Baculovirus dual promoter expression vector

This invention relates to specifically designing and genetically engineering recombinant baculovirus for producing, in a compatible insect system, a desired protein, virus, protein hybrid, or virus hybrid. In particular aspects, this invention relates to the use of different baculovirus promoters for the ultimate purpose of constructing a recombinant baculovirus designed for the investigator's specific need. For example, the recombinant baculovirus of this invention can be designed to produce a viral pesticide. This invention also describes the construction of a genetically engineered virus or virus hybrid (e.g. animal or human pathogen) which is not capable of replicating itself but is essentially identical to the authentic pathogen in terms of structure and antigenicity. This baculovirus is constructed such that the non-structural viral genes are truncated, mutated or both and are located 3' and directly under the control of an early baculovirus gene promoter and the structural viral genes are located 3' and directly under the control of a late baculovirus gene promoter. This genetically engineered baculovirus is therefore capable of temporal regulation and successive synthesis of non-structural and structural proteins. The truncated or mutated non-structural viral genes creates the non-replicative aspect of this invention. Since the genetically produced virus or virus hybrid is essentially identical to the authentic pathogen, the product is thereby highly antigenic and potent in terms of efficacy and efficiency. This invention enables the design and constructure of a virus particle or virus hybrid with specific antigenic properties which further allows for the safe and inexpensive production of vaccines or diagnostics.

BACKGROUND OF THE INVENTION 
A. Field of the Invention 
This invention relates to specifically designing and genetically 
engineering recombinant baculovirus for producing, in a compatible insect 
system, a desired protein, virus, protein hybrid, or virus hybrid. In 
particular aspects, this invention relates to the use of different 
baculovirus promoters for the ultimate purpose of constructing a 
recombinant baculovirus designed for the investigator's specific need. 
B. Description of the Related Art 
The capabilities of producing genetically engineered proteins, through the 
use of recombinant DNA technologies, have dramatically increased in the 
last decade. However, problems have accompanied these new capabilities. 
One of the many problems involves constructing and genetically engineering 
the desired protein product. The operative words in the last sentence are 
"engineering the desired protein product." All too often, when it comes to 
creating a construct specifically designed for the production of a desired 
protein or virus, comprises have to be made. It is not uncommon that many 
intervening and tedious steps are involved in the creation of a desired 
construct for the purpose of producing a desired product. It is also not 
uncommon to have "to settle" for the production of an almost perfect 
construct because of technical difficulties in designing compatible sites 
for insertion of desired genes. 
It is the intent of this invention to detail methods and compositions for 
designing and creating a baculovirus construct. This construct is designed 
in such a manner that few if any compromises have to be made in order to 
create the desired product. This baculovirus construct allows for the 
production of virtually any protein, vaccine, therapeutic, diagnostic, 
viral pesticide, or virus, as well as, the creation of virtually any new 
combination of the above. Combinations of proteins or viruses are termed 
"protein hybrid" or "virus hybrid." These terms imply there are multiple 
sources from which the product is created or that the product itself is 
made up of different components. 
This invention will allow the investigator to design and create desired 
proteins, vaccines, viruses, therapeutics, diagnostics, viral pesticides, 
hybrids of the above, or functional and active domains from any of the 
above as well. Therefore, the potential use of this vector is endless. 
The success of this invention, is in part, due to the unique use of 
multiple baculovirus promoters (both early and late) along with homologous 
recombination into a non-essential region(s) of the AcMNPV genome. For 
optimal flexibility, this invention employs three baculovirus promoters: 
the natural polyhedrin promoter mapped to the Eco-RI site of Fragment I; 
an early baculovirus promoter and a late baculovirus promoter. 
The present invention also describes the production of recombinantly 
generated virus or virus hybrid particles or proteins involving the novel 
use of an early baculovirus gene promoter to temporally drive the 
expression of truncated or mutated non-structural genes of a virus 
particle as a unit, 5' to a late baculovirus gene promoter controlling the 
expression of cDNA for structural genes from the same or a different 
virus. The recombinantly expressed virus or virus hybrid, obtained from 
this construct, is essentially intact, virtually identical to the 
authentic parent virus particle, highly antigenic yet non-replicative and 
is therefore exceedingly functional in terms of use as a vaccine or 
diagnostic reagent. 
This invention allows the investigator the flexibility to design and create 
a baculovirus construct that produces either a desired protein, vaccine, 
virus, therapeutic, diagnostic, viral pesticide, hybrid of the above, or 
functional and active domain from any of the above as well. This invention 
will be accomplished efficiently and without undue experimentation. 
SUMMARY OF THE INVENTION 
In general and overall scope, the present invention provides a method for 
creating a recombinant baculovirus construct for the purpose of 
specifically producing a desired protein, vaccine, diagnostic, 
therapeutic, viral pesticide, or hybrid thereof and presenting it to the 
cell or organism. The vector described in this invention may also be used 
to produce a variety of different combinations of protein hybrids, as well 
as proteins or peptides that can be specifically produced at a particular 
time in the infection cycle to optimize the effect of that protein or 
peptide. Also, this invention describes designing and creating a construct 
for the purpose of producing functional and active domains from a specific 
protein, vaccine, diagnostic, therapeutic, viral pesticide, or hybrid 
thereof. The exact regions that encompass the functional and active domain 
will vary from protein to protein, from vaccine to vaccine, from 
diagnostic to diagnostic, etc. 
More particularly, the present invention describes the use of multiple 
baculovirus promoters representative of different classes of promoters, as 
well as homologous recombination to a non-essential region of the AcMNPV 
genome, to produce the desired protein, vaccine, diagnostic, therapeutic, 
viral pesticide, or hybrid thereof. 
This invention also pertains to designing and creating a desired 
recombinant baculovirus construct for the specific purpose of producing a 
desired protein, vaccine, diagnostic, therapeutic, viral pesticide, or 
hybrid thereof. Further in accordance with this invention, hybrids of 
either a desired protein, vaccine, diagnostic, therapeutic, or viral 
pesticide are defined and designed. 
In addition to the above, the recombinant DNA vector includes a DNA region 
comprising a cDNA sequence coding for a desired protein. Sequences coding 
for a variety of different genes are known to those skilled in the art and 
are commercially available from American Type Culture Collection (ATCC, 
Rockville, Md.). For example, the following is a brief list representing 
the range of cloned genes or probes available from ATCC: epidermal growth 
factor receptor, beta-glucuronidase, Y-mos M1 Maloney sarcoma virus, 
tissue-type plasminogen activator, arginosuccinate synthetase, insulin (A 
and B chain), prolactin, interleukin 1 and 2, colony stimulating factor, 
tumor necrosis factor, beta-hemoglobulin, interferon, leutinizing hormone, 
beta-hexosaminadase, coagulation factor VIIIC, transferrin, esterase D, 
adenosine deaminase, etc. This cDNA sequence is further comprised of 
nucleotide sequences coding for a desired protein having a deleted 5' 
untranslated region, and a deleted or mutated translational initiation 
site. In terms of this invention, amino acid and nucleotide numbers will 
be used interchangeably with the appropriate conversion factor employed. 
For enhanced expression and production of the desired protein, this 
recombinant DNA vector, in addition to the above stated components, 
includes a DNA region comprising unique signals for the initiation of 
transcription and translation positioned 5'. 
In terms of infecting or transfecting yeast or eukaryotic cells with a 
recombinant DNA vector, these techniques are standard and known to those 
skilled in the art of recombinant DNA technology. In terms of transfecting 
cells with the recombinant DNA vector described above, this invention 
could also be applied for the production of stable cell lines which are, 
by definition, continuously producing the specific chimera protein. The 
production of cell lines with stably integrated recombinant DNA vectors 
has been described extensively in the literature, practiced for years, and 
is therefore known to those skilled in the art. 
It should be noted that genes may be structurally similar but that a 
variety of regulatory processes, which occur at the transcriptional and 
translational levels and function in a manner consistent with the biology 
of the organism or the cell, makes it impossible to predict the exact 
process by which the gene will be expressed and/or regulated. 
The present invention also provides a method for producing a highly 
antigenic non-replicative virus or virus hybrid, to be used as a vaccine, 
a protein producing system, or a diagnostic reagent, by employing the 
baculovirus expression vector system (BEVS). The production of a highly 
antigenic non-replicative virus or virus hybrid particle involves the 
construction of, for example, a recombinant baculovirus expression vector 
containing two or three promoters genetically engineered to include 
foreign genes (e.g. non-structural and structural) inserted under the 
transcriptional regulation of both early and late baculovirus gene 
promoters. Thus, truncated or mutated non-structural genes will be 
expressed early to provide genomic, non-replicative RNA (or DNA) followed 
by late expression of structural genes. This strategy will ensure the 
availability of genomic material, which is needed for proper assembly of 
the virus particle of choice. Below, Sindbis virus and Rubella virus 
(Alphavirus) or combinations thereof will serve as examples for the 
clarification of this concept. It should be emphasized that the invention 
can be adapted or applied to a large variety of viral pathogens, insect 
pesticides, protein production systems and that the examples described 
below serve only as models. The insect pesticides can be designed to 
produce a desired protein, peptide, as well as an mRNA or DNA fragment 
which could result in insecticidal affects. 
