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Contents lists available at ScienceDirect

Vaccine

jou rn al hom ep age: www.elsev ier .com/ locat e/vacc ine

anoparticle formulation enhanced protective immunity provoked byYGPI8p-transamidase related protein (PyTAM) DNA vaccine inlasmodium yoelii malaria model�

ahamoud Sama Cherif a,c,f,1, Mohammed Nasir Shuaibua,c,1, Yukinobu Kodamab,ideon Kofi Helegbea, Mihoko Kikuchia, Akitoyo Ichinosed, Tetsuo Yanagie,itoshi Sasakib,c, Katsuyuki Yuig, Nguyen Huy Tienh, Juntra Karbwangh,enji Hirayamaa,c,∗

Department of Immunogenetics, Institute of Tropical Medicine (NEKKEN), Nagasaki University, JapanDepartment of Hospital Pharmacy, Nagasaki University, JapanGlobal COE Program, Institute of Tropical Medicine (NEKKEN), Nagasaki University, JapanElectron Microscopy Central Laboratory, Institute of Tropical Medicine (NEKKEN), Nagasaki University, JapanAnimal Research Center for Tropical Medicine, Nagasaki, JapanInstitut National de Santé Publique, Université de Conakry, GuineaDivision of Immunology, Department of Molecular Microbiology and Immunology, Graduate School of Biomedical Sciences, Nagasaki University, JapanDepartment of Product Development, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan

r t i c l e i n f o

rticle history:eceived 9 September 2013eceived in revised form3 November 2013ccepted 2 January 2014vailable online xxx

eywords:anoparticle

a b s t r a c t

We have previously reported the new formulation of polyethylimine (PEI) with gamma polyglutamicacid (�-PGA) nanoparticle (NP) to have provided Plasmodium yoelii merozoite surface protein-1 (PyMSP-1) plasmid DNA vaccine with enhanced protective cellular and humoral immunity in the lethal mousemalaria model. PyGPI8p-transamidase-related protein (PyTAM) was selected as a possible candidate vac-cine antigen by using DNA vaccination screening from 29 GPI anchor and signal sequence motif positivegenes picked up using web-based bioinformatics tools; though the observed protection was not complete.Here, we observed augmented protective effect of PyTAM DNA vaccine by using PEI and �-PGA complexas delivery system. NP-coated PyTAM plasmid DNA immunized mice showed a significant survival rate

PI8p-transamidaseolyethylimine (PEI)amma polyglutamic acid (�-PGA)lasmodium yoeliiNA vaccine

from lethal P. yoelii challenge infection compared with naked PyTAM plasmid or with NP-coated emptyplasmid DNA group. Antigen-specific IgG1 and IgG2b subclass antibody levels, proportion of CD4 andCD8T cells producing IFN-� in the splenocytes and IL-4, IFN-�, IL-12 and TNF-� levels in the sera and inthe supernatants from ex vivo splenocytes culture were all enhanced by the NP-coated PyTAM DNA vac-cine. These data indicates that NP augments PyTAM protective immune response, and this enhancement

ased

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was associated with incre

. Introduction

Please cite this article in press as: Cherif MS, et al. Nanoparticle formulationrelated protein (PyTAM) DNA vaccine in Plasmodium yoelii malaria model.

Malaria caused 216 million clinical episodes in 2010 [1]. Par-site and mosquito’s resistance remains a real and ever-present

� This is an open-access article distributed under the terms of the Creative Com-ons Attribution-NonCommercial-No Derivative Works License, which permits

on-commercial use, distribution, and reproduction in any medium, provided theriginal author and source are credited.∗ Corresponding author at: Department of Immunogenetics, Institute of Tropicaledicine (NEKKEN), Nagasaki University, Japan. Tel.: +81 95 819 7818;

ax: +81 95 819 7821.E-mail addresses: hiraken@nagasaki-u.ac.jp, hirakenhiraken@gmail.com

K. Hirayama).1 These authors contributed equally to this work.

264-410X/$ – see front matter © 2014 The Authors. Published by Elsevier Ltd. All rights ttp://dx.doi.org/10.1016/j.vaccine.2014.01.005

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DC activation and concomitant IL-12 production.© 2014 The Authors. Published by Elsevier Ltd. All rights reserved.

danger to successful malaria control. Vaccines are often the mostcost-effective tools for public health, and DNA vaccination is a sim-ple method for eliciting an immune response [2] and therefore apromising tool for malaria vaccine development.

