WO2019246520A1 - Aminobisphosphonates as latency reversing agents and combination treatments for hiv cure - Google Patents

Abstract

The present disclosure provides novel methods to cure HIV. In particular aminobisphosphonates are disclosed as latency reversing agents (LRAs), and methods to cure HIV by administering to a subject a combination of bisphosphonates and γδ T cells are provided. In some embodiments, these interventions may be combined with other latency reversing agents, e.g., histone deacetylase (HD AC) inhibitors, and other immunotherapeutics for HIV clearance (e.g., broadly HIV -neutralizing antibodies).

Description

AMINOBISPHOSPHONATES AS LATENCY REVERSING AGENTS AND

COMBINATION TREATMENTS FOR HIV CURE

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Serial No. 62/687,936 filed June 21, 2018, Natalia Soriano-Sarabia, Atty. Dkt. 150-30-PROV, which is hereby incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under Grant Number AI125097, AI126619, AI50410, and CA016086 awarded by the National Institutes of Health. The government has certain rights in the invention.

1. FIELD

[0003] The present disclosure provides novel methods to cure HIV. In particular aminobisphosphonates are disclosed as latency reversing agents (LRAs), and methods to cure HIV by administering to a subject a combination of bisphosphonates and gd T cells are provided. In some embodiments, these interventions may be combined with other latency reversing agents, e.g., histone deacetylase (HD AC) inhibitors, and other immunotherapeutics for HIV clearance (e.g., broadly HIV-neutralizing antibodies).

2. BACKGROUND

2.1. Introduction

[0004] The“background” description provided herein is for the purpose of generally presenting the context of the disclosure. Work of the presently named inventors, to the extent it is described in this background section, as well as aspects of the description which may not otherwise qualify as prior art at the time of filing, are neither expressly or impliedly admitted as prior art against the present disclosure.

[0005] The latent human immunodeficiency vims (HIV) reservoir within resting memory CD4⁺ T cells is the major barrier to efforts to eradicate persistent infection (1-4). Today the standard of care for patients with HIV is long term antiretroviral therapy (ART) to suppress active viral replication and new infection of cells. However, therapeutic approaches that allow the clearance of latent but replication-competent HIV are needed. Current strategies are based on the use of ongoing ART and concurrently employ latency reversal agents (LRA) to induce viral antigen expression (5, 6) to allow immunological clearance. This clearance part has been mainly based on CD8 T cells (7, 8). However, CD8-based therapies can be challenging due to insufficient HIV antigen expression on latently-infected cells, diminished function of specific CD8 T cells (9, 10) and escape HIV variants (7).

[0006] In contrast to the majority of ab T cells that recognize antigen peptides bound to major histocompatibility complex (MHC) class I or II, gd T cells, including the most prevalent peripheral subset, V52 cells, mainly recognize non-peptidic phosphorylated metabolites of isoprenoid biosynthesis (11). These metabolites include (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) (12, 13) and isopentenyl pyrophosphate (IPP) (14), and their recognition does not require conventional MHC-antigen presentation (14, 15).

[0007] Isoprenoids are a class of organic chemicals derived from terpenes. Terpenoids are modified terpenes with added or removed methyl groups or oxygen atoms added. The use terpene can be used more broadly to include terpenoids. These compounds can be classified according to the number of isoprene units that contain the parent terpene in hemiterpenoids, mono terpenoids, sesquiterpenoids, diterpenoids, sesterterpenoids, triterpenoids, tetraterpenoids, poly terpenoids. These chemicals are phosphorylated intermediates of the non-mevalonate (or MEP pathway) and mevalonate pathway of isoprenoid biosynthesis (Figure 1). See Jomaa, H. et al. Inhibitors of the nonmevalonate pathway of isoprenoid biosynthesis as antimalarial drugs. Science (New York,

N. Y. ) 285, 1573-1576 (1999); Hintz, M. et al. Identification of (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate as a major activator for human gammadelta T cells in Escherichia coli. FEBS letters 509, 317-322 (2001). These intermediate isoprenoids include but are not limited to (E)-4- hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), isopentenyl pyrophosphate (IPP), dimethyl-allyl-pyrophosphate (DMAPP). Bromohydrin pyrophosphate (BrHPP) and 2-methyl-3- butenyl-l -pyrophosphate (2M3B1PP) are examples of synthetic isoprenoids. See Espinosa, E. et al. Chemical synthesis and biological activity of bromohydrin pyrophosphate, a potent stimulator of human gamma delta T cells. The Journal of biological chemistry 276, 18337-18344, doi:l0.l074/jbc.Ml00495200 (2001); and Kobayashi, H. et al. Safety profile and anti-tumor effects of adoptive immunotherapy using gamma-delta T cells against advanced renal cell carcinoma: a pilot study. Cancer immunology, immunotherapy: C// 56, 469-476,

doi:l0.l007/s00262-006-0l99-6 (2007).

3. SUMMARY OF THE DISCLOSURE [0008] This disclosure provides a method for inducing expression of HIV viral antigen(s) in latently HIV-infected cells which comprises exposing the latently HIV-infected cells to a bisphosphonate so as to reverse latency and to induce the expression of the HIV viral antigen(s). The bisphosphonate may be an aminobisphosphonate. The aminobisphosphonate may be an alkyl aminobisphosphonate, a substituted alkyl aminobisphosphonate, a bisphosphonate with a nitrogen containing heterocycle, or a bisphosphonate containing a cyclic alkane or cycloaminoalkane. In some embodiments, the aminobisphosphonate is Alendronate (Fosamax™), Ibandronate (Boniva™ or Bonviva™), Neridronate (Nerixia™), Olpadronate, Pamidronate (APD/Aredia™), Risedronate (Actonel™), or Zoledronate (Zometa™ /Aclasta™).

[0009] The invention also provides a method of inducing expression of HIV viral antigens in latently HIV-infected cells in a subject which comprises administering to the subject a bisphosphonate so as to reverse latency and induce expression of the HIV viral antigens in the cells in the subject. The latently infected cells may be rCD4 cells.

[0010] The disclosure also provides a method to boost gd T cell functions and reverse viral latency in a subject infected with HIV which comprises administering to the subject a bisphosphonate.

[0011] In another embodiment, the disclosure provides for the use of an aminobisphosphonate as a latency reducing agent. The aminobisphosphonate may be Alendronate (Fosamax™), Ibandronate (Boniva™ or Bonviva™), Neridronate (Nerixia™), Olpadronate, Pamidronate (APD/Aredia™), Risedronate (Actonel™), or Zoledronate (Zometa™ /Aclasta™).

[0012] The present disclosure also provides a method for eliminating latently infected cells harboring quiescent HIV provirus that comprises reversing HIV latency by exposure to a bisphosphonate, and clearing these infected cells via the direct antiviral activity of gd T cells and/or adjuvant function over other effector cells such as CD8 T cells or Natural Killer (NK) cells.

[0013] In another embodiment, the disclosure provides a method of treating/curing a subject infected with HIV that comprises administering to the subject ex vivo expanded gd T cells and a bisphosphonate.

[0014] These methods further may comprise exposing the infected cells to a second HIV latency reversing agent, that may include but are not limited to an epigenetic modifier, an NFkB agonist, a PI3K Akt pathway inhibitor, a protein kinase C agonist, a TCR activator, a TLR agonist, a second mitochondrial-derived activator of caspases (SMAC) mimetic, an inhibitor of IAP (Inhibitor of Apoptosis Protein) family of proteins, or a stimulator of interferon genes protein (STING) agonists.

[0015] The expanded gd T cells and the bisphosphonate may be administered concurrently or sequentially. [0016] In yet another embodiment, the disclosure provides a method of treating HIV infection in a subject, comprising: (a) isolating peripheral blood mononuclear (PBMC) cells from the subject; (b) culturing the isolated PBMC cells ex vivo with an effective amount of a bisphosphonate or antibodies and suitable cytokines/chemokines so as to expand gd T cells; (c) optionally genetically modifying the gd T cells before or after expansion, e.g., CAR T cells; (d) infusing the expanded gd T cells into the subject; and (e) administering to the subject a bisphosphonate so as to activate quiescent HIV pro virus, allowing elimination of HIV infection.

[0017] The method further may comprise administering to the subject in step (e) a second HIV latency reversing agent such as an epigenetic modifier, an NFkB agonist, a PI3K/Akt pathway inhibitor, a protein kinase C agonist, SMAC mimetic, a TCR activator, a STING agonist or a TLR agonist. An example of an epigenetic modifier LRA is a histone deacetylase (HD AC) inhibitor.

[0018] In most embodiments of the methods above, the subject is receiving ongoing, standard antiretroviral therapy (ART).

[0019] The PBMCs are expanded in the presence of the bisphosphonate for 5 to 30 days or 7 to 21 days. Alternatively, the bisphosphonate may be washed off after initial exposure of the cells to the drug.

[0020] In the methods disclosed herein, the expanded gd T cells and the bisphosphonate are administered concurrently or sequentially.

[0021] As used herein, the bisphosphonate may be Alendronate (Fosamax™), Ibandronate (Boniva™ or Bonviva™), Neridronate (Nerixia™), Olpadronate, Pamidronate (APD/Aredia™), Risedronate (Actonel™), or Zoledronate (Zometa™ /Aclasta™). The HDAC inhibitor may be belinostat (PXD101), entinostat (MS-275), mocetinostat (MGCD0103), panobinostat (LBH589), romidepsin or vorinostat (SAHA). Alternatively, the bisphosphonate may be used in conjunction with a TLR agonist, such as the TLR9 agonist Lefitolimod/MGNl703.

[0022] N-BPs are structurally related to pyrophosphates generated in the isoprenoid biosynthesis pathways and are shown herein to be both HIV latency reversing agents to reactivate latency and induce activation of gd T cell functions, including direct cytotoxic capacity and adjuvant functions over other effector cell populations. Other inhibitors of the mevalonate pathway may also be effective. Examples of terpenoids include but are not limited to (E)-4-hydroxy-3-methyl-but-2- enyl pyrophosphate (HMBPP), isopentenyl pyrophosphate (IPP), dimethyl-allyl-pyrophosphate (DMAPP), bromohydrin pyrophosphate (BrHPP) and 2-methyl-3-butenyl-l -pyrophosphate (2M3B1PP). Preliminary data indicates that HMBPP was effective as an HIV latency reversing agent. HMG-CoA inhibitors including statins may also be useful as HIV reversing agents. Non limiting examples of statins include atorvastatin, Lipitor ®; fluvastatin, Lescol®, Lescol® XL; lovastatin, Mevacor®; mevastatin; pitavastatin, Livalo®; pravastatin, Pravachol®; rosuvastatin, Crestor®; simvastatin, Zocor®; simvastatin & ezetimibe, Vytorin®; lovastatin + niacin extended- release, Advicor®; atorvastatin + amlodipine, Caduet®; and simvastatin + niacin extended-release, Simcor®.

[0023] The disclosure also provides pharmaceutical compositions and uses as HIV latency reversing agents of a bisphosphonate and a second HIV latency reversing agent. Known HIV reversing agents are enumerated in the application, for example a histone deacetylase inhibitor such as vorinostat (SAHA).

[0024] In addition, the disclosure provides pharmaceutical compositions comprising an HMG- CoA reductase inhibitor and an HIV latency reversing agent such as a histone deacetylase. The disclosure also provides for pharmaceutical compositions comprising either (i) a synthetic analogue of a component of the mevalonate pathway, (ii) an inhibitor of the mevalonate pathway (beyond bisphosphonates or HMG-CoA reductase inhibitors), or (iii) an intermediate from the mevalonate pathway in combination with HIV latency reversing agent such as a histone deacetylase. These combination pharmaceutical compositions are useful as part of an HIV treatment and/or cure protocol.

4. BRIEF DESCRIPTION OF THE FIGURES

[0025] FIG. 1 : The mevalonate pathway produces famesyl pyrophosphate. Nitrogen- containing bisphosphonates (N-BP) inhibit the enzyme famesyl pyrophosphate synthase preventing farnesyl pyrophosphate groups to be synthesized. These groups are used by Famesyl transferase enzymes for prenylation downstream. Inhibition of the farnesyl pyrophosphate synthase induces accumulation of geranyl pyrophosphate and isopentenyl pyrophosphate, which are specifically recognized by gd T cells that become activated.

[0026] FIG.2A. Quantification of HIV gag RNA levels in HIV-infected suppressed donors.

A total of 18 donors were analyzed. Each graph represents one single donor. Each dot represents one replicate well containing lxlO⁶ rCD4 cells. Six to 15 wells were assayed per participant depending on cell availability. HIV gag cell-associated RNA (caRNA) levels were compared between untreated cells, the positive control (2pg/mL PHA and lOOU/mL IL-2), Vorinostat (335nM), and N-BPs PAM (25pg/mL and 2.5pg/mL) and Zol (ImM) in different experiments depending on cells availability. [0027] FIG. 2B. Normalized levels of HIV gag RNA copies to the media condition. PAM

(DL2415) significantly induces HIV expression compared to the control, as well as positive controls PHA and IL-2 and SAHA. Wilcoxon signed rank test.

[0028] FIG. 2C-2D. N-BPs reactivate HIV from latency. N-BPs were used at concentrations that are comparable to the in vivo plasma concentrations reported for their use to treat bone-related diseases (PAM at 2.5pg/mL and Zol at ImM). Isolated rCD4 cells from nine HIV-infected donors on stable ART and suppressed for at least one year were assayed. Each dot represents one donor, which is the mean of 6-15 replicates of lxlO⁶ cells. FIG. 2C) PAM and Zol induced HIV gag caRNA copies/l0⁵ resting CD4 cells compared to the untreated condition (p=0.004 and p=0.03, respectively). Mean HIV caRNA was comparable between the PHA and PAM and PHA and Zol (p>0.05 in both cases, Wilcoxon paired signed rank test). FIG. 2D) Fold change induction of HIV caRNA copies normalized to the untreated condition was comparable between the positive control (PHA and IL-2) and the conditions treated with PAM and Zol (p>0.05, Wilcoxon paired signed rank test).

[0029] FIG. 3A: PAM induces production of replication-competent HIV in cultures of isolated rCD4 cells. PHA reactivated latent HIV in 9 of the 10 patients analyzed by QVOA. SAHA induced HIV production in six of nine patients and finally PAM at 25pg/mL reactivated latent HIV in seven of the 10 patients while VOR (SAHA) reactivated in 6 out of 9 individuals.

[0030] FIG. 3B. PAM at 2.5pg/mL induce production of replication-competent HIV comparable to the positive control PHA and IL-2. rCD4 cells from seven HIV-infected donors on stable ART and suppressed for at least one year were assayed. Cells were cultured in limiting dilution from lxlO⁶ to O.lxlO⁶ cells to perform quantitative viral outgrowth assays (QVOA). Frequency of infection expressed as infectious units per million (IUPM) rCD4 cells is represented. IUPM cells was 0.291 after PAM treatment compared to 0.227 after treatment with the positive control PHA and IL-2. Both the positive control and PAM at 2.5pg/mL reactivated above the untreated condition (p=0.03 in both cases). Wilcoxon paired signed rank test.

[0031] FIG. 4A-4B: Toxicity of N-BPs to cell populations. Total PBMC or isolated rCD4 cells from HIV-infected participants were exposed to different concentrations of PAM. In parallel, controls with lOOU/mL IL-2 were ran. FIG. 4A) Percentage of 7-AAD in total PBMCs. No significant differences between media and increasing concentrations of PAM (DL-2415) were detected in any of the cell populations analyzed: total CD4 cells, CD8 cells, NK cells or gd T cells. FIG. 4B) Expression of CD95 in isolated rCD4 cells. PAM (DL-2415) is not toxic to rCD4 cells.

[0032] FIG. 5A: Effect of PAM (DL-2415) on cell proliferation. Representative histograms of one donor is shown. PAM does not promote proliferation of isolated rCD4 cells. PAM does not promote proliferation of total CD4 or CD8 T cells. As expected, V52 cells proliferated in response to PAM and IL-2 (bottom histograms).

[0033] FIG. 5B: N-BPs do not induce proliferation of CD4, CD8 or gd T cells. Percentage of proliferation measured by carboxyfluorescein diacetate succinimidyl ester (CFSE) for varying concentration of PAM (upper graph) and Zol (lower graph). Percentage of CFSE positive cells (non proliferated) was comparable to the untreated condition for all concentrations analyzed. PAM at 2.5pg/mL and Zol at lOng/mL an lOOU/mL IL-2 were used as the positive control for V52 cell expansion. Bars represent the mean ±SEM of three to 5 different donors.

[0034] FIG. 5C. N-BPs do not promote activation of lymphocyte populations. Isolated PBMC were exposed to different concentrations of PAM (graphs on the left) and Zol (graphs on the right). PHA was used as the positive control, and untreated cells (media alone) were used as the negative control. Expression of activation markers CD69 and HLA-DR by flow cytometry was increased in the positive control, but activation levels remained comparable to the untreated condition in all other experimental conditions. Cells from three to five individuals were assayed in triplicate. Multiple t-test Holm-Sidak analysis p>0.05.

[0035] FIG. 6A-6C. N-BPs mechanism of action. RNA from isolated rCD4 cells was extracted and RNA-seq performed. FIG. 6A) The principal component analysis represents a multidimensional association showing that the greatest source of variation in gene expression is due to the treatment. A heatmap showed differentially expressed genes in the treated and the untreated conditions. Upregulated and downregulated were split according to the treatment (data not shown). FIG. 6B) Examples of pathways altered by PAM treatment analyzed by gene set enrichment using GSVA and curated gene modules (C2). Genes involved in the positive control, cholesterol biosynthesis pathway were upregulated in the untreated condition compared to the PAM-treated condition. Comparison of modules of genes involved in p53 DNA damage, methylation targets and HDAC7 targets between untreated and PAM-treated conditions is shown. FIG. 6C) Publicly available RNA-seq from HIV infected CD4+ T cell line and RNA-seq from PAM treatment showed that pathway coefficients correlated (r=0.44) suggesting that pathways are altered similarly.

[0036] FIG. 7A-7B:N-BPs reverse latency in vivo. ART-treated patients received the N-BP alendronate (Arm A) or placebo (Arm B). FIG.7A) Seven of the ten patients in arm A who received alendronate, showed an effect on HIV caRNA. In four of them the effect was a decrease compared to baseline, and in the other three, there was an increase compared to baseline. FIG. 7B) In the five patients analyzed that received placebo, HIV caRNA was comparable between all the time points analyzed. Wilcoxon signed rank test. [0037] FIG. 8A-8C. Expansion of V52 cells after six days of culture. FIG.8A) Greater V52 cell frequency in uninfected donors. PBMC of uninfected (N=l0) and HIV-infected donors (N=l3) were stained for CD3 and V52 and analyzed by flow cytometry. As expected, uninfected individuals showed a statistically higher percentage of V52 cells compared to HIV-infected donors Data represent mean ±SEM (Mann-Whitney U-test, p<0.00l). FIG. 8B) Representative histograms showing V52 cell expansion. PBMC from uninfected (left histogram) or ART- suppressed HIV-infected donors (right panel) were incubated for six days using HMBPP+IL-2, PAM+IL-2, or IL-2 alone. FIG. 8C) V52 cells from HIV-infected individuals expand in response to Pamidronate (PAM) and IL-2. V52 cell fold change relative to basal cell numbers is represented. HIV-infected donors’ response to HMBPP was lower, not statistically significant after FDR adjustment, compared to uninfected individuals (FDR p=0.ll). Response to PAM and IL-2 was similar between uninfected and HIV-infected donors (FDR p=0.29). Response to HMBPP and PAM in uninfected donors was comparable (FDR p= 0.22), while response to HMBPP in HIV- infected donors was statistically lower (FDR p=0.04). Uninfected donors (n=9) are represented with grey circles and HIV-infected donors (n=ll) with squares. Uninfected and HIV-infected donors were compared using Mann Whitney U-test, *FDR-adjusted p<0.05. HMBPP, PAM and IL-2 conditions in uninfected donors and in HIV-infected donors were compared using Wilcoxon signed rank test, * FDR-adjusted p<0.05, ** FDR-adjusted p<0.005.

[0038] FIG. 9A-9B. Percentage of V52 cells after six days of culture. FIG. 9A) Basal and expanded V52 cell frequency. V52 cells frequencies from ART-suppressed HIV-infected donors achieve greater values when expanded with pamidronate (PAM)+IL-2. FIG. 9B) Detailed comparison of V52 cell numbers after exposure to HMBPP and PAM in HIV-infected donors. Each symbol shape represents one condition and each individual donor is represented by a different color. Mann Whitney U-test. *p<0.05, **p<0.005, ***p<0.0005.

