key: cord-0720871-e0m0h926
authors: van Oers, Monique M.
title: Vaccines for Viral and Parasitic Diseases Produced with Baculovirus Vectors
date: 2006-09-22
journal: Adv Virus Res
DOI: 10.1016/s0065-3527(06)68006-8
sha: 2ff451eed16e4189c7c7cc680bfbb1aabf1e6294
doc_id: 720871
cord_uid: e0m0h926

The baculovirus–insect cell expression system is an approved system for the production of viral antigens with vaccine potential for humans and animals and has been used for production of subunit vaccines against parasitic diseases as well. Many candidate subunit vaccines have been expressed in this system and immunization commonly led to protective immunity against pathogen challenge. The first vaccines produced in insect cells for animal use are now on the market. This chapter deals with the tailoring of the baculovirus–insect cell expression system for vaccine production in terms of expression levels, integrity and immunogenicity of recombinant proteins, and baculovirus genome stability. Various expression strategies are discussed including chimeric, virus‐like particles, baculovirus display of foreign antigens on budded virions or in occlusion bodies, and specialized baculovirus vectors with mammalian promoters that express the antigen in the immunized individual. A historical overview shows the wide variety of viral (glyco)proteins that have successfully been expressed in this system for vaccine purposes. The potential of this expression system for antiparasite vaccines is illustrated. The combination of subunit vaccines and marker tests, both based on antigens expressed in insect cells, provides a powerful tool to combat disease and to monitor infectious agents.

in occlusion bodies, and specialized baculovirus vectors with mammalian promoters that express the antigen in the immunized individual. A historical overview shows the wide variety of viral (glyco)proteins that have successfully been expressed in this system for vaccine purposes. The potential of this expression system for antiparasite vaccines is illustrated. The combination of subunit vaccines and marker tests, both based on antigens expressed in insect cells, provides a powerful tool to combat disease and to monitor infectious agents.

Historically, vaccines have been one of the most cost-effective and easily administered means of controlling infectious diseases in humans and animals. Vaccine development has its roots in the work of Edward Jenner (1749-1823) who discovered that man could be protected from smallpox by inoculation with cowpox (Fenner, 2000) and the work of Louis Pasteur (1822-1895) who developed the first rabies vaccine (Fu, 1997) . These pioneering efforts led to vaccines against diseases that had once claimed millions of lives worldwide (Andre, 2003) . Childhood vaccination programs are now common practice and elaborate vaccination programs have been set up by the World Health Organization (WHO), leading to the official eradication of smallpox in 1979 (Fenner, 2000) . Today large parts of the world are also declared poliomyelitis free, and measles is the next target for eradication. Vaccines have controlled major bacterial and viral diseases in humans, and effective vaccines are available against many more (Andre, 2003; Hansson et al., 2000b) . Vaccination also protects our livestock and pet animals (Pastoret et al., 1997) . For some diseases, however, such as malaria and acquired immunodeficiency syndrome (AIDS), vaccines are desparately sought.

Most human and animal vaccines are based on killed or live-attenuated pathogens. Killed vaccines require the production of large amounts of often highly virulent pathogens and these types of vaccines are therefore risky to produce. Another risk lies in the potential for incomplete inactivation of the pathogens. Inactivation on the other hand affects the immunogenic properties of the pathogen, and hence the efficacy as a vaccine, and it is often difficult to find the balance between efficient inactivation and conservation of immunogenicity. Live-attenuated vaccines consist of pathogens that are reduced in virulence or have been attenuated either by growing them in alternative hosts or under unfavorable growing conditions, or by recombinant DNA technology. These live-attenuated vaccines can potentially replicate in their host, but are typically attenuated in their pathogenicity to avoid the development of severe disease. Live vaccines elicit humoral and cellular immunity, and may provide lifelong protection with a single or a few doses (Dertzbaugh, 1998; Hansson et al., 2000b; Schijns, 2003) . Such long-term protection is advantageous in developing countries where individuals are often only immunized once. A drawback of live-attenuated vaccines is that they can cause side effects, which may be dangerous when used for prophylaxis in immunocompromised persons such as the elderly or individuals with genetic or acquired diseases of the immune system (e.g., AIDS or severe combined immunodeficiency; SCID). Live-attenuated vaccines may also convert to virulent strains and spread to nonimmunized persons as observed during recent poliomyelitis outbreaks (Kew et al., 2004) . Adverse effects with both killed and liveattenuated vaccines can also be due to allergic reactions to components of the vaccine such as residual egg proteins in the case of influenza vaccines (Kelso and Yunginger, 2003) or gelatin in the measles-mumps-rubella (MMR) vaccine (Patja et al., 2001) .

The development of vaccines is not easy for all infectious diseases and the medical and veterinary world is challenged frequently by the emergence of novel diseases such as AIDS, severe acute respiratory syndrome (SARS), and West Nile virus infection. The vaccine industry is under constant pressure for rapidly changing pathogens, for which large amounts of vaccines are needed annually, such as influenza viruses (Palese, 2004) , and flexible vaccine production techniques are required. For several infectious diseases vaccines cannot be developed using conventional approaches, for instance due to a lack of appropriate animal production systems or the high-mutation frequency of the pathogen (Human immunodeficiency virus (HIV), malaria). A vaccine against the H5N1 influenza strain that is currently epidemic in Asian poultry could not be produced the classical way, by using embryonized chicken eggs without reducing the virulence of the virus by reverse genetics, due to high-mortality rates of the chicken embryos (Horimoto et al., 2006) .

Recombinant protein production systems may provide good alternatives for the development of vaccines that are more difficult to produce in vivo for manufacture of so-called subunit vaccines. A pathogen consists of many proteins, frequently with carbohydrate moieties, but these are not all equally important for generation of an adequate immunological response. Subunit vaccines contain the immunodominant components of a pathogen and in the case of viral vaccines these are often (glyco)proteins of the viral coat or envelope such as the hepatitis B surface antigen (Valenzuela et al., 1982) or the classical swine fever virus (CSFV) E2 glycoprotein (Bouma et al., 1999) . Viral coat proteins sometimes form virus-like particles (VLPs) when expressed in heterologous systems (Brown et al., 1991) , which are often immunogenic and may induce both humoral and cellular responses. The subunit vaccine against hepatitis B produced in yeast is highly succesful. An extreme example of subunit vaccines are peptide-based vaccines which consist of small amino acid chains harboring the part of the antigenic protein that is recognized by antibodies. Typically, subunit vaccines do not contain the genetic material of the pathogen or only a small part thereof. Therefore, these vaccines cannot cause disease and do not introduce pathogens into nonendemic regions. An additional advantage of subunit vaccines is that they can be used in combination with specific marker tests, which make it possible to differentiate infected from vaccinated animals, the so-called DIVA vaccines (Capua et al., 2003; van Oirschot, 1999) ; an important issue in monitoring virus prevalence and virus-free export of animals and their products.

Immunogenic subunits can be isolated chemically from the pathogen, such as the purified capsular polysaccharides present in the Streptococcus pneumonia vaccine (Pneumovax23; Merck). This process still requires the production of virulent pathogens, which is not without risk. An alternative is the use of recombinant DNA technology to produce protein subunits in a heterologous system, and a variety of expression systems are available (Clark and Cassidy-Hanley, 2005; Hansson et al., 2000b ). The yeast system Saccharomyces cerevisiae for instance is used to produce the hepatitis B subunit vaccine (Valenzuela et al., 1982) , which is currently the only licensed recombinant subunit vaccine for human use. The yeast Pichia pastoris is used for production of the antitick vaccine Gavac TM (Canales et al., 1997) , which protects cattle against the tick Boophilus microplus, the transmitter of Babesia and Anaplasma parasite species. Insect cells are used to produce vaccines against classical swine fever or hog cholera (Depner et al., 2001; van Aarle, 2003) . For the production of recombinant proteins in higher eukaryotics, mammalian, insect, and plant expression systems are available that either use trangenes or viral vectors for protein expression. Plants have been recognized for the production of so-called edible subunit vaccines to be administered by ingestion of vegetable foods (Ma et al., 2005; Streatfield and Howard, 2003) .

This chapter concentrates on the use of cultured insect cells or larvae in combination with baculovirus expression vectors for the production of subunit vaccines. The baculovirus expression system is an accepted and well-developed system for the production of viral antigens with vaccine potential (Dertzbaugh, 1998; Hansson et al., 2000a; Vlak and Keus, 1990) . This system has also been explored for development of vaccines against protozoan parasites (Kaba et al., 2005) and for therapeutic vaccines against tumors. A vaccine against prostate-cancer (Provenge) is in phase II/III clinical trials and is based on combining recombinant prostatic acid phosphatase (characteristic of 95% of prostate cancers) with the patient's own dendritic cells before immunization (Beinart et al., 2005; Rini, 2002) . Trials have also been initiated for a prophylactic vaccine using VLPs produced in insect cells against cervical cancer caused by Human papillomavirus (HPV) 16 (Mao et al., 2006) .

Each expression system has advantages and drawbacks (Table I ) and the system of choice depends very much on the specific requirements for a particular vaccine and is often based, at least partly, on trial and error. Before a definitive choice can be made, the expression levels achieved, the adequacy of posttranslational modifications, the immunological performance, the possibilites for scale-up, the costs, the risk of contamination, the method of administration, and legal aspects must all be taken into account. 

The baculovirus-insect cell expression system (Smith et al., 1983) has been developed for the production of biologically active (glyco) proteins in a well-established and safe eukaryotic environment (Kost et al., 2005) . The family Baculoviridae contains rod-shaped, invertebrate-infecting viruses, which have large double-stranded, covalently closed circular DNA genomes (Table II) . The members of this large virus family are taxonomically divided into the genera Nucleopolyhedrovirus (NPV) and Granulovirus (GV), based on occlusion body morphology (Theilmann et al., 2005) . NPVs express two genes, polyhedrin and p10, at very high levels in the very late phase of infection. The polyhedrin protein forms the viral occlusion bodies or polyhedra and p10 is present in fibrillar structures, which function in polyhedron morphology and in breakdown of infected cell-nuclei to release the polyhedra (Okano et al., 2006; Van Oers and Vlak, 1997) . These two genes are not essential for virus replication in cell culture and, therefore, their promoters are exploited to drive foreign gene expression, which forms the basis for the baculovirus-insect cell expression system. Since baculoviruses are rod-shaped, large amounts of foreign DNA can be accommodated within the virus particle, in contrast to vaccinia and especially adenovirus expression vectors (Table II) .