Following a genome-wide search for a blood stage malaria DNA-based vaccine using web-based bioinformatics tools, we identifiedPyGPI8p-transamidase related protein (PyTAM) as secretory pro-tein containing GPI-anchor motif with protective immune response[3]. During the erythrocytic schizogony, the merozoite enters itshost red blood cell by an active invasion process that is mediatedby a glycosylphosphatidyl inositol (GPI)-anchored protein complex

enhanced protective immunity provoked by PYGPI8p-transamidaseVaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.01.005

which is distributed around the parasite surface and is composedof fragments of merozoite surface protein-1 (MSP1) plus associ-ated partner proteins [4–6]. Among several components of GPIcomplex, GPI8 is one of the catalytic components responsible for

reserved.

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leavage of GPI-attachment signal sequences [7,8]. Our identifiedyGPI8p-transamidase related protein (PyTAM) is considered asatalytic component responsible for cleavage of GPI attachment sig-al sequences in Plasmodium yoelii. We have previously shown thatyTAM DNA vaccine was partially protective with low antibodyroduction and the PyTAM recombinant protein could produceigh antibody titer but no observable protective effect [3]. Weave also reported that a novel nanoparticle (NP) delivery system

or malaria antigens is a promising strategy capable of promot-ng both cellular and humoral immune responses [3,9]. It has beeneported that nanoparticle complexed with �-PGA could deliverntigen directly to dendritic cells (DCs) and induce their maturation10,11].

In this study, we adopted mouse model of malaria (lethal P. yoeliind C57BL/6 mice), PyTAM DNA vaccine and the newly developedelivery system of PEI and �-PGA complex. The aim of this studyas to observe the effect of nanoparticle coating on the partiallyrotective PyTAM plasmid on induction of better immune response

n mice. This strategy may be a promising approach to augment theeported insufficient immune stimulation of many antigens thatre vaccine candidates.

. Methods

.1. Plasmid DNA construction and formulation with nanoparticle

Sequence of P. yoelii GPI8p transamidase-related proteinPY03470) from the Plasmodium genome database, PlasmoDBwww.plasmodb.org) [12] or PY03470 hypothetical protein (ID:431730) from the NCBI (www.ncbi.nlm.nih.gov) [13], was used forrimer design as described previously [3]. The PCR-amplified prod-ct was directly cloned into a BglII and BamHI restriction sites oflasmid VR1020 (Vical, San Diego, CA, USA) to obtain the constructVR1020-TAM. Plasmid DNA was analyzed by restriction digestionnd DNA sequence was confirmed by automated DNA sequenc-ng. Nanoparticle (PEI/�-PGA)-coated plasmid was formulated asreviously described [14].

.2. Recombinant protein (rPyTAM) expression and purification

The rPyTAM expression and purification was previouslyescribed [3]. Protein concentration was measured by BCA Proteinssay Reagent Kit (Pierce, Rockford, IL).

.3. Mice immunization and challenge schedule

Groups of six week old female C57BL/6 mice (SLC, Lab, Japan)ere used immunized intraperitoneally with different formula-

ions of the DNA vaccine designated as NP-formulated TAM plasmidNA, Naked-TAM plasmid DNA or NP-formulated empty plasmidNA. Mice were prime-immunized at day 0 and two subsequentoosters of 100 �g/mouse of plasmid DNA were given at days 21nd 42. Two weeks after the last immunization, sera were collectedrom all the mice and three mice per group were then sacrificedor flow cytometry and cytokines analysis. The remain mice werehallenged intraperitoneally with 105 of lethal strain of P. yoelii7XL-parasitized red blood cells (pRBCs) in 200 �l volume. A dailyhin blood smear was examined by microscopy for the presence ofarasites after Giemsa staining, from day 3 post challenge infec-

Please cite this article in press as: Cherif MS, et al. Nanoparticle formulationrelated protein (PyTAM) DNA vaccine in Plasmodium yoelii malaria model.

ion, until clearance of parasitaemia or otherwise death of mouse.ll animal experiments were approved by the Nagasaki University,oard of Animal Research, according to Japanese Guideline for usef experimental animals (Permit Number 0811130716).

