Peter D. Katsikis, MD, PhD - US grants

[unreadable] DESCRIPTION (provided by applicant): Although cytotoxic CD8+ T cells (CTL) play an important protective role during HIV infection, a number of studies have suggested that these cells may suffer from functional defects that impair their efficiency in controlling HIV virus. Studies from our laboratory have revealed that HIV-specific CD8+ T cells exhibit increased susceptibility to CD95/Fas-induced apoptosis and that HIV-infected macrophages can kill HIV- specific CD8+ T cells by a CD95/Fas-dependent mechanism. In addition, HIV-specific CD8+ T cells lack terminal differentiation and CD95/Fas apoptosis may be involved in this skewing of differentiation. The ability of HIV-specific CD8+ T cells therefore to kill infected cells and their differentiation may be compromised due to CD95/Fas-mediated apoptosis of these cells. Recently, we have found a decrease in the anti-apoptotic molecules Bcl-2 and Bcl-xL in HIV-specific CD8+ T cells, and this may account at least partially for their increased apoptosis sensitivity. Interleukin 15 (IL-15) we found can inhibit CD95/Fas- mediated apoptosis of HIV-specific CD8+ T cells and can restore the levels of Bcl-2 and Bcl-xL in these cells. Based on our findings we hypothesize that an imbalance of pro- and anti-apoptotic factors combined with increased mitochondria! mass and a pro-apoptotic CD95/Fas signaling pathway result in the apoptosis sensitivity of HIV-specific CD8+ T cells. We hypothesize that IL-15 inhibits CD95/Fas-mediated apoptosis of HIV-specific CD8+ T cells by acting at a very early step of CD95/Fas death receptor signaling. In the current proposal we will investigate the molecular mechanism(s) involved in the increased apoptosis sensitivity of HIV-specific CD8+ T cells and how IL-15 inhibits this apoptosis. Understanding the mechanism behind this defect of HIV-specific CD8+ T cells may allow for the restoration of survival or function of HIV- specific CD8+ T cells which in turn would have a profound effect in controlling or clearing HIV. Finally, such an understanding will be critical for the development of vaccines that provide long lasting CTL immunity. [unreadable] Lay language description: These studies will investigate why immune cells that can kill HIV infected cells are sensitive to dying and ways to increase the survival and function of these cells. These killer cells play a critical role in controlling HIV infection therefore increasing their survival would benefit patients with HIV infection. These studies may lead to new treatments for HIV infection a serious public health problem. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant):HIV-specific cytotoxic CD8+ T cell responses play an important protective role against HIV infection. A number of studies, however, have indicated that HIV-specific CDS+ T cells may be functionally impaired. In addition, we recently have shown that HIV-specific CDS+ T cells exhibit increased susceptibility to undergo CD95/Fas-mediated apoptosis. This is of particular importance as HIV upregulates FasL on infected cells, while at the same time inhibits CD95/Fas-mediated apoptosis of infected cells. Indeed we have recently shown that HIV-infected macrophages can induce apoptosis of HIV-specific CDS+ T cells and this is largely due to CD95/Fas-mediated apoptosis. Furthermore HIV-specific CDS+ T cells lack the terminally differentiated CD45RA+CD62L- effector memory phenotype and this may affect the function of these cells in vivo. We recently have found evidence that CD95/Fas-mediated apoptosis may be responsible for this lack of differentiation. Thus HIV, by manipulating CD95/Fas-induced apoptosis, may be setting up an intricate CTL evasion and counterattack mechanism. Enhancing effector function, differentiation and/or survival of HIV-specific CDS+ T cells would enhance the efficiency of HIV-specific CD8+ T cells to control or clear HIV virus. Interleukin 15 (IL-15) is a pluripotent cytokine that expands in vivo memory CD8+ T cells, enhances their survival, and inhibits CD95/Fas-induced apoptosis. To date, no in depth investigation has been carried out on the effect of IL-15 on HIV-specific CD8+ T cells. We hypothesize that IL-15 treatment can enhance cytotoxic CDS+ T cells immunity against HIV This hypothesis is based on the known effects of lL-15 on mouse memory CDS+ T cells, and our own preliminary data that show that IL-15 can inhibit apoptosis and enhance effector function of HIV-specific CDS+ T cells. We propose to investigate the in vitro effect of IL-15 on effector function, differentiation, proliferation, and apoptosis of HIV-specific CDS+ T cells. We will also investigate the potential mechanism of action of IL-15 by examining the ASK1 signaling pathway and also examining gene expression using gene arrays. The studies in this proposal are novel and will provide valuable information on potential approaches to enhance the CD8+ T cell response against HIV in infected individuals. Information yielded from these studies will fill a knowledge gap as to the effect of IL-15 on anti-viral cytotoxic CDS+ T cell responses, and may prove useful for both novel therapeutic strategies and vaccine development for HIV infection and other viral infections.

