Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Insect cell – per se
Reexamination Certificate
2000-05-24
2002-04-30
Saucier, Sandra E. (Department: 1651)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Insect cell, per se
C435S948000
Reexamination Certificate
active
06379958
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to cell lines which are generated from differentiated tissue of insects and which are susceptible to baculoviruses and may be used to replicate such viruses. The invention further relates to the use of such cell lines to replicate large numbers of baculoviruses and particularly, to replicate large numbers of genetically engineered baculoviruses, and to thus express large quantities of recombinant proteins. In addition, the invention relates to cell lines which tolerate the expression of a wide range of recombinant proteins including known toxins.
2. Description of the Relevant Art
Baculoviruses are considered a potentially important tool for managing insect pests and cell culture is a desired means of producing them. In nature, insects can become infected with baculovirus particles as a result of consuming food contaminated with baculovirus particles. These food-borne baculovirus particles are typically in the form of occlusion bodies (OB) which are composed of multiple viral particles embedded within a virus-encoded proteinaceous crystal. After ingestion, the protein crystal of the occlusion bodies dissolves, releasing individual virus particles which invade the epithelial cells that line the midgut. Viral replication takes place in the nuclei of the cells, and usually two forms of baculovirus, occluded and extracellular virus (ECV), are generated during replication. ECV is produced first and acquires an envelope as it buds out from the surface of the cell. The ECV can then infect other cells within the insect, including fat body cells, epidermal cells, and hemocytes. Following this initial stage of infection, virions are produced which are occluded in OB. OB formation continues until the cell ultimately dies or lyses. Some baculoviruses can infect virtually every tissue in the host insect so that at the end of the infection process, the entire insect is liquified, releasing extremely large numbers of OB which are then responsible for spreading the infection to other insects (1986. The Biology of Baculoviruses, Vol. I and II. Granados et al., Eds. CRC Press, Boca Raton, Fla.).
The first attempts to replicate baculoviruses in cell culture were in lines from the homologous insect species (Grace, T. D. C. 1967. In Vitro 3: 104-117; Goodwin et al. 1970
. J. Invertebr. Pathol
. 16: 284-288). However, Sohi et a. (1972
. J. lnvertebr. Pathol
. 19: 51-61) and Granados et al. (1976. In: Invertebrate Tissue Culture, Applications in Medicine, Biology, and Agriculture, Kurstak et al., Eds. Academic Press, New York, N. Y. , pages 379-389) soon demonstrated the replication of baculoviruses in cell lines from insect species other than the ones from which the viruses were isolated. Quiot (1976
. C. R. Acad. Sci. Ser. D
. 282: 465-467) reported a gypsy moth cell line that replicated an iridovirus and seven baculoviruses, but not the gypsy moth nuclear polyhedrosis virus (NPV). Goodwin et aL (1978
. In Vitro
14: 485-494) reported that several cell lines developed from gypsy moth by very similar methods were quite different in their replication of baculoviruses. Some cell lines replicated the gypsy moth NPV; whereas, others did not replicate gypsy moth NPV but did replicate the
Autographa californica
nuclear polyhedrosis virus (AcNPV).
AcNPV, in particular, has a broad in vivo host range and will multiply in cell lines from a number of species of insects. Some of the cell lines in which AcNPV has been replicated are shown in Table 1. In addition to 11 genera and 12 species, the listed cell lines are from a number of different tissues such as blood cells and minced whole larvae. The relative merits of this large variety of cell diversity has not been adequately screened to categorize its potential for virus production or gene expression.
