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Comparative Immunology, Microbiology and Infectious Diseases 34 (2011) 335– 345

Contents lists available at ScienceDirect

Comparative Immunology, Microbiologyand Infectious Diseases

j o ur nal homep age : w ww.elsev ier .com/ locate /c imid

ystemic and mucosal immunity induced by attenuated Salmonellanterica serovar Typhimurium expressing ORF7 of porcineeproductive and respiratory syndrome virus

oung Woo Hana, Seong Bum Kima, Masudur Rahmana, Erdenebileg Uyangaaa,yung Min Leea, Jin Hyoung Kimb, Ki In Parkb, Jin Tae Hongc, Sang-Bae Hanc,eong Kug Eoa,∗

College of Veterinary Medicine and Bio-Safety Research Institute, Chonbuk National University, Jeonju 561-756, Republic of KoreaDepartment of Biology, College of Natural Science, Chonbuk National University, Jeonju 561-756, Republic of KoreaCollege of Pharmacy and Medical Research Center, Chungbuk National University, 410 Seongbong, Heungduk, Cheongju, Chungbuk 361-763, Republic oforea

r t i c l e i n f o

rticle history:eceived 12 December 2010eceived in revised form 22 March 2011ccepted 4 April 2011

eywords:ttenuated Salmonella vaccineorcine reproductive and respiratoryyndromeucosal immunity

ystemic immunity

a b s t r a c t

Oral administration of attenuated Salmonella vaccine may provide valuable advantagessuch as low cost, easy preparation, and safety. Attenuated Salmonella vaccines also serveas carriers of foreign antigens and immunomodulatory cytokines. Presently, an attenuatedSalmonella enterica serovar Typhimurium strain was used as a carrier for open readingframe 7 (ORF7) protein of porcine reproductive and respiratory syndrome virus (PRRSV), aswine pathogen of significant global economic importance. Initially, an attenuated S. enter-ica serovar Typhimurium expressing ORF7 gene derived from PRRSV Korean isolate wasconstructed. Following oral administration of a single dose of the attenuated Salmonellavaccine expressing PRRSV ORF7, humoral and cell-mediated immune responses specificfor ORF7 were induced at both systemic and mucosal sites including spleen, mesentericlymph node, Peyer’s patch, and laminar propria, as evaluated by determining serum ORF7-specific IgG and mucosal IgA responses, as well as Th1- and Th2-type cytokine production

from antigen-stimulated T cells. The induced humoral responses were sustained for atleast 12 weeks post-immunization. In particular, the immunized mice displayed immuneresponses to both the foreign ORF7 antigen and Salmonella itself. The results indicate thevalue of attenuated S. enterica serovar Typhimurium as an oral carrier of PRRSV antigenicproteins to induce effective systemic and mucosal immunity.

. Introduction

Orally administered Salmonella enterica serovaryphimurium colonizes the gut-associated lymphoidissue (Peyer’s patch) as well as secondary lymphatic

∗ Corresponding author. Tel.: +82 63 270 3882; fax: +82 63 270 3780.E-mail address: vetvirus@chonbuk.ac.kr (S.K. Eo).

147-9571/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.oi:10.1016/j.cimid.2011.04.001

© 2011 Elsevier Ltd. All rights reserved.

tissue (spleen), thereby eliciting systemic and mucosalimmune responses against Salmonella [1]. AttenuatedSalmonella vaccines constructed by crippling genesrequired for virulence are genetically modified to expressimportant antigen(s) of the target pathogen [2,3]. Theresulting recombinant Salmonella vaccines induce immu-nity to the expressed antigenic protein, as well as to

Salmonella itself.

It is essential that the foreign antigen-specifying plas-mids in Salmonella vaccines be stably maintained forthe expression of foreign protein during the in vivo

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colonization process. As well, the plasmid must lack antibi-otic resistance genes because of a reduced reliance onin-feed antibiotics, if the vaccine is destined for use inlive-stock. To this end, aspartate �-semialdehyde dehy-drogenase (asd) has been targeted as an essential factorfor a balanced-lethal host vector that functions to stablymaintain such multicopy plasmids [4,5]. Asd+ plasmids areretained in vivo in attenuated Salmonella vaccine strainswith the asd gene deleted [4,5]. Furthermore, an Asd+

plasmid was designed to translocate foreign proteins intothe periplasmic space of the Salmonella vaccine strain,and the synthesized proteins subsequently reached thesupernatant fluid without cell lysis [6]. This recombinantattenuated Salmonella vaccine that secretes the target for-eign protein may be a promising means of modulatingvarious infectious diseases in livestock.

Porcine reproductive and respiratory syndrome (PRRS)caused by infection with PRRS virus (PRRSV) manifestsas reproductive failure in pregnant sows and respiratorytract illness in young piglets, exerting significant economiclosses on the global swine industry [7,8]. The virus has alinear, single-stranded RNA genome of about 15 kb thatcontains nine open reading frames (ORFs). Two of theORFs encode polymerase (ORF1a and ORF1b) and sevenencode structural genes (ORF2a, 2b, 3, 4, 5, 6, and 7) [9–12].Vaccine-mediated control of PRRSV is challenging due tothe significant antigenic diversity known to exist amongall PRRSV RNA, which results in insufficient developmentof protective immunity against heterologous PRRSV strains[13]. Experimental vaccination strategies against PRRSVhave included the use of live-attenuated virus vaccines[14–16], inactivated whole virus vaccine [17], and DNAvaccines encoding ORFs of PRRSV [18,19]. ORF4 and ORF5,which encode GP4 and GP5, respectively, are representa-tive antigens that induce neutralizing antibodies, whichmay be one of the factors contributing to protection againstPRRSV [20,21]. Also, the most abundant and immunogenicprotein in the virion is the 15 kDa nucleocapsid (N) protein,which is encoded by ORF7 [11,22]. ORF7 is an important tar-get for virological and serological detection by polymerasechain reaction (PCR) and enzyme-linked immunosorbentassay (ELISA) [23]. The ORF7 protein may have the capac-ity to induce humoral and cell-mediated immunity, andto reduce the viral load in infected animals [11,19,24].Furthermore, ORF7 epitopes are more conserved withinisolates within and between continents, compared to otherORFs [25,26].

