key: cord-1036585-1db5qvi0
authors: Fabre, M. Laura; Arrías, Paula N.; Masson, Tomás; Pidre, Matías L.; Romanowski, Víctor
title: Baculovirus-Derived Vectors for Immunization and Therapeutic Applications
date: 2019-09-20
journal: Emerging and Reemerging Viral Pathogens
DOI: 10.1016/b978-0-12-814966-9.00011-1
sha: cc05521634a830e5f599bb3c11935e78e0db048f
doc_id: 1036585
cord_uid: 1db5qvi0

Baculoviruses are arthropod-specific, enveloped viruses with circular, supercoiled double-stranded deoxyribonucleic acid genomes. While many viruses are studied to seek solutions for their adverse impact on human, veterinary, and plant health, the study of baculoviruses was stimulated initially by their potential utility to control insect pests. Later, the utility of baculovirus as gene expression vectors was evidenced leading to numerous applications. Several strategies are employed to obtain recombinant viruses that express large quantities of heterologous proteins. A major step forward was the development of bacmid technology (the construction of bacterial artificial chromosomes containing the genome of the baculovirus) which allows the manipulation of the baculovirus genome in bacteria. With this technology, foreign genes can be introduced into the bacmid by homologous and site-directed recombination or by transposition. Baculoviruses have been used to explore fundamental questions in molecular biology such as the nature of programmed cell-death. Moreover, the ability of baculoviruses to transduce mammalian cells led to the consideration of their use as gene-therapy and vaccine vectors. Strategies for genetic engineering of baculoviruses have been developed to meet the requirements of new application areas. Display of foreign proteins on the surface of virions or in nucleocapsid structures, the assembly of expressed proteins to form virus-like particles or protein complexes have been explored and validated as vaccines. The aim of this chapter is to update the areas of application of the baculoviruses in protein expression, alternative vaccine designs and gene therapy of infectious diseases and genetic disorders. Finally, we review the baculovirus-derived products on the market and in the pipeline for biomedical and veterinary use.

Baculoviruses are insect-specific pathogens that have been isolated from about 600 species belonging to the orders Lepidoptera, Hymenoptera, Diptera, Orthoptera, Coleoptera, Neuroptera, Thysaneura, and Trichoptera. Taxonomy of Baculoviridae family includes four genera: Alphabaculovirus (nucleopolyhedroviruses specific of lepidopterans), Betabaculovirus (granuloviruses specific of lepidopterans), Gammabaculovirus (nucleopolyhedroviruses specific of hymenopterans), and Deltabaculovirus (granuloviruses specific of dipterans) (Jehle et al., 2006; Herniou et al., 2011) . These viruses infect arthropod larvae and possess circular doublestranded deoxyribonucleic acid (DNA) genomes that range in size from 80 to 180 kbp. The baculoviral genome is packed inside a distinct rod-shaped nucleocapsid (NC) . The life cycle of baculoviruses is biphasic, and each phase is characterized by a different viral phenotype. In the environment, baculoviruses are found as occlusion bodies (OBs). A protein matrix encloses virions and provides protection against harsh environmental conditions such as temperature, dehydration, and ultraviolet light until a susceptible larva ingests the OBs. Briefly, the baculovirus infection cycle of insect hosts is as it follows: OBs are ingested together with foliage by larvae and dissolved in the midgut-releasing occlusion-derived viruses (ODVs), and the damaged peritrophic membrane allows the receptormediated endocytosis of the ODV by epithelial cells (Adams and McClintock., 1991) . ODVs may contain one or multiple rod-shaped NCs depending on the baculovirus (or less frequently on growth conditions). The NCs enter the cytoplasm and translocate to the nucleus where the viral DNA is transcribed and replicated. Newly synthesized genomic DNA assembles with specific viral proteins to form the NCs in the nucleus. The NCs travel to the plasma membrane where budding takes place to yield the budded viruses (BVs), the second viral phenotype, responsible for the systemic infection of the larvae.

Gene expression is temporally regulated; early genes are transcribed directly by host ribonucleic acid (RNA) polymerase II, whereas late and very-late gene transcription is dependent on viral RNA polymerase. Late genes code mostly for structural proteins characteristic of the beginning of systemic infection while very-late genes code for most ODV structural proteins including the major OB matrix protein (Horton and Burand, 1993; Haas-Stapleton et al., 2004; Au et al., 2013) . BVs can infect both in vivo tissues and insect cell cultures (Laakkonen et al., 2008; Long et al., 2006a,b) . The possibility to propagate these viruses in cell culture has made baculoviruses an accessible tool for genetic engineering (Goodwin et al., 1970) , both for the study of their biology and for the development of a recombinant protein expression system (BEVS: baculovirus expression vector system) (Smith et al., 1983) . This system is considered a useful, efficient, and robust tool in many areas, including but not limited to biotechnology, clinical biochemistry, and molecular biology ( Fig. 11 .1).

