WO2010099576A1

WO2010099576A1 - Compositions and methods for enhancing immune responses

Info

Publication number: WO2010099576A1
Application number: PCT/AU2010/000248
Authority: WO
Inventors: Nitin Kumar Saksena; Bin Wang
Original assignee: Sydney West Area Health Service
Priority date: 2009-03-04
Filing date: 2010-03-04
Publication date: 2010-09-10

Abstract

The invention relates generally to the field of immunotherapy. Specifically, the invention relates to compositions and methods for enhancing the differentiation of antigen presenting precursor cells. More specifically, the invention relates to the treatment of conditions and diseases responsive to enhanced differentiation of antigen presenting precursor cells and/or the suppression of viral replication (e.g. HIV infection and cancer).

Description

Compositions and Methods for Enhancing Immune Responses

Technical Field

The invention relates generally to the field of immunotherapy. Specifically, the invention relates to compositions and methods for enhancing the differentiation of antigen presenting precursor cells. More specifically, the invention relates to the treatment of conditions and diseases responsive to enhanced differentiation of antigen presenting precursor cells and/or the suppression of viral replication.

Background

The mammalian immune system involves humoral and cell-mediated immunity. Humoral immunity is mediated by secreted antibodies and is predominantly directed to extracellular pathogens (e.g. bacteria). In contrast, cell-mediated immunity protects against intracellular pathogens (e.g. viruses) by eliminating infected cells and eliciting the production of cytokines. Cell-mediated immunity is also instrumental in the elimination of cancer cells.

In general, cellular immune responses are triggered by antigens displayed on the surface of antigen presenting cells (APCs). APCs are a functionally defined group of cells which are able to take up, process and display antigens to other immune cells such as T lymphocytes. "Professional" APCs (e.g. macrophages, dendritic cells (DCs) and B cells) are the most efficient APCs, and have been demonstrated to use both the MHC class I and MHC class II pathways of antigen presentation. Professional APCs also express costimulatory molecules which provide the second signal required for naive T cell activation via MHC bound antigen. In contrast, non-professional APCs (e.g. fibroblasts, vascular endothelial cells, and glial cells) do not express the costimulatory molecules necessary for the stimulation of naϊve T cells.

The generation of protective immunity against pathogens and tumours requires the efficient presentation of foreign or altered self antigens to T lymphocytes. Professional APCs such as DCs and macrophages are essential to this process. DCs are considered to be the most potent antigen-presenting cells (APCs) as they efficiently acquire and process antigen for presentation via MHC proteins and express high levels of T cell costimulatory ligands, both of which are necessary to trigger complete differentiation of naϊve T cells into competent effector cells. Although dendritic cells are important in presenting antigen, particularly to initiate primary immune responses, macrophages are the professional APC type most prominent in inflammatory sites and specialized for clearing necrotic and apoptotic material. B lymphocytes also play a role in antigen presentation, and have the additional capability of responding to antigens presented by other APCs.

A number of diseases and conditions of mammals are characterised by reduced numbers of APCs and/or impaired APC activity. For example, the quantity and function of DCs are significantly compromised during Human Immunodeficiency Virus (HIV) infection, which is also a characteristic feature of cancer patients. Macrophages are direct targets of HIV infection and this significantly reduces their ability to mount effective immune responses. Similarly, the anti-tumour activity of macrophages is also defective in cancer patients. The inability of APCs to efficiently process and present antigens to other immune cells in patients suffering from such conditions suppresses the host immune response and contributes significantly to morbidity and mortality.

There is an ongoing need for more effective means of stimulating the host immune system in response to infectious diseases and cancer. In particular, there is a need for treatments that induce differentiation of APC precursors to increase the number of functional APCs. hi the case of infectious diseases, treatments that increase the number of functional APCs with concurrent suppression of the infectious agent are particularly desirable.

Summary of the Invention

In a first aspect, the invention provides a method for producing an antigen presenting cell from an antigen presenting precursor cell, the method comprising contacting said precursor cell with a combination of:

(i) at least one CC chemokine; (ii) interferon-gamma; and

(iii) interleukin-2, wherein said contacting differentiates the precursor cell into an antigen presenting cell.

In a second aspect, the invention provides a method for producing an antigen- specific lymphocyte, the method comprising the steps of:

(i) producing an antigen presenting cell according to the method of the first aspect,

(ii) contacting the antigen presenting cell with a substance comprising an antigen to produce a loaded antigen presenting cell, and (iii) contacting the loaded antigen presenting cell with a lymphocyte, wherein said contacting produces the antigen-specific lymphocyte.

In one embodiment of the second aspect, the substance comprising the antigen is a pathogen or a cancer cell, or derived from a cell infected with a pathogen or a cancer cell. The pathogen may be a virus. The virus may be human immunodeficiency virus.

In one embodiment of the second aspect, the lymphocyte is a helper CD4+ T lymphocyte or a cytotoxic T lymphocyte.

In one embodiment of the first or second aspect, the precursor cell is a myeloid precursor cell. In one embodiment of the first or second aspect, the precursor cell is a CD14⁺ monocyte. hi one embodiment of the first or second aspect, the antigen presenting cell is a dendritic cell, a macrophage, or a B lymphocyte. hi one embodiment of the first or second aspect, the method further comprises contacting the precursor cell with interleukin-6. hi a third aspect, the invention provides a method for preventing or treating a disease or condition characterised by:

(i) reduced numbers of antigen presenting cells,

(ii) impaired antigen presenting cell activity, or (iii) both (i) and (ii) above, said method comprising administering to a subject a therapeutically effective amount of a combination of cytokines comprising at least one CC chemokine, interferon- gamma and interleukin-2. hi one embodiment of the third aspect, the disease or condition is selected from the group consisting of human immunodeficiency virus infection, acquired immune deficiency syndrome and cancer.

In a fourth aspect, the invention provides a method for enhancing an immune response in a subject, the method comprising administering to the subject a therapeutically effective amount of a combination of cytokines comprising interferon- gamma, interleukin-2 and at least one CC chemokine.

In one embodiment of the fourth aspect, the immune response is an antigen-specific immune response mediated by T lymphocytes.

In a fifth aspect, the invention provides a method for treating or preventing human immunodeficiency virus infection in a subject, the method comprising administering to the subject a therapeutically effective amount of a combination of cytokines comprising at least one CC chemokine, interferon-gamma and interleukin-2.

In a sixth aspect, the invention provides a method for suppressing human immunodeficiency virus replication in a subject, the method comprising administering to the subject a therapeutically effective amount of a combination of cytokines comprising at least one CC chemokine, interferon-gamma and interleukin-2.

In one embodiment of the third, fourth, fifth or sixth aspect, the method further comprises administering to the subject a therapeutically effective amount of interleukin-6.

In one embodiment of the fourth, fifth or sixth aspect, the method is used as an adjunct to highly active antiretroviral therapy (HAART).

In one embodiment of the first, second, third, fourth, fifth or sixth aspect, the at least one CC chemokine is one or more of CCL2 (MCP-I), CCL3 (MIPl alpha), CCL4 (MlPlbeta) and CCL8 (MCP-2).

In one embodiment of the first, second, third, fourth, fifth or sixth aspect, the combination of cytokines comprises CCL2 (MCP-I), CCL3 (MIPl alpha), CCL8 (MCP- 2), interferon-gamma and interleukin-2.

In one embodiment of the first, second, third, fourth, fifth or sixth aspect, the combination of cytokines comprises CCL2 (MCP-I), CCL4 (MlPlbeta), CCL8 (MCP-2), interferon-gamma and interleukin-2. In one embodiment of the first, second, third, fourth, fifth or sixth aspect, the combination of cytokines comprises CCL2 (MCP-I), CCL3 (MIPl alpha), CCL4 (MlPlbeta), CCL8 (MCP-2), interferon-gamma and interleukin-2.

In one embodiment of the first, second, third, fourth, fifth or sixth aspect, the combination of cytokines comprises CCL2 (MCP-I), CCL3 (MIPl alpha), CCL4 (MlPlbeta), interferon-gamma and interleukin-2. hi one embodiment of the first, second, third, fourth, fifth or sixth aspect, the combination of cytokines further comprises a CXC chemokine. The CXC chemokine may be CXCLlO (IP-IO).

In one embodiment of first, second, third, fourth, fifth or sixth aspect, the combination of cytokines comprises interleukin-6.

In a seventh aspect, the invention provides use of a combination of cytokines comprising at least one CC chemokine, interferon-gamma and interleukin-2 for the manufacture of a medicament for the treatment or prevention of a disease or condition characterised by: (i) reduced numbers of antigen presenting cells,

(ii) impaired antigen presenting cell activity, or

(iii) both (i) and (ii) above.

In an eighth aspect, the invention provides a combination of cytokines comprising of at least one CC chemokine, interferon-gamma and interleukin-2 for the treatment or prevention of a disease or condition characterised by:

(i) reduced numbers of antigen presenting cells,

(ii) impaired antigen presenting cell activity, or

(iii) both (i) and (ii) above. hi one embodiment of the seventh or eighth aspect, the at least one CC chemokine is one or more of CCL2 (MCP-I), CCL3 (MIP-I alpha), CCL4 (MlPlbeta) and CCL8 (MCP-2).

In one embodiment of the seventh or eighth aspect, the combination of cytokines comprises CCL2 (MCP-I), CCL3 (MIP-I alpha), CCL8 (MCP-2), interferon-gamma and interleukin-2.

In one embodiment of the seventh or eighth aspect, the combination of cytokines comprises CCL2 (MCP-I), CCL4 (MlPlbeta), CCL8 (MCP-2), interferon-gamma and interleukin-2.

In one embodiment of the seventh or eighth aspect, the combination of cytokines comprises CCL2 (MCP-I), CCL3 (MIPl alpha), CCL4 (MlPlbeta), interferon-gamma and interleukin-2.

In one embodiment of the seventh or eighth aspect, the combination of cytokines further comprises a CXC chemokine. The CXC chemokine may be CXCLlO (IP-IO).

In one embodiment of the seventh or eighth aspect, the combination of cytokines further comprises interleukin-6. hi a ninth aspect, the invention provides a composition for producing an antigen presenting cell from an antigen presenting precursor cell, the composition comprising at least one CC chemokine, interferon-gamma and interleukin-2.

In one embodiment of the ninth aspect, the at least one CC chemokine is one or more of CCL2 (MCP-I), CCL3 (MIPl alpha), CCL4 (MlPlbeta) and CCL8 (MCP-2).

In one embodiment of the ninth aspect, the composition comprises CCL2 (MCP-I), CCL3 (MIPl alpha), CCL8 (MCP-2), interferon-gamma and interleukin-2. hi one embodiment of the ninth aspect, the composition comprises CCL2 (MCP-I), CCL4 (MlPlbeta), CCL8 (MCP-2), interferon-gamma and interleukin-2. In one embodiment of the ninth aspect, the composition comprises CCL2 (MCP-I), CCL3 (MIPl alpha), CCL4 (MlPlbeta), interferon-gamma and interleukin-2.

In one embodiment ninth aspect, the composition further comprises a CXC chemokine. The CXC chemokine maybe CXCLlO (IP-IO). hi one embodiment of the ninth aspect, the composition comprises interleukin-6.

In a tenth aspect, the invention provides a kit for producing an antigen presenting cell from an antigen presenting precursor cell, the kit comprising at least one CC chemokine, interferon-gamma and interleukin-2.

In one embodiment of the tenth aspect, the at least one CC chemokine is one or more of CCL2 (MCP-I), CCL3 (MIPl alpha), CCL4 (MlPlbeta) and CCL8 (MCP-2).

In one embodiment of the tenth aspect, the kit comprises CCL2 (MCP-I), CCL3 (MIP-I alpha), CCL8 (MCP-2), interferon-gamma and interleukin-2. hi one embodiment of the tenth aspect, the kit comprises CCL2 (MCP-I), CCL4 (MlPlbeta), CCL8 (MCP-2), interferon-gamma and interleukin-2. In one embodiment of the tenth aspect, the kit comprises CCL2 (MCP-I), CCL3

(MIPl alpha), CCL4 (MlPlbeta), interferon-gamma and interleukin-2.

In one embodiment of the tenth aspect, the kit further comprises a CXC chemokine. The CXC chemokine may be CXCLlO (IP-IO).

In one embodiment of the tenth aspect, the kit comprises interleukin-6. In an eleventh aspect, the invention provides an antigen presenting cell produced by the method of the first aspect.

In a twelfth aspect, the invention provides an antigen-specific lymphocyte produced by the method of the second aspect.

A cytokine combination, composition or kit as contemplated in the invention may, for example, comprise a combination of cytokines selected from the group consisting of: (i) CCL2 (JE/MCP- 1 ), CCL4 (MIP lbeta), CCL8 (MCP-2), IFNγ, and IL-2.

(ii) CCL2 (JE/MCP-1), CCL3 (MIPl alpha), CCL4 (MlPlbeta), CCL8 (MCP-2),

IFNγ, and IL-2.

(iii) CCL2 (JE/MCP-1), CCL3 (MIPl alpha), CCL4 (MlPlbeta), IFNγ, and IL-2. (iv) CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL8 (MCP-2), IFNγ, and IL-2.

(v) CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL4 (MIP-I beta), IFNγ, and IL-2.

(vi) CCL2 (JE/MCP-1), CCL4 (MlPlbeta), CCL8 (MCP-2), IFNγ, IL-2, and

CXCLlO. (vii) CCL2 (JE/MCP-1), CCL3 (MIPl alpha), CCL4 (MlPlbeta), CCL8 (MCP-2),

IFNγ, IL-2, and CXCLlO. (viii) CCL2 (JE/MCP-1), CCL3 (MIPl alpha), CCL4 (MlPlbeta), IFNγ, IL-2, and

CXCLlO. (ix) CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL8 (MCP-2), IFNγ, IL-2, and

CXCLlO. (x) CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL4 (MIP-I beta), IFNγ, IL-2, and

CXCLlO.

(xi) CCL2 (JE/MCP-1), CCL4 (MlPlbeta), CCL8 (MCP-2), IFNγ, IL-2, and IL-6. (xii) CCL2 (JE/MCP-1), CCL3 (MIPl alpha), CCL4 (MlPlbeta), CCL8 (MCP-2),

IFNγ, IL-2, and IL-6. (xiii) CCL2 (JE/MCP-1), CCL3 (MIPl alpha), CCL4 (MlPlbeta), IFNγ, IL-2, and

IL-6. (xiv) CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL8 (MCP-2), IFNγ, IL-2, and IL- 6.

(xv) CCL2 (JE/MCP-1), CCL3 (MP-I alpha), CCL4 (MP-I beta), IFNγ, IL-2, and

IL-6. (xvi) CCL2 (JE/MCP-1), CCL4 (MlPlbeta), CCL8 (MCP-2), IFNγ, IL-2, CXCLlO and IL-6. (xvii) CCL2 (JE/MCP-1), CCL3 (MPl alpha), CCL4 (MPlbeta), CCL8 (MCP-2),

IFNγ, IL-2, CXCLlO and IL-6. (xviii) CCL2 (JE/MCP-1), CCL3 (MPl alpha), CCL4 (MlPlbeta), IFNγ, IL-2,

CXCLlO and IL-6.

(xix) CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL8 (MCP-2), IFNγ, IL-2, CXCLlO and IL-6.

(xx) CCL2 (JE/MCP-1), CCL3 (MP-I alpha), CCL4 (MP-I beta), IFNγ, IL-2,

CXCLlO and IL-6.

Brief Description of the Drawings

A preferred embodiment of the present invention will now be described, by way of an example only, with reference to the accompanying drawings wherein:

Figure 1 is a panel of microscopy images showing the effect of supernatant derived from p24 stimulated PBMCs of the study patient on the differentiation of CD 14+ monocytes derived from HIV(+) patients at 0 hours (top row), 4 hours (middle row) and 8 hours (bottom row). Panels labelled 1 show the effect of bioactive mix from the study patient on peripheral blood cells derived from patients experiencing below detection to low plasma viral loads (<50-5000 copies /ml plasma). Panels labelled 2 show the effect of active supernatant on cells derived from patients with intermediate plasma viral loads (<50,000 copies/ml). Panels labelled 3 show the effect of active supernatant on cells derived from patients with high plasma viral loads (> 100,000 copies/ml).

Figure 2 is a panel of microscopy images showing CD 14+ monocytes treated with control (no stimulation) (panels 1 and 2), supernatant derived from p24-stimulated CD4+T cells of the study patient (panels 3 and 4), or a mix of 5 cytokines (IL-2, IFNgamma, CCL2, CCL4 and CCL8) used at physiologic levels (Panels 5 and 6).

Definitions As used in this application, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a cytokine" also includes a plurality of cytokines.

As used herein, the term "comprising" means "including." Variations of the word "comprising", such as "comprise" and "comprises," have correspondingly varied meanings. Thus, for example, a combination of cytokines "comprising" two particular types of cytokines may consist exclusively of those two types of cytokines or may include one or more additional types of cytokines.

As used herein, the term "antigen presenting precursor cell" includes any cell capable of developing into a cell exhibiting the characteristics of an antigen presenting cell. An "antigen presenting precursor cell" may or may not be capable of processing antigens and displaying their peptide fragments on the cell surface (i.e. antigen presentation). The term encompasses "dendritic precursor cells" which are cells capable of differentiating into mature dendritic cells. Dendritic precursor cells typically have a non-dendritic morphology and are not competent to elicit a primary immune response as antigen presenting cells. The term also encompasses "macrophage precursor cells" which are cells capable of differentiating into macrophages. Macrophage precursor cells typically are not competent to elicit a primary immune response as antigen presenting cells. The term also encompasses "B lymphocyte precursor cells" which are cells capable of differentiating into B lymphocytes. B lymphocyte precursor cells typically are not competent to elicit a primary immune response as antigen presenting cells.

As used herein, the term "antigen presenting cell" (APC) encompasses any cell that can process antigens and display their peptide fragments on the cell surface, thereby providing a means of activating other immune cells. The term encompasses any antigen presenting cell, including but not limited to, macrophages, B cells and dendritic cells

(DCs).

As used herein, the term "dendritic cell" (DC) has its ordinary meaning in the field. Non-limiting examples of dendritic cells include Langerhans cells, dermal dendritic cells, interstitial dendritic cells, interdigitating dendritic cells, follicular dendritic cells, blood dendritic cells, veiled cells, plasmacytoid dendritic cells, myeloid dendritic cells, CDIa+ dendritic cells and DC-SIGN-expressing dendritic cells.

