Baculovirus expression vectors

A method and composition for improving the level of expression of a gene or collection of genes by providing a novel baculovirus promoter to which a heterologous gene is attached. The novel promoter may contain a modification of a natural viral promoter or may be totally synthetic. The novel baculovirus promoter may also comprise a combination promoter such as a promoter with a double start site or a combination of two different promoters. Foreign gene placement in a novel location or genomic orientation is also included.

TECHNICAL FIELD 
The present invention relates to methods and compositions for improving the 
expression of a gene. More particularly, the present invention relates to 
classes of novel promoters that improve the expression of a heterologous 
gene in a baculovirus system. 
BACKGROUND OF THE INVENTION 
As used throughout this specification, the following definitions apply for 
purposes of the present invention: 
The term "expression" may be characterized in the following manner. A cell 
is capable of synthesizing many proteins. At any given time, many proteins 
which the cell is capable of synthesizing are not being synthesized. When 
a particular polypeptide, coded for by a given gene, is being synthesized 
by the cell, that gene is said to be expressed. In order to be expressed, 
the DNA sequence coding for that particular polypeptide must be properly 
located with respect to the control region of the gene. The function of 
the control region is to permit the expression of the gene under its 
control. 
The term "vector" refers to an extra-chromosomal molecule of duplex DNA 
comprising an intact replicon that can be replicated in a cell. Generally, 
vectors are derived from viruses or plasmids of bacteria and yeasts. A 
baculovirus vector comprises a baculovirus replicon. 
The term "gene" refers to those DNA sequences which transmit the 
information for and direct the synthesis of a single protein chain. 
The term "infection" refers to the invasion by agents (e.g., viruses, 
bacteria, etc.) of cells where conditions are favorable for their 
replication and growth. 
The term "transfection" refers to a technique for infecting cells with 
purified nucleic acids of viruses. 
The term "heterologous gene" in reference to the baculovirus vectors 
hereof, refers to DNA that encodes polypeptides ordinarily not produced by 
the virus from which the vector is derived, but which is introduced into 
the cell as recombinant DNA or within viruses carrying recombinant DNA 
genomes. The terms "passenger gene" or "passenger DNA" as used herein are 
equivalent to the term "heterologous gene." "Exogenous," as used herein, 
has the same meaning as heterologous. 
The term "transplacement plasmid" means a bacterial vector which is used as 
an intermediate in the construction of a virus vector. A transplacement 
plasmid facilitates the transfer of exogenous genetic information, such as 
the combination of a novel promoter and a heterologous structural gene 
under the regulatory control of that promoter, to a specific site within 
the viral genome by homologous recombination. That homologous 
recombination occurs via the DNA sequences flanking the chimeric gene. 
The science of genetic engineering has advanced to a stage wherein certain 
biologically useful products can be produced in large quantities. For 
example, two commercially successful drugs, human growth hormone and 
tissue plasminogen activator (t-PA), are now being produced in large 
quantities and are being used to treat a variety of pathological 
conditions. However, scientists are constantly trying to discover new and 
more efficient systems for producing the proteins and other products in 
various biological systems. 
The technology of transferring genes from one species and expressing them 
in another is made possible because the DNA of all living organisms is 
chemically similar in that it is composed of long chains containing the 
same four nucleotides. Nucleotide sequences are arranged in codons 
(triplets) which code for specific amino acids with the coding 
relationship between the amino acid and nucleotide sequence being 
essentially the same for all species of organisms. The DNA is organized 
into genes which are comprised of control regions which mediate initiation 
of expression of the gene and coding regions. These control regions are 
commonly referred to as "promoters." An enzyme, called RNA polymerase, 
binds to the promoter region and is either activated or in some way is 
signalled so that it travels along the coding region and transcribes the 
encoded information from the DNA into messenger ribonucleic acid (mRNA). 
The mRNA contains recognition signals: signals for ribosome binding, 
signals for translational start and stop, and for polyadenylation. 
Cellular ribosomes then translate the nucleotide codon information of the 
mRNA into protein with an amino acid sequence specified by the nucleotide 
codon sequence. 
The general use of restriction endonucleases and the ability to manipulate 
DNA sequences has been greatly improved by the availability of chemically 
synthesized double stranded oligonucleotides containing desired nucleotide 
sequences including useful restriction site sequences. Virtually, any 
naturally occurring, cloned, genetically altered or chemically synthesized 
segment of DNA can be coupled to any other segment by attaching an 
oligonucleotide containing the appropriate sequences or recognition sites 
to the ends of the DNA molecule. Subjecting this product to the hydrolytic 
action of the appropriate restriction endonuclease produces the requisite 
complementary ends for coupling the DNA molecules. While there are many 
possible variations in gene transfer schemes, it is important to note that 
the techniques are available for inserting DNA sequences in the proper 
location and orientation with respect to a promoter region to allow 
expression of those sequences. 
Potentially, any DNA sequence can be inserted into a vector molecule to 
construct an artificial recombinant molecule or composite, sometimes 
called a chimera or hybrid DNA. For most purposes, the vector utilized is 
a duplex extra-chromosomal DNA molecule comprising an intact replicon such 
that the recombinant DNA molecule can be replicated when placed into 
bacteria or yeast by transformation. Vectors commonly in use are derived 
from viruses or plasmids associated with bacteria and yeast. 
Because of the nature of the genetic code, the inserted gene or portions 
thereof will direct the production of the amino acid sequence for which it 
codes if the gene or gene portion is attached to a control region 
(promoter) which is capable of regulating expression in the cell in which 
the vector replicates. The general techniques for constructing expression 
vectors with cloned genes located in the proper relationship to promoter 
regions are described in the literature (e.g., See T. Maniatis, et al. 
(1982) Molecular Cloning A Laboratory Manual, Cold Spring Harbor 
Laboratory, Cold Spring Harbor, N.Y.). 
A number of vector systems utilizing the above-described general scheme and 
techniques have been developed for use in the commercial or experimental 
synthesis of proteins by genetically modified organisms. Many of these 
vector systems utilize prokaryotic bacterial hosts for vector replication 
and heterologous gene expression. Additionally, systems have been utilized 
which employ eukaryotic cells for vector replication and heterologous gene 
expression. Such systems are employed for hepatitis B virus surface 
antigen synthesis and for human tissue plasminogen activator synthesis. 
Eukaryotic hosts are preferred for the production of some eukaryotic 
proteins which require modification after synthesis (i.e., glycosylation) 
to become biologically active. Prokaryotic cells are generally incapable 
of such modifications. 
The use of virus vectors in eukaryotic hosts has been the subject of a 
considerable amount of recent investigation. Viral vector systems may 
suffer from significant disadvantages and limitations which diminish their 
utility. For example, some viral vectors are not able to achieve high 
enough levels of gene expression for economic protein production in costly 
eukaryotic cell culture systems. Some eukaryotic viral vectors are either 
pathogenic or oncogenic in mammalian systems, creating the potential for 
serious health and safety problems associated with accidental infection. 
Some virus vectors have severe limitations on the size of the heterologous 
gene that can be stably inserted into the virus particles. 
As the genetic engineering technology becomes more sophisticated, there 
will be an increased interest in inserting more than one heterologous 
gene, i.e., genes coding for more than one protein, into a host cell to 
achieve coordinated expression and possibly to obtain coordinated activity 
of the various gene products. 
An ideal viral vector should be capable of stably carrying a large segment 
of heterologous DNA, efficiently infecting cells and converting virtually 
all the protein biosynthesis of the infected cell to the high level 
expression of the foreign gene. A virus that appears to be well suited as 
a vector for the propagation and high level expression of many 
heterologous genes in a higher eukaryotic environment is the baculovirus 
Autographa californica nuclear polyhedrosis virus (AcMNPV). (See Miller, 
L. K. (1981) "Virus Vector for Genetic Engineering in Invertebrates," In 
Genetic Engineering in the Plant Sciences, N. Panopolous (ed.), Praeger 
Publ., N.Y., pp. 203-224; U.S. Pat. No. 4,745,051.) 
