Patent Application
20130121958
The present invention provides an innovative versatile system, which allows delivery of one or several antigens or biologically active molecules onto and/or into subset of cells and in particular onto and/or into dendritic cells (DC) or subsets of DC.
This two-component system combines (i) a fusion polypeptide comprising a streptavidin or avidin polypeptide and effector molecule(s), which is capable of binding biotin molecules, and (ii) biotinylated targeting molecules, which are capable of targeting subset(s) of cells and/or cell surface molecules(s).
Using this system, the invention allows for efficient and specific delivery of effector molecules, in particular antigens, onto and/or into subset(s) of cells which have been targeted via biotinylated targeting molecules.
The invention is more particularly directed to a combination of compounds and in particular to a composition comprising the aforementioned components (i) and (ii).
The combination and the composition of the invention are suitable for use for diagnosing or immunomonitoring a disease in a mammal or for use in prophylactic or curative treatment and especially in vaccination and in therapy including in immunotherapy.
The invention also relates to the use of a fusion polypeptide as defined above, in combination with biotinylated targeting molecule as defined above, for targeting, in vivo, in vitro or ex vivo, of one or several effector molecule(s) to subset(s) of cells and/or cell surface molecules(s).
The invention also relates to the use of a fusion polypeptide as defined above, in combination with biotinylated targeting molecule as defined above, in vivo or ex vivo, for inducing a T cell immune response in bone marrow of naive donors before transplantation or for activation and/or expansion before transplantation of the already present antigen-specific T cell immune response(s) in the bone marrow grafts from already immunized donors.
The invention also relates to methods for the production of a fusion polypeptide of the invention and to a kit for a diagnostic test of a disease in a mammal, for immunomonitoring a disease in a mammal or for the prevention or treatment of a disease in a mammal.
Hence, a first object of the invention is directed to a combination of compounds (or kit-of-parts) which comprises or consists of at least two components:
According to the invention, components (i) and (ii) are present either in distinct compositions or in the same (i.e., in a single) composition.
Hence, in a particular embodiment, the invention also relates to a composition comprising or consisting of components (i) and (ii).
Unless otherwise indicated, each embodiment disclosed in this application is applicable independently of and/or in combination with any or several of the other described embodiments.
By “composition”, it is meant herein in particular a pharmaceutical or an immunological composition.
By “fusion polypeptide” it is meant herein that the SA or avidin polypeptide is genetically fused to the polypeptidic structure of one or several effector molecule(s). One or several (in particular 2, 3, 4, 5 or more) effector molecule(s) can be fused at the N-terminal end, at the C-terminal end or at both ends of the SA or avidin polypeptide.
In a particular embodiment of the invention, this fusion polypeptide is the expression product of a recombinant polynucleotide, which can be expressed in a cell, for example an Escherichia coli (E. coli) cell, which is transformed (as a result of recombination) to comprise said recombinant polynucleotide or a plasmid or a recombinant vector comprising said polynucleotide.
In a particular embodiment of the invention, the fusion polypeptide also comprises one or several linker(s) (or spacer(s)), in particular one or several flexible linker(s), which is(are) located, for example, between the SA or avidin polypeptide (more specifically, between a SA or avidin monomer) and an effector molecule.
In a particular embodiment of the invention, a linker is located between two effector molecules present in the fusion polypeptide.
A “linker” as used herein consists of a polypeptide product having an amino acid sequence of at least 2 amino acid residues, preferably at least 4 residues, for example 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 residues.
By “SA or avidin polypeptide”, it is meant herein a full length native SA or avidin protein, a variant thereof or a derivative of this native protein or variant thereof, which variant and derivative retain the property of the native protein to bind biotin, and in particular to selectively bind biotin.
In a particular embodiment of the invention, a SA polypeptide is used to build the fusion polypeptide. This SA polypeptide is for example derived from the SA protein obtainable from Streptomyces avidinii.
In a particular embodiment of the invention, a “variant” of a SA or avidin protein consists of an amino acid sequence having at least 50%, preferably at least 70% and more preferably at least 90% identity with the sequence of a native full length SA or avidin protein, for example SA from Steptomyces avidinii (SEQ ID NO.: 1).
In a particular embodiment of the invention, a “variant” of a SA or avidin protein consists of a polypeptide variant having an amino acid sequence which differs from that of the full length SA or avidin protein (for example from that of SA from Steptomyces avidinii) by insertion, deletion and/or substitution, preferably by insertion and/or substitution of one or several amino acid residues, for example 1, 2, 3, 4, 5, or 6 amino acid residues. Hence, a “variant” includes a portion of a native full-length SA or avidin protein.
In a particular embodiment of the invention, said variant is a fragment of the full-length SA or avidin protein, which retains the capacity of binding biotin. The invention especially relates in this respect, to such a variant which is a fragment devoid of the N-terminal and C-terminal regions of the native full-length SA or avidin protein.
In a particular embodiment of the invention, the SA polypeptide present in the polypeptide fusion is the portion called natural core, which ranges from amino acid residues 13 to 139 or 14 to 139 in the SA protein from Steptomyces avidinii (SEQ ID NO.: 2 and 41 respectively).
Alternatively, a variant consisting in a sequence having at least 70%, preferably at least 80% and more preferably at least 90% or 95% identity with the amino acid sequence of this natural core and retaining the property to bind biotin can be used. For example, a polypeptide comprising or consisting of polypeptides stv-25 or stv-13 (Sano et al., 1995), of sequence SEQ ID NO.: 3 and SEQ ID NO.: 4 respectively, can be used as SA polypeptide in the fusion polypeptide of the invention.
A “derivative” of a SA or avidin polypeptide as used herein designates a polypeptide modified chemically, for example by deglycosylation, or by PEGylation of a SA or avidin full-length protein or a variant thereof. An example of a deglycosylated version of a avidin polypeptide is the protein called neutravidin.
Preferably, the “variant” and “derivative” of a SA or avidin native protein also retain the property of these proteins to form a tetramer.
Indeed, in a particular embodiment of the invention, the fusion polypeptide is in the form of a tetramer. This tetrameric fusion polypeptide can be a homotetramer or a heterotetramer, i.e., comprises or consists of either four identical monomers or two or more (two, three or four) different monomers respectively. Every monomer of this tetramer (homotetramer or heterotetramer) comprises at least a monomer of the SA or avidin polypeptide.
Hence, when the fusion polypeptide of the invention is in the form of a heterotetramer, at least one monomer of this tetramer comprises or consists of (i) a monomer of the SA or avidin polypeptide and (ii) one or several effector molecule(s). The other monomers of the tetramer then comprise or consist of a monomer of the SA or avidin polypeptide, and optionally one or several effector molecule(s).
In a particular embodiment of the invention, each monomer of the tetrameric fusion polypeptide of the invention (homotetramer or heterotetramer) comprises or consists of both a monomer of the SA or avidin polypeptide and one or several effector molecule(s) (preferably several effector molecules).
In a particular embodiment of the invention, each monomers of a tetramer comprise the same effector molecule(s).
In another particular embodiment of the invention, monomers of the tetramer (at least two monomers of the tetramer) have a different content in effector molecule(s).
When the fusion polypeptide of the invention is in the form of a heterotetramer, at least one monomer of this tetramer (i.e., one, two, three or four monomer(s) of this tetramer) retains the property of SA and avidin proteins to bind biotin. The other monomers of this tetramer can retain or not the property of SA and avidin proteins to bind biotin. Hence, a tetrameric fusion polypeptide of the invention can have one, two, three or four functional (or active) biotin binding subunits.
In a particular embodiment of the invention, the fusion polypeptide is in the form of a heterotetramer, wherein one, two or three monomers of this tetramer are non-functional, i.e., do not retain the property of SA and avidin proteins to bind biotin, for example due to one or several mutation(s) in the SA or avidin polypeptide. For example, the fusion polypeptide can include a SA or avidin tetramer which comprises only one functional biotin subunit that retains the property of SA or avidin to bind biotin, and in particular a monovalent SA tetramer as disclosed in Howarth et al., 2006.
Alternatively, in a particular embodiment of the invention, the fusion polypeptide of the invention is in the form of a monomer, which comprises or consists of a monomeric SA or avidin polypeptide and one or several effector molecule(s), preferably several effector molecules. This monomeric SA or avidin polypeptide can be for example a variant of a SA or avidin wild-type protein, which has an increased biotin binding affinity, an in particular a monomeric SA polypeptide as disclosed in Wu and Wong, 2005.
By “effector molecule”, it is meant herein a molecule which has a biologically activity (i.e., a biologically active molecule) which comprises or consists of one or several polypeptidic structures, i.e., a biologically active polypeptidic molecule. Said polypeptide may especially be chosen for its properties for the purpose of preparing prophylactic product or in a therapeutic product i.e., may have a prophylactic or a therapeutic activity, or may enhance a prophylactic or therapeutic activity.
In particular embodiments of the invention, the effector molecule(s) or some of the effector molecule(s) is(are) selected in the group comprising: polypeptides including peptides, glycopeptides and lipopeptides.
Additionally or alternatively, one or several elements chosen in the group of lipids, sugars, nucleic acids (in particular DNAs and RNAs and for example cDNA or siRNA), chemical moieties, and chemical molecules, for example radioelements, dyes or immunostimulant, for example Poly I:C (polyinosinic:polycytidylic acid or polyinosinic-polycytidylic acid sodium salt), can be grafted onto the fusion polypeptide and in particular onto one or several effector molecule(s) present in the fusion polypeptide. This(these) element(s) can be attached onto the fusion polypeptide either by chemical coupling, or by adding into the fusion polypeptide an aptamer or another recombinant ligand that would bind (especially with high affinity) said element(s). Said element(s) can be grafted for example on the polypeptidic structure of an effector molecule.
In particular embodiments of the invention, the effector molecule(s) or some of the effector molecule(s) is(are) a polypeptide and especially a peptide.
In a particular embodiment of the invention, the “effector molecule” does not interfere with the folding of the SA or avidin polypeptide, and in particular with tetramerization of these polypeptides. Alternatively, in case an effector molecule which interferes with tetramerization of the SA or avidin polypeptide is used, it is possible to insert, between the SA or avidin polypeptide and said effector molecule, one or several linker(s), in particular one or several flexible linker(s), in order that the SA or avidin polypeptide still forms a tetramer despite the presence of this effector molecule in the fusion polypeptide.
In a specific embodiment of the invention, the effector molecule(s) or some of the effector molecule(s) comprise or consist of 2 to 1000, preferably 5-800, 5 to 500, 5 to 200, 5 to 100, 8 to 50, 5 to 25, 5 to 20 or 8 to 16 amino acid residues.
In a particular embodiment of the invention, the effector molecule(s) or at least some of the effector molecule(s) is(are) chosen among the following group:
In a particular embodiment of the invention, the fusion polypeptide comprises as effector molecule(s) at least one recombinant ligand as disclosed herein.
As used herein, the term “epitope” refers to a polypeptide and especially a peptide that can elicit an immune response, when presented in appropriate conditions to the immune system of a host. In particular, such an epitope can comprise or consist of a stretch of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid residues.
The polypeptidic molecule suitable for eliciting an immune response is especially one eliciting a T-cell immune response, including a CTL response or a T helper response. The polypeptidic molecule suitable for eliciting an immune response can also be one eliciting a B-cell immune response.
In specific embodiments, the immunogen is derived from an allergen, a toxin, a tumor cell, or an infectious agent, in particular a bacteria, a parasite, a fungus or a virus.
In a particular embodiment of the invention, the effector molecule(s) or at least some effector molecule(s) comprise or consist of an antigen selected from the group consisting of a Chlamydia antigen, a Mycoplasma antigen, a Mycobacteria antigen (for example, an antigen from Mycobacterium tuberculosis or Mycobacterium leprae), a Plasmodia antigen (for example, an antigen from Plasmodium berghei, Plasmodium vivax or Plasmodium falciparum), a hepatitis virus antigen, a poliovirus antigen, an HIV virus antigen (for example, a HIV protein), a human papillomavirus (HPV) virus antigen, especially an antigen of HPV16 or HPV18 (for example, a E7 antigen of a HPV virus, especially the E7 antigen of HPV16 or HPV18), a CMV virus antigen (for example, the phosphoprotein 65 (pp65)), an influenza virus antigen, a choriomeningitis virus antigen, or a tumor-associated antigen, or comprise or consist of a part of an amino acid sequence of any these antigens which comprises at least one epitope.
In a particular embodiment of the invention, the effector molecule(s) or at least some effector molecule(s) are from Mycobacterium tuberculosis (also called MTB herein). For example, they can comprise or consist of an amino acid sequence chosen from the ones of the following proteins:
In a particular embodiment of the invention, the effector molecule(s) or at least some effector molecule(s) are from a CMV virus, for example, pp65 or the immediate early protein-1 (IE-1) of a CMV virus, or comprise or consist of a part of any of these proteins to the extent that the amino acid sequence of said part comprises at least one epitope.
In a particular embodiment of the invention, the effector molecule(s) or at least some effector molecule(s) are from a human papilloma virus, for example, the E7 protein antigen of a HPV virus, or comprise or consist of a part of any of these proteins to the extent that the amino acid sequence of said part comprises at least one epitope.
In a particular embodiment of the invention, the effector molecule or at least one of the effector molecule(s) comprises or consists of sequence SEQ ID NO.: 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 64, or 66.
In a particular embodiment of the invention, the effector molecule(s) or at least some effector molecule(s) are any of the effector molecule(s) disclosed in the example part of the application, or a variant of an effector molecule disclosed herein, which variant comprises or consists of a sequence having at least 70%, preferably at least 80% and more preferably at least 90% or 95% identity with the amino acid sequence of the effector molecule from which it is derived, said variant retaining the immunogenic properties (if any) of said effector molecule. In particular, a variant of an effector molecule can be devoid of cysteine residues or contain a reduced number of cysteine residues in comparison with the sequence of the effector molecule from which it is derived. By “x % identity” it is meant herein x % identity calculated over the entire length of the sequence of the polypeptide (global alignment calculated for example by the Needleman and Wunsch algorithm).
In a particular embodiment of the invention, the effector molecule(s) or at least some effector molecule(s) are a tumor associated antigen (TAA). Tumor-associated antigens have been characterized for a number of tumors such as for example: Melanoma, especially metastatic melanoma; Lung carcinoma; Head & neck carcinoma; cervical carcinoma, Esophageal carcinoma; Bladder carcinoma, especially infiltrating Bladder carcinoma; Prostate carcinoma; Breast carcinoma; Colorectal carcinoma; Renal cell carcinoma; Sarcoma; Leukemia; Myeloma. For these various histological types of cancers, it has been shown that antigenic peptides are specifically expressed on tumor samples and are recognized by T cells, especially by CD8+ T cells or CD4+ T cells.
