Patent Publication Number: US-6699465-B2

Title: Covalent attachment of polymer to cell to prevent virus bonding to receptor

Description:
BACKGROUND OF THE INVENTION 
     1. Technical Field 
     The present invention relates generally to covalent modification of surface protein or carbohydrate for protecting an animal against viral attack. 
     2. Related Art 
     FIG. 1 illustrates a cellular cross-sectional view of viral disease pathogenesis, in accordance with the related art. FIG. 1 shows cells  10  and  20  within an extracelluar environment  15 . The cell  10  comprises a cell interior  12 , and a nucleus  11  within the cell interior  12 . A viral receptor  14  is coupled to a membrane surface  13  of the cell  10 . The cell  20  comprises a cell interior  22  and a nucleus  21  within the cell interior  22 . A viral receptor  24  is coupled to a membrane surface  23  of the cell  20 . 
     An extracellular virus  1  in the extracellular environment  15  enters the cell  10  through the viral receptor  14 . While within the cell interior  12  of the cell  10 , the virus  1  undergoes multiple rounds of replication, resulting in the replication of viral DNA, RNA, and protein from viruses  2 ,  3 ,  4 , and  5 , which: are packaged into their envelopes to become viruses  6 ,  7 ,  8 , and  9 , respectively; and pass through the membrane surface  13  into the extracellular environment  15 . 
     The virus  9  enters the cell  20  through the viral receptor  24 . While within the cell interior  22  of the cell  20 , the virus  9  undergoes multiple rounds of replication (not shown) in the cell interior  22  of the cell  20 , and subsequently passes through the membrane surface  23  enters the extracellular environment  15  as replicated viruses  27 ,  28 , and  29 . 
     Unfortunately, the viral replication in the cells  10  and  20 , as described supra, causes destruction of the cells  10  and  20  and possible consequent viral disease of an animal (i.e., a human or non-human animal) that comprises the cells  10  and  20 . Thus, there is a need to prevent such viral disease from occurring in the animal. 
     SUMMARY OF THE INVENTION 
     The present invention provides a chemo-physiological structure, comprising: 
     a membrane surface of a cell of an animal; 
     a viral receptor coupled to the membrane surface; and 
     a linker molecule covalently bonded to a tissue member selected from the group consisting of the membrane surface, the viral receptor, and a combination thereof, wherein a polymer is covalently attached to the linker molecule, and wherein the polymer prevents an extracellular virus from bonding to the viral receptor. 
     The present invention provides a method for forming a chemo-physiological structure, comprising: 
     providing a membrane surface of a cell of an animal and a viral receptor coupled to the membrane surface; and 
     covalently bonding a linker molecule to a tissue member selected from the group consisting of the membrane surface, the viral receptor, and a combination thereof, wherein a polymer is covalently attached to the linker molecule, and wherein the polymer prevents an extracellular virus from bonding to the viral receptor. 
     The present invention provides a chemo-physiological structure, comprising: 
     a virus having a capsid; and 
     a linker molecule covalently bonded to the capsid, wherein a polymer is covalently attached to the linker molecule, and wherein the polymer envelops the virus in a manner that prevents the virus from bonding to a cell of an animal. 
     The present invention provides a method for forming a chemo-physiological structure, comprising: 
     providing a virus having a capsid; and 
     covalently bonding a linker molecule to the capsid, wherein a polymer is covalently attached to the linker molecule, and wherein the polymer envelops the virus in a manner that prevents the virus from bonding to a cell of an animal. 
     The present invention prevents a virus from recognizing the viral receptors or the cell membrane of an animal cell, and thus from entering an interior portion of the cell. Accordingly, the present invention protects the animal cell against viral attack and prevents viral infection of the animal. The present invention may be used to prevent viral infection in both human animals and non-human animals. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 depicts a cellular cross-sectional view of viral disease pathogenesis, in accordance with the related art. 
     FIG. 2 depicts a cellular cross-sectional view of how viral disease may be prevented by using polymerated linker chemicals, in accordance with the present invention. 
     FIG. 3 is enlarged view of a virus of FIG.  2  and its surrounding environment, in accordance with embodiments of the present invention. 
