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IL-15 HIV Cure Research
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"thein vivoactivation of NK cells, integral mediators of the innate immune response, by treatment with an IL-15 superagonist increases their anti-HIV activity and enables them to potently suppress acutein vivoHIV-1 infection. These results indicate thatin vivoactivation of NK cells may represent a new immunotherapeutic approach to suppress acute HIV-1 infection."
Proof of Principle Study of Pulse Dosing of IL-15 to Deplete the Reservoir in HIV Infected People (ALT-803)on Optimized ART With Undetectable Plasma HIV RNA....Assess the impact of ALT-803 on the size of the inducible reservoir....Assess the safety and tolerability of ALT-803.....https://clinicaltrials.gov/ct2/show/NCT02191098
from Jules: In Gilead's recent announcements of cure research grants they awarded a grant to support research into IL-15 & cure:Foundation for the National Institutes of Health, National Cancer Institute, Center for Cancer Research - George N. Pavlakis, M.D., Ph.D. - Efficacy of Heterodimeric IL-15 Treatment Regimens in Reducing SIV Reservoir; as well to support Larry Corey researching Car T Cells in HIV cure research. TLR9 cure research is also funded in the Gilead grant [see separate report].....Gilead Awards More Than $22 Million in Grants to Support HIV Cure Research - (01/13/17)
Use of heterodimeric IL-15 in immune therapeutic regimens against HIV-1 or SIV infection http://www.jimmunol.org/content/194/1_Supplement/144.4 Interleukin-15 (IL-15) promotes the growth of NK and memory CD8 T cells, which makes it an attractive candidate in immune therapeutic strategies to eradicate HIV-1 infection. In conclusion, the combination of CD8 effector cells expansion and lack of viral activation makes IL-15 an ideal component in immune therapeutic strategies for the treatment and potential eradication of HIV-1 infection.
Therapeutic Vaccination with Simian Immunodeficiency Virus (SIV)-DNA+IL-12 or IL-15 Induces Distinct CD8 Memory Subsets in SIV-Infected Macaques
IL-15 Treatment during Acute Simian Immunodeficiency Virus (SIV) Infection Increases Viral Set Point and Accelerates Disease Progression despite the Induction of Stronger SIV-Specific CD8+ T Cell Responses
Our data show that IL-15 treatment increases viral set point by 3 logs and leads to accelerated disease progression to simian AIDS in two of six animals with one developing SIV meningoencephalitis......http://www.jimmunol.org/content/180/1/350.full
Levels of Interleukin-15 in Plasma May Predict a Favorable Outcome of Structured Treatment Interruption in Patients with Chronic Human Immunodeficiency Virus Infection.....STI responders showed a higher baseline concentration of interleukin (IL)–15 in plasma than did STInon responders.....These results indicate that higher levels of IL-15 in plasma correlate with a delayed or an absent rebound of HIV viremia, a delay or absence that results from the ability to control viral replication. To determine a better outcome of STI, baseline levels of IL-15 in plasma could therefore be used as markers. Moreover, an inability to spontaneously control viral replication—an inability that is due to a selective impairment of HIV-specific immune response—could be caused by defective IL-15 production. Accordingly, these findings may support the hypothesis that, in most patients with HIV who, in the absence of ART, are unable to control viral replication, treatment with IL-15 may represent a useful strategy to enhance HIV-specific CD8 T cell response [17, 18]. Altogether, our data indicate that, in patients with chronic HIV infection, levels of IL-15 in plasma may predict a favorable outcome of STI, a finding suggesting that, in patients unable to spontaneously control viral replication, treatment with IL-15 could be useful in attempts to improve the antiviral immune responsehttp://jid.oxfordjournals.org/content/188/5/661.full
In Vivo Activation of Human NK cells by Treatment with an IL-15 Superagonist Potently Inhibits Acute In Vivo HIV-1 Infection in Humanized Mice
