Vaccine 27 (2009) 28162822

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Vaccine
journal homepage: www.elsevier.com/locate/vaccine

The recombinant Hc subunit of Clostridium botulinum neurotoxin serotype A is an effective botulism vaccine candidate
Yun-Zhou Yu, Na Li, Heng-Qi Zhu, Rui-Lin Wang, Yun Du, Shuang Wang, Wei-Yuan Yu, Zhi-Wei Sun
Beijing Institute of Biotechnology, 20 Dong Dajie Street, Fengtai District, Beijing 100071, China

a r t i c l e

i n f o

a b s t r a c t
Vaccination with recombinant His-tagged isoforms of the Clostridium botulinum Hc domain of neurotoxin serotype A (rAHc) have effectively protected against challenge with active botulinum neurotoxin serotype A. To establish a formulation suitable for human use, rAHc was expressed in Escherichia coli without a His-tag and puried by sequential chromatography on ion-exchange and hydrophobic-interaction resins. Puried rAHc was used to vaccinate mice and survival was evaluated following challenge with active toxin. rAHc-vaccinated mice were protected against an active toxin challenge in mouse models of disease and a doseresponse relationship was observed between the dose of rAHc administered and protection. Vaccination with rAHc in the presence or absence of adjuvants was also tested following intramuscular or subcutaneous vaccination to determine the optimal route of vaccination in the context of active toxin challenge. The data presented in the report suggested that rAHc administered with or without adjuvants functioned effectively over time in protecting mice against challenge with neurotoxin suggesting that this form of rAHc may be developed into a human vaccine candidate designed for the prevention of botulism. 2009 Elsevier Ltd. All rights reserved.

Article history: Received 1 December 2008 Received in revised form 9 February 2009 Accepted 25 February 2009 Available online 11 March 2009 Keywords: Clostridium botulinum Botulism Recombinant AHc Neurotoxin Vaccine

1. Introduction The Clostridium botulinum neurotoxins (BoNTs) produced by C. botulinum are among the most potent toxins affecting humans and can be divided into seven serotypes (AG) which possess similar structures but are antigenically distinct. Serotype A is the most toxic but human botulism can also result from C. botulinum isolates producing serotype B, E or F. Each BoNT is 150 kDa and consists of a heavy (100 kDa) and a light (MW 50 kDa) chain connected by a single disulde bond and each BoNT contains three functional domains e.g., an N-terminal catalytic domain (light chain), an internal heavy chain translocation domain (HN domain, 50 kDa) and the C-terminal heavy chain receptor-binding domain (Hc domain, 50 kDa) [1,2]. Botulism can be effectively prevented by the presence of neutralizing antibodies (as a consequence of vaccination) against the botulinum neurotoxin. The most commonly used human botulism vaccine is a formalin-inactivated pentavalent vaccine (PBT) that is protective against serotypes AE. However, this formulation is expensive, difcult to produce and involves a hazardous detoxication process [3,4]. Therefore the development of novel vaccine-design strategies for the prevention of botulism

based on the production of nontoxic recombinant BoNT proteins is necessary [4,5]. More recently, a synthetic oligonucleotide form of the Hc domain of C. botulinum neurotoxin serotype A (AHc) was generated and a soluble recombinant AHc His-tag fusion was expressed in E. coli by our laboratory that conferred protection in mice against challenge with active botulinum neurotoxin serotype A [6,7]. To produce a vaccine suitable for human use, a non-His-tagged rAHc isoform was puried using sequential chromatography with ionexchange (SP and Q) and hydrophobic-interaction (HIC) resins. The rAHc was evaluated as a subunit vaccine candidate in mouse models of botulism. The data presented in this report demonstrate that rAHc administered in the presence or absence of adjuvant protected mice against a lethal botulinum neurotoxin serotype A challenge and elicited high anti-rAHc antibodies that were long-lived, suggesting that this form of rAHc may be suitable for use in humans.

2. Materials and methods 2.1. Expression of rAHc in E. coli The oligonucleotide primers for PCR amplication of the synthetic AHc gene (amino acids 868 through 1296, 50 kD) using pGEM-AHc as a template were (underlined sequence indicate restriction enzyme recognition sites) forward primer, F-

Corresponding author. Tel.: +86 10 63855329; fax: +86 10 63833521. E-mail address: yunzhouyu@163.com (Z.-W. Sun). 0264-410X/$ see front matter 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2009.02.091

