EE

RS

a

ARRA

KERV

I

iiwawSp

gpaTHSatta

I1

(

0d

Immunobiology 216 (2011) 485–490

Contents lists available at ScienceDirect

Immunobiology

journa l homepage: www.e lsev ier .de / imbio

nhanced expression of soluble recombinant tetanus neurotoxin Hc inscherichia coli as a tetanus vaccine candidate

ui Yu, Lihua Hou ∗, Changming Yu, Shuling Liu, Jun Ren, Ting Fang, Xiaoyan Zhang, Wei Chen ∗

tate Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, 20 Dongdajie Street, Fengtai, Beijing 100071, China

r t i c l e i n f o

rticle history:eceived 23 June 2010eceived in revised form 6 September 2010ccepted 6 September 2010

eywords:

a b s t r a c t

The expression of the carboxyl fragment of the heavy chain of tetanus neurotoxin (TeNT-Hc) in Escherichiacoli has been hampered by the unusually high AT content and the presence of rarely used codons byClostridium. The gene encoding TeNT-Hc was optimized for E. coli by replacing rare codons and decreas-ing the AT pairs from 72.57% to 52.47%. The reconstructed gene was expressed in E. coli BL21(DE3)and resulted in a soluble product which was about 46% of the total bacterial protein. TeNT-Hc pro-

nhanced expressionecombinant TeNT-Hcaccine

duced in a 42 L fermentor was purified to >95% at 87 g/kg of cell paste (approximately 333 mg/L). BALB/cmice vaccinated with three bi-weekly doses of TeNT-Hc with Freund’s adjuvant were fully protectedagainst an intraperitoneally challenge of 2 × 103 50% lethal doses (LD50s) of tetanus neurotoxin. NIHmice vaccinated with TeNT-Hc adsorbed to aluminum hydroxide gel adjuvant demonstrated a potency of168 IU/mL, which was 2 times higher than the national standard for tetanus vaccines. These results suggestthat TeNT-Hc may be a promising second-generation vaccine candidate for clinical use against tetanus

neurotoxin.

ntroduction

Tetanus is characterized by the action of its neurotoxin whichs produced during the growth of the anaerobic bacterium Clostrid-um tetani (Walter and Talbot 1996). The neurotoxin is very toxic

ith the estimated human lethal dose at <2.5 ng/kg (WHO 2010)nd the dramatic muscular spasms of tetanus make it one of theorld’s most feared diseases especially in the developing countries.

eeking for effectively and safely preventive vaccines of tetanus isulled off in many countries.

The tetanus neurotoxin (TeNT) is synthesized as a 150-kDa sin-le polypeptide (Eisel et al. 1986) and is cleaved by clostridialroteases into a heavy chain of 100 kDa linked by a disulfide bond tolight chain of 50 kDa (Eisel et al. 1986; Matsuda and Yoneda 1974).he carboxyl-terminal domain of the heavy chain (fragment C orc; 50 kDa) binds to nerve terminals at the neuromuscular junction.ubsequently, the amino-terminal domain of the heavy chain (HN)

ids in the translocation of the light chain into the cytosol wherehe light chain prevents the release of the inhibitory neurotransmit-ers. The Hc domain of TeNt (TeNT-Hc), which retains full bindingffinities (Rummel et al. 2003), shows considerable promise as a

∗ Corresponding author at: Department of Applied Molecular Biology, Beijingnstitute of Microbiology and Epidemiology, 20 Dongdajie Street, Fengtai, Beijing00071, China. Tel.: +86 10 63815273; fax: +86 10 63815273.

E-mail addresses: houlihua@sina.com (L. Hou), cw789661@yahoo.comW. Chen).

171-2985/$ – see front matter © 2010 Elsevier GmbH. All rights reserved.oi:10.1016/j.imbio.2010.09.001

© 2010 Elsevier GmbH. All rights reserved.

possible next-generation sub-unit vaccine against tetanus (Heltingand Nau 1984; Maassen et al. 1999).

