IMPROVED POXVIRAL VACCINES.

专利摘要:
The present application relates to novel administration regimes for poxviral vectors comprising nucleic acid constructs encoding antigenic proteins and invariant chains. In particular, the use of said poxviral vectors for priming or for stimulating an immune response is described.
公开号:BE1021192B1
申请号:E2014/0166
申请日:2014-03-14
公开日:2016-10-21
发明作者:Stefano Colloca；Riccardo Cortese；Antonella. FOLGORI；Alfredo Nicosia
申请人:Glaxosmithkline Biologicals S.A.；Okairos Ag；
IPC主号:

专利说明:

Enhanced poxviral vaccines
The present application relates to improved poxviral vaccines comprising nucleic acid constructs encoding antigenic proteins and invariant chains and novel delivery regimes for such poxviral vectors. In particular, the use of various poxviral vectors for priming or stimulating an immune response is described.
Context of the invention
Infectious diseases still represent a major threat to humanity. One way to prevent or treat infectious diseases is the artificial induction of an immune response by vaccination which is the administration of antigenic material to an individual such that an adaptive immune response to the respective antigen develops. . The antigenic material may be pathogens (e.g., microorganisms or viruses) that are structurally intact but inactivated (i.e., non-infectious) or attenuated (i.e. , with reduced infectivity), or purified components of the pathogen that have been found to be highly immunogenic. Another approach for the induction of an immune response against a pathogen is the provision of expression systems comprising one or more vectors encoding immunogenic proteins or peptides of the pathogen. Such a vector may be in the form of naked plasmid DNA, or the immunogenic proteins or peptides may be delivered using viral vectors, for example on the basis of modified vaccinia viruses (e.g. vaccinia Ankara, MVA) or adenoviral vectors. Such expression systems have the advantage of comprising well-characterized components with low sensitivity to environmental conditions.
A particular goal in the development of vector-based expression systems is that the application of these expression systems to a patient triggers an immune response that is protective against infection by the respective pathogen. However, while inducing an immunogenic response against the pathogen, some expression systems are not capable of triggering an immune response strong enough to fully protect against infections by the pathogen. Therefore, there is still a need for improved expression systems that are capable of inducing a protective immune response against a pathogen as well as new delivery regimes for known expression systems that elicit amplified immune responses.
Antigens are peptide fragments presented on the surface of antigen presenting cells by MHC molecules. The antigens can be of foreign origin, that is to say, pathogenic, or from the organism itself, the latter being called autoantigens. There are two classes of MHC molecules, MHC class I (MHC-I) and MHC class II (MHC-II). The MHC-I molecules have peptide fragments that are synthesized within the respective cell. MHC-II molecules have peptide fragments that are absorbed by phagocytosis and then digested in the endosome. Generally, MHC-II molecules are only expressed by "professional" antigen presenting cells such as macrophages or dendritic cells. Antigens bound to MHC-II molecules are recognized by helper T cells. T cell receptor binding of an helper T cell to an antigen presented by a MHC-II molecule, together with cytokines secreted by the antigen presenting cells, induces the maturation of an immature helper T cell of the THo phenotype in various types of effector cells.
MHC-II molecules are membrane-bound receptors that are synthesized in the endoplasmic reticulum and leave the endoplasmic reticulum in a class II MHC compartment. In order to prevent endogenous peptides, ie self-antigens, from binding to the MHC-II molecule, the nascent MHC-II molecule combines with another protein, the invariant chain, which which blocks the peptide binding gap of the MHC-II molecule. When the class II MHC compartment fuses with a late endosome containing phagocytosed and degraded proteins, the invariant chain is cleaved to leave only the CLIP region bound to the MHC-II molecule. In a second step, the CLIP region is removed by an HLA-DM molecule leaving the free MHC-II molecule to bind fragments of the foreign antigen. Said fragments are presented on the surface of the antigen presenting cell once the class II MHC compartment merges with the plasma membrane, thus presenting foreign antigens to other cells, mainly helper T cells.
It has been previously discovered (WO 2007/062656, which published as US 2011/0293704 and incorporated by reference for the purpose of describing invariant chain sequences) that the fusion of the invariant chain to an antigen which is included in a Expression system used for vaccination increases the immune response against said antigen, if administered with an adenovirus. In addition, said adenoviral construct has been found useful for initiating an immune response in the context of a primary-boosting regimen (WO 2010/057501, which published as US 2010/0278904 and incorporated by reference for the purpose of describe adenoviral vectors encoding invariant chain sequences). The present inventors have surprisingly found that the immune response against a given antigen can be even amplified if, in place of an adenovirus, a poxvirus is used for the production of invariant-chain fusion. In this way, an immune response can be generated. It is particularly surprising that the poxviral vectors have triggered this effect also when used to initiate an immune response. Summary of the invention
In a first aspect, the present invention relates to a poxviral vector comprising a nucleic acid construct for use in priming or stimulating an immune response, the nucleic acid construct comprising: nucleic acid sequence encoding at least one antigenic protein or antigenic fragment thereof operably linked to (ii) a nucleic acid encoding at least one invariant chain.
In another aspect, the present invention relates to a vaccine combination comprising: (a) a poxviral vector comprising a nucleic acid construct, the nucleic acid construct comprising: (i) a nucleic acid sequence encoding at least one first antigenic protein or an antigenic fragment thereof operably linked to (ii) a nucleic acid encoding at least one invariant chain and (b) a viral vector comprising a nucleic acid sequence encoding at least one second protein an antigenic fragment or an antigenic fragment thereof or a second antigenic protein or an antigenic fragment thereof wherein at least one epitope of the first antigenic protein or antigenic fragment thereof is immunologically identical to the second antigenic protein or a fragment of it.
In yet another aspect, the present invention relates to the vaccine combination described above for use in a primary-boost regimen. Methods of using such poxviral vectors and such combinations are also provided.
Detailed description of the invention
Unless otherwise defined, all technical and scientific terms used in this document have the same meanings as those commonly understood by a person of average skill in the field.
For example, the terms used in this document are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, H.G.W, Nagel, B. and Klbl, H. eds. (1995), Helvetica Chimica
Acta, CH-4010 Bay, Switzerland).
Throughout the description and the claims that follow, unless the context requires otherwise, the term "understand", and variations such as "includes" and "comprising", will be understood to imply the inclusion of an integer or a specified step or a group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
Several documents are quoted throughout the text of the description. Each of the documents cited in this application (including all patents, patent applications, scientific publications, manufacturers' specifications, instructions, etc.), whether above or below, is incorporated by reference in its entirety. Nothing in this application shall be construed as an admission that the invention shall not be considered to predate such disclosures by the effect of a prior invention. All the definitions provided herein in the context of one aspect of the invention also apply to other aspects of the invention.
The underlying problem of the present invention is solved by the embodiments characterized in the claims and in the description below.
In a first aspect, the present invention relates to a poxviral vector comprising a nucleic acid construct for use in priming or stimulating an immune response, the nucleic acid construct comprising: (i) a sequence of nucleic acid encoding at least one antigenic sequence or antigenic fragment thereof operably linked to (ii) a nucleic acid encoding at least one invariant chain.
As used herein, the term "poxviral vector" refers to a naturally occurring member of the poxvirid family or a viral vector derived therefrom that is capable of introducing the poxvirus product. nucleic acid assembly in a cell of an individual. In the context of the present invention, it is contemplated that the antigen and invariant chain encoded by the introduced nucleic acid construct is expressed within said cell after introduction by the poxviral vector.
The family of poxvirids is characterized by a genome consisting of double-stranded DNA. Suitably, the poxviral vector belongs to the chordopoxvirine subfamily, more preferably to a genus in said subfamily selected from the group consisting of orthopox, parapox, yatapox, avipox (preferably canarypox (ALVAC) or fowlpox (FPV)) and molluscipox. Even more preferably, the poxviral vector belongs to the genus orthopox and is selected from the group consisting of vaccinia virus, NYVAC virus (derived from the Copenhagen strain of vaccinia), modified vaccinia virus Ankara (MVA) , the cowpox virus and the monkeypox virus. Most preferably, the poxviral vector is MVA.
A description of MVA can be found in Mayr A, Stickl H, Müller HK, Danner K, Singer H. "The smallpox vaccination strain MVA: marker, genetic structure, experience gained with parenteral vaccination and behavior in a debilitated disease mechanism "Abstammung, Eigenschaften und Verwendung des attenuierten Vaccinia-Stammes MVA." Zentralbl Bakteriol B. 1978 Dec; 167 (5-6): 375-90 and in Mayr, A., Hochstein-Mintzel, V. & Stickl, H (1975).
Infection 3, 6-14.
MVA is a highly attenuated strain of vaccinia virus that has undergone multiple fully characterized deletions during more than 570 passages in chicken embryo fibroblast cells. These included genes from the host spectrum and genes encoding cytokine receptors. The virus is unable to replicate effectively in humans and in most cells of other mammals, but the abnormality of replication occurs at a late stage of virion assembly such that gene expression viral and recombinant is not impaired, making MVA an effective expression vector in a single series unable to cause infection in mammals.
