Vaccine 27 (2009) 473–482

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Adjuvanticity and protective immunity of Plasmodium yoelii nigeriensisblood-stage soluble antigens encapsulated in fusogenic liposome

Varun Dwivedia, Azevedo Vascob, Satish Vedic, Anil Dangic, Khan Arifd,Shailja Mishra Bhattacharyac, Mohammad Owaisd,∗

a Department of Biochemistry, J.N. Medical College, Aligarh Muslim University, Aligarh, Indiab Department de Biologia, University Federal de Minas Gerais, CP 486, Brazilc Department of Parasitology, Central Drug Research Institute, Lucknow, Indiad Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh, India

a r t i c l e i n f o

Article history:Received 1 October 2007Received in revised form 13 October 2008Accepted 20 October 2008Available online 7 November 2008

Keywords:SaccharosomeAdjuvantCellular immunity

a b s t r a c t

In our previous studies we established fusogenic properties of lipids isolated from edible yeast Saccha-romyces cerevisiae (S. cerevisiae). We demonstrated that liposomes prepared from S. cerevisiae membranelipid (saccharosome) can deliver encapsulated antigen into cytosol of the antigen presenting cells and elicitantigen specific cell mediated as well as humoral immune responses. In this study, we evaluated immuno-logical behavior of saccharosome encapsulated cytosolic proteins (sAg) of Plasmodium yoelii nigeriensis inBALB/c mice. Immunization with antigen (sAg) encapsulated in saccharosome resulted in enhancementof CD4+ and CD8+ T cell populations and also up-regulated the expression of CD80 and CD86 moleculeson the surface of antigen presenting cells. Further, immunization with saccharosome-encapsulated sAg-induced elevated levels of both IFN-� and IL-4 cytokines in the immunized mice when compared to egg PCliposome encapsulated sAg or its IFA emulsified form. Saccharosome-mediated immunization resulted in

Humoral immunity

Vaccine induction of high level of total antibody response with preponderance of IgG2a isotype as well. The dataof this study suggest that saccharosome-based vehicle can emerge as an effective vaccine in imparting

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. Introduction

Malaria is one of the major public health problems with sig-ificant economic and social consequences for many developingountries [1]. Global incidences of the malaria range from 300o 500 million cases per year, out of which 2–3 million suc-umbed to death [2]. In fact majority of the deaths involvinghildren, under the age of 5 years, have been linked with oner other form of the disease. Despite many preventive efforts,orbidity and mortality continue to rise. Ironically, chemother-

py and vector control programs against the dreadful disease areot adequate due to the emergence of drug resistant Plasmod-

um spp. and spread of insecticide-resistant mosquito vectors.eeping into consideration the non-effectiveness of chemother-

py, it is always imperative to deal this problem by developingrophylactic measures using effective vaccines. Interestingly, itas been observed that some adults living in endemic areas doot exhibit clinical manifestation of malaria even when parasites

∗ Corresponding author. Tel.: +91 5712720388; fax: +91 5712721776.E-mail address: owais lakhnawi@yahoo.com (M. Owais).

Pyeprarc

264-410X/$ – see front matter © 2008 Elsevier Ltd. All rights reserved.oi:10.1016/j.vaccine.2008.10.054

cellular pathogens including Plasmodium yoelii nigeriensis.© 2008 Elsevier Ltd. All rights reserved.

re present in systemic circulation; suggestive of effectiveness ofmmune system of the host in containment of the infection [3].n order to develop an effective vaccine, the complexity of themmune response against Plasmodia spp. poses a major hurdle andequires better insight of the operative immune defenses againsteadly parasite. An effective immunization protocol is ought to

nvolve identification of immunogenic protein capable of evok-ng parasite specific antibodies. Besides, development of suitabledjuvant/antigen vehicle is also of strategic interest to achieveesirable cell-mediated immune responses. Most of the avail-ble adjuvants offer limited protective immune responses againstlood-stage Plasmodium yoelii parasite [4–8]. For example, theommonly used adjuvant alum is not appropriate against malariaarasite as it stimulates a Th-2 type immune response mainly [9].rotective immunity against intracellular pathogens including P.oelii is generally dependent on Th1 type immune responses. How-ver, because of the lack of MHC-I molecules on erythrocyte surface,

rotective immunity against blood-stage malaria is complex andequires a concerted effort by a Th1 type cellular immune responsend Th 2-mediated humoral immunity [10,11]. Earlier studies onodent models have revealed a role for CD4+ T lymphocytes, Bells, as well as antigen-specific antibody in mediating naturally

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nduced immunity against primary blood-stage P. yoelii infection10,11].

We have earlier shown that liposomes made of lipids isolatedrom Saccharomyces cerevisiae (Bakers yeast) have strong tendencyo fuse with plasma membrane of target cells thereby deliveringhe entrapped contents into their cytosol [12]. Our findings wereurther first confirmed by Stubbs et al. and recently by Bernstein etl., where they demonstrated that saccharosome-mediated deliv-ry of antigens ensued in the activation of antigen specific humorals well as cellular immune responses [12–15].

