2011 Insect Cell Technology is a Versatileand Robust Vaccine Manufacturing Platform

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Baculovirus & insect cell technologyAutographa californica multiple nucleopoly hedro-virus (AcMNPV) is an enveloped baculovirus with a double-stranded, circular DNA genome of 134 kb that infects insect cells as its natural host. The baculovirus was originally studied and devel-oped for the biological control of agricultural pests. In the early 1980s, AcMNPV was genetically mod-ified to express heterologous recombinant proteins in insect cell cultures [1]; since then, the baculo-virus expression system (BEVS) has been adopted by many molecular and biochemical laboratories as the most efficient and versatile tool to produce large quantities of target proteins very rapidly [2], including complex structures such as monoclonal antibodies [3]. In the mid-1990s, Hofmann et al. demonstrated the efficient BEVS transduction of hepatocytes, which led to broader use of the BEVS to express foreign proteins in mammalian cells [4,5]. For almost three decades, the BEVS has served the research and development community well, especially in drug discovery and structural ana lysis departments.

The popularity of the system and its broad use for the transient expression of recombinant pro-teins were essentially due to its intrinsic advan-tages, which included vector construct stability, high expression yields, eukaryotic-like post-translational modification abilities and a large genome capacity. Over the years, advanced BEVS technology with multiple promoters, usually p10 and polyhedrin, and carrying special features has been developed to facilitate and accelerate the cloning process [6].

Combined with these multiple advantages, the BEVS benefited from the early development of tissue culture technology, mostly in the field of biopesticides. Many continuous cell lines, including Spodoptera frugiperda cells (Sf-21or Sf-9 clones) and Trichoplusia ni cells (for exam-ple, the High Five™ clone), have been isolated and characterized for the industrial manufactur-ing of biopesticides. These cell lines, and many others [7,8], were rapidly developed for suspension culture and serum-free media [9] to minimize production costs.

Jimmy A Mena1 and Amine A Kamen†1

1Animal Cell Technology, Bioprocess Center, Biotechnology Research Institute, National Research Council, Canada †Author for correspondence:Tel.: +1 514 496 2264 Fax: +1 514 496 6785 [email protected]

Baculovirus and insect cell culture technologies have mostly been limited to research laboratories for the transient expression of target proteins for drug development purposes. With the renaissance of the vaccine field and the regulatory acceptance of recombinant DNA technology, the baculovirus expression system has been more broadly adopted for the development of subunit vaccines, including virus-like particles. In the numerous clinical trials extensively discussed and cross-referenced in this article, product quality, safety and efficacy have been demonstrated for many candidate vaccines targeting infectious diseases. The 2007 market authorization of Cervarix™, a bivalent human papillomavirus virus-like particle vaccine against cervical cancer, was a critical milestone for the regulatory acceptance of insect cell technology in manufacturing human vaccines, opening the door to the approval of more baculovirus-derived vaccines. Insect cell technology is now a dominant platform for veterinary vaccines. This article covers the application of recombinant baculovirus as vectored vaccines to mediate systemic and mucosal immune responses through the display or expression of foreign antigens. We will probably observe increasingly more baculovirus-derived products and market licensing of safe and efficacious vaccines.

Keywords: baculovirus • BEVS • clinical trials • immunogenicity • insect cells • subunit vaccines • vectored vaccines • virus-like particles • VLP

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In the early 1990s, because insect cell cultures were easily acces-sible to many researchers and had become ‘easier’ to culture in standard biochemical engineering set-ups, insect cells and, more specifically, S. frugiperda-derived insect cell clones were adopted by many bioengineers to study the effect of shear-stress in bioreac-tors and address the critical challenges for large-scale operations. Infection and viral production kinetics were also the focus of many studies, resulting in advanced mathematical models [10].

These efforts contributed to the rapid development and scale up of insect cell culture [11] and animal cell technology in gen-eral. Over the years, BEVS/insect cell suspension and serum-free cultures developed into a mature and robust technology, which can be successfully operated in a large scale and with high cell densities [12,13].

Vaccines development & the BEVS As a consequence of rapid development, the emerging BEVS and insect cell culture technology was accepted as an alternative for the commercial production of recombinant proteins, including subunit vaccines. As early as the middle of 1988, only 5 years after the discovery of HIV by a team at Institut Pasteur (Paris, France), clinical testing of the first HIV candidate vaccine was sponsored by MicroGeneSys [14]. The gp160 HIV envelope recombinant vaccine was produced in insect cell culture using the BEVS. The insect cell culture-derived vaccine ‘preparation stimulated a strong immune response in animals’ [14], revealing that the adjuvant effect of insect cell- and baculovirus-derived proteins might be effective in vaccination strategies. Recently published data on the adenovirus-based vaccine trial [15] and other unsuccessful HIV-related clinical trials concluded that the traditional vaccination approach targeting a strong immune response might not be an adequate strategy in the continuing quest for an HIV vaccine. Although not successful, the early HIV clinical trials established insect cell technology as a reliable platform for vaccine manufac-turing and set the stage for further developments and the use of the BEVS in the field of vaccine manufacturing.

Among the vaccine candidates, subunit vaccines, including recombinant proteins, virus-like particles (VLPs) and vec-tored vaccines have been emerging rapidly. They can expose and express proteins from foreign pathogens and induce spe-cific immunological responses against these antigens in vivo. Generally, these vaccines can stimulate potent humoral and cellular immune responses.

The BEVS/insect cell technology is highly versatile to serve as a platform in the manufacturing of subunit vaccine candidates with desired characteristics. BEVS can be used to efficiently pro-duce monomeric or oligomeric recombinant proteins as well as complex protein structures, such as enveloped or nonenveloped VLPs. Also, because of the well-documented safety profile of the baculovirus vector and its ability to efficiently transduce mam-malian cells, recombinant baculoviruses are being evaluated as alternative vectors to deliver antigens for vaccination [16].

For all these vaccination strategies, the BEVS/insect cell tech-nology does not require a high biocontainment facility. With recent developments integrating advanced production strategies

[17,18], the technology is now cost effective and meets the eco-nomical requirements for manufacturing modern vaccines for large populations. The BEVS has also been used in the efficient manufacturing of adeno-associated vectors for gene delivery and vaccination [10].

In the past decade, vaccines manufactured using bacu-lovirus/insect cell technology to prevent human and animal diseases have been licensed. For example, GlaxoSmithKline’s Cervarix™ (GlaxoSmithKline [GSK], Rixensart, Belgium), a bivalent human papillomavirus (HPV) VLP vaccine against cervi-cal cancer, was approved by the EMA in 2007 and the US FDA in 2009. Other candidate human vaccines in the late stages of clinical development include FluBlok®, a recombinant hemagglu-tinin (HA)-based trivalent seasonal influenza vaccine developed by Protein Sciences Corporation (PSC; Meriden, Connecticut, USA), and Diamyd®, a therapeutic vaccine for the treatment of Type 1 diabetes mellitus developed by Diamyd Medical AB (Stockholm, Sweden).

A significant commercial potential exists for BEVS technology in manufacturing different types of vaccine for animal use. Indeed, vaccines such as Ingelvac® CircoFLEX (Boehringer Ingelheim Vetmedia Inc., USA) or Porcilis® PCV (Intervet/Schering-Plough, The Netherlands) against porcine circovirus type 2 produced by BEVS technology were granted marketing authorization by the regulatory agencies in Europe and North America, thus paving the way to the licensing of many other BEVS-derived veterinary vaccines.

Here, we review the progress of the baculovirus/insect cell tech-nology platform in manufacturing vaccines and vaccine candi-dates for human and animal use. This article is divided in three sections, broadly corresponding to three different type of vaccine candidates as illustrated in Figure 1. First, we describe the prog-ress of baculovirus-expressed monomeric or oligomeric recombi-nant proteins as immunodominant antigens for prophylactic or therapeutic vaccination. Second, we extensively review the use of nonenveloped and enveloped VLPs as promising prophylactic subunit vaccines. Although conceptually the candidate vaccines described in these first two sections are similar with regard to the vaccination strategy, for clarity, they have been segregated because they pose different manufacturing challenges. Finally, we introduce the use of baculoviruses as vectors to expose and/or express antigens in vivo. We have cross-referenced many publica-tions with data from government clinical trial sites and regulatory agencies to provide a snapshot of the current situation within a field that is progressing at an extremely rapid pace.

