research article evaluation of protective immune responses...

Research ArticleEvaluation of Protective Immune ResponsesInduced by Recombinant TrxLp and ENO2 Proteins againstToxoplasma gondii Infection in BALBc Mice

Meng Wang Xiao-Yu Yang Nian-Zhang Zhang De-Lin Zhang and Xing-Quan Zhu

State Key Laboratory of Veterinary Etiological Biology Key Laboratory of Veterinary Parasitology of Gansu ProvinceLanzhou Veterinary Research Institute Chinese Academy of Agricultural Sciences Lanzhou Gansu 730046 China

Correspondence should be addressed to Nian-Zhang Zhang nianzhang919163com

Received 4 July 2016 Revised 18 August 2016 Accepted 7 September 2016

Academic Editor Roberto Amerigo Papini

Copyright copy 2016 Meng Wang et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Toxoplasma gondii is an obligate intracellular parasitic protozoan that can infect almost all species of warm-blooded animals As anychemical-based drugs could not act against the tissue cyst stage of T gondii vaccination may be one of the ideal control strategiesIn the present study two new vaccine candidates named TgENO2 and TgTrxLp were purified from Escherichia coli with pET-30a(+) expression system and then were injected into BALBc mice to evaluate the protective efficacy against acute and chronictoxoplasmosis The results showed that both the recombinant proteins either alone or in combination could elicit strong humoraland cellular immune responses with a higher level of IgG antibodies IFN-120574 IL-2 CD4+ and CD8+ T cells as compared to thosein mice from control groups After acute challenge with tachyzoites of the GJS strain mice immunized with rTgTrxLp (8 plusmn 277 d)rTgENO2 (74 plusmn 181 d) and rTgTrxLp + rTgENO2 (838 plusmn 457 d) proteins showed significantly longer survival time than thosethat received Freundrsquos adjuvant (678 plusmn 208 d) and PBS (638 plusmn 465 d) (1205942 = 9687 df = 4 119875 = 0046) The protective immunity ofrTgTrxLp rTgENO2 and rTgTrxLp + rTgENO2 proteins against chronic T gondii infection showed 6977 5814 and 2093brain cyst reduction as compared to mice that received PBS The present study suggested that both TgENO2 and TgTrxLp werepotential candidates for the development of multicomponent vaccines against toxoplasmosis

1 Introduction

Toxoplasma gondii is a worldwide prevalent pathogen inall the warm-blooded animals including humans [1 2] Tgondii infection in immune-competent individuals is rarelysymptomatic However the infection occurring in the fetus orimmunocompromised patients (HIV patients) could result insevere diseases or even death [3ndash5] Infection of domestic ani-mals with the parasite can cause substantial economic lossesand also pose a considerable threat to public health [6ndash10]

The infection begins from ingestion of oocysts or cystsof T gondii Once the tachyzoites invade into the intestinalepithelial cells the parasites rapidly proliferate by intracel-lular endodyogeny [1] During the division of tachyzoitesT gondii enolase (TgENO2) exhibits robust nuclear labelingwith the ability to bind promoters and to regulate the geneexpression [11ndash13]

Subsequent to the infection of the intestinal epithelialcells T gondii disseminate throughout the organism Duringthe migration the protozoan is exposed to reactive oxygenspecies (ROS) generated by inflammatory cells T gondiievolved a series of antioxidant proteins to relieve the oxidativestress produced by the host immune system Thioredoxin(Trx) is one of the important antioxidants against the ROSdestruction that can convert hydrogen peroxide (H

2O2) to

water [14ndash16]The thioredoxin-like protein (TrxL) inT gondiiwas discovered as a component of a microtubule-associatedcomplex but not directly interacting with the microtubuleswhich would support numerous vital cellular functions ineukaryotes [17]

In our previous study both of the enolase and Trx pro-teins were identified in T gondii excretedsecreted antigens(TgESA) from mice enterocelia after being infected by theparasite (unpublished data) The crude ESAs of T gondii

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 3571962 8 pageshttpdxdoiorg10115520163571962

2 BioMed Research International

are considered attractive vaccine candidates that have beenwidely studied in animal models [18ndash20] However it isyet to be clarified whether TgENO2 and TgTrxLp as theconstituents of TgESAs could induce protective immuneresponses against T gondii infection in the mouse modelIn the present study the immunogenicity of TgENO2 andTgTrxLp recombinant proteins was examined in mice Fur-thermore the immunoprophylaxis efficacy against acute andchronic toxoplasmosis was also estimated

2 Materials and Methods

21 Animals Thespecific-pathogen-free (SPF) grade BALBcand Kunming mice (6ndash8 weeks old) were purchased fromLanzhou Veterinary Research Institute Laboratory AnimalCenter (Lanzhou China) All animals were strictly handledaccording to the Good Animal Practice Requirements ofthe Animal Ethics Procedures and Guidelines of the Peo-plersquos Republic of China The present study was approvedby the Animal Ethics Committee of Lanzhou VeterinaryResearch Institute ChineseAcademyofAgricultural Sciences(Approval no LVRIAEC2012-011)

22 Parasites Tachyzoites of T gondiiGJS (Genotype DB9)strain were maintained in Kunming mice by a series ofintraperitoneal infections and obtained from the peritonealexudates followed by purification by centrifugation asdescribed by Qu et al [21 22] Kunming mice infected withthe low virulent PRU strain (Genotype II) were used to collectthe toxoplasma cysts that were orally passaged by infection ofthe brain homogenate

23 Prokaryotic Expression of TgTrxLp and TgENO2 Proteinsand Purification The full-length coding sequences of theTgTrxLp (GenBank access number XM 0023697031) andTgENO2 (GenBank accession number AF1234571) geneswere amplified using one-step reverse transcription-PCR(RT-PCR) following themanufacturerrsquos instructions (TakaraChina) The primers for the amplification of TgTrxLp frag-ment were 51015840-GG119866119866119879119860119862119862ATGGCGCCTCTGCGTGT-GTGCGCGTTC-31015840 (forward) and 51015840-CC119860119860119866119862119879119879TT-ACAGTTCGTCCTTCTTGTCTGCCTT-31015840 (reverse) andfor TgENO2 fragment were 51015840-CG119866119860119860119879119879119862ATGGTGGC-CATCAAGGACATCACTGCT-31015840 (forward) and 51015840-CC-119860119860119866119862119879119879TTAGTTGGGATGGCGGAAGCCAGCGCC-31015840(reverse) The restriction sites induced in the two pairs ofprimers were italicized

