Cofactor-independent phosphoglycerate mutase is an essential gene in procyclic form Trypanosoma brucei

Research ArticleCofactor Independent Phosphoglycerate Mutase ofBrugia malayi Induces a Mixed Th1Th2 Type ImmuneResponse and Inhibits Larval Development in the Host

Prashant K Singh Susheela Kushwaha Ajay K Rana and Shailja Misra-Bhattacharya

Division of Parasitology CSIR-Central Drug Research Institute BS 101 Sector 10 PO Box 173Jankipuram Extension Sitapur Road Lucknow Uttar Pradesh 226031 India

Correspondence should be addressed to Shailja Misra-Bhattacharya shailja cdrirediffmailcom

Received 23 February 2014 Revised 6 May 2014 Accepted 20 May 2014 Published 1 July 2014

Academic Editor Amogh A Sahasrabuddhe

Copyright copy 2014 Prashant K Singh et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Lymphatic filariasis is a major debilitating disease endemic in 72 countries putting more than 139 billion people at risk and120 million are already infected Despite the significant progress in chemotherapeutic advancements there is still need for othermeasures like development of an effective vaccine or discovery of novel drug targets In this study structural and immunologicalcharacterization of independent phosphoglycerate mutase of filarial parasite Brugia malayi was carried out Protein was found tobe expressed in all major parasite life stages and as an excretory secretory product of adult parasites Bm-iPGM also reacted to allthe categories of human bancroftian patientrsquos sera including endemic normals In vivo immunological behaviour of protein wasdetermined in immunized BALBc mice followed by prophylactic analysis in BALBc mice and Mastomys coucha Immunizationwith Bm-iPGM led to generation of a mixed Th1Th2 type immune response offering 582 protection against larval challenge inBALBc and 65ndash68 protection in M coucha In vitro studies confirmed participation of anti-Bm-iPGM antibodies in killing ofB malayi infective larvae and microfilariae through ADCC mechanism The present findings reveal potential immunoprotectivenature of Bm-iPGM advocating its worth as an antifilarial vaccine candidate

1 Introduction

Lymphatic filariasis (LF) is one of the oldest andmostmorbidand debilitating parasitic diseases [1] caused by three thread-like nematode worms Wuchereria bancrofti Brugia malayiandB timori An estimated 130million people in 72 countriesare currently infected and around 134 billion are at riskof infection [2 3] Diethylcarbamazine (DEC) principallymicrofilaricidal is the only drug of choice for the treatmentof LF [4] Mass drug administration (MDA) using DECivermectin (IVN) and albendazole (ALB) combination hasbeen quite successful however several rounds of MDA arerequired to reduce the level of infection to sustain trans-mission [5] As no drug is available against adult parasitealternative strategies like vaccine development need to beexplored in order to improve the effectiveness of diseasecontrol and prevention programmes With the availability of

the draft genome sequence of B malayi [6] and the sequenc-ing of endosymbiont Wolbachia mosquito vectors (Aedesand Anopheles) and the human host the ability to carryout large-scale comparative genomics presents opportunitiesto understand the molecular basis of parasitism definingmolecules and pathways unique to nematode developmentand parasitism that can be characterised as the novel antifi-larial drug targets or vaccine candidates

In the current study molecular biophysical and immunecharacterisation of independent phosphoglycerate mutase(Bm-iPGM) of human filarial parasite B malayi was carriedout Phosphoglycerate mutases the key enzymes in theglycolytic and gluconeogenic pathways exist in two differentforms having different mechanism of action and struc-ture and that are either cofactor (23-diphosphoglycerate)dependent or cofactor-independent The independent formis predominant in plants nematodes bacteria and archaea

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 590281 19 pageshttpdxdoiorg1011552014590281

2 BioMed Research International

[7] All experimentally characterised iPGMs from eubacteriaplants and invertebrates are monomers with a molecularmass of 55ndash75 kDa [8 9]The absence of iPGM from humansand being indispensable in all nematodes including thefilariids [10] advocate its potential as anthelminthic drugtarget Bm-iPGM was purified successfully using bacterialhostE coli Circular dichroism (CD) andfluorescence spectraof the recombinant protein were obtained to determine itssecondary structure and native conformation The abundantsharing of Bm-iPGM by all the major life-forms of B malayiand its release in the form of excretory-secretory products[11] pointed towards its immunogenic nature In silico analysisof Bm-iPGM predicted it to be highly antigenic with thepresence of both MHC I and MHC II binding peptidesThe antigenic nature was further validated by the cross-reactivity of Bm-iPGM with human bancroftian sera ofdifferent categories of LF which persuaded us to examine theimmunoprophylactic efficacy of the recombinant protein inanimal models of LF

In vivo immune characterisation of Bm-iPGM in BALBcmice revealed it to invoke a mixed type of Th1Th2 immuneresponse The immunised animals (BALBc and Mastomys)that were challenged with the infective larvae displayedreduced worm establishment As active filarial infectionsare accompanied with downregulation of the host immunesystem skewing the helper immune response of host toTh2 type we propose that immunization with Bm-iPGM iscapable of generating a mixed Th1Th2 type response that isunfavourable for parasite establishment and was responsiblefor providing considerable protection against LF in mousemodels thus validating Bm-iPGM to be possible vaccinecandidate against LF

2 Materials and Methods

21 Experimental Animals Randomly bred 6ndash8-week-oldmale BALBc (35) and Mastomys coucha (36) were usedin the experiment The animals were maintained in properhousing condition at Laboratory Animal Facility at CSIR-Central Drug Research Institute (CDRI) Lucknow IndiaAnimalswere fed on standard pellet diet andwater ad libitumThe animals and the animal experimental procedures wereapproved by the Animal Ethics Committee of CDRI dulyconstituted under the provisions of CPCSEA (Committeefor the Purpose of Control and Supervision on Experimentson Animals) Government of India The study bears theIAEC number 8309ParaIAEC dated 270409 All theexperiments were performed in duplicate (both forMastomysandBALBc) and almost similar results were obtained in boththe experiments and therefore pooled

22 Parasites Infective larvae (L3) of B malayi were recov-ered from the laboratory bred vector mosquitoes (Aedesaegypti) fed on donor Mastomys 9 plusmn 1 day back [12] L3were isolated from gently crushed mosquitoes by Baermanntechnique washed and counted in Ringerrsquos solution AdultB malayi worms and microfilariae (Mf) were collected from

the peritoneal cavities of the infected jirds on day 80ndash85 afterL3 inoculation

23 Homology Modelling of Bm-iPGM and Amino AcidSequences The homology model for Bm-iPGM was gener-ated using Phyre server [13] Bm-iPGM structure was gener-ated with 100 precision and 41 identity using structure ofBacillus anthracis cofactor-independent 2 phosphoglyceratemutase as template (PDB id c2ifyA length 508AA)Thedatagenerated was analysed by The PyMOL Molecular GraphicsSystem Version 13 Schrodinger LLC and the cartoonstructure was generated Amino acid sequence of Bm-iPGMwas also aligned with iPGM from B anthracis (accessionnumber 2IFY A) using Clustal W programme

24 In Silico Antigenicity Prediction The antigenicity ofBm-iPGM was determined by Kolaskar and Tongaonkarmethod [14] This semiempirical method predicts antigenicdeterminants based on the physicochemical properties ofamino acid residues and the frequencies of their occurrencein experimentally known segmental epitopes Prediction ofimmunodominant T cell antigenic sites from the primarysequence of Bm-iPGM was determined by ProPred-I andProPred MHC class-II binding peptide prediction serverswhich are online web tools for the prediction of peptidebinding to MHC class-I (HLA-A1 HLA-A2 HLA-A0201HLA-A0205 HLA-A1101 HLA-A3101 HLA-A3302 HLA-B2102 HLA-A3501 HLA-A4403 and HLA-5101) and class II(HLA-DRB1 0101 HLA-DRB1 0301 HLA-DRB1 0401 HLA-DRB1 0701 andHLA-DRB1 0801) alleles [15 16]The highestranking MHC I and MHC II binding peptides were high-lighted in the cartoon structure of Bm-iPGMobtained earlier

25 Cloning Expression and Purification of Bm-iPGMExpression and purification of Bm-iPGM was done asdescribed elsewhere with minor modifications [17] Brieflygene specific forward (51015840AGTCGGATCCATGGCCGA-AGCAAAGAATCG-31015840) and reverse (51015840ATGCCTCGA-GGGCTTCATTACCAATGGC31015840) primers having restric-tion sites for the enzymes BamHI (F) and XhoI (R) weresynthesised Amplification of gene was carried out using1 120583M of each primer 200120583M of each dNTP (FermentasUSA) 05 unit taq DNA polymerase (Invitrogen USA)1xPCR buffer and 15 120583M of MgCl

2with the following

reaction conditions 95∘C for 2min followed by 29 cyclesof 95∘C for 1min 58∘C for 1min 72∘C for 2min and 1cycle at 72∘C for 10min The 1548 bp amplified gene wascloned into pTZ57RT (2886 bp) vector as permanufacturerrsquosinstructions (Fermentas USA) Plasmid DNA was isolatedand the insert was verified by sequencing The 1548 bp genewas subcloned into pET28a expression vector for proteinexpression and purificationThe conditions like temperatureisopropyl 120573-D-thiogalactopyranoside (IPTG) concentrationand duration of induction were standardized for optimalexpression of the protein in soluble form Five mL cultures(E coli strain BL21 (DE3)) were grown at 37∘C in an incubatorshaker at 220 rpm and induced (at OD

600of 05-06) for

4 h with 02 05 and 10mM IPTG After induction cells

BioMed Research International 3

were harvested by centrifugation at 7000 rpm for 5min andlysed in 5mL sample buffer (0313M Tris-HCl pH 68 50glycerol 10 SDS and 005 bromophenol blue) for analysison 10 SDS-PAGE (Laemmli 1970) along with uninducedvector control culture To observe the solubility of recombi-nant protein the cell pellet was resuspended in 1mL of lysisbuffer (50mM Tris-HCl pH 75 200mM NaCl and 100mMDTT) sonicated at 10 db10s in a Soniprep 150 sonicator incold The cell lysate was centrifuged at 14000 rpm for 30minutes to collect the supernatant containing soluble fractionand the remaining pellet containing insoluble fraction wasresuspended in 1mL of lysis buffer Soluble and insolublefractions were then analyzed in parallel on 10 SDS-PAGEand visualized by Coomassie blue staining For purificationof the expressed recombinant protein the supernatant wasrun on Ni-NTA agarose affinity column preequilibrated with50mM Tris-HCl buffer (pH 75) along with 200mM NaCland 10mM imidazole The column was subsequently washedwith the samebuffer containing 25mMand40mMimidazoleand the recombinant protein was eluted with 250mM imi-dazole analyzed on SDS-PAGE and protein expression wasconfirmedusing anti-His antibody inWestern blot Followingpurification the exact mass of Bm-iPGM was determinedthrough matrix-assisted laser desorptionionization time-of-flight mass spectrometry (MALDI-TOF) The protein wasdialysed in water (ON) to remove any buffer content anddiluted in 30 acetonitrile (ACN) and 01 trifluoroaceticacid (TFA) solution in a matrix of sinapinic acid Ten 120583Lof the prepared sample was then pipetted on the plate andwas air-dried to allow cocrystallization of the protein andthe matrix the target plate was loaded in the MALDI-TOF spectrometer (Applied Biosystems MDS Sciex 4800Plus MALDI TOFTOF Foster City CA USA) To checkwhether the recombinant protein was biochemically activethe activity of recombinant Bm-iPGM was measured (datanot shown) as described previously using a standard one-stepenzyme-coupled assay [17]

26 Biophysical Investigations CDmeasurements were madeon JASCO J810 Spectropolarimeter calibrated with ammo-nium (+)-10-camphorsulfonate with 6 120583M protein in 10mMcitrate glycine and HEPES (CGH) buffer of desired pH witha 2mm path length cell at 25∘C using the following param-eters 1 sec response 50 nmmin scan speed 01 nm dataacquisition interval 3 accumulations and 2 nm bandwidthThe spectra were recorded over a range of 190ndash250 nm Thevalues obtained were normalized by subtracting the baselinerecorded for the buffer under similar conditionsThe elliptic-ity was reported as molar ellipticity (120579) (mdegsdotcmminus2sdotmolminus1)Fluorescence spectra were recordedwith Perkin Elmer LS50Bluminescence spectrometer in a quartz cell of 5mm pathlength 6 120583M concentration of protein in 50mM phosphatebuffer (pH 70) was incubated at 25∘C before recording thespectra Excitation wavelength was 280 nm and the spectrawere recorded between 290 nm and 400 nm

27 Production of Polyclonal Antibodies to Analyze StageSpecific Expression of Bm-iPGM Five BALBc mice were

administered subcutaneously with the recombinant Bm-iPGM (25 120583ganimal) in three doses at 2-week intervals Firstdose was given in Freundrsquos complete adjuvant (FCA) whilethe remaining two in Freundrsquos incomplete adjuvant (FIA)The animals were euthanized a week after the last proteinbooster and blood was collected for serum separation Forpreparation of soluble extracts adult parasites L3 andMf were homogenized in sterile PBS (pH 72) containingprotease inhibiter cocktail (Sigma USA) in cold and leftfor overnight (ON) extraction at 4∘C and were furthersonicated and centrifugedThe protein content was estimatedin the supernatant by Bradford method [18] Protein wasloaded on to 10 SDS-PAGE and transferred to nitrocel-lulose membrane (NC) Membrane strips were incubatedwith Bm-iPGM specific polyclonal antibody raised in mouse(1 5000) After washing membranes were incubated withHRP-goat anti-mouse antibody and were developed withsubstrate o-phenylenediamine dihydrochloride (OPD) [1920] Bm-iPGM gene expression in various stages of Bmalayi was also observed using cDNA Adult worms L3and Mf were recovered as mentioned above RNA wasextracted from all the three life stages using TRIzol reagent(Invitrogen USA) and quantified with a GeneQuant (Bio-Rad) After treatment with DNase I to eliminate genomicDNA contamination 2120583g of total RNAs from each life stagewas used for the first cDNA synthesis using a first-strandcDNA synthesis kit (InvitrogenUSA) cDNAswere amplifiedwith specific primer pairs under the conditions mentionedabove

28 Analysis of Bm-iPGM in Excretory and Secretory (ES)Product Adult worms (4 wormsmL) were maintained invitro in serum-free RPMI 1640 (GIBCO) supplementedwith antibiotic antimycotic (Invitrogen 100UmL penicillin100mgmL streptomycin and 025mgmL of amphotericinB) and 25mM HEPES at 37∘C in 5 CO

2in air The

utilizedmedia was collected and replaced with fresh mediumevery 24 h continuously up to 7 days The medium collectedwas filtered through 02mM filters (Millipore USA) andstored pooled and concentrated using 3 kDa cut-off mem-branes filters (Millipore USA) Concentrated ES productand recombinant Bm-iPGM were individually run on 10SDS-PAGE and transferred to NC membrane Membranewas blocked with 3 skimmed milk for 1 h and incubatedat room temperature (RT) with 1 200 dilution of anti-Bm-iPGM antibodies raised in BALBc mice The membranewas reincubated with goat 1 10000 dilution of anti-mouseIgG-HRP antibody for 1 h at RT and the reaction wasdeveloped with the substrate 33-diaminobenzidine (DAB)tetrahydrochloride

29 Reactivity of Bm-iPGMwithHumanBancroftianAntibod-ies Reactivity of recombinant enzymewas observed with theantibody present in the sera of human subjects by Westernblotting and ELISA For serum blood was collected fromW bancrofti endemic area in the outskirts of LucknowIndia and was categorized as endemic normal asymp-tomatic microfilaria carriers microfilaraemic symptomatic


and amicrofilaraemic symptomatic Sera from humans livingin filaria free zones like Jammu and Kashmir India servedas nonendemic control Mf presence or absence was earlierdetermined in the 2mL night blood by membrane filtrationtechnique [21] Purified recombinant protein along withprestainedmolecular weightmarker was run on a preparative10 SDS-PAGE transferred toNCmembrane and processedfor immune-recognition with human sera pools (1 200) of 10subjects per category Goat anti-human IgG-HRP (1 10000dilutions) was used as secondary antibody and reaction wasdeveloped by the DAB substrate

IgG antibodies in individual sera sample (10 sera of eachcategory) belonging to microfilaraemic amicrofilaraemicsymptomatic endemic normal (EN) and nonendemic nor-mal (NEN) categoriesweremeasured using recombinant Bm-iPGMas an antigen in ELISA as stated aboveThe human serasamples were added at 1 200 dilutions as primary antibodywhile goat anti-human IgG antibody-HRP (1 10000) wasused as secondary antibody

210 Immunization of BALBc and Mastomys with Recom-binant Bm-iPGM Immune characterization of recombinantBm-iPGM was carried out in BALBc while prophylacticefficacy was investigated both in BALBc and in MastomysBALBc mice do not support full development of L3 topreadult or adult stage nor develop microfilaraemia whileMastomys being highly susceptible supports full developmentfrom L3 to adult with the release of Mf Mice are idealfor immune characterization of an antigen We divided theanimals into three different treatment groups (ten BALBcmice and twelve Mastomys per group were used) whichreceived three equal immunization doses on day 0 day15 and day 23 Animals in treatment group 1 receivedonly PBS (unimmunized control group) while animals intreatment group 2 received equivalent volume of FCA (day15) and FIA (day 23) in PBS (adjuvant group) Animals inthe last treatment group 3 were administered with 25 120583grecombinant protein along with the adjuvant (FCA on day15 and with FIA on day 23) respectively Preimmunized serawere collected from the retroorbital plexus of each mouseprior to immunization and thereafter on days 14 and 20 afterfirst antigen dose One week following final booster dose halfof the BALBc from each group received 50 L3 of B malayieach into the peritoneal cavity and were euthanized on day 15after L3 challenge to assess the recovery of developing L3Theremaining 5 mice from each group were kept unchallengedand euthanized on day 30 post infection (pi) for investi-gating the immune responses generated by the recombinantprotein

Similarly Mastomys from all the three groups were chal-lenged with 100 L3 of B malayi subcutaneously (sc) oneweek after the final booster dose Half of the animals fromeach group were euthanized on day 30 after L3 challengeand the remaining animals on day 180 after L3 challengerespectively to investigate the prophylactic efficacy and cel-lular proliferation in Bm-iPGM in immunized and controlanimals

211 Bm-iPGM Specific Antibody and Isotype Levels in Sera byELISA IgG antibody titre and antibody isotypes were meas-ured by ELISA For measuring IgG antibody titre the wellsof ELISA plate (Nunc Denmark) were coated with 1120583gmLof Bm-iPGM (100 120583Lwell) in carbonate buffer pH 96 at4∘C overnight (ON) blocked (1 gelatin in PBS containingTween-20) for 2 hours at 37∘C and washed thrice withPBS-T with each single washing for 5min Pooled serum ofimmunized and control group of animals (BALBc) was usedas primary antibody using serial twofold dilutions startingfrom 1 50 to 1 102400 while goat anti-mouse IgG-horseradish peroxidase (HRP) was added (1 10000) after washingand plate was incubated at 37∘C for another 1 h Reaction wasdeveloped by adding OPD substrate prepared fresh (20mg ofOPD in 25mL citrate buffer of pH 50 and 20120583L of H