TeNT-Hc, which would be expected to have clear advantagesover toxoid production in regards to production, characterization,and homogeneity (Qazi et al. 2006), has not been evaluated in clini-cal trials. Due to the AT-rich (Fairweather and Lyness 1986) genomeof C. tetani and the absence of rare codon tRNAs in Escherichia coli,TeNT-Hc showed poor levels of expression and was often expressedin an insoluble form, which limited its development as a vaccineagainst tetanus. In this paper, we expressed high levels of non-tagged TeNT-Hc in E. coli in a soluble form and evaluated its efficacyas a sub-unit vaccine in a murine model exposed to 2 × 103 50%lethal doses (LD50s) of TeNT. Its potency as a human vaccine wasalso determined as 168 IU/mL which was higher than the nationalstandard for tetanus vaccines.

Materials and methods

Reagents

The Q Sepharose XL, Phenyl-Sepharose FF, SP Sepharose XL andHiPrep 26/10 Desalting column were purchased from Pharmacia

Biotech. DNA endonuclease EcoRI, XhoI and T4 DNA ligase were pur-chased from TaKaRa Company (Dalian, China). The E. coli plasmidextraction kit was purchased from Omega Company. Isopropylthio-d-galactoside (IPTG), Freund’s adjuvant (complete, incomplete) andganglioside GT1b were purchased from Sigma. Al(OH)3 gel adjuvant

4 iology

wspoiwCpwh

A

atngB

C

CArtaa3ITs

(wwflttapNaS

W

btmitmT0sowr

P

w

goat anti-mouse IgG antibody for 1 h at 37 ◦C. The plates were

86 R. Yu et al. / Immunob

as purchased from Brenntag Biosector (Denmark). The expres-ion vector pET-32a(+) and its host strain E. coli BL21 (DE3) wereurchased from Novagen. The anti-tetanus neurotoxin mouse mon-clonal antibody and human polyclonal antiserum were preparedn our lab. The goat anti-mouse and anti-human IgG conjugated

ith peroxidase were purchased from Zhongshan Goldenbridgeompany (Beijing, China). Western blotting luminol reagent wasurchased from SANTA CRUZ. All other chemicals and reagentsere obtained from other commercial sources and were of theighest purity available.

nimals and neurotoxin

BALB/c mice (female, 6–8 weeks old) and NIH mice (half malend half female, 14–16 g) were purchased from Beijing Labora-ory Animal Center. Tetanus neurotoxin, adsorbed tetanus toxoidational reference materials and formalin-inactivated toxoid wereifts from National Institute for the Control of Pharmaceutical andiological Products.

loning and expression of TeNT-Hc

The gene coding Hc fragment of tetanus neurotoxin fromlostridium tetani strain No. 64008 (GenBank accession number:F154828) was used in this work. Its sequence was optimized byeplacing rare codons with frequent ones in E. coli and decreasinghe content of AT pairs from 72.57% to 52.47%. With additional EcoRInd XhoI sites at 5′ and 3′ ends, respectively, and a TAA sequencefter the EcoRI site to end the expression of thioredoxin on pET-2a(+), the new sequence was fully synthesized in Invitrogen Inc.

t was ligated to pET-32a(+) and transformed into E. coli DH5�.he resulting plasmid named pET-32a-TeNT-Hc was proved withequencing and introduced to E. coli BL21 (DE3).

A single transformant was inoculated into 5 mL Luria–BertaniLB) medium supplemented with ampicillin (100 mg/L) and grownith 220 rpm shaking overnight at 37 ◦C. Three milliliter cultureas transferred to 300 mL fresh LB medium in a 1000 mL shakeask. The culture was grown with 220 rpm shaking at 37 ◦C untilhe OD600 reached at 0.8–1.0. IPTG was then added to a final concen-ration of 0.2 mM and the culture was grown with 220 rpm shakingt 28 ◦C for induction. The pellete was harvested after 4 h and resus-ended in 50 mL PBS (20 mM phosphate-buffered saline, 0.15 MaCl, pH 7.4). After lysed by sonication, the sample was centrifugedt 20,000 × g for 30 min and the supernatant was analyzed by 12%DS-PAGE and stained by Coomassie blue R-250.