The type member of the NPVs is Autographa californica multiple nucleopolyhedrovirus (AcMNPV), a virus with a genome of 133 kilobase pairs (Ayres et al., 1994) . This baculovirus is routinely used for foreign gene expression. The baculovirus Bombyx mori NPV is being used for vaccine purposes to a much lesser extent (Choi et al., 2000; Mori et al., 1994) . Baculovirus expression vectors replicate in cultured insect cells or larvae and high yields of heterologous protein are generally obtained when the strong viral polyhedrin and p10 promoters are exploited (King and Possee, 1992; O'Reilly et al., 1992) . The insect cell lines used in the baculovirus expression system are derived from lepidopteran insects (moths) and are most often Spodoptera frugiperda lines (Sf9 or Sf21) and Trichoplusia ni (High Five TM ) cells, which can be used in combination with AcMNPV-based vectors. B. mori cells (e.g., Bm5) are used for BmNPV. Insect cell lines vary in their characteristics in terms of growth rate, protein production, secretion efficiency and glycosylation pattern, and interference with viral genome stability (Pijlman et al., 2003b; Vlak et al., 1996) . These insect cells are relatively easy to maintain and many grow equally well in suspension in large volumes (up to 2000 L reactions) and at high densities as on solid supports, and can be cultivated in serum-free media which facilitates purification of recombinant proteins. Unlike mammalian cells, they do not require CO 2 and can easily withstand temperature fluctuations. An extra advantage is that the chance of contamination with human or mammalian viruses, especially in serum-free cultures, is small compared to mammalian production systems because these vertebrate viruses do not replicate in lepidopteran cells. These cells do not support the growth of mammalian mycoplasmas either. Instead of insect cells, whole insect larvae may be used as live bioreactors for vaccine production. The use of whole insect larvae has the advantage that the simple insect-rearing technology and downstream processing can be exploited. Such in vivo production could be performed by small-scale local industries, especially if the larvae can be fed directly to animals such as for an experimental Newcastle disease vaccine for chickens (Mori et al., 1994) . Such vaccines are less well defined however and quality control may therefore be more difficult to achieve. Expression of proteins in insect cells allows for appropriate folding, posttranslational modification, and oligomerization and therefore, biological activity is normally preserved. Protein glycosylation in insects and mammals is not identical though: the N-glycan-processing pathway in insects results in glycoproteins with paucimannose glycan groups, in contrast to mammalian glycoproteins which contain complex sialylated glycans (see also Harrison, this volume, . The exact glycan composition varies between different insect cell lines (Kost et al., 2005; Tomiya et al., 2004) . In general, glycan groups are not very immunogenic and therefore this does not seem to be a major disadvantage for subunit vaccines. In situations where more authentic glycosylation is required, for instance for preserving functional activity, transformed "humanized" insect cell lines expressing mammalian glycosylation enzymes are available (Jarvis, 2003; Kost et al., 2005; Tomiya et al., 2004) . For some insect cell lines it has been reported that fucose groups are added to N-glycans. The impact of this remains to be determined, but since fucans may cause allergic reactions, it may be a point for consideration when choosing an insect cell line for vaccine production (Long et al., 2006; Tomiya et al., 2004) .

Originally, the baculovirus expression system was based on the allelic exchange of the baculovirus polyhedrin gene for a heterologous gene by recombination in insect cells (Smith et al., 1983) . In a similar way, baculovirus vectors have since been developed which exploit the nonessential very late baculovirus p10 promoter Weyer and Possee, 1991) . Vectors that leave the polyhedrin gene intact can be used for the production of recombinant proteins in insect larvae (Fig. 1) . The in vivo recombination protocol was improved by using linearized viral DNA in the allelic replacement, which resulted in dominant selection and much higher percentages of recombinant viruses (Kitts et al., 1990; Martens et al., 1995) . In vectors of this type (BacPAK TM vectors, BaculoGold TM , Bac-N-Blue TM ) the linearized viral DNA carries a lethal deletion (ORF1609) and becomes replication competent only after recombination with a transfer plasmid carrying the foreign gene, thereby restoring the deletion (Kitts and Possee, 1993) . Baculovirus vectors based on Gateway technology (BaculoDirect TM ) are linear baculovirus vectors in which foreign genes are introduced through site-specific in vitro recombination.

At about the same time, another efficient and rapid method for generation of recombinant baculoviruses was developed (Luckow et al., 1993) that employed transposition of a foreign gene expression cassette from a donor plasmid into a bacterial artificial chromosome (BAC) which contains the entire AcMNPV genome (bacmid). In this system (Bac-to-Bac TM ) recombinant baculovirus genomes are generated in Escherichia coli and then used to transfect insect cells to obtain recombinant baculovirus particles. After generating high-titer virus stocks, insect cells are infected to produce recombinant proteins. With the bacmid-based methodology the time to generate recombinant viruses is reduced considerably. Another advantage is that the recombinant bacmid can be stored in E. coli and recovered when needed. A disadvantage is that the bacterial gene cassette present in bacmidderived viruses may easily be lost during virus passaging (Pijlman et al., 2003a) . In addition to AcMNPV, bacmids have also been constructed for Spodoptera exigua MNPV and Helicoverpa armigera SNPV (Pijlman et al., 200 2; Wa n g et al., 2003) . T h e m os t r ec en t m et ho d c om bi ne s b ac mi d technologies with allelic replacement (FlashBac TM ; Oxford Expression Technologies) and thereby removes the BAC sequences from the viral genome. This latter system is especially suitable for high-throughput screening.

Over the years, novel baculovirus vectors have been developed with special features and for more specific applications: transfer vectors have been modified to express polyhistidine-tagged proteins for easy purification (pFastBac-His TM ). Transfer vectors with dual, triple, or quadruple promoters usually p10 and polyhedrin, have been developed for allelic replacement (Belyaev and Roy, 1993; Weyer and Possee, 1991) ; dual (pFastBacDual TM ) and quadruple vectors (Tareilus et al., 2003) have also been developed for bacmid technology. Such multiple vectors can be used to express various proteins simultaneously, and hence are useful for producing multimeric complexes, including viral capsids consisting of more than one viral protein (Belyaev and Roy, 1993) . Balancing expression levels is sometimes a problem in these vectors and may require coinfection with a vector expressing only the dominant protein, or a modification of the promoters. One of the promoters in multiple promoter vectors may be used to express a reporter gene, such as green fluorescent protein (GFP), which makes it easy to follow the infection process in cells, perform virus titrations, and track baculovirus infection in the insect (Cha et al., 1997; Kaba et al., 2003) . The recently developed vector system (UltraBac) uses the baculovirus late basic protein (P6.9) promoter to express GFP together with the foreign gene to allow earlier monitoring of infection (Philipps et al., 2005) .

Baculovirus surface display vectors (Grabherr et al., 2001 ) expose the antigen on the surface of budded baculovirus particles. This is achieved by fusing the foreign antigen to the baculovirus envelope glycoprotein GP64 (Monsma et al., 1996) . The chimeric protein is transported to the cell membrane and is taken up in the viral envelope during budding. This system has also been combined with bacmid technology (Kaba et al., 2003) . The recombinant budded virus (BV) particles and lysates of cells infected with a display vector have been shown to evoke protective immune responses (Kaba et al., 2005; Tami et al., 2004; Yoshida et al., 2003) . Baculovirus vectors that express foreign genes in fusion with polyhedrin along with wild-type polyhedrin allow for incorporation of antigens into baculovirus occlusion bodies (Je et al., 2003) . These occlusion bodies are stable and easy to purify and can be used directly for immunization (Wilson et al., 2005) .

Expression of surface (glyco)proteins that go through the export pathway is in general more difficult than expression of soluble cytoplasmic proteins and results in much lower yields (van Oers et al., 2001 ). To increase the production level, surface proteins are often expressed as secreted proteins by removing hydrophobic transmembrane regions (TMR) that serve to anchor the protein to cell membranes. Removing these domains by recombinant DNA technology leads to secreted proteins which can then be purified from the culture medium. However, some caution is needed because this approach may affect folding and vaccine efficacy.

Not all proteins present at the surface under native conditions are automatically transported to the cell surface when expressed in insect cells, such as the p67 surface protein of the bovine parasite Theileria parva (Nene et al., 1995) . When the original signal peptide was replaced with an insect analogue, such as the honeybee mellitin signal peptide (Tessier et al., 1991) , p67 was properly routed to the cell surface (Kaba et al., 2004a) . A similar routing of p67 to the export pathway could be obtained by fusion to GP64 in a surface display vector (Kaba et al., 2002) , where the GP64 signal peptide directed the protein to the cell surface.

Membrane and secreted proteins pass through the endoplasmic reticulum (ER) and the Golgi apparatus on their way to the cell surface and may become glycosylated during this process. The abundant baculovirus protein chitinase is also transported to the ER and accumulates there due to a KDEL retention sequence (Saville et al., 2004; Thomas et al., 1998) . Chitinase is expressed in the late phase of baculovirus infection and is involved in the dissolution of the insect chitinous cuticle to enhance the spread of viral occlusion bodies (Hawtin et al., 1997) . Deletion of chitinase from the baculovirus vector resulted in higher levels of secreted recombinant protein (Possee et al., 1999) possibly because chitinase "cloggs up" the protein translocation machinery and competes with recombinant secretory proteins. The FlashBac system described earlier lacks this chitinase gene. Another baculovirus protein, v-cathepsin also accumulates in the ER and is activated on cell death by proteolytic cleavage (Hom et al., 2002) . Processing of pro-v-cathepsin into active cathepsin is also triggered by chaotropic agents, such as sodium dodecyl sulfate, and this may result in proteolysis of recombinant proteins during extraction and purification (Hom and Volkman, 1998) . A bacmid vector that lacked both chitinase and v-cathepsin (AcBacÁCC) improved the stability of a secreted recombinant protein, thereby increasing the yield of full-length protein molecules (Kaba et al., 2004a) .

Folding of complicated transmembrane glycoproteins can be improved by coexpression of molecular chaperones. The serotonin transporter (SERT) protein is a brain glycoprotein with 12 predicted transmembrane domains. Coexpression of the chaperones calnexin and, to a lesser extent, of immunoglobulin heavy chain-binding protein (BiP) or calreticulin increased the yield of functional SERT threefold. The foldase ERp57 did not have this effect (Tate et al., 1999) . Calreticulin and calnexin were also shown to increase the level of active lipoprotein lipase when coexpressed in insect cells, and to stimulate dimerization of the recombinant protein (Zhang et al., 2003) . Expression of calnexin and calreticulin in a stable transgenic insect cell line, which was than infected with a recombinant baculovirus, resulted in a lower ratio of secreted versus intracellular recombinant protein than when cells were coinfected with two baculoviruses, one carrying the gene of interest and the other a chaperone (Kato et al., 2005) . This result suggests that chaperone expression levels should be of the same order as recombinant protein levels.

Another special adaptation is the incorporation of mammalian promoters in baculovirus vectors to drive foreign gene expression. Baculovirus vectors with mammalian promoters (BacMam TM viruses) have the potential to serve as gene delivery vectors in gene therapy (Huser and Hofmann, 2003; Kost and Condreay, 2002 ) and have also been tested for vaccination purposes (Abe et al., 2003; Aoki et al., 1999; Facciabene et al., 2004; Poomputsa et al., 2003) . In this case, a mammalian promoter or a viral promoter active in mammalian cells, such as the human cytomegalovirus (HCMV) IE1 promoter, drives intracellular expression of the antigen. Exposure on the cell surface via the major histocompatibility complex (MHC) activates the cellular immune system and in this respect, these types of vaccines resemble DNA vaccines. BacMam TM vectors are produced in insect cells and are replication incompetent in mammalian cells (Table II) . A further advantage is that multiple genes can be inserted simultaneously into the baculovirus genome allowing for multivalent vaccines. Expression of multiple proteins is an advantage of the baculovirus expression system over other systems, especially adenovirus vectors, where the maximal increase in genome size is more limited due to packaging restrictions.