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HcE (EcoRI): 5 -GCCGGAATTCTAATGGAATACATCAAGAACATCATC3 , and reverse primer R-HcX (XhoI): 5 -GTTGGGGTGAACGTCCACTGATTATCCTCGAGCTAG-3 . The PCR products were digested with EcoRI and XhoI to excise the AHc DNA fragment which was subcloned into a pTIG-Trx expression vector [6] (cut with the same restriction enzymes) resulting in recombinant plasmid pTIGTrx-AHc1 that was conrmed by sequencing. pTIG-Trx-AHc1 was subsequently transformed into E. coli strain BL21 (DE3) (Stratagene, La Jolla, CA) and grown in Luria Bertani (LB) medium containing 100 g/ml ampicillin at 37 C until an optical density at 600 nm was 0.5. Isopropyl--d-thiogalactopyranoside (IPTG, Sigma) (1 M) was added to a nal concentration of 0.2 mM for an additional 3 h. Cells (2 l cultures) were centrifuged at 6000 rpm for 10 min, resuspended in buffer A (20 mM sodium phosphate, pH 7.4) and then stored at 20 C until used. 2.2. Purication of recombinant AHc E. coli expressing AHc was thawed in 200 ml of buffer A at 4 C and disrupted by sonication. The resulting lysates were centrifuged at 15,000 g for 30 min at 4 C and small-scale rAHc purication using conventional chromatography was carried out as described by the manufacturer. Briey, HiTrapTM SP FF (5 ml) (Pharmacia Biotech, Sweden) was used in the rst purication step. The column and buffers were maintained at ambient temperature and the sample was kept on ice during loading. The column was equilibrated with 10 column volumes (CV) of buffer (20 mM sodium phosphate, pH 7.4) at a rate of 100 cm/h and then loaded with the ltered lysates at a rate of 60 cm/h and then washed with 10 CV of equilibration buffer at a rate of 100 cm/h. Recombinant product was eluted from the column using step elution with 6 CV of 300 mM NaCl in equilibration buffer at a rate of 60 cm/h. The column was stripped with 1 M NaCl in equilibration buffer and then cleaned with a mixture of 1 M NaOH and 1 M NaCl. The fraction obtained from the SP column was dialyzed at 4 C against 20 vol of 20 mM TrisHCl, pH 7.4 to decrease the sample conductivity in preparation for the negative purication step. Negative purication was carried out using HiTrapTM Q FF (5 ml, Pharmacia). The column was equilibrated with 10 CV of buffer (20 mM TrisHCl, pH 7.4) at a rate of 100 cm/h prior to loading the dialyzed SP product pool. After loading was complete, the column was washed with 6 CV of equilibration buffer and the ow-through fraction containing the rAHc product was collected. Bound proteins were eluted with increasing concentrations of NaCl up to 1 M in sodium phosphate buffer and the column was cleaned with a mixture of 1 M NaOH and 1 M NaCl. In the nal chromatography step, the Q product fraction was adjusted to 2.6 M NaCl and applied to a HiTrapTM octyl (high sub) FF (5 ml, Pharmacia). The column was pre-equilibrated with 10 CV of high-ionic-strength buffer (2.6 M NaCl in 20 mM sodium phosphate, pH 7.4) at a rate of 100 cm/h. After loading was complete, the column was washed with 10 CV of equilibration buffer and product was eluted with 50 mM NaCl in sodium phosphate buffer. The column was cleaned with a mixture of 250 mM NaOH and 250 mM NaCl. The HIC product fraction was dialyzed at 4 C against 20 mM sodium phosphate buffer, pH 7.4, to remove residual salt. The product was frozen at 70 or 20 C for storage. The soluble fraction and puried rAHc were subjected to 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) and Western blot analysis using hyperimmune horse anti-neurotoxin A antiserum (National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China). Following SDSPAGE proteins were transferred onto a nitrocellulose membrane (Hybond-C, Amersham) and blocked with 5% nonfat dry milk for 2 h. The blots were washed with 0.1% Tween 20 in phosphate