The baculovirus group includes the subgroups of nuclear polyhedrosis 
viruses (NPV) and granulosis viruses (GV); baculoviruses infect only 
arthropod hosts. The virus particles of NPV and GV are occluded in 
proteinaceous crystals. In occluded forms of baculoviruses, the virions 
(enveloped nucleocapsids) are embedded in a crystalline protein matrix. 
This structure, referred to as an inclusion or occlusion body, is the form 
found extraorganismally in nature and is responsible for spreading the 
infection between organisms. The subgroup NPV produces many virions 
embedded in a single, large (up to 5 micrometers) polyhedral crystal, 
whereas the subgroup GV produces a single virion embedded in a small 
crystal. The crystalline protein matrix in either form is primarily 
composed of a single 25 to 33 kDa polypeptide which is known as polyhedrin 
or granulin in NPV or GV, respectively. 
More general information on the subject of baculovirus structure and the 
process of infection is available in the following reviews: Carstens 
(1980) "Baculoviruses--Friend of Man, Foe of Insects? ," Trends and 
Biochemical Science, 52:107-10; Harrap and Payne (1979) "The Structural 
Properties and Identification of Insect Viruses" in Advances in Virus 
Research, Vol. 25, M. A. Lawer et al. (eds.), Academic Press, New York, 
pp. 273-355; and Miller, L. K. (1981) supra. 
Baculovirus helper-independent viral vectors are particularly useful for 
the high-level production of biologically active eukaryotic proteins. 
Expression levels for some foreign genes have been reported to be 10% to 
25% of the total protein of the recombinant infected cell. Appropriate 
post-translational modifications, including signal peptide cleavage, 
glycosylation, phosphorylation, oligomerization, complex formation, 
isolation and proteolysis, have been reported for a variety of different 
heterologous proteins produced using this expression system. 
The baculovirus expression vectors described to date use very late 
promoters, such as the polyhedrin or polypeptide 10 (p10) promoters to 
drive foreign gene expression. (Reviewed by Luckow and Summers (1988) 
"Trends in the Development of Baculovirus Expression Vector," 
Bio/Technology :47-55 Miller, L. K. (1988) "Baculoviruses as Gene 
Expression Vectors," Ann. Review of Microbiology, 42:177-199.) These 
promoters are regulated during the course of virus infection and are 
activated very late in the infectious process usually beginning 18 to 24 
hours post-infection. The polyhedrin and p10 genes are not essential for 
replication in cell culture, so the gene can be replaced with the 
heterologous gene of interest without interfering with the production of 
the budded form of the virus. The replacement of the polyhedrin gene, 
however, does interfere with the formation of the occluded form of the 
virus. The absence of the occluded virus in recombinant plaques provides a 
useful although somewhat tedious phenotypic selection for the recombinant 
viruses. It also places limitations on the ability to use the recombinant 
viruses for less expensive mass protein production in insect larvae. 
The economic value and general utility of a baculovirus vector is strongly 
dependent on the nature of the promoter used to drive heterologous gene 
expression. The polyhedrin promoter is currently the promoter of choice 
for the production of high levels of protein. However, even higher levels 
of protein production are often necessary for economic feasibility. In 
addition to the need for high productivity, vectors are needed which can 
express more than one heterologous gene. The disadvantage to such vectors 
is that they are often genomically unstable if promoter sequences are 
duplicated within the vector. To minimize such instability, a different 
promoter must be employed for each heterologous gene to be expressed. 
These promoters must be different (nonhomologous) from naturally occurring 
viral promoters within the vector to more fully avoid genomic instability 
problems. Vectors should also have the ability to include promoters that 
express genes at an earlier stage of protein production to improve protein 
quality and to ensure that the protein has the necessary 
post-translational modifications to confer biological activity or 
immunological authenticity. Such post-translational modifications appear 
to decline during the very late phase of infection when the very late 
promoters of AcMNPV vectors, such as polyhedrin and p 10, are activated. 
What are needed are modified or synthetic promoter regions which will cause 
significantly increased expression of the heterologous gene in the 
baculovirus system or which will allow the production of high quality 
protein. The new promoters should be flexible enough so that either a 
single heterologous gene or a series of heterologous genes can be inserted 
into the vector system so that various proteins can be produced at the 
same or at different times. In addition, the polyhedrin gene should be 
present if the virus is to be administered orally to the appropriate 
insect host. 
SUMMARY OF THE INVENTION 
The present invention provides an improved means of employing baculovirus 
gene expression vectors for expression of heterologous genes by placing 
the genes under the control of novel or modified promoters; preferably a 
novel promoter which promotes high level expression of a gene and allows 
the proper post-translational modifications of the gene product in insect 
cells. More specifically, in a preferred embodiment, the present invention 
comprises providing a modified baculovirus promoter to which a 
heterologous gene is attached. The promoter is inserted into a virus via a 
transplacement plasmid or by direct insertion to produce a recombinant 
virus vector which can be used to infect suitable host cells. The infected 
host cells are then used to produce the heterologous gene product. 
In accordance with the present invention, a method and composition are 
provided for improving the level of expression of a heterologous gene or a 
collection of genes. The present invention includes a novel promoter which 
contains a modification of a natural baculovirus promoter or a synthetic 
modified promoter functional in a baculovirus expression system. The new 
promoter may be a combination promoter such as a promoter with a double 
start site (i.e., two ATAAG's) or a combination of two different promoters 
such as an early/very late promoter or a late/very late promoter. It is 
contemplated that the present invention also includes foreign gene 
placement in a novel location or genomic orientation. All the 
aforementioned promoters are termed "modified baculovirus promoters" 
herein. 
A modified baculovirus promoter of the present invention has one or more 
features selected from the group consisting of an ATAAG sequence flanked 
by a very A+T rich region, an upstream activator sequence, similar to 
those of linker scan promoters LSXVI, LSXIV and LSXVII, of about 10bp of 
GC-rich sequence, placed from about 10 to about 30bp upstream of the 
transcription start site, a polyadenylation site (A.sub.2 UA.sub.3 site) 
in the orientation opposite to desired transcription to block antisense 
transcripts which may originate from fortuitous TAAG sequences in the 
foreign gene, and selected components of one or more of three very strong 
late promoters (vp39, p10, and p6.9), and/or of the very strongly 
expressed very late genes (polyhedrin and p10). The selection of those 
components is based on their presence or conservation in untranslated 
leader sequences of abundantly expressed late and very late genes. An 
upstream activator sequence of a modified promoter is a sequence which 
acts to increase the level of transcription at a transcription start site 
located downstream therefrom. For use with a late or very late promoter in 
a baculovirus expression vector, the upstream activating sequence is a 
GC-rich sequence, with a nucleotide composition of at least about 60% G+C. 
The modified promoter of the present invention shares only limited sequence 
homology or continuity with any other AcMNPV promoter. A major advantage 
of the novel promoter is that it can be stably incorporated at any region 
in the AcMNPV genome without concern for recombination with other regions 
of the AcMNPV genome. The modified promoter may also be designed to be 
stronger than the polyhedrin promoter. 
The modified promoter of the present invention has been incorporated into 
several different transplacement plasmids by methods well known to those 
of ordinary skill in the art. For example, plasmid pSynVI contains the 
synthetic promoter in place of the EcoRV to KpnI fragment within AcMNPV 
sequences between 3.1 to 6.16 map units. According to one aspect of the 
present invention, the orientation of the promoter is such that the 
direction of expression will be opposite that of normal polyhedrin gene 
expression, thus taking advantage of the potential increase in gene 
expression when foreign genes are placed in this orientation. Foreign 
genes can be placed within the multicloning site of this plasmid and 
recombinant viruses can be identified on the basis of their occlusion 
negative phenotype. 
A second transplacement plasmid, pSynVI+wtp, contains the synthetic 
promoter (including a multicloning site for passenger gene insertion) as 
well as an intact polyhedrin gene under the control of the wild-type 
polyhedrin promoter. When cotransfected with DNA from a 
polyhedrin-deficient mutant virus, this transplacement plasmid allows 
construction (or formation) of a recombinant virus that directs the 
production of both polyhedrin and the foreign gene product. The benefit is 
two fold: the recombinant virus has an easily visible and rapidly 
selectable (occlusion positive) phenotype, and the recombinant virus can 
infect insects orally. Thus it can be easily employed for mass production 
in insect larvae. 