As used herein, the term "administering" and variations of that term including "administer", and "administration", include contacting, applying, delivering or providing a cytokine combination or composition of the invention to a subject by any appropriate method. This includes, for example, administration by standard routes such as parenteral (e.g. intravenous, intraspinal, subcutaneous or intramuscular), topical, oral, or intranasal administration, and/or delivering to the cells of a subject a vector construct comprising a nucleic acid encoding one or more components of a cytokine combination of the invention, which may then be used to express the components within the cell.

As used herein the terms "effective amount" and "therapeutically effective amount" each include within their meaning a non-toxic but sufficient amount of an agent or compound to provide the desired therapeutic effect. The exact amount required will vary from subject to subject depending on factors such as the species being treated, the age and general condition of the subject, the severity of the condition being treated, the particular agent being administered and the mode of administration and so forth. Thus, it is not possible to specify an exact "effective amount". However, for any given case, an appropriate "effective amount" may be determined by one of ordinary skill in the art using only routine experimentation. As used herein, the term "nucleic acid" refers to a deoxyribonucleotide or ribonucleotide polymer in either single-or double-stranded form, and unless otherwise limited, encompasses known analogues of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides. As used herein, the term "polypeptide" means a polymer made up of amino acids linked together by peptide bonds. The terms "polypeptide" and "protein" are used interchangeably herein, although for the purposes of the present invention a "polypeptide" may constitute a portion of a full length protein. As used herein, the term "polynucleotide" refers to a single- or double-stranded polymer of deoxyribonucleotide bases, ribonucleotide bases or known analogues or natural nucleotides, or mixtures thereof.

As used herein, the term "subject" includes humans and individuals of any mammalian species of social, economic or research importance including but not limited to members of the genus ovine, bovine, equine, porcine, feline, canine, primates, and rodents.

As used herein the term "cytokine" encompasses chemokines.

As used herein, the term "kit" refers to any delivery system for delivering materials.

Such delivery systems include systems that allow for the storage, transport, or delivery of reaction reagents (for example labels, reference samples, supporting material, etc. in the appropriate containers) and/or supporting materials (for example, buffers, written instructions for performing the assay etc.) from one location to another. For example, kits include one or more enclosures, such as boxes, containing the relevant reaction reagents and/or supporting materials. The term "kit" includes both fragmented and combined kits. As used herein, the term "fragmented kit" refers to a delivery system comprising two or more separate containers that each contains a subportion of the total kit components. The containers may be delivered to the intended recipient together or separately. Indeed, any delivery system comprising two or more separate containers that each contains a subportion of the total kit components are included in the term "fragmented kit". In contrast, a "combined kit" refers to a delivery system containing all of the components of a reaction assay in a single container (e.g. in a single box housing each of the desired components).

It will be understood that use the term "about" herein in reference to a recited numerical value includes the recited numerical value and numerical values within plus or minus ten percent of the recited value.

Any description of prior art documents herein, or statements herein derived from or based on those documents, is not an admission that the documents or derived statements are part of the common general knowledge of the relevant art. For the purposes of description all documents referred to herein are hereby incorporated by reference in their entirety unless otherwise stated.

Detailed Description The present inventors have conducted investigations on an individual infected with

HIV for at least 21 years exhibiting an undetectable viral load and a stable CD4+ T lymphocyte count over the period of infection. Experiments conducted on immune cells derived from this individual led to the isolation of a number of soluble factors with potent anti-HIV activity. It was determined that the soluble factors responsible for this anti-HIV activity are a combination of cytokines which act in tandem to induce the rapid differentiation of antigen presenting precursor cells (APPCs) into antigen presenting cells (APCs). The cytokine combination thus provides a means of enhancing host immune responses. Advantageously, it was demonstrated that the cytokine combination induces differentiation of APPCs into APCs far more rapidly than existing commercial reagents. Accordingly, the invention described herein provides compositions (e.g. laboratory reagents, therapeutic compositions) and methods for enhancing the differentiation of APCs from APPCs. Also provided are methods for the production of antigen-specific lymphocytes. Additionally provided are compositions and methods for preventing or treating diseases and conditions associated with reduced numbers of APCs and/or compromised APC activity.

Analyses conducted by the inventors revealed that the cytokine combination significantly inhibits HIV replication. The combination was shown to suppress the replication of CCR5 tropic HIV strains, CXCR4 tropic HIV strains and CCR5/CXCR4 dual tropic HIV strains. Accordingly, the invention provides compositions and methods for inhibiting HIV replication. Also provided are compositions and methods for preventing or treating HIV infection and acquired immune deficiency syndrome (AIDS).

Cytokine combination The invention provides a combination of cytokines capable of enhancing the number and activity of APCs. It will be understood that the term "cytokine" as used herein also encompasses "chemokines".

The inventors have determined that the combination of interferon-gamma, interleukin-2 and at least one CC chemokine is capable of inducing the rapid differentiation of APC precursor cells into APCs. The combination also provides potent antiviral activity in that it significantly suppresses HIV replication. Without being bound to a particular mechanism or mode of action, it is thought that the cytokines of the combination function in a synergistic manner to achieve these beneficial effects. The cytokine combination may comprise a CC chemokine. A CC chemokine as provided herein is one in which the arrangement of the first two of the four invariant cysteine residues at the amino terminus are adjacent. Isoforms and variants of CC chemokines having similar or identical biological activity to conventional CC chemokines are also contemplated. CC chemokines of the cytokine combination may be derived from any source, including humans and other mammalian species (e.g. mice, rats, primates, pigs, horses, sheep, cows). In one embodiment, the CC chemokine is a human CC chemokine. Examples of suitable CC chemokines include, but are not limited to, CCLl (I-309/TCA-3), CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL3L1 (MIP-I alpha isoform LD78 beta), CCL4 (MIP-I beta), CCL4L1 (LAG-I), CCL5 (RANTES), CCL6 (ClO), CCL7 (MCP-3/MARC), CCL8 (MCP-2), CCL9/10 (MIP-I gamma), CCLI l (Eotaxin), CCLl 2 (MCP-5), CCLl 3 (MCP-4), CCLl 4a (HCC-I), CCLHb (HCC-3), CCLl 5 (MIP- 1 delta), CCL16 (HCC-4), CCL17 (TARC), CCL18 (PARC), CCL19 (MIP-3 beta), CCL20 (MIP-3 alpha), CCL21 (6Ckine), CCL22 (MDC), CCL23 (MPIF-I), CCL24 (Eotaxin-2), CCL25 (TECK), CCL26 (Eotaxin-like), CCL26-like (Eotaxin-3-like), CCL27 (CTACK), CCL28, MCK-2, TAFAl (FAM19A1), TAF A3 (FAM19A3), TAF A4 (FAMl 9A4) and TAFA5 (FAMl 9A5).

In general, CC chemokines of the cytokine combination are capable of binding to multiple seven-transmembrane, G-protein coupled CC chemokine receptors. Examples of such receptors include, but are not limited to, CCRl, CCR2, CCR3, CCR4, CCR5, CCR7, CCR8, CCR9, CCRlO and CCRL2/LCCR/CRAM-A/B. CC chemokines of the cytokine combination may also be capable of binding CC chemokine receptor homologues, non- limiting examples of which include CCI, MCV-type II, MIP-I, MIP-II, and MIP-III.

In one embodiment of the invention, the cytokine combination comprises one or more of CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL4 (MIP-I beta), and CCL8 (MCP- 2). The CCL2, CCL3, CCL4 or CCL8 may be derived from a human. The human CCL2 may have the amino acid sequence set forth in GenBank accession No. EAW90827.1 or GenBank accession No. EAW80212.1. The human CCL3 may have the amino acid sequence set forth in GenBank accession No ABK41952.1 or GenBank accession No AAH71834.1. The human CCL4 may have the amino acid sequence set forth in GenBank accession no. AAX07305.1 or GenBank accession no.AAX07292.1. The human CCL8 may have the amino acid sequence set forth in GenBank accession no. AAI26243.1 or GenBank accession no. EAW80208.1.

In preferred embodiments, the cytokine combination comprises each of CCL2 (JE/MCP-1), CCL4 (MIP-I beta), and CCL8 (MCP-2).

In other preferred embodiments, the cytokine combination comprises each of CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), and CCL8 (MCP-2).

In other preferred embodiments, the cytokine combination comprises each of CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL4 (MIP-I beta), and CCL8 (MCP-2). In other preferred embodiments, the cytokine combination comprises CCL2 (MCP-

1), CCL3 (MIPl alpha) and CCL4 (MlPlbeta).

The cytokine combination may comprise interferon gamma (IFNγ). The IFNγ may be derived from any source including humans and other mammalian species (e.g. mice, rats, primates, pigs, horses, sheep, cows). In one embodiment, the IFNγ is human IFNγ. The human IFNγ may have the amino acid sequence set forth in GenBank accession No. AAB59534.1, GenBank accession No. AAM28885.1, GenBank accession No. CAP 17327.1, or GenBank accession No. CAP 17327.1. Variants and muteins of IFNγ having similar or identical biological activity to conventional IFNγ are also contemplated. For example, IFNγ variants with modified PEG and/or glycosylation sites may be included in the cytokine combination, such as those described in U.S. Patent No. 7230081 and U.S. Patent No. 7232562. Other examples of suitable IFNγ variants include those described in U.S. publication No. 2006099175, U.S. Patent No. 6531122, U.S. Patent No. 7144574, U.S. Patent No. 7238344, U.S. Patent No. 7338788 and U.S. Patent No. 7431921. The cytokine combination may comprise interleukin 2 (IL-2). The IL-2 may be derived from any source, including humans and other mammalian species (e.g. mice, rats, primates, pigs, horses, sheep, cows). In one embodiment, the IL-2 is human IL-2. The human IL-2 may have the amino acid sequence set forth in GenBank accession No. CAAOl 199.1, GenBank accession No. AAA59140.1, GenBank accession No. AAA98792.1 , or GenBank accession No. ABI20697. Variants and muteins of IL-2 having the same or similar biological activity to conventional IL-2 may also be used in the cytokine combination. Examples of suitable IL-2 variants and/or muteins are described in U.S. patent No. 4931543, U.S. patent No. 4752585, U.S. patent No. 4766106, U.S. publication No. 2006269515 and U.S. publication No. US2006160187. In some embodiments, the cytokine combination comprises IFNγ and IL-2.

In other embodiments, the cytokine combination comprises IFNγ and at least one

CC chemokine. The CC chemokine may be one or more of CCL2 (JE/MCP-1), CCL3

(MIP-I alpha), CCL4 (MIP-I beta) and CCL8 (MCP-2). In one embodiment, the cytokine combination comprises IFNγ, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), and CCL8

(MCP-2). In one embodiment, the cytokine combination comprises IFNγ, CCL2

(JE/MCP-1), CCL4 (MIP-I beta), and CCL8 (MCP-2). In one embodiment, the cytokine combination comprises IFNγ, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL4 (MIP-I beta), and CCL8 (MCP-2). In one embodiment, the cytokine combination comprises IFNγ, CCL2 (MCP-I), CCL3 (MIPl alpha), and CCL4 (MlPlbeta).

In other embodiments, the cytokine combination comprises IL-2 and at least one CC chemokine. The CC chemokine may be one or more of CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL4 (MIP-I beta) and CCL8 (MCP-2). In another embodiment, the cytokine combination comprises IL-2, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL4 (MIP-I beta), and CCL8 (MCP-2). hi one embodiment, the cytokine combination comprises IL-2, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), and CCL8 (MCP-2). hi one embodiment, the cytokine combination comprises IL-2, CCL2 (JE/MCP-1), CCL4 (MIP- 1 beta), and CCL8 (MCP-2). hi one embodiment, the cytokine combination comprises IL- 2, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL4 (MIP-I beta), and CCL8 (MCP-2). In one embodiment, the cytokine combination comprises IL-2, CL2 (MCP-I), CCL3 (MIPl alpha) and CCL4 (MlPlbeta).

In preferred embodiments, the cytokine combination comprises IFNγ, IL-2 and at least one CC chemokine. The CC chemokine may be one or more of CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL4 (MIP-I beta) and CCL8 (MCP-2). hi particularly preferred embodiments, the cytokine combination comprises IFNγ,

IL-2, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), and CCL8 (MCP-2).

In other particularly preferred embodiments, the cytokine combination comprises IFNγ, IL-2, CCL2 (JE/MCP-1), CCL4 (MIP-I beta), and CCL8 (MCP-2). hi still other particularly preferred embodiments, the cytokine combination comprises IFNγ, IL-2, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL4 (MIP-I beta), and CCL8 (MCP-2). hi other particularly preferred embodiments, the cytokine combination comprises IFNγ, IL-2, CCL2 (MCP-I), CCL3 (MIPl alpha), and CCL4 (MlPlbeta). The cytokine combination may comprise a CXC chemokine. A CXC chemokine as provided herein is one in which the two N-terminal cysteines are separated by a single amino acid. Isoforms and variants of CC chemokines having similar or identical biological activity to conventional CC chemokines are also contemplated. CC chemokines in the cytokine combination of the invention may be derived from any source, including humans and other mammalian species (e.g. mice, rats, primates, pigs, horses, sheep, cows). Examples of suitable CC chemokines include, but are not limited to, CXCLl,

CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCLlO,

CXCLI l, CXCLl 2, CXCLl 3, CXCL14, CXCLl 5, CXCLl 6 and CXCLl 7. Preferably, the CXC chemokine is CXCLl 0 (IP-10).

Accordingly, in some embodiments a cytokine combination of the invention comprises IFNγ, IL-2, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL8 (MCP-2), and CXCLlO.

In other embodiments the cytokine combination comprises IFNγ, IL-2, CCL2 (JE/MCP-1), CCL4 (MIP-I beta), CCL8 (MCP-2) and CXCLlO (IP-10).

In other embodiments the cytokine combination comprises IFNγ, IL-2, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL4 (MIP-I beta), CCL8 (MCP-2), and CXCLlO (IP-10).

In still other embodiments the cytokine combination comprises IFNγ, IL-2, CCL2 (MCP-I), CCL3 (MIPl alpha), CCL4 (MlPlbeta) and CXCLlO (IP-10).

In further embodiments CXCLlO production is induced by the presence of another component of the cytokine combination, such as IFNγ.

The cytokine combination may comprise interleukin-6 (IL-6).

Accordingly, in some embodiments a cytokine combination of the invention comprises IFNγ, IL-2, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL8 (MCP-2), and IL- 6. The combination may further comprise CXCLlO.

In other embodiments the cytokine combination comprises IFNγ, IL-2, CCL2 (JE/MCP-1), CCL4 (MIP-I beta), CCL8 (MCP-2) and IL-6. The combination may further comprise CXCLlO (IP-10). In other embodiments the cytokine combination comprises IFNγ, IL-2, CCL2

(JE/MCP-1), CCL3 (MIP-I alpha), CCL4 (MIP-I beta), CCL8 (MCP-2), and IL-6. The combination may further comprise CXCLlO (IP-10). In still other embodiments the cytokine combination comprises IFNγ, IL-2, CCL2 (MCP-I), CCL3 (MIPl alpha), CCL4 (MIPl beta) and IL-6. The combination may further comprise CXCLlO (IP-IO).

It will be understood that no particular limitation exists regarding the particular proportions of each cytokine component within a given cytokine combination of the invention.

Components of the cytokine combination may act in a synergistic manner. In general, a synergistic effect as used herein refers to interaction/s between the components of the cytokine combination that enhance an effect beyond that which would be achieved by adding the effect of each component taken in isolation. By way of example only, two or more individual components of the cytokine combination may act synergistically to enhance the differentiation of antigen presenting precursor cells into APCs. Additionally or alternatively, two or more individual components of the cytokine combination may act synergistically to suppress the replication of HIV. Cytokines included in the cytokine combination may be obtained or produced using any suitable method known in the art. For example, almost all commonly known cytokines (including IFNγ, IL-2, CC chemokines (such as CCL3 (MIP-I alpha), CCL4 (MIP-I beta), CCL8 (MCP-2), CXC chemokines (such as CXCLlO) and IL-6 are readily available from commercial sources (e.g. BD Biosciences, Cayman Chemical, Cell Sciences, and R&D Systems).

Alternatively, standard recombinant protein production techniques may be utilised to produce cytokines such as IFNγ, IL-2, CC chemokines, CXC chemokines and IL-6. Recombinant protein production techniques will typically involve the cloning of a gene encoding the cytokine into a plasmid/expression vector for subsequent overexpression in a suitable microorganism.

Suitable methods for the construction of expression vectors or plasmids are described in detail in standard texts such as Sambrook et al, "Molecular Cloning : A Laboratory Manual", (1989), Cold Spring Harbor, New York; and Ausubel et al., "Current Protocols in Molecular Biology", (2007), John Wiley and Sons. Methods suitable for producing recombinant cytokines for combinations and compositions of the invention are described in standard texts in the field such as Coligan et al., "Current Protocols in Protein Science", (Chapter 5), (2007), John Wiley and Sons, Inc.; and Pharmacia Biotech., "The Recombinant Protein Handbook', (1994), Pharmacia Biotech. US Patent No. 488903 describes a specific method of producing IFNγ using CHO cells transformed with plasmid constructs comprising the human IFNγ gene under the control of an SV40 promoter.

Commonly used expression systems suitable for the production of cytokines include, for example, bacterial (e.g. E. colϊ), yeast (e.g. Saccharomyces cerevisiae, Aspergillus, Pichia pastorisis), viral (e.g. baculovirus and vaccinia), cellular (e.g. mammalian and insect) and cell-free systems. Non-limiting examples of cell-free systems include eukaryotic rabbit reticuloctye, wheat germ extract systems, and the prokaryotic E. coli cell-free system (see for example, Madin et al., 2000, "A highly efficient and robust cell-free protein synthesis system prepared from wheat embryos: plants apparently contain a suicide system directed at ribosomes", Proc. Natl. Acad. Sci. U.S.A. 97:559- 564; Pelham and Jackson, 1976, "An efficient mRNA-dependent translation system from reticulocyte lysates", Εur. J. Biochem., 67: 247-256; Roberts and Paterson, 1973, "Efficient translation of tobacco mosaic virus RNA and rabbit globin 9S RNA in a cell- free system from commercial wheat germ", Proc. Natl. Acad. Sci., 70: 2330-2334; Zubay, 1973, "In vitro synthesis of protein in microbial systems", Ann. Rev. Genet., 7: 267; Lesley et al., 1991, "Use of in vitro protein synthesis from polymerase chain reaction- generated templates to study interaction of Escherichia coli transcription factors with core RNA polymerase and for epitope mapping of monoclonal antibodies ", J. Biol. Chem., 266(4): 2632-2638; Baranov et al., 1989, "Gene expression in a cell-free system on the preparative scale", Gene, 84: 463-466; and Kudlicki et al, 1992, "High efficiency cell-free synthesis of proteins: refinement of the coupled transcription/translation system ", Analyt. Biochem., 206: 389-393).

