Patent Grant
10376576
This application is the National Phase of International Patent Application No. PCT/NL2015/050390, filed Jun. 1, 2015, published on Dec. 10, 2015 as WO 2015/187009 A1, which claims priority to European Patent Application No. 14170733.1, filed Jun. 2, 2014. The contents of these applications are herein incorporated by reference in their entirety.
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-WEB and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 8, 2017, is named 069818_2180SequenceListing.txt and is 412 KB.
The present invention relates to the fields of medicine and immunology. In particular, it relates to novel peptides that may be used in the treatment and/or prevention of a Hepatitis B viral infection and/or an Hepatitis B related disease or condition.
Chronic infection with the hepatitis B virus (HBV) is a major global health problem. HBV is the prototype member of the Hepadnaviridae family, which have a strong preference for infecting liver cells (Ganem et al, 2004).
Despite the availability since three decades of an efficacious preventive vaccine for the protection against hepatitis B, an estimated two billion people have nevertheless been infected with HBV and more than 240 million currently have chronic (long-term) hepatitis B infection, with a geographical predominance in regions outside Western Europe and North America (World Health Organization, July 2013).
Transmission of the virus between people occurs by direct blood-to-blood contact or via semen or vaginal fluid of an infected person. In endemic areas, the infection occurs characteristically by perinatal transmission from mother to child. Thus, although HBV is not transmitted casually, the virus—via similar modes of entry as human immunodeficiency virus (HIV) but being at least 50 times more infectious—can be easily transmitted by perinatal, percutaneous or sexual exposure. Frequent person-to-person contact with infected individuals accordingly poses a serious risk to groups like health workers.
Infection with HBV can develop as an acute viral hepatitis, an illness that begins with general ill-health, loss of appetite, nausea, vomiting, body aches, mild fever, and dark urine, and then progresses to development of jaundice. The illness lasts for a few weeks and then gradually improves in most affected adults. A few people may have more severe liver disease (fulminant hepatic failure), and may die as a result. The infection may be entirely asymptomatic and may go unrecognized. Chronic infection with hepatitis B virus either may be asymptomatic or may be associated with a chronic inflammation of the liver (chronic hepatitis), leading to cirrhosis over a period of many years. This type of infection dramatically increases the incidence of hepatocellular carcinoma (liver cancer), also with a latency of many years.
Treatment of chronically HBV-infected individuals with antiviral drugs such as nucleoside/nucleotide analogues (e.g. Entecavir and Tenofovir) or interferon (IFN)α efficiently decreases serum viral loads. However, antiviral therapy rarely leads to a sustained virological response and drug resistance occurs (Zoulim et al., 2012). Moreover, the great majority of HBV carriers remains untreated.
Approximately 15-40% of chronic HBV carriers will develop clinically significant liver diseases in their lifetime with a high risk of death from liver cirrhosis and associated liver failure or hepatocellular carcinoma (HCC) (Lok, 2002; and Huang et al., 2011). Yearly up to one million people die worldwide due to the acute or chronic consequences of hepatitis B (Michel et al, 2001; Grimm et al, 2013). Due to the failure of antiviral drugs to eradicate infection, and consequently the need for long-term if not lifelong antiviral therapy with its drawbacks such as toxic side-effects and high costs, there is an urgent need for novel therapeutic approaches (Grimm et al., 2013).
The present invention is meant to enable efficacious therapeutic vaccination against chronic HBV infection. Therapeutic vaccination constitutes a promising strategy to treat chronic hepatitis B (Michel et al., 2011).
Next to the humoral immune response against HBV, which is predominantly involved in the protection against HBV infection by the current prophylactic vaccines (Lok, 2002), the cellular immune response is unequivocally involved in the natural resistance against HBV infection.
Perinatal transmission of HBV from mothers to neonates and infections during the first years of life result in persistent infection in more than 90% of children. By contrast, infection during adulthood clears spontaneously in more than 90% of cases and results in lifelong protective immunity (Rehermann et al., 2005).
In acute, self-limited hepatitis B virus infection, vigorous polyclonal and multispecific CD8+ cytotoxic T cell (CTL) and CD4+ T-helper (Th) cell responses to many HBV antigens are readily demonstrable in the peripheral blood (Michel et al., 2011).
These T cell responses are crucial in HBV clearance and control. Experiments in HBV-infected chimpanzees have shown the essential role of HBV-specific CD8+ T cells as effector cells in this process (Thimme et al., 2003). In contrast to the response in patients with resolved HBV infections, in patients with chronic hepatitis B the T cell responses are usually very weak, focused on only a few epitopes and functionally impaired (Michel et al., 2011). The goal of therapeutic vaccination is to install vigorous and robust multivalent CTL and T-helper cell responses directed to many HBV antigens, thereby pursuing viral clearance, hepatitis control and cure.
Despite the fact that great progress has been made in understanding the etiology and epidemiology of the disease, there is still a need for an effective therapeutic HBV vaccine.
The inventors identified a selection of HBV antigens to be used for efficacious therapeutic vaccination. Based on HLA class I and class II binding capacities of HBV protein-derived peptides and analysis of the generation of these HLA class I binding peptides by cleavages made by the proteasome, the most immunogenic regions, covering a very high percentage of all possible T cell epitopes in the global hepatitis B patient population, have been uncovered in the HBV polymerase protein, core protein, X protein and large surface protein. These regions contain high numbers of T cell epitopes and when administered to the hepatitis B patient—either as chemically synthesized long peptide or via genetic approaches—such a vaccination is envisioned to induce a vigorous T cell response, resolving the HBV infection.
The use of relatively short peptides is highly preferred for medical purposes as these can be efficiently synthesized in vitro, which is not possible or uneconomical for native proteins larger than approximately 100, i.e. 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105 amino acids. Chemical synthesis of peptides is routine practice and various suitable methods are known to the skilled person. Chemical synthesis of peptides also overcomes the problems associated with recombinant production of intact proteins, which is difficult to standardize and requires extensive purification and quality control measures. Peptides with a length that exceeds the length of human leukocyte antigen (HLA) class I and class II epitopes (e.g. having a length as indicated below herein) are particularly advantageous for use as vaccine component because they are large enough to be taken up by professional antigen presenting cells (APC), in particular Dendritic cell (DC), as explained in WO02/070006, and processed in the DC before cell surface presentation of the contained HLA class I-presented and HLA class II-presented epitopes takes place. Therefore, the disadvantageous induction of T cell tolerance by the systemic presentation of minimal HLA class I-presented epitopes on non-antigen presenting cells (as shown in Toes et al., 1996a, and Toes et al., 1996b), is prevented by the application of peptides exceeding the length of human leukocyte antigen (HLA) class I and class II epitopes (as shown in Zwaveling et al., 2002).
The present invention relates to novel peptides of about 15 to about 100 amino acids in length, also denominated herein as long peptides, that each exceed the length of human leukocyte antigen (HLA) class I and class II presented epitopes and that induce a combined CD4+ and CD8+ T cell response that is therapeutically successful and inducing cure in a high percentage of patients. Preferably, the long peptides of the invention are synthetic peptides, denominated herein as synthetic long peptides (SLPs). As compared to vaccination with the peptides of the present invention, therapeutic vaccination with full length HBV proteins is likely to be less potent (Rosalia et al., 2013). From the viewpoint of manufacture and administration of peptides to patients, immunization with the complete set of overlapping long peptides or SLPs spanning the full length HBV polymerase, HBV core protein, HBV X protein and HBV large surface protein is not feasible. To narrow the number of peptides in a vaccine, it is needed to incorporate the most immunogenic SLPs that are recognized by the highest percentage of patients. The present invention provides for peptides and peptide vaccines to meet this need. Using a stepwise sophisticated selection procedure based on bioinformatics analyses that are experimentally underpinned, the long peptide and/or SLP sequences with the highest coverage of HLA class I-restricted cytotoxic T lymphocyte epitopes and HLA class II-restricted T helper epitopes were identified. The selections as described herein identify the long peptide and/or SLP sequences that incorporate HBV-derived T cell epitopes that are presented on all predominantly expressed HLA class I and class II alleles. By covering the vast majority of worldwide expressed HLA haplotypes (Bui et al., 2006), the long peptide and/or SLP vaccine composition can be used in all HBV infected individuals. The present invention describes the identification and selection of HBV-derived long peptides, preferably SLPs, that are highly immunogenic and capable of inducing a potent combined HBV-directed CD4+ T helper and CD8+ cytotoxic T cell response, when administered as a vaccine composition to patients. Such highly immunogenic long peptides from HBV have not been disclosed in the prior art. The HBV-derived long peptides of the invention were identified based on a putative immunogenicity score developed and validated by the inventors and as disclosed herein. The putative immunogenicity is quantified herein using the T cell Regional Immunogenicity Assessment (TRIA) score. The TRIA score is based on the cumulative Class I-BCI score of said peptide, which is indicative for their immunogenic CTL activating capacity, and the cumulative Class II-B score of said peptide, which is indicative for their immunogenic Th-cell activating capacity. Calculation of the cumulative Class I-BCI score and the cumulative Class II-B score is described in detail herein in the Examples section. The TRIA score is calculated as the sum of the cumulative Class I-BCI score and the cumulative Class II-B score. A strong correlation was found between this TRIA score and the T cell responses found in PBMC of HBV-immune donors. Therefore, the TRIA score enables the selection of optimal immunogenic long peptides.
In a first aspect, the present invention provides a peptide derived from an HBV protein. Preferably the peptide of the invention comprises or consists of a peptide selected from the group consisting of SEQ ID NO: 51-79, 1142-1145 and 1468-1471, more preferably the peptide of the invention comprises or consist of a peptide selected from the group consisting of SEQ ID NO: 51, 55, 60, 63, 64, 68, 71, 74, 75, 76, 77, 1142 and 1469, more preferably selected from the group consisting of SEQ ID NO: 51, 55, 60, 63, 64, 68, 71, 74, 75, 77, 1142 and 1469, even more preferably selected from the group consisting of SEQ ID NO: 55, 60, 63, 64, 68, 71, 74, 75, 76, 77 and 1469, even more preferably selected from the group consisting of SEQ ID NO: 55, 60, 63, 64, 68, 71, 74, 75, 77 and 1469, even more preferably selected from the group consisting of SEQ ID NO: 60, 63, 71, 74, 75 and 1469, most preferably selected from the group of SEQ ID NO: 75, 1469 and 63. Further preferred is a peptide of the invention that comprises or consists of a peptide selected from the group consisting of SEQ ID NO: 51, 60, 63, 64, 68, 71, 74-77. Also preferred is a peptide of the invention that comprises or consists of a peptide selected from the group consisting of SEQ ID NO: 63, 71 and 75. Preferably, the peptide of the invention comprises at least about 70 predicted T-cell epitopes. More preferably, the peptide of the invention comprises at least about 70 predicted T-cell epitopes and at least about 3 proteasomal cleavage sites. Preferably, the peptide if the invention comprises at least about 70 predicted HLA class I-restricted CD8+ cytotoxic T-cell epitopes, at least about 1 predicted HLA class II-restricted CD4+ T-helper epitope. More preferably, the peptide if the invention comprises at least about 70 predicted HLA class I-restricted CD8+ cytotoxic T-cell epitopes, at least about 1 predicted HLA class II-restricted CD4+ T-helper epitope and at least about 3 proteasomal cleavage sites. HLA class I-restricted CD8+ cytotoxic T-cell epitope is also denominated herein as CTL epitope and HLA class II-restricted CD4+ T-helper epitope is also denominated herein as Th-cell epitope. Preferably, the peptide of the invention comprises at least about 70 predicted CTL epitopes, at least about 15 predicted Th-cell epitopes. More preferably, the peptide of the invention comprises at least about 70 predicted CTL epitopes, at least about 15 predicted Th-cell epitopes and at least about 3 proteasomal cleavage sites. Preferably, the peptide of the invention comprises at least about 95 predicted CTL epitopes, at least about 25 predicted Th-cell epitopes. More preferably, the peptide of the invention comprises at least about 95 predicted CTL epitopes, at least about 25 predicted Th-cell epitopes and at least about 3 proteasomal cleavage sites. Preferably, the peptide of the invention comprises at least about 125 predicted CTL epitopes, at least about 50 predicted Th-cell epitopes. More preferably, a peptide of the invention comprises at least about 125 predicted CTL epitopes, at least about 50 predicted Th-cell epitopes and at least about 3 proteasomal cleavage sites. Preferably, a peptide of the invention has a TRIA score of at least about 6300, at least about 8000, at least about 9000, at least about 10000, or preferably at least about 14000.
A peptide of the invention can advantageously be used in the prevention and/or treatment of an HBV related disease or condition in a subject, preferably a mammal, more preferably a human. Preferably, the peptide of the invention comprises or consists of an amino acid sequence, preferably a contiguous amino acid sequence, of any of the proteins selected from the group consisting of HBV protein polymerase, HBV core protein, HBV X-protein and HBV large surface protein. Preferably, said peptide comprises or consists of a peptide selected from the group consisting of SEQ ID NO: 51-79, 1142-1145 and 1468-1471. A peptide of this group is characterized in that it has a TRIA score of at least 6300, indicating the high immunogenic capacity for CD4+ and CD8+ T cell activation. Furthermore, a peptide of this group is characterized in that it comprises at least 70 predicted HLA class I-restricted CD8+ cytotoxic T-cell epitopes, at least 1 predicted HLA class II-restricted CD4+ T-helper epitope. Preferably, a peptide of this group comprises at least 3 proteasomal cleavage sites.
More preferably, the peptide of the invention comprises or consists of a peptide selected from the group consisting of SEQ ID NO: 51-53, 55-57, 60-66, 68-78, 1142-1145 and 1468-1471. A peptide of this group is characterized in that it has a TRIA score of at least 8000.
More preferably, the peptide of the invention comprises or consists of a peptide selected from the group consisting of SEQ ID NO: 51-53, 55-57, 60-66, 68, 69, 71-79, 1142-1145 and 1468-1471. A peptide of this group is characterized in that it comprises at least 70 predicted CTL epitopes, at least 15 predicted Th-cell epitopes. Preferably, a peptide of this group comprises at least 3 proteasomal cleavage sites. Preferably, the peptide of the invention comprises or consists of a peptide selected from the group consisting of SEQ ID NO: 51-53, 55, 57, 60, 63, 64, 66, 68, 71, 72, 74-78, 1142, 1145, 1468-1471.
More preferably, the peptide of the invention comprises or consists of a peptide selected from the group consisting of SEQ ID NO: 53, 55-57, 60-66, 68, 69, 71, 73-78, 1142-1145, 1468-1471. A peptide of this group is characterized in that it has a TRIA score of at least 9000.
