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Introduction
Baculoviruses make up a family of
viruses and are grouped into nuclear polyhedrosis viruses (NPV) and
granulosis viruses. More than 500 different types of baculoviruses
have been discovered and the host range is restricted to
invertebrates, mostly to insects (eg, moths and butterflies); thus,
several baculoviruses (eg, Helicoverpa zea, Orgya
pseudotsugata, and Lymantria dispar) have been registered
for use as biological pesticides by the US Environmental Protection
Agency[1]. Among the numerous baculoviruses,
Autographa californica multiple NPV (AcMNPV) is the most well
studied and most extensively used. AcMNPV has a circular
double-stranded DNA genome of approximately 130 kb, which is
condensed with a protamine-like protein into the core and packed
into the nucleocapsids. Nucleocapsids are synthesized in the nucleus
of infected cells (typically 40 nm-50 nm in diameter and
200 nm-400 nm in length). Membrane-enveloped nucleocapsids are
referred to as virus particles or virions. In nature, AcMNPV are
occluded in a polyhedron (2 mm-15 mm in size) mainly consisting of
polyhedrin protein. After ingestion by insects, the polyhedrin
matrix is dissolved in the alkaline midgut, thus releasing the
embedded virions, which subsequently infect the epithelial cells of
the intestine. Early in the infection cycle, the DNA genome is
replicated and transcribed in the nucleus and the nucleocapsids are
assembled. The budding of nucleocapsids through plasma membrane
results in the release of budded virus, which is responsible for
systemic transmission within an infected insect. Late in the
infection cycle, progeny nucleocapsids become membrane-bound within
the nucleus and are embedded into the polyhedra. After cell death
and lysis, the polyhedra are released in the wild to spread the
infection. Budded virus is highly infectious to cultured insect
cells and is the primary form used in the laboratory as an
expression vector.
Baculovirus infection of insect
cells
The baculovirus has been widely used
for the production of numerous recombinant proteins in insect cells[2-4]
because it has the following advantages: (i) proper
post-translational modification, because insect cells are higher
eukaryotes; (ii) a high capacity for multiple genes or a large
insert, because of the huge and flexible viral genome (130 kb);
(iii) biosafety, because baculovirus naturally does not infect
humans; and (iv) a very high yield driven by the strong promoters
polyhedrin or p10. Among the natural host cells, Sf-9 and Sf-21,
derived from the ovarian tissue of Spodoptera frugiperda, and
BTI-TN-5B1-4 (High-Five; Invitrogen, Carlsbad, CA, USA), derived
from Trichoplusia ni, which is suitable for the expression of
secreted proteins[5], are most popular. In addition to
cultured cell lines, insect larvae are also excellent hosts for
protein production. For example, T ni insect larvae can be
readily infected by recombinant AcMNPV[6,7], and
silkworms can be readily infected by recombinant Bombyx mori
NPV[8]. Both systems have been employed for the
production of numerous recombinant proteins[6,9]. The
products synthesized in insect larvae are almost identical to those
produced in insect cells and mammalian cells and the yields are
generally very high (eg 13 mg luciferase per larva[10]).
Therefore, baculovirus infection of live insects may represent an
inexpensive and rapid approach for protein production[11],
but consistent infection and qua-lity control are more difficult to
achieve.
The large cloning capacity renders
baculovirus an ideal tool for the synthesis of virus-like particles
(VLP), which generally require simultaneous expression of multiple
viral structural proteins for self-assembly. VLP are empty particles
composed of viral structural proteins but devoid of viral nucleic
acids; thus, they are non-infectious. VLP can generally induce broad
and strong immune responses thanks to the preservation of many
essential epitopes[12,13]; therefore, VLP have gained
increasing attention as potential vaccine candidates[14,15]
or diagnostic reagents[16]. Extended applications of VLP
as gene delivery vehicles have also been reported[17,18].
To date, numerous VLP of different viruses, including HIV[19],
herpes simplex virus[20], human papilloma-virus[21],
polyomavirus[22], parvovirus[23], infectious
bursal disease virus[24], hepatitis C virus (HCV)[16],
and enterovirus 71[25], have been expressed using the
baculovirus/insect cell expression system. Very recently, the
synthesis of severe acute respiratory syndrome coronavirus (SARS-CoV)
VLP using the baculovirus/insect cell system has also been reported[26,27].
The assembly of VLP using baculovirus enables the investigation of
virus assembly process as well as the minimal requirements for viral
capsid assembly. For example, Mortola and Roy demonstrated that
coexpression of SARS-CoV envelope and membrane proteins is required
and sufficient for the assembly of spikeless VLP[27], yet
additional coexpression of the spike (S) protein results in the
incorporation of the S protein. The resultant VLP possesses
distinctive spike projections, making it morphologically
indistinguishable from the authentic virus. Through co-infection
with different recombinant baculoviruses, one can manipulate the
composition of the particles, obtaining higher specific
immunogenicity[28], or other biological functions by
incorporating enzymes[29] or affinity tags for downstream
purification[24]. In this regard, Conner et al
successfully demonstrated chimeric VLP formed by structural proteins
of viruses isolated from different animals (bovine and simian)[30].