The specific aims of this study were to provideinsights into the values of attenuated S. enterica serovarTyphimurium as a carrier for PRRSV ORF7 proteinto induce immunity against PRRSV using a murinemodel. Initially, we constructed an attenuated S. enter-ica serovar Typhimurium expressing PRRSV ORF7 genederived from PRRSV Korean isolate. A single administra-tion of this vaccine induced humoral and cell-mediatedimmune responses against PRRSV ORF7 at both sys-temic and mucosal lymphoid tissues. Therefore, a useful

value of attenuated S. enterica serovar Typhimuriumas carrier for PRRSV antigenic proteins contributing toinduce immune responses against PRRSV was discussedherein.

gy and Infectious Diseases 34 (2011) 335– 345

2. Materials and methods

2.1. Bacterial strains, plasmids, media, and growthconditions

Escherichia coli �6212 (F−�−�80�(lacZYA-argF) endA1recA1 hsdR17 deoR thi-1 glnV44 gyrA96 relA1 �asdA4) [5]was used as the host strain for construction of the Asd+

vectors. Attenuated S. enterica serovar Typhimurium �8501(hisG �crp-28 �asdA16), which was kindly provided byDr. H.Y. Kang (Pusan National University, Korea) [6], wasused for the delivery of PRRSV ORF7 protein. pYA3560Asd+ plasmid was derived from pYA3493 Asd+ plasmid [6]by changing pBR ori gene (origin of replication of pBR322plasmid) with p15A ori gene (origin of replication of p15Aplasmid) to allow stable maintenance in bacteria. E. coliand S. enterica serovar Typhimurium cultures were grownat 37 ◦C in Lennox broth [27], Luria–Bertani (LB) broth oron LB agar [28]. Diaminopimelic acid (DAP; Sigma–Aldrich,St. Louis, MO) was added (50 �g/ml) to induce the growthof Asd-negative bacterial strains [5]. Phosphate-bufferedsaline (PBS, pH 7.4) containing 0.01% gelatin (BSG) wasused for the suspension of Salmonella vaccines that wereconcentrated by centrifugation at 7000 × g at 4 ◦C for 5 min.

2.2. Construction of attenuated S. enterica serovarTyphimurium expressing PRRSV ORF7

Viral RNA was extracted from PRRSV Korean isolate3722 propagated in MARC-145, and employed to amplifythe gene coding for PRRSV ORF7 with RT-PCR using spe-cific primer pairs (Forward: 5′-GAA TTC CCA AAT AAC AACGGC AAG CAG-3′, Reverse: 5′-AAG CTT TTA TCA TGC TGAGGG TGA TGC TGT-3′) containing restriction sites of EcoRIand HindIII. The start codon of ORF7 in specific primerpairs was not included, since the initiating codon of the�-lactamase gene encoded by pYA series expression vec-tors of Salmonella bacteria will drive the translation ofthe PRRSV ORF7 protein. The PCR products were theninserted into pGEMT vector (Promega, Madison, WI) andthe PRRSV ORF7 gene was sequenced to identify ORFs.Subsequently, the pGEMT vector encoded with the PRRSVORF7 gene was digested with EcoRI and HindIII, after whichthe released fragment containing the PRRSV ORF7 genewas subcloned into the EcoRI and HindIII sites of pYA3493and pYA3560 expression vectors using E. coli �6212 hostsgrown in the presence of DAP. The positive colonies of E.coli �6212 harboring pYA3493 or pYA3560 were selectedin the absence of DAP. To construct Salmonella vaccineexpressing PRRSV ORF7 protein, freshly S. enterica serovarTyphimurium �8501 (1 × 108 cfu) washed extensively withsterilized ice-cold WB (WB = 10% ultra pure glycerol, 90%distilled water, v/v) were mixed with 10 pg to 0.1 �g ofpYA3493 or pYA3560 plasmid DNA on ice in a 0.2 cmcuvette and electroporated using a Gene pulser (Bio-Rad,Hercules, CA) at 12.5 kV/cm (2.5 kV, 25 �F and 200 �).The bacteria were removed from the cuvette into a ster-

ile culture tube containing 1 ml of LB broth medium andincubated with moderate shaking for 60 min at 37 ◦C. Thetransformed culture (100 �l) was then plated onto LB agarin the absence of DAP. Finally, colonies of the attenuated

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Y.W. Han et al. / Comparative Immunology, Mi

. enterica serovar Typhimurium harboring PRRSV ORF7-ncoding pYA3493 or pYA3560 vector were cultured andtored after confirmation of the coding sequences.

