Technical Field of the Invention

The present invention relates in general to immunity against pathogens, and more particularly, to delivering antigens to human dendritic cells (DCs) via Dectin-1 to enhance pathogen-specific Thl 7 cells in memory pools. Background Art

Without limiting the scope of the invention, its background is described in connection with targeting antigens to enhance Thl 7 cells and immunity against pathogens. United States Patent Application No. 2010/0166784 (Murphy et al., 2010) describes a method to modulate the development of Thl 7 or Treg cells. The Murphy invention provides methods of modulating an immune response in a host by providing a nucleic acid sequence that modulates the development of Thl 7 or Treg cells.

United States Patent Application No. 2008/0233140 (Banchereau et al., 2008) includes compositions and methods for binding Dectin-1 on immune cells with anti-Dectin-1 -specific antibodies or fragments thereof capable of activating the immune cells. Disclosure of the Invention

The present invention describes compositions and methods for enhancing pathogen-specific T cell responses using human dendritic cells. The method describes an anti-Dectin-1 -specific antibody or binding fragment thereof fused with one or more antigens, that may be used in the presence or absence of TLR2 ligands to enhance Thl and Thl7 cell responses and at the same time decrease Th2 cell responses. Methods for treating pathogenic infections using the compositions described herein are also presented that drive the immune response to a Thl and Thl7 helper T cells responses.

The instant invention in one embodiment provides a method for enhancing antigen-specific T cell responses in a Dectin-1 -expressing antigen presenting cell (APC) comprising: (i) loading the APC with an anti-Dectin-1 -specific antibody or binding fragment thereof conjugated or fused with one or more antigens, (ii) contacting the antigen-loaded APC with T cells, and (iii) isolating T cells that proliferate when contacted with the antigen-loaded APC wherein the antigen-specific T cell response is enhanced to secrete IL-23.

In one aspect of the method provided hereinabove the one or more antigens comprise bacterial, fungal or viral antigens. In a specific aspect of the method above the antigen is a HA1 subunit of an influenza virus. In another aspect the composition optionally comprises one or more TLR2 ligands. In another aspect the one or more TLR2 ligands comprise heat- killed bacteria, lipoglycans, lipopolysaccharide, lipoteichoic acids, peptidoglycans, synthetic lipoproteins, zymosan or combinations and modifications thereof. In yet another aspect the TLR2 ligand comprises lipopolysaccharides comprising P. gingivalis LPS or E.coli LPS. In a related aspect the method enhances Thl7 and Thl and reduces Th2 cell responses.

Another embodiment of the instant invention describes a method for enhancing antigen- specific T cell responses in a Dectin-1 -expressing antigen presenting cell (APC) comprising the step of contacting the APC with an anti-Dectin-l -specific antibody or fragment thereof fused with one or more antigens and one or more TLR2 ligands. The one or more antigens of the method comprise bacterial, fungal or viral antigens. In one aspect the antigen is a HA1 subunit of an influenza virus. In another aspect the one or more TLR2 ligands comprise heat- killed bacteria, lipoglycans, lipopolysaccharide, lipoteichoic acids, peptidoglycans, synthetic lipoproteins, zymosan or combinations and modifications thereof. In yet another aspect the composition comprises lipopolysaccharides comprising P. gingivalis LPS or E.coli LPS. In another aspect the method increases secretion of IL-Ι β, IL-6, and IL-23 thereby leading to an enhanced Thl7 response. In another aspect the method reduces Th2 cell responses.

In yet another embodiment the instant invention relates to an influenza vaccine composition for prophylaxis, treatment, amelioration of symptoms or combinations thereof comprising: an anti-Dectin-l -specific antibody or binding fragment thereof fused with a HA1 subunit of an influenza virus and one or more optional pharmaceutically acceptable excipients or adjuvants. In one aspect the composition optionally comprises one or more TLR2 ligands. In another aspect the one or more TLR2 ligands comprise heat-killed bacteria, lipoglycans, lipopolysaccharide, lipoteichoic acids, peptidoglycans, synthetic lipoproteins, zymosan or combinations and modifications thereof. The TLR2 ligands of the instant invention comprise lipopolysaccharides comprising P. gingivalis LPS or E.coli LPS. In one aspect the composition enhances Thl7 and Thl responses by a secretion of IL-23. In another aspect the composition reduces Th2 cell responses. In another aspect the composition is administered by an oral route, a parenteral route or an intra-nasal route.

An influenza vaccine composition for prophylaxis, treatment, amelioration of symptoms or combinations thereof is described in one embodiment of the present invention. The vaccine of the present invention comprises: an anti-Dectin-1 -specific antibody or binding fragment thereof fused with a HA1 subunit of an influenza virus, one or more TLR2 ligands comprising P. gingivalis LPS or E.coli LPS or combinations thereof, and one or more optional pharmaceutically acceptable excipients or adjuvants. In one aspect the composition increases secretion of IL-Ιβ, IL-6, and IL-23 thereby leading to an enhanced Thl7 response and reduces Th2 cell responses. The composition of the present invention is administered by an oral route, a parenteral route or an intra-nasal route.

Another embodiment of the instant invention discloses a method for treating, prophylaxis or amelioration of symptoms of influenza in a human subject comprising the steps of: identifying the subject in need of the treatment, prophylaxis or amelioration of symptoms of the influenza and administering a therapeutically effective amount of a pharmaceutical composition or a vaccine comprising an anti-Dectin-1 -specific antibody or binding fragment thereof fused with a HA1 subunit of an influenza virus and one or more optional pharmaceutically acceptable excipients or adjuvants in an amount sufficient for the treatment, prophylaxis or amelioration of the symptoms of the influenza. In one aspect the composition is administered by an oral route, a parenteral route or an intra-nasal route.

In yet another embodiment the present invention discloses a method for treating, prophylaxis or amelioration of symptoms of influenza in a human subject comprising the steps of: identifying the subject in need of the treatment, prophylaxis or amelioration of symptoms of the influenza and administering a therapeutically effective amount of a pharmaceutical composition or a vaccine comprising an anti-Dectin-1 -specific antibody or binding fragment thereof fused with a HA1 subunit of an influenza virus, TLR2 ligands comprising P. gingivalis LPS or E.coli LPS or combinations thereof, and one or more optional pharmaceutically acceptable excipients or adjuvants in an amount sufficient for the treatment, prophylaxis or amelioration of the symptoms of the influenza. In one aspect of the method described above the composition is administered by an oral route, a parenteral route or an intra-nasal route. One embodiment of the present invention relates to a composition for enhancing antigen- specific T cell responses in a Dectin-1 -expressing antigen presenting cell (APC) comprising an anti-Dectin-1 -specific antibody or binding fragment thereof fused with one or more antigens. The APC of the present invention comprises an isolated dendritic cell (DC), a peripheral blood mononuclear cell (PBMC), a monocyte, a B cell, a myeloid dendritic cell or combinations thereof. In one aspect the APC comprises an isolated dendritic cell (DC), a peripheral blood mononuclear cell, a monocyte, a B cell, a myeloid dendritic cell or combinations thereof that have been cultured in vitro with GM-CSF and IL-4, IFNa, antigen, and combinations thereof. In another aspect the one or more antigens comprises bacterial, fungal or viral antigens, wherein the antigen is a HA1 subunit of an influenza virus and optionally comprises one or more TLR2 ligands. In one aspect the one or more TLR2 ligands comprise heat-killed bacteria, lipoglycans, lipopolysaccharide, lipoteichoic acids, peptidoglycans, synthetic lipoproteins, zymosan or combinations and modifications thereof. In another aspect the TLR2 ligand comprises lipopolysaccharides comprising P. gingivalis LPS or E.coli LPS. In another aspect the composition results in a proliferation of CD4+ T cells. In yet another aspect the CD4+ T secrete one or more cytokines selected from the group consisting of ΓΕΝγ, IL-13, IL-10, IL-17, and IL-21. In one aspect the composition enhances Thl7 and Thl responses by a secretion of IL-23. In another aspect the composition reduces Th2 cell responses. In another embodiment the instant invention presents a composition for enhancing antigen- specific T cell responses in a Dectin-1 -expressing antigen presenting cell (APC) comprising an anti-Dectin-1 -specific antibody or binding fragment thereof fused with one or more antigens and one or more TLR2 ligands. In one aspect the APC comprises an isolated dendritic cell (DC), a peripheral blood mononuclear cell (PBMC), a monocyte, a B cell, a myeloid dendritic cell or combinations thereof. In another aspect the one or more antigens comprise bacterial, fungal or viral antigens. In a specific aspect the antigen is a HA1 subunit of an influenza virus. In another aspect the one or more TLR2 ligands comprise heat-killed bacteria, lipoglycans, lipopolysaccharide, lipoteichoic acids, peptidoglycans, synthetic lipoproteins, zymosan or combinations and modifications thereof. In yet another aspect the composition comprises lipopolysaccharides comprising P. gingivalis LPS or E.coli LPS. In one aspect the composition increases secretion of IL-Ιβ, IL-6, and IL-23 thereby leading to an enhanced Th-17 response. In another aspect the composition reduces Th2 cell responses. Description of the Drawings