By including viral proteins from different strains, referred to as
phenotypic mixing, one can broaden the spectrum of the chimeric
particles as a vaccine[31].
There are drawbacks associated with
the baculovirus/insect cell expression system. First, baculovirus
infection ultimately results in cell death and lysis in a few days.
Because the expression driven by a polyhedrin or p10 promoter
usually reaches the maximum near the death of infected cells,
protein processing is likely to be suboptimal at that time because
of the compromise of post-translational machinery and secretory
pathway. This may seriously affect the processing of proteins
destined for plasma membrane or for secretion. Efforts have been
directed towards alleviating this problem by the use of early
baculovirus promoters (eg, IE1) in either transiently or stably
transformed cells[32]. The expression driven by early
promoters is continuous and stable, and confers more efficient
processing of the glycoprotein (eg tissue plasminogen activator),
but the expression level is lower than those obtained with the lytic
baculovirus system. Such a system is a feasible approach for
expression in insect cells for products that are relatively poorly
processed in lytic insect cells, and may be useful for functional
studies of recombinant receptor proteins. To circumvent the protein
degradation problem associated with cell lysis, a novel non-lytic
baculovirus has recently been developed by random mutagenesis of
viral genomes[33] and subsequent selection. At 5 d
post-infection, the non-lytic baculovirus shows only 7% cell lysis,
in contrast to 60% cell lysis by lytic baculovirus. This system is
thus a convenient alternative for the production of proteins
vulnerable to degradation using lytic baculoviruses.
The second disadvantage is that
glycosylation in insect cells differs in many aspects from in
mammalian cells. For example, truncated oligosaccharides containing
just 3 or even 2 mannose residues and sometimes fucose have been
found on proteins expressed in insect cells. Besides, there appears
to be no significant sialylation of N-glycans in insect cells[34].
The glycoproteins lacking sialic acids have extremely short
half-lives in vivo; therefore, the truncated N-glycans
of glycoproteins produced in insect cells constitute a barrier to
their use as therapeutics. One example to engineer the
N-glycosylation pathway is the construction of transformed
Sf-9 cells that constitutively express bovine b1,
4-galacto-syltransferase under hr5-ie1 control[35,36].
Baculo-virus infection of this cell line results in the production
of terminally galactosylated recombinant proteins. Other attempts
and strategies to "mammalianize" or "humanize" the
N-glycosylation capacity of insect cells have been reviewed
previously[37-39]. Another limitation of the baculovirus/insect
cell system is its inefficiency to properly process proteins that
are initially synthesized as large inactive precursor proteins, such
as peptide hormones, matrix metalloproteases, and fusogenic viral
envelope glycopro-teins. A family of mammalian proprotein
convertases has been characterized to be involved in the cleavage,
and it has been shown that coexpression of the prohormone convertase
furin with transforming growth factor b1 (TGF b1) results in a
7.8-fold increase in the production of mature TGF b1[40].
Baculovirus transduction of
mammalian cells
Baculovirus was originally regarded
as infecting solely insects and invertebrates; but in 1983 Volkman
and Goldsmith found that baculovirus can be internalized by
non-target vertebrate cells (eg, human lung carcinoma cell line
A427)[41]. Carbonell et al further confirmed the
entry of baculovirus into mammalian cells and very low level
reporter gene expression under the control of polyhedrin and Rous
sarcoma virus (RSV) promoters in mammalian cells[42].
However, virus replication was not detected in either study. Their
discovery was not widely noted until 10 years later when two pioneer
groups reported that recombinant baculoviruses harboring a
cytomegalovirus (CMV) promoter-luciferase gene cassette[43]
or an RSV long terminal repeat (LTR) promoter-b galactosidase
(b-gal) gene cassette[44] can efficiently transduce
mammalian cells. Their data indicate a strong preference for
baculovirus to enter hepatocytes of different origins, and suggest
that the block to expression in less susceptible cells does not
appear to result from the ability to be internalized by the target
cells but rather by events subsequent to viral entry because high
and low expressing cell lines internalize similar amounts of virus[44].
