FIELD

[0001] The present disclosure teaches in the field of treatment and prognosis of human immunodeficiency virus (HIV). In one particular aspect the disclosure relates to the stratification of subjects with respect to HIV disease progression and therapy, and to treatment of HIV infected subjects, including slowing HIV disease progression and assisting in immune recovery. The present disclosure also enables methods of reducing persistent unwanted T cell activation and/or inflammation in subjects with HIV including immunological non-responders, or in subject that do not have HIV.

BACKGROUND

[0002] Bibliographic details of the publications referred to by author in this specification are collected at the end of the description.

(0003] Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.

[0004] HIV infections continue to cause millions of deaths around the world every year and since the epidemic was recognised in 1990 nearly 30 million people have died from AlDS-related causes. The World Health Organisation estimates thai there were 34 million people living with HIV/AIDS in 2010 with 2.7 million people newly infected in that year of whom 1 % were children. The mainstay of treatment is combination anti- retroviral drug therapy (cART) which can slow disease progression through viral suppression but has unwanted side effects. The five different types of available chemotherapeutic agents are nucleoside or non-nucleoside reverse transcriptase inhibitors, protease inhibitors, fusion or early entry inhibitors and most recently, integrase inhibitors.

[0005] HIV infections in vivo are characterized by chronic immune activation, inflammation and increased oxidative stress. These facets of infection are poorly undersiood yet collectively they play an important role in the pathogenesis of HIV disease. Residual and uncontrolled HIV replication, even in the context of viral suppression with cART, is characterized by persistent T cell activation and inflammation, measured by CD38 and HLA-DR expression on peripheral CD4+ and CD8+ T cells (Kitchen et al, 2011), and increased percentages of CD8+Ki67+ and CD4+Ki67+proliferat»ng T cells (Fleury et al, 2000, Gordon et al, 2010).

[0006] There is a need for methods for monitoring CD4 T cell levels, diagnosing prognosing and treating HIV infection and HIV associated disease progression in a subject and for monitoring, treating or preventing persistent CD4 T cell immune activation, and reducing CD4 T cell inflammatory markers in non-HIV infected subjects in need thereof.

SUMMARY

[0007] In one embodiment, the present description enables an assay to stratify a subject as a progressing or not-progressing with respect to HIV disease progression, the assay comprising determining the level or activity of CD4+ T cell Glutl in a biological sample from the HIV infected subject, wherein the level or activity of CD4+ T cell Glutl is different between a subject who is progressing and a subject who is not progressing and wherein the level or activity of Glutl in the subject relative to a control identifies the subject as progressing or not progressing.

[0008] In some embodiments, the HIV infected subject is a non-immunological responder. Subjects are preferably human subjects, but animal models and other mammalian or vertebrate animals are also contemplated.

[0009] In some embodiments, the assay comprises comparing the level or activity of CD4+ T cell GJutl in the subject to the level or activity of CD4+ T cell Glutl in a control subject selected from a non-progressor or a progressor, wherein a similarity in the level or activity of CD4+ T cell Glutl between the subject and the control non-progressor indicates that the subject is not progressing, a difference between the level or activity of CD4+ T cell Glutl between the subject and the control non-progressor indicates that the subject is progressing a similarity between the level or activity of CD4+ T cell Glutl in the subject and a control progressor indicates that the subject is progressing, and a difference between the level or activity of CD4+ T cell Glutl in the subject and a control progressor indicates that the subject is not progressing.

[0010) As determined herein the level of CD4+ T cell Glutl correlates positively with markers of CD4+ T cell immune activation and inflammation indicative of a positive risk of HIV associated disease progression. Disease progression includes AIDS and AIDS related conditions known in the art and includes a propensity to develop conditions associated with chronic immune activation and inflammation.

[0011) As determined herein the level of CD4+ T cell Glutl correlates negati ely with CD4+ T cell numbers, a known marker of HIV disease progression. Disease progression includes AIDS and AIDS related conditions known in the art and includes a propensity to develop conditions associated with chronic immune activation and inflammation.

[0012] In some embodiments, the assay comprises determining the level of Glutl and the level of CD4 on CD3 T cells of the subject.

[0013) In some embodiments of the assay, the level or activity of CD4+ T cell Glutl is compared to a reference level or activity which indicates the concentration of Glutl +CD4+ T cell cells or activated CD4+T cells in the subject or control subject. As determined herein Glutl expression on CD4+ T cells is associated with markers of T cell activation and low CD4 T cell counts. Prolongation of high CD4 T cell glycolytic activity, as illustrated herein, is proposed to contribute to low CD4+ T cell recovery in some subjects on cART.

[0014] In some embodiments of the assay, the level of CD4+ T cell Glutl is compared to a reference level which indicates the concentration of GlutI+CD4+ T cell cells and the concentration of activated CD4+T cells for any given level of CD4+T cell Glutl in the sample from the subject or control subject.

[0015] The level or activity of Glutl may be determined by any method known in the art and as described herein. In some embodiments, the level of Glutl is determined using an antibody that specifically recognises Glutl, or using a labelled glucose analog, such as a fluorescent labelled glucose analog, or a primers/probe that specifically recognises RNA encoding Glutl. In some embodiments, a fluorescent (or similar for example chemiluminescent) glucose analog is used to assess the level or the activity of Glutl in CD4 T cells.

[0016) In another embodiment, the present description enables an assay of a biological sample from a subject for T cell immune activation and/or inflammation status, the assay comprising determining the level of CD4+ T cell Glutl in the biological sample, wherein the level of CD4+ T cell Glutl correlates positively with CD4+ T cell immune activation and/or inflammation. Various markers of T cell activation and inflammation are exemplified herein and others are known to those of skill in the art. Illustrative markers are CD38, HDA-DR and TNF.

[0017] The level of Glutl in CD4 T cells is also a functional marker and as determined herein agents that suppress Glutl levels also reduce inter alia, the levels of CD4+ T cell activation and CD4+ T cell inflammatory markers.

[0018] Accordingly, in another aspect a method is enabled of reducing CD4+ T cell immune activation and/or inflammation in a subject in need thereof comprising administering to the subject an effective amount of an antiglycolytic agent or Glut] antagonist for a time and under conditions sufficient to suppress markers of T cell immune activation or inflammation, or normalise Glutl levels, glucose uptake or glycolysis in CD4+ T cells in the subject. Illustrative markers include inter alia, HLR-DR, CD38, TNF, and PDl .

[0019] In some embodiments, the subject is an HIV infected subject.

[0020] In some embodiments, the HIV infected subject is on cART or another treatment regime. In some embodiments, the HIV infected subject on cART is classified as an immunological non-responder.

[0021] In some embodiments, a method of treatment or prophylaxis of an HIV infected subject or treatment or prophylaxis of disease progression in an HIV infected subject is enabled herein. In some embodiments, the method comprises stratifying the subject with respect to progression or non-progression by determining the level or activity of CD4+ T cell Glutl, wherein the level or activity of CD4+ T cell Glutl relative to a control provides a correlation as to the state, classification, progression of HIV infection/ AIDS in the subject, and then administering a therapeutic agent.

[0022] In some embodiments, a method of treatment or prophylaxis of an HIV infected subject or treatment or prophylaxis of disease progression in an HIV infected subject is enabled herein. In some embodiments, the method comprises stratifying the subject by determining the level or activity of CD4+ T cell Glutl, wherein the level or activity of CD4+ T cell Glutl relative to a control provides a correlation as to the state, classification, progression of HIV infection AIDS in the subject, and then administering a therapeutic agent.

[0023] A method of differentially treating or monitoring a population with respect to HIV infection is enabled, the method comprising determining a level or activity of CD4+ T cell Glutl in the population, wherein subjects in the population are stratified as progressing or not progressing with respect to HIV infection based on the level or activity of C 4+ T cell Glutl, and subjects stratified as progressing receive different treatment regimes and/or monitoring regimes than subjects stratified as progressing.

[0024] In some embodiments, the treatment regime includes or the therapeutic agent is cART, an anti-HIV vaccine, an antiglycolytic agent or a CD4+ T cell Glutl antagonist, or a combination of one or two or more of these.

[0025] In some embodiments, the CD4+ T cell Glutl antagonist is a PDK-mTOR inhibitor.

[0026] In other embodiments, the PI3l0-mTOR inhibitor targets ΡΙ3Κγ.

[0027] In particular embodiments, the Pl3K-mTOR inhibitor targets mTORC 1.

[0028] In some embodiments, two PI3K-mTOR inhibitors are administered which target ΡΪ3Κγ and mTORC 1 , respectively. [0029] In some embodiments, the PDK-mTOR inhibitor is administered as a pharmaceutical composition comprising non-cytotoxic amounts of the inhibitor.

[0030] In some embodiments the combination includes cART and a CD4 T cell Glull antagonist or an HIV vaccine and a CD4 T cell Glutl antagonist. By combination, administration does not have to be at the same time but should be consider to be effective, at least to some extent, in concert.

[0031] In some embodiments, the present description enables a method of treatment or prevention of an HIV infection in a subject, or treatment or prevention of HIV associated disease progression in an HIV infected subject, said method comprising administering to the subject a therapeutically effective amount of a CD4+ T cell Glutl antagonist or anti- glycolytic agent for a time and under conditions to normalise CD4+ T cell Glutl level or activity and glycolysis in CD4+ T cells in the subject.

[0032] In some embodiments, the treatment regime includes or the therapeutic agent is cART, an anti-HiV vaccine, an antigJycolytic agent or a CD4+ T cell Glutl antagonist, or a combination of one or two or more of these.

[0033] In some embodiments, the CD4+ T cell Glutl antagonist is a PDK-mTOR inhibitor. ^

[0034J In other embodiments, the PDK-mTOR inhibitor targets ΡΙ3Κγ.

[0035] In particular embodiments, the PDK-mTOR inhibitor targets mTORC 1.

[0036] In some embodiments, two PI3K-mTOR inhibitors are administered which target ΡΙ3Κγ and mTORC 1 , respectively.

[0037] In some embodiments, the PDK-mTOR inhibitor is administered as a pharmaceutical composition comprising non-cytotoxic amounts of the inhibitor.

[0038] A method of reducing T cell immune activation and/or inflammation in a subject in need thereof comprising administering to the subject a non-cytotoxic amount of an antiglycolytic agent or Glutl antagonist for a time and under conditions sufficient to effectively suppress markers of T cell immune activation or inflammation, or normalise Glutl levels, glucose uptake or glycolysis in CD4+ T cells in the subject.

[0039) In other embodiments, the Pl3 -mTOR inhibitor targets ΡΙ3 γ.

[0040] In particular embodiments, the PI3 -mTOR inhibitor targets mTORCl.

[0041] In some embodiments, two Pl3 -mTOR inhibitors are administered which target ΡΠΚγ and mTORC 1 , respectively.

[0042] In some embodiments, the Pl3K-mTOR inhibitor is administered as a pharmaceutical composition comprising non-cytotoxic amounts of the inhibitor.

[0043] A pharmaceutical composition for use or when used in any of the methods described herein comprising a unit dose amount of PI3 -mTOR inhibitor suitable to providing in a subject a non-cytotoxic blood concentration of about 0.1 nM to about 500nM Pl3K-mTOR inhibitor, or InM to 300nM PI3K-mTOR inhibitor and a pharmaceutically acceptable carrier or diluent.

[0044] In other embodiments, the PI3K-mTOR inhibitor is a Ρΐ3 γ inhibitor.

[0045] In particular embodiments, the PI3K-mTOR inhibitor is an mTORCl inhibitor.

[0046] In some embodiments, the two Pi3K-mTOR inhibitors are a ΡΙ3Κγ inhibitor and an mTORCl inhibitor, respectively.

[0047] In some embodiments, the PI3K-mTOR inhibitor is administered as a pharmaceutical composition comprising non-cytotoxic amounts of the inhibitor.

[0048] A method of screening a potential therapeutic agent, selected from an antiglycolytic agent and an Glutl antagonist (or a potential antiglycolytic agent or putative Glutl antagonist), and a CD4 T- cell or a suitable other model cell, comprising contacting the agent with the cell and measuring the uptake of a labelled glucose analog by the cell relative to a control, wherein the ability of the agent to reduce uptake of the glucose analog is an indication of the ability of the agent to reduce CD4 T cell immune activation or CD4 T ceil inflammation.

[0049] A method of screening an agent, selected from a putative or known antiglycolytic agent and an Glutl antagonist, and a CD4 T- cell or a suitable other cell, comprising contacting the agent with the cell and measuring the uptake of a labelled glucose analog by the cell relative to a control, wherein the ability of the agent to selectively reduce uptake of the glucose analog is an indication of the ability of the agent to selectively reduce CD4 T cell immune activation or CD4 T cell inflammation.

[0050] A non-cytotoxic amount is an amount that, when administered to a CD4 T cell or subject, is able to reduce Glutl levels in CD4 T cells but does not kill CD4 T cells. In an illustrative embodiment, the Glutl antagonist is a PI3 gamma (such as AS-605240) or an mTORCl inhibitor (such as temsirolimus) wherein a non-cytotoxic amount is sufficient to provide n concentrations of the inhibitor (e.g., approximately about InM (say O.lnM to ΙΟηΜ) to approximately about 200nM (say ΙΟΟηΜ to 500 500nM) amounts as described herein) which, when administered to a CD4 T cell or subject is able to reduce Glutl levels in CD4 T cells but does not induce CD4 T cell death.

[0051] A composition comprising an non-cytotoxic amount of an antiglycolytic agent or a Glutl antagonist effective to reduce Glutl level or activity in CD4 T cells.

[0052] A composition comprising an non-cytotoxic amount of an anti-glycolytic agent or a Glutl antagonist effective to reduce one or more markers of CD4+ T cell activation and/or one or more markers of CD4+ T cell inflammation.

[0053] A composition comprising a anti-glycolytic agent or a Glutl antagonist for use in, or in the manufacture of a medicament for, the treatment or prevention of HIV infection or the treatment or prevention of HIV associated disease progression in an HIV infected subject.

[0054] In some embodiments, the agent or antagonist is administered in an amount effective to reduce Glutl level or activity in CD4+ T cells, or one or more markers of CD4+ T cell activation and/or one or more markers of CD4+ T cell inflammation as described herein. [00SS] In some embodiments, the antiglycolytic agent or a Glutl antagonist is used to reduce or prevent HIV infection in a subject. ,

[0056] Use of a composition comprising an antiglycolytic agent or a Glutl antagonist in, or in the manufacture of a medicament for, the treatment or prevention of HIV infection or HIV associated disease progression. As described herein, by reducing glycolysis or Glutl in activated CD4+ T cells, levels of glycolysis and Glutl are normalised and markers of immune activation and inflammation reduced without suppressing HIV antigen specific immune responses (see Figures 18 to 26).

(0057J The present invention further contemplates kits for monitoring Glutl levels, kits comprising the therapeutic agents in suitable pharmaceutical unit dosage form as described herein described herein, and diagnostic kits. Illustrative kits include kits for monitoring CD4 T cell numbers by measuring the level or activity of Glutl in CD4 T cells and comparing the results with a control or reference value enabling the number of CD4 T cells to be determined, preferably visually.

[0058] Each embodiment in this specification is to be applied mutatis mutandis to every other embodiment unless expressly stated otherwise.

[0059] The above summary is not and should not be seen as an exhaustive recitation of all the embodiments of the present description.

BRIEF DESCRIPTION OF FIGURES AND TABLES

[0060] Some figures contain colour representations or entities. Coloured versions of the figures are available from the Patentee upon request or from an appropriate Patent Office. A fee may be imposed if obtained from a Patent Office.

[0061] Figure 1 A through I are graphical representations of Glutl expression on CD4+ and CD8+ T cells from HIV+ individuals and uninfected controls. Whole blood were analyzed for Glutl expression on CD4+ and CD8+ T cells within lh of blood collection. (A) Lymphocytes (circled) were defined using side scatter (SSC) and forward scatter (FSC). (B) Representative flow cytometric dot plot illustrating the distribution of CD3+Glutl+ T cells in peripheral blood from representative HIV- and HIV+ subjects. (C) Percentage of CD3+GJutI+ lymphocytes in peripheral blood from HIV-, HIV+ and HIV+/cART subjects. (D-E) Gating strategy showing CD3+ T cells gated on the lymphocyte population which is then defined based on CD4 and CD8 surface expression. (F) Representative flow cytometric dot plot of Glutl expression on CD4+ T cells in peripheral blood from HIV+ subjects. (G) % CD4+Glutl+ T cells in peripheral blood of HIV-, HIV+ and HIV+/ cART subjects. (H) A representative flow cytometric dot plot of Glutl expression on CD8+ T cells in peripheral blood from HIV+ subjects. (I) Percentage of CDS-*- T cells that are Glutl + and MFI of Glutl on CD8+ T cells. Nonparametric T test (Mann-Whitney) was used to evaluate significant differences between the median values of each group.

(0062) Figure 2 A through C are graphical representations showing expression of Glutl on functional subsets of CD4+ T cells. (A) Representative flow cytometric plot of gated CD3+CD4+ T cell population further analysed by their expression of CD45RA and CD27 to identify effector (E, CD45RA+CD27-), naWe (N. CD45RA+CD27+), memory (M, CD45RA-CD27+) and memory-effector cells (ME, CD45RA-CD27-). (B) Median % of CD4+ T cell subsets that are Glutl+. (C) Median % CD4+Glutl+ T cells expressing markers of functional subsets. Significant difference between subsets was evaluated using the Paired T Test.