In one embodiment, the MVA is derived from the 460 MG viral seed lot obtained from the 571st passage of vaccinia virus on CEF cells. In another embodiment, MVA is derived from viral seed lot MVA 476 MG / 14/78. In another embodiment, the MVA is derived or produced prior to December 31, 1978 and is free of prion contaminations. Other poxviral vectors for use of the invention possess properties similar to MVA. In particular, they are infectious but incompetent for replication in humans. Because of this trait, it may be necessary to express the proteins in trans for replication. In general, these proteins are stably or transiently expressed in a virus producing cell line, thereby allowing replication of the virus.
The term "nucleic acid" refers to a polymeric macromolecule made of nucleotide monomers. The nucleotide monomers are composed of a nucleobase, a five-carbon sugar (such as, but not limited to, ribose or 2'-deoxyribose), and one to three phosphate groups. In general, a polynucleotide is formed via phosphodiester bonds between the individual nucleotide monomers. In the context of the present invention, the nucleic acid molecules include, but are not limited to, ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). In addition, the term "polynucleotide" also includes artificial analogs of DNA or RNA, such as a peptide nucleic acid (PNA).
The term "nucleic acid construct" refers to a nucleic acid that encodes at least one antigenic protein and at least one invariant chain. Suitably, said nucleic acid further comprises elements that direct the transcription and translation of polypeptides encoded by the nucleic acid construct. Such elements include promoter and enhancer elements for directing the transcription of mRNA in an acellular system or a cell-based system, for example a cell-based system. Suitably, such a promoter and / or enhancer is an endogenous promoter and / or enhancer of the poxviral vector. If the nucleic acid construct is provided in the form of translatable RNA, it is contemplated that the nucleic acid construct comprises those elements that are necessary for the translation and / or stabilization of the RNAs encoding the less an immunogenic polypeptide, e.g., a polyA tail, IRES, cap structures, etc.
The term "substantially similar", when used in conjunction with nucleic acid sequences or amino acid sequences, refers to a degree of sequence identity of greater than 80%, more than 85%, more of 90%, more than 95%, more than 98%, or more than 99% with the indicated nucleotide or amino acid sequence, respectively.
The residues in two or more polypeptides are said to "match" with each other if the residues or a group of residues occupy one or more analogous positions in the polypeptide structures. As is well known in the art, analogous positions in two or more polypeptides can be determined by aligning the polypeptide sequences based on the similarities of the amino or structural amino acid sequences. Such alignment tools are well known to those skilled in the art and can be obtained, for example, on the Internet, for example, ClustalW (www.ebi.ac.uk/clustalw) or Align (www.ebi. ac.uk/emboss/aliqn/index.html) using standard settings, eg for Align, Printing: Needle, Matrix: Blosum62, Gap Opening 10.0, Gap Extension 0.5. Those skilled in the art will appreciate that it may be necessary to introduce gaps in either sequence to produce a satisfactory alignment. The residues are said to "match" if the residues are aligned in the best alignment of the sequences. The "best sequence alignment" between two polypeptides is defined as the alignment that produces the largest number of aligned identical residues. The "best sequence alignment region" terminates and thus determines the bounds and length limits of the comparison sequence for the purpose of determining the similarity score, if the similarity or sequence identity between two aligned sequences falls to less than 30%, less than 20%, or less than 10% over a length of 10, 20 or 30 amino acids.
As pointed out above, it is contemplated that the vector of the present invention is a poxviral vector. Thus, if said poxviral vector is competent for replication, the nucleic acid construct is composed of a larger nucleic acid molecule that further comprises nucleic acid sequences that are required for replication of the viral vector and or regulatory elements directing the expression of the encoded polypeptide by the nucleic acid construct.
In one embodiment of the present invention, the antigenic protein or an antigenic fragment thereof and the invariant chain are comprised by a single open reading frame such that transcription and translation of said open reading frame results in a fusion protein comprising the antigenic protein or an antigenic fragment thereof and the invariant chain.
The term "open reading frame" (ORF) refers to a sequence of nucleotides, which can be translated into amino acids. In general, such an ORF contains a start codon, a subsequent region usually having a length which is a multiple of 3 nucleotides, but which does not contain a stop codon (TAG, TAA, TGA, UAG, UAA, or UGA). in the given reading frame. In general, ORFs exist naturally or are artificially constructed, i.e., by gene-based technological means. An ORF encodes a protein where the amino acids into which it can be translated form a peptide link chain.
The terms "protein", "polypeptide" and "peptide" are used interchangeably herein and refer to any peptide-bonded amino acid chain, regardless of length, cotranslational or posttranslational modifications.
The term "posttranslational" as used herein refers to events that occur after the translation of a nucleotide triplet into an amino acid and the formation of a peptide bond to the preceding amino acid in the sequence. Such posttranslational events may occur after formation of the entire polypeptide or already during the translation process on those portions of the polypeptide that have already been translated. Generally, posttranslational events alter or modify the chemical or structural properties of the resulting polypeptide. Examples of posttranslational events include, but are not limited to, events such as glycosylation or phosphorylation of amino acids, or cleavage of the peptide chain, for example, by an endopeptidase.
The term "cotranslational" used in this document refers to events that occur during the process of translating a nucleotide triplet into an amino acid chain. Generally, these events alter or alter the chemical or structural properties of the resulting amino acid chain. Examples of cotransportational events include, but are not limited to, events that can completely stop the translation process or interrupt the formation of peptide bonds resulting in two distinct translation products. .
Proteins useful in the present invention (including protein derivatives, protein variants, protein fragments, protein segments, protein epitopes, and protein domains) can be further modified by chemical modification. Therefore, such a chemically modified polypeptide may comprise chemical groups other than the residues found among the 20 naturally occurring amino acids. Examples of these other chemical groups include, but are not limited to, glycosylated amino acids and phosphorylated amino acids. The chemical modifications of a polypeptide may provide advantageous properties as compared to the parent polypeptide, for example, one or more of stability enhancement, an increase in the biological half-life, or an increase in solubility in water. The chemical modifications applicable to the variants usable in the present invention include, without limitation: PEGylation, glycosylation of non-glycosylated parent polypeptides, or modification of the glycosylation profile present in the parent polypeptide. Such chemical modifications applicable to variants usable in the present invention may be cotranslational or posttranslational.
An "antigenic protein" as referred to herein is a polypeptide as defined above which contains at least one epitope. An "antigenic fragment" of an antigenic protein is a partial sequence of said antigenic protein comprising at least one epitope. For immunization purposes, only those parts of a protein that trigger an immune response are relevant. Therefore, the nucleic acid construct need not encode the complete antigenic protein as found in a pathogen or a cancer cell. A shorter fragment of such a protein is sufficient as long as its amino acid sequence comprises the epitope or epitopes responsible for the recognition of the antigenic protein by the immune system.
The term "epitope" also known as an antigenic determinant, as used in the context of the present invention, is part of a polypeptide that is recognized by the immune system. Suitably, this recognition is mediated by the binding of antibodies, B cells, or T cells to the epitope in question. Epitopes linked by antibodies or B cells are referred to as "B cell epitopes" and T cell-linked epitopes are termed "T cell epitopes". In this context, the term "binding" refers to a specific binding, which is defined as a binding with an association constant between the antibody or T cell receptor (TCR) and the respective epitope of 1 x 10 5 M '. 1 or higher, or 1 x ΙΟ6 Μ-1, 1 x ΙΟ7 Μ-1, 1 x 108 1VT1 or greater. Those skilled in the art are well aware of how to determine the association constant (see, for example, Caoili, S.E. (2012) Advances in Bioinformatics Vol 2012). Suitably, the specific binding of antibodies to an epitope is mediated by the Fab region (fragment, antigen binding) of the antibody, the specific binding of a B cell is mediated by the Fab region of the antibody cell-B receptor-specific binding and T cell-specific binding is mediated by the variable region (V) of the T-cell receptor. T-cell epitopes are presented on the surface of a cell presenting the antigen, or they are linked to major histocompatibility complex (MHC) molecules. There are at least three different classes of MHC molecules called MHC Class I, II, and III molecules, respectively. Epitopes presented via the MHC-I pathway trigger a response by cytotoxic T lymphocytes (CD8 + cells), whereas epitopes presented via the MHC-II pathway trigger a response by cells T helper (CD4 + cells). The T cell epitopes exhibited by the MHC class I molecules are generally peptides of between 8 and 11 amino acids in length and the T cell epitopes exhibited by MHC class II molecules are generally a length between 13 and 17 amino acids. MHC class III molecules also have non-peptide epitopes such as glycolipids. Therefore, the term "T cell epitope" refers to a peptide of length 8 to 11 or 13 to 17 amino acids that can be presented by a molecule of either class I of MHC or class II of the CMH.
