Guido Silvestri - US grants

[unreadable] DESCRIPTION (provided by applicant): The pathogenesis of AIDS likely involves both the direct killing of infected CD4+ T cells and the induction of functional defects and apoptosis within the pool of uninfected lymphocytes. A consistent abnormality of lymphocytes from HIV-infected patients is a perturbation of the normal cell cycle control, which we have termed "cell cycle disease" (CCD), that consists of (i) increased activation of the cyclin B/p34-cdc2 complex, and (ii) abnormal nucleolar structure with deregulation of nucleolin turnover. Since these abnormalities of CCD are exacerbated in vitro by mitogens and are associated with induction of apoptosis, we have proposed that the cell cycle dysregulation represents a link between increased activation/turnover and high levels of apoptosis of uninfected T lymphocytes observed in HIV-infected patients. Our preliminary data indicate that CCD can be corrected in vitro by treatment with IL-2. In addition, we have found that CCD is most prominent in vivo in the subset of patients who manifest persistent lymphocyte depletion despite HAART-induced suppression of viral replication. These observations suggest that analysis of the cell cycle perturbations might be able to help identify patients that could benefit from alternative, immune-based interventions, such as IL-2 and others, in addition to standard HAART, The overall goal of this project is to test the hypothesis that the CCD is a significant contributor to the pathogenesis of AIDS. We will perform: (i) a detailed cross-sectional study of HIV-infected individuals to study the relationship between cell cycle abnormalities and the main markers of HIV-disease progression, including the response to HAART; (ii) a study of CCD in non-human primates with both pathogenic (rhesus macaques) and non-pathogenic (sooty mangabeys) SIV-infection; and (iii) a longitudinal analysis of CCD inpatients treated with IL-2 plus HAART or HAART alone, to test the hypothesis that IL-2 therapy recapitulates in vivo the beneficial effects of in vitro IL-2 administration on the cell cycle abnormalities. These analyses could provide relevant information on the role played by the loss of cell cycle regulation in the pathogenesis of AIDS, and could help defining the rationale for additional, immune-based interventions in a subset of HAART-treated HIV-infected patients. [unreadable] [unreadable]

PROJECT 3 DESCRIPTION: The generation of an effective AIDS vaccine is complicated by our limited knowledge of the correlates of immune protection during HIV and SIV infection. Here we propose to study the cellular immune responses generated by different immunization strategies that include MVA- and Adenovirus/MVA-based HIV and SIV vaccines, and to compare them with those generated during natural HIV and experimental SIV infection. An improved understanding of the determinants and generation of protective cellular immune responses against a model virus infection have emerged from studies of LCMV infection in mice. These studies elucidated the crucial distinction between central memory T cells (Tcm) and effector memory T cells (Tem), and described the predominant role of Tcm cells in protecting from re-challenge. Using these insights, in combination with emerging insights into functional defects of memory T cells following HIV infection, we seek to determine if responses elicited by vaccination result in a better ability to develop and maintain functional, durable Tcm-mediated responses to HlV and SlV antigens. The phenotypes and functions of the HIV-specific Tem and Tcm cell populations induced by different vaccination regimens will be defined, and compared to the memory T cell responses that arise following HIV and SIV infections as well as those that will be observed after SIV-challenge of previously vaccinated macaques. In this way, we will explore potential mechanisms by which vaccine-induced HIV- and SIV-specific memory T cell responses may be more effective in controlling virus replication than those following infection, and less likely to precipitate immunopathologic consequences. In all, these studies are aimed at defining markers that will predict vaccine effectiveness in inducing host responses that can contain virus replication, prolong disease-free survival, and decrease secondary transmission. Definition of the characteristics of such effective immune responses will ideally advance our understanding of the correlates of protection to be pursued in future efforts of AIDS vaccine development.

lymphocyte proliferation; homeostasis; immunoregulation; T lymphocyte; Primates; animal colony;

