Veterinary Microbiology 171 (2014) 93–101

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Rabies-virus-glycoprotein-pseudotyped recombinant baculovirus vaccine confers complete protection against lethal rabies virus challenge in a mouse model Qunfeng Wu a,1, Fulai Yu a,1, Jinfang Xu a, Yang Li a, Huanchun Chen a, Shaobo Xiao a, Zhen F. Fu a,b,**, Liurong Fang a,* a

Division of Animal Infectious Diseases, State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China Departments of Pathology, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA

b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 23 October 2013 Received in revised form 23 March 2014 Accepted 25 March 2014

Rabies virus has been an ongoing threat to humans and animals. Here, we developed a new strategy to generate a rabies virus vaccine based on a pseudotyped baculovirus. The recombinant baculovirus (BV-RVG/RVG) was pseudotyped with the rabies virus glycoprotein (RVG) and also simultaneously expressed another RVG under the control of the immediate early CMV promoter. In vitro, this RVG-pseudotyped baculovirus vector induced syncytium formation in insect cells and displayed more efficient gene delivery into mammalian cells. Mice immunized with BV-RVG/RVG developed higher levels of virus-neutralizing antibodies, and conferred 100% protection against rabies viral challenge. These data indicate that the RVG-pseudotyped baculovirus BV-RVG/RVG can be used as an alternative strategy to develop a safe and efficacious vaccine against the rabies virus. ß 2014 Elsevier B.V. All rights reserved.

Keywords: Rabies virus Glycoprotein Pseudotyped baculovirus Vaccine

1. Introduction Rabies, an ancient disease, remains an important global zoonosis. More than 55,000 human fatalities are reported annually, and millions of other cases require post-exposure

* Corresponding author at: College of Veterinary Medicine, Huazhong Agricultural University, 1 Shi-zi-shan Street, Wuhan 430070, China. Tel.: +86 27 8728 6884; fax: +86 27 8728 2608. ** Corresponding author at: Department of Pathology, College of Veterinary Medicine, University of Georgia, 501 D.W. Brooks Drive, Athens, GA 30602, USA. Tel.: +1 706 542 7021; fax: +1 706 542 5828. E-mail addresses: [email protected] (Z.F. Fu), [email protected] (L. Fang). 1 Both authors made equal contributions to the work. http://dx.doi.org/10.1016/j.vetmic.2014.03.037 0378-1135/ß 2014 Elsevier B.V. All rights reserved.

treatment (Shwiff et al., 2013). Stray dogs, cats and wild carnivores are natural reservoirs of rabies virus (RABV) and thus the major sources for human exposure with RABV in China (Yin et al., 2013). Prophylactic vaccination is the most effective and cost-saving method to prevent RABV infection. In China, the only available domestic-made vaccines for pet and stray dogs are live-attenuated rabies virus vaccines generated from the Evelyn-Rokitnicki-Abelseth (ERA) (Guo et al., 2009) or the low egg passage (LEP) strain (Ren, 2010). However, live attenuated rabies vaccine ERA can revert to virulent phenotype due to RNA nucleotide mutation (Fehlner-Gardiner et al., 2008). Thus, safer and more cost-effective rabies vaccines are needed for dog immunization in China.

