Induction of antigen-specific immune responses in mice by recombinant baculovirus expressing premembrane and envelope proteins of West Nile virus
© Zhu et al; licensee BioMed Central Ltd. 2012
Received: 21 December 2011
Accepted: 16 July 2012
Published: 16 July 2012
West Nile Virus (WNV) is an emerging arthropod-born flavivirus with increasing distribution worldwide that is responsible for a large proportion of viral encephalitis in humans and horses. Given that there are no effective antiviral drugs available for treatment of the disease, efforts have been directed to develop vaccines to prevent WNV infection. Recently baculovirus has emerged as a novel and attractive gene delivery vehicle for mammalian cells.
In the present study, recombinant baculoviruses expressing WNV premembrane (prM) and envelope (E) proteins under the cytomegalovirus (CMV) promoter with or without vesicular stomatitis virus glycoprotein (VSV/G) were constructed. The recombinant baculoviruses designated Bac-G-prM/E and Bac-prM/E, efficiently express E protein in mammalian cells. Intramuscular injection of the two recombinant baculoviruses (at doses of 108 or 109 PFU/mouse) induced the production of WNV-specific antibodies, neutralizing antibodies as well as gamma interferon (IFN-γ) in a dose-dependent pattern. Interestingly, the recombinant baculovirus Bac-G-prM/E was found to be a more efficient immunogen than Bac-prM/E to elicit a robust immune response upon intramuscular injection. In addition, inoculation of baculovirus resulted in the secretion of inflammatory cytokines, such as TNF-α, IL-2 and IL-6.
These recombinant baculoviruses are capable of eliciting robust humoral and cellular immune responses in mice, and may be considered as novel vaccine candidates for West Nile Virus.
West Nile Virus (WNV), a mosquito-born virus, belongs to the Japanese encephalitis serogroup of flaviviruses  and is an outstanding example of a zoonotic pathogen as its geographic range spans North and South America, Europe, the Middle East, Africa, Western Asia and Australia [2, 3]. Zoonotic transmission of WNV involves Culex mosquitos and birds as the natural reservoir hosts, with humans and horses as dead-end hosts . WNV infections are ranging from a sub-clinical illness, to a self-limiting febrile syndrome or a lethal neuroinvasive disease, and the incidence of severe disease and death increases with age [5, 6]. Since the emergence of WNV in north American in 1999, WNV has been responsible for over 12,000 cases of meningitis/encephalitis, and over 1,100 fatalities in humans and mass mortality of resident birds . The arbovirus infection of humans and animals is becoming a major public health and veterinary concern . The WNV outbreak in North America coupled with the absence of specific therapy against WNV infection has triggered vaccine development to prevent the infection of humans and animals. It has been shown that virion envelope is embedded with viral envelope (E) and premembrane/membrane (prM/M) proteins. The prM protein helps proper folding of the E protein by forming heterodimers  and is cleaved into M by furin [9, 10]. The glycoprotein E is responsible for many properties of the virus including host range, replication, assembly, and stimulation of humoral and cellular immune responses . A variety of WNV vaccine candidates based on the glycoprotein E, which evokes the majority of the neutralizing antibodies, have been developed. Three equine vaccines have been successfully commercialized in the USA, including InnovatorTM, formulated based on formalin-inactivated virus, RecombitekTM, a recombinant replicative canarypoxvirus vaccine, and pCBWN, a recombinant plasmid DNA vaccine . Novel recombinant subunit vaccine candidates such as virus-like particles (VLPs), vectoring vaccine candidates, DNA vaccine candidates , and TripliVAX JE, a chimeric vaccine expressing prM/E and NS1 gene of Japanese encephalitis virus , have shown protective immunity in animal model. However, there are no vaccines currently available for humans. With the growing volume of international travel and commerce, exotic pathogens can spread quickly between continents. Therefore, safe and effective vaccines of WNV are needed for humans.