Even more preferably, the peptide of the invention is a peptide that comprises or consists of a peptide selected from the group consisting of SEQ ID NO: 55-57, 60-65, 68, 69, 71, 74, 75, 77, 78, 1142-1145, 1468, 1469 and 1471. A peptide of this group is characterized in that it has a TRIA score of at least 10000.
Even more preferably, the peptide of the invention comprises or consists of a peptide selected from the group consisting of SEQ ID NO: 52, 53, 55, 57, 60, 61, 63, 64, 68, 69, 71, 72, 75, 77, 78, 1142-1145, 1468, 1469 and 1471. A peptide of this group is characterized in that it comprises at least 95 predicted CTL epitopes, at least 25 predicted Th-cell epitopes. Preferably, a peptide of this group comprises at least 3 proteasomal cleavage sites. Preferably, the peptide of the invention comprises or consists of a peptide selected from the group consisting of SEQ ID NO: 55, 60, 63, 64, 68, 71, 75, 77, 1142, 1469.
Most preferably, the peptide of the invention comprises or consists of a peptide selected from the group consisting of SEQ ID NO: 63, 75, 1143-1145, 1468 and 1469. A peptide of this group is characterized in that it has a TRIA score of at least 14000. Furthermore, a peptide of this group is characterized in that it comprises at least 125 predicted CTL epitopes, at least 50 predicted Th-cell epitopes. Preferably, a peptide of this group comprises at least 3 proteasomal cleavage sites.
A “T-cell epitope” is defined herein as a linear fragment of a polypeptide antigen, which is recognized and bound by a T-cell receptor, preferably a human T-cell receptor, after being made accessible to a T-cell receptor by intracellular proteolytic processing of the polypeptide antigen and subsequent presentation by an HLA class I or HLA class II molecule on the cell surface of an antigen-presenting cell. A “predicted T-cell epitope” is to be understood herein as a linear fragment of a polypeptide antigen for which liberation from the source protein or peptide by proteolytic cleavage and T-cell receptor recognition and/or binding has been predicted using bioinformatics analyses based on algorithms that predict HLA class I and II peptide binding and C-terminal generation by the proteasome of all possible HLA class I binding peptides (with a length of a HLA class I ligand; 8-12 aa) contained in the HBV proteins. A “confirmed T-cell epitope” is to be understood herein as a linear fragment of a polypeptide antigen for which liberation from the source protein or source polypeptide by proteolytic cleavage and T-cell receptor recognition and/or binding, and more preferably CD4+ or CD8+ T cell activation capability, have been established experimentally as disclosed herein. A “linear fragment” is understood herein to be a contiguous amino acid sequence of a polypeptide antigen, said polypeptide antigen preferably being an HBV protein, more preferably a protein selected from the group consisting of HBV protein polymerase, HBV core protein, HBV X-protein and HBV large surface protein. An identical linear fragment of a polypeptide antigen showing binding affinity to a second or further type of HLA class I or HLA class II molecule is to be understood herein as a second or further T-cell epitope. In other words, a specific linear fragment of a polypeptide antigen being capable to bind to two types of HLA molecules is understood herein to be two separate or distinct T-cell epitopes, and is scored twice within the cumulative BCI Class I- and/or Class II-B score. A T-cell epitope typically comprises or consists of at least 8 amino acids and up to 20 or (exceptionally) even more amino acids. A T-cell epitope can be an HLA class I-restricted CD8+ cytotoxic T cell (CTL) epitope or an HLA class II-restricted CD4+ T-helper (Th-)cell epitope. HLA class I restricted epitopes (also referred to as CTL epitopes) are typically presented via the classical proteasome dependent HLA class I processing route, whereas HLA class-II molecules are typically loaded with linear fragments in the late endosomal compartment. Preferably, a peptide according to the invention comprises T-cell epitopes that are selected from the group consisting of SEQ ID NO: 80-276, 278-314, 316-429, 432-483, 486-545, 548-636, 638-1140, and 1146-1466 (see Tables 4-7). A preferred peptide according to the invention comprises at least 70, 71, 72, 73, 74, 75, 79, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225 or from about 230 to about 233 predicted T-cell epitopes from HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein. A more preferred peptide according to the invention comprises at least 95, 96, 97, 98, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225 or from about 230 to about 233 predicted T-cell epitopes from HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein. An even more preferred peptide according to the invention comprises at least 70, 71, 72, 73, 74, 75, 79, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225 or from about 230 to about 233 predicted T-cell epitopes selected from the group consisting of SEQ ID NO: 80-276, 278-314, 316-429, 432-483, 486-545, 548-636, 638-1140, and 1146-1466. An even more preferred peptide according to the invention comprises at least 95, 96, 97, 98, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225 or from about 230 to about 233 predicted T-cell epitopes selected from the group consisting of SEQ ID NO: 80-276, 278-314, 316-429, 432-483, 486-545, 548-636, 638-1140, and 1146-1466. Preferably, the predicted T-cell epitopes of the present invention are confirmed experimentally as disclosed herein. A preferred peptide according to the invention comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or 70 confirmed T-cell epitopes from HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein. A more preferred peptide according to the invention comprises at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95 confirmed T-cell epitopes from HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein. An even more preferred peptide according to the invention comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or 70 confirmed T-cell epitopes selected from the group consisting of SEQ ID NO: 80-276, 278-314, 316-429, 432-483, 486-545, 548-636, 638-1140, and 1146-1466. An even more preferred peptide according to the invention comprises at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95 confirmed T-cell epitopes selected from the group consisting of SEQ ID NO: 80-276, 278-314, 316-429, 432-483, 486-545, 548-636, 638-1140, and 1146-1466.
A “proteasomal cleavage site” is understood herein as a site in a protein or polypeptide that is cleaved by the proteasome, preferably a human proteasome/proteasome naturally present in a human cell. A specific proteasomal cleavage site liberating the C-terminus of the epitope is preferably present exactly after the C-terminus of the epitope amino acid sequence, in order to allow the epitope's C-terminal residue to be liberated from the larger peptide and to be presented by the HLA class I molecule. The first important event that defines an HLA class I-restricted CD8+ cytotoxic T-cell (CTL) epitope is the release of the epitope (or the epitope-precursor) from its flanking protein regions through enzymatic cleavage by cytosolic peptidases.
The multicatalytic proteasome is the primary enzyme complex required for the generation of the exact C-terminus of the vast majority of CTL epitopes (Rock et al., 2004). Proteasomes are multicatalytic enzyme complexes abundantly present intracellularly and are considered responsible for the generation of the C terminus of the vast majority of CTL epitopes (Craiu et al, 1997; Stoltze et al., 1998; Mo et al., 1999). The generation of the amino-terminus of a CTL epitope, on the other hand, is much more flexible because several amino-terminal exo-peptidases (like ERAP1, puromycin sensitive aminopeptidase, bleomycin hydrolase and others) reside in the cytosol and endoplasmic reticulum and these trimming enzymes have the capacity to shorten an N-terminal elongated epitope-precursor to its precise length. In contrast, C-terminal trimming has not been reported. Therefore the identification of the proteasome-mediated cleavage sites in a protein or in a polypeptide, like a peptide of the invention, can be used as an important identifier of almost every CTL epitope, because the proteasomal cleavages determine and enable C-terminal epitope generation (Kessler et al., 2001; Kessler and Melief, 2007). The assessment of proteasomal cleavage sites in the HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein identifies the C-termini of intracellularly produced HBV peptide fragments, specifically for HLA class I presented peptide fragments. Length requirements are much less strict for loading of HLA class II with peptide fragments. Therefore, precise enzymatic generation of the HLA class II binding peptide fragment is not needed. These T-cell epitope requirements have been used in the present invention to localize and design long peptides derived from the full length sequences of an HBV protein which comprises preferred CD8+ cytotoxic T cell (CTL) and CD4+ T-helper (Th-)cell epitopes and/or combinations thereof and are thus highly immunogenic and therefore suitable peptides for synthesis and (therapeutic) vaccination purposes.
Proteasome mediated proteolytic cleavages can be predicted in silico using a prediction algorithm. Cleavage as performed by the proteasome can be verified in a proteasome mediated cleavage assay as disclosed herein, which measures the C-terminal liberation of the epitope from its flanking regions (Kessler et al., 2001; Kessler and Melief, 2007). A cell free proteasome cleavage assay identifying and quantitatively measuring the amino acid (aa) positions and the abundancy of cleavages by the proteasome in a polypeptide can be used to determine which peptides are generated from the source protein (or source polypeptide), thereby establishing the peptide pool available for epitope generation. The cell free proteasome cleavage assay involves the co-incubation of a polypeptide (preferably having a length of 28-40 aa, more preferably having a length of 30-39 aa) with a preparation of purified proteasomes in an appropriate buffer solution. Two main forms of proteasomes exist, the immunoproteasomes, which are mainly expressed in professional antigen presenting cells, like e.g. Dendritic Cells, and the constitutive proteasomes, which are expressed mainly in other cell types. These types contain variant catalytic subunits with slightly different catalytic activity. Although most epitopes are liberated by both types of proteasomes, sometimes differential epitope generation occurs dependent on proteasome type (Morel et al., 2000; Chapiro et al., 2006). Accordingly, proteasome-mediated cleavage assays may be performed separately with these two proteasome types. Preferably, a constitutive 20S-proteasome or immune 20S-proteasome is used as disclosed herein. The reaction mixture comprising the peptides to be cleaved and either of the two proteasome types (purified proteasome preparations) is incubated at 37° C. and samples are drawn at 1 h, 3 h, 6 h and 24 h time points as detailed in the Examples herein. Subsequently, generated peptide cleavage fragments and the remaining source polypeptide are identified and quantified by mass spectrometry (Kessler et al., 2001). This assay reveals both the positions in the polypeptide (and thus in the source protein) where the proteasome cleaves and the cleavage efficiency (abundancy) at these positions. A cleavage site can be confirmed by detection of fragments containing as COOH terminus the residue NH2-terminal from the cleavage site together with the (possible) complementary fragment(s), as calculated from the intensities of the fragment peaks in the mass spectra (preferably present for >1%, more preferably present for ≥7%, at 24 h incubation), in both the digestion with constitutive proteasomes and the digestion with immunoproteasomes. Preferably, a peptide of the invention comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more, preferably at least 3, proteasomal cleavage site as defined herein. More preferably, a peptide of the invention comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more, preferably at least 3, proteasomal cleavage site as assessed and verified in a proteasomal cleavage assay as described above.
As indicated above, examples of T-cell epitopes are HLA class I-restricted CD8+ cytotoxic T cell (CTL) epitopes and HLA class II-restricted CD4+ T-helper (Th-)cell epitopes. A “CTL epitope” is understood herein as a linear fragment of a polypeptide antigen that is liberated from the source protein by proteasome mediated proteolytic cleavage and subsequently presented by an HLA class I molecule on the cell surface of an antigen presenting cell (APC), preferably a human antigen presenting cell. A “predicted CTL epitope” is understood herein as a linear fragment of a polypeptide antigen for which liberation from its source protein by proteolytic cleavages and HLA class I molecule binding have been predicted using bioinformatics analyses based on algorithms that predict HLA class I peptide binding and C-terminal generation by the proteasome of all HLA class I binding short peptides (with a length of a CTL epitope; 8-12 aa) contained in the HBV proteins. Preferably, a predicted CTL epitope of the present invention is confirmed experimentally as disclosed herein. A CTL epitope of the invention is preferably capable of activating a CD8+ T cell response. A “confirmed CTL epitope” is understood herein as a linear fragment of a polypeptide antigen for which liberation by proteolytic cleavages and HLA class I molecule binding, more preferably CD8+ T cell activation, have been established experimentally as disclosed herein. A CTL epitope of the invention is preferably capable of activating a CD8+ T cell response. A CTL epitope typically comprises at least 8 up to 12, or exceptionally up to 13 or 14 amino acids. Preferably a CTL epitope consists of 8-14 amino acids, i.e. has a length of at least 8 up to 14 amino acids.
A CTL epitope is defined by two important intracellular events being (i) proteasome mediated proteolytic cleavage and (ii) binding to an HLA class I molecule, which takes place in the endoplasmic reticulum (ER). The stronger a linear peptide fragment binds and the slower the off-rate, the more likely that this linear peptide fragment will become a cell surface presented immunogenic CTL epitope (Van der Burg et al., 1996). Analysis of proteasome mediated proteolytic cleavages can be performed as indicated above. Preferably, specific binding to an HLA class I molecule is predicted using an in silico prediction algorithm and established by using an HLA class I peptide binding assay as known by the person skilled in the art (Kessler and Melief, 2007; Kessler et al., 2003). Preferably, the HLA class I-restricted epitope in a long peptide according to the invention is predicted to be generated at its C-terminus by the proteasome and preferably has a predicted high affinity binding capacity for the HLA class I molecule using an assay as described in van der Burg et al., 1995 and Kessler et al., 2003; e.g. IC50≤about 5 μM may be considered high affinity binding, about 5 μM<IC50≤about 15 μM may be considered intermediate affinity binding, about 15 μM<IC50≤100 μM may be considered low affinity binding and IC50>about 100 μM may be considered as no binding. To measure class I binding affinity of a peptide or fragments thereof, various HLA class I binding assays are available. The assays can be divided into cell-free assays (using soluble HLA) versus cellular assays (using HLA class I molecules on the cell surface), and competitive assays (resulting in semi-quantitative data) versus assays that do not use a labeled reference peptide and are therefore quantitative (Kessler and Melief, 2007; Viatte et al., 2006). The assays have in common that the HLA class I peptide binding affinity is reliably assessed.
The actual presentation of a CTL epitope on the cell surface, i.e. the net result of both proteasomal cleavage, possible other proteolytic events like N-terminal trimming, and binding and presentation by an HLA class I molecule, which events together define a CTL epitope as indicated above, can be demonstrated by a biochemical approach or by a functional approach using cytotoxic T cells with a T-cell receptor specific for the epitope and HLA class I molecule (geno)type, as known by the person skilled in the art (Kessler and Melief, 2007).
The biochemical approach involves the biochemical purification of HLA-epitope complexes from cells expressing the HBV antigen of the invention together with the presenting HLA class I molecule (geno)type, followed by the mass spectrometric search for the epitope in the eluted HLA class I-bound CTL receptor ligands as known by the person skilled in the art (Schirle et al., 2000; Schirle et al., 2001).