[0039] FIG. 10A-10C. Expansion of V52 cells from ART -suppressed HIV-infected donors in response to pamidronate (PAM) FIG. 10A) PAM exposure significantly increase V52 cell frequency. V52 cells from suppressed HIV+ donors (N=2l) significantly expandeded in response to PAM. Patients treated in the acute infection (N=9) are represented with green triangles and patients treated in chronic infection (N=l2) are represented with purple squares. FIG. 10B) Decreased V52 cell numbers in donors treated in the chronic phase of the infection. HIV- infected donors treated in the chronic phase of the infection (N=l2) showed significantly reduced number of V52 cells (FDR p=0.007) compared to those treated in the acute phase of the infection (N=9). After PAM expansion, V52 cells remained significantly lower in donors treated in chronic infection. Mean ±SEM. Mann-Whitney U-test, FDR p=0.02. FIG. 10C) Comparable expansion capacity between patients who initiated ART in the acute or chronic phase of HIV infection.

Fold change of V52 cell expansion with PAM was similar in patients treated in the acute and chronic infection. Mean ±SEM. Mann Whitney U-test.

[0040] FIG. 11A-11C: Phenotype of pamidronate (PAM)-expanded V52 cells in ART- suppressed HIV-infected donors. Phenotype of V 52 cells was analyzed by flow cytometry in eight HIV-infected individuals after expansion. Mean ±SEM is represented. FIG. 11A) Memory populations defined as central memory (TCM): CD45-/CD27+/CCR7+); transitional memory (TTM: CD45-/CD27+/CCR7-); effector memory (TEM: CD45-/CD27-/CCR7-). FIG. 11B) Expression of cytotoxic markers CD8, CD56 and CD16, and FIG. 11C) Expression of activation markers CD69, CD25 and HLA-DR, and exhaustion markers PD-l and CTLA-4.

[0041] FIG. 12A-12C. V52 T cells inhibit active HIV replication. FIG. 12A) Ex vivo isolated V52 T cells reduce HIV p24 production. HIV p24 production from autologous superinfected CD4 T cells was significantly reduced in the presence of V52 (N=8 for the 1:1 effectordarget ratio). Bars represent average viral production normalized to the condition where only superinfected CD4 cells were cultured. Ratios expressed as effector: target cells. FIG. 12B) Pamidronate (PAM)-expanded V52 T cells retain their capacity to inhibit viral replication. After 14 days of exposure to PAM, gd T cells were cocultured with autologous superinfected CD4 cells. V52 T cells (N=10 for the 1 : 1 effector: target ratio) significantly reduced HIV p24 production. FIG. 12C) Comparable inhibition capacity between basal and PAM-expanded V52 T cells. Data from basal HIV inhibition assays were compared to their respective inhibition capacity after PAM exposure. Inhibition capacity was similar in V52 cells (N=7 for the 1:1 effector: target ratio). Mean ±SEM is represented. Mann Whitney U-test. *p<0.05, **p<0.005, ***p<0.0005.

[0042] FIG. 13A) Examples of individual HIV p24 production by isolated V52 T cells expanded with pamidronate (PAM) compared to CD4 cells cultured alone. FIG. 13B) Comparison of inhibition capacity between basal and expanded V52 cells with CD8 T cells at 1:1 effector: target cell (N= 4, Mann-Whitney U-test).

[0043] FIG. 14A-14B. Cytotoxic assays. FIG. 14A) V52 cells degranulate in response to

PHA. CD107a production from V52 cells cocultured with PHA-activated HIV- superinfected CD4 cells is comparable to that produced after coculture with PHA-activated but not superinfected CD4 cells. FIG. 14B) MHC-blocking experiments. Expanded V52 cells were incubated with a pan- HLA monoclonal antibody prior to coculture with autologous HIV- superinfected CD4 cells.

[0044] FIG. 15A-15C. V52 cells degranulate in the presence of autologous HIV-infected

CD4 T cells. FIG. 15A) Flow cytometry plots showing an example of CD107a detection in cocultures of expanded V52 cells with autologous CD4 cells (left) and with autologous JR-CSF- superinfected CD4 cells (right). FIG. 15B) Greater CD107a production in the presence of HIV- infected cells. CDl07a production was statistically higher when V52 cells were cocultured with HIV-superinfected CD4 cells compared to cocultures of autologous isolated CD4 cells (FDR p=0.006). CD 107 a production was the highest when V52 cells were cocultured with PH A- activated HIV-CD4 cells (FDR p=0.02) but without statistical differences compared to cells infected using polybrene. Mean ±SEM is represented. p=0.08, Wilcoxon matched-pairs signed rank test. FIG. 15C) Comparable degranulation capacity of V52 cells between donors treated in acute and chronic HIV infection. CDl07a production was not statistically different between acute and chronic patients. Both groups of patients showed statistically higher CDl07a expression in cocultures of V52 cells and superinfected CD4 target cells than in cultures of V52 cells cocultured with ex vivo isolated CD4 cells. Effector: target ratio (1:1). Mann Whitney U-test.

[0045] FIG. 16. gd T cells clear latently infected cells after latency reversal with vorinostat (VOR). Isolated resting CD4 (r-CD4) cells from ART-suppressed HIV-infected donors were reactivated with 0.5mM VOR. After washing, r-CD4 cells were cultured alone or with gd T cells, which were removed from the culture after 24 hours. The same number of replicate cultures of lxlO⁶ from each condition were then cultured in parallel for 19 days. VOR efficiently reactivated latent HIV in six (represented in the graph) of the eight patients analyzed in the condition where r- CD4 cells were cultured alone. Frequency of HIV recovery (number of positive wells for HIVp24 measured by ELISA) decreased significantly (p=0.03, Wilcoxon signed-rank test.) in cultures of r- CD4 cells in the presence of gd T cells, demonstrating that gd T cells can recognize and clear latently HIV infected cells upon latency reversal.

5. DETAILED DESCRIPTION OF THE DISCLOSURE

5.1. Definitions

[0046] While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.

[0047] Throughout the present specification, the terms“about” and/or“approximately” may be used in conjunction with numerical values and/or ranges. The term“about” is understood to mean those values near to a recited value. For example,“about 40 [units]” may mean within ± 25% of 40 (e.g., from 30 to 50), within ± 20%, ± 15%, ± 10%, ± 9%, ± 8%, ± 7%, ± 6%, ± 5%, ± 4%, ± 3%, ± 2%, ± 1%, less than ± 1%, or any other value or range of values therein or there below. Alternatively, depending on the context, the term“about” may mean ± one half a standard deviation, ± one standard deviation, or ± two standard deviations. Furthermore, the phrases“less than about [a value]” or“greater than about [a value]” should be understood in view of the definition of the term “about” provided herein. The terms “about” and “approximately” may be used interchangeably.

[0048] Nonlimiting examples of bisphosphonates are non-nitrogenous bisphosphonates are Etidronate (Didronel™), Clodronate (Bonefos™, Loron™) or Tiludronate (Skelid™). Examples of nitrogenous bisphosphonates are Alendronate (Fosamax™), Ibandronate (Boniva™ or Bonviva™), Neridronate (Nerixia™), Olpadronate, Pamidronate (APD/Aredia™), Risedronate (Actonel™), or Zoledronate (Zometa™ /Aclasta™).

[0049] "Disease" refers to any disease, disorder, condition, symptom, or indication.

[0050] "Host" preferably refers to a human. It also includes other mammals including the equine, porcine, bovine, feline, and canine families.

[0051] Non-limiting examples of latency reversing agents (LRAs) are an epigenetic modifier, an NFkB agonist, a PI3K/Akt pathway inhibitor, a protein kinase C agonist, a TCR activator or a TLR agonist. See Darcis et ah, 2017, Trends Immun 38(3) 217-228; Rasmussen and Lewin 2016 Curr Opin HIV AIDS 11:394-401; and Kim et al. 2018 Cell Host Microbe 23 14-26.

[0052] The epigenetic modifier may be (i) a bromodomain or BET inhibitor (e.g., CPI- 203, CPI-0610, 1-BET 151 (GSK1210151A), I-BET 762 (GSK525762), JQ1, LY294002, olinone, OTX- 015, TEN-010); (ii) a crotonylation agent, e.g., sodium crotonate or PEP005, see Jiang et al. 2018 “HIV latency is reversed by ACSS2-driven histone crotonylation” J Clin Invest, doi.org/l0.H72/JCI9807l published February 19, 2018 (iii) a histone deacetylase inhibitor (e.g., 4SC-202, Abexinostat (PCI-24781), ACY-1215, AR-42, Belinostat (PXD101), CG200745, Chidamide, CHR-2845 , CHR-3996, CUDC-101, Entinostat (MS-275), Givinostat (ITF2357), HBI- 8000, (a benzamide HDI), Kevetrin (selective for HDAC2), ME-344, Mocetinostat (MGCD0103), Panobinostat, Quisinostat (JNJ-26481585), Resminostat (4SC-201), Romidepsin, sulforaphane, Valproic acid, Vorinostat); (iv) a methylation inhibitor (e.g., 5-azacytidine, 5-aza-2-deoxycytidine (5-Aza-CdR), 5-fluoro-2-deoxycytidine, (-)-epigallocatechin-3-gallate, hydralazine, procainamid and zebularine); (v) a methyltransferase inhibitor, see Pechalrieu et al. 2017, Biochem Pharmacol 129 1-13, (e.g., 5-Aza-2'-deoxycytidine (5azadC), 5-Azacytidine (5azaC), DC_05 analogues, Dichlone, Flavonoid derivatives (Kazinol Q, chloro-nitroflavanones), Guadecitabine (SGI- 110), Indole derivatives, Isoxazoline and oxazoline derivatives, Laccaic acid A, MG98, Nanaomycin A, Procainamide conjugates, Propiophenone derivatives, Pyrrolopyridine derivatives, Quinazoline derivatives, RG108 analogues, SGI-1027, SW155246, Zebularine); (vi) a pTEF-b activator, see

Wang et al 2017 Sci Rep 7(9451) Aug. 25, 2017 (e.g., ingenol derivative, EK-16A). [0053] Nonlimiting examples of NFkB agonists are the SMAC (second mitochondria-derived activator of caspases) mimetics (e.g., AZD5582 (Sampey et al. 2018, bioRxiv May 2, 2018), BV6, birinapant, LCL161).

[0054] Nonlimiting examples of a PI3K/Akt pathway inhibitors are disulfiram, mTor inhibitors (e.g., RAD rapamycin (sirolimus) and derivatives/analogs thereof such as everolimus or RAD001; ABT578, AP23573, AP23841, ascomycin (an ethyl analog of FK506), AZD08055, CCI-779, EX2044, EX3855, EX7518, INK-128, KU-0063794, OSI027, SAR543).

[0055] Nonlimiting examples of protein kinase C agonists are bryostatin, ingenol B/PEP005, prostratin).

[0056] Nonlimiting examples of a TLR agonist are TLR7 and TLR9 agonists (e.g., TLR9 agonist, Lefitolimod/MGNl703 or 1V270 or SD-101, see Sato-Kaneko et ah, 2017 JCI Insight 2(18) e93397).

[0057] gd T cells (gamma delta T cells) represent a small subset of T cells that possess a distinct T cell receptor (TCR) on their surfaces. A majority of T cells have a TCR composed of two glycoprotein chains called a- and b- TCR chains. However, in gd T cells, the TCR is made up of one g-chain and one d-chain. In humans they represent under 10% of total T cells) gd T cells are not MHC-restricted. As used herein, the gd T cells may be endogenous cells, e.g., native cells or expanded native cells from a patient. In other embodiments the gd T cells may be genetically engineered, e.g., CCR5 and/or CXCR4 knockouts. See Delhove and Qasim, 2017,“Genome-Edited T Cell Therapies” Curr Stem Cell Rep 3:124-136; Schumann et ah, 2015, "Generation of knock-in primary human T cells using Cas9 ribonucleoproteins". Proceedings of the National Academy of Sciences USA. 112 (33): 10437-10442; Zhang et al. 2017,“Gene editing in T cell therapy” J Genetics and Genomics 44 (2017) 415-422. In other embodiments, the gd T cells are adopted T cells. See Ruella and Kalos, 2014,“Adoptive immunotherapy for cancer.” Immunol Rev. 257(1): 14- 38; Pankrac et al., 2017,“Eradication of HIV-l latent reservoirs through therapeutic vaccination” AIDS Res Ther 14:45 DOI 10.1186/S12981-017-0177-4.

[0058] "Pharmaceutically acceptable salts" refer to derivatives of the disclosed compounds wherein the parent compound is modified by making acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of the basic residues. The pharmaceutically acceptable salts include the conventional quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include, but are not limited to, those derived from inorganic and organic acids selected from l,2-ethanedisulfonic, 2-acetoxybenzoic, 2- hydroxyethanesulfonic, acetic, ascorbic, benzenesulfonic, benzoic, bicarbonic, carbonic, citric, edetic, ethane disulfonic, ethane sulfonic, fumaric, glucoheptonic, gluconic, glutamic, glycolic, glycollyarsanilic, hexylresorcinic, hydrabamic, hydrobromic, hydrochloric, hydroiodide, hydroxymaleic, hydroxy naphthoic, isethionic, lactic, lactobionic, lauryl sulfonic, maleic, malic, mandelic, methanesulfonic, napsylic, nitric, oxalic, pamoic, pantothenic, phenylacetic, phosphoric, polygalacturonic, propionic, salicyclic, stearic, subacetic, succinic, sulfamic, sulfanilic, sulfuric, tannic, tartaric, and toluenesulfonic. Such conventional and non-toxic salts also include inorganic or organic bases such as lithium, magnesium, sodium, potassium, calcium, aluminum, zinc, arginine, lysine, benzathine, chloroprocaine, choline, diethanolamine, ethanolamine, ethylenediame, histadine, meglumine, procaine, and triethylamine. A detailed description of salt formation in pharmaceutically active compounds can be found in the following book: Stahl, P H, and Camille G Handbook of Pharmaceutical Salts: Properties, Selection, and Use. Ziirich: Verlag Helvetica Chimica Acta, 2011.

[0059] "Therapeutically effective amount" includes an amount of a compound of the present invention that is effective when administered alone or in combination to treat the desired condition or disorder. "Therapeutically effective amount" includes an amount of the combination of compounds claimed that is effective to treat the desired condition or disorder. The combination of compounds is preferably a synergistic combination. Synergy, as described, for example, by Chou and Talalay, Adv. Enzyme Regul. 1984, 22:27-55, occurs when the effect of the compounds when administered in combination is greater than the additive effect of the compounds when administered alone as a single agent. In general, a synergistic effect is most clearly demonstrated at sub-optimal concentrations of the compounds. Synergy can be in terms of lower cytotoxicity, increased antiviral effect, or some other beneficial effect of the combination compared with the individual components.

[0060] "Treating" or "treatment" covers the treatment of a disease-state in a mammal, and includes: (a) preventing the disease-state from occurring in a mammal, in particular, when such mammal is predisposed to the disease-state but has not yet been diagnosed as having it; (b) inhibiting the disease-state, e.g., arresting its development; and/or (c) relieving the disease-state, e.g., causing regression of the disease state until a desired endpoint is reached. Treating also includes the amelioration of a symptom of a disease (e.g., lessen the pain or discomfort), wherein such amelioration may or may not be directly affecting the disease (e.g., cause, transmission, expression, etc.).

[0061] Throughout the present specification, numerical ranges are provided for certain quantities. It is to be understood that these ranges comprise all subranges therein. Thus, the range“from 50 to 80” includes all possible ranges therein (e.g., 51-79, 52-78, 53-77, 54-76, 55-75, 60-70, etc.). Furthermore, all values within a given range may be an endpoint for the range encompassed thereby (e.g., the range 50-80 includes the ranges with endpoints such as 55-80, 50-75, etc.). [0062] As used herein, the verb“comprise” as used in this description and in the claims and its conjugations are used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded.

[0063] Throughout the specification the word“comprising,” or variations such as“comprises” or “comprising,” will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. The present disclosure may suitably“comprise”,“consist of’, or “consist essentially of’, the steps, elements, and/or reagents described in the claims.

[0064] It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely", "only" and the like in connection with the recitation of claim elements, or the use of a "negative" limitation.

[0065] Unless defined otherwise, 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 disclosure belongs. Preferred methods, devices, and materials are described, although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure. All references cited herein are incorporated by reference in their entirety.

5.2. Pharmaceutically Acceptable Compositions

[0066] Provided herein are pharmaceutical compositions comprising a compound disclosed herein as an active ingredient, or a pharmaceutically acceptable salt, solvate or hydrate thereof in combination with a pharmaceutically acceptable vehicle, carrier, diluent, or excipient, or a mixture thereof.

[0067] The compound provided herein may be administered alone, or in combination with one or more other compounds provided herein. The pharmaceutical compositions that comprise a compound disclosed herein can be formulated in various dosage forms for oral, parenteral, and topical administration. The pharmaceutical compositions can also be formulated as modified release dosage forms, including delayed-, extended-, prolonged-, sustained-, pulsatile-, controlled-, accelerated- and fast-, targeted-, programmed-release, and gastric retention dosage forms. These dosage forms can be prepared according to conventional methods and techniques known to those skilled in the art (see, Remington: The Science and Practice of Pharmacy. 2lst Ed., Lippincott, Williams & Wilkins, Baltimore, M.D., 2006; Modified-Release Drug Delivery Technology, Rathbone et ah, Eds., Drugs and the Pharmaceutical Science, Marcel Dekker, Inc.: New York, N.Y., 2003; Vol. 126).

[0068] In one embodiment, the pharmaceutical compositions are provided in a dosage form for oral administration, which comprise a compound provided herein, e.g., a compound disclosed herein or a pharmaceutically acceptable salt, solvate or hydrate thereof; and one or more pharmaceutically acceptable excipients or carriers.

[0069] In another embodiment, the pharmaceutical compositions are provided in a dosage form for parenteral administration, which comprise a compound disclosed herein or a pharmaceutically acceptable salt, solvate or hydrate thereof; and one or more pharmaceutically acceptable excipients or carriers.

[0070] In yet another embodiment, the pharmaceutical compositions are provided in a dosage form for topical administration, which comprise a compound disclosed herein or a pharmaceutically acceptable salt, solvate or hydrate thereof; and one or more pharmaceutically acceptable excipients or carriers.

[0071] The pharmaceutical compositions provided herein can be provided in a unit-dosage form or multiple-dosage form. A unit-dosage form, as used herein, refers to physically discrete a unit suitable for administration to a human and animal subject, and packaged individually as is known in the art. Each unit-dose contains a predetermined quantity of an active ingredient(s) sufficient to produce the desired therapeutic effect, in association with the required pharmaceutical carriers or excipients. Examples of a unit-dosage form include an ampoule, syringe, and individually packaged tablet and capsule. A unit-dosage form may be administered in fractions or multiples thereof. A multiple-dosage form is a plurality of identical unit-dosage forms packaged in a single container to be administered in segregated unit-dosage form. Examples of a multiple-dosage form include a vial, bottle of tablets or capsules, or bottle of pints or gallons. The pharmaceutical compositions provided herein can be administered at once, or multiple times at intervals of time. It is understood that the precise dosage and duration of treatment may vary with the age, weight, and condition of the patient being treated, and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test or diagnostic data. It is further understood that for any particular individual, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the formulations.

[0072] In one embodiment, the therapeutically effective dose is from about 0.1 mg to about 2,000 mg per day of a compound provided herein. The pharmaceutical compositions therefore should provide a dosage of from about 0.1 mg to about 2000 mg of the compound. In certain embodiments, pharmaceutical dosage unit forms are prepared to provide from about 1 mg to about 2000 mg, from about 10 mg to about 1000 mg, from about 20 mg to about 500 mg or from about 25 mg to about 250 mg of the essential active ingredient or a combination of essential ingredients per dosage unit form. In certain embodiments, the pharmaceutical dosage unit forms are prepared to provide about 10 mg, 20 mg, 25 mg, 50 mg, 100 mg, 250 mg, 500 mg, 1000 mg or 2000 mg of the essential active ingredient.

5.2.1. Parental Administration

[0073] The pharmaceutical compositions provided herein can be administered parenterally by injection, infusion, or implantation, for local or systemic administration· Parenteral administration, as used herein, include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular, intrasynovial, intravesical, and subcutaneous administration·

[0074] The pharmaceutical compositions provided herein can be formulated in any dosage forms that are suitable for parenteral administration, including solutions, suspensions, emulsions, micelles, liposomes, microspheres, nanosystems, and solid forms suitable for solutions or suspensions in liquid prior to injection. Such dosage forms can be prepared according to conventional methods known to those skilled in the art of pharmaceutical science (see, Remington: The Science and Practice of Pharmacy, supra).

[0075] The pharmaceutical compositions intended for parenteral administration can include one or more pharmaceutically acceptable carriers and excipients, including, but not limited to, aqueous vehicles, water-miscible vehicles, non-aqueous vehicles, antimicrobial agents or preservatives against the growth of microorganisms, stabilizers, solubility enhancers, isotonic agents, buffering agents, antioxidants, local anesthetics, suspending and dispersing agents, wetting or emulsifying agents, complexing agents, sequestering or chelating agents, cryoprotectants, lyoprotectants, thickening agents, pH adjusting agents, and inert gases.

