**6. Baculovirus and complement**

Complement is an important component of the innate immune system and plays an important role in the recognition and elimination of pathogens. Complement can be activated by three separate pathways: the classical, alternative, and lectin pathways (Ricklin et al., 2010). The classical activation pathway begins with the binding of the complement protein C1q to the pathogen surface or to antibody-antigen complex. The alternative complement activation 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) (Figure 6.a.b).

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

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

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

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

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

Complement is an important component of the innate immune system and plays an important role in the recognition and elimination of pathogens. Complement can be activated by three separate pathways: the classical, alternative, and lectin pathways (Ricklin et al., 2010). The classical activation pathway begins with the binding of the complement protein C1q to the pathogen surface or to antibody-antigen complex. The alternative complement activation

proteins promoting both IFN secretion and cytotoxicCD8+ T cells activation.

CD8+ T cell response, but was very effective to induce a CD4+ T and B cell responses.

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

(Molinari et al., 2011).

with the BV-OVA protect against this tumor model.

powders (Chen et al., 2011; Song et al., 2010).

**6. Baculovirus and complement**

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

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 Kaikkonnen et al. 2011.

#### **6.1. Complement activation by baculoviruses**

As particulated antigens, baculoviruses are vulnerable to the action of the complement. This fact was observed in several studies which demonstrate that baculovirus-mediated gene transfer into hepatocytes is strongly reduced in the presence of untreated human serum.

Kim et al., 2010; Kim et al., 2006). Although serum stability of the PEG-coatedbaculovi‐ ruses was not directly studied, these results support the notion that PEG coating can be used to protect baculovirus vectors against the immune system and prolong its surviv‐

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Nevertheless, it is necessary adjust the ratiosof PEI or PEG and virus particles, and the polymer

Pseudotyping consists in a process in which the natural envelope proteins of the virus are replaced with surface proteins from another virus. This strategy has been shown to mitigate the problem of complement attack (Tani et al., 2003). Unlikechemical engineering which is limited and requires extensive optimization to retain virus infectivity, pseudotyping conserves virus infectivity and allows virusevasion ofcomplement-mediated destruction.The most widely used method of pseudotyping of baculoviruses relies on the employ of the VSV-Gprotein. Several researches have shown that VSV-G is capable to improve transduction efficiency of baculovirus in vertebrate cells (Barsoum et al., 1997; Pieroni et al., 2001; Tani et al., 2003; Tani et al., 2001). VSV-G can alsoreplace GP64 and allow productive infection, replication, and propagation of thevirus in Sf9 insect cells (Kitagawa et al., 2005; Mangor et al., 2001). However, pseudotyping is typically performed by co-expressing both the desired

Other reportsalso demonstrated increased gene delivery into mouse after direct intramuscular injection of VSV-Gpseudotypedbaculovirus (Pieroni & La Monica, 2001; Pieroni et al., 2001). Additionall, Tani et al. found that the VSV-Gmodified baculovirusexhibited greater resistance to human, rabbit, guinea pig, rat, hamster and mouse serum inactivation compared to the unmodified control baculovirus (Tani et al., 2003). Furthermore, co-display of a short trans‐ membrane fragment of VSV-G was found to give similar complement protection as intact VSV-G (Kaikkonen et al., 2010). These results suggest that envelope modification of the baculovirus can change its immunogenic properties and protect them for complement inactivation.

The last strategy for complement inactivation that will be discussed in this section consists in the baculovirus surface display of eukaryotic complement inhibitors. Several reports showed that genetic modificatedviruses expressing complement regulators presented an improved survival rate, unlike wild type controls. In this context, the most promising results to increase the serum stability of baculovirus vectors by genetic means have been attained by displaying complement regulating proteins fused to Gp64 on the virion surface (Huser et al., 2001; Kaikkonen et al., 2010). The first described research generated a recombinant baculovirus which expressed on its surface the DAF complement regulator. Kaikkonen et al. have recently verified the protective nature of DAF-display and studied the efficacy of other complement regulatory proteins (FHL-1, C4BP and MCP) and their combinations for complement inacti‐ vation and consequently baculovirus survival rates. (Kaikkonen et al., 2010). Their resultscon‐

al time in circulation (Jevsevar et al., 2010).

