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Vaccine 29 (2011) 1455–1462

Contents lists available at ScienceDirect

Vaccine

journa l homepage: www.e lsev ier .com/ locate /vacc ine

n ocular mucosal administration of nanoparticles containing DNA vaccineRSC-gD-IL-21 confers protection against mucosal challenge with herpesimplex virus type 1 in mice

ai Hua,b,1, Jun Doua,∗,1, Fangliu Yua,1, Xiangfeng Hea, Xianwen Yuana, Yaqing Wanga,hunsheng Liua, Ning Guc,∗∗

Department of Pathogenic Biology and Immunology, Medical School, Southeast University, Nanjing 210009, ChinaDepartment of Ophthalmology, Affiliated Zhong Da Hospital, Southeast University, Nanjing 210009, ChinaSchool of Biological Science & Medical Engineering, Southeast University, Nanjing 210096, China

r t i c l e i n f o

rticle history:eceived 24 August 2010eceived in revised form9 November 2010ccepted 9 December 2010vailable online 24 December 2010

eywords:

a b s t r a c t

Herpes stromal keratitis (HSK) is a chronic inflammatory process caused by the infection of herpes sim-plex virus type 1 (HSV-1). Development of a HSV-1 vaccine is a priority because these infections arecommon and cannot be well prevented. It appears that the potential of nanocarriers in DNA vaccinationwill be required to augment the immune response to DNA vaccines. Therefore, in the study, nanoparticlesFe3O4 coated with glutamic acid, DNA vaccine pRSC-gD-IL-21 and polyethylenimine were prepared andimmunized in the mice by ocular mucosal administration. The immune responses and protection effi-ciency against HSV-1 challenge were also tested. The results showed that the nanoparticles containing

erpes stromal keratitisNA vaccineanoparticles interleukin-21lycoprotein D

DNA vaccine pRSC-gD-IL-21 induced mice to generate higher levels of specific neutralizing antibody, sIgAin tears, and IFN-�, IL-4 in serum, and to enhance the cytotoxicities of NK cells and splenocytes as wellas splenocyte proliferative response to glycoprotein D compared with those of the control mice. Moreimportantly, the mice immunized with the experimental vaccine showed less HSK degree than that ofthe control mice after HSV-1 challenge of the murine ocular mucosa. In conclusion, an ocular mucosaladministration of nanoparticles containing DNA vaccine confers strong specific immune responses and

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effective inhibition of HS

. Introduction

Herpes stromal keratitis (HSK) is the most common cause oforneal blindness in the world. Herpes simplex virus type 1 (HSV-) first infects corneal epithelium, followed by entering the sensoryerve endings, travelling along axons, and getting to the trigem-

nal ganglion where it becomes latent. It is widely believed thathe recurrent keratitis is due to the reactivation of HSV-1 from theatent state in the proximate sensory ganglia. The re-infection ratef ocular HSV-1 within 2 years ranges from 23% to 33%, and about0–25% of those develop T-cell-mediated HSK, the most frequent

ause of unilateral blindness [1]. Although antiviral or immuneegulatory therapy can reduce the incidences of reactivation andsymptomatic viral shedding, it cannot cure HSV-1 infection [2]. Argent need exists for safe and effective prophylactic treatments to

∗ Corresponding author. Tel.: +86 25 83272454; fax: +86 25 83272295.∗∗ Corresponding author.

E-mail addresses: njdoujun@yahoo.com.cn (J. Dou), guning@seu.edu.cn (N. Gu).1 Authors Hu, Dou and Yu contributed equally.

264-410X/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.oi:10.1016/j.vaccine.2010.12.031

HSV-1 infected murine model.© 2010 Elsevier Ltd. All rights reserved.

prevent or minimize the corneal scarring, vision loss, discomfort,and glaucoma that often result from HSK recurrences [3].