More particularly, the present invention describes the production of 
recombinantly generated virus or virus hybrid particles or proteins 
involving the novel use of an early baculovirus gene promoter to 
temporally drive the expression of truncated or mutated non-structural 
genes of a virus particle as a unit, 5' to a late baculovirus gene 
promoter controlling the expression of cDNA for structural genes from the 
same or a different virus. The recombinantly expressed virus or virus 
hybrid, obtained from this construct, is essentially intact, virtually 
identical to the authentic parent virus particle, highly antigenic yet 
non-replicative and is therefore exceedingly functional in terms of use as 
a vaccine or diagnostic reagent. 
The recombinant baculovirus produced from this invention is controlled 
through transcriptional regulation of both early and late baculovirus gene 
promoters to temporally and successively synthesize both non-structural 
and structural proteins. Due to the temporal regulation and subsequent 
expression of genes under the control of different baculovirus promoters, 
the truncated or mutated non-structural genes are expressed first (because 
of control by an early baculovirus gene promoter) and the structural genes 
are subsequently expressed thereby allowing for the accurate assembly of a 
viral or viral hybrid particle. Thus, the different portions of the 
pathogen (non-structural versus structural) will be transcriptionally 
regulated by both early and late baculovirus gene promoters to produce 
e.g. Sindbis virus particles. 
In terms of this invention, "recombinant baculovirus expression vector" or 
"recombinant baculovirus" refer to vectors or baculoviruses which have the 
ability to genetically produce both non-structural and structural proteins 
derived from either the same or different viral sources. It is the final 
vector or baculovirus described in the production scheme that contains the 
genetic material to produce different recombinant proteins (non-structural 
and structural). 
Additionally, in terms of this invention, "bacterial transfer vector" shall 
be defined as the bacterial plasmid vector optimally containing the 
following: viral flanking sequences essential for optimal homologous 
recombination to occur, bacterial plasmid sequences, either an early or 
late baculovirus gene promoter, cDNA and genomic DNA encoding for both 
non-structural or structural viral genes and a multiple cloning site 
adjacent to the specific promoter. Genomic DNA is defined as DNA isolated 
from the chromosome of an organism or virus. The bacterial plasmid 
sequences may be derived from any one of the many different vectors that 
are commercially available and known to those skilled in the art of 
recombinant DNA technology. For the purpose of this invention, pUC8 is 
used as a matter of preference, however, other vectors would be equally 
effective. 
These bacterial plasmid transfer vectors are utilized in cotransfection 
experiments, and through the process of homologous recombination or by any 
process in which the vectors may serve as a vehicle to deliver the desired 
gene or gene product, allows for the insertion or integration of the gene 
of interest into the baculovirus. The process of homologous recombination 
is standard and known to those skilled in the art. The detailed procedures 
are available in many different protocol texts. 
In addition to the temporal transcriptional regulation, this invention also 
enables the design, construction and assembly of a non-replicative virus 
particle or virus hybrid, which is highly antigenic and functionally 
identical to the authentic parent virus particle. Due to the truncation or 
mutation of the RNA encoding for the non-structural viral genes, this 
novel and genetically engineered virus is therefore not capable of 
replicating itself, and yet, it is structurally, and antigenically, 
essentially identical to the authentic pathogen. The truncation or 
mutation must not, however, alter the genomic RNA in such a way that the 
genomic sequences needed for encapsidation are deleted. 
An advantage and novel aspect of this invention is the freedom to design a 
recombinant baculovirus expression vector that can be used for the 
expression and production of a completely different non-replicative virus 
hybrid of choice. The basic concept is to genetically engineer different 
portions of genomic DNAs or cDNAs from different pathogens (human, animal 
or plant) for which a vaccine or diagnostic is needed. The non-structural 
gene will be derived from one viral source while the structural gene will 
be derived from a different viral source. This freedom in designing 
virtually any combination of viruses should allow for the production of 
highly antigenic, "special-order", potent and very specific vaccines or 
diagnostics. 
In one embodiment of this invention, an example of a double recombinant 
virus hybrid is described which combines non-structural genes from Sindbis 
virus and structural genes from Rubella virus. The virus hybrid produced 
from this example is one of many different potential combinations 
involving an animal and a human pathogen. From this point of view, the 
combinations are therefore endless. 
Another advantage of this invention is that it enables the production of 
"natural," highly potent, non-infectious antigens or combinations of 
assembled antigens. Generally, an intact virus particle is a better 
antigen and or immunogen than is a single purified protein or derivatives 
thereof. The product from this method for producing a vaccine or 
diagnostic of choice, is more efficient, safe, inexpensive and effective 
as compared to the more traditional procedures (i.e. attenuating the virus 
or virus subunit production). Thus, this invention represents an important 
potential improvement in the area of vaccine production compared to the 
available traditional vaccine production protocols. 
The genetically engineered virus produced from this invention may be 
derived from a human, animal or plant pathogen or combinations thereof. 
The pathogen or viral source for the production of a recombinant 
baculovirus expression vector can be from the family of arboviruses which 
includes, but is not limited to: Sindbis virus, bluetongue virus, rabies 
virus, yellow fever virus, St. Louis encephalitis virus, Colorado tick 
fever virus or dengue fever virus. 
The viral source for the production of a virus or virus hybrid may also be 
derived from, but is not limited to include: poliovirus, influenza virus, 
hepatitis B virus, human immunodeficiency virus, polyoma virus, Punta Toro 
phlebovirus, Simian rotavirus or Simian virus. 
In accordance with the present invention, the recombinant baculovirus 
expression vector which will be used to produce a foreign virus or virus 
hybrid will minimally include a DNA region comprising a transcriptional 
regulator, a cDNA region or genomic DNA encoding a truncated or mutated 
non-structural gene for a desired viral particle, a DNA region comprising 
a different transcriptional regulator and a cDNA or genomic DNA region 
encoding a structural gene for a desired viral particle. These components 
are properly spaced and only use one open reading frame. 
In terms of this invention, "transcriptional regulator" is defined as a 
promoter which controls and drives the expression of a gene located 
downstream (3') the promoter itself. 
In accordance with the present invention, "transcriptional regulator" shall 
be further defined as a promoter derived from either an early or late 
baculovirus gene. For the purpose of this invention, an "early" 
baculovirus gene promoter is meant to include promoters derived from 
intermediate- early as well as delayed-early baculovirus genes. Examples 
of intermediate- and delayed-early baculovirus genes are IE1, IE0 and IEN, 
respectively. The promoters derived from these two early baculovirus genes 
have previously been shown to be strong promoters with respect to driving 
the expression of the specific genes located 3' to the promoter itself. 
Other promoters would probably be effective, however, the inventors prefer 
to employ the promoters derived from IE1, IE0 or IEN early baculovirus 
genes. 
Additionally, for the purpose of this invention, promoters derived from 
"late" baculovirus genes include those promoters derived from the 
polyhedrin or p10 genes. Other promoters would probably be effective, 
however, the inventors prefer to employ the promoters derived from 
polyhedrin or p10 late baculovirus genes. 
Further in accordance with this invention, the recombinant baculovirus 
expression vectors as well as the promoters employed to drive the 
expression of the viral genes, may be derived from Autographa californica 
nuclear polyhedrosis virus, Trichoplusia ni nuclear polyhedrosis virus, 
Rachiplusia ou nuclear polyhedrosis virus or Galleria mellonella nuclear 
polyhedrosis virus, Heliothis zea nuclear polyhedrosis virus, Mamestra 
brassica nuclear polyhedrosis virus, Spodoptera exigua nuclear 
polyhedrosis virus, Spodoptera frugiperda nuclear polyhedrosis virus, 
Orgyia pseudosugata nuclear polyhedrosis virus, Anisota senatoria nuclear 
polyhedrosis virus, or any one of the more than 500 additional baculovirus 
species. Although the invention described herein employs the use of 
baculoviruses and promoters from Autographa californica nuclear 
polyhedrosis virus as a matter of preference, any of the above mentioned 
polyhedrosis viruses would be as effective. 
In terms of this invention, "directionally positioned", "in an appropriate 
open reading frame", "from 5' to 3' with appropriate spacing" and 
"adjacent to" are interchangeable terms referring to the positional 
placement of certain components in the baculovirus expression vector or 
the bacterial transfer vector to maintain the necessary requirements 
(positioning and open reading frame) for efficient and accurate 
transcription. 
The various techniques which have been successfully applied to the cloning 
and expression of many heterologous genes in a variety of host systems, 
employing many different promoters and expression vectors, are known to 
those skilled in the art of recombinant DNA technology and could be 
applied to the embodiments described herein. Appropriate positional 
spacing between the numerous recombinant DNA vector components 
(directionally positioned 5' to 3') would be determined for each specific 
recombinant baculovirus vector or bacterial transfer vector and are 
included to further optimize the expression and production of the desired 
virus or virus hybrid. 