     FIG. 4 depicts an animal and modes of delivering a polymerated linker chemical therein, in accordance with embodiments of the present invention. 
     FIG. 5 lists exemplary viruses of human significance and of veterinary significance, in accordance with embodiments of the present invention. 
     FIG. 6 depicts an exemplary chemistry of coupling the polymerated linker chemical of FIG. 2 or FIG. 3 to a protein, in accordance with embodiments of the present invention. 
     FIG. 7 lists exemplary polymeric linker compounds and associated protein or carbohydrate targets that can be covalently reacted with the exemplary polymeric linker compounds, for use in conjunction with FIG.  2  and in accordance with embodiments of the present invention. 
     FIG. 8 is a bar graph showing the effect of covalent modification of monkey kidney epithelial cells on the rate at which the cells become infected with a virus. 
     FIG. 9 is a bar graph showing the effect of covalent modification of Simian Vacuolating Agent (SV40) virus on the rate of viral infection of monkey kidney epithelial cells located near the SV40 viruses. 
     FIG. 10 depicts a densitometry curve for a control sample for the SV40 virus of FIG.  9 . 
     FIG. 11 depicts a densitometry curve for the covalently modified SV40 virus of FIG.  9 . 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     FIG. 2 illustrates a cellular cross-sectional view of how viral disease may be prevented, in accordance with the present invention. FIG. 2 shows cells  30  and  40  within an extracelluar environment  45 . The cell  30  comprises a cell interior  32 , and a nucleus  31  within the cell interior  32 . A viral receptor  34  is coupled to a membrane surface  33  of the cell  30 . The cell  40  comprises a cell interior  42 , and a nucleus  41  within the cell interior  42 . A viral receptor  44  is coupled to a membrane surface  43  of the cell  40 . 
     Also shown in FIG. 2 are extracellular viruses  55  and  56 , which are unable to access the viral receptors  34  and  44  because of a blocker layer  54  and blocker envelopes  57  which are formed in accordance with the present invention. By being so prevented from accessing the viral receptors  34  and  44 , the extracellular viruses  55  and  56  are said to be “inactivated.” The blocker layer  54  results from covalent bonding of a polymerated linker chemical  50  to the viral receptors  34  and the membrane surface  33  of the cell  30 , and also to the viral receptors  44  and the membrane surface  43  of the cell  40 . The polymerated linker chemical  50  includes a linker molecule  51  with a covalently attached polymer  52 . The polymerated linker chemical  50  is said to represent an activated form of the polymer  52  (e.g., if the polymer is methylpolyethylene glycol (mPEG), then then “activated mPEG” is exemplified by having mPEG covalently bonded to the linker molecule of cyanuric chloride). The linker molecule  51  is covalently bonded to proteins or carbohydrates in the viral receptors  34  and  44 , and to proteins or carbohydrates in the membrane surfaces  33  and  43 . The covalent linking of the linker molecule  51  to a protein may include a covalent linking of the linker molecule  51  to an amino acid in the protein or to a sulfhydryl group in the protein. Thus, the linker molecule  51 , together with the covalently attached polymer  52 , is disposed between the virus  55  (or  56 ) and the viral receptors  34  and  44 . The polymer  52  has a “long chain length;” i.e., a chain length that is of sufficient magnitude to fill the space around itself to create the blocker layer  54 . Thus, the blocker layer  54  constitutes a barrier that prevents the viruses  55  and  56  from having access to the viral receptors  34  and  44 . In addition, the polymer  52  within the blocker layer  54  prevents the approach and binding of viruses by steric hindrance. Additionally, the polymer  52  may be highly hydrophillic so as to create a hydration zone around itself to alternatively create the blocker layer  54 . Inasmuch as the viruses  55  and  56  would covalently bond to the viral receptors  34  and  44  via a charge-charge coupling mechanism, the hydration zone encompassed by the blocker layer  54  effectively camouflages molecular charge sites and thus prevents the viruses  55  and  56  from having access to the viral receptors  34  and  44 . Thus, the polymer  52  effectively prevents the viruses  55  and  56  from recognizing the viral receptors  34  and  44  and thus from entering an interior portion of the cell  30  and of the cell  40 . 