PRESSxxx (2014) xxx– xxx

2.4. PyTAM specific IgG and its subtype antibody assessment(ELISA)

ELISA was performed to assess the immunoglobulin G and itssubtypes antibody responses from the individual sera collectedfrom immunized mice 14 days after last immunization as describedpreviously [15].

2.5. Flow cytometric analysis of spleen and lymph node cells

Spleens and systematic lymph nodes were removed asepticallyfrom each mouse and cell suspension prepared. After several wash-ing, a tube containing 106 splenocytes or lymph node cells werestained with Fc-� blocking antibody, and incubated for 15 min at4 ◦C and then stained with APC-CD11c and FITC-MHC-II and PEconjugated with CD40, CD80 and CD86 and respective isotype con-trols (eBioscience, San Diego, CA) then washed and resuspended in500 �l of FACs buffer and acquired on the FACS machine (BeckmanCoulter, Fullerton, CA, USA). Phenotype analyses were performedusing Flowjo software (Tree Star, Inc., OR, USA). Proportion of DCsamong the gated CD11c+ MHC II+ cells, then conventional DC(expressing MHC-II and CD11chigh) and others cells (expressingMHC-II and CD11cint) and costimulatory molecules were analyzed.

2.6. IFN-� intracellular staining

One million (106) splenocyte/ml were cultured in RPMI-1640medium supplemented with 10% FCS-RPMI for 6 h at 37 ◦C, under5% CO2 in the presence of 50 ng/ml phorbol-myristate-acetate(PMA) and 250 ng/ml ionomycin (Sigma, St Louis, MO, USA) as pos-itive control, 5 �g/ml of rPyTAM and medium as negative control.After 6 h incubation, cells were washed twice in PBS, and subse-quently stained for 30 min at 4 ◦C with CD3, CD4 and CD8 mAbs (BDBiosciences, CA, USA), respectively. Cells were then permeabilizedwith 0.1% saponin (Sigma, St Louis, MO, USA). Finally, cells wereincubated for 30 min at 4 ◦C with PE-labeled anti-IFN-� antibody.IFN-� producing CD4 and CD8 phenotype analysis was performedusing Flowjo software (Tree Star, Inc., OR, USA).

2.7. Analysis of plasma levels and antigen specific cytokinesproduction from spleen cells cultured supernatants

Cell suspensions obtained from C57BL/6 vaccinated mice fol-lowing their sacrifice, were adjusted to a final concentration about106 cells/ml and cultured in 10% FCS-RPMI 1640, at 37 ◦C in ahumidified incubator with 5% CO2 for 48 h with either 0.5 �g/mlof recombinant PyTAM protein, concanavalin A or medium onlyand then, the supernatant was collected and stored at −80 ◦C untiluse.

IL-4, IFN-�, IL-12 and TNF-� levels in the supernatants ofantigen-stimulated splenocyte and sera from immunized micewere measured using Procarta® Cytokine Assay Kit (Affymetrix,Inc., Santa Clara, CA, USA) according to the manufacturer’s instruc-tions and previously described [9].

2.8. Immunofluorescence and confocal imaging

Slide wells were smeared with about 10% hematocrit of 15–20%pRBC and fixed in cold acetone or methanol/acetone (1:1) thenincubated with blocking buffer and forty times diluted PyTAM DNAvaccine -immunized mice serum was added and incubated for 3 hat room temperature. Slides were washed and then incubated for

enhanced protective immunity provoked by PYGPI8p-transamidaseVaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.01.005

30 min with 25 �l of FITC-conjugated goat anti-mouse IgG serum(Sigma Chemical Company, St. Louis, MO) diluted 1:50 in PBS. Slideswere visualized under conventional wide field optical epifluores-cence or confocal laser microscope (Leica Microsystems).

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.9. Immunoelectron microscopy

Blood stage P. yoelii from C57BL/6 mice at about 30–40% par-sitaemia was washed three times in PBS and then fixed in 4%araformaldehyde, 0.5% glutaraldehyde and 2.5% sucrose in PBSpH 7.4) for 2 h at 4 ◦C. The fixed cells were then dehydratedy ascending ethanol concentrations. Thin sections with thick-ess of 80 nm were made then blocked with 5% skimmed milk

n PBST and incubated with immune serum from PyTAM DNAaccine-immunized mice at 4 ◦C overnight. The sections were thenncubated with goat-anti-mouse IgG gold particles and then stained

ith 1% osmium for about 5 min and then with 6% uranyl acetate for min and the rinsed with distilled water. The sections were carbonoated then examined in Digital Transmission Electron MicroscopeDTEM) – JEOL JEM-1230 (JEOL Ltd., Tokyo, Japan).