Even though in the last three decades, different strategies have been developed for the generation of recombinant baculoviruses, two remain the most widely used. The more traditional approach consists of cotransfecting insect cells with both a baculoviral genome and a transfer plasmid. The transfer plasmid carries the genes of interest flanked by two regions containing sequences for homologous recombination; meanwhile, the viral genome provides the genetic information of a wild-type virus. Isolation of the recombinant event was made by selecting the OB-phenotype (nonoccluded viruses) observed by microscopy. However, due to a low recombination frequency ranging from 0.1% to 1%, this original technique yielded a high proportion of nonrecombinant wild-type virus (Fig. 11 .2A) (O'Reilly and Miller, 1991) . This contamination with parental virus makes isolation and purification of recombinant viruses laborious since it involves multiple plaque FIGURE 11.1 Baculovirus expression vector system (BEVS). The recombinant baculovirus (rBV) enters the cell via endocytosis. Following this event, the nucleocapsid (NC) is released into the cytoplasm and translocated into the nucleus, where viral DNA will be expressed. A simplified scheme of the viral DNA is presented, highlighting the promoter and the coding sequence of the gene of interest (goi). Alternative promoters: Immediate early protein ie1, polh, p10, gp64, p6.9, vp39 promoters from baculovirus genomes are used for protein expression in insect cells and promoters such as Simian Virus SV40, immediate early protein ie1 cytomegalovirus (CMV), elongation factor (EF)-1α, hybrid CAG promoter, human ubiquitin C, U6, Pol III H1, and Drosophila melanogaster hsp70, among others, are commonly used for expression in mammalian cells. On the left of the schematic: different applications of the baculovirus expression system. selection steps that are time consuming. In order to reduce the parental virus progeny, the baculoviral genome was linearized in a unique restriction site (Bsu36I) in the accessory gene polh, which codes for the major protein of the nucleopolyhedroviruses' OB, polyhedrin (POLH) and has a strong baculoviral promoter for high expression in insect cells. Since linear genome cannot replicate (noninfective), only the genomes that become circular after homologous recombination with the transfer vector are able to produce progeny. This modification to the system provides a recombination frequency near 30%, but the (A) Classic methodology. rBV are generated by homologous recombination between a baculoviral wild-type genome and a transfer plasmid containing the gene of interest (goi) in insect cells; (B) Linearized viral genome approach. The viral genome is linearized and the essential gene orf1629 is truncated. This approach facilitates the recombinant virus' selection steps. Homologous recombination reconstitutes orf1629; (C) Bac-to-Bac transposition-based technology. The goi is inserted by site-specific transposition into a bacmid inside Escherichia coli cells; (D) FlashBAC system. Homologous recombination occurs between a bacmid and a transfer plasmid in insect cells. Bacterial sequences are eliminated after the recombination event.

possibility of isolating wild-type virus still exists due to genetic repair events or incomplete digestion ( Fig. 11 .2B) (Possee et al., 1991) . Further modifications have been made to this system, by inserting additional unique restriction sites, one of which is inserted in the essential gene orf1629, which will be disrupted during linearization of the viral DNA by restriction enzyme digestion. This open reading frame (ORF) codes for a phosphoprotein that is a key component of the NC and its deletion renders undesired recircularization events nonviable (Kitts and Possee, 1993) .

The second and most widely used methodology of site-specific transposition of Tn7 transposon into a bacmid containing the baculoviral genome (Fig. 11 .2C). This system is commercially available as Bac-to-Bac Expression System (Invitrogen, Inc.). Shortly, this technique for generating recombinant baculoviruses consists in the site-specific transposition of a transfer vector (pFastBac) that carries the goi under a strong baculoviral promoter, and a bacmid. The bacmid contains Tn7 sites and a bacterial origin of replication (mini-F) and is propagated in DH10Bac Escherichia coli. This E. coli strain contains a helper plasmid that codes for Tn7 transposase. When both bacmid and transfer plasmid are transformed into bacteria containing the helper plasmid, the site-specific transposition takes place generating a recombinant baculoviral genome. The selection of clones is made by white and blue colony screening. The bacmid can be purified from bacterial cultures and transfected into Sf9 cells inside of which it can replicate, thus generating recombinant baculoviruses. The downside to this system when developing therapeutic products for both veterinary and human use is the presence of bacterial genomic sequences and selection markers such as antibiotic resistance.

Oxford Expression Technologies has made a safer approach with its flashBAC platform, which combines the two most widely used methodologies for the generation of recombinant baculoviruses ( Fig. 11 .2D). This platform is based on a bacmid where an essential gene such as orf1629 has been partially deleted (other accessory genes such as p74, p10, p26 have been also knocked out) and homologous recombination that occurs in insect cells with transfer plasmid containing part of orf1629 restores the complete sequence of this essential gene, while eliminating bacterial sequences (Je et al., 2003; Hitchman et al., 2010) . The flashBAC platform also offers variants to increase yields for specific target proteins (GOLD, ULTRA, and PRIME). More recently, a new platform known as MultiBac was developed (Geneva Biotech) for the expression of multiple polypeptides, especially suitable for the production of eukaryotic multiprotein complexes (Berger et al., 2013) . This system allows the insertion of foreign genes into two different loci of an engineered baculoviral genome: polyhedrin locus and chiA/v-cath locus.

The advantages of MultiBac are the tandem recombining that permits expression of proteins with various subunits and the reduction of proteolytic activity in the cell.

Expression systems for the production of therapeutic proteins have been established using recombinant DNA technology in mammalian, plant, insect, fungal, and bacterial cells, as well as transgenic multicellular organisms (Wu et al., 2002; Steinbach et al., 2002; Chen et al., 2003; Katagiri et al., 2003; Hammonds et al., 2007) .

The BEVS can be divided into two stages: the first is the generation and isolation of a recombinant baculovirus carrying the goi and the second is the infection of a susceptible host (cultured cells or larvae) with this recombinant virus (O'Reilly and Miller, 1991) . The very first recombinant protein that was produced using BEVS was human interferon beta (Smith et al., 1983) , but to date thousands of proteins have been expressed using this system. By far the most widely used virus is Autographa californica nucleopolyhedrovirus (AcMNPV); however, Bombyx mori nucleopolyhedrovirus is also used due to the higher protein expression using silkworm larvae or its pupae (Motohashi et al., 2005) . The most commonly used promoters for the expression of heterologous proteins in BEVS are the POLH promoter (polh) and p10 promoter (p10). These promoters have a high expression capacity and they become active following the late stages of infection.

