**5.1. Baculovirus and mammalian cell transduction**

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

This method conserves the advantages of the baculovirus surface display using the entire

Apart from infecting insect cells, baculoviruses are able to transduce different types of animal cells such as human, rodent, rabbit, porcine, bovine, fish and avian cells (Hu, 2005; Hu, 2006) In addition, baculovirus can transduce embryonic stem cells, adult stem cells and induced

Baculovirus are safer than other transduction vectors because they don´t integrate its DNA into host genome, nor replicates it inside the transduced cells (Chen et al., 2011; Merrihew et al., 2001). It has been demonstrated that humans do not possess pre-existing antibodies and specific T-cells against baculoviruses (Strauss et al., 2007). For this reason, baculoviruses may

Thus, the coding sequence of a protein of interest can be cloned into the viral genome under the control of a suitable promoter. Then, the inoculation of an animal with the recombinant virus results in the expression of the heterologous protein inside different cell types. The expression of a foreign protein in the cytoplasm trigger the MHC class I antigen presentation of proteasome processed peptides of the recombinant protein. In this way, joined to the adjuvancy showed by baculoviruses, transduction of animal cells may induce a strong cellular

Yoshida et al. have developed a baculovirus based dual expression system, with the aim to develop multifunctional vaccines capable of inducing strong humoral and cellular immune responses. In this study a chimeric protein was constructed with the display necessary sequences of Gp64 and the entire open reading frame of the*Plasmodium berghei* circumsporo‐ zoite protein (PbCSP) under the control of polyhedron and CMV Ie1 promoters. ELISPOT assays with splenocytes from immunized mice with the recombinant baculovirus showed significative IFN-γ secretion compared with the results for immunization with a recombinant AcMNPV without the CMV Ie1 promoter when the splenocytes was stimulated with a PbCSP synthetic peptide. In addition, this baculovirus based dual system showed to be more protec‐

On the other hand, Hervas-Stubbs et al. demonstrated that baculoviruses induced strong humoral and cellular immune responses by co-administration of AcMNPV wt. and a purified antigen.They showed that budded baculoviruses had strong adjuvant properties, promoting humoral and CTL responses against coadministered antigen. They observed also that baculo‐ virus could induced DC maturation, and the production of inflammatory mediators through mechanisms primarily mediated by IFN-α and IFN-β.It has been shown previously that type

Gp64, reducing significantly possible cloning troubles (Figure 5.b).

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

avoid the pre-existing immunity problem caused by other viral vectors.

tive than the simple baculovirus display system (Yoshida et al., 2009)

**5. Baculovirus and cellular immunity**

pluripotent stem cells (Chen et al., 2011).

immune response (Figure 4.c).

Xu et al., 2009).

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 a role (Chen et al., 2011).

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

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 recombinant proteins could be expressed.

### **5.2. Baculovirus capsid display**

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 diseases.

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 (Molinari et al., 2011).

In the section 5.1 it were described the possible mechanisms of entry of baculovirus in mammalian cells. Besides the complete mechanism diverge in different cell types, endosome trafficking and Gp64 mediated fusion are always involved. Under these circumstances, it seems unlikely that the antigen displayed on the BV envelope would be able to efficiently reach the cytoplasm and consequently would be preferentially presented by MHC class II pathway. For this reason, antigen displayed on the envelope of baculovirus failed to produce a robust CD8+ T cell response, but was very effective to induce a CD4+ T and B cell responses.

pathway is initiated by spontaneous hydrolysis of the C3 protein into C3a and C3b and the subsequently attaching of C3b to amine and carbohydrate groups on the target surface. Finally, the lectin pathway is activated by the recognition of specific carbohydrate patterns on the pathogen surface by mannose-binding proteins. Once complement was activated, a cascade of proteolysis events of complement proteins leads to the recruitment of the membrane attack complex (MAC) and the subsequently target membrane perforation (Kaikkonen et al., 2011)

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On the other hand, complement must be regulated. There are two different types of comple‐ ment regulators: Surface-bound regulators, and soluble regulators. Surface-bound regulators consists in a group of molecules integratedby factors that accelerate decay of the convertases (complement receptor 1, CR1; decay accelerating factor, DAF), act as a cofactor for the factor I-mediated degradation of C3b and C4b (CR1; membrane cofactor protein, MCP), or prevent the formation of the membrane attack complex (CD59) (Hourcade et al., 2000; Ricklin et al., 2010). Soluble regulators also mediate the first two functions of surface-bound regulators. C4bbinding protein (C4BP), factor H (FH) and FH like protein-1 (FHL-1) are examples of the

In this context, baculovirus engineering with the aim to confer it resistance to complement inactivation results very attractive to improve the efficiency of baculoviruses for gene delivery.

**Figure 6.** The two major complement activation pathways in baculoviruses: The classical pathway is triggered by the binding of C1 to antigen-bound antibody molecules. The classical pathway utilize C2 and C4 to generate the C3-con‐ vertase C4b2a. The alternative pathway is initiated by the spontaneous hydrolysis of C3. Then, the complete comple‐ ment cascade (from C3 proteolysis to formation of membran attack complex (MAC)) proceeds. Adapted from

members of this group (Kaikkonen et al., 2011) (Figure 6.c).

(Figure 6.a.b).

Kaikkonnen et al. 2011.

However, antigen capsid display should be able to reach the cytosol and preferentially trigger MHC class I presentation pathway and mount a strong CD8+ T cell response (Molinari et al., 2011).

In this context, Molinari et al. developed a capsid display system and probed it fusing OVA with VP39 (BV-OVA) and showed that OVA could enter into the MHC class I pathway. Consequently, it was observed that inoculation of an animal model with the recombinant baculovirus triggered the activation of naive CD8+ T cells inducing an OVA-specific cytotoxic response. Though the mechanism involved in OVA MHC class I presentation was not elucidated, all these data suggest that capsid display is more convenient over envelope surface display for CTL activation. One of the proposed hypothesis consists of the possibility of the entire baculovirus capsid digestion by proteasome generating MHC class I binding peptides.

In summary, baculovirus are internalized by DCs and induce their maturation and the production of the pro-inflammatory cytokines IL-6 and IL-12 and are able to mount a type I IFN response (Section 3). Finally, Molinari et al. also examined the efficacy of the strong CTL and innate immune response elicited by baculovirus by the capacity of BV-OVA to confer protection against the classical MO5 melanoma tumor model. It was observed that inoculation with the BV-OVA protect against this tumor model.

Other researchers used capsid display as an alternative for mammalian cells transduction. In the work presented by Song et al. the ZnO binding peptide has been fused to the N-terminus of VP39 while retaining the viral infectivity and conferring the ability to bind nanosizedZnO powders (Chen et al., 2011; Song et al., 2010).

In conclusion, capsid display results in a very attractive alternative for cells transduction and for triggering MHC class I presentation of antigenic peptides. In this way, capsid display showed to be strongly effective to mount a robust cellular response against heterologous proteins promoting both IFN secretion and cytotoxicCD8+ T cells activation.