Recently, a growing body of literatures shows that an immuno-prophylaxis of primary herpetic infection is a rational approachto deal with recurrent HSK. Several attempts have been made todevelop new types of vaccines including DNA vaccines and sub-unit types of vaccines [4–6]. DNA vaccine is a very powerful andeasy method for the induction of strong humoral and cell-mediatedimmune (CMI) responses in mice. By virtue of its strong capac-ity to induce CMI responses, this vaccine approach is particularlyattractive for the prophylaxis of intracellular pathogens, such asMycobacterium tuberculosis, hepatitis C virus and HSV [7–9]. How-ever, one of the drawbacks of DNA vaccine encoding the singleantigen is that it does not produce a great protective effect. Alsoan immunogenicity of the DNA vaccine in primates is lower thanthat in rodents [10]. DNA transport through the cell membrane

is an essential requirement for efficient DNA vaccination, there-fore, another drawback of DNA vaccine is that the cell membranetransport of DNA is an inefficient process, and the mechanisms ofthis process is not clear [11]. In these regards, most efforts relatedto DNA vaccines are currently focusing on utilization of molecu-

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ar adjuvant that can significantly improve the immunogenicitynd efficacy of DNA vaccines. This adjunctive modality could bemportant to obtain the optimal responses in DNA vaccine strategy12–14]. Accumulating evidence has also shown that the barrier ofhe membrane transport of DNA can be overcome by properly beingtructured nano sized particles (nanoparticles) that may avoid DNAegradation and facilitate targeted delivery to antigen presentingells [11,15,16]. The use of nanoparticles for vaccine delivery is alatform technology, applicable to delivery of a variety of existingnd potential vaccines [17].

In previous study from our laboratory, we showed that theNA vaccine expressing HSV glycoprotein D (gD) antigen and

nterleukin-21 (IL-21) (incorporated as a molecular adjuvant)nduced an comparable better immune protection efficacy againstSV-1 challenge on cornea in mice than that of the DNA vaccinexpressing HSV gD alone [18]. IL-21 was selected as a moleculardjuvant because IL-21 plays a role in a wide range of relevantiological activities, including the activation and proliferation ofD8 T cells and NK cells, and it is also a costimulator for enhanc-

ng memory lymphocyte responses and modulating immunologicalomeostasis [19–21]. IL-21 enhances immunogenicity of DNA vac-ine eliciting protection efficacy against Mycobacterium tuberculosishallenge in mouse model [13]. In this study, we have extended ourtudy of the DNA vaccine pRSC-gD-IL-21 by the development ofanoparticles Fe3O4 coated with glutamic acid, DNA vaccine pRSC-D-IL-21 and polyethylenimine (PEI), which could be capable ofvercoming the barrier to the cell membrane transport of DNA.mportantly, we found that the use of nanoparticles containing DNAaccine improved its immunogenicity and obviously prevented arimary ocular HSV-1 infection in mice.

. Materials and methods

.1. Mice

Balb/c mice, female, 5–6 weeks, which were ordered from thenimal Center of Yang Zhou University of China. The mice wereaised at the Experimental Animal Center, Medical College, South-ast University, Nanjing, China, under pathogen-free conditionsn air filtered containers. All animal experiments were conductedollowing the guidelines of the Animal Research Ethics Board ofoutheast University, China.

.2. Cell lines and viruses

Vero cells derived from African green monkey kidney, SP2/0 cellserived from Balb/c mice myeloma cells and YAC-1 cells derivedrom Moloney leukemia-induced T-cell lymphoma, were obtainedrom Cellular Institute of China in Shanghai and were grown at7 ◦C in 5% CO2 atmosphere in RPMI 1640 supplemented with 10%etal bovine serum containing 100 units/ml penicillin G sodium and00 �g/ml streptomycin sulfate. HSV-1 strain F was ordered fromhe Institute of Virology in Wuhan, China, propagated and plaquessayed on Vero cells in the study.

.3. Preparation of PEI-plasmid DNA-Fe3O4-Glu nanoparticles

Nanoparticles of Fe3O4 coated with glutamic acid (nano-Fe3O4-lu) were a gift from Dr. Chen Zhongping in School of Biologicalcience & Medical Engineering, Southeast University of China,hich has no cytotoxicity to cell lines in vitro [22]. PEI was obtained

rom Sigma (Steinheim, Germany). The genes of gD and IL-21 wereonstructed under two different expression cassettes with two dif-erent promoters in the same vector to form the plasmid DNAaccine pRSC-gD-IL-21 that encodes the gD and IL-21 separately.he plasmid DNA vaccine pRSC-gD encodes the gD alone. These