In terms of this invention, a foreign virus or virus hybrid is defined as 
any viral particle produced in the baculovirus expression system which 
normally would not be made in that system. 
For the purpose of this invention, virus hybrid or viral hybrid particles 
will be used interchangeably and will be defined as any particles produced 
in the recombinant baculovirus expression vector system, whereby the 
non-structural and structural genes are derived from two different viral 
sources (e.g. non-structural genes derived from Sindbis virus and 
structural genes derived from Rubella virus). 
For the purpose of this invention, non-structural viral genes shall refer 
to those viral genes that encode for essential viral proteins needed for 
authentic viral replication and assembly. Structural viral genes will 
refer to those genes that encode for capsid, core and/or envelope proteins 
(antigenic or immunogenic epitopes). Non-structural may as well refer to a 
limited nucleic acid sequence needed for the formation or assembly of the 
capsid or core structure. 
In addition to the above mentioned features, for added ease in handling and 
manipulating, this recombinant baculovirus expression vector could include 
a DNA region comprising a multiple cloning cassette sequence, 
appropriately spaced and in frame, between the transcriptional regulator 
and the non-structural viral genes or the structural viral genes. Multiple 
cloning cassette sequence cartridges are commercially available from 
several different companies (Promega, New England Biolabs, etc). A typical 
cassette sequence cartridge would include restriction sites for 8-11 
different enzymes (i.e. Eco R1, Sac1, Sma 1, Ava 1, Bam H1, Xba 1, Hinc 
II, Acc 1, Sal 1, Pst 1, Hind III, etc.). The availability of these 
cassette cartridges are known to those skilled in the art. 
Further in accordance with this invention, the recombinant baculovirus 
expression vector infects a suitable cell line in order to produce a 
functional virus or virus hybrid. Lepidopteran insect cells derived from 
e.g. Spodoptera frugiperda, Heliothis virescens, Heliothis zea, Mamestra 
brassicae, Estigmene acrea or Trichoplusia ni are used. Although cell 
lines derived from the above mentioned species would be effective, cell 
lines derived from Spodoptera frugiperda are preferred. Sf9 cells or Sf9 
IE1-helper cells are two commonly available Spodoptera frugiperda cell 
lines which are routinely utilized. 
Thus, optimal production of a specific virus or virus hybrid could 
potentially be enhanced if the recombinant baculovirus expression vector 
was engineered to include an IE1 or IEN early baculovirus gene promoter 
positioned 5' to cDNA and genomic DNA encoding truncated or mutated 
non-structural genes for a specific virus, both of which are located 
adjacent to a polyhedrin or p10 late baculovirus gene promoter positioned 
next to cDNA encoding structural genes for a specific virus. The above 
transfer vectors and appropriate baculoviral DNA would then be 
cotransfected into Sf9 or Sf9 IE1-helper Lepidopteran insect cells. This 
optimal expression vector, as described above, would also include the 
appropriate transcriptional start and stop signals as well as multiple 
cloning sites located 3' to the promoters for ease in inserting the cDNA 
encoding for the desired viral genes. 
Once cotransfection and homologous recombination occurs in the above 
mentioned experiments, baculoviral DNA containing the recombinant will be 
selected by visual selection of white occlusion negative plaques. Plaque 
selection and purification are known procedures familiar to those skilled 
in this art. 
The non-replicative recombinant will then be purified from the cells or 
culture medium by techniques known to those skilled in the art and the 
virus or virus hybrid is ready for further use as a vaccine or as a 
diagnostic reagent. 
The viral or viral hybrid products obtained from this invention are 
inexpensive to produce, safe to use, highly antigenic and potent, and 
functionally very similar to the authentic parent virus.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
Autographa Californica Nuclear Polyhedrosis Virus (AcMNPV) 
Autographa californica nuclear polyhedrosis virus (AcMNPV), the prototype 
virus of the family Baculoviridae, has a wide host range and infects more 
than 30 species of Lepidopteran insects. During AcMNPV infection, two 
forms of viral progeny are produced: extracellular virus particles (ECV) 
and occluded virus particles (OV). The latter are embedded in 
proteinaceous viral occlusions, called polyhedra. A polyhedrin protein, 
with a molecular weight of 29,000 Daltons, is the major viral encoded 
structural protein of the viral occlusions. 
Since the viral occlusions provide a means for stable horizontal 
transmission, they are an important part of the natural virus life cycle. 
When infected larvae die, millions of polyhedra are left within the 
decomposing tissue. The viral occlusions aid in protecting the embedded 
virus particles from environmental factors that would otherwise rapidly 
inactivate ECV. When larvae feed on contaminated plants, they ingest the 
polyhedra. The occlusions dissolve in the alkaline environment of the 
insect gut, releasing virus that invade and replicate in the cells of the 
midgut tissue. 
Secondary infection spreads to other insect tissues by the extracellular 
viral (ECV) route. Virus particles enter the cell by endocytosis or fusion 
and the viral DNA is uncoated. DNA replication occurs at about 6 hours 
post-infection (pi) and by 10 hours pi, extracellular virus is released 
from the cell by budding. Polyhedrin protein can be detected by 12 hours 
pi but viral occlusions are not readily detected until 18-24 hours pi. 
Extracellular virus levels reach a maximum between 36-48 hours pi and the 
polyhedrin protein continues to accumulate for 4-5 days until the infected 
cells lyse. 
Baculovirus Expression Vectors (BEVs) 
Baculovirus expression vectors (BEVs) have become extremely important tools 
for the expression of foreign genes, both for basic research and for the 
production of proteins with direct clinical applications in human and 
veterinary medicine. BEVs are recombinant insect viruses in which the 
coding sequence for a desired foreign gene has been inserted 3' to a 
select baculovirus promoter, e.g. the polyhedrin promoter in place of the 
non-essential viral gene, polyhedrin, thereby promoting the expression of 
the chosen foreign gene. (V. A. Luckow and M. D. Summers, Bio/Technology, 
6:47-55 (1988a); M. D. Summers, Curr. Commun. in Molec. Biol., Cold Spring 
Harbor Press. Cold Spring Harbor, N.Y. (1987), Summers, M. D. and Smith, 
G. E., TAES Bulletin No. 1555, (1988); Smith and Summers, U.S. Pat. Nos. 
4,745,051 and 4,040,367); Webb and Summers, In Press, Techniques, 1990). 
Several advantages may be enjoyed when employing the exemplary baculovirus 
expression vector (BEV) system. One of these advantages is the strong 
polyhedrin promoter which directs a high level of expression of the 
foreign insert (protein of choice). The newly expressed protein 
accumulates in large amounts within these infected insect cells or as 
secreted products. Thus, as a result of the relative strength of the 
polyhedrin promoter, many different desired foreign gene inserts can be 
expressed at very high levels. 
In addition to providing a high expression level, another advantage of the 
BEV system, is the ease with which these baculoviruses are produced and 
identified. This process begins by co-transfecting wild-type viral DNA and 
a "transfer vector" into susceptible host cells. A transfer vector is 
defined as a bacterial plasmid which contains a foreign gene directly 3' 
to a desired baculovirus promoter, e.g. the polyhedrin promoter, as well 
as long viral sequences flanking the promoter on the 5' side and the 
foreign gene on the 3' side. During cotransfection, homologous 
recombination occurring between viral and transfer vector DNA will produce 
a small percentage of viral genomes in which the polyhedrin gene has been 
replaced by the foreign gene (less than 5%). The wild-type progeny can be 
differentiated from the recombinant progeny by a conventional viral plaque 
assay. Recombinants in which the polyhedrin gene has been replaced, can be 
identified by their occlusion-negative plaque phenotype observed on a 
background of occlusion-positive wild-type plaques. 
Because the polyhedrin gene is a non-essential gene for productive viral 
infection, another advantage of baculovirus expression vectors is that the 
recombinants are viable, helper-independent viruses. Also, baculoviruses 
are known to infect arthropods of which the Lepidopteran insects comprise 
the largest group of susceptible species. Thus, they are noninfectious for 
vertebrates, and are therefore relatively safe genetic manipulation 
agents. 
Thus, baculoviruses have gained popularity as expression vectors because of 
the advantages presented above. The BEV system is currently being employed 
by numerous investigators for the over expression and production of many 
different foreign gene products. To date, more than 175 different genes 
have now been expressed by employing this system (Luckow, V. A., and M. D. 
Summers, Bio/Technology 6:47-55 (1988)). 
Recombinant proteins produced in the BEV system retain many of their 
authentic biological properties including intracellular targeting, 
secretion and receptor binding (Luckow and Summers, 1988, Id.; Webb and 
Summers, In Press, Techniques, 1990). In terms of processing, most 
recombinant protein products appear to undergo normal post-translational 
modifications such as proteolytic processing of polyprotein precursors, 
removal of signal sequences and chemical modifications including 
glycosylation and phosphorylation. 
The recombinant baculovirus infected insect cells would therefore serve as 
an ideal model for the production of a variety of different types of 
proteins, vaccines, antigens or immunogens. 