     The blocker envelope  57  results from covalent bonding of a polymerated linker chemical  59  with the virus  56 . The polymerated linker chemical  59  includes a linker molecule  61  with a covalently attached polymer  62 . The polymerated linker chemical  59  may be the same as (i.e., chemically identical to), or different from, the polymerated linker chemical  50 . The linker molecule  61  is covalently bonded to proteins or carbohydrates in an outer portion (i.e., the capsid) of the virus  56 . The polymer  62  has a “long chain length;” i.e., a chain length that is of sufficient magnitude to fill the space around itself to create the blocker envelope  57 . Thus, the blocker envelope  57  constitutes a barrier that prevents the virus  56  from having access to the viral receptors  34  and  44  even if the blocker layer  54  were absent. In addition, the polymer  52  within the blocker layer  54  prevents, by steric hindrance, the virus  56  from approaching, and binding to, animal cells. Additionally, the polymer  62  may be highly hydrophillic so as to create a hydration zone around itself to alternatively create the blocker envelope  57 . Inasmuch as the virus  56  would covalently bond to the viral receptors  34  and  44  via a charge-charge coupling mechanism, the hydration zone encompassed by the blocker envelope  57  effectively camouflages molecular charge sites and thus prevents the virus  56  from having access to the viral receptors  34  and  44  even if the blocker layer  54  were absent. Thus, the polymer  62  effectively prevents the virus  56  from recognizing the viral receptors  34  and  44  and thus from entering an interior portion of the cell  30  and of the cell  40 . 
     FIG. 3 is enlarged view of the virus  56  and blocker envelope  57  of FIG. 2, in accordance with embodiments of the present invention. The virus  56  includes a viral core  47  and a capsid  48 . The viral core  47  includes genetic material (i.e., DNA or RNA). The capsid  48  is a shell comprising protein. Some viruses additionally include an outer lipid envelope (not shown) that surrounds the capsid. FIG. 3 shows that the linker molecule  61  of the polymerated linker chemical  59  is covalently bonded to the capsid  48 . In particular, the polymerated linker chemical  59  may be covalently bonded to an amino acid (e.g., lysine), a sulfhydryl group, or a carbohydrate at the capsid  48 . The polymer  62  of the polymerated linker chemical  59  envelops the virus  56  in a manner that prevents the virus  56  from bonding to a cell (and from entering the cell) of an animal. 
     The cells  30  and  40  of FIG. 2 may be treated in vivo within an animal  60  (see FIG. 4) with the polymerated linker chemical  50  or  59  (or both) for clinical purposes such for preventing or treating a viral infection. FIG. 4 shows the animal  60  having an epithelium  17  (i.e., membranous cellular tissue at external surfaces of the animal  60  or “skin”), an interior  18 , openings  63  and  64 , an organ  65  coupled to the opening  63 , a muscle  66 , and a blood vessel  67 . The animal  60  may be a human animal (e.g., a human being or a fetus) or a veterinary animal. A veterinary animal is a non-human animal of any kind such as, inter alia, a domestic animal (e.g., dog, cat, etc.), a farm animal (cow, sheep, pig, etc.), a wild animal (e.g., a deer, fox, etc.), a laboratory animal (e.g., mouse, rat, monkey, etc.), an aquatic animal (e.g., a fish, turtle, etc.), etc. The openings  63  and  64  include a cell  73  and  74 , respectively, the organ  65  includes a cell  75 , the muscle  66  includes a cell  76 , and the blood vessel  67  includes a cell  77 . The blood vessel  67  is part of a systemic vascular system (not shown) capable of transporting polymerated linker chemical  50  or  59  (or both) to cells distributed throughout the animal  60 . The openings  63  and  64  include any opening that pertains to the animal  60 . If the animal  60  is a human being, for example, then the openings  63  and  64  may include, inter alia, a nasal cavity, a mouth, a vagina if the animal  60  is female, etc. The organ  65  includes any organ that pertains to the animal  60 . If the animal  60  is a human being, for example, then the organ  65  may include, inter alia, a lung, a stomach, a kidney, a liver, etc. The organ  65  may be coupled to the opening  63  or  64 , or may rather be coupled to the blood vessel  67  through the systemic vascular system of the animal  60 . FIG. 4 also shows viruses  35 ,  36 ,  37 ,  38 , and  39  in the opening  63 , the opening  64 , the muscle  66 , the blood vessel  67 , and the organ  65 , respectively. 
