Botulisum+Vaccine+Journal

8/13/2019 Botulisum+Vaccine+Journal

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Y.-Z. Yu et al. / Vaccine 27 (2009) 28162822 2817

HcE (EcoRI): 5 -GCCGGAATTCTAATGGAATACATCAAGAACATCATC-3 , and reverse primer R-HcX (XhoI): 5 -GTTGGGGTGAACG-TCCACTGATTATCCTCGAGCTAG-3. The PCR products were digestedwith EcoRI and XhoI to excise the AHc DNA fragment which wassubcloned into a pTIG-Trx expression vector [6] (cut with thesame restriction enzymes) resulting in recombinant plasmid pTIG-Trx-AHc1 that was conrmed by sequencing. pTIG-Trx-AHc1 wassubsequently transformed into E. coli strain BL21 (DE3)(Stratagene,La Jolla, CA) and grown in Luria Bertani (LB) medium containing100 g/ml ampicillin at 37 C until anopticaldensity at600nm was0.5. Isopropyl- -d -thiogalactopyranoside (IPTG, Sigma) (1 M) wasadded to a nal concentration of 0.2 mM for an additional 3 h. Cells(2 l cultures) were centrifuged at 6000rpm for10min, resuspendedin 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 Cand disruptedby sonication. The resulting lysates were centrifugedat 15,000 g for 30min at 4 C and small-scale rAHc puricationusingconventionalchromatography wascarriedout as describedbythe manufacturer. Briey,HiTrap TM SPFF (5 ml)(PharmaciaBiotech,Sweden) was used in the rst purication step. The column andbuffers were maintained at ambient temperature and the samplewas kept on ice during loading. The column was equilibrated with10 column volumes (CV) of buffer (20 mM sodium phosphate, pH7.4) at a rate of 100cm/h and then loaded with the ltered lysatesat a rate of 60 cm/h and then washed with 10 CV of equilibrationbuffer at a rate of 100 cm/h. Recombinant product was eluted fromthe column using step elution with 6 CV of 300mM NaCl in equili-bration buffer at a rate of 60 cm/h. The column was stripped with1 M NaCl in equilibration buffer and then cleaned with a mixtureof 1 M NaOH and 1 M NaCl. The fraction obtained from the SP col-umn was dialyzed at 4 C against 20vol of 20 mM TrisHCl, pH 7.4to decrease the sample conductivity in preparation for the negativepurication step.

Negative purication was carried out using HiTrap TM Q FF(5ml,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 thedialyzed SP product pool. After loading was complete, the columnwas washedwith 6 CV of equilibration buffer and the ow-throughfraction containing the rAHc product was collected. Bound pro-teins were eluted with increasing concentrations of NaCl up to 1Min sodium phosphate buffer and the column was cleaned with amixture of 1 M NaOH and 1M NaCl.

In the nal chromatography step, the Q product fraction wasadjusted to 2.6 M NaCl and applied to a HiTrap TM octyl (high sub)FF (5 ml, Pharmacia). The column was pre-equilibrated with 10CV of high-ionic-strength buffer (2.6 M NaCl in 20mM sodiumphosphate, pH 7.4) at a rate of 100 cm/h. After loading was com-

plete, the column was washed with 10 CV of equilibration bufferand product was eluted with 50mM NaCl in sodium phosphatebuffer. The column was cleaned with a mixture of 250mM NaOHand 250 mM NaCl. The HIC product fraction was dialyzed at4 C against 20 mM sodium phosphate buffer, pH 7.4, to removeresidual salt. The product was frozen at 70 or 20 C for stor-age.

The soluble fraction and puried rAHc were subjected to12% sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDSPAGE) and Western blot analysis using hyperimmune horseanti-neurotoxin A antiserum (National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China). FollowingSDSPAGE proteins were transferred onto a nitrocellulose mem-brane (Hybond-C, Amersham) and blocked with 5% nonfatdry milk

for 2 h. The blots were washed with 0.1% Tween 20 in phosphate

buffered saline (PBST) three times (5min each) between each incu-bations step. The blots were subsequently incubated with 1:200dilution of horse anti-serum for 1 h at room temperature. Afterwashing theblots were then incubatedwith 1:2000 dilution of goatanti-horse IgGHRP (Santa Cruz Biotechnology, Inc.), washed and5 ml of DAB substratewere added to visualizebinding. Blots probedwith secondary antibody only did not revel any non-specic bind-ing. Dilutions of the primary and secondary antibody were donein blocking buffer. Protein concentrations were estimated by usingBCAproteinassay(Sigma) accordingto themanufacturersprotocol.