For a more complete understanding of the features and advantages of the present invention, reference is now made to the detailed description of the invention along with the accompanying figures and in which: FIGS. 1A-1F show antigen targeting to DCs via hDectin-1 resulting in HA 1 -specific CD4 ⁺ T cell responses: FIG. 1A reduced SDS-gel analysis of recombinant fusion proteins (Lane 1 : anti-hDectin-1, Lane 2: anti-hDectin-l-HAl, and Lane 3: IgG4-HAl), FIG. IB 293F cells transfected with full length of hDectin-1 and IFNDCs, FIG. 1C loading with different concentrations of anti-hDectin-1 -HA 1 or IgG4-HAl, and then stained with anti-human IgG- PE, FIG. ID CFSE-labeled purified autologous CD4 ⁺ T cells were co-cultured with IFNDCs loaded with 10 or 1 μg/ml recombinant fusion proteins. Cell proliferation was measured on day 7. Three independent runs showed similar results, FIG. IE CD4 ⁺ T cells restimulation after 7 days with 15 peptide pools (10 μΜ for each pool) for 4 h in the presence of Brefeldin A, and then stained with 7-AAD, anti-CD4, and anti-IFNy antibodies (upper panels). Individual peptides (0.5 μΜ) in pool 8 were further tested (lower panels). Pep 32 from pool 2 was tested as a control, and FIG. IF CD4 ⁺ T cells were restimulated with indicated peptides for 36h, and then cytokines in culture supernatants were assessed. Error bars represent SD of triplicate assay. Two independent runs resulted in similar data;

FIG. 2 shows antigen targeting to DCs via hDectin-1 allows the detection of antigen-specific Thl7 cells in healthy donors: Purified autologous CD4 ⁺ T cells were co-cultured IFNDCs loaded with 1 μg/ml anti-hDectin-1 -HA 1 for 7 days. CD4 ⁺ T cells were then restimulated with 0.5 μΜ peptides indicated for 36h. Cytokines in the culture supernatants were measured. Peptide epitopes for seven healthy donors were determined by performing intracellular IFNy staining with peptide pools and then individual peptides as described in FIG. IE. Pep 18 and 32 were used as controls. Error bars represent SD of triplicate assay;

FIG. 3 shows a total 2 xl05 CD4+ T cells co-cultured with 5 x 103 IFNDCs targeted with 1 mg/ml anti-hDectin-1 -HA 1 for one week. Different concentrations of Pam3 was added into the co-culture of DCs and CD4+ T cells. CD4+ T cells were restimulated with indicated peptides (1 mM) for 48h. Cytokines in culture supernatants were measured by Luminex; FIGS. 4A-4D shows antigen targeting to DCs via hDectin-1 enhance antigen-specific Thl7 cell responses by activating pre-existing antigen-specific Thl7 memory cells. Purified autologous CD4+ T cells (CD45RA+CD45RO- and CD45RA-CD45RO+) were co-cultured with IFNDCs loaded with 1 μ^ηιΐ anti-hDectin-l-HAl for 7-8 days. CD4+ T cells were then restimulated with HA 1 -derived peptides for 36h. Cytokines in culture supernatants were measured: FIG. 4A cells from four healthy donors were tested. Each line represents the data acquired with one donor, FIG. 4B data from three independent studies using cells from healthy donor. P values in FIGS. 4A and 4B were acquired by t-test, FIG. 4C IL-23 secreted by IFNDCs loaded with 1 μg/ml anti-hDectin-l-HAl, and FIG. 4D purified autologous total CD4+ T cells were co-cultured with IFNDCs loaded with 1 μg/ml anti-hDectin-l-HAl for 7- 8 days. CD4+ T cells were then restimulated with HAl-derived peptides for 36h. Cytokines in culture supernatants were measured;

FIGS. 5A-5C show P. gingivalis LPS can promote antigen-specific Thl7 cell responses elicited by IFNDCs targeted with anti-hDectin-l-HAl : FIG. 5 A purified autologous CD4 ⁺ T cells were co-cultured IFNDCs loaded with 1 μg/ml anti-hDectin-l-HAl for 7 days in the presence of 200 ng/ml P. gingivalis LPS (PG-LPS), 500 ng/ml poly I:C, 100 ng/ml E. coli LPS, or 200 ng/ml R848. CD4 ⁺ T cells were then restimulated with 0.5 μΜ peptides indicated for 36h. Cytokines in the culture supernatants were measured, FIG. 5B different concentrations of P. gingivalis LPS were tested, and FIG. 5C 40 ng/ml PG-LPS were tested using cells from healthy donors;

FIGS. 6A and 6B show that Pam3 can promote antigen-specific Thl7 cell responses elicited by IFNDCs targeted with anti-hDectin-l-HAl : FIG. 6A purified autologous CD4 ⁺ T cells were co-cultured IFNDCs loaded with 1 μg/ml anti-hDectin-l-HAl for 7 days in the presence of different concentrations of Pam3. CD4 ⁺ T cells were then restimulated with 0.5 μΜ peptides indicated for 36h. Cytokines in the culture supernatants were measured and FIG. 6B 40 ng/ml PG-LPS were tested using cells from healthy donors; FIGS. 7A-7E show TLR2-mediated enhancement of antigen-specific memory Thl7 cell responses are through IL-Ι β and is due to the activation of pre-existing memory Thl7 cells, but not the induction of antigen-specific Thl7 cells: FIG. 7A purified autologous CD4+ T cells (CD45RA+CD45RO- and CD45RA-CD45RO+) were co-cultured IFNDCs loaded with 1 μg/ml anti-hDectin-l-HAl for 7 days in the presence or absence of 40 ng/ml P. gingivalis LPS (PG-LPS). CD4+ T cells were then restimulated with 0.5 μΜ pep43 (donor #1), pep7 (donor #2), pep22 (donor #4), and pep22 (donor #5) for 36h. Cytokines in the culture supernatants were measured. P values were acquired by t-test, FIG. 7B total CD4+ T cells were co-cultured with IFNDCs loaded with 1 ug/ml anti-hDectin-l-HAl in the presence or absence 40 ng/'ml PG-LPS for seven days. Cells were then stimulated with PMA and ionomycin, and stained for intracellular IFNy and IL-17, FIG. 7C total RNA was extracted from CD4+ T cells in FIG. 7B. Relative expression levels of T-bet, Rorc, and GATA-3 were measured by RT-PCR. β-actin was used as a control. Three independent runs resulted in similar results and error bars are SD of the data from three runs, FIG. 7D 1 x 105 IFNDCs loaded with 1 μg/ml anti-hDectin-l-HAl, 40 ng/ml P. gingivalis LPS or 1 μg/ml anti- hDectin-l-HAl plus 40 ng/'ml PG-LPS, and then incubated overnight. IL-Ιβ and IL-6 levels in culture supernatants were measured, and FIG. 7E total CD4+ T cells were co-cultured with IFNDCs loaded with 1 ug/ml anti-hDectin-l-HAl in the presence 40 ng/ml PG-LPS with indicated antibodies (10 g/ml of each) for seven days. CD4+ T cells were then restimulated with pep43 (donor #1), pep7 (donor #2), pep22 (donor #4), and pep22 (donor #5) for 36h and IFNy and IL-17 levels in the culture supernatants were measured. P values were acquired by t-test; and FIGS. 8A and 8B show the phenotype of HAl-specific Thl and Thl7 CD4 ⁺ T cells elicited by DCs targeted with anti-hDectin-l-HAl: FIG. 8 A purified autologous CD4 ⁺ T cells were co-cultured with IFNDCs loaded with 1 ug/ml anti-hDectin-l-HAl. Cells were restimulated with pep43 (donor #1) and stained for intracellular IFNy and IL-17. Expression levels of CCR4, CCR5, CCR6, CCR9, CXCR3, integrin b7, and CD161 on both IFNy ⁺ and IL-17 ⁺ HA 1 -specific CD4 ^~ T cells were measured by flow cytometry, and FIG. 8B cells were restimulated with PMA/ionomycin and then stained for intracellular IFNy and IL-17, and surface receptors.

Description of the Invention

While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention.

To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as "a", "an," and "the" are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not delimit the invention, except as outlined in the claims.

As used herein, the term "Antigen Presenting Cells" (APC) refers to cells that are capable of activating T cells, and include, but are not limited to, certain macrophages, B cells and dendritic cells. "Dendritic cells" (DCs) refers to any member of a diverse population of morphologically similar cell types found in lymphoid or non-lymphoid tissues. These cells are characterized by their distinctive morphology, high levels of surface MHC-class II expression (Steinman, et al, Ann. Rev. Immunol. 9:271 (1991); incorporated herein by reference for its description of such cells). These cells can be isolated from a number of tissue sources, and conveniently, from peripheral blood, as described herein. Dendritic cell binding proteins refers to any protein for which receptors are expressed on a dendritic cell. Examples include GM-CSF, IL-1, TNF, IL-4, CD40L, CTLA4, CD28, and FLT-3 ligand.