A review of peptides found as tumor-associated antigens in these types of tumors is made by Van der Bruggen P. et al (Immunological Reviews, 2002, vol 188:51-64). Especially, the disclosure of the peptides contained in table 3 of said review is referred to herein as providing examples of such tumor-associated antigens and said table 3 is incorporated by reference to the present application.
The following antigens are cited as examples of tumor-associated antigens recognized by T cells, according to Kawakami Y. et al (Cancer Sci, October 2004, vol. 95, no. 10, p 784-791) that also provides methods for screening these antigens or further one: antigens shared by various cancers, including MAGE (especially in Melanoma), NY-ESO-1, Her2/neu, WT1, Survivin, hTERT, CEA, AFP, SART3, GnT-V, antigens specific for some particular cancers such as βbeta-catenin, CDK4, MART-2, MUM3, gp100, MART-1, tyrosinase for Melanoma; bcr-abl, TEL-AML1 for Leukemia; PSA, PAP, PSM, PSMA for prostate cancer; Proteinase 3 for myelogenous leukemia; MUC-1 for breast, ovarian or pancreas cancers; EBV-EBNA, HTLV-1 tax for lymphoma, ATL or cervical cancer; mutated HLA-A2 for Renal cell cancer; HA1 for leukemia/lymphoma. Tumor-associated antigens in animals have also been described such as Cycline D1 and Cycline D2 in tumors affecting cats or dogs.
Tumor-associated antigens recognized by T cells have also been disclosed in Novellino L. et al (Immunol Immunother 2004, 54:187-207).
More generally, TAA of interest in the present invention are those corresponding to mutated antigens, or to antigens that are overexpressed on tumor cells, to shared antigens, tissue-specific differenciation antigens or to viral antigens.
In a particular embodiment of the invention, the tumor-associated antigen is an antigen of papillomavirus (HPV) or is tyrosinase. In a particular embodiment of the invention, the fusion polypeptide further comprises one or several ligand(s), in particular one or several recombinant ligand(s), for example one or several protein scaffold(s). Especially, protein scaffolds allowing binding and targeting of co-receptors of T cells or cytokines (for example IL-2 or IFNγ), or delivery of nucleic acids (in particular RNAs and DNAs and for example cDNAs or siRNA) or of adjuvant molecules (for example CpG) could be used.
Hence, according to this particular embodiment of the invention, the fusion polypeptide comprises or consists of:
Examples of ligands include ABD (wild type human serum albumin domain of protein G)-derived protein scaffolds as disclosed in the example part of the application, or a variant of any of these ligands, which variant comprises or consists of a sequence having at least 70%, preferably at least 80% and more preferably at least 90% or 95% identity with the amino acid sequence of the ligand from which it is derived, said variant retaining the binding and targeting properties of said ligand.
Other examples of adjuvant molecules include the ones that are disclosed herein.
Hence, the invention allows co-delivery of effector molecule(s) as defined herein (in particular effector molecule(s) suitable for eliciting an immune response) and of molecules bound to the above-mentioned ligand(s) (in particular cytokines, nucleic acids or adjuvant molecules) to the same subset(s) of cells and in particular to subset(s) of cells as disclosed herein (for example DC subset(s)).
In a particular embodiment of the invention, the fusion polypeptide further comprises a domain that enables to increase the level of production (in particular the level of expression) of the fusion polypeptide in an E. coli cell.
An example of an appropriate domain includes the TRP sequence (MKAIFVLNAQHDEAVDA; SEQ ID NO.:42). Said TRP sequence can be located for example between the SA or avidin polypeptide (e.g., at the C-terminal end of the SA or avidin polypeptide) and one of the effector molecule(s) ((e.g., at the N-terminal end of this effector molecule).
Another example of an appropriate domain is the sequence MASIINFEKL (SEQ ID NO.:43). This sequence can be located for example at the N-terminal end of the SA or avidin polypeptide and/or at the N-terminal end of the fusion polypeptide. It allows to increase the stability of the fusion polypeptide and thus to increase its level of expression in an E. coli cell. In addition, the sequence SIINFEKL (SEQ ID NO.:44) which is present in this sequence is an epitope for CD8+ T cells, which can be useful for example as a marker for analysis of antigen delivery capacity into DC in vitro as well as in vivo.
By “targeting molecule(s)” it is meant herein a molecule which is capable of targeting subset(s) of cells and/or cell surface molecule(s), and in particular capable of specifically interacting with targeted subset(s) of cells and/or cell surface molecule(s) and especially binding to such cells and/or cell surface molecule(s).
In a particular embodiment of the invention, the biotinylated targeting molecule(s) enable targeting of subset(s) of cells by interacting with surface molecule(s) of these cells.
By “subset(s) of cells”, it is meant herein in particular antigen presenting cells (APC) and/or subset(s) of APC. In a particular embodiment of the invention, the terms “subset(s) of cells” and “APC” designate dendritic cells (DC) or subset(s) of DC and/or B lymphocytes or subset(s) of B lymphocytes. In a more particular embodiment of the invention, the term “subset(s) of cells” and “APC” designate DC or subset(s) of DC.
In a particular embodiment of the invention, by “subset(s) of cells”, it is meant herein in particular cells or subset(s) of cells (e.g. DC or subset(s) of DC as disclosed herein) generated from bone marrow precursors.
By “cell surface molecule(s)”, it is meant herein any molecule which is expressed at the cell surface, and in particular cell surface receptor(s) and/or toll-like receptor(s) (TLR). These molecules include in particular the ones which are expressed at the surface of APC, and in particular at the surface of DC and/or B lymphocytes and/or T lymphocytes (more preferably at the surface of DC).
Hence, by “cell surface receptor(s)”, it is meant herein in particular APC surface receptor(s), preferably DC and/or B lymphocytes and/or T lymphocytes surface receptor(s) and more preferably DC surface receptor(s).
DC subset(s) can be in particular chosen among the following group: plasmacytoid DC, blood-derived lymphoid tissue resident DC, peripheral migratory DC, monocyte-derived inflammatory DC.
DC subset(s) can also be in particular Bone Marrow-derived DC (BM-DC).
In a particular embodiment of the invention, the total DC population, i.e. CD11c+ cells, are targeted, or DC subset(s) are chosen among CD11b+ and/or CD205+ DC.
In a particular embodiment of the invention, DC subset(s) are lung DC or lung DC subset(s).
In a particular embodiment of the invention, a biotinylated targeting molecule comprises or consists of one or several polypeptide especially peptidic structure(s) wherein the meaning of “polypeptide” is as disclosed herein.
In a particular embodiment of the invention, a biotinylated targeting molecule is a polypeptide.
Different biotinylated targeting molecule(s) can be used in the invention and in particular in the combination or the composition of the invention.
In a particular embodiment of the invention, the biotinylated targeting molecule(s) are capable of specifically targeting cells, and in particular of specifically interacting with, cells or subset of cells (in particular DC or B lymphocytes or subset(s) of DC or B lymphocytes), which induce a CD4+ T-cell immune response and/or a CD8+ T-cell immune response or which induce essentially a CD4+ or a CD8+ T-cell immune response.
In a particular embodiment of the invention, the biotinylated targeting molecule(s) or at least some of the biotinylated targeting molecule(s) present in the combination or the composition of the invention is(are) capable of specifically interacting with one or several cell surface receptor(s) chosen from the following group:
Another example of members of the mannose receptor family that can be used according to the invention is DEC 206.
In a particular embodiment of the invention, at least one of the biotinylated targeting molecule(s) present in the combination or the composition of the invention is(are) capable of specifically targeting and in particular interacting with CD11b, CD11c or CD205.
Receptor CD207 enables to target in particular DC of the dermis and epidermis and in epithelium lining the human airways, and is thus particularly appropriate for use for example in the prevention or treatment tuberculosis.
In a particular embodiment of the invention, biotinylated targeting molecule(s) capable of targeting and in particular interacting with CD205 are used, in conjunction with a fusion polypeptide as defined herein which comprises at least one effector molecule (e.g., a protective antigen or a fragment thereof comprising or consisting of at least one epitope) derived from Mycobacterium tuberculosis.
An example of an antibody specific to the C-type lectin endocytic receptor CD205 is the monoclonal antibody NLDC-145 (Celldex Therapeutics; Needham, USA).
An example of an antibody specific to the mannose receptor CD206 is the monoclonal antibody disclosed in the example part of the application.
In a particular embodiment of the invention, the biotinylated targeting molecule(s) are chosen from:
By “antibodies” it is meant herein any type of antibody and in particular monoclonal antibodies, which are specific to subset(s) of cells and/or cell surface molecule(s), as defined herein or antibody-like molecules.
The term “monoclonal antibody” encompasses:
The term “antibody-like molecule” refers to a molecule having all or part of the variable heavy and light domains of an antibody, but devoid of the conventional structure of a four-chain antibody, and conserving nevertheless the capacity to interact with and bind an immunogen. In a particular embodiment of the invention, an antibody-like molecule is a fragment of an antibody and in particular comprises the CDR1, CDR2 and CDR3 regions of the VL and/or VH domains of a full length antibody.
The term “antibody-like molecule” encompasses in particular:
trispecific molecules i.e., molecules in which the two antigen binding sites of a Fab3 fragment (variable and CH1 domains of light and heavy chains) interact with different immunogens);
In a particular embodiment of the invention, the combination or the composition further comprises one or several biotinylated, non-targeting molecule(s), which can be for example chosen from the following group: biotinylated immunogens as defined herein, biotinylated protoxins, biotinylated nucleic acids (in particular RNAs, DNAs or cDNAs), biotinylated adjuvant molecules and biotinylated cytokines (for example IL-2, IL-10, IL-12, IL-17, IL-23, TNFα or IFNγ).
Examples of adjuvants that can be used as biotinylated, non-targeting molecule(s) include the ones disclosed herein, and in particular biot-CL264.
Hence, the invention allows co-delivery of effector molecule(s) as defined herein (in particular effector molecule(s) suitable for eliciting an immune response) and biotinylated, non-targeting molecule(s) (in particular biotinylated adjuvant or biotinylated cytokines) to the same subset(s) of cells and in particular to subset(s) of cells as disclosed herein (for example DC subset(s)).
These biotinylated, non-targeting molecule(s), as well as the biotinylated targeting molecule(s) and the effector molecule(s) used to carry out the invention can be humanized, in particular for use in vivo, in a human host, or ex vivo, on a sample of human cells.
In a particular embodiment of the invention,
In a particular embodiment of the invention, the composition of the invention comprises or consists of a complex formed between the fusion polypeptide and biotinylated targeting molecule(s). Hence, in a particular embodiment, the invention relates to a composition (or complex) in which the fusion polypeptide is complexed to biotinylated targeting molecule(s) present in the composition, and optionally to biotinylated, non-targeting molecule(s) as defined herein.
By “complex”, it is meant herein that the fusion polypeptide associates with biotinylated molecule(s) (in particular with biotinylated targeting molecule(s) and, when present, with biotinylated non-targeting molecule(s)) via non-covalent interactions that occur between the SA or avidin polypeptide and the biotin moiety of biotinylated molecule(s) present in the composition of the invention. This complex can include one, two, three or four biotins molecules, and in particular one, two, three or four biotinylated targeting molecule(s) as defined herein.
In a particular embodiment of the invention, this complex includes at least one biotinylated molecule(s) as defined herein, for example one, two, three or four biotinylated molecules.
In a particular embodiment of the invention, the fusion polypeptide and the complex comprising the fusion polypeptide are watersoluble.
In a particular embodiment of the invention, the effector molecule(s) or at least one effector molecule comprises or consists of the amino acid sequence of the ESAT-6 protein from Mycobacterium tuberculosis. In this case, a soluble fusion polypeptide is preferably produced by co-expression with the CFP-10 protein from Mycobacterium tuberculosis, in a cell, and in particular in a E. coli cell, for example an E. coli BL21 λDE3 cell, more preferably at 20° C.
In a particular embodiment of the invention, the composition of the invention or the composition(s) which are present in the combination of the invention is free or substantially free of biotinylated molecules (in particular of biotinylated targeting molecule(s)) not bound to the fusion polypeptide.
In a particular embodiment of the invention, the combination the invention and/or the composition of the invention further comprise(s) a pharmaceutically acceptable carrier, and optionally an adjuvant, an immunostimulant, for example Poly I:C, and/or another molecule which is therapeutically active or suitable to have a prophylactic effect, which is(are) combined with (i.e., present in the same composition as) the fusion polypeptide and/or the biotinylated targeting molecule(s), and/or, if present, biotinylated, non-targeting molecule(s).
In the context of the present invention a “therapeutically active molecule” can be one which may be beneficial to the condition of a human or non-human host to which it is administered. It is especially an active principle suitable for use in the manufacturing of a drug. It may be a compound suitable to either, potentiate increase or modulate the effect of an therapeutically active principle.
The invention is further directed to of a fusion polypeptide as defined herein, a composition of the invention, or a combination of the invention, for use in prophylaxis and/or in therapy, and in particular for use to elicit a T-cell immune response and/or a B-cell immune response in vivo, in a human or non-human host in need thereof. By “immune response”, it is meant herein a single immune response or several immune responses, and in particular a humoral and/or cellular immune response.
In a particular embodiment of the invention, said immune response comprises or consists of a T-cell immune response, including a CTL response or a T helper (Th) response. Additionally or alternatively, said immune response can comprise or consist of a B-cell immune response.
In a particular embodiment of the invention, an “immune response” as recited herein (in particular a “T-cell immune response” or “T-cell response” as recited herein) comprises or consists of a mucosal immune response (in particular a mucosal T-cell immunity).
In a particular embodiment of the invention, a “T-cell immune response” (or “T-cell response”) comprises or consists of an IFN-γ and/or an IL-2 and/or an IL-17 (preferably an IFN-γ) T-cell immune response.
The fusion polypeptide of the invention is in particular appropriate for use in prophylactic vaccination and/or in immunotherapy protocols or in diagnostic proliferative recall response assay, a T cell cytokine recall response assay, or a T cell cytotoxic recall response assay, respectively, detecting the presence of antigen-specific T cells. It can be used especially as priming reagent or as boosting reagent, i.e. after the host or the cells has(have) been primed with an effector molecule, for example using a construct comprising a CyaA protein and said effector molecule.
The invention relates in particular to a fusion polypeptide as defined herein or to a combination of the invention and in particular a composition of the invention, for use for the prevention or the treatment of a disease selected from neoplasia, cancers and infectious diseases selected from viral-, retroviral-, bacterial-, parasite- or fungal-induced diseases.
In a particular embodiment, the invention is intended for the induction of a protective anti-mycobacterial immunity, and in particular is intended for anti-tuberculosis vaccination.
In a particular embodiment, the invention is intended for the induction of a protective anti-viral immunity, and in particular is intended for vaccination against CMV.