DESCRIPTION (provided by applicant): Understanding the reasons why SIV-infected sooty mangabeys (SMs) remain healthy despite high viremia is a key unanswered question in contemporary AIDS research, with important ramifications in terms of HIV pathogenesis, therapy, and vaccines. In recent studies, we have sorted central memory CD4+ T cells (TCM) and effector-memory CD4+ T cells (CD4+ TEM) from SIV-infected SMs and rhesus macaques (RMs), and that, while CD4+ TEM were similarly infected in both species, CD4+ TCM of SMs show significantly (>1 log) fewer SIV-DNA copies in vivo than CD4+ TCM of RMs. Based on this result, we hypothesize that protection of CD4+ TCM from virus infection is a key mechanisms by which SIV-infected SMs avoid CD4+ T cell depletion, chronic immune activation, and progression to AIDS. To test this hypothesis we will expand upon this previous work and propose a series of studies that will clarify the main features of in vivo and in vitro SIV infection in CD4+ TCM of both SMs and RMs, and elucidate the mechanisms by which CD4+ TCM of SMs are protected from SIV infection. In the first Aim, we will conduct a systematic comparative analysis of in vivo SIV replication in different CD4+ T cell subsets of SIV-infected SMs and RMs. We will examine different tissues (blood, lymph nodes, mucosal tissues) and CD4+ T cell subsets (Th1, Th2, Th17, and Tregs), and establish correlations with the course of disease progression. In the second Aim, we will study the relationship between SIV replication in central memory CD4+ T cells of LNs and the presence of LN immunopathology, including loss of CD4+ T cells, immune activation, disruption of architecture, fibrosis, and follicular DC virus trapping. In the third Aim, we will assess how SIV replicates in vivo in CD4+ TCM and CD4+ TEM of SMs and RMs by (i) conducting a detailed analysis of the level of integrated proviral DNA, the frequency of SIV-infected cells and the number of RNA copies/cell by limiting dilution analysis, and by (ii) performing sequence analysis of the virus. In the fourth Aim, we will assess the intrinsic susceptibility to SIV infection by CD4+ TCM and CD4+ TEM of uninfected SMs and RMs by conducting experiments of in vitro infection, in which we will seek to identify what stage(s) of virus replication are blocked in CD4+ TCM of SMs. As part of this Aim, we will also study the expression of host restriction factors (i.e., TRIM-5a, APOBECs, and Tetherin) as well as the global transcriptional profile in CD4+ TCM and CD4+ TEM of SMs and RMs. We believe that these studies will advance significantly our understanding of how naturally SIV-infected SMs are resistant to AIDS despite high viremia. We envision that answering this question will provide clues to AIDS pathogenesis in humans that will have ultimately an impact on the prevention and treatment of HIV infection. PUBLIC HEALTH RELEVANCE: Despite a huge effort by the scientific community, there is still neither a cure nor an effective vaccine for AIDS. A major obstacle to achieve these goals is our incomplete understanding of how infection with the human immunodeficiency virus (HIV) causes AIDS. In previous work, others and we have shown that certain African monkeys, such as sooty mangabeys, do not progress to AIDS despite being infected with a virus, the Simian Immunodeficiency Virus (SIV) that is closely related to HIV. The proposed studies are aimed at understanding why the sooty mangabeys are able to remain healthy when infected with SIV. We believe that these studies will improve our comprehension of AIDS pathogenesis in humans and that this knowledge will ultimately translate in better prevention and therapies for the infection.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This study is focused on comparative studies of the defects in the hematologic and immunological function of the bone marrow as well as other immunological sites during pathogenic and non-pathogenic Simian Immunodeficiency Virus (SIV) infections of rhesus macaques (RMs) and sooty mangabeys (SMs), respectively. As part of this study, and to elucidate the mechanisms underlying the benign phenotype of SIV infection of SMs, we have used high-density oligonucleotide microarrays to longitudinally assess host gene expression in SIV-infected SMs and RMs. We found that acute SIV infection of SMs was consistently associated with a robust innate immune response, including widespread upregulation of IFN-stimulated genes (ISGs). While SMs exhibited a rapid resolution of ISG expression and immune activation, both responses were observed chronically in RMs. Systems biology analysis indicated that expression of LAG3, a marker of T cell exhaustion, correlated with immune activation in SIV-infected RMs but not SMs. Our findings suggest that active immune regulatory mechanisms, rather than intrinsically attenuated innate immune responses, underlie the low levels of immune activation characteristic of SMs chronically infected with SIV. We are hopeful that this study will provide information useful in treating the chronic aberrant immune activation of HIV-infected humans.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. This project focuses on understanding the reasons why natural hosts for SIV infection, such as the sooty mangabeys (SMs), in striking contrast to HIV-infected individuals, are not susceptible to AIDS despite high viremia. We recently showed that CCR5 expression is significantly lower in central memory CD4+ T cells of SMs when compared to humans and macaques, and that this lower expression results in decreased susceptibility to SIV infection. This work is now submitted for publication. In addition, as part of the studies sponsored by R01-AI66998, we have made three novel observation that are all now published (see below). The first observation is that the conclusion of a five-years longitudinal survey of naturally SIV-infected SMs prompted us to realize that the infection is associated with a slow but progressive decline of CD4+ T cells without increasing viral load or immune activation. The second observation is that 5-7% of naturally SIV-infected SMs are homozygous for a 2bp deletion in the second extracellular loop of CCR5 that results in abrogated surface expression of thsi molecule. Interestingly, these rare CCR5 knock-out SMs are infected with SIV albeit at lower levels. These animals do not develop viruses using CXCR4, but other SIV co-receptors such as GPR-1 and GPR-15. The third observation is that, in naturally SIV-infected SMs, the level of autologous neutralizing antibodies is very low, and does not correlate with either viral load or CD4+ T cell loss. We believe that these data improve our understanding of the pathophysiology of SIV infection in SMs.