In this study, we evaluated antigen delivery potential of sac-harosomes in the immunized animals. The data of this studyighlights the ability of saccharosome to modify immunologicalehavior of soluble cytosolic antigen of Plasmodium yoelii nigerien-is. Of the various adjuvants tested, protective immune responsesere driven primarily by immunization with antigens encapsu-

ated in saccharosome-based formulation. Whereas other forms ofhe antigen (free or egg PC liposome encapsulated) failed to impartrotective immune responses.

. Materials and methods

.1. Animal and infection

Inbred female BALB/c mice (8–10 weeks old) of 20 ± 2 geight were obtained from the Institute

′s Animal House Facil-

ty. The Plasmodium yoelii nigeriensis MDR strain was obtainedrom Division of Parasitolgy, Central Drug Research Insti-ute, Lucknow. The parasite was fully resistant to chloroquine28 mg/kg × 4 dose and quinine 600 mg/kg × 4 dose and meflo-uine 256 mg/kg × 4 doses and was maintained in mice by routineassage [16].

Blood-stage infection was initiated by intra-peritoneal injectionf parasitized erythrocytes obtained from donor mice. Enumer-ting parasitized erythrocytes in thin blood smears stained withiemsa’ stain monitored resulting parasitemia in the infected mice

17]. Routine surveillance was conducted through out the studyo ensure that mice were free of any infection with common

ouse pathogens. The immunization protocols including tech-iques used for bleeding were strictly followed as per mandatespproved by the Animal Ethics Committee (Committee for the Pur-ose of Control and Supervision of Experimental, Government of

ndia).

.2. Chemicals and reagents

Egg phosphatidyl choline (EPC) was isolated and purified usinghe standard method [18]. Cholesterol was purchased from Centronesearch Laboratory, Mumbai, India. [3H]-Thymidine was boughtrom Bhabha Atomic Research Center, Mumbai, India. Monoclonalnti mouse CD4+ and CD8+, CD80 and CD86 FITC conjugates andts purified control (Rat IgG2a isotype) were from Sigma Immunohemicals, St. Louis, USA.

.3. S. cerevisiae lipid

S. cerevisiae was procured from MTCC, Chandigarh and culturedn YPD (yeast extract, peptone and dextrose) medium. The cells

ere harvested from mid-log phase (18–20 h) and phospholipids

ere isolated by the method of Bligh & Dyer as modified in our lab

19]. Briefly, cell suspension was treated with 2:1 chloroform andethanol mixture to extract lipid. Subsequently, the organic phaseas extensively washed with normal saline and evaporated under

acuum to finally isolate S. cerevisiae lipid.

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27 (2009) 473–482

.4. Antigen preparation

Blood was collected from BALB/c mice infected with P.oelii (MDR) (average 35–40% parasitemia). The leucocytes wereemoved by passage of blood over microcrystalline CF11 column20]. The isolated blood contained a mixture of ring, trophozoite-nd schizont-stage parasite as described earlier [21]. The infectedBCs were lysed with 0.01% saponin. The whole parasites were re-uspended in 10 volume of 50 mM KCl–5 mM MgCl2 followed by 10ycles of rapid freezing and thawing, followed by brief sonication.oluble antigen (sAg) fractions were recovered by centrifugation for5 min at 200,000 × g (4 ◦C). Of the 60 mg of total protein obtainedn each preparation, 50–60% was recovered in the soluble fraction.

.5. Preparation of liposomes

Dried reconstituted vesicles (DRVs) were prepared using pub-ished procedure as standardized in our lab [22]. Briefly, PC/Chol2:1 molar ratio, total lipid 20 mg) or S. cerevisiae membrane lipid20 mg) was reduced to thin dry film in clean round bottomedask. The dried lipid film was hydrated with pyrogen-free normalaline and sonicated in a bath type sonicator for 1 h at 4 ◦C underitrogen atmosphere. The vesicles thus formed were mixed withqual volume of sAg (20 mg/ml) solution. The mixture was flashrozen and thawed (three cycles) followed by the lyophilization. Theree flowing, dried powder obtained was re-hydrated with distilledater (120 �l) and finally reconstituted with PBS. The preparationas washed at least three times with PBS to remove traces of un-

ntrapped protein.

.6. Estimation of protein content in Saccharosomes

The vesicular formulations (DRVs) were separated from the freeun-entrapped) antigen by pelleting them at 10,000 × g for 10 mint 4 ◦C. The washing was repeated thrice with a fresh phosphateuffer saline (pH 7.4) to accomplish complete removal of free formf antigen. To determine the protein content, an aliquot of vesic-lar fraction was mixed with minimum amount of Triton X-1000.5%, w/v) to disrupt the vesicles and released protein (antigen)as estimated using BCA (bicinchoninic acid) protein assay. Theercent antigen entrapment in DRVs was calculated as describedarlier [12].

.7. Immunization

BALB/c mice (15 animals per group) were immunized subcuta-eously with 100 �g of various forms of sAg (free-sAg, sAg-IFA, sAgntrapped in PC liposomes (PC-sAg) and sAg entrapped in saccha-aosomes (YL-sAg), and empty saccharosomes (Sham-YL)), on days, 21 and 28. For protection studies, BALB/c mice (n = 10) were chal-

enged on day 35 post-first immunization, intra-peritoneally with05 parasitized RBC of P. yoelii nigeriensis (MDR) strain.