2.3. Vaccinations

Specic pathogen-free female Balb/c mice (purchased fromBeijing Laboratory Animal Center, Beijing) 6 weeks of age wererandomly assigned to different treatment groups. Groups of eightmice were vaccinated with puried rAHc via intramuscular (i.m.),intraperitoneal (i.p.) or subcutaneous (s.c.)routes. rAHcwas dilutedin 25% (v/v) Alhydrogel (aluminum hydroxide, Sigma, St. Louis,MO) or aluminum phosphate adjuvant suspensions (containing 2%AlPO4 , Sigma)and injections were administeredat 2 week intervals(100 l/injection). In some experiments, rAHc was administeredwithout adjuvant. PBS formulated with the different adjuvants wasused to vaccinate mice in the negative control groups and bloodfrom all groups was collected via the tail vein before each vacci-nation or neurotoxin challenge and the serum isolated for rAHcantibody reactivity. Mice from all groups were challenged i.p. withdifferent dosages of pure botulinum neurotoxin serotype A dilutedin 20 mM sodium phosphate buffer (pH 6.5) containing 0.2% (w/v)gelatin (Wako, Japan) 3 weeks after the last vaccination. The micewere observed for 1 week and percent survival was determined foreach vaccination group.

In addition,the durationof the protective response wasassessedin 6-week-old Balb/c mice (6 groups, 16 mice/group). Mice in groupI were vaccinated i.m. with 1 g rAHc only. Groups IIIV mice werevaccinated i.m.with0.2, 1 or 5 g rAHc formulated with aluminumhydroxideadjuvant respectively. Group V micewere vaccinated i.m.

with 1 g rAHc formulated with aluminum phosphate adjuvant.Group VI mice (negative control) were vaccinated i.m. with PBSformulated with aluminum hydroxide. All groups were vaccinatedi.m. three times (days0, 15,and 30) atthe indicateddosagesof 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 werebled at 15-day intervals after the second inoculation (1 month) andat each indicated time point.

2.4. Antibody titer measurements

Sera from mice in the different treatment groups were screenedforanti-rAHcantibodies byan ELISA. ELISA plates (Corning Incorpo-rated, 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 beginningat 1:100 and 100 l was added to each well for 1 h at 37 C. Afterwashing, 100 l of a 1:2000 dilution of goat anti-mouse IgGHRP(SantaCruz Biotechnology,Inc.)was added for30 minat 37 C. Afterwashing,anti-rAHc reactivitywasvisualized byadding100 lofcit-rate buffer (pH 5.0) containing 0.04% (w/v) of o-phenylenediamineand 0.02% (v/v) hydrogen peroxide for 5 min at 37 C. The reactionwas stopped with 50 l o f 2 M H 2 SO4 and the absorbance was readat 492 nm using a Thermo Labsystems (Frannklin, MA) microplatereader. Antibody titerswere estimatedas thereciprocalof themax-imum dilution of serum giving an absorbance reading greater than0.5 units following subtraction of non-specic binding detected incontrol sera and twofold greater than that of the matched dilution

of control sera. Serum samples from individual mice were assayed


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2818 Y.-Z. Yu et al. / Vaccine 27 (2009) 28162822

in duplicate and their mean was used to calculate the geometricmean titer (GMT) for each group.

2.5. BoNT/A neutralization assay

Sera from mice vaccinated with two or three doses of AHc inthe rst vaccination study were pooled for the toxin neutralizationassay as described previously [7,8] . In brief, mixtures of serial dilu-tions of sera in phosphate buffer (50 mM Na

2HPO

4) containing 1%

gelatin with 10 LD 50 of botulinum neurotoxin serotypeA wereincu-bated 0.5h at room temperature andthe mixtures were injected i.p.into Balb/c mice (1622g) using a volume of 500 l/mouse (fourmice in each group). The mice were observed for 1 week, and deathor not was recorded. The concentration of neutralizing antibody inthe serum was calculated relative to a World Health Organizationbotulinum 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 10,000mouse i.p. LD 50 of botulinum neurotoxin serotype A.

