Joseph B. Margolick - US grants

Phagocytosis-inducing factor (PIF) is a newly described human lymphokine which we have recently described, partially purified, and shown to be distinct from interferon-gamma and other lymphokines. PIF is defined biologically by stimulation of phagocytosis by cells of the human monocytoid line, U937. Preliminary data suggest that PIF may be a differentiation- inducing factor for U937 cells and possibly other cells and that PIF may play an important role in the regulation of immunological interactions between T lymphocytes and mononuclear phagocytes. Our ultimate goal is to determine the importance of PIF in the pathogenesis of diseases characterized by deficient or excessive immune functioning, such as AIDS or autoimmune disease, and the potential usefulness of PIF int he treatment of such diseases. The goal of the present proposal is to determine the biochemical characteristics, molecular actions, and immunologic functions of PIF. This goal will be accomplished in four subprojects. In the first project, we will utilize a specialized cell culture system, along with well-established methods for protein purification, to biochemically purify PIF, which is produced constitutively by certain T cell lines we have identified. The second project will be to prepare monoclonal antibodies specific for PIF and use these for affinity-purification of PIF and development of a specific immunoassay for PIF, according to well-established procedures. We will then delineate the molecular characteristics of PIF, including molecular weight, isoelectric point, and degree and functional importance of glycosylation. In the third project we will use purified PIF and monoclonal antibodies to PIF to define the effects of PIF on cell so the immune system and cells from subjects infected with human immunodeficiency virus, in particular testing the hypotheses that PIF is an amplifier of antigen-induced reactions and that PIF potentiates T cell activation and susceptibility of T cells to infection with the human retroviruses HTLV-I and HIV; and 3) to develop a solid- phase immunoassay for PIF. In the final stage, we will use this immunoassay to characterized the subset of T4 positive lymphocytes that produces PIF and to determine its relationship of this subset to the subsets of T4 positive cells that 1) proliferate in response to soluble antigen and 2) can be transformed with HTLV-I. These studies will provide a rational basis for determining if PIF, or neutralizing antibodies to it, may be potentially useful in the treatment of diseases of deficient immune function.

We have recently developed an in vitro system in which normal PBMC are stimulated with soluble antigen, exposed to infectious HIV, and cultured for an additional 2-3 weeks with monitoring of their responsiveness to further immunologic stimuli as well as their production of HIV. This system exhibits many of the features of acquired immunodeficiency syndrome (AIDS), in that a single exposure of PBMC to the soluble antigen tetanus toxoid amplified the susceptibility of PBMC to productive HIV infection by a factor of 10-100, and exposure of antigen-stimulated PBMC to low levels of HIV can inhibit the responsiveness of these cells to further immunologic stimuli. The goal of this proposal is to define the cellular and immunologic mechanisms responsible for these phenomena. First, we will define the factors that determine the magnitude of such antigen- induced amplification of HIV replication, including the time interval between exposure to antigen and exposure to HIV, the dose of HIV, and the dose of antigen. We will then investigate the immunological basis for this amplification by determining a) the identity of the cell in which increased HIV replication occurs after antigenic stimulation (i.e., CD4+ lymphocyte or macrophage, or both), b) whether the responsiveness of these cell populations to antigenic stimulation, as reflected in the production of cytokines and soluble receptors for interleukin 2, correlates with the magnitude of antigen-induced amplification of HIV replication, and c) whether cyclosporin A can inhibit antigen- induced amplification of HIV infection. Next, by analyzing the responses of PBMC that can proliferate in response to 2 or more antigens, we will determine whether antigen-activated CD4+ subpopulations are preferentially infected by HIV, compared to resting populations of specific for other antigens. Finally, we will address the mechanism of HIV-induced inhibition of antigen- and mitogen-induced lymphoproliferative responses, determining a) if it is also amplified by prior antigen-induced activation, as indicated by some preliminary data; and b) whether this effect requires live (infectious) HIV, and, if so, whether a low level productive infection or a reactivated latent infection is involved. These studies will provide insight into the mechanisms that determine susceptibility of human PBMC to infection with HIV and lead to the immunosuppressive effects of such infection, and will provide basic data that may be useful in the development of approaches to limit replication of HIV in infected hosts.

CD4 lymphocyte levels have been shown to correlate well with stage of HIV-1 infection, but their predictive value for future rates of CD4 decline or for time to development of AIDS is much poorer. We propose here that station measurements of CD4 levels do not take into account the dynamics of T cell loss and replacement. We will therefore, refine currently available methods for identifying newly generated T cells and use them to assess T cell turnover rates in HIV-1 infected individuals, much as reticulocyte counts are used to evaluate erythrocyte turnover. We will test the hypothesis that individuals with similar levels of circulating T cell subsets may be at very different risk for AIDS, depending on the extent of HIV-induced lymphocyte replacement. We hypothesize that the increased prevalence of T cells bearing surface determinants that are conventionally interpreted as memory T cell activation markers in HIV-1 infection represents an accumulation of immature T cells rather than the activation of memory cells. Despite the phenotypic similarity between the two developmental stages, as shown in our preliminary data as well as data from other laboratories, activated memory cells would be immunologically competent whereas immature cells would not. Criteria used to distinguish immature from activated T cells will be confirmed on the basis of phenotypic and functional profiles expressed by cells obtained from two types of patients known to be generating large numbers of new lymphocytes, namely bone marrow transplant recipients and neonates. The characteristics of newly generated lymphocytes in these individuals will be compared with those of circulating T lymphocytes from HIV-1-seropositive and - seronegative homosexual men being followed in the Baltimore center of the Multicenter AIDS Cohort Study (MACS). This information will provide an initial validation of our immature T cell phenotypic profile in HIV-1 infected individuals. Next, the different T cell maturational components will be quantified longitudinally in selected members of the Baltimore cohort before and at intervals after seroconversion. Fresh and cryopreserved specimens from individuals of known clinical outcome will be tested. The data will be used to construct a mathematical model for assessing HIV-1 disease progression based on the maturational composition of T cells in individuals with similar CD4 cell counts (i.e., the estimation of the effect of CD4 cell immaturity after controlling for the absolute CD4 cell number). These analyses will be conducted in seroprevalent and seroconverter members of the Baltimore cohort using techniques which we have previously applied to lymphocyte population shifts in MACS. The data obtained may add to our understanding of the natural history and pathogenesis of HIV-1 infection.

