Human Vaccines & Immunotherapeutics
ISSN: 2164-5515 (Print) 2164-554X (Online) Journal homepage: http://www.tandfonline.com/loi/khvi20
Production and evaluation of a recombinant subunit vaccine against botulinum neurotoxin serotype B using a 293E expression system YunZhou Yu, DanYang Shi, Si Liu, Zheng-Wei Gong, Shuang Wang & ZhiWei Sun To cite this article: YunZhou Yu, DanYang Shi, Si Liu, Zheng-Wei Gong, Shuang Wang & ZhiWei Sun (2015) Production and evaluation of a recombinant subunit vaccine against botulinum neurotoxin serotype B using a 293E expression system, Human Vaccines & Immunotherapeutics, 11:2, 468-473, DOI: 10.4161/hv.29714 To link to this article: http://dx.doi.org/10.4161/hv.29714
Accepted online: 01 Nov 2014.
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Date: 17 September 2015, At: 09:42
RESEARCH PAPER Human Vaccines & Immunotherapeutics 11:2, 468--473; February 2015; © 2015 Taylor & Francis Group, LLC
Production and evaluation of a recombinant subunit vaccine against botulinum neurotoxin serotype B using a 293E expression system YunZhou Yu*, DanYang Shi, Si Liu, Zheng-Wei Gong, Shuang Wang, and ZhiWei Sun* Beijing Institute of Biotechnology; Beijing, PR China
Downloaded by [University of Manitoba Libraries] at 09:42 17 September 2015
Keywords: Botulinum neurotoxin serotype B, Hc, expression, vaccine, binding activity
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.1-3 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.3-5 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.3,5-7 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.8 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
*Correspondence to: YunZhou Yu; Email: [email protected]; ZhiWei Sun; Email: [email protected] Submitted: 04/28/2014; Revised: 06/10/2014; Accepted: 06/24/2014 http://dx.doi.org/10.4161/21645515.2014.974991
468
Human Vaccines & Immunotherapeutics
Volume 11 Issue 2
Downloaded by [University of Manitoba Libraries] at 09:42 17 September 2015
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 1. Analysis of purified recombinant BHc protein by SDS-PAGE (A) and immunoblot (B). Lane 1, the protein standards; lane 2, 1 mg of recombinant BHc expressed and purified in one experiment; lanes 3 and 4, 2 mg of recombinant BHc expressed and purified in another experiment. Arrows indicate the position of the recombinant BHc protein.
pABE293 expression vector containing foreign gene.8,9 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 (Fig. 1). 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. The ganglioside is regarded a component of the double-receptor system of botulinum neurotoxins.10-12 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 (Fig. 2) indicates that
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 mg/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.
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Efficacy of BHc subunit vaccine against BoNT/B in mice The immunogenicity of recombinant BHc subunit vaccine was evaluated in mice. As shown in Table 1, 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 mg. 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 (Fig. 3), the first step in BoNT/B intoxication of neurons. 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 (Table 2). Immunizations of two injections of 0.2 mg or three injections of 0.04 mg 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 mg or three injections of 0.2 mg BHc antigen in mice afforded completely protective potency against a challenge with 10,000 LD50 of BoNT/B. 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
Human Vaccines & Immunotherapeutics
469
Table 1. Survival, sera antibody titers, and neutralizing titers of mice following immunization with 1 or 10 mg recombinant BHc formulated with aluminum hydroxide adjuvant Number Alivea
Number Aliveb
Log10 GMT(SD)C
Sera neutralizing titer (IU/ml)d
Vaccination
1e
2e
3e
2e
3e
1e
2e
3e
2e
3e
1 mg 10 mg Control
0 4 0
8 8 0
8 8 0
3 8 ND
8 8 ND
2.67(0.27) 2.90(0.32)
ISSN: 2164-5515 (Print) 2164-554X (Online) Journal homepage: http://www.tandfonline.com/loi/khvi20
Production and evaluation of a recombinant subunit vaccine against botulinum neurotoxin serotype B using a 293E expression system YunZhou Yu, DanYang Shi, Si Liu, Zheng-Wei Gong, Shuang Wang & ZhiWei Sun To cite this article: YunZhou Yu, DanYang Shi, Si Liu, Zheng-Wei Gong, Shuang Wang & ZhiWei Sun (2015) Production and evaluation of a recombinant subunit vaccine against botulinum neurotoxin serotype B using a 293E expression system, Human Vaccines & Immunotherapeutics, 11:2, 468-473, DOI: 10.4161/hv.29714 To link to this article: http://dx.doi.org/10.4161/hv.29714
Accepted online: 01 Nov 2014.
Submit your article to this journal
Article views: 30
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=khvi20 Download by: [University of Manitoba Libraries]
Date: 17 September 2015, At: 09:42
RESEARCH PAPER Human Vaccines & Immunotherapeutics 11:2, 468--473; February 2015; © 2015 Taylor & Francis Group, LLC
Production and evaluation of a recombinant subunit vaccine against botulinum neurotoxin serotype B using a 293E expression system YunZhou Yu*, DanYang Shi, Si Liu, Zheng-Wei Gong, Shuang Wang, and ZhiWei Sun* Beijing Institute of Biotechnology; Beijing, PR China
Downloaded by [University of Manitoba Libraries] at 09:42 17 September 2015
Keywords: Botulinum neurotoxin serotype B, Hc, expression, vaccine, binding activity
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.1-3 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.3-5 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.3,5-7 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.8 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
*Correspondence to: YunZhou Yu; Email: [email protected]; ZhiWei Sun; Email: [email protected] Submitted: 04/28/2014; Revised: 06/10/2014; Accepted: 06/24/2014 http://dx.doi.org/10.4161/21645515.2014.974991
468
Human Vaccines & Immunotherapeutics
Volume 11 Issue 2
Downloaded by [University of Manitoba Libraries] at 09:42 17 September 2015
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 1. Analysis of purified recombinant BHc protein by SDS-PAGE (A) and immunoblot (B). Lane 1, the protein standards; lane 2, 1 mg of recombinant BHc expressed and purified in one experiment; lanes 3 and 4, 2 mg of recombinant BHc expressed and purified in another experiment. Arrows indicate the position of the recombinant BHc protein.
pABE293 expression vector containing foreign gene.8,9 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 (Fig. 1). 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. The ganglioside is regarded a component of the double-receptor system of botulinum neurotoxins.10-12 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 (Fig. 2) indicates that
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 mg/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.
www.tandfonline.com
Efficacy of BHc subunit vaccine against BoNT/B in mice The immunogenicity of recombinant BHc subunit vaccine was evaluated in mice. As shown in Table 1, 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 mg. 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 (Fig. 3), the first step in BoNT/B intoxication of neurons. 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 (Table 2). Immunizations of two injections of 0.2 mg or three injections of 0.04 mg 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 mg or three injections of 0.2 mg BHc antigen in mice afforded completely protective potency against a challenge with 10,000 LD50 of BoNT/B. 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
Human Vaccines & Immunotherapeutics
469
Table 1. Survival, sera antibody titers, and neutralizing titers of mice following immunization with 1 or 10 mg recombinant BHc formulated with aluminum hydroxide adjuvant Number Alivea
Number Aliveb
Log10 GMT(SD)C
Sera neutralizing titer (IU/ml)d
Vaccination
1e
2e
3e
2e
3e
1e
2e
3e
2e
3e
1 mg 10 mg Control
0 4 0
8 8 0
8 8 0
3 8 ND
8 8 ND
2.67(0.27) 2.90(0.32)