.3. Salmonella subcellular fractionation andmmunoblot anaylses

The periplasmic fraction was prepared by the lysozyme-smotic shock method with some modifications [29]. S.nterica serovar Typhimurium expressing PRRSV ORF7as cultured in LB broth to an optical density at 600 nm

OD600) of 0.8. The cells were recovered by centrifuga-ion at 7000 × g for 5 min, after which the supernatantuid was saved for analysis of secreted proteins. The cellellets were resuspended in 800 �l of 100 mM Tris–HCluffer (pH 8.6) containing 500 mM sucrose and 0.5 mMDTA. Lysozyme (Sigma–Aldrich) was added to a finaloncentration of 200 �g/ml, followed immediately by theddition of 3.2 ml of 50 mM Tris–HCl buffer (pH 8.6) con-aining 250 mM sucrose, 0.25 mM EDTA, and 2.5 mM MgCl2.fter gentle agitation, the suspension was incubated for5 min in an ice bath. Bacteria were removed by cen-rifugation at 7000 × g for 5 min followed by filtration ofhe supernatant through a 0.45 �m pore-size filter. Theltered fluid constituted the periplasmic fraction. Bac-eria resuspended in 4 ml of 20 mM Tris–HCl (pH 8.6)ere disrupted by two passages through a French pres-

ure cell (American Instrument Company, Silver Spring,D). Bacterial cell lysates were centrifuged at 7000 × g

t 4 ◦C for 6 min to remove unbroken cells, and theupernatant fraction was used for protein samples of cellysates. The original culture supernatants were filteredhrough a 0.22 �m pore-size filter and PRRSV ORF7 pro-ein secreted in each supernatant was precipitated with0% trichloroacetic acid (TCA) at 4 ◦C for 1 h. Prepared pro-ein samples were boiled for 5 min and then separated byodium dodecyl sulfate-polyacrylamide gel electrophore-is (SDS-PAGE). The protein bands were then visualized bytaining with 0.1% Coomassie brilliant blue R-250 (Elpis-iotech, Deajeon, Korea). For immunoblotting, proteinseparated by SDS-PAGE were transferred eletrophoreti-ally to nitrocellulose membranes. The membranes werehen blocked with a blocking buffer (PBS [pH 7.4], 3% bovineerum albumin, and 0.1% Tween 20) and incubated withouse polyclonal antibody specific for PRRSV ORF7 protein

nd then with a peroxidase-conjugated goat anti-mousemmunoglobulin G (IgG) (Southern Biotech, Birmingham,L). Immunoreactive bands were detected by the additionf chemiluminescence dye using a WEST-one TM Westernlot Detection System (iNtRON Biotech, Seoul, Korea) in theresence of hydrogen peroxide (H2O2).

.4. Immunization and sample collection

Groups of 6-week-old female BALB/c mice (n = 7 perroup) were deprived of food and water for 4 h beforehe administration of Salmonella vaccines. The attenuated

. enterica serovar Typhimurium expressing PRRSV ORF7rown in LB broth was resuspended in PBS containing 0.01%elatin. The suspension was orally administered to BALB/cice (1 × 109 cfu/mouse). Attenuated S. enterica serovar

gy and Infectious Diseases 34 (2011) 335– 345 337

Typhimurium carrying empty pYA3493 or pYA3560 vec-tor was used as a vector control. Serum samples werecollected by retro-orbital bleeding at biweekly intervals.Following centrifugation at 4000 × g for 5 min, the serumwas removed from the whole coagulated leukocytes andstored at −20 ◦C. Fecal samples collected at the indicatedweeks were weighed and suspended at a concentration of100 mg/ml in PBS containing 0.1% sodium azide. The vagi-nal lavage fluid was obtained by introducing 100 �l PBS(pH 7.2) into the vaginal canals and recovering it with amicropipette. Each sample was stored at −80 ◦C until use.

2.5. ELISA for detecting systemic and mucosal antibodies

A standard enzyme-linked immunosorbent assay(ELISA) was used to determine the levels of PRRSV ORF7-specific antibodies in the serum, feces, and vaginal lavagefluids [total immunoglobulin G (IgG), IgG1, IgG2a, andfecal and vaginal IgA]. Briefly, ELISA plates were coatedovernight at 4 ◦C with an optimal dilution (100 ng/well) ofPRRSV ORF7 protein purified from E. coli by His6-Ni NTAchelating agarose column (Peptron, Daejeon, Korea), andgoat anti-mouse IgG/IgG1/IgG2a (Southern Biotechnology)or rabbit anti-mouse IgA (Zymed, San Francisco, CA)in standard wells. The plates were washed three timeswith PBS-0.05% Tween 20 (PBST) and blocked with 3%nonfat-dehydrated milk. The samples were serially dilutedtwofold, incubated for 2 h at 37 ◦C, and then incubatedwith horseradish peroxidase-conjugated goat anti-mouseIgG/IgG1/IgG2a for 1 h. To measure the IgA levels in thefecal and vaginal lavage fluid samples, biotinylated goatanti-mouse IgA was added for 2 h at 37 ◦C, followed by theaddition of peroxidase-conjugated streptavidin (JacksonImmunoResearch Laboratories, West Grove, PA). The colorwas developed by the addition of a suitable substrate[11 mg of 2,2-azinobis-3-ethylbenzothiazoline-6-sulfonicacid (ABTS) in a mixture of 25 ml of 0.1 M citric acid,25 ml of 0.1 M sodium phosphate, and 10 �l of H2O2].The concentration of PRRSV ORF7-specific antibodies wasdetermined using a Spectra MAX340 automated ELISAreader and the SOFTmax Pro4.3 program (MolecularDevices, Sunnyvale, CA).