     A polymerated linker chemical (PLC)  68  may be delivered to any cell of the animal  60  where viral infection is possible such as, inter alia, to any of the cells  73 - 77 , or to extracellular viruses in any opening (e.g., the openings  63  and  64 ), in any organ (e.g., the organ  65 ), in any muscle (e.g., the muscle  66 ), in any blood vessel (e.g., the blood vessel  67 ), or in any other relevant location such as a peritoneal cavity, etc. Said delivery of the PLC  68  may be accomplished in any manner known to one of ordinary skill in the art such as, inter alia, via spray bottle  70  into the opening  63 , via syringe  71  into the opening  64 , via needle  72  into the muscle  66 , and via intravenous delivery apparatus  69  into the blood vessel  67 . A spray of the PLC  68  from the spray bottle  70  may be, inter alia, aerosol activated. 
     There are numerous examples of how the PLC  68  may be delivered to cells of the animal  60  or to viruses within the animal  60 . As a first example, the PLC  68  may be packaged within the spray bottle  70  and sprayed into a nasal cavity as represented by the opening  63 , where the PLC  68  generates a blocker layer (see, e.g., the blocker layer  54  of FIG. 2) on the nasal epithelial cell  73  in the nasal cavity  63 , and a blocker envelope (see, e.g., the blocker envelope  57  of FIG. 2) over any extracellular virus that is present in the nasal cavity  63 . The PLC  68  from the spray bottle  70 , after being sprayed into the nasal cavity represented by the opening  63 , may be inhaled into a lung as represented by the organ  65 , where the PLC  68  generates a blocker layer on the pulmonary cell  75  in the lung, and a blocker envelope over any extracellular virus that is present in the lung. As a second example, the PLC  68  in the spray bottle  70  may be sprayed into a mouth as represented by the opening  63 , and may be inhaled into a lung as represented by the organ  65 , where the PLC  68  generates a blocker layer on the cell  75  in the lung, and a blocker envelope over any extracellular virus that is present in the lung. As a third example, the PLC  68  in the syringe  71  may be delivered to a vagina as by the opening  64 , where the PLC  68  generates a blocker layer on the vaginal cell  74  in the vagina, and a blocker envelope over any extracellular virus that is present in the vagina. Any mechanism discussed supra in conjunction with FIG. 2 for inactivating any of the viruses in FIG. 2 may be utilized for inactivating any of the viruses in FIG.  4 . 
     The cells  30  and  40  of FIG. 2 may be alternatively removed from the animal  60  of FIG.  4  and treated in vitro (i.e., outside of the animal) with the PLC  50  or  59 , or both (see FIG.  2 ), such as in a laboratory setting for such purposes as, inter alia, research or testing. The PLC  50  or  59 , or both may be delivered in vitro to any cell of the animal  60  that has been so removed from any portion of the animal  60 , such as to, inter alia, any of the cells  73 - 77  of FIG. 4, in any manner known to one of ordinary skill in the art such as, inter alia, by spraying the PLC  50  or  59 , or both on the cells, or by immersion of the cells into a liquid that includes the PLC  50  or  59 , or both, to form a blocker layer on the cells. In addition, the PLC  50  or  59 , or both, may be delivered in vitro to viruses in the vicinity of the cells so removed from the animal  60  of FIG. 4, in any manner known to one of ordinary skill in the art such as, inter alia, by spraying the PLC  50  or  59 , or both, on or near the viruses to form blocker envelopes around the viruses. 
     FIGS. 2,  3 , and  4  show “chemo-physiological structures.” A chemo-physiological structure is defined herein as an organic structure that includes at least one organism (e.g., an animal, a cell, a virus, or any portion thereof) and any chemical that is covalently bonded to any organism of the at least one organism. 
