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Potent tetravalent replicon vaccines against botulinum neurotoxins using DNA-based Semliki Forest virus replicon vectors
Vaccine 31 (2013) 2427–2432
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Potent tetravalent replicon vaccines against botulinum neurotoxins using DNA-based Semliki Forest virus replicon vectors Yun-Zhou Yu a,∗,1 , Jin-Peng Guo b,1 , Huai-Jie An c,1 , Shu-Ming Zhang d,∗ , Shuang Wang a , Wei-Yuan Yu a , Zhi-Wei Sun a,∗ a
Beijing Institute of Biotechnology, Beijing, China Institute for Disease Prevention and Control, Academy of Military Medical Sciences, Beijing, China c Wound Care & Research Center of Sea Battle of PLA China, Navy General Hospital, Beijing, China d National Institutes of Food and Drug Control, Beijing, China b
a r t i c l e
i n f o
Article history: Received 21 August 2012 Received in revised form 7 February 2013 Accepted 28 March 2013 Available online 10 April 2013 Keywords: Botulinum neurotoxin Tetravalent vaccine Replicon vector Dual-expression
a b s t r a c t Human botulism is commonly associated with botulinum neurotoxin (BoNT) serotypes A, B, E and F. This suggests that the greatest need is for a tetravalent vaccine that provides protection against all four of these serotypes. In current study, we investigated the feasibility of generating several tetravalent vaccines that protected mice against the four serotypes. Firstly, monovalent replicon vaccine against BoNT induced better antibody response and protection than that of corresponding conventional DNA vaccine. Secondly, dual-expression DNA replicon pSCARSE/FHc or replicon particle VRP-E/FHc vaccine was well resistant to the challenge of BoNT/E and BoNT/F mixture as a combination vaccine composed of two monovalent replicon vaccines. Finally, the dual-expression DNA replicon or replicon particle tetravalent vaccine could simultaneously and effectively neutralize and protect the four BoNT serotypes. Protection correlated directly with serum ELISA titers and neutralization antibody levels to BoNTs. Therefore, replicon-based DNA or particle might be effective vector to develop BoNT vaccines, which might be more desirable for use in clinical application than the conventional DNA vaccines. Our studies demonstrate the utility of combining dual-expression DNA replicon or replicon particle vaccines into multi-agent formulations as potent tetravalent vaccines for eliciting protective responses to four serotypes of BoNTs. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction The botulinum neurotoxins (BoNTs) are the most toxic proteins and can be classed into seven serotypes (A–G) which own similar structures but are antigenically distinct. Human botulism illness generally occurs in three forms: food-borne botulism, infant botulism, and wound botulism [1,2]. Clostridium botulinum neurotoxin serotypes A, B, E and F can cause disease in human, as opposite to serotypes C and D that can cause disease in cattle and horses [3,4]. Each BoNT contains three functional domains e.g., the N-terminal catalytic domain (light chain), the internal heavy chain translocation domain (Hn domain, 50 kDa) and the C-terminal heavy chain receptor-binding domain (Hc domain, 50 kDa). Botulism can
∗ Corresponding authors at: Beijing Institute of Biotechnology, 20 Dongdajie Street, Fengtai District, Beijing 100071, China. Tel.: +86 10 63855329; fax: +86 10 63833521. E-mail addresses: [email protected] (Y.-Z. Yu), [email protected] (S.-M. Zhang), [email protected] (Z.-W. Sun). 1 These authors contributed equally to this work. 0264-410X/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.vaccine.2013.03.046
be effectively prevented by the presence of neutralizing antibodies induced by vaccination. The formalin-inactivated pentavalent vaccine (PBT) commonly used in human is protective against serotypes A–E. However, it has met with some drawbacks, including feasibility, dangers associated with handling neurotoxins, and secondary complications due to residual formaldehyde contamination [5–8]. Therefore, the improved vaccines designed to overcome above disadvantages are needed. A novel vaccine strategy for the prevention of botulism based on the production of nontoxic recombinant BoNT proteins has been developed [5,6]. The Hc domains of BoNTs produced in Escherichia coli and Pichia pastoris were shown to elicit protective immune responses in mice and other animals [9–22]. DNA vaccines encoding the Hc domains of serotypes A and F were described as the next generation of botulinum vaccines [23–26]. Candidate vaccines against BoNT serotypes A and C were also developed by using a Venezuelan equine encephalitis (VEE) virus replicon vector [27,28]. Further, individual and bivalent candidate replicon vaccines against BoNT serotypes A and B were developed using DNA-based Semliki Forest virus (SFV) vectors by us in recent studies [29,30]. In this current study, we developed several tetravalent replicon vaccines for BoNT serotypes A, B, E, and
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F using the DNA-based SFV replicon vectors, and evaluated their immunogenicity and protective capability against challenge with the BoNTs mixture in mice. 2. Materials and methods 2.1. Construction of DNA replicon vaccines Two plasmid DNA replicon vaccines (pSCARSEHc and pSCARSFHc) and a dual-expression plasmid DNA replicon vaccine (pSCARSE/FHc) encoding the Hc domain of BoNT/E (EHc) and BoNT/F (FHc) were constructed in this study. Briefly, the AHc of pSCARSAHc was replaced with EHc (one synthetic gene encoding the Hc domain of BoNT/E, strain NCTC 11219, amino acids 841 to 1252), which has been submitted to Genbank under the accession number HM231315) or FHc (EU386771, one synthetic FHc gene encoding the Hc domain of BoNT/F) [16] by using the Nde I and Sma I cloning sites, resulting in pSCARSEHc or pSCARSFHc (Fig. 1C and D), respectively, and the AHc and BHc of pSCARSA/BHc was replaced with EHc and FHc resulting in pSCARSE/FHc (Fig. 1F) as described previously [30]. To generate two corresponding conventional DNA vaccines encoding the EHc or FHc, the DNA fragments were isolated by digesting pSCARSEHc or pSCARSFHc with BamH I and Spe I, and then inserting them into the vector pVAX1 to create pVAX1SEHc or pVAX1SFHc (Fig. 1G and H), respectively. All plasmids were prepared and purified using Endofree Mega-Q kits (QIAGEN GmbH, Hilden, Germany) for transfection and immunization. BHK-21 cells were transfected with pSCARSEHc, pSCARSFHc, pSCARSE/FHc, pVAX1SEHc or pVAX1SFHc, and the expression of EHc or FHc was analyzed as described previously [29,30].
approved by Institution Animal Care and Use Committee of our Institution. 2.4. Antibody titer measurements and BoNTs neutralization assays Sera from mice in different treatment groups were screened for anti-AHc, BHc, EHc (recombinant EHc was expressed and purified as other Hc proteins) [29,30] or FHc antibodies by ELISA as described previously [16,31]. Briefly, the total IgG titers were determined using HRP-conjugated goat anti-mouse antibodies. Antibody ELISA titers represent the highest dilution of samples showing a 2-fold OD492 value over controls. Sera obtained 5 days before challenge from individual mouse were assayed and their geometric mean titers (GMT) were 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 BoNTs neutralization assay as described previously [18,31,32]. Briefly, pooled sera from each group of mice above were diluted initially 1:8 and then diluted twofold for serum neutralization titers. Each sample dilution was incubated with a standard concentration of corresponding BoNTs. After incubated 0.5 h at room temperature, the mixtures were injected i.p. into Balb/c mice (18–22 g) using a volume of 500 l/mouse (four mice in each group). The mice were observed for one week, and death or survival was recorded. The concentration of neutralizing antibody in the sera was calculated relative to a World Health Organization BoNTs antitoxin and neutralizing antibody titers of sera were reported as international units per milliliter (IU/ml). Due to the limited amount of serum available, serum from each group of mice was pooled, and so only the average neutralization titer could be assayed.
