The Immunogenicity of the C Fragment of Tetanus Neurotoxin...

Research ArticleThe Immunogenicity of the C Fragment of Tetanus Neurotoxinin Production of Tetanus Antitoxin

Rui Yu 1 Chong Ji23 Junjie Xu1 Denghai Wang2 Ting Fang1 Yue Jing 45

Clifton Kwang-Fu Shen 45 andWei Chen 1

1Beijing Institute of Biotechnology 20 Dongdajie Street Fengtai District Beijing China2Gaotai Tianhong Biochemical Technology Development Co Ltd Gaotai Gansu China3Jiangxi Institute of Biological Products Jian Jiangxi China4Shenzhen Qianhai Tianzheng (SQT) Biotechnology Ltd Shenzhen Guangdong China5Shenzhen JinRuiFeng (Golden Harvest) Biotechnology Ltd Shenzhen Guangdong China

Correspondence should be addressed to Yue Jing yj443nyuedu Clifton Kwang-Fu Shen cliftonshengmailcomand Wei Chen cw0226foxmailcom

Received 16 November 2018 Revised 10 December 2018 Accepted 16 December 2018 Published 31 December 2018

Guest Editor Deguang Song

Copyright copy 2018 Rui Yu et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The demand of tetanus antitoxin (TAT) as tetanus treatment in developing and underdeveloped countries is still great since it isrelatively easy to achieve and affordable However there are still issues in the preparation of highly effective TAT with tetanustoxoid (TT) as the immunogenThe tetanus toxin native C-fragment (TeNT-Hc) retains many properties and it is a very promisingcandidate for the development of tetanus human vaccine In this study we tested the immunogenicity of TeNT-Hc in the preparationof tetanus antibodies by TeNT-Hc alone or in different combinations with TT The antibody titers and components in horseserum or plasma in different groups were analyzed and compared with those immunized by the conventional TT and it showedcomparability with the results of traditional methods The plasma efficacy and in vivo tetanus toxin neutralization were also testedAfter two stages of immunizations the average potency in plasma of all groups reached more than 1000 IU mL except that ingroup 4 In group 5 the first two basic immunizations with TT and the subsequent immunizations with TeNT-Hc it showedslightly higher antibody titers and potency This study demonstrated that TeNT-Hc is a safe effective and yet easy-to-producelow-cost immunogen and suitable for TT replacement in tetanus antitoxin production

1 Introduction

Tetanus is an acute lethal infectious disease and neurologicaldisorder with fatality rate up to 40 [1] Any injury or traumahas the possibility of getting Clostridium tetani (C tetani)infection that causes tetanus toxin production [2] Tetanusantibodies are the only effective intervention for preventingand treating tetanus on the first two-week period of infections[3 4] Tetanus antitoxin (TAT) and tetanus immunoglobulin(TIG) are currently available on the market [5 6] TIGpurified human tetanus antibodies is manufactured fromplasma of blood donors immunized with hepatitis B vaccineand then tetanus toxoid (TT) [7] Due to its human originTIG can be applied directly without skin test However sinceit is a human blood product potential risk of infecting human

viruses such as hepatitis C AIDS and other infectiousdiseases still remains In addition due to source restrictionsand unstable supplies TIG products are generally ratherexpensive have hard-to-find and have only 20 or lowerefficacy per vial compared to TAT which requires multipledoses Therefore it is very difficult for developing andunderdeveloped countries to afford TIG for the preventionand treatment of tetanus [8] On the contrary utilization ofTAT as tetanus treatment in those countries is relatively easyto achieve and affordableThus we canpredict it will continuebeing in clinical applications in the near future [9]

TAT is made of toxin-neutralizing immunoglobulin frag-ments F (abrsquo)

2 extracted and purified from tetanus toxoid-

immunized horse blood [10] At present there are stillissues in the preparation of highly effective TAT (1) due to

HindawiBioMed Research InternationalVolume 2018 Article ID 6057348 9 pageshttpsdoiorg10115520186057348

2 BioMed Research International

its high toxicity it is difficult to purify raw tetanus toxinand afford its high-purity form even after formaldehydeinactivationdetoxification During the initial immunizationand subsequent hyperimmunization processes interferenceof impurities within TT antigen results low-grade antibodiesagainst tetanus toxin (2) since TT still has residue toxicityfrequent and high dose injection of TT antigen into blood-harvesting horses induces the gradual hepatic degenerationand necrosis of their liver cells This effect is generallyquite visible after prolonged repeated immunization cycleDuring this time the horse liver continues enlarging anddeteriorating and finally it ruptures and causes internalbleeding and the death of the horse (3) the amount of TTused in horse immunization processes is considerably largeand results in rather high cost Therefore antigen qualityimprovement toxicity reduction and lower production costsare all essential to achieve better TAT [11]

Tetanus toxin is an enormously potent neurotoxinsecreted by the anaerobic bacteriumC tetani After collectingculture supernatants and inactivating with formaldehydetetanus toxoid (TT) is simply harvested by filtration Despiteits good immunogenicity in horse inactivated toxoid gen-erally contaminates with residual formaldehyde which isstill toxic Additionally C tetani can form spores that resistheat and chemical treatment and post certain risks in TTproduction Furthermore inactivation step with formalde-hyde sometimes could not guarantee complete detoxifica-tion of tetanus toxin that could be harmful to the horses[12]

The tetanus toxin native C-fragment (TeNT-Hc) retainsmany properties such as intact binding to gangliosidesimmunogenic potency comparable to native toxin low toxic-ity and low allergenicity [13] It is a very promising candidatefor the development of tetanus subunit vaccines and geneticengineering vaccines which have been used to in Phase Iclinical trial as replacement of TT vaccine conjugates withbacterial and viral vectors mucosal vaccines and many more[14ndash17] Our group has successfully developed a recombinanthuman tetanus vaccine with TeNT-Hc as an antigen in thepast few years [18ndash20] This study aimed at animal immuno-genicity and toxicological pharmacology demonstrated thatTeNT-Hc is a safe effective and yet easy-to-produce low-cost immunogen and suitable for TT replacement in tetanusantitoxin production

In this study we tested the immunogenicity of TeNT-Hcin the preparation of tetanus antitoxins by TeNT-Hc alone orin different combinations with TT to exploit its potential as areplacement immunogen of TT

2 Materials and Methods

Animals Adult male horses (4 to 10 years old 250-400kgbody mass) without tetanus natural antibodies were pur-chased from Datong area in Qinghai Province China ICRmice (17-19g male and female) were purchased from HunanSJA Laboratory Animal Co Ltd The horse and mice studieswere carried out in accordance with the recommendationsof SQT Biotech Antitoxin Production Council on Equine

