**3. Baculovirus as immunogens**

The innate immune system provides the first line of host defense against infection. It is extremely important to mount a strong specific immune response by expressing co-stimulating factors necessary for the activation of adaptative immunity cells.

It was shown in previous articles that inoculation of a murine macrophage cell line with budded baculovirus induces the secretion of inflammatory cytokines, such as tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and IL-12 (Abe et al., 2005; Chimeno Zoth et al., 2012; Han et al., 2010; Hervas-Stubbs et al., 2007).

AcMNPV induces pro-inflammatory cytokines secretion through a MyD88/TLR9-dependent signaling pathway, while other signaling molecules may participate in IFN-α production in response to AcMNPV.

Toll-like receptors (TLRs) are a family of transmembrane proteins that recognize and bind endogenous and exogenous ligands. Signaling through TLR generally culminates in the production of pro-inflammatory cytokines resulting in modulation of several aspects of the innate immune response (Han et al., 2010). In the case of baculovirus, it has been reported that BVs could induce cytokine production through the TLR9 signaling pathway in mammals.

TLR9 was shown to be responsible in vivo for immune system stimulation by oligodeox‐ ynucleotides containing unmethylated CpG motifs. Like bacteria, AcMNPV contains a significant number of potentially bioactive CpG motifs. Indeed, a number of studies demonstrate that AcMNPV can stimulate professional Antigen Presenting Cells (APCs) by this pathway. Furthermore, Abe et al. demonstrated that internalization and endoso‐ mal maturation are required for TLR9 activation by CpG-rich DNA. They showed that the inhibition of endosomal maturation abolishes the immune system activation of AcMNPV in a dose-dependent manner. These results imply that immune system activation by AcMNPV through TLR9 requires membrane fusion via Gp64 as well as the liberation of the viral genome into cytoplasmic TLR9-containing vesicles (Figure 4.a)

Budded virions of baculoviruses enter cells by endocytosis. Gp64 is the major compo‐ nent of the viral envelope, and the unique protein with fusogenic activity in AcMNPV. Gp64 is triggered to induce the fusion at the low pH of endosomes. In addition Gp64 is distinguished from any other fusion protein in its ability of going through a reversi‐ ble conformational change, unlike class I and class II fusion proteins, for which the postfusion conformation is thermodynamically more stable and the conformational

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

Gp64 is expressed early and late in the infection of an insect cell. It is a 64 kDa protein which forms trimmers and locates in the BV envelope with a polarized distribution. As Gp64 is a transmembrane protein that exposes an outer domain, it can be used to display a selected protein on the BV surface. A chimeric Gp64 can be constructed to contain the protein of interest allowing it to be incorporated in the BV structure upon infection of

In order to facilitate the construction of a chimeric protein it was shown that is not necessary to conserve the complete structure of Gp64. The signal peptide (SP), the multimerization domain, the transmembrane (TM) and the cytoplasmic tail domain (CTD)were shown to be enough for the surface display, whereas the rest of the protein can be eliminated. This strategy avoids the need of dealing with large transfer vectors as well as permitting to increase the

The innate immune system provides the first line of host defense against infection. It is extremely important to mount a strong specific immune response by expressing co-stimulating

It was shown in previous articles that inoculation of a murine macrophage cell line with budded baculovirus induces the secretion of inflammatory cytokines, such as tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and IL-12 (Abe et al., 2005; Chimeno Zoth et al.,

AcMNPV induces pro-inflammatory cytokines secretion through a MyD88/TLR9-dependent signaling pathway, while other signaling molecules may participate in IFN-α production in

Toll-like receptors (TLRs) are a family of transmembrane proteins that recognize and bind endogenous and exogenous ligands. Signaling through TLR generally culminates in the production of pro-inflammatory cytokines resulting in modulation of several aspects of the innate immune response (Han et al., 2010). In the case of baculovirus, it has been reported that BVs could induce cytokine production through the TLR9 signaling pathway in mammals.

factors necessary for the activation of adaptative immunity cells.

2012; Han et al., 2010; Hervas-Stubbs et al., 2007).

rearrangement is irreversible.

**2.2. Gp64 for protein display**

insect cells (Grabherr & Ernst, 2010).

number of displayed proteins.

response to AcMNPV.

**3. Baculovirus as immunogens**

On the other hand, despite BVs cannot replicate in mammalian or other vertebrate animal cells (Via et al., 1983), recent studies showed that BVs have strong adjuvant properties in mice, promoting potent humoral and CD8+ T cell adaptive responses (Abe et al., 2003; Gronowski et al., 1999). In addition, BVs induce the production of inflammatory cytokines by the *in vivo* maturation of dendritic cells (Figura 4.c).