The functional approach involves a CTL line or clone that is specifically recognizing the HLA-epitope, which is used as a tool to demonstrate the natural processing and actual presentation of the epitope by HLA class I molecules. In this methodology, using a CTL induction assay as known in the art, either the synthetically generated minimal (i.e. exact length) epitope or the peptide sequence of interest encompassing the epitope, for instance a peptide, long peptides and/or SLP as defined herein, is used to stimulate and select HLA-epitope-specific cytotoxic T cells. To that end, briefly, a multivalent CD8+ T cell population, or a multivalent mixed CD8+ and CD4+ T cell population, is stimulated with autologous target cells of which the HLA class I molecules on the cell surface are either exogenously loaded with the precise synthetic epitope or endogenously loaded with intracellularly generated CTL epitopes derived from the exogenously loaded long peptide of the invention after its uptake by the antigen presenting target cells. In case the autologous target cells are loaded with a peptide, e.g. the synthetic long peptide of the invention, or fragments thereof, encompassing the epitope, the epitope is generated after cellular uptake of the peptide and its intracellular processing by the proteasome together with other N-terminal trimming peptidases. Subsequently, using a T cell recognition assay, the HLA-epitope-specific CTL is used to demonstrate the intracellular generation and natural presentation of the epitope of the invention by HLA class I molecules on the surface of HBV-infected cells. Specific recognition of an HLA class I restricted epitope by a CTL demonstrates the cell surface expression of the epitope and reveals its immunogenicity, i.e. the presence of epitope-specific T cells in the (T-cell receptor) repertoire of a selected donor. Preferably, the CD8+ T cell activating capability has been demonstrated ex vivo and/or in vivo, in T cells from human healthy control individuals or even more preferably in T cells from a human patient with an HBV related disease or condition and/or from a healthy control. The activation is preferably assessed ex vivo or in vivo, more preferably in a human patient with an HBV related disease. A CTL epitope for which liberation by proteolytic cleavage and HLA class I molecules presentation, or preferably CD8+ T cell activating capability, has been demonstrated experimentally is denominated herein as a confirmed CTL epitope.
A peptide of the invention preferably comprises at least 70, 71, 72, 73, 74, 75, 79, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230 and to up to 233 predicted CTL epitopes as defined herein. Preferably, a peptide of the invention comprises at least 70, 71, 72, 73, 74, 75, 79, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230 and to up to 233 predicted CTL epitopes as defined herein. Preferably, a peptide according to the invention comprises at least 70, 71, 72, 73, 74, 75, 79, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230 and to up to 233 predicted CTL epitopes from the HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein. More preferably a peptide according to the invention comprises at least 70, 71, 72, 73, 74, 75, 79, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230 and to up to 233 predicted CTL epitopes from the HBV core protein, HBV polymerase, HBV X protein or HBV large surface protein. Even more preferably, a peptide according to the invention comprises or consists of a contiguous amino acid sequence of any of the proteins selected from the group consisting of HBV core protein, HBV polymerase, HBV X protein and HBV large surface protein, wherein said contiguous amino acid sequence comprises at least 70, 71, 72, 73, 74, 75, 79, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230 and to up to 233 predicted CTL epitopes. Preferably, a peptide according to the invention comprises at least 70, 71, 72, 73, 74, 75, 79, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230 and to up to 233 CTL predicted epitopes selected from the group consisting of SEQ ID NO: 80-276, 278-314, 316-429, 432-483, 486-545, 548-636, 638-685; 846-923, 959-1090, and 1146-1395 (see Tables 4a, 5a, 6a and 7a). Even more preferably, a peptide of the invention preferably comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, or preferably at least 95 confirmed CTL epitopes as defined herein and verified using a biochemical or functional assay as described above. Most preferred is a peptide of the invention that comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or 70 or preferably at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95 confirmed CTL epitopes as defined herein and verified using a functional assay as described above. Preferably, a peptide of the invention comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or 70 or preferably at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95 confirmed CTL epitopes selected from the group consisting of SEQ ID NO: 80-276, 278-314, 316-429, 432-483, 486-545, 548-636, 638-685; 846-923, 959-1090, and 1146-1395.
A “Th-cell epitope” is understood herein to be a linear peptide fragment that is recognized by an HLA class II molecule. A “predicted Th-cell epitope” is understood herein as a linear fragment of a polypeptide antigen for which HLA class II molecule recognition has been predicted using sophisticated bioinformatics analyses that are experimentally underpinned. Preferably, a predicted Th-cell epitope of the present invention is confirmed experimentally as disclosed herein. A Th-cell epitope of the invention is preferably capable of inducing a CD4+ T cell response. A “confirmed Th-cell epitope” is understood herein as a linear fragment of a polypeptide antigen for which HLA class II molecule recognition has been established experimentally as known by the person skilled in the art and further detailed herein.
An HLA class II-restricted CD4+ T-helper cell (Th-cell) epitope typically comprises 15 up to 20, or exceptionally even more, amino acids. Preferably, an HLA class II-restricted T-helper cell epitope comprises or 10-20 or 10-15 amino acids. Specific recognition of a predicted HBV-derived Th-cell epitope can be tested and/or verified in a Th-cell induction assay. To this end the peptide or fragment thereof, long peptide and/or SLP sequence of interest comprising the predicted Th-cell epitope is exogenously loaded on the surface of target cells and subsequently these peptide-loaded target cells are co-incubated with a multivalent autologous T helper cell population. After several rounds of stimulation, epitope-specific T helper cells can be selected and can be back-tested for the recognition of the T helper cell epitope contained in the peptide or SLP thereby proving its natural cell surface presentation. Preferably, an HLA class II-restricted CD4+ T-helper cell epitope comprised in a peptide according to the invention is capable of inducing or activating a CD4+ T-helper cell in a human patient with an HBV related disease or condition. The induction or activation is preferably assessed ex vivo or in vivo, more preferably in a human patient with an HBV related disease. Most preferably, the HLA class II-restricted epitope is capable of activating a CD4+ T-helper memory and/or CD4+ T-helper effector response, i.e. activation of a CD45RO-positive CD4+ T-helper cell. This will lead, by virtue of the ‘license to kill’ signal through CD40-triggering of DC (Lanzavecchia, 1998) to a more robust CD8+ effector and memory cytotoxic T cell response. In another setting the activated CD4+ T-helper cells may activate non-HLA restricted killer cells of the immune system. A Th-cell epitope for which recognition by an HLA class II molecule, or preferably CD4+ activating capability, has been demonstrated experimentally is denominated herein as a confirmed Th-cell epitope.
Preferably, a peptide according to the invention comprises at least one predicted Th-cell epitope from HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein. Preferably, a peptide of the invention comprises at least 1, 2, 3, 4, 5, 6, 8, 7, 8, 9, 10 or preferably at least 15 predicted Th-cell epitope(s) as defined herein. Preferably, a peptide according to the invention comprises at least 1, 2, 3, 4, 5, 6, 8, 7, 8, 9, 10, or preferably at least 15 predicted Th-cell epitope(s) from the HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein. Even more preferably, a peptide according to the invention comprises or consists of a contiguous amino acid sequence of any of the proteins selected from the group consisting of HBV core protein, HBV polymerase, HBV X protein and HBV large surface protein, wherein said contiguous amino acid sequence comprises at least 1, 2, 3, 4, 5, 6, 8, 7, 8, 9, 10, or preferably at least 15 predicted Th-cell epitope(s). Preferably, a peptide according to the invention comprises at least 1, 2, 3, 4, 5, 6, 8, 7, 8, 9, 10, or preferably at least 15 predicted Th-cell epitope(s) selected from the group consisting of SEQ ID NO: 686-845; 924-958, 1091-1140, and 1396-1466 (see Tables 4b, 5b, 6b, and 7b). More preferably, a peptide of the invention comprises at least 1, 2, 3, 4, 5, 6, 8, 7, 8, 9, 10, or preferably at least 15 confirmed Th-cell epitope(s) as defined herein. Even more preferably, a peptide of the invention comprises at least 1, 2, 3, 4, 5, 6, 8, 7, 8, 9, 10, or preferably at least 15 confirmed Th-cell epitope(s) as defined herein and verified using an T helper cell induction assay as described above. Preferably, a peptide of the invention comprises at least 1, 2, 3, 4, 5, 6, 8, 7, 8, 9, 10, or preferably at least 15 confirmed Th-cell epitope selected from the group consisting of SEQ ID NO: 686-845; 924-958, 1091-1140, and 1396-1466.
Preferably, a peptide according to the invention comprises both at least 70 predicted CTL epitopes and at least one predicted Th-cell epitope from the HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein. More preferably, a peptide according to the invention is a peptide derived from the HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein, preferably is a fragment of the HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein, that comprises at least 70 predicted CTL epitopes, at least one predicted Th-cell epitope and at least 3 proteasomal cleavage sites. The presence of at least 3 proteasomal cleavage sites, at least 70 predicted CTL epitopes and at least 1 Th epitope within a single peptide according to the invention, being a continuous amino acids fragment of an antigen protein of interest, has been observed to be particularly advantageous due to synergy between the Th response and the CTL response in mounting and maintaining an effective CD8+ cytotoxic T cell response. Several published studies have demonstrated that CD4+ T-helper cells upon interaction with HLA class II epitope presenting dendritic cells (DC) upregulate CD40 ligand. The interaction of the Th-cell by its CD40 ligand with the CD40 molecule on the DC leads to activation of the DC. Activated DCs display upregulated costimulatory molecules and secrete CTL-promoting cytokines. This allows both a more robust CD8+ CTL response induced by such an activated DC that presents HLA class I restricted epitopes and a much more robust CTL memory response (Ridge et al., 1998; Schoenberger et al., 1998; Sun et al., 2004). The need for CD40 expression on DC for robust CD8+ CTL responses following vaccination with synthetic long peptides (length of 35 aa.) has been demonstrated in Zwaveling et al. (2002).
Accordingly, a preferred peptide according to the invention comprises at least 70 predicted CTL epitopes and at least one predicted Th-cell epitope; preferably at least 70 predicted CTL epitopes selected from the group consisting of SEQ ID NO: 80-276, 278-314, 316-429, 432-483, 486-545, 548-636, 638-685; 846-923, 959-1090, and 1146-1395 and at least one predicted Th-cell epitope selected from the group consisting of SEQ ID NO: 686-845; 924-958, 1091-1140, and 1396-1466. A more preferred peptide according to the invention comprises at least 70 predicted CTL epitopes and at least 15 predicted Th-cell epitopes; preferably at least 70 predicted CTL epitopes selected from the group consisting of SEQ ID NO: 80-276, 278-314, 316-429, 432-483, 486-545, 548-636, 638-685; 846-923, 959-1090, and 1146-1395 and at least 15 predicted Th-cell epitopes selected from the group consisting of SEQ ID NO: 686-845; 924-95, 1091-1140, and 1396-1466. More preferably, a peptide according to the invention comprises at least 71, 72, 73, 74, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170 up to 175 predicted CTL epitopes selected from the group consisting of SEQ ID NO: 80-276, 278-314, 316-429, 432-483, 486-545, 548-636, 638-685; 846-923, 959-1090, and 1146-1395 and at least 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, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 up to 96 predicted Th-cell epitopes selected from the group consisting of SEQ ID NO: 686-845; 924-958, 1091-1140, and 1396-1466. Even more preferably, a peptide according to the invention comprises at least 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170 up to 175 predicted CTL epitopes selected from the group consisting of SEQ ID NO: 80-276, 278-314, 316-429, 432-483, 486-545, 548-636, 638-685; 846-923, 959-1090, and 1146-1395 and at least 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, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 up to 96 predicted Th-cell epitopes selected from the group consisting of SEQ ID NO: 686-845; 924-958, 1091-1140, and 1396-1466. Preferably, a peptide according to the invention comprises at least 95 predicted CTL epitopes as defined herein and at least 25 predicted Th-cell epitopes as defined herein.
More preferably, a preferred peptide according to the invention comprises at least 5 confirmed CTL epitopes and at least one confirmed Th-cell epitope; preferably at least 5 confirmed CTL epitopes selected from the group consisting of SEQ ID NO: 80-276, 278-314, 316-429, 432-483, 486-545, 548-636, 638-685; 846-923, 959-1090, and 1146-1395 and at least one confirmed Th-cell epitope selected from the group consisting of SEQ ID NO: 686-845; 924-958, 1091-1140, and 1396-1466. A more preferred peptide according to the invention comprises at least 15 confirmed CTL epitopes and at least one confirmed Th-cell epitope; preferably at least 15 confirmed CTL epitopes selected from the group consisting of SEQ ID NO: 80-276, 278-314, 316-429, 432-483, 486-545, 548-636, 638-685; 846-923, 959-1090, and 1146-1395 and at least one confirmed Th-cell epitope selected from the group consisting of SEQ ID NO: 686-845; 924-95, 1091-1140, and 1396-1466. More preferably, a peptide according to the invention comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or 70 confirmed CTL epitopes selected from the group consisting of SEQ ID NO: 80-276, 278-314, 316-429, 432-483, 486-545, 548-636, 638-685; 846-923, 959-1090, and 1146-1395 and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 confirmed Th-cell epitopes selected from the group consisting of SEQ ID NO: 686-845; 924-958, 1091-1140, and 1396-1466. Even more preferably, a peptide according to the invention comprises at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95 confirmed CTL epitopes selected from the group consisting of SEQ ID NO: 80-276, 278-314, 316-429, 432-483, 486-545, 548-636, 638-685; 846-923, 959-1090, and 1146-1395 and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 confirmed Th-cell epitopes selected from the group consisting of SEQ ID NO: 686-845; 924-958, 1091-1140, and 1396-1466. Preferably, a peptide according to the invention comprises at least 15 confirmed CTL epitopes as defined herein and at least 5 confirmed Th-cell epitopes as defined herein.