The term "vaccine composition" as used in the present invention is intended to indicate a composition which can be administered to humans or to animals in order to induce an immune system response; this immune system response can result in a production of antibodies or simply in the activation of certain cells, in particular antigen-presenting cells, T lymphocytes and B lymphocytes. The vaccine composition can be a composition for prophylactic purposes or for therapeutic purposes or both. As used herein the term "antigen" refers to any antigen which can be used in a vaccine, whether it involves a whole microorganism or a subunit, and whatever its nature: peptide, protein, glycoprotein, polysaccharide, glycolipid, lipopeptide, etc. They may be viral antigens, bacterial antigens or the like; the term "antigen" also comprises the polynucleotides, the sequences of which are chosen so as to encode the antigens whose expression by the individuals to which the polynucleotides are administered is desired, in the case of the immunization technique referred to as DNA immunization. They may also be a set of antigens, in particular in the case of a multivalent vaccine composition which comprises antigens capable of protecting against several diseases, and which is then generally referred to as a vaccine combination or in the case of a composition which comprises several different antigens in order to protect against a single disease, as is the case for certain vaccines against whooping cough or the flu, for example. The term "antibodies" refers to immunoglobulins, whether natural or partially or wholly produced artificially, e.g. recombinant. An antibody may be monoclonal or polyclonal. The antibody may, in some cases, be a member of one or a combination immunoglobulin classes, including: IgG, IgM, IgA, IgD, and IgE.

The term "adjuvant" refers to a substance that enhances, augments or potentiates the host's immune response to a vaccine antigen. The term "gene" is used to refer to a functional protein, polypeptide or peptide-encoding unit. As will be understood by those in the art, this functional term includes both genomic sequences, cDNA sequences or fragments or combinations thereof, as well as gene products, including those that may have been altered by the hand of man. Purified genes, nucleic acids, protein and the like are used to refer to these entities when identified and separated from at least one contaminating nucleic acid or protein with which it is ordinarily associated

As used herein, the term "nucleic acid" or "nucleic acid molecule" refers to polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments generated by the polymerase chain reaction (PCR), and fragments generated by any of ligation, scission, endonuclease action, and exonuclease action. Nucleic acid molecules can be composed of monomers that are naturally-occurring nucleotides (such as DNA and RNA) or analogs of naturally-occurring nucleotides (e.g., a-enantiomeric forms of naturally- occurring nucleotides) or a combination of both. Modified nucleotides can have alterations in sugar moieties and/or in pyrimidine or purine base moieties. Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azido groups, or sugars can be functionalized as ethers or esters. Moreover, the entire sugar moiety can be replaced with sterically and electronically similar structures, such as aza- sugars and carbocyclic sugar analogs. Examples of modifications in a base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines or other well-known heterocyclic substitutes. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the like. The term "nucleic acid molecule" also includes so-called "peptide nucleic acids," which comprise naturally-occurring or modified nucleic acid bases attached to a polyamide backbone. Nucleic acids can be either single stranded or double stranded. As used in this application, the term "amino acid" refers to the one of the naturally occurring amino carboxylic acids of which proteins are comprised. The term "polypeptide" as described herein refers to a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as "peptides." A "protein" is a macromolecule comprising one or more polypeptide chains. A protein may also comprise non-peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to a protein by the cell in which the protein is produced, and will vary with the type of cell. Proteins are defined herein in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless.

As used herein, the term "in vivo" refers to being inside the body. The term "in vitro" used as used in the present application is to be understood as indicating an operation carried out in a non-living system.

As used herein, the term "treatment " or "treating" includes any administration of a compound of the present invention and includes (1) inhibiting the disease in an animal that is experiencing or displaying the pathology or symptomatology of the diseased (i.e., arresting further development of the pathology and/or symptomatology), or (2) ameliorating the disease in an animal that is experiencing or displaying the pathology or symptomatology of the diseased (i.e., reversing the pathology and/or symptomatology). The instant invention describes methods and compositions for enhancing Thl7 cell responses by targeting antigens to human dendritic cells (DCs) via Dectin-1.

IL-17-producing T cells (Thl7 cells) are crucial components of protective immunity against bacterial, fungal, and viral infections. Thus, the enhancement of pathogen-specific Thl7 cells in memory pools is of importance for protection against subsequent infections. However, pathogen-specific human memory Thl7 cells have been poorly understood because of their low frequencies in healthy individuals. Dectin-1, a c-type lectin-like pattern-recognition receptor, has been associated with Thl7 cell responses during bacterial and fungal infections. The present invention demonstrates that healthy individuals maintain broad ranges of pathogen (Influenza viruses)-specific Thl7 cells. This was achieved by targeting antigens (HA1 subunit, A/PR8/34) to human dendritic cells (DCs) via Dectin-1 using recombinant proteins of agonistic anti-hDectin-1 fused to HA1 (anti-hDectin-l-HAl). HAl-specific Thl7 cell responses elicited with anti-hDectin-l-HAl was further enhanced by P. gingivalis lipopolyssaccharide (LPS) and Pam3, but not poly I:C, E.coli LPS, or R848. The TLR2 ligand-mediated enhancement of Thl7 cell responses were mainly dependent on IL-lb secreted by DCs. The findings of the present invention demonstrate that HA 1 -specific Thl7 cell responses elicited by anti-hDectin-l-HAl alone or anti-hDectin-l-HAl plus TLR2 were not the results of priming naive CD4+ T cells, but the results of activation of pre-existing HA 1 -specific memory Thl7 cells.

IL-17-producing Thl7 CD4+ T cells (Thl7 cells) has been broadly linked to the pathogenesis of multiple autoimmune diseases (1-3). However, recent compelling evidence indicates that Thl7 cells are crucial for protective immunity against many mucosal and systemic infections of bacteria (5-7)(4), fungi (8-1 1), viruses (12-14), and parasites (15). Thl7 cells also play an important role in vaccine-induced protective immunity against infections (6, 12, 13, 16-18). Thus, understanding the pathways for the enhancement of pathogen-specific Thl7 cells is important to mount potent protective immunity against such infections. Early activation and expansion of pre-existing pathogen-specific Thl7 cells in memory pools are also thought to be an efficient way to mount protective immunity against subsequent infections by the same pathogens or pathogens sharing antigenic epitopes.

The induction of mouse Thl7 cells from naive T cells is initially dependent on the presence of TGF-β, IL-21, and IL-6, and at later stages on IL-23 (19). In humans, the differentiation of naive T cells into Thl7 cells is associated with IL-1, IL-6 (20, 21) and TGF-β (22-24). In addition, IL-23 and IL-Ιβ induce the production of IL-17 from human memory CD4+ T cells (25, 26). However, many of these studies have been conducted in limited experimental conditions, such as using APC-free cultures with anti-CD3/CD28 stimuli, addition of exogenous cytokines, and neutralization of IFN-7/IL-4. In addition, Thl7 cell responses elicited by polyclonal stimuli or by activating T cells via allogeneic recognitions may not always represent the pathogen-specific Thl7 cell responses elicited during and after infections. Furthermore, it is still not clear that memory Thl7 cells specific for pathogen- derived peptide-MHC class II exist as discrete Thl7 subset in vivo because such cells are difficult to detect in normal hosts. In the present invention the inventors tested the presence of pathogen (Influenza viruses)- specific Thl7 cells in healthy donors by targeting antigens (HA1 subunit) to DCs via human Dectin-1 (hDectin-1). Antigen targeting to DCs is an efficient way to elicit antigen-specific T cell responses (28, 29). The inventors demonstrate that healthy individuals maintain broad ranges of HA 1 -specific memory Thl7 cells that could be greatly enhanced by TLR2 ligands. The findings of the present invention also indicate that TLR2 ligand-mediated enhancement of HA 1 -specific Thl7 responses was the results of activation of pre-existing memory Thl7 cells.

Cells: Peripheral blood mononuclear cells (PBMCs) of healthy volunteers were fractionated by elutriation, according to Institutional Review Board guidelines. IL-4DCs and IFNDCs were generated by culturing monocytes from healthy donor in serum free media (Cellgenix, Germany) supplemented with GM-CSF (100 ng/ml) and IFNa a (500 U/ml) (IFNDCs) or GM-CSF (100 ng/ml) and IL-4 (50 ng/ml). The medium was replenished with cytokines on day 1 for IFNDCs and on day 3 for IL-4DCs. IFNa, IL-4 and GM-CSF were from the Pharmacy in Baylor University Medical Center (Dallas, TX). Autologous CD4+ T cells were purified using EasySep Human CD4+ T Cell Enrichment Kit (Stemcell, CA). Monocytes and B cells from PBMCs were purified with EasySep Human CD4 ⁺ T Cell Enrichment Kit (StemCell, CA). Naive (CD45RA+CD45RO-) and memory CD4+ T cells (CD45RA- CD45RO+) (purity>99.2%) were purified by FACS Aria (BD Biosciences).