In recent years, baculovirus has emerged as a vector for gene delivery and vaccine development, with promising newcomer being Autographa californica multiple nucleopolyhedrovirus (AcMNPV), a member of baculoviridae family. The host specificity of baculovirus was originally thought to be restricted to cells derived from a few taxonomically related insect species . However, it has been shown to infect a number of mammalian cells without replication [15–18]. Additionally, accumulated evidence has revealed that baculovirus express foreign proteins not only in insect cells, but also in mammalian cells under the transcriptional control of mammalian promoters [14, 16, 19]. Subsequently, it has been reported that a baculovirus pseudotyped with the glycoprotein (G) of the vesicular stomatitis virus (VSV/G) appeared to help the virus escape from the endosomes, which increases transduction efficiency in mammalian cells [20, 21]. Furthermore, the baculovirus has advantages of being a strong adjuvant and thus can induce inflammatory cytokines and interferons [22–24]. To this end, several studies have demonstrated that direct vaccination with recombinant baculovirus can induce high-level humoral and cell-mediated immunity against various antigens, including pseudorabies virus glycoprotein B (gB) , GP5 and M protein of porcine reproductive and respiratory syndrome virus (PRRSV) , capsid protein of procine circovirus type2 , rabies virus glycoprotein  and Japanese encephalitis virus (JEV) envelope protein .
In the present study, recombinant baculoviruses expressing WNV prM and E protein were constructed with or without pseudotyping with VSV/G. Protein expression was characterized in transduced mammalian cells and its immunogenicity was investigated in a mouse model. The results showed that direct immunization with the recombinant baculoviruses induced a higher level of WNV-specific antibody, neutralizing antibody and gamma interferon compared with E-DIII protein vaccine, suggesting that the recombinant baculoviruses expressing WNV prM and E protein could be developed as new vaccine candidates for WNV.
Construction of recombinant baculoviruses expressing WNV protein
Characterization of WNV-specific antibodies elicited by recombinant baculoviruses in mice
Neutralizing antibody titers in mice immunized with recombinant baculoviruses (mean values of groups of 4 mice)
WNV-specific cellular immune responses elicited by recombinant baculoviruses in mice
To validate this observation, mRNA level of IFN-γ produced in splenocytes was further analyzed by real-time PCR in parallel with the housekeeping gene. Similar to the results of IFN-γ determined by ELISA, the mean relative level of IFN-γ mRNA expression showed significant difference between Bac-G-prM/E- or Bac-prM/E-immunized mice and Bac-G-EGFP- or E-DIII protein-inoculated mice (Figure 4B). All these results demonstrate that immunization with recombinant baculoviruses can stimulate cellular immune responses.
Recombinant baculoviruses induce inflammatory cytokines in mice
WNV continues to present public health threat to the western hemisphere, yet, there is neither effective therapy nor vaccines available for humans. Thus developing a safe and effective vaccine is a priority. Several immunization strategies have been described, some of which utilize vectored vaccines expressing WNV glycoprotein E resulted in 80%-100% protective immunity in animal model [30–34]. In this present study, attempts were made by using recombinant baculovirus approach for WNV vaccine development since baculovirus exhibit low toxoxicity in mammalian cells and can induce interferon production . Two recombinant baculoviruses, Bac-G-prM/E and Bac-prM/E, expressing the major immunogenic protein, glycoprotein E, were constructed,and the humoral and cellular immune responses were determined in mice. WNV-specific humoral (WNV-specific antibodies and neutralizing antibodies) and cellular immunity (WNV-specific IFN-γ and inflammatory cytokines) were induced in mice inoculated with recombinant baculoviruses. These data are in concordance with previous studies that showed robust humoral and cellular immune responses against PRRSV, PCV, or JEV by recombinant baculoviruses .