AIDS; AIDS Vaccines; Acquired Immune Deficiency; Acquired Immune Deficiency Syndrome; Acquired Immuno-Deficiency Syndrome; Acquired Immunodeficiency Syndrome; Animals; CRISP; Computer Retrieval of Information on Scientific Projects Database; Disease Progression; Funding; Genetic; Grant; Immune response; Immunologic Deficiency Syndrome, Acquired; Institution; Investigators; Macaca mulatta; Modification; Modified Vaccinia Ankara; Modified Vaccinia Virus Ankara; NIH; National Institutes of Health; National Institutes of Health (U.S.); Protocol; Protocols documentation; Research; Research Personnel; Research Resources; Researchers; Resources; Rhesus; Rhesus Macaque; Rhesus Monkey; SIV; Safety; Series; Simian Immunodeficiency Viruses; Source; Testing; United States National Institutes of Health; Vaccination; base; host response; immunogenicity; immunoresponse; vector

In contrast to HIV-1 infection of humans and SIV infection of rhesus macaques (RMs), which are almost invariably associated with AIDS, natural lentiviral infections of African primates, such as SIVsmm infection of sooty mangabeys (SM), are typically non-pathogenic. The mechanisms underlying this non-pathogenic phenotype remain largely unknown;however, we have previously shown that a key feature of nonpathogenic SIV infection is the absence of chronic, generalized immune activation in the context of low cellular immune responses to the virus. As the HIV-associated immune activation (IA) is thought to be a key factor in the pathogenesis of AIDS, we proposed that the lack of IA protects SIV-infected SMs from disease progression. In further studies, we observed that SM-derived plasmacytoid dendritic cells (pDCs) produce significantly lower levels of type 1 interferon (IFNs) in response to toll-like receptor (TLR)-7 or -9 stimulation than human or RM-derived pDCs. These findings led us to hypothesize that a blunted type 1 IFN responses to TLR-7/9 stimulation mediated by SIV or its products protects SIV-infected SMs from developing the generalized immune activation associated with pathogenic HIV/SIV infections. The aim of this project is to directly test this hypothesis by treating naturally SIV-infected SMs with IFN-a and TLR-7/-9 stimulation, as well as treating SIV-infected RMs with anti-IFN-a antibody and TLR-7/-9 inhibitors. These interventions will allow us to determine whether and to what extent differences in the level of pDCs responsiveness to TLR-7/-9 stimulation and/or IFN-a production between RMs and SMs are responsible for the striking differences in the level of immune activation observed in these two species after SIV infection. The results of these studies will improve our understanding of the mechanisms underlying the lack of disease in SIV-infected SMs and determine whether targeting TLR-7/9 activation and/or IFN-a production are promising targets for therapeutic interventions aimed at reducing the HIV-associated chronic immune activation.

DESCRIPTION (provided by applicant): Several observations indicate that CD8+ T cells are important in controlling virus replication during chronic HIV and SIV infection. Paramount among these observations is that experimental in vivo depletion of CD8+ lymphocytes in SIV-infected rhesus macaques (RM) is followed by a dramatic increase in virus replication. However, the mechanisms by which CD8+ T cells mediate this antiviral effect are still poorly understood, as emphasized by the negative result of the Merck Adenovirus-based, cytotoxic T lymphocyte (CTL)-inducing candidate AIDS vaccine in a large phase IIb clinical trial. In a recent study (Klatt et al., PLoS Pathogens, in press), we sought to assess the mechanisms underlying the antiviral effect of CD8+ lymphocytes during chronic SIVmac239 infection of RMs by treating two groups of animals (i.e., CD8+ lymphocyte-depleted or controls) with antiretroviral therapy (ART). Using a well-accepted mathematical model to calculate the in vivo lifespan of productively infected cells, we found that, in both early and late SIV infection, depletion of CD8+ lymphocytes did not change the in vivo lifespan of infected cells. This result indicates that SIV suppression mediated by CD8+ lymphocytes goes above and beyond the direct killing of cells producing virus, and suggests that more studies are needed to fully understand the antiviral role of CD8+ T cells during SIV infection. Here we propose to conduct a series of experiments of in vivo CD8+ lymphocyte depletion aimed at better elucidating the antiviral effects of these cells during chronic SIV mac239 infection of RMs. In the first Aim, we will determine how CD8+ lymphocytes impact the average lifespan of in vivo infected cells in Mamu-B*08/17+ RMs that show better control of virus replication upon SIV mac239 infection. In the second Aim, we will determine how CD8+ lymphocyte depletion induces changes in the fraction, type, and location of SIV infected cells as well as the burst size of virus production. In the third Aim, we will assess how CD8+ lymphocyte depletion affects the production of soluble antiviral factors (i.e., cytokines and chemokine) and alters the intrinsic susceptibility of CD4+ T cells to SIV infection by changing their level of activation, proliferation, CCR5 expression, and intracellular levels of host restriction factors. We believe that these experiments will provide important insights into CD8+ lymphocyte inhibition of virus replication during SIV infection. Ultimately, we hope that this knowledge will favor the rational design of an effective CTL-based AIDS vaccine. Our current inability to design immunogens that induce broadly reactive neutralizing antibodies against the HIV-1 Envelope protein has shifted the focus of the AIDS vaccine development effort to the design of immunogens that induce high levels of HIV-specific CD8+ T cells. The experiments included in this proposal are aimed at elucidating the mechanisms by which CD8+ lymphocytes suppress virus replication in SIV-infected RMs. The results generated here will provide correlates of CD8+ lymphocyte-mediated protection to be used to rank and prioritize the development of candidate AIDS vaccines for use in humans.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The generation of an effective AIDS vaccine is greatly complicated by our incomplete knowledge of the correlates of immune protection during HIV infection. This project focuses on the immunogenicity and protection from SIV challenge conferred by chimpanzee adenovirus (AdC)-based candidate AIDS vaccines. Over the past year we concluded the analysis and published the results of an immunization and challenge study using an AdC6SIVgag vector boosted with AdC7-gDSIVgag (a vector expressing Gag as a fusion protein within the immunostimulatoryHSV-1gD molecule). In addition, we made significant progress on an additional large study of the safety, immunogenicity and protection from pathogenic SIV challenge of two combinations of the SIVgag/tat expressing AdC vectors, AdC6 and AdC7, that we used in a sequential heterologous prime-boost regimen in 30 RMs (i.e., AdC6-SIVgag/tat followed by AdC7-SIVgag/tat and AdC7-SIVgag/tat followed by AdC6-SIVgag/tat). We have completed the immunization phase, and we are now in the challenge phase of the experiment, which involves up to 15 low-dose intra-rectal challenges with SIVmac251 that were administered every two weeks with weekly monitoring of SIV viremia. Currently 28 out of 30 RMs have been infected, and thus we expect to complete the challenge phase within the next month. At that point the animals will be followed for an additional 6 months after infection to monitor their level of disease progression (i.e., set point viral load, CD4 depletion, survival). Ultimately we hope that this study will allow us to assess the efficacy in the SIV NHP model of gag/tat expressing AdC-based candidate AIDS vaccines