.10. Statistical analyses

GraphPad Prism software version 5.0 was used andann–Whitney test for direct comparison between groups of

mmunized mice. Data are presented as mean ± standard deviationSD). Kaplan–Meier test for survival rate comparison between theroups of immunized mice. P < 0.05 was considered statisticallyignificant for the two-tailed test.

. Results

.1. NP-formulated TAM plasmid DNA vaccine suppressedarasitemia and prolonged survival

Please cite this article in press as: Cherif MS, et al. Nanoparticle formulationrelated protein (PyTAM) DNA vaccine in Plasmodium yoelii malaria model.

Parasitemia determined in Giemsa-stained thin blood smearshowed that group of mice immunized with NP-formulated emptylasmid DNA (Fig. 1A) and naked TAM plasmid DNA (Fig. 1B) devel-ped high parasitemia and were not able to control the challenge

ig. 1. Six weeks old C57BL/6 mice were divided in three groups and were intraperitoneanjection of 105 pRBCs and parasitemia monitored daily. Each line in Fig. 1A–C representNA, (B) NP-formulated empty plasmid DNA or (C) NP-formulated TAM plasmid DNA. (Designated as p < 0.05. This is a representative result of 3 different independent experime

PRESSxxx (2014) xxx– xxx 3

infection (Supporting Fig. 1) as compared to the group of mice

immunized with NP-formulated TAM plasmid DNA (Fig. 1C). Theaverage of survival rate in three different experiments showedan increase of mortality in the group of mice immunized withNP-formulated empty plasmid DNA (27.5% of average survival ofthe three independent experiments) and naked TAM plasmid DNAimmunized mice (12.5%) as compared to those mice immunizedwith NP-formulated TAM plasmid DNA (78.9%) (Supporting Table1). The survival rate in the NP-formulated empty plasmid DNAimmunized group could be explained by the fact that nanopar-ticles are often, after being up-taken, stimulate innate immunitythrough the innate signaling pathways. Kaplan–Meier test showeda significant difference of survival rate between the groups of miceimmunized with NP-formulated TAM plasmid DNA as compared tothose mice immunized with naked TAM plasmid DNA (p < 0.0001)or NP-formulated empty plasmid DNA group (p < 0.001) (Fig. 1D).

3.2. NP-formulated TAM plasmid DNA vaccine elicited IgG1 and2b against P. yoelii infection

Two weeks after the last immunization, sera were collected andantigen-specific IgG titers determined by ELISA. We observed thatthe group of mice vaccinated with NP-formulated TAM plasmidDNA showed significantly high titer of IgG and its subclass spe-cific to recombinant PyTAM as compared to those mice immunizedwith Naked TAM plasmid DNA. IgGl and IgG2b Abs were predom-inant subclass with comparatively higher levels in the group ofNP-formulated TAM plasmid DNA immunized mice (Table 1).

3.3. Antigen specific cytokines production

enhanced protective immunity provoked by PYGPI8p-transamidaseVaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.01.005

To further explore the effectiveness of the NP-formulation andmalaria DNA vaccine on cytokines production, we investigated thelevels of cytokines (IL-4, IFN-�, IL-12, and TNF-�) in the immune

lly immunized 3 times at 3-week intervals, and then challenged by intraperitoneals individual parasitemia profile of mice immunized with (A) Naked-TAM plasmid) Survival curves analyzed by using Kaplan–Meier test. Statistical significance wasnts.

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Table 1Effect of nanoparticle coating on IgG and its subclass antibody production.

