HK1118077A - Method for removal of viruses from blood by lectin affinity hemodialysis - Google Patents

Description

Method for removing virus in blood by lectin affinity hemodialysis

This application claims priority to U.S. provisional application 60/440771, filed on 17/1/2003, the contents of which are incorporated herein by reference.

Technical Field

The present invention relates to the field of therapeutic methodologies for treating viral infections.

Background

Many viruses have been reported to be pathogenic to humans, and many of these viruses are currently neither pharmaceutical nor vaccine-free. In the case of viruses currently having therapeutic drugs, the therapeutic effects are often limited by the occurrence of resistance mutations and drug side effects; examples of such viruses include hepatitis c and Human Immunodeficiency Virus (HIV).

HIV is the causative agent of acquired immunodeficiency syndrome (AIDS). It selectively infects cells of the immune system thereby impairing the immune response of the infected individual. It is estimated that there are more than one million HIV-infected individuals in the United states and more than 1300 million HIV-infected individuals worldwide. The clinical manifestations of HIV infection include a prolonged asymptomatic state followed by a loss of T4 lymphocytes that predisposes the individual to opportunistic infections and tumors.

HIV-1 replication occurs primarily in CD4+ lymphocytes, most of which are located in lymphoid organs such as peripheral lymph nodes and the spleen. HIV-1 is also found in macrophages and macrophage-like cells, such as microglia (microroglia) in the central nervous system (Cohen et al. Immunol Rev 159: 31-48, 1997).

HIV-1 plasma concentrations are closely related to the presence of HIV-1 infected peripheral blood lymphocytes, such as the clinical status of HIV-1 infected individuals. (Ferre et al J Acquir Immune Defic Syndr Hum Retrovirol10 (supplement 2): S51-56, 1995; O' Brien et al N Engl J Med 334 (7): 426-. The half-life of circulating virions is 6 hours, whereas that of infected cells HIV-1 in the peripheral blood is 1.6 days. Is administered at a rate of 10 days¹⁰The above virions are released into circulating blood (Ho et al J biol Regul Homeost Agents 9 (3): 76-77, 1995; Ho et al Nature 3/3 (6510): 125-. The ability of the host immune system to maintain suppression of HIV infection and limit clinical symptoms, is proportional to viral load. Antiretroviral therapy, nucleoside congeners, non-nucleoside reverse transcriptase inhibitors and protease inhibitors are intended to reduce the viral load so that the immune system can control and clear residual infection (Fauci, Harrisons Principles of Medicine: 1791-1856, 1998).

HIV infection is mediated by gp120, which binds to CD4 as well as to surface chemokine receptors. In the cell, the virion is uncoated and the viral RNA is reverse transcribed to double stranded DNA. Proviral DNA enters the nucleus, integrates into the host genome, and is transcribed into viral RNA which is translated into viral proteins. Mature virions are assembled and released from the cell body by budding. (Fauci et al Ann Intern Med 124 (7): 654-. Dying cells can also release all of their cellular contents, including intact viral particles and fragments, into the blood, so that circulating blood of HIV-infected individuals contains intact viral particles and viral proteins (especially virulent viral surface proteins).

Aids is marked by the gradual loss of CD4+ T cells, eventually disabling the immune system from fighting opportunistic infections. Although the mechanism by which HIV causes aids is poorly understood, clinical data suggest that in addition to the loss of infected T cells, a number of uninfected T cells are imminent to death, and HIV envelope proteins appear to be closely related.

The major envelope glycoprotein gp120 of HIV has been shown to have potent biological activity in vitro. gp120 causes apoptosis of CD4+ T cells and directs cellular clearance when gp120 is anchored to CD4+ cells and opsonized by complement in the presence of anti-viral envelope antibodies. The combined effect is the destruction of uninfected immune cells. Furthermore, HIV envelope proteins are involved in elevated c-globulin blood (c-globulin. The average gp120 level in AIDS patients was found to be 29 ng/ml, which is several orders of magnitude higher than the virus concentration.

Currently there is no cure for HIV infection. Reverse transcriptase and protease inhibitors have been approved for the treatment of HIV. A typical treatment regimen employs an approved combination of drugs known as HAART (highly active antiretroviral therapy). Although more than 16 drugs and pharmaceutical compositions are approved by the U.S. Food and Drug Administration (FDA) for the treatment of HIV infection, the emergence of drug-resistant mutants and the presence of non-treatable viral reservoirs (e.g., within memory T cells) limit the use of these drugs. Unfortunately, no effective HIV vaccine has been developed as expected, in part because of the rapid mutation of the HIV genome, which is not as accessible as immunogenic epitopes for viral proteins. Therefore, new therapeutic approaches are urgently needed.