The HLA class I epitopes in the peptides according to the invention are preferably capable of being presented on HLA molecules being encoded by HLA alleles that are predominant in the population of human subjects to be treated. Preferred HLA class I epitopes in peptides according to the invention are epitopes capable of binding to: HLA-A0101; HLA-A0201; HLA-A0206; HLA-A0301; HLA-A1101; HLA-A2301; HLA-A2402; HLA-A2501; HLA-A2601; HLA-A2902; HLA-A3001; HLA-A3002; HLA-A3101; HLA-A3201; HLA-A3303; HLA-A6801; HLA-A6802; HLA-A7401; HLA-B0702; HLA-B0801; HLA-B1301; HLA-B1302; HLA-B1402; HLA-B1501; HLA-B1502; HLA-B1525; HLA-B1801; HLA-B2702; HLA-B2705; HLA-B3501; HLA-B3503; HLA-B3701; HLA-B3801; HLA-B3901; HLA-B4001; HLA-B4002; HLA-B4402; HLA-B4403; HLA-B4601; HLA-B4801; HLA-B4901; HLA-B5001; HLA-B5101; HLA-B5201; HLA-B5301; HLA-B5501; HLA-B5601; HLA-B5701; HLA-B5801 and HLA-B5802. In a preferred embodiment, a peptide of the invention, covers at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the HLA class I molecules that are encoded by HLA alleles predominant in the population of human subjects to be treated, wherein “Cover an HLA class I molecule” is understood herein as comprising a CTL epitope that shows binding affinity, preferably intermediate binding affinity, more preferably high binding affinity to said HLA class I molecule. Preferably, a peptide of the invention covers at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of group of HLA class I molecules consisting of: HLA-A0101; HLA-A0201; HLA-A0206; HLA-A0301; HLA-A1101; HLA-A2301; HLA-A2402; HLA-A2501; HLA-A2601; HLA-A2902; HLA-A3001; HLA-A3002; HLA-A3101; HLA-A3201; HLA-A3303; HLA-A6801; HLA-A6802; HLA-A7401; HLA-B0702; HLA-B0801; HLA-B1301; HLA-B1302; HLA-B1402; HLA-B1501; HLA-B1502; HLA-B1525; HLA-B1801; HLA-B2702; HLA-B2705; HLA-B3501; HLA-B3503; HLA-B3701; HLA-B3801; HLA-B3901; HLA-B4001; HLA-B4002; HLA-B4402; HLA-B4403; HLA-B4601; HLA-B4801; HLA-B4901; HLA-B5001; HLA-B5101; HLA-B5201; HLA-B5301; HLA-B5501; HLA-B5601; HLA-B5701; HLA-B5801 and HLA-B5802.
The HBV genome (SEQ ID NO: 3; see Table 1) consists of a partially double-stranded circular DNA molecule having four overlapping open reading frames (ORFs) that are responsible for the transcription and expression of seven different hepatitis B proteins through the use of multiple in-frame start codons. The HBV proteins are the core protein and the e antigen (HBeAg) encoded by the C gene, the HBV polymerase encoded by the P gene, the viral surface proteins (small (S), middle (M), and large (L)) encoded by the S gene, and X protein encoded by the X gene. There is an outer shell (or envelope) composed of several proteins known collectively as HBs or surface Proteins. This outer shell is frequently referred to as the surface coat. The outer surface coat surrounds an inner protein shell, composed of HBc protein. This inner shell is referred to as the core particle or capsid. Finally the core particle surrounds the viral DNA and the enzyme DNA polymerase.
The HBV core protein is the major component of the viral nucleocapsid. The amino acid sequences of the HBV polymerase, HBV core protein, HBV X protein and HBV large surface protein are represented by SEQ ID NO: 1, 4, 45 and 1141 respectively (see Table 1).
A preferred amino acid sequence of a human HBV polymerase protein is a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity with the sequence depicted in SEQ ID NO: 1; a preferred coding sequence is a sequence having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity with the sequence depicted in SEQ ID NO: 2. A preferred amino acid sequence of an HBV core protein is a sequence having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity with the sequence depicted in SEQ ID NO: 4; a preferred coding sequence is a sequence having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity with the sequence depicted in SEQ ID NO: 5. A preferred amino acid sequence of an HBV large surface protein is a sequence having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity with the sequence depicted in SEQ ID NO: 1141, a preferred coding sequence is a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity with the sequence depicted in SEQ ID NO: 1467.
The full length consensus amino acid sequence of the X protein was obtained by deducing the optimal sequence from the 39 published and reviewed full length (154 amino acid) HBV X protein amino acid sequences in the UniProt database (at: www.uniprot.org). These 39 sequences were first aligned and subsequently for each aa position the most frequently occurring aa was selected for that position in the consensus sequence. The 39 sequences with the following entries were included in the analysis: P69713; P03165; P0C686; P69714; Q8JMY5; Q69604; Q05499; O91531; Q9PX75; P20976; P20975; P20977; P24026; Q9PXA2; Q67923; P0C685; P0C678; O93195; Q9E6S8; P12936; Q91C38; Q913A9; Q8JMY3; Q8JN06; Q8JMZ5; Q69607; Q91B15; Q801U5; Q4R1S9; Q4R1S1; Q9YZR6; P0C687; Q9QMI3; P0C681; Q801U8; Q99HR6; P17102; Q67877; and Q69027 (see Table 2). A preferred consensus amino acid sequence of a human HBV X protein is a sequence having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity with the sequence depicted in SEQ ID NO: 45. The consensus amino acid sequence may be encoded by any coding sequence known or designed; the person skilled in the art knows how to design a coding sequence from a known amino acid sequence; such coding sequence may be a codon-optimized sequence. The terms “HBV X protein” and “consensus HBV X protein” are used interchangeably herein.
Percentage of identity is herein determined by calculating the ratio of the number of identical nucleotides/amino acids in the sequence divided by the length of the total nucleotides/amino acids of said sequence, minus the lengths of any gaps. Identity with a given SEQ ID NO means identity based on the full length of said sequence (i.e. over its whole length or as a whole).
Within the context of the present invention, “a peptide derived from an HBV protein” means that the peptide comprises at least 15 and at most 100 consecutive amino acids originating from the HBV core protein, HBV polymerase, HBV X consensus protein and/or HBV large surface protein. In other words, “a peptide derived from the HBV polymerase protein” comprises at most 100 consecutive amino acids of SEQ ID NO: 1, “a peptide derived from the HBV core protein” comprises at most 100 consecutive amino acids of SEQ ID NO: 4, “a peptide derived from the HBV X consensus protein” comprises at most 100 consecutive amino acids of SEQ ID NO: 45, and “a peptide derived from the HBV large surface protein” comprises at most 100 consecutive amino acids of SEQ ID NO: 1141. Preferably, “a peptide derived from the HBV polymerase protein” consists of at most 100 consecutive amino acids of SEQ ID NO: 1, “a peptide derived from the HBV core protein” consists of at most 100 consecutive amino acids of SEQ ID NO: 4, “a peptide derived from the HBV X consensus protein” consists of at most 100 consecutive amino acids of SEQ ID NO: 45, and “a peptide derived from the HBV large surface protein” consists of at most 100 consecutive amino acids of SEQ ID NO: 1141. Therefore, by definition, a peptide according to the invention is distinct from a full length HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein, as these full length proteins are all longer than 100 amino acids. Preferably, the peptide of the present invention is from about 15 to about 100 amino acids in length. More preferably the length of the peptide is from 15 up to 100 length indicated herein as the length of the peptide is 15-100 amino acids, or preferably the length of the peptide is 15-95 amino acids, or 15-90 amino acids, or 15-85 amino acids, or 15-70 amino acids, or 15-65 amino acids, or 15-60 amino acids, or 15-55 amino acids, or 15-50 amino acids, even more preferably 15-45 amino acids, even more preferably, 15-40 amino acids, even more preferably 17-39, even more preferably 19-43 amino acids, even more preferably 22-40 amino acids, even more preferably 28-40 and even more preferably 30-39 amino acids. Within the context of the present invention “a peptide which comprises at most 100 amino acids derived from an HBV protein” preferably means that the number of consecutive amino acids originating from an HBV protein, preferably being a protein selected from the group consisting of HBV core protein, HBV polymerase, HBV X consensus protein and HBV large surface protein and present in a peptide as defined herein, is 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, or 30 amino acids or less. Within the context of the present invention “a peptide which comprises at least 15 amino acids derived from an HBV protein” preferably means that the number of consecutive amino acids originating from a protein selected from the group consisting of HBV core protein, HBV polymerase, HBV X consensus protein and HBV large surface protein and present in a peptide as defined herein, is at least 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 or 45 amino acids. Within the context of the present invention “a peptide which comprises 15-100 amino acids derived from an HBV protein” preferably means that the number of consecutive amino acids originating from a protein selected from the group consisting of HBV core protein, HBV polymerase, HBV X consensus protein and HBV large surface protein and present in a peptide as defined herein, is at least 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 or 45 amino acids and no more than 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, or 30 amino acids. Within the context of the present invention “a peptide which comprises 15-100 amino acids derived from an HBV protein” preferably means that the number of consecutive amino acids originating from a protein selected from the group consisting of SEQ ID NO: 1, 4, 45 and 1141 and present in a peptide as defined herein, is at least 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 or 45 amino acids and no more than about 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, or 30 amino acids. More preferably, the length of the contiguous amino acid sequence from HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein comprised within the peptide is 15-100 amino acids, or preferably 15-95 amino acids, or 15-90 amino acids, or 15-85 amino acids, or 15-70 amino acids, or 15-65 amino acids, or 15-60 amino acids, or 15-55 amino acids, or 15-50 amino acids, even more preferably 15-45 amino acids, even more preferably, 15-40 amino acids, even more preferably 17-39, even more preferably 19-43 amino acids, even more preferably 22-40 amino acids, even more preferably 28-40 and even more preferably 30-39 amino acids. Even more preferably, the length of the contiguous amino acid sequence from the sequences selected from the group consisting of SEQ ID NO: 1, 4, 45 and 1141 comprised within the peptide is 15-100 amino acids, or preferably 15-95 amino acids, or 15-90 amino acids, or 15-85 amino acids, or 15-70 amino acids, or 15-65 amino acids, or 15-60 amino acids, or 15-55 amino acids, or 15-50 amino acids, even more preferably 15-45 amino acids, even more preferably, 15-40 amino acids, even more preferably 17-39, even more preferably 19-43 amino acids, even more preferably 22-40 amino acids, even more preferably 28-40 and even more preferably 30-39 amino acids. A peptide according to the invention may comprise additional amino acids than the ones originating from an HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein or may entirely be made of or consist of an amino acid sequence originating from a protein selected from the group consisting of HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein. A peptide according to the invention may comprise several parts of non-contiguous amino acid sequences from an HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein, wherein it is to be understood that said peptide has a length, a TRIA score and/or amount and type of T cell epitopes as defined herein.
According to one embodiment, a peptide according to the invention consists of any of the contiguous amino acid sequence from HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein as defined herein and indicated by its representing SEQ ID NO, whereby it is understood that no amino acids are appended to either end of the said peptide.
According to another embodiment, the peptide according to the invention comprises any of the contiguous amino acid sequences from HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein as defined herein and indicated by its representing SEQ ID NO and further may comprise a modified amino acid and/or a covalently linked functional group such as a fluorinated group, a human toll-like receptor ligand and/or agonist, an oligonucleotide conjugate, PSA, a sugar chains or glycan, a pam3cys and/or derivative thereof, preferably a pam3cys lipopeptide or variant or derivative thereof, preferably such as described in WO2013051936A1, CpG oligodeoxynucleotides (CpG-ODNs), Cyclic dinucleotides (CDNs), 2-aminoisobutyric acid (Abu), Muramyl dipeptide (MDP), a DC pulse cassette, a tetanus toxin derived peptide.
In an embodiment, the peptide of the invention comprises or consists of a non-naturally occurring sequence as a result of the synthesis of non-natural lengths or as a result of comprising additional amino acids not originating from an HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein or as a result of comprising non-contiguous amino acid sequences from an HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein, and/or as a result of comprising a modified amino acid and/or a non-naturally occurring amino acid and/or a covalently linked functional group such as a fluorinated group, a fluorcarbon group, a human toll-like receptor ligand and/or agonist, an oligonucleotide conjugate, PSA, a sugar chains or glycan, a pam3cys and/or derivative thereof preferably such as described in WO2013051936A1, CpG oligodeoxynucleotides (CpG-ODNs), Cyclic dinucleotides (CDNs), a DC pulse cassette, a tetanus toxin derived peptide, a human HMGB1 derived peptide; either within the peptide or appended to the peptide, as indicated above. The peptide of the invention may comprise 2-aminoisobutyric acid (Abu, an isostereomer of cysteine). A cysteine of the peptide of the invention may be replaced by Abu. Encompassed within the present invention is a peptide of SEQ ID NO: 77, wherein the N-terminal cysteine has been replaced by Abu.
Preferably, a peptide of the invention is an isolated peptide, wherein “isolated” does not reflect the extent to which the peptide is purified, but indicates that the peptide has been removed from its natural milieu (i.e., that has been subject to human manupilation), and may be a recombinantly produced peptide or a synthetically produced peptide.
Preferably, the invention relates to a peptide that can be effectively used in the prevention, partial clearance and/or treatment or full clearance of a HBV related disease or condition in a subject, preferably as detectable by:
In all embodiments of the present invention, a subject is preferably a mammal, more preferably a human. A subject may be an animal model, preferably a non-human mammalian model with humanized HLA class I and class II molecules, or a mammalian, preferably human, organ, such as a liver.
In all embodiments of the present invention, the term “HBV related disease or condition” is preferably defined as acute HBV infection, chronic HBV infection and other conditions where the hepatitis virus is found in the blood or body fluids containing blood of a subject, such as liver cirrhosis and liver cancer, or optionally of an asymptomatic subject that is characterized by the presence of the virus in the body of said subject.
In the context of the invention, a patient may survive and may be considered as being disease free as a consequence of treatment according to the invention. Alternatively, the disease or condition may have been stopped or regressed (i.e. cleared or partially cleared infection). A significant increase of HBV-specific activated CD4+ or CD8+ cells in peripheral blood at least one week after vaccination is preferably at least a 5%, 10%, 20%, 30% increase or more. An inhibition of the proliferation of HBV infected cells is preferably at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% inhibition or more. An induction of HBV infected cell death may be at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, or more. HBV infection may be inhibited at least 5%, 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70% or 75% induction, or more. HBV infected cells may be reduced by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 100%.
In each embodiment, within this or further aspects disclosed herein, wherein the effect of a peptide according to the invention, a composition according to the invention, a polynucleotide according to the invention, a viral vector comprising a polynucleotide according to the invention and/or a cell according to the invention and/or a cell obtained or obtainable by a method according to the invention, is quantified, the assay may be carried out by comparison to a subject not treated or to the same subject before treatment.
Acute and chronic HBV infection can be treated using the present invention. A peptide according to the invention comprising epitopes which are to be presented to T-cell receptors of CD8+ cytotoxic T cells and/or CD4+ T-helper cells preferably fulfill a number of structural requirements as defined herein. In vitro and ex vivo T cell experiments are preferably used to confirm the capability of peptides according to the invention to induce substantial CD4+ T-helper and CD8+ cytotoxic T cell responses. The peptides of the present invention thereby provide a marked improvement in the selection of relatively short peptides that may be chemically synthesized, comprising the most potent and most widely applicable HLA class I and/or class II presented T cell epitopes derived from HBV.
In an embodiment, a peptide is distinct from a contiguous sequence of amino acids of HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein.
A peptide according to the invention comprising a T-cell epitope from HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein, may be modified by deletion or substitution of one or more amino acids, by extension at the N- and/or C-terminus with additional amino acids or functional groups, which may improve bio-availability, targeting to T-cells, or comprise or release immune modulating substances that provide adjuvant or (co) stimulatory functions. The optional additional amino acids at the N- and/or C-terminus are preferably not present in the corresponding positions in the native amino acid sequence of HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein.