Temporal Regulation of Baculovirus Genes 
Baculovirus genes are expressed in a sequential, temporally-regulated 
fashion during one or more of the different phases of the viral 
replication cycle (L. A. Guarino, and M. D. Summers, Journal of Virology, 
57(2):563-571 (1986); Guarino, L. A. and Summers, M. D., Journal of 
Virology, 61(7):2091-2099 (1987)). Therefore, depending on their temporal 
expression during viral infection, different baculovirus genes are 
classified as immediate-early, delayed-early, late or very late. 
The expression of these genes occurs sequentially, probably as the result 
of a `cascade` mechanism of transcriptional regulation. In other words, 
the immediate-early genes are expressed immediately after infection and 
expression may occur in the absence of other viral functions. One or more 
of the resulting gene products in turn induces transcription of the 
delayed-early genes. Continuing in the cascade, some delayed-early gene 
products induce transcription of late genes and finally the very late 
genes are expressed under the control of the previously expressed gene 
products from one or more of the earlier temporal classes of genes. 
One relatively well defined component of this regulatory cascade is IE1, an 
immediate-early gene of Autographa californica nuclear polyhedrosis virus. 
IE1 is expressed in the absence of other viral functions and encodes a 
product that stimulates the transcription of several genes of the 
delayed-early class, including 39K gene, as well as late genes (early 
genes: L. A. Guarino and M. D. Summers, J. Virol., 57:563-571 (1986a); J. 
Virol., 61:2091-2099 (1987); late genes: L. A. Guarino and M. D. Summers, 
Virol., 162:444-451 (1988)). 
In contrast to the IE1 gene, the polyhedrin gene is classified as a very 
late gene. Thus, transcription from the polyhedrin promoter requires the 
previous expression of an unknown number of other viral and/or cellular 
gene products. Thus, the exemplary BEV system described by Smith and 
Summers (U.S. Pat. No. 4,745,051) will express foreign genes only as a 
result of gene expression from the earlier expressed portion of the viral 
genome and only after the viral infection is well underway. 
Construction of Recombinant Baculovirus Expression Vectors 
General methods for handling and preparing baculovirus vectors and 
baculoviral DNA, as well as insect cell culture procedures, are outlined 
in A Manual of Methods for Baculovirus Vectors and Insect Cell Culture 
Procedures (Summers, M. D. and G. E. Smith, TAES Bulletin No. 1555, 1988). 
Homologous recombination, plaque selection, purification and propagation 
procedures as well as cotransfection protocols are known to those familiar 
and skilled in this art. 
Bacterial transformation, screening by restriction mapping, extraction, 
construction of bacterial transfer vectors, purification of bacterial 
plasmid DNA, as well as other standard molecular biology procedures, will 
be accomplished by standard recombinant DNA techniques (Maniatis, et al, 
Molecular cloning: A laboratory manual, Cold Spring Harbor Press, Cold 
Spring Harbor, N.Y. (1982)). 
It should be emphasized that the experimental procedures described below 
represent examples of how genes are cloned and expressed at different time 
points post infection (p.i.) in baculovirus infected insect cells. 
Standard recombinant procedures are used for the construction of 
recombinant plasmids (Maniatis et al, 1982) as well as for the generation 
and propagation of recombinant baculovirus strains (Summers and Smith, 
1987). 
The examples which follow are illustrative of laboratory techniques found 
by the present inventor to constitute preferred modes for practicing 
various aspects of the invention. However, those of skill in the art, in 
light of the present disclosure, will appreciate that various 
modifications and alterations can be made in the structuring and carrying 
out of the invention, and still remain within the spirit and scope of the 
invention. 
EXAMPLE 1 
Model Baculovirus Expression Vector Containing Three Baculovirus Promoters 
for Either Recombinant Protein, Virus, Protein Hybrid or Virus Hybrid 
Production 
The construction of a model expression vector which would be employed for 
recombinant protein, virus, protein hybrid or virus hybrid production is 
diagramed. 
FIG. 1 outlines the minimal essential features needed for the production of 
either a recombinant virus, protein, virus hybrid or protein hybrid. 
Desired genes are inserted 3' to either the late or the early baculovirus 
promoters. This bacterial transfer vector will recombine homologously at 
13.0 map units in the nonessential region of wild type baculovirus. The 
potential combinations for the different genes and their modifications are 
numerous. 
For the production of different viruses or vaccines, the transfer vector 
can be designed to contain cDNA encoding truncated non-structural genes 
positioned 3' to an early baculovirus gene promoter while the genes coding 
for the structural proteins are positioned adjacent the late baculovirus 
promoter, e.g. the polyhedrin gene promoter. The details for the 
production of different viruses, vaccines, or virus hybrids are described 
below. 
In general, recombinant baculovirus DNA of choice is produced by 
cotransfection of Sf9 cells with a specifically designed and constructed 
transfer vector and wild type viral DNA (FIG. 1). This specifically 
designed transfer vector can be used for the production of many different 
proteins, viruses, protein hybrids, or virus hybrids. One of the many 
novel concepts for using a specifically designed recombinant baculovirus 
expression vector involves combining structural and non-structural genes 
from two different viral sources for the purpose of designing, and thereby 
producing, an essentially different hybrid viral particle. The 
specifically designed transfer vector may be used for producing many 
different compositions. 
Minimal essential features of the model baculovirus expression vector are: 
1. Natural Polyhedrin Promoter/desired gene: designates a late baculovirus 
polyhedrin promoter with or without a desired gene of choice inserted 3' 
to the polyhedrin promoter. 3' is defined as the region located downstream 
of the compared component. This natural polyhedrin promoter is located 
within Eco-RI Fragment I and is useful for stabilizing the virus in the 
environment by conferring resistance to inactivation. 
2. Late Promoter/desired gene: designates a baculovirus late promoter with 
a desired gene of choice inserted 3' to the polyhedrin promoter. 3' is 
defined as the region located downstream of the compared component. 
This promoter and gene complex will be recombined at a nonessential region 
into wild type baculovirus. A non-essential region of the baculovirus 
genome is defined as a place where insertion or modification of natural 
viral DNA sequence or gene structure has no effect on infectivity of the 
modified virus in cell culture. The site for recombination can be any one 
available, however, the inventors prefer the non-essential 13.0 map unit 
site of wild type baculovirus. 
Many different combinations of a late promoter with a desired gene are 
available. For example, for the late promoter, any baculovirus late 
promoter will suffice, however, the inventors prefer to employ the 
promoter from either polyhedrin or p10 gene. The desired gene can feasibly 
be derived from any eukaryote or prokaryote. 
3. Early Promoter/desired gene: designates a baculovirus early promoter 
with a desired gene of choice inserted 3' to the polyhedrin promoter. 3' 
is defined as the region located downstream of the compared component. 
This promoter and gene complex will be recombined at a nonessential region 
into wild type baculovirus (as described above). A non-essential region of 
the baculovirus genome is defined as a place where insertion or 
modification of natural viral DNA sequence or gene structure has no effect 
on infectivity of the modified virus in cell culture. The site for 
recombination can be any one available, however, the inventors prefer the 
13.0 non-essential map unit region of wild type baculovirus. 
Many different combinations of an early promoter with a desired gene are 
available. The early gene promoter region isolated from baculovirus may be 
an immediate-early gene of the virus such that no additional viral gene or 
gene product is needed in order to get constitutive expression of the 
heterologous gene. The immediate-early gene from which the promoter region 
is derived may be either IE1 or IEN. In a preferred embodiment, the gene 
promoter region is isolated from the immediate-early gene of baculovirus, 
IE1 or IEN. 
IE1 may be expressed in the absence of other viral functions and encodes a 
product that stimulates the transcription of several genes of the 
delayed-early class, including the 39K gene (L. A. Guarino and M. D. 
Summers, J. Virol., 57:563-571 (1986a); J. Virol., 61:2091-2099 (1987)). 
An immediate-early gene as described above is used in combination with a 
baculovirus gene promoter region of the delayed-early category. Unlike the 
immediate-early genes, such delayed-early genes require the presence of 
other viral genes or gene products such as those of the immediate-early 
genes. The combination of immediate-early genes can be made with any of 
several delayed-early gene promoter regions such as 39K or one of the 
delayed-early gene promoters found on the HindIII-k fragment of the 
baculovirus genome. In a preferred embodiment, the 39K promoter region is 
linked to the heterologous gene of interest and expression is further 
controlled by the presence of IE1. 
Additionally, when a combination of immediate-early genes with a 
delayed-early gene promoter region is used, enhancement of the expression 
of heterologous genes can be realized by the presence of an enhancer 
sequence in direct cis linkage with the delayed-early gene promoter 
region. Such enhancer sequences are characterized by their enhancement of 
delayed-early gene expression in situations where the immediate-early gene 
or its product is limited. In a preferred embodiment, the hr5 enhancer 
sequence is linked directly (in cis) to the delayed-early gene promoter 
region, 39K, thereby enhancing the expression of the cloned heterologous 
DNA. 
Again, the desired gene positioned to the early baculovirus promoter can 
feasibly be derived from any eukaryote or prokaryote. 
This model baculovirus expression vector is essentially comprised of three 
baculovirus promoters. Two of the three promoters are homologously 
recombined into the 13.0 map unit non-essential region of wild type 
baculovirus. 