Naive Naked-TAM plasmid DNA NP-formulated empty plasmid DNA NP-formulated TAM plasmid DNA

IgG 0.02 ± 0.01 0.04 ± 0.01 0.03 ± 0.01 0.08 ± 0.04*

IgG1 0.14 ± 0.01 (56%) 0.14 ± 0.04 (48.2%) 0.15 ± 0.06 (44.1%) 0.52 ± 0.20* (48.5%)IgG2a 0.03 ± 0.02 (12%) 0.02 ± 0.01 (6.8%) 0.03 ± 0.02 (8.8%) 0.06 ± 0.04* (5.6%)IgG2b 0.08 ± 0.01 (32%) 0.13 ± 0.03 (45%) 0.16 ± 0.08 (47.1%) 0.49 ± 0.29* (45.9%)

Two weeks after the last immunization, sera were collected from immunized mice and specific IgG and its subclass antibody titers measured by ELISA as OD450nm at 1:40dilution using recombinant PyTAM as antigen. Values are from three independent measurements. Man Whitney test was used to find difference between NP-formulatedTAM plasmid DNA vs Naked-TAM plasmid DNA group and NP-formulated TAM plasmid DNA vs NP-formulated empty plasmid DNA. Statistical significance was designatedaN isotyp

AM pl

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s p < 0.05.B: Values are OD, shown as mean ± SD (. . .). The percentage contribution of each

* Significant difference between NP-formulated TAM plasmid DNA and Naked–T

era collected two weeks after the last boost. Plasma levels of theseeasured cytokines showed low level of IL-4 and IFN-� and high

evel of IL-12 and TNF-�; and increase of IL-12 and TNF-� levelsn the group of mice immunized with NP-formulated TAM plasmidNA as compared to those immunized with Naked-TAM plasmidNA. However, no significant difference of cytokines in the seraas observed between NP-formulated TAM plasmid DNA and NP-

oated empty plasmid DNA group (Fig. 2).We further investigated the levels of the cytokines in the super-

atant from splenocytes and LN cells incubated for 48 h eitherith concanavalin A (as positive control), recombinant PyTAM

r medium (as negative control). We observed that, a signifi-ant increase of IL-4 and IFN-� levels in the cultured splenocytesupernatant co-incubated with rTAM from mice vaccinated withP-formulated TAM plasmid DNA as compared to those from mice

mmunized with Naked TAM plasmid DNA or NP-formulated emptylasmid DNA. Whereas, IL-12 and TNF-� were increased in the cul-

Please cite this article in press as: Cherif MS, et al. Nanoparticle formulationrelated protein (PyTAM) DNA vaccine in Plasmodium yoelii malaria model.

ured splenocytes supernatants from mice immunized with eitherP-formulated TAM plasmid DNA or NP-formulated empty plas-id DNA compared with Naked TAM plasmid. This suggested that

anoparticle itself stimulated the production of those cytokines

ig. 2. Plasma levels of T-cell-derived cytokines (IL-4 and IFN-�) and APC-derived cytokinfter the last boost. The graphs represent the amount of cytokine in the immune sera beetween NP-formulated TAM plasmid DNA vs Naked TAM plasmid DNA or NP-formulated

s p < 0.05 (*p ≤ 0.05; **p ≤ 0.01 and ***p ≤ 0.001).

e.asmid DNA or NP-formulated empty plasmid DNA group.

in an antigen-independent manner. This phenomenon is prob-ably associated with the IL-12 production by DC or TNF-� bymacrophage (Supporting Table 2). However, antigen-driven effectis also very clear as there was significant difference between theNP-formulated TAM plasmid DNA and NP-formulated empty plas-mid DNA (Supporting Table 2). This observation suggests that anonspecific stimulation could be induced by NP coating (e.g. in theNP-plasmid group). The mechanisms by which this nanoparticleformulation interacts with the immune system are not well under-stood, however, this observation suggests that NP could induceinnate immune stimulation in antigen-independent manner.

3.4. T cell population and IFN-� production

To determine whether the NP has any effect on splenic T cellpopulations, we investigated IFN-� production using intracellularstaining of spleen cells collected two weeks after the last boost.

enhanced protective immunity provoked by PYGPI8p-transamidaseVaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.01.005

A significant increased proportion of IFN-� producing CD8T cellsas well as CD4T cells, were observed in the group of mice immu-nized with NP-coated TAM plasmid DNA as compared to the groupof mice immunized with Naked TAM plasmid DNA or NP-coated

es (IL-12 and TNF-�) determined by multiplex assays from sera collected 2-weeksfore challenge and express as mean ± SD. Student test was used to find differenceTAM plasmid DNA group of immunized mice. Statistical significance was designated

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Fig. 3. Intracellular cytokine staining analysis for IFN-� production from splenocytes of immunized mice. A flow cytometry analysis using cell surface markers (CD4-FITCa 0 ng/mT ucingC

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nd CD8-PE) and intracellular IFN-�- APC staining in cells stimulated either with 5AM (5 �g/ml) or without any stimulation (Medium). (A) Percent of CD4 T cell prodD4 or CD8 positive cells producing IFN-�.

mpty plasmid DNA (Fig. 3). This observation suggests that NP-oated TAM plasmid DNA induced type one CD4 helper T cells andFN-� producing CD8T cells.