Insect cell-expressed recombinant proteins as subunit vaccinesThe BEVS is currently the preferred production platform for vari-ous recombinant proteins used in different vaccination strategies. Several of these vaccines for human or veterinary use are com-mercially available or in advanced stages of development (Table 1).

This section focus on reviewing the progress in subunit vaccines produced using the BEVS to express antigens as full or trun-cated recombinant proteins. For clarity, VLPs that form complex

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structures involving the baculovirus expres-sion of more than one recombinant protein will be the subject of the next section.

Human vaccinesFluBlok and Diamyd are the most advanced human vaccine candidates. Both are in Phase III clinical trials in the USA and Europe [19–21]. FluBlok is a recombinant vaccine based on trimers of the HA pro-tein from the influenza virus [22]. Diamyd is a therapeutic vaccine composed of a recombinant glutamic acid decarboxylase enzyme (rGAD65) for the treatment of Type 1 diabetes mellitus [19,23]. Both vac-cine candidates are produced in a S. fru-giperda cell line or derived clones such as expresSF+®, a proprietary cell line from PSC [22,24]. In successive clinical trials, FluBlok was well tolerated in both elderly individuals over 65 years and younger sub-jects (6–58 months), thus demonstrating its safety [20,21]. In addition, FluBlok led to a similar immune response in elderly subjects as the conventional trivalent inac-tivated vaccine produced in eggs [21], but a weaker immune response was observed in children aged between 6 and 35 months [20]. Safety was also demonstrated for the vaccine candidate Diamyd and no severe adverse effects were reported in clinical trials [19,23]. The therapeutic vaccine candidate showed a delayed onset of b-cell malfunction for about 30 months in patients suffering from Type 1 diabetes mellitus [19].

Provenge® is a recently FDA-approved cellular immunother-apy for prostate cancer, which was developed by Dendreon (WA, USA). A recombinant fusion protein produced by the BEVS is used as an antigen [25,26]. The PA2024 antigen is a chimeric protein formed by the human prostatic acid phosphatase and granulocyte–macrophage colony-stimulating factor (GM-CSF). Throughout clinical trials, PA2024 in conjunction with autolo-gous antigen-presenting cells was demonstrated to be safe and well tolerated by patients, although mild adverse effects were observed [25–27]. Survival was increased by 4.1–4.5 months among the sub-jects treated with Provenge, though no significant reduction in disease progression was observed [26,27].

The hepatitis E vaccine and H5N1 influenza virus vac-cine are vaccine candidates in early developmental stages (Phase I/II) (Table 1). The hepatitis E vaccine is a recombinant ORF2-truncated capsid protein (Sar 56 kDa) expressed in Sf9 cells [28,29]. The vaccine candidate showed an efficacy of 88.5% after the first dose, and a cumulative efficiency of 95.5% after three doses [30]. The vaccine was well tolerated among the sub-jects and no severe side effects were observed [30]. PanBlok™, which was developed by PSC, is a candidate vaccine against the pandemic H5N1 influenza virus. Similar to FluBlok®,

the seasonal flu vaccine, the PanBlok vaccine is composed of recombinant HA (rHA) protein trimers (rH5) [31]. Although the PanBlok clinical trial is still in progress (NCT01147068), a preliminary study demonstrated a suboptimal immune response among the vaccinated subjects, although the vaccine candidate was extremely well tolerated [31]. The authors hypothesized that HA from avian viruses might be less immunogenic in compari-son with HA from human viruses [31] and they suggested the addition of coadjuvants to improve the immunogenicity of the PanBlok vaccine. In an independent study, Wei et al. demon-strated that the trimeric and oligomeric forms of rHA, from H5N1 influenza virus, elicited a better immune response than its monomeric form [32]. Additionally, they demonstrated similar immunogenicity of the rHA trimeric forms produced in mam-malian or insect cells despite different glycosylation patterns in the two expression systems. Throughout numerous clinical trials, the aforementioned vaccine candidates and therapeutic vaccine strategies have demonstrated that recombinant proteins produced using BEVS technology in insect cells are safe and well tolerated, showing efficacy in the prevention or treatment of severe diseases.

Many other full or truncated recombinant proteins produced in insect cells are currently in early development stages (Table 1). These vaccine candidates are against different human diseases such as severe acute respiratory syndrome (SARS) virus [33–35], avian influenza B virus [36], West Nile virus [37], herpes simplex

Gene of interest

rBaculovirus

Antigen

Baculovirus vaccine

Insect cell

VLP vaccine

rProtein vaccine

Expert Rev. Vaccines © Future Science Group (2011)

Figure 1. Schematic description of vaccine production by baculovirus/insect cell technology.r: Recombinant; VLP: Virus-like particle.


Review Mena & Kamen

virus [38,39] and varicella zoster virus [40]. One example of these vaccine candidates is the recombinant SARS coronavirus spike (S) protein. Full or truncated S protein induced a strong bind-ing and neutralizing antibody response in mice in the pres-ence or absence of adjuvants [33–35]. The rHA and recombinant neuraminidase (rNA) proteins of avian influenza B virus are other vaccine candidates for the prevention of acute human respiratory diseases [36]. The rHA and rNA proteins have been expressed in Sf9 cells and their immunogenicity was tested in a murine model. Their efficacies were compared with the conventional inactivated vaccines (CIV) and live-attenuated vaccines (LAIV) [36]. The immune and protective responses with the combination of equal immunogenic rHA and rNA proteins outperformed both LAIV and CIV. This combination provided better cross-reactive immunity than LAIV and CIV when the immunized mice were challenged by an anti genically drifted virus. The recombinant truncated West Nile virus enve-lope protein (rWNV-E) [37] is another vaccine candidate; this protein has been produced by expresSF+ cells and tested in different animal models. A single dose of 10 mg of rWNV-E induced high titers of neutralizing antibodies and gave full protection (100% survival rate) after challenge with a lethal

dose of West Nile virus in a murine model. The toxicity test in rats showed no adverse effects related to the administration of rWNV-E. In addition, rWNV-E elicited similar antibody titers to the commercial Fort Dodge vaccine when their boost-ing capacities were tested in pre-immunized horses and naive foals, demonstrating the potential of this vaccine candidate for human vaccination [37].

Baculovirus expression system technology has been used recently to produce recombinant proteins as vaccine candi-dates against tropical parasitic diseases such as malaria [41–43]. FALVAC-1, a truncated merozoite surface protein (MSP-1

19), was

evaluated in monkeys and showed acceptable immune and pro-tective responses when formulated with complete or incomplete Freund’s adjuvant. In a comparative study, Arnot et al. demon-strated the superior immunogenicity of MSP-1

19 produced in

insect cell cultures over the same protein produced by Pichia pastoris [41]. Lyon et al. compared another truncated recombinant protein (MSP-1

42) produced by insect cells and Escherichia coli

with different adjuvants [43]. All vaccine candidate formulations elicited adequate immune and protective responses in monkeys; however, the E. coli variant gave a slightly better performance over the baculovirus variant.

Table 1. Baculovirus-expressed recombinant proteins for human vaccination.

Subunit Virus or disease Vaccine name/animal model (manufacturer)

Status Ref.

