Title:  Channel forming toxins as antiviral agents

United States Patent:  6,495,315

Issued:  December 17, 2002

Inventors:  Hildreth; James E. K. (Woodstock, MD); Nguyen; Dzung H. (Baltimore, MD); Buckley; James Thomas (Victoria, CA)

Assignee:  The Johns Hopkins University (Baltimore, MD)

Appl. No.:  758248

Filed:  January 12, 2001

The infectivity of a population of enveloped viruses which comprise a glycosylphosphatidylinositol-anchored protein in their membrane can be reduced by employing certain toxins such as aerolysin, alpha toxin of Clostridium septicum, or enterolobin. Toxins which bind to glycosylphosphatidylinositol-anchored proteins inactivate such viruses. The toxins can be used to produce attenuated viral vaccines, to purge blood products, cells, or tissues of such viruses, and to detect viruses in samples.

DETAILED DESCRIPTION OF THE INVENTION

It is a discovery of the inventors that certain toxins bind with high affinity to viruses such as HIV-1 whose envelopes comprise GPI-anchored proteins. It is a further discovery of the inventors that toxins which oligomerize and form ion channels upon binding to such viruses are capable of neutralizing the infectivity of the viruses.

These discoveries make possible the treatment of certain viruses with a toxin to reduce the infectivity of the viruses. Viruses which can be employed in the invention are enveloped viruses containing GPI-anchored proteins in their membrane, such as HIV-1. Enveloped viruses are viruses which are released from eukaryotic cells by a process of budding, during which the viral particles become surrounded by a portion of membrane from the host cell that includes viral proteins. Examples of enveloped viruses include retroviruses such as HIV-1, Rous sarcoma virus, Semliki forest virus, vesicular stomatitis virus, herpes virus, influenza virus, flavivirus, and bunyavirus. Enveloped viruses for use with the invention have GPI-anchored protein in their membrane. Preferably the envelopes are unilamellar. GPI-anchored proteins are common in eukaryotic cells and can be obtained by a virus from the host cell membrane during budding. Examples of viruses whose envelopes contain GPI-anchored proteins are HIV-1, simian immunodeficiency virus (SIV), human cytomegalovirus (HCMV), and human T cell leukemia/lymphoma oncovirus type I (HTLV-1).

A population of viruses containing GPI-linked proteins in their membranes can be contacted with a toxin which binds to the GPI-linked proteins. A "population" of such viruses refers to a plurality of the viruses existing in any form consistent with treatment by a toxin to reduce their infectivity. For example, a population of viruses can be a suspension of virus particles present in a cell culture medium or other solution. A population of viruses can also be a pellet or a freeze-dried preparation containing the viruses. The population of viruses can be either in pure or impure form. If desired the viruses can be purified by any appropriate method known in the art either prior to or subsequent to treatment of the population of viruses with the toxin.

The population of viruses can be treated by certain toxins to reduce the infectivity of the population of viruses. The population of viruses is treated by exposure to the toxin in any manner which renders the infectivity of the population of viruses less than it would have been without the treatment. For example, the treatment can consist of contacting the population of viruses with the toxin by adding the toxin to a suspension containing the population of viruses in a culture medium under conditions which promote the binding of the toxin to the viruses. Once the toxin has bound to the viruses, their infectivity will be reduced by the bound toxin. Alternatively, the toxin can be added to cells from which the population of viruses are subsequently released, such that upon release, the viruses contain the bound toxin.

A variety of toxins are suitable for use in the invention A suitable toxin has the property of binding with high affinity to GPI-anchored protein in the membrane of the virus or host cell containing the virus and thereby inactivating the virus or rendering the infectivity of a population of such viruses less than it would have been without the bound toxin. While the invention is not limited to any particular mechanism, the loss of infectivity may be related to the formation of ion channels by the toxin, for example by oligomerization of the toxin to yield one or more pore-forming structures in the viral envelope. Alternatively, the loss of infectivity may be due to steric inhibition of virus binding to cellular receptors. Suitable toxins include any of the aerolysins of Aeromonas species (Parker et al., 1996), alpha toxin of Clostridium septicum Ballard et al., 1995), enterolobin of Enterolobium contortisiliquum (Fontes et al., 1997; Sousa et al., 1994; Sousa & Morhy, 1989), and variants of them which have the property of binding with high affinity to GPI-anchored protein in the membrane of the virus or host cell containing the virus and thereby inactivating the virus. Proaerolysin and some aerolysin variants bind GPI-anchored proteins but do not form channels. They can be used to bind, but they do not reduce infectivity of the virus.