One factor accounting for the low apparent transduction efficiency
in other cell types is promoter strength. Shoji et al showed
that cells that are not transduced by a baculovirus expressing b-gal
under the control of a CMV promoter can be efficiently transduced by
a baculovirus expressing the same reporter protein under the
transcriptional control of a stronger CAG promoter[45] (a
composite promoter consisting of the CMV immediate early enhancer,
chicken b-actin promoter and rabbit b-globin polyadenylation
signal). Thus, it is of interest to examine different promoters of
viral and cellular origins in the baculovirus context in mammalian
cells. Following these findings, subsequent studies have rapidly
expanded the list of permissive cells that include cell lines
originating from human (eg, HeLa, Huh-7, HepG2, keratinocytes, and
bone marrow fibroblasts), rodent (eg, CHO, BHK), porcine (eg, CPK,
PK-15), bovine (eg, BT)[46], and even fish sources (eg,
EPC, CHH-1)[47]. Besides, baculovirus is capable of
transducing non-dividing cells[48]. Transduction of
primary cells, such as human neural cells[49], pancreatic
islet cells[50], and rat articular chondrocytes[51],
has also been observed. In addition, Wagle and Jesuthasan recently
showed that baculovirus can successfully transduce the embryos of
zebrafish[52]. By injecting the baculovirus expressing
ephrinB2a into specific tissues, ephrinB2a is normally expressed in
the posterior region of developing somite and baculovirus-mediated
misexpression causes abnormal somite boundary formation. Despite the
rapidly growing list of susceptible cells, however, baculovirus
transduction of cell lines of hematopoietic origin, such as U937,
K562, Raw264.7[53], LCL-cm, and Raji[54], is
inefficient.
The transduction efficiencies vary
considerably depending on cell types and can be up to 95% for BHK
cells or lower than 10% for NIH-3T3 cells[54]. One
possibility accounting for the high transduction efficiency in
certain cell lines is the activation of mammalian promoters (eg, CMV
promoter and heat shock promoter) by a DNA sequence upstream from
the polyhedrin (pu) and a homologous region (hr) of
AcMNPV[55]. The hr1 sequence enhances
transcription from polyhedrin and Drosophila heat shock
protein (hsp70) promoter in Sf cells[56] and
functions as an origin of replication (ori), but does not
support ori activities in mammalian cells[57].
pu and hr together function synergistically, resulting in
as much as 18 000-fold promoter activation[55]. The hr1
can also function in mammalian cells as an enhancer when present
in trans[57]. The insertion of an additional copy of
the hr1 region in the AcMNPV genome thus represents an
attractive approach to promoting overexpression of foreign proteins
in mammalian cells as has been demonstrated in insect cells[56].
The additional hr1 can also help maintain the genetic
stability of the bacmid-derived baculoviruses, because spontaneous
deletion of the heterologous gene(s) in the foreign bacterial
artificial chromosome sequences readily occurs[58,59].
The transduction efficiency can be
markedly enhanced by the addition of sodium butyrate, trichostatin A[53],
and valproic acid[60]. These compounds are histone
deacetylase inhibitors, inducing a hyperacetylation of the chromatin
and enhancement of transcription, thus highlighting the importance
of the chromatin state of the baculovirus genome in the transduced
cells for transgene expression. However, cytotoxicity is often
associated with the use of these drugs[60]. Another
approach to enhance the transduction efficiency is to alter the
transduction protocol. For transduction, typically the virus is
concentrated by ultracentrifugation and resuspended in
phosphate-buffered saline (PBS). The cells are then incubated with
the virus for 1 h at 37 ¡ãC using growth medium (eg, DMEM) as the
surrounding solution[44,45,61]. Recently, we found that
incubation of the unconcentrated virus (ie virus supernatant) at a
lower temperature (eg, 25 ¡ãC) for 4 h-6 h using PBS as the
surrounding solution results in gene transfer into HeLa and
chondrocytes[51,62] with efficiencies comparable or
superior to those using the traditional protocols. This is in part
because of prolonged virus uptake provided that sufficient amount of
the virus is present. One key determinant accounting for the high
transduction efficiency is PBS, which is superior to DMEM or TNM-FH
(the medium for baculovirus production) as the surrounding solution
in terms of enhancing the transduction efficiency and transgene
expression[51,62], although the exact mechanism awaits
further elucidation. The same protocol also confers gene delivery
into human mesenchymal stem cells (MSC) isolated from umbilical cord
blood (uMSC) and bone marrow (bMSC) with efficiencies of up to
approximately 72.8% and 41.1%, respectively[63]
(Figure 1). This protocol eliminates the need for virus
ultracentrifugation; thus, it not only represents a simpler
approach, but also considerably reduces possible virus inactivation
during ultracentrifugation.
Baculovirus transduction is
generally considered non-toxic to mammalian cells and does not
hinder cell growth even at high multiplicity of infection[43,64].