buffered saline (PBST) three times (5 min each) between each incubations step. The blots were subsequently incubated with 1:200 dilution of horse anti-serum for 1 h at room temperature. After washing the blots were then incubated with 1:2000 dilution of goat anti-horse IgGHRP (Santa Cruz Biotechnology, Inc.), washed and 5 ml of DAB substrate were added to visualize binding. Blots probed with secondary antibody only did not revel any non-specic binding. Dilutions of the primary and secondary antibody were done in blocking buffer. Protein concentrations were estimated by using BCA protein assay (Sigma) according to the manufacturers protocol. 2.3. Vaccinations Specic pathogen-free female Balb/c mice (purchased from Beijing Laboratory Animal Center, Beijing) 6 weeks of age were randomly assigned to different treatment groups. Groups of eight mice were vaccinated with puried rAHc via intramuscular (i.m.), intraperitoneal (i.p.) or subcutaneous (s.c.) routes. rAHc was diluted in 25% (v/v) Alhydrogel (aluminum hydroxide, Sigma, St. Louis, MO) or aluminum phosphate adjuvant suspensions (containing 2% AlPO4 , Sigma) and injections were administered at 2 week intervals (100 l/injection). In some experiments, rAHc was administered without adjuvant. PBS formulated with the different adjuvants was used to vaccinate mice in the negative control groups and blood from all groups was collected via the tail vein before each vaccination or neurotoxin challenge and the serum isolated for rAHc antibody reactivity. Mice from all groups were challenged i.p. with different dosages of pure botulinum neurotoxin serotype A diluted in 20 mM sodium phosphate buffer (pH 6.5) containing 0.2% (w/v) gelatin (Wako, Japan) 3 weeks after the last vaccination. The mice were observed for 1 week and percent survival was determined for each vaccination group. In addition, the duration of the protective response was assessed in 6-week-old Balb/c mice (6 groups, 16 mice/group). Mice in group I were vaccinated i.m. with 1 g rAHc only. Groups IIIV mice were vaccinated i.m. with 0.2, 1 or 5 g rAHc formulated with aluminum hydroxide adjuvant respectively. Group V mice were vaccinated i.m. with 1 g rAHc formulated with aluminum phosphate adjuvant. Group VI mice (negative control) were vaccinated i.m. with PBS formulated with aluminum hydroxide. All groups were vaccinated i.m. three times (days 0, 15, and 30) at the indicated dosages of rAHc. All of the groups were challenged i.p. with the indicated dose of BoNT/A (LD50 ) 6 or 12 months after the last vaccination. Mice were bled at 15-day intervals after the second inoculation (1 month) and at each indicated time point. 2.4. Antibody titer measurements Sera from mice in the different treatment groups were screened for anti-rAHc antibodies by an ELISA. ELISA plates (Corning Incorporated, Corning, NY) were coated overnight at 4 C with 100 l rAHc (2 g/ml). Plates were washed with PBST between all incubations. Serum samples were serially diluted at 1:2 increments beginning at 1:100 and 100 l was added to each well for 1 h at 37 C. After washing, 100 l of a 1:2000 dilution of goat anti-mouse IgGHRP (Santa Cruz Biotechnology, Inc.) was added for 30 min at 37 C. After washing, anti-rAHc reactivity was visualized by adding 100 l 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 l of 2 M H2 SO4 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 0.5 units following subtraction of non-specic binding detected in control sera and twofold greater than that of the matched dilution of control sera. Serum samples from individual mice were assayed

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in duplicate and their mean was used to calculate the geometric mean titer (GMT) for each group. 2.5. BoNT/A neutralization assay Sera from mice vaccinated with two or three doses of AHc in the rst vaccination study were pooled for the toxin neutralization assay as described previously [7,8]. In brief, mixtures of serial dilutions of sera in phosphate buffer (50 mM Na2 HPO4 ) containing 1% gelatin with 10 LD50 of botulinum neurotoxin serotype A were incubated 0.5 h at room temperature and the mixtures were injected i.p. into Balb/c mice (1622 g) using a volume of 500 l/mouse (four mice in each group). The mice were observed for 1 week, and death or not was recorded. The concentration of neutralizing antibody in the serum was calculated relative to a World Health Organization botulinum serotype A antitoxin and neutralizing antibody titers of serum were reported as international units per milliliter (IU/ml). One IU is dened as the amount of antibody neutralizing 10,000 mouse i.p. LD50 of botulinum neurotoxin serotype A. 2.6. Statistical analysis Differences in antibody titers were analyzed statistically using the Students t-test or the paired t-test between group differences. Two-factor ANOVA was also used to determine whether the data were statistically signicant. Fishers exact test was used to determine statistical differences in survival between the treatment groups. For all tests only data resulting in p values of p < 0.05 were regarded as statistically signicant. 3. Results 3.1. Expression and purication of rAHc from E. coli DNA encoding AHc was synthesized with optimal codon usage for expression in E. coli [6]. Recombinant AHc (50 kDa) expression in E. coli (BL21) was conrmed by Western blot analysis (Fig. 1) that demonstrated a high level of expression (approximately 25% of the cell protein). Small-scale purication of rAHc was carried out using sequential chromatography using ion-exchange (SP and Q) and hydrophobicinteraction (HIC) resins. SP cation-exchange chromatography efciently captured the rAHc fragment and the SP product fraction contained approximately 57% rAHc fragment based on SDSPAGE analysis (Fig. 1, lane 5). The second chromatography step (negative purication step) removed numerous contaminants and the purication process was facilitated by the basic charge of rAHc (pI 9.1) that was easily identiable in the column ow-through. The Q product fraction contained approximately 85% rAHc fragment based on SDSPAGE analysis (Fig. 1, lane 4). The nal chromatography
Table 1 Survival and antibody titers after i.m. vaccination with rAHc. Vaccination dose Number alivea 1e 5 g 1 g 0.2 g 0.04 g
a b

