO:430), RTAFLVRNVARSIARSVTIZXASVVH (SEQ ID NO:431), RTAFLVRNVARRSIARSVTIZXASVVH (SEQ ID NO:432), RRTAFLVRNVARSIARSVTIZXASVVH (SEQ ID NO:433), RRTAFLVRNVARRSIARSVTIZXASVVH (SEQ ID NO:434), RRTAFLVRNVARRSIARSVTIZXASVVHR (SEQ ID NO:435), RRTAFLVRNVARRSIARSVTIZXASVVHRR (SEQ ID NO:436), RGDpr(Aoa)TPI(Har)QDWGNRANRGTPTRQEWDCRIS (SEQ ID NO:437), RGDpr(Aoa)TPI(Har)QDWGNRANRGTPTRQEWDARIS (SEQ ID NO:438), RGTPI(Har)QDWGNRANRGTPTRQEWDCRIS (SEQ ID NO:439), RGTPI(Har)QDWGNRANRGTPTRQEWDARIS (SEQ ID NO:440), RGCTPI(Har)QDWGNRANRGTPTRQEWDCRIS (SEQ ID NO:441), RGCTPI(Har)QDWGNRANRGTPTRQEWDARIS (SEQ ID NO:442), RGKTPI(Har)QDWGNRANRGTPTRQEWDCRIS (SEQ ID NO:443), RGKTPI(Har)QDWGNRANRGTPTRQEWDARIS (SEQ ID NO:444), RGLys(Me)TPI(Har)QDWGNRANRGTPTRQEWDCRIS (SEQ ID NO:445), RGLys(Me)TPI(Har)QDWGNRANRGTPTRQEWDARIS (SEQ ID NO:446), RGDTPI(Har)QDWGNRANRGTPTRQEWDCRIS (SEQ ID NO:447), RGDTPI(Har)QDWGNRANRGTPTRQEWDARIS (SEQ ID NO:448), RGETPI(Har)QDWGNRANRGTPTRQEWDCRIS (SEQ ID NO:449), RGETPI(Har)QDWGNRANRGTPTRQEWDARIS (SEQ ID NO:450), RGDpr(Ser)TPT(Har)NGWDVKLSRGTPI(Har)QEW(Har)SL(Nle)NQEW (SEQ ID NO:451), RGTPT(Har)NGWDVKLSRGTPI(Har)QEW(Har)SL(Nle)NQEW (SEQ ID NO:452), RGKTPT(Har)NGWDVKLSRGTPI(Har)QEW(Har)SL(Nle)NQEW (SEQ ID NO:453), RGCTPT(Har)NGWDVKLSRGTPI(Har)QEW(Har)SL(Nle)NQEW (SEQ ID NO:454), RGLys(Me)TPT(Har)NGWDVKLSRGTPI(Har)QEW(Har)SL(Nle)NQEW (SEQ ID NO:455), RGDTPT(Har)NGWDVKLSRGTPI(Har)QEW(Har)SL(Nle)NQEW (SEQ ID NO:456), RGETPT(Har)NGWDVKLSRGTPI(Har)QEW(Har)SL(Nle)NQEW (SEQ ID NO:457), RRLECVYCKQQLLRREVYDFAFRDLC (SEQ ID NO:458), RRLECVYCKQQLLRRRGEVYDFAFRDLC (SEQ ID NO:459), RRRLECVYCKQQLLRRGEVYDFAFRDLC (SEQ ID NO:460), RRRLECVYCKQQLLRRRGEVYDFAFRDLC (SEQ ID NO:461), RRRGLECVYCKQQLLRRRGEVYDFAFRDLC (SEQ ID NO:462), RRGVYDFAFRDLCRRGFAFRDLCIVYR (SEQ ID NO:463), RRGVYDFAFRDLCRRRGGFAFRDLCIVY (SEQ ID NO:464), RRRGVYDFAFRDLCRRGGFAFRDLCIVYR (SEQ ID NO:465), RRRGVYDFAFRDLCRRRGGFAFRDLCIVY (SEQ ID NO:466), RRRGGVYDFAFRDLCRRRGGFAFRDLCIVYR (SEQ ID NO:467), RRGVFDYAFRDINRRGFAYRDINLAYR (SEQ ID NO:468), RRGVYDFAFRDLCRRRGGFAFRDLCIVY (SEQ ID NO:469), RRRGVYDFAFRDLCRRGGFAFRDLCIVYR (SEQ ID NO:470), RRRGVYDFAFRDLCRRRGGFAFRDLCIVY (SEQ ID NO:471), RRRGGVYDFAFRDLCRRRGGFAFRDLCIVYR (SEQ ID NO:472), RRVDIRTLEDLLRRGTLGIVCPIGR (SEQ ID NO:473), RRVDIRTLEDLLRRRGGTLGIVCPIG (SEQ ID NO:474), RRRVDIRTLEDLLRRGGTLGIVCPIGR (SEQ ID NO:475), RRRVDIRTLEDLLRRRGGTLGIVCPIG (SEQ ID NO:476), RRRGVDIRTLEDLLRRRGGTLGIVCPIGR (SEQ ID NO:477), RGNIVPZVVTARRIGDLIVQAV (SEQ ID NO:479), RRNIVPZVVTARRIGDLIVQAV (SEQ ID NO:480), RRRNIVPZVVTARRIGDLIVQAV (SEQ ID NO:481), and RRNIVPZVVTARRRIGDLIVQAV (SEQ ID NO:482).


59. The isolated peptide according to any one of embodiments 1-58, which peptide is not specifically disclosed in any one PCT application with application numbers WO2000N000075, WO2011DK050460, or WO2012DK050010.


60. The isolated peptide according to any one of embodiments 1-59, which peptide is not a peptide selected from RRGYIPLVGAPLGBGRVARALAHGVRV (SEQ ID NO:47), RGYIPLVGAPLGRRVARALAHGVRV (SEQ ID NO:48), RGYIPLVGAPLGRRRVARALAHGVRVR (SEQ ID NO:49), RRGYIPLVGAPLGRRVARALAHGVRV (SEQ ID NO:50), RRGYIPLVGAPLGRRRVARALAHGVRV (SEQ ID NO:51), BRGYIPLVGAPLGRRVARALAHGVRV (SEQ ID NO:52), RRRGYIPLVGAPLGBRVARALAHGVRV (SEQ ID NO:53), RGYIPLVGAPLGKKKVARALAHGVRV (SEQ ID NO:54), RGYIPLVGAPLGRRRVARALAHGVRV (SEQ ID NO:55), KKGYIPLVGAPLGKKVARALAHGVRV (SEQ ID NO:56), WGYIPLVGAPLGRRVARALAHGVRV (SEQ ID NO:57), WWGYIPLVGAPLGRRVARALAHGVRV (SEQ ID NO:58), EEGYIPLVGAPLGEEVARALAHGVRV (SEQ ID NO:59), GGGYIPLVGAPLGGGVARALAHGVRV (SEQ ID NO:60), EEGYIPLVGAPLGRRVARALAHGVRV (SEQ ID NO:61), RRGYIPLVGAPLGLRRVARALAHGVRV (SEQ ID NO:62), WWGYIPLVGAPLGRRVARALAHGVRV (SEQ ID NO:63), WWGYIPLVGAPLGRRRVARALAHGVRV (SEQ ID NO:64), WWGYIPLVGAPLGRVARALAHGVRV (SEQ ID NO:65), RGYIPLVGAPLGRRVARALAHGVRV (SEQ ID NO:66), RRGYLPAVGAPIGBRVIRVIAHGLRL (SEQ ID NO:67), RRGYIPLVGAPLGBRVARALAHGVRV (SEQ ID NO:68), GYIPLVGAPLGGVARALAHGVRV (SEQ ID NO:69), WWGYLPAVGAPIRRVIRVIAHGLRL (SEQ ID NO:70), GYIPLVGAPLGGVARALAHGVRV (SEQ ID NO:71), RRGYIPLVGAPLGBGRVARALAHGVRV (SEQ ID NO:72), RGYIPLVGAPLGRRVARALAHGVRV (SEQ ID NO:73), RGYIPLVGAPLGRRRVARALAHGVRV (SEQ ID NO:74), RRGYIPLVGAPLGRRVARALAHGVRV (SEQ ID NO:75), RRGYIPLVGAPLGRRRVARALAHGVRV (SEQ ID NO:76), BRGYIPLVGAPLGRRVARALAHGVRV (SEQ ID NO:77), RRRGYIPLVGAPLGBRVARALAHGVRV (SEQ ID NO:78), RGYIPLVGAPLGKKKVARALAHGVRV (SEQ ID NO:79), RGYIPLVGAPLGRRRVARALAHGVRV (SEQ ID NO:80), KKGYIPLVGAPLGKKVARALAHGVRV (SEQ ID NO:81), WGYIPLVGAPLGRRVARALAHGVRV (SEQ ID NO:82), WWGYIPLVGAPLGRRVARALAHGVRV (SEQ ID NO:83), RRGYIPLVGAPLGLRRVARALAHGVRV (SEQ ID NO:84), RRNYVTGNIPGBRGITFSIFLIVS (SEQ ID NO:85), WWNYATGNLPGRRCSFSIFLLAL (SEQ ID NO:86), WWNYVTGNIPGBRGITFSIFLIVS (SEQ ID NO:87), WWNYVTGNIPGRRGITFSIFLIVS (SEQ ID NO:88), RRNYATGNLPGRRGCSFSIFLLAL (SEQ ID NO:89), RRVTGNIPGSTYSGBRGITFSIYLIVS (SEQ ID NO:90), RRIRNLGRVIETLTGBRLNleGYIPLIGA (SEQ ID NO:91), RRSRNLGKVIDTLTCBRLMGYIPLVGA (SEQ ID NO:92), SRNLGKVIDTLTCGFADLMGYIPLVGA (SEQ ID NO:93), WWIRNLGRVIETLTRRLNleGYIPLIGA (SEQ ID NO:94), WWSRNLGKVIDTLTCRRLMGYIPLVGA (SEQ ID NO:95), RRGGGQIIGGNYLIPRBPBIGVRATB (SEQ ID NO:96), GGGQIVGGVYLLPRRGPRLGVRATR (SEQ ID NO:97), RRGGGQIVGGVYLLPRRGPRLGVRATR (SEQ ID NO:98), WWGGGQIVGGVYLLPRRGPRLGVRAT (SEQ ID NO:99), BRLIFLARSALIVRGSVAHKS (SEQ ID NO:100), EDLIFLARSALILRGSVAHKS (SEQ ID NO:101), BRLIFLARSALILBGRSALILRGSVAHK (SEQ ID NO:102), SAYERMCNILKGKFQTAAQRAMM (SEQ ID NO:103), SAYERNleVNILKGKFQTAAQRAVNle (SEQ ID NO:104), BRTAYERNleCNILBRGRFQTVVQBA (SEQ ID NO:105), BRIAYERMCNILLBRGKFQTAAQRA (SEQ ID NO:106), IAYERMCNILKGKFQTAAQRA (SEQ ID NO:107), LFFKClYRLFKHGLKRGPSTEGVPESM (SEQ ID NO:108), BRRLFFKTITRLFBHGLRRLLSTEGVPNSNle (SEQ ID NO:109), BRGLEPLVIAGILARRGSLVGLLHIVL (SEQ ID NO:110), BRGSDPLVVAASIVRRASIVGILHLIL (SEQ ID NO:111), RNLVPMVATVRRNLVPMVATVB (SEQ ID NO:112), RNLVPMVATVBRRNLVPMVATVB (SEQ ID NO:113), RNIVPNleVVTARRNIVPNleVVTAB (SEQ ID NO:114), PEVIPMFSALSEGATPQDLNTMLN (SEQ ID NO:115), RFIIPXFTALSGGRRALLYGATPYAIG (SEQ ID NO:116), KALGPAATLEEMMTACQGVG (SEQ ID NO:117), RRGPVVHLTLRRRGQAGDDFS (SEQ ID NO:118), RRGPVVHLTLRRRGQAGDDFS (SEQ ID NO:119), RRGPVVHLTLRGRRGQAGDDFS (SEQ ID NO:120), RRLECVYCKQQLLRREVYDFAFRDLC (SEQ ID NO:121), RRGVYDFAFRDLCRRGFAFRDLCIVYR (SEQ ID NO:122), RRGVFDYAFRDINRRGFAYRDINLAYR (SEQ ID NO:123), RRGATPVDLLGARRGALNLCLPMR (SEQ ID NO:124), RRGVTPAGLIGVRRGALQIBLPLR (SEQ ID NO:125), RGYLPAVGAPIGRRRVIRVIAHGLRLR (SEQ ID NO:196), RRSRNLGKVIDTLTCRRLMGYIPLVGA (SEQ ID NO:197), RRIRNLGRVIETLTLNleGYIPLIGARRIRNLGRVIETLTLNleGYIPLIGAR (SEQ ID NO:199), X1-NYVTGNIPG-X3-GITFSIYLIVS; X1-IRNLGRVIETLT-X3-LNleGYIPLIGA; X1-GYLPAVGAPI-X3-VIRVIAHGLRL; X1-GGGQIIGGNYLIP-X3-PBIGVRATB; X1-NYATGNLPG-X3-GCSFSIFLLAL; X1-SRNLGKVIDTLTC-X3-LMGYIPLVGA; X1-GYIPLVGAPL-X3-VARALAHGVRV; X1-GGGQIVGGVYLLP-X3-PRLGVRATR; X1-LTFLVRSVLL1-X3-GSVLIVRGSLVH; X1-TAYERNleCNIL-X3-GRFQTVVQBA; X1-SDPLVVAASIV-X3-ASIVGILHLIL; X1-LIFLARSALIL-X3-SALILRGSVAH; X1-IAYERMCNIL-X3-GKFQTAAQRA; and X1-LEPLVIAGILA-X3-GSLVGLLHIVL; X1-NLVPMVATV-X3-NLVPMATV; X1-GYLPAVGAPIG-X3-VIRVIAHGLRL; X1-IRNLGRVIETLTG-X3-LNleGYIPLIGA; X1-GVYDFAFRDLC-X3-GFAFRDLCIVYR, X1-GVFDYAFRDIN-X3-GFAYRDINLAYR, X1-GATPVDLLGA-X3-GALNLCLPMR, X1-GVTPAGLIGV-X3-GALQIBLPLR, and X1-IRNLGRVIETLTLNleGYIPLIGA-X3-IRNLGRVIETLTLNleGYIPLIGA; optionally with an X5 in the C-terminal of the peptide; wherein X1 and X3 and X5 refers to X′, X3, and X5 of formula II.


61. An isolated multimeric, such as dimeric peptide comprising two or more monomeric peptides, each monomeric peptide independently comprising the following structure





(Z1-Z2)1-Z3-(Z4-Z5)2-Z6-(Z7-Z8)3-Z9-(Z10-Z11)4-Z12


wherein Z1, Z4, and optional Z7 and Z10 defines a linear sequence of one, two, or three arginine residues or derivatives thereof optionally followed by a glycine (G) or an alanine (A); Z2, Z5, Z8 and Z11 defines an optional amino acid selected from cysteine (C), lysine (K), aspartic acid (D), asparagine (N), glutamic acid (E), glutamine (Q), 2,3-Diaminopropionic acid (Dpr), tryptophan (W), or tyrosine (Y) or a derivative thereof; Z3, and optional Z6, Z9 and Z12 defines any chemical moiety, such as a linear amino acid sequence, said monomeric peptides being covalently joined by one or more intermolecular bond.


62. The isolated multimeric, such as dimeric peptide according to embodiment 61, wherein two or more monomeric peptides are identical in sequence.


63. The isolated multimeric, such as dimeric peptide according to embodiment 61, wherein two or more monomeric peptides are different in sequence.


64. The isolated multimeric, such as dimeric peptide according to any of embodiments 61-63, comprising at least two peptides monomers, each peptide monomer independently being as defined in any one of embodiments 1-58.


65. The isolated multimeric, such as dimeric peptide according to any one of embodiments 61-64, wherein one or more peptide strands of the multimeric, such as dimeric peptide has delayed proteolytic degradation in the N-terminal, such as by incorporation of the first 1, 2, or 3 amino acids in the N-terminal in the D-form, or by incorporation of the first 1, 2, or 3 amino acids in the N-terminal in beta or gamma form.


66. The isolated multimeric, such as dimeric peptide according to any one of embodiments 61-65, which multimeric, such as dimeric peptide contain a helper epitope of at least 12 amino acids, such as at least 13, 14, 15 or 17 amino acids, which helper epitope consist of a combined sequence of amino acids, which is a sequence of amino acids from a first specific continuous antigenic peptide sequences, and a sequence of amino acids from at least one second specific continuous antigenic peptide sequence of the same or different protein derived from the same virus, any different virus, or any disease antigen, such as between 2-12 amino acids from the first specific continuous antigenic peptide sequences and 2-12 amino acids from the at least one second specific continuous antigenic antigenic peptide sequence.


67. The isolated multimeric, such as dimeric peptide according to any one of embodiments 61-66, wherein said intermolecular bond is a disulfide (S—S) bond between two Cys residues.


68. The isolated multimeric, such as dimeric peptide according to any one of embodiments 61-67, wherein said intermolecular bond is a thioether bond between a Cys residue in the first monomeric peptide and a modified Lys residue in the at least one second monomeric peptide.


69. The isolated multimeric, such as dimeric peptide according to any one of embodiments 61-68, wherein said intermolecular bond is an oxime bond between a derivatized Lys residue in the first monomeric peptide and a derivatized Ser residue in the at least one second monomeric peptide.


70. The isolated multimeric, such as dimeric peptide according to any one of embodiments 61-69, wherein said intermolecular bond is a peptide bond between a N-methylated Lys side-chain in the first monomeric peptide and the side-chain of an Asp or Glu residue in the at least one second monomeric peptide.


71. The isolated multimeric, such as dimeric peptide according to any one of embodiments 61-70, wherein said intermolecular bond is an oxime bond between an aldehyde moiety, produced by oxidation of a serine residue in the first monomeric peptide and a free aminooxy group of a modified amino acid (aminooxy acid), such as derivatized diaminopropionic acid, Lysine or Ornithine in in the second monomeric peptide


72. The isolated multimeric, such as dimeric peptide according to any one of embodiments 61-71, wherein said monomeric peptides are linked by a polyethylene glycol (PEG) linker, such as through an Asp or a Glu residue in the first monomeric peptide and an Asp or a Glu residue in the at least one second monomeric peptide, or by a polyLys core.


73. The isolated multimeric, such as dimeric peptide according to any one of embodiments 61-72, wherein a C residue in Z2 of the first peptide monomer is linked to an amino acid selected from a K or a C residue in Z2 of the second monomer.


74. The isolated multimeric, such as dimeric peptide according to any one of embodiments 61-73, wherein a K residue in Z2 of the first peptide monomer is linked to an amino acid selected from a C, D or E residue in Z2 of the second monomer.


75. The isolated multimeric, such as dimeric peptide according to any one of embodiments 61-74, wherein a D residue in Z2 of the first peptide monomer is linked to an amino acid selected from a N or Q residue in Z2 of the second monomer.


76. The isolated multimeric, such as dimeric peptide according to any one of embodiments 61-75, wherein a E residue in Z2 of the first peptide monomer is linked to an amino acid selected from a N or Q residue in Z2 of the second monomer.


77. The isolated multimeric, such as dimeric peptide according to any one of embodiments 61-76, wherein a N residue in Z2 of the first peptide monomer is linked to a D or E residue in Z2 of the second monomer.


78. The isolated multimeric, such as dimeric peptide according to any one of embodiments 61-77, wherein a Q residue in Z2 of the first peptide monomer is linked to a D or E residue in Z2 of the second monomer.


79. The isolated multimeric, such as dimeric peptide according to any one of embodiments 61-78, wherein a Dpr(Aao) residue in Z2 of the first peptide monomer is linked to an Dpr(Ser) residue in Z2 of the second monomer.


80. The isolated multimeric, such as dimeric peptide according to any one of embodiments 61-79, wherein a W residue in Z2 of the first Z1-Z2 peptide repeat is linked to an Y residue in Z2 of the second Z1-Z2 peptide repeat.


81. The isolated multimeric, such as dimeric peptide according to any one of embodiments 61-80, wherein a Y residue in Z2 of the first Z1-Z2 peptide repeat is linked to an W residue in Z2 of the second Z1-Z2 peptide repeat.


82. Composition comprising two or more compounds selected from a monomeric peptide is as defined in any one of embodiments 1-60, and an isolated multimeric, such as dimeric peptide as defined in any one of embodiments 61-81.


83. Use of a peptide selected from a monomeric peptide is as defined in any one of embodiments 1-60, and an isolated multimeric, such as dimeric peptide as defined in any one of embodiments 61-81 for inducing an immune response in a subject, such as a humoral or Cell Mediated Immune (CMI) response.