2011) 1455–1462

plasmid DNA vaccines were constructed by our laboratory [13,18].The nanoparticles combined with DNA vaccine pRSC-gD-IL-21 wereprepared by vortexing the mixtures of 5 ml of the nano-Fe3O4-Gluto 1 ml PEI (5:1 ratio) for 10 min firstly and then vortexing themixtures with 2 �g/1 �l of DNA vaccine pRSC-gD-IL-21 (2:1 ratio)for another 10 min. The solution of nanoparticles mixtures was pH7.2–7.4 [23]. The nano-Fe3O4-Glu particles were photographed bythe transmission electron microscope (TEM) as shown in A of Fig. 1.

2.4. Transfection in cultured cells and Western blot

For transfection experiments, 20 �l of the nanoparticles mix-ture suspensions were incubated with 1 × 106 SP2/0 cells in6-well plates (10 �g DNA vaccine pRSC-gD-IL21/well) in completemedium for 4 h at 37 ◦C. The cells were washed with RPMI 1640 toremove the unbound nanoparticle mixtures and were further incu-bated for 48 h. As a reference, 1 × 106 SP2/0 cells were transfectedwith the 10 �g DNA vaccine pRSC-gD-IL-21 or 10 �g mock plasmidpRSC by LipofectamineTM 2000 reagent (Invitrogen, USA) accordingto the manufacturers instructions. Gene expressions were con-firmed by Western blot. The membranes were rinsed for 5 minwith antibody (Ab) wash solution 3 times before the rabbit anti-mouse gD Ab or a goat anti-mouse IL-21 Ab were added, followedby the incubating for another 1 h. The following steps were per-formed according to the Western-Breeze Kit’s protocol (Invitrogen)[22,24,25].

2.5. Immunization and challenge

For the vaccination experiments, Balb/c mice were randomlydivided into pRSC-group (mock plasmid), pRSC-gD group, pRSC-gD-IL-21 group and pRSC-gD-IL-21+ nanoparticles group in order.12 mice per group were used routinely. In the nanoparticles group,20 �l (40 �g DNA) of the nanoparticles containing DNA vaccinepRSC-gD-IL-21 were topically dropped into the eye of each mouseon days 0, 14 and 28. In other groups, each mouse was inoculatedtopically into the eye with 40 �g DNA vaccine pRSC-gD-IL-21 orpRSC-gD or mock plasmid in 20 �l PBS 3 times at two week inter-vals. Three weeks after the final immunization, 6 mice per groupwere sacrificed and used for detecting immune efficiency, and theother 6 mice were used for HSV-1 challenge experiments. Micewere anesthetized by intraperitoneal injection of sodium pento-barbital (1.5 mg/0.15 ml) and the corneas of mice were scarified10 times with 1 ml needle and the ocular mucosa of each eyewere dropped with HSV-1 (10 �l of 5 × 106 plaque forming unit(PFU)/ml). After the inoculation of the virus into the eyes, mice wereevaluated for clinical signs of epithelial or stromal keratitis from day1 to day 15 with an ophthalmological slit lamp microscope. Epithe-lial lesions were scored as follows: 0, no epithelial lesion; 1, stellatekeratitis; 2, dendritic keratitis occupying less than one quarter ofthe cornea; 3, dendritic keratitis occupying one quarter to one halfof the cornea; and 4, dendritic keratitis extending over more thanone half of the cornea. Stromal lesions also were scored as follows:0, normal; 1, slight opacity of the stroma; 2, opacity and edema ofthe stroma confined to less than one half the diameter of the cornea;3, opacity and edema of the stroma extending over more than onehalf the diameter of the cornea; and 4, severe stromal opacity andedema [6,26,27].