[0063] Figure 3 A through C are graphical representations showing expression of activation markers on Glutl+ T cells. (A) Percentage of CD4+CD38+HL A-DR+ and CD8+CD38+HLA-DR+ T cells in peripheral blood from HIV-, HIV+ and HlV+/cART subjects. (B) Percentage of peripheral CD4+GIutl+ and CD4+Glutl- cells that are CD38+ HLA-DR+. (C) MFI of CD38 and HLA-DR on peripheral CD4+GIutl+ and CD4+Gluil- T cells. Significant differences between the means of subject groups was evaluated using the nonparametric T test (Mann- Whitney) while significant difference between Glutl+ and Glutl - subsets was evaluated using the Paired T Test.

(0064] Figure 4 A through C are graphical representations showing Glutl cell surface expression on proliferating CD4+ T cells. Lymphocytes were identified in forward and sideward scatter and were gated for CD3+ cells and further separated into CD3+CD4+ T cells. (A) Mean frequencies of Ki67+ cells within the CD4+Glutl+ and CD4+Glutl- subsets. (B) Representative flow cytometric plot showing the SSC and FSC of lymphocytes from a HIV- subject unstimulated or stimulated with PHA (10 μ£/πι1)/Ιί-2 (5 ng/ml) for 4 days (red and blue dots, respectively). (C) Representative plots of ΜΠ of CFSE-labelled CD4+ in PBMCs after 4 days of incubation with PHA IL-2 (blue line) or untreated (red line). The bar charts indicate the M of Glutl on CD4+ cell within the corresponding peaks.

[0065] Figure S A through K are graphical representations showing the effects of HIV-1 status on glucose uptake and glycolysis in CD4+ T cells. (A) Kinetics of glucose uptake by CD4+ T cells from HIV-, HIV+ and HIV+/cART subjects. Uptake of 2-NBDG in CD4+ T in PBMCs incubated at different time points with 15 μΜ 2-NBDG (n^ per group). (B) Uptake of 2-NBDG in CD4+ T in PBMCs incubated for 60 min with 15 μΜ 2-NBDG (n=8). (C) Uptake of 2-NBDG by CD4+GJutl+ or CD4+Glutl- T cells. (D) A representative dot plot of cells gated within the CD4+ T population to identify CD4+Glutl+ cells in unstimulated (left panel) or anti-CD3 28 stimulated (right panel) PBMCs from an HIV- subject. (E) A representative overlay between 2-NBDG uptake and Glutl expression on CD4+ T cells in stimulated PBMCs from an HIV- subject. (F) Basal concentrations of intracellular glucose-6-phosphate (G-6-P) in Jurkat cells cultured in glucose-containing (1 ImM) and glucose-deprived (0 raM) RPMI-1640 medium. (G) Basal concentrations of intracellular G-6-P in CD4+ T cells incubated in glucose containing RPMI-1640 for 4h. (H) Basal secretion of L-lactate in culture medium containing CD4+ T cells rested for 24h from a cohort of HIV-, H.V1+ and HIV+/cART. (I) Representative histogram showing intracellular lactate levels in Jurkat celts cultured for 2 h in the absence (blue) or presence (brown) of glucose (left panel). (J) Representative histogram showing intracellular lactate levels in CD4+Glutl+ and CD4+Glutl- T cells in PBMCs stimulated for 24h with anti-CD3/28 microbeads. (K) Intracellular L-lactate staining (left panel) in Glutl- (black) and Glut 1+ (red) cells in CD4+ T cells from HIV+ subjects. Levels of intracellular L-lactate were significantly higher in Glutl- and Glutl+ CD4+ T cells compared to Glutl- CD4+ T cells in HIV+ subjects (n=5, p=0.0l ), (right panel). [0066] Figure 6 A through I are graphical representations showing levels of activation of Akt (T308) and PTEN expression in untreated and activated CD4+ T cells from HIV- and HIV+ subjects. (A-B) Representative dot plot of Akt (T308) phosphorylation on Glutl+ cells gated in CD3+CD4+ T cells from unstimulated or anti- CD3/28 stimulated PBMCs from HIV- or HIV+ Subjects. (C) Percentage of Glutl+p-Akt (T308)+ T cells within the CD3+CD4+ T cell populations of unstimulated or anti-CD3/28 stimulated PBMCs from HIV- and HIV+ subjects (n=5). (D) Geometric MFI of p-Akt (T308) and PTEN on Glutl- and Glutl+ cells within the CD3+CD4+ T cell populations of PBMCs from HIV- or HIV+ subjects. (E) Representative flow cytometric dot plot showing Glutl - and Glutl+ cells gated on CD3+CD4+ T cells from anti-CD3/28 stimulated PBMCs from HIV- subjects. (F) Representative flow cytometric histogram comparing the shift of fluorescence intensity of PTEN-AF648 and p-Akt (T308)-PE in Glutl- and Glutl + cells gated on CD3+CD4+ T cells from anti~CD3 28 stimulated PBMCs from HIV- subjects. (G-H) Representative flow cytometric dot plot of Akt (T473) phosphorylation in GlutH- T cells gated on CD3+CD4+ T cells from unstimulated or anti-CD3 28 stimulated PBMCs from HIV- or HIV+ Subjects. (I) Percentage of Glutl+p-Akt (T473)+ T cells within the CD3+CD4+ T cell populations of untreated or anti-CD3/28 stimulated PBMCs from HIV- and HIV+ subjects (n=5). Differences between the mean of unstimulated and stimulated cells were analyzed using the two- tailed Paired T test.

[0067] Figure 7 A through C are graphical representations showing the relationship between percentage CD3+CD4+ T cells and CD4 T cell count in HIV-1 infected patients. (A) Spearman's correlations between percentage CD3+CD4+ T cells and the percentage (left panel) of CD4+Glutl+ T cells in peripheral blood from HIV+ and HIV+/cART subjects and between percentage CD4+Glutl+ T cells and absolute CD4 T cell count (right panel). (B) Changes in percentage of CD4+Glutl+ T cells, T cell activation markers and percentage of CD4+ T cells over time in a single, untreated HIV+ non-progressing and (C) a single disease progressing donor.

[0068] Figure 8 A through F representations showing that CD4+GJutl+ cells harbours high levels of total HIV-1. (A) Comparison between total HIV DNA in CD4+ T cell populations in HIV+ subjects with high and low frequency of GlutH cells with the CD4+ T cell population (n=4 per group). (B) Representative dot plot showing OX40 and Glutl expression within the CD4+ T cell population of PBMCs obtained from virally suppressed HIV+/cART subjects after incubation with GFP-labelled HIV for 3 days. (C-E) Representative dot plots showing the frequency of HIV-GFP+ cells within different population of CD4+ T cells based on their cell surface expression of Glutl and OX40. (F) Proposed model illustrating HIV-induced activation of CD4+ T cells, which leads to the activation of the PI3 pathway, increased Glutl expression, aerobic glycolysis which increases susceptibility to HIV infection which feeds back onto CD4+T cell activation.

[0069] Figure 9 A through B are graphical representations showing Glutl mRNA expression is higher in CD4+ T cells from HIV+ subjects. (A) Glutl mRNA expression in HIV-, HIV+ and HIV+/cART subjects. (B) Correlation between Glutl mRNA expression in CD4+ T cells and the number of CD4+ T cells expressing Glutl. Red and blue dots represent HIV+ and HIV+/cART subjects, respectively. Nonparametric T test (Mann- Whitney) was used to evaluate significant differences between the median values of each group. Horizontal bars represent median values.

(0070] Figure 10 A through £ are graphical representations showing Glutl expression on N subsets during chronic HIV-1 infection. Lymphocytes were defined using side scatter (SSC) and forward scatter (FSC) as previously described. (A-B) CD56+ cells are gated within the CD3- population and then characterized on the basis of Glu l expression. (C) The percentage of CD56+ NK cells expressing Glutl and the MFI of Glutl on CD56+ NK cells in peripheral blood from HIV-, HIV-f- and HIV+/cART subjects. Significant differences between the median of each groups were evaluated using the nonparametric T test (Mann- hitney). (D) A representative flow cytometry dot plot of CD16 and CD56 expression to define NK cell subsets. (E) The percentage of Glutl+ cells on different NK subsets in peripheral blood from HIV-, HIV+ and HIV+/cART subjects. Significant differences between each subset were evaluated using the One-tailed Paired T Test.

[0071] Figure 11 A through F are graphical representations showing expression of Glutl on activated primary CD4 and CD8+ T cells. (A) Frequency of CD4+Glutl+ T cells in untreated (UT) PBMCs and PBMCs stimulated with anti-CD3/CD28 microbeads (beads.cells, 1 :2 ratio) or lOOng/ml PMA + lug/ml ionomycin for the times shown. (B) Representative example of a flow cytometry histogram showing the shift in fluorescence intensity for Glutl-FITC staining on CD4+ T cells after stimulating PBMCs for 24h. (C) MFI of Glutl on CD4+ T cells from untreated (UT) PBMCs and PBMCs stimulated with anti-CD3/CD28 microbeads or PMA+Ionomycin for the times shown. (D) Frequency of CD8+Glutl+ T cells in untreated (UT) PBMCs and PBMCs stimulated with anti- CD3/CD28 microbeads or PMA+ Ionomycin for the times shown. (E) Representative example of a flow cytometry histogram showing the shift in fluorescence intensity for Glutl-FITC staining on CD8+ T cells after stimulating PBMCs for 24h. (F) MFI of Glutl on CD3+CD8+ T cells from untreated (UT) PBMCs and those stimulated with anti- CD3/CD28 microbeads or PMA+Ionomycin for the times shown. The results are pooled data from 6 independent experiments with 3 replicates per experiment. Bars graphs represent mean ± SD. Differences between individual groups were analyzed using the non- parametric two-tailed Mann- Whitney U test. Statistical differences are indicated by p values above the plots.

[0072] Figure 12 A through C are graphical representations showing effects of inhibitors of signaling pathways on Glutl expression and glycolysis in CD4+ T cells. (A) Frequency of CD4+Glutl+ cells and the MFI of Glutl on CD3+CD4+T cells in unstimulated PBMCs, PBMCs stimulated for 24h with anti-CD3/CD28 microbeads with or without inhibitors. The concentrations of the inhibitors used are given in Table S 1. The results are pooled data from 5 independent experiments. Bars graphs represent mean ± SD. Differences between individual groups were analysed using the non-parametric two-tailed Mann- Whitney U test. (B) Jurkat cells were seeded into 48-well plates and were left untreated or treated chronically with inhibitors for 48h. 500,000 viable Jurkat cells were resuspended in glucose free XF modified DMEM Assay medium and seeded into 24-well XF cell culture microplates and glycolytic analysis was conducted. Basal extracellular acidification rate (ECAR) measurements demonstrated glycolytic inhibition of cells treated with AS-605240 and temsirolimus. (C) Glycolytic function of cells was measure by sequential addition of glucose, oligomycin (ATP synthase inhibitor) and 2-deoxygIucose. Graphs illustrate that cells treated with AS-605240 and temsirolimus (blue and pink line respectively), had reduced ECAR, indicative of glycolytic inhibition.

(0073] Figure 13 A through G are representations showing effects of ΡΙ3Κ _γ and mTOR inhibition on HIV Infection of CD4+ T cells. Purified T lymphocytes were pretreated for 48h with AS-605240 or Temsirolimus and activated with anti-CD3/CD28 microbcads for 24h prior to viral infection with a CXCR or a CCR5 tropic virus. (A) PB Cs untreated with HIV. (B) PB Cs with no inhibitors and incubated with CXCR tropic virus. (C D) PBMCs pretreated with ASr605240 (AS) and temsirolimus (terns) respectively prior to incubating with CXCR tropic virus. (E-G) PBMCs pretreated with AS-605240 (AS) and temsirolimus (terns) respectively prior to incubating with CCR5 tropic virus.

[0074] Figure 14 shows the relationship between percentage CD3+CD4+ T cells and CD4 T cell count in HIV-1 infected patients. (A) Spearman's correlations between percentage CD3+CD4+ T cells and the percentage (left panel) of CD4+Glutl+ T cells in peripheral blood from HIV+ and HIV+/cART visually suppressed subjects and between percentage CD4+Glutl+ T cells and absolute CD4 T cell count (right panel). (B) Comparative relationship between the percentage of CD4+Glutl+ T cells and maikers of CD4+ T cell activation, and the percentage of CD3+CD4+ T cells in cART-nai've HIV+ and (C) HIV+/cART subjects. % of CD4+ Glutl T cells is strongest predictor of total CD4 cell percent.

[0075] Figure 15 A through B shows the intracellular Glutl pool is also increased in CD4+ T cells from HIV- infected subjects. (A) shows a comparison of the percentage of Glutlc-term CD4+ T cells from HIV-, HiV+ and HIV+/cART subjects. (B) shows a comparison of the MFI of c-terminal Glutl expression in CD4+ T cells from HIV-, HIV+ and HIV+/cART subjects. The antibody used here detected intracellular Glutl expression in CD4+ T cells from HIV+ subjects. The data suggest for the first time that not only docs HIV affects Glutl cell surface expression but also the intracellular machinery thai synthesize Glutl . Antibody was obtained from Abeam, MA, USA.

[0076] Figure 16 provides data confirming that the R&D antibody detects Glutl . (A through B) transfection of HE 293T cells and Glutl staining. (C) shows Western blot analysis of Glutl. (Lane 1) Protein extracts from HEK293T cells transfected with pQCXIP-huGlutl. (Lane 2) Protein extracts from HE 293T cells transfected with empty pQCXIP vector.

[0077J Figure 17 shows confirmation that the R&D antibody detects Glutl . (A through B) shows cell surface expression of Glutl on NIH3T3 cells transfected with a lentiviral vector expressing Glutl . (A) Dual analysis of cell surface Glutl and eGFP. (B) Histogram illustrating fluorescent shift using R&D System Glutl-APC conjugated antibody in cells transfected with empty of Glutl -expressing lentiviral vector.

[0078] Figure 18 is a graphical representation of data showing inhibitors of Ρΐ3Κγ and mTORCl suppress markers of HIV immune activation and general inflammation. Inhibitors of PI3K _T (AS-605240, 200 nM) and mTORCl (temsirolimus, 10 nM) suppress markers of immune activation and inflammation. PBMCs were either left untreated (UT) or pre-treated with AS-605240 or temsirolimus alone or in combination (Comb.) for 48h prior to activation with antiCD3/CD28 microbeads for an additional 24h. Some PBMCs were not treated with inhibitors but were activated with antiCD3/CD28 microbeads (act). (A-B) The T cell activation marker HLADR and CD38 were evaluated by flow cytometry. (C) Secreted TNF in culture was detected by ELISA. Data was analysed using the Wilcoxon matched-pairs signed rank test.

[0079) Figures 19 A through B show inhibitors of ΡΙ3 γ and mTORCl significantly suppress glucose uptake and L-lactate (glycolytic output) in purified and activated CD4+ T cells. Inhibitors of Pl3K _y (AS-605240, 200 nM) and mTORCl (temsirolimus, 100 nM, everolimus, 20nM) suppress glucose uptake and glycolysis in CD4+ T cells. CD4+ T cells were purified by negative selection from HIV- healthy donors and stimulated with an activation cocktail consisting of PMA (100 ng/ml), lonomycin (1 Hg ml) and 1L-2 (5ng/ml). Cells were activated with the activation cocktail for 48h, replaced with fresh media, and were left untreated (Act) or treated with either, AS-605240, temsirolimus, a combination of AS-605240 and temsirolimus (Comb.), everolimus (20 nM) or SB201290 (lOuM, p38 inhibitor) for 48h. UT represent unactivated cells. (A) Glucose and (B) L- lactate concentrations in culture media after 48h of treatment. Glucose concentration in media represents the concentration of glucose in RPMI-1640 supplemented with 10% human serum, L-glutamine and penicillin and streptomycin. 500,000 activated cells were cultured in 500 μΐ of media. Note that the inhibitors suppress glucose uptake hence those media contain higher glucose levels compared to the untreated activated (Act.) cells. Data was analysed using the Wilcoxon matched-pairs signed rank test. CD4+Glutt+ T cells from HIV+ subjects have high glucose uptake and glycolytic metabolism and this was associated with low CD4 T cell count. Enabled therefore is the use of inhibitors of the ΡΙ3 γ mTO Cl pathway to normalize the glucose metabolic activity in activated CD4 Glutl+ T cells.

[0080] Figure 20 shows 2 nM of everolimus was sufficient to reduce L-lactate production in activated CD4+ T cells. Relatively low concentrations of the mTOR inhibitor everolimus suppresses L-lactate production in activated CD4+ T cells. CD4+ T cells were purified by negative selection from HIV- healthy donors and stimulated with an activation cocktail consisting of PMA (100 ng/ml), lono ycin (1 μ§ π-1) and IL-2 (5ng/ml). Cells were left inactivated (UT), activated with the activation cocktail for 48h. replaced with fresh media, and were left untreated (Act) or treated with different concentrations of everolimus for 72h. Activated cells (500,000) were cultured in 500 μΐ of media. L-lactate concentrations are in cell media after 48h of treatment.

[0081] Figure 21 shows the inhibitory effect of temsirolimus on activated CD4+ T cells is prolonged in its absence for up to 4 days. The inhibitory effect of temsirolimus is maintained in its absence. CD4+ T cells were purified by negative selection from HIV- healthy donors and stimulated with an activation cocktail consisting of PMA (100 ng ml), lonomycin (1 μg ml) and IL-2 (5ng ml). Cells were activated with the activation cocktail for 48h, replaced with fresh media, and were left untreated (Act) or treated with either, AS-605240, temsirolimus, a combination of AS-605240, temsirolimus (Comb.), everolimus (20 nM) or SB201290 (lOuM, p38 inhibitor) for 48h. Cells were washed three times in fresh culture media and 500,000 activated cells were seeded in 500 μΙ of media in a 48-well culture plate. L-lactate concentrations were measured in cell media after 4 days. Data was analysed using the Paired T lest. (0082] Figure 22 shows inhibitors of ΡΙ3Κγ and mTORCl significantly suppress the basal extracellular acidification rate (a proxy for the rate of glycolysis) of activated CD4+ T cells.

