WO2013017545A1 - Souris transgéniques humanisées hla-a2 / hla-dp4 et leurs utilisations en tant que modèle expérimental pour la recherche biomédicale et le développement biomédical - Google Patents

Souris transgéniques humanisées hla-a2 / hla-dp4 et leurs utilisations en tant que modèle expérimental pour la recherche biomédicale et le développement biomédical Download PDF

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WO2013017545A1
WO2013017545A1 PCT/EP2012/064813 EP2012064813W WO2013017545A1 WO 2013017545 A1 WO2013017545 A1 WO 2013017545A1 EP 2012064813 W EP2012064813 W EP 2012064813W WO 2013017545 A1 WO2013017545 A1 WO 2013017545A1
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hla
mouse
response
antigen
antigens
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PCT/EP2012/064813
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Yu Chun LONE
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Institut National De La Sante Et De La Recherche Medicale (Inserm)
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70514—CD4
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0278—Knock-in vertebrates, e.g. humanised vertebrates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/15—Animals comprising multiple alterations of the genome, by transgenesis or homologous recombination, e.g. obtained by cross-breeding
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention concerns transgenic mice comprising non-functional H2 class I and H2 class II genes and comprising a functional HLA-A2 transgene and a functional HLA-DP4 transgene.
  • the present invention also relates to methods of identifying the presence of one or more epitopes in a candidate antigen or group of antigens, methods of determining the immune response of a mouse following its immunization with an antigen or a vaccine or following its treatment with an immunotherapeutic compound, methods of comparing the efficiency of a response induced by two or more vaccinal or immunotherapeutic compositions, and methods of determining whether a vaccinal or an immunotherapeutic composition poses a risk of induction of an autoimmune disease when administered to a human.
  • the invention further concerns transgenic mouse cells comprising non-functional H2 class I and H2 class II genes and comprising a functional HLA-A2 transgene and a functional HLA-DP4 transgene, and kits for identifying the presence of one or more epitopes in a candidate antigen or group of antigens.
  • MHC molecules play a pivotal role in the shaping of both the specificity and the functional issue of adaptive immunity. Indeed, adaptive immunity establishment relies on specific selecting of presentable MHC restricted epitopes.
  • HLA class I or HLA class II transgenic mice have been developed for mapping the potential pathogenic and tumoral epitopes for human clinical use. Identification of HLA-restricted epitopes is particularly important for boosting the development of HLA restricted epitope-based vaccine candidates and for developing reagents to monitor both CD8 and CD4 specific T-cellular responses. Further validation of documented epitopes is still needed to identify candidate immunologic biomarkers inducing clinical responses. In addition, considerable effort focused on specific induction of T-cytotoxic lymphocytes (CTL) to prevent or control viral diseases, or specific induction of T-helper lymphocytes to control or cure autoimmune diseases.
  • CTL T-cytotoxic lymphocytes
  • Promising protective antiviral immunity using CTL epitope-based vaccines have been demonstrated in several experimental models of infection.
  • Promising protective strategy using CD4 restricted epitope-based vaccines have also been designed in several experimental autoimmune and pathogenic diseases. However, in human preclinical or clinical trial, these vaccines did not induce significant or sufficient activities. Inefficacity is probably due to no simultaneous activation of those human specific B cells, cytotoxic T cells and helper T cells for protective immunity.
  • this advanced model could merely represent around 3-9 % of the human population (30-50% for HLA-A2.1 , 6-18% for DR1 ), which highly limits its usage in pharmaceutical development, as it is difficult to find HLA-A2/DR1 individuals to confirm obtained results. For this reason, it would be helpful to obtain strains of mice co-expressing HLA-A2.1 with other HLA class II molecules, even if the binding of peptides to HLA class II molecules is less restrictive than to class I molecules.
  • the HLA-DP4 locus has been demonstrated to be one of the most abundant HLA alleles (20-60%) in the world, even higher than HLA-A2 (30-50%). It consists of two subtypes, DP0401 and DP0402, which differ from each other by only 3 amino acids. Preliminary reports show that DP0401 and DP0402 molecules together take up the frequency of 50% in Europe, 60% in south America, 80% in north America, 60% in India, 40% in Xinjiang district in China, 25% in Africa and 15% in Japan. More interestingly, some evidence suggests that antigen presentation by HLA-DP4 molecules may be critical for virus elimination and plays an important role in the pathogenesis of chronic hepatitis B.
  • HLA-DP4 in pathogen infection and some autoimmunity diseases.
  • WO2006/092515 describes the construction of a transgenic mice HLA-DP4 and HLA-DP4 hCD4 + mCD4°.
  • HLA-DP4-restricted HTL epitopes There are a limited number of documented HLA-DP4-restricted HTL epitopes, which were identified, in viral and tumoral cells. Therefore, the inventors determined that it would be useful to take advantage of the HLA-DP4 importance and high frequency in the world population to develop tools allowing the identification and/or the validation of new HLA- DP4 epitope based vaccine that could induce efficient protection in a large population.
  • HLA-A2, HLA-DP4, hCD4 transgenes expression and the immunological activities
  • HLA-A2, HLA-DP4, hCD4 immunological activities
  • they have screened for HLA-DP4 restricted epitopes derived from Ag-HBs by using this A2/DP4 mouse.
  • Four new HLA-DP4 restricted epitopes (S 25 6- 2 68, S 326 - 33 8, S 34 7- 3 58, S 352 -36 4 ) have been demonstrated to trigger similar T-helper cells response in both vaccinated A2/DP4 mice and HBV vaccinated HLA-DP4-positive donors.
  • this animal model can facilitate the identification of novel HLA-A2 or/and DP4 restricted epitopes that may be used for developing monitoring biomarkers, evaluating innovative vaccines and immunotherapies, as well as decrypting the correlation between some DP4-restricted response and disease prevention or pathogenesis.
  • the present invention concerns a transgenic mouse comprising non-functional H2 class I and H2 class II genes and comprising a functional HLA-A2 transgene and a functional HLA-DP4 transgene.
  • the invention provides a transgenic mouse comprising a disrupted H2 class I gene, a disrupted H2 class II gene, a functional HLA class I transgene, and a functional HLA class II transgene.
  • a "non-functional" gene is a gene that does not produce an mRNA transcript, or does not produce a properly processed protein.
  • a non-functional gene is a disrupted gene.
  • a “disrupted” gene is a gene that has been mutated using homologous recombination or other approaches known in the art.
  • the mutation present in the disrupted gene may correspond to an insertion, a deletion, or a substitution of one or more nucleic acids as compared to the non-disrupted corresponding gene.
  • "Homologous recombination” is a general approach for targeting mutations to a preselected, desired gene sequence of a cell in order to produce a transgenic animal.
  • the term “recombination” relates to a process by which a molecule of nucleic acid (usually DNA or RNA) is broken and then joined to a different one.
  • An “homologous recombination” is a type of recombination in which nucleotide sequences flanked by two similar or identical molecules of nucleic acid are exchanged.
  • Gene targeting involves the use of standard recombinant DNA techniques to introduce a desired mutation into a cloned DNA sequence of a chosen locus. That mutation is then transferred through homologous recombination to the genome of a pluripotent, embryo-derived stem (ES) cell.
  • ES embryo-derived stem
  • the altered stem cells are microinjected into mouse blastocysts and are incorporated into the developing mouse embryo to ultimately develop into chimeric animals.
  • germ line cells of the chimeric animals will be derived from the genetically altered ES cells, and the mutant genotypes can be transmitted through breeding.
  • the gene of interest In order to utilize the "gene targeting" method, the gene of interest must have been previously cloned, and the intron-exon boundaries determined. The method results in the insertion of a marker gene into a translated region of a particular gene of interest. Thus, use of the gene targeting method results in the gross destruction of the gene of interest.
  • the chimeric or transgenic animal cells of the present invention are prepared by introducing one or more DNA molecules into a cell, which may be a precursor pluripotent cell, such as an ES cell, or equivalent.
  • a cell which may be a precursor pluripotent cell, such as an ES cell, or equivalent.
  • the term "precursor” is intended to denote only that the pluripotent cell is a precursor to the desired ("transfected") pluripotent cell, which is prepared in accordance with the teachings of the present invention.