In a particular embodiment, the invention is used (in vivo, in vitro or ex vivo) for the detection and/or induction (i.e. activation and/or expansion) of immune responses and in particular of T cell responses (especially of specific immune responses and in particular of specific T cell responses) directed against the effector molecule(s) or against at least one effector molecule(s) present in the fusion polypeptide of the invention. In a particular embodiment of the invention, an immune response is induced against one of the effector molecules disclosed in the example part of the application, or against a variant of one said effector molecules (as disclosed herein).
In a particular embodiment, the invention is intended for in vivo, in vitro or ex vivo detection and/or induction (i.e. activation and/or expansion) of immune responses and in particular of T cell responses (especially of specific immune and T cells responses) directed against the allergen, toxin, tumor cell, infectious agent (in particular the bacteria (for example a mycobacteria, especially Mycobacterium tuberculosis or Mycobacterium leprae), the parasite, the fungus or the virus (for example the CMV or the HTLV) from which the effector molecule(s) or at least one effector molecule(s) are derived.
In a particular embodiment of the invention, an “activation” (or “induction”) consists in a re-activation of immune response(s) and in particular of memory T cell immune response(s).
The immune responses mentioned herein can be a protective immune response and/or a prophylactic immune response, which can be directed for example against any allergen, toxin, tumor cell or infectious agent disclosed herein, and more particularly against Mycobacterium tuberculosis, HPV or CMV.
The invention is also directed to the use of a fusion polypeptide as defined herein or a combination of the invention and in particular a composition of the invention, for the preparation of a vaccine or a medicament intended for the prevention and/or the treatment of a disease selected from neoplasia, cancers and infectious diseases selected from bacterial-, parasite-, fungus, viral- or retroviral-induced diseases especially resulting from infection with agents among those disclosed herein.
In a particular embodiment of the invention, a fusion polypeptide as defined herein or a combination of the invention and in particular a composition of the invention, is used in vivo, ex vivo or in vitro,
In a particular embodiment, the invention is used for the preparation of a booster vaccine intended for induction (or expansion) of immune responses as disclosed herein (in particular a T cell immune response and especially a mucosal T cell immunity) specific for the effector molecule(s) or against at least one effector molecule(s) present in the fusion polypeptide of the invention.
Another aspect of the invention relates to the use of a fusion polypeptide as defined herein or a combination of the invention and in particular a composition of the invention, in particular in vitro, ex vivo or in vivo, to select (or target) cell or subset(s) of cells, for use for diagnosing or immunomonitoring a disease in a mammal or for use in recall response assays from a sample (for example, from a sample of whole blood) from a human or a non-human mammal. Recall response assays can be performed for example in the case of following up the efficacy of an anti-tuberculosis or anti-CMV vaccine application.
Recall response assays can also be performed for example in the case of following up the efficacy of an anti-HPV vaccine application.
The invention relates in particular to the use in vitro, in vivo or ex vivo of a fusion polypeptide as defined herein or a combination of the invention (in particular a composition of the invention), for diagnosing or immunomonitoring an infection by an infectious agent as disclosed herein, in particular an infection by MTB, a CMV or a HPV, in a human or non-human mammal.
The invention also relates to a fusion polypeptide as defined herein, for use in vivo, in combination with:
By “recall assays”, it is meant herein an in vitro stimulation of T lymphocytes present in a sample of PBMC or whole blood from a human or non-human host by one or several immunogens presented by APCs, to detect specific T cell responses.
The invention also relates to the use of a fusion polypeptide as defined herein, in combination with one or several biotinylated targeting molecule(s) as defined herein and, optionally, one or several additional elements as defined above, for targeting, in particular in vitro or ex vivo, one or several effector molecule(s) of the fusion polypeptide to subset(s) of cells (in particular to DC or subset(s) of DC) and/or to cell surface molecule(s), in particular to cell surface receptor(s) (including DC surface receptor(s)).
The invention enables direct in vivo, ex vivo or in vitro, targeting of cells or subset(s) of cells and in particular DC subset(s) through their specific surface markers (particularly surface receptors).
Indeed, the use of a fusion polypeptide as defined herein in combination (for example in a composition or a composition of the invention) with one or several biotinylated targeting molecule(s) as defined herein and optionally, one or several additional elements as defined above, enables the delivery (or transfer) of one or several effector molecule(s) to the surface of cells or of subset(s) of cells, which have been selectively targeted via the biotinylated targeting molecule(s). In a preferred embodiment of the invention, these effector molecule(s) are then delivered into target cells, for example via endocytosis. Optionally they can be processed for MHC (in particular MHC-I or II) molecule-mediated antigen presentation.
Hence the invention enables the delivery, in vivo, ex vivo or in vitro, of one or several effector molecule(s) (in particular one or several immunogen(s)) onto and/or into (preferably into) subset(s) of cells, by the use of individual biotinylated targeting molecule of specificity against subset(s) of cells and/or against surface receptor(s) expressed on said subset(s) of cells.
The fusion polypeptide as defined herein or a combination (in particular a composition) of the invention may be formulated for administration enterally, parenterally (intravenously, intramuscularly or subcutaneously), transcutaneously (or transdermally or percutaneously), cutaneously, orally, mucosally, in particular nasally, orally, ophtalmically, otologically, vaginally, rectally, or by intragastric, intracardiac, intraperitoneal, intrapulmonary or intratracheal delivery. In a particular embodiment of the invention, they are administered intravenously or via the mucosal route, in particular intra-nasally or orally.
In a particular embodiment, the invention enables to raise, especially to prime or to boost, or to enhance antibody responses and/or T-cell responses, especially CD4+ and/or CD8+ systemic responses and/or mucosal T-cell responses, and/or lymphoproliferative responses, and/or to enhance resistance to tumor growth or to viral, parasitic or bacterial infection, in a cell or in a host (in vitro, ex vivo or in vivo).
T-cell responses as used herein can include CD4+ and/or CD8+ T cells responses, and in particular Th1, Th2, Th17, Treg and/or CD8+ T-cell responses.
The invention is also directed to a method for targeting one or several effector molecule(s) to cells, subsets of cells and/or to cell surface molecule(s) as defined herein, said method comprising:
(i) contacting said cells with one or several biotinylated targeting molecule(s) as defined herein, and
(ii) contacting cells with a fusion polypeptide as defined herein; and
(iii) optionally, contacting said cells with one or several additional elements chosen from biotinylated, non-targeting molecule(s) as defined herein, a pharmaceutically acceptable carrier, an adjuvant, an immunostimulant (for example Poly I:C), and another therapeutically active molecule as defined herein.
These two or three steps can be replaced by a single step consisting of contacting the cells with a composition of the invention.
Alternatively, steps (i) and (ii) can be performed separately, the fusion polypeptide and the biotinylated targeting molecule(s) being present in different compositions.
In a particular embodiment of the invention, steps (i) and (ii) are performed separately, and step (i) is performed before step (ii).
This method, which can be used for the delivery of one or several effector molecule(s) onto and/or into (preferably into) cells or subsets of cells as defined herein, can be performed in particular in vivo, in vitro or ex vivo.
In a particular embodiment, this method is performed in vitro or ex vivo, and cells are contacted either first with one or several biotinylated targeting molecule(s) as defined herein, and then with a fusion polypeptide as defined herein, or preferably with a composition of the invention.
In another particular embodiment, this method is performed in vivo. In this case, the fusion polypeptide, the biotinylated targeting molecule(s) and optionally, additional elements as defined herein, are contacted to cells of a human or non-human host by administration of these compounds or of composition(s) comprising them to said host. Preferably, a composition of the invention is administered to the host. Alternatively, the fusion polypeptide and the biotinylated targeting molecule(s) can be administered as separate compositions, but preferably extemporaneously.
By “contacting cells” or “exposing cells”, it is meant herein that a sample comprising said cells or consisting of said cells is contacted or exposed. In a particular embodiment of the invention, said sample comprises different types of cells and/or different types of subset(s) of cell(s), and in particular it does not comprise only the type(s) of cells or the subset(s) of cells which are said to be “contacted” or “exposed”. In another particular embodiment of the invention, said sample comprises only the type(s) of cells or the subset(s) of cells which are said to be “contacted” or “exposed”.
Hence, when it is mentioned herein that subset(s) of cell(s) are “targeted”, “contacted” or “exposed”, this does not necessarily mean that the fusion polypeptide as defined herein or a combination (in particular a composition) of the invention is applied to a sample comprising only said subset(s) of cell(s).
By “sample” it is meant herein any sample containing cells or subsets of cells as disclosed herein (especially a sample containing APC, for example DC cells or subset(s) of DC as disclosed herein, and/or cells or subset of cell(s) generated from bone marrow precursors), and in particular a sample of whole blood or of PBMC, or a sample of cells or subsets of cells as defined herein. In a particular embodiment of the invention, said sample is from a human or non-human host, in particular a human or a non-human mammal.
The invention also provides a method for preventing or treating a human disease, by contacting one or several effector molecule(s) with human cells, or subset(s) of human cells, in vivo or ex vivo. This method, which requires the use of a fusion polypeptide as defined herein, one or several biotinylated targeting molecule(s) as defined herein, and optionally one or several additional elements as defined herein, can be performed by the method for targeting one or several effector molecule(s) to cells, subsets of cells and/or to cell surface molecule(s) disclosed herein.
In a particular embodiment of the invention, one or several pico moles (for example 2, 3, 4, 5, 6, 7, 8, 9 or 10 pico moles) of an effector molecule are administered to a human or non human host.
In a particular embodiment of the invention, the cells (for example human or non human cells) which are contacted with the fusion polypeptide and the effectors molecule(s) or the host (for example the human or non human host) to which the fusion polypeptide and the effectors molecule(s) are administered have(has) been previously primed with effector molecule(s) which is (are) identical to effector molecule(s) that are present in the fusion polypeptide.
In a further aspect, the invention relates to the use of a SA or avidin polypeptide as defined herein for preparing a fusion polypeptide as defined herein.
The invention is also directed to methods for the production of a polypeptide comprising a SA or avidin polypeptide, and in particular a fusion polypeptide as defined herein. A first method comprises: expressing said polypeptide in a cell, for example an E. coli cell, at a temperature of 20° C. or less than 20° C., from a gene construct (in particular a polynucleotide, a plasmid or a vector) encoding said polypeptide. The E. coli cell can be for example an E. coli BL21 λDE3 cell or preferably an E. coli Artic Express DE3 cell.
E. coli Artic Express DE3 cell enables production of a polypeptide, and in particular of a polypeptide (for example a tetrameric fusion polypeptide) at a temperature of less than 20° C., for example at a temperature ranging from 10 to 15° C. or from 10 to 20° C., and in particular at 10° C. or 15° C. The produced polypeptide can then be solubilized in a 2M urea buffer, without having to use denaturing urea concentrations (which are above 4M).
This method enables direct production of a soluble tetrameric polypeptide or fusion polypeptide comprising a SA or avidin polypeptide, in the cytoplasm of E. coli. In contrast, methods disclosed in the prior art only allow production of a fusion polypeptide comprising a SA or avidin polypeptide as inclusion bodies, from which fusion polypeptide has to be extracted under denaturing conditions, for example with urea or guanidine solutions, and subsequently renaturated and refolded to form tetramers in vitro. Other methods disclosed in the prior art enable production of a fusion polypeptide comprising a SA or avidin polypeptide as a soluble fusion polypeptide but which is exported into periplasmic space of E. coli cells, which results in reduced yields.
In a particular embodiment of the invention, the method for the production of a polypeptide comprising a SA or a avidin polypeptide further comprises a step wherein the expressed polypeptide, in particular the expressed fusion polypeptide is purified using one or several IminoBiotin-Agarose columns (from Sigma).
By way of illustration, the affinity purification step can be performed as follows:
A second method for the production of a polypeptide comprising a SA or avidin polypeptide, and in particular a fusion polypeptide as defined herein comprises or consists of:
a) expressing said polypeptide in a cell, for example an E. coli cell (for example, an E. coli cell as disclosed herein), from a gene construct (in particular a polynucleotide, a plasmid or a vector as defined herein) encoding said polypeptide;
b) extracting said polypeptide from cytosolic extract or from cell debris with solubilizing or denaturing concentrations of urea (e.g., 2M or 8M urea);
c) diluting out polypeptide solution containing urea, said dilution being optionally performed in the presence of biotin.
In a particular embodiment of the invention, step b) consists in extracting said polypeptide upon cell disruption from cytosolic extract or from cell debris (with solubilizing or denaturing concentrations of urea, e.g., 2M or 8M urea), for example by sonication or cell lysis (especially enzymatic cell lysis);
In a particular embodiment of the invention, in step c), polypeptide solution containing urea is diluted out using a solution comprising biotin, in particular a solution comprising biotinylated targeting molecule(s) as defined herein, for example biotinylated-conjugated targeting antibodies as defined herein or a solution comprising biotinylated beads.
Alternatively or cumulatively, in a particular embodiment of the invention, in step c), polypeptide solution containing urea is diluted out using a solution, for example a buffer solution, said dilution being performed on a biotilylated surface, for example in a recipient, especially in a well (e.g. an ELISA (Enzyme-linked immunosorbent assay) well), a plate (e.g. a microtiter plate), or a tube, in which at least one surface is biotinylated.
In a particular embodiment of the invention, in step c), a dilution of at least 1:5 or 1:10, preferably 1:100, into said solution is performed.
Alternatively or cumulatively, in a particular embodiment of the invention, in step c), a dilution below 2M urea is performed.
During step c), interactions with biotin promote folding of the polypeptide and thus facilitate its tetramerization.
In a particular embodiment of the invention, step c) enables refolding and tetramerization of the polypeptide (only folded formed tetramers being bound strongly to biotin).
In a particular embodiment of the invention, the second method for the production of a polypeptide disclosed above further comprises a step d) wherein the polypeptide is purified using one or several IminoBiotin-Agarose column(s) (from Sigma), as disclosed herein.
Hence, the invention is also directed to a method for the preparation, of a polypeptide comprising a SA or avidin polypeptide (and in particular a fusion polypeptide as defined herein), in the form of a tetramer.
In a particular embodiment of the invention, said method comprises or consists of diluting said polypeptide or a composition or a complex as defined herein in the presence of biotin, for example:
in a solution comprising biotin, in particular a solution comprising biotinylated targeting molecule(s) as defined herein, for example biotinylated-conjugated targeting antibodies as defined herein or a solution comprising biotinylated beads; and/or
on a biotinylated surface, for example in a recipient, especially a well (e.g. an ELISA well), a plate (e.g. a microtiter plate), or a tube, in which at least one surface is biotinylated.
In a particular embodiment of the invention, said method comprises or consists in performing a method for the production of a polypeptide as disclosed herein.