2.2. Preparation of DNA-based recombinant SFV replicon particles
2.5. Statistical analysis
Recombinant SFV replicon particles (VRP) were prepared as described previously [30]. Briefly, BHK-21 cells were co-transfected with DNA-based replicon expression vectors and helper vector (pSHCAR), and the VRP were harvested at 24–36 h after transfection. VRP were concentrated and purified by centrifugation, and the virus pellets were resuspended in PBS buffer. For titer detection, BHK-21 cells were infected with serial dilutions of activated virus and monitored for expression of antigen by X-gal staining (VRP-lacZ) or immunofluorescence assay (IFA).
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. 3. Results 3.1. Effect of individual DNA replicon vaccine on antibody responses and protection against BoNTs
2.3. Vaccination and challenge of mice Specific pathogen-free female Balb/c mice (purchased from Beijing Laboratory Animal Center, Beijing, China) six weeks of age were randomly assigned to different treatment groups and vaccinated with DNA or VRP. Dosage used in the immunized groups was optimized by a series of preliminary experiments. For DNA vaccination, groups of eight mice were injected with 10 g of plasmid DNA replicon vaccines or conventional DNA vaccine intramuscularly (bilaterally in the quadriceps) in a total volume of 0.1 ml for three or four times with 3-week intervals. As a negative control, mice were vaccinated with 10 g of pSCAR or pVAX1 as above. For VRP vaccination, 106 infectious particles (one infectious unit = 1 IU) of activated VRP-Hc or VRP-lacZ (negative control) in a total volume of 0.2 ml were inoculated subcutaneously for twice or three times with 3-week intervals. Mice from all groups were challenged i.p. with different dosages of pure BoNTs (BoNT/A from strain 62A, BoNT/B from strain Okra, BoNT/E from Iwanai and BoNT/F Langeland strain) 4 weeks after the last vaccination. The mice were observed two times every day for one week on intoxication symptoms or death after challenge. Survival was determined for each vaccination group. The animal protocols in this study were
The immunogenicity and protective ability of DNA replicon vaccines against BoNT/E or BoNT/F were investigated and compared with the corresponding conventional DNA vaccines encoding the same antigen (Table 1). Mice vaccinated three times with pSCARSEHc or pSCARFBHc were completely protected against 1000 50% lethal dose (LD50 ) of each BoNTs, while three doses of pVAX1SEHc or pVAX1SFHc provided partly protection against 1000 LD50 of each BoNTs. Additionally, mice that received three injections of pSCARSFHc were completely protected against 10,000 LD50 of BoNT/F, while no protection was observed in mice that received three injections of pVAX1SFHc against 10,000 LD50 of BoNT/F. Mice in the negative control groups succumbed to botulism and died within 5 h. As shown in Table 1, the mean antibody titers to each Hc in the DNA replicon vaccination mice groups were higher than those obtained from mice immunized with the conventional DNA vaccines. There is statistically significant difference between three doses of DNA replicon (pSCARSEHc or pSCARSFHc) and conventional DNA (pVAX1SEHc or pVAX1SFHc)-vaccinated groups (P < 0.05). And higher titers of neutralizing antibodies against each BoNTs were observed in the sera of mice vaccinated with the DNA
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Fig. 1. Schematic diagrams of plasmids used for DNA vaccination in this study. The individual transcriptional control elements comprising the DNA-based replicon vaccine plasmids based on SFV replicon (pSCARSAHc, pSCARSBHc, pSCARSEHc, pSCARSFHc, pSCARSA/BHc and pSCARSE/FHc) and the conventional DNA vaccine plasmid (pVAX1SEHc and pVAX1SFHc) are indicated. CMV, cytomegalovirus immediate early (CMV IE) enhancer/promoter. TT/pA, BGH transcription termination and polyadenylation signal. HDV, HDV antigenomic ribozyme sequence. 26S, the subgenomic promoter of SFV. AHc, BHc, EHc and FHc, four completely synthetic genes encoding the Hc domains of BoNT serotypes A, B, E and F, respectively. S, Ig leader sequence. pSCARSA/BHc, a dual-expression plasmid DNA replicon vaccine that respectively expressed the Hc domains of BoNT serotypes A and B under two separate 26S promoters. pSCARSE/FHc, a dual-expression plasmid DNA replicon vaccine that respectively expressed the Hc domains of BoNT serotypes E and F under two separate 26S promoters.
replicon vaccines and no neutralizing antibodies were observed in the negative control groups. These results indicated that immunization with the plasmid DNA replicon vaccines could induce stronger humoral responses and produce more protective efficacy against each BoNTs than the conventional DNA vaccines. 3.2. Effect of individual RVP vaccine on antibody responses and protection against BoNTs To evaluate the immunogenicity and protective efficacy of individual VRP, mice were inoculated subcutaneously two or three times with VRP-EHc or VRP-FHc, respectively. A
dose-dependent protective effect was observed against each BoNTs (Table 2). For VRP-EHc, mice that received three injections of VRP were completely protected were against challenge with BoNT/E, and two injections of VRP provided partial protection. For VRP-FHc, mice that received three injections of VRP were completely protected against challenge with 1000 or 10,000 LD50 of BoNT/F, and two injections of VRP provided partial protection. In contrast, mice in the negative control groups vaccinated with VRPlacZ succumbed to botulism and died within 24 h. The results of this study demonstrated that the VRP vaccines were capable of inducing efficient protection against lethal BoNT/E or BoNT/F challenge. A dose-dependent antibody response to vaccination with VRP-EHc or
Table 1 Survival, sera antibody titers, and neutralization titers of mice following vaccination i.m. with pSCARSEHc, pVAX1SEHc, pSCARSFHc, or pVAX1SFHc. Vaccine
Log10 GMT(SD)a e
pSCARSEHc pVAX1SEHc pSCARSFHc pVAX1SFHc
Neutralization titer (IU/ml)b e
e
e
Number alivec e
Number alived e
3
4
3
4
3
4
3e
4e
4.03(0.27) 3.57(0.27)* 4.03(0.27) 3.34(0.28)#
4.48(0.21) 4.44(0.19) 4.56(0.16) 4.26(0.16)@
0.64 0.16 1.28 0.32
2.56 1.28 5.12 2.56
8 4 8 2$
8 8 8 8
ND ND 8 0&
ND ND 8 8
ND, not done. a Sera antibody titers (the log10 GMT for each group) to EHc or FHc were shown in table, respectively. b Sera neutralization titers against BoNT/E or BoNT/F were shown in table, respectively. c Mice alive (8 mice/group) after i.p. challenge with a dose 103 LD50 of biologically active each BoNTs (BoNT/E or BoNT/F) 4 weeks after the last injection, respectively. d Mice alive (8 mice/group) after i.p. challenge with 104 LD50 of BoNT/F 4 weeks after the last injection. e Number of vaccinations. * P = 0.025 indicates significant titer difference between three doses of pSCARSEHc and pVAX1SEHc-vaccinated groups. # P = 0.005 indicates significant titer difference between three doses of pSCARSFHc and pVAX1SFHc-vaccinated groups. @ P = 0.011 indicates significant titer difference between four doses of pSCARSFHc and pVAX1SFHc-vaccinated groups. $ P = 0.007 indicates significant survival difference between three doses of pSCARSFHc and pVAX1SFHc-vaccinated groups when challenged with a dose of 103 LD50 of BoNT/F. & P = 0.00016 indicates significant survival difference between three doses of pSCARSFHc and pVAX1SFHc-vaccinated groups when challenged with a dose of 104 LD50 of BoNT/F.