Each RT-PCR product was purified (Tiangen China) andligated into the prokaryotic expression vector pET-30a(+)via the respective restriction sites forming the recombinantplasmids pET-TrxLp and pET-ENO2 respectively Subse-quently the two recombinant plasmids were transformedinto Escherichia coli strain BL21(DE3) and induced with10mmolL isopropyl 120573-d-1-thiogalactopyranoside (SangonChina) shaking for 6 h at 30∘CThe rTgTrxLp and rTgENO2proteins were purified on a Ni2+ column (Novagen USA)following ultrasonic bacterial lysis on the ice The purifiedprotein samples were analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

24 Western Blot Analysis The proteins were resolved onSDS-PAGE and the purified proteins were transferred tonitrocellulose (NC) membranes (Pall USA)Then the mem-branes were blocked with 5 bovine serum albumin (BSA) inPBST (005 Tween-20 in PBS) at room temperature (RT)After 1 h the membrane was washed 4 times with PBSTThe swine sera against T gondii that were collected at 60days after the onset of symptoms as described previously[23] were diluted 1 1000 as the first antibody probed onthe membranes for 1 h at RT Then the membranes werewashed 4 times with PBST and incubated with horseradishperoxidase- (HRP-) conjugated goat anti-pig IgG (1 5000)(Sigma USA) The immunogens were developed with ECLreagents A and B (TIANGEN China) according to themanufacturerrsquos instructions

25 Immunization and Challenge The female BALBc micewere randomly divided into five groups (26 mice in eachgroup) The animals were subcutaneously injected with100 120583g rTgTrxLp + rTgENO2 (G1) rTgTrxLp (G2) orrTgENO2 (G3) proteins emulsified in 100120583L Freundrsquos com-plete adjuvant (FCA) respectively Two weeks following theprimary immunization mice in G1 G2 and G3 were inoc-ulated with the same dose of each antigen plus incompleteFreundrsquos adjuvant respectively The mice injected with equaladjuvant (G4) or PBS alone (G5) served as negative controls

Fifteen mice in each group were subjected to acuteinfection by the intraperitoneal administration with 103 Tgondii tachyzoites of GJS strain at 2 weeks after the thirdimmunization The challenged mice were monitored dailyuntil total mortality Six mice from each group were orallychallenged with 10 cysts of T gondii PRU strain at 2 weeksafter the final immunization Thirty days later the animalswere sacrificed to examine the number of brain cysts

26 Collection of the Sera and Lymphocyte Samples Theblood samples ofmice fromall the groupswere collected fromthe tail vein prior to each vaccinationThe serawere harvestedby centrifugation at 2000timesg for 20min and stored at minus20∘Cuntil assayed for antibody titers and cytokines

Two weeks after the final immunization 3 mice pergroup were sacrificed to aseptically harvest the spleen Thespleens were pooled and filtered through a nylon membraneto obtain the splenocytes Then the cells were purified usingerythrocyte lysis buffer (Solarbio China) to remove the redblood cells Subsequently the harvested splenocytes wereresuspended in DMEM medium supplemented with 10fetal calf serum (FCS) and were used for the analysis oflymphocyte proliferation and the percentage of CD4+ andCD8+ T cells

27 IgG ELISA The specific humoral immune responsesanti-rTgTrxLp rTgENO2 or the mixtures were evaluated byELISA using SBA Clonotyping System-HRP Kit (SouthernBiotech Co Ltd Birmingham USA) according to the man-ufacturerrsquos instructions The preparation of rTgTrxLp andrTgENO2 proteins was adjusted to 50 120583gmL The 96-wellmicrotiter plates were coated with 100 120583L of each protein at37∘C for 2 h 100 120583L of the prepared serum samples (1 10

BioMed Research International 3

dilution) from mice of each group was the added andincubated at RT for 1 h The secondary antibody goat anti-mouseHRP-IgG (Sigma) at 1 250 was incubated on the plateat RT for 1 h After 3 rinses the plates were visualized byincubating with substrate solution (pH 40) (105 citratesubstrate buffer 15 ABTS 003 H

2O2) for 15min in

dark and then the reaction was stopped with 2M H2SO4

The absorbance of each well was measured at 450 nm Allestimations were performed in triplicate

28 Lymphocyte Proliferation Assays by MTS The puri-fied splenocytes per group were stimulated with the cor-responding antigens (CAS) and concanavalin A (ConASigma) after the density of cells was adjusted to 2 times 105The cells cocultured with medium alone served as thenegative control Four days later the proliferation activ-ity was measured by MTS method (Promega USA) Thestimulation index (SI) was calculated using the formulaOD490CASOD490M OD

490ConAOD490M

29 FlowCytometry Analysis Thepurified splenocytes resus-pended in DMEM medium plus 10 FCS were incubatedwith fluorescently in-labeled anti-mouse IgG antibodiesincluding PE-CD3 APC-CD4 and FITC-CD8 (BioLegendUSA) for 30min at 4∘C After PBS washes the cells were fixedwith FACSbuffer (1FCSplus 01 sodiumazide in PBS) and2 paraformaldehyde under dim light Data were collectedand analyzed by System II software (Coulter)

210 Cytokine Assays The collected sera from mice in eachgroup were used to examine the levels of IL-2 IL-4 and IFN-120574 in flat-bottom 96-well microtiter plates at 1 10 dilutionThe detection was performed using commercial ELISA kitsaccording to the manufacturerrsquos instructions (BioLegendUSA) The data from three independent experiments wereanalyzed

211 Statistical Analysis One-way ANOVA was used forcomparing the differences in antibody responses percentagesof CD4+ and CD8+ T cells and the reduction in braincysts The difference of each variable in lymphoproliferationassays between the two groups was calculated by the 119905-testThe difference in survival time was calculated by the chi-square testThe figures were prepared by the GraphPad Prismstatistical program version 50 (San Diego CA USA) Avalue of 119875 lt 005 was considered significant