It has been controversial as to the relative role of the humoral versus cellular immune responses in the WNV-protective immunity. High antibody titers are believed to protect animals against flavivirus infections through direct neutralization of receptor binding, inhibition of viral fusion, and/or complement-mediated viral clearance [35, 36]. Neutralizing antibody titers of >1:10 were reported to be sufficient to afford protection in humans against JE . Schneeweiss et al. also reported that mice immunized once with pT-WNV-E produced neutralizing antibodies titer of 1:16 which can protect 100% of mice from WNV challenge . However, others have argued that cellular immune responses such as robust anti-CD8+ T cell responses are essential for clearing WNV [39, 40]. In our study, the antibody responses were evaluated in mice immunized with recombinant baculoviruses. Majority of mice immunized with two dosages of Bac-G-prM/E or Bac-prM/E developed neutralizing antibodies titers of >1:16 (Table 1). Furthermore, these baculovirus-derived vaccines can also induce anti-JEV cross-neutralizing antibodies although the titers of the antibodies were lower than antibodies against WNV. These results strongly demonstrate that the antibody response induced by WNV protein could partially cross-neutralize JEV, as has been reported in previous studies . It is reasonable to anticipate that immunization with these recombinant baculoviruses expressing WNV antigens will not only provide protection against WNV infection, but also provide partial protection against JEV infection. Cellular immunity such as IFN-γ production and inflammatory cytokines responses were also determined in mice after immunization with these baculoviruses since these cytokines play a critical role in directing the cell-mediated immune responses . Bac-G-prM/E- or Bac-prM/E-immunized mice produced higher levels of IFN-γ than any other group (Figure 4) and promoted the release of inflammatory cytokines, including tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and interleukin-2 (IL-2) (Figure 5). In this case, secretion of IFN-γ, TNF-α, or IL-2 are indicative of a Th1 response, whereas a Th2 response is characterized by induction of IL-6 . These indicate that the recombinant baculoviruses can stimulate both humoral and cellular immune responses which are essential to control dissemination of infection and to inhibit the entry of virus into brain.
Our results also demonstrate that Bac-G-prM/E exhibited better immunogenicity than Bac-prM/E, as indicated by the higher levels of antibody titers, IFN-γ and inflammatory cytokines production. It is highly probable that the expression of VSV/G was associated with enhancement of the immune responses by augmenting transduction efficiency of baculovirus into mammalian cells . In addition, the VSV/G-modified baculovirus exhibited greater resistance to animal serum inactivation than the unmodified baculovirus , which may contribute to the better immunogenicity of Bac-G-prM/E.
It has been reported that recombinant E-DIII protein induces high neutralizing antibody titers, as well as protection against lethal WNV and partial protection against lethal JEV . Therefore, the recombinant E-DIII protein of WNV expressed in E.coli was used as a positive control in the present study. However, intramuscular injection of mice with E-DIII protein elicited lower levels of neutralization antibody titers than mice immunized with the recombinant baculoviruses even at a low dosage (108 PFU/mouse) (Table 1), although the total IgG level was high (Figure 3). This could be due to differences in experimental designs such as the route of immunization, with or without adjuvant, and mouse strain. In addition, it has been shown that E-DIII protein immunization elicits low level of neutralizing antibodies with relatively high IgG responses , which is consistent with our results. It is noticed that E-DIII protein also induced lower levels of cellular immune response than recombinant baculoviruses, since E protein expressed by recombinant baculovirus contains more T cell epitopes than its domain III , and the baculovirus augment cellular immunity.
Baculovirus has been shown to possess a strong adjuvant activity and to promote humoral and cellular immune responses for foreign antigens, maturation of dendritic cells, and production of inflammatory cytokines and IFN . The transduction of macrophages in vitro by baculovirus led to the induction of significant levels of IL-6 and TNF-α . It has been proposed that baculovirus genome, especially its CpG motifs, could be recognized by macrophages and DCs. Furthermore, baculoviruses enter into the cells through mannose receptor (MR)-mediated endocytosis or phagocytosis, leading to the secretion of inflammatory cytokines through a MyD88/TLR9-dependent signaling pathway [23, 24]. However, Chen et al. reported that recombinant baculoviruses are able to induce a robust secretion of inflammatory cytokines through a TLR3-dependent pathway . In our study, induction of a measurable IFN-γ and inflammatory cytokines, including TNF-α, IL-6, and IL-2 by control baculovirus (Bac-G-EGFP and Bac-EGFP) was observed, demonstrating that this virus can function as a stimulator for production of cell-mediate immunity in an antigen- independent manner (Figure 4). Similar results have been reported previously that injection with control baculovirus is capable of inducing IFN-γ responses [26, 27, 29]. This is probably due to direct interaction of baculovirus with dendritic cells and macrophages in the spleen. Since it has been reported that splenic dendritic cells and macrophages play a central role in baculovirus-induced inflammatory responses in mice . These baculovirus- induced non-specific inflammatory responses raise concerns that whether they will compromise the use of baculovirus vectors for in vivo gene therapy in humans and animals. However, Bac-G-E2 inoculated mice released inflammatory cytokines as early as 6h post-injection, and returned to background levels by 48h post-injection . Furthermore, there was no cytokine-induced acute toxicity or mortality in baculovirus-immunized mice, which suggests that non-specific inflammatory responses induced by baculovirus vectors may be minimized by host. Nevertheless, more investigations are needed to ensure the safety of baculovirus vectors.
