**4. Baculovirus display**

**a**.

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

**b**.

**c**.

**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.

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 (Grabherr & Ernst, 2010).

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 displayed proteins (Kost et al., 2005).

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

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

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

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. These studies will be discussedlater.

the full length gp64 coding region. (Boublik et al., 1995; Grabherr & Ernst, 2010). The conser‐ vation of the biological function of several proteins when they were expressed by the baculo‐ virus display system, e.g HIV gp120, indicated that large, complex proteins could be displayed

Baculovirus Display: A Novel Tool for Vaccination

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The mechanism of incorporation into the viral particle was proposed to be due to oligomeri‐ zation of the chimeric Gp64 with wild-type Gp64. In addition the CTD of the chimeric Gp64 may play an important role in the nucleocapsid recognition for budding process (Figure 5.a)

For the purpose of antigen display various epitopes were presented and shown to induce

The advantages of this method reside in that all needed sequences for glycoprotein transport and maturation are present in the entire sequence of Gp64. Complete Gp64 fused antigens will be synthetized through the glycoprotein synthesis pathway and will be directed to plasmatic

However, the utilization of entire Gp64 may cause some problems in the cloning process due

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

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‐

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

on the surface of baculovirus particles in a functional form.

immune response in mice.

**4.2. Peptide insertion on Gp64**

er et al., 2002).

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

membrane and also budded virus envelope.

to the length of the subsequent transfer vector.

gp64 with a maximum size of 23 amino acids (Figure 5.c).

**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 recombinant Gp64 expressing a small peptide.

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

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 the full length gp64 coding region. (Boublik et al., 1995; Grabherr & Ernst, 2010). The conser‐ vation of the biological function of several proteins when they were expressed by the baculo‐ virus display system, e.g HIV gp120, indicated that large, complex proteins could be displayed on the surface of baculovirus particles in a functional form.

The mechanism of incorporation into the viral particle was proposed to be due to oligomeri‐ zation of the chimeric Gp64 with wild-type Gp64. In addition the CTD of the chimeric Gp64 may play an important role in the nucleocapsid recognition for budding process (Figure 5.a)

For the purpose of antigen display various epitopes were presented and shown to induce immune response in mice.

The advantages of this method reside in that all needed sequences for glycoprotein transport and maturation are present in the entire sequence of Gp64. Complete Gp64 fused antigens will be synthetized through the glycoprotein synthesis pathway and will be directed to plasmatic membrane and also budded virus envelope.

However, the utilization of entire Gp64 may cause some problems in the cloning process due to the length of the subsequent transfer vector.