In vitro therapy provides a treatment modality that can be used for systemic diseases. In vitro plasma perfusion, plasma replacement and lymph replacement of protein A have all been used as immunomodulatory treatments for HIV infection, but have resulted in thrombocytopenia (Kiprov et al, Curr student Blood transfuses 57: 184-. These therapies are proposed to remove immune complexes and other humoral mediators generated during HIV infection. They do not directly remove the HIV virus. The mechanism by which in vitro plasma replacement limits viral replication has been tested in preliminary experiments (Bisaccia et al J Acquir Immune DefficSyndr 6 (4): 386-. However, none of these therapies can remove both virus and viral proteins.

It has been proposed to remove HIV from blood products using chromatographic techniques. In 1997, Motomura et al proposed the use of sulfonate salt porous ion exchangers to remove HIV and related substances from body fluids (us patent 5667684). Takashima and colleagues (U.S. Pat. No. 5041079) provide ion exchangers containing a solid matrix of slightly acidic or slightly basic surfaces for the ex vivo removal of HIV from a patient's body fluids. Both methods are similar to those of Porath and Janson (U.S. Pat. No. 3925152), and both describe a method for separating a mixture of charged colloidal particles, such as virus variants, by passing the mixture through an adsorbent consisting of an insoluble organic integration of basic nitrogen-containing groups and acidic carboxyl or sulfo amphoteric components (U.S. Pat. No. 3925152). However, none of these chromatographic materials is selective for viruses and will therefore remove many other important components and therefore not be useful for in vivo blood purification.

Also, immunoabsorption techniques have been proposed to treat viral infections. Terman et al, 1980, proposed a plasmapheresis device for the extracorporeal treatment of disease, which contained an immunoadsorbent immobilized on a large surface area spiral membrane to remove pathogens (U.S. patent 4215688). The device does not envision a method of directly treating blood and requires a toxic reagent that is immunoreactive. In 1987 to 1988, Ambrus and Horvath proposed a blood purification system based on antibodies or antigen capture matrices bound to the outer surface of asymmetric toxin-permeable membranes (U.S. Pat. No. 4714556; U.S. Pat. No. 4787974), however, no examples have shown that pathogens can be removed. Lopukhin et al, 1991, reported that serum from rabbit anti-HIV proteins, when coupled to Sepharose4B or silica, can be used to remove HIV proteins from in vitro rabbit blood injected with recombinant HIV proteins (Lopukhim et al Vestn Akad Med Nauk SSSR 11: 60-63, 1991). This strategy is ineffective, however, because it requires extracorporeal blood adsorption and does not provide a mechanism for removing free HIV virions from blood (Lopukhin et al, 1991, supra) U.S. patent 6528057 describes methods for removing viruses and viral nucleic acids using antibodies and antisense DNA.

Lectins are proteins that selectively bind to polysaccharides and glycoproteins, and are widely distributed in plants and animals. Although many lectins are not specific enough for use, some have recently been found to be highly selective for enveloped viruses (De Clercq. et al Med Res Rev 20 (5): 323-. Among the lectins having the above-mentioned properties are the lectins from snowflake (Galanthus nivalis), namely the snowflake lectin (GNA, Galanthus nivalis agglutinin), the lectins from the yellow Narcissus (Narcissus pseudo Narcissus), namely the yellow Narcissus agglutinin (NPA, Narcissus pseudo Narcissus agglutinin), and the lectins from the blue-green algae known as Nostoc ellipsosporum, namely the cyanovirin (Boyd et al, Antimicrob Agents adhesives chemi 41 (7): 1. 1530, 1997; Hadamar et al, Annmm Acad Sci 724: 166. 169, 1994; Kaku et al. Arch biom Biophys279 (2): 304, 1990). GNA is non-toxic and it is safe enough 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 J15 (4): 469-477.1998). These lectins bind to glycoproteins with high mannose content, such as the surface proteins of HIV (Chervenak et al Biochemistry 34 (16): 5685-5695, 1995). GNA has been used in enzyme-linked immunosorbent assays (ELISA) to detect HIV gp120 in human plasma (Hinkula et al J Immunol Methods 175 (1): 37-46, 1994; Mahmood et al J Immunol Methods151 (1-2): 9-13, 1992; Sibille et al Vet Microbiol45 (2-3): 259-42-267, 1995) and the Feline Immunodeficiency Virus (FIV) envelope protein in serum (Sibille et al Vet Microbiol45 (2-3): 259-267, 1995). Although GNA binds HIV (type 1 and type 2), the envelope glycoprotein of Simian Immunodeficiency Virus (SIV) (Gilljam et al AIDS Res Hum Retroviruses9 (5): 431- > 438, 1993), and inhibits pathogenic growth in culture (Amin et al Apmis103 (10): 714- > 720, 1995; Hammer et al AIDS Res Hum Retroviruses11 (1): 87-95, 1995), these in vitro studies fail to reflect the complex, proteinaceous mediators seen in HIV-infected blood samples. Thus, it is not known whether lectins that bind mannose-rich glycoproteins in vitro are able to bind such molecules in HIV-infected blood samples. In contrast, it is believed that the high concentration of gp120 antibodies typically present in HIV-infected individuals may block the binding sites for lectins such as GNA in mannose-rich glycoproteins.