FIG. 1B demonstrates one of the many different combinations that may be 
employed in this model baculovirus expression vector. This model 
expression vector contains three baculovirus promoters adjacent to desired 
genes and serves as an example only. The first polyhedrin promoter appears 
in its natural site within Eco-RI Fragment I. The second promoter is a 
polyhedrin promoter, adjacent and 5' to a desired gene, inserted through 
homologous recombination into a non-essential region of the baculovirus 
genome. The arrow indicates the direction the promoter expresses the 
adjacent DNA. Also, inserted through homologous recombination into this 
non-essential region of the genome, is a third promoter. This third 
promoter is an early or immediate early baculovirus promoter, for example 
IE1, 39K or IEN. In this example, the immediate early baculovirus 
promoter, IE1, is adjacent to a desired gene and the promoter directs the 
expression of the desired gene (indicated by the arrow). The two promoters 
in the non-essential region of the genome are expressed in opposite 
directions. Non-essential region of the baculovirus genome is defined as 
above. 
EXAMPLE 2 
Construction of New AcMNPV Transfer Vectors Containing Both Early and Late 
Baculovirus Promoters 
The development of recombinant viruses that are optimally infectious in 
insect larvae requires encapsulation of these viruses into viral 
occlusions. The integrity of the polyhedrin gene in the recombinant virus 
must therefore be left intact. A region of the AcMNPV genome which is 
separate from the polyhedrin gene and which is non-essential for the 
replication and infection of AcMNPV in vivo and in vitro has been 
identified (Gonzalez, Smith and Summers, 1989, Virology 170: 160-175). 
FIG. 2A schematically represents a transcription map of AcMNPV. The gene 
products are indicated above the horizontal line. At least 15 different 
gene products are indicated by the bold, filled in arrow heads. Examples 
of gene products are: Polyhedrin, egt, V-ubi, 39K, ETL, 25K, DNA 
polymerase, capsid, etc. 
Arbitrary designation of map units for the fragments generated from the 
restriction enzyme Eco-RI are indicated by small numbers below the solid 
horizontal line. Zero map units start at the right of "HR1" and 100 map 
units ends at the left of "HR1." 
Enhancer sequences are numbered numerically HR1, HR2, HR3, HR4, or HR5 for 
convenience. When a combination of immediate-early genes and a 
delayed-early gene promoter region is employed, enhancement of the 
expression of heterologous genes can be realized by the presence of an 
enhancer sequence in direct cis linkage with the delayed-early gene 
promoter region. Such enhancer sequences are characterized by their 
enhancement of delayed-early gene expression in situations where the 
immediate-early gene or its product is limited. In a preferred embodiment, 
the hr5 enhancer sequence is linked directly (in cis) to the delayed-early 
gene promoter region, 39K, thereby enhancing the expression of the cloned 
heterologous DNA. 
Fragments generated by the restriction enzyme Eco-RI are assigned letters 
according to size ("Fragment A" is the largest, "B" is the next largest, 
"C" is the next largest, etc.; I, R, D, A, J, K, T, M, N, F, V, U, C, G, 
W, D, Q, L, E, H, S, X, P, and B) 
The region where pSfHindIII-L will recombine into the AcMNPV [.d10A] 
polyhedrin deletion mutant is designated by the wedged pie area and 
designated FIG. 2B. 
Recombination of pSfHindIII-L into the AcMNPV.d10A polyhedrin deletion 
mutant is diagramed in FIG. 2B. Only AcMNPV.EcoR1-A and the physical 
alterations of the fragment are shown. The majority of Pst1-O was deleted 
and a 3.7 kb fragment from pSfHindIII-L is inserted in its place. 
Horizontal lines represent transcripts and their orientation. Dashed lines 
indicate possible termination sites. Genomic map units (m.u.) are shown at 
specific sites beneath correlating fragment dimensions. The wedged pie 
area is magnified in subsequent figures to present more detail. 
FIG. 2B describes the recombination of pSfHindIII-L into the AcMNPV.d10A 
polyhedrin deletion mutant. Only AcMNPV.EcoR1-A and the physical 
alterations of the fragment are shown. This figure also depicts the 
non-essential region that has been mapped to the EcoRI-A fragment 
(13.4-14.7 map units) of the ACMNPV genome. The insertion of foreign DNA 
into this region results in occlusion-positive viruses which are 
infectious in vivo (Gonzalez et al, 1989). This nonessential 13.2 map unit 
region of the AcMNPV genome is designated the "13.0 map unit region" for 
simplicity. The definition of the 13.0 map unit region of the AcMNPV 
genome is meant to include 13.2 map units. 
The majority of Pst1-O was deleted and a 3.7 kb fragment from pSfHindIII-L 
is inserted in its place (FIG. 2B). Horizontal lines represent transcripts 
and their orientation. Dashed lines indicate possible termination sites. 
Genomic map units (m.u.) are shown at specific sites beneath correlating 
fragment dimensions. 
Isolation of the Ac/Sf Hybrid Virus 
The AcMNPV mutant d 10A was used to construct a recombinant virus 
containing a heterologous polyhedrin gene. The AcMNPV d 10A lacks 
approximately 25% of the polyhedrin coding region and produces a truncated 
polyhedrin protein of 20 kDa (Smith et al., 1983b), thus, producing 
occlusion-negative (occ-) plaques. The SfMNPV polyhedrin gene was 
localized to the 4.0-kb Hindlll-L fragment of SfMNPV DNA by Southern 
hybridization using the AcMNPV -Hindlll-V fragment as a probe. A 
recombinant plasmid containing the SfMNPV Hindlll-L fragment was then 
cotransfected with AcMNPV d 10A DNA. This resulted in occlusion-positive 
plaques which were marked and further plaque-purified. Occlusive-positive 
plaques were detected at a frequency of 1 in every 10,000 plaques. 
Cloning Strategy 
Foreign DNA may be directed into specific regions of the AcMNPV genome by 
flanking the foreign DNA with viral sequences that are homologous to the 
targeted region. The foreign DNA is inserted into the targeted region by 
homologous recombination. To construct transfer vectors that will direct 
homologous recombination to the 13.2 map-unit region of the AcMNPV genome, 
a 4.3 kb Bgl II fragment derived from the EcoR1-A fragment was cloned into 
a pUC9 plasmid vector. A unique XbaI site located in this fragment is used 
for the insertion of foreign DNAs so that 2.4 kb and 1.9 kb of EcoR1-A 
sequence flank the 5' and 3' ends of the inserted DNA. 
Expression of foreign genes may be temporally regulated by using viral 
promoters which are active at different times during the course of 
infection. To express proteins both early and late in infection, the 
immediate early IE1 promoter and the very late polyhedrin promoter were 
chosen for the new transfer vector constructs. 
EXAMPLE 3 
Construction of Transfer Vector RIA-1392 
FIG. 3 depicts transfer vector RIA-1392. For late expression, foreign genes 
which contain a translation start signal may be inserted into the Bgl II 
site of RIA-1392. The line drawing indicates that RIA-1392 will 
homologously recombine into the non-essential 13.0 map unit region of 
Fragment A of the AcMNPV genome. 
RIA-1392 contains a unique Bgl II site for the insertion of foreign genes 
under the control of the polyhedrin promoter. Sequences containing a 
modified polyhedrin promoter, a portion of the polyhedrin open reading 
frame, and the polyhedrin polyadenylation signal are inserted at the Xbal 
site in the transfer vector construct designated "RIA-1392." RIA-1392 
contains a unique Bgl II cloning site for the insertion of foreign genes 
located at +35 in relation to a mutated polyhedrin translation start 
signal. RIA-1392 also contains the B-glucoronidase gene under the control 
of the polyhedrin promoter for use as a selectable marker. This transfer 
vector contains 1.9 kb (PstI-XbaI 1.9 kb non-essential region of AcMNPV 
genome at 13.4 map unit of Eco-RI-A viral flanking sequences) and 2.4 kb 
(Bam HI-XbaI non-essential region of AcMNPV genome at 13.4 map units of 
EcoR1-A viral flanking sequences) of Eco-RI-A viral flanking sequences. 
The cloning steps for construction of this vector are: 
1. Subclone the 4.3 kb BGIII fragment of the AcMNPV EcoRI-A region into 
BamH1-digested pUC9. This construct is designated pUC9/RIA-BgIII. 
2. Mutate the Xbal site of pVLI392 by digesting with Xbal, filling in with 
Klenow, and religating. The resulting construct is designated 1392(-Xba). 
3. Partially digest 1392(-Xba) with Hindlll and completely digest with 
EcoRV. Isolate the 1.1 kb HindIII-EcoRV fragment, which contains the 
polyhedrin promoter, a polylinker for the insertion of foreign genes 
located 35 bp downstream from a mutated translation start signal, a 
portion of the polyhedrin open reading frame, and a polyadenylation 
signal. Insert this fragment into HindIII and Hindll-digested Bluescript. 
The resulting construct is designated Bst/1392. 