[0076] Suitable aqueous vehicles include, but are not limited to, water, saline, physiological saline or phosphate buffered saline (PBS), sodium chloride injection, Ringers injection, isotonic dextrose injection, sterile water injection, dextrose and lactated Ringers injection. Non-aqueous vehicles include, but are not limited to, fixed oils of vegetable origin, castor oil, com oil, cottonseed oil, olive oil, peanut oil, peppermint oil, safflower oil, sesame oil, soybean oil, hydrogenated vegetable oils, hydrogenated soybean oil, and medium-chain triglycerides of coconut oil, and palm seed oil. Water- miscible vehicles include, but are not limited to, ethanol, l,3-butanediol, liquid polyethylene glycol (e.g., polyethylene glycol 300 and polyethylene glycol 400), propylene glycol, glycerin, N- methyl-2-pyrrolidone, N,N-dimethylacetamide, and dimethyl sulfoxide.

[0077] Suitable antimicrobial agents or preservatives include, but are not limited to, phenols, cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoates, thimerosal, benzalkonium chloride (e.g., benzethonium chloride), methyl- and propyl-parabens, and sorbic acid. Suitable isotonic agents include, but are not limited to, sodium chloride, glycerin, and dextrose. Suitable buffering agents include, but are not limited to, phosphate and citrate. Suitable antioxidants are those as described herein, including bisulfite and sodium metabisulfite. Suitable local anesthetics include, but are not limited to, procaine hydrochloride. Suitable suspending and dispersing agents are those as described herein, including sodium carboxymethylcelluose, hydroxypropyl methylcellulose, and polyvinylpyrrolidone. Suitable emulsifying agents include those described herein, including polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate 80, and triethanolamine oleate. Suitable sequestering or chelating agents include, but are not limited to EDTA. Suitable pH adjusting agents include, but are not limited to, sodium hydroxide, hydrochloric acid, citric acid, and lactic acid. Suitable complexing agents include, but are not limited to, cyclodextrins, including a-cyclodextrin, b-cyclodextrin, hydroxypropyl-b- cyclodextrin, sulfobutylether^-cyclodextrin, and sulfobutylether 7^-cyclodextrin (CAPTISOL®, CyDex, Lenexa, Kans.).

[0078] The pharmaceutical compositions provided herein can be formulated for single or multiple dosage administration· The single dosage formulations are packaged in an ampoule, a vial, or a syringe. The multiple dosage parenteral formulations must contain an antimicrobial agent at bacteriostatic or fungistatic concentrations. All parenteral formulations must be sterile, as known and practiced in the art.

[0079] In one embodiment, the pharmaceutical compositions are provided as ready-to-use sterile solutions. In another embodiment, the pharmaceutical compositions are provided as sterile dry soluble products, including lyophilized powders and hypodermic tablets, to be reconstituted with a vehicle prior to use. In one embodiment, the lyophilized nanoparticles are provided in a vial for reconstitution with a sterile aqueous solution just prior to injection. In yet another embodiment, the pharmaceutical compositions are provided as ready-to-use sterile suspensions. In yet another embodiment, the pharmaceutical compositions are provided as sterile dry insoluble products to be reconstituted with a vehicle prior to use. In still another embodiment, the pharmaceutical compositions are provided as ready-to-use sterile emulsions. The pharmaceutical compositions provided herein can be formulated as immediate or modified release dosage forms, including delayed-, sustained, pulsed-, controlled, targeted-, and programmed-release forms.

[0080] The pharmaceutical compositions can be formulated as a suspension, solid, semi-solid, or thixotropic liquid, for administration as an implanted depot.

5.2.2. Oral Administration Compositions

[0081] The pharmaceutical compositions provided herein can be provided in solid, semisolid, or liquid dosage forms for oral administration. As used herein, oral administration also includes buccal, lingual, and sublingual administration· Suitable oral dosage forms include, but are not limited to, tablets, fastmelts, chewable tablets, capsules, pills, troches, lozenges, pastilles, cachets, pellets, medicated chewing gum, bulk powders, effervescent or non-effervescent powders or granules, solutions, emulsions, suspensions, wafers, sprinkles, elixirs, and syrups. In addition to the active ingredient(s), the pharmaceutical compositions can contain one or more pharmaceutically acceptable carriers or excipients, including, but not limited to, binders, fillers, diluents, disintegrants, wetting agents, lubricants, glidants, coloring agents, dye-migration inhibitors, sweetening agents, and flavoring agents.

[0082] Binders or granulators impart cohesiveness to a tablet to ensure the tablet remaining intact after compression. Suitable binders or granulators include, but are not limited to, starches, such as corn starch, potato starch, and pre-gelatinized starch (e.g., STARCH 1500); gelatin; sugars, such as sucrose, glucose, dextrose, molasses, and lactose; natural and synthetic gums, such as acacia, alginic acid, alginates, extract of Irish moss, panwar gum, ghatti gum, mucilage of isabgol husks, carboxymethylcellulose, methylcellulose, polyvinylpyrrolidone (PVP), Veegum, larch arabogalactan, powdered tragacanth, and guar gum; celluloses, such as ethyl cellulose, cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethyl cellulose, methyl cellulose, hydroxyethylcellulose (HEC), hydroxypropylcellulose (HPC), hydroxypropyl methyl cellulose (HPMC); microcrystalline celluloses, such as AVICEL-PH-101, AVICEL-PH-103, AVICEL RC- 581, AVICEL-PH-105 (FMC Corp., Marcus Hook, Pa.); and mixtures thereof. Suitable fillers include, but are not limited to, talc, calcium carbonate, microcrystalline cellulose, powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof. The binder or filler may be present from about 50 to about 99% by weight in the pharmaceutical compositions provided herein.

[0083] Suitable diluents include, but are not limited to, dicalcium phosphate, calcium sulfate, lactose, sorbitol, sucrose, inositol, cellulose, kaolin, mannitol, sodium chloride, dry starch, and powdered sugar. Certain diluents, such as mannitol, lactose, sorbitol, sucrose, and inositol, when present in sufficient quantity, can impart properties to some compressed tablets that permit disintegration in the mouth by chewing. Such compressed tablets can be used as chewable tablets.

[0084] Suitable disintegrants include, but are not limited to, agar; bentonite; celluloses, such as methylcellulose and carboxymethylcellulose; wood products; natural sponge; cation-exchange resins; alginic acid; gums, such as guar gum and Veegum HV; citrus pulp; cross-linked celluloses, such as croscarmellose; cross-linked polymers, such as crospovidone; cross-linked starches; calcium carbonate; microcrystalline cellulose, such as sodium starch glycolate; polacrilin potassium; starches, such as com starch, potato starch, tapioca starch, and pre-gelatinized starch; clays; aligns; and mixtures thereof. The amount of a disintegrant in the pharmaceutical compositions provided herein varies upon the type of formulation, and is readily discernible to those of ordinary skill in the art. The pharmaceutical compositions provided herein may contain from about 0.5 to about 15% or from about 1 to about 5% by weight of a disintegrant. [0085] Suitable lubricants include, but are not limited to, calcium stearate; magnesium stearate; mineral oil; light mineral oil; glycerin; sorbitol; mannitol; glycols, such as glycerol behenate and polyethylene glycol (PEG); stearic acid; sodium lauryl sulfate; talc; hydrogenated vegetable oil, including peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, com oil, and soybean oil; zinc stearate; ethyl oleate; ethyl laureate; agar; starch; lycopodium; silica or silica gels, such as AEROSIL® 200 (W. R. Grace Co., Baltimore, Md.) and CAB-O-SIL® (Cabot Co. of Boston, Mass.); and mixtures thereof. The pharmaceutical compositions provided herein may contain about 0.1 to about 5% by weight of a lubricant.

[0086] Suitable glidants include colloidal silicon dioxide, CAB-O-SIL® (Cabot Co. of Boston, Mass.), and asbestos-free talc. Coloring agents include any of the approved, certified, water soluble FD&C dyes, and water insoluble FD&C dyes suspended on alumina hydrate, and color lakes and mixtures thereof. A color lake is the combination by adsorption of a water-soluble dye to a hydrous oxide of a heavy metal, resulting in an insoluble form of the dye. Flavoring agents include natural flavors extracted from plants, such as fruits, and synthetic blends of compounds which produce a pleasant taste sensation, such as peppermint and methyl salicylate. Sweetening agents include sucrose, lactose, mannitol, syrups, glycerin, and artificial sweeteners, such as saccharin and aspartame. Suitable emulsifying agents include gelatin, acacia, tragacanth, bentonite, and surfactants, such as polyoxyethylene sorbitan monooleate (TWEEN® 20), polyoxyethylene sorbitan monooleate 80 (TWEEN® 80), and triethanolamine oleate. Suspending and dispersing agents include sodium carboxymethylcellulose, pectin, tragacanth, Veegum, acacia, sodium carbomethylcellulose, hydroxypropyl methylcellulose, and polyvinylpyrrolidone. Preservatives include glycerin, methyl and propylparaben, benzoic add, sodium benzoate and alcohol. Wetting agents include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate, and polyoxyethylene lauryl ether. Solvents include glycerin, sorbitol, ethyl alcohol, and syrup. Examples of non- aqueous liquids utilized in emulsions include mineral oil and cottonseed oil. Organic acids include citric and tartaric acid. Sources of carbon dioxide include sodium bicarbonate and sodium carbonate.

[0087] It should be understood that many carriers and excipients may serve several functions, even within the same formulation.

[0088] The pharmaceutical compositions provided herein can be provided as compressed tablets, tablet triturates, chewable lozenges, rapidly dissolving tablets, multiple compressed tablets, or enteric-coating tablets, sugar-coated, or film-coated tablets. Enteric-coated tablets are compressed tablets coated with substances that resist the action of stomach acid but dissolve or disintegrate in the intestine, thus protecting the active ingredients from the acidic environment of the stomach.

Enteric-coatings include, but are not limited to, fatty acids, fats, phenyl salicylate, waxes, shellac, ammoniated shellac, and cellulose acetate phthalates. Sugar-coated tablets are compressed tablets surrounded by a sugar coating, which may be beneficial in covering up objectionable tastes or odors and in protecting the tablets from oxidation. Film-coated tablets are compressed tablets that are covered with a thin layer or film of a water-soluble material. Film coatings include, but are not limited to, hydroxyethylcellulose, sodium carboxymethylcellulose, polyethylene glycol 4000, and cellulose acetate phthalate. Hydrophilic polymer formulations have been widely used for improved oral availability such as ethylene oxides, hydroxy propyl methyl cellulose (HPC), poly (ethylene oxide) (PEO), polyvinyl alcohol (PVA), poly(hydroxyethylmethyl acrylate) methyl methacrylate (PHEMA), or vinyl acetate (PCT Pub. No. WO1999/37302 (Alvarez et al. ); Dimitrov & Lambov, 1999, Int J Pharm 189 105-111; Zhang et al., 1990, Proc Int. Symp Controlled Release Bioact. Mater. 17, 333, the contents of which are hereby incorporated by reference in their entirety). Film coating imparts the same general characteristics as sugar coating. Multiple compressed tablets are compressed tablets made by more than one compression cycle, including layered tablets, and press- coated or dry-coated tablets.

[0089] The tablet dosage forms can be prepared from the active ingredient in powdered, crystalline, or granular forms, alone or in combination with one or more carriers or excipients described herein, including binders, disintegrants, controlled-release polymers, lubricants, diluents, and/or colorants. Flavoring and sweetening agents are especially useful in the formation of chewable tablets and lozenges.

[0090] The pharmaceutical compositions provided herein can be provided as soft or hard capsules, which can be made from gelatin, methylcellulose, starch, or calcium alginate. The hard gelatin capsule, also known as the dry-filled capsule (DFC), consists of two sections, one slipping over the other, thus completely enclosing the active ingredient. The soft elastic capsule (SEC) is a soft, globular shell, such as a gelatin shell, which is plasticized by the addition of glycerin, sorbitol, or a similar polyol. The soft gelatin shells may contain a preservative to prevent the growth of microorganisms. Suitable preservatives are those as described herein, including methyl- and propyl parabens, and sorbic acid. The liquid, semisolid, and solid dosage forms provided herein may be encapsulated in a capsule. Suitable liquid and semisolid dosage forms include solutions and suspensions in propylene carbonate, vegetable oils, or triglycerides. Capsules containing such solutions can be prepared as described in U.S. Pat. Nos. 4,328,245; 4,409,239; and 4,410,545, the contents of which are hereby incorporated by reference in their entirety. The capsules may also be coated as known by those of skill in the art in order to modify or sustain dissolution of the active ingredient.

[0091] The pharmaceutical compositions provided herein can be provided in liquid and semisolid dosage forms, including emulsions, solutions, suspensions, elixirs, and syrups. An emulsion is a two-phase system, in which one liquid is dispersed in the form of small globules throughout another liquid, which can be oil-in-water or water-in-oil. Emulsions may include a pharmaceutically acceptable non-aqueous liquid or solvent, emulsifying agent, and preservative. Suspensions may include a pharmaceutically acceptable suspending agent and preservative. Aqueous alcoholic solutions may include a pharmaceutically acceptable acetal, such as a di(lower alkyl) acetal of a lower alkyl aldehyde, e.g., acetaldehyde diethyl acetal; and a water-miscible solvent having one or more hydroxyl groups, such as propylene glycol and ethanol. Elixirs are clear, sweetened, and hydroalcoholic solutions. Syrups are concentrated aqueous solutions of a sugar, for example, sucrose, and may also contain a preservative. For a liquid dosage form, for example, a solution in a polyethylene glycol may be diluted with a sufficient quantity of a pharmaceutically acceptable liquid carrier, e.g., water, to be measured conveniently for administration.

[0092] Other useful liquid and semisolid dosage forms include, but are not limited to, those containing the active ingredient(s) provided herein, and a dialkylated mono- or poly-alkylene glycol, including, l,2-dimethoxymethane, diglyme, triglyme, tetraglyme, polyethylene glycol-350- dimethyl ether, polyethylene glycol-550-dimethyl ether, polyethylene glycol-750-dimethyl ether, wherein 350, 550, and 750 refer to the approximate average molecular weight of the polyethylene glycol. These formulations can further comprise one or more antioxidants, such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), propyl gallate, vitamin E, hydroquinone, hydroxycoumarins, ethanolamine, lecithin, cephalin, ascorbic acid, malic acid, sorbitol, phosphoric acid, bisulfite, sodium metabisulfite, thiodipropionic acid and its esters, and dithiocarbamates.

[0093] The pharmaceutical compositions provided herein for oral administration can be also provided in the forms of liposomes, micelles, microspheres, or nanosystems. Micellar dosage forms can be prepared as described in U.S. Pat. No. 6,350,458, the content of which is hereby incorporated by reference in its entirety.

[0094] The pharmaceutical compositions provided herein can be provided as non-effervescent or effervescent, granules and powders, to be reconstituted into a liquid dosage form. Pharmaceutically acceptable carriers and excipients used in the non-effervescent granules or powders may include diluents, sweeteners, and wetting agents. Pharmaceutically acceptable carriers and excipients used in the effervescent granules or powders may include organic acids and a source of carbon dioxide.

[0095] Coloring and flavoring agents can be used in all of the above dosage forms. The pharmaceutical compositions provided herein can be formulated as immediate or modified release dosage forms, including delayed-, sustained, pulsed-, controlled, targeted-, and programmed- release forms. [0096] The pharmaceutical compositions provided herein can be co-formulated with other active ingredients which do not impair the desired therapeutic action, or with substances that supplement the desired action.

5.2.3. Topical Administration

[0097] The pharmaceutical compositions provided herein can be administered topically to the skin, orifices, or mucosa. The topical administration, as used herein, includes (intra)dermal, conjunctival, intracorneal, intraocular, ophthalmic, auricular, transdermal, nasal, vaginal, urethral, respiratory, and rectal administration·

[0098] The pharmaceutical compositions provided herein can be formulated in any dosage forms that are suitable for topical administration for local or systemic effect, including emulsions, solutions, suspensions, creams, gels, hydrogels, ointments, dusting powders, dressings, elixirs, lotions, suspensions, tinctures, pastes, foams, films, aerosols, irrigations, sprays, suppositories, bandages, dermal patches. The topical formulation of the pharmaceutical compositions provided herein can also comprise liposomes, micelles, microspheres, nanosystems, and mixtures thereof.

[0099] Pharmaceutically acceptable carriers and excipients suitable for use in the topical formulations provided herein include, but are not limited to, aqueous vehicles, water-miscible vehicles, non-aqueous vehicles, antimicrobial agents or preservatives against the growth of microorganisms, stabilizers, solubility enhancers, isotonic agents, buffering agents, antioxidants, local anesthetics, suspending and dispersing agents, wetting or emulsifying agents, complexing agents, sequestering or chelating agents, penetration enhancers, cryoprotectants, lyoprotectants, thickening agents, and inert gases.

[00100] The pharmaceutical compositions can also be administered topically by electroporation, iontophoresis, phonophoresis, sonophoresis, or microneedle or needle-free injection, such as POWDERJECT™ (Chiron Corp., Emeryville, CA), and BIOJECT™ (Bioject Medical Technologies Inc., Tualatin, OR).

[00101] The pharmaceutical compositions provided herein can be provided in the forms of ointments, creams, and gels. Suitable ointment vehicles include oleaginous or hydrocarbon vehicles, including lard, benzoinated lard, olive oil, cottonseed oil, and other oils, white petrolatum; emulsifiable or absorption vehicles, such as hydrophilic petrolatum, hydroxystearin sulfate, and anhydrous lanolin; water-removable vehicles, such as hydrophilic ointment; water-soluble ointment vehicles, including polyethylene glycols of varying molecular weight; emulsion vehicles, either water-in-oil (W/O) emulsions or oil-in-water (O/W) emulsions, including cetyl alcohol, glyceryl monostearate, lanolin, and stearic acid (see, Remington: The Science and Practice of Pharmacy. supra). These vehicles are emollient but generally require addition of antioxidants and preservatives. [00102] Suitable cream base can be oil-in-water or water-in-oil. Cream vehicles may be water-washable, and contain an oil phase, an emulsifier, and an aqueous phase. The oil phase is also called the“internal” phase, which is generally comprised of petrolatum and a fatty alcohol such as cetyl or stearyl alcohol. The aqueous phase usually, although not necessarily, exceeds the oil phase in volume, and generally contains a humectant. The emulsifier in a cream formulation may be a nonionic, anionic, cationic, or amphoteric surfactant.

[00103] Gels are semisolid, suspension-type systems. Single -phase gels contain organic macromolecules distributed substantially uniformly throughout the liquid carrier. Suitable gelling agents include crosslinked acrylic acid polymers, such as carbomers, carboxypolyalkylenes, CARBOPOL®; hydrophilic polymers, such as polyethylene oxides, polyoxyethylene- polyoxypropylene copolymers, and polyvinylalcohol; cellulosic polymers, such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, and methylcellulose; gums, such as tragacanth and xanthan gum; sodium alginate; and gelatin. In order to prepare a uniform gel, dispersing agents such as alcohol or glycerin can be added, or the gelling agent can be dispersed by trituration, mechanical mixing, and/or stirring.

[00104] The pharmaceutical compositions provided herein can be administered rectally, urethrally, vaginally, or perivaginally in the forms of suppositories, pessaries, bougies, poultices or cataplasm, pastes, powders, dressings, creams, plasters, contraceptives, ointments, solutions, emulsions, suspensions, tampons, gels, foams, sprays, or enemas. These dosage forms can be manufactured using conventional processes as described in Remington: The Science and Practice of Pharmacy, supra.

[00105] Rectal, urethral, and vaginal suppositories are solid bodies for insertion into body orifices, which are solid at ordinary temperatures but melt or soften at body temperature to release the active ingredient(s) inside the orifices. Pharmaceutically acceptable carriers utilized in rectal and vaginal suppositories include bases or vehicles, such as stiffening agents, which produce a melting point in the proximity of body temperature, when formulated with the pharmaceutical compositions provided herein; and antioxidants as described herein, including bisulfite and sodium metabisulfite. Suitable vehicles include, but are not limited to, cocoa butter (theobroma oil), glycerin- gelatin, carbowax (polyoxyethylene glycol), spermaceti, paraffin, white and yellow wax, and appropriate mixtures of mono-, di- and triglycerides of fatty acids, hydrogels, such as polyvinyl alcohol, hydroxyethyl methacrylate, polyacrylic acid; glycerinated gelatin. Combinations of the various vehicles may be used. Rectal and vaginal suppositories may be prepared by the compressed method or molding. The typical weight of a rectal and vaginal suppository is about 2 to about 3 g. [00106] The pharmaceutical compositions provided herein can be administered ophthalmically in the forms of solutions, suspensions, ointments, emulsions, gel-forming solutions, powders for solutions, gels, ocular inserts, and implants.