Therefore, although it is known that lectins can bind to viral envelope glycoproteins, no technique has been developed for directly adsorbing HIV or other enveloped viruses in blood by in vivo lectin dialysis or plasmapheresis. Thus, there is a need for new therapeutic approaches to treat HIV and other viral infections. In particular, there is a need to develop new methods to reduce viral load to increase the efficacy of other therapies and/or immune responses.

Summary of The Invention

The present invention relates to a method for removing mannose-rich glycoproteins or fragments of mannose-rich pathogens from infected blood or infected plasma using lectins that bind such pathogens in an extracorporeal device. Accordingly, the present invention provides a method of reducing the viral load of an individual, the method comprising the steps of: obtaining blood or plasma from an individual; passing it through a porous hollow fiber membrane having lectin molecules immobilized in the outer portion of the pores of the membrane; the blood or plasma that passes through is collected and returned to the individual.

The passage of blood through hollow fibers immobilized with lectin, mannose glycoprotein rich viral particles and fragments thereof bind to lectin, thereby reducing the viral load in the effluent. In one embodiment, the invention employs lectins that bind to the envelope proteins of many subtypes of type 1, type 2 HIV and SIV viruses. The method of the present invention can reduce the number of virus particles in blood and can rapidly and effectively reduce the level of virus surface protein which may be toxic in infected blood. It will be appreciated by those skilled in the art that this method can help clear other common infections that occur with HIV-1, such as Hepatitis C Virus (HCV) (Fauci et al, 1998, supra).

Accordingly, it is an object of the invention to provide a method for reducing the viral load in the blood of an individual infected with a virus. In one embodiment, the method is to remove the viral particles or protein fragments or a mixture of both from the blood of an individual infected with the virus.

It is another object of the present invention to provide a method for reducing the viral load in blood by extracorporeally circulating blood through hollow fibers having immobilized thereon lectins having affinity for mannose-rich glycoproteins of viruses.

It is a further object of the present invention to provide a device comprised of hollow fibers having an outer surface intimately associated with an immobilized lectin having specific affinity for mannoprotein-rich proteins of viruses or other pathogens.

Drawings

FIG. 1 is a schematic longitudinal section of an affluent strain.

Fig. 2 is a schematic horizontal section through plane 2 in fig. 1.

Fig. 3 is a schematic representation of a channel in fig. 2, the hollow fiber membrane structure 40 being comprised of a section of pipe comprising a relatively dense ultrafiltration membrane 42 and a relatively porous outer section 44 in which affinity molecules 46, such as lectins, are immobilized.

FIG. 4 illustrates the removal of gp120 from HIV-containing physiological saline. Initially, gp120 was made up to a concentration of 500 ng/ml (1.6 ml per run) with phosphate buffered saline. Gp120 was passed through a column containing 0.2 ml of GNA Sepharose or Sepharose4B (control) at room temperature at a flow rate of 0.5-058 ml/min.

FIG. 5 illustrates the removal of gp120 immune complexes from the plasma of HIV-infected persons. The initial gp120 concentration of HIV-positive human plasma was 500 ng/ml (1.6 ml per run). Gp120 immune complexes were detected with goat anti-human IgG antibodies using a 10. mu.g/well GNA/NPA plate capture. The plasma was recirculated to the Glen Research column containing 0.2 ml of GNA agarose or control Sepharose4B at a flow rate of 0.5-058 ml/min at room temperature. Theoretical best fit index R of experimental curve (. smallcircle.)²The control line (□) is a straight line, 0.91.