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RABV is a single-stranded, negative-sense RNA virus belonging to the family Rhabdoviridae (Jackson, 2002; Zhang et al., 2013). The glycoprotein (RVG) encoded by RABV is the only surface protein that induces virusneutralizing (VN) antibodies (Johnson et al., 2010). Therefore, RVG is the leading target in the development of a genetically engineered vaccine against rabies (Ge et al., 2011; Gupta et al., 2005; Lees et al., 2002; Saxena et al., 2009). Traditionally, the baculovirus Autographa californica multiple nucleopolyhedrovirus has been used as an efficient vehicle to overexpress recombinant proteins in insect cells. Recent evidence has revealed that baculovirus carrying mammalian-cell-active promoters can mediate foreign gene expression in mammalian cells and animal hosts (Hofmann et al., 1995; Prabakaran et al., 2010). Furthermore, it has been reported that a pseudotyped baculovirus displaying the glycoprotein of vesicular stomatitis virus (VSVG) on the envelope has an extended host range and increased efficiency of transduction in mammalian cells and in mouse skeletal muscles (Barsoum et al., 1997; Facciabene et al., 2004; Park et al., 2001; Pieroni et al., 2001). With a highly efficient gene delivery mechanism and bio-safety profile plus unique immunestimulatory capacities, direct vaccination with recombinant baculovirus can induce high levels of humoral and cell-mediated immunity against animal and human diseases, including influenza virus, porcine reproductive and respiratory syndrome virus, porcine circovirus type 2, West Nile virus, RABV and hepatitis C virus (Chen et al., 2010; Facciabene et al., 2004; Fan et al., 2008a; Huang et al., 2011; Wu et al., 2013; Zhu et al., 2012). Because VSV is also a member of the family Rhabdoviridae and RVG has a membrane fusion function similar to that of VSVG, we propose a new strategy for the development of a baculovirus-vectored vaccine against rabies in which RVG is displayed on the envelope of pseudotyped baculovirus and another RVG is expressed under the control of the immediate early cytomegalovirus (CMV) promoter in vivo. Theoretically, this design includes the use of two types of RVG antigen to induce RVG-specific immune responses, baculovirus-expressed RVG and RVG expressed in vivo in the host, which should improve the immune effects of the vaccine. To test the feasibility of our strategy in this study, we first constructed an RVG-pseudotyped recombinant baculovirus expressing the enhanced green fluorescent protein (EGFP) under the CMV promoter, BV-RVG/EGFP, and demonstrated that the RVG-pseudotyped baculovirus more efficiently delivered gene into mammalian cells than the non-pseudotyped baculovirus. We then constructed another RVG-pseudotyped baculovirus, BV-RVG/RVG, expressing an additional RVG via the CMV promoter and evaluated its immunogenicity in a mouse model. 2. Materials and methods 2.1. Cells and viruses Spodoptera frugiperda 9 (Sf9) insect cells were propagated in Grace’s medium (Invitrogen) containing 10% (v/v)

heat-inactivated fetal bovine serum (FBS; Invitrogen). Baby hamster kidney (BHK-21) cells and HeLa cells were grown in Dulbecco’s modified Eagle’s medium (Invitrogen) containing 10% FBS. Mouse neuroblastoma (NA) cells were maintained in RPMI 1640 medium supplemented with 10% FBS. Recombinant baculoviruses BV-CMV-EGFP (BV-EGFP, nonpseudotyped) and BV-VSVG/EGFP (pseudotyped with VSVG) were generated as previously described (Fig. 1A) (Fan et al., 2008a; Pan et al., 2009). RABV strain CVS-24 was propagated in suckling mouse brains and strains ERA and CVS-11 were propagated in NA cells (Chen et al., 2013). 2.2. Plasmids construction and generation of recombinant baculoviruses To construct the RVG-pseudotyped recombinant baculovirus BV-RVG/EGFP expressing EGFP, the full-length cDNA of the RVG gene was amplified from RABV strain ERA (GenBank: J02293.1) and inserted under the control of the polyhedrin promoter in the pFastBac1 vector (Invitrogen). The CMV-EGFP expression cassette was then introduced into downstream of RVG-poly(A) (Fig. 1A). To generate the RVG-pseudotyped baculovirus, BV-RVG/RVG, which expresses another RVG under the control of the CMV promoter, a CMV-RVG expression cassette was inserted into BV-RVG/EGFP by replacing the CMV-EGFP expression cassette (Fig. 1A). The recombinant baculoviruses BV-RVG/ EGFP and BV-RVG/RVG were generated using the Bac-toBac1 Baculovirus Expression System (Invitrogen), and purified by sucrose density gradient centrifugation (Wu et al., 2011). The infectious units per milliliter (IFU/ml) were determined using the BacPAK Baculovirus Rapid Titer Kit (Clontech, Mountain View, CA, USA). 2.3. Infection of Sf9 cells and transduction of mammalian cells by baculoviruses Sf9 cells, BHK-21 cells, and HeLa cells were infected or transduced with BV-EGFP, BV-VSVG/EGFP, BV-RVG/EGFP, and/or BV-RVG/RVG at a multiplicity of infection of 0.05 or 50 (Pan et al., 2009). To detect the fusogenic activity of RVG expressed in Sf9 cells, the infected cells were directly examined under confocal microscopy 48 h after infection. To determine whether RVG was incorporated into the recombinant baculovirus, the purified baculovirus virions were analyzed with immunoblotting with a monoclonal antibody (mAb, 1:2000 dilution) against RVG (Chen et al., 2013). To analyze the expression of RVG in the transduced BHK-21 cells, the cells were collected 48 h after transduction and subjected to immunoblotting with the anti-RVG mAb. The expression of gp64 and b-actin was used as the control. 2.4. Flow-cytometric analysis The baculovirus-transduced BHK-21 and HeLa cells were detached with trypsin, washed with phosphatebuffered saline (PBS), and resuspended in PBS for flowcytometric analysis (FACSCalibur, BD BioSciences). The percentage of EGFP-positive cells in each sample was analyzed with the CellQuest software (Becton Dickinson).

