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J 



Europaisches Patentamt 
® ^Jjj European Patent Office © Publication number: 0 155 476 

Office europeen des brevets 



® EUROPEAN PATENT APPLICATION 

@ Application number: 85101009.0 @ Int.CI*: C 12 N 15/00 

^ C 12 N 7/00, C 12 N 5/00 

^ Date of filing: 31.01,85 //C12R1 '91 



@ Priority: 31.01.84 US 575453 


© Applicant: IDAHO RESEARCH FOUNDATION 




Moscow Idaho(US) 


@ Date of publication of application: 


@ inventor: Miller, Lois K. 


25.09.B5 Bulletin 85/39 


@ Designated Contracting States: 


1187 Foot Hill Road 


Moscow Idaho(US) 


AT BE OH DE FR GB IT LI LU ML SE 


@ Representative: Dipl.-lng. Schwabe, Dr. Dr. Sandmair, 






Dr. Marx 




Stuntzstrasse 16 




D-8000 Munchen 80(DE) 



Qj) Production of polypeptides in insect cells. 

@ A novel expression vector is employed in the expression 
of exogenous genes in insect cells utilizing recombinant DNA 
techniques. Exogenous genes under the control of strong 
promoters are inserted into viral chromosomes through the 
use of novel plasmids to form e viral vector which, in 
conjunction with the insect host is capable of expressing 
large segments of eulcaryotic, prokaryotic or viral DNA in an 
eukaryotic environment. 



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Oovdon Pnniinp Companv lid 



SCHWABE • SANDMAIR • MARX 
PATENTANWALTE 



6TUN7Z6TRASSE 16 ' 8000 MONCHEN 60 P'?S47l5 



Anwaltsakte 50 512 



Production of Polypeptides In Insect cells 

The present invention relates to methods and products 
thereof for expressing an exogenous gene in insect cells, 
and more particularly to the insertion of an exogenous 
gene which codes for a desired gene product into the viral 
chromosome of an insect virus such that it is under the 
control of a strong gene promoter as intergrated therein, 
introducing the viral vector into insect cells, and 
expressing the desired gene product. 

Interest in microbial insecticides has arisen as a result 
of the problems associated with the use of chemical pesti- 
cides. Chemical pesticides generally affect beneficial as 
well as non-beneficial species, and insects tend to 
acquire resistance to such chemicals so that new insect 
populations rapidly develop that are resistant to the 
chemical pesticides. Furthermore, chemical residues pose 
environmental hazards and possible health concerns. Thus, 
microorganisms that are pathogenic to insects (entomo- 
pathogens) present an opportunity for an alternative means 
of pest control and can play a role in integrated pest 
management and reduce dependence on chemical pesticides. 

Naturally occurring microorganisms or microbial by-products 
have been identified as potentially useful insecticidal 
agents. A number of entomopathogens have relatively 
narrow host ranges, which makes it possible to reduce 
specific pest populations while natural predators and bene- 
ficial insects are preserved or given the opportunity to 
become reestablished. Entomopathogens which are useful for 
insect control include certain bacteria, viruses and 

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fungi. 

Viruses that cause natural epizootic diseases within 
insect populations have been commercially developed as 
pesticides. One such family of viruses that has been 
extensively studied is the Baculoviridae . Baculoviruses 
possess large (about 100 to 200 kilobases) , double- 
stranded, circular, covalently closed DNA genomes that are 
packaged in enveloped, rod-shaped capsids approximately 
4 0 to 140 by 250 to 4 00 nanometers. The term "baculovirus" 
is derived from the rod-shaped nucleocapsid structure which 
is characteristic of this family. A nucleocapsid is a unit 
of viral structure, consisting of a capsid (protein coat) 
with an enclosed nucleic acid. 

The baculoviruses include the subgroups of nuclear poly- 
hedrosis viruses (NPV) and granulosis viruses (GV) , The 
virus particles of NPV and GV are occluded in proteina- 
ceous crystals. In occluded forms of baculoviruses, the 
virions (enveloped nucleocapsids) are embedded in a 
crystalline protein matrix. This structure, also referred 
to as a polyhedral inclusion body, is the form found 
extracellular ly in nature and is responsible for spreading 
the infection between organisms. The subgroup NPV contains 
many virions embedded in a single, large (up to 15 micro- 
meters) polyhedral crystal, whereas the subgroup GV 
contains a single virion embedded in a small crystal. The 
crystalline protein matrix in both forms is primarily 
composed of a single 25,000 to 33,000 dalton polypeptide. 

The NPV and GV baculovirus subgroups have been investi- 
gated for use as entomopathogens . The advantages of using 
viruses from the family Baculoviridae include: (1) they 
are known to cause lethal infections only in invertebrates; 
(2) they have a relatively narrow host range; (3) they 

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produce sufficient progeny virus per insect to allow 
conunercial production; and (4) the virus particles of NPV 
and GV are occluded in proteinaceous crystals which 
renders the viruses more stable in the environment, in- 
creases the shelf life as commercially prepared micro- 
bial pesticides, and facilitates combination with other 
pesticide formulations. 

When used as pesticides, occluded viruses are usually 
sprayed on foliage. Insects that consume the contaminated 
foliage acquire the virus-induced disease. The ingested 
virus passes through the foregut of the insect to the mid- 
gut where the alkaline pH solubilizes the crystal. The 
virions are released from the matrix and begin the in- 
fection of the midgut columnar cells by fusion with micro- 
villar membrane. Upon the death of the insect and the 
disintegration of the integument, the occluded viruses 
are released into the surrounding environments, and, if 
consumed by susceptible hosts, spread the infection. 

The most extensively studied baculovirus is the Auto- 
qrapha californica nuclear polyhedrosis virus (AcNPV) 
which has a relatively broad host range. Viruses of this 
type replicate into two mature forms that are referred to 
as the non-occluded virus (NOV) form and the occluded 
virus (OV) or polyhedral inclusion body (PIB) form. 

In both the NOV and OV forms, the nucleocapsids are enve- 
loped by a membrane, but the precise nature of the enve- 
lopes is different. The nucleocapsids of the NOV acquire 
an envelope by budding through cellular membranes, gene- 
rally the plasma membrane, whereas the nucleocapsids of 
the OV subgroup acquire a membrane envelope within the 
nucleus. The AcNPV nucleocapsids are usually encl-osed 
within a single envelope when envelopement occurs in the 

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nucleus. Enveloped nucleocapsids within the cell nucleus may 
be embedded in a crystalline protein matrix. The resulting 
protein crystals, containing many enveloped nucleocapsids, 
are known as occlusion bodies, polyhedral inclusion bodies 
(PIBs) , or polyhedra. The occlusion bodies of AcNPV are 
approximately one to four micrometers in diameter, allowing 
visualization with light microscopy. Each virion consists 
of one or more nucleocapsids enveloped in a single membrane. 
The morphological feature, whereby there may be more than 
one nucleocapsid per envelope, is characteristic of a type 
of NPV known as multiple-embedded virus to distinguish it 
from NPVs in which a single nucleocapsid is individually 
enveloped. From a genetic engineering standpoint it is 
important that the presence of multiple nucleocapsids per 
envelope is chara'cteristic of only the occluded form of an 
NPV and not the non-occluded form, since it is possible to 
clone and genetically manipulate the virus more easily 
using the non-occluded form. 