Our recent studies again confirm this notion because transduction
with a wild-type baculovirus does not cause any observable adverse
effect to chondrocytes or MSC[51,63]. From the growth
curves, however, we noted a slower cell proliferation on expression
of enhanced green fluorescent protein (EGFP) under the
transcriptional control of a CMV promoter. The slightly retarded
cell growth is attributed to the EGFP overexpression because: (i)
EGFP overexpression could be toxic and might even induce apoptosis
in some cells[65,66]; and (ii) the transcriptional
activation of CMV promoter could repress other viral or cellular
gene expression[67]. Fortunately, the cell growth rate
may be restored after several passages, as EGFP expression
attenuates[63].
Mechanisms of baculovirus entry
into mammalian cells Baculovirus enters insect cells via
endocytosis followed by low pH-induced fusion of a viral envelope
protein with the endosomal membrane, thus allowing viral entry into
the cytoplasm and nucleus[68]. Likewise, baculovirus is
generally considered to follow the same route to enter mammalian
cells, as gene expression is inhibited by lysosomotropic agents (eg,
chloroquine), which block endosomal matura-
tion[43,44]. Initially it was suggested that the
transduction is liver specific and the asialoglycoprotein could be
involved in virus binding[43,44]. However, van Loo
et al showed that Pk1 cells, which do not express the
asialoglycoprotein rece-ptors, could be successfully transduced,
thus asialoglyco-protein is not a key determinant[48]. It
was also shown that electrostatic interactions and the heparan
sulfate moieties might be necessary for baculovirus binding to the
mammalian cell surface[69]. However, no follow-up report
has confirmed their findings and the nature of the receptors remains
unclear. In contrast, the viral envelope glycoprotein gp64 is shown
to be essential for virus attachment and endosomal escape[70].
Supporting this is the finding that a baculovirus over-expressing
gp64 in addition to the endogenous copy of gp64 can incorporate
approximately 1.5-fold the normal amount of gp64 on the virion
surface and exhibit 10- to 100-fold more reporter gene expression in
a variety of mammalian cells compared with similar viruses with a
normal amount of gp64[71]. In the same study,
phospholipids on the cell surface are suggested to serve as an
important docking point for gp64, thus facilitating viral entry into
mammalian cells. By transient depletion of calcium using EGTA
pretreatment, Bilello et al demonstrated that paracellular
junction complexes are important for baculoviral entry into primary
hepatocytes[72].
It is generally assumed that the
escape from the endo-somes sets the block for transduction of some
mammalian cells by baculovirus[44,73]; however, a recent
study showed that the transduction block lies not in the endosomal
escape, but rather in the cytoplasmic trafficking or nuclear import
of the virus capsids[74]. In the cytoplasm, the
nucleocapsids appear to induce formation of actin filaments, which
probably facilitate the transport of viral nucleocapsids into the
nucleus, as cytochalasin D strongly reduces the transduction
efficiency but not the delivery of nucleocapsids to the cytoplasm[48].
In contrast, we recently found that incubation of cells with PBS
during transduction results in significantly higher virus entry and
higher transduction efficiency compared with incubation with DMEM[63],
suggesting that virus internalization may be a bottleneck as well.
Overall, the events responsible for virus uptake and detailed
mechanisms of intracellular movement and nuclear entry of the virus
are still largely unknown.
Baculovirus display The
tropism and transduction efficiency of baculovirus has been
manipulated by modifying the envelope protein. The modification can
be performed by fusing a heterologous protein (or peptide) in-frame
at the
N-terminus of the gp64 gene under the control of a polyhedrin or p10
promoter. The fusion protein, after expression as an additional
copy, is translocated to the plasma membrane and incorporated into
the viral envelope on virus budding. Proof of this principle was
first approached by fusing HIV-1 envelope proteins; the modified
virus binds the CD4 receptor on T cells[75]. A similar
strategy has been applied to construct avidin-displaying baculovirus,
which shows a 5-fold increase in transduction efficiency in rat
malignant glioma cells and a 26-fold increase in rabbit aortic
smooth muscle cells compared with the wild-type baculovirus[76].
Baculovirus displaying heterologous envelope proteins, such as
vesicular stomatitis virus G protein (VSVG), has also been
constructed, which transduces human hepatoma and rat neuronal cells
at efficiencies roughly 10- to 100-fold greater than baculovirus
lacking VSVG[73]. This pseudotyped virus also transduces
cell lines that are transduced at very low levels or not at all by
the unmodified baculovirus, thus broadening the tropism. The
enhanced transduction efficiency and wider tropism are attributed to
increasing transport of baculovirus DNA into nuclei rather than to
increasing cell binding or virus uptake[73]. In contrast,
specific targeting of baculovirus to mammalian cells by displaying a
single-chain antibody fragment specific for the carcinoembryonic
antigen (CEA) or synthetic IgG binding domains is also demonstrated[77,78].