.5. DC profile in the spleen and lymph nodes

Because of the high level of IL-12 production observed, weurther explored whether DC maturation could be induced byanoparticle formulation. The phenotype of DC is both CD11chigh+nd MHC-II+ on their surface. As shown in Fig. 4, FACS analy-is revealed that group of mice immunized with NP-formulatedlasmid DNA showed no significant difference in number of DCopulation (expressing CD11chigh+ and MHC-II+) in the spleenFig. 4A). However, an increased number of CD11c+ and MHC-II+,articularly DC population (expressing CD11chigh+ and MHC-II+)as observed in the lymph nodes (Fig. 4B–D). The phenotype ofD11cint+ and MHC-II+ expressing cells are considered as non-onventional DC and may be a mixture of plasmocytoid DC and

cells. Additionally, we have observed a significant increase ofD86 expression among the LN cells expressing double CD11c hind MHC-II (Fig. 4D).

.6. Immunofluorescent staining and immuno-electronicroscopy of PyTAM

To characterize the localization of PyTAM during the blood stagenfection, we performed immunofluorescent staining and confocalmaging using mouse anti-PyTAM anti-serum. We found that theyTAM protein was revealed to be expressed at the ring and earlychizont stage, and located in the cytosol near the parasite nucleusFig. 5A). However, at the late schizont stage, it diffuses on the para-itophorous vacuole, as observed by immuno-electron microscopicmages (Fig. 5B).

. Discussion

Please cite this article in press as: Cherif MS, et al. Nanoparticle formulationrelated protein (PyTAM) DNA vaccine in Plasmodium yoelii malaria model.

As a vaccine delivery system, nanoparticle with PEI plus �-PGAi-layered has been reported to have several significant characteris-ics. Firstly, the antigens or DNA can be encapsulated and protected

l of Phorbol-Myristate-Acetate (PMA) and 250 ng/ml of Ionomycin, recombinant IFN-�. (B) CD8T cell producing IFN-�. Results are expressed as proportion of gated

from biological degradation and can be delivered by various routesof administration. Secondly, the nanometer scale and chemicalcharacteristics of �-PGA make it easy to be up-taken by immunecells. Finally, the endosome disruption enables cross presentationof antigen for eliciting both CD4 and CD8T cell responses [16]. Theseare the major reasons why we used NP for our malaria DNA vaccinedevelopment.

In the series of DNA vaccine studies, NP-formulation wasrevealed to have several advantages. (1) Relatively easy and simplepreparation of the recombinant plasmid DNA, (2) simple procedureof NP formation, (3) efficient way to stimulate DC, and both type 1and 2 T cells [1,16,17]. In the present study, we have confirmed theenhancement of survival of NP-formulated TAM plasmid DNA com-pared with naked TAM plasmid DNA vaccine. This enhancement iscorrelated with a significant increase of DCs expressing CD11chigh

and MHC-II and concomitant induction of APC-derived cytokine,IL-12 and T cells-derived IFN-� in NP-formulated TAM plasmidDNA-immunized mice in contrast to the group of mice immunizedwith the Naked-TAM plasmid DNA and NP-coated empty plasmidDNA. Therefore, NP enhanced both humoral and cellular immu-nity in an antigen-specific manner. Gene delivery systems basedon NPs formulation is considered as a novel vaccination tool thatoffers substantial flexibility for several disease models [20–22].