The AcNPV DNA genome (128 kilobases) has been mapped with 
respect to restriction sites for various restriction endo- 
nucleases, and is primarily composed of unique nucleotide 
sequences. See L.K. Miller et al.. Science Vol. 219:715 - 
721 (11 Feb. 1983) , 

The dV form of AcNPV is not infestious in cell culture 
unless virions are released from the occlusion body by al- 
kaline disruption. Thus, OVs formed in cell culture are 
non-infectious in cell-cultures. Rather, NOVs are re- 
sponsible for the spread of infection from cell to cell. 
As the infection becomes more extensive the virus repli- 
cates in the midgut cells and produces NOVs that bud 
through the basement membrane into the hemolyraph of the 
insect. The non-occluded virions that are released into 
*ae hemolymph are responsible for the systemic infection 
in the insect, as well as for spreading the infection in 



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cell cultures. 

The synthesis of virus induced proteins in AcNPV infected 
cells is a temporally controlled process. There are four 
stages of viral-induced protein synthesis: (1) an early 
alpha phase approximately two to three hours after in- 
fection; (2) an intermediate beta phase (six to seven hours 
after infection) requiring functional protein and DNA 
synthesis; (3) a gamr^a phase (at ten hours) including syn- 
thesis of virion structural proteins; and (4) a late delta 
phase (at fifteen hours) associated with occlusion. 

Infectious non-occluded virions of AcNPV are found in the 
cuture medium approximately ten hours after infection. The 
synthesis of polyhedrin, the major protein of the occlusion 
matrix, has been detected by twelve hours after infection 
and this preceeds the appearance of occluded virus at 
sixteen or eighteen hours after infection. 

Temporal control of transcription is probably responsible 
for the temporal control of AcNPV protein synthesis. * The 
transcription of the polyhedrin gene is first observed at 
twelve hours after infection, and the 1.2 kilobase trans- 
cript progressively increases in quantity up to twenty- 
four hours or more after infection. In cell culture, NOV 
release usually preceeds occlusion. In the case of AcNPV 
in nature, the infection is so pervasive that as much as 
10% of the weight of a dead infected host organism may 
be due to occluded virus. The 10% weight relationship 
indicates truly massive production of the occluded form 
of the virus, and in conjunction therewith, massive 
production of polyhedrin protein. 

From a genetic engineering standpoint, the genes controlling 
occlusion are not necessary for infection in cell culture. 



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and the elimination or replacement of these genes pro- 
vides an opportunity for substitution of passenqer DKA 
into the regions controlling occlusion and this could 
provide a high level of expression of passenger genes 
late times in the infection cycle. The crystalline poly- 
hedrin protein constituting the matrix of the occlusion 
boyd of AcNPV is predominately a single protein of 
approximately 30,000 daltons. About 95% of the weight of 
an occlusion body consists of polyhedrin. Thus, polyhedrin 
IS -synthesized in extremely large quantities as evidenced 
by the high proportion of the virus in the body weight of 
the infected host, m addition, the increased level of 
synthesis of this single protein is illustrated bv cells 
infected with wild type strain AcNPV which produces an 
average of over 60 occlusion bodies per cell, each occlu- 
sion body being approximately 4 micrometers in diameter. 
Thus the promoter for polyhedrin mRNA synthesis appears to 
be exceptionally strong. 

A. California nuclear polyhedrosis virus has been proposed 
as a vector for recombinant DNA resear-ch and genetic 
engineering in invertebrates. See L.K. Miller, j. vjroi 
39:973, the disclosure of which is incorporated herein by 
reference. However, until the achievement of the present 
invention, it has not been possible to use that virus, or 
any other insect virus, as a vector. 

Accordingly, it is an object of the present invention to 
provide a vector which is useful in expression of exoqer.ou. 
materials in insect cells. 

Summary of the Invention 

m accordance with the present invention, an expression 
vector is employed for the first time in expressing ex.- 



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genous gene products by insect cells utilizing recombinant 
DNA techniques. As used in this application, the term 
"gene product^ means the end product of a gene's expression 
and includes RNAs whose role may be structural or enzymatic 
in nature and the translational products of mRNAs, i.e. 
polypeptides, whether or not glycosylated, whose role may 
be structural or enzymatic in nature. This definition is 
also meant to include RNAs and polypeptides which play a 
regulatory role in gene expression. This invention provides 
novel means of employing expression vectors, e.g. plasmids 
or viral vectors, preferably an insect or entomopathogen 
virus expression vector, for expression of an exogenous 
gene product which is under the control of an effective 
promoter, preferably a promoter which promotes expression 
of a gene product in insect cells. More specifically, in a 
preferred embodiment, the present invention comprises -cloning 
the region of an NPV (such as AcNPV) surrounding the poly- 
hedrin gene, providing a plasmid which includes the poly- 
hedrin gene region, inserting an appropriate passenger 
gene into the polyhedrin gene or replacing at laast a 
portion of the polyhedrin gene with an appropriate passenger 
DNA while in plasmid form, co-transf ecting the resulting 
passenger plasmid DNA with wild type AcNPV DNA into 
suitable host cells and use of the transformed host to 
produce the exogenous gene .product. Suitably transformed 
host cells can be identified, e.g. by selecting for a 
virus having a phenotype where an allelic replacement of 
the polyhedrin gene with passenger DNA has resulted in a 
virus defective in occlusion body formation. 

Another promoter for use in connection with the present 
invention is the promoter controlling the synthesis by 
A. californica of a 7,200 dalton protein, termed simply 
the ••7.2K protein". See Adang and Miller, J. Virol 44:782 - 
793, 1982. Like the polyhedrin protein, this protein is 
expressed late in infection during the occlusion phase of 



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virus replication. While the exact function of this protein 
in occlusion is not yet known, the gene which produces the 
protein has been located and identified, and thus is 
available for use. See Figure 1. As used in this application 
the term "7.2K promoter" means the promoter which controls 
the gene which is responsible for expression of that 7.2 
Kd protein. Other promoters, from AcNPV or elsewhere, can 
also be used to produce exogenous gene products in insects, 
including other promoters from Baculoviridae, as well as 
other viruses, bacteria, yeasts, other fungal, insect and 
even maimnalian promoters. Those promoters which are functio- 
nal in insect cells may be identified, e.g. by replacement 
of the AcNPV promoter pMC 874 described infra, transfected 
into insect ceHs of interest, and beta-galactosidase 
production monitered, as disclosed herein. 

Thus, valuable host/vector systems are provided which can 
be employed for propagating and e:tpressing large segments 
of eukaryotic, prokaryotic or viral passenger DNA in a 
eukaryotic environment. Thus^ the present invention permits 
exogenous passenger DNAs, including those with gene products 
causing cell death, to be propagated and expressed at a 
high level in an invertebrate environment. 

Brief Description of the Drawings 

This invention can be more clearly understood by referring 
to the accompanying drawings wherein: 

Fig. 1 is the physical map and gene organization of the 
baculovirus AcNPV. 

Fig. 2 shows a linear representation of the restri-cticn 
endonuclease recognition sites in the circular DNA of 
the L-1 variant of AcNPV. 



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Pig. 3 ifi a physical map of the pEXX94 2 plaemia DMA. 
FiG. 4 is a physical map of the. pGP-B6874/SAL plasmid DNA. 
Pig, 5 is a physical map of the pGP-B6874 plasmid DNA. 
Fig. 6 shows the PstI and BamHl patterns of AcNPV 
L1GP-Gal3. 

