Vaccine 26 (2008) 5997–6005

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Vaccination with 73 kDa recombinant heavy chain myosin generates high levelof protection against Brugia malayi challenge in jird and mastomys models

Satish Vedia, Anil Dangia, Krishnan Hajelab, Shailja Misra-Bhattacharyaa,∗

a Division of Parasitology, Central Drug Research Institute, Post Box 173, M.G. Marg, Chattar Manzil Palace, Lucknow (U.P.) 226001, Indiab School of Life Sciences, Devi Ahilya VishvaVidhayalaya, Indore (M.P.) 452001, India

a r t i c l e i n f o

Article history:Received 20 June 2008Received in revised form 19 August 2008Accepted 19 August 2008Available online 24 September 2008

Keywords:Recombinant myosinFilarial vaccinationBrugia malayiImmune response

a b s t r a c t

We have earlier reported identification, expression and purification of a 2.0 kb cDNA clone coding forBrugia malayi heavy chain myosin which exhibited strong immuno-reactivity with bancroftian sera fromendemic normal (EN) human subjects which are considered to be putatively immune. In the presentstudy, immunoprophylactic characterization of B. malayi recombinant myosin was carried out in rodentmodels and the protective efficacy was evaluated by assessing the microfilarial burden and adult wormcounts in vaccinated host after an infective larval challenge. Data indicates that immunization resultedin to a significant reduction in microfilarial burden (∼76%) and adult worm establishment (54–58%),accompanied with embryostatic effect (70–75%) in both the animal models. The findings suggest thatimmune-protection by recombinant myosin was conferred through both humoral and cellular arms ofimmunity as indicated by an increased antibody titer with predominance of IgG2a and IgG2b isotypesalong with elevated level of IgG1 apart from significant proliferation of lymphocytes, increased nitric oxide

production and profound adherence of splenocytes causing cytotoxicity to microfilariae and infectivelarvae. The present study indicates that the recombinant B. malayi myosin is a promising vaccine candidate

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. Introduction

Human lymphatic filariasis (LF) caused by Wuchereria bancrofti,rugia malayi and Brugia timori, is endemic in over 100 coun-ries and place more than 1.1 billion people (20% of the world’sopulation) at risk of infection [1]. Approximately, 130 millioneople living in the tropics and sub-tropics are infected withhese parasites, with 115 million infected with W. bancrofti and3 million with B. malayi [2]. India alone accounts for 40% of thelobal disease burden with approximately 420 million people resid-ng in endemic areas and 48.11 million infected [3]. W. bancrofticcounts for more than 90% of the total lymphatic filariasis casesorldwide and in India [3,4]. Forty-four million infected persons

re incapacitated and/or disfigured by LF, while another 76 mil-

ion have internal damage to their renal and lymphatic systems5], which makes this disease second leading cause of perma-ent and long-term disability in developing countries including

ndia [3,4,6]. Diethylcarbamazine (DEC), since its discovery, is

∗ Corresponding author. Tel.: +91 522 2612411–18x4224/4221;ax: +91 522 2623938/2623405.

E-mail addresses: Shailja cdri@rediffmail.com, shailjacdri@yahoo.comS. Misra-Bhattacharya).

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264-410X/$ – see front matter © 2008 Elsevier Ltd. All rights reserved.oi:10.1016/j.vaccine.2008.08.073

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he only drug of choice for the treatment of LF, although it isrincipally microfilaricidal [7,8]. World health organization has rec-mmended the use of albendazole in combination with DEC whichxerts partial macrofilaricidal efficacy [9,10]. Although diethylcar-amazine (DEC), ivermectin, and albendazole are the commonlysed drugs to treat LF, they have the inherent disadvantage ofequiring repeated and prolonged treatment for years leading tootential drug resistance [11]. Recently, with the emergence ofrug resistance against the mainstay drugs like ivermectin orlbendazole, the situation is becoming bleaker now [12–14]. Feweports have also demonstrated the re-occurrence of microfilar-ae even after 10–20 years of post-transmission blockage [15,16].

ass drug administration in disease endemic countries may havead significant impact in morbidity control [17], however, reducingransmission due to constant reinfections have not been adequatelyddressed. Thus, a long-term disease control strategy is neededhich possibly combines mass chemotherapy with a protective

accine.Several examples of successful helminth vaccines for live-stock

o exist [18] and currently, a vaccine against human hookworm dis-ase is progressing towards human trial [19]. The filarial parasitesave many developmental stages (mf-larvae-adult) in their life-ycle which poses a major setback in developing an effective vaccinei.e. a vaccine candidate effective against a specific stage may not

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e equally effective on other stage). Vaccination with irradiated3 in the cattle filarial infection of O. ochengi most closely resem-ling human onchocerciasis, have resulted to highly significantrotection during subsequent field exposure [20], demonstratingvidently that an epidemiologically relevant protection in the fieldan be achieved through a vaccine. Recent vaccination study withrradiated larvae also reports long-term protection against L3 [21],mplicating that surface antigen/s have important role in generationf protective immunity. Consequently, characterization of protec-ive response generated by surface antigen/s that can be used asaccine is worth considerable. A few body wall antigens have earlierenerated protection [22–24] and are potential vaccine candidatesgainst filarial infections. The crude or partially purified antigenserived mostly from animal filarial parasites have failed to offerignificant level of protection in the past [25], suggesting that puri-ed native protein or recombinant filarial proteins might be moreseful for achieving the desired results.