Purification of cytokines produced by such methods may be achieved using standard techniques in the art such as those described in Coligan et al., "Current Protocols in Protein Science", (Chapter 6), (2007), John Wiley and Sons, Inc. For example, if the protein is in a soluble state, it may be isolated using standard methods such as column chromatography. Cytokines may be genetically engineered to contain various affinity tags or carrier proteins that aid purification. For example, the use of histidine and protein tags engineered into an expression vector containing a nucleic acid sequence encoding the cytokine may facilitate purification by, for example, metal-chelate chromatography (MCAC) under either native or denaturing conditions. Purification may be scaled-up for large-scale production purposes.

In one embodiment, the invention provides a laboratory reagent comprising a cytokine combination of the invention for stimulating the differentiation of APCs (e.g. macrophages, dendritic cells) from antigen presenting precursor cells (e.g. CDl 4+ monocytes). Advantageously, the cytokine combination may induce differentiation of antigen presenting precursor cells into APCs more rapidly than existing commercial reagents. In preferred embodiments, the laboratory reagent comprises IFNγ, IL-2, CCL2

(JE/MCP-1), CCL3 (MIP-I alpha), and CCL8 (MCP-2). The laboratory reagent may further comprise CXCLlO (IP-10) and/or interleukin 6 (IL-6).

In other preferred embodiments, the laboratory reagent comprises IFNγ, IL-2, CCL2 (JE/MCP-1), CCL4 (MIP-I beta), and CCL8 (MCP-2). The laboratory reagent may further comprise CXCLl 0 (IP- 10) and/or interleukin 6 (IL-6).

In other preferred embodiments, the laboratory reagent combination comprises IFNγ, IL-2, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL4 (MIP-I beta), and CCL8 (MCP-2). The laboratory reagent may further comprise CXCLlO (IP-10) and/or interleukin 6 (IL-6). In other preferred embodiments, the laboratory reagent combination comprises

IFNγ, IL-2, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), and CCL4 (MIP-I beta). The laboratory reagent may further comprise CXCLlO (IP-10) and/or interleukin 6 (IL-6). The invention also provides compositions comprising cytokine combination(s) of the invention. In some embodiments, the composition is a laboratory reagent for stimulating the differentiation of APCs from antigen presenting precursor cells, thus finding broad application in a laboratory setting.

Cytokine combination(s) of the invention may be included in a pharmaceutical composition (e.g. a therapeutic agent). The pharmaceutical composition may comprise a pharmaceutically acceptable carrier, excipient and/or diluent. The carriers, excipients and diluents must be "acceptable" in terms of being compatible with the other ingredients of the composition, and not deleterious to the recipient thereof. Examples of pharmaceutically acceptable carriers or diluents are demineralised or distilled water; saline solution; vegetable based oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oil, arachis oil or coconut oil; silicone oils, including polysiloxanes, such as methyl polysiloxane, phenyl polysiloxane and methylphenyl polysolpoxane; volatile silicones; mineral oils such as liquid paraffin, soft paraffin or squalane; cellulose derivatives such as methyl cellulose, ethyl cellulose, carboxymethylcellulose, sodium carboxymethylcellulose or hydroxypropylmethylcellulose; lower alkanols, for example ethanol or iso-propanol; lower aralkanols; lower polyalkylene glycols or lower alkylene glycols, for example polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1,3- butylene glycol or glycerin; fatty acid esters such as isopropyl palmitate, isopropyl myristate or ethyl oleate; polyvinylpyrolidone; agar; gum tragacanth or gum acacia, and petroleum jelly. Typically, the carrier or carriers will form from 10% to 99.9% by weight of the compositions.

Compositions of the invention, and in particular pharmaceutical compositions of the invention, may comprise additional components such as chemotherapeutic drugs and/or anti-HIV agents. Any chemotherapeutic drug may be included in a composition of the invention.

Non-limiting examples of chemotherapeutic drugs include, but are not limited to, adenosine analogues (e.g. pentostatin and cladribine); alkyl sulfonates (e.g. busulfan); alkylators (e,g, dacarbazine, altretamine, temozolamide and procarbazine); anthracycline antibiotics (e.g. doxorubicin); antimicrotubule agents, (e.g., vindesine, vincristine, vinorelbine, and other vinca alkaloids); antitumor antibiotics (e.g. daunorubicin, doxorubicin, mitomycin, dactinomycin, bleomycin, idarubicin, epirubicin and mitoxantrone); aziridines (e.g. thiotepa); camptothecin analogues (e.g. topotecan and irinotecan); epipodophyllotoxins (e.g. teniposide and etoposide); folate analogues (e.g. methotrexate); nitrogen mustards, (e.g. melphalan, chlorambucil, estramustine, cyclophosphamide, ifosfamide, and mechlorethamine); nitrosoureas (e.g. lomustine, streptozocin, and carmustine); platinum complexes (e.g. cisplatin and carboplatin); purine analogues (e.g. mercaptopurine, thiogaunine,and fludarabine); pyrimidine analogs (e.g. cytarabine, capecitabine, floxuridine, fluorouracil, and gemcitabine); substituted ureas (e.g. hydroxyurea); taxanes (e.g. docetaxel (taxotere) and paclitaxel (taxol)); and topoisomerase inhibitors (e.g. topoisomerase I inhibitors such as camptothecin and topoisomerase II inhibitors such as amsacrine, daunorubicin, doxorubicin, mitoxantrone and etoposide).

Any anti-HIV agent may be included in a composition of the invention. Non- limiting examples of anti-HIV agents include, but are not limited to, protease inhibitors such as Amprenavir (APV), Atazanavir (ATV), Indinavir (IDV), Ritonavir (RTV), Lopinavir/Ritonavir (LPV/RTV), Nelfmavir (NFV) and Saquinavir (SQV); non- nucleoside reverse transcriptase inhibitors such as Delavirdine (DLV), Efavirenz (RFV) and Nevirapine (NVP); nucleoside/nucleotide analogue reverse transcriptase inhibitors such as Abacavir (ABC), Didanosine (ddl), Emtricitabine (FTC), Lamivudine (3TC), Stavudine (d4T), Tenofovir (TDF), Zalcitabine (ddC) and Zidovudine (AZT); integrase inhibitors such as Raltegravir, Elvitegravir; and fusion inhibitors such as Enfuvirtide (T20).

Compositions of the invention may be in a form suitable for administration by injection, in the form of a formulation suitable for oral ingestion (such as capsules, tablets, caplets, elixirs, for example), in the form of an ointment, cream or lotion suitable for topical administration, in a form suitable for delivery as an eye drop, in an aerosol form suitable for administration by inhalation, such as by intranasal inhalation or oral inhalation, in a form suitable for parenteral administration, that is, subcutaneous, intramuscular or intravenous injection.

For administration as an injectable solution or suspension, non-toxic parenterally acceptable diluents or carriers can include, Ringer's solution, isotonic saline, phosphate buffered saline, ethanol and 1 ,2 propylene glycol.

Some examples of suitable carriers, diluents and excipients for oral use include peanut oil, liquid paraffin, sodium carboxymethylcellulose, methyl cellulose, sodium alginate, gum acacia, gum tragacanth, dextrose, sucrose, sorbitol, mannitol, gelatine and lecithin. Other examples include emollients, emulsifiers, thickening agents, preservatives, and buffering agents. In addition these oral formulations may contain suitable flavouring and colourings agents. When used in capsule form the capsules may be coated with compounds such as glyceryl monostearate or glyceryl stearate which delay disintegration.

Solid forms for oral administration may contain binders acceptable in human and veterinary pharmaceutical practice, sweeteners, disintegrating agents, diluents, flavourings, coating agents, preservatives, lubricants and/or time delay agents. Suitable binders include gum acacia, gelatine, corn starch, gum tragacanth, sodium alginate, carboxymethylcellulose or polyethylene glycol. Suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharine. Suitable disintegrating agents include corn starch, methylcellulose, polyvinylpyrrolidone, guar gum, xanthan gum, bentonite, alginic acid or agar. Suitable diluents include lactose, sorbitol, mannitol, dextrose, kaolin, cellulose, calcium carbonate, calcium silicate or dicalcium phosphate. Suitable flavouring agents include peppermint oil, oil of wintergreen, cherry, orange or raspberry flavouring. Suitable coating agents include polymers or copolymers of acrylic acid and/or methacrylic acid and/or their esters, waxes, fatty alcohols, zein, shellac or gluten. Suitable preservatives include sodium benzoate, vitamin E, alpha-tocopherol, ascorbic acid, methyl paraben, propyl paraben or sodium bisulphite. Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc. Suitable time delay agents include glyceryl monostearate or glyceryl distearate.

Liquid forms for oral administration may contain, in addition to the above agents, a liquid carrier. Suitable liquid carriers include water, oils such as olive oil, peanut oil, sesame oil, sunflower oil, safflower oil, arachis oil, coconut oil, liquid paraffin, ethylene glycol, propylene glycol, polyethylene glycol, ethanol, propanol, isopropanol, glycerol, fatty alcohols, triglycerides or mixtures thereof.

Suspensions for oral administration may further comprise dispersing agents and/or suspending agents. Suitable suspending agents include sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose, poly-vinyl-pyrrolidone, sodium alginate or acetyl alcohol. Suitable dispersing agents include lecithin, polyoxyethylene esters of fatty acids such as stearic acid, polyoxyethylene sorbitol mono-or di-oleate, -stearate or- laurate, polyoxyethylene sorbitan mono-or di-oleate, -stearate or-laurate and the like.

The emulsions for oral administration may further comprise one or more emulsifying agents. Suitable emulsifying agents include dispersing agents as exemplified above or natural gums such as guar gum, gum acacia or gum tragacanth.

Methods for preparing parenterally administrable compositions are known to those skilled in the art, and are described in more detail in, for example, Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa., the contents of which are incorporated herein by reference in their entirety.

Topical formulations of the present invention may comprise an active ingredient (e.g. one or more cytokine combinations of the invention) together with one or more acceptable carriers, and optionally any other therapeutic ingredients. Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin to the site of where treatment is required, such as liniments, lotions, creams, ointments or pastes, and drops suitable for administration to the eye, ear or nose.

Drops according to the present invention may comprise sterile aqueous or oily solutions or suspensions. These may be prepared by dissolving the active ingredient/s in an aqueous solution of a bactericidal and/or fungicidal agent and/or any other suitable preservative, and optionally including a surface active agent. The resulting solution may then be clarified by filtration, transferred to a suitable container and sterilised. Sterilisation may be achieved by autoclaving or maintaining at 90⁰C-IOO⁰C for half an hour, or by filtration, followed by transfer to a container by an aseptic technique. Examples of bactericidal and fungicidal agents suitable for inclusion in the drops are phenylmercuric nitrate or acetate (0.002%), benzalkonium chloride (0.01%) and chlorhexidine acetate (0.01%). Suitable solvents for the preparation of an oily solution include glycerol, diluted alcohol and propylene glycol. Lotions according to the present invention include those suitable for application to the skin or eye. An eye lotion may comprise a sterile aqueous solution optionally containing a bactericide and may be prepared by methods similar to those described above in relation to the preparation of drops. Lotions or liniments for application to the skin may also include an agent to hasten drying and to cool the skin, such as an alcohol or acetone, and/or a moisturiser such as glycerol, or oil such as castor oil or arachis oil.

Creams, ointments or pastes according to the invention are semi-solid formulations of the active ingredient for external application. They may be made by mixing the active ingredient in finely-divided or powdered form, alone or in solution or suspension in an aqueous or non-aqueous fluid, with a greasy or non-greasy basis. The basis may comprise hydrocarbons such as hard, soft or liquid paraffin, glycerol, beeswax, a metallic soap; a mucilage; an oil of natural origin such as almond, corn, arachis, castor or olive oil, wool fat or its derivatives, or a fatty acid such as stearic or oleic acid together with an alcohol such as propylene glycol or macrogols.

A composition of the invention may incorporate any suitable surfactant such as an anionic, cationic or non-ionic surfactant such as sorbitan esters or polyoxyethylene derivatives thereof. Suspending agents such as natural gums, cellulose derivatives or inorganic materials such as silicaceous silicas, and other ingredients such as lanolin, may also be included.

For sustained action, cytokine combinations of the invention can be microencapsulated and administered to patients, for example, orally or systemically. Alternatively, cytokine combinations of the invention may be delivered using a nanoparticle coating approach for sustained action, for example, by systemic delivery.

Compositions of the invention may also be administered in the form of liposomes. Liposomes are generally derived from phospholipids or other lipid substances, and are formed by mono-or multi-lamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolisable lipid capable of forming liposomes can be used. The compositions in liposome form may contain stabilisers, preservatives, excipients and the like. The preferred lipids are the phospholipids and the phosphatidyl cholines (lecithins), both natural and synthetic. Methods to form liposomes are known in the art, and in relation to this specific reference is made to: Prescott, Ed., "Methods in Cell Biology", (1976), Volume XIV, Academic Press, New York, N.Y. p.33 et seq., the contents of which are incorporated herein by reference in their entirety.

Antigen presenting cells

The invention provides methods for enhancing the development of antigen presenting cells from antigen presenting precursor cells. In general, the methods comprise contacting an antigen presenting precursor cell with a cytokine combination of the invention or a composition comprising the same.

It will be understood that contacting an antigen presenting precursor cell (APPC) with a cytokine combination of the invention does not necessarily require that the APPC is contacted with each cytokine component of the combination simultaneously.

Accordingly, the APPC may be administered some component(s) of the combination prior to being administered other component(s) of the combination.

Accordingly, contacting an APPC with a cytokine combination of the invention as contemplated herein includes administering each component of the combination to the APPC simultaneously or, administering some component(s) of the combination to the APPC separately from other component(s) of the combination. Preferably, when the APPC is administered components of a given combination of the invention separately, the components are administered in an order and/or over a time period that does not substantially compromise the development of an antigen presenting cell from the APPC.

It will also be understood that no particular limitation exists regarding the relative proportions of each component within a cytokine combination of the invention used to contact the APPC.

In some embodiments, the cytokine combination comprises IFNγ and IL-2.

In other embodiments, the cytokine combination comprises IFNγ and at least one CC chemokine. The CC chemokine may be one or more of CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL4 (MIP-I beta) and CCL8 (MCP-2). The cytokine combination may comprise IFNγ, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), and CCL8 (MCP-2). The cytokine combination may comprise IFNγ, CCL2 (JE/MCP-1), CCL4 (MIP-I beta), and CCL8 (MCP-2). The cytokine combination may comprise IFNγ, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL4 (MIP-I beta), and CCL8 (MCP-2). The cytokine combination may comprise IFNγ, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), and CCL4 (MIP-I beta).

In other embodiments, the cytokine combination comprises IL-2 and at least one CC chemokine. The CC chemokine may be one or more of CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL4 (MIP-I beta) and CCL8 (MCP-2). The cytokine combination may comprise IL-2, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL4 (MIP-I beta), and CCL8 (MCP-2). The cytokine combination may comprise IL-2, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), and CCL8 (MCP-2). The cytokine combination may comprise IL-2, CCL2 (JE/MCP-1), CCL4 (MIP-I beta), and CCL8 (MCP-2). hi one embodiment, the cytokine combination comprises IL-2, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL4 (MIP-I beta), and CCL8 (MCP-2). The cytokine combination may comprise IL-2, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), and CCL4 (MIP-I beta).

In preferred embodiments, the cytokine combination comprises IFNγ, IL-2 and at least one CC chemokine. The CC chemokine may be one or more of CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL4 (MIP-I beta) and CCL8 (MCP-2).

In particularly preferred embodiments, the cytokine combination comprises IFNγ, IL-2, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), and CCL8 (MCP-2).

In other particularly preferred embodiments, the cytokine combination comprises IFNγ, IL-2, CCL2 (JE/MCP-1), CCL4 (MIP-I beta), and CCL8 (MCP-2). In still other particularly preferred embodiments, the cytokine combination comprises IFNγ, IL-2, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL4 (MIP-I beta), and CCL8 (MCP-2).

In other preferred embodiments, the cytokine combination comprises IFNγ, IL-2, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha) and CCL4 (MIP-I beta). The cytokine combination may comprise a CXC chemokine. A CXC chemokine as provided herein is one in which the two N-terminal cysteines are separated by a single amino acid. Isoforms and variants of CC chemokines having similar or identical biological activity to conventional CC chemokines are also contemplated. CC chemokines of the cytokine combination may be derived from any source, including humans and other mammalian species (e.g. mice, rats, pigs, primates, horses, sheep, cows). Examples of suitable CC chemokines include, but are not limited to, CXCLl, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCLlO, CXCLI l, CXCLl 2, CXCL13, CXCL14, CXCL15, CXCL16 and CXCL17. Preferably, the CXC chemokine is CXCLlO (IP-IO). In some embodiments the cytokine combination comprises IFNγ, IL-2, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL8 (MCP-2), and CXCLlO.

In other embodiments the cytokine combination comprises IFNγ, IL-2, CCL2 (JE/MCP-1), CCL4 (MIP-I beta), CCL8 (MCP-2) and CXCLlO (IP-IO). In other embodiments the cytokine combination comprises IFNγ, IL-2, CCL2

(JE/MCP-1), CCL3 (MIP-I alpha), CCL4 (MIP-I beta), CCL8 (MCP-2), and CXCLlO (IP-10).

In other embodiments the cytokine combination comprises IFNγ, IL-2, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL4 (MIP-I beta) and CXCLlO (IP-IO). In some embodiments, CXCLlO production is induced by the presence of another component of the cytokine combination, such as IFNγ.

The cytokine combination may comprise interleukin-6 (IL-6).