estern blot analysis of TeNT-Hc

The identification of the expressed protein was carried outy Western blot analysis. The proteins in the supernatant con-ained TeNT-Hc from SDS-PAGE were transferred to nitrocellulose

embrane (0.2 �M). The membrane was incubated in the block-ng buffer (3% BSA in PBS) with gentle shaking for 1 h at roomemperature, and then incubated with 1:1000 of dilution of

ouse anti-tetanus neurotoxin monoclonal antibodies for 1 h.he membrane was washed 3 times with PBST (PBS containing.1% Tween 20) and then incubated in a 1:5000 of dilution ofecondary antibody (goat anti-mouse IgG conjugated with per-xidase) for 1 h at room temperature. The colorimetric detectionas carried out by being developed with chemiluminescence

eagents.

urification of TeNT-Hc from fermentor cultures

Forty-two liters fermentor containing 30 L LB culture mediumas inoculated to a starting OD600 of 0.1 from an overnight

216 (2011) 485–490

culture supplemented with ampicillin (100 mg/L). Cultures weregrown with 300 rpm shaking at 37 ◦C and then induced with0.2 mM IPTG (final concentration) at 28 ◦C and the OD600 of 0.8–1.0for 4 h. Cells were harvested by centrifugation at 10,000 × g for15 min and stored at −20 ◦C. Cell paste (115 g) was resuspendedin 1150 mL of 20 mM Tris·Cl buffer (pH 8.5) and lysed with highpressure homogenizer (900 bar, 1 time). The supernatant collectedafter centrifugation at 20,000 × g for 30 min was loaded in a QSepharose XL column with volume of approximately 300 mL resinpre-equilibrated with 20 mM Tris·Cl buffer (pH 8.5). The columnwas washed with the equilibrating buffer until the absorbance at280 nm was below 0.01 and then eluted by using a linear gradient of0–0.5 M NaCl in 20 mM Tris·Cl buffer (pH 8.5). The ammonium sul-fate powder was added to the above elution liquid containing theeluted target protein to the concentration of 0.5 M. The above elu-tion was loaded in the Phenyl-Sepharose FF column with a volumeof approximately 300 mL resin pre-equilibrated with 20 mM Tris·Cl(pH 8.0) supplemented with 0.5 M ammonium sulfate. The columnwas washed with the equilibrating buffer until the absorbance at280 nm was below 0.01 and then eluted by using a linear gradient in20 mM Tris·Cl buffer (pH 8.0) from 0.5 M ammonium sulfate to 0 Mammonium sulfate. The elution peak from the Phenyl-SepharoseFF column was exchanged buffer with 20 mM NaAc (pH 4.0) usinga HiPrep 26/10 Desalting column and loaded to the SP SepharoseXL with a volume of approximately 300 mL resin. The column waswashed with 20 mM NaAc (pH 4.0) until the absorbance at 280 nmwas below 0.01 and then eluted by using a linear gradient of 0–0.5 MNaCl in 20 mM NaAc buffer (pH 4.0). The target protein peak wascollected and analyzed by 12% SDS-PAGE. The final purity of theproduct was judged by means of SEC-HPLC. The final product wasdegermed through the 0.22 �m filter and stored at −80 ◦C in 20 mMPB buffer.

For detecting the stability of purified TeNT-Hc, the protein waskept at 4 ◦C, −20 ◦C, −80 ◦C, respectively. The stability of TeNT-Hcwas measured by 12% SDS-PAGE after being kept at different times.

Vaccination of mice

Six- to eight-week-old female BALB/c mice were used in thisstudy and every six mice were classified in one group. Each BALB/cmouse received an intraperitoneal (i.p.) injection of 1 �g, 5 �g and10 �g of TeNT-Hc in 100 �L of PBS emulsified in an equal vol-ume of complete Freund’s adjuvant. After intervals of 2 and 4weeks, booster injections were given as outlined above except thatincomplete Freund’s adjuvant was used instead. The sera from tailswere collected 2 weeks after each injection. One group of miceinjected by PBS and the adjuvant was carried out as the negativecontrol.