For manufacturing subunit vaccines, large-scale production units will be needed, for instance for the production of malaria or the annual influenza subunit vaccines. Baculovirus-insect cell systems have been scaled-up to large-scale cultures in either fermentors (bioreactors) or cellbag devices (WAVE reactors). Insect cell bioreactors up to 2000 L have been reported. The bioprocess technology behind this large scale production has been reviewed by others (Hunt, 2005; Ikonomou et al., 2003; Vlak et al., 1996) . A problem repeatedly encountered when expres sing recom binan t protein s wit h baculo virus vectors is a drop in expres sion lev els wit h increas ing virus passag e (review ed in Kre ll, 1996 ) . This so-called "passag e effect" is intrinsic to bac ulovirus replication in cell culture , but is less critical for small la borator y-scal e prot ein produ ction when the number of viru s pass ages is low (< 10). It is a signifi cant problem thoug h for large-sca le indust rial produ ction of vaccines in insect cell bioreact ors (Van Lier et al., 1996) and prevents the use o f continous bioreact ors. The maj or cau ses of loss o f recom binant prote in expres sion ar e (1) mutati ons in the FP25K gene, reduc ing the activity of the poly hedrin promoter ( Harrison et al. , 1996 ) , (2) th e gene ration of defe ctive interf ering partic les (DIs) whic h replica te at th e expense of the full-leng th recombin ant virus (Kool et al., 1991 ; Pijlman et al. , 2001; Wick ham et al., 1991 ) , (3) the intrace llula r accumul ation of concate nated viral sequ ences, for exam ple, non-hr (homo log ous repeat ) origins o f DNA replic ation) whic h in terfere wit h replic ation of full-length geno mes ( Lee and Krell, 1994 ; Pijlm an et al. , 2002 ) , and (4) sponta neous deleti on of the heterol ogou s gene from th e baculov irus vector. The latter aspect is espe cially seen in bac mid-deri ved vec tors, whic h are extrem ely sens itive to sponta neous removal of the expres sion cassette, a large piece of DNA which is not under selection (Pijlman et al., 2003a) . To prevent the amplification of DIs, baculovirus vectors must be used at low multiplicities of infection (MOI) (de Gooijer et al., 1992; Wickham et al., 1991) and it is now common practice to keep the number of viral passages to a minimum and establish low-passage virus banks as seed stocks for production purposes.

In recent years, several approaches have been used to improve the stability of the baculovirus genome. The accumulation of non-hr-containing sequences can easily be prevented by removing this sequence from the baculovirus backbone (Pijlman et al., 2002) . Reducing the distance between origins of replication in the bacmid system by insertion of an extra hr sequence within the expression cassette also resulted in prolonged foreign gene expression in a test bioreactor (Pijlman et al., 2004) . In the FlashBac system, all destabilizing bacterially derived sequences are removed on recombination with the transfer vector. To prevent loss of the foreign gene cassette, a bicistronic vector was developed that contained the foreign gene and the baculovirus essential gene GP64 on a single bicistronic transcriptional unit linked by an internal ribosome entry site (IRES). GP64 was deleted from its original locus. In this bicistronic vector, loss of the foreign gene would automatically result in loss of expression of the essential gene, which is needed for the generation of complete virus particles as well as for DIs. GFP expression levels were kept at a high level for at least 20 passages with this vector providing dominant selection for GP64 (Pijlman et al., 2006) . This system awaits testing for expression of proteins of medical importance. By combining several of the methods described in this section, it is likely that genome stability will be further improved.

Since its recognition as a production system for subunit vaccines (Vlak and Keus, 1990) , the baculovirus-insect cell expression system has been used extensively for the expression of candidate vaccine antigens. A comprehensive overview of the viral antigens from viruses of vertebrates that have been expressed in this system is provided in Table III . Only those antigens that were tested for their ability to induce protective immune responses are included. In addition, many viral antigens have successfully been expressed in insect cells for the development of diagnostics, and to perform structural and functional studies, but these studies are excluded from this chapter. Various viral antigens ranging from capsid and envelope proteins to nonstructural proteins have been chosen for the development of subunit vaccines. These viral antigens can be divided into those that are expressed as single or oligomeric protein subunits, and those that self-assemble into VLPs. Different approaches to vaccination are described in the examples later, with special attention paid to influenza subunit vaccines.

Envelope proteins are synthesized as single or oligomeric subunits. The expressed envelope proteins are often functionally active and have been reported to oligomerize, an indication that they are correctly folded (Crawford et al., 1999) . Commonly, viral envelope glycoproteins are glycosylated in insect cells. Examples of baculovirus-produced subunit vaccine candidates (Table III) are the fusion proteins and hemagglutinins of paramyxoviruses, such as Newcastle disease virus, and the E proteins of Flaviviridae, including West Nile virus, dengue viruses, and CSFV. Two commercially available veterinary subunit vaccines against classical swine fever (BAYOVAC CSF E2 TM and PORCILIS PESTI TM ) are based on the CSFV E2 glycoprotein produced in insect cells (Ahrens et al., 2000; Bouma et al., 1999 Bouma et al., , 2000 Depner et al., 2001; van Aarle, 2003) . The E2 envelope glycoprotein of CSFV was expressed as a secreted protein by removing the TMR and this resulted in a threefold increase in expression levels, and allowed for purification of E2 from the culture medium (Hulst et al., 1993) . In a similar way, the related Bovine diarrhea virus (BVDV) E2 protein was expressed in insect cells (Bolin and Ridpath, 1996) . Recent research showed that the BVDV E2 protein needs to be glycosylated to be effectively secreted from baculovirus-infected cells (Pande et al., 2005) and that the glycosylated protein was able to block BVDV infection better in an in vitro assay. Whether the glycosylated E2 protein also performs better as a vaccine is not known. The spike glycoprotein of the SARS coronavirus is one of the most recently expressed proteins in insect cells and protected mice against intranasal SARS infection (Bisht et al., 2005) . Influenza presents a serious risk for both human and animal health. The single-stranded RNA of the influenza virus changes quickly through an accumulation of mutations and frequent recombination events, requiring annual vaccine updates (Palese, 2004) . The most threatening recent example is the outbreak of avian influenza of the H5N1 serotype which has killed birds and humans in the Far East since 2003 (WHO) and which caused the first human casualties outside this area in East Turkey in January 2006. The big fear is that such an avian virus will change into a virus that can be transmitted directly from man to man, which may then lead to an influenza outbreak of pandemic dimensions (Palese, 2004) . The most widely used influenza vaccines, e g., Fluzone (Sanofi Pasteur) and Fluvirin (Chiron), consist of chemically inactivated split virus or purified virus subunits. These vaccines have several disadvantages which have recently been reviewed (Cox, 2005; Cox et al., 2004) , including reduced efficacy in the elderly, where vaccination does reduce mortality rates but is not very effective in preventing disease. In addition, an enormous number of eggs are needed each year (one egg per dose) which will very likely lead to a shortage of vaccine in the event of a pandemic; some strains grow poorly in eggs requiring coinfections with other strains or genetic adaptations (e.g., H5N1) (Horimoto et al., 2006) ; and these vaccines can cause strong allergic reactions in some individuals. Live, attenuated influenza vaccines have the advantage of inducing secretory and systemic immunity and are applied intranasally, preventing virus replication in the respiratory tracts (Cox et al., 2004) . However, all of these vaccines still need to be grown in chicken embryos, which are ironically also the target for a potentially pandemic virus like H5N1.

To overcome these drawbacks, various cell-based vaccines for influenza are under development as well as recombinant protein vaccines. Clinical trials of vaccines based on influenza virus produced in mammalian cell cultures, such as Madin Darby canine kidney (MDCK) cells, have been described (Brands et al., 1999; Percheson et al., 1999) and trials with influenza vaccines produced in the human retina cell line Per.C6 (Pau et al., 2001) are ongoing. These products still require inactivation of the influenza virus which may reduce immunogenicity as seen for inactivated vaccines. In response to human casualties of H5 and H7 influenza viruses in Asia in the late 1990s, the immunogenicity and safety of baculovirus recombinant H5 and H7 hemagglutinin (HA) proteins was tested in chickens and resulted in 100% protection against disease symptoms (Crawford et al., 1999) . The immunogenicity of the baculovirus-derived H5 vaccine was subsequently evaluated in over 200 healthy human adults. The vaccine was well tolerated and provided neutralizing antibody responses equivalent to those observed in convalescent sera in $50% of the individuals after two doses (Treanor et al., 2001) . A clinical trial with baculovirus-produced recombinant H3 antigens in 127 adult volunteers showed protective neutralizing antibody levels and a reduction in influenza rates in the following epidemic season compared to a placebo group (Powers et al., 1995) . This HA-based vaccine induced both B and T memory cells (Powers et al., 1997) . A clinical study of 399 individuals with an average age of 70 years was completed in 2003-2004 with an experimental vaccine (FluBlØk, Protein Sciences corporation) containing the same three HA antigen variants as present in the licensed inactivated vaccine of that flu season (Treanor et al., 2006) . Compared to the licensed vaccine, the recombinant vaccine produced higher antibody titers against the H3 strain, the strain responsible for the majority of influenza deaths each year (Cox, 2005) . This result suggests that this vaccine can be especially useful for reduction of the annual number of influenza-related deaths in the elderly, where H3 antibody titers induced by conventional vaccines are too low to be protective. Phase III trials in healthy adults have been completed and showed a 100% protective effcicacy even against H3N2 influenza viruses (Manon Cox, personal communication) (http://www.proteinsciences.com/, Jan 2006). Preparation of a recombinant influenza virus vaccine cocktail for the coming flu season may take about 3 months to complete from the moment the new vaccine composition is announced by the World Health Organization (WHO).

The inactivated conventional vaccine and the trivalent recombinant HA-based vaccine under development are based on antibody responses against the HA surface protein and require annual modifications to the vaccine due to antigenic drift of the influenza virus. A baculovirus recombinant vaccine with both HA and neuraminidase (NA) subunits resulted in a bivalent seroconversion with antibodies against both HA and NA (Johansson, 1999) . The efficacy of an H3N2 vaccine based on both HA and NA produced with a recombinant baculovirus was analyzed in a murine model and compared with a conventional killed and a live-attenuated vaccine preparation and an HA single-subunit vaccine (Brett and Johansson, 2005) . The NA in the baculovirus-derived vaccine was much more immunogenic than in the conventional vaccines. The advantage of inducing an immune response to both surface proteins is illustrated by the fact that the recombinant vaccine containing both HA and NA did not only prevent infection with homotypic and closely related viruses, but also showed a strong reduction in pulmonary virus titers in infections with a more distantly related virus (H3N2 A/Panama/2007/99 versus A/Fuijan/411/2002), in contrast to a vaccine based on HA only. These results suggest that a vaccine containing intact NA tolerates more antigenic drift, thereby reducing the chance of virus escaping the immune system during the flu season.