2.6. Histopathology

The typical cornea pathological changes in mice were pho-tographed. 15 days after HSV-1 challenge, mice were sacrificed andthe eye tissues were fixed in 10% formalin, and then embedded inparaffin. Serial thin tissue sections (4 �m) were cut and mountedon SuperFrost Plus glass slides, fixed in methanol, and stained in

K. Hu et al. / Vaccine 29 (2011) 1455–1462 1457

Fig. 1. Image of the nanoparticles-Fe3O4-Glu and expressions of gD and IL-21 in transfected SP2/0 cells as well as influence of nanoparticles containing DNA on the corneaand conjunctiva in mice. (A) TEM image of the nanopartieles-Fe3O4-Glu. (B) Lane 1 shows the successful protein expressions of gD and IL-21 in SP2/0 cells transfected withthe nanoparticles containing pRSC-gD-IL-21: lane 2 or lane 3 shows the expressions of gD and IL-21 in SP2/0 cells transfected with pRSC-gD-IL-21 or mock plasmid pRSCr corneap (D) Tha panea unctiv

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espectively using Lipofectamine. (C) There were no inflammatory reactions on theanel, day 20 post-administration) compared to the normal cornea (bottom panel).fter an ocular administration of pRSC-gD-IL-21 in nanoparticles formulation (toprrows: corneal epithelium: middle arrows: corneal stroma; short arrows: subconj

ematoxylin and eosin (H&E). The slides were viewed under a Zeissxioplan light microscope at a magnification of 200× [28,29].

.7. Measurement of anti-HSV neutralizing antibody, sIgAntibody and cytokines

Three weeks after the final immunization, blood was taken fromice by retro-orbital bleeding. Sera were separated for detecting

eutralizing antibody or cytokines IL-4 and IFN-� respectively. Foreutralizing Ab assay, a twofold series dilutions of mouse sera were

ncubated with an equal volume of the virus (3 × 103 PFU/ml) forh at 37 ◦C. Residual plaque-forming units of the infective virusere assayed on Vero cell monolayers. The plates were incubated

t 37 ◦C for 72 h and the medium was removed, and stained with 1%resyl violet. The plaques were counted. Anti-virus neutralizing Abiter was determined as the reciprocal of the dilution, causing 50%laques reduction [27,28]. For secretory IgA (sIgA) Ab assay, tearamples were collected by carefully inserting a strip of swab insidehe lower lid of eyes of immunized mouse for 2 h and repeateduintic. The tears in swab were expelled into the 5 mm internaliameter tubes in 0.1 ml PBS and the tubes were put overnight at◦C, and then twofold dilutions of the tears were used in detecting

IgA by enzyme-linked immunosorbent assay (ELISA). Briefly, 96-ell flat-bottom plates were coated with a HSV-1 gD (10 �g/ml) in

00 �l NaHCO3 buffer. A 1:2 dilution of tears was added to the wellnd incubated overnight at 4 ◦C. The next steps were performed asreviously described in references [30,31]. Plates were read at unitD490 using a Fusion or Victor 3 1420 plate reader (Perkin Elmer,ellesley, MA).IL-4 and IFN-� were measured by ELISA according to the man-

facturer’s protocol (eBioscience Company, USA). To determinerecise concentrations, dilutions of the test samples were alwaysested (in triplicate). The serum samples were tested (in tripli-ate) starting at a dilution of 1:100. The cutoff value used was.1 units/ml for IFN-� and IL-4, corresponding to three standardeviations above the mean values found in the standard negativeontrol cultures [32].

.8. Measurement of cellular immune responses

The cytotoxic activities of natural killer (NK) cells andplenocytes were detected using 3-(4,5-dimethylthiazol-2-yl)-2,5-

after an ocular administration of pRSC-gD-IL-21 in nanoparticles formulation (topere were no inflammatory cell infiltrations (arrows) in the cornea and conjunctival, day 20 post-administration), the same as the normal eye (bottom panel). Longa.

diphenyltetrazolium bromide (MTT) colorimetry method [32,33].Splenocyte proliferative responses to gD were detected by thestandard [3H] thymidine incorporation assay technique. All pro-liferation assays were performed in triplicate. Data were describedas the mean counts per minute (CPM) [27,34].

2.9. Statistics analysis

Significant differences in the immune responses betweengroups were determined by Student’s t test. Bonferroni correc-tion was used where multiple comparisons were made. The meanand standard deviations of the result between groups was usedand p value <0.05 was taken as statistically significant. Analyseswere performed with the SAS9.1 statistical software package. TheKruskal–Wallis H test was used for the statistic of clinical data.