Accordingly, in some embodiments the cytokine combination comprises IFNγ, IL- 2, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL8 (MCP-2), and IL-6. The combination may further comprise CXCLlO.

In other embodiments the cytokine combination comprises IFNγ, IL-2, CCL2 (JE/MCP-1), CCL4 (MIP-I beta), CCL8 (MCP-2) and IL-6. The combination may further comprise CXCLlO (IP-10).

In other embodiments the cytokine combination comprises IFNγ, IL-2, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL4 (MIP-I beta), CCL8 (MCP-2), and IL-6. The combination may further comprise CXCLlO (IP-10).

In still other embodiments the cytokine combination comprises IFNγ, IL-2, CCL2 (MCP-I), CCL3 (MIPl alpha), CCL4 (MIPl beta) and IL-6. The combination may further comprise CXCLlO (IP-10). An antigen presenting precursor cell as described herein includes any cell capable of developing into a cell exhibiting the characteristics of an antigen presenting cell. Non- limiting examples of antigen presenting precursor cells include dendritic precursor cells, macrophage precursor cells and B lymphocyte precursor cells.

A dendritic precursor cell as described herein includes any cell capable of differentiating into a mature dendritic cell. Examples of dendritic precursor cells include, but are not limited to, myeloid dendritic precursor cells, lymphoid dendritic precursor cells and plasmacytoid dendritic precursor cells. Phenotypic surface markers expressed by various subsets of dendritic precursor cells are well known in the art and may be used for the purpose of identification, for example, by flow cytometry or using immunohistochemical techniques.

In general, a myeloid precursor cell may be identified by the expression of markers such as CD 13 and CD33. Myeloid dendritic precursor cells may differentiate into mature dendritic cells via CD 14 or CDIa pathways. Accordingly, a dendritic precursor cell of the invention may be a CDH⁺CDIa^" dendritic precursor cell or a CD 14 CD Ia⁺ dendritic precursor cell. In certain embodiments of the invention, a myeloid dendritic precursor cell may be characterised by a CD34⁺CD33⁺CD7^~CD10^" phenotype. In a preferred embodiment, the myeloid dendritic precursor cell is a CD14⁺ monocyte. The CD14⁺ monocyte may also express the GM-CSF receptor.

In general, lymphoid dendritic precursor cells develop from CD34⁺ Lin^" CDlO⁺ progenitor cells and may be identified by the expression of CD34 and CD7. In certain embodiments of the invention, a lymphoid dendritic precursor cell may be characterised by a CD34⁺CD33^+/"CD7⁺CD10⁺ phenotype. Plasmacytoid dendritic precursor cells are of lymphoid origin and generally exhibit a distinct plasma cell like morphology. Plasmacytoid dendritic precursor cells may be identified by a CDl lc^lowB220⁺MHC-II^lo/" phenotype. In certain embodiments, a plasmacytoid dendritic precursor cell may be characterised by a CDl Ic^" CD45RA^hiCDl lb^"MHC-II^loIL-3R^hiCD4⁺ phenotype. Phenotypic surface markers expressed by various macrophage precursor cells are well known in the art. In general, a macrophage precursor cell in accordance with the invention encompasses antigen precursor cells of myeloid origin. A myeloid precursor cell may be identified by the expression of markers such as CD 13 and CD33. Macrophage precursor cells include monoblasts, promonocytes, and myeloblasts. Monoblasts may be identified by the surface expression of one or more of HLA-DR, CD4, CDl Ib, CDl Ic, CD33, CD64, CD65 and CD36. Promonocytes may be identified by the surface expression of CD4¹⁰, CDl Ic and CD 14, and are generally CD34^" and CDl 17^". Myeloblasts may be identified by the surface expression of CD 13, CD33, and CDl 17 and may also express CD34, CD36, CD64, and/or HLADR. Other examples of macrophage precursor cells include microglia, which may be identified by the surface expression of one or more of CD 14, CD45, CCR5, CXCR4, and CCR3. In a preferred embodiment of the invention, the macrophage precursor cell is a CD14⁺ monocyte.

B lymphocyte precursor cells include, but are not limited to, pro B cells, pre-B cells, and immature B cells. Phenotypic surface markers expressed by various B lymphocyte precursors are known in the art. For example, pro B cells may be identified by the surface expression of one or more of CD34, CD45 (B220), CD 19, CD43 and TdT. Pre B cells may be identified by the expression of one or more of CD9, CD 19, CD45 (B220), CDlO, CD43 and CD24. The cytokine combination or composition comprising the same may be used to enhance the development of antigen presenting cells from antigen presenting precursor cells in vivo, ex vivo or in vitro.

For ex vivo and in vitro applications, a cytokine combination of the invention or a composition comprising the same may be administered to a sample comprising antigen presenting precursor cells. The antigen presenting precursor cells may be derived from a biological sample (e.g. a blood sample). Alternatively, the antigen presenting precursor cells may be in the form of a cell line. If so desired, samples enriched for antigen presenting precursor cells may be obtained by various methods known in the art. Examples of suitable techniques include density gradient separation, fluorescence activated cell sorting (FACS), flow filtration, immunological cell separation techniques such as panning (e.g. antibody panning), complement lysis, resetting, magnetic cell separation techniques, nylon wool separation, and combinations of such methods (see, for example, O'Doherty et ai, "Dendritic cells freshly isolated from human blood express CD4 and mature into typical immunostimulatory dendritic cells after culture in monocyte- conditioned medium ", (1993), J Exp. Med. 178: 1067-76; Young and Steinman, "Dendritic cells stimulate primary human cytolytic lymphocyte responses in the absence ofCD4+ helper T cells ", (1990), J. Exp. Med. 171: 1315-32; Freudenthal and Steinman, "The distinct surface of human blood dendritic cells, as observed after an improved isolation method", (1990), Proc. Natl. Acad. Sci. USA 87: 7698-702). Methods for immuno-selecting antigen presenting cell precursors include, for example, using antibodies to cell surface markers associated with the precursors, such as anti-CD34 and/or anti-CD 14 antibodies coupled to a substrate.

For the purpose of exemplification only, a cytokine combination or composition comprising the same used to enhance the development of an antigen presenting cell from an antigen presenting precursor cell (APPC) in ex vivo and in vitro applications may be administered to the APPC as follows: IFNγ (about 500pg/ml culture - about 1500pg/ml culture, preferably about 800pg/ml culture - about 1200 pg/ml culture); and IL-2 (about 50pg/ml culture - about 200pg/ml culture, preferably about lOOpg/ml culture - about 200 pg/ml culture); and at least one CC chemokine (e.g. CCL2, CCL3, CCL4, and/or CCL8) (about lOOOpg/ml culture - about 3000 pg/ml culture, preferably about 1500pg/ml culture - about 2500 pg/ml culture); and optionally CXCLlO (IP-10) (about 2500pg/ml culture - about 4500pg/ml culture, preferably about 3000pg/ml culture - about 4000pg/ml culture); and optionally IL-6 (about 50pg/ml culture - about 300pg/ml culture, preferably about 100pg/ml culture - about 185pg/ml culture). It will be understood that "culture" in this context includes biological materials comprising APPC, with or without the addition of additional reagents (e.g. culture reagents such as culture media and the like).

For in vivo applications the cytokine combination or composition may be administered to a subject, for example, by standard parenteral routes, such as subcutaneously, intravenously, or intramuscularly. Administration can be performed daily as a single dose, multiple doses, or in continuous dose form. Alternatively, some or all of the components of the cytokine combination may be administered by delivery of genes encoding the component/s. Alternative methods for delivery of the cytokine combination in vivo include, but are not limited to, localized injection at a specific site, administration by implantable pump or continuous infusion, liposomes, gene therapy, and therapeutic vaccines.

For the purpose of exemplification only, a cytokine combination or composition comprising the same used to enhance the development of an antigen presenting cell from an antigen presenting precursor cell (APPC) for an in vitro application may be administered to a subject comprising the APPC such that individual components within the combination are administered in one or more of the dosages as set out below in the section entitled "Dosages and routes of administration".

In accordance with the methods of the invention, contacting antigen presenting precursor cells with the cytokine combination of the invention or a composition comprising the same provides a means of enhancing the differentiation of antigen presenting cells. Accordingly, the invention provides a means of enhancing the number of antigen presenting cells. In cases where antigen presenting cell function is compromised or impaired (e.g. when a subject is suffering from a disease), enhancing the differentiation of antigen presenting cells in accordance with the methods of the invention may also provide a means of improving APC function. It will be understood that individual cytokine components of the cytokine combination provided herein may be administered to antigen presenting precursor cells simultaneously or sequentially.

Maturation of antigen presenting cells can be monitored using methods known in the art. For example, cell surface markers characteristic of mature antigen presenting cells may be detected using techniques such as flow cytometry, immunohistochemistry, and the like.

In one embodiment of the invention, the differentiated antigen presenting cell expresses one or more of CD83, CDl Ic, CD 14, CD86 and CD40. Differentiated dendritic cells may be identified by the detection of cell surface markers including CDl Ic, CD19, CD83, CD86, and HLA-DR (MHC II). Mature dendritic cells may also express major histocompatability protein I (MHC I). Mature dendritic cells may also be negative for characteristic cell surface markers such as CD3, CD4, CD8, CD14, CD16 and CD20. Cell surface markers characteristic of differentiated macrophages include, but are not limited to, CCR5 (2F9), CD 16, HLA-DR (MHC II), CD32, Mac-1, Mac-2, CXCLl 3 and ICAM-I. Cell surface markers characteristic of differentiated B lymphocytes include, but are not limited to, CDl 9, CD20, CD21, CD22, CD23, CD40, and HLA-DR (MHC II).

The maturation of antigen presenting cells may be monitored by assessing cytokine production (e.g. by ELISA, another immune assay, or by use of an oligonucleotide array), or assessing the expression of genes associated with antigen presentation and antigen uptake (e.g. HLA-DQAl, HLA-DOA, HLA-DPAl, HLA-DMA, HLA-DQBl and TAP- 2). Maturation of B lymphocytes may be monitored by detecting the secretion of specific immunoglobulin subtypes. Mature DCs generally lose the ability to uptake antigen. Accordingly, mature DCs may be identified using uptake assays known to those of ordinary skill in the art. Suitable techniques include testing for the uptake of antigens by macropinocytosis (for example lucifer-yellow) and/or receptor-mediated endocytosis (for example FITC-labeled dextran), the presentation of soluble antigens to autologous T cells (for example TT peptide) or the ability to stimulate a MLR. Also provided herein are antigen presenting cells produced by the methods of the invention.

Antigen-specific cells

The invention provides methods for producing antigen-specific lymphocytes. In general, the methods involve the production of an antigen presenting cell in accordance with the methods of the invention (see section above entitled "Antigen presenting cells").

The antigen presenting cell is then contacted with a substance or material comprising an antigen, thereby producing a loaded antigen presenting cell. The loaded antigen presenting cell is then brought into contact with a lymphocyte, thereby generating an antigen-specific lymphocyte.

Suitable antigens for use in the methods of the invention include any antigen for which lymphocyte activation is desired. Such antigens may include, for example, viral particles or preparations comprising viral antigens, tumour specific or tumour-associated antigens (e.g. whole tumour or cancer cells, tumour cell lysates, tumour cell membrane preparations, isolated or partially isolated antigens from tumours, fusion proteins, liposomes, and the like), bacterial cells, or preparations comprising bacterial antigens, and any other antigen or fragment of an antigen (e.g. a peptide or polypeptide antigen). In certain embodiments, the antigen is a human immunodeficiency virus antigen or a derivative thereof. The HIV antigen or derivative thereof may be derived from HIV-I or HIV-2. Examples of suitable HIV antigens and derivatives include, but are not limited to, one or more antigens encoded by the HIV viral genes gag, pro, pol and env. It will be understood that an HIV antigen or a derivative thereof as used herein is a reference to any component of HIV or derivative thereof. In this context, a derivative includes fragments, parts, portions, equivalents, analogues, mutants, homologues and mimetics from natural, synthetic or recombinant sources including fusion proteins. Derivatives may be derived from insertion, deletion or substitution of amino acids. Derivatives also include fragments having particular epitopes or parts of the entire protein fused to peptides, polypeptides or other proteinaceous or non-proteinaceous molecules.

Antigens for use in the method may also be derived from a sample of a subject. For example, the antigen/s may be present in a sample obtained by biopsy or by surgical resection. Alternatively, a membrane preparation of cells from a subject (e.g. a cancer patient) or an established cell line may also be used as a source of antigen. Alternatively, the antigen can be encoded by nucleic acids (e.g. RNA or DNA) that may be purified or amplified from a tumour cell.

In one embodiment of the invention, a tumour cell lysate obtained at biopsy is used as a source of the antigen. For example, a sample of a cancer patient's own tumour may be used directly as a source of antigen, or to provide a cell lysate or nucleic acids for antigen presentation. Alternatively, a membrane preparation of tumour cells from a cancer patient may be used. The tumour cell may be, for example, lung, melanoma, prostatic, colon, breast, ovarian, brain, or any other type of tumour cell. A lysate and membrane preparation can be prepared from isolated tumour cells using methods known in the art. In another embodiment of the invention, one or more cells (e.g. PBMCs) infected with a virus (e.g. human immunodeficiency virus) is obtained from a subject and used directly as a source of antigen, or to provide a cell lysate or nucleic acids for antigen presentation. Alternatively, a membrane preparation of infected cells from the subject may be used as a source of antigen.

In another embodiment of the invention, one or more cells (e.g. PBMCs) infected with a virus (e.g. human immunodeficiency virus) is obtained from a subject and used directly as a source of antigen by stimulating the infected cells in vitro with externally sourced antigen (e.g. commercially available HIV antigens). Additionally or alternatively, the antigen-stimulated cell may be used to provide a cell lysate or nucleic acids for antigen presentation. Additionally or alternatively, a membrane preparation of the antigen-stimulated cell may be used as a source of antigen.

The antigen may be expressed or produced recombinantly. For example, a recombinant antigen may be expressed on the surface of a host cell (e.g. bacteria, yeast, insect, vertebrate or mammalian cells). Alternatively, a recombinant antigen may be present in a lysate, or may instead be purified from the lysate.

The methods of the invention may be used to generate antigen specific lymphocytes cells in vivo, ex vivo or in vitro.

For example, antigen specific lymphocytes may be generated in vitro or ex vivo by culturing an antigen presenting precursor cell with a cytokine combination of the invention (or a composition comprising the same) under suitable conditions in the presence of a predetermined antigen. Alternatively, the antigen presenting precursor cell may initially be cultured under suitable conditions with a cytokine combination of the invention (or a composition comprising the same) in the absence of the predetermined antigen, which may then be added to the culture subsequently.

Methods for loading macrophages with antigens are known in the art, and are described for example in Hilburger, and Zwilling, "Antigen presentation by macrophages from bacille Calmette-Guerin (BCG)-resistant and -susceptible mice", (1994), Clin. Exp. Immunol. 96: 225-229. Methods for contacting dendritic cells with antigen are also known in the art, and described for example in Steel and Nutman, "Helminth antigens selectively differentiate unsensitized CD45RA+ CD4+ human T cells in vitro ", (1998), J. Immunol. 160: 351-60; Tao et al, "Induction of IL-4-producing CD4+ T cells by antigenic peptides altered for TCR binding", (1997), J. Immunol. 158: 4237-44; and Dozmorov and Miller, "In vitro production of antigen-specific T cells from unprimed mice: role of dexamethasone and anti-IL-10 antibodies ", (1997), Cell Immunol. 178: 187-96. General methods for the culture of antigen presenting cells are described in Coligan et al. (Eds) "Current protocols in Immunology", (1991-2008), John Wiley and Sons, Inc.; and Bonifacino et al. (Eds) "Current protocols in Cell Biology", (2007), John Wiley and Sons, Inc.

A mature, loaded antigen presenting cell may be mixed and incubated with any type of lymphocyte. T lymphocytes (e.g. naive T lymphocytes, cytotoxic T lymphocytes, CD4+ T lymphocytes, helper CD4+ T lymphocytes, CD8+ T lymphocytes, memory T lymphocytes) and/or B lymphocytes may be obtained from various lymphoid tissues for use as responder cells. Examples of suitable tissues include, but are not limited to, lymph nodes and spleen. Additionally or alternatively, lymphocytes may be obtained from the peripheral blood. Contacting the lymphocytes with the loaded antigen presenting cell (e.g. loaded macrophage, loaded dendritic cell or loaded B lymphocyte) leads to the stimulation of lymphocytes specific for the antigen which may mature into antigen- specific lymphocytes which may then undergo clonal expansion. Accordingly, the generation of antigen specific lymphocytes provides a means of enhancing an immune response.

Also provided herein are antigen-specific lymphocytes produced by the methods of the invention.

Methods of treatment

The invention provides methods for preventing or treating diseases and conditions by administering to a subject a cytokine combination of the invention or a composition comprising the same. It will be understood that administering a cytokine combination of the invention to a subject does not necessarily require that each cytokine component of the combination be administered simultaneously.

Accordingly, administering a cytokine combination of the invention to a subject as contemplated herein includes administering the components of the combination simultaneously or, administering one or more component(s) of the combination separately to other component(s) of the combination.

Preferably, different components of a given combination of the invention when administered to a subject separately are administered in an order and/or over a time period that does not substantially compromise the therapeutic benefits of the combination. It will also be recognised that different components of a given combination of the invention when administered to a subject separately may be administered by different routes (e.g. by parenteral (e.g., intravenous, intraspinal, subcutaneous or intramuscular), topical, oral, and intranasal administration). In some embodiments, the cytokine combination comprises IFNγ and IL-2.

