**8. Perspectives and conclusions**

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

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

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

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

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

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

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)

plexus in ventricles and the obtained transduction efficiency was very high.

for RNA interference therapies (Hu, 2008; Ong et al., (2005).

gene delivery vectors.

**In vivo gene therapy**

appears to be a significant barrier.

system present in animal sera.

**7.2. Libraries**

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 (Kost et al., 2005).

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 using different mammalian expression cassettes.

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 response against specific antigens (Xu et al., 2009). The ability of baculoviruses to activate innate immune system cells guarantees the mount of a robust immune response and the generation of immunological memory. More specifically, AcMNPV induces pro-inflammatory cytokines secretion through a MyD88/TLR9-dependent signaling pathway (Abe et al., 2005; Chimeno Zoth et al., 2012).

[2] Abe, T, Takahashi, H, Hamazaki, H, Miyano-kurosaki, N, Matsuura, Y, & Takaku, H. (2003). Baculovirus induces an innate immune response and confers protection from

Baculovirus Display: A Novel Tool for Vaccination

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[3] Airenne, K. J, Hiltunen, M. O, Turunen, M. P, Turunen, A. M, Laitinen, O. H, Kulo‐ maa, M. S, & Yla-herttuala, S. (2000). Baculovirus-mediated periadventitial gene

[4] Backovic, M, & Jardetzky, T. S. (2009). Class III viral membrane fusion proteins. *Cur‐*

[5] Barsoum, J, Brown, R, Mckee, M, & Boyce, F. M. (1997). Efficient transduction of mammalian cells by a recombinant baculovirus having the vesicular stomatitis virus

[6] Blissard, G. W, & Wenz, J. R. (1992). Baculovirus gp64 envelope glycoprotein is suffi‐

[7] Bon, L, & Lucchetti, C. (2006). Auditory environmental cells and visual fixation effect

[8] Boublik, Y. Di Bonito, P. & Jones, I. M. ((1995). Eukaryotic virus display: engineering the major surface glycoprotein of the Autographa californica nuclear polyhedrosis vi‐ rus (AcNPV) for the presentation of foreign proteins on the virus surface. *Biotechnolo‐*

[9] Chen, C, Lin, Y, Chen, C. -Y, & Hu, G. -Y. Y.-C. ((2011). Baculovirus as a gene deliv‐ ery vector: Recent understandings of molecular alterations in transduced cells and

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241-247.

It was showed by several authors that baculovirus surface display induced high specific antibody titers against various virus families and parasitic pathogens (Jordan et al., 2009; Meng et al., 2011; Prabakaran et al., 2010; Yoshida et al., 2009). Furthermore, it was demonstrated that many of these titers had neutralizing properties.

On the other hand, it was discussed before that baculoviruses could also transduce mammalian cells (Kost et al., 2005). This feature results very interesting because it allows intracellular expression of heterologous proteins and its subsequent presentation through the MHC class I pathway. In this context, several authors demonstrate that baculoviruses can also induce a specific cellular immune response either by cloning the desired antigen under the control of a suitable promoter, or through the capsid display technique. CTL activation and IFN-γ secretion was detected in all of these researches (Yoshida et al., 2009).

Finally, baculovirus were shown to be useful as gene therapy vectors so as to create libraries of binding proteins.

For all these reasons, we conclude that baculoviruses represent a very useful tool in biotech‐ nology as vaccination vectors. Its adjuvant capacity makes baculoviruses in a promising alternative for the generation of immunological memory.