Human vaccine candidates in clinical trials

Hemagglutinin Influenza virus FluBlok® Phase IIINCT00395174NCT00336453NCT00539864NCT00539981NCT00328107

[20–22,205]

Hemagglutinin H5N1 influenza virus PanBlok™ (Protein Sciences Corporation)

Phase IINCT01147068

[31,205]

Glutamic acid decarboxylase Diabetes Diamyd® (Diamyd® Inc.) Phase IIINCT00723411NCT00751842

[23,24,146,206]

Prostatic acid phosphatase fusion protein (PA2024)

Prostate cancer Provenge® (Dendreon) ApprovedPhase IIINCT00027599NCT00005947NCT00065442NCT00715078

[25–27,147,207]

ORF2 truncated protein (Sar 56 kDa) Hepatitis E virus (GlaxoSmithKline; Novavax; NIAID)

Phase IINCT00287469

[28–30]

Human vaccine candidates under development

Full or truncated virus protein SARS Mouse [33–35,148]

Truncated virus envelope protein West Nile virus Mouse, hamster, horse [37]

Glycoprotein D2 Herpes Simplex virus Mouse, guinea pig [38,39]

Merezoite surface protein 1 Malaria (Plasmodium falciparum) Mouse, rabbit, monkey [41,42]

Hemagglutinin and M proteins Influenza B virus Mouse [32,36]

SARS: Severe acute respiratory syndrome.



Veterinary vaccinesVeterinary vaccines have been a profitable niche for baculovirus-expressed recombinant proteins (Table 2). The first two licensed vaccines produced in insect cells (Porcilis® Pesti and Bayovac® CSF) were for veterinary applications. Both recombinant vaccines contain the envelope glycoprotein (rE2) of the classical swine fever virus as the antigen [44–46]. Porcilis Pesti is manufactured by Intervet/Schering-Plough and has rE2 from a Brescia strain [46], meanwhile Bayovac CSF is manufactured by Bayer Leverkeussen Inc. and uses rE2 from a virulent Alfort/Tubingen strain [46]. Both vaccines have proven records of safety and efficacy preventing horizontal and vertical infections after 4 weeks postvaccination [46,47]. However, in a comparative study of both vaccines, Bayovac CSF provided better protection for vertical infection after 2 weeks postinfection in comparison with Porcilis Pesti [45]. These marker vaccines were specifically designed to allow serological differentia-tion between infected and vaccinated animals, thus avoiding the killing of healthy animals in the case of outbreaks [44,46]. With the aim of reducing production costs, classical swine fever virus vaccine (rE2) production by the baculovirus/larva system has been tested and promising results concerning immunogenicity and protection were obtained, although further studies are needed [48]. In February 2008, Ingelvac CircoFLEX was approved by the EMA [201]. This vaccine is formulated with the recombinant cap-sid protein of PCV-2, the causing agent of the porcine respiratory disease complex. Additionally, PCV-2 has been associated with porcine circovirus diseases [49]. The vaccine is referred to in release documents from the Canadian Food Inspection Agency and the US Department of Agriculture as a ‘killed baculovirus vector’ because the vaccine consists of filtered and inactivated recombinant baculovirus- infected insect cell culture supernatant. Although, the main immunogenic component of the vaccine is the recombinant capsid protein of the PCV-2 produced in the supernatant, the true mechanism of immunization may be far more complex.

Rotavirus [50], bovine viral diarrhea virus (BVDV) [51], Newcastle disease virus [52], Japanese encephalitis virus (JEV) [53] and avian influenza virus (H5N1) [54] are among the target infec-tious diseases for the development of veterinary marker vaccines with insect cell-expressed recombinant proteins (Table 2). Vaccine candidates have been produced by either S. frugiperda or T. ni cell lines and have been tested in calf, avian and murine models. In the case of rotavirus, the recombinant VP6 protein did not elicit a strong immune response when pregnant cows were vaccinated, but it provided a satisfactory passive protection when newborn colostrum-fed calves were challenged with virulent rotavirus [50]. However, the immunization mechanism involving the VP6 vac-cine candidate is not yet fully understood [50]. In the case of BVDV, recombinant baculovirus-expressed envelope glycoprotein E2 (brE2) prevented pyrexia, reduced leukopenia and delayed virus shedding in a similar fashion to mammalian-expressed pro-tein (mrE2) in calves after challenge with virulent BVDV [51]. However, this protection was dose dependent, requiring 100 µg of brE2 versus 5 µg of mrE2 for a similar response. This dose–response effect might be due to different glycosylation patterns of the two expression systems [51]. By contrast, for Newcastle disease virus, the recombinant fusion (rND F) and HA-neuraminidase (rND HN) glycoproteins induced 100% protection in chickens after challenge, and diminished the virus shedding even after a single immunization [52]. A similar efficacy was achieved with the recombinant glycoproteins of JEV in a murine model [53]. Recombinant pre-membrane envelope (prME) and envelope (E) glycoproteins provided a survival rate of 100 and 92.3%, respec-tively, in comparison with a survival rate of 7.7% in nonimmu-nized mice after a lethal challenge of JEV, even though prME and E elicited low levels of neutralizing antibodies [53]. In the case of avian influenza vaccines for veterinary purposes, the rHA from highly pathogenic H5N1 inhibited virus shedding and showed high protective efficacy in chickens after challenge [54]. Moreover,

Table 2. Baculovirus-expressed recombinant proteins for veterinary immunization.

Subunit Virus Vaccine name/animal model (manufacturer) Ref.

Vaccines commercially available

Envelope glycoprotein E2 Classic swine fever virus Porcilis® Pesti (Intervet/Schering-Plough) [44,45,47,149,208]

Envelope glycoprotein E2 Classic swine fever virus Bayovac® CSF (Bayer) [45,46,149,150]

ORF2 Capsid protein Porcine circovirus type 2 Ingelvac® CircoFLEX™ (Boehringer Ingelheim) [49,151,152,209]

Vaccine candidates in field trials

Envelope glycoprotein E2 Classic swine fever virus Pig [48]

VP6 protein Rotavirus Calf [50]

Q-like protein Canine visceral leishmaniasis Dog [56]

Envelope glycoprotein E2 Bovine diarrhea virus Calf [51]

HA and M proteins Avian influenza virus Chicken [54]

HA and F proteins Newcastle disease virus Chicken [52]

Virus proteins Japanese encephalitis virus Mouse [53]

p67 protein East coast fever virus Cow [55]

F: Fusion; HA: Hemagglutinin; M: Matrix protein.


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full protection was provided at vaccine doses between 0.5 and 10 µg of protein, although lower doses led to mild clinical signs due to the challenge and high doses showed no signs of disease [54].

Interestingly, the BEVS has also been used for the production of veterinary subunit vaccines against parasitic diseases such as East Coast fever (Theileria parva) [55] and canine visceral leish-maniasis (CanVL) [56]. A recombinant fusion protein between the baculovirus envelope gp64 protein and Theileria parva p67 protein (GP64:p67) provoked a strong immune response and provided an acceptable neutralization of the sporozoite infection (76%) after challenge with a lethal dose of T. parva in cattle [55]. However, higher sporozoite neutralization (89%) and antibody titers were achieved with recombinant fusion proteins of GFP and p67 protein (GFP:p67). The authors hypothesized that these differences could be related to either misfolding due to the gp64 size or protein impurities found in GP64:p67 preparation [55]. In the case of CanVL [56], a recombinant chimeric protein (rJPCM5_Q), containing five antigenic fragments of acidic ribosomal and histone H2A pro-teins of Leshmania infantum, proved to be safe and induced adequate humoral and cellular responses in a canine model [56]. Nevertheless, CanVL provided no immunity when vac-cinated dogs were challenged with L. infantum promastigotes and the authors suggested further studies in adjuvant selection to improve the vaccine efficacy [56].