     As discussed supra in conjunction with FIGS. 2 and 3, the present invention uses a polymerated linker chemical  50  or  59  to generate the blocker layer  54  and the blocker envelope  57 , respectively, to inactivate the extracellular viruses  55  and  56  by preventing the extracellular viruses  55  and  56  from bonding with viral receptors  33  and  44  which are coupled to cells  30  and  40 , respectively. The use of the blocker layer  54  and the blocker envelope  57  is non-specific as to the type of virus that is inactivated and any virus that can infect an animal (human or non-human) can be inactivated in accordance with the present invention. FIG. 5 tabulates examples of viruses that can be inactivated in accordance with the present invention. Each listed virus in FIG. 5 is classified as to whether said listed virus is of human significance or of veterinary significance. A virus is of human significance if the virus is known to one of ordinary skill in the art as being capable of infecting a human animal. A virus is of veterinary significance if the virus known to one of ordinary skill in the art as being capable of infecting a non-human animal. The list of viruses in FIG. 5 is merely exemplary. Numerous viruses other than those listed in FIG. 5 can be inactivated in accordance with the present invention. 
     FIG. 6 illustrates an exemplary chemistry of coupling the polymerated linker chemical, as depicted in FIG. 2 or FIG. 3, to a protein, in accordance with embodiments of the present invention. In FIG. 6, two chemical reactions are illustrated. In the first chemical reaction shown in FIG. 6, a polymer  80  reacts with a linker molecule  81  to form a polymeric linker chemical (PLC)  82  in which the polymer  80  is covalently bonded to the linker molecule  81 . Specifically in FIG. 6, the polymer  80  is methoxypolyethylene glycol (mPEG) having the chemical structure of CH 3 (—O—CH 2 —CH 2 ) n —OH wherein n≧2. The linker molecule  81  is an alkyl halide (namely, cyanuric acid) and the resultant PLC  82  is 2-O-mPEG-4,6-dichloro-s-triazine. In the first chemical reaction, the hydroxyl group (OH − ) is a nucleophile that reacts generally with an alkyl halide (specifically, cyanuric chloride), resulting in displacement and release of the chlorine ion (CL − ) in position 2 of the cyanuric chloride triazine ring as well as release of the hydrogen ion (H − ) from the hydroxy group of the mPEG. The first chemical reaction may be implemented in any manner known to one of ordinary skill in the art such as in, inter alia, anhydrous benzene at a temperature of about 25° C. Formation of the PLC  82  of 2-O-mPEG-4,6-dichloro-s-triazine is well-known in the art and may be obtained commercially. 
     In the second chemical reaction shown in FIG. 6, a protein  83  reacts with the PLC  82  to form a protein-polymer complex  84 . Specifically in FIG. 6, the protein  83  includes lysine, wherein H 3 N + —(CH 2 ) 4  is a portion of the lysine that reacts with the PLC  82 , and wherein X represents a remaining portion of the protein  83  including a remaining portion of the lysine. The remaining portion of the lysine has a carbon atom covalently bonded to H, H 3 N + , and a carboxyl group. As shown in FIG. 6, a hydrolysis of the chlorine in position 4 of the cyanuric chloride triazine ring has replaced said chlorine in position 4 with the H 3 N + —(CH 2 ) 4  portion of the lysine of the protein  83 , to form the protein-polymer complex  84 . Specifically in FIG. 6, the protein-polymer complex  84  is 2-O-mPEG-4-Y-6-chloro-s-triazine, wherein Y is the protein H 3 N + —(CH 2 ) 4 —X. More generally, FIG. 6 shows generation of a PEG-conjugated protein with attachment of an activated PEG (e.g., the PLC  82 ) to an ε-amino group (e.g., the lysine or another amino acid such as arginine). The second chemical reaction may be implemented in an alkaline phosphate buffer (e.g., 50 mM of K 2 HPO 4  and 105 mM of NaCl, wherein mM denotes millimoles). The second reaction can be efficiently accomplished in a wide range of media including, inter alia, saline, phosphate buffered saline, blood plasma, blood serum, albumin containing buffers, Hanks Balanced Salt Solution (HBSS), N-[2-hydroxyethyl]piperazine-N′-2-ethanesulfonic acid (“HEPES”), Roswell Park Memorial Institute 1640 (“RPMI 1640”), etc. 