In addition, the baculovirus display technology has been utilized to
construct and screen a eukaryotic epitope library[79]. In
this context, the HIV-gp41 epitope "ELDKWA" specific for the
neutralizing MAb 2F5 is inserted into the antigenic site of
influenza virus hemagglutinin, flanked by additional random amino
acids. This pool of hemagglutinin genes is cloned into baculovirus,
and an individual clone displaying the epitope with markedly
increased binding capacity out of a pool of 8000 variants is
screened rapidly by fluorescence activated cell sorting.
Note, however, that the expression
of gp64-fusion under the control of a late promoter may lead to a
heterogeneous virus population that possesses various amounts of
fusion protein and hence varied properties. This might be resolved
by: (i) inserting the gp64 fusion gene into the genomic gp64 locus
so that all gp64 copies contain the fusion partner; and (ii)
expressing the gp64 fusion protein under an early promoter. More
recently, baculovirus capsid display has also been developed in
which the fusion can be made to the N- or
C-terminus of the major capsid protein vp39 rather than to gp64
without compromising the viral titer or functionality[74].
It has been demonstrated that the incorporation of green fluorescent
protein (GFP) into the virus capsid does not interfere with the
capsid assembly and allows for visualization of the virus
biodistribution in vivo[74]. This capsid-modified
baculovirus holds great promise for the nuclear and subcellular
targeting of the transgenes.
Viral vector generation and viral
protein production Thanks to the large DNA genome, recombinant
baculovirus offers an attractive means for the production of other
viral vectors for gene delivery/gene therapy purposes because the
production of many viral vectors requires either plasmid
transfection into producer cell lines or helper virus infection. For
instance, all genes essential for replication and packaging of the
adenoviral vector (38 kb) can be delivered and expressed by a single
baculovirus, which is co-transfected with a plasmid vector harboring
only the inverted terminal repeats (ITR) and packing signal of the
wild-type virus as well as a reporter gene[80]. The
co-delivery results in the generation of a fully deleted adenovirus
vector free of helper virus. This technique is also exploited for
the production of adeno-associated virus (AAV), whose use is
hampered by the lack of a simple and efficient vector production
method. The mammalian producer cells are co-transduced with 3
viruses: 1 baculovirus harboring the reporter gene flanked by AAV
ITR, 1 baculovirus expressing the AAV rep gene, and a helper
adenovirus expressing the AAV cap gene[81]. The
simultaneous delivery produces infectious recombinant AAV particles
and constitutes an option for improving large-scale recombinant AAV
vector production. Besides virus preparation, Ramos et al
recently investigated the feasibility of using the baculovirus/mammalian
cell system for protein production. They investigated two protocols
for optimal cell culture and virus transduction and achieved
volumetric yields for 3 secreted proteins (SAF-3-Fc, CD40-hexa his,
and Asp 2-Fc) at 4 mg/L-25 mg/L[82]. In our laboratory,
the application of baculovirus is further expanded to the production
of hepatitis delta virus (HDV) VLP. HDV can not replicate in
cultured cell lines, and thus is difficult to propagate; therefore,
studies of HDV particle assembly mainly rely on cDNA transfection,
which suffers, however, from low efficiency and hinders subsequent
structural and immunological studies of HDV VLP. We demonstrated
that the co-transduction of hepatoma cell lines with 2 recombinant
baculoviruses, one expressing hepatitis large delta antigen and
another expressing hepatitis B surface antigen (HBsAg), leads to
self-assembly and secretion of the HDV VLP[83]. The yield
(~150 ng HBsAg per mL in the secreted particle) is approximately
2-fold of that achieved by plasmid transfection. VLP synthesis has
also been demonstrated in BHK cells and the process is transferred
to a novel oscillating bioreactor for production on a larger scale[84].
One bioreactor run (500 mL working volume) can yield over 400 mg
HBsAg, which substantially increases the total particle yield and
thus enables future structural and immunological studies of the VLP.
In comparison with stable cell lines, the gene expression levels and
the VLP composition can be easily manipulated simply by varying the
virus concentration. In addition, the expressed proteins are
obtained in a few days; therefore, this baculovirus/mammalian cell
expression system may serve as a transient expression system for
early evaluation of proteins of interest while stable cell
lines are being generated and evaluated.
Studies of gene functions and
viral infections Gene delivery into primary cells using
transfection or electroporation either suffers from low efficiency
or causes extensive membrane damage. Because baculovirus is
generally non-toxic and non-replicative in mammalian cells, and the
transduction efficiencies into various human primary cells are high,
cell-based assays have been developed for gene function studies.