Insect cell-expressed VLPs as subunit vaccinesVirus-like particles are recombinant structures morphologically similar to native viruses (enveloped and nonenveloped) but lack-ing viral genomes. Therefore they are not infectious and can induce similar or better immune responses than inactivated virus vaccines. Numerous VLPs have been produced with bacu-lovirus/insect cell technology [6,57], mainly because of the high expression levels of insect cell expression systems in comparison to mammalian cell expression systems, and the versatility of the BEVS for expressing VLPs formed by up to five different pro-teins [57]. Furthermore, the BEVS can efficiently produce VLPs from either enveloped or nonenveloped viruses. In influenza VLP manufacturing, standard procedures such as cloning, baculovi-rus amplification and quantification (plaque assays or median tissue culture infections dose [TCID

50] assays) are performed

exclusively in S. frugiperda cell lines, so these cells have been the preferred host for influenza VLP production [58]. However, in recent studies [59,60], T. ni cells have become an outstanding alternative for influenza VLP production, reaching HA yields between 1.5- to 15-times higher than S. frugiperda cell lines with-out compromising the VLP size, morphology or immunogenicity properties [59]. Indeed, H1N1 influenza VLPs, produced in BTI-TN5B1-4 cells, were more dense than their Sf9 counterpart, and the authors hypothesized that more HA proteins were assembled into VLPs, thus exhibiting a higher density [60]. Furthermore, baculovirus production in the supernatant by BTI-TN5B1-4 was 2-log lower in comparison with Sf9 cells, thus VLP downstream processing may be simplified and baculovirus contamination of VLP preparations reduced [59].

Although the BEVS has been widely adopted as the main system for VLP production, there are still several manufacturing issues to be addressed to achieve robust VLP upstream and downstream processing. These challenges are essentially associated with the intrinsic characteristics of the VLP vaccine candidate, such as the stoichiometry and assembly efficiency of the structural proteins or the budding process of enveloped VLPs. Palomares and Ramírez extensively reviewed the challenges for the production of rotavirus-like particles, a relevant model for nonenveloped VLP vaccines [57]. The manufacturing of enveloped VLP structures might be even more challenging because of complex budding mechanisms associ-ated with the release of the VLPs in the culture supernatant. Among the challenges identified, in the case of complex VLP structures, the potential encapsidation of host cell DNA during VLP assem-bly appears as a critical parameter that might impact the safety of the final product. Different approaches, specific to each type of VLP vaccine, have been considered to overcome these challenges [57,61,62]. Recently, it was suggested to use single-round replication baculovirus constructs to avoid the release of progeny baculovirus in the culture supernatant [62]. Removal of baculovirus remains a critical step in the downstream processing of VLPs. Baculovirus contamination in the final product poses a critical regulatory issue, especially for VLP-based human vaccine candidates [63]. Different approaches have been used for the concentration, capture, purifica-tion and polishing of nonenveloped, as well as enveloped, VLPs. Such approaches include ultrafiltration, ultracentrifugation, liquid chromatography and aqueous two-phase systems [63–65].

This section describes the current status of different VLPs vaccines and vaccine candidates for human and veterinary use and provides, whenever possible, details on the upstream and downstream processing solutions implemented to address the aforementioned manufacturing challenges.

Human vaccinesCervarix (GSK) against HPV, a baculovirus-expressed VLP vac-cine has been approved and is currently commercially available; in addition, several other VLPs vaccine candidates are at different stages of development (Table 3).

Human papilloma virus is a nonenveloped virus and five HPV serotypes (16, 18, 31, 33 and 45) are the causing agent of 87% of the cervical cancer cases in North America [61]. Although approved in Europe in September 2007, Cervarix, the first human recombinant vaccine produced by insect cells, was approved by the FDA in October 2009 for vaccination against HPV type 16 and 18 in women between 10 and 25 years old [202]. The delay of approval between the two agencies was speculated to be mainly related to the use of a new adjuvant in the formula-tion of the vaccine. In any case, Cervarix approval is certainly a critical milestone for the acceptance of the BEVS by regulators and bio manufacturers as a production platform for either human therapeutics or vaccines. HPV VLP, which is composed of types 16 and 18 L1 protein [61] (the antigen component of Cervarix [66]), is produced by the proprietary T. ni Hi-5 Rix4446 cell line [61]. In 1992, Kirnbauer et al. demonstrated that baculovirus-expressed HPV VLP (type 16) elicited high titers of neutralizing antibodies



in rabbits and the full L1 protein self-assembled spontaneously into VLPs [7]. Further studies confirmed that the full L1 pro-tein self-assembly occurs in the nucleus and cytoplasm of insect cells [67]. However, Cervarix contains a C-terminal-truncated L1 protein to prevent intracellular self-assembly, thus avoiding the potential encapsidation of foreign DNA and also facilitating the purification process [61]. Throughout clinical trials, Cervarix proved to be safe and well tolerated among the subjects [68,69] and induced high neutralizing antibody titers [66,68], demonstrating high efficacy (90%) against cervical intraepithelial neoplasia [69].

A comparative study between Cervarix and Gardasil®, a similar VLP vaccine produced in yeast and commercialized by Merck and Co., demonstrated similar performances for both vaccines in terms of immunogenicity, efficacy and tolerance; however, serum neutralizing antibody titers and the frequency of HPV-specific memory B-cells were higher after 7 months with Cervarix, though the authors suggested further studies are needed to determine the relevance of these findings [66,70]. HPV VLP vaccine candidates against other HPV types (31 and 58) are in preclinical stages with promising results with regard to immune response and long-term

Table 3. Baculovirus-expressed virus-like particles for human vaccination.

VLPs Virus/parasite Vaccine name/animal model (manufacturer)

Status Ref.

Human vaccine candidates in clinical trials

Papillomavirus-like particle

Human papillomavirus Cervarix™ (GlaxoSmithKline) Commercially available [7,61,66–68, 70,153,210]

Influenza VLP H1N1 influenza virus H1N1 2009 VLP vaccine (Novavax) Phase IINCT01072799

[111]

Influenza VLP Influenza virus Influenza VLP vaccine (Novavax) Phase IINCT01014806NCT00903552NCT00754455

[105,107]

Influenza VLP H5N1 influenza virus H5N1 influenza vaccine (Novavax) Phase I/IINCT00519389

[108,109]

Parvovirus B19 VLP Parvovirus B19 MEDI-149 (Meridian Life Sciences; MedImmune; NIAID)

Phase II [75,77,78,154]

Norwalk-VLP Norwalk virus Norwalk VLP vaccine (LigoCyte Inc.) Phase INCT00806962NCT00973284

[79,81–

83,155]

Human vaccine candidates under development

Papillomavirus VLP; serotypes 31 and 58

Human papillomavirus Mouse, rabbit [71,72]

Rotavirus-like particles Rotavirus Mouse, rabbit, pig [86,92,96, 98,156,157]

Influenza VLP 1918 influenza virusH3N2 influenza virusH5N1 influenza virusH1N1 influenza virus

Mouse [59,60,106, 110,112,158]

Influenza pseudotyped Gag VLP

H3N2 influenza virusH5N1 influenza virus

Mouse, ferret [159]

HIV-VLP HIV-1 Mouse, baboon [160–163]

SARS VLP SARS coronavirus Mouse [114,116, 117,164]

Ebola and Marburg VLP Ebola virus Mouse [118–120]

Hepatitis VLP Hepatitis E virus Monkey [101]

West Nile VLP West Nile virus Mouse [165]

Polyomavirus JC VLP Polyomavirus JC Rabbit [103]

Enterovirus 71 Hand, foot and mouth disease

Mouse [3,102]

Influenza VLP Mycobacterium tuberculosis Mouse [113]

JC: John Cunningham; SARS: Severe acute respiratory syndrome; VLP: Virus-like particle.


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humoral immunity [71,72]. Deschuyteneer et al. briefly described the Cervarix purification scheme [61]. As the L1 protein (HPV 16 and HPV 18) does not self assemble into VLP, it remains in the cytoplasm. The producing cells are disrupted by osmotic shock to release the L1 protein into the supernatant, and then the L1 protein is purified through a series of chromatographic steps. As final steps, the L1 capsomers are concentrated and placed under special buffer conditions to promote the assembly of the HPV-VLPs. An independent study proposed a purification scheme of HPV-VLP (serotype 33) based on two chromatographic steps [73]. The approach includes a cation exchange chromatography step for the removal of major protein contaminants and a ceramic hydroxyapatite chromatography as a polishing step of the final product achieving a recovery yield of 58.7% and a final product purity of 97%.