CD4 lymphocyte levels have been shown to correlate well with stage of HIV-1 infection, but their predictive value for future rates of CD4 decline or for time to development of AIDS is much poorer. We propose here that station measurements of CD4 levels do not take into account the dynamics of T cell loss and replacement. We will therefore, refine currently available methods for identifying newly generated T cells and use them to assess T cell turnover rates in HIV-1 infected individuals, much as reticulocyte counts are used to evaluate erythrocyte turnover. We will test the hypothesis that individuals with similar levels of circulating T cell subsets may be at very different risk for AIDS, depending on the extent of HIV-induced lymphocyte replacement. We hypothesize that the increased prevalence of T cells bearing surface determinants that are conventionally interpreted as memory T cell activation markers in HIV-1 infection represents an accumulation of immature T cells rather than the activation of memory cells. Despite the phenotypic similarity between the two developmental stages, as shown in our preliminary data as well as data from other laboratories, activated memory cells would be immunologically competent whereas immature cells would not. Criteria used to distinguish immature from activated T cells will be confirmed on the basis of phenotypic and functional profiles expressed by cells obtained from two types of patients known to be generating large numbers of new lymphocytes, namely bone marrow transplant recipients and neonates. The characteristics of newly generated lymphocytes in these individuals will be compared with those of circulating T lymphocytes from HIV-1-seropositive and - seronegative homosexual men being followed in the Baltimore center of the Multicenter AIDS Cohort Study (MACS). This information will provide an initial validation of our immature T cell phenotypic profile in HIV-1 infected individuals. Next, the different T cell maturational components will be quantified longitudinally in selected members of the Baltimore cohort before and at intervals after seroconversion. Fresh and cryopreserved specimens from individuals of known clinical outcome will be tested. The data will be used to construct a mathematical model for assessing HIV-1 disease progression based on the maturational composition of T cells in individuals with similar CD4 cell counts (i.e., the estimation of the effect of CD4 cell immaturity after controlling for the absolute CD4 cell number). These analyses will be conducted in seroprevalent and seroconverter members of the Baltimore cohort using techniques which we have previously applied to lymphocyte population shifts in MACS. The data obtained may add to our understanding of the natural history and pathogenesis of HIV-1 infection.

CD4 lymphocyte levels have been shown to correlate well with stage of HIV-1 infection, but their predictive value for future rates of CD4 decline or for time to development of AIDS is much poorer. We propose here that station measurements of CD4 levels do not take into account the dynamics of T cell loss and replacement. We will therefore, refine currently available methods for identifying newly generated T cells and use them to assess T cell turnover rates in HIV-1 infected individuals, much as reticulocyte counts are used to evaluate erythrocyte turnover. We will test the hypothesis that individuals with similar levels of circulating T cell subsets may be at very different risk for AIDS, depending on the extent of HIV-induced lymphocyte replacement. We hypothesize that the increased prevalence of T cells bearing surface determinants that are conventionally interpreted as memory T cell activation markers in HIV-1 infection represents an accumulation of immature T cells rather than the activation of memory cells. Despite the phenotypic similarity between the two developmental stages, as shown in our preliminary data as well as data from other laboratories, activated memory cells would be immunologically competent whereas immature cells would not. Criteria used to distinguish immature from activated T cells will be confirmed on the basis of phenotypic and functional profiles expressed by cells obtained from two types of patients known to be generating large numbers of new lymphocytes, namely bone marrow transplant recipients and neonates. The characteristics of newly generated lymphocytes in these individuals will be compared with those of circulating T lymphocytes from HIV-1-seropositive and - seronegative homosexual men being followed in the Baltimore center of the Multicenter AIDS Cohort Study (MACS). This information will provide an initial validation of our immature T cell phenotypic profile in HIV-1 infected individuals. Next, the different T cell maturational components will be quantified longitudinally in selected members of the Baltimore cohort before and at intervals after seroconversion. Fresh and cryopreserved specimens from individuals of known clinical outcome will be tested. The data will be used to construct a mathematical model for assessing HIV-1 disease progression based on the maturational composition of T cells in individuals with similar CD4 cell counts (i.e., the estimation of the effect of CD4 cell immaturity after controlling for the absolute CD4 cell number). These analyses will be conducted in seroprevalent and seroconverter members of the Baltimore cohort using techniques which we have previously applied to lymphocyte population shifts in MACS. The data obtained may add to our understanding of the natural history and pathogenesis of HIV-1 infection.