4. Insert the 2.2 kb BamHI fragment from pVLIO62, which contains the 
13-glucuronidase open reading frame, into the Bgl II site of Bst/1392. A 
subclone containing the B-glucoronidase insert in the proper orientation 
in relation to the polyhedrin promoter is designated Bst/1392-Bgluc. 
5. In a three-way ligation, combine the .about.1.1 kb Xbal-Apal fragment of 
Bst/1392, the .about.3.3 kb Spe-ApaI fragment of Bst/1392-Bgluc and 
Xbal-digested pUC9/RIA-BgIII. The resulting construct is designated 
RIA-1392. 
The direction in which the promoter directs transcription of the adjacent 
gene is indicated by arrows. 
To facilitate the selection and purification of recombinant occ+ 
baculoviruses, the gene encoding B-glucuronidase (Jefferson, Burgess, and 
Hirsch, 1986, pNAS 83:8447-8451) under the control of the polyhedrin 
promoter is inserted into the transfer vectors in the opposite orientation 
to the foreign gene. Because the foreign gene is on the same plasmid as 
B-glucuronidase, cells which acquire the foreign gene will also acquire 
the marker gene. Thus, the addition of the chromogenic dye 
5-bromo-4-chloryl-3-indolyl-B-D-galactopyranoside (X-gal) may be used to 
identify recombinant plaques in a baculovirus plaque assay. 
EXAMPLE 4 
Construction of Transfer Vector RIA-IEI 
Construction of transfer vector RIA-IE1 is depicted in FIG. 4. For 
immediate early expression, foreign genes which contain a translation 
start signal may be inserted into the Bgl II site of RIA-IE1. The line 
drawing indicates that RIA-IE1 will homologously recombine into the 
non-essential 13.0 map unit region of Fragment A of the AcMNPV genome. 
Sequences containing a modified IE1 promoter, a portion of the IE1 open 
reading frame, and the IE1 polyadenylation signal are inserted at the XbaI 
site in the transfer vector construct designated "RIA-IEI." RIA-IE1 
contains a unique Bgl II site for the insertion of foreign genes under the 
control of the IE1 promoter. Foreign genes are inserted at -39 in relation 
to the IEI translation start ATG. RIA-IE1 also contains the 
B-glucoronidase gene under the control of the polyhedrin promoter for use 
as a selectable marker. This transfer vector contains 1.9 kb (PstI-XbaI 
1.9 kb non-essential region of AcMNPV genome at 13.4 map unit of Eco-RI-A 
viral flanking sequences) and 2.4 kb (Bam HI-XbaI non-essential region of 
AcMNPV genome at 13.4 map units of EcoR1-A viral flanking sequences) of 
Eco-RI-A viral flanking sequences. 
The cloning steps for construction of this vector are: 
1. Isolate the .about.1.8 kb BamHI-SpeI fragment from IE1(-)39Bgl II. This 
fragment contains a modified IE1 promoter containing a Bgl II site 35 bp 
upstream from the IE1 translation start signal, a portion of the IE1 open 
reading frame, and a polyadenlyation signal. Subclone this fragment into 
BamHI and SpeI digested Bluescript. The resulting construct is designated 
Bst/IE1. 
2. Replace the 1.1 kb Apa-SpeI fragment of RIA-1392 with the 1.8 kb Apa-Spe 
fragment from Bst/IE1. The resulting construct is designated RIA-IE1. 
The direction in which the promoter directs transcription of the adjacent 
gene is indicated by arrows. 
To facilitate the selection and purification of recombinant occ+ 
baculoviruses, the gene encoding B-glucuronidase (Jefferson, Burgess, and 
Hirsch, 1986, PNAS 83:8447-8451) under the control of the polyhedrin 
promoter is inserted into the transfer vectors in the opposite orientation 
to the foreign gene. Because the foreign gene is on the same plasmid as 
B-glucuronidase, cells which acquire the foreign gene will also acquire 
the marker gene. Thus, the addition of the chromogenic dye 
5-bromo-4-chloryl-3-indolyl-B-D-galactopyranoside (X-gal) may be used to 
identify recombinant plaques in a baculovirus plaque assay. 
EXAMPLE 5 
Construction of Transfer Vector RIA-39K 
The construction of transfer vector RIA-IE1 is depicted in FIG. 5. The line 
drawing indicates that RIA-39K will homologously recombine into the 
non-essential 13.0 map unit region of Fragment A of the AcMNPV genome. 
RIA-39K contains a unique Bgl II site for the insertion of foreign genes 
under the control of the 39K promoter. Foreign genes are inserted at -80 
in relation to the 39K translation start codon. RIA-39K also contains the 
B-glucoronidase gene under the control of the polyhedrin promoter for use 
as a selectable marker. This transfer vector contains 1.9 kb (PstI-XbaI 
1.9 kb non-essential region of AcMNPV genome at 13.4 map unit of Eco-RI-A 
viral flanking sequences) and 2.4 kp (Bam HI-XbaI non-essential region of 
AcMNPV genome at 13.4 map units of EcoR1-A viral flanking sequences) of 
Eco-RI-A viral flanking sequences. The direction in which the promoter 
directs transcription of the adjacent gene is indicated by arrows. 
The cloning steps for construction of this vector are: 
1. Digest 39CAT with BamHI, fill-in with Klenow, and ligate to Bgl II 
linkers. Digest the resulting DNA with Bgl II and FspI. Isolate the 330 bp 
Bgl II-Fsp fragment, which contains a modified 39K promoter and 3' viral 
sequences. Excise the ApaI-Bgl II fragment from RIA- 1392 and replace it 
with the 330 bp 39K fragment. 
To facilitate the selection and purification of recombinant occ+ 
baculoviruses, the gene encoding B-glucuronidase (Jefferson, Burgess, and 
Hirsch, 1986, PNAS 83:8447-8451) under the control of the polyhedrin 
promoter is inserted into the transfer vectors in the opposite orientation 
to the foreign gene. Because the foreign gene is on the same plasmid as 
B-glucuronidase, cells which acquire the foreign gene will also acquire 
the marker gene. Thus, the addition of the chromogenic dye 
5-bromo-4-chloryl-3-indolyl-B-D-galactopyranoside (X-gal) may be used to 
identify recombinant plaques in a baculovirus plaque assay. 
EXAMPLE 6 
Construction of Transfer Vector RIA-1392CAT 
The construction of transfer vector RIA-1392CAT is depicted in FIG. 6. The 
line drawing indicates that RIA-1392CAT will homologously recombine into 
the non-essential 13.0 map unit region of Fragment A of the AcMNPV genome. 
The direction in which the promoter directs transcription of the adjacent 
gene is indicated by arrows. 
Construct RIA-1392CAT contains the chloramphenicol acetyl transferase gene 
(CAT) under the control of one polyhedrin gene and the b-glucoronidase 
gene under the control of a second polyhedrin gene for use as a selectable 
marker. This transfer vector contains 1.9 kb (PstI-XbaI 1.9 kb 
non-essential region of AcMNPV genome at 13.4 map unit of Eco-RI-A viral 
flanking sequences) and 2.4 kp (Bam HI-XbaI non-essential region of AcMNPV 
genome at 13.4 map units of EcoR1-A viral flanking sequences) of Eco-RI-A 
viral flanking sequences. 
Initial studies to examine the ability of this transfer vector to direct 
the insertion of foreign genes into the 13 map unit region of the AcMNPV 
genome, and to evaluate the stability of recombinant viruses obtained from 
these transfer vectors are planned. The expression level produced by these 
transfer vectors will be compared to the expression obtained from 
conventional transfer vectors (i.e. pVL941) using the reporter gene CAT. 
To facilitate the selection and purification of recombinant occ+ 
baculoviruses, the gene encoding B-glucuronidase (Jefferson, Burgess, and 
Hirsch, 1986, pNAS 83:8447-8451) under the control of the polyhedrin 
promoter is inserted into the transfer vectors in the opposite orientation 
to the foreign gene. Because the foreign gene is on the same plasmid as 
B-glucuronidase, cells which acquire the foreign gene will also acquire 
the marker gene. Thus, the addition of the chromogenic dye 
5-bromo-4-chloryl-3-indolyl-B-D-galactopyranoside (X-gal) may be used to 
identify recombinant plaques in a baculovirus plaque assay. 
EXAMPLE 7 
Construction of Vector RIA-IE1CAT 
The construction of transfer vector RIA-IE1CAT is depicted in FIG. 7. The 
line drawing indicates that RIA-IE1CAT will homologously recombine into 
the non-essential 13.0 map unit region of Fragment A of the AcMNPV genome. 
The direction in which the promoter directs transcription of the adjacent 
gene is indicated by arrows. 
Construct RIA-IE1CAT contains the chloramphenicol acetyl transferase gene 
(CAT) under the control of the IE1 promoter and the B-glucoronidase gene 
under the control of the polyhedrin promoter for use as a selectable 
marker (FIG. 7). This transfer vector contains 1.9 kb (PstI-XbaI 1.9 kb 
non-essential region of AcMNPV genome at 13.4 map unit of Eco-RI-A viral 
flanking sequences) and 2.4 kp (Bam HI-XbaI non-essential region of AcMNPV 
genome at 13.4 map units of EcoR1-A viral flanking sequences) of Eco-RI-A 
viral flanking sequences. Linear representation of vector RIA-IE1CAT is 
shown in FIG. 8. 