Fig. 1. Analysis of rAHc during the various purication steps by SDSPAGE (A) and Western blot (B). Lanes 1 and 8, pTIG-Trx-AHc1 transformed BL21 cell lysates; lanes 2 and 9, puried, dialyzed rAHc; lane 3, HIC rAHc product; lane 4, Q rAHc product; lane 5, SP rAHc product; lane 7, pTIG-Trx vector-transformed cell lysates and lane 6, MW standards 112, 66, 45, 35 and 25 kDa. An arrow indicates the position of the rAHc.

HiTrapTM octyl purication step removed lower molecular weight band contaminants. SDSPAGE analysis of the HIC product fractions showed approximately 98% purity after this step (Fig. 1, lane 3) and the product band (50 kDa) was immunoreactive by Western blot analysis (Fig. 1, lane 9). The purication process described consistently produced 95% pure product based on HLPC and 98% pure based on Coomassie staining. Final product yields ranged from 10 to 15 mg of puried protein per liter of culture. 3.2. rAHc subunit vaccine efcacy To assess the immunogenicity of puried rAHc, an efcacy study was carried out in Balb/c mice. Mice were vaccinated i.m. once, twice, or three times using four different doses of rAHc and subsequently challenged with a dose 100,000-fold of the 50% mouse lethal dose (100,000 LD50 ) of biologically active neurotoxin serotype A. A rAHc dose- and frequency-dependent protective effect was observed (Table 1). Mice that received two injections of 0.2 g and three injections of 0.04 g were completely protected (100% survival) following challenge with neurotoxin serotype A. In contrast, mice in the negative control groups succumbed to botulism and died within 5 h. To determine the protective effects of rAHc vaccination against a higher BoNT/A challenge doses, i.m.vaccinated mice were challenged with a dose 1,000,000 LD50 of

Number aliveb 3e 8 8 8 8 2e 8 7 4 2 3e 8 8 8 6

log10 GMT(SD)c 1e 3.19(0.33) 2.96(0.35) 2.69(0.39) 2.58(0.32) 2e 4.41(0.45) 4.17(0.35) 3.87(0.31) 3.61(0.32) 3e 5.19(0.23) 4.78(0.31) 4.40(0.36) 4.10(0.32)

Serum neutralization titer (IU/ml)d 2e 5.12 2.56 0.64 0.32 3e 40.96 20.48 5.12 2.56

2e 8 8 8 7

5 4 2 1

Balb/c mice alive (8 mice/group) after i.p. challenge with a dose 100,000 LD50 of BoNT/A 3 weeks after the last injection. Mice alive (8 mice/groups) after i.p. challenge with a dose 1,000,000 LD50 of BoNT/A 3 weeks after the last injection. c Sera obtained 2 days before challenge from individual mice was assayed in duplicate and used to calculate the log10 GMT for each group. Standard deviations (SD) of GMT are in parenthesis. d Pooled sera from each group of mice vaccinated with two or three doses of AHc were diluted initially 1:10 and then twofold for serum neutralization titers. e Number of vaccinations. Mice were vaccinated either once, twice, or three times at four different dosages of rAHc range from 0.04 to 5 g. rAHc was diluted in 25% (v/v) Alhydrogel (aluminum hydroxide) and injections were administered at 2 weeks intervals (100 l/injection).

Y.-Z. Yu et al. / Vaccine 27 (2009) 28162822 Table 2 Balb/c and KM survival and antibody titers following i.m. rAHc vaccination. Mouse strain Number alivea 2 Balb/c KM
c

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Table 3 Survival and antibody titers following i.m. rAHc vaccination. Adjuvant Number alivea 2
** c

log10 GMT(SD)b 3
c

log10 GMT(SD)b 2c 4.07(0.25) 3.95(0.23) 3c 4.70(0.36) 4.63(0.28)

6 5

8 8

4.10(0.28) 4.03(0.27)

4.74(0.34) 4.55(0.28)*

Aluminum hydroxide Aluminum phosphate

7 6

8 8

a Mice alive (8 mice/group) after an i.p. challenge with a dose 1,000,000 LD50 of BoNT/A 3 weeks after the last injection. b Sera obtained 2 days before challenge from individual mice were assayed in duplicate and used to calculate the log10 GMT for each group. GMT SD reported in parenthesis. c Number of vaccinations. Balb/c and KM mice were vaccinated twice or three times with 1 g of rAHc. rAHc was diluted in 25% (v/v) Alhydrogel and injections were administered at 2 weeks intervals (100 l/injection). * P = 0.02 < 0.05 indicates signicant titer difference between two- and three-dose groups. ** P = 0.0017 < 0.01 indicates signicant titer difference between two- and threedose groups.

