A large number of viruses have been described which are pathogenic for humans. Among these viruses are many for which neither drugs nor vaccines are available. In cases where drug treatments are available, the occurrence of resistant mutations and drug side effects often limit the effectiveness of therapy. Examples of such viruses include Hepatitis C and human immunodeficiency virus (HIV).
HIV is the etiological agent of acquired immunodeficiency syndrome (AIDS). It infects selected cells of the immune system thereby compromising the infected individual's immune response. It is estimated that there are over 1 million HIV infected individuals in the United States and over 13 million worldwide. The clinical course of HIV infection typically consists of a prolonged asymptomatic state, followed by a depletion of T4 lymphocytes making the individual susceptible to opportunistic infections and neoplasms.
HIV-1 replication occurs predominantly in CD4+ lymphocytes, the majority of which are located in lymphoid organs, such as peripheral lymph nodes and spleen. HIV-1 can also be found in macrophages and macrophage-like cells, such as microglia in the central nervous system (Cohen et al. Immunol Rev 159: 31-48, 1997).
Plasma HIV-1 levels and presence of HIV-1 infected lymphocytes in peripheral blood strongly correlate with the clinical status of HIV-1 infected patients (Ferre et al. J Acquir Immune Defic Syndr Hum Retrovirol 10(Suppl 2): S51-56, 1995; O'Brien et al. N Engl J Med 334(7): 426-431, 1996). The half-life of circulating virions is 6 hours, while the half-life of HIV-1 infected cells in peripheral blood is 1.6 days. Greater than 1010 virions may be released into the circulation each day (Ho et al. J Biol Regul Homeost Agents 9(3): 76-77, 1995; Ho et al. Nature 373(6510): 123-126, 1995; Wei et al. Nature 373(6510): 117-122, 1995). The ability of the host immune system to keep HIV infection in check, and limit clinical symptoms, is directly proportional to the viral burden. Anti-retroviral therapies, nucleoside analogues, non-nucleoside reverse transcriptase inhibitors, and protease inhibitors, aim to reduce the viral burden so that the immune system can control or clear residual infection (Fauci, Harrisons Principles of Internal Medicine: 1791-1856, 1998).
HIV infection is mediated by gp120, which binds to CD4 as well as to a surface chemokine receptor. Inside the cell the virion is uncoated and the viral RNA is reverse transcribed into double-stranded DNA. Proviral DNA enters the cell nucleus, integrates into the host genome and is transcribed into viral RNAs, which are translated into viral proteins. Mature virions are assembled and released from the cell by budding. (Fauci et al. Ann Intern Med 124(7): 654-663, 1996). A dying cell may also release all its contents including intact virions, and fragments thereof into the blood. Thus, circulating blood of HIV-infected individuals contains intact virions, and viral proteins, in particular toxic viral surface proteins.
The hallmark of AIDS is the gradual loss of CD4+ T cells, which ultimately leaves the immune system unable to defend against opportunistic infections. While the mechanism through which HIV causes AIDS is imperfectly understood, the clinical data suggest that in addition to the loss of infected T-cells, a large number of uninfected T-cells are dying and that HIV derived envelope proteins appear to be intimately involved.
The major HIV envelope glycoprotein gp120 has been shown to have profound biological effects in vitro. Gp120 causes CD4+ T cells to undergo apoptosis and binding of gp120 to CD4+ cells in the presence of anti-envelope antibodies and complement opsoninizes the cells, targeting them for clearance. The combined effect is the destruction of uninfected immune cells. In addition, HIV envelope proteins have been implicated in HIV related hyper-gammaglobulinemia. In AIDS patients, gp120 levels have been measured at an average of 29 ng/ml which is orders of magnitude higher than the concentration of the virus.
Currently there is no cure for HIV infection. Reverse transcriptase and protease inhibitors have been approved for the treatment of HIV. Typical treatment regimes use combinations of approved drugs and are termed HAART (highly active antiretroviral therapy). While more than 16 drugs and drug combinations have been approved by the FDA for treating HIV infection, the emergence of drug resistant mutants and the presence of the untreatable virus reservoirs (e.g. in memory T cells) has limited their usefulness. Unfortunately, no effective HIV vaccine has been forthcoming due, in part, to the rapid mutation of the HIV genome and the inaccessibility of immunogenic epitopes of viral proteins. Thus there is an urgent need for new treatments.
Extracorporeal treatments provide a therapeutic modality which may be used to treat systemic disease. Extracorporeal perfusion of plasma over protein A, plasmapheresis and lymphapheresis have all been used as immunomodulatory treatments for HIV infection, and the thrombocytopenia resulting from it (Kiprov et al. Curr Stud Hematol Blood Transfus 57: 184-197, 1990; Mittelman et al. Semin Hematol 26(2 Suppl 1): 15-18, 1989; Snyder et al. Semin Hematol 26(2 Suppl 1): 31-41, 1989; Snyder et al. Aids 5(10): 1257-1260, 1991). These therapies are all proposed to work by removing immune complexes and other humoral mediators, which are generated during HIV infection. They do not directly remove HIV virus. Extracorporeal photopheresis has been tested in preliminary trials as a mechanism to limit viral replication (Bisaccia et al. J Acquir Immune Defic Syndr 6(4): 386-392, 1993; Bisaccia et al. Ann Intern Med 113(4): 270-275, 1990). However, none of these treatments effectively remove both virus and viral proteins.
