**7. Other applications**

The use of baculoviruses as vectors for the generation of immunity is not the only possible application for these viruses. Their ability to transduce mammalian cells and their capacity to allow the introduction of large amounts of heterologous DNA in their genomes represent remarkable advantages. In addition to the biosafety benefits of baculovirus in comparison with other viral vectors, these features make baculoviruses as adequate vectors for in vivo animal transduction. The absence of preliminary immune cells against baculoviruses makes them a promising tool for human treatment.

In this section, two different novel applications for baculoviruses will be discussed. In first place, the use of baculoviruses for gene therapy and the goals and limitations of this practice will be analyzed. Then, the construction of displaying libraries using baculovirus display system will be exemplified.

#### **7.1. Gene therapy**

There are two different categories in which gene therapy vectors can be classified: nonviral and viral vectors. Non-viral vectors consist in polycation conjugated polymers that allow delivery of the DNA. Positively charged liposomes are one example of this type of vectors. Although these vectors are advantageous in biosafety, its application is restricted by the low efficiency in the delivery and expression of transgenes. (Verma & Somia, 1997). On the other hand, viral vectors, such as retroviral, lentiviral, adenoviral, and adeno-associated viral (AAV) vectors, have a higher efficiency in cell entry and transduction by expressing different transgenes. Advantages and disadvantages depend on each particular viral vector. The mechanism used for replication and protein expression, and the biological hazard inherent in its use are some of the features to be analyzed at the moment in which a viral vector is chosen for gene therapy. In comparison with these common viral vectors, baculoviruses possess a number of advantages.

In first place, baculovirus-mediated transduction does not present any toxic effect against mammalian cells and does not disturb cell growth even at high MOI (Gao et al., 2002; Hofmann et al., 1995). In contrast, cell proliferation may be retarded by transgene products because they could be toxic and even induce apoptosis in some cells (Detrait et al., 2002; Liu & Carstens, 1999). Furthermore, baculoviruses do not replicate in transduced mammalian cells (Kost & Condreay, 2002). These features of baculoviruses are particularly important because other viral vectors are human pathogens, and consequently represent a biological risk.

cluded that serum stability was dependent on the displayed complement regulatory protein

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

In general, the complement regulators DAF and MCP gave the best results. Conversely, simultaneous co-display of soluble complement regulatory proteins did not provide further benefit.Best protection was gained in mouse serum (70%), while the worst protection rate was obtained with rat serum (13%). In the case of human serum, about 30% of the viral particles were still competent to transduce mammalian cells after 1 h preincubation with serum

All these data suggest that engineering of baculoviral vectors for complement inactivation result very convenient not only to reduce the number of necessary inoculations for an efficient transduction, but also to avoid the undesired mortality induced by high doses of non-modified

The use of baculoviruses as vectors for the generation of immunity is not the only possible application for these viruses. Their ability to transduce mammalian cells and their capacity to allow the introduction of large amounts of heterologous DNA in their genomes represent remarkable advantages. In addition to the biosafety benefits of baculovirus in comparison with other viral vectors, these features make baculoviruses as adequate vectors for in vivo animal transduction. The absence of preliminary immune cells against baculoviruses makes them a

In this section, two different novel applications for baculoviruses will be discussed. In first place, the use of baculoviruses for gene therapy and the goals and limitations of this practice will be analyzed. Then, the construction of displaying libraries using baculovirus display

There are two different categories in which gene therapy vectors can be classified: nonviral and viral vectors. Non-viral vectors consist in polycation conjugated polymers that allow delivery of the DNA. Positively charged liposomes are one example of this type of vectors. Although these vectors are advantageous in biosafety, its application is restricted by the low efficiency in the delivery and expression of transgenes. (Verma & Somia, 1997). On the other hand, viral vectors, such as retroviral, lentiviral, adenoviral, and adeno-associated viral (AAV) vectors, have a higher efficiency in cell entry and transduction by expressing different transgenes. Advantages and disadvantages depend on each particular viral vector. The mechanism used for replication and protein expression, and the biological hazard inherent in its use are some of the features to be analyzed at the moment in which a viral vector is chosen for gene therapy. In comparison with these common viral vectors, baculoviruses possess a

and the source of serum.

(Kaikkonen et al., 2010).

**7. Other applications**

promising tool for human treatment.

system will be exemplified.

**7.1. Gene therapy**

number of advantages.

vectors.

Another advantage of baculoviruses as gene therapy vectors consists in its large cloning capacity. The baculovirus (AcMNPV) genome is a large circularized DNA molecule with 130 kb of length and a maximum cloning capacity of at least 38 kb. This flexibility results partic‐ ularly advantageous in contrast to retroviral and AAV vectors whose cloning capacities are limited (Hu, 2008).