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Fig. 1. Construction and characterization of RVG-pseudotyped baculoviruses. (A) Schematic illustration and theoretical structure of BV-EGFP (Pan et al., 2009), BV-VSVG/EGFP (Fan et al., 2008a), BV-RVG/EGFP, and BV-RVG/RVG. PPH, the polyhedrin promoter of the baculovirus; RVG, rabies virus glycoprotein; VSVG, the glycoprotein of vesicular stomatitis virus; EGFP, enhanced green fluorescent protein; gp64, baculovirus envelope glycoprotein gp64; CMV, cytomegalovirus immediate-early promoter; poly (A), polyadenylation signal. In the theoretical pseudotyped baculoviruses at the bottom of the panel, the interested genes shown here are only driven by the CMV promoter. (B) Syncytium formation in Sf9 cells induced by the expression of RVG and VSVG. (C) Incorporation of RVG into the recombinant baculovirus particles.

2.5. Mice immunization and challenge experiments Groups of 6-week-old female BALB/c mice (14 mice/ group) (Laboratory Animals Center, Institute of Medicine, Hubei Province, China) were immunized intramuscularly with 1  108 IFU of BV-VSVG/EGFP, BVRVG/EGFP, or BVRVG/RVG at 3-week interval (Fig. 3B). The control group was immunized with PBS only. Sera samples were collected 3 and 5 weeks after the primary immunization

for serological tests. Two weeks after the booster immunization, 6 mice from each group were euthanized with isoflurane and their splenocytes were isolated for interferon g (IFN-g) analysis. The remaining mice were challenged intracerebrally with 50 mouse 50% lethal doses (50 LD50) of rabies virus CVS-24 according to the National Institutes of Health (NIH) potency testing (Seligmann, 1973) and observed for clinical signs for 14 days. All animal experiments were performed following protocols

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approved by the Hubei Provincial Animal Care and Use Committee. 2.6. Serological tests Mouse sera were tested for the presence of VN antibodies against CVS-11 with a rapid fluorescent focus inhibition test (Chen et al., 2013; Zavadova et al., 1996). Briefly, 50 ml of serial dilutions of serum was prepared in Lab-Tek Chamber slides (Nalge Nunc International, Rochester, NY). Fifty 50% fluorescing focus doses (FFD50) of CVS-11 were added to each chamber, and the slides were incubated for 90 min at 37 8C. NA cells (5  105 cells) were added into each chamber, and the slides were incubated at 37 8C for 20 h. Then, the cells were fixed with ice-cold 80% acetone and stained with FITC-conjugated anti-RABV N antibody (Chen et al., 2013) for 1 h at 37 8C. Twenty fields in each chamber were observed under a fluorescence microscope, and the 50% endpoint titers were calculated according to the ReedMuench formula (Reed and Muench, 1938). The values were compared with those obtained with the reference serum (National Institute for Biological Standards and Control, Herts, United Kingdom) and normalized to international units (IU)/ml. RVG-specific antibodies were assessed with a direct enzyme-linked immunosorbent assay (ELISA) using 2 mg/ ml purified RVG as the capture antigen (Saxena et al., 2008). Antigen-coated 96 well plates were blocked with 1% BSA and then incubated with the two-fold serial dilution of serum samples beginning at 1:20. HRPconjugated goat anti-mouse IgG (1:5000) (Southern Biotech, USA) was used as the secondary antibody and 3,30 ,5,50 -tetramethylbenzidine (TMB) was added as substrate to visualize the reaction. The results were expressed as the ratio of OD490 nm produced by the serum samples compared with negative control serum. Sera with a ratio value higher than 2.1 were considered to be positive sera. The titers were expressed as the reciprocal of the highest dilution of the sera producing ratio values of 2.1 (Fan et al., 2008b). 2.7. Analysis of IFN-g expression with ELISA IFN-g expression was determined as in previous studies (Wu et al., 2009; Wu et al., 2011; Wu et al., 2013). Briefly, mouse splenocytes (2  106 cells/ml) were isolated and cultured in 24-well plates in RPMI 1640 supplemented with 10% FBS, and containing 10 mg/ml RVG protein. After stimulation for 72 h, the presence of IFN-g in the supernatant was evaluated with a mouse IFN-g ELISA kit (R&D Systems). 2.8. Statistical analysis All data were analyzed with one-way analysis of variance with the Origin 8 software (Origin Lab). A value of P < 0.05 was considered to indicate a statistically significant difference. The survival curves were analyzed with the Kaplan-Meier method and compared with a logrank test (x2 test).