2.6. Statistical analysis

Differences in antibody titers were analyzed statistically usingthe Students t -test or the paired t -test between group differ-ences. Two-factor ANOVA was also used to determine whether thedata were statistically signicant. Fishers exact test was used todetermine statistical differences in survival between the treatmentgroups. For all tests only data resulting in p values of p < 0.05 wereregarded as statistically signicant.

3. Results

3.1. Expression and purication of rAHc from E. coli

DNA encoding AHc was synthesized with optimal codon usagefor expression in E. coli [6] . Recombinant AHc (50kDa) expressionin E . coli (BL21) was conrmed by Western blot analysis ( Fig. 1 ) thatdemonstrated a high level of expression (approximately 25% of thecell protein).

Small-scale purication of rAHcwas carried outusingsequentialchromatography using ion-exchange (SP and Q) and hydrophobic-interaction (HIC) resins. SP cation-exchange chromatographyefciently captured the rAHc fragment and the SP product fractioncontained approximately 57% rAHc fragment based on SDSPAGEanalysis ( Fig. 1, lane 5). The second chromatography step (negativepurication step) removed numerous contaminants and the puri-cation process was facilitated by the basic charge of rAHc (pI 9.1)thatwas easily identiable in thecolumn ow-through.The Q prod-uct fraction contained approximately 85% rAHc fragment basedon SDSPAGE analysis ( Fig. 1, lane 4). The nal chromatography

Fig. 1. Analysis of rAHc during the various purication steps by SDSPAGE (A) andWesternblot (B). Lanes 1 and 8, pTIG-Trx-AHc1transformed BL21 cell lysates;lanes2 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 therAHc.

HiTrap TM octyl purication step removed lower molecular weightbandcontaminants.SDSPAGEanalysis of the HIC product fractionsshowed approximately 98%purity after this step ( Fig. 1 , lane 3)andthe product band (50 kDa) was immunoreactive by Western blotanalysis ( Fig. 1, lane 9). The purication process described consis-tently produced 95% pure product based on HLPC and 98% purebased on Coomassie staining. Final product yields ranged from 10to 15mg of puried protein per liter of culture.

3.2. rAHc subunit vaccine efcacy

To assess the immunogenicity of puried rAHc, an efcacystudy was carried out in Balb/c mice. Mice were vaccinated i.m.once, twice, or three times using four different doses of rAHcand subsequently challenged with a dose 100,000-fold of the 50%mouse lethal dose (100,000 LD 50 ) of biologically active neuro-toxin serotypeA. A rAHcdose-and frequency-dependentprotectiveeffect was observed ( Table 1 ). Mice that received two injections of 0.2 g andthree injections of 0.04 g werecompletelyprotected(100% survival) following challenge with neurotoxin serotype A.In contrast, mice in the negative control groups succumbed tobotulism and died within 5 h. To determine the protective effectsof rAHc vaccination against a higher BoNT/A challenge doses, i.m.-vaccinated mice were challenged with a dose 1,000,000 LD 50 of

Table 1Survival and antibody titers after i.m. vaccination with rAHc.

Vaccination dose Number alive a Number alive b log 10 GMT(SD)c Serum neutralization titer (IU/ml) d

1e 2e 3e 2e 3e 1e 2e 3e 2e 3e

5 g 5 8 8 8 8 3.19(0.33) 4.41(0.45) 5.19(0.23) 5.12 40.961 g 4 8 8 7 8 2.96(0.35) 4.17(0.35) 4.78(0.31) 2.56 20.480.2 g 2 8 8 4 8 2.69(0.39) 3.87(0.31) 4.40(0.36) 0.64 5.120.04 g 1 7 8 2 6 2.58(0.32) 3.61(0.32) 4.10(0.32) 0.32 2.56

a Balb/c mice alive (8 mice/group) after i.p. challenge with a dose 100,000 LD 50 of BoNT/A 3 weeks after the last injection.b Mice alive (8 mice/groups) after i.p. challenge with a dose 1,000,000 LD 50 of BoNT/A 3 weeks after the last injection.c Sera obtained2 daysbefore challenge from individual mice was assayed in duplicateand used to calculate thelog 10 GMT foreachgroup. 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 dif ferent 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).


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Table 2Balb/c and KM survival and antibody titers following i.m. rAHc vaccination.