3 Results

31 Identification of rTgTrxLp and rTgENO2 Proteins by SDS-PAGE and Western Blot After respective transformationof pET-TrxLp and pET-ENO2 constructs into E coli BL21(DE3) the recombinant bacteria were lysed and proteinsresolved using SDS-PAGE and stained with CoomassieBrilliant Blue The rTgTrxLp and rTgENO2 proteins wereidentified as approximately 49 kDa which coincided with thecorrespondingly theoretical molecular mass (Figure 1) TheWestern blot results showed that rTgTrxLp and rTgENO2proteins could be identified by the sera from swine that was

M 1 2 3 4

70 kDa

55 kDa

35 kDa

Figure 1 Identification of rTgTrxLp and rTgENO2 proteins withsera from pigs infected with Toxoplasma gondii GJS strain byWestern blot M protein molecular weight lanes 1 and 3 rTgTrxLpand rTgENO2 identified by sera from pigs infected with T gondiiGJS strain lanes 2 and 4 rTgTrxLp and rTgENO2 reacted with Tgondii negative sera from pigs

00

05

10

15

20

25

OD

490

G5 G4 G3 G2 G1

NS

NS

lowastlowastlowastlowastlowastlowast

lowastlowastlowast


lowastlowastlowast



lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0weeks2weeks

4weeks6weeks

Figure 2 IgG antibodies induced by rTgTrxLp + rTgENO2 (G1)rTgTrxLp (G2) rTgENO2 (G3) adjuvant (G4) or PBS alone (G5) inthe sera of mice at 0 2 and 8 weeks Each bar represents the meanOD (plusmnSE 119899 = 3) lowastlowastlowast119875 lt 00001 NS not significant compared tocontrols

infected with T gondii as assessed by the positive band atnearly 49 kDa (Figure 1)

32 Humoral Immune Responses To analyze the specifichumoral immune responses induced by various vaccines theserum samples collected prior to each immunization wereexamined by ELISA (Figure 2) The results revealed that theIgG antibody elicited by each protein vaccine was continu-ously increased with successive immunization and reacheda maximum level at 2 weeks after the final immunization[119865(4 25) = 4329 119875 lt 00001] compared with that in the


00

05

10

15

20

25St

imul

atio

n in

dex

(SI)

rTgTrxLp(5120583gmL)

ConA(5120583gmL)

rTgTrxLp(15 120583gmL)

lowast

lowastlowastlowast

lowastlowastlowast

PBSFreundrsquos adjuvantrTgTrxLp

(a)

Stim

ulat

ion

inde

x (S

I)

ConA(5120583gmL)

lowast

lowastlowastlowast

lowastlowastlowast

PBSFreundrsquos adjuvant

0

1

2

3

4

(5120583gmL)rTgENO2

(15 120583gmL)rTgENO2

rTgENO2

(b)

Stim

ulat

ion

inde

x (S

I)

ConA(5120583gmL)



NS

rTgTrxLp + rTgENO2

rTgTrxLp +rTgENO2(5120583gmL)

rTgTrxLp +rTgENO2

(15120583gmL)

0

1

2

3

(c)

Figure 3 Splenocyte proliferation responses in immunized mice lowast119875 lt 005 lowastlowastlowast119875 lt 00001 NS not significantly represented splenocytesfrommice immunized with rTgTrxLp (a) rTgENO2 (b) or rTgTrxLp + rTgENO2 (c) that was stimulated with 5120583gmL and 15 120583gmL of eachprotein compared with those in mice from Freundrsquos adjuvant and PBS Each bar represents the mean stimulation index (plusmn SE 119899 = 3)

control groups However after the third immunization thesignificantly highest IgG levels were detected inmice fromG3as compared to that in mice fromG1 and G2 [119865(2 15) = 1487119875 = 00003] No statistically significant differences in levels ofIgG antibodies in the sera from controls at 2 weeks after thelast immunization were observed [119905(10) = 1937 119875 = 0082]

33 Splenocyte Proliferation The proliferation of splenocytesstimulated by antigens or ConAwas examined using theMTSassay The splenocytes frommice in G1 G2 and G3 were sig-nificantly proliferative after stimulation by the correspondingantigen proteins compared to that from mice in G4 and G5(119875 lt 00001 Figure 3) The levels of SI were significantlyelevated with the increased concentration of rTgENO2 [119905(4)= 2782119875 = 00497] and rTgTrxLp [119905(4)=4056119875 = 00154]

However no significant differencewas detected in the spleno-cytes in mice from G1 after coculturing with 15 120583gmL and5 120583gmL rTgTrxLp + rTgENO2 respectively [119905(4) = 1490119875 = 0210]

34 Flow Cytometry Analysis Percentages of CD4+ andCD8+ T cells in mice from each group were examined byflow cytometric analysis for the specific surface marker Thepercentages of CD4+ T cells in total splenocytes in micefrom G1 [244 plusmn 137 119865(2 6) = 2933 119875 = 00008]G2 [2807 plusmn 186 119865(2 6) = 4690 119875 lt 00002] or G3[2783 plusmn 07 119865(2 6) = 5904 119875 = 00001] were signifi-cantly higher than those in the controls which varied from13 to 1417 (Figure 4(a)) Compared with the control groupsremarkably high levels of CD8+ T cells in mice from the G1[119865(2 6) = 1595 119875 = 0004] G2 [119865(2 6) = 3113 119875 = 00007]


0

10

20

30

40

PBS Freundrsquos rTgTrxLp rTgENO2 rTgTrxLp +rTgENO2


lowastlowastlowast

Perc

ent o

f CD4+

T ce

lls

adjuvant

(a)


0

5

10

15

20


lowastlowastlowast

Perc

ent o

f CD8+

T ce

lls

adjuvant

(b)

Figure 4 Percentages of CD4+ (a) and CD8+ (b) T cells in micelowastlowastlowast119875 lt 0001 NS not significant compared to controls Mice

that received PBS and Freundrsquos adjuvant were treated as controls instatistical analysis

and G3 [119865(2 6) = 3946 119875 = 00004] groups were detectedcompared to that in the controls (Figure 4(b))

