Abstract
Although Escherichia coli and yeast were commonly used to express recombinant Hc of botulinum neurotoxins, as an alternative, in current study, a 293E expression system was used to express the Hc of botulinum neurotoxin serotype B (BHc) as soluble recombinant protein for experimental vaccine evaluation. Our results demonstrated that the 293E expression system could produce high level of recombinant secreted BHc protein, which was immunorecognized specifically by anti-botulinum neurotoxin serotype B (BoNT/B) sera and showed ganglioside binding activities. The serological response and efficacy of recombinant BHc formulated with aluminum hydroxide adjuvant were evaluated in mice. Immunization with Alhydrogel-formulated BHc subunit vaccine afforded the effective protection against BoNT/B challenge. A frequency- and dose-dependent effect to immunization with BHc subunit vaccine was observed and the ELISA antibody titers correlated well with neutralizing antibody titers and protection. And a solid-phase assay showed that the neutralizing antibodies from the BHc-immunized mice inhibited the binding of BHc to the ganglioside GT1b. Our results also show that the plasmid pABE293SBHc derived of the 293E expression system as DNA vaccine is capable of inducing stronger humoral response and protective efficacy against BoNT/B than the pVAX1SBHc. In summary, immunization with the 293E-expressed BHc protein generates effective immune protection against BoNT/B as E. coli or yeast-expressed BHc, so the efficient expression of botulinum Hc protein for experimental vaccine can be prepared using the 293E expression system.
Introduction
The botulinum neurotoxins (BoNTs) produced by bacteria of the genus Clostridium are the most toxic proteins and can be classed into seven serotypes (A-G). BoNT serotypes A, B, E and F can cause disease in human.Citation1-3 BoNTs are synthesized as single-chain polypeptides of ∼150 kDa composed of three domains, each of approximately 50 kDa, e.g., the N-terminal catalytic domain (light chain), the internal heavy chain translocation domain (Hn domain) and the C-terminal heavy chain receptor-binding domain (Hc domain). The Hc domain, which alone is nontoxic, mediates the binding to target neurons and has demonstrated the ability to elicit protective immune responses in animals challenged with homologous botulinum neurotoxin.Citation3-5 The Hc domains of BoNTs produced in E. coli and P. pastoris have been shown to elicit protective immune responses in mice and other animals and demonstrated the feasibility of this strategy for the development of the next generation of vaccines against botulism.Citation3,Citation5-7
As an alternative, the transient transfection of mammalian cells grown in monolayers can generate significant amounts of recombinant active proteins. The FreeStyleTM 293 Expression System (Invitrogen, CA) is designed to allow transfection of suspension 293E cells in a defined, serum-free medium and produce high level of recombinant secreted protein in the supernatants.Citation8 Therefore, in the present study we tested the feasibility of designing a second generation of botulinum neurotoxin vaccine based on recombinant Hc domain expressed in a scalable FreeStyleTM 293 Expression System. Indeed, high level of recombinant secreted BHc protein was expressed by transient transfection of suspension-growing human 293E cells with the pABE293 vector containing the BHc gene. The 293E-expressed active BHc protein was immunorecognized specifically by anti-BoNT/B sera, and mice immunized with the recombinant BHc subunit vaccine were protected from a high dose of BoNT/B challenge. Finally, the plasmid pABE293SBHc derived of the 293E expression system as DNA vaccine induced stronger humoral response and protective efficacy against BoNT/B than the pVAX1SBHc.
Results
Purification and analysis of recombinant BHc protein expressed in 293E cells
High level of recombinant protein was produced by transient transfection of suspension-growing human 293E cells with the pABE293 expression vector containing foreign gene.Citation8,9 To express recombinant BHc protein in 293E cells, a plasmid expression vector pABE293SBHc containing the BHc gene was constructed in this study. The plasmid was transfected to suspension 293E cells for instantaneous expression. Secret BHc protein in supernatants was purified and the recombinant BHc was confirmed by both SDS-PAGE and reaction with specific antibodies against BoNT/B in immunoblot (). Expression of the secreted BHc protein was also considerable, as it was produced at levels exceeding 10 mg purified recombinant BHc per liter of culture.
Figure 1. Analysis of purified recombinant BHc protein by SDS-PAGE (A) and immunoblot (B). Lane 1, the protein standards; lane 2, 1 μg of recombinant BHc expressed and purified in one experiment; lanes 3 and 4, 2 μg of recombinant BHc expressed and purified in another experiment. Arrows indicate the position of the recombinant BHc protein.