Mouse st rain Number al ive a log 10 GMT(SD)b

2c 3c 2c 3c

Balb/c 6 8 4.10(0.28) 4.74(0.34) **

KM 5 8 4.03(0.27) 4.55(0.28) *

a Mice alive (8 mice/group) after an i.p. challenge with a dose 1,000,000 LD 50 of BoNT/A 3 weeks after the last injection.

bSera obtained 2 days before challenge from individual mice were assayed in

duplicate and used to calculate the log 10 GMT for each group. GMT SD reported inparenthesis.

c Number of vaccinations. Balb/c and KM mice were vaccinated twice or threetimes with 1 g of rAHc. rAHc was diluted in 25% (v/v) Alhydrogel and injectionswere administered at 2 weeks intervals (100 l/injection).

* P = 0.02< 0.05indicates signicant titerdifference betweentwo- andthree-dosegroups.

** P = 0.0017 < 0.01 indicates signicant titer difference between two- and three-dose groups.

neurotoxin serotype A ( Table 1 ). Two injections partially or com-pletely protected the mice at all dose tested with 100% survivalobserved at a dose of 5 g. Three injections of 0.2 g affordedcomplete protection (100% survival).

Serum antibody titers for individual mice were analyzed bythe ELISA and the GMTs for each group in the study were deter-mined. A dose-dependent antibody response to vaccination withrAHc was observed and the group GMT correlated well with pro-tection ( Table 1 ). The group geometric mean titer of mice injectedwith a single dose of rAHc was relative low (2.583.19) dependingon the immunization dose and these mice were partiallyprotected.Following secondand third vaccinations the GMTs increased signif-icantly ( 3.87, P < 0.01) and correlated with100% survival followinga challengewith a 100,000 LD 50 of neurotoxin serotypeA. Similarly,the four treatment groups that were 100% protected had geometricmean titers of 4.4 when challenged with a dose 1,000,000 LD 50 of neurotoxin serotype A.

Serumfrom theELISA studywas used in a BoNT/Aneutralization

assay. Due to the limited amount of serum available, serum fromeach group of mice vaccinated with two or three doses of AHc waspooled, and so only the average neutralization titer of the groupcould be assayed. As shown in Table 1 , neutralizing antibody titersincreased with thenumber anddose of vaccinations and correlatedwell with group ELISA antibody titers and protection. Groups withpartial survival against a challenge with a dose 1,000,000 LD 50 of neurotoxin serotypeA all hadneutralizing titers of 2.56IU/ml, andgroups with 100% survival allhad neutralizing titers of 5.12IU/ml.

The immunogenicity and protective efcacy of rAHc was alsoevaluated in a KM (Chinese Kun Ming) mouse model. Mice werevaccinated i.m. two or three times at a dose of 1 g per mouseand subsequently challenged with a dose 1,000,000 LD 50 of bio-logically active neurotoxin serotype A. Survival and groupantibodyELISA titers of the Balb/c and KM mice are shown in Table 2 .Results revealed that the immunogenicity and protective efcacyof rAHc vaccine in KM mice was comparable to that of Balb/cmice. These results indicated that soluble rAHc was a highly effec-tive immunogen and protected against a lethal dose of biologicallyactive botulinum neurotoxin serotypeA in mice of different geneticbackgrounds.

3.3. rAHc subunit vaccine efcacy in the presence or absence of adjuvant

Two types of adjuvants (aluminum hydroxide and aluminumphosphate) were tested in rAHc vaccine efcacy trials. Mice werevaccinated 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 LD 50

Table 3Survival and antibody titers following i.m. rAHc vaccination.

Adjuvant Number alive a log 10 GMT(SD)b

2c 3c 2c 3c

Aluminum hydroxide 7 8 4.07(0.25) 4.70(0.36)Aluminum phosphate 6 8 3.95(0.23) 4.63(0.28)

a Balb/c mice (8 mice/group) were challenged i.p. with a dose1,000,000 LD 50 of BoNT/A 3 weeks after the last immunization.

bSera obtained 2 days before challenge from individual mice were assayed in

duplicate and used to calculate the log 10 GMT for each group. SD reported in paren-thesis.

c Numberof vaccinations. Balb/cmice were vaccinated twiceorthreetimesat 1 gdosageof rAHc. Vaccinesweredilutedin 25%(v/v)Alhydrogel(aluminum hydroxide)or aluminumphosphate adjuvant suspensions (containing 2% AlPO 4 ) and injectionswere administered at 2 week intervals (100 l/injection).

of biologically active neurotoxin serotype A. Survival and groupantibody ELISA titers are shown in Table 3 . Results revealed thatvaccination withrAHc formulated withaluminumhydroxide or alu-minum phosphate adjuvant elicited similar anti-rAHc antibodiesthat conferred equal levels of protection against botulinum neuro-toxin serotype A challenge.