In other embodiments, the cytokine combination comprises IFNγ and at least one CC chemokine. The CC chemokine may be one or more of CCL2 (JE/MCP-1),CCL3 (MIP-I alpha), CCL4 (MIP-I beta) and CCL8 (MCP-2). The cytokine combination may comprise IFNγ, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), and CCL8 (MCP-2). The cytokine combination may comprise IFNγ, CCL2 (JE/MCP-1), CCL4 (MIP-I beta), and CCL8 (MCP-2). The cytokine combination may comprise IFNγ, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL4 (MIP-I beta), and CCL8 (MCP-2). The cytokine combination may comprise IFNγ, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), and CCL4 (MIP-I beta). In other embodiments, the cytokine combination comprises IL-2 and at least one

CC chemokine. The CC chemokine may be one or more of CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL4 (MIP-I beta) and CCL8 (MCP-2). The cytokine combination may comprise IL-2, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL4 (MIP-I beta), and CCL8 (MCP-2). The cytokine combination may comprise IL-2, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), and CCL8 (MCP-2). The cytokine combination may comprise IL-2, CCL2 (JE/MCP-1), CCL4 (MIP-I beta), and CCL8 (MCP-2). The cytokine combination may comprise IL-2, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL4 (MIP-I beta), and CCL8 (MCP-2). The cytokine combination may comprise IL-2, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha) and CCL4 (MIP-I beta). In preferred embodiments, the cytokine combination comprises IFNγ, IL-2 and at least one CC chemokine. The CC chemokine may be one or more of CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL4 (MIP-I beta) and CCL8 (MCP-2).

In particularly preferred embodiments, the cytokine combination comprises IFNγ, IL-2, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), and CCL8 (MCP-2). In other particularly preferred embodiments, the cytokine combination comprises

IFNγ, IL-2, CCL2 (JE/MCP-1), CCL4 (MIP-I beta), and CCL8 (MCP-2).

In still other particularly preferred embodiments, the cytokine combination comprises IFNγ, IL-2, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL4 (MIP-I beta), and CCL8 (MCP-2). In other particularly preferred embodiments, the cytokine combination comprises IFNγ, IL-2, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha) and CCL4 (MIP-I beta).

The cytokine combination may comprise a CXC chemokine. A CXC chemokine as provided herein is one in which the two N-terminal cysteines are separated by a single amino acid. Isoforms and variants of CC chemokines having similar or identical biological activity to conventional CC chemokines are also contemplated. CC chemokines of the cytokine combination may be derived from any source, including humans and other mammalian species (e.g. mice, rats, primates, pigs, horses, sheep, cows). Examples of suitable CC chemokines include, but are not limited to, CXCLl, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCLlO, CXCLI l, CXCLl 2,

CXCLl 3, CXCL14, CXCLl 5, CXCLl 6 and CXCLl 7. Preferably, the CXC chemokine is

CXCLlO (IP-IO).

Accordingly, in some embodiments the cytokine combination comprises IFNγ, IL- 2, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL8 (MCP-2), and CXCLlO. In other embodiments the cytokine combination comprises IFNγ, IL-2, CCL2

(JE/MCP-1), CCL4 (MIP-I beta), CCL8 (MCP-2) and CXCLlO (IP-10).

In other embodiments the cytokine combination comprises IFNγ, IL-2, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL4 (MIP-I beta), CCL8 (MCP-2), and CXCLlO (IP-10). In other embodiments the cytokine combination comprises IFNγ, IL-2, CCL2

(JE/MCP-1), CCL3 (MIP-I alpha), CCL4 (MIP-I beta), and CXCLlO (IP-10).

In some embodiments, CXCLlO production is induced by the presence of another component of the cytokine combination, such as IFNγ.

The cytokine combination may comprise interleukin-6 (IL-6). Accordingly, in some embodiments the cytokine combination comprises IFNγ, IL-

2, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL8 (MCP-2), and IL-6. The combination may further comprise CXCLlO.

In other embodiments the cytokine combination comprises IFNγ, IL-2, CCL2 (JE/MCP-1), CCL4 (MIP-I beta), CCL8 (MCP-2) and IL-6. The combination may further comprise CXCLl 0 (IP- 10).

In other embodiments the cytokine combination comprises IFNγ, IL-2, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL4 (MIP-I beta), CCL8 (MCP-2), and IL-6. The combination may further comprise CXCLlO (IP-10). In still other embodiments the cytokine combination comprises IFNγ, IL-2, CCL2 (MCP-I), CCL3 (MIPl alpha), CCL4 (MlPlbeta) and IL-6. The combination may further comprise CXCLlO (IP-IO).

For the purpose of exemplification only, a cytokine combination or composition comprising the same used in the treatment methods of the invention may be administered to a subject such that individual components within the combination are administered in one or more of the dosages as set out below in the section entitled "Dosages and routes of administration".

In one aspect, the disease or condition is one which is characterised by reduced numbers of antigen presenting cells, impaired antigen presenting cell activity, or both. The disease or condition may be any disease or condition provided that it is characterised by at least one of the above features.

In a preferred embodiment of the invention, the disease or condition is cancer. Non- limiting examples of cancer which may be treated or prevented include carcinoma, sarcoma, melanoma, glioma, glioblastoma, brain cancer, lung cancer, thyroid follicular cancer, pancreatic cancer, breast cancer, anaplastic astrocytoma, bladder cancer, myelodysplasia, prostate cancer, testicular cancer, colon and rectal cancer, lymphoma, leukemia, or mycosis fungoides.

In another preferred embodiment of the invention, the disease or condition is HIV infection or AIDS. The HIV infection may be HIV-I infection, HIV-2 infection, infection by recombinant HIV strains, infection by CCR5-tropic HIV strains, infection by CXCR4- tropic strains, infection by CCR5/CXCR4 dual tropic strains or infection by drug-resistant

HIV strains. The HIV infection may be characterised by a high viral load (greater than

100,000 viral RNA copies/ml blood), medium viral load (between 10,000 and 100,000 viral RNA copies/ml blood), low viral load (between 50 and 10,000 viral RNA copies/ml blood), or undetectable viral load (less than 50 viral RNA copies/ml blood).

Administration of a cytokine combination of the invention or a composition comprising the same is demonstrated herein to significantly suppress HIV replication. Accordingly, there is provided a method for suppressing human immunodeficiency virus replication in a subject, the method comprising administering to a subject a therapeutically effective amount of a cytokine combination comprising at least one CC chemokine, and/or interferon-gamma and/or interleukin-2, or a composition comprising the same.

In some embodiments, the cytokine combination comprises IFNγ and IL-2. In other embodiments, the cytokine combination comprises IFNγ and at least one CC chemokine. The CC chemokine may be one or more of CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL4 (MIP-I beta) and CCL8 (MCP-2). The cytokine combination may comprise IFNγ, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), and CCL8 (MCP-2). The cytokine combination may comprise IFNγ, CCL2 (JE/MCP-1), CCL4 (MIP-I beta), and CCL8 (MCP-2). The cytokine combination may comprise IFNγ, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL4 (MIP-I beta), and CCL8 (MCP-2). The cytokine combination may comprise IFNγ, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha) and CCL4 (MP- 1 beta). In other embodiments, the cytokine combination comprises IL-2 and at least one

IFNγ, IL-2, CCL2 (JE/MCP-1), CCL4 (MIP-I beta), and CCL8 (MCP-2).

In still other particularly preferred embodiments, the cytokine combination comprises IFNγ, IL-2, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL4 (MIP-I beta), and CCL8 (MCP-2). In other particularly preferred embodiments, the cytokine combination comprises

IFNγ, IL-2, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha) and CCL4 (MIP-I beta).

The cytokine combination may comprise a CXC chemokine. A CXC chemokine as provided herein is one in which the two N-terminal cysteines are separated by a single amino acid. Isoforms and variants of CC chemokines having similar or identical biological activity to conventional CC chemokines are also contemplated. CC chemokines of the cytokine combination may be derived from any source, including humans and other mammalian species (e.g. mice, rats, primates, pigs, horses, sheep, cows). Examples of suitable CC chemokines include, but are not limited to, CXCLl, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCLlO, CXCLI l, CXCLl 2, CXCL13, CXCL14, CXCL15, CXCL16 and CXCL17. Preferably, the CXC chemokine is CXCLlO (IP-IO).

Accordingly, in some embodiments the cytokine combination of the invention comprises IFNγ, IL-2, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL8 (MCP-2), and CXCLlO.

In other embodiments the cytokine combination comprises IFNγ, IL-2, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL4 (MIP-I beta), and CXCLlO (IP-10).

In some embodiments, CXCLlO production is induced by the presence of another component of the cytokine combination, such as IFNγ. The cytokine combination may comprise interleukin-6 (IL-6).

In other embodiments the cytokine combination comprises IFNγ, IL-2, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL4 (MIP-I beta), CCL8 (MCP-2), and IL-6. The combination may further comprise CXCLlO (IP-10). In still other embodiments the cytokine combination comprises IFNγ, IL-2, CCL2

(MCP-I), CCL3 (MIPl alpha), CCL4 (MlPlbeta) and IL-6. The combination may further comprise CXCLlO (IP-10).

The HIV infection may be HIV-I infection, HIV-2 infection, infection by recombinant HIV strains, infection by CCR5-tropic HIV strains, infection by CXCR4- tropic strains, infection by CCR5/CXCR4 dual tropic strains or infection by drug-resistant HIV strains.

The suppression of HIV replication can be determined using standard techniques known in the art. For example, the viral load of a subject may be measured using commercially available RT-PCR based kits (e.g. the AMPLICOR™ HIV-I Monitor® Test vl .5 kit, the COBAS AMPLICOR™ HIV-I Monitor^® Test vl.5 kit, the TaqMan HIV-I Monitor Test vl.5 kit, and the AMPLICOR™ HIV-I DNA Test vl.5 kit, all available from Roche Pharmaceuticals).

The methods for treating or preventing HIV infection and AIDS provided herein may be used as an adjunct to other anti-HIV therapies. For example, the methods may be used in conjunction with the administration of one or more anti-HIV drugs. Non-limiting examples of anti-HIV drugs include protease inhibitors such as Amprenavir (APV), Atazanavir (ATV), Indinavir (IDV), Ritonavir (RTV), Lopinavir/Ritonavir (LPV/RTV), Nelfmavir (NFV) and Saquinavir (SQV); non-nucleoside reverse transcriptase inhibitors such as Delavirdine (DLV), Efavirenz (RFV) and Nevirapine (NVP); nucleoside/nucleotide analogue reverse transcriptase inhibitors such as Abacavir (ABC), Didanosine (ddl), Emtricitabine (FTC), Lamivudine (3TC), Stavudine (d4T), Tenofovir (TDF), Zalcitabine (ddC) and Zidovudine (AZT); integrase inhibitors such as Raltegravir, Elvitegravir; and fusion inhibitors such as Enfuvirtide (T20). In preferred embodiments of the invention, the methods for treating or preventing

HIV infection and AIDS provided herein are used in tandem with a combination of the anti-HIV drugs. In a particularly preferred embodiment, the combination of anti-HIV drugs is administered as a highly active antiretroviral therapy (HAART) regimen.

In accordance with the methods of prevention/treatment provided herein, the cytokine combination or a composition comprising the same may be used as a therapeutic vaccine (i.e. one that is given to a subject who already has a disease or condition, wherein the therapeutic vaccine can elicit an immune response or boost the individual's existing immune response to the disease or condition). This includes an immune response to a diseased cell, such as a cancer cell or a cell infected by a pathogen such as a virus, bacterium, protozoa, or fungus.

In accordance with the methods of prevention/treatment provided herein, the components of the cytokine combination may be administered to a subject in combination or sequentially. The cytokine combination or composition may be administered to a subject, for example, by parenteral (e.g., intravenous, intraspinal, subcutaneous or intramuscular), topical, oral, and intranasal administration. Administration may be systemic, regional or local. Administration may be chronic or acute. Administration may be performed daily as a single dose, multiple dose, or in continuous dose form. Alternative methods for delivery of the cytokine combination in vivo include, but are not limited to, localized injection at a specific site, administration by implantable pump or continuous infusion, liposomes, gene therapy, and therapeutic vaccines.

Alternatively, the some or all of the components of the cytokine combination may be administered by delivery of genes encoding the component/s. In certain embodiments, polynucleotides encoding a component of the cytokine combination may be administered to a subject. Typically, the encoding polynucleotide is operably linked to a promoter such that the appropriate polypeptide sequence is produced following administration of the polynucleotide to the subject. The polynucleotide may be administered to the subject in a vector. The vector may be a plasmid vector, a viral vector, or any other suitable vehicle adapted for the insertion of foreign sequences, their introduction into eukaryotic cells and the expression of the introduced sequences. The nucleic acid construct to be administered may comprise naked DNA or may be in the form of a composition, together with one or more pharmaceutically acceptable carriers.

Typically the vector is a eukaryotic expression vector and may include expression control and processing sequences such as a promoter, an enhancer, ribosome binding sites, polyadenylation signals and transcription termination sequences. The expression of a gene encoding a component of the cytokine combination may be increased in cells of a subject using various methods of gene delivery known in the art. For example, an expression vector comprising a nucleic acid sequence encoding IL-2, IFNγ, a CC chemokine or a CXC chemokine operably linked to an expression control sequence such as an inducible promoter may be administered to a subject to increase the production of the protein in cells of the subject.

Alternatively, viral vectors (for example retroviral and adenoviral vectors) containing a nucleic acid sequence encoding IL-2, IFNγ, a CC chemokine, a CXC chemokine or IL-6 may be administered to a subject in order to elicit the production of said protein in target cells and tissues (gene therapy). Non-limiting examples of suitable vectors for use in gene therapy include retroviral vectors, adenoviruses, adeno-associated viral (AAV) vectors and lentiviruses. The viral vector selected should be capable of infecting the target cell and the transferred gene (e.g. genes encoding IL-2, IFNγ, CC chemokines such as CCL2, CCL3, CCL4 and CCL8, CXC chemokines such as CXCLlO, or IL-6) and also be capable of being expressed and persisting in the cell for an extended period of time. Other virus vectors that may be used for gene transfer into cells include retroviruses such as Moloney murine leukemia virus (MoMuLV), papovaviruses such as JC, SV40, polyoma, adenoviruses, Epstein-Barr Virus (EBV), papilloma viruses such as bovine papilloma virus type I (BPV), vaccinia and poliovirus.

Alternatively, the delivery of a gene encoding IL-2, IFNγ, a CC chemokine, a CXC chemokine or IL-6 may also be achieved by extracting cells from a subject, administering a vector containing the gene of interest, and then re-introducing the cells to the subject.

Dosages and routes of administration

Cytokine combinations of the invention and compositions comprising the same may be administered by standard routes. In general, the compositions may be administered parenterally (e.g. intravenous, intraspinal, subcutaneous or intramuscular). More preferably the compositions may be administered topically, orally, or intranasally. Administration may be systemic, regional or local. The particular route of administration to be used at any given time will depend on a number of factors, including the nature of the condition to be treated, the severity and extent of the condition, the required dosage of the particular composition to be delivered and the potential side-effects of the composition. Cytokine combinations of the invention and compositions comprising the same may be administered either therapeutically or preventively. In a therapeutic application, the administration is to a patient already suffering from a disease or condition, in an amount sufficient to cure or at least partially arrest the disease or condition and its complications. The cytokine combination or composition should provide a quantity of the agent sufficient to effectively treat the patient.

The therapeutically effective dose level for any particular patient will depend upon a variety of factors including: the disorder being treated and the severity of the disorder; activity of the compound or agent employed; the composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of sequestration of the agent or compound; the duration of the treatment; drugs used in combination or coincidental with the treatment, together with other related factors well known in medicine. A person of ordinary skill in the art would be able, by routine experimentation, to determine an effective, non-toxic amount of a cytokine combination or composition of the invention which would be required to treat applicable diseases or conditions.

Generally, an effective dosage of a given cytokine component present in a combination of the invention (or composition comprising the same) is expected to be in the range of about O.OOOlmg to about lOOOmg per kg body weight per 24 hours; typically, about O.OOlmg to about 750mg per kg body weight per 24 hours; about O.Olmg to about 500mg per kg body weight per 24 hours; about O.lmg to about 500mg per kg body weight per 24 hours; about O.lmg to about 250mg per kg body weight per 24 hours; about l.Omg to about 250mg per kg body weight per 24 hours. More typically, an effective dose range is expected to be in the range about l.Omg to about 200mg per kg body weight per 24 hours; about l.Omg to about lOOmg per kg body weight per 24 hours; about l.Omg to about 50mg per kg body weight per 24 hours; about l.Omg to about 25mg per kg body weight per 24 hours; about 5.0mg to about 50mg per kg body weight per 24 hours; about 5.0mg to about 20mg per kg body weight per 24 hours; about 5.0mg to about 15mg per kg body weight per 24 hours.

Alternatively, an effective dosage of a given cytokine component present in a combination of the invention (or composition comprising the same) may be up to about 500mg/m². Generally, an effective dosage of a given cytokine component present in a combination of the invention is expected to be in the range of about 25 to about 500mg/m², preferably about 25 to about 350mg/m², more preferably about 25 to about 300mg/m , still more preferably about 25 to about 250mg/m , even more preferably about 50 to about 250mg/m², and still even more preferably about 75 to about 150mg/m².

Typically, in therapeutic applications, the treatment would be for the duration of the disease state or condition. Further, it will be apparent to one of ordinary skill in the art that the optimal quantity and spacing of individual dosages will be determined by the nature and extent of the disease state or condition being treated, the form, route and site of administration, and the nature of the particular individual being treated. Also, such optimum conditions can be determined by conventional techniques. It will also be apparent to one of ordinary skill in the art that the optimal course of treatment, such as, the number of doses of the composition given per day for a defined number of days, can be ascertained by those skilled in the art using conventional course of treatment determination tests. Medicaments

The invention also provides the use of a cytokine combination of at least one CC chemokine, interferon-gamma, interleukin-2, optionally a CXC chemokine, and optionally IL-6, for the manufacture of a medicament for the treatment or prevention of a disease or condition. The disease or condition is characterised by reduced numbers of antigen presenting cells, impaired antigen presenting cell activity, or both. For example, the disease or condition may be human immunodeficiency virus, acquired immune deficiency syndrome, or cancer.

In some embodiments, the cytokine combination comprises IFNγ and IL-2. In other embodiments, the cytokine combination comprises IFNγ and at least one

CC chemokine. The CC chemokine may be one or more of CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL4 (MIP-I beta) and CCL8 (MCP-2). The cytokine combination may comprise IFNγ, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), and CCL8 (MCP-2). The cytokine combination may comprise IFNγ, CCL2 (JE/MCP-1), CCL4 (MIP-I beta), and CCL8 (MCP-2). The cytokine combination may comprise IFNγ, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL4 (MIP-I beta), and CCL8 (MCP-2). The cytokine combination may comprise IFNγ, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), and CCL4 (MIP-I beta).