Measurement of antibody responses by ELISA

Using a formalin-inactivated tetanus toxoid as the solid-phaseantigen to measure the titer of the antibody induced by TeNT-Hcimmunization, 96-well plates (Costar) were coated with 1 �g/mL ofantigen in PBS at 4 ◦C and left overnight; then they were blocked for1 h with 3% BSA. The serum samples collected from mice were seriesdiluted from 1:800 to 1:409,600 in PBS, then added to the plate,and incubated for 2 h at 37 ◦C. The plates were washed with PBSTand then incubated with a 1:5000 dilution of peroxidase-labeled

washed again and the peroxidase substrate, o-phenylenediaminedihydrochloride, was added to each well and incubated at roomtemperature. The o-phenylenediamine dihydrochloride reactionwas stopped with 2 M H2SO4, and the absorbance at 490 nm wasread with a microplate reader.

iology 216 (2011) 485–490 487

G

sc2a1swbwHPt1wIdf

T

ibca

Ph

HAow

2wNiatvwactrH

R

Sp

owtp

harboring pET32a-TeNT-Hc were treated with IPTG with final con-centration of 0.2 mM at 28 ◦C. The expression of a 50 kDa proteincorresponding to the predicted size was induced in the pres-ence of IPTG (Fig. 2A). The soluble recombinant protein existed in

R. Yu et al. / Immunob

T1b binding inhibition ELISAs

The inhibition of TeNT-Hc binding to ganglioside GT1b by theerum containing anti-tetanus antibodies was measured by aompetitive ELISA. 96-Well plates were coated with 100 �L of�g/mL GT1b in methanol and plates were left at room temper-ture overnight to allow evaporation of methanol. Beginning at:100 of dilution, the sera from immunized mice were diluted inerial two-fold dilutions in PBST to 1:25,600 of dilution. Diluted seraere mixed with an equal volume of 5 �g/mL TeNT-Hc and incu-

ated for 0.5 h at 37 ◦C. After the ganglioside GT1b-coated wells wereashed with PBST, 100 �L of each TeNT-Hc/serum mix or TeNT-c alone was added to wells in triplicate and incubated for 1 h.lates were washed 4 times in PBST and incubated for 1 h at roomemperature with 1:100 of diluted human polyclonal antiserum at00 �L/well. The plates were washed with PBST and then incubatedith a 1:5000 of dilution of peroxidase-labeled goat anti-human

gG antibody for 1 h at 37 ◦C. Plates were washed and developed asescribed above. The inhibition efficiency (IE) was counted as theollowing formula:

IE (%) = (1 − OD490 of TeNT − Hc/serum mix well/OD490 of

TeNT − Hc control well) × 100%

eNT challenge of mice

Mice were challenged with tetanus neurotoxin by subcutaneousnjection of 2 × 103 LD50s of toxin with a volume of 0.5 mL in borate-uffered saline (0.5 g of borax, 4.5 g of boric acid, and 8.5 g of sodiumhloride in 1 L of distilled water). Mice were observed for 10 days,nd those alive after that time were scored as survivors.

reparation and potency of TeNT-Hc adsorbed to aluminumydroxide adjuvant

The preparation of human used vaccine: the purified TeNT-c protein (final concentration of 20 �g/mL) was added to thel(OH)3 gel adjuvant with final concentration of 1.5 mg/mL. Aftervernight adsorption at 4 ◦C, the vaccine suspension was preparedith 0.5 mL per human dose.