Initial studies to examine the ability of these transfer vectors to direct 
the insertion of foreign genes into the 13 map unit region of the AcMNPV 
genome, and to evaluate the stability of recombinant viruses obtained from 
these transfer vectors are planned. The expression level produced by these 
transfer vectors will be compared to the expression obtained from 
conventional transfer vectors (i.e. pVL941) using the reporter gene CAT. 
To facilitate the selection and purification of recombinant occ+ 
baculoviruses, the gene encoding B-glucuronidase (Jefferson, Burgess, and 
Hirsch, 1986, PNAS 83:8447-8451) under the control of the polyhedrin 
promoter is inserted into the transfer vectors in the opposite orientation 
to the foreign gene. Because the foreign gene is on the same plasmid as 
B-glucuronidase, cells which acquire the foreign gene will also acquire 
the marker gene. Thus, the addition of the chromogenic dye 
5-bromo-4-chloryl-3-indolyl-B-D-galactopyranoside (X-gal) may be used to 
identify recombinant plaques in a baculovirus plaque assay. 
EXAMPLE 8 
Homologous Recombination of a Desired Recombinant Baculovirus Transfer 
Vector into the 13.0 Map Unit Nonessential Region of Wild Type Baculovirus 
FIG. 9 diagrams the homologous recombination of a desired recombinant 
baculovirus transfer vector into the 13.0 map unit nonessential region of 
wild type baculovirus. The construction of different bacterial transfer 
vectors are described in the examples above. The transfer vectors 
described above (RIA-1392, RIA-IE1, RIA-39K, RIA-1392 CAT, RIA-IE1 CAT) 
are used as examples only. Any other desired promoter and gene complex may 
be employed with equal success. The polyhedrin-beta-glucoronidase 
containing recombinant baculovirus produces blue plaques. This selectable 
marker allows for easy identification of desired recombinants (via 
observing blue plaques). Once Sf9 cells are infected with the desired 
recombinant, this desired recombinant may further be propagated, isolated, 
and purified. These protocols are known and standard for one of ordinary 
skill in the art. 
EXAMPLE 9 
Genetic Engineering of a Non-Replicative Virus or Virus Hybrid Using the 
Baculovirus as a Tool 
Viral Infections and Vaccines 
The number of known viral infections that infect humans and animals is too 
numerous to list. The necessity to control or abate viral infections has 
been a major medical concern for many decades. Basically, the eradication 
and/or control of viral infections involves the production of a variety of 
different and highly potent vaccines. Quantitative production of pure and 
potent viral antigens or immunogens is difficult as well as expensive. 
This is usually due to the fact that many viruses replicate very slowly, 
if at all, in controlled cell culture environments. If the virus of choice 
seldom replicates in the test tube, then the production of the specific 
viral particle becomes less favorable and effective as well as more 
expensive. 
The classical method for producing vaccines against viral pathogens is to 
"attenuate" the virus. Attenuation is a process of diminution of viral 
virulence in an organism. This diminution is generally obtained through 
the selection of variants which occur naturally or through experimental 
manipulation. With attenuation, the virus remains infective and 
self-replicative but does not cause the symptomatic disease. Because 
attenuation does not generally alter the replicative capacity of the 
virus, drawbacks arise. Attenuation is therefore not favored because the 
introduction of live attenuated vaccines into humans, as well as are the 
risks involved in handling the attenuated vaccines, may be hazardous. 
Another approach for the production of viral antigen and/or immunogen is to 
synthesize single proteins of the derived viral particle or portions 
thereof. Thus, the aim is to elicit an appropriate immune response or 
reaction without the danger of viral replication (viremia). The production 
of so called "Subunit" vaccines or diagnostic reagents has been achieved 
by using a variety of different expression vector systems as well as by 
chemical synthesis of the corresponding peptide sequences of importance 
(putative antigenic or immunogenic epitopes). However, subunit vaccines 
are for most of the part much less potent and less protective as compared 
to attenuated whole virus particles. 
Thus, there is a strong need for the production of vaccines and diagnostics 
that are potent, safe to use and also economical to produce. Therefore, it 
is the intent of this invention to produce vaccines and diagnostics which 
are potent, safe to use as well as economical to produce, thereby filling 
the void in the area of clinical medicine and research. Production of 
potent vaccines is a necessity for this field to advance. The synthesized 
vaccine must closely represent the authentic pathogen (e.g. mimic the 
virus particle) without being harmful in clinical use. These 
characteristics are naturally advantageous also when diagnostic reagents 
are concerned. 
Genetic Engineering of a Non-Replicative Virus Using the Baculovirus as a 
Tool 
FIG. 10A depicts the construction of a non-replicative baculovirus virus 
wherein the non-structural genes are truncated or mutated. The source of 
the viral genome structural and non-structural genes are the same. Sindbis 
virus is used as an example only. The non-structural genes are mutated or 
truncated in such a manner that capsid formation is possible whereas the 
structural proteins are intact (capsid, E1, E2). 
Wild-type replicative Sindbis RNA is represented by a darker line as 
compared to the line representing truncated non-replicative Sindbis RNA. 
The structural Sindbis proteins E1 and E2 are represented by large open 
circles. Since the respective proteins are successively synthesized, and 
therefore temporally regulated, the truncated non-replicative 
non-structural RNA, whose synthesis is driven by an early baculovirus 
promoter, is synthesized prior to the structural proteins (whose synthesis 
is driven by a late baculovirus promoter). This temporal regulation of 
transcription allows for the proper assembly of the virus particle 
constructed from one recombinant baculovirus. 
Strategy for Synthesizing a Non-Replicative Sindbis Virus Particle in an 
Insect Cell Using Baculovirus Vectors 
The genes encoding the non-structural proteins (ns) have to be modified to 
either inactive the translation protein products or simply alter the 
translation start site of the intact messenger by mutation (e.g. mutation 
of the AUG). An example of inactivation would be deletion of essential 
sequences within single genes. This is merely one example and those 
skilled in this art know that alternate methods exist. although The latter 
possibility would make an almost identical genome as compared to the 
wild-type but the messenger can not be translated. Consequently, 
non-structural proteins would not be synthesized and the genome would be 
non-replicative. In addition the RNA sequence of the genome specifying the 
26S subgenomic RNA, i.e.d the coding sequence for the structural proteins 
would be deleted. The modifications, however, have to be made so that the 
capsid protein still is capable of forming the complex with the "remaining 
mutated genomic RNA", formation of the capsid itself, in order for the 
final virus particle to be assembled. The result is a piece of genomic RNA 
that can not replicated within the cell but still has the properties 
needed for capsid formation. 
The "remaining genomic cDNA" is placed under the transcriptional regulation 
of e.g. the baculovirus IE1 promoter and the complete 26S cDNA is driven 
by the baculovirus polyhedrin gene promoter. Thus the result is a Sindbis 
virus particle which is synthesized during recombinant baculovirus 
infection. The particle has the same antigenic properties as its authentic 
infectious counterpart, but can not replicate in the cell. 
Genetic Engineering of a Non-Replicative Virus Hybrid Using the Baculovirus 
as a Tool 
FIG. 10B HYBRID is a schematic representation of constructing a 
non-replicating virus hybrid. The non-replicative aspect results from 
truncating the RNA encoding the non-structural genes. The virus hybrid is 
constructed by producing non-structural genes from Virus A and structural 
genes from Virus B. Truncated Sindbis non-structural genes and structural 
Rubella genes are used as examples only. 
As depicted in FIG. 10B HYBRID, the non-structural genes are from Sindbis 
virus and the structural genes are from Rubella virus. The Sindbis 
non-structural genes are mutated or truncated in such a manner that 
encapsidation is possible. The Sindbis capsid protein is represented by 
small closed circles. The structural genes encoding for the Sindbis capsid 
protein and Rubella virus envelope glycoproteins remain intact, while the 
genomic RNA is genetically mutated or truncated by standard techniques 
known to those skilled in the art. Wild-type replicative Sindbis RNA is 
represented by a darker line as compared to the line representing 
truncated non-replicative Sindbis RNA. 
The structural Sindbis envelope proteins E1 and E2 are represented by large 
open circles whereas the corresponding envelope proteins of Rubella virus 
are illustrated by large shaded circles thereby generating what is termed 
the virus hybrid particle. Since the respective proteins are temporally 
regulated, and therefore successively synthesized, the truncated 
non-replicative Sindbis non-structural RNA, whose synthesis is driven by 
an early baculovirus promoter, is synthesized prior to the structural 
Sindbis and Rubella proteins (whose synthesis is driven by a late 
baculovirus promoter). This temporal regulation of transcription allows 
for the proper assembly of the virus hybrid particle comprised of 
non-structural genes from Sindbis virus and Rubella virus structural 
genes. 