DESCRIPTION (provided by applicant): Recently it has become apparent that innate immunity can regulate aspects of the adaptive immune response. The aim of this proposal is to determine how dendritic cells (DC), cells of innate immunity, regulate cytotoxic CD8+ T cell responses to pathogens. To date much has been elucidated in terms of the role DC play during the initiation phase of adaptive immune responses. In particular these very potent antigen-presenting cells are critical for the initiation of antiviral CD8+ T cells responses. Mature DC expressing appropriate co-stimulation molecules are required to present antigen in order to initiate the activation of naive CD8+ T cells. What is not known, however, is whether DC play a role in the later expansion and contraction phases of the CD8+ T cell response. We have preliminary data showing that a great influx of DC occurs in the lungs of influenza type A virus infected animals 2-5 days postinfection and that costimulation is required during the later phases of the virus-specific CD8+ T cell response. By using DTR-CD11c transgenic animals we plan to deplete DC during different phases of the CD8+ T cell response and thus start to understand what role DC are playing during the later phases of CD8+ T cell response. The requirement of costimulation molecules such as CD28 or CD27 will also be investigated to delineate the mechanism of action of DC at these later stages of the response. Finally, the role of DC and whether costimulation is required in secondary CD8+ T cell responses is largely unknown. To date the classical costimulation pathway through CD28 has been extensively studied during the initiation phase of the immune response and found to be required for the maximal anti-viral CD8+ T cell response to take place. Its involvement in secondary responses is however not known even though some memory CD8+ T cells are known to express CD28. We will investigate the role of DC and the contribution of CD28 and CD27 in the secondary response and the generation of memory. In order to understand what the role of these DC is, we will investigate what are the characteristics of these pulmonary DC, whether they are infected, whether they present antigen and what cytokines they make. Finally, the mechanism that controls the migration of DC into the lungs will be investigated. The studies proposed here are novel as no previous study has examined how DC regulate expansion, contraction and the generation of memory during the later phases of the cytotoxic CD8+ T cell response. Elucidating the DC and costimulation requirements of cytotoxic CD8+ T cell responses may ultimately lead to the development of novel therapeutic and vaccine strategies against tumors and viral infections.

Cytotoxic CD8+ T cell (CTL) responses play a critical protective role against HIV virus. A number of studies, however, have indicated that HIV-specific CD8+ T cells may be functionally impaired. Furthermore, we have demonstrated that HIV-specific CD8+ T cells exhibit increased susceptibility to undergo CD95/Fas-mediated apoptosis and HIV-infected macrophages can kill these cells. This apoptosis of HIV-specific CD8+ T cells therefore may impair their ability to function as serial killers. Augmenting the effector function and the survival of HIV-specific CD8+ T cells would greatly enhance the efficiency of these cells to control or clear HIV virus. Interleukin 15 (IL-15) is a pluripotent cytokine that we have shown potently inhibits CD95/Fas-mediated apoptosis of HIV-specific CD8+ T cells and upregulates the anti-apoptotic Bcl-2 and Bcl-xL molecules in these cells. Furthermore IL-15 enhances their cytotoxic activity and IFNgamma production. SIV-specific CD8+ T cells from SIV-infected rhesus macaques also show increased sensitivity to CD95/Fas-mediated apoptosis and IL-15 potently inhibits this apoptosis and upregulates Bcl-2 and Bcl-xL molecules. Based on these observations, we recently performed a short pilot study in chronically SIV-infected cynomolgus macaques to investigate the potentially beneficial effect of IL-15 treatment in vivo. In vivo treatment with IL-15 significantly increased peripheral blood CD8-t- T cell and NK cell numbers by more than 2-fold. This increase was mainly due to proliferation of CD8+ T cells, as proliferating Ki67^ CD8+ T cells increased with treatment. The expanded CD8+ T cells were of the CD45RA- CD62L- and CD45RA+ CD62L- effector memory phenotype. We hypothesize that IL-15 treatment in vivo can enhance cytotoxic CD8+ T cells immunity against SIV and enhance antiviral immunity. We propose to examine the effect of in vivo treatment with recombinant simian IL-15 on primary and chronic SIV infection of rhesus macaques. Synergy with ART treatment will also be evaluated in chronic infection studies. Viral load, immunological changes, apoptosis and Bcl-2/Bcl-xL molecule expression will be evaluated. In particular the effect of in vivo IL-15 treatment on SIV-specific CTL response will be investigated. The studies in this proposal are novel and will provide valuable information on the potential therapeutic value of IL-15. Information yielded from these studies may prove useful for the design of novel therapeutic strategies against HIV infection and other viral infections.