*6.2.2. Pseudotyping*

molecule and Gp64.

*6.2.3. Display of complement inhibitors*

sizeto preserve virus infectivity and minimize cytotoxicity.

The complement cascade is usually activated to protect the host from foreign elements. The complement activating properties of various gene transfer vectors was demonstrated. The mechanism of complement activation by liposomes and synthetic DNA complexes depends mainly on the formulation, charge and size. Murine retroviruses are effectively lysed by primate complement triggered by the classical pathway, involving direct binding of C1q and C1s to the envelope and/or to antibody-antigen complexes. Comparatively, Hoffmann et al. found baculovirus survival in C1q-depleted human serum indicating baculovirus-mediated activation of the complement cascade through the classical pathway.

Given that there is no evidence of pre-existing anti baculovirus antibodies in human sera, this data suggests that baculoviruses activate the complement cascade by an antibody-independent activation of the classical pathway (Hofmann & Strauss, 1998).

#### **6.2. Strategies for complement inactivation**

At present there are different strategies which help to avoid complement attack during baculovirus treatment (Huser et al., 2001; Kaikkonen et al., 2010). As noted previously, the surface of baculovirus particles can easily be engineered. As an example,desired peptides or proteins can be displayed as fusion proteins (Boublik et al., 1995; Makela & Oker-Blom, 2008; Oker-Blom et al., (2003). The most widely used technique for surface engineering makes use of the trimeric major baculoviral envelope glycoprotein GP64 as a fusion partner (Kadlec et al., 2008). In this section, diverse strategies for complement inactivation mediated by baculo‐ viruses will be discussed. In particular, the discussion will be focused in the use of polymers for baculovirus surface coating, pseudotyping of baculoviruses by the expression on VSV-G protein and surface display of eukaryotic complement inhibitors.

#### *6.2.1. Polymer coating*

With the aim to protect baculoviral vectors against complement inactivation, using polymers should be appropriated. The coating is based on the electrostatic interaction between the virus particle and the polymer. In the case of baculovirus, the negative charge of its surface allows coating with positively charged polymers such aspolyethylenimine (PEI) (Yang et al., 2009). It was observed that the 25 kDa PEI protected the virions against complement destruction resulting in a 10% to nearly 100% of vector survival in samples treated with human and rat serum, respectively. In addition,Kim et al. observed thatafter intraportal delivery the PEItreated viruses exhibited improved transduction of liver and spleen compared to non-coated virions(Kim et al., 2009).

Additionally, another polymer, PEG (Mw 5000), has also been reported to increasebacu‐ lovirus transduction efficiency in vitro and in mouse brain and lung (Kim et al., 2007; Kim et al., 2010; Kim et al., 2006). Although serum stability of the PEG-coatedbaculovi‐ ruses was not directly studied, these results support the notion that PEG coating can be used to protect baculovirus vectors against the immune system and prolong its surviv‐ al time in circulation (Jevsevar et al., 2010).

Nevertheless, it is necessary adjust the ratiosof PEI or PEG and virus particles, and the polymer sizeto preserve virus infectivity and minimize cytotoxicity.

## *6.2.2. Pseudotyping*

**6.1. Complement activation by baculoviruses**

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

As particulated antigens, baculoviruses are vulnerable to the action of the complement. This fact was observed in several studies which demonstrate that baculovirus-mediated gene transfer into hepatocytes is strongly reduced in the presence of untreated human serum.