2.6. ELISPOT for enumerating cells producing PRRSVORF7-specific IgA

The numbers of PRRSV ORF7-specific IgA-producingcells in mesenteric lymph node (MLN), Peyer’s patch (PP),and laminar propria (LP) were determined by an ELISPOTassay, as described previously [30]. Briefly, ELISPOT plates(Millipore, Billerica, MA) were coated with PRRSV ORF7proteins (200 ng/well) and incubated overnight at 4 ◦C.After blocking with RPMI medium containing 10% FBS for3 h, the cells were prepared from MLN, PP, and LP withsome modifications [31], loaded in 100 �l volumes, andincubated for 72 h at 37 ◦C. The plates were then washedthree times with PBS and subsequently with PBST, fol-

lowed by incubation with biotinylated goat anti-mouseIgA (Zymed) for 1 h at 37 ◦C. The spots were developedwith nitro blue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate substrates after incubation with alkaline

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phosphatase–streptavidin conjugate for 1 h and counted24 h later under a stereomicroscope.

2.7. Th1- and Th2-type cytokine ELISA following in vitrostimulation of T cells

Four weeks after immunization, mice were sacrificedto prepare splenocytes and cells obtained from MLN, PP,and LP, after which prepared cells were used as respon-der cells [31]. The enriched antigen-presenting cell (APC)populations, which were obtained as described previ-ously [32], were used as stimulators following pulsingwith PRRSV ORF7 protein (100 �g/ml) for 3 h. The respon-der cells and the PRRSV ORF7-pulsed APC stimulatorswere combined at responder-to-stimulator ratios of 5:1,2.5:1, and 1.25:1 in 200 �l of RPMI medium. The culturesupernatants were harvested after 3 days of incubation. Asimilar number of responder cells were stimulated withconcanavalin A (Con A, 5 �g/ml) as a polyclonal positivestimulator for 48 h. ELISA was used to determine cytokinelevels in the culture supernatants. The ELISA plates werecoated with interleukin (IL)-2 (JES6-1A12), IL-4 (11B11),and interferon-gamma (IFN-�; R4-6A2) (BD Pharmingen,San Diego, CA) and incubated overnight at 4 ◦C. The plateswere washed three times with PBST and blocked with 3%nonfat-dehydrated milk for 2 h at 37 ◦C. The culture super-natant and standards for recombinants IL-2, IL-4, and IFN-�proteins (BD Pharmingen) were added to the plates andincubated overnight at 4 ◦C. Biotinylated IL-2 (JES6-5H4),IL-4 (BVD6-24G2), and IFN-� (XMG1.2) antibodies (BDPharmingen) were then added and further incubated for 2 hat 37 ◦C. The plates were then washed and incubated withperoxidase-conjugated streptavidin (BD Pharmingen) for1 h, followed by color development with a substrate (ABTS)solution. The concentrations of cytokines were determinedusing a Spectra MAX340 automated ELISA reader and theSOFTmax Pro4.3 program (Molecular Devices).

2.8. Statistical analysis

Where specified, the data were analyzed for statisti-cal significance using Student’s t-test. A p-value < 0.05 wasconsidered significant.

3. Results

3.1. Construction of attenuated S. enterica serovarTyphimurium expressing PRRSV ORF7

To test whether the attenuated S. enteria serovarTyphimurium could be a suitable carrier of ORF7 fororal immunization against PRRSV, we constructed atten-uated S. enterica serovar Typhimurium expressing ORF7protein of PRRSV. PRRSV ORF7 DNA fragment (372 bp)derived from PRRSV Korean isolate 3722 by RT-PCRwas revealed to belong to the North American geno-type [26], and was subsequently cloned into the EcoRI

and HindIII sites of pYA3493 and pYA3560 Asd+ plasmidvectors (Fig. 1A) that were transformed into S. enter-ica serovar Typhimurium �8501 by electroporation. Toidentify the expression of PRRSV ORF7 protein by the

gy and Infectious Diseases 34 (2011) 335– 345

attenuated S. enterica serovar Typhimurium harboringpYA3493 (pYA3493/ORF7) or pYA3560 (pYA3560/ORF7)encoding PRRSV ORF7, periplasmic, cell lysate, and cul-ture supernatant fractions of cultured S. enterica serovarTyphimurium harboring pYA3493/ORF7 or pYA3560/ORF7were prepared and examined to detect ORF7 protein ineach fraction using immunoblot analysis. The expressionof PRRSV ORF7 from S. enterica serovar Typhimurium har-boring pYA3493/ORF7 or pYA3560/ORF7 was detected at amolecular mass of 17 kDa as early as 18 h post-incubation,and gradually increased thereafter (Fig. 1B). Although alarge amount of PRRSV ORF7 protein resided in the cyto-plasm fraction (cell lysate), about half of the cellular PRRSVORF7 protein was detected in the periplasmic fraction andan appreciable amount of the protein was secreted intothe culture supernatant. In particular, S. enterica serovarTyphimurium harboring pYA3560/ORF7 showed a higherexpression of ORF7 protein in the cytoplasm and moreenhanced amount of ORF7 protein in the culture super-natant than S. enterica serovar Typhimurium harboringpYA3493/ORF7 (Fig. 1B). The result suggested that theattenuated S. enterica serovar Typhimruium harboringpYA3493/ORF7 or pYA3560/ORF7 successfully expressedPRRSV ORF7 protein, which was actively secreted insteadof being elaborated as a result of nonspecific membraneleakage or lytic cell death.