In the present study, we have evaluated the prophylactic effi-acy of purified recombinant B. malayi protein myosin that waselected based on high immune reactivity with bancroftian serarom human endemic normal subject [26]. The efficacy was eval-ated by employing the natural course of infection in two rodentodels (mastomys and jird) and higher degree of protection was

chieved.

. Materials and methods

.1. Expression and purification of B. malayi recombinant myosin

Expression and purification of recombinant myosin was donesing a recombinant construct of B. malayi recombinant myosin

n pET28b was maintained in DH5� E. coli cells (Qiagen) asescribed earlier [26]. In brief, the recombinant plasmid was trans-ormed into E. coli BL21 (DE3) cells (Qiagen). At an OD600 of 0.6,PTG (0.5 mM, Sigma, USA) was added and incubated for 3 h. Thenduced cells were harvested and disrupted in buffer A (50 mMaH2PO4, 300 mM NaCl, pH 6.5) containing imidazole (10 mM)

upplemented with 1.0 mM PMSF protease inhibitor. Total pro-eins from the supernatant were separated on SDS–PAGE 10% (w/v)els, and the presence of histidine-tagged protein was confirmedsing an anti-histidine mouse antibody (Qiagen). Subsequently,he supernatant containing the histidine-tagged recombinant pro-ein was subjected to affinity purification through Ni-NTA ColumnSepharose, Fast Flow with Ni4+ metal coupled, Qiagen) and allowedo bind for 30 min. After washing with buffer-A containing 10 mM,0 mM, 50 mM Imidazole, protein was eluted by buffer-A contain-

ng 250 mM Imidazole. Eluates were collected as 1 ml fractionshich were then analyzed on 10% SDS–PAGE. The fractions con-

aining purified protein were pooled and dialyzed over nightgainst 50 mM NaH2PO4 at 4 ◦C. Dialyzed protein was freezeried. The endotoxin level of the resulting protein preparationas <1 EU/mg, as determined by the E-toxate assay (Sigma, Poole,K).

.2. Experimental animals

Purpose-bred, parasite naive, 6-week-old male mastomys (Mas-omys coucha) and jirds (Meriones unguiculatus) were used for thexperiments (n = 6). Throughout the experimentation animals were

aintained in proper housing condition at animal house facility

t Central Drug Research Institute (CDRI), Lucknow, India. Animalsere fed on standard pellet diet and water ad libitum. All the ani-al experiments and procedures were duly approved by the animal

thical committee of the institute.

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2008) 5997–6005

.3. Parasites

Infective larvae (L3) of B. malayi were recovered from theaboratory bred vector mosquitoes (Aedes aegypti) fed on donor

astomys 9 ± 1 day back [27]. Larvae were isolated from gentlyrushed mosquitoes by Baermann technique, washed and countedn Ringer’s solution. All the animal groups (control and immunizedroups of mastomys and jirds) were challenged with 100 activeotile L3 by subcutaneous (s.c.) route in the back region post-

mmunization.

.4. Vaccination and challenge infection

Immunoprophylactic potential of the recombinant proteingainst B. malayi infective larvae challenge was determined inoth mastomys and jird models. Immunization was done asescribed earlier [26]. Briefly, animals were divided into fourroups: unimmunized control (cont gp), adjuvant alone group (adjp), recombinant myosin immunized group (rMyo gp) and B. malayidult antigen immunized group (BmAg gp). For priming immuniza-ion animals were subcutaneously injected with 25 �g of proteinmulsified with Freund’s complete adjuvant (FCA; Sigma, USA) onay 0, followed by two booster doses of equivalent quantity of pro-ein in Freund’s incomplete adjuvant (FIA) on 14th and 21st dayf first immunization. BmAg gp received 100 �g adult B. malayiomatic antigen/animal emulsified in FCA/FIA, s.c., at the sameime intervals. Age and weight matched adj gp received equiva-ent amounts of FCA/FIA alone in PBS (Phosphate buffer saline, pH.2) and normal control gp received PBS. One week following finalooster dose, all groups of mastomys and jird were challenged with00 larvae of B. malayi s.c.

.5. Estimation of microfilaraemia, worm recovery and fecundity

Twelve weeks post-challenge (p.c.), observation of microfila-aemia was initiated in control and immunized groups of mastomysnd continued fortnightly up to 20th week, by blood smear methods described earlier [28]. Briefly, 10 �l tail blood smear were pre-ared and stained with 2% Giemsa stain and microfilariae countedicroscopically (10×). Microfilariaemia observation could only be

arried out in mastomys due to experimental convenience. Mas-omys and jirds were euthanized 20th week p.c. for the recoveryf adult worms. Various tissues viz; heart, lungs, testes and lymphodes were teased in normal saline, adult worms were isolated andounted. Female worms were teased on glass slide in saline andbserved microscopically to assess the effect of immunization onorm fecundity. Parasite recoveries and counting were performedithout knowledge of the experimental status of each animal.rithmetic means were calculated for the total worm burdensnd the percentage protection was calculated as [(u − v)/u × 100],here u is the mean value for the control group and v is the mean

alue for the experimental group. Female worm sterilization wasalculated as [number of sterile worms/total worm teased × 100].

