Impact of Molecular Modification on the Efficiency of Recombinant Baculovirus Vector Invasion to Mammalian Cells and Its Immunogenicity in Mice

The baculovirus display system (BDS), an excellent eukaryotic surface display technology that offers the advantages of safety, efficiency, and economy, is widely used in biomedicine. A previous study using rBacmid-Δgp64-ires-gp64 expressed in low copy numbers of the gp64 gene achieved high-efficiency expression and co-display of three fluorescent proteins (GFP, YFP, and mCherry). However, low expression of GP64 in recombinant baculoviruses also reduces the efficiency of recombinant baculovirus transduction into mammalian cells. In addition, the baculovirus promoter has no expression activity in mammalian cells and thus cannot meet the application requirements of baculoviral vectors for the BDS. Based on previous research, this study first determined the expression activity of promoters in insect Spodoptera frugiperda 9 cells and mammalian cells and successfully screened the very early promoter pie1 to mediate the co-expression of multiple genes. Second, utilizing the envelope display effect of the INVASIN and VSVG proteins, the efficiency of transduction of recombinant baculovirus particles into non-host cells was significantly improved. Finally, based on the above improvement, a recombinant baculovirus vector displaying four antigen proteins with high efficiency was constructed. Compared with traditional BDSs, the rBacmid-Δgp64 system exhibited increased display efficiency of the target protein by approximately 3-fold and induced an approximately 4-fold increase in the titer of serum antibodies to target antigens in Bal B/c mice. This study systematically explored the application of a new multi-gene co-display technology applicable to multi-vaccine research, and the results provide a foundation for the development of novel BDS technologies.


Introduction
Insect baculoviruses are double-stranded circular DNA viruses with a typical envelope structure in nature [1]. The typical characteristic of insect baculoviruses is that the virus particle has two different structural forms, namely BV (budded virus) and ODV (occlusion-derived virus) [2]. BVs and ODVs are responsible for cell and oral infections, respectively, and they are widely used as high-efficiency expression vectors and biological pesticides because of their high safety, environmental friendliness, and simplicity of genetic manipulation [3][4][5]. Baculoviral vectors have been widely used in the fields of antigen presentation, gene therapy, and DNA vaccines [6,7]. Based on the baculovirus display system (BDS), a target protein is displayed on the surface of a baculovirus capsule and cell membrane with high efficiency and activity using a co-expression technology [8]. The classic display technology involves inserting the target gene between the signal peptide and the extracellular domain of the envelope protein gene, which can display the target stably and efficiently without affecting the life cycle of the baculovirus [9,10]. Proteins widely used for display include low pH-mediated membrane-promoting fusion proteins, including GP64 (group I type baculovirus), F protein (group II type baculovirus), and non-polar protein VSVG (vesicular stomatitis virus G protein) [11][12][13].
When a baculovirus infects a host cell, the virus binds to the cell surface in a nonspecific manner to complete the mutual recognition of receptors and then enters the cell via endocytosis, pinocytosis, or membrane fusion [14]. Studies have found that dynein and clathrin can mediate the endocytosis of membrane proteins under low-pH conditions, which in turn triggers fusion between the capsule membrane and the cell membrane [15]. Furthermore, protein inhibitors can significantly reduce the invasion efficiency of pseudotype recombinant viruses, indicating that receptor recognition involves BV particle envelope proteins (such as GP64) [15]. Further studies have shown that a potential cholesterol recognition amino acid sequence is located in the specific recognition region of the GP64 protein [16]. Moreover, membrane phospholipids and heparan sulfate proteoglycan sites also mediate the invasion process of BV particles [17].
To date, many envelope proteins in the BDS system have been studied, including VSVG, INVASIN (Invasins protein), and influenza A virus H protein [18]. A feature common to these proteins is that they play a role in clathrin-dependent endocytosis in the invasion processes of viruses and bacteria [19]. VSVG is a non-polar protein on the surface of the vesicular stomatitis virus envelope and directs the process of vesicular stomatitis virus invasion of host cells [20]. Vesicular stomatitis virus can invade cells in a wide range of hosts, including mammals, birds, fish, and insects. VSVG is often used as an immune enhancement factor because it induces immune responses and neutralizes specific antibodies. Functionally, VSVG is similar to GP64, which exhibits changes in the spatial conformation of the protein under low-pH conditions (pH < 6.1) [20]. The INVASIN protein is encoded by the inv gene of Yersinia pseudotuberculosis and binds to the β1 chain of integrin to specifically recognize αβ1 family proteins [21]. In previous research, we elucidated the process of INVASIN-mediated direct invasion of Spodoptera frugiperda 9 (Sf9) and BmN cells by Escherichia coli Sw106 [8]. Therefore, we speculated that INVASIN may also increase the efficiency and range of baculovirus infection. In addition, INVASIN is a mature immune adjuvant that can significantly increase the titer of specific antibodies.
In previous studies, we achieved the efficient display of three fluorescent proteins on the surface of the baculovirus capsule and insect cell membrane through the low expression of GP64 mediated by deletion of the gp64 and internal ribosome entry site (ires) genes in Bacmid. However, the baculoviral promoter cannot be expressed efficiently in mammalian cells, and low expression of GP64 also reduces the invasion efficiency of recombinant baculoviruses [8]. In this study, we conducted a screening for promoters that can be efficiently expressed in mammalian and insect cells in combination with surface display of non-homologous envelope proteins (INVASIN and VSVG) in order to construct a recombinant baculovirus particle with high display and transduction efficiency. In addition, the immunogenicity of the co-displayed antigen was explored in mice. The results of this research could lead to an improved BDS and lay an experimental foundation for the development of insect baculovirus vectors for multiple vaccines.