  • the pluripotent (precursor or transfected) cell can be cultured in vivo in a manner known in the art to form a chimeric or transgenic animal. Any ES cell can be used in accordance with the present invention. It is, however, preferred to use primary isolates of ES cells.
  • Such isolates can be obtained directly from embryos, such as the CCE cell line, or from the clonal isolation of ES cells from the CCE cell line.
  • Such clonal isolation can be accomplished according to the method of E. J. Robertson (In: Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, (E. J. Robertson, Ed.), IRL Press, Oxford, 1987), which reference and method are incorporated herein by reference.
  • the purpose of such clonal propagation is to obtain ES cells, which have a greater efficiency for differentiating into an animal.
  • Clonally selected ES cells are approximately 10-fold more effective in producing transgenic animals than the progenitor cell line CCE.
  • clonal selection provides no advantage.
  • ES cell lines which have been clonally derived from embryos, are the ES cell lines, AB1 (hprt.sup.+) or AB2.1 (hprt.sup.-).
  • the ES cells are preferably cultured on stromal cells (such as STO cells (especially SNC4 STO cells) and/or primary embryonic fibroblast cells). Methods for the production and analysis of chimeric mice are well known by the skilled in the art.
  • the stromal (and/or fibroblast) cells serve to eliminate the clonal overgrowth of abnormal ES cells.
  • the cells are cultured in the presence of leukocyte inhibitory factor ("lif"). Since the gene encoding lif has been cloned, it is especially preferred to transform stromal cells with this gene, by means known in the art, and to then culture the ES cells on transformed stromal cells that secrete lif into the culture medium.
  • leukocyte inhibitory factor leukocyte inhibitory factor
  • transgene refers to a nucleic acid sequence, which is partly or entirely heterologous, i.e., foreign, to the "transgenic" animal or cell into which it is introduced, or, is homologous to an endogenous gene of the "transgenic” animal or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the animal's genome in such a way as to alter the genome of the cell into which it is inserted (e.g., it is inserted at a location which differs from that of the natural gene or its insertion results in the disruption of a natural gene).
  • a transgene can be operably linked to one or more transcriptional regulatory sequences and any other nucleic acid, such as introns, that may be necessary for optimal expression of a selected nucleic acid.
  • Exemplary transgenes of the present invention encode, for instance an HLA polypeptide.
  • Other exemplary transgenes are directed to disrupting one or more H2 genes by homologous recombination with genomic sequences of an H2 gene.
  • a “functional transgene” is one that produces an mRNA transcript, which in turn produces a properly processed protein in at least one cell of the mouse comprising the transgene.
  • One of skill will realize that the diverse set of known transcriptional regulatory elements and sequences directing post-transcriptional processing provide a library of options from which to direct the expression of a transgene in a host mouse.
  • expression of an HLA transgene under the control of an H2 gene regulatory element may be preferred.
  • HLA is the human MHC complex
  • H2 the mouse MHC complex
  • the human complex comprises three class I a-chain genes, HLA-A, HLA-B, and HLA-C, and three pairs of MHC class II a- and ⁇ -chain genes, HLA-DR, -DP, and -DQ.
  • the three class I a-chain genes are H2-L, H2-D, and H2-K.
  • the mouse MHC class II genes are H2-A and H2-E.
  • embodiments of the invention disclosed herein may substitute one polymorphic HLA antigen for another or one HLA allele for another.
  • the HLA-A2 transgene comprised by the transgenic mouse according to the invention is an HLA-A2.1 transgene and/or the HLA-DP4 transgene is an HLA-DP4.1 transgene.
  • the present invention also concerns a transgenic mouse comprising non-functional H2 class I and H2 class II genes and comprising a functional HLA-A2.1 transgene and a functional HLA-DP4.1 transgene.
  • An example of an HLA-A2.1 transgene is one that comprises the HLA-A2.1 sequence SEQ ID NO: 1 provided in the sequence listing.
  • An example of an HLA-DP4.1 transgene is one that comprises the HLA-DP4.1 sequence SEQ ID NO: 2 and SEQ ID NO: 3 provided in the sequence listing.
  • the transgenic mouse according to the invention further comprises a functional human CD4 (hCD4) transgene.
  • the transgenic mouse further comprises a non-functional murine CD4 (mCD4) gene.
  • the human CD4 (hCD4) transgene is one that comprises the hCD4 sequence SEQ ID NO: 4 provided in the sequence listing.
  • the HLA-A2 transgene of the invention comprises the sequence SEQ ID NO: 1 and/or the HLA-DP4 transgene of the invention comprises the sequence SEQ ID NO: 2 and SEQ ID NO: 3 and/or the functional human CD4 (hCD4) transgene of the invention comprises the sequence SEQ ID NO: 4.
  • the invention provides a transgenic mouse deficient for both H2 class I and class II genes, wherein the transgenic mouse comprises a functional HLA-A2 transgene and a functional HLA-DP4 transgene.
  • the HLA-A2/HLA-DP4 transgenic, H2 class l-/class ll-KO mice express, in a ⁇ 2 ⁇ - ⁇ context, a HLA-A2.1 monochain in which the human 32m is covalently linked by a peptidic arm to the HLA-A2.1 heavy chain. They further lack cell surface expression of conventional H2 IA and IE class II molecules as a result of the inactivation of the H2 IA3 b gene, since H2 ⁇ is a pseudogene in the H2 b haplotype.
  • the transgenic mouse has the genotype HLA-A2 + HLA- DP4 + 32m° ⁇ °. In other embodiments, the transgenic mouse has the genotype HLA-A2 + HLA-DP4 + hCD4 + 32m° ⁇ ° mCD4°.
  • the mouse of the invention is an optimized, humanized transgenic mouse, whose H2 class I (mouse 32m) and class II (H2 ⁇ 13 ) genes have been deleted and replaced with equivalent human genes HHD (HLA-A * 0201 : SEQ ID NO: 1 ), HLA-DPA * 0103 (SEQ ID NO: 2) and HLA-DPB * 0401 (SEQ ID NO: 3).
  • mice of the invention which comprise a knock-out for both H2 class I and class II genes, and express HLA class I transgenic molecules and HLA class II transgenic molecules represent a completely humanized experimental mouse that can be used to simultaneously detect the presence of antigen-specific antibodies, an antigen-specific HLA-DP4 restricted T cell response, and an antigen-specific HLA-A2 restricted T cell response. These mice are useful to study how mutual coordination operates between a T- cytotoxic response, a T-helper response and, optionally, a humoral response. These mice represent an optimized tool for basic and applied vaccinology studies.
  • HLA-A2.1 -/HLA-DP4-transgenic H2 class l-/class ll-KO mice is completely restricted by the human HLA molecules, with a complete absence of immune responses restricted by the murine MHC molecules.
  • the absence of competition between murine MHC and human (transgenic) HLA immune responses allows for use of these mice to characterize epitopes in human vaccines that require collaboration between H LA- restricted CD4+ T helper and HLA-restricted CD8+ T cytolytic cells.
  • another aspect of the invention is a method of identifying the presence of one or more epitopes in a candidate antigen or group of antigens, wherein the epitope elicits a specific humoral response, a T-helper HLA-DP4 restricted response, and/or a T- cytotoxic HLA-A2 restricted response, said method comprising:
  • HLA-A2 restricted T-cytotoxic response in the mouse identifies an epitope which elicits a HLA-A2 restricted T-cytotoxic response in the antigen.
  • An antigen is a substance or a molecule that is able to induce an adaptive immune response.
  • An adaptive immune response may for instance be a humoral and/or cell- mediated immune response.
  • a “humoral immune response” or “humoral response” refers to antibody-mediated specific immunity.
  • a “cell-mediated immune response” refers to specific immunity mediated by T cells.
  • T-cells play a central role in many aspects of acquired immunity, carrying out a variety of regulatory and defensive functions.
  • T cells designated cytotoxic T- cells
  • helper T-cells respond to perceived foreign antigens by stimulating B cells to produce antibodies, or by suppressing certain aspects of a humoral or cellular immune response.
  • a "T-helper response” is an immune response mediated by T-helper cells
  • a "HLA-DP4 restricted T-helper response” is a T-helper response that involves at least one HLA-DP4 molecule.
