**4.2. Peptide insertion on Gp64**

Another strategy consists in peptides directly engineered into the native Gp64 of AcMNPV in order to increase the avidity of the displayed target. In this case a short peptide is inserted into the sequence of the wild type Gp64, being this protein the only variant expressed in the virion, in contrast to the previous approach where both wt and the modified versions coexisted in the BV surface. It has been reported that this method resulted very efficient to mount a robust specific antibody response against the inserted peptide with a significantly increased avidity.

However, manipulating the native gp64 envelope protein may cause some problems. Given that no wild-type gp64 exists in order to guarantee functional cell fusion and virus budding, it is possible that the overall incorporation of the recombinant protein into cell membrane or viral envelope as well as viral titers decrease considerably. For this reason, insertion sites for foreign fragments must be chosen carefully. Moreover, the size of the peptides for insertion results in a limiting condition. Indeed, only small peptides have been inserted into the native gp64 with a maximum size of 23 amino acids (Figure 5.c).

Alternatively, expressing a second copy of gp64 displaying the target peptide in addition to the wild type Gp64 represents an effective solution (Grabherr & Ernst, 2010; Speng‐ er et al., 2002).

#### **4.3. SP, TM and CTD display systems**

**4.1. Chimeric proteins using the entire Gp64**

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

recombinant Gp64 expressing a small peptide.

Gp64 can serve as a fusion partner that together with a chosen target protein gets incorporated into the cell membrane and into budded virions. In the first reports of baculovirus display proteins were fused to the complete gp64. In these works the target proteins were cloned into a vector providing N-terminal fusion with the gp64 signal peptide and C-terminal fusion with

**Figure 5. Different kinds of baculovirus display. a.** Baculovirus surface display using the entire Gp64. **b.** Baculovirus surface display using only TM, MMD and CTD as fusion partner of the antigenic target. c. Baculovirus display using

> More recently, several reports demonstrated that using only the signal peptide region (SP),transmembrane region (TM) and the cytoplasmic tail domain (CTD) was enough for surface display on the insect cell surface as well as on the budded virions. The resulting smaller transfer vectors represented a significant improvement for the increased cloning efficiencies

and the number of displayed chimeric proteins (Grabherr & Ernst, 2010; Spenger et al., 2002; Xu et al., 2009).

I IFNs act directly on naive B cells and CD4+ and CD8+ T cells, promoting clonal expansion

Baculovirus Display: A Novel Tool for Vaccination

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

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Baculovirus entry into mammalian cells represents an important goal for immune response induction and most recently for different genic therapies. It was initially suggested that baculovirus entry depended on electrostatic interactions, heparin sulfate and phospholipids (Duisit et al., 1999; Tani et al., 2001), but the exact cell surface molecules and the involved mechanism remained unknown. Based on the mechanism of Gp64 mediated membrane fusion and the entry pathway of baculoviruses in insect cells, it was also proposed that clathrinmediated endocytosis and macropinocytosis play roles in baculovirus entry (Long et al., 2006; Matilainen et al., 2005) In contrast, Laakkonen et al.(2008) discovered that baculovirus could enter some types of mammal cells, such as hepatic cells, by a pathway independent of clathrin-mediated endocytosis and macropinocytosis suggesting that phagocytosis might play

These data suggest that baculovirus entry pathway varies with cell types and will be necessary more studies to elucidate the complete mechanisms. Nevertheless, all studies determined that baculovirus envelope protein gp64 is pivotal for entry and for the activation of dendritic cells

Once inside the cells, baculovirus is transported to the endosome. Then, virions are released by the acid-triggered gp64 fusion (Kukkonen et al., 2003) and subsequently transported into the nucleus (Laakkonen et al., 2008; van Loo et al., 2001) reorganizing the actin cytoskeleton (Matilainen et al., 2005); Salminen et al., 2005). A major component of type III intermediate filaments, vimentin, also participates in intracellular trafficking (Mahonen et al., 2010). Inside the nucleus, baculoviral DNA could be recognized by the cellular transcription machinery and

As was described before in this section, several authors reported strategies in which the coding sequence of an antigen was cloned driven by the cytomegalovirus (CMV) promoter to obtain antigen specific T cell immune responses, resulting in high levels of protection against parasitic

In addition to the baculovirus surface display on the envelope, heterologous protein has been displayed on the capsid by fusion with the major capsid protein VP39 without any interference in the virus assembly (Molinari et al., 2011). Kukkonen et al. fused the enhanced green fluorescent protein (EGFP) with VP39 with the aim to improve the nuclear traffic of BV in mammalian cells, and shown no interference with virus titer (Kukkonen et al., 2003). This finding suggested the possibility of performing insertions into the inner capsid of the BV particle. VP39 is the most abundant protein of the nucleocapsid and consist in a 39 KDa polypeptide with monomers arranged in stacked rings around the nucleoprotein core

and differentiation (Curtinsger 2005; (Bon & Lucchetti, 2006).

(DCs) (Abe et al., 2005; Niu et al., 2008; Schutz et al., 2006).

recombinant proteins could be expressed.

**5.2. Baculovirus capsid display**

diseases.

(Molinari et al., 2011).

**5.1. Baculovirus and mammalian cell transduction**

a role (Chen et al., 2011).

This method conserves the advantages of the baculovirus surface display using the entire Gp64, reducing significantly possible cloning troubles (Figure 5.b).