To our knowledge, this study is the first reported demonstration that in vivoactivation of NK cells can inhibit acute HIV-1 infection.The NK cells were activated byin vivo treatment with a superagonist of IL-15, a multifunctional cytokine which plays acrucial role in NK cell and CD8+ T cell development, activation, proliferation,differentiation and functional activity (12, 15, 45)......This demonstrated that human NK cells were the crucial effector cellsactivated by in vivo treatment with the IL-15-agonist that mediated inhibition of acute invivo HIV-1 infection......The functional activity of NK cells is impaired during chronic HIV-1 infection(9), and it is possible that this impairment may be reversed by treatment with the IL-15superagonist. Further studies in humanized mice infected with HIV-1 and treated withthe IL-15 superagonist, should enable us to further delineate the effects of NK cellactivation mediated by IL-15 superagonist treatment on inhibiting in vivo HIV-1 infection.Treatment of SHIV-infected monkeys with broadly neutralizing HIV-specific antibodiesimproved the function of their HIV-specific CD8+ T cells and lowered their viral-load setpoints, suggesting that neutralizing antibody therapy could increase anti-viral immuneresponses to enable better control of SHIV infection (61). It is possible that NK cellmediated antibody-dependent cellular cytotoxicity (ADCC) may contribute to thereported reduction in rebound viremia in humanized mice treated with broadlyneutralizing HIV-specific antibodies (62). Thus, an intriguing possibility is that increasingNK cell activity by treatment with the IL-15 superagonist may further facilitate the antiHIV-1 activity of neutralizing antibody therapy by augmenting ADCC activity. Furtherstudies in humanized mice infected with HIV-1 and treated with the IL-15 superagonist,should enable us to further delineate the effects of IL15R-agonist-mediated NK cellactivation on directly inhibiting in vivo HIV-1 infection and further suppressing HIV-1infection through ADCC when combined with broadly neutralizing HIV-specific antibodytherapy.
Natural killer (NK) cells with anti-HIV-1 activity may inhibit HIV-1 replication and dissemination during acute HIV-1 infection. We hypothesized that the NK cell capacity to suppress acute in vivo HIV-1 infection would be augmented by activating them via treatment with an IL-15 superagonist, IL-15 bound to soluble IL-15Rα, an approach that potentiates human NK cell-mediated killing of tumor cells. In vitro stimulation of human NK cells with a recombinant IL-15 superagonist significantly induced their expression of cytotoxic effector molecules granzyme B and perforin, their degranulation upon exposure to K562 cells as indicated by cell-surface expression of CD107a and their capacity to lyse K562 cells and HIV-1-infected T cells. The impact of IL-15 superagonist-induced activation of human NK cells on acute in vivo HIV-1 infection was investigated using hu-spl-PBMC-NSG mice, NOD-SCID-IL2rγ-/- mice intrasplenically injected with human PBMCs, which develop productive in vivo infection after intrasplenic inoculation with HIV-1. IL-15 superagonist treatment potently inhibited acute HIV-1 infection in hu-spl-PBMC-NSG mice even when delayed until three days after intrasplenic HIV-1 inoculation. Removal of NK cells from the human PBMCs prior to intrasplenic injection into NSG mice completely abrogated IL-15 superagonist-mediated suppression of in vivo HIV-1 infection. Thus, the in vivo activation of NK cells, integral mediators of the innate immune response, by treatment with an IL-15 superagonist increases their anti-HIV activity and enables them to potently suppress acute in vivo HIV-1 infection. These results indicate that in vivo activation of NK cells may represent a new immunotherapeutic approach to suppress acute HIV-1 infection.
Importance Epidemiological studies have indicated that NK cells contribute to the control of HIV-1-infection and in vitro studies have demonstrated that NK cells can selectively kill HIV-1-infected cells. We demonstrated that in vivo activation of NK cells by treatment with an IL-15 superagonist that potently stimulates the anti-tumor activity of NK cells markedly inhibited acute HIV-1 infection in humanized mice, even when activation of the NK cells by IL-15 superagonist treatment is delayed until 3 days after HIV-1 inoculation. NK cell-depletion from the PBMCs prior to their intrasplenic injection abrogated the suppression of in vivo HIV-1-infection observed in humanized mice treated with the IL-15 superagonist, demonstrating that the activated human NK cells were mediating IL-15 superagonist-induced inhibition of acute HIV-1 infection. Thus, in vivo immunostimulation of NK cells, a promising therapeutic approach for cancer therapy, may represent a new treatment modality for HIV-1-infected individuals particularly in the earliest stages of infection.