The goal of this application is to define the factors that have a positive or negative effect on the risk of transmission of HIV-1 through sexual activity in gay men. To address this question, we will conduct a non-concurrent prospective study utilizing specimens obtained from two groups of gay couples. The first group remained discordant for HIV, i.e., one member of the couple is HIV-1-seropositive (HIV+) and the other is seronegative (HIV), despite high-risk sexual activity. The second group of couples were initially discordant, but then HIV infection was transmitted from the HIV+ to the HIV- partner. Our preliminary data show that the discordant couples maintained their discordant status over several years in which they engaged in high-risk sex, and this status was confirmed by all available diagnostic tests (HIV culture, serology, and polymerase chain reaction (PCR)). In this proposal we will attempt to delineate specific factors that could explain the lack of transmission of HIV in these couples, including characteristics of the HIV+ partner (HIV properties or host immunological factors) or the HIV- partner (host immunological or susceptibility factors). Using a repository of banked specimens of PBMC, semen, serum, and HIV isolates collected at times when high-risk behaviors were occurring in both sets of couples, we will test the hypotheses that lack of transmission of HIV is due to 1) low viral load in semen or peripheral blood, and/or reduced viral infectiousness in the non-transmitting HIV+ partners; 2) strong specific or non-specific anti-HIV immune responses (neutralizing antibody, activated CD8+ T lymphocytes, activated natural killer cells) in the HIV+ partner; 3) genetically resistant CD4+ lymphocytes in the HIV- partner; or 4) strong specific or non-specific anti-HIV immune responses (activated CD8+ T lymphocytes, activated natural killer cells) in the HIV- partner. These hypotheses will be tested by comparing the measurements obtained in the nontransmitting couples to those in the transmitting couples. In these studies, special advantage will be taken of the ability to test the HIV- partner's immune responsiveness against a specific HIV isolate to which he has been exposed, namely that of the HIV+ partner. Whether the HIV- men have immunological memory for HIV antigens will also be tested, as evidence that they have been exposed and may have a protective immunologic response, by measuring cytokine production. The data obtained should clarify the pathogenesis of HIV infection and the relative importance of factors that contribute to the risk of transmission of HIV.

This application requests continuation of a Multi-center AIDS Cohort Study (MACS) Pathogenesis Research Laboratory (MPRL) which consists of a consortium of investigators at John Hopkins University, the University of Pittsburgh, and the University of Washington. The scientific agenda for this application is based on the accomplishments of the current funding period, especially the finding of an ordered cascade of events which characterized the 3-4 years before the onset of clinically defined AIDS in those HIV-1 infected MACS participants who progressed to advanced HIV-1 disease. The hallmarks of this cascade included increases in viral intra-timepoint diversity and divergence from the founder strain, which then stabilized or declined; detection of X4 (CXCR4-utilizing) strains of HIV-1 in the peripheral blood, at least transiently; and loss of normal of circulating total T (CD3+) lymphocytes (failure of T cell homeostasis), including preferential loss of naive CD4+ T cells. The specific aims of the present application, therefore, are: 1) To confirm this cascade and the stages of asymptomatic HIV-1 infection it defines; 2) To determine the specific mutations and patterns of evolution required for outgrowth of X4 viruses; 3) To elucidate the role of CD8+ T cells in controlling HIV replication and evolution; and 4) To use the above information to determine the impact of virologic and immunologic status at the initiation of highly active anti-retroviral therapy (HAART) on the response to HAART. Both retrospective and real-time study designs will be used, to take advantage of the excellent MACS cohort follow-up as well as repository of lymphocyte, plasma, and clinical data. Results from these studies should help to improve understanding of a) mechanisms influencing the rate of HIV-1 disease progression and b) optimal ways to balance the benefits and risks of HAART.

The proposal is a response to Program AnnounCement 96-060, Acute Infection and Early Disease Research Network We propose to recruit 30-50 individuals, primarily injection drug users, with recent HIV- l infection for detailed studies of the effect of early, aggressive treatment of HIV-1 infection. The treatments that will be given include newly developed, highly potent antiretroviral drug combinations; study participants will be randomized to receive or not receive the immunostimulatory cytokine, interleukin-2 (IL-2). The effect of these treatments on the natural history and pathogenesis of HIV-1 infection will be studied. Hypotheses to be tested include l) antiretroviral therapy will suppress, but not eradicate, infectious and latent HIV in PBMC; 2) the extent of suppression of HIV-1 will correlate with the fitness of the early HIV-1 isolate, and with the resiStanCe of the virus to drug therapy; 3) IL-2 will enhance the degree of viral suppression and will augment HIV-specific CLT memory pools; 4) suppression of HIV replication by the drugs will correlate with establishment of in vitro resistance of PBMC to infection with HIV; 5) IL-2 reduces the extent of HIV-l acute damage to the T Cell repertoire and accelerates the recovery from this damage, which is reflected in depletion of memory Cells and development of "holes" in the T Cell repertoire; and 6) production of IL-12, a key stimulater of all- mediated immunity, is defective from the onset of HIV infection, and this defect is mediated by a complement-CD46 interaction on mononuclear phagocytes. These hypotheses will be tested in an integrated design using several newly developed methods for assessing latent infection of cells with HIV-1, diversity of T Cell receptor expression, phenotypic expression of CD4+ lymphocytes, viral fitness, and cytokine levels in vivo and in vitro. The data obtained will help define the optimal use of the new, potent therapies for HIV-1 infection, and will also help to understand the mechanism of immune system damage in HIV-l infection.

The Study to help AIDS Research Effort (SHARE) studies the natural history of infection with human immuno deficiency virus, type 1 (HIV- 1). SHARE, along with similar sites in Chicago, Pittsburgh, and Los Angeles, forms the Multicenter AIDS Cohort Study (MACS). MACS participants, including 1447 enrolled in SHARE, have been followed semiannually since 1984 and have provided questionnaire data, physical exam data, laboratory data ( including HIV-1 serostatus and T - cell subset measurements), and a large repository of plasma, serum, cryopreserved peripheral blood mononuclear cells and other specimens. Evaluating and following the prevalent and incident cases of HIV-1 infection in the MACS including 584 in SHARE, has provided key insights into risk factor s for infection with HIV-1, progression of HIV-1 infection once it is established, host defense against HIV- 1, genetic factors affecting HIV-1 pathogenesis, and use and efficacy of different forms of therapy for HIV-1 infection and for opportunistic pathogens. SHARE and the MACS have made key contributions to defining the importance of the measurement of t- helper (CD4+) lymphocytes, plasma viral load, and immune activation as pathogenic and prognostic factors in HIV-1 infection. SHARE has also played a leading role in the MACS neuropsychological studies. The current project request the continuation of the follow up of the SHARE cohort from 1999 to 2003. Expected survival and enrollment of the HIV-1 infected cohort members through this time period is 89% Specific Aims of the renewal include defining the long-term efficacy and safety of newer highly active antiretroviral treatments (HAART) and new clinical outcomes associated with HAART - induced increases in survival (e.g, neurological and oncological diseases with long incubation periods); defining prognostic markers for people taking HAART; and following cohort members for full characterization of outcomes. These aims can be addressed only with continued follow up of this extremely well-characterized cohort. Other studies that depend on such follow up, but will be carried out with collaborators rather than being directly addressed in this application, include virological and immunological mechanisms of HIV-1 pathogenesis, and laboratory correlates of disease progression or non-progression. The MACS should continue to play a leading role in studies designed to lead to better treatments and preventive vaccines for HIV-1 infection.