3. Results

3.1. Analysis of protein expressions in SP2/0 cells and influence ofthe nanoparticles containing plasmid DNA on cornea andconjunctiva in mice

Fig. 1A shows the TEM image of the nanoparticles-Fe3O4-Glu,whose size is around 12 nm. The nanoparticles combining DNAvaccine pRSC-gD-IL-21 were modified by PEI and then transfectedinto SP2/0 cells. We identified the expressions of gD and IL-21 inSP2/0 cells transfected with the nanoparticles containing DNA vac-cine pRSC-gD-IL-21 by Western blot. Lane 1 in Fig. 1B shows thatthe gD and IL-21 were respectively expressed in its actual sizesof 17 kDa (IL-21) and 48 kDa (gD) in the transfected SP2/0 cells. Itwas found that the transfection efficiency using nanoparticles con-taining DNA vaccine was similar to that of pRSC-gD-IL-21 usingLipofectamine as shown in Lane 2 of Fig. 1B. The mechanism ofnanoparticles containing DNA vaccine entering into the cells maybe that the nanoparticles modified by PEI with positive charge canbind DNA vaccine tightly by electrostatic interaction, and then a

penetrating power of nanoparticles facilitates itself together withDNA vaccine entering into cells easily [35]. The results suggestedthat the limitation of the cell membrane transport of plasmid DNAwas overcome by our prepared nanoparticles containing DNA vac-cine pRSC-gD-IL-21.

1458 K. Hu et al. / Vaccine 29 (2011) 1455–1462

Fig. 2. Serum neutralizing antibody and tear sIgA in immunized mice as determined by ELISA. (A) Serum neutralizing antibody titers in immunized mice. The average liner rs of md roni an

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epresents geometric mean (n = 6). (B) Exhibits the sIgA levels (OD490 values) in teaifferences between two groups judged by Student’s t test and corrected by Bonfer

In order to evaluate the influence of the nanoparticles con-aining plasmid DNA on the cornea and conjunctiva of mice inivo, we topically dropped the nanoparticles containing DNA vac-ine into the eyes of the mice and kept observing the mice dailyrom day 1 to day 20 post-administration. We did not find anynflammation reactions or other side effect signs on the eyesf mice through this continuous observation. The representative

ictures in Fig. 1C and D show no differences compared withhe eye of normal mouse. This data suggested that the nanopar-icles containing DNA vaccine pRSC-gD-IL-21 was developedppropriately.

ig. 3. Specific cytotoxic activities and splenocyte proliferative responses of immunized mean percentages of OD value in (A). Mean counts per minute (CPM) values represented t

For black histogram of A and B). #p < 0.05, ##p < 0.03 (For white histogram of A).

ice immunized with the different vaccines. Asterisks show statistically significantalysis ***p < 0.003, **p < 0.03, *p < 0.05.

3.2. Induction of HSV-1 neutralizing antibody and sIgA afterimmunization in mice

A higher level of anti-HSV-1 neutralizing Ab was detected in themice immunized with nanoparticles containing DNA vaccine threeweeks after the last immunization and the average titer was 1:181(Fig. 2A). Serum average neutralizing Ab titers were also elevated

significantly in both pRSC-gD-IL-21 (1:72) and pRSC-gD (1:29)immunized mice compared with the mock plasmid immunizedmice (1:2). Additionally, mice in the pRSC-gD-IL-21+ nanoparti-cles group generated the highest level of tear specific sIgA, whose

ice in vitro. The cytotoxic activities of NK cells and splenocytes were shown as thehe splenocyte proliferative responses shown in (B). ***p < 0.003, **p < 0.03, *p < 0.05

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K. Hu et al. / Vaccin

D490 value was significantly different compared with the pRSC-D-IL-21 (p < 0.05) or the pRSC-gD group (p < 0.03) or the mocklasmid group (p < 0.003) in Fig. 2B. The above mentioned datahow that the experimental vaccine elevated the specific humoralmmune responses in mice.