In terms of cell lines, those derived from Spodoptera frugiperda ovary (Sf9, Sf21, and ExpressSF 1 ) and Trichoplusia ni (HighFive, BTI-TN-5B1-4, Invitrogen, Carlsbad, CA, United States) are the ones most frequently chosen by the users of BEVS. All these lines are able to grow both adherent or in suspension, in serum-free media and without requirements of CO 2 (Grace, 1962; Granados, 1994; Vaughn et al., 1997; Lynn, 2001; Ikonomou et al., 2003; Agathos, 2010) . It has been reported that HighFive cells have the highest heterologous protein expression yield Yamaji et al., 2006) . Larvae belonging to the order Lepidoptera, such as the silkworm, can also be used for the expression, with similar protein yields, the higher rate of production and, lower costs compared to those of cell culture. However, the requirements of Good Manufacturing Practice and quality control using larvae are more difficult to achieve, which makes the use of these insects not as common. Although insect cell lines are more secure than mammalian ones they have to be screened for adventitious agents that pose a risk to human and veterinary health, such as Alphanodavirus and Rhabdovirus that have been isolated from Helicoverpa armigera and S. frugiperda cells, respectively (Bai et al, 2011; Ma et al., 2014) . A disadvantage associated with the use of both cell culture and larvae is that after the first round of infection, cells lyse, and the entire culture has to be replaced, which does not allow a continuous expression of the goi. However, the use of low-cost serum-free media has somewhat helped overcome this disadvantage.

The recombinant protein production has also been improved by the deletion of baculoviral accessory genes such as chitinase and cathepsin that had a positive effect on the integrity of both the intracellular and secreted recombinant protein (Kaba et al., 2004; Hitchman et al., 2010) .

There are many advantages of BEVS over other expression systems. It provides a eukaryotic platform for adequate folding, disulfide bridge establishment, oligomerization, and other posttranslational modifications needed for biological activity of proteins. Moreover, high protein yields can be obtained using strong viral promoters, and the system allows the simultaneous expression of multiple genes due to the baculoviral genome's capacity to insert DNA fragments up to 34 kbp (O'Reilly et al, 1994) . It is important to mention that the production of recombinant proteins using this system has great scaleup potential and low-associated cost; low biosecurity requirements are needed since baculoviruses have a very limited host range and are nonpathogenic to humans.

In terms of posttranslational modifications of recombinant proteins, such as the eukaryotic glycosylation, BEVS presents simple protein glycosylation patterns. However, the N-glycosylation of proteins in insect cells does not resemble fully that of mammalian cell culture, while insect N-glycans have terminal mannose residues, and mammalian N-glycans often have terminal sialic acid residues. This difference may result in a lower or even total loss of biological function of the protein of interest Jarvis, 2006, 2016) . In order to overcome this problem, humanized insect cells have been developed that express the mammalian glycosylating enzymes (Harrison and Jarvis, 2006; Jarvis et al., 1998; Mabashi-Asazuma et al., 2014; Okada et al., 2010) . Another strategy consists in modifying the baculoviral genome in order to express the goi along with these glycosylating enzymes (SweetBac) (Palmberger et al., 2012) .

Product contamination with baculoviruses might be a problem when using BEVS for development of human and veterinary vaccines. Marek et al. have developed a system called Bacfree. This methodology uses vp80 deficient baculovirus that can only be propagated in cells that provides VP80 in trans. Deletion of this essential gene prevents the assembly of the viral phenotypes BV and ODV (Marek et al., 2011) .

The rapid, scalable, and low-cost production using this technology is particularly important in terms of confronting sanitary emergencies such as the influenza pandemic of 2009. Fedson and Dunnill (2007) reported that if just 25% of global bioreactor capacity was used in the generation of anti-flu vaccines with the BEVS, 425 million vials of 10 μg/dose could be produced in only 1 month, as opposed to the classic platform used for this purpose, which involves embryonated chicken eggs and takes about 6 months (Wong and Webby, 2013) . This means that nowadays, the production of influenza vaccines begins months before the exact prevalent strain of influenza is know for certain. If BEVS platform was used instead, the time required for the production would be just 45 days (Cox, 2012) .

Over the last 30 years, the scientific community has used this platform for the production of recombinant proteins at a laboratory scale. However, in the last decade, the BEVS has started to gain priority in the biotechnological industry. To date, five veterinary vaccines and four products for human use exist on the market including, vaccines, immunotherapy, and gene therapy vectors (Table 11 .1).

FluBlok is the first recombinant vaccine to be licensed by the US Food and Drug Administration for the prevention of seasonal influenza and is manufactured using BEVS in expresSF 1 insect cells. It is a trivalent recombinant hemagglutinin (HA) vaccine, produced by Protein Sciences Corporation, that contains HA antigen derived from three influenza virus strains, selected for inclusion in the annual influenza vaccine by the World Health Organization and updated on an annual basis (Holtz et al., 2003; Wang et al., 2006) . Since in this system, there cannot be replication, subsequent inactivation steps of the live influenza virus that are crucial in the licensed egg-based vaccines are not needed using this alternative.

Diamyd is a therapeutic vaccine produced using BEVS in Sf9 cells aimed to treat type 1 diabetes. Diamyd is a recombinant 65 kDa glutamate decarboxylase corresponding to human glutamate decarboxylase 65 that is the major autoantigen in type 1 diabetes. Diamyd is developed by Diamyd Medical and produced by Protein Sciences Corporation (Hinke, 2008) .

In summary, the BEVS is a valuable platform for the manufacture of a great variety of biotechnological products since with just one insect cell line and a single method for the construction of recombinant baculoviruses it is possible to express a large variety of products. 

The baculovirus/insect cell expression system has been widely used for the generation of antigens and the development of viral vaccines both for human and veterinary use (Cox, 2012; Mena and Kamen, 2011; van Oers, 2006; Yamaji, 2014) . Classic viral vaccines (first-generation vaccines), such as those for measles, rubella, rabies, and yellow fever, use attenuated viral strains or inactivated viruses. Attenuation corresponds with limited viral replication after vaccination, and although the immune response induced by this kind of vaccines is similar to natural infection adverse, reactions cannot be dismissed. Conversely, inactivated strains cannot replicate since the genetic material has been destroyed, making them more secure than the attenuated ones. However, the immune response generated by these formulations is not as efficient, and three to five doses are generally required to attain optimal antibody titers; in addition, risks of incomplete inactivation can lead to events such as the Cutter incident, in which about 40,000 polio cases were caused by a defective polio vaccine in the United States (Fitzpatrick, 2006) .