Viral capsid proteins produced in insect cells often self-assemble into VLPs. The advantage of VLPs is that they resemble the natural virus but are not infectious because they lack genetic material. VLPs are also an excellent tool for study of virus structure. VLPs can easily be purified by extraction, centrifugation, or precipitation (Brown et al., 1991) and often give strong immune reactions even in the absence of adjuvants due to their particulate nature. In addition, humoral, cellmediated, and mucosal immune responses have been reported (Roy, 1996) . An example of a vaccine consisting of recombinant VLPs produced with a baculovirus vector is a patented Canine parvovirus vaccine Valdes et al., 1999) . Sometimes the expression of more than one viral coat protein is needed to make immunogenic VLPs, either due to the complexity of the capsid structure (Bluetongue virus: BTV, Reoviridae) or presence of crucial epitopes on several coat proteins. Multicomponent VLPs can be produced by using vectors with multiple promoters or by coinfections with several baculovirus vectors that each encode one or more viral proteins. One difficulty in making complex VLPs is to achieve appropriate expression levels of each protein present in the viral capsid.

The capsid protein of Hepatitis E virus (Caliciviridae) forms VLPs and these VLPs induce both systemic and mucosal immunity after oral administration in a mouse model. They also protect cynomolgus monkeys when challenged with HEV against infection and hepatitis (Li et al., 2001 (Li et al., , 2004 . Infectious bursal disease (IBDV, Birnaviridae) of birds can also be prevented by vaccination with single component VLPs (Martinez-Torrecuadrada et al., 2003; Wang et al., 2000) . The major capsid protein L1 of Papovaviridae forms VLPs and has been shown to protect cottontail rabbits against Cottontail rabbit papillomavirus. Combinations of the HPV-16 L1 and L2 capsid proteins and the oncogenic protein E7 protected against tumor formation in a mouse model (Greenstone et al., 1998) . Multivalent VLP preparations containing BTV (Reoviridae) VP2 subunits of various serotypes were made by coinfections of several baculovirus vectors and induced longlasting protection in sheep (Roy et al., 1994) . Vaccine candidates in the form of VLPs with up to four different VPs have also been successfully produced for BTV (Pearson and Roy, 1993; van Dijk, 1993) as well as for several other Reoviridae (Conner et al., 1996a,b) . Immunization of mice with an influenza VLP containing the two matrix proteins M1 and M2, and the surface proteins HA and NA showed almost complete protection against an H3N2 virus via both intramuscular and intranasal immunization routes (Galarza et al., 2005a) .

The rationale for using VLPs as vaccine candidates is obvious for nonenveloped viruses, because in these viruses the capsid proteins are directly exposed to the immune system. However, they may also be useful for displaying epitopes of enveloped viruses. HIV is an enveloped virus and in this case VLPs have been produced based on gp55 (gag) to which immunogenic segments of the envelope protein gp120 were coupled (Arico et al., 2005; Buonaguro et al., 2002; Tobin et al., 1997) . An extension of these chimeric VLP-based vaccines is to use VLPs of one virus to display epitopes of heterologous proteins that do not form VLPs by themselves. Examples of such systems are Human parvovirus B19 VLPs which carry linear epitopes in fusion with the viral VP2 protein. This system was used to display epitopes of Murine hepatitis virus A59 (MHV; Coronaviridae) and Herpes simplex virus (HSV; Herpesviridae) (Brown et al., 1994) . Such chimeric VLPs protected mice against a lethal challenge with MHV of HSV. Epitopepresenting chimeric VLPs have also been developed based on Mouse papillomavirus (Tegerstedt et al., 2005) and Flock house virus VLPs (Scodeller et al., 1995) .

Mono-and oligomeric protein subunits are often less potent and need to be formulated carefully before administration to extend their half-life and to present them in a proper form to the immune system, for instance by uptake by antigen-presenting cells (APCs) (Dertzbaugh, 1998; Schijns, 2003) . Adjuvant possibilities for human application are very limited because of safety considerations and this may limit the application of monomeric subunit vaccines in humans. VLPs on the other hand have been shown to induce protection even without the addition of adjuvants (Li et al., 2004; Roy, 1996) . An alternative way to modulate the immune response is by the addition of recombinant cytokines as vaccine adjuvants. Cytokines can either be added separately to the vaccine or may be included in VLPs. This approach may not only modulate the magnitude but also the type of immune response (Lofthouse et al., 1996) . By carefully choosing which cytokine is added the immune response can be driven in a certain direction. Interferon gamma (IFN-) may be added to stimulate macrophages, while addition of interleukin-12 (IL-12) promotes cellmediated adaptive immunity (Abbas and Lichtman, 2005) . IL-12 can be efficiently produced with baculovirus vectors as functional dimers that shift the immunogenic balance to Th1 cells in bovine calves (Takehara et al., 2002) . IL-12 added to influenza VLPs enhances antibody responses but in this case VLPs alone already result in 100% protection (Galarza et al., 2005a) . Immune reactions to helminths involve Th2 responses. Interleukin-4 (IL-4) drives the immune response to differentiation of Th2 cells and to the production of heminth-specific IgE antibodies (Abbas and Lichtman, 2005) . Addition of IL-4 may therefore be helpful for vaccines against helminths (Lofthouse et al., 1996) . For baculovirus-derived products the addition of cytokines has not been fully exploited, but it is commonly used for DNA vaccines. The addition of costimulators, such as B7 or CD40, to baculovirus-produced vaccines has not been reported.

Baculoviral vectors with mammalian promoters driving the expression of viral genes have been used in a limited number of vaccine trials. A candidate Pseudorabies virus vaccine expressing its glycoprotein B from a recombinant baculovirus vector with a mammalian promoter resulted in seroconversion in immunized mice (Aoki et al., 1999) . Intramuscular injection with baculovirus BVs expressing the E2 glycoprotein of Hepatitis C virus controlled by the CMV immediate-early promoter-enhancer provided specific humoral and cellular responses (Facciabene et al., 2004) . Similar results were obtained with the carcinoembryonic antigen (CEA) indicating that these types of vaccines can also be effective against tumors. The addition of the Vesicular stomatis virus (VSV) G protein to the baculovirus envelope increased immunogenicity in this experiment, possibly by enhancement of virus fusion. BacMam TM vectors have also been used to produce mutated, attenuated influenza virus in mammalian cells by delivery of an altered NS1 gene (Poomputsa et al., 2003) . Surprisingly, a baculovirus vector with the influenza hemagglutinin gene (H1) controlled by the chicken betaactin promoter gave a similar level of protection as a wild-type baculovirus against a lethal influenza challenge in intranasally immunized mice (Abe et al., 2003) . The authors ascribe this to the induction of a strong innate immune response by the baculovirus, protecting the mice from a subsequent lethal challenge with influenza virus.

A possible drawback to the use of baculoviruses directly for vaccination and possibly also for surface display and polyhedra-incorporation vectors is the accumulation of anti-baculovirus antibodies upon repeated vaccination, resulting in rapid inactivation of subsequent vaccines of the same type. Pigs for instance have been shown to produce high levels of baculovirus-neutralizing antibodies after injection of baculovirus BVs (Tuboly et al., 1993) . A solution to this problem could be to design multivalent vaccines, since baculoviruses can take up a large amount of foreign DNA. Another problem with the use of baculovirus particles as vaccines is rapid degradation by the complement system which also affects gene therapy applications. Pseudotyping of BVs with the VSV glycoprotein instead of GP64 resulted in a reduction in complement inactivation (Tani et al., 2003) . Incorporation of human decayaccelerating factor (DAF), a complement-regulatory protein, in the BV envelope has been shown to protect baculovirus gene therapy vectors against complement-mediated inactivation (Huser et al., 2001) . This strategy could also be applied for vaccine purposes.

HIV VLPs containing only gp55 (gag) have been used to boost immunization induced by a gp55 DNA vaccine (Jaffray et al., 2004) . In this case, a capsid protein of an enveloped virus is a reasonable subunit for vaccination because DNA vaccines are expressed intracellularly and fragments of the resulting proteins are presented by MHC complexes to induce cellular immune responses. In this way the natural situation of intracellular expression of viral genes is mimicked. T cell responses, especially the action of cytotoxic T lymphocytes (CTL) are crucial in defense against HIV. HIV combination vaccines where adenovirus, vaccinia (HIVAC-1e), or vesicular stomatitis virus vectors were used for immunization in combination with a boost with a protein subunit (gp160, gp41) have been tested in phase I trials in humans (Cooney et al., 1993; Graham et al., 1993; Lubeck et al., 1994; Luo et al., 2006; Perales et al., 1995; Zheng, 1999) . Viral carriers also express HIV antigens inside cells, and these antigens are displayed either by specialized APCs or other cells to the immune system. The aim of this regimen with a DNA/carrier vaccine and a protein boost regimen is therefore to induce both neutralizing antibodies and T cell responses.

Nonvaccination policies exist for many animal diseases because of the risk that vaccinated animals may be protected against disease but may still be carriers of the virus. In cases of outbreaks, ring vaccination is sometimes applied, but large-scale vaccination is generally not allowed. A prerequisite for the broader use of animal vaccines is the development of marker vaccines that enable differentiation between vaccinated and infected animals. This is especially important for endemic diseases in animals where monitoring is of crucial importance to avoid spread of the virus to nonendemic regions or to other host species such as humans or wild animals. Marker vaccines also have good prospects for eradication of animal diseases in general (van Aarle, 2003) . For such purposes, marker vaccines do not have to give 100% herd immunity, because reducing susceptibility and transmission can be sufficient to have a major effect in controlling animal disease (Henderson, 2005) . Marker vaccines need to be accompanied by specific diagnostic tests that are commonly based on determining serum titers of a viral component that is absent in the vaccine but present in the pathogen, to which antibodies can be raised. The baculovirus expression system has proven to be useful in producing not only the protein subunits for marker vaccines but also the recombinant polypeptides for these diagnostic tests. The commercially available CSFV vaccine based on the E2 glycoprotein is a marker vaccine because only antibodies against E2 are generated. An enzyme-linked immunosorbent assay (ELISA) test for serum antibodies against the other immunogenic surface protein E RNS can be used to discriminate immunized animals from virus carriers (Langedijk et al., 2001; van Aarle, 2003) . Another possibility is to use only some of the epitopes of an antigen for immunization and others for the diagnostic analysis, as demonstrated for CSFV (van Rijn et al., 1999) . The coexistence of a subunit vaccine and a discriminative diagnostic test enabled registration of CSFV vaccines in Europe. Marker vaccines will also be very useful for immunization of poultry against avian influenza (Crawford et al., 1999) , where monitoring for the presence of virus is essential. Animals immunized with HA or HA/NA vaccines could be screened for antibodies against the viral matrix proteins. Similar assays have been developed for other subunit vaccines, such as one that discriminates between VSV-infected and immunized animals, where the marker vaccine is based on the glycoprotein and the assay on the nucleocapsid protein produced in insect larvae .

Parasites of the genera Plasmodium, Theileria, and Babesia are protozoan blood parasites causing malaria, theileriosis, and babesiosis. These parasites have a complex life cycle and are transmitted by either mosquito or tick vectors. Candidate subunit vaccines against these parasites can be roughly separated into preblood (preerythrocyte or prelymphocyte) stage vaccines, blood stage vaccines, transmissionblocking vaccines, and multistage vaccines. An overview of parasite subunits expressed in the baculovirus insect cell system for vaccine purposes is given in Table IV .