.3. Cellular immune responses in immunized mice

To assess the CMI responses in the immunized mice, we inves-igated the cytotoxic activities of NK cells and splenocytes as wells splenocyte proliferative responses to gD. Fig. 3A shows the NKctivity in pRSC-gD-IL-21+ nanoparticles group was the highest53.2 ± 8.17%) among four groups and the pRSC-gD-IL-21 groupanked second (39.89 ± 2.95%), followed by the pRSC-gD group19.15 ± 3.36%), and the lowest one was pRSC group (5.3 ± 1.27%).here was a significant difference between the nanoparticles groupnd the pRSC-gD-IL-21 group (p < 0.05), between the nanoparticlesroup and the pRSC-gD group (p < 0.03), and between the nanopar-icles group and the mock plasmid group (p < 0.003). There werelso a significantly powerful NK activities in the mice of pRSC-D-IL-21 group versus the mock plasmid group (p < 0.03), and theRSC-gD group versus the mock plasmid group (p < 0.05).

The splenocyte cytotoxic activity was obviously increased inhe pRSC-gD-IL-21+ nanoparticles group versus the pRSC-gD-IL-21roup (statistical significance p < 0.05), the pRSC-gD-IL-21 groupersus the pRSC-gD group (p < 0.05) and the pRSC-gD-IL-21 groupersus the pRSC group (p < 0.03), which is shown in Fig. 3A.

To know whether the immune cells of spleen in mice wereensitized by gD or not after the mice were immunized withhe different vaccines, we measured the splenocyte proliferativeesponses to gD in vitro. Fig. 3B shows that the splenocyte prolifera-ive responses to gD was increased significantly in mice immunizedith the nanoparticles containing DNA vaccine compared with theice immunized with the pRSC-gD-IL-21 (p < 0.05) or the pRSC-gD

p < 0.03) or the mock plasmid (p < 0.003).Our results suggested that the nanoparticles containing DNA

accine pRSC-gD-IL-21 could induce the immunized mice to cre-te an effective CMI that includes the elevated cytotoxic activitiesf NK cells and splenocytes as well as vigorous splenocyte prolifer-tive responses to gD compared with the pRSC-gD-IL-21 group orRSC-gD group.

.4. Cytokine production in immunized mice

In Fig. 4 (black histogram), the highest serum level of IFN-� wasignificantly enhanced in the pRSC-gD-IL-21+ nanoparticles groupp < 0.003), in the pRSC-gD-IL-21 group (p < 0.03) and in the pRSC-D group (p < 0.05) in contrast to the levels in the mock plasmidroup as well as in the pRSC-gD-IL-21+ nanoparticles group versushe pRSC-gD-IL-21 group (p < 0.05). Fig. 4 also indicates that the IL-level was elevated in mice immunized with the pRSC-gD-IL-21+anoparticles compared with the pRSC-gD-IL-21 (p < 0.05) or theRSC-gD (p < 0.03) or the pRSC group (p < 0.03). This results indi-ated that the nanoparticles containing DNA vaccine could elicitice to generate a high level cytokines in mice, in particular IFN-�,hich was increased more obvious than IL-4.

.5. Viral challenge in vaccine immunized mice

As shown in Fig. 5F, the clinical mean score of herpes keratitis inice immunized with mock plasmid pRSC was the highest among 4

roups, which means the most serious keratitis exhibits severe sign,corneal turbid, edema, vascularization and blepharitis. Keratitis

ot peaked on day 7 after viral challenge and maintained till day 15hen we sacrificed them; while the mice immunized with pRSC-

D-IL-21+ nanoparticles showed the least score of keratitis, only

in this experiment. An asterisk shows statistically significant differences betweentwo groups as judged by Student’s t test and corrected by Bonferroni analysis.***p < 0.003, **p < 0.03, *p < 0.05 (For black histogram). #p < 0.05, ##p < 0.03 (For whitehistogram).

having slight lesions and getting totally recovered by day 10; alsothe mean score of the mice immunized with pRSC-gD-IL-21 wasless than that of mice immunized with pRSC-gD or mice immunizedwith pRSC-gD respectively, which indicated the former had lessserious keratitis.

Also from the representative clinical pictures taken on day 10after viral challenge, we could not find any keratitis in the mouseof pRSC-gD-IL-21+ nanoparticles group (Fig. 5B), almost the sameas normal mouse (Fig. 5A). The mouse of pRSC-gD-IL-21 group hadsome degree keratitis (Fig. 5C) while the mouse of pRSC-gD grouphad much lager area keratitis (Fig. 5D). The mouse of pRSC grouphad the most serious keratitis with the whole cornea edema, vas-cularized (Fig. 5E).