In comparison with other viral vectors, baculoviruses are easy to produce. Retroviral, lentivi‐ ral, and AAV vectors require transfection of plasmids encoding essential genes into packaging cells for its production. In contrast, baculovirus can be easily propagated by infecting insect cells in suspension culture or monolayer and harvesting the supernatant 3–4 days postinfec‐ tion. In addition, the construction, propagation, and handling of baculoviruses can be per‐ formed in Biosafety Level 1 laboratories without the need for specialized equipment.

Finally, one of the most important advantages is that baculoviruses do not present preexisting immunity in mammalian. One of the problems associated with other viral vectors is that most people are exposed to these viruses and develop specific humoral response. Circulating antibodies can significantly reduce the efficiency of transduction with the viral vector. The use of baculovirus vectors in gene therapy, therefore, may avoid the problem of preexisting immunity (Hu, 2008).

However, baculoviruses have a number of disadvantages as gene therapy vectors. One of these is that baculovirus induce a transient expression in mammalian cells. In vivo, transgene expression typically declines by day 7 and disappears by day 14 (Airenne et al., 2000; Lehto‐ lainen et al., 2002). The duration of in vitro transgene expression using baculoviruses is significantly shorter than expression mediated by retroviral, lentiviral, and AAV vectors.

Baculoviral vectors differ mainly than other viral vectors in the time that the carried genes can persist in the host nucleus. In the case of retroviral, lentiviral and adenoviral vectors, viral DNA can remain into the nucleus either in an integrated or episomal form, for a longer period. In fact, Tjia et al. demonstrated that baculoviral DNA persists in the nuclei of transduced mammalian cells for only 24–48 h (Tjia et al., 1983).

Another disadvantage of using baculovirus as gene therapy vector is the inactivation by complement. As described in previous sections, contact between baculoviruses and serum complement results in rapid inactivation of budded virions. There are need several modifica‐ tions for reduce the negative effect of complement in baculovirus-mediated transduction. However, the complement system is not a problem only for baculovirus. It is also a potent barrier to in vivo administration of other gene delivery systems such as liposomes, murine retrovirus, and various synthetic DNA complexes (Hu, 2008).

Additionally, baculoviruses as enveloped virus are very fragile. The envelope structure is essential for virus infectivity because of the anchored Gp64, responsible of viral and cellular membrane fusion. (Blissard & Wenz, 1992). For this reason it renders virus vulnerable to mechanical force and results in relatively low virus stability, a common problem also observed for other enveloped viruses such as retrovirus. Ultracentrifugation is often necessary for budded virions purification, but also leads to significant loss of infectivity probably because of the viral envelopes damage. Labile thermal stability, in conjunction with the tendency to be inactivated by serum complement, may further restrict the in vivo application of baculovirus gene delivery vectors.

Eukaryotic expression libraries, in contrast, are a powerful tool for finding new ligands, identification of cellular interaction partners and affinity maturation of antibody and antibody

Baculovirus Display: A Novel Tool for Vaccination

http://dx.doi.org/10.5772/55572

157

As discussed before, the expression of foreign proteins on the surface of insect cells, in occlusion bodies and on the baculovirus surface make baculoviruses an important resource in biotechnology. Moreover, fusion proteins with the baculoviral envelope protein Gp64 as well as different foreign membrane proteins such as the influenza virus hemagglutinin or VSV-G protein have shown to be targeted to the surface of infected insect cells in several researches about baculovirus display. Then, it is possible take advantage of baculovirus display systems

Ernst et al. expressed a specific antibody epitope in the context of the influenza virus hemag‐ glutinin, randomizing the adjacent amino acid. This procedure results in the construction of a baculovirus surface display library capable to allow the selection of the displayed peptide with optimal antigenicity. Furthermore, baculovirus surface display libraries served to identify

In comparison with bacterial phage display in which cross infection does not occur and every infected cell just propagates one individual phage, in baculovirus surface display cross infection is very probably. The situation may result advantageous or disadvantageous depending the aim of the library. For the assembly of a multisubunit protein, this fact is highly advantageous. However, when the library is performed to screening different proteins, these cross infections have to be considered (Grabherr & Ernst, 2010). Adjusting the multiplicity of

In conclusion, baculovirus insect cell system consists in a highly useful tool for constructing and screening of surface display libraries, specially for the expression of eukaryotic complex

There are many biotechnological uses for baculoviruses. One of the most widespread is the use of baculoviruses as insecticide agents. Moreover, recombinant baculoviruses have been extensively used as expression vectors in insect cell cultures. A variety of technological improvements have eliminated the tedious procedures to isolate the recombinant viruses turning the baculovirus-based expression system in a safe, easy to use and scale up system

In addition, protein expression in larvae or cell culture is not the only application of baculo‐ viruses. In fact, baculoviruses are widely used in the development of strategies for displaying foreign peptides and proteins on the virus surface as well as mammalian cell transduction

As described in this chapter, baculovirus surface display based on the generation of Gp64 chimeric proteins result in a very efficient technology capable to induce a strong immune

with the aim to generate a surface display library for high trhoughput screening.

infection (moi) usually result convenient for avoid the cross infection problem.