A comprehensive record of all subunit and recombinant carrier vaccines under development for human malaria is maintained by WHO (Reed, 2005) . Most of these vaccines are still in a preclinical stage but several Plasmodium falciparum vaccines are in phase I and phase II trials in malaria endemic countries. Plasmodium is transmitted in the form of sporozoites by Anopheles mosquitoes. A vaccine candidate based on the circumsporozoite protein (CSP) is aimed at blocking these sporozoites. Both B and T cell responses appear to be essential for protective immunity based on the CSP protein. CSP has been produced in insect cells but was minimally immunogenic when tested in 20 volunteers (Herrington et al., 1992) . Alternative approaches to experimental CSP vaccines, which facilitate T cell responses, are in phase II trials (Ballou et al., 2004) . These vaccines include CSP displayed on HBsAg VLP particles, modified vaccinia Ankara virus as recombinant carrier, or DNA vaccines.

The merozoite is the extracellular, erythrocyte-invasive form of the Plasmodium parasite. Merozoite surface proteins are promising -Mitchell et al., 1997 vaccine candidates due to their accessibility for antibodies and their expected role in erythrocyte invasion. The major merozoite surface protein (MSP-1) is an important prebloodstage candidate vaccine with homology to epidermal growth factor (EGF) and antibodies directed against this protein block erythrocyte invasion (Holder and Blackman, 1994) . Plasmodium cynomolgi functions as a model system for the highly similar Plasmodium vivax in humans. An active, C-terminally processed form of P. cynomolgi MSP-1, was produced in insect cells and protected primates in a challenge experiment (Perera et al., 1998) . The C-terminally mature MSP-1 of P. falciparum was also successfully expressed in insect cells and used for ultrastructural studies (Chitarra et al., 1999; Pizarro et al., 2003) , but has not been tested in human trials. Meanwhile, E. coli-expressed MSP-1 has entered phase II clinical trials (Ballou et al., 2004) . Plasmodium parasites in the ookinete stage are taken up by mosquitoes and antigens specific for this stage can function as transmission-blocking subunit vaccines. The major ookinete surface antigen Pbs21 (P28) of Plasmodium berghei was expressed in B. mori larvae, preserving conformational B cell epitopes which were lost upon expression in E. coli. The recombinant Pbs21 antigen produced in insect cells blocked oocyte formation in Anopheles mosquitoes fed on immunized mice (Matsuoka et al., 1996) . The immunogenicity of the recombinant protein was strongly reduced when the protein was expressed as a secreted protein by removing its glycosylphosphatidylinositol (GPI) anchor signal (Martinez et al., 2000) . This protein provides a good example of loss of immunogenicity by expressing a membrane protein in a secreted form. Subunit vaccines based on the Plasmodiuminduced erythrocyte membrane protein 1 (EMP-1) are aimed at blocking vertical transmission from mother to child (maternal malaria) via the placenta. This transmission involves the sequestration of P. falciparum-infected erythrocytes through EMP-1, which binds to chondroitin sulphate A in the placenta. EMP-1 expressed in the baculovirus system induces inhibitory antibodies which react with both homologous and heterologous EMP-1 proteins (Costa et al., 2003) .

T. parva is the causative agent of East Coast fever, a deadly cattle disease endemic in large parts of Africa. Immunization with recombinant sporozoite surface protein p67 is aimed at blocking invasion of lymphocytes by this parasite. P67 is an example of a protein that was not easy to express in a native form in insect cells as well as in many other systems. In insect cells, it was expressed at low levels and in contrast to expectations was not present on the cell surface. Similar to E. coli-expressed p67, it did not react with a monoclonal antibody against native p67, indicating that the folding of the protein was not correct (Nene et al., 1995) . Several adaptations were therefore made to the expression system. Expression of p67 coupled to the honeybee mellitin signal instead of the original signal peptide resulted in correct routing of this protein to the cell surface, but the folding was still not optimal (Kaba et al., 2004a) . Fusion of p67 to the C terminus of GFP drastically increased expression levels and resulted in recognition by the conformation-sensitive monoclonal antibody (Kaba et al., 2002) . A similar effect was also seen when parts of this protein were fused to the baculovirus GP64 glycoprotein in a surface display vector, which led to expression on the cell surface and on baculovirus BVs (Kaba et al., 2003) . The recombinant GFP-p67 protein and the C terminal half of p67 coupled to GP64 induced high levels of sporozoiteneutralizing serum antibodies and showed up to 80% protection against lethal T. parva challenge in a double-blind placebo-controlled experiment (Kaba et al., 2004b (Kaba et al., , 2005 . The next phase will be to evaluate the quality of these experimental vaccines under field conditions in countries in which East Coast fever is endemic.

Babesia species are a major cause of parasitemias in cattle and dogs. Babesia divergens is the major cause of bovine babesiosis in Europe and the increased incidence of this disease is correlated with an increase in the numbers of ticks (Ixodus ricinus) that transmit this parasite. B. divergens is also responsible for zoonotics in immunocompromised humans (Zintl et al., 2003) . The soluble parasite antigen (SPA) of bovine and canine Babesia species has been developed as a vaccine against clinical manifestations in dogs and is produced in mammalian cells infected with Babesia (Schetters, 2005) . Several other Babesia antigens have been expressed in the baculovirus expression system to develop ELISA tests for diagnosis. The baculovirusexpressed Babesia radhaini P26 protein was shown to induce protection against the disease in rats (Igarashi et al., 2000) .

Theileria and Babesia parasites are transmitted by ioxidic ticks and in the future candidate antiparasite vaccines may be combined with vaccines directed against the tick vector (Bishop et al., 2004) . These vaccines may be tick antigens directly exposed to the host immune system, such as vitellin, the most abundant B. microplus egg protein (Tellam et al., 2002) or cement proteins involved in the attachment of the tick to the skin of the host. Concealed antigens can also give good results, such as the B. microplus Bm86 gut antigen (Gavac TM ), which results in binding of antibodies taken up from immunized animals to a gut transmembrane protein (Willadsen et al., 1995) . Ticks are known to immunomodulate their host by secreting specific immunomodulators and transmitted parasites also profit from the reduction in immune response. Tick vaccines may therefore be aimed at reducing the chance of transmission by directly affecting the feeding process, by interacting with immunomodulators, or by reducing tick populations.

Chagas' disease in the Americas is caused by Trypanosoma cruzi and is found in humans, dogs, cats, and rodents. T. cruzi is a macrophageinvading protozoan, and complicating factors in the development of vaccines are immune escape and autoimmunity due to molecular mimickry (Girones et al., 2005) . Macrophages are also the target for Leishmania parasites, which use complex immune evasion strategies that affect host cell signaling (Olivier et al., 2005) . Immunization with the T. cruzi Tol A-like protein (TolT) expressed in insect cells resulted in T cell-dependent antiparasitic activity (Quanquin et al., 1999) . For Leishmania several candidate subunit vaccine antigens with protective potential have been identified, including the surface protein gp63 (Coler and Reed, 2005) , but these proteins have not been expressed with baculovirus vectors in insect cells.

Helminths or parasitic worms that are a serious threat to human and animal health worldwide, are divided into the Annelida (segmented worms), Platyhelminthes (flatworms including flukes), and Nematoda (roundworms). These worms have complex life cycles, which often involve more than one host. Because helminths vary widely among populations, vaccines are not competitive so far with chemical broad spectrum antihelminths (Bos and Schetters, 1990) . Helminths may also modulate the immune system as exemplified by the filarial nematodes, which are present in 150-200 million humans worldwide and cause river blindness for example. These filarial nematodes are difficult to combat because they modulate T cell responses leading to chronic helminth infections (reviewed by Hoerauf et al., 2005) . This T cell modulation is not restricted to the response to filarial larvae but also affects allergies, the response to other pathogens and vaccines through modulation of not only antigen-specific T cells but also APCs, which affects immune responses in general.

There are a few examples of baculov irus-ex pressed helminth proteins but few of these have bee n tested in immuniz ation stud ies ( Table IV ) . How ever, the bacu lovirus expres sion syste m may be a valuable tool for these parasitic wor ms as illustr ated by th e following examples : a baculov irus-deri ved subu nit vaccine agains t liver fluke ( Fasciola hepati ca ) based on proc atheps in L3 confer red 50% prot ection to rats, in contras t to the yeas t-express ed protein whic h did not confer any prote ction (Reszk a et al ., 2005) . Bilhar zia or schistos omias is is caused by Sch istosoma spp. (Platyh elminthe s), blood parasites of humans in trop ical areas. Several Sch istoso ma gene s have been expressed in i nsect cells , but prim arily for anal ysis of en zymatic functi ons. The large sub unit of calpain (SmP80 ) prod uced in the bac ulovirus expres sion system reduc ed the worm burden i n mice ( Hota-Mitchell et al., 1997) . Antigens of the tapeworm Taenia solium have been expressed with baculovirus vectors for diagnostic purposes (Lee et al., 2005; Levine et al., 2004) .

There are many examples in the literature where immunization with recombinant proteins produced in the baculovirus-insect cell expression system conferred good protection against infectious disease. Subunit vaccine development begins with careful identification of the antigen, which is related to whether neutralizing adaptive immune responses are raised against the particular protein in natural infections. Once selected, the open reading frame (ORF) of the antigen is cloned while keeping flanking DNA sequences to a minimum, which can best be achieved with a proof-reading PCR enzyme. The sequence around the ATG translational start site is best modified to that of the polyhedrin or p10 gene in the wild-type baculovirus, with at least an adenosine residue at the À3 position (Chang et al., 1999) . Tags may be added to facilitate purification, preferably in such a way that they can subsequently be removed. Tags are not required for VLPs because they can be purified by centrifugation. Transmembrane (glyco) proteins are best produced in a secreted form by removal of TMRs to increase expression levels. However, this may occasionally affect the folding of the protein. An alternative approach is to fuse the immunogenic domains of envelope proteins to GP64. Vectors that lack chitinase and v-cathepsin genes are preferable for expression of envelope proteins. During the preparation of seed stocks, care should be taken to keep the virus passage number low and to use a multiplicity of infection of <0.1 to minimize the formation of DIs. Careful checking of the purity of recombinant bacmids or plaque purified recombinant viruses by PCR is crucial to avoid loss of recombinant virus in subsequent passages due to empty vectors that out-compete the recombinants.

Only two baculovirus-produced products are approved for veterinary practice, namely, two vaccines against classical swine fever consisting of the E2 surface glycoprotein. With these vaccines in the market (although a nonvaccination policy still exists) the confidence in this type of subunit vaccine will grow as well as the possibilities for registration, thereby increasing the likelihood that more vaccines of this kind will appear in the market. This expectation of an increasing number of products may be expanded to human applications when registration of a trivalent recombinant influenza vaccine, for which phase III clinical trials in humans in the United States have been completed recently, can be achieved. Therapeutic anticancer vaccines, such as a vaccine against prostate cancer, may be more readily accepted, in view of the severe side effects of anticancer drugs and irradiation techniques. The application of baculovirus display or BacMam TM vectors for vaccines against infectious disease may be applied in the future for animal use. Because more foreign proteins are incorporated into the vaccine than just the targeted antigen, such vaccines for human use are likely to be in the more distant future because of safety considerations.