Welfare Guidelines (GS-P009-01 and GS-P001-01) The pro-tocol was approved by the SQTBiotech Antitoxin ProductionCouncil (OS-P003-01 to OS-P008-01 GS-P006-01 and GS-P015-01) All animals used in this study were raised underhumanitarian conditions with free access to food and waterAll efforts were made to minimize suffering After injectionmice were followed for the five days to check for any signsof paralysis or death Loss of righting reflex was used as thehumane end point of the experiment Mice were monitoredthree times a day for their condition and for the occurrenceof end point

21 Materials

Incomplete Freundrsquos Adjuvant Liquid paraffin (ShanghaiZhongqin Chemical Reagent Co Ltd) and lanolin (Medicalgrade China Huating Lanolin Plant) were mixed (volumetricratio of 2 1) to afford the adjuvant Antigen and AdjuvantTeNT-Hc (molecular weight 45 kD prepared by the Depart-ment of vaccine and antibody engineering Beijing Instituteof Biotechnology) was mixed with incomplete Freundrsquos adju-vant into a water-in-oil emulsion with protein content of0625mgmL Tetanus toxoid (TT) purchased from ChengduOlymvax Biopharmaceuticals China wasmixedwith incom-plete Freundrsquos adjuvant into a water-in-oil emulsion with aprotein content of 0625mgmL A mixed antigen solutionwas prepared by mixing TeNT-Hc and TT with incompleteFreundrsquos adjuvant with incomplete Freundrsquos adjuvant into awater-in-oil emulsion with a protein content of 0625mgmLThe SDS-PAGE electrophoresis analysis of TeNT-Hc and TTis shown in Figure 1 In comparison with TT TeNT-Hc wasshown as single component and has higher purity and smallermolecular weight Secondary Antibody HRP-conjugated anti-horse IgG and IgM as secondary antibodies was purchasedfrom Abcam Standard Standard tetanus toxins and anti-tetanus serum were purchased from the China NationalInstitutes of Food andDrugControlBorate BufferTheboratesolution was made of 1L of water 85g of NaCl 45g ofH3BO3 and 05g of Na

2B4O7sdot10H2O and adjusted pH to

70-72

22 Horse Grouping Immunization Schedule and SerumPlasma Separation Eighteen horses with no natural anti-bodies against TeNT were divided into 6 groups (3 horseseach group) according to age body weight and health con-dition Following the immunization schedule different dosesof TeNT-Hc TT or mixed antigens (Mix Ag) were injectedintramuscularly into the neck and back of horses A three-stage immunization schedule was applied (1) the first stagecontains two basic immunization phases The first phase isbasic immunization with two shots and 7-day interval Beforethe second phase there is a rest of 56 days The second phaseis hyperimmunization with 7 shots and 7-day interval (2) thesecond stage begins after a rest of 16 days with 3 shots and 7-day interval (3) the third stage begins after a rest of 18 dayswith 3 shots and 7-day interval The specific types of antigensand immunization dose administrated was summarized inTable 1 The schedule and the shots were summarized in

BioMed Research International 3

kDa

120100

8060

50

40

30

M

(a)

120100

806050

40

30

20

10

kDa M

(b)

Figure 1 Nonreducing (a) and reducing (b) SDSndashPAGE analysis of TT and TeNT-Hc Lane M molecular weight markers lane 1 TT lane 2TeNT-Hc

Table 1 Horse groups and immunization schedule and doses

Schedule Numberof shots

Group 1(mg)

Group 2(mg)

Group 3(mg)

Group 4(mg)

Group 5(mg)

Group 6(mg)

Routine TTimmunization

(mg)

Stage 1 Phase 1 (Basic)1 TeNT-Hc

0625TeNT-Hc

125TeNT-Hc0625

TeNT-Hc0625 TT 0625 Mix Ag

0625 TT 0625

2 TeNT-Hc1875

TeNT-Hc375

TeNT-Hc1875

TeNT-Hc1875 TT 1875 Mix Ag 1875 TT 1875

Stage 1 Phase 2(Hyperimmunization)

1 TeNT-Hc125

TeNT-Hc1875 TT 125 TeNT-Hc

125TeNT-Hc

125 Mix Ag 125 TT 125

2 TeNT-Hc25

TeNT-Hc375 TT 25 TeNT-Hc 25 TeNT-Hc 25 Mix Ag 25 TT 25

3 TeNT-Hc375


375TeNT-Hc

375 Mix Ag 375 TT 375

4 TeNT-Hc 5 TeNT-Hc 75 TeNT-Hc 5 TT 5 TeNT-Hc 5 Mix Ag 5 TT 55 TeNT-Hc 5 TeNT-Hc 75 TeNT-Hc 5 TT 5 TeNT-Hc 5 Mix Ag 5 TT 5

6 TeNT-Hc 75 TeNT-Hc1125 TeNT-Hc 75 TeNT-Hc 75 TeNT-Hc 75 Mix Ag 75 TT 75

7 TeNT-Hc 10 TeNT-Hc 15 TeNT-Hc 10 TeNT-Hc 10 TeNT-Hc 10 Mix Ag 10 TT 10

Stage 21 TeNT-Hc

375TeNT-Hc

375TeNT-Hc

375TeNT-Hc

375TeNT-Hc

375 Mix Ag 375 TT 375

2 TeNT-Hc 75 TeNT-Hc 75 TeNT-Hc 75 TeNT-Hc 75 TeNT-Hc 75 Mix Ag 75 TT 753 TeNT-Hc 15 TeNT-Hc 15 TeNT-Hc 15 TeNT-Hc 15 TeNT-Hc 15 Mix Ag 15 TT 15

Stage 31 TeNT-Hc

375TeNT-Hc

375TeNT-Hc

375TeNT-Hc

375TeNT-Hc

375 Mix Ag 375 TT 375


Figure 2 In different stagesphases of immunization sched-ule various samples of corresponding horse blood werecollected and analyzed Horse blood samples were collectedfrom the jugular veins and the corresponding plasma wasseparated using a modified human blood apheresis machineThe corresponding serum was collected after the horse bloodclotted and centrifugation

23 SDS-PAGE Analysis of Plasma Antibodies The plasmacollected after the final immunization of the third stage fromeach group or collected after routine TT immunization wasdiluted 10 times with normal saline After being added into2 times loading buffer the samples were detected by a 12SDS-PAGE and the differences plasma components betweendifferent immune groups were analyzed


Day0

Day7

Day54

Day61

Day68

Day75

Day82

Day89

Day96

Day111

Day118

Day125

Day143

Day150

Day157

Stage 1Phase 1 (Basic)