84. An isolated nucleic acid or polynucleotide encoding a peptide according to any one of embodiments 1-61.


85. A vector comprising the nucleic acid or polynucleotide according to embodiment 84.


86. A host cell comprising the vector according to embodiment 85.


87. An immunogenic composition comprising at least one monomeric peptide according to any one of embodiments 1-61, an isolated multimeric, such as dimeric peptide according to any one of embodiments 61-81, a peptide composition according to embodiment 82, the nucleic acid or polynucleotide according to embodiment 84, or the vector according to embodiment 85; in combination with a pharmaceutically acceptable diluent or vehicle and optionally an immunological adjuvant.


88. The immunogenic composition according to embodiment 87 in the form of a vaccine composition.


89. A method for inducing an immune response in a subject against an antigen which comprises administration of at least one monomeric peptide according to any one of embodiments 1-60, an isolated multimeric, such as dimeric peptide according to any one of embodiments 61-79, a peptide composition according to embodiment 82, the nucleic acid or polynucleotide according to embodiment 84, or the vector according to embodiment 85; or the composition according to any one of embodiments 87-88.


90. A method for reducing and/or delaying the pathological effects of a disease antigen, such as an infectious agent in a subject infected with said agent or having said disease caused by said antigen, the method comprising administering an effective amount of at least one monomeric peptide according to any one of embodiments 1-60, an isolated multimeric, such as dimeric peptide according to any one of embodiments 61-81, a peptide composition according to embodiment 82, the nucleic acid or polynucleotide according to embodiment 84, or the vector according to embodiment 85; or the composition according to any one of embodiments 87-88.


91. A peptide according to any one of embodiments 1-81 for use as a medicament.


92. A peptide according to any one of embodiments 1-81 for treating the pathological effects of a disease antigen, such as an infectious agent in a subject infected with said agent or having said disease caused by said antigen.


93. A peptide according to any one of embodiments 1-81 for use in an in vitro assay, such as an ELISA assay, such as for diagnostic purposes.


94. Use of a peptide according to any one of embodiments 1-81 for in vitro assay, such as an ELISA assay, such as for diagnostic purposes.


Sequence list (amino acids in bold represents suitable antigenic sequences that may be used as any of Z3, and optional Z6, Z9 and Z12 as defined in formula I of the present invention)










SEQ ID NO: 1:



Accession no AF009606; Hepatitis C virus subtype


is polyprotein gene, complete cds.


MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRL





GVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGYPWPLYGNEGCGWAGWLLSPRG





SRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAARALAHGVRVLED





GVNYATGNLPGCSFSIFLLALLSCLTVPASAYQVRNSSGLYHVTNDCPNSSIVYEAAD





AILHTPGCVPCVREGNASRCWVAVTPTVATRDGKLPTTQLRRHIDLLVGSATLCSALY





VGDLCGSVFLVGQLFTFSPRRHWTTQDCNCSIYPGHITGHRMAWDMMMNWSPTAALVV





AQLLRIPQAIMDMIAGAHWGVLAGIAYFSMVGNWAKVLVVLLLFAGVDAETHVTGGSA





GRTTAGLVGLLTPGAKQNIQLINTNGSWHINSTALNCNESLNTGWLAGLFYQHKFNSS





GCPERLASCRRLTDFAQGWGPISYANGSGLDERPYCWHYPPRPCGIVPAKSVCGPVYC





FTPSPVVVGTTDRSGAPTYSWGANDTDVFVLNNTRPPLGNWFGCTWMNSTGFTKVCGA





PPCVIGGVGNNTLLCPTDCFRKHPEATYSRCGSGPWITPRCMVDYPYRLWHYPCTINY





TIFKVRMYVGGVEHRLEAACNWTRGERCDLEDRDRSELSPLLLSTTQWQVLPCSFTTL





PALSTGLIHLHQNIVDVQYLYGVGSSIASWAIKWEYVVLLFLLLADARVCSCLWMMLL





ISQAEAALENLVILNAASLAGTHGLVSFLVFFCFAWYLKGRWVPGAVYAFYGMWPLLL





LLLALPQRAYALDTEVAASCGGVVLVGLMALTLSPYYKRYISWCMWWLQYFLTRVEAQ





LHVWVPPLNVRGGRDAVILLMCVVHPTLVFDITKLLLAIFGPLWILQASLLKVPYFVR





VQGLLRICALARKIAGGHYVQMAIIKLGALTGTYVYNHLTPLRDWAHNGLRDLAVAVE





PVVFSRMETKLITWGADTAACGDIINGLPVSARRGQEILLGPADGMVSKGWRLLAPIT





AYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTATQTFLATCINGVCWTVYHGAGTR





TIASPKGPVIQMYTNVDQDLVGWPAPQGSRSLTPCTCGSSDLYLVTRHADVIPVRRRG





DSRGSLLSPRPISYLKGSSGGPLLCPAGHAVGLFRAAVCTRGVAKAVDFIPVENLETT





MRSPVFTDNSSPPAVPQSFQVAHLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATL





GFGAYMSKAHGVDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHS





TDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVSHPNIEEVALSTTGEIPFYGK





AIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVS





TDALMTGFTGDFDSVIDCNTCVTQTVDFSLDPTFTIETTTLPQDAVSRTQRRGRTGRG





KPGIYRFVAPGERPSGMFDSSVLCECYDAGCAWYELTPAETTVRLRAYMNTPGLPVCQ





DHLEFWEGVFTGLTHIDAHFLSQTKQSGENFPYLVAYQATVCARAQAPPPSWDQMWKC





LIRLKPTLHGPTPLLYRLGAVQNEVTLTHPITKYIMTCMSADLEVVTSTWVLVGGVLA





ALAAYCLSTGCVVIVGRIVLSGKPAIIPDREVLYQEFDEMEECSQHLPYIEQGMMLAE





QFKQKALGLLQTASRQAEVITPAVQTNWQKLEVFWAKHMWNFISGIQYLAGLSTLPGN





PAIASLMAFTAAVTSPLTTGQTLLFNILGGWVAAQLAAPGAATAFVGAGLAGAAIGSV





GLGKVLVDILAGYGAGVAGALVAFKIMSGEVPSTEDLVNLLPAILSPGALVVGVVCAA





ILRRHVGPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARVTAILSSLTVTQLLR





RLHQWISSECTTPCSGSWLRDIWDWICEVLSDFKTWLKAKLMPQLPGIPFVSCQRGYR





GVWRGDGIMHTRCHCGAEITGHVKNGTMRIVGPRTCRNMWSGTFPINAYTTGPCTPLP





APNYKFALWRVSAEEYVEIRRVGDFHYVSGMTTDNLKCPCQIPSPEFFTELDGVRLHR





FAPPCKPLLREEVSFRVGLHEYPVGSQLPCEPEPDVAVLTSMLTDPSHITAEAAGRRL





ARGSPPSMASSSASQLSAPSLKATCTANHDSPDAELIEANLLWRQEMGGNITRVESEN





KVVILDSFDPLVAEEDEREVSVPAEILRKSRRFARALPVWARPDYNPPLVETWKKPDY





EPPVVHGCPLPPPRSPPVPPPRKKRTVVLTESTLSTALAELATKSFGSSSTSGITGDN





TTTSSEPAPSGCPPDSDVESYSSMPPLEGEPGDPDLSDGSWSTVSSGADTEDVVCCSM





SYSWTGALVTPCAAEEQKLPINALSNSLLRHHNLVYSTTSRSACQRQKKVTFDRLQVL





DSHYQDVLKEVKAAASKVKANLLSVEEACSLTPPHSAKSKFGYGAKDVRCHARKAVAH





INSVWKDLLEDSVTPIDTTIMAKNEVFCVQPEKGGRKPARLIVFPDLGVRVCEKMALY





DVVSKLPLAVMGSSYGFQYSPGQRVEFLVQAWKSKKTPMGFSYDTRCFDSTVTESDIR





TEEAIYQCCDLDPQARVAIKSLTERLYVGGPLTNSRGENCGYRRCRASGVLTTSCGNT





LTCYIKARAACRAAGLQDCTMLVCGDDLVVICESAGVQEDAASLRAFTEAMTRYSAPP





GDPPQPEYDLELITSCSSNVSVAHDGAGKRVYYLTRDPTTPLARAAWETARHTPVNSW





LGNIIMFAPTLWARMILMTHFFSVLIARDQLEQALNCEIYGACYSIEPLDLPPIIQRL





HGLSAFSLHSYSPGEINRVAACLRKLGVPPLRAWRHRARSVRARLLSRGGRAAICGKY





LFNWAVRTKLKLTPIAAAGRLDLSGWFTAGYSGGDIYHSVSHARPRWFWFCLLLLAAG





VGIYLLPNR





SEQ ID NO: 2:


HCV core protein, H77, Accession AF009606


Genbank number: 2316097


>gi|2316098|gb|AAB66324.1| polyprotein [Hepatitis C virus


subtype 1a]


MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARR





PEGRTWAQPGYPWPLYGNEGCGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAAR





ALAHGVRVLEDGVNYATGNLPGCSFSIFLLALLSCLTVPASA





SEQ ID NO: 3:


Hepatitis C virus mRNA, complete cds;


ACCESSION M96362 M72423;


Hepatitis C virus subtype 1b


MSTNPKPQRKTKRNTNRRPQDIKFPGGGQIVGGVYLLPRRGPRL





GVRATRKTSERSQPRGRRQPIPKARRPEGRAWAQPGYPWPLYGNEGLGWAGWLLSPRG





SRPSWGPTDPRRKSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGVARALAHGVRVLED





GVNYATGNLPGCSFSIFLLALLSCLTTPVSAYEVRNASGMYHVTNDCSNSSIVYEAAD





MIMHTPGCVPCVREDNSSRCWVALTPTLAARNASVPTTTLRRHVDLLVGVAAFCSAMY





VGDLCGSVFLVSQLFTESPRRHETVQDCNCSIYPGRVSGHRMAWDMMMNWSPTTALVV





SQLLRIPQAVVDMVTGSHWGILAGLAYYSMVGNWAKVLIAMLLFAGVDGTTHVTGGAQ





GRAASSLTSLFSPGPVQHLQLINTNGSWHINRTALSCNDSLNTGFVAALFYKYRFNAS





GCPERLATCRPIDTFAQGWGPITYTEPHDLDQRPYCWHYAPQPCGIVPTLQVCGPVYC





FTPSPVAVGTTDRFGAPTYRWGANETDVLLLNNAGPPQGNWFGCTWMNGTGFTKTCGG





PPCNIGGVGNNTLTCPTDCFRKHPGATYTKCGSGPWLTPRCLVDYPYRLWHYPCTVNF





TIFKVRMYVGGAEHRLDAACNWTRGERCDLEDRDRSELSPLLLSTTEWQVLPCSFTTL





PALSTGLIHLHQNIVDIQYLYGIGSAVVSFAIKWEYIVLLFLLLADARVCACLWMMLL





VAQAEAALENLVVLNAASVAGAHGILSFIVFFCAAWYIKGRLVPGAAYALYGVWPLLL





LLLALPPRAYAMDREMAASCGGAVFVGLVLLTLSPHYKVFLARFIWWLQYLITRTEAH





LQVWVPPLNVRGGRDAIILLTCVVHPELIFDITKYLLAIFGPLMVLQAGITRVPYFVR





AQGLIRACMLARKVVGGHYVQMVFMKLAALAGTYVYDHLTPLRDWAHTGLRDLAVAVE





PVVFSDMETKVITWGADTAACGDIILALPASARRGKEILLGPADSLEGQGWRLLAPIT





AYSQQTRGLLGCIITSLTGRDKNQVEGEVQVVSTATQSFLATCINGVCWTVFHGAGSK





TLAGPKGPITQMYTNVDQDLVGWPAPPGARSLTPCTCGSSDLYLVTRHADVIPVRRRG





DGRGSLLPPRPVSYLKGSSGGPLLCPSGHAVGILPAAVCTRGVAMAVEFIPVESMETT





MRSPVFTDNPSPPAVPQTFQVAHLHAPTGSGKSTRVPAAYAAQGYKVLVLNPSVAATL





GFGAYMSKAHGIDPNLRTGVRTITTGAPITYSTYGKFLADGGGSGGAYDIIMCDECHS





TDSTTIYGIGTVLDQAETAGARLVVLSTATPPGSVTVPHLNIEEVALSNTGEIPFYGK





AIPIEAIKGGRHLIFCHSKKKCDELAAKLSGLGLNAVAYYRGLDVSVIPTSGDVVVVA





TDALMTGFTGDFDSVIDCNTCVTQTVDFSLDPTFTIETTTVPQDAVSRSQRRGRTGRG





RAGIYRFVTPGERPSGMFDSSVLCECYDAGCAWYELTPAETSVRLRAYLNTPGLPVCQ





DHLEFSEGVFTGLTHIDAHFLSQTKQAGENFPYLVAYQATVCARAQAPPPSWDEMWRC





LIRLKPTLHGPTPLLYRLGAVQNEVTLTHPITKFIMTCMSADLEVVTSTWVLVGGVLA





ALAAYCLTTGSVVIVGRIILSGKPAIIPDREVLYQEFDEMEECASHLPYFEQGMQLAE





QFKQKALGLLQTATKQAEAAAPVVESKWRALETFWAKHMWNFISGIQYLAGLSTLPGN





PAIRSPMAFTASITSPLTTQHTLLFNILGGWVAAQLAPPSAASAFVGAGIAGAAVGTI





GLGKVLVDILAGYGAGVAGALVAFKIMSGEMPSAEDMVNLLPAILSPGALVVGIVCAA





ILRRHVGPGEGAVQWMNRLIAFASRGNHVSPRHYVPESEPAARVTQILSSLTITQLLK





RLHQWINEDCSTPCSSSWLREIWDWICTVLTDFKTWLQSKLLPRLPGVPFFSCQRGYK





GVWRGDGIMHTTCPCGAQITGHVKNGSMRIVGPKTCSNTWYGTFPINAYTTGPCTPSP





APNYSKALWRVAAEEYVEVTRVGDFHYVTGMTTDNVKCPCQVPAPEFFTEVDGVRLHR





YAPACRPLLREEVVFQVGLHQYLVGSQLPCEPEPDVAVLTSMLTDPSHITAETAKRRL





ARGSPPSLASSSASQLSAPSLKATCTTHHDSPDADLIEANLLWRQEMGGNITRVESEN





KVVILDSFDPLRAEDDEGEISVPAEILRKSRKFPPALPIWAPPDYNPPLLESWKDPDY





VPPVVHGCPLPPTKAPPIPPPRRKRTVVLTESTVSSALAELATKTFGSSGSSAIDSGT





ATAPPDQASGDGDRESDVESFSSMPPLEGEPGDPDLSDGSWSTVSEEASEDVVCCSMS





YTWTGALITPCAAEESKLPINPLSNSLLRHHNMVYATTSRSAGLRQKKVTFDRLQVLD





DHYRDVLKEMKAKASTVKAKLLSVEEACKLTPPHSAKSKFGYGAKDVRSLSSRAVTHI





RSVWKDLLEDTETPISTTIMAKNEVFCVQPEKGGRKPARLIVFPDLGVRVCEKMALYD





VVSTLPQAVMGSSYGFQYSPKQRVEFLVNTWKSKKCPMGFSYDTRCFDSTVTENDIRV





EESIYQCCDLAPEAKLAIKSLTERLYIGGPLTNSKGQNCGYRRCRASGVLTTSCGNTL





TCYLKATAACRAAKLRDCTMLVNGDDLVVICESAGTQEDAASLRVFTEAMTRYSAPPG





DPPQPEYDLELITSCSSNVSVAHDASGKRVYYLTRDPTTPLARAAWETARHTPVNSWL





GNIIMYAPTLWARMILMTHFFSILLAQEQLEKTLDCQIYGACYSIEPLDLPQIIERLH





GLSAFSLHSYSPGEINRVASCLRKLGVPPLRAWRHRARSVRAKLLSQGGRAATCGKYL





FNWAVRTKLKLTPIPAASRLDLSGWFVAGYSGGDIYHSLSRARPRWFMLCLLLLSVGV





GIYLLPNR





SEQ ID NO: 4,



nucleocapsid protein of influenza A virus



  1 MASQGTKRSY EQMETSGERQ NATEIRASVG RMVGGIGRFY IQMCTELKLS DHEGRLIQNS





 61 ITIERMVLSA FDERRNKYLE EHPSAGKDPK KTGGPIYRRR DGKWMRELIL YDKEEIRRIW





121 RQANNGEDAT AGLTHMMIWH SNLNDATYQR TRALVRTGMD PRMCSLMQGS TLPRRSGAAG





181 AAVKGVGTMV MELIRMIKRG INDRNFWRGE NGRRTRIAYE RMCNILKGKF QTAAQRAMMD





241 QVRESRNPGN AEIEDLIFLA RSALILRGSV AHKSCLPACV YGLAVASGYD FEREGYSLVG





301 IDPFRLLQNS QVFSLIRPNE NPAHKSQLVW MACHSAAFED LRVSSFIRGT RVVPRGQLST





361 RGVQIASNEN METMDSSTLE LRSRYWAIRT RSGGNTNQQR ASAGQISVQP TFSVQRNLPF





421 ERATIMAAFT GNTEGRTSDM RTEIIRMMEN ARPEDVSFQG RGVFELSDEK ATNPIVPSFD





481 MSNEGS





SEQ ID NO: 5



>gi|73919153|ref|YP_308840.1| matrix protein 2



[Influenza A virus (A/New York/392/2004(H3N2))]









MSLLTEVETPIRNEWGCRCNDSSDPLVVAASIIGILHLILWILDRLFFKCVYRLEKHGLKRGPSTEGVPE
70







SMREEYRKEQQNAVDADDSHFVSIELE













SEQ ID NO: 6



>gi|73919147|ref|YP_308843.1| nucleocapsid protein



[Influenza A virus (A/New York/392/2004(H3N2))]









MASQGTKRSYEQMETDGDRQNATEIRASVGKMIDGIGRFYIQMCTELKLSDHEGRLIQNSLTIEKMVLSA
 70






FDERRNKYLEEHPSAGKDPKKTGGPIYRRVDGKWMRELVLYDKEEIRRIWRQANNGEDATAGLTHIMIWH
140





SNLNDATYQRTRALVRTGMDPRMCSLMQGSTLPRRSGAAGAAVKGIGTMVMELIRMVKRGINDRNFWRGE
210





NGRKTRSAYERMCNILKGKFQTAAQRAMVDQVRESRNPGNAEIEDLIFLARSALILRGSVAHKSCLPACA
280





YGPAVSSGYDFEKEGYSLVGIDPFKLLQNSQIYSLIRPNENPAHKSQLVWMACHSAAFEDLRLLSFIRGT
350





KVSPRGKLSTRGVQIASNENMDNMGSSTLELRSGYWAIRTRSGGNTNQQRASAGQTSVQPTFSVQRNLPF
420





EKSTIMAAFTGNTEGRTSDMRAEIIRMMEGAKPEEVSFRGRGVFELSDEKATNPIVPSFDMSNEGSYFFG
490





DNAEEYDN









--






SEQ ID NO: 7



>gi|56583270|ref|NP_040979.2| matrix protein 2



[Influenza A virus (A/Puerto Rico/8/34(H1N1))]


MSLLTEVETPIRNEWGCRCNGSSDPLAIAANIIGILHLILWILDRLFFKCIYRREKYGLKGGPSTEGVPK






SMREEYRKEQQSAVDADDGHFVSIELE






SEQ ID NO: 8



>gi|8486130|ref|NP_040982.1| nucleocapsid protein



[Influenza A virus (A/Puerto Rico/8/34(H1N1))]


MASQGTKRSYEQMETDGERQNATEIRASVGKMIGGIGRFYIQMCTELKLSDYEGRLIQNSLTIERMVLSA





FDERRNKYLEEHPSAGKDPKKTGGPIYRRVNGKWMRELILYDKEEIRRIWRQANNGDDATAGLTHMMIWH





SNLNDATYQRTRALVRTGMDPRMCSLMQGSTLPRRSGAAGAAVKGVGTMVMELVRMIKRGINDRNFWRGE





NGRKTRIAYERMCNILKGKFQTAAQKAMMDQVRESRDPGNAEFEDLTFLARSALILRGSVAHKSCLPACV





YGPAVASGYDFEREGYSLVGIDPFRLLQNSQVYSLIRPNENPAHKSQLVWMACHSAAFEDLRVLSFIKGT





KVVPRGKLSTRGVQIASNENMETMESSTLELRSRYWAIRTRSGGNTNQQRASAGQISIQPTFSVQRNLPF





DRTTVMAAFTGNTEGRTSDMRTEIIRMMESARPEDVSFQGRGVFELSDEKAASPIVPSFDMSNEGSYFFG





DNAEEYDN


--





SEQ ID NO: 9



>gi|73912687|ref|YP_308853.1| membrane protein M2



[Influenza A virus (A/Korea/426/68(H2N2))]