  • a "T-cvtotoxic response” is an immune response mediated by T-cytotoxic cells
  • a "HLA-A2 restricted T-cvtotoxic response” is a T- cytotoxic response that involves at least one HLA-A2 molecule.
  • An “epitope” is a site on an antigen that is recognized by the immune system.
  • An antibody epitope is a site on an antigen recognized by an antibody.
  • a T-cell epitope is a site on an antigen that binds to an MHC molecule.
  • a T-helper epitope is one that binds to an MHC class II molecule.
  • a T-cytotoxic epitope is one that binds to an MHC class I molecule.
  • Th T-helper cells
  • Th1 cells detect invading pathogens or cancerous host cells through a recognition system referred to as the T cell antigen receptor.
  • Th1 -related processes generally involve the activation of non-B cells and are frequently characterized by the production of IFN-gamma.
  • Th1 system is primarily independent from the production of humoral antibodies, Th1 cytokines do promote immunoglobulin class switching to the lgG 2a isotype.
  • Th1 cells Upon detection of a foreign antigen, most mature Th1 cells direct the release of IL-2, IL-3, IFN-gamma, TNF-beta, GM-CSF, high levels of TNF-alpha, MIP-1 alpha, MIP-1 beta, and RANTES. These cytokines promote delayed-type hypersensitivity and general cell- mediated immunity.
  • IL-2 for instance, is a T cell growth factor that promotes the production of a clone of additional T cells sensitive to the particular antigen that was initially detected. The sensitized T cells attach to and attack cells or pathogens containing the antigen.
  • Th2 cells tend to promote the secretion of IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, GM-CSF, and low levels of TNF-alpha.
  • the Th2 response promotes humoral immunity by activating B cells, stimulating antibody production and secretion, and inducing class switching to IgA, IgG, and IgE isotypes.
  • Another aspect of the invention concerns a method of identifying the presence of one or more epitopes in a candidate antigen or group of antigens, wherein the epitope elicits a T-helper HLA-DP4 restricted response, said method comprising:
  • Th1 -specific response in the mouse to the antigen identifies an epitope which elicits a Th1 -specific response in the mouse to the antigen
  • Th2-specific response in the mouse to the antigen identifies an epitope which elicits a Th2-specific response in the mouse to the antigen.
  • an “antigen” comprises: 1 ) at least one HTL epitope, or 2) at least one CTL epitope or, 3) at least one B cell epitope, or 4) at least one HTL epitope and at least one CTL epitope, or 5) at least one HTL epitope and at least one B cell epitope, or 6) at least one CTL epitope and at least one B cell epitope, or 7) at least one HTL epitope and at least one CTL epitope and at least one B cell epitope.
  • a “candidate antigen” is a molecule that is under investigation to determine whether it functions as an antigen.
  • the antigen can comprise a polypeptide sequence or a polynucleotide sequence, which can comprise RNA, DNA, or both.
  • the antigen comprises at least one polynucleotide sequence operationally encoding one or more antigenic polypeptides.
  • the word "comprises” intends that at least one antigenic polypeptide is provided by the transcription and/or translation apparatus of a host cell acting upon an exogenous polynucleotide that encodes at least one antigenic polypeptide.
  • Antigens of the invention can be any antigenic molecule.
  • Antigenic molecules include: proteins, lipoproteins, and glycoproteins, including viral, bacterial, parasitic, animal, and fungal proteins such as albumins, tetanus toxoid, diphtheria toxoid, pertussis toxoid, bacterial outer membrane proteins (including meningococcal outer membrane protein), RSV-F protein, malarial derived peptide, B-lactoglobulin B, aprotinin, ovalbumin, lysozyme, and tumor associated antigens such as carcinoembryonic antigen (CEA), CA 15-3, CA 125, CA 19-9, prostrate specific antigen (PSA), and the TM complexes of U.S. Pat. No.
  • CEA carcinoembryonic antigen
  • CA 15-3 CA 15-3
  • CA 125 CA 19-9
  • PSA prostrate specific antigen
  • TM complexes of U.S. Pat. No.
  • carbohydrates including naturally-occurring and synthetic polysaccharides and other polymers such as ficoll, dextran, carboxymethyl cellulose, agarose, polyacrylamide and other acrylic resins, poly (lactide-co-glycolide), polyvinyl alcohol, partially hydrolyzed polyvinyl acetate, polyvinylpryrolidine, Group B Steptococcal and Pneumococcal capsular polysaccharides (including type III), Pseudomonas aeruginosa mucoexopolysaccharide, and capsular polysaccharides (including fisher type I), and Haemophilus influenzae polysaccharides (including PRP); haptens, and other moieties comprising low molecular weight molecules, such as TNP, saccharides, oligosaccharides, polysaccharides, peptides, toxins, drugs, chemicals, and allergens; and haptens, and other moieties comprising low molecular weight molecules, such as
  • influenzae S. pneumoniae, E. Coli, Klebsiella, S. aureus, S. epidermidis, N. meningiditis, Polio, Mumps, measles, rubella, Respiratory Syncytial Virus, Rabies, Ebola, Anthrax, Listeria, Hepatitis A, B, C, Human Immunodeficiency Virus I and 1 1 , Herpes simplex types 1 and 2, CMV, EBV, Varicella Zoster, Malaria, Tuberculosis, Candida albicans, and other Candida, Pneumocystis caringi, Mycoplasma, Influenzae virus A and B, Adenovirus, Group A streptococcus, Group B streptococcus, Pseudomonas aeryinosa, Rhinovirus, Leishmania, Parainfluenzae, types 1 , 2 and 3, Coronaviruses, Salmonella, Shigella, Rotavirus, Toxoplasma, Enterovirus
  • a “substance or a compound of the invention” may be an antigen, a group of antigen, a vaccine comprising one or more antigens, a vaccine composition, an immunotherapeutic compound or an immunotherapeutic composition.
  • an “immunotherapeutic compound” is a compound capable of inducing, enhancing, or suppressing an immune response.
  • the immunotherapeutic compound may be of any kind; it may for instance be an interleukin (such as IL-2,IL-7,IL-12%), a cytokine (such as interferons, G-CSF, imiquimod%), a chemokine, or a compound of other nature such as cytosine phosphate-guanosine, oligodeoxynucleotides, glucans...
  • An immunotherapeutic compound may also refers to one or more immune effector cells such as lymphocytes, macrophages, dendritic cells, natural killer cells (NK), cytotoxic T lymphocytes (CTL)..
  • an immunotherapeutic compound may also refers to one or more immune effector cells such as lymphocytes, macrophages, dendritic cells, natural killer cells (NK), cytotoxic T lymphocytes (CTL).
  • administering a substance or a compound of the invention to a mouse, may be performed by any method familiar to those of ordinary skill in the art, commonly including oral and intranasal routes, and intravenous, intramuscular, and subcutaneous injections, but also encompassing, intraperitoneal, intracorporeal, intra-articular, intraventricular, intrathecal, topical, tonsillar, mucosal, transdermal, intravaginal administration and by gavage.
  • the amount of a substance or a compound to be administered can be determined empirically and will take into consideration the age and size of the mouse to whom it is to be administrated.
  • An appropriate dose is within the range of 50 ⁇ g to 100 ⁇ g per inoculum for a DNA vaccine and of 1 ⁇ g to 5 ⁇ g for a recombinant protein vaccine, but higher and lower amounts may also be indicated.
  • Secondary booster administrations can be given at intervals ranging from one day to many months later.
  • Assaying for an immune response or an autoimmune response following administration of a substance or a compound of the invention may be performed by evaluating any directly, indirectly, or statistically observable or measurable increase or other desired change in the immune response in a host.
  • Observation of an immune response may for instance correspond to observation of a measurable increase in a humoral or cellular immune response to at least one epitope of the antigen as compared to the response obtained if the antigen is administered to the mouse without prior treatment with the vaccine.
  • observation of an immune response can correspond to observation of the production of antibodies directed against an antigenic epitope of interest or stimulate a detectable protective effect against a pathogenic or allergenic challenge or to promote a protective CTL response against an antigenic epitope of interest.
  • Assaying for an immune response may for instance include an ex vivo tissue culture host, comprising at least one cell of the immune system or cell line derived therefrom.