A peptide according to the invention comprising a T-cell epitope is obtainable by chemical synthesis and subsequent purification according to methods well-known in the art. (see e.g. Atherton et al., 1989; Barany et al., 1979; Fields et al., 1997). A peptide according to the invention is preferably soluble in physiologically acceptable watery solutions (e.g. PBS) comprising no more than 35, 20, 10, 5 or 0% DMSO. In such a solution the peptide according to the invention is preferably soluble at a concentration of at least 0.5, 1, 2, 4, or 8 mg peptide per ml. More preferably, a mixture of more than one different peptide according to the invention is soluble at a concentration of at least 0.5, 1, 2, 4, or 8 mg peptide per ml in such solutions.
The peptides according to the invention may be easily synthesized and are large enough to be taken up by professional antigen presenting cells, in particular dendritic cells (DC), processed by the proteasome and/or the endosomal/lysosomal degradation and antigen processing system and preferably have sufficient length to contain at least 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 73, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170 to preferably up to 175 CTL epitopes and/or at least 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 to preferably up to 96 Th-cell epitopes as defined herein. Optionally, a peptide according to the invention may comprise N- or C-terminal extensions, which may be amino acids, modified amino acids or other functional groups that may for instance enhance bio-availability, cellular uptake, processing and/or solubility.
Preferably, a peptide according to the invention is a peptide comprising a peptide with an amino acid sequence selected from the group consisting of:
wherein the length of the preferably contiguous amino acid sequence is preferably at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33 amino acids and/or preferably no more than 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15 amino acids, most preferably a length of 30-39 amino acids.
In a second aspect, the invention provides a polynucleotide encoding a peptide according to the invention, preferably a peptide as defined herein above. A polynucleotide may be any polynucleotide comprising e.g. RNA, DNA, and/or cDNA; a polynucleotide may be single stranded or double stranded and may comprise nucleotide analogues and/or nucleotide equivalents such as a peptide nucleic acid (PNA) and a morpholino nucleotide analogue. A polynucleotide may be codon optimized for a host of choice to facilitate expression of the encoded subject matter.
The polynucleotide according to the invention does not encode a wild-type full length HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein, but rather encode a peptide according to the invention as such, or flanked by amino acid sequence that are not contiguous with a wild-type HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein. Such flanking amino acids may be from proteins other than a wild-type HBV and/or they may be from other locations within a wild-type HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein that are not contiguous with the peptide they flank. Preferably, the polynucleotide encodes two or more peptides according to the invention arranged as beads-on-string, whereby the peptides according to the invention (the beads) are linked directly together and/or are linked through linker sequences that are from proteins other than a wild-type HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein, and/or from other locations within a wild-type HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein, that are not contiguous with the peptide they flank. The amino acid sequences flanking or linking the peptides may comprise proteolytic cleavage sites. A polynucleotide according to the invention may be applied to deliver a peptide according to the invention in various ways. A polynucleotide according to the invention may e.g. be used in the production of recombinant protein or peptide in a suitable host cell (e.g. a bacterial host cell such as E. coli, a suitable yeast host cell such as S. cerevisiae, a suitable filamentous fungal such as an Aspergillus or mammalian host cell) from which the recombinant protein or peptide may be purified. Alternatively the polynucleotide may be operably linked to expression regulatory sequences (promoters and the like) and incorporated in an expression construct for human cells. Such (autologous) cells may be transfected or transduced ex vivo to be (re)-administered to a subject in need thereof. Alternatively such expression construct according to the invention may be incorporated into a suitable gene therapy vector. Viral vectors (based on a defective virus) are more efficient agents for gene transfer as compared to non-viral agents. Suitable viral expression constructs include e.g. vectors that are based on adenovirus, adeno-associated virus (AAV), retroviruses or modified vaccinia Ankara (MVA). The polynucleotide according to the invention may also be operably linked to a sequence encoding and adjuvant such as a Toll-like receptor (TLR) ligand, a NOD ligand, or a RIG-I ligand.
In a third aspect, the present invention provides a cell comprising the polynucleotide according to the second aspect of the invention. Such cell can be used for e.g. production of a peptide according to the invention or for medical purposes such as prevention and/or treatment of an HBV related disease as defined elsewhere herein. Said cell may be any host cell. For the specific applications such as described here above, the selection of the host cell may be made according to such use. The host cell may be a prokaryote or may be a eukaryote. A preferred prokaryote cell is E. coli. When the cell is a eukaryote, the cell preferably is a mammalian, insect, plant, fungal, or algal cell. Preferred mammalian cells include e.g. Chinese hamster ovary (CHO) cells, COS cells, 293 cells, PerC6 cells, and antigen presenting cells such as dendritic cells. Preferred insect cells include e.g. Sf9 and Sf21 cells and derivatives thereof. Preferred fungal cells include Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, Yarrowia and filamentous fungal cells. Most preferably, the eukaryotic cell is a human antigen presenting cell, preferably a dendritic cell.
Methods to introduce a polynucleotide into a cell are known to the person skilled in the art. When expression of the polynucleotide is desired, the person skilled in the art knows how to achieve such; the polynucleotide may e.g. be provided with proper control sequences such as a promoter and terminator sequence and may be inserted into a proper vector such as a plasmid or a method described in the second aspect of the invention may be used.
The present invention also provides for an antigen presenting cell such as a dendritic cell as defined earlier herein that has been contacted and/or loaded with a peptide according to the invention, preferably a peptide according to the first aspect of the present invention. Such preferably autologous dendritic cell may be used for immune therapeutic treatment of a subject in need thereof. Such dendritic cell can be isolated from the subject, loaded with at least one peptide according to the invention and used for treatment.
In a fourth aspect, the present invention provides a method for the preparation of an HBV specific T-cell, said method comprising contacting a T-cell with an antigen presenting cell expressing a polynucleotide according to the invention and/or contacting a T-cell with an antigen presenting cell loaded with a peptide according to the invention; and, optionally, culturing said T-cell. The T-cell is preferably a CD8+ cytotoxic T-cell or a CD4+ T-helper cell.
Contacting a cell with a polynucleotide may be performed using any method known to the person skilled in the art, preferably a polynucleotide according to the invention is introduced into the antigen presenting cell (APC), preferably a dendritic cell, using transfection. Before contacting, the polynucleotide according to the invention may be provided with proper control sequences, or be comprised into a proper vector such as described elsewhere herein.
Contacting a T-cell with a peptide according to the invention can be performed by any method known to the person skilled in the art. Preferably, a peptide or an epitope comprised in a peptide is presented to the CD8+ cytotoxic T-cell or CD4+ T-helper cell by an HLA class I or an HLA class II molecule on the surface of an antigen presenting cell, preferably a dendritic cell. The person skilled in the art knows how to load an antigen presenting cell with a peptide.
Culturing said T-cell may be performed using any method known by the person skilled in the art. Maintaining a T-cell under conditions to keep the cell alive is herein also to be construed to be culturing.
Preferably, the T-cell according to this aspect of the invention is contacted with a peptide according to the invention as defined in the first aspect of the invention.
In a fifth aspect, the present invention provides a T-cell obtainable by the method depicted in the fourth aspect of the present invention. Preferably, such T-cell is a T-cell that is obtained by the method according to the fourth aspect of the invention. The T-cell is preferably a CD8+ cytotoxic T-cell or a CD4+ T-helper cell.
Preferably, the T-cell according to this aspect of the invention has been contacted with a peptide according to the invention as defined in the first aspect of the invention.
In a sixth aspect, the present invention provides a composition useful for the prevention and/or treatment of an HBV related disease or related condition, comprising a peptide according to the invention and/or a polynucleotide according to the invention and/or a cell, preferably a T-cell, according to the invention and/or a cell, preferably a T-cell, obtained by the method according to the fourth aspect of the invention and a pharmaceutically acceptable carrier.
When comprising a peptide according to the invention, the composition according to the invention preferably comprises at least 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 and up to 33 different peptides according to the invention. Preferably, a composition according to the invention comprises a peptide according to the invention as defined in the first aspect of the invention. In a preferred embodiment, a composition of the invention comprises a combination of peptides wherein said combination of peptides covers at least 70%, 80%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the HLA class I molecules that are encoded by HLA alleles predominant in the population of human subjects to be treated as defined herein above.
When comprising a polynucleotide according to the present invention, the composition according to the invention preferably comprises at least 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 and up to 33 different polynucleotides according to the invention. Preferably, a composition according to the invention comprises a polynucleotide according to the invention as defined in the second aspect of the invention.
When comprising a cell according to the invention, the composition according to the invention preferably comprises at least 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 and up to 33 different cells, preferably T-cells that have been contacted with a peptide according to the invention. Preferably, said T-cells have been contacted with a peptide according to the invention as defined in the first aspect of the invention. The T-cell is preferably a CD8+ cytotoxic T-cell or a CD4+ T-helper cell.
In a preferred embodiment, the composition of the invention comprises at least 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 and up to 33 different peptides of the peptides consisting of or comprising of a peptide selected from the group consisting of SEQ ID NO: 51-79, 1142-1145 and 1468-1471, more preferably selected from the group consisting of SEQ ID NO: 51, 55, 60, 63, 64, 68, 71, 74, 75, 76, 77, 1142 and 1469, more preferably selected from the group consisting of SEQ ID NO: 51, 55, 60, 63, 64, 68, 71, 74, 75, 77, 1142 and 1469, even more preferably selected from the group consisting of SEQ ID NO: 55, 60, 63, 64, 68, 71, 74, 75, 76, 77 and 1469, even more preferably selected from the group consisting of SEQ ID NO: 55, 60, 63, 64, 68, 71, 74, 75, 77 and 1469, even more preferably selected from the group consisting of SEQ ID NO: 60, 63, 71, 74, 75 and 1469, most preferably selected from the group of SEQ ID NO: 75, 1469 and 63. Further preferred is a composition that comprises at least 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 and up to 33 different peptides of the peptides consisting of or comprising of a peptide selected from the group consisting of SEQ ID NO: 51, 60, 63, 64, 68, 71, 74-77, more preferably selected from the group consisting of SEQ ID NO: 63, 71 and 75.
In a preferred embodiment, the composition of the invention comprises at least a peptide that comprises or consists of a peptide of SEQ ID NO: 63 and peptide that comprises or consists of a peptide of SEQ ID NO: 1143.
Also preferred is a composition that comprises at least a peptide that comprises or consists of a peptide of SEQ ID NO: 63 and peptide that comprises or consists of a peptide of SEQ ID NO: 75.
Also preferred is a composition that comprises at least a peptide that comprises or consists of a peptide of SEQ ID NO: 1143 and peptide that comprises or consists of a peptide of SEQ ID NO: 75.
Also preferred is a composition that comprises at least a peptide that comprises or consists of a peptide of SEQ ID NO: 71 and peptide that comprises or consists of a peptide of SEQ ID NO: 75.
Also preferred is a composition that comprises at least a peptide that comprises or consists of a peptide of SEQ ID NO: 71 and peptide that comprises or consists of a peptide of SEQ ID NO: 63.
Also preferred is a composition that comprises at least a peptide that comprises or consists of a peptide of SEQ ID NO: 1144 and peptide that comprises or consists of a peptide of SEQ ID NO: 63.
Also preferred is a composition that comprises at least a peptide that comprises or consists of a peptide of SEQ ID NO: 1144 and peptide that comprises or consists of a peptide of SEQ ID NO: 75.
Also preferred is a composition that comprises at least a peptide that comprises or consists of a peptide of SEQ ID NO: 1144 and peptide that comprises or consists of a peptide of SEQ ID NO: 1143.
Also preferred is a composition that comprises at least a peptide that comprises or consists of a peptide of SEQ ID NO: 63, a peptide that comprises or consists of a peptide of SEQ ID NO: 1143, and a peptide that comprises or consists of a peptide of SEQ ID NO: 75.
Also preferred is a composition that comprises at least a peptide that comprises or consists of a peptide of SEQ ID NO: 63, a peptide that comprises or consists of a peptide of SEQ ID NO: 1143, a peptide that comprises or consists of a peptide of SEQ ID NO: 75, and a peptide that comprises or consists of a peptide of SEQ ID NO: 1144. Also preferred is a composition that comprises at least a peptide that comprises or consists of a peptide of SEQ ID NO: 75, and a peptide that comprises or consists of a peptide of SEQ ID NO: 1469. Also preferred is a composition that comprises at least a peptide that comprises or consists of a peptide of SEQ ID NO: 63, and a peptide that comprises or consists of a peptide of SEQ ID NO: 1469.
Also preferred is a composition that comprises at least a peptide that comprises or consists of a peptide of SEQ ID NO: 75, a peptide that comprises or consists of a peptide of SEQ ID NO: 1469, and a peptide that comprises or consists of a peptide of SEQ ID NO: 63. Preferably, said composition further comprising a peptide that comprises or consists of a peptide of SEQ ID NO: 60 and/or that comprises or consists of a peptide of SEQ ID NO: 71, and/or that comprises or consists of a peptide of SEQ ID NO: 74.
A preferred composition of the invention comprises a peptide that consists of or comprises a peptide of SEQ ID NO: 75, a peptide that consists of or comprises a peptide of SEQ ID NO: 63, and a peptide that consists of or comprises a peptide of SEQ ID NO: 1469
A preferred composition of the invention comprises a peptide that consists of or comprises a peptide of SEQ ID NO: 75, a peptide that consists of or comprises a peptide of SEQ ID NO: 63, and a peptide that consists of or comprises a peptide of SEQ ID NO: 71.
A pharmaceutically acceptable carrier can be any such carrier known to the person skilled in the art, e.g. buffered aqueous solutions at physiological ionic strength and/or osmolarity (such as e.g. PBS).
Preferably, a composition according to the present invention further comprises at least one adjuvant. Such adjuvant may be any adjuvant known to the person skilled in the art. Preferred adjuvants are defined later herein.
A preferred use of a peptide, polynucleotide, composition, cell and/or T-cell according to the invention or a T-cell obtainable or obtained by a method according to the invention is the use as a medicament. A specific preferred use of a peptide, polynucleotide, composition, cell and/or T-cell according to the invention or a T-cell obtainable or obtained by a method according to the invention is for the treatment and/or prevention of an HBV related disease or condition. Accordingly, the invention provides for the use of a peptide, polynucleotide, composition, cell and/or T-cell according to the invention or a T-cell obtainable or obtained by a method according to the invention for the manufacturing of a medicament for the treatment and/or prevention of an HBV related disease.
The invention further provides a method for the prevention and/or treatment of an HBV related disease or condition comprising administering to a subject an effective amount of a peptide, polynucleotide, composition, cell or T-cell according to the invention and/or a T-cell obtainable or obtained by a method according to the invention.