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The design of AIDS vaccines is complicated by the fact that virtually every HIV/SIV-specific adaptive immune response aimed at controlling the infection will generate activated HIV/SIV-specific CD4+ T-cells that may facilitate virus transmission or disease progression. This project is aimed at testing the hypothesis that the best correlate of protection from SIV challenge is the induction of robust and persistent SIV-specific mucosal CD8+ T-cell responses in the presence of low levels of mucosal activated CD4+CCR5+ T-cells. In this first experiment of this project, we are characterizing, in the macaque model of SIV vaccination and challenge, the immunogenicity and protection from low-dose intra-rectal challenge of vector combinations that include the most promising platforms available at this time (see table). Group-A: 6 RM Mamu-A*01+ AdHu5 AdHu5 AdHu5 Group-B: 6 RM Mamu-A*01+ DNA/EP AdC6 AdC7 Group-C: 6 RM Mamu-A*01+ Vaccinia AdC6 AdC7 Group-D: 6 RM Mamu-A*01+ DNA/EP Vaccinia AdC6 Group-E: 6 RM Mamu-A*01+ DNA/EP Vaccinia AdC7 Table-1: sequence of the vectors to be used in the experiments of Aim #1. At this time all animals included in this study have been identified, quarantined, and assigned to the study. The immunization phase of this study is currently in progress, and all RMs are assessed in terms of: (i) presence of systemic and mucosal CD8+ T cell responses to SIV;and (ii) levels of systemic and mucosal activated CD4+ T cells. We expect that these studies will advance our understanding of the correlates of immune protection at the level of mucosal tissues.

DESCRIPTION (provided by applicant): The goal of this R24 application is to generate whole transcriptome reference databases for several immune cell types at baseline and during HIV and SIV infection, with emphasis on the comparative models of pathogenic and non-pathogenic infections. We envision that these databases will serve as novel and valuable resources for studies of AIDS pathogenesis, prevention, and therapeutics. The project is an outgrowth of the revolutionary advances associated with next-generation sequencing, and leverages the complementary expertise of Dr. Katze in systems biology and bioinformatics and of Dr. Silvestri in AIDS research. In Aim 1, we will generate baseline reference transcriptomes for several key immune cell subsets (immunome) in four primate species (humans, rhesus macaques, RMs, sooty mangabeys, SMs, and African green monkeys, AGMs) that represent the most used models for AIDS research. We will sort cell types from each species, and we will isolate RNA that will be comprehensively sequenced by.the Katze lab. In Aim 2, we will generate reference transcriptomes for the same immune cells but in the context of acute SIV infection in the pathogenic RM and the non-pathogenic AGM models. In Aim 3, we will generate reference transcriptomes for the same immune cells but in the context of chronic HIV and SIV infection in humans, RMs, SMs, and AGMs. We believe that the potential impact of these resources is substantial. Resource applications include: (i) improvements to gene models for already sequenced species and assistance for genome assembly/annotation for new species (i.e., SMs and AGMs); (ii) development of tools (e.g., species-specific gene probes or microarrays) for AIDS-related systems biology research; (iii) investigation ofthe interaction between lentiviruses and the immune system during acute arid chronic infection. The obtained reference transcriptome information will be used to generate new hypotheses, design new experiments, and advance basic, translational, and clinical research in the fields of HIV prevention and therapy.