  • Host cells can be derived from animal peripheral blood, lymph nodes or the like.
  • Preferred tissue culture hosts include freshly isolated T cells, B cells, macrophages, oligodendrocytes, NK cells, and monocytes, each of which can be isolated or purified using standard techniques.
  • Observable or measurable responses include, B or T cell proliferation or activation; increased antibody secretion; isotype switching; increased cytokine release, particularly the increased release of one or more of IL-1 , IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12, IL-13, GM-CSF, IFN- ⁇ , TNF-a, TNF- ⁇ , GM-CSF, MIP-1 a, MIP-1 ⁇ , or RANTES; increased antibody titer or avidity against a specific antigen; reduced morbidity or mortality rates associated with a pathogenic infection; promoting, inducing, maintaining, or reinforcing viral latency; suppressing or otherwise ameliorating the growth, metastasis, or effects of malignant and non-malignant tumors; and providing prophylactic protection from a disease or the effects of a disease.
  • observationable or measurable humoral response may for instance include, without being limited to, B cell proliferation or activation increased antibody secretion, isotype switching, increased antibody titer or avidity against a specific antigen.
  • Observable or measurable T-helper response may for instance include, without being limited to, T-helper cell proliferation or activation, increased cytokine release, particularly the increased release of one or more of IL-1 , IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12, IL-13, GM-CSF, IFN- ⁇ , TNF-a, TNF- ⁇ , GM-CSF, MIP-1 a, MIP-1 ⁇ , or RANTES.
  • Observable or measurable T-cvtotoxic response may for instance include, without being limited to, T-cytotoxic cell proliferation or activation, or increase of the cytotoxic activity of T-cytotoxic lymphocytes.
  • Assaying an humoral response may for instance be performed by measuring the amount of specific antibodies directed against the substance or the compound administrated to a mouse present in the serum of the mouse, using the ELISA technique as illustrated in Example 1 .5, or any other specific dosage method.
  • the amount of the same antibodies present in the serum of the mouse before administration of the substance or the compound to the mouse may be used as a negative control.
  • Assaying a T-helper response or a T-cytotoxic response may for instance be performed by measuring the amount of specific cytokines (such as e.g. IFN- ⁇ ) produced following administration to a mouse of the substance or the compound, using the ELISA or the ELISPOT techniques as illustrated in Examples 1 .5 et 1 .6.
  • the amount of specific cytokines detected in the mouse before administration of the substance or the compound to a mouse may be used as a negative control.
  • Assaying a T-helper response or a T-cytotoxic response may also be performed by quantifying T cell proliferation as illustrated in Examples 1 .8 and 1 .9.
  • Assaying a T-cytotoxic response may also be performed by quantifying the cytotoxic activity of the T-cytotoxic cells, using a specific CTL-mediated lysis assay.
  • a non-specific CTL-mediated lysis assay, using cells that have been infected with an antigen different from that administered to the mouse, may be used as a negative control.
  • assaying a T-helper response or a T-cytotoxic response may be performed by measuring the amount of T cells specific of one or more epitopes present in the substance or the compound administrated to the mouse according to the invention.
  • Quantify ex vivo antigen-specific T cells may be performed in flow cytometry following binding of antigen-specific T cells to soluble tetramer (or pentamer) MHC-peptide complexes bound to fluorochrome.
  • the interaction of T cell receptors on T lymphocytes with tetrameric MHC-peptide complexes mimics the situation on the cell surface, and allows for reliable binding.
  • Tetramers, or pentamers may for instance consist of four, respectively five, biotinylated HLA-peptide epitope complexes bound to streptavidin conjugated with fluorescent dye, or of four, respectively five, phycoerythrin-labeled HLA- peptide epitope complexes. Fluorescence of the tetramers or pentamers bound to antigen-specific T cells can then be detected by flow cytometry, as illustrated in Example 1 .7, allowing quantification of the percentage of antigen-specific T cells in a cellular population. The percentage of the same antigen-specific T cells present in the mouse before administration of the substance or the compound to the mouse may be used as a negative control.
  • Another aspect of the invention is a method of determining the humoral response, the T-helper response, and/or the T-cytotoxic response of a mouse following administration of an antigen or of a vaccine comprising one or more antigens or of an immunotherapeutic compound to the mouse, said method comprising:
  • the T-helper response is a HLA-DP4 restricted response and/or the T cytotoxic cell response is a HLA-A2 restricted response.
  • Another aspect of the invention pertains to a method of comparing the immune responses induced by two or more vaccinal or immunotherapeutic compositions, said method comprising:
  • each candidate vaccinal or immunotherapeutic composition determines the relative efficiency of each candidate vaccinal or immunotherapeutic composition to induce a response by comparing the responses to each of the vaccinal or immunotherapeutic compositions to be compared with each other, wherein said response is a humoral response, and/or a T-helper response, and/or a
  • the T-helper response is a HLA-DP4 restricted response and/or the T cytotoxic cell response is a HLA-A2 restricted response.
  • a “candidate vaccine” is a substance, a compound or a molecule that is under investigation to determine whether it functions as a vaccine.
  • An “immunotherapeutic compound” is a compound that is under investigation to determine whether it capable of inducing, enhancing, or suppressing an immune response.
  • a “vaccine” or an “immunotherapeutic compound” comprises at least one immunological composition, which can be dissolved, suspended, or otherwise associated with a pharmaceutically acceptable carrier or vehicle.
  • a pharmaceutically acceptable carrier can be employed for administration of the composition. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, 18th Edition (A. Gennaro, ed., 1990) Mack Pub., Easton, Pa.
  • Carriers can be sterile liquids, such as water, polyethylene glycol, dimethyl sulfoxide (DMSO), oils, including petroleum oil, animal oil, vegetable oil, peanut oil, soybean oil, mineral oil, sesame oil, and the like. Carriers can be in the form of mists, sprays, powders, waxes, creams, suppositories, implants, salves, ointments, patches, poultices, films, or cosmetic preparations.
  • DMSO dimethyl sulfoxide
  • the composition is preferably water soluble, and saline is a preferred carrier.
  • penetrants appropriate to the barrier to be permeated can be included in the formulation and are known in the art.
  • the active ingredient can be combined with carriers suitable for inclusion into tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like.
  • Time-sensitive delivery systems are also applicable for the administration of the vaccine or of the immunotherapeutic compound of the invention.
  • Representative systems include polymer base systems, such as poly(lactide-glycoside), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid and polyanhydrides. These and like polymers can be formulated into microcapsules according to methods known in the art.
  • Aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable or aerosol solutions.
  • suitable propellants can be added as understood by those familiar with the art.
  • the immunological composition can also be formulated with solubilizing agents; emulsifiers; stabilizers; dispersants; flavorants; adjuvants; carriers; topical anesthetics, such as lidocaine, xylocaine, and the like; antibiotics; and known or suspected anti-viral, anti-fungal, anti-parasitic, or anti-tumor compounds.
  • an “adjuvant” is a composition that promotes or enhances an immune response to a target antigen.
  • an adjuvant for use in practicing the present invention in view of the disclosure herein.
  • the amount of an antigen-containing composition to be administered and the frequency of administration can be determined empirically and will take into consideration the age and size of the mouse to whom it is to be administrated.
  • An appropriate dose is within the range of 0.01 ⁇ g to 100 ⁇ g per inoculum, but higher and lower amounts may also be indicated.
  • Secondary booster immunizations can be given at intervals ranging from one week to many months later.
  • the two or more vaccinal or immunotherapeutic compositions to be compared by the method of the invention may in particular differ by the type of adjuvant present in the compositions or the ratio of antigen to adjuvant present in the compositions.
  • Another aspect of the invention is a method of optimizing two or more candidate vaccinal or immunotherapeutic compositions for administration to a human, based on preselected criteria, the method comprising:
  • the preselected criteria of the method of optimizing two or more candidate vaccine compositions may in particular be the type of adjuvant or the ratio of antigen to adjuvant present in the vaccinal or immunotherapeutic composition.
  • Another aspect of the invention is a method of determining whether a vaccinal or an immunotherapeutic composition poses a risk of induction of an autoimmune disease when administered to a human, said method comprising:
  • an "autoimmune disease” a condition that occurs when the immune system mistakenly attacks and destroys healthy body tissue.