35 Cytokine Assays The cytokines in serum samples fromeach group were quantified by ELISA As shown in Figure 5levels of IFN-120574 in mice that received rTgTrxLp [119865(2 12) =9982 119875 = 00028] rTgENO2 [119865(2 12) = 6518 119875 = 00121]or rTgTrxLp + rTgENO2 [119865(2 12) = 1206 119875 = 00013] weresignificantly increased as compared to that in the controlsAlso the levels of IL-2 were significantly elevated in miceimmunized with rTgTrxLp [119865(2 12) = 9394 119875 = 00035]rTgENO2 [119865(2 12) = 1051 119875 = 00023] or rTgTrxLp +rTgENO2 [119865(2 12) = 1224 119875 = 00013] as compared to thatin the controls However any substantial differences in IL-4were not seen among the groups (119875 = 007)

36 Protection against Acute and Chronic T gondii InfectionAs shown in Figure 6 the acute T gondii infection in micefrom G1 (838 plusmn 457 d) G2 (8 plusmn 277 d) and G3 (74 plusmn 181 d)survived significantly longer than the infected mice from G4(678 plusmn 208 d) and G5 (638 plusmn 465 d) (1205942 = 9687 df = 4 119875 =0046)

To evaluate whether rTgTrxLp rTgENO2 and rTgTrxLp+ rTgENO2 proteins could generate protective immunityagainst chronic T gondii infection mice from each groupwere challenged with 10 cysts of T gondii PRU strain follow-ingwhich the number of brain cysts was counted 30 days afterchallenged with infection The number of brain cysts in micevaccinated with rTgTrxLp (300 plusmn 10954) [119865(2 15) = 9752119875 = 00019] and rTgTrxLp + rTgENO2 (21667 plusmn 11690)[119865(2 15) = 1416 119875 = 00004] was significantly lower thanthat in the controls (Figure 7) However the brain cysts inmice that received rTgENO2 (56667 plusmn 10954) were notsignificantly different from that in the controls [119865(2 15) =0955 119875 = 0407] The brain cysts in mice from G1 G2 andG3 were reduced to 6977 5814 and 2093 respectivelycompared to that in the mice from G5

4 Discussion

The immunogenicity of thioredoxin and enolase proteinsfrom several protozoa andhelminths has beenwell-evaluatedand both proteins were considered as potential vaccinecandidates against those pathogensrsquo infection [24 25] In thepresent studymice immunizedwith rTgTrxLp and rTgENO2proteins either alone or in combination induced stronghumoral and cellular immune responses with significantlonger survival time and lower brain cyst loadings afteracute and chronic infection compared to that of the controlsThis phenomenon indicated that the two antigens would befurther used in the development of epitope peptide-basedvaccines against T gondii infection

T cell-mediated immune response especially the cyto-toxic activity of CD8+ T cells is critical for mediatingresistance to T gondii infection [26] Herein the T cellsubclasses were stained by the surface markers CD4 andCD8 molecules and were analyzed by flow cytometry Theincreased levels of CD4+ andCD8+ Tcell in themice fromG1G2 and G3 compared to that in controls would be essentialfor chronic toxoplasmosis

During the acute stage of T gondii infection IFN-120574-mediated immune responses play a major role in resistanceto the proliferation of tachyzoites [27ndash29] After immuniza-tion with rTgTrxLp rTgENO2 and rTgTrxLp + rTgENO2proteins the levels of IFN-120574 were significantly higher thanthose in the controls which would result in longer survivaland lower numbers of brain cysts IL-2 is another importantcytokine that can stimulate CD8+ T cell proliferation afterantigen presentation and also play the crucial role in thedevelopment of CD8+ T cells [30] The levels of IL-2 inmice vaccinated with rTgTrxLp rTgENO2 and rTgTrxLp+ rTgENO2 proteins were significantly higher than that inthe controls which would also contribute to the protectiveefficacy against acute and chronic infection T gondii infec-tion could induceTh1-dominant immune responses [31] Theincreased level of IFN-120574 and IL-2 in mice immunized withrTgTrxLp and rTgENO2 indicated that both the proteinscould induce aTh1-biased immune response

IL-4 exerts diverse functions in regulating the prolif-eration and differentiation of activated B cells [32] and is



0

100

200

300IF

N-120574

prod

uctio

n (p

gm

L) NSNS

NS

lowastlowast

lowastlowast

lowast

adjuvant

(a)


IL-2

pro

duct

ion

(pg

mL)

NSNS

NS

lowastlowastlowastlowast

lowastlowast

0

50

100

150

200

adjuvant

(b)


IL-4

prod

uctio

n (p

gm

L)

NSNS

NSNS

NS NS

0

10

20

30

adjuvant

(c)

Figure 5 Cytokine production in sera ofmice immunizedwith rTgTrxLp + rTgENO2 rTgTrxLp and rTgENO2 compared to that in controls(a) Levels of IFN-120574 (b) levels of IL-2 (c) levels of IL-4 lowast119875 lt 005 and lowastlowast119875 lt 001 NS not significant compared to controls Mice that receivedPBS and Freundrsquos adjuvant were treated as controls in statistical analysis Each bar represents the mean OD (plusmnSE 119899 = 5)


rTgENO2

rTgTrxLp + rTgENO2

0

20

40

60

80

100

Perc

ent o

f sur

viva

l

0 2 4 6 8 10 12

Days

Figure 6 Survival rates of mice immunized with various proteinvaccines followed by challenge BALBc mice were challenged with1 times 103 tachyzoites of Toxoplasma gondiiGJS strain 2 weeks after thefinal immunization

recognized as the Th2-type cytokine Herein the secretionof IL-4 in mice immunized with rTgTrxLp rTgENO2 andrTgTrxLp + rTgENO2 proteins was not significantly higherthan that in the controls

IgGs were also considered critical in controlling theacute infection by T gondii through opsonizing the parasitefor phagocytosis and activating the classical complementpathway [33] The immunization of mice with rTgTrxLprTgENO2 or rTgTrxLp + rTgENO2 could induce signifi-cantly high levels of IgG antibodies than that in the controls(119875 lt 0001) which would contribute to the strong protectiveefficacy against T gondii infection The results were inagreement with previous studies of vaccinating mice withplasmids codingROM4ROM5 [20] andCDPK3 [34] aswellas the recombinant ROM1 [35] ROP18 [21] and ROP38 [36ndash38]

