Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of...
Reexamination Certificate
1998-12-16
2003-09-09
Celsa, Bennett (Department: 1639)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
C514S672000, C562S041000, C562S607000, C564S510000
Reexamination Certificate
active
06617157
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to methods and compositions for the selective modification of nucleic acids in biological compositions.
Transmission of viral diseases (e.g., hepatitis A and B, acquired immunodeficiency syndrome (HIV), cytomegalovirus infections) by blood or blood products is a significant problem in medicine. While donor selection criteria and screening of donor blood for viral markers helps reduce the transmission of viruses to recipients, screening methods are incomplete or less than 100% sensitive, as most are directed to only a few discrete viruses. Even in such cases, their sensitivity is insufficient. In addition, other biological compositions, e.g., mammalian and hybridoma cell lines, products of cell lines, milk, colostrum and sperm, can contain infectious viruses.
It is desirable to inactivate any virus contained in donor blood, blood products, or other biological compositions. At the same time, it is important to leave the structure and function of valuable constituents, such as red blood cells, platelets, leukocytes, and plasma biopolymers, such as proteins and polysaccharides relatively unchanged.
In addition, it is often unknown whether compositions containing blood, blood products, or products of mammalian cells contain infectious viruses. In this case it would be valuable to have compositions and methods to treat such compositions to inactivate any infectious viruses present.
Furthermore, the manufacture of maximally safe and effective killed vaccines for human or veterinary use requires methods which completely and reliably render live microorganisms, e.g., viruses and bacteria, noninfectious (“inactivated”) but which have minimal effects on their immunogenicity. Methods typically used for the inactivation of viruses, such as those useful in the preparation of viral vaccines, generally alter or destroy the function and structure of cells, cell components, proteins and other antigens.
Current inactivation methods, including the use of formalin, beta-propiolactone, and ultraviolet radiation, have been developed empirically, with little basis in fundamental chemical or structural principles. For example, ethyleneimine monomers have been used to inactivate the foot-and-mouth disease virus (Russian patent no. SU 1915956). Ethyleneimine monomers have also been used to inactivate Mycoplasma and Acholeplasma (WO 92/18161) and avian infections (Romania patent no. RO 101400). Binary ethyleneimine (i.e., ethyleneimine monomer generated by a combination of two reagents) has been used for the inactivation of feline enteric coronavirus, FECV, (EP 94200383). Polyethyleneimine has been used as a plant virus control agent (JP 7882735). The foregoing methods and compounds modify microorganisms, such as viruses and bacteria, nonspecifically, and are difficult to standardize and apply reproducibly.
In addition, ignorance of which chemical alterations render the microorganism noninfectious makes the process difficult to apply reproducibly. Periodic outbreaks of disease resulting from inadequate inactivation or reversion following inactivation are the result. Major outbreaks of paralytic poliomyelitis,, foot and mouth disease and Venezuelan equine encephalitis have occurred due to this problem.
In general, multiple components of the microorganism, including important surface antigenic determinants such as viral capsid proteins, are affected by currently used inactivating agents. These agents significantly modify not only nucleic acids but also other biopolymers such as proteins, carbohydrates and lipids, thereby impairing their function. Altered antigens or the inactivation of protective epitopes can lead to reduced immunogenicity and hence low potency (e.g., inactivated polio vaccine), to altered antigenicity and hence immunopotentiation of disease instead of disease prevention (e.g., respiratory syncytial virus and inactivated measles vaccines produced by formnalin inactivation), or to the appearance of new antigens common to another killed vaccine prepared with the same inactivant.
For example, in the preparation of hepatitis B virus vaccine, it is common practice to heat preparations at temperatures in excess of 80° C. and to treat with formaldehyde. These treatments not only inactivate viral infectivity, but also damage proteins and other antigens. Carrier substances added to the vaccine as stabilizers also may be unintentionally modified, producing allergic reactions, as occurs with human serum albumin in rabies vaccine inactivated with beta-propiolactone.
The problems of inactivation of viruses in biological mixtures are distinct from the problems of inactivation of the viruses alone due to the co-presence of desirable biopolymers such as proteins, carbohydrates, and glycoproteins in the plasma. While it is possible to inactivate the hepatitis B virus by using agents such as formaldehyde or oxidizing agents, these methods are not suitable for the inactivation of viruses in blood, due to the observation that most of these inactivating agents impair the biological activity of biopolymers in plasma or cellular components of blood. For example, the use of ultraviolet light has been shown to inactivate viruses in a platelet concentrate. However, severe platelet damage resulted from higher doses. Beta-propiolactone reacts with nucleic acid and protein at similar rates; thus, while viruses can be inactivated, more than half of the factor VIII activity of plasma is lost.
Yet another problem is that some of the viruses contaminating blood or other biological fluids are contained within the cell, either as a fully formed virus, viral genome fragments, or viral nucleic acid integrated into the host genome. For instance, the HIV virus is contained within leukocytes. It is a special concern to be able to inactivate both cell-free and cell-contained forms of virus, while retaining the structural integrity of cells.
Problems may also exist in obtaining valuable biopolymers from non-blood sources since pathogenic viruses may also contaminate such compositions.
SUMMARY OF THE INVENTION
The invention features a method of selectively modifying nucleic acid molecules in a biological composition; the method includes the step of contacting the composition with an inactivating agent having the formula:
where each of R
1
, R
2
, R
3
, R
4
, R6, R
7
, and R
8
is, independently, H or a monovalent hydrocarbon moiety containing between 1 and 4 carbon atoms, inclusive, provided that R
1
, R
2
, R
3
, R
4
, R
6
, R
7
, and R
8
cannot all be H; R
5
is a divalent hydrocarbon moiety containing between 2 and 4 carbon atoms, inclusive; X is a pharmaceutically acceptable counter-ion; and n is an integer between 1 and 10, inclusive. Preferably, R
5
is alkylene and each of R
1
, R
2
, R
3
, R
4
, R
6
, R
7
, and R
8
is H or alkyl, and n is 2, 3, or 4.
The invention further features a method for selectively inactivating a virus by contacting the biological composition with this inactivating agent, where the nucleic acid molecules are contained within an infectious vertebrate virus. This method may be used for both enveloped and non-enveloped viruses. The inactivated viruses can then be included in killed vaccines.
The invention also features a method for selectively modifying nucleic acids that are contained within a transforming DNA fragment, using this inactivating agent.
The invention also features a killed vaccine that includes an effective amount of inactivated vertebrate virus and a pharmaceutically acceptable carrier, where the inactivated vertebrate virus is made by a process of incubating the virus with an inactivating agent under viral inactivating conditions. The inactivating agent has the formula:
where each of R
1
, R
2
, R
3
, R
4
, R
6
, R
7
, and R
8
is, independently, H or a monovalent hydrocarbon moiety containing between 1 and 4 carbon atoms, inclusive, provided that R
1
, R
2
, R
3
, R
4
, R
6
, R
7
, and R
8
cannot all be H; R
5
is a divalent hydrocarbon moiety containing between 2 and 4 carbon atoms, inclusive; X is a pharmaceutically acceptable
Ackerman Samuel K.
Budowsky Edward I.
Edson Clark M.
Purmal Andrei A.
Celsa Bennett
V.I. Technologies, Inc.
Wolf Greenfield & Sacks P.C.
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