Label Primer Sequences Genes
Rluc F: 5 -tctagaatgatgtacatagaagatt-3 (Xba I) R: 5 -ctgcagttacacttgatacatgc-3 (Pst I) Renilla-luciferase   To determine the invasion efficiency of AcBV-∆gp64-ires-gp64 in insect and mammalian cells, the vsvg and invasin genes were fused to the recombinant plasmid pFBDM-p64gfp-ires-gp64. The promoter pie1 fused with the gp64 signal peptide was cloned using the primers pie1-F and pie1-R shown in Table 1 and introduced into the recombinant plasmid pFBDM-ie1-p64-gfp-ires-gp64 via the SpeI and XmaI restriction sites. The envelope gene vsvg was amplified using the primers vsvg-F and vsvg-R and integrated downstream of pie1 via the XmaI and XhoI restriction sites to obtain the recombinant plasmid pFBDM-pie1vsvg-p64-gfp-ires-gp64 ( Figure 2). The invasin gene, lacking a stop codon, was amplified using the primers invasin-F and invasin-R (Table 1) and then ligated into the plasmid via the XmaI and XhoI restriction sites. The fused gp64 gene (64orf, amplified using the primers 64ORF-F and 64ORF-R) was inserted via the XhoI and SphI restriction sites to generate the recombinant plasmid pFBDM-pie1-invasin-64orf-p64-gfp-ires-gp64 ( Figure 2).

Production of Recombinant Baculovirus
Escherichia coli Sw106 cells harboring the rBacmid encoding foreign genes were cultured until the optical density at 600 nm reached 0.5-1. The bacteria were collected by centrifugation (3000× g) and resuspended in serum-free insect medium. Aliquots of the bacterial suspension were adjusted to different densities (10 5 -10 8 cells/mL) using serumfree Grace's insect medium [22]. Next, Sf9 cells were cultured overnight in a 12-well plate until the cell density was approximately 70-80%. The supernatant was discarded, and different concentrations of bacteria were added to the corresponding wells. After culturing at 28 • C for 4-5 h, the bacteria in each well were washed out using serum-free Grace's insect medium, and then, 500 µL of fresh insect medium (with 10% FBS and 0.075% penicillin) was added and incubated for 4-5 days post infection (dpi). Infected Sf9 cells were identified by examining the fluorescence in the corresponding wells under a fluorescence microscope (Eclipse Ti-S, Nikon, Tokyo, Japan). In order to obtain a stable titer of recombinant virus, the supernatant was collected and used to infect Sf9 cells again. Fluorescence appeared again at 3-4 dpi, indicating that the recombinant baculovirus was successfully constructed and distributed in the cell supernatant.

Determination of Recombinant Baculovirus Titer
Early exponential phase Sf9 cells were diluted to 10 6 cells/mL with SF-900 II serumfree medium, and then, a 100-µL working volume was placed into different micro-wells in a 96-well plate. Serial 10-fold baculovirus dilutions were also prepared to 10 −8 using SF-900 II insect medium. The cell culture medium was removed, and 100 µL of each virus dilution was added (12 wells per dilution) to the cell monolayer and then incubated at 28 • C. Following 4 h of incubation, the supernatant in each well was replaced with 100 µL of fresh insect medium. Plates were checked once daily for 4-5 days until the fluorescence was observed to reach a maximum. The baculovirus titer is expressed as the 50% tissue culture infective dose according to the standard method of Reed and Muench.