Parvovirus B19 VLPs and Norwalk VLPs are other vaccine candidates of nonenveloped viruses currently in late development stages (Table 3). Parvovirus B19 VLP vaccine candidate is a relevant example of BEVS versatility, where its capsid composition can be modulated to increase the immune response [74,75]. The native parvovirus B19 capsid is composed of VP1 (5%) and VP2 (95%) proteins [75]. However, parvovirus B19 VLPs with between 25 and 40% of VP1 induced a higher immune response than the vaccine candidates with a similar ratio to the native virus [74]. The parvovirus B19 VLP vaccine candidate (MEDI-491) was developed by the National Health Lung and Blood Institute of the NIH and MedImmune (MD, USA) under a cooperative research agreement, whereas Meridian Life Sciences (TN, USA) developed and licensed the production process as well as delivered material for clinical trials [76]. The parvovirus B19 VLP production process consists of coinfection of Sf9 cells with baculoviruses bacVP1 and bacVP2 at multiplicity of infection of 1 and 0.5 PFU/cell, respectively, producing self-assembled parvovirus B19 VLP with 20–40% of VP1 [76]. In contrast to influenza VLPs, parvo virus B19 VLPs are not released into the supernatant, requiring strin-gent conditions such as pH 9 and 50°C for their extraction with VLP recovery yields approximately 20% [77]. In clinical trials [78], MEDI-149 formulated with MF59C.1 adjuvant proved to be safe and well tolerated by the subjects, and induced serum neutralizing antibody titers that lasted at least 6 months after vaccination [78]. MedImmune developed a purification method for parvovirus B19 VLP based mainly on microfluidization for cell disruption and ion exchange chromatography for capturing, purification and polishing, although recovery yield and purity were not provided [76]. However, MedImmune has stopped further development of MEDI-491 vaccine and has returned its rights to the NIH; the NIH, National Institute of Allergy and Infectious Disease in col-laboration with Meridian Life Science is continuing parvovirus B19 VLP vaccine development [76].

Two clinical trials are currently in progress to evaluate the safety, immunogenicity and efficacy of a Norwalk VLP vaccine candidate manufactured by the US company LigoCyte (Table 3). Originally considered as a valuable tool for cell–virus interaction research [79], Norwalk VLPs expressed in Sf9 cells are now being considered as a vaccine candidate, which proved to be highly

immunogenic in mice even without adjuvant [80]. Norwalk VLPs are composed of 180 monomers of VP1 (58 kDa) organized in icosahedra with a diameter of 35 nm [81]. The production of Norwalk VLPs takes advantage of the BEVS technology because a single-layered capsid formed by one kind of protein requires a single baculovirus construct [81]; in addition, the high expression level of a self-assembled capsid that is released into the supernatant greatly simplifies the downstream processing [79]. Interestingly, these particles have shown great stability under acidic conditions (pH 3–7) with the desired characteristics of an oral vaccine candi-date [81]. The ability of Norwalk VLPs to induce high neutralizing antibody titers and satisfactory humoral, mucosal and cellular responses has been demonstrated in animals [82] and humans [83] regardless of adjuvant addition or the administration route (oral or intranasal).

Rotavirus-like particles (RLPs) may be the next VLPs vaccine candidate to be evaluated in clinical trials as an alternative to live-attenuated virus vaccines [84,85]. Rotavirus is a nonenveloped virus and the causing agent of 440,000 infant deaths by severe gastroentiritis worldwide [86]. Although rotavirus vaccines based on attenuated or inactivated viruses are highly immunogenic, the first rotavirus vaccine (Rotashield™) showed important safety concerns, such as intussusception among vaccinated infants [87]; recently, one of the commercially available vaccines showed cross-contamination with porcine circovirus type 1 [203]. These safety issues are inherent to the attenuated vaccine technology that rely on infectious viruses for vaccine production, but these problems can be avoided with RLP vaccines due to the lack of a viral genome [85]. Rotavirus capsids are composed of four viral proteins (VP2, VP6, VP7 and VP4), so different self-assembled RLPs can be formed when such proteins are expressed simul-taneously in insect cells [57]. Single-layered (VP2) [88], double-layered (VP2/VP6, VP6/VP7)[88,89], triple-layered (VP2/VP6/VP7, VP2/VP6/VP4) [90] and complete (VP2/VP6/VP7/VP4) RLPs are produced by the BEVS [57] and their immunogenicity profiles have been widely investigated in different animal mod-els. Double-layered [86,91–93], triple-layered [89,94,95] and complete [96–98] RLPs induced high rotavirus specific antibody titers in serum when they were administered by intranasal, intrarectal or intramuscular routes in mice and rabbits. Large-scale RLP bio-processing in insect cells is complex and some challenges still need to be overcome [57]. Such challenges are inherent to the rotavirus capsid morphology, requiring the stoichiometric and simultane-ous production of up to four recombinant viral proteins [57,88]. Similarly to other nonenveloped viral capsids expressed in insect cells, RLP self-assembly occurs intracellularly, so each cell should express the recombinant proteins simultaneously [88]. Protein co-expression can be achieved by two strategies: coinfection at an optimal multiplicity of infection ratio with monocistronic bacu-loviruses expressing the viral proteins [11,95,99], or infection with a multicistronic baculovirus expressing the capsid proteins under different promoters to modulate their level of expression [91,100]. Downstream processing of rotavirus VLP poses difficult chal-lenges owing to the different structures produced during the cell culture. The typical purification approaches for RLP purification



are based on ultracentrifugation with cesium chloride gradi-ent. However, different cost-effective and less time-consuming approaches such as ultrafiltration, size exclusion chromatography and aqueous two-phase systems have been applied to purify dif-ferent rotavirus VLP structures with remarkable success [64,65]. Peixoto et al. proposed a purification scheme of the triple-layered VLP (VP2/VP6/VP7) based on depth filter clarification, ultra-filtration/diafiltration by hollow fiber and size exclusion chroma-tography for polishing [64]. This approach allowed to segregate rotavirus triple-layered VLP from double-layered VLP, as well as complete removal of the baculovirus contamination. An accept-able recovery yield was achieved (37%) with removal of between 95 and 99% DNA contamination.

Baculovirus-expressed VLPs of nonenveloped viruses such as hepatitis E virus (HEV) [101], enterovirus 71 (EV71) [3,102] and human JC polyomavirus [103] are also vaccine candidates in preclinical trials for human vaccination (Table 3). Currently, no vaccines are available against HEV or EV71. However, HEV VLPs [101] have shown outstanding performances, inducing high titers of neutralizing antibodies and providing 100% immune protection after lethal challenge in animals (monkeys and mice) without adjuvant. In the case of EV71 VLPs, immunized mother mice conferred higher immune protection (89%) to newborn mice against lethal challenge than inactivated virus vaccine (60%) [3,102]. EV71 VLP manufacturing presents a challenge to the baculovirus system because of its requirement for the proteo-lytic processing of the P1 precursor by the viral 3CD protease; therefore, these proteins must be coexpressed in each cell [104]. Both coinfection with monocistronic baculoviruses or single infection with a bicistronic baculovirus led to self-assembled EV71 VLPs, although the latter give higher EV71 VLP yields [104]. Further improvements in the bicistronic construct and upstream processing led to a 43-fold increase of the EV71 VLP yield (1.5–64 mg/l) [102].