a Balb/c mice (8 mice/group) were challenged i.p. with a dose1,000,000 LD50 of BoNT/A 3 weeks after the last immunization. b Sera obtained 2 days before challenge from individual mice were assayed in duplicate and used to calculate the log10 GMT for each group. SD reported in parenthesis. c Number of vaccinations. Balb/c mice were vaccinated twice or three times at 1 g dosage of rAHc. Vaccines were diluted in 25% (v/v) Alhydrogel (aluminum hydroxide) or aluminum phosphate adjuvant suspensions (containing 2% AlPO4 ) and injections were administered at 2 week intervals (100 l/injection).

neurotoxin serotype A (Table 1). Two injections partially or completely protected the mice at all dose tested with 100% survival observed at a dose of 5 g. Three injections of 0.2 g afforded complete protection (100% survival). Serum antibody titers for individual mice were analyzed by the ELISA and the GMTs for each group in the study were determined. A dose-dependent antibody response to vaccination with rAHc was observed and the group GMT correlated well with protection (Table 1). The group geometric mean titer of mice injected with a single dose of rAHc was relative low (2.583.19) depending on the immunization dose and these mice were partially protected. Following second and third vaccinations the GMTs increased significantly (3.87, P < 0.01) and correlated with 100% survival following a challenge with a 100,000 LD50 of neurotoxin serotype A. Similarly, the four treatment groups that were 100% protected had geometric mean titers of 4.4 when challenged with a dose 1,000,000 LD50 of neurotoxin serotype A. Serum from the ELISA study was used in a BoNT/A neutralization assay. Due to the limited amount of serum available, serum from each group of mice vaccinated with two or three doses of AHc was pooled, and so only the average neutralization titer of the group could be assayed. As shown in Table 1, neutralizing antibody titers increased with the number and dose of vaccinations and correlated well with group ELISA antibody titers and protection. Groups with partial survival against a challenge with a dose 1,000,000 LD50 of neurotoxin serotype A all had neutralizing titers of 2.56 IU/ml, and groups with 100% survival all had neutralizing titers of 5.12 IU/ml. The immunogenicity and protective efcacy of rAHc was also evaluated in a KM (Chinese Kun Ming) mouse model. Mice were vaccinated i.m. two or three times at a dose of 1 g per mouse and subsequently challenged with a dose 1,000,000 LD50 of biologically active neurotoxin serotype A. Survival and group antibody ELISA titers of the Balb/c and KM mice are shown in Table 2. Results revealed that the immunogenicity and protective efcacy of rAHc vaccine in KM mice was comparable to that of Balb/c mice. These results indicated that soluble rAHc was a highly effective immunogen and protected against a lethal dose of biologically active botulinum neurotoxin serotype A in mice of different genetic backgrounds. 3.3. rAHc subunit vaccine efcacy in the presence or absence of adjuvant Two types of adjuvants (aluminum hydroxide and aluminum phosphate) were tested in rAHc vaccine efcacy trials. Mice were vaccinated i.m. two or three times at a dose of 1 g rAHc per mouse and subsequently challenged with a dose of 1,000,000 LD50

of biologically active neurotoxin serotype A. Survival and group antibody ELISA titers are shown in Table 3. Results revealed that vaccination with rAHc formulated with aluminum hydroxide or aluminum phosphate adjuvant elicited similar anti-rAHc antibodies that conferred equal levels of protection against botulinum neurotoxin serotype A challenge. Furthermore, rAHc administered via different vaccination routes e.g., i.m., s.c. or i.p. and then challenged with a dose of neurotoxin 1,000,000 LD50 of biologically active neurotoxin serotype A demonstrated that protection against toxin challenge was not affected by the route of vaccination (Table 4). The potency of the rAHc subunit vaccine in the absence of adjuvant was also studied in the context of the vaccination route. Mice vaccinated i.m. or s.c. with rAHc as described above in the absence or presence of an adjuvant and challenged with a dose of 100,000 of LD50 of biologically active neurotoxin serotype A showed that in the absence of the adjuvant rAHc elicited a modest IgG response (P < 0.001 compared to negative control group) and in the presence of the adjuvant this response was signicantly elevated (P < 0.01 compared to rAHc-only treated mice) (Table 5). Mice vaccinated i.m. or s.c. with rAHc in the presence of an adjuvant were fully protected against neurotoxin challenge, in contrast, mice vaccinated i.m. or s.c. with two doses of 1 g rAHc without adjuvant were partially protected (P < 0.05 compared to vaccinations with adjuvant) and mice vaccinated i.m. or s.c. with three doses of 1 g rAHc in the absence of an adjuvant showed nearly complete protection (P = 0.007 compared to the negative control i.m. group and P = 0.0014 compared to the negative s.c. control group). The surviving mice were given a second challenge with much higher neurotoxin doses (1,000,000 LD50 ). Most of the surviving mice were protected except that of vaccination with two doses of AHc in the absence of an adjuvant (data not shown). These results indicated that rAHc in the absence or presence of adjuvant was effective in protecting against botulism depending on the number of immunizations.
Table 4 Survival and antibody titers of following i.m., s.c. or i.p. rAHc vaccinations. Vaccination route Number alivea 2c i.m. s.c. i.p. 5 7 7 3c 8 8 8 log10 GMT(SD)b 2c 4.10(0.28) 4.18(0.27) 4.14(0.25) 3c 4.70(0.32) 4.78(0.31) 5.82(0.28)