.6. Lymphocyte proliferation assay

The proliferation assay was performed post-vaccination asescribed earlier [29]. Spleen from immunized and control mas-omys that were aseptically recovered, crushed with sterile glassrusher in RPMI-1640 (Sigma, USA) and filtered with sterile nylon

ell strainer (40 �m pore size; BD Falcon, USA) to prepare singleell suspension. Splenocyte suspension (100 �l/well) from stock5 × 106 cells/ml) were plated in 96 well Nunc culture plate inriplicate and were stimulated with 100 �l rMyo (2.5 �g/ml; opti-

ized earlier) or 100 �l concanavalin A (2.5 �g/ml; Sigma, USA) for

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2 h and pulsed with 1.0 �Ci/well of [3H] thymidine (3H-Tdr, spe-ific activity 18Ci/m mole, BARC, India) for 18 h preceding harvest.he radioactive incorporation was measured by standard liquidcintillation counting (Beckman Instruments, Palo Alto, CA). Thetimulation index (SI) was calculated as mean cpm (counts perinute) values of stimulated culture/mean cpm values of unstim-

lated culture. An SI of ≥2 was considered significant.

.7. Macrophage function assay

Peritoneal exudate cells (PEC) from immunized and controlroup of mastomys were lavaged in sterile RPMI-1640 mediumontaining heparin (5 U/ml) to prepare a final concentration of× 106 cells/ml. PEC’s (100 �l/well) from stock were plated in 96ell Nunc culture plate in triplicate and were stimulated with

00 �l rMyo (2.5 �g/ml;) or 100 �l LPS (1.0 �g/ml; Sigma, USA) andncubated for 24 h (37 ◦C, 5% CO2). Nitric oxide (NO) productionas measured in cell supernatant using the Griess reagent and by

aking the optical density at 540 nm. A standard curve was plottedsing sodium nitrate to quantitate the test results and the concen-ration of NO expressed in �M NO/106 cells. Real-time monitoringf intracellular reactive oxygen species in PEC’s was determinedhrough a fluorometric assay using 2′,7′-dichlorofluorescin diac-tate (DCF-DA) as described earlier [30]. Briefly, freshly harvestedEC’s (immunized and control animals) at a concentration of× 106 cells/ml in PBS, were washed with PBS × 3 and distributed in

ACS tubes (1 × 106 cells/tube). For probe loading, PEC’s were incu-ated with the DCF-DA at a final concentration of 1 �M, for 15 mint 37 ◦C, 5% CO2 and then washed twice in PBS. The Reactive oxy-en species (ROS) level in individual living cells was determinedy sequentially measuring their fluorescence intensity (FI) on FACSalibur (Becton Dickinson, USA). Data was analyzed by CellQuestoftware (Becton Dickinson, USA) and mean ROS values were eval-ated for cell populations.

.8. Determination of rMyo specific IgG isotypes by ELISA

Immunized and control mastomys were bled for sera throughetro-orbital plexus prior to vaccination, 7 days post-final booster,5 days post L3 challenge and on final day of autopsy to mon-tor presence of antibodies as described earlier [26]. Briefly, 96

ell microtitre polystyrene plates (Nunc, USA) were coated with.0 �g/ml solution of rMyo diluted in carbonate buffer, pH 9.6100.0 �l/well) and kept overnight at 4 ◦C. At each step hereafter,lates were washed thrice with PBST (PBS containing 0 × 05%v/v) Tween-20). Uncoated surface were blocked with 5% (v/v)kim milk (in PBS) at 37 ◦C for 2 h. Sera were serially dilutedn the wells starting from 1:100 dilution in dilution buffer (1%kimmed milk and 0.01% Tween 20 in PBS) and incubated at7 ◦C for 1 h. Bound primary antibodies were detected with HRPhorseradish peroxidase)-conjugated goat anti-mouse IgG (Sigma,SA) at 1:5000 dilution in dilution buffer and substrate OPD (o-henyl diamine, Sigma, USA) and H2O2. The reaction was stoppedy adding 40 �l of 2.5 M H2SO4. The OD was determined at 492 nmInfinite Series M200, Tecan, Switzerland).

Antibody isotypes were determined by ELISA kit (Sigma, USA)s per manufacturer’s protocol. Briefly coating and blocking wereone as described above. Following blocking, sera diluted to 1:100

as reacted with coated antigen at 37 ◦C for 1 h. Primary antibodyas reacted with isotype specific antibodies (goat anti-mouse IgG1,

gG2a, IgG2b and IgG3) at 1:1000 dilution for 1 h at 37 ◦C in tripli-ate. Detection of bound isotype specific antibodies was done witheroxidase labeled rabbit anti-goat IgG (1:5000) and OPD substrate.he optical density (OD) was measured at 492 nm.