3.2. Humoral responses induced by attenuated S.enterica serovar Typhimurium expressing PRRSV ORF7

To assess humoral responses induced by attenuatedS. enterica serovar Typhimurium expressing PRRSV ORF7,a single dose of S. enterica serovar Typhimurium har-boring pYA3493/ORF7 or pYA3560/ORF7 (1 × 109 cfu) wasorally administered to 6-week-old female BALB/c mice. Agroup of mice orally administered a single dose of S. enter-ica serovar Typhimurium harboring empty pYA3493 orpYA3560 vector (1 × 109 cfu) was used as the vector con-trol. Because attenuated S. enterica serovar Typhimuriumis expected to persist in the intestinal tract for at least4 weeks [33], mice orally administered S. enterica serovarTyphimurium expressing PRRSV ORF7 were monitored foras long as 90 days. All mice orally administered a sin-gle dose of S. enterica serovar Typhimurium harboring theplasmid vector did not display any signs of disease andsurvived for the 90-day monitoring period. The sera IgGsagainst foreign antigen ORF7 was detected at 2 weeks post-immunization and gradually increased thereafter, whenthe ORF7-specific IgG levels in sera of immunized micewas measured (Fig. 2A). Notably, the ORF7-specific IgG lev-els in sera were saturated at 8 weeks post-immunizationand were sustained until 12 weeks post-immunization.Also, mice orally administered with S. enterica serovarTyphimurium harboring pYA3560/ORF7 showed highersaturated ORF7-specific IgG levels (1.5- to 2-fold increase)than mice that received S. entrica serovar Typhimuriumharboring pYA3493/ORF7 (Fig. 2A). In contrast, the anti-

body response to Salmonella lipopolysaccharide (LPS) wasnot significantly different between mice administered S.enterica serovar Typhimurium harboring pYA3493/ORF7and pYA3560/ORF7, suggesting that S. enterica serovar

Y.W. Han et al. / Comparative Immunology, Microbiology and Infectious Diseases 34 (2011) 335– 345 339

Fig. 1. Construction of live attenuated S. enterica serovar Typhimurium expressing PRRSV ORF7. (A) Diagram of periplasmic secretion Asd+ vector pYA3493and pYA3560. The expression Asd+ pYA3493 and pYA3560 vectors differ in the origin site of replication (pBR322 ori for pYA3493 and p15A ori for pYA3560).A DNA fragment encoding the �-lactamase signal sequence and 12 amino acid residues of the N terminus of mature �-lactamase of plasmid pBR322was positioned under the control of the Ptrc promoter. The nucleotide sequences of the Ptrc promoter region, �-lactamase signal sequence (bla SS) andmulticloning sites are shown. The Ptrc sequences for -35, -10 (RNA polymerase-binding site), and Shine-Dalgarno box (SD, ribosomal binding site) areindicated by blue and green boldface, and the translocation start codon (ATG) is in red boldface. An arrow within the sequence indicates the signalpeptidase cleavage site. Unique restriction enzyme sites in the multicloning site are indicated. 5ST1T2 is a transcriptional terminator. (B) Identificationof PRRSV ORF7 expression by immunoblot analysis. The PRRSV ORF7 protein expressed by S. enterica serovar Typhimurium carrying pYA3493/ORF7 andpYA3560/ORF7 was detected by imunoblotting with polyclonal PRRSV ORF7-specific antibody in culture supernatants, periplasmic space, and cell lysates.Attenuated S. enterica serovar Typhimurium carrying empty pYA3493 or pYA3560 cultured for 18 h was used as negative control. (For interpretation of thereferences to color in this figure legend, the reader is referred to the web version of the article.)

4000

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Fig. 2. Serum IgG responses to PRRSV ORF7 and S. enterica serovar Typhimurium LPS following administration of attenuated S. enterica serovar Typhimuriumexpressing PRRSV ORF7. Groups of BALB/c mice (n = 7) were orally immunized with S. enterica serovar Typhimurium expressing PRRSV ORF7, and serum IgGlevels against PRRSV ORF7 (A) and LPS (B) were determined by conventional ELISA at the indicated weeks after immunization. The graphs are expressed bythe average and standard deviation from each group. ***, p < 0.001 compared with the levels of group orally administered S. enterica serovar Typhimuriumharboring pYA3493/ORF7.

340 Y.W. Han et al. / Comparative Immunology, Microbiology and Infectious Diseases 34 (2011) 335– 345

Table 1Distribution of ORF7-specific Ig isotypes in sera of mice administered S. enterica serovar Typhimurium expressing PRRSV ORF7.

Treatmenta 4 weeks 12 weeks

IgG1b IgG2a Ratio of IgG1/IgG2a IgG1 IgG2a Ratio of IgG1/IgG2a

pYA3493 2.6 ± 1.3 0.2 ± 0.7 – 23.9 ± 11 26.3 ± 23 –pYA3560 3.8 ± 4.2 2.9 ± 1.9 – 57.1 ± 33 22.5 ± 29 –pYA3493-ORF7 451 ± 35 184 ± 29 2.45 881 ± 121 1125 ± 209 0.78pYA3560-ORF7 477 ± 67 224 ± 71 2.13 2416 ± 201 2153 ± 198 1.12

a Groups of mice (n = 7) were orally immunized with S. enterica serovar Typhimurium expressing PRRSV ORF7, and IgG isotypes, IgG1 and IgG2a, specifics after

up.

for PRRSV ORF7 were determined by conventional ELISA at 4 and 12 week

b The data represent the average and standard deviation from each gro

Typhimurium harboring different vectors for ORF7 expres-sion can elicit comparable antibody responses to SalmonellaLPS (Fig. 2B). Furthermore, mice orally administered S.enterica serovar Typhimurium harboring pYA3493/ORF7or pYA3560/ORF7 showed a similar ratio of IgG isotypes(IgG1 to IgG2a) against ORF7 protein (Table 1). The IgG1isotype against ORF7 protein was produced more than theIgG2a isotype at 4 weeks post-immunization, but the twoIgG isotypes against ORF7 displayed same level (around a1:1 ratio of IgG1 to IgG2a) at 8 weeks post-immunization.These results indicate that a single administration ofthe attenuated S. enterica serovar Typhimurium harbor-ing pYA3493/ORF7 or pYA3560/ORF7 successfully inducesantibody responses against foreign antigen ORF7 withoutboost immunization.