Epitopes usually consist of groups of chemically active surface molecules such as amino acids or side chains of sugars and usually have specific three-dimensional structural characteristics as well as specific charge characteristics. The term "epitope" refers to both conformational and non-conformational epitopes. The conformational and non-conformational epitopes are distinguished in that the binding to the first but not the last is lost in the presence of denaturing solvents. T cell epitopes are nonconformational, i.e. they are linear, whereas B cell epitopes can be conformational or nonconformational. Epitopes of linear B cells generally range from 5 to 20 amino acids in length.
An antigenic protein according to the present invention is derived from a pathogen selected from the group consisting of viruses, bacteria, protozoa and multicellular parasites. In an alternative embodiment of the present invention, the antigenic protein is a polypeptide or fragment of a polypeptide expressed by a cancer cell.
The antigenic proteins or antigenic fragments thereof induce a B cell response or a T cell response or a B cell response and a T cell response. Therefore, the antigenic proteins or antigenic fragments comprise at least one T cell epitope and / or at least one B cell epitope.
In a certain exemplary embodiment of the present invention, the antigenic protein encoded by the vector is derived from hepatitis C virus (HCV). The HCV genome consists of a single RNA strand about 9.5 kb in length that encodes a precursor polyprotein of about 3000 amino acids. (Choo et al (1989) Science 244, 362-364, Choo et al (1989) Science 244, 359-362, Takamizawa et al (1991) J. Virol 65, 1105-1113). The HCV polyprotein contains the viral proteins in the order: C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B. Individual viral proteins are produced by proteolysis of the HCV polyprotein. The host cell proteases release the putative structural proteins C, E1, E2, and p7, and create the N-terminus of NS2 at amino acid 810. (Mizushima et al., (1994) J. Virol 65, 2731-2734, Hijikata et al (1993) PNAS USA 90, 10773-10777).
The NS3, NS4A, NS4B, NS5A and NS5B nonstructural proteins are likely to be the virus replication machinery and are released from the polyprotein. A zinc-dependent protease associated with NS2 and the N-terminus of NS3 is responsible for cleavage between NS2 and NS3. (Grakoui et al (1993) J. Virol 67, 1385-1395, Hijikata et al (1993) P.N.A.S. USA 90, 10773-10777). A distinct serine protease localized in the N-terminal domain of NS3 is responsible for proteolytic cleavages at the NS3 / NS4A, NS4A / NS4B, NS4B / NS5A and NS5A7NS5B junctions. (Bartenschlager et al., (1993) J. Virol 67, 3835-3844, Grakoui et al., (1993) Proc Natl Acad Sci USA 90, 1058310587, Tomei et al (1993) Virol 67, 4017-4026). NS4A provides a cofactor for NS3 activity. (Failla et al (1994) J. Virol 68, 3753-3760, De Francesco et al., US Patent No. 5,739,002). NS5A is a highly phosphorylated protein conferring resistance to interferon. (From Francesco et al., (2000) Semin, Liver Dis., 20 (1), 69-83, Pawlotsky (1999) Viral Hepat Suppl 1, 47-48). NS5B provides an RNA-dependent RNA polymerase. (De Francesco et al., International Publication Number WO 96/37619, Behrens et al., EMBO 15, 12-22, 1996, Lohmann et al., Virology 249, 108-118, 1998).
In a non-limiting exemplary embodiment, the antigenic protein is a Met-NS3-NS4A-NS4B-NS5A-NS5B polypeptide containing an NS5B inactive RNA-dependent RNA polymerase region. For example, said antigenic protein has an amino acid sequence substantially similar to the sequence defined by SEQ ID NO: 11 and has sufficient protease activity to induce the production of at least one polypeptide substantially similar to the NS5B present in SEQ ID NO: 11. The sequence of this antigenic protein was published in WO 2003/031588, published as US 2004/0247615 and incorporated by reference for the purpose of describing HCV polypeptides. The product polypeptide corresponding to NS5B is inactive from an enzymatic point of view. In another embodiment, the HCV polypeptide has sufficient protease activity to produce polypeptides substantially similar to the NS3, NS4A, NS4B, NS5A, and NS5B regions present in SEQ ID NO: 11.
In one embodiment, the degree of sequence identity with the sequence according to SEQ ID NO: 11 is more than 80%, more than 85%, more than 90%, more than 95% or more than 98% relative to to the sequence defined by SEQ ID NO: 11. However, in some embodiments, the sequence of the antigenic protein has greater than 99% sequence identity with the sequence defined by SEQ ID NO: 11 or is identical to that -this.
The term "invariant chain", also known as "li" or "CD74", refers to a non-polymorphic type II membrane integral protein. The protein has multiple functions in the maturation of lymphocytes and adaptive immune responses; in particular, li provides targeting of newly synthesized MHC-II to the endocytic pathway, where the complex may encounter antigenic peptides. (Pieters J. (1997) Curr Opin Immunol., 9: 8996). In addition, it has been shown that li functions as a class I MHC chaperone (Morris et al (2004) Immunol Res., 30: 171-179) and, by its endosomal targeting sequence, facilitates the stimulation of CD4 + T cells, but not CD8 + T cells directed against the covalently bound antigen (Diebold et al (2001) Gene Ther 8: 487-493).
For the human invariant chain, four different isoforms are known, generally named p33, p35, p41 and p43 (Strubin et al., 1986, EMBO Journal, 5: 3483-3488). SEQ ID NO: 1 and SEQ ID NO: 2 correspond to the amino acid sequence and the nucleic acid sequence of the p35 isoform of the human invariant chain. Compared with human p33 and p41, human p35 and p43 isoforms contain 16 additional residues at the N-terminus due to a translation initiation variant. Compared with human p33 and p35, human p41 and p43 isoforms comprise an additional domain (splice variant of exon 6b) inserted into the frame in the C-terminal region of the invariant chain. SEQ ID NO: 5 and SEQ ID NO: 6 correspond to the amino acid sequence and the nucleic acid sequence of the p43 isoform of the human invariant chain. The sequence of an additional human isoform c that lacks two exons compared to human p33 and p35 is available from Genbank (Accession BC024272). SEQ ID NO: 9 and SEQ ID NO: 10 correspond to the amino acid sequence and nucleic acid sequence of the c-isoform of the human invariant chain.
For the murine invariant chain, only two isoforms (p31 and p41) are known, which correspond to the p33 and p41 isoforms of the human invariant chain. SEQ ID NO: 3 and SEQ ID NO: 4 correspond to the amino acid sequence and the nucleic acid sequence of the p31 isoform of the murine invariant chain. SEQ ID NO: 7 and SEQ ID NO: 8 correspond to the amino acid sequence and the nucleic acid sequence of the p41 isoform of the murine invariant chain. An overall diagram of the different isoforms is shown in Figure 4.
In one embodiment, the invariant chain used in the present invention is substantially similar to the invariant chain according to SEQ ID NO: 1 or 3.
The invariant chain comprises several domains: a cytosolic domain which comprises a peptide of sorting (targeting) (also known as the lysosomal targeting sequence) (positions 17 to 46 in the human invariant chain SEQ ID NO: 1, positions 1 to 29 in the murine invariant chain SEQ ID NO: 3) preceded by a retention signal in the ER in the p35 and p43 variants of the human invariant chain (positions 1 to 16 in the human invariant chain SEQ ID NO: 1), a domain transmembrane (signal anchor, positions 47 to 72 in the human invariant chain SEQ ID NO: 1, positions 30 to 55 in the murine invariant SEQ ID NO: 3), and a luminal domain which in itself comprises a KEY region ( positions 93 to 96 in the human invariant chain SEQ ID NO: 1, positions 76 to 79 in the murine invariant chain SEQ ID NO: 3), an adjacent CLIP region (positions 97 to 120 in the human invariant chain SEQ ID NO: 1 , positions 80 to 103 in the murine invariant chain SEQ ID NO: 3). The CLIP region comprises a central CLIP peptide (positions 103 to 117 in the human invariant chain SEQ ID NO: 1, positions 86 to 100 in the murine invariant chain SEQ ID NO: 3) and a trimerization domain (positions 134 to 208 in the human invariant chain SEQ ID NO: 1, positions 117 to 191 in the murine invariant chain SEQ ID NO: 3, Mittendorf et al., (2009) Expert Opin. Biol. Ther., 9: 71-78; Strumptner-Cuvelette and
Benaroch, 2002, Biochem. Biophys. Acta, 1542: 1-13). The remainder of the luminal domain comprises two highly flexible regions located between the transmembrane region and the KEY region (positions 73 to 92 in the human invariant chain SEQ ID NO: 1, positions 56 to 75 in the murine invariant chain SEQ ID NO: 3) or downstream the trimerization domain (positions 209 to 232 in the human invariant chain SEQ ID NO: 1, positions 192 to 215 in the murine invariant chain SEQ ID NO: 3). The invariant chain has been characterized in several organisms such as chicken, cow, dog, mouse, rat and human being.