Stage 1 Phase 2 (Hyperimmunization)

Stage 2

Stage 3

Figure 2 The summary of the immunization schedule and the shots

24 Antibody Level Analysis Horse serum and plasma werecollected respectively at the end of the first stage (basicimmunization hyperimmunization) the second stage andthe third stage The anti-TT and anti-TeNT-Hc antibodytiters were determined by ELISA The specific method wassummarized as following Each well on the 96-well ELISAplate (Costar) was coated with 2 120583g mL TT or TeNT-Hc at4∘C overnight and washed 4 times with PBST (PBS + 01Tween-20) Horse serum or plasma was diluted (1 20000 vvfor IgG detection or 1 500 for IgM detection) incubated at37∘C for 1 h washed 4 times in PBST for 5min added 1100000 dilution of HRP-anti horse IgG (1100000 dilution)or HRP-anti horse IgM (1 20000 dilution) incubated at37∘C for 40min washed with PBST 4 times and finallyadded chromogenic solution (TMB Sigma) After colordevelopment 2MH

2SO4was applied to stop the reaction and

the final readout was performed at 450 nm

25 Agar Diffusion Test to Determine Plasma Antibody Titer225g of agarose was added 150mL of purified water It washeated to boil and poured into petri dishes After cooling 9sample reservoirs were made by a hole puncher (one hole inthe center with equal distance to the rest eight holes) To thecenter hole 100120583L of TeNT-Hc (017mgmL) was added andfor the rest of holes in the clockwise fashion 100120583L of serialdiluted immunized horse sera (vv 1 5 110 120 140 1801160 1320 and 1640) was added respectively The loadedpetri dish was placed in a moisture control chamber andincubated at 37∘C for 48 hours before recording the resultingprecipitation lines

26 Flocculation Method to Determine Plasma EfficacyAccording to the method in Chinese Pharmacopoeia (2015edition Method 3506) different volumes (100 LfmL) oftetanus toxin standard solution were precisely-measured andadded to the corresponding reaction tubes Then dilutedhorse plasma (1mL) was added quickly into those tubes andmixed thoroughly The tubes were immersed in a water bath(45-50∘C) and observed closely and the volume of standardsolution for first occurrence of flocculation was recordedAfter repeating three times the horse plasma efficacy (inflocculation unit (LfmL) = V times n times 100 [V is the volumeof the tetanus toxin standard solution used in the firstflocculation (mL) n is the dilution of the horse plasma]) canbe determined

27 In Vivo Tetanus Toxin Neutralization Test Accordingto the method in Chinese Pharmacopoeia (2015 editionMethod 3508) 02mL of the tetanus antitoxin standard (ca05 IUmL) and different concentrations of horse plasma(diluted by borate buffer) were mixed with 02mL of tetanustoxin standard respectively The mixtures were incubatedat 37∘C for 1 h and were then injected into mice intraperi-toneally There were three mice in each group and each groupwas observed at least twice a day for the first five days Thecontrol group should all die within 72 to 120 hours Theefficacy of the horse serum was evaluated as the highestdilution which is most likely to die of the same symptoms asthe control mice

28 Statistical Analysis Unpaired two-tailed Studentrsquos t-testwas used to determine the significance of the differences inantibody titers and neutralizing potency between the groupsProbability (P) values lt005 were considered to be significantandmarked aslowast Plt001 was considered to be very significantand marked as lowastlowast

3 Results

31 Comparison of Plasma Composition after Different Immu-nizationMethods As shown in Figure 1 TeNT-Hcwas shownas single component and has higher purity and smallermolecular weight in comparison with TT However SDS-PAGE (Figure 3) showed no significant difference betweenthe main components (as well as antibodies) in the horseplasma prepared by different immunizationmethods (lanes 1-6) and those immunized byTT (lane 7)Themolecularweightand proportion of the main components of each group arevery similar

32 Anti-Tetanus Toxin Antibody Levels Induced by TeNT-HcSerum titers of anti-TT anti-TeNT-Hc IgG or IgM induced byTeNT-Hc alone or TeNT-Hc and TT co-immunization weretested As shown in Figure 4 the titers of anti-TT IgG inserum of groups 5 and 6 were significantly higher than othergroups one week post the basal immunization Howeverfurther along the immunization schedule there was nosignificant difference (pge005) of anti-TT IgG antibody titersin serum among the groups except group 4 one week afterthe first second and third immunization The lowest anti-TTIgG antibody titer in group 4 has significant difference with


120100

806050

4030

20

10

M kDa

(a)

120100

806050

4030

20

10

M kDa

(b)

Figure 3 Nonreducing (a) and reducing (b) SDSndashPAGE analysis of horse plasmas Lane M molecular weight markers lanes 1 to 6 plasmafrom horses in groups 1 to 6 lane 7 plasma from routine TT immunized horses

8

7

6

5

4

3

2

1

0

Anti-T

T IgG

tiers (log1

0)

Basicimmunizationof first stage

Superimmunizationof first stage

Second stage ird stage

Serum antibody titers

Group 1Group 2Group 3


(a)

8

7

6

5

4

3

2

1

0




Serum antibody titersAnti-T

eNT-Hc IgG

titers

(log

10)



(b)




Serum antiody titers

5

4

3

2

1

0

Anti-T

T Ig

M titer (log1

0)



(c)





5

4

3

2

1

0Anti-T

eNT-Hc IgM

titers

(log

10)



(d)

Figure 4 Serum antibody titers aer different immunization stages (a) Anti-TT IgG titers in serum (b) anti-TeNT-Hc IgG titers inserum (c) anti-TT IgM titers in serum (d) anti-TeNT-Hc IgM titers in serum Unpaired two-tailed Studentrsquos t-test was used to determine thesignificance of the differences in antibody titers between the groups P values le 005 were considered to be significant


Anti-T

T IgG

titers

(log

10)

Plasma antibody titers7

6

5

4

3

2

1

0

Second stageFirst stage ird stage



(a)

Plasma antibody titers

Anti-T

eNT-Hc IgG

titers

7

6

5

4

3

2

1

0




(b)

Anti-T

T IgG

titers

(log

10) Plasma antibody titers

68

66

64

62

6

58

56

54

52

5

Group

1

Group

2

Group

3

Group

4

Group

5

Group

6

TT ro

utine immun

ization

(c)


Anti-T

eNT-Hc T

iters

(log

10)

66

64

62

6

58

56

54

52

5

48

Group

1

Group

2

Group

3

Group

4

Group

5

Group

6

TT ro

utine immun

ization

lowastlowast

(d)