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2008) 5997–6005 5999

.9. In vitro antibody-dependant cellular adhesion andytotoxicity assay

Cellular adhesion to microfilariae was carried out as describedarlier [29]. Briefly 200 live mf isolated from infected jird by mem-rane filtration (5.0 �m pore size) were cultured with normalastomys splenocytes (1 × 106) in 96-well plate containing RPMI-

640 supplemented with sera of normal and immunized mastomys.ach well contained 100 �l splenocytes (1 × 106), 50 �l of serum1:8, 1:16, 1:32 and 1: 64 dilutions), 25 �l Guinea pig serum as aource of complement and 25 �l mf (200). Plates were kept at 37 ◦Cn a CO2 incubator (Binder, Germany). Cell-adherence on the sur-ace of mf affecting their motility was noted after 1, 3, 6 and 24 hf incubation. Percent microfilarial with adherence (5 or more cellsdhered/mf) as well as intensity of cells adhered to single mf wasssessed by microscopic examination of 100 mf in each well. Cellu-ar adherence was also observed with L3 (50/well) and adult wormsone male or female/well) as described above.

.10. Statistical analysis

Data were analyzed using Student’s t-test and one-way anal-sis of variance (ANOVA) as appropriate. Individual comparisonsollowing ANOVA were made using the Student–Newman–Keul

ethod with the help of statistical software PRISM 3.0. Resultsf worm recovery, microfilarial density and absorbance values ofLISA have been presented as mean ± S.D. The criterion for statis-ical significance between the results of immunized and controlroups were as follows: P-value <0.05 was considered significant,0.01 as highly significant, <0.001 as very highly significant. Unlesstherwise indicated, all values were derived from the mean of threeeplicates and the standard deviation is presented.

. Results

.1. Protective efficacy of B. malayi myosin in terms oficrofilaraemia and adult worm recovery

Microfilaria started appearing in blood circulation at 12th week.c., therefore assessment of microfilaraemia density in peripherallood of mastomys was initiated at 12th week p.c. and continuedp to 20th week p.c. following which all animals were euthanizedor the recovery of adult worms. Initially, at 12th week the densityf microfilaria was low in all experimental group of animals, sub-equently there was a sharp rise in mf density in control groups asompared to rMyo immunized group (Fig. 1). Mf density remainedignificantly low in rMyo immunized group throughout the obser-ation period. Microfilarial density in rMyo gp (63.0 ± 40.28) was6.29%, 78.97%, 64.96% lower than cont gp (265.8 ± 62.9), adj gp299.6 ± 87.3) and BmAg gp (179.8 ± 43.35), respectively on the dayf autopsy. The difference in microfilarial density in rMyo group andontrol group was statistically significant (P < 0.05) throughout thebservation period except for the initial assessment at 12th weekFig. 1).

Twenty-one weeks p.c. with L3, recombinant myosin and adult. malayi soluble somatic antigen immunized and control groupsf jird and mastomys were euthanized humanly through termi-al anesthesia and worms were recovered from heart, lung, lymphodes and testis and counted (Table 1). Vaccination with recombi-

ant myosin had a profound effect on adult worm recovery from

mmunized group animals as significant reduction in adult wormounts in rMyo group in both the mastomys and jird was observed.he B. malayi adult worms recovery from experimental groupsf mastomys viz; cont gp (21.0 ± 7.0), adj gp (22.5 ± 11.7), BmAg

6000 S. Vedi et al. / Vaccine 26 (

Fig. 1. Microfilarial kinetics in immunized and control group of mastomys. M. couchawere immunized with rMyo in FCA/FIA (�), B. malayi adult somatic antigen inFCA/FIA (�), FCA/FIA alone (©) or PBS alone (×). Blood microfilaraemia was deter-mined after 12 weeks–20th week p.c. Microfilarial density was low at initial weekbut exhibited a sharp increasing trend in the following weeks in all experimentalgroup except rMyo vaccinated where level remained low throughout the observa-tion period. rMyo group exhibited 76.29%, 78.97%, 64.96% reduction in microfilarialburden compared to normal infected PBS alone, adjuvant alone and B. malayi vac-cinated groups respectively at final reading day. The reduction in microfilarial loadigdt

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n rMyo gp was significant (P < 0.05) compared to controls and BmAg vaccinatedroups throughout the experiment. Experiment was performed in duplicate andata of both were later merged as one. Each point represents a value obtained inwelve animals. Mean ± S.D. are shown.

p (16.8 ± 5.9) were significantly higher (P < 0.05) than rMyo gp8.5 ± 5.1), implying that rMyo immunized group exhibited a wormeduction of 59.52%, 62.35% and 49.4% against cont gp, adj gp andmAg gp respectively. In jird model also worm recoveries from

ont gp (23.17 ± 4.72), adj gp (20.3 ± 4.2) was significantly higherP < 0.05) than rMyo gp (8.6 ± 3.6), thus generating reduction inorm count to 62.8% and 57.63% against control and adjuvant

roups respectively (Table 2). Since worm recoveries in BmAg gp inastomys did not generate significant protection against control

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able 1dult worm recovery and female worm fecundity

roups No. of animals/survivinganimals

Adult worm counts in individual animal Adu(me

xperiment 1: mastomysa

PBS alone 12/12 ♀ 17, 15, 25, 14, 8, 19, 12,6, 12, 10, 11, 19 14♂ 9,8, 6, 9, 6, 8, 5, 0, 7, 6, 11, 10 7.

Adjuvant alone 12/12 ♀ 14, 15, 9, 7, 14, 13, 21, 8, 5, 4, 8, 28 12♂ 13, 29, 1, 12, 5, 7, 10, 8, 2, 15, 13, 10 10.