Furthermore,rAHc administeredvia differentvaccination routese.g., i.m., s.c. or i.p. and then challenged with a dose of neurotoxin1,000,000LD 50 of biologically active neurotoxin serotypeA demon-strated that protection against toxin challenge was not affected bythe route of vaccination ( Table 4 ).

The potency of the rAHc subunit vaccine in the absence of adju-vant was also studied in the context of the vaccination route. Micevaccinated i.m. or s.c. with rAHc as described above in the absenceor presence of an adjuvant and challenged with a dose of 100,000of LD50 of biologically active neurotoxin serotype A showed that inthe absence of the adjuvant rAHc elicited a modest IgG response(P < 0.001 compared to negative control group) and in the presenceof the adjuvant this response was signicantly elevated ( P < 0.01comparedto rAHc-onlytreated mice)( Table5 ). Micevaccinated i.m.or s.c. with rAHc in thepresence of an adjuvant were fully protected

againstneurotoxinchallenge,in contrast,mice vaccinatedi.m.ors.c.with two doses of 1 g rAHc without adjuvant were partially pro-tected ( P < 0.05 compared to vaccinations with adjuvant) and micevaccinated i.m. or s.c. with three doses of 1 g rAHc in the absenceof an adjuvant showed nearly complete protection ( P = 0.007 com-pared to the negative control i.m. group and P = 0.0014 comparedto the negative s.c. control group). The surviving mice were givena second challenge with much higher neurotoxin doses (1,000,000LD50 ). 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 absenceor presence of adjuvant waseffective in protecting against botulismdepending on the number of immunizations.

Table 4Survival and antibody titers of following i.m., s.c. or i.p. rAHc vaccinations.

Vaccination route Number alive a log 10 GMT(SD)b

2c 3c 2c 3c

i.m. 5 8 4.10(0.28) 4.70(0.32)s.c. 7 8 4.18(0.27) 4.78(0.31)i.p. 7 8 4.14(0.25) 5.82(0.28)

a Balb/c mice (8 mice/group) were challenged i.p. with a dose 1,000,000 LD 50 of BoNT/A 3 weeks after the last injection.

b Sera obtained 2 days before challenge from individual mice were assayed induplicate andusedto calculate thelog 10 GMT foreachgroup.SD indicatedin paren-theses.

c Number of vaccinations. Mice were vaccinated twice or three times with 1 gof rAHc. Vaccines were diluted in 25% (v/v) Alhydrogel and injections were admin-istered i.m., s.c. or i.p. to Balb/c mice at 2 weeks intervals (100 l/injection).


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subunit vaccines, synthesized in E. coli [912] or yeast [1321]expression vectors are less expensive and hazardous to producethanthe traditional toxin-inactivated vaccines. In addition,the pureand concentrated immunogens may increase immunogenicity andprotection efcacy in humans. Recombinant botulism vaccines areunder development with the aim of providing protection againstbotulism.A milestonewas reached when the rstrecombinantsub-unit vaccine [rBV A/B( Pichia pastoris ) vaccine]wastested inhumansduring a phase I clinical trial [5] .

As an appropriate vaccine candidate, the carboxyl-terminal frag-ment of botulinum neurotoxin heavy chain (Hc) is responsiblefor toxin binding to the nerve cells, and the recombinant Hc hasbeen proved to be a safe and efcient immunogen to generateneutralizing antibodies for immunotherapy [2] . Recently, our lab-oratory and others have expressed recombinant Hc of BoNTs inE. coli at levels sufcient for vaccine development [7,2226] . Inthis study we describe the production of a recombinant untaggedHc subunit vaccine of serotype A as a candidate subunit vaccinefor human use in the prevention of botulism. A synthetic geneencoding the Hc domain of BoNT/A was designed, expressed andthe gene product puried from E. coli using conventional chro-matography. Recombinant AHc was assessed as a subunit vaccineagainst the botulinum neurotoxin serotype A in mouse models of disease. We performed the efcacy study to understand the rela-tionship between multiple vaccinations and protection at variousdose levels. Clearly, multiple injections of immunogen protectedmice better than a single injection. Mice given two injections of 0.2 g or three injections of 0.04 g were completely protectedwhen challenged i.p. with a 100,000 LD 50 dose of biologicallyactive botulinum neurotoxin serotype A. Two injections partiallyor completely protected mice at all dose levels and three injec-tions afforded even greater protection with a higher challenge of BoNT/A (1,000,000 LD 50 dose of botulinum neurotoxin serotypeA). Protective efcacy against botulinum neurotoxin serotype A inmice vaccinated with the untagged rAHc in this study was similarto that in mice vaccinated with the rAHc with His-tag as previ-ously reported [7] . A dose-response effect was also observed with