Pig. 7 shows the schematic construction of pGP-B6874/SAL 
utilized as a transplacement vector wherein the exoge- 
nous passenger DNA of the transplacement vector expresses 
chloramphenicol acetyl transferase and is under the 
control of the exogenous promoter RSV-LTR, obtained from 
Rous sarcoma virus, incorporated therein. 

Detailed Description of the Invention 

In one embodiment of this invention, a method of using 
Autographa California nuclear polyhedrosis virus (AcNPV) 
as an expression vector has been developed wherein a eeg- 
ment of AcNPV DNA encompassing the polyhedrin gene is 
cloned in E. coli using a plasmid vector. As mentioned 
above, AcNPV has been extensively studied. Samples of 
AcNPV may be obtained from numerous sources, including 
the Yale Arbovirus Research Unit (YARU) located in New 
Haven, Conneticut. A restriction enzyme is used to 
open up the polyhedrin gene on the recombinant plasmid 
DNA to create a site for insertion of passenger DNA. 
The passenger DNA is selected and inserted into the 
cloned plasmid at the site opened by the restriction 
enzyme. The plasmid is then introduced, e.g. transf acted, 
along with wild type AcNPV DNA into suitable host cells. 
Thereafter, either by gene conversion or a double recombi- 
nation event , the DNA from the recombinant plasmid 
replaces its allelic counterpart, that is, the poly- 
hedrin gene, on the full length wild type AcNPV chromosome 
Hence, when RNA sythesis occurs in the virus, the messen- 
gerRNA read from the inserted passenger DNA serves to 



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express a new gene product under its direction. Specif: ' 
viruses containing the passenger DNA may be selected by 
any appropriate technique. The selected virus may then 
be propagated to exploit the ability of this passenger 
DNA to direct the sythesis of the desired gene product. 

Fig. 1 represents the physical map and gene organization 
of the baculovirus AcNPV. The physical map of the re- 
striction endonuclease sites of the 128 kilobase circu- 
lar, double-stranded DNA genome is presented in the 
inner eight concentric rings. The outer concentric circle 
presents information on the gene organization of AcNPV. 
The stippled areas refer to the position of some of the 
temperature- sensitive mutations, and the hatched areas 
refer to regions encoding various structural and non- 
structural proteins of AcNPV with the size of the prote- 
ins given in the number of kilodaltons. Note that the 
approximately 33,000 dalton gene for polyhedrin protein 
is located in the outer ring at the zero to approximately 
the 10% position, and the gene for an approximately 
7,200 dalton protein ("7.2"), which is also expressed 
in late stages, is located in the outer ring at about 
the 85-90% position. 

Fig. 2 is a linear representation of the restriction 
endonuclease recognition sites in the L-1 variant of 
AcNPV. The L-i variant has an additional Hind III site 
within Hind III-B and the two fragments are referred to 
as B1 and B2 with the size of the total genome being 
approximately 128 kilobases. 

Fig. 3 represents a physical map of the pEXS94 2 plasmid 
DNA which contains the 0.0 to 8.7 region of the AcNPV 
L-1 DNA, including EcoRI-I, K and 0 fragments. The vector 
segment was derived by EcoRl and Sail digeistion of 



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pBR325. 

Fig. 4 is a physical map of the pGP-B6874/Sal plasmid 
DNA having a total weight of 12.3 kilobases and con- 
taining a gene for kanamycin resistance. 

Fig. 5 is a physical map of the pGP-B6874 plasmid DNA 
having a total weight of 25.2 kilobases and containing 
a gene from E. coli which codes for the protein beta- 
galactosidase. 

It has been found that in accordance with one aspect 
of the present invention, baculoviruses are useable as 
vectors for propagating and expressing exogenous DNA in an 
invertebrate eukaryotic environment. Baculoviruses are 
highly advantageous as recombinant DNA vector systems 
because they possess such features as: (1) a covalently- 
closed, circular nuclear-replicating DNA genome, (2) 
an extendable rod-shaped capsid which may increase in 
size to accept an additional 20 kilobase or larger DNA 
segment, (3) a group of genes that are involved in 
occlusion yet are non-essential for infectious virus 
production and thus may be deleted, and (4) one or more 
strong promoters which are turned on after infectious 
virus production and control the synthesis of proteins 
involved in occlusion (e.g. polyhedrin and 7.2K protein) 
which constitute 10% or more of the protein of infected 
cells. AcNPV is employed in the method of this invention 
by using a plasmid vector containing a passenger DNA 
to replace the viral DNA genome that codes for occlusion 
protein synthesis. Thus the passenger DNA, once it is 
included in the viral DNA, has the advantage of the 
strong polyhedrin and/or 7.2K protein promoter, which, 
once turned on, will express the gene product encoded for 
in the passenger DNA in desirable amounts. It has also 
been found that, where desirable, the passenger DNA may 



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C15547C! ' 

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alternatively be under the control of its own or other 
exogenous promoters as discussed herein below. Finally, 
viruses that have expressed the passenger DNA may be 
easily selected and propagated. 

In a preferred embodiment, the first step in utilizing 
the polyhedrin promoter to express exogenous DNA is to 
clone a vector containing the AcNPV polyhedrin gene into 
an appropriate host. The location of the 3' terminal 6 2% 
of the 1.2 kilobase polyhedrin gene of AcNPV is located 
in the Hind Ill-V region of AcNPV as indicated in Figs. 
1 and 2. Thus a plasmid for the replacement of the poly- 
hedrin gene with a passenger DNA is begun by cloning 
into E. coli , using a plasmid vector a segment of 
AcNPV DNA from 0.0 to approximately 6.0 Kd. This will 
encompass at least the 3' terminal segment of the poly- 
hedrin gene (which occurs at about 3.3 to 4.0 Kd) , if not 
the entire gene. A restriction endonuclease that recog- 
nizes a site or sites whithin the polyhedrin gene region 
may then be used to open the recombinant plasmid DNA,^ 
creating a site for the insertion of passenger DNA. It 
is noted that the restriction endonuclease BamHI, has 
some useful sites in this region. Once the passenger DNA 
is inserted into the cloned plasmid the plasmid can be 
used in a "marker rescue" fashion. See L.K. Miller, 
Journal of Virology Vol. 39:973 (1981). Using this 
technique, full length AcNPV DNA is co-transf ected with 
the fragment of AcNPV DNA containing the passenger DNA 
presented by the plasmid. Allelic replacement occurs 
either by gene corA'ersion or a double recombination 
event resulting in the replacement of DNA in the 
full lenoth viral DNA with DNA from the fragment pre- 
sented by the plasmid. Thus, the natural polyhedrin gene 
is replaced with the engineered polyhedrin gene passenger 
DNA, and selection for passenger D>3A-containing viruses 
may be performed by screening plaques for absence of 



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occlusion bodies, e.g. using visual observation of virus 
plaques under the light microscope. An exact size re- 
placement of any deletions of the polyhedrin gene is not 
necessary since the rod-shaped nucleocapsid of baculo- 
viruses is extendable to greater lengths. 

In many instances, it is preferable to have the poly- 
eptide or other gene product which is expressed in 
accordance with this invention secreted outside the cell 
membrane of the insect cells in which it is expressed. 
This can be accomplished in known manners, e.g. by encoding 
en appropriate leader sequence which is expressed in 
conjunction with the polypeptide sought, and which enables 
that product to be secreted by the cell. 