The transplacement plasmids can be further manipulated in any number of 
additional ways which may increase expression in different situations. For 
example, it may be useful to include a polyadenylation signal within the 
multicloning site and downstream from the foreign gene insert so that 
efficient polyadenylation occurs if an effective polyadenylation site is 
not included in the foreign gene insert. Portions of the multicloning site 
which are not used in transplacement plasmid construction may be deleted 
to improve expression since an A+T rich leader and an AACAAT sequence near 
the ATG seem to be preferred by highly expressed baculovirus genes (i.e., 
vp39, p10 and p6.9). 
The ability to construct a modified promoter from linker scan modifications 
or from component parts of other promoters is a novel approach to promoter 
design. Most of the components of the novel promoter have been placed in 
one logical order. It is likely that the order of the components of the 
untranslated leader has flexibility. 
Thus in accordance with the present invention, a modified promoter is 
provided that will allow multiple heterologous genes to be expressed with 
one vector. 
An object of the present invention is to provide baculovirus vectors with 
modified promoters that will allow more than one heterologous genes to be 
expressed within one vector. 
It is yet another object of the present invention to provide a modified 
promoter that is sufficiently different in sequence from other promoters 
in the virus so as to substantially eliminate the possibility of 
homologous recombination and subsequent deletion or inversion of a portion 
of the viral genome. 
It is yet another object of the present invention to provide a recombinant 
baculovirus expression vector that can be fed orally to the appropriate 
insect host, thereby allowing the insect host-viral vector system to 
produce the desired heterologous protein. 
It is yet another object of the present invention to provide an expression 
vector system in the baculovirus system that will allow increased 
expression of heterologous proteins when compared to the polyhedrin 
system. 
These and other object features and advantages of the present invention 
will become apparent after a review of the following detailed descriptions 
of the disclosed embodiments and the appended claims. 
Any baculovirus capable of replication in cultured host cells is useful for 
conversion into a vector of this invention. Preferably the baculovirus 
used is a nuclear polyhedrous virus, and more preferably is 
Autographa/californica. 
The vectors of this invention may be prepared by genetic engineering 
technologies known to the art, preferably insertion of chimeric genes 
comprising the modified promoters of this invention in combination with 
heterologous genes placed under regulatory control of said promoters into 
the genome of a baculovirus by homologous recombination in an area of said 
genome able to tolerate said insertion without interference with the 
replication functions of said baculovirus, all as will be readily apparent 
to those skilled in the art. 
The host cells useful for expression of the heterologous genes under 
control of the modified promoters of this invention are insect cells in 
which the baculovirus vectors of this invention are capable of replication 
and expression, all as known to the art, and include cultured insect cells 
in vitro and insect larvae. 
The conserved sequences of the late and very late promoters of this 
invention may be readily identified by those skilled in the art and are 
exemplified, e.g., in Table 1. 
Any upstream activating sequence known to those skilled in the art may be 
used to enhance expression of the chimeric genes of this invention. A 
preferred upstream activating sequence of this invention is a G+C-rich 
sequence of about 10 bp, more preferably a HindIII site linker as 
exemplified by the sequence CCAAGCTTGG. 
Any transplacement plasmid known to the art which has sequences flanking 
the chimeric gene(s) of the present invention which are homologous to the 
baculovirus which is to serve as the expression vector can be used in the 
practice of the present invention. It is understood that those homologous 
flanking sequences must be sufficiently long to mediate the homologous 
recombination of the chimeric gene from the transplacement plasmid to the 
baculovirus vector. The transplacement plasmids exemplified by the present 
invention are pEV55 and derivatives thereof.

DETAILED DESCRIPTION OF THE DISCLOSED EMBODIMENTS 
In accordance with the present invention, a method and composition are 
provided for improving the expression of a gene or collection of genes in 
a baculovirus expression vector. More particularly, the present invention 
provides a plasmid containing a novel promoter that is used to drive 
foreign gene expression and improve the quality of the proteins produced 
during infection. The novel promoter may optionally contain a synthetic 
promoter, modifications of a natural promoter, and/or combinations of 
natural, modified or synthetic promoters. The novel promoter may also 
comprise a combination promoter, including but not limited to, a promoter 
with a double start site (i.e., two ATAAG's) or a combination of two 
baculovirus different promoters such as an early/very late promoter or a 
late/very late promoter. It is contemplated that the present invention 
also includes promoter-foreign gene placement in a novel location or a 
genomic orientation. 
The insect baculovirus Autographa californica nuclear polyhedrosis virus 
(AcMNPV) is now widely employed as a gene expression vector (reviewed by 
Luckow and Summers, "Trends in the Development of Baculovirus Expression 
Vectors," Bio/Technology 6:47-55, 1988; Miller, L. K., Baculoviruses as 
Gene Expression Vectors, Ann. Review of Microbiology, 42:177-199, 1988). 
The helper-independent viral vector systems generally involve the 
insertion of the foreign gene to be expressed into the AcMNPV genome under 
the control of a very late viral promoter, either the polyhedrin promoter 
or the p10 promoter. These two novel promoters are considered particularly 
useful because they mediate high level transcription of their respective 
genes, resulting in high steady state levels of mRNA during the last phase 
of the infection process, the occlusion phase. Occlusion, the embedding of 
viral particles in a paracrystalline protein matrix, is nonessential for 
virus propagation in cell culture although it is required for the 
efficient oral infection of host larvae. Thus, the replacement of 
occlusion phase-specific genes and the high level expression of 
heterologous genes instead of the occlusion genes have no observable 
impact on nonoccluded virus (budded virus) production which is responsible 
for infection in cell cultures. 
The baculovirus expression system was originally developed with very little 
knowledge of baculovirus gene organization or the nature of baculoviral 
promoters (Miller, L. K., 1981 supra. One of the first transplacement 
plasmids developed for a polyhedrin promoter-based vector system was 
pAc373; it was widely used before it was reported that some of the 
sequences essential for normal levels of gene expression were absent in 
this vector (Matsuura et al. (1987) "Baculovirus expression vectors: The 
requirements for high level expression of proteins, including 
glycoproteins" J. Gen. Virol. 68:1233-1250). Transplacement plasmids, 
which include the nucleotides immediately upstream of the +1 (A of the 
ATG) position of the polyhedrin gene are now recommended for vector use 
(Luckow and Summers (1988) supra: Miller, L. K. (1988) supra). Nucleotides 
downstream of the +1 position (i.e., within the polyhedrin open reading 
frame) are not required for optimal transcriptional efficiency (Carbonell 
et al. (1988) "Synthesis of a gene encoding an insect-specific scorpion 
neurotoxin and attempts to express it using baculovirus vectors," Gene, 
73:409-418). 
Linker scan mutational analysis of the region immediately upstream of the 
polyhedrin coding sequence by the present inventor has revealed several 
unique features of this region. The primary determinant of promoter 
activity resides within the sequence TAAGTATT at the transcriptional start 
site located at -50 with respect to the ATG (+1, +2, +3) at the 
translation start site. The sequences flanking the transcriptional start 
site influence the levels of transcription and reporter gene expression in 
more subtle ways. Linker replacement of the sequences 10 to 30 bases 
upstream of the TAAGTATT increases the levels of transcription and gene 
expression as much as 50% while linker replacements between TAAGTATT and 
the +1 position (i.e., within the sequences specifying the untranslated 
leader region) down-regulate promoter activity by 2 to 10 fold. Reversing 
the genomic direction of the reporter gene and 93 nucleotides of the 
polyhedrin "promoter" (i.e., +1 to -92) provided equivalent or slightly 
higher levels of reporter gene expression than that observed in the 
natural direction. None of the mutations altered the temporal regulation 
of gene expression; all gene expression from the modified polyhedrin 
promoters was restricted to the very late phase (Rankin et al. (1988) 
"Eight Base Pairs Encompassing the Transcriptional Start Point and the 
Major Determinants for Baculovirus Polyhedrin Gene Expression," Gene 
70:39-49, incorporated herein by reference). 