Zoth et al. evaluated the effect of baculovirus administration on the innate immune response of chickens. They found an upregulation of IFN-γ and IL-6 in the baculovirustreated chicken spleens and a decrease of the TGF-β gene expression. These facts indicated a strong proinflammatory immune response. Moreover, they demonstrated that BV induced modifications in the mononuclear cells pattern of different organs using flow cytometry.

The duration of the BV-induced response is very limited. This fact represents one of the many interesting benefits of the use of baculovirus for stimulating innate immunity, because the potential damage for a strong inflammatory immune response on an extended time period could be avoided (Chimeno Zoth et al., 2012)

On the other hand, it could be presumed that baculovirus inoculation produced an indirect effect on monocytes/macrophages. Zoth et al. also showed an increase of both the mRNA and the protein levels of IFN-γ, and a priming effect of Nitric Oxyde (NO) response in splenocytes of chickens treated with baculoviruses. NO acts as a multi-functional mediator with diverse physiological and pathological roles in host defense, (MacMicking et al., 1997). The production of NO by activated monocytes/macrophages is an important innate immune response sign of cellular antiviral and bactericidal activity.

Moreover, Kitajima et al. demonstrated that AcMNPV inoculation of mice induced NK cells activation. They observed that in AcMNPV inoculated animals there was up to fourfold increase in the number of NK cells in spleen, liver, bone marrow and thymus. Furthermore, it was analyzedt he antitumor ability of AcMNPV-induced NK cells and they concluded that AcMNPV injection induces a NKT cell and IFN-γ independent NK cell cytotoxicity against tumor cells in mice (Kitajima et al., 2007) These findings will be approached in section 7.

In conclusion, the strong immune response induced by AcMNPV makes it a promising candidate for a novel, adjuvant- containing vaccine vehicle against infectious diseases (Abe et al., 2005).

**4. Baculovirus display**

(Grabherr & Ernst, 2010).

displayed proteins (Kost et al., 2005).

These studies will be discussedlater.

humoral response against the antigen displayed (Figure 4.b).

Eukaryotic systems represent a highly interesting model for the study of higher eukaryotic structures and interaction mechanisms because they provide posttranslation‐ al modifications and complex protein folding, in contrast to prokaryotic systems. Moreover, displaying a protein on the surface of a cell or a virus is a very successful strategy, for recreating and maturing binding properties such as antigenic recognition

Baculovirus Display: A Novel Tool for Vaccination

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

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Several strategies have been developed for displaying heterologous peptides or pro‐ teins on the baculovirus envelope by fusing the peptide or protein to gp64. In most instances the vector is designed with the aim of obtaining baculovirus particles that contain both wild-type gp64 and chimeric gp64 molecules. Furthermore, baculoviruses displaying proteins fused to Gp64 have proven to be very effective immunogens and they have been used successfully to generate antibody responses to a variety of

Given that baculoviruses are able to mount a robust innate immune response by activating professional APCs, it is expected that baculovirus expressing an heterologous antigen on its surface could generate a specific response against this antigen. In fact, several works showed that baculoviruses expressing chimeric Gp64 on its surface were able to mount a very strong

Xu et al. demonstrated in several works that baculovirus surface display of different proteins of Japanese Encephalitis Virus and swine fever virus generated high titers of specific antibodies useful for the protection against the disease. More specifically, they found that inoculation with recombinant baculoviruses produced a specific IgG response comparable with the response mounted by the preexistent attenuated vaccine and high neutralizing antibody titers

Furthermore, numerous studies used baculovirus display for the development of new generation vaccines and obtained similar results to those showed by Xu et al. In this context, baculovirus surface display conferred protection and induced a strong humoral response against avian reovirus (Lin et al., 2008), human enterovirus (Meng et al., 2011), influenza (Jin

In the next sub-sections the different strategies for efficient baculovirus display will be discussed. These include baculovirus display using the entire Gp64 for the generation of the chimeric proteins, baculovirus display based on single peptide insertion in Gp64 and a truncated Gp64 system with several cloning advantages will be considered (Figure 5).

Baculovirus display strategies have also been used for modification of the viral surface to command baculovirus mediated transduction of mammalian cells. In addition, capsid modifications may allow novel approaches for enhancing baculovirus mediated gene delivery.

against the virus (Xu et al., 2008; Xu & Liu, 2008; Xu et al., 2009; Xu et al., 2011).

et al., 2008; Prabakaran et al., 2010), malaria (Yoshida et al., 2009), etc.

**Figure 4. Immune response induced by baculovirus summarized. a.** Activation of immune cells by inoculation with AcMNPV wild type. **b.** Immune response triggered by AcMNPV displaying a Gp64 fused antigen. **c.** Immune response generated by antigen coding AcMNPV under the control of CMV Ie1 promoter.