(0083J Figure 23 shows inhibitors of ΡΙ3Κγ and mTORCl significantly suppress PI3K and mTOR activity in activated CD4+ T cells. Western Blot Analysis showing the effects of inhibitors on PI3K (Phos - Ser473A t) and mTOR (Phos -p70S6 ) activation.

(0084) Figure 2 shows that 1 nM of temsirolimus was sufficient to lower glucose metabolism in activated CD4+ T cells. Relatively low concentrations of ΡΙ3Κγ (AS- 605240) and mTORCl (temsirolimus) inhibitors suppress glucose uptake. CD4+ T cells were purified by negative selection from HIV- healthy donors and stimulated with an activation cocktail consisting of PMA (100 ng/ml), Ionomycin (I μ ml) and IL-2 (5ng ml). Cells were activated with the activation cocktail for 48h, replaced with fresh media, and were left untreated (Act) or treated with different concentrations of (A) AS- 605240 or (B) temsirolimus for 72h. 500,000 activated cells were cultured in 500 μΐ of media. Note that the inhibitors suppress glucose uptake hence the media contain higher glucose levels compared to the untreated activated (Act.) cells. Data was analysed using the Wilcoxon matched-pairs signed rank test.

[0085] Figures 25 A through C show inhibition of ΡΙ3Κγ and mTORCl does not suppress HIV or other antiviral responses in cells from patients on cART. Inhibition of ΡΙ3 γ and mTORCl does not suppress HIV-GAG specific T cellular immune responses in HIV+ subjects on cART. PBMCs were obtained from HIV+ subjects on cART. Cells were pre-treated with AS-605240 (200 nM), temsirolimus (100 nM) or everolimus (20nM) for 48h and then stimulated with HIV-GAG peptide pool or CEF peptides (pool of CMV, EBV and Flu). ELISPOT assay to detect INF _y secreting cells was conducted as previously described by Palmer et al, 2008.

(0086] Figure 26 shows inhibition of Pl3 y and mTORCl suppresses glycolytic gene expression in activated CD4+ T cells. Inhibitors of ΡΙ3 γ (AS-605240, 200 nM) and mTORCl (temsirolimus, 100 nM, everolimus, 20nM) suppress the expression of genes within the glycolytic pathway. CD4+ T cells were purified by negative selection from HIV- healthy donors and stimulated with an activation cocktail consisting of P A (100 ng/ml), Ionomycin (1 pg ml) and IL-2 (5ng ml). Cells were activated for 48h, left untreated (Act) or treated with either, AS-605240, temsirolimus, everolimus (20 nM) or SB201290 (lOuM, p38 inhibitor) for 48h. Gene expression was normalized relative to β-actin. The sequence of primers used is provided in the sequence listing (see Table 4), and SED ID NOs: 7 to 14.

[0087] Table 1 provides clinical characteristics of study groups.

[0088] Table 2 provides markers associated with the frequency of CD3+CD4+GIW 1 + T cells.

[0089] Table 3 provides effect of inhibition of selected pathways on Glutl expression on CD4+ T cells.

[0090J Table 4 provides a brief description of the SEQ ID NOs. DETAILED DESCRIPTION OF SOME EMBODIMENTS

(0091) The subject invention is not limited to particular screening procedures for agents, specific formulations of agents and various medical methodologies, as such may vary.

[0092] Unless otherwise stated, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Any materials and methods similar or equivalent to those described herein can be used to practice or test the present invention. All publications cited herein are incorporated herein by reference in their entirety for the purpose of describing and disclosing the methods, reagents, tools reported in the publications that might be used in connection with the invention.

[0093] The practice of the invention employs, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, cell biology, genetics, immunology and pharmacology. Such techniques are fully described in the literature. Practitioners are particularly directed to Sambrook el al. Molecular Cloning: A Laboratory Manual (2nd ed.). Cold Spring Harbour Laboratory, Cold Spring Harbour, NY, Sections 13, 16 and 17, 1989, Coligan et al, Current Protocols In Protein Science, John Wiley & Sons, Inc., 1995-1997, in particular Chapters 1, 5 and 6. and Ausubel et al, Current Protocols in Molecular Biology, Supplement 47, John WiJey & Sons, New York, 1999; Colowick and Kaplan, eds., Methods In Enzymology, Academic Press, Inc.; Weir and BJackwell, eds., Handbook of Experimental Immunology. Vols. I-IV, Blackwell Scientific Publications, 1986 ^" ; Joklik ed., Virology, 3rd Edition, 1988; Fields and Knipe, eds, Fundamental Virology, 2nd Edition, 1991; Fields et al, eds, Virology, 3rd Edition, Lippincon-Raven, Philadelphia, Pa, 1996, Gcnnaro A. R. ed. (1990); Remington's Pharmaceutical Sciences, I ' ^h ed, Mack Publishing Co, Easton, PA, U.S.A.

[0094] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers r steps but not the exclusion of any other integer or step or group of integers or steps. Thus, use of the term "comprising" and the like indicates that the listed elements arc required or mandatory, but that other elements are optional and may or may not be present. By "consisting of is meant including, and limited to, whatever follows the phrase "consisting of. Thus, the phrase "consisting of indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

[0095J As used herein the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a pad" includes a single pad, as well as two or more pads; reference to "an elastomer" includes one elastomer, as well as two or more elastomers; and so forth. (0096] The present disclosure applies a metabolic approach to identify strategies for ameliorating the chronic immune activity and inflammation seen in HIV infected subjects. As a result of this approach it has been determined that the level or activity of the glucose transporter, Glutl is significantly elevated on circulating CD4+ T cells, but riot CD8+ T cells, in chronically HIV infected subjects and is decreased but remained significantly elevated following cART. As described in the Examples, the increased level or activity of Glutl was found to be significantly associated with several immunologically and clinically relevant parameters of HIV disease progression. Glutl level or activity were inversely associated with CD4 T cell counts and positively with markers of T cell activation and inflammation. Glutl represents a sensitive and functional marker of T cell activation of CD4+ T cells. Non-immunological responders have particularly high levels of CD4 T cell Glut. Proposed herein is a method to attentuate Glutl expression or glycolysis in CD4 T cells to target overactive T cells and immune activation in HIV infected subjects.

[0097] The identification of Glutl as a prognostic metabolic marker for disease progression in HIV/AIDS should facilitate the delivery of critical early and appropriate treatment. As described herein Glutl has been found to be a prognostic marker for CD4 T cells and not CD8 T cells, making tests for Glutl protein particularly useful tools in pharmacotranslational studies and in the clinical management of HIV infected subjects.

{0098] Glutl mRNA was also significantly increased in CD4+ T cells from HIV infected subjects. Accordingly, in some embodiments Glutl the level or activity of Glutl is determined by measuring the level or activity of Glutl nucleic acid.

[0099] Gluts comprise a family of at least 13 members, Glutl -12 plus the proton- myoinositol co-transporter HMIT. Class 1 Gluts such as Glutl have high affinity for glucose and Glutl is the main glucose transporter on T cells (Wieman et al 2007, Wofford et al, 2008, Maciver et al, 2008). The transportation of glucose by Glutl across the hydrophobic cells membrane is the first rate-limiting step of glucose metabolism. The post transcriptional regulation of Glutl is controlled in part by the PI3K-Akt-mTOR axis, although it can also be transcriptionally regulated by the stress responsive hypoxia inducible factor ! alpha (HIF-la) (Noch and Khailli, 2012). [0100] Olutl is a downstream target of Akt/PI3K pathway and when activated, Glutl is translocated from the cytoplasm to the cell surface membrane to facilitate increased glucose uptake and metabolism. Class IB PI3K comprises the pi 10γ catalytic and pi 105 regulatory subunits. These isoforms differ from the other P13 subunits in that they have a 'tissue-restricted expression' being predominantly expressed in white blood cells. Although several studies have confirmed the role of Akt PI3K activation in Glutl expression, the contribution of the specific PI3K subunits in its regulation on CD4+ T cells has not previously been described.

(0101) Few studies have evaluated the role of HIV-1 infection on glucose metabolism in leukocytes and these have been conducted exclusively in vitro (Sorbara el al, 1996, Hollenbugh et al, 2011, Loisel-Meyer et al, 2012). Furthermore, glucose metabolism in immune cells in the setting of an inflammatory response and immune activation to HIV in vivo has never been evaluated.

[0102] Accordingly, in one aspect, assays assessing the level or activity of Glutl protein or nucleic acid is described herein to be useful in the prognosis of HIV infection.

[0103] Identification of subjects with early signs of progression will enable better treatment. The tests will also be useful to identifying whether or not a subject is likely to respond to particular drugs, for identifying types of subjects that respond well to specific medications and as a guide for new drug discovery and testing.

[0104] Prognosis in this context refers to obtaining an indication and particularly an early indication whether or not an HIV infected subject is developing HIV induced disease (AIDS). "Progressor" or "progressing" are interchangeable and indicate that the subject is developing or likely to develop AIDS and the clinical and immunological signs thereof including reducing CD4 T cell levels. "Not progressing" or "non-progressor" are used interchangeably and indicate that the subject is not yet developing AIDS and the clinical and immunological signs thereof including reducing CD4 T cell levels.

[0105] The level or activity of Glutl or the level or activity of Glutl RNA is usually assessed in a biological sample from the subject. [0106] In some embodiments a method of treatment or prophylaxis of an HIV infected subject is provided, said method comprising stratifying the subject with respect to progression or non-progression (including degree of progression) by determining the level or activity of Glutl on CD4+ T cells, wherein the level or activity of Glutl relative to a control provides a correlation as to the state, classification, progression of HIV infection/AlDS in the subject and then administering a therapy.

(0107) In some embodiments, the therapy is cART, an anti-HlV vaccine, an antiglycolytic agent or a Glutl antagonist, or a combination of one or two or more of these.

[0108] In another embodiment, the invention provides a web-based system where data on the level or activity of Glutl are provided by a client server to a central processor which analyses and compares to a control and optionally considers other information such as patient age, sex, weight and other medical conditions and then provides a report such as a report on the likelihood of progression or non-progression in HIV infection AIDS, Hence, knowledge-based computer software and hardware also form part of the invention.

[0109] In one embodiment there is provided an assay to stratify a subject as a progressing or not-progressing with respect to HIV infection, the assay comprising determining the level or activity of CD4+ T cell Glutl (or Glutl RNA), wherein the level or activity of Glutl is different between a subject who is progressing and a subject who is not progressing and wherein the level or activity of Glutl in the subject relative to a control identifies the subject as progressing or not progressing.

[0110] In some embodiments the assays comprise comparing the level or activity of Glutl in the subject to the level or activity of Glutl in a control subject selected from a non-progressor or a progressor, wherein a similarity in the level or activity of Glutl between the subject and the control non-progressor indicates that the subject is not progressing, a difference between the level or activity of Glutl between the subject and the control non-progressor indicates that the subject is progressing a similarity between the level or activity of Glutl in the subject and a control progressor indicates that the subject is progressing, and a difference between the level or activity of Glutl in the subject and a control progressor indicates that the subject is not progressing.

(0111) The term "stratification" or "stratify" includes identification, diagnosing or clarification of the stage of HIV infection AlDS development including determining susceptibility to developing more severe signs of AIDS.

[0112] In one embodiment, the present invention identifies a correlation between the level or activity of CD4 T cell Glutl and progression or non-progression in HIV infected subjects. The present invention enables, therefore, an AIDS risk profile (also referred to as "status") to be determined for a test subject based on assessing the level or activity CD4 T cell Glutl. The profiling enables early diagnosis, conformation of a clinical diagnosis, treatment monitoring and treatment selection.

[0113] Reference to a "control" broadly includes data that the skilled person would use to facilitate the accurate interpretation of technical data. In an illustrative example, the level or activity of Glutl from a subject is compared to the level or activity of Glutl in one or more cohorts (populations/groups) of control or reference subjects. In some assays, control or reference subjects include a non-progressing cohort wherein the subjects are HIV infected but in whom AIDS is not progressing i.e., they are stable with respect to AIDS over a follow up period. Also, control or reference levels may be established using a progressing subject cohort wherein the subjects have an HIV infection and are progressively developing symptoms of AIDS over a follow up period). In some embodiments, the control may be the level or activity of Glutl in a sample from a test subject taken at an earlier time point. Thus, a temporal change in analyte levels can be used to identify progression or a degree of progression or likelihood of progression or provide a correlation as to the state of HIV/ AIDS. In some embodiments, the relative levels of two or more markers (such as Glut) and CD4) provides a useful control.

[0114] In some embodiments, a control subject is a group of control subjects. The level of Glutl in a control subject group may be a mean value or a preselected level, threshold or range of levels that define, characterize or distinguish a particular group. Thresholds may be selected that provide an acceptable ability to predict diagnostic or prognostic risk, treatment success, etc. In illustrative examples, receiver operating characteristic (ROC) curves are calculated by plotting the value of one or more variables versus its relative frequency in- two populations (called arbitrarily "progressor" and "non HIV-infected" or "non-progressor" and "treatment naive" groups for example). The area under the curve provides the C-statistic which is a measure of the probability the measurement will allow correct identification of a condition or risk. The distribution of level(s) for subjects in two control populations will likely overlap. Under such conditions, a test level may not absolutely distinguish between groups with 100% accuracy, and the area of overlap indicates where the test cannot distinguish between groups. Accordingly, in some embodiments, a threshold or range is selected, within which the test is considered to be "indicative" i.e., able to discriminate between disease status and without which the test is considered to be "non-indicative" i.e., unable to discriminate.

[0115] Alternatively, or in addition, thresholds may be established by obtaining a level or activity of Glutl from the same patient, to which later results may be compared. In these embodiments, the individual in effect acts as their own "control group". An increase in Glutl level or activity over time in the same patient can indicate a development of risk of progression or progression or a failure of a treatment regimen, while a decrease over time can indicate remission of risk or success of a treatment regimen. Various further controls will be routinely applied by the skilled artisan.

(0116) The present invention provides a kit of pre-mixed reagents that allow for the simple enumeration of Glutl + cells or Glutl+ and CD4+ T cells.

(0117) In some illustrative embodiments, CD4.T cells are isolated, for example, by positive selection with anti-CD4 magnetic beads and the amount of Glutl measured, for example, by ELISA using monoclonal or polyclonal antibodies to Glutl, or by metabolic assays using substrates of the Glutl transporter such as the fluorescent glucose analog, 2- (N-(7-nitrobenz-2-oxa-l, 3-diazol-4-yl) amino)-2 deoxyglucose (2-NBDG). The total amount of Glutl activity detected would then be proportional to the number of Glutl- positive CD4+ T cells.

(0118] In another embodiment, the CD4 T cells could also be isolated by negative selection to remove monocytes, CD8+ T cells, NK cells, B-cells and other leukocytes using, for example, a cocktail of antibodies on magnetic beads, or a cocktail of antibodies in the form of RosetteSep (STEMCELL Techologies, Vancouver) that would result in rosetting and removal of the unwanted white blood cells with unwanted red blood cells. Total Gl tl remaining in the sample would then be measured in the same way.

[0119J Alternatively, the test may be conducted by Flow cytometry where cells arc stained for CD4 and Glutl using monoclonal antibodies, as described in Figure 1.

[0120] In some embodiments of the methods, a "difference" typically represents an at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%, or even an at least about 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000% increase, or an at least about 10%, 20%, 30% 40%, 50%, 60%, 70%, 80%, 90%, 92%, 94%, 96%, 97%, 98% or 99%, or even an at least about 99.5%, 99.9%, 99.95%, 99.99%, 99.995% or 99.999% decrease in the level or activity of Glutl as compared to the level or activity in a control.

[0121] In some embodiments, the level or activity of Glutl is determined by measuring the level or activity of RNA encoding Glutl (e.g., mRNA) or a DNA copy of the Glutl RNA whose level is measured using at least one nucleic acid probe that hybridises under at least low, medium, or high stringency conditions to the Glutl RNA or to the DNA copy, wherein the nucleic acid probe comprises at least 15 contiguous nucleotides of an Glutl polynucleotide. In some embodiments, the measured level or abundance of the Glutl RNA or its DNA copy is normalized to the level or abundance of a reference RNA or a DNA copy of the reference RNA that is present in the same sample. Suitably, the nucleic acid probe is immobilized on a solid or semi-solid support. In illustrative examples of this type, the nucleic acid probe forms part of a spatial an ay of nucleic acid probes. In some embodiments, the level of nucleic acid probe that is bound to the target RNA or to the DNA copy is measured by hybridization (e.g., using a nucleic acid array). In other embodiments, the level of nucleic acid probe that is bound to the target RNA or to the DNA copy is measured by nucleic acid amplification (e.g., using a polymerase chain reaction (PCR)). In still other embodiments, the level of nucleic acid probe that is bound to the target RNA or to the DNA copy is measured by nuclease protection assay. An illustrative cDNA sequence of Glutl is available at Genbank Accession No. BC1 1 S590 ( see aJso SEQ ID NO:6)

(0122) In other embodiments, the level or activity of Glutl is measured using at least one antigen-binding molecule that is immuno-interactive with Glutl. Suitably, the antigen- binding molecule is immobilized on a solid or semi-solid support. In illustrative examples of this type, the antigen-binding molecule forms part of a spatial array of antigen-binding molecule. In some embodiments, the level of antigen-binding molecule that is bound to the target polypeptide is measured by immunoassay (e.g., using an ELISA) or assessed using flow cytometry as described herein.