Figure 5 Plasma antibody titers aer different immunization stages (a) Anti-TT IgG titers in plasma from group 1-6 (b) anti-TeNT-HcIgG titers in plasma (c) anti-TT IgG titers in plasma after three stages immunization and TT routine immunized plasma (d) anti-TeNT-HcIgG titers in plasma after three stages immunization and TT routine immunized plasma Unpaired two-tailed Studentrsquos t-test was used todetermine the significance of the differences in antibody titers between the groups P values le 005 were considered to be significant Plt001was considered to be very significant and marked as lowastlowast

the highest antibody titer in group 5 (p lt005) Likewise therewere no significant difference in anti-TeNT-Hc IgG antibodytiter anti-TT and TeNT-Hc IgM antibody titers betweengroups observed in immunization progress (pgt 005) Allimmune methods can induce humoral immunity based onIgG antibody

In addition to serum antibodies antibody titers of anti-TT and anti-TeNT-Hc IgG in horse plasma one week aftereach stage of immunization were also studied (Figure 5) Asthe whole the anti-TT IgG antibody titer in group 5 wasgenerally higher than those in other groups and significantlyhigher than those in groups 1 and 4 after the first stageimmunization (p lt005) However there was no significantdifference of anti-TT IgG titers among the groups after thesecond and the third stage immunization (pge005) The titerof anti-TeNT-Hc IgG antibody in group 4 was slightly lowerand significantly lower than those in group 5 after the first andthird stage immunization (plt005) No significant differenceof anti-TeNT-Hc IgG titerswas found among the other groups

(pge005)The anti-TT and anti-TeNT-Hc IgG titers in plasmaafter three stages of immunization with TeNT-Hc werecompared with those collected after routine immunizationwith TTThe levels of anti-TT IgG antibodies among differentgroups had no significant difference (pge005) However thetiters of anti-TeNT-Hc IgG antibody in the plasma of theTT conventional immunized group were significantly lowerthan those of the six groups of horse plasma immunized withTeNT-Hc (p lt001)

33 Specific Antibody Levels in Horse Plasma Tested byAgarose Diffusion The titers of antitoxins specific bindingto TeNT-Hc in different groups of horse plasma were eval-uated of at different immunization stages by an agarosediffusion method According to the result shown in Fig-ure 6 the titers between group 3 and 4 had significantdifference after the second immunization (plt005) Therewas no significant difference in antibody titers among


First stageSecond stageird stage

Antito

xin titers (log4)

lowast

Group

1Group

2Group

3Group

4Group

5Group

6

Group

1Group

2Group

3Group

4Group

5Group

6Group

1Group

2Group

3Group

4Group

5Group

6

Figure 6 Plasma antibody titers tested by agarose diffusionmethod Unpaired two-tailed Studentrsquos t-test was used to determinethe significance of the differences in antibody titers between thegroups P le 005 was considered to be significant and marked as lowast

Lfm

L

Group

1

Group

2

Group

3

Group

4

Group

5

Group

6

lowast

Figure 7 Antitoxin titers in plasma measured by flocculationUnpaired two-tailed Studentrsquos t-test was used to determine thesignificance of the differences in antibody titers between the groupsPle 005 was considered to be significant and marked as lowast

groups after the first and the third stage of immunization(pge005)

34 Antitoxin Titers in Plasma Measured by Flocculation Inour preliminary experiments the potency of TeNT-Hc witha protein concentration of 017mg mL was determined tobe equivalent to the titer of tetanus toxoid (TT) of 100 Lf mL The potency of each group after three immunizationstages was compared by using the TeNT-Hc (017mg mL)as the antigen As shown in Figure 7 the antitoxin flocculentunits in group 3 and group 5 were slightly higher thanthose in other groups The flocculent units in group 4 weresignificantly lower than those in group 3 (p lt005) Therewas no significant difference among groups 1 2 3 5 and 6(pge005)

35 In Vivo Neutralization of the Antitoxin in Mice The invivo neutralization test in mice was used to test the antitoxinpotency of the plasma in each group one week after each stageof immunization As shown in Figure 8 at one week after thefirst stage of immunization the average titers of the plasmaantitoxins in groups 2 3 5 and 6 were all higher than 1000 IUmL while the average titers of in groups 1 and 4 were lessthan 1000 IU mL One week after the second and third stageof immunization the average antitoxin in each group washigher than 1000 IUmL except that in group 4 According tostatistical analysis there was no significant difference amongthe antitoxin titers in groups 1 2 3 5 and 6 (pge005)

4 Discussion and Conclusion

As an effective immunogen TT has played a critical role inthe preparation of TAT However there were still inherentdrawbacks of using TT the production scale of toxin andbacterial strains were limited due to safety concern theformaldehyde detoxification is generally incomplete andthe large-scale waste could cause environmental issues therepeated immunization with considerable high-dose to theblood-harvesting horses will sooner or later induce the livertoxicity resulting in reduced product quality and increasedproduction costs TeNT-Hc the nontoxic fragment from TTand recombinant expressed in E coli has good immuno-genicity is low-cost is easy to scale up and is purified notmention it is completely safe and nontoxic to both humansand horses TeNT-Hc as a candidate for human tetanusvaccine is currently well underway and sued as a potentialreplacement of TT in TAT production In this paper usingan established immunization procedures and doses the invivo immunogenicity of TeNT-Hc in horses was tested aloneor in combination with TT antigen The antibody titers inserum or plasma in different groups were compared withthose immunized by the conventional TT and it showedvery similar results from those using TT immunization Afteradditional two stages of immunization the average potencyin plasma of all groups reached more than 1000 IU mLexcept that in group 4 With refining the immunizationmethods and adjust the dosage we have confidence that theresults could easily exceed the potency required for tetanusantitoxin production The purpose of this study is to evaluatethe possibility of TeNT-Hc as an immunogen to replace TT inthe tetanus antitoxin production The difference in immuneprograms will affect the titers of antibodies and antitoxinsThe immunization programused in this study is a routine andoptimized procedure for the preparation of tetanus antitoxinby TT immunization For TeNT-Hc this programmay be notthe optimal program and it is necessary to further explore thebest immunization program in the follow-up study

As the experimental results show there was no significantdifference between the groups In group 5 the first two basicimmunizations with TT and the subsequent immunizationwith TeNT-Hc it showed slightly higher antibody titersand potency In group 4 which utilized the sequence ofhyperimmunization with TT and the rest immunization withTeNT-Hc it resulted in lower antibody titers and potency


Neutr

aliz

ation Po

tenc

y (I

Um

L)