a Balb/c mice (8 mice/group) were challenged i.p. with a dose 1,000,000 LD50 of BoNT/A 3 weeks after the last injection. b Sera obtained 2 days before challenge from individual mice were assayed in duplicate and used to calculate the log10 GMT for each group. SD indicated in parentheses. c Number of vaccinations. Mice were vaccinated twice or three times with 1 g of rAHc. Vaccines were diluted in 25% (v/v) Alhydrogel and injections were administered i.m., s.c. or i.p. to Balb/c mice at 2 weeks intervals (100 l/injection).

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Y.-Z. Yu et al. / Vaccine 27 (2009) 28162822 Table 6 Long term protection against neurotoxin challenge following rAHc vaccinationa . Group I II III IV V VIf
d

Table 5 Survival and antibody titers after i.m. or s.c. vaccination with rAHc. Vaccination route i.m. i.m. s.c. s.c. Adjuvant + + Number alivea 2c 3c 8** 2 8* 3 8 6 8 7 log10 GMT(SD)b 2c 3c 4.07(0.25) 2.77(0.32) 4.14(0.25) 3.00(0.28) 4.67(0.30) 3.64(0.33) 4.74(0.30) 3.80(0.23)

Dose (g) 1 0.2 1 5 1 0

Adjuvant AHe AHe AHe APe AHe

Challenge dose 10 106 106 106 106 102

5

Survivalb 6/8 8/8** 8/8** 8/8** 8/8** 0/8

*

Survivalc 4/8 6/8* 8/8** 8/8** 8/8** 0/8

a Balb/c mice were challenged i.p. with a dose 100,000 LD50 of BoNT/A 3 weeks after the last immunization. b Sera obtained 2 days before challenge from individual mice were assayed in duplicate and used to calculate the log10 GMT for each group. SD indicated in parentheses. c Number of vaccinations. Mice were vaccinated twice or three times at 1 g dose of rAHc. Vaccines were diluted in 25% (v/v) Alhydrogel (+) or PBS () and injections were administered to Balb/c mice at 2 weeks intervals (100 l/injection). * P = 0.026 < 0.05 indicates signicant survival difference between the s.c.vaccinated groups receiving two doses of 1 g rAHc in the absence of an adjuvant and the s.c.-vaccinated group vaccinated with two doses of 1 g rAHc in the present of an adjuvant. ** P = 0.007 < 0.01 indicates signicant survival difference between i.m. vaccination groups receiving two doses of 1 g rAHc in the absence of an adjuvant and the i.m.-vaccinated group vaccinated with two doses of 1 g rAHc in the presence of adjuvant.

a Survival of mice vaccinated i.m. three times (days 0, 15, and 30) with rAHc. Comparisons between experimental groups to control group. b Mice were challenged i.p. with the indicated dose of BoNT/A (LD50 ) 6 months after the last immunization. c Mice were challenged i.p. with the indicated dose of BoNT/A (LD50 ) 12 months after the last immunization. d Mice vaccinated with rAHc without Alhydrogel adjuvant (). e AH, aluminum hydroxide; AP, aluminum phosphate. f PBS vaccinated mice (negative control). * P = 0.007 < 0.01. ** P = 0.00016 < 0.001.