3. Results 3.1. Construction and characterization of RVG-pseudotyped baculovirus expressing EGFP The strategy underlying this study was that the RVG protein driven by the polyhedrin promoter in insect cells would be incorporated into the baculovirus via its transmembrane and cytoplasmic domains, thereby broadening the baculovirus tropism in mammalian cells and improving the baculovirus transduction in vitro and in vivo (Barsoum et al., 1997; Tani et al., 2003). To examine whether the RVG expressed in insect cells from BV-RVG/EGFP has the biological activity to mediate cell membrane fusion, as does the VSVG protein, we investigated the fusogenic properties of RVG directly with microscopy after infection. Syncytium formation was clearly observed in Sf9 cells infected with BV-RVG/EGFP (Fig. 1B), although it was unlike the intensive syncytium formation induced by VSVG expressed from BVVSVG/EGFP. However, no syncytium formation was observed in Sf9 cells infected with the wild-type baculovirus or with BV-EGFP, an unmodified baculovirus. To investigate whether the RVG protein expressed in Sf9 cells could be incorporated into the baculovirus particles, purified virions were subjected to immunoblotting assay. The RVG envelope glycoprotein was clearly detected by anti-RVG mAb in BV-RVG/EGFP but not in purified BV-VSVG/EGFP particles (Fig. 1C). 3.2. RVG modification improves gene delivery efficiency in mammalian cells After transduction of cells with BV-EGFP, BV-RVG/EGFP, and BV-VSVG/EGFP, all these recombinant baculoviruses (pseudotyped with VSVG or RVG or unmodified) efficiently mediated EGFP expression in both BHK-21 and HeLa cells 48 h after transduction (Fig. 2A). However, BV-RVG/EGFP showed a clearly higher transduction efficiency than BVEGFP. The flow-cytometric data confirmed that 86% of BHK-21 cells were transduced by BV-RVG/EGFP, compared with 63% of cells transduced by the unmodified baculovirus BV-EGFP (P < 0.01; Fig. 2B). Similar results were also observed in HeLa cells (Fig. 2B). It should be noted that BVVSVG/EGFP displayed the highest transduction efficiency among the three baculoviruses tested in both BHK-21 and HeLa cells (P < 0.05). 3.3. Generation and characterization of RVG-pseudotyped baculovirus BV-RVG/RVG Encouraged by the enhanced gene delivery efficiency mediated by RVG-pseudotyped baculovirus in mammalian cells, we used this RVG-modified baculovirus vector to construct the recombinant baculovirus BV-RVG/RVG, in which another RVG is expressed under the control of the CMV promoter (Fig. 1A). Immunoblotting analysis showed that RVG was successfully incorporated into virion particles (Fig. 1C) and BV-RVG/RVG efficiently expressed RVG under the CMV promoter in BHK-21 cells, whereas no RVG expression was detected in the BV-VSVG/EGFP- and BV-RVG/EGFP-transduced cells (Fig. 3A).

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Fig. 2. Gene delivery into mammalian cells by recombinant baculoviruses. (A) BHK-21 and HeLa cells were transduced with BV-EGFP, BV–RVG/EGFP, or BVVSVG/EGFP. The expression of EGFP was examined directly under a confocal microscope. (B) The percentage (%) of EGFP-positive cells was analyzed by flow cytometry. Data (average values from three independent experiments) represent means  SD. *P < 0.05 and **P < 0.01.