The protection is an important index to evaluate the efficiency of vaccine. However, the ability of our recombinant baculoviruses to protect against lethal challenge of WNV was not determined in consideration of the safety problem since WNV has not been found in China. Nevertheless, the neutralizing antibodies and the cellular immune responses induced by recombinant baculoviruses may provide the basis for protection. Immunization with baculovirus expressing JEV antigens have been shown to confer protection against JEV challenge, demonstrating that cellular immunity plays a crucial role against JEV infection . Thus it is well supported that recombinant baculoviruses expressing WNV prM/E proteins could provide protective immunity against WNV challenge.
Cells, plasmids and virus
Spodoptera frugiperda 9 (Sf9) cells were propagated at 28°C in Grace’s media (Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS, 100 μg/ml streptomycin and 100 IU/ml penicillin. BHK- 21 cells, used for transduction and neutralization tests, were grown and maintained in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% heated-inactivated fetal bovine serum (FBS), 100 μg/ml streptomycin and 100 IU/ml penicillin, at 37°C with 5% CO2.
The prM and E genes region (GenBank accession number DQ211652) corresponding to nt 466–2469, derived from a North American NY99 strain of WNV, were synthesized and cloned into pcDNA3.1 vector backbone, to generate plasmid pcDNA3.1-prM/E. The construction of baculovirus transfer vector, pFastBac-VSV/G, in which the VSV/G gene is under the control of the polyhedrin promoter of pFastBacTM1 (Invitrogen), was described previously . Bac-G-EGFP is a pseudotype baculovirus containing a CMV promoter that controls the expression of EGFP  and was used as a control strain of the baculovirus.
Protein and monoclonal antibodies
WNV envelope protein (E protein) domain III (the sequence corresponding to nt 1858–2211) was expressed in E.coli as an inclusion body, and then purified and refolded in appropriate buffer . Protein concentrations were determined at an absorbance of 280 nm by spectrophotometry. Anti-E-DIII monoclonal antibody (MAb) was produced from the mice immunized with E-DIII protein.
Construction of recombinant baculoviruses
To generate the recombinant transfer plasmid pFastBac-G-prM/E, the DNA fragment containing the prM/E expression cassette, which was controlled under the CMV promoter, was released from pcDNA3.1-prM/E and inserted into pFastBac-VSV/G. To generate the recombinant transfer plasmid pFastBac-prM/E the same prM/E expression cassette was digested and clone into pFastBac1 vector. The recombinant baculoviruses Bac-G-prM/E and Bac-prM/E were subsequently generated by using Bac-to-Bac system (Invitrogen) following the manufacturer’s instructions. The resultant viruses were further amplified by propagation in Sf9 cells and purified as described . The virus pellet was resuspended in phosphate-buffer saline (PBS, PH7.4) and infectious titers were determined by a plaque assay as described in the Bac-to-Bac system, and then stored in aliquots at −80°C until needed.
Detection of E protein in Baculovirus-transduced cells
The transduction procedure was performed as described previously with minor modifications [27, 29]. BHK-21 cells were seeded into six-well plates at concentration of 1.5 × 105 cells/well. At approximately 70-80% confluence, culture medium was removed and cells were washed three times with PBS and incubated with baculoviruses for 4h at 28°C. After removal of the inoculum, fresh medium was added and cultures incubated at 37°C for 48h. Duplicate wells were processed in parallel for indirect immunofluorescence assay and Western blotting. At 48h after transduction, the cells were fixed with absolute methanol and processed for the indirect immunofluorescence assay using monoclonal antibodies (MAb) against WNV E-DIII protein, followed by fluorescein isocyanate-conjugated goat anti-mouse immunoglobulin (Ig) G. For Western blot analysis, transduced cells were collected at 48h post-transduction and lysed in SDS sample buffer. Cell extracts were separated by sodium dodecyl sulfate −10% polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto a nitrocellulose membrane. The nonspecific antibody binding sites were blocked with 1% bovine serum albumin (BSA) in TBST buffer (10mM Tris–HCl PH 8.0, 150mM Nacl, and 0.05% Tween-20), and finally the membrane were reacted with anti-E-DIII Mab to detect the expressed E protein.