.8. Immunoblot analysis

The plasmodial cytosolic antigens were subjected to SDS–PAGE10%) analysis under standard conditions as described earlier23,24]. For immunoblot analysis, the resolved proteins were elec-rophoretically transferred to nitrocellulose membrane and thessay was performed according to the published procedure. Briefly,

itrocellulose strip was blocked overnight with 5% BSA solution in00 mM Tris buffered saline, pH 7.6, and containing 0.1% Tween-20Tween-TBS). Next morning, the strips were washed with Tween-BS solution and incubated with the mouse sera (1:200 dilutions)or 90 min at 37 ◦C. The strips were subsequently washed with TBS-T

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nd incubated with a 1: 1000 dilution of HRP-conjugated goat anti-ouse immunoglobulin (Sigma Immunologicals, USA) for 90 min

t 37 ◦C. The strips were washed thrice, incubated with substrate0.3% DAB (Sigma) in PBS with 0.4% H2O2) till development of color,nd finally washed thoroughly with distilled water.

.9. IgG response in immunized mice

The production of antigen specific total IgG as well as their iso-ypes was monitored in the sera of immunized mice bled on day 35ost-first immunization using ELISA method [25,26]. Briefly, 96-ell microtitre plate was incubated overnight with 50 �l of sAg

ntigen (25 �g/ml) in carbonate–bicarbonate buffer (0.05 M, pH.6) at 4 ◦C. After usual steps of washing and blocking, the plateas finally incubated with 1:500 dilutions of various experimen-

al as well as pre-immune sera at 37 ◦C for 2 h. After excessiveashing of the plate, it was further incubated with 50 �l of biotiny-

ated goat anti-mouse IgG1, IgG2a and IgG2b antibodies. The plateas incubated at 37 ◦C for 1 h. After usual washing steps, 50 �l of

treptavidin-HRP was added to each well and the plate was incu-ated at 37 ◦C for 1 h. The plate was washed extensively followedy addition of 50 �l ABTS and was finally incubated at 37 ◦C for0 min. The reaction was terminated by the addition of 50 �l of0% H2SO4. The absorbance was read at 492 nm with an ELISA plateeader (Eurogenetics, Torino, Italy).

.10. Assessment of delayed-type hypersensitivity (DTH) response

The DTH response was determined in the immunized mice asn index of cell-mediated immunity. Various forms of sAg anti-en (50 �g in 50 �l of vehicle) were injected subcutaneously to theeft footpad of the animals (belonging to matching immunizationroup); and for comparison the right footpad of the individual ani-al was injected with same volume of saline. The thickness of both

ootpads was measured using a digital Vernier Calliper. Percentagef increase in footpad thickness was calculated as follows:

Percent increase in footpad thickness

= left footpad thickness-right foot pad

thickness/right footpad thickness × 100

.11. Lymphocyte proliferation assay

The BALB/c mice belonging to various experimentalroups were sacrificed on day 35 post-primary immuniza-ion and their spleens were taken out aseptically. The CD4+

-cells (2 × 104cells/well) were incubated with macrophages6 × 104 cells/well) and pulsed with increasing amount of match-ng form of antigen (free-sAg; PC-sAg or YL-sAg). The experimentalultures were incubated for 72 h (37 ◦C, 5% CO2) and subse-uently pulsed with labeled thymidine (1 �Ci, 3H-thymidinespecific activity 18 Ci/mmole, BARC, India)] during last 18 h of

ncubation to monitor DNA synthesis. The radioactive incorpo-ation in the proliferating cells was measured by standard liquidcintillation spectroscopy. The stimulation index (S.I.) was cal-ulated as mean cpm values of stimulated culture/mean cpm ofn-stimulated culture.

.12. Saccharosome-mediated induction of Th1/Th2 cytokines

To assess the effect of YL-sAg-mediated immunization on pro-uction of various cytokines; spleens of the immunized miceere removed aseptically on day 35 post-primary immunization.

pleenic tissues were grounded gently to get primary cell culture.

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27 (2009) 473–482 475

ell suspension was prepared in RPMI 1640 (Life Technologies),upplemented with 10% heat inactivated FCS (Life Technologies).solated spleen cells were cultured by seeding them at density of× 106 cells per well in 100 �l of the medium in 24-well tissue cul-

ure plates. The cells were incubated with matching form of thentigen (sAg) for 48 h at 37 ◦C in humidified 5% CO2 incubator toonitor cytokine production.Culture supernatants were analyzed for the presence of cytokine

y ELISA method. The production of IFN-� and IL-4 was measuredn triplicate using commercial ELISA kits as described earlier [27]ollowing the manufacturer’s instructions. The quantitative esti-

ation was done using standard curves obtained for individualytokines.