Group

1

Group

2

Group

3

Group

4

Group

5

Group

6

First stage

(a)

Neutr

aliz

ation Po

tenc

y (I

Um

L)

Group

1

Group

2

Group

3

Group

4

Group

5

Group

6

Second stage

(b)

Neutr

aliz

ation Po

tenc

y (I

Um

L)

Group

1

Group

2

Group

3

Group

4

Group

5

Group

6

ird stage

(c)

Figure 8Neutralizing potency of the corresponding antibodies in plasma from group 1-6 (a) Plasma collected after the first immunizationstage (b) plasma collected after the second stage (c) plasma collected after the third stage Unpaired two-tailed Studentrsquos t-test was used todetermine the significance of the differences in antibody titers between the groups

The result that there was no significant difference in immuno-genicity between the different dose groups and the differentantigen combination groupsmay indicate that the lowest doseof TeNT-Hc used in the experiment was sufficient for thepreparation of antitoxinHowever due to the small number ofsamples and large variations between the individual horses alarger scale experiment is warranted to figure out the optimalimmunization program By optimizing the immunizationschedule and the corresponding doses TeNT-Hc may evenshow better immunogenicity

Without the accumulated liver toxicity from TT immu-nization TeNT-Hc positions itself as a completely nontoxicrecombinant replacement while retaining all the effectivenessof TT as a long-term safety study could easily reveal Inconclusion this study further confirmed the validity of usingTeNT-Hc to be replaced or used in conjugation with TT toproduce TAT

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

Authors Chong Ji and Denghai Wang were employed byGaotai Tianhong Biochemical Technology DevelopmentCo Ltd Authors Yue Jing and Clifton Kwang-Fu Shenwere employed by Shenzhen JinRuiFeng (Golden Harvest)Biotechnology Ltd which was a subsidiary of ShenzhenQianhai Tianzheng (SQT) Biotechnology Ltd All otherauthors declare no conflicts of interest The authors declarethat they have no conflicts of interest with the contents of thisarticle

References

[1] I H Mallick andM CWinslet ldquoA review of the epidemiologypathogenesis andmanagement of tetanusrdquo International Journalof Surgery vol 2 no 2 pp 109ndash112 2004

[2] L Zibners ldquoDiphtheria pertussis and tetanus evidence-basedmanagement of pediatric patients in the emergency depart-mentrdquo Pediatric Emergency Medicine Practice vol 14 no 2 pp1ndash24 2017


[3] I Lukic E Marinkovic A Filipovic et al ldquoKey protectionfactors against tetanus Anti-tetanus toxin antibody affinity andits ability to prevent tetanus toxin - Ganglioside interactionrdquoToxicon vol 103 pp 135ndash144 2015

[4] K E Vollman N M Acquisto and R P Bodkin ldquoResponse toldquoprotective effect of tetanus antibodiesrdquordquoThe American Journalof Emergency Medicine vol 32 no 9 pp 1128-1129 2014

[5] Y Wu Y Gao B Zhu et al ldquoAntitoxins for diphtheria andtetanus decline more slowly after vaccination with DTwP thanwith DTaP A study in a Chinese populationrdquo Vaccine vol 32no 22 pp 2570ndash2573 2014

[6] S Ghafourian M Raftari N Sadeghifard and Z SekawildquoToxin-antitoxin systems Classification biological functionand application in biotechnologyrdquo Current Issues in MolecularBiology vol 16 no 1 pp 9ndash14 2014

[7] H E de Melker and E W Steyerberg ldquoFunction of tetanusimmunoglobulin in case of injury administration often unnec-essaryrdquo Nederlands Tijdschrift voor Geneeskunde vol 148 no 9pp 429ndash433 2004

[8] S Wendt I Eder R Wolfel P Braun N Lippmann and ARodloff ldquoBotulism Diagnosis and Therapyrdquo Deutsche Medi-zinische Wochenschrift vol 142 no 17 pp 1304ndash1312 2017

[9] W Xu L Ohanjanian J Sun et al ldquoA systematic review andmeta-analysis of preclinical trials testing anti-toxin therapies forB anthracis infection A need formore robust study designs andresultsrdquo PLoS One vol 12 no 8 p e0182879 2017

[10] I Al-Abdulla N R Casewell and J Landon ldquoSingle-reagentone-step procedures for the purification of ovine IgG F(ab1015840)2and Fab antivenoms by caprylic acidrdquo Journal of ImmunologicalMethods vol 402 no 1-2 pp 15ndash22 2014

[11] S Kodihalli A Emanuel T Takla et al ldquoTherapeutic efficacyof equine botulism antitoxin in Rhesus macaquesrdquo PLoS ONEvol 12 no 11 2017

[12] F Hirano S Imamura Y Sasaki et al ldquoEstablishment of anequine tetanus antitoxin reference standard for veterinary usein Japanrdquo Biologicals vol 44 no 5 pp 374ndash377 2016

[13] M Yousefi F Tahmasebi V Younesi et al ldquoCharacterizationof neutralizing monoclonal antibodies directed against tetanustoxin fragment Crdquo Journal of Immunotoxicology vol 11 no 1pp 28ndash34 2014

[14] A Rummel S Bade J Alves H Bigalke and T Binz ldquoTwocarbohydrate binding sites in the HCC-domain of tetanusneurotoxin are required for toxicityrdquo Journal of MolecularBiology vol 326 no 3 pp 835ndash847 2003

[15] J L Halpern and A Loftus ldquoCharacterization of the receptor-binding domain of tetanus toxinrdquo The Journal of BiologicalChemistry vol 268 no 15 pp 11188ndash11192 1993

[16] J Herreros G Lalli and G Schiavo ldquoC-terminal half oftetanus toxin fragment C is sufficient for neuronal bindingand interaction with a putative protein receptorrdquo BiochemicalJournal vol 347 no 1 pp 199ndash204 2000

[17] J MWells P W Wilson P M NortonM J Gasson and R WF Le Page ldquoLactococcus lactis high-level expression of tetanustoxin fragment C and protection against lethal challengerdquoMolecular Microbiology vol 8 no 6 pp 1155ndash1162 1993

[18] R Yu T Fang S Liu et al ldquoComparative Immunogenicity ofthe TetanusToxoid andRecombinant TetanusVaccines inMiceRats and Cynomolgus Monkeysrdquo Toxins vol 8 no 7 p 1942016

[19] R Yu S Yi C Yu et al ldquoA conformational change of C fragmentof tetanus neurotoxin reduces its ganglioside-binding activity

but does not destroy its immunogenicityrdquo Clinical and VaccineImmunology vol 18 no 10 pp 1668ndash1672 2011