Said method for the preparation, of a polypeptide comprising a SA or avidin polypeptide, in the form of a tetramer can in particular be applied before performing an ELISA analysis.
The invention is also directed to a method for the production of a composition or complex as defined herein, which comprises or consists of the following steps:
In a particular embodiment of the invention, this method comprises or consists of the following steps:
In a particular embodiment of the invention, the fusion polypeptide is contacted first with one or several biotinylated targeting molecule(s) in condition enabling said biotinylated targeting molecules to interact and complex with the fusion polypeptide and, optionally, with non-targeting molecule(s) as defined herein.
In a particular embodiment of the invention, the fusion polypeptide is contacted with biotinylated targeting or non-targeting molecules by mixing a composition comprising said fusion polypeptide with a composition comprising said biotinylated targeting or non-targeting molecules.
The invention also relates to a method for the stimulation of specific T lymphocytes by targeting an antigen or fragment thereof comprising at least one T-cell epitope to antigen presenting cells, wherein said method comprises the steps of:
Said method can be performed in vivo, in vitro or ex vivo, but it is preferably performed in vitro or ex vivo.
The invention also relates to a method for the in vitro or ex vivo selection of a subset of APC to which the targeting of an antigen or a fragment thereof comprising at least one T-cell epitope can induce a T-cell immune response directed against said antigen or fragment thereof, wherein said method comprises the steps of:
In a particular embodiment of the invention, the above-mentioned method comprises the steps of:
a) exposing a subset of APC to (i) biotinylated targeting molecules as defined herein, which are capable of targeting these APCs and in particular of interacting with one or several cell receptor(s) present on the surface of these subset of APCs, and to (ii) a fusion polypeptide as defined herein, which comprises the antigen or fragment thereof,
wherein optionally said fusion polypeptide has been previously produced by the method of the invention; and
b) exposing T cells, in particular CD8+ or CD4+ T cells, to the subset of APCs provided by step a); and
c) detecting in vitro a change in activation of the T cells.
Step a) provides a subset of APC binding the fusion polypeptide through the biotinylated targeting molecule.
A “change in activation of the T cell(s)” as used herein can be for example a change in IL-2, IL-4, IL-5, IL-17 or IFN-γ production.
In a particular embodiment of the invention, the detection of a change in T cell activation is achieved with the EPLISPOT assay, ELISA, or other assay to detect T cell activation, for example a proliferation assay.
In a particular embodiment of the invention, the test sample used in theses methods is peripheral blood mononuclear cells (PBMC), whole blood, or a fraction of whole blood.
The invention also relates to a polynucleotide encoding a fusion polypeptide as defined herein, and to a plasmid or a recombinant vector (in particular a recombinant expression vector) comprising said polynucleotide.
In a particular embodiment of the invention, the polynucleotide of the invention or the plasmid or a recombinant vector of the invention comprises or consists of SEQ ID NO.: 41.
The invention also relates to a cell comprising the polynucleotide of the invention or a plasmid or a recombinant vector of the invention.
In a particular embodiment of the invention, the cell of the invention is able to express the fusion polypeptide as defined herein.
The invention is also directed to a kit, in particular a kit for a diagnostic test of a disease in a mammal, for immonomonitoring a disease in a mammal and/or for the prevention and/or the treatment of a disease in a mammal, which comprises:
Other characterizing features of the invention will become apparent from the examples and from the figures and they apply, individually or in combination, to the above disclosed elements of the invention.
A. The amino acid sequences of the following constructs are given:
In these sequences, the methionine residue which is underlined corresponds to the one which is located at the junction between the antigen and linker-encoded sequences (these sequences are N-terminal to the methionine residue) and the streptavidin polypeptide (residues 14-139 of the streptavidin preprotein from Streptomyces avidinii) (these sequences are C-terminal to the methionine residue). In addition, a linker of sequence leucine-glutamic acid (L-E), which is optional, is located at the C terminal end of these sequences.
B. Sequence of pET28b-CFP-10:Esat-6-SA (vector for co-expression of CFP-10 with ESAT6-SA) (SEQ ID NO.: 40). The sequences indicated (i) in bold, (ii) in italics and underlined, (iii) in bold and italics and (iv) the sequence which is underlined correspond respectively to sequences of the (i) T7 promoter, (ii) the lac operon, (iii) the ribosome binding site, and (iv) the M. tuberculosis antigens CFP-10 and Esat-6 and to the SA polypeptide (residues 14-139).
and
=differences statistically significant, respectively, p<0.05 and 0.005, between BCG-unprimed mice and mice immunized with TB10.4-SA targeted to different DC markers. * and **=differences statistically significant, respectively, p<0.05 and 0.005, between BCG-primed mice and BCG-primed mice and boosted with TB10.4-SA targeted to different DC markers. (C) CD8+ T-cell cross priming in BCG-primed BALB/mice boosted with TB10.4-SA targeted to CD205. Representative CD8+ T-cell responses to H-2Kd-restricted GYAGTLQSL epitope, shared by TB10.3 and TB10.4, detected by cytofluorometry by use of a combination of FITC-anti-CD8α, allophycocyanine-anti-CD44 and PE-conjugated H-2Kd pentamer complexed with TB10.3/4:20-28 peptide. Results are representative of at least two independent experiments.
Expression: mm—molecular marker;
Extraction: 3: cytosolic extract (100° C., 5 min.);
Purification (after extraction from cell debris with 2 M urea):
The results shown were obtained for the soluble tetrameric SI-SA-ABD223 fusion polypeptide. Similar results were obtained for the soluble tetrameric SI-SA-ABDwt fusion polypeptide (data not shown).
Expression: mm—molecular marker;
Extraction: 3: cytosolic extract (100° C., 5 min.);
Purification (after extraction with 8 M urea):
The results shown were obtained for the insoluble monomeric SI-SA-ABD275 fusion polypeptide. Similar results were obtained for the insoluble monomeric SI-SA-ABD29 and SI-SA-ABD35 fusion polypeptides (data not shown).
Expression, extracts:
Purification on Iminobiotin Agarose:
Dialysis, Endotoxin removal, concentration:
Expression, extracts:
Purification on DEAE sepharose:
Purification on Phenyl sepharose:
The codon-optimized synthetic gene encoding for expression in E. coli of residues 14-139 of the streptavidin protein from Streptomyces avidinii (Sano et al, 1995) was obtained from GenScript (NJ, USA) and inserted into the pET28b expression vector (Novagen, Darmstadt, Germany). The reason for using only the natural core of strepavidin without N- and C-terminal part was the previously reported proteolytical processing of streptavidin during the cultivation and purification of SA from E. coli (Pahler A. et al 1987, Bayer E. A. et al, 1989). To avoid cleavage of the streptavidin fusion proteins, the natural core SA without the processed sequences was used, where the truncation of SA did not perturb tetramerization capacity of the fusion constructs.
The genes for appropriate antigens were PCR-amplified using pairs of PCR primers indicated in Table 1 and genetically fused to the 5′- or 3′ end of the streptavidin gene by insertion into appropriate restriction sites (Table 1). The exact sequence of the cloned inserts was verified by DNA sequencing. The plasmids were transformed in to E. coli cells for IPTG inducible production of proteins.
CFP-10 fusion proteins (CFP-10-SA, CFP-10-Esat-6-SA, CFP-10-Esat-6-SA-Tb7.7) were produced in E. coli BL21(λDE3) cells (Stratagene, La Jolla, USA) at 20° C., while E. coli Artic Express DE3 cells (Stratagene, La Jolla, USA) was used for production of other SA fusion proteins. To express the CFP-10-SA fusion proteins the transformed E. coli strain BL21(λDE3) was grown at 20° C. in LB medium (containing 60 μg/ml kanamycin), which was inoculated with 0/N culture to the OD600˜0.8 and subsequently induced with IPTG to final concentration of 0.5 mM. The cells were harvested 8 hours later, washed one time in 50 mM CH3COONH4 buffered to pH 9 by 25% NH3*H2O (AC buffer) and stored at −20° C.
The ESAT-6-SA fusion polypeptide on its own is rather poorly soluble in E. coli cells. However, when CFP-10, which is a chaperon for ESAT-6, is co-expressed with ESAT-6, or with a fusion polypeptide comprising ESAT-6 (for example the fusion polypeptide ESAT-6-SA), it enhances the solubility of ESAT-6 or of said fusion polypeptide in E. coli cytoplasm.
Other SA-fusion expression vectors were transformed to E. coli strain Artic Express DE3, where the newly synthesized proteins were stabilized by chaperons cpn 10 and cpn 60, induced at low growth temperatures. The proteins of interest were produced in soluble form in bacterial cytosol. 500 ml LB medium (60 μg/ml kanamycin and 20 μg/ml gentamycin) was inoculated with 0/N culture, cells were grown at 28° C. to A600˜0.8 before temperature was lowered to 10° C. and production of proteins was induced by addition of IPTG to 0.5 mM final concentration. The culture was harvested 24 hours later, washed one time in 50 mM AC buffer and stored at −20° C.
E. coli Arctic express DE3 cells have cold-inducible expression of cpn10 and cpn60 chaperons, which assist protein folding at low temperatures (around 10° C.) and help maintain recombinant proteins soluble. Use of these cells enables to obtain folded tetramers of fusion polypeptides, which only precipitate in E. coli cytoplasm, without forming true inclusion bodies, and can be extracted as in native tetramers forms, for example using 2 M urea, which is not denaturing the tetramers. Hence, no in vitro refolding of the produced fusion polypeptides is necessary under these conditions.
The frozen cells were resuspended in AC buffer and lyzed by ultrasonic disruption. CFP-10-SA fusion proteins were purified directly from soluble cytosolic extract, while the other SA fusion tetramers were solubilized from cell debris by extraction with 2 M Urea in AC buffer without tetramer disruption, respectively. The extracts were loaded on IminoBiotin-Agarose (Sigma) columns equilibrated in AC buffer with 0.5 M NaCl (pH 9). The columns were first washed with several bed volumes of equilibration buffer, followed by 0.1 M acetic acid pH 2.9 with 0.5 M NaCl. Elution was achieved with 0.1 M acetic acid pH 2.9 without salt. Eluted fractions were immediately buffered by addition of 1/50 of fraction volume of 25% NH3*H2O to reach a final pH 9 and a soluble stable protein was obtained. In turn, elution with 50 mM ammonium or sodium acetate pH 4.0 recommended within IminoBiotin agarose datasheet of Sigma-Aldrich, resulted in elution of precipitated Ag-SA fusion proteins.
The unfused core streptavidin eluted already in 0.1 M acetic acid 0.5 M NaCl. In contrast, the washing step with 0.1 M acetic acid 0.5 M NaCl pH 2.9 was crucial for stabilization and retention of the Ag-SA fusion tetramers on the column during the decrease of pH from equilibration buffer (pH 9) to elution buffer (pH 2.9) to prevent precipitation. Subsequent elution with 0.1 M acetic acid pH 2.9 without salt allowed to recover soluble proteins.
The Ag-SA fusion proteins were concentrated on spin columns (Millipore, Bedford, Mass., USA) and contaminating lipopolysacharide was removed by passage through EndoTrap column (Profos, Regensburg, Germany), to reduce LPS levels below 50 EU/mg of protein, as assessed using the endotoxin chromogenic LAL test assay kit (Lonza, Walkersville, Md., USA). Formation of Ag-SA tetramers was controlled using Tris-Tricine SDS-PAGE gels (15%). Biotin binding was controlled in Western blots by detection of biotinylated marker proteins with antigen-SA fusions that were themselves detected by a sandwich of antigen-specific polyclonal sera and anti-rabbit-peroxidase conjugate.
Construct CFP-10:ESAT-6-SA enables co-expressing the CFP-10 protein and the ESAT-6-SA fusion polypeptide in the same cell (for example in E. coli cell), from the same expression vector. CFP-10, which is a chaperon for ESAT-6, associates with the ESAT-6-SA fusion polypeptide and hence enhances its solubility in E. coli cytoplasm.
CFP-10:ESAT-6-SA fusion protein binds efficiently to the surface of mouse bone-marrow-derived dendritic cells (BM-DC) via biotinylated mAbs specific to DC surface markers (see
Following injection of CFP-10:ESAT-6-SA, complexed to biot-mAbs specific to DC surface markers, it is possible to detect this fusion protein specifically at the surface of the targeted DC subset (see
CFP-10:ESAT-6-SA fusion protein, targeted in vitro to the surface of BM-DC via biot-mAbs specific to different DC surface markers, gains access to the MHC-II processing/presentation pathway, leading to the presentation of immunodominant epitopes by MHC-II molecules to specific TCR (see
One-third of the Earth's population is infected with Mycobacterium tuberculosis, making the pulmonary tuberculosis the most widely spread infectious disease, leading to 1.6 million deaths annually. The only vaccine in use against infection with M. tuberculosis, the live attenuated M. bovis BCG (Bacillus Calmette-Guérin), is not able to protect efficiently against the adult pulmonary tuberculosis in endemic zones. Moreover, with the resurgence of tuberculosis in immuno-compromised individuals and the rapid expansion of multi-drug resistant and extensively drug-resistant tuberculosis, the need of a better rational design of new strategies of anti-tuberculosis vaccines is reinforced (WHO, 2007). Despite intense research on live attenuated and/or sub-unit anti-tuberculosis vaccines, a very few vaccine candidates display only slightly improved protective effect, with limited success compared to BCG (Kaufmann, 2006).
In the present study, we sought to drive the extensive knowledge available on the properties of DC and antigen targeting to DC subsets, towards the practical in vivo antigen delivery to DC in anti-tuberculosis vaccination. Indeed, so far, addressing M. tuberculosis-derived protein antigens to DC subset(s) and/or DC surface receptor(s), for the induction of protective anti-mycobacterial immunity, has not been investigated.
Our strategy for the rational design of a new anti-mycobacterial vaccine was to target prominent mycobacterial antigens, i.e., proteins of ESX family, to diverse DC subsets with specialized activities. Indeed, mobilization of the latter by their direct in vivo targeting through their specific surface markers represents a promising pathway to dictate and to control differentiation of T cells. To this end, we developed a versatile in vivo approach by genetic fusion of selected ESX antigens to streptavidin, thus able to be complexed to individual biotin-conjugated mAbs of a wide-ranging panel of specificities against DC surface receptors and to be readily carried in vivo to different DC subsets. By use of this strategy, we showed that minute amounts, i.e, several pmoles, of ESX antigens targeted to 132 integrins, PDCA-1 or diverse C-type lectins were highly efficiently captured, endocytosed and presented by MHC molecules in vitro and in vivo and induced ESX-specific Th1 and Th17—but not Th2—responses. Moreover, in BCG-primed mice, boosting with ESX antigen targeting to DC subsets led to a remarkable improvement of Th1 and Th17 responses in the case of targeting to C-type lectins or to PDCA-1. In BCG-primed mice, TB10.4 targeting to CD205 endocytic C-type lectin also induced a significant cross-priming of specific CD8+ T cells.