Fig. 6 shows the representative pathological changes of thecornea and conjunctiva in mice immunized with the different vac-cines 15 day after HSV-1 strain F challenge. From the mice ofpRSC-gD-IL-21+ nanoparticles group to the mouse of pRSC group,we could tell the inflammatory cell infiltrations got more andmore in the cornea and subconjunctiva. The former had the mostminimized infiltrations while the latter had the most serious infil-trations. From the above mentioned data, we demonstrated thatthe pRSC-gD-IL-21+ nanoparticles vaccine was efficient to inhibitHSK in mouse model, which was more efficient than that of thevaccine pRSC-gD-IL-21 or pRSC-gD.

4. Discussion

The major goal of vaccine design is to induce protective immuneresponses in a naïve host so that the occurrence of infection willresult in a dramatically shortened illness to the individual and asignificantly reduced disease burden on the population [36]. In thisstudy, we used gD candidate as target antigen (a major target for

virus neutralization) and IL-21 as immune adjuvant in an attemptto enhance the immunogenicity of gD. We developed the nanopar-ticles Fe3O4 coated with glutamic acid, DNA vaccine pRSC-gD-IL-21and PEI, and evaluated the immunogenicity of DNA vaccines as wellas the protective efficacy against HSV-1 infection.

1460 K. Hu et al. / Vaccine 29 (2011) 1455–1462

Fig. 5. Different degrees of HSk in different vaccine immunized mice after HSV-1 challenge. Panel A–E: representative clinical pictures of different groups on day 10 afterinfection. A (normal control mouse): no cornea edema and opacity, with some normal tear film reflection (arrow); B (nanoparticles containing pRSC-gD-IL-21 group): nocornea edema and opacity, with some normal tear film reflection (arrow); C (pRSC-gD-IL-21 group): there is some cornea edema and opacity. The line marked the area; D( ; E (pRT ne immT was u

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pRSC-gD group): there is severe cornea edema and opacity. The line marked the areahe line marked the area. Panel F: the dynamic clinical HSK scores of different vaccihe HSK degree was scored (see Section 2). Data are means ± SD. Kruskal–wallis H

From the experiment data, we found that the nanoparticlesontaining DNA vaccine could be transported successfully intoP2/0 cells and the gD and IL-21 were expressed correctly, and

hat the transfection efficiency using nanoparticles containing DNAaccine was same as that of DNA vaccine pRSC-gD-IL-21 usingipofectamineTM 2000 reagent (Fig. 1A). The results suggested thathe barriers to plasmid DNA transport through the cell membrane

ig. 6. Different inflammation degrees in the cornea and subconjunctiva of immunized mornea and conjunctiva tissues respectively. A (normal mouse): no inflammatory cell infiew inflammatory cell infiltrations (arrows); C (pRSC-gD-IL-21-immunized mouse): manumber of, densely, inflammatory cell infiltrations (arrows) with some conjunctiva structroma and subconjunctiva were fully filled with inflammatory cell infiltrations (arrows)

SC group): there is the most severe cornea edema and opacity with vascularization.unized mice after infection. Mice were monitored daily till 15 days after infection.

sed for statistics. ***p < 0.003, **p < 0.03, *p < 0.05.

was overcome by our prepared nanoparticles, which was furtherverified by our experiment results that, mice immunized with thenanoparticles containing DNA vaccine generated the robust serum

neutralizing antibody and tear sIgA responses, the high levels ofIFN-� and IL-4, and the powerful CMI responses.

The activated NK cells and spleen lymphocytes in the immu-nized mice may play a crucial role in protection against HSV

ice 15 days after HSV-1 challenge (HE, 200×). The top and bottom panels show theltrations (arrows); B (nanoparticles containing pRSC-gD-IL-21 immunized mouse):y inflammatory cell infiltrations (arrows); D (pRSC-gD-immunized mouse): a largeture abnormality (bottom arrow); E (pRSC-immunized mouse): the whole cornealwith the utmost conjunctiva structure abnormality (bottom arrow).