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Table 2 Survival, sera antibody titers, and neutralization titers of mice following vaccination s.c. with VRP-EHc or VRP-FHc. Vaccine
Log10 GMT(SD)a e
RVP-EHc RVP-FHc
Neutralization titer (IU/ml)b e
e
e
Number alivec e
Number alived e
2
3
2
3
2
3
2e
3e
3.42(0.32) 3.59(0.48)
3.96(0.16)* 4.14(0.25)#
0.16 0.32
1.28 1.28
6 7
8 8
ND 4
ND 8
ND, not done. a Antibody titers (the log10 GMT for each group) to EHc or FHc were shown in table, respectively. b Sera neutralization titers against BoNT/E or BoNT/F were shown in table, respectively. c Mice alive (8 mice/group) after i.p. challenge with 103 LD50 of biologically active each BoNTs (BoNT/E or BoNT/F) 4 weeks after the last injection, respectively. d Mice alive (8 mice/group) after i.p. challenge with 104 LD50 of BoNT/F 4 weeks after the last injection. e Number of vaccinations. * P = 0.001 indicates significant titer difference between two doses of VRP-EHc and three doses of VRP-EHc-vaccinated groups. # P = 0.011 indicates significant titer difference between two doses of VRP-FHc and three doses of VRP-FHc-vaccinated groups.
VRP-FHc was also observed and the group GMTs or neutralization antibody levels correlated well with protection (Table 2). Control mice did not show serological reactivity to either EHc or FHc and showed no protection against BoNT/E or BoNT/F. 3.3. Bivalent replicon vaccines for BoNT serotypes E and F We also evaluated two types of bivalent vaccine candidates: a dual-expression DNA replicon vaccine pSCARSE/FHc and a mixture vaccine composed of pSCARSEHc and pSCARSFHc. As shown in Table 3, antisera from mice immunized with the mixture and dualexpression DNA replicon vaccines demonstrated high IgG titers to each of the individual Hc based on ELISA and sufficient neutralizing antibody levels based on BoNTs neutralization assay. And vaccination of mice with either the dual-expression DNA replicon vaccine or a mixture of DNA replicon vaccines resulted in complete protection of the animals against challenge with 1000 LD50 of BoNT serotypes E and F mixture. To determine the protective effects of the bivalent vaccine against a higher neurotoxin challenge dose, vaccinated mice were challenged with a dose of 10,000 LD50 of BoNT/F, and complete immune protection was also observed (Table 3). Additionally, dual-expression replicon particle VRP-E/FHc was also developed using DNA-based SFV vectors. The immunogenicity of the single bivalent replicon VRP-E/FHc vaccine was compared with another bivalent vaccine composed of a mixture of individual VRP-EHc and VRP-FHc. Antisera from mice immunized with the mixture and dual-expression VRP vaccines also showed high IgG titers and sufficient neutralizing antibody levels. Vaccination with either dual-expression VRP vaccine or a mixture of VRP vaccines completely protected the mice against challenge with a dose of 1000 LD50 of BoNT serotypes E and F mixture and mostly protected the mice against challenge with a higher neurotoxin challenge dose (10,000 LD50 of BoNT/F) (Table 3). Meanwhile, mice were inoculated with individual pSCARSEHc, pSCARSFHc, VRP-EHc or VRP-FHc as control vaccine groups and challenged similarly. These single vaccines only induced detectable antibody titers to the respective EHc or FHc antigen and did not protected the vaccinated mice with a challenge of mixtures of BoNT/E and BoNT/F (P = 0.00016 < 0.001). These results revealed that the immunogenicity and protective efficacy of dual-expression replicon bivalent vaccines were comparable to that of the mixture of two individual replicon vaccines against BoNT/E and BoNT/F. 3.4. Tetravalent replicon vaccines for BoNT serotypes A, B, E, and F Further, tetravalent replicon vaccines against BoNTs were developed and the protective capability was evaluated against challenge with mixture of BoNTs in mice. We evaluated four types of tetravalent replicon vaccine candidates: two dual-expression DNA replicon vaccines combination (pSCARSA/BHc + pSCARSE/FHc), a mixture vaccine composed of four single DNA replicon vaccines
(pSCARSAHc + pSCARSBHc + pSCARSEHc + pSCARSFHc), two dualexpression VRP vaccines combination (VRP-A/B + VRP-E/F), and another mixture vaccine composed of four single VRP vaccines (VRP-AHc, VRP-BHc, VRP-EHc and VRP-FHc). As shown in Table 4, antisera from mice immunized with these different tetravalent replicon vaccines demonstrated high IgG titers to each Hc based on ELISA and efficacious neutralizing antibody levels against each BoNTs. And the vaccination resulted in complete protection of the animals against challenge with 100 LD50 of BoNT serotypes A, B, E and F mixture and mostly protected the mice following challenge with a higher neurotoxin challenge dose (a mixture of 1000 LD50 of each BoNTs) (Table 4). Also, mice were inoculated with bivalent replicon vaccines as control vaccine groups and challenged similarly. The vaccinated mice succumbed to botulism and died within 24 h with a challenge of mixtures of BoNTs (P = 0.00016 < 0.001). Our findings revealed that vaccination with these different tetravalent replicon vaccines elicited similar antibody responses that conferred equal levels of protection against the challenge with mixture of BoNTs (P = 0.4667 > 0.05). Therefore, our studies demonstrate the utility of combining dual-expression plasmid DNA replicon or replicon particle vaccines into multiagent formulations as tetravalent vaccines for eliciting protective immune responses to four serotypes of BoNTs. 4. Discussion BoNTs are the most toxic proteins for humans and have been described as a potential toxic agent for malicious applications. The current formalin-inactivated pentavalent vaccine is protective against serotypes A–E, but not against the serotype F. Of seven BoNT serotypes (A–G), serotypes A, B, E and F usually cause human botulism. Therefore, there is currently a demand for a tetravalent vaccine or preparation that provides protection against all four of these serotypes [33]. This also promoted the current study to produce tetravalent vaccines to neutralize the four BoNTs serotype and prevent botulism. Previously, Pushko et al. reported that bivalent vaccines based on VEE virus replicon protected guinea pigs against infection with Lassa and Ebola viruses [34]. Smith et al. used the alphavirus replicon system to elicit an immune response in mice that protected against the Marburg virus, anthrax toxin, and BoNT/C [28]. In our recent studies, bivalent candidate replicon vaccines against BoNT serotypes A and B were developed using DNA-based SFV vectors [30]. The current study extends these findings to show the feasibility of generating multi-agent replicon vaccines that protects against the four serotypes of BoNT. In this study, the DNA replicon vaccines pSCARSEHc or pSCARSFHc can induce stronger humoral response and provide more effective protection against BoNT/E or BoNT/F than the corresponding conventional DNA vaccines encoding the same antigens, as previously described by using the DNA replicon vaccines
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Table 3 Survival, sera antibody titers, and neutralization titers of mice following vaccination three times with bivalent replicon vaccines against BoNTs. Vaccine
10 g(each) 10 g 106 IU(each) 106 IU
pSCARSEHc + pSCARFBHc pSCARSE/FHc RVP-EHc + RVP-FHc RVP-E/FHc a b c d
Log10 GMT(SD)a
Dose
Neutralization titer (IU/ml)b
Number alive
EHc
FHc
BoNT/E
BoNT/F
103 c
104 d
4.03(0.27) 4.11(0.32) 3.96(0.16) 3.88(0.14)
4.11(0.23) 4.10(0.32) 4.10(0.32) 3.80(0.28)
0.64 0.64 1.28 0.64
1.28 0.64 1.28 1.28
8 8 8 8
8 8 8 6
Sera antibody titers to EHc or FHc (the log10 GMT for each group) were shown in table, respectively. Sera neutralization titers against to BoNT/E or BoNT/F were shown in table, respectively. Mice alive (8 mice/group) after i.p. challenge with 103 LD50 of biologically active BoNT/E and BoNT/F mixture 4 weeks after the last injection. Mice alive (8 mice/group) after i.p. challenge with 104 LD50 of BoNT/F 4 weeks after the last injection.