Baculovirus expression systems compete with other cheaper production systems, which are more widely used and which scale-up more easily, such as E. coli and yeast (Table I) , and once a good protection level is achieved there is no commercial interest to switch to the more expensive insect cell system. For those cases though, where folding or posttranslational modifications are crucial to epitope formation the baculovirus-insect cell system is a versatile expression system and many candidate vaccines have been successfully tested. Alternative methods are provided by the development of DNA vaccine technology and recombinant carrier vaccines based on vaccinia, adenovirus, or BacMam TM vectors (Table II) . These methods are more prone to induce cellular immune responses than many protein subunit vaccines. The combination of a primary vaccine with an intracellular delivery system (recombinant carrier vaccines, DNA vaccines, BacMam TM vectors) followed by a boost immunization with recombinant protein subunits appears to be a promising approach, aimed at both cellular and humoral immune responses. This approach can be further strengthened through the addition of recombinant cytokines that drive the immune response in a specific direction. A major challenge now is to broaden the array of viral vaccines produced in insect cells and to develop effective vaccines against more complex organisms, such as protozoan parasites and multicellular worms, for which the baculovirus expression system also holds promise. With the increasing insight into immunology leading to new methods of vaccine production and delivery, accompanied by a wealth of genomic and proteomic data, many new generation vaccines are expected within the foreseeable future. ACKNOWLEDGMENTS I thank Manon Cox, Dick Schaap, and Stephen Kaba for assistance with the sections on viral vaccines, parasite vaccines, and malaria vaccines respectively; Christina van Houte for verifying Table III , and Just Vlak for review of the manuscript and for helpful suggestions.

Cellular and Molecular Immunology

Baculovirus induces an innate immune response and confers protection from lethal influenza virus infection in mice

Immunological characterization of the VSV nucleocapsid (N) protein expressed by recombinant baculovirus in Spodoptera exigua larva: Use in differential diagnosis between vaccinated and infected animals

Efficacy of the classical swine fever (CSF) marker vaccine Porcilis Pesti in pregnant sows

HIV experimental vaccines based on the iscom technology using envelope and GAG gene products

Vaccinology: Past achievements, present roadblocks and future promises

Induction of antibodies in mice by a recombinant baculovirus expressing pseudorabies virus glycoprotein B in mammalian cells

Immature monocyte derived dendritic cells gene expression profile in response to virus-like particles stimulation

Immunogenicity of hepatitis B surface antigen derived from the baculovirus expression vector system: A mouse potency study

The complete DNA sequence of Autographa californica nuclear polyhedrosis virus

Induction of protective cytotoxic T cells with viral proteins

Recombinant Norwalk virus-like particles as an oral vaccine

Oral immunization with recombinant Norwalk virus-like particles induces a systemic and mucosal immune response in mice

Recombinant Norwalk virus-like particles given orally to volunteers: Phase I study

Update on the clinical development of candidate malaria vaccines

Antigenic and immunogenic properties of a chimera of two immunodominant African swine fever virus proteins

Immunological responses of mice and cattle to baculovirus-expressed F and H proteins of rinderpest virus: Lack of protection in the presence of neutralizing antibody

Antigen-presenting cells 8015 (Provenge) in patients with androgen-dependent, biochemically relapsed prostate cancer

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

Analysis of a recombinant dengue-2 virus-dengue-3 virus hybrid envelope protein expressed in a secretory baculovirus system

Theileria: Intracellular protozoan parasites of wild and domestic ruminants transmitted by ixodid ticks

Neutralizing antibody and protective immunity to SARS coronavirus infection of mice induced by a soluble recombinant polypeptide containing an N-terminal segment of the spike glycoprotein

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

Glycoprotein E2 of bovine viral diarrhea virus expressed in insect cells provides calves limited protection from systemic infection and disease

Is there a future for vaccines against gastrointestinal helminths?

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

Determination of the onset of the herd-immunity induced by the E2 sub-unit vaccine against classical swine fever virus

Influvac: A safe madin darby canine kidney (MDCK) cell culture-based influenza vaccine

Immunization with viruslike particles from cottontail rabbit papillomavirus (CRPV) can protect against experimental CRPV infection

Immunization against influenza A virus: Comparison of conventional inactivated, live-attenuated and recombinant baculovirus produced purified hemagglutinin and neuraminidase vaccines in a murine model system

Protection of cotton rats against human respiratory syncytial virus by vaccination with a novel chimeric FG glycoprotein

Protection of cotton rats against human parainfluenza virus type 3 by vaccination with a chimeric FHN subunit glycoprotein

Immunotherapy of SV40 induced tumours in mice: A model for vaccine development

Analysis of murine antibody responses to baculovirus-expressed human immunodeficiency virus type 1 envelope glycoproteins

Assembly of empty capsids by using baculovirus recombinants expressing human parvovirus B19 structural proteins

Chimeric parvovirus B19 capsids for the presentation of foreign epitopes

Induction of neutralizing antibodies and cytotoxic T lymphocytes in Balb/c mice immunized with virus-like particles presenting a gp120 molecule from a HIV-1 isolate of clade A

Large-scale production in Pichia pastoris of the recombinant vaccine Gavac against cattle tick

Development of a DIVA (differentiating infected from vaccinated animals) strategy using a vaccine containing a heterologous neuraminidase for the control of avian influenza

Severe acute respiratory syndrome vaccine development: Experiences of vaccination against avian infectious bronchitis coronavirus

Simplification of titer determination for recombinant baculovirus by green fluorescent protein marker

Modulation of translational efficiency by contextual nucleotides flanking a baculovirus initiator AUG codon

The crystal structure of C-terminal merozoite surface protein 1 at 1.8 A resolution, a highly protective malaria vaccine candidate

High-level expression of canine parvovirus VP2 using Bombyx mori nucleopolyhedrovirus vector

Production of mink enteritis parvovirus empty capsids by expression in a baculovirus vector system: A recombinant vaccine for mink enteritis parvovirus in mink

Immunization with viruslike particles induces long-term protection of rabbits against challenge with cottontail rabbit papillomavirus

Recombinant subunit vaccines: Potentials and constraints

Second-generation vaccines against leishmaniasis

Rotavirus subunit vaccines

Viruslike particles as a rotavirus subunit vaccine

Cotton rats previously immunized with a chimeric RSV FG glycoprotein develop enhanced pulmonary pathology when infected with RSV, a phenomenon not encountered following immunization with vaccinia-RSV recombinants or RSV

Enhanced immunity to human immunodeficiency virus (HIV) envelope elicited by a combined vaccine regimen consisting of priming with a vaccinia recombinant expressing HIV envelope and boosting with gp160 protein

Immunization with recombinant duffy binding-like-gamma3 induces pan-reactive and adhesion-blocking antibodies against placental chondroitin sulfate A-binding plasmodium falciparum parasites

Cell-based protein vaccines for influenza

Influenza virus: Immunity and vaccination strategies. Comparison of the immune response to inactivated and live, attenuated influenza vaccines

Baculovirus-derived hemagglutinin vaccines protect against lethal influenza infections by avian H5 and H7 subtypes

Evaluation of rubella virus E2 and C proteins in protection against rubella virus in a mouse model

Fasciola hepatica cathepsin L-like proteases: Biology, function, and potential in the development of first generation liver fluke vaccines

A structured dynamic model for the baculovirus infection process in insect cell reactor configurations

Molecular analysis of astacin-like metalloproteases of Ostertagia ostertagi

An aspartyl protease inhibitor of Ostertagia ostertagi: Molecular cloning, analysis of stage and tissue specific expression and vaccine trial

Protective efficacy in mice of a secreted form of recombinant dengue-2 virus envelope protein produced in baculovirus infected insect cells

Classical swine fever (CSF) marker vaccine. Trial II. Challenge study in pregnant sows

Recombinant neuraminidase vaccine protects against lethal influenza

Genetically engineered vaccines: An overview

Recombinant baculoviruses expressing yellow fever virus E and NS1 proteins elicit protective immunity in mice

Induction of HIV-1 envelope (gp120)-specific cytotoxic T lymphocyte responses in mice by recombinant CHO cell-derived gp120 is enhanced by enzymatic removal of N-linked glycans

Is there an advantage to including the nucleoprotein in a rabies glycoprotein subunit vaccine

A prototype recombinant vaccine against respiratory syncytial virus and parainfluenza virus type 3

Cell mediated immunity induced in mice by HPV 16 L1 virus-like particles

Immunization of monkeys with baculovirus-dengue type-4 recombinants containing envelope and nonstructural proteins: Evidence of priming and partial protection

Baculovirus vectors elicit antigen-specific immune responses in mice

Dengue type-2 virus envelope protein made using recombinant baculovirus protects mice against virus challenge

Adventures with poxviruses of vertebrates

Vaccination against hepatitis delta virus infection: Studies in the woodchuck (Marmota monax) model

Serum antibody responses to equine herpesvirus 1 glycoprotein D in horses, pregnant mares and young foals

Assembly of double-shelled, viruslike particles of bluetongue virus by the simultaneous expression of four structural proteins

Rabies and rabies research: Past, present and future

Oral vaccination of racoons (Procyon lotor) with baculovirus-expressed rabies virus glycoprotein

Murine HIV-1 p24 specific T lymphocyte activation by different antigen presenting cells: B lymphocytes from immunized mice present core protein to T cells

Virus-like particle (VLP) vaccine conferred complete protection against a lethal influenza virus challenge

Virus-like particle vaccine conferred complete protection against a lethal influenza virus challenge

Recombinant poxviruses as mucosal vaccine vectors

Immunoselection of recombinant baculoviruses expressing high levels of biologically active herpes simplex virus type 1 glycoprotein D

Expression and characterization of baculovirus expressed herpes simplex virus type 1 glycoprotein L

Local expression of tumor necrosis factor alpha and interleukin-2 correlates with protection against corneal scarring after ocular challenge of vaccinated mice with herpes simplex virus type 1

Vaccination with a cocktail of seven recombinantly expressed HSV-1 glycoproteins protects against ocular HSV-1 challenge more efficiently than vaccination with any individual glycoprotein

Vaccination with different HSV-1 glycoproteins induces different patterns of ocular cytokine responses following HSV-1 challenge of vaccinated mice

Antibody-dependent enhancement of HSV-1 infection by anti-gK sera

Trypanosoma cruzi-induced molecular mimicry and Chagas' disease

Characterization of tick-borne encephalitis virus-specific human T lymphocyte responses by stimulation with structural TBEV proteins expressed in a recombinant baculovirus

Lymphocyte proliferative responses following immunization with human immunodeficiency virus recombinant GP160. The NIAID AIDS vaccine clinical trials network

Vaccineinduced antibodies to native and recombinant human immunodeficiency virus type 1 envelope glycoproteins. NIAID AIDS vaccine clinical trials network

Developments in the use of baculoviruses for the surface display of complex eukaryotic proteins

Augmentation of human immunodeficiency virus type 1 neutralizing antibody by priming with gp160 recombinant vaccinia and boosting with rgp160 in vaccinia-naive adults. The NIAID AIDS vaccine clinical trials network

Chimeric papillomavirus virus-like particles elicit antitumor immunity against the E7 oncoprotein in an HPV16 tumor model

Protection of swine against footand-mouth disease with viral capsid proteins expressed in heterologous systems

Recombinant Norwalk virus-like particles administered intranasally to mice induce systemic and mucosal (fecal and vaginal) immune responses

The bovine parainfluenza virus type-3 (BPIV-3) hemagglutinin/neuraminidase glycoprotein expressed in baculovirus protects calves against experimental BPIV-3 challenge

Protection of cotton rats by immunization with the human parainfluenza virus type 3 fusion (F) glycoprotein expressed on the surface of insect cells infected with a recombinant baculovirus

Design and production of recombinant subunit vaccines

Design and production of recombinant subunit vaccines

Molecular characterization and baculovirus expression of the glycoprotein B of a seal herpesvirus (phocid herpesvirus-1)

The role of the AcMNPV 25K gene 'FP25' in baculovirus polh and p10 expression

Safety and immunogenicity trial in adult volunteers of a human papillomavirus 16 L1 virus-like particle vaccine

Liquefaction of Autographa californica nucleopolyhedrovirus-infected insects is dependent on the integrity of virus-encoded chitinase and cathepsin genes

Overview of marker vaccine and differential diagnostic test technology

Safety and immunogenicity in volunteers of a recombinant Plasmodium falciparum circumsporozoite protein malaria vaccine produced in lepidopteran cells

Antigenicity and vaccine potential of Marburg virus glycoprotein expressed by baculovirus recombinants

Immunomodulation by filarial nematodes

Vaccine efficacy of a cell lysate with recombinant baculovirus-expressed feline infectious peritonitis (FIP) virus nucleocapsid protein against progression of FIP

What is the function of MSP-I on the malaria merozoite?