Clay et al demonstrated that co-transduction of cells with 2
recombinant baculoviruses, one expressing an estrogen receptor and
another expressing a reporter gene controlled by an estrogen
receptor-responsive promoter,
results in ligand-induced reporter gene activity, and this approach
is successfully applied to assay development in Saos-2 human
osteosarcoma (HOS) cells[85]. Similarly, a cell/cell
fusion assay that mimics the HIV viral/cell fusion process was
developed based on the baculovirus system[86]. In the
assay, HOS cells stably expressing CCR5, CD4, and LTR-luciferase
serve as the recipient host cell. An HEK-293 cell line transduced
with a baculovirus expressing the viral proteins gp120, gp41, tat,
and rev represents the virus. Interaction of gp120 with CCR5/CD4
results in the fusion of 2 cells and transfer of tat to the
cytoplasm of the HOS cell; tat, in turn, binds to the LTR region on
the luciferase reporter and activates transcription, leading to an
increase in cellular luciferase activity. This assay has been
demonstrated to be a robust and reproducible high-throughput
surrogate assay for evaluating the effects of compounds on
gp120/CCR5/CD4-mediated viral fusion into host cells[86].
This system could also facilitate the assessment of different
promoters for their strength as well as temporal and spatial
regulation across mammalian cells of different origins.
Similar to HDV, hepatitis B virus (HBV)
and HCV are not capable of replication in cultured cells; thus, the
evaluation of novel antiviral strategies have been hindered by the
lack of a convenient and reliable in vitro culture system. To
overcome this problem, recombinant baculoviruses are constructed to
carry and deliver the genomes of HBV[87] and HCV[88]
into hepatoma cells lines. With this approach, replication of HBV
DNA and RNA, and expression of viral genes are detected[87].
Viral replication is evidenced by the presence of high levels of
intracellular replicative intermediates and protected HBV DNA in the
medium. The successful HBV replication thus allows for the
establishment of a highly flexible system for studying: (i) the
effects of (-)-b-2',3'-dideoxy-3'-thiacytidine on HBV replication
and accumulation of covalently closed circular DNA[89];
and (ii) the cross-resistance profiles of HBV mutants against
antihepadnaviral compounds[90]. The HCV full-length and
minigenome under the tetracycline-inducible promoter are also
delivered into HepG2 cells and regulable transcription and viral
polypeptide processing are demonstrated[91], thus
providing a novel tool for the analysis of HCV replication and
host-cell interactions.
Generation of stable cell lines
Normally, baculovirus-mediated gene expression lasts
approximately 1-2 weeks as a result of cell division. The gradual
extinction of transgene expression is also attributed to the
degradation of baculoviral DNA, as degradation has been revealed in
hepatoma cells[83] and articular chondrocytes[51].
However, it was found that transduction of CHO cells with a
baculovirus expressing neomycin phosphotransferase results in the
isolation of CHO cells stably expressing GFP by selection using the
antibiotic G418[53]. The frequency of G418 resistant
colony formation is approximately 1 in 50-100 of the transduced
cells. The same group further confirmed that discrete portions of
baculovirus DNA are randomly integrated as single-copy fragments
ranging in size from 5 kb to 18 kb[92]. Periodic assays
on the stable clones show no loss of transgene expression over a
5 month period. Coupled with high transduction efficiency, the high
frequency of emergence of stable transductants makes baculovirus a
valuable tool to derive human cell lines that are either difficult
to isolate in large numbers or are difficult to transfect by other
methods.
In vivo gene therapy
Thanks to highly efficient gene delivery, baculovirus has captured
increasing interest as a vector for in vitro and in vivo
gene delivery. However, reports of successful in vivo
applications are limited. The complement system appears to be a
potent barrier for in vivo gene therapy because the
efficiency of gene transfer into hepatocytes of complement-deficient
mice is greatly enhanced[70]. Inactivation of baculovirus
in human plasma and whole blood is prevented by treatment with cobra
venom factor[93]. An alternative approach is adopted by
avoiding contact of baculovirus vectors with blood components. By
using a silastic collar, transduction of adventitial cells in rabbit
carotid arteries is achieved and the efficiencies are comparable to
those obtained with adenoviral vectors[94]. The gene
expression is transient and the arterial structure and endothelium
remain intact after baculovirus transduc-tion, although signs of
inflammation are detected. Unmodified baculovirus vectors have also
been injected into the rodent brain where the complement proteins
may not reach in the presence of an intact blood-brain barrier
and/or the complement level in the brain is insufficient to affect
the gene transfer[95]. After in vivo injection
into the brain, baculovirus specifically transduces the epithelium
of the choroids plexus in ventricles and the transduction efficiency
is as high as 76%¡À14%; thus, baculovirus seems to be especially
useful for the targeting of choroids plexus cells[95].