1. To determine the extent of suppression of early HIV infection achieved by highly active antiretroviral therapy (HAART) compared to combined HAART and immunotherapy with ultralow-dose interleukin-2 (IL-2). Hypotheses: a. Early HAART will eliminate the unintegrated proviral reservoir and slow the establishment of the integrated, potentially infectious latent reservoir HIV in PBMC and lymph nodes. b. IL-2 immunotherapy will augment and prolong HIV-specific CTL. c. The magnitude of viral load (plasma RNA, integrated DNA, and unintegrated DNA) correlates with the fitness (replicative capacity) and drug resistance of the early HIV isolate. d. Suppression of HIV replication, and disease progression, will correlate with establishment of in vitro resistance of PBMC to endogenous and exogenous virus growth. 2. To evaluate the extent of immune system damage that occurs in early HIV infection, and the effect of HAART alone vs. combined HAART plus ultralow-dose IL-2 on immune system recovery. Hypotheses: a. Acute HIV infection leads to depletion of memory cells and development of "holes" in the T cell repertoire. b. IL-12 production is defective even in early HIV infection, and this defect is mediated by a complement-CD46 interaction similar to that seen in measles infection. c. IL-2 reduces the extent of damage and accelerates immune system recovery. 3. To establish a repository of specimens of plasma, serum, peripheral blood and lymph node mononuclear cells, and HIV-1 isolates that can be used for additional studies of HIV pathogenesis.

The Study to Help the AIDS Research Effort (SHARE) along with three other clinical sites (Northwestern University, Chicago, Illinois; UCLA School of Public Health, Los Angeles, California; Pittsburgh-Graduate School of Public Health, Pittsburgh, Pennsylvania) collectively know as MACS was funded by National Institute of Allergy and Infectious Disease (NIAID) in 1983 and began recruiting AIDS-free homosexual and bisexual men in 1984 for a natural history study of AIDS, subsequently redefined as the natural history of HIV-1 infection. The original goals of the MACS were to define risk factors for acquisition of HIV-1 infection, mechanisms of AIDS pathogenesis and specific incidences and risk factors for particular clinical manifestations of HIV-1 infection, such as infections, malignancies and neuropsychological impairments. Funding for the SHARE study was renewed for four years in April 1999. The study consists of several components working together to achieve the following specific aims: -To determine the long-term effectiveness, at the population level, of highly active antiretroviral therapy (HAART) -To delineate the determinants of individual response to therapy -To further characterize host genetic factors, immune responses and virologic characteristics specific to participants who demonstrate progression of HIV-1 infection, in contrast to those who are on progressors, with particular emphasis on characteristics pertinent to development of vaccines and therapy -To further characterize the nature of host-mediated resistance in highly exposed HIV-1 uninfected participant -To continue efforts to refine and extend prognostic markers of disease progression -To determine factors that contribute to specific disease outcomes -To determine the epidemiology and pathogenesis of HIV-1 malignancies Cohort maintenance is paramount to achieving the specific aims. Ninety-five percent of participant visits take place in the GCRC during regular clinic hours on Tuesday and Friday evenings. There are a small percentage of men who, for a number of reasons (illness, schedule conflict) are not able to attend clinic. Study staff continue to create ways to meet the needs of the study and the participants. Utilizing regular clinic hours, non-clinic hours and off-site facilites, we have been able to maintain a follow-up rate of 75% among the seropositive cohort.

This application addresses the need at Johns Hopkins University for increased capacity to perform high speed cell sorting of specimens infected with human immunodeficiency virus (HIV) type 1 and related retroviruses. It specifically requests support for the purchase of a Cytomation MoFlo cell sorter, to be house in a biosafety level 3 laboratory in the Johns Hopkins School of Hygiene and Public Health. The request is necessary for research programs at Johns Hopkins to take advantage of recent exciting findings and methodological advances that promise important progress in understanding disease mechanisms in AIDS. The cell sorting core facility to be modernized with this purchase was begun in 1989 with construction of new laboratory space designed specifically for HIV research, and acquisition of a cell sorter (Coulter ELITE) that was close to state-of-the-art t the time. Both of these enterprises were supported by grants obtained for the specific purpose of establishing a cell sorting core facility for biohazardous specimens. The facility has been successful in that it has been increasingly well used for AIDS research. However, we have reached the point where the sorting capacity of the ELITE (2500-3500 cells/second for most applications is severely limiting research that could otherwise be done. The Cytomation MoFlo, based on data from several laboratories, could increase sorting speed and throughput by at least 5-fold and represents the most cost-effective method for alleviating the current bottleneck. Particularly benefitted by the improved sorting capacity requested would be research in 3 areas: host defense against HIV, the establishment of viral reservoirs that cna persist despite potent new anti-retroviral therapies,a nd the effect of HIV infection on turnover and renewal of human immune cells. Specific research projects of 7 investigators that would so benefit are described. Procedures and plans for the cell sorting facility are described for continuing it service and productivity in the future. The instrumentation requested will be well utilized by the facility, overseen by a full time cytometer operator, a facility director, and an internal advisory committee. This instrumentation will also allow the Johns Hopkins AIDS research community to meet its research agenda for the next several years as we struggle for treatments and vaccines against this scourge.