TABLE 1
Insect Cell Lines Susceptible to AcNPV
Cell Line
Tissue of Origin
Species of Origin
IPLB-Ld-652Y
Pupal ovary
Lymantria dispar
IPLB-Sf-21
Pupal ovary
Spodoptera frugiperda
BCIRL-Hz-AM3
Pupal ovary
Helicoverpa zea
BCIRL-Hv-AM1
Pupal ovary
Heliothis virescens
Tn-368
Adult ovary
Trichoplusia ni
IZD-MB-0503
Hemocytes
Mamestra brassicae
IPRI-Md-108
Hemocytes
Malocosoma disstria
NIAS-MaBr-85
Larval Fat Body
Mamestra brassicae
NIAS-LeSe-11
Larval Fat Body
Leucania separata
IPLB-Tn-RR
Embryo
Trichoplusia ni
BTI-TN5B1-4
Embryo
Trichoplusia ni
IPLB-LdEIt
Embryo
Lymantria dispar
UFL-Ag-286
Embryo
Anticarsia gemmatalis
UCR-Se-1
Neonate larvae
Spodoptera exigua
FPMI-Ms-5
Neonate larvae
Manduca sexta
IPRI-Cf-1
Neonate larvae
Choristoneura fumiferana
The first comparison of AcNPV replication in cell lines from several species of insects was made by Lynn and Hink in 1980 (
J. Invertebr. Pathol
. 35: 234-240). Five cell lines known to be susceptible to AcNPV were evaluated using several parameters to measure performance. The cell line used in any in vitro system is an important element both in terms of the quality and the quantity of the final product. The selected cell line must be capable of growth in suspension in large volumes. The IZD-MB0503 line from
Mamestra brassicae
was the best of those tested based upon the yield of active polyhedra over several passages. However, cell lines from
Trichoplusia ni
Tn-368, produced polyhedra with specific activity closest to that of polyhedra produced in insect larvae. The line Sf-1254, from
Spodoptera frugiperda
, produced only about one-tenth the amount of ECV produced by most of the other cell lines. Thus, it would be difficult to use this line for production as either large volumes of culture supernatant would be required for seed virus or the virus would have to be concentrated.
The NPV of the gypsy moth is registered as a pesticide by EPA under the name GYPCHEK. Production of the virus for pest management is in insect larvae as the yields from cell lines developed by Goodwin (Goodwin et al. 1978, supra) have been too low and the process too costly. Therefore, cell lines for different gypsy moth tissues, embryonic and fat body, were developed by Lynn and screened for their production capabilities (Lynn et al. 1989
. Appl Environ. Microbiol
. 55: 1049-1051). A line from fat body, IPLB-LdFB, and one from embryos, IPLB-Elt, were compared with Goodwin's line from pupal ovary, IPLB-Ld-652Y. OBs produced in the three lines are shown in FIG.
1
. Three strains of the virus were used in the studies. The LdFB cells produced the highest number of OBs regardless of the virus strain used, with the LdFB-Ab combination producing significantly more than any other combination. ECV was not produced in high titers in any cell-virus combination. TCID
50
s/ml ranged from 3.83×10
3
to 2.61×10
5
in IPLB-652Y depending on the virus strain used. These results were obtained in attached cultures and based upon them, the LdFB-Ab virus combination was considered the best for in vitro production. However, attempts to scale up suspension cultures of the LdFB cells have not been successful as the cells are very fragile, especially after infection. Yields in suspension culture were considerably less than in static culture. Volkman et al. (1984
. Appl. Environ. Microbiol
. 44:227-233) made a comparison similar to the Lynn studies using an immunoassay to detect infected cells. All of the cell lines tested responded linearly to virus dose; however, the dose range for Tn-368 cells, the most sensitive, was 4.5-5.5 log
10
and the range for the least sensitive cells, from
L. dispar
, was 1.3-2.3 log
10
. Cell lines from
Bombyx mori
and
L. dispar
produced more than 99% single cell foci of infection. One possible interpretation of these results is that these cell lines do not produce much ECV, making them less suitable for production.
In the most extensive screening of variation in virus susceptibility, Miltenburger et al. (1984
.Z. Naturforsch
39: 993-1002) challenged over 80 primary cultures derived from
Cydia pomonella
, apple codling moth, with
Choristoneura murinana
NPV and
C. pomonella
granulosis virus (GV). The primary cultures
Deming Clay
Hackett Kevin J.
Vaughn James L.
Afremova Vera
Fado John D.
Rabin Evelyn M.
Saucier Sandra E.
Silverstein M. Howard
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