In other embodiments, the cytokine combination comprises IL-2 and at least one CC chemokine. The CC chemokine may be one or more of CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL4 (MIP-I beta) and CCL8 (MCP-2). The cytokine combination may comprise IL-2, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL4 (MIP-I beta), and CCL8 (MCP-2). The cytokine combination may comprise IL-2, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), and CCL8 (MCP-2). The cytokine combination may comprise IL-2, CCL2 (JE/MCP-1), CCL4 (MIP-I beta), and CCL8 (MCP-2). The cytokine combination may comprise IL-2, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL4 (MIP-I beta), and CCL8 (MCP-2). The cytokine combination may comprise IL-2, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha) and CCL4 (MIP-I beta).

In preferred embodiments of, the cytokine combination comprises IFNγ, IL-2 and at least one CC chemokine. The CC chemokine may be one or more of CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL4 (MIP-I beta) and CCL8 (MCP-2).

In a particularly preferred embodiments, the cytokine combination comprises IFNγ, IL-2, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), and CCL8 (MCP-2). In other particularly preferred embodiments, the cytokine combination comprises IFNγ, IL-2, CCL2 (JE/MCP-1), CCL4 (MIP-I beta), and CCL8 (MCP-2).

In still other particularly preferred embodiments, the cytokine combination comprises IFNγ, IL-2, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL4 (MIP-I beta), and CCL8 (MCP-2).

In other particularly preferred embodiments, the cytokine combination comprises IFNγ, IL-2, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), and CCL4 (MIP-I beta).

The cytokine combination may comprise a CXC chemokine. A CXC chemokine as provided herein is one in which the two N-terminal cysteines are separated by a single amino acid. Isoforms and variants of CC chemokines having similar or identical biological activity to conventional CC chemokines are also contemplated. CC chemokines of the cytokine combination may be derived from any source, including humans and other mammalian species (e.g. mice, rats, primates, pigs, horses, sheep, cows). Examples of suitable CC chemokines include, but are not limited to, CXCLl, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCLlO, CXCLI l, CXCL12,

CXCL13, CXCL14, CXCL15, CXCL16 and CXCL17. Preferably, the CXC chemokine is

CXCLlO (IP-IO).

Accordingly, in some embodiments the cytokine combination comprises IFNγ, IL- 2, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL8 (MCP-2), and IL-6. The combination may further comprise CXCLlO- In other embodiments the cytokine combination comprises IFNγ, IL-2, CCL2 (JE/MCP-1), CCL4 (MIP-I beta), CCL8 (MCP-2) and IL-6. The combination may further comprise CXCLlO (IP-IO). hi other embodiments the cytokine combination comprises IFNγ, IL-2, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL4 (MIP-I beta), CCL8 (MCP-2), and IL-6. The combination may further comprise CXCLlO (IP-IO). hi still other embodiments the cytokine combination comprises IFNγ, IL-2, CCL2 (MCP-I), CCL3 (MIPl alpha), CCL4 (MIPl beta) and IL-6. The combination may further comprise CXCLlO (IP-10). It will be understood that no particular limitation exists regarding the particular proportions of each cytokine component used in the preparation of a medicament as described herein.

For the purpose of exemplification only, the cytokine components may be formulated in the medicaments to facilitate the dosages set out below in the section entitled "Dosages and routes of administration".

Kits

The invention provides kits for producing an antigen presenting cell from an antigen presenting precursor cell. In some embodiments, the kit comprises IFNγ and IL-2.

In other embodiments, the cytokine combination comprises IFNγ and at least one CC chemokine. The CC chemokine may be one or more of CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL4 (MIP-I beta) and CCL8 (MCP-2). The cytokine combination may comprise IFNγ, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), and CCL8 (MCP-2). The cytokine combination may comprise IFNγ, CCL2 (JE/MCP-1), CCL4 (MIP-I beta), and CCL8 (MCP-2). The cytokine combination may comprise IFNγ, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL4 (MIP-I beta), and CCL8 (MCP-2). The cytokine combination may comprise IFNγ, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), and CCL4 (MIP-I beta). In other embodiments, the cytokine combination comprises IL-2 and at least one

CC chemokine. The CC chemokine may be one or more of CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL4 (MIP-I beta) and CCL8 (MCP-2). The cytokine combination may comprise IL-2, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL4 (MIP-I beta), and CCL8 (MCP-2). The cytokine combination may comprise IL-2, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), and CCL8 (MCP-2). The cytokine combination may comprise IL-2, CCL2 (JE/MCP-1), CCL4 (MIP-I beta), and CCL8 (MCP-2). The cytokine combination may comprise IL-2, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL4 (MIP-I beta), and CCL8 (MCP-2). The cytokine combination may comprise IL-2, CCL2 (JE/MCP-1), CCL3 (MDP-I alpha), and CCL4 (MIP-I beta).

In other particularly preferred embodiments, the cytokine combination comprises IFNγ, IL-2, CCL2 (JE/MCP-1), CCL4 (MIP-I beta), and CCL8 (MCP-2).

CXCL13, CXCL14, CXCL15, CXCL16 and CXCL17. Preferably, the CXC chemokine is

CXCLlO (IP-IO).

In other embodiments the cytokine combination comprises IFNγ, IL-2, CCL2 (JE/MCP-1), CCL4 (MIP-I beta), CCL8 (MCP-2) and CXCLlO (IP-10). In other embodiments the cytokine combination comprises IFNγ, IL-2, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL4 (MIP-I beta), CCL8 (MCP-2), and CXCLlO (IP-IO).

In other embodiments the cytokine combination comprises IFNγ, IL-2, CCL2 (JE/MCP-1), CCL3 (MIP-I alpha), CCL4 (MIP-I beta), and CXCLlO (IP-IO).

The cytokine combination may comprise interleukin-6 (IL-6).

(JE/MCP-1), CCL3 (MIP-I alpha), CCL4 (MIP-I beta), CCL8 (MCP-2), and IL-6. The combination may further comprise CXCLlO (IP-10).

In still other embodiments the cytokine combination comprises IFNγ, IL-2, CCL2 (MCP-I), CCL3 (MIPl alpha), CCL4 (MlPlbeta) and IL-6. The combination may further comprise CXCLlO (IP-10).

It will be understood that no particular limitation exists regarding the particular proportions of each cytokine component included in kits of the invention.

For the purpose of exemplification only, the cytokine components may be included in the kits to facilitate their administration at the dosages set out below in the section entitled "Dosages and routes of administration" .

Components of the kits may be administered by standard routes including, but not limited to parenteral (e.g. intravenous, intraspinal, subcutaneous or intramuscular), topical, oral, and intranasal routes. Administration may be systemic, regional or local. Administration may be chronic or acute. In alternative embodiments, the kits described herein comprise an agent capable of increasing the level of at least one CC chemokine, interferon-gamma, interleukin-2, and/or a CXC chemokine in a subject. For example, polynucleotides encoding one or more of the cytokine components may be included in the kit. Typically, the encoding polynucleotide is operably linked to a promoter such that the appropriate polypeptide sequence is produced following administration of the polynucleotide to the subject. The polynucleotide may be administered to the subject in a vector. The vector may be a plasmid vector, a viral vector, or any other suitable vehicle adapted for the insertion of foreign sequences, their introduction into eukaryotic cells and the expression of the introduced sequences. The nucleic acid construct to be administered may comprise naked DNA or may be in the form of a composition, together with one or more pharmaceutically acceptable carriers.

Typically the vector is a eukaryotic expression vector and may include expression control and processing sequences such as a promoter, an enhancer, ribosome binding sites, polyadenylation signals and transcription termination sequences. Alternatively, viral vectors (for example retroviral and adenoviral vectors) containing a nucleic acid sequence encoding component/s of the cytokine combination may included in the kits of the invention for administration to a subject in order to elicit the production of the protein. Viral vectors may be used to transform cells extracted from a subject which may then be re-introduced to the subject.

Kits according to the invention may also include other components required to conduct the methods of the present invention, such as buffers and/or diluents, means to extract and/or process biological samples (e.g. blood samples), reference samples, labels, and written instructions for using the kit components in the methods of the invention. Kits according to the invention may be combined kits or fragmented kits.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Examples

The invention will now be described with reference to specific examples, which should not be construed as in any way limiting. EXAMPLE 1: Identification of cytokine combination

MATERIALS AND METHODS 1. Patient clinical details

The male subject, presumed infected in December 1988, acquired HIV-I infection most likely through homosexual transmission from his partner, who died of AIDS on January 22, 1993. He was diagnosed seropositive, both by ELISA and Western blot in 1993. The subject has since remained asymptomatic, and plasma viral loads have remained undetectable (<50 copies/ml) since the first sample (1993) was tested. Both CD4+ and CD8+ T lymphocyte counts have remained elevated throughout the study period (around 1000, and > 2000 cells/μl, respectively), and PBMC have remained virus culture-negative. Apart from steroid-associated lymphopenia in 1992, there were no clinical abnormalities or signs of disease progression. Until 2008, the patient has maintained below detectable levels of HIV with high CD4 and CD8 counts (690 CD4/ul blood and 1300 CD8/ul blood) and continues to display antiviral and cellular differentiation activity.

2. Isolation andp24 Antigen Stimulation of PBMCs 50-60 ml of freshly collected blood was centrifuged at 2000rpm for 10 minutes at room temperature in order to isolate the plasma fraction. After removing the plasma, the remaining blood cells were diluted in 1 :1 ratio with sterile PBS to maximize cell separation. PBS mimics human conditions and protects cells against rupture. Peripheral blood mononuclear cells (PBMCs) were separated from blood by density gradient sedimentation overlayed on 15ml of flcoll-paque in 50ml tubes. The tubes were centrifuged for 20 minutes at 4°C for 1800rpm with a break off to avoid interruption of the gradient. Interface containing PBMCs was collected in fresh 50ml tube by plastic pipette and washed twice with sterile PBS at room temperature. The PBMC pellets were resuspended in RPMIl 640 to give a final concentration of 2X10⁶ cells /ml. Subsequently the PBMCs were transferred into a 6-well tissue culture plate, the p24 antigen (2ng/ml) added, and then incubated at 37°C, with 5% CO₂ for 24 hours. After incubation for 24 hours, PBMCs were collected from the culture plate and the cells were washed twice with PBS. All washes were performed by centrifugation at 1200rpm for 10 minutes. These cells were then used for CD 14+ monocyte and CD4+ T cell isolation. 3. Separation of CD14+ monocytes and CD4+ T lymphocytes from the PBMC stimulated fraction

CD 14+ monocytes and CD4+ T cells were isolated from donors PBMCs by positive sorting using anti-CD 14 and anti-CD4 conjugated magnetic microbeads from Miltenyi (Miltenyi Biotech., GMBH, Germany). PBMC was prepared as described under point 2 above and the cells were washed in MACS (magnetic-activated cell sorter) buffer by centrifuging for 10 minutes at 1200rpm. The cell numbers were counted, re-suspended in MACS buffer (80μl per 10⁷ total cells) and required volume of CD14-microbeads were pippeted (20μl per 10⁷ total cells) into 15 ml tubes. The mixture of PBMC and CD 14 microbeads was incubated for 15 minutes at 4-8°C. After incubation, the cells were washed in MACS buffer (l-2ml per 10⁷ cells) by centrifuging for 10 minutes at 300xg and resuspended in 500μl of the same buffer. Before proceeding to magnetic separation, the MS column (Miltenyi Biotech., GMBH, Germany) was pre-wetted, placed in magnetic MACS separator and the cells were allowed to pass through the column. The unlabelled cells were collected, followed by washing the column three times with 500μl of MACS buffer. After removing the column from the separator, ImI of MACS buffer was added to the column and the plunger was used to flush out CD 14+ cell fraction. The above step was repeated for the unlabelled cell fraction to extract CD4+ T cell isolation by using CD4 microbeads. The collected cells were then centrifuged at 1200rpm for 5 minutes at room temperature and stored at -70°C or processed immediately for RNA extraction. Also, an unstimulated fraction was always saved aside as a control to be tested in assays for gene expression.

4. Superarray studies 4.1. RNA isolation and purification

All superarray experiments comprised of unstimulated and stimulated CD4 + T cell and CD 14+ monocyte fractions of the study patient. RNA was extracted from both fractions for gene expression assays. RNA extraction and purification were conducted using RNeasy mini kit and RNase-free DNase set produced by Qiagen (Qiagen, Australia). 5x 10⁶- 1 Ox 10⁶ of cells were mixed with 600μl of buffer RLT (lysis buffer) and 6μl of BME in 1.5ml eppendorf tube by vortexing for 15 seconds. 6Q0μl of 70% ethanol was added to the sample and mixed well by vortexing. The mixture was transferred to an RNeasy mini spin column placed in a 2ml collection tube and centrifuged at lOOOOrpm for 15 seconds. 350μl of RWl buffer (washing buffer) was added into the spin column and centrifuged at lOOOOrpm for 15 seconds to wash the spin column membrane. The DNAse I incubation mix (lOμl of DNAse I stock solution and 70μl of buffer RDD supplied by the kit) was prepared and added directly to the spin column. This was followed by 15 minutes incubation at room temperature and washed once more with 350μl of RWl buffer. After the DNAse digestion step, 500μl of buffer RPE (supplied as a concentrate) was added and centrifuged at lOOOOrpm for 15 seconds. The Rneasy spin column was washed again by passing through 500μl of buffer RPE at lOOOOrpm for 2 minutes, hi order to eliminate any possible carryover of buffer RPE, the RNeasy spin column was placed in a new 2ml collection tube and spun at 15000rpm for 1 minute. RNA was eluted by placing RNeasy spin column in a clean 1.5ml collection tube and adding 30-50μl of RNAse free H₂O and centrifuged at lOOOOrpm for 1 minute. The RNA eluted was kept at -20⁰C or at -80⁰C for long-term storage.

4.2. Quantification of total RNA RNA quality assurance and quantitative analysis was carried out using Agilent

RNA 6000 nano kit and Agilent 2100 bioanalyzer (Agilent Technologies, USA) to check the final concentration, quality and purity of total sample RNA. Before running the chip, the RNA 6000 Nano dye concentrate and a filter gel (supplied by the kit) were allowed to equilibrate to room temperature for 30 minutes. After vortexing the dye for 10 seconds, 0.5μl of the dye was added into a 32.5 μl of filtered gel followed by spinning the tube at 13000g for 10 minutes at room temperature. The Agilent 2100 bioanalyzer was also cleaned by adding 350μl of RNaseZAP and RNase-free H₂O for 1 minute and 10 seconds respectively. The RNA samples were then denatured by placing sample tubes in a heating block for 2 minutes at 65°C. Furthermore, after loading 9μl of gel dye mix, 5μl of RNA 6000 Nano marker, lμl of prepared ladder, and lμl of each sample RNA in the appropriate wells, the chip was vortexed for 1 minute at 2000rpm before running it in the Agilent 2100 bioanalyzer for 20-30 minutes. The output was a scan of mass versus size. The 28S:18S rRNA ratio was calculated by integrating the areas of 18S and 28S rRNA peaks, followed by dividing the area of the 18S rRNA peak into the area of the 28S rRNA peak. As each RNA sample was analyzed, the software generated both a gel-like image and an electrophoretogram. Furthermore, the RNA Integrity Number (RIN) was assigned to estimate the integrity of total RNA. The algorithm assigns a RIN score of 1 to 10 RIN, where by level 10 RNA is completely intact. 4.3. DNase digestion

DNase digestion of RNA samples is required prior to RT-PCR as DNA contamination will be amplified along with cDNA products during PCR analysis. 25ng to 5μg of total RNA was mixed with 2μl of 5x gDNA elimination buffer and the remaining volume was made up to lOμl using RNAse-free H₂O. The contents were mixed gently, incubated at 42°C for 15 minutes and were immediately chilled on ice for at least one minute.

4.4. cDNA synthesis This step involved the conversion of RNA into cDNA according to the reaction mix described in Table 1 below.

Table 1: Mixture of DNAse-treated RNA and RT cocktail for the conversion of RNA into cDNA.

Reverse transcription was performed by adding lOμl of RT cocktail into lOμl of

DNAse-treated RNA with total of 25ng to 5μg of RNA. The mixture was gently mixed, incubated at 42°C for 15 minutes and denaturized at 95°C for 5 minutes. This allowed the

RNA to be degraded and also to inactivate the reverse transcriptase. 9 lμl of ddH₂O was added to each 20μl of cDNA synthesis reaction and finally the mixture was kept at -20°C. 4.5. Amplification ofcDNA by Real-Time PCR cDNA was amplified using real-time PCR using the reaction mix described in Table 2 below.

Table 2. Mixture of diluted first strand cDNA and Super Array 's RT² qPCR Master Mix containing SYBR Green.

After reverse transcription of RNA to cDNA, diluted first strand cDNA were mixed with 2x RT² SYBR Green qPCR Master Mix and ddH₂O to a final volume of 2550 μl

(Table 2). 25μl of this experimental cocktail was added to each well of the 96-well PCR

Array and the amplifications were performed using Stratagene Mx3005p Real-Time RT-

PCR. The thermocycler parameters were as follows: 1 cycle of 95°C for 10 minutes (in order to activate the DNA polymerase), followed by 40 cycles of 95°C for 15 seconds; 60⁰C for 1 minute; and lastly, 1 cycle of 95°C for 1 minute; 55⁰C for 30 seconds; and

95°C for 30 seconds to obtain the dissociation or melting curve. After the data collection, the amplification plots, dissociation curves, and threshold cycles (Ct) values were generated by the Mx3000P software. The results obtained were then analyzed using software provided at Strategene PCR Array Data Analysis web portal http://www.superarray.com/pcrarraydataanalysis.php.