The adsorbed tetanus toxoid national reference materials were4, 48, 96, 192-fold diluted and the prepared TeNT-Hc vaccinesere 30, 60, 120, 240-fold diluted with normal saline, respectively.IH mice were used in this study and every 14 mice were classified

n one group with half male and half female. Each mouse receivedn abdominal subcutaneous injection of different diluted adsorbedetanus toxoid national reference materials and TeNT-Hc humanaccines. Four weeks after the vaccination, mice were challengedith 50 LD50s of tetanus neurotoxin and were observed for 5 days,

nd those alive after that time were scored as survivors. A standardurve was drawn according to the result of the adsorbed tetanusoxoid national reference materials and the method of qualitativeesponses parallel was used to determine the potency of the TeNT-c human vaccine.

esults

equence optimization and the construction of the recombinantlasmid

The gene coding TeNT-Hc was fully synthesized after sequenceptimization by replacing the rare codons with frequent ones,hich showed a decrease in the content of AT pairs from 72.57%

o 52.47%. TeNT-Hc cDNA was cloned into the expression vectorET-32a(+) (Fig. 1) and the recombinant plasmid containing accu-

Fig. 1. The construction of expression vector of pET32a-TeNT-Hc. After adding addi-tional EcoRI and XhoI sites at 5′ and 3′ ends, respectively, and a TAA sequence afterthe EcoRI site to end the expression of thioredoxin on pET-32a(+), the optimizedgene of TeNT-Hc was ligated to pET-32a(+) to construct the expression vector.

rate TeNT-Hc gene sequence was further analyzed by restrictionenzymatic pattern and finally confirmed by DNA sequencing.

Expression and identification of TeNT-Hc

When the culture reached the mid-log phase, E. coli cells

Fig. 2. SDS-PAGE (A) and western blotting (B) analysis of TeNT-Hc expression.Lane M, low molecular protein marker; lane 1, the supernatants of sonicated bac-teria BL21(DE3)/pET32a-TeNT-Hc; lane 2, the supernatants of sonicated bacteriaBL21(DE3)/pET32a(+).

488 R. Yu et al. / Immunobiology 216 (2011) 485–490

FSa

tso

pa

P

4HaSDtpTT−

I

1Fr1i1

T

e1dp

5

5.5

6

og10

)

3.5

4

4.5

xoid

tite

r(lo

2

2.5

3

teta

nus

tox

1μ5μ

1

1.5

6420

Ant

i-t

g of TeNT-Hc10μ

Weeks post first immunization

g of TeNT-Hcg of TeNT-Hc

Fig. 4. Kinetics of antibody response in BALB/c mice following vaccination. BALB/cmice were vaccinated with either 1 �g, 5 �g, or 10 �g of TeNT-Hc/Freund’s adjuvanton weeks 0, 2, and 4 and bleed 2 weeks after each vaccination. Each point is theaverage of six mice.

80

100

40

60

0

20Inhi

bitio

n (%

)

1 1.5 2 2.5 3 3.5 4 4.5Anti-tetanus serum dilution (Log10)

Fig. 5. GT1b binding inhibition ELISA. Wells of microtiter plates coated with GT1b

TP

ig. 3. SDS-PAGE analysis of TeNT-Hc purification after Q-Sepharose FF, Phenyl-epharose FF, and SP-Sepharose XL (Coomassie blue staining). The separate lanesre protein samples eluted from the column used in the last step of purification.

he supernatants of sonicated bacteria. Measured by densitometrycanning, the amount of synthesized TeNT-Hc was about up to 46%f the total cellular protein.

The result of western blotting analysis showed that the 50 kDrotein could specifically bind to the mouse monoclonal antibodygainst tetanus (Fig. 2B).

urification of TeNT-Hc

115 g of wet cell paste was collected after fermentation with2 L fermentor containing 30 L of LB medium. Recombinant TeNT-c could be purified from the supernatant of the bacteria lysatefter a combination of Q-Sepharose XL (data not shown), Phenyl-epharose FF (data not shown), and SP-Sepharose XL (Fig. 3).etected by SDS-PAGE, the purity increased step by step and up

o 95% of purity was achieved finally (Fig. 3). The summary of eachurification step is presented in Table 1. The final yield of purifiedeNT-Hc from each liter of culture was about 333 mg. The purifiedeNT-Hc in PBS buffer was stable when stored at 4 ◦C, −20 ◦C and80 ◦C for 6 months.