An alternative that is set to outclass this type of vaccines consists of subunit or recombinant protein formulations, which include virus-like particles (VLPs). These recombinant vaccines carry a limited number of viral proteins, either complete or truncated, in a nonpathogenic viral vector. VLPs are self-assembling, complex protein structures that resemble wild-type viral capsids or enveloped virions. The difference with the latter is that VLPs do not contain pathogenic (replicative) genomes. Vaccines formulated with these structures combine the advantages of attenuated vaccines with those of subunit vaccines since the epitopes can be recognized by host's immune system and efficiently stimulate the adaptive immune response while avoiding the possible reversion to virulent phenotypes.

VLPs are classified in two types based on structure: enveloped VLPs, that include a host-derived lipid membrane and naked or nonenveloped (simply referred to as VLPs). Likewise, they are also classified as simple or multiple VLPs depending on the number of different recombinant proteins included in the structure (Noad and Roy, 2003; Roldão et al., 2011) . Some difficulties may be associated with VLP production: for example, protein stoichiometry can be a problem since the incorporation of adequate proportions of each protein may not occur (Latham and Galarza, 2001) . However, even if the VLPs do not fully resemble the wild-type virus, they can stimulate the host immune system to an extent high enough to meet the requirements of a vaccine candidate.

VLPs represent a significant advance in terms of developing preventive tools against viruses that cannot be propagated in cell lines or ex vivo cultures, such as the hepatitis B virus and enteric adenoviruses types 40 and 41.

VLP production using BEVS can be accomplished by one of two alternatives, or by the combination of both. The first consists of the coinfection of insect cells with as many recombinant viruses as proteins the manufacturer wants to assemble in the VLP structure (monocistronic baculoviruses) or with a single polycistronic virus that codes for all the proteins. Complex multiprotein VLPs present difficulties for their production since the infection with multiple recombinant baculoviruses are not homogenous. Vieiria et al. compared both strategies for the production of rotavirus VLPs containing VP2, VP6, and VP7. They observed that the coexpression strategy was superior to the coinfection one; meanwhile Roldão et al. for the same VLPs found the opposite (Vieira et al., 2005; Roldão et al., 2006) . To this day, a vast array of taxonomically and structurally different VLPs has been produced using BEVS. Many of these already are in preclinical and clinical trial stages, and seven VLP-based vaccines have already been approved and are currently being commercialized (Table 11 .2). One of the most promising products already on the market is Cervarix, a VLP-based vaccine against human papillomaviruses 16 and 18, which are responsible for 70% of cervical cancer cases, produced by GlaxoSmithKline (Muñ oz et al., 2003; Smith et al., 2007) .

As mentioned earlier, the BEVS can be used to produce substantial amounts of heterologous proteins for their use as immunogens. This strategy, although key to subunit vaccines development, can be replaced by peptide display on virion surfaces. Baculovirus surface display refers to the display of heterologous peptides or proteins on the surface of BVs by different strategies all of which involve a translational fusion of a peptide to GP64 or a portion of it (Kost et al., 2005; Xu et al., 2011) . GP64 is the major surface protein and a class III integral membrane glycoprotein of budded virions that is able to mediate membrane fusion under acidic pH conditions. AcMNPV GP64 is a 512-amino acid long phosphoglycoprotein with 15 cysteine residues that participate in disulfide bridge formation required for stability and biological activity (Hefferon et al., 1999; Garry and Garry, 2008) .

The surface display of the heterologous polypeptide can be achieved by constructing a chimera with complete wild-type GP64 or only the transmembrane, multimerization, and carboxy-terminal (cytoplasmic tail) domains to the target protein, or by expression of a recombinant GP64 fused to a short or long heterologous peptide (Grabherr and Ernst, 2010) . Baculovirus display of heterologous proteins by fusion to GP64 was demonstrated to be very effective immunogens, successfully able to induce antibody response to a variety of displayed proteins (Kost et al., 2005) . In addition, baculoviruses by themselves are able to trigger a robust innate immune response by activation of professional antigenpresenting cells.

Baculoviruses displaying heterologous envelope proteins, such as vesicular stomatitis virus (VSV)-G protein, have also been constructed. Several studies have shown that VSV-G is capable of improving transduction efficiency of baculovirus in vertebrate cells (Barsoum et al., 1997) . Different strategies have been developed to increase the effectiveness of virus display (Chapple and Jones 2002) . The polypeptides displayed by VSV-G exhibited higher densities of recombinant proteins than GP64 fusions because this protein is homogenous distributed in the BV compared with GP64 that is mostly apical.

In contrast to surface display, heterologous protein can be displayed on the NC of the virion. The NC core is composed of a circular, covalently closed dsDNA genome and the major basic core protein P6.9, and the NC sheath is formed by stacked ring-like subunits consisting of one major polypeptide, VP39, and several minor proteins. The NC display strategy involves the translational fusion of a peptide to VP39 (Kukkonen et al., 2003; Molinari et al., 2011; Pidre et al., 2013) .

Depending on the host immune response that is targeted, one strategy might be chosen over the other. Antigen displayed on the NC should be able to reach the cytosol and preferentially trigger major histocompatibility complex class I presentation pathway and mount a strong CD8 1 T-cell response (Molinari et al., 2011) . In turn, the BV displaying antigens on the surface elicit a humoral response (Gronowski et al., 1999; Abe et al., 2005) . Several studies have shown that baculovirus display can generate high titers of specific antibodies that protect against different pathogens such as Japanese encephalitis virus, avian reovirus, human enterovirus, influenza, and malaria (Xu et al., 2011; Lin et al., 2014; Marek et al., 2011; Jin et al., 2008; Madhan et al., 2010) .