.13. FACS analysis

The development of CD4 + as well as CD8+ T-cell populationas determined in splenic cells isolated from various experimen-

al groups by direct immuno-fluorescence staining. Spleen cells1 × 106) of mouse (n = 5 from each group) were stained withuorescein isothiocynate (FITC)-conjugated monoclonal antibod-

es specific for mouse CD4 and CD8a T-cell receptors. The cellsere incubated for 30 min at 4 ◦C, washed three times with dilu-

ion buffer (0.01 M phosphate buffer saline, pH 7.4 containing% bovine serum albumin) and re-suspended in 500 �l of 2%ara-formaldehyde. The percentage of CD4/CD8 positive cells waseasured using fluorescence-activated cell sorter (Becton Dick-

nson) and data were analyzed by Cell Quest software (Bectonickinson). The expression of CD80 was detected on the surface of

timulated macrophages isolated (by Panning method) from groupf animals immunized with various forms of sAg following pub-ished protocol [26]. The recovered adherent cells were consisted of99% macrophages as judged by histochemistry and FACS analysissing labeled anti F4/80 antibodies. The macrophages were incu-ated with PC-sAg or YL-sAg form of antigen. The macrophagesere first incubated with Fc block and subsequently stained with

ITC conjugated hamster anti-mouse CD80/86 diluted in FACSuffer. The stained cells were acquired on FACScan and popula-ions of macrophages were analyzed on macrophage/monocyteone using CELLQUEST software. The exclusion of debris and lym-hocytes from cell suspension by suitable gating allowed analysisf scattering events consistent with macrophages size range.

The analysis of mean fluorescence intensity (MFI) was per-ormed on histograms in which the abscissa and ordinate denoteog FITC fluorescence and relative cell count respectively.

.14. Flow cytometric-based detection of ROI

To measure the saccharosome-mediated ROI in macrophages;e used cell-permeable redox sensitive dye CM-H2DCFDA. The

educed form of dye is non-fluorescent, while its oxidized forms fluorescent active permitting its detection by flow cytometricnalysis as described elsewhere [28–30]. Briefly cells (106) werencubated with various forms of sAg (10 �g/ml) for 24 h at 37 ◦Collowed by the addition of 10 �M H2DCFDA and further incubatedor 15 min at 37 ◦C and 5% CO2. The change in fluorescence wasssessed using with a FACS Calibur flow cytometer (Becton Dickin-on, Franklin Lakes, and NJ).

.15. Statistics

The data were analyzed by Bonferroni test or Mann–Whitneyest as per specification of the analysis. P values <0.05 were consid-red statistically significant.

4 ccine 27 (2009) 473–482

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. Results

.1. Immunoblot analysis

Immune reactivity of the host against sAg (P. yoelii cytosolicntigen) was determined by analysis of sera isolated from variousroups of immunized animals. Reactivity of serum obtained frommmunized animals indicated a strong antibody reaction againstarious cytosolic proteins of Plasmodium yoelii. Further, the blotnalysis revealed that induction of antibody formation was mainlyegulated by specific adjuvant co-administration along with sAg.he antibodies were reactive to cytosolic polypeptides of P. yoeliiemonstrating the multiband patterns in the animals immunizedith YL-sAg while pre-immune sera did not recognize any antigen

Fig. 1).

.2. Antibody response

We determined production of total antibody in response tommunization with various forms of P. yoelii sAg (Fig. 2). Animalsmmunized with sAg entrapped in saccharosomes (YL-sAg) elicitedsignificantly high total IgG response after a single booster when

ompared to sAg-PC formulation (p < 0.001). Immunization with

Ag-IFA induced relatively less IgG response when compared toL-sAg or PC-sAg group. The antibody titres were moderate in theroups of animals immunized with the free form of antigen or whenham liposomes were administered (p < 0.001). The isotype analy-

ig. 1. Immunoblot analysis of antibodies induced in the animals immunizedith cytosolic antigens of P. yoelii: the Plasmodium yoelii blood-stage antigens

10 �g/lane) were separated by SDS–PAGE (10% gel) and subsequently transferredo membrane. The reactivity of various protein components with sera obtainedrom mice immunized with YL-sAg (lane 2), PC-sAg (lane 3) or sAg-IFA (lane 4)as analyzed by Western blot technique as described in Section 2. The reactivity of

lasmodium yoelii blood-stage antigens with pre-immune sera (lane 1) was used asnegative control.

Fig. 2. IgG profile in animals immunized with various formulations of sAg: theimmunized animals were examined for the presence of total IgGs (A) and theirisotype (B) responses by ELISA method. Sera (1:500 dilution) obtained from nor-mal as well as experimental immunized animals were analyzed for the presence ofmalaria-specific IgG isotype by ELISA method as described in Section 2. The level ofIgG isotypes was expressed as absorbance (A492) of the colored complex developediov

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n the immunosorbant assay. The values expressed are the mean of the absorbancef the sera of the five different animal’s ± S.D. PC-sAg vs YL-sAg, p < 0.001. IFA-sAgs PC-sAg, p < 0.001; free-sAg vs IFA-sAg, p > 0.1; IFA-sAg vs PC-sAg, p > 0.1

is of the immunized mice showed highest level of IgG1 in the micemmunized with YL-sAg immunized group when compared to PC-Ag as well as IFA-sAg combination. Besides IgG1, YL-sAg inducedubstantial level of IgG2a isotype antibody as well. In contrast, PC-Ag induced very less IgG2a antibody when compared to YL-sAgpYL-sAg vs PC-sAg p > 0.001). No significant rise in IgG2b levelas observed in mice immunized with other forms of antigens

p < 0.001).