  • the invention also pertains to a transgenic mouse cell comprising non-functional H2 class I and H2 class II genes, and comprising a functional HLA-A2 transgene and a functional HLA-DP4 transgene.
  • the invention also provides isolated transgenic mouse cells.
  • the cell according to the invention comprises a disrupted H2 class I gene, a disrupted H2 class II gene, a functional HLA class I transgene, and a functional HLA class II transgene.
  • the HLA class I transgene can be an HLA-A2 transgene and the HLA class II transgene can be an HLA-DP4 transgene.
  • the HLA-A2 transgene comprised by the transgenic mouse cell according to the invention is an HLA-A2.1 transgene and/or the HLA-DP4 transgene is an HLA-DP4.1 transgene.
  • An example of an HLA-A2.1 transgene is one that comprises the HLA-A2.1 sequence SEQ ID NO: 1 provided in the sequence listing.
  • An example of an HLA-DP4.1 transgene is one that comprises the HLA-DP4.1 sequence SEQ ID NO: 2 and SEQ ID NO: 3 provided in the sequence listing.
  • the transgenic mouse cell according to the invention further comprises a functional human CD4 (hCD4) transgene, and optionally comprises a non-functional murine CD4 (mCD4) gene.
  • hCD4 human CD4
  • mCD4 non-functional murine CD4
  • the human CD4 (hCD4) transgene is one that comprises the hCD4 sequence SEQ ID NO: 4 provided in the sequence listing.
  • the HLA-A2 transgene of the cell of the invention comprises the sequence SEQ ID NO: 1 and/or the HLA-DP4 transgene of the cell of the invention comprises the sequence SEQ ID NO: 2 and SEQ ID NO: 3 and/or the functional human CD4 (hCD4) transgene of the cell of the invention comprises the sequence SEQ ID NO: 4.
  • the HLA-A2/HLA-DP4 transgenic, H2 class l-/class ll-KO mice cell express, in a ⁇ 2 ⁇ - ⁇ context, a HLA-A2.1 monochain in which the human 32m is covalently linked by a peptidic arm to the HLA-A2.1 heavy chain. They further lack cell surface expression of conventional H2 IA and IE class II molecules as a result of the inactivation of the H2 IA3 b gene, since H2 ⁇ is a pseudogene in the H2 b haplotype.
  • the invention provides an isolated transgenic mouse cell deficient for both H2 class I and class II molecules, wherein the transgenic mouse cell comprises a functional HLA class I transgene and a functional HLA class II transgene.
  • the transgenic mouse cell has the genotype HLA-A2 + HLA-DP4 + 32m° ⁇ °. In other embodiments, the transgenic mouse cell has the genotype HLA-A2 + HLA-DP4 + hCD4 + 32m° ⁇ ° mCD4°.
  • the isolated mouse cells of the invention can be obtained from a mouse or mouse embryo.
  • the mouse or mouse embryo has the same genotype as the cell to be obtained.
  • the mouse or mouse embryo has a different genotype than the cell to be obtained.
  • a gene of the cell can be disrupted by, for example, homologous recombination.
  • a functional transgene can be introduced into the genome of the cell by, for example, transfection.
  • any suitable method known in the art can be applied to modify the genome of the cell to thereby obtain an isolated mouse cell having the desired genotype.
  • kits that are useful in the above methods.
  • kits comprise means for detecting the amount of specific antibodies and/or of specific HLA- DP4 restricted T-helper lymphocytes and/or of specific HLA-A2 restricted T-cytotoxic lymphocytes against a given antigen or group of antigens. They can be used, e.g. for identifying the presence of one or more epitopes in a candidate antigen or group of antigens.
  • the kit according to the invention comprises, in addition to the means for detecting the amount of specific antibodies and/or of specific HLA-DP4 restricted T-helper lymphocytes and/or of specific HLA-A2 restricted T-cytotoxic lymphocytes against a candidate antigen or group of antigens, a negative control sample indicative of the amount of specific antibodies and/or of specific HLA-DP4 restricted T- helper lymphocytes and/or of specific HLA-A2 restricted T-cytotoxic lymphocytes against the antigen or group of antigens in a mouse which have received no administration of the antigen or group of antigens.
  • kits according to the invention may for example comprise, in addition to the means for detecting the amount of specific antibodies and/or of specific HLA-DP4 restricted T-helper lymphocytes and/or of specific HLA-A2 restricted T-cytotoxic lymphocytes against a candidate antigen or group of antigens, one of (1 ) to (3) below:
  • a positive control sample indicative of the amount of specific antibodies and/or of specific HLA-DP4 restricted T-helper lymphocytes and/or of specific HLA-A2 restricted T- cytotoxic lymphocytes against a known antigen or group of antigens in a mouse which have received an administration of said known antigen or group of antigens;
  • a negative control sample indicative of the amount of specific antibodies and/or of specific HLA-DP4 restricted T-helper lymphocytes and/or of specific HLA-A2 restricted T- cytotoxic lymphocytes against the candidate antigen or group of antigens in a mouse which have received no administration of said candidate antigen or group of antigens;
  • kit may for example comprise (1 ) and (2), (1 ) and (3), (2) and (3), or (1 ), (2) and (3).
  • kits for identifying the presence of one or more epitopes in a candidate antigen or group of antigens, wherein the epitope elicits a specific immune response comprising:
  • a positive control sample indicative of the amount of specific antibodies and/or of specific HLA-DP4 restricted T-helper lymphocytes and/or of specific HLA-A2 restricted T- cytotoxic lymphocytes against a known antigen or group of antigens in a mouse which have received an administration of said known antigen or group of antigens
  • a negative control sample indicative of the amount of specific antibodies and/or of specific HLA-DP4 restricted T-helper lymphocytes and/or of specific HLA-A2 restricted T-cytotoxic lymphocytes against said candidate antigen or group of antigens in a mouse which have received no administration of said candidate antigen or group of antigens
  • Means for detecting the amount of specific antibodies and/or of specific HLA-DP4 restricted T-helper lymphocytes and/or of specific HLA-A2 restricted T-cytotoxic lymphocytes against a candidate antigen or group of antigens are well-known in the art. They include, e.g. reagents useful for performing a dosage assay, an ELISA assay, or a flow cytometry assay.
  • Such reagents may be:
  • specific HLA-DP4 restricted T-helper lymphocytes may be used in proliferation test, Elispot test onT4 (secretion cytokine); the intracellular secretion intracellulaire of cytokine may be detected using specific antibodies + Facs (Fluorescence Activated Cell Sorting) ; the specific T4 may be quantified using specific tetramers + Facs
  • T-cytotoxic lymphocytes may be used in cellular toxicity test, Elispot test on T8 (secretion cytokine); the intracellular secretion intracellulaire of cytokine may be detected using specific antibodies + Facs; the specific T8 may be quantified using specific tetramers + Facs.
  • the means for detecting the amount of specific antibodies and/or of specific HLA- DP4 restricted T-helper lymphocytes and/or of specific HLA-A2 restricted T-cytotoxic lymphocytes against a candidate antigen or group of antigens may also include reagents such as e.g. reaction, binding and/or washing buffers.
  • the means may be present, e.g., in vials or microtiter plates, or be attached to a solid support such as a microarray.
  • Fig. 1 Percentages of peripheral CD8+ T lymphocytes observed in HLA-A2/DP4/hCD4+ transgenic mouse and wild type BABL/c mouse. 1 .5-2.3% CD3+ positive T cells are mCD8+ T lymphocytes (left and middle panels), compared with 17.2% mCD8+ T cells detected in the wild type BABL/c mouse (right panel).
  • Fig. 2 Percentage of CD4+T lymphocytes and hCD4+ expression observed in HLA- A2/DP4/hCD4+ transgenic and wild type BABL/c mouse. 89-90% CD3+ T cells express hCD4 molecule (left and middle panels, Fig 2b) and 0-0.3% of residual express mCD4 (left and middle panels, Fig 2a), in contrast, 77.3% CD3+ T cells express mCD4+ (right panel, Fig 2a) and no hCD4 expression (right panel, Fig 2b) in wild type BABL/c mouse.
  • Fig. 3 (a) M1 specific antibody responses in HLA-A2.1/DP4 transgenic mice.