5.3. Aerosol Administration

[00107] The pharmaceutical compositions provided herein can be administered intranasally or by inhalation to the respiratory tract. The pharmaceutical compositions can be provided in the form of an aerosol or solution for delivery using a pressurized container, pump, spray, atomizer, such as an atomizer using electrohydrodynamics to produce a fine mist, or nebulizer, alone or in combination with a suitable propellant, such as 1,1,1, 2-tetrafluoroethane or 1, 1,1, 2, 3,3,3- heptafluoropropane. There are many examples in the literature of metered dose inhalers (MDIs) or pressurized metered dose inhalers (pMDIs). See Kleinstreuer et al. 2015 World J Clin Cases 2(12) 742-756. The pharmaceutical compositions can also be provided as a dry powder for insufflation, alone or in combination with an inert carrier such as lactose or phospholipids; and nasal drops. Many pulmonary drugs are delivered by dry powder inhalers (DPIs) with differing arrangements such as single dose, multi-dose with the pharmaceutical composition in bulk, or multi-dose with individual blister packs. See Kleinstreuer et al. 2015; Weer and Miller 2015 J Pharm Sci 104 3259- 3288. For intranasal use, the powder can comprise a bioadhesive agent, including chitosan or cyclodextrin. As mentioned above, the pharmaceutical composition may be delivered by nebulizer such as an atomizer (jet nebulizer), an ultrasonic wave nebulizer, or a vibrating mesh nebulizer. See Kleinstreuer et al. 2015.

[00108] Alternatively, the pharmaceutical composition may be dissolved in glycerol, propane 1,2 diol gycol (PG), water or a mixture thereof and vaporized at relatively low temperature (>100 °C, typically 40-65 °C) in an e-cigarette. See Bertholon et al. 2013 Respiration 86 433-438; Brown and Cheng 2014 Tob Control May;23 Suppl 2:ii4- 10; and Famele 2015 Nicotine Tob Res 271-279; European Patent Appn. No. EP2641490A1 (Liu); European Patent Nos. EP1618803B1 and EP1736065B1 (Hon L., Best Partners Worldwide Limited); U.S. Appn. Nos. 20050016550 Al (Katase), 20110265806 (Alarcon and Healy), 20110277780A1 (Terry and Minskoff), 20130213418 Al (Tucker et al., Altria Client Service Inc.), 20130192621 (Li et al., Altria Client Service Inc.), 20130192623 (Tucker et al., Altria Client Service Inc.), 20130213419 (Tucker et al., Altria Client Service Inc.), 20130220315 (Conley, Fuma International); U.S. Patent Nos. 8,490,628 (Hon, Ruyan Investment Limited), 8,528,569 (Newton), 8,550,069 (Alelov).

[00109] Solutions or suspensions for use in a pressurized container, pump, spray, atomizer, or nebulizer can be formulated to contain ethanol, aqueous ethanol, or a suitable alternative agent for dispersing, solubilizing, or extending release of the active ingredient provided herein, a propellant as solvent; and/or a surfactant, such as sorbitan trioleate, oleic acid, or an oligolactic acid.

[00110] The pharmaceutical compositions provided herein can be micronized to a size suitable for delivery by inhalation, such as about 50 micrometers or less, or about 10 micrometers or less. Particles of such sizes can be prepared using a comminuting method known to those skilled in the art, such as spiral jet milling, fluid bed jet milling, supercritical fluid processing to form nanoparticles, high pressure homogenization, or spray drying.

[00111] Capsules, blisters and cartridges for use in an inhaler or insufflator can be formulated to contain a powder mix of the pharmaceutical compositions provided herein; a suitable powder base, such as lactose or starch; and a performance modifier, such as l-leucine, mannitol, or magnesium stearate. The lactose may be anhydrous or in the form of the monohydrate. Other suitable excipients or carriers include dextran, glucose, maltose, sorbitol, xylitol, fructose, sucrose, and trehalose. The pharmaceutical compositions provided herein for inhaled/intranasal administration can further comprise a suitable flavor, such as menthol and levomenthol, or sweeteners, such as saccharin or saccharin sodium.

[00112] The pharmaceutical compositions provided herein for topical administration can be formulated to be immediate release or modified release, including delayed-, sustained-, pulsed-, controlled-, targeted, and programmed release.

5.4. Modified Release Formulations

[00113] The pharmaceutical compositions provided herein can be formulated as a modified release dosage form. As used herein, the term“modified release” refers to a dosage form in which the rate or place of release of the active ingredient(s) is different from that of an immediate dosage form when administered by the same route. Modified release dosage forms include delayed-, extended-, prolonged-, sustained-, pulsatile-, controlled-, accelerated- and fast-, targeted-, programmed-release, and gastric retention dosage forms. The pharmaceutical compositions in modified release dosage forms can be prepared using a variety of modified release devices and methods known to those skilled in the art, including, but not limited to, matrix controlled release devices, osmotic controlled release devices, multi-particulate controlled release devices, ion- exchange resins, enteric coatings, multilayered coatings, microspheres, liposomes, and combinations thereof The release rate of the active ingredient(s) can also be modified by varying the particle sizes and polymorphism of the active ingredient(s).

[00114] Examples of modified release include, but are not limited to, those described in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,639,480; 5,733,566; 5,739,108; 5,891,474;

5,922,356; 5,972,891; 5,980,945; 5,993,855; 6,045,830; 6,087,324; 6,113,943; 6,197,350; 6,248,363; 6,264,970; 6,267,981; 6,376,461; 6,419,961; 6,589,548; 6,613,358; and 6,699,500, the contents of which are hereby incorporated by reference in their entirety.

5.4.1. Matrix Controlled Release Devices

[00115] The pharmaceutical compositions provided herein in a modified release dosage form can be fabricated using a matrix controlled release device known to those skilled in the art (see, Takada et al. in“Encyclopedia of Controlled Drug Delivery,” Vol.2, Mathiowitz Ed., Wiley, 1999).

[00116] In one embodiment, the pharmaceutical compositions provided herein in a modified release dosage form is formulated using an erodible matrix device, which is water- swellable, erodible, or soluble polymers, including synthetic polymers, and naturally occurring polymers and derivatives, such as polysaccharides and proteins.

[00117] Materials useful in forming an erodible matrix include, but are not limited to, chitin, chitosan, dextran, and pullulan; gum agar, gum arabic, gum karaya, locust bean gum, gum tragacanth, carrageenans, gum ghatti, guar gum, xanthan gum, and scleroglucan; starches, such as dextrin and maltodextrin; hydrophilic colloids, such as pectin; phosphatides, such as lecithin; alginates; propylene glycol alginate; gelatin; collagen; and cellulosics, such as ethyl cellulose (EC), methylethyl cellulose (MEC), carboxymethyl cellulose (CMC), CMEC, hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), cellulose acetate (CA), cellulose propionate (CP), cellulose butyrate (CB), cellulose acetate butyrate (CAB), CAP, CAT, hydroxypropyl methyl cellulose (HPMC), HPMCP, HPMCAS, hydroxypropyl methyl cellulose acetate trimellitate (HPMCAT), and ethylhydroxy ethylcellulose (EHEC); polyvinyl pyrrolidone; polyvinyl alcohol; polyvinyl acetate; glycerol fatty acid esters; polyacrylamide; polyacrylic acid; copolymers of ethacrylic acid or methacrylic acid (EUDRAGIT®, Rohm America, Inc., Piscataway, N.J.); poly(2-hydroxyethyl- methacrylate); polylactides; copolymers of L-glutamic acid and ethyl-L-glutamate; degradable lactic acid-glycolic acid copolymers; poly-D-(-)-3-hydroxybutyric acid; and other acrylic acid derivatives, such as homopolymers and copolymers of butylmethacrylate, methylmethacrylate, ethylmethacrylate, ethylacrylate, (2-dimethylaminoethyl)methacrylate, and

(trimethylaminoethyl)methacrylate chloride.

[00118] In further embodiments, the pharmaceutical compositions are formulated with a non- erodible matrix device. The active ingredient(s) is dissolved or dispersed in an inert matrix and is released primarily by diffusion through the inert matrix once administered. Materials suitable for use as a non-erodible matrix device included, but are not limited to, insoluble plastics, such as polyethylene, polypropylene, polyisoprene, polyisobutylene, polybutadiene, polymethylmethacrylate, polybutylmethacrylate, chlorinated polyethylene, polyvinylchloride, methyl acrylate-methyl methacrylate copolymers, ethylene-vinyl acetate copolymers, ethylene/propylene copolymers, ethylene/ethyl acrylate copolymers, vinyl chloride copolymers with vinyl acetate, vinylidene chloride, ethylene and propylene, ionomer polyethylene terephthalate, butyl rubber epichlorohydrin rubbers, ethylene/vinyl alcohol copolymer, ethylene/vinyl acetate/vinyl alcohol terpolymer, and ethylene/vinyloxyethanol copolymer, polyvinyl chloride, plasticized nylon, plasticized polyethylene terephthalate, natural rubber, silicone rubbers, polydimethylsiloxanes, silicone carbonate copolymers, and; hydrophilic polymers, such as ethyl cellulose, cellulose acetate, crospovidone, and cross-linked partially hydrolyzed polyvinyl acetate; and fatty compounds, such as carnauba wax, microcrystalline wax, and triglycerides.

[00119] In a matrix-controlled release system, the desired release kinetics can be controlled, for example, via the polymer type employed; the polymer viscosity; the particle sizes of the polymer and/or the active ingredient(s); the ratio of the active ingredient(s) versus the polymer, and other excipients or carriers in the compositions.

[00120] The pharmaceutical compositions provided herein in a modified release dosage form can be prepared by methods known to those skilled in the art, including direct compression, dry or wet granulation followed by compression, melt-granulation followed by compression.

5.4.2. Osmotic Controlled Release Devices

[00121] The pharmaceutical compositions provided herein in a modified release dosage form can be fabricated using an osmotic controlled release device, including one-chamber system, two- chamber system, asymmetric membrane technology (AMT), and extruding core system (ECS). In general, such devices have at least two components: (a) the core which contains the active ingredient(s); and (b) a semipermeable membrane with at least one delivery port, which encapsulates the core. The semipermeable membrane controls the influx of water to the core from an aqueous environment of use so as to cause drug release by extrusion through the delivery port(s).

[00122] In addition to the active ingredient(s), the core of the osmotic device optionally includes an osmotic agent, which creates a driving force for transport of water from the environment of use into the core of the device. One class of osmotic agents water-swellable hydrophilic polymers, which are also referred to as“osmopolymers” and“hydrogels,” including, but not limited to, hydrophilic vinyl and acrylic polymers, polysaccharides such as calcium alginate, polyethylene oxide (PEO), polyethylene glycol (PEG), polypropylene glycol (PPG), poly (2-hydroxy ethyl methacrylate), poly(acrylic) acid, poly(methacrylic) acid, polyvinylpyrrolidone (PVP), crosslinked PVP, polyvinyl alcohol (PVA), PVA/PVP copolymers, PVA/PVP copolymers with hydrophobic monomers such as methyl methacrylate and vinyl acetate, hydrophilic polyurethanes containing large PEO blocks, sodium croscarmellose, carrageenan, hydroxy ethyl cellulose (HEC), hydroxypropyl cellulose (HPC), hydroxypropyl methyl cellulose (HPMC), carboxymethyl cellulose (CMC) and carboxyethyl, cellulose (CEC), sodium alginate, polycarbophil, gelatin, xanthan gum, and sodium starch glycolate.

[00123] The other class of osmotic agents is osmogens, which are capable of imbibing water to affect an osmotic pressure gradient across the barrier of the surrounding coating. Suitable osmogens include, but are not limited to, inorganic salts, such as magnesium sulfate, magnesium chloride, calcium chloride, sodium chloride, lithium chloride, potassium sulfate, potassium phosphates, sodium carbonate, sodium sulfite, lithium sulfate, potassium chloride, and sodium sulfate; sugars, such as dextrose, fructose, glucose, inositol, lactose, maltose, mannitol, raffinose, sorbitol, sucrose, trehalose, and xylitol, organic acids, such as ascorbic acid, benzoic acid, fumaric acid, citric acid, maleic acid, sebacic acid, sorbic acid, adipic acid, edetic acid, glutamic acid, p- toluenesulfonic acid, succinic acid, and tartaric acid; urea; and mixtures thereof

[00124] Osmotic agents of different dissolution rates can be employed to influence how rapidly the active ingredient(s) is initially delivered from the dosage form. For example, amorphous sugars, such as MANNOGEM™ EZ (SPI Pharma, Lewes, DE) can be used to provide faster delivery during the first couple of hours to promptly produce the desired therapeutic effect, and gradually and continually release of the remaining amount to maintain the desired level of therapeutic or prophylactic effect over an extended period of time. In this case, the active ingredient(s) is released at such a rate to replace the amount of the active ingredient metabolized and excreted.

[00125] The core can also include a wide variety of other excipients and carriers as described herein to enhance the performance of the dosage form or to promote stability or processing.

[00126] Materials useful in forming the semipermeable membrane include various grades of acrylics, vinyls, ethers, polyamides, polyesters, and cellulosic derivatives that are water-permeable and water-insoluble at physiologically relevant pHs, or are susceptible to being rendered water- insoluble by chemical alteration, such as crosslinking. Examples of suitable polymers useful in forming the coating, include plasticized, unplasticized, and reinforced cellulose acetate (CA), cellulose diacetate, cellulose triacetate, CA propionate, cellulose nitrate, cellulose acetate butyrate (CAB), CA ethyl carbamate, CAP, CA methyl carbamate, CA succinate, cellulose acetate trimellitate (CAT), CA dimethylaminoacetate, CA ethyl carbonate, CA chloroacetate, CA ethyl oxalate, CA methyl sulfonate, CA butyl sulfonate, CA p-toluene sulfonate, agar acetate, amylose triacetate, beta glucan acetate, beta glucan triacetate, acetaldehyde dimethyl acetate, triacetate of locust bean gum, hydroxylated ethylene-vinylacetate, EC, PEG, PPG, PEG/PPG copolymers, PVP, HEC, HPC, CMC, CMEC, HPMC, HPMCP, HPMCAS, HPMCAT, poly(acrylic) acids and esters and poly- (methacry lie) acids and esters and copolymers thereof, starch, dextran, dextrin, chitosan, collagen, gelatin, polyalkenes, polyethers, polysulfones, polyethersulfones, polystyrenes, polyvinyl halides, polyvinyl esters and ethers, natural waxes, and synthetic waxes.

[00127] Semipermeable membrane can also be a hydrophobic microporous membrane, wherein the pores are substantially filled with a gas and are not wetted by the aqueous medium but are permeable to water vapor, as disclosed in U.S. Pat. No. 5,798,119. Such hydrophobic but water- vapor permeable membrane are typically composed of hydrophobic polymers such as polyalkenes, polyethylene, polypropylene, polytetrafluoroethylene, polyacrylic acid derivatives, polyethers, polysulfones, polyethersulfones, polystyrenes, polyvinyl halides, polyvinylidene fluoride, polyvinyl esters and ethers, natural waxes, and synthetic waxes.

[00128] The delivery port(s) on the semipermeable membrane can be formed post-coating by mechanical or laser drilling. Delivery port(s) can also be formed in situ by erosion of a plug of water-soluble material or by rupture of a thinner portion of the membrane over an indentation in the core. In addition, delivery ports can be formed during coating process, as in the case of asymmetric membrane coatings of the type disclosed in U.S. Pat. Nos. 5,612,059 and 5,698,220, the contents of which are hereby incorporated by reference in their entirety.

[00129] The total amount of the active ingredient(s) released and the release rate can substantially by modulated via the thickness and porosity of the semipermeable membrane, the composition of the core, and the number, size, and position of the delivery ports.

[00130] The pharmaceutical compositions in an osmotic controlled-release dosage form can further comprise additional conventional excipients or carriers as described herein to promote performance or processing of the formulation.

[00131] The osmotic controlled-release dosage forms can be prepared according to conventional methods and techniques known to those skilled in the art. See, Remington: The Science and Practice of Pharmacy, supra ; Santus and Baker, J. Controlled Release 1995, 35, 1-21; Verma et ak, Drug Development and Industrial Pharmacy 2000, 26, 695-708; Verma et ak, J. Controlled Release 2002, 79, 7-27, the contents of which are hereby incorporated by reference in their entirety.

[00132] In certain embodiments, the pharmaceutical compositions provided herein are formulated as AMT controlled-release dosage form, which comprises an asymmetric osmotic membrane that coats a core comprising the active ingredient(s) and other pharmaceutically acceptable excipients or carriers. See, U.S. Pat. No. 5,612,059 and WO 2002/17918, the contents of which are hereby incorporated by reference in their entirety. The AMT controlled-release dosage forms can be prepared according to conventional methods and techniques known to those skilled in the art, including direct compression, dry granulation, wet granulation, and a dip-coating method. [00133] In certain embodiments, the pharmaceutical compositions provided herein are formulated as ESC controlled-release dosage form, which comprises an osmotic membrane that coats a core comprising the active ingredient(s), a hydroxylethyl cellulose, and other pharmaceutically acceptable excipients or carriers.

5.4.3. Multiparticulate Controlled Release Devices

[00134] The pharmaceutical compositions provided herein in a modified release dosage form can be fabricated as a multiparticulate controlled release device, which comprises a multiplicity of particles, granules, or pellets, ranging from about 10 pm to about 3 mm, about 50 pm to about 2.5 mm, or from about 100 pm to about 1 mm in diameter. Such multiparticulates can be made by the processes known to those skilled in the art, including wet-and dry-granulation, extrusion/spheronization, roller-compaction, melt-congealing, and by spray-coating seed cores. See, for example, Multiparticulate Oral Drug Delivery: Marcel Dekker: 1994; and Pharmaceutical Pelletization Technology; Marcel Dekker: 1989.

[00135] Other excipients or carriers as described herein can be blended with the pharmaceutical compositions to aid in processing and forming the multiparticulates. The resulting particles can themselves constitute the multiparticulate device or can be coated by various film forming materials, such as enteric polymers, water-swellable, and water-soluble polymers. The multiparticulates can be further processed as a capsule or a tablet.

5.5. DOSAGE

[00136] The pharmaceutical compositions that are provided can be administered for prophylactic and/or therapeutic treatments. An "effective amount" refers generally to an amount that is a sufficient, but non- toxic, amount of the active ingredient (i.e., a compound disclosed herein) to achieve the desired effect, which is a reduction or elimination in the severity and/or frequency of symptoms and/or improvement or remediation of damage. A "therapeutically effective amount" refers to an amount that is sufficient to remedy a disease state or symptoms, or otherwise prevent, hinder, retard or reverse the progression of a disease or any other undesirable symptom. A "prophylactically effective amount" refers to an amount that is effective to prevent, hinder or retard the onset of a disease state or symptom.

[00137] In general, toxicity and therapeutic efficacy of the compound disclosed herein can be determined according to standard pharmaceutical procedures in cell cultures and/or experimental animals, including, for example, determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compositions that exhibit large therapeutic indices are preferred. [00138] The data obtained from cell culture and/or animal studies can be used in formulating a range of dosages for humans. The dosage of the active ingredient typically lines within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized.

[00139] The effective amount of a pharmaceutical composition comprising a compound disclosed herein to be employed therapeutically or prophylactically will depend, for example, upon the therapeutic context and objectives. One skilled in the art will appreciate that the appropriate dosage levels for treatment, according to certain embodiments, will thus vary depending, in part, upon the molecule delivered, the indication for which the compound disclosed herein is being used, the route of administration, and the size (body weight, body surface or organ size) and/or condition (the age and general health) of the patient. A clinician may titer the dosage and modify the route of administration to obtain the optimal therapeutic effect. Typical dosages range from about 0.1 pg/kg to up to about 100 mg/kg or more, depending on the factors mentioned above. In certain embodiments, the dosage may range from 0.1 pg/kg up to about 150 mg/kg; or 1 pg/kg up to about 100 mg/kg; or 5 pg/kg up to about 50 mg/kg.

[00140] The dosing frequency will depend upon the pharmacokinetic parameters of the compound disclosed herein in the formulation. For example, a clinician will administer the composition until a dosage is reached that achieves the desired effect. The composition may therefore be administered as a single dose or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via an implantation device or catheter. Treatment may be continuous over time or intermittent. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them. Appropriate dosages may be ascertained through use of appropriate dose-response data.

[00141] In this disclosure, we demonstrate gd T cell targeting and elimination of reactivated HIV- infected resting CD4 T cells ex vivo. To our knowledge, our work is the first to comprehensively analyze the potential of gd T cells from ART-suppressed infected donors to clear persistent HIV infection in resting CD4 cells. We have performed ex vivo studies demonstrating cytotoxic and antiviral capacities of gd T cells to specifically target and inhibit viral replication. Further, using autologous systems we demonstrate the specific clearance of latently HIV-infected cells by gd T cells after latency reversal. This disclosure supports the use of gd T cells in combination with bisphosphonates in HIV cure strategies particularly for ART-suppressed infected individuals. [00142] Current strategies aimed to cure HIV infection are based on combined efforts to reactivate the vims from latency and improve immune effector cell function to clear infected cells. These strategies are primarily focused on CD8 T cells and approaches are challenging due to insufficient HIV antigen production from infected cells and poor HIV-specific CD8+ T cell gd T cells represent a unique subset of effector T cells that can traffic to tissues, and selectively target cancer or virally-infected cells without requiring MHC presentation. We analyzed whether gd T cells represent a complementary/alternative immunotherapeutic approach towards HIV cure strategies.