Adeno-associated viruses (AAV) have become relevant for in vivo gene-therapy (Nayerossadat et al., 2012; Flotte, 2013; Wang and Gao, 2014) . This therapy involves the incorporation of genes into cells. AAV used as viral a vector is the tool that allows the safe, efficient, and specific incorporation of the gene inside the organism. The first group of hereditary diseases for which gene-therapy was investigated included hemophilia A and B, Leber's macular degeneration, and Duchenne's muscular dystrophy, but nowadays there are many studies focused on central nervous system diseases such as Parkinson's, different types of cancers, cardiovascular afflictions, and bone regeneration (Flotte et al., 1996; Kay et al., 2000; Kaplitt et al. 2007; Jiang et al., 2011 ). An estimate of 70%À80% of human population has been exposed to at least one event of infection with AAV, and no adverse consequences have been associated to the infection and latency of these viruses (Boutin et al., 2010; Erles et al., 1999; Tobiasch et al., 1998) . AAVs have a singlestranded DNA genome and are nonenveloped viruses of 18À25 nm of diameter. They belong to the Parvovirus family and are classified as members of the Dependovirus genus. Twenty human and nonhuman primate strains have been isolated, and all serotypes except for serotype 5 share a very similar capsid structure, genome size, and organization. AAV genome has a size of approximately 4.7 kb and contains two ORFs that code for four regulatory proteins. These ORFs are flanked by two inverted terminal repeats (ITRs) that contain cis-regulatory sequences needed for viral infection. Major proteins Rep78 and Rep68 are involved in genome excision, rescue, replication, and integration. Integration into the host genome is site-specific and takes place in chromosome 19, more specifically into locus AAVS1 of the human genome, without adverse consequences. Minor proteins Rep52 and Rep40 are responsible for the accumulation of DNA and packaging. VP1, VP2, and VP3 are structural proteins encoded in the Cap ORF.

Recombinant AAVs are constructed by replacement of rep and cap genes with the goi flanked by ITR regions. This viral vector is able to transduce both active division and quiescent cells. It is very important to highlight that, in contrast to wt AAV, the recombinant AAV has no site-specific integration capacity since rep genes are absent; thus the integration occurs at random. However, the estimated integration frequency of these recombinant adeno-associated viruses (rAAVs) is 0.1%À0.5% in mammalian cell lines (McCarty et al., 2004) . In view of this, to establish stable and persistent expression in patients, an alternative to genome integration has been developed consisting in double-stranded DNA that persists as an episome inside the cells (Yang et al., 1999) . To date, adeno-associated vectors have been modified to be tissue-specific, the preferred organs being those that are immuneprivileged such as the eye or the brain.

The classic technique for the production of AAVs involves HEK293, A549, or HeLa cells cotransfected with two plasmids, one containing the recombinant DNA (recombinant adeno-associated virus plasmid) and the other the rep and cap genes (pHelper). The cells are then infected with a helper virus such as an adenovirus or a herpes simplex virus to allow the replication of the recombinant AAV (Samulski et al., 1987) . The downside to this methodology is the possibility of undesired recombination events between plasmids and that purification of the helper virus is time consuming, and the scale-up process is difficult to achieve. A good alternative has been the use of BEVS platform [revised in Galibert and Merten (2011) ], which involves the use of three recombinant baculoviruses and Sf9 cells. Each of the three recombinant baculoviruses contains one of the following genes: rep, cap, and the construct of interest flanked by the ITRs. Alternatives involving a polycistronic baculovirus containing rep and cap genes are also used (Smith et al., 2009 ). The OneBac system involves transgenic insect cell lines that express the genes rep and cap of the AAV and only one recombinant baculovirus. The efficiency of this technique and the scale-up feasibility are higher than the previous alternatives, and it provides a more flexible strategy since it allows the production of other AAV serotypes by simply changing cell lines (Mietzsch, 2014) . Such as other products derived from BEVS, the contamination of rAAVs with baculovirus particles has to be reduced. There is already one rAAV produced by BEVS on the market commercialized as Glybera (alipogene tiparvovec) developed and marketed by uniQure that compensates for lipoprotein lipase deficiency, which received regulatory approval by the European Medicines Agency in 2012, although now it has been discontinued due to lack of demand.

It was demonstrated in the 1990s that the baculoviral genome was capable of transducing mammalian cells and that if heterologous genes were under control of adequate mammalian promoters, the ORFs carried by this baculoviral genome were transiently expressed in these cells. The viral promoters such as Simian Viruses SV40, immediate early protein ie1 CMV, elongation factor-1α, hybrid CAG promoter, human ubiquitin C, U6, Pol III H1, and Drosophila melanogaster hsp70 among others are commonly used for expression in mammalian cells. Following the first days, the baculoviral genome is degraded and unable of insertion in the mammalian genome in the absence of selection pressure (Volkman and Goldsmith, 1983; Carbonell et al., 1985; Boyce and Bucher, 1996) . At present, the baculoviral transduction ability has been documented both in vitro and in vivo for a variety of cell types including embryonic stem cells and pluripotent stem cells (Kost et al., 2005; Ho et al., 2005; Airenne et al., 2010; Chen et al., 2011) . It is suggested that the mechanism by which the baculoviruses ingress mammalian cells is clathrin and dynamin-dependent endocytosis and macropinocytosis. Makkonen showed that this process also requires the host cell proteins heparan sulfate proteoglycans and syndecan-1 that serves as a receptor (Long et al., 2006a,b; Kataoka et al., 2012; Makkonen et al., 2013 Makkonen et al., , 2014 Nasimuzzaman, 2014) . Once inside the cell, the baculovirus virions are transported into the endosome, where acidic conditions promote membrane fusion mediated by GP64, and the genetic material is thus liberated into the cytoplasm. Actin cytoskeleton reorganization promotes translocation of the baculoviral genome into the nucleus where transcription occurs by host machinery.