Despite their shared morphology, abundance in T-cell areas of lymphoid tissues, high MHC-II expression and outstanding potential to continuously probe the environment, process and present antigens to T cells, DC are divided into different subsets, according to their ontogenic origin, phenotype, maturation programs and specialized functions. Although the well-established classification of the mouse DC subsets cannot been directly transposed to the human DC populations, plasmacytoid DC, blood-derived lymphoid tissue resident DC, peripheral migratory DC and monocyte-derived inflammatory DC have been distinguished in both mice and humans (Reis e Sousa, 2006, Shortman, 2007, Randolph, 2008). In the mouse spleen, three major DC subsets are distinguished: (i) CD11c+ B220+ Plasmacytoid DC Antigen (PDCA)-1+ plasmacytoid DC, specialized in the production of type-I IFNs, (ii) CD11c+ CD11b− CD8α+ conventional DC, with high potential to take up notably dead cells, to process and to cross-present the derived antigens and to activate T cells via IL-12p70 production, and (iii) CD11c+ CD11b+ CD8α− conventional DC, considered as potent inducers of MHC-II-restricted T-cell responses against exogenous antigens (Reis e Sousa, 2006). In the mouse intestine-associated lymphoid organs, at least two functionally distinct DC subsets have been described, according to their expression of the integrin CD103. Only the CD103+ DC population displays properties at inducing Foxp3+ Treg from FoxP3− T cells via a TFG-β- and retinoic acid-dependent mechanism (Coombes, 2007; Sun, 2007). Another level of specialization of DC subsets has been recently evidenced in the mouse skin. Indeed, in addition to the resident DC of the lymph nodes, the skin contains epidermis-derived CD205hi CD8α− Langerhans cells, CD207+ CD205int CD8α− conventional dermal DC and a CD207+ CD103+ dermal DC population. Only the latter is able to cross-present viral and self antigens to naive CD8+ T cells (Bedoui, 2009). In the mouse lungs and conducting airways, different DC subsets with functional specialization have been identified, as well. At the steady state, the trachea contains intraepithelial CD11b− CD207+ CD103+ DC. Under conditions of inflammation, the submucosa of the airways may contain CD11b+ CD103− conventional DCs with potential capacity to prime and/or restimulate effector CD4+ T cells. In the lung parenchyma CD11c+ CD11b+ and CD11c+ CD11b− DCs are present, can migrate to the alveolar lumen or to mediastinal lymph nodes. Like in the spleen, in the lung parenchyma plasmacytoid DC are detectable, display a CD11cint CD11b− PDCA-1+ phenotype and produce large amounts of IFN-α upon in vitro TLR triggering (de Heer, 2005).
Numerous evidences argue that the magnitude of adaptive immune responses, as well as differentiation and specialization of CD4+ T cells into Th1, Th2 or Th17, are dictated by different DC subsets with specialized activities (Villadangos, 2007) (Steinman, 2008) (Steinman, 2007). Mobilization of different DC subsets by their direct in vivo targeting through their specific surface markers represents a promising pathway to design well-controlled immunization strategies for the development of preventive and/or therapeutic vaccines (Steinman, 2008; Shortman, 2009). In this domain, the most significant strategy has been elegantly developed by the teams of Steinman and Nussenzweig, through antigen coupling to antibodies specific for DC surface receptors. In this approach, the ovalbumin (OVA) model antigen or pathogen-derived antigens are coupled or genetically inserted to the NLDC-145 mAb, specific to the C-type lectin endocytic receptor CD205. A single, low dose of this vector, together with an appropriate DC maturation signal, are able to induce robust and long-lasting antibody responses, CD4+ and CD8+ systemic and mucosal T-cell responses, correlated with an enhanced resistance to tumor growth or to viral infection (Bonifaz, 2002; Boscardin, 2006; Dudziak, 2007) (Trumpfheller, 2006). More recently, another endocytic C-type lectin, i.e., DC, NK lectin Group Receptors-1 (DNGR-1, Clec9A), has been identified by two independent teams. DNGR-1, specifically expressed on mouse CD8α+ splenic DC, has been used as targeted DC surface marker, (i) in the absence of adjuvant, for efficient induction of humoral immunity (Caminschi, 2008) and, (ii) in the presence of anti-CD40 agonistic mAb as adjuvant, for induction of OVA-specific CD4+and CD8+ T cells, with successful preventive and therapeutic effect against OVA-expressing melanoma tumor cells (Sancho, 2008). Another C-type lectin Clec12A, highly expressed on splenic CD8+ DC and plasmacytoid DC has been used as targeted DC surface receptor and induces Ab responses, in the presence of minimal amounts of adjuvant (Lahoud, 2009).
So far, the antigen targeting strategy is limited by the requirement of individual chemical coupling or genetic insertion of each immunogen of interest to mAbs specific to each of the numerous DC surface receptors, candidate for antigen addressing. Even though it is largely admitted that DC translate information from different surface receptors into an activation program that orients the Th cell differentiation, since all the rules governing functions of DC subsets are not yet understood, it remains difficult to predict which DC subsets/DC surface receptors are the most appropriate to be targeted in order to optimize the protective immunity against a given pathogen. Therefore, comparison of the properties and impacts of various DC subsets on the generation of pathogen-specific adaptive responses may help identify the most adapted DC subset(s), able to tailor the most adapted and protective adaptive immunity. To this end, here we designed a versatile approach to identify the most appropriate DC receptor(s) to which the targeting of relevant M. tuberculosis-derived immunogens can induce optimized immune responses with anti-tuberculosis protective potential. In this approach, prominent mycobacterial immunogens are genetically fused to streptavidin (SA). The resulting fusion proteins are tetramerized to optimize their high affinity interaction with biotin (biot). Such SA fusion tetramers are then complexed to biot-conjugated mAbs, specific to diverse DC surface receptors. Therefore, in this flexible model, once such antigen-SA fusion proteins are produced, they can be readily carried and delivered to different DC subsets by simple use of individual biot-mAbs of a large panel of specificities against DC surface receptors, with expression profiles restricted to given DC subsets.
Potent mycobacterial antigens included in this study were selected among highly-conserved, low-molecular weight immunogens belonging to the Early Secreted Antigenic Target, 6 kDa (ESAT-6) protein family (ESX) of M. tuberculosis (Brodin, 2004). These proteins, actively secreted by the type VII secretion system of mycobacteria (Simeone, 2009), are known for their marked immunogenicity in mice, guinea pig and in ethnically different human populations, and for their protective potential in animal tuberculosis models (Brodin, 2004). Moreover, the presence of CD4+ and CD8+ effector T cells specific to such proteins is directly correlated to the natural anti-mycobacterial protection in M. tuberculosis-infected humans. We characterized the immunogenicity of several ESX proteins fused to SA (ESX-SA), targeted to different DC surface receptors by complexing them to biot-mAbs specific to MHC-II molecules, CD11b or CD11c β2 integrins, PDCA-1 or diverse C-type lectins. The latter were chosen from: (i) mannose receptor family, i.e., CD205 (DEC205), (ii) asialoglycoprotein receptor family, i.e., CD207 (Langerin, Clec4K), or CD209 (DC-Specific ICAM3-Grabbing Non-integrin, DC-SIGN), or (iii) DC Immunoreceptor (DCIR) subfamily of asialoglycoproteoin receptor, i.e., DCIR-2 (Clec4A) (Geijtenbeek, 2009). We explored this model to select the most appropriate DC subsets or DC surface receptors to target in anti-tuberculosis vaccination on the basis of capture/endocytosis/processing and presentation of ESX antigens by MHC molecules, in vivo outcome of the ESX-specific Th1, Th2, Th17 Treg or CD8+ T-cells responses, boost effect of such immunization subsequent to BCG priming and protective potential in the mouse model of M. tuberculosis infection.
The E. coli codon-optimizeds synthetic gene encoding residues 14-139 of streptavidin from Streptomyces avidinii was obtained from GenScript (NJ, USA) and inserted into the pET28b expression vector (Novagen, Darmstadt, Germany). The genes for TB antigens cfp-10, esat-6 and tb10.4 were PCR-amplified using pairs of PCR primers indicated in Table 1 and genetically fused to the 5′-end of the streptavidin gene by insertion into the NcoI, NheI and EcoRI sites. The exact sequence of the cloned inserts was verified by DNA sequencing. The plasmids were transformed in to E. coli cells for IPTG inducible production of proteins.
CFP-10-SA protein was produced in E. coli BL21 λDE3 cells (Stratagene, La Jolla, Canada) at 20° C., while E. coli Artic Express DE3 cells (Stratagene, La Jolla, Canada) was used for production of ESAT-6-SA and TB10.4-SA proteins. In the latter case, cells were grown at 28° C. to A600 0.8 before temperature was lowered to 10° C. and production of proteins was induced by addition of IPTG to 0.5 mM final concentration. Cells were grown in LB medium containing 60 μg/ml kanamycin and 20 μg/ml gentamycin (for E. coli Arctic express only).
The cells were harvested and lyzed by ultrasonic disruption. CFP-10-SA was purified directly from soluble cytosolic extract, while ESAT-6-SA and TB10.4-SA tetramers were solubilized from cell debris by extraction with 2 M Urea, respectively. The extracts were loaded on IminoBiotin-Agarose (Sigma) columns equilibrated in 50 mM CH3COONH4 buffered with NH3*H2O to pH 9. The columns were washed with several bed volumes of 0.1 M acetic acid, 0.5 M NaCl pH 3 and eluted using 0.1 M acetic acid without salt pH 3, with immediate neutralization of acetic acids by addition of 1/50 of fraction volume of 25% NH3*H2O to reach a final pH 9. The proteins were concentrated on spin columns (Millipore, Bedford, Mass., USA) and contaminating lipopolysacharide was removed by passage through EndoTrap column (Profos, Regensburg, Germany) to reduce its level below 50 EU of LPS/mg of protein, as assessed using the endotoxin chromogenic LAL test assay kit (Lonza, Walkersville, Md., USA). Formation of the tetramers was controlled using Tris-Tricine SDS-PAGE gels (15%). Biotin binding was controlled in Western blots by detection of biotinylated marker proteins with antigen-SA fusions that were themselves detected by a sandwich of antigen-specific polyclonal sera and anti-rabbit-peroxidase conjugate.
The synthetic peptides ESAT-6:1-20 (Brandt, 1996), Culture Filtered Protein, 10 kDa (CFP-10):11-25 (Kamath, 2004), TB10.3/4:20-28 (Majlessi, 2003), and TB10.4:74-88 (Hervas-Stubbs, 2006) peptides were all synthesized by NeoMPS (Strasbourg, France).
Biotinylated mAbs Specific to DC Surface Receptors
mAbs specific to CD11b (clone M1/70.15.11.5.HL, rat IgG2b, ATTC-TIB-12), CD11c (clone N418, Armenian hamster IgG, ATTC-HB-224), DCIR-2 (clone 33D1, rat IgG2b, ATCC-TIB-227) or to MHC-II (I-A/I-E) (clone M5/114.15.2, rat IgG2b) or the control Ig (clone R187, rat IgG, ATCC-CRL-1912) were prepared from supernatants of B-cell hybridomas, cultured in serum-free, synthetic HL-1 medium (Lonza BioWhittaker, Walkersville, Md.) complemented with 2 mM L-glutamax, 5×10−5 M β-mercapto-ethanol, 100 IU/ml penicillin and 100 μg/ml streptomycin. Supernatants were treated with (NH4)2SO4, prepared in sterile water for injection (Baxter, Maurepas, France), at 50% final concentration at 4° C. in endotoxin-free conditions, as described elsewhere (Jaron, 2008). The precipitated proteins were extensively dialyzed against PBS and sterilized by filtration through 0.2 μm filters. Absence of endotoxins in the Ig preparations was then checked by use of “Limulus Amebocytes Lysate” kit (Cambrex, Emerainville, France), with a detection limit of 0.01 IU/ml. Ig were biotinylated by use of EZ-Link Sulfo-NHS-LC kit (Pierce, Rockford, Ill.), according to the manufacture's protocol, and under stoechiometric conditions leading to fixation of 2 moles of biotine per mole of Ig. Biot-anti-CD205 mAb (clone NLDC-145, rat IgG2a) was purchased from Celldex Therapeutics (Needham, USA). (Czech Republic). Biot-mAbs specific to CD207 (Langerin) (clone eBioL31, rat IgG2a), CD209 (DC-SIGN) (clone LWC06, rat IgG2a) or CD317 (PDCA-1) (clone eBio927, rat IgG2b) were purchased from eBioscience (San Diego, Calif.).
Conventional or plasmacytoid DC were generated from femur-derived hematopoietic precursors, respectively, in the presence of GM-CSF or Flt3L, as previously described (Mouries, 2008). BM-DC (1×106 cells/well) were incubated at 4° C. with 1.5 μg/ml of biot-mAbs specific to DC surface markers or of biotin-conjugated control Ig isotypes. Cells were then washed at 4° C. and incubated with 1 μM (=21 μg/ml) of ESAT-6-SA for 1 h at 4° C. Cells were washed three times at 4° C. and were then either left at 4° C. or incubated for 3 h at 37° C. to evaluate the possible internalization. The presence of ESAT-6 at the cell surface was detected by cytofluorometry, by use of the anti-ESAT-6 mAb (clone 11G4) (Antibody Shop, Gentoft, Denmark), labeled with the pH sensitive Alexa647H, by use of FluoProbs protein labeling kit (Interchim, Montluçon, France). Percentages of the reduction in the MFI of the cell surface bound ESAT-6 signal was calculated as 100−[(MFIbiot-mAb+ESAT-6-SA 37° C.)−(MFIblot-control Ig+ESAT-6-SA 4° C.)/(MFIbiot-mAb+ESAT-6-SA 4° C.)−(MFIbiot-control Ig+ESAT-6-SA 4° C.)]×100.
In vivo binding of ESAT-6 to spleen DC subsets was studied at different time points after i.v. injection of ESAT-6-SA, complexed to biot-mAbs or to biot-control Ig, in the presence of Poly Inosinic:Poly Cytidylic acid (Poly I:C). Spleen low density cells were prepared by use of iodixanol gradient medium (OptiPrep, Axis-Shield, Dundee, UK). Briefly, collagenase-DNase-treated spleens were homogenized and splenocytes were suspended in 15% iodixanol and layered with 11.5% iodixanol. After centrifugation, low density cells recovered from the top of the gradient were stained with a combination of DC-specific and Alexa647H-anti-ESAT-6 mAbs, prior to analysis by cytofluorometry.