Although it has been assumed that the very late polyhedrin and p10 
promoters probably represent the optimal promoters for foreign gene 
expression, the comparative ability of late promoters to drive foreign 
gene expression has never been tested directly. The two most abundantly 
expressed late virus structural genes encoding vp6.9 (core) and vp39 
(capsid) have been located, sequenced, and transcriptionally mapped 
(Wilson et al. (1987) "Location, transcription, and sequence of a 
baculovirus gene encoding a small arginine-rich polypeptide," J. Virology, 
61:661-666). These genes are transcribed abundantly at 12 hrs 
post-infection, and approximately 12 hrs before transcription from the 
strong polyhedrin promoter. The products of the vp6.9 and vp39 genes are 
required for both the budded and occluded forms of the virus. Thus, their 
expression would be expected to continue through very late times 
post-infection and provide a longer period for foreign gene expression 
than the polyhedrin or p10 promoters. Specific expression of foreign gene 
products at very late times has been considered to be preferable for 
expression of those foreign genes whose products might interfere with 
budded virus synthesis (Miller, L. K. (1981) supra). 
However, many heterologous gene products do not adversely affect virus 
synthesis, and earlier high level expression would be advantageous from a 
production perspective. Expression during the late phase is also 
preferable to the very late phase for efficient post-translational 
modification. Host nuclear mRNA transcripts decrease dramatically between 
12 and 24 hrs post-infection and the post-translational modification, 
which is probably mediated primarily by host gene products, appears to 
become inefficient during the very late phase of expression. The inventor 
has found that the late capsid promoter, i.e., the vp39 promoter, is 
excellent for driving foreign gene expression during the late phase; it 
outperforms the polyhedrin promoter between 12 and 24 hours post-infection 
by approximately 5 to 8 fold. 
The present invention includes the improvement of baculovirus gene 
expression vectors to provide higher levels of foreign gene expression 
than currently provided by very late promoters such as natural viral 
polyhedrin and p10 promoters. The present invention also includes two 
different approaches: (1) modification of the upstream sequences flanking 
the transcriptional start point of the polyhedrin promoter and (2) 
utilization of the late vp39 (capsid) promoter or late/very late promoter 
combinations to drive foreign gene expression. Reversing the genomic 
orientation of the promoter and foreign gene seems to have very little 
impact on the levels of gene expression and has allowed the design of 
plasmids for the simultaneous transplacement and the expression of two or 
more foreign genes directed by a single baculovirus expression vector. 
In addition, according to the present invention, novel synthetic promoters 
have been designed which share minimal DNA sequence homology with other 
natural baculoviral promoters. 
Such promoters are useful for the expression of more than one foreign gene 
using a single recombinant virus and eliminate the need for duplicating a 
natural viral promoter sequence within the vector, thereby minimizing the 
potential for genomic instability due to homologous recombination between 
the repeated promoter sequences. 
As part of the present invention, transplacement plasmids are described 
which mediate the formation of baculovirus expression vectors capable of: 
(1) elevated levels of gene expression, (2) simultaneous expression of two 
passenger genes, (3) expression of passenger genes during the late as well 
as very late phases, and/or (4) simultaneous expression of both a 
passenger gene and the polyhedrin protein. 
Transplacement plasmids facilitate the insertion of a foreign gene into the 
baculovirus genome by providing useful restriction sites for inserting the 
foreign gene(s) (i.e., a multicloning site at the appropriate site 
downstream of a promoter), and flanking sequences of viral DNA from the 
region of the viral genome into which the foreign gene(s) is to be 
integrated. The flanking viral sequences provide sites for cell-mediated 
homologous recombination between nonrecombinant viral DNA and recombinant 
transplacement plasmids to yield the recombinant viral vector. 
The inventor has found that certain linker scan mutations upstream of the 
polyhedrin TAAGTATT increase transcription and increase foreign gene 
expression relative to the wild-type polyhedrin promoter (Rankin et al. 
(1988) supra). The plasmids phcLSXIV, shown schematically in FIG. 1, and 
pEVmodXIV, which carries the same promoter and basic multi-cloning site as 
phcLSXIV but lacks the CAT gene, contain a linker scan mutant promoter, 
currently referred to as the LSXIV promoter. Viruses derived from 
peEVmodXIV, or derived from phcLSXIV, provide higher levels of reporter 
gene (CAT) expression than other known linker scan modifications. 
Expression from viruses carrying the CAT reporter gene under the control 
of the LSXIV promoter has been shown by the inventor to be 50% higher than 
that from the polyhedrin promoter (i.e., compared to CAT gene expression 
from recombinant viruses derived from phcwt, shown schematically in FIG. 
10, which contains a wild-type polyhedrin promoter). 
Thus, preferred embodiments of the present invention include several 
plasmids which contain promoters having various modifications upstream of 
the TAAGTATT sequences. The plasmid designated phcLSXIV, shown 
schematically in FIG. 1 is an example of a plasmid containing such 
modified promoters. Other plasmids of this nature include phcLSXVI and 
phcLSXVII (as shown in Table 5). These plasmids contain upstream modified 
promoters which causes elevated levels of foreign gene expression when 
incorporated into recombinant viruses. 
As another preferred embodiment, the present invention includes plasmids 
containing a synthetic promoter. Synthetic promoters provide diversity in 
the nucleotide sequence of promoters used to drive heterologous gene 
expression and provide differences in the levels of foreign genes 
expressed. A sequence for a preferred synthetic promoter is described best 
by the DNA sequence shown in Table 1. (SEQ ID NO: 6). The arrow in the DNA 
sequence shows the start point and the direction of transcription. It is 
to be understood that there is much flexibility possible in arranging and 
ordering the components of the promoter and that the sequence shown in 
Table 1 (SEQ ID NO: 6) is only one example of an embodiment of the present 
invention. 
An example of a transplacement plasmid of the second preferred embodiment 
which includes the above-referenced synthetic promoter is shown in the 
general construction in FIG. 2. This plasmid, designated pSynVI-, is a 
transplacement plasmid carrying the synthetic promoter shown in Table 1 
(SEQ ID NO: 6) and the multi-cloning site (MCS) termed MCS#2 herein. The 
MCS#2 sequence of pSynVI- is shown in Table 2 and in SEQ ID NO: 7 from 
nucleotide 93 through nucleotide 159. The orientation of the synthetic 
promoter is such that foreign gene expression will occur in the genomic 
orientation opposite that of the polyhedrin gene. This promoter construct 
drives heterologous gene expression at approximately 10%-20% of the level 
of the wild-type polyhedrin promoter. 
Another example of the second preferred embodiment of the present invention 
is the plasmid designated pSynVI+wtp. The schematic representation of this 
promoter is shown in FIG. 3. This construct is a transplacement vector 
containing the synthetic promoter (SEQ ID NO: 6) described above and MCS#2 
(see SEQ ID NO: 7, ) nucleotides as in the foregoing pSynVI- but also 
expressing the polyhedrin gene under wild-type polyhedrin promoter 
control. It is an example of a plasmid which is able to express two genes 
simultaneously. This plasmid provides the selectable occlusion positive 
phenotype and, because recombinant viruses are occlusion positive, they 
can be used to orally infect insects. This plasmid construction drives 
expression of the heterologous gene at approximately 10%-20% of the 
natural polyhedrin promoter. 
Another plasmid that is included as an example of the second preferred 
embodiment of the present invention is designated pSynwtVI- and is shown 
in FIG. 4. This is a transplacement plasmid containing both the polyhedrin 
promoter (with an MCS site, MCS#1) and a synthetic promoter (with a second 
MCS site, MCS#2), features which allow for the expression of two foreign 
genes. This transplacement plasmid is therefore an example of a two-gene 
expression vector. However, it is to be understood that it is not 
necessary to use the polyhedrin promoter because other synthetic promoters 
can be designed to replace the polyhedrin promoter based on principles 
noted for the synthetic promoter described above. 