DESCRIPTION (provided by applicant): This is a revised application for a proposal to conduct a prospective, randomized trial of daily, low-dose administration of lnterleukin 2 (1L2) in the treatment of early HIV infection (i.e., infection that was identified, and treated with highly active antiretroviral therapy (HAART), soon after establishment of infection). The long-range of this research is to develop treatments for HIV infection that minimize the need for chronic therapy with antiretroviral medications. The major hypothesis to be tested in this application is that Interleukin 2 (IL2) given with highly active antiretroviral therapy (HAART) will enable the host immune system to suppress HIV replication more effectively than will HAART alone. This hypothesis will be tested by administering low doses of IL2 subcutaneously daily to a population of subjects whose HIV infection was diagnosed, treated with HAART, and suppressed to undetectable levels in the blood (<50copies/ml) within one year of the time of infection as part of the Acute Infection and Early Disease Research Project (AIEDRP). The study design contains two treatment cycles, in each of which HAART will be stopped for up to 12 weeks and the viral set point that is achieved will be measured. Each study participant will receive IL2 during one of the treatment cycles. If the hypothesis is true, viral set points in a given treatment cycle should be lower in those who received IL2 in that cycle than in those who did not. We further hypothesize that the extent of HIV suppression of HIV will correlate with the magnitude of the CD4+ and CD8+ T cell responses to recurrent viremia. To test this hypothesis, short-term lymphocyte cultures will be stimulated with HIV peptides that span all HIV genes and the proportion of cells that are activated will be measured by flow cytometry using CD69 and intracellular production of interferon-gamma as indicators of cellular activation. The data obtained will help determine if early treatment of HIV is clinically beneficial, and whether direct measurement of HIV-reactive cells can predict the ability of the host immune response to control HIV replication. The revision addresses all points in the critique of the original application, in particular distinguishing the proposed research from previous research in the use of lL2 to treat early HIV infection.

DESCRIPTION (provided by applicant): The Study to Help the AIDS Research Effort (SHARE) was funded by NAID and NCI in 1983 to study the natural history of infection with human immunodeficiency virus, type 1 (HIV). SHARE, along with similar sites in Chicago, Pittsburgh, and Los Angeles, forms the Multicenter AIDS Cohort Study (MACS). MACS participants, including 1447 enrolled in SHARE, have been followed semiannually since 1984 and have provided questionnaire data, physical exam data, laboratory data (including HIV serostatus and T-cell subset measurements), and a large repository of plasma, serum, cryopreserved peripheral blood mononuclear cells and other specimens. Evaluating and following the prevalent and incident cases of HIV-infection in SHARE and the MACS, has provided key insights into risk factors for infection with HIV-1, monitoring and mechanisms of progression of HIV infection once it is established, host defense against HIV, genetic factors affecting HIV pathogenesis, and use and efficacy of different therapies for HIV infection and for opportunistic pathogens. SHARE and MACS have just completed the recruitment of 1326 additional participants, including 352 in SHARE, who are younger and more non-Caucasian than the cohort previously recruited. This application requests the continuation of the follow-up of the SHARE cohort from 2004 to 2008. Expected survival and continuation in the study of HIV-infected cohort members through this time period is 88%. Specific Aims of the renewal include: determine the long-term effectiveness of highly active antiretroviral therapy (HAART); define determinants of individual responses to HAART including host genetic characteristics, co-infections (hepatitis viruses, HHV-8), immune responses to HIV, adherence, race/ethnicity; define mechanisms of host resistance to HIV infection and progression of the infection; and serve as a platform for independently funded pathogenesis studies and other collaborative research, including virological and immunological mechanisms of HIV-1 pathogenesis, laboratory correlates of disease progression or non-progression, and biological interactions between co-infections and HIV. These aims can be addressed only with continued follow-up of this extremely well-characterized cohort. SHARE and the MACS should continue to play a leading role in studies designed to lead to better treatments and preventive vaccines for HIV infection.

DESCRIPTION (provided by applicant): The overall objective of the study in this revised application is to improve clinical decision-making regarding the appropriate time to initiate highly active antiretroviral therapy (HAART) among persons with early HIV infection. Current guidelines suggest that antiretroviral therapy be considered for HIV-infected people who are identified within the first 6-12 months of infection. However, there are no randomized studies that have provided data supporting early treatment of HIV infection as a valid therapeutic strategy for the long-term management of the disease, nor are there are data to justify a cut-off that would establish how "early" infection should be best defined for the purposes of this strategy. Therefore, Specific Aim 1 of this application is to conduct a randomized trial of HAART vs. no therapy in people who are identified as HIV-infected within one year of becoming infected, and divided into periods of acute (<2 months) and early (2-12 months) infection, to determine whether therapy is superior to no therapy in terms of subsequent virologic control of HIV and whether treatment within 2 months of infection is superior to treatment with 2-12 months. In response to reviewers' concern about our statistical power to address these questions, we have increased the number of sites at which we will accrue participants from two to five (Baltimore, MD and Vancouver, BC, Victoria, BC, Montreal, PQ, and Toronto, ON, Canada) and the anticipated enrollment from 120 to 180 subjects over a 2-year period, yielding substantially improved statistical power. Enrollees will be randomized to receive HAART for one year or no therapy. After the initial year of follow-up, subjects will be followed for up to 4 more years and the principal study endpoint will be a comparison of the plasma viral load 24 months after initial presentation in all treated vs. untreated subjects. If our hypothesis is correct, early treatment will lead to viral suppression and preservation of host immune responses that will be reflected in lower viral loads in subsequent years. (These lower viral loads would be expected to translate into improved AIDS-free survival and time remaining therapy-free according to current guidelines for therapy of chronic HIV infection, although these endpoints because they would likely occur later than the 5-year term of this application.) Such findings would have a major impact on treatment of and surveillance for acute and early HIV infection and would result in reduced costs and morbidity of therapy as well as a much-needed databased approach to treatment of acute/early HIV infection for a limited period of time. Since our study population will also include a high proportion of injecting drug users (IDUs), the findings will also help in the design of new approaches for treatment of acute/early HIV infection in this important group of people at risk for HIV infection. In Specific Aim 2, we will monitor the prevalence of primary drug resistance in all study subjects and monitor its stability over time in untreated subjects. Comparison with untreated subjects carrying susceptible isolates will allow us to evaluate the contribution (positive or negative) of primary drug resistance on HIV disease progression.

