Abstract:
A composition and method for enhancing cell growth and increasing the density of cell cultures containing lentivirus-infected host cells comprises adding a suitable quantity of an antibiotic to the culture to destroy harmful bacteria.

Description:
FIELD OF THE INVENTION 
     The present invention relates to novel cell cultures. In particular, the invention is directed to new cell cultures infected with lentiviruses, such as feline immunodeficiency virus (FIV), which contain a suitable antibiotic to increase the cell density and enhance the growth of the culture. The invention also relates to new methods for increasing the density of cell cultures using certain antibiotics. 
     BACKGROUND OF THE INVENTION 
     Lentiviruses constitute a class of viruses which can lead to a variety of diseases in both humans and animals. These diseases are often preceded by several months or even years of incubation. For example, the pathologies associated with AIDS are caused by the human immunodeficiency viruses (HIVs) and result from a chronic progression of the disease, often times causing cachexia and death in the patient several years after infection. 
     Lentiviruses have also been associated with various pathologies in such species as apes and monkeys, as well as domesticated animals such as horses, burros, cattle, goats, and sheep. Among these, equine infectious anemia (EIA) has been characterized as the most important infectious disease of horses occurring throughout the world. Lentiviruses of sheep are relatively common pathogens in most parts of the world, and include Maedi-visna virus and progressive pneumonia virus as two predominate types. Bovine immunodeficiency virus is an important cause of illness in cattle. 
     Other lentiviruses are vital indicators of pathology in animals such as cats. Feline immunodeficiency virus, structurally similar to HIV, can cause death in house cats. Increasingly, doctors, veterinarians, and researchers are devoting considerable time and resources to preventing and treating diseases caused by these viruses. 
     Part of the research involves growing tissue cultures containing cells, for example lymphocytes, which have been infected with one or more of the known lentiviruses. Lymphocyte cultures or anchorage dependent epithelium-like cells such as feline kidney cell cultures may be grown in T-flasks, roller bottles, spinner flasks and bioreactors using media such as Minimal Essential Media (MEM), Roswell Park Memorial Institute (RPMI), Dulbecco&#39;s MEM (DMEM), and AIM V (Gibco/LTI, Grand Island, N.Y.) supplemented with bovine serum up to about 20% and up to about 5% bovine serum albumin (BSA). Large-scale cultures may be grown in large spinners, fermentor, and bioreactors in the presence of shear protective chemical, thickener, emulsifiers or compounds such as methylcellulose, carboxylmethyl cellulose, and surfactants such as the Pluronic series, e.g. PLURONIC® F-68, manufactured by BASF Corporation of Wyandotte, Mich. 
     In maintaining tissue cultures containing viruses, researchers seek to destroy harmful bacteria within the culture so that the cells containing the virus can grow and propagate. At the same time, a concomitant goal is to maximize growth of the cell culture. To facilitate growth and destroy bacteria, it has been accepted practice to add antibiotics, usually a combination thereof, to the cell culture. For example, U.S. Pat. No. 5,958,423 sets forth a cell culture of Madin-Darby Bovine Kidney (MDBK) cells in which up to 30 mcg/mL of polymixin B and neomycin, and up to 2.5 mcg/mL of amphotericin B is utilized. 
     What is now needed in the art are new cell culture compositions containing cells infected with lentiviruses. Especially needed are novel cell cultures in which cell growth can be maximized and the presence of harmful organisms such as bacteria can be simultaneously minimized. Also needed are new methods of growing cell cultures in which the density thereof can be increased through the promotion of tissue growth. Further needed are new additives which can optimize the density of cell cultures by enabling and enhancing the growth of the cells which comprise the culture. 
     SUMMARY OF THE INVENTION 
     In one embodiment, the present invention is directed to a cell culture comprising at least one lentivirus-infected host cell and a growth-promoting amount of an antibiotic consisting essentially of neomycin or a biologically compatible salt thereof. 
     The invention is also directed to a cell culture which contains at least one lentivirus-infected host cell and neomycin or a biologically compatible salt thereof, such that the neomycin is present substantially without another antibiotic in an amount which is effective at inhibiting bacterial growth and increasing the density of the cell culture. 
     Also provided as part of the invention is a method for increasing the density of a cell culture in which cells therein have been infected with a lentivirus, which involves adding an antibiotic consisting essentially of neomycin or a biologically acceptable salt thereof to the cell culture. 
     The invention also provides a composition suitable for addition to a cell culture infected with lentivirus. The composition contains a cell density enhancing quantity of an antibiotic consisting essentially of neomycin or a biologically salt thereof, along with at least one cell culture supplement. The cell culture supplement is desirably bovine-derived sera. 
     The foregoing and other features and advantages of the invention will become more apparent from the detailed description of the preferred embodiments of the invention given below. 
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     The present invention is directed to cell cultures. Those contemplated for use herein are those suitable for research and study which are capable of harboring lentiviruses and permitting the growth thereof. Such cell cultures would therefore include lymphocytes and other types of cells which can become infected with one or more lentiviruses. Other suitable cell cultures would include feline lymphocytes, fibroblast-like and epithelium-like cells such as feline kidney cells, Crandell Feline Kidney or CRFK cells, FL6, FL72 and FL 74 (Feline Lymphocyte) cells. T-cell lymphocytes may be preferred for use herein, as well as IL-2 independent FetJ and FL6 lymphocytes. 
     The term “lentiviruses’ is used herein to encompass all known and yet-to-be-discovered lentiviruses, including without limitation equine infectious anemia virus, Maedi-visna virus, progressive pneumonia virus, caprine arthritis-encephalitis virus, feline immunodeficiency virus (FIV), simian immunodeficiency viruses infecting such species as the macaque, and African monkeys and baboons, and the human immunodeficiency virus (HIV) Types I and II. The term “lentiviruses” also includes analogs, derivatives and peptide sequences of any of the foregoing. Preferred for use herein are the non-human lentiviruses, and in particular, feline immunodeficiency virus (FIV). 