     Time and temperature for performing the second reaction are very flexible. For example, a reaction between mPEG and amino acid of cell membranes or cell viral receptors may be accomplished in 4 minutes or longer at 4° C. if the pH is about 9. If the pH is lower (e.g., about 8), the reaction may proceed at room temperature for a longer period (e.g., 60 minutes or longer) so that the cells are not stressed by temperature and not stressed by harsh alkaline conditions. As to pH, it is useful to have a pH of about 8 when reacting mPEG with lysine. When reacting mPEG with a virus, weakly acidic to alkaline conditions should be used with a representative pH range of about 6.0 to about 9.0. When reacting mPEG with a living cell, a suitable pH range is cell specific for the particular type of living cell being reacted. 
     Effective doses of the PLC in the second reaction depend on several variables, including: linker chemistry, the polymer being used, surface area of cell membranes being modified, density of viral receptors, geometric factors such as available volume above the cells being modified (e.g., a higher dose may be needed to cover an upper nasal cavity than a low nasal cavity), etc. 
     It should be noted that the chlorine in position 6 of the cyanuric chloride triazine ring is quite unreactive and thus unavailable to react with either an amino acid or with a second polymerated linker chemical. 
     FIG. 6 illustrates a mechanism of the covalent attachment of the PLC of cyanuric chloride coupled mPEG with membrane proteins, and potentially membrane carbohydrates. Virtually all cells and proteins can be similarly modified (e.g., red blood cells, platelets, endothelial cells, epithelial cells, stromal cells) with only slight variations in pH, temperature and time. Indeed, the pH, time and temperature conditions at which the modification reaction can be done at are very malleable, thus making this invention applicable to a wide variety of cell types. Other polymers may be utilized instead of mPEG, such as, inter alia, polyethylene glycol, ethoxypolyethylene glycol, dextran, ficoll, and arabinogalactan. Other linker molecules may be utilized instead of cyanuric chloride, such as, inter alia, imidazolyl formate, succinimidyl succinate, succinimidyl glutarate, N-hydroxysuccinimide, 4-Nitrophenol, 2,4,5-trichlorophenol, and a chloroformate. FIG. 7 lists exemplary polymeric linker compounds (PLCs) that may be used with the present invention and associated targets that can be reacted with the PLCs. Most of the listed targets in FIG. 7 are proteins. The thiol groups in FIG. 7 include sulfhydryl groups which are protein components. Any of the PLCs that react with the hydroxyl group can be reacted with a carbohydrate. Note that the PLC of phospholipid PEG interacts with a lipid by intercalation rather than by covalent bonding. 
     The present invention is illustrated by the following non-limiting examples. 
     EXAMPLE 1 
     Epithelial monolayers of monkey kidney CV1 cells were covalently modified with activated mPEG (i.e., mPEG covalently bonded to a cyanuric chloride linker molecule). In particular, the cells were confluently grown on glass slides. The cells were then exposed to a solution of activated mPEG, followed by exposure to Simian Vacuolating Agent (SV40) virus for 72 hours in a medium of Minimum Essential Medium (MEM). It should be noted that the SV40 virus has veterinary significance, but does not have human significance. 
     FIG. 8 is a bar graph that shows the percentage of CV1 cells infected after 24 hours, as assayed via T antigen staining. Concentrations of 12 and 25 milligrams (mg) of mPEG per milliliter (ml) of medium were each analyzed. Control cells, which are not mPEG-modified, were infected at a rate of nearly 50% at 24 hours of exposure to the SV40 virus. In contrast, the 12 and 25 mg/ml samples of mPEG-modified cells were infected at a rate of only 5% and 1%, respectively, at 24 hours of exposure to the SV40 virus. 
     The results of this test support covalently bonding a polymerated linker chemical (e.g., activated mPEG) to membrane cell surfaces to prevent viral infection of the cells. While this test utilized mPEG as a polymer in the polymerated linker chemical, any other polymer discussed herein could have been used instead of mPEG. Similarly, while this test utilized cyanuric chloride as a linker molecule in the polymerated linker chemical, any other linker molecule discussed herein could have been used instead of cyanuric chloride. Although this test utilized monkey kidney CV1 cells, cells of other animal species (or cells of a monkey other than monkey kidney cells), could have been used instead of the monkey kidney CV1 cells. 