Fig. 6A and 6B show that GNA agarose removes native HIV. FIG. 6A is a graph showing an exponential profile of plasmapheresis, where R is²0.90 (except at the point of 22 hours). FIG. 6B shows a logarithmic plot of the initial removal (virus) rate, with half the removal time being about 0.9 hours. Masterflex membrane pump was used, #14 silicon tubing (1.1 ml/min). The initial amount of plasma sample was 3 ml (100000 Copies Per Ml (CPM) BBIER 8-03030-. Taking a 250 microliter aliquot of plasma for RNA isolation; realtome RTPCR and Sybr green tracking dyes; thermocycling temperatures 95, 60, 72, 83 ℃ (15, 30, 60 seconds, 6 second readings, respectively); the Ct value of the initial curve at T-20 is calculated.

FIG. 7 illustrates the removal of gp120 from HIV-positive human blood. Initial gp120 in HIV-positive human plasma was 100ng/ml and the immune complex after lysis with acid/detergent before assay was detected with 10. mu.g/well GNA/NPA plates. The blood was repeatedly circulated through a Microkros column containing 0.6 ml of GNA agarose or control Sepharose 4B. Masterflex pump (1rpm) and Parmed6485-16 tubing were used at 37 ℃ and flow rate of 0.9 ml/min. Experimental Curve (□) (t)_1/222 minutes) of the theoretical best fit index R²The control curve (∘) is a straight line.

FIG. 8 illustrates the removal of hepatitis C virus from infected blood. The blood was repeatedly circulated through a Micrrokros column containing 0.6 ml of GNA agarose or control Sepharose 4B. The flow rate was 0.5 ml/min at room temperature using a Masterflex pump (1rpm) and Pharmed 6485-16 tube. The theoretical best-fit index of the curve is R²＝0.85。

Detailed Description

The term "viral load" as used herein for purposes of the specification and claims refers to the amount of virus particles or toxic fragments thereof in a biological fluid such as blood or plasma. Viral load is thus related to the number of viral particles in the body. The viral load may thus be a measure of any of a number of indicators of the presence of the virus, such as the number of copies of the virus per unit of blood or plasma, or the amount of the viral protein or fragment thereof per unit of blood or plasma.

The term "mannose-rich glycoprotein" as used herein for purposes of the specification and claims refers to a glycoprotein comprising a mannose-mannose linkage in a linked form of alpha-1- > 3 or alpha-1- > 6 mannose-mannose linkage. Some examples of such lectins include GNA, NPA, cyanovirin, and concanavalin a (cona).

The present invention relates to a method for removing pathogenic microorganisms and fragments thereof from infected blood or plasma using lectins in an extracorporeal device. Accordingly, the present invention provides a method of reducing the viral load in an individual, the method comprising the steps of: obtaining blood or plasma from an individual; passing it through a porous hollow fibre membrane having lectin particles immobilised in the extracellular portion of the membrane; the blood or plasma that passes through is collected and returned to the individual.

In a preferred embodiment, the method of the invention is carried out using an affinity column of the device shown in FIG. 1. Common types of such devices are described in U.S. patents 4714556, 4787974, and 6528057, the contents of which are incorporated herein by reference. In this device, blood is passed through a lumen of a hollow fiber ultrafiltration membrane, intimately connected on the side of the membrane not wetted by the blood, to immobilized lectins forming means capable of receiving and immobilizing viruses and their toxic and/or infectious fragments. Thus, the device is able to retain intact virus particles and virus glycoproteins bound by lectins while allowing other components to pass through the lumen.

HIV is the prototype virus described in the present invention, but the present invention can be used to remove any blood-borne virus. The apparatus is shown in detail in fig. 1-3 and comprises multiple channels of mesoporous fibrous ultrafiltration membranes, which form filtration chambers. An inlet and an outlet are in communication with the chamber. Preferably, the hyperosmotic membrane is an anisotropic membrane, having a tight or retentive side facing the blood stream. The membrane may be conveniently prepared from any of the polymeric materials known in the art, for example, polysulfone, polyethersulfone, polyamide, polyimide, cellulose acetate, and polyacrylamide. Preferably, the membrane pore size is 200-500nm, which allows intact viruses and virus particles and fragments (e.g., HIV virus particles with a diameter of 110 nm) to pass through, but does not allow most blood cells (red blood cells with a diameter of 2000 nm, lymphocytes with a diameter of 7000-12000 nm, macrophages with a diameter of 10000-18000 nm) to pass through. The apparatus is schematically shown in FIG. 1. The plug body 10 of the device comprises a chamber 12 for treating blood, which is formed by a glass inner wall 14. Surrounding the chamber 12 is an optional outer chamber 16, into which chamber 16 is fed via an inlet port 18, and from which temperature-controlled liquid is circulated via an outlet port 20. The device includes a blood inlet port 32 and an outflow port 34. The device also provides one or more ports 48 and 50 that communicate with the outer passage space in the column. As shown in fig. 1 and 2, the chamber 12 is equipped with a multi-layer ultrafiltration (tubular) membrane 22. These membranes preferably have an inner diameter of 0.3 mm and an outer diameter of 0.5 mm. FIG. 3 is a representative cross-sectional view of the channel 22 showing the anisotropy of the film; as shown in fig. 3, the hollow fiber membrane structure 40 is comprised of a tubular body formed of a polymeric material that includes a relatively dense ultrafiltration membrane 42 and a relatively porous outer portion 44, with lectin 46 immobilized within the portion 44. In operation of the device, a solution containing the lectin is added through inlet 48 to immobilize the lectin at the membrane exterior 22, as shown in FIG. 2. The non-immobilized lectin may be collected from outlet 50 by washing with saline or other solution.