The novel construction of a non-replicative virus hybrid offers the 
following advantages: Abundant production of potent non-infectious viral 
antigen (in this case Rubella) to be used as a vaccine or as a diagnostic 
reagent. 
Other potential candidates for combinations to produce a variety of virus 
hybrids, for subsequent vaccine production, include Rubella virus, 
poliovirus, bluetongue virus, hepatitis B, HIV, etc. Some of these viruses 
will be briefly outlined below. This brief list is not meant to be 
inclusive nor limiting. 
Sindbis Virus 
Sindbis virus (SV) is a small RNA virus (about 12 kb) that belongs to the 
Alphavirus genus within the Togaviridae family. The nucleocapsid 
containing the single stranded viral RNA complexed with a basic capsid 
protein (C) is surrounded by a lipid bilayer containing two integral viral 
envelope spike glycoproteins designated E1 and E2. The translation of 
these structural proteins is initiated at a single site on a subgenomic 
26S messenger RNA. The features of this virus are comprehensively studied 
at the molecular level (Schlesinger and Schlesinger, 1986) and was 
therefore chosen to serve as an example. 
Rubella Virus 
Rubella virus (RV), a major human pathogen, is the single member of the 
Rubivirus genus within the Togaviridae family (Porterfield et al, 1978 
reference from Rubella paper). The virion contains three structural 
proteins E1, E2 and C. The capsid protein (C) is associated with the 
single stranded 40S genomic RNA. The E1 and E2 envelope glycoproteins form 
the viral spikes. 
A cloned cDNA encoding the RV envelope glycoproteins E1 and E2 was used to 
study the ability of infected Spodoptera frugiperda (Sf9) cells to 
synthesize and process these glycoproteins. Oker-Blom et al (Virology, 
172:82-91, 1989) demonstrated that Sf9 cells infected with the recombinant 
baculovirus synthesize polypeptides of the size and the antigenicity 
similar to those isolated from RV particles grown in VERO cells. These 
results clearly indicate that the BEVS is a good candidate vector for the 
abundant expression of recombinant RV structural glycoproteins. 
Poliovirus 
Urakawa et al (1989) demonstrated the synthesis of immunogenic, but 
non-infectious, poliovirus (PV) particles in insect cells by employing a 
baculovirus expression vector driven by the polyhedrin gene promoter. As 
demonstrated by use of the appropriate antibodies, infected insect cells 
made poliovirus proteins that included the structural proteins VP0, VP1 
and VP3. These data suggested that processing of the poliovirus gene 
product by the AcLeon construct was catalyzed by the poliovirus - encoded 
proteases. These data demonstrate that antigenic and immunogenic 
poliovirus proteins and empty particles can be made in insect cells by 
recombinant baculoviruses. 
Bluetongue Virus 
Bluetongue virus is a prototype virus of the Orbivirus genus (Reoviridae 
family). The virus contains a genome consisting of 10 double-stranded RNA 
molecules (segments) each of which is unique and is believed to code for a 
single polypeptide product. Bluetongue virus proteins have been produced 
in an Sf9-baculovirus expression system. DNA sequences corresponding to 
the gene that codes for the bluetongue virus neutralization antigen VP2 
and for the group-specific antigen VP3 have been inserted (under the 
transcriptional regulation of the polyhedrin gene promoter) into a 
baculovirus transfer vector and expressed (European Patent Application 
#0279661, Bishop and Roy). 
Hepatitis B Virus 
Bishop and Kang described the expression of human hepatitis B virus 
antigens in insects and in cultured insect cells. Permissive insects and 
cells were infected with recombinant baculoviruses that have the requisite 
human hepatitis B virus genes inserted into the baculovirus genome under 
the control of the baculovirus polyhedrin gene promoter and in lieu of the 
initial 5' coding sequences of the viral polyhedrin protein (European 
Application #0260090). 
EXAMPLE 10 
Homologous Recombination of a Non-Replicative Sindbis Virus into the 13.0 
Map Unit Nonessential Region of Wild Type Baculovirus 
FIG. 11 depicts one of the many different kinds of virus or vaccines that 
can be produced with this specifically designed vector. 
The Sindbis virus genomic RNA may be mutated, truncated or both, all in a 
manner that makes it non-replicative. However, the regions in the RNA that 
are important for encapsidation must remain intact. Oker-Blom and Summers 
(Journal of Virology, 63:3, 1256-1264, (1988)) used cloned cDNA for 
Sindbis virus 26S, encoding the structural proteins of the virus, to study 
protein processing of the Sindbis virus polyprotein in Sf9 cells. The 
transcription of the 26S mRNA, which normally occurs in the cytoplasm of 
Sindbis virus infected vertebrate cells, takes place in the nucleus of 
baculovirus infected invertebrate cells. The authors demonstrated that Sf9 
cells infected with a recombinant baculovirus synthesize polypeptides that 
are similar to those synthesized in Sindbis virus infected BHK cells. The 
results thus show that Sindbis virus structural proteins that normally are 
encoded by non-nuclear RNAs are expressed and proteolytically processed 
similarly, if not identically, in Sf9 cells and BHK cells. 
Homologous Recombination of a Non-Replicative Sindbis Virus into the 13.0 
Map Unit Nonessential Region of Wild Type Baculovirus 
FIG. 11 diagrams the homologous recombination of a non-replicative Sindbis 
virus into the 13.0 map unit nonessential region of wild type baculovirus. 
Truncated genomic non-structural Sindbis virus cDNA and 26S are used as an 
example only. This is achieved by cloning a mutant (truncated) 
non-structural Sindbis virus gene under the control of an IE1 early 
baculovirus promoter (into the unique Bgl-II site adjacent and in the 
proper orientation to the IE1 promoter). The completed bacterial transfer 
vector is generated in a two-step fashion. First the truncated Sindbis 
virus non-structural gene is inserted under the control of an early 
baculovirus promoter (for example, IE1; FIG. 11). The next step involves 
inserting genes encoding intact structural Sindbis viral 26S proteins 
under the transcriptional regulation of a late baculovirus gene promoter 
(for example, polyhedrin). This insertion is performed on the recombinant 
baculovirus which already contains non-structural genes under the 
regulation of an early baculovirus gene promoter. 
Mutant Sindbis viral non-structural genome is inserted 3' and therefore 
under the control of the IE1 early baculovirus promoter. The 
non-structural Sindbis viral genome must first be truncated, mutated or 
both prior to replacing the B-galactosidase gene. Truncation or mutation 
is achieved with standard methodology known to those skilled in the art. 
This is done in a way that makes the non-structural protein products 
inactive, thus, prohibiting replication of Sindbis genomic RNA by means of 
self replication (the non-structural proteins are responsible for 
replication of genomic Sindbis virus 49S RNA). 
This new transfer vector is then used for transfection into Lepidopteran 
insect Sf9 cells together with wild type baculovirus DNA. Homologous 
recombination occurs at the non-essential 13.0 map unit region of the 
AcMPNV genome. Isolation, purification, and propagation of the 
extracellular recombinant baculovirus, containing the truncated 
non-structural Sindbis virus genes under the control of the early IE1 
baculovirus promoter and Sindbis virus 26S structural gene under the 
control of the polyhedrin promoter, is achieved by employing standard 
protocols known to those skilled in the art. 
These constructs are then be selected by employing the pVL1392/1393 vector 
which allows for insertion by homologous recombination into the Eco-RI-I 
fragment. The desired constructs are then verified by Southern 
hybridization with an appropriate piece of DNA. 
During the baculovirus infection, because of the temporal transcriptional 
regulation of the genes, the mutated or truncated non-replicative genomic 
RNA of the Sindbis virus, which is under the control of an early 
baculovirus gene promoter, is therefore synthesized before the structural 
proteins, which are under the control of the late polyhedrin gene 
promoter. The capsid (C) protein, is expressed from the 26S cDNA and able 
to form nucleocapsids together with the truncated genomic RNA. The 
nucleocapsids become enveloped at the plasma membrane by the process of 
budding (a natural result of Sindbis virus infection). The budded 
particles can be found in the extracellular space of the recombinant 
baculovirus infected Sf9 cells and can be purified by conventional methods 
known to those skilled in the art. 
While the compositions and methods of this invention have been described in 
terms of preferred embodiments, it will be apparent to those of skill in 
the art that variations and modifications may be applied to the 
composition, methods and in the steps or in the sequence of steps of the 
methods described herein without departing from the concept, spirit and 
scope of the invention. More specifically, it will be apparent that 
certain agents which are both chemically and physiologically related may 
be substituted for the agents described herein while the same or similar 
results would be achieved. The method outlined in the Examples presented 
supra describe constructing a genetically engineered recombinant 
baculovirus capable of expression and assembly of non-replicative Sindbis 
virus particles in Sf9 cells. Sindbis virus serves only as an example. 
Thus, this invention allows for potential abundant production of human, 
animal or plant pathogens that can not replicate but which do posses a 
morphology (molecular structure) and immunogenicity similar or identical 
to their authentic counterparts. All such similar substitutes and 
modifications apparent to those skilled in the art are deemed to be within 
the spirit, scope and concept of this invention as defined by the appended 
claims.