mmunization of mice by use of TeNT-Hc as a sub-unit vaccine

The anti-tetanus titers of BALB/c mice vaccinated with either�g, 5 �g, or 10 �g of TeNT-Hc at 0, 2, 4 and 6 weeks are shown inig. 4. As the result shown, the TeNT-Hc could elicit strong immuneesponses against tetanus toxoid in mice after immunization once.�g of TeNT-Hc could induce as high as 1:204,800 of antibody titers

n the sera of mice after 3 times vaccination, as well as 5 �g and0 �g of TeNT-Hc did.

he inhibition of TeNT-Hc binding to GT1b

TeNT-Hc specifically binds to neurons via gangliosides (Schiavot al. 1994; Montecucco and Schiavo 1995; Halpern and Neale995). In our study, a binding inhibition ELISA was performed toetect it. As shown in the result (Fig. 5), the serum containingolyclonal antibodies against tetanus could effectively inhibit the

able 1urification of TeNT-Hc from fermentor cultures (115 g of wet cell paste).

Purification step Total protein (mg) Percentage y

E. coli cell lysate 30,617 100Q-Sepharose FF 14,436 82Phenyl-Sepharose FF 11,737 75SP-Sepharose XL 10,417 71

were pre-incubated with BALB/c mouse anti-TeNT-Hc sera and TeNT-p Hc. TeNT-Hcbound to GT1b was detected by using human anti-tetanus antibody. Each point isthe average of three wells.

specific binding between TeNT-Hc and one of its receptor GT1b. Theless dilution of the serum, the stronger inhibition could be observed.This in vitro experiment suggested that TeNT-Hc elicited high titerserum that specifically blocked binding of TeNT-Hc to GT1b.

In vivo protection against tetanus neurotoxin

The mice vaccinated with TeNT-Hc 3 times were challengedwith tetanus neurotoxin and the protections were evaluated. Whenchallenged with 2 × 103 LD50s of tetanus neurotoxin, TeNT-Hc pro-vided excellent protection. All the mice immunized with TeNT-Hcsurvived while all the mice immunized with PBS died in 48 h(Table 2).

Potency as a human vaccine

Aluminum salts are the most commonly utilized adjuvants foruse in clinical vaccine products (Lindblad 2004) and aluminumhydroxide adjuvant has been approved for human use by the Food

ield (%) Purity (%) Total protein of interest (mg)

46 14,08480 11,54990 10,56396 10,010

R. Yu et al. / Immunobiology 216 (2011) 485–490 489

Table 2In vivo protection of TeNT-Hc used as sub-unit vaccine.

Vaccine Numbers of mice Tetanus toxin challenged (LD50s) Survival rate

1 �g TeNT-Hc + adjuvant 6 2 × 103 6/65 �g TeNT-Hc + adjuvant 6 2 × 103 6/610 �g TeNT-Hc + adjuvant 6 2 × 103 6/6PBS + adjuvant 6 2 0/6

Table 3Potency comparison between tetanus toxoid national reference standard and TeNT-Hc/Alhydrogel vaccines.

Vaccine Group (-fold diluted) Number of vaccinations Tetanus toxin challenged (LD50s)c Survival rate

Adsorbed tetanus toxoid national reference materials a 24 1 50 12/1448 1 50 4/1496 1 50 0/14

192 1 50 0/14

TeNT-Hc human vaccine b 30 1 50 14/1460 1 50 13/14

120 1 50 8/14240 1 50 3/14

rationoteins neu

aptmtplwwuttTu

D

taesposro(efawcsTsrrladh

a The adsorbed tetanus toxoid national reference materials had an initial concentb The TeNT-Hc human vaccine was prepared by 20 �g/mL of purified TeNT-Hc prc All the mice were vaccinated once and were challenged with 50 LD50s of tetanu

nd Drug Administration of the U.S. The efficacy of TeNT-Hc pre-ared using aluminum hydroxide adjuvant was evaluated againsthe adsorbed tetanus toxoid national reference material in NIH

ice. Four weeks after vaccinated with different-fold diluted ofhe adsorbed tetanus toxoid national reference materials and therepared TeNT-Hc human vaccines, the NIH mice were all chal-

enged by 50 LD50s of active tetanus neurotoxin and the survivalsere recorded as Table 3. The potency of TeNT-Hc human vaccineas calculated as 168 IU/mL (95% confidence interval: 250–116)sing the method of qualitative parallel line analysis. Accordingo the requirement in the Chinese Pharmacopoeia, the potency ofetanus vaccines should be achieved 80 IU/mL and the recombinanteNT-Hc human vaccine could fully meet the demand for humanse.