Antibodies and reagents: Anti-CD4, anti-IFNy, anti-CCR6, and anti-CXCR3 were purchased from Biolegend (CA). Anti-CCR4, anti-CCR5, anti-CCR9, anti-IL-lRI, and anti-CCR7 were from R&D Systems (MN). Αηίί-β7 integrin, anti-CD161, anti-CD45RA, and anti-CD45RO were purchased from BD Biosciences (CA). Anti-IL-17 (eBioscience, CA) and anti-human IgG (Jackson ImmunoResearch Laboratories, PA) were used. Neutralizing anti-IL-23pl9 and control IgG were purchased from R&D Systems (CA). GolgiPlug was purchased from BD Pharmingen (CA). CFSE (Molecular probes, Oregon) was used for measuring CD4 ⁺ T cell proliferation. LPS from P. gingivalis, LPS from E. coli, Pam3CSK4, poly I:C, and R848 were purchased from Invivogen (OR).

Peptides: Overlapping (staggered by 11 amino acids) 17-mer peptides spanning the entire HA1 subunit of HA (A/PR/8/34 H1N1) were synthesized by Biosynthesis (TX).

DCs and CD4+ T cell co-cultures: 1-2 xl05 CFSE-labeled purified CD4+ T cells were co- cultured with 5x103 DCs in complete RPMI 1640 (GIBCO, NY) supplemented with 25 mM HEPES buffer, 2 mM L-glutamine, 1% nonessential amino-acids, ImM sodium pyruvate, 50 units/ml penicillin, 50 μg/ml streptomycin, and 10% AB serum (GemCell, CA). DCs were loaded with recombinant fusion proteins indicated for at least 6h before mixing with the CD4+ T cells. After 7 days, CD4+ T cell proliferation was tested by measuring CFSE- dilution. In some studies, anti-IL-23pl9, anti-IL-6 and anti-IL-6R, anti-IL-lb or control IgG (10 mg/ml) was added into the co-cultures of DCs and CD4+ T cells. Assessment of antigen-specific CD4+ T cell responses: CD4+ T cells were restimulated with indicated HA1 -derived peptides (2 niM) for 4h in the presence of Brefeldin A, and then stained with 7-AAD, anti-CD4 and anti-IFNg antibodies labeled with fluorescent dyes. CD4+ T cells expressing IFNg were detected by flow cytometry. CD4+ T cells were also stained for both IL-17 and IFNg during restimulation with 50 ng/ml phorbol 12-myristate 13-acetate (PMA) and 1 mg/ml ionomycin. In separate experiments, CD4+ T cells were stimulated with indicated peptides for 36h, and then culture supernatants were harvested for measuring cytokines and chemokines. Cytokine multiplex analysis was carried out using the Beads cytokine assay kit (seromap) as per the manufacturer's protocol. Cytokine concentrations were measured with a Bio-Plex Luminex instrument (Biorad, CA). To measure IL-23 secreted from DCs loaded with recombinant fusion proteins, 1x105 DCs were loaded with 1 mg/ml anti-hDectin-l-HAl or IgG4-HAl . After 24h, IL-23 in culture supernatants was measured using human IL-23 ELISA KIT (eBiosciences).

Expression and purification of chimeric recombinant mAbs fused to HA1 : Total RNA was prepared from hybridoma cells using RNeasy kit (Qiagen, CA) and used for cDNA synthesis and PCR (SMART RACE kit, BD Biosciences). PCR products were then cloned (pCR2.1 TA kit, Invitrogen) and characterized by DNA sequencing (MC Lab, CA). Using the derived sequences for the mouse heavy (H) and light (L) chain variable (V)-region cDNAs, specific primers were used to PCR amplify the signal peptide and V-regions while incorporating flanking restriction sites for cloning into expression vectors encoding downstream human IgG4H regions. The vector for expression of chimeric mVK-hlgK was built by amplifying residues 401-731 (gi|63101937|) flanked by Xho I and Not I sites and inserting this into the Xho I - Not I interval of pIRES2-DsRed2 (BD Biosciences). PCR was used to amplify the mAb Vk region from the initiator codon, appending a Nhe I or Spe I site then CACC, to the region encoding (e.g., residue 126 of gi|76779294|), appending a distal Xho I site. The PCR fragment was then cloned into the Nhe I - Not I interval of the above vector. The control human IgGK sequence corresponds to gi|49257887| residues 26-85 and gi|21669402| residues 67-709. The control human IgG4H vector corresponds to residues 12-1473 of gi|19684072| with S229P and L236E substitutions, which stabilize a disulphide bond and abrogate residual Fc interaction (30), inserted between the Bgl II and Not I sites of pIRES2-DsRed2 while adding the sequence 5'gctagctgattaattaa 3' (SEQ ID NO: 7) instead of the stop codon. PCR was used to amplify the mAb VH region from the initiator codon, appending CACC then a Bgl II site, to the region encoding residue 473 of gi| 19684072|. The PCR fragment was then cloned into the Bgl II - Apa I interval of the above vector.

The Flu HA1 antigen coding sequence is a CipA protein [Clostridium thermocellum] gi|479126| residues 147-160 preceding hemagglutinin [Influenza A virus (A/Puerto Rico/8/34(HlNl))] gi| 126599271 ) residues 18-331 with a P321L change and with 6 C- terminal His residues was inserted between the H chain vector Nhe I and Not I sites to encode recombinant antibody-HAl fusion proteins. Stable CHO-S cells were grown in GlutaMAX and HT media (Invitrogen) and recombinant proteins were purified by protein A column chromatography. Purified proteins were confirmed by reduced-SDS gel analysis.

Binding of recombinant fusion proteins to hDectin-l and APCs: 2x105 cells (293F cells transfected with full length of hDectin-l and IFNDCs) were incubated with different concentrations of recombinant fusion proteins (anti-hDectin-l-HAl and IgG4-HAl) for 20 min at 4°C Cells were then washed twice with 2% FCS in PBS, and then stained with secondary antibody, anti-human IgG-PE, for 20 min. Cells were analyzed by flow cytometry.

RT-PCR: Total RNA was isolated from cell lysates using QIAGEN RNeasy "Mini" spin columns according to the instructions of the manufacturer and then subjected to a 20mL cDNA synthesis reaction (Promega). Random primers were used as primer. 2.5mL cDNA was used for PCR amplification. The primer sequences and PCR temperature profiles for T- bet, RORC, GATA-3, and b-actin is provided in Table 1. A total of 4μΕ of the reverse transcriptase (RT)-PCR reactions was electrophoresed through a 4-12% Bis-Tris gel and stained with ethidium bromide for visualization under ultraviolet light. Table 1 : Primer sequences and PCR temperature profiles.

Statistical Analysis: Statistical significance was determined using the Student's t-test and significance was set at PO.05. Spearman's correlations statistics were used.

Anti-hDectin-l-HAl can target hDectin-1 molecules expressed on DCs: To target HAl to DCs via hDectin-1, recombinant proteins of an agonistic anti-hDectin-1 mAb (Ni et al. 2010) fused to HAl subunit of influenza viral hemagglutinin (A/PR8/34, H1N1) (anti-hDectin-l- HAl) were generated and analyzed in reduced SDS-gel (FIG. 1A). A human IgG4-HAl fusion protein was made as a control. Anti-hDectin-1 mAb was engineered as a chimera containing mouse V-region and human IgG4 Fc with two site mutations to abrogate residual non-specific binding capacity to Fc receptors (30).

Binding capacity of the two recombinant fusion proteins to hDectin-1 molecules were assessed. Anti-hDectin-l-HAl, but not IgG4-HAl, bound efficiently to 293F cells transfected with full length of hDectin-1 molecules in a concentration dependent manner (FIG. IB). Similarly, anti-hDectin-l-HAl bound to IFNDCs more efficiently than IgG4-HAl (FIG. 1C), suggesting that anti-hDectin-l-HAl target hDectin-1 molecules expressed on DCs. In addition, IFNDCs loaded with anti-hDectin-l-HAl induced greater proliferation of the purified autologous CD4+ T cells than IgG4-HAl did (FIG. ID). IFNDCs loaded with either 10 or 1 mg/ml anti-hDectin-l-HAl induced similar levels of CD4+ T cell proliferation (>38%). In contrast, 10 mg/ml IgG4-HAl induced only 7.9 % of CD4+ T cell proliferation. 1 mg/ml IgG4-HAl induced background levels of CD4+ T cell proliferation. Thus, it can be concluded that anti-hDectin-l-HAl can target hDectin-1 molecules expressed on DCs, and this resulted in the enhanced proliferation of autologous CD4+ T cells. Table 2: Predicted binding scores of individual peptide to corresponding MHC class II in each donor tested in this study.