MSLLTEVETPIRNEWGCRCNDSSDPLVVAASIIGILHFILWILDRLFFKCIYRFEKHGLKRGPSTEGVPE






SMREEYRKEQQSAVDADDSHFVSIELE






SEQ ID NO: 10



>gi|73921307|ref|YP_308871.1| nucleoprotein [Influenza A virus



(A/Korea/426/68(H2N2))]


MASQGTKRSYEQMETDGERQNATEIRASVGKMIDGIGRFYIQMCTELKLSDYEGRLIQNSLTIERMVLSA





FDERRNKYLEEHPSAGKDPKKTGGPIYKRVDGKWMRELVLYDKEEIRRIWRQANNGDDATAGLTHMMIWH





SNLNDTTYQRTRALVRTGMDPRMCSLMQGSTLPRRSGAAGAAVKGVGTMVMELIRMIKRGINDRNFWRGE





NGRKTRSAYERMCNILKGKFQTAAQRAMMDQVRESRNPGNAEIEDLIFLARSALILRGSVAHKSCLPACV





YGPAIASGYNFEKEGYSLVGIDPFKLLQNSQVYSLIRPNENPAHKSQLVWMACNSAAFEDLRVLSFIRGT





KVSPRGKLSTRGVQIASNENMDTMESSTLELRSRYWAIRTRSGGNTNQQRASAGQISVQPAFSVQRNLPF





DKPTIMAAFTGNTEGRTSDMRAEIIRMMEGAKPEEMSFQGRGVFELSDEKATNPIVPSFDMSNEGSYFFG





DNAEEYDN





SEQ ID NO: 11



>gi|330647|gb|AAA45994.1| pp65 [Human herpesvirus 5]










MASVLGPISGHVLKAVFSRGDTPVLPHETRLLQTGIHVRVSQPSLILVSQYTPDSTPCHRGDNQLQVQHT
 70






YFTGSEVENVSVNVHNPTGRSICPSQEPMSIYVYALPLKMLNIPSINVHHYPSAAERKHRHLPVADAVIH
140





ASGKQMWQARLTVSGLAWTRQQNQWKEPDVYYTSAFVFPTKDVALRHVVCAHELVCSMENTRATKMQVIG
210





DQYVKVYLESFCEDVPSGKLFMHVTLGSDVEEDLTMTRNPQPFMRPHERNGFTVLCPKNMIIKPGKISHI
280





MLDVAFTSHEHFGLLCPKSIPGLSISGNLLMNGQQIFLEVQAIRETVELRQYDPVAALFFFDIDLLLQRG
350





PQYSEHPTFTSQYRIQGKLEYRHTWDRHDEGAAQGDDDVWTSGSDSDEELVTTERKTPRVTGGGAMAGAS
420





TSAGRKRKSASSATACTAGVMTRGRLKAESTVAPEEDTDEDSDNEIHNPAVFTWPPWQAGILARNLVPMV
490






ATVQGQNLKYQEFFWDANDIYRIFAELEGVWQPAAQPKRRRHRQDALPGPCIASTPKKHRG

541











SEQ ID NO: 12



>gi|33330937|gb|AAQ10712.1| putative transforming protein E6



[Human papillomavirus type 16]









MHQKRTAMFQDPQERPGKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDLCIVYRDGNPYAVC
 70






DKCLKFYSKISEYRHYCYSVYGTTLEQQYNKPLCDLLIRCINCQKPLCPEEKQRHLDKKQRFHNIRGRWT
140





GRCMSCCRSSRTRRETQL












SEQ ID NO: 13



>gi|56583270|ref|NP_040979.2| matrix protein 2



[Influenza A virus (A/Puerto Rico/8/34(H1N1))]


MSLLTEVETPIRNEWGCRCNGSSDPLAIAANIIGILHLILWILDRLFFKCIYRRFKYGLKGGPSTEGVPK





SMREEYRKEQQSAVDADDGHFVSIELE





SEQ ID NO: 14



>gi|8486139|ref|NP_040987.1| PB2 protein



[Influenza A virus (A/Puerto Rico/8/34(H1N1))]


MERIKELRNLMSQSRTREILTKTTVDHMAIIKKYTSGRQEKNPALRMKWMMAMKYPITADKRITEMIPER





NEQGQTLWSKMNDAGSDRVMVSPLAVTWWNRNGPMTNTVHYPKIYKTYFERVERLKHGTFGPVHFRNQVK





IRRRVDINPGHADLSAKEAQDVIMEVVFPNEVGARILTSESQLTITKEKKEELQDCKISPLMVAYMLERE





LVRKTRFLPVAGGTSSVYIEVLHLTQGTCWEQMYTPGGEVKNDDVDQSLIIAARNIVRRAAVSADPLASL





LEMCHSTQIGGIRMVDILKQNPTEEQAVGICKAAMGLRISSSFSFGGFTFKRTSGSSVKREEEVLTGNLQ





TLKIRVHEGYEEFTMVGRRATAILRKATRRLIQLIVSGRDEQSIAEAIIVAMVFSQEDCMIKAVRGDLNF





VNRANQRLNPMHQLLRHFQKDAKVLFQNWGVEPIDNVMGMIGILPDMTPSIEMSMRGVRISKMGVDEYSS





TERVVVSIDRFLRVRDQRGNVLLSPEEVSETQGTEKLTITYSSSMMWEINGPESVLVNTYQWIIRNWETV





KIQWSQNPTMLYNKMEFEPFQSLVPKAIRGQYSGFVRTLFQQMRDVLGTFDTAQIIKLLPFAAAPPKQSR





MQFSSFTVNVRGSGMRILVRGNSPVFNYNKATKRLTVLGKDAGTLTEDPDEGTAGVESAVLRGFLILGKE





DRRYGPALSINELSNLAKGEKANVLIGQGDVVLVMKRKRDSSILTDSQTATKRIRMAIN





SEQ ID NO: 15



>gi|8486137|ref|NP_040986.1| polymerase PA



[Influenza A virus (A/Puerto Rico/8/34(H1N1))]


MEDFVRQCFNPMIVELAEKTMKEYGEDLKIETNKFAAICTHLEVCFMYSDFHFINEQGESIIVELGDPNA





LLKHRFEIIEGRDRTMAWTVVNSICNTTGAEKPKFLPDLYDYKENRFIEIGVTRREVHIYYLEKANKIKS





EKTHIHIFSFTGEEMATKADYTLDEESRARIKTRLFTIRQEMASRGLWDSFRQSERGEETIEERFEITGT





MRKLADQSLPPNFSSLENFRAYVDGFEPNGYIEGKLSQMSKEVNARIEPFLKTTPRPLRLPNGPPCSQRS





KFLLMDALKLSIEDPSHEGEGIPLYDAIKCMRTFFGWKEPNVVKPHEKGINPNYLLSWKQVLAELQDIEN





EEKIPKTKNMKKTSQLKWALGENMAPEKVDFDDCKDVGDLKQYDSDEPELRSLASWIQNEFNKACELTDS





SWIELDEIGEDVAPIEHIASMRRNYFTSEVSHCRATEYIMKGVYINTALLNASCAAMDDFQLIPMISKCR





TKEGRRKTNLYGFIIKGRSHLRNDTDVVNFVSMEFSLTDPRLEPHKWEKYCVLEIGDMLLRSAIGQVSRP





MFLYVRTNGTSKIKMKWGMEMRRCLLQSLQQIESMIEAESSVKEKDMTKEFFENKSETWPIGESPKGVEE





SSIGKVCRTLLAKSVFNSLYASPQLEGFSAESRKLLLIVQALRDNLEPGTFDLGGLYEAIEECLINDPWV





LLNASWFNSFLTHALS





SEQ ID NO: 16



>gi|8486133|ref|NP_040984.1| nonstructural protein NS1



[Influenza A virus (A/Puerto Rico/8/34(H1N1))]


MDPNTVSSFQVDCFLWHVRKRVADQELGDAPFLDRLRRDQKSLRGRGSTLGLDIETATRAGKQIVERILK





EESDEALKMTMASVPASRYLTDMTLEEMSREWSMLIPKQKVAGPLCIRMDQAIMDKNIILKANFSVIFDR





LETLILLRAFTEEGAIVGEISPLPSLPGHTAEDVKNAVGVLIGGLEWNDNTVRVSETLQRFAWRSSNENG





RPPLTPKQKREMAGTIRSEV





SEQ ID NO: 17



>gi|8486132|ref|NP_040983.1| nonstructural protein NS2



[Influenza A virus (A/Puerto Rico/8/34(H1N1))]


MDPNTVSSFQDILLRMSKMQLESSSEDLNGMITQFESLKLYRDSLGEAVMRMGDLHSLQNRNEKWREQLG





QKFEEIRWLIEEVRHKLKVTENSFEQITFMQALHLLLEVEQEIRTFSFQLI





SEQ ID NO: 18



>gi|8486128|ref|NP_040981.1| neuraminidase



[Influenza A virus (A/Puerto Rico/8/34(H1N1))]


MNPNQKIITIGSICLVVGLISLILQIGNIISIWISHSIQTGSQNHTGICNQNIITYKNSTWVKDTTSVIL





TGNSSLCPIRGWAIYSKDNSIRIGSKGDVFVIREPFISCSHLECRTFFLTQGALLNDRHSNGTVKDRSPY





RALMSCPVGEAPSPYNSRFESVAWSASACHDGMGWLTIGISGPDNGAVAVLKYNGIITETIKSWRKKILR





TQESECACVNGSCFTIMTDGPSDGLASYKIFKIEKGKVTKSIELNAPNSHYEECSCYPDTGKVMCVCRDN





WHGSNRPWVSFDQNLDYQIGYICSGVFGDNPRPKDGTGSCGPVYVDGANGVKGFSYRYGNGVWIGRTKSH





SSRHGFEMIWDPNGWTETDSKFSVRQDVVAMTDWSGYSGSFVQHPELTGLDCIRPCFWVELIRGRPKEKT





IWTSASSISFCGVNSDTVDWSWPDGAELPFTIDK





SEQ ID NO: 19



>gi|8486126|ref|NP_040980.1| haemagglutinin [Influenza A virus



(A/Puerto Rico/8/34(H1N1))]


MKANLLVLLCALAAADADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCRLKGIAPLQLG





KCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSW





PNHNTTKGVTAACSHAGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPSNSKDQQNIYQ





NENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPRYAFALSRGFGS





GIITSNASMHECNTKCQTPLGAINSSLPFQNIHPVTIGECPKYVRSAKLRMVTGLRNIPSIQSRGLFGAI





AGFIEGGWTGMIDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNIQFTAVGKEFNKLEKR





MENLNKKVDDGFLDIWTYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCDN





ECMESVRNGTYDYPKYSEESKLNREKVDGVKLESMGIYQILAIYSTVASSLVLLVSLGAISFWMCSNGSL





QCRICI





SEQ ID NO: 20



>gi|8486123|ref|NP_040978.1| matrix protein 1 [Influenza A virus



(A/Puerto Rico/8/34(H1N1))]


MSLLTEVETYVLSIIPSGPLKAEIAQRLEDVFAGKNTDLEVLMEWLKTRPILSPLTKGILGFVFTLTVPS





ERGLQRRRFVQNALNGNGDPNNMDKAVKLYRKLKREITFHGAKEISLSYSAGALASCMGLIYNRMGAVTT





EVAFGLVCATCEQIADSQHRSHRQMVTTTNPLIRHENRMVLASTTAKAMEQMAGSSEQAAEAMEVASQAR





QMVQAMRTIGTHPSSSAGLKNDLLENLQAYQKRMGVQMQRFK





SEQ ID NO: 21



>gi|83031685|ref|YP_418248.1| PB1-F2 protein [Influenza A virus



(A/Puerto Rico/8/34(H1N1))]


MGQEQDTPWILSTGHISTQKRQDGQQTPKLEHRNSTRLMGHCQKTMNQVVMPKQIVYWKQWLSLRNPILV





FLKTRVLKRWRLFSKHE





SEQ ID NO: 22



>gi|8486135|ref|NP_040985.1| polymerase 1 PB1 [Influenza A virus



(A/Puerto Rico/8/34(H1N1))]


MDVNPTLLFLKVPAQNAISTTFPYTGDPPYSHGTGTGYTMDTVNRTHQYSEKARWTTNTETGAPQLNPID





GPLPEDNEPSGYAQTDCVLEAMAFLEESHPGIFENSCIETMEVVQQTRVDKLTQGRQTYDWTLNRNQPAA





TALANTIEVFRSNGLTANESGRLIDFLKDVMESMKKEEMGITTHFQRKRRVRDNMTKKMITQRTIGKRKQ





RLNKRSYLIRALTLNTMTKDAERGKLKRRAIATPGMQIRGFVYFVETLARSICEKLEQSGLPVGGNEKKA





KLANVVRKMMTNSQDTELSLTITGDNTKWNENQNPRMFLAMITYMTRNQPEWFRNVLSIAPIMFSNKMAR





LGKGYMFESKSMKLRTQIPAEMLASIDLKYFNDSTRKKIEKIRPLLIEGTASLSPGMMMGMFNMLSTVLG





VSILNLGQKRYTKTTYWWDGLQSSDDFALIVNAPNHEGIQAGVDRFYRTCKLHGINMSKKKSYINRTGTF





EFTSFFYRYGFVANFSMELPSFGVSGSNESADMSIGVTVIKNNMINNDLGPATAQMALQLFIKDYRYTYR





CHRGDTQIQTRRSFEIKKLWEQTRSKAGLLVSDGGPNLYNIRNLHIPEVCLKWELMDEDYQGRLCNPLNP





FVSHKEIESMNNAVMMPAHGPAKNMEYDAVATTHSWIPKRNRSILNTSQRGVLEDEQMYQRCCNLFEKFF





PSSSYRRPVGISSMVEAMVSRARIDARIDFESGRIKKEEFTEIMKICSTIEELRRQK





SEQ ID NO: 23



>gi|8486130|ref|NP_040982.1| nucleocapsid protein



[Influenza A virus (A/Puerto Rico/8/34(H1N1))]


MASQGTKRSYEQMETDGERQNATEIRASVGKMIGGIGRFYIQMCTELKLSDYEGRLIQNSLTIERMVLSA





FDERRNKYLEEHPSAGKDPKKTGGPIYRRVNGKWMRELILYDKEEIRRIWRQANNGDDATAGLTHMMIWH





SNLNDATYQRTRALVRTGMDPRMCSLMQGSTLPRRSGAAGAAVKGVGTMVMELVRMIKRGINDRNFWRGE





NGRKTRIAYERMCNILKGKFQTAAQKAMMDQVRESRDPGNAEFEDLTFLARSALILRGSVAHKSCLPACV





YGPAVASGYDFEREGYSLVGIDPFRLLQNSQVYSLIRPNENPAHKSQLVWMACHSAAFEDLRVLSFIKGT





KVVPRGKLSTRGVQIASNENMETMESSTLELRSRYWAIRTRSGGNTNQQRASAGQISIQPTFSVQRNLPF





DRTTVMAAFTGNTEGRTSDMRTEIIRMMESARPEDVSFQGRGVFELSDEKAASPIVPSFDMSNEGSYFFG





DNAEEYDN





SEQ ID NO: 24



>gi|73918826|ref|YP_308855.1| polymerase 2 [Influenza A virus



(A/Korea/426/1968(H2N2))]


MERIKELRNLMSQSRTREILTKTTVDHMAIIKKYTSGRQEKNPSLRMKWMMAMKYPITADKRITEMVPER





NEQGQTLWSKMSDAGSDRVMVSPLAVTWWNRNGPMTSTVHYPKIYKTYFEKVERLKHGTFGPVHFRNQVK





IRRRVDINPGHADLSAKEAQDVIMEVVFPNEVGARILTSESQLTITKEKKEELQDCKISPLMVAYMLERE





LVRKTRFLPVAGGTSSVYIEVLHLTQGTCWEQMYTPGGEVRNDDVDQSLIIAARNIVRRAAVSADPLASL





LEMCHSTQIGGTRMVDILRQNPTEEQAVDICKAAMGLRISSSFSFGGFTFKRTSGSSIKREEEVLTGNLQ





TLKIRVHEGYEEFTMVGKRATAILRKATRRLVQLIVSGRDEQSIAEAIIVAMVFSQEDCMIKAVRGDLNF





VNRANQRLNPMHQLLRHFQKDAKVLFQNWGIEHIDNVMGMIGVLPDMTPSTEMSMRGIRVSKMGVDEYSS





TERVVVSIDRFLRVRDQRGNVLLSPEEVSETQGTEKLTITYSSSMMWEINGPESVLVNTYQWIIRNWETV





KIQWSQNPTMLYNKMEFEPFQSLVPKAIRGQYSGFVRTLFQQMRDVLGTFDTTQIIKLLPFAAAPPKQSR





MQFSSLTVNVRGSGMRILVRGNSPVFNYNKTTKRLTILGKDAGTLTEDPDEGTSGVESAVLRGFLILGKE





DRRYGPALSINELSTLAKGEKANVLIGQGDVVLVMKRKRDSSILTDSQTATKRIRMAIN





SEQ ID NO: 25



>gi|73919145|ref|YP_308850.1| hemagglutinin [Influenza A virus



(A/Korea/426/68(H2N2))]


MAIIYLILLFTAVRGDQICIGYHANNSTEKVDTILERNVTVTHAKDILEKTHNGKLCKLNGIPPLELGDC





SIAGWLLGNPECDRLLSVPEWSYIMEKENPRYSLCYPGSFNDYEELKHLLSSVKHFEKVKILPKDRWTQH





TTTGGSWACAVSGKPSFFRNMVWLTRKGSNYPVAKGSYNNTSGEQMLIIWGVHHPNDEAEQRALYQNVGT





YVSVATSTLYKRSIPEIAARPKVNGLGRRMEFSWTLLDMWDTINFESTGNLVAPEYGFKISKRGSSGIMK





TEGTLENCETKCQTPLGAINTTLPFHNVHPLTIGECPKYVKSEKLVLATGLRNVPQIESRGLFGAIAGFI





EGGWQGMVDGWYGYHHSNDQGSGYAADKESTQKAFNGITNKVNSVIEKMNTQFEAVGKEFSNLEKRLENL





NKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRMQLRDNVKELGNGCFEFYHKCDNECMD





SVKNGTYDYPKYEEESKLNRNEIKGVKLSSMGVYQILAIYATVAGSLSLAIMMAGISFWMCSNGSLQCRI





CI





SEQ ID NO: 26



>gi|73912688|ref|YP_308854.1| membrane protein M1



[Influenza A virus (A/Korea/426/68(H2N2))]


MSLLTEVETYVLSIVPSGPLKAEIAQRLEDVFAGKNTDLEALMEWLKTRPILSPLTKGILGFVFTLTVPS





ERGLQRRRFVQNALNGNGDPNNMDRAVKLYRKLKREITFHGAKEVALSYSAGALASCMGLIYNRMGAVTT





EVAFAVVCATCEQIADSQHRSHRQMVTTTNPLIRHENRMVLASTTAKAMEQMAGSSEQAAEAMEVASQAR





QMVQAMRAIGTPPSSSAGLKDDLLENLQAYQKRMGVQMQRFK





SEQ ID NO: 27



>gi|73912687|ref|YP_308853.1| membrane protein M2



[Influenza A virus (A/Korea/426/68(H2N2))]


MSLLTEVETPIRNEWGCRCNDSSDPLVVAASIIGILHFILWILDRLFFKCIYRFFKHGLKRGPSTEGVPE





SMREEYRKEQQSAVDADDSHFVSIELE





SEQ ID NO: 28



>gi|73912685|ref|YP_308852.1| polymerase PA [Influenza A virus



(A/Korea/426/68(H2N2))]


MEDFVRQCFNPMIVELAEKAMKEYGEDLKIETNKFAAICTHLEVCFMYSDFHFINEQGESIMVELDDPNA





LLKHRFEIIEGRDRTMAWTVVNSICNTTGAEKPKFLPDLYDYKENRFIEIGVTRREVHIYYLEKANKIKS





ENTHIHIFSFTGEEMATKADYTLDEESRARIKTRLFTIRQEMANRGLWDSFRQSERGEETIEERFEITGT





MRRLADQSLPPNFSCLENFRAYVDGFEPNGYIEGKLSQMSKEVNAKIEPFLKTTPRPIRLPDGPPCFQRS





KFLLMDALKLSIEDPSHEGEGIPLYDAIKCMRTFFGWKEPYIVKPHEKGINPNYLLSWKQVLAELQDIEN





EEKIPRTKNMKKTSQLKWALGENMAPEKVDFDNCRDISDLKQYDSDEPELRSLSSWIQNEFNKACELTDS





IWIELDEIGEDVAPIEHIASMRRNYFTAEVSHCRATEYIMKGVYINTALLNASCAAMDDFQLIPMISKCR





TKEGRRKTNLYGFIIKGRSHLRNDTDVVNFVSMEFSLTDPRLEPHKWEKYCVLEIGDMLLRSAIGQMSRP





MFLYVRTNGTSKIKMKWGMEMRPCLLQSLQQIESMVEAESSVKEKDMTKEFFENKSETWPIGESPKGVEE





GSIGKVCRTLLAKSVFNSLYASPQLEGFSAESRKLLLVVQALRDNLEPGTFDLGGLYEAIEECLINDPWV





LLNASWFNSFLTHALR





SEQ ID NO: 29



>gi|73921833|ref|YP_308877.1| PB1-F2 protein [Influenza A virus



(A/Korea/426/68(H2N2))]