The ganglioside is regarded a component of the double-receptor system of botulinum neurotoxins.Citation10-12 Therefore, the BHc protein binding with the ganglioside (GT1b) was performed to assess if the recombinant 293E-expressed BHc protein had the GT1b binding capacity. The recognition of ganglioside by the purified BHc in ganglioside binding assays () indicates that the recombinant BHc protein can well bind to GT1b and has a functionally active conformation. In addition, the quantitative ganglioside binding assays show concentration-dependent binding responses between recombinant BHc protein and GT1b.
Figure 2. Enzyme-linked immunosorbent assay of binding activity of the recombinant BHc protein to ganglioside (GT1b). Wells were coated with different concentration of ganglioside (GT1b), and incubated with 50 μg/mL recombinant BHc protein or BSA. The same amount of BSA was coated on the plate as negative control. Values represent means from 4 separate experiments with bars representing standard mean of deviations.
Efficacy of BHc subunit vaccine against BoNT/B in mice
The immunogenicity of recombinant BHc subunit vaccine was evaluated in mice. As shown in , a dose-dependent immune response to the BHc antigen formulated with aluminum hydroxide adjuvant was observed and the anti-BHc ELISA antibody titers correlated well with protective potency against BoNT/B. The geometric mean titer (GMT) of mice with single BHc vaccination was relative low (2.67–2.90) dependence upon the injection doses and these immunized mice were partially protected against BoNT/B. The once or twice boost immunizations obviously increased the group GMT (≥ 3.51) and produced 100% survival against a challenge with a 1000 50% mouse lethal dose (LD50) of active BoNT/B. The above survival mice were rechallenged with 10,000 LD50 of BoNT/B a week later and we observed no mice deaths in the BHc-immunized groups except for two vaccinations with 1 μg.
Table 1. Survival, sera antibody titers, and neutralizing titers of mice following immunization with 1 or 10 μg recombinant BHc formulated with aluminum hydroxide adjuvant
Additionally, the neutralizing antibody titers augmented with the frequency and dose of immunizations and associated well with the anti-BHc ELISA antibody titers and protection. And a solid-phase ganglioside binding assay was performed, which allowed the assessment of the ability of anti-BHc antibodies to block BHc binding to ganglioside GT1b. The sera of negative control group did not interfere with the binding of BHc to GT1b, while the sera antibodies from BHc-immunized mice showed dose-dependent inhibition of BHc binding to GT1b (), the first step in BoNT/B intoxication of neurons.
Figure 3. Immune sera antibodies blocked BHc binding to ganglioside. The sera from mice immunized with three doses of 1 or 10 μg recombinant BHc formulated with aluminum hydroxide adjuvant were used to block BHc binding to GT1b. Sera from mice injected with PBS and Alhydrogel were used as negative control. Values represent means from 4 separate sera samples of mice with bars representing standard mean of deviations.
To further confirm the potency of the BHc subunit vaccine, another efficacy study against BoNT/B was performed in mice. Mice were immunized intramuscularly (i.m.) once, twice or three times with four various doses of recombinant BHc formulated with aluminum hydroxide adjuvant and challenged with various doses of BoNT/B. A BHc antigen frequency- and dose-dependent protective potency against BoNT/B was observed in the immunized mice (). Immunizations of two injections of ≥ 0.2 μg or three injections of ≥ 0.04 μg BHc antigen in mice afforded completely protective potency (100% survival) against a challenge with 1000 LD50 of BoNT/B. Immunizations of two injections of ≥ 5 μg or three injections of ≥ 0.2 μg BHc antigen in mice afforded completely protective potency against a challenge with 10,000 LD50 of BoNT/B.
Table 2. Survival of mice following vaccination once, twice or three times with different doses of recombinant BHc formulated with aluminum hydroxide adjuvant
In sum, the recombinant BHc is proved to bind with ganglioside and the Alhydrogel-formulated BHc subunit vaccine affords effective immune protection against BoNT/B, indicating that the 293E-expressed and purified BHc protein has a functionally active conformation and can be used as a human subunit candidate vaccine against BoNT/B.