In a third preferred embodiment of the present invention, the plasmids 
contain a promoter or promoters in an orientation opposite to that of the 
naturally occurring promoter. The plasmids pSPLSXIVVI+CAT and pLSXIVVI+CAT 
(FIGS. 5 and 6) are plasmids with the pLSXIV promoter (see SEQ ID NO: 5) 
in the opposite orientation (with respect to the original polyhedrin 
direction). These plasmids are used to test CAT expression from the LSXIV 
promoter in opposite orientations. The pLSXIVVI+CAT plasmid has a tandem 
double promoter. CAT gene expression from the LSXIV promoter (SEQ ID NO: 
5) in the opposite orientation has been found by the inventor to be 
similar to the levels achieved in the original orientation. 
It is contemplated as part of the present invention that the promoters can 
be positioned in different genomic positions/orientations to give elevated 
levels of expression. The plasmids pSPLSXIVVI+CAT (FIG. 5) and 
pLSXIV3VI+(FIG. 13) are two examples of high level expression promoters in 
opposite orientation. 
A fourth preferred embodiment of the present invention includes plasmids 
containing the vp39 (capsid) promoter sequence. The DNA sequence of this 
promoter is shown in Table 3 SEQ ID NO: 8. The transplacement plasmids 
which can be used for testing reporter CAT expression include phc39 and 
pEVmod39. The vp39 promoter provides approximately 5 to 120 fold higher 
levels of expression during the period of time from 12 to 24 hrs 
post-infection than the naturally occurring polyhedrin promoter. It is 
contemplated that the present invention also includes a combination of 
late/very late promoters such as the polyhedrin and vp39 promoters which 
would provide earlier gene expression and higher final levels (total 
levels) of heterologous proteins. One obtains higher levels of expression 
at 12 hrs post-infection from the late promoters because they are being 
expressed earlier in the infection process (i.e., they turn on at 6 hrs 
post-infection rather than at 18 hrs post-infection as in the case of 
polyhedrin). The combination late/very late promoter ideally turns on at 
approximately 6 hrs post-infection and continues through 70 hr 
post-infection thus providing both late and very late gene expression. 
Design of a synthetic promoter and a transplacement plasmid carrying the 
synthetic promoter 
The inventor has discovered that the major determinant for polyhedrin gene 
expression appears to be located within the sequence ATAAGTATT. 
Transcription of this abundantly transcribed late gene also initiates 
within an ATAAG sequence flanked by an A+T rich region (Wilson et al., 
1987, supra). A novel baculovirus promoter has been designed which lacks 
minimal extended sequence homology to other viral promoters by combining 
short sequence motifs common to other abundantly expressed late and very 
late genes. Two additional features incorporated in the design of the 
novel promoter include: an LSXIV linker, in the context of a G+C-rich 
region upstream of the ATAAG and a TTTATT sequence within the untranslated 
region near the ATG. The TTTATT serves as a polyadenylation signal for any 
transcripts initiating in the opposite orientation from ATAAG sequences 
which occur by chance in the foreign gene insert. 
The inventor has found that late and very late promoters contain an ATAAG 
at the most abundant transcriptional start points. This sequence is 
important for polyhedrin expression and promoter activity. In addition, 
linker scan mutational analysis of the polyhedrin promoter shows that 
nucleotides throughout the untranslated leader region appear to contribute 
to optimum gene expression. The sequences specifying the untranslated 
leader regions of four promoters of the most abundantly expressed genes of 
AcMNPV, namely the late gene encoding the major capsid protein (p39), the 
late gene encoding the basic core protein (p6.9), the very late gene 
encoding p10 and the polyhedrin gene have been aligned. All contain ATAAGs 
in the context of an A+T-rich region. 
Several short segments found in two or more of the promoters have been 
chosen as component parts of the synthetic promoter (Table 1, SEQ ID NO: 
6). The longest segment common to two or more promoters is nine 
nucleotides, TAAATTACA, found in both the p10 and p6.9 promoters. The 
sequence ACAAT has been found near the ATG of 3 of 4 promoters but not 
polyhedrin), TACTGT was found in both the polyhedrin and p10 promoters, 
TTTGTA has been found in both the p10 and polyhedrin promoters, TTTGTA has 
been found in both the p10 and polyhedrin promoters, and TCAANTCA has been 
found in the p10 and the p39 promoters. In addition, all the promoters 
contain stretches of at least 3 T's, usually flanked by A's, and stretches 
of at least 3 A's, usually preceded by a pyrimidine. All these components 
have been designed into the model synthetic promoter. The components have 
been placed to minimize length; for example, the T stretches and A 
stretches have been placed just downstream of the ATAAG to also serve as 
an A+T rich region downstream of the ATTAG. However, the ACAAT sequence 
has been placed near the 3' restriction site (BglII) which serves to link 
the promoter to a multicloning site and the TTTATT (polyadenylation signal 
in reverse) has been positioned flanking this sequence to encourage 
termination of reverse transcripts as far from the ATAAG as possible, 
thereby minimizing antisense RNA interference at the 5' end. The sequences 
of the two oligonucleotides which have been synthesized to construct the 
model synthetic promoter, referred to as the Syn promoter, are shown in 
Table 1, (SEQ ID NO: 6) along with the designations of the component 
parts. 
The transplacement plasmid pSynVI- (FIG. 2) has the Syn promoter (SEQ ID 
NO: 6) driving expression in the opposite direction as the polyhedrin 
promoter. It also has a multicloning site (MCS#2) (SEQ ID NO: 7, 
nucleotides 93-159) with numerous useful restriction sites. It lacks 627 
nucleotides encoding the N-terminus of polyhedrin, and thus the use of 
this transplacement plasmid results in recombinants with occlusion 
negative phenotypes. 
The synthetic promoter and attached multicloning site (MCS#2) can also be 
moved to other plasmids for transplacement into other AcMNPV genomic 
locations using flanking restriction endonucleases (e.g., SacI downstream 
of MCS#2 and KpnI or SmaI upstream of the promoter). 
This invention is further illustrated by the following examples, which are 
not to be construed in any way as imposing limitations upon the scope 
thereof. On the contrary, it is to be clearly understood that resort may 
be had to various other embodiments, modifications, and equivalents 
thereof which, after reading the description herein, may suggest 
themselves to those skilled in the art without departing from the spirit 
of the present invention and/or the scope of the appended claims. 
EXAMPLES 
Example I 
All viruses are derived originally from AcMNPV L-1 (Lee and Miller (1978) 
"Isolation of genotypic variants of A. californica nuclear polyhedrosis 
virus," J. Virol. 27:754-767); and are plaque-purified and propagated in 
the Spodoptera frugiperda IPLB-SF-21 cell line (Vaughn et al. (1977) 
"Establishment of Two Insect Cell Lines From the Insect Spodoptera 
frugiperda (Lepidoptera: Noctuidae)" In Vitro 13:213-217 using methods 
described previously (Lee and Miller (1978); Miller et al. (1986) supra; 
"An insect baculovirus host-vector system for high-level expression of 
foreign genes," in Genetic Engineering, Principles and Methods, Vol. 8 J. 
Setlow and A. Hollaender (eds.), Plenum Press, N.Y., pp. 277-298, 1986. 
CAT assays are performed and specific activities calculated as described 
previously (Carbonell et al. (1985) "Baculovirus-Mediated Expression of 
Bacterial Genes in Dipteran and Mammalian Cells," J. Virol. 56:153-160; 
Rankin et al. (1988) supra;. The CAT gene for all CAT-containing plasmids 
used in these studies was derived from pCM1CAT (LKB Biotechnology, 
Piscataway, N.J.) in which the CAT gene is inserted as a SalI cassette. 
*428 
Example II 
Construction of the pEVmodXIV transplacement plasmid (FIG. 9) is described. 
The previously described transplacement plasmid pEV55 (Miller et al. 