Baculovirus Purification and Transmission Electron Microscopy (TEM)
The supernatant of Sf9 cells infected with recombinant baculovirus (multiplicity of infection (MOI) = 1) was collected at 4 dpi, and baculoviruses in the supernatant were purified by two rounds of sucrose gradient ultracentrifugation according to standard methods. The purified baculoviruses were adsorbed onto glow discharge-activated carboncoated grids for 2 min. The sample-coated grids were washed three times with distilled water, following by negative staining with 1% uranyl acetate for 45 s. Images were acquired using an Talos F200S transmission electron microscope (FEI, Hillsboro, OR, USA).

Dual-Glo Luciferase Assay System
Insect cells or mammalian cells were cultured overnight in a 12-well plate and expanded to the density of 80-90%. Recombinant baculovirus was directly added at a MOI of 1.0 and incubated for 6-8 h, after which the medium was replaced with fresh cell culture medium. The cells were then cultured for 48-96 h to allow for infection, and the cell infection rate was determined based on fluorescence. The cells were pelleted and washed with 1× PBS, and evaluation of Renilla and Firefly luciferase expression was determined according to the manufacturer's instructions.

ELISA
A standard curve of absorbance versus concentration of antibody/antigen standard was prepared for each ELISA. According to the assay instructions, diluted sample (mouse serum and purified recombinant baculovirus) was fully mixed with HRP-conjugated reagent and incubated in the dark at 37 • C for 60 min. The sample was then mixed with chromogen solution and stop solution for 10 min, and the optical density at 400 nm was determined for each well.

Statistical Analysis
All values are presented as the mean ± standard deviation. GraphPad Prism 7 software (GraphPad Software, San Diego, CA, USA) was used for data analyses. One-way analysis of variance followed by Tukey's post-hoc test was used to determine significant difference. p < 0.05 was considered statistically significant.

Determination of Promoter Expression in Insect and Mammalian Cells
The Sf9 cells infected with rBacmid-∆gp64-pX-Fluc-p64-Rluc-p64-gfp-ires-gp64 (pX: p10, pvp39, pcmv, pie1, and psv40) turned green (Figure 4). Within 48 to 120 h post infection (h.p.i), the number of green cells gradually increased, and the level of fluorescence reached a maximum at 120 h.p.i (Figure 4), indicating that the baculoviruses were propagating. The culture supernatant was collected and centrifuged at 80,000× g, and the cell pellet was observed by TEM, which indicated that the recombinant baculovirus particles AcBV-∆gp64-pX-Fluc-p64-Rluc-p64-gfp-ires-gp64 (pX: p10, pvp39, pcmv, pie1, and psv40) were constructed successfully (Figure 4). When the viruses were collected and used to re-infect Sf9 cells (MOI = 1), green cells were observed at 24 h.p.i (Figure 4). Similar to infection with the first-generation strain, fluorescence gradually increased within 24-96 h.p.i and reached a maximum at 96 h.p.i (Figure 4). A standard curve of absorbance versus concentration of antibody/antigen standard was prepared for each ELISA. According to the assay instructions, diluted sample (mouse serum and purified recombinant baculovirus) was fully mixed with HRP-conjugated reagent and incubated in the dark at 37 °C for 60 min. The sample was then mixed with chromogen solution and stop solution for 10 min, and the optical density at 400 nm was determined for each well.

Statistical Analysis
All values are presented as the mean ± standard deviation. GraphPad Prism 7 software (GraphPad Software, San Diego, CA, USA) was used for data analyses. One-way analysis of variance followed by Tukey's post-hoc test was used to determine significant difference. p < 0.05 was considered statistically significant.