SEQ ID NO: 13 SEQ ID NO: 13

SEQ ID NO 9

SEQ ID NO 10 -DREI

VVYAFALSReFGSGBTS SEQ ID NO 9

SEQ ID NO 10 -DRBrrS

VYAFALSBGFGSGBTS 1

i¾A MA

HLA-DQS'!'£« SEQ ID NO 12

LEPGDTEFEANSNUA. SEQ ID NO 9 as-usa SEQ ID NO 12

Ocsior LE GOTi ISrffi DA ^" lOOO lDO SEQ ID NO 9

-DRB

i tCS1.Q5

-DGBV33 f<iA WA

HA1 targeted to DCs via hDectin-1 activate different types of HA 1 -specific CD4+ T cells: Antigen specificity of the proliferating CD4+ T cells (FIG. ID) was tested by measuring intracellular IFNg expression. Fifteen clusters of HA 1 -derived peptides (1 1-12 peptides in one cluster, 17-mers overlapping by 11 amino acids) were first screened (upper panels in FIG. IE), and then individual peptides in the positive cluster (cluster 8) were further tested (lower panels in FIG. IE). Significant numbers of CFSE-CD4+ T cells expressed intracellular IFNg during restimulation with 1 mM peptides 43 and 45. Pep32 from pool 2 was tested as a negative control. The inventors then subsequently measured the amount of cytokines (IFNg, IL-13, IL-10, IL-17, and IL-21) secreted from the CD4+ T cells stimulated with HA 1 -derived pep43, pep45, and pep32 (FIG. IF). Both pep43 and pep45 induced CD4+ T cells to secrete significant amounts of the cytokines tested. This suggests that HA1 targeted to DCs via liDectin-1 can elicit HA 1 -specific CD4+ T cell responses. Although monocyte-derived DCs, B cells, and monocytes in peripheral blood mononuclear cells (PBMC) express similar levels of hDectin-1, DCs were far more efficient than other APCs for inducing CD4+ T cell proliferation as well as activating HA 1 -specific CD4+ T cells (data not shown). Influenza viral infections induce IFNa secretion from immune cells, including DCs (31), and IFNa can induce monocyte differentiation into DCs (32). IFNDCs generated in the presence of IFNa and GM-CSF were more potent than IL-4DCs generated with IL-4 and GM-CSF for proliferation and activation of HA 1 -specific CD4+ T cells (data not shown).

To extend the findings described in FIG. IF, the inventors assessed the types of HA 1 -specific CD4+ T cells present in 7 healthy individuals (FIG. 2). First of all, all healthy individuals maintained significant levels of HA 1 -specific CD4+ T cells, including IL-17-producing cells. The magnitudes, as measured by the levels of cytokines secreted, of different types of HA1- specific CD4+ T cells were highly variable among peptide epitopes as well as among individuals. For example, all IFNg-inducing peptides (pep7, pep45, pep46, and pep52) in donor #2 also induced CD4+ T cells to secrete significant amounts of IL-13. However, pep52-specific CD4+ T cells produced higher amounts of both IL-10 and IL-21 than CD4+ T cells specific for pep7, 45, and pep46. In addition, the magnitudes of HA 1 -derived peptide specific Thl7 responses were not correlated to the magnitudes of other types of CD4+ T cell responses that were specific for the same HAl-derived peptides (FIG. 3). As an example, HA 1 -specific CD4+ T cells in donor #2 secreted greater amounts of IFNg, IL-13, IL-10, and IL-21 than did HA 1 -specific CD4+ T cells in donor #3, but CD4+ T cells in donor #3 secreted greater amount of IL-17 than did CD4+ T cells in donor #2. Detailed information for HLA types of healthy donors tested and predicted binding scores of individual peptides to corresponding class II types are summarized in Table 2.

Based on the data in FIGS. IF and 2, it can be assumed that healthy individuals maintain pathogen-specific memory Thl7 cells. The inventors then tested if the types of HA 1 -specific CD4+ T cell responses observed were the results of the activation of pre-existing HA1- specific memory T cells. Two populations of CD4+ T cells (CD45PvA+CD45RO- and CD45RA-CD45RO+) were separately tested. The possibility that the responses observed with CD45RA+CD45RO- CD4+ T cell population might also be the results of the activation of contaminated HA 1 -specific memory T cells was not eliminated. However, the inventors assumed that the responses observed with CD45RA-CD45RO+ T cells are mainly due to the activation of memory T cells. FIG. 4A shows that both populations of CD4+ T cells (CD45RA+CD45RO- and CD45RA-CD45RO+) resulted in similar levels of IFNg-, IL-13-, IL-10, and IL-21 -producing HAl-specific responses. In contrast, significant levels of HAl- specific Thl 7 responses were observed only from CD45RA-CD45RO+ CD4+ T cells. This suggestes that HAl-specific Thl 7 cell responses observed in healthy donors were mainly due to the activation of pre-existing HAl-specific Thl7 memory cells. FIG. 4B presents the data from three independent studies using cells from the same donor.

Anti-hDectin-l-HAl could activate DCs to secrete IL-23 (FIG. 4C) that can contribute to the enhanced Thl 7 and Thl, and reduced Th2 cell responses (FIG. 4D). However, it was important to note that the magnitudes of IL-17 cell responses observed in FIG. 2 were not correlated with the amounts of IL-23 secreted by DCs from the same donors (data not shown). For example, DCs from donor #2 and #5 secreted higher levels of IL-23 (~ 80 pg/ml) than DCs from donor #1, but HAl-specific CD4+ T cells in donor #1 secreted greater amount of IL-17 than CD4+ T cells in donor #2 or #5. Taken together, the data demonstrates that DCs targeted with ant-hDectin-l-HAl could enhance HAl-specific Thl7 cell responses by activating pre-existing memory Thl 7 cells.

TLR2 ligands can promote the enhancement of HAl-specific memory Thl7 cell responses: The inventors tested whether TLR ligands could further enhance the HAl-specific Thl 7 cell responses elicited by DCs targeted with anti-hDectin-l-HAl (FIG. 5 A). Only P. gingivalis LPS and E. coli LPS significantly enhanced HAl-specific Thl 7 cell responses. Neither poly I:C nor R848 (TLR7/8 ligand) enhanced Thl7 cell responses. Although E. coli LPS enhanced Thl7 cell responses, it also promoted IL-10-producing HAl-specific CD4+ T cell responses. P. gingivalis LPS was further titrated using cells from donor #1 (FIG. 5B). Both Thl and Thl 7 responses peaked at 40 ng/ml P. gingivalis LPS. 40 ng/ml P. gingivalis LPS also enhanced HAl-specific Th21 CD4+ T cell responses. Interestingly, P. gingivalis LPS, at high dose (200 ng/ml), resulted in decreased HAl-specific Th2 type CD4+ T cell responses. It was also important to note that a low dose of . gingivalis LPS (8 ng/ml) could enhanceThl7 responses, but not Thl responses. The inventors then extended the studies by testing cells from other healthy donors tested in FIG. 2. FIG. 5C shows that P. gingivalis LPS resulted in enhanced HAl-specific Thl7 cell responses in all 6 donors. P. gingivalis LPS also promoted both Thl and Th21 CD4+ T cell responses in donors tested except for donor #2. Both IL-13 and IL-10-producing CD4+ T cell responses were variable among donors. Data in FIG. 3 show that P. gingivalis LPS enhanced the correlations between Thl 7 and Thl responses as well as Thl 7 and Th21 responses to the same peptide epitopes tested. In addition to P. gingivalis LPS, the inventors tested another TLR2 ligand, Pam3 (FIGS. 6A and 6B). Both P. gingivalis LPS and Pam 3 resulted in enhanced HAl-specific Thl7 cell responses. P. gingivalis LPS can bind to TLR2 (36, 37).

TLR2 ligands promote antigen-specific memory Thl 7 cell responses by inducing DCs to produce IL-lb: To test if the TLR2 ligands-mediated enhancement of HAl-specific Thl7 cell responses were due to the activation of pre-existing memory Thl7 cells, purified CD45RA+CD45RO- and CD45RA-CD45RO+ populations were tested (FIG. 7A). P. gingivalis LPS significantly enhanced HAl-specific Thl7 cell responses in the studies using CD45RA+CD45RO-, but not CD45RA-CD45RO+ population. This suggested that the TLR2 ligands-mediated HAl-specific Thl7 cell responses were mainly due to the activation of pre- existing memory Thl7 cells. FIG. 7B shows that memory Thl7 cells activated in the presence of P. gingivalis LPS express either IL-17 alone or IL-17 and IFNg. Pam3 also resulted in a similar response (data not shown). Consistently, T cells cultured in the presence of TLR2 ligands showed a significant increase in the expression of Rorc (FIG. 7C).