  • HLA- A2/DP4 mice were immunized by intramuscular injection of M1 -encoding plasmid DNA (solid column) or vector plasmid (hollow column). The sera were collected after 0, 10, 20, 30 days and the antibody against M1 was determined in an ELISA assay,
  • Fig. 4 Tetramer binding to CTLs in non-immunized and DNA immunized HLA-A2/DP4 transgenic mice.
  • the tetramer synthesized with influenza matrix 58-66 epitope was used to count the number of tetramer-positive (tet+) CTL cells.
  • HLA-A2/DP4 mice were immunized by intramuscular injection of HBs-encoding plasmid DNA (solid column) or PBS (hollow column), (a) The sera were collected at 10 days after third immunization and the antibody (IgG) titers against HBs particles were determined in an ELISA assay, (b) HBs derived epitope-specific IFN- ⁇ production by CTLs cells were determined by HLA-A2 restricted epitopes HBsAg348-357 and HBsAg335-343 as well as H2 K b restricted HBsAg371-378 epitope.
  • Fig. 6. HBs specific antibody production and proliferative responses following pCMV-S2S immunization in both HLA-A2/DP4/hCD4 transgenic and H2 class l/class N/mCD4+ KO mice. H2-class II KO mice were used as control.
  • HLA-A2/DP4 transgenic mice solid column
  • H2 class II KO mice high-density mice
  • IgG The antibody
  • titers against HBs particles and against the preS2 1 09 -i 34 peptide were determined in an ELISA assay
  • SEQ ID NO: 1 shows the sequence of the human gene HHD (HLA-A * 0201 ).
  • SEQ ID NO: 2 shows the sequence of the human gene HLA-DPA * 0103.
  • SEQ ID NO: 3 shows the sequence of the human gene HLA-DPB * 0401 .
  • SEQ ID NO: 4 shows the sequence of the functional human CD4 (hCD4) transgene.
  • SEQ ID NO: 5 shows the sequence of the HHD forward primer.
  • SEQ ID NO: 6 shows the sequence of the HHD reverse primer.
  • SEQ ID NO: 7 shows the sequence of the DP0103a forward primer.
  • SEQ ID NO: 8 shows the sequence of the DP0103a reverse primer.
  • SEQ ID NO: 9 shows the sequence of the DP0401 ⁇ forward primer.
  • SEQ ID NO: 10 shows the sequence of the DP0401 ⁇ forward primer.
  • SEQ ID NO: 1 1 shows the sequence of the hCD4 forward primer.
  • SEQ ID NO: 12 shows the sequence of the hCD4 reverse primer.
  • SEQ ID NO: 13 shows the sequence of the mCD4 forward primer.
  • SEQ ID NO: 14 shows the sequence of the mCD4 reverse primer.
  • SEQ ID NO: 15 shows the sequence of the Neo 715 primer.
  • SEQ ID NO: 16 shows the sequence of the 32m forward primer.
  • SEQ ID NO: 17 shows the sequence of the 32m reverse primer.
  • SEQ ID NO: 18 shows the sequence of the Neo 55 primer.
  • SEQ ID NO: 19 shows the sequence of the ⁇ forward primer.
  • SEQ ID NO: 20 shows the sequence of the the ⁇ reverse primer.
  • SEQ ID NO: 21 shows the sequence of the Mag-3 2 43-258 peptide
  • SEQ ID NO: 22 shows the sequence of the S109-121 peptide
  • SEQ ID NO: 23 shows the sequence of the S165-177 peptide
  • SEQ ID NO: 24 shows the sequence of the S181 -192 peptide
  • SEQ ID NO: 25 shows the sequence of the S197-209 peptide
  • SEQ ID NO: 26 shows the sequence of the S256-268 peptide
  • SEQ ID NO: 27 shows the sequence of the S318-331 peptide
  • SEQ ID NO: 28 shows the sequence of the S319-331 peptide
  • SEQ ID NO: 29 shows the sequence of the S326-338 peptide
  • SEQ ID NO: 30 shows the sequence of the S347-358 peptide
  • SEQ ID NO: 31 shows the sequence of the S352-364 peptide
  • SEQ ID NO: 32 shows the sequence of the S362-374 peptide
  • SEQ ID NO: 33 shows the sequence of the S376-388 peptide.
  • HLA-DP4 transgenic H2 class ll-KO ( ⁇ °) mice were obtained at the INSERM by crossing our established HLA-DP4-transgenic mice with H2 class ll-KO ( ⁇ °) mice (described in WO 2006/092515)
  • the HLA-A2.1 transgenic mice expressing a chimeric monochain (HHD molecule: a1-a2 domains of HLA-A2.1 , a3 to cytoplasmic domains of H2 D b , linked at its N terminus to the C terminus of human ⁇ 2 ⁇ by a 15-amino-acid peptide linker), were created in our laboratory (Pascolo S. et al., J Exp Med 1997, 185:2043-2051 ).
  • HLA-A2.1 (HHD) transgenic H2 class I KO mice and HLA-DP4 transgenic H2 class II KO ( ⁇ °) mice were intercrossed and progenies screened until HLA-A2.1 + / HLA-DP4+ double-transgenic H2 class I ( ⁇ 2 ⁇ 0 ) / class II ( ⁇ °) KO animals were obtained and used for the experiments described in this report.
  • Mice were bred in the animal facilities at INSERM (Paris, France) U1014 under barrier conditions and fed commercial mouse chow and water ad libitum. All operations followed the related rule in INSERM to comply with the French and European regulations on Animal Welfare and Public Health Service recommendations.
  • HLA-A2.1 (HHD) transgenic H2 class I KO and DP4 transgenic H2 class II KO were identified by means of PCR.
  • Mice genomes were extracted by genomic DNA isolation protocols. Briefly, tails were digested by incubation with 100 mM NaCI, 50 mM Tris-HCI pH 7.2, 100 mM EDTA, 1 % SDS and 0.5 mg/ml proteinase K (Merck, Darmstadt, Germany) overnight, at 56°C, followed by the addition of 250 ⁇ _ of saturated NaCI solution and isopropanol precipitation. Pellets were washed 2 times by 70% ethanol and resuspended in 100 ⁇ _ deionized water. After homogenization of DNA concentration, PCR were administrated for HHD by different pairs of primers:
  • - DP0103a forward : 5'-TAATACAAAGTCTGCAGCTGGC-3' (SEQ ID NO: 7), reverse : 5'-AGCAATGTTAGCCAGCC-3' (SEQ ID NO: 8);
  • - hCD4 forward : 5'-TCAGTGCAATGTAGGAGTCCAAG-3' (SEQ ID NO: 1 1 ), reverse : 5'- CACGATGTCTATTTTGAACTCCAC-3' (SEQ ID NO: 12);
  • - mCD4 forward : 5'-GGAGTTGTGGGTGTTCAAAGTG-3' (SEQ ID NO: 13), reverse : 5'- AGAGTTGCTATCCAAGGTCAGGG-3' (SEQ ID NO: 14), Neo 715 : 5'- GCTTCCTCGTGCTTTACGGTATC-3' (SEQ ID NO: 15);
  • Splenic cells were separated by using Ficolls (GE Lifesciences, Uppsala, Sweden).
  • the CD4+/CD8+ positive lymphocytes were first labeled by APC anti-CD3, percentage of single mCD8+, mCD4+ and hCD4+ lymphocytes were labeled by using PE-labeled anti- mouse CD8, FITC-labeled anti-mouseCD4 and PECy7-labeled anti-human CD4 respectively. Wild BABL/c was chose as control.
  • mice were used to verify consistency between the transgenic mice and human in cellular response in vivo.
  • transgenic mice were pre-immunized by cardiotoxin. After 5 days, mice were immunized 3 times intramuscularly at 10-day intervals and each time with 100 ⁇ g DNA vaccine injection per mouse. Ten days after the last immunization, mice were used for further analyses. 1.5. ELISA
  • ELISA was used to measure the production of HBs and preS2 specific antibodies in the sera. Sera were obtained every 10 days by centrifugation (3000 rpm/min for 15 minutes) at 4°C and stored at -20 ' ⁇ for later use in serologic tests. HBs antigen and preS2 proteins were diluted in coating buffer (0.1 M Na2C03, PH9.6) respectively and dispensed 50 ⁇ of diluted solution (10 g ml) to each well of a 96-well plate (Nunc, Roskilde, Denmark.), 4 ⁇ ⁇ overnight.