The complement cascade is usually activated to protect the host from foreign elements. The complement activating properties of various gene transfer vectors was demonstrated. The mechanism of complement activation by liposomes and synthetic DNA complexes depends mainly on the formulation, charge and size. Murine retroviruses are effectively lysed by primate complement triggered by the classical pathway, involving direct binding of C1q and C1s to the envelope and/or to antibody-antigen complexes. Comparatively, Hoffmann et al. found baculovirus survival in C1q-depleted human serum indicating baculovirus-mediated

Given that there is no evidence of pre-existing anti baculovirus antibodies in human sera, this data suggests that baculoviruses activate the complement cascade by an antibody-independent

At present there are different strategies which help to avoid complement attack during baculovirus treatment (Huser et al., 2001; Kaikkonen et al., 2010). As noted previously, the surface of baculovirus particles can easily be engineered. As an example,desired peptides or proteins can be displayed as fusion proteins (Boublik et al., 1995; Makela & Oker-Blom, 2008; Oker-Blom et al., (2003). The most widely used technique for surface engineering makes use of the trimeric major baculoviral envelope glycoprotein GP64 as a fusion partner (Kadlec et al., 2008). In this section, diverse strategies for complement inactivation mediated by baculo‐ viruses will be discussed. In particular, the discussion will be focused in the use of polymers for baculovirus surface coating, pseudotyping of baculoviruses by the expression on VSV-G

With the aim to protect baculoviral vectors against complement inactivation, using polymers should be appropriated. The coating is based on the electrostatic interaction between the virus particle and the polymer. In the case of baculovirus, the negative charge of its surface allows coating with positively charged polymers such aspolyethylenimine (PEI) (Yang et al., 2009). It was observed that the 25 kDa PEI protected the virions against complement destruction resulting in a 10% to nearly 100% of vector survival in samples treated with human and rat serum, respectively. In addition,Kim et al. observed thatafter intraportal delivery the PEItreated viruses exhibited improved transduction of liver and spleen compared to non-coated

Additionally, another polymer, PEG (Mw 5000), has also been reported to increasebacu‐ lovirus transduction efficiency in vitro and in mouse brain and lung (Kim et al., 2007;

activation of the complement cascade through the classical pathway.

activation of the classical pathway (Hofmann & Strauss, 1998).

protein and surface display of eukaryotic complement inhibitors.

**6.2. Strategies for complement inactivation**

*6.2.1. Polymer coating*

virions(Kim et al., 2009).

Pseudotyping consists in a process in which the natural envelope proteins of the virus are replaced with surface proteins from another virus. This strategy has been shown to mitigate the problem of complement attack (Tani et al., 2003). Unlikechemical engineering which is limited and requires extensive optimization to retain virus infectivity, pseudotyping conserves virus infectivity and allows virusevasion ofcomplement-mediated destruction.The most widely used method of pseudotyping of baculoviruses relies on the employ of the VSV-Gprotein. Several researches have shown that VSV-G is capable to improve transduction efficiency of baculovirus in vertebrate cells (Barsoum et al., 1997; Pieroni et al., 2001; Tani et al., 2003; Tani et al., 2001). VSV-G can alsoreplace GP64 and allow productive infection, replication, and propagation of thevirus in Sf9 insect cells (Kitagawa et al., 2005; Mangor et al., 2001). However, pseudotyping is typically performed by co-expressing both the desired molecule and Gp64.

Other reportsalso demonstrated increased gene delivery into mouse after direct intramuscular injection of VSV-Gpseudotypedbaculovirus (Pieroni & La Monica, 2001; Pieroni et al., 2001). Additionall, Tani et al. found that the VSV-Gmodified baculovirusexhibited greater resistance to human, rabbit, guinea pig, rat, hamster and mouse serum inactivation compared to the unmodified control baculovirus (Tani et al., 2003). Furthermore, co-display of a short trans‐ membrane fragment of VSV-G was found to give similar complement protection as intact VSV-G (Kaikkonen et al., 2010). These results suggest that envelope modification of the baculovirus can change its immunogenic properties and protect them for complement inactivation.

#### *6.2.3. Display of complement inhibitors*

The last strategy for complement inactivation that will be discussed in this section consists in the baculovirus surface display of eukaryotic complement inhibitors. Several reports showed that genetic modificatedviruses expressing complement regulators presented an improved survival rate, unlike wild type controls. In this context, the most promising results to increase the serum stability of baculovirus vectors by genetic means have been attained by displaying complement regulating proteins fused to Gp64 on the virion surface (Huser et al., 2001; Kaikkonen et al., 2010). The first described research generated a recombinant baculovirus which expressed on its surface the DAF complement regulator. Kaikkonen et al. have recently verified the protective nature of DAF-display and studied the efficacy of other complement regulatory proteins (FHL-1, C4BP and MCP) and their combinations for complement inacti‐ vation and consequently baculovirus survival rates. (Kaikkonen et al., 2010). Their resultscon‐ cluded that serum stability was dependent on the displayed complement regulatory protein and the source of serum.