BmAg gp 12/12 ♀ 13, 7, 3, 9, 10, 12, 12, 6, 23, 6, 10, 10 10.♂ 8, 7, 2,3, 6, 8, 6, 10, 10, 6, 10, 5 6.

rMyosin 12/12 ♀ 3, 7, 15, 10, 0, 7, 5, 5, 5, 3, 6, 7 7.♂ 0, 1, 4, 2, 0, 0, 0, 1, 3, 0, 4, 2 1

ignificantly different from values obtained from control group, **P < 0.01; ***P < 0.001.a Values represented are mean ± S.D.

able 2dult worm recovery and sterilization of female worms recovered from experimental jird

roups No. of animals/survivinganimals

Adult worm counts inindividual animal

Male/femaladult worm

xperiment 2: Jirdsa

PBS gp 6/6 ♀ 5, 20, 11, 13, 17, 16 15.83 ± 4.3♂ 8, 6, 4, 9, 5, 7 7.33 ± 1.6

Adj gp 6/6 ♀ 10, 14, 19, 10, 17, 13 14.33 ± 3.2♂ 4, 8, 9, 7, 3, 5 6.0 ± 2.3

rMyo gp 6/6 ♀ 4, 8, 8, 5, 5, 3 5.5 ± 2.0♂ 4, 3, 4, 1, 3, 0 2.50 ± 1.6

ignificantly different from values obtained for control groups, **P < 0.01; ***P < 0.001.a Values represented are mean ± S.D.

2008) 5997–6005

ence the group was not included in jird studies. Worm recover-es from the respective groups in mastomys models was reflectivef their microfilarial status as control groups demonstrated higherorm recoveries followed by BmAg group and lowest in rMyo

roup. Upon dissecting total worm population in male and female,t was observed that the effect of vaccination was not equal acrossoth the populations in mastomys model. Percent reduction of maleorm in vaccinated gp of mastomys was very high (80–86%) com-ared to female (40–50%). However, the ratio of male and femaleorm reduction in jird was comparable, i.e. 65%.

Apart from the reduction in worm count, the recovered adultemale worms in both the animal models from rMyo groups alsoxhibited higher sterilization (Table 1). The sterilization percent-ge of adult females worms (Table 2) recovered from rMyo gp70.89 ± 12.23%) was significantly higher (P < 0.05) than the nor-

al control gp (19.34 ± 8.86%), adj gp (17.68 ± 10.79%) and BmAgp (25.32 ± 12.71%). The difference in female worm sterility wastatistically significant (P < 0.001). Female worm sterilization inMyo immunized group of jirds was also high (76 ± 17.24) in com-arison to that of normal control gp (19.59 ± 12.12) and adj gp23.75 ± 11.67) of worms which was also statistically significantP < 0.001).

.2. Cellular immune response in immunized and control groupsfter L3 challenge

The proliferative response of splenocytes harvested from recom-inant myosin immunized mastomys was compared with that ofastomys receiving B. malayi crude soluble somatic adult antigen

nd control group on day 90 after L3 challenge. Control groups andmAg immunized group exhibited a suppressed cellular immuneesponse post-challenge (Fig. 2). The rMyo vaccinated mastomysfter L3 challenge revealed higher SI value (6.64 ± 1.09) as com-ared to BmAg immunized or control groups and the difference

lt wormsan ± S.D.)

Adult worm recovery(mean ± S.D.)

Worm burden reductionwith normal control (%)

Female wormsterilization (%)

.0 ± 5.34 21.0 ± 7.09 0 19.34 ± 8.8608 ± 2.87

.17 ± 6.96 22.5 ± 11.7 0.00 17.68 ± 10.7942 ± 7.31

08 ± 5.01 16.8 ± 5.9 25.19 25.32 ± 12.7175 ± 2.63

08 ± 4.14** 8.5 ± 5.1** 64.28** 70.89 ± 12.23**.41 ± 1.56***

s

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Total adult wormrecovery

Reduction in worm countagainst PBS control (%)

Female wormsterilization

5 23.17 ± 4.72 – 19.59 ± 12.123

0 20.33 ± 4.22 – 23.75 ± 11.673

7** 8.0 ± 3.26** 62.62 76.53 ± 17.24**4***

S. Vedi et al. / Vaccine 26 (2008) 5997–6005 6001

Fig. 2. T cell proliferation in presence of recombinant B. malayi myosin (rMyo;2.5 �g/ml) or Con A (2.5 �g/ml). M. coucha were immunized with rMyo, B. malayiadult somatic antigen (BmAdAg) in FCA/FIA, FCA/FIA alone or PBS alone. The assaywas performed 150 days post-vaccination or 90 days post-challenge of L3. Con-trol (open and light shade bars) and adult antigen (dark shade bar) vaccinatedgroup splenocytes did not show much proliferation on stimulation with ConA afterthe establishment of adult worms, in contrast, rMyo vaccinated group (close bar)splenocytes exhibited marked cellular proliferation. Proliferation was quantified byt*

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Fig. 3. Nitric oxide production by macrophages of immunized and control group ofmastomys post-vaccination and post-challenge after in vitro stimulation with LPS(1.0 �g/ml) or rMyo (2.5 �g/ml). NO determination was done at two time points, i.e.post-vaccination (day 7 of final booster) and post-challenge (at the time of wormrecovery). Each bar represents mean ± S.D. of six animals. NO production was sig-nificantly higher in rMyo immunized group macrophages (close bars) than PBScontrol (open bar), adjuvant control (light shade bar) and BmAg immunized group(wac

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hymidine incorporation. Each bar represent mean ± S.D. of six animals. *P < 0.05;*P < 0.01. Analysis done by ANOVA, Newman–Keuls multiple comparison test.

etween the SI values of control and rMyo immunized gp was sta-istically significant (P < 0.01).