In the case of VLP vaccine candidates for enveloped viruses, baculovirus-expressed influenza VLPs are the most advanced vaccine candidates currently in late development stages (Table 3). In contrast with the influenza vaccine candidates composed only of trimers of HA protein, which were described in the ‘Insect cell-expressed virus-like particles as subunit vaccines’ section. Influenza VLPs are complex structures with similar morphol-ogy and size (80–120 nm in diameter) to the native virus [63,105]. Such particles are formed by HA, neuraminidase (NA), matrix 1 (M1) and matrix 2 (M2) recombinant proteins. HA and NA are glycoproteins whereas M1 and M2 are the matrix structural pro-teins of the influenza VLPs [63,105,106]. Five clinical trials (Phase I and II) are ongoing to evaluate three different influenza VLP vaccine candidates manufactured by the US company Novavax (influenza VLPs, H5N1 VLPs and H1N1 2009 VLPs). These influenza vaccine candidates are formed by HA, NA and M1 proteins of different serotypes and clades and are expressed in the Sf9 cell line. The production of these influenza VLPs exempli-fies the versatility of the BEVS where three or four recombinant proteins are expressed by one multicistronic baculovirus with independent expressions cassettes [105,106]. Infection of insect

cells with the multicistronic baculovirus led to self-assembled influenza VLPs with similar morphology and size to the native influenza virus [105]. These VLPs are released into the supernatant by the infected insect cells at 72 h post-infection, facilitating the recovery and downstream processing of influenza VLPs [105]. In preclinical trials, avian H9N2 VLPs were well tolerated and elicited similar antibody titers in mice to those obtained with inactivated H9 influenza virus. These VLPs immunized the ani-mals, avoiding virus replication after challenge with live H9 virus [105]. In the case of H3N2 VLPs, also manufactured by Novavax, Bright et al. obtained similar neutralizing antibody titers in com-parison with both inactivated virus and rHA in ferrets and mice [107]. However, H3N2 VLPs elicited broader immune responses, including expression of antibodies against five different viral isolates, while both rHA and inactivated virus elicited antibod-ies against only two viral isolates [107]. The H5N1 VLP vaccine candidate (clade 1 and 2) also showed an ability to elicit broader immune responses and cross-clade immunogenicity in mice [108] and ferrets [109]. An independent study demonstrated that an unadjuvanted H5N1 VLP vaccine not only provided 100% pro-tection to vaccinated mice but also gave long-lasting immune protection (up to 14 months) in comparison with an inactivated virus vaccine (up to 8 months) [110].

The advantages of the BEVS over other production systems were highlighted by the H1N1 2009 pandemic VLP vaccine candidate with its rapid generation, production and evaluation in animals. It induced high titers of HA neutralizing anti bodies in ferrets, providing full protection against infection when the animals were challenged with virulent H1N1 2009 influenza virus [111]. Two other influenza VLP vaccine candidates are under devel-opment by TechnoVax (NY, USA) [5,106,112]. Galarza et al. dem-onstrated that an influenza VLP, formed by HA and M1 proteins and formulated without adjuvant, provided 100% of protection in mice against a lethal H3N2 influenza virus challenge regardless of the administration route (intramuscular or intranasal) [112]. TechnoVax is also developing a VLP vaccine against the pandemic 1918 H1N1 influenza virus [106]. The 1918 VLP is formed by the HA, NA, M1 and M2 proteins and is expressed in Sf9 cells by a multicistronic baculovirus [106]. Immunized mice with the 1918 VLP showed high HA antibody titers in serum, and the VLP vaccine provided similar protective efficacy to the vaccination with attenuated virus [106].

Influenza VLPs downstream processing is a particular example of the specific challenges posed by enveloped VLPs. Influenza VLPs have irregular size and structure in contrast with the well-defined shape of nonenveloped VLPs. Moreover, as influenza VLPs and baculovirus co-bud from the producing insect cells by different mechanisms but through similar cellular paths, it is very likely that both structures might have envelope proteins such as gp64 or HA and other cellular proteins on their membrane [63]. In addition, as both particles are enveloped structures, any treatment to selectively inactivate the progeny of baculovirions will very likely impact the influenza VLPs structure as well. At small scale, influenza VLP purification protocols relied almost exclusively on ultracentrifugation techniques such as sucrose


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cushion, discontinuous sucrose or iodixanol density gradients [59,105,106,113]. Although these techniques provide VLPs ready to use for preclinical trials, baculovirus removal is incomplete with baculovirus concentrations in the final product between 1.0 × 106 PFU/ml to 1.0 × 108 PFU/ml depending on the cell line used for production [59,105,113]. However, Pincus et al. reported a successful large-scale purification step sequence for influenza VLP industrial manufacturing [63]. The approach is a combination of ultrafil-tration and chromatographic steps carried on in the following order: tangential flow filtration for cell removal; concentration/diafiltration for cell debris and media component removal; ion exchange chromatography for baculovirus and DNA removal; and finally size exclusion chromatography for residual host compo-nents removal and polishing. This scheme facilitates the removal of the baculovirus and DNA/RNA contamination, because under the matrix conditions both contaminants are negatively charged and bind onto the resin whereas the influenza VLPs are collected in the flowthrough. This purification scheme achieved VLP purity between 78 and 90% and the residual baculovirus is inactivated by treatment with b-propiolactone. Overall these steps are very common in the vaccine industry and the sequence of steps can be reasonably extrapolated to other enveloped VLP vaccines processing.

Baculovirus/insect cell technology has been used to develop VLP vaccines against another pandemic threat, the syndrome of acute respiratory syndrome coronavirus (SARS CoV). SARS CoV is an enveloped virus formed by a positive-stranded RNA (29 Kb) and four structural proteins: spike (S), E, membrane (M) and nucleo-capsid (N) [114]. The CoV VLPs are composed only of the S, M and E proteins of SARS CoV [114,115]. CoV VLP production is based on the coinfection of Sf21 cells with two baculoviruses, one bicistronic for expressing M and E proteins, and another monocistronic for expressing S protein. Coexpression of M and E proteins is suf-ficient to obtain self-assembled CoV VLPs in the cytoplasm of infected cells [114,115], although highly immunogenic CoV VLPs are achieved by inclusion of S protein [114,116]. Moreover, CoV VLP immunogenicity can be modulated by changing the S protein from

different CoVs [116]. Lu et al. demonstrated that adjuvanted CoV VLP induced a satisfactory humoral and cellular response with high neutralizing antibody titers in mice regardless of a subcutaneous, intranasal or intraperitoneal administration route [117].

Filoviruses, such as Ebola and Marburg, are other enveloped viruses also among the targets for the development of vaccines based on VLPs (Table 3). Ebola (eVLP) [118–120] and Marburg (mVLP) [120] virus-like particles highlight the advantages of vaccine manufacturing with the BEVS technology because no operation is required under high containment levels for vaccine production [121]; moreover, five- to 20-times higher VLP yields are obtained in comparison with those of mammalian cell expres-sion systems [120,121]. Ye et al. produced self-assembled eVLPs by simultaneously expressing VP40 and surface glycoprotein (GP) in Sf9 cells [118]. However, the sole expression of GP protein led to pleomorphic structures, which were very dissimilar to the native virus. Comparison of the immunogenic profile of eVLP produced by insect and mammalian cells in mice revealed a similar dendritic cell response for both VLPs regardless of the different glycosyl-ation patterns [118,120]. Moreover, two independent studies recently obtained encouraging results when eVLP-vaccinated mice showed 100% protection against a lethal challenge of the Zaire strain of the Ebola virus [119,120]. Warfield et al. demonstrated full protec-tion with eVLPs formed by VP40, GP and nucleoprotein and expressed in BTI-Tn5B1-4 cells [120], whereas Sun et al. achieved 100% efficacy with eVLPs formed by VP40 and GP proteins and expressed in Sf9 cells [119]. These results again showed the outstanding robustness of the BEVS technology where excellent vaccine performance can be achieved regardless of the cell line used for manufacturing.

Animal vaccinesThe development of baculovirus-expressed VLP vaccines for vet-erinary use is relatively recent (Table 4). In January 2009, Porcilis PCV vaccine was approved by the EMA [204] . This vaccine is formulated with VLPs of PCV-2, which is a nonenveloped virus and the causing agent of the postweaning multisystemic wasting

syndrome in pigs [122]. In contrast to the Ingelvac® CircoFLEX vaccine, the immu-nogen of Porcilis PCV is the VLP formed by the ORF2 capsid protein of PCV-2 [122]. Porcilis PCV is safe, highly immuno-genic and effective against PCV-2 infec-tion. Moreover, it induces broad immune protection against different genotypes (1 and 2) and various geographic isolates [122,123]. ORF2 protein expression in either Sf9 [124] or Tn-5B1–4 [125] cells leads to self-assembled PCV-2 VLPs with similar morphologies and diameters (20 nm) as the native virus. However, contrary to Sf9 cells, Tn-5B1-4 cells produce higher yields and PCV-2 VLPs are released to the cell culture. The PCV-2 VLP self-assembly process remains unclear in both cell lines

Table 4. Baculovirus-expressed virus-like particles for veterinary immunization.