[00143] gd T cells from HIV-infected virologically suppressed donors were expanded with the bisphosphonate pamidronate (PAM) and cells were used in autologous cellular systems ex vivo. These cells are i) are potent cytotoxic effectors able to efficiently inhibit HIV replication ex vivo, ii) degranulate in the presence of autologous infected CD4 T cells and iii) specifically clear latently infected cells after latency reversal with VOR.

[00144] This is the first proof of concept showing that gd T cells target and clear autologous HIV reservoir upon latency reversal. Our results open new insights of the immunotherapeutic use of gd T cells for current interventions in HIV eradication strategies.

[00145] The following Examples further illustrate the disclosure and are not intended to limit the scope. In particular, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

6. EXAMPLES

6.1. Aminobisphosphonates as Latency Reversing Agents [00146] BACKGROUND

[00147] The human immunodeficiency virus (HIV) infects CD4+ cells and integrates its viral genome into the host cell genome. Some of these cells enter a resting state where latent but replication-competent HIV remains invisible to current antiretroviral therapy (ART) and the immune system. This quiescent provirus within long-lived resting CD4 (rCD4) cells represent the main barrier to cure HIV infection, as demonstrated by HIV rebound when ART is interrupted. Current research efforts to cure HIV infection aim to get HIV out of latency using small molecule

HIV latency-reversing agents (LRAs) and inducing potent immune responses to eliminate infected cells. Histone deacetylase (HD AC) inhibitors have been the most widely LRAs studied in different clinical studies (Archin et al., 2014). However, the effectiveness on single agent LRA use has shown modest efficacy in inducing reactivation sufficient to enable the immune system to target and clear infected cells (Martin and Siliciano, 2016). LRAs must reactivate the latent HIV without activating the cells and without disturbing functions from immune effector cells.

[00148] In addition to HDAC inhibitors, farnesyl transferase inhibitors (FTi) have also been reported to modestly induce the expression of latent HIV in cell model systems and in cells from patients. The magnitude of viral expression was substantially increased when delivered in combination with the HDAC inhibitor vorinostat (SAHA), currently undergoing several clinical trials worldwide. The discovery of FTi as LRAs was the result of an ultra-high throughput screen of 2.9 million compounds conducted in the presence of sub-optimal concentrations of SAHA (Hazuda D. HIV Persistence Workshop, Miami 2013). However, the specific mechanisms underlying HIV induction by FTi are poorly understood.

[00149] The enzyme famesyl transferase is required for prenylation, which transfers the farnesyl pyrophosphate (FPP) groups to proteins. These FPP groups are essential for several protein post- translational modifications such as prenylation. Protein prenylation is critical to the functions of a select group of proteins that have crucial functions in biological regulation, such as protein localization and regulatory capacity of cell signaling (Wang and Casey, 2016). FTi inhibit protein prenylation. These FPP groups are synthesized in the isoprenoid biosynthesis pathway also called the mevalonate pathway.

[00150] The mevalonate pathway, that produces FPP, can be manipulated using different drugs such as statins and bisphosphonates (BP) (FIG. 1). BP are a class of drugs currently approved for the treatment of several diseases involving bone density loss including treatment of osteoporosis, multiple myeloma, bone metastases lesions, and Paget’s disease, among others. BP can be divided into two classes: non-nitrogen-containing (Non-N-BP) class and nitrogen-containing class or aminobisphosphonates (N-BPs). Among the clinically available N-BPs that can be tested, we can find pamidronate, zoledronate, alendronate, ibandronate, risedronate, neridronate, and olpadronate.

Contrary to non-nitrogen-containing BPs that are metabolized in the cell, N-BPs are not metabolized, are more potent than their non-nitrogen containing counterparts, and function by inhibiting the enzyme famesyl pyrophosphate synthase (FDPS) in the mevalonate pathway (FIG.

1). Inhibition of the enzyme FPDS prevents the formation of FPP therefore protein prenylation is indirectly inhibited as well. Another indirect effect of the inhibition of the mevalonate pathway using N-BPs is the accumulation of the intermediate isopentenyl pyrophosphate (IPP), which in turn specifically activates V52 cells (Morita et al., 2007; Tanaka et ak, 1995; Wang et ak, 2011).

These V52 cells can be largely expanded in vitro using N-BPs to become potent cytotoxic effectors against tumor cell lines (Poonia and Pauza, 2012). In addition, we have demonstrated that expanded V52 cells from ART- suppressed HIV-infected patients can be expanded in vitro using PAM. These expanded cells are i) potent cytotoxic effectors that inhibit active viral replication ex vivo, ii) degranulate in the presence of autologous infected CD4 T cells and iii) specifically target and eliminate latent HIV-infected cells after latency reversal with SAHA (Garrido et al., JCI Insight 2018 published June 21, 2018).

[00151] As N-BP inhibit the formation of FPP groups that are essential for protein prenylation, we hypothesized that N-BP may also reactivate HIV from latency. Induction of latent HIV by N- BP was analyzed using two different approaches: i) quantitation of HIV gag RNA copies in real time quantitative reverse PCR and ii) quantification of replication-competent HIV in quantitative viral outgrowth assays (QVOA). The use of N-BPs as LRA constitute a novel use of the clinically available N-BPs as a novel strategy towards HIV eradication.

[00152] Immunotherapy using ex vivo expanded gd T cells alone or in combination with N-BPs administration has been exploited in the oncology field (Kobayashi and Tanaka, 2015; Van Acker et ah, 2016; Zou et ak, 2017). We propose the use of N-BPs alone or in combination with other compounds classified as LRAs to reactivate the virus, and concomitant or subsequent adoptive transfer of ex vivo expanded gd T cells (autologous or allogeneic) as a novel approach to eliminate persistent HIV infection. Once activated, gd T cells also exert critical adjuvant functions to induce proper adaptive immune responses including induction of dendritic cell maturation and activation of HIV-specific T cell responses (Lafont et ak, 2014). Therefore, our strategy includes the novel use of N-BPs as LRAs and take advantage of the specific activation of gd T cell functions to eradicate persistent HIV infection.

[00153] Among the methods and uses disclosed herein are:

[00154] ex vivo expansion of gd T cells. PBMC from uninfected individuals will be isolated and gd T cells expanded in vitro using N-BPs and a combination of cytokines that will generate potent cytotoxic effector gd T cells, as per previously optimized protocols. Expansions will last at least 14 days, to obtain sufficient number of cells to analyze phenotype and ex vivo function.

[00155] ii) N-BP administration. ART-suppressed HIV-infected individuals will receive a dose of N-BPs alone or in combination with other LRAs, according to standard procedures to reactivate the latent virus. In an alternative embodiment, the methods and compositions may be used for treatment of acute HIV infection or patients that have not received ART therapy.

[00156] iii) Infusion of gd T cells. Previously expanded gd T cells will be infused concomitant to LRAs administration or as a two-step approach. The gd T cells may be administered with external cytokines such as IL-2, IL-7 or IL-15. In some embodiments, the expanded gd T cells may be genetically engineered.

[00157] RESULTS

[00158] N-BPs induce HIV gag RNA production from isolated rCD4 cells. For HIV RNA analyses, 18 patients were analyzed. In each patient, a total of 24-72 million isolated rCD4 cells were studied using between 6-15 million cells per condition, depending on cell availability. Following in vitro exposure of isolated rCD4 cells to 25pg/mL PAM, in five out of the 11 patients analyzed, HIV gag RNA copies/lxlO⁵ cells were significantly quantified above the untreated control, the same patients whom showed positive HIV gag RNA copies using the positive control PHA and IL-2 (FIG. 2A). Importantly, in some patients HIV RNA expression levels induced by PAM were comparable to PHA control. The HDACi SAHA was also used as a control at the physiological concentration of 335nM in seven patients showing significant gag HIV expression in one patient. Altogether, in eleven patients following six hours of culture in media alone analyzed without stimulation, HIV gag RNA was quantifiable at a mean level of 70 HIV gag RNA copies, compared to 901 HIV gag RNA copies after PHA stimulation, 174 HIV gag RNA after VOR stimulation and 260 HIV gag RNA copies after 25pg/mL PAM stimulation. The lower concentration of 2.5pg/mL in two patients, induced a mean viral production of 149 HIV gag RNA copies compared to 97 HIV gag RNA copies after PHA and IL-2 stimulation and 92 gag HIV gag RNA copies after 25pg/mL PAM.

[00159] Overall, normalized RNA data to the untreated condition from 11 HIV-infected donors showed the capacity of PAM to induce latency reversal, measured as HIV gag RNA copies, from latent HIV in isolated rCD4 cells from durable suppressed individuals (FIG. 2B). As expected, the positive control PHA and IL-2 significantly induced HIV production compared to the untreated condition (p=0.004). In addition, compared to the untreated condition, PAM efficiently and significantly reversed latency in all patients (p=0.004), without statistical differences with the clinically used HDACi SAHA (p=0.2).

[00160] Ex vivo exposure of rCD4 cells to PAM 2.5pg/mL induces comparable HIV caRNA levels to that of a positive control

[00161] Next, as the exposure of 25pg/mL may translate into higher concentrations than what is achieved in the blood using FDA approved doses, in nine subsequent experiments we exposed isolated rCD4 cells to 2.5pg/mL showing that the lO-fold lower concentration effectively reversed latency compared to the untreated condition (FIG. 2C). After exposure of rCD4 cells to 2.5pg/mL

PAM, we detected a mean of 24.7 HIV gag copies in the untreated condition, 65.2 mean copies after PHA and IL-2 treatment, and 66.4 mean HIV RNA copies. Interestingly, mean HIV copy numbers were comparable between the PHA and IL-2 condition and the PAM at 2.5pg/mL condition (p>0.05, Wilcoxon signed rank test). These HIV copy numbers were translated into a comparable fold change induction of HIV cell-associated RNA (caRNA) (FIG. 2D).

[00162] Finally, to corroborate that the capacity to reactivate latent HIV was translatable to other N-BPs, we tested whether ex vivo exposure to ImM Zol was capable of reversing HIV latency (FIG. 3A-3B). Untreated rCD4 cells showed a mean HIV gag RNA copies of 15.8, compared to a mean of 76.2 copies in the PHA condition, 52.9 copies when we used 2.5pg/mL PAM, 42.5 mean HIV RNA copies after exposure to ImM Zol. Our results demonstrate the capacity of Zol to reactivate HIV from latency. Comparison between the capacity to induce HIV caRNA of the positive control and N-BPs was comparable (p>0.05, Wilcoxon signed rank test).

[00163] Our results demonstrate that N-BPs are capable of inducing the production of HIV gag RNA from isolated rCD4 cells from HIV-infected donors on suppressive ART.

[00164] N-BPs induce replication-competent HIV production from isolated rCD4 cells in QVOA

[00165] To test whether PAM was inducing production of non-defective, replication-competent HIV, 12 HIV+ donors were analyzed. rCD4 cells were isolated, plated in limiting dilution and incubated in parallel with 2pg/mL PHA and lOOU/mL IL-2, 25pg/mL PAM, 335nM SAHA or 5U/mL IL-2 to perform QVOA. HIV latency was reversed and replication-competent vims produced in nine of 10 patients with a mean value of IUPM rCD4 cells of 0.426. SAHA induced viral production in six of nine patients tested with a mean IUPM value of 0.159. Finally, in cultures of rCD4 cells incubated with PAM, HIV was induced to produce replication-competent virus in seven of 10 patients with a mean IUPM rCD4 cells value of 0.180. The results are shown in FIG. 3A.

[00166] Ex vivo exposure of rCD4 cells to PAM 2.5pg/mL induce replication-competent HIV to levels comparable to the positive control

[00167] Following the rationale and experimental approach described in 00154, in seven additional experiments, we performed QVOA exposing the cells to 2.5pg/mL PAM. Our results show the capacity of PAM to produce replication-competent HIV from rCD4 cells with a similar frequency than the positive control PHA and IL-2 (FIG. 3B).

[00168] N-BPs are not toxic to immune cells

[00169] In order to analyze whether N-BPs are toxic to immune cells, dose response curves using PAM concentrations from 25pg/mL to 200pg/mL were performed to analyze the expression of 7- AAD in total CD4, CD8, gd and NK cells (FIG. 4A), and CD95 expression was used in rCD4 cells (FIG. 4B). Results show that N-BPs are not toxic even at the highest concentration of 200pg/mL to any of the populations analyzed.

[00170] N-BPs do not promote proliferation of rCD4 cells, total CD4 cells or CD8 cells [00171] Proliferation analysis using CFSE assays demonstrate that N-BPs used at different concentrations, from 25mg/mL to 200pg/mL do not promote proliferation of rCD4 cells. PHA and IL-2 were used as the positive control, media alone as the negative control and 335nM SAHA as a comparator, as this HDACi do not promote proliferation either. Our results show that PAM did not induce proliferation of rCD4 cells at any of the concentrations analyzed (FIG. 5A-5B). As an additional control we tested the effect of PAM on V52 cells, that specifically induces expansion of V52 cells. As expected, our results demonstrate that PAM in combination with IL-2 significantly induces expansion of V52 cells. The results are shown in FIG. 5A-5B.

[00172] N-BPs do not promote activation of lymphocyte populations

[00173] We analyzed the expression of the activation markers CD69 and HLA-DR on total CD4 cells, CD8 T cells, gd T cells and rCD4 cells after exposure to different doses of the N-BPs PAM and Zol. PHA and IL-2 was used as the positive control and VOR was used to compare with N- BPs. As expected, PHA and IL-2 induced significant increase expression of both markers in all cell populations analyzed. Our results showed no increase on activation markers CD69 and HLA-DR expression after exposure to N-BPs compared to the untreated condition in total CD4 cells, CD8 cells and gd T cells (FIG. 5C).

[00174] N-BPs impact mRNA expression of genes implicated in chromatin regulation

[00175] In order to investigate the mechanism by which N-BPs induce reactivation of latent HIV, RNA-seq was performed on total RNA isolated from rCD4 cells from four of the same patients in whom caRNA experiments were assayed. Whole-transcriptome analysis with total RNA sequencing was performed in PAM-treated samples and compared to the untreated conditions. The principal component analysis demonstrated that treatment with PAM was the responsible for the largest source of gene expression variation (FIG. 6A). Results showed that 3,953 genes were differentially expressed (q- value <0.1 and base mean >10) showing a good split of downregulated and upregulated genes according to treatment (data not shown). Gene set enrichment analysis was performed using GSVA and curated modules (C2). First, the effect of N-BPS on cholesterol biosynthesis pathway was analyzed as our positive control and as expected, treatment with N-BPs decreased expression of genes related to this pathway (FIG. 6B). Example pathways regulated by N-BPs included DNA damage, cell cycle, metabolism and epigenetics. Comparison between untreated and PAM-treated conditions in DNA damage, methylation targets and HDAC7 targets are shown in figure 6B as examples. Interestingly, we found a positive correlation between the genes that are altered during HIV infection and those impacted by treatment with N-BPs (FIG. 6C). This result show that pathways are altered similarly and strongly suggest that N-BPs may have a specific function over HIV reactivation. Finally, we performed a comparison with the HDACi vorinostat (SAHA) showing that 67% of the genes are altered with this treatment, showing the non specific effect over HIV reactivation (data not shown). The top ten genes that were significantly upregulated in the treated samples and showed greater than 2-fold upregulation compared to the untreated cells were as follows: LOC100507548 (3.17), RGS4 (2.60), PROKR2 (2.45), TACR3 (2.16), OPTC (2.15), KRTAP4-2 (2.14), RFPL3 (2.12), PRAMEF13 (2.11), SFTPA2 (2.04), BTNL8 (2.02). Importantly, some of these genes are involved in chromatin regulation and may have a critical role enabling HIV transcription. The current understanding of latency maintenance is that transcriptional silencing occurs through several complementary molecular mechanisms, including transcription initiation and elongation, and epigenetic silencing via histone deacetylases (HDACs) and histone methyltransferases Thus, N-BPs may be acting at the chromatin level to reverse HIV latency. Specifically, an example of the impact at the chromatin level is the downregulation of the expression of HDA7 targets and methyltransferases (FIG. 6B).

[00176] In vivo evidence of the capacity of N-BPs to reactivate the latent virus and to activate gd T cell’s functions as to eliminate infected cells.

[00177] About 67% of HIV-infected individuals suffer from osteopenia or osteoporosis¹. See,

Brown, T. T. & Qaqish, R. B. Antiretroviral therapy and the prevalence of osteopenia and osteoporosis: a meta-analytic review. AIDS (London, England) 20, 2165-2174, doi:l0.l097/QAD.0b0l3e3280l022eb (2006). A clinical trial was performed in 2004 to evaluate the safety and effectiveness of the oral N-BP alendronate in the treatment of HIV-associated osteopenia and osteoporosis. See McComsey, G. A. et al. Alendronate with calcium and vitamin D supplementation is safe and effective for the treatment of decreased bone mineral density in HIV.

AIDS (London, England) 21, 2473-2482, doi:l0T097/QAD.0b0l3e3282ef96ld (2007). The study consisted of HIV-infected individuals receiving stable ART. One arm (Arm A) of the study received alendronate once weekly and vitamin D, and the other arm (Arm B) received placebo instead of alendronate and vitamin D. Authors reported that once weekly the N-BP alendronate was safe and effective in the treatment of decreased bone mineral density. In 2018 we contacted the AIDS

Clinical Trial Groups (ACTG) and obtained some frozen PBMCs and plasma of the HIV-infected individuals enrolled in that clinical trial. Following the same protocols used for the ex vivo analysis, we quantified HIV gag caRNA levels in total PBMCs at baseline (prior to treatment) and then at weeks 2, 24 and 48 during the treatment with alendronate. We analyzed ten individuals from Arm

A and five from Arm B. Seven of the ten patients from Arm A showed a significant effect of the alendronate treatment in vivo (FIG. 7A). Interestingly, in four of them, we measured a decrease in

HIV caRNA from basal to week 48, and in three of the seven, we detected an increase compared to the basal. We hypothesized that in the four individuals who experienced a decrease in caRNA compared to baseline, alendronate induced the activation of gd T cells. Activated gd T cells may have exerted a direct cytotoxic function eliminating HIV-infected reactivated cells and/or adjuvant functions that included the induction of dendritic cells, and anti-HIV specific responses helping in the clearance of the infected cells. These results constitute the first proof of concept of the capacity of N-BPs to induce HIV from latency. In the placebo arm, HIV caRNA levels were comparable across the time-points in all individuals analyzed (FIG. 7B).

[00178] CONCLUSIONS

[00179] Our results demonstrate that N-BPs are novel LRAs capable of inducing HIV expression from latency. N-BPs are not toxic to immune cells, i.e., CD4 cells, CD8 cells, gd cells or NK cells, do not induce proliferation of infected cells and do not alter CD4 cell subpopulations, i.e., naive, TCM, TTM, TD (data not shown). We have identified N-BPs as LRAs to be used as a novel tool in HIV cure strategies. We propose that N-BPs can be used to reactivate latent HIV and these compounds may be used alone, in combination with other LRAs, or combined with immunotherapeutic strategies to augment immune capacity to eliminate the reactivated HIV. In this regard, this is of critical importance as V52 cells are potent effectors capable of targeting and eliminating infected cells (Garrido et al, JCI Insight 2018). The data disclosed herein also confirm the beneficial specific activation of V52 cells after incubation with N-BPs. Importantly, as demonstrated in HIV-infected patients treated for low bone mineral density often associated with HIV infection, N-BP can be safely used in combination with ART. Importantly, we were able to obtain samples from a clinical trial performed with the only objective to analyze the effect of one N-BP, alendronate, on bone density of HIV-infected individuals. Our results demonstrate that in vivo administration of N-BPs leads to the reactivation of the latent provirus and may induce cytotoxic functions from effector cells that lead to the killing of those reactivated cells. In summary, we have demonstrated that N-BPs are novel latency reversing agents and we propose their novel use in the field of HIV latency and persistence.

[00180] METHODS

[00181] Reagents

[00182] To test our hypothesis, we analyzed the ability of the N-BP pamidronate (PAM, Sigma- Aldrich) at two different concentrations: 25pg/mL and 2.5pg/mL to induce HIV reactivation from latency. The lower concentration translates into what has been predicted in plasma of cancer patients (2.6lpg/mL). Phytohaemagglutinin (PHA) was used as a positive control at a concentration of 2pg/mL for QVOA and at 5pg/mL for PCR experiments. VOR was used at the clinically relevant concentration of 335-500nM.

[00183] Donors [00184] HIV-infected donors under suppressive antiretroviral treatment (ART) with undetectable plasma viral load (<50 copies/mL) for at least one consecutive year before inclusion were analyzed. Patients had initiated ART either in the acute or in the chronic phase of HIV infection.