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nfection. However, there are some reports that, an antiviral Tells becoming nonresponsive to viral antigens either are physi-ally deleted or persist in a nonfunctional “exhausted” state or cellunctions are rapidly aborted during chronic infection, preventingiral clearance, such as chronic lymphocytic choriomeningitis virus,uman immunodeficiency virus, etc. [37,38]. Thus, in the murineSK model, the immune defense induced by the nanoparticles con-

aining DNA vaccine pRSC-gD-IL-21 was able to halt the spread ofhe virus from the corneal epithelium to the stroma, to efficiently

inimize the HSV-1 infected keratocytes and stromal cells, and tobviously reduce corneal stromal and subconjunctival inflamma-ory cell infiltrations. Therefore, the HSK was not severe and theymptom vanished quickly in mice (Figs. 5B and 6B). The effect ofnhibiting HSK in experimental vaccine immunized mice was bet-er than that of pRSC-gD group (Figs. 5D and 6D) or pRSC-gD-IL-21roup (Figs. 5C and 6C).

It is known that the helper T lymphocyte type 1 immuneesponse provides protection from chronic virus infection, and thathe IL-21 could induce secondary cytokine production, particu-arly IFN-� [21]. The increased IFN-� can efficiently enhance theytotoxic activities of T lymphocytes and NK cells, enhancing theymphocyte immune responses to target cells [21,39]. For this rea-on, we detected the serum IFN-� concentration in vaccinated mice.he results indicated that the serum IFN-� concentration was theighest in the pRSC-gD-IL-21+ nanoparticles immunized mice, anduggested that the increased IFN-� may be secreted by the NK cellsnd the splenocytes in turn the IFN-� may also react on the spleno-ytes and the NK cells, enhancing their cytotoxic activities, playingkey biological role in suppressing the infection of HSV-1.

To our knowledge, the IL-4 level is closely related to antibodyeneration. Further immunologic analysis clearly revealed that theevels of serum IL-4, neutralizing Ab and sIgA Ab were elevated in

ice immunized with the pRSC-gD-IL-21+ nanoparticles comparedith other groups. Although the Ab biological function of clearingSV infection is not as efficient as CMI, it is still associated with CMI

esponses to HSV-1 infection and inhibition of HSK.It is also known that the mucosal immune system provides an

mportant primary defense against mucosal infection and one ofajor components in lacrimal fluid is sIgA that contributes to the

rst line of defense against infection of the ocular surface [40,41].he pathogen-specific sIgA in tears may be accepted as first mucosalmmune response characteristic in HSV infected mice, therefore,

e detected the sIgA However, the specificity of these sIgA in tearsas not investigated in much detail. From the detected sIgA, weay have a hypothesis that the high level of sIgA in tears (Fig. 2B)akes a key function of preventing HSV-1 infection and inhibitingSK in mouse model.

Given the growing use of plasmid-based immune adjuvants tomprove the immunogenicity and efficacy of DNA vaccines, moredjuvant agents need to be further detailed studied. Using a mouseodel of HSV-1 infection, we demonstrated that IL-21 is an essen-

ial component of NK cell and splenocyte activation. In the absencef IL-21 DNA vaccine group (pRSC-gD), though anti-HSV Ab andytokines as well as CMI were augmented, this immune responsesere insufficient to suppress the HSK with respect to clinical scores

Fig. 5D and F). Thus, IL-21 specifically sustains humoral and CMIctivities and provides an important new immune molecule adju-ant for DNA vaccines.

In summary, our findings show that the developed nanopar-icles effectively transported plasmid DNA vaccine into cells andhat an ocular mucosal administration of nanoparticles containingNA vaccine pRSC-gD-IL-21 indicates a promise in the inhibitionf HSK. Also we concluded that IL-21 is a promising immune adju-

ant to enhance immunogenicity of DNA vaccine eliciting powerfulmmune responses. This approach will facilitate the developmentf more effective vaccines to inhibit or eliminate HSK in the future.

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2011) 1455–1462 1461

Acknowledgments

We are grateful to Professor Yuexi Li (Institute of MilitaryMedicine, Nanjing Military Command, China) for kindly gifting theGlycoprotein D of Herpes simplex virus type 1. This study was sup-ported by the Program for Top Researchers in Six Fields in JiangsuProvince, China (No. D14) and in part by the 973 Program of China(No. 2011CB933500).

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