Preventing proteolytic artifacts in the baculovirus expression system

Autographa californica M nucleopolyhedrovirus ProV-CATH is activated during infected cell death

Development of a novel subunit vaccine that protects cotton rats against both human respiratory syncytial virus and human parainfluenza virus type 3

The development and characterization of H5 influenza virus vaccines derived from a 2003 human isolate

Protection against Schistosoma mansoni infection with a recombinant baculovirus-expressed subunit of calpain

Protection of macaques against SIV infection by subunit vaccines of SIV envelope glycoprotein gp160

Glycoprotein E1 of hog cholera virus expressed in insect cells protects swine from hog cholera

From gene to protein: A review of new and enabling technologies for multi-parallel protein expression

Baculovirus vectors: Novel mammalian cell genedelivery vehicles and their applications

Incorporation of decay-accelerating factor into the baculovirus envelope generates complement-resistant gene transfer vectors

Immunization with recombinant surface antigens p26 with Freund's adjuvants against Babesia rodhaini infection

Insect cell culture for industrial production of recombinant proteins

Characterization of pseudorabies virus neutralization antigen glycoprotein gIII produced in insect cells by a baculovirus expression vector

Human immunodeficiency virus type 1 subtype C Gag viruslike particle boost substantially improves the immune response to a subtype C gag DNA vaccine in mice

Developing baculovirus-insect cell expression systems for humanized recombinant glycoprotein production

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

Immunization with hepatitis C virus-like particles induces humoral and cellular immune responses in nonhuman primates

Immunization with influenza A virus hemagglutinin and neuraminidase produced in recombinant baculovirus results in a balanced and broadened immune response superior to conventional vaccine

A nasal proteosome influenza vaccine containing baculovirus-derived hemagglutinin induces protective mucosal and systemic immunity

Fusion to green fluorescent protein improves expression levels of Theileria parva sporozoite surface antigen p67 in insect cells

Baculovirus surface display of Theileria parva p67 antigen preserves the conformation of sporozoite-neutralizing epitopes

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

Improved immunogenicity of novel baculovirus-derived Theileria parva p67 subunit antigens

Novel baculovirus-derived p67 subunit vaccines efficacious against East Coast fever in cattle

Self-assembled B19 parvovirus capsids, produced in a baculovirus system, are antigenically and immunogenically similar to native virions

Partial control of hepatitis delta virus superinfection by immunisation of woodchucks (Marmota monax) with hepatitis delta antigen expressed by a recombinant vaccinia or baculovirus

Improvement of the production of GFP uv -ß1,3-N-acetylglucosaminyltransferase 2 fusion protein using a molecular chaperone-assisted insect-cell-based expression system

Pathogenesis of and immunity to avian influenza A H5 viruses

Human immunodeficiency virus (HIV-1) gp160-specific lymphocyte proliferative responses of mononuclear leukocytes from HIV-1 recombinant gp160 vaccine recipients

Studies of high doses of a human immunodeficiency virus type 1 recombinant glycoprotein 160 candidate vaccine in HIV type 1-seronegative humans

Purified dengue 2 virus envelope glycoprotein aggregates produced by baculovirus are immunogenic in mice

Immunization of egg-allergic individuals with egg-or chicken-derived vaccines

Circulating vaccine-derived polioviruses: Current state of knowledge

Protection of mice with recombinant influenza virus neuraminidase

The Baculovirus Expression System: A Laboratory Guide

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

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

A method for producing recombinant baculovirus expression vectors at high frequency

Immunogenic and protective properties of chicken anaemia virus proteins expressed by baculovirus

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

Recombinant baculoviruses as mammalian cell gene-delivery vectors

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

Passage effect of virus infection in insect cells

Expression of glycoprotein D of herpes simplex virus type 1 in a recombinant baculovirus: Protective responses and T cell recognition of the recombinant-infected cell extracts

Protective effects of equine herpesvirus-1 (EHV-1) glycoprotein B in a murine model of EHV-1-induced abortion

Recombinant baculovirus influenza A hemagglutinin vaccines are well tolerated and immunogenic in healthy adults

Enzyme-linked immunosorbent assay using a virus type-specific peptide based on a subdomain of envelope protein Erns for serologic diagnosis of pestivirus infections in swine

Expression of muscovy duck parvovirus capsid proteins (VP2 and VP3) in a baculovirus expression system and demonstration of immunity induced by the recombinant proteins

Feasibility of baculovirus-expressed recombinant 10-kDa antigen in the serodiagnosis of Taenia solium neurocysticercosis

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

Comparison of soluble and secreted forms of human parainfluenza virus type 3 glycoproteins expressed from mammalian and insect cells as subunit vaccines

Partial protection by vaccination with recombinant feline immunodeficiency virus surface glycoproteins

Characterization, cloning, and expression of two diagnostic antigens for Taenia solium tapeworm infection

Oral administration of hepatitis E virus-like particles induces a systemic and mucosal immune response in mice

Protection of cynomolgus monkeys against HEV infection by oral administration of recombinant hepatitis E virus-like particles

Immunization strategies to block the herpes simplex virus type 1 immunoglobulin G Fc receptor

Cytokines as adjuvants for ruminant vaccines

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

Recombinant vaccine for canine parvovirus in dogs

Expression of the outer capsid protein VP5 of two bluetongue viruses, and synthesis of chimeric double-shelled virus-like particles using combinations of recombinant baculoviruses

Immunogenicity of recombinant adenovirus-human immunodeficiency virus vaccines in chimpanzees following intranasal administration

Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli

Autoreactivity in HIV-infected individuals does not increase during vaccination with envelope rgp160

Chimeric gag-V3 virus-like particles of human immunodeficiency virus induce virus-neutralizing antibodies

Induction of neutralizing antibody against human immunodeficiency virus type 1 (HIV-1) by immunization with gp41 membrane-proximal external region (MPER) fused with porcine endogenous retrovirus (PERV) p15E fragment

Molecular farming for new drugs and vaccines. Current perspectives on the production of pharmaceuticals in transgenic plants

Expression and properties of feline herpesvirus type 1 gD (hemagglutinin) by a recombinant baculovirus

Efficacy of human papillomavirus-16 vaccine to prevent cervical intraepithelial neoplasia: A randomized controlled trial

Antibodies to the major linear neutralizing domains of cytomegalovirus glycoprotein B among natural seropositives and CMV subunit vaccine recipients

Development of a baculovirus vector that facilitates the generation of p10-based recombinants

The roles of the glycosylphosphatidylinositol anchor on the production and immunogenicity of recombinant ookinete surface antigen Pbs21 of Plasmodium berghei when prepared in a baculovirus expression system

Production of porcine parvovirus empty capsids with high immunogenic activity

Structure-dependent efficacy of infectious bursal disease virus (IBDV) recombinant vaccines

Induction of anti-malarial transmission blocking immunity with a recombinant ookinete surface antigen of Plasmodium berghei produced in silkworm larvae using the baculovirus expression vector system

Protection of mice against lethal Japanese encephalitis with a recombinant baculovirus vaccine

Immune responses elicited by recombinant vaccinia-human immunodeficiency virus (HIV) envelope and HIV envelope protein: Analysis of the durability of responses and effect of repeated boosting

Comparison of the protective efficacy of DNA and baculovirus-derived protein vaccines for EBOLA virus in guinea pigs

The GP64 envelope fusion protein is an essential baculovirus protein required for cell-to-cell transmission of infection

Serum antibodies to HIV-1 in recombinant vaccinia virus recipients boosted with purified recombinant gp160. NIAID AIDS vaccine clinical trials network

Expression of the Newcastle disease virus (NDV) fusion glycoprotein and vaccination against NDV challenge with a recombinant baculovirus

Vaccination against newcastle disease with a recombinant baculovirus hemagglutinin-neuraminidase subunit vaccine

Neutralizing antibodies to African swine fever virus proteins p30, p54, and p72 are not sufficient for antibody-mediated protection

Characterization of an insect cell-derived Theileria parva sporozoite vaccine antigen and immunogenicity in cattle

Recombinant virus-like particles of a norovirus (genogroup II strain) administered intranasally and orally with mucosal adjuvants LT and LT(R192G) in BALB/c mice induce specific humoral and cellular Th1/Th2-like immune responses

Baculovirus Expression Vectors, A Laboratory Manual

Vaccination with a heterologous respiratory syncytial virus chimeric FG glycoprotein demonstrates significant subgroup cross-reactivity

Conserved molecular systems of the Baculoviridae

Expression of bovine herpesvirus 1 glycoprotein gIII by a recombinant baculovirus in insect cells

Subversion mechanisms by which Leishmania parasites can escape the host immune response: A signaling point of view

Immunogenicity and efficacy of baculovirus-expressed and DNA-based equine influenza virus hemagglutinin vaccines in mice

Control of Theileria sergenti infection by vaccination

Immune responses and protective efficacy of recombinant baculovirus-expressed glycoproteins of equine herpesvirus 1 (EHV-1) gB, gC and gD alone or in combinations in BALB/c mice

Influenza: Old and new threats

The glycosylation pattern of baculovirus expressed envelope protein E2 affects its ability to prevent infection with bovine viral diarrhoea virus

Veterinary Vaccinology

Allergic reactions to measles-mumps-rubella vaccination

The human cell line PER.C6 provides a new manufacturing system for the production of influenza vaccines

Genetically engineered multi-component virus-like particles as veterinary vaccines

Comparison of nucleic acid and protein immunization for induction of antibodies specific for HIV-1 gp120

Protection from La Crosse virus encephalitis with recombinant glycoproteins: Role of neutralizing anti-G1 antibodies

A vaccinia-gp160-based vaccine but not a gp160 protein vaccine elicits anti-gp160 cytotoxic T lymphocytes in some HIV-1 seronegative vaccinees

A phase I, randomized controlled clinical trial to study the reactogenicity and immunogenicity of a new split influenza vaccine derived from a non-tumorigenic cell line

Baculovirus merozoite surface protein 1 C-terminal recombinant antigens are highly protective in a natural primate model for human Plasmodium vivax malaria