In one embodiment, the invariant chain is derived from vertebrates, avian origin or mammals, or furthermore, it is selected from the group consisting of invariant chains derived from chicken, cow, dog, mouse , the rat, a non-human primate and the human being. In another embodiment, it is of human or murine origin, for example, the human invariant chain has an amino acid sequence as defined by SEQ ID NO: 1.
Said polypeptide is, in one embodiment, encoded by a nucleic acid sequence as given in SEQ ID NO: 2. In another embodiment, the murine invariant chain has an amino acid sequence as defined by SEQ ID NO: 3. Said polypeptide is, in one embodiment, encoded by a nucleic acid sequence as given in SEQ ID NO: 4.
The term " invariant chain " also includes variants of the polypeptides described above characterized by deletions of naturally occurring invariant chain amino acid sequence portions substantially similar to naturally occurring invariant chains or by their substitution by other sequences. Examples of variants are given below.
In a particular variant of the invariant chain, the endogenous KEY region which consists of amino acid residues LRMK is deleted or substituted by a different amino acid sequence. For example, LRMK amino acid residues or corresponding residues are deleted. Deletion of LRMK amino acid residues can be complete (involving all amino acid residues LRMK) or partial (involving at least one amino acid residue of LRMK). Complete deletion of all amino acid residues LRMK is contemplated. In addition, at least one or all of the amino acid residues are substituted with different amino acid residues.
In yet another example of a variant, the methionines in the positions 107 and 115 of the human invariant chain according to SEQ ID NO: 1 or the methionines in the 90 and 98 positions of the murine invariant chain according to SEQ ID NO: 3 or the methionines corresponding to these positions in other invariant chains are substituted by other amino acids. Suitably, methionine is substituted.
In yet another variant example, the invariant chain is truncated at the N-terminus, for example to such an extent that the N-terminus to the transmembrane region is removed. Therefore, in another embodiment, the invariant chain according to SEQ ID NO: 1, 46 amino acids or less of the N-terminus are truncated, 41 amino acids or less are truncated, or 36 amino acids or less are truncated. Therefore, it is also contemplated that for the invariant chain according to SEQ ID NO: 3, 30 amino acids or less of the N-terminus are truncated, 25 amino acids or less are truncated, or 20 amino acids or less truncated . For an embodiment of the invariant chain according to SEQ ID NO: 1, the first 16 amino acid residues of the human invariant chain are deleted. It is also possible that at least one, but not all, of the first 16 amino acid residues are deleted. In addition, it is possible that at least one, or all, of the first 16 amino acid residues of the human invariant chain (SEQ ID NO: 1) are substituted with other amino acid residues.
In yet another variant, at least one signal peptide for expression in the endoplasmic reticulum lumen is added to the N-terminus of the invariant chain, for example to a truncated version at the N-terminus of the invariant chain to which - because of the N-terminal truncation - the transmembrane region is missing.
In yet another variant of the invariant string, at least one CLIP region is added to or replaces the endogenous CLIP region of the respective invariant string. In the human invariant chain according to SEQ ID NO: 1, the CLIP region covers positions 97 to 120 and in the murine invariant chain according to SEQ ID NO: 3, it covers positions 80 to 103. Thus, the person skilled in the art can easily determine the amino acid residues corresponding to the CLIP region in the invariant chain according to SEQ ID NO: 1 and 3. In another embodiment, the complete endogenous CLIP region is deleted or replaced. However, the deletion or replacement of at least one amino acid residue belonging to the endogenous CLIP region is also contemplated.
The term "invariant chain" also refers to fragments of the invariant chains and their variants described above, for example, invariant chains having the amino acid sequences according to SEQ ID NO: 1 or 3 or encoded by the sequences nucleic acid according to SEQ ID NO: 2 or 4. It should be understood that because of the degenerate nature of the genetic code, an amino acid may be encoded by more than one codon. For example, the amino acid isoleucine may be encoded by AUU, AUC or AUA codons. Therefore, the present invention also contemplates all variants of the aforementioned nucleic acid sequences that encode the amino acid sequences defined by SEQ ID NO: 1 or 3 without regard to the specific nucleic acid sequence. Since different organisms use different codons with different efficiency, it may be advantageous to adapt the nucleotide sequence to the intended host organism. In one embodiment, the fragment is a fragment of at least 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 or 210. amino acid residues of a wild-type invariant chain or a variant thereof as defined above.
In addition, the term "invariant chain" refers to polypeptides having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8% or 99.9% sequence identity with any of the wild-type invariant chains described above, variants or fragments thereof. Methods for determining sequence identity between two different polypeptides are well known in the art. The similarity of nucleotide and amino acid sequences, i.e., percent sequence identity, can be determined using sequence alignment. Such alignments can be made with several algorithms known in the art, for example with the mathematical algorithm of Karlin and Altschul (Karlin & Altschul (1993) Proc Natl Acad Sci USA 90: 5873-5877), with hmmalign (HMMER package, hmmer.wustl.edu/) or with the CLUSTAL algorithm (Thompson, JD, Higgins, DG & Gibson, TJ (1994) Nucleic Acids Res 22, 4673-80) available, for example, at www.ebi.ac.uk/Tools/clustalw/ or at www.ebi.ac.uk/Tools/clustalw2/index.html or at npsa-pbil.ibcp.fr/cgi -bin / npsa_automat.pl page = / SNPA / npsa_clustalw.html. The preferred settings used are the default settings as presented at www.ebi.ac.uk/Tools/clustalw/ or www.ebi.ac.uk/Tools/clustalw2/index.html. The rank of sequence identity (sequence matching) can be calculated using, for example, BLAST, BLAT or BlastZ (or BlastX). A similar algorithm is incorporated in the BLASTN and BLASTP programs of Altschul et al. (1990) J. Mol. Biol. 215: 403-410.
To obtain alignments with gaps for comparison purposes, Gapped BLAST is used as described in Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402. When BLAST and Gapped BLAST programs are used, the default settings of the respective programs are used. Sequence mapping analysis can be complemented by established homology mapping techniques such as Shuffle-LAGAN (Brudno M., Bioinformatics 2003b, Suppl.1: 154-162) or random Markov fields. When reference is made to percentages of sequence identity in the present application, these percentages are calculated with respect to the full length of the longer sequence, if not otherwise indicated.
The term "priming of an immune response" refers to the first encounter of the immune system with the at least one antigenic protein or antigenic fragment thereof and the subsequent induction of an antigen-specific immune response. within a defined period of time. The said period of time is, for example, at least 1 year, at least 2 years, at least 3 years, at least 5 years or at least 10 years prior to initiation. In one embodiment, the immune system encounters of the individual or subject with the antigenic protein or an antigenic fragment thereof that do not induce an antigen-specific immune response are not considered "priming". an immune response. For example, encounters of the individual's immune system with the antigenic protein or an antigenic fragment thereof that do not induce lasting immunity are not considered to "initiate an immune response" according to the present invention. In another embodiment, the induction of lasting immunity is mediated by the generation of memory B cells and / or memory T cells. In the case of cancer, for example, a specific antigen can be expressed by the cancer cells without triggering an immune response. The mere presence of this antigen is not a "priming of an immune response" against said antigen as understood by the present application. In one embodiment, the individual or subject has not been deliberately immunized with the antigenic protein or an antigenic fragment thereof or a vector comprising a nucleic acid encoding such a protein or fragment for the purpose of to treat or prevent a disease in the given period of time.
However, the term "priming of an immune response" refers to any prior contact of the individual with a pathogen naturally comprising said antigenic protein or an antigenic fragment thereof, provided that said contact has not occurred. not artificially induced for medical purposes. In particular, it is contemplated by the present application that the individual may have already been infected with the above-mentioned pathogen, provided that said infection has not been artificially induced for medical purposes. At the moment when the priming of the immune response takes place, the infection of the individual may still be present or it may have already been eliminated. Similarly, in the case of cancer, it is contemplated that the individual or subject to be immunized with the poxviral vector of the present invention already suffers from cancer expressing the antigenic protein or an antigenic fragment thereof which is understood by the nucleic acid construct of the present invention.
The patient or subject to be immunized with a poxviral vector according to the present invention is, for example, a mammal or a bird, more specifically a primate, a mouse, a rat, a sheep, a goat, a cow, a pig, a horse, goose, chicken, duck or turkey and, most specifically, a human being. .
The poxviral vector comprising a nucleic acid construct as defined above is, for example, used in a primary-boost regimen.
In many cases, a single administration of a vaccine is not sufficient to generate the number of long-lasting immune cells that are required for effective protection in the event of future infection with the pathogen in question, to protect against diseases including tumor diseases or to treat therapeutically a disease, such as a tumor disease. Therefore, repeated stimulation with a biological preparation specific for a pathogen or specific disease is required in order to establish an immunity that lasts and protects against said pathogen or disease or to cure a given disease. A dosing regimen comprising repeated administration of a vaccine directed against the same pathogen or disease is referred to herein as a "primary-boost regimen". In one embodiment, a primary-boost regimen involves at least two administrations of a vaccine or vaccine composition directed against a specific pathogen, a group of pathogens or diseases. The first administration of the vaccine is referred to as "priming" and any subsequent administration of the same vaccine or vaccine directed against the same pathogen as the first vaccine is referred to as a "booster". Thus, in another embodiment of the present invention, the primary-boost regimen involves administering the vaccine to prime the immune response and at least one subsequent administration to stimulate the immune response (boost). It should be understood that 2, 3, 4 or even 5 administrations for stimulating the immune response are also contemplated by the present invention.