Formulation of medicaments, ways of administration and the use of pharmaceutically acceptable excipients are known and customary in the art and for instance described in Remington; The Science and Practice of Pharmacy, 21st Edition 2005, University of Sciences in Philadelphia. Pharmaceutical compositions and medicaments according to the invention are preferably formulated to be suitable for intravenous or subcutaneous, or intramuscular administration, although other administration routes can be envisaged, such as mucosal administration or intradermal and/or intracutaneous administration, e.g. by injection. Intradermal administration is preferred herein. Advantages and/or preferred embodiments that are specifically associated with intradermal administration are later on defined in a separate section entitled “intradermal administration”.
It is furthermore encompassed by the present invention that the administration of a peptide, a polynucleotide, a composition and/or a cell according to the invention and/or a cell obtainable or obtained by a method according to the invention with an appropriate pharmaceutical excipient such as an adjuvant and/or a carrier may be carried out as a single administration. Alternatively, the administration may be repeated if needed and/or distinct peptides, polynucleotides, compositions and/or cells according to the invention and/or cells obtainable or obtained by a method according to the invention with appropriate pharmaceutical excipients such as adjuvants and/or carriers, may be sequentially administered.
The peptide, polynucleotide, composition and/or cell according to the invention and/or cell obtainable or obtained by a method according to the invention (also referred to as medicaments according to the invention) may preferably comprise at least one immune response stimulating compound or adjuvant. Advantageously the medicaments according to the invention may additionally comprise one or more synthetic adjuvants. Such adjuvant may be admixed to the medicament according to the invention or may be administered separately to the subject, mammal or human, to be treated. Particularly preferred are those adjuvants that are known to act via the Toll-like receptors and/or via a RIG-I (Retinoic acid—Inducible Gene-1) protein and/or via an endothelin receptor. Immune modifying compounds that are capable of activation of the innate immune system can be activated particularly well via Toll like receptors (TLRs), including TLRs 1-10. Compounds capable of activating TLR receptors and modifications and derivatives thereof are well documented in the art. TLR1 may be activated by bacterial lipoproteins and acetylated forms thereof, TLR2 may in addition be activated by Gram positive bacterial glycolipids, LPS, LPA, LTA, fimbriae, outer membrane proteins, heat shock proteins from bacteria or from the host, and Mycobacterial lipoarabinomannans. TLR3 may be activated by dsRNA, in particular of viral origin, or by the chemical compound poly(I:C). TLR4 may be activated by Gram negative LPS, LTA, Heat shock proteins from the host or from bacterial origin, viral coat or envelope proteins, taxol or derivatives thereof, hyaluronan containing oligosaccharides and fibronectins. TLR5 may be activated with bacterial flagellae or flagellin. TLR6 may be activated by mycobacterial lipoproteins and group B Streptococcus heat labile soluble factor (GBS-F) or Staphylococcus modulins. TLR7 may be activated by imidazoquinolines, such as imiquimod, resiquimod and derivatives imiquimod or resiquimod. TLR9 may be activated by unmethylated CpG DNA or chromatin—IgG complexes. In particular TLR3, TLR7 and TLR9 play an important role in mediating an innate immune response against viral infections, and compounds capable of activating these receptors are particularly preferred for use in the methods of treatment and in the compositions or medicaments according to the invention. Particularly preferred adjuvants comprise, but are not limited to, synthetically produced compounds comprising dsRNA, poly(I:C), unmethylated CpG DNA which trigger TLR3 and TLR9 receptors, IC31, a TLR 9 agonist, IMSAVAC, a TLR 4 agonist, Montanide ISA-51, Montanide ISA 720 (an adjuvant produced by Seppic 7, France). RIG-I protein is known to be activated by dsRNA just like TLR3 (Kato et al, 2005). A particularly preferred TLR ligand is a pam3cys and/or derivative thereof, preferably a pam3cys lipopeptide or variant or derivative thereof, preferably such as described in WO2013051936A1. Further preferred adjuvants are Cyclic dinucleotides (CDNs), Muramyl dipeptide (MDP) and poly-ICLC. In a preferred embodiment, the adjuvants of the invention are non-naturally occurring adjuvants such as the pam3cys lipopeptide derivative as described in WO2013051936A1, Poly-ICLC, imidazoquinoline such as imiquimod, resiquimod or derivatives thereof, CpG oligodeoxynucleotides (CpG-ODNs) having a non-naturally occurring sequence, and peptide-based adjuvants, such as muramyl dipeptide (MDP) or tetanus toxoid peptide, comprising non-naturally occurring amino acids.
In another preferred embodiment, the synthetic adjuvant compounds are physically linked to the peptides of the invention. Physical linkage of adjuvants and costimulatory compounds or functional groups to the HLA class I and HLA class II epitope comprising peptides provides an enhanced immune response by improved targeting to antigen-presenting cells, in particular dendritic cells, that internalize, metabolize and display antigen and by simultaneously stimulating such cells to upregulate expression of a variety of co-stimulatory molecules, thereby becoming efficient T cell response inducing and enhancing cells. Another preferred immune modifying compound is an inhibitor of an endothelin receptor such as BQ-788 (Buckanovich R J et al., 2008; Ishikawa K, 1994). BQ-788 is N-cis-2,6-dimethylpiperidinocarbonyl-L-gamma-methylleucyl-D-1-methoxycarbonyltryptophanyl-D-norleucine. However any derivative of BQ-788 or modified BQ-788 compound is also encompassed within the scope of this invention. Another preferred immune response stimulating compound or adjuvant is Interferon alpha (IFNα), more preferably pegylated Interferon alpha, which may be admixed to the medicament according to the invention, or may be administered separately to the subject as an immuno-modulatory agent. It is to be construed herein that when an immune response stimulating compound is admixed to the medicament according to the invention, it is depicted as an adjuvant; when administered separately, it is depicted as an immuno-modulatory agent, or an immuno-modulator, which terms are used herein interchangeably. Furthermore, the use of antigen presenting cell (co)stimulatory molecules, as set out in WO99/61065 and in WO03/084999, in combination with the peptides and compositions of the invention is preferred. In particular the use of 4-1BB and/or CD40 ligands, agonistic antibodies, OX40 ligands, CD27 ligands or functional fragments and derivatives thereof, as well as synthetic compounds with similar agonistic activity are preferably administered separately or combined with the peptides of the invention to subjects to be treated in order to further stimulate the mounting of an optimal immune response in the subject.
In addition, a preferred embodiment comprises delivery of the medicaments according to the invention, with or without additional immune stimulants such as TLR ligands and/or anti CD40/anti-4-1BB/anti-OX-40 or anti-CD27 antibodies in a slow release vehicle such as mineral oil (e.g. Montanide ISA 51) or PLGA. Alternatively, the medicament according to the invention may be delivered intradermally, e.g. by injection, with or without immune stimulants (adjuvants and/or immuno-modulators). Preferably, for intradermal delivery the medicaments according to the invention are administered in a composition consisting of the medicaments and one or more immunologically inert pharmaceutically acceptable carriers, e.g. buffered aqueous solutions at physiological ionic strength and/or osmolarity (such as e.g. PBS).
In a preferred embodiment, a medicament according to the invention as defined herein is formulated to be suitable for intradermal administration or application. Intradermal is known to the skilled person. In the context of the invention, intradermal is synonymous with intradermal and is distinct from subcutaneous. A most superficial application of a substance is epicutaneous (on the skin), then would come an intradermal application (in or into the skin), then a subcutaneous application (in the tissues just under the skin), then an intramuscular application (into the body of the muscle). An intradermal application is usually given by injection. An intradermal injection of a substance can be done to test a possible reaction, allergy and/or cellular immunity to it, but can also be performed to elicit a specific antibody or T cell immune response. A subcutaneous application is usually also given by injection: a needle is injected in the tissues under the skin.
The advantage of intradermal administration is that the formulation procedure can be simplified and be made more robust. Furthermore, intradermal vaccine delivery has been repeatedly shown to allow significant dose sparing for eliciting vaccine-induced antibody or T cell responses when compared to conventional administration methods such as intramuscular and subcutaneous administration. This effect is attributed to the relatively dense network of immune cells present in the skin. This was also shown with the HPV16 synthetic long peptides in a human study published by Van der Burg et al. (2007). In this study it was shown that intradermal injection of pools of HPV16 synthetic long peptides is safe and results in the migration of HPV16-specific T cells into the skin as well as in an increase in the number of HPV16-specific T cells circulating in the blood.
In an embodiment, a medicament according to the invention does not comprise any adjuvant such as Montanide ISA-51, and specifically Montanide ISA-51. This means that the formulation of the medicament is more simple: an oil-water based emulsion is preferably also not present in a medicament according to the invention. Accordingly, a medicament according to the invention preferably does not comprise an adjuvant such as Montanide ISA-51 and specifically Montanide ISA-51 and/or does not comprise an oil-in-water based emulsion; more preferably a medicament according to the invention comprises neither of these to adjuvant and even more preferably comprises no adjuvant at all. Therefore, in an embodiment, the medicament according to the invention is a, preferably buffered, aqueous solution, preferably at physiological ionic strength and/or osmolality, such as e.g. PBS (Phosphate Buffer Saline) or water for injection (WFI), comprising or consisting of one or more medicaments as defined earlier herein. The skilled person knows how to prepare such a solution.
A medicament according to the invention has another advantage, which is that by intradermally administering low amounts of a medicament, preferably a peptide as earlier herein defined, an immunogenic effect may still be achieved. The amount of each peptide used is preferably ranged from 1 and 1000 μg, more preferably from 5 and 500 μg, even more preferably from 10 and 100 μg.
In an embodiment, the medicament according to the invention comprises a peptide as earlier defined herein and at least one adjuvant, said adjuvant being not formulated in an oil-in water based emulsion and/or not being of an oil-in-water emulsion type as earlier defined herein. This type of medicament according to the invention may be administered as a single administration. Alternatively, the administration of a peptide as earlier herein defined and/or an adjuvant may be repeated if needed and/or distinct peptides and/or distinct adjuvants may be sequentially administered. It is further encompassed by the present invention that a peptide according to the invention is administered intradermally whereas an adjuvant as defined herein is sequentially administered. The adjuvant may be intradermally administered. However any other way of administration may be used for the adjuvant. Intradermal administration of a peptide may be attractive since typically and depending on the disease, the injection of the vaccine is realized at or as close by as possible to the site of the disease resulting in the local activation of the disease draining lymph node, resulting in a stronger local activation of the immune system. A preferred immune response stimulating compound (immuno-modulator) or adjuvant for intradermal administration is Interferon alpha (IFNα), more preferably pegylated Interferon alpha, which may be admixed to the medicament according to the invention, or may be administered separately, for example by subcutaneous injection to the subject. When administered separately the Interferon alpha is preferably also administered subcutaneously and is preferably administered at a dose of 1 microgram/kilogram of body weight within 10 cm proximity to the site where the medicament according to the invention is administered, such described in Zeestraten et al, 2013.
Another typical advantage of the medicaments according to the invention is that relatively low amounts of peptides may be used, in one single shot, in a simple formulation and without any adjuvant known to give undesired side-effects as Montanide ISA-51.
The medicament for intradermal administration may be any medicament according to the invention as defined herein. A medicament according to the invention used for subcutaneous administration may be the same as the one used for intradermal administration and may thus be any medicament according to the invention as defined herein. The skilled person knows how to formulate a medicament suited for subcutaneous administration.
Preferably, a medicament according to the invention for subcutaneous administration comprises a peptide as already herein defined in combination with an adjuvant. Preferred adjuvants or immune modulators have already been mentioned herein. Other preferred adjuvants are of the type of an oil-in water emulsions such as incomplete Freund's adjuvant or IFA, Montanide ISA-51 or Montanide ISA 720 (Seppic France). In a further preferred embodiment, a medicament according to the invention suited for subcutaneous administration comprises one or more peptides according to the invention, an adjuvant or immune modulator as earlier defined herein and an inert pharmaceutically acceptable carrier and/or excipients all as earlier defined herein. Formulation of medicaments, and the use of pharmaceutically acceptable excipients are known and customary in the art and for instance described in Remington; The Science and Practice of Pharmacy, 21st Edition 2005, University of Sciences in Philadelphia. A preferred immune response stimulating compound or adjuvant for subcutaneous administration is Interferon alpha (IFNα), more preferably pegylated Interferon alpha, which may be admixed to the medicament according to the invention, or may be administered separately to the subject. When administered separately, the Interferon alpha is preferably also administered subcutaneously and is preferably administered at a dose of 1 microgram/kilogram of body weight within 10 cm proximity to the site where the medicament according to the invention is administered, such described in Zeestraten et al., 2013.
In an embodiment, the medicament according to the invention suited for intradermal administration may be simultaneously administered with a medicament according to the invention suited for subcutaneous administration. Alternatively, both medicaments may be sequentially intradermally and subsequently subcutaneously administered or vice versa (first subcutaneous administration followed by intradermal administration). In this embodiment as in the earlier described embodiment dedicated to the intradermal administration, the intradermal and/or subcutaneous administration of a medicament according to the invention, preferably a peptide according to the invention, and/or of an adjuvant may be repeated if needed and/or of distinct medicament, preferably peptides and/or of distinct adjuvants may be sequentially intradermally and/or subcutaneously administered. It is further encompassed by the present invention that a medicament according to the invention, preferably a peptide is administered intradermally and/or subcutaneously whereas an adjuvant as defined herein is sequentially administered as immune-modulator. The adjuvant or immune-modulator may be intradermally and/or subcutaneously administered. However any other way of administration may be used for the adjuvant or immune-modulator.
We expect the combination of an intradermal and a subcutaneous administration of a medicament according to the invention is advantageous. DC in the epidermis are clearly different from DC in the dermis and in the subcutis. The intracutaneous (intradermal) immunization will cause antigen processing and activation of epidermal DC (Langerin-positive Langerhans cells) that through their dendritic network are in close contact with the keratinocytes. This will also optimally activate inflammatory pathways in the interactions between Langerhans cell and keratinocytes, followed by trafficking of antigen loaded and activated Langerhans cell to the skin-draining lymph nodes. The subcutaneous administration will activate other DC subsets, that will also become loaded with antigen and travel independently to the skin-draining lymph nodes. Conceivably, the use of a medicament which may be administered both intradermally and subcutaneously may lead to a synergistic stimulation of T-cells in these draining nodes by the different DC subsets.
A medicament according to the present invention and the methods of treatment described herein using a medicament according to the invention may advantageously be combined with other medicaments and methods of treatment. As such, a medicament according to the invention or a method of treatment according to the invention may be combined with e.g. therapy, and/or antibody therapy against an HBV related disease or may be combined with e.g. immunotherapy and/or antibody therapy against another than HBV related disease, or may be combined with immunotherapy against another antigen than HBV to treat an HBV related disease.