DCs loaded with anti-hDectin-l-HAl plus TLR2 ligands secreted greater amounts of IL-lb and IL-6 than DCs loaded with either anti-hDectin-l-HAl or P. gingivalis LPS alone (FIG. 7D). Thus, the inventors tested whether IL-lb or IL-6 could contribute to the TLR2 ligand- mediated enhancement of HAl-specific memory Thl7 cell responses. Blocking IL-lb in the co-culture of DCs and CD4+ T cells resulted in decreased levels of IL-17 production from T cells stimulated with HAl-derived peptides, suggesting that IL-lb plays a crucial role in the enhancement of HAl-specific memory Thl7 cell responses. Blocking IL-6 resulted in decreased Thl 7 cell responses. Taken together, the data obtained herein demonstrated that, in an IL- lb-dependent manner, TLR2 ligand-mediated enhancement of HAl-specific Thl 7 cell responses are mainly due to the activation of memory Thl 7 cells. In FIG. 7E total CD4 ⁺ T cells were co-cultured with IFNDCs loaded with 1 ug/ml anti-hDectin-l-HAl in the presence 40 ng/ml PG-LPS with indicated antibodies (10 g/ml of each) for seven days. CD4 ⁺ T cells were then restimulated with pep43 (donor #1), pep7 (donor #2), pep22 (donor #4), and pep22 (donor #5) for 36h and IFNy and IL-17 levels in the culture supernatants were measured. HAl-specific Thl7 CD4 ⁺ T cells express high levels of CCR4, CCR6, and CCR9, but low levels of CD161 and β7 integrin. Phenotype of HA 1 -specific Thl7 and Thl cells expanded with anti-hDectin-l-HAl was tested. Flow cytometry analysis shows that a large fraction of the HA 1 -specific Thl 7 cells express CCR4 and CCR6, whereas HAl-specific Thl cells expressed CCR4 and CXCR3 (FIG. 8A). Compared to HAl-specific Thl cells, Thl7 cells expressed lower levels of β7 integrin, but slightly higher levels of CD161 (33). Importantly, significant fractions of HAl-specific Thl7 cells expressed high levels of CCR9. The inventors then compared the phenotype of HAl-specific CD4 ⁺ T cells vs. total CD4 ⁺ T cells in the same culture (FIG. 8B). Both HAl-specific and total Thl cells expressed CCR4, CXCR3, and β7 integrin. However, a subset of only total Thl cells expressed significant levels of CD161. Similarly, compared to total Thl7 cells, HAl-specific Thl7 cells expressed lower levels of CD 161. The data obtained by the present inventors also show that only fractions of HAl-specific Thl7 cells express high levels of CCR9 though the expression levels of CCR6 or CCR9 were not correlated to the capacity of IL-17 secretion (34). Addition of TLR2 ligands in the co-culture of DCs and CD4 ⁺ T cells did not enhance the expression levels of the chemokines receptors tested (data not shown).

The types of antigen-specific CD4+ T cells primed or boosted during infections and after vaccinations could determine the potency of protective immunity in the hosts (38). Thl7 cells are now recognized as crucial components for protective immunity against infections of many microbial pathogens (4-18), including influenza viruses (39-42), and for the protection against subsequent infections. Thus, the proper activation and enhancement of pre-existing pathogen-specific Thl 7 cells is thought to be an efficient way to mount protective immunity. This study is the first demonstration that healthy individuals maintain pathogen (influenza)- specific Thl 7 cells and that such pathogen-specific memory Thl 7 cell responses can be further enhanced by targeting antigens to DCs via hDectin-1 in the presence of TLR2 ligands. Dendritic cells (DCs) are the major antigen-presenting cells that can induce and control the quality of immune responses (43, 44). Thus, the study of Thl 7 cell responses elicited by DCs is more physiologically relevant than by T cells coupled with limited experimental conditions, such as APC-free cultures with anti-CD3/CD28 stimuli, exogenous cytokines, and neutralization of IFNg and IL-4. Delivering antigens to DC via a surface lectin, DEC205, has demonstrated an efficient way to elicit potent and broad spectrum antigen-specific T cell responses (28, 29). One such lectin-like receptors expressed on DCs, Dectin-1, is strongly associated with the induction and promotion of Thl7 CD4+ T cells (10, 35, 45, 46). Signaling via Dectin-1 activates DCs to secrete IL-lb, IL-6, and IL-23 that contribute to the enhanced Thl7 cell responses (10, 20, 47). Carter et al, also showed that antigens delivered to mouse DCs via Dectin-1 resulted in antigen-specific CD4+ T cell responses (48). The present inventors have previously reported that antigen targeting to human DCs via Dectin-1 using recombinant proteins of agonistic anti-hDectin-1 fused to antigens resulted in potent antigen- specific CD8+ T cell responses in vitro (Ni et al. 2010). Therefore, hDectin-1 expressed on DCs is considered to be a prominent target molecule to deliver antigens to DCs. In support of this, the strategy employed in this study, targeting HA1 to DCs via hDectin-1, allowed the inventors to characterize multiple HA 1 -derived peptide epitopes that have not been previously described.

Most importantly, antigen targeting to DCs via hDectin-1 permitted the inventors to detect pathogen (HA1 of influenza viruses)-specific memory Thl7 cell responses in healthy individuals. It has not been easy to detect Thl7 memory T cells specific for pathogen-derived peptides in vivo, and this was partly due to the low frequency of such Thl7 cells in healthy hosts. A recent study showed that pathogen-specific Thl7 cells are shorter-lived than Thl cells in mice infected with Listeria monocytogene (27). Taking the advantages of the strategy described herein, targeting antigens to DCs via hDectin-1, the inventors first demonstrated that healthy individuals maintain influenza viral peptide epitope-specific memory Thl 7 cells. The agonistic property of anti-hDectin-1 fused to HA1 resulted in IL-23 induction from DCs, and this contributed to the amplification of HAl-specific memory Thl7 cell responses in vitro. Although IL-23 promoted Thl7 cell responses, as previously described (33-35), IL-23 alone may not be sufficient to mount potent pathogen-specific Thl 7 cell responses.

The magnitudes of HAl-specific memory Thl 7 cell responses were not correlated with the magnitudes of other types of HAl-specific CD4+ T cell responses. However, there was a correlation between the magnitudes of HAl-specific Thl cell responses and those of HAl- specific Th2 cell responses. Additionally, the magnitudes of Thl7 cell responses observed were highly variable among individuals and among peptide epitopes. These findings, the presence of HAl-specific memory Thl 7 cells in healthy individuals, are of fundamental importance because of the potential to promote such memory Thl 7 cell responses in healthy individuals.

The roles of TLR2 ligands in the expansion of Thl7 CD4+ T cell responses are not clearly elucidated. TLR2 deficiency results in increased Thl7 immunity associated with diminished expansion of regulatory T cells (49). It is also known that TLR2 promote regulatory T cell responses that inhibited autoimmunity in mice (50). In contrast, TLR2 engagement on DCs promotes influenza viral specific memory CD4+ T cell responses (41). In addition, activation of hDCs via Dectin-1 and TLR2 resulted in enhanced Thl7 responses (51, 52). Those discrepancy could be dependent on several factors, such as the strength of signals delivered to DCs via TLR2, integration of different signals delivered to DCs at the same time, subsets of DCs or T cells (memory vs. nai ^' ve), and distinct specie differentiation (i.e. human vs. non- human models). However, the role of TLR2 ligands in the enhancement of HA 1 -specific memory Thl7 responses was solid and generic. TLR2 ligands were capable to enhance memory Thl7 responses and Thl in a less extent in all healthy donors tested. A previous study (53) showed that freshly isolated circulating human Thl7 cells secrete IL-17 alone or with IL2, but those activated by DCs co-express IL-17 and IFNg. In combination with anti- liDectin-l-HAl, TLR2 ligands did not significantly enhance HA 1 -specific IL-10-producing CD4+ T cell responses. E. coli LPS could also enhance HA1 -specific Thl 7 cell responses, but it also enhanced IL-10-producing CD4+ T cell responses.

It is also important to note that the majority of HA 1 -specific Thl 7 cell responses were not the results of priming HA 1 -specific T cells, but the results of the activation of memory CD4+ T cells. IL-23 and IL-Ιβ secreted by DCs enhanced HAl-specific Thl7 cell responses, but did not result in the induction of HA 1 -specific Thl7 cells in vitro. While testing HAl-specific T cell responses, the inventors also assessed the allogeneic nai ^' ve CD4+ T cell responses induced by DCs, as many studies employ allogeneic systems to test the types of T cell responses induced in vitro. Indeed, the inventors observed the induction of allogeneic Thl 7 cell responses, which were further enhanced by activating DCs with anti-hDectin-1 mAb or curdlan, a fungal b-glucan (data not shown). The disparity observed between allogeneic T cells and antigen-specific T cells needs to be considered carefully, particularly when the induction of Thl 7 cell responses are assessed. The findings presented herein suggest that other factors, including signals from other immune cells and the strength of signaling via T cell receptors, are involved in the induction of pathogen-specific Thl 7 cells in vivo.