Effect of plasmid pABE293SBHc as DNA vaccine against BoNT/B
To determine effect of the plasmid pABE293SBHc as DNA vaccine against BoNT/B, mice were vaccinated i.m. with pABE293SBHc or pVAX1SBHcCitation13 and challenged with various doses of BoNT/B. The pABE293SBHc vaccination afforded effective protection against BoNT/B, while the pVAX1SBHc vaccination only afforded partial protection against 100 LD50 of BoNT/B, and no protection against 1000 LD50 of BoNT/B (). The anti-BHc GMT in the pABE293SBHc-immunized mice was significantly higher than that of the pVAX1SBHc- immunized mice (P < 0.01). Our data indicate that the plasmid pABE293SBHc as BHc-expressing DNA vaccine elicits stronger antibody response and protective potency against BoNT/B than the pVAX1SBHc.
Table 3. Survival and sera antibody titers following vaccination i.m. with pABE293SBHc or pVAX1SBHc
Discussion
BoNTs, which can cause human botulism, have been described as biological weapons for malicious applications.Citation4 Botulism can be effectively prevented by vaccination, which induces specific neutralizing antibodies against BoNTs. The Hc domain of BoNTs was found to carry most of the neutralizing epitopesCitation14,15 and is the leading candidate for recombinant botulinum vaccine preparation.Citation7,16 These findings give rise to interest toward preparing a safe and effective second generation of botulinum subunit vaccine, which replaces the formalin-inactivated toxoid with the Hc domain.Citation4,5,7 Extensive studies aimed to produce recombinant Hc of BoNTs as candidate vaccine are performed. The expression and purification of these proteins using E. coliCitation17-21 and yeastCitation22-25 are the systems used most widely. As an alternative, the Hc domain of BoNT/A as a subunit vaccine antigen was expressed using a bi-cistronic baculovirus-Sf21 insect cell expression system.Citation26
In current study, a 293E expression system was used to express the Hc of BoNT/B as soluble recombinant protein for experimental vaccine evaluation. High level of recombinant secreted BHc protein produced by the 293E expression system was immunorecognized specifically by anti-BoNT/B sera. The recognition of ganglioside by a 293E-expressed BHc indicates that the recombinant BHc protein has a functionally active conformation as active neurotoxins or recombinant E. coli and yeast-expressed BHc.Citation11,12,19,27,28 Thus, our study shows that an immunogenically active BHc protein can be expressed using the 293E expression system.
Then, we evaluated its immunity and protective capacity against BoNT/B challenge in mice. The immunization with recombinant BHc antigen formulated with aluminum hydroxide adjuvant afforded the effective protection against neurotoxin challenge in Balb/c mice. A frequency- and dose-dependent effect to immunization with the BHc subunit vaccine was observed and the ELISA antibody titers correlated well with neutralizing antibody titers and protection. Multiple immunizations of the BHc subunit vaccine provided better protection than a single immunization. The efficacy study further resolved the question of how well the 293E-expressed BHc protein protected mice against a high-dose BoNT/B challenge.
The high toxic potency of BoNTs is mainly due to their neuro-specific binding which is mediated by the interaction with two receptor components. Ganglioside is a component of the double-receptor system of BoNTs.Citation10-12 The first step in BoNT/B intoxication of neurons starts with binding of C-terminus subdomain of Hc to ganglioside on presynaptic membranes.Citation29,30 To further elucidate the mechanism of BHc-immunized sera inhibition, a solid-phase ganglioside binding assay was performed. Our results show that the anti-BHc antibodies effectively block BHc binding to ganglioside, which indicates that the immune sera neutralize or inhibit the first step in BoNT/B intoxication of neurons, i.e., the neutralizing sera block the interaction of BoNT/B or BHc with its cognate ganglioside receptor.
In this study, we also reported on the effect of the plasmid pABE293SBHc expression vector from the 293E expression system as DNA vaccine against BoNT/B. Our results show that the plasmid pABE293SBHc as DNA vaccine is capable of inducing stronger antibody responses and protective potency against BoNT/B than the pVAX1SBHc, which further indicates that the expression vector derived of the 293E expression system is effective to express foreign proteins in vitro and in vivo. Thus, our study provided supportive evidence for easy expression of recombinant BHc protein as vaccine antigen using a 293E expression system.