In first place, baculovirus-mediated transduction does not present any toxic effect against mammalian cells and does not disturb cell growth even at high MOI (Gao et al., 2002; Hofmann et al., 1995). In contrast, cell proliferation may be retarded by transgene products because they could be toxic and even induce apoptosis in some cells (Detrait et al., 2002; Liu & Carstens, 1999). Furthermore, baculoviruses do not replicate in transduced mammalian cells (Kost & Condreay, 2002). These features of baculoviruses are particularly important because other viral

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Another advantage of baculoviruses as gene therapy vectors consists in its large cloning capacity. The baculovirus (AcMNPV) genome is a large circularized DNA molecule with 130 kb of length and a maximum cloning capacity of at least 38 kb. This flexibility results partic‐ ularly advantageous in contrast to retroviral and AAV vectors whose cloning capacities are

In comparison with other viral vectors, baculoviruses are easy to produce. Retroviral, lentivi‐ ral, and AAV vectors require transfection of plasmids encoding essential genes into packaging cells for its production. In contrast, baculovirus can be easily propagated by infecting insect cells in suspension culture or monolayer and harvesting the supernatant 3–4 days postinfec‐ tion. In addition, the construction, propagation, and handling of baculoviruses can be per‐

Finally, one of the most important advantages is that baculoviruses do not present preexisting immunity in mammalian. One of the problems associated with other viral vectors is that most people are exposed to these viruses and develop specific humoral response. Circulating antibodies can significantly reduce the efficiency of transduction with the viral vector. The use of baculovirus vectors in gene therapy, therefore, may avoid the problem of preexisting

However, baculoviruses have a number of disadvantages as gene therapy vectors. One of these is that baculovirus induce a transient expression in mammalian cells. In vivo, transgene expression typically declines by day 7 and disappears by day 14 (Airenne et al., 2000; Lehto‐ lainen et al., 2002). The duration of in vitro transgene expression using baculoviruses is significantly shorter than expression mediated by retroviral, lentiviral, and AAV vectors.

Baculoviral vectors differ mainly than other viral vectors in the time that the carried genes can persist in the host nucleus. In the case of retroviral, lentiviral and adenoviral vectors, viral DNA can remain into the nucleus either in an integrated or episomal form, for a longer period. In fact, Tjia et al. demonstrated that baculoviral DNA persists in the nuclei of transduced

Another disadvantage of using baculovirus as gene therapy vector is the inactivation by complement. As described in previous sections, contact between baculoviruses and serum complement results in rapid inactivation of budded virions. There are need several modifica‐ tions for reduce the negative effect of complement in baculovirus-mediated transduction. However, the complement system is not a problem only for baculovirus. It is also a potent barrier to in vivo administration of other gene delivery systems such as liposomes, murine

formed in Biosafety Level 1 laboratories without the need for specialized equipment.

vectors are human pathogens, and consequently represent a biological risk.

limited (Hu, 2008).

immunity (Hu, 2008).

mammalian cells for only 24–48 h (Tjia et al., 1983).

retrovirus, and various synthetic DNA complexes (Hu, 2008).

In general, the complement regulators DAF and MCP gave the best results. Conversely, simultaneous co-display of soluble complement regulatory proteins did not provide further benefit.Best protection was gained in mouse serum (70%), while the worst protection rate was obtained with rat serum (13%). In the case of human serum, about 30% of the viral particles were still competent to transduce mammalian cells after 1 h preincubation with serum (Kaikkonen et al., 2010).

All these data suggest that engineering of baculoviral vectors for complement inactivation result very convenient not only to reduce the number of necessary inoculations for an efficient transduction, but also to avoid the undesired mortality induced by high doses of non-modified vectors.