In conclusion the present study demonstrated that boththe rTgTrxLp and rTgENO2 proteins can generate humoraland cellular immune responses in a mouse model and cansignificantly prolong the survival time and reduce brain cystnumber Mice immunized with the combination of the twoproteins showed the longest survival time (838 plusmn 457 d)and the lowest brain cyst number (6977) when comparingcontrols and mice immunized with a single protein Theseresults indicated that rTgTrxLp and rTgENO2 proteins couldbe used as potential candidates in the development of multi-component vaccines against toxoplasmosis



0

500

1000

1500

Num

ber o

f bra

in cy

sts

NSlowastlowast

lowastlowastlowast

adjuvant

Figure 7 Number of tissue cysts per brain after Toxoplasma gondiichallenge in mice from all groups Mice immunized with rTgTrxLp+ rTgENO2 rTgTrxLp and rTgENO2 were challenged with 10tissue cysts of Toxoplasma gondii PRU strain 2 weeks after thefinal booster lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 00001 NS not significantcompared to controls Mice that received PBS and Freundrsquos adjuvantwere treated as controls in statistical analysis Each bar represents themean number (plusmnSE 119899 = 6)

Competing Interests

The authors declare that they have no competing interests

Authorsrsquo Contributions

Meng Wang and Xiao-Yu Yang contribute equally to thiswork

Acknowledgments

The project support was provided by the National NaturalScience Foundation of China (Grant no 31230073)

References

[1] J P Dubey Toxoplasmosis of Animals and Humans CRC PressBoca Raton Fla USA 2nd edition 2010

[2] F Robert-Gangneux and M-L Darde ldquoEpidemiology of anddiagnostic strategies for toxoplasmosisrdquo Clinical MicrobiologyReviews vol 25 no 2 pp 264ndash296 2012

[3] S A Elmore J L Jones P A Conrad S Patton D SLindsay and J P Dubey ldquoToxoplasma gondii epidemiologyfeline clinical aspects and preventionrdquo Trends in Parasitologyvol 26 no 4 pp 190ndash196 2010

[4] J P Dubey E G Lago S M Gennari C Su and J L JonesldquoToxoplasmosis in humans and animals in Brazil high preva-lence high burden of disease and epidemiologyrdquo Parasitologyvol 139 no 11 pp 1375ndash1424 2012

[5] P Zhou Z G ChenH-L Li et al ldquoToxoplasma gondii infectionin humans in Chinardquo Parasites ampVectors vol 4 article 165 2011

[6] J P Dubey ldquoToxoplasmosis in pigs-the last 20 yearsrdquoVeterinaryParasitology vol 164 no 2ndash4 pp 89ndash103 2009

[7] J P Dubey D E Hill J L Jones et al ldquoPrevalence of viableToxoplasma gondii in beef chicken and pork from retail meat

stores in the United States risk assessment to consumersrdquoJournal of Parasitology vol 91 no 5 pp 1082ndash1093 2005

[8] E A Innes P M Bartley S W Maley S E Wright andD Buxton ldquoComparative host-parasite relationships in ovinetoxoplasmosis and bovine neosporosis and strategies for vac-cinationrdquo Vaccine vol 25 no 30 pp 5495ndash5503 2007

[9] H V Fajardo S DrsquoAvila R R Bastos et al ldquoSeroprevalence andrisk factors of toxoplasmosis in cattle from extensive and semi-intensive rearing systems at Zona da Mata Minas Gerais stateSouthern Brazilrdquo Parasites and Vectors vol 6 no 1 article 1912013

[10] R Verin L Mugnaini S Nardoni et al ldquoSerologic molecularand pathologic survey of Toxoplasma gondii infection in free-ranging red foxes (Vulpes vulpes) in central Italyrdquo Journal ofWildlife Diseases vol 49 no 3 pp 545ndash551 2013

[11] S Feo D Arcuri E Piddini R Passantino and A GiallongoldquoENO1 gene product binds to the c-myc promoter and acts asa transcriptional repressor relationship with Myc promoter-binding protein 1 (MBP-1)rdquo FEBS Letters vol 473 no 1 pp 47ndash52 2000

[12] A Subramanian and D M Miller ldquoStructural analysis of 120572-enolase mapping the functional domains involved in down-regulation of the c-myc protooncogenerdquoThe Journal of Biologi-cal Chemistry vol 275 no 8 pp 5958ndash5965 2000

[13] T Mouveaux G Oria EWerkmeister et al ldquoNuclear glycolyticenzyme enolase of Toxoplasma gondii functions as a transcrip-tional regulatorrdquo PLoS ONE vol 9 no 8 Article ID e1058202014

[14] S M Kanzok A Fechner H Bauer et al ldquoSubstitution ofthe thioredoxin system for glutathione reductase in Drosophilamelanogasterrdquo Science vol 291 no 5504 pp 643ndash646 2001

[15] J H Ahn J H Choi J M Song et al ldquoIncrease in Trx2Prx3redox system immunoreactivity in the spinal cord and hip-pocampus of aged dogsrdquo Experimental Gerontology vol 46 no11 pp 946ndash952 2011

[16] D A Drechsel and M Patel ldquoRespiration-dependent H2O2

removal in brain mitochondria via the thioredoxinperoxire-doxin systemrdquoThe Journal of Biological Chemistry vol 285 no36 pp 27850ndash27858 2010

[17] J Liu LWetzel Y Zhang et al ldquoNovel thioredoxin-like proteinsare components of a protein complex coating the corticalmicrotubules of Toxoplasma gondiirdquo Eukaryotic Cell vol 12 no12 pp 1588ndash1599 2013

[18] A Decoster F Darcy and A Capron ldquoRecognition of Toxo-plasma gondii excreted and secreted antigens by human serafrom acquired and congenital toxoplasmosis identification ofmarkers of acute and chronic infectionrdquo Clinical and Experi-mental Immunology vol 73 no 3 pp 376ndash382 1988

[19] I Prigione P Facchetti L Lecordier et al ldquoT cell clones raisedfrom chronically infected healthy humans by stimulation withToxoplasma gondii excretory-secretory of the clones and impli-cations for vaccine developmentrdquo The Journal of Immunologyvol 164 no 7 pp 3741ndash3748 2000

[20] N-Z Zhang Y Xu M Wang et al ldquoProtective efficacy of twonovel DNA vaccines expressing Toxoplasma gondii rhomboid 4and rhomboid 5 proteins against acute and chronic toxoplasmo-sis in micerdquo Expert Review of Vaccines vol 14 no 9 pp 1289ndash1297 2015