To practice the method of the invention, blood containing viral particles and/or fragments thereof is drawn from a patient into contact with an ultrafiltration membrane. In a preferred embodiment, blood is separated into plasma and cellular components; contacting the plasma with a lectin to remove viral particles or fragments thereof by binding between the lectin and mannose-rich glycoproteins; the plasma and cellular components are then recombined and returned to the patient. Or separate return of the organelle components to the patient. This treatment is repeated periodically until the desired effect is achieved, for example 4 hours of treatment once a week.

Techniques for immobilizing enzymes, chelating agents and antibodies in dialysis columns have been developed (Ambrus et al Science 201 (4358): 837-839, 1978; Ambrus et al Ann Intern Med 106 (4): 531-537, 1987; Kalghatgi et al Res Commun Chem Pathol Pharmacol27 (3): 551 561, 1980) which are incorporated herein by reference. These dialysis columns can be used to prime a patient's blood via a direct connection and then returned to the patient without further manipulation. Blood can also be separated into plasma and cellular components using standard techniques. The cellular fraction may be combined with the plasma and returned, or the cellular fraction may be returned separately. The viral load of the column effluent can be determined by standard methods, such as the ELLSA method and nucleic acid amplification detection techniques. Prototype samples have been used to metabolically remove excess phenylalanine (Kalghatgi et al, 1980, supra; Ambrus, 1978, supra) or excess aluminum (Anthonone et al, J Amer soc Nephrol 6: 1271-. Techniques for immobilizing proteins on hollow fibers for use in the methods of the invention are described in U.S. Pat. Nos. 4,714,556, 4,787,974, and 5,528,057.

To immobilize the lectin on the ultrafiltration membrane, the ultrafiltration membrane polymer is first activated, i.e., chemically made to readily bind to the protein using techniques known in the art. A variety of different polymers may be used. For example, to prepare reactive acrylic polymers, carbodiimides can be used (Valuev et al, 1998, Biomaterials, 19: 41-3). Once the polymer is activated, the lectin may bind directly or through a linker, both of which may form an affinity matrix. Suitable linkers include, but are not limited to, avidin, streptavidin, biotin, protein a, and protein G. Alternatively, the lectin may be bound directly to the ultrafiltration membrane polymer using a coupling agent, such as a bifunctional agent; or may be directly bonded. In a preferred embodiment, GNA is covalently coupled to agarose to form an affinity matrix.

The following examples are intended to illustrate the invention without limiting its scope.

Detailed Description

Example one

This example shows the preparation of GNA covalently coupled agarose affinity matrices using cyanogen bromide. Direct coupling with cyanogen bromide (CNBr) -activated agarose was performed essentially as described by Cuarecasas et al (Cuatracasas et al Proc Natl Acad Sci USA 61 (2): 636-. Briefly, 1ml of NaHCO was dissolved in 0.1M at pH9.5₃GNA at a solution concentration of 10 mg/ml was added to 1ml CNBr activated agarose (Sigma, st. louis, MO) and allowed to react overnight at low temperature. After completion of the reaction, the unreacted substance was aspirated off, and the lectin-coupled agarose was thoroughly washed with sterile PBS. The lectin-agarose affinity matrix is then refrigerated for use. GNA agarose is also available from Vector Lab (Burlingame, ca, usa).

