**4. Mammalian cells transduction and BacMam systems**

#### **4.1. Introduction**

Initial interest in baculoviruses as gene delivery vectors for mammalian cells was driven by their good biosafety profile [84]. Compared to other human‐derived viral gene delivery vectors, the safety requirements for handling baculoviruses are relatively low. Baculoviruses are so exceptionally adapted to their natural hosts that they pose no threat to vertebrate organisms. They are unable to replicate in mammalian cells, can be manipulated in laboratories at BSL1/2 levels and can be easily inactivated [85]. Moreover, insect larvae in the wild are infected via the gut by occluded baculoviruses and polyhedrin‐deleted recombinant virus used to transduce mammalian cells does not efficiently infect larvae. The viruses are unstable outside of the laboratory, so they are environmentally contained as well.

Baculovirus entry into mammalian cells was suggested to depend on electrostatic interactions, heparin sulfate and phospholipids, but the exact cell surface molecules for baculovirus docking remained unknown [86]. It was also proposed that clathrin‐mediated endocytosis and macropinocytosis play roles in baculovirus entry [87, 88]. Contradictorily, a recent study [89] discovered that (1) baculovirus entered cells into vesicles devoid of clathrin; (2) macropino‐ cytosis‐related regulators imparted no significant effects on virus transduction and (3) the internalization and nuclear uptake were affected by the regulators of clathrin‐independent entry. These data unveiled a baculovirus entry pathway independent of clathrin‐mediated endocytosis and macropinocytosis and suggested that phagocytosis might play a role, which echoed the observations reported previously [90]. Moreover, other recent studies reported that baculovirus transduction related to direct fusion pathway induced by a short pH trigger [91]. Nevertheless, one consensus is that baculovirus envelope protein gp64 is pivotal for entry because blocking gp64 can abrogate the baculovirus ability to transduce mammalian cells and activate dendritic cells [92]. Very recently, it has been demonstrated that 6‐O‐ and N‐sulfated syndecan‐1 promotes baculovirus binding and entry into mammalian cells. [93].

Numerous cell lines have been transduced [94], including primary cells in vitro and human livers ex vivo and the capability of baculovirus as a gene therapy vector has been studied. More recent studies have described the use of AcMNPV vectors in the form of BVs for in vivo targeting of different organs including brain and liver [95], and stem cells for tissue engineering [96].

transduction efficiencies near 100% can be reached. With this high transduction efficiency and flexibility, the technology easily enables coexpression of several genes with multiple baculo‐ viruses and modulation of expression level by dosing and timing. This flexibility is especially relevant in studies of multimeric complex functional proteins and also in assays of processes where mix‐and‐match coexpression experiments with a number of cofactors and interacting

**Figure 5.** Production and application of BacMam viruses. Target gene sequences cloned into a transfer plasmid con‐ taining a mammalian cell‐active expression cassette are transferred to baculovirus DNA via recombination. The viral DNA is transfected into insect cells where virus production occurs. Budded virus are clarified from the insect cell cul‐ ture medium. The stock virus is used to transduce mammalian cells and expression of the recombinant protein(s) is usually validated 24–48 h later. The effects of the expression can be tested with this transient assay. BacMam vectors

Recombinant baculovirus

Transduced animal

Transduced mammalian cells

Infected insect cells

95

Genetic Engineering of Baculoviruses http://dx.doi.org/10.5772/56976

Purified budded virus

The vectors used for the development of BacMams are derivatives from AcMNPV transfer vectors. The most widely used system for the generations of BacMam are based on the Bac‐to‐ Bac system (InvitrogenTM) for baculovirus generation. With this system the recombinant baculoviral genome is constructed in *E. coli*, via a transfer vector. The gene of interest is first subcloned into a BacMam transfer vector, which is then transformed into a special *E. coli* strain DH10Bac to generate the recombinant viral DNA. The viral DNA is then used to transfect insect cells in order to generate the recombinant virus. The entire process is simple and easy to perform, allowing generation of multiple viruses simultaneously. With the procedure, recombinant BacMams can be generated in less than 2 weeks. The Bac‐to‐Bac system [102] uses

partners are necessary.

Transfer plasmid containig target genes

Expressed multimeric complex

**4.2. Available vectors for BacMam development**

can be also used to transduce mammalian organisms.

The term BacMam refers to baculoviruses in which a mammalian promoter is used to drive heterologous gene expression in mammalian cells following viral transduction (Figure 5). Since the viral genome can stably accommodate an insert sequence of at least 40 kb, BacMams are particularly suitable for expression of multimeric complexes. Unless a selection force is applied, gene expression in transduced cells is transient and can usually last for up to 4 days. However, the expression can even be prolonged to 16 days. For viruses carrying a selectable marker, stable cell lines can also be established upon selection [97].