[21] D Qu J Han and A Du ldquoEnhancement of protective immuneresponse to recombinant Toxoplasma gondii ROP18 antigen byginsenoside Rerdquo Experimental Parasitology vol 135 no 2 pp234ndash239 2013


[22] D Qu J Han and A Du ldquoEvaluation of protective effectof multiantigenic DNA vaccine encoding MIC3 and ROP18antigen segments of Toxoplasma gondii in micerdquo ParasitologyResearch vol 112 no 7 pp 2593ndash2599 2013

[23] Y Wang G Wang D Zhang H Yin and M Wang ldquoIdentifi-cation of novel B cell epitopes within Toxoplasma gondiiGRA1rdquoExperimental Parasitology vol 135 no 3 pp 606ndash610 2013

[24] G Anugraha J Madhumathi P R Prince et al ldquoChimeric epi-tope vaccine from multistage antigens for lymphatic filariasisrdquoScandinavian Journal of Immunology vol 82 no 4 pp 380ndash3892015

[25] R Gupta V Kumar P K Kushawaha et al ldquoCharacterizationof glycolytic enzymesmdashrAldolase and rEnolase of Leishmaniadonovani identified as Th1 stimulatory proteins for theirimmunogenicity and immunoprophylactic efficacies againstexperimental visceral leishmaniasisrdquo PLoS ONE vol 9 no 1Article ID e86073 2014

[26] S J Parker C W Roberts and J Alexander ldquoCD8+ T cells arethe major lymphocyte subpopulation involved in the protectiveimmune response to Toxoplasma gondii in micerdquo Clinical andExperimental Immunology vol 84 no 2 pp 207ndash212 1991

[27] Y Suzuki M A Orellana R D Schreiber and J S RemingtonldquoInterferon-120574 the major mediator of resistance against Toxo-plasma gondiirdquo Science vol 240 no 4851 pp 516ndash518 1988

[28] C R Sturge A Benson M Raetz et al ldquoTLR-independentneutrophil-derived IFN-120574 is important for host resistance tointracellular pathogensrdquo Proceedings of the National Academyof Sciences of the United States of America vol 110 no 26 pp10711ndash10716 2013

[29] K H Ely L H Kasper and I A Khan ldquoAugmentation of theCD8+ T cell response by IFN-gamma in IL-12-deficient miceduring Toxoplasma gondii infectionrdquo The Journal of Immunol-ogy vol 162 no 9 pp 5449ndash5454 1999

[30] N Zhang and M J Bevan ldquoCD8+ T cells foot soldiers of theimmune systemrdquo Immunity vol 35 no 2 pp 161ndash168 2011

[31] Y Suzuki Q Sa M Gehman and E Ochiai ldquoInterferon-gamma- and perforin-mediated immune responses for resis-tance against Toxoplasma gondii in the brainrdquo Expert Reviewsin Molecular Medicine vol 13 article e31 2011

[32] M-H Bessieres B Swierczynski S Cassaing et al ldquoRole ofIFN-120574 TNF-120572 IL4 and IL 10 in the regulation of experimentalToxoplasma gondii infectionrdquo Journal of Eukaryotic Microbiol-ogy vol 44 no 6 p 87s 1997

[33] P C Sayles G W Gibson and L L Johnson ldquoB cells are essen-tial for vaccination-induced resistance to virulent Toxoplasmagondiirdquo Infection and Immunity vol 68 no 3 pp 1026ndash10332000

[34] N-Z Zhang S-Y Huang D-H Zhou et al ldquoProtective immu-nity against Toxoplasma gondii induced by DNA immunizationwith the gene encoding a novel vaccine candidate calcium-dependent protein kinase 3rdquo BMC Infectious Diseases vol 13article 512 2013

[35] J Li Q Han P Gong et al ldquoToxoplasma gondii rhomboidprotein 1 (TgROM1) is a potential vaccine candidate againsttoxoplasmosisrdquo Veterinary Parasitology vol 184 no 2ndash4 pp154ndash160 2012

[36] Y Xu N-Z Zhang M Wang et al ldquoA long-lasting protectiveimmunity against chronic toxoplasmosis in mice induced byrecombinant rhoptry proteins encapsulated in poly (lactide-co-glycolide) microparticlesrdquo Parasitology Research vol 114 no 11pp 4195ndash4203 2015

[37] N-Z Zhang J Chen M Wang E Petersen and X-Q ZhuldquoVaccines against Toxoplasma gondii new developments andperspectivesrdquo Expert Review of Vaccines vol 12 no 11 pp 1287ndash1299 2013

[38] N-Z Zhang M Wang Y Xu E Petersen and X-Q ZhuldquoRecent advances in developing vaccines against Toxoplasmagondii an updaterdquo Expert Review of Vaccines vol 14 no 12 pp1609ndash1621 2015

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


are considered attractive vaccine candidates that have beenwidely studied in animal models [18ndash20] However it isyet to be clarified whether TgENO2 and TgTrxLp as theconstituents of TgESAs could induce protective immuneresponses against T gondii infection in the mouse modelIn the present study the immunogenicity of TgENO2 andTgTrxLp recombinant proteins was examined in mice Fur-thermore the immunoprophylaxis efficacy against acute andchronic toxoplasmosis was also estimated

2 Materials and Methods

21 Animals Thespecific-pathogen-free (SPF) grade BALBcand Kunming mice (6ndash8 weeks old) were purchased fromLanzhou Veterinary Research Institute Laboratory AnimalCenter (Lanzhou China) All animals were strictly handledaccording to the Good Animal Practice Requirements ofthe Animal Ethics Procedures and Guidelines of the Peo-plersquos Republic of China The present study was approvedby the Animal Ethics Committee of Lanzhou VeterinaryResearch Institute ChineseAcademyofAgricultural Sciences(Approval no LVRIAEC2012-011)

22 Parasites Tachyzoites of T gondiiGJS (Genotype DB9)strain were maintained in Kunming mice by a series ofintraperitoneal infections and obtained from the peritonealexudates followed by purification by centrifugation asdescribed by Qu et al [21 22] Kunming mice infected withthe low virulent PRU strain (Genotype II) were used to collectthe toxoplasma cysts that were orally passaged by infection ofthe brain homogenate