2O2)

in dark for 10ndash15 minutes at RT and terminated by adding25 N H

2SO4 Absorbance was read at 492 nm in an ELISA

plate reader For antibody isotyping pooled sera (BALBc1 100) was used as primary antibody while goat-anti-mousemonoclonal antibodies to IgM IgA IgG1 IgG2a IgG2band IgG3 (1 1000) and rabbit anti goat-IgG-HRP (1 5000)(Sigma antibody isotype kit USA) served as secondary andtertiary antibodies respectively Reactions were measuredafter adding the substrate OPD as mentioned above Meanof the triplicate OD values was calculated and was used forplotting the graph

212 Oxidative Burst in Peritoneal Macrophages Real-timemonitoring of intracellular reactive oxygen species (ROS) inperitoneal exudate cells (PEC) of BALBc was determinedthrough a fluorometric assay using 2101584071015840-dichlorofluoresceindiacetate (DCF-DA) as described earlier [22] with minormodifications [20] Briefly freshly harvested PECrsquos (fromimmunized and control animals) at 1 times 106 cellstube wereprobe loadedwith theDCF-DA at final concentration of 1 120583Mfor 15min at 37∘C in CO

2incubator ROS levels in individual

living cells were determined by sequentially measuring theirfluorescence intensity (FI) on FACSCalibur (BD USA) Datawas analyzed by CellQuest Software (BD USA) and meanROS values were evaluated for cell populations

213 Immunophenotyping of T and B Lymphocyte Popula-tion Splenocytes from PBSadjuvant control and Bm-iPGMimmunized groups of BALBc were used for immunophe-notyping to assess lymphocyte subset population on a flowcytometer (FACSCalibur BD USA) using fluorochrome(FITC or PE) conjugated anti-mouse antibodies (SerotecUK) directed against receptors to CD4 CD8 and CD19[21] Splenocytes (1 times 106) were initially blocked with MouseSeroblock FcR at RT for 10min washed and divided intodifferent tubes for labelling with monoclonals to CD4+ andCD8+ T cells or CD 19+ B cells for 10min at RT Cells werewashed and finally suspended in sheath fluid for analysis byFACSCalibur using CellQuest analysis software (BD USA)after gating the forward and side-scatter settings to excludedebris For each determination 20000 cells were analyzedand the results are reported as percentage of each cell pop-ulation


214 Intracellular Th1 and Th2 Cytokine Response in Immu-nized BALBc The measurement of intracellular cytokinesin the spleen was done as per manufacturerrsquos (BD USA)protocol as mentioned earlier [21] Briefly splenocytes (4times 106mL) were incubated with brefeldin A (10 120583gmL)(Serotec UK) in dark for 6 h at 37∘C and reincubated withmouse Seroblock FcR at RT for another 10min Cells werewashed and incubated with FITC-rat anti-mouse CD4+antibody LeucopermA and LeucopermB (Serotec UK)wereadded at RT for 15min and cells were dispensed in fourtubes each containing 1 times 106 cells100 120583L PE-rat anti-mousemonoclonal antibodies to cytokines interleukin- (IL-) 2 IL-4 IL-10 and IFN-120574 were added to separate tubes and cellswere finally suspended in 500 120583L of 05 paraformaldehydefor flow cytometer readings

215 Cellular Immune Response in Immunized and ControlMastomys Groups The proliferation of splenocytes isolatedfrom the control and experimental Mastomys after vaccina-tion and challenge was performed from both the batchesas described earlier [23] In brief spleen was asepticallyremoved and cells were passed through a sterile nylon cellstrainer (40120583m pore size BD Falcon USA) to prepare singlecell suspension Cells (100 120583Lwell) from the stock (5 times 106cellsmL) were plated in a 96-well culture plate in triplicateand stimulated with 100 120583L Bm-iPGM (optimal concentra-tion 25 120583gmL) or concanavalin A (25120583gmL Sigma USA)for 72 h and pulsed with 10 120583Ciwell of [3H] thymidine (3H-Tdr specific activity 18 Cim mole BARC India) for 18 hpreceding harvest The radioactive incorporation in cells wasmeasured in a 120573-counter (Beckman Instruments Palo AltoCA) using scintillation fluid The stimulation index (SI) wasassessed as a ratio of mean cpm (counts per minute) values ofstimulated and unstimulated cultures

216 Effect of Bm-iPGM on Parasitaemia and Parasite Burdenin BALBc andMastomys TheBALBcmice were euthanizedon day 15 after L3 challenge to observe effect of vaccinationon development of L3 to L4 On the other hand half of theMastomys from all the three groups were euthanized on day30 to assess effect of immunization on development of youngadults and remaining half on day 180 after L3 challenge tomonitor microfilaraemia as well as effect on the adult wormestablishment [12] Various tissues namely heart lungstestes and lymph nodes were isolated and teased gently inPBS to recover adult worms Female worms were teased onglass slide in a drop of PBS and observed microscopically toobserve the effect of protein on worm fecundity Data werecompared with that of controls and arithmetic means werecalculated for blood Mf density worm burden and femaleworm reproductive potential

217 In Vitro Antibody-Dependent Cellular Adhesion andCytotoxicity Adherence of PECs to the surface of Mf andL3 was observed as described earlier [20] Mf (100) andL3 (10) were individually cocultured with 1 times 106 PECsisolated from normal Mastomys in 96-well plate in presenceof serum collected from immunized and normal Mastomys

Each well contained 100 120583L PECs 50120583L serum (1 32) and25 120583L guinea pig serum as a source of complement Plateswere kept at 37∘C in a CO

2incubator (Binder Germany)

and cell adherence on the surface of parasite and furthercytotoxicity was noted microscopically after 1 3 6 24 and48 h of incubation Cytotoxicity was expressed by consideringthe number of immobile or dead parasites by adherence ofeffector cells against the total number of parasites recoveredwithin 48 h The percentage of cytotoxicity was calculatedby subtracting the number of deadimmobile parasites fromthe total parasites taken dividing the result by total numberof parasites and finally multiplying by 100 Furthermore thepresence of Bm-iPGM antigen on the surface of Mf andL3 was also investigated by fluorescence microscopy usingpolyclonal antibody raised against Bm-iPGM In brief 10L350Mf were incubated with pooled serum (1 500 dilution)from Bm-iPGM immunizedMastomys (collected on day 30)for 4 h at 37∘C in 48-well flat-bottom tissue culture platesThe parasites were washed and reincubated with secondaryantibody (goat anti-mouse IgG-FITC 1 10000) for 2 h at RTon a rotor-shaker and parasites were finally transferred toglass slide for fluorescence microscopy (Nikon Japan)

3 Statistical Analysis

Data were analyzed using one-way analysis of variance(ANOVA) Individual comparisons following ANOVA weremade using the Newman-Keuls method with the help ofstatistical software PRISM 30 Results of flow cytometry andworm recovery have been presented as mean plusmn SE Thecriterion for statistical significance between the results ofimmunized and control groups were as follows 119875 lt 005was considered as significant 119875 lt 001 was considered ashighly significant 119875 lt 0001 was considered as very highlysignificant and 119875 gt 005 was considered as nonsignificant

4 Results

41 Bm-iPGM is Composed of Two Identical Domains andIs Highly Antigenic Amino acid sequence alignment ofBm-iPGM with iPGM from B anthracis using Clustal Wshowed 41 identity (Figure 1(a)) Bm-iPGM structure wasgenerated with 100 precision and 41 identity using thesame structure of B anthracis cofactor-independent 2 phos-phoglycerate mutase taken as template (PDB id c2ifyAlength 508 AA) The data analysed by ldquoThe PyMOL Molec-ular Graphics Systemrdquo showed Bm-iPGM to be composedof two identical domains connected by two linkers Boththe domains show similar folds containing central 120573 sheetstructure which are flanked on both sides by 120572 helices(Figure 1(b)) The in silico prediction showed 21 antigenicdeterminants in the protein with an average propensitybeing 10233 (see Supplementary Table 1 available online athttpdxdoiorg1011552014590281) A number of MHCbinding peptides were identified for the alleles used inanalysis and Table 1 shows the best predicted binding peptidefor each allele used in analysis and their log score These


lowast lowastlowastlowastlowastlowastlowast lowastlowast

MRKPTALIILDGFGLREETYGNAVAQAKKPNFDGYWNKFPHTTLTACGEAVGLPEGQMAEAKNRVCLVVIDGWGISNETKGNAILNAKTPVMDELCVMNSHP IQAHGLHVGLPEGL

FATISGRYYSMDRDKRWDRVEKCYRAMVN GEGPTYKSAEECVEDSYANGIYDEFVLPSISTIVGRYYAMDRDKRWERIRVCYDALIGGVGEKTTIDKAIDVIKGRYAKDETDEFLKPI

VIVNEDNTPVATINDDDAVIFYNFRPDRAIQIARVFTNGDFREFDRGEKVPHIPEFVCMTILSDEGRT KDGDTLIFFDYRADRMREITECMGMERYKDLNSNIKHPKNMQVIGMT

HFSETVDGYVAFKPMNLDNTLGEVVAQAGLKQLRIAETEKYPHVTFFFSGGREAEFPGEEQYKAEFTFPALFPPESHKNVLAEWLSVNGLTQFHCAETEKYAHVTFFFNGGVEKQFANEE

RRILINSPKVATYDLKPEMSIYEVTDALVNEIENDKHDVIILNFANCDMVGHSGMMEPTIKCLVVSPKVATYDLEPPMSSAAVADKVIEQLHMKKHPFVMCNFAPPDMVGHTGVYEAAVK

AVEATDECLGKVVEAILAKDGVALITADHGNADEELTSEGEPMTAHTTNPVPFIVTKNDVAVEATDIAIGRIYEACKKNDYILMVTADHGNAEKMMAPDGSKHTAHTCNLVPFTCSSMKY

ELRE DGILGDIAPTMLTLLGVEQPKEMTGKTIIKKFMDKLPDREMALCDVAPTVLKVMGVPLPSEMTGQPLVNEA

lowastlowast lowast lowast lowast lowast lowast lowastlowastlowastlowastlowastlowast

lowast lowastlowast lowastlowastlowastlowast lowast lowastlowast lowast lowast lowast lowast lowastlowast lowastlowastlowastlowast lowast lowastlowastlowastlowast

lowast

lowastlowastlowastlowast

lowast lowast lowast lowastlowast lowast lowast

lowastlowastlowast lowast lowast

lowast lowast lowast lowast

lowast lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowastlowastlowast

lowast

lowast

lowastlowast lowastlowastlowastlowastlowast lowast lowastlowastlowastlowastlowastlowastlowastlowast lowastlowastlowast

lowastlowastlowastlowast lowastlowastlowastlowast lowast lowastlowastlowastlowastlowast lowastlowastlowastlowastlowastlowastlowastlowastlowast lowast

lowast lowastlowast lowastlowastlowastlowast lowast lowastlowastlowastlowastlowast

lowast

lowast lowastlowast lowast lowast lowastlowast

lowastlowastlowastlowast lowast lowastlowastlowastlowast

DGGVHSHMNHMFALLRLAAKEGVEKVYIHAFLDGRDVGPKTAQSYIDATNEVIKETGVGQDGGVHSHIDHLFALITALKQLKVPKLYIQFFGDGRDTSPTSGVGFLQQLIDFVNKEQYGE

lowastlowast lowastlowastlowast lowast lowast lowast lowastlowastlowastlowast lowastlowastlowastlowastlowastlowastlowastlowastlowast lowast

MGNSEVGHLNIGAGRIVYQSLTRVNVAIREGEFDKNETFQSAIKSVKEKGTALHLFGLLSMGNSEVGHLNIGAGRVVYQDIVRINLAVKNKTLVENKHLKEAAERAIKGNGRMHLCGLVS

lowastlowastlowast lowast lowastlowast lowastlowastlowast lowast lowast lowast lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

-

- - - - -

-

- - - - - - -

-

---Ba-iPGMBm-iPGM

Ba-iPGMBm-iPGM

Ba-iPGMBm-iPGM

Ba-iPGMBm-iPGM

Ba-iPGMBm-iPGM

Ba-iPGMBm-iPGM

Ba-iPGMBm-iPGM

Ba-iPGMBm-iPGM

Ba-iPGMBm-iPGM

5759

117119

177179

235239

295294

355354

415414

475474

509515

(a)

Linkers

Transferase domain Phosphatase domain

(b)

Linkers


(c)

Figure 1 Amino acid sequence alignment and in silico structure of Bm-iPGM (a) Multiple sequence alignment of the deduced amino acidsequence of Bm-iPGM Bm-iPGM sequence was aligned with Bacillus anthracis cofactor-independent 2 phosphoglycerate mutase taken astemplate (Accession no 2IFY A) using Clustal W Bm-iPGM showed 41 identity with amino acid sequence of B anthracis iPGM Regionsof identity (lowast) strong similarity () and weak similarity () are displayed (b) In silico cartoon structure of Bm-iPGM Helical content matcheswith the experimental CDdata (c) Bm-iPGM in silico cartoon structure showing quantitatively predictedMHCI andMHCII binding stretches(regions in red are presented by MHCI while the dark blue are presented by MHCII)

peptides were also visualised and highlighted in the cartoonstructure of Bm-iPGM (Figure 1(c))

42 Bm-iPGM Was Cloned Recombinant Protein OptimallyExpressed as a Single Band of sim60 kDa The 1548 bp genewas successfully cloned into pTZ57RT (2886 bp) vectorgene sequence verified by sequencing and subcloned intoexpression vector pET 28a The maximal protein expressionwas obtained after four hours of 05mM IPTG induction at37∘CAsim60 kDa recombinant protein band authenticated theexpressed protein to be recombinant protein in-frame withthe N-terminal 6x-His Tag (Figures 2(a) and 2(b)) whichwas found to be biochemically active The exact mass of

recombinant protein was found to be 61779 kDa as analyzedby MALDI-TOF (Figure 2(c))

43 Secondary Structure Analysis by Spectroscopy RevealedBm-iPGM to Be 120572120573 Type Protein Far-UV CD spectrum canbe used empirically as ldquoblueprintrdquo of a particular proteinproviding information about the polypeptide backbone andthe protein conformation in terms of its secondary structure[24] The secondary structure of Bm-iPGM as characterizedby far-UV CD shows that it is 120572120573 type protein As depictedin Figure 2(d) Bm-iPGM has two negative peaks around222 nm and 208 nm and a stronger positive peak near 190 nmwhich is a characteristic of predominant 120572-helical protein


Table1Ad

ultp

arasite

recovery

andfemalew

orm

fecund

ityfro

mcontroland

Bm-iP

GM

immun

ized

Mastomys

Animalgrou

psNum

bero

fanimals

Adultp

arasite

coun

tsanim

alAd

ultw

orm

recovery

(meanplusmnSE)

redu

ctionin

worm

burden

femalep

arasite

sterilization

Day

30pc

Day

180pc

Day

30pc

Day

180pc

Day

30pc

Day

180pc

Day

180pc

PBS

66

302732281824

C242515142120

D9788952650plusmn2022750plusmn195

00

00

1850

Adjuvant

66

182226272331

C221918122115

D131011811102450plusmn1832833plusmn215

754

minus301

2051

Bm-iP

GM

66

88105912

C696745

D433262866plusmn095lowastlowastlowast

95plusmn067lowastlowastlowast

6729

6545

6997

Statisticallysig

nificantvaluesw

ereo

btainedin

Bm-iP

GM

immun

isedgrou

psas

comparedto

thec

ontro

lgroup

slowastlowastlowast119875lt0001V

aluesrepresented

arem

eanplusmnSE


(kDa)116

662

45

35

25

184

144

Bm-iPGM

L1 L2 L3 L4 L5 L7L6

(a)

L1 L2

(kDa)

98

64

50

(b)

100

90

80

70

60

50

40

30

20

10

0199600 320978 442356 563734 685112 806490

2074210

23396203090429

IPGM 61799571230

Inte

nsity

()

Mass (mz)(c)

10

0

minus10

minus20

minus30

minus40

200 210 220 230 240 250

Wavelength (nm)

[120579](times10

3de

gmiddotcm

2middotd

mol

minus1 )

(d)

300 320 340 360 380 400100

150

200

250

300

350

400

Fluo

resc

ence

(au

)

Wavelength (nm)

(e)

Figure 2 Cloning expression and purification of Bm-iPGM (a) Purification of Bm-iPGM L1 flow through L2-L4 wash 1ndash3 L5 and L6 elute1-2 L7 standard protein marker (kDa) (b) Western blot analysis using anti-His mAb L1 prestained protein marker L2 purified Bm-iPGM(c) MALDI-TOF analysis of the molecular mass of recombinant Bm-iPGM A single major peak confirmed the mass of recombinant Bm-iPGM to be 61799 kDa (d) Far-UV CD spectra of Bm-iPGM CD measurements were made on JASCO J810 spectropolarimeter calibratedwith ammonium (+)-10-camphorsulfonate with 6120583M protein in 10mM CGH buffer (e) Fluorescence emission spectra of Bm-iPGM andspectra of Bm-iPGM in 50mM phosphate buffer were recorded with Perkin Elmer LS50B luminescence spectrometer On excitation at280 nm maximum emission spectra were noted at 340 nm

secondary structure [25] Analysis of the averaged far-UVCDspectrum gave an estimate of 5626 120572-helix and 569 120573strands The intrinsic fluorescence of Bm-iPGM was studiedto disclose the microenvironment surrounding the residuesof tyrosine and tryptophan When excited at 280 nm themaximum emission of Bm-iPGM was recorded at 340 nm(Figure 2(e)) revealing that tyr and trp residues were mainlylocated in hydrophobic environment and the Bm-iPGM waspurified in its native form

44 Bm-iPGM Is Expressed by All the Major Life-Forms of Bmalayi and Is Excreted Out by Adult Worms The polyclonalantibodies raised against the recombinant Bm-iPGM reactedwith the native protein in lysates of adult parasites Mfand L3 (Figure 3(a)) Bm-iPGM gene was also amplified

from cDNA of three major life stages of B malayi usinggene specific primers (Figure 3(b)) The results demonstratepresence of Bm-iPGM in all the three life stages analysed thusdemonstrating it to be an abundant protein Polyclonal anti-bodies raised against Bm-iPGM reacted with recombinantBm-iPGM and with the ES product demonstrating it to bepresent in the in vitro excretory-secretory products of femaleB malayi worms (Figure 3(c))

45 Human W bancrofti Patients Harbour Serum IgG Anti-bodies to Bm-iPGM The recombinant protein showed goodimmunoreactivity in Western blot with bancroftian humansera belonging to different clinical categories namely nonen-demic normals (NEN) endemic normals (EN) asymp-tomatic microfilaraemic (Mf+ve) carrier microfilaraemic