The time period between initiation and recall is, possibly, 1 week, 2 weeks, 4 weeks, 6 weeks or 8 weeks. More particularly, it is 4 weeks or 8 weeks. If more than one booster is given, the following reminder is given 1 week, 2 weeks, 4 weeks, 6 weeks or 8 weeks after the previous booster. For example, the interval between any two reminders is 4 weeks or 8 weeks.
Primary-vaccination regimens may be homologous or heterologous. In homologous primary-boost regimens, both priming and at least one booster are performed using the same means of administration of the antigenic protein or antigenic fragment thereof, i.e. say, priming and boosting are performed using a polypeptide or priming and boosting are performed using a nucleic acid construct included in the same vector. In the context of the present invention, a homologous primary-boost regimen will include the use of the poxviral vector of the invention for both priming and stimulating the immune response (boosting). A heterologous priming-booster diet involves the use of different means for priming and for stimulating the immune response (booster). In the context of the present invention, a heterologous priming-boosting regimen will comprise a poxviral vector as described above for priming an immune response and a different vector or peptide vaccine for stimulating the immune response ( recall).
Alternatively, a heterologous priming-boosting regimen will comprise a different peptide vector or vaccine for priming an immune response and a poxviral vector as described above for stimulating the immune response (boost).
In one embodiment of the present invention, the primary-boost regimen is homologous.
In another embodiment of the present invention, the primary-boost regimen is heterologous.
In a heterologous priming-boosting regimen, a poxviral vector as described above is used for stimulation of the immune response (booster) and a different peptide vector or vaccine is used for priming the immune response. In another embodiment of the heterologous priming-boost regimen, a poxviral vector as described above is used for priming the immune response and a different peptide vector or vaccine is used for stimulation of the immune response. (recall).
In another embodiment, the heterologous priming-boosting regimen will comprise an adenoviral vector for priming an immune response and a poxviral vector as described above for stimulating the immune response (boosting).
In yet another embodiment, the heterologous priming-boosting regimen will comprise a poxviral vector as described above for priming an immune response and an adenoviral vector for stimulating the immune response (boosting).
For all primary-boost regimens, it is contemplated that the antigenic proteins or antigenic peptides used to stimulate the immune response are immunologically identical to the antigenic protein or antigenic fragment thereof used for priming the the immune response. It should be understood that the antigenic protein or an antigenic fragment thereof can be administered as a polypeptide ("peptide vaccine") or that it can be encoded by a molecule of nucleic acid administered to the subject individual. In the latter case, the antigenic protein or antigenic peptide that triggers the desired immune response is expressed in the cells of the immunized individual.
Two or more antigenic proteins or antigenic fragments thereof are "immunologically identical" if they are recognized by the same antibody, T-cell or B-cell. Recognition of two immunogenic polypeptides or more by the same antibody, the same T cell or B cell is also known as "cross-reactivity" of said antibody, T cell or B cell. In one embodiment, recognition of two or more immunologically identical polypeptides by the same antibody, the same T cell or B cell is due to the presence of identical or similar epitopes in all the polypeptides. Similar epitopes share sufficient structural and / or charge characteristics to be bound by the Fab region of the same antibody or B cell receptor or by the V region of the same T cell receptor. The binding characteristics of an antibody, a T cell receptor or a B cell receptor are defined, for example, by the binding affinity of the receptor to the epitope in question. Two immunogenic polypeptides are "immunologically identical" as understood by the present application if the affinity constant of the polypeptide with the lower affinity constant is at least 30%, at least 40%, at least 50%, at least minus 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 98% of the affinity constant of the polypeptide with the higher affinity constant. Methods for determining the binding affinity of a polypeptide to a receptor such as equilibrium dialysis or ELISA (enzyme linked immunosorbent assay) are well known in the art.
In one embodiment, two "immunologically identical" polypeptides comprise at least one identical epitope. The strongest vaccination effects can usually be achieved if the immunogenic polypeptides comprise identical epitopes or if they have an identical amino acid sequence.
In one embodiment, the use of the poxviral vector as described above for priming an immune response will establish protective immunity against a pathogen or disease or will lead to the eradication of the disease.
In one embodiment, the poxviral vector is administered intranasally, intramuscularly, subcutaneously, intradermally, intragastrically, orally, and topically.
"Intranasal administration" is the administration of a vector of the present invention to the mucosa of the complete respiratory tract including the lung. More particularly, the composition is administered to the lining of the nose. In one embodiment, intranasal administration is achieved by means of instillation, spraying or aerosol. In another embodiment, said administration does not involve perforation of the mucosa by mechanical means such as a needle.
The term "intramuscular administration" refers to the injection of a vector into any muscle of an individual. Examples of intramuscular injections are given in the deltoid, large external muscle or ventroglual and dorsogluteal areas.
The term "subcutaneous administration" refers to the injection of a vector into the hypodermis.
The term "intradermal delivery" refers to the injection of a vector into the dermis between the layers of the skin.
The term "oral administration" refers to the administration of a vector through the mouth to the gastric system.
"Topical administration" is the administration of the vector to any part of the skin with a needle or comparable device. The vector can also be administered topically to the lining of the mouth, nose, genital area and rectum.
In another aspect, the present invention relates to a vaccine combination comprising: (a) a poxviral vector comprising a nucleic acid construct, the nucleic acid construct comprising: (i) a nucleic acid sequence encoding at least one first antigenic protein or antigenic fragment thereof operably linked to (ii) a nucleic acid encoding at least one invariant chain and (b) a vector comprising a nucleic acid sequence encoding at least one second antigenic protein or an antigenic fragment thereof or a second antigenic protein or an antigenic fragment thereof or pseudoviral particles wherein at least one epitope of the first antigenic protein or antigenic fragment thereof is immunologically identical to the at least one second antigenic protein or fragment thereof.
The term "vaccine" refers to a biological preparation that induces or enhances immunity to a specific disease. The preparation may comprise a killed or live attenuated pathogen. It may also include one or more compounds derived from a pathogen suitable for eliciting an immune response. In one embodiment, said compound is a polypeptide that is substantially identical or immunologically identical to a polypeptide of said pathogen. In another embodiment, the vaccine comprises a nucleic acid construct that encodes an immunogenic polypeptide that is substantially identical or immunologically identical to a polypeptide of said pathogen. In the latter case, it is also contemplated that the polypeptide is expressed in the individual treated with the vaccine. The underlying principle of vaccination is the generation of an immunological "memory". Stimulation of an individual's immune system with a vaccine induces formation and / or propagation of immune cells that specifically recognize the compound included in the vaccine. At least a portion of said immune cells remain viable for a period of time that may exceed 10, 20 or 30 years after vaccination. If the individual's immune system encounters the pathogen from which the compound capable of eliciting an immune response has been derived within the aforementioned period of time, the immune cells generated by the vaccination are reactivated and enhance the immune response. against the pathogen compared to the immune response of an individual who has not been stimulated with the vaccine and who encounters the immunogenic compounds of the pathogen for the first time.
As used herein, the term "vector" refers to at least one polynucleotide or a mixture of at least one polynucleotide and at least one protein that is capable of introducing the polynucleotide included therein into a cell. In addition, the term "vector" may also refer to at least one polynucleotide formulated with a liposome or lipid nanoparticle preparation that is capable of transfecting a cell with at least one polynucleotide, as described, for example, by Geall et al., 2012, PNAS, 109: 14604-14609.
At least one polynucleotide included in the vector consists of or comprises at least one nucleic acid construct coding for at least one immunogenic protein. In addition to the polynucleotide consisting of or comprising the nucleic acid construct of the present invention, additional polynucleotides and / or polypeptides may be introduced into the cell. The addition of additional polynucleotides and / or polypeptides is also contemplated if said additional polynucleotides and / or polypeptides are required to introduce the nucleic acid construct of the present invention into the cell or the introduction of polynucleotides and / or polypeptides. Additional enhances the expression of the immunogenic polypeptide encoded by the nucleic acid construct of the present invention.
In the context of the present invention, it is contemplated that the antigenic protein or antigenic fragment thereof encoded by the introduced nucleic acid construct is expressed within the cell after introduction of the vector or vectors. Examples of suitable vectors include, but are not limited to, plasmids, cosmids, phages, viral vectors, lipid nanoparticles, or artificial chromosomes.