A more cutting-edge approach to
alleviate the complement inactivation problem is the generation of
complement-resistant baculovirus by displaying decay-accelerating
factor, a regulator that blocks complement at the central step of
both the classical and alternative pathways[96]. Such a
complement-resistant baculovirus vector expressing additional human
coagulation factor IX (hFIX) allows for a substantial improvement of
gene transfer into complement-sufficient neonatal rats in vivo
after local injection into the liver parenchyma. Gene expression is
transient probably as a result of the generation of antibodies
directed against the transgene product hFIX, which might lead to
clearance of either expressed hFIX protein and/or positively
transduced cells. Alternatively, baculovirus can be pseudotyped by
displaying VSVG on the envelope. The VSVG-modified virus enhances
gene transfer efficiencies into mouse skeletal muscle in vivo
and the transgene expression lasts 178 d in DBA/2J mice and 35 d in
BALB/c and C57BL/6 mice[97]. The VSVG-modified
baculovirus also exhibits greater resistance to inactivation by
animal sera and can transduce the cerebral cortex and testis of mice
by direct inoculation in vivo[61].
Another potential barrier may be the
presence of heparin or heparin-like factors, which block baculovirus-mediated
gene delivery in cell culture[93]. Intercellular
junctions may be an additional hurdle because transient disruption
of these junctions by EDTA treatment prior to transduction improves
the gene delivery efficiency into long-term cultures of primary
hepatocytes but does not lead to permanent alternations in
hepatocyte ultrastructure, albumin mRNA, and protein expression
profiles[98]. Their studies also suggest the importance
of the basolateral surface for virus entry at least for some cell
types[72]. In our laboratory, however, the transient
disruption of cell junctions fails to effectively enhance the
baculovirus-mediated gene transfer into chondrocytes and HepG2 cells
that are cultured to over-confluence, implying that other factors in
addition to the paracellular junction complexes might be involved in
the transduction of these cells.
Another problem associated with
baculovirus as a gene delivery vector is that purification by
ultracentrifugation often leads to virus aggregation[99].
In addition, the ultracentrifugation process is time-consuming,
labor intensive, and difficult to scale up. To resolve these
problems, we construct a recombinant baculovirus with a
hexahistidine (His6) tag displayed on the viral envelope,
which enables virus purification by a simple immobilized metal
affinity chromatography with high purity (~87%). This methodology
obviates the need for successive ultracentrifugation steps[100]
and may be employed to purify other viral vectors for gene therapy
studies.
Ex vivo gene therapy
To date, most gene therapy studies using baculovirus vectors
have focused on in vivo experiments, yet relatively little is
known about the potential of baculovirus for ex vivo therapy.
One relevant report was published by Sandig et al[101]
who established an ex vivo perfusion model for human liver
segments. The recombinant baculovirus is perfused through the liver
segments for 15 min and reasonable transduction rates are achieved
in all perfused parts of the liver tissue. This study verifies for
the first time that baculovirus-mediated gene transfer is possible
in the liver tissue and encourages future studies including in
situ perfusion of intact livers with baculovirus vectors in
animal models.
Recently, we have also demonstrated
the highly efficient baculovirus-mediated gene transfer into
articular chondro-cytes[51] and human MSC[63],
both being candidate cell sources for cartilage tissue engineering.
Importantly, normal differentiation states of chondrocytes and MSC
are retained on baculovirus transduction. Our experiments regarding
MSC suggest that the phenotypic changes along the adipogenic
differentiation pathway positively influence the baculovirus
transduction and may implicate two possible applications: (i) MSC
may be transduced with baculovirus expressing specific factors (eg
b-FGF or BMP-2) to promote the expansion or regulate the
differentiation via autocrine and paracrine effects of the factors
so that supplementation of these factors in the medium may be
omitted; and (ii) differentiation of MSC towards a specific lineage
pathway may be induced first and the committed progenitor cells can
be transduced ex vivo by the recombinant baculovirus
expressing appropriate growth factors (eg TGFb1 or IGF-1), seeded
onto polymeric scaffolds, and implanted into animal models for
tissue regeneration. Although sustained transgene expression is
restricted by the degradation of baculoviral DNA within the cells,
the non-replication and degradation characteristics prove that
baculovirus may be a safe gene delivery vehicle for tissue
engineering in which long-term expression is not critical.
Immunological response and
potential as a vaccine vector Host responses to baculovirus
uptake, either in vitro or in vivo, were not evaluated
until Gronowski et al reported that administration of
baculovirus in vitro induces interferon (IFN) production from
human and murine cell lines and induces in vivo protection of
mice from encephalomyocarditis virus infection[102]. A
more recent study discovered that the presence of baculovirus
disrupts the phenobarbital gene induction, a potent transcriptional
activation event characteristic of highly differentiated hepatocytes,
and represses expression of the albumin gene, but neither cAMP nor
PKA activities are affected by the virus[102].
Baculovirus transduction also induces expression of cytokines such
as TNF-a, IL-1a, and IL-1b in primary hepatocytes[103].
