Patients infected with HIV are at increased risk of developing malignant B cell lymphoproliferative diseases (LPD). The majority of these LPDs are positive for the Epstein-Barr virus (EBV), run an aggressive clinical course and are associated with a poor prognosis. EBV is an oncogenic, human herpes virus that infects more than 90% of adults worldwide and is associated with a wide spectrum of malignancies. The incidence of EBV- LPD in HIV-infected patients is, in part, related to the inadequate quantity and or quality of EBV-specific T lymphocyte response. We hypothesize that deficiency of cellular immunity against EBV lytic and latent antigens leads to increased risk of EBV-LPD, and vaccine-enhanced EBV-specific immunity will restore protection and prevent the development of EBV-LPD in this high risk population. We utilized a SCID mouse model which, when engrafted with human leukocytes from healthy EBV seropositive donors, spontaneously develops human EBV-LPD. We discovered several EBV latent and lytic antigens recognized by human CD8(+) T cells when the disease is controlled in vivo. Furthermore, we validated the recognition of these same antigens in iatrogenically immune suppressed patients with EBV-LPD while successfully controlling their disease in vivo following the reduction of their immune suppressive regimen. We cloned several EBV genes into an adeno-associated and adenoviral constructs, infected human antigen presenting cells and demonstrated robust autologous T cell expansion. Using HLA-specific tetramers loaded with immune dominant peptides derived from BZLF, we documented that In vivo expansion of BZLF1-specific T cells occurring in these patients correlated with EBV+ tumor regression and improved patient survival. With the support of Leukemia Lymphoma Society and corporate partners, we have recently completed a large scale purification of BZLF1 and are initiating preclinical toxicity studies for a planned pre IND meeting over the next year. Because a comprehensive EBV vaccine will likely require multiple EBV protein targets, we propose to complete a characterization of 4 additional target proteins and perform preclinical analysis to identify promising immunogens that may be further developed clinically. This Grand Opportunities grant will accomplish three specific aims: (1) characterize the human cellular response across various HLA types in response to each of the 5 proposed full length gene products in vitro in order to identify the optimal candidate gene product(s) to include in the vaccination strategy;(2) assess the efficacy of full length purified viral polypeptide preparations in stimulating and maintaining EBV specific memory T cell expansion and preventing fatal human EBV- associated lymphoproliferative disorder in our chimeric mouse-human model of EBV-LPD and (3) scale up production of full length candidate polypeptides for further characterization, evaluation of optimal delivery and adjuvant activity, toxicity evaluation, biomarker validation and immunogenicity studies. Once we have completion the above basic exploratory studies with the support of this grant, we will have identified the optimal viral target(s) that are "seen" across a broad HLA spectrum and be in a position to apply for an IND to conduct Phase I trials for safety of delivering specific vaccine preparations to HIV+ patients who are at high risk of developing EBV-LPD. Ultimately, a preventive approach for such viral-driven malignancies will likely prove to be more effective, less expensive, and hold great promise for benefitting more people worldwide by further reducing the incidence EBV-LPD and improving the quality of life of patients infected with HIV. PUBLIC HEALTH RELEVANCE: People infected with the Human Immune Deficiency virus (HIV) are at increased risk of developing malignant B cell lymphoproliferative diseases (LPD). Most of these LPDs are positive for Epstein-Barr virus (EBV-LPD), run an aggressive clinical course and are associated with a poor prognosis. Healthy, immune competent people do not develop EBV-LPD because of a highly efficient, adaptive cellular, antigen-specific immune response. The incidence of EBV-LPD in HIV-infected patients is, in part, related to the inadequate quantity and or quality of EBV-specific T lymphocyte response. Our laboratory program has focused on developing efficient strategies to "boost" cellular immune surveillance mechanisms to treat and prevent EBV-LPD in patients with underlying immune deficiency. We have developed a reproducible small animal model that closely mimics human EBV-LPD, several experimental therapeutic strategies to deliver EBV-encoded polypeptide target proteins, and partnerships with industry and not-for profit organizations to synthesize a vaccine to prevent EBV-LPD. Here we propose to utilize tools that we have developed over the past several years to systematically test the efficacy of vaccinating with selective EBV lytic and latent full length polypeptides to prevent EBV-LPD. This Grand Opportunities Award will provide funding to complete all basic scientific work to identify the key EBV targets, confirm biomarkers to measure immune response to the vaccine, and perform preclinical testing to identify promising vaccine candidate proteins that can then, with the help of our partners, move on towards clinical development to vaccinate HIV+ patients and, perhaps other immune suppressed patient groups. The project has great potential to make an impact on public health, job creation and economic growth.