[0123] As used herein, the term "binds specifically," "specifically immuno- interactive" and the like when referring to an antigen-binding molecule refers to a binding reaction which is determinative of the presence of an antigen (Glutl) in the presence of a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antigen-binding molecules bind to a pellicular antigen and do not bind in a significant amount to other proteins or antigens present in the sample. Specific binding to an antigen under such conditions may require an antigen- binding molecule that is selected for its specificity for a particular antigen. For example, antigen-binding molecules can be raised to a selected protein antigen, which bind to that antigen but not to other proteins present in a sample. The amino acid sequence of the 492 amino acid sequence of Glutl is available from UniProt Acc. No. PI 1 166. A variety of immunoassay formats may be used to select antigen-binding molecules specifically immuno-interactive with a Glutl or part of Glutl . For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immuno- interactive with a protein. See Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity. Antibodies can fuither or alternatively be tested for their ability to antagonise Gluil activity using methods known in the ar (0124} The term "biological sample" or "sample from a subject" etc as used herein refers to a sample that may be extracted, untreated, treated, diluted or concentrated from a human subject. The biological sample may include a biological fluid such as whole blood, serum, plasma, saliva, urine, sweat, ascitic fluid, peritoneal fluid, synovial fluid, amniotic fluid, cerebrospinal fluid, tissue biopsy, and the like. In certain embodiments, the sample or biological sample is blood, especially peripheral blood.

[0125] In still other embodiments, the activity of Glutl is measured using at least one substrate for Glutl with which it reacts to produce a reaction product. In some embodiments, the measured functional activity is normalized to the functional activity of a reference polypeptide that is present in the same sample.

[0126] In some embodiments, a system is used to perform the prognostic methods as broadly described herein, which suitably comprises at least one end station coupled to a base station. The base station is suitably caused (a) to receive subject data from the end station via a communications network, wherein the subject data represents parameter values corresponding to the measured or normalized level or functional activity of at least one expression product in the sample from a subject, and (b) to compare the subject data with predetermined data representing the measured or normalized level or functional activity of at least one corresponding expression product in the reference sample to thereby determine any difference in the level or functional activity of the expression product in the biological sample as compared to the level or functional activity of the corresponding expression product in the reference sample. Desirably, the base station is further caused to provide a diagnosis or prognosis for the presence, absence or degree of progression of AIDS. In these embodiments, the base station may be further caused to transfer an indication of the diagnosis/prognosis to the end station via the communications network..

[0127] The present invention relates to primers and probes for use in measuring the level or activity of Glutl nucleic acid in CD4 T cells a sample. Illustrative primers and the cDNA sequence of human glutl is set out in the Examples. The requisite hybridisation or amplification conditions are routine to those of skill in the art. [01 8] Nucleic acid used in polynucleotide-based assays can be isolated from cells contained in the biological sample, according to standard methodologies (Sambrook, et al., 1989, supra; and Ausubel et al., 1994, supra). The nucleic acid is typically fractionated (e.g., poly A RNA) or whole cell RNA. Where RNA is used as the subject of detection, it may be desired to convert the RNA to a complementary DNA. In some embodiments, the nucleic acid is amplified by a template-dependent nucleic acid amplification technique. A number of template dependent processes are available to amplify the Glut I marker sequences present in a given template sample. An exemplary nucleic acid amplification technique is the polymerase chain reaction (referred to as PCR) which is described in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159, Ausubel et al. {supra), and in Jnnis et al, ("PCR Protocols", Academic Press, Inc:, San Diego Calif., 1990). A reverse transcriptase PCR amplification procedure may be performed in order to quantify the amount of mRNA amplified. Methods of reverse transcribing RNA into cDNA are well known and described in Sambrook et al, 1989, supra. Alternative methods for reverse transcription utilize thermostable, RNA-dependent DNA polymerases. These methods are described in WO 90/07641. Polymerase chain reaction methodologies are well known in the art. In certain advantageous embodiments, the template-dependent amplification involves the quantification of transcripts in real-time. For example, RNA or DNA may be quantified using the Real-Time PCR technique (Higuchi, 1992, et al, Bioiechnology 10: 413- 17). By determining the concentration of the amplified products of the target DNA in PCR reactions that have completed the same number of cycles and are in their linear ranges, it is possible to determine the relative concentrations of the specific target Glutl sequence in the original DNA mixture. If the DNA mixtures are cDNAs synthesized from RNAs isolated from different tissues or cells, the relative abundance of the specific mRNA from which the target sequence was derived can be determined for the respective tissues or cells. This direct proportionality between the concentration of the PCR products and the relative mRNA abundance is only true in the linear range of the PCR reaction. The final concentration of the target DNA in the plateau portion of the curve is determined by the availability of reagents in the reaction mix and is independent of the original concentration of target DNA. (0129] In certain embodiments, Glutl nucleic acids are quantified using blotting techniques, which are well known to those of skill in the art. Southern blotting involves the use of DNA as a target, whereas Northern blotting involves the use of RNA as a target. Each provide different types of information, although cDNA blotting is analogous, in many aspects, to blotting or RNA species. Briefly, a probe is used to target a DNA or RNA species that has been immobilized on a suitable matrix, often a filter of nitrocellulose. The different species should be spatially separated to facilitate analysis. This often is accomplished by gel electrophoresis of nucleic acid species followed by "blotting" on to the filter. Subsequently, the blotted target is incubated with a probe (usually labeled) under conditions that promote denaturation and rehybridisation. Because the probe is designed to base pair with the target, the probe will bind a portion of the target sequence under renaturing conditions. Unbound probe is then removed, and detection is accomplished as described above. Following detection quantification, one may compare the results seen in a given subject with a control reaction or a statistically significant reference group of normal subjects or of subjects lacking an Glutl -related condition. In this way, it is possible to correlate the amount of a Glutl nucleic acid detected with the progression of HIV/AIDS.

{0130] Consistent with the present invention, the presence of an increased or decreased concentration of Glutl protein relative to a control is indicative of the presence, degree, or stage of HIV/ AIDS. Glutl level or activity in a sample can be assayed using any suitable method known in the art. as described herein. For example, Glutl protein can be quantified based upon its transporter activity or based upon the number of molecules of the protein contained in a sample. Antibody-based techniques may be employed, such as, for example, immunohistological and immunohistochemical methods for measuring the level of a Glutl in a blood sample. For example, specific recognition is provided by a primary antibody (polyclonal or monoclonal) and a secondary detection system is used to detect presence (or binding) of the primary antibody. Detectable labels can be conjugated to the secondary antibody, such as a fluorescent label, a radiolabel, or an enzyme (e.g., alkaline phosphatase, horseradish peroxidase) which produces a quantifiable, e.g., coloured, product. In another suitable method, the primary antibody itself can be detectably labeled. In some embodiments, a protein extract is produced from a biological sample (e.g., tissue, cells) for analysis. Such an extract (e.g., a detergent extract) can be subjected to western- blot or dot/slot assay of the level of the protein of interest, using routine immunobJotting methods (Jalkanen et al., 1985, J. Cell. Biol. 101: 976-985; Jalkanen et al, 1 87, J. Cell. Biol. 105: 3087-3096).

(0131) Other useful antibody-based methods include immunoassays, such as the enzyme-linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). For example, a protein-specific monoclonal antibody, can be used both as an immunoadsorbent and as an enzyme-labeled probe to detect and quantify an Giutl marker protein of interest. The amount of such protein present in a sample can be calculated by reference to the amount present in a standard preparation using a linear regression computer algorithm (see Lacobilli et al., 1988, Breast Cancer Research and Treatment 11: 19-30). In other embodiments, two different monoclonal antibodies to the protein of interest can be employed, one as the immunoadsorbent and the other as an enzyme-labeled probe.

[0132J Additionally, recent developments in the field of protein capture arrays permit the simultaneous detection and/or quantification of a large number of proteins. For example, low-density protein arrays on filter membranes, such as the universal protein array system (Ge, 2000 Nucleic Acids Res. 28(2):e3) allow imaging of arrayed antigens using standard ELISA techniques and a scanning charge-coupled device (CCD) detector. Immuno-sensor arrays have also been developed that enable the simultaneous detection of clinical analytes. It is now possible using protein arrays, to profile protein expression in bodily fluids, such as in sera of healthy or diseased subjects, as well as in subjects pre- and post-drug treatment.

[0133] Protein capture arrays typically comprise a plurality of protein-capture agents each of which defines a spatially distinct feature of the array. The protein-capture agent can be any molecule or complex of molecules which has the ability to bind a protein and immobilize it to the site of the protein-capture agent on the array. The protein-capture agent may be a protein whose natural function in a cell is to specifically bind another protein, such as an antibody or a receptor. Alternatively, the protein-capture agent may instead be a partially or wholly synthetic or recombinant protein which specifically binds a protein. Alternatively, the protein-capture agent may be a protein which has been selected in vitro from a mutagenized, randomized, or completely random and synthetic library by its binding affinity to a specific protein or peptide target. The selection method used may optionally have been a display method such as ribosome display or phage display, as known in the art. Alternatively, the protein-capture agent obtained via in vitro selection may be a DNA or 'RNA aptamer which specifically binds a protein target (see, e.g., Potyrailo er al, 1998 Anal. Chem. 70:3419-3425; Cohen et al., 1998, Proc. Natl. Acad. Set USA 95:1 272-14277; Fukuda, et al., 1997 Nucleic Acids Symp. Ser. 37:237-238; available from SomaLogic). An alternative to an array of capture molecules is one made through 'molecular imprinting' technology, in which peptides (e.g., from the C -terminal regions of proteins) are used as templates to generate structurally complementary, sequence-specific cavities in a polymerisable matrix; the cavities can then specifically capture (denatured) proteins which have the appropriate primary amino acid sequence (e.g., available from ProteinPrint™ and Aspira Biosystems).

[0134] Exemplary protein capture arrays include arrays comprising spatially addressed antigen-binding molecules, commonl referred to as antibody arrays, which can facilitate extensive parallel analysis of numerous proteins defining a proteome or subproteome. Antibody arrays have been shown to have the required properties of specificity and acceptable background, and some are available commercially (e.g., BD Biosciences, Clontech, Bio ad and Sigma). Individual spatially distinct protein-capture agents are typically attached to a support surface, which is generally planar or contoured. Common physical supports include glass slides, silicon, microwells, nitrocellulose or PVDF membranes, and magnetic and other microbeads.

[0135) Particles in suspension can also be used as the basis of arrays, providing they are coded for identification; systems include color coding for microbeads (e.g., available from Luminex, Bio-Rad and Nanomics Biosystems) and semiconductor nanocrystals (e.g., QDots™, available from Quantum Dots), and barcoding for beads (UltraPlex™, available from Smartbeads) and multimetal microrods (Nanobarcodes™ particles, available from Surromed). Beads can also be assembled into planar arrays on semiconductor chips (e.g., available from LEAPS technology and BioArray Solutions). Where particles are used, individual protein-capture agents are typically attached to an individual particle to provide the spatial definition or separation of the array. The particles may then be assayed separately, but in parallel, in a compartmentalized way, for example in the wells of a microtiter plate or in separate test tubes.

[0136] In operation, a protein sample, which is optionally fragmented to form peptide fragments (see, e.g., U.S. Pat. App. Pub. 2002/0055186), is delivered to a protein-capture array under conditions suitable for protein or peptide binding, and the array is washed to remove unbound or non-specifically bound components of the sample from the array. Next, the presence or amount of protein or peptide bound to each feature of the array is detected using a suitable detection system. The amount of protein bound to a feature of the array may be determined relative to the amount of a second protein bound to a second feature of the array. In certain embodiments, the amount of the second protein in the sample is already known or known to be invariant. f0l37] For analyzing differential expression of proteins between two cells or cell populations, a protein sample of a first cell or population of cells is delivered to the array under conditions suitable for protein binding. In an analogous manner, a protein sample of a second cell or population of cells to a second array, is delivered to a second array which is identical to the first array. Both arrays are then washed to remove unbound or non- specifically bound components of the sample from the arrays. In a final step, the amounts of protein remaining bound to the features of the first array are compared to the amounts of protein remaining bound to the corresponding features of the second array. To determine the differential protein expression pattern of the two cells or populations of cells, the amount of protein bound to individual features of the first array is subtracted from the amount of protein bound to the corresponding features of the second array. ·

[0138] In specific embodiments, the diagnostic prognostic method is implemented using a system comprising at least one end station coupled to a base station. The base station attempts to identify individuals having similar parameter values to the test subjeci and once the status has been determined on the basis of that identification, the base station provides an indication of the diagnosis/prognosis to the end station. [0139] All the essential materials and reagents required for detecting and quantifying Glutl protein or gene expression products may be assembled together in a kit. The kits may also optionally include appropriate reagents for detection of labels, positive and negative controls, washing solutions, blotting membranes, microtiter plates dilution buffers and the like. For example, a nucleic acid-based detection kit may include (i) a Glutl marker polynucleotide (which may be used as a positive control), (ii) a primer or probe that specifically hybridizes to a Glutl marker polynucleotide. Also included may be en2ymes suitable for amplifying nucleic acids including various polymerases (Reverse Transcriptase, Taq, Sequenase™ DNA ligase etc. depending on the nucleic acid amplification technique employed), deoxynucleotides and buffers to provide the necessary reaction mixture for amplification. Such kits also generally will comprise, in suitable means, distinct containers for each individual reagent and enzyme as well as for each primer or probe.

[0140] Alternatively, a protein-based detection kit may include (i) an Glutl marker polypeptide (which may be used as a positive control), (ii) an antigen-binding molecule that is immuno-interactive with an Glutl protein, and optionally an antigen-binding molecule that is immuno-interactive with CD4. The kit can also feature various devices and reagents for performing one of the assays described herein; and/or printed instructions for using the kit to assess the level or activity of a Glutl protein.

[0141] While it is not known how markers of T cell activation such as CD38 and HLA-DR are regulated, the Glutl signalling pathway is well understood making direct targeting possible. As described herein, glucose uptake, glucose-6-phosphate, L-lactate, intracellular p-Akt (T308) and p-Akt (S473) were significantly higher in CD4+Glut+ cells compared to CD4+Glut- cells. This corresponded with an overall increased glucose uptake and glycolysis and lower level or activity of the phosphatase and tensin homologue deleted on chromosome 10 (PTEN) expression in CD4+ T cells from HIV-1 positive compares seronegative subjects. TCR-triggered Glutl expression on CD4+ T cells was found to be sensitive to specific inhibition of the Class IB PI3K-y and mTORCl pathways which were also anti-glycolytic and blocked HIV-1 infection of CD4+ T cells in vitro. Total HIV-1 DNA was higher in CD4+ T cells from patients who had a higher frequency of circulating CD4+Gluil+ T cells. Furthermore CD4+Glutl+ T cells were more susceptible to HIV-1 infection than CD4+Glutl- T cells in vitro but infection was primarily restricted to the CD4+Glutl+OX40+ vs the CD4+Glutl+OX40- subset.

[0142] Accordingly, CD4+ T cells from HIV-1 infected patients have increased glucose uptake and glycolytic activity mediated, at least in part, by the PI3k-y-mTORCl pathway. In some embodiments, Glutl over expression or elevated glycolysis in CD4+T cells may be normalised using P13K-mTOR inhibitors or anti-glycolytic drugs to slow HIV- 1 disease progression and aid immune recovery.

[0143] In another related aspect, the description provides a method of treatment of an HIV infection in a subject, said method comprising administering a Glutl antagonist or an anti-glycolytic agent. The agents are administered for a time and under conditions suitable to reduce the level or activity of Glutl in CD4+ T cells, or to normalise Glutl level or activity and glycolysis in CD4+ T cells in the subject.

[01 4] In some embodiments, the treatment regime includes or the therapeutic agent is cART, an anti-HIV vaccine, an antiglycolytic agent or a CD4+ T cell Glutl antagonist, or a combination of one or two or more of these.

[0145] In some embodiments, the CD4+ T cell Glutl antagonist is a PI3K-mTOR inhibitor.

[0146] In other embodiments, the Pl3K-mTOR inhibitor targets ΡΙ3Κγ.

[0147] In particular embodiments, the PBK-mTOR inhibitor targets mTORC 1.

[0148] In some embodiments, two PI3K-mTOR inhibitors are administered which target PI3Ky and mTORC 1, respectively.

[0149] In some embodiments, the PBK-mTOR inhibitor is administered as a pharmaceutical composition comprising non-cytotoxic amounts of the inhibitor.

[0150J In another related aspect, the description provides a method of treatment of HIV in a subject, said method comprising administering an anti-glycolytic agent to reduce the level or activity of Glutl in CD4+ T cells. It is proposed reducing the level of aerobic glycolysis in CD4+ T cells will feedback to provide reduced levels of Glutl expressed on the surface of CD4+ T cells and reduce levels of HIV infection.

[0151] The term "to reduce the level or activity of Glutl" includes, therefore, direct and indirect modes of antagonising Glutl. Directly acting antagonists interact with the transporter and reduce its activity by preventing transport of glucose. Inhibitory R A molecules directly antagonise Glutl by reducing the expression of Glutl polypeptide thereby reducing the amount of glucose uptake by the cell. Indirect methods of antagonising Glutl activity will bp known generally to those skilled in the art and include, as specifically determined herein reducing P13K-Akt mTORl activity or reducing the transport of Glutl into the cell membrane. Indirect methods of antagonising Glutl activity include the use of anti-glycolytic agents to reduce aerobic glycolysis in CD4+ T cells bearing Glutl transporters.

(0152] Once Glutl is recognised as a useful target in the treatment or prevention of HIV/AIDS progression the skilled person will be aware of various agents that will act as antagonists of Glutl levels or activity or of downstream aerobic glycolysis in CD4+ T cells. Furthermore, Glutl is identified as a target for screening assays to identify a new class of anti-HIV agents directed to normalising glycolysis. Many methods of screening will be apparent to the skilled person.

[0153] In one illustrated embodiment, the Glutl antagonist is an inhibitor of mTORC 1 or PI3 -p 110γ activity.

[0154] In some embodiments, the Glutl antagonist is Fasentin or sodium valproate or similar agents that modulate the level of Glutl .