MGQEQDTPWTQSTEHINIQKRGSGQQTRKLERPNLTQLMDHYLRTMNQVDMHKQTASWKQWLSLRNHTQE





SLKIRVLKRWKLFNKQEWTN





SEQ ID NO: 30



>gi|73912683|ref|YP_308851.1| PB1 polymerase subunit



[Influenza A virus (A/Korea/426/68(H2N2))]


MDVNPTLLFLKVPAQNAISTTFPYTGDPPYSHGTGTGYTMDTVNRTHQYSEKGKWTTNTETGAPQLNPID





GPLPEDNEPSGYAQTDCVLEAMAFLEESHPGIFENSCLETMEVIQQTRVDKLTQGRQTYDWTLNRNQPAA





TALANTIEVFRSNGLTANESGRLIDFLKDVIESMDKEEMEITTHFQRKRRVRDNMTKKMVTQRTIGKKKQ





RLNKRSYLIRALTLNTMTKDAERGKLKRRAIATPGMQIRGFVHFVETLARNICEKLEQSGLPVGGNEKKA





KLANVVRKMMTNSQDTELSFTITGDNTKWNENQNPRVFLAMITYITRNQPEWFRNVLSIAPIMFSNKMAR





LGKGYMFESKSMKLRTQIPAEMLASIDLKYFNESTRKKIEKIRPLLIDGTVSLSPGMMMGMFNMLSTVLG





VSILNLGQKKYTKTTYWWDGLQSSDDFALIVNAPNHEGIQAGVNRFYRTCKLVGINMSKKKSYINRTGTF





EFTSFFYRYGFVANFSMELPSFGVSGINESADMSIGVTVIKNNMINNDLGPATAQMALQLFIKDYRYTYR





CHRGDTQIQTRRSFELKKLWEQTRSKAGLLVSDGGSNLYNIRNLHIPEVCLKWELMDEDYQGRLCNPLNP





FVSHKEIESVNNAVVMPAHGPAKSMEYDAVATTHSWTPKRNRSILNTSQRGILEDEQMYQKCCNLFEKFF





PSSSYRRPVGISSMVEAMVSRARIDARIDFESGRIKKEEFAEIMKICSTIEELRRQK





SEQ ID NO: 31



>gi|73921567|ref|YP_308869.1| non-structural protein NS2



[Influenza A virus (A/Korea/426/68(H2N2))]


MDSNTVSSFQDILLRMSKMQLGSSSEDLNGMITQFESLKLYRDSLGEAVMRMGDLHSLQNRNGKWREQLG





QKFEEIRWLIEEVRHRLKITENSFEQITFMQALQLLFEVEQEIRTFSFQLI





SEQ ID NO: 32



>gi|73921566|ref|YP_308870.1| non-structural protein NS1



[Influenza A virus (A/Korea/426/68(H2N2))]


MDSNTVSSFQVDCFLWHVRKQVVDQELGDAPFLDRLRRDQKSLRGRGSTLDLDIEAATRVGKQIVERILK





EESDEALKMTMASAPASRYLTDMTIEELSRDWFMLMPKQKVEGPLCIRIDQAIMDKNIMLKANFSVIFDR





LETLILLRAFTEEGAIVGEISPLPSLPGHTIEDVKNAIGVLIGGLEWNDNTVRVSKTLQRFAWRSSNENG





RPPLTPKQKRKMARTIRSKVRRDKMAD





SEQ ID NO: 33



>gi|73921307|ref|YP_308871.1| nucleoprotein [Influenza A virus



(A/Korea/426/68(H2N2))]


MASQGTKRSYEQMETDGERQNATEIRASVGKMIDGIGRFYIQMCTELKLSDYEGRLIQNSLTIERMVLSA





FDERRNKYLEEHPSAGKDPKKTGGPIYKRVDGKWMRELVLYDKEEIRRIWRQANNGDDATAGLTHMMIWH





SNLNDTTYQRTRALVRTGMDPRMCSLMQGSTLPRRSGAAGAAVKGVGTMVMELIRMIKRGINDRNFWRGE





NGRKTRSAYERMCNILKGKFQTAAQRAMMDQVRESRNPGNAEIEDLIFLARSALILRGSVAHKSCLPACV





YGPAIASGYNFEKEGYSLVGIDPFKLLQNSQVYSLIRPNENPAHKSQLVWMACNSAAFEDLRVLSFIRGT





KVSPRGKLSTRGVQIASNENMDTMESSTLELRSRYWAIRTRSGGNTNQQRASAGQISVQPAFSVQRNLPF





DKPTIMAAFTGNTEGRTSDMRAEIIRMMEGAKPEEMSFQGRGVFELSDEKATNPIVPSFDMSNEGSYFFG





DNAEEYDN





SEQ ID NO: 34



>gi|73921304|ref|YP_308872.1| neuraminidase [Influenza A virus



(A/Korea/426/68(H2N2))]


MNPNQKIITIGSVSLTIATVCFLMQIAILVTTVTLHFKQHECDSPASNQVMPCEPIIIERNITEIVYLNN





TTIEKEICPEVVEYRNWSKPQCQITGFAPFSKDNSIRLSAGGDIWVTREPYVSCDPGKCYQFALGQGTTL





DNKHSNDTIHDRIPHRTLLMNELGVPFHLGTRQVCVAWSSSSCHDGKAWLHVCVTGDDKNATASFIYDGR





LMDSIGSWSQNILRTQESECVCINGTCTVVMTDGSASGRADTRILFIEEGKIVHISPLSGSAQHVEECSC





YPRYPDVRCICRDNWKGSNRPVIDINMEDYSIDSSYVCSGLVGDTPRNDDRSSNSNCRNPNNERGNPGVK





GWAFDNGDDVWMGRTISKDLRSGYETFKVIGGWSTPNSKSQINRQVIVDSNNWSGYSGIFSVEGKRCINR





CFYVELIRGRQQETRVWWTSNSIVVFCGTSGTYGTGSWPDGANINFMPI





SEQ ID NO: 35



>gi|73919213|ref|YP_308844.1| nonstructural protein 2



[Influenza A virus (A/New York/392/2004(H3N2))]


MDSNTVSSFQDILLRMSKMQLGSSSEDLNGMITQFESLKIYRDSLGEAVMRMGDLHLLQNRNGKWREQLG





QKFEEIRWLIEEVRHRLKTTENSFEQITFMQALQLLFEVEQEIRTFSFQLI





SEQ ID NO: 36



>gi|73919212|ref|YP_308845.1| nonstructural protein 1



[Influenza A virus (A/New York/392/2004(H3N2))]


MDSNTVSSFQVDCFLWHIRKQVVDQELSDAPFLDRLRRDQRSLRGRGNTLGLDIKAATHVGKQIVEKILK





EESDEALKMTMVSTPASRYITDMTIEELSRNWFMLMPKQKVEGPLCIRMDQAIMEKNIMLKANFSVIFDR





LETIVLLRAFTEEGAIVGEISPLPSFPGHTIEDVKNAIGVLIGGLEWNDNTVRVSKNLQRFAWRSSNENG





GPPLTPKQKRKMARTARSKV





SEQ ID NO: 37



>gi|73919207|ref|YP_308839.1| hemagglutinin [Influenza A



virus (A/New York/392/2004(H3N2))]


MKTIIALSYILCLVFAQKLPGNDNSTATLCLGHHAVPNGTIVKTITNDQIEVTNATELVQSSSTGGICDS





PHQILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNES





FNWTGVTQNGTSSACKRRSNNSFFSRLNWLTHLKFKYPALNVTMPNNEKFDKLYIWGVHHPGTDNDQISL





YAQASGRITVSTKRSQQTVIPSIGSRPRIRDVPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGK





SSIMRSDAPIGKCNSECITPNGSIPNDKPFQNVNRITYGACPRYVKQNTLKLATGMRNVPEKQTRGIFGA





IAGFIENGWEGMVDGWYGFRHQNSEGTGQAADLKSTQAAINQINGKLNRLIGKTNEKFHQIEKEFSEVEG





RIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFERTKKQLRENAEDMGNGCFKIYHKCD





NACIGSIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWILWISFAISCFLLCVALLGFIMWACQKGNI





RCNICI





SEQ ID NO: 38



>gi|73919153|ref|YP_308840.1| matrix protein 2 [Influenza A



virus (A/New York/392/2004(H3N2))]


MSLLTEVETPIRNEWGCRCNDSSDPLVVAASIIGILHLILWILDRLFFKCVYRLFKHGLKRGPSTEGVPE





SMREEYRKEQQNAVDADDSHFVSIELE





SEQ ID NO: 39



>gi|73919152|ref|YP_308841.1| matrix protein 1 [Influenza A



virus (A/New York/392/2004(H3N2))]


MSLLTEVETYVLSIVPSGPLKAEIAQRLEDVFAGKNTDLEALMEWLKTRPILSPLTKGILGFVFTLTVPS





ERGLQRRRFVQNALNGNGDPNNMDKAVKLYRKLKREITFHGAKEIALSYSAGALASCMGLIYNRMGAVTT





EVAFGLVCATCEQIADSQHRSHRQMVATTNPLIKHENRMVLASTTAKAMEQMAGSSEQAAEAMEIASQAR





QMVQAMRAVGTHPSSSTGLRDDLLENLQTYQKRMGVQMQRFK





SEQ ID NO: 40



>gi|73919150|ref|YP_308848.1| PB1-F2 protein [Influenza A



virus (A/New York/392/2004(H3N2))]


MEQEQDTPWTQSTEHTNIQRRGSGRQIQKLGHPNSTQLMDHYLRIMSQVDMHKQTVSWRLWPSLKNPTQV





SLRTHALKQWKSFNKQGWTN





SEQ ID NO: 41



>gi|73919149|ref|YP_308847.1| polymerase PB1 [Influenza A



virus (A/New York/392/2004(H3N2))]


MDVNPTLLFLKVPAQNAISTTFPYTGDPPYSHGTGTGYTMDTVNRTHQYSEKGKWTTNTETGAPQLNPID





GPLPEDNEPSGYAQTDCVLEAMAFLEESHPGIFENSCLETMEVVQQTRVDKLTQGRQTYDWTLNRNQPAA





TALANTIEVFRSNGLTANESGRLIDFLKDVMESMDKEEMEITTHFQRKRRVRDNMTKKMVTQRTIGKKKQ





RVNKRGYLIRALTLNTMTKDAERGKLKRRAIATPGMQIRGFVYFVETLARSICEKLEQSGLPVGGNEKKA





KLANVVRKMMTNSQDTELSFTITGDNTKWNENQNPRMFLAMITYITKNQPEWFRNILSIAPIMFSNKMAR





LGKGYMFESKRMKLRTQIPAEMLASIDLKYFNESTRKKIEKIRPLLIDGTASLSPGMMMGMFNMLSTVLG





VSVLNLGQKKYTKTTYWWDGLQSSDDFALIVNAPNHEGIQAGVDRFYRTCKLVGINMSKKKSYINKTGTF





EFTSFFYRYGFVANFSMELPSFGVSGINESADMSIGVTVIKNNMINNDLGPATAQMALQLFIKDYRYTYR





CHRGDTQIQTRRSFELKKLWDQTQSRAGLLVSDGGPNLYNIRNLHIPEVCLKWELMDENYRGRLCNPLNP





FVSHKEIESVNNAVVMPAHGPAKSMEYDAVATTHSWNPKRNRSILNTSQRGILEDEQMYQKCCNLFEKFF





PSSSYRRPIGISSMVEAMVSRARIDARIDFESGRIKKEEFSEIMKICSTIEELRRQK





SEQ ID NO: 42



>gi|73919147|ref|YP_308843.1| nucleocapsid protein



[Influenza A virus (A/New York/392/2004(H3N2))]


MASQGTKRSYEQMETDGDRQNATEIRASVGKMIDGIGRFYIQMCTELKLSDHEGRLIQNSLTIEKMVLSA





FDERRNKYLEEHPSAGKDPKKTGGPIYRRVDGKWMRELVLYDKEEIRRIWRQANNGEDATAGLTHIMIWH





SNLNDATYQRTRALVRTGMDPRMCSLMQGSTLPRRSGAAGAAVKGIGTMVMELIRMVKRGINDRNFWRGE





NGRKTRSAYERMCNILKGKFQTAAQRAMVDQVRESRNPGNAEIEDLIFLARSALILRGSVAHKSCLPACA





YGPAVSSGYDFEKEGYSLVGIDPFKLLQNSQIYSLIRPNENPAHKSQLVWMACHSAAFEDLRLLSFIRGT





KVSPRGKLSTRGVQIASNENMDNMGSSTLELRSGYWAIRTRSGGNTNQQRASAGQTSVQPTFSVQRNLPF





EKSTIMAAFTGNTEGRTSDMRAEIIRMMEGAKPEEVSFRGRGVFELSDEKATNPIVPSFDMSNEGSYFFG





DNAEEYDN





SEQ ID NO: 43



>gi|73919136|ref|YP_308842.1| neuraminidase



[Influenza A virus (A/New York/392/2004(H3N2))]


MNPNQKIITIGSVSLTISTICFFMQIAILITTVTLHFKQYEFNSPPNNQVMLCEPTIIERNITEIVYLTN





TTIEKEMCPKLAEYRNWSKPQCDITGFAPFSKDNSIRLSAGGDIWVTREPYVSCDPDKCYQFALGQGTTL





NNVHSNDTVHDRTPYRTLLMNELGVPFHLGTKQVCIAWSSSSCHDGKAWLHVCVTGDDKNATASFIYNGR





LVDSIVSWSKKILRTQESECVCINGTCTVVMTDGSASGKADTKILFIEEGKIIHTSTLSGSAQHVEECSC





YPRYPGVRCVCRDNWKGSNRPIVDINIKDYSIVSSYVCSGLVGDTPRKNDSSSSSHCLDPNNEEGGHGVK





GWAFDDGNDVWMGRTISEKLRSGYETFKVIEGWSKPNSKLQINRQVIVDRGNRSGYSGIFSVEGKSCINR





CFYVELIRGRKEETEVLWTSNSIVVFCGTSGTYGTGSWPDGADINLMPI





SEQ ID NO: 44



>gi|73919134|ref|YP_308846.1| polymerase PA



[Influenza A virus (A/New York/392/2004(H3N2))]


MEDFVRQCFNPMIVELAEKAMKEYGEDLKIETNKFAAICTHLEVCFMYSDFHFINEQGESIVVELDDPNA





LLKHRFEIIEGRDRTMAWTVVNSICNTTGAEKPKFLPDLYDYKENRFIEIGVTRREVHIYYLEKANKIKS





ENTHIHIFSFTGEEIATKADYTLDEESRARIKTRLFTIRQEMANRGLWDSFRQSERGEETIEEKFEISGT





MRRLADQSLPPKFSCLENFRAYVDGFEPNGCIEGKLSQMSKEVNAKIEPFLKTTPRPIKLPNGPPCYQRS





KFLLMDALKLSIEDPSHEGEGIPLYDAIKCIKTFFGWKEPYIVKPHEKGINSNYLLSWKQVLSELQDIEN





EEKIPRTKNMKKTSQLKWALGENMAPEKVDFDNCRDISDLKQYDSDEPELRSLSSWIQNEFNKACELTDS





IWIELDEIGEDVAPIEYIASMRRNYFTAEVSHCRATEYIMKGVYINTALLNASCAAMDDFQLIPMISKCR





TKEGRRKTNLYGFIIKGRSHLRNDTDVVNFVSMEFSLTDPRLEPHKWEKYCVLEIGDMLLRSAIGQISRP





MFLYVRTNGTSKVKMKWGMEMRRCLLQSLQQIESMIEAESSIKEKDMTKEFFENKSEAWPIGESPKGVEE





GSIGKVCRTLLAKSVFNSLYASPQLEGFSAESRKLLLVVQALRDNLEPGTFDLGGLYEAIEECLINDPWV





LLNASWFNSFLTHALK





SEQ ID NO: 45



>gi|73919060|ref|YP_308849.1| polymerase PB2



[Influenza A virus (A/New York/392/2004(H3N2))]


MERIKELRNLMSQSRTREILTKTTVDHMAIIKKYTSGRQEKNPSLRMKWMMAMKYPITADKRITEMVPER





NEQGQTLWSKMSDAGSDRVMVSPLAVTWWNRNGPVASTVHYPKVYKTYFDKVERLKHGTFGPVHFRNQVK





IRRRVDINPGHADLSAKEAQDVIMEVVFPNEVGARILTSESQLTITKEKKEELRDCKISPLMVAYMLERE





LVRKTRFLPVAGGTSSIYIEVLHLTQGTCWEQMYTPGGEVRNDDVDQSLIIAARNIVRRAAVSADPLASL





LEMCHSTQIGGTRMVDILRQNPTEEQAVDICKAAMGLRISSSFSFGGFTFKRTSGSSVKKEEEVLTGNLQ





TLKIRVHEGYEEFTMVGKRATAILRKATRRLVQLIVSGRDEQSIAEAIIVAMVFSQEDCMIKAVRGDLNF





VNRANQRLNPMHQLLRHFQKDAKVLFQNWGIEHIDSVMGMVGVLPDMTPSTEMSMRGIRVSKMGVDEYSS





TERVVVSIDRFLRVRDQRGNVLLSPEEVSETQGTERLTITYSSSMMWEINGPESVLVNTYQWIIRNWEAV





KIQWSQNPAMLYNKMEFEPFQSLVPKAIRSQYSGFVRTLFQQMRDVLGTFDTTQIIKLLPFAAAPPKQSR





MQFSSLTVNVRGSGMRILVRGNSPVFNYNKTTKRLTILGKDAGTLIEDPDESTSGVESAVLRGFLIIGKE





DRRYGPALSINELSNLAKGEKANVLIGQGDVVLVMKRKRDSSILTDSQTATKRIRMAIN





SEQ ID NO: 46: CMV Protein IE122:


>gi|39841910|gb|AAR31478.1| UL122 [Human herpesvirus 5]


MESSAKRKMDPDNPDEGPSSKVPRPETPVTKATTFLQTMLRKEVNSQLSLGDPLFPELAEESLKTFEQVT





EDCNENPEKDVLAELGDILAQAVNHAGIDSSSTGHTLTTHSCSVSSAPLNKPTPTSVAVTNTPLPGASAT





PELSPRKKPRKTTRPFKVIIKPPVPPAPIMLPLIKQEDIKPEPDFTIQYRNKIIDTAGCIVISDSEEEQG





EEVETRGATASSPSTGSGTPRVTSPTHPLSQMNHPPLPDPLARPDEDSSSSSSSSCSSASDSESESEEMK





CSSGGGASVTSSHHGRGGFGSAASSSLLSCGHQSSGGASTGPRKKKSKRISELDNEKVRNIMKDKNTPFCTPNVQTRRG





RVKIDEVSRMFRNTNRSLEYKNLPFTIPSMHQVLDEAIKACKTMQVNNKGIQIIYTRNHEVKSEVDAVRCRLGTMCNLA





LSTPFLMEHTMPVTHPPEVAQRTADACNEGVKAAWSLKELHTHQLCPRSSDYRNMIIHAATPVDLLGALNLCLPLMQKF





PKQVMVRIFSTNQGGFMLPIYETAAKAYAVGQFEQPTETPPEDLDTLSLAIEAAIQDLRNKSQ





SEQ ID NO: 126:


>gi|4927721|gb|AAD33253.1|AF125673_2 E7


[Human papillomavirus type 16]


MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQ





STHVDIRTLEDLLMGTLGIVCPICSQKP





SEQ ID NO: 200: Influensa M2


>gi|21693176|gb|AAM75162| /Human/M2/H1N1/Puerto


Rico/1934/// matrix protein M2 [Influenza A virus


(A/Puerto Rico/8/34/Mount Sinai(H1N1))]



MSLLTEVETPIRNEWGCRCNGSSDPLAIAANIIGILHLTLWILDRLFFKCIYRRFKYGLKGGPSTEGVPK






SMREEYRKEQQSAVDADDGHFVSIELE





SEQ ID NO: 201: >gi|1906383|gb|AAB50256.1| tat protein


[Human immunodeficiency virus 1]


MEPVDPRLEPWKHPGSQPKTACTNCYCKKCCFHCQVCFITKALGISYGRKKRRQRRRAHQNSQTHQASLS





KQPTSQPRGDPTGPKE





SEQ ID NO: 202: >B.FR.1983.HXB2-LAI-IIIB-BRU (gp120)


MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACV





PTDPNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPCVKLTPLCVSLKCTDLKNDTNTNSSSGRMIME





KGEIKNCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYKLTSCNTSVITQACPKVSFEPIPIHYCAPAGFAI





LKCNNKTFNGTGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSVNFTDNAKTIIVQLNTSVEINCTRPN





NNTRKRIRIQRGPGRAFVTIGKIGNMRQAHCNISRAKWNNTLKQIASKLREQFGNNKTIIFKQSSGGDPEIVTHS





FNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTITLPCRIKQIINMWQKVGKAMYAPPISGQIRCSSNIT





GLLLTRDGGNSNNESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKRRVVQREKR





SEQ ID NO: 203: HIV gp41


>B.FR.1983.HXB2-LAI-IIIB-BRU (ACC No. K03455)


AVGIGALFLGFLGAAGSTMGAASMTLTVQARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERY





LKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNHTTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELL





ELDKWASLWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSIVNRVRQGYSPLSFQTHLPTPRGPDRPEGIEEE






GGERDRDRSIRLVNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKYWWNLLQYWSQELKNSA






VSLLNATAIAVAEGTDRVIEVVQGACRAIRHIPRRIRQGLERILL





SEQ ID NO: 204: >1b._._.AB016785._ (HCV-E1)


YEVRNVSGVYHVTNDCSNSSIVYGAADMIMHTPGCVPCVRENNSSRCWVALTPTLAARNRSIPTTTIRRHVDLLV





GAAAFCSAMYVGDLCGSVFLVSQLFTFSPRRYETVQDCNCSLYPGHVSGHRMAWDMMMNWSPTAALVVSQLLRIP





QAVVDMVTGAHWGVLAGLAYYSMVGNWAKVLIVMLLFAGVDG





SEQ ID NO: 205: >1b._._.AB016785.AB016785


TTHVTGGQTGRTTLGITAMFAFGPHQKLQLINTNGSWHINRTALNCNDSLNTGFLAALFYARKFNSSGCPERMAS





CRPIDKFVQGWGPITHAVPDNLDQRPYCWHYAPQPCGIIPASQVCGPVYCFTPSPVVVGTTDRFGAPTYTWGENE





TDVLLLNNTRPPQGNWFGCTWMNGTGFAKTCGGPPCNIGGVGNNTLTCPTDCFRKHPEATYTKCGSGPWLTPRCM





VDYPYRLWHYPCTVNFTIFKVRMYVGGVEHRLTAACNWTRGERCDLEDRDRSELSPLLLSTTEWQVLPCSFTTLP





ALSTGLIHLHQNIVDVQYLYGVGSAVVSIVIKWEYILLLFLLLADARVCACLWMMLLIAQAEA





SEQ ID NO: 309: >gi|52139259|ref|YP_081534.1| major


capsid protein [Human herpesvirus 5]


MENWSALELLPKVGIPTDFLTHVKTSAGEEMFEALRIYYGDDPERYNIHFEAIFGTFCNRLEWVYFLTSG





LAAAAHAIKFHDLNKLTTGKMLFHVQVPRVASGAGLPTSRQTTIMVTKYSEKSPITIPFELSAACLTYLR





ETFEGTILDKILNVEAMHTVLRALKNTADAMERGLIHSFLQTLLRKAPPYFVVQTLVENATLARQALNRI





QRSNILQSFKAKMLATLFLLNRTRDRDYVLKFLTRLAEAATDSILDNPTTYTTSSGAKISGVMVSTANVM





QIIMSLLSSHITKETVSAPATYGNFVLSPENAVTAISYHSILADFNSYKAHLTSGQPHLPNDSLSQAGAH





SLTPLSMDVIRLGEKTVIMENLRRVYKNTDTKDPLERNVDLTFFFPVGLYLPEDRGYTTVESKVKLNDTV





RNALPTTAYLLNRDRAVQKIDFVDALKTLCHPVLHEPAPCLQTFTERGPPSEPAMQRLLECRFQQEPMGG





AARRIPHFYRVRREVPRTVNEMKQDFVVTDFYKVGNITLYTELHPFFDFTHCQENSETVALCTPRIVIGN





LPDGLAPGPFHELRTWEIMEHMRLRPPPDYEETLRLFKTTVTSPNYPELCYLVDVLVHGNVDAFLLIRTF





VARCIVNMFHTRQLLVFAHSYALVTLIAEHLADGALPPQLLFHYRNLVAVLRLVTRISALPGLNNGQLAE





EPLSAYVNALHDHRLWPPFVTHLPRNMEGVQVVADRQPLNPANIEARHHGVSDVPRLGAMDADEPLFVDD





YRATDDEWTLQKVFYLCLMPAMTNNRACGLGLNLKTLLVDLFYRPAFLLMPAATAVSTSGTTSKESTSGV





TPEDSIAAQRQAVGEMLTELVEDVATDAHTPLLQACRELFLAVQFVGEHVKVLEVRAPLDHAQRQGLPDF





ISRQHVLYNGCCVVTAPKTLIEYSLPVPFHRFYSNPTICAALSDDIKRYVTEFPHYHRHDGGFPLPTAFA





HEYHNWLRSPFSRYSATCPNVLHSVMTLAAMLYKISPVSLVLQTKAHIHPGFALTAVRTDTFEVDMLLYS





GKSCTSVIINNPIVTKEERDISTTYHVTQNINTVDMGLGYTSNTCVAYVNRVRTDMGVRVQDLFRVFPMN





VYRHDEVDRWIRHAAGVERPQLLDTETISMLTFGSMSERNAAATVHGQKAACELILTPVTMDVNYFKIPN





NPRGRASCMLAVDPYDTEAATKAIYDHREADAQTFAATHNPWASQAGCLSDVLYNTRHRERLGYNSKFYS





PCAQYFNTEEIIAANKTLFKTIDEYLLRAKDCIRGDTDTQYVCVEGTEQLIENPCRLTQEALPILSTTTL





ALMETKLKGGAGAFATSETHFGNYVVGEIIPLQQSMLFNS





SEQ ID NO: 310: >gi|52139266|ref|YP_081541.1|


tegument protein UL16 [Human herpesvirus 5]


MAWRSGLCETDSRTLKQFLQEECMWKLVGKSRKHREYRAVACRSTIFSPEDDGSCILCQLLLFYRDGEWI





LCLCCNGRYQGHYGVGHVHRRRRRICHLPTLYQLSFGGPLGPASIDFLPSFSQVTSSMTCDGITPDVIYE





VCMLVPQDEAKRILVKGHGAMDLTCQKAVTLGGAGAWLLPRPEGYTLFFYILCYDLFTSCGNRCDIPSMT





RLMAAATACGQAGCSFCTDHEGHVDPTGNYVGCTPDMGRCLCYVPCGPMTQSLIHNEEPATFFCESDDAK





YLCAVGSKTAAQVTLGDGLDYHIGVKDSEGRWLPVKTDVWDLVKVEEPVSRMIVCSCPVLKNLVH





SEQ ID NO: 311: >gi|52139212|ref|YP_081485.1|


tegument protein UL26 [Human herpesvirus 5]


MTSRRAPDGGLNLDDFMRRQRGRHLDLPYPRGYTLFVCDVEETILTPRDVEYWKLLVVTQGQLRVIGTIG





LANLFSWDRSVAGVAADGSVLCYEISRENFVVRAADSLPQLLERGLLHSYFEDVERAAQGRLRHGNRSGL





RRDADGQVIRESACYVSRALLRHRVTPGKQEITDAMFEAGNVPSALLP





SEQ ID NO: 312: >gi|52139244|ref|YP_081517.1|


multifunctional expression regulator [Human herpesvirus 5]


MELHSRGRHDAPSLSSLSERERRARRARRFCLDYEPVPRKFRRERSPTSPSTRNGAAASEYHLAEDTVGA





ASHHHRPCVPARRPRYSKDDDTEGDPDHYPPPLPPSSRHALGGTGGHIIMGTAGFRGGHRASSSFKRRVA





ASASVPLNPHYGKSYDNDDGEPHHHGGDSTHLRRRVPSCPTTFGSSHPSSANNHHGSSAGPQQQQMLALI





DDELDAMDEDELQQLSRLIEKKKRARLQRGAASSGTSPSSTSPVYDLQRYTAESLRLAPYPADLKVPTAF





PQDHQPRGRILLSHDELMHTDYLLHIRQQFDWLEEPLLRKLVVEKIFAVYNAPNLHTLLAIIDETLSYMK





YHHLHGLPVNPHDPYLETVGGMRQLLFNKLNNLDLGCILDHQDGWGDHCSTLKRLVKKPGQMSAWLRDDV





CDLQKRPPETFSQPMHRAMAYVCSFSRVAVSLRRRALQVTGTPQFFDQFDTNNAMGTYRCGAVSDLILGA





LQCHECQNEMCELRIQRALAPYRFMIAYCPFDEQSLLDLTVFAGTTTTTASNHATAGGQQRGGDQIHPTD





EQCASMESRTDPATLTAYDKKDREGSHRHPSPMIAAAAPPAQPPSQPQQHYSEGELEEDEDSDDASSQDL





VRATDRHGDTVVYKTTAVPPSPPAPLAGVRSHRGELNLMTPSPSHGGSPPQVPHKQPIIPVQSANGNHST





TATQQQQPPPPPPVPQEDDSVVMRCQTPDYEDMLCYSDDMDD






Example 1
Preparation of Dimeric Peptides According to the Invention

Amino acids that link two monomeric peptide sequences are underlined.


Influenza (M2e):

Constructs derived from the extracellular domain on influenza protein M2 (M2e-domain)


Native Domain:

MSLLTEVETPIRNEWGCRCNDSSD


The following sequences was prepared or are under preparation. The different parts, Z1-Z7, are divided by brackets.




embedded image


This construct links the monomeric peptides via a Dpr(Aoa) in the first peptide to an oxidized by NaIO4 Dpr(Ser) residue in the second.


Dpr(Aoa)=N-α-Fmoc-N-β-(N-t.-Boc-amino-oxyacetyl)-L-diaminopropionic acid


Explanation:

The brackets used in the sequences are meant to indicate the different parts/boxes. For the BI155 monomeric parts, the boxes will have the following amino-acid sequences (A/B monomer):


Part Z1 RG
Part Z2 Dpr(Aoa)/Dpr(Ser)
Part Z3 TPI(Har)QDWGNRAN/TPT(Har)NGWDVKLS
Part Z4 RG

Part Z5-, means not present in these peptides


Part Z6 TPTRQEWDCRIS/TPI(Har)QEW(Har)SL(Nle)NQEW

Part Z7 not present (optional)


The boxes on part of the other sequences can be found in a similar manner




embedded image


Examples of disulfide linked constructs can be, but are not restricted to, the following linked peptide sequences:




embedded image


The above disulfide linked constructs may e.g. be synthesised by titration of 2-pyridinesulfenyl (SPyr)-protected cysteine-containing peptides with thiol-unprotected peptides. This has proven to be a superior procedure to selectively generate disulfide-linked peptide heterodimers preventing the formation of homodimers (Schutz A et al., Tetrahedron, Volume 56, Issue 24, 9 Jun. 2000, Pages 3889-3891). Similar dimeric constructs may be made with the other monomeric peptides according to the invention.




embedded image


Examples of thio-esther linked constructs can be, but are not restricted to, the following linked peptide sequences:




embedded image


The Cys-Lys linker is typically established in the form of a thioether bond between a cysteine in one peptide and a bromoacetyl derivatized lysine in the other peptide.


Examples of other linked constructs can be, but are not restricted to, the following linked peptide sequences, N-ε-methylated Lys may be linked to Asp or Glu by a side-chain to side-chain peptide bond, wherein the N methylation makes the bond more stable (Lys(Me) refers to an N-ε-methylated Lys residue).




embedded image


Example 3
Immunological Studies
Rabbit Immunizations

New Zealand White female rabbits (n=3) is immunized intradermally at weeks 0, 2 & 6 with 1 ml of BI400-B vaccine consisting of 500 μg BI400-B in 50% V/V Freund's adjuvant (i.e. Complete Freund's adjuvant used for priming, followed by boostings with Incomplete Freund's adjuvant). Individual blood serum is isolated for ELISA.


Direct ELISA for Human or Rabbit Sera

50-100 μl of BI400-B (pre-incubated in Coating buffer—0.05M Na2CO3 pH9.6; denoted CB—in cold at 16 μg/ml for each peptide 1-3 days prior to coating) or just CB (background control) is used for coating wells in microtiter plates at 4° C. overnight. The microtiter plates are then washed 3× with washing buffer (PBS+1% v/v Triton-X100; denoted WB), followed by 2 h blocking at room temperature (RT) with 200 μl/well of blocking buffer (PBS+1% w/v BSA). Plates are then washed 3× with WB, followed by 1 h incubation at 37° C. with 50-70 ul/well of added human (or rabbit) sera (serial dilutions ranging from 1:1-1:250 in dilution buffer (PBS+1% v/v Triton-X100+1% w/v BSA; denoted DB)). Plates are then washed 6× with WB, followed by 1 h incubation at RT with 70 μl/well of Alkaline Phosphatase-conjugated Protein G (3 μg/ml in DB; Calbiochem 539305). Plates are then washed 6× with WB, followed by 10-60 min incubation at room temperature with 100 μl/well of 0.3% w/v of Phenophtalein monophosphate (Sigma P-5758). Plates are finally quenched by adding 100 μl/well of Quench solution (0.1M TRIS+0.1M EDTA+0.5M NaOH+0.01% w/v NaN3; pH14), followed by ELISA reader (ASYS UVM 340) at 550 nm.


Example 4
Virus Specific Response by ELISPOT Assay

At day one, PBMC samples from blood donors are thawed, washed with warm medium and incubated in flasks (250000PBMCs/cm2) for 24 hours at 37° C., 5% CO2 in covering amount of culture media (RPMI 1640 with ultra-glutamine, Lonza, BE12-702F701; 10% Foetal Bovine serum (FBS), Fisher Scientific Cat. No. A15-101; Penicillin/Streptomycin, Fisher Scientific Cat. No. P11-010) to allow the cells to recover after thawing. At day two, the cells are added to a Falcon Microtest Tissue Culture plate, 96 well flat bottom, at 500 000 cells per well in a volume of 200 μl total medium. Parallel wells are added the indicated stimuli in duplicate or left with medium as a control for 6 days at 37° C., 5% CO2. After the six day of incubation, 100 μl of the cell suspension are transferred to an ELISPOT (Millipore multiscreen HTS) plate coated with 1 μg/ml native influenza M2e protein. After a 24 hour incubation, the plate is washed four times with PBS+0.05% Tween20, and a fifth time with PBS, 200 μl/well. A mouse Anti-human IgG or IgM biotin (Southern Biotech 9040-08 and 9020-08) is diluted in PBS with 0.5% FBS and incubated for 90 minutes at 37° C. The washing is repeated as described, before 80 μl Streptavidin-Alkaline-Phosphatase (Sigma Aldrich, S289) is added each well and incubated at 60 minutes in the dark, at room temperature. The wells are then washed 2 times with PBS+0.05% Tween20 and 4 times with PBS, 200 μl/well, before the substrate, Vector Blue Alkaline Phosphatase Substrate kit III (Vector Blue, SK-5300) is added and let to develop for 7 minutes at room temperature. The reaction is stopped with running water, the plates let dry and the sport enumerated by an ELISPOT reader (CTL-ImmunoSpot® S5 UV Analyzer).


Virus Specific Response by ELISA

100 μl of antigen as indicated (pre-incubated in Coating buffer—0.05M Na2CO3 pH9.6; denoted CB—in cold at 8 μg/ml 1-3 days) or just CB (background control) is used for coating wells in microtiter plates at 4° C. The microtiter plates are then washed 3× with washing buffer (PBS+1% v/v Triton-X100; denoted WB), followed by 2 h blocking at room temperature (RT) with 200 μl/well of blocking buffer (PBS+1% w/v BSA). Plates are then washed 3× with WB, followed by 1 h incubation at 37° C. with 50-70 ul/well of added human (or rabbit or sheep) sera (serial dilutions ranging from 1:5-1:250 in dilution buffer (PBS+1% v/v Triton-X100+1% w/v BSA; denoted DB)). Plates are then washed 6× with WB, followed by 1 h incubation at RT with 70 μl/well of Alkaline Phosphatase-conjugated Protein G (3 μg/ml in DB; Calbiochem 539305) or goat anti-mouse IgG biotin (1 μg/ml, Southern Biotech, 1030-08. In case of the goat anti-mouse IgG biotin, the plates are washed one extra step as described, before addition of 100 μl Streptavidin-Alkaline-Phosphatase (1 μg/ml, Sigma Aldrich, S289) and incubated 1 hour at RT. Plates are then washed 6× with WB, followed by 10-60 min incubation at room temperature with 100 μl/well of 0.3% w/v of Phenophtalein monophosphate (Sigma P-5758). Plates are finally quenched by adding 100 μl/well of Quench solution (0.1M TRIS+0.1M EDTA+0.5M NaOH+0.01% w/v NaN3; pH14), followed by a measurement with a ELISA reader (ASYS UVM 340) at 550 nm. The strength of the sera, i.e. the magnitude of the humoral immune response, is then reported as the dilution of sera that result in the described Optical Density (OD) value, or the OD value at the indicated dilution of sera.


Example 5

The peptides according to the invention used in the following examples are synthesized by Schafer-N as c-terminal amides using the Fmoc-strategy of Sheppard, (1978) J. Chem. Soc., Chem. Commun., 539.


BI100-190e, BI100-190f, BI100-260b, BI100-260c, BI100-260d, BI100-260e, and BI100-260f were synthezised by Schafer-N with and without Biotin in the C-terminal tested:


Cell Penetration Assay

A set of peptides were biotinylated on C-terminal, and different combinations of aminoacids, with respect to length and type, were added to the sequence box Z1, Z4 and Z7 in the peptides according to the present invention, formula I. The peptides were tested on cells grown from one individual blood donor.


Schematic diagram of amino acid sequence of the peptides according to the invention (Each Z here defines a sequence of amino acids):




embedded image


Intracellular Staining for Biotinylated Peptides

96-well U-bottom polystyrene plates (NUNC, cat no: 163320) were used for staining of human PBMCs. Briefly, 8 ul of N- or C-terminally biotinylated peptides according to table 1 or table 2 (i.e. 5 mM, 2.5 mM & 1.25 mM tested for each peptide) were incubated at 37° C. for 2 h with 40 ul of PBMC (12.5×106 cells/ml) from blood donors. Cells were then washed 3× with 150 ul of Cellwash (BD, cat no: 349524), followed by resuspension of each cell pellet with 100 ul of Trypsin-EDTA (Sigma, cat no: T4424), then incubated at 37° C. for 5 min. Trypsinated cells were then washed 3× with 150 ul of Cellwash (BD, cat no: 349524), followed by resuspension with BD Cytofix/Cytoperm™ plus (BD, cat no: 554715), then incubated at 4° C. for 20 min according to manufacturer. Cells were then washed 2× with 150 ul PermWash (BD, cat no: 554715). Cells were then stained with Streptavidin-APC (BD, cat no: 554067) & Anti-hCD11c (eBioscience, cat no: 12-0116) according to manufacturer at 4° C. for 30 min aiming to visualize biotinylated peptides & dendritic cells, respectively. Cells were then washed 3× with 150 ul PermWash, followed by resuspension in staining buffer (BD, cat no: 554656) before flow cytometry. Dendritic cells were gated as CD11c+ events outside lymphocyte region (i.e. higher FSC & SSC signals than lymphocytes). 200 000 total cells were acquired on a FACSCanto II flow cytometer with HTS loader, and histograms for both total cells & dendritic cells with respect to peptide-fluorescence (i.e. GeoMean) were prepared.


Extracellular Staining for Biotinylated Peptides

96-well U-bottom polystyrene plates (NUNC, cat no: 163320) were used for staining of human PBMCs. Briefly, 8 ul of N- or C-terminally biotinylated peptides according to table 1 or table 2 (i.e. 5 mM, 2.5 mM & 1.25 mM tested for each peptide; all peptides manufactured by Schafer) were incubated at 37° C. for 2 h with 40 ul of PBMC (12.5×106 cells/ml) from blood donors. Cells were then washed 3× with 150 ul of Cellwash (BD, cat no: 349524), then stained with Streptavidin-APC (BD, cat no: 554067) & Anti-hCD11c (eBioscience, cat no: 12-0116) according to manufacturer at 4° C. for 30 min aiming to visualize biotinylated peptides & dendritic cells, respectively. Cells were then washed 3× with 150 ul of Cellwash (BD, cat no: 349524), followed by resuspension in staining buffer (BD, cat no: 554656) before flow cytometry. Dendritic cells were gated as CD11c+ events outside lymphocyte region (i.e. higher FSC & SSC signals than lymphocytes). 200 000 total cells were acquired on a FACSCanto II flow cytometer with HTS loader, and histograms for both total cells & dendritic cells with respect to peptide-fluorescence (i.e. GeoMean) were prepared.