[00185] Quantitative Polymerase Chain Reaction (qPCR)

[00186] qPCR constitutes the gold standard method to test latency reversal capacity. Isolated rCD4 cells are cultured in bulk using positive and negative controls: in the presence of positive controls PHA and IL-2 and VOR, untreated cells, or in the presence of the experimental condition: PAM at 25pg/mL or 2.5pg/mL for six hours. After six hours cells are washed and plated in replicates of lxlO⁶ cells. After further washes, cells are pelleted and stored at -80°C until RNA is extracted. Then, cDNA is synthesized using well-standardized protocols and gag HIV PCR performed using specific and validated primers to amplify Gag (Archin et al., 2012).

[00187] QVOA

[00188] HIV p24 protein production (replication-competent vims as measure of infectious particles production) in quantitative viral outgrowth assays (QVOA) after 24 hours of rCD4 cells exposure to PAM was analyzed. This assay has been extensively previously reported. Briefly, isolated rCD4 cells are maintained 24 hours in antiretrovirals to avoid de novo infection of potentially spontaneous reactivation. Then, cells are washed, they are counted and plated in limiting dilution. We performed 8 replicates at lxlO⁶, 8 replicates at 0.5xl0⁶ and 10 replicates at O.lxlO⁶ of isolated rCD4 cells. Cells are exposed for 24 hours to positive controls PHA and IL-2 or VOR, PAM, and in parallel to 5U/mL IL-2 as a negative control. Then, cells are washed and PHA- activated PBMC-depleted of CD8 T cells are added at days 5 and 8 to the culture as targets of new infections to outgrowth the virus. After 15 days supernatants are harvested and HIVp24 ELISA performed, and confirmed at day 19 of culture. Then, using a previously well-established algorithm, the infectious units per million cultured cells (IUPM) are calculated.

[00189] Toxicity

[00190] The viability dye 7-Aminoactinomycin D (7-AAD) is a non-permanent dye that can be used to identify non- viable cells as it can penetrate in cells with non-intact membranes. Once inside the cell, 7-AAD binds to the DNA producing fluorescence that identifies cells as non-viable. 7- AAD was used to identify the toxic effect of PAM using the above-mentioned concentrations on PBMC after 6 and 24 hours of incubation. To analyze toxicity of PAM in rCD4 cells we used the expression of CD95 as a marker of apoptosis.

[00191] Cell activation

[00192] Using the same experimental conditions and controls, rCD4 cells and PBMC were incubated with different PAM concentrations for 6 or 24 hours, harvested, washed, stained with monoclonal antibodies and analyzed by flow cytometry. The expression of the activation markers CD69 and HLA-DR was analyzed in subpopulations of PBMC (total CD4 cells and CD8 cells). For rCD4 cells CD69, HLA-DR and CD25 were used.

[00193] Cell proliferation

[00194] Cell proliferation was analyzed using carboxyfluorescein diacetate succinimidyl ester (CFSE)-based assays. CFSE is a fluorescent dye that covalently binds to amine residues and importantly, it halves in each cell division making this dye an excellent tracker for proliferation assays. In these assays, matched PBMC and rCD4 cells from HIV-infected donors are stained with CFSE following standard optimized protocols. After CFSE staining and washing, cells are cultured with the experimental condition for 6 and 24 hours. After washing, cells are cultured for five additional days to test the effect of PAM on proliferation. Controls for proliferation assays included 2pg/mL or 5pg/mL PHA and lOOU/mL IL-2, and 335nM VOR as positive controls, and 5U/mL and lOOU/mL as negative controls. In addition, we also included the effect of 25pg/mL and lOOU/mL IL-2 on V52 cells as a control, as these cells specifically proliferate in response to PAM and high IL-2 doses. Cells are then harvested and washed and stained with monoclonal antibodies to analyze specific proliferation in different T cell subpopulations that include: Total Lymphocytes (CD3+ cells), CD3+CD4+ cells, CD3+CD8+ cells, CD3+y5TCR+ cells, and CD3-CD56+ (NK) cells. This set of experiments included dose-response curve analysis using increasing PAM concentrations ranging from 0.5pg/mL to 200pg/mL (0.5, 1.5, 2.5, 5, 10, 12.5, 25, 50,100 and 200 pg/mL).

[00195] RNA sequencing

[00196] Isolated total RNA was quantified using a NanoDrop® Spectrophotometer. RNA Integrity Number (RIN) was then analyzed using the RNA6000 assay (Agilent) lpg of total RNA was converted to RNAseq libraries using the KAPA Stranded mRNA-Seq Kit (Illumina) and sequenced on an Illumina HiSeq 4000 using a 2x50bp configuration. Quality-control-passed reads were aligned to the human reference genome CGRh38/hg38 using STAR. See Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, Chaisson M, and Gingeras TR. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013;29(1):15-21. Transcript abundance estimates for each sample were performed using Salmon, an expectation-maximization algorithm using the UCSC gene definitions. See Patro R, Duggal G, Love MI, Irizarry RA, and Kingsford C. Salmon provides fast and bias-aware quantification of transcript expression. Nat Methods. 2017;14(4):417- 9. Raw read counts for all RNAseq samples were normalized to a fixed upper quartile. See Bullard JH, Purdom E, Hansen KD, and Dudoit S. Evaluation of statistical methods for normalization and differential expression in mRNA-Seq experiments. BMC bioinformatics. 20l0;ll(l):94. Differential gene expression analysis comparing treated (DL2415) versus untreated samples was performed using DESeq2 (4). See Love MI, Huber W, and Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biology. 2014;15(12):550. We run principal component analysis (PCA) to visualize sample-to-sample distances and examine the relationship between untreated and treated paired samples. See Venables WN, and Ripley BD. Modern Applied Statistics with S. Springer- Verlag New York; 2002.

6.2. REFERENCES (Section 6.1)

Archin, N.M., Liberty, A.L., Kashuba, A.D., Choudhary, S.K., Kuruc, J.D., Crooks, A.M., Parker, D.C., Anderson, E.M., Kearney, M.F., Strain, M.C., et al. (2012). Administration of vorinostat disrupts HIV-l latency in patients on antiretroviral therapy. Nature 487, 482-485.

Archin, N.M., Sung, J.M., Garrido, C., Soriano-Sarabia, N., and Margolis, D.M. (2014). Eradicating HIV-l infection: seeking to clear a persistent pathogen. Nat Rev Microbiol 12, 750-764.

Kobayashi, H., and Tanaka, Y. (2015). gammadelta T Cell Immunotherapy-A Review. Pharmaceuticals (Basel, Switzerland) 8, 40-61.

Lafont, V., Sanchez, F., Laprevotte, E., Michaud, H.A., Gros, L., Eliaou, J.F., and Bonnefoy, N. (2014). Plasticity of gammadelta T Cells: Impact on the Anti-Tumor Response. Frontiers in immunology 5, 622.

Martin, A.R., and Siliciano, R.F. (2016). Progress Toward HIV Eradication: Case Reports, Current Efforts, and the Challenges Associated with Cure. Annual review of medicine 67, 215-228.

Morita, C.T., Jin, C., Sarikonda, G., and Wang, H. (2007). Nonpeptide antigens, presentation mechanisms, and immunological memory of human Vgamma2Vdelta2 T cells: discriminating friend from foe through the recognition of prenyl pyrophosphate antigens. Immunological reviews 215, 59-76.

Poonia, B., and Pauza, C.D. (2012). Gamma delta T cells from HIV+ donors can be expanded in vitro by zoledronate/interleukin-2 to become cytotoxic effectors for antibody-dependent cellular cytotoxicity. Cytotherapy 14, 173-181.

Tanaka, Y., Morita, C.T., Tanaka, Y., Nieves, E., Brenner, M.B., and Bloom, B.R. (1995). Natural and synthetic non-peptide antigens recognized by human gamma delta T cells. Nature 375, 155- 158.

Van Acker, H.H., Anguille, S., Willemen, Y., Smits, E.L., and Van Tendeloo, V.F. (2016). Bisphosphonates for cancer treatment: Mechanisms of action and lessons from clinical trials. Pharmacology & therapeutics 158, 24-40. Wang, H., Sarikonda, G., Puan, K.J., Tanaka, Y., Feng, L, Giner, J.L., Cao, R., Monkkonen, L, Oldfield, E., and Morita, C.T. (2011). Indirect stimulation of human Vgamma2Vdelta2 T cells through alterations in isoprenoid metabolism. Journal of immunology (Baltimore, Md. : 1950) 187, 5099-5113.

Wang, M., and Casey, P.J. (2016). Protein prenylation: unique fats make their mark on biology. Nature reviews. Molecular cell biology 17, 110-122.

Zou, C., Zhao, P., Xiao, Z., Han, X., Fu, F., and Fu, L. (2017). gammadelta T cells in cancer immunotherapy. Oncotarget 8, 8900-8909.

6.3. dg T Cells: Immunotherapeutic Approach for HIV Cure Strategies [00197] Introduction

[00198] Aminobisphosphonates (N-BP) such as pamidronate (PAM) and zoledronate (Zol) are drugs that modulate the isoprenoid metabolism indirectly augmenting IPP production and activating V52 cells (16). Once activated, V52 cells are potent cytotoxic effectors (20) against malignancies (17, 18) and HIV-infected cells (19-23). We and others have previously demonstrated that V52 T cells can inhibit active viral replication (20, 24). In addition, expanded V52 T cell immune adjuvant functions include induction of dendritic cell maturation and activation of HIV-specific T cell responses (25), which are of special interest as a future complementary use of gd T cells in adoptive cell transfer strategies. In this regard, a small clinical trial with ART-naive HIV-infected donors reported expansion of V52 cells, dendritic cell activation and, increased HIV-specific T cell responses after in vivo administration of Zol and IL-2 (26). Altogether, these studies suggest that gd T cells have potential in interventions aimed to eradicate HIV infection taking advantage of the knowledge generated from the cancer field (27-29).

[00199] Comparison of V52 cell expansion in HIV-infected and uninfected individuals

[00200] We compared different ex vivo experimental conditions to expand nd2 cells from ART- suppressed HIV-infected donors (N=l3) and uninfected donors (N=l0). In this first approach we expanded the cells for six days and conditions included i) HMBPP and IL-2; ii) PAM and IL-2; or iii) IL-2 alone. Basal nd2 cell percentages within CD3+ cells were analyzed by flow cytometry showing wide interindividual differences in uninfected individuals, and expected (30) profound depletion in HIV-infected donors (mean 4.0% vs 0.7%, respectively, FIG. 8A).

[00201] In uninfected individuals, HMBPP was a more potent inducer of nd2 cell expansion compared to PAM, while cells from HIV-infected donors expanded better after PAM treatment

(FIG.8B, FIG. 8C and FIG. 9A). Importantly, the fold change expansion induced by PAM in HIV- infected donors was comparable to uninfected donors (FIG.8B, FIG. 8C). In HIV-infected donors, after six days of culture, a mean of 5.3% of the total CD3 cells present in the culture were V52 cells after HMBPP treatment, compared to 11.0% after PAM treatment (FIG 9B). In summary, PAM allows efficient V52 cell expansion in HIV-infected donors.

[00202] V52 T cells from ART-treated HIV-infected individuals successfully expand after exposure to PAM

[00203] Based on these results, in further experiments PBMC were exposed to 25 pg/mL PAM and 100 U/mL IL-2 for a total of 14 days, to increase V52 cell numbers. We expanded V52 cells from 21 ART-suppressed HIV-infected donors, to be used for different functional assays. Expansions were performed in the presence of antiretrovirals to avoid spread of infection. The average V52 cell expansion rate was 11.9% and was variable between subjects (FIG. 10A). As we previously reported (24) patients treated in the acute phase of the infection had greater basal V52 cell numbers compared to chronically treated patients (0.7% vs. 0.3%, p=0.007). After expansion, percentages of V52 cells from patients treated in the acute phase of HIV infection were also higher compared to patients treated in the chronic HIV infection (mean 15.0 vs. 6.8, p=0.02, FIG. 10B), but with comparable fold change expansion (p=0.56, FIG. 10C). Overall, mean fold expansion was 28.4, ranging from 1.7-fold to more than 124-fold increase. Altogether, V52 T cells from suppressed HIV-infected donors were successfully expanded in response to ex vivo exposure to PAM and IL- 2.

[00204] Phenotype of expanded V52 cells

[00205] The phenotype of expanded V52 cells after 14 days of exposure to PAM was analyzed in a subgroup of eight ART-treated suppressed HIV-infected donors (six patients treated in chronic infection and two patients treated in acute infection) by measuring the expression of markers of memory, cytotoxicity, activation and immune exhaustion by flow cytometry. V52 memory cell populations were defined as follows: Central Memory (TCM, CD45RA-/CD27+/CCR7+),

Transitional Memory (TTM, CD45RA-/CD27+/CCR7-) and Effector Memory (TEM, CD45RA-

/CD27-/CCR7-). The majority of expanded V52 cells expressed a TTM phenotype (65%), followed by TCM (23%) and finally by TEM (8%) (FIG. 11A). Cytotoxic markers including CD8, CD56 and CD16 were expressed by 37%, 32% and 45% of expanded V52 cells, respectively (FIG. 11B).

Altogether, 50% of the expanded V52 cells displayed a cytotoxic phenotype (V52+CD56+) and around 30% displayed an ADCC-like phenotype characterized by the expression of CD16.

Activation markers CD69, CD25 and HLA-DR were expressed in a mean of 37.2%, 14.8% and

16.6% V52 cells, respectively. The expression of the exhaustion markers PD-1 was observed in a mean of 19.8% and CTLA-4 was observed in 4.3% of the expanded V52 cells (FIG. 11C). [00206] PAM-expanded V52 cells inhibit active HIV replication

[00207] We previously reported that gd T cells from uninfected donors are potent inhibitors of viral replication in vitro (24). To confirm and extend these results, we used an autologous cellular system to measure the capacity of ex vivo isolated V52 T cells from ART-suppressed HIV-infected donors to inhibit viral replication in CD4 T cells infected with the JR-CSF strain in vitro. A mean reduction of 85% in HIV p24 production was detected at 1 : 1 effector: target cell ratio, 64% at a 1 : 10 and 54% at 1:100 (FIG. 12A). HIV p24 production was significantly reduced at 1:1 and 1: 10 effectordarget cell ratio.

[00208] The capacity of PAM-expanded V52 T cells to inhibit HIV replication was demonstrated, using the same functional assays of viral inhibition. Our results show that PAM- expanded V52 cells retain their capacity to significantly inhibit viral replication (FIG. 12 B, FIG. 13A). Expanded nd2 cells at the 1:1 ratio showed a mean inhibition of viral replication of 71%, 57% at the 1:10 ratio, and 18% at 1: 100. To avoid inter-individual differences in the comparison of basal and expanded nd2 cells, we performed a side-by-side analysis using the same donors. Basal mean viral inhibition mediated by ex vivo nd2 T cells was comparable to that measured after expansion with PAM (FIG. 12C). We conclude that exposure of nd2 T cells to PAM and IL-2 for 14 days does not impair the antiviral function of these cells. Finally, in four patients, we compared the inhibition capacity of nd2 cells with that of CD8 T cells, showing comparable inhibition capacity between both cell types (FIG. 13B).

[00209] V52 T cells degranulate in the presence of HIV-infected CD4 cells

[00210] The ability of nd2 cells to target HIV-infected CD4 T cells was measured analyzing

CD107a expression. Total CD4 T cells from nine ART-suppressed HIV-infected individuals were isolated and superinfected with the HIV strain JR-CSF to act as targets in cocultures with autologous expanded nd2 cells. In preliminary experiments, isolated CD4 cells were activated with PHA prior to superinfection with JR-CSF. However, we observed that cytotoxic nd2 cells were activated when

CD4 cells were exposed to PHA, regardless of HIV superinfection (FIG 14A). Therefore, we used an alternative protocol to infect isolated CD4 cells using polybrene rather than PHA. We detected a significant increase of CD107a expression in expanded nd2 cells after co-culture with JR-CSF- infected-CD4 cells, but not when cultured with CD4 cells without superinfection or alone (mean=

13.2%, 8.5% and 8.3%, respectively, p= 0.006, FIG. 15A, FIG. 15B). Expression of CD107a was also statistically higher in the HIV-CD4 group activated with PHA (mean= 18.7%, p= 0.02) but compared to polybrene-infected targets, there were not significant differences (p=0.08). Finally, we determined if the state of HIV infection (acute or chronic) at the time of ART initiation, had an impact on gd T cell effector function. CD 107a expression was comparable in cells from acute and chronic treated donors, suggesting that the cytotoxic function of V52 cells may be recovered after ART initiation (FIG. 15C). In summary, we demonstrate that expanded V52 cells specifically degranulate in the presence of HIV-infected CD4 cells.

[00211] gd T cells can efficiently clear latently HIV-infected cells upon latency disruption

[00212] gd T cells reduce the recovery of replication-competent HIV after reactivation of r-CD4 T cells with VOR. We made a modification to the previously reported latency clearance assay protocol used for CD8 T cells (8), which is itself a modification of viral outgrowth assays. This assay provides evidence of the capacity of effector cells to deplete r-CD4 cells producing replication-competent HIV following latency reversal. Our modification of the assay consisted on depleting gd T cells after 24 hours of coculture, and it is critical to allow evaluation of the specific clearance by gd T cells. This modification avoids measurement of unspecific effects over allogeneic uninfected cells added later to outgrowth the virus. Briefly, r-CD4 cells from eight ART- suppressed donors (six treated in chronic infection and two treated in acute infection) were isolated, exposed to VOR and co-cultured with or without autologous isolated expanded V52 T cells. After 24 hours of culture, gd T cells were depleted from the cultures, plated in replicates, and uninfected allogeneic CD4 cells were added to amplify replication-competent HIV. After 15/19 days of culture, viral outgrowth from r-CD4 cells cultured alone was detected in six out of eight HIV-infected donors, as measured by the number of HIV p24 positive wells. Interestingly, when gd T cells were present in the co-culture system, no virus was recovered in four donors, and viral recovery was reduced from 5 to 2, and 4 to 3 culture wells in the other two participants (FIG. 16). These results demonstrate that expanded V52 T cells can clear latently infected cells at the time of latency disruption by VOR.

[00213] DISCUSSION

[00214] Here we demonstrate the function, and ex vivo expansion capability of gd T cells from ART- suppressed HIV-infected individuals. These cells kill autologous HIV-infected CD4 T cells. In addition, V52 T cells were able to expand up to 120 fold in response to PAM/IL2 ex vivo and reduce up to 80% viral replication in autologous co-culture systems. Overall, this disclosure supports the important finding that gd T cell possess antiviral capabilities that are maintained in virologically-suppressed individuals. Further, such antiviral gd T cells can be expanded ex vivo to target latently infected cells induced to express HIV. The present work constitutes the first proof- of-concept showing that in the context of durable suppression of HIV infection, gd T cells are capable of eliminating HIV-infected targets, suggesting that gd T cells should be explored as a novel effector population to clear HIV infection from latent and active reservoirs. [00215] As previously reported for the N-BP Zol (21), we show that PAM induces expansion of V52 cells in HIV-infected individuals. Although V52 cell numbers were higher in patients treated in the acute phase of the infection compared to chronic infection, expansion capacity was comparable between both groups. Unfortunately, due to the difficulty recruiting women (31) this disclosure only included men. However, we acknowledge that gender differences may exist given controversial findings in previous works according to gd T cell frequencies (32-34), that may potentially translate into functional differences. Current work is focused towards this end. PAM- expanded V52 cells were mostly in a transitional memory state, characterized by elevated IFN-g production (35, 36). This phenotype has also been reported both after in vitro exposure to different N-BP (37) and in vivo after Zol treatment in cancer patients (38). In addition, cytotoxic phenotype in gd T cells has been associated with the expression of CD16, CD56 and CD8 (34). Frequencies of expression of cytotoxic markers in expanded nd2 cells described herein are in accordance to those reported in cancer and HIV settings (25, 39, 40). Finally, although PD-l and CTLA-4 are well- known markers of immune exhaustion CD8 T cells (41), the significance of their expression on expanded nd2 cells remains to be elucidated (41).

[00216] PAM-expanded nd2 cells showed increased CD107a production after coculture with autologous HIV-superinfected CD4 cell targets compared to cocultures of autologous CD4 cells without superinfection, demonstrating specific degranulation triggered by HIV infection. CD107a production, although statistically significant, was not very high, suggesting the involvement of other pathways in HIV recognition (42). In addition, as only a fraction of the CD4 cells used as targets may be infected, CDl07a production may be potentially diminished compared to an assay were 100% of targets were activating gd T cells. Antigen recognition by the gd TCR is generally not restricted to major MHC molecules (14, 15), although gd T cell recognition of peptides loaded on MHC molecules has been reported (43). Interestingly, our MHC blocking experiments showed a moderate decrease of CD107a expression in nd2 cells that need to be further investigated (FIG 14A-14B). This disclosure also highlights the importance of carefully controlling for external factors in culture systems that might provide confounding results— such as the use of PHA to activate prior to infection, as cytotoxic nd2 cells were activated by cells treated with PHA independently of HIV infection. Even when isolated CD4 cells were rested for six to seven days after PHA activation, nd2 cells showed increased CD 107 a production, suggesting that PHA binding to glycosylated surface proteins produces long-term, and perhaps irreversible, changes at the surface of cells that are recognized by gd T cells.