BacMams have been used as delivery vehicles to mammalian cells for many polypeptide genes, including secreted [98] and transmembrane proteins [99, 100, 101]. When high MOIs are used,

**Figure 5.** Production and application of BacMam viruses. Target gene sequences cloned into a transfer plasmid con‐ taining a mammalian cell‐active expression cassette are transferred to baculovirus DNA via recombination. The viral DNA is transfected into insect cells where virus production occurs. Budded virus are clarified from the insect cell cul‐ ture medium. The stock virus is used to transduce mammalian cells and expression of the recombinant protein(s) is usually validated 24–48 h later. The effects of the expression can be tested with this transient assay. BacMam vectors can be also used to transduce mammalian organisms.

transduction efficiencies near 100% can be reached. With this high transduction efficiency and flexibility, the technology easily enables coexpression of several genes with multiple baculo‐ viruses and modulation of expression level by dosing and timing. This flexibility is especially relevant in studies of multimeric complex functional proteins and also in assays of processes where mix‐and‐match coexpression experiments with a number of cofactors and interacting partners are necessary.

#### **4.2. Available vectors for BacMam development**

**4. Mammalian cells transduction and BacMam systems**

94 Current Issues in Molecular Virology - Viral Genetics and Biotechnological Applications

outside of the laboratory, so they are environmentally contained as well.

syndecan‐1 promotes baculovirus binding and entry into mammalian cells. [93].

marker, stable cell lines can also be established upon selection [97].

Numerous cell lines have been transduced [94], including primary cells in vitro and human livers ex vivo and the capability of baculovirus as a gene therapy vector has been studied. More recent studies have described the use of AcMNPV vectors in the form of BVs for in vivo targeting of different organs including brain and liver [95], and stem cells for tissue engineering

The term BacMam refers to baculoviruses in which a mammalian promoter is used to drive heterologous gene expression in mammalian cells following viral transduction (Figure 5). Since the viral genome can stably accommodate an insert sequence of at least 40 kb, BacMams are particularly suitable for expression of multimeric complexes. Unless a selection force is applied, gene expression in transduced cells is transient and can usually last for up to 4 days. However, the expression can even be prolonged to 16 days. For viruses carrying a selectable

BacMams have been used as delivery vehicles to mammalian cells for many polypeptide genes, including secreted [98] and transmembrane proteins [99, 100, 101]. When high MOIs are used,

Initial interest in baculoviruses as gene delivery vectors for mammalian cells was driven by their good biosafety profile [84]. Compared to other human‐derived viral gene delivery vectors, the safety requirements for handling baculoviruses are relatively low. Baculoviruses are so exceptionally adapted to their natural hosts that they pose no threat to vertebrate organisms. They are unable to replicate in mammalian cells, can be manipulated in laboratories at BSL1/2 levels and can be easily inactivated [85]. Moreover, insect larvae in the wild are infected via the gut by occluded baculoviruses and polyhedrin‐deleted recombinant virus used to transduce mammalian cells does not efficiently infect larvae. The viruses are unstable

Baculovirus entry into mammalian cells was suggested to depend on electrostatic interactions, heparin sulfate and phospholipids, but the exact cell surface molecules for baculovirus docking remained unknown [86]. It was also proposed that clathrin‐mediated endocytosis and macropinocytosis play roles in baculovirus entry [87, 88]. Contradictorily, a recent study [89] discovered that (1) baculovirus entered cells into vesicles devoid of clathrin; (2) macropino‐ cytosis‐related regulators imparted no significant effects on virus transduction and (3) the internalization and nuclear uptake were affected by the regulators of clathrin‐independent entry. These data unveiled a baculovirus entry pathway independent of clathrin‐mediated endocytosis and macropinocytosis and suggested that phagocytosis might play a role, which echoed the observations reported previously [90]. Moreover, other recent studies reported that baculovirus transduction related to direct fusion pathway induced by a short pH trigger [91]. Nevertheless, one consensus is that baculovirus envelope protein gp64 is pivotal for entry because blocking gp64 can abrogate the baculovirus ability to transduce mammalian cells and activate dendritic cells [92]. Very recently, it has been demonstrated that 6‐O‐ and N‐sulfated

**4.1. Introduction**

[96].