4.6. Gene arrays

Two arrays were used to analyse the expression of genes in CD4+ T lymphocytes and CD 14+ monocytes derived from the HIV positive patients (including the study subject) and HIV negative donors:

For antigen presentation, the study subjects included in the study consisted of the main study subject, HIV (+) patients and healthy HIV (-) control subjects. Firstly, all three groups of study subjects PBMCs were isolated by Ficoll separation (see section 2 above). The PBMCs were then divided into two groups: p24 stimulated and unstimulated. After 24 hours of culturing at 37°C, the supernatant from both groups were collected (see Section 2 above). Furthermore, both CD4+ T cells and CD 14+ monocytes were isolated from both groups of PBMCs by CD4 and CD14-specific microbeads (see Section 3 above). This was followed by RNA extraction and RNA integrity check by Agilent Bioanalyzer (see Section 4.1 above). Finally both CD4+ T cells and CD 14+ monocytes from all three groups of study subjects were subjected to Super Array (SuperArray Biosciences Corp., USA) to study the differential expression of host genes in response to p24 antigen (both in HIV Infection and Host Immune Response Array and Innate and Adaptive Immune Response Array)

4.6. I Human Innate & Adaptive Immune Response Array:

A list of genes analyzed for innate and adaptive immune response in p24 antigen- stimulated vs unstimulated CD4+ T cells and CD 14+ monocytes is provided below. These genes were assessed differentially by comparing between a pair (for instance- stimulated vs unstimulated). The Array consists of four classes of genes: IL-IR / TLR members and related genes, host defense to bacteria, innate immune response, and septic shock.

- IL-IR / TLR Members and Related Genes:

Detection of Pathogens: TLRl, TLR3, TLR4, TLR6, TLR8.

Interleukin-1 Receptors: ILlRl, IL1R2, ILlRAP, IL1RAPL2, IL1RL2.

Other Genes Involved in the IL-IR Pathway: IKBKB, MAPK14, MAPK8.

Inflammatory Response: ILIA, ILlB, ILlFlO, IL1F5, IL1F6, IL1F8, ILlRl, ILlRN, IRAK2, MYD88, NFKBl, TLRl, TLRlO, TLR2, TLR3, TLR4, TLR6, TLR8, TLR9,

TNF, TOLLIP.

Apoptosis: ILIA, ILlB, NFKBl, NFKBIA, TGFBl, TNF.

Cytokines: IFNAl, IFNBl, ILIA, ILlB, ILlFlO, IL1F5, IL1F6, IL1F7, IL1F8, IL1F9,

IL6, TNF. Genes Involved in NFKB Signaling: CHUK, IRAK2, MYD88, TLRl, TLR3, TLR4,

TLR6, TLR8, TRAF6. Host Defense to Bacteria:

Detection of Bacteria: CDlD, PGLYRPl, PGLYRP2, PGLYRP3, TLRl, TLR3, TLR6.

LSP Receptor: CD14, CXCR4, DAF.

Acute-phase Response: CRP, FNl, LBP. Complement Activation: C5, C8A, DAF.

Inflammatory Response: C5, CCL2, CD14, CRP, CYBB, LY96, NFKBl, NOS2A,

S100A12, TLRl, TLR3, TLR6, TLR9.

Cytokines. Chemokines. and their Receptors: C5, CCL2, CXCR4, IFNGRl, IFNGR2,

ILl 2RB2, PPBP. Antibacterial Humoral Response: COLEC12, CYBB, LY96, NFKBl .

Defense Response to Bacteria: CAMP, DEFB4, LALBA, LBP, LTF, LYZ, NOS2A,

PGLYRPl, PGLYRP2, PGLYRP3, PPBP, S100A12, TLR3, TLR6, TLR9.

Other Genes Involved in the Host Defense Against Bacteria: CARD12, DMBTl, IRFl,

NCF4, NFKBIA.

Innate Immune Response:

Innate Immune Response: COLEC12, DMBTl, PGLYRPl, PGLYRP2, PGLYRP3,

SFTPD, TLR8

Other Genes Involved in the Innate Immune Response: CDlD, IFNBl, TLRlO.

Septic Shock:

Apoptosis: ADORA2A, CASPl, CASP4, ILlO, ILlB, NFKBl, PROC, TNF, TNFRSFlA. Cytokines and Growth Factors: ILlO, ILlB, IL6, MIF, TNF.

Inflammatory Response: ADORA2A, CCR3, ILlO, ILlB, ILlRN, MIF, NFKBl, PTAFR, TLR2, TLR4, TNF.

Other Genes Involved in Septic Shock: HMOXl, IRAKI, NFKB2, SERPINAl, SERPINE1, TREM1.

4.6.2 Human HIV Infection and Host Response Array: A list of genes analyzed for human genes specific for HIV infection in p24 antigen- stimulated vs unstimulated CD4+ T cells and CD 14+ monocytes is provided below. These genes were compared in a differential manner (for instance between stimulated and unstimulated cells). HIV Receptors and Natural Ligands: CCL2, CCL4, CCL5, CCR5, CD4, CXCLl 2, CXCR4.

Cellular Cofactors Involved in HIV Infection: Chetnokine Receptors: CCR2, CCR3, CCR4.

G-protein Coupled Receptors: CCR2, CCR3, CCR4.

Protein Kinases: CDK7, CDK9, HCK, PTK2B.

Transcription Factors and Regulators: APEXl, BCLI lB, CCNTl, CDK7, CDK9,

CREBBP, EP300, HMGAl, HTATSFl, NFATCl, RBL2, SMARCBl, TFCP2, TSGlOl, YYl.

Apoptosis Genes: EP300, LTBR, PTK2B.

Cell Cycle Regulators: CCNTl, CDK7, CDK9, EP300, RBL2, SMARCBl.

Genes Regulating Cell Proliferation: CDK7, CDK9, PTK2B.

Inflammatory Response: CCR2, CCR3, CCR4. Antimicrobial Humoral Response: CCR2. YYl.

Other Immune Response Genes: APOBEC3G, CD209, LTBR.

Genes Involved in Cell Adhesion: CCR3, CD209, PTK2B.

Viral Genome Replication: APOBEC3G, CD209, HTATSFl.

Other Cellular Cofactors: APOBEC3F, BANFl, BTRC, CBX5, CD247 (CD3Z), COPS6, ELA2, PPIA, TRIM5, VPS4A, XPOl .

Genes Involved in Innate Immune Response:

Acute-phase Response: SERPINAl.

Antimicrobial Humoral Response: IL12B, ILlB, KLRDl, XCLl. Response to Virus: CCL4, CCL5, CCL8, CXCLl 2, IFNBl, TNF.

Response to Pest. Pathogen or Parasite: CCL2, TNF.

Inflammatory Response: CCL2, CCL4, CCL5, CCL8, CXCL12, ILlO, ILlB, IL8, TNF.

Other Genes Involved in the Immune Response: CD74, CR2, CX3CL1, IFNG, ILl 6, IL2,

MBL2. Natural Killer Cell Activation: IFNBl, ILl 2B, IL2.

Defense Response Against Pathogens: CD69, CX3CL1, IFNAl, IFNBl, IFNG.

Genes Involved in Cell Adhesion: CCL2, CCL4, CCL5, CD44, CX3CL1, CXCLl 2, IL8,

SELL, TNF. Other Genes Involved in the Innate Response: PRDXl, SERPINCl, SLPI, TGFBl, TNFRSFlB.

Immune Evasion: CD4, FCAR, MAP3K5.

Cellular Proteins Induced or Activated by HIV Infection:

Apoptosis: BAD, BAX, BCL2, CASP3, CASP8, CDKNlA, GADD45A, NFKBIA, STATl, TNFSFlO.

Cell Cycle Regulators: BAX, BCL2, CDK9, CDKNlA, GADD45A, IRFl, STATl. Regulators of Cell Proliferation: BCL2, CDK9, CDKNlA, IRF2.

Transcription Factors and Regulators: CDK9, CEBPB, FOS, IRFl, IRF2, NFATCl, NFKBIA, STATl, STAT3.

4.7. Data Analysis Data obtained in each samples were normalized using the average Ct value of all house keeping genes that were not influenced by the experimental conditions. The average Ct value of the house keeping genes was subtracted from the gene of interest to give changes in Ct (dCt). Furthermore, ddCt value for each gene across two PCR Arrays was obtained by subtracting dCt value of control group from those for stimulated or experimental group, and χ^άάCi was calculated. The fold-change in gene expression (differences in ddCt) was then determined as 1Og₂ relative units.

5. Quantitative determination of cytokines using LUMINEX assay in study subject supernatant produced by inducing study patient CD4+ T cells with p24 antigen Beadlyte Human 22-plex Multi-cytokine Detection System (Millipore Inc., St.

Charles, MO, USA) was used for quantitative assessment of cytokines in the culture supernatant of the study patient as per the manufacturer's protocol. The panel of cytokines used in the analysis included IL-Ia, IL-IB, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-IO, IL-12 (p40), IL12 (p70), IL-13, IL-15, GM-CSF, IFNr, TNF-alpha, Eotaxin, MCP-I, RANTES, MIPl -alpha and IP-IO, respectively. The results were converted to pg/ml using the equation coefficient of standards as prescribed by the manufacturer. RESULTS

1. Superarray studies: Differential Expression of Genes Related to HIV Infection and Host Immune Responses in CD4+ T Cells and CD14+ monocytes in response to p24 antigen stimulation

1.1 CD4+ T cells

The expression of genes related to human innate and adaptive immune responses (see Section 4.6.1) and HIV host responses (see Section 4.6.2) were studied in response to p24 antigen stimulation. Gene expression in CD4+ T cells from the study subject and in CD4+ T cells from HIV-I infected patients 68 and 70 were analyzed in response to p24 antigen. In response to p24, CD4+ T cells of the study subject showed upregulation of the IL-2, CCL2, CCL4, CCL8, and IFNgamma cytokine genes (IFN-gamma - 41.2 fold; IL-2 - 32 fold; CCL8 - 30.4 fold; CCL4 - 10.2 fold; CCL2 - 14.7 fold).

1.2 CD 14+ monocytes

A significant number of genes in CD14+ monocytes of the study subject showed changes in expression during differentiation, while fewer changes in gene expression were observed in the control HIV patients (particularly patient 70). Healthy HIV (-) donor CD 14+ monocytes showed few changes in gene expression in response to p24 antigen. The overlapping genes expressed between study subject and patient 68 included ILlRN, CCL2, TNF, CD 14, and TREMl. A major difference was that CD 14+ monocytes from the study patient displayed an 11.8 fold upregulation in the IL-6 gene when stimulated with HIV p24 antigen, a feature that was not observed in the other patients tested. However, it is important to note that TREMl was down-regulated in CD 14+ monocytes of patient 68 compared to CD14+ monocytes of the study subject in response to p24 stimulation, indicating a possible role of TREMl in controlling HIV infection. In addition, CD14 down-regulation was observed in both the study subject and patient 68. 2. Luminex assays: quantitative determination of cytokines using LUMINEX assay in study subject supernatant produced by inducing study patient CD4+ T cells with p24 antigen

Quantitative cytokine estimation in culture supernatant derived from p24-stimulated s CD4+ T cells of the study subject using the LUMINEX assay showed increased production of IFN-gamma, CCL2, CCL4, IL2 at levels comparable to those observed in the Superarray experiments, and increased production of CCL3 and CXCLlO (IP-IO). An 8.3 fold increase in IL-6 production was observed (22 picograms/ml in unstimulated CD4+ T cells increased to 183 picograms/ml in HIV-p24 antigen-stimulated CD4+To cells). CCL8 was not included in the panel of cytokines in the LUMINEX assay. LUMINEX quantitation was used as a basis to generate a biomix containing each cytokine for testing on HIV patients.

A dose suitable for cell culture was derived for each cytokine in the combination referred to above (i.e. IFNγ, IL-2, CCL2, CCL3, CCL4, CCL8 and CXCLlO) bys comparing cytokine levels in supernatant of unstimulated CD4+ T cells with cytokine levels present after stimulation of the CD4+ T cells, as detected by Luminex ELISA. The concentrations derived were taken directly for in vitro testing because they were the most appropriate physiologic doses. The following doses were used in the biomix: 0 CCL2 (JE/MCP-1): 2464 pg/ml culture CCL3 (MIP-I alpha): 2008pg/ml culture IFNγ: 1098pg/ml culture IL-2: 142pg/ml culture CXCLlO (IP-10): 3408pg/ml culture 5

As mentioned above, CCL8 was not in the panel of cytokines tested in the LUMINEX assay. Given that CCL8 is a CC chemokine, the doses observed for other CC chemokines seen in the stimulated supernatant fraction were mimicked. Thus, CCL8 was included in the biomix at the dose of 2000pg/ml. 0

DISCUSSION

The purpose of the study was to analyze the genetic basis of HFV p24 antigen specific immune responses in a unique HIV-I infected true long-term non-progressor using quantitative multiplex SuperArray and Luminex assay. The immune responses were studied in CD4+ T cells and CD 14+ monocytes with and without stimulation to HIV-I p24 antigen at the level of all host genes known in the context of HIV disease and all the genes known in the context of innate and adaptive immunity.

Virus-specific CD4+ T lymphocytes, which are undetectable in chronic HIV-I infection, are crucial to the maintenance of effective immunity in HIV infection. Second, the sheer quantity and quality of antigen-specific cells in HIV patients guide the progression and non-progression of HIV disease. The study subject (LTNP) showed strong HIV-I specific proliferative responses to p24 antigen resulting in the elaboration of IFN-γ and antiviral β-chemokines. The strong expression of IL-2 and IFN-γ observed in the study subject suggests the presence of intact IFN-γ-producing CD4+ T cells directed against HIV-I. High expression of both IL-2 and IFN-γ were consistent with the previous findings showing one third of study subject's IFN-γ producing CD4+ T cells also synthesized IL-2 in response to HIV-I p24 antigen. In addition, they also showed that ThI cytokines are important in cell-mediated immunity. Abnormal production of cytokines in high viremic patients and impaired cell mediated immunity, together contribute to HIV disease. This forms a strong basis of antiviral protection mediated by the cytokine combination of the invention.

High up-regulation of CCL2 (MCP-I), CCL4 (MIP-I β), CCL8 (MCP-2) were observed in CD4+ T cells of the study patient in response to p24 antigen. CCL-2, CCL-4 and CCL-8 may have antiviral function against HIV. The HIV-specific immune responses to p24 antigen in CD4+ T cells of the study subject clearly demonstrated the upregulation of a distinct cytokine combination comprising 5 cytokines (IL2, IFNgamma, CCL2, CCL4 and CCL8) in the study patient's CD4+ T cells. Together, these cytokines can provide antiviral activity, cellular differentiation activity to antigen presenting cells and immune enhancement.

The luminex assay also revealed an 8.3 fold increase in IL-6 production. IL-6 is a multifunctional cytokine that acts as an immune, inflammatory and metabolic mediator. IL-6 plays a pivotal role in the initial response to infection because it influences both innate and acquired immunity. As both types of immune responses are involved in the first steps of HIV-I infection, it is conceivable that IL-6 plays the role of modulator on the vulnerability to HIV-I infection. The data presented herein indicates that IL-6 quantities (in Luminex Assay) were up-regulated in the HIV-p24 antigen-stimulated CD4+ T cell fraction (183 pico-grams) as opposed to unstimulated fraction (22 pico- grams), but not seen at the gene expression level. Given that the subject patient is a long- term non-progressor with high CD4 and CD8+ T cell counts and no viral load, the higher IL-6 secretion by antigen-stimulated CD4+T cells may be protective, as it is secreted together with elevated levels of MIP-lbeta (CCL4), CCL2 (MCP-I), CCL8 (MCP-2), IL- 2 and IFN-gamma. The elevation of IL-6 levels observed after antigenic stimulation of the subject's

CD4+ T cells in conjunction with elevation of the other protective cytokines identified could be related to innate and adaptive immune responses which assist in controlling HIV infection.

The present results identify a key group of cytokines from HIV-antigen stimulated CD4+ T cells and show the antigen-presenting cells and viral antigenic products that can promote the polarization of human HIV+ CD4+ T cells from non-progressors. Since the group of cytokines, including IL-6, are produced quickly following HIV-antigenic stimulation, they certainly relate to host defense. Given the role of IL-6 in cell differentiation, it is likely that IL-6 is a potent regulator of DC differentiation in vivo, and IL-6-gpl30-STAT3 signaling in DCs may represent a critical target for controlling T cell- mediated immune responses in vivo. The cytokines identified may participate in polarizing monocytes to HIV-specific antigen presenting dendritic cells and macrophages in combating HIV.

EXAMPLE 2: Treatment of CD14+ monocytes with cytokine combination and antigen presentation - mini-trial on HIV-infected patients

MATERIALS AND METHODS

1. Patient selection

HIV patients at low (A), intermediate (B) and high (c) viral loads were analysed in a mini clinical trial to assess the efficacy of bioactive mix on HIV patient's CD 14+ monocytes.

2. Isolation andp24 Antigen Stimulation of study patient's PBMCs

50-60 ml of freshly collected blood was centrifuged at 2000rpm for 10 minutes at room temperature in order to isolate the plasma fraction. After removing the plasma, the remaining blood cells were diluted in 1:1 ratio with sterile PBS to maximize cell separation. PBS mimics human conditions and protects cells against rupture. Peripheral blood mononuclear cells (PBMCs) were separated from blood by density gradient sedimentation overlayed on 15ml of ficoll-paque in 50ml tubes. The tubes were centrifuged for 20 minutes at 4°C for 1800rpm with a break off to avoid interruption of the gradient. Interface containing PBMCs was collected in fresh 50ml tube by plastic pipette and washed twice with sterile PBS at room temperature. The PBMC pellets were resuspended in RPMIl 640 to give a final concentration of 2X10⁶ cells /ml. Subsequently the PBMCs were transferred into a 6-well tissue culture plate, added required p24 antigen (2ng/ml) and then incubated at 37°C, with 5% CO₂ for 24 hours (control PBMCs were incubated without P24). After incubation for 24 hours, culture supernatants containing the cytokine combination were collected.

3. Separation ofCD14+ monocytes CD 14+ monocytes were isolated from HIV+ and HIV- PBMC samples by positive sorting using anti-CD 14 conjugated magnetic microbeads from Miltenyi (Miltenyi Biotech., GMBH, Germany) as described in Example 1 above.

4. Treatment of CD 14+ monocytes derived from HIV+ and HIV- PBMCs with supernatant derived from p24-stimulated PBMCs of the study patient

Supernatant was obtained from culturing the study patient's PBMC with p24 viral antigen (see Example 1, Section 2 above). 1x10⁶ CD 14+ monocytes from HIV(-) and HIV(+) patients at various viral loads (low, medium and high) were cultured with the 500μl of supernatant derived from study patient. The cell differentiation was monitored for 24 hours and the cultures were terminated. The effect of supernatant differentiation of CD 14+ monocytes from HIV(+) (low, medium and high viral load) donors was recorded at 0, 4 and 16 hrs. The effect of supernatant differentiation of CD 14+ monocytes from HIV(-) patients donors was recorded at 0 and 12 hrs.