One of the advantages of using baculovirus as vectors for mammalian cell transduction is that in contrast to other viruses used to the same end, they are not human pathogens, they do not replicate in mammalian cells, no integration in the genome has been documented, and there is no preexisting immunity to baculoviruses (Strauss et al., 2007; Jin et al., 2008) . A human study was made in which volunteers consumed OBs in their diet; the results were promising since no inflammatory response, allergies, or adverse effects were observed in a 5-day follow-up (Heimpel et al., 1973) . These studies have been extended to animal models to evaluate possible side-effects such as skin irritation and cytotoxicity, all of which have been negative (Kost and Condreay, 2002; Jin et al., 2008) .

Even though a downside to the use of baculovirus as viral vectors for gene delivery is the lack of long-term expression of the transgene, alternatives to overcome this have been developed. For example, Lo et al. engineered a baculovirus vector system with Cre/loxP sites. In their approach, the construct is excised and remains as an episome in the cells that prolong expression (Lo et al., 2017) .

All this evidence, together with the fact that baculoviruses infect no other organisms besides those of the phylum Arthropoda, shows they are safe vectors for developing DNA vaccines, immunotherapy, and gene therapy. A reflection of this is the increasing number of peerreviewed publications that use recombinant baculoviruses over other viral vectors. For example, Swift et al. (2013) used a baculovirus with a prodrug approach to mediate cell-death of prostate cancer cells with success, even observing the good capacity of cell-layer penetration compared to other viral vectors. In the last few years, gene silencing by RNA interference has become more relevant for gene regulation. Baculoviral vectors have been engineered to deliver microribonucleic acid and short hairpin ribonucleic acid into insect and mammalian cells successfully. Gottardo and Pidre showed that baculovirusmediated gene silencing of human in increased survival and reduced the progression of pituitary tumor growth in vivo (Gottardo et al., 2018) . This type of strategy can be also applied to interfere with viral pathogens infecting humans and other animals (Makkonen et al., 2015) .

For over 30 years, baculoviruses have been exploited in molecular biology laboratories, and the trend has never stopped increasing. Since they are a highly versatile tool, with low-associated costs and scalable production, pharmaceutical giants such as Merck are taking advantage of them. Baculoviruses are not mere biological control agents anymore but are also key in the state of the art of gene-therapy and immunization departments. One of the most relevant advantages of BEVS is the rapid response under an epidemiological emergency such as an influenza pandemic. Meanwhile, the classical first-generation, egg-based approach for developing flu vaccines can be nonsuitable for allergic users and takes about six months to develop; the BEVS recombinant technology approach can give a fully functional vaccine within the first 45 days after the outbreak. Using the BEVS is not only inexpensive and direct, but it also provides unique plasticity. Together with the fact that almost no special biosafety measures have to be taken into account for its application, it makes an excellent platform when compared to other systems.

Involvement of the Toll-like receptor 9 signaling pathway in the induction of innate immunity by baculovirus

Baculoviridae, nuclear polyhedrosis viruses Part 1. Nuclear polyhedrosis viruses of insects

Insect cell culture

In vivo application and tracking of baculovirus

Baculovirus nuclear import: open, nuclear pore complex (NPC) sesame. Viruses 5

Isolation and characterization of a novel Alphanodavirus

Efficient transduction of mammalian cells by a recombinant baculovirus having the vesicular stomatitis virus G glycoprotein

Development of baculovirus triple and quadruple expression vectors: co-expression of three or four bluetongue virus proteins and the synthesis of bluetongue virus-like particles in insect cells

The multiBac protein complex production platform at the EMBL

Protection of swine against post-weaning multisystemic wasting syndrome (PMWS) by porcine circovirus type 2 (PCV2) proteins. Vaccine 21

Prevalence of serum IgG and neutralizing factors against adeno-associated virus (AAV) types 1, 2, 5, 6, 8, and 9 in the healthy population: implications for gene therapy using AAV vectors

Broad cross-protection is induced in preclinical models by a human

Baculovirus-mediated gene transfer into mammalian cells

Formation of poliovirus-like particles by recombinant baculoviruses expressing the individual VP0, VP3, and VP1 proteins by comparison to particles derived from the expressed poliovirus polyprotein

High efficient production of Pr55gag virus-like particles expressing multiple HIV-1 epitopes, including a gp120 protein derived from an Ugandan HIV-1 isolate of subtype A

Structural requirements of astrovirus virus-like particles assembled in insect cells

Baculovirus-mediated expression of bacterial genes in dipteran and mammalian cells

Non-polar distribution of green fluorescent protein on the surface of Autographa californica nucleopolyhedrovirus using a heterologous membrane anchor

PROVENGE (sipuleucel-T) in prostate cancer: the first FDA-approved therapeutic cancer vaccine

Biosynthesis of 3-O-sulfated heparan sulfate: unique substrate specificity of heparan sulfate 3-O-sulfotransferase isoform 5

Biosafety assessment of human mesenchymal stem cells engineered by hybrid baculovirus vectors

Expression, purification and characterization of enterovirus-71 virus-like particles

Recombinant protein vaccines produced in insect cells

Characterization of virus-like particles produced by the expression of rotavirus capsid proteins in insect cells

Expression of an RSV-gag virus-like particle in insect cell lines and silkworm larvae

Assembly of feline calicivirus-like particle and its immunogenicity

Update on the prevalence of serum antibodies (IgG and IgM) to adeno-associated virus (AAV)

The effect of vaccination against porcine circovirus type 2 in pigs suffering from porcine respiratory disease complex

New approaches to confronting an imminent influenza pandemic

Immune responses to AAV-vectors, the Glybera example from bench to bedside

The cutter incident: how America's first polio vaccine led to the growing vaccine crisis

Birth of a new therapeutic platform: 47 years of adeno-associated virus biology from virus discovery to licensed gene therapy

A phase I study of an adeno-associated virus-CFTR gene vector in adult CF patients with mild lung disease

Latest developments in the large-scale production of adeno-associated virus vectors in insect cells toward the treatment of neuromuscular diseases

Generation of porcine reproductive and respiratory syndrome (PRRS) virus-likeparticles (VLPs) with different protein composition

Proteomics computational analyses suggest that baculovirus GP64 superfamily proteins are class III penetrenes