[20] R Yu L Hou C Yu et al ldquoEnhanced expression of solublerecombinant tetanus neurotoxin Hc in Escherichia coli as atetanus vaccine candidaterdquo Immunobiology vol 216 no 4 pp485ndash490 2011

Hindawiwwwhindawicom

International Journal of

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Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of



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The Scientific World Journal

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MicrobiologyHindawiwwwhindawicom

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Submit your manuscripts atwwwhindawicom


its high toxicity it is difficult to purify raw tetanus toxinand afford its high-purity form even after formaldehydeinactivationdetoxification During the initial immunizationand subsequent hyperimmunization processes interferenceof impurities within TT antigen results low-grade antibodiesagainst tetanus toxin (2) since TT still has residue toxicityfrequent and high dose injection of TT antigen into blood-harvesting horses induces the gradual hepatic degenerationand necrosis of their liver cells This effect is generallyquite visible after prolonged repeated immunization cycleDuring this time the horse liver continues enlarging anddeteriorating and finally it ruptures and causes internalbleeding and the death of the horse (3) the amount of TTused in horse immunization processes is considerably largeand results in rather high cost Therefore antigen qualityimprovement toxicity reduction and lower production costsare all essential to achieve better TAT [11]

Tetanus toxin is an enormously potent neurotoxinsecreted by the anaerobic bacteriumC tetani After collectingculture supernatants and inactivating with formaldehydetetanus toxoid (TT) is simply harvested by filtration Despiteits good immunogenicity in horse inactivated toxoid gen-erally contaminates with residual formaldehyde which isstill toxic Additionally C tetani can form spores that resistheat and chemical treatment and post certain risks in TTproduction Furthermore inactivation step with formalde-hyde sometimes could not guarantee complete detoxifica-tion of tetanus toxin that could be harmful to the horses[12]

The tetanus toxin native C-fragment (TeNT-Hc) retainsmany properties such as intact binding to gangliosidesimmunogenic potency comparable to native toxin low toxic-ity and low allergenicity [13] It is a very promising candidatefor the development of tetanus subunit vaccines and geneticengineering vaccines which have been used to in Phase Iclinical trial as replacement of TT vaccine conjugates withbacterial and viral vectors mucosal vaccines and many more[14ndash17] Our group has successfully developed a recombinanthuman tetanus vaccine with TeNT-Hc as an antigen in thepast few years [18ndash20] This study aimed at animal immuno-genicity and toxicological pharmacology demonstrated thatTeNT-Hc is a safe effective and yet easy-to-produce low-cost immunogen and suitable for TT replacement in tetanusantitoxin production

In this study we tested the immunogenicity of TeNT-Hcin the preparation of tetanus antitoxins by TeNT-Hc alone orin different combinations with TT to exploit its potential as areplacement immunogen of TT

2 Materials and Methods

Animals Adult male horses (4 to 10 years old 250-400kgbody mass) without tetanus natural antibodies were pur-chased from Datong area in Qinghai Province China ICRmice (17-19g male and female) were purchased from HunanSJA Laboratory Animal Co Ltd The horse and mice studieswere carried out in accordance with the recommendationsof SQT Biotech Antitoxin Production Council on Equine

Welfare Guidelines (GS-P009-01 and GS-P001-01) The pro-tocol was approved by the SQTBiotech Antitoxin ProductionCouncil (OS-P003-01 to OS-P008-01 GS-P006-01 and GS-P015-01) All animals used in this study were raised underhumanitarian conditions with free access to food and waterAll efforts were made to minimize suffering After injectionmice were followed for the five days to check for any signsof paralysis or death Loss of righting reflex was used as thehumane end point of the experiment Mice were monitoredthree times a day for their condition and for the occurrenceof end point

21 Materials

Incomplete Freundrsquos Adjuvant Liquid paraffin (ShanghaiZhongqin Chemical Reagent Co Ltd) and lanolin (Medicalgrade China Huating Lanolin Plant) were mixed (volumetricratio of 2 1) to afford the adjuvant Antigen and AdjuvantTeNT-Hc (molecular weight 45 kD prepared by the Depart-ment of vaccine and antibody engineering Beijing Instituteof Biotechnology) was mixed with incomplete Freundrsquos adju-vant into a water-in-oil emulsion with protein content of0625mgmL Tetanus toxoid (TT) purchased from ChengduOlymvax Biopharmaceuticals China wasmixedwith incom-plete Freundrsquos adjuvant into a water-in-oil emulsion with aprotein content of 0625mgmL A mixed antigen solutionwas prepared by mixing TeNT-Hc and TT with incompleteFreundrsquos adjuvant with incomplete Freundrsquos adjuvant into awater-in-oil emulsion with a protein content of 0625mgmLThe SDS-PAGE electrophoresis analysis of TeNT-Hc and TTis shown in Figure 1 In comparison with TT TeNT-Hc wasshown as single component and has higher purity and smallermolecular weight Secondary Antibody HRP-conjugated anti-horse IgG and IgM as secondary antibodies was purchasedfrom Abcam Standard Standard tetanus toxins and anti-tetanus serum were purchased from the China NationalInstitutes of Food andDrugControlBorate BufferTheboratesolution was made of 1L of water 85g of NaCl 45g ofH3BO3 and 05g of Na

2B4O7sdot10H2O and adjusted pH to

70-72

22 Horse Grouping Immunization Schedule and SerumPlasma Separation Eighteen horses with no natural anti-bodies against TeNT were divided into 6 groups (3 horseseach group) according to age body weight and health con-dition Following the immunization schedule different dosesof TeNT-Hc TT or mixed antigens (Mix Ag) were injectedintramuscularly into the neck and back of horses A three-stage immunization schedule was applied (1) the first stagecontains two basic immunization phases The first phase isbasic immunization with two shots and 7-day interval Beforethe second phase there is a rest of 56 days The second phaseis hyperimmunization with 7 shots and 7-day interval (2) thesecond stage begins after a rest of 16 days with 3 shots and 7-day interval (3) the third stage begins after a rest of 18 dayswith 3 shots and 7-day interval The specific types of antigensand immunization dose administrated was summarized inTable 1 The schedule and the shots were summarized in


kDa

120100

8060

50

40

30

M

(a)

120100

806050

40

30

20

10

kDa M

(b)




Group 1(mg)

Group 2(mg)

Group 3(mg)

Group 4(mg)

Group 5(mg)

Group 6(mg)


(mg)