     EXAMPLE 2 
     SV40 virus was covalently modified with a polymerated linker chemical of activated mPEG (i.e., mPEG covalently bonded to a cyanuric chloride linker molecule) in Minimal Essential Medium (MEM) (a Cellgro® cell media product by Mediatech, Inc.), supplemented with 5% fetal bovine serum (FBS) and MEM vitamins and mineral supplement. The SV40 viruses were modified at room temperature for a period of either 30 minutes or 60 minutes. Next, epithelial monolayers of monkey kidney CV1 cells were exposed to the covalently modified SV40 virus for 72 hours in a medium of MEM. 
     FIG. 9 is a bar graph that shows the percentage of CV1 cells infected after 24 hours, 48 hours, and 72 hours of SV40 virus exposure, as assayed via T antigen staining. The “I” above and below each bar denotes a standard deviation. Concentration of 0.1, 0.5, 1.0, 2.0, 3.0, 5.0, 10.0, and 20.0 mg/ml of mPEG, at a pH of 8.0, were each analyzed. C1 and C2 represent control cells not mPEG-modified, having a pH of 7.4 and 8.0 respectively. The control cells had a rate 35%-40% infection rate at 24 hours and nearly a 100% infection rate at 72 hours. The mPEG modified cells had an infection rate that decreased with concentration of mPEG. At the highest mPEG concentration of 20 milligrams/milliliter, the infection rate was only about 10% at 72 hours of SV40 virus exposure. 
     FIGS. 10 and 11 depict densitometry curves, based on sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, that show an extent to which the SV40 virus has been covalently mPEG modified in the tests of FIG.  9 . FIG. 10 depicts a densitometry curve for a control sample (C1 or C2 of FIG. 9) for the SV40 virus of FIG.  9 . As stated supra, the control samples have not been mPEG modified. The “A” portion of the densitometry curve of FIG. 10 represents a VP1 protein of the SV40 viral capsid, as detected by an anti-VP1 antibody. The indicated value of 3816 represents the area under the curve of the “A” portion that denotes the VP1 antibody response and serves as a reference value for subsequent comparison purposes. 
     FIG. 11 depicts a densitometry curve for the covalently mPEG-modified SV40 virus of FIG.  9 . The “A” portion of the densitometry curve of FIG. 11 represents a VP1 protein of the SV40 viral capsid and the indicated area of 3235 represents a small decrease in VP1 antibody response”. The “B1”, “B2”, and “B3” portions of the densitometry curve of FIG. 11 respectively represents an antibody response to 1 mPEG, 2 mPEGs, and 3 mPEGs, covalently bonded to a single protein. The indicated values of 2557, 406, and 724 for the areas under the B1, B2, and B3 curves, respectively, denote relative abundances of the 1 mPEG-modified proteins, 2 mPEG-modified proteins, and 3 mPEG-modified proteins. The presence of the B1, B2, and B3 portions of the densitometry curve of FIG. 11, and the absence of B1, B2, and B3 portions in the control sample of FIG. 10, demonstrates that covalent bonding of the SV40 virus with activated mPEG indeed occurred for the tests of FIG.  9 . 
     The results of this test support covalently bonding a polymerated linker chemical (e.g., activated mPEG) to a virus so as to inactivate an ability of the virus to infect adjacent or nearby cells of an animal. While this test utilized mPEG as a polymer in the polymerated linker chemical, any other polymer discussed herein could have been used instead of mPEG. Similarly, while this test utilized cyanuric chloride as a linker molecule in the polymerated linker chemical, any other linker molecule discussed herein could have been used instead of cyanuric chloride. Although this test utilized monkey kidney CV1 cells, cells of other animal species (or cells of a monkey other than monkey kidney cells), could have been used instead of the monkey kidney CV1 cells. 
     While particular embodiments of the present invention have been described herein for purposes of illustration, many modifications and changes will become apparent to those skilled in the art. Accordingly, the appended claims are intended to encompass all such modifications and changes as fall within the true spirit and scope of this invention.