250

130

95

72

55

36

28

17

M Mf L3 Ad(kDa)

(a)

MMF L3 Ad (Da)

3000

2000

1500

1200

1000

900800700600

400

500

300

200

100

(b)

P1 P2 M250

130

95

72

55

36

28

17

11

(kDa)

(c)

Figure 3 Stage specific expression of Bm-iPGM (a) Western blot displaying expression of Bm-iPGM M standard protein marker Mfmicrofilariae L3 infective larvae and Ad adult parasites (b) 10 agarose gel displaying Bm-iPGM amplification Bm-iPGM gene wasamplified from cDNA of three major life stages of B malayi using specific primers Mf microfilariae L3 infective larvae Ad adultparasites and M standard DNA marker (c) Bm-iPGM in excretory-secretory products of adult parasite Western blot was done to confirmpresenceabsence of iPGM enzyme in the ES product of B malayi Anti-Bm-iPGM antibody raised in mouse showed reactivity with thepurified recombinant protein as well as the ES product of adult parasite P1 purified recombinant Bm-iPGM P2 adult worm ES product

symptomatic (MFC) and amicrofilaraemic symptomatic(AMFCS) demonstrating the presence of Bm-iPGMin thetarget human parasiteW bancrofti (Figure 4(a))The individ-uals fromNEN category who are not exposed to filarial larvaedid not display any reactivity with the recombinant enzymeshowing filarial specificity of the expressed protein

Bm-iPGM specific IgG ELISA was also carried outto determine the seroreactivity of individual categoryof serum samples from microfilaraemic amicrofilaraemicsymptomatic EN and NEN individuals All the former threegroups analysed revealed elevated levels of anti-Bm-iPGMIgG antibody with amicrofilaraemic symptomatic patientsdisplaying highest antibody titre which was significant overthe other groups (119875 lt 001) (Figure 4(b)) However nonen-demic normals did not react to Bm-iPGM

46 Bm-iPGM Generates Vigorous Antibody Response inBALBc Mice Antibody levels were measured in the seraobtained when the animals were euthanized on day 30post infection (pi) The Bm-iPGM group developed higherlevels of Bm-iPGM specific antibodies compared to PBScontrol and FCAFIA group Anti-Bm-iPGM antibody levelremained higher in Bm-iPGMgroup even at 1 6400 dilutionNone of the controls developed Bm-iPGM specific antibodyresponse (Figure 5(a)) Measurement of Bm-iPGM specificIgG isotypes in experimental groups revealed that animalsimmunized with recombinant Bm-iPGM induced predom-inantly elevated level of IgG1 IgG2a IgG2b IgG3 IgM andIgA (Figure 5(b)) IgG2aIgG1 ratio was indicative of a mixed

type of Th1Th2 immune response Animals from controlgroups did not develop Bm-iPGM specific antibody isotypes

47 Bm-iPGM Activates the Antigen Presenting Cells (APCs)Upregulating the Production of Reactive Oxygen Species Areal time monitoring of oxidative burst generated fromperitoneal macrophages of immunized and controls group ofBALBc was done Flow cytometry data indicate that Bm-iPGM immunization led to the generation of significantlyhigher oxidative burst (119875 lt 001) in macrophages from Bm-iPGM group as compared to the controls which might haveplayed important role in parasite death (Figures 6(a) and6(b))

48 Increased Number of Both T And B Cell Population WasObserved Bm-iPGM administration into BALBc mice ledto expansion of both cellular and humoral immune responseand a significant rise in CD4+ (119875 lt 0001) and CD8+ (119875 lt001) T cells (Figures 6(c) and 6(d)) CD 19+ B cell populationalso increased significantly (119875 lt 005) (Figure 6(e)) The PBSand FCAFIA control groups of animals did not showed suchheightened cellular and humoral immune response Experi-ments were carried out to illustrate the in vitro proliferationof splenocytes from both the batches ofMastomys euthanizedon day 30 and day 180 after larval challenge in presenceof Bm-iPGM or mitogen Con A Spleen cells from Bm-iPGM immunized animals exhibited noticeable proliferationwhether stimulated with Con A or Bm-iPGM at both timepoints (Figures 7(a) and 7(b))


M L2 L3 L4 L5 L6

1160

662

450

350

250

(kDa)

(a)

00

05

10

15

20

Human subjectsMFC AMFCS EN NEN

OD

at4

92

nm

(b)

Figure 4 Seroreactivity of Bm-iPGMwith human bancroftian sera pooled from 10 filarial patients of each category (a)Western blots showingcross-reactivity of recombinant Bm-iPGM with various categories of human bancroftian sera L1 standard protein marker L2 nonendemicnormal L3 endemic normal L4 microfilaraemic asymptomatic L5 microfilaraemic symptomatic L6 amicrofilaraemic symptomaticpatients (b) Bm-iPGM specific ELISA showing reactivity of Bm-iPGM with human bancroftian sera taken from human subjects belongingto various categories MFC microfilaraemic carrier AMFCS amicrofilaraemic symptomatic EN endemic normal and NEN nonendemicnormal individuals Serum (1 200) from ten individuals per clinical category was tested in ELISA for reactivity with recombinant Bm-iPGMAll the three groups from filarial endemic area revealed elevated levels of anti-Bm-iPGM antibodies

0

01

02

03

04

05

06

07

08

09

50 100

200

400

800

1600

3200

6400

12800

51200

102400

DilutionControlAdjuvantBm-iPGM

OD

at4

92

nm

(a)

ControlAdjuvant

4

3

2

1

0

OD

at4

92

nm

P lt 0001 P lt 0001 P lt 0001

P lt 0001

P lt 0001P lt 0001

P lt 0001 P lt 0001P lt 0001P lt 0001P lt 0001P lt 0001

Antibody isotypesIgG1 IgG2a IgG2b IgG3 IgM IgA

Bm-iPGM

(b)

Figure 5 Bm-iPGM specific IgG antibody and antibody isotypes (a) Antibodies were detected by ELISA in the pooled sera of BALBc miceadministered with Bm-iPGM along with adjuvant and PBS only Elevated IgG level was maintained in the Bm-iPGM immunized group(b) Anti-Bm-iPGM antibody isotype levels (IgG1 IgG2a IgG2b IgG3 IgM and IgA) in the pooled sera of Bm-iPGM immunized adjuvantimmunized and control groups Considerable increase in the levels of all the isotypes was noticed Each bar represents mean of triplicate ODvalues taken at 492 nm each obtained with pooled sera of five experimental animals

49 Immunization with Bm-iPGM Elicits a Mixed Th1Th2Immune Response with Marked Reduction in Larval Devel-opment in BALBc While in Mastomys Considerably ReducedMicrofilarial Density Adult Worm Recovery and FemaleWorm Fecundity Were Observed The levels of both proin-flammatory and anti-inflammatory cytokines were deter-mined intracellularly in the splenic cell population of immu-nized BALBc mice There was an up regulation in the levels

of proinflammatory cytokines IL-2 (119875 lt 0001) and IFN-120574(119875 lt 001) as well as anti-inflammatory cytokines IL-4 (119875 lt001) and IL-10 (119875 lt 001) (Figures 8(a) 8(b) 8(c) and 8(d))as analysed by flow cytometrywhich indicated generation of amixed Th1Th2 immune response Immunization of BALBcmice with Bm-iPGM resulted in a significant reduction inworm establishment in Bm-iPGM (number of parasites 86 plusmn11) immunized animals (119875 lt 0001) as compared to the PBS


0

400

100

200

300

Cou

nts

100 101 102 103 104

Control

Adjuvant

Bm-iPGM

FL1-HDCFDA

(a)

ROS generation500

400

300

200

100

Animal groups

Fluo

resc

ence

inte

nsity

P lt 001

0

Control Adjuvant Bm-iPGM

P gt 005

(b)

30

20

10

0

Animal groups

P lt 0001

P lt 0001


P gt 005

CD4+ cells

Gat

ed C

D4+

cells

()

(c)

P lt 001P lt 001

30

40

20

10

0

Animal groupsControl Adjuvant Bm-iPGM

P gt 005

CD8+ cells

Gat

ed C

D8+

cells

()

(d)

15

10

5

0


P lt 005P gt 005

CD19+ cells

Gat

ed C

D19+

cells

()

(e)

Figure 6 Flow cytometric analysis of ROS generation by peritoneal macrophages and splenic lymphocyte surface staining for T and B cells(a) Representative FACS histogram for ROS generation from each group is displayed and values closer to mean are represented (b) Reactiveoxygen species produced by the peritoneal macrophages (1 times 106mL) isolated from for all groups were loaded with probe DCF-DA and ROSgeneration was evaluated on day 30 when the animals were euthanized Bm-iPGM immunization led to activation of macrophages whichsignificantly generated reactive oxygen species (119875 lt 001) (c) CD4+ T cell marker (d) CD8+ T cell marker and (e) CD19+ B cell markerSignificant increase was noticed in the number of CD4+T cells (119875 lt 0001) and CD8+T cells (119875 lt 001) fromBm-iPGM immunized animalsThough a marginal expansion in B cell population was observed it was statistically significant (119875 lt 005)

control (number of parasites 2060plusmn 12) and FCAFIA (2240plusmn 143) groups Thus immunization with Bm-iPGM resultedin up to 5825 reduction in parasite establishment whenBALBc mice were euthanized on day 15 pc (Figure 9(a))

Immunization of Mastomys with Bm-iPGM had pro-found adverse effect on the Mf density and adult wormestablishment when compared with the nonimmunized con-trols Though Mf appeared in all the three groups by day90 (Figure 9(b)) their density was much lower (108 plusmn 2534)in Bm-iPGM immunized group in contrast to 3913 plusmn 7732and 293 plusmn 1006 in PBS and adjuvant groups respectivelyat the time of euthanization demonstrating 724 reductionover that of control (119875 lt 005) Considerable reduction(6545ndash6729) in adult worm recovery was noticed in Bm-iPGM immunized groups when the animals were euthanizedon days 30 and 180 after larval challenge (Table 1) In contrastto immunized groups (8ndash10 wormsanimal) the averagerecovery of adult worms ranged between 2450 and 280 in thetwo control groups Vaccination also led to significant adverseeffect on the female worm fecundity as observed on day 180

(Table 1) The percentage of sterile adult females recoveredfrom Bm-iPGM gp was 6997 plusmn 4234 which was significantlyhigher (119875 lt 0001) than that of the normal control gps (PBS1850 plusmn 2023 and adjuvant gp 2051 plusmn 1543)

410 Bm-iPGMSpecific Cellular Adherence and Cytotoxicity toMf and L3 Profound in vitro complement mediated cellularadherence and cytotoxicity to both Mf and L3 was noticed(Supplementary Figures 1(a) and 1(b)) in the presence of Bm-iPGM specific antibody Percentage cytotoxicity was calcu-lated by counting the number of immobile or dead parasitesby adherence of effector cells against the total number oflive parasites recovered which resulted in 614 and 520death of Mf and L3 respectively (Figure 10(a)) which wasstatistically highly significant (119875 lt 0001) when comparedto normal Mastomys serum where no cell adhesion wasseen Interaction of anti-Bm-iPGMantibodies with B malayiMf (Figure 10(b)) and L3 (Figure 10(c)) was confirmed byfluorescence microscopy


8

6

4

2

0Con A

Sim

ulat

ion

inde

x (S

I)

Mastomys (30 d pc)Bm-iPGM

P lt 0001

P lt 0001

ControlAdjuvantBm-iPGM

(a)

8

6

4

2

0Con A

Sim

ulat

ion

inde

x (S

I)

Bm-iPGMMastomys (180 d pc)

P lt 001P lt 005


(b)

Figure 7 In vitro lymphocyte proliferation Proliferation was assessed in the splenocytes isolated from immunized adjuvant and controlgroup of Mastomys by radioactive incorporation of [3H]-thymidine after stimulation with either concanavalin A (25120583gmL) or Bm-iPGM(25 120583gmL) Radioactive incorporation in the cells was measured and results are expressed as stimulation index (a) Batch A euthanized onday 30 after larval challenge (b) Batch B euthanized on day 180 after larval challenge

5 Discussion

Phosphoglycerate mutases are the enzyme that catalyzesthe reversible interconversion of 3-phosphoglycerate and2-phosphoglycerate in both glycolysis and gluconeogene-sis (Ulrike and Peter 2007) B malayi possesses cofactor-independent form of PGM while the dependent form ispresent in mammals that present iPGM as an attractiveantifilarial drug or vaccine candidate Raverdy [17] carriedout the biochemical characterization of B malayi iPGMand emphasized its worth as an antifilarial drug target Noinformation is however available on whether this proteinplays any role in parasite immunobiology or host-parasiteinteractions

In the present investigation molecular and immunecharacterization studies of B malayi iPGM have been carriedout Bm-iPGMwas cloned expressed and purified to homo-geneity as a single band protein of sim60 kDa Recombinantprotein was found to be biochemically active in its nativeform as observed by circular dichroism and fluorescencespectroscopy which demonstrated 120572120573 type topology havingmore than 50 120572-helix and around 5-6120573 strands whichwasconsistent with the previous report on the three-dimensionalcrystal structure of iPGM of B anthracis [26] The homologymodel predicted the recombinant enzyme to be composedof a globular structure with two domains termed as thetransferase and phosphatase interconnected by two shortlinker peptides Both the domains demonstrate similar foldscontaining central 120573 sheet structure which are flanked onboth sides by 120572-helices thus further confirming 120572-120573 typetopology Till date there are no known inhibitors of iPGMand the homology model generated can thus be utilisedto design a series of inhibitors providing us with possibleantifilarial drugs

A small fragment of antigen can induce immune responseagainst the whole antigen thus locating promiscuous bindingregions from the whole protein sequence can be useful indesigning vaccine candidates Bm-iPGMgene sequence in sil-ico showed presence of 21 antigenic determinants carrying anaverage antigenic propensity of 10284 which points towardsthe high antigenicity of this protein Further computationalanalysis of the target gene sequence predicted binding prop-erties of the peptides to be driven by both MHC I and MHCII immune pathways The alleles with high frequency withinhuman population and with significant binding data wereselected and the best binding peptides for each allele wereidentified and highlighted on the cartoon structure of Bm-iPGM and these were mainly located in the 120572-helix region ofthe structure

Bm-iPGM seems indispensable for the parasite as it wasfound to be expressed by Mf L3 and adult parasites andis also present in the excretory-secretory (ES) product ofadult parasites ES products released by live parasites caninterferewith every aspect of host immunity [27] and requiresfunctional characterization of their role in parasite and host-parasite interactions Many of these proteins could serveas drug targets and can also be evaluated for prophylacticefficacy [11] Bm-iPGM is one such protein and could serve tobe major protein targeting all the important parasitic stages

The serum collected from human bancroftian subjectsand endemic normal individuals contained anti-Bm-iPGMantibodies which was demonstrated by reactivity againstrecombinant Bm-iPGM in blots In ELISA amicrofilaraemicsymptomatic sera showed higher antibody titre to Bm-iPGM than endemic normals or microfilaraemic carrierswhile none of the 10 individual sera collected from filarialnonendemic area reacted with Bm-iPGM demonstratingfilarial specificity of the recombinant protein which may also


(a)

(b)

(c)

(d)

Animal groups

P lt 001P lt 001

3

2

1

0

P gt 005

Con

trol

Adju

vant

Bm-iP

GM

CD4+IL-4+

Gat

ed C

D4+

IL-4+

cells

()

P lt 001P lt 001

4

3

2

1

0

P gt 005

Animal groups

Con

trol

Adju

vant

Bm-iP

GM

CD4+IL-10+

Gat

ed C

D4+

IL-10+

cells

()

IL-2

PE

Isotype control

100

101

102

103

104

100 101 102 103 104

006

Control

100 101 102 103 104

084

Adjuvant

100 101 102 103 104

101

Bm-iPGM

100 101 102 103 104

222

100 101 102 103 104

108

100 101 102 103 104

114

100 101 102 103 104

398

100

101

102

103

104

100 101 102 103 104

IL-4

PE

014

100 101 102 103 104

104

100 101 102 103 104

102

100 101 102 103 104

22

100

101

102

103

104

100 101 102 103 104

CD4 FITC

IL-10

PE

018

100 101 102 103 104

CD4 FITC

132

100 101 102 103 104

CD4 FITC

151

100 101 102 103 104

CD4 FITC

274

100

101

104

100 101 102 103 104

016016

102

103

IFN

-120574PE

Gat

ed C

D4+

IL-2+

cells

()

Animal groups

3

2

1

0

P lt 0001P lt 001

P lt 005

Con

trol

Adju

vant

Bm-iP

GM

CD4+IL-2+

Animal groups

P lt 001P lt 0058

6

4

2

0

P gt 005

Con

trol

Adju

vant

Bm-iP

GM

CD4+

IFN

-120574+

cells

() CD4+IFN-120574

Figure 8 Flow cytometric analysis of intracellular IL-2 IFN-120574 IL-10 and IL-4 production in CD4+ T cells Splenocytes were stained andprocessed as described in Section 2 Numbers in the upper right quadrant of dot plot represent the mean percentage of CD4+ T cells positivefor (a) IL-2 (b) IFN-120574 (c) IL-4 and (d) IL-10 in particular group Bar graph was generated for percentage of CD4+ T cells positive forIL-2 IFN-120574 IL-4 and IL-10 Statistical significance of the differences between mean values of immunized and control groups is depicted aslowast

119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001


Worm recoveryRe

cove

red

wor

ms

30

20

10

0

Control Adjuvant Bm-iPGMAnimal groups

P lt 0001

P lt 0001

P gt 005

(a)

0

50

100

150

200

250

300

350

400

450

500

90 120 150 180

Days (pc)

Microfilarial density in control adjuvant and immunized groups

AdjuvantControlImmunized

lowastlowast

Mf i

n10

120583L

bloo

d(b)

Figure 9 (a) Recovery of B malayi preadults from different groups of Bm-iPGM immunized and control mice Parasites were collectedby washing the peritoneal cavity of infected BALBc mice Immunization of mice with Bm-iPGM resulted in marked reduction in wormestablishment (119875 lt 0001) Statistical analysis was done using one-way ANOVA followed by Newman-Keuls Multiple Comparison TestEach bar represents mean plusmn SE of worms recovered from five animals (b) Assessment of microfilarial density in tail blood of Bm-iPGMimmunized adjuvant and control groups Mf count was initiated from day 90 till day 180 pc Control and adjuvant groups exhibited elevatedlevels of bloodMf density However Mf levels remained low in Bm-iPGM immunized group and significant reduction inmicrofilarial density(119875 lt 00172396) on day 180 (pc) was observed in immunized animals Each point represents a value obtained from six animals

find its use in LF diagnosis Seroreactivity with EN sera is ofprime importance because EN are considered to be putativelyimmunoprotective and despite being continuously exposedto filarial larvae remain infection-free and do not develop thedisease [28] Few such antigens reacting stronglywith EN serahave earlier been shown to offer protective immunity [29ndash32] We further investigated the nature of immune responsegenerated after administration of recombinant Bm-iPGM inBALBc mice followed by prophylactic evaluation both inBALBc and Mastomys It is worth mentioning that BALBcis immunologically a well dissected model and thereforehas been used in the current investigation to decipher thetype of immune responses triggered by the recombinantprotein while Mastomys is a susceptible model that supportsestablishment of adult parasites mimicking the life cycle ofparasite in human host L3 are the most important stagesin the life cycle of filariid that initiate an infection andfurther establish as adult parasites Immunized mice weretherefore challenged with L3 whose further development intoL4 stage or adulthoodwas observed Infection of BALBcwithB malayi or B pahangi L3 has earlier provided importantinsights into host-parasite biology in spite of the nonpermis-siveness of immunocompetent mice to Brugia species [33]