.3. In vitro stimulation of macrophages with rMyo led to highO and oxidative burst in immunized group

Peritoneal macrophages of rMyo immunized animals generatedigher NO in comparison to controls gp of mastomys (P < 0.05)pon rMyo stimulation post-vaccination as well as post-challengeFig. 3). Although, the production of NO, post-challenge (after 20eeks p.c.) was lower than at the end of vaccination, the differenceas not statically significant. A real time monitoring of oxidativeurst generated from peritoneal macrophages of immunized andontrols group of mastomys was done on day 7 post-vaccinationn order to determine true in vivo milieu of macrophage activ-ty (Fig. 4). FACS data indicate that rMyo immunization led tohe generation of significantly higher oxidative burst (P < 0.01) in

acrophages from rMyo gp as compared to the controls. ROS gen-ration by rMyo gp PEC’s was twofold higher than BmAg gp andhreefold higher than adj gp/cont gp. a

ig. 4. Representative FACS figures demonstrating total oxidative burst in the freshly leaaccination. Significantly high oxidative burst was observed in PEC’s from rMyo vaccinate

dark shade bar) both at both time period. NO production by rMyo gp macrophagesas not hampered even after L3 challenge. Each bar represents mean ± S.D. of six

nimals. **P < 0.01; ***P < 0.001. Analysis done by ANOVA, Newman–Keuls multipleomparison test.

.4. Vaccination generated high IgG titer with a predominance ofgG2a/IgG2b isotypes

The rMyo group developed higher levels of rMyo specificntibodies compared to BmAg immunized and control group ofastomys (Fig. 5). The titer of rMyo specific antibodies showed

ose dependent increase, reaching maximum on day7 post-finalooster dose. Antibody titer remained higher in rMyo gp even after5 days p.c. of L3. None of the controls developed rMyo specificntibody response. Measurement of rMyo specific IgG isotypes inxperimental group of post-immunization revealed that mastomysmmunized with recombinant myosin induced predominantly ele-ated level of IgG2a and IgG2b isotypes followed by IgG1 isotypehile levels of IgG3 remained low (Fig. 6) which was indicative of

h1 response by rMyo immunization. BmAg immunized group seraemonstrated the presence of low level of rMyo specific IgG1, IgG2a,

gG2b and IgG3 isotypes, while adjuvant controls did not developedMyo specific antibody isotype.

Markedly increased cellular adherence was observed againstll the three stages used in the experiment in presence of

ched peritoneal exudates cells of control and vaccinated group of mastomys post-d group mastomys.

6002 S. Vedi et al. / Vaccine 26 (2008) 5997–6005

Fig. 5. Kinetics of rMyo specific IgG antibody titer in sera of mastomys immunizedwith recombinant B. malayi myosin. Groups of mastomys were immunized withrMyo (©), BmAg (�), FCA/FIA alone (�) or PBS alone (�), and rMyo specific antibodylevels were detected by ELISA at different time intervals. All animals were free ofrMyo specific antibodies on the day of initiation of vaccination (A). Vaccination withrMyo resulted in high titer of rMyo specific IgG post-vaccination (B), 15 days postL3 challenge (C) and remained high even up to the day of autopsy (D). BmAg gp andcv

rLnmdBcLm

Fig. 6. Anti-rMyo IgG isotypes levels in sera of mastomys of different groups. Mas-tomys were immunized with rMyo (close bar), BmAg with FCA/FIA (dark shade bar),FCA/FIA alone (light shade bar) or PBS alone (open bars) and challenged with B.malayi L3. IgG1, IgG2a, IgG2b and IgG3 antibodies were checked by ELISA for spe-cific antibodies against rMyo. Each point represents a value obtained with sera ofsix experimental animals. Mean ± S.D. are shown.

Table 3Antibody-dependant cellular cytotoxicity to L3, microfilariae and adult femaleworms induced by sera of mastomy immunized with rMyo

Experimental group Percentage cytotoxicitya Adult worm

Microfilariae L3

Control group (n = 5) 7.33 ± 3.0 5.3 ± 2.0 −Ar

F2mw

ontrol group did not develop rMyo specific IgG response. Each point represents aalue obtained with sera of six animals. Mean ± S.D. are shown.