ESAT-6:1-20-specific, I-Ab-restricted, NB11 T-cell hybridoma has been recently described (Frigui, 2008). TB10.4:74-88-specific T-cell hybridomas were generated from BALB/c (H-2d) mice, immunized s.c. with 1×107 CFU of BCG. Two weeks after the immunization, total splenocytes and inguinal lymph node cells were pooled and stimulated in vitro with 10 μg/ml of TB10.4:74-88 peptide. At day 4, viable cells were harvested on Lympholyte M (Cedarlane Laboratories) and were fused, at 1:1 ratio, with BW51-47 thymoma cells by use of polyethylene glycol 1500 (Roche Diagnostics), as previously described (Majlessi, 2006). T-cell hybridomas were first individually expanded and screened for their capacity to release IL-2 upon recognition of TB10.4:74-88 peptide, presented by syngenic BM-DC. The positive T-cell hybridomas were then screened for their capacity to recognize BM-DC incubated with the recombinant TB10.4 protein (Hervas-Stubbs, 2006) or BM-DC infected with BCG for 24 h at m.o.i of 1, in antibiotic-free conditions. The presence of IL-2 in the supernatants of the co-cultures of BM-DC and T-cell hybridomas was assessed by a standard IL-2-specific ELISA. L fibroblasts, transfected with I-Ad, I-Ed or I-Ab restricting elements, were used as peptide presenting cells, in the same type of assay to determine the H-2 restriction of the presentation to the T-cell hybridomas. A selected T-cell hybridoma (1H2), specific to TB10.4:74-88 and restricted by I-Ad, was used in in vitro and ex vivo presentation assays of TB10.4 antigen delivery to DC.
BM-derived macrophages (fully adherent CD11c− CD11b+ cells), BM-derived conventional DC (semi-adherent CD11c+ CD11b+ cells) or BM-derived plasmacytoid DC (CD11cintB220+ PDCA-1+), as previously described (Mouries, 2008), (1×105 cells/well) were incubated with 1.5 μg/ml of biot-mAbs specific to diverse markers of DC for 30 min at 4° C. Cells were then washed and incubated with various concentrations of ESX-SA fusion proteins. Cells were then washed again extensively at 4° C. and co-cultured overnight with 1×105 cells/well of appropriate T-cell hybridomas. The efficiency of antigen presentation was judged by the evaluation of IL-2 produced in the co-culture supernatants by ELISA. When indicated, the efficiency of ESX antigen presentation was measured by use of polyclonal T cells from M. tuberculosis infected C57BL/6 mice, prepared by positive magnetic sorting of Thy-1.2+ T splenocytes by use of anti-Thy-1.2-mAb-conjugated magnetic microbeads and AutoMacs Pro (Miltenyi Biotec, Bergisch-Gladbach, Germany) and by use of Possel-D program. In this case, the supernatants of co-cultures were assessed for IFN-γ by ELISA.
For ex vivo antigen presentation assays, BALB/c mice were injected i.v. with 50 pmoles (=1 μg)/mouse of TB10.4-SA, complexed to biot-mAbs, in the presence of 25 μg/mouse of Poly I:C. At different time points post-injection, low density cells were prepared from the spleen of the injected mice, as detailed above, and were stained with anti-biot mAb-coupled to magnetic microbeads
(Miltenyi Biotec) for further positive selection of cells targeted in vivo by TB10.4-SA-biot-mAb complex. Cells were then magnetically sorted on AutoMacs Pro by use of Possel-S program. Various numbers of cells contained in positive or negative fractions were co-cultured with anti-TB10.4:74-88 1H2 T-cell hybridoma and IL-2 was assessed in their co-culture supernatants after 24 h incubation.
Female BALB/c (H-2d), C57BL/6 (H-2b) and C3H(H-2k) mice were purchased from Charles Rivers (Arbresle, France) and were immunized at 6-12-week-old. Tetramers of ESX-SA fusion proteins and biot-conjugated mAbs or appropriate biot-conjugated control Ig were mixed at a ratio of 2:1 at molar basis, at the indicated doses, and were complexed by incubation at 4° C. for 1 h. The final mixture was injected i.v. in 200 μl/mouse in the presence of 25 μg/mouse of Poly I:C. Immunization with BCG (Pasteur 1173P2 strain) was performed by s.c. injection at the basis of the tail. C57BL/6 FcγRo/o mice, deficient for activating FcγRI, III, IV receptors (Takai, 1994), were kindly provided by Pierre Bruhms and Marc Daeron (Institut Pasteur, Paris). C57BL/6 CD11c YFP mice (Lindquist, 2004) were kindly provided by Philippe Bousso (Institut Paster, Paris). Treg attenuation was performed by an i.p. injection of 1 mg/mouse of anti-CD25 mAb (clone PC61) or of control Ig at day −2 before immunization. All animal studies were approved by the Institut Pasteur Safety Committee, in accordance with the national law and European guidelines.
CD4+ T-cell assays were performed on splenocytes from individual immunized mice. Cells were cultured in complete HL-1 medium in the presence of various concentrations of appropriate ESX-derived peptides, harboring MHC-II-restricted immunodominant T-cell epitopes or mycobacterial-derived Ag85A:101-120 or Ag85A:241-260, as negative control peptides, respectively in H-2d or H-2b haplotype. Supernatants of such cultures were assessed at 24 h post-incubation for the presence of IL-2 and at 72 h for IL-5 and IFN-γ, as previously described (Jaron, 2008). IL-17A was also quantified at 72 h by ELISA by use of anti-IL-17 mAb (clone 50104) for coating and biot-anti-IL-17 mAb (clone BAF421) from R&D system for the detection.
To evaluate CD8+ T-cell responses, total splenocytes from immunized mice were stimulated in vitro with 10 μg/ml of TB10.3/4:20-28 peptide in RPMI, complemented with 2 mM L-glutamax, 5×10−5 M β-mercapto-ethanol, 100 IU/ml penicillin, 100 μg/ml streptomycin and 10% FCS. At day 6, detection of CD8+ T lymphocytes, specific to the TB10.3/4:20-28 epitope, was performed by cytofluorometry, by use of a PE-conjugated pentamer of H-2Kd, complexed to TB10.3/4:20-28 peptide (Proimmune, Oxford, UK), in the presence of FITC-conjugated anti-CD8α (clone 53-6.7) and allophycocyanin-conjugated anti-CD44 (clone IM7) mAbs, purchased from BD/PharMingen, (Le Pont de Claix, France). Dead cells were excluded by gating out the PI+ cells. Cells were analyzed in a FacsCalibur system (Becton Dickinson, Grenoble, France) by use of FlowJo program.
Protection Assay Against Infection with M. Tuberculosis
M. tuberculosis H37Rv was grown at 37° C. in Dubos broth (Difco, Becton Dickinson, Sparks, Md.), complemented with albumin, dextrose and catalase (ADC, Difco). BALB/c (H-2d) mice (n=6) were primed by BCG (1×104 CFU/mouse, s.c.) at day 0 and then boosted at day 14 and 21 by 50 pmoles (=1 μg)/mouse of TB10.4-SA complexed with 25 pmoles (=3.6 μg)/mouse of biot-control Ig or biot-mAbs specific to various DC surface receptors, in the presence of 25 μg of Poly I:C. Mice were challenged at day 28 with M. tuberculosis H37Rv, via the aerosol route, by use of a home-made nebulizor. Five ml of a suspension containing 5×106 CFU/ml were aerosolized to obtain an inhaled dose ranged from 100 to 200 CFU/mouse. Four weeks post-challenge, lungs and spleens were homogenized by use of 2.5-mm diameter glass beads and an MM300 organ homogenizer (Qiagen, Courtaboeuf, France). Serial 5-fold dilutions of homogenates were seeded on 7H11 Agar, supplemented with Ovalbumin ADC(OADC, Difco). CFU were counted after 18 days of incubation at 37° C. Mice infected with M. tuberculosis H37Rv were housed in isolator and manipulated in A3 animal facilities at Institut Pasteur.
The complete polyeptide sequences of mycobacterial antigens from the ESX protein family, i.e., ESAT-6 (ESX A, Rv3875), CFP-10 (ESX B, Rv3874) or TB10.4 (ESX H, Rv0288), were genetically fused to SA to generate protein that formed tetramers and could be combined with biot-mAbs specific to DC surface markers. For our purpose we used only residues 14 to 139 of streptavidin from Streptomyces avidinii. The N-terminal part of SA was replaced by fused MTB antigens. Importantly, the presence of the complete sequence of ESAT-6, CFP-10 or TB10.4 at the N-terminal part of the SA did not perturb the tetramerization capacity of the fusion constructs (
C.2. Highly Efficient Ab-Mediated Binding of ESX-SA Fusion Proteins to DC Surface Receptors and their Marked Endocytosis
We first evaluated the binding of tetramerized ESAT-6-SA fusion protein to DC, through biot-conjugated mAbs specific to various DC surface receptors. Conventional BM-DC, pre-incubated at 4° C. with biot-mAbs specific to CD11b, CD11c, MHC-II or DCIR-2, and then with ESAT-6-SA, displayed a marked binding of ESAT-6 at their cell surface, as detected by Alexa647H-anti-ESAT-6 mAb (
We then investigated the capacity of BM-derived APC, to which ESX-SA fusion proteins were delivered via CD11b, CD11c, MHC-II or PDCA-1, to present immunodominant MHC-II-restricted ESX epitopes to specific TCR. C57BL/6 (H-2b)-derived BM-DC were incubated with biot-mAbs specific to DC markers or with biot-control Ig, and then incubated with various concentrations of ESAT-6-SA at 4° C. The cells were extensively washed before being cultured with ESX-specific T cells in order to evaluate the presentation of ESAT-6 bound to the targeted DC surface receptors. The BM-DC initially coated with biot-anti-CD11b, —CD11c or -MHC-II, were able to present efficiently the immunodominant ESAT-6 epitope to the I-Ab-restricted, ESAT-6:1-20-specific NB11 T-cell hybridomas (
C.4. In Vivo Binding of ESX Antigens to the Surface of DC Subsets, Targeted by Minute Amounts of ESX-SA and their Highly Efficient Ex Vivo Presentation
We then evaluated in vivo the specificity of ESX antigen binding to DC subsets, as well as the kinetics and efficiency of their presentation to T cells. CD11c YFP C57BL/6 mice were injected i.v. with 500 pmoles (=10 μg/mouse) of ESAT-6-SA, complexed to biot-anti-CD11c mAb or to biot-control Ig, at a molar ratio of 2:1, in the presence of 25 μg of the TLR3 agonist Poly I:C. At different time points post injection, low-density cells were prepared from the spleen and analyzed for the presence of ESAT-6 at the surface of DC by use of Alexa647H-anti-ESAT-6 mAb. CD11c YFP cells from the recipients of ESAT-6-SA complexed to biot-anti-CD11c mAb—but not from their counterparts injected with ESAT-6-SA complexed to biot-control Ig-stained positively for ESAT-6 at 24 h and, at a lesser extent, at 48 h post injection (
To study in vivo the efficacy of antigen presentation and the specificity of antigen presentation by the targeted DC subsets, after injection of ESX-SA-biot-mAb complexes, targeted APC were purified and co-cultured with ESX-specific, MHC-II-restricted T-cell hybridoma. BALB/c mice were injected with low dose of 50 pmoles (=1 μg)/mouse of TB10.4-SA, complexed at a molar ratio of 1:1, to mAbs specific to different DC surface receptors. The molar ratio of 1:1, used in this complex formation left free one of the two moles of biot previously fixed per mole of mAb, making possible the ex vivo magnetic sorting of biot-mAb-coated DC by use of an anti-biot mAb coupled to magnetic beads. At 3 h post-injection, no TB10.4 presentation was detected with total low-density spleen cells recovered from mice injected with TB10.4-SA complexed to biot-control Ig. In contrast, positive fractions of cells from mice injected with TB10.4-SA complexed to biot-mAbs specific to CD11b or CD11c, sorted by use of anti-biot beads, were able to markedly stimulate the anti-TB010.4:74-88, I-Ad-restricted, 1H2 T-cell hybridoma (
We then evaluated the potential of the ESX antigen targeting to DC subsets in immunization of mice. C57BL/6 (H-2b) mice were immunized i.v. by a single injection of 50 pmole (=1 μg)/mouse of ESAT-6-SA, without Ig, or complexed at a molar ratio of 2:1, to blot-control Ig or biot-mAbs specific to CD11b or CD11c integrins, to CD205, CD207 or CD209 C-type lectins or to PDCA-1, in the presence of Poly I:C. Control groups immunized with ESAT-6-SA, either without biot-Ig or complexed to biot-control Ig, did not develop T-cell responses to ESAT-6. In contrast, mice immunized with ESAT-6-SA complexed to biot-anti-CD11b, -CD11c or -CD205 mounted specific, intense and sensitive IFN-γ (
We excluded the possibility that the biot-mAbs operated in vivo via the FcR, rather than by their specific DC surface ligands, since blot-control Ig, complexed to ESAT-6-SA, did not induce T-cell responses (
We then determined the lowest dose of the antigen complexed to biot-mAb, able to induce significant T-cell responses. Immunization with ESAT-6-SA, complexed to biot-anti-CD11b mAb, at different doses, ranging from 250 to 5 pmoles (=5 to 0.1 μg)/mouse showed that 50 pmoles (=1 μg/mouse) were enough to induce IFN-γ, IL-2 and IL-17—but not IL-5—responses at their maximal intensities. Moreover, an injection dose as low as 5 pmoles (=0.1 μg)/mouse of ESAT-6-SA was still able to trigger a significant Th1 and Th17 responses, showing the marked efficiency of the antigen delivery approach.