3.4. Duration of protective immunity To evaluate the duration of immunity induced by rAHc vaccinations mice were vaccinated i.m. with rAHc on days 0, 15, and 30 and then serologically monitored for 1 year (Fig. 2). The results of group antibody ELISA titers were obtained at the times indicated in Fig. 2. Following the third vaccination (day 30) the GMT was signicantly increased (P < 0.01). A dose-response effect was also observed with larger GMTs correlating with higher concentrations (or dosages) of rAHc administered during vaccinations. The antibody responses in mice vaccinated with 1 g rAHc plus adjuvant tended to be always higher than in mice vaccinated in the absence of an adjuvant (P < 0.01). After 2 months, these three vaccination groups (IIIV) receiving 1 and 5 g AHc in the presence of aluminum hydroxide and 1 g rAHc with aluminum phosphate showed a trend towards relatively stable GMTs over a 12-month period. The GMTs resulting from vaccinations with rAHc adsorbed with aluminum hydroxide were equivalent to those observed in mice immunized

with rAHc adsorbed in aluminum phosphate. The peak antibody response in mice (group II) vaccinated at a dose of 0.2 g rAHc formulated with aluminum hydroxide was 4.56 (6 months post the last vaccination) and the peak antibody response in group I mice vaccinated with 1 g rAHc without aluminum hydroxide was 3.92 (5 months post the last vaccination). Different vaccination groups were challenged either 6 or 12 months post the last vaccination with different dose of biologically active neurotoxin serotype A as indicated in Table 6. Three experimental groups (IIIV) vaccinated with 1 or 5 g rAHc plus adjuvant were 100% protected 6 and 12 months post the last vaccination when challenged i.p. with a dose of 1,000,000 LD50 of biologically active neurotoxin serotype A. The II experimental group receiving a dose of 0.2 g rAHc plus aluminum hydroxide afforded complete protection at month 7 following an i.p. challenge of 1,000,000 LD50 of biologically active neurotoxin. Group I vaccinated with 1 g rAHc without aluminum hydroxide was signicantly protected against challenge with 100,000 LD50 of neurotoxin at month 7 (P = 0.007 compared to negative control group) following an i.p. challenge. The results of the i.p. challenge experiments were identical to those described above. Groups I and II 12 months post the last vaccination showed a relative decrease in potency, which correlated well with the group GMT 12 months post the last vaccination. All surviving mice challenged at month 7 or 13 later remained healthy without overt signs of BoNT intoxication. The mice in the negative control group (VI) succumbed to toxin challenge and demonstrated typical accid paralysis and died within 5 h. These results indicated that rAHc could induce a long-term effective and protective antibody response with the capacity of preventing botulism. 4. Discussion Botulism is caused in humans primarily by BoNTs serotypes A, B, E, and F that have also been described as biological weapons [4]. Concern about the potential biological warfare use of the toxin has strengthened the need for novel vaccination strategies. At present the human botulinum vaccine has met with some obstacles including feasibility (toxin yields from C. botulinum are relative low), danger associated with toxoiding large quantities of toxin, the toxoided product consists of a crude extract of clostridial proteins that may inuence vaccine efcacy and the toxoided vaccine can elicit secondary complications resulting from residual formaldehyde contamination [3]. There is therefore a need for improved vaccines designed to prevent botulism. Current research efforts to develop new botulism vaccines have focused on the production of nontoxic recombinant BoNTs [3,4]. These recombinant Hc

Fig. 2. Duration of rAHc-induced immunity in Balb/c mice. Groups (I through V) were vaccinated i.m. with rAHc vaccines. Groups I was vaccinated i.m. with rAHc only. Vaccines in four groups (II through V) were diluted in 25% (v/v) Alhydrogel (aluminum hydroxide) or aluminum phosphate adjuvant (100 l/injection). Mice were vaccinated i.m. three times on days 0, 15, and 30 (indicated by solid arrows) at the indicated doses. Group VI mice (negative control) were vaccinated i.m. with PBS mixed with aluminum hydroxide. Mice were bled at 15-day intervals after the second immunization (1 month) and at each indicated time point. Sera from individual mice were assayed in duplicate and used to calculate the log10 GMT for each group. The data are expressed as the mean SD of duplicate GMT values.