[0155] In one illustrative embodiment, the Glutl antagonist is AS-605240 or an analog which targets the mTOR (mTORCl) pathway. Reference to AS-605240 includes its salts, homologs, analog, isoforms and enantiomers including stereoisomers. Functional equivalent forms of AS-605240 are also contemplated herein. AS-605240 has the chemical formula C12H7N302S (CAS No. 648450-29-7). A functional equivalent includes a molecule sterically similar to AS-605240 and having at least the same ability to inhibit TORC1. An example of a functional equivalent includes the change or modification of a substituent on the AS-605240 molecule, selecting a particular isoform, naturally occurring or prepared by physical or chemical manipulation and selecting a molecule with the same physical or chemical properties as described herein.

[0156] In another illustrative embodiment, the agent is a rapalogue (rapamycin analogue) such as CCI-779 (temsirolimus), sirolimus and everolimus or an analog which targets the Class IB ΡΙ3Κ-γ (pi lOy) pathway. Temsirolimus (CCI-779, torisel) has the formula C56H87N016 (CAS No. 162635-04-3). Reference to temsirolimus or everolimus includes its salts, homologs, analog, isoforms and enantiomers including stereoisomers. Functional equivalent forms of temsirolimus or everolimus are also contemplated herein. A functional equivalent includes a molecule sterically similar to temsirolimus or everolimus and having at least the same ability to inhibit Class IB ΡΙ3 -γ (pHOy) pathway. An example of a functional equivalent includes the change or modification of a substituent on the temsirolimus or everolimus molecule, selecting a particular isoform, naturally occurring or prepared by physical or chemical manipulation and selecting a molecule with the same physical or chemical properties as described herein.

[0157] Accordingly in some embodiments, the Glutl antagonist is an anti-glycolytic (an inhibitor of glycolysis) agents such as those aforementioned or as used in the treatment of cancer, optionally in combination with other agents such as those that reduce non-target effects. Illustrative anti-glycolylic agents include 3-bromopyryvatc and D-fructose-6- phosphate. Another illustrative anti-cancer agent useful in the present invention to reduce Glutl activity is a 2-Deoxyglucose (2-DG).

[0158] In some embodiments, the Glutl antagonist is an antibody or antigen-binding part thereof that specifically recognise Glutl.

[0159] In some embodiments, the Glutl antagonist is a small chemical molecule, a peptide such as a stapled peptide or foldamer or an inhibitory RNA molecules typically inhibit Glutl gene translation or transcription. Translation may be suppressed by reducing translational efficiency or reducing message stability or a combination of their effects. ethods of producing chimeric constructs capable of inducing RNA interference in eukaryotic cells are described in the art.

[0160] Furthermore, Glutl is identified as a target for screening assays to identify a new class of anti-HIV agents. Many methods of screening will be apparent to the skilled person. In one embodiment screening assays identify antibodies or antigen binding fragments that bind specifically to Glutl and reduces its activity.

[0161] Typically, the therapeutic agents will be administered in pharmaceutical compositions together with a pharmaceutically acceptable carrier and in an effective amount to achieve their intended purpose. The dose of active compounds administered to a subject should be sufficient to achieve a beneficial response in the subject over time such as a reduction in, or relief from, the symptoms of HIV infection/AlDS. Efficacy may be measured however by assessing the level of Glutl, normalisation of Glutl levels and of aerobic glycolysis. The quantity of the pharmaceutically active compounds(s) to be administered may depend on the subject to be treated inclusive of the age, sex, weight and general health condition thereof. In this regard, precise amounts of the active compound(s) for administration will depend on the judgement of the practitioner. In determining the effective amount of the active compound(s) to be administered in the treatment of HIV, the medical practitioner may evaluate severity of any symptom associated with HIV infection or treatment thereof including tachycardia, fever, chills, vomiting, diarrhoea, skin rash, headaches, confusion, muscle aches, seizures. In any event, those of skill in the art may readily determine suitable dosages of the therapeutic agents and suitable treatment regimens without undue experimentation.

[0162] The term "treat ¹ is meant to include both therapeutic and prophylactic treatment.

[0163] By "effective amount", in the context of treating or preventing a condition is meant the administration of that amount of active to an individual in need of such treatment or prophylaxis, either in a single dose or as part of a series, that is effective for the prevention of incurring a symptom, holding in check such symptoms, and/or treating existing symptoms, of HIV/AIDS. In addition, the effective amount may be one which reduces the various markers described herein such as CD4+ T cell Glutl, glycolysis, CD4+ T cell activation markers, CD4+T cell inflammation markers, lactate levels, levels of ΡΙ3Κγ and mTORCl pathway activity, etc. The effective amount will vary depending upon the health and physical condition of the individual to be treated, the formulation of the composition, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.

[0164] The therapeutic agents and pharmaceutical compositions of the present description can be used in combination with one or more additional agents such as described herein. The dose of the second agent can be appropriately selected based on a clinically employed dose. The proportion of the compound of the present invention and the second agent can be appropriately determined according to the administration subject, the administration route, the target disease, the clinical condition, the combination, and other factors. In cases where the administration subject is a human, for instance, the second agent may be used in an amount of 0.01 to 100 parts by weight per part by weight of the antiglycolytic agent or Glutl antagonist. The second compound of the pharmaceutical combination formulation or dosing regimen preferably has complementary activities to the compound of this invention such that they do not adversely affect each other. Such agents, such as cART or other antiglycolytic agent or Glutl antagonist are suitably present in combination in amounts that are effective for the purpose intended. Accordingly, another aspect of the present invention provides a composition comprising an antiglycolytic agent or Glutl antagonist in combination with a second agent, such as described herein. An antiglycolytic agent or Glutl antagonist and the additional pharmaceutically active agent(s) may be administered together in a unitary pharmaceutical composition or separately and, when administered separately this may occur simultaneously or sequentially in any order. Such sequential administration may be close in time or remote in time. The amounts of the antiglycolytic agent or Glutl antagonist and the second agent(s) and the relative timings of administration will be selected in order to achieve the desired combined therapeutic effect. The combination therapy including combinations of antiglycolytic agent or Glutl antagonist such as temsirolimus and AS-605240 may provide "synergy" and prove "synergistic", i.e., the effect achieved when the active ingredients used together is greater than the sum of the effects that results from using the agents separately. A synergistic effect may be attained when the active ingredients are: (1) co-formulated and administered or delivered simultaneously in a combined, unit dosage formulation; (2) delivered by alternation or in parallel as separate formulations; or (3) by some other regimen.

[0165] The compounds of the invention may be administered by any route appropriate to the condition to be treated. Suitable routes include oral, parenteral (including subcutaneous, intramuscular, intravenous, intraarterial, intradermal, intrathecal and epidural), transdermal, rectal, nasal, topical (including buccal and sublingual), vaginal, intraperitoneal, intrapulmonary and intranasal. It will be appreciated that the preferred route may vary with for example the conditio of the recipient. Where the compound is administered orally, it may be formulated as a pill, capsule, tablet, etc. with a pharmaceutically acceptable carrier, diluent or excipient. Where the compound is administered parenterally, it may be formulated with a pharmaceutically acceptable parenteral vehicle and in a unit dosage injectable form, as detailed below.

[0166] In order to use a antiglycolytic agent or Glutl antagonist for the therapeutic treatment (including prophylactic treatment) of mammals/vertebrates including humans, it is normally formulated in accordance with standard pharmaceutical practice as a pharmaceutical composition. According to this aspect of the invention there is provided a pharmaceutical composition that comprises a therapeutic agent as described herein.

[0167] Pharmaceutical compositions are formulated, dosed and administered in a fashion, i.e., amounts, concentrations, schedules, course, vehicles and route of administration, consistent with good medical practice. The antiglycolytic agent or Glutl antagonist is typically formulated into pharmaceutical dosage forms to provide an easily controllable dosage of the agent and to enable patient compliance with the prescribed regimen..

[0168] Pharmaceutical formulations of the antiglycolytic agent or Glutl antagonist may be prepared for various routes and types of administration. For example, an agent having the desired degree of purity may optionally be mixed with pharmaceutically acceptable diluents, carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences (1 80) 16th edition, Osol, A. Ed.), in the form of a lyophiJized formulation, a milled powder, or an aqueous solution. Formulation may be conducted by mixing at ambient temperature at the appropriate pH, and at the desired degree of purity, with physiologically acceptable carriers, i.e., carriers that are non-toxic to recipients at the dosages and concentrations employed. The pH of the formulation depends mainly on the particular use and the concentration of compound, but may range from about 3 to about 8. Formulation in an acetate buffer at pH 5 is a suitable embodiment. The formulations may be prepared using conventional dissolution and mixing procedures. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0169] Formulations suitable for parenteral administration include aqueous and nonaqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The compositions may also be in a form suitable for oral use (for example as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs), for topical use (for example as creams, ointments, gels, or aqueous or oily solutions or suspensions), for administration by inhalation (for example as a finely divided powder or a liquid aerosol), for administration by insufflation (for example as a finely divided powder). Suitable pharmaceutically- acceptable excipients for a tablet formulation include, for example, inert diluents such as lactose, sodium carbonate, calcium phosphate or calcium carbonate, granulating and disintegrating agents such as com starch or algenic acid; binding agents such as starch; lubricating agents such as magnesium stearate, Stearic acid or talc; preservative agents such as ethyl or propyl p-hydroxybenzoate, and anti-oxidants, such as ascorbic acid. Tablet formulations may be uncoated or coated either to modify their disintegration and the subsequent absorption of the active ingredient within the gastrointestinal tract, or to improve their stability and/or appearance, in either case, using conventional coating agents and procedures well known in the art. Compositions for oral use may be in the form of hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules in which the active ingredient is mixed with water or an oil such as peanut oil, liquid paraffin, or olive oil. Aqueous suspensions generally contain the active ingredient in finely powdered form together with one or more suspending agents, such as sodium carboxyrnethylcellulose, methylcellulose, hydroxypropylmethylccllulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents such as lecithin or condensation products of an alkylene oxide with fatty acids (for example polyoxethylene stearate), or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The amount of a compound of this invention that is combined with one or more excipients to produce a single dosage form will necessarily vary depending upon the subject treated, the severity of the disorder or condition, the rate of administration, the disposition of the compound and the discretion of the prescribing physician. In one embodiment, a suitable amount of a compound of this invention is administered to a mammal in need thereof. Administration in one embodiment occurs in an amount to provide between about 0.1 nM to 800nM amounts in the subject, or between about between about 0.5n and 300n amounts in the blood stream, liver etc. In some instances, dosage levels below the lower limit of the aforesaid range may be more than adequate, while in other cases still larger doses may be employed without causing any harmful side effect, provided that such larger doses are first divided into several small doses for administration throughout the day. For further information on routes of administration and dosage regimes, see Chapter 25.3 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press 1990, which is specifically incorporated herein by reference.

[0170] In another embodiment of the invention, an article of manufacture, or "kit", containing materials useful for the treatment of HIV associated disease progression is provided. Suitable containers include, for example, bottles, vials, syringes, blister pack, etc. The container may be formed from a variety of materials such as glass or plastic. In one embodiment, the kit comprises a container comprising a compound of this invention. The container may hold a compound of this invention or a formulation thereof which is effective for treating the condition and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). In another embodiment, the kit comprises a container comprising a system for assaying the level or activity of Glutl on CD4 T cells. The kit may further comprise a label or package insert on or associated with the container. The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such.

[0171] The study described herein is the first to evaluate glucose metabolism in leucocytes from HIV+ patients. As determined herein, CD4+ T cells from HIV-1 infected patients are characterized by a substantial increase in expression of the glucose transporter Glutl. The increase in CD4+Glutl+T cells correlates strongly with low absolute CD4+ T cell count and is partially normalized by cART. However even in the settin of controlled viral replication there was a significant correlation between CD4+ T cell count and the frequency of CD4+Glutl+ T cells. CD4+Glutl+ T cells had higher expression of CD38 and HLA-DR than CD4+Glutl- cells but the majority of CD4+Glutl+ T cells did not co- express these activation markers. The circulating CD4+Glutl+ T cells are preferentially distributed within memory and effector CD4+ T cell populations. CD4+GIutl+ have higher glycolytic activity than Ct>4+Glutl-T cells and increased permissiveness to HIV-1 infection with are partially attributed to PI3Ks-mTORCl signalling. f0172] The relationship between percentage CD3+CD4+ T cells and CD4 T cell count in HIV-1 infected patients is shown in Figure 14. (A) Spearman's correlations between percentage CD3+CD4+ T cells and the percentage (left panel) of CD4+Glutl+ T cells in peripheral blood from HIV+ and HIV+/cART subjects and between percentage CD4+Glutl+ T cells and absolute CD4 T cell count (right panel). (B) Comparative relationship between the percentage of CD4+Glutl+ T cells and markers of CD4+ T cell activation, and the percentage of CD3+CD4+ T cells in cART-naYve HIV+ and (C) HIV+/CART subjects. (0173) A comparison of the percentage of Glutlc-term CD4+ T cells from H1V-, HIV+ and HlV+/cART subjects was undertaken and the results are illustrated in Figure 15. Figure 15 (B) shows a comparison of the MFI of c-terminal Glutl expression in CD4+ T cells from HIV-, HIV+ and HIV+/cART subjects. The antibody used here detected intracellular Glutl expression in CD4+ T cells from H1V+ subjecis. The data suggest for the first time that not only does HIV affects Glutl cell surface expression but also the intracellular machinery that synthesize Glutl. Antibody was obtained from Abeam, MA, USA.

[0174] Inhibitors of ΡΙ3Κγ and mTORCl suppress markers of HIV immune activation and general inflammation (see Figures 18 A through C). Inhibitors of Ρΐ3Κ _γ (AS-605240, 200 nM) and mTORCl (temsirolimus, 10 nM) suppress markers of immune activation and inflammation. PBMCs were either left untreated (UT) or pre-treated with AS-605240 or temsirolimus alone or in combination (Comb.) for 48h prior to activation with antiCD3/CD28 microbeads for an additional 24h. Some PBMCs were not treated with inhibitors but were activated with antiCD3/CD28 microbeads (act). (A-B) The T cell activation marker HLADR and CD38 were evaluated by flow cytometry. (C) Secreted T F in culture was detected by ELISA. Data was analysed using the Wilcoxon matched- pairs signed rank test.

[0175] Inhibitors of ΡΙ3Κγ and mTORCl significantly suppress glucose uptake and L-lactate (glycolytic output) in purified and activated CD4+ T cells as shown in Figures 1 A through B. Inhibitors of Pl3K _y (AS-605240, 200 nM) and mTORCl (temsirolimus, 100 nM, everolimus, 20nM) suppress glucose uptake and glycolysis in CD4+ T cells. CD4+ T cells were purified by negative selection from HIV- healthy donors and stimulated with an activation cocktail consisting of PMA (100 ng/ml), Ionomycin (1 g ml) and IL-2 (5ng/ml). Cells were activated with the activation cocktail for 48h, replaced with fresh media, and were left untreated (Act) or treated with either, AS-605240, temsirolimus, a combination of AS-605240 and temsirolimus (Comb.), everolimus (20 nM) or SB201290 (lOuM, p38 inhibitor) for 48h. UT represent unactivated cells. (A) Glucose and (B) L- lactate concentrations in culture media after 48h of treatment. Glucose concentration in media represents the concentration of glucose in RPMI-1640 supplemented with 10% human serum, L-glutamine and penicillin and streptomycin. 500,000 activated cells were cultured in 500 μΐ of media. Note that the inhibitors suppress glucose uptake hence those media contain higher glucose levels compared to the untreated activated (Act.) cells. Data was analysed using the Wilcoxon matched-pairs signed rank test. CD4+Glutl+ T cells from HIV+ subjects have high glucose uptake and glycolytic metabolism and this was associated with low CD4 T cell count. It is proposed that these inhibitors could be explored to normalize the glucose metabolic activity in these cells.

[0176J 2 nM of everolimus was sufficient to reduce L-lactate production in activated CD4+ T cells as illustrated in Figure 20. Relatively low concentrations of the mTOR inhibitor everolimus suppresses L-lactate production in activated CD4+ T cells. CD4+ T cells were purified by negative selection from HIV- healthy donors and stimulated with an activation cocktail consisting of PMA (100 ng/ml), Ionomycin (1 μg/ml) and IL-2 (5ng/ml). Cells were left inactivated (UT), activated with the activation cocktail for 48h, replaced with fresh media, and were left untreated (Act) or treated with different concentrations of everolimus for 72h. 500,000 activated cells were cultured in 500 μΐ of media. L-lactate concentrations in cell media after 48h of treatment.

[0177] The inhibitory effect of temsirolimus on activated CD4+ T cells is prolonged in its absence for up to 4 days as shown in Figure 21. The inhibitory effect of temsirolimus is maintained in its absence. CD4+ T cells were purified by negative selection from HIV- healthy donors and stimulated with an activation cocktail consisting of PMA (100 ng/ml), Ionomycin (1 μ /ml) and IL-2 (5ng/ml). Cells were activated with the activation cocktail for 48h, replaced with fresh media, and were left untreated (Act) or treated with either, AS-605240, temsirolimus, a combination of AS-605240, temsirolimus (Comb.), everolimus (20 nM) or SB201290 (lOuM, p38 inhibitor) for 48h. Cells were washed three times in fresh culture media and 500,000 activated cells were seeded in 500 μΐ of media in a 48-well culture plate. L-lactate concentrations were measured in cell media after 4 days. Data was analysed using the Paired T test.