In order to insert a passenger DNA into AcNPV L-1, the 
0.0 to 8.7 region of AcPV L-1 DNA in E. coli is cloned 
using PBR325 as a vector. The AcNPV L-1 is a wild type 
virus which may be used In this recombinant DNA construc- 
tion and its physical map is shown in Fig. 2. See Miller, 
et al. Virology Vol. 126:376-380 (1983), the disclosure ' 
of which is incorporated herein by reference. The plasmid 
PBR325 (described by Bolivar in Gene Vol. 4, p. 121 
(1978) is a derivative of plasmid pBR322 and carries 
unique EcoRI sites for selection of EcoRI generated 
recombinant DNA molecules. A recombinant plasmid may be 
constructed by digesting pBR325 with EcoRI and Sail 
restriction endonucleases which cleave within the chlor- 
arrphenicol resistance and tetracycline resistance genes, 
respectively, leaving the ampi<:illin resistance gene and 
replication origin intact with one EcoRI cohesive end 
and one Sail cohesive end. AcNPV L-1 DNA may be digested 
simcultaneously with EcoRI and Xhol. Following ligation 
of the EcoRI, Xhol, or Sail digested viral/pBR325 
fragmentDNAs, E.coli is transformed. Ap'^ CM^ tet^ colonies 



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may be screened for plasmids containing BamF. During 
screening procedures, pEXS942 has been found and, it is 
believed, arose due to the co-clonlng of an AcNPV Xho-EcoRI 
fragment and an EcoRI partial digestion product containing 
EcoRI-I, R and 0. Since pEXS942 contains the region from 
0.0 to 8.7, rather than 1.9 to 5.9, there is provided 
a larger region of the AcNPV genome and more DNA on both 
sides of the Hind III-V region (3.3 to 4.0), It is be- 
lieved that this larger region facilitates the double 
recombination (or gene conversion event) necessary for 
marker rescue between plasmid DNA and AcNPV DNA in vivo. 
Another advantage is the presence of the viral PstI site 
at 8.0 which is useful in further constructions to Insert 
passenger DNA into the polyhedrin region. Finally, the 
0.0 to 8.7 region totally encompasses the Hind III-N 
region of AcNPV which includes tsB113 which is a 
temperature sensitive -mutant. Thus, tsB113 and pEXS942 can 
be used together as additional selection for allelic 
replacement. 

To determine if the region of AcNPV L-1 encompassing 
BamF could be deleted in its entirety, the pEXS94 2 DNA 
was digested with BamHI and religated so as to delete 
the BamF fragment. Among the plasmids obtained, a novel 
construction was isolated and hereafter referred to as 
pEXS942 B6. This plasmid is not deleted for Bam F, but 
rather lacks the BamHI site at 4.5. Thus, pEXS942 B6 has 
only a single BamHI site at 3.0 map units, since the 
BamHI site was eliminated at 4,5. In this procedure, the 
region of AcNPV surrounding the polyhedrin gene is cloned 
in E. coli and from that a plasmid pEXS94 2 B6 may be 
isolated- This plasmid includes the AcNPV region from 
0.0 to 8.7 map units, thus encompasising at least the 
polyhedrin 3' terminal 62% between 3.3 and 4.0. Thus, 
the polyhedrin region in pEXS94 2 B6 or its derivatives 



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can be replaced with an appropriate passenger DNA while 
in plasmid form. The resulting pEXS942 B6 passenger 
plasmid DNA is then co-transf ected with the wild type 
AcNPV genome. A double recombination or gene conversion 
event between the recombinant plasmid DNA and wild type 
AcNPV results in allelic replacement of the polyhedrin 
gene of AcNPV with passenger DNA. Successful allelic 
replacement of the polyhedrin gene with passenger DNA 
results in viruses defective in occlusion body formation. 
This phenotype may be selected with appropriate plaque 
assays. In addition, a mutation in an AcNPV mutant, 
tsB113, maps adjacent to the polyhedrin gene so that 
successful allelic replacement within this region is 
selectable by decreased temperature sensitivity. The 
host/vector system may be employed for a variety of 
purposes once an appropriate restriction site for 
passenger DNA insertion has been identified. 

The advantages of the AcNPV vector system include the 
ability to package large segments of passenger DNA in 
the rod-shaped viral capsid and the availability of 
strong promoters such as the polyhedrin and 7.2K protein 
promoters which are induced following the production of 
infectious non-occluded virus. An additional advantage 
of placing an exogenous passenger DNA in this position 
is that sythesis for these promoters occur late in the 
infection cycle, subsequent to the onset of NOV release, 
and more than 24 hours prior to cytolysis. As a result, 
infectious virus production is not impaired by any 
active passenger gene product. Long periods of cell 
viability have been achieved, which suggests continued 
metabolic functioning during passenger protein synthesis . 

The genome of AcNPV is a double stranded, circular, 
covalently-closed DNA molecule of approximately 



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0155476 

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82,000,000 daltons. These features include a rod-shaped 
capsid that may accommodate 20 kilobases or more of 
passenger DNA and a sequential expression of viral 
genes resulting in the early production of infectious 
virus followed by a lengthy period of virus directed 
protein synthesis from a number of promoters. 

In another embodiment of the present invention, the promoter 
controlling the synthesis of the 7.2K protein may also 
be utilized for passenger gene expression. The 7.2K 
protein gene is located between 88 and 91 map units in 
Figure 1. See Adang and Miller, J, of Virology 44:782 
(1982), the disclosure of which is incorporated herein by 
reference. Synthesis of the mRNA encoding the 7.2K 
protein extends from Hind III-Q through Hind III-P. A 
plasmid containing most of the DNA encoding the 7.2K 
protein has been cloned (Adang and Miller, supra) and 
could be used for passenger gene insertion and allelic 
replacement within the 7.2K protein gene. 

In other embodiments of the invention, it is possible to 
introduce passenger genes under the control of exo- 
genous (non-baculovirus) promoters, such as promoters 
from other viruses, bacteria, fungae, insects, mammals, 
etc. In this approach, the passenger gene is attached to 
an exogenous promoter and together the exogenous promoter- 
gene combination is inserted into an appropriate "trans- 
placement vector". The transplacement vector is designed 
so as to permit allelic replacement of an appropriate 
region of viral DNA. The plasmid pGP-B6874/Sal (Fig. 4) 
is one such transplacement vector. This plasmid has a 
single Pst I site in a non-essential region of the plasmid. 
An exogenous promoter gene can be inserted into this Pst I 
site. The resulting recombinant plasmid is co-transf ected 



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01 55476 



with wild type viral DNA and recombinants selected by an 
appropriate technique. In the case of pGP-B6874/Sal , the 
vector is designed so that a blue color is produced for 
recoinbinant viruses resulting from allelic replacement. 
One example of an exogenous promoter is the long terminal 
repeat promoter of Rous sarcoma virus, an avian sarcoma 
virus which is widely available. This promoter (RSV-LTR) 
has been inserted into AcNPV and shown to direct the 
synthesis in insect cells of an exogenous protein, such 
as the chloramphenicol acetyl transferase gene of E. coli . 
One advantage of the use of an exogenous promoter of this 
nature is that the promoter is activated at early as well 
as late times in the viral replication process* Early 
expression may be useful in some applications of genetic 
engineering of baculoviruses and/or insect cells, parti- 
cularly with respect to the use of baculoviruses as 
biological pesticides. Where large scale production of 
polypeptides using insect cells is the goal, early 
producers would be advantageous, e.g. where the protein 
produced would not adversely affect cell viability or 
productivity. 

The following examples are given to illustrate embodiments 
of the invention as it is presently preferred to practice 
it. It will be understood that these examples are illus- 
trative, and the invention is not to be considered as 
restricted thereto except as indicated in the appended 
claims. 