Table 4 Survival, sera antibody titers, and neutralization titers of mice following vaccination three times with tetravalent replicon vaccines against BoNTs. Vaccine
pSCARSAHc + pSCARSBHc + pSCARSEHc + pSCARSFHc pSCARSA/BHc+ pSCARSE/FHc RVP-AHc + RVP-BHc+ RVP-EHc + RVP-FHc RVP-A/BHc + RVP-E/FHc a b c
Dose
Log10 GMT(SD)a
Neutralization titer (IU/ml)b
Number alivec
AHc
BHc
EHc
FHc
BoNT/A
BoNT/B
BoNT/E
BoNT/F
102
103
10 g(each)
3.75(0.14)
3.64(0.27)
3.96(0.16)
3.92(0.16)
0.16
0.32
0.64
0.64
8
8
10 g(each)
3.51(0.27)
3.65(0.28)
3.81(0.40)
3.76(0.60)
0.32
0.64
0.64
1.28
8
7
106 IU(each)
3.72(0.28)
3.61(0.51)
3.82(0.51)
4.14(0.30)
0.32
0.64
1.28
1.28
8
6
106 IU(each)
3.56(0.23)
3.57(0.27)
3.72(0.28)
3.78(0.36)
0.32
0.64
0.64
0.64
8
8
Sera antibody titers (the log10 GMT for each group) to AHc, BHc, EHc or FHc were shown in table, respectively. Sera neutralization titers against to BoNT/A, BoNT/B, BoNT/E or BoNT/F were shown in table, respectively. Mice alive (8 mice/group) after i.p. challenge with the indicated dose of BoNT serotypes A, B, E and F (102 or 103 LD50 , each) mixture 4 weeks after the last injection.
pSCARSAHc or pSCARSBHc against BoNT/A or BoNT/B, respectively [29,30]. And, our results demonstrated that the VRP vaccines VRP-EHc or VRP-FHc as an alternative vaccine format were capable of inducing efficient protection against challenge with BoNT/E or BoNT/F, just as candidate VEE virus replicon vaccines against BoNT/A and BoNT/C [27,28]. Moreover, we constructed and tested a dual-expression DNA replicon vaccine pSCARSE/FHc as well as a dual-expression RVP Vaccine VRP-E/FHc using DNA-based SFV vectors as previously described for BoNT/A and BoNT/B [30]. Mice immunized with the bivalent replicon vaccines were protected against challenge with mixture of BoNT/E and BoNT/F as a combination vaccine composed of two monovalent replicon vaccines (Table 3). Protective effect was consistent with the serological reactivity observed above. These results showed that alphavirus replicon vaccines could induce adequate immune responses and protective effects without the need for high doses of DNA/VRP or demanding immunization regimens. No different efficacy against BoNTs was observed in two types of vaccination methodologies in this study and further studies are underway to assess and better define their utility. Finally, tetravalent replicon vaccines against BoNT serotypes A, B, E and F were developed and the protective capability was evaluated against challenge with BoNTs. An effective tetravalent vaccine must simultaneously neutralize and protect against the four BoNT serotypes. We evaluated four types of tetravalent replicon vaccine candidates. Mice immunized with the tetravalent replicon vaccines were resistant to challenge with mixture of BoNT serotypes A, B, E and F as multivalent subunit vaccines against BoNTs [14,15]. The antibody response, neutralization antibody level and protective efficacy of tetravalent vaccines composed of two dual-expression replicon vaccines were comparable to that of mixture of four individual replicon vaccines against four BoNT serotypes. As shown in our results, protective potency against BoNTs correlated well with anti-Hc and BoNTs neutralization antibody levels, which is consistent with the concept that antibodies play a key role in protection against toxins. In addition to the high degree of observed protection against the four serotypes of BoNTs simultaneously, dual-expression replicon tetravalent
vaccines for BoNTs may offer other advantages, including simplification of experimental immunization regimens and cost savings in manufacturing one product instead of two or two instead of four. To our knowledge, this is the first report to date demonstrating the preparation of tetravalent DNA or RVP vaccines for eliciting protective immune responses to all four serotypes of BoNTs. In summary, experimental individual replicon vaccines for BoNT/E or BoNT/F and bivalent replicon vaccines for both BoNT/E and BoNT/F were developed using DNA-based SFV vectors. More importantly, our results demonstrated the utility of combining individual DNA replicon or replicon particle vaccines into multiagent formulations as tetravalent vaccines for eliciting protective immune responses against four serotypes of BoNTs. Note of, the immunogenicity of DNA vaccine against BoNT/A was enhanced by the dual-expression replicon vector expressing GM–CSF adjuvant [35], and one new dual-expression replicon vector vaccine simultaneously expressed PA and LF of anthrax are being assessed in our laboratory. Except for simplification of immunization regimens and cost savings in manufacturing, this type of dual-expression vector could enhance the potency of vaccine as mixture of individual PA and LF-DNA vaccines [36]. Also, multivalent smallpox DNA vaccine composed of eight different DNA plasmids developed by Hirao et al. [37] can be replaced with our four dual-expression vectors. Currently, there also is a demand for a new generation of safe and highly potent multivalent vaccines, which could simultaneously provide efficient protection against several important pathogens [38]. Thus, we described a new and efficient strategy to design and develop multivalent vaccines against multi-agent pathogens or specifically certain pathogen using DNA-based SFV replicon vectors.
Acknowledgments This work was partly supported by grants from the National Natural Science Foundation of China (30901375) and Beijing Natural Science Foundation (7102125).