  • the plates were washed three times with phosphate buffered saline (PBS), blocked with 1 % BSA-PBS for 1 h at 37 ⁇ €, and washed twice. Added 50 ⁇ sera serial dilutions from 1 :15 for HBs and 1 :10 for preS2 in wells for 1 h at 37°C.
  • PBS phosphate buffered saline
  • the plates were washed three times with 0.05% Tween 20-phosphate buffered saline (PBS), and after 45 min incubation with 50 ⁇ _ goat anti-mouse IgG conjugated to HRP (Serotec, Cergy-Saint-Christophe, France) diluted solution (1 :1000) in every well, washed three times with 0.05% Tween 20-PBS. After washing, 50 ⁇ _ ready-to-use ELISA substrate (Roche, Mannheim, Germany) was dispensed in every well. Fifteen minutes later; the reaction was stopped by addition of 50 ⁇ of 3 M HCI and measured at 405 nm by using a Bioserv (BIOSERV, Rostock, Germany).
  • ELISPOT assay was implemented to detect IFN- ⁇ secreted by CD8+ T lymphocyte. Briefly, membrane-backed 96-well ELISPOT plates (Millipore, Bedford, MA) were coated with anti-IFN- ⁇ mAb (Diaclone, Besancone, France) overnight at 4°C and then blocked with 1 % skimmed milk. Lymphocytes (2 x10 5 /well) were added to every well and cultured with 20 ⁇ g/ml synthetic peptides and incubated for 20 h at 37°C, 5% C02.
  • the IFN- ⁇ - secreting cells were captured by coating anti-IFN- ⁇ mAb and detected by incubation with biotinylated anti-mouse IFN- ⁇ Ab (Diaclone) for 1 h30 at 37°C, followed by incubation with Streptavidin-HRP for 1 h. Finally, the plates were developed using substrate BEC (Diaclone, ready to use), washed, and dried. Spots were counted using the ELISPOT reader (CTL, Germany).
  • Lymphocytes were separated by Ficolls and resuspended in phosphate buffered saline (PBS) / 2% bovine serum albumin (BSA). PE-labeled HLA-A2.1 restricted tetramer influenza M1 :58-66 staining was performed as follows. Lymphocytes (2x10 5 ) were incubated with tetramers at the concentration of 100 ⁇ g ml, 4°C for 1 h.
  • PBS phosphate buffered saline
  • BSA bovine serum albumin
  • lymphocytes were washed by 2% bovine serum albumin (BSA)-PBS, followed by labelling by FITC-anti-CD8 mAb at 4°C for " l O min. After washed twice with phosphate buffered saline (PBS) / 2% bovine serum albumin (BSA), the lymphocytes were analyzed by FACS.
  • BSA bovine serum albumin
  • red blood cell-depleted, Ficoll-purified splenocytes (5 ⁇ 10 6 cells /25cm 2 culture flask) were co-cultured with peptide-pulsed (20 g/ml) in HL1 serum free medium supplemented with 10 mM Hepes, 1 mM sodium pyruvate, 5 ⁇ 10 "5 ⁇ 2-mercaptoethanol,100 lU/ml penicillin and 100 Ig streptomycin for 72h at 37°C in 5% C02.
  • PBMC Human peripheral blood mononuclear cell
  • the HLA-A2.1 +/+ HLA-DP4 +/+ hCD4 +/+ mCD4 / IA3 / 32m A (A2/DP4) mice obtained by crossing both the parental ⁇ _ ⁇ - ⁇ 2 +/+ ⁇ 2 ⁇ /_ (A2) mice and the HLA- DP4 +/+ hCD4 +/+ mCD4 /" IA3 /" (DP4) mice. Then, the inventors checked the genotype and cell surface expression of HLA-A2.1 , HLA-DP4 on splenocytes of A2/DP4 mice by PCR and flow cytometry, respectively (data not shown). The inventors also confirmed non- expression of H2 ⁇ and 32m in HLA-A2/DP4 mice.
  • HLA-A2 and HLA-DP4 were lower than the expression of endogenous H2 class I and class II molecules, but similar as previously reported in parental A2 mice and DP4 mice.
  • CD4+ and CD8+ splenic T cell numbers were determined by immunostaining and flow cytometry analysis.
  • T lymphocytes were labeled by anti-CD3-APC antibody and subsequently labeled by anti-mCD8-PE and hCD4-PECy7 antibodies as illustrated in Fig. 1 -2.
  • HLA-A2/DP4 mice To evaluate the immunological potential of HLA-A2/DP4 mice and their reliable prediction of human responses, the inventors observed humoral response and the immunodominant HLA-A2 restricted response that have been reported in natural infected patients or immunized human in HLA-A2/DP4 mice. In this purpose, they immunized the HLA-A2/DP4 mice with 2 vaccines that are well documented in human and in A2 transgenic mice: 1 ) the Influenza virus H5N1 pJW4303-M1 DNA vaccine, this plasmid encodes M1 protein.
  • HBsAg DNA plasmid encodes two HBV envelope proteins (preS2/S middle and S/small) that self-assemble in particles carrying HBsAg and they are the two protein components of the currently used vaccine against hepatitis B.
  • HLA-A2/DP4 mice were functionally restricted by the transgenic human class I molecules as reported in HLA-A2 mice (Pascolo S. et al., J Exp Med 1997, 185:2043-2051 )
  • the inventors examined the influenza M1 -specific and HBsAg-specific CD8+ CTL response by monitoring, the immunodominant HLA-A2.1 -restricted epitope responses directed at the influenza M1 58 - 6 6, HBsAg 348 -357 and HBsAg 335 -343, respectively.
  • M1 specific humoral response and influenza M1 58 - 6 6 CTL responses were monitoring by ELISA, ELISPOT and tetramer labeling assays.
  • the M1 specific antibody could be detected at 10 days after first immunization (Fig 3a) and the titer of M1 specific antibody increased up to peak after the third immunization (solid column).
  • solid column In contrast, in the group immunized with vector plasmid pJW4303, no specific antibody was detected (hollow column).
  • HLA-A2 restricted M1 58 - 6 6 and M1 2 -i o peptides were applied to observe M1 derived epitope-specific IFN- ⁇ production for CTLs (Fig 3b).
  • Fig 3b M1 derived epitope-specific IFN- ⁇ production for CTLs
  • the inventors examined the HBsAg specific humoral response in each tested mouse (Fig. 5a) and A2 restricted CD8+ T cell response in HLA-A2/DP4 transgenic mice.
  • the immunodominant HLA-A2.1 - restricted epitope was directed at the HBsAg 348 _357 and HBsAg 335 _ 343 peptides, while in C57BL/6 mice, the H2 Kb-restricted HBsAg-specific CTL response was directed at the HBsAg 3 7i_378 peptide.
  • HLA-A2/DP4 humanized mice had the same HLA-A2 restricted CTL response as HLA-A2/DR1 mice and humans
  • splenic T cells were stimulated with relevant HLA-A2.1 -restricted peptide HBsAg 348 - 3 57, HBsAg 3 35_34 3 and control (HBsAg 3 7i_37 8 , H2 Kb-restricted) peptides to detect the secretion of IFN- ⁇ .
  • HLA-A2.1 -restricted peptide HBsAg 348 - 3 57, HBsAg 3 35_34 3 and control (HBsAg 3 7i_37 8 , H2 Kb-restricted) peptides to detect the secretion of IFN- ⁇ .
  • HBsAg DNA immunization elicited a significant HBsAg 34 8-357 and HBsAg 335 _ 343 specific CTL response and no response against
  • HLA-A2/DP4 mice To evaluate the CD4+ T immunological behavior of HLA-A2/DP4 mice, the inventors immunized these mice with hepatitis B virus DNA vaccine pCMV-S2S by intramuscular injection. For the convenience of observation, H2-class II KO mice were used as control. As shown in Fig 6a, HBs protein and PreS2 antigen specific antibodies could be induced in the HLA-A2/DP4 transgenic mouse (solid column) ten days after the third immunization, and conversely, no antigen specific antibodies were detected in H2- class II KO mice. This result is consistent with previously evaluation in HLA-DP4 mice and revealed that potent humoral response requires the help of CD4+ T cells.