.3. DTH responses

Cell-mediated immunity plays crucial role in elimination ofntracellular pathogens. It is evident from Fig. 3 that YL-sAg form ofntigen induced 64% DTH response as compared to PC-sAg (37.5%)n the immunized animals. While insignificant DTH response wasbserved in control (PBS only) animals (14.75%) (p < 0.001).

.4. T-lymphocyte proliferation in response to immunization withaccharosome encapsulated sAg

The lymphocytes obtained from the animals immunized withaccharosome entrapped sAg underwent significantly better prolif-

V. Dwivedi et al. / Vaccine 27 (2009) 473–482 477

Fig. 3. Delayed type hypersensitivity (DTH) response in animals upon immuniza-tion with various forms of sAg: The DTH response in animals vaccinated with variousforms of antigen was determined by injecting 50 �g of sAg antigen in 50 �l of vehi-cle (saline, Sham, IFA, free antigen, PC-liposome and YL) in left hind footpad ofmice. An equal volume of saline was injected into opposite footpad. The thicknessof footpad was measured by Vernier calipers and DTH was expressed as the per-cmp

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Fig. 5. Induction of cytokines (A) IFN-�, (B) Il-4 in animals immunized with sac-charosome encapsulated sAg: the CD4+ T cells obtained from various group of

entage increase in footpad thickness at 24 h post-immunization, the time at whichaximum reaction was observed. Free-sAg vs IFA-sAg, p < 0.001; PC-sAg vs YL-sAg,< 0.001; Sham-YL vs free-sAg, p < 0.01.

ration when compared with lymphocytes obtained from animalsmmunized with PC-sAg or sAg emulsified with IFA (Fig. 4).he T-lymphocyte proliferation was found to depend on dosef antigen (data not shown). Maximum proliferation occurredhen cells were stimulated with 10 �g/ml antigen. The responseas significantly higher for YL-sAg (S.I. > 6) as compared to PC-

Ag (S.I. < 3) and IFA-sAg (S.I. < 2.5) groups of animals. Controlxperimental sets containing cells obtained from animals immu-

ized with free-sAg, PBS or Sham-YL (empty liposomes with noAg), induced background level of <2000 cpm of 3H-thymidinencorporation.

ig. 4. Saccharosome encapsulated sAg induces higher T-cells proliferation in themmunized mice: proliferation of T cells in response to saccharosome encapsu-ated sAg. CD4+ T-cells (/well) were isolated from various groups of mice, platedt 2 × 104 cells/well on 96-well round-bottom plates, and tested for their abilityo proliferate in response to various forms of sAg. The isolated T-cells were andultured with free-sAg, YL-sAg and PC-sAg pulsed macrophages (6 × 104 cells/well).fter 72 h, [3H]-thymidine was added, and its incorporation was measured 16 h latery liquid scintillation spectroscopy. The stimulation index (S.I.) was calculated asean cpm values of stimulated culture/mean cpm values of un-stimulated culture.

ontrol cultures containing cells obtained from either free-sAg immunized animalsr the groups immunized with PBS or fusogenic lipids only (Sham liposome with noAg), gave background levels of <2000 cpm of [3H]-thymidine incorporation. Eachata point represents the mean cpm of triplicate wells; error bars represent the S.D.he data are representative of three different experiments.

immunized animals were co-cultured in presence of various forms of P. yoelii sAgas described in Section 2. The culture supernatants from the wells were collectedafter, 24 h from animals of experimental groups (PC-sAg or YL-sAg). Cytokine levelswere measured by sandwich ELISA as described in Section 2. The levels of cytokineswere calculated by use of standard curves that were plotted using recombinantcytokines and are expressed as picograms per milliliter. IFN-�: uninduced free-sAg vs IFA-sAg, p < 0.001; PC-sAg vs YL-sAg, p < 0.001; Sham-YL vs free-sAg, p < 0.05;IFA-sAg vs PC-sAg, p < 0.001 induced saline vs Sham-YL, p < 0.001; free-sAg vs IFA-sPp

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Ag, p < 0.001; PC-sAg vs YL-sAg, p < 0.001; saline vs free-sAg, p < 0.1 IFA-sAg vsC-sAg, p < 0.001; Il-4: (induced) IFA-sAg vs PC-sAg, p < 0.001; PC-sAg vs YL-sAg,< 0.001

.5. Effect of saccharosome-mediated immunization on inductionf Th1/Th2 cytokines

The cytokine production was assessed as a measure of cel-ular sensitization of the immunized mice (Fig. 5 A and B). Thenimals immunized with YL-sAg induced significantly higherxpression of IFN-� and IL-4 (60.65 pg/ml IFN-� and 58.15 pg/mlL-4, respectively) when compared to animals immunized with PC-Ag (35.8 pg/ml IFN-�, 35.4 pg/ml IL-4) and sAg emulsified with IFA27.15 pg/ml IFN-�, 25.3 pg/ml IL-4) (p < 0.001).