Baculovirus-based gene silencing of Humanin for the treatment of pituitary tumors

Establishment of four strains of cells from insect tissues grown in vitro

Baculovirus for eukaryotic protein display

Trichoplusia ni cell line which supports replication of baculoviruses

Baculovirus stimulates antiviral effects in mammalian cells

P74 mediates specific binding of Autographa californica multiple nucleopolyhedrovirus occlusion-derived virus to primary cellular targets in the midgut epithelia of Heliothis virescens larvae

Advances in methods for the production, purification, and characterization of HIV-1 Gag-Env pseudovirion vaccines

Protein N-glycosylation in the baculovirus-insect cell expression system and engineering of insect cells to produce 'Mammalianized' recombinant glycoproteins

Transforming lepidopteran insect cells for continuous recombinant protein expression

Host cell receptor binding by baculovirus GP64 and kinetics of virion entry

The presence of nuclear polyhedrosis viruses of Trichoplusia ni on cabbage from the market shelf

Virus Taxonomy: Classification and Nomenclature of Viruses: Ninth Report of the International Committee on Taxonomy of Viruses

Diamyd, an alum-formulated recombinant human GAD65 for diabetes and the prevention of autoimmune diabetes

Genetic modification of a baculovirus vector for increased expression in insect cells

Assembly of human severe acute respiratory syndrome coronavirus-like particles

Transgene expression and differentiation of baculovirus-transduced human mesenchymal stem cells

Production of a recombinant influenza vaccine using the baculovirus expression vector system

Saturable attachment sites for polyhedron-derived baculovirus on insect cells and evidence for entry via direct membrane fusion

Chimeric L2-based virus-like particle (VLP) vaccines targeting cutaneous human papillomaviruses (HPV)

Insect cell culture for industrial production of recombinant proteins

Engineering N-glycosylation pathways in the baculovirus-insect cell system

Baculovirus expression vectors that incorporate the foreign protein into viral occlusion bodies

On the classification and nomenclature of baculoviruses: a proposal for revision

Immunogenicity and safety of virus-like particle of the porcine encephalomyocarditis virus in pig

Expression, self-assembly, and antigenicity of the Norwalk virus capsid protein

Synergistic antitumor effect of AAV-mediated TRAIL expression combined with cisplatin on head and neck squamous cell carcinoma

Safety and Immunogenicity of H5N1 influenza vaccine based on baculovirus surface display system of Bombyx mori

Development of a chitinase and v-cathepsin negative bacmid for improved integrity of secreted recombinant proteins

Safety and tolerability of gene therapy with an adeno-associated virus (AAV) borne GAD gene for Parkinson's disease: an open label, phase I trial

All six modules of the gelatin-binding domain of fibronectin are required for full affinity

Baculovirus GP64-mediated entry into mammalian cells

Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector

Papillomavirus L1 major capsid protein self-assembles into virus-like particles that are highly immunogenic

A method for producing recombinant baculovirus expression vectors at high frequency

Recombinant baculoviruses as mammalian cell genedelivery vectors

Baculovirus as versatile vectors for protein expression in insect and mammalian cells

Purification and characterization of virus-like particles and pentamers produced by the expression of SV40 capsid proteins in insect cells

Baculovirus capsid display: a novel tool for transduction imaging

Baculovirus-mediated immediate-early gene expression and nuclear reorganization in human cells

Formation of wild-type and chimeric influenza virus-like particles following simultaneous expression of only four structural proteins

Update on baculovirus as an expression and/or delivery vehicle for vaccine antigens

Efficient production of type 2 porcine circovirus-like particles by a recombinant baculovirus

Rift valley fever virus structural proteins: expression, characterization and assembly of recombinant proteins

A virus-like particle vaccine for coxsackievirus A16 potently elicits neutralizing antibodies that protect mice against lethal challenge. Vaccine

Use of baculovirus expression system for generation of virus-like particles: successes and challenges

Enhanced critical-size calcarial bone healing by ASCs engineered with Cre/loxP-based hybrid baculovirus

Function, oligomerization and N-linked glycosylation of the Helicoverpa armigera single nucleopolyhedrovirus envelope fusion protein

Functional entry of baculovirus into insect and mammalian cells is dependent on clathrin-mediated endocytosis

Novel techniques to establish new insect cell lines

Identification of a novel rhabdovirus in Spodoptera frugiperda cell lines

A novel baculovirus vector for the production of non fucosylated recombinant glycoproteins in insect cells

Baculovirus as vaccine vectors

6-O-and N-sulfated syndecan-1 promotes baculovirus binding and entry into mammalian cells

Reply to "Heparan sulfate in baculovirus binding and entry of mammalian cells

Baculovirus-mediated gene delivery and RNAi applications

Expression of the major core structural proteins VP3 and VP7 of African horse sickness virus, and production of core-like particles

Engineering of baculovirus vectors for the manufacture of virion-free biopharmaceuticals

Production of porcine parvovirus empty capsids with high immunogenic activity

Integration of adeno-associated virus (AAV) and recombinant AAV vectors

Insect cell technology is a versatile and robust vaccine manufacturing platform

Effective chikungunya virus-like particle vaccine produced in insect cells

Adeno-Associated Virus Vectors for Gene Therapy: From Scalable Production Systems via Serotype-Specific Glycan Interaction Patterns to Gene Transfer Applications. Submitted to the Department of Biology

Development of foot-and-mouth disease virus (FMDV) serotype O virus-like-particles (VLPs) vaccine and evaluation of its potency

Baculovirus capsid display potentiates OVA cytotoxic and innate immune responses

Efficient largescale protein production of larvae and pupae of silkworm by Bombyx mori nuclear polyhedrosis virus bacmid system

Epidemiologic classification of human papillomavirus types associated with cervical cancer

Immune responses in mice vaccinated with virus-like particles composed of the GP5 and M proteins of porcine reproductive and respiratory syndrome virus