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[21] Boles J, West M, Montgomery V, Tammariello R, Pitt ML, Gibbs P, et al. Recombinant C fragment of botulinum neurotoxin serotype B (rBoNTB(Hc)) immune response and protection in the rhesus monkey. Toxicon 2006;47:877–84. [22] Webb RP, Smith TJ, Wright PM, Montgomery VA, Meagher MM, Smith LA. Protection with recombinant Clostridium botulinum C1 and D binding domain subunit (Hc) vaccines against C and D neurotoxins. Vaccine 2007;25:4273–82. [23] Clayton J, Middlebrook JL. Vaccination of mice with DNA encoding a large fragment of botulinum neurotoxin serotype A. Vaccine 2000;18:1855–62. [24] Shyu RH, Shaio MF, Tang SS, Shyu HF, Lee CF, Tsai MH, et al. DNA vaccination using the fragment C of botulinum neurotoxin type A provided protective immunity in mice. J Biomed Sci 2000;7:51–7. [25] Bennett AM, Perkins SD, Holley JL. DNA vaccination protects against botulinum neurotoxin type F. Vaccine 2003;21:3110–7. [26] Jathoul AP, Holley JL, Garmory HS. Efficacy DNA vaccines expressing the type F botulinum toxin Hc fragment using different promoters. Vaccine 2004;22:3942–6. [27] Lee JS, Pushko P, Parker MD, Dertzbaugh MT, Smith LA, Smith JF. Candidate vaccine against botulinum neurotoxin serotype A derived from a Venezuelan equine encephalitis virus vector system. Infect Immun 2001;69:5709–15. [28] Lee JS, Groebner JL, Hadjipanayis AG, Negley DL, Schmaljohn AL, Welkos SL, et al. Multiagent vaccines vectored by Venezuelan equine encephalitis virus replicon elicits immune responses to Marburg virus and protection against anthrax and botulinum neurotoxin in mice. Vaccine 2006;24:6886–92. [29] Yu YZ, Zhang SM, Sun ZW, Wang S, Yu WY. Enhanced immune responses using plasmid DNA replicon vaccine encoding the Hc domain of Clostridium botulinum neurotoxin serotype A. Vaccine 2007;25:8843–50. [30] Yu YZ, Yu JY, Li N, Wang RL, Wang S, Yu WY, et al. Individual and bivalent vaccines against botulinum neurotoxin serotypes A and B using DNA-based Semliki Forest virus vectors. Vaccine 2009;27:6148–53. [31] Yu YZ, Zhang SM, Ma Y, Zhu HQ, Wang WB, Du Y, et al. Development and evaluation of candidate vaccine and antitoxin against botulinum neurotoxin serotype F. Clin Immunol 2010;137:271–80. [32] Yu YZ, Wang WB, Li N, Wang S, Yu WY, Sun ZW. Enhanced potency of individual and bivalent DNA replicon vaccines or conventional DNA vaccines by formulation with aluminum phosphate. Biologicals 2011;38:658–63. [33] Torii Y, Tokumaru Y, Kawaguchi S, Izumi N, Maruyama S, Mukamoto M, et al. Production and immunogenic efficacy of botulinum tetravalent (A, B, E, F) toxoid. Vaccine 2002;20:2556–61. [34] Pushko P, Geisbert J, Parker M, Jahrling P, Smith J. Individual and bivalent vaccines based on alphavirus replicons protect guinea pigs against infection with Lassa and Ebola viruses. J Virol 2001;75:11677–85. [35] Li N, Yu YZ, Yu WY, Sun ZW. Enhancement of the immunogenicity of DNA replicon vaccine of Clostridium botulinum neurotoxin serotype A by GM–CSF gene adjuvant. Immunopharmacol Immunotoxicol 2011;33:211–9. [36] Hermanson G, Whitlow V, Parker S, Tonsky K, Rusalov D, Ferrari M, et al. A cationic lipid-formulated plasmid DNA vaccine confers sustained antibodymediated protection against aerosolized anthrax spores. Proc Natl Acad Sci USA 2004;101:13601–6. [37] Hirao LA, Draghia-Akli R, Prigge JT, Yang M, Satishchandran A, Wu L, et al. Multivalent smallpox DNA vaccine delivered by intradermal electroporation drives protective immunity in nonhuman primates against lethal monkeypox challenge. J Infect Dis 2011;203:95–102. [38] Merkel TJ, Perera PY, Kelly VK, Verma A, Llewellyn ZN, Waldmann TA, et al. Development of a highly efficacious vaccinia-based dual vaccine against smallpox and anthrax, two important bioterror entities. Proc Natl Acad Sci USA 2010;107:18091–6.
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Potent tetravalent replicon vaccines against botulinum neurotoxins using DNA-based Semliki Forest virus replicon vectors Yun-Zhou Yu a,∗,1 , Jin-Peng Guo b,1 , Huai-Jie An c,1 , Shu-Ming Zhang d,∗ , Shuang Wang a , Wei-Yuan Yu a , Zhi-Wei Sun a,∗ a
Beijing Institute of Biotechnology, Beijing, China Institute for Disease Prevention and Control, Academy of Military Medical Sciences, Beijing, China c Wound Care & Research Center of Sea Battle of PLA China, Navy General Hospital, Beijing, China d National Institutes of Food and Drug Control, Beijing, China b
a r t i c l e
i n f o
Article history: Received 21 August 2012 Received in revised form 7 February 2013 Accepted 28 March 2013 Available online 10 April 2013 Keywords: Botulinum neurotoxin Tetravalent vaccine Replicon vector Dual-expression
a b s t r a c t Human botulism is commonly associated with botulinum neurotoxin (BoNT) serotypes A, B, E and F. This suggests that the greatest need is for a tetravalent vaccine that provides protection against all four of these serotypes. In current study, we investigated the feasibility of generating several tetravalent vaccines that protected mice against the four serotypes. Firstly, monovalent replicon vaccine against BoNT induced better antibody response and protection than that of corresponding conventional DNA vaccine. Secondly, dual-expression DNA replicon pSCARSE/FHc or replicon particle VRP-E/FHc vaccine was well resistant to the challenge of BoNT/E and BoNT/F mixture as a combination vaccine composed of two monovalent replicon vaccines. Finally, the dual-expression DNA replicon or replicon particle tetravalent vaccine could simultaneously and effectively neutralize and protect the four BoNT serotypes. Protection correlated directly with serum ELISA titers and neutralization antibody levels to BoNTs. Therefore, replicon-based DNA or particle might be effective vector to develop BoNT vaccines, which might be more desirable for use in clinical application than the conventional DNA vaccines. Our studies demonstrate the utility of combining dual-expression DNA replicon or replicon particle vaccines into multi-agent formulations as potent tetravalent vaccines for eliciting protective responses to four serotypes of BoNTs. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction The botulinum neurotoxins (BoNTs) are the most toxic proteins and can be classed into seven serotypes (A–G) which own similar structures but are antigenically distinct. Human botulism illness generally occurs in three forms: food-borne botulism, infant botulism, and wound botulism [1,2]. Clostridium botulinum neurotoxin serotypes A, B, E and F can cause disease in human, as opposite to serotypes C and D that can cause disease in cattle and horses [3,4]. Each BoNT contains three functional domains e.g., the N-terminal catalytic domain (light chain), the internal heavy chain translocation domain (Hn domain, 50 kDa) and the C-terminal heavy chain receptor-binding domain (Hc domain, 50 kDa). Botulism can
∗ Corresponding authors at: Beijing Institute of Biotechnology, 20 Dongdajie Street, Fengtai District, Beijing 100071, China. Tel.: +86 10 63855329; fax: +86 10 63833521. E-mail addresses: [email protected] (Y.-Z. Yu), [email protected] (S.-M. Zhang), [email protected] (Z.-W. Sun). 1 These authors contributed equally to this work. 0264-410X/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.vaccine.2013.03.046
be effectively prevented by the presence of neutralizing antibodies induced by vaccination. The formalin-inactivated pentavalent vaccine (PBT) commonly used in human is protective against serotypes A–E. However, it has met with some drawbacks, including feasibility, dangers associated with handling neurotoxins, and secondary complications due to residual formaldehyde contamination [5–8]. Therefore, the improved vaccines designed to overcome above disadvantages are needed. A novel vaccine strategy for the prevention of botulism based on the production of nontoxic recombinant BoNT proteins has been developed [5,6]. The Hc domains of BoNTs produced in Escherichia coli and Pichia pastoris were shown to elicit protective immune responses in mice and other animals [9–22]. DNA vaccines encoding the Hc domains of serotypes A and F were described as the next generation of botulinum vaccines [23–26]. Candidate vaccines against BoNT serotypes A and C were also developed by using a Venezuelan equine encephalitis (VEE) virus replicon vector [27,28]. Further, individual and bivalent candidate replicon vaccines against BoNT serotypes A and B were developed using DNA-based Semliki Forest virus (SFV) vectors by us in recent studies [29,30]. In this current study, we developed several tetravalent replicon vaccines for BoNT serotypes A, B, E, and
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F using the DNA-based SFV replicon vectors, and evaluated their immunogenicity and protective capability against challenge with the BoNTs mixture in mice. 2. Materials and methods 2.1. Construction of DNA replicon vaccines Two plasmid DNA replicon vaccines (pSCARSEHc and pSCARSFHc) and a dual-expression plasmid DNA replicon vaccine (pSCARSE/FHc) encoding the Hc domain of BoNT/E (EHc) and BoNT/F (FHc) were constructed in this study. Briefly, the AHc of pSCARSAHc was replaced with EHc (one synthetic gene encoding the Hc domain of BoNT/E, strain NCTC 11219, amino acids 841 to 1252), which has been submitted to Genbank under the accession number HM231315) or FHc (EU386771, one synthetic FHc gene encoding the Hc domain of BoNT/F) [16] by using the Nde I and Sma I cloning sites, resulting in pSCARSEHc or pSCARSFHc (Fig. 1C and D), respectively, and the AHc and BHc of pSCARSA/BHc was replaced with EHc and FHc resulting in pSCARSE/FHc (Fig. 1F) as described previously [30]. To generate two corresponding conventional DNA vaccines encoding the EHc or FHc, the DNA fragments were isolated by digesting pSCARSEHc or pSCARSFHc with BamH I and Spe I, and then inserting them into the vector pVAX1 to create pVAX1SEHc or pVAX1SFHc (Fig. 1G and H), respectively. All plasmids were prepared and purified using Endofree Mega-Q kits (QIAGEN GmbH, Hilden, Germany) for transfection and immunization. BHK-21 cells were transfected with pSCARSEHc, pSCARSFHc, pSCARSE/FHc, pVAX1SEHc or pVAX1SFHc, and the expression of EHc or FHc was analyzed as described previously [29,30].