Although the bacterium–E. coli and the yeast– P. pastoris are commonly used to express recombinant Hc protein of BoNTs, our results demonstrate that the 293E cells can also effectively produce an immunogenically active BHc protein. Immunization with the 293E-expressed BHc protein formulated with aluminum hydroxide adjuvant generates effective immune protection against BoNT/B as E. coli or yeast-expressed BHc,Citation18,19,23,24 which makes it able to be prepared into an effective human subunit vaccine candidate against BoNT/B. In summary, our study demonstrates the proof of concept for efficient expression of botulinum Hc protein for experimental vaccine preparation using a 293E expression system.
Previously, the recombinant BHc was expressed in E. coli, but it was insoluble in the form of inclusion bodies as previous reportedCitation27,31 and had no biological activity in our laboratory. Meanwhile, we also demonstrated that secretion of BHc protein from yeast resulted in the unwanted glycosylation of expressed product and the glycosylated BHc protein failed to elicit protective immunity in mice as previous reported.Citation24,32 Therefore, as an alternative to solve above problem, a 293E expression system was used to express the Hc of BoNT/B. This is the first report on manufacturing a recombinant active fragment of BoNTs using mammalian cells.
Even though a mammalian cell expression system for expressing certain protein is more expensive, time-consuming and needs more experienced techniques of cell culture, the 293E cells can still be considered as an alternative host for recombinant bacterial protein or vaccine production because some soluble and active proteins can be effectively expressed in them, but not expressed in E. coli or yeast. Moreover, in contrast to lower eukaryotes (i.e., yeast) or prokaryotes (i.e., E. coli), mammalian cells provide active recombinant proteins with functional conformations that possess relevant post-translational medications.
Materials and Methods
Construction of the plasmid vector
To express recombinant BHc protein in 293E cells, a plasmid expression vector pABE293SBHc was constructed in this study. Briefly, the BHc gene was amplified using PCR from pGEM-BHc (containing a completely synthetic gene encoding the Hc domain of BoNT/B, amino acids 853 through 1291, ∼50 kDa)Citation33 with primer pairs CATGGAATTC GCCGCCACCC ATGGAGACAG ACACACACTC C and CATGGGATCC TTAGTGGTGG TGGTGGTGGT GGGAACCCTC GGTCCAACCC TCGTCTTTT. The PCR products were digested with EcoR I and BamH I to excise the BHc DNA fragment and subcloned into the designed pABE293 expression vector having an improved cytomegalovirus expression cassette, which derived of plasmid H293.Citation8,9 The resulting recombinant plasmid pABE293SBHc contains the BHc gene fused with an Ig κ signal peptide sequence in the N-terminus and an His-tag in the C-terminus. The plasmid pABE293SBHc was prepared and purified using Endofree Mega-Q kits (QIAGEN GmbH, Hilden, Germany) for transfection and immunization.
Purification and analysis of recombinant protein expressed in 293E cells
Small-scale transient transfection of suspension 293E cells with the plasmid pABE293SBHc was performed in a defined serum-free medium according to the standard procedures (Invitrogen, CA). Supernatants containing secreted protein BHc were collected and purified by nickel affinity column chromatography (GE Healthcare, Piscataway, NJ) for the six His-tag fusion protein according to the recommendation of the manufacturer. A His-tag was fused to the C-terminus of recombinant BHc. The purified protein was verified by 12% SDS-PAGE and immunoblot using hyperimmune horse BoNT/B antiserum.
Vaccinations and challenge
Specific pathogen-free female Balb/c mice 6 wk (purchased from Beijing Laboratory Animal Center, Beijing) were randomly assigned to different treatment groups (8 mice in each group). For preparation of recombinant BHc subunit vaccine, the different doses of recombinant BHc antigen in PBS were formulated with 0.33% (w/w) alhydrogel (Aluminum hydroxide Gel, 1.3%, Sigma, St. Louis, MO) and injections were administered at 2 wk intervals (100 μl/injection). In the first vaccination study, mice were i.m. vaccinated once, twice or three times with 1 or 10 μg recombinant BHc antigen per injection, respectively. In the second vaccination study, mice were i.m. vaccinated either one (1 ×), two (2 ×), or three (3 ×) doses of 0.04, 0.2, 1 or 5 μg BHc antigen per injection, respectively. For DNA immunization, mice were i.m. (bilaterally in the quadriceps) injected with 30 μg of plasmid pABE293SBHc or pVAX1SBHc in a total volume of 0.1 ml three times with 2-wk intervals between each injection. Mice from all groups were challenged i.p. with different doses of pure BoNT/B diluted in 20 mM sodium phosphate buffer (pH 6.5) 3 wk after the last vaccination. The mice were observed for 1 wk after challenge, and survival was determined for each vaccination group. The animal protocols in this study were approved by Institution Animal Care and Use Committee of our Institution.