Virus-like particles Virus Vaccine name/animal model (manufacturer)

Ref.

Veterinary vaccines commercially available

Circovirus VLPs PCV-2 Porcilis® PCV (Intervet/Schering-Plough)

[122,123]

Veterinary vaccines in field trials

Circovirus VLPs PCV-2 Mouse, pig [124–126]

Parvovirus VLPs Porcine parvovirus Pig [127]

Bluetongue VLPs Bluetongue virus Sheep [128]

Foot and mouth VLPs Foot-and-mouth disease virus Cow [131,166]

Influenza VLPs Avian influenza virus Chicken, duck [129,130]

PCV: Porcine circovirus; VLP: Virus-like particle.



[124,125]. With the aim to have a cost-effective alternative, T. ni larvae have recently been evaluated as a manufacturing platform instead of traditional cell culture [126]. The resulting PCV-2 VLPs induced an adequate immune response, although not as strong as the immune response induced by typical PCV-2 VLPs, requiring the addition of adjuvant to improve their immunogenicity [126].

Porcine parvovirus (PPV) [127], Bluetongue virus (BTV) [128], avian influenza virus [129,130] and foot and mouth disease virus [131] have been evaluated as VLP vaccine candidates for animals (Table 4). PPV and BTV VLPs are the most advanced and widely investigated. PPV VLP consists of 60 monomers of VP2 protein and the expression of VP2 in Sf9 cells leads to self-assembled VLPs in the cellular cytoplasm [132]. A cost-effective PPV VLP manufacturing process has been described by Maranga et al. [13]. In addition, Antonis et al. performed a comparative field study to evaluate the immunogenicity and potency of different PPV VLP formulations in comparison with a live attenuated com-mercial vaccine [127]. The PPV VLP vaccine candidate outper-formed the commercially available vaccine in both immunogenic-ity and efficacy, and also provided transplacental protection to piglets. After rotavirus, BTV is probably the most studied system related to baculovirus-expressed VLPs. Both viruses belong to the Reoviridae family and have similar capsid architecture and size. Therefore, both vaccine candidates share similar advantages and challenges for their manufacturing in insect cells. BTV VLP immunogenicity has been widely tested in animals with encour-aging results [84]. For veterinary use, the vaccine should be cost effective and with a broad spectrum covering several of the 24 different serotypes. With this in mind, Stewart et al. designed three VLP vaccine candidates sharing the same serotype for the inner capsid proteins (VP3 and VP7) but differing in serotypes for the outer capsid proteins (VP2 and VP5) [128]. The immuno-genicity and efficacy study of one of these VLPs demonstrated safety and showed 100% protection after challenge in vaccinated sheep. This approach would facilitate the manufacturing of VLP vaccines against different serotypes [128].

Recombinant baculovirus vectors as vaccine candidatesIn the previous sections, we described the current status of several baculovirus-expressed proteins and VLPs used as subunit vac-cines. However, the use of recombinant baculoviruses as a delivery system for immunogens has also been explored with very promis-ing results in animal experiments and these BEVS applications have been extensively reviewed [133–135]. Consequently, this section briefly summarizes the most important advances in the area.

Baculoviruses are able to infect mammalian cells, but the lack of mammalian-specific promoters impedes replication. Therefore baculoviruses are generally considered as safe, noninfectious and nonpathogenic for humans. By inserting mammalian expression cassettes into the baculovirus genome, recombinant baculoviruses have been developed as efficient delivery systems in mammalian cells for gene therapy and protein expression [134].

Glycoprotein gp64 is the main component of the baculovirus envelope and is responsible for the baculovirus interaction with the cell membrane. The baculovirus envelope is susceptible to

modifications without affecting its infectivity. To improve bacu-lovirus infectivity and trafficking in mammalian cells, its envelope has been modified to display the vesicular stomatitis virus glycopro-tein (VSV-G) instead of gp64 [136,137]. Recombinant baculoviruses combining mammalian expression cassettes with VSV-G pseu-dotyping have been widely evaluated as vaccines candidates [134].

Published process development studies on BEVS-derived prod-ucts have mainly focussed on either recombinant protein or VLPs rather than the baculovirus vector as a final product. However, recent developments of baculovirus vectors for vaccination and gene therapy generated a renewed interest in this topic. Aucoin et al. reviewed the state of the art of the baculovirus bioprocessing, highlighting recent advances in both upstream and downstream processing [58]. For the latter, purity and functionality are impor-tant aspects to consider for baculovirus vectors as vaccine candi-dates for human use. Different downstream processing strategies have been successfully used for baculovirus purification. These include ultracentrifugation steps for baculovirus concentration alongside chromatographic techniques such as anion exchange, size exclusion and affinity chromatography for baculovirus captur-ing and polishing [138–141]. These purification processes provided relatively pure material (87–98%) while retaining the baculovirus infectivity and overall recovery yields from 25 up to 78% [138–141].

Table 5 summarizes the results from several studies that evaluated the immunogenicity and effectiveness of baculovirus vaccines in different animal models. Baculovirus vaccines can be grouped into three categories:

• Recombinant baculoviruses that express the immunogen under the control of mammalian expression cassettes, including VSV-G-pseudotyped baculoviruses;

• Recombinant baculoviruses that display on the surface the anti-gens fused to the gp64 of the baculovirus envelope;

• A dual action with recombinant baculoviruses that combine mammalian expression cassettes with surface display technology.

Chen et al. compared these three strategies for vaccination with baculovirus vaccines against H5N1 influenza virus [142]. All three vectors induced a potent immune humoral response, resulting in high titers of neutralizing antibodies. However, the immune response depended on the vector type and immuniza-tion route. By intranasal or subcutaneous administration, vec-tors displaying HA protein in the baculovirus envelope elicited higher titers than a baculovirus vector expressing HA under the control of a cytomegalovirus immediate–early promoter. By intramuscular injection, HA-expressing baculoviruses vectors triggered stronger immune responses than a baculovirus vector only displaying HA. These results suggest that a dual display and expression strategy with baculovirus vectors might provide more efficient vaccine candidates, although the authors recom-mended further studies to confirm the vaccine efficacy [142]. In addition, dual-action baculovirus vaccines have been tested against Plasmodium berghei [143] and Plasmodium falciparum [144] in murine models. Yoshida et al. evaluated the immunoge-nicity and efficacy of a dual baculovirus vaccine that displayed


Review Mena & Kamen

and expressed the P. berghei circumporozoite protein (PbCSP) [143]. This vaccine induced high PbCSP-specific antibody titers in combination with Th1 and Th2 cellular responses but, more importantly, the vaccine provided 100% protection against challenge with a lethal dose of P. berghei sporozoites.

Baculovirus vector vaccines have been exclusively designed based on AcMNPV. However, Jin et al. designed and evaluated a baculovirus vaccine using Bombix mori multiple nucleopoly-hedrovirus [145]. The baculovirus vaccine displaying HA protein (H5N1 influenza virus) on the baculovirus envelope proved to be safe in vaccinated monkeys, and induced a long-lasting immune response with high HA antibody titers up to 14 weeks postvac-cination [145].

Expert commentaryFor years, BEVS and insect cell technology have been used as tools in transient recombinant protein expression for drug discovery purposes.

In the biopharmaceutical field, due to the well-established mammalian cell culture platforms with Chinese hamster ovary cells dominating the commercial manufacturing of recombi-nant proteins and monoclonal antibodies, insect cell technol-ogy remained confined to research and development laborato-ries. As a consequence, with the exception of some small and medium entreprises and bioengineering governmental labora-tories, industries associated with biopharmaceutical manufac-turing, including culture media manufacturers and contract manufacturing organizations, invested limited resources in the development of baculovirus/insect technology for commercial manufacturing. As an example, no commercial chemically defined medium for insect cell culture is available. Most of the serum-free media rely on supplementation of yeastolate to the basal IPL41 formulation [9].