.6. Sacchaarosome encapsulated sAg induces specific CD4+ andD8+ T-cell immunity in BALB/c mice

There was a substantial increase in both CD4+ (27.45%) and CD8+

24.92%) T-cell populations upon immunization with the YL-sAgorm of antigen (Fig. 6). On the other hand, mice immunized withC-sAg showed a relatively less increase in CD4+ T-cells (17.75%).hile unimmunized control animals (PBS only) had 12.53% of CD4+

-cells (p < 0.05). IFA-sAg immunized animals also showed only

478 V. Dwivedi et al. / Vaccine 27 (2009) 473–482

Fig. 6. Determination of CD4+ and CD8+ T cell populations in animals immunized with sAg: spleen cells (1 × 106) of mouse were stained with fluorescein isothiocynate (FITC)-c rs. Thec n Dic

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onjugated monoclonal antibodies specific for mouse CD4 and CD8a T-cell receptoell sorter (Becton Dickinson) and data were analyzed by Cell Quest software (Becto

arginal increase in both types of T cell populations (CD4+ 13.53%nd CD8+ 14.64% respectively).

.7. Saccharosome encapsulated sAg upregulates CD80/86olecules on antigen presenting cells

The minimum requirements for activation of CD4+ T cellsnclude co-presentation of processed peptide along with classI MHC, and its subsequent recognition by T-cell receptor. Thectivation of macrophages and dendritic cells should be accom-anied with expression of the appropriate co stimulatory markers

CD80 and CD86) on their surface [31]. The data of this studylearly revealed that macrophages isolated from animals vac-inated with YL-sAg showed significantly higher expression ofoth CD80 (MFI) and CD86 (MFI) on their surface. In con-rast, macrophages isolated from animals vaccinated with PC-sAg

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percentage of CD4/CD8 positive cells was measured using fluorescence-activatedkinson).

xpressed relatively low levels of CD80 and CD86 co-stimulatoryignals (Fig. 7).

.8. Saccharosomes induced reactive oxygen species in theacrophages of immunized animals

The ability of YL-sAg to activate macrophages for productionf reactive oxygen/NO species in the immunized animals was alsostablished by using CM-H2DCFDA dye. The released free radicalsave ability to oxidize CM-H2DCFDA that gets converted into itsuorescent active oxidized form (DCFDA). The histogram shown

n the Fig. 4 clearly demonstrate that macrophage isolated fromhe group of animals immunized with YL-sAg induced higherroduction of ROI MFI (112.08%) when compared to animals immu-ized with sAg-IFA (80.12%) or PC-sAg (78.98%) form of antigenFig. 8).

V. Dwivedi et al. / Vaccine 27 (2009) 473–482 479

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ig. 7. Expression of co stimulatory molecules (CD80 and CD 86) on macrophages isn macrophages of animals immunized with PC-sAg and YL-sAg were evaluated usuest software (Becton Dickinson).

.9. Potential of saccharosome-based sAg vaccine in impartingrotection against P. yoelii infection in Balb/c mice

To compare the efficacy of various sAg-based vaccines in con-erring protective immunity, the immunized mice were challengedntraperitonealy with drug resistant isolate of P. yoelii (MDR) on

ay 35 post-first immunization. In a manner similar to controlroup (PBS only), mice immunized with free-sAg, IFA-sAg or PC-sAguffered an abrupt increase in parasitemia with high mortal-ty (Figs. 9 and 10). Mice immunized with YL-sAg adapted lessevere courses of infection with significantly low parasitemia levels

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ig. 8. Determination of ROI level using flow cytometric analysis. The level of ROI was dete2DCFDA with macrophages isolated from animals immunized with YL-sAg and variousith IFA). The ROS has the ability to oxidize H2DCFDA to its fluorescent active form (DCFDA

Becton Dickinson) and data were analyzed by Cell Quest software (Becton Dickinson).

from animals immunized with liposomised sAg: co-expression of CD80 and CD86orescence-activated cell sorter (Becton Dickinson) and data were analyzed by Cell

p < 0.001 and p < 0.001, respectively). There was a delay of at least–3 days in attainment of peak parasitemia level in mice immu-ized with PC-sAg, or IFA-sAg when compared to non-immunizedice. As shown in Fig. 9, mice immunized with PBS or Sham lipo-

ome developed a progressive infection. On day 6 post-challengeith infection, parasitic burdens in all the control groups were

ound almost similar (2.7–3.9%), while in the YL-sAg immunizednimals, no parasitic load was detected.