Antibody titer measurement and BoNT/B neutralization assay
Sera from mice in the different treatment groups were screened for anti-BHc antibodies by ELISA. Briefly, ELISA plates (Corning Inc., Corning NY) were coated overnight at 4°C with 100 μl recombinant BHc (2 μg/ml). Serum samples were serially diluted at 1:2 increments beginning at 1: 100 and 100 μl was added to each well for 1 h at 37C°. After washing, 100 μl of 1: 2000 dilution of goat anti-mouse IgG-HRP (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was added for 30 min at 37°C. After washing, anti-BHc reactivity was visualized by adding 100 μl of citrate buffer (pH 5.0) containing 0.04% (w/v) of o–phenylenediamine and 0.02% (v/v) hydrogen peroxide for 5 min at 37°C. The reaction was stopped with 50 μl of 2 M H2SO4 and the absorbance was read at 492 nm using a Thermo Labsystems (Frannklin, MA) microplate reader. Antibody titers were estimated as the reciprocal of the maximum dilution of serum giving an absorbance reading greater than 0.3 units following subtraction of non-specific binding detected in control sera and 2-fold greater than that of the matched dilution of control sera. Serum samples from individual mice were assayed and their geometric mean titer (GMT) was used to calculate the log10 GMT for each group. Standard deviations (SD) of the GMT are in parenthesis.
Neutralization potency of above sera was assayed by using BoNT/B neutralization assay as described previously.Citation13,34 Briefly, mixtures of serial dilutions of mice pooled sera from mice in the different treatment groups with a standard concentration of BoNT/B were incubated 0.5 h at room temperature and the mixtures were injected i.p. into mice (18–22 g) using a volume of 500 μl /mouse (four mice in each group). The mice were observed for one week, and death or not was recorded. The concentration of neutralizing antibody in the sera was calculated relative to a World Health Organization BoNT/B antitoxin and neutralizing antibody titers of sera were reported as international units per milliliter (IU/ml).
Ganglioside binding
Ganglioside binding assays were performed as described previously.Citation35 Briefly, ELISA plates were coated with 100 μl of 40 μg/ml bovine ganglioside GT1b (Sigma) per well and blocked with PBS containing 1% Casein (Sigma) at 25°C for 3 h. After incubation, 100 μl of purified BHc (50 μg /mL) or BSA (50 μg/mL) was added to different wells for 3 h at 25°C. Unbound BHc protein was removed by three washes each with PBS containing 0.05% Tween-20 (PBST). Plates were incubated for 8 h at 4°C with 100 μl of 1:2,000 dilutions of polyclonal mouse anti-BHc antibodies, washed, and incubated for 0.5 h with goat anti-mouse IgG-HRP. The amount of bound BHc protein was detected by the polyclonal mouse anti-BHc antibodies. Detection was at 492 nm by adding 100 μl of citrate buffer (pH 5.0) containing 0.04% (w/v) of o–phenylenediamine and 0.02% (v/v) hydrogen peroxide for 5 min at 37°C. A quantitative binding analysis was performed using the same procedure with different concentrations—160, 40, 10, 2.5, 0.625, 0.156, 0.039 and 0 μg/ml of GT1b.
To observe the ability of anti-BHc antibodies to block BHc binding to ganglioside GT1b, sera from mice immunized with three doses of 1 or 10 μg BHc were incubated with the BHc protein in above ganglioside binding assays. In brief, ganglioside (4 μg /well) was immobilized on ELISA plates. After BHc (2 μg /well) had been pre-incubated for 2 h at 25°C with either different dosages of sera from mice in the negative control group or sera from BHc- immunized mice range from 50 μl to 0.4 μl/well, they were added to different wells for 3 h at 25°C as above to continue performing ganglioside binding assays. 1:2,000 dilutions of polyclonal mouse anti-BHc antibodies were used as primary antibody for the experiment.
Statistical analysis
Differences in antibody titers were analyzed statistically using the Student's t test between group differences. Fisher's exact test was used to determine statistical differences in survival between the treatment groups. For all tests only data resulting in P values < 0.05 were regarded as statistically significant.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
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