23 Prokaryotic Expression of TgTrxLp and TgENO2 Proteinsand Purification The full-length coding sequences of theTgTrxLp (GenBank access number XM 0023697031) andTgENO2 (GenBank accession number AF1234571) geneswere amplified using one-step reverse transcription-PCR(RT-PCR) following themanufacturerrsquos instructions (TakaraChina) The primers for the amplification of TgTrxLp frag-ment were 51015840-GG119866119866119879119860119862119862ATGGCGCCTCTGCGTGT-GTGCGCGTTC-31015840 (forward) and 51015840-CC119860119860119866119862119879119879TT-ACAGTTCGTCCTTCTTGTCTGCCTT-31015840 (reverse) andfor TgENO2 fragment were 51015840-CG119866119860119860119879119879119862ATGGTGGC-CATCAAGGACATCACTGCT-31015840 (forward) and 51015840-CC-119860119860119866119862119879119879TTAGTTGGGATGGCGGAAGCCAGCGCC-31015840(reverse) The restriction sites induced in the two pairs ofprimers were italicized

Each RT-PCR product was purified (Tiangen China) andligated into the prokaryotic expression vector pET-30a(+)via the respective restriction sites forming the recombinantplasmids pET-TrxLp and pET-ENO2 respectively Subse-quently the two recombinant plasmids were transformedinto Escherichia coli strain BL21(DE3) and induced with10mmolL isopropyl 120573-d-1-thiogalactopyranoside (SangonChina) shaking for 6 h at 30∘CThe rTgTrxLp and rTgENO2proteins were purified on a Ni2+ column (Novagen USA)following ultrasonic bacterial lysis on the ice The purifiedprotein samples were analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

24 Western Blot Analysis The proteins were resolved onSDS-PAGE and the purified proteins were transferred tonitrocellulose (NC) membranes (Pall USA)Then the mem-branes were blocked with 5 bovine serum albumin (BSA) inPBST (005 Tween-20 in PBS) at room temperature (RT)After 1 h the membrane was washed 4 times with PBSTThe swine sera against T gondii that were collected at 60days after the onset of symptoms as described previously[23] were diluted 1 1000 as the first antibody probed onthe membranes for 1 h at RT Then the membranes werewashed 4 times with PBST and incubated with horseradishperoxidase- (HRP-) conjugated goat anti-pig IgG (1 5000)(Sigma USA) The immunogens were developed with ECLreagents A and B (TIANGEN China) according to themanufacturerrsquos instructions

25 Immunization and Challenge The female BALBc micewere randomly divided into five groups (26 mice in eachgroup) The animals were subcutaneously injected with100 120583g rTgTrxLp + rTgENO2 (G1) rTgTrxLp (G2) orrTgENO2 (G3) proteins emulsified in 100120583L Freundrsquos com-plete adjuvant (FCA) respectively Two weeks following theprimary immunization mice in G1 G2 and G3 were inoc-ulated with the same dose of each antigen plus incompleteFreundrsquos adjuvant respectively The mice injected with equaladjuvant (G4) or PBS alone (G5) served as negative controls

Fifteen mice in each group were subjected to acuteinfection by the intraperitoneal administration with 103 Tgondii tachyzoites of GJS strain at 2 weeks after the thirdimmunization The challenged mice were monitored dailyuntil total mortality Six mice from each group were orallychallenged with 10 cysts of T gondii PRU strain at 2 weeksafter the final immunization Thirty days later the animalswere sacrificed to examine the number of brain cysts

26 Collection of the Sera and Lymphocyte Samples Theblood samples ofmice fromall the groupswere collected fromthe tail vein prior to each vaccinationThe serawere harvestedby centrifugation at 2000timesg for 20min and stored at minus20∘Cuntil assayed for antibody titers and cytokines

Two weeks after the final immunization 3 mice pergroup were sacrificed to aseptically harvest the spleen Thespleens were pooled and filtered through a nylon membraneto obtain the splenocytes Then the cells were purified usingerythrocyte lysis buffer (Solarbio China) to remove the redblood cells Subsequently the harvested splenocytes wereresuspended in DMEM medium supplemented with 10fetal calf serum (FCS) and were used for the analysis oflymphocyte proliferation and the percentage of CD4+ andCD8+ T cells

27 IgG ELISA The specific humoral immune responsesanti-rTgTrxLp rTgENO2 or the mixtures were evaluated byELISA using SBA Clonotyping System-HRP Kit (SouthernBiotech Co Ltd Birmingham USA) according to the man-ufacturerrsquos instructions The preparation of rTgTrxLp andrTgENO2 proteins was adjusted to 50 120583gmL The 96-wellmicrotiter plates were coated with 100 120583L of each protein at37∘C for 2 h 100 120583L of the prepared serum samples (1 10



2O2) for 15min in




490ConAOD490M




3 Results


M 1 2 3 4

70 kDa

55 kDa

35 kDa


00

05

10

15

20

25

OD

490

G5 G4 G3 G2 G1

NS

NS


lowastlowastlowast


lowastlowastlowast



lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0weeks2weeks

4weeks6weeks





00

05

10

15

20

25St

imul

atio

n in

dex

(SI)


ConA(5120583gmL)


lowast

lowastlowastlowast

lowastlowastlowast


(a)

Stim

ulat

ion

inde

x (S

I)

ConA(5120583gmL)

lowast

lowastlowastlowast

lowastlowastlowast


0

1

2

3

4

(5120583gmL)rTgENO2


rTgENO2

(b)

Stim

ulat

ion

inde

x (S

I)

ConA(5120583gmL)



NS

rTgTrxLp + rTgENO2


rTgTrxLp +rTgENO2

(15120583gmL)

0

1

2

3

(c)







0

10

20

30

40



lowastlowastlowast

Perc

ent o

f CD4+

T ce

lls

adjuvant

(a)


0

5

10

15

20


lowastlowastlowast

Perc

ent o

f CD8+

T ce

lls

adjuvant

(b)







4 Discussion







0

100

200

300IF

N-120574

prod

uctio

n (p

gm

L) NSNS

NS

lowastlowast

lowastlowast

lowast

adjuvant

(a)


IL-2

pro

duct

ion

(pg

mL)

NSNS

NS


lowastlowast

0

50

100

150

200

adjuvant

(b)