In one embodiment of the present invention, the viral vector is selected from the group consisting of adenoviral vectors, adeno-associated virus (AAV) vectors (e.g., type 5 and type 2 AAV), alphavirus vectors. (eg Venezuelan equine encephalitis virus (VEE), sindbis virus (SIN), semliki forest virus (SFV), and VEE-SIN chimeras), herpesvirus vectors (eg , vectors derived from cytomegalovirus, such as the rhesus monkey cytomegalovirus (RhCMV), arenavirus vectors (e.g., lymphocytic choriomeningitis virus (LCMV) vectors), measles virus vectors, poxviral vectors , paramyxovirus vectors, baculovirus vectors, vesicular stomatitis virus vectors, retroviruses, lentiviruses, pseudoviral particles, and bacterial spores.
In another embodiment, the vectors are adenoviral vectors, in particular adenoviral vectors derived from humans or a large non-human monkey. Examples of great apes from which adenoviruses are derived are the chimpanzee (Pan), the gorilla (Gorilla) and the orangutan (Pongo), for example the Bonobo (Pan paniscus) and the common chimpanzee (Pan troglodytes) . In general, naturally occurring non-human large apeno adenoviruses are isolated from the stool samples of the respective large monkey. Specifically, the vectors are adenoviral vectors that do not replicate based on the vectors hAd5, hAd11, hAd26, hAd35, hAd49, ChAd3, ChAd4, ChAd5, ChAd6, ChAd7, ChAd8, ChAd9, ChAd10, ChAd11, ChAd16, ChAd17. , ChAd19, ChAd20, ChAd22, ChAd24, ChAd26, ChAd30,
ChAd31, ChAd37, ChAd38, ChAd44, ChAd55, ChAd63, ChAd73,
ChAd82, ChAd83, ChAdl46, ChAdl47, PanAdl, PanAd2, and PanAd3 or competent vectors for Ad4 and Ad7 replication. Human adenoviruses hAd4, hAd5, hAd7, hAd11, hAd26, hAd35 and hAd4 9 are well known in the art. The vectors based on ChAd3, ChAd4, ChAd5, ChAd6, ChAd7, ChAd8, ChAd9, ChAd10, ChAd11, ChAd16, ChAd17, ChAd19, ChAd20, ChAd22, ChAd24, ChAd26, ChAd30, ChAd31, ChAd37, ChAd38, ChAd44, ChAd63 and ChAd82. Existing naturally occurring are described in detail in WO 2005/071093, published as US 20110217332 and incorporated by reference with respect to the adenoviral vectors described therein. The naturally-based PanAd1, PanAd2, PanAd3, ChAd55, ChAd73, ChAd83, ChAdl46, and ChAdl47 based vectors are described in detail in WO 2010/086189, published as US 20120027788 and incorporated by reference by compared to the adenoviral vectors described in.
In another embodiment, the second antigenic protein or an antigenic fragment thereof is immunologically identical to the antigenic protein or antigenic fragment thereof encoded by the nucleic acid construct comprised in the vector. poxvirus.
In another embodiment of the present invention, the vector is present in the form of naked DNA. The term "naked DNA" refers to any nucleic acid molecule, DNA or RNA, that does not encode proteins of a viral vector but encodes at least one antigenic protein or fragment thereof. It is contemplated that naked DNA is not associated with any polypeptides, particularly not with viral polypeptides. For example, naked DNA is present as a plasmid, cosmid or as an artificial chromosome. In another embodiment, the naked DNA encodes a polypeptide that is immunologically identical to the antigenic protein or antigenic fragment thereof encoded by the nucleic acid construct comprised in the poxviral vector.
The term "pseudoviral particles" (PPV) refers to assemblies comprising viral proteins but not nucleic acids. PPVs can be produced by expressing viral surface proteins in appropriate producer cell lines. Lack of nucleic acid, and thus genetic information, makes PPVs non-infectious, creating a safe vaccine. For example, PPV comprises a polypeptide that is immunologically identical to the antigenic protein or antigenic fragment thereof encoded by the nucleic acid construct comprised in the poxviral vector.
In one embodiment of the present invention, the vaccine combination described above is used in a primary-boost regimen. In a first embodiment of this primary-boost regimen, the poxviral vector is used to prime the immune response and the viral vector or antigenic protein or an antigenic fragment thereof is used to stimulate the immune response. (recall) . In another embodiment of the primary-boost regimen, the viral vector or antigenic protein or antigenic fragment thereof is used to prime the immune response and the poxviral vector is used to stimulate the immune response ( recall).
In one embodiment of the present invention, the immune response is initiated by intranasal administration and the immune response is stimulated by at least one intramuscular administration; the immune response is initiated by intranasal administration and the immune response is stimulated by at least subcutaneous administration; the immune response is initiated by intranasal administration and the immune response is stimulated by at least one intradermal administration; the immune response is initiated by intranasal administration and the immune response is stimulated by at least one intragastric administration; the immune response is initiated by intranasal administration and the immune response is stimulated by at least one oral administration; the immune response is initiated by intranasal administration and the immune response is stimulated by at least one topical administration; the immune response is initiated by intranasal administration and the immune response is stimulated by at least one intranasal administration; the immune response is initiated by intramuscular administration and the immune response is stimulated by at least one intramuscular administration; the immune response is initiated by intramuscular administration and the immune response is stimulated by at least subcutaneous administration; the immune response is initiated by intramuscular administration and the immune response is stimulated by at least one intradermal administration; the immune response is initiated by intramuscular administration and the immune response is stimulated by at least one intragastric administration; the immune response is initiated by intramuscular administration and the immune response is stimulated by at least one oral administration; the immune response is initiated by intramuscular administration and the immune response is stimulated by at least one topical administration; the immune response is initiated by intramuscular administration and the immune response is stimulated by at least one intranasal administration; the immune response is initiated by subcutaneous administration and the immune response is stimulated by at least one intramuscular administration; the immune response is initiated by subcutaneous administration and the immune response is stimulated by at least subcutaneous administration; the immune response is initiated by subcutaneous administration and the immune response is stimulated by at least one intradermal administration; the immune response is initiated by subcutaneous administration and the immune response is stimulated by at least one intragastric administration; the immune response is initiated by subcutaneous administration and the immune response is stimulated by at least one oral administration; the immune response is initiated by subcutaneous administration and the immune response is stimulated by at least one topical administration; the immune response is initiated by subcutaneous administration and the immune response is stimulated by at least one intranasal administration; the immune response is initiated by intradermal administration and the immune response is stimulated by at least one intramuscular administration; the immune response is initiated by intradermal administration and the immune response is stimulated by at least subcutaneous administration; the immune response is initiated by intradermal administration and the immune response is stimulated by at least one intradermal administration; the immune response is initiated by intradermal administration and the immune response is stimulated by at least one intragastric administration; the immune response is initiated by intradermal administration and the immune response is stimulated by at least one oral administration; the immune response is initiated by intradermal administration and the immune response is stimulated by at least one topical administration; the immune response is initiated by intradermal administration and the immune response is stimulated by at least one intranasal administration; the immune response is initiated by intragastric administration and the immune response is stimulated by at least one intramuscular administration; the immune response is initiated by intragastric administration and the immune response is stimulated by at least subcutaneous administration; the immune response is initiated by intragastric administration and the immune response is stimulated by at least one intradermal administration; the immune response is initiated by intragastric administration and the immune response is stimulated by at least one intragastric administration; the immune response is initiated by intragastric administration and the immune response is stimulated by at least one oral administration; the immune response is initiated by intragastric administration and the immune response is stimulated by at least one topical administration; the immune response is initiated by intragastric administration and the immune response is stimulated by at least one intranasal administration; the immune response is initiated by oral administration and the immune response is stimulated by at least one intramuscular administration; the immune response is initiated by oral administration and the immune response is stimulated by at least subcutaneous administration; the immune response is initiated by oral administration and the immune response is stimulated by at least one intradermal administration; the immune response is initiated by oral administration and the immune response is stimulated by at least one intragastric administration; the immune response is initiated by oral administration and the immune response is stimulated by at least one oral administration; the immune response is initiated by oral administration and the immune response is stimulated by at least one topical administration; the immune response is initiated by oral administration and the immune response is stimulated by at least one intranasal administration; the immune response is initiated by topical administration and the immune response is stimulated by at least one intramuscular administration; the immune response is initiated by topical administration and the immune response is stimulated by at least subcutaneous administration; the immune response is initiated by topical administration and the immune response is stimulated by at least one intradermal administration; the immune response is initiated by topical administration and the immune response is stimulated by at least one intragastric administration; the immunological response is initiated by topical administration and the immune response is stimulated by at least one oral administration; the immune response is initiated by topical administration and the immune response is stimulated by at least one topical administration; the immune response is initiated by topical administration and the immune response is stimulated by at least intranasal administration.
In one embodiment, the immune response is initiated by intranasal administration and the immune response is stimulated by at least one intramuscular administration.
In yet another embodiment, the immune response is initiated by intranasal administration and the immune response is stimulated by at least intranasal administration.
In yet another embodiment, the immune response is initiated by intramuscular administration and the immune response is stimulated by at least one intramuscular administration.