approved by Institution Animal Care and Use Committee of our Institution. 2.4. Antibody titer measurements and BoNTs neutralization assays Sera from mice in different treatment groups were screened for anti-AHc, BHc, EHc (recombinant EHc was expressed and purified as other Hc proteins) [29,30] or FHc antibodies by ELISA as described previously [16,31]. Briefly, the total IgG titers were determined using HRP-conjugated goat anti-mouse antibodies. Antibody ELISA titers represent the highest dilution of samples showing a 2-fold OD492 value over controls. Sera obtained 5 days before challenge from individual mouse were assayed and their geometric mean titers (GMT) were 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 BoNTs neutralization assay as described previously [18,31,32]. Briefly, pooled sera from each group of mice above were diluted initially 1:8 and then diluted twofold for serum neutralization titers. Each sample dilution was incubated with a standard concentration of corresponding BoNTs. After incubated 0.5 h at room temperature, the mixtures were injected i.p. into Balb/c mice (18–22 g) using a volume of 500 l/mouse (four mice in each group). The mice were observed for one week, and death or survival was recorded. The concentration of neutralizing antibody in the sera was calculated relative to a World Health Organization BoNTs antitoxin and neutralizing antibody titers of sera were reported as international units per milliliter (IU/ml). Due to the limited amount of serum available, serum from each group of mice was pooled, and so only the average neutralization titer could be assayed.
2.2. Preparation of DNA-based recombinant SFV replicon particles
2.5. Statistical analysis
Recombinant SFV replicon particles (VRP) were prepared as described previously [30]. Briefly, BHK-21 cells were co-transfected with DNA-based replicon expression vectors and helper vector (pSHCAR), and the VRP were harvested at 24–36 h after transfection. VRP were concentrated and purified by centrifugation, and the virus pellets were resuspended in PBS buffer. For titer detection, BHK-21 cells were infected with serial dilutions of activated virus and monitored for expression of antigen by X-gal staining (VRP-lacZ) or immunofluorescence assay (IFA).
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. 3. Results 3.1. Effect of individual DNA replicon vaccine on antibody responses and protection against BoNTs
2.3. Vaccination and challenge of mice Specific pathogen-free female Balb/c mice (purchased from Beijing Laboratory Animal Center, Beijing, China) six weeks of age were randomly assigned to different treatment groups and vaccinated with DNA or VRP. Dosage used in the immunized groups was optimized by a series of preliminary experiments. For DNA vaccination, groups of eight mice were injected with 10 g of plasmid DNA replicon vaccines or conventional DNA vaccine intramuscularly (bilaterally in the quadriceps) in a total volume of 0.1 ml for three or four times with 3-week intervals. As a negative control, mice were vaccinated with 10 g of pSCAR or pVAX1 as above. For VRP vaccination, 106 infectious particles (one infectious unit = 1 IU) of activated VRP-Hc or VRP-lacZ (negative control) in a total volume of 0.2 ml were inoculated subcutaneously for twice or three times with 3-week intervals. Mice from all groups were challenged i.p. with different dosages of pure BoNTs (BoNT/A from strain 62A, BoNT/B from strain Okra, BoNT/E from Iwanai and BoNT/F Langeland strain) 4 weeks after the last vaccination. The mice were observed two times every day for one week on intoxication symptoms or death after challenge. Survival was determined for each vaccination group. The animal protocols in this study were
The immunogenicity and protective ability of DNA replicon vaccines against BoNT/E or BoNT/F were investigated and compared with the corresponding conventional DNA vaccines encoding the same antigen (Table 1). Mice vaccinated three times with pSCARSEHc or pSCARFBHc were completely protected against 1000 50% lethal dose (LD50 ) of each BoNTs, while three doses of pVAX1SEHc or pVAX1SFHc provided partly protection against 1000 LD50 of each BoNTs. Additionally, mice that received three injections of pSCARSFHc were completely protected against 10,000 LD50 of BoNT/F, while no protection was observed in mice that received three injections of pVAX1SFHc against 10,000 LD50 of BoNT/F. Mice in the negative control groups succumbed to botulism and died within 5 h. As shown in Table 1, the mean antibody titers to each Hc in the DNA replicon vaccination mice groups were higher than those obtained from mice immunized with the conventional DNA vaccines. There is statistically significant difference between three doses of DNA replicon (pSCARSEHc or pSCARSFHc) and conventional DNA (pVAX1SEHc or pVAX1SFHc)-vaccinated groups (P < 0.05). And higher titers of neutralizing antibodies against each BoNTs were observed in the sera of mice vaccinated with the DNA
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Fig. 1. Schematic diagrams of plasmids used for DNA vaccination in this study. The individual transcriptional control elements comprising the DNA-based replicon vaccine plasmids based on SFV replicon (pSCARSAHc, pSCARSBHc, pSCARSEHc, pSCARSFHc, pSCARSA/BHc and pSCARSE/FHc) and the conventional DNA vaccine plasmid (pVAX1SEHc and pVAX1SFHc) are indicated. CMV, cytomegalovirus immediate early (CMV IE) enhancer/promoter. TT/pA, BGH transcription termination and polyadenylation signal. HDV, HDV antigenomic ribozyme sequence. 26S, the subgenomic promoter of SFV. AHc, BHc, EHc and FHc, four completely synthetic genes encoding the Hc domains of BoNT serotypes A, B, E and F, respectively. S, Ig leader sequence. pSCARSA/BHc, a dual-expression plasmid DNA replicon vaccine that respectively expressed the Hc domains of BoNT serotypes A and B under two separate 26S promoters. pSCARSE/FHc, a dual-expression plasmid DNA replicon vaccine that respectively expressed the Hc domains of BoNT serotypes E and F under two separate 26S promoters.