0625TeNT-Hc

125TeNT-Hc0625


0625 TT 0625

2 TeNT-Hc1875

TeNT-Hc375

TeNT-Hc1875



1 TeNT-Hc125


125TeNT-Hc

125 Mix Ag 125 TT 125

2 TeNT-Hc25


3 TeNT-Hc375


375TeNT-Hc

375 Mix Ag 375 TT 375




Stage 21 TeNT-Hc

375TeNT-Hc

375TeNT-Hc

375TeNT-Hc

375TeNT-Hc

375 Mix Ag 375 TT 375


Stage 31 TeNT-Hc

375TeNT-Hc

375TeNT-Hc

375TeNT-Hc

375TeNT-Hc

375 Mix Ag 375 TT 375





Day0

Day7

Day54

Day61

Day68

Day75

Day82

Day89

Day96

Day111

Day118

Day125

Day143

Day150

Day157



Stage 2

Stage 3









3 Results




120100

806050

4030

20

10

M kDa

(a)

120100

806050

4030

20

10

M kDa

(b)


8

7

6

5

4

3

2

1

0

Anti-T

T IgG

tiers (log1

0)







(a)

8

7

6

5

4

3

2

1

0





eNT-Hc IgG

titers

(log

10)



(b)





5

4

3

2

1

0

Anti-T

T Ig

M titer (log1

0)



(c)





5

4

3

2

1

0Anti-T

eNT-Hc IgM

titers

(log

10)



(d)



Anti-T

T IgG

titers

(log

10)


6

5

4

3

2

1

0




(a)


Anti-T

eNT-Hc IgG

titers

7

6

5

4

3

2

1

0




(b)

Anti-T

T IgG

titers

(log


68

66

64

62

6

58

56

54

52

5

Group

1

Group

2

Group

3

Group

4

Group

5

Group

6

TT ro

utine immun

ization

(c)


Anti-T

eNT-Hc T

iters

(log

10)

66

64

62

6

58

56

54

52

5

48

Group

1

Group

2

Group

3

Group

4

Group

5

Group

6

TT ro

utine immun

ization

lowastlowast

(d)








Antito

xin titers (log4)

lowast

Group

1Group

2Group

3Group

4Group

5Group

6

Group

1Group

2Group

3Group

4Group

5Group

6Group

1Group

2Group

3Group

4Group

5Group

6


Lfm

L

Group

1

Group

2

Group

3

Group

4

Group

5

Group

6

lowast









Neutr

aliz

ation Po

tenc

y (I

Um

L)

Group

1

Group

2

Group

3

Group

4

Group

5

Group

6

First stage

(a)

Neutr

aliz

ation Po

tenc

y (I

Um

L)

Group

1

Group

2

Group

3

Group

4

Group

5

Group

6

Second stage

(b)

Neutr

aliz

ation Po

tenc

y (I

Um

L)

Group

1

Group

2

Group

3

Group

4

Group

5

Group

6

ird stage

(c)




Data Availability




References

























Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



kDa

120100

8060

50

40

30

M

(a)

120100

806050

40

30

20

10

kDa M

(b)




Group 1(mg)

Group 2(mg)

Group 3(mg)

Group 4(mg)

Group 5(mg)

Group 6(mg)


(mg)


0625TeNT-Hc

125TeNT-Hc0625


0625 TT 0625

2 TeNT-Hc1875

TeNT-Hc375

TeNT-Hc1875



1 TeNT-Hc125


125TeNT-Hc

125 Mix Ag 125 TT 125

2 TeNT-Hc25


3 TeNT-Hc375


375TeNT-Hc

375 Mix Ag 375 TT 375




Stage 21 TeNT-Hc

375TeNT-Hc

375TeNT-Hc

375TeNT-Hc

375TeNT-Hc

375 Mix Ag 375 TT 375


Stage 31 TeNT-Hc

375TeNT-Hc

375TeNT-Hc

375TeNT-Hc

375TeNT-Hc

375 Mix Ag 375 TT 375





Day0

Day7

Day54

Day61

Day68

Day75

Day82

Day89

Day96

Day111

Day118

Day125

Day143

Day150

Day157



Stage 2

Stage 3









3 Results




120100

806050

4030

20

10

M kDa

(a)

120100

806050

4030

20

10

M kDa

(b)


8

7

6

5

4

3

2

1

0

Anti-T

T IgG

tiers (log1

0)







(a)

8

7

6

5

4

3

2

1

0





eNT-Hc IgG

titers

(log

10)



(b)





5

4

3

2

1

0

Anti-T

T Ig

M titer (log1

0)



(c)





5

4

3

2

1

0Anti-T

eNT-Hc IgM

titers

(log

10)



(d)



Anti-T

T IgG

titers

(log

10)


6

5

4

3

2

1

0




(a)


Anti-T

eNT-Hc IgG

titers

7

6

5

4

3

2

1

0




(b)

Anti-T

T IgG

titers

(log


68

66

64

62

6

58

56

54

52

5

Group

1

Group

2

Group

3

Group

4

Group

5

Group

6

TT ro

utine immun

ization

(c)


Anti-T

eNT-Hc T

iters

(log

10)

66

64

62

6

58

56

54

52

5

48

Group

1

Group

2

Group

3

Group

4

Group

5

Group

6

TT ro

utine immun

ization

lowastlowast

(d)








Antito

xin titers (log4)

lowast

Group

1Group

2Group

3Group

4Group

5Group

6

Group

1Group

2Group

3Group

4Group

5Group

6Group

1Group

2Group

3Group

4Group

5Group

6


Lfm

L

Group

1

Group

2

Group

3

Group

4

Group

5

Group

6

lowast









Neutr

aliz

ation Po

tenc

y (I

Um

L)

Group

1

Group

2

Group

3

Group

4

Group

5

Group

6

First stage

(a)

Neutr

aliz

ation Po

tenc

y (I

Um

L)

Group

1

Group

2

Group

3

Group

4

Group

5

Group

6

Second stage

(b)

Neutr

aliz

ation Po

tenc

y (I

Um

L)

Group

1

Group

2

Group

3

Group

4

Group

5

Group

6

ird stage

(c)




Data Availability




References

























Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



Day0

Day7

Day54

Day61

Day68

Day75

Day82

Day89

Day96

Day111

Day118

Day125

Day143

Day150

Day157



Stage 2

Stage 3









3 Results




120100

806050

4030

20

10

M kDa

(a)

120100

806050

4030

20

10

M kDa

(b)


8

7

6

5

4

3

2

1

0

Anti-T

T IgG

tiers (log1

0)







(a)

8

7

6

5

4

3

2

1

0





eNT-Hc IgG

titers

(log

10)



(b)





5

4

3

2

1

0

Anti-T

T Ig

M titer (log1

0)



(c)