Example 1 

AcNPV can be successfully employed as a recombinant D^lh 
vector by stably propagatinn a 9.2 Kb passenger .DNA in- 
serted into the polyhedrin gene. In addition the polyhe- 
drin promoter drives the high level expretision of a 
passenger gene, such as K.coli beta- 



-18- 



-18- 



Q155476' 



galactosidase. 

The plasmid pEXS94 2 B6 contains the 0.0 to 8.7 map unit 
region of AcNPV but lacks the BamHIsite at the BamF/C 
junction, and thus contains only a single BamHI site near 
or within the polyhedrin gene. 

The pMC874 plasmid possesses a single BamHI site at the 
eight codon of the E. ^ coli beta-galactosidase gene in 
the same reading frame as the BamHI site of the polyhedrin 
gene of beta-galactosidase. See Casadaban et al., J. Bact. 
143:971-80 (1980). In addition to the beta-galactosidase 
gene, the 9.2 Kb pMC874 plasmid contains the lac y gene, 
part of the lac A gene, a portion of the ampicillin 
resistance gene, a Col El replication origin, and a 
gene for kanamycin resistance. A fusion of BamHI digested 
pMC874 and plasmid pEXS942 B6 DNAs by in vitro ligation 
results* in the formation of a 25.2 Kb plasmid, pGP-B6874 
shown in Fig. 5, and hereafter called the fusion plasmid. 

The 25.2 Kb plasmid pGP-B6874 was constructed by digesting 
pEXS942 B6 and pMC874 with BamHI followed by ligation at 
high DMA concentrations to favor bimolecular fusions. See 
Maniatis et al.. Molecular Cloning, CoJd Spring Harbor 
Laboratory (1982), the disclosure of which is incorpo- 
rated herein by reference. Following transformation of 
E. coll HB 101 with the ligation products, the plasmid 
fusions were selected by their resistance to ampicillin 
and kanamycin. The ampicillin resistance gene is supplied 
by pEXS94 2 B6 and the kanamycin resistance gene is 
supplied from the pMCB74. Blue colonies on plates con- 
taining ampicillin, kanamycin and X-gal (a colorimetric 
indicator for beta-galactosidase enzymatic activity) were 
selected. 

For plaque assays involving blue color production, a 



Qlfi5476 

-19- 

procedure similar to plaque assay procedures invoXvin^ 
neutral red staining of lepidopteran cell monolayers 
was adapted. See Lee and Miller, J. Virol . 27:754-^67 
(1978). The procedure used for blue color production was 
similar except that 120 micrograms per milliliter of 
X-gal was included in the agarose overlay media. Instead 
of neutral red staining, the plaques are visualized by 
their blue color which is enhanced by warming the plates 
at 37 degrees C. for 4 - 6 hours, or allowing the color 
to develop overnight at room temperature. Alternatively 
for quick visualization, the tissue culture dishes are 
frozen in liquid nitrogen for five to ten seconds, thawed 
at room temperature, and the color allowed to develop 
for two to three hours. Infectious viruses were picked 
from the plaques, and replaqued directly or grown into 
a virus stock. 

The colonies which contained the fusion plasmi4 pGP-B 
6874 (Fig. 5) with the beta-^alactosidase gene fused to 
the polyhedrin N-terminus were demonstrated by f^stric- 
tion endonuclease digestion with Sal I, Eco RI and Hind 
III enzymes. These enzyme analyses distinguish tHe two 
possible orientations of the fused plasmids. 

TO transfer the polyhedrin/pMC874 fusion sequences (now 
represented by the fusion plasmid pGP-B6874) into the 
AcNPV DNA genome, allelic replacement or"transplacement" 
technique is employed. See L.K. Miller, J. Virol. 
39:973-76 (1981), the disclosure of which is incorporated 
by reference. Monolayers of s. frugiperda cells were co- 
transf acted with intact wild type AcNPV and a 1Qt)-fold 
molar excess of pGP-B6874 DNA using a calcium phosphate 
co-precipation technique. See Potter and Miller, 
J. Invert. Path. 36:431-2 (1980), the disclosure of which 



-20- 



• • • ' 



-20- • 

P155476 

is incorporated herein by reference. Viruses expressing 
the beta-galactosidase gene were detected as blue colored 
plaques in the presence of X-gal. 

Plasinid DNAs were prepared by a rapid clear lysate pro- 
cedure. See Holmes and Quigley, Anal. Blochem. 114:193 
(1981). AcKPV DNA was prepared from extracellular virus 
which was partially purified from infected culture media 
by centrifugation through a 20% sucrose solution at 
90,000 X g for 90 minutes at The pellet was re- 

suspended in 200 microliters of 10 mM Tris-HCl pE 7.6, 
10 mM EDTA and 0.25% SDS. Proteinase K was added to a 
final concentration of 0.5 mg/ml and the mixture was 
incubated for 30 minutes at 30»C and then phenol extrac- 
ted twice before the DNA was precipitated. 

The wild type virus used in the recombinant DNA con- 
struction was the L-1 variant of AcNPV referred to 
above and isolated and described in Lee and Miller, 
^' Virol. 27:754-767 (1978), the disclosure of which is 
incorporated herein by reference. A continuous cell line 
of a lepidopteran noctuid Trichoplusia nl . TN-368, was 
propagated in TC-100 media (Microbioloqical Associates) 
supplemented with 0.26% Tryptose broth, 10% fetal bovine 
serum, 2 mM L-glutamine and an antibiotic-antimycotic 
preparation (Gibco) . 

Comparison of neutral red-stained monolayers and X-gal- 
stained monolayers indicated that approximately one in 
a thousand of the plaques resulting from co-transfection 
of AcNPV and pGP-B6874 expressed beta-galactosidase. 
Several blue plaques were chosen and directly replaqued 
on fresh S. frugiperda cell monolayers. 

Transfections were performed by the method of Potter and 
Miller, supra , using the calcium phosphate precipitation 



-21- 



-21- ^''^5476 

method without the glycerol boost step. Following the 
20 minute incubation of the DNA precipitate, the S. 
frugiperda cells were overlaid with liquid niediuin~for 
two to three hours and this overlay was then replaced 
with either fresh liquid medium or agarose-containinq 
medium for plague development. For transplacement ex- 
periments, a molecular ratio of 100 plasmid DNAs 
per viral DNA was routinely used. The DNAs to be used 
in the transfection procedures were stored in water 
rather than a Tris-EDTA solution. 

Blue plaques, free of occlusion bodies, were picked, 
developed into virus stocks, and characterized by 
restriction endonuclease analysis. The Pst I and BamHI 
patterns of one of these viruses, AcNPV LlGP-gal3, is 
presented in Fig. 6. Pst-D is missing in LlGP-gal3, 
and two new fragments are present. One new fragment is 
found between Pst-C and D and the other is found below 
Pst-F. The latter fragment co-migrates with a Pst I 
fragment of pGP-B6874 from the ApS region of pMC874 to 
the Eco EI-0 region of AcNPV. The new Pst fragment 
between Pst C and Pst D corresponds to the Pst I-O/N 
junction in Eco RI-B throuqh Eco Rl-i of AcNPV (i) and 
the E. coli beta -gal, lac Y and lac A region. The BamHI 
pattern also confirms that the entire pMC874 is properly 
inserted into AcNPV. No BamHI fragments from the wild 
type strain AcNPV are present in Ll<3P-gal3 but a new 
9.2 kilobase fragment, co-migrating with pMC874, is 
found. BamF has also been observed in the LlGP-galB virus, 
indicating that homologous recombination between AcNPV 
and PGP-B6874 occurred near the pMC874 and the Eco RI-i 
(s) junction. Thus, the virus AcNPV LlGp-gal3 expresses 
beta-galactosidase activity, indicating that thebeta- 
galactosidase gene originating from pMC874 has been 
found to recombine into this virus. 