In another aspect, the present invention relates to a vaccine composition comprising a poxviral vector for priming an immune response as defined above or a vaccine combination comprising a poxviral vector and an agent selected from the group consisting of ) a vector comprising a nucleic acid sequence encoding at least a second antigenic protein or an antigenic fragment thereof, (ii) a second antigenic protein or antigenic fragment thereof, and (iii) pseudoviral particles.
The term "composition" refers to the combination comprising an antigenic protein or fragment thereof or a pseudoviral particle or vector comprising a nucleic acid construct and at least one other compound selected from the group consisting of pharmaceutically acceptable carriers pharmaceutical excipients and excipients. "Pharmaceutically Acceptable" means approved by a federal or state regulatory agency or listed in the US Pharmacopoeia or other pharmacopoeia generally recognized for use in animals, and more specifically in humans.
The term "carrier" as used herein refers to a pharmacologically inactive substance such as, but not limited to, a diluent, excipient, or vehicle with which the therapeutically active ingredient is administered. Such pharmaceutical carriers can be liquid or solid. A liquid carrier includes, but is not limited to, sterile liquids, such as saline solutions in water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soy soybean oil, mineral oil, sesame oil and the like. Saline solutions and aqueous solutions of dextrose and glycerol can also be used as liquid carriers, particularly for injectable solutions. Saline is a preferred carrier when the pharmaceutical composition is administered intravenously or intranasally by a nebulizer.
Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc. sodium chloride, dried skimmed milk, glycerol, propylene glycol, water, ethanol and the like.
Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin.
The term "adjuvant" refers to agents that enhance, stimulate, activate, potentiate or modulate the immune response to the active ingredient of the composition at either the cellular or humoral level, for example, immunological adjuvants stimulate the immune system response to the immune system. antigen in question, but have no immunological effect themselves. Examples of such adjuvants include, but are not limited to, inorganic builders (e.g., inorganic metal salts such as aluminum phosphate or aluminum hydroxide), organic builders (eg, saponins, or the like). squalene), oil-based adjuvants (e.g., Freund's complete adjuvant and Freund's incomplete adjuvant), cytokines (e.g., IL-Ιβ, IL-2, IL-7, IL-12 , IL-18, GM-CSF, and INF-γ), particulate adjuvants (e.g., immunostimulatory complexes (ISCOMs), liposomes, or biodegradable microspheres), virosomes, bacterial adjuvants (e.g., monophosphoryl lipid A, or muramyl peptides), synthetic adjuvants (e.g., nonionic block copolymers, muramyl-peptide analogs, or synthetic lipid A), or synthetic polynucleotide adjuvants (e.g., polyarginine or polylysine).
Brief description of the figures
Figure 1: The MVA coding for an invariant chain (1i) - NS antigen induces a stronger response of T cells in mice. Two groups of mice were immunized with 2 x 10 5 MVA plate (pfu) units including NS antigen (left image) and MVA including NS linked to an invariant chain (right image). The total T cell response to the NS antigen was measured by an ELISpot assay for IFNy and the numbers on the ordinate represent stain forming cells (SFC) / million splenocytes.
Figure 2: the MVA coding for an invariant (li) -antigen chain NS induces a stronger response of T cells than the corresponding adenovirus in mice. Two groups of mice were immunized with MVA encoding the NS antigen (MVA wt) or the human invariant chain-linked antigen (MVAli). Two additional groups of mice were immunized with a comparable dose of ChAd3 encoding NS (ChAd3 wt) or NS linked to a human invariant chain (ChAd31i). The total T cell response to the NS antigen was measured by an ELISpot assay for IFNy and the numbers on the ordinate represent stain forming cells (SFC) / million splenocytes.
Figure 3: MVA booster comprising NS antigen bound to an invariant chain increases the generation of HCV-NS specific T cells in macaques.
Two groups of 4 macaques were sensitized with ChAd31iNS and 50 weeks later, they were recalled with MVA-NS (gray bars) or with MVA-liNS (black bars). Image A shows the response by an ELISpot test for IFNy one week (peak of the booster) or 3 months after the booster (memory). The numbers on the ordinate represent the stain forming cells (SFC) / million PBMCs. Image B shows a higher frequency of CD8 by IFNy ICS one week after recall with MVAliNS (black bars). The numbers on the ordinate represent the% of CD8 T cells specific for the antigen producing IFNy.
Figure 4: Schematic diagram showing the four isoforms of the human invariant chain (p33, p35, p41, p43, isoform c) and the two isoforms of the murine invariant chain (p31, p41). In human p35 and p43, 16 additional residues are present at the N-terminus because of a translation initiation variant. In the human p41 and p43 isoforms and the murine p41 isoform, an additional domain is present because of a splice variant. At human isoform c, two exons are missing from human p33 and p35 (three exons compared to human p41 and p43) leading to frame shift.
Examples
Example 1 - Priming with MVA comprising the invariant chain-linked NS antigen (MVA-hli NS) increases the generation of HCV-NS specific T cells in mice.
Two groups of Balb / c mice were immunized intramuscularly with 2 x 105 pfu (plaque forming units) of MVA encoding the NS antigen or with the same dose of MVA comprising NS antigen bound to a human invariant chain. . The NS region encompasses two-thirds of the HCV genome and encodes five different proteins (NS3, NS4A, NS4B, NS5A and NS5B) that result from the proteolytic cleavage of the HCV polyprotein by the encoded NS3 protease. Ten days after immunization, splenocytes were removed and the HCV-NS specific T cell response was assayed by an ELIspot assay for IFNy using peptide groups spanning the NS region. The response was evaluated by summing the reactivities against the six individual peptide groups and subtracting the background (spots counted in the control wells without any peptide). The level of specific T cells targeting the NS antigen was higher in the mice sensitized with the MVA-based vaccine (FIG. 1).
Example 2 - Priming with MVA comprising invariant chain-linked NS antigen (MVA-hli NS) induces a stronger T-cell response in mice than the corresponding adenoviral vector.
Two groups of Balb / c mice were immunized intramuscularly with 2 × 10 5 pfu of MVA encoding the NS antigen or with the same dose of MVA comprising NS antigen bound to a human invariant chain. Two additional groups of mice were immunized with 2 x 10Λ5 μi (infectious units) of ChAd3 encoding the NS antigen or with the same dose of ChAd3 comprising NS antigen bound to a human invariant chain. The peak of the immune response was assessed on splenocytes taken 10 and 21 days after immunization with the MVA and ChAd3 vector vaccines, respectively. T cell response was assessed by an IFNγ ELIspot assay using peptide groups covering the NS antigen. The results (Figure 2) show that the MVA-based vaccine elicits a superior response to the corresponding li-based ChAd3 vaccine.
Example 3 - MVA booster comprising the invariant chain-linked NS antigen (MVA-hli NS) increases the generation of HCV-NS specific T cells in macaques.
Two groups of 4 macaques were sensitized with ChAd31iNS and 50 weeks later, they were recalled with MVA-NS (gray bars) or with MVA-liNS (black bars). The injected dose was 1 x 1010 vp for the adenoviral vectors, and 2 x 108 pfu for the MVA vectors. The immune response was assessed on PBMCs taken 1 week (response peak) and 3 months (memory response) after priming with an IFNγ ELIspot assay and intracellular IFNy staining (ICS) using groups of peptides covering the NS antigen. As illustrated in FIG. 3, the upper response of the ELISpot test was induced in the group receiving the MVAliNS at the two time points (black bars). Image A shows the response by the ELIspot test for IFNy a week or 3 months after the recall. Image B shows the higher frequency of CD8 T cells producing IFNγ by ICS one week after the MVAliNS boost (black bars).
Materials and processes
Adenoviral vectors and MVA
The ChAd3 vector expressing the entire NS3-5B (NS) region of HCV from genotype 1b, strain bk, has been previously described (Colloca et al., Sci Transi Med 4 (115), 115rall2, 2012). The MVA vector expressing the same cassette was derived and prepared as previously described (Cottingham, M.G. et al., PLoS ONE 3, el638, 2008, Di Lullo, G. et al., Virol.
Methods 156, 37-43, 2009). The human liII insert (p35, NCBI reference sequence: NM_004355) was synthesized by GeneArt (Life Technologies, Paisley, UK) and then cloned at the N-terminus of the NS transgene under the control of HCMV and BGHpA.
Animals and vaccinations
All experimental procedures were carried out in accordance with national and international laws and policies (European Council Directive 86/609, Italian Legislative Decree 116/92). The ethics committee of the Italian Ministry of Health approved this research. Animal handling procedures were performed under anesthesia and every effort was made to minimize suffering and reduce numbers of animals. Six week old Balb / c or C57B1 / 6 female mice were purchased from Charles River (Horn, Italy), and experimental groups of 5 mice each were formed. The ChAd3 and MVA vectors were administered intramuscularly in the quadriceps by delivering a volume of 50 μΐ per site (100 μΐ final volume).