An active filarial infection is characterised by downreg-ulated Th1 immune response in the form of suppressed Tcell proliferation decreased production of proinflammatorycytokines such as IFN-120574 and IL-2 along with Th2 dominatedprofile indicated by increased production of the IgE andTh2 cytokines IL-4 IL-5 IL-10 and IL-13 with expansion

and greater mobilization of effector cells such as mast cellseosinophils and basophils [27 34ndash38] Treg cells are inducedby the parasite to evade the human immune system and areconsidered to be the important regulators of the immuneresponse to filarial nematodes in experimental animals [3940] CD4+ T cells express increased levels of CD25 CTLA-4 and glucocorticoid-induced TNF receptor family-relatedgene (GITR) with increased Treg functionality in microfila-raemic individuals Treatment with antibodies to CD25 andGITR reverses this hyporesponsiveness with reduced parasiteestablishment [41] while depletion of Treg cells has shown torestore T cell as well as B cell proliferation [42]

Immunization with recombinant Bm-iPGM activatedboth the cellular and humoral arms of immunity Profoundantibody response was observed in BALBc with high IgGtiters in addition to IgG1 IgG2a IgG2b IgG3 IgM andIgA demonstrating induction of both Th1 and Th2 immuneresponse which was supported by the equal ratio of IgG1 andIgG2a Antifilarial antibodies have been reported to play animportant role in protective immunity evidenced by studieswhere passive transfer of immune sera from resistant tonaive animals showed reduced adult worm establishment[43] B cell-deficient mice that lacked antibody displayedsuppressed vaccine-induced protection against murine filar-iasis [44] Antibodies directed against the surface of L3 andMf have also been shown to be protective and an inversecorrelation between adult worms and circulating antibodieshas been noticed IgG is believed to be the predominantantibody involved in antibody dependent cellular cytotoxicity


ADCCMf75

50

25

0Control Immunized

In v

itro

cyto

toxi

city

()

ADCCL375

50

25

0Control Immunized

In v

itro

cyto

toxi

city

()

(a)

(b) (c)

Figure 10 Antibody dependent cellular adhesion to Mf and L3 of B malayi Ten L3 and 100 Mf were taken per well and were incubated withPEC isolated from normal Mastomys in the presence of sera from Bm-iPGM immunized animals (a) Sera of Bm-iPGM immunized micepromoted adherence of PEC to Mf and L3 larvae and induced significant death of Mf (6140 cytotoxicity) and L3s (52) Photographs werecaptured on phase contrastmicroscope (Nikon Japan) at 40xmagnification Data are presented asmeanplusmn SE values fromfive different wellsInteraction of anti-Bm-iPGM antibodies with B malayi Mf (b) and L3 (c) as shown by fluorescence microscopy Parasites were incubatedwith anti-Bm-iPGM sera for 4 h and further incubated with FITC labelled anti-mouse IgG for 2 h Images were captured under fluorescentmicroscope at 20X for Mf and 10X for L3

(ADCC) mechanism involving adherence of neutrophilsmacrophages and eosinophils to Mf and L3 [45ndash48] In ourADCC experiment peritoneal exudates cells got adhered onthe surface of both L3 and Mf in presence of immunizedsera causing parasite immobility and death In a study onL sigmodontis model of filariasis it has been proposedthat the cell recruitment depends on many factors like hostsusceptibility immune response and cell adhesion properties[49] Immunofluorescence staining of L3 and Mf carriedout with serum from immunized Mastomys also confirmedattachment of anti-Bm-iPGM antibodies to their surfacesconfirming presence of iPGM on parasite surface and itsinteraction with antibodies The reduction in parasite recov-ery and sterilization of recovered female worms from Bm-iPGM immunized animals could be due to this observedcytotoxicity to inoculated L3 which was apparent in BALBcor both of the groups ofMastomyswhether euthanized on day30 after larval challenge or on day 180 pc

We observed significant levels of IgA which remainsundefined in human bancroftian filariasis in spite of the fact

that studies in other helminths have indicated a protectiverole for parasite specific IgA restricting infection intensity[50ndash53] Recently in human bancroftian filariasis the roleof IgA in protective immunity has been demonstrated [54]IgA levels in mice have directly been found to be associatedwith raised interferon gamma (IFN-120574) production by T cells[10 54] The role of IgM remains undefined in nematodeshowever in vitro it has been shown to play a major rolein adherence of host immune cells to filarial L3 and Mfcausing cytotoxicity and their death [55] Thus elevated levelof Bm-iPGM specific IgM might also have adversely affectedthe survival of challenged B malayi L3 and their furtherdevelopment inMastomys

Raised reactive oxygen species (ROS) levels might havebeen one of the mechanisms responsible for providingprotection [56 57] that could be correlated with the IFN-120574levels in the immunized mice since macrophage activationlargely depends upon IFN-120574 produced byTh1 (CD8+T cells)Animals from both the batches of Bm-iPGM immunizedMastomys revealed higher T cell proliferation in presence


of recombinant Bm-iPGM or mitogen Con A conferringthat Bm-iPGM was equally effective in causing cellularproliferation as Con A Mice deficient in T and B cells havebeen shown to be permissive to filarial infection therebystressing the importance of T and B cells in preventing filarialestablishment [58ndash60] An expansion in B cell populationwasalso noticed after Bm-iPGM administration in the animalsas observed by the increased levels of CD19+ B cells Lowmicrofilaraemia in antigen immunized group which wasapparent from day 120 onwards may also be an attributefor T cell proliferation as state of hyporesponsiveness hasbeen directly linked to high number of circulating Mfs [61]Significant upregulation in CD4+ and CD8+ cells populationwas noticed in Bm-iPGM immunized animals which arein general used for defining helper and cytotoxic T cellsubpopulations respectively [62 63] It is generally believedthat a vaccine will have at its core instigation of an antigenspecific CD4+ T cell response which plays an important rolein development of protective immunity against infection Anumber of studies have shown thatCD4+Tcells play a criticalrole in regulating the immune response to nematode parasiteswhere depletion of CD4+ T cells in infected mice has beenshown to enhance adult worm and microfilarial burden

Contradictions to the studies that consider immuneresponses to helminth parasites including filariids to be Th2type do exist and reports either impairment of both Th1and Th2 pathways and domination of Th1 response [6465] these differing observations might have been due todifferent life stages examined Past studies in murine modelsadvocate the involvement of both Th1 and Th2 arms ofimmunity in resistance to filarial parasites [66 67] thusdownregulation inTh1Th2 effector function would certainlyfacilitate the establishment and maintenance of filarial infec-tions Therefore any vaccine regimen that would help toovercome downregulation might be useful in impairing theestablishment of filarial parasites

Immunization with Bm-iPGM led to generation of aneffective immune mechanism mediated through an upreg-ulated Th1 (IFN-120574 IL-2) and Th2 (IL-4 IL-10) cytokineproduction that could provide considerable protection (58BALBc 65ndash68Mastomys) against challenged larval devel-opment possibly by combating the immune downregulationcaused by the challenged larvae Since BALBc were euth-anized on day 15 after L3 challenge all the L3 stages hadconverted into advanced L4 stages and none of the recoveredlarva was L3 However this period could demonstrate wellthat there was noticeable killing of the L3 in Bm-iPGMimmunized mice as was observed in the Mastomys whichdisplayed reduction in parasite establishment both on day 30or day 180 pc

IL-4 and IL-5 have been shown to play critical roles inthe host resistance to L loa infection in knock out BALBcmice (Nicholas 2012) Th1 cytokine IFN-120574 controls B malayiinfection in murine models IL-5 controls adult worm devel-opment in primary infection and IL-4 mediated pathwaysare necessary for the control of Mf and the developmentof adult worms [68] Studies in IL-4 knockout mice haverevealed an undeniable role of IL-4 in countering larvalestablishment in murine modelof Litomosoides sigmodontis

diminished Th2-type responses with failure to produce par-asite specific IgG1in Trichuris muris infection [69ndash71] IL-4 dependent effector mechanisms have been shown to bedependent on IL-10 in mice that were knocked out for IL-4IL-10 displaying antagonistic activity between IL-4 and IL-10 [68] Recently levels of IL-10 have been directly linked toparasite survival overcome resistance and allow full patencyin murine filariasis [72] Immunity in human infections hasbeen reported to be associated with an elevated level of IL-2and IFN-120574 [73 74] EN and chronic patients develop strongerimmune response raised IFN-120574 level as compared to that ofpatients carrying active filarial infection [75 76] B malayiMf and L3 have been shown to be killed in vitro by IFN-120574activated macrophages via production of nitric oxide (NO)and ROS [77ndash79] A mixed Th1Th2 response as observed inthe current investigation has been ascribed to exert profoundimmune protective function [80 81] The present findingsclearly suggests that on vaccinationwith Bm-iPGM a correctmilieu with a mixed type of Th1Th2 immune responseaccompanied with innate immunity was maintained whichwas efficient in providing significant degree of protectionagainst establishment of B malayi in the immunized hostThe different molecular events that are required tomaintain abalanced cytokine levels need careful investigation to furtherfacilitate vaccine development programme

In summary the overall immune response generated byBm-iPGM correlated with the percentage level of protectionachieved in terms of low adult worm recovery reducedmicrofilaraemia and embryostatic effect in female wormsThe independent phosphoglycerate mutase of filarial parasiteB malayi appears to be an immunogenic protein with diag-nostic potential which considerably impairs filarial parasiteestablishment and presents a promising vaccine candidateImmunization studies with Bm-iPGMusing human adminis-trable adjuvants are underway to further improve its efficacyand usefulness

Conflict of Interests

The authors declare that they have no conflict of interests

Acknowledgments

Theauthors are grateful toMr A L Vishwakarma (SAIF divi-sion CSIR-CDRI) for his help in acquiring flow cytometrydata and to Mr A K Roy and Mr R N Lal for experimentalmaintenance of B malayi They are grateful to CSIR andUGC for providing financial assistance to Prashant K SinghSusheela Kushwaha and Ajay K Rana in the form of researchfellowships The authors also acknowledge CSIR for financialassistance in the form of CSIR Network Project SPLenDID(BSC 0104) This papers bears CSIR-CDRI communicationno 8701

References

[1] E Palumbo ldquoFilariasis diagnosis treatment and preventionrdquoActa Biomedica de lAteneo Parmense vol 79 no 2 pp 106ndash1092008


[2] Progress Report 2000ndash2009 and Strategic Plan 2010ndash2020 of theGlobal Programme to Eliminate Lymphatic filariasis HalfwayTowards Eliminating Lymphatic filariasis 2010

[3] S SabesanM Palaniyandi P K Das and EMichael ldquoMappingof lymphatic filariasis in IndiardquoAnnals of Tropical Medicine andParasitology vol 94 no 6 pp 591ndash606 2000

[4] D H Molyneux ldquoFilaria control and elimination diagnosticmonitoring and surveillance needsrdquo Transactions of the RoyalSociety of TropicalMedicine andHygiene vol 103 no 4 pp 338ndash341 2009

[5] WHO ldquoGlobal programme to eliminate lymphatic filariasisprogress report onmass drug administration in 2008rdquo inWeeklyEpidemiological Record vol 42 pp 437ndash444 World HealthOrganization Geneva Switzerland 2009

[6] E Ghedin S Wang D Spiro et al ldquoDraft genome of the filarialnematode parasite Brugia malayirdquo Science vol 317 no 5845 pp1756ndash1760 2007

[7] J van der Oost M A Huynen and C H Verhees ldquoMolecularcharacterization of phosphoglycerate mutase in archaeardquo FEMSMicrobiology Letters vol 212 no 1 pp 111ndash120 2002

[8] U Johnsen and P Schonheit ldquoCharacterization of cofactor-dependent and cofactor-independent phosphoglycerate muta-ses from Archaeardquo Extremophiles vol 11 no 5 pp 647ndash6572007

[9] M W Nowicki B Kuaprasert I W McNae et al ldquoCrystalstructures of Leishmania mexicana phosphoglycerate mutasesuggest a one-metal mechanism and a new enzyme subclassrdquoJournal of Molecular Biology vol 394 no 3 pp 535ndash543 2009

[10] Y Zhang S Pacheco C L Acuna et al ldquoImmunoglobulin A-deficientmice exhibit altered T helper 1-type immune responsesbut retain mucosal immunity to influenza virusrdquo Immunologyvol 105 no 3 pp 286ndash294 2002

[11] S Bennuru R Semnani Z Meng J M C Ribeiro T DVeenstra and T B Nutman ldquoBrugia malayi excretedsecretedproteins at the hostparasite interface stage- and gender-specific proteomic profilingrdquo PLoS Neglected Tropical Diseasesvol 3 no 4 article e410 2009

[12] U Singh S Misra P K Murthy J C Katiyar A Agrawal andA R Sircar ldquoImmunoreactive molecules of Brugia malayi andtheir diagnostic potentialrdquo Serodiagnosis and Immunotherapy inInfectious Disease vol 8 no 3-4 pp 207ndash212 1997

[13] L A Kelley andM J E Sternberg ldquoProtein structure predictionon the web a case study using the Phyre serverrdquo Nature Proto-cols vol 4 no 3 pp 363ndash371 2009

[14] A S Devi T Rehana A S Kolaskar and M W PanditldquoHydrophilicity and antigenicity of proteinsmdasha case study ofmyoglobin and hemoglobinrdquo Journal of Biosciences vol 14 no2 pp 133ndash142 1989

[15] H Singh andG P S Raghava ldquoProPred prediction of HLA-DRbinding sitesrdquo Bioinformatics vol 17 no 12 pp 1236ndash1237 2002

[16] H Singh and G P S Raghava ldquoProPred1 prediction ofpromiscuousMHC class-I binding sitesrdquo Bioinformatics vol 19no 8 pp 1009ndash1014 2003

[17] S Raverdy Y Zhang J Foster and C K S Carlow ldquoMolecularand biochemical characterization of nematode cofactor inde-pendent phosphoglyceratemutasesrdquoMolecular and BiochemicalParasitology vol 156 no 2 pp 210ndash216 2007

[18] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[19] H Towbin T Staehelin and J Gordon ldquoElectrophoretic trans-fer of proteins frompolyacrylamide gels to nitrocellulose sheetsprocedure and some applicationsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 76 no9 pp 4350ndash4354 1979

[20] S Vedi A Dangi K Hajela and S Misra-Bhattacharya ldquoVacci-nation with 73 kDa recombinant heavy chain myosin generateshigh level of protection against Brugia malayi challenge in jirdand mastomys modelsrdquo Vaccine vol 26 no 47 pp 5997ndash60052008

[21] M Singh S Shakya V K Soni A Dangi N Kumar and S-MBhattacharya ldquoThe n-hexane and chloroform fractions of Piperbetle L trigger different arms of immune responses in BALBcmice and exhibit antifilarial activity against human lymphaticfilarid Brugia malayirdquo International Immunopharmacology vol9 no 6 pp 716ndash728 2009

[22] N Zurgil Y Shafran E Afrimzon D Fixler A Shainbergand M Deutsch ldquoConcomitant real-time monitoring of intra-cellular reactive oxygen species and mitochondrial membranepotential in individual living promonocytic cellsrdquo Journal ofImmunological Methods vol 316 no 1-2 pp 27ndash41 2006

[23] S Misra M Mukherjee M Dikshit and R K ChatterjeeldquoCellular immune response of mastomys and gerbils in exper-imental filariasisrdquo Tropical Medicine and International Healthvol 3 no 2 pp 124ndash129 1998

[24] A Ayuso-Sacido C Graham J P Greenfield and J A Boock-var ldquoThe duality of epidermal growth factor receptor (EGFR)signaling and neural stem cell phenotype cell enhancer or celltransformerrdquo Current Stem Cell Research ampTherapy vol 1 no3 pp 387ndash394 2006

[25] J T Yang C-S C Wu and H M Martinez ldquoCalculationof protein conformation from circular dichroismrdquo Methods inEnzymology vol 130 pp 208ndash269 1986

[26] M Nukui L V Mello J E Littlejohn et al ldquoStructureand molecular mechanism of Bacillus anthracis cofactor-independent phosphoglycerate mutase a crucial enzyme forspores and growing cells ofBacillus speciesrdquoBiophysical Journalvol 92 no 3 pp 977ndash988 2007

[27] J P Hewitson J R Grainger and R M Maizels ldquoHelminthimmunoregulation the role of parasite secreted proteins inmodulating host immunityrdquo Molecular and Biochemical Para-sitology vol 167 no 1 pp 1ndash11 2009

[28] E A Ottesen ldquoThe Wellcome Trust Lecture Infection anddisease in lymphatic filariasis an immunological perspectiverdquoParasitology vol 104 pp S71ndashS79 1992

[29] D O Freedman T B Nutman and E A Ottesen ldquoProtectiveimmunity in bancroftian filariasis Selective recognition of a43-kD larval stage antigen by infection-free individuals in anendemic areardquo Journal of Clinical Investigation vol 83 no 1 pp14ndash22 1989

[30] S Lustigman E R James W Tawe and D Abraham ldquoTowardsa recombinant antigen vaccine against Onchocerca volvulusrdquoTrends in Parasitology vol 18 no 3 pp 135ndash141 2002

[31] A J Macdonald W Tawe O Leon et al ldquoOv-ASP-1 theOnchocerca volvulus homologue of the activation associatedsecreted protein family is immunostimulatory and can induceprotective anti-larval immunityrdquo Parasite Immunology vol 26no 1 pp 53ndash62 2004

[32] S Ramachandran M P Kumar R M V Rami et al ldquoThe larvalspecific lymphatic filarial ALT-2 induction of protection usingprotein or DNA vaccinationrdquo Microbiology and Immunologyvol 48 no 12 pp 945ndash955 2004


[33] A Horauf and B Fleischer ldquoImmune responses to filarial infec-tion in laboratorymicerdquoMedicalMicrobiology and Immunologyvol 185 no 4 pp 207ndash215 1997

[34] E A Ottesen P F Weller and L Heck ldquoSpecific cellularimmune unresponsiveness in human filariasisrdquo Immunologyvol 33 no 3 pp 413ndash421 1977