(1986) supra), which serves as the basis for constructing phcwt (FIG. 7), 
also serves as the starting plasmid for pEVmodXIV construction. The pEV55 
plasmid contains AcMNPV DNA from 3.18 to 7.3 map units (mu). A 
multicloning site replaces polyhedrin coding sequences from +1 to +635. 
The numerous restriction endonuclease sites at the junction between the 
pUC vector portion of pEV55 and the SalI site of AcMNPV DNA at 3.18 limit 
the types of restriction sites which can be utilized in the multicloning 
site. Therefore, the sites at this junction are removed by digesting pEV55 
with SmaI, which cuts at the vector junction, and by deleting 
approximately 70 nucleotides at the junction by the combined action of 
ExoIII and mung bean nuclease. Ligation at this deletion site results in 
the plasmid pEVdel. The viral sequences between 6.16 mu and 7.3 mu in 
pEVdel are deleted by digestion with BamHI (6.16 mu) and NotI (7.3 mu) at 
the right vector junction. Blunt end ligation at the deleted junction 
resulted in pEVmod (FIG. 8). The polyhedrin promoter (from the EcoRV at 
-92 to the BglII at +1) (see SEQ ID NO: 5) of pEVmod is replaced with the 
small EcoRV-BglII fragment of phcLSXIV (FIG. 1) resulting in the improved 
and convenient transplacement plasmid pEVmodXIV. Virus vectors derived 
from pEVmodXIV-based transplacement plasmids provide approximately 50% 
higher levels of expression than virus vectors derived from pEV55 or any 
other known plasmids. 
Example III 
Construction of the phc39 (FIG. 15) and pEVmod39 transplacement plasmids is 
described. A plasmid containing the vp39 promoter is first constructed as 
follows. The NarI to EcoRV fragment containing the vp39 promoter between 
57.6 to 57.9 map units on the AcMNPV genome is cloned into the AccI and 
EcoRV sites of the plasmid vector Bluescript KS- (Stratagene, San Diego, 
Calif.). The resulting plasmid, pSTVNM, is cut at the XhoI and ApaI sites. 
The N-terminal sequences of the vp39 coding sequence are deleted with 
Exonuclease III and mung bean nuclease digestion. A BolII linker 
oligonucleotide is inserted into the gap using DNA ligase. One plasmid is 
selected which contains the BolII site at -2 relative to the ATG (+1, +2, 
+3) of vp39. To construct p39pro, the 458 bp capsid promoter region is 
excised from the previously selected plasmid using KpnI and EcoRV and is 
ligated into the plasmid Bluescript KS+ (Stratagene, San Diego, Calif.) 
which is also cut with KonI and EcoRV. To construct the transplacement 
plasmid phc39, the 458 bp EcoRV to BglII fragment of p39pro containing the 
vp39 promoter is excised and inserted into EcoRV and BglII digested phcwt, 
thus replacing the polyhedrin promoter of phcwt with the vp39 promoter 
region. The resulting transplacement plasmid, phc39 (FIG. 15) see also SEQ 
ID NO: 8 is used to construct viral vector vhc39. CAT gene expression 
using this vector is 5 to 10 times higher between 12 and 24 hours post 
infection than CAT gene expression from virus vectors carrying the CAT 
gene under polyhedrin promoter control. The earlier expression of the 
foreign gene provides for more efficient post-translational modification 
of the protein product. More convenient transplacement plasmids, such as 
pEVmod39 (not shown), can be derived easily from phc39 by replacing the 
CAT gene with a MCS containing BclII and KpnI termini (e.g., MCS#1). 
Example IV 
To construct the plasmid pSynVI- (FIG. 2), which contains the Syn promoter 
(SEQ ID NO: 6) and an adjoining multicloning site (MCS#2) (SEQ ID NO: 7, 
nucleotides 93-159) in place of the polyhedrin promoter, the two Syn 
promoter oligonucleotides shown in Table 1 are annealed, the duplex is 
purified by polyacrylamide electrophoresis, and is inserted into the KpnI- 
and EcoRV-digested Bluescript plasmid vector pBSKS- (Stratagene, San 
Diego, Calif.), which lacks the SmaI restriction site in the multicloning 
site. This allows the forced cloning of the oligonucleotide into the 
multicloning site of the Bluescript plasmid and the regeneration of both 
the EcoRV and KpnI sites. 
The portion of the multicloning site of this plasmid from the EcoRV to the 
SacI site contains, in order, EcoRV, EcoRI, PstI, BamHI, SpeI, XbaI, NotI, 
SacII, and SacI sites. The synthetic promoter and this multicloning site 
are removed from the Bluescript vector by ordered digestion with SacI, 
mung bean nuclease, and KpnI. The small fragment is gel purified and 
inserted into the large, gel-purified fragment of KpnI- and EcoRV digested 
pEVmod (FIG. 8). The sequence of the promoter from the SmaI site through 
the flanking multicloning site, referred to as MCS#2, is determined by 
sequencing. The sequence is shown in SEQ ID NO: 7 and Table 2. The SacI 
site is regenerated unexpectedly and fortuitously during the cloning 
process; the action of mung bean nuclease downstream of the SacI site 
accounts for the observed junction. 
A pSynVI- plasmid containing the reporter CAT gene is constructed to 
measure the level of gene expression of the pSynVI- plasmid by measuring 
the CAT gene expression of the constructed CAT-containing plasmid. 
In constructing the pSynVI-CAT plasmid (FIG. 10), the CAT gene is excised 
from pCM1CAT with SalI, blunt-ended with mung bean nuclease, and inserted 
into pSynVI- cut with EcoRV. Orientation is checked using the EcoRI site 
in the N-terminal portion of the CAT gene. The corresponding viral vector, 
vSyn VI-CAT, provides CAT expression at levels approximately 10-20% of the 
levels observed for viral vectors expressing CAT under polyhedrin promoter 
control. Although not as high as polyhedrin promoter expression, this 
level of expression from a component synthetic promoter is significant. 
The Syn promoter and others derived by the combinatorial approach will be 
useful for balanced expression of two different foreign genes and/or the 
expression of genes encoding proteins controlling specific 
post-translational modifications. 
Example V 
For some applications, it is useful for a transplacement plasmid to 
transfer genetic information to form a recombinant virus which allows both 
foreign gene expression and polyhedrin gene expression. Expression of both 
genes results in occlusion, which permits efficient oral infection of 
insect larvae for inexpensive, bulk protein production. Furthermore, 
production in insect larvae allows expression in differentiated insect 
cells, including the fat body which constitutes a major secretory tissue 
of the insect and may provide more efficient post-translational 
modification than that found in available lepidopteran cell lines such as 
SF9 cells. 
Thus, the pSynVI- (FIG. 2) is modified to produce a transplacement plasmid, 
pSynVI+wtp (FIG. 3), which allows simultaneous, high level expression of 
both polyhedrin and the foreign gene. An additional advantage of 
pSynVI+wtp is the ability to select recombinant viruses as occlusion 
positive plaques if the DNA of a polyhedrin deletion mutant (or a 
polyhedrin/.beta.-galactosidase fusion mutant) is used in the initial 
co-transfections of host cells with viral DNA and transplacement plasmid. 
The pSynVI+wtp transplacement plasmid is constructed by digesting a plasmid 
carrying the EcoRI-I fragment of the AcMNPV genome, with EcoRV and KpnI to 
remove the polyhedrin promoter and N-terminal polyhedrin region. The 
approximately 0.7kb EcoRV/KpnI fragment is gel-purified and inserted into 
pSynVI- which had been digested with SmaI and KpnI. Ligation eliminates 
the SmaI and EcoRV sites while regenerating the KpnI site found in the 
polyhedrin coding region. More specifically, the polyhedrin promoter and 
N-terminal coding sequences from EcoRV (-92) to KpnI (+635) are inserted 
into pSynVI- to produce the transplacement plasmid pSynVI+wtp. Polyhedrin 
mRNA is thus transcribed in its normal orientation under the control of 
its normal promoter while the foreign gene is inserted at the multicloning 
site under synthetic promoter control and is transcribed in the opposite 
direction to polyhedrin. The features of the plasmid are illustrated in 
FIG. 3. The sequence of the EcoRV/SmaI junction, the synthetic promoter, 
and the multicloning site (MCS#2) are shown in Table 1and 2 (SEQ ID NOS: 6 
and ). 