DESCRIPTION (provided by applicant): Despite many advances in AIDS research, including the availability of potent anti-retroviral therapy (ART) that effectively controls virus replicatio in a large proportion of HIV-infected patients, a treatment that can cure the infection remains elusive. To this end, new approaches are required to eradicate the reservoirs of latently infected cells that persist during ART and are the source of virus reactivation when therapy is interrupted. In the R21 phase of this grant application we propose to use the existing, well-established non-human primate model of SIVmac infection of rhesus macaques (RMs) to validate studies of HIV eradication/functional cure by developing an experimental system in which virus replication is fully and persistently suppressed in vivo by a potent ART regimen (Aim 1). We will then use this validated model to investigate directly in vivo and in multiple organs th anatomic and phenotypical nature of the persistent reservoirs of latently infected cells, with specific focus on the relationship between expression of co-inhibitory molecules (i.e. PD-1, CTLA-4, TIM-3, and LAG-3) and size of the persistent reservoirs (Aim 2). The results of the studies proposed in the R21 part of this application will pave the way for further experiments, to be conducted in the R33 phase of this proposal, in which we will test, in ART-treated SIV-infected RMs with full suppression of virus replication, immune-based interventions aimed at reducing and possibly eliminating in vivo the persisting reservoirs of latently infected cells. The key proposed intervention consists of a blockade of the co-inhibitory pathway most closely associated with SIV latency, which will be performed as a stand-alone therapy or in combination with a non-specific virus reactivating agent (i.e. the histone deacytelase inhibitor, SAHA). We believe that the proposed studies will provide unprecedented insights into the biology of persistent virus reservoirs of latently infected cells, and elucidate the potential of targeting co inhibitory pathways to reduce the reservoir during SIV infection.

? DESCRIPTION: Despite many major advances in AIDS research, including the development of anti-retroviral drugs that suppress virus replication and greatly reduce the mortality and morbidity of HIV infection, a treatment that can cure the infection is still not available. Indeed, combination antiretroviral therapy (ART) must be taken for life, thus posing significant challenges in terms of costs and clinical safety, and interruption of therapy results in a rapid rebound of viremia in the majority of HIV-infected individuals. To this end, new approaches are required to eradicate the reservoirs of latently infected cells that persist during ART and are the source of virus reactivation when therapy is interrupted. The overarching Aim of this proposal is to explore the therapeutic potential of type I interferon (IFN-I), that activates a very potent natural antiviral molecular system, in reducing the reservoirs of virus-infected cells that persist under ART. In the R21 phase of this grant application we propose to use the existing, well-established nonhuman primate model of SIVmac infection of rhesus macaques (RMs) to evaluate, in a relatively small pilot study, the potential impact of pegylated IFN-?2a (pIFN-?2a) on the overall size, anatomic location, and cellular distribution of the reservoirs of latently infected cells in ART-treated, SIV-infected RMs. We will use this very robust model to investigate directly in vivo and in multiple organs (i.e., blood, lymph nodes, spleen, mucosal tissues, etc.) and cell types (i.e., memory CD4+ T cell subsets and macrophages) whether and to what extent pIFN-?2a administration enhances the effect of ART on the virus reservoir. The results of the studies proposed in the R21 part of this application will pave the way for further experiments, to be conducted in the R33 phase of this proposal, in which we will test, in a larger cohort of SIV-infected RMs treated with long-term ART and exhibiting full suppression of virus replication, the effect of two consecutive cycles of pIFN-?2a treatment on (i) the size of the persisting reservoirs of latently infected cells, and (ii) the time of rebound of plasma viremia afer ART interruption. We believe that the proposed studies will provide unprecedented insights into the role of type I interferon in reducing and/or altering the cellular and anatomic distribution of the persistent virus reservoirs of latently infected cells in an in vivo model of pathogenic lentivral infection in which active virus replication is fully suppressed by ART. We believe that these results will be crucial to determine the potential of IFN-I therapy in HIV-infected individuals.