respect to antibody titers and the dosage andfrequency of the rAHcvaccinations and the group GMT correlated well with group neu-tralizing antibody titers and protection. The potency of the rAHcvaccine was also evaluated in a KM mouse model. Results revealedthat the immunogenicity and protective efcacy of a rAHc vac-cine 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/Achallenge.

Many studies have contributed to the observation that the Hcdomain of BoNTs administered with an adjuvant evokes a strongimmune response [5,17,25,26] . Vaccines based on recombinantproteins often require adjuvants in order to obtain an adequateimmune response. Aluminum salts are the most commonly uti-

lizedadjuvants foruse in humanvaccineproducts [27] . Twogeneraltypes of aluminum adjuvants approved for human use by FDAare aluminum hydroxide and aluminum phosphate. In the currentstudy these two types of adjuvants were tested. Antibody titers andprotection against botulinum neurotoxinserotypeA challengeweresimilar in mice vaccinated with rAHc adsorbed in either aluminumhydroxide or aluminum phosphate. The results revealed that thetwo types of adjuvants worked effectively in facilitating the elicita-tion of anti-rAHc immunity. The efcacy of rAHc administered viadifferent routes in the absence or presence of an adjuvant was alsoassessed. Vaccine efcacy following administration rAHc via threedifferent routes i.e., i.m., s.c., and i.p. was assessed, demonstratingthat efcacy was independent of the vaccination route. Further-more, 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 showednearly complete protection against a 100,000 LD 50 dose challengeof biologically active neurotoxin serotype A. These results indicatedthat rAHc administeredin the absence or presence of adjuvantcon-ferred protection against a lethal challenge but that fewer doseswere needed to reach full protection if administered with adjuvant.

The duration of immunity and protection induced by rAHc vac-cination was also evaluated. Although twogroups (I and II) showedattenuation of antibody titers over the course of a year and wereless efciently protected against challenge 13 months later, mice inthree experimentalgroups(IIIV)were completelyprotected fromaBoNT/Achallenge 6 and12 months after the last vaccination andnoappreciable decrease in antibody titers was noted over a period of 12months in micefrom thesegroups suggestinga direct correlationwith antibody titers and protection.

Since rAHc is a vaccine candidate for use in humans, large quan-tities will ultimately be needed. Therefore, the purication schemefor vaccine production should be designed for high throughputproduction and include as few steps as possible do decrease thechances of altering the vaccines native conformation. The puri-cation scheme described in this report repeatedly produced purerAHc that remained stable following processing steps. This is therst report demonstrating that a rAHc isoform not expressed witha His-tag could be puried from E. coli to produce large quantitiesproduct with the potential of being used in humans. This type of vaccine has advantages over traditional toxin-inactivated vaccinesin that it is cheaper, less hazardous to produce andcan be generatedin large quantities [3,4] .

The results presented in this report also indicated that a geneti-cally engineered form of the rAHc produced in an E. coli expressionvector was highly effective in protecting mice against challengedoses of biologically active botulinum neurotoxin serotype A. Ourultimate goal is to license and further develop this rAHc formula-tion for human use. We have made progress in the developmentof recombinant Hc vaccines against four serotypes of BoNTs ( i.e.,A, B, E and F) commonly associated with human botulism. Theapproach to produce the recombinant Hc vaccines involves the

cloning and expression of nontoxic Hc domains of BoNTs, evalu-ation of immunogenicity and protective efcacy of the Hc domains.The Hc domains of botulinum neurotoxins serotypes B, E andF havealso been successfully expressed in E. coli by our laboratory [28]demonstratingthe efcacy of thisexpressionand purication strat-egy in the development of the next generation of vaccines againstbotulism.

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