These findings raise concerns as to whether these properties will
compromise the use of baculovirus vectors for in vivo gene
therapy in humans, and more investigations will be needed to ensure
the safety of baculovirus vectors. Despite these, the ability of
baculovirus to induce immune responses has been exploited by Abe
et al, who found that intranasal inoculation with a wild-type
baculovirus elicits a strong innate immune response that protects
mice from a lethal challenge of influenza virus[104]. The
feasibility of using baculovirus as a vaccine carrier was also
demonstrated by Aoki et al[105], who found that a
recombinant baculovirus expressing glycoprotein gB of pseudorabies
virus induces antibodies against gB protein in mice, thus suggesting
that this recombinant baculovirus could serve as a vaccine candidate
for pseudo-rabies. Similarly, in vivo administration of a
baculo-virus expressing hFIX elicits antibodies against hFIX. The
antibody titers are proportional to the amount of protein produced
by the virus[96]. More recently, Facciabene et al
demonstrated that the intramuscular injection of a baculovirus
expressing CEA induces measurable anti-CEA-specific CD4+
T-cell response[106]. The immunogenic property of baculo-virus
is not restricted to CEA because the intramuscular injection of
another baculovirus (Bac-E2) expressing E2 glycoprotein of HCV
induces anti-E2 CD8+ T-cell response[106].
Interestingly, when Bac-E2 is pseudotyped to display VSVG on the
envelope, the minimal dose required to elicit a measurable T-cell
response is 10-fold less, indicating that the VSVG-pseudotyped
Bac-E2 appears to be a more potent immunogen than the unmodified
virus. This finding agrees with the previous statement that
baculovirus displaying VSVG confers a more efficient immunogen
expression in transduced cells.
It is also likely that baculovirus
can mediate an immune response against an antigen when it is
displayed on the viral surface. In this regard, the immunodominant
antigenic site of foot-and-mouth disease virus (FMDV) has been
displayed on the baculovirus envelope[107], which is able
to bind a panel of FMDV-specific MAb and indicates the preservation
of antigenic epitopes on the virus envelope. A more recent study
further showed that immunization with adjuvant-free baculovirus
displaying rodent malaria Plasmodium berghei circumsporozoite
protein (PbCSP) on the envelope induces high levels of antibodies
and g-interferon-secreting cells against PbCSP, and protects 60% of
mice against sporozoite challenge[108]. These studies
substantiate the potential of baculovirus displaying immunogens as a
vaccine candidate.
Conclusions and future prospects
The values of baculovirus for
recombinant protein expression in insect cells have long been
proven. The extremely high yield by baculovirus-infected insect
cells or larvae makes it an attractive tool for pharmaceutical
protein production. Despite its inability to process some proteins
correctly, rapid advances in cell engineering may overcome these
drawbacks and render baculovirus a proper system for large-scale
protein production. To the best of my knowledge, no insect
cell-derived recombinant protein has gained official approval from
the US Food and Drug Administration. Nonetheless, more and more
proteins are produced in insect cells in the preclinical stage.
Particularly promising is the field of viral vaccines, where the
formation of virus-mimicking multimers can be essential for
eliciting protective immune responses. An HCV vaccine might be the
first human therapeutic expressed in insect cells[109].
The broad range of susceptible
mammalian cells, coupled with its non-toxic and non-replication
nature, makes baculovirus a useful tool for studying the expression
and function of gene products. As a gene therapy vector, baculovirus
possesses the following extra advantages: (i) the AcMNPV genome is
large, thus rendering the virus flexible to carry multiple genes or
large inserts; (ii) recombinant baculoviruses are easy to construct
and produce to high titers simply by infecting insect cells; and
(iii) the purification of baculovirus can be readily performed by
ultracentrifugation[110] or, alternatively, by a newly
developed affinity chromatographic technique[100]. The
last two features greatly simplify the large-scale preparation of
high titer baculoviral vectors. In addition to therapeutic genes,
baculo-virus may serve as a promising vector for the delivery of
cDNA of infectious RNA viruses towards antisense therapy approaches.
The same concept may be applied to the delivery of siRNA to silence
specific target genes. The combination of ex vivo gene
transfer and tissue engineering holds great promise as well. One
bottleneck to the application of baculovirus lies in the short
duration of transgene expres-sion. This may be overcome by selecting
a proper promoter that is not silenced, and developing a proper
superinfection protocol that can effectively deliver fresh genes
into cells. The baculovirus may also be applied in conjunction with
other viral vectors to escape either pre-existing or therapy-induced
antiviral immunity. In summary, baculovirus can be used as an
efficient vector for gene delivery into insect and mammalian
cells for a wide variety of applications.
Acknowledgment
The author gratefully acknowledges
the financial support of the National Science Council (Grant NSC
93-2214-E-007-016) and Ministry of Economic Affairs (Technology
Development Program for Academia Grant 93-EC-17A-17S1-0009), Taiwan,
China.
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