It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, kit, reagent or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention. It may be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims.

All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

The term "or combinations thereof as used herein refers to all permutations and combinations of the listed items preceding the term. For example, "A, B, C or combinations thereof is intended to include at least one of: A, B, C, AB, AC, BC or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, MB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context. All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it may be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

REFERENCES

United States Patent Application No. 2010/0166784: Method and Compositions for Modulating Thl7 Cell Development.

United States Patent Application No. 2008/0233140: Therapeutic Applications of Activation of Human Antigen-Presenting Cells Through Dectin-1.

1. Miossec, P., T. Kom, and V.K. Kuchroo. 2009. Interleukin-17 and type 17 helper T cells. N Engl J Med 361 :888-898.

2. Fouser, L.A., J.F. Wright, K. Dunussi-Joannopoulos, and M. Collins. 2008. Thl7 cytokines and their emerging roles in inflammation and autoimmunity. Immunol Rev 226:87- 102.

3. Mills, K.H. 2008. Induction, function and regulation of IL-17-producing T cells. Eur J Immunol 38:2636-2649.

4. O'Connor, W., L.A. Zenewicz, and R.A. Flavell. The dual nature of TH17 cells: shifting the focus to function. Nat Immunol 11 :471-476.

5. Zenaro, E., M. Donini, and S. Dusi. 2009. Induction of Thl/Thl7 immune response by Mycobacterium tuberculosis: role of dectin-1, Mannose Receptor, and DC-SIGN. J Leukoc Biol 86: 1393-1401.

6. Khader, S.A., and A.M. Cooper. 2008. IL-23 and IL-17 in tuberculosis. Cytokine 41:79-83.

7. Schulz, S.M., G. Kohler, N. Schutze, J. nauer, R.K. Straubinger, A.A. Chackerian,

E. Witte, K. Wolk, R. Sabat, Y. Iwakura, C. Holscher, U. Muller, R.A. astelein, and G.

Alber. 2008. Protective immunity to systemic infection with attenuated Salmonella enterica serovar enteritidis in the absence of IL-12 is associated with IL-23 -dependent IL-22, but not

IL-17. J Immunol 181:7891-7901.

8. Conti, H.R., F. Shen, N. Nayyar, E. Stocum, J.N. Sun, M.J. Lindemann, A.W. Ho,

J.H. Hai, J.J. Yu, J.W. Jung, S.G. Filler, P. Masso-Welch, M. Edgerton, and S.L. Gaffen.

2009. Thl7 cells and IL-17 receptor signaling are essential for mucosal host defense against oral candidiasis. J Exp Med 206:299-311. 9. Acosta-Rodriguez, E.V., L. Rivino, J. Geginat, D. Jarrossay, M. Gattorno, A. Lanzavecchia, F. Sallusto, and G. Napolitani. 2007. Surface phenotype and antigenic specificity of human interleukin 17-producing T helper memory cells. Nat Immunol 8:639- 646.

10. Leibundgut-Landmann, S., O. Gross, MJ. Robinson, F. Osorio, E.C. Slack, S.V. Tsoni, E. Schweighoffer, V. Tybulewicz, G.D. Brown, J. Ruland, and E.S.C. Reis. 2007. Syk- and CARD9-dependent coupling of innate immunity to the induction of T helper cells that produce interleukin 17. Nat Immunol 8:630-638.

11. Milner, J.D., J.M. Brenchley, A. Laurence, A.F. Freeman, B.J. Hill, K.M. Elias, Y. Kanno, C. Spalding, H.Z. Elloumi, M.L. Paulson, J. Davis, A. Hsu, A.I. Asher, J. O'Shea,

S.M. Holland, W.E. Paul, and D.C. Douek. 2008. Impaired T(H)17 cell differentiation in subjects with autosomal dominant hyper-IgE syndrome. Nature 452:773-776.

12. Williman, J., E. Lockhart, L. Slobbe, G. Buchan, and M. Baird. 2006. The use of Thl cytokines, IL-12 and IL-23, to modulate the immune response raised to a DNA vaccine delivered by gene gun. Vaccine 24:4471-4474.

13. Kohyama, S., S. Ohno, A. Isoda, O. Moriya, M.L. Belladonna, H. Hayashi, Y. Iwakura, T. Yoshimoto, T. Akatsuka, and M. Matsui. 2007. IL-23 enhances host defense against vaccinia virus infection via a mechanism partly involving IL-17. J Immunol 179:3917-3925.

14. Smiley, K.L., M.M. McNeal, M. Basu, A.H. Choi, J.D. Clements, and R.L. Ward. 2007. Association of gamma interferon and interleukin- 17 production in intestinal CD4+ T cells with protection against rotavirus shedding in mice intranasally immunized with VP6 and the adjuvant LT(R192G). J Virol 81 :3740-3748.

15. Kelly, M.N., J.K. Kolls, K. Happel, J.D. Schwartzman, P. Schwarzenberger, C. Combe, M. Moretto, and LA. Khan. 2005. Interleukin- 17/ ^' interleukin- 17 receptor-mediated signaling is important for generation of an optimal polymorphonuclear response against Toxoplasma gondii infection. Infect Immun 73:617-621.

16. Huang, W., L. Na, P.L. Fidel, and P. Schwarzenberger. 2004. Requirement of interleukin- 17A for systemic anti-Candida albicans host defense in mice. J Infect Dis 190:624-631.

17. Khader, S.A., G.K. Bell, J.E. Pearl, J.J. Fountain, J. Rangel-Moreno, G.E. Cilley, F. Shen, S.M. Eaton, S.L. Gaffen, S.L. Swain, R.M. Locksley, L. Haynes, T.D. Randall, and A.M. Cooper. 2007. IL-23 and IL-17 in the establishment of protective pulmonary CD4+ T cell responses after vaccination and during Mycobacterium tuberculosis challenge. Nat Immunol 8:369-377.

18. Pitta, M.G., A. Romano, S. Cabantous, S. Henri, A. Hammad, B. Kouriba, L. Argiro, M. el heir, B. Bucheton, C. Mary, S.H. El-Safi, and A. Dessein. 2009. IL-17 and IL-22 are associated with protection against human kala azar caused by Leishmania donovani. J Clin Invest 119:2379-2387.

19. Korn, T., E. Bettelli, M. Oukka, and V.K. Kuchroo. 2009. IL-17 and Thl7 Cells. Annu Rev Immunol 27:485-517.

20. Acosta-Rodriguez, E.V., G. Napolitani, A. Lanzavecchia, and F. Sallusto. 2007. Interleukins lbeta and 6 but not transforming growth factor-beta are essential for the differentiation of interleukin 17-producing human T helper cells. Nat Immunol 8:942-949.

21. Wilson, N.J., K. Boniface, J.R. Chan, B.S. McKenzie, W.M. Blumenschein, J.D. Mattson, B. Basham, K. Smith, T. Chen, F. Morel, J.C. Lecron, R.A. Kastelein, D.J. Cua, T.K. McClanahan, E.P. Bowman, and R. de Waal Malefyt. 2007. Development, cytokine profile and function of human interleukin 17-producing helper T cells. Nat Immunol 8:950- 957.

22. Manel, N., D. Unutmaz, and D.R. Liftman. 2008. The differentiation of human T(H)-17 cells requires transforming growth factor-beta and induction of the nuclear receptor RORgammat. Nat Immunol 9:641-649.

23. Volpe, E., N. Servant, R. Zollinger, S.I. Bogiatzi, P. Hupe, E. Barillot, and V.

Soumelis. 2008. A critical function for transforming growth factor-beta, interleukin 23 and proinflammatory cytokines in driving and modulating human T(H)-17 responses. Nat Immunol 9:650-657.

24. Yang, L., D.E. Anderson, C. Baecher-Allan, W.D. Hastings, E. Bettelli, M. Oukka, V.K. Kuchroo, and D.A. Hafler. 2008. IL-21 and TGF-beta are required for differentiation of human T(H)17 cells. Nature 454:350-352.

25. van Beelen, A.J., Z. Zelinkova, E.W. Taanman-Kueter, F.J. Muller, D.W. Hommes, S.A. Zaat, M.L. Kapsenberg, and E.C. de Jong. 2007. Stimulation of the intracellular bacterial sensor NOD2 programs dendritic cells to promote interleukin- 17 production in human memory T cells. Immunity 27:660-669.

26. Liu, H., and C. Rohowsky-Kochan. 2008. Regulation of IL-17 in human CCR6+ effector memory T cells. J Immunol 180:7948-7957. 27. Pepper, M., J.L. Linehan, A.J. Pagan, T. Zell, T. Dileepan, P.P. Geary, and M.K. Jenkins. Different routes of bacterial infection induce long-lived TH1 memory cells and short-lived TH17 cells. Nat Immunol 11 :83-89.