[0178) Inhibitors of ΡΙ3Κγ and mTORCl significantly suppress the basal extracellular acidification rate (a proxy for the rate of glycolysis) of activated CD4+ T cells as shown in Figure 22. Cell viability assessment illustrated no significant toxicity of the inhibitors to CD4+T cells (data not shown), however reduced cell growth and cell size was observed (data not shown) in cultures treated with the inhibitors. To demonstrate that the changes in glucose metabolism and Lactate production was not simply due to cell growth inhibition 500,000 purified and activated CD4+ T cells and cells pre-treated with the PDKy inhibitor AS-605240 (Selleck, TX, USA) and the mTORCl inhibitor temsirolimus (Selleck) was incubated for 48h, resuspended them in XF modified DMEM Assay medium and seeded them in 24-well Seahorse XF-24 plates (Seahorse Bioscience, MA, USA), pre-coated with 0.01% collagen. The glycolytic capacity of cells were analysed using the Seahorse XF analyser according to manufacturer's instruction (Seahorse Bioscience). The basal extracellular acidification rate (ECAR) was measured after approximately 25 min, well before cell proliferation. As shown the inhibitors of ΡΓ3Κγ and mTORCl significantly suppress the basal ECAR.

[01791 Inhibitors of ΡΒΚγ and mTORCl significantly suppress PI3K and mTOR activity in activated CD4+ T cells as shown in Figure 23. Western Blot Analysis showing the effects of inhibitors on PI3K (Phos - Ser473A t) and mTOR (Phos -p70S6 ) activation. Samples were lysed and protein concentration determined. Lysates were solubilised and 10 μg protein was loaded and resolved by SDS-PAGE on polyacrylamide gels, transferred to membranes and blocked with 5% BSA. Immunoblotling was performed using the following primary antibodies: phosphorylated Akt (Ser473), Total Akt, phosphorylated p70S6K, total p70 S6 kinase and β-actin (all from Cell Signaling Technology, Danvers, MA, USA). After incubation with appropriate secondary antibody, the immunoreactivc proteins were detected with enhanced chemi luminescence and quantified by densitometry.

[0180] 1 nM of temsirolimus was sufficient to lower glucose metabolism in activated CD4+ T cells as shown in Figure 24. Relatively low concentrations of ΡΙ3 γ (AS-605240) and mTORCl (temsirolimus) inhibitors suppress glucose uptake. CD4+ T cells were purified by negative selection from HIV- healthy donors and stimulated with an activation cocktail consisting of PMA (100 ng/ml), lonomycin (1 g mj) and lL-2 (5ng/ml). Cells were activated with the activation cocktail for 48h _> replaced with fresh media, and were left untreated (Act) or treated with different concentrations of (A) AS-605240 or (B) temsirolimus for 72h. 500,000 activated cells were cultured in 500 μΐ of media. Note that the inhibitors suppress glucose uptake hence the media contain higher glucose levels compared to the untreated activated (Act.) cells. Data was analysed using the Wilcoxon matched-pairs signed rank test.

[0181] Inhibition of ΡΙ3 γ and mTORCl does not suppress HIV or other antiviral responses in cells from patients on cART as shown in Figures 25. Inhibition of ΡΙ3Κγ and mTORCl does not suppress HIV-GAG specific T cellular immune responses in HIV+ subjects on cART. PBMCs were obtained from HIV+ subjects on cART. Cells were prc- treated with AS-605240 (200 nM), temsirolimus (100 nM) or everolimus (20nM) for 48h and then stimulated with HIV-GAG peptide pool or CEF peptides (pool of CMV, EBV and Flu). ELISPOT assay to detect INF _r secreting cells was conducted as previously described by Palmer et al, 2008.

[0182] Inhibition of PI3Ky and mTORCl suppresses glycolytic gene expression in activated CD4+ T cells as shown in Figure 26. Inhibitors of ΡΙ3Κγ (AS-605240, 200 nM) and mTORCl (temsirolimus, 100 nM, everolimus, 20nM) suppress the expression of genes within the glycolytic pathway. CD4+ T cells were purified by negative selection from HIV- healthy donors and stimulated with an activation cocktail consisting of PMA (100 ng/ml), lonomycin (1 μg/ml) and IL-2 (5ng/ml). Cells were activated for 48h, left untreated (Act) or treated with either, AS-605240, temsirolimus, everolimus (20 nM) or SB201290 (lOuM, p38 inhibitor) for 48h. Gene expression was normalized relative to β-actin. Amplification was done on the Stratagene MX-3000 system and analysis was conducted using the comparative threshold method using β-actin as the reference gene. PCR conditions were 50°C for 2 min, 95°C for 10 min and 40 cycles of (95°C/30 sec; 60°C, 1 min).

[0183) Illustrative methods and materials used in the Examples are described as follows. Participant recruitment and blood separation

(0184] The study population included 45 untreated HIV-1 infected individuals (38 progressors and 7 long term non-progressors, LTNPs), 35 HIV-1 infected subjects on combination antiretroviral therapy (cART) and 25 HIV seronegative controls. LTNPs and described as those with >10 years HIV- 1 infection, cART-naive, CD4 count >350 cells/ul

Φ

and viral load <20,000 copies/ml. Subjects were recruited from the Infectious Diseases Unit at The Alfred Hospital, Melbourne and from the community. Samples were also obtained from the Clinical Research Core (CRC) Repository or Specimen Collection Service, Center for AIDS Research, University of Washington, USA. Informed consent was obtained from all participants and the research was approved by The Alfred hospital Research and Ethics Committee and University of Washington Ethics Committee. Fresh blood samples from patients recruited in Melbourne were collected in EDTA, citrate or heparin anticoagulant tubes; cryopreserved PBMCs were shipped to Melbourne from Washington. Male participants were predominantly recruited because HIV-1 patients in Australia are predominantly male (>90%). The main exclusion criteria included co- infection with HCV, vaccination, physical trauma or surgery within 3 weeks prior to participation.

Phenotyping

[0185] Blood collected into anticoagulant was mixed with a 20 volumes of 1 * FACS lysing solution (BD Biosciences), incubated on ice for 10 min, then washed twice at 4°C with FACS wash (0.5% BSA in calcium- and magnesium-free phosphate-buffered saline (Invitrogen)). Fresh cells or thawed PBMCs (> 90% viability) were stained on ice for 30 min in the dark using the following pre-titrated antibodies: Glutl-FITC/APC (R and D Systems, MN, USA), CD3-PE, CD4-PerCP, CD 8- APC CD14-APC, CD16-PE.Cy7, CD27- APC, CD28-APC, CD45-RA-PE, CD38-PE, CDS 6- APC, Ki67-APC, CD57-FITC, CCR5- APC, HLA-DR-FITC (all from BD Biosciences). In parallel, samples were stained with appropriate conjugated immunoglobulin isotype control antibodies and used to set gates for defining positive staining. Cells were washed twice in FACS wash and fixed in 0.5% formaldehyde prior to analysis. Analysis was performed on a FACSCalibur flow cytometer (BD Biosciences). At least 100,000 events were acquired within the lymphocyte or monocyte gates and analyzed using FlowJo software, version 8;8 (Tree Star, Inc, Ashland OR).

PBMC Preparation

[0186] Blood was obtained by venipuncture into tubes containing citrate or EDTA anticoagulant and peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation (Ficoll-paque, Pharmacia Uppsala, Sweden) as previously described (Palmer et al, 2008) and cryopreserved in 10% dimethlyl sulfoxide (DMSO, Sigma-Aldrich, St- Louis, MO) and 90% autologous plasma.

Plasma cytokine and biochemical analysis

[0187] Cytokines were assayed using a high sensitivity Milliplex cytokine panel (Millipore) and read using a Luminex 100 instrument. 25 OH Vitamin D was assayed using the IDS iSYS assay (Immuno diognistic systems Inc, AZ, USA) as per manufacturers recommendations. Insulin, high sensitivity CRP and D-dimer were assayed using immunoturbidimetric assays ( amiya) and read on the Cobas Mira instrument. Glucose, ALT, creatinine, triglyceride total and HDL cholesterol was measured on the Beckman DXi 600 instrument. AH these analysis were conducted by Cardinal Bio-research Pty Ltd (Queensland, Australia).

Proliferation assay

[0188] Peripheral blood mononuclear cells were resuspended at 1 * 10 ⁶ cells/mL in xPBS and incubated at 37 °C for 7 min with 2.5 μΜ carboxyfluorcscein diacetate succinimidy] ester (CFSE, Invitrogen Life Technologies, Australia). CFSE labelling was terminated by washing the cells three times with cold xPBS/0.5% FCS (y/v).

Inhibition studies

[0189) Inhibitor (Table SI) stocks in dimethyl sulfoxide (DMSO) were stored at -20°C and diluted in sterile PBS when required. Vehicle controls received the same final volume of DMSO concentration as all agent-treated incubations. Glucose uptake assay

[01 0J The fluorescently labelled glucose analogue 2-(N-(7-nitrobenz-2-oxa-l, 3- diazol-4-yJ) amino)-2 deoxyglucose (2-NBDG) (Invitrogen, Vic, Australia) was used to measure the activity of glucose transporters in T cells. PBMCs were thawed and allowed to recover for 2h in RPMI-1640 medium supplemented with 10% human serum, penicillin streptomycin (Invitrogen), 2mM L-glutamine (Invitrogen) at 37 ^* C, 5% CO ₂ . Cells were then treated with 2-NBDG incubated for the indicated times, washed twice with x lPBS, stained for cell surface markers, washed and resuspended in PBS and analysed within 15 minutes on a FACSCalibur (BD Biosciences, NSW, Australia). ,

Gene expression analysis

[0191] Cells were lysed in lysis buffer (0.-1 M Tris HC1, pH 7.5 containing 1 % lithium dodecyl sulphate, 0.5 M LiCl, 10 mM EDTA, 5 mM DTT) and mRNA isolated using oligo(dT) magnetic beads (Invitrogen). cDNA was prepared using a Transcriptor First Strand cDNA Synthesis Kit (Roche) and Glutl gene expression was evaluated by quantitative real-time PCR using Brilliant® II SYBR® Green QPCR Master Mix (Stratagene) and employing the following primer sets: Primers for β-actin:

Forward: 5'-3': AGGCATCCTCACCCTGAAGT (SEQ ID NO: 1) Reverse: 5'-3' : GCGTACAGGGATAGCACAGC (SEQ ID NO: 2) and

Glutl primers:

Forward Primer TCTGGCATCAACGCTGTCTTC (SEQ ID NO. 3) Reverse Primer CGATACCGGAGCCAATGGT (SEQ ID NO: 4).

[0192] The amino acid sequence for an illustrative Glutl is the 492 aa human glucose transporter type 1 (Glutl; UniProt Acc. No. PI 1 166):

MEPSS KLTGRLMLAVGGAVLGSLQFGYNTGVINAPQKVIEEFYNQTWVHRYGE

SILPTTLTTLWSLSVAIFSVGGMIGSFSVGLFVNRFGRRNSMLMMNLLAFVSAVLM

GFSKLGKSFEMLILGRFHGVYCGLTTGFVPMYVGEVSPTALRGALGTLHQLGIVV

GILIAQVFGLDSIMGN DLWPLLLSIIFIPALLQCIVLPFCPESPRFLLINRNEENRAK

SVLKKLRGTADVTHDLQEMKEESRQMMRE VTILELFRSPAYRQPILIAVVLQLS QQLSGINAVFYYSTSIFEKAGVQQPVYATIGSGIVNTAFTVVSLFVVERAGR TLHL IGLAGMAGCAILMTIALALLEQLPWMSYLSIVAIFGFVAFFEVGPGPIPWFIVAELI-S QGPRPAAIAVAGFSNWTSNFIVGMCFQYVEQLCGPYVFIIFTVLLVLFFIFTYFKVP ET GRTFDEIASGFRQGGASQSDKTPEELFHPLGADSQV (SEQ ID NO: 5).

[0193) Illustrative natural variants are described at uniprot.org.

(0194] Illustrative nucleic acid sequences encoding Glutl include the cDNA sequence (Genbank Acc. No. BC118590):

atggagcccagcagcaagaagctgacgggtcgcctcatgctggccgtgggaggagcagtg cttggctccctgcagtttggctac aacactggagtcatcaatgccccccagaaggtgatcgaggagttctacaaccagacatgg gtccaccgctatggggagagcatc ctgcccaccacgctcaccacgctctggtccctctcagtggccatcttttctgttgggggc atgattggctccttctctgtgggccttttc gttaaccgctttggccggcggaattcaatgctgatgatgaacctgctggccttcgtgtcc gccgtgctcatgggcttctcgaaactgg gcaagtcctttgagatgctgatcctgggccgcttcatcatcggtgtgtactgcggcctga ccacaggcttcgtgcccaigtatgtgg gtgaagtgtcacccacagcccttcgtggggccctgggcaccctgcaccagctgggcatcg tcgtcggcatcctcatcgcccaggt gttcggcctggactccatcatgggcaacaaggacctgtggcccctgctgctgagcatcat cttcatcccggcccigctgcagtgcat cgtgctgcccttctgccccgagagtccccgcttcctgctcatcaaccgcaacgaggagaa ccgggccaagagtgtgctaaagaa gctgcgcgggacagctgacgtgacccatgacctgcaggagatgaaggaagagagtcggca gatgatgcgggagaagaaggt caccatcctggagctgttccgctcccccgcctaccgccagcccatcctcatcgctgtggt gctgcagctgtcccagcagctgtctg gcatcaacgctgtcttctattactccacgagcatcttcgagaaggcgggggtgcagcagc ctgtgtatgccaccattggctccggta tcgtcaacacggccncactgtcgtgtcgctgtttgtggtggagcgagcaggccggcggac cctgcacctcataggcctcgctgg catggcgggttgtgccatactcatgaccatcgcgctagcactgctggagcagciaccctg gatgtcctatctgagcatcgtggccat ctttggctttgtggccttctttgaagtgggtcctggccccatcccaiggttcatcgtggc tgaactcttcagccagggtccacgtccag ctgccattgccgttgcaggcttctccaactggacctcaaatttcattgtgggcatgtgct tccagtatgtggagcaactgtgtggtccc tacgicttcatcatcttcactgtgctcciggttctgttcttcatcttcacctacttcaaa gttcctgagactaaaggccggaccncgatga gatcgcttccggcttccggcaggggggagccagccaaagtgacaagacacccgaggagct gttccatcccctgggggctgattc ccaagtgtga (SEQ ID NO: 6).

HIV-l infection and ON A amplification

(0195] HIV-l infection of T cells were conducted using the CXCR4 tropic NL4.3-

GFP lab strain and the CCR5 tropic lab strain NL4.3(AD8)-GFP at lCPM/ccll. Samples were treated with the virus for 2 hours at 37 °C, wash twice with cold * 1PBS and cells were resuspend at concentration of 2.5 * 10 ⁶ in supplemented RPMI-1640 containing 5 ng/ml IL-2. Viral infection was determined by the detection of GFP+ cells within the FL1 channel of a FACSCalibur (BD Biosciences, NSW, Australia).

[0196] For HIV-1 detection in clinical samples, CD4+ T cells were purified from patient's PBMCs using the Human EasySep CD4+ T cell enrichment kit (Stem Cell, Technology Inc, Vancouver, BC, Canada), and lysates were analysed for total HIV DNA and integrated DNA using real-time RT-PC as previously described (Butler, 2001, O'Doherty, 2002, Lewin, 2008).

Pltosflow and intracellular stain analysis

[0197] PBMCs were thawed and resuspended in RPMI-1640 medium supplemented with 10% human serum, penicillin streptomycin (Invitrogen), 2mM L- glutamine (Invitrogen) and allowed to rest for 24h at a concentration of 1 *10 ⁶ cell/ml at 37°C, 5% COz. Cells were fixed and stained using the Intrastain Kit (DAKO). Antibodies employed were anti-pAkt (pS473), anti-pAkt (T308) and PTEN (BD Biosciences). Cells were washed twice with 0.5% paraformaldehyde and analysed.

Metabolism assays

(0198] For Glucose-6-phosphate (G-6-P) assay 5 * 10 ⁶ cell were rested in supplemented RPMI-1640 medium described above with or without glucose (glucose- limiting) for 4h. Cells were pelleted, washed once in ice cold *1 BS and resuspended in 50 μΐ of ice cold *1PBS. Cells were sonicated for 30 min on the 'high setting" of a Soniciean sonicator (Soniciean PTY Ltd, SA, Australia). Cell lysates were spun at 10,0000 rpm at 4°C for 5 min and G-6-P levels were determined using a G-6-P assay kit according to manufacturer's protocol (Biovision, CA, USA).

(0199) Secreted lactate concentrations in cell-free culture supernatants were determined by using the Glycolysis Cell-Based Assay Kit according to manufacturer's protocol (Cayman Chemical Company, MI, USA). To measure intracellular levels of lactate, cells were rested for 24h in supplemented RPMI-1640 medium and suspensions were stained using the Glycolysis Cell-Based Assay Kit according to manufacturer's protocol. Stained cells were washed once in wash buffer (0.5% FCS PBS), stained with cell surface markers and resuspended in *1 PBS prior to analysis. The highly coloured intracellular formazan was detected in the FL3 channel on a FACSCalibur.

Seahorse XF analyser

[0200] 500,000 Jurkat cells pre-treated with the PI3K* inhibitorAS-605240

(Selleck, TX, USA) and the mTORCl inhibitor temsirolimus (Selleck) for 48h were resuspended in XF modified DMEM Assay medium seeded in 24-well Seahorse XF-24 plates (Seahorse Bioscience, MA, USA), pre-coated with 0.01% collagen. The glycolytic capacity of cells were analysed using the Seahorse XF analyser according to manufacturer's instruction (Seahorse Bioscience). Extracellular acidification rate (ECAR) (a proxy for the rate of glycolysis) were measured basally as well as after sequential injection of the glucose (10 mM), ATP synthase inhibitor oligomycin (Ι Μ) and the glucose uptake inhibitor 2-deoxyglucose (50 mM).