Cynomolgus (Macaca fascicularis) macaques female, naive, 11 to 19 years old (weight range 3.2 to 6.5 kg) from a colony raised for this purpose housed at the Institute of Cell Biology and Neurobiology (National
Research Council of Italy, Rome), were assigned to experimental groups of four animals each. All immunizations were delivered intramuscularly into the deltoid muscle by injecting 0.5 ml of diluted virus into stabilization buffer. The injected dose was 1 x 1010 vp for the adenoviral vectors, and 2 x 108 pfu for the MVA vectors. During handling, the animals were anesthetized by i.m. 10 mg / kg ketamine hydrochloride.
peptides
A set of 494 peptides, 15 amino acids long, overlapping 11 amino acids and covering the NS3-NS5B (1985 aa) open reading frame of the HCV genotype lb strain BK was obtained from BEI Resources (Manassas, VA).
ELISpot test for ex vivo IFNy with mouse and macaque samples
S4510 MSIP plates (Millipore) were sensitized with 10 μρ / μιτιΐ of anti-mouse IFNγ or anti-mouse IFNγ antibodies (both from U-CyTech Utrecht, The Netherlands) overnight at 4 ° C. After washing and blocking, mouse splenocytes or peripheral blood mononuclear cells
(CMSP) were deposited in duplicate at two different densities (2 x 10 5 and 4 x 10 5 cells / well) and stimulated overnight with groups of overlapping sea peptides at a final concentration of 4 μg / ml. ml of each single peptide. DMSO peptide diluents (Sigma-Aldrich, Milan, Italy) and ConA (Sigma-Aldrich, Milan, Italy) were used respectively as negative and positive controls. Plates were developed by subsequent incubations with biotinylated anti-mouse IFNγ or anti-IFNγ antibody (both from U-CyTech Utrecht, The Netherlands), streptavidin-alkaline phosphatase conjugate (BD).
Biosciences, NJ) and finally with a BCIP / NBT 1-Step solution (Thermo Fisher Scientific, Rockford, IL). The plates were acquired and analyzed by an automated plate reader A.EL.VIS. The ELISpot response was considered positive when all of the following conditions were met: production of IFNγ present in wells stimulated with Con-A; at least 50 spots / million of splenocytes or PBMCs specific for at least one group of peptides; the number of spots observed in the positive wells was three times the number detected in the control wells (DMSO); and that the responses have decreased with cell dilutions. Data from the ELISpot assay were expressed in IFNy (SFC) staining-forming cells per million splenocytes or PBMCs.
Intracellular cytokine staining (ICS) and FACS analysis with macaque samples
Briefly, 2 x 10 6 Mono PBMCs were stimulated at 37 ° C in 5% CO 2 for 15 to 20 hours using peptide groups as an antigen at 2 μg / ml of each final concentration of peptides in the presence human anti-CD28 / CD49d costimulatory antibodies (BD Biosciences, NJ) and Brefeldin A (Sigma-Aldrich, Milan, Italy). DMSO (Sigma-
Aldrich, Milan, Italy) was used as a negative control, and staphylococcal enterotoxin B (SEB, Sigma-Aldrich, Milan, Italy) was used as a positive control. After overnight stimulation, PBMCs were stained with the following surface antibodies: Monkey CD3 anti-CD3, clone SP34-2; PerCp-Cy5.5 anti-CD4 monkey, clone L200; PE anti-human CD8, clone RPA-T8 (all from BD Biosciences, NJ). Intracellular staining was performed after treatment with Cytofix / Cytoperm and in the presence of PermWash (BD Biosciences, NJ) using the FITC-labeled human anti-IFNy MD-1 clone (U-CyTech Utrecht, The Netherlands). The stained cells were acquired on a FACS Canto flow cytometer, and analyzed using DIVA software (BD Biosciences, NJ). At least 30,000 events dependent on CD8 +, CD3 + were acquired for each sample.

权利要求:
Claims (20)
[1]
A poxviral vector comprising a nucleic acid construct for use in priming or stimulating an immune response, the nucleic acid construct comprising: (i) a nucleic acid sequence encoding at least one protein antigenically or an antigenic fragment thereof operably linked to (ii) a nucleic acid encoding at least one invariant chain.
[2]
A poxviral vector according to claim 1, wherein at least one encoded invariant chain is derived from a mammal.
[3]
A poxviral vector according to claims 1 or 2, wherein at least one encoded invariant chain is characterized by at least one of the following characters: (i) the endogenous KEY region is deleted or substituted by a different sequence; (ii) methionine in positions 107 and 115 (human invariant chain) or positions 90 and 98 (murine invariant chain) or corresponding positions in another invariant chain is substituted by another amino acid; (iii) the first 16 amino acids of the sequence of the wild-type human invariant chain are deleted; (iv) at least one sorting peptide is added to, removed from or replaces the endogenous chain sorting peptide of the invariant chain, and / or (v) at least one CLIP region is added to, removed from or replaces the endogenous CLIP region of at least one invariant chain.
[4]
A poxviral vector according to any one of claims 1 to 3, wherein at least one encoded invariant chain is a fragment of SEQ ID NO: 1 or SEQ ID NO: 3 of at least 40 consecutive amino acids or at least 85% sequence identity with the same fragment of SEQ ID NO: 1 or SEQ ID NO: 3.
[5]
A poxviral vector according to any one of claims 1 to 4, wherein at least one antigenic protein is a protein of a pathogenic organism, a cancer-specific protein, or a protein associated with an abnormal physiological response.
[6]
A poxviral vector according to claim 5, wherein the pathogenic organism is a multicellular virus, bacterium, protist or parasite.
[7]
7. Poxviral vector according to any one of claims 1 to 6, wherein the poxvirus is selected from a viral vector of orthopox, parapox, yatapox, avipox and molluscipox.
[8]
A poxviral vector according to claim 7, wherein said orthopox viral vector is a poxviral monkey vector, a cow poxviral vector or a vaccinia virus vector, preferably a modified vaccinia virus Ankara (MVA).
[9]
A poxviral vector according to any one of claims 1 to 7, wherein the priming of the immune response is part of a homologous primary-boost regimen.
[10]
A poxviral vector according to any one of claims 1 or 2, wherein the priming of the immune response is part of a heterologous priming-boost regimen.
[11]
A poxviral vector according to any one of claims 1 to 10, wherein the poxviral vector is administered intranasally, intramuscularly, subcutaneously, intradermally, intragastrically, orally and topically.
[12]
A vaccine combination comprising: (a) a poxviral vector comprising a nucleic acid construct, the nucleic acid construct comprising: (i) a nucleic acid sequence encoding at least a first antigenic protein or antigenic fragment of it is operably linked to (ii) a nucleic acid encoding at least one invariant chain and (b) a vector comprising a nucleic acid sequence encoding at least a second antigenic protein or an antigenic fragment thereof or a second antigenic protein or an antigenic fragment thereof or pseudoviral particles wherein at least one epitope of the first antigenic protein or antigenic fragment thereof is immunologically identical to the second antigenic protein or fragment thereof. it.
[13]
The vaccine combination according to claim 12, wherein the viral vector is selected from an adenoviral vector, a poxviral vector, an adeno-associated viral vector, a lentiviral vector, an alphavirus vector, a measles virus vector, an arenavirus vector, a paramyxovirus vector, a baculovirus vector, naked DNA and pseudoviral particles.
[14]
The vaccine combination according to claim 13, wherein the adenoviral vector is an adenoviral vector derived from a large non-human monkey, preferably an adenoviral vector of chimpanzee or bonobo.
[15]
15. A vaccine combination according to any one of claims 12 to 14 for use in a primary-boost regimen.
[16]
The vaccine combination according to claim 15, wherein the poxviral vector (a) is used for priming the immune response and the viral vector or antigenic protein of (b) is used for stimulating the immune response.
[17]
The vaccine combination of claim 15, wherein the viral vector or antigenic protein of (b) is used for priming the immune response and the poxviral vector (a) is used for stimulating the immune response.
[18]
The vaccine combination according to claims 16 or 17, wherein the immune response is initiated by a route of administration selected from the group consisting of intranasal administration, intramuscular administration, subcutaneous administration, intradermal administration, administration. intragastric, oral and topical administration; and the immune response is stimulated by a route of administration selected from the group consisting of intranasal administration, intramuscular administration, subcutaneous administration, intradermal administration, intragastric administration, oral administration and topical administration.
[19]
19. A composition comprising the poxviral vector of claim 1 for stimulating an immune response in a subject.
[20]
The composition of claim 19 for stimulating an immune response in a subject by further administering (i) a vector comprising a nucleic acid sequence encoding at least a second antigenic protein or antigenic fragment thereof. -this ; (ii) a second antigenic protein or an antigenic fragment thereof; and pseudoviral particles.

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法律状态:
2019-12-05| MM| Lapsed because of non-payment of the annual fee|Effective date: 20190331 |

优先权:

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申请号 | 申请日 | 专利标题

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PCT/EP2013/055409|WO2014139587A1|2013-03-15|2013-03-15|Improved poxviral vaccines|

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WO201355409|2013-03-15|

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