Determination of Promoter Expression in Insect and Mammalian Cells
The Sf9 cells infected with rBacmid-Δgp64-pX-Fluc-p64-Rluc-p64-gfp-ires-gp64 (pX: p10, pvp39, pcmv, pie1, and psv40) turned green (Figure 4). Within 48 to 120 h post infection (h.p.i), the number of green cells gradually increased, and the level of fluorescence reached a maximum at 120 h.p.i (Figure 4), indicating that the baculoviruses were propagating. The culture supernatant was collected and centrifuged at 80,000× g, and the cell pellet was observed by TEM, which indicated that the recombinant baculovirus particles AcBV-Δgp64-pX-Fluc-p64-Rluc-p64-gfp-ires-gp64 (pX: p10, pvp39, pcmv, pie1, and psv40) were constructed successfully ( Figure 4). When the viruses were collected and used to re-infect Sf9 cells (MOI = 1), green cells were observed at 24 h.p.i (Figure 4). Similar to infection with the first-generation strain, fluorescence gradually increased within 24-96 h.p.i and reached a maximum at 96 h.p.i (Figure 4).  The dual-luciferase reporter results showed that the promoters pie1, p10, and pvp39 exhibited different expression timing and high expression activity in Sf9 cells compared with the p64 promoter in the early stage. The expression efficiency of pie1 was the highest in the early stage (24 h) of infection but decreased rapidly with the continuation of infection ( Figure 5A). In addition, pcmv and psv40 also mediated the expression of target genes in Sf9 cells, but the expression efficiency was insufficient ( Figure 5A). Within 72 h of recombinant baculovirus invasion of 293T and BHK-21 cells, three mammalian cell promoters successfully expressed active luciferase. In particular, pie1 and psv40 exhibited higher early expression efficiency and longer expression time ( Figure 5B,C). Interestingly, the expression rate of pie1 in BHK-21 cells initially increased and then decreased, with maximum expression efficiency occurring within 36 h of infection ( Figure 5C). Furthermore, the expression efficiency of the pie1 promoter was better than that of pcmv and psv40, thus meeting the BDS requirements for high display efficiency and cross-host expression of foreign genes. The dual-luciferase reporter results showed that the promoters pie1, p10, and pvp39 exhibited different expression timing and high expression activity in Sf9 cells compared with the p64 promoter in the early stage. The expression efficiency of pie1 was the highest in the early stage (24 h) of infection but decreased rapidly with the continuation of infection ( Figure 5A). In addition, pcmv and psv40 also mediated the expression of target genes in Sf9 cells, but the expression efficiency was insufficient ( Figure 5A). Within 72 h of recombinant baculovirus invasion of 293T and BHK-21 cells, three mammalian cell promoters successfully expressed active luciferase. In particular, pie1 and psv40 exhibited higher early expression efficiency and longer expression time ( Figure 5B,C). Interestingly, the expression rate of pie1 in BHK-21 cells initially increased and then decreased, with maximum expression efficiency occurring within 36 h of infection ( Figure 5C). Furthermore, the expression efficiency of the pie1 promoter was better than that of pcmv and psv40, thus meeting the BDS requirements for high display efficiency and cross-host expression of foreign genes. Viruses 2022, 14, x 10 of 17

Determination of the Co-Display Efficiency of Multiple Bacmid-Δgp64 Genes
Increases in cell infection rate and gene expression indicate that rBacmids encoding foreign fluorescent-protein genes (gfp and mCherry) successfully invaded Sf9 cells ( Figure  9). Baculovirus particles in the supernatant were collected and analyzed by TEM and then used to re-infect normal Sf9 cells (Figure 9) to obtain the stable titer of baculoviruses.

Determination of the Co-Display Efficiency of Multiple Bacmid-∆gp64 Genes
Increases in cell infection rate and gene expression indicate that rBacmids encoding foreign fluorescent-protein genes (gfp and mCherry) successfully invaded Sf9 cells (Figure 9). Baculovirus particles in the supernatant were collected and analyzed by TEM and then used to re-infect normal Sf9 cells (Figure 9) to obtain the stable titer of baculoviruses. Similarly, the transduction efficiency of baculoviruses carrying INVASIN and VSVG to 293T cells increased to 31.0% and 25.6%, respectively, compared with the control (p < 0.05) (Figure 8). To sum up, the results indicated that envelope display of INVASIN and VSVG could increase the visual invasion rate in non-host cells, significantly.

Determination of the Co-Display Efficiency of Multiple Bacmid-Δgp64 Genes
Increases in cell infection rate and gene expression indicate that rBacmids encoding foreign fluorescent-protein genes (gfp and mCherry) successfully invaded Sf9 cells ( Figure  9). Baculovirus particles in the supernatant were collected and analyzed by TEM and then used to re-infect normal Sf9 cells (Figure 9) to obtain the stable titer of baculoviruses.  To further investigate the difference in display efficiency between the two rBacmids, antigen ELISAs were performed, and the results showed that the display efficiency of PCV2 was increased by 2.93-fold, E2 by 3.04-fold, and GP5 by 3.53-fold compared with the traditional BDS ( Figure 10). To further investigate the difference in display efficiency between the two rBacmids, antigen ELISAs were performed, and the results showed that the display efficiency of PCV2 was increased by 2.93-fold, E2 by 3.04-fold, and GP5 by 3.53-fold compared with the traditional BDS ( Figure 10).