DESCRIPTION (provided by applicant): The Study to Help the AIDS Research Effort (SHARE) was funded by NIAID and NCI in 1983 to study the natural history of infection with human immunodeficiency virus, type 1 (HIV). SHARE, along with similar sites in Chicago, Pittsburgh, and Los Angeles, forms the Multicenter AIDS Cohort Study (MACS). MACS participants, including 1447 enrolled in SHARE, have been followed semiannually since 1984 and have provided questionnaire data, physical exam data, laboratory data (including HIV serostatus and T-cell subset measurements), and a large repository of plasma, serum, cryopreserved peripheral blood mononuclear cells and other specimens. Evaluating and following the prevalent and incident cases of HIV-infection in SHARE and the MACS, has provided key insights into risk factors for infection with HIV-1, monitoring and mechanisms of progression of HIV infection once it is established, host defense against HIV, genetic factors affecting HIV pathogenesis, and use and efficacy of different therapies for HIV infection and for opportunistic pathogens. SHARE and MACS have just completed the recruitment of 1326 additional participants, including 352 in SHARE, who are younger and more non-Caucasian than the cohort previously recruited. This application requests the continuation of the followup of the SHARE cohort from 2004 to 2008. Expected survival and continuation in the study of HIV-infected cohort members through this time period is 88%. Specific Aims of the renewal include: determine the long-term effectiveness of highly active antiretroviral therapy (HAART);define determinants of individual responses to HAART including host genetic characteristics, co-infections (hepatitis viruses, HHV-8), immune responses to HIV, adherence, race/ethnicity;define mechanisms of host resistance to HIV infection and progression of the infection;and serve as a platform for independently funded pathogenesis studies and other collaborative research, including virological and immunological mechanisms of HIV-1 pathogenesis, laboratory correlates of disease progression or non-progression, and biological interactions between co-infections and HIV. These aims can be addressed only with continued followup of this extremely well-characterized cohort. SHARE and the MACS should continue to play a leading role in studies designed to lead to better treatments and preventive vaccines for HIV infection.

DESCRIPTION (provided by applicant): Continued training in The Molecular and Cellular Bases of Infectious Diseases (MCBID) is proposed for 8 PhD students and 3 postdoctoral fellows selected from large pools of highly qualified applicants. The training program is uniquely situated in the Molecular Microbiology and Immunology Department (MMI) within the Johns Hopkins Bloomberg School of Public Health. The 29 training faculty have a wide range of experience and expertise in viruses, bacteria and parasites causing human disease and in the vectors and environmental factors associated with emergence and transmission of these pathogens. The training program has been funded since 1994 and has produced scientists working in many areas of academia and government on problems related to infectious diseases, vaccine development and the public's health. The goal of the MCBID training program is to provide students with both a firm foundation in the basic disciplines necessary for the study of infectious diseases and a perspective that will enable them to apply their knowledge creatively to public health problems. Each student is expected to complete 1) a series of required courses in the basic disciplines of cell and molecular biology, biochemistry, and immunology, 2) courses in virology, bacteriology, parasitology, and disease ecology, 3) courses in research ethics and public health perspectives, and 4) elective courses relevant to thesis topic and long-term career goals. Elective courses are chosen from among courses available in MMI, other departments in the School of Public Health, or in other Divisions of the University. Students will also complete 3 11-week laboratory rotations during the first year. Student progress is monitored by a Thesis Advisory Committee and the Graduate Program Committee. The goals of the postdoctoral training program are 1) to provide focused training in those areas of the molecular and cellular basis of infectious diseases in which program faculty have special expertise;2) to provide an opportunity for doctoral degree holders trained in more traditional environments to broaden their exposure to problems of public health importance and to evaluate their career goals in terms of public health issues;and 3) to prepare the PDF for an independent career in the biological sciences. RELEVANCE : This program is highly relevant to national interests in the areas of emerging infectious diseases, as it trains students and postdoctoral fellows broadly not only in both the molecular aspects of pathogen biology and disease pathogenesis, but also in the ecology of disease emergence and the role of vectors in pathogen transmission.