[00217] To further analyze the role of gd T cells in a context more relevant to HIV eradication in vivo, we performed a modification of the previously reported latency clearance assay (8, 44). In this assay, the capacity of effector cells to recognize latent HIV reactivated by latency reversal agents is evaluated. In our modified assay, we depleted gd T cells from the coculture before the addition of uninfected target cells to ensure specific clearance of r-CD4 infected cells. Our results demonstrate that gd T cells reduce viral recovery following latency reversal of r-CD4 cells with VOR. Immunotherapy with ex vivo expanded gd cells has been used in oncology with little toxicity and overall good tolerability (27). Of great significance for future strategies aimed to cure HIV, the use of haploidentical expanded nd2 cells from relatives in adoptive transfer strategies has been successful (45, 46). This type of intervention may be valuable for future use in HIV eradication strategies, as gd T cells can harbor replication-competent HIV (24). In this regard, supernatants from expansions showed a consistent negative HIV p24 detection. However, even if cells are expanded in the presence of antiretrovirals avoiding new rounds of infection, we still do not completely understand the overall importance of defective viruses that may be accumulating, and their modulating effector responses (47).

[00218] In summary, gd T cells from HIV-infected individuals retain their functionality after expansion and constitute an attractive immunotherapeutic alternative or complementary tool to current approaches aimed to cure HIV. Our work has opened novel and intriguing questions regarding the basic biology, function and specificities of gd T cells. Here, we show the first proof- of-concept of the potential clinical use of gd T cells in cellular therapy strategies for HIV cure.

[00219] METHODS

[00220] Participants

[00221] All HIV-infected donors included were on ART and virologically suppressed (<50 copies/mL) for at least one year prior to inclusion. Characteristics and inclusion criteria of these donors have been previously described (24, 48). HIV-infected donors treated in the acute phase of HIV infection started therapy within 45 days of the estimated date of infection. Buffy coats from uninfected donor volunteers were obtained from the New York Blood Center (Long Island City, NY, USA).

[00222] Isolation of cell populations

[00223] Peripheral blood mononuclear cells (PBMC) from HIV-infected individuals were isolated from leukapheresis products, and cells from uninfected individuals were isolated from buffy coats, all by ficoll-gradient centrifugation. nd2 cells and CD8 T cells were isolated by fluorescent activated cell sorting (FACS) using a FACSAria II (BD). PBMC were stained with monoclonal antibodies against CD3 (clone SK7), V52 (clone B6), CD4 (clone SK3), and CD8 (clone SK1) (all from Biolegend, San Diego, CA). nd2 T cells were defined as CD3⁺ nd2⁺ and CD8 T cells were defined by CD3⁺ ndT nd2 CD4 CD8⁺. CD4 T cells were isolated from the same donor using a commercially available enrichment kit that contains antibodies against CD8, CD14, CD16, CD19, CD20, CD36, CD56, CD66b, CD123, TCR-g/d and glycophorin A (StemCell Technologies, Vancouver). Resting CD4 (r-CD4) cells were isolated using a custom StemCell Technologies cocktail that contained the following antibodies: CD8, CD14, CD16, CD19, CD20, CD36, CD56, CD123, ybTCR, Glycophorin A, CD66b, CD25, HLADR and CD69. Purity of the FACS-isolated populations and resting CD4 T cells was >99%, and magnetically isolated CD4 T cells showed a purity >96%.

[00224] Expansion of V52 cells

[00225] To compare V52 cell response to pyrophosphates and N-BP from HIV-infected and uninfected donors, lxlO⁶ PBMC were incubated in the presence of 100hM (E)-4-hydroxy-3- methyl-but-2-enyl pyrophosphate (HMBPP) (kindly provided by Dr. H. Jomaa, Justus-Liebig University, Giessen, Germany) and lOOU/mL IL-2, or 25pg/mL PAM and lOOU/mL IL-2, or lOOU/mL IL-2 alone. After six days, cells were stained with monoclonal antibodies against CD3 and V52 to analyze gd T cell frequency by flow cytometry. Briefly, cells were harvested, washed, resuspended in staining buffer, incubated on ice in the dark for 20 minutes and finally washed and resuspended in 2% paraformaldehyde solution. Acquisition and analysis was performed on the Attune NxT instrument (Applied Biosystems).

[00226] Fifty to 70 million PBMC from HIV-infected individuals were incubated in complete IMDM containing antiretrovirals (lOmM raltegravir and either lOmM abacavir or ImM efavirenz, depending on the patient’s regimen), 25pg/mL pamidronate (PAM, Sigma) and 200U/mL IL-2 during 14 days. Every three-four days, media containing lOOU/mL IL-2 was refreshed. HIV p24 quantification (ABL_mc, Rockville, MA, USA) at days 7 and 14 was consistently under the limit of detection of the assay.

[00227] Phenotypic analysis of expanded V52 cells

[00228] At day 14 of expansion, an aliquot of PBMC was harvested, washed and resuspended in staining buffer to analyze the expression of different markers within expanded nd2 cells by flow cytometry i) Memory: CD45RA (clone HI100), CD27 (clone MT271) and CCR7 (clone G043H7); ii) cytotoxic: CD8 (clone SK1), CD56 (clone 5.1H11) and CD16 (clone 3G8); iii) activation: CD69 (clone FN50), CD25 (clone BC96) and HLA-DR (clone L243), and exhaustion: PD-l (clone EH12.2H7) and CTLA-4 (clone BNI3). Cells were stained for 20 min on ice in the dark, washed, fixed in a 2% paraformaldehide solution, acquired on FACS Aria II (BD) and analyzed using FlowJo v.lO.l.

[00229] HIV infection of isolated CD4 T cells [00230] Isolated CD4 T cells from HIV-infected donors were super- infected with the viral strain JR-CSF using two different approaches i) CD4 cells were activated with 4pg/mL PHA and lOOU/mL IL-2 for 24h, washed twice and super-infected by spinoculation at 2500rpm for 2 hours. Cells were then extensively washed to remove free virions and then used for further experiments ii) Isolated CD4 cells were spinoculated at 2500rpm for 4 hours in the presence of 8 pg/mL polybrene. Cells were resuspended in complete media containing 20 U/mL IL-2 and without washing the virus were further cultured for 7 days. Then, cells were extensively washed, resuspended in suitable media and experiments were performed. As a control, isolated CD4 cells were mock infected following the same two approaches. Level of infection of isolated CD4 cells was routinely tested quantifying HIV p24 production in culture supernatants by ELISA. Results showed a consistent efficacy of infection with a mean of 264.1 ng/mL for cells activated with PHA and 128.9 ng/mL for cells infected with polybrene. HIV p24 production was below the limit of detection of the assay for non-superinfected CD4 T cells.

[00231] Viral inhibition assays

[00232] Viral inhibition assays using V52 cells from HIV-infected individuals as effectors, were performed as previously described for uninfected individuals (24), with some modifications. Fifty thousand infected CD4 T cells were co-cultured in triplicate at different effector: target ratios of 1 : 1 , 1:10 and 1:100, unless otherwise noted. Infected CD4 cells alone were also cultured in triplicate as control of HIV production. In some experiments, ex vivo isolated CD8 T cells were used as effector cells. Supernatants were harvested at day 7 and stored at -20°C until HIVp24 ELISA quantification (ABLinc., Rockville, MA, USA) was performed. Results are expressed as percent of viral inhibition normalized to HIV p24 production when target CD4 T cells were cultured alone.

[00233] Degranulation assays

[00234] CD 107 a was used as a functional marker of cytotoxicity. FACS -sorted expanded V52 T cells were co-cultured with JR-CSF-infected autologous CD4 cells as targets. CD4 T cells were infected following the two different approaches described above. At least 100,000 effector cells were co-cultured at a 1 : 1 ratio with CD4 target cells in 96-well plates for 5 hours in the presence of GolgiStop (BD) and the monoclonal antibody CDl07a (clone H4A3, BD). In some experiments, MHC expression was blocked by incubating the cells with a pan-HLA monoclonal antibody (clone W6/32, Biolegend). Cells were then harvested, washed with staining buffer, stained with V52-FITC (Biolegend) for 20 minutes on ice in the dark, washed twice, re-suspended in staining buffer and analyzed in the Attune Focusing Cytometer (Applied Biosystems).

[00235] Assays to analyze clearance of latent HIV after reactivation [00236] These experiments are based on the latency clearance assay reported for CD8 T cells (8) and more recently for NK cells (44). Briefly, r-CD4 cells from HIV-infected donors were isolated as described above, and exposed to 0.5 mM VOR for 18 hours. After washing, r-CD4 cells were split into two conditions, one cultured alone and the other co-cultured with isolated expanded gd T cells at a 2: 10 ratio (gd cell:r-CD4 cell). After 24 hours of culture, gd cells were removed using a depletion magnetic bead-based kit according to manufacturer’s instructions (Miltenyi Biotech). The same treatment was applied to both conditions in parallel. This gd T cell depletion after the initial 24 hours of culture constitutes our modification of the original latency clearance assay reported for CD8 T cells, providing definitive proof that the effect observed at the end of the culture is due to clearance of the reactivated r- CD4 cells by gd T cells. After depletion, r-CD4 cells were plated at lmillion cells/well. Same number of wells (from 7 to 23 replicates) were assayed for the condition of r-CD4 cells alone and r-CD4 cells cocultured with gd T cells. As target cells to amplify viral signal, we added isolated PHA-activated total CD4 cells from uninfected donors at days three and eight of culture. Supernatants were harvested at day 15 and 19 by HIV p24 ELISA. Depletion of gd T cells after co-culture was analyzed by flow cytometry showing >99.9% efficacy. Results are expressed as number of positive wells for HIVp24, comparing cultures of r-CD4 cells with and without gd T cells.

[00237] Study approval

[00238] All patients provided written informed consent prior to inclusion in the study, and studies were approved by the UNC Institutional Review Board.

[00239] Statistics

[00240] Non-parametric tests were used. Comparison between different groups were performed using the Mann Whitney U-test and repeated measures within same groups were analyzed using Wilcoxon matched-pairs signed rank test. Where indicated, multiple comparisons were accounted for using FDR-adjusted p values (49). Statistical significance was considered for p<0.05.

6.4. REFERENCES (Sections 2.1 and 6.3)

1. Archin NM, Sung JM, Garrido C, Soriano-Sarabia N, and Margolis DM. Eradicating HIV- 1 infection: seeking to clear a persistent pathogen. Nat Rev Microbiol. 2014;12(11):750- 64.

2. Chun TW, Carruth L, Finzi D, Shen X, DiGiuseppe JA, Taylor H, Hermankova M,

Chadwick K, Margolick J, Quinn TC, et al. Quantification of latent tissue reservoirs and total body viral load in HIV-1 infection. Nature. 1997;387(6629):183-8.

3. Wong JK, Hezareh M, Gunthard HF, Havlir DV, Ignacio CC, Spina CA, and Richman DD. Recovery of replication-competent HIV despite prolonged suppression of plasma viremia. Science ( New York, NY). 1997;278(5341):1291-5.

4. Finzi D, Blankson J, Siliciano JD, Margolick JB, Chadwick K, Pierson T, Smith K,

Lisziewicz J, Lori F, Flexner C, et al. Latent infection of CD4+ T cells provides a mechanism for lifelong persistence of HIV-l, even in patients on effective combination therapy. Nature medicine. 1999;5(5):512-7.

Siliciano JD, and Siliciano RF. Recent developments in the effort to cure HIV infection: going beyond N = 1. The Journal of clinical investigation. 2016;126(2):409-14.

Margolis DM, Garcia JV, Hazuda DJ, and Haynes BF. Latency reversal and viral clearance to cure HIV-l. Science ( New York, NY). 20l6;353(6297):aaf65l7.

Deng K, Pertea M, Rongvaux A, Wang L, Durand CM, Ghiaur G, Lai J, McHugh HL,

Hao H, Zhang H, et al. Broad CTL response is required to clear latent HIV-l due to dominance of escape mutations. Nature. 2015.

Sung JA, Lam S, Garrido C, Archin N, Rooney CM, Bollard CM, and Margolis DM. Expanded cytotoxic T-cell lymphocytes target the latent HIV reservoir. The Journal of infectious diseases. 2015;212(2):258-63.

Day CL, Kaufmann DE, Kiepiela P, Brown JA, Moodley ES, Reddy S, Mackey EW, Miller JD, Leslie AJ, DePierres C, et al. PD-1 expression on HIV-specific T cells is associated with T-cell exhaustion and disease progression. Nature. 2006;443(7l09):350-4. Shan L, Deng K, Shroff NS, Durand CM, Rabi SA, Yang HC, Zhang H, Margolick JB, Blankson JN, and Siliciano RF. Stimulation of HIV-l -specific cytolytic T lymphocytes facilitates elimination of latent viral reservoir after virus reactivation. Immunity.

2012;36(3):491-501.

Morita CT, Jin C, Sarikonda G, and Wang H. Nonpeptide antigens, presentation mechanisms, and immunological memory of human Vgamma2Vdelta2 T cells:

discriminating friend from foe through the recognition of prenyl pyrophosphate antigens. Immunological reviews. 2007;215(59-76.

Jomaa H, Wiesner J, Sanderbrand S, Altincicek B, Weidemeyer C, Hintz M, Turbachova I, Eberl M, Zeidler J, Lichtenthaler HK, et al. Inhibitors of the nonmevalonate pathway of isoprenoid biosynthesis as antimalarial drugs. Science (New York, NY).

1999;285(5433): 1573-6.

Hintz M, Reichenberg A, Altincicek B, Bahr U, Gschwind RM, Kollas AK, Beck E, Wiesner J, Eberl M, and Jomaa H. Identification of (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate as a major activator for human gammadelta T cells in Escherichia coli. FEBS letters. 2001;509(2):317-22.

Tanaka Y, Morita CT, Tanaka Y, Nieves E, Brenner MB, and Bloom BR. Natural and synthetic non-peptide antigens recognized by human gamma delta T cells. Nature.

1995;375(6527): 155-8.

Vavassori S, Kumar A, Wan GS, Ramanjaneyulu GS, Cavallari M, El Daker S, Beddoe T, Theodossis A, Williams NK, Gostick E, et al. Butyrophilin 3A1 binds phosphorylated antigens and stimulates human gammadelta T cells. Nature immunology. 2013;14(9):908-

16.

Wang H, Sarikonda G, Puan KJ, Tanaka Y, Feng J, Giner JL, Cao R, Monkkonen J, Oldfield E, and Morita CT. Indirect stimulation of human Vgamma2Vdelta2 T cells through alterations in isoprenoid metabolism. Journal of immunology (Baltimore, Md : 1950). 2011 ; 187(10):5099- 113.

Lo Presti E, Dieli F, and Meraviglia S. Tumor-Infiltrating gammadelta T Lymphocytes: Pathogenic Role, Clinical Significance, and Differential Programing in the Tumor Microenvironment. Frontiers in immunology. 2014;5(607.

Silva-Santos B, Serre K, and Norell H. gammadelta T cells in cancer. Nature reviews Immunology. 2015;15(11):683-91.

Wallace M, Bartz SR, Chang WL, Mackenzie DA, Pauza CD, and Malkovsky M. Gamma delta T lymphocyte responses to HIV. Clinical and experimental immunology.

1996; 103(2): 177-84.

Poccia F, Battistini L, Cipriani B, Mancino G, Martini F, Gougeon ML, and Colizzi V. Phosphoantigen-reactive Vgamma9Vdelta2 T lymphocytes suppress in vitro human immunodeficiency virus type 1 replication by cell-released antiviral factors including CC chemokines. The Journal of infectious diseases. 1999;180(3):858-61.

Poonia B, and Pauza CD. Gamma delta T cells from HIV+ donors can be expanded in vitro by zoledronate/interleukin-2 to become cytotoxic effectors for antibody-dependent cellular cytotoxicity. Cytotherapy. 2012;14(2):173-81.

Fausther-Bovendo H, Wauquier N, Cherfils-Vicini J, Cremer I, Debre P, and Vieillard V. NKG2C is a major triggering receptor involved in the V [delta] 1 T cell-mediated cytotoxicity against HIV-infected CD4 T cells. AIDS (London, England ). 2008;22(2):2l7- 26.

Hudspeth K, Fogli M, Correia DV, Mikulak J, Roberto A, Della Bella S, Silva-Santos B, and Mavilio D. Engagement of NKp30 on Vdeltal T cells induces the production of CCL3, CCL4, and CCL5 and suppresses HIV-l replication. Blood. 2012;119(17):4013-6. Soriano-Sarabia N, Archin NM, Bateson R, Dahl NP, Crooks AM, Kuruc JD, Garrido C, and Margolis DM. Peripheral Vgamma9Vdelta2 T Cells Are a Novel Reservoir of Latent HIV Infection. PLoS pathogens. 20l5;l l(l0):el00520l.

Lafont V, Sanchez F, Laprevotte E, Michaud HA, Gros L, Eliaou JF, and Bonnefoy N. Plasticity of gammadelta T Cells: Impact on the Anti-Tumor Response. Frontiers in immunology. 20l4;5(622.

Poccia F, Gioia C, Martini F, Sacchi A, Piacentini P, Tempestilli M, Agrati C, Amendola A, Abdeddaim A, Vlassi C, et al. Zoledronic acid and interleukin-2 treatment improves immunocompetence in HIV-infected persons by activating Vgamma9Vdelta2 T cells. AIDS ( London , England). 2009;23(5):555-65.

Kobayashi H, and Tanaka Y. gammadelta T Cell Immunotherapy-A Review.

Pharmaceuticals (Basel, Switzerland). 2015 ;8(l):40-6l .

Zou C, Zhao P, Xiao Z, Han X, Fu F, and Fu L. gammadelta T cells in cancer

immunotherapy. Oncotarget. 20l7;8(5):8900-9.

Van Acker HH, Anguille S, Willemen Y, Smits EL, and Van Tendeloo VF.

Bisphosphonates for cancer treatment: Mechanisms of action and lessons from clinical trials. Pharmacology & therapeutics. 20l6;l58(24-40.

Poccia F, Boullier S, Lecoeur H, Cochet M, Poquet Y, Colizzi V, Fournie JJ, and Gougeon ML. Peripheral V gamma 9/V delta 2 T cell deletion and anergy to nonpeptidic mycobacterial antigens in asymptomatic HIV-l -infected persons. Journal of immunology (Baltimore, Md : 1950). 1996; 157(1):449-61.

Loutfy MR, V LK, Mohammed S, Wu W, Muchenje M, Masinde K, Salam K, Soje L, Gregorovich S, and Tharao W. Recruitment of HIV-Positive Women in Research:

Discussing Barriers, Facilitators, and Research Personnel's Knowledge. The open AIDS journal. 20l4;8(58-65.

Caccamo N, Dieli F, Wesch D, Jomaa H, and Eberl M. Sex-specific phenotypical and functional differences in peripheral human Vgamma9/Vdelta2 T cells. Journal of leukocyte biology. 2006;79(4):663-6.

Cairo C, Armstrong CL, Cummings JS, Deetz CO, Tan M, Lu C, Davis CE, and Pauza CD. Impact of age, gender, and race on circulating gammadelta T cells. Human immunology. 2010;71(10):968-75.

Ryan PL, Sumaria N, Holland CJ, Bradford CM, Izotova N, Grandjean CL, Jawad AS, Bergmeier LA, and Pennington DJ. Heterogeneous yet stable Vdelta2(+) T-cell profiles define distinct cytotoxic effector potentials in healthy human individuals. Proceedings of the National Academy of Sciences of the United States of America. 2016; 113(50): 14378- 83.

Dieli F, Poccia F, Lipp M, Sireci G, Caccamo N, Di Sano C, and Salerno A.

Differentiation of effector/memory Vdelta2 T cells and migratory routes in lymph nodes or inflammatory sites. The Journal of experimental medicine. 2003;l98(3):39l-7. Fritsch RD, Shen X, Sims GP, Hathcock KS, Hodes RJ, and Lipsky PE. Stepwise differentiation of CD4 memory T cells defined by expression of CCR7 and CD27. Journal of immunology ( Baltimore , Md : 1950). 2005;l75(l0):6489-97.

Ferlazzo V, Sferrazza C, Caccamo N, Di Fede G, Di Lorenzo G, D'Asaro M, Meraviglia S, Dieli F, Rini G, and Salerno A. In vitro effects of aminobisphosphonates on

Vgamma9Vdelta2 T cell activation and differentiation. International journal of immunopathology and pharmacology. 2006;l9(2):309-l7.

Dieli F, Gebbia N, Poccia F, Caccamo N, Montesano C, Fulfaro F, Arcara C, Valerio MR, Meraviglia S, Di Sano C, et al. Induction of gammadelta T-lymphocyte effector functions by bisphosphonate zoledronic acid in cancer patients in vivo. Blood. 2003;l02(6):23l0-l. Alexander AA, Maniar A, Cummings JS, Hebbeler AM, Schulze DH, Gastman BR, Pauza CD, Strome SE, and Chapoval AI. Isopentenyl pyrophosphate-activated CD56+

{ gamma} {delta} T lymphocytes display potent antitumor activity toward human squamous cell carcinoma. Clinical cancer research : an official journal of the American Association for Cancer Research. 2008;l4(l3):4232-40.