As we have reported, PyTAM conferred partial protection inmice with low antibody production, following their administrationas DNA vaccine; and no protection was observed following theiradministration as recombinant protein, even though high anti-body production was observed [18]. We therefore conclude that thePyTAM mechanism of protection was not dependent on the anti-body levels but more on an effective T cell immunity. In human,Pombo et al. [24]; showed that immunity in people is character-

ized by a T helper 1 like response, with induction of interferon �,greatly raised concentrations of nitric oxide synthase in peripheralblood mononuclear cells, in the absence of detectable antibodies[24]. This suggests that, the splenic destruction of infected RBCs

enhanced protective immunity provoked by PYGPI8p-transamidaseVaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.01.005

is a major mechanism of parasite clearance involving macrophageand other immune cells than blocking the invasion of merozoiteinto RBCs by specific antibody as was strongly suggested by histor-ical experiments of adoptive transfer of anti-cell surface proteins

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Fig. 4. DC profile in the spleen and lymph nodes. A representative profiles of the three independent experiments of DC surface markers (CD11chigh and MHC class II) analysisusing spleen cells (A) and lymph node cells (B). Proportions of conventional DC (expressing CD11c high/MHC-II+) in the LN (C) and proportion of other cells expressingCD11cint/MHC-II+ (D) and the proportion of cells expressing costimulatory molecules among the gated CD11chi+ MHC II+ cells population in the LN (D). Spleens and LN werecollected two week after the last boost. Values are shown as mean ± SD values from the three independent experiments. Student test was used to find difference betweenN ted TAd ning n

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P-formulated TAM plasmid DNA vs Naked-TAM plasmid DNA group or NP-formulaesignated as p < 0.05 (*p ≤ 0.05; ** p ≤ 0.01; ***p ≤ 0.001 and ****p ≤ 0.0001; ns mea

f merozoites monoclonal antibodies [24–27]. Recently, cellularmmunity is getting more attention in the development of novelaccines either for the disease therapy or for protection [28,29].arasites and parasite-infected red blood cells activating dendriticells through pathogen recognition receptors (PRRs) are phagocy-osed and their antigens are presented to T cells. PRR signalingeads to the secretion of cytokines and direct Th1 cell differenti-tion. Th1 cells provide help for B cell differentiation and antibodyecretion. IFN-�-activated macrophages phagocytose opsonized-arasites and infected red blood cells, and subsequently kill themy NO- and O2-dependent pathways [23].

Many strategies have been tried to improve immunogenicity ofntigens including the use of adjuvants [32], cytokines [33], incor-oration of immuno-stimulatory sequences in the backbone of thelasmid [34,35], co-expression of stimulatory molecules [36–38],nd utilization of the appropriate delivery system [14,39]. Theeason why we conducted this study was to improve the immuno-

Please cite this article in press as: Cherif MS, et al. Nanoparticle formulationrelated protein (PyTAM) DNA vaccine in Plasmodium yoelii malaria model.

enicity of PyTAM previously known to be weakly immunogenic.n fact, the major problem encountered in recent malaria vaccineevelopment is a low immunogenicity. Therefore, DNA plasmidoating by nanoparticle may be a good solution to overcome serious

M plasmid DNA vs NP-formulated empty plasmid DNA. Statistical significance wason-significant).

problem of insufficient immune response after the normal vaccina-tion protocol.

Nanoparticle interaction with immune system, in general, isstill not clearly understood. In this study, we observed that anti-body production, interferon-� producing CD4 and CD8T cells aswell as DC population in the LN are well associated with protection.However, the non-specific immune response observed in cytokineslevels in the plasma and cultured splenocytes supernatant, couldbe explained by the fact that nanoparticles are often first picked upby the phagocytic cells of the immune system (e.g. macrophages,dentritic cells), after being up-taken, stimulate innate immunitythrough the innate signaling pathways; and any inadvertent recog-nition of nanoparticles as foreign by the immune cells may resultin a multilevel immune response, such as immunosuppression,immunostimulation or nonspecific responses [16,40–42]. On theother hand, some studies suggested that the �-PGA coating onPEI/DNA complexes is known to significantly enhance their cellu-

enhanced protective immunity provoked by PYGPI8p-transamidaseVaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.01.005

lar uptake via a gamma-PGA-specific receptor-mediated pathway[43]. Also, after internalization, a large part of this PEI/DNA/�-PGAcomplex avoids entry into lysosomes. Very small percentage ofinternalized ternary complex was observed to co-localized in the

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M.S. Cherif et al. / Vaccine xxx (2014) xxx– xxx 7