[35] J L Grogan P G Kremsner A M Deelder and M Yaz-danbakhsh ldquoElevated proliferation and interleukin-4 releasefrom CD4+ cells after chemotherapy in human Schistosomahaematobium infectionrdquo European Journal of Immunology vol26 no 6 pp 1365ndash1370 1996

[36] R M Maizels A Balic N Gomez-Escobar M Nair MD Taylor and J E Allen ldquoHelminth parasitesmdashmasters ofregulationrdquo Immunological Reviews vol 201 pp 89ndash116 2004

[37] J M Behnke C J Barnard and D Wakelin ldquoUnderstandingchronic nematode infections evolutionary considerations cur-rent hypotheses and the way forwardrdquo International Journal forParasitology vol 22 no 7 pp 861ndash907 1992

[38] R M Maizels and M Yazdanbakhsh ldquoImmune regulation byhelminth parasites cellular andmolecularmechanismsrdquoNatureReviews Immunology vol 3 no 9 pp 733ndash744 2003

[39] Y Belkaid ldquoRegulatory T cells and infection a dangerousnecessityrdquo Nature Reviews Immunology vol 7 no 11 pp 875ndash888 2007

[40] M D Taylor N van der Werf A Harris et al ldquoEarly recruit-ment of natural CD4+Foxp3+ Treg cells by infective larvaedetermines the outcome of filarial infectionrdquo European Journalof Immunology vol 39 no 1 pp 192ndash206 2009

[41] M D Taylor L LeGoff A Harris E Malone J E Allen andR M Maizels ldquoRemoval of regulatory T cell activity reverseshyporesponsiveness and leads to filarial parasite clearance invivordquo Journal of Immunology vol 174 no 8 pp 4924ndash49332005

[42] L J Wammes F Hamid A E Wiria et al ldquoRegulatory T cellsin human lymphatic filariasis stronger functional activity inmicrofilaremicsrdquo PLoS Neglected Tropical Diseases vol 6 no 5Article ID e1655 2012

[43] V Chenthamarakshan K Cheirmaraj M V R Reddy and B CHarinath ldquoImmunoprophylactic studies with a 43 kDa humancirculating filarial antigen and a cross reactive 120 kDa Brugiamalayi sodium dodecyl sulphate soluble antigen in filariasisrdquoJournal of Biosciences vol 22 no 1 pp 91ndash98 1997

[44] C Martin M Saeftel P N Vuong et al ldquoB-cell deficiencysuppresses vaccine-induced protection against murine filariasisbut does not increase the recovery rate for primary infectionrdquoInfection and Immunity vol 69 no 11 pp 7067ndash7073 2001

[45] N Weiss and M Tanner ldquoStudies on Dipetalonema viteae(Filarioidea) 3 Antibody-dependent cell-mediated destructionof microfiliariae in vivordquo Tropenmedizin und Parasitologie vol30 no 1 pp 73ndash80 1979

[46] R Chandrashekar U R Rao P B Parab and D Subrah-manyam ldquoBrugia malayi serum dependent cell-mediated reac-tions to microfilariaerdquo Southeast Asian Journal of TropicalMedicine and Public Health vol 16 no 1 pp 15ndash21 1985

[47] R Chandrashekar U R Rao and D Subrahmanyam ldquoSerumdependent cell-mediated immune reactions to Brugia pahangiinfective larvaerdquo Parasite Immunology vol 7 no 6 pp 633ndash6411985

[48] B K L Sim B H Kwa and J W Mak ldquoImmune responsesin human Brugia malayi infections serum dependent cell-mediated destruction of infective larvae in vitrordquo Transactions

of the Royal Society of Tropical Medicine and Hygiene vol 76no 3 pp 362ndash370 1982

[49] T Attout C Martin S A Babayan et al ldquoPleural cellularreaction to the filarial infection Litomosoides sigmodontis isdetermined by the moulting process the worm alteration andthe host strainrdquoParasitology International vol 57 no 2 pp 201ndash211 2008

[50] D C Lloyd R J Purrott E J Reeder A A Edwards andG W Dolphin ldquoChromosome aberrations induced in humanlymphocytes by radiation from 252Cfrdquo International Journal ofRadiation Biology vol 34 no 2 pp 177ndash186 1978

[51] N M Almond and R M E Parkhouse ldquoImmunoglobulinclass specific responses to biochemically defined antigens ofTrichinella spiralisrdquo Parasite Immunology vol 8 no 4 pp 391ndash406 1986

[52] J-M Grzych D Grezel J-L Neyrinck et al ldquoIgA antibodies toa protective antigen in human Schistosomiasis mansonirdquo Journalof Immunology vol 150 no 2 pp 527ndash535 1993

[53] H M Khalil M H Abd el Baki M M Abd el MawlaK M Maklad S A Sharaf and A E Saad ldquoInterleukin-4immunoglobulin E and immunoglobulin A and resistance tore-infection with Schistosoma haematobium before and afterchemotherapyrdquo Journal of the Egyptian Society of Parasitologyvol 29 no 2 pp 395ndash408 1999

[54] B R Sahu M C Mohanty P K Sahoo A K Satapathy and BRavindran ldquoProtective immunity in human filariasis a role forparasite-specific IgA responsesrdquo Journal of Infectious Diseasesvol 198 no 3 pp 434ndash443 2008

[55] B Rajan T Ramalingam and T V Rajan ldquoCritical role for IgMin host protection in experimental filarial infectionrdquo Journal ofImmunology vol 175 no 3 pp 1827ndash1833 2005

[56] H L Callahan R K Crouch and E R James ldquoHelminthanti-oxidant enzymes a protective mechanism against hostoxidantsrdquo Parasitology Today vol 4 no 8 pp 218ndash225 1988

[57] PM Brophy andD I Pritchard ldquoImmunity to helminths readyto tip the biochemical balancerdquo Parasitology Today vol 8 no12 pp 419ndash422 1992

[58] S Babu L D Shultz T R Klei and T V Rajan ldquoImmunity inexperimental murine filariasis roles of T and B cells revisitedrdquoInfection and Immunity vol 67 no 6 pp 3166ndash3167 1999

[59] A L Vincent A C Vickery A Winters and W A SodemanJr ldquoLife cycle of Brugia pahangi (Nematoda) in nude miceC3HHeN (nunu)rdquo Journal of Parasitology vol 68 no 4 pp553ndash560 1982

[60] R R Suswillo D G Owen and D A Denham ldquoInfections ofBrugia pahangi in conventional and nude (athymic) micerdquoActaTropica vol 37 no 4 pp 327ndash335 1980

[61] R A OConnor J S Jenson J Osborne and E Devaney ldquoAnenduring association Microfilariae and immunosupression inlymphatic filariasisrdquo Trends in Parasitology vol 19 no 12 pp565ndash570 2003

[62] W Ellmeier S Sawada and D R Littman ldquoThe regulationof CD4 and CD8 coreceptor gene expression during T celldevelopmentrdquo Annual Review of Immunology vol 17 pp 523ndash554 1999

[63] A Dhur P Galan P Preziosi and S Hercberg ldquoLymphocytesubpopulations in the thymus lymph nodes and spleen of iron-deficient and rehabilitated micerdquo Journal of Nutrition vol 121no 9 pp 1418ndash1424 1991

[64] S Babu C P Blauvelt V Kumaraswami and T B NutmanldquoRegulatory networks induced by live parasites impair bothTh1


andTh2pathways in patent lymphatic filariasis implications forparasite persistencerdquo Journal of Immunology vol 176 no 5 pp3248ndash3256 2006

[65] S Babu and T B Nutman ldquoProinflammatory cytokines domi-nate the early immune response to filarial parasitesrdquo Journal ofImmunology vol 171 no 12 pp 6723ndash6732 2003

[66] S Babu L M Ganley T R Klei L D Shultz and T V RajanldquoRole of gamma interferon and interleukin-4 in host defenseagainst the human filarial parasite Brugia malayirdquo Infection andImmunity vol 68 no 5 pp 3034ndash3035 2000

[67] T V Rajan P Porte J A Yates L Keeper and L D Shultz ldquoRoleof nitric oxide in host defense against an extracellular metazoanparasite Brugia malayirdquo Infection and Immunity vol 64 no 8pp 3351ndash3353 1996

[68] S Specht L Volkmann TWynn and A Hoerauf ldquoInterleukin-10 (IL-10) counterregulates IL-4-dependent effector mecha-nisms in murine filariasisrdquo Infection and Immunity vol 72 no11 pp 6287ndash6293 2004

[69] T A Wynn A Reynolds S James et al ldquoIL-12 enhancesvaccine-induced immunity to schistosomes by augmentingboth humoral and cell-mediated immune responses against theparasiterdquo Journal of Immunology vol 157 no 9 pp 4068ndash40781996

[70] A J Bancroft D Artis D D Donaldson J P Sypek andR K Grencis ldquoGastrointestinal nematode expulsion in IL-4 knockout mice is IL-13 dependentrdquo European Journal ofImmunology vol 30 no 7 pp 2083ndash2091 2000

[71] L Le Goff T J Lamb A L Graham Y Harcus and J E AllenldquoIL-4 is required to prevent filarial nematode developmentin resistant but not susceptible strains of micerdquo InternationalJournal for Parasitology vol 32 no 10 pp 1277ndash1284 2002

[72] S Specht M D Taylor M A Hoeve J E Allen R Lang and AHoerauf ldquoOver expression of IL-10 by macrophages overcomesresistance to murine filariasisrdquo Experimental Parasitology vol132 no 1 pp 90ndash96 2012

[73] L H Elson M H Calvopina W Y Paredes et al ldquoImmunityto onchocerciasis putative immune persons produce aTh1-likeresponse to Onchocerca volvulusrdquo Journal of Infectious Diseasesvol 171 no 3 pp 652ndash658 1995

[74] K A Dimock M L Eberhard and P J Lammie ldquoTh1-likeantifilarial immune responses predominate in antigen-negativepersonsrdquo Infection and Immunity vol 64 no 8 pp 2962ndash29671996

[75] R M Maizels E Sartono A Kurniawan F Partono ME Selkirk and M Yazdanbakhsh ldquoT-cell activation and thebalance of antibody isotypes in human lymphatic filariasisrdquoParasitology Today vol 11 no 2 pp 50ndash56 1995

[76] E Sartono Y C M Kruize A Kurniawan et al ldquoElevatedcellular immune responses and interferon-120574 release after long-term diethylcarbamazine treatment of patients with humanlymphatic filariasisrdquo Journal of Infectious Diseases vol 171 no6 pp 1683ndash1687 1995

[77] M J Taylor H F Cross A AMohammed A J Trees and A EBianco ldquoSusceptibility of Brugia malayi andOnchocerca lienalismicrofilariae to nitric oxide and hydrogen peroxide in cell-freeculture and from IFN120574-activated macrophagesrdquo Parasitologyvol 112 part 3 pp 315ndash322 1996

[78] G R Thomas M McCrossan and M E Selkirk ldquoCytostaticand cytotoxic effects of activated macrophages and nitric oxidedonors on Brugia malayirdquo Infection and Immunity vol 65 no7 pp 2732ndash2739 1997

[79] A W Pfaff H Schulz-Key P T Soboslay S M Geiger and WH Hoffmann ldquoThe role of nitric oxide in the innate resistanceto microfilariae of Litomosoides sigmodontis in micerdquo ParasiteImmunology vol 22 no 8 pp 397ndash405 2000

[80] A El Bouhdidi C Truyens M-T Rivera H Bazin andY Carlier ldquoTrypanosoma cruzi infection in mice induces apolyisotypic hypergammaglobulinaemia and parasite-specificresponse involving high IgG2a concentrations and highly avidIgG1 antibodiesrdquo Parasite Immunology vol 16 no 2 pp 69ndash761994

[81] B Pulendran J L Smith G Caspary et al ldquoDistinct dendriticcell subsets differentially regulate the class of immune responsein vivordquo Proceedings of the National Academy of Sciences of theUnited States of America vol 96 no 3 pp 1036ndash1041 1999


[7] All experimentally characterised iPGMs from eubacteriaplants and invertebrates are monomers with a molecularmass of 55ndash75 kDa [8 9]The absence of iPGM from humansand being indispensable in all nematodes including thefilariids [10] advocate its potential as anthelminthic drugtarget Bm-iPGM was purified successfully using bacterialhostE coli Circular dichroism (CD) andfluorescence spectraof the recombinant protein were obtained to determine itssecondary structure and native conformation The abundantsharing of Bm-iPGM by all the major life-forms of B malayiand its release in the form of excretory-secretory products[11] pointed towards its immunogenic nature In silico analysisof Bm-iPGM predicted it to be highly antigenic with thepresence of both MHC I and MHC II binding peptidesThe antigenic nature was further validated by the cross-reactivity of Bm-iPGM with human bancroftian sera ofdifferent categories of LF which persuaded us to examine theimmunoprophylactic efficacy of the recombinant protein inanimal models of LF

In vivo immune characterisation of Bm-iPGM in BALBcmice revealed it to invoke a mixed type of Th1Th2 immuneresponse The immunised animals (BALBc and Mastomys)that were challenged with the infective larvae displayedreduced worm establishment As active filarial infectionsare accompanied with downregulation of the host immunesystem skewing the helper immune response of host toTh2 type we propose that immunization with Bm-iPGM iscapable of generating a mixed Th1Th2 type response that isunfavourable for parasite establishment and was responsiblefor providing considerable protection against LF in mousemodels thus validating Bm-iPGM to be possible vaccinecandidate against LF

2 Materials and Methods

21 Experimental Animals Randomly bred 6ndash8-week-oldmale BALBc (35) and Mastomys coucha (36) were usedin the experiment The animals were maintained in properhousing condition at Laboratory Animal Facility at CSIR-Central Drug Research Institute (CDRI) Lucknow IndiaAnimalswere fed on standard pellet diet andwater ad libitumThe animals and the animal experimental procedures wereapproved by the Animal Ethics Committee of CDRI dulyconstituted under the provisions of CPCSEA (Committeefor the Purpose of Control and Supervision on Experimentson Animals) Government of India The study bears theIAEC number 8309ParaIAEC dated 270409 All theexperiments were performed in duplicate (both forMastomysandBALBc) and almost similar results were obtained in boththe experiments and therefore pooled

22 Parasites Infective larvae (L3) of B malayi were recov-ered from the laboratory bred vector mosquitoes (Aedesaegypti) fed on donor Mastomys 9 plusmn 1 day back [12] L3were isolated from gently crushed mosquitoes by Baermanntechnique washed and counted in Ringerrsquos solution AdultB malayi worms and microfilariae (Mf) were collected from

the peritoneal cavities of the infected jirds on day 80ndash85 afterL3 inoculation

23 Homology Modelling of Bm-iPGM and Amino AcidSequences The homology model for Bm-iPGM was gener-ated using Phyre server [13] Bm-iPGM structure was gener-ated with 100 precision and 41 identity using structure ofBacillus anthracis cofactor-independent 2 phosphoglyceratemutase as template (PDB id c2ifyA length 508AA)Thedatagenerated was analysed by The PyMOL Molecular GraphicsSystem Version 13 Schrodinger LLC and the cartoonstructure was generated Amino acid sequence of Bm-iPGMwas also aligned with iPGM from B anthracis (accessionnumber 2IFY A) using Clustal W programme

24 In Silico Antigenicity Prediction The antigenicity ofBm-iPGM was determined by Kolaskar and Tongaonkarmethod [14] This semiempirical method predicts antigenicdeterminants based on the physicochemical properties ofamino acid residues and the frequencies of their occurrencein experimentally known segmental epitopes Prediction ofimmunodominant T cell antigenic sites from the primarysequence of Bm-iPGM was determined by ProPred-I andProPred MHC class-II binding peptide prediction serverswhich are online web tools for the prediction of peptidebinding to MHC class-I (HLA-A1 HLA-A2 HLA-A0201HLA-A0205 HLA-A1101 HLA-A3101 HLA-A3302 HLA-B2102 HLA-A3501 HLA-A4403 and HLA-5101) and class II(HLA-DRB1 0101 HLA-DRB1 0301 HLA-DRB1 0401 HLA-DRB1 0701 andHLA-DRB1 0801) alleles [15 16]The highestranking MHC I and MHC II binding peptides were high-lighted in the cartoon structure of Bm-iPGMobtained earlier

25 Cloning Expression and Purification of Bm-iPGMExpression and purification of Bm-iPGM was done asdescribed elsewhere with minor modifications [17] Brieflygene specific forward (51015840AGTCGGATCCATGGCCGA-AGCAAAGAATCG-31015840) and reverse (51015840ATGCCTCGA-GGGCTTCATTACCAATGGC31015840) primers having restric-tion sites for the enzymes BamHI (F) and XhoI (R) weresynthesised Amplification of gene was carried out using1 120583M of each primer 200120583M of each dNTP (FermentasUSA) 05 unit taq DNA polymerase (Invitrogen USA)1xPCR buffer and 15 120583M of MgCl

2with the following

reaction conditions 95∘C for 2min followed by 29 cyclesof 95∘C for 1min 58∘C for 1min 72∘C for 2min and 1cycle at 72∘C for 10min The 1548 bp amplified gene wascloned into pTZ57RT (2886 bp) vector as permanufacturerrsquosinstructions (Fermentas USA) Plasmid DNA was isolatedand the insert was verified by sequencing The 1548 bp genewas subcloned into pET28a expression vector for proteinexpression and purificationThe conditions like temperatureisopropyl 120573-D-thiogalactopyranoside (IPTG) concentrationand duration of induction were standardized for optimalexpression of the protein in soluble form Five mL cultures(E coli strain BL21 (DE3)) were grown at 37∘C in an incubatorshaker at 220 rpm and induced (at OD

600of 05-06) for

4 h with 02 05 and 10mM IPTG After induction cells







2in air The









2O2)


















4 Results













lowast





lowast lowast

lowastlowast

lowastlowast

lowastlowast


lowast

lowast




lowast







-

- - - - -

-

- - - - - - -

-

---Ba-iPGMBm-iPGM

Ba-iPGMBm-iPGM

Ba-iPGMBm-iPGM

Ba-iPGMBm-iPGM

Ba-iPGMBm-iPGM

Ba-iPGMBm-iPGM

Ba-iPGMBm-iPGM

Ba-iPGMBm-iPGM

Ba-iPGMBm-iPGM

5759

117119

177179

235239

295294

355354

415414

475474

509515

(a)

Linkers


(b)

Linkers


(c)







Table1Ad

ultp

arasite

recovery

andfemalew

orm

fecund

ityfro

mcontroland

Bm-iP

GM

immun

ized

Mastomys

Animalgrou

psNum

bero

fanimals

Adultp

arasite

coun

tsanim

alAd

ultw

orm

recovery

(meanplusmnSE)

redu

ctionin

worm

burden

femalep

arasite

sterilization

Day

30pc

Day

180pc

Day

30pc

Day

180pc

Day

30pc

Day

180pc

Day

180pc

PBS

66

302732281824

C242515142120

D9788952650plusmn2022750plusmn195

00

00

1850

Adjuvant

66

182226272331

C221918122115

D131011811102450plusmn1832833plusmn215

754

minus301

2051

Bm-iP

GM

66

88105912

C696745



6729

6545

6997

Statisticallysig

nificantvaluesw

ereo

btainedin

Bm-iP

GM

immun

isedgrou

psas

comparedto

thec

ontro

lgroup


aluesrepresented

arem

eanplusmnSE


(kDa)116

662

45

35

25

184

144

Bm-iPGM

L1 L2 L3 L4 L5 L7L6

(a)