The design of an AIDS vaccine is complicated by the fact that any HIV/SIV-specific immune responses aimed at preventing transmission or disease progression inevitably result in the generation of activated CD4+ T cells that may paradoxically facilitate transmission and/or disease progression. Indeed, increased HIV acquisition in the ineffective Step/Phambili trials and the lack of efficacy in HVTN-505 indicate that vaccine-elicited CD4+ T-cell responses can mitigate protection and, in some cases, increase acquisition. The overarching goal of this Program Project grant is to test the hypothesis that durable and balanced HIV-specific humoral immune responses are needed to provide effective protection from mucosal challenge, and avoid paradoxical effects that may enhance virus acquisition. This project focuses on the question of whether vaccine-induced changes in the mucosal micro-environment are factors influencing, and often undermining, the protective efficacy of humoral immunity in rhesus macaques (RMs) and by extension, humans. Three main aspects of the mucosal environment will be examined: (1) the role of activated CD4+ T cells as potential targets for the virus; (2) the role of the innate immune response, and in particular type I interferon (IFN-I) and interferon-stimulated gene (ISG)- mediated pathways, as antiviral but also pro-inflammatory factors; and (3) the potential role of abortive infection and induction of pyroptotic cell death in quiescent mucosal CD4+ T cells as a source of inflammatory/activating signals facilitating spread of the transmitted founder virus populations. The studies proposed in in this project leverage animals and samples from Project 1 (which will elucidate the humoral determinants for persistent anti- gp120 responses) and Project 2 (in which the protection conferred by passive administration of an anti-gp120 antibody will be tested in the setting of concomitant exposure to a vector-based vaccine known to induce strong mucosal cellular immune responses to HIV/SIV). There are three specific aims. Aim 1: To examine how the in vivo interventions in Projects 1 and 2 affect the number, phenotype, activation state, histological location, and gene expression of mucosal CD4+ T cells. Aim 2: To examine how the in vivo interventions in Projects 1 and 2 affect the innate immune environment of mucosal tissues, with specific focus on IFN-I and ISGs. Aim 3: To determine whether abortive infection and inflammatory pyroptosis occur in mucosal CD4+ T cells of vaccinated macaques and promote expansion and dissemination of SHIV infection. This work will provide a comprehensive picture of the mucosal micro-environment in the macaque vaccine model, within the context of settings where protective anti-gp120 antibodies are present. This information will validate hypotheses regarding the causal relationships between balanced mucosal immune profiles and protective efficacy via humoral immunity. Such information will have significant translational impact on the development of future HIV vaccine strategies.

? DESCRIPTION (provided by applicant): The use of antiretroviral therapy (ART) typically results in reduction of plasma viral loads to below detectable levels in human immunodeficiency virus (HIV)-infected individuals. However, infection with HIV persists despite suppressive ART and treatment interruption results in rapid viral rebound. In absence of ART, CD8+ T cells have been shown to inhibit virus replication during HIV infection and depletion of CD8+ lymphocytes in vivo during simian immunodeficiency virus (SIV) infection of rhesus macaques (RMs) results in increased viral loads. The precise role of CD8+ lymphocytes in controlling virus replication and/or production during continuous, highly active ART is unknown. Understanding the mechanisms controlling HIV/SIV reservoir dynamics under ART, and particularly the role of host immune responses, is critical to design effective strategies to reduce the size of these reservoirs and promote HIV/SIV remission. In this proposal, we will build upon our preliminary data indicating that CD8+ lymphocytes act in concert with ART to maintain virus suppression. Using the highly relevant SIV/RM model, we will answer three important questions regarding the mechanism(s) of CD8+ lymphocyte-mediated virus suppression. First (Aim 1), we will determine if the antiviral effect of CD8+ lymphocytes is present in SIV-infected ART-treated RMs with prolonged suppression of viremia (as an extension of our preliminary results demonstrating this effect in the setting of short-term virus suppression). In this study, we will perform CD8 depletio in SIV-infected RMs treated with ART for at least one year to more closely mimic long-term ART-treated HIV-infected individuals with a stable virus reservoir. Second (Aim 2), we will determine if the observed antiviral effect of CD8+ lymphocytes under ART is mediated by CD8+ T cells vs. CD8+ NK cells. This critical experiment is made possible by a newly available monoclonal antibody (mAb) that targets cells expressing CD8? (i.e., CD8??+ T cells, but not CD8?+ NK cells) for depletion. Third (Aim 3), we will quantify the contribution of CD4+ T cell activation/proliferation to the increase in viremia that follows in vivo CD8 depletion. By using a neutralizing anti- IL-15 mAb together with CD8 depletion we can selectively block homeostatic CD4+ T cell activation and measure subsequent virologic outcomes. This work will allow us to understand how the host antiviral cellular immune response works in concert with ART to suppress SIV replication and/or production. These results will refine and clarify the evidence to explore cure-directed interventions, such as therapeutic vaccination and checkpoint blockade inhibitors, aimed at boosting the virus-specific CD8+ lymphocyte response in ART-treated HIV-infected individuals.