replicon vaccines and no neutralizing antibodies were observed in the negative control groups. These results indicated that immunization with the plasmid DNA replicon vaccines could induce stronger humoral responses and produce more protective efficacy against each BoNTs than the conventional DNA vaccines. 3.2. Effect of individual RVP vaccine on antibody responses and protection against BoNTs To evaluate the immunogenicity and protective efficacy of individual VRP, mice were inoculated subcutaneously two or three times with VRP-EHc or VRP-FHc, respectively. A
dose-dependent protective effect was observed against each BoNTs (Table 2). For VRP-EHc, mice that received three injections of VRP were completely protected were against challenge with BoNT/E, and two injections of VRP provided partial protection. For VRP-FHc, mice that received three injections of VRP were completely protected against challenge with 1000 or 10,000 LD50 of BoNT/F, and two injections of VRP provided partial protection. In contrast, mice in the negative control groups vaccinated with VRPlacZ succumbed to botulism and died within 24 h. The results of this study demonstrated that the VRP vaccines were capable of inducing efficient protection against lethal BoNT/E or BoNT/F challenge. A dose-dependent antibody response to vaccination with VRP-EHc or
Table 1 Survival, sera antibody titers, and neutralization titers of mice following vaccination i.m. with pSCARSEHc, pVAX1SEHc, pSCARSFHc, or pVAX1SFHc. Vaccine
Log10 GMT(SD)a e
pSCARSEHc pVAX1SEHc pSCARSFHc pVAX1SFHc
Neutralization titer (IU/ml)b e
e
e
Number alivec e
Number alived e
3
4
3
4
3
4
3e
4e
4.03(0.27) 3.57(0.27)* 4.03(0.27) 3.34(0.28)#
4.48(0.21) 4.44(0.19) 4.56(0.16) 4.26(0.16)@
0.64 0.16 1.28 0.32
2.56 1.28 5.12 2.56
8 4 8 2$
8 8 8 8
ND ND 8 0&
ND ND 8 8
ND, not done. a Sera antibody titers (the log10 GMT for each group) to EHc or FHc were shown in table, respectively. b Sera neutralization titers against BoNT/E or BoNT/F were shown in table, respectively. c Mice alive (8 mice/group) after i.p. challenge with a dose 103 LD50 of biologically active each BoNTs (BoNT/E or BoNT/F) 4 weeks after the last injection, respectively. d Mice alive (8 mice/group) after i.p. challenge with 104 LD50 of BoNT/F 4 weeks after the last injection. e Number of vaccinations. * P = 0.025 indicates significant titer difference between three doses of pSCARSEHc and pVAX1SEHc-vaccinated groups. # P = 0.005 indicates significant titer difference between three doses of pSCARSFHc and pVAX1SFHc-vaccinated groups. @ P = 0.011 indicates significant titer difference between four doses of pSCARSFHc and pVAX1SFHc-vaccinated groups. $ P = 0.007 indicates significant survival difference between three doses of pSCARSFHc and pVAX1SFHc-vaccinated groups when challenged with a dose of 103 LD50 of BoNT/F. & P = 0.00016 indicates significant survival difference between three doses of pSCARSFHc and pVAX1SFHc-vaccinated groups when challenged with a dose of 104 LD50 of BoNT/F.
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Table 2 Survival, sera antibody titers, and neutralization titers of mice following vaccination s.c. with VRP-EHc or VRP-FHc. Vaccine
Log10 GMT(SD)a e
RVP-EHc RVP-FHc
Neutralization titer (IU/ml)b e
e
e
Number alivec e
Number alived e
2
3
2
3
2
3
2e
3e
3.42(0.32) 3.59(0.48)
3.96(0.16)* 4.14(0.25)#
0.16 0.32
1.28 1.28
6 7
8 8
ND 4
ND 8
ND, not done. a Antibody titers (the log10 GMT for each group) to EHc or FHc were shown in table, respectively. b Sera neutralization titers against BoNT/E or BoNT/F were shown in table, respectively. c Mice alive (8 mice/group) after i.p. challenge with 103 LD50 of biologically active each BoNTs (BoNT/E or BoNT/F) 4 weeks after the last injection, respectively. d Mice alive (8 mice/group) after i.p. challenge with 104 LD50 of BoNT/F 4 weeks after the last injection. e Number of vaccinations. * P = 0.001 indicates significant titer difference between two doses of VRP-EHc and three doses of VRP-EHc-vaccinated groups. # P = 0.011 indicates significant titer difference between two doses of VRP-FHc and three doses of VRP-FHc-vaccinated groups.
VRP-FHc was also observed and the group GMTs or neutralization antibody levels correlated well with protection (Table 2). Control mice did not show serological reactivity to either EHc or FHc and showed no protection against BoNT/E or BoNT/F. 3.3. Bivalent replicon vaccines for BoNT serotypes E and F We also evaluated two types of bivalent vaccine candidates: a dual-expression DNA replicon vaccine pSCARSE/FHc and a mixture vaccine composed of pSCARSEHc and pSCARSFHc. As shown in Table 3, antisera from mice immunized with the mixture and dualexpression DNA replicon vaccines demonstrated high IgG titers to each of the individual Hc based on ELISA and sufficient neutralizing antibody levels based on BoNTs neutralization assay. And vaccination of mice with either the dual-expression DNA replicon vaccine or a mixture of DNA replicon vaccines resulted in complete protection of the animals against challenge with 1000 LD50 of BoNT serotypes E and F mixture. To determine the protective effects of the bivalent vaccine against a higher neurotoxin challenge dose, vaccinated mice were challenged with a dose of 10,000 LD50 of BoNT/F, and complete immune protection was also observed (Table 3). Additionally, dual-expression replicon particle VRP-E/FHc was also developed using DNA-based SFV vectors. The immunogenicity of the single bivalent replicon VRP-E/FHc vaccine was compared with another bivalent vaccine composed of a mixture of individual VRP-EHc and VRP-FHc. Antisera from mice immunized with the mixture and dual-expression VRP vaccines also showed high IgG titers and sufficient neutralizing antibody levels. Vaccination with either dual-expression VRP vaccine or a mixture of VRP vaccines completely protected the mice against challenge with a dose of 1000 LD50 of BoNT serotypes E and F mixture and mostly protected the mice against challenge with a higher neurotoxin challenge dose (10,000 LD50 of BoNT/F) (Table 3). Meanwhile, mice were inoculated with individual pSCARSEHc, pSCARSFHc, VRP-EHc or VRP-FHc as control vaccine groups and challenged similarly. These single vaccines only induced detectable antibody titers to the respective EHc or FHc antigen and did not protected the vaccinated mice with a challenge of mixtures of BoNT/E and BoNT/F (P = 0.00016 < 0.001). These results revealed that the immunogenicity and protective efficacy of dual-expression replicon bivalent vaccines were comparable to that of the mixture of two individual replicon vaccines against BoNT/E and BoNT/F. 3.4. Tetravalent replicon vaccines for BoNT serotypes A, B, E, and F Further, tetravalent replicon vaccines against BoNTs were developed and the protective capability was evaluated against challenge with mixture of BoNTs in mice. We evaluated four types of tetravalent replicon vaccine candidates: two dual-expression DNA replicon vaccines combination (pSCARSA/BHc + pSCARSE/FHc), a mixture vaccine composed of four single DNA replicon vaccines
(pSCARSAHc + pSCARSBHc + pSCARSEHc + pSCARSFHc), two dualexpression VRP vaccines combination (VRP-A/B + VRP-E/F), and another mixture vaccine composed of four single VRP vaccines (VRP-AHc, VRP-BHc, VRP-EHc and VRP-FHc). As shown in Table 4, antisera from mice immunized with these different tetravalent replicon vaccines demonstrated high IgG titers to each Hc based on ELISA and efficacious neutralizing antibody levels against each BoNTs. And the vaccination resulted in complete protection of the animals against challenge with 100 LD50 of BoNT serotypes A, B, E and F mixture and mostly protected the mice following challenge with a higher neurotoxin challenge dose (a mixture of 1000 LD50 of each BoNTs) (Table 4). Also, mice were inoculated with bivalent replicon vaccines as control vaccine groups and challenged similarly. The vaccinated mice succumbed to botulism and died within 24 h with a challenge of mixtures of BoNTs (P = 0.00016 < 0.001). Our findings revealed that vaccination with these different tetravalent replicon vaccines elicited similar antibody responses that conferred equal levels of protection against the challenge with mixture of BoNTs (P = 0.4667 > 0.05). Therefore, our studies demonstrate the utility of combining dual-expression plasmid DNA replicon or replicon particle vaccines into multiagent formulations as tetravalent vaccines for eliciting protective immune responses to four serotypes of BoNTs. 4. Discussion BoNTs are the most toxic proteins for humans and have been described as a potential toxic agent for malicious applications. The current formalin-inactivated pentavalent vaccine is protective against serotypes A–E, but not against the serotype F. Of seven BoNT serotypes (A–G), serotypes A, B, E and F usually cause human botulism. Therefore, there is currently a demand for a tetravalent vaccine or preparation that provides protection against all four of these serotypes [33]. This also promoted the current study to produce tetravalent vaccines to neutralize the four BoNTs serotype and prevent botulism. Previously, Pushko et al. reported that bivalent vaccines based on VEE virus replicon protected guinea pigs against infection with Lassa and Ebola viruses [34]. Smith et al. used the alphavirus replicon system to elicit an immune response in mice that protected against the Marburg virus, anthrax toxin, and BoNT/C [28]. In our recent studies, bivalent candidate replicon vaccines against BoNT serotypes A and B were developed using DNA-based SFV vectors [30]. The current study extends these findings to show the feasibility of generating multi-agent replicon vaccines that protects against the four serotypes of BoNT. In this study, the DNA replicon vaccines pSCARSEHc or pSCARSFHc can induce stronger humoral response and provide more effective protection against BoNT/E or BoNT/F than the corresponding conventional DNA vaccines encoding the same antigens, as previously described by using the DNA replicon vaccines
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Table 3 Survival, sera antibody titers, and neutralization titers of mice following vaccination three times with bivalent replicon vaccines against BoNTs. Vaccine
10 g(each) 10 g 106 IU(each) 106 IU
pSCARSEHc + pSCARFBHc pSCARSE/FHc RVP-EHc + RVP-FHc RVP-E/FHc a b c d
Log10 GMT(SD)a
Dose
Neutralization titer (IU/ml)b
Number alive
EHc
FHc
BoNT/E
BoNT/F
103 c
104 d
4.03(0.27) 4.11(0.32) 3.96(0.16) 3.88(0.14)
4.11(0.23) 4.10(0.32) 4.10(0.32) 3.80(0.28)
0.64 0.64 1.28 0.64
1.28 0.64 1.28 1.28
8 8 8 8
8 8 8 6
Sera antibody titers to EHc or FHc (the log10 GMT for each group) were shown in table, respectively. Sera neutralization titers against to BoNT/E or BoNT/F were shown in table, respectively. Mice alive (8 mice/group) after i.p. challenge with 103 LD50 of biologically active BoNT/E and BoNT/F mixture 4 weeks after the last injection. Mice alive (8 mice/group) after i.p. challenge with 104 LD50 of BoNT/F 4 weeks after the last injection.