A toxin binds to GPI-anchored proteins if, when contacted with cells or enveloped viruses, the toxin molecules associate with or non-covalently bond to GPI-anchored protein molecules or regions of the cell or viral membranes containing GPI-anchored proteins. A toxin binds selectively to GPI-anchored proteins if it binds to such proteins with higher affinity than to other proteins or binds to membrane regions containing GPI-anchored proteins with higher affinity than to other membrane regions. Typically a toxin will bind at least 2-fold, 3-fold, 5-fold, 10-fold, 100-fold, or 1000-fold more avidly to a GPI-anchored protein than to other proteins.

The infectivity of a population of viruses can be measured by determining the percentage of cells which become infected when exposed to a given dose of virus from the population of viruses. The multiplicity of infection for such measurements will preferably be less than 1 and more preferably less than 10^-1 or 10^-2. Infectivity can be expressed as the fraction or percentage of host cells which become infected. Alternatively, infectivity can be expressed as the amount of virus or viral protein produced by a given number of host cells, or as the number, fraction, or percentage of cells that die upon exposure to the virus. The infectivity of the population of viruses can be reduced according to the method of the invention by any amount. Preferably, a treatment will reduce the infectivity of a population of viruses by at least 50%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%.

An attenuated viral vaccine according to the invention is a vaccine which comprises attenuated virus and which induces an immune response when administered to a human patient or an animal. The virus contained in the vaccine can be attenuated according to the invention by treatment with a toxin which binds to GPI-anchored proteins in the viral membrane. This can be accomplished by any of the methods described above. The virus is attenuated when the infectivity of a population of the virus is reduced by at least 30%, at least 50%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%. The vaccine can be administered therapeutically to an infected individual or prophylactically to uninfected individuals. The vaccine can be administered by any suitable route, for example by oral or intranasal administrtion, or by intramuscular, intravenous, or transdermal injection. The vaccine can be lyophylized to enhance its stability. The vaccine can comprise an adjuvant, a potentiator, a stabilizer, a buffer or other substances suitable for safe and effective administration. The vaccine can also be combined with other vaccines into a combination vaccine.

A biological sample can be purged of enveloped viruses that comprise a GPI-anchored protein in their membrane. A biological sample can be a blood product, a cell suspension or other arrangement of cells obtained from any organism, or a tissue obtained from any organism. A blood product is whole blood or any cell, protein, or liquid fraction obtained from the whole blood or serum of a human or an animal, or any product derived from it. The sample can contain the virus to be purged, located in the extracellular portion of the sample. The biological sample can be subjected to the purging process whether or not the sample is known to contain enveloped viruses that comprise a GPI-anchored protein in their membrane.

Purging a sample that contains or possibly contains such viruses can be accomplished by contacting the sample with a toxin which binds GPI-anchored proteins so as to either functionally or physically remove the viruses from the biological sample. The sample is functionally purged if the infectivity of the viruses in the sample is reduced by at least 50%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%. The sample is physically purged if the toxin binds to at least 50%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% of the viruses in the sample, and at least 50%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% of the bound viruses are then separated or removed from the sample. A variety of methods can be used to separate the viruses from the sample. These include binding the viruses to immobilized toxin which is bound to a solid support, such as the matrix of a chromatographic material, magnetic particles, the surfaces of a plastic container, polymer fibers, and the like. Alternatively, the toxin can be non-immobilized, e.g., added free in solution to bind to the viruses, and the toxin can then be separated using an immobilized receptor for the toxin, such as an antibody to the toxin. Alternatively, no separation step need be performed if the viruses contained in the sample are rendered non-infective upon binding to the toxin. The virus is rendered non-infective upon binding to the toxin if, at least 50%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% of the viruses are bound to the toxin, and the infectivity is reduced by at least 50%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%.