3.3. Mucosal responses induced by attenuated S. entericaserovar Typhimurium expressing PRRSV ORF7

To evaluate mucosal antibody responses in miceorally administered a single dose of S. enterica serovarTyphimurium expressing PRRSV ORF7, we determined thelevels of ORF7-specific IgA in fecal and vaginal lavage fluidsamples. Mice that received attenuated S. enterica serovarTyphimurium harboring pYA3493/ORF7 or pYA3560/ORF7showed significant production of ORF7-specific IgA in fecesand vaginal lavage fluid, compared with mice adminis-tered S. enterica serovar Typhimurium harboring emptypYA3493 or pYA3560 vector (Fig. 3A and B). Also, S. entericaserovar Typhimurium harboring pYA3560/ORF7 inducedhigher levels of ORF7-specific IgA than S. enterica serovarTyphimurium harboring pYA3493/ORF7. Furthermore, theproduction of ORF7-specific IgA by S. enterica serovarTyphimurium harboring pYA3493/ORF7 or pYA3560/ORF7at mucosal lymphoid tissues was confirmed by enumer-ating cells producing ORF7-specific IgA by an ELISPOTassay. Once again, it was evident that a single admin-istration of S. enterica serovar Typhimurium harboringpYA3493/ORF7 or pYA3560/ORF7 induced ORF7-specificIgA producers in mucosal lymphoid tissues including MLN,PP, and LP (Fig. 3C). Notably, mice orally administered S.enterica serovar Typhimurium harboring pYA3560/ORF7displayed significantly higher numbers of ORF7-specific

IgA-producing cells in MLN and LP than mice receiving S.enterica serovar Typhimurium harboring pYA3493/ORF7.These results further demonstrated that a single admin-istration of S. enterica serovar Typhimurium expressing

immunization.

PRRSV ORF7 via the oral route can induce mucosal IgAresponses against foreign protein ORF7, along with sys-temic antibody responses.

3.4. Cellular immune responses induced by attenuated S.enterica serovar Typhimurium expressing PRRSV ORF7

CD4+ T cell responses play an important role in induc-ing optimal antibody responses, as well as a CD8+ T cellresponse [34,35]. Therefore, we examined CD4+ T cellresponses in mice administered PRRSV ORF7-expressingS. enterica serovar Typhimurium. To this end, we deter-mined Th1/Th2-type cytokine production from enrichedT cells stimulated with ORF7 protein, which is knownto induce the predominant expansion of immune CD4+

T cells [36,37]. Following a single administration of S.enterica serovar Typhimurium harboring pYA3493/ORF7or pYA3560/ORF7, immune T cells enriched from spleenand mucosal lymphoid tissues (MLN, PP, and LP) of immu-nized mice were stimulated with syngeneic APC previouslypulsed with PRRSV ORF7 protein, and the levels of Th1/Th2-type cytokines in the supernatants of stimulated T cellswere then determined. There was no significant produc-tion of IFN-�, IL-2, and IL-4 from ORF7 protein-stimulatedCD4+ T cells in mice administered S. enterica serovarTyphimurium harboring empty pYA3493 or pYA3560 plas-mid vector. However, Th1- and Th2-type cytokines weresignificantly produced by CD4+ T cells obtained from miceadministered S. enterica serovar Typhimurium expressingPRRSV ORF7 following stimulation of PRRSV ORF7 pro-tein (Fig. 4). Notably, it is worthy to note that Th1- andTh2-type cytokines were detectably produced by stimu-lated CD4+ T cells derived from spleen as well as mucosallymphoid tissues including MLN, PP, and LP (Fig. 4). Thissuggests that a single administration of attenuated S. enter-ica serovar Typhimurium expressing PRRSV ORF7 via theoral route induces cellular immune responses in systemicand mucosal lymphoid tissues.

4. Discussion

Oral administration of attenuated invasive bacteria suchas Salmonella vaccine may provide valuable advantages

of low cost, easy preparation, and safety. Furthermore,Salmonella egress from the gut lumen via the M cells of PP,and migrate eventually into visceral lymphoid tissues suchas LN and spleen, thereby stimulating all types of immune

Y.W. Han et al. / Comparative Immunology, Microbiology and Infectious Diseases 34 (2011) 335– 345 341

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***

**

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A

C

B

Fig. 3. Mucosal IgA responses induced by S. enterica serovar Typhimurium expressing PRRSV ORF7. (A and B) PRRSV ORF7-specific IgA levels in feces andvaginal tract of mice administered with S. enterica serovar Typhimurium expressing PRRSV ORF7. Groups of BALB/c mice (n = 7) were orally immunized withS. enterica serovar Typhimurium expressing PRRSV ORF7, and PRRSV ORF7-specific IgA levels in fecal samples (A) and vaginal lavage fluids (B) collected atthe indicated weeks following immunization were determined by ELISA. (C) Enumeration of cells producing PRRSV ORF7-specific IgA in mucosal lymphoidt teric lymd hs reprep s.

rittuaep[orstSisIadcmToot

issues. The number of PRRSV ORF7-specific IgA-producing cells in mesenetermined by ELISPOT 2 and 4 weeks following immunization. The grap