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subunit vaccines, synthesized in E. coli [912] or yeast [1321] expression vectors are less expensive and hazardous to produce than the traditional toxin-inactivated vaccines. In addition, the pure and concentrated immunogens may increase immunogenicity and protection efcacy in humans. Recombinant botulism vaccines are under development with the aim of providing protection against botulism. A milestone was reached when the rst recombinant subunit vaccine [rBV A/B (Pichia pastoris) vaccine] was tested in humans during a phase I clinical trial [5]. As an appropriate vaccine candidate, the carboxyl-terminal fragment of botulinum neurotoxin heavy chain (Hc) is responsible for toxin binding to the nerve cells, and the recombinant Hc has been proved to be a safe and efcient immunogen to generate neutralizing antibodies for immunotherapy [2]. Recently, our laboratory and others have expressed recombinant Hc of BoNTs in E. coli at levels sufcient for vaccine development [7,2226]. In this study we describe the production of a recombinant untagged Hc subunit vaccine of serotype A as a candidate subunit vaccine for human use in the prevention of botulism. A synthetic gene encoding the Hc domain of BoNT/A was designed, expressed and the gene product puried from E. coli using conventional chromatography. Recombinant AHc was assessed as a subunit vaccine against the botulinum neurotoxin serotype A in mouse models of disease. We performed the efcacy study to understand the relationship between multiple vaccinations and protection at various dose levels. Clearly, multiple injections of immunogen protected mice better than a single injection. Mice given two injections of 0.2 g or three injections of 0.04 g were completely protected when challenged i.p. with a 100,000 LD50 dose of biologically active botulinum neurotoxin serotype A. Two injections partially or completely protected mice at all dose levels and three injections afforded even greater protection with a higher challenge of BoNT/A (1,000,000 LD50 dose of botulinum neurotoxin serotype A). Protective efcacy against botulinum neurotoxin serotype A in mice vaccinated with the untagged rAHc in this study was similar to that in mice vaccinated with the rAHc with His-tag as previously reported [7]. A dose-response effect was also observed with respect to antibody titers and the dosage and frequency of the rAHc vaccinations and the group GMT correlated well with group neutralizing antibody titers and protection. The potency of the rAHc vaccine was also evaluated in a KM mouse model. Results revealed that the immunogenicity and protective efcacy of a rAHc vaccine in KM mice was comparable to that observed for Balb/c mice. The efcacy study also addressed the question of how well the E. coli-expressed rAHc protected mice following a high-dose BoNT/A challenge. Many studies have contributed to the observation that the Hc domain of BoNTs administered with an adjuvant evokes a strong immune response [5,17,25,26]. Vaccines based on recombinant proteins often require adjuvants in order to obtain an adequate immune response. Aluminum salts are the most commonly utilized adjuvants for use in human vaccine products [27]. Two general types of aluminum adjuvants approved for human use by FDA are aluminum hydroxide and aluminum phosphate. In the current study these two types of adjuvants were tested. Antibody titers and protection against botulinum neurotoxin serotype A challenge were similar in mice vaccinated with rAHc adsorbed in either aluminum hydroxide or aluminum phosphate. The results revealed that the two types of adjuvants worked effectively in facilitating the elicitation of anti-rAHc immunity. The efcacy of rAHc administered via different routes in the absence or presence of an adjuvant was also assessed. Vaccine efcacy following administration rAHc via three different routes i.e., i.m., s.c., and i.p. was assessed, demonstrating that efcacy was independent of the vaccination route. Furthermore, in the absence of the adjuvant, vaccination with rAHc alone elicited a modest IgG response and mice vaccinated i.m. or s.c. with

three doses of 1 g rAHc in the absence of an adjuvant showed nearly complete protection against a 100,000 LD50 dose challenge of biologically active neurotoxin serotype A. These results indicated that rAHc administered in the absence or presence of adjuvant conferred protection against a lethal challenge but that fewer doses were needed to reach full protection if administered with adjuvant. The duration of immunity and protection induced by rAHc vaccination was also evaluated. Although two groups (I and II) showed attenuation of antibody titers over the course of a year and were less efciently protected against challenge 13 months later, mice in three experimental groups (IIIV) were completely protected from a BoNT/A challenge 6 and 12 months after the last vaccination and no appreciable decrease in antibody titers was noted over a period of 12 months in mice from these groups suggesting a direct correlation with antibody titers and protection. Since rAHc is a vaccine candidate for use in humans, large quantities will ultimately be needed. Therefore, the purication scheme for vaccine production should be designed for high throughput production and include as few steps as possible do decrease the chances of altering the vaccines native conformation. The purication scheme described in this report repeatedly produced pure rAHc that remained stable following processing steps. This is the rst report demonstrating that a rAHc isoform not expressed with a His-tag could be puried from E. coli to produce large quantities product with the potential of being used in humans. This type of vaccine has advantages over traditional toxin-inactivated vaccines in that it is cheaper, less hazardous to produce and can be generated in large quantities [3,4]. The results presented in this report also indicated that a genetically engineered form of the rAHc produced in an E. coli expression vector was highly effective in protecting mice against challenge doses of biologically active botulinum neurotoxin serotype A. Our ultimate goal is to license and further develop this rAHc formulation for human use. We have made progress in the development of recombinant Hc vaccines against four serotypes of BoNTs (i.e., A, B, E and F) commonly associated with human botulism. The approach to produce the recombinant Hc vaccines involves the cloning and expression of nontoxic Hc domains of BoNTs, evaluation of immunogenicity and protective efcacy of the Hc domains. The Hc domains of botulinum neurotoxins serotypes B, E and F have also been successfully expressed in E. coli by our laboratory [28] demonstrating the efcacy of this expression and purication strategy in the development of the next generation of vaccines against botulism. References
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