The design of an AIDS vaccine is complicated by the fact that any HIV/SIV-specific immune responses aimed at preventing transmission or disease progression inevitably result in the generation of activated CD4+ T cells that may paradoxically facilitate transmission and/or disease progression. Indeed, increased HIV acquisition in the ineffective Step/Phambili trials and the lack of efficacy in HVTN-505 indicate that vaccine-elicited CD4+ T-cell responses can mitigate protection and, in some cases, increase acquisition. The overarching goal of this Program Project grant is to test the hypothesis that durable and balanced HIV-specific humoral immune responses are needed to provide effective protection from mucosal challenge, and avoid paradoxical effects that may enhance virus acquisition. This project focuses on the question of whether vaccine-induced changes in the mucosal micro-environment are factors influencing, and often undermining, the protective efficacy of humoral immunity in rhesus macaques (RMs) and by extension, humans. Three main aspects of the mucosal environment will be examined: (1) the role of activated CD4+ T cells as potential targets for the virus; (2) the role of the innate immune response, and in particular type I interferon (IFN-I) and interferon-stimulated gene (ISG)- mediated pathways, as antiviral but also pro-inflammatory factors; and (3) the potential role of abortive infection and induction of pyroptotic cell death in quiescent mucosal CD4+ T cells as a source of inflammatory/activating signals facilitating spread of the transmitted founder virus populations. The studies proposed in in this project leverage animals and samples from Project 1 (which will elucidate the humoral determinants for persistent anti- gp120 responses) and Project 2 (in which the protection conferred by passive administration of an anti-gp120 antibody will be tested in the setting of concomitant exposure to a vector-based vaccine known to induce strong mucosal cellular immune responses to HIV/SIV). There are three specific aims. Aim 1: To examine how the in vivo interventions in Projects 1 and 2 affect the number, phenotype, activation state, histological location, and gene expression of mucosal CD4+ T cells. Aim 2: To examine how the in vivo interventions in Projects 1 and 2 affect the innate immune environment of mucosal tissues, with specific focus on IFN-I and ISGs. Aim 3: To determine whether abortive infection and inflammatory pyroptosis occur in mucosal CD4+ T cells of vaccinated macaques and promote expansion and dissemination of SHIV infection. This work will provide a comprehensive picture of the mucosal micro-environment in the macaque vaccine model, within the context of settings where protective anti-gp120 antibodies are present. This information will validate hypotheses regarding the causal relationships between balanced mucosal immune profiles and protective efficacy via humoral immunity. Such information will have significant translational impact on the development of future HIV vaccine strategies.

Project Summary: Virology and Molecular Biomarkers Core (Core J) The Virology and Molecular Biomarkers Core of the Emory CFAR provides state-of-the-art viral quantification and characterization services in support of the extensive basic, clinical, and translational HIV/SIV research being conducted at Emory University. Core J has been extremely successful in its provision of these services because 1) it limits costs to researchers through an economy of scale, 2) it is effective at responding to the constantly evolving needs of HIV/SIV researchers, and 3) it serves as a resource for training and consultation in molecular virologic assays. This proposal provides a detailed blueprint for the Core's future service provision as well as for a Tier-2 funded expansion of services and expertise that will support the growing number of investigators focused on HIV cure research. Core J will be divided into three main laboratories. The Translational Virology (TV) laboratory develops and implements quantitative viral assays that support research using nonhuman primate models of AIDS pathogenesis, prevention, and therapy. Additionally, the Core's expertise in molecular viral diagnostics has led to the validation and implementation of several new assays in response to the needs of AIDS researchers at Yerkes, including notably, cell-associated HIV and SIV DNA assays with single copy sensitivity and viral amplicon deep sequencing. The primary focus of the Core Clinical Virology (CV) laboratory is to provide virologic and molecular diagnostic assays for clinical studies of HIV infection, STIs, and co-infections in a CLIA/CAP-certified environment, as well as microbiome sequencing. These highly utilized functions will be maintained in the next project period. During the next funding period, the Core will expand its scope and menu of services in order to meet the demands of HIV/SIV/AIDS researchers that require new tools to study the establishment, maintenance, and eradication of the persistent reservoir of latently infected cells during antiretroviral therapy. It will establish a third Core laboratory, the Viral Reservoir (VR) Laboratory, with the purpose of validating and implementing quantitative, cell culture-based assays of replication competent virus in HIV- and SIV-infected CD4+ T cells. In addition, Core J will expand its menu of tests utilizing its recently acquired Illumina MiSeq deep sequencing platform to provide virus and more detailed microbiome sequencing services in support of initiatives to characterize virus integration sites, viral evolution in response to new therapies, and the relationship between the genital/gut microbiomes and HIV transmission/pathogenesis. The new activities proposed herein will significantly impact the outcomes of a large number of new and ongoing research programs at Emory University by providing the state-of-the-art virological services that are crucial for characterizing the latent viral reservoir and evaluating novel HIV cure treatment strategies.