To test the efficacy of the transplacement vector pSynVI+wtp, the reporter 
CAT gene is inserted into the MCS of pSynVI+wtp (FIG. 3) at the EcoRV site 
resulting in the plasmid pSynVI+wtpCAT1 (FIG. 11). To construct 
pSynVI+CAT1, the blunt-ended CAT-containing SalI fragment of pCM-1CAT is 
inserted into EcoRV-digested pSynVI+wtp. The ability of the Syn promoter 
to drive reporter CAT gene expression at the same time as polyhedrin gene 
expression is tested by isolating the recombinant virus vSynVI+wtpCAT1 and 
comparing levels of CAT expression with the levels obtained with 
vSynVI-CAT. No significant difference in CAT gene expression is observed 
between these two virus vectors showing that simultaneous expression of 
the polyhedrin gene did not impair foreign gene expression. The polyhedrin 
gene is also being abundantly expressed since the recombinant virus 
vSynVI+wtpCAT1 produces high levels of occlusion bodies. 
Example VI 
Construction of transplacement plasmid pSynwt VI-(FIG. 4) is described. 
This plasmid has two promoters in a back-to-back relationship allowing 
simultaneous transplacement of two heterologous genes and concerted 
expression of the two genes by a single vector. The pEV55 EcoRV-KpnI 
fragment, containing the polyhedrin promoter and multicloning site MCS#1, 
was inserted into pSynVI- which had been digested with SmaI and KpnI. 
A pSynwtVI- plasmid containing the reporter CAT gene is constructed to 
provide a measure of gene expression of pSynwtVI-. The plasmid is 
pSynwtVI-CAT1 (FIG. 12). To construct pSynwtVI-CAT1, the blunt ended 
CAT-containing SalI fragment of pCM-1CAT is inserted into EcoRV-digested 
pSynwtVI-. The equivalent virus vector vSynwtVI-CAT1 expresses CAT 
activity at 10-20% of the levels observed for vectors expressing CAT under 
polyhedrin promoter control. 
Example VII 
Construction of the pSPLSXIVVI+CAT and pLSXIVVI+CAT transplacement plasmids 
(FIG. 5 and 6) are described. To construct pSPLSXIVVI+CAT, the CAT gene 
and attached LSXIV promoter (SEQ ID NO: 5) is removed from phcLSXIV (FIG. 
1) by KpnI digestion (and blunt ended with mung bean nuclease) and EcoRV 
digestion, and then inserted into EcoRV-digested pSynVI+wtp. This places 
the LSXIV promoter in tandem with the synthetic promoter (SEQ ID NO: 6). 
The plasmid is then cut with EcoRV and KpnI and the large vector fragment 
is gel-purified. The EcoRV/KpnI fragment of AcMNPV EcoRI-I fragment 
containing the wild-type polyhedrin promoter (SEQ ID NO: 6) and N-terminus 
is gel-purified and inserted into the vector fragment (EcoRV/KpnI) of 
pSPLSXIVVI+CAT to yield pLSXIVVI+CAT which contains the CAT gene under 
LSXIV promoter control and the intact polyhedrin gene under polyhedrin 
promoter control. 
The viral vectors derived from these plasmids (vSPLSXIVVI+CAT and 
vLSXIVV+CAT) express equivalent or slightly higher levels of CAT than 
viruses expressing CAT under wild-type polyhedrin promoter control. An 
additional advantage of virus vectors derived from pLSXIVVI+CAT is that 
they produce polyhedrin at high levels. 
Example VIII 
Construction of pLSXIV3VI+ and pLSXIV2 transplacement plasmids (FIG. 13 and 
14) is described. Plasmid pLSXIVVI+CAT is digested with Bg1II and EcoRI; 
the sticky ends removed with mung bean nuclease. The DNA is then cut with 
SacI and the large fragment is gel-purified and ligated to a multicloning 
site (MCS#3) having EcoRV and SacI termini to form pLSXIV3VI+lacking the 
CAT gene. This plasmid is then digested with EcoRV and KpnI. The large 
fragment is isolated and the EcoRV/KpnI fragment of pEVmodXIV is inserted 
into the large fragment resulting in pLSXIV2 which has two back-to-back 
LSXIV promoters, each with its own MCS. The pLSXIV3VI+plasmid allows 
superior levels of foreign gene expression using the powerful LSXIV 
promoter (SEQ ID NO: 5) as well as polyhedrin gene expression. The pLSXIV2 
plasmid allows superior expression of two foreign genes, each under LSXIV 
promoter control. 
Example IX 
Transplacement plasmid phc39 (FIG. 15) is constructed with the CAT reporter 
gene under the control of the vp39 promoter (SEQ ID NO: 6). Expression of 
CAT from vhc39 shows approximately 8 fold higher levels of CAT expression 
at 12 hrs post-infection than vhcwt, the vector carrying CAT under 
wild-type promoter control. The CAT gene of phc39 can be replaced with a 
heterologous gene of interest. Earlier expression during the infection 
process may allow more efficient post-translational modification of the 
heterologous protein product. 
Example X 
Transplacement plasmids are constructed to allow the simultaneous 
transplacement of two foreign genes into the virus vector and allow the 
recombinant viruses to simultaneously and abundantly express both foreign 
genes. The transplacement plasmid pSynwtVI- (FIG. 4) is one example of 
such a dual gene transplacement plasmid. This plasmid is constructed by 
digesting pSynVI- with SmaI and KpnI and inserting the pEV55 EcoRV/KpnI 
fragment containing the wild-type polyhedrin promoter and MCS#1. This 
plasmid thus contains two different multicloning sites, each downstream 
from back-to-back Syn and wild-type polyhedrin promoters. 
Example XI 
Another example which allows even higher levels of the second gene is 
pLSXIV2 (FIG. 14). This plasmid provides back-to-back LSXIV promoters, 
each with a multicloning site (MCS#1 and #2) containing useful restriction 
sites for inserting two different foreign genes, each under the control of 
an LSXIV promoter. The efficacy of back-to-back promoters is tested using 
vLSXIVVI+CAT, a recombinant virus carrying the pLSXIVVI+CAT construct in 
which the LSXIV promoter drives CAT expression back-to-back with the 
polyhedrin promoter, which in turn drives polyhedrin gene expression. CAT 
expression by the vLSXIVVI+CAT vector was equivalent to or slightly higher 
than that observed with phcwt alone which expresses only CAT. 
Example XII 
Construction of vp39/polyhedrin promoter fusions is described. HindIII 
linkers are inserted into the 462 bp promoter of vp39. The DNA sequence of 
vp39 is shown in Table 3 (SEQ ID NO: 8) (see Rankin et al. (1988) supra, 
for general linker scan approach). The HindIII linkers serve to allow 
interface with the HindIII sites of the linker-scan-modified promoters to 
generate late/very late combination promoters. Combination promoters are 
constructed which contain the OCT-A and OCT-B regions of vp39 (e.g., from 
-65 to approximately -350) fused to the +1 to approximately -60 of the 
polyhedrin promoter of the LSXIV promoter (SEQ ID NO: 5) of Rankin et al. 
(1988) supra. To do this, a plasmid is selected (e.g., pvp39-65) 
containing a HindIII linker at an appropriate site (e.g., -65). The 
HindIII to Bg1II fragment is replaced with the HindIII to BolII fragment 
of phcLSXIV. The entire BolII to EcoRV fragment containing the combination 
vp39/LSXIV promoter, can then be transferred to pEV55 to construct a 
transplacement plasmid, pEVvp39/LSXIVCAT (FIG. 16) with the combination 
promoter controlling CAT expression. 