  • HBsAg DP4-restricted CD4+ T cells responded to whole HBs antigen and previously reported DP4-restricted peptide S181 -S192. No responses were showed in the H2-class II KO mice (hollow column) and the control HLA-DP4-restricted peptide Mage-3 24 3-258- The result demonstrated that HBsAg could induce DP4 restricted CD4+ T cells proliferation in the inventors' new HLA-A2/DP4 transgenic mice. 2.4.2. Identification of new HLA-DP4 epitopes in response to the HBsAg DNA vaccine
  • HTL and CTL epitopes are related to their MHC-binding capacities, with good HTL inducers generally having a high affinity for MHC class II molecule.
  • the inventors therefore scanned the hepatitis B envelope protein according HLA-DP4 peptide-binding motif and selected 1 1 candidate peptides. Affinities for each of the candidate peptides were evaluated (Table 1 ).
  • HLA-A2/DP4 transgenic mice were immunized intramuscularly with pCMV- S2S, as described in Material and Methods.
  • the splenocytes derived from the primed mice were separated and then stimulated in vitro with 12 H LA- DP4- restricted peptides included HBs 1 8 i -i 92 reported DP4 epitope.
  • Peptides S109-121 , S256-268, S326-338, S347-358 and S352-364 induced proliferative responses in HLA-A2/DP4 mice, while no responses were observed in control H2 class ll-deficient mice (data not shown).
  • the HLA-A2/DP4 transgenic H2 class I /class II deficient mice allowed the inventors to identify 5 novel HLA-DP4 epitopes from the hepatitis B envelope proteins.
  • HLA-DP4 restricted epitopes stem from HBs proteins and the selected candidate epitopes immunogenicity in HLA- A2/DP4/hCD4transgenic H2 class l/class N/mCD4 KO mice following immunization with pCMV-S2-S. Twelve HLA-DP4 restricted epitopes were predicted by scanning the whole HBs protein and synthesized. Exp IC50 (nM) represent the predictive affinity according algorithms, Obs (nM) represent the affinity obtained from MHC class II binding assay. [ 3 H]- thymidine incorporation assay was used to measure CD4+ T cells proliferation in DNA vaccine immunized mice with 12 synthesized peptides, including the previously reported epitope S181 -192. SI (Stimulation index).
  • HLA-DP4-transgenic H2 class ll-deficient mice were utilized for analyzing their DP4 restricted CD4 + T cell response in humans.
  • the result of proliferation showed that 3 out of 4 immunized subjects responded to the Celis (S181 -S192) peptide (Table 2).
  • Two donors displayed response directed to S326-338 and S256-268 peptides, respectively.
  • One response was observed with the S352-364 and S347-358 peptides.
  • Non significant response against the S109-121 peptide (which stem from S2 part of the antigen) was observed in the two donors who had received HBV vaccine.
  • HLA transgenic mouse models have been created to study anticancer, antiviral, antiparasite and autoimmune T responses, as well as a functional comparison among innovative immunotherapeutic methods.
  • nearly all of them are single HLA-class I or HLA-class II mouse models and limited to evaluate either CTLs response or Th response.
  • CTLs response the interaction among B cells, CTLs and Th cells response would be ignored, leading to lower fidelity to human response.
  • This shortcoming highlighted the significance of improving mouse model in simultaneously monitoring human CD4+ and CD8+ response.
  • the inventors disclose another novel HLA class I and II double humanized mouse model in the context of deletion of the whole H2 system. It means that both the development of CTLs and Th cells repertoire and the mobilization of effector cells in the periphery would be restricted only by HLA molecules rather than murine H2 molecules.
  • the HLA restricted immunological properties were successfully confirmed by HLA-A2 restricted CTL response and HLA-DP4 restricted Th response after H5N1 and HBV immunization.
  • this new HLA-A2/DP4 transgenic mouse model combined the most frequent HLA class I allele A * 0201 and the most popular class II allele DP4, probably becoming one of the best model to predict human response.
  • this novel mouse model could greatly facilitate the study of DP4-related immuno- pathogenesis and with the help of a large CD4+ T cell repertoire that has undergone thymic education on the HLA-DP4 molecule, these mice have capability to measure human DP4-restricted responses, especially used to identify new DP4 restricted epitopes and rank their immunogenicity.
  • this mouse model is able to assess the efficiency and safety of novel vaccines, what's more, it may also be developed into viruses chronic carrier or spontaneous tumor models by expressing target antigens.
  • HLA-A2/DP4/hCD4+ transgenic and H2-class l/class N/mCD4+ knockout mice disclosed here by the inventors has proved to be an effective and versatile animal model to identify immunodominant epitopes and rank their immunogenicity in priming CTLs and Th cells, unscramble the molecular mechanism of HLA-DP4-associated pathogen infection as well as autoimmunity diseases. Meanwhile, these animal models represent a promising surrogate to study the immune response in human clinical trials.

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Abstract

La présente invention concerne des souris transgéniques comprenant des gènes non fonctionnels H2 de classe I et H2 de classe II et comprenant un transgène fonctionnel HLA-A2 et un transgène fonctionnel HLA-DP4. La présente invention concerne également des procédés d'identification de la présence d'un ou plusieurs épitopes dans un antigène candidat ou d'un groupe d'antigènes, des procédés de détermination de la réponse immunitaire d'une souris à la suite de son immunisation par un antigène ou un vaccin ou à la suite de son traitement par un composé immunothérapeutique, des procédés de comparaison de l'efficacité d'une réponse induite par au moins deux compositions de vaccin ou immunothérapeutiques, et des procédés de détermination de savoir si une composition de vaccin ou immunothérapeutique pose un risque d'induction d'une maladie auto-immune lorsqu'elle est administrée à un être humain. L'invention concerne en outre de cellules murines transgéniques comprenant des gènes non fonctionnels H2 de classe I et H2 de classe II et comprenant un transgène fonctionnel HLA-A2 et un transgène fonctionnel HLA-DP4, et des trousses pour l'identification de la présence d'un ou plusieurs épitopes dans un antigène candidat ou un groupe d'antigènes.
PCT/EP2012/064813 2011-07-29 2012-07-27 Souris transgéniques humanisées hla-a2 / hla-dp4 et leurs utilisations en tant que modèle expérimental pour la recherche biomédicale et le développement biomédical WO2013017545A1 (fr)

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WO2020053304A2 (fr) 2018-09-14 2020-03-19 Scancell Limited Épitopes
CN112575037A (zh) * 2019-09-29 2021-03-30 上海市公共卫生临床中心 一种嵌合人hla-dp基因组区域的人源化转基因小鼠模型的构建方法
WO2021214022A1 (fr) 2020-04-21 2021-10-28 Scancell Limited Peptides de nucléophosmine citrulinés utilisés en tant que vaccins anticancéreux
DE102020120377A1 (de) 2020-08-03 2022-02-03 Hamm Ag Ummantelung für eine Bodenbearbeitungswalze
WO2022106696A2 (fr) 2020-11-23 2022-05-27 Scancell Limited Réponses antitumorales à des cytokératines

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020053304A2 (fr) 2018-09-14 2020-03-19 Scancell Limited Épitopes
CN112575037A (zh) * 2019-09-29 2021-03-30 上海市公共卫生临床中心 一种嵌合人hla-dp基因组区域的人源化转基因小鼠模型的构建方法
CN112575037B (zh) * 2019-09-29 2024-05-24 上海市公共卫生临床中心 一种嵌合人hla-dp基因组区域的人源化转基因小鼠模型的构建方法
WO2021214022A1 (fr) 2020-04-21 2021-10-28 Scancell Limited Peptides de nucléophosmine citrulinés utilisés en tant que vaccins anticancéreux
DE102020120377A1 (de) 2020-08-03 2022-02-03 Hamm Ag Ummantelung für eine Bodenbearbeitungswalze
EP3951065A1 (fr) 2020-08-03 2022-02-09 Hamm AG Gaine pour un rouleau de traitement du sol
WO2022106696A2 (fr) 2020-11-23 2022-05-27 Scancell Limited Réponses antitumorales à des cytokératines

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