Statistical Analysis

[0201) Statistical analysis was performed using OraphPad Prism statistical software (GraphPad Software, San Diego, CA). The non-parametric Mann- Whitney U was used for comparison tests of unpaired data and the Wicoxon matched pairs signed rank test used for paired data. Spearman Rank test was used for correlation analyses. Measures of central tendency are expressed as median and inter-quartile range (IQR 25th, 75th percentile). Linear regression was applied to assess the relationship between % of CD4+Glutl+ and other covariates. Percentage of CD4+GIutl+ was log transformed in order to meet the normal distribution assumption. The markers significant in the univariable analyses were entered in a multivariable linear regression model and the final model was derived through a process of backward elimination. P- values <0.05 were considered significant. All statistical analyses were performed using Stata (version 11).

[0202] The present invention is further described by the following non-limiting Examples. ' EXAMPLE 1

[0203] Subject clinical characteristics - Demographic characteristics and clinical parameters of patients and controls recruited into this study are summarized in Table 1. A total of 105 participants including 45 HIV-1+ treatment naive (HIV+, 7 of whom were LTNP), 35 HIV-1+ combination antiretroviral treatment experienced (HIV+/cART) and 25 HIV seronegative (HIV-) control subjects were recruited. Subjects within each group were matched with respect to sex, body mass index (BMI) and age. When possible, participants were also matched on the basis of sexual preferences and lifestyle, including alcohol intake, exercise regimen and smoking. There were no significant differences between BMI and age between the groups and 11/25 (44%) of the HIV- control subjects were self-reported MSM. At the time of recruitment the mean CD4 cell count in the HIV+ and HlV+/cART groups was 397 ±193 and 496 ± 256 cells/μΐ cells/μΐ, p=0.06, respectively. There were no significant differences in the fasting levels of plasma glucose, insulin, total cholesterol, creatine and vitamin D between the 3 groups. Plasma concentrations of T F were significantly elevated in the HIV+ (p=0.005) and HIV+/cART (p=0.02) groups vs the HIV- group and there was a trend towards increased hsCRP (p=0.07) and D-dimer (p=0.06) in HIV+ vs HIV- group.

[0204] It was proposed that T cells would increase glucose transport and upregulate Glutl expression in HIV-infected individuals. Further, that increased glucose uptake would be required for HIV-1 replication. The key steps in glucose metabolism in immune cells from HIV-1 infected patients were analysed. Since glucose uptake is a rate-limiting step in glucose metabolism the cell surface expression of glucose transporter 1 (Glutl) was measured on lymphocyte subpopulations. Functional assays such as glucose uptake, glycolytic analysis and pharmacological inhibition were employed to determine the different points and signal pathways that may be modified during HIV-1 infection. Univariable and multivariable models were used to elucidate the associations between glucose metabolic abnormalities in immune cells and clinical and immunologic characteristics in HIV-1 infected patients. (0205] HIV- J Infection in vivo is associated' with increased percentages of CD3+ lymphocytes expressing Glutl in the peripheral blood - Glutl cell surface expression on T cell subpopulations from 45 HIV+, 35 HIV/cA T and 25 HIV- control subjects was measured by flow cytometry. Figure 1A-B illustrates the gating strategy used for a representative HIV+ and HIV- subjects. The proportion of CD3+Glutl+ T cells in HIV+ subjects (median: 50.5%, range: 27.8-86.2%) was significantly higher than in HIV- controls (median: 25.4%, range: 12.4-55.4%; p<0.0001; (Figure 1C). Suppression of viral replication by cART only partially normalized the proportion of peripheral CD3+Glutl+ T cells which was still elevated above the levels in HIV- subjects (median: 40.50% range 15.0-63.2%; p<0.0001).

(0206] It was next determined whether Glutl was differentially expressed on CD4+ and CD8+ T cells. Figure 1D-E illustrates the gating strategy used and Figure IF is a representative dot plot from an H1V+ subject showing the percentage of CD3+CD4+ T cells that expressed Glutl (referred to as CD4+Glutl+ T cells). The proportion of CD4+Glutl+ T cells in HIV+ subjects (median: 20.0%, range, 3.3-90.0%) was significantly higher than in HIV- controls (median: 5.2%, range: 1.6-10.7%; pO.0001) (Figure 1G, left panel). The proportion of CD4+Glutl+ T cells in the HIV+/cART subjects remained significantly elevated above that of HIV- controls after commencing cART (median: 11.7%, range: 2.25-53.50%, p=0.0002). In a group of 17 individuals commencing cART and analysed after 2.1 ±1,3 years the proportion of CD4+Glutl+ T cells that expressed Glutl significantly decreased from a median of 30.9% (range: 10.10- 59.0%) to a median of 16.5% (range: 2.3-36.6%, p=0.002) (Figure 1G, right panel). Over this time the median CD4 cell count in these 17 patients increased from a median of 233 cells/μΐ (range: 11-488) to 433 cells/μΐ (range: 123-1090). Seven patients in the HIV+ group were long term non-progressofs (LT Ps: infected for >10 years, CD4 cell count >350 and viral load <20,000 copies/ml and have not initiated cART). Of interest, the median percentage of CD4+GIutl+ T cells in these LTNPs was only 1 1.6%, (range: 4.95- 19.2, n=7). Figure lH-I illustrates that Glutl was expressed on virtually all CD8+ T cells (median: 98%, range: 90-100%), irrespective of HIV or treatment status. Taken together, these data show that Glutl is elevated in CD3+ T cells from HIV-infected individuals and that this was due to differences in expression on CD4+ T cells. Furthermore suppression of HIV replication fails to completely normalize Glutl expression on these cells.

[0207] Glutl mRNA expression in CD4+ T cells isolated by negative selection was quantified by RT-PCR. The level of Glutl mRNA was significantly increased in CD4+ T cells from HIV+ subjects and correlated strongly with the number of CD4+Glutl+ T cells (Figure 9).

(0208) The frequencies of Glutl + T cells are higher in CD4+T effector cell populations - Glutl expression was measured on CD4+ effector, naive, memory and effector-memory cells in a subset of HIV+ and HIV- subjects. Figure 2 A shows a representative example of a flow cytometry dot plot illustrating the CD4+ T cells subsets, defined by their expression of CD45RA and CD27; effector (E, CD45RA+CD27-), naive (N, CD45RA+CD27-t-), memory (M, CD45RA-CD27+) and memory-effector cells (ME, CD45RA-CD27-) (Schweneker et al., 2008). The CD4+ naive and memory subset in HIV- subjects have minimal co-expression of Glutl (median: 5.2%, range: 2.50-23.0% and median: 10.3, range: 2.40-23.0%, respectively). However the proportion of naive and memory T cells that expressed Glutl were significantly increased during HIV-1 infection (median: 13.25, range: 3.20-39.40%, p=0.02 and median: 25.50, range: 5.10-40.1%, p-0.006, respectively). The proportion of effector and effeclor-memory subsets expressing Glutl+ was similar between HIV- subjects and HIV+ subjects (Figure 2B). As illustrated in Figure 2C, a similar pattern existed between the distribution of CD4+Glutl+ T cells in the different CD4+T cell subsets in HIV- controls and HIV+ subjects suggesting that HIV- induced increases in Glutl on total CD4+ T cells is not due to altered expression of Glutl+ cells in these subsets.

[0209] Glutl expression in other lymphocytes subsets - Unlike CD4+ T cells, there were no significant differences in the proportion of CD56+GIutl+ NK cells between HIV+ subjects and HIV- controls. However similar to CD8+ T cells, the vast majority of C 56+ NK cells expressed Glutl and there were no significant differences in levels of expression of Glutl on CD56+ NK cells irrespective of HIV or treatment status. NK cells are classified based on their surface expression of CD56 and CD16, the major subsets being the more mature CD56+CD16+ NK cells. Examination of Glutl expression on the different subsets showed that the CD56+CD16+ subsets comprised the highest percentage of Glutl + cells. The proportion of Glutl+ cells within the different subsets were unchanged by HIV-1 or treatment status (Figure 10).

[0210] CD4+Glutl+ T cells have high expression of activation markers - HIV-1 infection is associated with immune activation, which is shown by the elevated plasma concentrations of TNF among HIV-1 seropositive patients compared with uninfected controls (Table 1) and the increased frequency of peripheral blood CD4+CD38+HLA-DR+ and CD8+CD38+HLA-DR+ cells in HIV-1 infected subjects (Figure 3 A). Immune activation persisted but was reduced in patients receiving cART. The proportion of CD4+ T cells that co-expressed CD38 and HLA-D and the MFI of both CD38 and HLA-DR was significantly higher in the Glut 1+ population than in the Glutl- population (Figure 3B, C) in all study groups, showing that activated T cells express more Glutl consistent with previous reports (Manel et al, 2003, Maratou et al, 2007, Kinet et al, 2007).

[0211] Glutl expression on CD4+ T cells is associated with markers of proliferation - Glutl mediates tumor cell proliferation in cancer (Furuta et al, 2010). To determine if Glutl expression is associated with proliferation of CD4+ T cells in the context of HIV-1 infection the expression of i67 was measured. The percentage of cells expressing Ki67 was significantly higher in Glutl+ compared to Glutl- CD4+ T cells irrespective of HIV status but HIV-1 infection results in a significant expansion of GD4+Glutl+Ki67+ T cells in peripheral blood (Figure 4A). Since Glutl expression is induced by TCR engagement (Kinet et al, 2007, Jacobs et at, 2008) anti-CD3/28 microbeads were used to stimulate T cells and quantify Glutl expression on proliferating and non-proliferating CD4+ T cells isolated from HIV- control subjects. The majority of CD4+ T cells undergoing proliferation (Ki67+) in response to CD3/CD28 cross linking for 2 days expressed Glutl (data not shown). The kinetics of Glutl expression was determined over time in dividing T cells. Using CFSE-labelled PBMCs activated with PI-IA and IL-2 for 4 days it was demonstrated that CD4+ cells that had undergone replication had increased cell surface Glutl (Figure 4B-C) but lost expression during extended periods of stimulation for 6 days (Data not shown). Collectively, these data suggest that Glutl expression is associated with CD4+ T cells entering the cell cycle. [0212) HIV-I infection increases glucose uptake in CD4+ T cells - To determine the impact of increased Glutl expression on glucose metabolism, glucose uptake in PBMCs was monitored by multicolour flow cytometry using the fluorescent glucose analogue 2- NBDG. The kinetics of glucose uptake were compared using 4 representative subjects from each study group and demonstrated that CD4+ T cells from HIV+ subjects take up more glucose over time compared to CD4+ T cells from HIV- and HIV+/cART subjects (Figure 5A). The 60 min time point was selected to evaluated glucose uptake in a larger sample size. As shown in Figure 5B, the mean fluorescence intensity of intracellular 2- NBDG was significantly higher in the CD4+ T cells from HIV+ than in cells from HIV- or HIV-l+/cART subjects and this correlated significantly with Glutl expression on CD4+ T cells (r=0.70, p=0.005, n ^« =24, data not shown). Further, 2-NBGD uptake in Glutl+ cells was higher than in Glutl- cells (Figure 5C), and these observations were confirmed in our T cell activation model where overlay analysis showed a direct association between Glut) expression and 2-NBDG uptake (Fig 5D-E). In contrast to CD4+ cells, glucose uptake in CD8+ T cells was similar between the groups, although there was a trend of reduced 2- NBDG uptake by CD8+ T cells from HIV-1+ subjects (Data not shown). Collectively, these data show that increased Glutl expression on CD4+ T cells during HIV-1 infection is associated with increased glucose uptake in CD4+ T cells.

(0213) Intracellular retention of glucose occurs by phosphorylation of glucose to glucose-6-phosphate (G-6-P) by hexokiriases, which drives glucose transportation via Glutl . The intracellular concentrations of G-6-P was measured in purified CD4+ T cells which were incubated in glucose containing media for 4h. Jurkat cells were used for a positive control, which have high basal levels of glycolysis. Figure 5F shows significantly higher basal levels of intracellular G-6-P in Jurkat cells incubated for 4h in culture medium containing 1 1 mM glucose vs compared with those cultured in glucose limiting medium (n-4). Basal levels of intracellular G-6-P were significantly higher in CD4+ T cells from HIV+ subjects (median: 76.5 μΜ) compared to HIV- (median: 4.92 μΜ; p^0.0009, n=8) or and when compared to HIV-l+/cART (median: 20.25 μΜ; p=0.005, n=8) (Figure 5G), consistent with an increased transport of glucose in these cells. [0214] CD4+ T cells in HIV~1 infected patients have increased glycolytic activity - These observations were extended to show that CD4+ T cells from HIV+ subjects secreted significantly more L-lactate into the medium than CD4+ T cells from the other groups (Figure 5H), demonstrating that the increased rate of glucose transport results in increased glycolytic flux. To verify that the increased glycolytic activity was associated with Glutl expression multicolour flow cytometry was used to measure the intracellular concentration of L-lactate in both Glutl+ and Glutl - cells. Jurkat cells, which have a high glycolytic rate, and anti CD3/CD28-stimulated PBMCs were used as positive controls (Figure 5I-J). Figure 5K (left panel) shows higher intracellular L-lactate concentrations in GlutH- compared to Glutl- CD4+ T cells of a single representative HIV+ donor. Levels of intracellular L-lactate were significantly higher in Glutl + compared to Glutl - CD4+ T cells in HIV-1 infected individuals (n=5, p=0.01, right panel).

{0215) PI3K~mTORCl signalling plays regulate Glutl expression on CD4+ T cells - Activation of T cells is known to induce PI3K. to support Glutl translocation from the cytoplasm to the cell surface membrane. Another important regulator of Glutl expression is mTOR pathway (comprised of mTORCl and mTORC2 subunits) that serves as a nutrient and inflammatory sensor that regulate glucose uptake and cellular biosynthesis. Using the activation models of anti-CD3/CD28 microbeads and PMA+ionomycin stimulation, there was a time dependent expansion of CD4+Glutl+ T cells (Fig I I , A) and expression of Glutl on the cell surface membrane of CD4+T cells (Figure 1 1, B-C). By contrast, Glutl expression on CD8+ T cells was less responsive to anti-CD3/28 stimulation but responded to PMA+ ionomycin (Figure 1 1 D-F).

(0216) To address the mechanism of Glutl expression in CD4+ T cells its expression was evaluated on CD4+ T cells in purified lymphocytes pre-treated with inhibitors for PI3K.-5, mitochondrial hexokinase, p38 Kinase, mTORCl, Akt kinase, MAPK, Ca ² *calmodulin-dependent kinase II, JN l/2/3, ME l/2, and ERK1 prior to anti- CD3/CD28 microbead stimulation for 24h. Figure 12 illustrates the effects of inhibiting selected targeted pathways and illustrates that inhibition of Class IB Pl3 -pl 10γ with AS- 605340 and mTORCl with temsirolimus resulted in the greatest suppression of Glutl on CD4+ T cells (2 and 41%, respectively), at non-cytotoxic concentrations of 200 and 10 nM, respectively. Furthermore ΡΙ3Κ-ρ1 10γ and mTORCl inhibition demonstrated antiglycolytic activity in CD4 (Jurkat) cell lines (Figure 12 B-C)

[0217) The PBK-mTOR pathway is activated in CD4+GlutI+ T cells - Using phos- flow it was demonstrated that the vast majority of cells that contained phosphorylated Akt (308), a downstream effector of the PI3 kinase pathway, were Glutl + and that CD4+ T cells from HIV+ subjects have augmented basal PI3K activity assessed using p-Akt (308) staining (Figure 6A and C). Furthermore CD4+ T cells from HIV+ patients have significantly more phosphorylated Akt (308) in response to CD3/CD28 co-stimulation (Fig 6.B-C). The phosphatase and tensin homologue deleted on chromosome 10 (PTEN) inhibits the effector molecules of the PI3K pathway that contain a pleckstrin homology domain. To determine whether increased glucose metabolic activity in CD4+Glutl+ T cell from HIV+ subjects was attributed to elevated PI3K activity or reduced PTEN levels intracellular levels of PTEN were compared in Glutl- vs GlutH- cells. As shown in Figure 6D, PTEN expression was significantly lower in the Glutl+ vs Glutl- cells irrespective of HIV status. Overall there were no significant differences in PTEN levels in CD4+ T cells between HIV- and MIV+ subjects/Therefore the increase in metabolic activity in CD4+ T cells for HIV+ subjects is most likely due to increased PI3 activity and not decreased PTEN levels. CD4+T cells were activated via anti-CD3 28 stimulation and it was confirmed that CD4+Glutl+ cells have lower levels of PTEN and higher levels of p-Akt (308) than CD4+Glutl+ cells. The mTOR pathway serves as a nutrient and inflammatory sensor to augment many of the metabolic activities that regulate glucose uptake and cellular biosynthesis. mTOR complex 2 phosphorylates Akt at a separate site S473. Phosphorylation at this site was used as a measure of mTOR activation and showed that it is higher in CD4+ T cells from HIV-1 infected individuals (Figure 6G) although this was not restricted to Glutl + cells. Similarly to Akt (308), Akt (473) was hyper-phosphorylated in response to T cell activation in cells from HIV+ subjects (Figure 6H-I). Taken together these data suggests that the increased frequency of CD4+Glutl+ T cells in HIV+ subjects is driven at least in part by increased PI3K _r mTOR pathway.