Example two

This example shows the preparation of lectin affinity matrices using cyanoborohydride to covalently couple GNA to glass beads via schiff base and a reducing reagent. Silica lectin affinity matrices were prepared using a modified Hermanson method (Hermanson. bioconjugate Techniques: 785, 1996). The GNA lectin was dissolved in 0.1M sodium borate solution buffer ph9.5 to a final protein concentration of 10 mg/ml and added to the aldehyde silica glass (Bioconnexant, Austin TX). This reaction is most efficient in alkaline pH environments but at pH7-9, typically the concentration of GNA is 2-4 times higher than the coupling site. To each ml of this reaction mixture was added 10. mu.l of 5M NaCNBH3 (Bioconnoxant, Austin TX) crosslinking solution formulated with 1M NaOH, and the mixture was allowed to react at room temperature for 2 hours. At the end of the reaction, the residual unreacted aldehyde groups on the glass surface were blocked with 20. mu.l of 3M ethanolamine pH9.5 per ml of the reaction solution. After reaction at room temperature for 15 minutes, the reaction solution was discarded, and unbound proteins and reagents were removed by washing with PBS. The affinity matrix was refrigerated in a refrigerator for future use.

EXAMPLE III

This example illustrates the coupling of GNA to an aminonitrilo (aminoceite) using glutaraldehyde. Aminonitriloplastics (diatomaceous earth containing silicates) were prepared by reacting a nitriloplast (celite) in 5% aqueous aminopropyltriethoxysilane overnight. The aminoplast was washed with water and ethanol to remove excess reagent and dried overnight to give a white powder. 1 g of the powder was suspended in 5ml of 5% glutaraldehyde (Sigma) and reacted for 30 minutes. Filtration and washing with water removes excess glutaraldehyde until no residual aldehyde is detected in the wash with Schiff's reagent. The filter cake was resuspended in 5ml Sigma borohydride coupling buffer containing 2-3 mg/ml GNA and reacted overnight at room temperature. After the reaction was terminated, unreacted GNA was washed off and the unreacted aldehyde group was amidated with ethanolamine as described above. The material was cryopreserved for use after final washing with sterile PBS.

Example four

This example illustrates the preparation of an exemplary lectin plasmapheresis device. To a small volume filter plug (Glen Research, Silverton, Va.) was added 0.2 ml of lectin resin for use, sealed and equilibrated with 5-10 column volumes of sterile PBS. The plug is used immediately.

EXAMPLE five

This example illustrates how a GNA lectin affinity hemodialysis device can be prepared. The viral blood purification apparatus (Hemopurifier) injects a microparticle slurry of GNA-immobilized agar beads or hypo-acetyl plastic beads prepared in sterile PBS buffer into the external compartment of a hollow fiber dialysis column using a syringe. For up to 15 ml of blood samples, a Microdros polyethersulfon hollow fiber dialysis column was used, which was equipped with Luer parts (200. mu. IDx 240. mu. OD, pore size 200-. The column filled with the affinity resin was equilibrated with 5-10 column volumes of sterile PBS.

EXAMPLE six

This example illustrates the removal of HIV gp120 from physiological saline using an affinity plasma exchange device. The plasmapheresis device described in example 4 was equilibrated with 5-10 column volumes of sterile PBS. Approximately 1.5ml of gp 120-containing (typically 500 ng/ml) sample was circulated at room temperature through the cartridge at a flow rate of 0.5-0.6 ml/min. The circulating fluid was tested for the presence of gp120 and gp120 immune complexes at various time intervals.

Gp120 quantitative ELISA assays for HIV-1 were performed using a modified Weiler method (Weiler et al J Virol methods 32 (2-3): 287-301, 1991). GNA/NPA plates were prepared on a Greiner C plate, 100. mu.l of protein (1-100. mu.g/ml each of GNA and NPA in PBS) was added dropwise to each well, and incubated at 37 ℃ for 2 hours. The plates were then washed with PBST (PBS containing 0.01% Tween 20) and blocked with casein blocking buffer for 1 hour. Plates not immediately used can be stored for 2 weeks at 4 ℃.

To detect free gp120, 100 microliters of test sample was incubated for 1-2 hours at 37 ℃. After capturing gp120, it was washed with PBS and 100. mu.l of an anti-gp 120 antibody appropriately labeled with HRP (horseradish peroxidase) (1: 2500 in blocking buffer) was added. After incubation for 1 hour at 37 ℃, the antiserum was aspirated, the plates were washed with 4x300 μ l PBSTA, and bound horseradish peroxidase was detected with a stabilized Trimethylbenzidine (TMB) enzyme substrate (BioFx) solution. To determine the formation of immune complexes and immune complexes, after capture, plates were washed with PBS and 100. mu.l of affinity purified horseradish peroxidase-labeled goat anti-human IgG antibody (1: 2500 in blocking buffer) was added. After incubation for 1 hour at 37 ℃, the antiserum was aspirated and washed four times with 300 microliters of PBSTA. Bound horseradish peroxidase was detected with trimethylbenzidine (BioFx).