< 0.01; ***, p < 0.001 compared between the levels of the indicated group

esponse including mucosal, antibody- and cell-mediatedmmunity [1]. Attenuated Salmonella vaccines were ini-ially developed as a live vaccine for human and animalso diseases caused by Salmonella infection. Recently, these of attenuated Salmonella vaccine as a carrier for foreignntigens as well as immunomodulatory cytokines has beenxtended to provide effective immune responses againstathogens at both mucosal and systemic lymphoid tissues38,39]. The present results also demonstrate the valuef attenuated S. enterica serovar Typhimurium as a car-ier for foreign antigen ORF7 of PRRSV, which exerts aignificant economic impact on the global swine indus-ry. In particular, a single oral administration of attenuated. enterica serovar Typhimurium expressing PRRSV ORF7nduced immune responses at both systemic and mucosalites, which lasted at least 12 weeks post-immunization.mmune responses to both foreign antigen PRRSV ORF7nd Salmonella antigen were evident, as evaluated byetermining anti-LPS and ORF7 IgG levels. Furthermore,ell-mediated immunity was induced at both systemic anducosal lymphoid tissues such as spleen, MLN, PP, and LP.

herefore, our results provide insight into the valuable usef attenuated S. enterica serovar Typhimurium as a carrierf PRRSV-derived foreign antigens that may play a role inhe induction of protective immunity against PRRSV.

ph node (MLN), Peyer’s patch (PP), and laminar propria (LP) of mice wassent the average and standard deviation from each group. *, p < 0.05; **,

In the development of attenuated Salmonella-basedmultivalent vaccines, the stable maintenance of antigen-encoding plasmid DNAs in Salmonella vaccines during thein vivo colonization process and translocation of specifiedantigens into the cell envelope or secretion from bacteria isrequired to increase the strength of immune responses. Forthe former issue, we used two types of expression vectors(pYA3493 and pYA3560) that are distinct from each otherconcerning their origin site of replication. The pYA3560Asd+ plasmid was derived from pYA3493 Asd+ plasmidDNA by replacing the pBR ori gene (replication origin ofpBR322) with the p15A ori gene (replication origin of p15Aplasmid) to allow more stable maintenance of the plas-mid in bacteria (Fig. 1A). Attenuated S. enterica serovarTyphimurium harboring pYA3560/ORF7 more abundantlyproduced ORF7 protein (2–5-fold increase) than S. enter-ica serovar Typhimurium harboring pYA3493/ORF7, whichelicited stronger immune responses, especially in ORF7-specific antibody levels (Fig. 2A). A signal sequence plusan additional 12 amino acids of mature �-lactamase arerequired to translocate �-lactamase through the cytoplas-

mic membrane of gram-negative bacteria [40,41]. Thus,fusion of a protein to the �-lactamase signal peptide isexpected to promote the secretion of the fusion proteininto the bacterial periplasm [41,42]. For the latter issue,

342 Y.W. Han et al. / Comparative Immunology, Microbiology and Infectious Diseases 34 (2011) 335– 345

g/m

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Fig. 4. Th1- and Th2-type cytokines produced from T cells of mice administered with S. enterica serovar Typhimurium expressing PRRSV ORF7. Groupsof BALB/c mice (n = 4) were orally immunized with S. enterica serovar Typhimurium expressing PRRSV ORF7. After 4 weeks, T cells enriched from spleen,MLN, PP, and LP of immunized mice were stimulated by APCs that had been previously pulsed with purified ORF7 protein (100 �g/ml). The Th1- and Th2-cytokine levels in the supernatants of cultures were determined by ELISA after 72 h of stimulation. A similar number of responder cells were stimulatedwith concanavalin A (Con A, 5 �g/ml) as a polyclonal positive stimulator for 48 h. The graphs represent the average and standard deviation of cytokine levelsobtained from three individual experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with group orally administered S. enterica serovar Typhimuriumharboring empty pYA3493 or pYA3560 vector.

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he presently-constructed pYA3493 and pYA3560 plasmidNAs were designed to use for the periplasmic secre-

ion of recombinant antigens for antigen delivery by thealmonella vaccine. We reasoned that PRRSV ORF7 pro-ein attached to the �-lactamase signal peptide shoulde secreted into the periplasm of Salmonella vaccinetrains. Indeed, about half of the cellular RRSV ORF7rotein was detected in the periplasmic fraction and aignificant amount of PRRSV ORF7 protein was secretednto the culture supernatant (Fig. 1B). Notably, moreRRSV ORF7 protein was detected in the culture super-atant of attenuated S. enterica harboring pYA3560/ORF7,hich showed higher expression of ORF7 protein in the

ytoplasm.The immunogenicity and appropriate subcellular loca-

ion of PRRSV ORF7 in the Salmonella vaccine mayontribute to an augmented immune response by facili-ating adequate exposure of PRRSV ORF7 antigen to APCs43]. Mice orally immunized with a single dose of S. enter-ca serovar Typhimurium expressing PRRSV ORF7 showedrolonged immune responses to both PRRSV ORF7 antigennd Salmonella itself. In particular, mucosal ORF7-specificgA responses were observed, as evaluated by determiningRF7-specific IgA levels in feces and vaginal lavage flu-

ds, and enumerating cells producing ORF7-specific IgA inucosal lymphoid tissues (MLN, PP, and LP) (Fig. 3). Detec-

ion of PRRSV ORF7-specific IgG in sera from mice orallymmunized with S. enterica serovar Typhimurium express-ng PRRSV ORF7 (Fig. 2) indicates that the Salmonellaaccine likely reached appropriate lymphoid tissues totimulate a systemic immune response. It is notewor-hy that a single administration of attenuated S. entericaerovar Typhimurium expressing PRRSV ORF7 via the oraloute induced ORF7-specific antibody responses at bothystemic and mucosal sites, because systemic IgG anducosal IgA immunity may act as primary contributors to