DESCRIPTION (provided by applicant): Continued training in The Molecular and Cellular Bases of Infectious Diseases (MCBID) is proposed for 8 PhD students and 3 postdoctoral fellows selected from large pools of highly qualified applicants. The training program is uniquely situated in the Molecular Microbiology and Immunology Department (MMI) within the Johns Hopkins Bloomberg School of Public Health. The 29 training faculty have a wide range of experience and expertise in viruses, bacteria and parasites causing human disease and in the vectors and environmental factors associated with emergence and transmission of these pathogens. The training program has been funded since 1994 and has produced scientists working in many areas of academia and government on problems related to infectious diseases, vaccine development and the public's health. The goal of the MCBID training program is to provide students with both a firm foundation in the basic disciplines necessary for the study of infectious diseases and a perspective that will enable them to apply their knowledge creatively to public health problems. Each student is expected to complete 1) a series of required courses in the basic disciplines of cell and molecular biology, biochemistry, and immunology, 2) courses in virology, bacteriology, parasitology, and disease ecology, 3) courses in research ethics and public health perspectives, and 4) elective courses relevant to thesis topic and long-term career goals. Elective courses are chosen from among courses available in MMI, other departments in the School of Public Health, or in other Divisions of the University. Students will also complete 3 11-week laboratory rotations during the first year. Student progress is monitored by a Thesis Advisory Committee and the Graduate Program Committee. The goals of the postdoctoral training program are 1) to provide focused training in those areas of the molecular and cellular basis of infectious diseases in which program faculty have special expertise; 2) to provide an opportunity for doctoral degree holders trained in more traditional environments to broaden their exposure to problems of public health importance and to evaluate their career goals in terms of public health issues; and 3) to prepare the PDF for an independent career in the biological sciences. RELEVANCE : This program is highly relevant to national interests in the areas of emerging infectious diseases, as it trains students and postdoctoral fellows broadly not only in both the molecular aspects of pathogen biology and disease pathogenesis, but also in the ecology of disease emergence and the role of vectors in pathogen transmission.

DESCRIPTION (provided by applicant): This application addresses the ongoing need at Johns Hopkins University (JHU) for high-speed cell sorting of specimens infected with organisms which are biohazardous to humans. It requests support to upgrade a Beckman Coulter MoFlo Legacy flow cytometer, housed in a biosafety level 3 (BSL3) laboratory in the Johns Hopkins Bloomberg School of Public Health, to the functional equivalent of a MoFlo XDP flow cytometer. The MoFlo Legacy cytometer in question is more than 11 years old, is tenuous as a critical instrument to support a large number of research projects that depend on it (specifically projects in the areas of HIV/AIDS, cancer, neurodegenerative diseases, and other diseases of human cells where sorting in a protected environment is appropriate), and will no longer be supported by the manufacturer as of late 2013. As most cell sorting facilities at JHU refer to us the sorting of human samples that may contain agents such as hepatitis viruses or other pathogens, and our laboratory is the main biosafety level 3 (BSL3) cell sorting facility at JHU, a reliable and adequately sophisticated sorter is critical for the continued success of this facility, which has contributed data to at least 70 publications, including important advances in understanding and care of HIV infection, since the acquisition of the current cytometer. The laboratory team is well experienced in management of a BSL3 core facility, with the principal investigator of this application having directed this facility since 1989. With the current MoFlo cytometer now reaching its life expectancy, upgrading and modernizing it are a) needed to sustain and expand the current scale of research, and b) much more cost-effective for this purpose than purchasing a new sorting cytometer. Among the HIV-related research areas that depend on this facility are understanding the generation and possible mechanisms of eradication of HIV reservoirs in vivo, immune pathogenesis of HIV infection, and pathogenesis of HIV-related infections. Other areas of human investigation include pathogenesis of neurological disease, role of stem cells in cancer and other diseases, and aspects of gene regulation in many human diseases. Specific research projects of 5 major investigators and numerous other users of the core that would benefit from continued availability and reliability of this facility are described, as are the specific features of the MoFlo upgrade that justify its acquisition. Many other projects would also use the new sorter over its lifetime. Procedures and plans for sustaining the cell sorting facility's ability to support HIV and human research at JHU for the future are described, as are financial plans for operation of the facility including institutional support comprised of covering the service contract for the upgraded cytometer and any financial shortfalls that may develop. The facility will be operated by an experienced full-time cytometer operator and a new operator to be trained and supervised by the facility director, with oversight by an internal advisory committee. With the requested upgrade, the facility will continue to make unique contributions to the treatment, management, and prevention of HIV infection and other human diseases for many years.

Project Summary This application is for the renewal of the Study to Help the AIDS Research Effort (SHARE), which has been the Baltimore-Washington DC site of the Multicenter AIDS Cohort Study (MACS) since 1984. The MACS was established with sites in Baltimore-Washington, Chicago, Pittsburgh, and Los Angeles, initially to study the natural history of HIV infection in men who have sex with men and later, with the advent of effective combination antiretroviral therapy (cART), the treated history of HIV infection, including the relationship between long-term controlled HIV infection and chronic diseases and co-morbidities associated with aging. To reflect these changing priorities in HIV research, SHARE has had subsequent enrollments to balance proportions of HIV- and HIV+ men and to reflect the HIV epidemic more accurately. Men enrolled have been followed semi annually since 1984 or enrollment and have a) provided questionnaire data, physical exam data, laboratory data (including HIV serostatus, T cell subset measurements, and HIV viral load measurements), and a large repository of plasma, serum, cryopreserved peripheral blood mononuclear cells, and other specimens; and these have permitted SHARE and MACS to make major contributions to the understanding of treated and untreated HIV disease, and comorbidities associated with increased survival due to antiretroviral treatment. The present application, in response to RFA-HL-19-008, aims to extend followup of the SHARE cohort (about 640 men) from 2019 to 2026, to allow merger of SHARE (and the other MACS sites) with the nine sites of the Women?s Interagency HIV Study (WIHS) into a single cohort, termed the MACS-WIHS Combined Cohort Study (CCS), that will include both men and women. The specific aims of the CCS are outlined in a parallel application from the putative Data and Coordinating Center (DACC) of the CCS, and the 10 Specific Aims of that single cohort are outlined in the DACC application for the CCS; these include Cardiovascular (CV), Pulmonary/Sleep (PS), Neurocognitive NC), Aging (AG), Cancer, HIV Pathogenesis (PA), Psychosocial (PS), Health Disparities, Platform (PL), and Career Development (CD) Aims. The specific aims of the SHARE Clinical Research Site (CRS) are to: 1) play a leading role in the achievement 7 of these Aims by the CCS (i.e., CV, PS, AG, PA, PS, PL, and CD), including serving as a reading center for echocardiography and sleep studies; 2) to contribute to the achievement of the other 3 CCS aims, and 3) to implement all applicable CCS protocols and support the scientific goals of the CCS. Local initiatives on inflammation and physical activity will also support these aims, which take full advantage of the extensive follow-up and repository of biological specimens already available from this cohort and which will be extended under this proposal, enabling further contributions to our understanding and care of comorbidities and other health issues faced by people living with HIV today.