-22- 



-22- 



0155476 



Regulation of Expression of Beta-Galactosi dase Activity 

To determine If the expression of the beta-galactosidase 
gene was under the same temporal regulation as polyhedrin 
gene expression, monolayers of S. frugiperda were in- 
fected with wild type AcNPV or AcNPV LlGP-gal3 at a 
multiplicity of infection of 20 plaque forming units 
(PFU) per cell. At various times post infection, the 
infected cells were assayed for beta-galactosidase 
activity and total protein content. Beta-galactosidase 
activity first appears between 18 and 24 hours and then 
dramatically increases through 48 hours after infection. 

Levels of beta-galactosidase were determined using the 
ortho-nitrophenylgalactoside (ONPG) assay described by 
Miller in Experiments in Molecular Genetics, Cold Spring 
Harbor Laboratory, New York (1972). Insect cells tested 
were infected with a virus at a multiplicity of infection 
of 20 PFU/cell. The virus was allowed to adsorb for 1 hour 
at room temperature -with gentle rocking. The innoculum 
was removed and replaced with appropriate media and 
incubation was carried out at 27 degrees centrigrade 
for the time indicated. The cells were washed with a 
phosphate-buffered saline solution (Lee and Miller, 
supra ) and cells were scraped from the plate and pelleted 
at 200 x g for five minutes. The supernatant was discar- 
ded and the cell pellet frozen in liquid nitrogen and 
stored at minus 70°C until assayed. The cells were 
resuspended in distilled water and then diluted 
appropriately (from 1:1 to 1:1000) in Z buffer so a 
faint yellow color developed between 15 minutes and 
1 hour after ONPG addition. The cells were disrupted 
using chloroform and 0.1% SDS as recommended by Miller, 
1972. Specific activity was defined as n moles ONPG 
cleaved/min/mg protein. 



-23- 



"23- 



0165476' 



The observed specific activity at 48 hours post infection 
is 8,000 n moles of ONPG cleaved 'per min/per mg. protein, 
which represents an approximate 900-fold increase in 
beta-galactosidase activity. A specific activity of 
8,000 means that at least 3% of the total cellular 
protein is beta-galactosidase since pure beta-galacto- 
sidase has a specific activity of 3 x 10^ units per mg. 
At 72 hours post-infection, the specific activity drops 
probably due to the onset of cytolysis. 

Regulation of polyhedrin/beta-galactosidase gene 
expression was also analyzed by pulse-labelling infected 
cells with 35g methionine at various times post infection. 
The 35g labelled proteins are analyzed by SDS-poly- 
acrylamide gel electrophoresis. Coomassie blue staining 
of the proteins shows that early in the infection there 
is no significant difference between the wild type or 
the LlGP-gal3 profiles. From IB to 72 hours post in- 
fection however, the 33,000 dalton polyhedrin protein 
is synthesized in wild type infected cells but not in 
LlGP-gal3 infected cells. 

LlGP-gal3 profiles show a new protein of approximately 
120,000 daltons as well as several smaller proteins in 
the 95,000 dalton range at about 18 hours post infection 
that are not found in wild type infected cells. The lar- 
gest protein is the appropriate size for a polyhedrin/ 
beta-galactosidase fusion polypeptide. The intensity of 
the Coomassie blue stain in the 120,000 dalton region 
corroborates the specific activity estimate that active 
be*-a-gaBctosidase constitutes at least 3% of the 
total cellular protein at 48 hours post infection. 
Coomassie blue stain also reflects the reduction in the 
amount of the polyhedrin/beta-galactosidase fusion protein 
at 72 hours post infection. 



-24- 



-24- 



0155'476 



The procedure of Miller, et al, 1983, supra is used for 
analysis of virus-induced proteins in infected 
fruqiperda cells except that 1 x 10^ cells per 35 laili- 
meter plate were used and the cells v/ere washed with 
media after pelleting at low speed. 

Immune Precipitation with Polyhedrin Antibody 

To further explore the nature of the polypeptides pro- 
duced at 48 hours in wild type and the LlGP-gal3-in- 
fected cells, the 35 S-labeled proteins at 48 hours post 
infection were precipitated with antiserum raised to 
purified polyhedrin. Immune precipitation using polyhe- 
drin antiserum was performed by a slight modification 
of the procedure of Kessler, Immunology 117:1482-1490 
(1976). Forty microliters of 35 S-labeled protein from 
■ 48 hours post infection were incubated with 40 micro- 
liters of polyhedrin antiserum for 1 hour at room 
temperature and then 160 microliters of a 2% suspension 
of the Staphylococcus aureus (Sigma) in precipitation 
buffer (0.15 M NaCl, 5 nuM EDTA, 50 mM"Tris-Hcl, 0.02% 
NaN3, 0.05% NP40, and 0.1% BSA) was added. The mixture 
was incubated at room temperature for 20 minutes and 
the resulting precipitate was collected by ce^trif ugation 
at 15,000 x g for 1 minute. The supernatant was discar- 
ded and the pellet was washed with 0.5 milliliters of 
precipitation buffer. The pellet was then resusnended 
in solubilization buffer (1% SDS, 0.5% beta-mercapto- 
ethanol and 0.5 M urea) with vortexing and heated for 
5 minutes at 100°C. The debris was pelleted at 15,000 
X g for five minutes and the supernatant was analyzed by 
SDS-polyacrylamide gel electrophoresis. 

The polyhedrin antiserum precipitated the 33,000 dalton 
polyhedrin protein from wild type infected cells as wel3 
as several polypeptides in the 20,000 to 25,000 



-2S- 



-25- 



dalton region. Polypeptides of this size were not 
precipitated by polyhedrin antiserum in the LlGP-gal3- 
infected cells. Instead, the 120,000 protein was the 
predominant precipitant as well as the series of poly- 
peptides in the 95,000 dalton region noted previously 
and several other smaller polypeptides of discreet size 
between 40,000 to 95,000 daltons. Thus the 120,000 protein 
is a polyhedrin fusion product and the smaller poly- 
peptides represent alternate forms of the fusion product. 
The specific activity of beta-galactosidase in LlGP- 
gal3-infected cells is exceptionally high. The highest 
specific activity reported in yeast is one-half that ob- 
served in Ll<3P-gal3-infected cells at 48 hours post in- 
fection. The 33,000 dalton protein found in wild type 
infected cells is replaced in LlGP-gal3-infect€d cells 
by a polyhedrin/beta-galactosidase fusion protein of 
approximately 120,000 daltons as well as a series of 
other proteins of smaller molecular weight. The smaller 
proteins are individually less abundant than the 120,000 
dalton protein and may arise by proteolytic cleavage of 
the 120,000 dalton fusion protein. 