Example 6

Positive CTL response may alternatively be assayed by ELISPOT assay.


Human IFN-Gamma Cytotoxic T-Cell (CTL) Response by ELISPOT Assay

Briefly, at day 1, PBMC samples from HCV patients were incubated in flasks (430 000 PBMCs/cm2) for 2 h at 37° C., 5% CO2 in covering amount of culture media (RPMI 1640 Fisher Scientific; Cat No. PAAE15-039 supplemented with L-Glutamine, (MedProbe Cat. No. 13E17-605E, 10% Foetal Bovine serum (FBS), Fisher Scientific Cat. No. A15-101) and Penicillin/Streptomycin, (Fisher Scientific Cat. No. P11-010) in order to allow adherence of monocytes. Non-adherent cells were isolated, washed, and frozen in 10% V/V DMSO in FBS until further usage. Adherent cells were carefully washed with culture media, followed by incubation at 37° C. until day 3 in culture media containing 2 μg/ml final concentration of hrGM-CSF (Xiamen amoytop biotech co, cat no: 3004.9090.90) & 1 μg/ml hrIL-4 (Invitrogen, Cat no: PHC0043), and this procedure is then repeated at day 6. At day 7, cultured dendritic cells (5 000-10 000 per well) were added to ELISPOT (Millipore multiscreen HTS) plates coated with 0.5 μg/well anti-human γ Interferon together with thawed autologous non-adherent cells (200 000 per well), antigen samples (1-8 ug/ml final concentration for peptide antigens; 5 ug/ml final concentration for Concanavalin A (Sigma, Cat no: C7275) or PHA (Sigma, Cat no: L2769)) and optionally, anti-Anergy antibodies (0.03-0.05 ug/ml final concentration for both anti-PD-1 (eBioscience, cat no: 16-9989-82) & anti-PD-L1 (eBioscience, cat no: 16-5983-82)). Plates were incubated overnight and spots were developed according to manufacturer. Spots were read on ELISPOT reader (CTL-ImmunoSpot® S5 UV Analyzer).


Example 7
The REVEAL & ProVE® Rapid Epitope Discovery System in Detail

Binding properties to HLA for the ninemers listed are tested for the following HLA-classes: HLA-A1, HLA-A2, HLA-A3, HLA-A11, HLA-A24, HLA-A29, HLA-B7, HLA-B8, HLA-B14, HLA-B15, HLA-B27, HLA-B35, HLA-B40.


The peptides are synthesized as a Prospector PEPscreen®: Custom Peptide Library. Peptides 8-15 amino acids in length are synthesized in 0.5-2 mg quantities with high average purity. Quality control by MALDI-TOF Mass Spectrometry is carried out on 100% of samples.


The REVEAL™ binding assay determined the ability of each candidate peptide to bind to one or more MHC class I alleles and stabilizing the MHC-peptide complex. By comparing the binding to that of high and intermediate affinity T cell epitopes, the most likely immunogenic peptides in a protein sequence can be identified. Detection is based on the presence or absence of the native conformation of the MHC-peptide complex.


Each peptide is given a score relative to the positive control peptide, which is a known T cell epitope. The score of the test peptide is reported quantitatively as a percentage of the signal generated by the positive control peptide, and the peptide is indicated as having a putative pass or fail result. Assay performance is confirmed by including an intermediate control peptide that is known to bind with weaker affinity to the allele under investigation.


Example 8
Intracellular Staining

Peptides as described herein with Z3 and Z6 derived from HCV, Influenza, or CMV are prepared and tested for intracellular staining in an experiment as described above in the “Cell penetration assay”.


Average over results from buffy coats from ten donors, normalized to N-biotin for each donor is illustrated in FIGS. 3 and 4.


Example 9









TABLE 10







Peptides used as controls and not part to the invention,


but carrying the same epitopes (Z3, Z6, Z9) linked by


glycines and serines, for comparison to peptides of the


invention. (Z = Norleucine, X = Homoarginine, biotc


indicates that a biotinylated lysine residue has been


added to the C-terminal).














Peptide
-
Z3
-
Z6
-
Z9
C-ter tag





BI330-72-2-
GS
VITYSIFLIVS
GS
GGNVIGGIYZIPR


biotin-


ns-biotc






NH2





BI330-83-ns-
GS
TANWARVIS
GS
ANWAKVIL
S
NWAKVI
biotin-


biotc






NH2





BI310-511-ns-
S
GYLPAVGAPI
GS
VIRVIAHGLRL


biotin-


biotc






NH2





BI100-330-ns-
GS
TAYERZCNIL
GS
GLEPLVIAGILA


biotin-


biotc






NH2





BI100-270-ns-
GS
TVIGASZIPLL
GS
TPIXQDWENRAN


biotin-


biotc






NH2





BI100-130-ns-
GS
AAFEEZXITS
GS
VAFEDLXZZSFI


biotin-


biotc






NH2









Results

Biotinylated versions of scaffold peptides were tested for intracellular and extracellular uptake. All tested peptides had stronger intracellular and extracellular uptake compared to the control peptide N-biotin (N-bio), as seen from FIGS. 1-2. Also when comparing the uptake of peptides according to the invention to peptides carrying the same epitopes linked by Glycine and Serine residues instead (Table 11), tested peptides according to the invention generally had a higher uptake. Many of the peptides tested show very strong uptake and potentially we are seeing saturation of the cell assay system for these.


Values represent averages over readouts from buffy coats from ten (five) donors and three (four) concentrations of peptide each, normalized by value for N-biotin for each donor for scaffold (non-scaffold) peptides respectively.









TABLE 11







Intracellular and extracellular uptake of peptides of


the invention (Bold) compared to peptides containing


the same epitopes linked by Gly and Ser residues (non-


bold Italics). Median readouts from buffy coats from ten (five)


donors and three (four) concentrations of peptide each, normalized


by value for N-biotin for each donor for scaffold (non-scaffold) peptides.









Peptide
Intracellular
Extracellular


(biotinylated)
Uptake
Uptake













BI100-270


2.05


20.47




BI100-270b


3.35


16.54




BI100-270c


2.91


9.56




BI100-270d


5.73


4.77




BI100-270e


10.26


3.54




BI100-270ns


1.30


1.29




BI100-330


70.36


655.35




BI100-330b


76.42


744.11




BI100-330c


880.85


244.29




BI100-330d


80.82


592.82




BI100-330e


23.89


529.05




BI100-330ns


1.82


416.04




BI310-511


22.62


227.46




BI310-511b


67.29


466.71




BI310-511c


31.83


203.62




BI310-511d


70.64


267.15




BI310-511e


44.59


473.80




BI310-511f


26.85


178.61




BI310-511g


66.74


171.31




BI310-511ns


3.85


4.56




BI330-83


194.69


364.04




BI330-83b


120.10


518.60




BI330-83c


154.43


435.66




BI330-83d


52.14


267.38




B1330-83ns


63.51


380.25










Example 10

Effect of peptide based influenza vaccine in protection of HLA A2 mice against influenza virus challenge


C57/B6/Tg HLA A2 mice (n=10 mice per group) were immunized week 0 and week 2 by subcutaneous administration (2×50 μl; each side of base of tail), of a solution containing 50 μg of each peptide, or 0.07 μg HA of inactivated influenza A/PR8 (H1N1) virus given as vaccine control.


At week 4 the mice was infected with live influenza virus in order to measure the immune response to viral infection. The challenge was done with a mouse adapted strain of influenza A at a dose of 1×10̂5TCID50/mouse which is enough to reliably infect the animals without mortality as determined by titration in the same mouse strain. The animals were then monitored for 7 days by weight loss at the start of challenge and daily from day three before they were sacrificed and serum collected. Individual serum for mice in all groups were collected before start of experiment, and day of sacrifice.










TABLE 12





Group
Treatment







1
Vaccinate with peptides + adjuvant Provax (week 0, 2)


2
Vaccinate with peptides + adjuvant ISA 51 (week 0, 2)


3
Vaccinate with inactivated conventional vaccine (week 0, 2)


4
Naïve mice.
















TABLE 13







Survival (n)











Day













Group
1-4
5
6
7

















1 Peptide, Provax
10
10
10
7



2 Peptide, ISA 51
10
10
10
9



3 PR8
10
9
9
6



4 Naïve
10
10
10
8










Results

Following the weight loss after challenge a clear protective effect is seen for both groups receiving the peptide vaccine with either ISA51 or Provax as adjuvant, as compared to the standard inactivated viral vaccine, PR8, or naïve mice (FIG. 5).