3.4. Humoral immune responses elicited by RVGpseudotyped baculoviruses in immunized mice As shown in Fig. 3C, after primary immunization, the RVG-specific ELISA antibodies titers reached detectable levels in groups BV-RVG/EGFP and BV-RVG/RVG. Further increases in the antibody titers were observed 2 weeks after booster immunization. However, the mean ELISA antibody titer in the group immunized with BV-RVG/RVG was significantly higher than that in the group immunized with BV-RVG/EGFP (P < 0.01). As expected, the sera from mice immunized with BV-VSVG/EGFP or PBS displayed no detectable antibody throughout the experiment. Serum samples were further evaluated for their ability to neutralize RABV. Both RVG-pseudotyped baculoviruses BV-RVG/EGFP and BV-RVG/RVG induced strong VN antibodies against RABV (Fig. 3D). BV-RVG/RVG induced a much better antibody response than BV-RVG/EGFP did, especially following booster immunization, when the VN

titer increased to over 12.6 IU/ml. It is noteworthy that the VN titer induced by RVG-pseudotyped baculovirus BVRVG/EGFP reached 3.5 IU/ml. 3.5. Cellular immune responses elicited by RVG-pseudotyped baculoviruses in immunized mice The cellular immune response was also evaluated by measuring the expression of IFN-g in splenocytes following recall stimulation with the RVG antigen in vitro. As shown in Fig. 3E, the BV-RVG/RVG group secreted the highest concentration of IFN-g, which was at least 4-fold, 8-fold, and 42-fold higher than those secreted by the BVRVG/EGFP, BV-VSVG/EGFP, and PBS groups, respectively. Moreover, even though BV-VSVG/EGFP carries no antigen directed against RABV, mice immunized with it also expressed a certain level of IFN-g, indicating that the recombinant baculovirus can act as a unique adjuvant to elicit nonspecific cellular immune responses in vivo.

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Fig. 3. Evaluation of immunogenicity of RVG-pseudotyped baculoviruses in mice. (A) RVG expression in BHK-21 cells. (B) The time point of mice experiments. (C) ELISA antibodies test. (D) VN antibodies test. (E) IFN-g production by splenocytes from immunized mice during the recall response to the RVG antigen in vitro. Differences between groups were considered significant and are represented as *P < 0.05, **P < 0.01; NS, no significant difference (P > 0.05).

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Fig. 4. Weight changes and survival in mice after lethal challenge. (A) Weight loss was evaluated for 14 days after challenge and is expressed as the mean (SD) percentage of the original body weight. (B) Survival curves for the immunized groups. The Kaplan–Meier survival curves were analyzed with the log-rank test. For PBS versus BV-VSVG/EGFP, P < 0.05; for BV-VSVG/ EGFP versus BV-RVG/EGFP, P < 0.01; and for BV-RVG/EGFP versus BV-RVG/ RVG after viral challenge, P > 0.05.

3.6. Protective immune responses induced by RVGpseudotyped baculoviruses in immunized mice To investigate whether a higher immune response correlated with better protection, immunized mice were challenged with 50 LD50 of RABV 2 weeks after booster immunization. As shown in Fig. 4A, the mice immunized with PBS or BV-VSVG/EGFP showed serious weight loss and signs of illness, including listlessness and neurological disorders or death. Very slight signs of illness were observed in some mice in the BV-RVG/EGFP group. In contrast, the BV-RVG/RVG group displayed no obvious weight loss after infection with RABV. As shown in Fig. 4B, significantly more survivors were observed among the mice immunized with BV-RVG/RVG (100%) or BV-RVG/ EGFP (85.7%) than among the control groups. In additional, 1/8 mice survived in the BV-VSVG/EGFP group after challenge. 4. Discussion In this study, RVG was successfully incorporated into the baculovirus vector to develop a novel vaccine BV-RVG/ RVG, mediated by the RVG-pseudotyped baculovirus