Table 4 Survival, sera antibody titers, and neutralization titers of mice following vaccination three times with tetravalent replicon vaccines against BoNTs. Vaccine
pSCARSAHc + pSCARSBHc + pSCARSEHc + pSCARSFHc pSCARSA/BHc+ pSCARSE/FHc RVP-AHc + RVP-BHc+ RVP-EHc + RVP-FHc RVP-A/BHc + RVP-E/FHc a b c
Dose
Log10 GMT(SD)a
Neutralization titer (IU/ml)b
Number alivec
AHc
BHc
EHc
FHc
BoNT/A
BoNT/B
BoNT/E
BoNT/F
102
103
10 g(each)
3.75(0.14)
3.64(0.27)
3.96(0.16)
3.92(0.16)
0.16
0.32
0.64
0.64
8
8
10 g(each)
3.51(0.27)
3.65(0.28)
3.81(0.40)
3.76(0.60)
0.32
0.64
0.64
1.28
8
7
106 IU(each)
3.72(0.28)
3.61(0.51)
3.82(0.51)
4.14(0.30)
0.32
0.64
1.28
1.28
8
6
106 IU(each)
3.56(0.23)
3.57(0.27)
3.72(0.28)
3.78(0.36)
0.32
0.64
0.64
0.64
8
8
Sera antibody titers (the log10 GMT for each group) to AHc, BHc, EHc or FHc were shown in table, respectively. Sera neutralization titers against to BoNT/A, BoNT/B, BoNT/E or BoNT/F were shown in table, respectively. Mice alive (8 mice/group) after i.p. challenge with the indicated dose of BoNT serotypes A, B, E and F (102 or 103 LD50 , each) mixture 4 weeks after the last injection.
pSCARSAHc or pSCARSBHc against BoNT/A or BoNT/B, respectively [29,30]. And, our results demonstrated that the VRP vaccines VRP-EHc or VRP-FHc as an alternative vaccine format were capable of inducing efficient protection against challenge with BoNT/E or BoNT/F, just as candidate VEE virus replicon vaccines against BoNT/A and BoNT/C [27,28]. Moreover, we constructed and tested a dual-expression DNA replicon vaccine pSCARSE/FHc as well as a dual-expression RVP Vaccine VRP-E/FHc using DNA-based SFV vectors as previously described for BoNT/A and BoNT/B [30]. Mice immunized with the bivalent replicon vaccines were protected against challenge with mixture of BoNT/E and BoNT/F as a combination vaccine composed of two monovalent replicon vaccines (Table 3). Protective effect was consistent with the serological reactivity observed above. These results showed that alphavirus replicon vaccines could induce adequate immune responses and protective effects without the need for high doses of DNA/VRP or demanding immunization regimens. No different efficacy against BoNTs was observed in two types of vaccination methodologies in this study and further studies are underway to assess and better define their utility. Finally, tetravalent replicon vaccines against BoNT serotypes A, B, E and F were developed and the protective capability was evaluated against challenge with BoNTs. An effective tetravalent vaccine must simultaneously neutralize and protect against the four BoNT serotypes. We evaluated four types of tetravalent replicon vaccine candidates. Mice immunized with the tetravalent replicon vaccines were resistant to challenge with mixture of BoNT serotypes A, B, E and F as multivalent subunit vaccines against BoNTs [14,15]. The antibody response, neutralization antibody level and protective efficacy of tetravalent vaccines composed of two dual-expression replicon vaccines were comparable to that of mixture of four individual replicon vaccines against four BoNT serotypes. As shown in our results, protective potency against BoNTs correlated well with anti-Hc and BoNTs neutralization antibody levels, which is consistent with the concept that antibodies play a key role in protection against toxins. In addition to the high degree of observed protection against the four serotypes of BoNTs simultaneously, dual-expression replicon tetravalent
vaccines for BoNTs may offer other advantages, including simplification of experimental immunization regimens and cost savings in manufacturing one product instead of two or two instead of four. To our knowledge, this is the first report to date demonstrating the preparation of tetravalent DNA or RVP vaccines for eliciting protective immune responses to all four serotypes of BoNTs. In summary, experimental individual replicon vaccines for BoNT/E or BoNT/F and bivalent replicon vaccines for both BoNT/E and BoNT/F were developed using DNA-based SFV vectors. More importantly, our results demonstrated the utility of combining individual DNA replicon or replicon particle vaccines into multiagent formulations as tetravalent vaccines for eliciting protective immune responses against four serotypes of BoNTs. Note of, the immunogenicity of DNA vaccine against BoNT/A was enhanced by the dual-expression replicon vector expressing GM–CSF adjuvant [35], and one new dual-expression replicon vector vaccine simultaneously expressed PA and LF of anthrax are being assessed in our laboratory. Except for simplification of immunization regimens and cost savings in manufacturing, this type of dual-expression vector could enhance the potency of vaccine as mixture of individual PA and LF-DNA vaccines [36]. Also, multivalent smallpox DNA vaccine composed of eight different DNA plasmids developed by Hirao et al. [37] can be replaced with our four dual-expression vectors. Currently, there also is a demand for a new generation of safe and highly potent multivalent vaccines, which could simultaneously provide efficient protection against several important pathogens [38]. Thus, we described a new and efficient strategy to design and develop multivalent vaccines against multi-agent pathogens or specifically certain pathogen using DNA-based SFV replicon vectors.
Acknowledgments This work was partly supported by grants from the National Natural Science Foundation of China (30901375) and Beijing Natural Science Foundation (7102125).
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