FIG. 4 shows that GNA agarose is 99% efficient at removing gp120 from the buffer in 15 minutes. Because gp120 is a highly glycosylated protein, it can bind non-specifically to a variety of surfaces; it was not surprising that 85% of the gp120 input in the control column was also bound.

EXAMPLE seven

This example illustrates the removal of HIV gp120 from infected plasma using a lectin affinity plasmapheresis device. The plasmapheresis device described in example 4 was equilibrated with 5-10 column volumes of sterile PBS. 1.5ml of plasma containing gp120 (typically at a concentration of 500 ng/ml) was circulated through the column at a rate of 0.5-0.6/min at sample temperature. The circulating fluid was tested for the presence of gp120 and gp120 immune complexes at different suitable time intervals as in example 4.

Because anti-gp 120 antibodies are in HIV⁺Plasma is generally abundant and gp120 removal from infected plasma is expected to be more difficult than removal from buffer alone. Due in part to these antibodies, HIV⁺The amount of gp120 usually measured in plasma and blood is very low. To determine the amount removed, gp120 must be added to the plasma of infected patients to provide a test sample. This sample was confirmed by ELISA detection that all gp120 added to the sample formed a complex with anti-gp 120 antibody (values not published).

Figure 5 shows that GNA agarose affinity resin efficiently removed gp120 from the immune complex from HIV-infected plasma samples. The removal process is rapid with a significant half removal reaction time of 20 minutes. Partial gp120 signal (about 10% of the original gp120 immune complex) was not removed even after 7 hours, which appears to represent background binding of IgG in this assay.

Example eight

This example illustrates the use of GNA plasma placementAlternatively, HIV viral particles are removed from the infected plasma. HIV-infected plasma samples (ER8-03030-0002 native HIV, Boston Biomedia, Boston MA) containing 100000 copies (cpm) of virus per ml were taken and placed on 0.2 ml GNA agar columns for circulation as described in example 4. Aliquots of 250. mu.l of plasma were removed intermittently and viral RNA was extracted using TRI-LS reagent (MRC corporation) according to the manufacturer's instructions. Then using real-time RT PCR and Access 1 step reagent set (Madison, Wis.) from Promega, in 25 microliters gag gene primers containing 400nM SK432 and SK461, Sybr green (1: 10000), 1xSCA blocking buffer, 3mM MgCl₂Viral RNA of HIV was quantitatively determined in reaction volumes of 400. mu.M dNTPs and 10. mu.l of unknown RNA from an armored RNA standard (Ambion Austin TX) or HIV-1 RNA. The amplification reactions were carried out in a SmartCycler real-time thermal cycler (Cepheid, Sunnyvale, Calif.) for the following time periods, essentially following the manufacturer's instructions: RT (45 min at 48 ℃), 40 cycles of PCR (94 ℃/15 sec; 62 ℃/30 sec; 72 ℃/60 sec; 83 ℃/reading). When amplification is to be confirmed, aliquots of 10 ml of amplification mixture prepared in 0.5 XTBE buffer, pH8.3, containing 0.25. mu.g/ml ethidium bromide are run on a 2% (w/v) agarose (Sigma, molecular biology grade) gel for 45 minutes at room temperature, 120 VCD. Images of the gel were taken with a uv conversion illuminator and then digitized and analyzed with ImageJ.

Fig. 6A, 6B show that GNA agarose effectively removes HIV virions from infected plasma. FIG. 6A is a data curve (R) fitted to an exponential decay²0.9) diagram. The curve predicts the amount of substantial HIV removal in about 10 hours, fig. 6B is a log plot of the HIV removal rate, and the time to HIV half removal is estimated to be 0.9 hours. Virus removal is expected to be a kinetic first order mechanical equation when GNA is much more abundant than virus. CPM represents the number of copies of HIV per ml.

Example nine

This example illustrates the removal of gp120 from HIV-infected blood using a GNA lectin-affinity hemodialysis unit. Since most HIV + plasma samples are low in gp120 or undetectable, 5ml of fresh packed red blood cells of O + type were mixed with 5 mmLiter of HIV-infected plasma (typically 10)⁵cpm) HIV infected blood samples were prepared and a sufficient amount of gp120IIIB was added to adjust the concentration to 100 ng/ml.

The affinity hemodialysis device described in example 5 was equilibrated with 5-10 cylinder volumes of sterile PBS. 10 ml gp 120-containing infected blood samples were circulated at 37 ℃ through the column at a flow rate of 0.9 ml/min using a Masterflex roller pump (rpm) and Parmed6485-16 silicon tubing. The circulating fluid was taken at various time intervals and the immune complex was denatured and neutralized with acid to destroy the immune complex and to detect the presence of free gp 120.