In this document and in its claims, the verb “to comprise” and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article “a” or “an” does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article “a” or “an” thus usually means “at least one”. The word “about” or “approximately” when used in association with a numerical value (e.g. about 10) preferably means that the value may be the given value (of 10) more or less 0.1% of the value.
The sequence information as provided herein should not be so narrowly construed as to require inclusion of erroneously identified bases. The skilled person is capable of identifying such erroneously identified bases and knows how to correct for such errors. In case of sequence errors, the sequence of the HBV core, HBV polymerase and HBV large surface protein polypeptides obtainable by expression of the gene present in SEQ ID NO: 5, 2 and 1467 containing the respective nucleic acid sequence coding for the polypeptides should prevail.
All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.
Unless stated otherwise, the practice of the invention will employ standard conventional methods of molecular biology, virology, microbiology or biochemistry. Such techniques are described in Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual (2nd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press; in Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY; in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA; and in Volumes I and II of Brown (1998) Molecular Biology LabFax, Second Edition, Academic Press (UK); Oligonucleotide Synthesis (N. Gait editor); Nucleic Acid Hybridization (Hames and Higgins, eds.).
The invention is further explained in the following examples. These examples do not limit the scope of the invention, but merely serve to clarify the invention.
Introduction
In the current invention, we developed an optimal T cell inducing vaccine composition consisting of synthetic long peptides to treat chronically HBV-infected patients. A selection of 37 long peptides (Table 3; sequences ranging from 30 to 39 amino acids) was made encompassing the regions of the HBV Polymerase, Core protein, Surface Antigen and X-protein with the highest putative T cell immunity inducing capacity. To this end first putative HLA class I restricted cytotoxic T cell (CTL) epitopes and putative HLA class II restricted T helper epitopes were identified in these proteins using algorithms that predict HLA class I and II peptide binding and C-terminal generation by the proteasome of all HLA class I binding short peptides (with a length of a CTL epitope; 8-12 aa) contained in the HBV proteins. Numerical values were assigned to all putative CTL epitopes and T helper epitopes reflecting their immunogenicity quality. The quality of any CTL epitope was assessed using the so-called Class I-BCI score (Class I-Binding-Cleavage-Immunogenicity score), incorporating the combination of the HLA class I binding capacity of the epitope in conjunction with the likelihood of its C-terminal liberation from the source protein by the proteasome. Putative T helper epitopes were assessed using the so-called Class II-B score (Class II-Binding score) reflecting their binding capacity and thus predicted immunogenic quality. The combined cumulative value, i.e. the sum of the cumulative Class I-BCI score and the cumulative Class II-B score, was calculated for all possible long peptides, reflecting the cumulative quantity and quality of all CTL epitopes and T helper epitopes, and is expressed in the T cell Regional Immunogenicity Assessment (TRIA) score. Accordingly, the TRIA score enabled the assessment of the total T cell immunogenicity of any possible long peptide (length 30-39 aa) in the HBV proteins. The 37 HBV-derived long peptides with the highest TRIA scores were selected. Next, we assessed whether naïve donors and donors that have cleared an HBV infection in the past are able to respond to one or more of a subset of 13 long peptides with varying TRIA scores selected from the broad set of 37 long peptides.
PBMC were obtained from buffy coats of twelve healthy donors, six of which were HBV-naïve and six others had cleared an HBV-infection in the past. Using long-term T cell induction assays, we found responses against 12 out of 13 peptides in naïve donors (eleven of which induced responses in multiple donors), confirming the ability of these SLPs to induce T cell responses in a great majority of individuals that had not encountered the antigens before. The strength of T cell responses found in PBMC of HBV-immune donors against these 13 long vaccine peptides correlated with their predicted strength of overall T cell immunogenicity, as expressed in their TRIA scores, validating the predictive value of the TRIA score for selecting immunogenic peptides. Thus the TRIA score is a reliable criterion for the selection of optimal immunogenic long peptides, invigorating us to select the long peptides with the highest TRIA scores into our HBV SLP-based vaccine.
Material and Methods
Peptide Synthesis
Peptides were synthesized using solid phase Fmoc/tBu chemistry on a PTI Prelude peptide synthesizer and purified on a Gilson preparative HPLC system to >95% purity. The identity and purity of the peptides were confirmed with UPLC-MS on a Waters Acquity UPLC/TQD system.
Prediction of CTL and T Helper Cell Immunity Inducing Capacity in Selected HBV-Derived SLPs by Net-Based Algorithm
The putative CTL immunity inducing capacity per SLP was predicted by calculating the cumulative Class I-BCI score per SLP. As detailed below, the cumulative BCI score is based on the Class I-B score, which is a measure for HLA class I peptide binding, and the C-score, which is a measure for proteosomal epitope liberation.
The putative T helper cell immunity inducing capacity per SLP was predicted by calculating the cumulative Class II-B score per SLP. As detailed below, the cumulative Class II-B score is based on the Class II-B score, which is a measure for HLA class II peptide binding.
The total putative immunity inducing capacity per SLP was predicted by summation of the cumulative Class I-BCI score and the cumulative Class II-B score, which value is denominated herein as the TRIA score.
Class I-B Score
Peptide binding to 50 HLA class I molecules (see text) of HBV polymerase-derived peptides, HBV Core protein-derived peptides, HBV Surface Antigen-derived peptides and HBV X protein-derived peptides was assessed in silico using a proprietary algorithm. The upper 1.5th percentile of the predicted binding peptides to each HLA class I molecule was selected. The ‘Class I-Binding score (Class I-B score)’ is derived from the ranking of the predicted binding affinity of the peptides. Briefly, the ranking was first reversed and subsequently normalized to 100, so that the peptide predicted to bind best has a score of 100. Example: five peptides were selected (5 within the 1.5th percentile). Peptides were first assigned the ‘reverse ranking score’ 5 to 1 (5 for the best binding peptide). Subsequently, each reverse ranking score is normalized to the number of peptides within the upper 1.5th percentile, so that the best binder scores 100. To that end, the ranking score for each peptide is multiplied with 100/5 (=20). The best binder then obtains a Class I-B score: 5×20=100, the second best binder has a Class I-B score of 4×20=80, etc. In general, the ranking score is multiplied with 100/n (n=number of peptide within the 1.5th percentile). As a consequence, the best predicted binder (to a certain HLA class I molecule) always scores 100, irrespective of the precise number of peptides within the 1.5th percentile that are selected.
C-Score
C-terminal generation by the proteasome of the upper 1.5th percentile of predicted high affinity binding peptides of HBV polymerase, HBV Core protein, HBV Surface Antigen and HBV X-protein (for each HLA class I molecule) was assessed using two proprietary algorithms, which predict the likelihood of a proteasomal cleavage after a certain amino acid position in HBV polymerase, HBV Core protein, HBV Surface Antigen and HBV X-protein, respectively, and can score between 0 and 1, where a higher value represents a higher likelihood of cleavage after the amino acid. The value 0.5 may be used as an arbitrary threshold value: >0.5 the cleavage is likely to occur, and <0.5 the cleavage is likely to not occur. Accordingly, a value close to 1 indicates a high likelihood of cleavage after the specific residue. Because great differences between the predictions by both different algorithms occur, we developed the Cleavage score (C-score) that takes into account the prediction results of both proprietary algorithms. The C-score is the summation of the separate scores by both methods. Therefore, the C-score for each position in HBV polymerase is maximally (close to) 2, and minimally (close to) 0, where close to 2 reflects a very high likelihood by BOTH methods that the cleavage after the residue will be produced by the proteasome, and a C-score close to 1 is considered as an indifferent tendency for cleavage by the proteasome (as predicted on average by both network methods).
Class I-BCI Score
To incorporate in one quantitative measure both the Class I-B score and the C-score, which are the measures indicative for the likelihood that a peptide will bind with high affinity to HLA class I molecules and will be C-terminally produced, the Class I-Binding-Cleavage-Immunogenicity (Class I-BCI) score was developed. The Class I-BCI score is the Class I-B score multiplied by the C-score. As such the Class I-BCI can attain a maximal value of 200 (100×2) (arbitrary units).
Cumulative Class I-BCI Score
The cumulative Class I-BCI score for each long peptide according to the invention was used as (one of two) selection criterion to identify the peptides of the invention. The cumulative Class I-BCI score is a quantitative reflection of both the total number of CD8+ cytotoxic T cell epitopes that are contained in a long peptide according to the invention and their predicted quality, in terms of binding capacity and likelihood of intracellular generation by the proteasome, and is as such indicative for the CD8+ cytotoxic T cell-inducing power of each peptide according to the invention (its CD8+ T cell immunogenicity). A relatively high cumulative Class I-BCI score of a peptide according to the invention indicates a high CD8+ T cell immunogenicity.
Class II-B Score
Peptide binding to 13 prevalent HLA class II molecules of HBV polymerase-derived peptides, HBV Core protein-derived peptides, HBV Surface Antigen-derived peptides and HBV X protein-derived peptides was assessed in silico using a proprietary algorithm. The ‘Class II-Binding score’ (Class II-B score) is derived from the ranking of the predicted binding affinity of the peptides. Briefly, the ranking was first reversed and subsequently normalized to 100, so that the peptide predicted to bind best has a score of 100. To reduce the number of peptides in the list, all length variants of peptides predicted to bind to a particular HLA class II molecule with a lower predicted binding (lower Class II-B score) are discarded in the list.
Cumulative Class II-B Score
The cumulative Class II-B score for each long peptide according to the invention was used as the second selection criterion to identify the peptides of the invention. The cumulative Class II-B score is a quantitative reflection of both the total number of CD4+ T-helper cell epitopes that are contained in a long peptide according to the invention and their predicted quality, in terms of binding capacity, and is as such indicative for the CD4+ T cell-inducing power of each peptide according to the invention (its CD4+ T cell immunogenicity). A relatively high cumulative Class II-B score of a peptide according to the invention indicates a high CD4+ T cell immunogenicity
T Cell Regional Immunogenicity Assessment (TRIA) Score
The TRIA score for a particular peptide of the invention (SLP) is the sum of cumulative Class I-BCI score and the cumulative Class II-B score of that particular long peptide of the invention (SLP).
Proteasomal Digestion
Dithiotreitol (DTT; Sigma-Aldrich) was freshly dissolved in UPLC-grade water and added to a 2× concentrated proteasome digestion buffer (60 mM Trizma-base; pH 7.5; Sigma-Aldrich, 20 mM KCl; Sigma-Aldrich, 10 mM MgCl2; Sigma-Aldrich, 10 mM NaCl; Sigma-Aldrich) to an end concentration DTT of 2 mM. Then, 130 μl UPLC-grade water was added to 3 reaction vials, along with 150 μl of the 2× concentrated proteasome digestion buffer containing 2 mM DTT, and 10 μl of the peptide to be tested (stock concentration 300 nmol/ml). After vortexing the vials, 10 μl of water was added to vial 1 (mock control digest), 1 μg (10 μl) of constitutive 20S-proteasome (stock 0.1 mg/ml; Enzo Life Sciences) to vial 2, and 1 μg (10 μl) of immune 20S-proteasome (stock 0.1 mg/ml; Enzo Life Sciences) to vial 3. A 50 μl sample for T=0 was taken directly after vortexing and 4 μl of Formic acid (Sigma-Aldrich) was added to stop the reaction. The reaction vials were vortexed and incubated at 37° C. Samples of 50 μl were collected after 1 h, 3 h, 6 h and 24 h incubation. The reactions were stopped with 4 μl Formic acid, and all samples were stored at 20° C.
Mass Spectrometry Analysis of Digested Fragments
A Q-TOF1 mass spectrometer (Waters) equipped with an online nanoelectrospray interface with an approximate flow rate of 250 nl/min was used for electrospray ionization-mass spectrometry. Peptide-digestion samples were trapped on a precolumn (MCA-300-05-C18; Dionex) and were eluted with a steep gradient of 70-90% buffer B over 10 min (buffer A, water, acetonitrile and formic acid, 95:3:1 (vol/vol/vol); buffer B, water, acetonitrile and formic acid, 10:90:1 (vol/vol/vol)). Mass spectra were recorded from a mass of 50-2000 daltons. In tandem mass spectrometry mode, ions were selected with a window of 3 daltons. The collision gas was argon (4×10−5 mbar), and the collision voltage was ˜30 V. For peptide digestion by purified constitutive proteasome and immunoproteasome, peaks in the mass spectra were searched in source substrate peptides with BioLynx software (Waters) and the abundance of a specific digestion fragment was assessed quantitatively as its percentage of the total summed intensities, including undigested substrate.
Cells
Peripheral blood mononuclear cells (PBMC) from healthy donors were isolated by centrifugation over a Ficoll gradient. To generate dendritic cells (DCs), approximately 50*106 PBMC were brought to a concentration of 3*106 cells/ml complete medium (IMDM, Lonza, supplemented with 8% HS, Seralab; penicillin/streptomycin, Lonza; L-glutamin, Lonza) and seeded 3 ml/well in a 6-wells plate (Corning). After incubation for 1.5 hours at 37° C., non-adherent cells were washed away in three washing steps using complete medium (day 0). The adherent cells were cultured for three days at 37° C. in 2 ml/well of complete medium containing 800 U/ml GM-CSF and 500 U/ml IL-4 (Peprotech). On day 3, 1 ml of complete medium containing 2400 U/ml GM-CSF and 1500 U/ml IL-4 was added to each well and cultured for another three days at 37° C.
Induction of T Cells
On day 6, long peptides distributed over 3 pools were added to monocyte-derived DCs of naïve donors at a 3 nmol/ml concentration and incubated overnight at 37° C. Pool 1 comprises SLP 26 (SEQ ID NO: 76), SLP 24 (SEQ ID NO: 74), SLP 1 (SEQ ID NO: 51) and SLP 30 (SEQ ID NO: 1142); Pool 2 comprises Abu-SLP 27 (SEQ ID NO: 77, wherein the cysteine on amino acid position 1 is replaced by Abu), SLP 25 (SEQ ID NO: 75), SLP 10 (SEQ ID NO: 60) and SLP 34 (SEQ ID NO: 1469); and Pool 3 comprises SLP 5 (SEQ ID NO: 55), SLP 13 (SEQ ID NO: 63), SLP 14 (SEQ ID NO: 64) and SLP 21 (SEQ ID NO: 21). On day 7, the cells were washed twice with complete medium to remove peptides. DC and autologous PBMC were co-cultured in a 1:10 ratio for 10 days at 37° C. in the presence of 10 ng/ml IL-7 and 100 pg/ml IL-12p70. The T cell lines generated by this process were checked every 2-3 days and split when necessary.