MHC class I and II mimotopes (Grabherr & Ernst, 2010; Wang, 2005).

fragments (Grabherr & Ernst, 2010).

proteins (Ernst et al., 1998).

(Kost et al., 2005).

**8. Perspectives and conclusions**

using different mammalian expression cassettes.

#### **In vivo gene therapy**

Due to their ability to transduce various cell types, baculoviruses have captured increasing interest as vectors for in vivo gene delivery. Baculovirus-mediated gene delivery was tested in different tissues that including rabbit carotid artery, rat liver, rat brain, mouse brain, mouse skeletal muscle, mouse cerebral cortex and testis, and mouse liver (Hu, 2006). However, for baculovirus-mediated in vivo gene therapy in all of these tissues the complement system appears to be a significant barrier.

Baculovirus vectors have also been injected into the rodent brain where complement proteins may be absent because of the blood–brain barrier (Hu, 2008; Lehtolainen et al., 2002). After injection into the brain, baculoviruses specifically transduced the epithelium of the choroids plexus in ventricles and the obtained transduction efficiency was very high.

As discussed in previous sections, baculoviruses can be alternatively pseudotyped by displaying VSVG on the envelope. This modified virus enhanced gene transfer efficien‐ cies into mouse skeletal muscle and the transgene expression in mice. The VSVGmodified baculovirus also exhibited greater resistance to inactivation by the complement system present in animal sera.

Moreover, it has been shown that transduction of different cell lines with a baculovirus expressing shRNAs (short-hairpin RNAs) effectively knocked down expression of the target mRNA and protein (Nicholson et al., 2005). Additionally, baculoviruses have been used to mediate RNA interference (RNAi). The recombinant baculovirus encoding RNAi sequence was efficient in suppressing expression of the target gene by 95% in cultured cells and by 82% in vivo in rat brain. These data suggest that baculoviruses may be also used as delivery vectors for RNA interference therapies (Hu, 2008; Ong et al., (2005).

#### **7.2. Libraries**

Surface display libraries represent a very useful methodology for selecting binding proteins out of defined pools of protein variants. Although prokaryotic expression systems such as phage display technology or protein targeting to the cellular surface of Escherichia coli are widely used, they fail allowing the functional display of complex proteins such as eukaryotic glycoproteins which require a high degree of modification and processing. (Ernst 1998)

Eukaryotic expression libraries, in contrast, are a powerful tool for finding new ligands, identification of cellular interaction partners and affinity maturation of antibody and antibody fragments (Grabherr & Ernst, 2010).

As discussed before, the expression of foreign proteins on the surface of insect cells, in occlusion bodies and on the baculovirus surface make baculoviruses an important resource in biotechnology. Moreover, fusion proteins with the baculoviral envelope protein Gp64 as well as different foreign membrane proteins such as the influenza virus hemagglutinin or VSV-G protein have shown to be targeted to the surface of infected insect cells in several researches about baculovirus display. Then, it is possible take advantage of baculovirus display systems with the aim to generate a surface display library for high trhoughput screening.

Ernst et al. expressed a specific antibody epitope in the context of the influenza virus hemag‐ glutinin, randomizing the adjacent amino acid. This procedure results in the construction of a baculovirus surface display library capable to allow the selection of the displayed peptide with optimal antigenicity. Furthermore, baculovirus surface display libraries served to identify MHC class I and II mimotopes (Grabherr & Ernst, 2010; Wang, 2005).

In comparison with bacterial phage display in which cross infection does not occur and every infected cell just propagates one individual phage, in baculovirus surface display cross infection is very probably. The situation may result advantageous or disadvantageous depending the aim of the library. For the assembly of a multisubunit protein, this fact is highly advantageous. However, when the library is performed to screening different proteins, these cross infections have to be considered (Grabherr & Ernst, 2010). Adjusting the multiplicity of infection (moi) usually result convenient for avoid the cross infection problem.

In conclusion, baculovirus insect cell system consists in a highly useful tool for constructing and screening of surface display libraries, specially for the expression of eukaryotic complex proteins (Ernst et al., 1998).