FIG. 7 shows circulation of blood samples in the column with initial gp120 concentrations within 4-6 hours (significant t)_1/222 minutes) from 100ng/ml to background level. The control column removed gp120 very slowly.

Example ten

This example illustrates the removal of HCV (hepatitis c virus) from infected blood by GNA lectin affinity hemodialysis. To show the high specificity of GNA lectin to eliminate virus, we performed lectin affinity hemodialysis on HCV-infected blood. The lectin affinity hemodialysis device described in example 4 was equilibrated with 5-10 column volumes of sterile PBS. HCV-infected blood samples were prepared by mixing 1ml of fresh packed erythrocytes type O with 1ml of HCV-infected plasma. The infected blood samples were cycled repeatedly through the column at room temperature at a rate of 0.5 ml/min using a Masterflex roller pump (1rpm) and Pharmed 6485-16 tubing. The circulating fluid was taken at different time intervals to test for the presence of HCV viral RNA.

Viral RNA was isolated from 10. mu.l of plasma using TRI-LS (MRC) reagent according to the manufacturer's instructions. Viral RNA was then assayed for HCV by quantitative RT PCR using an ImpromII reagent set of Promeg (Madison, Wis.) containing 400nM EY80 and EY78HCV specific primers, Sybr green (1: 10000), 1XSCA blocking buffer, 3mM MgCl in a 25 microliter reaction volume₂400uM dNTPs, Tfl polymerase and AMV reverse transcriptase 0.2 units/microliter each. Typically, 50. mu.l of the mixture is used to solubilize RNA isolated from 100. mu.l of plasmaThe resolved mixture was equally divided into two identical samples. The amplification reaction was carried out in a SmartCycler real-time thermal cycler (Cepheid, CA) for the following reaction times, essentially according to the manufacturer's instructions: RT (45 min at 48 ℃), 40 cycles of PCR (94 ℃/15 sec; 62 ℃/30 sec; 72 ℃/60 sec; 87 ℃ reading). The amount of viral RNA was estimated by comparing the signal intensity of the viral RNA standard at the initial stage of the amplification reaction (Ct ═ 20).

Figure 8 shows that blood is repeatedly circulated in the column, with an initial HCV reduction of about 50% over 3 hours (significant t)_1/23 hours). The curve is reasonably fit to the exponential decay.

From the foregoing, it will be apparent to those skilled in the art that various modifications can be made in the above-described methods and compositions without departing from the spirit and scope of the invention. Accordingly, the present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. Accordingly, the present examples and embodiments are to be considered as illustrative and not restrictive, and all changes coming within the meaning and equivalency range of the appended claims are intended to be embraced therein.

Claims (13)

1. A method for reducing the blood count of virally infected individuals for viral particles and lectin-binding fragments thereof, comprising the steps of:

a) obtaining blood from an infected individual;

b) passing the blood through a porous hollow fiber membrane having lectin molecules immobilized in the porous outer portion of the membrane, said lectin molecules being capable of binding mannose-rich glycoproteins.

c) Collecting the blood passing through;

d) the blood is returned to the individual.

2. The method of claim 1, wherein said lectin is selected from the group consisting of Galanthamine (GNA), Narcissus (NPA), cyanovirin, and concanavalin a.

3. The method of claim 2, wherein the lectin is GNA.

4. The method of claim 1, wherein the lectin molecule binds to a viral envelope protein or a fragment thereof.

5. The method of claim 3, wherein the virus is HIV-1.

6. The method of claim 1, wherein the virus is HCV.

7. A method for reducing viral load in the plasma of a virally infected individual, the method comprising:

a) obtaining plasma of the subject;

b) passing the blood through a porous hollow fiber membrane having lectin molecules immobilized in the porous outer portion of the membrane, said lectin molecules being capable of binding mannose-rich glycoproteins.

c) Collecting the blood passing through;

d) the blood is returned to the individual.

8. The method of claim 7, wherein unbound plasma is mixed with the cellular component and returned to the subject.

9. The method of claim 7, wherein the lectin molecule is selected from the group consisting of GNA, NPA, concanavalin A, and cyanovirin.

10. The method of claim 9, wherein the lectin molecules are GNAs.

11. The method of claim 7, wherein the lectin molecule is capable of binding to a viral envelope protein or a fragment thereof.

12. The method of claim 7, wherein the virus is HIV-1.

13. The method of claim 7, wherein the virus is HCV.