L1 L2

(kDa)

98

64

50

(b)

100

90

80

70

60

50

40

30

20

10

0199600 320978 442356 563734 685112 806490

2074210

23396203090429

IPGM 61799571230

Inte

nsity

()

Mass (mz)(c)

10

0

minus10

minus20

minus30

minus40

200 210 220 230 240 250

Wavelength (nm)

[120579](times10

3de

gmiddotcm

2middotd

mol

minus1 )

(d)

300 320 340 360 380 400100

150

200

250

300

350

400

Fluo

resc

ence

(au

)

Wavelength (nm)

(e)







250

130

95

72

55

36

28

17

M Mf L3 Ad(kDa)

(a)

MMF L3 Ad (Da)

3000

2000

1500

1200

1000

900800700600

400

500

300

200

100

(b)

P1 P2 M250

130

95

72

55

36

28

17

11

(kDa)

(c)









M L2 L3 L4 L5 L6

1160

662

450

350

250

(kDa)

(a)

00

05

10

15

20


OD

at4

92

nm

(b)


0

01

02

03

04

05

06

07

08

09

50 100

200

400

800

1600

3200

6400

12800

51200

102400


OD

at4

92

nm

(a)

ControlAdjuvant

4

3

2

1

0

OD

at4

92

nm

P lt 0001 P lt 0001 P lt 0001

P lt 0001

P lt 0001P lt 0001



Bm-iPGM

(b)





0

400

100

200

300

Cou

nts

100 101 102 103 104

Control

Adjuvant

Bm-iPGM

FL1-HDCFDA

(a)

ROS generation500

400

300

200

100

Animal groups

Fluo

resc

ence

inte

nsity

P lt 001

0


P gt 005

(b)

30

20

10

0

Animal groups

P lt 0001

P lt 0001


P gt 005

CD4+ cells

Gat

ed C

D4+

cells

()

(c)

P lt 001P lt 001

30

40

20

10

0


P gt 005

CD8+ cells

Gat

ed C

D8+

cells

()

(d)

15

10

5

0


P lt 005P gt 005

CD19+ cells

Gat

ed C

D19+

cells

()

(e)







8

6

4

2

0Con A

Sim

ulat

ion

inde

x (S

I)


P lt 0001

P lt 0001


(a)

8

6

4

2

0Con A

Sim

ulat

ion

inde

x (S

I)


P lt 001P lt 005


(b)


5 Discussion







(a)

(b)

(c)

(d)

Animal groups

P lt 001P lt 001

3

2

1

0

P gt 005

Con

trol

Adju

vant

Bm-iP

GM

CD4+IL-4+

Gat

ed C

D4+

IL-4+

cells

()

P lt 001P lt 001

4

3

2

1

0

P gt 005

Animal groups

Con

trol

Adju

vant

Bm-iP

GM

CD4+IL-10+

Gat

ed C

D4+

IL-10+

cells

()

IL-2

PE

Isotype control

100

101

102

103

104

100 101 102 103 104

006

Control

100 101 102 103 104

084

Adjuvant

100 101 102 103 104

101

Bm-iPGM

100 101 102 103 104

222

100 101 102 103 104

108

100 101 102 103 104

114

100 101 102 103 104

398

100

101

102

103

104

100 101 102 103 104

IL-4

PE

014

100 101 102 103 104

104

100 101 102 103 104

102

100 101 102 103 104

22

100

101

102

103

104

100 101 102 103 104

CD4 FITC

IL-10

PE

018

100 101 102 103 104

CD4 FITC

132

100 101 102 103 104

CD4 FITC

151

100 101 102 103 104

CD4 FITC

274

100

101

104

100 101 102 103 104

016016

102

103

IFN

-120574PE

Gat

ed C

D4+

IL-2+

cells

()

Animal groups

3

2

1

0

P lt 0001P lt 001

P lt 005

Con

trol

Adju

vant

Bm-iP

GM

CD4+IL-2+

Animal groups

P lt 001P lt 0058

6

4

2

0

P gt 005

Con

trol

Adju

vant

Bm-iP

GM

CD4+

IFN

-120574+

cells

() CD4+IFN-120574




Worm recoveryRe

cove

red

wor

ms

30

20

10

0


P lt 0001

P lt 0001

P gt 005

(a)

0

50

100

150

200

250

300

350

400

450

500

90 120 150 180

Days (pc)



lowastlowast

Mf i

n10

120583L

bloo

d(b)







ADCCMf75

50

25

0Control Immunized

In v

itro

cyto

toxi

city

()

ADCCL375

50

25

0Control Immunized

In v

itro

cyto

toxi

city

()

(a)

(b) (c)















Acknowledgments


References





























































































2in air The









2O2)


















4 Results













lowast





lowast lowast

lowastlowast

lowastlowast

lowastlowast


lowast

lowast




lowast







-

- - - - -

-

- - - - - - -

-

---Ba-iPGMBm-iPGM

Ba-iPGMBm-iPGM

Ba-iPGMBm-iPGM

Ba-iPGMBm-iPGM

Ba-iPGMBm-iPGM

Ba-iPGMBm-iPGM

Ba-iPGMBm-iPGM

Ba-iPGMBm-iPGM

Ba-iPGMBm-iPGM

5759

117119

177179

235239

295294

355354

415414

475474

509515

(a)

Linkers


(b)

Linkers


(c)







Table1Ad

ultp

arasite

recovery

andfemalew

orm

fecund

ityfro

mcontroland

Bm-iP

GM

immun

ized

Mastomys

Animalgrou

psNum

bero

fanimals

Adultp

arasite

coun

tsanim

alAd

ultw

orm

recovery

(meanplusmnSE)

redu

ctionin

worm

burden

femalep

arasite

sterilization

Day

30pc

Day

180pc

Day

30pc

Day

180pc

Day

30pc

Day

180pc

Day

180pc

PBS

66

302732281824

C242515142120

D9788952650plusmn2022750plusmn195

00

00

1850

Adjuvant

66

182226272331

C221918122115

D131011811102450plusmn1832833plusmn215

754

minus301

2051

Bm-iP

GM

66

88105912

C696745



6729

6545

6997

Statisticallysig

nificantvaluesw

ereo

btainedin

Bm-iP

GM

immun

isedgrou

psas

comparedto

thec

ontro

lgroup


aluesrepresented

arem

eanplusmnSE


(kDa)116

662

45

35

25

184

144

Bm-iPGM

L1 L2 L3 L4 L5 L7L6

(a)

L1 L2

(kDa)

98

64

50

(b)

100

90

80

70

60

50

40

30

20

10

0199600 320978 442356 563734 685112 806490

2074210

23396203090429

IPGM 61799571230

Inte

nsity

()

Mass (mz)(c)

10

0

minus10

minus20

minus30

minus40

200 210 220 230 240 250

Wavelength (nm)

[120579](times10

3de

gmiddotcm

2middotd

mol

minus1 )

(d)

300 320 340 360 380 400100

150

200

250

300

350

400

Fluo

resc

ence

(au

)

Wavelength (nm)

(e)







250

130

95

72

55

36

28

17

M Mf L3 Ad(kDa)

(a)

MMF L3 Ad (Da)

3000

2000

1500

1200

1000

900800700600

400

500

300

200

100

(b)

P1 P2 M250

130

95

72

55

36

28

17

11

(kDa)

(c)









M L2 L3 L4 L5 L6

1160

662

450

350

250

(kDa)

(a)

00

05

10

15

20


OD

at4

92

nm

(b)


0

01

02

03

04

05

06

07

08

09

50 100

200

400

800

1600

3200

6400

12800

51200

102400


OD

at4

92

nm

(a)

ControlAdjuvant

4

3

2

1

0

OD

at4

92

nm

P lt 0001 P lt 0001 P lt 0001

P lt 0001

P lt 0001P lt 0001



Bm-iPGM

(b)





0

400

100

200

300

Cou

nts

100 101 102 103 104

Control

Adjuvant

Bm-iPGM

FL1-HDCFDA

(a)

ROS generation500

400

300

200

100

Animal groups

Fluo

resc

ence

inte

nsity

P lt 001

0


P gt 005

(b)

30

20

10

0

Animal groups

P lt 0001

P lt 0001


P gt 005

CD4+ cells

Gat

ed C

D4+

cells

()

(c)

P lt 001P lt 001

30

40

20

10

0


P gt 005

CD8+ cells

Gat

ed C

D8+

cells

()

(d)

15

10

5

0


P lt 005P gt 005

CD19+ cells

Gat

ed C

D19+

cells

()

(e)







8

6

4

2

0Con A

Sim

ulat

ion

inde

x (S

I)


P lt 0001

P lt 0001


(a)

8

6

4

2

0Con A

Sim

ulat

ion

inde

x (S

I)


P lt 001P lt 005


(b)


5 Discussion







(a)

(b)

(c)

(d)

Animal groups

P lt 001P lt 001

3

2

1

0

P gt 005

Con

trol

Adju

vant

Bm-iP

GM

CD4+IL-4+

Gat

ed C

D4+

IL-4+

cells

()

P lt 001P lt 001

4

3

2

1

0

P gt 005

Animal groups

Con

trol

Adju

vant

Bm-iP

GM

CD4+IL-10+

Gat

ed C

D4+

IL-10+

cells

()

IL-2

PE

Isotype control

100

101

102

103

104

100 101 102 103 104

006

Control

100 101 102 103 104

084

Adjuvant

100 101 102 103 104

101

Bm-iPGM

100 101 102 103 104

222

100 101 102 103 104

108

100 101 102 103 104

114

100 101 102 103 104

398

100

101

102

103

104

100 101 102 103 104

IL-4

PE

014

100 101 102 103 104

104

100 101 102 103 104

102

100 101 102 103 104

22

100

101

102

103

104

100 101 102 103 104

CD4 FITC

IL-10

PE

018

100 101 102 103 104

CD4 FITC

132

100 101 102 103 104

CD4 FITC

151

100 101 102 103 104

CD4 FITC

274

100

101

104

100 101 102 103 104

016016

102

103

IFN

-120574PE

Gat

ed C

D4+

IL-2+

cells

()

Animal groups

3

2

1

0

P lt 0001P lt 001

P lt 005

Con

trol

Adju

vant

Bm-iP

GM

CD4+IL-2+

Animal groups

P lt 001P lt 0058

6

4

2

0

P gt 005

Con

trol

Adju

vant

Bm-iP

GM

CD4+

IFN

-120574+

cells

() CD4+IFN-120574




Worm recoveryRe

cove

red

wor

ms

30

20

10

0


P lt 0001

P lt 0001

P gt 005

(a)

0

50

100

150

200

250

300

350

400

450

500

90 120 150 180

Days (pc)



lowastlowast

Mf i

n10

120583L

bloo

d(b)







ADCCMf75

50

25

0Control Immunized

In v

itro

cyto

toxi

city

()

ADCCL375

50

25

0Control Immunized

In v

itro

cyto

toxi

city

()

(a)

(b) (c)















Acknowledgments


References





























































































2O2)


















4 Results













lowast





lowast lowast

lowastlowast

lowastlowast

lowastlowast


lowast

lowast




lowast







-

- - - - -

-

- - - - - - -

-

---Ba-iPGMBm-iPGM

Ba-iPGMBm-iPGM

Ba-iPGMBm-iPGM

Ba-iPGMBm-iPGM

Ba-iPGMBm-iPGM

Ba-iPGMBm-iPGM

Ba-iPGMBm-iPGM

Ba-iPGMBm-iPGM

Ba-iPGMBm-iPGM

5759

117119

177179

235239

295294

355354

415414

475474

509515

(a)

Linkers


(b)

Linkers


(c)







Table1Ad

ultp

arasite

recovery

andfemalew

orm

fecund

ityfro

mcontroland

Bm-iP

GM

immun

ized

Mastomys

Animalgrou

psNum

bero

fanimals

Adultp

arasite

coun

tsanim

alAd

ultw

orm

recovery

(meanplusmnSE)

redu

ctionin

worm

burden

femalep

arasite

sterilization

Day

30pc

Day

180pc

Day

30pc

Day

180pc

Day

30pc

Day

180pc

Day

180pc

PBS

66

302732281824

C242515142120

D9788952650plusmn2022750plusmn195

00

00

1850

Adjuvant

66

182226272331

C221918122115

D131011811102450plusmn1832833plusmn215

754

minus301

2051

Bm-iP

GM

66

88105912

C696745



6729

6545

6997

Statisticallysig

nificantvaluesw

ereo

btainedin

Bm-iP

GM

immun

isedgrou

psas

comparedto

thec

ontro

lgroup


aluesrepresented

arem

eanplusmnSE


(kDa)116

662

45

35

25

184

144

Bm-iPGM

L1 L2 L3 L4 L5 L7L6

(a)

L1 L2

(kDa)

98

64

50

(b)

100

90

80

70

60

50

40

30

20

10

0199600 320978 442356 563734 685112 806490

2074210

23396203090429

IPGM 61799571230

Inte

nsity

()

Mass (mz)(c)

10

0

minus10

minus20

minus30

minus40

200 210 220 230 240 250

Wavelength (nm)

[120579](times10

3de

gmiddotcm

2middotd

mol

minus1 )

(d)

300 320 340 360 380 400100

150

200

250

300

350

400

Fluo

resc

ence

(au

)

Wavelength (nm)

(e)







250

130

95

72

55

36

28

17

M Mf L3 Ad(kDa)

(a)

MMF L3 Ad (Da)

3000

2000

1500

1200

1000

900800700600

400

500

300

200

100

(b)

P1 P2 M250

130

95

72

55

36

28

17

11

(kDa)

(c)









M L2 L3 L4 L5 L6

1160

662

450

350

250

(kDa)

(a)

00

05

10

15

20


OD

at4

92

nm

(b)


0

01

02

03

04

05

06

07

08

09

50 100

200

400

800

1600

3200

6400

12800

51200

102400


OD

at4

92

nm

(a)

ControlAdjuvant

4

3

2

1

0

OD

at4

92

nm

P lt 0001 P lt 0001 P lt 0001

P lt 0001

P lt 0001P lt 0001



Bm-iPGM

(b)





0

400

100

200

300

Cou

nts

100 101 102 103 104

Control

Adjuvant

Bm-iPGM

FL1-HDCFDA

(a)

ROS generation500

400

300

200

100

Animal groups

Fluo

resc

ence

inte

nsity

P lt 001

0


P gt 005

(b)

30

20

10

0

Animal groups

P lt 0001

P lt 0001


P gt 005

CD4+ cells

Gat

ed C

D4+

cells

()

(c)

P lt 001P lt 001

30

40

20

10

0


P gt 005

CD8+ cells

Gat

ed C

D8+

cells

()

(d)

15

10

5

0


P lt 005P gt 005

CD19+ cells

Gat

ed C

D19+

cells

()

(e)







8

6

4

2

0Con A

Sim

ulat

ion

inde

x (S

I)


P lt 0001

P lt 0001


(a)

8

6

4

2

0Con A

Sim

ulat

ion

inde

x (S

I)


P lt 001P lt 005


(b)


5 Discussion







(a)

(b)

(c)

(d)

Animal groups

P lt 001P lt 001

3

2

1

0

P gt 005

Con

trol

Adju

vant

Bm-iP

GM

CD4+IL-4+

Gat

ed C

D4+

IL-4+

cells

()

P lt 001P lt 001

4

3

2

1

0

P gt 005

Animal groups

Con

trol

Adju

vant

Bm-iP

GM

CD4+IL-10+

Gat

ed C

D4+

IL-10+

cells

()

IL-2

PE

Isotype control

100

101

102

103

104

100 101 102 103 104

006

Control

100 101 102 103 104

084

Adjuvant

100 101 102 103 104

101

Bm-iPGM

100 101 102 103 104

222

100 101 102 103 104

108

100 101 102 103 104

114

100 101 102 103 104

398

100

101

102

103

104

100 101 102 103 104

IL-4

PE

014

100 101 102 103 104

104

100 101 102 103 104

102

100 101 102 103 104

22

100

101

102

103

104

100 101 102 103 104

CD4 FITC

IL-10

PE

018

100 101 102 103 104

CD4 FITC

132

100 101 102 103 104

CD4 FITC

151

100 101 102 103 104

CD4 FITC

274

100

101

104

100 101 102 103 104

016016

102

103

IFN

-120574PE

Gat

ed C

D4+

IL-2+

cells

()

Animal groups

3

2

1

0

P lt 0001P lt 001

P lt 005

Con

trol

Adju

vant

Bm-iP

GM

CD4+IL-2+

Animal groups

P lt 001P lt 0058

6

4

2

0

P gt 005

Con

trol

Adju

vant

Bm-iP

GM

CD4+

IFN

-120574+

cells

() CD4+IFN-120574




Worm recoveryRe

cove

red

wor

ms

30

20

10

0


P lt 0001

P lt 0001

P gt 005

(a)

0

50

100

150

200

250

300

350

400

450

500

90 120 150 180

Days (pc)



lowastlowast

Mf i

n10

120583L

bloo

d(b)







ADCCMf75

50

25

0Control Immunized

In v

itro

cyto

toxi

city

()

ADCCL375

50

25

0Control Immunized

In v

itro

cyto

toxi

city

()

(a)

(b) (c)















Acknowledgments


References

































































































4 Results













lowast





lowast lowast

lowastlowast

lowastlowast

lowastlowast


lowast

lowast




lowast







-

- - - - -

-

- - - - - - -

-

---Ba-iPGMBm-iPGM

Ba-iPGMBm-iPGM

Ba-iPGMBm-iPGM

Ba-iPGMBm-iPGM

Ba-iPGMBm-iPGM

Ba-iPGMBm-iPGM

Ba-iPGMBm-iPGM

Ba-iPGMBm-iPGM

Ba-iPGMBm-iPGM

5759

117119

177179

235239

295294

355354

415414

475474

509515

(a)

Linkers


(b)

Linkers


(c)







Table1Ad

ultp

arasite

recovery

andfemalew

orm

fecund

ityfro

mcontroland

Bm-iP

GM

immun

ized

Mastomys

Animalgrou

psNum

bero

fanimals

Adultp

arasite

coun

tsanim

alAd

ultw

orm

recovery

(meanplusmnSE)

redu

ctionin

worm

burden

femalep

arasite

sterilization

Day

30pc

Day

180pc

Day

30pc

Day

180pc

Day

30pc

Day

180pc

Day

180pc

PBS

66

302732281824

C242515142120

D9788952650plusmn2022750plusmn195

00

00

1850

Adjuvant

66

182226272331

C221918122115

D131011811102450plusmn1832833plusmn215

754

minus301

2051

Bm-iP

GM

66

88105912

C696745



6729

6545

6997

Statisticallysig

nificantvaluesw

ereo

btainedin

Bm-iP

GM

immun

isedgrou

psas

comparedto

thec

ontro

lgroup


aluesrepresented

arem

eanplusmnSE


(kDa)116

662

45

35

25

184

144

Bm-iPGM

L1 L2 L3 L4 L5 L7L6

(a)