Example 2 

Expression of Polvhedrin/Beta-nalactosidase 
in other Cell Lines 

The polyhedrin/beta-galactosidase fusion protein is also 
expressed by other insect cells which are permissive of 
AcNPV replication. For example, a cell line derived from 
the lepidopteran noctuid Trichoplusia ni, TM-368, was 
propagated in TC-100 media (Microbiological Associates) 
supplemented with 0.26% tryptose broth, 10% fetal bovine 
serum, 2mM L-glutamine and an antibiotic-antimycotic 
preparation «3ibco) . Monolayers of this cell line were 
infected with various dilutions of the reconfcinant virus 



-26- 01 65476 

LlGP-gal3 and overlaid with agarose-inedia containing 120 
nticrograms per milliliter of the B-galactosidase color 
indicator, X-gal. The monolayers were incubated at 27»C 
for 4 days and then color was developed by warming the 
plates to 37»C for 4-6 hours. The expression o' the 
polyhedrin/B-galactosidase fusion protein was demonstrated 
by the vivid blue plaques on the T^ monolayers. The 
titers indicated that the recombinant virus was approxi- 
mately equally infectious on the TNni monolayers as on 
S. frugiperda monolayers. 

Example 3 

A Generalized TranEolaceme nt Vector 

For some types of genetic engineering applications it 
will be preferred to insert passenger genes under their 
own promoter control into the vector. In one such appli- 
cation, the transplacement vector plasmid pGP-B6874/Sal 
was constructed. This plasmid has been inserted in E^^oli 
HE 101, a sample of which has been deposited at the 
American Type Culture Collection, and is he3d under ATCC 

accession number • This vector provides a single 

Pst I site for the insertion of a passenger DKA under 
the control of its own or other exogenous promoter and 
allows the recombinant plasmid to be inserted into 
AcNPV using "blue" plaques as the means for selecting 
recombinant viruses. 

The pGP-B6874/Sal plasmid illustrated in Fig. 4, was 
constructed by partially digesting pGP-B6874 with the 
restriction endonuclease Sal I and religating at low DNA 
concentration. Following transformation of E. coli HB 101 
blue colonies were selected on kanamycin plates and then 
tested for sensitivity to ampicillin. The plasmids of 



-27- 



-27- 



Q15547!B; 



Amp^i Kan , blue colonies were characterized by restriction 
endonuclease analysis. The pGP"B6874/Sal plasmid was 
shown to contain the regions beta-gal including the 
lac 2 (beta-ga]actosidase gene, "Z") lac V, ("Y"), Rep 
and Kan^ genes to the Sal I site of Eco RI-1 (S). The 
pGP-B6874/Sal plasmid is essentially pMC8 7 4 bordered by 
the N terminus and C terminus of the polyhedrin gene as 
in pGP-B6874. The Pst I site is not essential for beta- 
galactosidase expression or replication in E> coli. 
E, coli containing pCP-B6874/Sal are blue on x-gal plates 
and red on Maconkey plates. 

Thus a transplacement plasmidi pGP-B6874/Sal has been 
constructed and may be used to facilitate the insertion 
and selection of recombinant viruses carrying passenger 
genes which are under the control of their own promoters. 
Passenger genes can be inserted at the unique Pst 1 site 
next to the E.coli replication genes of the plasmid and 
cloned in £. coli . The co-transf ection of the resulting 
plasmid with wild type AcNPV and subsequent selection 
for blue viruses, should facilitate the selection of re- 
combinant viruses. 

To demonstrate the utility of pGP-B6874/SAL as a trans- 
placement vector and to demonstrate the effective use of 
an exogenous promoter ^ the E. coli gene encoding chlor- 
amphenicol acetyl transferase (CAT) attached to the exo- 
genous promoter, RSV-LTR, was inserted into pGP-B6874/SAL. 
The scheme for this construction is found in Figure 7. 
The plasmid pRSV-CAT «k>rman et a3 , Proc. Natl Acad Sci . 
79: 6777 (1982)) was digested with PstI and BamHI. The 
transplacement plasmid pGP-B6874/SAL was digested with 
PstI and partially digested with BamHI. The two re- 
striction endonuclease-digested plasmids were mixed and 
ligated. The resulting recombinant plasmids were intro- 



^28^ 



-28- 



0165476 



duced into E. coli by tr5.nE^orrr.?.tion and lac*^ colonie . 
were selected on minimal media. The nlasmid pJ.C-l (se.^ 
Fig. 7) was isolated and its structure verified by rr 
striciton endonuclease analysis. 

S. frugiperda cell monolayers were transfected with a 
mixture of AcNPV L-1 DMA and pLC-1 DNA in a ratio of 1 
to 100. Viruses producing plaques with blue color and 
lacking OV were selected. A virus, AcHPV LI-RSV-CAT, was 
isolated and shown to contain the pLC-1 genes lac Z, 
lac at least part of A, ("A"), ori (origin of repli- 
cation, sometimes referred to as "rep") and RSV-LTR- 
CAT by restriction endonuclease analysis. 

The AcNPV-Ll-RSV-CAT virus expressed the CAT gene as 
early as 2 hours post-infection as shown by assaying 
the CAT activity by the procedure of Gorman et al supra . 
Activity was first observed at 2 hours post-infection 
and continued to increase through 6 hours and 12 hours 
post infection. Thus an exogenous promoter, the RSV-LTR 
can be used to express exogenous genes in insect cells. 
Futhermore, an exogenous promoter can be used to express 
exogenous genes early in infection. 

Many features of AcNPV make this virus a good host/ 
vector for the propagation and expression of passenger 
genes in a metazoan environment. The rod-shaped capsid 
extends to accomodate additional DNA sequences and vector 
capacity may exceed 100 kilobases. Since extracellular 
virus is the infectious form in cel.T culture, the genes 
for occlusion are non-essential. The genes encoding poly- 
hedrin or the 7.2K protein are ideal sites for passenger 
gene insertion since they are abundantly expressed bet- 
ween 18 and 72 hours post infection. At late times in 

-29- 



BAD ORIGINAL 



-29- 



Q1554Zi 



infection these proteins may constitute 20% or more of 
the total protein of the infected cell. The locations of 
the polyhedrin arid 7.2K protein genes on the AcNPV physi- 
cal map and the directions of the transcription are 
known. The synthesis of the 1.2 kilobase polyhedrin 
messenger RNA is temporally controlled and is first 
detected at twelve hours post infection. The late ex^ 
pression (18 hours post infection) from the polyhedrin 
promoter is particularly advantageous in expressing cyto^ 
toxic gene products. 

The above examples demonstrate that AcNPV can be success- 
fully genetically engineered and employed as a recombi- 
nant DNA vector. The examples also demonstrate that the 
polyhedrin promoter and other promoters can drive the 
high level expression of a passenger gene such as E. coli 
beta-galactosidase. The specific activities of beta- 
galactosidase achieved are significantly higher than 
any previously reported in other prokaryotic or eukary- 
otic expression systems to the best, of the applicant's 
knowledge. 

Additional advantages and modifications will readily occur 
to those skilled in the art from a consideration of this 
disclosure or practice of this invention, and it is 
intended that this invention be limited only by the scope 
of the appended claims. 



0155476 

What iB claimed ir: 

1. A vector for production of a gene product by ir':;ect 
cellEr compriBing an exogenous gene segment which encocies the 
expression of the gene product, the gene segment being under th^ 
control of a promoter which promotes the expression of a gene 
product by insect cells. 

2. The vector of claim 1, wherein the vector comprise? ; 
baculovirus. 

3. The vector of claim 2, wherein the baculovirus 
comprises a nuclear polyhedrosis virus. 

4. The vector of claim 3, wherein the nuclear 
polyhedrosis virus comprises an Autoorapha californiqa nuclear 
polyhedrosis virus or variant thereof. 

