Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Genetically modified micro-organism – cell – or virus
Reexamination Certificate
1996-07-25
2002-03-12
Mosher, Mary E. (Department: 1648)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Genetically modified micro-organism, cell, or virus
C435S069100, C435S235100, C435S320100
Reexamination Certificate
active
06355240
ABSTRACT:
FIELD OF INVENTION
This invention relates to a method of enhancing the efficacy of recombinant insect viruses, such as baculoviruses, for use as insecticides. This invention relates to recombinant insect viruses and vectors for use therewith in which the expression of a heterologous gene or fragments thereof (preferably encoding an insect controlling substance or modifying substance, such as an insect toxin) is operably linked to an early promoter.
BACKGROUND OF THE INVENTION
The following abbreviations are used throughout this application:
AcMNPV—
Autographa californica
nuclear polyhedrosis virus
bp—base pairs
BEVS—baculovirus expression vector system
ECV—extracellular virus
GV—granulosis virus
kD—kilodaltons
NPV—nuclear polyhedrosis virus
occ
−
—occlusion negative virus(es)
occ
+
—occlusion positive virus(es)
OV—occluded virus
PCR—polymerase chain reaction
pfu—plaque forming unit
p.i.—post-infection
PIB—polyhedron inclusion body (also known as occlusion body)
5′ UTR: The mRNA or gene sequence corresponding to the region extending from the start site of gene transcription to the last base or base pair that precedes the initiation codon for protein synthesis.
3′ UTR: The mRNA or gene sequence corresponding to the region extending from the first base or basepair after the termination codon for protein synthesis to the last gene-encoded base at the 3′ terminus of the mRNA.
(+)strand: Refers to the DNA strand of a gene and its flanking sequences which has the same sense as the RNA that is derived from that gene.
(−)strand: Refers to the DNA strand of a gene and its flanking sequences that is complementary to the (+)strand.
Since the advent of recombinant DNA technology, there has been steady growth in the number of systems available for the regulated expression of cloned genes in prokaryotic and eukaryotic cells. One eukaryotic system that has gained particularly widespread use is the baculovirus expression vector system, or BEVS, developed by Smith and Summers (1). This system utilizes a nuclear polyhedrosis virus isolated from the alfalfa looper,
Autographa californica
, as a vector for the introduction and high level expression of foreign genes in insect cells.
Autographa californica
multicapsid nuclear polyhedrosis virus (AcMNPV) is the prototype virus for the Family Baculoviridae. These viruses have large, circular, double-stranded DNA genomes (at least 90-230 kilobases (2)). There are two Subfamilies, Nudibaculovirinae, which do not form occlusion bodies, and the Eubaculovirinae, which are characterized by their ability to form occlusion bodies in the nuclei of infected insect cells. The structural properties of the occlusion bodies are used to further classify the members of this Subfamily into two genera: the nuclear polyhedrosis viruses (NPVs) and the granulosis viruses (GVs).
As exemplified by AcMNPV, the occlusion bodies formed by NPVs are 1-3 microns in diameter and typically contain several hundred virions embedded in a para-crystalline matrix. Occlusion bodies are also referred to as either polyhedra (polyhedron is the singular term) or as polyhedron inclusion bodies (PlBs). The major viral-encoded structural protein of the occlusion bodies is polyhedrin, which has a molecular weight of 29 kilodaltons (kD) (1,3). More than a hundred such occlusions can frequently be found in the nucleus of a single infected cell. GVs are distinguished from NPVs by the fact that their occlusions are much smaller and contain only one virion, which is embedded in a matrix of the viral protein granulin. Nevertheless, the fundamental principles of GV replication are similar to those described below for AcMNPV.
Viral occlusion bodies play an essential role in the horizontal (insect to insect) transmission of Eubaculovirinae. When a larva infected with AcMNPV dies, large numbers of occlusion bodies are left in the decomposing tissues. In neutral or acidic conditions (pH<10), the protein matrix and outer calyx of the occlusion body protect the embedded virions against chemical degradation in the environment and provide limited protection against UV radiation. However, when the occlusion bodies are ingested by another larva, they dissolve rapidly in the larval midgut, which is strongly alkaline (pH 10.5-12), and the embedded virions are released. These virions then adsorb to and infect various types of midgut cells.
Infected midgut cells synthesize few if any new occlusion bodies. Instead, they produce a second form of the virus, known as extracellular virus (ECV). Whereas the occluded form of the virus is responsible for the horizontal transmission of the virus among larvae, the ECV is used to spread the infection from tissue to tissue internally. This is an essential aspect of normal viral pathogenesis and continues until most tissues of the larva have been infected and lysed. As the virus spreads internally, many of the infected cells, especially hemocytes and fat body cells, produce not only more ECV, but also copious amounts of occluded virus (OV) in the form of occlusion bodies. When the larva dies, the occlusion bodies are deposited in the environment and the cycle begins anew.
Although ECV and OV are genetically identical, they are biochemically distinct. Shortly after the AcMNPV infects a cell, the nucleocapsid structure (which contains the DNA genome) migrates to the nucleus of the cell, where it is uncoated. This sets in motion a regulated cascade of viral gene expression which leads to the onset of viral DNA synthesis (at about 6-12 hours post-infection (p.i.)) and the formation of many new nucleocapsids. ECV production begins at about 10-13 hours p.i. with the budding of the nucleocapsids through the cytoplasmic surface of the cell. During the budding process, the nucleocapsids acquire a lipid membrane, or envelope, which is decorated with a viral glycoprotein known as gp64. This protein is specific to the ECV form of the virus and is required for ECV infectivity. The formation of occlusion bodies begins much later (24-36 hours p.i.) and requires the concerted action of numerous specialized viral gene products, the most prominent of which is polyhedrin.
The polyhedrin gene plays a central role in the BEVS technology. Because large amounts of polyhedrin are required for occlusion body formation, the polyhedrin gene is one of the most actively transcribed genes in the viral genome during the very late phases of virus replication. Smith and Summers (1) show that expression of a heterologous gene can be achieved by substituting the coding region of the polyhedrin gene with the coding region of a heterologous gene of interest. Since polyhedrin is not required for ECV formation, the resulting virus is able to replicate normally in cultured insect cells. However, it is no longer able to produce polyhedrin for occlusion body formation and is therefore occlusion-negative (occ
−
).
The BEVS has been used successfully to express foreign genes isolated from a wide range of prokaryotic and eukaryotic organisms and viruses. Some representative examples include the human &agr;- and &bgr;-interferons, the Drosophila Krueppel gene product,
E. coli
&bgr;-galactosidase, various HIV structural proteins, and a
Neurospora crassa
site-specific DNA binding protein (3). In general, these genes may encode cytosolic proteins, nuclear proteins, mitochondrial proteins, secreted proteins or membrane-bound proteins. In most cases, the proteins are biologically active and undergo appropriate post-translational modification, including proteolytic processing, glycosylation, phosphorylation, myristylation and palmitylation. Hence, this system has proven to be a highly valued tool for both fundamental molecular research and for the production of proteins for commercial purposes. Using BEVS technology, recombinant viruses are produced in cultured insect cells by homologous DNA recombination between AcMNPV DNA and a plasmid-based transfer (or transplacement) vector containing the heterologous gene of interest under the control of the polyhedrin gene promo
BASF Aktiengellschaft
Mosher Mary E.
Sale Elaine
LandOfFree
Enhanced insecticidal insect virus through the expression of... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Enhanced insecticidal insect virus through the expression of..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Enhanced insecticidal insect virus through the expression of... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2816944