L1 L2

(kDa)

98

64

50

(b)

100

90

80

70

60

50

40

30

20

10

0199600 320978 442356 563734 685112 806490

2074210

23396203090429

IPGM 61799571230

Inte

nsity

()

Mass (mz)(c)

10

0

minus10

minus20

minus30

minus40

200 210 220 230 240 250

Wavelength (nm)

[120579](times10

3de

gmiddotcm

2middotd

mol

minus1 )

(d)

300 320 340 360 380 400100

150

200

250

300

350

400

Fluo

resc

ence

(au

)

Wavelength (nm)

(e)







250

130

95

72

55

36

28

17

M Mf L3 Ad(kDa)

(a)

MMF L3 Ad (Da)

3000

2000

1500

1200

1000

900800700600

400

500

300

200

100

(b)

P1 P2 M250

130

95

72

55

36

28

17

11

(kDa)

(c)









M L2 L3 L4 L5 L6

1160

662

450

350

250

(kDa)

(a)

00

05

10

15

20


OD

at4

92

nm

(b)


0

01

02

03

04

05

06

07

08

09

50 100

200

400

800

1600

3200

6400

12800

51200

102400


OD

at4

92

nm

(a)

ControlAdjuvant

4

3

2

1

0

OD

at4

92

nm

P lt 0001 P lt 0001 P lt 0001

P lt 0001

P lt 0001P lt 0001



Bm-iPGM

(b)





0

400

100

200

300

Cou

nts

100 101 102 103 104

Control

Adjuvant

Bm-iPGM

FL1-HDCFDA

(a)

ROS generation500

400

300

200

100

Animal groups

Fluo

resc

ence

inte

nsity

P lt 001

0


P gt 005

(b)

30

20

10

0

Animal groups

P lt 0001

P lt 0001


P gt 005

CD4+ cells

Gat

ed C

D4+

cells

()

(c)

P lt 001P lt 001

30

40

20

10

0


P gt 005

CD8+ cells

Gat

ed C

D8+

cells

()

(d)

15

10

5

0


P lt 005P gt 005

CD19+ cells

Gat

ed C

D19+

cells

()

(e)







8

6

4

2

0Con A

Sim

ulat

ion

inde

x (S

I)


P lt 0001

P lt 0001


(a)

8

6

4

2

0Con A

Sim

ulat

ion

inde

x (S

I)


P lt 001P lt 005


(b)


5 Discussion







(a)

(b)

(c)

(d)

Animal groups

P lt 001P lt 001

3

2

1

0

P gt 005

Con

trol

Adju

vant

Bm-iP

GM

CD4+IL-4+

Gat

ed C

D4+

IL-4+

cells

()

P lt 001P lt 001

4

3

2

1

0

P gt 005

Animal groups

Con

trol

Adju

vant

Bm-iP

GM

CD4+IL-10+

Gat

ed C

D4+

IL-10+

cells

()

IL-2

PE

Isotype control

100

101

102

103

104

100 101 102 103 104

006

Control

100 101 102 103 104

084

Adjuvant

100 101 102 103 104

101

Bm-iPGM

100 101 102 103 104

222

100 101 102 103 104

108

100 101 102 103 104

114

100 101 102 103 104

398

100

101

102

103

104

100 101 102 103 104

IL-4

PE

014

100 101 102 103 104

104

100 101 102 103 104

102

100 101 102 103 104

22

100

101

102

103

104

100 101 102 103 104

CD4 FITC

IL-10

PE

018

100 101 102 103 104

CD4 FITC

132

100 101 102 103 104

CD4 FITC

151

100 101 102 103 104

CD4 FITC

274

100

101

104

100 101 102 103 104

016016

102

103

IFN

-120574PE

Gat

ed C

D4+

IL-2+

cells

()

Animal groups

3

2

1

0

P lt 0001P lt 001

P lt 005

Con

trol

Adju

vant

Bm-iP

GM

CD4+IL-2+

Animal groups

P lt 001P lt 0058

6

4

2

0

P gt 005

Con

trol

Adju

vant

Bm-iP

GM

CD4+

IFN

-120574+

cells

() CD4+IFN-120574




Worm recoveryRe

cove

red

wor

ms

30

20

10

0


P lt 0001

P lt 0001

P gt 005

(a)

0

50

100

150

200

250

300

350

400

450

500

90 120 150 180

Days (pc)



lowastlowast

Mf i

n10

120583L

bloo

d(b)







ADCCMf75

50

25

0Control Immunized

In v

itro

cyto

toxi

city

()

ADCCL375

50

25

0Control Immunized

In v

itro

cyto

toxi

city

()

(a)

(b) (c)















Acknowledgments


References


































































































lowast





lowast lowast

lowastlowast

lowastlowast

lowastlowast


lowast

lowast




lowast







-

- - - - -

-

- - - - - - -

-

---Ba-iPGMBm-iPGM

Ba-iPGMBm-iPGM

Ba-iPGMBm-iPGM

Ba-iPGMBm-iPGM

Ba-iPGMBm-iPGM

Ba-iPGMBm-iPGM

Ba-iPGMBm-iPGM

Ba-iPGMBm-iPGM

Ba-iPGMBm-iPGM

5759

117119

177179

235239

295294

355354

415414

475474

509515

(a)

Linkers


(b)

Linkers


(c)







Table1Ad

ultp

arasite

recovery

andfemalew

orm

fecund

ityfro

mcontroland

Bm-iP

GM

immun

ized

Mastomys

Animalgrou

psNum

bero

fanimals

Adultp

arasite

coun

tsanim

alAd

ultw

orm

recovery

(meanplusmnSE)

redu

ctionin

worm

burden

femalep

arasite

sterilization

Day

30pc

Day

180pc

Day

30pc

Day

180pc

Day

30pc

Day

180pc

Day

180pc

PBS

66

302732281824

C242515142120

D9788952650plusmn2022750plusmn195

00

00

1850

Adjuvant

66

182226272331

C221918122115

D131011811102450plusmn1832833plusmn215

754

minus301

2051

Bm-iP

GM

66

88105912

C696745



6729

6545

6997

Statisticallysig

nificantvaluesw

ereo

btainedin

Bm-iP

GM

immun

isedgrou

psas

comparedto

thec

ontro

lgroup


aluesrepresented

arem

eanplusmnSE


(kDa)116

662

45

35

25

184

144

Bm-iPGM

L1 L2 L3 L4 L5 L7L6

(a)

L1 L2

(kDa)

98

64

50

(b)

100

90

80

70

60

50

40

30

20

10

0199600 320978 442356 563734 685112 806490

2074210

23396203090429

IPGM 61799571230

Inte

nsity

()

Mass (mz)(c)

10

0

minus10

minus20

minus30

minus40

200 210 220 230 240 250

Wavelength (nm)

[120579](times10

3de

gmiddotcm

2middotd

mol

minus1 )

(d)

300 320 340 360 380 400100

150

200

250

300

350

400

Fluo

resc

ence

(au

)

Wavelength (nm)

(e)







250

130

95

72

55

36

28

17

M Mf L3 Ad(kDa)

(a)

MMF L3 Ad (Da)

3000

2000

1500

1200

1000

900800700600

400

500

300

200

100

(b)

P1 P2 M250

130

95

72

55

36

28

17

11

(kDa)

(c)









M L2 L3 L4 L5 L6

1160

662

450

350

250

(kDa)

(a)

00

05

10

15

20


OD

at4

92

nm

(b)


0

01

02

03

04

05

06

07

08

09

50 100

200

400

800

1600

3200

6400

12800

51200

102400


OD

at4

92

nm

(a)

ControlAdjuvant

4

3

2

1

0

OD

at4

92

nm

P lt 0001 P lt 0001 P lt 0001

P lt 0001

P lt 0001P lt 0001



Bm-iPGM

(b)





0

400

100

200

300

Cou

nts

100 101 102 103 104

Control

Adjuvant

Bm-iPGM

FL1-HDCFDA

(a)

ROS generation500

400

300

200

100

Animal groups

Fluo

resc

ence

inte

nsity

P lt 001

0


P gt 005

(b)

30

20

10

0

Animal groups

P lt 0001

P lt 0001


P gt 005

CD4+ cells

Gat

ed C

D4+

cells

()

(c)

P lt 001P lt 001

30

40

20

10

0


P gt 005

CD8+ cells

Gat

ed C

D8+

cells

()

(d)

15

10

5

0


P lt 005P gt 005

CD19+ cells

Gat

ed C

D19+

cells

()

(e)







8

6

4

2

0Con A

Sim

ulat

ion

inde

x (S

I)


P lt 0001

P lt 0001


(a)

8

6

4

2

0Con A

Sim

ulat

ion

inde

x (S

I)


P lt 001P lt 005


(b)


5 Discussion







(a)

(b)

(c)

(d)

Animal groups

P lt 001P lt 001

3

2

1

0

P gt 005

Con

trol

Adju

vant

Bm-iP

GM

CD4+IL-4+

Gat

ed C

D4+

IL-4+

cells

()

P lt 001P lt 001

4

3

2

1

0

P gt 005

Animal groups

Con

trol

Adju

vant

Bm-iP

GM

CD4+IL-10+

Gat

ed C

D4+

IL-10+

cells

()

IL-2

PE

Isotype control

100

101

102

103

104

100 101 102 103 104

006

Control

100 101 102 103 104

084

Adjuvant

100 101 102 103 104

101

Bm-iPGM

100 101 102 103 104

222

100 101 102 103 104

108

100 101 102 103 104

114

100 101 102 103 104

398

100

101

102

103

104

100 101 102 103 104

IL-4

PE

014

100 101 102 103 104

104

100 101 102 103 104

102

100 101 102 103 104

22

100

101

102

103

104

100 101 102 103 104

CD4 FITC

IL-10

PE

018

100 101 102 103 104

CD4 FITC

132

100 101 102 103 104

CD4 FITC

151

100 101 102 103 104

CD4 FITC

274

100

101

104

100 101 102 103 104

016016

102

103

IFN

-120574PE

Gat

ed C

D4+

IL-2+

cells

()

Animal groups

3

2

1

0

P lt 0001P lt 001

P lt 005

Con

trol

Adju

vant

Bm-iP

GM

CD4+IL-2+

Animal groups

P lt 001P lt 0058

6

4

2

0

P gt 005

Con

trol

Adju

vant

Bm-iP

GM

CD4+

IFN

-120574+

cells

() CD4+IFN-120574




Worm recoveryRe

cove

red

wor

ms

30

20

10

0


P lt 0001

P lt 0001

P gt 005

(a)

0

50

100

150

200

250

300

350

400

450

500

90 120 150 180

Days (pc)



lowastlowast

Mf i

n10

120583L

bloo

d(b)







ADCCMf75

50

25

0Control Immunized

In v

itro

cyto

toxi

city

()

ADCCL375

50

25

0Control Immunized

In v

itro

cyto

toxi

city

()

(a)

(b) (c)















Acknowledgments


References
























































































Table1Ad

ultp

arasite

recovery

andfemalew

orm

fecund

ityfro

mcontroland

Bm-iP

GM

immun

ized

Mastomys

Animalgrou

psNum

bero

fanimals

Adultp

arasite

coun

tsanim

alAd

ultw

orm

recovery

(meanplusmnSE)

redu

ctionin

worm

burden

femalep

arasite

sterilization

Day

30pc

Day

180pc

Day

30pc

Day

180pc

Day

30pc

Day

180pc

Day

180pc

PBS

66

302732281824

C242515142120

D9788952650plusmn2022750plusmn195

00

00

1850

Adjuvant

66

182226272331

C221918122115

D131011811102450plusmn1832833plusmn215

754

minus301

2051

Bm-iP

GM

66

88105912

C696745



6729

6545

6997

Statisticallysig

nificantvaluesw

ereo

btainedin

Bm-iP

GM

immun

isedgrou

psas

comparedto

thec

ontro

lgroup


aluesrepresented

arem

eanplusmnSE


(kDa)116

662

45

35

25

184

144

Bm-iPGM

L1 L2 L3 L4 L5 L7L6

(a)

L1 L2

(kDa)

98

64

50

(b)

100

90

80

70

60

50

40

30

20

10

0199600 320978 442356 563734 685112 806490

2074210

23396203090429

IPGM 61799571230

Inte

nsity

()

Mass (mz)(c)

10

0

minus10

minus20

minus30

minus40

200 210 220 230 240 250

Wavelength (nm)

[120579](times10

3de

gmiddotcm

2middotd

mol

minus1 )

(d)

300 320 340 360 380 400100

150

200

250

300

350

400

Fluo

resc

ence

(au

)

Wavelength (nm)

(e)







250

130

95

72

55

36

28

17

M Mf L3 Ad(kDa)

(a)

MMF L3 Ad (Da)

3000

2000

1500

1200

1000

900800700600

400

500

300

200

100

(b)

P1 P2 M250

130

95

72

55

36

28

17

11

(kDa)

(c)









M L2 L3 L4 L5 L6

1160

662

450

350

250

(kDa)

(a)

00

05

10

15

20


OD

at4

92

nm

(b)


0

01

02

03

04

05

06

07

08

09

50 100

200

400

800

1600

3200

6400

12800

51200

102400


OD

at4

92

nm

(a)

ControlAdjuvant

4

3

2

1

0

OD

at4

92

nm

P lt 0001 P lt 0001 P lt 0001

P lt 0001

P lt 0001P lt 0001



Bm-iPGM

(b)





0

400

100

200

300

Cou

nts

100 101 102 103 104

Control

Adjuvant

Bm-iPGM

FL1-HDCFDA

(a)

ROS generation500

400

300

200

100

Animal groups

Fluo

resc

ence

inte

nsity

P lt 001

0


P gt 005

(b)

30

20

10

0

Animal groups

P lt 0001

P lt 0001


P gt 005

CD4+ cells

Gat

ed C

D4+

cells

()

(c)

P lt 001P lt 001

30

40

20

10

0


P gt 005

CD8+ cells

Gat

ed C

D8+

cells

()

(d)

15

10

5

0


P lt 005P gt 005

CD19+ cells

Gat

ed C

D19+

cells

()

(e)







8

6

4

2

0Con A

Sim

ulat

ion

inde

x (S

I)


P lt 0001

P lt 0001


(a)

8

6

4

2

0Con A

Sim

ulat

ion

inde

x (S

I)


P lt 001P lt 005


(b)


5 Discussion







(a)

(b)

(c)

(d)

Animal groups

P lt 001P lt 001

3

2

1

0

P gt 005

Con

trol

Adju

vant

Bm-iP

GM

CD4+IL-4+

Gat

ed C

D4+

IL-4+

cells

()

P lt 001P lt 001

4

3

2

1

0

P gt 005

Animal groups

Con

trol

Adju

vant

Bm-iP

GM

CD4+IL-10+

Gat

ed C

D4+

IL-10+

cells

()

IL-2

PE

Isotype control

100

101

102

103

104

100 101 102 103 104

006

Control

100 101 102 103 104

084

Adjuvant

100 101 102 103 104

101

Bm-iPGM

100 101 102 103 104

222

100 101 102 103 104

108

100 101 102 103 104

114

100 101 102 103 104

398

100

101

102

103

104

100 101 102 103 104

IL-4

PE

014

100 101 102 103 104

104

100 101 102 103 104

102

100 101 102 103 104

22

100

101

102

103

104

100 101 102 103 104

CD4 FITC

IL-10

PE

018

100 101 102 103 104

CD4 FITC

132

100 101 102 103 104

CD4 FITC

151

100 101 102 103 104

CD4 FITC

274

100

101

104

100 101 102 103 104

016016

102

103

IFN

-120574PE

Gat

ed C

D4+

IL-2+

cells

()

Animal groups

3

2

1

0

P lt 0001P lt 001

P lt 005

Con

trol

Adju

vant

Bm-iP

GM

CD4+IL-2+

Animal groups

P lt 001P lt 0058

6

4

2

0

P gt 005

Con

trol

Adju

vant

Bm-iP

GM

CD4+

IFN

-120574+

cells

() CD4+IFN-120574




Worm recoveryRe

cove

red

wor

ms

30

20

10

0


P lt 0001

P lt 0001

P gt 005

(a)

0

50

100

150

200

250

300

350

400

450

500

90 120 150 180

Days (pc)



lowastlowast

Mf i

n10

120583L

bloo

d(b)







ADCCMf75

50

25

0Control Immunized

In v

itro

cyto

toxi

city

()

ADCCL375

50

25

0Control Immunized

In v

itro

cyto

toxi

city

()

(a)

(b) (c)















Acknowledgments


References
























































































(kDa)116

662

45

35

25

184

144

Bm-iPGM

L1 L2 L3 L4 L5 L7L6

(a)

L1 L2

(kDa)

98

64

50

(b)

100

90

80

70

60

50

40

30

20

10

0199600 320978 442356 563734 685112 806490

2074210

23396203090429

IPGM 61799571230

Inte

nsity

()

Mass (mz)(c)

10

0

minus10

minus20

minus30

minus40

200 210 220 230 240 250

Wavelength (nm)

[120579](times10

3de

gmiddotcm

2middotd

mol

minus1 )

(d)

300 320 340 360 380 400100

150

200

250

300

350

400

Fluo

resc

ence

(au

)

Wavelength (nm)

(e)







250

130

95

72

55

36

28

17

M Mf L3 Ad(kDa)

(a)

MMF L3 Ad (Da)

3000

2000

1500

1200

1000

900800700600

400

500

300

200

100

(b)

P1 P2 M250

130

95

72

55

36

28

17

11

(kDa)

(c)









M L2 L3 L4 L5 L6

1160

662

450

350

250

(kDa)

(a)

00

05

10

15

20


OD

at4

92

nm

(b)


0

01

02

03

04

05

06

07

08

09

50 100

200

400

800

1600

3200

6400

12800

51200

102400


OD

at4

92

nm

(a)

ControlAdjuvant

4

3

2

1

0

OD

at4

92

nm

P lt 0001 P lt 0001 P lt 0001

P lt 0001

P lt 0001P lt 0001



Bm-iPGM

(b)





0

400

100

200

300

Cou

nts

100 101 102 103 104

Control

Adjuvant

Bm-iPGM

FL1-HDCFDA

(a)

ROS generation500

400

300

200

100

Animal groups

Fluo

resc

ence

inte

nsity

P lt 001

0


P gt 005

(b)

30

20

10

0

Animal groups

P lt 0001

P lt 0001


P gt 005

CD4+ cells

Gat

ed C

D4+

cells

()

(c)

P lt 001P lt 001

30

40

20

10

0


P gt 005

CD8+ cells

Gat

ed C

D8+

cells

()

(d)