Notably, lymphoproliferative (
TB10.4-SA or CFP-10-SA, complexed to biot-anti-CD11b or -CD205 mAbs were also able to induce strong Th1 responses, respectively in BALB/c (H-2d) (
Priming with BCG, or with its improved recombinant variants, followed by boosting with efficient subunit vaccines, is considered as the most promising prophylactic anti-tuberculosis vaccination strategy (Kaufmann, 2006). We therefore sought to evaluate and to compare the T-cell responses in BCG-primed mice which were then boosted by ESX antigens targeted to different DC surface receptors by the developed approach. BALB/c mice, unprimed or primed s.c. with 1×106 CFU/mouse of BCG at day 0, were boosted i.v., at days 14 and 21, with 50 pmoles (=1 μg)/mouse of TB10.4-SA complexed to biot-control Ig or to different biot-mAbs specific to C-type lectins or PDCA-1, in the presence of Poly I:C. We then analyzed IFN-γ CD4+ T-cell responses, analyzed at day 28 (
Th17 CD4+ T-cell responses (
We then analyzed TB10.4-specific CD8+ T-cell responses in BALB/c mice, by use of the H-2Kd pentamer complexed with TB10.3/4:20-28 GYAGTLQSL epitope, shared by TB10.3 and TB10.4 (TB10.3/4:20-28) (Majlessi, 2003). In mice immunized with two injections of TB10.4-SA complexed to biot-mAbs specific to CD11b, CD207, CD209 or PDCA-1, in the presence of Poly I:C, we did not detect specific CD8+ T cells (data not shown). In their counterparts immunized by TB10.4 targeted to CD205, we barely detected specific CD8+ T cells (<1% pentamer+ cells in the CD8+ T-cell compartment) (
Rational design of anti-tuberculosis vaccines is restrained by our lack of exhaustive knowledge in the type of protective immune effectors and in the reasons of the limited efficiency of adaptive T-cell responses in eradication of intracellular tubercle bacilli. Considering that the differentiation and specialization of T-cell effectors is dictated by different DC subsets with specialized activities, immunization by mycobacterial antigen targeting to different DC subsets may provide new insights into the type of the anti-tuberculosis adaptive immunity with protective potential. However, as all the mechanisms governing functions of DC subsets are not yet thoroughly understood, it remains difficult to predict which DC subsets/DC surface receptors are the most appropriate to be targeted in order to optimize the protective immunity against mycobacterial infection. To compare the properties and impacts of various DC subsets on the generation of mycobacteria-specific adaptive immune responses and protection, we developed a versatile approach allowing addressing of selected mycobacterial immunogens to different DC subsets/DC surface receptors. Our experimental approach consists of the genetic fusion of full-length sequences of highly immunogenic mycobacterial antigens from the ESX family, i.e., ESAT-6, CFP-10 and TB10.4, to SA, followed by tetramerization of the resulted fusion proteins which leads to the possibility of complex formation between them and individual biot-mAbs of a wide-ranging panel of specificities against DC surface receptors. Such complexes can thus be used to deliver the ESX immunogens to the selected DC surface receptors for direct comparison of the adaptive immune responses, established via the action of different DC subsets.
We first showed in vitro that this approach allows delivery of ESX immunogens to various APC surface receptors, either integrins, C-type lectins, MHC-II or PDCA-1. Importantly, ESX antigens bound to CD11b, CD11c, DCIR-2, MHC-II or PDCA-1 were efficiently endocytosed, most probably due to the cross-linking of the targeted surface receptors by the biot-mAbs, leading to their capping together with the bound ESX-SA cargo. The ESX antigens were then processed and the derived epitopes were loaded on MHC-II molecules and presented to specific TCR, in a highly sensitive and efficient manner. Furthermore, the antigen delivery was also highly specific in vivo, as only the DC subsets, targeted with ESX-SA complexed to selected biot-mAbs: (i) displayed ESX antigens at their surface, as detected from 3 h to 24 h after i.v. injection by cytofluorometry using anti-ESX mAb, and (ii) when positively sorted ex vivo, were able to present ESX antigens to specific MHC-II-restricted T-cell hybridomas.
Besides efficient presentation of prominent mycobacterial antigens by DC, an appropriate activation of the latter is crucial for proper induction of T-cell responses. A priori it is conceivable that agonistic biot-mAbs carrying the ESX-SA, could by themselves give the DC maturation signal to the targeted subset. CD11b and CD11c, heterodimerized with CD18, form respectively αmβ2 and αxβ2 integrins which are phagocytic receptors for complement coated particles. These β2 integrins are both signal transducing receptors, as their ligation by their natural ligands or by specific mAbs induces phosphorylation of Mitogen-Activated Protein (MAP) kinases and upregulation of DNA-binding activity of NF-κκB, leading notably to the transcription and secretion of IL-1β, Macrophage Inflammatory Proteins (MIP)-1α and MIP-1β and thus may have an important role in the recruitment of other inflammatory cells during initiation of the immune response (Ingalls, 1995, Rezzonico, 2000, Rezzonico, 2001). However, full activation of DC subsequent to surface ligation of CD11b or CD11c with mAbs has not been reported. Hence, it is known that partially activated DC are rather tolerogenic or inducer of Treg (Joffre, 2009). Interaction of C-type lectins with their different natural ligands, i.e., pathogen-derived carbohydrates, may activate the signal transduction pathways and NF-κB nuclear translocation and thereby expression of pro-inflammatory cytokines, i.e., IL-6, IL-12p70 and IL-23 or alternatively may negatively affect TLR-mediated DC activation, leading to IL-10 production and an anti-inflammatory microenvironment (Geijtenbeek, 2009) (Gringhuis, 2009). In contrast, to triggering of C-type lectins by their natural ligands, only few information are available on in vivo activation/maturation of DC by mAbs specific to C-type lectins. So far, mAb-mediated antigen targeting to CD205, without co-stimulatory signal, induces a T-cell division followed by T-cell peripheral deletion and tolerance (Bonifaz, 2002), while mAb-mediated antigen targeting to DNGR-1 (Clec9A) is immunogenic and induces high titers of IgG antibody responses (Caminschi, 2008). In contrast, in a parallel investigation, in antigen targeting to DNGR-1, an anti-CD40 agonistic mAb has been systematically used as adjuvant for the generation of CD4+ and CD8+ T-cell responses (Sancho, 2008). In the absence of more detailed information on the potential of DC activation by mAbs specific to DC surface receptors, for further immunizations by ESX antigen targeting to DC subsets, we opted for systematic use of a DC maturation signal, i.e., the synthetic analog of dsRNA, Poly I:C. This choice was based on the well-established structural interaction of Poly I:C with endosomal TLR3 or with cytosolic dsRNA sensors, i.e., Retinoic acid-Inducible Gene-I (RIG-I) and Melanoma Differentiation-Associated gene-5 (MDA5) RNA helicases, probably expressed by different cell types (Kawai, 2008). Moreover, Poly I:C displays a marked capacity, not only to induce type I IFN, necessary to induce both Th1 and CTL responses, but also to activate NK cells. DC triggered via TLR3 are able to derive Th17 differentiation, as well (Veldhoen, 2006).
By use of different biot-mAbs specific to β2 integrins, diverse C-type lectins or PDCA-1, in the presence of Poly I:C, we directly compared the efficiencies of ESX targeting to different DC subsets/DC surface receptors in the induction of T-cell responses. Remarkably, a single injection of only 1 μg (=50 pmoles)/mouse of ESX-SA complexed to biot-mAbs specific to CD11b, CD11c or CD205, induced specific, intense and highly sensitive Th1—but not Th2—responses. ESX-SA complexed to biot-mAbs specific to DC207 or PDCA-1 induced less intense and less sensitive, yet still marked Th1 responses. The efficient induction of Th1 responses by ESX antigen targeting to β2 integrins is in accordance with: (i) the substantial potential of other CD11b targeting delivery vectors, such as the recombinant adenylate cyclase CyaA of Bordetella pertussis in the induction of T-cell immunity against diverse pathogens, including mycobacteria, or tumor antigens (Guermonprez, 2001) (Majlessi, 2006) (Hervas-Stubbs, 2006) (Preville, 2005), and (ii) the notable efficiency of mAb-mediated OVA antigen targeting to CD11c, a much more specific marker of DC, albeit expressed at low levels on activated CTL, NK cells and macrophages of marginal zones. Highly efficient CD4+ and CD8+ T cell triggering in this case has been explained by the delivery of the antigen towards CD11c+ cells both in the marginal zones and to CD11c+ cross-presenting DC in the T-cell zone (Kurts, 2008).
Among the C-type lectins evaluated in the present study, CD205 was the most efficient at inducing Th1 cells in primary responses to ESX antigens. This endocytic integral transmembrane mannose receptor, is expressed at high levels by cortical thymic epithelium and DC subsets, including the splenic CD8+ DC population. CD205 also may act as a receptor for necrotic and apoptotic cells (Shrimpton, 2009). CD205 is rapidly taken up after binding with carbohydrates. Its cytosolic domain mediates highly efficient endocytosis and recycling through the late endosomes and MHC-II rich compartments, compared to the most of the other surface endocytic receptors, whose ligation induces endocytosis through early and more peripheral endosomes (Jiang, 1995).
The significant efficiency of ESX antigen targeting to CD207 (Langerin, Clec4K) C-type lectin is also in accordance with the results obtained with OVA antigen targeting to this C-type lectin leading to strong proliferative responses of both OT-I or OT-II TCR transgenic T cells (Valladeau, 2002) (Idoyaga, 2008). CD207 is a type II transmembrane endocytic receptor which is highly expressed by the skin immature Langerhans cells and dermal DC, and at much lower levels by spleen CD11c+ CD8α+ DC. CD207 is detected in an endosomal recycling compartment and is potent inducer of organelles consisting of typical superimposed pentalamellar membranes, i.e. Birbeck granules, and routes endocytosed antigens into these organelles. Maturation of Langerhans DC is concomitant with downregulation of CD207 and disappearance of Birbeck granules (Kissenpfennig, 2005). Most importantly for the anti-M. tuberculosis vaccination, CD207 mRNA is detectable in the lungs in mice and in epithelium lining the human airways (Valladeau, 2002) and therefore can be of particular interest in the induction of T cells directly close to the potential site of potential mycobacterial infection.
In contrast to ESX antigen targeting to CD205 and CD207, a single dose injection of ESX-SA complexed to biot-anti-CD209 (DC-SIGN) in C57BL/6 mice failed to induce specific Th1 responses but was inefficient in BALB/c mice. The reason of this discrepancy is not yet elucidated. CD209 is expressed by myeloid
DC and is involved in upregulation of TLR-induced IL-10 production, yet as a function of its different natural ligands, i.e., mannose or fucose, can induce or inhibit production of IL-12 and IL-6 in human DC (Gringhuis, 2009). Absence of Th1 responses subsequent to immunization with ESX-SA complexed to anti-CD209 mAb ESX suggests that the interaction of the used mAb with mouse CD209 would be anti-inflammatory and not appropriate for the induction of Th1 and Th17 responses.
We also show that ESX antigen targeting to PDCA-1 (CD317) allowed ESX antigen routing to MHC-II machinery of BM-derived plasmacytoid DC in vitro and induced marked specific Th1 responses in vivo. This surface marker is predominantly expressed by plasmacytoid DC in naive mice. Following viral stimulation, due to the production of type-I/II IFN, PDCA-1 become detectable on other DC subsets, myeloid CD11b+ cells, NK, NKT, T and B cells (Blasius, 2006). Thus, we, cannot exclude that in the presence of Poly I:C, and thereby efficient production of type I IFN, PDCA-1 would be expressed on other cells than plasmacytoid DC, enlarging the spectrum of cells to which biot-anti-PDCA-1 mAb could deliver ESX.
A large body of data has long established the necessary—but not sufficient—role of Th1 cells and IFN-γ production in the control of mycobacterial infections, while the contribution of Th17 cells and IL-17 remains debatable. Besides the early production of IL-17 by lung TCRy□ T cells (Lockhart, 2006), CD4+ Th17 cells can be readily detected in mice and humans exposed to mycobacteria (Umemura, 2007) (Scriba, 2008). However, IL-23p19o/o mice, with normal Th1 but decreased Th17 responses, develop tuberculosis symptoms similar to those observed in WT mice, with comparable mycobacterial loads (Chackerian, 2006). Moreover, IL-17o/o and WT mice control in a similar manner the growth of M. bovis BCG, given at high dose by aerosol route (Umemura, 2007). Therefore, according to these data, compared to Th1 responses, Th17 cells do not seem to contribute directly to the control of primary mycobacterial infections. Nevertheless, in C57BL/6 mice vaccinated with the ESAT-6:1-20 peptide, adjuvanted with a strong inducer of Th17 responses, and then challenged with M. tuberculosis, Th17 cells populate the lungs 3-4 days before the wave of Th1-cell recruitment and trigger the production of CXCL9, CXCL10 and CXCL11 chemokines, which certainly contribute to the chemo-attraction of Th1 cells (Khader, 2007). These data support at least an indirect role of Th17 in the set up of anti-mycobacterial immunity subsequent to vaccination. Taking in account this observation, in parallel to antigen-specific IFN-γ responses, we followed IL-17-producing specific CD4+ T cells in mice immunized by ESX antigen targeting to DC subsets. In mice immunized with a single injection of ESX-SA complexes in the presence of Poly I:C, only targeting to CD11b and CD11c integrins or to CD205 C-type lectin, and in a lesser extent to CD207 or PDCA-1, was able to induce Th17 responses. It is interesting to note that the good inducers of Th17 responses were also inducers of the highest Th1 responses, in accordance with the hypothesis that Th17 cells may pave the way for the recruitment/activation of Th1 cells (Khader, 2007).
As priming with live attenuated mycobacteria followed by boosting with subunit vaccines, is of the most promising prophylactic anti-tuberculosis vaccination strategies (Kaufmann, 2006), we also analyzed the boosting potential of ESX antigen targeting to DC subsets by use of TB10.4 (Rv0288, ESX-H) antigen, another promising protective ESX antigen (Hervas-Stubbs, 2006; Dietrich, 2005). This antigen is of higher interest in the development of innovative sub-unit vaccine candidate compared to ESAT-6 and CFP-10, due to the importance of the latter in the diagnostic tests. In mice primed with BCG and then boosted with TB10.4-SA targeted to CD205, CD207, CD209 or DCIR-2, a comparable boost effect of IFN-γ responses was obtained. The best boost effect at the level of Th17 response was obtained with TB10.4 targeting to CD205, followed by CD207 and PDCA-1.
We investigated several immunization protocols, i.e., single injection or boost immunization after BCG priming, with TB10.4 antigen targeting to different DC subsets to induce CD8+ T-cell priming. Among all the conditions evaluated, we only detected efficient TB10.4-specific CD8+ T-cell cross priming, in mice primed with BCG and then boosted with TB10.4 targeted to CD205. In addition to its capacity to shuttle antigens from the extracellular space into a specialized MHC-II rich lysosomal compartments, CD205 is also able to efficiently introduce antigens to the MHC-I processing machinery, in a Transporters of Antigen Presentation (TAP)-dependent manner. So far, compared to the critical role of CD4+ T cells, the contribution of CD8+ T cells to the protection in experimental tuberculosis was underestimated, probably due to the absence, in mice, of several CD8+ T-cell populations, including CD1-restricted CD8+ T cells. A recent study, performed in the sensitive model of rhesus macaques, described a previously unappreciated contribution of CD8+ T cells. Indeed, Ab-mediated depletion of CD8+ T cells in BCG-vaccinated and then M. tuberculosis-challenged macaques leads to a marked increase in mycobacterial burden and remarkably less-organized and necrotic granulomas versus well-contained granluomas in their control isotype-treated counterparts (Chen, 2009). Therefore, our observation that BCG priming followed by TB10.4-SA targeting to CD205 trigger efficiently CD8+ T-cell responses is of major importance in the design of subunit anti-tuberculosis booster vaccine.