(0218) A negative correlation exists between Glutl -expressing CD4+ T cells and circulating CD4+ T cells - It was next determined whether there is an association between the frequencies of CD4+Glutl+ T cells and markers of disease progression. A significant inverse correlation was found between the percentage of CD4+Glutl+ T cells and the percentage of CD4+ T cells (r=-0.75, r O.0001, n=80) and absolute CD4 count (r=-0.43, p=0.0002, n-63) in peripheral blood of H1V+ patients (Figure 7A). This was also true when cART treated HIV+ patients were specifically analyzed (r=-0.53, p=0.0005, n=35 for percentage CD4+ T cells and r=-0.50, p=0.004, n= 27 for absolute CD4 count; data not shown). This association in the context of viral suppression suggests that Glutl expression on CD4+ T cells may be driven predominantly by immunological rather than virological factors. There were no significant correlations between plasma concentrations of glucose and insulin and the percentage of CD4+Glutl+ T cells suggesting that peripheral glucose homeostasis is an unlikely factor influencing Glutl expression on CD4+ T cells in this setting.

[0219] Linear regression analysis was employed to evaluate the relationship between the percentage of CD4+Glutl+ T cells and other immunological and clinically relevant parameters (Table 2). Univariable analysis revealed that HIV status, several immunological parameters (CD4/CD8 ratio, % CD4+CD38+HLA-DR+ , % CD8+CD38+HLA-DR+ T cells and plasma concentrations of D-dimer) and HIV viral load were significantly associated with the proportion of CD4+Glutl+ T cells. Within a multivariable model, the percentage of CD4+T cells and MFI of HLA-DR on CD4+ T cells were independently associated with frequency of CD4+Glutl+ T in the peripheral blood of subjects (Table 2).

(0220) To examine the stability of the frequency of CD4+GIutH- T cells over the percentage of CD4 T cells was measured expressing Glutl in fresh blood samples taken at 3 month intervals for 9 months from two HIV+ treatment naive individuals one of whom maintained a steady CD4 count and one who showed a steady decline in T cell numbers and was placed on antiretroviral therapy. In the non-progressing subject the percentage of CD4+Glutl+ T cells remained stable between 14-18% over a 9 month period (Figure 7B). Conversely, the percentage of CD4+Glutl+ T cells in the progressor increased from 21% at baseline and peaked at 40- 44% before decreasing to 14.5% following 8 weeks on cART (Figure 7C). These data are consistent with and extend the data presented in Fig 1 G showing the potential of CD4+Glut]+ T cells as a marker of disease progression. Importantly, changes in the proportion of Glutl+CD4+ T cells reflected changes in markers of activation on CD8+ T cells as measured by the levels of expression of CD38 and HLA-DR.

(02211 Total HIV-1 is predominantly found within CD4+Glutl+OX40+ T cells - In order to determine if CD4+Glutl+ T cells are more susceptible to HIV-1 infection in vivo the levels of HIV-1 R A was compared in purified CD4+ T cells from HIV-1 infected patients with either a low (14.1 ± 9.5%, n=4) or high (55.15 ± 23.49%, n=4) frequency of CD4+Glutl+ T cells. Higher copies of HIV-1 RNA were observed in CD4+ T cells from patients who have a high proportion of CD4+Glutl+ cells (Figure 8 A). It was next determined whether CD4+Glutl+ cells from patients have a sufficient activation state to support HIV-1 infection in vitro. PB Cs were selected from virally suppressed HIV+/cART subjects who had a high percentage of CD4+Glutl+ T cells and exposed them to GFP-tagged HIV-1 in culture in the absence of activating stimuli. Approximately 18.0 ± 5.0% of CD4+Glutl+ T cells were HIV-1-GFP+ vs 3.5 ± 0.5 % for CD4+Glutl- T cells (n=3, data not shown).

|0222J The specific inhibitors of PI3K and mTOR signalling inhibited HIV- 1 infection of CD4+ T cells in vitro (Figure 13). Now it was tested whether that CD4+Glutl + T cells from patients with the highest activity of PI3K would be preferential targets for HIV-1 infection. OX40 (CD 134), a member of the TNFR superfamily, primarily functions to augment Akt signaling in T cells by enhancing the amount of PI3K and Akt available to the TCR. Hence OX40 expression correlates with PI3K-Akt activation in T cells (So ei al, 201 1). PBMCs from HIV+/cART subjects who had a high frequency of CD4+Glull+ T cells were exposed to GFP labelled virus in the absence of activating stimuli. Multicolour flow cytometry was employed to identify CD4+Glutl+T cells with high PI3K-Akt activity using OX40 expression as a surrogate marker and it was found that approximately 85% of CD4+OX40+ T cells were Glutl-t- (data not shown). Fig 8 B-E illustrates that HIV-GFP+ cells were predominantly found within the CD4+GlutI+OX40+ T cells. Taken together, the data suggests that both Glut] expression and PI3 -Akt signalling arc required to mediate HIV-1 infection and that additional activating stimuli are not required in vitro to support HlV-1 infection and replication.

[0223] As described herein the proportion of CD4+Glutl+ T cells in HIV-1 infected patients was positively associated with key markers of T cell activation, inflammation and immunological deterioration. The frequency of CD4+Glutl+ T cells was independently associated with augmented levels of HLA-DR on CD4+ T cells, suggesting chronic immune activation may be linked with modified glucose metabolism in CD4+T cells. Furthermore the induction of Glutl on CD4+ T cells is unlikely to be nutrient dependent since no significant associations were observed between plasma concentrations of glucose, insulin or lipids and the frequency of circulating CD4+Glutl+ T cells. At least in the resting stages, the expression of Glutl on T cells and monocytes was unresponsive to insulin (Dimitriadis et al, 2005, aratou et al, 2007). This is in contrast to the adipocytes and muscle cells in which the predominant glucose transporter Glut4 is highly responsive to insulin (Berengucr et al, 2010, Habegger et al, 2012, Bogan 2012), and nutrients such as lipids (Pu et al, 201 1).

[0224] Further characterization of CD4+Glutl+ T cells showed that they have increased glucose uptake and increased intracellular G-6-P and L-lactate and increased PI3k-mTOR signalling, a metabolic profile aconsistent with aerobic glycolysis exhibited by proliferating tissues and tumour cells (Vander-Heiden et al, 2009). It is proposed herein that down-regulation of PI3 -mTOR signalling and aerobic glycolysin will down- modulate this associated immune activation and inflammation.

[0225J Phosflow analysis showed a high level of phosphorylation of Akt at T308 and S473, (indicative of activation) of Akt in CD4+Glutl + T cells, suggesting the PI3K-mTOR pathway contributes to increased glucose metabolism in CD4+ T cells during HIV-1 - infection in vivo. The activation of Akt is considered as an important switch to activate metabolic programs characteristic of proliferating cells (Fruman and Bismuth et al, 2009) and is likely to contribute to the homeostatic response of the immune system that stimulates activation and proliferation of specific populations of CD4+ T cells to replenish that compartment. Unfortunately, this homeostatic-driven CD4+ T cell increased proliferation may provide further activated targets cells for viral infection (d'Ettorre et al, 2011). A subject was selected who was virally suppressed but had a low CD4 T cell recovery and high percentage of CD4+Glutl+ T cells and employed an unconventional approach to infect CD4+T cells in vitro by subjecting PBMCs to HIV infection in vitro in the absence of exogenous activating stimuli to determine if CD4+Glutl+ T cells possessed the necessary components to facilitate infection. The results demonstrated that the most susceptible CD4+Glutl+ cells to HIV-1 infection were those that had a high PI3 activity.

[0226] The PI3K pathway may be regulated by PTEN, the en2yme that counteracts PI3 signalling. In the absence of PTEN, the PH-domain containing kinases, such as Akt, are localized basically to the membrane and become constitutively activated. In vitro the COOH-terminally truncated form of HIV-1 pi 7 induced activation of P K/Akt by maintaining PTEN in an inactive phosphorylated form promoting B cell growth and tumorgenesis (Giagullic, marsicos, 2011) and HIV Tat protein negatively regulated PTEN resulting in increased PI3K activity (Y kim et al, 2011). However it is more likely that increased glycolytic activity in CD4+ T cells in HIV-1 infected subjects is due to increased PI3 activity rather than decreased PTEN levels, given the marked levels of phosphorylated Akt (308) in these cells.

(0227] Regulation of cell surface expression of Glutl is complex and is controlled at the level of transcription, translation or post-transcriptionally in part through the activation of the PI3 -Akt pathway. The observation herein of increased expression of Glutl mR A in CD4+ T cells from chronically HIV-1 infected subjects may be mediated at least in part through the transcriptional activation of Glutl and other key glycolytic genes (Deshmanc et al, 2009). It has previously been demonstrated that HIV-1 Vpr protein activates the oxidative stress pathway, thus leading to the induction of HIF-Ια which is a transcriptional regulator of the PI3K-Akt-mTOR axis (Deshmane et al, 2011). As disclosed herein CD4+Glutl+ T cells also have increased expression of CCR5, a co-receptor for HIV-1 and thus inhibition of Glutl may also directly reduce HIV infection rates.

(0228] Several mechanisms have bee proposed in the literature to explain the inexorable depletion of CD4+ T cells. The existing paradigm for HIV-associated homeostatic failure of CD4+ T cells are complex and in addition to direct viral infeciion, include several effects of HlV-induced immune activation such as increased proliferative senescence, disruption of anatomic environment and bystander apoptosis.

[0229] HIV-1 infection increases the proportion of CD4+ T cells that displays high glycolytic activity, a profile similar to that of cancer cells. A considerable increase in Glutl expression on CD4+ T cells is described that is strongly associated with CD4+ cell depletion in HIV+ patients. This supports a model where increased glycolytic environment may provide a niche for HIV-1 viral infection and drives CD4+ T cell destruction. Whether through direct glycolytic inhibition or modification of cell signalling pathways that regulate glycolytic flux, therapies aimed at countering glycolytic imbalance in CD4+ T cells are proposed to reduce the progression of HIV-1 disease. Although several therapeutic approaches have been discussed to reduce HIV-1 associated chronic immune activation perhaps the most important limitation of this approach is our limited understanding of the molecular mechanisms responsible for this phenomenon. This has hampered the design of immunological interventions that target specific signalling pathways, therefore leaving advocates of this approach with only "generic" immune suppressive agents (d'Ettorre et al, 2011). For example in the setting of HIV-1 infection, the immune suppressive agents hydroxychloroquine and chloroquine suppress immune activation by a number of mechanisms including inhibition of TLR signalling and inflammatory cytokine secretion (Piconi et al, 2011), and reduced the frequency of CD38+HLA-DR+CD8+ T cells as well as Ki-67 expression on CDS and CD4+T cells (Murray et al, 2010). The herein described metabolic approach and metabolic components of HIV-1 associated immune activation provides a rationale for exploratory studies thai integrate both approaches for therapeutic interventions. From a metabolic perspective, therapies may function at a variety of levels, ranging from the inhibition of Glutl cell surface expression and normalization of glucose uptake in CD4+T cells by targeting the PI3 -mTOR pathway which may be achieved using rapalogues such as the mTOR inhibitors sirolimus and everolimus that also inhibit cellular lactate production and aerobic glycolysis (Noch and Khalili, 2012). EXAMPLE 2

Glucose metabolism is an integral process in CD4+ Tcell activation and inflammation in fflV-l infected subjects

[0230] Background - CD4+ T cells undergo rapidly glucose uptake in respond to immune challenges. Glucose uptake is mediated by glucose transporter 1 (Glutl), the major glucose transporter in T cells. As shown herein, Glutl expression is increased on CD4+ T in HIV+, subjects. Described herein is an investigation of the role Glutl expression on CD4+ T cell activation.

[0231] Results - The proportion of CD4+Glutl+ T cells in HIV+ subjects was significantly higher than in HIV- controls and remained elevated in HIV+/cART subjects. The frequency of CD4+Glutl+ T cells was independently associated with the percentage of total CD4+ T cells and the MFI of HLA-DR on CD4+ T cells irrespective of treatment status. CD4+Glutl+ T cells had higher levels of CD38 and HLA-DR, increased basal glucose uptake, intracellular G-6-P, L-lactate, ECAR, and PI3K-mTOR signalling than CD4+Glutl- T cells. These activation and metabolic phenotypes were sensitive to Class IB PI3 _T and mTORCl inhibitors following T cell activation in vitro. CD4+T cells with high PI3 -mTORCl activity were more susceptible to HIV-1 infection.

[0232] Conclusion - Glutl expression on CD4+ T cells is positively associated with markers of T cell activation and inflammation. Importantly, normal glucose metabolism in CD4+ T cells was not restored by cART. Thus, Glutl represents a sensitive and functional marker of T cell activation of CD4+ T cells. Approaches to attenuate Glutl expression or glycolysis in CD4+T cells are an attractive platform to target overactive T cells and immune activation in H1V+ subjects.

[0233] Many modifications will be apparent to those skilled in the art without departing from the scope of the present invention. TABLE 1: Clinical characteristics of study groups

TABLE 2: Markers associated wdk the frequency ofCD3+CD4+Gluil+ T cells

Univariable analyses Multivariable model

P

n Coefficient 95% CI Coefficient 95% CI

value value

HIV

HIV-

HlV+ off 25 Ref _

treatment

45 1.38 1.02, 1.74 <0.001

HIV+ on

treatment 35 0.80 0.42, 1.18 0.001

Age 93 -0.01 -0.02, 0.004 0.190

CD4 count 63 -0.002 -0.002, -0.001 <0.001

Viral Load 60 4.21e-06 1.67e-06, 6.76e-06 0.002

TNF-a 52 0.02 -0.01, 0.06 0.201

D-dimer 48 0.35 0.03, 0.67 0.034

% CD3-K}lutl+T cells 105 0.04 0.03, 0.04 <0.001

% CD3+CD4+ 105 -0.04 -0.04, -0.03 <0.001 -0.027 -0.035, -0.019 <0.001

% CD3+CD8+ 104 0.03 0.03, 0.0 «0.001

CD4/CD8 ratio 104 -0.58 -0.75, -0.42 <0.001

MFI of Glutl

(CD3-HCD4+T cells) 82 0.09 0.07, 0.12 O.OOl

% CD4+CD38+HLA-DR+ 90 0.09 0.07, 0.12 <0.001

% CD8+CD38+HLA-DR+ 77 0.03 0.01, 0.05 0.001

MFI of CD38

(CD3÷CD8+T cells) 77 0.03 0.004, 0.05 0.021

MFI ofHLADR

(CD3+CD8+T cells) 77 0.03 0.01, 0.04 <0.001

MFI ofCD38

(CD3+CD4+T cells) 89 0.008 -0.003, 0.02 0.134

MFI ofHLADR

(CD3+C 4+T cells) 89 0.05 0.03, 0.06 O.001 0.027 0.013, 0.040 <0.001

Plasma Glucose 82 0.12 -0.08, 0.32 0244

Plasma Insulin 48 -0.003 -0.02, 0.02 0.796

Plasma Triglyceride 64 -0.12 -0.33, 0.09 0.262

Plasma HDLCH 65 -0.53 -1.14, 0.09 0.097

Plasma Cholesterol 65 -0.19 -0.40, 0.02 0.073

Plasma Vitamin D 48 -0.003 -0.008, 0.007 0.937

N = 89; R ² „s = 0.571; AIC= 149.85

TABLE 3: Effect of inhibition of selected pathways on Glutl expression on CD4+ T cells

Inhibitors Target pathways % Glutl inhibition*

Akt inhibitor IV

Akt protein kinase 7.4 ± 17.9

(500 urn, CaIbiochem,CA, USA)

AS-605240

Class IB PI3K-y (pllOy) 24. ± 10.0

(200 nM Selleck, TX, USA)

IC87114

PI3K-5 1.8 ± 8.6

(5 μΜ, Calbiochem,CA, USA)

Lonidamine

Mitochondrial hexokmase 8.3 ± 5.8

(60 μΜ Tocric Bioscience, Bristol, UK)

BIRB 796

p38 MAPK 5.1 ± 8.7

(100 nM, Selleck, TX, USA)

SB202190

p38 MAPK

(10 μΜ, Selleck, TX, USA) 8.8 ± 9.4

CCI-779 (temsirolimus)

mTOR (mTORCl)

(10 nM, Selleck, TX, USA) 41.0 ± 12.11

Perifosine

Akt protein kinase

(20 μΜ, Selleck, TX, USA) 11.3 ± 7.2

U-0126

MAPK (MKK, MEK) kinase 2.9 ± 8.9

(2.5 μΜ, Selleck, TX, USA)

KN-93

Ca2+/calmodulin-dependent kinase II (CaMKII

(5 μΜ, Cayman Chemicals, MI, USA) 18.6 ± 9.8

AS- 1269574

(5 μΜ, Cayman Chemicals, MI, USA) GPR119 agonist NI

SP 600125

JNK1,2,3 3.1 ± 10.0

(10 μΜ, Cayman Chemicals, MI, USA)

AICAR

AMPK activator 16.8 ± 7.5

( 12.2 μΜ, Cayman Chemicals, MI, USA)

PD98059 _λ , _ρ „. ^

(50 μΜ, Selleck, ΤΧ, USA) ^Λ1 ^ ^{ι> Μ} ^ Nl

CAY10561

(5 μΜ, Cayman Chemicals, MI, USA) 15.6±9.7 lymphocytes pre-trealed with inhibitors for 48h followed by activation with

antiCD3 28 microbeads (1 cell: I bead ratio)

•Results expressed as meartfcSD of 5 independent experiments with the exception of

AS-605240 and CCI-779 (temsirolimus) where 6 independent experiments were conducted.

TABLE 4

Summary of sequence identifiers

SEQUENCE ID NO: DESCRIPTION

] β-actin Primer

2 β-actin Primer

3 Glutl Primer

4 Glutl Primer

5 Amino acid sequence encoded by SEQ ID NO: 6

6 Nucleotide sequence encoding human Glutl

. 7 Hexokinase II Primer

8 Hexokinase II Primer

9 Phosphofructokinase I Primer

10 Phosphofi'uctokinase I Primer

1 1 Pyruvate Kinase Primer

12 Pyruvate Kinase Primer

13 Lactate dehydrogenase Primer

14 Lactate dehydrogenase Primer

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