Female BALB/c mice (6–8 week old) were purchased from the Animal Centre, Institute of Medicine, Hubei province, China. Mouse studies were performed according to the guidelines of this institution (No. 00020502) and all experimental protocols were approved by the Research Ethics Committee of College of Veterinary Medicine, Huazhong Agricultural University, Hubei, China. Then the mice were randomly divided into eight groups (6 mice per group). Two groups were injected intramuscularly (i.m.) with 100μl of PBS containing 1 × 108 or 1 × 109 PFU of Bac-G-prM/E and another two groups were immunized with Bac-prM/E. The other three groups were injected intramuscularly with 100μl of PBS containing 1 × 109 PFU of Bac-G-EGFP, Bac-EGFP, and 100μl of PBS containing 50μg of E-DIII protein, respectively. The last group was used as a negative control by intramuscularly injecting 100μl of PBS. Booster immunizations were identically performed 3 weeks later. Serum samples were collected from the tail vein of 6 individual mice before the second injection and from the retro-orbital plexus at 6 weeks after primary immunization and then store at −20°C for serological tests. At 6 weeks after the primary immunization, three mice of each group were euthanized, and splenocytes were harvested for immunological assay.
Induction of E protein-specific antibody was determined by using indirect enzyme linked immunosorbent assay (ELISA). Microplates were coated with E-DIII protein (20ng/well) overnight at 4°C . After blocking for 1h at 37°C with PBST (2% bovine serum albumin, PBS, 0.05% Tween 20) to avoid nonspecific binding, serum diluted in PBST (1:500) were added to each well and incubated at 37°C for 1h. After washing with PBST, bounded proteins were detected with HRP conjugated with goat anti-mouse IgG (Boster, China). Serum antibody titer was monitored versus optical density values. The endpoint titers of the neutralizing antibodies against WNV and JEV were determined by plaque-reduction neutralization tests (PRNTs) using WNV strain NY99 and JEV strain P3, in biosafety level–3 conditions,as described earlier . Briefly, serum samples were heat-inactivated for 45min at 56°C and then serially diluted and mixed with an equal volume containing 100 PFU of virus. The mixtures were incubated for 1h at 37°C and then inoculated onto BHK-21 cell monolayers in 12-well plates. After adsorption for 1h at 37°C, the wells were overlaid with medium containing 1.5% carboxymethyl cellulose, and the plates were incubated in a 5% CO2 incubator at 37°C for 3–4 days for plaque formation, then fixed with 10% formalin in PBS. Plates were washed and stained with 0.01% crystal violet. The percent plaque reduction was calculated relative to virus controls without serum. The PRNT titers were given as the reciprocal of the highest serum dilutions which resulted in > 50% reduction of the plaques.
Mouse splenocytes were prepared from immunized mice and resuspended in RPMI 1640 (Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS, 2mM L-glutamine, 100μg/ml streptomycin and 100 IU/ml penicillin. Splenocytes (1 × 106/ml) were cultured in 24-well plates at 37°C in the presence of 20μg/ml WNV E-DIII or JEV NS3 protein. After 72h incubation, culture supernatant was harvested and the presence of IFN-γ was measured with commercial mouse IFN-γ immunoassay ELISA kits (eBioscience Inc., San Diego, CA) according to the manufacture’s guidelines. The concentrations of IFN-γ in the samples were calculated from the standard curves.
Analysis of cytokine mRNA expression by Real-time PCR
Relative quantitative real-time PCR primers for targeted transcripts mRNAs
Primer sequence (5’to 3’)
50°C 2min; 94°C 10min 40 cycles (94°C 15s then 60°C 1min)
Where specified, an unpaired t-test was used to compare the humoral and cellular immune responses between the different groups. All comparisons were made using two-tailed and P-values of 0.05 or less were considered statistically significant.
This research work was supported by 863 (Grant No: 2011AA10A2) and Special Fund for Agro-scientific Research in the Public Interest (200903037). We deeply appreciate Keqian Bio Inc. (Wuhan, China) for providing experimental assistance.
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