Chromatographic techniques for the removal of HIV from blood products have been proposed. In 1997, Motomura et al., proposed salts of a sulfonated porous ion exchanger for removing HIV and related substances from body fluids (U.S. Pat. No. 5,667,684). Takashima and coworkers (U.S. Pat. No. 5,041,079) provide ion exchange agents comprising a solid substance with a weakly acidic or weakly alkaline surface for extracorporeal removal of HIV from the body fluids of a patient. Both are similar to the work of Porath and Janson (U.S. Pat. No. 3,925,152) who described a method of separating a mixture of charged colloidal particles, e.g. virus variants by passing the mixture over an adsorbent constituted of an insoluble, organic polymer containing amphoteric substituents composed of both basic nitrogen-containing groups and acidic carboxylate or sulphonate groups (U.S. Pat. No. 3,925,152). However, none of these chromatographic materials are selective for viruses and will clearly remove many other essential substances. Thus they are not useful for in vivo blood purification.
Immunosorptive techniques have also been proposed for the treatment of viral infections. In 1980, Terman et al. described a plasmapheresis apparatus for the extracorporeal treatment of disease including a device having an immunoadsorbent fixed on a large surface area spiral membrane to remove disease agents (U.S. Pat. No. 4,215,688). The device envisioned no method for directly treating blood and required the presence of an immunologically reactive toxic agent. In 1987 and 1988, Ambrus and Horvath described a blood purification system based on antibody or antigen capture matrices incorporated onto the outside surface of an asymmetric, toxin permeable membrane (U.S. Pat. Nos. 4,714,556; 4,787,974), however, no examples of pathogen removal were given therein. In 1991, Lopukhin et al. reported that rabbit antisera raised against HIV proteins, when coupled to Sepharose 4B or silica, could be used for extracorporeal removal of HIV proteins from the blood of rabbits which had been injected with recombinant HIV proteins (Lopukhin et al. Vestn Akad Med Nauk SSSR 11: 60-63, 1991). However, this strategy was inefficient as it required extracorporeal absorption of blood and did not provide for a mechanism to remove free HIV viral particles from the blood (Lopukhin et al., 1991, supra). U.S. Pat. No. 6,528,057 describes the removal of virus and viral nucleic acids using antibodies and antisense DNA.
Lectins are proteins that bind selectively to polysaccharides and glycoproteins and are widely distributed in plants and animals. Although many are insufficiently specific to be useful, it has recently been found that certain lectins are highly selective for enveloped viruses (De Clercq. et al Med Res Rev 20(5): 323-349, 2000). Among lectins which have this property are those derived from Galanthus nivalis in the form of Galanthus nivalis agglutinin (“GNA”), Narcissus pseudonarcissus in the form of Narcissus pseudonarcissus agglutinin (“NPA”) and a lectin derived from blue green algae Nostoc ellipsosporum called “cyanovirin” (Boyd et al. Antimicrob Agents Chemother 41(7): 1521-1530, 1997; Hammar et al. Ann N Y Acad Sci 724: 166-169, 1994; Kaku et al. Arch Biochem Biophys 279(2): 298-304, 1990). GNA is non-toxic and sufficiently safe that it has been incorporated into genetically engineered rice and potatoes (Bell et al. Transgenic Res 10(1): 35-42, 2001; Rao et al. Plant J 15(4): 469-477, 1998). These lectins bind to glycoproteins having a high mannose content such as found in HIV surface proteins (Chervenak et al. Biochemistry 34(16): 5685-5695, 1995). GNA has been employed in ELISA to assay HIV gp120 in human plasma (Hinkula et al. J Immunol Methods 175(1): 37-46, 1994; Mahmood et al. J Immunol Methods 151(1-2): 9-13, 1992; Sibille et al. Vet Microbiol 45(2-3): 259-267, 1995) and feline immunodeficiency virus (FIV) envelope protein in serum (Sibille et al. Vet Microbiol 45(2-3): 259-267, 1995). While GNA binds to envelope glycoproteins from HIV (types 1 and 2), simian immunodeficiency virus (SIV) (Gilljam et al. AIDS Res Hum Retroviruses 9(5): 431-438, 1993) and inhibits the growth of pathogens in culture, (Amin et al. Apmis 103(10): 714-720, 1995; Hammar et al. AIDS Res Hum Retroviruses 11(1): 87-95, 1995) such in vitro studies do not reflect the complex, proteinacious milieu found in HIV infected blood samples. It is therefore not known if lectins capable of binding high mannose glycoproteins in vitro would be able to bind such molecules in HIV infected blood samples. On the contrary, it is generally considered that the high concentrations of antibodies to gp120 typically present in individuals infected with HIV would sequester the high mannose glycoprotein sites to which lectins such as GNA bind.
Accordingly, although lectins are known to bind viral envelope glycoproteins, no previous technologies have been developed using lectins to directly adsorb HIV or other enveloped viruses from the blood using in vivo dialysis or plasmapheresis. Therefore, there is an ongoing need for novel therapeutic approaches to the treatment of HIV and other viral infections. In particular, there is a need for the development of novel approaches to reduce viral load so as to increase the effectiveness of other treatments and/or the immune response.
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