Fig. 5. (A) Immuno-fluorescent staining images of blood stage fixed parasites showing ring and early schizont stages. All the panels depict PyTAM staining (FITC) in green colora Immua he gol

loi[iahssiioGinac

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nd nuclear staining (PI) in red color as analyzed by confocal laser microscopy. (B)nd accumulation of PyTAM. Dark spots indicating the localization of PyTAM onto t

ysosome [16]; and therefore, this might explain the promotionf transfection efficiency of the complex. Another crucial questions the safety issue of this delivery system. As previously reported14], this nanoparticle was shown to be safer and compatible withmmune system except for the fact that PEI itself is highly toxicnd stable in the body. Because the safety issues are critical foruman application, as such short- and long-term toxicity should beeriously considered. The significant finding here, is that NP couldignificantly enhance the protective effect of PyTAM DNA vaccinen murine model, and this enhancement was associated with thencreased of INF-� producing T cells and CD11c+ and high levelsf MHC class II+ expressing cell populations in the lymph nodes.uermonprez et al. suggested that an effective vaccine is the one

Please cite this article in press as: Cherif MS, et al. Nanoparticle formulationrelated protein (PyTAM) DNA vaccine in Plasmodium yoelii malaria model.

nducing DCs maturation and its migration to the regional lymphodes [44]. Maturation of DCs is required for antigen-processingnd presentation to other immune cells. This final maturation isharacterized by the ability to process antigen, express CD11c+ and

no-electron microscopy images accumulation depicting stage specific expressiond particles. Mz and Hz are meroite and hemozoin, respectively.

high levels of MHC class II+ (antigen presentation) and costimula-tory molecules such as CD40 (TNF-� family), CD80 (B7-1) and CD86(B7-2) and an abundant IL-12 production [45–47].

Among the cell populations that appear to be present in lymphnodes and spleen, two main subsets CD11c expressing cells havebeen defined. The first is CD11clow/int DC, including plasmocytoidDC and/or B cells [12,13] and the second subset is CD11chigh DC,identified as conventional DC. In this study we use CD11c and itsrelative expression level (high and low/intermediate CD11c expres-sion). Mouse CD11chigh DC have been best studied in spleen andcorrespond to the original Steinman DC identified 30 years ago [50]and have been shown to be necessary for activation of naive CD4 (+)CD8 (+) T cells in vivo [51] and capable of releasing large amounts

enhanced protective immunity provoked by PYGPI8p-transamidaseVaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.01.005

of IL-12, which can stimulate a Th1 cell differentiation [52,53].Another important question is what PyGPI8p-transamidase

related protein (PyTAM) functionally and anatomically does duringthe infection? It is not immediately clear whether it is expressed on

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he surface of parasite or parasitized red blood cell and whether itas a similar enzymatic activity as transamidase. The GPI transami-ase (GPI8p) that mediates GPI attachment has not been clearlyharacterized [7]. GPI glycolipid serves to anchor many surface pro-eins to the plasma membrane such as the abundant MSP-1 proteinshat is directly involved in erythrocyte invasion by the merozoites54]. However, using mouse anti-PyTAM antibody, this protein wasevealed to be expressed in the schizont stage parasite and locatedn the parasitophorous vacuole by immuno-electron microscopyFig. 7B). Therefore, targeting this GPI8p protein will hamper thisritical step in the life cycle of the parasite.

. Conclusion

NP delivery system was shown to significantly enhance the pro-ective effect of PyTAM DNA vaccine in murine model, and thisnhancement was associated with the increase of DCs activation.herefore, by using this system, PyTAM is proven to be a promisinglood stage vaccine candidate antigen in animal model. However,hether the human malaria orthologue (PF3D7 1128700) [25] of

yTAM could be a good candidate for blood stage human malariaaccine, that remains to be properly explored.

ncited references

[19,30,31,48,49].

cknowledgments

This work was supported by Grants-in-Aid for the GCOE pro-ram, Nagasaki University, Japan. We would like to thank Evaristushibunna Mbanefo for some technical assistance and Vical incorpo-ated for providing the vector VR1020 (Vical, San Diego, CA, USA).

Conflict of interest: The author(s) declared no conflicts of interestith respect to the authorship and/or publication of this article.

ppendix A. Supplementary data

Supplementary data associated with this arti-le can be found, in the online version, atttp://dx.doi.org/10.1016/j.vaccine.2014.01.005.

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