Myo immunized mastomys sera (at 1:32 dilution; mf-77.56%,3-62.44%) compared to that occurred in presence of BmAg immu-ized mastomys sera (mf-30.06%, L3-28.44%) and adjuvant controlastomys sera (mf-2.20%, L3-5.33%) within 24 h (Table 3). The

ifference in the percent cytotoxicity by rMyo immunized withmAg immunized sera and control sera were statistically signifi-ant (P < 0.001). Apart from cell cytotoxicity and death of mf and3 by adhered cells, rMyo immunized mastomys serum also pro-oted high cellular adherence to adult female worms (Fig. 7)

wawa

ig. 7. Antibody dependent cellular adhesion to (a) microfilariae, (b) L3, (c) adult female o4 h. Cellular adhesion with mf and L3 led to their death within 48 h. Adherence of cells totility to a great extent. Photographs were captured on phase contrast florescent microorm and black arrow indicate adhered cells.

dult ag immunized gp (n = 5) 33.50 ± 8.8 28.4 ± 10.5 ++Myo vaccinated group (n = 5) 77.56 ± 4.06 62.4 ± 5.14 ++++

a Values shown are mean ± S.D.

hich was quite minor with BmAg gp sera and negligible withdj/control gp sera. Here the adhesion did not lead to death of adultorms (observed up to 96 h), but retarded their motility consider-

bly.

f B. malayi. Adhesion of splenocytes with different life stages was observed withino male and female though could not lead to their death up to 96 h but retarded thescope (Nikon, Japan) at 40× magnification. White arrow indicates mf, L3 or adult

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. Discussion

In the perspective of absence of fully efficient chemotherapeuticontrol measures against lymphatic filariasis, vaccination approachs sought to combat. It is imperative to exploit the immunogenicotential of newer antigens eliciting protective immunity by bothumoral as well as cellular response. In our earlier study [26], weave reported expression, purification and immunological charac-erization of a 73 kDa B. malayi antigen myosin which was selectedased on its high immune-recognition with hyper immune seragainst female worm somatic antigen. The purified myosin demon-trated strong blot reaction with the human sera from endemicormal’s (EN) which are considered to be putatively immune [31]s they do not develop disease even in the endemicity of the dis-ase. Furthermore, antigens displaying strong reaction with sera ofhis category have demonstrated to be protective in nature [32–34].

The present study unequivocally demonstrated for the first timehat immunization with recombinant B. malayi myosin elicits highevel of protection (∼63%) against filarial parasite B. malayi. Inur laboratory two animal models (mastomys and jird) are rou-inely used for the experimental B. malayi infection, therefore inhe present study protective efficacy of recombinant protein wasvaluated in both these models for observing the reproducibilitynd consistency of the protectivity data of recombinant myosin.he real time monitoring of blood microfilaraemia is more conve-ient in mastomys, however, jirds have been more commonly used

n filarial vaccination studies in the past (35–38). Nevertheless, theommercially available mouse immunological reagents can also beonveniently used with mastomys rather than jirds. Experimentnd vaccination protocol for the evaluation of protective potentialf the protein myosin were designed in such a way that it closelyimic the situation as occurs clinically. Effectiveness of vaccine was

etermined on each stage for an extended duration of 5 monthsost-vaccination which has yet not been implicated in lymphaticlariasis by any previous vaccination study. In contrast, majorityf vaccination studies in past either directly introduced L3/mf ormployed micropore chambers laden with these into the peritonealavity for the evaluation of the protective potential of the vaccineandidate [24,34–38]. Since intraperitoneal inoculation or micro-ore chambers restricts the movement of inoculated/challenged

arvae, the probability of killing of L3/mf in a confined space byhe augmented immune response in immunized animals would ofourse, be very high in comparison to controls which do not havexaggerated immune response. The primary action of killing of L3r mf (when administered intraperitoneally) would be through theells of innate immunity i.e., macrophages which are numeroust this site. Our in vitro cytotoxicity assay also substantiates thisssumption where peritoneal exudates cells (macrophages) whenncubated with splenocytes in presence of myosin specific masto-

ys serum led to high percentage of cytotoxicity/death of L3 andf. Furthermore, ADCC assay also revealed significantly higher cel-

ular adherence against adult worms, however worm death couldot be observed up to 96h thus correlating with the protectivityegree. On contrary, L3 entering subcutaneously do not encounterpecific/nonspecific cells immediately, giving them a brief oppor-unity to escape and/or to modulate immune response.

For an immunoprophylactic agent to be effective, it is impera-ive that vaccine candidate should induce both humoral and cellularrms of immunity. Myosin vaccination led to high humoral and cel-ular immune response that is evident from the higher antibody

evel as well as high proliferative response in rMyo immunized

astomys post-vaccination and post-challenge. The suppressedellular proliferation of splenocytes in mastomys receiving B. malayirude soluble somatic adult antigen could possibly be due to pres-nce of large number of antigens in the crude extract include

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2008) 5997–6005 6003

mmunosuppressive ones. Along with higher cellular proliferation,Myo vaccinated group also demonstrated high humoral responsehat was high even up to the day of autopsy. Antibody isotypeevealed the predominant presence of Th1 antibodies IgG2a andgG2b, although increased level of IgG1 in the rMyo immunizedera. It has been shown that predominance of IgG2a antibodyogether with IgG1 exerts greatest functional activity [39]. Th1esponses are usually cellular mediated, antibodies albeit at lowerevels are also produced [40]. In the murine system, Th1 response isssociated with the production of IgG2a antibody isotype, whereash2 response is associated with IgG1 isotype [41]. It is believed thegG2a allows for more potent complement activation, as well aspsonization and antibody dependent cellular cytotoxicity (ADCC)han IgG1 [42], and this is in consistency with our data of cyto-oxicity assay. Thus, the antibody responses during Th1 responseynergize the cellular components to allow for more effectivelearance of the pathogen. Results obtained with the rodent mod-ls used in the present study illustrates distinctive resemblanceith the studies involving filarial-endemic population where true