Heparan sulfate in baculovirus binding and entry of mammalian cells

Viral and nonviral delivery systems for gene delivery

Virus-like particles as immunogens

N-Glycosylation engineering of lepidopteran insect cells by the introduction of the β1,4-Nacetylglucosaminyltransferase III gene

Improvement of a baculovirus pesticide by deletion of the egt gene

Baculovirus Expression Vectors: A Laboratory Manual

Sweetbac: a new approach for the production of mammalianised glycoproteins in insect cells

Baculovirus display: a novel tool for vaccination

Nucleotide sequence of the Autographa californica nuclear polyhedrosis 9.4 kbp EcoRI-I and -R (polyhedrin gene) region

Mono-and quadri-subtype virus-like particles (VLPs) containing H10 subtype elicit protective immunity to H10 influenza in a ferret challenge model

Intracellular dynamics in rotavirus-like particles production: evaluation of multigene and monocistronic infection strategies

Quality control and analytical methods for baculovirus-based products

A recombinant plasmid from which an infectious adeno-associated virus genome can be excised in vitro and its use to study viral replication

Recombinant parvovirus-like particles as an antigen carrier: a novel nonreplicative exogenous antigen to elicit protective antiviral cytotoxic T cells

Immunotherapy of hormone-refractory prostate cancer with antigen-loaded dendritic cells

Human papillomavirus type distribution in invasive cervical cancer and high-grade cervical lesions: a meta-analysis update

A simplified baculovirus-AAV expression vector system coupled with one-step affinity purification yields high-titer rAAV stocks from insect cells

Recombinant equine interferons: expression cloning and biological activity

Baculovirus-based vaccination vectors allow for efficient induction of immune responses against plasmodium falciparum circumsporozoite protein

Protection against lethal challenge by Ebola virus-like particles produced in insect cells

Evaluating baculovirus as a vector for human prostate cancer gene therapy

Virus-like particle vaccine comprised of the HA, NA, and M1 proteins of an avian isolated H5N1 influenza virus induces protective immunity against homologous and heterologous strains in mice

Assembly of herpes-simplex virus type-1 capsids using a panel of recombinant baculoviruses

Expression of feline leukaemia virus gp85 and gag proteins and assembly into virus-like particles using the baculovirus expression vector system

Discrimination between different types of human adeno-associated viruses in clinical samples by PCR

Suitability of an E2 subunit vaccine of classical swine fever in combination with the E(rns)-marker-test for eradication through vaccination

Vaccines for viral and parasitic diseases produced with baculovirus vectors

The establishment of two cell lines from the insect Spodoptera frugiperda (Lepidoptera; Noctuidae)

Triple layered rotavirus VLP production: kinetics of vector replication, mRNA stability and recombinant protein production

In vitro survey of Autographa californica nuclear polyhedrosis virus interaction with nontarget vertebrate host cells

State-of-the-art human gene therapy: Part I. Gene delivery technologies

Studies on serum-free culture of insect cells for virus propagation and recombinant protein production

Expression and purification of an influenza hemagglutinin-one step closer to a recombinant protein-based influenza vaccine

The next step in gene delivery: molecular engineering of adeno-associated virus serotypes

Duck hepatitis A virus structural proteins expressed in insect cells self-assemble into virus-like particles with strong immunogenicity in ducklings

Filovirus-like particles produced in insect cells: immunogenicity and protection in rodents

Homologous and heterologous protection of nonhuman primates by ebola and sudan virus-like particles

Traditional and new influenza vaccines

Expression of biologically active recombinant equine interferon-gamma by two different baculovirus gene expression systems using insect cells and silkworm larvae

Baculovirus surface display of E envelope glycoprotein of Japanese encephalitis virus and its immunogenicity of the displayed proteins in mouse and swine models

Suitability and perspectives on using recombinant insect cells for the production of virus-like particles

Efficient production of recombinant protein in immobilized insect cell culture using serum-free basal media after baculovirus infection

Concatamerization of adeno-associated virus circular genomes occurs through intermolecular recombination

Ebola virus-like particles produced in insect cells exhibit dendritic cell stimulating activity and induce neutralizing antibodies

Development of a VLP-based vaccine in silkworm pupae against rabbit hemorrhagic disease virus

A novel mechanism for nuclear import by actin-based propulsion used by the baculovirus nucleocapsid

Production of adeno-associated viral vectors in insect cells using triple infection: optimization of baculovirus concentration ratios

Bioprocessing of baculovirus vectors: a review

Efficacy and stability of a subunit vaccine based on glycoprotein E2 of classical swine fever virus

Technology transfer and scale-up of the Flublok recombinant hemagglutinin (HA) influenza vaccine manufacturing process

Preclinical in vivo evaluation of pseudotyped adeno-associated virus vectors for liver gene therapy

A largely random AAV integration profile after LPLD gene therapy

Linearization of baculovirus DNA enhances the recovery of recombinant virus expression vectors

Detection and analysis of Autographa californica nuclear polyhedrosis virus mutants with defective interfering properties

Engineering adeno-associated viruses for clinical gene therapy

Reiterated DNA fragments in defective genomes of Autographa californica nuclear polyhedrosis virus are competent for AcMNPV-dependent DNA replication

Adeno-associated virus vectors in clinical trials

Baculovirus as a gene delivery vector for cartilage and bone tissue engineering

Baculovirus entry into human hepatoma cells baculovirus entry into human hepatoma cells

OneBac: platform for scalable and high-titer production of adeno-associated virus serotype 1À12 vectors for gene therapy

Enveloped virus-like particles as vaccines against pathogenic arboviruses

Autographa californica baculoviruses with large genomic deletions are rapidly generated in infected insect cells

Safety profile of recombinant adeno-associated viral vectors: focus on alipogene tiparvovec (Glybera(s))

Adeno-associated virus (AAV) vectors in cancer gene therapy

Co-expression vs. co-infection using baculovirus expression vectors in insect cell culture: benefits and drawbacks

Insect cells as a factory to produce adenoassociated virus type 2 vectors. Hum

Adeno-associated virus serotypes: vector toolkit for human gene therapy