Restimulation of T Cell Lines
Three days after T cell induction (day 10), a second batch of autologous DC was differentiated and loaded with peptide pools as described above. On day 10 after initiation of the T cell line (day 17), the peptide-loaded DC were washed twice with complete medium and added to the T cells in a 1:10 (DC:T cell) ratio in the presence of 10 ng/ml IL-7 and 100 pg/ml IL-12p70. The cells were co-cultured for 7 days. On day 17, also a new batch of DC was differentiated and loaded with peptide pools as described above. For this second restimulation, starting on day 24, DC and T cells were co-cultured in a 1:10 ratio. Two days after restimulation, 30 IU/ml IL-2 (Peprotech) and 5 ng/ml IL-15 (Peprotech) were added to the culture medium. A third restimulation, starting on day 31, was performed identically to the second restimulation.
T Cell Proliferation and IFNγ Production
To measure T cell activation and proliferation, autologous DC were loaded for 6 hours with each of the 13 HBV-derived peptides separately on day 23, 30 and 37 of the T cell induction protocol described above. The DCs were washed and 5,000 peptide-loaded DC were co-cultured with 50,000 T cells for 48 hours. Then, supernatant was collected for ELISA (IFNγ ELISA, Diaclone) and culture medium containing 3H thymidine was added to all wells. Radioactive 3H thymidine is incorporated in the DNA of newly formed (proliferated) cells, which is measured after 16 hours of incubation on a MicroBeta liquid scintillation counter (Wallac/Perkin Elmer).
IFNγ-ELISpot
To detect antigen-specific IFNγ-producing human T cells, the PBMC were first pre-stimulated with 3 nmol/ml of the indicated peptide for 72 hours at 37° C. During this stimulation, ELISpot PVDF plates (Mabtech) were coated with 5 ug/ml anti-human IFNγ mAb 1-D1K coating antibody (Mabtech) in PBS and incubated overnight at 4° C. After stimulation of the PBMC, the coating antibody was aspirated from the plate, and washed 4 times with PBS. To block aspecific binding, 100 μl of IMDM containing 8% FCS was added to all wells and incubated at 37° C. for 1 hour. In the meantime, stimulated PBMC were harvested, centrifuged, resuspended in X-vivo 15 medium (Lonza) and counted. All PBMC samples were brought to a concentration of 1.5*106 cells/ml in X-vivo 15 medium. The medium in the wells of the PVDF plate was aspirated and 100 μl of each PBMC sample was added to the plate in quadruplicates. The plates were incubated at 37° C. overnight. The next day, the supernatant was discarded and plates are washed 6 times with PBS/Tween20 0.05%. The biotinylated anti-human IFNγ mAb 7-B6-1 (Mabtech) was added to all wells (100 μl/well) at a 0.3 μg/ml concentration in PBS with 1% FBS, and incubated for 2 hours at RT. Next, plates were washed 6 times using PBS/Tween20 0.05% and 1 μg/ml Extravidin-Alkaline phosphatase (ALP) (Sigma-Aldrich) was added to all wells (100 μl/well) in PBS with 1% FBS. The plates were incubated for 1 hour at RT. ALP substrate solution BCIP/NBT-plus (Mabtech) was prepared and 100 μl/well was added to all wells after the plates were washed 4 times with PBS/Tween20 0.05%. To terminate the colorimetric reaction (after 1-20 minutes), tap water was used to wash the plates extensively. After drying, the formed spots were measured on a Biosys Bioreader 5000.
Results
Selection of Long Peptides Based on HLA Class I Peptide Binding Prediction and Predicted C-Terminal Generation of all Possible CD8+ T Cell Epitopes Contained in the Long Peptides
A high quality CD8+ T cell epitope is defined as a peptide that possesses both a predicted high affinity for the HLA class I molecule to which it binds and is also predicted to be generated at its C-terminus by a proteolytic cleavage of the proteasome. Peptides according to the present invention were selected in HBV protein regions that contain optimally high numbers of high quality CD8+ and CD4+ T cell epitopes. To this end, first the HLA class I binding and C-terminal generation of all possible CD8+ T cell epitopes was assessed using a proprietary HLA class I peptide binding algorithm and two proprietary algorithms predicting the cleavages by the proteasome. Subsequently, we devised a single quantitative measure, the so-called binding-cleavage-immunogenicity (BCI) Class I-score, that for each short peptide (8-13 amino acids) incorporates both its predicted binding affinity for the HLA class I molecule to which it binds and the likelihood that the peptide is generated by the proteasome in the cells. The Class I-BCI score is calculated from (1) the binding Class I-score (Class I-B score), which is derived from the results of the in silico prediction of HLA class I peptide binding using the aforementioned algorithm, and (2) the cleavage score (C-score), which is derived from the results of the in silico prediction of the proteasome-mediated C-terminal generation of the peptide by the proteasome using the aforementioned algorithms. Tables 4a, 5a, 6a, and 7a present the Class I-BCI score for all possible CD8+ T cell epitopes of these Polymerase, Core protein, Surface Antigen or X-protein derived SLPs, respectively, together with the cumulative BCI Class I score. Tables 4b, 5b, 6b and 7b present the Class II-B score for all possible CD4+ T cell epitopes of these Polymerase, Core protein, Surface Antigen or X-protein derived SLPs, respectively, together with the cumulative Class II-B score. Together the cumulative Class I-BCI score (for CTL epitopes) and the cumulative B Class II-score (for T helper epitopes) resulted in one quantitative value, the so-called Total Regional Immunogenicity Assessment (TRIA) score, reflecting the overall T cell immunogenicity of a long vaccine peptide (Table 3). Based on the highest TRIA scores, 37 SLPs derived from HBV Polymerase, Core protein, Surface Antigen or X-protein were selected for further evaluation (Table 3). From these, to validate the predictive power of the TRIA score, we chose a representative set of 13 SLPs, which included SLP with relatively low and high TRIA scores, for in vitro immunogenicity assessment. These 13 peptides were divided over three peptide pools for T cell induction assays, as described (see below).
Peptide Fragment Analysis after Proteasomal Digestion of SLPs Reveals High Accuracy of in Silico Predictions
An important component of the identification of putative CTL epitopes is the prediction of their C-terminal generation by a proteasome-mediated cleavage. To validate the reliability of this prediction, proteasomal digestion patterns were experimentally assessed for all but 3 of the 13 functionally tested long vaccine peptides (three long peptides were not being cleaved due to technical reasons).
Digestion experiments were performed separately with 20S constitutive proteasomes and 20S immuno proteasome preparations. The combined analysis of cleavages produced by both types of proteasomes allows the assessment of the C-terminal generation of CTL epitopes that are expressed both on the surface of antigen presenting cells (mainly dendritic cells), containing immunoproteasomes, and on the surface of cancer cells, especially from solid tumors, which mainly express constitutive proteasomes. For vaccination purposes these epitopes are preferred because vaccination with such epitopes will allow the induction of CTL by vaccination and the subsequent eradication of cancer cells by these CTL after recognition of the epitopes on the surface of cancer cells.
As indicated in
The observed cleavage sites observed after 24 h incubation are indicated with arrows. Only cleavage sites that were observed in both the digestion with constitutive proteasomes and the digestion with immunoproteasomes are shown. Major cleavage sites and minor cleavages sites at 24 h digestion are depicted with bold and thin arrows, respectively, according to the following classification:
Major cleavage site: fragments containing as COOH terminus the residue NH2-terminal from the cleavage site together with the (possible) complementary fragment(s) are present for ≥7% at 24 h incubation, as calculated from the intensities of the fragment peaks in the mass spectra.
Minor cleavage site: fragments containing as COOH terminus the residue NH2-terminal from the cleavage site together with the (possible) complementary fragment(s) are present for <7% at 24 h incubation. Cleavage sites with a cumulative fragment abundance of <1% are not shown.
As indicated by
Induction of T Cell Responses Against 12 Out of 13 Selected Peptides in a Naïve Population
To assess whether the 13 selected peptides are able to induce a T cell response in naïve donors, PBMC were isolated from buffy coats derived from six healthy donors that had not experienced an HBV-infection. These PBMC were restimulated with either of the 3 peptide pools to obtain T cell lines, that were subsequently stimulated with the selected 13 peptides. The production of IFNγ and T cell proliferation (3H thymidine incorporation) were measured as a read-out for T cell activation. Results are shown in
Strength of Pre-Existing T Cell Responses in HBV-Immune Donors Correlates with TRIA Score
Subjects that have gone through an HBV infection and successfully cleared it, possess circulating memory T cells specific for HBV. To assess the relevance of the selected vaccine peptides for the clearance of a naturally occurring HBV infection, we tested the presence of T cell responses against the 13 selected HBV peptides in PBMC derived from six HBV-immune donors. After isolation of the PBMC, cells were stimulated with each of the 13 peptides and an IFNγ-ELISpot was performed to detect T cell responses. PBMC samples from 3 out of 6 tested donors showed a positive IFNγ response against one or more of the 13 peptides (SI>3). Responses were observed against 11 (SLP 5, 10, 13, 14, 18, 21, 24, 25, 26, 27 and 34, represented by SEQ ID NO: 55, 60, 63, 64, 68, 71, 74, 75, 76, 77 and 1469, respectively) out of the 13 peptides, 6 of which induced responses in multiple donors (SLP 10, 13, 21, 24, 25 and 34, represented by SEQ ID NO: 60, 63, 71, 74, 75 and 1469, respectively). The results are summarized in
Discussion
The experimental results presented herein validate, and therefore support, the selection and underscore the immunological relevance of the HBV-derived long vaccine peptides of the present invention. These long vaccine peptides encompass the HBV protein regions with the highest number of high quality HLA class I and HLA class II binding epitopes in an outbred population. A preferred combination of the vaccine peptides will be incorporated in a novel HBV SLP vaccine composition to treat chronically HBV-infected patients. Using algorithms to predict the peptide binding affinity of short peptides (8-12 aa) for all prevalent HLA class I molecules and the likelihood of the C-terminal generation of these short peptides by cleavage by the proteasome, in combination with the identification of the putative T helper epitopes, we identified highly immunogenic regions from which the optimal 37 long vaccine peptides were selected.
To enable proper selection of long vaccine peptides in the present invention, a quantitative measure was developed, the TRIA score. Without such a quantitative measure attributed to all possible long HBV peptides, a proper selection of the optimal long peptides is not possible. The TRIA score is a quantitative representation of the quality and quantity of all putative HLA class I restricted CD8+ cytotoxic T cell epitopes and HLA class II restricted CD4+ T helper epitopes contained in a long peptide. The TRIA score was calculated for all possible HBV peptides with a length of 30-39 aa, which is the optimal peptide length for vaccination purposes, enabling the rational selection of a set of highly immunogenic long vaccine peptides.
For further testing and validation of the immunological relevance, we selected a set of 13 SLP with varying TRIA scores. First, we experimentally digested these SLP using either constitutive proteasome or immunoproteasome. The generated fragments showed a clear overlap with the predicted C-terminal cleavage sites, expressed in the BCI score.
Thereafter, T cell assays were performed using PBMC from both naïve and HBV-immune donors. Nearly all 13 selected SLPs were able to induce T cell responses in PBMC derived from naïve healthy donors, which proves that the selected SLP set has the potential to induce responses by the T cell repertoire that had not been stimulated before. Within the same set of vaccine peptides, a strong correlation was observed between the TRIA score of a certain vaccine peptide and the strength of the IFNγ-response in HBV-immune donors, indicating that the TRIA score is a predictive value for in vivo immunogenicity and thus functionality of the vaccine peptides.
A Cumulative Class I-BCI score: See Material and Methods (Examples section).
BCumulative Class II-B score: See Material and Methods (Examples section).
CTRIA score is the sum of the Cumulative Class I-BCI score and the Cumulative Class II-B score.
APeptide amino acid sequence. Each HLA class I binding peptide of HBV polymerase is listed separately for each HLA class I molecule to which it is predicted to bind, and can be listed multiple times for that reason.
BClass I-B score. See Material and Methods (Examples section).
CC-score. See Material and Methods (Examples section).
DClass I-BCI score. See Material and Methods (Examples section).
ECumulative Class I-BCI score. See Material and Methods (Examples section).
APeptide amino acid sequence. Each HLA-DRB1 binding peptide of HBV polymerase is listed separately for each HLA class II molecule to which it is predicted to bind, and each peptide can be listed multiple times for that reason.
BClass II-B score. See Material and Methods (Examples section).
CCumulative Class II-B score. See Material and Methods (Examples section).
APeptide amino acid sequence. Each HLA class I binding peptide of HBV core protein is listed separately for each HLA class I molecule to which it is predicted to bind, and can be listed multiple times for that reason.
BClass I-B score. See Material and Methods of Examples section.
CC-score. See Material and Methods of Examples section.
DClass I-BCI score. See Material and Methods (Examples section).
ECumulative Class I-BCI score. See Material and Methods (Examples section).
BClass II-B score. See Material and Methods (Examples section).
CCumulative Class II-B score. See Material and Methods (Examples section).
APeptide amino acid sequence. Each HLA class I binding peptide of HBV X protein is listed separately for each HLA class I molecule to which it is predicted to bind, and can be listed multiple times for that reason.
BClass I-B score. See Material and Methods (Examples section).
CC-score. See Material and Methods (Examples section).
DClass I-BCI score. See Material and Methods (Examples section).
ECumulative Class I-BCI score. See Material and Methods (Examples section).
APeptide amino acid sequence. Each HLA-DRB1 binding peptide of HBV X protein is listed separately for each HLA class II molecule to which it is predicted to bind, and each peptide can be listed multiple times for that reason.
BClass II-B score. See Material and Methods (Examples section).
APeptide amino acid sequence. Each HLA class I binding peptide of HBV large surface protein is listed separately for each HLA class I molecule to which it is predicted to bind, and can be listed multiple times for that reason.
BClass I-B score. See Material and Methods (Examples section).
CC-score. See Material and Methods (Examples section).
DClass I-BCI score. See Material and Methods (Examples section).
ECumulative Class I-BCI score. See Material and Methods (Examples section).
APeptide amino acid sequence. Each HLA-DRB1 binding peptide of HBV large surface protein is listed separately for each HLA class II molecule to which it is predicted to bind, and each peptide can be listed multiple times for that reason.
BB-score. See Material and Methods (Examples section).
CCumulative B-score. See Material and Methods (Examples section).
| Number | Date | Country | Kind |
|---|---|---|---|
| 14170733 | Jun 2014 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/NL2015/050390 | 6/1/2015 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2015/187009 | 12/10/2015 | WO | A |
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| Number | Date | Country | |
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| 20170246293 A1 | Aug 2017 | US |
