Kaposi's sarcoma is the most common cancer associated with HIV infection. It is caused by infection with Kaposi's sarcoma-associated herpesvirus (KSHV). In patients with HIV infection as well as in non-HIV-infected organ transplant recipients who are immunocompromised, Kaposi's sarcoma can be an aggressive and life- threatening disease. KSHV can be transmitted by blood transfusion and from organ transplant donors to recipients. While serological assays have been devised to detect KSHV infection, they have been largely restricted to research settings and none are of adequate sensitivity and specificity and in a suitable high- throughput format to facilitate screening of the general population. The goal of Epiphany Biosciences is to develop a sensitive and specific serological assay for the detection of circulating KSHV antibodies that can be used in a high-throughput manner for purposes such as screening the blood supply. A combination of optimized antibody-capture antigens is envisaged for the optimal detection of KSHV-specific antibodies. The specific objectives of our Phase I research and development efforts are to make major improvements to the immunoreactivity of two of three critical KSHV serodiagnostic capture antigens. These antigens are derived from orf K8.1, a spliced mRNA encoding the virion envelope glycoprotein gp35-37, and orf 73, a major latency antigen (LANA1) found in the nucleus of latently infected cells. Our hypothesis is that K8.1 and LANA1 produced in mammalian cells and purified under non-denaturing conditions will exhibit vastly superior immunoreactivity to previously used recombinant antigens and will be key ingredients of a multi-antigen, high throughput combination EIA assay of high sensitivity and specificity to detect KSHV antibodies circulating in infected individuals. In Aim 1, we will express K8.1 in mammalian cells and purify it under non-denaturing conditions to produce native, correctly-folded glycoprotein. It's immunoreactivity will be compared with conventional bacterial-derived k8.1 antigens by measuring the dilutional sensitivity of serum samples obtained from KSHV-infected patients and blood donors. The binding affinities of antibodies from such individuals will also be measured to both types of antigen. Classic, immunofluorescence assays for KSHV antibodies using cell lines latently- and lytically-infected will be used to confirm positive samples. In Aim 2, we will perform similar experiments for LANA1. The long-term objective, which will be the focus of subsequent Phase II and Phase III applications, is to produce permanent cell-lines expressing these native antigens and then to combine these purified antigens with a synthetic peptide derived from the KSHV orf 65 nucleocapsid protein, in order to develop and commercialize a high-throughput enzyme immunoassay suitable for screening the blood supply so as to prevent infection of organ recipients, HIV-infected individuals and blood recipients, in general.
Public Health Relevance: Infection with Kaposi's sarcoma-associated herpes virus, which is present in blood donors in the United States and is transmissible by transfusion, has the potential to cause cancer in immunocompromised patients, including those with HIV infection, who receive transfusions. There is currently no suitable test to screen the blood supply for this virus. The goal of this project is to develop a test that can be used to screen for Kaposi's sarcoma-associated herpes virus and make the blood supply much safer for high-risk individuals and blood recipients, in general.
Public Health Relevance: This Public Health Relevance is not available.
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