Determination of Immunogenicity of Multiple-Vaccines Encoded by Bacmid-Δgp64
A total of thirty Bal B/c mice were divided into three groups, including a control and two experimental groups (rBacmid and rBacmid-Δgp64). Mice were injected intraperitoneally with the same titer of recombinant baculoviruses AcBV-E64-G64-I64-P64-G-M (as control, virus injection titer 1 × 10 6 PFU) and AcBV-Δgp64-E64-G64-I64-P64-G-M-iresgp64 (virus injection titer 1 × 10 6 PFU), and the control mice were injected with the same titer of AcBV-Δgp64-p64-gfp-ires-gp64. Serum antibody ELISAs conducted on days 14, 28, and 42 after immunization showed that the expression of the three specific antibodies initially increased and then decreased. Further analyses indicated that the titers of the specific antibodies induced by recombinant Bacmid-Δgp64 were significantly higher than those obtained using traditional BDS (p < 0.05), with the greatest difference in titer (~4 times the control level) observed 28 days after immunization ( Figure 11).

Determination of Immunogenicity of Multiple-Vaccines Encoded by Bacmid-∆gp64
A total of thirty Bal B/c mice were divided into three groups, including a control and two experimental groups (rBacmid and rBacmid-∆gp64). Mice were injected intraperitoneally with the same titer of recombinant baculoviruses AcBV-E64-G64-I64-P64-G-M (as control, virus injection titer 1 × 10 6 PFU) and AcBV-∆gp64-E64-G64-I64-P64-G-M-iresgp64 (virus injection titer 1 × 10 6 PFU), and the control mice were injected with the same titer of AcBV-∆gp64-p64-gfp-ires-gp64. Serum antibody ELISAs conducted on days 14, 28, and 42 after immunization showed that the expression of the three specific antibodies initially increased and then decreased. Further analyses indicated that the titers of the specific antibodies induced by recombinant Bacmid-∆gp64 were significantly higher than those obtained using traditional BDS (p < 0.05), with the greatest difference in titer (~4 times the control level) observed 28 days after immunization ( Figure 11). To further investigate the difference in display efficiency between the two rBacmids, antigen ELISAs were performed, and the results showed that the display efficiency of PCV2 was increased by 2.93-fold, E2 by 3.04-fold, and GP5 by 3.53-fold compared with the traditional BDS ( Figure 10).

Determination of Immunogenicity of Multiple-Vaccines Encoded by Bacmid-Δgp64
A total of thirty Bal B/c mice were divided into three groups, including a control and two experimental groups (rBacmid and rBacmid-Δgp64). Mice were injected intraperitoneally with the same titer of recombinant baculoviruses AcBV-E64-G64-I64-P64-G-M (as control, virus injection titer 1 × 10 6 PFU) and AcBV-Δgp64-E64-G64-I64-P64-G-M-iresgp64 (virus injection titer 1 × 10 6 PFU), and the control mice were injected with the same titer of AcBV-Δgp64-p64-gfp-ires-gp64. Serum antibody ELISAs conducted on days 14, 28, and 42 after immunization showed that the expression of the three specific antibodies initially increased and then decreased. Further analyses indicated that the titers of the specific antibodies induced by recombinant Bacmid-Δgp64 were significantly higher than those obtained using traditional BDS (p < 0.05), with the greatest difference in titer (~4 times the control level) observed 28 days after immunization ( Figure 11).

Discussion
The BDS is a eukaryotic surface display system dependent on insect cell gene expression [9]. Due to simplicity of operation and high expression and safety, the BDS is widely used for antigen presentation, antibody preparation, and receptor screening [10]. Previous research has shown that competition between GP64 and the fusion protein at the binding site on the surface of the baculovirus envelope is the primary factor affecting display efficiency [8]. The Bacmid-Δgp64 constructed in the previous study did not promote complete germination and maturation of progeny viruses due to the gp64 gene deletion, so ires was used to mediate the low expression of GP64. The ires gene sequence effectively recruited ribosomes, resulting in complete gene transcription and protein translation. Another study showed that ires mediates target gene expression in HeLa and CHO cells, with 20-50% of expression due to the promoter, and this phenomenon also occurs in insect Sf9 cells [26]. The promoter p64, which mediates GP64 expression, is a typical very early promoter (12-24 h post infection); therefore, this result also used the early promoter pie1 to reduce competition and improve display efficiency [27]. In addition, the pie1 promoter exhibits high expression activity in both insect and mammalian cells, which could facilitate gene expression by recombinant baculovirus vectors in non-host cells [28][29][30].
Other reports have shown that the envelope protein GP64 primarily mediates baculovirus cell-invasion processes, including electrostatic adsorption, membrane protein