The presence of an enveloped virus in a sample can be detected by means of the selective binding of a toxin to GPI-anchored proteins in the viral envelope. The toxin can optionally be labeled, for example by incorporation of a suitable radiolabel into the toxin molecule or by covalently or non-covalently attaching to the toxin molecule a suitable fluorescent label. The binding of the toxin to the enveloped virus can be detected and used as an indication of the presence of the enveloped virus in the sample. Any biological sample suspected of harboring an enveloped virus can be used. For example, the sample can be blood, plasma, tissue, urine or other bodily fluid from a patient or an animal. The sample can also be a cell from a cell culture or cell suspension, or it can be an organ intended for transplantation. The entire sample or a portion of the sample can be processed to prepare a cell-free extract. The cell-free extract should be substantially free of whole cells and cell fragments which contain GPI-anchored proteins but should preferably contain a measurable fraction of enveloped virus present in the original sample. A measurable fraction of an enveloped virus is an amount which can be detected or quantified by the chosen method of detecting the virus, examples of which are described below. Any method known in the art can be used to produce a cell-free extract, such as homogenization of the sample followed by centrifugation, ultrafiltration, trapping the viral particles or non-viral contaminants on a column or other solid support, or any combination of such methods.

In order to detect the virus in the cell-free extract, the extract is contacted with toxin under conditions which favor the binding of the toxin to GPI-anchored proteins in the viral envelope. For quantitative determination of the amount of virus in a sample, the toxin is preferably labeled and in molar excess over the number of toxin binding sites in the sample, so that the amount of labeled toxin that binds provides an accurate representation of the number of enveloped viruses in the sample. For example, the toxin can be in at least 1.2-fold, 1.5-fold, 2-fold, 3-fold, 5-fold, 10-fold, 30-fold, or 100-fold excess over the number of viral toxin binding sites. The appropriate level of excess labeled toxin can be determined by adding increasing amounts of toxin to an extract containing a constant amount of enveloped virus. The level of toxin is sufficient if further increases in toxin do not alter the amount of virus detected.

In some embodiments, detection or quantification of the amount of virus-bound toxin employs separation of virus-bound toxin from unbound toxin, or from toxin bound to non-viral structures. Any separation technique known in the art can be used, such as centrifugation, ultrafiltration, trapping the viral particles or non-viral contaminants on a column or other solid matrix, or any combination of such methods. Following separation of virus-bound toxin, the toxin is detected. If the toxin is radiolabeled, for example using ³ H, ¹⁴ C, ³² P, ³5 S, ¹²⁵ I or some other suitable radioisotope, then a method of detection is chosen which is suitable to detect the emitted radiation (e.g., scintillation counting or gamma counting). If the toxin is fluorescently labeled, then a suitable spectrofluorometric method can be used. In one embodiment, the binding of fluorescently labeled toxin to the enveloped virus is determined by quenching of fluorescence as the labeled toxin molecules oligomerize within the viral membrane. Immunological and enzyme-based methods can also be used to detect toxin bound to virus.

The method of detection can either be used non-quantitatively, to detect the presence or absence of the virus in the cell-free extract, or quantitatively, to detect the amount of virus in the cell-free extract. In either case, the sample under investigation should be compared to a control sample which is known to be free of enveloped viruses that bind the toxin. The amount of bound toxin in the control sample can be subtracted from the amount of label in the sample under investigation. The remaining toxin is representative of the presence and amount of enveloped virus in the cell-free extract. Based on the number of toxin molecules bound, and the number of toxin binding sites per virus, the number of virus particles in the extract and in the original sample can be readily determined.

Because the method of detecting enveloped viruses described above will give a cummulative measure of enveloped viruses containing GPI-anchored proteins, in some embodiments it will be desirable to subsequently perform other analyses to determine the type of viruses present. This can be accomplished by performing standard analyses such as immunological or electron microscopic analyses.

Claim 1 of 11 Claims

We claim:

1. A method of treating a population of enveloped viruses which comprise a glycosylphosphatidylinositol-anchored protein in their membrane, the method comprising the step of:

contacting the population of enveloped viruses with an amount of a channel forming toxin which binds to glycosylphosphatidylinositol-anchors, wherein the amount is sufficient to reduce infectivity of the population of viruses by at least 50%.
 

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