Further, immunization with PC-sAg resulted in partial pro-ection (30%) on day 15, while 25% protection was observed inFA-sAg immunized group. On day 15 post-challenge with infec-

rmined using flow cytometric analysis. It was measured after 15 min incubation ofother forms of sAg (sAg entrapped in PC liposome or saccharosomes or emulsified). The fluorescence intensity was evaluated using fluorescence-activated cell sorter

480 V. Dwivedi et al. / Vaccine

Fig. 9. Prophylactic effect of various sAg vaccines against blood parasitic load.BALB/c mice were immunized with various forms of P. yoelii cytosolic antigen viz.YL-sAg or PC-sAg, or emulsified form of sAg with IFA (IFA-sAg). Control animalswere immunized with empty liposome (Sham) or PBS only. The immunized animalswere challenged with 1 × 106 P. yoelii (MDR) parasitized erythrocytes via tail veinon day 35 post-first immunization. The parasitic load in the experimental animalswas determined as described in Section 2. p-Value on day 10: IFA-sAg vs PC-sAg,p < 0.001; PC-sAg vs YL-sAg, p < 0.001.

Fig. 10. Prophylactic potential of saccharosome in terms of survival rate of immu-nized animals after challenging them with P. yoelii (MDR) infection. The animalswere immunized with various sAg-based vaccine as described in Section 2. Theappd

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nimals were subsequently challenged with 1 × 106 parasitized RBCs on day 35ost-first immunization. The animals were monitored for survival till day 20thost-challenge. Each group consisted of 10 animals. Data are representative of threeifferent experiments. p-Value on day 16 PC-sAg vs YL-sAg, p < 0.001.

ion, increased protection (80.7%) of animals was achieved in theroup of animals that were immunized with YL-sAg. The results ofhe study indicate that YL-sAg was the best combination for con-erring effective protection against blood-stage malaria in terms ofnd high survival rate of the immunized animals.

. Discussion

In general, humoral immune responses are mediated by circulat-ng antibodies that help in recognition and subsequent eliminationf the pathogen from systemic circulation. Some pathogens adapt

ntracellular parasitism as a strategy to avoid antibody-mediatedilling. To eliminate such pathogens the host immune system reliesn cell-mediated immunity. The cytotoxic T lymphocytes recognizeathogen derived processed peptide co-expressed along with MHColecules ensuing in killing of intracellular parasite.

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27 (2009) 473–482

As per their residential requirements, various life cycle stagesf P. yoelii have adapted themselves against both arms of hostmmune system. Among the liver and blood stages of P. yoelii, theatter seems to be of more strategic importance, as it is capable ofvoiding both humoral as well as cell-mediated immune responses.evertheless, attempts had been made to develop vaccinesgainst various stages of Plasmodium spp. Earlier studies basedn malaria parasite blood-stage antigens have produced mixedesults [5–8,32–37]. There seems to be further scope in improvinghe vaccine potential of cytosolic fraction of blood-stage malariaarasite.

It can be speculated that encapsulation of soluble antigens inusogenic liposome will result in both Type I and Type II antigenrocessing steps simultaneously that could favorably influence theut come of immune responses [12,13,21]. In an attempt to con-rm this hypothesis we specifically evaluated adjuvant potential of

iposome made up of the membrane lipid of S. cerevisiae.The first part of the study was conducted to examine potential

f saccharosomes to generate effective cell-mediated immunity inhe host. In contrast to conventional PC liposome, the enhancedmmunological responses elicited in the animals immunized withaccharosome encapsulated form of antigen may be due to itsccess to the cytosol of the APCs that eventually leads to activationf significant CD8+ CTL response. Besides, the fraction of anti-en delivered into endo-lysosome, entered into MHC-II restrictedntigen processing pathway that eventually helped in activa-ion of CD4+ T cells as well. In concordance with this fact, webserved that beside Th1-mediated activation of CD8+ T lympho-ytes; saccharosomes-based immunization leads to the generationf CD4+ Th-2 type cells as well.

The potential of saccharosome to induce cell-mediated immuneesponse was also assessed by determining DTH response in themmunized animals. The footpad thickness significantly increasedn the animals that were immunized with YL-sAg in comparisonf other control groups including those immunized with egg PC-Ag. Interestingly, there was a good correlation between elevatedootpads swelling in the group of animals immunized with YL-sAgo induction of Th1 cytokines viz. IFN-� (Fig. 5A). The cytokine IFN-

activate monocytes and macrophages and under the influencef some specific chemokines help in induction of DTH response38,39].

CD4+ Th1 and Th2 subsets have crucial role in B cell differ-ntiation, proliferation and isotype regulation [40]. Interestingly,n concordance with the above fact, the immunization of mice

ith YL-sAg was found to elicit both IgG1 and IgG2a responses;owever, the level of IgG2a was significantly higher when com-ared with groups of animals that were immunized with otherorms of sAg. This is clear indication that saccharosome-basedAg has the potential to activate both Th1/Th2 subpopulationsf the T helper cells. The ability of liposome to evoke antigenpecific immune response has been attributed primarily to anncreased uptake of antigen by antigen presenting cells and pro-essing and presentation of internalized antigen along with MHC/II surface molecules [41]. Normally, liposomes injected via sub-utaneous route do not have direct access to bloodstream sincehe permeability of blood capillaries is restricted to water-solublemall molecules [42]. Instead liposome are absorbed by lym-hatic capillaries and drained to site where antigen-presentingells are present. More importantly, lymphatic absorption stronglyepends on physical attributes of liposome-based particulate

elivery systems such as their size, charge and fusogenicity,tc.