ABSTRACT ? Nonhuman Primate (NHP) Core (Core 1, Leader: Dr. Guido Silvestri, Yerkes National Primate Research Center, Emory University) The most appropriate animal model to define the origin, kinetics, and impact of humoral and cellular immune- based strategies on viral rebound in infants following postnatal HIV-1 infection and treatment is simian immunodeficiency virus (SIV)/simian-human immunodeficiency virus (SHIV) infection of rhesus macaques. Indeed, this model is particularly well-suited for our proposed studies because pediatric models of postnatal transmission have been developed and used to define the pathogenesis of infant HIV-1 infection and prevention strategies. However, NHP studies require extensive infrastructure and unique expertise. Thus, we the Program's Nonhuman Primate (NHP) Core (Core 1) will coordinate and implement all the NHP experiments proposed by the Program's two Projects: Project 1: ?Origin and predictors of viral rebound in infants?, (PI: Dr. Ann Chahroudi, Emory University), and Project 2: ?Impact of immune-based intervention on viral rebound in orally SHIV-infected infant monkeys? (PI: Dr. Genevieve Fouda, Duke University). The Core will support the Program through the following Specific Aims: Aim 1 - Organize, coordinate, and conduct studies of infant SHIV reservoir and rebound; Aim 2 - Administer and monitor antiretroviral treatment (ART) in orally SHIV-infected infant monkeys; and Aim 3 - Perform pharmacokinetic analysis of polyclonal antibody infusions in infant macaques to determine the dose and administration interval for treatment of infant SHIV infection. Led by Dr. Guido Silvestri, the NHP Core will ensure the following that are essential to the successful completion of these projects: 1) adherence to regulatory procedures relevant to research with NHPs; 2) coordination and implementation of all animal-related procedures, including housing, immunizations of infants, nursery-rearing of infants, antiretroviral treatments, and sample collections for weekly viral load measurement and monthly blood counts; 3) proper storage and/or shipment of samples to the Projects and Cores; and 4) maintenance of a database of clinical health parameters. Dr. Silvestri will ensure all NHP supported research efforts are tightly integrated and that evolving needs of the research projects will be met. As an integral component of this Program, the NHP Core will provide critical support to achieve the shared goals of understanding the source, kinetics, and impact of immune interventions on HIV-1 rebound in postnatally infected infants.  

Abstract The presence of a reservoir of cells harbouring integrated, replication-competent virus that persists under long- term, fully suppressive antiretroviral therapy (ART) and the inability of the host immune responses to control the initial events of viral replication that follow ART interruption are critical barriers to curing HIV infection. Thus, novel therapeutic strategies to remove these barriers are critically needed. The overarching hypotheses of ERASE HIV are: (i) decreased and/or dysfunctional CD8+ T and NK cell antiviral functions, combined with the recently-described CD8+ T-cell-mediated transcriptional silencing of HIV, favour HIV persistence under ART and prevent the control of viremia if ART is stopped; and (ii) novel approaches to elicit effective CD8+ T-cell, NK cell, and antibody-dependent cellular cytotoxicity (ADCC) functions while inhibiting the CD8+ T-cell-mediated virus silencing will promote remission and/or eradication of HIV. The overarching goal of ERASE HIV is to identify novel mechanisms of HIV persistence and to test them in the most relevant pre-clinical animal models through mechanistically-oriented, community-supported therapeutic strategies that can be ultimately translated to cure HIV infection in humans. ERASE HIV includes three highly integrated Research Foci (RFs). RF1 is aimed at identifying the molecular and cellular mechanisms underlying the two distinct antiviral activities of CD8+ T-cells: the MHC-restricted, Ag-specific response that directly eliminates virus-infected cells, and the non-MHC restricted, non-cytolytic silencing of HIV transcription. As such, RF1 will provide the conceptual basis for the interventions tested in RF2 and RF3. RF2 will use animal models of ART-treated HIV infection to (i) restore CD8+ T and NK cell function with a combined ?-IL-10 and IL-15 superagonist (N-803) strategy; (ii) target rebounding virus by using a CD4-mimetic compound (CD4mc) to enhance antibody recognition of cells expressing HIV Env and their elimination via ADCC; and (iii) determine if improving CD8 T and NK cell function via ?-IL-10 and N- 803 synergizes with CD4mc to clear infected cells. RF3 will determine if suppression of the latency-promoting activity of CD8+ T-cells, coupled with N-803 and interventions to promote apoptosis (Bcl-2 inhibitors) or immune- mediated removal (CD4mc) of cells that have reactivated virus, will reduce the reservoir size. In all, we will exploit the synergy between the mechanistic data generated in RF1 and the in vivo interventions in RF2 and RF3 to validate a strategy that targets both HIV persistence during ART and HIV recrudescence after ART interruption. ERASE HIV is supported by experts in HIV advocacy (SisterLove); recognition and killing of HIV Env-expressing cells (Finzi/Sodroski); T and NK cell biology (Sekaly/Ribeiro/Deleage/Parsons); reservoir assays and latency models (Kulpa/Jones/Litchterfeld/Howell); pre-clinical animal studies (Paiardini/Silvestri/Garcia/Saez- Cirion/Keele/Kumar); mathematical modelling (Davenport); and therapeutics development (Merck and ImmunityBio) for HIV cure. We believe that the proposed mechanistically-oriented pre-clinical work will inform strategies that can be translated in clinical trials to achieve prolonged viral remission in PWH.