5. Treatment of CD 14+ monocytes derived from HIV+ and HIV- PBMCs with 5-cytokine mix

1x10⁶ CD 14+ monocytes from HIV(-) patients were cultured with 500μl of a cytokine mix consisting of the following cytokines (IFNg 2000pg/ml, IL-2 142 pg/ml, CCL2 2000pg/ml, CCL3 2000pg/ml, CCL4 2000pg/ml). The cell differentiation was monitored for 24 hours and the cultures were terminated. The effect of the cytokine mix on the differentiation of CD 14+ monocytes from HIV(-) and HFV(+) (low, medium and high viral load) donors was recorded at 0 and 12 hrs.

6. Preparation of CD J 4+ monocytes for FACS analysis

CD 14+ cells were prepared for flow cytometric analysis according to the following steps:

1. Wash CD 14+ cells twice in FACS Buffer to remove the media, by adding 2ml of FACS Buffer into each tube and centrifuge at 1500 rpm for 5 mins.

2. Decant the supernatant carefully without disturbing the pellet, 3. Cells would be in approximately lOOul of FACS Buffer.

4. Add cell surface markers (volume: CD14-APC-2ul, CD83-PE-2ul) per tube. Vortex tubes gently/resuspend it by flicking the tube.

5. Incubate at 4C/on ice for 30 mins in dark.

6. Wash with 2ml of FACS Buffer at 1500 rpm for 5 mins at RT. 7. Decant the supernatant gently.

8. Resuspend in 250ul of FACS Buffer /paraformaldehyde.

9. Keep at 4°C until analysis step on Flow machine.

RESULTS The effect of study patient supernatant on HIV+ patient cells patients at various viral loads (low, medium and high) is shown at 0, 4 and 12 hrs is shown in Figure 1. Panel A shows the effect of bioactive mix from the study patient on peripheral blood cells derived from patients experiencing below detection to low plasma viral loads (<50-5000 copies/ml plasma). Panel B shows the effect of active supernatant on cells derived from patients with intermediate plasma viral loads (<50,000 copies/ml). Panel C shows the effect of active supernatant on cells derived from patients with high plasma viral loads (> 100,000 copies/ml). VL = Plasma Viral Load.

In both panel A and B cell differentiation activity could be detected, although less in the case of intermediate viral loads (between 0-12 hours) and in Panel C from patients with high viral loads (> 100,000 copies/ml) showed no effect suggesting the patients with high viral loads harbor impaired antigen presenting activity possibly due to lack of monocytes at that stage of viremia. Hence, study patient supernatant was effective at low viral loads and moderately effective at intermediate viral loads, where robust to moderate cell differentiation was seen. However, it failed to have an effect at high viral loads, as no differentiation was seen (Figure 1).

The effect of supernatant from the study patient and the 5 cytokine mix on CD 14+ monocytes from HIV(-) cells is shown in Figure 2. No differentiation effect was observed in unstimulated healthy HIV (-) CD 14+ monocytes (Figure 2 - panels 1 and 2), while healthy HIV(-) CD 14+ monocytes showed potent cellular differentiation in response to both supernatant derived from the study patient (Figure 2 - panels 3 and 4) and the 5 cytokine mix (Figure 2 - panels 5 and 6).

Differentiated cells from supernatant-treated and cytokine mix-treated samples were typed by flow cytometry and the actual phenotype was assessed for resulting cell types upon differentiation. Immunophenotypic characterization was gated on CD3-CD16- CD19-cells. All CDl Ic cells were CD14+HLA-DR+, whereas the control sample showed CD14+HLA-DR+ cells. Adherent cells showed high expression of CD40, CD38, CD83 and CD86 markers, respectively. CD40, CDI lC, CD83 and CD86 are some of the markers expressed on antigen presenting cells-monocytes, DCs, and macrophages.

This indicates that the bioactive supernatant derived from p24 stimulation of the study patient's PBMCs was able to act on CD 14+ monocyte differentiation in an HIV- antigen specific manner (p24 antigen) in addition to inducing cellular differentiation in healthy donor CD 14+ monocytes. This demonstrated that the cytokine mix mimicked the components present in the cell supernatant of study patient. Clearly, the cellular differentiation of CD 14+ monocytes derived from HIV(-) patients (at 0 and 12 hours) can be seen in CD 14+ monocytes treated with the cytokine mix. This activity was absent in control panel A and B.

A flow cytometric assessment of dendritic cell populations only (flow cytometry protocol outlined above), was conducted using CD 14+ monocytes treated and untreated with cytokine mix containing IFNg 2000pg/ml, IL-2 142 pg/ml, CCL2 2000pg/ml, CCL3 2000pg/ml and CCL4 2000pg/ml. Cellular phenotype upon differentiation of CD 14+ monocytes following treatment with cytokine mix achieved comparable phenotypic differentiation at the level of DC populations what was observed with CD 14+ monocytes upon treatment with bioactive supernatant, validating the authenticity of cytokine mix. Upon treatment with cytokine mix for 16 hours, a shift was observed which resulted in CD83+ DC populations in the treated fraction. This shift was almost 12% in comparison to untreated control samples. Example 3: Validation of antigen presentation at the gene level in treated and untreated monocytes of HIV patient cells

MATERIALS AND METHODS

1. Patient clinical details

Study subjects in this study included a healthy HIV (-) control subject and four HIV (+) patients. Peripheral blood (PB) samples of the HIV (-) control subject were obtained from Australian Red Cross Blood Service (Australian Red Cross, Sydney, Australia), while HIV (+) patients' blood samples were obtained from the Westmead Hospital (Westmead, Sydney, Australia). Prior to sample collection, the informed consent was secured on each occasion. HIV (+) patients were randomly picked (named patient 1, 2, 5 and 6). All four patients are currently receiving highly active antiretroviral therapy (HAART) and are experiencing below detectable levels of plasma viremia (<50 copies viral RNA/ml of plasma).

2. CD 14+ monocyte separation and culture with study patient's supernatant.

Patient PBMCs were obtained and CD 14+ monocytes isolated as described in the Examples above. CD 14+ monocytes derived from HFV(+) patients and HIV(-) controls were cultured with supernatant derived from treatment of the study patient's PBMC with p24 viral antigen (see Example 1, Section 2 above). The effect of supernatant (derived from the study patient) was tested on CD 14+ monocytes from HIV(+) and HIV(-) patients by culturing for 24 hours. Controls (i.e. no supernatant added) were also used for CD 14+ monocytes from each HIV (+) and HIV (-) patient.

3. Human dendritic & antigen presenting cell array

CD 14+ monocytes (with and without supernatant treatment) were assessed using a human dendritic & antigen presenting cell array in accordance with the manufacturer's instructions. The genes involved in antigen presentation and uptake are shown below.

Human Dendritic & Antigen Presenting Cell Array

A list of classes of genes included in the Dendritic and Antigen Presenting Cell Array is provided below. The array consists of five classes of genes: cytokines, chemokines and their receptors, antigen uptake, antigen presentation, cell surface receptors, and signal transduction.

Cytokines, Chemokines and Their Receptors: CCLl 1, CCLl 3, CCLl 6, CCLl 9, CCL2, CCL3, CCL3L1, CCL4, CCL5, CCL7, CCL8, CCRl, CCR2, CCR3, CCR5, CXCLl, CXCLlO, CXCLl 2, CXCL2, CXCR4, ERBB2, IFNG, IFNGRl, ILl 2 A, ILl 2B, ILl 6, IL2, IL8, IL8RA, INHBA, LYN, MDK, MIF, TNF, TNFSFI l, TRAPl.

Antigen Uptake: CD44, CDC42, ICAMl, ICAM2, RACl, STK4, TAP2.

Antigen Presentation: B2M, CDlA, CDlB, CDlC, CDlD, CD209, CD28, CD4, CD40, CD40LG, CD74, CD80, CD86, CD8A, HLA-A, HLA-DMA, HLA-DOA, HLA-DPAl, HLA-DQAl, HLA-DQBl, TAPBP.

CeU Surface Receptors: CD2, CD40, FCERlA, FCER2, FCGRlA, LRPl , TLRl , TLR2.

Signal Transduction: CDKNlA, CEBPA, CSFlR, FAS, FCAR, IFIT3, ITGAM, ITGB2, NFKBl, NFKB2, PDIA3, RELA, RELB, VCL.

RESULTS AND DISCUSSION

Table 3 shows that the study patient's supernatant can induce antigen presentation in healthy cells. Further, HIV(+) patient cells were tested with the study patient's supernatant. CD 14+ monocytes were treated with the supernatant and subjected to antigen presentation superarray. Consistent and comparable patterns of gene expression in all 4 HIV (+) patients CD 14+ monocytes treated with supernatant were observed. Also similar trends of gene expression, particularly in genes related to antigen presentation, cytokines and chemokines, were a consistent feature in all four HIV+ patients. The degree of the gene expression was slightly different between the four patients, but the overall trends were highly similar. Important to note is that all four HIV (+) patients had below detectable plasma viral loads, and hence the differences in the level of the gene expression between patients, which were minimal, could be attributed to T cell counts and the ratio of CD4+ and CD8+ T cells. Furthermore, up-regulation of genes related to antigen uptake (such as TAP2) was observed in patients 2, 5, and 6, while genes related to antigen presentation (i.e. HLA- DPAl, HLA-DQAl, HLA-DOA, HLA-DMA, CD74, and CDlB) were present in all four patients (Table 4). Nevertheless, these results clearly demonstrate that upon treatment with the study patient supernatant, CD 14+ monocytes from all four HIV (+) patients were able to effectively present and uptake antigen. In addition, other genes related to cytokines, chemokines (i.e. CXCLlO and CCL8), surface receptors and signal transduction were also expressed in all 4 patients CD 14+ monocytes treated with active mix, which may have immense value in antigen presenting function of these cells and the antigen presentation restoration in HIV (+) patients.

Healthy HIV (-) CD14+ monocytes treated with control mix vs CD 14+ monocytes treated with bioactive mix using Human Dendritic and

Antigen Presenting Cells Array

Up-regulated

HLA-DOA 23.7

CCL8 19.3

IFNG 19.1

HLA-DQBl 16.1

HLA-DPAl 15.5

CD74 11.1

CCLl 9 10.5

CXCLlO 9.6

HLA-DMA 8.9

TNF 4.9

FCGRlA 4.6

CD40 4.0

HLA-DQAl 3.6

CCL3 3.3

ICAM2 3.2

CCL4 3.1

Down-regulated

CXCR4 -4.6

LRPl -3.9

CCR2 -3.7

TABLE 3: Healthy HIV (-) CD14+ monocytes treated with control mix vs CD14+ monocytes treated with bioactive mix using Human Dendritic and Antigen Presenting Cells Array

TABLE 4: Antigen presentation gene expression upon treatment of CD14+ monocytes from 4 HIV+ patients with the bioactive mix, demonstrating that the bioactive mix can induce antigen presentation in HIV+ patients at different stages of plasma viremia. Example 4: Antiviral assays

CD4+ T cells from HIV(-) healthy donors were infected with an HIV strain (HIV BAL-I) in the presence and absence of cytokine mix containing IFNg 2000pg/ml, IL-2 142 pg/ml, CCL2 2000pg/ml, CCL3 2000pg/ml and CCL4 2000pg/ml. The cultures were followed up every 3 days from Day 0 to Day 11. HIV replication was determined by measuring Reverse transcriptase activity (Enzchek RT Assay Kit) in culture supernatant at each 3-day interval. No RT activity was detected in cells treated with study patient supernatant.

Incorporation by reference

This application claims priority from Australian provisional application number 2009900926 filed on 4 March 2009, the entire contents of which are incorporated herein by reference.

Claims

CLAIMS:

1. A method for producing an antigen presenting cell from an antigen presenting precursor cell, the method comprising contacting said precursor cell with a combination of cytokines comprising: s (i) at least one CC chemokine;

(ii) interferon-gamma; and

(iii) interleukin-2, wherein said contacting differentiates the precursor cell into an antigen presenting cell. o

2. A method for producing an antigen-specific lymphocyte, the method comprising the steps of:

(i) producing an antigen presenting cell according to the method of claim 1 ,

(ii) contacting the antigen presenting cell with a substance comprising an antigen to produce a loaded antigen presenting cell, and s (iii) contacting the loaded antigen presenting cell with a lymphocyte, wherein said contacting produces said antigen-specific lymphocyte.

3. The method according to claim 2, wherein the substance comprising the antigen is derived from a cell infected with a pathogen or a cancer cell.

4. The method according to claim 3, wherein said pathogen is a virus. 0

5. The method according to claim 4, wherein said virus is a human immunodeficiency virus.

6. The method according to any one of claims 2 to 5, wherein said lymphocyte is a helper CD4+ T lymphocyte or a cytotoxic T lymphocyte.

7. The method according to any one of claims 1 to 6, wherein said precursor cell5 is a myeloid precursor cell.

8. The method according to any one of claims 1 to 7, wherein said precursor cell is a CD14⁺ monocyte.

9. The method according to any one of claims 1 to 8, wherein said antigen presenting cell is a dendritic cell, a macrophage, or a B lymphocyte. 0 10. A method for preventing or treating a disease or condition characterised by:

(i) reduced numbers of antigen presenting cells, (ii) impaired antigen presenting cell activity, or (iii) both (i) and (ii) above, said method comprising administering to a subject a therapeutically effective amount of a combination of cytokines comprising at least one CC chemokine, interferon- gamma and interleukin-2.

11. The method according to claim 10, wherein said disease or condition is selected from the group consisting of human immunodeficiency virus infection, acquired immune deficiency syndrome and cancer.

12. A method for enhancing an immune response in a subject, the method comprising administering to the subject a therapeutically effective amount of a combination of cytokines comprising interferon-gamma, interleukin-2 and at least one CC chemokine.

13. The method according to claim 12, wherein said immune response is an antigen-specific immune response mediated by T lymphocytes.

14. A method for treating or preventing human immunodeficiency virus infection in a subject, the method comprising administering to the subject a therapeutically effective amount of a combination of cytokines comprising at least one CC chemokine, interferon-gamma and interleukin-2.

15. A method for suppressing human immunodeficiency virus replication in a subject, the method comprising administering to the subject a therapeutically effective amount of a combination of cytokines comprising at least one CC chemokine, interferon- gamma and interleukin-2.

16. The method according to any one of claims 11 to 15, wherein said method is used as an adjunct to highly active antiretroviral therapy (HAART).

17. The method according to any one of claims 1 to 16, wherein the method further comprises administering to the subject a therapeutically effective amount of interleukin-6.

18. The method according to any one of claims 1 to 17, wherein said at least one CC chemokine is one or more of CCL2 (MCP-I), CCL3 (MIPl alpha), CCL4 (MlPlbeta) and CCL8 (MCP-2).

19. The method according to any one of claims 1 to 18, wherein said combination of cytokines comprises CCL2 (MCP-I), CCL3 (MIPl alpha), CCL8 (MCP-2), interferon- gamma and interleukin-2.

20. The method according to any one of claims 1 to 18, wherein said combination of cytokines comprises CCL2 (MCP-I), CCL4 (MlPlbeta), CCL8 (MCP-2), interferon- gamma and interleukin-2.

21. The method according to any one of claims 1 to 18, wherein said combination of cytokines comprises CCL2 (MCP-I), CCL3 (MIPl alpha), CCL4 (MlPlbeta), CCL8 (MCP-2), interferon-gamma and interleukin-2.

22. The method according to any one of claims 1 to 21, wherein said combination 5 of cytokines further comprises a CXC chemokine.

23. The method according to claim 22, wherein said CXC chemokine is CXCLlO (IP-IO).

24. Use of a combination of cytokines comprising at least one CC chemokine, interferon-gamma and interleukin-2 for the manufacture of a medicament for theo treatment or prevention of a disease or condition characterised by: (i) reduced numbers of antigen presenting cells, (ii) impaired antigen presenting cell activity, or (iii) both (i) and (ii) above.

25. The use according to claim 24, wherein said at least one CC chemokine iss one or more of CCL2 (MCP-I), CCL3 (MIP-I alpha), CCL4 (MlPlbeta) and CCL8

(MCP-2).

26. The use according to claim 24 or claim 25, wherein said combination of cytokines comprises CCL2 (MCP-I), CCL3 (MIP-I alpha), CCL8 (MCP-2), interferon- gamma and interleukin-2. o

27. The use according to claim 24 or claim 25, wherein said combination of cytokines comprises CCL2 (MCP-I), CCL4 (MlPlbeta), CCL8 (MCP-2), interferon- gamma and interleukin-2.

28. The use according to any one of claims 24 to 27, wherein said combination of cytokines further comprises interleukin-6 and/or CXCLlO. 5

29. A composition for producing an antigen presenting cell from an antigen presenting precursor cell, the composition comprising at least one CC chemokine, interferon-gamma and interleukin-2.

30. The composition according to claim 29, wherein said at least one CC chemokine is one or more of CCL2 (MCP-I), CCL3 (MIPl alpha), CCL4 (MlPlbeta) and0 CCL8 (MCP-2).

31. The composition according to claim 29 or claim 30, wherein said composition comprises CCL2 (MCP-I), CCL3 (MIPl alpha), CCL8 (MCP-2), interferon-gamma and interleukin-2.

32. The composition according to claim 29 or claim 30, wherein said composition comprises CCL2 (MCP-I), CCL4 (MIPl beta), CCL8 (MCP-2), interferon-gamma and interleukin-2.

33. The composition according to any one of claims 29 to 32, wherein said composition further comprises interleukin-6 and/or CXCLlO.

34. A kit for producing an antigen presenting cell from an antigen presenting precursor cell, the kit comprising at least one CC chemokine, interferon-gamma and interleukin-2.

35. The kit according to claim 34, wherein said kit comprises CCL2 (MCP-I), CCL3 (MIP-I alpha), CCL8 (MCP-2), interferon-gamma and interleukin-2.

36. The kit according to claim 34, wherein said kit comprises CCL2 (MCP-I), CCL4 (MlPlbeta), CCL8 (MCP-2), interferon-gamma and interleukin-2.

37. The kit according to any one of claims 34 to 36, wherein the kit further comprises interleukin-6 and/or CXCLlO.

38. An antigen presenting cell produced by the method of claim 1.

39. An antigen-specific lymphocyte produced by the method of claim 2.