Example XIII 
Construction of the transplacement plasmid pEVvp39/LSXIV (FIG. 16) is 
achieved in accordance with the method described in Example XII above. The 
sequence of the hybrid vp39/LSXIV promoter is shown in SEQ ID NO: 9 and 
Table 4. The sequence at the junction of the vp39/LSXIV combination 
promoter is marked by "1-10 CCAAGCTTG" which indicates a HindIII site at 
the junction (CCAAGCTTG) and approximately 1 to 10 nucleotides (N1-10) of 
the vp39 at the junction. 
It should be understood, of course, that the foregoing relates only to 
preferred embodiments of the present invention and that numerous 
modifications or alterations may be made therein without departing from 
the spirit and the scope of the invention as set forth in the appended 
claims. 
TABLE 1 
__________________________________________________________________________ 
DNA sequence for model synthetic baculovirus promoter 
of pSynVI (SEQ ID NO: 6) 
__________________________________________________________________________ 
##STR1## 
##STR2## 
##STR3## 
__________________________________________________________________________ 
TABLE 2 
__________________________________________________________________________ 
DNA sequence including the sequence of multicloning 
site #2 (MCS #2) (SEQ ID NO: 7) 
__________________________________________________________________________ 
##STR4## 
##STR5## 
##STR6## 
__________________________________________________________________________ 
TABLE 3 
__________________________________________________________________________ 
vp39 (capsid) promoter sequence in phc39 SEQ ID NO: 8 
__________________________________________________________________________ 
##STR7## 
GTT CAA ATT TGA TTT CAA TTT TAT CGT GTT GGT AAA CGT ACA CTT TAA TTA TTT 
TAC TCA 112 
##STR8## 
GCA CCA ACG CGT TGG TAT CTT TAG GCC AAT AAA CAA ATT TTT TGT GTT TGG AAT 
TAG TCT 242 
##STR9## 
##STR10## 
##STR11## 
##STR12## 
__________________________________________________________________________ 
TABLE 4 
__________________________________________________________________________ 
vp39/LSXIV hybrid promoter sequence SEQ ID NO: 9 
__________________________________________________________________________ 
##STR13## 
GTT CAA ATT TGA TTT CAA TTT TAT CGT GTT GGT AAA CGT ACA CTT TAA TTA TTT 
TAC TCA 
##STR14## 
##STR15## 
##STR16## 
##STR17## 
##STR18## 
##STR19## 
__________________________________________________________________________ 
TABLE 5 
__________________________________________________________________________ 
Nucleotide sequences of the wild-type and mutant polyhedrin 
promoter/leader region. 
__________________________________________________________________________ 
##STR20## 
##STR21## 
##STR22## 
GAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGT. . . 
XhoI/SalI 
B 
##STR23## 
##STR24## 
phcLSXVIGATATCATGGAGccaagcttggTGATAACCATCTCGCAAATAA 
ATAAGTATTTTACTGTTTTCGTAACAGTTTTGTAATAAAAAAACCTATAAATAG 
phcLSXVIIGATATCATGGAGATAATTAAAAccaagcttggCTCGCAAATAA 
ATAAGTATTTTACTGTTTTCGTAACAGTTTTGTAATAAAAAAACCTATAAATAG 
phcLSXIVGATATCATGGAGATAATTAAAATGccaagcttggCGCAAATAA 
ATAAGTATTTTACTGTTTTCGTAACAGTTTTGTAATAAAAAAACCTATAAATAG 
__________________________________________________________________________ 
##STR25## 
##STR26## 
##STR27## 
indicates the start point of transcription. This sequence is now 
identified as SEQ ID No 1. 
(B) Nucleotide sequences of the wt and LS promoter/leader region. For the 
LS mutants, the linker replacement sequences are in 
lower-case letters. The asterisk at nt -50 indicates the start point of 
transcription. All nucleotides are numbered so that the last digit of 
the number corresponds to the given nucleotide. 
phcwt, phcLSXVI, phcLSXVII and phcLSXIV are now identified as SEQ ID NO: 
2, SEQ ID NO: 3, SEQ ID NO: 4 and 
SEQ ID NO: 5, respectively. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 9 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 187 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
GATATCA TGGAGATAATTAAAATGATAACCATCTCGCAAATAAATAAGTATTTTACTGTT60 
TTCGTAACAGTTTTGTAATAAAAAAACCTATAAATAGATCTCGACGAGATTTTCAGGAGC120 
TAAGGAAGCTAAAATGGAGAAAAAAATCACTGGATATACCACCGTTGATATA TCCCAATG180 
GCATCGT187 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 97 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
GATATCATGGAGATAATTAAAATGATAACCATCTCGCAAATAAATAAGTATTTTACTGTT60 
TTCGTAACAGTTTTGTAATAAAAAAACCTATAAATAG97 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 97 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
GATATCATGGAGCCAAGCTTGGTGATAACCATCTCGCAAATAAATAAGTATTTTACTGTT60 
TTCGTAACAGTTTTGTAATAAAAAAA CCTATAAATAG97 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 97 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
GATATCATGGAGATAATTAAAACC AAGCTTGGCTCGCAAATAAATAAGTATTTTACTGTT60 
TTCGTAACAGTTTTGTAATAAAAAAACCTATAAATAG97 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 97 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
GATATCATGGAGATAATTAAAATGCCAAGCTTGGCGCAAATAAATAAGTATTTTACTGTT60 
TTCGTAACAGTTTTGTAATAAAAAAACCTATAAATAG97 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 95 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
GTACCTCCCGGGCCAAGCTTGGCGTTATTGAATAAGAATTTAAAAATCAATCATTTGTAT60 
ACTGTAAATTACATACTGTTTTATTTAACAATAGA95 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 162 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(ix) FEATURE: 
(A) NAME/KEY: miscfeature 
(B) LOCATION: 92..160 
(D) OTHER INFORMATION: /note="mcs #Microsoft Corp 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
GATGTCCCGGGCCAAGCTTGGCGTTATTGAATAAGAATTTAAAAATCAATCATTTGTATA60 
CTGTAAATTACATACTGTTTTATTTAACAATAGATATCGAATTCCTG CAGCCGGGGGATC120 
CACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAGCTCCAATC162 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 473 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
( ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
GATATCTTGTTCGCCATCGTGGAATCAAATAGATCAATGTCACTTTTCGAAAAATATACA60 
TGTTCAAATTTGATTTCAATTTTATCGTGTTGGTAAACGTACACTTTAATTATTTTACTC120 
AAGTTGTGCGAAAGAGTCTTGT AAGGCAGTTTGATTTCTTTGCTTTCTCTCCACACCAAC180 
GGCACCAACGCGTTGGTATCTTTAGGCCAATAAACAAATTTTTTGTGTTTGGAATTAGTC240 
TTTTTCACGCTTGATATTATGTTATTGCAAGCGCTCTGAATAGGTATACGAGTGCGAAAG300 
CCGTTTTCGTCGTACAAATCGAAATATTGTTTGCCAGCGAATAATTAGGAACAATATAAG360 
AATTTAAAATTTTATACAACAAATCTTGGCTAAAATTTATTGAATAAGAGATTTCTTTCT420 
CAATCACAAAATCGCCGTAGTCCATATTTATAACGGCAACAATAT GCAGATCT473 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 448 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
GATATCTTGTTCGCCATCGTGGAATCAAATAGATCAATGTCAC TTTTCGAAAAATATACA60 
TGTTCAAATTTGATTTCAATTTTATCGTGTTGGTAAACGTACACTTTAATTATTTTACTC120 
AAGTTGTGCGAAAGAGTCTTGTAAGGCAGTTTGATTTCTTTGCTTTCTCTCCACACCAAC180 
GGCACCAACGCGTTGGTATC TTTAGGCCAATAAACAAATTTTTTGTGTTTGGAATTAGTC240 
TTTTTCACGCTTGATATTATGTTATTGCAAGCGCTCTGAATAGGTATACGAGTGCGAAAG300 
CCGTTTTCGTCGTACAAATCGAAATATTGTTTGCCAGCGAATAATTAGGAACAATATAAG360 
AATTTAAAATTTTCCAAGCTTGCGCAAATAAATAAGTATTTTACTGTTTTCGTAACAGTT420 
TTGTAATAAAAAAACCTATAAATAGATC448