This application proposes studies to evaluate the potential for development of hypericin (HY) and of numerous structurally related aromatic polycyclic diones, as virus inactivators in red cell concentrates for transfusion. The studies are based on encouraging preliminary data which show lack of adverse effects to red blood cells (RBC) at effective virucidal doses.
They aim to define the optimal conditions for obtaining complete inactivation of very high titers of cell free virus in a blood environment. Hypericin is an effective virucidal agent which directly inactivates a broad range of enveloped viruses. Hypericin is a photodynamic molecule, which is also active in vivo, presumably due to a low red/ox potential. Thus, HY is currently being evaluated in clinical trials in AIDS patients, and phase I of the trials confirmed the transfusibility of HY. The problem of dual transmission of HIV infection via cell-free virus and through latently infected PBMC will be addressed by evaluating combinations of HY and leukocyte filtration in achieving complete sterility, and the possibility of inflicting photodynamic lesions to infected PBMC. The application will address aspects of the mechanism of the virucidal action of HY. The modifications in viral proteins HIV p55gag, p24 and RT, which are targeted for photodynamic cross-linking by HY will be characterized, and other affected virus proteins identified. The potential involvement of viral RNA in RNA- protein covalent complexes will be evaluated. The investigators propose clarify whether the action of HY covalently cross links viral genomic RNA, with RT and other core proteins. Amplification of RNA from HY treated virions using RNA PCR will be utilized to identify genomic regions cross linked by HY. The question whether HY itself or only photodynamically generated excited oxygen species affect the capsids will be examined by isolation of intact HIV cores and examination of their susceptibility to HY and light. These studies have implications for potential inactivation of non enveloped viruses (such as B19 parvovirus) in blood by HY. RT activity and capsid protein migration patterns on SDS-PAGE and Western blots will be used as assays. Structural HY analogs with methyl group substitutions and alterations in the polycyclic aromatic skeleton will be evaluated for potentially improved efficacies and for reduced binding to RBC. The virucidal activities of HY ion pairs with cationic metals, basic amines and amino acids, and their interactions with physiological transport proteins, will be studied in comparison with the standard HY-Na+. Potential for adverse effects to blood components including clinically significant parameters of red cell surface membrane integrity, (2,3-DPG, hemolysis, normal expression of ABO, Rh, minor blood group antigens and possible deposition of IgG or complement on RBC), by treatment with HY will be examined. Potential methods for removal of HY from blood after virus inactivation, incorporating hydrophobic resins and other methods will be evaluated. The applicant organization will collaborate with the following sites: 1) New York Blood Center, 2) Oklahoma Blood Institution, and 3) Weizmann Institute of Science.
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