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vector. We demonstrated in vitro that the RVG-pseudotyped baculovirus has an enhanced capacity to deliver gene into mammalian cells. In vivo, the RVG-pseudotyped baculovirus can induce a substantial protective immune responses against RABV as a subunit vaccine. More importantly, the recombinant baculovirus BV-RVG/RVG conferred 100% protection against lethal challenge in a mouse model. During the development of a vaccine against rabies, it has previously been accepted that neutralizing antibodies are responsible for the vaccine-induced protective immunity against RABV infection (Both et al., 2012; Gupta et al., 2005; Johnson et al., 2010). However, recent studies have shown that collaboration between antibody production and the cell-mediated immune response is necessary for protective immunity against RABV (Chopy et al., 2011; Hooper et al., 1998; Saxena et al., 2009; Wen et al., 2011). Therefore, the simultaneous induction of humoral immunity and cellular immunity should be considered when designing a new rabies vaccine. In this study, all the mice immunized with BV-RVG/RVG displayed higher humoral and cellular immune responses and achieved complete protection against lethal challenge compared with those immunized with the subunit vaccine BV-RVG/EGFP. This enhanced immunogenicity may be attributable to the high level of antigen delivered by BV-RVG/RVG in vivo. First, after immunization, the RVG incorporated into the baculovirus vector is immediately recognized by the immunocompetent cells and presented to dendritic cells (DCs) and activated B cells, which then secrete specifically functional VN antibodies. Second, the addition of the CMVRVG expression cassette causes the expression of further antigen in vivo, allowing RVG to be processed via an endogenous antigen-presentation pathway as in a naı¨ve infection, which will once again activate the immune system to produce persistent and specific immune responses. Third, the RVG incorporated into the baculovirus particles confers on the baculovirus a new receptorbinding protein, which mediates the efficient penetration of the baculovirus into the target immune cells for antigen presentation (Tani et al., 2003). In this study, we also observed that 1 out of the 8 mice immunized with baculovirus BV-VSVG/EGFP survived the challenge. This result indicates that inoculation with baculovirus might activate antiviral innate immune response in vivo. It has been reported that baculovirus protected mice against lethal challenge with encephalomyocarditis virus (Gronowski et al., 1999), influenza H1N1 virus (Abe et al., 2003), and foot-and-mouth disease virus (Molinari et al., 2010). These studies suggest that IFN production and the secretion of other inflammatory cytokines induced by the baculovirus are involved in protective immune responses (Abe et al., 2003; Gronowski et al., 1999; Molinari et al., 2010). In this study, we detected a higher level of IFN-g secretion in the BV-RVG/ RVG group and a certain level of IFN-g production (55.84 pg/ml) in the BV-VSVG/EGFP group. Robust IFN-g expression may profoundly enhance the action of this vaccine against RABV in vivo, especially because previous studies have shown that IFN-g can inhibit the replication of RABV in cultured cells (Brzozka et al., 2006).

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The enhanced cell-mediated immune responses may have been induced by both the high level of antigen delivered in vivo and the adjuvant activity offered by the baculovirus vector, which is different from parainfluenza virus type 5 (PIV5)-vectored rabies vaccines (Chen et al., 2013). It has been reported that the immunization of mice with ovalbumin plus baculovirus genomic DNA was sufficient to promote humoral and cytotoxic T-lymphocyte responses against coadministered antigens (HervasStubbs et al., 2007). Furthermore, baculovirus has been shown to infect DCs and B cells preferentially, and to promote natural killer cell proliferation in vivo. It then triggers signaling cascade pathways in these immunocompetent cells, causing them to secrete various cytokines, including members of the IFN family, which play vital roles against RABV (Brzozka et al., 2006). Baculovirus has abundant unmethylated CpG motifs, and induces innate immune responses through at least two different signaling pathways, the TLR9/MyD88-dependent endosomal-recognition pathway and the pathway mediated by DNAdependent activator of interferon-regulatory factors (Abe et al., 2009; Suzuki et al., 2010). CpG sites have been reported to have immunostimulatory effects in mice and dogs and significantly enhance the effects of an inactivated rabies vaccine by protecting mice from disease development after viral challenge (Ren et al., 2010). It is well known that street RABV infections stimulate low-level innate immune responses, with extremely low levels of IFN and inflammatory chemokine production (Wang et al., 2005). These weak innate immune responses after RABV infection are associated with weak adaptive immune responses, which prolong the clinical course of the rabies disease. It is possible that the innate immune response and RABV-specific antibody response elicited by BV-RVG/RVG offer more efficient resistance to RABV infection because the innate immune response is the first line of the host’s defenses against viral pathogens. In summary, the strong immunogenicity of BV-RVG/ RVG in mice plus the nonspecific antiviral immunity induced by the baculovirus vector makes the RVGpseudotyped baculovirus vector a novel tool for the future development of a vaccine against RABV. Acknowledgment This work was supported by State 863 High Technology R&D Project of China (2011AA10A212), and the Fundamental Research Funds for the Central Universities (2013PY043).

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Rabies-virus-glycoprotein-pseudotyped recombinant baculovirus vaccine confers complete protection against lethal rabies virus challenge in a mouse model.

Rabies virus has been an ongoing threat to humans and animals. Here, we developed a new strategy to generate a rabies virus vaccine based on a pseudot...
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