     The cell cultures of the invention may be cultivated using methods known in the art. For example, the host cells such as lymphocytes may be chronically infected with one or more of the foregoing lentiviruses using accepted procedures, and then grown in suitable media. Preferably, the media is substantially liquid media. Even more preferably, the media is initially provided as substantially serum-free media. The host cells may be suspended in the liquid media, for example. Cell density of the cultivated cell culture may vary according to the particular host cells, the media, and the growth chamber, but can be within the range of about 1×10 4  to about 1×10 6  suitable cells per milliliter (mL) of cell culture (including media). More preferably, the cell density is about 2×10 5  to about 5×10 5  cells per milliliter. 
     As a further part of the invention, the cell cultures contain a suitable antibiotic which is effective at inhibiting the growth of bacteria within the culture, while at the same time increasing the growth of the cells and thereby increasing the density of the cell culture. Preferred for use herein is the antibiotic neomycin, which would include all biologically compatible salts and derivatives thereof, such as neomycin sulfate. By “biologically compatible” it is meant that the salt or derivative thereof has substantially no adverse biological effects upon the cell. 
     The quantity of neomycin included in the cell culture may vary according to the needs of the skilled artisan, but is typically included in an amount that will increase the density of the cell culture. An amount of neomycin within the range of from about 5 micrograms/mL of cell culture to about 60 micrograms/mL of cell culture (including media) is usually preferred. In a more preferred embodiment, neomycin is included in the cell culture in a quantity of at least about 10 micrograms/mL, and more preferably at least about 20 micrograms/mL. Even more desirably, the quantity of neomycin will be within about 30 micrograms/mL to about 60 micrograms/mL. 
     It is preferred to utilize neomycin to enhance cell culture growth and density without the inclusion of such other antibiotics as polymixin B and gentamycin, for example. Polymixin B may be derived from polymixin B 1  and B 2 , which are produced by the growth of  Bacillus polymyxa  (Prazmowski) Migula (Fam. Bacillaceae). It has now been found that including both neomycin and polymixin B in cell culture can, in many instances, result in a significantly smaller increase in cell density when compared with the use of neomycin alone. 
     Other components of the cell culture of the invention would typically include at least one culture supplement. The culture supplement may be bovine-derived, such as from bovine sera, and can include bovine serum and bovine serum albumin (BSA). The culture supplement may be included in amounts of from about 0.1 to about 10% by volume of the final cell culture. 
     The use of neomycin as herein described may increase cell density by at least about 20%, and more preferably by at least about 33 ⅓% over an identical cell culture of lentivirus-infected host cells not containing any antibiotic. Cell density may be assessed by acceptable methods, including the use of trypan blue exclusion on hemacytometer. 
     The following example is provided to illustrate one preferred aspect of the invention, but should not be construed as limiting the scope thereof. 
     EXAMPLE 
     In this example, Fet-J cells chronically infected with feline immunodeficiency virus (FIV) were grown in serum free media such as modified DMEM:F12 Media or AIM V media, supplemented with 2.5 mg/mL of ALBUMAX®, which is derived from BSA. Cells were grown in suspension in Erlenmeyer flasks on a rotary shaker at 150 rpm at 37° C. Cells were planted at a cell density of 3×10 5  viable cells/mL. Cell densities were determined by trypan blue exclusion on hemacytometer. Gp 120 expression determination was accomplished by enzyme linked immunosorbent assay (ELISA) using anti-FIV Gp 120 monoclonal antibodies. The antibiotics assessed for FIV supplementation included gentamycin, neomycin and polymixin B. Media were spiked with respective antibiotics at a concentration of 30 micrograms/mL. Cell densities were determined on a 24 hour basis by the method described above. The results are shown in TABLE 1. 
                                                               TABLE 1                           Antibiotic           Concentration            Experimental   (micrograms/   Day 0           Day   Day   Day       Group   mL)   (cell/mL)   Day 1   Day 2   3   4   5               Control   N/A   3.0 × 10 5     3.6 × 10 5     5.5 × 10 5     1.0 × 10 6     1.2 × 10 6     1.2 × 10 6         Gentamycin   30 μg/mL   3.0 × 10 5     2.4 × 10 5     3.2 × 10 5     6.5 × 10 5     1.1 × 10 6     1.1 × 10 6         Neomycin   30 μg/mL   3.0 × 10 5     4.7 × 10 5     7.8 × 10 5     1.2 × 10 6     1.3 × 10 6     1.8 × 10 6         Polymixin B   30 μg/mL   3.0 × 10 5     2.9 × 10 5     4.6 × 10 5     5.8 × 10 5     6.6 × 10 6     6.6 × 10 5         Polymixin B +   30 μg/mL +   3.0 × 10 5     2.9 × 10 5     5.0 × 10 5     7.0 × 10 5     7.7 × 10 5     7.8 × 10 5         Neomycin   30 μg/mL                    
The results from Table 1 show that the cell culture suspensions supplemented with neomycin had the best overall growth and increase in cell densities. Gentamycin had no significant effect, with daily cell densities being substantially the same as the controls. Cultures supplemented with Polymixin B, or neomycin together with polymixin B actually suppressed cell density as compared to the cultures in which no antibiotic was utilized and thus were also much less dense than those wherein neomycin by itself was used.
 
     Although the invention has been described with reference to particular embodiments thereof, it should be appreciated that many changes and modifications can be made without departing from the spirit or scope of the invention. Accordingly, the invention is not to be considered as limited by the foregoing description, but is only limited by the scope of the appended claims.