Cytotoxic CD8+ T cell (CTL) responses play a critical protective role against HIV virus. A number of studies, however, have indicated that HIV-specific CD8+ T cells may be functionally impaired. Furthermore, we have demonstrated that HIV-specific CD8+ T cells exhibit increased susceptibility to undergo CD95/Fas-mediated apoptosis and HIV-infected macrophages can kill these cells. This apoptosis of HIV-specific CD8+ T cells therefore may impair their ability to function as serial killers. Augmenting the effector function and the survival of HIV-specific CD8+ T cells would greatly enhance the efficiency of these cells to control or clear HIV virus. Interleukin 15 (IL-15) is a pluripotent cytokine that we have shown potently inhibits CD95/Fas-mediated apoptosis of HIV-specific CD8+ T cells and upregulates the anti-apoptotic Bcl-2 and Bcl-xL molecules in these cells. Furthermore IL-15 enhances their cytotoxic activity and IFNgamma production. SIV-specific CD8+ T cells from SIV-infected rhesus macaques also show increased sensitivity to CD95/Fas-mediated apoptosis and IL-15 potently inhibits this apoptosis and upregulates Bcl-2 and Bcl-xL molecules. Based on these observations, we recently performed a short pilot study in chronically SIV-infected cynomolgus macaques to investigate the potentially beneficial effect of IL-15 treatment in vivo. In vivo treatment with IL-15 significantly increased peripheral blood CD8-t- T cell and NK cell numbers by more than 2-fold. This increase was mainly due to proliferation of CD8+ T cells, as proliferating Ki67^ CD8+ T cells increased with treatment. The expanded CD8+ T cells were of the CD45RA- CD62L- and CD45RA+ CD62L- effector memory phenotype. We hypothesize that IL-15 treatment in vivo can enhance cytotoxic CD8+ T cells immunity against SIV and enhance antiviral immunity. We propose to examine the effect of in vivo treatment with recombinant simian IL-15 on primary and chronic SIV infection of rhesus macaques. Synergy with ART treatment will also be evaluated in chronic infection studies. Viral load, immunological changes, apoptosis and Bcl-2/Bcl-xL molecule expression will be evaluated. In particular the effect of in vivo IL-15 treatment on SIV-specific CTL response will be investigated. The studies in this proposal are novel and will provide valuable information on the potential therapeutic value of IL-15. Information yielded from these studies may prove useful for the design of novel therapeutic strategies against HIV infection and other viral infections.

DESCRIPTION (provided by applicant): Developing interventions that inhibit the transmission of HIV infection are critical for halting the HIV epidemic. Topical prevention strategies usually termed microbicides have been proposed as one strategy to halt or slow down the HIV epidemic. We have identified novel lead microbicides that potently inhibit HIV and SIV infection/replication in vitro. During our previous submission we reported an oligonucleotide with a phosphorothioate backbone (OPB) that could inhibit HIVBaL or SIVmac251 infection and/or replication in human or simian PBMC, respectively. OPB also inhibited infection/replication in cell-free infections of P4-R5 MAGI cells by HIVBaL and HIVIIIB. OPB exhibited no toxicity against PBMC or P4-R5 MAGI cells after 24h continuous exposure. Preliminary data suggested that OPB may also inhibit other viruses as it was also effective against influenza type A virus. Thus, our first generation OPB may be a potent microbicide against HIV that prevents infection at mucosal sites when topically applied. Our preliminary studies were carried out with a 13mer Poly T or Poly A oligonucleotide of OPB and this suggested that the effect was sequence independent and may even be mediated by the phosphorothioate deoxyribose sugar backbone. Indeed in our current re-submission we present data on our next generation compound, a baseless phosphorothioate 2'deoxyribose backbone (PDB) that has more potent HIV inhibitory activity than OPB. A 14mer PDB we show here has no toxicity, is a potent inhibitor of HIV and has the advantage of being a TLR7/9 antagonist that inhibits HIV-induced IFN[unreadable] production. This later property is important as the establishment of HIV infection may depend on HIV-induced mucosal inflammation triggered by TLR. Importantly, we show that PDB is active when formulated in hydroxyethylcellulose (HEC) gel at pH 4.4, survives pH transition to a neutral pH, and in retains its activity in HEC for long periods. We hypothesize that PDB binds enveloped viruses and inhibits their infectivity by acting as a "chemical lectin". We further hypothesize that PDB can act as a microbicide against HIV and can prevent SIV vaginal infection of rhesus macaques. The studies planned in the R21 phase will further optimize and characterize the safety and effectiveness of PDB in vitro and its safety in the Swiss Webster mouse vaginal/cervical model of irritation. They will determine the optimal size and composition that remains effective against HIV and exhibits no toxicity. Finally, the mechanism of action of PDB will be investigated, the effect of inclusion into hydroxyethylcellulose gel will be tested and PDB's effect on the growth of commensal lactobacilli will be determined. Five specific milestones have been set for the progression from the R21 Phase to the R33 Phase. The R33 phase will test the effectiveness of PDB in preventing vaginal SIV infection, investigate the effect of seminal plasma and pH transition on the efficacy of OPB, determine its safety with human genital epithelial tissue, and investigate its effectiveness against HSV-2. The current application will allow for an extensive evaluation of PDB as possible novel microbicide candidates. The studies proposed here address the important public health problem of developing treatments that inhibit the transmission of HIV infection. The current application investigates a novel chemical that may be used to inhibit infection with HIV.