Claims
  • 1. An isolated monomeric peptide comprising the following structure (Z1-Z2)1-Z3-(Z4-Z5)2-Z6-(Z7-Z8)3-Z9-(Z10-Z11)4-Z12
  • 2. The isolated monomeric peptide according to claim 1, wherein said chemical moiety of Z3, and optional Z6, Z9 and Z12 is a linear amino acid sequence of 8-30 amino acids.
  • 3. The isolated monomeric peptide according to claims 1 or 2, wherein Z2 defines an amino acid selected from cysteine (C), lysine (K), aspartic acid (D), asparagine (N), glutamic acid (E), glutamine (Q), 2,3-Diaminopropionic acid (Dpr), tryptophan (W), or tyrosine (Y) or a derivative thereof.
  • 4. The isolated monomeric peptide according to any one of claims 1-3, wherein Z5 defines an amino acid selected from cysteine (C), lysine (K), aspartic acid (D), asparagine (N), glutamic acid (E), glutamine (Q), 2,3-Diaminopropionic acid (Dpr), tryptophan (W), or tyrosine (Y) or a derivative thereof.
  • 5. The isolated monomeric peptide according to claims 1-4, wherein Z8 defines an amino acid selected from cysteine (C), lysine (K), aspartic acid (D), asparagine (N), glutamic acid (E), glutamine (Q), 2,3-Diaminopropionic acid (Dpr), tryptophan (W), or tyrosine (Y) or a derivative thereof.
  • 6. The isolated monomeric peptide according to claims 1-5, wherein Z11 defines an amino acid selected from cysteine (C), lysine (K), aspartic acid (D), asparagine (N), glutamic acid (E), glutamine (Q), 2,3-Diaminopropionic acid (Dpr), tryptophan (W), or tyrosine (Y) or a derivative thereof.
  • 7. The isolated monomeric peptide according to any one of claims 1-6, wherein Z7 defines a linear sequence of one, two, or three arginine residues or derivatives thereof optionally followed by a glycine (G) or an alanine (A).
  • 8. The isolated monomeric peptide according to any one of claims 1-7, wherein Z10 defines a linear sequence of one, two, or three arginine residues or derivatives thereof optionally followed by a glycine (G) or an alanine (A).
  • 9. The isolated monomeric peptide according to any one of claims 1-8, wherein Z6 defines any chemical moiety, such as a linear amino acid sequence.
  • 10. The isolated monomeric peptide according to any one of claims 1-9, wherein Z9 defines any chemical moiety, such as a linear amino acid sequence.
  • 11. The isolated monomeric peptide according to any one of claims 1-10, wherein Z12 defines any chemical moiety, such as a linear amino acid sequence.
  • 12. The isolated monomeric peptide according to any one of claims 1-11, wherein Z1, Z4, and optional Z7 and Z10 is followed by a glycine (G) or an alanine (A).
  • 13. The isolated monomeric peptide according to any one of claims 1-12, wherein Z3, and optional Z6, Z9 and Z12 is a linear amino acid sequence of 8-30 amino acids derived from an antigen with more than 40%, such as more than 45%, such as more than 50%, such as more than 55%, such as more than 60%, such as more than 65%, such as more than 70%, such as more than 75%, such as more than 80%, such as more than 85%, such as more than 90%, such as more than 95%, such as more than 96%, such as more than 97%, such as more than 98%, such as more than 99%, such as 100% sequence identity to a specific natural antigen.
  • 14. The isolated monomeric peptide according to any one of claims 1-13, wherein Z3, and optional Z6, Z9 and Z12 defines a specific natural antigen of a protein or peptide sequence derived from a disease antigen, such as an infectious agent, such as bacteria, virus, parasite, fungus, or cancer antigens such as oncogene (lung, stomach, breast cancer) or an antigen causing an autoimmune disease such as diabetes, multiple sclerosis (MS), celiac disease, Myalgic Encephalomyelitis (ME), psoriasis, and/or Crohn's Disease.
  • 15. The isolated monomeric peptide according to claim 14, wherein said specific natural antigen is a viral protein, such as a structural protein, such as a capsid protein, a regulatory protein, an enzymatic protein, and a proteolytic protein.
  • 16. The isolated monomeric peptide according to any one of claims 14-15, wherein said viral protein is selected from a core protein or an envelope protein, of a virus selected from the Hepatitis C virus, influenza virus, such as an M2 protein, human immunodeficiency virus (HIV), cytomegalovirus (CMV), and Human papillomavirus (HPV).
  • 17. The isolated monomeric peptide according to claim 16, wherein said viral protein is a viral protein of Hepatitis C virus selected from any one HCV consensus sequence of a specific genotype, such as 1, such as subtypes 1a and 1b, genotype 2, such as 2a and 2b, genotype 3, such as 3a, genotype 4, genotype 5, and genotype 6.
  • 18. The isolated monomeric peptide according to any one of claims 1-17, wherein a sequence of amino acids defined by (Z1-Z2)1-Z3-(Z4-Z5)2-Z6-(Z7-Z8)3-Z9-(Z10-Z11)4-Z12 is not found in the native sequence of a natural antigen.
  • 19. The isolated monomeric peptide according to any one of claims 1-18, which monomeric peptide is of 10-60 amino acids, such as of 11-60 amino acids, such as of 12-60 amino acids, such as of 13-60 amino acids, such as of 14-60 amino acids, such as of 15-60 amino acids, such as of 16-60 amino acids, such as of 17-60 amino acids, such as of 18-60 amino acids, such as of 19-60 amino acids, such as of 20-60 amino acids, such as of 21-60 amino acids, such as of 22-60 amino acids, such as of 23-60 amino acids, such as of 24-60 amino acids, such as of 25-60 amino acids, such as of 26-60 amino acids, such as of 27-60 amino acids, such as of 28-60 amino acids, such as of 29-60 amino acids, such as of 30-60 amino acids, such as of 31-60 amino acids, such as of 32-60 amino acids, such as of 33-60 amino acids, such as of 34-60 amino acids, such as of 35-60 amino acids, such as of 36-60 amino acids, such as of 37-60 amino acids, such as of 38-60 amino acids, such as of 39-60 amino acids, such as of 40-60 amino acids, such as of 42-60 amino acids, such as of 44-60 amino acids, such as of 46-60 amino acids, such as of 48-60 amino acids, such as of 50-60 amino acids, such as of 52-60 amino acids, such as of 54-60 amino acids, such as of 56-60 amino acids, such as of 58-60 amino acids.
  • 20. The isolated monomeric peptide according to any one of claims 1-19, which monomeric peptide is of 10-60 amino acids, such as 10-58 amino acids, such as 10-56 amino acids, such as 10-54 amino acids, such as 10-52 amino acids, such as 10-50 amino acids, such as 10-48 amino acids, such as 10-46 amino acids, such as 10-44 amino acids, such as 10-42 amino acids, such as 10-40 amino acids, such as 10-39 amino acids, such as 10-38 amino acids, such as 10-37 amino acids, such as 10-36 amino acids, such as 10-35 amino acids, such as 10-34 amino acids, such as 10-33 amino acids, such as 10-32 amino acids, such as 10-31 amino acids, such as 10-30 amino acids, such as 10-29 amino acids, such as 10-28 amino acids, such as 10-27 amino acids, such as 10-26 amino acids, such as 10-25 amino acids, such as 10-24 amino acids, such as 10-23 amino acids, such as 10-22 amino acids, such as 10-21 amino acids, such as 10-20 amino acids, such as 10-19 amino acids, such as 10-18 amino acids, such as 10-17 amino acids, such as 10-16 amino acids, such as 10-15 amino acids, such as 10-14 amino acids, such as 10-13 amino acids, such as 10-12 amino acids, such as 10-11 amino acids.
  • 21. The isolated monomeric peptide according to any one of claims 1-20, which monomeric peptide consist of not more than about 55 amino acids, such as not more than about 50 amino acids, such as not more than about 45 amino acids, such as not more than about 40 amino acids, such as not more than about 38 amino acids, such as not more than about 36 amino acids, such as not more than about 34 amino acids, such as not more than about 32 amino acids, such as not more than about 30 amino acids, such as not more than about 28 amino acids, such as not more than about 26 amino acids, such as not more than about 24 amino acids, such as not more than about 22 amino acids, such as not more than about 20 amino acids, such as not more than about 18 amino acids, such as not more than about 16 amino acids, such as not more than about 14 amino acids, such as not more than about 12 amino acids, such as not more than about 10 amino acids.
  • 22. The isolated monomeric peptide according to any one of claims 1-21, which monomeric peptide consist of at least about 10 amino acids, such as at least about 12 amino acids, such as at least about 14 amino acids, such as at least about 16 amino acids, such as at least about 18 amino acids, such as at least about 20 amino acids, such as at least about 22 amino acids, such as at least about 24 amino acids, such as at least about 26 amino acids, such as at least about 28 amino acids, such as at least about 30 amino acids, such as at least about 32 amino acids, such as at least about 34 amino acids, such as at least about 36 amino acids, such as at least about 38 amino acids, such as at least about 40 amino acids, such as at least about 45 amino acids, such as at least about 50 amino acids, such as at least about 55 amino acids, such as at least about 60.
  • 23. The isolated monomeric peptide according to any one of claims 1-22, wherein the overall net charge of (Z1-Z2)1-Z3-(Z4-Z5)2-Z6-(Z7-Z8)3-Z9-(Z10-Z11)4-Z12 is equal to or above 0, such as above 1, 2, 3, 4, or 5.
  • 24. The isolated monomeric peptide according to any one of claims 1-23, wherein said monomeric peptide is capable of inducing a humoral immune response.
  • 25. The isolated monomeric peptide according to any one of claims 1-24, wherein said monomeric peptide comprises at least one amino acid selected from a Cys, a Lys, an Asp, and a Glu residue, or derivatives thereof.
  • 26. The isolated monomeric peptide according to any one of claims 1-25, which monomeric peptide contain one or more intramolecular bond, such as one or more Cys-Cys bond.
  • 27. The isolated monomeric peptide according to any one of claims 1-26, which monomeric peptide has delayed proteolytic degradation in the N-terminal, such as by incorporation of the first 1, 2, or 3 amino acids in the N-terminal in the D-form, or by incorporation of the first 1, 2, or 3 amino acids in the N-terminal in beta or gamma form.
  • 28. The isolated peptide according to any one of claim 1-27, wherein said peptide is demonstrated to translocate across a plasma membrane in the assay based on biotinylation of peptides as described in example 5.
  • 29. The isolated peptide according to any one of claims 1-28, wherein said peptide is capable of inducing a T lymphocyte response.
  • 30. The isolated peptide according to any one of claims 1-29, wherein the net charge of Z3, and/or optional Z6, Z9 and Z12 is below or equal to 0.
  • 31. The isolated peptide according to any one of claims 1-30, wherein the net charge of Z3 is below or equal to 0; and wherein the net charge of Z6 and/or optional Z9 and Z12 is above or equal to 1.
  • 32. The isolated peptide according to any one of claims 1-31, wherein the net charge of Z3, and/or optional Z6, Z9 and Z12 are above or equal to 1.
  • 33. The isolated peptide according to any one of claims 1-32, wherein the net charge of Z3 is above or equal to 1; and wherein the net charge of Z6 and/or optional Z9 and Z12 is below or equal to 0.
  • 34. The isolated peptide according to any one of claims 1-33, wherein the peptide comprises one or more cysteine.
  • 35. The isolated peptide according to any one of claims 1-34, wherein the N- and/or C-terminal amino acid in Z3, and/or optional Z6, Z9 and Z12 is a hydrophilic or polar amino acid.
  • 36. The isolated peptide according to any one of claims 1-35, wherein Z3, and/or optional Z6, Z9 and Z12 defines a sequence of 8-25 amino acids, such as 8-20 amino acids, such as 8-15 amino acids.
  • 37. The isolated peptide according to any one of claims 1-36, wherein Z3, and/or optional Z6, Z9 and Z12 defines a sequence of less than 25, such as less than 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7 or 6 amino acids.
  • 38. The isolated peptide according to any one of claims 1-37, wherein Z3, and/or optional Z6, Z9 and Z12 defines a sequence of more than 8, such as more than 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 amino acids.
  • 39. The isolated peptide according to any one of claims 1-38, which does not consist of the following sequence RFIIP[Nle]FTALSGGRRALLYGATPYAIG, where Nle denotes a nor-leucine.
  • 40. The isolated peptide according to any one of claims 1-39, wherein Z3, and/or optional Z6, Z9 and Z12 is not derived from HIV.
  • 41. The isolated peptide according to any one of claims 1-40, wherein Z3, and/or optional Z6, Z9 and Z12 is a linear sequence of less than 12 amino acids.
  • 42. The isolated peptide according to any one of claims 1-41, wherein Z3, and/or optional Z6, Z9 and Z12 is a linear sequence of less than 12 amino acids.
  • 43. The isolated peptide according to any one of claims 1-42, wherein Z3, and/or optional Z6, Z9 and Z12 do not contain nor-leucine.
  • 44. The isolated peptide according to any one of claims 1-43, wherein Z3, and/or optional Z6, Z9 and Z12 do not contain nor-leucine.
  • 45. The isolated peptide according to any one of claims 1-44, wherein Z3, and/or optional Z6, Z9 and Z12 only contains natural amino acids.
  • 46. The isolated peptide according to any one of claims 1-45, wherein Z3, and/or optional Z6, Z9 and Z12 only contains natural amino acids.
  • 47. The isolated peptide according to any one of claims 1-46, wherein Z3, and/or optional Z6, Z9 and Z12 only contains natural amino acids if derived from HIV.
  • 48. The isolated peptide according to any one of claims 1-47, wherein Z3, and/or optional Z6, Z9 and Z12 is derived from HCV, CMV, HPV, Influenza, adenoviruses, herpesviruses, or picornaviruses.
  • 49. The isolated peptide according to any one of claims 1-48, wherein Z1 is as defined in any one of table 3, table 4, table 5, or table 7, such as any one selected from R, RR, RRR, RG, RRG and RRRG.
  • 50. The isolated peptide according to any one of claims 1-49, wherein Z2 is as defined in any one of table 3, table 4, table 5, or table 7, such as any one selected from Dpr(Aoa), C, K, Lys(Me), D, E, Dpr(Ser).
  • 51. The isolated peptide according to any one of claims 1-50, wherein Z3 is as defined in any one of table 3, table 4, table 5, or table 7, such as any one selected from GGQLIGGIYLIPG (SEQ ID NO:313), VITYSIFLIVS (SEQ ID NO:314), TANWARVIS (SEQ ID NO:315), GYLPAVGAPI (SEQ ID NO:316), NIVPZVVTA (SEQ ID NO:317), VTPADLIGA (SEQ ID NO:318), PRPEGYTLFF (SEQ ID NO:319), LPYPRGYTLFV (SEQ ID NO:320), ETILTPRDV (SEQ ID NO:321), SSTSPVYDL (SEQ ID NO:322), TAYERZCNIL (SEQ ID NO:323), TVIGASZIPLL (SEQ ID NO:324), AAFEEZXITS (SEQ ID NO:325), GLEPLVIAGILA (SEQ ID NO:326), TAFLVRNVA (SEQ ID NO:327), TPI(Har)QDWGNRAN (SEQ ID NO:328), TPT(Har)NGWDVKLS (SEQ ID NO:329), LECVYCKQQLL (SEQ ID NO:330), GVYDFAFRDLC (SEQ ID NO:331), GVFDYAFRDIN (SEQ ID NO:332), and VDIRTLEDLL (SEQ ID NO:333).
  • 52. The isolated peptide according to any one of claims 1-51, wherein Z4 is as defined in any one of table 3, table 4, table 5, or table 7, such as any one selected from R, RR, RRR, RG, RRG and RRRG.
  • 53. The isolated peptide according to any one of claims 1-52, wherein Z5 is as defined in any one of table 3, table 4, table 5, or table 7, such as any one selected from Dpr(Aoa), C, K, Lys(Me), D, E, Dpr(Ser).
  • 54. The isolated peptide according to any one of claims 1-53, wherein Z6 is as defined in any one of table 3, table 4, table 5, or table 7, such as any one selected from EVYDFAFRDLC (SEQ ID NO:334), GFAFRDLCIVY (SEQ ID NO:335), GFAYRDINLAY (SEQ ID NO:336), GTLGIVCPIG (SEQ ID NO:337), GLEPLVIAGILA (SEQ ID NO:338), TPIXQDWENRAN (SEQ ID NO:339), VAFEDLXZZSFI (SEQ ID NO:340), RFQTVVQBA (SEQ ID NO:341), GSLVGLLHIVL (SEQ ID NO:342), SIARSVTIZXASVVH (SEQ ID NO:343), TPTRQEWDCRIS (SEQ ID NO:344), TPTRQEWDARIS (SEQ ID NO:345), TPI(Har)QEW(Har)SL(Nle)NQEW (SEQ ID NO:346), IGDLIVAQV (SEQ ID NO:347), QYNPVAVZF (SEQ ID NO:348), GYTLFFTS (SEQ ID NO:349), GYTLFVSD (SEQ ID NO:350), NTLZTPRDV (SEQ ID NO:351), SSTSPVYNL (SEQ ID NO:352), VITFSIYLIVS (SEQ ID NO:353), GGNVIGGIYZIPR (SEQ ID NO:354), ANWAKVIL (SEQ ID NO:355), VIRVIAHGLRL (SEQ ID NO:356), and IGDLIVQAV (SEQ ID NO:478).
  • 55. The isolated peptide according to any one of claims 1-54, wherein Z7 is as defined in any one of table 3, table 4, table 5, or table 7, such as any one selected from R, RR, RRR, RG, RRG and RRRG.
  • 56. The isolated peptide according to any one of claims 1-55, wherein Z8 is as defined in any one of table 3, table 4, table 5, or table 7, such as any one selected from Dpr(Aoa), C, K, Lys(Me), D, E, Dpr(Ser).
  • 57. The isolated peptide according to any one of claims 1-56, wherein Z9 is as defined in any one of table 3, table 4, table 5, or table 7, such as NWAKVI.
  • 58. The isolated peptide according to any one of claims 1-57, which peptide consist of (Z1-Z2)1-Z3-(Z4-Z5)2-Z6-(Z7-Z8)3-Z9 as defined in any one of table 3, table 4, table 5, or table 7, such as any one selected from RRGGQLIGGIYLIPGRRVITFSIYLIVS (SEQ ID NO:357), RRRGGQLIGGIYLIPGRRVITFSIYLIVS (SEQ ID NO:358), RRGGQLIGGIYLIPGRRRVITFSIYLIVS (SEQ ID NO:359), RRGGQLIGGIYLIPGRRVITFSIYLIVSR (SEQ ID NO:360), RRGGQLIGGIYLIPGRRVITFSIYLIVSRR (SEQ ID NO:361), RRVITYSIFLIVSRRGGNVIGGIYZIPR (SEQ ID NO:362), RRRVITYSIFLIVSRRGGNVIGGIYZIPR (SEQ ID NO:363), RRVITYSIFLIVSRRRGGNVIGGIYZIPR (SEQ ID NO:364), RRRVITYSIFLIVSRRRGGNVIGGIYZIPR (SEQ ID NO:365), RRGTANWARVISRANWAKVILRNWAKVI (SEQ ID NO:366), RGTANWARVISRRANWAKVILRNWAKVI (SEQ ID NO:367), RGTANWARVISRANWAKVILRNWAKVI (SEQ ID NO:368), RGTANWARVISRGANWAKVILRNWAKVI (SEQ ID NO:369), RRGTANWARVISRANWARVILRNWAKVI (SEQ ID NO:370), RGTANWARVISRRANWARVILRNWAKVI (SEQ ID NO:371), RGTANWARVISRANWARVILRNWAKVI (SEQ ID NO:372), RGTANWARVISRGANWARVILRNWAKVI (SEQ ID NO:373), RGYLPAVGAPIRRRVIRVIAHGLRLR (SEQ ID NO:374), RRGYLPAVGAPIRRVIRVIAHGLRLR (SEQ ID NO:375), RRGYLPAVGAPIRRRVIRVIAHGLRL (SEQ ID NO:376), RRGYLPAVGAPIRRVIRVIAHGLRL (SEQ ID NO:377), RGYLPAVGAPIRRVIRVIAHGLRLR (SEQ ID NO:378), RGYLPAVGAPIRVIRVIAHGLRLR (SEQ ID NO:379), RGYLPAVGAPIRRVIRVIAHGLRL (SEQ ID NO:380), RGNIVPZVVTARRIGDLIVAQV (SEQ ID NO:381), RRNIVPZVVTARRIGDLIVAQV (SEQ ID NO:382), RRRNIVPZVVTARRIGDLIVAQV (SEQ ID NO:383), RRNIVPZVVTARRRIGDLIVAQV (SEQ ID NO:384), RGVTPADLIGARRQYNPVAVZF (SEQ ID NO:385), RRVTPADLIGARRQYNPVAVZF (SEQ ID NO:386), RRRVTPADLIGARRQYNPVAVZF (SEQ ID NO:387), RRVTPADLIGARRRQYNPVAVZF (SEQ ID NO:388), RRGPRPEGYTLFFRGYTLFFTSR (SEQ ID NO:389), RGPRPEGYTLFFRRGYTLFFTSR (SEQ ID NO:390), RRGPRPEGYTLFFRRGYTLFFTSR (SEQ ID NO:391), RRGPRPEGYTLFFRRRGYTLFFTSR (SEQ ID NO:392), RRRGPRPEGYTLFFRRGYTLFFTSR (SEQ ID NO:393), RGLPYPRGYTLFVRRGYTLFVSDR (SEQ ID NO:394), RRGLPYPRGYTLFVRRGYTLFVSDR (SEQ ID NO:395), RRGLPYPRGYTLFVRRRGYTLFVSDR (SEQ ID NO:396), RRRGLPYPRGYTLFVRRGYTLFVSDR (SEQ ID NO:397), RRGLPYPRGYTLFVRRGYTLFVSDR (SEQ ID NO:398), RRGETILTPRDVRNTLZTPRDVR (SEQ ID NO:399), RGETILTPRDVRRNTLZTPRDVR (SEQ ID NO:400), RGETILTPRDVRNTLZTPRDVR (SEQ ID NO:401), RGETILTPRDVRGNTLZTPRDVR (SEQ ID NO:402), RRSSTSPVYDLRRSSTSPVYNLR (SEQ ID NO:403), RRSSTSPVYDLRRRSSTSPVYNLR (SEQ ID NO:404), RRRSSTSPVYDLRRSSTSPVYNLR (SEQ ID NO:405), RRRSSTSPVYDLRRRSSTSPVYNLR (SEQ ID NO:406), RRTAYERZCNILRRGLEPLVIAGILA (SEQ ID NO:407), RRRTAYERZCNILRRGLEPLVIAGILA (SEQ ID NO:408), RRTAYERZCNILRRRGLEPLVIAGILA (SEQ ID NO:409), RRTAYERZCNILRRGLEPLVIAGILAR (SEQ ID NO:410), RRTAYERZCNILRRGLEPLVIAGILARR (SEQ ID NO:411), RRTVIGASZIPLLRGTPIXQDWENRAN (SEQ ID NO:412), RRRTVIGASZIPLLRGTPIXQDWENRAN (SEQ ID NO:413), RRTVIGASZIPLLRRGTPIXQDWENRAN (SEQ ID NO:414), RRRTVIGASZIPLLRRGTPIXQDWENRAN (SEQ ID NO:415), RRRTVIGASZIPLLRRGTPIXQDWENRANR (SEQ ID NO:416), RRAAFEEZXITSRRVAFEDLXZZSFI (SEQ ID NO:417), RRRAAFEEZXITSRRVAFEDLXZZSFI (SEQ ID NO:418), RRRAAFEEZXITSRRGVAFEDLXZZSFI (SEQ ID NO:419), RRRAAFEEZXITSRRRVAFEDLXZZSFI (SEQ ID NO:420), RRRAAFEEZXITSRRRVAFEDLXZZSFIGR (SEQ ID NO:421), RRTAYERZCNILRRGRFQTVVQBA (SEQ ID NO:422), RRTAYERZCNILRRGRFQTVVQBAR (SEQ ID NO:423), RTAYERZCNILRGRFQTVVQBAR (SEQ ID NO:424), RRTAYERZCNILRGRFQTVVQBA (SEQ ID NO:425), BRGLEPLVIAGILARRGSLVGLLHIVL (SEQ ID NO:426), RRGLEPLVIAGILARRGSLVGLLHIVL (SEQ ID NO:427), RRGLEPLVIAGILARRGSLVGLLHIVLR (SEQ ID NO:428), RRGLEPLVIAGILARRRGSLVGLLHIVL (SEQ ID NO:429), RRGLEPLVIAGILARRRGSLVGLLHIVLR (SEQ ID NO:430), RTAFLVRNVARSIARSVTIZXASVVH (SEQ ID NO:431), RTAFLVRNVARRSIARSVTIZXASVVH (SEQ ID NO:432), RRTAFLVRNVARSIARSVTIZXASVVH (SEQ ID NO:433), RRTAFLVRNVARRSIARSVTIZXASVVH (SEQ ID NO:434), RRTAFLVRNVARRSIARSVTIZXASVVHR (SEQ ID NO:435), RRTAFLVRNVARRSIARSVTIZXASVVHRR (SEQ ID NO:436), RGDpr(Aoa)TPI(Har)QDWGNRANRGTPTRQEWDCRIS (SEQ ID NO:437), RGDpr(Aoa)TPI(Har)QDWGNRANRGTPTRQEWDARIS (SEQ ID NO:438), RGTPI(Har)QDWGNRANRGTPTRQEWDCRIS (SEQ ID NO:439), RGTPI(Har)QDWGNRANRGTPTRQEWDARIS (SEQ ID NO:440), RGCTPI(Har)QDWGNRANRGTPTRQEWDCRIS (SEQ ID NO:441), RGCTPI(Har)QDWGNRANRGTPTRQEWDARIS (SEQ ID NO:442), RGKTPI(Har)QDWGNRANRGTPTRQEWDCRIS (SEQ ID NO:443), RGKTPI(Har)QDWGNRANRGTPTRQEWDARIS (SEQ ID NO:444), RGLys(Me)TPI(Har)QDWGNRANRGTPTRQEWDCRIS (SEQ ID NO:445), RGLys(Me)TPI(Har)QDWGNRANRGTPTRQEWDARIS (SEQ ID NO:446), RGDTPI(Har)QDWGNRANRGTPTRQEWDCRIS (SEQ ID NO:447), RGDTPI(Har)QDWGNRANRGTPTRQEWDARIS (SEQ ID NO:448), RGETPI(Har)QDWGNRANRGTPTRQEWDCRIS (SEQ ID NO:449), RGETPI(Har)QDWGNRANRGTPTRQEWDARIS (SEQ ID NO:450), RGDpr(Ser)TPT(Har)NGWDVKLSRGTPI(Har)QEW(Har)SL(Nle)NQEW (SEQ ID NO:451), RGTPT(Har)NGWDVKLSRGTPI(Har)QEW(Har)SL(Nle)NQEW (SEQ ID NO:452), RGKTPT(Har)NGWDVKLSRGTPI(Har)QEW(Har)SL(Nle)NQEW (SEQ ID NO:453), RGCTPT(Har)NGWDVKLSRGTPI(Har)QEW(Har)SL(Nle)NQEW (SEQ ID NO:454), RGLys(Me)TPT(Har)NGWDVKLSRGTPI(Har)QEW(Har)SL(Nle)NQEW (SEQ ID NO:455), RGDTPT(Har)NGWDVKLSRGTPI(Har)QEW(Har)SL(Nle)NQEW (SEQ ID NO:456), RGETPT(Har)NGWDVKLSRGTPI(Har)QEW(Har)SL(Nle)NQEW (SEQ ID NO:457), RRLECVYCKQQLLRREVYDFAFRDLC (SEQ ID NO:458), RRLECVYCKQQLLRRRGEVYDFAFRDLC (SEQ ID NO:459), RRRLECVYCKQQLLRRGEVYDFAFRDLC (SEQ ID NO:460), RRRLECVYCKQQLLRRRGEVYDFAFRDLC (SEQ ID NO:461), RRRGLECVYCKQQLLRRRGEVYDFAFRDLC (SEQ ID NO:462), RRGVYDFAFRDLCRRGFAFRDLCIVYR (SEQ ID NO:463), RRGVYDFAFRDLCRRRGGFAFRDLCIVY (SEQ ID NO:464), RRRGVYDFAFRDLCRRGGFAFRDLCIVYR (SEQ ID NO:465), RRRGVYDFAFRDLCRRRGGFAFRDLCIVY (SEQ ID NO:466), RRRGGVYDFAFRDLCRRRGGFAFRDLCIVYR (SEQ ID NO:467), RRGVFDYAFRDINRRGFAYRDINLAYR (SEQ ID NO:468), RRGVYDFAFRDLCRRRGGFAFRDLCIVY (SEQ ID NO:469), RRRGVYDFAFRDLCRRGGFAFRDLCIVYR (SEQ ID NO:470), RRRGVYDFAFRDLCRRRGGFAFRDLCIVY (SEQ ID NO:471), RRRGGVYDFAFRDLCRRRGGFAFRDLCIVYR (SEQ ID NO:472), RRVDIRTLEDLLRRGTLGIVCPIGR (SEQ ID NO:473), RRVDIRTLEDLLRRRGGTLGIVCPIG (SEQ ID NO:474), RRRVDIRTLEDLLRRGGTLGIVCPIGR (SEQ ID NO:475), RRRVDIRTLEDLLRRRGGTLGIVCPIG (SEQ ID NO:476), RRRGVDIRTLEDLLRRRGGTLGIVCPIGR (SEQ ID NO:477), RGNIVPZVVTARRIGDLIVQAV (SEQ ID NO:479), RRNIVPZVVTARRIGDLIVQAV (SEQ ID NO:480), RRRNIVPZVVTARRIGDLIVQAV (SEQ ID NO:481), and RRNIVPZVVTARRRIGDLIVQAV (SEQ ID NO:482).