This revised R21 addresses the role of T-cell responses to cytomegalovirus (CMV) in the pathogenesis of frailty, chronic inflammation, and immune activation in people with and without HIV infection, all of which are key areas of this RFA. Frailty is a syndrome characterized by diminished physiologic reserve and increased vulnerability to stressors, leading to adverse outcomes. We and others have identified that chronic inflammation and immune activation play an important role in aging and frailty, but the etiology of chronic inflammation and immune activation in aging HIV+ and HIV- populations remains obscure. Chronic CMV infection, which is highly prevalent in both the geriatric population and HIV+ persons and causes a very large expansion of T-cells, has long been thought to contribute to shrinking of the T cell repertoire and chronic inflammation and immune activation. We have published that: 1) presence of CMV viral DNA in peripheral monocytes, which is detected in about 60% of CMV-seropositive elderly persons, is strongly correlated with frequency of CMV pp65-specific CD8+ T cells and chronic inflammation (elevated serum neopterin and IL-6 levels); and 2) T-cell responses to CMV are very broad, with responses to CMV pp65 or IE1 (the two most studied CMV antigens) constituting only <12% of the total CD4+ and <40% of the total CD8+ CMV-directed T- cell responses measured using overlapping peptides covering 19 CMV open reading frames (ORFs). Moreover, our published pilot data in men who have sex with men (MSM) in the Multicenter AIDS Cohort Study (MACS) indicate that the total IL-2 response of CD4 T cells to these CMV ORFs (but not to only pp65 and IE1) is strongly correlated with markers of chronic inflammation and immune activation and predicts onset of frailty. While these pilot findings could have broad implications, the precision of the estimated correlations is limited by the small sample size studied to date. Thus, this R21 is designed to confirm, and improve the precision of, these observations in a larger sample of MACS men, both HIV- and HIV+. Our specific aims are to: 1) test the hypothesis that the magnitude of the total CMV-specific T-cell response is strongly correlated with the level of chronic inflammation and immune activation in frail and nonfrail HIV- and virologically suppressed HIV+ men; and 2) explore whether the magnitude of the total CMV-specific T-cell response predicts incident frailty in nonfrail HIV- and HIV+ men. The data obtained will provide needed insight into the impact of chronic CMV infection on chronic inflammation and immune activation, and onset of frailty, in aging HIV+ and HIV- MSM. The data obtained will provide an important foundation for future studies to a) determine if chronic CMV infection is correlated with chronic inflammation and immune activation in HIV- and HIV+ women and non-MSM men; b) clarify the biologic basis of CMV-induced inflammation and immune activation; and, c) if appropriate, design clinical trials of anti- CMV and/or anti-inflammatory therapy for prevention of age-related conditions (e.g., frailty) in aging HIV- and HIV+ individuals.

Project Summary (from U01HL146201 cooperative agreement) This application is for the renewal of the Study to Help the AIDS Research Effort (SHARE), which has been the Baltimore-Washington DC site of the Multicenter AIDS Cohort Study (MACS) since 1984. The MACS was established with sites in Baltimore-Washington, Chicago, Pittsburgh, and Los Angeles, initially to study the natural history of HIV infection in men who have sex with men and later, with the advent of effective combination antiretroviral therapy (cART), the treated history of HIV infection, including the relationship between long-term controlled HIV infection and chronic diseases and co-morbidities associated with aging. To reflect these changing priorities in HIV research, SHARE has had subsequent enrollments to balance proportions of HIV- and HIV+ men and to reflect the HIV epidemic more accurately. Men enrolled have been followed semi annually since 1984 or enrollment and have a) provided questionnaire data, physical exam data, laboratory data (including HIV serostatus, T cell subset measurements, and HIV viral load measurements), and a large repository of plasma, serum, cryopreserved peripheral blood mononuclear cells, and other specimens; and these have permitted SHARE and MACS to make major contributions to the understanding of treated and untreated HIV disease, and comorbidities associated with increased survival due to antiretroviral treatment. The present application, in response to RFA-HL-19-008, aims to extend followup of the SHARE cohort (about 640 men) from 2019 to 2026, to allow merger of SHARE (and the other MACS sites) with the nine sites of the Women?s Interagency HIV Study (WIHS) into a single cohort, termed the MACS-WIHS Combined Cohort Study (CCS), that will include both men and women. The specific aims of the CCS are outlined in a parallel application from the putative Data and Coordinating Center (DACC) of the CCS, and the 10 Specific Aims of that single cohort are outlined in the DACC application for the CCS; these include Cardiovascular (CV), Pulmonary/Sleep (PS), Neurocognitive NC), Aging (AG), Cancer, HIV Pathogenesis (PA), Psychosocial (PS), Health Disparities, Platform (PL), and Career Development (CD) Aims. The specific aims of the SHARE Clinical Research Site (CRS) are to: 1) play a leading role in the achievement 7 of these Aims by the CCS (i.e., CV, PS, AG, PA, PS, PL, and CD), including serving as a reading center for echocardiography and sleep studies; 2) to contribute to the achievement of the other 3 CCS aims, and 3) to implement all applicable CCS protocols and support the scientific goals of the CCS. Local initiatives on inflammation and physical activity will also support these aims, which take full advantage of the extensive follow-up and repository of biological specimens already available from this cohort and which will be extended under this proposal, enabling further contributions to our understanding and care of comorbidities and other health issues faced by people living with HIV today.