Intramuscular delivery of heterodimeric IL-15 DNA in macaques produces systemic levels of bioactive cytokine inducing proliferation of NK and T cells
Interleukin-15 (IL-15) supports the development, proliferation, survival and trafficking of several lymphocyte subsets, including NK, CD8+ and γσ T cells......
Gene Therapy (2015)
CBergamaschi1, VKulkarni1, MRosati2, CAlicea1, RJalah1, SChen2, JBear1, N YSardesai3, AValentin2, B KFelber1 and G NPavlakis2 1Human Retrovirus Pathogenesis Section, Vaccine Branch, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD, USA2Human Retrovirus Section, Vaccine Branch, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD, USA3Inovio Pharmaceuticals, Plymouth Meeting, PA, USA
Interleukin-15 (IL-15) is a common γ-chain cytokine that has a significant role in the activation and proliferation of T and NK cells and holds great potential in fighting infection and cancer. We have previously shown that bioactive IL-15 in vivo comprises a complex of the IL-15 chain with the soluble or cell-associated IL-15 receptor alpha (IL-15Rα) chain, which together form the IL-15 heterodimer. We have generated DNA vectors expressing the heterodimeric IL-15 by optimizing mRNA expression and protein trafficking. Repeated administration of these DNA plasmids by intramuscular injection followed by in vivo electroporation in rhesus macaques resulted in sustained high levels of IL-15 in plasma, with no significant toxicity. Administration of DNAs expressing heterodimeric IL-15 also resulted in an increased frequency of NK and T cells undergoing proliferation in peripheral blood. Heterodimeric IL-15 led to preferential expansion of CD8+NK cells, all memory CD8+ T-cell subsets and effector memory CD4+ T cells. Expression of heterodimeric IL-15 by DNA delivery to the muscle is an efficient procedure to obtain high systemic levels of bioactive cytokine, without the toxicity linked to the high transient cytokine peak associated with protein injection.
Cytokines of the γ-chain family have the potential to be used in different clinical settings, such as AIDS and cancer immunotherapy, due to their ability to regulate the homeostasis of the immune system and to boost host responses against pathogens and tumor antigens. Interleukin-15 (IL-15) supports the development, proliferation, survival and trafficking of several lymphocyte subsets, including NK, CD8+ and γσ T cells. It has also a non-redundant role in the establishment and maintenance of CD8+ T-cell memory.1, 2, 3, 4, 5 The efficient secretion of IL-15 requires co-expression of the IL-15-binding protein named IL-15 receptor alpha (IL-15Rα) in the same cell.6, 7, 8 Co-production of the two molecules leads to intracellular association of IL-15 and IL-15Rα in the endoplasmic reticulum, stabilization of both molecules and efficient transport to the cell surface.9, 10, 11 This complex anchored to the cell surface is trans-presented to cells expressing the β/γ subunits comprising the low-affinity IL-2 receptor and IL-15 receptor,12 or is secreted in a soluble form upon cleavage of the transmembrane domain of the IL-15Rα.13 Indeed, the circulating form of IL-15 in biological fluids is in complex with soluble IL-15Rα (sIL-15Rα) in both mice and humans.14 Several studies in mice showed that the soluble heterodimeric IL-15:sIL-15Rα (hetIL-15) has superior pharmacokinetics and a 10- to 100-fold increase in agonistic activity over single-chain IL-15 in vivo.10,13,15, 16, 17, 18