  • 59. The isolated peptide according to any one of claims 1-58, which peptide is not specifically disclosed in any one PCT application with application numbers WO2000N000075, WO2011DK050460, or WO2012DK050010.
  • 60. The isolated peptide according to any one of claims 1-59, which peptide is not a peptide selected from RRGYIPLVGAPLGBGRVARALAHGVRV (SEQ ID NO:47), RGYIPLVGAPLGRRVARALAHGVRV (SEQ ID NO:48), RGYIPLVGAPLGRRRVARALAHGVRVR (SEQ ID NO:49), RRGYIPLVGAPLGRRVARALAHGVRV (SEQ ID NO:50), RRGYIPLVGAPLGRRRVARALAHGVRV (SEQ ID NO:51), BRGYIPLVGAPLGRRVARALAHGVRV (SEQ ID NO:52), RRRGYIPLVGAPLGBRVARALAHGVRV (SEQ ID NO:53), RGYIPLVGAPLGKKKVARALAHGVRV (SEQ ID NO:54), RGYIPLVGAPLGRRRVARALAHGVRV (SEQ ID NO:55), KKGYIPLVGAPLGKKVARALAHGVRV (SEQ ID NO:56), WGYIPLVGAPLGRRVARALAHGVRV (SEQ ID NO:57), WWGYIPLVGAPLGRRVARALAHGVRV (SEQ ID NO:58), EEGYIPLVGAPLGEEVARALAHGVRV (SEQ ID NO:59), GGGYIPLVGAPLGGGVARALAHGVRV (SEQ ID NO:60), EEGYIPLVGAPLGRRVARALAHGVRV (SEQ ID NO:61), RRGYIPLVGAPLGLRRVARALAHGVRV (SEQ ID NO:62), WWGYIPLVGAPLGRRVARALAHGVRV (SEQ ID NO:63), WWGYIPLVGAPLGRRRVARALAHGVRV (SEQ ID NO:64), WWGYIPLVGAPLGRVARALAHGVRV (SEQ ID NO:65), RGYIPLVGAPLGRRVARALAHGVRV (SEQ ID NO:66), RRGYLPAVGAPIGBRVIRVIAHGLRL (SEQ ID NO:67), RRGYIPLVGAPLGBRVARALAHGVRV (SEQ ID NO:68), GYIPLVGAPLGGVARALAHGVRV (SEQ ID NO:69), WWGYLPAVGAPIRRVIRVIAHGLRL (SEQ ID NO:70), GYIPLVGAPLGGVARALAHGVRV (SEQ ID NO:71), RRGYIPLVGAPLGBGRVARALAHGVRV (SEQ ID NO:72), RGYIPLVGAPLGRRVARALAHGVRV (SEQ ID NO:73), RGYIPLVGAPLGRRRVARALAHGVRV (SEQ ID NO:74), RRGYIPLVGAPLGRRVARALAHGVRV (SEQ ID NO:75), RRGYIPLVGAPLGRRRVARALAHGVRV (SEQ ID NO:76), BRGYIPLVGAPLGRRVARALAHGVRV (SEQ ID NO:77), RRRGYIPLVGAPLGBRVARALAHGVRV (SEQ ID NO:78), RGYIPLVGAPLGKKKVARALAHGVRV (SEQ ID NO:79), RGYIPLVGAPLGRRRVARALAHGVRV (SEQ ID NO:80), KKGYIPLVGAPLGKKVARALAHGVRV (SEQ ID NO:81), WGYIPLVGAPLGRRVARALAHGVRV (SEQ ID NO:82), WWGYIPLVGAPLGRRVARALAHGVRV (SEQ ID NO:83), RRGYIPLVGAPLGLRRVARALAHGVRV (SEQ ID NO:84), RRNYVTGNIPGBRGITFSIFLIVS (SEQ ID NO:85), WWNYATGNLPGRRCSFSIFLLAL (SEQ ID NO:86), WWNYVTGNIPGBRGITFSIFLIVS (SEQ ID NO:87), WWNYVTGNIPGRRGITFSIFLIVS (SEQ ID NO:88), RRNYATGNLPGRRGCSFSIFLLAL (SEQ ID NO:89), RRVTGNIPGSTYSGBRGITFSIYLIVS (SEQ ID NO:90), RRIRNLGRVIETLTGBRLNleGYIPLIGA (SEQ ID NO:91), RRSRNLGKVIDTLTCBRLMGYIPLVGA (SEQ ID NO:92), SRNLGKVIDTLTCGFADLMGYIPLVGA (SEQ ID NO:93), WWIRNLGRVIETLTRRLNleGYIPLIGA (SEQ ID NO:94), WWSRNLGKVIDTLTCRRLMGYIPLVGA (SEQ ID NO:95), RRGGGQIIGGNYLIPRBPBIGVRATB (SEQ ID NO:96), GGGQIVGGVYLLPRRGPRLGVRATR (SEQ ID NO:97), RRGGGQIVGGVYLLPRRGPRLGVRATR (SEQ ID NO:98), WWGGGQIVGGVYLLPRRGPRLGVRAT (SEQ ID NO:99), BRLIFLARSALIVRGSVAHKS (SEQ ID NO:100), EDLIFLARSALILRGSVAHKS (SEQ ID NO:101), BRLIFLARSALILBGRSALILRGSVAHK (SEQ ID NO:102), SAYERMCNILKGKFQTAAQRAMM (SEQ ID NO:103), SAYERNleVNILKGKFQTAAQRAVNle (SEQ ID NO:104), BRTAYERNleCNILBRGRFQTVVQBA (SEQ ID NO:105), BRIAYERMCNILLBRGKFQTAAQRA (SEQ ID NO:106), IAYERMCNILKGKFQTAAQRA (SEQ ID NO:107), LFFKClYRLFKHGLKRGPSTEGVPESM (SEQ ID NO:108), BRRLFFKTITRLFBHGLRRLLSTEGVPNSNle (SEQ ID NO:109), BRGLEPLVIAGILARRGSLVGLLHIVL (SEQ ID NO:110), BRGSDPLVVAASIVRRASIVGILHLIL (SEQ ID NO:111), RNLVPMVATVRRNLVPMVATVB (SEQ ID NO:112), RNLVPMVATVBRRNLVPMVATVB (SEQ ID NO:113), RNIVPNleVVTARRNIVPNleVVTAB (SEQ ID NO:114), PEVIPMFSALSEGATPQDLNTMLN (SEQ ID NO:115), RFIIPXFTALSGGRRALLYGATPYAIG (SEQ ID NO:116), KALGPAATLEEMMTACQGVG (SEQ ID NO:117), RRGPVVHLTLRRRGQAGDDFS (SEQ ID NO:118), RRGPVVHLTLRRRGQAGDDFS (SEQ ID NO:119), RRGPVVHLTLRGRRGQAGDDFS (SEQ ID NO:120), RRLECVYCKQQLLRREVYDFAFRDLC (SEQ ID NO:121), RRGVYDFAFRDLCRRGFAFRDLCIVYR (SEQ ID NO:122), RRGVFDYAFRDINRRGFAYRDINLAYR (SEQ ID NO:123), RRGATPVDLLGARRGALNLCLPMR (SEQ ID NO:124), RRGVTPAGLIGVRRGALQIBLPLR (SEQ ID NO:125), RGYLPAVGAPIGRRRVIRVIAHGLRLR (SEQ ID NO:196), RRSRNLGKVIDTLTCRRLMGYIPLVGA (SEQ ID NO:197), RRIRNLGRVIETLTLNleGYIPLIGARRIRNLGRVIETLTLNleGYIPLIGAR (SEQ ID NO:199), X1-NYVTGNIPG-X3-GITFSIYLIVS; X1-IRNLGRVIETLT-X3-LNleGYIPLIGA; X1-GYLPAVGAP1-X3-VIRVIAHGLRL; X1-GGGQIIGGNYLIP-X3-PBIGVRATB; X1-NYATGNLPG-X3-GCSFSIFLLAL; X1-SRNLGKVIDTLTC-X3-LMGYIPLVGA; X1-GYIPLVGAPL-X3-VARALAHGVRV; X1-GGGQIVGGVYLLP-X3-PRLGVRATR; X1-LTFLVRSVLL1-X3-GSVLIVRGSLVH; X1-TAYERNleCNIL-X3-GRFQTVVQBA; X1-SDPLVVAASIV-X3-ASIVGILHLIL; X1-LIFLARSALIL-X3-SALILRGSVAH; X1-IAYERMCNIL-X3-GKFQTAAQRA; and X1-LEPLVIAGILA-X3-GSLVGLLHIVL; X1-NLVPMVATV-X3-NLVPMATV; X1-GYLPAVGAPIG-X3-VIRVIAHGLRL; X1-IRNLGRVIETLTG-X3-LNleGYIPLIGA; X1-GVYDFAFRDLC-X3-GFAFRDLCIVYR, X1-GVFDYAFRDIN-X3-GFAYRDINLAYR, X1-GATPVDLLGA-X3-GALNLCLPMR, X1-GVTPAGLIGV-X3-GALQIBLPLR, and X1-IRNLGRVIETLTLNleGYIPLIGA-X3-IRNLGRVIETLTLNleGYIPLIGA; optionally with an X5 in the C-terminal of the peptide; wherein X1 and X3 and X5 refers to X′, X3, and X5 of formula II.
  • 61. An isolated multimeric, such as dimeric peptide comprising two or more monomeric peptides, each monomeric peptide independently comprising the following structure (Z1-Z2)1-Z3-(Z4-Z5)2-Z6-(Z7-Z8)3-Z9-(Z10-Z11)4-Z12
  • 62. The isolated multimeric, such as dimeric peptide according to claim 61, wherein two or more monomeric peptides are identical in sequence.
  • 63. The isolated multimeric, such as dimeric peptide according to claim 61, wherein two or more monomeric peptides are different in sequence.
  • 64. The isolated multimeric, such as dimeric peptide according to any of claims 61-63, comprising at least two peptides monomers, each peptide monomer independently being as defined in any one of claims 1-58.
  • 65. The isolated multimeric, such as dimeric peptide according to any one of claims 61-64, wherein one or more peptide strands of the multimeric, such as dimeric peptide has delayed proteolytic degradation in the N-terminal, such as by incorporation of the first 1, 2, or 3 amino acids in the N-terminal in the D-form, or by incorporation of the first 1, 2, or 3 amino acids in the N-terminal in beta or gamma form.
  • 66. The isolated multimeric, such as dimeric peptide according to any one of claims 61-65, which multimeric, such as dimeric peptide contain a helper epitope of at least 12 amino acids, such as at least 13, 14, 15 or 17 amino acids, which helper epitope consist of a combined sequence of amino acids, which is a sequence of amino acids from a first specific continuous antigenic peptide sequences, and a sequence of amino acids from at least one second specific continuous antigenic peptide sequence of the same or different protein derived from the same virus or a different virus, or any other disease antigen, such as an infectious agent, such as bacteria, virus, parasite, fungus, or cancer antigens such as oncogene (lung, stomach, breast cancer) or an antigen causing an autoimmune disease such as diabetes, multiple sclerosis (MS), celiac disease, Myalgic Encephalomyelitis (ME), psoriasis, and/or Crohn's Disease bacteria, or other disease causing agent, such as between 2-12 amino acids from the first specific continuous antigenic peptide sequences and 2-12 amino acids from the at least one second specific continuous antigenic antigenic peptide sequence.
  • 67. The isolated multimeric, such as dimeric peptide according to any one of claims 61-66, wherein said intermolecular bond is a disulfide (S—S) bond between two Cys residues.
  • 68. The isolated multimeric, such as dimeric peptide according to any one of claims 61-67, wherein said intermolecular bond is a thioether bond between a Cys residue in the first monomeric peptide and a modified Lys residue in the at least one second monomeric peptide.
  • 69. The isolated multimeric, such as dimeric peptide according to any one of claims 61-68, wherein said intermolecular bond is an oxime bond between a derivatized Lys residue in the first monomeric peptide and a derivatized Ser residue in the at least one second monomeric peptide.
  • 70. The isolated multimeric, such as dimeric peptide according to any one of claims 61-69, wherein said intermolecular bond is a peptide bond between a N-methylated Lys side-chain in the first monomeric peptide and the side-chain of an Asp or Glu residue in the at least one second monomeric peptide.
  • 71. The isolated multimeric, such as dimeric peptide according to any one of claims 61-70, wherein said intermolecular bond is an oxime bond between an aldehyde moiety, produced by oxidation of a serine residue in the first monomeric peptide and a free aminooxy group of a modified amino acid (aminooxy acid), such as derivataized diaminopropionic acid, Lysine or Ornithine in in the second monomeric peptide
  • 72. The isolated multimeric, such as dimeric peptide according to any one of claims 61-71, wherein said monomeric peptides are linked by a polyethylene glycol (PEG) linker, such as through an Asp or a Glu residue in the first monomeric peptide and an Asp or a Glu residue in the at least one second monomeric peptide, or by a polyLys core.
  • 73. The isolated multimeric, such as dimeric peptide according to any one of claims 61-72, wherein a C residue in Z2 of the first peptide monomer is linked to an amino acid selected from a K or a C residue in Z2 of the second monomer.
  • 74. The isolated multimeric, such as dimeric peptide according to any one of claims 61-73, wherein a K residue in Z2 of the first peptide monomer is linked to an amino acid selected from a C, D or E residue in Z2 of the second monomer.
  • 75. The isolated multimeric, such as dimeric peptide according to any one of claims 61-74, wherein a D residue in Z2 of the first peptide monomer is linked to an amino acid selected from a N or Q residue in Z2 of the second monomer.
  • 76. The isolated multimeric, such as dimeric peptide according to any one of claims 61-75, wherein a E residue in Z2 of the first peptide monomer is linked to an amino acid selected from a N or Q residue in Z2 of the second monomer.
  • 77. The isolated multimeric, such as dimeric peptide according to any one of claims 61-76, wherein a N residue in Z2 of the first peptide monomer is linked to a D or E residue in Z2 of the second monomer.
  • 78. The isolated multimeric, such as dimeric peptide according to any one of claims 61-77, wherein a Q residue in Z2 of the first peptide monomer is linked to a D or E residue in Z2 of the second monomer.
  • 79. The isolated multimeric, such as dimeric peptide according to any one of claims 61-78, wherein a Dpr(Aao) residue in Z2 of the first peptide monomer is linked to an Dpr(Ser) residue in Z2 of the second monomer.
  • 80. The isolated multimeric, such as dimeric peptide according to any one of claims 61-79, wherein a W residue in Z2 of the first Z1-Z2 peptide repeat is linked to an Y residue in Z2 of the second Z1-Z2 peptide repeat.
  • 81. The isolated multimeric, such as dimeric peptide according to any one of claims 61-80, wherein a Y residue in Z2 of the first Z1-Z2 peptide repeat is linked to an W residue in Z2 of the second Z1-Z2 peptide repeat.
  • 82. Composition comprising two or more compounds selected from a monomeric peptide is as defined in any one of claims 1-60, and an isolated multimeric, such as dimeric peptide as defined in any one of claims 61-81.
  • 83. Composition according to claim 82, comprising one or more compound selected from SEQ ID NO:357, SEQ ID NO:366, SEQ ID NO:377, SEQ ID NO:358, SEQ ID NO:367, SEQ ID NO:379, SEQ ID NO:359, SEQ ID NO:368, SEQ ID NO:380, SEQ ID NO:360, SEQ ID NO:369, SEQ ID NO:374, SEQ ID NO:361, SEQ ID NO:370, SEQ ID NO:375, SEQ ID NO:362, SEQ ID NO:371, SEQ ID NO:376, SEQ ID NO:363, SEQ ID NO:378, SEQ ID NO:364, SEQ ID NO:372, SEQ ID NO:365, and SEQ ID NO:373.
  • 84. Composition according to claim 82, comprising two or more compounds, wherein both compounds are selected in any one of the groups consisting of SEQ ID NO:357, SEQ ID NO:366, and SEQ ID NO:377;SEQ ID NO:358, SEQ ID NO:367, and SEQ ID NO:379;SEQ ID NO:359, SEQ ID NO:368, and SEQ ID NO:380;SEQ ID NO:360, SEQ ID NO:369, and SEQ ID NO:374;SEQ ID NO:361, SEQ ID NO:370, and SEQ ID NO:375;SEQ ID NO:362, SEQ ID NO:371, and SEQ ID NO:376;SEQ ID NO:362, SEQ ID NO:366, and SEQ ID NO:377;SEQ ID NO:363, SEQ ID NO:367, and SEQ ID NO:378;SEQ ID NO:364, SEQ ID NO:372, and SEQ ID NO:379; andSEQ ID NO:365, SEQ ID NO:373, and SEQ ID NO:380.
  • 85. Composition according to claim 82, comprising three compounds of any one of the groups consisting of SEQ ID NO:357, SEQ ID NO:366, and SEQ ID NO:377;SEQ ID NO:358, SEQ ID NO:367, and SEQ ID NO:379;SEQ ID NO:359, SEQ ID NO:368, and SEQ ID NO:380;SEQ ID NO:360, SEQ ID NO:369, and SEQ ID NO:374;SEQ ID NO:361, SEQ ID NO:370, and SEQ ID NO:375;SEQ ID NO:362, SEQ ID NO:371, and SEQ ID NO:376;SEQ ID NO:362, SEQ ID NO:366, and SEQ ID NO:377;SEQ ID NO:363, SEQ ID NO:367, and SEQ ID NO:378;SEQ ID NO:364, SEQ ID NO:372, and SEQ ID NO:379; andSEQ ID NO:365, SEQ ID NO:373, and SEQ ID NO:380.
  • 86. Composition according to claim 82, comprising one or more compounds selected from BI-155-5, SEQ ID NO:407, SEQ ID NO:412, SEQ ID NO:417, BI-155-4, SEQ ID NO:408, SEQ ID NO:413, SEQ ID NO:418, BI-155-3, SEQ ID NO:409, SEQ ID NO:414, SEQ ID NO:419, BI-155-2, SEQ ID NO:410, SEQ ID NO:415, SEQ ID NO:420, BI-155, SEQ ID NO:411, SEQ ID NO:416, and SEQ ID NO:421.
  • 87. Composition according to claim 82, comprising two or more compounds, wherein both compounds are selected in any one of the groups consisting of BI-155-5, SEQ ID NO:407, SEQ ID NO:412SEQ ID NO:417;BI-155-4, SEQ ID NO:408, SEQ ID NO:413, SEQ ID NO:418;BI-155-3, SEQ ID NO:409, SEQ ID NO:414, SEQ ID NO:419;BI-155-2, SEQ ID NO:410, SEQ ID NO:415, SEQ ID NO:420;BI-155, SEQ ID NO:411, SEQ ID NO:416, SEQ ID NO:421;BI-155-2, SEQ ID NO:411, SEQ ID NO:416, SEQ ID NO:421;BI-155-3, SEQ ID NO:410, SEQ ID NO:414, SEQ ID NO:419; andBI-155, SEQ ID NO:407, SEQ ID NO:415, SEQ ID NO:418.
  • 88. Composition according to claim 82, comprising three or more compounds, wherein each three compounds are selected in any one of the groups consisting of BI-155-5, SEQ ID NO:407, SEQ ID NO:412SEQ ID NO:417;BI-155-4, SEQ ID NO:408, SEQ ID NO:413, SEQ ID NO:418;BI-155-3, SEQ ID NO:409, SEQ ID NO:414, SEQ ID NO:419;BI-155-2, SEQ ID NO:410, SEQ ID NO:415, SEQ ID NO:420;BI-155, SEQ ID NO:411, SEQ ID NO:416, SEQ ID NO:421;BI-155-2, SEQ ID NO:411, SEQ ID NO:416, SEQ ID NO:421;BI-155-3, SEQ ID NO:410, SEQ ID NO:414, SEQ ID NO:419; andBI-155, SEQ ID NO:407, SEQ ID NO:415, SEQ ID NO:418.
  • 89. Composition according to claim 82, comprising four compounds of any one of the groups consisting of BI-155-5, SEQ ID NO:407, SEQ ID NO:412SEQ ID NO:417;BI-155-4, SEQ ID NO:408, SEQ ID NO:413, SEQ ID NO:418;BI-155-3, SEQ ID NO:409, SEQ ID NO:414, SEQ ID NO:419;BI-155-2, SEQ ID NO:410, SEQ ID NO:415, SEQ ID NO:420;BI-155, SEQ ID NO:411, SEQ ID NO:416, SEQ ID NO:421;BI-155-2, SEQ ID NO:411, SEQ ID NO:416, SEQ ID NO:421;BI-155-3, SEQ ID NO:410, SEQ ID NO:414, SEQ ID NO:419; andBI-155, SEQ ID NO:407, SEQ ID NO:415, SEQ ID NO:418.
  • 90. Use of a peptide selected from a monomeric peptide is as defined in any one of claims 1-60, and an isolated multimeric, such as dimeric peptide as defined in any one of claims 61-81 for inducing an immune response in a subject, such as a humoral or Cell Mediated Immune (CMI) response.
  • 91. An isolated nucleic acid or polynucleotide encoding a peptide according to any one of claims 1-61.
  • 92. A vector comprising the nucleic acid or polynucleotide according to claim 84.
  • 93. A host cell comprising the vector according to claim 85.
  • 94. An immunogenic composition comprising at least one monomeric peptide according to any one of claims 1-61, an isolated multimeric, such as dimeric peptide according to any one of claims 61-81, a peptide composition according to any one of claims 82-89, the nucleic acid or polynucleotide according to claim 91, or the vector according to claim 92; optionally in combination with a pharmaceutically acceptable diluent or vehicle and optionally an immunological adjuvant.
  • 95. The immunogenic composition according to claim 94 in the form of a vaccine composition.
  • 96. A method for inducing an immune response in a subject against an antigen which comprises administration of at least one monomeric peptide according to any one of claims 1-60, an isolated multimeric, such as dimeric peptide according to any one of claims 61-79, a peptide composition according to any one of claims 82-89, the nucleic acid or polynucleotide according to claim 91, or the vector according to claim 92; or the composition according to any one of claims 94-95.
  • 97. A method for reducing and/or delaying the pathological effects of a disease antigen, such as an infectious agent in a subject infected with said agent or having said disease caused by said antigen, the method comprising administering an effective amount of at least one monomeric peptide according to any one of claims 1-60, an isolated multimeric, such as dimeric peptide according to any one of claims 61-81, a peptide composition according to any one of claims 82-89, the nucleic acid or polynucleotide according to claim 91, or the vector according to claim 92; or the composition according to any one of claims 94-95.
  • 98. A peptide according to any one of claims 1-81, a peptide composition according to any one of claims 82-89, the nucleic acid or polynucleotide according to claim 91, or the vector according to claim 92; or the composition according to any one of claims 94-95, for use as a medicament.
  • 99. A peptide according to any one of claims 1-81, a peptide composition according to any one of claims 82-89, the nucleic acid or polynucleotide according to claim 91, or the vector according to claim 92; or the composition according to any one of claims 94-95, for treating the pathological effects of a disease antigen, such as an infectious agent in a subject infected with said agent or having said disease caused by said antigen.
  • 100. A peptide according to any one of claims 1-81, a peptide composition according to any one of claims 82-89, the nucleic acid or polynucleotide according to claim 91, or the vector according to claim 92; or the composition according to any one of claims 94-95 for use in an in vitro assay, such as an ELISA assay, such as for diagnostic purposes.
  • 101. Use of a peptide according to any one of claims 1-81, a peptide composition according to any one of claims 82-89, the nucleic acid or polynucleotide according to claim 91, or the vector according to claim 92; or the composition according to any one of claims 94-95, for in vitro assay, such as an ELISA assay, such as for diagnostic purposes.
PCT Information
Filing Document Filing Date Country Kind
PCT/EP2013/061751 6/6/2013 WO 00
Provisional Applications (1)
Number Date Country
61656256 Jun 2012 US