The saccharosomes, encapsulated sAg induced excellentytokine and antibody response and also greatly enhanced pro-ective immunity against lethal P. yoelii strain. This was best seen

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ith a discriminating dose of sAg, which failed to induce protectivemmunity in free form or in combination of reference adjuvant suchs PC-Lip and IFA. This is presumably due to inability of PC-Lip andFA-based adjuvant to switch on cytokine expression in desirableirection. In contrast, same amount of antigen induced good protec-ive immunity when administered in saccharosomes encapsulatedorm. The superior protective response upon immunization withaccharosomes correlates well with the induction of early increasen IFN-� and its potential to induce both IgG2a and IgG1 antibodyubclasses [43,44]. In addition, the saccharosome-mediated immu-ization was found to successfully activate macrophages to expressumor necrosis factor alpha levels that might help in clearance ofarasites (data not shown). It has been earlier demonstrated thatesides other factors, IFN-� and TNF-� are crucial for protectiongainst blood-stage P. chabaudi infections [45]. As evident from theresent data, it seems both Th-1 and Th-2 T-cell subsets play activeole in resolving parasitemia. Alternatively, it is possible that acti-ated Th0 cells produce both cytokines, as elevated IFN-� responsesreceded those of IL-4 by 2 days (personal observation). In a pre-ious report describing the case of the self-resolving P. chabaudinfection, it was found that an initial Th1 response is replaced byh2 response that ultimately ensued in suppression of parasitemia46,47].

Although the expression of Th1 and Th2 cytokines was initiallyonsidered to be mutually exclusive, T-helper cells expressing bothh1 as well as Th2 cytokines have been identified during differenti-tion of Th cells as well as in terminally differentiated cells [48]. Thebserved sacchrosomes-mediated strong CD8+ T cell generation; inpite of polarization of CD4+ T cells response in favor of Th2 cells;an be attributed to the physical targeting of entrapped antigen tohe cytosol of APCs.

Earlier studies [49] have also demonstrated that genes for both,ype I and type II classes of cytokines are independently regu-ated and that any combination of them can be observed without

clear distinction along the Th1 and Th2 phenotype. It is well-stablished fact that both CTLs and neutralizing antibodies aressential for imparting protective immunity against intracellularathogens. Hence peculiar behavior of the saccharosome-basedelivery system may help in activation of both cell mediated asell as humoral immune responses [50].

The FACS results showed enhanced expression of CD80 andD86 on the surface of macrophages isolated from saccharosome

mmunized animals. Expression of co-stimulatory molecules isrucial in stimulating the optimum activation of T-cell signaling51,52]. The ability of saccharosome encapsulated antigen to up-egulate co-stimulatory molecules on the surface of macrophagesstablishes their adjuvant potential.

It is believed that phagocytic cells such as monocytes are impor-ant in the clearance of blood-stage parasites [53,54]. However, the

echanism by which monocytes and other phagocytic cells affectilling of parasites is as yet unknown. Super oxide and its deriva-ives such as hydrogen peroxide (H2O2) and *OH, which collectivelyre referred to as reactive oxygen species are released into phago-ome with the involvement of NADPH oxidase and are essential inhe killing of the ingested pathogen [55,56].

Nitric oxide (NO) is another chemical entity that is generatedy phagocytes to kill invading pathogens. The importance of NOn the immune response to hepatic stages of malarial infectionas been reviewed [57]. A role for NO in the killing of blood-stagealarial parasites in mice is less clear [58–60]. We therefore con-

idered it of interest to demonstrate role of YL-sAg in inductionf NO and ROS that ultimately help in parasite clearance, as haseen shown to occur during the host response to infection with the

ntracellular bacterium Rhodococcus aqui [61]. The data of this studyemonstrate direct correlation between saccharosome-mediated

[

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27 (2009) 473–482 481

nduction of NO production and elimination of parasite from immu-ized animals.

Finally, to establish vaccine potential of saccharosome encapsu-ated sAg, we performed protection studies by assessing parasiteurden in blood of immunized mice. The parasitemia was deter-ined at various time points during the course of infection. Mice

accinated with saccharosome-encapsulated sAg (YL-sAg) showedignificantly higher protection than other control groups. For exam-le, the protection conferred by various control sAg vaccines inerms of blood parasitemia was around 3%, while no parasite wasetected in blood of the animals which were immunized with YL-Ag by the day 6 post-challenge with infection. Immunization withC-sAg resulted in partial protection on day 15 (30%), while 25%rotection was observed in IFA-sAg immunized group. Higher lev-ls of protection were achieved in YL-sAg immunized animals onay 15 as well.

In conclusion, data of this study establish potential of saccharo-ome as an effective antigen delivery system to induce both cellulars well as humoral immune responses in the host simultaneously.ore importantly we also infer that saccharosomes-based vaccines

re more crucial for imparting protection against pathogens, whichvoid antibody mediated on slaught by seeking intracellular shelternside the cytoplasm of the cells.

cknowledgements

The authors express their gratitude to Dr. C.M. Gupta, Direc-or, CDRI Lucknow and Prof. M. Saleemuddin, Co-ordinator, IB Unit,MU, Aligarh for allowing us to avail research facilities to conduct

his work.

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