Urban EM, Li H, Armstrong C, Focaccetti C, Cairo C, and Pauza CD. Control of CD56 expression and tumor cell cytotoxicity in human Vgamma2Vdelta2 T cells. BMC immunology. 2009; 10(50.

Teigler JE, Zelinskyy G, Eller MA, Bolton DL, Marovich M, Gordon AD, Alrubayyi A, Alter G, Robb ML, Martin JN, et al. Differential Inhibitory Receptor Expression on T Cells Delineates Functional Capacities in Chronic Viral Infection. Journal of virology. 20l7;9l(23).

Pauza CD, Poonia B, Li H, Cairo C, and Chaudhry S. gammadelta T Cells in HIV Disease: Past, Present, and Future. Frontiers in immunology. 20l4;5(687.

Born WK, Zhang L, Nakayama M, Jin N, Chain JL, Huang Y, Aydintug MK, and O'Brien RL. Peptide antigens for gamma/delta T cells. Cellular and molecular life sciences :

CMLS. 20ll;68(l4):2335-43.

Garrido C, Abad-Fernandez M, Tuyishime M, Pollara JJ, Ferrari G, Soriano-Sarabia N, and Margolis DM. IL-15 STIMULATED NATURAL KILLER CELLS CLEAR HIV-l INFECTED CELLS FOLLOWING LATENCY REVERSAL EX VIVO. Journal of virology. 2018.

Wilhelm M, Smetak M, Schaefer-Eckart K, Kimmel B, Birkmann J, Einsele H, and Kunzmann V. Successful adoptive transfer and in vivo expansion of haploidentical gammadelta T cells. Journal of translational medicine. 20l4;l2(45.

Norell H, Moretta A, Silva-Santos B, and Moretta L. At the Bench: Preclinical rationale for exploiting NK cells and gammadelta T lymphocytes for the treatment of high-risk leukemias. Journal of leukocyte biology. 20l3;94(6): 1123-39.

Bruner KM, Murray AJ, Pollack RA, Soliman MG, Laskey SB, Capoferri AA, Lai J,

Strain MC, Lada SM, Hoh R, et al. Defective proviruses rapidly accumulate during acute HIV-l infection. Nature medicine. 20l6;22(9): 1043-9.

Archin NM, Vaidya NK, Kuruc JD, Liberty AL, Wiegand A, Kearney MF, Cohen MS, Coffin JM, Bosch RJ, Gay CL, et al. Immediate antiviral therapy appears to restrict resting CD4+ cell HIV-l infection without accelerating the decay of latent infection. Proceedings of the National Academy of Sciences of the United States of America. 20l2;l09(24):9523- 8.

Benjamini Y, Drai D, Elmer G, Kafkafi N, and Golani I. Controlling the false discovery rate in behavior genetics research. Behavioural brain research. 200l;l25(l-2):279-84. 7. GENERALIZED STATEMENTS OF THE DISCLOSURE

[00241] The following numbered statements provide a general description of the disclosure and are not intended to limit the appended claims.

[00242] Statement 1: A method for inducing expression of HIV viral antigen(s) in latently HIV- infected cells which comprises exposing the latently HIV-infected cells to a bisphosphonate so as to reverse latency and to induce the expression of the HIV viral antigen(s).

[00243] Statement 2: The method of Statement 1, wherein the bisphosphonate is an aminobisphosponate.

[00244] Statement 3: The method of Statement 2, wherein the aminobisphosphonate is Alendronate (Fosamax™), Ibandronate (Boniva™ or Bonviva™), Neridronate (Nerixia™), Olpadronate, Pamidronate (APD/Aredia™), Risedronate (Actonel™), or Zoledronate (Zometa™ /Aclasta™).

[00245] Statement 4: A method of inducing expression of HIV viral antigens in latently HIV- infected cells in a subject which comprises administering to the subject a bisphosphonate so as to reverse latency and induce expression of the HIV viral antigens in the cells in the subject.

[00246] Statement 5: The method of Statement 4, wherein the latently infected cells are rCD4 cells.

[00247] Statement 6: The method of any of Statements 1-5, wherein the subject is receiving antiretroviral therapy (ART).

[00248] Statement 7 : A method to boost gd T cell expression and reverse viral latency in a subject infected with HIV which comprises administering to the subject a bisphosphonate.

[00249] Statement 8: The method of Statement 7, wherein the subject is receiving antiretroviral therapy (ART).

[00250] Statement 9: The use of an aminobisphosphonate as a latency reducing agent.

[00251] Statement 10: The use of Statement 9, wherein the aminobisphosphonate is Alendronate (Fosamax™), Ibandronate (Boniva™ or Bonviva™), Neridronate (Nerixia™), Olpadronate, Pamidronate (APD/Aredia™), Risedronate (Actonel™), or Zoledronate (Zometa™ /Aclasta™).

[00252] Statement 11 : A method for eliminating latently infected cells harboring quiescent HIV provirus that comprises exposing the infected cells with gd T cells and a bisphosphonate.

[00253] Statement 12: The method of Statement 11, further comprising exposing latently infected cells to a second HIV latency reversing agent.

[00254] Statement 13: The method of Statement 12, wherein the second HIV latency reversing agent is an epigenetic modifier, an NFkB agonist, a PI3K Akt pathway inhibitor, a protein kinase C agonist, a SMAC (second mitochondria-derived activator of caspases) mimetic, a TCR activator or a TLR agonist.

[00255] Statement 14: The method of Statement 13, wherein the epigenetic modifier is a histone deacetylase (HD AC) inhibitor.

[00256] Statement 15: A method of treating/curing a subject infected with HIV which comprises administering to the subject ex vivo expanded gd T cells and a bisphosphonate.

[00257] Statement 16: The method of Statement 15, further comprising administering to the subject a second HIV latency reversing agent.

[00258] Statement 17: The method of Statement 16, wherein the HIV latency reversing agent is an epigenetic modifier, an NFkB agonist, a PI3K/Akt pathway inhibitor, a protein kinase C agonist, a SMAC (second mitochondria-derived activator of caspases) mimetic, a TCR activator, a TLR agonist, an inhibitor of LAP (Inhibitor of Apoptosis Protein) family of proteins, or a stimulator of interferon genes protein (STING) agonist.

[00259] Statement 18: The method of Statement 17, wherein the epigenetic modifier is histone deacetylase (HD AC) inhibitor.

[00260] Statement 19: The method of any of Statements 11-18, wherein the expanded gd T cells and the bisphosphonate are administered concurrently.

[00261] Statement 20: The method of any of Statements 11-18, wherein the expanded gd T cells and the bisphosphonate are administered sequentially.

[00262] Statement 21: The method of Statement 20, wherein the expanded gd T cells and the bisphosphonate are administered over a 24 hour period.

[00263] Statement 22: The method of any of Statements 11-21, wherein the ex vivo expanded gd T cells are ndΐ T-cells.

[00264] Statement 23: The method of any of Statements 11-21, wherein the ex vivo expanded gd T cells are nd2 T-cells.

[00265] Statement 24: The method of any of Statements 11-23, wherein the ex vivo expanded gd T cells are autologous cells.

[00266] Statement 25: The method of any of Statements 11-23, wherein the ex vivo expanded gd T cells are heterologous cells.

[00267] Statement 26: The method of any of Statements 11-25, wherein the bisphosphonate is Alendronate (Fosamax™), Ibandronate (Boniva™ or Bonviva™), Neridronate (Nerixia™), Olpadronate, Pamidronate (APD/Aredia™), Risedronate (Actonel™), or Zoledronate (Zometa™ /Aclasta™). [00268] Statement 27: The method of any of Statements 14 or 18, wherein the HDAC inhibitor is belinostat (PXD101), entinostat (MS-275), mocetinostat (MGCD0103), panobinostat (LBH589), or vorinostat (SAHA).

[00269] Statement 28: A method of treating HIV infection in a subject, comprising: (a) isolating peripheral blood mononuclear (PBMC) cells from the subject; (b) culturing the isolated PBMC cells ex vivo with an effective amount of a bisphosphonate or antibodies and suitable cytokines so as to expand gd T cells; (c) or optionally genetically modifying the expanded gd T cells; (d) infusing the expanded gd T cells into the subject; and (e) administering to the subject a bisphosphonate so as to activate quiescent HIV provirus and treat the subject with HIV.

[00270] Statement 29: The method of Statement 28, further comprising administering to the subject in step (e) a second HIV latency reversing agent.

[00271] Statement 30: The method of Statement 29, wherein the second HIV latency reversing agent is an epigenetic modifier, an NFkB agonist, a PI3K/Akt pathway inhibitor, a protein kinase C agonist, a TCR activator, a TLR agonist, an inhibitor of LAP (Inhibitor of Apoptosis Protein) family of proteins, or a stimulator of interferon genes protein (STING) agonist.

[00272] Statement 31: The method of Statement 30, wherein the epigenetic modifier is a histone deacetylase (HDAC) inhibitor.

[00273] Statement 32: The method of any of Statements 28-31 , wherein the subject was receiving antiretroviral therapy (ART).

[00274] Statement 33: The method of any of Statements 28-32, wherein the PBMCs are expanded in the presence of the bisphosphonate for 5 to 30 days.

[00275] Statement 34: The method of any of Statements 28-33, wherein the PBMCs are expanded for 7 to 21 days.

[00276] Statement 35: The method of any of Statements 28-34, wherein the expanded gd T cells and the bisphosphonate are administered concurrently.

[00277] Statement 36: The method of any of Statements 28-34, wherein the expanded gd T cells and the bisphosphonate are administered sequentially.

[00278] Statement 37: The method of any of Statements 28-36, wherein the bisphosphonate is Alendronate (Fosamax™), Ibandronate (Boniva™ or Bonviva™), Neridronate (Nerixia™), Olpadronate, Pamidronate (APD/Aredia™), Risedronate (Actonel™), or Zoledronate (Zometa™ /Aclasta™).

[00279] Statement 38: The method of Statement 31, wherein the HDAC inhibitor is belinostat (PXD101), entinostat (MS-275), mocetinostat (MGCD0103), panobinostat (LBH589), or vorinostat (SAHA). [00280] Statement 39: A pharmaceutical composition comprising a bisphosphonate and a second HIV latency reversing agent.

[00281] Statement 40: The pharmaceutical composition of Statement 39, wherein the second HIV latency reversing agent is an epigenetic modifier, an NFkB agonist, a PI3K/Akt pathway inhibitor, a protein kinase C agonist, a TCR activator, a TLR agonist, an inhibitor of IAP (Inhibitor of Apoptosis Protein) family of proteins, or a stimulator of interferon genes protein (STING) agonist.

[00282] Statement 41 : The pharmaceutical composition of Statement 40, wherein the epigenetic modifier is a histone deacetylase (HD AC) inhibitor.

[00283] Statement 42: A pharmaceutical composition comprising an HMG CoA inhibitor and an HIV latency reversing agent.

[00284] Statement 45: The pharmaceutical composition of Statement 42, wherein the HMG CoA inhibitor is a statin.

[00285] Statement 45: The pharmaceutical composition of Statement 44, wherein the statin is torvastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin or a combination thereof.

[00286] Statement 46: A pharmaceutical composition comprising either (i) a synthetic analogue of a component of the mevalonate pathway, (ii) an inhibitor of the mevalonate pathway (beyond bisphosphonates or HMG-CoA reductase inhibitors), or (iii) an intermediate from the mevalonate pathway, and an HIV latency reversing agent.

[00287] Statement 47: The pharmaceutical composition of Statement 46, wherein the synthetic analogue, inhibitor, or intermediate is (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), isopentenyl pyrophosphate (IPP), dimethyl-allyl-pyrophosphate (DMAPP), bromohydrin pyrophosphate (BrHPP) or 2-methyl-3-butenyl-l-pyrophosphate (2M3B1PP).

[00288] Statement 48: The pharmaceutical composition of any of Statements 42-47, wherein the HIV latency reversing agent is an epigenetic modifier, an NFkB agonist, a PI3K/Akt pathway inhibitor, a protein kinase C agonist, a TCR activator, a TLR agonist, an inhibitor of IAP (Inhibitor of Apoptosis Protein) family of proteins, or a stimulator of interferon genes protein (STING) agonist.

[00289] Statement 49: The pharmaceutical composition of Statement 48, wherein the epigenetic modifier is a histone deacetylase (HD AC) inhibitor.

[00290] Statement 50: The pharmaceutical composition of Statement 49, wherein the HDAC inhibitor is belinostat (PXD101), entinostat (MS-275), mocetinostat (MGCD0103), panobinostat (LBH589), or vorinostat (SAHA). [00291] Statement 51: The use of the pharmaceutical composition of any of Statements 39-50 as an HIV latency reversing agent ex vivo.

[00292] Statement 52: The use of the pharmaceutical composition of any of Statements 39-50 as an HIV latency reversing agent when administered to a subject.

[00293] It should be understood that the above description is only representative of illustrative embodiments and examples. For the convenience of the reader, the above description has focused on a limited number of representative examples of all possible embodiments, examples that teach the principles of the disclosure. The description has not attempted to exhaustively enumerate all possible variations or even combinations of those variations described. That alternate embodiments may not have been presented for a specific portion of the disclosure, or that further undescribed alternate embodiments may be available for a portion, is not to be considered a disclaimer of those alternate embodiments. One of ordinary skill will appreciate that many of those undescribed embodiments, involve differences in technology and materials rather than differences in the application of the principles of the disclosure. Accordingly, the disclosure is not intended to be limited to less than the scope set forth in the following claims and equivalents.

[00294] INCORPORATION BY REFERENCE

[00295] All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entireties for all purposes. However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world. It is to be understood that, while the disclosure has been described in conjunction with the detailed description, thereof, the foregoing description is intended to illustrate and not limit the scope. Other aspects, advantages, and modifications are within the scope of the claims set forth below. All publications, patents, and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

Claims

CLAIMS What is claimed is:

1. A method for inducing expression of HIV viral antigen(s) in latently HIV-infected cells which comprises exposing the latently HIV-infected cells to a bisphosphonate so as to reverse latency and to induce the expression of the HIV viral antigen(s).

2. The method of claim 1, wherein the bisphosphonate is an aminobisphosponate.

3. The method of claim 2, wherein the aminobisphosphonate is Alendronate (Fosamax™), Ibandronate (Boniva™ or Bonviva™), Neridronate (Nerixia™), Olpadronate,

Pamidronate (APD/Aredia™), Risedronate (Actonel™), or Zoledronate (Zometa™ /Aclasta™).

4. A method of inducing expression of HIV viral antigens in latently HIV-infected cells in a subject which comprises administering to the subject a bisphosphonate so as to reverse latency and induce expression of the HIV viral antigens in the cells in the subject.

5. The method of claim 4, wherein the latently infected cells are rCD4 cells.

6. The method of claim 4, wherein the subject is receiving antiretroviral therapy (ART).

7. A method to boost gd T cell expression and reverse viral latency in a subject infected with HIV which comprises administering to the subject a bisphosphonate.

8. The method of claim 7, wherein the subject is receiving antiretroviral therapy (ART).

9. The use of an aminobisphosphonate as a latency reducing agent.

10. The use of claim 9, wherein the aminobisphosphonate is Alendronate (Fosamax™), Ibandronate (Boniva™ or Bonviva™), Neridronate (Nerixia™), Olpadronate,

Pamidronate (APD/Aredia™), Risedronate (Actonel™), or Zoledronate (Zometa™ /Aclasta™).

11. A method for eliminating latently infected cells harboring quiescent HIV pro virus that comprises exposing the infected cells to gd T cells and a bisphosphonate.

12. The method of claim 11, further comprising exposing latently infected cells to a second HIV latency reversing agent.

13. The method of claim 12, wherein the second HIV latency reversing agent is an epigenetic modifier, an NFkB agonist, a PI3K/Akt pathway inhibitor, a protein kinase C agonist, a SMAC (second mitochondria-derived activator of caspases) mimetic, a TCR activator, a TLR agonist, an inhibitor of IAP (Inhibitor of Apoptosis Protein) family of proteins, or a stimulator of interferon genes protein (STING) agonist.

14. The method of claim 13, wherein the epigenetic modifier is a histone deacetylase

(HD AC) inhibitor.

15. A method of treating/curing a subject infected with HIV which comprises administering to the subject ex vivo expanded gd T cells and a bisphosphonate.

16. The method of claim 15, further comprising administering to the subject a second HIV latency reversing agent.

17. The method of claim 16, wherein the second HIV latency reversing agent is an epigenetic modifier, an NFkB agonist, a PI3K/Akt pathway inhibitor, a protein kinase C agonist, a SMAC (second mitochondria-derived activator of caspases) mimetic, a TCR activator, a TLR agonist, an inhibitor of IAP (Inhibitor of Apoptosis Protein) family of proteins, or a stimulator of interferon genes protein (STING) agonist.

18. The method of claim 17, wherein the epigenetic modifier is histone deacetylase (HD AC) inhibitor.

19. The method of claim 15, wherein the expanded gd T cells and the bisphosphonate are administered concurrently.

20. The method of claim 15, wherein the expanded gd T cells and the bisphosphonate are administered sequentially.

21. The method of claim 20, wherein the expanded gd T cells and the bisphosphonate are administered over a 24 hour period.

22. The method of claim 15, wherein the ex vivo expanded gd T cells are V51 T-cells.

23. The method of claim 15, wherein the ex vivo expanded gd T cells are nd2 T-cells.

24. The method of claim 15, wherein the ex vivo expanded gd T cells are autologous cells.

25. The method of claim 15, wherein the ex vivo expanded gd T cells are heterologous cells.

26. The method of claim 15, wherein the bisphosphonate is Alendronate (Fosamax™), Ibandronate (Boniva™ or Bonviva™), Neridronate (Nerixia™), Olpadronate,

Pamidronate (APD/Aredia™), Risedronate (Actonel™), or Zoledronate (Zometa™ /Aclasta™).

27. The method of claim 18, wherein the HDAC inhibitor is belinostat (PXD101), entinostat (MS-275), mocetinostat (MGCD0103), panobinostat (LBH589), or vorinostat (SAHA).

28. A method of treating HIV infection in a subject, comprising:

(a) isolating peripheral blood mononuclear (PBMC) cells from the subject;

(b) culturing the isolated PBMC cells ex vivo with an effective amount of a

bisphosphonate or antibodies and suitable cytokines so as to expand gd T cells;

(c) or optionally genetically modifying the expanded gd T cells;

(d) infusing the expanded gd T cells into the subject; and

(e) administering to the subject a bisphosphonate so as to activate quiescent HIV provirus and treat the subject with HIV.

29. The method of claim 28, further comprising administering to the subject in step (e) a second HIV latency reversing agent.

30. The method of claim 29, wherein the second HIV latency reversing agent is an epigenetic modifier, an NFkB agonist, a PI3K/Akt pathway inhibitor, a protein kinase C agonist, a TCR activator, a TLR agonist, an inhibitor of LAP (Inhibitor of Apoptosis Protein) family of proteins, or a stimulator of interferon genes protein (STING) agonist.

31. The method of claim 30, wherein the epigenetic modifier is a histone deacetylase

(HDAC) inhibitor.

32. The method of claim 28, wherein the subject was receiving antiretroviral therapy (ART).

33. The method of claim 28, wherein the PBMCs are expanded in the presence of the

bisphosphonate for 5 to 30 days.

34. The method of claim 33, wherein the PBMCs are expanded for 7 to 21 days.

35. The method of claim 28, wherein the expanded gd T cells and the bisphosphonate are administered concurrently.

36. The method of claim 28, wherein the expanded gd T cells and the bisphosphonate are administered sequentially.

37. The method of claim 28, wherein the bisphosphonate is Alendronate (Fosamax™),

Ibandronate (Boniva™ or Bonviva™), Neridronate (Nerixia™), Olpadronate,

Pamidronate (APD/Aredia™), Risedronate (Actonel™), or Zoledronate (Zometa™ /Aclasta™).

38. The method of claim 31, wherein the HDAC inhibitor is belinostat (PXD101), entinostat (MS-275), mocetinostat (MGCD0103), panobinostat (LBH589), or vorinostat (SAHA).

39. A pharmaceutical composition comprising a bisphosphonate and a second HIV latency reversing agent.

40. The pharmaceutical composition of claim 39, wherein the second HIV latency reversing agent is an epigenetic modifier, an NFkB agonist, a PI3K/Akt pathway inhibitor, a protein kinase C agonist, a TCR activator, a TLR agonist, an inhibitor of IAP (Inhibitor of Apoptosis Protein) family of proteins, or a stimulator of interferon genes protein (STING) agonist.

41. The pharmaceutical composition of claim 40, wherein the epigenetic modifier is a histone deacetylase (HDAC) inhibitor.