5. The vector of claim 1, wherein the promoter is a 
viral promoter which is active in the late stages of viral growth 
to produce a viral gene product. 

6. A vector of claim 5# wherein the viral promoter 
comprises a polyhedrin promoter. 



7. The vector of claim 5, wherein the viral promotev 



-2- 



0165476'* ' 



conpriBee a 7.2K promoter. 

8. The vector of claim 1, wherein the promoter compriees 
a promoter which ie active in the early etagea of viral growth to 
produce a viral gene product. 

9. The vector of claim 1, wherein the promoter compriaee 
an exogenous promoter which is active in ineect cells. 

10. The vector of claim 1, wherein the exogenous segment 
conprises a gene segment which encodes the expression of a 
readily detectable gene product. 

11. The vector of claim 10. wherein the readily 
detectable gene product comprises a protein which governs 
resistance to an antibiotic or a protein which reacts with an 
indicator to produce a detectable physical change. 

12. The vector of claim 1, wherein the vector comprises 
the plasmid p6P'-B6874. 

13. The vector of claim 1, wherein the vector comprises 
the plasmid pGP-B6e74/SAL. 

14. The vector of claim 1, wherein the vector comprises 
the plasmid pLC-1 



15. A method of preparing an expression vector suitable 
for use in insect cells, comprising inserting an exoger^ous vtne 
segment into a DNA segment to form a vector, the insertion being 
made in a region of the DNA segment which is controlled by a 
promoter which promotes expression of a gene product by insect 
cells. 

16. The method of claim 15, wherein the vector comprieer; 
a nuclear polyhedrosis virus. 

17. The method of claim 15, wherein the DNA segment is 
derived from a virus which replicates in insect cells, the DNA 
segment comprises a gene region and a promoter region which 
controls the expression of the gene product encoded for in the 
gene region, and the insertion is made by providing a plasmid 
containing the DNA segment, and inserting the exogenous gene 
segment into the gene region of the DNA segment to form a 
recombinant plasmid vector. 

18. The method of claim 17, wherein a viral vector is 
:ormed by contacting the recombinant plasmid vector with a virus 

which replicates in insect cells, and selecting for resultant 
viruses which express a gene product coded for by exogenous gene 
i^^groent. 



Q1554Z6 

19. The method of claim 18, wherein the virus* is a 
nuclear polyhedrosie virusr the DNA segment comprieeB the 
polyhedrin promoter and at least a part of the polyhedrin gene, 
and the insertion occurs at a site within the polyhedrin gene 
region. 

20. The method of claim 19, wherein the nuclear 
polyhedrosis virus comprises an ^iitoyraphica californica nuclear' 
polyhedroBiB virus or variant thereof. 

21. The method of claim 15, wherein the promoter 
comprises a viral promoter. 

22. The method of claim 21, wherein the viral promoter 
is a promoter which is active in the late stages of viral growth 
to produce a viral gene product. 

23. The method of claim 15, wherein the promoter ie a 
viral promoter which is active in the early stages of viral 
growth. 

24. The method of claim 15. wherein the promoter is an 
exogenous promoter which is active in insect cells. 

25. The method of making a gene product comprising 
inserting a vector into insect cells, said vector compriBing an 



0155476 



exogenouE gene Beginent which codes for expresEion of the cane 
product, said exogenous gene segment being under the control of a 
promoter which promotes expression of a gene product in insect 
cells, culturing the insect cells under conditions favoring 
expression of the gene product, and recovering the gene product 
expressed. 

26. The method of claim 25 t wherein the vector comprirer 
a nuclear polyhedrosis virus. 

27. The method of claim 26, wherein the nuclear 
polyhedrosis virus comprises an Autographa California nuclear 
polyhedrosis virus or variant thereof. 

28. The method of claiin 25, wherein the promoter 
comprises a viral promoter. 

29. The method of claim 28, wherein the viral promoter 
is a promoter which is active in the late stages of viral growth 
to produce a viral gene product. 

30. The method of claim 29, wherein the viral promoter 
is a polyhedrin promoter* 



31. The method of claim 29 » wherein the promoter is 
7.2K promoter. 



.6. 



Q1554Z6 



32. The method of claim 25 r wherein the promoter i£ a 
viral promoter which is active in the early stages of viral 
growth to produce a viral gene product. 

33. The method of claim 25, wherein the promoter is an 
exogenous promoter which is active in insect cells. 

34. A gene product made in accordance with claim 25. 



► •• . . ^ • • ' *' ' 
• t)1 554/6 




PHYSICAL MAP AcMNPV-LI DNA 

^ f:i'> ^.^.'^ * ■ .^»{^^.^ HinDID 

^ , ^ ^ ■ ^ PI ° ■', ! L_!_ ss,i 

^r/, JL ° BotnHI 

A DC e A 
2 ■ I I I Smol 

I I I I II 1 1 1 1 1 

0 10 20 30 40 50 60 TO BO 90 100 

PIQ 2 Genome 



0155476 




0 b c 



0 b c 



*• •••• 
• * • • • 



Xrl 554/6 



Soli 



Bom HI 



HindX 



p6P-B6874/Sol 
i2.3kb 



Hind SI 
Bom HI 

HindllC 



.EcoRI 



FIG.4 



rep 



RstX 



Soil 



FIG.5 




m r ' r t 

0155476 




J 



European Patent 
OMic* 



EUROPEAN SEARCH REPORT 



01 55476 



. Appttcation Aumb«r 



DOCUMENTS CONSIDERED TO BE RELEVANT 



EP 85101009.0 



CiUtien of (iocum«ni with mdicaiion. wh«m appropnat*. 
of riiiv*ni pasu0M 



naiavant 
to Claim 



CLASSIFICATION OF THE 
APPLICATION (mt CM) 



D,A 



SCIENCE, vol. 219, no. 4585, 
February 11, 1983 
(Washington DC. ) 

L.K. MILLER et al . "Bacterial, 
Viral, and Fungal Insecticides** 
pages 715-721 

♦ Pages 718, 719 * 



JOURNAL OF VIROLOGY, vol. 44, 
no. 3, December 1982 
(Washington DC. ) 

M.J. ADANG et al. "Molecular 
Cloning of DNA Complementary to 
mRNA of the Baculovirus Auto- 
grapha californica Nuclear Poly- 
hedrosis Virus: Location and 
Gene Products of RNA Transcripts 
Found Late in Infection" 
pages 782-793 

* Abstract (page 782) * 



JOURNAL OF VIROLOGY, vol. 39, 
no. 3, September 1981 
(Washington DC. ) 

L.K. MILLER: "Construction of a 
Genetic Map of the Baculovirus 
Autographa californica Nuclear 
Polyhedrosis Virus by Marker 
Rescue of Temperature-Sensitive 
Mutants" 
pages 973-976 

* Totality * 



The present search repod hu t>een drawn up for all claimi 



1-4 



1-4 



1-4 



C 12 N 15/00 

C 12 N 7/00 

C 12 N 5/00 

//C 12 R 1:91 



TECHNICAL FIELDS 
SEARCHED (Inl CIA) 



C 12 N 



Place of search 

VIENNA 



Data ot completion of the search 

03-05-1985 



EKaminer 

WOLF 



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O : non-written disclosure 
P : intermediate document 



T : theory or principle underlying the invention 
E : earlier patent document, but published on. or 

after the filing date 
O : document cited in the application 
L ; document cited for other reasons 

A : member of the same patent family, corresponding 
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