For therapeutic purposes, hetIL-15 can be provided in vivo as recombinant protein or by gene delivery. Systemic delivery of protein may cause significant toxicity, as it has also been reported for single-chain IL-15 (refs 19, 20) and other cytokines, such as IL-2 (refs. 21, 22) or IL-12.23 Toxicity may be reduced or eliminated through the delivery of cytokine genes, which are expressed for short periods of time. Among gene therapy approaches, the use of naked DNA is promising because of its simplicity, flexibility and possibility of repeated applications owing to the absence of immunity against the vector (for review see Ferraro et al.24). Successful gene therapy depends largely on the optimization of gene expression and on the method of delivery (for recent reviews see Kutzler and Weiner,25 Felber et al.26). We have previously described methods to develop efficient expression vectors for IL-15, combining mRNA optimization (RNA/codon optimization) of the IL-15-coding sequences and substitution of the signal peptide with other efficient secretory signals to optimize trafficking of the molecules.27 We also produced vectors expressing IL-15 in combination with either membrane-associated or soluble IL-15Rα, which resulted in stable, secreted heterodimeric IL-15:sIL-15Rα, with a final improvement of more than 1000-fold in the systemic level of bioactive IL-15 compared with vectors expressing the wild-type IL-15 cDNA.10 Substantial improvement in gene delivery using plasmid DNAs has been achieved by intramuscular injection followed by in vivo electroporation (EP).28,29,30 In vivo EP of naked DNAs results in increased DNA uptake and in enhanced gene expression by the cells at the injection site. Intramuscular injection followed by in vivo EP (IM/EP) has been widely used as delivery method to improve the expression and immunogenicity of human/simian immunodeficiency virus (HIV/SIV) DNA vaccines in macaques31, 32, 33, 34, 35 and humans.36, 37, 38 In addition, DNA EP was used to deliver cytokine genes, including IL-12 and IL-15, as vaccine adjuvants in preventive and therapeutic SIV DNA immunization in macaques.34,39, 40, 41, 42 Cytokine gene delivery by in vivo EP has also been successfully employed as cancer treatment in several preclinical and clinical studies. Intratumoral delivery of single-chain IL-15-expressing DNA by EP resulted in the complete regression of established B16 melanoma tumors in mice.43 In melanoma patients, the delivery of the IL-12-expressing DNA by EP has been shown to be safe, with lower toxicity in comparison to the systemic delivery of the recombinant protein.44
In the present work, optimized DNA vectors encoding human heterodimeric IL-15 were delivered in rhesus macaques by the IM/EP method. Elevated levels of IL-15 were detected in the plasma and were associated with an increased proliferation of NK and T cells, with no adverse effects. These results demonstrate that intramuscular administration of optimized IL-15 vectors in non-human primates results in systemic bioactive levels of heterodimeric IL-15, suggesting possible applications in vaccination regimens and immunotherapy protocols.
Several investigators have tested the effect of single-chain IL-15, provided as recombinant protein, in non-human primates.19,47,53,54 Among these studies, recombinant Escherichia coli-derived single-chain human IL-15 has been tested in preclinical studies in macaques upon intravenous injection,53 subcutaneous injection and continuous intravenous infusion.47 The subcutaneous and continuous intravenous protocols are currently being evaluated in phase I clinical studies in patients with refractory metastatic melanoma and metastatic renal cell carcinoma (NCT01572493 and NCT01727076). Administration of single-chain IL-15 resulted in an increase in the absolute number and proliferation of peripheral NK and memory CD8+ T cells. Increased proliferation and migration to peripheral tissues of effector memory CD4+ T cells was also reported,51 although no significant effects on CD4+Foxp3+ T-regulatory cells were observed.19,53,54 It has been shown that high-dose IL-15 is able to expand Tregs in vitro, although these cells were characterized by weak suppressive activity.55,56 Single-chain IL-15 delivery to macaques did not result in a preferential proliferation of Tregs as compared with the general CD4+ T-cell population.19 Although administration of the single-chain IL-15 was overall well tolerated, transient neutropenia and hypotension were observed,19,20 effects possibly related to the spike levels of IL-15 in serum.