Time reduction and process optimization of the baculovirus expression system for more efficient recombinant protein production in insect cells

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

Pivotal role of the non-hr origin of DNA replication in the genesis of defective interfering baculoviruses

Spontaneous excision of BAC vector sequences from bacmid-derived baculovirus expression vectors upon passage in insect cells

Cell line-specific accumulation of the baculovirus non-hr origin of DNA replication in infected insect cells

Evaluation of baculovirus expression vectors with enhanced stability in continuous cascaded insect-cell bioreactors

Stabilized baculovirus vector expressing a heterologous gene and GP64 from a single bicistronic transcript

Insect cell-derived VP2 of infectious bursal disease virus confers protection against the disease in chickens

Failure of viral protein 3 of infectious bursal disease virus produced in prokaryotic and eukaryotic expression systems to protect chickens against the disease

Crystal structure of a Fab complex formed with PfMSP1-19, the C-terminal fragment of merozoite surface protein 1 from Plasmodium falciparum: A malaria vaccine candidate

Baculovirus expression of proteins of porcine reproductive and respiratory syndrome virus strain Olot/91. Involvement of ORF3 and ORF5 proteins in protection

Generation of recombinant influenza virus using baculovirus delivery vector

The use of baculovirus vectors for the production of membrane proteins in insect cells

Influenza A virus vaccines containing purified recombinant H3 hemagglutinin are well tolerated and induce protective immune responses in healthy adults

Humoral and cellular immune responses following vaccination with purified recombinant hemagglutinin from influenza A (H3N2) virus

Immunogenic and protective properties of rabies virus glycoprotein expressed by baculovirus vectors

Induction of sterilizing immunity against West Nile virus (WNV), by immunization with WNV-like particles produced in insect cells

Immunoreactivity and protective effects in mice of a recombinant dengue 2 Tonga virus NS1 protein produced in a baculovirus expression system

Immunization of mice with a TolA-like surface protein of Trypanosoma cruzi generates CD4(þ) T-cell-dependent parasiticidal activity

Expression of the fusion glycoprotein of human parainfluenza type 3 virus in insect cells by a recombinant baculovirus and analysis of its immunogenic property

Portfolio of candidate malaria vaccines currently in development

Fasciola hepatica procathepsin L3 protein expressed by a baculovirus recombinant can partly protect rats against fasciolosis

B-cell activation and differentiation by HIV-1 antigens among volunteers vaccinated with VaxSyn HIV-1

Technology evaluation: APC-8015, Dendreon

Synthesis of immunogenic hepatitis A virus particles by recombinant baculoviruses

Genetically engineered particulate virus-like structures and their use as vaccine delivery systems

Nature and duration of protective immunity to bluetongue virus infection

New generation of African horse sickness virus vaccines based on structural and molecular studies of the virus particles

Long-lasting protection of sheep against bluetongue challenge after vaccination with virus-like particles: Evidence for homologous and partial heterologous protection

A prime-boost immunization strategy with DNA and recombinant baculovirus-expressed protein enhances protective immunogenicity of glycoprotein D of equine herpesvirus 1 in naive and infection-primed mice

Functional and immunological properties of the baculovirus-expressed hemagglutinin of African swine fever virus

Update on adenovirus and its vectors

Canine parvovirus empty capsids produced by expression in a baculovirus vector: Use in analysis of viral properties and immunization of dogs

Expression of YAV proteins and vaccination against viral ascites among cultured juvenile yellowtail

Deletion of the Autographa californica nucleopolyhedrovirus chitinase KDEL motif and in vitro and in vivo analysis of the modified virus

Vaccination against canine babesiosis

Mechanisms of vaccine adjuvant activity: Initiation and regulation of immune responses by vaccine adjuvants

Protection against congenital cytomegalovirus infection and disease in guinea pigs, conferred by a purified recombinant glycoprotein B vaccine

Baculovirus expression of the M genome segment of Rift Valley fever virus and examination of antigenic and immunogenic properties of the expressed proteins

Antigenic subunits of Hantaan virus expressed by baculovirus and vaccinia virus recombinants

A new epitope presenting system displays a HIV-1 V3 loop sequence and induces neutralizing antibodies

Active immunity and T-cell populations in pigs intraperitoneally inoculated with baculovirus-expressed transmissible gastroenteritis virus structural proteins

Immune responses of lambs to the fusion (F) glycoprotein of bovine respiratory syncytial virus expressed on insect cells infected with a recombinant baculovirus

Immunization of mice with baculovirus-derived recombinant SV40 large tumour antigen induces protective tumour immunity to a lethal challenge with SV40-transformed cells

Development of a subunit vaccine for infectious pancreatic necrosis virus using a baculovirus insect/larvae system

Immune responses in goats to recombinant hemagglutinin-neuraminidase glycoprotein of Peste des petits ruminants virus: Identification of a T cell determinant

Recombinant hemagglutinin protein of rinderpest virus expressed in insect cells induces cytotoxic T-cell responses in cattle

Protection of mice against influenza A virus challenge by vaccination with baculovirus-expressed M2 protein

Production of human beta interferon in insect cells infected with a baculovirus expression vector

Active cross-protection induced by a recombinant baculovirus expressing chimeric infectious bursal disease virus structural proteins

Induction of protective immunity in chickens vaccinated with infectious bronchitis virus S1 glycoprotein expressed by a recombinant baculovirus

The production of a truncated form of baculovirus expressed EHV-1 glycoprotein C and its role in protection of C3H (H-2Kk) mice against virus challenge

The expression of the proteins of equine herpesvirus 1 which share homology with herpes simplex virus 1 glycoproteins H and L

High level expression of equine herpesvirus 1 glycoproteins D and H and their role in protection against virus challenge in the C3H (H-2Kk) murine model

Plant production systems for vaccines

Efficacy of vaccines in chickens against highly pathogenic Hong Kong H5N1 avian influenza

Production and in vivo testing of a recombinant bovine IL-12 as an adjuvant for Salmonella typhimurium vaccination in calves

Immunological properties of FMDV-gP64 fusion proteins expressed on SF9 cell and baculovirus surfaces

In vitro and in vivo gene delivery by recombinant baculoviruses

Molecular chaperones stimulate the functional expression of the cocaine-sensitive serotonin transporter

Murine polyomavirus virus-like particles (VLPs) as vectors for gene and immune therapy and vaccines against viral infections and cancer

Reduced oviposition of Boophilus microplus feeding on sheep vaccinated with vitellin

Enhanced secretion from insect cells of a foreign protein fused to the honeybee melittin signal peptide

Family Baculoviridae

Localization of a baculovirus-induced chitinase in the insect cell endoplasmic reticulum

Chimeric HIV-1 virus-like particles containing gp120 epitopes as a result of a ribosomal frameshift elicit Gag-and SU-specific murine cytotoxic T-lymphocyte activities

Comparing N-glycan processing in mammalian cell lines to native and engineered lepidopteran insect cell lines

Structure and expression in baculovirus of the Mokola virus glycoprotein: An efficient recombinant vaccine

Evaluation of a recombinant hemagglutinin expressed in insect cells as an influenza vaccine in young and elderly adults

Safety and immunogenicity of a recombinant hemagglutinin vaccine for H5 influenza in humans

Dose-related safety and immunogenicity of a tivalent baculvoirus-expressed infleunzavirus hemagglutinin vaccine in elderly adults

Potential viral vectors for the stimulation of mucosal antibody responses against enteric viral antigens in pigs

Active and passive protection against variant and classic infectious bursal disease virus strains induced by baculovirusexpressed structural proteins

Empty canine parvovirus capsids having CPV recombinant VP2 and vaccines having such capsids

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

Development of recombinant vaccines against bluetongue

Expression of bovine herpesvirus 1 glycoprotein gIV by recombinant baculovirus and analysis of its immunogenic properties

Protection of cattle from BHV-1 infection by immunization with recombinant glycoprotein gIV

Long-term semi-continuous production of recombinant baculovirus protein in a repeated (fed-) batch two-stage reactor system

The baculovirus 10-kDa protein

Secretory pathway limits the enhanced expression of classical swine fever virus E2 glycoprotein in insect cells

Diva vaccines that reduce virus transmission

An experimental marker vaccine and accompanying serological diagnostic test both based on envelope glycoprotein E2 of classical swine fever virus (CSFV)

Expression of biologically active and antigenically authentic parainfluenza type 3 virus hemagglutinin-neuraminidase glycoprotein by a recombinant baculovirus

Ex vivo stimulation and expansion of both CD4(þ) and CD8(þ) T cells from peripheral blood mononuclear cells of human cytomegalovirus-seropositive blood donors by using a soluble recombinant chimeric protein, IE1-pp65

Induction of protective immunity against Dengue virus type 2: Comparison of candidate live attenuated and recombinant vaccines

Immunity to St. Louis encephalitis virus by sequential immunization with recombinant vaccinia and baculovirus derived PrM/E proteins

Baculovirus expression vector system for production of viral vaccines

Expression of cauliflower mosaic virus gene I using a baculovirus vector based upon the p10 gene and a novel selection method

Current applications of cell culture engineering

Cloning of biologically active genomes from a Helicoverpa armigera singlenucleocapsid nucleopolyhedrovirus isolate by using a bacterial artificial chromosome

Selfassembly of the infectious bursal disease virus capsid protein, rVP2, expressed in insect cells and purification of immunogenic chimeric rVP2H particles by immobilized metal-ion affinity chromatography

Vaccination of cotton rats with a chimeric FG glycoprotein of human respiratory syncytial virus induces minimal pulmonary pathology on challenge

Comparison of simian virus 40 large T antigen recombinant protein and DNA immunization in the induction of protective immunity from experimental pulmonary metastasis

Immune response to baculovirus expressed protein fragment amino acids 190-289 of respiratory syncytial virus (RSV) fusion protein

A baculovirus dual expression vector derived from the Autographa californica nuclear polyhedrosis virus polyhedrin and p10 promoters: Co-expression of two influenza virus genes in insect cells

Characteristics of equine herpesvirus 1 glycoproteins expressed in insect cells

Baculovirus defective interfering particles are responsible for variations in recombinant protein production as a function of multiplicity of infection

Commercialisation of a recombinant vaccine against Boophilus microplus

Display of foreign proteins using recombinant baculovirus occlusion bodies: A novel vaccination tool

Characterization of pseudorabies virus glycoprotein gII expressed by recombinant baculovirus

Characterization of canine herpesvirus glycoprotein C expressed by a recombinant baculovirus in insect cells

Immunogenicity of baculovirus expressed recombinant proteins of Japanese encephalitis virus in mice

Production and characterization of simian-human immunodeficiency virus-like particles

Intranasal immunization with SIV virus-like particles (VLPs) elicits systemic and mucosal immunity

Baculovirus virions displaying Plasmodium berghei circumsporozoite protein protect mice against malaria sporozoite infection

Viral gene therapy strategies: From basic science to clinical applications

Calreticulin promotes folding/dimerization of human lipoprotein lipase expressed in insect cells (Sf21)

Immunization of mice with dengue structural proteins and nonstructural protein NS1 expressed by baculovirus recombinant induces resistance to dengue virus encephalitis

Technology evaluation: HIVAC-1e

Babesia divergens, a bovine blood parasite of veterinary and zoonotic importance