28. Bonifaz, L., D. Bonnyay, K. Mahnke, M. Rivera, M.C. Nussenzweig, and R.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. J Exp Med 196: 1627-1638.

29. Boscardin, S.B., J.C. Hafalla, R.F. Masilamani, A.O. Kamphorst, H.A. Zebroski, U. Rai, A. Morrot, F. Zavala, R.M. Steinman, R.S. Nussenzweig, and M.C. Nussenzweig. 2006. Antigen targeting to dendritic cells elicits long-lived T cell help for antibody responses. J Exp Med 203:599-606.

30. Reddy, M.P., C.A. Kinney, M.A. Chaikin, A. Payne, J. Fishman-Lobell, P. Tsui, P.R. Dal Monte, M.L. Doyle, M.R. Brigham-Burke, D. Anderson, M. Reff, R. Newman, N. Hanna, R.W. Sweet, and A. Truneh. 2000. Elimination of Fc receptor-dependent effector functions of a modified IgG4 monoclonal antibody to human CD4. J Immunol 164: 1925- 1933.

31. Diebold, S.S., M. Montoya, H. Unger, L. Alexopoulou, P. Roy, L.E. Haswell, A. Al-Shamkhani, R. Flavell, P. Borrow, and C. Reis e Sousa. 2003. Viral infection switches non-plasmacytoid dendritic cells into high interferon producers. Nature 424:324-328.

32. Blanco, P., A.K. Palucka, M. Gill, V. Pascual, and J. Banchereau. 2001. Induction of dendritic cell differentiation by IFN-alpha in systemic lupus erythematosus. Science 294: 1540-1543.

33. Oppmann, B., R. Lesley, B. Blom, J.C. Timans, Y. Xu, B. Hunte, F. Vega, N. Yu, J. Wang, K. Singh, F. Zonin, E. Vaisberg, T. Churakova, M. Liu, D. Gorman, J. Wagner, S. Zurawski, Y. Liu, J.S. Abrams, K.W. Moore, D. Rennick, R. de Waal-Malefyt, C. Hannum, J.F. Bazan, and R.A. Kastelein. 2000. Novel pl9 protein engages IL-12p40 to form a cytokine, IL-23, with biological activities similar as well as distinct from IL-12. Immunity 13:715-725.

34. Piskin, G., R.M. Sylva-Steenland, J.D. Bos, and M.B. Teunissen. 2006. In vitro and in situ expression of IL-23 by keratinocytes in healthy skin and psoriasis lesions: enhanced expression in psoriatic skin. J Immunol 176: 1908-1915.

35. Carmona, E.M., R. Vassallo, Z. Vuk-Pavlovic, J.E. Standing, T.J. Kottom, and A.H. Limper. 2006. Pneumocystis cell wall beta-glucans induce dendritic cell costimulatory molecule expression and inflammatory activation through a Fas-Fas ligand mechanism. J Immunol 177:459-467.

36. Darveau, R.P., T.T. Pham, K. Lemley, R.A. Reife, B.W. Bainbridge, S.R. Coats, W.N. Howald, S.S. Way, and A.M. Hajjar. 2004. Porphyromonas gingivalis lipopolysaccharide contains multiple lipid A species that functionally interact with both tolllike receptors 2 and 4. Infect Immun 72:5041-5051.

37. Burns, E., T. Eliyahu, S. Uematsu, S. Akira, and G. Nussbaum. TLR2-dependent inflammatory response to Porphyromonas gingivalis is MyD88 independent, whereas MyD88 is required to clear infection. J Immunol 184: 1455-1462.

38. Murphy, K.M., and S.L. Reiner. 2002. The lineage decisions of helper T cells. Nat

Rev Immunol 2:933-944.

39. Bermejo-Martin, J.F., R. Ortiz de Lejarazu, T. Pumarola, J. Rello, R. Almansa, P.

Ramirez, I. Martin-Loeches, D. Varillas, M.C. Gallegos, C. Seron, D. Micheloud, J.M. Gomez, A. Tenorio-Abreu, M.J. Ramos, M.L. Molina, S. Huidobro, E. Sanchez, M. Gordon, V. Fernandez, A. Del Castillo, M.A. Marcos, B. Villanueva, C.J. Lopez, M. Rodriguez- Dominguez, J.C. Galan, R. Canton, A. Lietor, S. Rojo, J.M. Eiros, C. Hinojosa, I. Gonzalez, N. Torner, D. Banner, A. Leon, P. Cuesta, T. Rowe, and D.J. Kelvin. 2009. Thl and Thl7 hypercytokinemia as early host response signature in severe pandemic influenza. Crit Care 13:R201.

40. McKinstry, K.K., T.M. Strutt, A. Buck, J.D. Curtis, J.P. Dibble, G. Huston, M.

Tighe, H. Hamada, S. Sell, RW. Dutton, and S.L. Swain. 2009. IX- 10 deficiency unleashes an influenza-specific Thl 7 response and enhances survival against high-dose challenge. J Immunol 182:7353-7363.

41. Chandran, S.S., D. Verhoeven, J.R. Teijaro, M.J. Fenton, and D.L. Farber. 2009. TLR2 engagement on dendritic cells promotes high frequency effector and memory CD4 T cell responses. J Immunol 183 :7832-7841.

42. Hamada, H., L. Garcia-Hernandez Mde, J.B. Reome, S.K. Misra, T.M. Strutt, K.K. McKinstry, A.M. Cooper, S.L. Swain, and RW. Dutton. 2009. Tcl7, a unique subset of CD8 T cells that can protect against lethal influenza challenge. J Immunol 182:3469-3481. 43. Dillon, S., A. Agrawal, T. Van Dyke, G. Landreth, L. McCauley, A. Koh, C.

Maliszewski, S. Akira, and B. Pulendran. 2004. A Toll-like receptor 2 ligand stimulates Th2 responses in vivo, via induction of extracellular signal-regulated kinase mitogen-activated protein kinase and c-Fos in dendritic cells. J Immunol 172:4733-4743. 44. Banchereau, J., B. Pulendran, R. Steinman, and K. Palucka. 2000. Will the making of plasmacytoid dendritic cells in vitro help unravel their mysteries? J Exp Med 192:F39-44.

45. Week, M.M., S. Appel, D. Werth, C. Sinzger, A. Bringmann, F. Grunebach, and P. Brossart. 2008. hDectin-1 is involved in uptake and cross-presentation of cellular antigens.

Blood 11 1:4264-4272.

46. Brown, G.D. 2006. Dectin-1 : a signalling non-TLR pattern-recognition receptor. Nat Rev Immunol 6:33-43.

47. Gross, O., A. Gewies, K. Finger, M. Schafer, T. Sparwasser, C. Peschel, I. Forster, and J. Ruland. 2006. Card9 controls a non-TLR signalling pathway for innate antifungal immunity. Nature 442:651-656.

48. Carter, R.W., C. Thompson, D.M. Reid, S.Y. Wong, and D.F. Tough. 2006. Preferential induction of CD4+ T cell responses through in vivo targeting of antigen to dendritic cell-associated C-type lectin- 1. J Immunol 177:2276-2284.

49. Loures, F.V., A. Pina, M. Felonato, and V.L. Calich. 2009. TLR2 is a negative regulator of Thl7 cells and tissue pathology in a pulmonary model of fungal infection. J Immunol 183 : 1279-1290.

50. Manicassamy, S., R. Ravindran, I. Deng, H. Oluoch, T.L. Denning, S.P. Kasturi, K.M. Rosenthal, B.D. Evavold, and B. Pulendran. 2009. Toll-like receptor 2-dependent induction of vitamin A-metabolizing enzymes in dendritic cells promotes T regulatory responses and inhibits autoimmunity. Nat Med 15:401-409.

51. Duraisingham, S.S., I. Hornig, F. Gotch, and S. Patterson. 2009. TLR-stimulated CD34 stem cell-derived human skin-like and monocyte-derived dendritic cells fail to induce Thl7 polarization of naive T cells but do stimulate Thl and Thl7 memory responses. I Immunol 183 :2242-2251.

52. Aliahmadi, E., R. Gramlich, A. Grutzkau, M. Hitzler, M. Kruger, R. Baumgrass, M. Schreiner, B. Wittig, R. Wanner, and M. Peiser. 2009. TLR2 -activated human langerhans cells promote Thl7 polarization via IL-lbeta, TGF-beta and IL-23. Eur J Immunol 39: 1221- 1230.

53. Dhodapkar, K.M., S. Barbuto, P. Matthews, A. Kukreja, A. Mazumder, D.

Vesole, S. Jagannath, and M.V. Dhodapkar. 2008. Dendritic cells mediate the induction of polyfunctional human IL17-producing cells (Thl7-1 cells) enriched in the bone marrow of patients with myeloma. Blood 1 12:2878-2885.