revent natural PRRSV infection [44]. However, the delayedesponses to ORF7 protein in mice administered orally. enterica serovar Typhimurium expressing PRRSV ORF7ere induced with the levels significantly detectable at

round 4 weeks post-immunization, compared with ORF7-pecific responses that appear between 1 and 2 weeksfter natural infection of PRRSV [11,22]. This result is sup-orted by a previous description of antibody responsesetectable at 4 weeks post-immunization using pneumo-occal PspA foreign antigen [6]. Conceivably, it is possiblehat this phenomenon might be caused by the intact naturef Salmonella vaccine as carrier of foreign antigens. Also, theomparable levels of anti-LPS IgG in sera from mice orallydministered S. enterica serovar Typhimurium harboringmpty vector and pYA3493/ORF7 or pYA3560/ORF7 sug-est that PRRSV ORF7-specific immunity did not interfereith immunity against the Salmonella vaccine itself.

PRRSV infection is manifested by persistence and theeterred clearance of virus due to the delay of cell-ediated immunity represented by IFN-� and neutralizing

ntibody production in infected pigs [45,46]. Results from

he use of PRRSV-modified live vaccines support the viewhat cell-mediated immune responses are the primary pro-ective immunity [47]. It has been previously reported thathe PRRSV ORF6 (M) protein induces the strongest T cell

gy and Infectious Diseases 34 (2011) 335– 345 343

responses, whereas the PRRSV ORF7 protein induces a rel-atively weak T cell-stimulating capacity [48]. However, therole of cell-mediated immunity against ORF7 in protec-tion remains controversial. While some investigators favora possible role of ORF7 in cell-mediated protection, oth-ers suggest no protection induced by ORF7 alone [18,24].A recent study supported the former view and suggestedthat immunity raised by ORF7 alone may confer partial pro-tection against lung lesions and decrease the number ofinfected cells in the lung [49]. Also, the recent identificationof immunodominant T-cell epitopes in the ORF7 proteinimplies that this protein performs an important functionin cell-mediated immunity to PRRSV [50]. However, thepresent data could not address the contribution of ORF7-specific immune responses raised by oral administrationof attenuated S. enterica serovar Typhimurium expressingPRRSV ORF7 to protection against PRRS, owing to the lack ofPRRSV challenge model using mouse. Further studies needto address the role of PRRSV-specific immune responsesinduced by Salmonella vaccine expressing PRRSV antigenssuch as ORF5, 6, and 7 in the protection against PRRSVinfection.

The antibody response during PRRSV infection, in par-ticular neutralizing antibodies, may be responsible for virusclearance and recovery from infection [45]. A previousstudy using DNA vaccines reported that ORF4, ORF5, ORF6,and ORF7 induce antibody responses [18]. Although anti-bodies to ORF7 are the most abundant, they generally donot have neutralizing activity and may function as a “decoy”to shift the host immune response to protein regions thatgenerate protective immunity [18,51]. Instead, antibodiesagainst ORF5 function as the most effective neutralizingantibodies [20]. However, a previous report showed thatcomplement-mediated virus neutralizing antibodies weredetectable following PRRSV challenge of pigs immunizedwith ORF7-encoding DNA vaccine [18]. Although this typeof antibody was not addressed in this study, it is conceiv-able that the ORF7-specific antibody induced by S. entericaserovar Typhimurium expressing PRRSV ORF7 may conferpartial protection against PRRSV challenge by this mech-anism. However, the collaborated antibodies induced byother ORFs such as ORF4, ORF5, and ORF6 may be requiredfor effective protective immunity against natural PRRSVinfection.

Attenuated S. enterica serovar Typhimurium is a well-characterized vaccine strain available to livestock industryfor the prevention of salmonellosis. This attenuatedSalmonella strain has potential for heterologous antigendelivery in livestock vaccination [52]. Furthermore, sincethe Salmonella bacteria used in this study were devoid ofthe asd gene that is essential for a balanced-lethal host-vector system, they may have been sufficiently attenuatedin their capacity to cause acute disease in immunized ani-mals. Another attenuated Salmonella strain (SL7207) hasbeen also reported to be a carrier for the delivery of DNAvaccine against PRRSV [53]. It is expected that more effec-tive immune responses will be induced if these strains are

employed for prime-boost vaccination against PRRSV. Anattenuated Salmonella vaccine designed to deliver PRRSVantigen is expected to persist in the intestine for at least4 weeks, thereby providing foreign antigens to induce

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344 Y.W. Han et al. / Comparative Immunology, Mi

immune responses in hosts [33], although the duration andstability of foreign protein expression after oral administra-tion of Salmonella bacteria was not addressed in this study.Conceivably, broad reactive immunity including antibody-mediated, cell-mediated, and mucosal immune responsesmay be primarily needed for the effective control of per-sistent PRRSV infection. Therefore, the cocktail vaccinationof attenuated S. enterica serovar Typhimurium expressingORF7 as well as other structural proteins (ORF4, ORF5, andORF6) may provide much more effective protective immu-nity against natural PRRSV infection.

Acknowledgments

This study was supported by Grant No. RTI05-03-02 from the Regional Technology Innovation Program ofthe Ministry of Commerce, Industry and Energy (MOCIE),Mid-career Research Program through NRF grant fundedby the MEST (2010-0000134, 2010-0029108), the Med-ical Research Center program (2010-0029480) throughthe National Research Foundation of Korea. This studywas supported in part by a research grant from the Bio-Safety Research Institute, Chonbuk National University.The authors thank Dr. H.Y. Kang (Pusan National Univer-sity, Korea) for supplying pYA3493 and pYA3560 vector andcritical suggestions.

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