Discussion
The BDS is a eukaryotic surface display system dependent on insect cell gene expression [9]. Due to simplicity of operation and high expression and safety, the BDS is widely used for antigen presentation, antibody preparation, and receptor screening [10]. Previous research has shown that competition between GP64 and the fusion protein at the binding site on the surface of the baculovirus envelope is the primary factor affecting display efficiency [8]. The Bacmid-∆gp64 constructed in the previous study did not promote complete germination and maturation of progeny viruses due to the gp64 gene deletion, so ires was used to mediate the low expression of GP64. The ires gene sequence effectively recruited ribosomes, resulting in complete gene transcription and protein translation. Another study showed that ires mediates target gene expression in HeLa and CHO cells, with 20-50% of expression due to the promoter, and this phenomenon also occurs in insect Sf9 cells [26]. The promoter p64, which mediates GP64 expression, is a typical very early promoter (12-24 h post infection); therefore, this result also used the early promoter pie1 to reduce competition and improve display efficiency [27]. In addition, the pie1 promoter exhibits high expression activity in both insect and mammalian cells, which could facilitate gene expression by recombinant baculovirus vectors in non-host cells [28][29][30].
Other reports have shown that the envelope protein GP64 primarily mediates baculovirus cell-invasion processes, including electrostatic adsorption, membrane protein recognition, and pinocytosis [31][32][33]. Although the rBacmid-∆gp64-ires-gp64 constructed in this study exhibited improved display efficiency, it also reduced the titer and infection rate of the recombinant baculovirus. In a previous study, we demonstrated direct invasion of E. coli into insect Sf9 and BmN cells via INVASIN [8]. In the present study, we also employed envelope display of protein INVASIN and VSVG to significantly improve the invasion efficiency of recombinant rBacmid-∆gp64-ires-gp64 in mammalian cells (p < 0.05). Further analyses indicated that VSVG is cytotoxic, which significantly promotes the fusion of infected Sf9 cells [34,35]. It has been reported that the Red-gam technology was used to delete the N-terminal ectodomain to prevent cell fusion, but this deletion abolished the VSVG function of improving the efficiency of baculovirus transduction [36,37].
As baculoviruses can stimulate the immune system, they are excellent recombinant virus vectors due to the vaccine adjuvant effect [38][39][40]. However, when a baculovirus vector enters a vertebrate animal, it is quickly recognized and inactivated by the complement system in serum or other body fluids [41]. Therefore, avoiding complement inactivation and prolonging the immunization duration are critical issues that have thus far limited the application of baculovirus vectors. Previous studies have shown that recombinant virus particles displaying Cobra venom factors on the envelope can bind to the alternative factor of complement C3, thus improving the efficiency of transduction and expression of viral vector genes in hepatocytes [42]. By co-displaying the C5α receptor and compstatinis, the recombinant baculovirus also significantly enhanced the antigen presentation efficiency, immune response, and cytoinflammatory factor expression of recombinant viral vectors [43]. In addition, DAF, factor H protein, C4b-binding protein, and cell membrane cofactor can also improve the anti-inactivation ability of virus particles [44,45]. Therefore, stable and efficient co-display of multiple target proteins and improvements in the complement-inactivation ability of the recombinant baculovirus will set a new stage for BDS in the field of multiple-vaccine development.

Conclusions
We have provided evidence that the BDS is a useful virus vector for genetic engineering of vaccines. We developed a recombinant baculovirus vector exhibiting higher display efficiency and higher virus titer compared with previous systems in addition to improved efficiency of baculovirus transduction into mammalian cells. Compared with traditional BDSs, the titers of specific antibodies against the three displayed antigens (PCV2-ORF2, PRRSV-GP5, and CSFV-E2) could be increased by approximately 4-fold, thereby maximizing the immunological advantages of recombinant baculovirus vector vaccines.