The Th1 and Th17 responses induced by ESX antigen targeting, at least to CD11b in the presence of Poly I:C, were under the negative control of Treg. We recently demonstrated that the Th1 responses induced by BCG vaccination were also negatively controlled by Treg and that attenuation of this subset in BCG-immunized BALB/c mice leads to weak, albeit significant and reproducible, improvement of the protection against M. tuberculosis aerosol challenge (Jaron, 2008). It will be of major interest to evaluate the Treg activity in the case of ESX antigen targeting to DC subsets in the presence of other co-stimulatory signals. Moreover, our recent observations in the OVA antigen model delivered by latex beads in the presence of a large panel of TLR2 to 9 agonists did not allow selection of an adjuvant minimizing the Treg induction, suggesting that Treg activity is probably not a consequence of the quality of inflammation. It has been hypothesized that indirect and partial maturation of DC induced by cytokines, in a bystander manner, therefore can be the cause of Treg induction, which for the rest, can be protective by avoiding excessive inflammation and tissue damage (Joffre, 2009).
ESX targeting to DC surface receptors allowed substantial reduction of the effective dose of antigen for immunization without impairment of T-cell immunity, as exemplified by the low dose of 5 pmoles (=0.1 μg)/mouse of ESX-SA, complexed to biot-anti-CD11b mAbs which induced highly significant ESX-specific Th1 and Th17 responses. It is noteworthy that except for the anti-CD11c mAb which is a hamster IgG, all the other mAbs used in this study were rat IgG, thereby minimizing the risk of introduction of different xenogeneic T helper determinants in the case of different antigen targeting assays and thus making possible the direct comparison of the effect of the different DC surface receptors targeted. Importantly, the facts that: (i) FcγRo/o and WT mice mounted comparable adaptive immune responses to mAb-mediated ESX targeting to DC and (ii) ESX-SA fusion proteins complexed to biot-control Ig did not induce detectable adaptive immune responses, show that the mechanism responsible of targeting, endocytosis and further antigen presentation does not involve FcγR.
IV.1. Constructs—Antigen and Capture Protein Fusions to Streptavidin (SA) for Antigen Delivery (See Table 1)
The recombinant Ag-SA fusion polypeptides disclosed in table 1 and
CMV epitopes from phosphoprotein 65 (pp65) and immediate early protein-1 (IE-1) (in bold and shadowed italics in the sequences of
Two fusion polypeptides were contructed:
pp65-SA (pET28b-SA-pp65): see
IE-1-SA (pET28b-SA-IE-1): see
The results obtained for the first experiments performed suggest potential use as vaccine for inducing of T cell responses in naive donors of bone marrow for transplantation, or for ex vivo induction/expansion of CMV-specific T cells (data not shown).
E7 antigen from human papillomavirus 16, in which all cysteine residues were replaced by glycine residues, was fused to streptavidin.
The following fusion polypeptide was contructed:
E7gly-SA (pET28b-SA-E7gly): see
c) Potential Use of SA Fusion Technology for Complex Stabilization Through Binding with Human Serum Albumin or Co-Delivery of Cytokines, Such as Human Inteferon Gamma (hIFNγ)
We speculated that one could fuse to SA a recombinant ligand that would capture an adjuvant, or a cytokine etc. and enable its co-delivery with the Ag-SA-biot-MAb complex.
Therefore, wild type human serum albumin domain of protein G (ABD), and its artificial scaffold-derivative that binds hIFNγ with nanomolar affinity, were fused to SA in order to stabilize the complex in human plasma, or deliver a cytokine, such as hIFNγ.
ELISA analysis were performed as indicated below:
We were able to produce streptavidin tetramers with (i) an OVA epitope (SIINFEKL) or a MTB antigen (ESAT6) genetically fused to the N-terminus of SA and (ii) an ABD-derived protein scaffold (a recombinant ligand) fused genetically to the C-terminus of SA, so that the produced fusion polypeptide binds with high affinity the human IFN-γ.
Examples of SA-ABD fusion polypeptides produced are given below:
SI-SA-ABDwt (pET28b-SA-ABDwt): see
SI-SA-ABD223 (pET28b-SA-ABD223): see
The results are presented in
The resulting SI-SA-ABD fusion polypeptides show a H-2Kd for IFN-γ in the nanomolar range, indicating that the produced tetramers can capture homodimers of human IFN-γ with high affinity.
In such a fusion polypeptide, the biotin-binding sites are free for binding one or several biotinylated molecules (in particular biotinylated targeting antibodies and/or biotinylated adjuvants), especially via non-covalent binding.
In addition, the resulting fusion polypeptides can be refolded in solution in the presence of biotinylated targets and captured on biotinylated ELISA plate wells, for example for use in ELISA for detection of IFN-γ.
Indeed, it should be noted that from all the produced SA-ABD fusion polypeptides, only the fusion polypeptides in which SA was fused to wt ABD or ABD223, remain soluble and form tetramers in bacterial cytosol. All other SA-ABD form inclusion bodies, and therefore have to be extracted from bacterial debris with 8 M urea, but can be refolded into active tetramers upon urea dilution, especially, if refolding is preformed in biotinylated wells of microtiter plates, or biotinylated tubes, or in presence of biotinylated antibody, for example, to drive and facilitate folding and tetramerization (thus, only folded formed tetramers are bound to biotin; the aggregated misfolded fusion polypeptides are washed out).
Hence, the SA core can carry both genetically fused elements as extensions of the SA core polypeptide, as well as effector molecules non-covalently bound, such as biotin-poly I:C, etc. . . . Such Ag-SA fusion polypeptides should therefore be particularly useful to achieve co-delivery of adjuvants and cytokines.
IV.2. Extension of the Developed Technology to the Chicken Ovalbumin Model Antigen for the Investigation of Different Aspects of Antigen Presentation by the Developed Technology
A novel OVA-SA fusion polypeptide was constructed to study different aspects of the delivery of the OVA model antigen by innate immune cells, targeted by the developed technology:
OVA-SA (OVA-derived MHC-I, MHC-II immunodominant epitopes) (pET28b-OVA-SA;
It contains in the C-terminal part a CD4+ T cell epitope restricted by H-2b MHC II molecules and presented to TCR of OT-II mice, which allows evaluation of in vitro/in vivo CD4+ T cell responses of OT-II mice.
IV.3. Evaluation of the Specific T-Cell Responses Subsequent to Intravenous Immunization by OVA-SA, Addressed to CD11c+ Cells
We then evaluated the immunogenicity of the OVA-SA tetramer in vivo, by immunizing mice intravenously with complexes formed with this construct and biot-anti-CD11bc mAb or biot-control Ig, in the presence of Poly I:C as adjuvant.
As shown in the
IV.4. Use of Monomers of SA-Ag Protein
New Technical Developments on the Side of the Antigen Delivery Technology
Often the antigen fusions to SA are insoluble in the producing E. coli, not forming the necessary tetramers capable of binding biotin. Therefore, it is of interest to be able to extract those proteins from inclusion bodies with denaturing concentrations of urea (e.g. 8 M) and refold them into active tetramers, by dilution out of urea into buffer, using the biot-conjugated targeting mAb as “catalyst”. The weak interaction with biotin would promote folding of the SA protein and facilitate its tetramerization, allowing high-affinity interaction with biotin (
Antigen Presenting Assay
OVA-SA (chicken ovalbumin epitope encoding sequences genetically fused to the 5′- and 3′-ends of the SA gene; see
Biot-mAbs specific to CD11c (as disclosed herein);
Biot-mAbs specific to DEC-206: a Rat IgG2a, immunoglobin recognizing the Mannose Receptor CRD4-7; commercially available (BioLegend, San Diego, Calif., USA).
BM-DC were stimulated with OVA-SA protein with or without mAb. Three hours later the specific T-cell hybridoma MF2.2D9 were added and after 16 hours the expression of the IL-2 was evaluated by ELISA (marker of the antigen presentation).
Soluble OVA-SA protein tetramers obtained from the first method for the production of polypeptide disclosed herein were compared to fusion polypeptides produced in insoluble form, extracted in 8 M urea and refolded by dilution at least 1:100 into biot-mAb solution (particular embodiment of the second method for the production of a polypeptide disclosed herein).
Comparison of antigen delivery potency for the soluble streptavidin tetramers and the insoluble OVA-SA monomers refolded from 8 M urea directly into biot-mAb solution:
The mixture of the antibody-streptavidin was prepared 2 hours before adding to the cells. The antibodies were diluted in PBS with 1% BSA and the OVA-SA protein was used at 0.001 nM to 1 nM (0.1-100 ng/ml) concentration, at mAb: OVA-SA ratios of 2:1, 1:1 and 1:2, respectively (
The best (signal/background) result was obtained using the biot-anti-CD11c mAb already at 0.1 nM concentration of soluble tetrameric OVA-SA, with the biot-anti-CD11cmAb and using a mA b:OVA-SA ratio of 2:1 (
The insoluble OVA-SA (refolded monomers) could deliver antigen via binding to the blot-anti-CD11c mAb, starting from 0.1 nM.
The expression of the mannose receptor (DEC 206, yellow labelled) is very low on mouse BM-DC, and no benefit of targeting with anti-DEC206 mAb was seen.
V.1. Study of The Expression Profile of The Dendritic Cell Surface Marker C-Type Lectin CD205 in the Lungs of Mice, at the Steady State or Subsequent to Adjuvant Injection
In the following investigation, we used the developed antigen delivery technology, based on antibody-mediated targeting of dendritic cell (DC) subsets, to induce mucosal T-cell immunity at the level of the lungs against Mycobacterium tuberculosis-derived ESAT-6 (Early-Secreted Antigenic Target, 6 kDa) immunogen, known for its protective potential.
One of the most promising DC surface markers, as target of antigen delivery, is the C-type lectine CD205, due to its high endocytic properties. At a first step, it was important to establish the expression profile of this DC surface marker in the lungs, at the steady state or subsequent to administration of adjuvant, for instance, Poly I:C that we previously used during the development of this technology for the induction of systemic immunity. To this end, mice were injected, as described in the legend to the
As shown in the
Taken together, these analyses demonstrated that CD205 was a suitable lung DC surface marker to be targeted for the delivery with M. tuberculosis immunogens of vaccinal interest.
V.2. Study of the Potential of CD11c or CD11c Beta2-Integrins or CD205 C-Type Lectin, as Mucosal Targets for the Delivery of Mycobacterial Antigens to the Lung DC Subsets in Order to Induce Specific T-Cell Responses and Further Protection
We then aimed to induce mucosal T-cell responses to the protective mycobacterial immunogen, ESAT-6, in the lungs of mice, by targeting this antigen to CD11c+, CD11b+ or CD205+ lung DC populations, by the use of the developed technology. It is noteworthy that the expression profile of CD11c and CD11b beta2-integrins in the lungs of mice is largely established. We further checked that the level of expression of these beta-2 integrins was not modified subsequent to Poly I:C injection (data not shown).
ESAT-6-SA tetramer was complexed to individual biot-mAs specific to CD11c, CD11b or CD205 and the complexes were used to immunize groups of mice by i.n. route, in the presence of Poly I:C, as detailed in the legend to the
The
Therefore, it is possible to efficiently induce specific mucosal T-cell immunity to mycobacterial ESAT-6 protective immunogen by use of the developed technology, applied to the lung DC subsets.
Based on the marked immune responses induced in the lungs by the use of the developed technology, we further investigated the possibility to induce mucosal protection against infection with virulent M. tuberculosis, in the lungs subsequent to intranasal immunization with TB10.4 antigen. This ESAT-6-related mycobacterial protein has been previously described as a strong protective immunogen in BALB/c (H-2d) mice.
We immunized BALB/c mice, with the gold standard BCG vaccine or by two i.n. injections of TB10.4-SA tetramer complexed to biot-anti-CD11b or anti-CD205 mAbs, or to biot-control Ig, as a negative control, as detailed in the legend to the
Therefore, mucosal immunization with protective mycobacterial immunogens by use of the developed strategy displays a high potential to trigger anti-mycobacterial protection in the lungs in the mouse model.
Free SA sites of the Ag-SA+biot-mAb complexes can be used to co-deliver other molecules to the targeted cells, for instance adjuvants for the activation of the innate cells, which is necessary for further stimulation of naïve T cells and induction of specific T-cell immunity. Therefore, we evaluated in vivo the possibility of co-delivery of the biotinylated adjuvant biot-CL264 (biotinylated form of the CL264 which is an Adenine analog and a TLR7 agonist) with the complex formed between TB10.4-SA and anti-CD11b mAb. Biot-CL264 is commercially available and can be purchased for example from InvivoGen. It is a 9-benzyl-8 hydroxyadenine derivative containing a glycine on the benzyl groupe (in para). CL264 is labeled with biotin on the acid group of the glycine via 3 HEX spacers. CL264 interacts with TLR7 and thereby activates innate cells like DC. As the biotinylated form preserves its activity, we combined it with the biot-mAb+Ag-SA to co-deliver the Ag and this adjuvant by the same complex to the same DC subset.
To evaluate the possibility of co-delivery of antrigen and an adjuvant to the same targeted cell subset, BALB/c mice were injected i.v. with TB10.4-SA: biot-anti-CD11b mAb: biot-CL264 ternary complex at a molar ratio of 4:3:1, as detailed in the legend to the
Note that the anti-CD11b mAb targets CD11c+ CD11b+ CD8α−, but not CD11c+ CD11b− CD8α+-DC subset.
At 18 hours post-injection, spleen DC were enriched and analyzed by cytofluorometry to evaluate their possible phenotypic maturation, as studied by the up-regulation of surface co-simulatory molecules.
As shown in the
Therefore, the developed technology allows concomitant delivery of biot-antigen and biot-adjuvant to the same DC subset, and represents a high potential for vaccine development, by requiring only minute levels of adjuvant to activate DC, which may considerably minimize the undesirable adjuvant side effects.
Colonna. 2006. Bone marrow stromal cell antigen 2 is a specific marker of type I IFN-producing cells in the naive mouse, but a promiscuous cell surface antigen following IFN stimulation. J Immunol 177:3260-3265.
M. Steinman. 2002. Efficient targeting of protein antigen to the dendritic cell receptor DEC-205 in the steady state leads to antigen presentation on major histocompatibility complex class I products and peripheral CD8+ T cell tolerance. The Journal of experimental medicine 196:1627-1638.
Engagement of CD11b and CD11c beta2 integrin by antibodies or soluble CD23 induces IL-1beta production on primary human monocytes through mitogen-activated protein kinase-dependent pathways. Blood 95:3868-3877.
| Number | Date | Country | Kind |
|---|---|---|---|
| 09290987.8 | Dec 2009 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IB2010/003497 | 12/12/2010 | WO | 00 | 2/4/2013 |