5

4

3

2

1

0Anti-T

eNT-Hc IgM

titers

(log

10)



(d)



Anti-T

T IgG

titers

(log

10)


6

5

4

3

2

1

0




(a)


Anti-T

eNT-Hc IgG

titers

7

6

5

4

3

2

1

0




(b)

Anti-T

T IgG

titers

(log


68

66

64

62

6

58

56

54

52

5

Group

1

Group

2

Group

3

Group

4

Group

5

Group

6

TT ro

utine immun

ization

(c)


Anti-T

eNT-Hc T

iters

(log

10)

66

64

62

6

58

56

54

52

5

48

Group

1

Group

2

Group

3

Group

4

Group

5

Group

6

TT ro

utine immun

ization

lowastlowast

(d)








Antito

xin titers (log4)

lowast

Group

1Group

2Group

3Group

4Group

5Group

6

Group

1Group

2Group

3Group

4Group

5Group

6Group

1Group

2Group

3Group

4Group

5Group

6


Lfm

L

Group

1

Group

2

Group

3

Group

4

Group

5

Group

6

lowast









Neutr

aliz

ation Po

tenc

y (I

Um

L)

Group

1

Group

2

Group

3

Group

4

Group

5

Group

6

First stage

(a)

Neutr

aliz

ation Po

tenc

y (I

Um

L)

Group

1

Group

2

Group

3

Group

4

Group

5

Group

6

Second stage

(b)

Neutr

aliz

ation Po

tenc

y (I

Um

L)

Group

1

Group

2

Group

3

Group

4

Group

5

Group

6

ird stage

(c)




Data Availability




References

























Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



120100

806050

4030

20

10

M kDa

(a)

120100

806050

4030

20

10

M kDa

(b)


8

7

6

5

4

3

2

1

0

Anti-T

T IgG

tiers (log1

0)







(a)

8

7

6

5

4

3

2

1

0





eNT-Hc IgG

titers

(log

10)



(b)





5

4

3

2

1

0

Anti-T

T Ig

M titer (log1

0)



(c)





5

4

3

2

1

0Anti-T

eNT-Hc IgM

titers

(log

10)



(d)



Anti-T

T IgG

titers

(log

10)


6

5

4

3

2

1

0




(a)


Anti-T

eNT-Hc IgG

titers

7

6

5

4

3

2

1

0




(b)

Anti-T

T IgG

titers

(log


68

66

64

62

6

58

56

54

52

5

Group

1

Group

2

Group

3

Group

4

Group

5

Group

6

TT ro

utine immun

ization

(c)


Anti-T

eNT-Hc T

iters

(log

10)

66

64

62

6

58

56

54

52

5

48

Group

1

Group

2

Group

3

Group

4

Group

5

Group

6

TT ro

utine immun

ization

lowastlowast

(d)








Antito

xin titers (log4)

lowast

Group

1Group

2Group

3Group

4Group

5Group

6

Group

1Group

2Group

3Group

4Group

5Group

6Group

1Group

2Group

3Group

4Group

5Group

6


Lfm

L

Group

1

Group

2

Group

3

Group

4

Group

5

Group

6

lowast









Neutr

aliz

ation Po

tenc

y (I

Um

L)

Group

1

Group

2

Group

3

Group

4

Group

5

Group

6

First stage

(a)

Neutr

aliz

ation Po

tenc

y (I

Um

L)

Group

1

Group

2

Group

3

Group

4

Group

5

Group

6

Second stage

(b)

Neutr

aliz

ation Po

tenc

y (I

Um

L)

Group

1

Group

2

Group

3

Group

4

Group

5

Group

6

ird stage

(c)




Data Availability




References

























Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



Anti-T

T IgG

titers

(log

10)


6

5

4

3

2

1

0




(a)


Anti-T

eNT-Hc IgG

titers

7

6

5

4

3

2

1

0




(b)

Anti-T

T IgG

titers

(log


68

66

64

62

6

58

56

54

52

5

Group

1

Group

2

Group

3

Group

4

Group

5

Group

6

TT ro

utine immun

ization

(c)


Anti-T

eNT-Hc T

iters

(log

10)

66

64

62

6

58

56

54

52

5

48

Group

1

Group

2

Group

3

Group

4

Group

5

Group

6

TT ro

utine immun

ization

lowastlowast

(d)








Antito

xin titers (log4)

lowast

Group

1Group

2Group

3Group

4Group

5Group

6

Group

1Group

2Group

3Group

4Group

5Group

6Group

1Group

2Group

3Group

4Group

5Group

6


Lfm

L

Group

1

Group

2

Group

3

Group

4

Group

5

Group

6

lowast









Neutr

aliz

ation Po

tenc

y (I

Um

L)

Group

1

Group

2

Group

3

Group

4

Group

5

Group

6

First stage

(a)

Neutr

aliz

ation Po

tenc

y (I

Um

L)

Group

1

Group

2

Group

3

Group

4

Group

5

Group

6

Second stage

(b)

Neutr

aliz

ation Po

tenc

y (I

Um

L)

Group

1

Group

2

Group

3

Group

4

Group

5

Group

6

ird stage

(c)




Data Availability




References

























Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018




Antito

xin titers (log4)

lowast

Group

1Group

2Group

3Group

4Group

5Group

6

Group

1Group

2Group

3Group

4Group

5Group

6Group

1Group

2Group

3Group

4Group

5Group

6


Lfm

L

Group

1

Group

2

Group

3

Group

4

Group

5

Group

6

lowast









Neutr

aliz

ation Po

tenc

y (I

Um

L)

Group

1

Group

2

Group

3

Group

4

Group

5

Group

6

First stage

(a)

Neutr

aliz

ation Po

tenc

y (I

Um

L)

Group

1

Group

2

Group

3

Group

4

Group

5

Group

6

Second stage

(b)

Neutr

aliz

ation Po

tenc

y (I

Um

L)

Group

1

Group

2

Group

3

Group

4

Group

5

Group

6

ird stage

(c)




Data Availability




References

























Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



Neutr

aliz

ation Po

tenc

y (I

Um

L)

Group

1

Group

2

Group

3

Group

4

Group

5

Group

6

First stage

(a)

Neutr

aliz

ation Po

tenc

y (I

Um

L)

Group

1

Group

2

Group

3

Group

4

Group

5

Group

6

Second stage

(b)

Neutr

aliz

ation Po

tenc

y (I

Um

L)

Group

1

Group

2

Group

3

Group

4

Group

5

Group

6

ird stage

(c)




Data Availability




References

























Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018
























Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


The Immunogenicity of the C Fragment of Tetanus Neurotoxin...

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