IL-4

prod

uctio

n (p

gm

L)

NSNS

NSNS

NS NS

0

10

20

30

adjuvant

(c)



rTgENO2

rTgTrxLp + rTgENO2

0

20

40

60

80

100

Perc

ent o

f sur

viva

l

0 2 4 6 8 10 12

Days







0

500

1000

1500

Num

ber o

f bra

in cy

sts

NSlowastlowast

lowastlowastlowast

adjuvant


Competing Interests




Acknowledgments


References

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



2O2) for 15min in




490ConAOD490M




3 Results


M 1 2 3 4

70 kDa

55 kDa

35 kDa


00

05

10

15

20

25

OD

490

G5 G4 G3 G2 G1

NS

NS


lowastlowastlowast


lowastlowastlowast



lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0weeks2weeks

4weeks6weeks





00

05

10

15

20

25St

imul

atio

n in

dex

(SI)


ConA(5120583gmL)


lowast

lowastlowastlowast

lowastlowastlowast


(a)

Stim

ulat

ion

inde

x (S

I)

ConA(5120583gmL)

lowast

lowastlowastlowast

lowastlowastlowast


0

1

2

3

4

(5120583gmL)rTgENO2


rTgENO2

(b)

Stim

ulat

ion

inde

x (S

I)

ConA(5120583gmL)



NS

rTgTrxLp + rTgENO2


rTgTrxLp +rTgENO2

(15120583gmL)

0

1

2

3

(c)







0

10

20

30

40



lowastlowastlowast

Perc

ent o

f CD4+

T ce

lls

adjuvant

(a)


0

5

10

15

20


lowastlowastlowast

Perc

ent o

f CD8+

T ce

lls

adjuvant

(b)







4 Discussion







0

100

200

300IF

N-120574

prod

uctio

n (p

gm

L) NSNS

NS

lowastlowast

lowastlowast

lowast

adjuvant

(a)


IL-2

pro

duct

ion

(pg

mL)

NSNS

NS


lowastlowast

0

50

100

150

200

adjuvant

(b)


IL-4

prod

uctio

n (p

gm

L)

NSNS

NSNS

NS NS

0

10

20

30

adjuvant

(c)



rTgENO2

rTgTrxLp + rTgENO2

0

20

40

60

80

100

Perc

ent o

f sur

viva

l

0 2 4 6 8 10 12

Days







0

500

1000

1500

Num

ber o

f bra

in cy

sts

NSlowastlowast

lowastlowastlowast

adjuvant


Competing Interests




Acknowledgments


References

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


00

05

10

15

20

25St

imul

atio

n in

dex

(SI)


ConA(5120583gmL)


lowast

lowastlowastlowast

lowastlowastlowast


(a)

Stim

ulat

ion

inde

x (S

I)

ConA(5120583gmL)

lowast

lowastlowastlowast

lowastlowastlowast


0

1

2

3

4

(5120583gmL)rTgENO2


rTgENO2

(b)

Stim

ulat

ion

inde

x (S

I)

ConA(5120583gmL)



NS

rTgTrxLp + rTgENO2


rTgTrxLp +rTgENO2

(15120583gmL)

0

1

2

3

(c)







0

10

20

30

40



lowastlowastlowast

Perc

ent o

f CD4+

T ce

lls

adjuvant

(a)


0

5

10

15

20


lowastlowastlowast

Perc

ent o

f CD8+

T ce

lls

adjuvant

(b)







4 Discussion







0

100

200

300IF

N-120574

prod

uctio

n (p

gm

L) NSNS

NS

lowastlowast

lowastlowast

lowast

adjuvant

(a)


IL-2

pro

duct

ion

(pg

mL)

NSNS

NS


lowastlowast

0

50

100

150

200

adjuvant

(b)


IL-4

prod

uctio

n (p

gm

L)

NSNS

NSNS

NS NS

0

10

20

30

adjuvant

(c)



rTgENO2

rTgTrxLp + rTgENO2

0

20

40

60

80

100

Perc

ent o

f sur

viva

l

0 2 4 6 8 10 12

Days







0

500

1000

1500

Num

ber o

f bra

in cy

sts

NSlowastlowast

lowastlowastlowast

adjuvant


Competing Interests




Acknowledgments


References

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

10

20

30

40



lowastlowastlowast

Perc

ent o

f CD4+

T ce

lls

adjuvant

(a)


0

5

10

15

20


lowastlowastlowast

Perc

ent o

f CD8+

T ce

lls

adjuvant

(b)







4 Discussion







0

100

200

300IF

N-120574

prod

uctio

n (p

gm

L) NSNS

NS

lowastlowast

lowastlowast

lowast

adjuvant

(a)


IL-2

pro

duct

ion

(pg

mL)

NSNS

NS


lowastlowast

0

50

100

150

200

adjuvant

(b)


IL-4

prod

uctio

n (p

gm

L)

NSNS

NSNS

NS NS

0

10

20

30

adjuvant

(c)



rTgENO2

rTgTrxLp + rTgENO2

0

20

40

60

80

100

Perc

ent o

f sur

viva

l

0 2 4 6 8 10 12

Days







0

500

1000

1500

Num

ber o

f bra

in cy

sts

NSlowastlowast

lowastlowastlowast

adjuvant


Competing Interests




Acknowledgments


References

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



0

100

200

300IF

N-120574

prod

uctio

n (p

gm

L) NSNS

NS

lowastlowast

lowastlowast

lowast

adjuvant

(a)


IL-2

pro

duct

ion

(pg

mL)

NSNS

NS


lowastlowast

0

50

100

150

200

adjuvant

(b)


IL-4

prod

uctio

n (p

gm

L)

NSNS

NSNS

NS NS

0

10

20

30

adjuvant

(c)



rTgENO2

rTgTrxLp + rTgENO2

0

20

40

60

80

100

Perc

ent o

f sur

viva

l

0 2 4 6 8 10 12

Days







0

500

1000

1500

Num

ber o

f bra

in cy

sts

NSlowastlowast

lowastlowastlowast

adjuvant


Competing Interests




Acknowledgments


References

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



0

500

1000

1500

Num

ber o

f bra

in cy

sts

NSlowastlowast

lowastlowastlowast

adjuvant


Competing Interests




Acknowledgments


References

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article evaluation of protective immune responses...

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