ndemic normal individuals have been reported to mount a Th1-ike antifilarial immune response [43,44] with generation of IgG2nd IgG3 isotype [45,46]. Data indicating significantly higher SIalues in lymphoproliferation assay, high cellular adhesion alongith increased macrophage function in rMyo immunized group

re all indicative of strong cellular immune response (Th1) andhis could be one of the reasons for killing of invading L3, lead-ng to reduced worm recovery from immunized group animals.nsignificant level of endotoxin (<1 EU/mg) in the finally dialyzedrotein preparation eliminated the possibility of its involvement

n high degree of lymphoproliferation in myosin vaccinated ani-als.The mechanisms by which mammalian hosts eliminate

icroparasites such as bacteria and viruses are well character-zed. In viral infections, these mechanisms include the interferoneutralizing and opsonizing antibodies and cytotoxic T lympho-ytes. In bacterial infections, polymorphonuclear leukocytes andacrophages are often facilitated by opsonizing antibodies and

ngest the infectious agent mediating host defense. In addition,omplement proteins in the presence of specific antibodies directedgainst surface antigens can lyse bacterial pathogens. The feasi-le killing mechanism in case of multicellular parasites such asematodes can only be antibody dependent cellular cytotoxicityr through action of external toxic mediator such as NO in theiricinity. In vitro evidence also suggests that larval stages are killedy nitric oxide [47–50].

The most intriguing observation in this study was the higherevel of sterilization in female worms recovered from myosinmmunized animals. It was quite hard for us to correlate the steril-ty of female worms with the vaccination. It is evident from earliertudies that endosymbiont Wolbachia is closely associated with theecundity of female parasites [51,52], therefore, we determined theacterial status in the sterile and fertile worms recovered from

mmunized animals through PCR studies which substantiated theresence of endosymbionts in both sterile and fertile worms thusxcluding this possibility as a reason for sterilization. A careful anal-sis of results revealed that the adult male worm recovery wasignificantly low in rMyo immunized group as compared to that ofontrols, which may be located in different tissues thus raising theossibility of unavailability of mate partner in the vicinity. Anotherossibility that cannot be overlooked for high rate of sterilization is

hrough emergence of nutrient limiting conditions for adult worms.t is well known that filarial nematodes do not have well-developedigestive system and fulfill their nutritional requirement throughurface absorption [53–56]. The cytotoxicity assay in our studyave demonstrated high cellular adherence with mf, L3 and adult

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emale worms. The same phenomenon if occurred in vivo wouldlock worm surface thus rendering hindrance in nutrient uptake,ebilitating the development of eggs and cause higher infertility.hus, it is apparent that immunization with myosin leads to higherterilization thereby decreasing mf density, imparting an additionaleature to the vaccine to be used as a transmission-blocking agent.

The magnificent prophylactic efficacy of B. malayi myosin vac-ination demonstrated in the present study which was effectiveven 5 months post-vaccination was dissimilar to the conclusionsf earlier study which reported failure of filarial myosin (O. volvulus)o provide protection against B. malayi challenge [57]. There werepparent differences in antigen preparation methods between thewo vaccination studies as unlike present study which utilized sin-le band purified recombinant protein, the former filarial myosinaccination study utilized crude protein (lysate of induced lysogen)n high quantity (200 �g/animal). Possibly, the quality of the recom-inant proteins (degraded) may have led to the key epitopes noteing presented, thus rendering protein inept for generating ade-uate protection. Similar discrepancy have earlier been reportedetween studies involving SmIrV-5, a recombinant 62 kDa frag-ent of S. mansoni myosin [58,59] and its S. japonicum homologue

Sj62), where SmIrV-5 induced strikingly high levels of protectionhile Sj62 could not generate synonymous protection [60,61]. The

ariation in protective efficacy in these studies was attributed toifference in antigen preparation methods.

Conclusively, the present study demonstrated that B. malayiyosin is a potent vaccine candidate for prophylaxis against human

ymphatic filarial parasite B. malayi exerting vaccination effectnequivocally on all the filarial life stages (mf, L3 and adult worms).accination studies involving homologue of W. bancrofti, the higherrevalent filariid would be have been more valuable than B. malayi,owever unavailability of suitable animal model for W. bancroftihich is strongly host-specific and does not establish in experi-ental animals except leaf monkeys [62,63] which have a restricted

se, is certainly a drawback. The B. malayi myosin expressed in theresent study has demonstrated strong cross-reactivity with ban-roftian sera of various categories [26] demonstrating the practicalonversion of the present findings to bancroftian infection also.urther studies to improve efficacy of recombinant myosin witharious other adjutants and delivery systems is underway. Vacci-ation studies should be carefully designed so that the protectiveotential of any vaccine candidate is properly deduced.

cknowledgements

Mr. A.K. Roy and R.N. Lal are duly acknowledged for techni-al assistance. The financial assistance in the form of the senioresearch fellowship from the Council of Scientific and Industrialesearch, New Delhi to two of us (SV and AD) is gratefully acknowl-dged.

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