iscussion

The Hc fragments of tetanus neurotoxin (TeNT-Hc) are non-oxic and have been studied as potential candidates for vaccinesgainst tetanus. TeNT-Hc has been cloned and expressed by oth-rs (Halpern et al. 1990; Fairweather et al. 1987), however, poorolubility resulted in low yields and difficulties associated with theurification of the expressed protein in E. coli limited the utilityf TeNT-Hc. Many studies have been exploited to get high expres-ion of TeNT-Hc. In Makoff et al.’s work, after complete sequenceeplacement and the promoter strength improved, the expressionf TeNT-Hc was increased to approximately 11–14% cell proteinMakoff et al. 1989; Oxer et al. 1991; Makoff and Oxer 1991). Ribast al. (2000) got a high level expression of TeNT-Hc-thioredoxinusion protein in E. coli using vector pET32a and the final yield wasbout 35 mg/L. In our study, the full-length sequence of TeNT-Hcas optimized by replacing rare codons with frequent ones in E.

oli and the content of AT pairs was decreased. The fully synthe-ized gene was cloned into pET32a and got excellent expression.he final yield of non-tagged TeNT-Hc was about 333 mg/L after 3-tep purification, which was about 10 times higher than the formereport (Ribas et al. 2000). The purification scheme described in this

eport repeatedly produced pure TeNT-Hc that remained stable fol-owing processing steps. This is the first report demonstrating that

non-tag TeNT-Hc isoform could be purified from E. coli to pro-uce large quantities product with the potential of being used inuman. This type of vaccine has advantages over traditional toxin-

of 44.3 IU/mL.and 1.5 mg/mL Al(OH)3 gel adjuvant.rotoxin in 0.5 mL of normal saline 4 weeks after the vaccination.

inactivated vaccines in that it is cheaper, less hazardous to produceand can be generated in large quantities.

Tetanus toxoid was used to coat the wells for the ELISA insteadof TeNT-Hc to measure the antibody response in mice vaccinatedwith TeNT-Hc with Freund’s adjuvant. Results showed that theBALB/c mice responded with high titers 2 weeks after the secondbooster vaccination. These results suggested that the TeNT-Hc wasexpressed and purified in an active form and that its conforma-tion was similar with what it is in the whole toxin. This conclusionwas further confirmed by the in vivo protection of mice challengedwith tetanus neurotoxin. Our ultimate goal is to license and fur-ther develop this TeNT-Hc vaccine for human use, so a vaccine thatcould be used in human bodies was prepared using Al(OH)3 gel asthe adjuvant and its potency was determined as 168 IU/mL whichwas 2 times higher than the national standard for tetanus vaccines.All the results showed that Hc domain of tetanus toxin generatedin our expression system should be useful as a potential sub-unitvaccine against human tetanus.

The present study of TeNT-Hc may lead to a novel and highlyeffective tetanus candidate human vaccine with fewer side effectsthan the currently available toxoid vaccines, which can be easy forhigh throughput production and purification.

Acknowledgments

We thank Drs Shumin Zhang in National Institute for the Controlof Pharmaceutical and Biological Products (China) for technical sup-ports and helpful discussions. This work was supported by NationalNatural Science Foundation of China (81025018).

References

Eisel, U., Jarausch, W., Goretzki, K., Henschen, A., Engels, J., Weller, U., et al., 1986.Tetanus toxin: primary structure, expression in E. coli, and homology withbotulinum toxins. EMBO J. 5 (10), 2495–2502.

Fairweather, N.F., Lyness, V.A., 1986. The complete nucleotide sequence of tetanustoxin. Nucleic Acids Res. 14 (19), 7809–7812.