The vectors used for the development of BacMams are derivatives from AcMNPV transfer vectors. The most widely used system for the generations of BacMam are based on the Bac‐to‐ Bac system (InvitrogenTM) for baculovirus generation. With this system the recombinant baculoviral genome is constructed in *E. coli*, via a transfer vector. The gene of interest is first subcloned into a BacMam transfer vector, which is then transformed into a special *E. coli* strain DH10Bac to generate the recombinant viral DNA. The viral DNA is then used to transfect insect cells in order to generate the recombinant virus. The entire process is simple and easy to perform, allowing generation of multiple viruses simultaneously. With the procedure, recombinant BacMams can be generated in less than 2 weeks. The Bac‐to‐Bac system [102] uses the Tn7‐mediated site‐specific transposition reaction to direct integration of expression cassettes contained in the transfer vector into a baculovirus backbone vector (bacmid) preex‐ isting in the *E. coli* DH10Bac strain. In this case, the bacmid is a mini‐F replicon with the baculovirus genome and has a kanamycin resistance marker. In addition, the *E. coli* strain contains a helper plasmid that expresses the Tn7 transposase gene. The system was designed in such a way that the recombinant Tn7 transposon from the transfer vector will be integrated into a mini‐attTn7 in the *lacZα* gene fragment contained within the recombinant viral genome, causing inactivation of the α‐complementation of *lacZ*. The desired recombinant transformants will be resistant to tetracycline, kanamycin, and gentamicin and can be easily distinguished from nonrecombinants by blue/white selection on X‐gal plates.

Serum complement proteins (e.g. C5b‐9) inactivate baculovirus, hence constituting a major hurdle in the in vivo use of baculovirus. The inactivation problem has been circumvented by the use of complement inhibitors [112] or by displaying human DAF (decay accelerating factor) via gp64 [113]. The DAF‐displaying baculovirus caused lower levels of inflammatory cytokines IL‐1β, IL‐6, and IL‐12p40 in macrophages and mitigated liver inflammation in mice when compared with the control virus. These results demonstrate that DAF display offers protection to the baculoviral vector against complement inactivation and attenuates complement‐

Genetic Engineering of Baculoviruses http://dx.doi.org/10.5772/56976 97

**4.3.2. Surface modification via capsid display, chemical coupling or electrostatic interactions**

Other than the display on the envelope, heterologous protein has been displayed on the capsid by fusion with the major capsid protein VP39. The VP39 fusion with enhanced green fluores‐ cent protein (eGFP) neither interferes with the virus assembly nor affects the virus titer, thereby enabling intracellular baculovirus trafficking and biodistribution monitoring [114]. Similarly, the ZnO binding peptide has been fused to the N‐terminus of VP39 while retaining the viral infectivity and conferring the ability to bind nanosized ZnO powders [115]. Besides, by fusing the protein transduction domain (PTD) of human immunodeficiency virus (HIV) TAT protein (a protein responsible for nuclear import of HIV genome) with VP39, the engineered baculo‐

Baculovirus can also be chemically conjugated with compounds such as polyethylene glycol (PEG) alone and folate [116] to improve the transduction of folate receptor‐positive KB cells. Additionally, baculoviral vectors have been coated with positively charged polyethylenimine (25 kDa) through electrostatic interactions. The modification imparts baculoviral vectors resistance to human and rat serum‐mediated inactivation in vitro and elevates in vivo

Recently, a novel molecular biology tool was established by the development of baculovirus surface display [117‐123], using different strategies for presentation of foreign peptides and proteins on the surface of budded virions. This eukaryotic display system enables presentation of large complex proteins on the surface of baculovirus particles and has thereby become a

The baculovirus system offers great potential as an eukaryotic surface display system, since the post‐translational modification of the recombinant proteins is efficient and high transfec‐ tion rates can be reached. These features are important for the generation of efficient surface display libraries. The principal applications of such strategies are ligand screening of surface expression libraries, for example epitope mapping, antigen display for induction of specific antibodies and presentation of proteins that increase binding to mammalian host cells.

virus results in improved transduction of various mammalian cells

transduction in the liver and spleen after tail vein injection into mice.

**5. Baculovirus display strategies**

versatile system in molecular biology.

**5.1. Introduction**

mediated inflammation injury.

The BacMam transfer vectors described here are derivatives of pFastBac1 of the Bac‐to‐Bac system (InvitrogenTM). Originally, the AcMNPV *polh* promoter of pFastBac1 was deleted for the introduction of cassettes containing a mammalian promoter. Later, the CMV immediate early promoter was inserted to allow expression of the cloned cDNA sequences in mammalian cells. The vector pFastBacMam‐1, in addition, contains a neomycin resistance gene driven by the SV40 promoter. The neomycin resistance marker allows selection of stable cell lines following BacMam transduction. Using this vector a new version was constructed (pFast‐ Backmam‐NA) to accommodate ORFs cloned in GatewayTM vectors (Invitrogen).