By contrast, the vaccine field, often described as very conser-vative, rapidly adopted the baculovirus/insect cell technology platform for manufacturing recombinant proteins and VLPs

Table 5. Baculovirus vaccines under development.

Protein Virus or disease Animal model

Approach Ref.

Human vaccine candidates

Hemagglutinin H5N1 Avian influenza Mouse, monkey Surface display technologyPseudotyped-MEPDual

[16,135,142, 145,167–170]

Plasmodium falciparum Pfs25 Malaria Mouse, rabbit Surface display technology [171]

P. falciparum CS protein Malaria Mouse Surface display technologyMEPDual

[144]

Plasmodium berghei CS protein Malaria Mouse Dual [143]

Plasmodium yoelii PyMSP1 protein Malaria Mouse Surface display technology [172]

Spike protein SARS coronavirus Mouse Surface display technologyMEP

[164,173]

Glycoprotein D Herpes simplex virus Mouse Surface display technology [174]

Toxoplasma gondii SAG1 protein Toxoplasmosis Mouse Pseudotyped-MEP [175]

Envelope protein Japanese encephalitis virus

Mouse Pseudotyped-MEP [176]

Mycobacterium tuberculosis polypeptide 85A TB Mouse Surface display technology [177]

Animal vaccine candidates

Envelope glycoprotein Classic swine fever virus Mouse Surface display technologyPseudotyped-MEP

[178,179]

NS3 protein Classic swine fever virus Mouse Surface display technology [180]

Hemagglutinin H5N1 Avian influenza Mouse Pseudotyped-MEP [181]

Glycoproteins B, C and D Pseudorabies virus Mouse Pseudotyped-MEP [182]

ORF2 protein Porcine circovirus type 2 Mouse Pseudotyped-MEP [183]

GP5 and M protein PRRS virus Mouse Pseudotyped-MEP [137]

Dual: Combination of mammalian expression promotor and surface display technology; M: Membrane; MEP: Mammalian expression promotor; ORF: Open reading frame; PPRS virus: Porcine reproductive and respiratory syndrome virus; SARS: Severe acute respiratory syndrome.



of modern vaccines. The two main reasons for selecting this technology over other expression technologies are associated with safety and cost–effectiveness. Safety is of paramount importance for vaccines, because many healthy people includ-ing children are vaccinated; however, cost–effectiveness is criti-cal to the successful commercialization of vaccines, especially for animal use. For the vectored vaccine approach, contrary to other viral vectors derived from human viruses such as adeno-virus or vaccinia virus, baculoviruses are generally considered as safe and do not require high-level bio-containment facilities. These characteristics contribute to both increased safety and cost–effectiveness.

The commercial licensing of Cervarix and at a lesser extent of Provenge contributed to the establishment of baculovirus/insect cell technology as a credible platform by demonstrating that the regulatory path is accessible for approval of safe biopharmaceuticals for human use.

Five-year viewThe pending approval of the FluBlok influenza vaccine will cer-tainly reinforce the positioning of the BEVS as a first-generation insect cell culture-derived flu recombinant vaccine for human use. Diamyd, a therapeutic vaccine for diabetes mellitus Type 1, might be the first BEVS-derived product with a very large market to address a major worldwide public health issue.

Further improvement with high cell density fed-batch opera-tions and the development of better and more robust media might

contribute to the cost–effectiveness of insect cell manufacturing processes and facilitate broader adoption of the technology by the biomanufacturing industry.

Other possible progresses are expected from better molecular design of specialized BEVS vectors to increase the production yield and facilitate the downstream processing of the vaccine bulk product by minimizing contamination with baculovirus.

To date, baculovirus/insect cell technology is preferred to produce VLP-type vaccines. Within the rapidly increasing vac-cine markets, more and more VLP vaccines, such as Rotavirus, Norwalk and pandemic influenza, are being developed, thus driving the success of the baculovirus/insect cell technology platform.Significant efforts continue to be invested in developing high-per-formance baculovirus vectors for gene delivery and vaccination, especially in Europe and Asia, which might ultimately translate to clinical applications.

Financial & competing interests disclosureThe authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.

Key issues

• The baculovirus/insect cell technology is a well-adopted platform for manufacturing novel vaccines using recombinant DNA technology. The technology has been successfully used for: subunit recombinant protein vaccines; virus-like particle (VLP) vaccines and baculovirus-vectored vaccines for display and/or expression of antigens.

• Multiple clinical trials of baculovirus expression system-derived vaccines have demonstrated quality, safety and efficacy, leading to regulatory approval and market authorization for the prevention and treatment of human diseases. The approval of Cervarix™, a VLP vaccine produced using insect cell technology, by the EMA in September 2007 and the US FDA in October 2009 set critical milestones for the regulatory acceptance of baculovirus expression system technology.

• In the case of veterinary vaccines, numerous recombinant proteins, VLPs and vectored vaccines are either commercially available or in late stages of development, indicating that insect cell technology is safe and cost effective.

• Continuous improvements of the baculovirus molecular design, such as multicistronic constructs, have led to flexible use of the system for efficient production of complex structures, such as VLPs requiring specific stoichiometry for assembly.

ReferencesPapers of special note have been highlighted as:•ofinterest••ofconsiderableinterest

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• GoodreferencepaperdescribingthesafetyprofileofinsectcellsandtheevaluationofFluBlok®vaccineinclinicaltrials.

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43 Lyon JA, Angov E, Fay MP et al. Protection induced by Plasmodium falciparum MSP1

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• InterestingpaperdescribingthebioprocessingchallengesofVLPmanufacturingbyinsectcells.

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•• Excellentpaperreviewinginsectcell/baculovirusupstreamanddownstreamprocessingandquantification.

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•• ExcellentpaperdescribingindetailthephysicochemicalcharacterizationoftheCervarix™vaccine.

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108 Bright RA, Carter DM, Crevar CJ et al. Cross-clade protective immune responses to influenza viruses with H5N1 HA and NA elicited by an influenza virus-like particle. PLoS ONE 3(1), e1501 (2008).

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110 Kang SM, Yoo DG, Lipatov AS et al. Induction of long-term protective immune responses by influenza H5N1 virus-like particles. PLoS ONE 4(3), e4667 (2009).

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• InterestingpaperreviewingtheuseofVLPvaccinesagainstEbolaandMarburgviruses.

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•• Interestingpaperreviewingthestateoftheartofbaculovirusasatoolforhumanvaccination.

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Websites

201 Ingelvac CircoFLEX Approval by EMA www.ema.europa.eu/ema/index.jsp?curl=pages/medicines/veterinary/medicines/000126/vet_med_000132.jsp&murl=menus/medicines/medicines.jsp&mid=WC0b01ac058001fa1c&jsenabled=true

202 Cervarix™ Approval by FDA www.fda.gov/downloads/BiologicsBloodVaccines/Vaccines/ApprovedProducts/UCM198333.pdf

203 Rotavirus vaccines safety update by FDA www.fda.gov/Safety/MedWatch/SafetyInformation/SafetyAlertsforHumanMedicalProducts/ucm205640.htm

204 Porcilis® PCV Approval by EMAwww.ema.europa.eu/ema/index.jsp?curl=pages/medicines/veterinary/medicines/000135/vet_med_000159.jsp&murl=menus/medicines/medicines.jsp

205 Protein Sciences Corporation www.proteinsciences.com

206 Diamyd Medical AB www.diamyd.com

207 Dendreon Corporation www.dendreon.com

208 Intervet/Schering-Plough www.intervet.com

209 Ingelvac® CircoFLEX™ www.circoflex.ca

210 Cervarix™ www.cervarix.ca

2011 Insect Cell Technology is a Versatileand Robust Vaccine Manufacturing Platform

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