Several reports suggested that gene therapy approaches could reduce or eliminate the toxicity associated with cytokine treatment. However, the major limitation of these preclinical and clinical studies was the undocumented efficiency of gene expression. This report explores the delivery, efficiency and safety of heterodimeric IL-15:sIL-15Rα gene therapy in rhesus macaques. Although single-chain IL-15 was shown to be bioactive in non-human primates,19,53 the use of heterodimeric IL-15 may provide additional advantages, as the heterodimeric IL-15:sIL-15Rα is a natural IL-15 form circulating in the blood,14 and is bioactive and more stable in vivo compared with monomeric IL-15.10,13 The physiological IL-15 plasma levels in rhesus macaques (~10–20 pg ml−1) were elevated 5- to 10-fold upon repeated delivery of DNA encoding heterodimeric IL-15:sIL-15Rα without any sign of toxicity. Elevated IL-15 levels were observed for more than 10 days, which suggests continuous production of IL-15 from the injected DNA vectors. Importantly, IL-15 delivered as DNA did not induce the high systemic cytokine spikes, likely responsible for the toxicity associated with that treatment, commonly observed after administration of recombinant proteins. Muscle is a physiologic location for IL-15 production57 and the IL-15 gene therapy did not cause any adverse effects in macaques. The achieved systemic levels of IL-15 were biologically relevant and had the expected effects on target populations. Heterodimeric IL-15 induced the proliferation of NK, γσ T cells, all memory CD8+ T cells, including stem cells memory, and effector memory CD4+ T cells in peripheral blood. Higher proliferation resulted in increased blood NK absolute cell counts. These results demonstrate that delivery of IL-15 as DNA or as recombinant protein induces similar effects in macaques. Interestingly, the systemic IL-15 peak levels achieved by DNA injections were similar to the ones obtained in macaques treated via the subcutaneous route with 1 μg kg−1 of purified human IL-15 heterodimer (unpublished data), but persisted for a longer period of time, indicative of continuous production of IL-15 from the injected DNA vectors in vivo. Therefore, heterodimeric IL-15 gene therapy using naked DNA delivered by IM/EP offers a versatile and effective way to obtain systemic-acting level of IL-15, without toxicity.
The use of SIV-infected macaques has also allowed us to evaluate the effects of IL-15 gene therapy on viral replication during chronic infection, in the absence of antiretroviral treatment. It has been reported that IL-15 administration during acute SIV infection resulted in increased viral set point and accelerated disease progression.58,59 However, other reports suggested that IL-15 treatment during chronic infection does not contribute to viral replication.42,46,51 In agreement with these findings, no differences were observed in the virological status of the animals before and after IL-15 DNA treatment, suggesting that IL-15 gene delivery is safe in the context of chronic HIV/SIV infection. In addition, heterodimeric IL-15 was able to promote the expansion of T cells, including effector memory CD4+ in infected animals.
In the present report, we have also compared the bioactivity of two different forms of heterodimeric IL-15. Heterodimeric IL-15:sIL-15RαFc has been shown to be superior to single-chain IL-15 in anti-cancer activity in several mouse studies.15,17,18,60,61 In macaques, both forms of the heterodimeric IL-15 expressed after DNA injection resulted in similar plasma levels and similar bioactivity. This is in contrast to the reported mouse data, where the IL-15:sIL-15RαFc form was characterized by increased bioactivity in comparison to the natural IL-15:sIL-15Rα as the result of its increased plasma half-life.15 The reason for this discrepancy could be either impaired entry into the blood stream of human IL-15:sIL-15RαFc from the site of production or, most likely, the high binding affinity of human IgG to the murine neonatal Fc receptor form.62,63 These results suggest important differences in the properties and biological activity of the two IL-15 heterodimers observed in mice and macaques.
In conclusion, improvements in vector design and delivery methods indicate that it is feasible to use IL-15 heterodimer-encoding DNA to produce systemic levels of bioactive cytokine, with minimal toxicity. This study provides a strong rationale for the evaluation of IL-15 heterodimer gene therapy for clinical applications, as an alternative to the recombinant protein administration.

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