Fairweather, N.F., Lyness, V.A., Maskell, D.J., 1987. Immunization of mice against

tetanus with fragments of tetanus toxin synthesized in Escherichia coli. Infect.Immun. 55 (11), 2541–2545.

Halpern, J.L., Neale, E.A., 1995. Neurospecific binding, internalization, and retrogradeaxonal transport. Curr. Top. Microbiol. Immunol. 195, 221–241.

Halpern, J.L., Habig, W.H., Neale, E.A., Stibitz, S., 1990. Cloning and expression offunctional fragment C of tetanus toxin. Infect. Immun. 58 (4), 1004–1009.

4 iology

H

L

M

M

M

M

M

90 R. Yu et al. / Immunob

elting, T.B., Nau, H.H., 1984. Analysis of the immune response to papain diges-tion products of tetanus toxin. Acta Pathol. Microbiol. Immunol. Scand. 92 (1),59–63.

indblad, E.B., 2004. Aluminum adjuvants—in retrospect and prospect. Vaccine 22(27–28), 3658–3568.

aassen, C.B., Laman, J.D., den Bak-Glashouwer, M.J., Tielen, F.J., van Holten-Neelen,J.C., Hoogteijling, L., et al., 1999. Instruments for oral disease-intervention strate-gies: recombinant Lactobacillus casei expressing tetanus toxin fragment C forvaccination or myelin proteins for oral tolerance induction in multiple sclerosis.Vaccine 17 (17), 2117–2128.

akoff, A.J., Oxer, M.D., 1991. High level heterologous expression in E. coliusing mutant forms of the lac promoter. Nucleic Acids Res. 19 (9),2417–2421.

akoff, A.J., Oxer, M.D., Romanos, M.A., Fairweather, N.F., Ballantine, S., 1989.

Expression of tetanus toxin fragment C in E. coli: high level expression by remov-ing rare codons. Nucleic Acids Res. 17 (24), 10191–10202.

atsuda, M., Yoneda, M., 1974. Dissociation of tetanus neurotoxin into two polypep-tide fragments. Biochem. Biophys. Res. Commun. 57 (4), 1257–1262.

ontecucco, C., Schiavo, G., 1995. Structure and function of tetanus and botulinumneurotoxins. Q. Rev. Biophys. 28 (4), 423–472.

216 (2011) 485–490

Oxer, M.D., Bently, C.M., Doyle, J.G., Peakman, T.C., Charles, I.G., Makoff, A.J., 1991.High level heterologous expression in E. coli using the anaerobically-activatednirB promoter. Nucleic Acids Res. 19 (11), 2889–2892.

Qazi, O., Sesardic, D., Tierney, R., Soderback, Z., Crane, D., Bolgiano, B., et al., 2006.Reduction of the ganglioside binding activity of the tetanus toxin HC fragmentdestroys immunogenicity: implications for development of novel tetanus vac-cines. Infect. Immun. 74 (8), 4884–4891.

Ribas, A.V., Ho, P.L., Tanizaki, M.M., Raw, I., Nascimento, A.L., 2000. High-level expres-sion of tetanus toxin fragment C–thioredoxin fusion protein in Escherichia coli.Biotechnol. Appl. Biochem. 31 (pt2), 91–94.

Rummel, A., Bade, S., Alves, J., Bigalke, H., Binz, J., 2003. Two carbohydrate bindingsites in the HCC-domain of tetanus neurotoxin are required for toxicity. J. Mol.Biol. 326 (3), 835–847.

Schiavo, G., Rossetto, O., Montecucco, C., 1994. Clostridial neurotoxins as tools to

investigate the molecular events of neurotransmitter release. Semin. Cell Biol. 5(4), 221–229.

Walter, J.B., Talbot, I.C., 1996. Wound Infections: The Clostridia. Walter and IsraelGeneral Pathology, Churchill Livingstone, pp. 320–322.

WHO/EPI/GEN/93.13 Immunological Basis for Immunization/Module 3: Tetanus,2010.