15

10

5

0


P lt 005P gt 005

CD19+ cells

Gat

ed C

D19+

cells

()

(e)







8

6

4

2

0Con A

Sim

ulat

ion

inde

x (S

I)


P lt 0001

P lt 0001


(a)

8

6

4

2

0Con A

Sim

ulat

ion

inde

x (S

I)


P lt 001P lt 005


(b)


5 Discussion







(a)

(b)

(c)

(d)

Animal groups

P lt 001P lt 001

3

2

1

0

P gt 005

Con

trol

Adju

vant

Bm-iP

GM

CD4+IL-4+

Gat

ed C

D4+

IL-4+

cells

()

P lt 001P lt 001

4

3

2

1

0

P gt 005

Animal groups

Con

trol

Adju

vant

Bm-iP

GM

CD4+IL-10+

Gat

ed C

D4+

IL-10+

cells

()

IL-2

PE

Isotype control

100

101

102

103

104

100 101 102 103 104

006

Control

100 101 102 103 104

084

Adjuvant

100 101 102 103 104

101

Bm-iPGM

100 101 102 103 104

222

100 101 102 103 104

108

100 101 102 103 104

114

100 101 102 103 104

398

100

101

102

103

104

100 101 102 103 104

IL-4

PE

014

100 101 102 103 104

104

100 101 102 103 104

102

100 101 102 103 104

22

100

101

102

103

104

100 101 102 103 104

CD4 FITC

IL-10

PE

018

100 101 102 103 104

CD4 FITC

132

100 101 102 103 104

CD4 FITC

151

100 101 102 103 104

CD4 FITC

274

100

101

104

100 101 102 103 104

016016

102

103

IFN

-120574PE

Gat

ed C

D4+

IL-2+

cells

()

Animal groups

3

2

1

0

P lt 0001P lt 001

P lt 005

Con

trol

Adju

vant

Bm-iP

GM

CD4+IL-2+

Animal groups

P lt 001P lt 0058

6

4

2

0

P gt 005

Con

trol

Adju

vant

Bm-iP

GM

CD4+

IFN

-120574+

cells

() CD4+IFN-120574




Worm recoveryRe

cove

red

wor

ms

30

20

10

0


P lt 0001

P lt 0001

P gt 005

(a)

0

50

100

150

200

250

300

350

400

450

500

90 120 150 180

Days (pc)



lowastlowast

Mf i

n10

120583L

bloo

d(b)







ADCCMf75

50

25

0Control Immunized

In v

itro

cyto

toxi

city

()

ADCCL375

50

25

0Control Immunized

In v

itro

cyto

toxi

city

()

(a)

(b) (c)















Acknowledgments


References
























































































250

130

95

72

55

36

28

17

M Mf L3 Ad(kDa)

(a)

MMF L3 Ad (Da)

3000

2000

1500

1200

1000

900800700600

400

500

300

200

100

(b)

P1 P2 M250

130

95

72

55

36

28

17

11

(kDa)

(c)









M L2 L3 L4 L5 L6

1160

662

450

350

250

(kDa)

(a)

00

05

10

15

20


OD

at4

92

nm

(b)


0

01

02

03

04

05

06

07

08

09

50 100

200

400

800

1600

3200

6400

12800

51200

102400


OD

at4

92

nm

(a)

ControlAdjuvant

4

3

2

1

0

OD

at4

92

nm

P lt 0001 P lt 0001 P lt 0001

P lt 0001

P lt 0001P lt 0001



Bm-iPGM

(b)





0

400

100

200

300

Cou

nts

100 101 102 103 104

Control

Adjuvant

Bm-iPGM

FL1-HDCFDA

(a)

ROS generation500

400

300

200

100

Animal groups

Fluo

resc

ence

inte

nsity

P lt 001

0


P gt 005

(b)

30

20

10

0

Animal groups

P lt 0001

P lt 0001


P gt 005

CD4+ cells

Gat

ed C

D4+

cells

()

(c)

P lt 001P lt 001

30

40

20

10

0


P gt 005

CD8+ cells

Gat

ed C

D8+

cells

()

(d)

15

10

5

0


P lt 005P gt 005

CD19+ cells

Gat

ed C

D19+

cells

()

(e)







8

6

4

2

0Con A

Sim

ulat

ion

inde

x (S

I)


P lt 0001

P lt 0001


(a)

8

6

4

2

0Con A

Sim

ulat

ion

inde

x (S

I)


P lt 001P lt 005


(b)


5 Discussion







(a)

(b)

(c)

(d)

Animal groups

P lt 001P lt 001

3

2

1

0

P gt 005

Con

trol

Adju

vant

Bm-iP

GM

CD4+IL-4+

Gat

ed C

D4+

IL-4+

cells

()

P lt 001P lt 001

4

3

2

1

0

P gt 005

Animal groups

Con

trol

Adju

vant

Bm-iP

GM

CD4+IL-10+

Gat

ed C

D4+

IL-10+

cells

()

IL-2

PE

Isotype control

100

101

102

103

104

100 101 102 103 104

006

Control

100 101 102 103 104

084

Adjuvant

100 101 102 103 104

101

Bm-iPGM

100 101 102 103 104

222

100 101 102 103 104

108

100 101 102 103 104

114

100 101 102 103 104

398

100

101

102

103

104

100 101 102 103 104

IL-4

PE

014

100 101 102 103 104

104

100 101 102 103 104

102

100 101 102 103 104

22

100

101

102

103

104

100 101 102 103 104

CD4 FITC

IL-10

PE

018

100 101 102 103 104

CD4 FITC

132

100 101 102 103 104

CD4 FITC

151

100 101 102 103 104

CD4 FITC

274

100

101

104

100 101 102 103 104

016016

102

103

IFN

-120574PE

Gat

ed C

D4+

IL-2+

cells

()

Animal groups

3

2

1

0

P lt 0001P lt 001

P lt 005

Con

trol

Adju

vant

Bm-iP

GM

CD4+IL-2+

Animal groups

P lt 001P lt 0058

6

4

2

0

P gt 005

Con

trol

Adju

vant

Bm-iP

GM

CD4+

IFN

-120574+

cells

() CD4+IFN-120574




Worm recoveryRe

cove

red

wor

ms

30

20

10

0


P lt 0001

P lt 0001

P gt 005

(a)

0

50

100

150

200

250

300

350

400

450

500

90 120 150 180

Days (pc)



lowastlowast

Mf i

n10

120583L

bloo

d(b)







ADCCMf75

50

25

0Control Immunized

In v

itro

cyto

toxi

city

()

ADCCL375

50

25

0Control Immunized

In v

itro

cyto

toxi

city

()

(a)

(b) (c)















Acknowledgments


References
























































































M L2 L3 L4 L5 L6

1160

662

450

350

250

(kDa)

(a)

00

05

10

15

20


OD

at4

92

nm

(b)


0

01

02

03

04

05

06

07

08

09

50 100

200

400

800

1600

3200

6400

12800

51200

102400


OD

at4

92

nm

(a)

ControlAdjuvant

4

3

2

1

0

OD

at4

92

nm

P lt 0001 P lt 0001 P lt 0001

P lt 0001

P lt 0001P lt 0001



Bm-iPGM

(b)





0

400

100

200

300

Cou

nts

100 101 102 103 104

Control

Adjuvant

Bm-iPGM

FL1-HDCFDA

(a)

ROS generation500

400

300

200

100

Animal groups

Fluo

resc

ence

inte

nsity

P lt 001

0


P gt 005

(b)

30

20

10

0

Animal groups

P lt 0001

P lt 0001


P gt 005

CD4+ cells

Gat

ed C

D4+

cells

()

(c)

P lt 001P lt 001

30

40

20

10

0


P gt 005

CD8+ cells

Gat

ed C

D8+

cells

()

(d)

15

10

5

0


P lt 005P gt 005

CD19+ cells

Gat

ed C

D19+

cells

()

(e)







8

6

4

2

0Con A

Sim

ulat

ion

inde

x (S

I)


P lt 0001

P lt 0001


(a)

8

6

4

2

0Con A

Sim

ulat

ion

inde

x (S

I)


P lt 001P lt 005


(b)


5 Discussion







(a)

(b)

(c)

(d)

Animal groups

P lt 001P lt 001

3

2

1

0

P gt 005

Con

trol

Adju

vant

Bm-iP

GM

CD4+IL-4+

Gat

ed C

D4+

IL-4+

cells

()

P lt 001P lt 001

4

3

2

1

0

P gt 005

Animal groups

Con

trol

Adju

vant

Bm-iP

GM

CD4+IL-10+

Gat

ed C

D4+

IL-10+

cells

()

IL-2

PE

Isotype control

100

101

102

103

104

100 101 102 103 104

006

Control

100 101 102 103 104

084

Adjuvant

100 101 102 103 104

101

Bm-iPGM

100 101 102 103 104

222

100 101 102 103 104

108

100 101 102 103 104

114

100 101 102 103 104

398

100

101

102

103

104

100 101 102 103 104

IL-4

PE

014

100 101 102 103 104

104

100 101 102 103 104

102

100 101 102 103 104

22

100

101

102

103

104

100 101 102 103 104

CD4 FITC

IL-10

PE

018

100 101 102 103 104

CD4 FITC

132

100 101 102 103 104

CD4 FITC

151

100 101 102 103 104

CD4 FITC

274

100

101

104

100 101 102 103 104

016016

102

103

IFN

-120574PE

Gat

ed C

D4+

IL-2+

cells

()

Animal groups

3

2

1

0

P lt 0001P lt 001

P lt 005

Con

trol

Adju

vant

Bm-iP

GM

CD4+IL-2+

Animal groups

P lt 001P lt 0058

6

4

2

0

P gt 005

Con

trol

Adju

vant

Bm-iP

GM

CD4+

IFN

-120574+

cells

() CD4+IFN-120574




Worm recoveryRe

cove

red

wor

ms

30

20

10

0


P lt 0001

P lt 0001

P gt 005

(a)

0

50

100

150

200

250

300

350

400

450

500

90 120 150 180

Days (pc)



lowastlowast

Mf i

n10

120583L

bloo

d(b)







ADCCMf75

50

25

0Control Immunized

In v

itro

cyto

toxi

city

()

ADCCL375

50

25

0Control Immunized

In v

itro

cyto

toxi

city

()

(a)

(b) (c)















Acknowledgments


References
























































































0

400

100

200

300

Cou

nts

100 101 102 103 104

Control

Adjuvant

Bm-iPGM

FL1-HDCFDA

(a)

ROS generation500

400

300

200

100

Animal groups

Fluo

resc

ence

inte

nsity

P lt 001

0


P gt 005

(b)

30

20

10

0

Animal groups

P lt 0001

P lt 0001


P gt 005

CD4+ cells

Gat

ed C

D4+

cells

()

(c)

P lt 001P lt 001

30

40

20

10

0


P gt 005

CD8+ cells

Gat

ed C

D8+

cells

()

(d)

15

10

5

0


P lt 005P gt 005

CD19+ cells

Gat

ed C

D19+

cells

()

(e)







8

6

4

2

0Con A

Sim

ulat

ion

inde

x (S

I)


P lt 0001

P lt 0001


(a)

8

6

4

2

0Con A

Sim

ulat

ion

inde

x (S

I)


P lt 001P lt 005


(b)


5 Discussion







(a)

(b)

(c)

(d)

Animal groups

P lt 001P lt 001

3

2

1

0

P gt 005

Con

trol

Adju

vant

Bm-iP

GM

CD4+IL-4+

Gat

ed C

D4+

IL-4+

cells

()

P lt 001P lt 001

4

3

2

1

0

P gt 005

Animal groups

Con

trol

Adju

vant

Bm-iP

GM

CD4+IL-10+

Gat

ed C

D4+

IL-10+

cells

()

IL-2

PE

Isotype control

100

101

102

103

104

100 101 102 103 104

006

Control

100 101 102 103 104

084

Adjuvant

100 101 102 103 104

101

Bm-iPGM

100 101 102 103 104

222

100 101 102 103 104

108

100 101 102 103 104

114

100 101 102 103 104

398

100

101

102

103

104

100 101 102 103 104

IL-4

PE

014

100 101 102 103 104

104

100 101 102 103 104

102

100 101 102 103 104

22

100

101

102

103

104

100 101 102 103 104

CD4 FITC

IL-10

PE

018

100 101 102 103 104

CD4 FITC

132

100 101 102 103 104

CD4 FITC

151

100 101 102 103 104

CD4 FITC

274

100

101

104

100 101 102 103 104

016016

102

103

IFN

-120574PE

Gat

ed C

D4+

IL-2+

cells

()

Animal groups

3

2

1

0

P lt 0001P lt 001

P lt 005

Con

trol

Adju

vant

Bm-iP

GM

CD4+IL-2+

Animal groups

P lt 001P lt 0058

6

4

2

0

P gt 005

Con

trol

Adju

vant

Bm-iP

GM

CD4+

IFN

-120574+

cells

() CD4+IFN-120574




Worm recoveryRe

cove

red

wor

ms

30

20

10

0


P lt 0001

P lt 0001

P gt 005

(a)

0

50

100

150

200

250

300

350

400

450

500

90 120 150 180

Days (pc)



lowastlowast

Mf i

n10

120583L

bloo

d(b)







ADCCMf75

50

25

0Control Immunized

In v

itro

cyto

toxi

city

()

ADCCL375

50

25

0Control Immunized

In v

itro

cyto

toxi

city

()

(a)

(b) (c)















Acknowledgments


References
























































































8

6

4

2

0Con A

Sim

ulat

ion

inde

x (S

I)


P lt 0001

P lt 0001


(a)

8

6

4

2

0Con A

Sim

ulat

ion

inde

x (S

I)


P lt 001P lt 005


(b)


5 Discussion







(a)

(b)

(c)

(d)

Animal groups

P lt 001P lt 001

3

2

1

0

P gt 005

Con

trol

Adju

vant

Bm-iP

GM

CD4+IL-4+

Gat

ed C

D4+

IL-4+

cells

()

P lt 001P lt 001

4

3

2

1

0

P gt 005

Animal groups

Con

trol

Adju

vant

Bm-iP

GM

CD4+IL-10+

Gat

ed C

D4+

IL-10+

cells

()

IL-2

PE

Isotype control

100

101

102

103

104

100 101 102 103 104

006

Control

100 101 102 103 104

084

Adjuvant

100 101 102 103 104

101

Bm-iPGM

100 101 102 103 104

222

100 101 102 103 104

108

100 101 102 103 104

114

100 101 102 103 104

398

100

101

102

103

104

100 101 102 103 104

IL-4

PE

014

100 101 102 103 104

104

100 101 102 103 104

102

100 101 102 103 104

22

100

101

102

103

104

100 101 102 103 104

CD4 FITC

IL-10

PE

018

100 101 102 103 104

CD4 FITC

132

100 101 102 103 104

CD4 FITC

151

100 101 102 103 104

CD4 FITC

274

100

101

104

100 101 102 103 104

016016

102

103

IFN

-120574PE

Gat

ed C

D4+

IL-2+

cells

()

Animal groups

3

2

1

0

P lt 0001P lt 001

P lt 005

Con

trol

Adju

vant

Bm-iP

GM

CD4+IL-2+

Animal groups

P lt 001P lt 0058

6

4

2

0

P gt 005

Con

trol

Adju

vant

Bm-iP

GM

CD4+

IFN

-120574+

cells

() CD4+IFN-120574




Worm recoveryRe

cove

red

wor

ms

30

20

10

0


P lt 0001

P lt 0001

P gt 005

(a)

0

50

100

150

200

250

300

350

400

450

500

90 120 150 180

Days (pc)



lowastlowast

Mf i

n10

120583L

bloo

d(b)







ADCCMf75

50

25

0Control Immunized

In v

itro

cyto

toxi

city

()

ADCCL375

50

25

0Control Immunized

In v

itro

cyto

toxi

city

()

(a)

(b) (c)















Acknowledgments


References
























































































(a)

(b)

(c)

(d)

Animal groups

P lt 001P lt 001

3

2

1

0

P gt 005

Con

trol

Adju

vant

Bm-iP

GM

CD4+IL-4+

Gat

ed C

D4+

IL-4+

cells

()

P lt 001P lt 001

4

3

2

1

0

P gt 005

Animal groups

Con

trol

Adju

vant

Bm-iP

GM

CD4+IL-10+

Gat

ed C

D4+

IL-10+

cells

()

IL-2

PE

Isotype control

100

101

102

103

104

100 101 102 103 104

006

Control

100 101 102 103 104

084

Adjuvant

100 101 102 103 104

101

Bm-iPGM

100 101 102 103 104

222

100 101 102 103 104

108

100 101 102 103 104

114

100 101 102 103 104

398

100

101

102

103

104

100 101 102 103 104

IL-4

PE

014

100 101 102 103 104

104

100 101 102 103 104

102

100 101 102 103 104

22

100

101

102

103

104

100 101 102 103 104

CD4 FITC

IL-10

PE

018

100 101 102 103 104

CD4 FITC

132

100 101 102 103 104

CD4 FITC

151

100 101 102 103 104

CD4 FITC

274

100

101

104

100 101 102 103 104

016016

102

103

IFN

-120574PE

Gat

ed C

D4+

IL-2+

cells

()

Animal groups

3

2

1

0

P lt 0001P lt 001

P lt 005

Con

trol

Adju

vant

Bm-iP

GM

CD4+IL-2+

Animal groups

P lt 001P lt 0058

6

4

2

0

P gt 005

Con

trol

Adju

vant

Bm-iP

GM

CD4+

IFN

-120574+

cells

() CD4+IFN-120574




Worm recoveryRe

cove

red

wor

ms

30

20

10

0


P lt 0001

P lt 0001

P gt 005

(a)

0

50

100

150

200

250

300

350

400

450

500

90 120 150 180

Days (pc)



lowastlowast

Mf i

n10

120583L

bloo

d(b)







ADCCMf75

50

25

0Control Immunized

In v

itro

cyto

toxi

city

()

ADCCL375

50

25

0Control Immunized

In v

itro

cyto

toxi

city

()

(a)

(b) (c)















Acknowledgments


References
























































































Worm recoveryRe

cove

red

wor

ms

30

20

10

0


P lt 0001

P lt 0001

P gt 005

(a)

0

50

100

150

200

250

300

350

400

450

500

90 120 150 180

Days (pc)



lowastlowast

Mf i

n10

120583L

bloo

d(b)







ADCCMf75

50

25

0Control Immunized

In v

itro

cyto

toxi

city

()

ADCCL375

50

25

0Control Immunized

In v

itro

cyto

toxi

city

()

(a)

(b) (c)















Acknowledgments


References
























































































ADCCMf75

50

25

0Control Immunized

In v

itro

cyto

toxi

city

()

ADCCL375

50

25

0Control Immunized

In v

itro

cyto

toxi

city

()

(a)

(b) (c)















Acknowledgments


References
































































































Acknowledgments


References




















































































































































































































































Cofactor-independent phosphoglycerate mutase is an essential gene in procyclic form Trypanosoma brucei

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